Chiral Separations: Methods and Protocols [3rd ed.] 978-1-4939-9437-3;978-1-4939-9438-0

Table of contents :
Front Matter ....Pages i-xiv
Recognition Mechanisms of Chiral Selectors: An Overview (Gerhard K. E. Scriba)....Pages 1-33
Enantioseparation by Thin-Layer Chromatography (Rituraj Dubey, Ravi Bhushan)....Pages 35-44
Enantioseparations by Gas Chromatography Using Porous Organic Cages as Stationary Phase (Sheng-Ming Xie, Jun-Hui Zhang, Li-Ming Yuan)....Pages 45-55
Chiral Metabolomics Using Triazine-Based Chiral Labeling Reagents by UPLC-ESI-MS/MS (Toshimasa Toyo’oka)....Pages 57-79
Chiral Mobile-Phase Additives in HPLC Enantioseparations (Lushan Yu, Shengjia Wang, Su Zeng)....Pages 81-91
Polysaccharide-Based Chiral Stationary Phases for Enantioseparations by High-Performance Liquid Chromatography: An Overview (Bezhan Chankvetadze)....Pages 93-126
HPLC Enantioseparations with Polysaccharide-Based Chiral Stationary Phases in HILIC Conditions (Roberto Cirilli)....Pages 127-146
Functional Cyclodextrin-Clicked Chiral Stationary Phases for Versatile Enantioseparations by HPLC (Jie Zhou, Jian Tang, Weihua Tang)....Pages 147-157
HPLC Enantioseparation on Cyclodextrin-Based Chiral Stationary Phases (Xiaoxuan Li, Yong Wang)....Pages 159-169
Hybrid Organic-Inorganic Materials Containing a Nanocellulose Derivative as Chiral Selector (Liang Zhao, Hui Li, Shuqing Dong, Yanping Shi)....Pages 171-181
Cyclofructans as Chiral Selectors: An Overview (Garrett Hellinghausen, Daniel W. Armstrong)....Pages 183-200
High-Performance Liquid Chromatography Enantioseparations Using Macrocyclic Glycopeptide-Based Chiral Stationary Phases: An Overview (István Ilisz, Tímea Orosz, Antal Péter)....Pages 201-237
Application of Sub-2 Micron Particle Silica Hydride Derivatized with Vancomycin for Chiral Separations by Nano-Liquid Chromatography (Chiara Fanali, Salvatore Fanali)....Pages 239-250
Cinchona Alkaloid-Based Zwitterionic Chiral Stationary Phases Applied for Liquid Chromatographic Enantiomer Separations: An Overview (István Ilisz, Attila Bajtai, Antal Péter, Wolfgang Lindner)....Pages 251-277
Enantioseparations by High-Performance Liquid Chromatography Based on Chiral Ligand Exchange (Federica Ianni, Lucia Pucciarini, Andrea Carotti, Roccaldo Sardella, Benedetto Natalini)....Pages 279-302
Applications of Chiral Supercritical Fluid Chromatography (Emmanuelle Lipka)....Pages 303-319
Chiral Separations by Countercurrent Chromatography (Sheng-Qiang Tong)....Pages 321-337
Cyclodextrins as Chiral Selectors in Capillary Electrophoresis Enantioseparations (Gerhard K. E. Scriba, Pavel Jáč)....Pages 339-356
Application of Dual-Cyclodextrin Systems in Capillary Electrophoresis Enantioseparations (Anne-Catherine Servais, Marianne Fillet)....Pages 357-364
Enantioseparation by Capillary Electrophoresis Using Cyclodextrins in an Amino Acid-Based Ionic Liquid Running Buffer (Joachim Wahl, Ulrike Holzgrabe)....Pages 365-371
Enantioseparations in Nonaqueous Capillary Electrophoresis Using Charged Cyclodextrins (Anne-Catherine Servais, Marianne Fillet)....Pages 373-381
Chiral Separation by NACE Using Polyol Derivative–Boric Acid Complexes (Lijuan Wang, Xu Hou, Fan Zhang, Ying Liu, Yimeng Ren, Hongyuan Yan)....Pages 383-389
Chiral Capillary Electrophoresis-Mass Spectrometry (María Castro-Puyana, María Luisa Marina)....Pages 391-405
Enantioseparation of Selected Imidazole Drugs Using Dual Cyclodextrin-Modified Micellar Electrokinetic Chromatography (Wan Aini Wan Ibrahim, Siti Munirah Abd Wahib, Dadan Hermawan, Mohd Marsin Sanagi)....Pages 407-416
Carbohydrate-Based Polymeric Surfactants for Chiral Micellar Electrokinetic Chromatography (CMEKC) Coupled to Mass Spectrometry (Vijay Patel, Shahab A. Shamsi)....Pages 417-444
Application of an (18-Crown-6)-2,3,11,12-Tetracarboxylic Acid-Based Chiral Stationary Phase in Capillary Electrochromatography (Wonjae Lee, Kyung Tae Kim, Jong Seong Kang)....Pages 445-452
Experimental Design Methodologies for the Optimization of Chiral Separations: An Overview (Luiz Carlos Klein-Júnior, Debby Mangelings, Yvan Vander Heyden)....Pages 453-478
Back Matter ....Pages 479-487

Citation preview

Methods in Molecular Biology 1985

Gerhard K. E. Scriba Editor

Chiral Separations Methods and Protocols Third Edition

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651

Chiral Separations Methods and Protocols Third Edition

Edited by

Gerhard K. E. Scriba Department of Pharmaceutical Chemistry, University of Jena, Jena, Germany

Editor Gerhard K. E. Scriba Department of Pharmaceutical Chemistry University of Jena Jena, Germany

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-9437-3 ISBN 978-1-4939-9438-0 (eBook) https://doi.org/10.1007/978-1-4939-9438-0 © Springer Science+Business Media, LLC, part of Springer Nature 2019 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana Press imprint is published by the registered company Springer Science+Business Media, LLC part of Springer Nature. The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.

Dedication To Beate, Sabrina, and Rebecca.

Preface What can more resemble my hand or my ear, and be more equal in all points, than its image in a mirror? And yet, I cannot put such a hand as is seen in the mirror in the place of its original; for if the one was a right hand, then the other in the mirror is a left, and the image of the right ear is a left one, which can never take the place of the former. —Immanuel Kant Prolegomena to Any Future Metaphysics That Will Be Able to Come Forward as Science (1783)

As stated by Immanuel Kant in 1783 in his discourse on metaphysics [1], a chiral object and its mirror image, although looking alike, are nevertheless incongruent. This epistemological analysis of a philosopher is also true in nature with regard to the interaction of chiral compounds with chiral biological target molecules. Thus, the enantiomers of chiral compounds often differ in their biological, pharmacological, toxicological, and/or pharmacokinetic profile. This has become evident specifically in pharmaceutical sciences, but it also affects chemistry, biology, food chemistry, forensics, etc. Consequently, analytical techniques capable of differentiating stereoisomers, specifically enantiomers, are required. Chromatographic and electromigration techniques are currently most often applied due to the fact that these techniques do not only separate enantiomers but also diastereomers and other chemically related compounds. For analytical enantioseparations, high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are most often employed for nonvolatile analytes, while gas chromatography (GC) is preferably applied in the case of volatile analytes. In recent years, sub- and supercritical chromatography (SFC) has gained importance as an environmentally friendly technique. HPLC and SFC can also be applied to preparative scale enantioseparations. While some compounds may only be enantioseparated with one technique based on the physicochemical properties, often the analyst can choose between two or more analytical techniques for a given analyte. This requires knowledge of the strengths and weaknesses of each technique in order to select the most appropriate method for the given problem. The focus of Chiral Separations: Methods and Protocols, Third Edition, is clearly on analytical separations by chromatographic and electrophoretic techniques, although a chapter on preparative countercurrent chromatography has been included. The book does not claim to comprehensively cover each possible chiral separation mechanism but rather gives an overview and especially practically oriented applications of the most important analytical techniques in chiral separation sciences as the “trademark” of the Methods in Molecular Biology series. Some review chapters give an overview of the current state of the art in the respective field. However, most chapters are devoted to the description of the typical analytical procedures providing reliable and established procedures for the user. Critical steps are addressed in the Notes section so that the user is enabled to transfer the described method to his/her actual separation problem. Sixty-three authors from 33 research laboratories in 17 countries have contributed by sharing their insight and expert knowledge of the techniques. I would like to take the opportunity to thank all the authors for their efforts and valuable contributions.

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Chiral Separations: Methods and Protocols, Third Edition, should be helpful for analytical chemists working on stereochemical problems in the fields of pharmacy, chemistry, biochemistry, food chemistry, molecular biology, forensics, environmental sciences, or cosmetics in academia, government, or industry. Jena, Germany

Gerhard K. E. Scriba

Reference 1. Kant I (1783) Prolegomena zu einer jeden ku¨nftigen Metaphysik die als Wissenschaft wird auftreten ko¨nnen. English translation: Hatfield G (1997) Prolegomena to any future metaphysics that will be able to come forward as science. Cambridge University Press, New York

Contents Dedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1 Recognition Mechanisms of Chiral Selectors: An Overview . . . . . . . . . . . . . . . . . . Gerhard K. E. Scriba 2 Enantioseparation by Thin-Layer Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . Rituraj Dubey and Ravi Bhushan 3 Enantioseparations by Gas Chromatography Using Porous Organic Cages as Stationary Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sheng-Ming Xie, Jun-Hui Zhang, and Li-Ming Yuan 4 Chiral Metabolomics Using Triazine-Based Chiral Labeling Reagents by UPLC-ESI-MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Toshimasa Toyo’oka 5 Chiral Mobile-Phase Additives in HPLC Enantioseparations . . . . . . . . . . . . . . . . . Lushan Yu, Shengjia Wang, and Su Zeng 6 Polysaccharide-Based Chiral Stationary Phases for Enantioseparations by High-Performance Liquid Chromatography: An Overview . . . . . . . . . . . . . . . . Bezhan Chankvetadze 7 HPLC Enantioseparations with Polysaccharide-Based Chiral Stationary Phases in HILIC Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Roberto Cirilli 8 Functional Cyclodextrin-Clicked Chiral Stationary Phases for Versatile Enantioseparations by HPLC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jie Zhou, Jian Tang, and Weihua Tang 9 HPLC Enantioseparation on Cyclodextrin-Based Chiral Stationary Phases . . . . . Xiaoxuan Li and Yong Wang 10 Hybrid Organic-Inorganic Materials Containing a Nanocellulose Derivative as Chiral Selector. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liang Zhao, Hui Li, Shuqing Dong, and Yanping Shi 11 Cyclofructans as Chiral Selectors: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Garrett Hellinghausen and Daniel W. Armstrong 12 High-Performance Liquid Chromatography Enantioseparations Using Macrocyclic Glycopeptide-Based Chiral Stationary Phases: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Istva´n Ilisz, Tı´mea Orosz, and Antal Pe´ter 13 Application of Sub-2 Micron Particle Silica Hydride Derivatized with Vancomycin for Chiral Separations by Nano-Liquid Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chiara Fanali and Salvatore Fanali

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Cinchona Alkaloid-Based Zwitterionic Chiral Stationary Phases Applied for Liquid Chromatographic Enantiomer Separations: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Istva´n Ilisz, Attila Bajtai, Antal Pe´ter, and Wolfgang Lindner 15 Enantioseparations by High-Performance Liquid Chromatography Based on Chiral Ligand Exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Federica Ianni, Lucia Pucciarini, Andrea Carotti, Roccaldo Sardella, and Benedetto Natalini 16 Applications of Chiral Supercritical Fluid Chromatography . . . . . . . . . . . . . . . . . . Emmanuelle Lipka 17 Chiral Separations by Countercurrent Chromatography . . . . . . . . . . . . . . . . . . . . . Sheng-Qiang Tong 18 Cyclodextrins as Chiral Selectors in Capillary Electrophoresis Enantioseparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gerhard K. E. Scriba and Pavel Ja´cˇ 14

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Application of Dual-Cyclodextrin Systems in Capillary Electrophoresis Enantioseparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anne-Catherine Servais and Marianne Fillet Enantioseparation by Capillary Electrophoresis Using Cyclodextrins in an Amino Acid-Based Ionic Liquid Running Buffer. . . . . . . . . . . . . . . . . . . . . . . Joachim Wahl and Ulrike Holzgrabe Enantioseparations in Nonaqueous Capillary Electrophoresis Using Charged Cyclodextrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anne-Catherine Servais and Marianne Fillet Chiral Separation by NACE Using Polyol Derivative–Boric Acid Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lijuan Wang, Xu Hou, Fan Zhang, Ying Liu, Yimeng Ren, and Hongyuan Yan Chiral Capillary Electrophoresis-Mass Spectrometry. . . . . . . . . . . . . . . . . . . . . . . . . Marı´a Castro-Puyana and Marı´a Luisa Marina Enantioseparation of Selected Imidazole Drugs Using Dual Cyclodextrin-Modified Micellar Electrokinetic Chromatography. . . . . . . . . . . . . . Wan Aini Wan Ibrahim, Siti Munirah Abd Wahib, Dadan Hermawan, and Mohd Marsin Sanagi Carbohydrate-Based Polymeric Surfactants for Chiral Micellar Electrokinetic Chromatography (CMEKC) Coupled to Mass Spectrometry . . . . Vijay Patel and Shahab A. Shamsi Application of an (18-Crown-6)-2,3,11,12-Tetracarboxylic Acid-Based Chiral Stationary Phase in Capillary Electrochromatography . . . . . . . . . . . . . . . . . Wonjae Lee, Kyung Tae Kim, and Jong Seong Kang Experimental Design Methodologies for the Optimization of Chiral Separations: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Luiz Carlos Klein-Ju´nior, Debby Mangelings, and Yvan Vander Heyden

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors DANIEL W. ARMSTRONG  Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX, USA ATTILA BAJTAI  Institute of Pharmaceutical Analysis, University of Szeged, Szeged, Hungary RAVI BHUSHAN  Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India ANDREA CAROTTI  Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy MARI´A CASTRO-PUYANA  Departamento de Quı´mica Analı´tica, Quı´mica Fı´sica e Ingenierı´a Quı´mica, Facultad de Ciencias, Universidad de Alcala´, Alcala´ de Henares, Madrid, Spain BEZHAN CHANKVETADZE  Institute of Physical and Analytical Chemistry, School of Exact and Natural Sciences, Tbilisi State University, Tbilisi, Georgia ROBERTO CIRILLI  National Institute of Health, Centre for the Control and Evaluation of Medicines, Rome, Italy SHUQING DONG  Key Laboratory of Chemistry of Northwestern Plant Resources of CAS and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou, People’s Republic of China RITURAJ DUBEY  Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India CHIARA FANALI  Department of Medicine, University Campus Bio-Medico of Rome, Rome, Italy SALVATORE FANALI  PhD School in Natural Science and Engineering, University of Verona, Verona, Italy MARIANNE FILLET  Laboratory for the Analysis of Medicines, Department of Pharmaceutical Sciences, CIRM, Quartier Hoˆpital, University of Lie`ge, Lie`ge, Belgium GARRETT HELLINGHAUSEN  Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX, USA DADAN HERMAWAN  Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Jenderal Soedirman, Purwokerto, Indonesia ULRIKE HOLZGRABE  Institute of Pharmacy and Food Chemistry, University of Wu¨rzburg, Wuerzburg, Germany XU HOU  Key Laboratory of Pharmaceutical Quality Control of Hebei Province, College of Pharmaceutical Sciences, Hebei University, Baoding, China; Key Laboratory of Medical Chemistry and Molecular Diagnosis, Ministry of Education, Hebei University, Baoding, China FEDERICA IANNI  Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy WAN AINI WAN IBRAHIM  Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, Johor Bahru, Johor, Malaysia ISTVA´N ILISZ  Institute of Pharmaceutical Analysis, University of Szeged, Szeged, Hungary PAVEL JA´Cˇ  Faculty of Pharmacy in Hradec Kra´love´, Department of Analytical Chemistry, Charles University, Hradec Kra´love´, Czech Republic JONG SEONG KANG  College of Pharmacy, Chungnam National University, Daejeon, Republic of Korea

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Contributors

KYUNG TAE KIM  Food Science and Technology Major, Dong-Eui University, Busan, Republic of Korea LUIZ CARLOS KLEIN-JU´NIOR  Pharmaceutical Chemistry Research Group, Universidade do Vale do Itajaı´ (UNIVALI), Itajaı´, SC, Brazil WONJAE LEE  College of Pharmacy, Chosun University, Gwangju, Republic of Korea XIAOXUAN LI  Department of Chemical Engineering, Chengde Petroleum College, Chengde, Hebei, People’s Republic of China HUI LI  Key Laboratory of Chemistry of Northwestern Plant Resources of CAS and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou, People’s Republic of China WOLFGANG LINDNER  Department of Analytical Chemistry, University of Vienna, Vienna, Austria EMMANUELLE LIPKA  Faculte´ de Pharmacie de Lille, Inserm, U995, LIRIC, Laboratoire de Chimie Analytique, Universite´ de Lille, BP 83, Lille Cedex, France YING LIU  Key Laboratory of Pharmaceutical Quality Control of Hebei Province, College of Pharmaceutical Sciences, Hebei University, Baoding, China; Key Laboratory of Medical Chemistry and Molecular Diagnosis, Ministry of Education, Hebei University, Baoding, China DEBBY MANGELINGS  Department of Analytical Chemistry, Applied Chemometrics and Molecular Modelling, Vrije Universiteit Brussel (VUB), Brussels, Belgium MARIA LUISA MARINA  Departamento de Quı´mica Analı´tica, Quı´mica Fı´sica e Ingenierı´a Quı´mica, Facultad de Ciencias, Universidad de Alcala´, Alcala´ de Henares, Madrid, Spain BENEDETTO NATALINI  Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy ´ TIMEA OROSZ  Institute of Pharmaceutical Analysis, University of Szeged, Szeged, Hungary VIJAY PATEL  Department of Chemistry, Georgia State University, Natural Science Center, Atlanta, GA, USA ANTAL PE´TER  Department of Inorganic and Analytical Chemistry, University of Szeged, Szeged, Hungary; Institute of Pharmaceutical Analysis, University of Szeged, Szeged, Hungary LUCIA PUCCIARINI  Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy YIMENG REN  Key Laboratory of Pharmaceutical Quality Control of Hebei Province, College of Pharmaceutical Sciences, Hebei University, Baoding, China; Key Laboratory of Medical Chemistry and Molecular Diagnosis, Ministry of Education, Hebei University, Baoding, China MOHD MARSIN SANAGI  Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, Johor Bahru, Johor, Malaysia ROCCALDO SARDELLA  Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy GERHARD K. E. SCRIBA  Department of Pharmaceutical Chemistry, University of Jena, Jena, Germany ANNE-CATHERINE SERVAIS  Laboratory for the Analysis of Medicines, Department of Pharmaceutical Sciences, CIRM, Quartier Hoˆpital, University of Lie`ge, Lie`ge, Belgium SHAHAB A. SHAMSI  Department of Chemistry, Georgia State University, Natural Science Center, Atlanta, GA, USA

Contributors

xiii

YANPING SHI  Key Laboratory of Chemistry of Northwestern Plant Resources of CAS and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou, People’s Republic of China JIAN TANG  Key Laboratory of Soft Chemistry and Functional Materials, Ministry of Education, Nanjing University of Science and Technology, Nanjing, People’s Republic of China WEIHUA TANG  Key Laboratory of Soft Chemistry and Functional Materials, Ministry of Education, Nanjing University of Science and Technology, Nanjing, People’s Republic of China SHENG-QIANG TONG  College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, People’s Republic of China TOSHIMASA TOYO’OKA  Laboratory of Analytical and Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan YVAN VANDER HEYDEN  Department of Analytical Chemistry, Applied Chemometrics and Molecular Modelling, Vrije Universiteit Brussel (VUB), Brussels, Belgium SITI MUNIRAH ABD WAHIB  Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, Johor Bahru, Johor, Malaysia JOACHIM WAHL  Institute of Pharmacy and Food Chemistry, University of Wu¨rzburg, Wuerzburg, Germany SHENGJIA WANG  Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, People’s Republic of China LIJUAN WANG  Key Laboratory of Pharmaceutical Quality Control of Hebei Province, College of Pharmaceutical Sciences, Hebei University, Baoding, China; Key Laboratory of Medical Chemistry and Molecular Diagnosis, Ministry of Education, Hebei University, Baoding, China YONG WANG  Tianjin Key Laboratory of Molecular Optoelectronic Science, Department of Chemistry, School of Science, Tianjin University, Tianjin, People’s Republic of China; Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin, People’s Republic of China SHENG-MING XIE  Department of Chemistry, Yunnan Normal University, Kunming, People’s Republic of China HONGYUAN YAN  Key Laboratory of Medical Chemistry and Molecular Diagnosis, Ministry of Education, Hebei University, Baoding, China LI-MING YUAN  Department of Chemistry, Yunnan Normal University, Kunming, People’s Republic of China LUSHAN YU  Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, People’s Republic of China SU ZENG  Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, People’s Republic of China FAN ZHANG  Key Laboratory of Pharmaceutical Quality Control of Hebei Province, College of Pharmaceutical Sciences, Hebei University, Baoding, China; Key Laboratory of Medical Chemistry and Molecular Diagnosis, Ministry of Education, Hebei University, Baoding, China JUN-HUI ZHANG  Department of Chemistry, Yunnan Normal University, Kunming, People’s Republic of China

xiv

Contributors

LIANG ZHAO  Key Laboratory of Chemistry of Northwestern Plant Resources of CAS and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou, People’s Republic of China JIE ZHOU  Key Laboratory of Soft Chemistry and Functional Materials, Ministry of Education, Nanjing University of Science and Technology, Nanjing, People’s Republic of China

Chapter 1 Recognition Mechanisms of Chiral Selectors: An Overview Gerhard K. E. Scriba Abstract Stereospecific recognition of chiral molecules plays an important role in nature as the basis of the interaction of chiral bioactive compounds with the chiral target structures. In separation sciences such as chromatographic and capillary electromigration techniques, interactions between chiral analytes and chiral selectors, i.e., the formation of transient diastereomeric complexes in thermodynamic equilibria, are the basis for chiral separations. Due to the large structural variety of chiral selectors, different structural features contribute to the overall chiral recognition process. This introductory chapter briefly summarizes the present understanding of the structural enantioselective recognition processes for various types of chiral selectors. Key words Chiral separation, Chiral recognition mechanism, Chiral selector, Enantiodifferentiation, Selector-selectand complex

1

Introduction “How would you like to live in looking-glass house, Kitty? I wonder if they’d give you milk in there? Perhaps looking-glass milk isn’t good to drink?” Alice asks her cat in the book Through the Looking-Glass and What Alice Found There, published in 1871 by the British writer Lewis Carroll [1]. The question reflects on the differentiation of enantiomers as a fundamental phenomenon in nature, i.e., the stereospecific interaction of chiral compounds with their chiral biological targets. Chiral molecules play an important role in many aspects of life sciences, medical sciences, synthetic chemistry, environmental chemistry, or food chemistry as well as many other fields. With regard to separation sciences, chromatographic techniques including gas chromatography (GC), (ultra) high-performance liquid chromatography (U)HPLC, as well as super- and subcritical fluid chromatography (SFC) or capillary electromigration techniques such as capillary electrophoresis (CE), electrokinetic chromatography (EKC), micellar electrokinetic chromatography (MEKC), microemulsion electrokinetic chromatography (MEEKC), and

Gerhard K. E. Scriba (ed.), Chiral Separations: Methods and Protocols, Methods in Molecular Biology, vol. 1985, https://doi.org/10.1007/978-1-4939-9438-0_1, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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capillary electrochromatography (CEC) are the most relevant techniques. In most cases, analyte stereoisomers, typically enantiomers, are separated via interaction with a chiral selector, which is either fixed to a solid support or added to the mobile phase or the background electrolyte. This approach is based on the formation of transient diastereomeric complexes between the analyte enantiomers and the selector in thermodynamic equilibria. This introductory chapter is intended to give a basic overview of the current understanding of mechanistic interactions between chiral analytes and the most important chiral selectors including novel developments, which are also addressed in chapters such as cyclofructans or molecular organic frameworks. Due to the multitude of compounds evaluated as chiral selectors in separation sciences, the chapter cannot be comprehensive. Further information as well as detailed discussions of the chiral recognition mechanisms in separation science have been summarized, for example, in a monograph [2], book chapters [3–5], as well as review papers [6–10].

2

Recognition Mechanisms of Chiral Selectors Depending on the selector, the formation of the transient diastereomeric complexes between analytes and a chiral selector is mediated via diverse interactions including ionic interactions, iondipole or dipole-dipole interactions, π-π interactions, van der Waals interactions, halogen bonds, or hydrogen bonds [11, 12]. Strong, long-range interactions, e.g., ionic interactions, are often considered in the initial binding, which may not be stereoselective, while short-range, directional interactions such as π-π interactions and direct or water-bridged hydrogen bonds are believed to be primarily involved in stereoselective binding. Moreover, steric factors due to the spatial arrangement of the binding site of the selector as well as a conformational change upon binding of the selectand may contribute to the stereorecognition process. In the case of concave or barrel-shaped molecules such as cyclodextrins or calixarenes, expulsion of high-energy water from the cavities may also play a role in complex formation. The cavities of these selectors contain water molecules, the number depending on the size of the cavity. These water molecules are “unorganized” because they can only form a limited number of stabilizing hydrogen bonds. Therefore, there is an enthalpic driving force for complex formation with molecules because this expels the water molecules into the bulk phase where they can form more hydrogen bonds [13]. For the elucidation of the mechanisms of chiral recognition, chromatographic and electrophoretic studies with variation of the structure of the analytes have been performed in order to establish structure-separation relationships. In addition, variations of the

Chiral Recognition Mechanisms

3

mobile phase conditions in chromatography or variations of the background electrolyte composition in electrophoresis can be performed. Spectroscopic techniques comprise UV spectroscopy, fluorimetry, IR spectroscopy, (electronic) circular dichroism and vibrational circular dichroism (VCD) spectroscopy, and NMR spectroscopy [2, 7, 14–16]. Especially NMR spectroscopy including various nuclear Overhauser effect (NOE) techniques allows conclusions about the spatial proximity of atoms and substituents. Moreover, they can often be performed under conditions comparable to the separation conditions. The structures of selectorselectand complexes in the solid state can be derived from X-ray crystallography. Chemoinformatics as well as molecular modeling and molecular dynamics simulations have been applied to study the binding thermodynamics as well as for the visualization of the selector-selectand complex structures [17–19]. The elucidation of the enantiomer elution order in chromatography by computational studies has been summarized [20]. 2.1 Polysaccharide Derivatives

Polysaccharide-based chiral stationary phases have been pioneered by Y. Okamoto and coworkers. To date, they represent the most widely applied chiral stationary phases in HPLC and SFC due to their broad applicability to many structurally diverse compounds. Commercial products with the selector either coated onto the silica gel support or covalently immobilized to it are available form Chiral Technologies under the trade names Chiralcel® or Chiralpak® or from Phenomenex under the trade name Lux®. It has been estimated that the two most popular chiral stationary phases containing cellulose tris(3,5-dimethylcarbamate) (e.g., Chiralcel® OD, Chiralcel® OD-H, Chiralpak® IB, Lux® Cellulose-1) and amylose tris(3,5-dimethylcarbamate) (e.g., Chiralpak® AD, Chiralpak® IA, Lux® Amylose-1, Lux® i-Amylose-1) account for about 2/3 of the published chiral separations achieved with polysaccharide-derived selectors [21]. These stationary phases can be operated in HPLC in the normal phase (hydrocarbon-alcohol) mode, the reversed phase (aqueous-organic) mode, and the polar organic mode as well as under SFC conditions [22–25]. Cellulose and amylose are linear helical polymers consisting of D-glucose molecules linked via β-1,4 (cellulose) or α-1,4 (amylose) glycosidic bonds. The hydroxy groups of the glucose molecules are derivatized with benzoate or phenylcarbamate moieties which contain methyl groups and/or chlorine substituents in various positions on the aromatic ring. The benzoate and phenylcarbamate residues are oriented toward the outside creating chiral helical grooves, with the polar groups located deep inside the grooves near the carbohydrate backbone, while the hydrophobic aromatic moieties are located outside. In the case of amylose tris (3,5-dimethylphenylcarbamate) a left-handed 4/3 helix has been derived from NMR and computational studies [26] as well as VCD [27]. In contrast, the structure of cellulose tris

4

Gerhard K. E. Scriba

(3,5-dimethylphenylcarbamate) appears to be somewhat controversial as VCD measurements indicated a right-handed helix of the polymer as a film but a left-handed helical structure in solution in dichloromethane [27]. The chiral groove of cellulose tris(3,5dimethylphenylcarbamate) appears to be slightly larger than the groove of amylose tris(3,5-dimethylphenylcarbamate). The structures of eight commercial cellulose- and amylose-based stationary phases, i.e., cellulose tribenzoate, cellulose tris(phenylcarbamate), cellulose tris(3,5-dimethylphenylcarbamate), cellulose tris(4-chlorophenylcarbamate), cellulose tris(4-methylphenylcarbamate), cellulose tris(4-methylbenzoate), amylose tris(3,5-dimethylphenylcarbamate), and amylose tris[(S)-α-methylbenzylcarbamate], have been studied by molecular modeling [28]. 6mers of the righthanded threefold helix of the cellulose derivatives and 8mers of the left-handed fourfold amylose helices revealed differences in size and shape of the chiral grooves depending on the derivatization of the polysaccharide. This results in different orientations and binding modes and, consequently, in different enantiomer elution orders of several chiral analytes on the respective chiral stationary phases. Selector-selectand complexes are thought to be primarily mediated via hydrogen bonds to the C¼O or NH of the carbamate groups and π-π interactions with the aromatic rings as well as van der Waals forces [21–23]. The carbamate groups are located deeply inside the cavities near the carbohydrate polymer backbone and are flanked by the aromatic substituents, which may affect the access to the binding pocket via steric factors. Furthermore, the carbamate linkage allows some flexibility of the aromatic rings for maximizing π-π interactions and van der Waals forces upon binding of the solute (induced fit). In the case of halogen-substituted analytes, halogen bonding interaction contributes to the stereorecognition of the analytes [12, 29]. Especially polarizable iodine substituents served as electron donor while the carbamate carbonyl group acted as halogen bonding acceptor. Molecular dynamics simulations of polyhalogenated 4,40 -bipyridines and cellulose tris(3,5-dimethylcarbamate) as well as amylose tris(3,5-dimethylcarbamate) have also been performed and correlated to the enantiomer elution order in HPLC experiments [30]. Finally, the mobile phase composition may modulate the recognition process due to changes of the structure of the selector by affecting intramolecular H-bonds [27, 31–33]. Molecular dynamics simulations of a 12mer of amylose tris(3,5-dimethylphenylcarbamate) in heptane/2-propanol (90:10, v/v) and pure methanol revealed that the polysaccharide maintained the 4/3 left-handed helical structure in both solvent systems but its structure was more extended in the heptane/2-propanol than in methanol due to differences in the distribution of solvent molecules close to the backbone resulting in changes in the dihedral angles of the glycosidic bonds between adjacent glucose molecules [34]. Simulations of the lifetime

Chiral Recognition Mechanisms

5

Fig. 1 Energy-minimized structures of complexes of amylose tris(3,5-dimethylphenylcarbamate) (ADMPC) with (a) (1S,2R)-(+)-norephedrine (+PPA) and (b) (1R,2S)-()-norephedrine (–PPA). The dotted lines indicate H-bonds, and π refers to π-π interactions (reproduced by permission of Elsevier from ref. 36 © 2008)

of hydrogen bonds corresponded to the enantiomer elution order of a flavanone as well as the chromatographic separation selectivities observed for both mobile phases. The binding mode between selectors and analyte enantiomers has been illustrated in several studies including spectroscopic and molecular modeling techniques, e.g., [26, 35–45]. As an example, Fig. 1 shows the energy-minimized structures of the complexes between amylose tris(3,5-dimethylphenylcarbamate) and enantiomers of norephedrine (2-amino-1-phenyl-1-propanol, PPA) [36]. The stronger retained (1R,2S)-configured ()-enantiomer

6

Gerhard K. E. Scriba

displayed three interactions, two H-bonds, i.e., (polymer) NH···OH(-PPA) and (polymer)C¼O···H2N(-PPA), and one π-π interaction. In the case of the weaker bound (1S,2R)-(+)-enantiomer only two interactions, one H-bond and one π-π interaction were observed. Interestingly, the situation was opposite for cellulose tris(3,5-dimethylphenylcarbamate). The stronger bound (+)enantiomer established one H-bond and two π-π interactions with the selector, while the weaker complexed ()-enantiomer formed only one H-bond and one π-π interaction. The modeling studies were in accordance with the reversed elution order of the analyte enantiomers for the two chiral stationary phases [36]. The separation selectivity of the polysaccharide-based chiral stationary phases as well as the elution order of analyte enantiomers depended on the polysaccharide backbone (cellulose vs. amylose) as well as the nature of the substituents and their position in the aromatic ring [22–25, 46]. The recently developed chlorinated polysaccharide-based chiral stationary phases displayed alternative and in some cases superior selectivities over only methyl groups containing selectors [25, 46]. Reversal of the enantiomer elution order depending on the chromatographic mode (normal phase, reversed phase, or polar organic mode) [25, 40, 46] composition of the mobile phase, for example, with regard to acidic or basic additives or the organic modifier [25, 47–49], the water content [50], or the temperature [51] has been observed. Moreover, it has been noted that separation selectivities obtained using a column with a coated chiral selector may differ from the selectivities observed on columns with the same but covalently immobilized chiral selector [52]. A model accounting for multivalent interactions, i.e., solute adsorption by the chiral selector interactions, solute-solvent interactions, and solute intramolecular hydrogen bonding, which allows a reliable estimation of the number of the potential binding sites between the selectand and the selector, has been described [38]. Further discussion and details of the current understanding of the chiral recognition mechanism of polysaccharide-derived chiral selectors can be found in [5, 7–9, 23–25, 53]. 2.2

Cyclodextrins

Cyclodextrins (CDs) are cyclic oligosaccharides consisting of α-1,4-linked D-glucose molecules produced by the digestion of starch by cyclodextrin glycosyltransferase of various bacteria such as Bacillus strains [54]. The most important industrially produced CDs differ in the number of D-glucopyranose units; that is, α-CD is composed of six glucose molecules, β-CD of seven molecules, and γ-CD of eight molecules. CDs are shaped like hollow toroids with a lipophilic cavity and a hydrophilic outside. The wider rim contains the secondary 2- and 3-hydroxy groups, while the narrower rim features the primary 6-hydroxy groups. The top to bottom dia´ meters of the cavity of the CDs are 4.7 and 5.3 A˚ for α-CD, 6.0 and

Chiral Recognition Mechanisms

7

´ ´ 6.5 A˚ for β-CD, and 7.5 and 8.3 A˚ for γ-CD [55]. The hydroxy groups can be chemically modified resulting in a large number of derivatives. CDs have found numerous pharmaceutical, chemical, (bio)technological, food, agrochemical, cosmetic, or textile applications [56–58]. With regard to separation sciences they have been used as chiral stationary phases in GC [59, 60], HPLC [59, 61], and SFC [61]. Commercial columns for GC include DEX® columns (Supelco), Lipodex® columns (Macherey-Nagel), or ChirasilDEX® columns (Agilent Technologies). In HPLC Cyclobond® columns from Astec, ChiraDex® columns from Merck, Ultron ES-CD® columns from Shinwa, or CDShell® RSP columns from AZYP are available. Furthermore, native CDs and CD derivatives are by far the most frequently used chiral selectors in CE, MEKC, and MEEKC [62–65]. CDs can be obtained from numerous companies including Sigma-Aldrich or Cydex Inc. and, especially, from CycloLab or Cyclodextrin-Shop. The complexation between CDs and analyte molecules has been studied extensively by numerous techniques including NMR spectroscopy, mass spectrometry, X-ray crystallography, molecular modeling, as well as chromatographic and CE investigations resulting in a fairly good understanding of the complexes. This may be attributed to the fact that CDs can be studied in solution under comparable conditions by spectroscopic methods such as NMR spectroscopy and by EKC so that direct correlations between complex structures and enantioseparations can be drawn. Specifically NMR techniques such as nuclear Overhauser enhancement spectroscopy (NOESY) and rotating frame Overhauser enhancement spectroscopy (ROESY) have contributed because these methods allow the evaluation of the proximity of atoms or functional groups of guest and host molecules [16, 66, 67]. Moreover, especially CE experimental enantioseparation conditions can be perfectly mimicked in the NMR experiments. The techniques for studying CD-solute complexes in aqueous solution [68] and in the solid state [69] have been summarized. Moreover, molecular modeling approaches in combination with EKC enantioseparation studies in an attempt to understand the chiral recognition mechanism have been discussed [19]. An overview of CD complexes can be found in a monograph [56] and a Web-based database for CD-solute complexes has been established [70]. In most cases, 1:1 complexes are formed but guest-host complexes with other ratios such as 2:1, 2:2, or higher order equilibria also exist. Complexation often involves the insertion of lipophilic moieties of the guest molecules into the cavity of the CD-displacing solvent molecules (typically high-energy water) from inside the cavity [13]. Van der Waals and hydrophobic interactions are believed to be primarily involved but hydrogen bonding with the hydroxy groups and steric effects also play a role. For derivatized CDs, additional interactions such as ionic interactions in the case of

8

Gerhard K. E. Scriba

a

b

CH3 CH3

OR 6 5

N 4

N H

HO

c

H2

O

R = H; β-CD R = SO3H, HS-β-CD

2

OH

7

d

H3

H3

H2

H1, H4

H1, H4 H5

H6

e

1 3

CH3

O

H2

H3

H3

H1, H4

H1, H4 H5

H5

H6

H6

H2

H5

H6

f

Fig. 2 Structures of (a) medetomidine and (b) β-CD and heptakis(6-O-sulfo)-β-CD, schematic structures of the complexes of medetomidine with (c) β-CD and (d) with heptakis(6-O-sulfo)-β-CD derived from NMR experiments and molecular modeling structures of the (S)-(+)-medetomidine enantiomer with (e) β-CD and (f) with heptakis(6-O-sulfo)-β-CD

CDs containing charged substituents or π-π interactions in the case of CDs containing aromatic substituents have to be considered as well. Depending on the analyte structure and the CD, inclusion can occur from the narrower or wider side of the CD. This has been demonstrated by numerous studies, as summarized in [13, 67]; for recent publications see [71–81]. As an example, the complex between medetomidine and β-CD as well as heptakis(6-O-sulfo)-β-CD (HS-β-CD) is shown in Fig. 2 [81]. ROESY NMR studies indicated insertion of the phenyl moiety into the β-CD cavity from the secondary side with the imidazole ring outside the cavity exposed to solvent molecules (Fig. 2b). This is also reflected

Chiral Recognition Mechanisms

9

in the molecular modeling structure β-CD complex shown in Fig. 2d. The (S)-(+)-enantiomer of the drug formed a stronger complex than the (R)-()-enantiomer which agreed with the enantiomer migration order observed in CE. In case of HS-β-CD, the compound is positioned “upside down” with the phenyl ring inside the cavity and the imidazole moiety interacting with the sulfate groups located at the primary rim as schematically shown in Fig. 2c and the modeled structure between the (S)-(þ)-enantiomer and HS-β-CD in Fig. 2e. NMR data also suggested stronger complexation in case of the (R)-()-enantiomer in agreement with the CE data. However, it should be kept in mind that a different binding mode does not necessarily translate into a different affinity toward analyte enantiomers or a different enantiomer migration in CE. Examples for both scenarios exist. A significant effect of the enantioseparation ability of β-CD immobilized to a solid support as a function of the orientation, i.e., immobilization via the primary or the secondary side, has been observed [82]. Higher enantioresolution was observed for most analytes for the column with β-CD immobilized via the primary side leaving the wider secondary side open for analyte complexation, although exceptions existed. In few cases, the highest resolution could be obtained with a commercial Cyclobond® column with random orientation of β-CD. Reversed enantiomer elution order depending on the CD orientation was found for flavanone. Molecular dynamics simulations indicated that formation of the inclusion complex from the wider or narrower side of the CD resulted in tighter interactions of the opposite enantiomer, which explained the observed opposite enantiomer elution order in chromatography [83]. Furthermore, it has been shown that formation of an inclusion complex is not a prerequisite for CD-mediated enantioseparation. In fact, many studies have shown that so-called external complexes with CDs can lead to effective enantioseparations in CE [72, 73, 83–87]. This is schematically shown for talinolol as an example in Fig. 3 [86]. As concluded from NMR experiments, the drug formed an external complex with heptakis(2,3-di-O-methyl-6-Osulfo)-β-CD (HDMS-β-CD) in aqueous buffers, while an inclusion complex was observed in the case of heptakis(2,3-di-O-acetyl-6-Osulfo)-β-CD (HDAS-β-CD). For both CDs, separation of the talinolol enantiomers was observed in CE. External complexes resulting in enantioseparations were also concluded for propranolol [84] and bupivacaine [85] in the presence of HDAS-β-CD or enilconazole in the presence of HDMS-β-CD [72]. Moreover, the structure of the compound can depend on the nature of the background electrolyte, i.e., aqueous or nonaqueous. For example, inclusion of the aliphatic side chain of propranolol into the cavity of HDAS-β-CD from the wider secondary side occurred in a methanolic background electrolyte, while only an external complex was formed with the CD in aqueous buffers [84]. In the case of

10

Gerhard K. E. Scriba

a

Talinolol / HDMS-β-CD

H2 H4

H3 H5 H6

H3 H5 H6

b H2

H4

H2 H4

Talinolol / HDAS-β-CD

H3 H5 H6

H3 H5

H2 H4

H6

Fig. 3 Schematic structures of the complexes of talinolol with (a) heptakis(2,3-di-O-methyl-6-O-sulfo)-β-CD (HDMS-β-CD) and (b) heptakis(2,3-di-O-acetyl-6-O-sulfo)-β-CD (HDAS-β-CD) in aqueous background electrolytes as derived from NMR experiments. The arrows indicate the observed intermolecular NOEs upon irradiation of the respective protons (reproduced by permission of Elsevier from ref. 86 © 2012)

HDMS-β-CD, an inclusion complex with insertion of the naphthyl moiety of propranolol via the narrower side was assigned in aqueous solutions, while an external complex was formed in a nonaqueous background electrolyte. Similarly, talinolol formed an inclusion complex with HDAS-β-CD in aqueous electrolyte solutions, while an external complex was concluded by NMR studies for nonaqueous solutions [86]. Sometimes such different complexation led to reversal of the enantiomer migration order but not in all cases. Finally, it has been shown that buffer components [88, 89] as well as surfactants such as SDS [90–92] may be complexed by CDs, thus affecting analyte complexation and consequently the enantioseparation. The effect of solvents on the enantioseparation of amino acids in the presence of β-CD has been studied by molecular dynamics simulation [93–95]. It should be noted, however, that up to now the molecular modeling and dynamic simulations have not taken into account the abovementioned presence of high-energy water molecules in the CD cavity although a modeling study described higher binding energies in the presence of water as compared to the gas phase, i.e., in the absence of solvents [96]. 2.3

Cyclofructans

Cyclofructans (CFs) are cyclic oligosaccharides composed of β-2,1-linked D-fructofuranose units. Native cyclofructans containing 6 (CF6) or 7 fructose units (CF7) as well as the corresponding O-alkyl derivatives, acyl derivatives, carbamoyl derivatives, or sulfated derivatives have been evaluated for chromatographic or CE enantioseparations [97–99]. The inner core of CFs is not a hydrophobic cavity as in the case of CDs but has the structure of a crown ether. The molecules possess a disklike shape with an electronegative side composed of the hydroxy groups in the 3- and 4-positions of the fructofuranose units and an electropositive side formed by the 1- and 6-methylene moieties [100]. Complex formation is

Chiral Recognition Mechanisms

11

Fig. 4 Molecular modeling structures of the complexes formed between CF6 and p-aminobenzoic acid in the (a) protonated stage, (b) in the deprotonated stage, and (c) in the zwitterionic stage. The dashed lines represent the distances between interacting groups in A´˚ (reproduced by permission of Elsevier from ref. 102 © 2015)

mediated via polar interactions such as dipole-dipole interactions or hydrogen bonds depending on the nature of the CF derivative, the solute, and the operation mode of the separation technique. Comparing the complexation of L-amino acids by CF6 and CF7 by mass spectrometry and NMR spectroscopy it was noted that only protonated amino acids formed complexes with the CFs most likely with a 1:1 stoichiometry [101]. Interaction occurred with the electronegative side of CF6. Hydrogen bonds as well as ion-dipole interactions were assumed as the major driving forces for complex formation. Although investigated for the achiral compound p-aminobenzoic acid (PABA), Wang et al. noted by UV and NMR spectroscopy as well as by mass spectrometry and molecular modeling that different complexes are formed between CF6 and PABA depending on the ionization of the selectand [102]. From MS and NMR data of the protonated species the formation of the complex was postulated to occur on the electronegative side of CF6 via hydrogen bonds between the ammonium group and the hydroxy groups of the CF (Fig. 4a). The negatively charged deprotonated carboxylate species formed only a weak

12

Gerhard K. E. Scriba

complex. The authors concluded from NMR data that the crown ether moiety is also associated with the binding of the solute in such a way that the carboxylate form can bind either at the electronegative or at the electropositive side of CF6. A sodium ion coordinates with the carboxyl group and inserts into the crown ether moiety via ion-dipole interactions as shown in Fig. 4b. In the case of neutral (zwitterionic) PABA a dimeric complex composed of two 1:1 PABA-CF6 complexes was postulated as illustrated in Fig. 4c. In one of the complexes PABA interacts with CF6 via the amino group whereas the interaction occurs via the carboxylate group in the other complex. Both “halves” interact via hydrogen bond between the amino group of one PABA molecule and the carboxyl group of the other PABA molecule. Despite the two studies mentioned here, further specific investigations for the elucidation of stereospecific differences in the complexation pattern of CFs with analyte enantiomers are still lacking to date. Furthermore, it is possible to transform a neutral CF into charged selectors by the binding of metal ions, which results in efficient complexation of anionic analytes [103]. Specifically, Ba2+ ions were bound to HPLC columns containing CF6 derivatives as chiral selectors for the enantioseparation of chiral sulfonic acids and phosphoric acids. Analyte retention also depended on the counterion of the Ba2+ salts. Thermodynamic investigations indicated that analyte retention may be enthalpy driven or entropy driven or both may apply. It was concluded that the selectivity of such selectors is based on the ion-pairing principle with ionic selector-selectand interactions occurring between the complexed Ba2+ ions and the negatively charged analyte. Further interactions may include hydrogen bonding, dipole-dipole interactions, as well as steric interactions. Cyclofructan-based chiral columns have been commercialized under the trade name Larihc® by AZYP. For a comparison of HPLC enantioseparations of amines applying columns based on superficially porous particles (core shell particles) with immobilized glycopeptides, a CD, or a CF see [104]. 2.4 Macrocyclic Glycopeptides

Macrocyclic glycopeptides have been frequently applied as chiral selectors in liquid-phase enantioseparations [105–107]. The most important compounds of this group are vancomycin, ristocetin A, teicoplanin, and teicoplanin aglycone. The common structural feature of these selectors is a set of interconnected amino acid-based macrocycles, each macrocycle containing two aromatic rings and a peptide sequence. Vancomycin contains three macrocycles while teicoplanin and ristocetin A are composed of four. The macrocycles form a three-dimensional, C-shaped basketlike structure. The carbohydrate moieties are positioned at the surface. Ionizable groups such as a carboxylic acid group or amino groups are present. Thus, a large number of interactions between analyte molecules and glycopeptide antibiotics are possible including hydrogen bonds, π-π,

Chiral Recognition Mechanisms

13

dipole-dipole, and ionic interactions depending on the experimental conditions [108]. Few studies have investigated the chiral recognition mechanisms of macrocyclic antibiotic selectors toward selected chiral solutes by molecular modeling techniques [109–111]. A comprehensive investigation on the enantioseparation of chiral xanthone derivatives by vancomycin, ristocetin A, teicoplanin, and teicoplanin aglycone in the normal-phase mode, the polar organic mode, the polar inorganic mode, and the reversed-phase mode in combination with molecular docking studies has been published recently [112]. The enantiomer elution order of the compounds depended on the type of the selector as well as on the elution mode. Teicoplanin and vancomycin proved to be the most universal selectors, because most enantiomers could be resolved using either of these selectors. Molecular docking revealed the binding mode of the enantiomers, which were mainly supported by π-π interactions and hydrogen bonding. Interestingly, most analyte enantiomers were docked within the basket structure of the glycopeptides as shown for a xanthone in Fig. 5a, while the enantiomers of a

c O H N

O

O

OH

O

d O H3C

O N H

OH

O CH3

Fig. 5 Molecular modeling of the structures of xanthone derivatives with teicoplanin showing the situation for (a) both enantiomers binding inside the basket structure and (b) enantiomers binding on opposite sides of teicoplanin. The selector is shown in gray with O atoms labeled in red, N atoms in blue, and chlorine atoms in green. (S)- and (R)-enantiomers of the xanthones are colored in magenta and yellow, respectively. Hydrogen bonds are shown as dashes and π–π stacking interactions as double arrows (reproduced by permission of MDPI and C. Fernandes from ref. 112 © 2018)

14

Gerhard K. E. Scriba

xanthone containing two chiral centers bound to opposite sides of the teicoplanin molecule (Fig. 5b). In HPLC, macrocyclic glycopeptide selectors are still the second most important group of chiral stationary phases after the polysaccharide derivatives. Commercial columns are sold under the trade names CHIROBIOTIC® V (vancomycin), CHIROBIOTIC® T (teicoplanin), CHIROBIOTIC® R (ristocetin), and CHIROBIOTIC® TAG (teicoplanin aglycone). Vancomycin and teicoplanin are also available with a different binding chemistry as CHIROBIOTIC® V2 and CHIROBIOTIC® T2. In VancoShell® and TeicoShell® columns from AZYP the macrocyclic glycopeptides are immobilized to superficially porous particles (core shell particles). 2.5

Proteins

2.6 Donor-Acceptor Chiral Selectors

The well-known stereoselective interaction between chiral compounds and proteins has led to their use as chiral selectors in separation sciences [113–116]. The binding of drugs to human serum albumin has been well characterized [117]. The protein is non-glycosylated and composed of three homologous domains. Each domain consists of ten helices, which can be further subdivided into subdomains consisting of six and four helices, respectively. The protein has two major binding sites, termed site 1 (warfarin-azapropazone site) and site 2 (indole-benzodiazepine site) as well as several minor sites binding a variety of drugs and other compounds. Due to the complexity of the protein selectors numerous molecular interactions including hydrogen bonds, π-π, dipole, and ionic interactions contribute to the complexation of analytes. Further proteins used as chiral selectors include α1-acid glycoprotein, ovomucoid, or cellobiohydrolase 1, many of which have been commercialized, e.g., Chiralpak® HSA (human serum albumin, Chiral Technologies or Regis), Resolvosil® BSA (bovine serum albumin, Macherey & Nagel), Chiralpak® AGP (α1-acid glycoprotein, Chiral Technologies or Regis), Ultron ES-OVM® (ovomucoid, Shinwa Chemical or Agilent Technologies), or Chiralpak® CBH (cellobiohydrolase 1, Chiral Technologies or Regis). Donor-acceptor chiral selectors are also termed brush-type or Pirkle-type selectors after William H. Pirkle, who pioneered this type of selectors. For a review on the development of these selectors see [118]. A summary of developments of small-molecule chiral selectors including Pirkle-type selectors has been published [119]. Commercial columns besides Whelk-O1® columns are ULMO® and DACH-DNB® from Regis or Chirex® from Phenomenex. Donor-acceptor selectors contain relatively small molecules, which enable donor-acceptor interactions such as hydrogen bonding, face-to-face or face-to-edge π-π interactions, or dipole-dipole stacking. Halogen bonds in the case of halogen-containing selectands should be considered as another type of interaction

Chiral Recognition Mechanisms

15

[12, 30]. Furthermore, rigid and bulky moieties as steric barriers may further amplify chiral recognition. Particularly the (S,S)-WhelkO1 selector has been studied in detail by molecular modeling and molecular dynamics simulation [120–124] as well as X-ray crystallography and NMR [125]. The electron-deficient 3,5-dinitrophenyl moiety may be involved in face-to-face π-π (acceptor) interactions, while the electron-rich phenanthryl group may establish edge-toface π-π (donor) interactions. The aromatic moieties are oriented perpendicular so that a cleft-like binding site results. The connecting amide bond may be involved in hydrogen bond interactions [121]. Figure 6 schematically illustrates the docking modes of the Whelk-O1 selector and an analyte containing an aromatic ring and a hydrogen-bonding site [124]. The preferred docking arrangement for a longer retained enantiomer (M1 docking) involves π-π interaction with the dinitrophenyl ring and a hydrogen bond with the amide hydrogen of the selector. This binding occurs typically within the cleft but for some analytes the dinitrophenyl ring rotates to allow interaction from the side. The so-called M2 docking of many less retained analytes inside the cleft is governed by a hydrogen bond with the amide hydrogen and a π-π interaction with the phenanthryl

Phenanthryl π–π CH–π/π–π

HB Amide

Tether

π–π Dinitrophenyl

M1 inside-of-cleft

π–π HB

HB

M2

M1 Side-of-Cleft

Silica

CH–π/π–π

HB

M3

π–π

π–π

HB

CH–π/π–π

M4

Fig. 6 Schematic representation of the docking arrangements of a solute containing an aromatic moiety (yellow hexagon), and a H-bonding site (yellow circle) and a side chain (white square) on Whelk-O1. The selector is composed of a tether (white rectangle), the phenanthryl group (blue rectangle), the dinitrophenyl moiety (blue hexagon), and the amide linker (green rectangle). The nitro substituents are only shown in the M4 docking arrangement. M1 docking is subdivided according to the location of the analyte, i.e., inside of cleft and side of cleft. Solid red arrows indicate hydrogen bonding and π-π stacking interactions, while dotted red arrows refer to secondary CH-π or edge-to-face π-π interactions (reproduced by permission of Elsevier from ref. 124 © 2009)

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moiety of the selector. In case of hydrogen bond donor groups of the analyte, formation of a hydrogen bond with the amide oxygen (M3 docking) or an oxygen of one of the nitro groups (M4 docking) is observed. 2.7 Chiral IonExchange Selectors

Chiral ion-exchange stationary phases are often considered a subgroup of the brush-type (Pirkle-type) phases. They interact with ionizable analytes via ionic interactions but π-π interactions and hydrogen bonding contribute to the stabilization of the complex. Commercial ion-exchange phases are based on quinine and quinidine carbamates immobilized to a silica gel support via a sulfide linker [126–128]. Weak anion-exchange selectors have been commercialized by Chiral Technologies under the trade names Chiralpak®QN-AX (quinine) and Chiralpak®QD-AX (quinidine). Further (noncommercial) anion-exchange selectors with different immobilization chemistry have been summarized in [126, 128]. Chiral cation exchangers for the separation of basic analytes feature sulfonic acid or carboxylic acid residues [126, 129]. Recently, zwitterionic ion exchangers have been developed expanding the use of this type of selectors to acidic, basic, as well as zwitterionic analytes [126, 127]. The so-far commercialized zwitterionic chiral phases are Chiralpak® ZWIX(+) (quinine and (1S,2S)-cyclohexyl-1-amino-2-solfonic acid bridged via a carbamoyl group) and Chiralpak® ZWIX() (quinidine and (1R,2R)cyclohexyl-1-amino-2-solfonic acid). For further zwitterionic selectors see [126]. The AX selectors and the ZWIX selectors form pseudo-enantiomeric complexes with analyte enantiomers so that reversed enantiomer elution order can be achieved when switching from the quinine-based selector to the quinidine-based selector and vice versa. The enantiorecognition mechanism of Cinchona alkaloidbased exchange selectors has been studied by chromatography, NMR spectroscopy, X-ray crystallography, and molecular dynamics simulations as summarized in [7–9, 128]. NMR investigations of the quinine-based selector revealed that after protonation of the quinuclidine nitrogen and when forming a complex with an acidic analyte, the conformation of the selector transforms preferentially into the “anti-open” conformation with the quinuclidine ring pointing away from the quinoline ring. This results in a cleft allowing a negatively charged analyte to freely access the protonated quinuclidine nitrogen for the primary ionic interaction. π-π interactions between aromatic moieties of the solutes and the quinoline ring of the selector as well as hydrogen bonds with the carbamate group may stabilize the complex [130]. Studies on structure-separation relationships, e.g., [131–133], and molecular modeling including molecular dynamics simulations [134–138] have been performed in order to understand the interactions between the selectors and solutes. The structure of the

Chiral Recognition Mechanisms

c O O

Si

CH3

H N

O

S

N H

SO3

d

NHBoc COOH O

O HN

H3CO HN N

17

NH O

O

e

Fig. 7 Structures of (a) ZWIX(+) selector, (b) the (S,S)-stereoisomer of Nα-Boc-N4-(hydroorotyl)-4-aminophenylalanine, and (c) molecular modeling of the ion-pair complex. Hydrogen bonds between selector (yellow) and selectand (blue) are shown as yellow dashes. The silica-gel layer is represented as thin sticks, and mercaptopropyl-functionalized silanols are colored in green (reproduced by permission of Elsevier from ref. 137 © 2015)

zwitterionic ZWIX(+) selector is schematically shown in Fig. 7a. At acidic conditions, the nitrogen of the quinuclidine ring is protonated (pKa ~9.8) acting as a positively charged anion exchanger. The sulfonic acid group is deprotonated acting as a cation exchanger. Consequently, two ion pairs may be formed by amphoteric solutes. In addition, π-π interactions between the quinolone ring and aromatic moieties of the solutes as hydrogen bonds with the carbamate NH and/or with the charged groups of the selector are possible. Finally, steric fit into the cleft of the selector may contribute to the chiral recognition. Acid or base additives to the mobile phase are required to displace the solutes from the selector. Consequently, the nature and concentration of acid or base additives to the mobile phase as well as the resulting pH affect the elution of analytes from the chiral stationary phase. Hydrogen bonds and π-π interactions may be modulated by the type and ratio of the organic modifier. The interactions derived from molecular modeling between the ZWIX(+) selector (Fig. 7a) and the

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(S,S)-stereoisomer of Nα-Boc-N4-(hydroorotyl)-4-aminophenylalanine (Fig. 7b), which formed the strongest complex with the selector and was the most retained stereoisomer in the chromatographic experiment, are illustrated in Fig. 7c [137]. In addition to the ionic interaction between the carboxylate group of the analyte and the protonated quinuclidine nitrogen, hydrogen bonds between the sulfonate group and two of the three quasi amide bonds of the hydroorotic acid moiety appear to be formed, while the carbamate linker of the selector and the N-Boc group do not seem to participate in H-bonding. 2.8 Chiral Ligand Exchange

Chiral ligand exchange methods are based on the reversible chelate coordination of a chiral analyte into the sphere of a metal ion, which is in turn complexed with a chelating chiral selector resulting in a selectand–metal ion–selector complex. The resulting diastereomeric complexes differ in their thermodynamic stabilities or formation ratios as well as spatial shape. Amino acid derivatives are often employed as chelating agents in combination with divalent metal ions such as Cu2+, Zn2+, Ni2+, or Mn2+, but hydroxy acids such as D-quinic acid, D-gluconic acid, or L-threonic acid have also been used. Boron has been applied as central ion in combination with diols as ligands such as L- or D-tartaric acid derivatives. Considering the complex composition, the method is restricted to analytes bearing two or three electron-donating groups such as amino acids, hydroxy acids, or amino alcohols. Developments and applications of chiral ligand exchange have been summarized in [139–141]. Computational methods have been applied to the elucidation of the enantiomer elution order in chiral ligandexchange chromatography [142]. For a molecular modeling study of complexes of the ternary complexes among L-proline or trans-4-L-hydroxy-proline, Cu2+, and amino acid enantiomers see [143]. Figure 8 illustrates the structures of the diastereomeric complexes between L-proline, Cu2+, and L-isoleucine (Fig. 8a) or D-isoleucine (Fig. 8b), respectively. In addition to the abovementioned chelating selectors, selectors exploiting the ion-exchange principle such as quinine and quinidine (as mobile-phase additives) [144] as well as chiral ionic liquids [145–147] have been applied as ligand exchanges for enantioseparations. Commercial HPLC columns are based on immobilized ligands such as N,N-dioctyl-L-alanine (Chiralpak® MA(+) by Chiral Technologies), L-hydroxyproline (Nucleosil® Chiral-1 by Macherey & Nagel or Chiralpak WH® by Chiral Technologies), or D-penicillamine (Chirex® (D)-penicillamine by Phenomenex). In CE, many combinations of the selectors described above and the corresponding metal ions have been applied.

Chiral Recognition Mechanisms

19

Fig. 8 Molecular modeling structure of the ternary L-proline–Cu(II)–isoleucine complex containing two chelated water molecules and (a) L-isoleucine or (b) D-isoleucine (reproduced by permission of Elsevier from ref. 143 © 2010) 2.9 Chiral Crown Ethers

Chiral crown ethers used in separation sciences include chiral moieties such as binaphthyl or tartaric acid units in a polyether macrocycle. They form complexes with protonated amines so that their application is somewhat limited to such analytes. The discrimination of amino acid enantiomers by (+)-(18-crown-6)-2,3,11,12tetracarboxylic acid has been studied by NMR [148, 149] and X-ray crystallography [150, 151]. Complex formation is due to the formation of hydrogen bonds between the protonated amines and oxygen atoms of the macrocycle. For chiral recognition, the crown ether has to adopt an asymmetric C1-type conformation resulting in a bowl-like shape with the N-H and Cα-H protons of the amino acid interacting with the oxygen atoms of the ring system as well as the carboxylate groups. The asymmetric C1-type shape is

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Gerhard K. E. Scriba

assumed to originate from a conformational sequence of successive rotations in the macrocycle [150]; see also [2, 5, 8]. Further interactions between analytes and the chiral crown ether may contribute to complex formation such as π-π interactions between aromatic moieties as recently shown for a chiral acridino-18-crown-6 ether and the enantiomers of 1-(1-naphthyl)ethylamine [152]. Complex formation between secondary amines and crown ethers involves only two N-H∙∙∙O hydrogen bonds between the protonated amine and the crown ether ring according to NMR studies [153]. However, the exact chiral recognition mechanism for the resolution of secondary amines has yet to be elucidated. Commercial HPLC columns based on (3,30 -diphenyl0 1,1 -binaphthyl)-20-crown-6 (Crownpak® CR(+) and CR() from Daicel) and (18-crown-6)-2,3,11,12-tetracarboxylic acid (ChiroSil® RCA(+)and RCA() from Regis) are available in both enantiomeric forms of the selector as indicated by the plus or minus sign. (+)- or ()-(18-crown-6)-2,3,11,12-tetracarboxylic acid is the most frequently used chiral crown ether in CE. Enantioseparations by HPLC [154–156] and by CE [156, 157] have been summarized. 2.10 Synthetic Polymers

Synthetic polymers such as polyacrylamides, polymethacrylates, polyacetylenes, or polyisocyanides have been applied as chiral stationary phases in chromatography. They can be obtained by polymerization of chiral monomers resulting in stereoregular polymers with defined helical conformation [23, 158]. Polymerization of the achiral triphenylmethacrylate results in a helically chiral polymer [158]. Furthermore, polymers with defined helicity can be obtained by the so-called helicity induction and memory approach in which a polymer adapts helical conformation upon complexation with a chiral compound [158, 159]. The induced helix remains unchanged when the chiral compound is removed. Hydrogen binding and π-π-interactions along with steric factors contribute to the chiral discrimination of analytes. For recent reviews on the synthesis and application of helical polymers for chromatographic enantioseparations see [23, 53]. Although these chiral selectors are not very often used in recent publications, commercial columns are available such as Chiralpak® OT(+)™ (Daicel), ChiraSpher® (Merck), AstecTM® P-CAP and AstecTM® P-CAP-DP™ (Sigma-Aldrich), or Kromasil® CHIDMB and CHI-TBB (EKA).

2.11

Metal-organic frameworks (MOFs) are microporous crystalline materials, which possess well-defined three-dimensional structures. They are built of metal ions (nodes) connected via di- or multidentate organic linkers. MOFs can be obtained by several synthetic routes [160–164]. For example, as the most reliable route, a homochiral MOF can be synthesized from metal-containing nodes and stereochemically pure chiral organic bridging ligands to ensure the

MOFs

Chiral Recognition Mechanisms

21

chirality of the resulting network structures. Another general route utilizes achiral metal nodes and bridging ligands, which form a chiral network in the presence of a chiral auxiliary reagent that does not participate itself in the formation of the network but forces the MOF to adopt a specific chiral topology. The third approach is based on the phenomenon of spontaneous resolution during crystal growth. Finally, a chiral MOF can be obtained by derivatization of an achiral MOF with chiral reagents. Homochiral MOFs have been employed as stationary phases in GC and HPLC as summarized, for example, in [161–167]. The chiral recognition mechanisms of MOFs have been explored primarily by structure-separation relationships and molecular modeling using simple chiral aromatic or aliphatic alcohols or amines as model compounds. Not only the chirality and surface chemistry of the MOF were important but also a contribution of the pore size with regard to a “match” between the size and shape of the framework and the selectand molecules. Depending on the structure of the MOF, further interactions such as hydrogen bonds or π-π interactions contribute to the stereoselective binding. For example, a MOF based on a C2-symmetric twisted tetracarboxylate ligand derived from a 1,10 -biphenol, which is coordinated by Mn2+ ions as schematically shown in Fig. 9, coordinated the enantiomers of 2-butylamine and 1-phenylethylamine within the microenvironment formed by the OH groups, the Mn2+ atoms, and the phenyl rings but in a different orientation. The binding of the 2-butylamine enantiomers is shown in Fig. 9c, d. The (S)-configured ligand favored the binding of the (R)-enantiomer of the analyte 2-butylamine over the (S)-enantiomer [168]. MIPs

Molecularly imprinted polymers (molecular imprinted polymers, MIPs) are synthetic polymers, which are obtained by polymerization of functional monomers and crosslinker molecules in the presence of a template, typically a stereochemically pure compound. The chiral recognition of a MIP is determined by the spatial arrangement of the interaction groups of the template and the polymer resulting in a better “fit” of one enantiomer over the other. As overall chiral recognition mechanism, the so-called weakest interaction model was proposed, which assumes that weaker interactions are primarily responsible for differentiating the solute enantiomers and not the strong interactions [169]. MIPs have been used for several applications including solid-phase extraction and chromatography [170, 171]. Because analyte specificity is determined by the structure of the template, the use of MIPs in enantioselective chromatography is limited.

2.13 Chiral Ionic Liquids

Chiral ionic liquids have been increasingly applied in separation sciences in recent years. Ionic liquids are salts that are liquid at or close to room temperature. Chiral ionic liquids are composed of

2.12

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c

d H3C H3 C

Mn Mn

Mn

CH3 H3 C OH HO

O

CH3

CH3 CH3

O

H3 C

O

O

O

O

Mn

e

O Mn

O

Mn Mn Mn

f

Fig. 9 (a) Schematic chemical structure of a metal-organic framework based on a C2-symmetric twisted tetracarboxylate ligand derived from a 1,10 -biphenol coordinated by Mn2+ ions. (b) X-ray structure of an infinite Mn-carboxylate chain. Binding sites in the framework of (c) (R)-2-butylamine and (d) (S)-2-butylamine (reproduced by permission of Springer Nature from ref. 168 © 2014)

either a chiral cation or a chiral anion. Because of the high solubility in aqueous and organic solvents, chiral ionic liquids have been used as chiral mobile-phase additives in chromatography as well as chiral background electrolyte additives or chiral selectors in CE. Often, ionic liquids are combined with other selectors such as CDs. Ionic and ion-pair interactions between the solutes and the ionic liquid are considered dominant with regard to the separation mechanism. The application of chiral ionic liquids in chromatography and CE has been summarized [145, 172, 173]. 2.14

Chiral Micelles

Although MEKC is an important electromigration technique, investigations of the chiral recognition mechanisms have been hampered by the flexibility of the selectors in a dynamic equilibrium between the detergent molecules associated with the micelles and the monomeric molecules in solution. Thus, investigation of selector-selectand interactions by spectroscopic techniques as well as molecular modeling has been challenging. This has been overcome by the introduction of chiral molecular micelles, which are

Chiral Recognition Mechanisms

23

obtained by polymerization of suitably functionalized surfactants via the hydrophobic tails [174, 175]. The chiral recognition of analytes by the dipeptide-type molecular micelles poly-(sodium undecyl-L-leucyl-L-valinate) (polySULV) and poly-(sodium undecyl-L-valyl-L-leucinate) (polySUVL) in aqueous solution has been studied by NMR [176] and molecular dynamics simulations [176–180]. The molecular micelles adopt an oval shape. In polySUVL, most of the dipeptide headgroups are oriented toward the core of the micelle so that a compact structure results. In contrast, the headgroups are typically oriented away from the core in the case of polySULV resulting in a more open shape. Besides yielding more potential binding pockets for chiral analytes, this open structure also results in the improved access of water molecules to the dipeptide headgroup region, which might be the reason for the observation that polySULV is generally a more potent chiral selector compared to polySUVL in enantioseparations. Moreover, higher dynamic monomer chain motions as well as higher solventaccessible surface are of polySULV compared to polySUVL. Docking studies of polySULV and the enantiomers of 1,10 -binaphthyl2,20 -diyl hydrogenphosphate (BHP) [178], the β-blockers atenolol and propranolol [179], as well as chlorthalidone and lorazepam [180] revealed four distinct binding pockets in the molecular micelle able to accommodate chiral solutes as shown for a polySULV micelle composed of 20 surfactant monomers in Fig. 10a, b. Pocket 1 is relatively narrow and deep compared to pockets 2–4 and contains more hydrophilic alpha spheres (shown in red) than the other pockets, thus offering more hydrogen-bond interactions in addition to hydrophobic interactions. The interactions between solute and selector as well as the stereoselective preferences of the polySULV micelle for analyte enantiomers were estimated from modeling of the distance between the solute enantiomers and the center of molecular micelle, hydrogen bond analysis, solvent-accessible surface area for the solutes during docking simulations, and free energy calculations. Each compound had preferred binding sites based on free energy calculations. Thus, both enantiomers of propranolol were bound almost exclusively in pocket 1 [179] and the enantiomers of BHP interacted preferably with pocket 2 [178]. In the case of atenolol the (S)-enantiomer interacted with pockets 1 and 4 at fractional populations of 0.73 and 0.27, respectively, while the (R)-enantiomer mainly populated pockets 1 and 3 with fractions of 0.58 and 0.42, respectively [179]. (S)-chlorthalidone was found exclusively in pocket 1, whereas the (R)-enantiomer occupied pocket 1 (fraction 0.82) and pocket 3 (fraction 0.14) [180]. Finally, (R)-lorazepam is preferentially bound to pocket 1, while (S)-lorazepam is found in pocket 1 (fraction 0.21) and pocket 2 (fraction 0.70) [180]. The enantiomers of the drugs also displayed differences in how deep they penetrated into the molecular micelle as

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Fig. 10 Molecular modeling of poly(SULV) showing (a) binding pocket 1 and (b) binding pockets 2, 3, and 4. Lipophilic and hydrophilic regions are lined in green and purple, respectively. Detailed view of (c) pocket one with docked (S)-propranolol and (d) (R)-propranolol. Hydrophobic regions of the pocket are colored in green and hydrophilic regions in red. Hydrogen bonds are indicated as blue dotted lines, and arene-hydrogen hydrophobic interactions as dotted yellow lines (adapted with permission of Elsevier from ref. 179 © 2015 Elsevier)

well as the strength and number of hydrogen bond and hydrophobic interactions formed with the chiral selector. As shown for propranolol in Fig. 10c, d for the complexation in binding pocket 1, both enantiomers are bound to polySULV with the aromatic rings inserted into the hydrocarbon core and the chiral side chains pointing toward the surface of the molecular micelle. (S)-propranolol formed a larger number of arene-hydrogen hydrophobic interactions and hydrogen bonds than (R)-propranolol indicating a stronger interaction with the molecular micelle, which is in accordance with CE enantioseparations of propranolol by polySULV [179]. 2.15 Miscellaneous Selectors

Calixarenes are basket-shaped synthetic molecules composed of phenol units linked by methylene groups. Chirality is introduced by modifications of the parent (achiral) calixarene by chiral molecules such as amino acids, ephedrine, Cinchona alkaloids, or cyclodextrins. Inclusion into the calixarene cavity as well as π-π

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25

interactions or ionic interactions with the chiral side chain moieties may contribute to the chiral recognition of analytes. Studies using calixarenes as selectors for enantioseparations have been performed, for example, by HPLC [181–184] and by CEC [185, 186]. While MOFs are macromolecules, chiral cages are relatively small individual molecules containing cavities or “pores,” which can form complexes in a stereoselective way. Depending on their structure, the molecular cages pack in window-to-window or window-to-face arrangements, thereby generating pore channel networks. Selector-selectand interactions are assumed to occur via hydrogen bonds, dipole-dipole, and π-π interactions as well as van der Waals forces or steric hindrance. Chiral cages have been used primarily as chiral selectors in GC demonstrating enantioselectivity for a variety of aliphatic and aromatic compounds [167, 187–190]. Aptamers are single-stranded RNA or DNA oligonucleotides obtained in vitro by the iterative process of systematic evolution of ligands by exponential enrichment (SELEX) [191, 192]. They possess a complex three-dimensional shape containing structural motifs such as stems, loops, bulges, hairpins, triplexes, or quadruplexes and can bind a large variety of target compounds with an affinity, specificity, and selectivity comparable to antibodies. Complexation is believed to occur via adaptive conformation changes of the aptamer in a multistep induced-fit process, folding from a relatively disordered structure into a defined binding pocket encapsulating the target molecule [193]. Hydrogen bonding, electrostatic interactions, stacking interactions, or hydrophobic interactions contribute depending on the structure of the target. Aptamers have been used as chiral selectors for enantioseparations by HPLC, CE, MEKC, and CEC [193, 194]. References 1. Carrol L (1871) Through the looking-glass and what Alice found there. Macmillan, London 2. Berthod A (2010) Chiral recognition in separation methods. Springer, Heidelberg 3. Scriba GKE (2013) Chiral recognition in separation science: an overview. In: Scriba GKE (ed) Chiral separations: methods and protocols, 2nd edn. Humana Press, New York 4. Ciogli A, Kotoni D, Gasparrini F et al (2013) Chiral supramolecular selectors for enantiomer differentiation in liquid chromatography. Top Curr Chem 349:73–106 5. Scriba GKE (2013) Differentiation of enantiomers by capillary electrophoresis. Top Curr Chem 340:209–276 6. Berthod A (2006) Chiral recognition mechanisms. Anal Chem 78:2093–2099

7. L€ammerhofer M (2010) Chiral recognition by enantioselective liquid chromatography: mechanisms and modern chiral stationary phases. J Chromatogr A 1217:814–856 8. Scriba GKE (2012) Chiral recognition mechanisms in analytical separation sciences. Chromatographia 75:815–838 9. Scriba GKE (2016) Chiral recognition in separation science – an update. J Chromatogr A 1467:56–78 10. Lang C, Armstrong DW (2017) Chiral surfaces: the many faces of chiral recognition. Curr Opin Colloid Interface Sci 32:94–107 11. Schneider HJ (2009) Binding mechanisms in supramolecular complexes. Angew Chem Int Ed 48:3924–3977 12. Peluso P, Mamane V, Cossu S (2015) Liquid chromatography enantioseparations of halogenated compounds on polysaccharide-based

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chiral stationary phases: role of halogen substituents in molecular recognition. Chirality 27:667–684 13. Biedermann F, Nau WM, Schneider JH (2014) The hydrophobic effect revisited studies with supramolecular complexes imply high-energy water as noncovalent driving force. Angew Chem Int Ed 53:11158–11171 14. Yang G, Xu Y (2011) Vibrational circular dichroism spectroscopy of chiral molecules. Top Curr Chem 298:189–236 15. Uccello-Barretta G, Vanni L, Balzano F (2010) Nuclear magnetic resonance approaches to the rationalization of chromatographic enantiorecognition processes. J Chromatogr A 1217:928–940 16. Salgado A, Chankvetadze B (2016) Applications of nuclear magnetic resonance spectroscopy for the understanding of enantiomer separation mechanisms in capillary electrophoresis. J Chromatogr A 1467:95–114 17. Lipkowitz KB (2001) Atomistic modeling of enantioselection in chromatography. J Chromatogr A 906:417–442 18. Del Rio A (2009) Exploring enantioselective molecular recognition mechanisms with chemoinformatic techniques. J Sep Sci 32:1566–1584 19. Elbashir AA (2012) Combined approach using capillary electrophoresis and molecular modeling for an understanding of enantioselective recognition mechanisms. J Appl Sol Chem Model 1:121–126 20. Sardella R, Ianni F, Macciarulo A et al (2018) Elucidation of the chromatographic enantiomer elution order through computational studies. Mini Rev Med Chem 18:88–97 21. Chen X, Yamamoto C, Okamoto Y (2007) Polysaccharide derivatives as useful chiral stationary phases in high-performance liquid chromatography. Pure Appl Chem 79:1561–1573 22. Ikai T, Okamoto Y (2009) Structure control of polysaccharide derivatives for efficient separation of enantiomers by chromatography. Chem Rev 109:6077–6101 23. Shen J, Okamoto Y (2016) Efficient separation of enantiomers using stereoregular chiral polymers. Chem Rev 116:1094–1138 24. Okamoto Y, Ikai T (2008) Chiral HPLC for efficient resolution of enantiomers. Chem Soc Rev 37:2593–2608 25. Chankvetadze B (2012) Recent developments on polysaccharide-based chiral stationary phases for liquid-phase separation of enantiomers. J Chromatogr A 1269:26–51

26. Yamamoto C, Yashima E, Okamoto Y (2002) Structural analysis of amylose tris (3,5-dimethylphenylcarbamate) by NMR relevant to its chiral recognition mechanism in HPLC. J Am Chem Soc 124:12583–12589 27. Ma S, Shen S, Lee H et al (2009) Mechanistic studies on the chiral recognition of polysaccharide-based chiral stationary phases using liquid chromatography and vibrational circular dichroism. Reversal of elution order of N-substituted alpha-methyl phenylalanine esters. J Chromatogr A 1216:3784–3793 28. Kim BH, Lee SU, Moon DC (2012) Chiral recognition of N-phthaloyl, N-tretrachlorophthaloyl, and N-naphthaloyl α-amino acids and their esters on polysaccharide-derived chiral stationary phases. Chirality 24:1037–1046 29. Peluso P, Mamane V, Aubert E et al (2016) Insights into halogen bond-driven enantioseparations. J Chromatogr A 1467:228–238 30. Dallocchio R, Dessi A, Solinas M et al (2018) Halogen bond in high-performance liquid chromatography enantioseparations: description, features and modelling. J Chromatogr A 1563:71–81 31. Wenslow RM, Wang T (2001) Solidstate NMR characterization of amylose tris(3,5-dimethylphenylcarbamate) chiral stationary-phase structure as a function of mobile-phase composition. Anal Chem 73:4190–4195 32. Wang T, Wenslow RM (2003) Effects of alcohol mobile-phase modifiers in the structure and chiral selectivity of amylose tris (3,5-dimethylphenylcarbamate) chiral stationary phase. J Chromatogr A 1015:99–110 33. Kasat RB, Zvinevich Y, Hillhouse HW et al (2006) Direct probing of sorbent-solute interactions for amylose tris(3,5-dimethylphenylcarbamate) using infrared spectroscopy, x-ray diffraction, solid-state NMR, and DFT modeling. J Phys Chem B 110:14114–14122 34. Zhao B, Oroskar PA, Wang X et al (2017) The composition of the mobile phase affects the dynamic chiral recognition of drug molecules by the chiral stationary phase. Langmuir 33:11246–11256 35. Layton C, Ma S, Wu L et al (2013) Study of enantioselectivity on an immobilized amylose carbamate stationary phase under subcritical fluid chromatography. J Sep Sci 36:3941–3948 36. Kasat RB, Wang NHL, Franses EI (2008) Experimental probing and modeling of key sorbent-solute interactions of norephedrine enantiomers with polysaccharide-based chiral stationary phases. J Chromatogr A 1190:110–119

Chiral Recognition Mechanisms 37. Kasat RB, Franses EI, Wang NHL (2010) Experimental and computational studies of enantioseparation of structurally similar chiral compounds on amylose tris(3,5-dimethylphenylcarbamate). Chirality 22:565–579 38. Tsui HW, Franses EI, Wang NHL (2014) Effect of alcohol aggregation on the retention factors of chiral solutes with an amylose-based sorbent: modeling and implications of the adsorption mechanism. J Chromatogr A 1328:52–65 39. Ortuso F, Alcaro S, Menta S et al (2014) A chromatographic an computational study on the driving force operating in the exceptionally large enantioseparation of N-thicarbamoyl-3-(40 -biphenyl)-5-phenyl-4,5-dihydro(1H ) pyrazole on a 4-methylbenzoate cellulose-based chiral stationary phase. J Chromatogr A 1324:71–77 40. Hu G, Huang M, Luo C et al (2016) Interactions between pyrazole derived enantiomers and Chiralcel OJ: prediction of enantiomer absolute configurations and elution order by molecular dynamics simulations. J Mol Graph Model 66:123–132 41. Tsui HW, Wang NHL, Franses EI (2013) Chiral recognition mechanism of acyloincontaining chiral solutes by amylose tris[(S)α-methylbenzylcarbamate]. J Phys Chem 117:9203–9216 42. Ma S, Tsui HW, Spinelli E et al (2014) Insights into chromatographic enantiomeric separation of allenes on cellulose carbamate stationary phase. J Chromatogr A 1362:119–128 43. Alcaro S, Bolasco A, Cirilli R et al (2014) Computer-aided molecular design of asymmetric pyrazole derivatives with exceptional enantioselective recognition toward the Chiralcel OJ-H stationary phase. J Chem Inf Model 52:649–654 44. Ali I, Al-Othman ZA, Al-Warthan A et al (2014) Enantiomeric separation and simulation studies on pheniramine, oxybutynin, cetirizine and brinzolamide chiral drugs on amylose-based columns. Chirality 26:136–143 45. Ali I, Sahoo DR, Al-Othman ZA et al (2015) Validated chiral high performance liquid chromatography separation method and simulation studies of dipeptides on amylose chiral column. J Chromatogr A 1406:201–209 46. Shedania Z, Kavaka R, Volonerio A et al (2018) Separation of enantiomers of chiral sulfoxides in high-performance liquid chromatography with cellulose-based chiral selectors using methanol and methanol-water mixtures as mobile phases. J Chromatogr A 1557:62–74

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47. Gogaladze K, Chankvetadze L, Tsintsadze M et al (2015) Effect of basic and acidic additives on the separation of some basic drug enantiomers on polysaccharide-based chiral columns with acetonitrile as mobile phase. Chirality 27:228–234 48. Matarashvili I, Chankvetadze L, Tsintsadze T et al (2015) HPLC separation of enantiomers of some chiral carboxylic acid derivatives using polysaccharide-based chiral columns and polar organic mobile phases. Chromatographia 78:473–479 49. Mosiashvili L, Chankvetadze L, Farkas T, Chankvetadze B (2013) On the effect of basic and acidic additives on the separation of the enantiomers of some basic drugs with polysaccharide-based chiral selectors and polar organic mobile phases. J Chromatogr A 1317:167–174 50. Matarashvili I, Ghughunishvili D, Chankvetadze L et al (2017) Separation of enantiomers of chiral weak acids with polysaccharidebased chiral columns and aqueous-organic mobile phases in high-performance liquid chromatography: typical reversed-phase behavior? J Chromatogr A 1483:86–92 51. Mskhiladze A, Karchkhadze M, Dadianidz A et al (2013) Enantioseparation of chiral antimycotic drugs by HPLC with polysaccharide-based chiral columns and polar organic mobile phases with emphasis on enantiomer elution order. Chromatographia 76:1449–1458 52. Beridze N, Tsutskiridze E, Takaishvili N et al (2018) Comparative enantiomer-resolving ability of coated and covalently immobilized versions of two polysaccharide-base chiral selectors in high-performance liquid chromatography. Chromatographia 81:611–621 53. Yashima E, Ida H, Okamoto Y (2013) Enantiomeric differentiation by synthetic helical polymers. Top Curr Chem 340:41–72 54. Biwer A, Antranikian G, Heinzle E (2002) Enzymatic production of cyclodextrins. Appl Microbiol Biotechnol 59:609–617 55. Rekharsky MV, Inoue Y (1998) Complexation thermodynamics of cyclodextrins. Chem Rev 98:1875–1917 56. Bilensoy E (2011) Cyclodextrins in pharmaceutics, cosmetics and biomedicine. In: Current and future industrial applications. John Wiley & Sons, Hoboken 57. Dodziuk H (2006) Cyclodextrins and their complexes: chemistry, analytical methods, applications. Wiley-VCH, Weinheim 58. Crini C (2014) A history of cyclodextrins. Chem Rev 114:10940–10975

28

Gerhard K. E. Scriba

59. Zhang X, Zhang Y, Armstrong DW (2012) Chromatographic separations and analysis: cyclodextrin-mediated HPLC, GC and CE enantiomeric separations. In: Carreira EM, Yamamoto H (eds) Comprehensive chirality, vol 8. Elsevier, Amsterdam, pp 177–199 60. Schurig V (2010) Use of derivatized cyclodextrins as chiral selectors for the separation of enantiomers by gas chromatography. Ann Pharm Franc 68:82–98 61. Xiao Y, Ng SC, Tan TT, Wang Y (2012) Recent development of cyclodextrin chiral stationary phases and their applications in chromatography. J Chromatogr A 1269:52–68 62. Rezanka P, Navratilova K, Rezanka M et al (2014) Application of cyclodextrins in chiral capillary electrophoresis. Electrophoresis 35:2701–2721 63. Escuder-Gilabert L, Martin-Biosca Y, Medina-Hernandez MJ, Sagrado S (2014) Cyclodextrins in capillary electrophoresis: recent developments and new trends. J Chromatogr A 1357:2–23 64. Saz JM, Marina ML (2016) Recent advances on the use of cyclodextrins in the chiral analysis of drugs by capillary electrophoresis. J Chromatogr A 1467:79–94 65. Zhu Q, Scriba GKE (2016) Advances in the use of cyclodextrins as chiral selectors in capillary electrokinetic chromatography: fundamentals and applications. Chromatographia 79:1403–1435 66. Dodziuk H, Kozinsky W, Ejchart A (2004) NMR studies of chiral recognition by cyclodextrins. Chirality 16:90–105 67. Chankvetadze B (2004) Combined approach using capillary electrophoresis and NMR spectroscopy for an understanding of enantioselective recognition mechanisms by cyclodextrins. Chem Soc Rev 33:337–347 68. Mura P (2014) Analytical techniques for characterization of cyclodextrin complexes in aqueous solution: a review. J Pharm Biomed Anal 101:238–250 69. Mura P (2015) Analytical techniques for characterization of cyclodextrin complexes in the solid state: a review. J Pharm Biomed Anal 113:226–238 70. Hazai E, Hazai I, Demko L et al (2010) Cyclodextrin knowledgebase a web-based service managing CD-ligand complexation data. J Comput Aided Mol Des 24:713–717 71. Salgado A, Tatunashvili E, Gologashvili A et al (2017) Structural rationale for the chiral separation and migration order reversal of clenpenterol enantiomers in capillary electrophoresis using two different

β-cyclodextrins. Phys Chem Chem Phys 19:27935–27939 72. Gogolashvili A, Tatunashvili E, Chankvetadze L et al (2017) Separation of enilconazole enantiomers in capillary electrophoresis with cyclodextrin-type chiral selectors and investigation of structure of selector-selectand complexes by using nuclear magnetic resonance spectroscopy. Electrophoresis 38:1851–1859 73. Fonseca MC, Santos da Silva RC, Soares Nascimento C Jr, Bastos Borges K (2017) Computational contribution to the electrophoretic enantiomer separation mechanism and migration order using modified β-cyclodextrins. Electrophoresis 38:1860–1868 74. Recio R, Elhalem E, Benito JM et al (2018) NMR study on the stabilization and chiral discrimination of sulforaphane enantiomers and analogues by cyclodextrins. Carbohyd Polym 187:118–125 75. Cucinotta V, Messina M, Contino A et al (2017) Chiral separation of terbutaline and non-steroidal anti-inflammatory drugs by using a new lysine-bridged hemispherodextrin in capillary electrophoresis. J Pharm Biomed Anal 145:734–741 76. Szabo ZI, Szo¨cs L, Horvath P et al (2016) Liquid chromatography with mass spectrometry enantioseparation of pomalidomide on cyclodextrin-bonded chiral stationary phases and the elucidation of the chiral recognition mechanisms by NMR spectroscopy and molecular modeling. J Sep Sci 39:2941–2949 77. Szabo ZI, Mohammadhassan F, Szo¨cs L et al (2016) Stereoselective interactions and liquid chromatographic enantioseparation of thalidomide on cyclodextrin-bonded stationary phases. J Incl Phenom Macrocycl Chem 85:227–236 78. Szabo ZI, Toth G, Vo¨lgyi G et al (2016) Chiral separation of asenapine enantiomers by capillary electrophoresis and characterization of cyclodextrin complexes by NMR spectroscopy, mass spectrometry and molecular modeling. J Pharm Biomed Anal 117:398–404 79. Yao Y, Song P, Wen X et al (2017) Chiral separation of 12 pairs of enantiomers by capillary electrophoresis using heptakis-(2,3-diacetyl-6-sulfato)-β-cyclodextrin as the chiral selector and the elucidation of the chiral recognition mechanism by computational methods. J Sep Sci 40:2999–3007 80. Fejo¨s I, Varga E, Benkovics G et al (2016) Comparative evaluation of the chiral recognition potential of single isomer sulfated betacyclodextrin synthesis intermediates in

Chiral Recognition Mechanisms non-aqueous capillary electrophoresis. J Chromatogr A 1467:454–462 81. Krait S, Salgado A, Chankvetadze B et al (2018) Investigation of the complexation between cyclodextrins and medetomidine enantiomers by capillary electrophoresis, NMR spectroscopy and molecular modeling. J Chromatogr A 1567:198–210 82. Li X, Yao X, Xiao Y, Wang Y (2017) Enantioseparation of single layer native cyclodextrin chiral stationary phases: effect of cyclodextrin orientation and a modeling study. Anal Chim Acta 990:174–184 83. Chankvetadze B, Burjanadze N, Maynard DM et al (2002) Comparative enantioseparations with native β-cyclodextrin and heptakis(2-O-methyl-3,6-di-O-sulfo)-β-cyclodextrin in capillary electrophoresis. Electrophoresis 23:3027–3034 84. Servais AC, Rousseau A, Fillet M et al (2010) Separation of propranolol enantiomers by CE using sulfated β-CD derivatives in aqueous and non-aqueous electrolytes: comparative CE and NMR study. Electrophoresis 31:1467–1474 85. Servais AC, Rousseau A, Dive G et al (2012) Combination of capillary electrophoresis, molecular modeling and nuclear magnetic resonance to study the interaction mechanism between single-isomer anionic cyclodextrin derivatives and basic drug enantiomers in methanolic background electrolyte. J Chromatogr A 1232:59–64 86. Chankvetadze L, Servais AC, Fillet M et al (2012) Comparative enantioseparation of talinolol in aqueous and non-aqueous capillary electrophoresis and study of related selectorselectand interactions by nuclear magnetic resonance spectroscopy. J Chromatogr A 1267:206–216 87. Lomsadze K, Salgado A, Calvo E et al (2011) Comparative NMR and MS studies on the mechanism of enantioseparation of propranolol with heptakis(2,3-diacetyl-6-sulfo)-β-cyclodextrin in capillary electrophoresis with aqueous and non-aqueous electrolytes. Electrophoresis 32:1156–1163 88. Riesova M, Svobodova J, Tosner Z et al (2013) Complexation of buffer constituents with neutral complexation agents: part I. Impact on common buffer properties. Anal Chem 85:8518–8525 89. Beni M, Riesova M, Svobodova J et al (2013) Complexation of buffer constituents with neutral complexation agents: part II. Practical impact in capillary zone electrophoresis. Anal Chem 85:8526–8534

29

90. Melani F, Giannini I, Pasquini B et al (2011) Evaluation of the separation mechanism of electrokinetic chromatography with a microemulsion and cyclodextrins using NMR and molecular modeling. Electrophoresis 32:3062–3069 91. Pasquini B, Melani F, Caprini C et al (2017) Combined approach using capillary electrophoresis, NMR and molecular modeling for ambrisentan related substances analysis: investigation of intermolecular affinities, complexation and separation mechanism. J Pharm Biomed Anal 144:220–229 92. Vargas C, Scho¨nbeck C, Heimann I, Keller S (2018) Extracavity effect in cyclodextrin/surfactant complexation. Langmuir 34:5781–5787 93. Alvira E (2013) Molecular dynamics study of the influence of solvents on the chiral discrimination of alanine enantiomers by β-cyclodextrin. Tetrahedron Asymmetry 24:1198–1206 94. Alvira E (2015) Theoretical study of the separation of valine enantiomers by β-cyclodextrin with different solvents: a molecular mechanics and dynamics simulation. Tetrahedron Asymmetry 26:853–860 95. Alvira E (2017) Influence of solvent polarity on the separation of leucine enantiomers by β-cyclodextrin: a molecular mechanics and dynamics simulation. Tetrahedron Asymmetry 28:1414–1422 96. Soares Nascimento C Jr, Fedoce Lopes J, Guimaraes L, Bastos Borges K (2014) Molecular modeling study of the recognition mechanism and enantioseparation of 4-hydroxypropranolol by capillary electrophoresis using carboxymethyl-β-cyclodextrin as the chiral selector. Analyst 139:3901–3910 97. Zhang Y, Breitbach ZS, Wang C, Armstrong DW (2010) The use of cyclofructans as novel chiral selectors for gas chromatography. Analyst 135:1076–1083 98. Sun P, Wang C, Breitbach ZS et al (2009) Development of new HPLC chiral stationary phases based on native and derivatized cyclofructans. Anal Chem 81:10215–10226 99. Jiang C, Tong MY, Breitbach ZS, Armstrong DW (2009) Synthesis and examination of sulfated cyclofructans as a novel class of chiral selectors for CE. Electrophoresis 30:3897–3909 100. Immel S, Schmitt RG, Lichtenthaler FW (1998) Cyclofructins with six to ten β(1!2)-linked fructofuranose units: geometries, electrostatic profiles, lipophilicity

30

Gerhard K. E. Scriba

pattern, and potential for inclusion complexation. Carbohydr Res 313:91–105 101. Wang L, Li Y, Yao L et al (2014) Evaluation and determination of the cyclofructans-amino acid complex binding pattern by electrospray ionization mass spectrometry. J Mass Spectrom 49:1043–1049 102. Wang L, Li C, Yin Q et al (2015) Construction the switch binding pattern of cyclofructans 6. Tetrahedron 71:3447–3452 103. Smuts JP, Hao XQ, Han Z et al (2014) Enantiomeric separations of chiral sulfonic and phosphoric acids with barium-doped cyclofructan selectors via an ion interaction mechanism. Anal Chem 86:1282–1290 104. Hellinghausen G, Roy D, Lee JT et al (2018) Effective methodologies for enantiomeric separations of 150 pharmacology and toxicology related 1 , 2 , and 3 amines with coreshell chiral stationary phases. J Pharm Biomed Anal 155:70–81 105. Dominguez-Vega E, Montealegre C, Marina ML (2016) Analysis of antibiotics by CE and their use as chiral selectors: an update. Electrophoresis 37:189–211 106. Genar M, Castro-Puyana M, Garcia MA, Marina ML (2018) Analysis of antibiotics by CE and CEC and their use as chiral selectors: an update. Electrophoresis 39:235–259 107. Ilisz I, Pataj Z, Aranyi A, Peter A (2012) Macrocyclic antibiotic selectors in direct HPLC enantioseparations. Sep Purif Rev 41:207–249 108. Berthod A (2009) Chiral recognition mechanisms with macrocyclic glycopeptide selectors. Chirality 21:167–175 109. Fernandes C, Tiritn ME, Cass Q et al (2012) Enantioseparation and chiral recognition mechanism of new chiral derivatives of xanthones on macrocyclic antibiotic stationary phases. J Chromatogr A 1241:60–68 110. Ravichandran S, Collins JR, Singh N, Wainer IW (2012) A molecular model of the enantioselective liquid chromatographic separation of (RS)-ifosfamide and its N-dechloroethylated metabolites on a teicoplanin aglycone chiral stationary phase. J Chromatogr A 1269:218–225 111. He X, Lin R, He H et al (2012) Chiral separation of ketoprofen on a Chirobiotic T column and its chiral recognition mechanisms. Chromatographia 75:1355–1363 112. Phyo YZ, Cravl S, Palmeira A et al (2018) Enantiomeric resolution and docking studies of chiral xanthonic derivatives on Chirobiotic columns. Molecules 23:E142. https://doi. org/10.3390/molecules23010142

113. Bertucci C, Tedesco D (2018) Human serum albumin as chiral selector in enantioselective high-performance liquid chromatography. Curr Med Chem 24:743–757 114. Bocain S, Skoczylas M, Biszewski B (2016) Amino acids, peptides, and proteins as chemically bonded stationary phases - a review. J Sep Sci 39:83–92 115. Haginaka J (2011) Mechanistic aspects of chiral recognition on protein-based stationary phases. In: Grushka E (ed) Advances in chromatography, vol 49. CRC Press, Boca Raton, pp 37–69 116. Haginaka J (2008) Recent progress in protein-based chiral stationary phases for enantioseparations in liquid chromatography. J Chromatogr B 875:12–19 117. Ghuman J, Zunszain PA, Petitpas I et al (2005) Structural basis of the drug-binding specificity of human serum albumin. J Mol Biol 353:38–52 118. Fernandes C, Tiritan ME, Pinto M (2013) Small molecules as chromatographic tools for HPLC enantiomeric resolution: Pirkletype chiral stationary phase evolution. Chromatographia 76:871–897 119. Fernandes C, Phyo YZ, Sulva AS et al (2018) Chiral stationary phases based on small molecules: an update of the last 17 years. Sep Purif Rev 47:89–123 120. Carraro ML, Palmeira A, Tiritan ME et al (2017) Resolution, determination of enantiomeric purity and chiral recognition mechanism of new xanthone derivatives on (S,S)Whelk-O1 stationary phase. Chirality 29:247–256 121. Fernandes C, Palmeira C, Santos A et al (2013) Enantioresolution of chiral derivatives of xanthones on (S,S)-whelk-O1 and L-phenylglycine stationary phases and chiral recognition mechanism by docking approach for (S,S)-Whelk-O1. Chirality 25:89–100 122. Zhao C, Cann NM (2007) The docking of chiral epoxides on the Whelk-O1 stationary phase: a molecular dynamics study. J Chromatogr A 1149:197–218 123. Zhao C, Cann NM (2008) Molecular dynamics study of chiral recognition for the WhelkO1 chiral stationary phase. Anal Chem 80:2426–2438 124. Zhao CF, Dimert S, Cann NM (2009) Rational optimization of the Whelk-O1 chiral stationary phase using molecular dynamics simulations. J Chromatogr A 1216:5968–5978 125. Koscho ME, Spence PL, Pirkle WH (2005) Chiral recognition in the solid state:

Chiral Recognition Mechanisms crystallographically characterized diastereomeric co-crystals between a synthetic chiral selector (Whelk-O1) and a representative chiral selector. Tetrahedron Asymmetry 16:3147–3153 126. Ilisz I, Bajtai A, Lindner W, Peter A (2018) Liquid chromatographic enantiomer separations applying chiral ion-exchangers based on Cinchona alkaloids. J Pharm Biomed Anal 159:127–152 127. L€ammerhofer M (2014) Liquid chromatographic enantiomer separation with special focus on zwitterionic chiral ion-exchangers. Anal Bioanal Chem 406:6095–6103 128. L€ammerhofer M, Lindner W (2008) Liquid chromatographic enantiomer separation and chiral recognition by Cinchona alkaloidderived enantioselective separation materials. Adv Chromatogr 46:1–107 129. Hoffmann CV, L€ammerhofer M, Lindner W (2007) Novel strong cation-exchange type chiral stationary phase for the enantiomer separation of chiral amines by highperformance liquid chromatography. J Chromatogr A 1161:242–251 130. Maier NM, Schefzick S, Lombardo GM et al (2002) Elucidation of the chiral recognition mechanism of Cinchona alkaloid carbamatetype receptors for 3,5-dinitrobenzoyl amino acids. J Am Chem Soc 124:8611–8629 131. Zhang T, Holder E, Franco P, Lindner W (2014) Zwitterionic chiral stationary phases based on cinchona and chiral sulfonic acids for the direct stereoselective separation of amino acids and other amphoteric compounds. J Sep Sci 37:1237–1247 132. Pell R, Sic S, Lindner W (2012) Mechanistic investigations of Cinchona alkaloid-based zwitterionic chiral stationary phases. J Chromatogr A 1269:287–296 133. Ianni F, Sardella R, Carotti A (2016) Quinine-based zwitterionic chiral stationary phase as a complementary tool for peptide analysis: Mobile phase effects on enantioand stereoselectivity of underivatized oligopeptides. Chirality 28:5–16 134. Sardella R, Macchiarulo A, Urbinati F et al (2018) Exploring the enantiorecognition mechanism of Cinchona alkaloid-based zwitterionic chiral stationary phases and the basic trans-paroxetine enantiomers. J Sep Sci 41:1199–1207 135. Ianni F, Pucciarini L, Carotti A et al (2018) Improved chromatographic diastereoresolution of cyclopropyl dafachronic acid derivatives using chiral anion exchangers. J Chromatogr A 1557:20–27

31

136. Grecso N, Kohout M, Carotti A et al (2016) Mechanistic considerations of enantiorecognition on novel Cinchona alkaloid-based zwitterionic chiral stationary phases from the aspect of the separation of trans-paroxetine enantiomers as model compounds. J Pharm Biomed Anal 124:164–173 137. Ianni F, Carotti A, Marinozzi M et al (2015) Diastereo- and enantioseparation of a Nα-Boc amino acid with a zwitterionic quinine-based stationary phase: focus on the stereorecognition mechanism. Anal Chim Acta 885:174–782 138. Sardella R, Lisanti A, Carotti A et al (2014) Ketoprofen enantioseparation with a Cinchona alkaloid based stationary phase: Enantiorecognition mechanism and release studies. J Sep Sci 37:2696–2703 139. Schmid MG, Gu¨bitz G (2011) Enantioseparation by chromatographic and electromigration techniques using ligand-exchange as chiral separation principle. Anal Bioanal Chem 400:2305–2316 140. Zhang H, Qi L, Mao L, Chen Y (2012) Chiral separation using capillary electromigration techniques based on ligand exchange principle. J Sep Sci 35:1236–1248 141. Hyun MH (2018) Liquid chromatographic ligand-exchange chiral stationary phases based on amino alcohols. J Chromatogr A 1557:28–42 142. Natalini B, Giacche N, Sardella R et al (2010) Computational studies for the elucidation of the enantiomer elution order of amino acids in chiral ligand-exchange chromatography. J Chromatogr A 1217:7523–7527 143. Mofaddel N, Adoubel AA, Morin CJ et al (2010) Molecular modelling of complexes between two amino acids and copper(II): correlation with ligand-exchange capillary electrophoresis. J Mol Struct 975:220–226 144. Echevarrı´a RN, Franca CA, Tascon M et al (2016) Chiral ligand-exchange chromatography with Cinchona alkaloids. Exploring experimental conditions for enantioseparation of α-amino acids. Microchem J 129:104–110 145. Kapnissi-Christodoulou CP, Stavrou IJ, Mavroudi MC (2014) Chiral ionic liquids in chromatographic and electrophoretic separations. J Chromatogr A 1363:2–10 146. He S, He Y, Cheng L et al (2018) Novel chiral ionic liquids stationary phases for the enantiomer separation of chiral acid by highperformance liquid chromatography. Chirality 30:670–679

32

Gerhard K. E. Scriba

147. Zhang Q (2018) Ionic liquids in capillary electrophoresis enantioseparations. Trends Anal Chem 100:145–154 148. Bang E, Jung JW, Lee W et al (2001) Chiral recognition of (18-crown-6)-tetracarboxylic acid as a chiral selector determined by NMR spectroscopy. J Chem Soc Perkin Trans 2:1685–1692 149. Lee W, Bang E, Baek CS, Lee W (2004) Chiral discrimination studies of (+)-(18-crown6)-2,3,11,12-tetracarboxylic acid by highperformance liquid chromatography and NMR spectroscopy. Magn Res Chem 42:389–395 150. Nagata H, Nishi H, Kamagauchi M, Ishica T (2008) Guest-dependent conformation of 18-crown-6 tetracarboxylic acid: relation to chiral separation of racemic amino acids. Chirality 20:820–827 151. Nagata H, Machida Y, Nishi H et al (2009) Structural requirement for chiral recognition of amino acid by (18-crown-6)-tetracarboxylic acid: binding analysis in solution and solid states. Bull Chem Soc Jpn 82:219–229 152. To´th T, Ne´meth T, Leveles I et al (2017) Structural characterization of the crystalline diastereomeric complex of enantiopure dimethylacridino-18-crown-6 ether and the enantiomers of 1-(1-napthyl)ethylamine hydrogen perchlorate. Struct Chem 28:289–296 153. Lovely A, Wenzel TJ (2008) Chiral NMR discrimination of amines: analysis of secondary, tertiary, and prochiral amines using (+)(18-crown-6)-2,3,11,12-tetracarboxylic acid. Chirality 20:370–378 154. Hyun MH (2015) Development of HPLC chiral stationary phases based in (+)(18-crown-6)-2,3,11,12-tetracarboxylic acid and their applications. Chirality 27:576–588 155. Hyun MH (2016) Liquid chromatographic enantioseparations on crown ether-based chiral stationary phases. J Chromatogr A 1467:19–32 156. Adhikari S, Lee W (2018) Chiral separation using chiral crown ethers as chiral selectors. J Pharm Invest 48:225–231 157. Elbashir AA, Aboul-Enein HY (2010) Application of crown ethers as buffer additives in capillary electrophoresis. Curr Pharm Anal 6:101–113 158. Yashima E, Maeda K, Idea H et al (2009) Helical polymers: synthesis, structures and functions. Chem Rev 109:6102–6211 159. Yashima E, Maeda K (2008) Chiralityresponsive helical polymers. Macromolecules 41:3–12

160. Cui Y, Li B, He H et al (2016) Metal-organic frameworks as platforms for functional materials. Acc Chem Res 49:483–493 161. Xue M, Li B, Qiu S, Chen B (2016) Emerging functional chiral microporous materials: synthetic strategies and enantioselective separations. Mater Today 19:503–515 162. Duerinck T, Denayer JFM (2015) Metalorganic frameworks as stationary phases for chiral chromatographic and membrane separations. Chem Eng Sci 124:179–187 163. Peluso P, Mamane V, Cossu S (2014) Homochiral metal-organic frameworks and their application in chromatography enantioseparations. J Chromatogr A 1363:11–16 164. Bhattacharjee S, Khan MI, Li X et al (2018) Recent progress in asymmetric catalysis and chromatographic separation by chiral metalorganic frameworks. Catalysts 8:120. https:// doi.org/10.3390/catal8030120 165. Li X, Chang V, Wang X et al (2014) Applications of homochiral metal-organic frameworks in the enantioselective adsorption and chromatography separation. Electrophoresis 35:2733–2743 166. Xie SM, Zhang M, Fei ZX, Yuan LM (2014) Experimental comparison of chiral metalorganic framework used as stationary phase in chromatography. J Chromatogr A 1363:137–143 167. Xie SM, Yuan LM (2017) Recent progress of chiral stationary phases for separation of enantiomers in gas chromatography. J Sep Sci 40:124–127 168. Peng Y, Gong T, Zhang K et al (2014) Engineering chiral porous metal-organic frameworks for enantioselective adsorption and separation. Nat Commun 5:4406. https:// doi.org/10.1038/ncomms5406 169. Rong F, Li P (2012) Study of the weakest interaction model for chiral resolution using molecularly imprinted polymer. Adv Mater Res 391-392:111–115 170. Cheong WJ, Ali F, Choi JH et al (2013) Recent applications of molecular imprinted polymers for enantioselective recognition. Talanta 106:45–59 171. Cheong WJ, Yang SH, Ali F (2013) Molecular imprinted polymers for separation science: a review of reviews. J Sep Sci 36:609–628 172. Greno M, Marina ML, Castro-Puyana M (2018) Enantioseparation by capillary electrophoresis using ionic liquids as chiral selectors. Crit Rev Anal Chem 48:429–446 173. Ding J, Armstrong DW (2005) Chiral ionic liquids. Synthesis and applications. Chirality 17:281–292

Chiral Recognition Mechanisms 174. Wang J, Warner IM (1994) Chiral separations using micellar electrokinetic capillary chromatography and a polymerized chiral micelle. Anal Chem 66:3773–3776 175. Dobashi A, Hamada M, Dobashi Y, Yamaguchi J (1995) Enantiomeric separation with sodium dodecanoyl-L-amino acidate micelles and poly(sodium(10-undecanoyl)-L-valinate) by electrokinetic chromatography. Anal Chem 67:3011–3017 176. Morris KF, Billiot EJ, Billiot FH et al (2012) Investigation of chiral molecular micelles by NMR spectroscopy and molecular dynamic simulation. Open J Phys Chem 2:240–251 177. Morris KF, Billiot EJ, Billiot FH et al (2013) A molecular dynamics simulation study of two dipeptide based molecular micelles: effect of amino acid order. Open J Phys Chem 3:20–29 178. Morris KF, Billiot EJ, Billiot FH et al (2014) A molecular dynamics simulation study of the association of 1,10 -binaphthyl-2,20 -diyl hydrogen phosphate enantiomers with a chiral molecular micelle. Chem Phys 439:36–43 179. Morris KF, Billiot EJ, Billiot FH et al (2015) Molecular dynamics simulation and NMR investigation of the association of the β-blockers atenolol and propranolol with a chiral molecular micelle. Chem Phys 457:133–146 180. Morris KF, Billiot EJ, Billiot FH et al (2018) Investigation of chiral recognition by molecular micelles with molecular dynamics simulations. J Disper Sci Technol 39:45–54 181. Yaghoubnejad S, Tabar Heydar K, Ahmadi SH, Zadmard R (2018) Preparation and evaluation of a chiral HPLC stationary phase based on cone calix[4]arene functionalized at the upper rim with L-alanine. Biomed Chromatogr 32:e4122 182. Chelvi SKT, Zhao J, Chen L et al (2014) Preparation and characterization of 4-isopropylcalix [4]arene-capped (3-(2-O-β-cyclodextrin)-2hydroxypropoxy)-propylsilyl-appended silica particles as chiral stationary phase for highperformance liquid chromatography. J Chromatogr A 1324:104–108 183. Chelvi SKT, Yong EL, Gong Y (2008) Preparation and evaluation of calyx[4]arene-capped β-cyclodextrin-bonded silica particles as chiral stationary phase for high-performance liquid

33

chromatography. J Chromatogr A 1203:54–58 184. Krawinkler KH, Maier NM, Sajovic E, Lindner W (2004) Novel urea-linked cinchonacalixarene hybrid-type receptors for efficient chromatographic enantiomer separation of carbamate-protected cyclic amino acids. J Chromatogr A 1053:119–131 185. Sanchez Pena M, Zhang Y, Warner IM (1997) Enantiomeric separations by use of calixarene electrokinetic chromatography. Anal Chem 69:3239–3242 186. Grady T, Joyce T, Smyth MR et al (1998) Chiral resolution of the enantiomers of phenylglycinol using (S)-di-naphthylprolinol calyx[4]arene by capillary electrophoresis and fluorescence spectroscopy. Anal Commun 35:123–125 187. Zhang JH, Xie SM, Wang BJ et al (2018) A homochiral porous organic cage with large cavity and pore window for the efficient gas chromatography separation of enantiomers and positional isomers. J Sep Sci 41:1385–1394 188. Zhang JH, Xie SM, Wang BJ et al (2015) Highly selective separation of enantiomers using a chiral porous organic cage. J Chromatogr A 1426:174–182 189. Xie SM, Zhang JH, Fu N et al (2016) A chiral porous organic cage for molecular recognition using gas chromatography. Anal Chim Acta 903:156–163 190. Chen LJ, Riss PS, Chong SY et al (2014) Separation of rare gases and chiral molecules by selective binding in porous organic cages. Nat Mater 13:954–960 191. Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346:818–822 192. Ellington AD, Szostak JW (1992) Selection in vitro of single-stranded DNA molecules that fold into specific ligand-binding structures. Nature 355:850–852 193. Peyrin E (2009) Nucleic acid aptamer molecular recognition principles and application in liquid chromatography and capillary electrophoresis. J Sep Sci 32:1531–1536 194. Ravelet C, Peyrin E (2006) Recent developments in the HPLC enantiomeric separation using chiral selectors identified by a combinatorial strategy. J Sep Sci 29:1322–1331

Chapter 2 Enantioseparation by Thin-Layer Chromatography Rituraj Dubey and Ravi Bhushan Abstract Despite the fact that high-performance liquid chromatography is the predominant technique for analytical and preparative enantioseparations, chiral thin-layer chromatography (TLC) may represent an alternative, especially if fast analysis with simple equipment is required. This chapter describes several approaches in chiral TLC for the separation of amino acids and basic drugs using DL-selenomethionine and β-adrenergic drugs as examples. Analytical approaches include the impregnation of the adsorbent with a chiral selector using pre-coated as well as custom-prepared TLC plates and addition of the selector to the mobile phase directly as well as in the form of copper metal complex. (
)-Quinine and L-amino acids were used as chiral selectors in different manners for enantioseparations. Key words Enantiomer separation, Chiral selector, Impregnation, Ligand-exchange, Thin-layer chromatography

1

Introduction The vast majority of analytical and preparative enantioseparations are carried out by high-performance liquid chromatography. However, thin-layer chromatography (TLC) has also been applied for the fast and cheap analysis as well as semi-preparative isolation of compounds in organic synthesis and natural product chemistry. TLC provides an easy technique for the resolution, isolation, and analytical control of the enantiomeric purity of a variety of compounds [1, 2]. Chiral TLC separations have been summarized [3–5]. The advantages of modern TLC in pharmaceutical and drug analysis in comparison to high-performance liquid chromatography have been reviewed [6]. In TLC, the chiral selector may be coated onto the adsorbent or added to the mobile phase. As only a few commercial TLC plates coated with chiral stationary phases are available, the coating of the chiral selector often has to be performed in the laboratory by the researcher himself/herself. This can be achieved by impregnating a commercial TLC plate containing, e.g., silica gel as adsorbent prior

Gerhard K. E. Scriba (ed.), Chiral Separations: Methods and Protocols, Methods in Molecular Biology, vol. 1985, https://doi.org/10.1007/978-1-4939-9438-0_2, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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to the use for an enantioseparation. This can be achieved by spraying a solution of the selector onto the TLC plate, by immersing or dipping the plate into the solution of the selector, or allowing a solution of the selector to ascend or descend in a normal manner of development. After drying, the TLC plate can be applied to an enantioseparation. Another approach is the use of laboratorymade TLC plates where the silica gel is mixed with a chiral selector and subsequently coated onto suitable supports. This approach offers the advantage that the thickness of the layer can be controlled so that analytical as well as semi-preparative TLC plates can be produced. Although modern laboratories most often use commercial pre-coated TLC plates that are expected to have a better uniformity of the adsorbent, the use of laboratory-made plates is also quite common for routine applications. Comparisons of various enantioseparations using either commercial or laboratory-made TLC plates from our laboratory did not reveal significant differences in terms of reproducibility and selectivity under the same conditions of mobile phase and temperature. Another alternative of chiral TLC is the use of a chiral mobilephase additive, in which the chiral selector is added to the mobile phase. The selector forms diastereomeric complexes with the analyte enantiomers which are subsequently separated on the (achiral) TLC plate. This chapter describes examples [7–9] for the approaches mentioned above, i.e., impregnation of TLC plates, the fabrication of laboratory-made TLC plates, and the use of chiral mobile-phase additives including the ligand-exchange principle. Analytes include DL-selenomethionine (SeMet) as an amino acid and the β-adrenergic drugs (RS)-atenolol, (RS)-propranolol, and (RS)salbutamol.

2

Materials

2.1 Instrumentation and Apparatus

1. Commercial pre-coated silica gel TLC plates (20 cm  20 cm  0.15 mm) from a standard international company (these may be cut into 20 cm  10 cm size for convenience and preliminary runs, the thickness of the layer remains the same). 2. For laboratory-made TLC (impregnated) plates: Glass plates (20  20 cm), silica gel G powder from a standard supplier (particle size 12 μm, pore diameter 60 A˚), and a suitable applicator (commonly known as Stahl-type applicator) to spread the slurry of silica gel. 3. TLC rectangular glass chambers (inside depth 07 cm, inside height 25 cm, inside width 27 cm; glass chamber of some other standard dimensions may be used depending upon the size of the TLC plate) with standard lid.

TLC Enantioseparations

37

4. A Hamilton syringe (25 μL) or graduated capillaries (with 5 μL markings) for spotting the sample on the plates. 5. A TLC sprayer for spraying with reagent solutions. 2.2 Solutions and Mobile Phases

2.2.1 Sample Solutions

Use HPLC-grade solvents and ultrapure water (conductivity not higher than 0.055 μS/cm at 25  C) for preparing solutions as per requirement. Store the standard solutions at 4  C. Follow appropriate safety regulations for the chemicals and solvents as well as waste disposal regulations. 1. SeMet sample solutions (SS-1A and SS-1B): Dissolve 2 mg of DLSeMet in 10 mL of water/sodium bicarbonate 1/8 (v/w) solution for SS-1A and 1 mg of L-(þ)-SeMet in 10 mL of water/sodium bicarbonate 1/8 (v/w) for SS-1B. 2. Atenolol sample solutions (SS-2A and SS-2B): Dissolve 5.2 mg of (RS)-atenolol in 10 mL methanol of SS-2A and 2.6 mg of either (R)-atenolol or (S)-atenolol in 10 mL methanol for SS-2B. 3. Propranolol sample solutions (SS-3A and SS-3B): Dissolve 5.2 mg of (RS)-propranolol in 10 mL methanol of SS-3A and 2.6 mg of either (R)-propranolol or (S)-propranolol in 10 mL methanol for SS-3B. 4. Salbutamol sample solutions (SS-4A and SS-4B): Dissolve 5.2 mg of (RS)-salbutamol in 10 mL methanol of SS-4A and 2.6 mg of either (R)-salbutamol or (S)-salbutamol in 10 mL methanol for SS-4B.

2.2.2 Solutions of Chiral Selector

1. Chiral selector solution CS-1: Dissolve 5 mg (
)-quinine in 10 mL methanol/water 8/2 (v/v) and adjust to pH 8.0 by addition of 0.1 M HCl. 2. Chiral selector solution CS-2: Dissolve 3.2 mg (
)-quinine in 10 mL ethanol. 3. Ligand-exchange solution CS-3: Dissolve 36 mg of copper acetate in 100.0 mL of water/methanol, 95/5 (v/v). Dissolve 72 mg of N,N-Me2-L-Phe in 100.0 mL of water/methanol, 95/5 (v/v). Mix 50 mL of the copper acetate solution and 100 mL of the N,N-Me2-L-Phe solution.

2.2.3 Mobile Phases

1. Mobile phase MP-1: Acetonitrile/methanol/dichloromethane/ water, 11/1/1/1.5 (v/v). 2. Mobile phase MP-2: Acetonitrile/methanol/dichloromethane/ water, 8/1/2/1.5 (v/v). 3. Mobile phase MP-3: Dissolve 35 mg (
)-quinine in 50 mL of acetonitrile/methanol/dichloromethane/water, 11/1/1/1.5 (v/v).

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4. Mobile phase MP-4: Acetonitrile/methanol/dichloromethane/ water, 10/1.5/0.5/1.5 (v/v). 5. Mobile phase MP-5: Acetonitrile/methanol/solution CS-3, 3/4/5 (v/v). 2.2.4 Detection Reagent

3

1. Ninhydrin solution: Dissolve 0.3 g ninhydrin in 97 mL nbutanol containing 3 mL glacial acetic acid.

Methods

3.1 General Procedure for TLC Separations

1. Use a clean and dry rectangular TLC chamber and line with Whatman number-1 filter paper or a similar filter paper up to a height of 15 cm. 2. Pour a few milliliters of one of the mobile phases into the TLC chamber so that the paper lining is properly wetted with the mobile phase and cover the chamber with the lid. See the respective example for the specific mobile phase (see Note 1). 3. Leave the chamber at room temperature for 10–15 min for equilibration and saturation with solvent vapors (see Note 2). 4. Spot 10 μL of the sample solution on TLC plate using a Hamilton syringe or a graduated capillary at a distance of 1 cm from the bottom of the plate. See the respective example for the specific sample solutions (see Note 3). 5. Remove the solvent mixture from the TLC chamber (the paper lining stays with the mobile phase soaked). 6. Pour a few milliliters of the fresh required mobile phase in the chamber. 7. Place the silica gel plate, duly spotted with the sample(s) in such a way that the spots are not dipped in the mobile phase present in the chamber. Cover the chamber properly with the lid. 8. Develop the plate (chromatogram) for 8 cm (note the required time). 9. Remove the TLC plate from the chamber and immediately mark the solvent front. 10. Dry the TLC plate at 40  C in an oven (for a few minutes) and cool to room temperature (see Note 4). 11. Place the TLC plate in an iodine chamber for visualization of the spots (see Note 5) or spray with a suitable detection reagent solution (e.g., ninhydrin solution in Example 4, see Note 6). 12. Calculate retention factors (Rf), separation selectivity (α), and resolution factor (RS) (see Note 7).

TLC Enantioseparations

3.2 Example 1: Enantioseparation of DL-SeMet on Laboratory-Made Chiral TLC Plates Prepared by Mixing Silica Gel and (
)Quinine as Chiral Selector

39

1. Prepare a slurry of 25 g silica gel G in 50 mL of selector solution CS-1. 2. Place six glass plates (10 cm height  5 cm width) side by side on the spreadsheet with the edges touching closely. 3. Spread the slurry on to the glass plates uniformly using a Stahltype applicator adjusted to a thickness of the layer of 0.5 mm. 4. Allow the slurry spread to settle and dry at room temperature. 5. Heat (activate) the plates in a laboratory drying oven for 6–8 h at 60  2  C. 6. Remove plates from the oven and allow them to cool at room temperature. 7. Apply sample solutions SS-1A and SS-1B as described in Subheading 3.1, step 4. 8. Use mobile phase MP-1 and follow the general procedure as described in Subheading 3.1, steps 1–12. Figure 1a shows a chromatogram of the separation of DL-SeMet using (
)-quinine as chiral selector on laboratory-made TLC plates. See also ref. 7.

3.3 Example 2: Enantioseparation of DL-SeMet on Laboratory-Made Chiral TLC Plates Prepared by Impregnation with the Chiral Selector (
)-Quinine by Ascending Development

1. Use commercial silica gel TLC plates. 2. Pour 10 mL of CS-2 in TLC chamber (see Note 8). 3. Place the TLC plate in the chamber with nearly 1 cm of the plate dipping in the solution; allow the solution to ascend on the plate up to 15 cm. 4. Remove plate from TLC chamber and dry at room temperature. 5. Use sample solutions SS-1A and SS-1B as well as mobile phase MP-2 and follow the general procedure according to Subheading 3.1, steps 1–12. A chromatogram of the separation of DL-SeMet using (
)quinine as chiral selector using impregnation by pre-coating of TLC plates is shown in Fig. 1b.

3.4 Example 3: Enantioseparation of DL-SeMet Using the Chiral Selector (
)-Quinine as MobilePhase Additive

1. Use commercial silica gel TLC plates. 2. Use samples solutions SS-1A and SS-1B as well as mobile phase MP-3 and follow the general procedure according to Subheading 3.1, steps 1 to 12. A chromatogram of the separation of DL-SeMet using (
)quinine as mobile-phase additive is shown in Fig. 1c.

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Fig. 1 Photographs of chromatograms showing resolution of DL-SeMet using (
)-quinine by the three approaches: (a): Using (
)-quinine-impregnated plates. Mobile phase: CH3CN–CH3OH–CH2Cl2–H2O (11:1:1:1.5, v/v). (b): Ascending development of the plate in the solution of chiral selector prior to spotting the racemate. Mobile phase: CH3CN–CH3OH–CH2Cl2–H2O (8:1:2:1.5, v/v). Development time, 9 min. (c): CS added to mobile phase. Mobile phase: CH3CN–CH3OH–CH2Cl2–H2O (9:1:1:1.5, v/v) having 0.07% of (
)quinine. Development time: 10 min. In all approaches, solvent front is 8 cm; temperature, 25  2  C; detection by ninhydrin or by iodine vapor; from left to right: Line 1, lower spot is of D-isomer and the upper spot is of L-isomer resolved from the mixture of DL-SeMet. Line 2, pure L-isomer. The plate acquires a light pink background due to ninhydrin treatment but the resolved spots are visible with greater intensity of characteristic color and sharpness (reproduced by permission of the Royal Society of Chemistry from ref. 7 © 2014) 3.5 Example 4: Enantioseparation of DL-SeMet Using (
)Quinine as ChiralInducing Reagent

1. Use commercial silica gel TLC plates. 2. Mix 1 mL of each of sample solution SS-1A and chiral selector solution CS-1. 3. Use this mixture as sample solution and mobile phase MP-4 and follow the general procedure according to Subheading 3.1, steps 1–12. 4. Instead of placing the plate in an iodine chamber, spray plate with ninhydrin reagent solution using a TLC sprayer (see Note 6). 5. Heat the plate in oven at 70  C for 10 min. 6. Remove plate, cool to room temperature, and calculate retention factors (Rf), separation selectivity (α), and resolution factor (RS) (see Note 7). Figure 2 shows a chromatogram of the separation of DL-SeMet using (
)-quinine as chiral-inducing agent. See also ref. 8.

TLC Enantioseparations

41

Fig. 2 Photograph of a chromatogram showing the resolution of DL-SeMet using (
)-quinine as CIR (Approach 3.2.4). Line 1: lower spot is D-(
)-isomer, upper spot is L-(þ)-isomer (from racemic mixture). Line 2: pure L-(þ)-isomer. Mobile phase: CH3CN–CH3OH–CH2Cl2–H2O (10:1.5:0.5:1.5, v/v). 8 cm; 10 min; 25  2  C; detection by ninhydrin (reproduced by permission of the Royal Chemical Society from ref. 8 © 2015)

Table 1 compares the methods described in Examples 1–4 for the enantioseparation of DL-SeMet using (
)-quinine as chiral selector. 3.6 Example 5: Enantioseparation of β-Adrenergic Drugs by Ligand-Exchange Method Using N,N-Me2-L-Phe as Chiral LigandExchange Reagent

1. Use commercial silica gel TLC plates. 2. Apply side by side 5–10 μL of sample solutions SS-2A and SS-2B, SS-3A and SS-3B, as well as SS-4A and SS-4B. 3. Use mobile phase MP-5 and follow the general procedure according to Subheading 3.1, steps 1–12. Figure 3 shows enantioseparation of the β-adrenergic drugs atenolol, propranolol, and salbutamol using Cu(II) complex of N, N-Me2-L-Phe as ligand-exchange chiral selector in the mobile phase. For further examples of mobile phases for the enantioseparation of β-adrenergic drugs see ref. 9.

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Table 1 Comparison of the methods described in Examples 1–4 for the enantioseparation of (
)-quinine as chiral selector

DL-SeMet

using

hRf

Mobile phase (solvent combinations, v/v)

a

S. No. Approach

From racemic mixture

Pure (L) (L) (D)

Time (min) RS

3.1

CS mixed in the slurry of CH3CN–CH3OH–CH2Cl2–H2O, silica gel before (11/1/1/1.5) (MP-1) making the plates

30

30

13

10

4.31

3.2

Ascending development of the plate in the solution of CS

CH3CN–CH3OH–CH2Cl2–H2O, (8/1/2/1.5) (MP-2)

40

40

19

9

2.84

3.3

CS added to mobile phase

CH3CN–CH3OH–CH2Cl2–H2O (9/1/1/1.5) having 0.07% of (
)-quinine (MP-3)

35

35

18

10

1.86

3.4

Premixing of CIR with analyte

CH3CN–CH3OH–CH2Cl2–H2O, (10/1.5/0.5/1.5) (MP-4)

13

26

13

10

1.69

Rs resolution hRf ¼ retardation factor  100 (Rf  100) CS chiral selector CIR chiral-inducing reagent a The serial number refers to the relevant section in the main text

4

Notes 1. Mix the solvents in the said ratio in a glass beaker, pour it into the glass chamber, and leave it for 15 min for equilibration period. After equilibration of the chamber with the mobile phase, quickly replace the solvent with a fresh lot of the same mobile phase for development of chromatogram. 2. Discard the mobile phase after one chromatographic run (i.e., a fresh lot of the mobile phase is to be used for saturating the TLC chamber with solvent and for developing the chromatogram each time). 3. Spot the solution of racemic and the pure isomer (10 μL) side by side with the help of 25 μL Hamilton syringe or a graduated capillary. 4. Take the developed chromatogram out of the glass chamber carefully, allow the solvent to evaporate at room temperature for a while, and then place it into the oven gently and for about 5 min at 40  C. Then take out the plates carefully. Do not forget to wear heat-resistant gloves.

TLC Enantioseparations

43

Fig. 3 Photograph of the chromatogram showing resolution of racemic atenolol, propranolol, and salbutamol using plain plate and Cu(II) complex of N,N-Me2-LPhe in the mobile phase as chiral additive (MP-5). Track 1 lower spot is (S)atenolol and the upper one is (R)-atenolol; track 2 is pure (S)-atenolol; track 3 lower spot is (S)-propranolol and the upper one is (R)-propranolol and track 4 is pure (S)-propranolol; in track 5 lower spot is (S)-salbutamol and the upper spot is (R)-salbutamol. Run time: 12 min. Detection: iodine vapor. The (R)enantiomers elute before (S)-enantiomers. For further examples of mobile phases for the enantioseparation of beta-adrenergic drugs see ref. 9. (reproduced by permission of Springer from ref. 9 © 2009)

5. Place 2–3 iodine granules in a glass chamber covered suitably with a greased glass plate. Place the dried and cooled chromatogram in the iodine chamber after you see yellow-browncolored fumes in the chamber. After 2–3 min, take the plate out of the chamber, observe, and mark the spots on the chromatograms. Capture a photograph of the same showing enantioseparation results. 6. Spray the ninhydrin reagent using an atomizer with hand bulb or air pump (in a uniform manner) on the plate by standing the plate in a fume hood. 7. The retention factor Rf is calculated by measuring the distance traveled by the center of the spot (ds) and the eluent front (dE) using Eq. 1:

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Rf ¼

ds dE

ð1Þ

The separation factor α is calculated according to Eq. 2:
Rf2
1
α¼ ð2Þ Rf1
1 The resolution factor RS is calculated according to Eq. 3: RS ¼ 2 

distance between the centers of the two spots sum of the widths of the two spots

ð3Þ

8. The total volume of the mobile phase is mentioned, as an example. Since the ratio of different solvents comprising that mobile phase is given the total volume of the mobile phase in the chamber may vary upon the size of the chamber and the TLC plate. If someone spots the sample at a distance different than 1 cm, the volume of mobile phase in the chamber will be different. The basic point is that the spots should not dip into the mobile phase taken inside the chamber. References 1. Bhushan R, Martens J (2010) Amino acids: chromatographic separation and enantioresolution. HNB Publishing, New York 2. Dixit S, Bhushan R (2014) Chromatographic analysis of chiral drugs. In: Komsta L, Waksmundzka-Hajnos M, Sherma J (eds) TLC in drug analysis. Taylor and Francis, Boca Raton, p 97 3. Kowalska T, Sherma J (eds) (2007) Thin layer chromatography in chiral separations and analysis. CRC Press, Boca Raton 4. Gunther K, Moeller K (2003) Enantiomer separations. In: Sherma J, Fried B (eds) Handbook of thin-layer chromatography, 3rd edn. Marcel Dekker Inc., New York, pp 471–533 5. Del Bubba M, Checchini L, Cincinelli A, Lepri L (2013) Enantioseparations by thin-layer chromatography. In: Scriba GKE (ed) Chiral separations, methods and protocols, 2nd edn. Humana Press, New York, pp 29–43

6. Sherma J (2001) Modern thin layer chromatography in pharmaceutical and drug analysis. Pharm Forum 27:3420–3431 7. Nagar H, Bhushan R (2014) Enantioresolution of DL-selenomethionine by thin silica gel plates impregnated with (
)-quinine and reversedphase TLC and HPLC of diastereomers prepared with difluorodinitrobenzene based reagents having L-amino acids as chiral auxiliaries. Anal Methods 6:4188–4198 8. Bhushan R, Nagar H, Martens J (2015) Resolution of enantiomers with both achiral phases in chromatography: conceptual challenge. RSC Adv 5:28316–28323 9. Bhushan R, Tanwar S (2009) Direct TLC resolution of the enantiomers of three β-blockers by ligand exchange with cu(II)–L-amino acid complex, using four different approaches. Chromatographia 70:1001–1006

Chapter 3 Enantioseparations by Gas Chromatography Using Porous Organic Cages as Stationary Phase Sheng-Ming Xie, Jun-Hui Zhang, and Li-Ming Yuan Abstract The resolution of chiral compounds into optically pure enantiomers is very important in various fields, such as pharmaceutical, chemical, agricultural, and food industries. Chiral gas chromatography (GC) is one of the efficient methods for enantioseparations of volatile compounds. In recent years, porous materials as stationary phases for chromatographic separations have achieved increasing attention. Porous organic cages (POCs) represent an emerging class of porous materials, which are assembled by discrete organic molecules with shape-persistent and permanent cavities through weak intermolecular forces. This chapter describes several chiral POCs as chiral stationary phases for GC enantioseparations of racemic compounds. Key words Enantioseparation, Chiral stationary phase, Gas chromatography, Porous material, Porous organic cage

1

Introduction Chirality is a fundamental property of the nature. The resolution of enantiomers in racemic mixtures is of great importance in many areas, especially in pharmaceutical industry, because different drug enantiomers will express different biological activity, pharmacology, or toxicity [1]. Among various chiral separation techniques, chiral chromatography seems to be the most efficient technique for chiral separation. Capillary gas chromatography (GC) as a chromatographic separation technique has been widely used for enantioseparation of volatile compounds due to its advantages including high efficiency, simplicity, sensitivity, speed, reproducibility, and so on. Chiral stationary phases (CSPs) are the key to enantiomer separation by chiral chromatography. Since the first example of separation enantiomers by GC on an N-trifluoroacetyl-L-isoleucine-based CSP was described by Gil-Av and co-workers in 1966 [2], enantiomeric separation by GC was rapidly developed. To date, the classical CSPs of GC are classified into three categories: (1) amino acid derivatives acting predominantly via hydrogen

Gerhard K. E. Scriba (ed.), Chiral Separations: Methods and Protocols, Methods in Molecular Biology, vol. 1985, https://doi.org/10.1007/978-1-4939-9438-0_3, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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bonding, (2) chiral metal coordination compounds via complexation, and (3) cyclodextrin derivatives via (inter alia) inclusion complex formation [3]. The enantioselectivity of amino acid derivativebased CSPs is relatively narrow, which is mainly used for the separation and analysis of chiral amino acid and amino alcohol derivatives. The use of metal coordination compound-based CSPs is limited due to their poor thermal stability. Among the available CSPs, cyclodextrin derivatives are without doubt the most widely employed CSPs in chiral GC and exhibit prominent resolution ability. At present, there are more than 20 kinds of commercial GC chiral columns based on cyclodextrin derivatives as stationary phases. However, the enantioselectivity of a single cyclodextrin derivative column is limited and the typically applied temperature should not exceed 230
C. Besides, the preparation process of cyclodextrin derivatives is more complex and, consequently, the price of such a column is relatively high. Therefore, the development of novel CSPs with high enantioselectivity, high temperature resistance, and low cost for GC is an important research topic. Porous materials are defined as substances that contain accessible voids and possess some unusual properties, such as diverse compositions and structures, high surface areas, and tunable pore diameters. In recent years, the utilization of porous materials such as metal-organic frameworks (MOFs) [4, 5], covalent organic frameworks (COFs) [6, 7], mesoporous silica [8, 9], and microporous organic polymers (MOPs) [10, 11] for chromatographic separations has attracted considerable attention. In particular, some of them have been explored as novel stationary phases for GC enantioseparations [6, 8, 9, 10–12]. However, the insolubility of these materials imposes significant limitations on the preparation of capillary GC columns. Porous molecular materials composed of discrete organic molecules have attracted increasing attention in recent years due to their unique properties that set them apart from porous materials with frameworks or networks, such as MOFs, COFs, zeolites, and MOPs [13–15]. For example, porous molecular materials are readily solution-processable because the discrete organic molecules assemble into solids or crystals by weak intermolecular interactions rather than covalent or coordination bonds. Moreover, porous molecules exhibit structural mobility due to the lack of intermolecular covalent bonding in assemblies, which allows cooperative interactions between the host and guests [16]. Porous organic cages (POCs) [17–19] represent novel porous molecular materials, which assemble from discrete organic cage molecules with shape-persistent, permanent, and accessible cavities and have become of significant interest in the past years owing to their potential applications in gas adsorption and separation [20, 21], molecular recognition [22, 23], sensing [24], and heterogeneous catalysis [25], and as molecular reaction containers [26, 27].

Chiral POCs for GC Enantioseparations

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In this chapter, the enantioseparation of chiral compounds by capillary GC using POCs as CSPs is described in detail. The fabricated columns exhibit excellent enantioselectivity for the resolution of racemic compounds [28–32]. Among these, a CC3-R-based column shows the most prominent resolution ability. A large number of racemates belonging to different classes have been resolved on the CC3-R-based column without derivatization, including chiral alcohols, diols, amines, esters, ketones, ethers, halohydrocarbons, organic acids, amino acid methyl esters, and sulfoxides [28]. Although the length of the coated column is only 15 m, most enantiomers were baseline separated with high-resolution values, especially for alcohols such as 2-butanol (RS ¼ 16.09), which are difficult to be well separated on commercially available chiral columns. A representative chromatogram is shown in Fig. 1. Compared with commercial β-DEX 120 and Chirasil-L-Val columns, the CC3-R-coated capillary column showed more preeminent enantioselectivity. The homochiral pentyl cages, CC9 and CC10, were able to achieve a number of separations that were not possible using CC3-R [30–32]. CC5 has large cavity volume and pore windows, so that analytes with relatively large size can be

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Fig. 1 Representative GC chromatograms on a CC3-R-based capillary column (15 m long  0.25 mm i.d.) for the separation of racemates. (a) 2-Butanol at 110
C under a N2 linear velocity of 16.5 cm s1; (b) 3-butene2-ol at 115
C under a N2 linear velocity of 12.5 cm s1; (c) 1-methoxy-2-propanol acetate at 145
C under a N2 linear velocity of 14.7 cm s1; (d) 2-ethylhexanoic acid at 210
C under a N2 linear velocity of 16.6 cm s1; (e) 1-methoxy-2-hydroxypropane at 150
C under a N2 linear velocity of 13.2 cm s1; (f) 1,2-epoxyoctane at 185
C under a N2 linear velocity of 16.6 cm s1; (g) 2-methyltetrahydrofuran at 115
C under a N2 linear velocity of 14.7 cm s1; (h) 2-methyltetrahydrofuran-3-one at 140
C under a N2 linear velocity of 15.6 cm s1 (reproduced by permission of the American Chemical Society from ref. 28 © 2015)

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separated on CC5-based columns but were not separated on the other POC-based columns [29]. These POC-based columns have a complementary role in chiral recognition.

2

Materials

2.1 Instrumentation and Materials

1. A commercial GC instrument, for example, a Shimadzu GC2014C system equipped with a flame ionization detector (FID) and suitable software for instrument control and data acquisition. 2. Fused silica capillaries (250 μm i.d.) with polyimide outer coating (400
C maximum temperature). 3. 0.22 μm Membrane filters. 4. A vacuum pump (allowing a pressure of 0.08 MPa). 5. A 1 μL syringe for sample injection.

2.2 Chemicals and Solutions 2.2.1 Chemicals for POC Synthesis

All solvents should be at least of analytical grade.

1. 1,3,5-Triformylbenzene. 2. (1R,2R)-1,2-diaminocyclohexane. 3. (1R,2R)-1,2-cyclopentanediamine dihydrochloride. 4. Tris(4-formylphenyl)amine. 5. (1R,2R)-1,2-diphenylethylenediamine. 6. (1R,2R)-1,2-bis(4-fluorophenyl)-1,2-ethanediamine dihydrochloride. 7. (1R,2R)-1,2-bis(2-hydroxyphenyl)-1,2-diaminoethane. 8. A polysiloxane OV-1701 column.

2.2.2 Sample Solutions (See Note 1)

3

Prepare solutions of the racemic analytes at a concentration of 5 mg/mL to 25 mg/mL in CH2Cl2.

Methods (See Note 2) All reactions are performed under nitrogen. Typically a doublenecked round-bottom flask with suitable connections to a nitrogen supply is used. Suitable protective gear should be worn when handling chemicals. Moreover all required safety precautions should be undertaken.

3.1 Synthesis of CC3-R

1. Weigh 1.0 g (6.17 mmol) 1,3,5-triformylbenzene into a 250 mL double-necked, round-bottom flask and dropwise add 20 mL dichloromethane under nitrogen.

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2. Add 20 μL trifluoroacetic acid to the solution. 3. Dissolve 1.0 g (8.77 mmol) (R,R)-1,2-diaminocyclohexane in 20 mL dichloromethane and slowly layer onto the above solution (see Note 3). 4. Cover the round-bottom flask and leave to stand at room temperature for 3 days. 5. Collect the white octahedron-like crystals by filtration and wash three times with 30 mL ethanol/dichloromethane (95:5, v/v). 3.2

Synthesis of CC5

1. Weigh 70 mg (0.40 mmol) (1R,2R)-1,2-cyclopentanediamine dihydrochloride into a 100 mL oven-dried double-necked, round-bottom flask, equipped with a magnetic stir bar under a nitrogen atmosphere. Add 33 mL dry methanol and 80 mg (0.79 mmol) dry triethylamine and stir at room temperature for 10 min to form a colorless solution. 2. Dissolve 88 mg (0.27 mmol) tris(4-formylphenyl)amine in 33 mL dry dichloromethane in a 100 mL double-necked, round-bottom flask under a nitrogen atmosphere. 3. Slowly add the solution of (1R,2R)-1,2-cyclopentanediamine dihydrochloride to the solution of tri(4-formylphenyl)amine. 4. Cover the round-bottom flask and leave to stand at room temperature. After 7 days, collect the crystalline product by filtration and wash three times with 50 mL dichloromethane to obtain pale yellow crystals of CC5.

3.3

Synthesis of CC9

1. Dissolve 1.50 g (7.06 mmol) of (1R,2R)-1,2-diphenylethylenediamine and 0.764 g (4.71 mmol) 1,3,5-triformylbenzene in 60 mL dry dichloromethane into a 150 mL double-necked, round-bottomed flask containing a magnetic stir bar under a nitrogen atmosphere. 2. Add 27 μL trifluoroacetic acid. 3. Continue stirring the reaction mixture at 15
C for 50 h. 4. Add an excess of NaHCO3 (approx. 100 mg) to quench the reaction. 5. Dilute the suspension by adding 60 mL dry dichloromethane. 6. Filter the reaction mixture using a sintered glass funnel. 7. Rinse the flask with 60 mL dry dichloromethane and add the rinse into the filter cake. 8. Concentrate the filtrate to about 40 mL using a rotatory evaporator. 9. Add 50 mL acetone into the 40 mL concentrate to form a suspension.

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10. Filter the suspension and wash the filter cake with 50 mL acetone. 11. Dry the filter cake under suction to afford CC9 as white solid. 3.4 Synthesis of CC10

1. Weigh 0.60 g (1.87 mmol) (1R,2R)-1,2-bis(4-fluorophenyl)1,2-ethanediamine dihydrochloride into a double-necked, round-bottom flask equipped with a magnetic stir bar under a nitrogen atmosphere. Add 40 mL deionized water and stir until all material is dissolved. 2. Add 0.54 mL triethylamine, stir for 15 min, then add 20 mL dichloromethane, and stir for another 30 min. 3. Settle and separate the two phases, retain the organic phase, and evaporate to dryness under reduced pressure at 100
C to afford (1R,2R)-1,2-bis(4-fluorophenyl)ethane-1,2-diamine as yellow oil product. 4. Dissolve 0.36 g (1.45 mmol) (1R,2R)-1,2-bis(4-fluorophenyl) ethane-1,2-diamine and 0.16 g (0.98 mmol) 1,3,5triformylbenzene in 5 mL dry dichloromethane in a doublenecked, round-bottom flask under a nitrogen atmosphere, and then add 0.25 g dried molecular sieves and 10 μL trifluoroacetic acid (see Note 4). 5. Continue stirring the reaction mixture for 96 h at room temperature. 6. Filter the reaction mixture, and add dropwise the filtrate to 75 mL acetonitrile to produce white precipitate. 7. Collect the white precipitate by centrifugation and wash with chloroform in a Soxhlet extractor. 8. Dry at 60
C under vacuum to yield CC10.

3.5 Synthesis of Homochiral Pentyl Cage

1. Add 2.0 g (8.2 mmol) (1R,2R)-1,2-bis(2-hydroxyphenyl)1,2-diaminoethane and 50 mL toluene into a 100 mL double-necked, round-bottomed flask equipped with a DeanStark trap and a magnetic stir bar at room temperature. 2. Add 2.46 mL (20.50 mmol) hexanal and reflux overnight with a Dean-Stark trap. 3. Remove the solvent under reduced pressure to obtain a viscous yellow oil product. 4. Purify the viscous yellow oil product by precipitation using methanol (60 mL) to afford a suspension. 5. Filter the suspension to afford (1S,2S)-N,N0 -bis(salicylidene)1,2-pentyl-1,2-diaminoethane as yellow solid. 6. Dissolve 2.13 g (2.6 mmol) (1S,2S)-N,N0 -bis(salicylidene)1,2-pentyl-1,2-diaminoethane in 12 mL THF and then add 0.78 mL 37% HCl solution and 12 mL THF. Add the mixture

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into a 50 mL double-necked, round-bottomed flask equipped with a magnetic stir bar under a nitrogen atmosphere. 7. Continue stirring the reaction mixture for 24 h at room temperature. 8. Dilute the mixture with 50 mL diethyl ether. Extract with 15 mL water three times and collect the aqueous phase. 9. Basify the water phase with 1.0 M NaOH solution. Extract with 30 mL of dichloromethane three times. Dry the combined dichloromethane over Na2SO4. 10. Evaporate the solvent under reduced pressure by using a rotary evaporator to afford (1S,2S)-1,2-pentyl-1,2-diaminoethane as a red liquid. 11. Dissolve 0.145 g (0.73 mmol) (1S,2S)-1,2-pentyl-1,2-diaminoethane in 3.3 mL chloroform and 0.06 g (0.40 mmol) 1,3,5-triformylbenzene in 2.3 mL chloroform, respectively. Mix the two solutions in a 25 mL double-necked, roundbottomed flask equipped with a magnetic stir bar. Add 10 μL trifluoroacetic acid. 12. Stir the reaction mixture at 60
C for 72 h. 13. Remove the solvent under reduced pressure and immediately add 20 mL acetone to the residue for preliminary purification by precipitation. Filter the suspension to obtain crude product of pentyl cage as a white solid. 14. Further purify the crude pentyl cage by diffusing acetone into a solution of the pentyl cage in dichloromethane. Dissolve the above-obtained crude product of the pentyl cage in dichloromethane in a small beaker and add acetone to a big beaker. Then, place the small beaker into the big beaker and seal the two beakers using fresh keeping film. Grow homochiral pentyl cage crystals by slowly diffusing acetone into the small beaker. 3.6 Coating Capillary GC Column 3.6.1 Preparation of CC3-R-, CC9-, CC10-, and Homochiral Pentyl CageBased Columns

The capillary GC columns were prepared by a static coating method. The detailed process is as follows: 1. Pretreat fused silica capillary columns (15 m long  250 μm i.d. for CC3-R, CC5, and homochiral pentyl cage columns; 30 m long  250 μm i.d. for CC9 and CC10 columns) according to the following process prior to coating: Attach the capillary columns to a vacuum pump (0.07 MPa pressure) by a connector, and then wash the columns with 1 M NaOH for 2 h, ultrapure water for 1 h, and 0.1 M HCl for 2 h, and again with ultrapure water for a period of time to ensure that the washings from the other end of the column were neutral. Finally, dry the columns with a nitrogen purge at 120
C for 6 h.

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2. Dissolve POC (CC3-R, CC9, CC10, homochiral pentyl cage) in dichloromethane to prepare 3.0 mg/mL solutions, and filter through 0.22 μm membrane filters. 3. Dissolve polysiloxane OV-1701 in dichloromethane to prepare a 4.5 mg/mL solution, and filter through a 0.22 μm membrane filter. 4. Mix 2 mL of the POC solution with 2 mL of the polysiloxane OV-1701 solution and degas by sonication (see Note 5). 5. Pump the mix of the solutions of POC and polysiloxane OV-1701 into the pretreated capillary column and seal one end of the column when the entire column is filled (see Note 6). 6. Connect the other end of the column to a vacuum bottle (0.07 MPa pressure) to gradually remove the solvent under reduced pressure at 36
C until the solvent is completely evaporated (see Note 7). 3.6.2 Preparation of CC5-Based Column

The preparation method of CC5-based column is similar to that described above. 1. Dissolve CC5 in chloroform to prepare 1.6 mg/mL solution, and filter through a 0.22 μm membrane filter. 2. Dissolve polysiloxane OV-1701 in chloroform to prepare a 4.5 mg/mL solution, and filter through a 0.22 μm membrane filter. 3. Follow steps 1 and 4–6 as described in Subheading 3.6.1 using a temperature of 46
C instead of 36
C in step 6.

3.6.3 GC Enantioseparations

1. Install the coated capillary in the GC instrument according to the instructions of the manufacturer. Set the experimental parameters of the GC instrument. In this example, the following settings are used: (a) Injector temperature: 300
C. (b) Detector temperature: 300
C. (c) Column temperature: see Fig. 1 for experimental conditions used for the respective analytes. 2. Condition the column at 200
C until a stable baseline is obtained (see Note 8). 3. Inject 0.1–0.2 μL of the solution of racemic analytes or 0.01–0.02 μL of the neat racemic analytes, and record the chromatograms. The split ratio is 1:40–1:100. 4. In case of insufficient separation, optimize the experimental conditions (see Note 9). For representative chromatograms using a CC3-R-based capillary column see Fig. 1. For examples of enantioseparations on other chiral POC columns see refs. [28–32].

Chiral POCs for GC Enantioseparations

4

53

Notes 1. Generally, inject the neat racemic analyte for GC enantioseparation if the racemic analyte is liquid. 2. In general, all chemical reactions should be performed under a well-ventilated fume hood. Some organic solvents and reagents such as dichloromethane, chloroform, and methanol are harmful if swallowed, inhaled, or absorbed through skin. Trifluoroacetic acid is highly corrosive and volatile. Thus, all organic solvents and reagents should be handled carefully. The syntheses of five POCs should be performed under nitrogen. To expediently add the reagents into the reactor under nitrogen, the double-necked, round-bottomed flask should be selected as reactor. 3. In order to get a higher yield, the solution should be slowly added along the wall of the flask and not stirred. 4. Solvents used in the synthetic procedure must be dried by suitable procedures. 5. Polysiloxanes, such as OV-1701, OV-17, OV-101, and OV-210, are a class of substituted polymers with various functional groups of different polarities, which have been widely used as diluents in the coating solutions of different separation materials. Mixing POC with polysiloxane OV-1701 as stationary phase can improve the column efficiency as well as selectivity due to the good film-forming ability and coating property of OV-1701. 6. Air bubbles should be excluded during the sealing process; otherwise it will affect the coating efficiency. 7. In order to prepare a POC-coated capillary column with uniform coating on the inner wall of capillary, the temperature and vacuum system must be stable during the process of coating. To keep the pressure stable and column still in the process of column coating, a large vacuum bottle was used to provide negative pressure. The vacuum bottle should be brought to a vacuum of 0.07 MPa through a vacuum pump before use. 8. Prior to the GC separation process, the coated column should be aged at 200
C under a flow of N2 to achieve a stable baseline. The column temperature cannot exceed the decomposition temperature of the stationary phase. The CC3-R, CC5, CC9, CC10, and homochiral pentyl cage stationary phases are stable up to 350
C, 300
C, 250
C, 230
C, and 290
C, respectively. 9. Optimization of a separation can be performed by changing the column temperature, linear velocity of carrier gas, split ratio, and quantity of sampling.

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Acknowledgments This work was supported by the National Natural Science Foundation (Nos. 21765025, 21705142, 21675141, 21365024) of China and Applied Basic Research Foundation of Yunnan Province (No. 2017FB013). References 1. Maier N, Franco P, Lindner W (2001) Separation of enantiomers: needs, challenges, perspectives. J Chromatogr A 906:3–33 2. Gil-Av E, Feibush B, Charles-Sigler R (1966) Separation of enantiomers by gas liquid chromatography with an optically active stationary phase. Tetrahedron Lett 7:1009–1015 3. Schurig V (2011) Separation of enantiomers by gas chromatography on chiral stationary phases, Chapter 9. In: Ahuja S (ed) Chiral separation methods. Wiley, Hoboken, pp 251–297 4. Gu ZY, Yan XP (2010) Metal-organic framework MIL-101 for high-resolution gas-chromatographic separation of xylene isomers and ethylbenzene. Angew Chem Int Ed 49:1477–1480 5. Zhang M, Pu ZJ, Chen XL, Gong XL, Zhu AX, Yuan LM (2013) Chiral recognition of a 3D chiral nanoporous metal-organic framework. Chem Commun 49:5201–5203 6. Qian HL, Yang CX, Yan XP (2016) Bottom-up synthesis of chiral covalent organic frameworks and their bound capillaries for chiral separation. Nat Commun 7:12104 7. Han X, Huang JJ, Yuan C, Liu Y, Cui Y (2018) Chiral 3D covalent organic frameworks for high performance liquid chromatographic enantioseparation. J Am Chem Soc 140:892–895 8. Zhang JH, Xie SM, Zhang M, Zi M, He PG, Yuan LM (2014) Novel inorganic mesoporous material with chiral nematic structure derived from nanocrystalline cellulose for highresolution gas chromatographic separations. Anal Chem 86:9595–9602 9. Li YX, Fu SG, Zhang JH, Xie SM, Li L, He YY, Zi M, Yuan LM (2018) A highly ordered chiral inorganic mesoporous material used as stationary phase for high-resolution gas chromatographic separations. J Chromatogr A 1557:99–106 10. Dong J, Liu Y, Cui Y (2014) Chiral porous organic frameworks for asymmetric heterogeneous catalysis and gas chromatographic separation. Chem Commun 50:14949–14952

11. Lu CM, Liu SQ, Xu JQ, Ding YJ, Ouyang GF (2016) Exploitation of a microporous organic polymer as a stationary phase for capillary gas chromatography. Anal Chim Acta 902:205–211 12. Xie SM, Zhang ZJ, Wang ZY, Yuan LM (2011) Chiral metal-organic frameworks for highresolution gas chromatographic separations. J Am Chem Soc 133:11892–11895 13. McKeown NB (2010) Nanoporous molecular crystals. J Mater Chem 20:10588–10597 14. Couderc G, Hulliger J (2010) Channel forming organic crystals: guest alignment and properties. Chem Soc Rev 39:1545–1554 15. Holst JR, Trewin A, Cooper AI (2010) Porous organic molecules. Nat Chem 2:915–920 16. Song Q, Jiang S, Hasell T, Liu M, Sun SJ, Cheetham AK, Sivaniah E, Cooper AI (2016) Porous organic cage thin films and molecularsieving membranes. Adv Mater 28:2629–2637 17. Mastalerz M (2010) Shape-persistent organic cage compounds by dynamic covalent bond formation. Angew Chem Int Ed 49:5042–5053 18. Zhang G, Mastalerz M (2014) Organic cage compounds-from shape-persistency to function. Chem Soc Rev 43:1934–1947 19. Tozawa T, Jones JTA, Swamy SI, Jiang S, Adams DJ, Shakespeare S, Clowes R, Bradshaw D, Hasell T, Chong SY, Tang C, Thompson S, Parker J, Trewin A, Bacsa J, Slawin AMZ, Steiner A, Cooper AI (2009) Porous organic cages. Nat Mater 8:973–978 20. Jin YH, Voss BA, Noble RD, Zhang W (2010) A shape-persistent organic molecular cage with high selectivity for the adsorption of CO2 over N2. Angew Chem Int Ed 49:6348–6351 21. Hasell T, Miklitz M, Stephenson A, Little MA, Chong SY, Clowes R, Chen L, Holden D, Tribello GA, Jelfs KE, Cooper AI (2016) Porous organic cages for sulfur hexafluoride separation. J Am Chem Soc 138:1653–1659 22. Mitra T, Jelfs KE, Schmidtmann M, Ahmed A, Chong SY, Adams DJ, Cooper AI (2013) Molecular shape sorting using molecular organic cages. Nat Chem 5:276–281

Chiral POCs for GC Enantioseparations 23. Chen L, Reiss PS, Chong SY, Holden D, Jelfs KE, Hasell T, Little MA, Kewley A, Briggs ME, Stephenson A, Thomas KM, Armstrong JA, Bell J, Busto J, Noel R, Liu J, Strachan DM, Thallapally PK, Cooper AI (2014) Separation of rare gases and chiral molecules by selective binding in porous organic cages. Nat Mater 13:954–960 24. Brutschy M, Schneider MW, Mastalerz M, Waldvogel SR (2012) Porous organic cage compounds as highly potent affinity materials for sensing by quartz crystal microbalances. Adv Mater 24:6049–6052 25. Sun JK, Zhan WW, Akita T, Xu Q (2015) Toward homogenization of heterogeneous metal nanoparticle catalysts with enhanced catalytic performance: soluble porous organic cage as a stabilizer and homogenizer. J Am Chem Soc 137:7063–7066 26. McCaffrey R, Long H, Jin Y, Sanders A, Park W, Zhang W (2014) Template synthesis of gold nanoparticles with an organic molecular cage. J Am Chem Soc 136:1782–1785 27. Uemura T, Nakanishi R, Mochizuki S, Kitagawa S, Mizuno M (2016) Radical polymerization of vinyl monomers in porous

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organic cages. Angew Chem Int Ed 55:6443–6447 28. Zhang JH, Xie SM, Chen L, Wang BJ, He PG, Yuan LM (2015) Homochiral porous organic cage with high selectivity for the separation of racemates in gas chromatography. Anal Chem 87:7817–7824 29. Zhang JH, Xie SM, Wang BJ, He PG, Yuan LM (2018) A homochiral porous organic cage with large cavity and pore windows for the efficient gas chromatography separation of enantiomers and positional isomers. J Sep Sci 41:1385–1394 30. Xie SM, Zhang JH, Fu N, Wang BJ, Chen L, Yuan LM (2016) A chiral porous organic cage for molecular recognition using gas chromatography. Anal Chim Acta 903:156–163 31. Zhang JH, Xie SM, Wang BJ, He PG, Yuan LM (2015) Highly selective separation of enantiomers using a chiral porous organic cage. J Chromatogr A 1426:174–182 32. Xie SM, Zhang JH, Fu N, Wang BJ, Hu C, Yuan LM (2016) Application of homochiral alkylated organic cages as chiral stationary phases for molecular separations by capillary gas chromatography. Molecules 21:1466

Chapter 4 Chiral Metabolomics Using Triazine-Based Chiral Labeling Reagents by UPLC-ESI-MS/MS Toshimasa Toyo’oka Abstract The determination of enantiomers of biological molecules is an important issue because a significant difference in the activity of the enantiomers is generally observed in biological systems. Chiral separations can be carried out by direct resolution using a chiral stationary column or by indirect resolution based on the derivatization with a chiral reagent. Many chiral-labeling reagents for ultraviolet-visible and fluorescence detections have been developed for various functional groups, such as amine and carboxylic acid. However, there are hardly any labeling reagents for LC-MS-specific detection. Based on this observation, we have developed several chiral-labeling reagents for LC-MS/MS analysis. This chapter describes methodologies and applications for the indirect LC-MS/MS determination of biological chiral molecules using triazine-based chiral-labeling reagents, i.e., (S and R)-1-(4,6-dimethoxy1,3,5-triazin-2-yl)pyrrolidin-3-amine (DMT-3(S and R)-Apy) for carboxylic acids and (S and R)-2,5dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidine-2-carboxylate (DMT-(S and R)Pro-OSu) for amines and amino acids. A reliable method for the non-targeted chiral metabolomics is also described in this chapter. Key words Enantioseparation, Indirect resolution, Chiral-labeling reagent, UHPLC separation, Triazine-based reagent, Mass spectrometry

1

Introduction Optically active (chiral) compounds are ubiquitous in life, and the biological activities of the enantiomers are sometimes dramatically different in the body. Thus, the separation of the enantiomers of chiral molecules has attracted intense interest in various fields such as the pharmaceutical industry for the past four decades. The enantiomer separation of chiral molecules has been mainly carried out by LC, GC, SFC, CEC, and CE [1]. Among them, HPLC is a major technique for the chiral separations of biologically important compounds. Many chiral compounds have been determined by direct resolution that employs chiral stationary-phase (CSP) column containing immobilized chiral selectors. The separation mechanism is due to the difference in the stability of the

Gerhard K. E. Scriba (ed.), Chiral Separations: Methods and Protocols, Methods in Molecular Biology, vol. 1985, https://doi.org/10.1007/978-1-4939-9438-0_4, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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diastereomeric complexes formed between the immobilized chiral selectors and the enantiomers in the flow system. Since the method requires no complex treatments such as derivatization, possible racemization during the separation seems to be negligible. However, the separation is highly influenced by the interaction between the CSP and the enantiomers. A lot of experience is thus required for the choice of the best column for the separation of each racemate. The elution order of a pair of enantiomers is also dependent upon the used CSP column and cannot be easily changed. Furthermore, the sensitivity of direct methods is often not adequate for trace analysis. The indirect resolution of enantiomers involving a derivatization step with a chiral-labeling reagent is known to be an alternative efficient technique for the separation of many racemates [2–6]. A pair of enantiomers is labeled with a chiral derivatization reagent to yield a pair of diastereomers which are subsequently separated by reversed-phase chromatography utilizing conventional achiral stationary-phase columns, such as an ODS column. The separation is based on the differences in the physicochemical properties of the diastereomers with an achiral stationary phase. As the separation is influenced by the distance between the two asymmetric carbons of the analyte and reagent, the distance should be minimized to get a good separation. The conformational rigidity around the chiral centers is another important factor for the separation. Although many considerations, such as optical purity of the reagent, stability of the reagent, racemization during the labeling reaction, and commercial availability of the reagent, are associated with the indirect method, the good sensitivity and selectivity of the indirect method coupled with an efficient detection system are attractive for the determination of chiral molecules. This derivatization method is suitable for trace analysis of enantiomers in biological samples such as blood and urine because a highly sensitive detection can be performed with the option of coupling analytes with suitable reagents which have a high molar ultraviolet-visible (UV-VIS) absorptivity, and a high fluorescence (FL) quantum yield. The labeling with chiral derivatization reagents is basically carried out by the reaction of reactive functional groups in the chiral molecules, e.g., amines (primary and secondary), carboxyl, carbonyl, hydroxyls (alcohol and phenol), and thiol. Various optically active labeling reagents for UV-VIS (e.g., 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate (GITC) and Marfey’s reagent) and FL (e.g., o-phthalaldehyde (OPA)/chiral thiols and 4-(N,N-dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (DBD-APy)) detections have been developed for each functional group in the chiral molecules [7–14]. These reagents are successfully applied to various chiral molecules in biological specimens and the efficiency of indirect resolution method is realized (see Note 1).

Chiral Triazine Labeling Reagents

59

Mass spectrometry (MS) is currently extensively used as a detector in various research fields due to the advancement of the hardware and software of the systems. Various MS instruments such as tandem quadrupole (TQ)-MS/MS and time-of-flight (TOF)MS/MS are gradually used as a reliable detector for HPLC in many analytical laboratories. The separation of diastereomers, derived from a chiral reagent, by reversed-phase chromatography and the following detection by MS/MS seem to be efficient for bioanalysis. However, the number of chiral-labeling reagents for the LC-MS/ MS analysis is currently very limited [15, 16]. Based on these observations, we have developed several chiral-labeling reagents for highly selective and sensitive detection by LC-MS/MS. An ideal chiral-labeling reagent for LC-MS/MS determination, that is effective for enhancing not only the MS/MS sensitivity but also the reversed-phase LC resolution, has to possess a highly protonaffinitive moiety [17–20], an asymmetric structure near the reactive functional group, and an adequate molecular mass (generally less than approximate 300). Other important points are that the labeling reaction proceeds by a one-step under mild conditions and the resulting derivative provides a characteristic product ion suitable for the selected reaction monitoring (SRM) (see Note 2). To meet such requirements, we synthesized several optically active derivatization reagents, such as L-pyroglutamic acid succinimidyl ester (L-PGAOSu), (S)-pyrrolidine-2-carboxylic acid N-(pyridine-2-yl)amide (PCP2), 1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3-amine (DMT-3-Apy), and 2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy1,3,5-triazin-2-yl)pyrrolidine-2-carboxylate (DMT-Pro-OSu), which produce the diastereomers corresponding to a pair of enantiomers for chiral carboxylic acids and amines [21–30] (see Note 3). This chapter deals with the methodologies for the indirect determination of targeted and/or non-targeted chiral molecules including metabolites based upon diastereomer formation using DMT-3(S and R)-Apy and DMT-(S and R)-Pro-OSu, as the representative chiral-labeling reagents for LC-MS/MS analysis [26–30]. The structures of the reagents and the labeling reactions with carboxylic acids and amines are shown in Fig. 1. The present methods using these reagents are applicable for the overall determination of chiral metabolites, named as “chiral metabolomics,” in biological specimens [31] (see Note 4). The application examples are also shown in this chapter.

2 2.1

Materials Instrumentation

1. A UHPLC system, for example, an ACQUITY ultraperformance liquid chromatograph (UPLC I-class) from Waters (Milford, MA, USA), equipped with a Xevo TQ-S

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Toshimasa Toyo’oka

Fig. 1 Derivatization reactions of carboxylic acids and amines by DMT-3-Apy and DMT-Pro-OSu (reproduced by permission from Elsevier from ref. 31 © 2015)

triple-quadrupole mass spectrometer (MS) controlled by the analytical software (MassLynx, Ver. 4.1). 2. A UHPLC reversed-phase column, for example an ACQUITY UPLC BEH C18 (1.7 μm, 100
2.1 mm i.d.) from Waters (Milford, MA, USA) (see Note 5). 3. Multivariate statistics software, for example MarkerLynx XS (Ver. 4.1) and Progenesis QI (Ver. 2.3) from Waters. 4. A centrifugal evaporator, for example EZ-2 from Genevac, UK. 5. A homogenizer for tissue homogenization, for example a bead beater-type homogenizer (ShakeMaster, Bio Medical Sciences, Tokyo, Japan). 6. 0.45 μm PTFE membrane filters. 2.2 Chemicals and Solutions

2.2.1 Sample Solutions and Solutions for Analyte Derivatization

Use HPLC-MS-grade solvents, trifluoroacetic acid (TFA), triethylamine (TEA), formic acid (FA), and ammonium acetate (CH3COONH4). All other reagents and solvents should be of analytical reagent grade. Use deionized and distilled water (e.g., purified by a PURELAB flex 3 Water Purification System from Elga, High Wycombe, UK). 1. Chiral carboxylic acids: DL-3-hydroxybutyric acid (DL-HA) and DL-lactic acid (DL-LA). Prepare 10 mM stock solutions of the carboxylic acids by dissolution of the compound in water. Prepare the working solutions at the appropriate concentration with acetonitrile.

Chiral Triazine Labeling Reagents

61

2. Chiral amines and amino acids: L-Amino acids, D-amino acids, DL-amino acids, (S)()-1-phenylethylamine (S-PEA), (R)(þ)1-phenylethylamine (R-PEA), (S)()-(1-naphthyl)ethylamine (S-NEA), (R)(þ)-(1-naphthyl)ethylamine (R-NEA), L-adrenaline (L-Ad), DL-adrenaline (DL-Ad), L-noradrenaline bitartrate monohydrate (L-NAd), and DL-noradrenaline bitartrate monohydrate (DL-NAd). Prepare 10 mM stock solutions of amines by dissolution of the compound in acetonitrile. Prepare 10 mM stock solutions of amino acids by dissolution of the compound in water. Prepare the working solutions at the appropriate concentration specified in the respective experiments by sequential dilution of the stock solutions with acetonitrile and methanol, respectively. 3. Chiral pharmaceuticals: RS-Ibuprofen (IBP), RS-naproxen (NAP) and RS-loxoprofen (LOX). Prepare 10 mM stock solutions of the carboxylic acids by dissolution of the compound in acetonitrile. Prepare the working solutions at the appropriate concentration specified in the respective experiments by sequential dilution of the stock solutions with acetonitrile. 4. Condensation reagents: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC), 3H-1,2,3-triazolo[4,5-b]pyridine-3-ol (HOAt), triphenylphosphine (TPP), and 2,20 -dipyridyl disulfide (DPDS) (see Note 6). Prepare 20 mM solutions of the condensation reagents by dissolution of the compounds in acetonitrile. 5. Chiral derivatization reagents: DMT-3(S)-Apy, DMT-3(R)Apy, DMT-(S)-Pro-OSu, and DMT-(R)-Pro-OSu (laboratory-made reagents) (see Note 7). Prepare 20 mM solutions of the derivatization reagents by dissolution of the compounds in methanol. 2.2.2 Mobile Phases

1. Mobile phase A: Prepare a solution of 0.1% (v/v) FA in water. 2. Mobile phase B: Prepare a solution of 0.1% (v/v) FA in acetonitrile. 3. Mobile phase C: Prepare a solution of 0.1% (v/v) FA in a mixture of water and acetonitrile. For the exact composition of the water-acetonitrile mixture for a specific analyte see Table 2. 4. Mobile phase D: Prepare a solution of 0.1% (v/v) FA in water. Mix 83 parts of this solution with 17 parts of methanol. 5. Mobile phase E: Prepare a solution of 0.1% (v/v) acetic acid in water. Mix 96 parts of this solution with four parts of acetonitrile-THF (9:1, v/v). 6. Mobile phase F: Prepare a solution of 20 mM ammonium acetate in water. Mix 97 parts of this solution with three parts of acetonitrile.

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7. Mobile phase G: Prepare a solution of 0.1% (v/v) acetic acid in water. Mix 95 parts of this solution with 5 parts of methanolTHF (9:1, v/v). Filter and degas all mobile phases before use.

3

Methods

3.1 General UHPLCMS Settings

1. Select appropriate mobile phase as described in the individual experiments or in Tables 1 and 2 and equilibrate column. 2. Set flow rate to 0.4 mL/min. 3. Set column temperature to 40  C. 4. Set the following MS parameters: positive-ion mode (ESI+), capillary voltage, 3.00 kV; cone voltage, 50 V; desolvation gas flow, 1000 L/h; cone gas flow, 150 L/h; nebulizer gas flow, 7.0 L/h; collision gas flow, 0.15 mL/min; collision energy, 20–35 eV; collision cell exit potential, 5 V; source temperature, 120  C; desolvation temp, 350–500  C.

Table 1 Separation and detection of chiral carboxylic acids Carboxylic acid

tR (min)

S-IBP

4.39

R-IBP

5.24

S-NAP

2.68

R-NAP

3.29

S-LOX

3.11

R-LOX

3.6

D-LA

L-LA

12.97

Mobile phase (A/B)

RSa

Precursor ion/ product ion (m/z)

CEb (eV)

55/45

5.14

412.2/226.3

32

15.6

D-HA

16.2

4.6 5.9

60/40

3.98

438.2/226.3

20

4.8 5.1

65/35

2.45

454.2/226.3

26

3.2 4.2

97/3

1.98

13.8

L-HA

LODc (amol)

97/3

1.65

298.2/209.2

22

12.0

298.2/226.3

26.2

298.2/209.2

11.5

298.2/226.3

25.8

312.2/226.3

t0 ¼ 0.21 min A: 0.1% (v/v) FA in water, B: 0.1% (v/v) FA in acetonitrile Reproduced with permission from Springer from ref. 26 © Elsevier 2015 a RS ¼ 2
(t2  t1)/(W1 + W2) b CE collision energy c LOD limit of detection (S/N ¼ 3)

23

15.2 15.8

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63

Table 2 Separation and detection of chiral amines and amino acids Precursor ion/product ion (m/z)

CEb (eV)

LODc (amol)

2.5

358.2[MþH]+/195.3, 209.3

25

26.1

+

Amine

Mobile phase

Retention time D/L (min)

RS

PEA

C (75/25)

R: 7.1/S: 6.6

a

NEA

C (65/35)

R: 6.0/S: 5.5

2.6

408.2[MþH] /195.3, 209.3

25

19.7

Nad

C (90/10)

6.5/7.0

2.3

406.2[MþH]+/195.3, 209.3

25

208

1.3

+

420.2[MþH] /195.3, 209.3

25

2900

5.0

+

326.1[MþH] /195.3, 209.3

20

61.4

+

Ad

C (88/12)

Ala

C (91/9)

10.9/10.2 8.1/6.9

His

C (91/9)

2.5/2.6

2.9

392.2[MþH] /195.3, 209.3

25

186.8

Met

C (85/15)

8.0/8.4

1.7

386.1[MþH]+/195.3, 209.3

25

63.2

2.4

+

25

157.5

Val

C (85/15)

8.6/8.0

354.2[MþH] /195.3, 209.3 2+

Cys

C (80/20)

8.3/8.8

1.7

357.1[Mþ2H] /195.3, 209.3

25

1200

Ile

C (80/20)

7.0/6.7

1.9

368.2[MþH]+/195.3, 209.3

25

83.2

+

Leu

C (80/20)

7.0/7.3

1.7

368.2[MþH] /195.3, 209.3

25

44.4

Lys

C (80/20)

6.2/5.7

2.4

619.3[MþH]+/209.6, 237.6

35

537.6

9.0

+

402.2[MþH] /195.3, 209.3

25

46.2

8.3

+

441.2[MþH] /195.3, 209.3

25

44.4

+

Phe Trp

C (80/20) C (80/20)

8.0/11.6 8.0/10.5

Tyr

C (80/20)

2.1/2.6

6.0

418.2[MþH] /195.3, 209.3

25

109.5

Pro

D (83/17)

32.3/37.4

2.0

352.2[MþH]+/195.3, 209.3

25

3294

1.8

+

356.2[MþH] /195.3, 209.3

25

63.2

+

Thr

D (83/17)

7.9/8.4

Asn

E (96/4)

9.2/9.5

1.4

369.1[MþH] /195.3, 209.3

20

1127

Gln

E (96/4)

10.8/11.2

1.2

383.2[MþH]+/195.3, 209.3

25

2316

Ser

E (96/4)

10.1

–

342.1[MþH]+/195.3, 209.3

25

ND

0.8

+

342.1[MþH] /195.3, 209.3

25

542.6

+

d

Ser

G (95/5)

16.9/17.4

Arg

F (97/3)

28.5/26.7

1.8

411.2[MþH] /195.3, 209.3

25

331.3

Asp

F (97/3)

5.2/4.5

3.1

370.1[MþH]+/195.3, 209.3

20

160.9

2.0

+

25

718.1

Glu

F (97/3)

6.0/5.1

384.1[MþH] /195.3, 209.3

C: 0.1% FA in H2O/CH3CN; D: 0.1% FA in H2O/CH3OH; E: 0.1% acetic acid in H2O/CH3CN-THF(9:1); F: 10 mM aqueous ammonium acetate/CH3CN; G: 0.1% CH3COOH in H2O/CH3OH-THF(9:1) Reproduced with permission from Elsevier from ref. 27 © Elsevier 2015 a RS ¼ 2
(t2  t1)/(W1 + W2) b CE collision energy c LOD limit of detection (S/N ¼ 3) d Column: ADME (100
2.1 mm, i.d., 2.7 μm)

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Toshimasa Toyo’oka

1. To 50 μL of a 40 μM solution of a chiral carboxylic acid (e.g., DL-LA, DL-HA, RS-IBP, RS-Lox, or RS-NAP) add 40 μL of a 20 mM EDC in acetonitrile and 40 μL of 20 mM HOAt in acetonitrile and mix.

3.2 Determination of Targeted Carboxylic Acid Enantiomers

2. Then add 20 μL of 20 mM solution of DMT-3(S)-Apy in methanol containing 0.1% TEA and thoroughly mix. 3. Leave at room temperature for 1.5 h in the dark. 4. Evaporate the reaction solution using a centrifugal evaporator. 5. Redissolve the residue in 50 μL of the initial mobile-phase solution (e.g., water-acetonitrile (93:7, v/v) containing 0.1% FA; see Table 1). 6. Inject an aliquot (e.g., 2 μL) into the UHPLC-MS/MS system. 7. Separate with the suitable mobile-phase solution and elution mode (e.g., isocratic elution of water:acetonitrile (55:45, v/v) containing 0.1% FA for IBP). See Table 1 for details.

relative intensity %

8. Detect the reagent characteristic product ions (i.e., m/z 226.3 and/or 209.2). See Fig. 2 as an example. Further conditions are listed in Table 1 (see Note 8).

226.3

100 185.1

O

H N

*

N *

O

O

%

N

N N

O

m/z 226.3

m/z 185.1 0 50

100

150

200

250 m/z

300

350

400

450

relative intensity

100

S-NAP derivative

R-NAP derivative

%

0

1.0

2.0

5.0 3.0 4.0 Time (min)

6.0

7.0

Fig. 2 MS/MS spectrum and SRM chromatogram obtained from the reaction of RS-NAP with DMT-3(S)-Apy. The MS/MS spectrum is recorded by the CID of m/z 438.2 [MþH]+ (precursor ion). The SRM chromatogram is obtained from the monitoring at m/z 438.2 ! m/z 226.3. The other UPLC-MS/MS conditions are described in Table 1 (reproduced by permission from Springer from ref. 26 © 2015)

Chiral Triazine Labeling Reagents

3.3 Determination of Targeted Chiral Amine and Amino Acid Enantiomers

65

1. To 50 μL of a 40 μM solution of the chiral amine or amino acid (e.g., DL-Ala) solution add 20 μL of 20 mM solution of DMT-(S)-Pro-OSu in methanol and mix. 2. Add 60 μL of 100 mM solution of TEA in acetonitrile and thoroughly mix (see Note 9). 3. Leave at room temperature for 3 h in the dark. 4. Dry the reaction solution using a centrifugal evaporator. 5. Redissolve the residue in 50 μL of initial mobile-phase solution (e.g., water:acetonitrile (8:2, v/v) containing 0.1% FA; see Table 2). 6. Inject an aliquot (e.g., 2 μL) into the UPLC-MS/MS system. 7. Detect at the reagent characteristic product ion (m/z 209.2). See Fig. 3 as an example. Further conditions are listed in Table 2 (see Note 10).

Fig. 3 SRM chromatograms of the derivatives obtained from authentic DL-amino acids (2 pmol each). The UPLC-MS/MS conditions are described in Table 2 (reproduced by permission from Elsevier from ref. 27 © 2015)

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3.4 Pretreatment of Biological Samples (See Note 11)

1. Add 0.95 mL acetonitrile to 50 μL of plasma or serum, mix, and let stand at room temperature for 15 min.

3.4.1 Plasma and Serum

3. React the supernatant solution and analyze as described in Subheadings 3.2, 3.3, or 3.5 depending on the intended analytes.

3.4.2 Saliva

1. Collect saliva (ca. 1 mL) in a tube without a collection device and store at 80  C until analysis.

2. Centrifuge at 3000
g for 10 min at 4  C.

2. Centrifuge at 3000
g for 10 min at 4  C to precipitate the denatured mucin. 3. Dilute 5 μL of the supernatant threefold with water, mix, add 285 μL acetonitrile, and mix. 4. After standing for 15 min at room temperature, centrifuge at 3000
g for 10 min at room temperature. 5. Collect supernatant and dry sample by a centrifugal evaporator. 6. Redissolve the residue in acetonitrile (e.g., 30 μL). 7. React the solution and analyze as described in Subheadings 3.2, 3.3, or 3.5 depending on the type of the analytes. 8. An example of the determination of carboxylic acids in saliva is shown in Fig. 4. 3.4.3 Brain Tissue

1. Collect brain tissue samples (specific brain region such as frontal lobe) and store at 80  C until use. 2. Place zirconia beads [2 (5.0 mm i.d.) and 3 (3.0 mm i.d.), total 5 beads] to a polypropylene tube, and then add 100 mg sample of brain tissue followed by 5 mL of MeOH:H2O (1:1, v/v). 3. Homogenize immediately for 20 min using a bead beater-type homogenizer or another suitable homogenizer. 4. Centrifuge at 14,000
g for 10 min, and then filtrate the supernatant through a 0.45 μm PTFE membrane filter. 5. Divide the supernatant into other tubes (250 μL each) and derivatize with the DMT-3(S)-Apy and/or DMT-(S)-ProOSu as described in Subheadings 3.2, 3.3, and 3.5 depending on the type of the intended analytes. 6. Analyze solutions as described in Subheadings 3.2, 3.3, or 3.5 depending on the intended analytes.

3.5 Non-targeted Chiral Metabolomics in Biological Samples (See Note 12)

1. Deproteinize biological sample, e.g., 50 μL plasma or saliva, by addition of 200 μL acetonitrile and centrifuge at 3000
g for 10 min at room temperature. 2. For the determination of chiral carboxylic acids, label the 50 μL of the supernatant solution by DMT-3(S)-Apy, according to the procedure of Subheading 3.2.

Chiral Triazine Labeling Reagents

67

Fig. 4 SRM chromatograms of biological carboxylic acids in saliva. DL-3-hydroxybutyric acid (DL-HA), acetoacetic acid (AA), α-ketoisocaproic acid (KCA), DL-lactic acid (DL-LA), 4-hydroxyphenylacetic acid (4HA), 4-hydroxyphenylpyruvic acid (4HP), propionic acid (PA), fumaric acid (FMA), butyric acid (BA), α-ketoisovaleric acid (KVA), malic acid (MA), α-ketoglutaric acid (KA), succinic acid (SA). Mobile phase: A (0.1% HCOOH in H2O), B (0.1% HCOOH in CH3CN); elution profile: isocratic of A:B (93:7) until 16.5 min and then linear increase to A:B (5:95) until 25.0 min (reproduced by permission from Springer from ref. 26 © 2015)

3. Label a second 50 μL portion of the supernatant using the reagent enantiomer DMT-3(R)-Apy as described in Subheading 3.2. 4. In the case of the determination of chiral amines and amino acids, label a third 50 μL portion by DMT-(S)-Pro-OSu, according to the procedure of Subheading 3.3. 5. Label a fourth 50 μL portion by the opposite reagent enantiomer DMT-(R)-Pro-OSu as described in Subheading 3.3. 6. After completion of the reactions, dry solutions by a centrifugal evaporator and redissolve in 50 μL of initial mobile-phase solution (e.g., water:acetonitrile (98:2, v/v) containing 0.1% FA). 7. Inject an aliquot (e.g., each 2 μL) into the UPLC-MS/MS system.

68

Toshimasa Toyo’oka

Fig. 5 Strategy of chiral metabolite extraction using a pair of reagent enantiomers

8. Run the following gradient using mobile phases A and B: A: B—98:2 (v/v) at 0 min, increase linear to A:B—80:20 (v/v) at 20 min, and increase linear to A:B—2:98 (v/v) at 50 min (Fig. 6). 9. Obtain the chromatograms from precursor ion scan (PIS) at m/z 209.2. 10. Statistically analyze the peaks in both chromatograms obtained from a pair of enantiomers (DMT-3(S)-Apy and DMT-3(R)Apy) (i.e., PCA and OPLS-DA). 11. Also statistically analyze the peaks on both chromatograms obtained from a pair of enantiomers (DMT-(S)-Pro-OSu and DMT-(R)-Pro-OSu) (i.e., PCA and OPLS-DA). 12. Search the increased and decreased peaks between both chromatograms (i.e., DMT-3(S)-Apy and DMT-3(R)-Apy or DMT-(S)-Pro-OSu and DMT-(R)-Pro-OSu) by the S-plot of OPLS-DA. See Figs. 5 and 6 as examples. 13. Identify the structures of the pairs of increased and decreased peaks based on the m/z value and the retention time of the derivatives by the authentic molecules if commercially available.

Chiral Triazine Labeling Reagents

69

Fig. 6 PCA and S-plot based on the PIS chromatogram of carboxylic acids and amines in human serum labeled with DMT-3(S and R)-Apy and DMT-(S and R)-Pro-OSu (reproduced by permission from Elsevier from ref. 31 © 2015)

4

Notes 1. The labeling of analytes with reagents that contain moieties absorbing in the UV or VIS region is the most popular means of derivatization because almost all laboratories possess a UV-VIS detector and the analysts are experienced in their use. Various chiral derivatization reagents for HPLC that provide UV-VIS absorption have been reported [4]. It is noted that the derivatives obtained from these reagents have a strong absorption in the long-wavelength region. Most endogenous substances in biological samples absorb at relatively short wavelengths. Since impurities interfere by absorbing in the detection wavelength of target analytes in real samples especially in complex matrices such as biological specimens, the reagents absorbing in the VIS region are preferable in terms of selectivity. Although a number of UV labels have been applied to the labeling of various functional groups, the sensitivity of the

70

Toshimasa Toyo’oka

derivatives is not good enough in some real samples. To solve this drawback, various types of FL labels are synthesized and reported in many papers. Fluorimetry is a sensitive and selective detection method due to setting of both excitation and emission wavelengths. Therefore, different types of FL labeling reagents have been developed for the enantioseparation of biologically important substances and these FL labels have been successfully applied to the analysis of real samples [3–5]. The FL properties of the resulting derivatives tend to be significantly affected by detection environment, e.g., temperature, viscosity of the solvent, and pH in the medium. It should also be noted that undesirable FL materials present as contaminants in the samples, especially in biological specimens, interfere with the determination. Thus, the selection of the labeling reagent significantly affects accuracy, precision, and repeatability of the quantitative analysis. 2. The derivatives labeled with some UV-VIS and FL reagents can also be determined by MS coupled to the HPLC system. HPLC-MS detection has become a powerful technique in various fields in the pharmaceutical, biotechnology, food, agriculture, and chemical industries. Many applications, such as a rapid analysis for drug discovery, impurity analysis, metabolite identification, regulatory science analysis, and combinatorial screening, are utilizing HPLC-MS as one of the major techniques. Among the various interfaces, the triple-quadrupole (TQ) MS provides the information not only of the m/z of the parent ion, but also of the fragments generated from the collision-induced dissociation (CID). The measurements from the multiple reaction monitoring (MRM) and precursor ion scanning available from a TQ MS are powerful means for highly sensitive detection and for simplifying complex samples, respectively. When a specific m/z peak appears by the CID, trace analyses of a pair of enantiomers are promising. TOF-MS instruments allow the generation of the exact mass information (generally 3–5 ppm error) with a greater accuracy and precision. The exact mass values can be used to speculate about the empirical formula of an unknown analyte, which significantly reduces the number of possible structures. Highly sensitive detection is generally performed using the positive ion mode in electrospray ionization (ESI)-MS. Therefore, the protonated structure before and/or after labeling is important for sensitive detection in MS analysis. Mild reaction condition is also important to avoid the racemization during the derivatization. The relatively low molecular mass of the derivatives is generally predominant for the detection by TQ MS. The production of a characteristic ion decomposed by CID is essential for sensitive determination by SRM. In

Chiral Triazine Labeling Reagents

71

addition, the separation efficiency of a pair of diastereomers by HPLC affects the distance of both asymmetric carbons in the resulting diastereomer. The compound possesses these properties and is thus recommended as the labeling reagent for HPLC-MS/MS determination. 3. Several important points worthy of consideration for the choice of the chiral derivatization reagent are as follows. (1) The optical purity of the reagent should be as high or the same as the chemical purity. Since the opposite enantiomer contaminating the reagent also produces a corresponding diastereomer, it is obvious that erroneous results will be obtained with the use of impure reagents. (2) The degree of racemization during the labeling reaction and storage of the reagent itself is another important issue for quantitative determinations. The chemical stability of the resulting diastereomers also influences the results. Good stability (at least 1 day) is required for many analyses, because overnight automated analysis is often performed. (3) The reactivity of the reagent for each enantiomer and the physicochemical properties of the resulting derivatives have to be essentially the same. When the reaction rates are different for both enantiomers, the reaction condition should be carefully optimized. If the reactivity is still different in both enantiomers, an individual calibration curve has to be constructed for each enantiomer. (4) The reagent should possess specificity for the target functional group and should quantitatively label the analyte under mild reaction conditions. (5) The resulting diastereomers should exhibit an adequate detector response for the sample analysis. (6) Another important point is the solubility of the reagent, whether it is freely soluble in water or miscible in aqueous solvents, such as methanol and acetonitrile, because many bioactive chiral molecules exist in aqueous solutions. (7) Another important practical point is that both enantiomers of the reagent are commercially available or easily obtained by simple synthesis, because the elution order can be controlled by the selection of the reagent enantiomer. This is necessary when the determination of a trace enantiomer is required in the presence of a large excess of the major enantiomer. These items listed here are general aspects for all chiral-labeling reagents, not only for MS reagent but also for UV-VIS and FL reagents. Since the optical purity of the chiral derivatization reagents is generally less than 99%, straightforward assay of a trace quantity of an enantiomer in the presence of a large excess of the major enantiomer is essentially difficult using the indirect method. Thus, the indirect methods described in the text are primarily recommended for bioanalysis, such as a metabolic study in biological specimens, because a few % error is usually in the acceptable range of bioanalysis.

72

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4. Metabolomics, which is a comprehensive analysis of lowmolecular-mass metabolites, is important for understanding the physiological functions in biological systems. Metabolomics is also a useful technique for the discovery of novel biomarker(s). Metabolome studies are thus important for understanding the physiological functions and discovering potential disease markers [32–34]. Some metabolites critically change based on the disease, for instance, the amounts of phenylalanine and tyrosine increase in uremic patients [35, 36]. Chiral metabolites are also found in a wide variety of living organisms and some of them are understood to be physiologically active compounds and biomarkers. For instance, D-serine (D-Ser) is related to the diseases with N-methyl-D-aspartate (NMDA) receptor dysfunctions such as schizophrenia and depression [37–39]. However, the overall analysis of chiral metabolomics is quite difficult due to the high number of metabolites, significant diversity in the physicochemical properties, and concentration range from metabolite to metabolite [40]. To solve this difficulty, we developed a novel approach for chiral metabolomics extraction, which is based on the labeling of a pair of enantiomers by chiral derivatization reagents (i.e., DMT-(S,R)-Pro-OSu and DMT-3(S,R)-Apy) and precursor ion scan chromatography of the derivatives. The multivariate statistics is also required for this strategy (Fig. 5). The proposed method was evaluated by the detection of a diagnostic marker (i.e., D-lactic acid) using the saliva of diabetic patients [26, 28]. This method was applied to the determination of biomarker candidates of chiral amines and carboxylic acids in Alzheimer’s disease (AD) brain homogenates [41]. Therefore, the proposed approach seems to be helpful for the determination of non-targeted chiral metabolomics possessing amines and carboxylic acids. 5. Many diastereomers are satisfactorily separated by HPLC utilizing conventional columns packed with 3–5 μm porous particles. However, the pharmaceutical industry is particularly interested in rapid and efficient procedures for qualitative and quantitative analyses in order to cope with a large number of samples. The simplest approach available to shorten an analytical run is to shorten the column length and increase the flow velocity. The approach utilizing a conventional stationary phase made of 3–5 μm particles is not adequate because the chromatographic performance is much lower and the separation of multicomponent mixtures will be insufficient. Another means to shorten the run time is to decrease the particle size. Methods utilizing small-particle-size columns allow rapid analysis with a high efficiency, but at the cost of high back pressure which cannot be tolerated by conventional

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HPLC instruments and columns. The problem is solved by both the development of a column packed with particle sizes less than 2 μm and of an instrument that can operate at a high pressure. Reducing the particle diameter was expected to result in an increased efficiency, speed, resolution, and sensitivity. Improvement in the chromatographic performance has been achieved by the introduction of ultrahigh-performance liquid chromatography (UHPLC or UPLC) systems utilizing 1.7 μm porous particles as the column. This technology provides higher peak capacity, greater resolution, increased sensitivity, and higher speed, compared to a 3 μm material [42]. This approach allows similar results as those previously obtained by a conventional HPLC, but in one-tenth of the run time. The radical shortening of the analytical time provides a relatively high-throughput separation not only for multiple component mixtures [43, 44] but also for chiral molecules. The use of a micro- or semimicro-separation column is further recommended to increase the detection sensitivity and decrease the sample volume. 6. Various condensation reagents, such as DPDS/TPP and EDC/HOAt, are basically applicable for the activation of carboxylic acids. The choice of the activation reagent essentially depends on the target carboxylic acid. However, the side reaction and the epimerization during labeling reaction are rarely observed in the use of inadequate condensation reagent use. The degree of epimerization sometimes depends on the condensation reagents used. Although various condensation reagents are adoptable for the reaction, care should be taken for the reagent selection. 7. The chiral-labeling reagents are synthesized according to the following manner [26]. (3S)-()-3-(tert.-butoxycarbonylamino)pyrrolidine (0.56 g, 3 mmol) dissolved in 30 mL CH3OH is added to 2-chloro-4,6-dimethoxy-triazine (CDMT) (0.35 g, 2 mmol) in 20 mL THF, and then mixed with 500 mL TEA for 6 h at room temperature. After that, the solution was evaporated under reduced pressure. The resulting residues are subjected to silica-gel column chromatography using dichloromethane/CH3OH (100:1) as the mobile phase. The white powders of tert.-butyl-(S)-(1-(4,6-dimethoxy-1,3,5triazin-2-yl)pyrrolidin-3-yl)carbamate (DMT-Boc-3(S)-Apy) are obtained. Then, a 500 mL TFA is added to the DMT-Boc3(S)-Apy (0.33 g) dissolved in 2 mL CH3OH and vigorously mixed for 6 h while cooling on ice water. After evaporation of the solution, 500 mL HCl in ether is added to the residues to form the salts. The precipitated crystals are washed three times with acetone, and (S)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3-amine hydrogen chloride (DMT-3(S)-Apy·HCl) is

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obtained. After the DMT-3(S)-Apy·HCl is dissolved in 1 mL CH3OH and neutralized by 1 mL ammonium hydroxide, the white powders of (S)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3-amine (DMT-3(S)-Apy) are obtained (yield: approximate 76%). ESI-MS: m/z 226.3 [MþH]+. 1HNMR in CD3OD (TMS): 3.91 ppm (6H, s, –OCH3), 3.26–3.32 ppm (4H, m, Hpy2, 5), 2.35–2.49 ppm (1H, m, Hpy3), 2.09–2.21 ppm (2H, q, Hpy4). (S)-Proline (0.35 g, 3 mmol) dissolved in 30 mL CH3OH is added to CDMT (0.35 g, 2 mmol) in 20 mL THF and then mixed with 500 mL TEA for 6 h at room temperature. After the reaction, the solution is evaporated under reduced pressure. The resulting residues are subjected to silica-gel column chromatography using dichloromethane/CH3OH (20:1) as the mobile phase. The white powders of (S)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidine-2-carboxylic acid (DMT-(S)-Pro-OH) are quantitatively obtained. To the DMT-(S)-Pro-OH (0.51 g) dissolved in 20 mL N,N-dimethylformamide (DMF)/CH3OH (1:9), EDC (0.42 g, 2.2 mmol) and HOSu (0.23 g, 2 mmol) are added and vigorously mixed for 6 h at room temperature. After that, the solution was dried under reduced pressure. The resulting residues are subjected to silica-gel column chromatography using hexane/ethyl acetate (4:1). The white powders of (S)-2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazin-2yl)pyrrolidine-2-carboxylate (DMT-(S)-Pro-OSu) are obtained (yield: approximate 33%). ESI-MS: m/z 352.1 [MþH]+. 1H NMR (500 MHz) in CDCl3 (TMS): 4.92–4.89 ppm (1H, t, Hpy2), 3.95 ppm (3H, s, –OCH3), 3.87 ppm (3H, s, –OCH3), 3.85–3.81 ppm (1H, m, Hpy5), 3.74–3.70 ppm (1H, m, Hpy5), 2.82 ppm (4H, s, Hsu), 2.52–2.40 ppm (2H, m, Hpy3), 2.21–2.06 ppm (2H, m, Hpy4). The opposite enantiomers of the reagents, i.e., DMT-3 (R)-Apy and DMT-(R)-Pro-OSu, are similarly synthesized from (3R)-(þ)-3-(tert.-butoxycarbonylamino)pyrrolidine and (R)-proline, respectively. 8. The chiral carboxylic acid is converted to the corresponding amide-type diastereomer. The reactivity of DMT-3(S)-Apy toward each enantiomer of the carboxylic acids (i.e., RSnaproxen) is comparable and the labeling reaction is completed after 90-min heating at 60  C. Thus, the reaction conditions at 60  C and 90 min in the presence of TPP and DPDS were adopted for the labeling of the chiral carboxylic acid pharmaceuticals. In the case of biological carboxylic acids, EDC and HOAt are used, because a higher sensitive labeling is observed under milder condition. The labeling reaction was completed at room temperature for 90 min. The optimal temperature and/or time seem to be dependent on the condensation

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reagents used. Although the condensation reagents used are different in pharmaceuticals and biological acids, the reaction manner is essentially the same. However, care should be taken for the choice of condensation reagents, because epimerization sometimes occurs during the labeling of some carboxylic acids by the unsuitable reagent use. Figure 2 shows the MS/MS spectrum and SRM chromatogram at m/z 226.3 of RS-naproxen. The separation and detection efficiencies of several chiral carboxylic acids are also listed in Table 1. The complete separation of enantiomers of carboxylic acids can be achieved by reversed-phase chromatography using ODS column. The resolution (RS) values of the diastereomers derived from pharmaceuticals and biological acids are in the range of 1.65–5.14. Highly sensitive detection (LOD, 3.2–26.2 atmol) is also obtained from SRM chromatograms at m/z 209.2 and/or 226.3 [26]. 9. The chiral reagents react with primary and secondary amines in the presence of triethylamine (TEA) to produce the corresponding amides. Instead of TEA, quinuclidine, 1,8-diazabicyclo[5.4.0]undecene (DBU) (organic base) and borate buffer (pH 9–10) (inorganic base) can also be used. 10. The RS values for the derivatives of amino acids by an ODS column are in the range of 1.2–9.0 (Table 2). The values obtained from neutral and/or aromatic amino acids tend to be higher values than those of basic and acidic amino acids, although the separation efficiency is different for each amino acid. Furthermore, the opposite elution order is obtained using the R-enantiomer of the reagent. The detection ability (i.e., LOD) is also dependent on the amines and amino acids (Table 2) [27]. Although the peaks of the derivatives of DL-Ser overlapped in the chromatograms using the ODS column, the separation was improved (RS, 0.8) by the use of a Capcell Pak adamantylethyl (ADME) column (Shiseido, Tokyo, Japan). However, the overall separation of chiral molecules using one labeling reagent and/or one column is generally difficult. Consequently, lineup of several reagents and columns seems to be required for the total analysis of the chiral molecules. 11. The pretreatment of samples is an important issue in trace analysis. During the analysis of real samples such as biological specimen and food products, the most significant and major part of the procedure involves how effectively the trace analytes of interest from a complicated matrix are obtained. The sample pretreatment, i.e., cleanup, deproteinization, and concentration of target analytes, is inevitable for the HPLC measurement with derivatization. The pretreatments in the LC-MS analysis are very important, because the MS intensity is sometimes

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decreased or increased by the matrix effect of intrinsic materials in the biological samples. The interference has to be carefully identified to avoid erroneous results. 12. Chiral metabolites are found in a wide variety of living organisms and some of them are understood to be the biomarkers of diseases. During the course of our study, we found that the ratio of D-lactic acid (D-LA) in the saliva of diabetic patients is significantly higher than that of healthy persons [21, 25, 26, 28]. Although several successful procedures including our results have been reported, the identification of non-targeted chiral molecules in biological specimens is very difficult in spite of using these optically active labeling reagents. To solve the difficulty, we developed a novel approach for chiral metabolomics extraction. An overview of the strategy is shown in Fig. 5. The sample solution is divided into two groups. One sample group is labeled with one enantiomer of the labeling reagent (e.g., DMT-3(S)-Apy), whereas the other one is also labeled with the opposite enantiomer of the labeling reagent (e.g., DMT-3(R)-Apy). The labeled molecules in both groups are determined by the precursor ion scan (PIS) chromatograms using UPLC-MS/MS system. The labeled molecules in the sample are identified by the PIS chromatograms; however, it is not obvious whether the molecules are chiral and achiral. Therefore, the peaks in both chromatograms are compared by multivariate statistics, i.e., principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). The chiral molecules are only extracted as increased and decreased markers by the S-plot of OPLS-DA (Fig. 6). The pair shows an enantiomer, but the optical structure (R or S conformation) is unknown by this result only. The absolute structure has to be identified by the authentic compound. This method is based on the fact that the elution order of an enantiomer is reversed with the use of the opposite reagent enantiomer. In contrast, an achiral molecule is eluted at the same retention time in the chromatograms, in spite of the use of the opposite reagent enantiomer. This strategy is based on the labeling of the pairs of the chiral derivatization reagents followed by LC-ESI-MS/MS determination (Fig. 5). The proposed method was verified by the determination of chiral carboxylic acids in the diabetic patient’s saliva and also applied to the detection of amines and carboxylic acids in the brain homogenates of Alzheimer’s disease (AD) patients [41].

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References 1. Ward TJ, Ward KD (2010) Chiral separations: Fundamental review 2010. Anal Chem 82:4712–4722 2. Toyo’oka T (2002) Resolution of chiral drugs by liquid chromatography based upon diastereomer formation with chiral derivatization reagents. J Biochem Biophys Methods 54:25–56 3. Toyo’oka T (2002) Development of chiral derivatization reagents having benzofurazan (2,1,3-benzoxadiazole) fluorophore for HPLC analysis and their application to the sensitive detection of biologically important compounds. Bunseki Kagaku 51:339–358 4. Toyo’oka T (1999) Derivatization for resolution of chiral compounds. In: Toyo’oka T (ed) Modern derivatization methods for separation sciences. Wiley, Chichester, pp 217–289 5. Toyo’oka T (1996) Recent progress in liquid chromatographic enantioseparation based upon diastereomer formation with fluorescent chiral derivatization reagents. Biomed Chromatogr 10:265–277 6. Sun XX, Sun LZ, Aboul-Enein HY (2001) Chiral derivatization reagents for drug enantioseparation by high-performance liquid chromatography based upon pre-column derivatization and formation of diastereomers: enantioselectivity and related structure. Biomed Chromatogr 15:116–132 7. Toyo’oka T (2002) Fluorescent tagging of physiologically important carboxylic acids, including fatty acids, for their detection in liquid chromatography. Anal Chim Acta 465:111–130 8. Liu YM, Schneider M, Sticha CM, Toyo’oka T, Sweedler JV (1998) Separation of amino acid and peptide stereoisomers by nonionic micellemediated capillary electrophoresis after chiral derivatization. J Chromatogr A 800:345–354 9. Ilisz I, Berkecz R, Peter A (2008) Application of chiral derivatization agents in the highperformance liquid chromatographic separation of amino acid enantiomers: a review. J Pharm Biomed Anal 47:1–15 10. Bhushan R, Kumar V (2008) Synthesis of chiral hydrazine reagents and their application for liquid chromatographic separation of carbonyl compounds via diastereomer formation. J Chromatogr A 1190:86–94 11. Bhushan R, Dixit S (2011) Application of hydrazine dinitrophenyl-amino acids as chiral derivatizing reagents for liquid chromatographic enantioresolution of carbonyl compounds. Chromatographia 74:189–196

12. Toyo’oka T, Ishibashi M, Terao T, Imai K (1993) 4-(N,N-Dimethylaminosulfonyl)-7(2-chloroformylpyrrolidine-1-yl)-2,1,3-benzoxadiazole: Novel fluorescent chiral derivatization reagents for the resolution of alcohol enantiomers by high-performance liquid chromatography. Analyst 118:759–763 13. Toyo’oka T, Liu YM, Hanioka N, Jinno H, Ando M (1994) Determination of hydroxyls and amines, labelled with 4-(N,N-dimethylaminosulfonyl)-7-(2-chloroformylpyrrolidine1-yl)-2,1,3-benzoxadiazole, by highperformance liquid chromatography with fluorescence and laser-induced fluorescence detection. Anal Chim Acta 285:343–351 14. Toyo’oka T, Liu YM, Hanioka N, Jinno H, Ando M, Imai K (1994) Resolution of enantiomers of alcohols and amines by highperformance liquid chromatography after derivatization with a novel fluorescent chiral reagent. J Chromatogr A 675:79–88 15. Nozawa Y, Sakai N, Arai K, Kawasaki Y, Harada K (2007) Reliable and sensitive analysis of amino acids in the peptidoglycan of actinomycetes using the advanced Marfey’s method. J Microbiol Methods 70:306–311 16. Fujii K, Ikai Y, Mayumi T, Oka H, Suzuki M, Harada K (1997) A nonempirical method using LC/MS for determination of the absolute configuration of constituent amino acids in a peptide: elucidation of limitations of Marfey’s method and of its separation mechanism. Anal Chem 69:3346–3352 17. Higashi T, Ichikawa T, Inagaki S, Min JZ, Fukushima T, Toyo’oka T (2010) Simple and practical derivatization procedure for enhanced detection of carboxylic acids in liquid chromatography-electrospray ionization-tandem mass spectrometry. J Pharm Biomed Anal 52:809–818 18. Xu L, Spink DC (2008) Analysis of steroidal estrogens as pyridine-3-sulfonyl derivatives by liquid chromatography electrospray tandem mass spectrometry. Anal Biochem 375:105–114 19. Shimbo K, Oonuki T, Yahashi A, Hirayama K, Miyano H (2009) Precolumn derivatization reagents for high-speed analysis of amines and amino acids in biological fluid using liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom 23:1483–1492 20. Inagaki S, Tano Y, Yamakata Y, Higashi T, Min JZ, Toyo’oka T (2010) Highly sensitive and positively charged precolumn derivatization reagent for amines and amino acids in liquid

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chromatography/electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom 24:1358–1364 21. Tsutsui H, Mochizuki T, Maeda T, Noge I, Kitagawa Y, Min JZ, Todoroki K, Inoue K, Toyo’oka T (2012) Simultaneous determination of DL-lactic acid and DL-3-hydroxybutyric acid enantiomers in saliva of diabetes mellitus patients by high-throughput LC–ESI-MS/MS. Anal Bioanal Chem 404:1925–1934 22. Mochizuki T, Taniguchi S, Tsutsui H, Min JZ, Inoue K, Todoroki K, Toyo’oka T (2013) Relative quantification of enantiomers of chiral amines by high-throughput LC-ESI-MS/MS using isotopic variants of light and heavy L-pyroglutamic acids as the derivatization reagents. Anal Chim Acta 773:76–82 23. Nagao R, Tsutsui H, Mochizuki T, Takayama T, Kuwabara T, Min JZ, Inoue K, Todoroki K, Toyo’oka T (2013) Novel chiral derivatization reagents possessing a pyridylthiourea structure for enantiospecific determination of amines and carboxylic acids in high-throughput liquid chromatography and electrospray-ionization mass spectrometry for chiral metabolomics identification. J Chromatogr A 1296:111–118 24. Mochizuki T, Todoroki K, Inoue K, Min JZ, Toyo’oka T (2014) Isotopic variants of light and heavy L-pyroglutamic acid succinimidyl esters as the derivatization reagents for DL-amino acid chiral metabolomics identification by liquid chromatography and electrospray ionization mass spectrometry. Anal Chim Acta 811:51–59 25. Kuwabara T, Takayama T, Todoroki K, Inoue K, Min JZ, Toyo’oka T (2014) Evaluation of a series of prolylamidepyridine as the chiral derivatization reagents for enantioseparation of carboxylic acids by LC-ESI-MS/ MS and the application to human saliva. Anal Bioanal Chem 406:2641–2649 26. Takayama T, Kuwabara T, Maeda T, Noge I, Kitagawa Y, Inoue K, Todoroki K, Min JZ, Toyo’oka T (2015) Profiling of chiral and achiral carboxylic acid metabolomics: synthesis and evaluation of triazine-type chiral derivatization reagents for carboxylic acids by LC-ESI-MS/ MS and the application to saliva of healthy volunteers and diabetic patients. Anal Bioanal Chem 407:1003–1014 27. Mochizuki T, Takayama T, Todoroki K, Inoue K, Min JZ, Toyo’oka T (2015) Towards the chiral metabolomics: liquid chromatography-mass spectrometry based DL-amino acid analysis after labeling with a new chiral reagent, (S)-2,5-dioxopyrrolidin-1yl-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)

pyrrolidine-2-carboxylate, and the application to saliva of healthy volunteers. Anal Chim Acta 875:73–82 28. Numako M, Takayama T, Noge I, Kitagawa Y, Todoroki K, Mizuno H, Min JZ, Toyo’oka T (2016) Dried saliva spot (DSS) as a convenient and reliable sampling for bioanalysis: an application for the diagnosis of diabetes mellitus. Anal Chem 88:635–639 29. Toyo’oka T (2016) Diagnostic approach to disease using non-invasive samples based on derivatization and LC-ESI-MS/MS. Biol Pharm Bull 39:1397–1414 30. Toyo’oka T (2017) Derivatization-based highthroughput bioanalysis by LC-MS. Anal Sci 33:555–564 31. Takayama T, Mochizuki T, Todoroki K, Min JZ, Mizuno H, Inoue K, Akatsu H, Noge I, Toyo’oka T (2015) A novel approach for LC-MS/MS-based chiral metabolomics fingerprinting and chiral metabolomics extraction using a pair of enantiomers of chiral derivatization reagents. Anal Chim Acta 898:73–84 32. Nishiumi S, Kobayashi T, Ikeda A, Yoshie T, Kibi M, Izumi Y, Okuno T, Hayashi N, Kawano S, Takenawa T, Azuma T, Yoshida M (2012) A novel serum metabolomics-based diagnostic approach for colorectal cancer. PLoS One 7(7):e40459 33. Nishiumi S, Shinohara M, Ikeda A, Yoshie T, Hatano N, Kakuyama S, Mizuno S, Sanuki T, Kutsumi H, Fukusaki E, Azuma T, Takenawa T, Yoshida M (2010) Serum metabolomics as a novel diagnostic approach for pancreatic cancer. Metabolomics 6:518–528 34. Vinayavekhin N, Homan EA, Saghatelian A (2010) Exploring disease through metabolomics. ACS Chem Biol 5:91–103 35. Gonzalez J, Willis MS (2010) Ivar Asbjorn folling discovered phenylketonuria (PKU). Lab medicine 41(2):118–119 36. Fu¨rst P (1989) Amino acid metabolism in uremia. J Am College Nutrition 8(4):310–323 37. Snyder SH, Kim PM (2000) D-amino acids as putative neurotransmitters: focus on D-serine. Neurochem Res 25:553–560 38. Kleckner NW, Dingledine R (1988) Requirement for glycine in activation of NMDAreceptors expressed in Xenopus oocytes. Science 241:835–837 39. Sakata K, Fukushima T, Minje L, Ogurusu T, Taira H, Mishina M, Shingai R (1999) Modulation by L- and D-isoforms of amino acids of the L-glutamate response of N-methyl-D-aspartate receptors. Biochemistry 38:10099–10106 40. Toyo’oka T (2008) Determination methods for biologically active compounds by ultra-

Chiral Triazine Labeling Reagents performance liquid chromatography coupled with mass spectrometry: application to the analyses of pharmaceuticals, foods, plants, environments, metabonomics, and metabolomics. J Chromatogr Sci 46:233–247 41. Inoue K, Tsutsui H, Akatsu H, Hashizume Y, Matsukawa N, Yamamoto T, Toyo’oka T (2013) Metabolic profiling of Alzheimer’s disease brains. Sci Rep 3:2364. https://doi.org/ 10.1038/srep02364 42. Wren SAC (2005) Peak capacity in gradient ultra-performance liquid chromatography (UPLC). J Pharm Biomed Anal 38:337–343

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43. Nguyen DTT, Guillarme D, Rudaz S, Veuthey JL (2006) Chromatographic behavior and comparison of column packed with sub-2 μm stationary phases in liquid chromatography. J Chromatogr A 1128:105–113 44. Guillarme D, Nguyen DTT, Rudaz S, Veuthey JL (2007) Recent developments in liquid chromatography - impact on qualitative and quantitative performance. J Chromatogr A 1149:20–29

Chapter 5 Chiral Mobile-Phase Additives in HPLC Enantioseparations Lushan Yu, Shengjia Wang, and Su Zeng Abstract High-performance liquid chromatography (HPLC) is one of the main separation techniques for chiral drugs. Among the chiral HPLC techniques available, the chiral mobile-phase additive (CMPA) technique is a valuable method for the direct enantioseparation of chiral chemical entities. In the CMPA method, the chiral selector is dissolved in the mobile phase while the stationary phase is achiral. Interaction with the analyte enantiomers results in the formation of transient diastereomeric complexes. These complexes differ in their formation constants and/or distribution between the (achiral) stationary phase and the mobile phase resulting in an enantioseparation. This chapter describes the HPLC separation applying CMPA methods by several most useful types of chiral selectors including chiral ligand exchangers, macrocyclic antibiotics, and cyclodextrins. Key words Chiral mobile-phase additive, Ligand-exchange, Macrocyclic antibiotics, Vancomycin, Cyclodextrin, Enantioseparation

1

Introduction For enantioseparations, HPLC is one of the most useful techniques. The separations can be carried out by so-called direct or indirect methods. In indirect enantioseparations, the analytes are derivatized with a stereochemically pure reagent, and the resulting diastereomers are subsequently separated on an achiral column. In direct enantioseparations, a chiral stationary phase (CSP) is either bound covalently or adsorbed dynamically onto a chromatographic support. Alternatively, a chiral mobile phase (CMP) can be used. The analyte enantiomers are separated due to stereospecific interactions with the chiral selectors present in the stationary phase or in the mobile phase. CSPs are relatively expensive so the use of a chiral selector in the mobile phase, the so-called chiral mobile-phase additive (CMPA), is an attractive alternative due to the simplicity and flexibility. The enantiorecognition mechanism in CMPA systems is widely recognized as quite complex. However, the chiral recognition is generally thought to require unique interactions due to the

Gerhard K. E. Scriba (ed.), Chiral Separations: Methods and Protocols, Methods in Molecular Biology, vol. 1985, https://doi.org/10.1007/978-1-4939-9438-0_5, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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stereogenic centers of both the chiral selector and the chiral analyte simultaneously in at least three positions [1, 2]. Physicochemically, the interactions include inclusion complexation, electrostatic interactions, π–π interactions, hydrogen bonding, and dipole–dipole interactions. In CMPA methods, the chiral selectors dissolved in the mobile phase form transient diastereomeric complexes with chiral analytes. Differences in the formation kinetics or relative stability of these transient diastereomeric complexes, as well as differences in their partitioning between the mobile phase and stationary phase, are the main driving forces for chiral separations [3]. Because of the multiplicity and complexity of the interactions between the enantiomers and a chiral selector, the surface of the stationary phase, and other components of the chromatographic system, the total separation efficiency can depend strongly on the composition (including the concentration of the chiral selector and other additives), pH, and temperature of the mobile phase [4]. Therefore, it is important to optimize all these parameters when developing a CPMA method. At present, a large number of chiral selectors have been investigated in CMPA methods, and more and more new chiral selectors are being synthesized or evaluated. According to their different separation mechanisms or according to their structure, CMPA can be divided into the following groups: ligand exchanger, macrocyclic antibiotics, cyclodextrin, chiral ion pair, etc. Despite the fact that some chiral selectors have demonstrated wide applicability, the existence of a truly universal chiral selector is unlikely. Hence, it is required to try another type of selector if a baseline separation could not be achieved with one chiral selector even at optimized experimental conditions. The separation mechanism of ligand-exchange chromatography (LEC) is based on the reversible formation of mixed ternary diastereomeric complexes composed of transition metal ions (Cu2+ is the most common ion used), a chiral selector ligand (generally amino acids and their derivatives), and the analyte enantiomers. The chromatographic resolution is due to differences in complex stability constants of the two ternary complexes with the analyte enantiomers. Typical analytes that can be resolved by this approach contain two or three electron-donating functional groups (e.g., carboxyl groups, amino groups of hydroxy groups), which can simultaneously enter the coordination sphere of the complexing metal ion and function as tridentate or bidentate chelating ligands. Accordingly, classes of organic compounds that can be resolved by LEC include derivatized and underivatized amino acids, hydroxy acids, amino alcohols, diamines, dicarboxylic acids, amino amides, or dipeptides. Since the analytes and the chiral selectors used in LEC contain strongly polar functional groups, they are usually better dissolved using water, alcohols, or other strong polar solvents as mobile phases. Thus, LEC is operated with aqueous or aqueous–organic mobile phases, i.e., in the reversed-phase mode.

Chiral Mobile Phase Additives in HPLC HO

OH

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A Fig. 1 Structures of vancomycin (a) and teicoplanin (b)

Another class of chiral selectors, the macrocyclic antibiotics (also known as macrocyclic glycopeptides), was first introduced as chiral selectors by Armstrong et al. in 1994 [5]. The most prominent compounds of this group are vancomycin and teicoplanin (Fig. 1). Glycopeptides have a large number of different functional groups, e.g., aromatic rings, hydroxy groups, amino groups, carboxylic acid moieties, amide linkages, and hydrophobic pockets, so that a large variety of intermolecular interactions can contribute to the chiral recognition ability of these selectors. The threedimensional molecular structures of macrocyclic glycopeptides show that they possess a characteristic “basket-shaped” aglycon, which consists of a peptide core of complex amino acids and linked phenolic moieties. The aglycon basket of all these molecules consists of either three or four fused macrocyclic rings and is responsible for their enantioselective properties. The unique structure of macrocyclic glycopeptides contributes to their wide applicability as chiral selectors. A large variety of anionic, neutral, and cationic compounds such as amino acids, neutral aromatic molecules, and nonsteroidal anti-inflammatory drugs can be separated with these chiral selectors. Macrocyclic antibiotics allow a wide variety of chiral separation modes including the normal-phase mode, the reversedphase mode, and the polar organic mode. Cyclodextrins (CDs) have been used as chiral selectors in HPLC [6] as well as capillary electrophoresis and represent the most frequently used type of chiral selectors for a broad application range. Most CDs possess sufficient solubility in the mobile phases and low UV absorbance. Moreover, several CD derivatives (native, methylated, and hydroxypropylated derivatives, etc.) are relatively cheap.

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Fig. 2 Structure of β-cyclodextrin

CDs are cyclic oligosaccharide molecules consisting of D-(þ)glucopyranose units connected via α-1,4-glycosidic bonds. The most commonly used CDs, α-CD, β-CD, and γ-CD, are composed of six, seven, or eight glucopyranose units, respectively. The structure of β-CD is shown in Fig. 2. The molecules possess the form of a truncated cone. The primary C-6 hydroxy groups are located at the narrower rim while the secondary C-2 and C-3 hydroxy groups are on the wider rim. CDs have numerous chiral centers (five in every glucose unit). The formation of inclusion host–guest complexes is thought to be a key interaction in the chiral recognition by CDs. In this case, hydrophobic groups of analyte are included into hydrophobic cavity of CD. Secondary interactions between analyte and hydroxy groups on the rims can also contribute to the chiral recognition. CDs have been shown to separate enantiomers with different functional groups including enantiomers with planar or axial chirality or those with heteroatoms (S, P, N, and Si) as chiral centers. It appears that there are no strict requirements for the structure of analytes for a successful chiral separation with CD-based chiral selectors; inclusion of aromatic and aliphatic moieties is possible. The separations using CDs as chiral selectors can be carried out in the polar organic mode, the normal-phase mode, and the reversed-phase mode. Chiral ion pair chromatography is also one method in CMPA. In the low polar organic mobile phase, the enantiomers and the chiral ion pair react to produce electrostatic, hydrogen, or hydrophobic reactions to form diastereomeric ion pairs. The two diastereomeric ion pairs have different stability, and the distribution behavior between organic phase and stationary phase is different, so they can be separated. The separation of enantiomers in this method is mainly based on counterions, so choosing suitable counterions is very important.

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The chiral recognition of counterion and enantiomeric solute should have three kinds of forces: the ion interaction between the solute and the chiral reagent, the hydrophobic interaction between different ring systems, the hydroxy group on the solute carbon chain, and the hydrogen bond between the reagent carbonyl group. If there are two functions, such as ionic interaction and hydrogen bonding, sometimes different forces can be produced to enantioseparate. Therefore, a good chiral counterion should have the following basic properties: 1. Relatively strong acid-base, because the ionic action is related to this. 2. There should be larger rigid groups near the counterion chiral center to enhance stereoselectivity. 3. There should be ionizable functional groups or hydrogen bonding groups near the counterionic chiral centers, such as hydroxy groups and carboxyl groups. 4. Chiral ions correspond to high optical purity. The commonly used chiral counterions include quinine, quinidine, 10-camphor sulfonic acid, N-benzoyl carbonyl-glycine-L-proline, tartaric acid derivatives, etc. (Fig. 3). Typical method development starts with the selection of achiral selector according to the structure (physicochemical property) of the analyte. Subsequently, a mobile-phase mode is chosen. Following the initial experiment, optimization is performed by variation of the experimental parameters such as the composition and pH of the mobile phase and column temperature until baseline separation is achieved. In this chapter, the enantioseparation of chiral compounds by CMPA methods using common chiral selectors as chiral additives is described in detail.

Fig. 3 Structures of examples of chiral compounds for chiral ion-pair formation

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Materials

2.1 Instrumentation and Materials

1. A commercial HPLC system with a UV or fluorescence detector. 2. A C18 HPLC column (e.g., 150 mm  4.6 mm I.D., 5 μm or 250 mm  4.6 mm I.D., 5 μm) (see Note 1). 3. 0.22 or 0.45 μm membrane filters (see Note 2). 4. A commercial sonication bath for degassing of the mobile phases.

2.2 Chemicals and Solutions

All chemicals should be of the highest purity commercially available. Organic solvents should be of HPLC grade. Use doubledistilled water or ultrapure water (Milli-Q water, 18 MΩ water) prepared by suitable water purification systems. Prepare and store all reagents at room temperature (unless indicated otherwise). 1. Mobile phase 1 (ligand-exchange chromatography): Prepare a 24 mM sodium phosphate buffer, pH 3.5 (see Note 3) containing 6 mM L-phenylalanine and 3 mM Cu(II) sulfate. Filter through a 0.22 μm membrane filter (see Note 2). Mix the buffer at a ratio of 86:14 (v/v) with methanol (see Note 4). Degas by sonication before use (see Note 5). 2. Mobile phase 2 (vancomycin as CMPA): Prepare a 20 mM ammonium acetate solution in water containing 2 mM vancomycin (see Note 6). Adjust pH to 5.5 using 0.1 M NaOH (see Note 7). Mix the buffer with methanol at a methanol/buffer ratio 45:55 (v/v) (see Note 8). Filter through a 0.22 μm membrane filter and degas by sonication before use (see Note 5). 3. Mobile phase 3 (carboxymethyl-β-CD (CM-β-CD) as CMPA): Prepare a 0.05 mol/L phosphate buffer, pH 1.8 (see Note 9), containing 22.9 mM CM-β-CD. Mix with methanol at a methanol/buffer ratio of 60:40 (v/v). Filter through a 0.45 μm membrane filter (see Note 2) and degas by sonication before use (see Note 5). 4. Sample solutions: Prepare sample solutions of ofloxacin, ketoprofen, and indanone derivatives at concentrations of 1000, 100, or 40 μg/mL (see Notes 10 and 11). Filter through 0.22 μm membrane filters (see Note 12).

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Methods

3.1 Example 1: Resolution of Chiral Analytes by Chiral Ligand-Exchange Chromatography

The present example describes the enantioseparation of ofloxacin using a L-phenylalanine-Cu(II) complex as CMPA [7]. Alternative ligands comprise L-proline or L-hydroxyproline as well as their derivatives. Suitable analytes are amino acids, amino acid derivatives, amino alcohols, etc. Instead of Cu(II) ions Zn(II) ions, Ni

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(min)

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(R)-ofloxacin

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Fig. 4 Chromatogram of the analysis of the enantiomers of ofloxacin. The enantiomers were analyzed separately. In case racemic compound is injected, overlap of the two individual chromatograms is observed

(II) ions, or Co(II) ions may be used (see Note 13). However, in these cases other experimental conditions may apply. 1. Install HPLC column in the HPLC instrument equipped with a fluorescence detector or a UV detector according to the instructions of the manufacturer. 2. Set flow rate to 1.0 mL/min and operate at ambient temperature. 3. Equilibrate the column with mobile phase 1 (see Note 14). 4. When using a fluorescence detector, set the emission excitation wavelength to 330 nm and the emission wavelength to 505 nm. When using a UV detector, set wavelength to 254 nm. 5. Inject ofloxacin sample solution and record chromatogram (see Note 15). A typical chromatogram of the ligand-exchange separation of the ofloxacin enantiomers is shown in Fig. 4 [7]. 3.2 Example 2: Resolution of Chiral Analytes Using Macrocyclic Glycopeptides as CMPA

The present example describes the enantioseparation of ketoprofen using vancomycin as CMPA. Alternative glycopepties comprise teicoplanin, ristocetin A, or the teicoplanin aglycon (see Note 16). Other acidic chiral analytes may be used. But different experimental conditions may apply. 1. Install HPLC column in the HPLC instrument equipped with UV detector according to the instructions of the manufacturer. 2. Set flow rate to 1.0 mL/min. 3. Equilibrate the column with mobile phase 2 (see Note 17). 4. Set detector wavelength to 300 nm. 5. Inject sample solution of ketoprofen and record chromatogram (see Notes 15 and 18).

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Fig. 5 Chromatograms of the enantioseparation of racemic indanone derivatives. (A) 2,3‐dihydro‐2‐(4‐ methoxyphenyl)‐2‐methylinden‐1‐one; (B) 3,4‐dihydro‐2‐(4‐methoxyphenyl)‐2‐methylnaphthalen‐1(2H)‐one; (C) 2‐(3‐fluorophenyl)‐3,4‐dihydro‐2‐methyl‐naphthalen 1(2H)‐one; (D) 3,4‐dihydro‐2‐(4‐methoxyphenyl)‐2‐ methylnaphthalen‐1 (2H)‐one 3.3 Example 3: Resolution of Chiral Analytes Using Cyclodextrins as CMPA

The present example describes the separation of indanone derivative enantiomers using carboxymethyl-β-CD (CM-β-CD) as CMPA [8]. Other CDs and analytes may be used but may require different analytical conditions. 1. Install HPLC column in the HPLC instrument equipped with UV detector according to the instructions of the manufacturer. 2. Set flow rate to 0.7 mL/min. 3. Equilibrate the column with mobile phase 3 (see Note 14). 4. Set detection wavelength to 240 nm. 5. Inject sample solutions of indanone derivatives and record chromatogram (see Note 15). The separation of the enantiomers of indanone derivatives using CM-β-CD as CMPA is shown in Fig. 5.

4

Notes 1. C18 columns with a length of 250 mm are the first choice. RP-HPLC column with different packing materials and different lengths can be used, but it should be realized that different packing materials may lead to different resolution results. As a common rule, columns with a higher carbon content usually display stronger retention and better resolution of low-polarity analytes and vice versa. At the same time, longer columns may lead to longer retention times and better resolutions.

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2. When filtering water or pure aqueous buffers, a watercompatible filter membrane should be used. Otherwise, the aqueous solution will not pass through the filter. When filtering organic solvents including aqueous buffers containing organic additives, a filter membrane compatible with organic solvents should be used. Membranes designated for use with pure aqueous solvents may dissolve in organic solvents. 3. If the pH of the mobile phase is not suitable for the formation of the ligand-exchange complex, a flocculent precipitate may appear in the mobile phase. If this happens, filter the mobile phase before use and readjust the pH. Alternatively, decrease the concentration of the chiral selector or change to a different chiral selector. 4. The retention and resolution factors can be strongly affected by changing the proportion of the organic modifier in the mobile phase. Generally, the retention time decreases with an increasing content of the organic modifier. The latter, however, should not exceed a limit of approximately 20% to obtain good resolution with suitable retention times. 5. Usually, sonication for 10–20 min is sufficient. A too long sonication time may lead to the evaporation of volatile components (e.g., MeOH, acetic acid) which leads to a variation of the composition of the mobile phase. Degassing can also be achieved by purging with helium for 20 min. 6. For the stability of the glycopeptide, store the stock solution in the refrigerator between runs and overnight. In aqueous solutions at a pH of 5.0–7.0, macrocyclic antibiotics such as vancomycin deteriorate within 2–4 days at room temperature. Vancomycin solutions are stable for 6–7 days when stored at 4  C. Teicoplanin or ristocetin A solutions can be stored for about 2 weeks at 4  C without deterioration. 7. As a general rule, better separations are obtained using acidic mobile phases below or close to the pI value of glycopeptide antibiotics [9]. For example, vancomycin has a pI of ca 7.2 and is reported to be unstable at pH below 4 or above 7.5. Therefore, the starting pH of the mobile phase is usually adjusted between pH 4.5 and 6.5 when using the compound as chiral selector. 8. Longer retention times and better resolution are usually observed in both high- and low-concentration regions of the organic modifier. A typical starting composition ratio of 20/80 (v/v) methanol/buffer or 10/90 (v/v) acetonitrile/buffer is recommended. The molecular masses of macrocyclic antibiotics are between 1000 and 2100. They are soluble in water, slightly soluble in methanol, and insoluble in higher alcohols.

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9. The pH of the buffer should be investigated during method development. A starting pH of the mobile phase is usually 2 units below the pKa value of the analyte. Ionization suppression by pH control usually results in longer retention times and increases the chances to achieve chiral separations. 10. A concentration of the analytes of 40 μg/mL can be detected and resolved in most cases. Typically, 1.0 mg/mL analyte stock solutions are prepared in order to determine chemical and instrumental parameters. If the analyte is insoluble in water, a few drops of methanol may be added to aid dissolution followed by dilution of the sample with water. 11. Besides the racemic analyte, at least one of the enantiomers should be acquired if the enantiomer elution order should be determined. However, when enantiomers are not available, computational methodology can assist because in the CMP process, the analyte enantiomer which forms a more stable complex with the selector in the mobile phase elutes first [10]. 12. Samples may also be centrifuged at 15,000  g for at least 15 min to separate from precipitated material. 13. Cu(II) ions have the ability to form thermodynamically stable and kinetically labile complexes with most chiral ligands used in LEC. Thus, Cu(II) is regarded as the preferred cation for enantioseparation of amino acids. The metal ion can be used as the sulfate, acetate, nitrate, or perchlorate salt, among which the nitrate and sulfate are the most frequently applied salts. 14. Wash the column first with methanol at a flow rate of 1 mL/ min for at least 15 min, followed by washing with methanolcontaining water at the concentration to the content of the intended mobile phase at 1 mL/min for 30 min, and finally with the mobile phase for at least 2 h or until a stable baseline is achieved. 15. The complexes formed between the analyte enantiomers and the chiral selector may exhibit different UV absorption properties in detection cell. Thus, quantitation of the enantiomers requires ideally separate calibration for each of the two enantiomers. 16. The macrocyclic antibiotics are complementary to one another. Thus, if a partial enantioresolution of a racemate is obtained with one glycopeptide, there is a high probability that a better or baseline separation can be obtained with another macrocyclic antibiotic selector [11]. Hence, it is worthwhile to try the same experimental conditions using another glycopeptide as chiral selector if necessary. 17. If the column is new, it should be first washed with 10 column volumes of organic solvent, i.e., acetonitrile or methanol, followed by 10 column volumes of a mixture of the organic

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solvent and water and finally with the mobile phase until a steady baseline is observed. Be sure to never store columns, not even for a short period of time, in buffers. This may cause clogging of the columns due to the crystallization of the buffer salts. Wash the column with a mobile phase with a high water content (but not higher than 95% or the column may be damaged) for 1 h after using buffers containing mobile phases. Subsequently, flush the column with pure organic solvent (methanol, isopropanol, or acetonitrile) or solvents recommended by the column manufacturer. 18. Generally, there is an increase in resolution with a decrease of the flow rate. For reversed-phase chromatography, flow rates of 0.5–1.5 mL/min are recommended when using conventional columns. Flow rates |ΔΔH |) whereas for temperatures below TISO the enantioseparation is enthalpy-controlled (|TΔΔS | 1.5) under reversed-phase HPLC conditions. The potential application of thiol-ene click reaction for CD-CSP preparation was also investigated by Huang et al. [37]. By immobilization of mono/di (10-undecenoyl)-perphenylaminocarbonyl-β-CD onto thiol silica, they obtained a novel phenylcabamoylated CD-CSP with a long hydrophobic spacer. The CD-CSP demonstrated reasonable chiral recognition ability toward 15 racemic compounds including pindolol, propranolol, and N-isopropyl-DL-noradrenaline with reversed-phase mode. Since the phenylcarbamoyl groups on the CD rims could provide more sites such as π-π stacking interaction, hydrogen bonding, and dipole-dipole interaction, the CD-CSP exhibited enhanced resolution toward some analytes in the normal-phase mode.

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Almost all CD-CSPs developed in recent years are still based on a single CD layer on the silica surface. This chapter describes the preparation of a bilayer dual CD-CSP via click chemistry and its application in chiral HPLC. The synthetic procedure is outlined in Fig. 1. In the first step, mono-6-toluenesulfonyl-β-CD (TsO–βCD) is reacted with propargylamine to afford mono-(6-deoxy-6-propargylamine)-β-CD (alkyne-β-CD) for the click reaction (Fig. 1a). Mono-azido-β-cyclodextrin (N3-β-CD) is thereafter prepared by reacting TsO-β-CD with sodium azide followed by immobilization onto silica surface via ether linkage (Fig. 1b). The final click step is then realized between alkyne-β-CD- and N3-β-CD-functionalized silica (Fig. 1c).

2

Materials

2.1 Instrumentation and Materials

1. A commercial HPLC instrument: In the present experiments, an Agilent 1100 HPLC system or a Lab Alliance HPLC system with a diode array detection (DAD) system (State College, PA, USA) was used. 2. A commercial HPLC column packing instrument. 3. A commercial Soxhlet extractor for the purification of the products. 4. Stainless steel columns (150 mm
4.6 mm I.D.). 5. A commercial pH meter and a glass electrode for pH adjustment of buffer solutions. 6. Kromasil spherical silica gel (5 μm, 100 A˚) (Eka Chemicals, Bohus, Sweden).

2.2 Chemicals and Solutions

Most chemicals must be considered as harmful. Follow all safety procedures required in a synthetic and analytical laboratory. Wear protective clothing, gloves, and safety glasses. Perform synthesis in a well-ventilated hood. In the chromatographic experiments, use only HPLC-grade solvents and ultrapure water purified by a suitable water purification system. 1. 1% Triethylammonium acetate buffer solution: Dissolve 1% (v/v) triethylamine in ultrapure water and adjust to pH 3.99 with acetic acid (see Note 1). 2. Mobile phase: Mix 60 volumes of methanol and 40 volumes of the 1% triethylammonium acetate buffer solution. Filter through a 0.45 μm membrane filter and degas before use. 3. Sample solutions: Dissolve racemic dansyl-amino acids, e.g., dansyl-DL-phenylalanine (Dns-Phe) and dansyl-DL-leucine (Dns-Leu) in MeOH/H2O (1:1, v/v) to obtain a concentration of 1 mg/mL. Filter all sample solutions through a 0.45 μm membrane before use.

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Fig. 1 Synthetic pathway of the bilayer CD-CSP. (a) Synthesis of mono-(6-deoxy-6-propargylamine)-β-CD, (b) synthesis of mono-6A-azido-β-cyclodextrin and immobilization, and (c) click reaction-mediated construction of the bilayer CD-CSP

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Methods

3.1 Synthesis of Mono-6A-Azido-βCyclodextrin (N3-β-CD)

1. Dissolve 3.0 g (2.34 mmol) mono-6A-tosyl-β-CD (TsO-β-CD) (see Note 2) in 30 mL water at 65  C into a 50 mL doublenecked, round-bottomed flask with a rubber septum, a Liebig condenser, and a Teflon-coated magnetic stir bar. 2. Add 0.76 g (11.69 mmol) sodium azide (see Note 3) to the solution and stir for 12 h at 95  C. 3. Cool to room temperature and pour the solution into 90 mL acetone. Stir for 15 min and filter. 4. Wash the obtained solid twice with 80 mL acetone to afford N3-β-CD (see Note 4).

3.2 Synthesis of Mono-(6-Deoxy-6Propargylamine)-β-CD

1. Add 6 g (4.7 mmol) TsO-β-CD to 15 mL propargylamine into a 50 mL double-necked, round-bottomed flask equipped with a rubber septum, a Liebig condenser, and a Teflon-coated magnetic stir bar. 2. Degas the whole reaction system with two cycles of nitrogen pumping and refilling. 3. Heat the reaction solution at 65  C for 24 h. 4. Cool to room temperature and pour into 90 mL acetonitrile. 5. Collect the light yellow precipitate by filtration and wash with acetone (2
50 mL) (see Note 5).

3.3 Synthesis of Mono-6A-Azido-βCyclodextrin-Modified Silica

1. Dissolve 1.13 g (0.97 mmol) N3-β-CD in 25 mL anhydrous DMF into a 50 mL double-necked, round-bottomed flask with a rubber septum, a Liebig condenser, and a Teflon-coated magnetic stir bar. 2. Degas the whole reaction system with two cycles of nitrogen pumping and refilling. 3. Add 46.8 mg (1.17 mmol) sodium hydride at room temperature and stir the reaction mixture at room temperature until no bubbles are generated any longer. 4. Filter excess sodium hydride and transfer filtrate into another 50 mL double-necked, round-bottomed flask with a rubber septum, a Liebig condenser, and a Teflon-coated magnetic stir bar. Add 0.26 mL (1.17 mmol) (3-glycidoxypropyl) trimethoxysilane and stir the resulting mixture at 90  C for 4 h. 5. Cool to room temperature and add 2.6 g activated silica gel (see Note 6). Stir the suspension at 110  C for 24 h under N2. 6. Collect solid by filtration and wash subsequently with DMF (2
10 mL), water (2
20 mL), methanol (2
10 mL), and acetone (2
10 mL) to afford the pure product.

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3.4 Synthesis of Bilayer CD CSP (D-CDCSP) via Click Chemistry

1. Add 0.6 g (0.44 mmol) mono-(6-deoxy-6-propargylamine)-β-CD into a suspension of 2.3 g N3-β-CD-modified silica in 30 mL DMF into a 50 mL double-necked, round-bottomed flask with a rubber septum, a Liebig condenser, and a Tefloncoated magnetic stir bar. 2. Add 9 mg (0.5 mmol%) CuI(PPh3) to the suspension at room temperature. 3. Heat suspension to 90  C and stir at this temperature for 48 h under N2. 4. Obtain crude product by filtration, and wash with DMF (2
10 mL). 5. Extract solid product with acetone (50 mL) in a Soxhlet extractor for 8 h. 6. Dry material under vacuum (0.1 mbar) at 60  C for 12 h to afford D-CD-CSP.

3.5 Packing of the Column

1. Add 3 g D-CD-CSP into a mixture composed of 15 mL dichloromethane and 15 mL methanol. 2. Sonicate the suspension until it is uniformly dispersed (approximately 10 min). 3. Attach empty stainless steel column (150 mm
4.6 mm I.D.) to HPLC column packing instrument. 4. Transfer 20 mL of the silica slurry into the packing reservoir of the column as soon as possible and start to pack into a stainless steel column at 6000 psi for 20–30 min. 5. Stop the pump and let the pressure drop to 0 psi (approximately 5 min). Unscrew the column and remove excess silica using a flat spatula or a razor blade. 6. Place end fitting and frit on the column, flush the column with methanol (10 mL), and seal the column.

3.6 Enantioseparations

1. Mount column into HPLC system. 2. Set flow rate to 1.0 mL/min and column temperature at 30  C. 3. Wash column with the mobile phase for column equilibration until a stable baseline is observed. 4. Set detector wavelength at 254 nm. 5. Inject 10 μL of the respective sample solution and record chromatogram. Representative chromatograms for the enantioseparation of the dipeptides Dns-Leu and Dns-Phe are shown in Fig. 2.

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Fig. 2 Chromatograms of the enantioseparation of (a) Dns-Leu and (b) Dns-Phe on the bilayer CD-CSP. Experimental conditions: methanol/1% TEAA pH 3.99 60:40 (v/v), 1.0 mL/min, 254 nm

6. Calculate the chromatographic parameters including retention factors (k1 and k2), enantioselectivity (α), and resolution (RS) according to k ¼ (tR  t0)/t0, α ¼ k2/k1, and Rs ¼ 1.18
(t2  t1)/(Wh1 + Wh2). Further examples of the analyte enantioseparations on the D-CD-CSP can be found in ref. 33.

4

Notes 1. The β-CD-CSP can be operated in the reversed-phase mode with organic solvents and water or buffer as the mobile phase. 2. TsO-β-CD is an important CD intermediate for the facile introduction of functional groups such as halides, azide, amine, and imidazole groups. The compound can be prepared as described in ref. 14 or obtained from commercial sources such as CycloLab (Budapest, Hungary). 3. Sodium azide is toxic and explosive. Handle with extreme care. 4. The successful conversion of tosyl group to azido group can be verified by FTIR and 1H-NMR spectroscopy. FTIR of N3-β-CD displays a representative peak of N3 at 2104 cm1 1 H-NMR (400 MHz, DMSO-d6) δ (ppm): 3.32–3.40 (m, 28 H, H-3, H-5, and H-6), 3.55–3.64 (m, 14 H, H-2, and H-4), 4.44–4.54 (m, 6 H, and OH-6), 4.83–4.87 (s br, 7 H, and H-1), and 5.62–5.77 (m, 14 H, OH-2, and OH-3). 5. The CD derivative can be characterized by 1H-NMR spectroscopy and mass spectrometry. 1H NMR (DMSO-d6) (ppm): 7.6–7.4 (2 H), 7.2–7.0 (2 H), 5.9–5.6 (14 H), 5.0–4.8 (7 H), 4.6–4.4 (6H), and 3.8–3.2 (42 H). ESI-MS (m/z):

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1172.4 (calcd.) and 1171.5 (found) for [M+], and 1343.4 (calcd.) and 1343.6 (found) for [M+TsO]. 6. Dry silica at 120  C for 24 h under vacuum to obtain activated silica gel.

Acknowledgments The financial support from the National Natural Science Foundation of China (no. 21575100), Tianjin Research Program of Application Foundation and Advanced Technology (17JCYBJC20500, 18JCZDJC37500), and National Program on Key Basic Research Project (2015CB856505) are gratefully acknowledged. References 1. Stalcup AM (2010) Chiral separations. Annu Rev Anal Chem 3:341–363 2. Ward TJ, Ward KD (2012) Chiral separations: a review of current topics and trends. Anal Chem 84:626–635 3. Dolowy M, Pyka A (2014) Application of TLC, HPLC and GC methods to the study of amino acid and peptide enantiomers: a review. Biomed Chromatogr 28:84–101 4. Zhou J, Tang J, Tang W (2015) Recent development of cationic cyclodextrins for chiral separation. Trends Anal Chem 65:22–29 5. Issaraseriruk N, Sritana-Anant Y, Shitangkoon A (2018) Substituent effects on chiral resolutions of derivatized 1-phenylalkylamines by heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-beta-cyclodextrin GC stationary phase. Chirality 30(7):900–906. https://doi. org/10.1002/chir.22856 6. Wang RQ, Ong TT, Tang W, Ng SC (2012) Recent advances in pharmaceutical separations with supercritical fluid chromatography using chiral stationary phases. Trends Anal Chem 37:83–100 7. Varga G, Tarkanyi G, Nemeth K, Ivanyi R (2010) Chiral separation by a monofunctionalized cyclodextrin derivative: from selector to permethyl-beta-cyclodextrin bonded stationary phase. J Pharm Biomed Anal 51:84–89 8. Vega ED, Lomsadze K, Chankvetadze L, Salgado A (2011) Separation of enantiomers of ephedrine by capillary electrophoresis using cyclodextrins as chiral selectors: comparative CE, NMR and high resolution MS studies. Electrophoresis 32:2640–2647 9. Zhou Z, Li X, Chen X, Hao X (2010) Synthesis of ionic liquids functionalized β-cyclodextrin-

bonded chiral stationary phases and their applications in high-performance liquid chromatography. Anal Chim Acta 678:208–214 10. Lai X, Tang W, Ng SC (2011) Novel β-cyclodextrin chiral stationary phases with different length spacers for normal-phase high performance liquid chromatography enantioseparation. J Chromatogr A 1218:3496–3501 11. Chen L, Zhang LF, Ching CB, Ng SC (2002) Synthesis and chromatographic properties of a novel chiral stationary phase derived from heptakis(6-azido-6-deoxy-2,3-di-O-phenylcarbamoylated)-β-cyclodextrin immobilized onto amino-functionalized silica gel via multiple urea linkages. J Chromatogr A 950:65–74 12. Han X, Yao T, Liu Y, Larock RC, Armstrong DW (2005) Separation of chiral furan derivatives by liquid chromatography using cyclodextrin-based chiral stationary phases. J Chromatogr A 1063:111–120 13. Ai F, Li L, Ng SC, Tan TT (2010) Sub-1micron mesoporous silica particles functionalized with cyclodextrin derivative for rapid enantioseparations on ultra-high pressure liquid chromatography. J Chromatogr A 1217:7502–7506 14. Xiao Y, Ng SC, Tan TT, Wang Y (2012) Recent development of cyclodextrin chiral stationary phases and their applications in chromatography. J Chromatogr A 1269:52–68 15. Li L, Cheng B, Zhou R, Cao Z, Zeng C, Li L (2017) Preparation and evaluation of a novel N-benzyl-phenethylamino-beta-cyclodextrinbonded chiral stationary phase for HPLC. Talanta 174:179–191 16. Rahim NY, Tay KS, Mohamad S (2017) Chromatographic and spectroscopic studies on β-cyclodextrin functionalized ionic liquid as

Cyclodextrin-Based Chiral Stationary Phases chiral stationary phase: Enantioseparation of NSAIDs. Adsorpt Sci Technol 36:130–148 17. Li X, Zhou Z (2014) Enantioseparation performance of novel benzimido-beta-cyclodextrins derivatized by ionic liquids as chiral stationary phases. Anal Chim Acta 819:122–129 18. Yao B, Yang X, Guo L, Kang S, Weng W (2014) Development of a composite chiral stationary phase from BSA and beta-cyclodextrin-bonded silica. J Chromatogr Sci 52:1233–1238 19. Li L, Zhang M, Wang Y, Zhou W, Zhou Z (2016) Preparation of chiral oxazolinylfunctionalized beta-cyclodextrin-bonded stationary phases and their enantioseparation performance in high-performance liquid chromatography. J Sep Sci 39:4136–4146 20. Lin C, Liu W, Fan J, Wang Y, Zheng S, Lin R, Zhang H, Zhang W (2012) Synthesis of a novel cyclodextrin-derived chiral stationary phase with multiple urea linkages and enantioseparation toward chiral osmabenzene complex. J Chromatogr A 1283:68–74 21. Lin C, Fan J, Liu WN, Tan Y, Zhang WG (2014) Comparative HPLC enantioseparation on substituted phenylcarbamoylated cyclodextrin chiral stationary phases and mobile phase effects. J Pharm Biomed Anal 98:221–227 22. Dr HCK, Prof MGF, Prof KBS (2001) ClickChemie: diverse chemische Funktionalit€at mit einer Handvoll guter Reaktionen. Angew Chem 113:2056–2075 23. Wang Y, Xiao Y, Yang Tan TT, Ng SC (2008) Click chemistry for facile immobilization of cyclodextrin derivatives onto silica as chiral stationary phases. Tetrahedron Lett 49:5190–5191 24. Zhang Y, Guo Z, Ye J, Xu Q, Liang X, Lei A (2008) Preparation of novel beta-cyclodextrin chiral stationary phase based on click chemistry. J Chromatogr A 1191:188–192 25. Zhou RD, Li LS, Cheng BP, Nie GZ, Zhang HF (2014) Enantioseparation and determination of propranolol in human plasma on a new derivatized β-cyclodextrin-bonded phase by HPLC. Chin J Anal Chem 42:1002–1009 26. Tang J, Pang L, Zhou J, Zhang S, Tang W (2016) Per(3-chloro-4-methyl)phenylcarbamate cyclodextrin clicked stationary phase for chiral separation in multiple modes highperformance liquid chromatography. Anal Chim Acta 946:96–103

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27. Tang J, Zhang S, Lin Y, Zhou J, Pang L, Nie X, Zhou B, Tang W (2015) Engineering cyclodextrin clicked chiral stationary phase for high-efficiency enantiomer separation. Sci Rep 5:11523 28. Lin Y, Zhou J, Tang J, Tang W (2015) Cyclodextrin clicked chiral stationary phases with functionalities-tuned enantioseparations in high performance liquid chromatography. J Chromatogr A 1406:342–346 29. Zhou J, Yang B, Tang J, Tang W (2016) Cationic cyclodextrin clicked chiral stationary phase for versatile enantioseparations in highperformance liquid chromatography. J Chromatogr A 1467:169–177 30. Hoyle CE, Bowman CN (2010) Thiol-ene click chemistry. Angew Chem 49:1540–1573 31. Zhou J, Pei W, Zheng X, Zhao S, Zhang Z (2015) Preparation and enantioseparation characteristics of a novel beta-cyclodextrin derivative chiral stationary phase in highperformance liquid chromatography. J Chromatogr Sci 53:676–679 32. Yao X, Zheng H, Zhang Y, Ma X, Xiao Y, Wang Y (2016) Engineering thiol-ene click chemistry for the fabrication of novel structurally welldefined multifunctional cyclodextrin separation materials for enhanced enantioseparation. Anal Chem 88:4955–4964 33. Zhao J, Lu X, Wang Y, Tan TT (2014) Surfaceup constructed tandem-inverted bilayer cyclodextrins for enhanced enantioseparation and adsorption. J Chromatogr A 1343:101–108 34. Li X, Li J, Kang Q, Wang Y (2018) Polarity tuned perphenylcarbamoylated cyclodextrin separation materials for achiral and chiral differentiation. Talanta 185:328–334 35. Li X, Jin X, Yao X, Ma X, Wang Y (2016) Thioether bridged cationic cyclodextrin stationary phases: effect of spacer length, selector concentration and rim functionalities on the enantioseparation. J Chromatogr A 1467:279–287 36. Yao X, Tan TT, Wang Y (2014) Thiol-ene click chemistry derived cationic cyclodextrin chiral stationary phase and its enhanced separation performance in liquid chromatography. J Chromatogr A 1326:80–88 37. Huang G, Ou J, Zhang X, Ji Y, Peng X, Zou H (2014) Synthesis of novel perphenylcarbamated beta-cyclodextrin based chiral stationary phases via thiol-ene click chemistry. Electrophoresis 35:2752–2758

Chapter 10 Hybrid Organic-Inorganic Materials Containing a Nanocellulose Derivative as Chiral Selector Liang Zhao, Hui Li, Shuqing Dong, and Yanping Shi Abstract Hybrid organic-inorganic materials (HOIM), with high mechanical stability, large surface area, tailored pore size, controlled morphology, and organic loading have shown superior chiral separation performance. In this chapter, the preparation of hybrid organic-inorganic materials of core-shell silica microspheres by a layer-by-layer self-assembly method is described. The enantioseparation performance by high-performance liquid chromatography is illustrated by various types of chiral compounds under normal- and reversedphase elution conditions. The chiral selector of nanocrystalline cellulose derivative hybrid organic-inorganic materials showed good performance in the separation of enantiomers. Key words Chiral selectors, Hybrid organic-inorganic materials, Nanocellulose, Enantioseparation, High-performance liquid chromatography

1

Introduction Chirality is an essential property of nature. When enantiomers were taken up by the human body or the ecological environment, there are significant differences in their pharmacological activities, metabolic processes and toxicity, and some may even have the opposite effect [1]. Thus far, chiral separations still remain one of the most significant issues in the field of analytical science. Over the last few decades, various analytical techniques have been developed for chiral separations including gas chromatography, highperformance liquid chromatography, capillary electrochromatography, and capillary liquid chromatography [2]. Compared to many methods, high-performance liquid chromatography using chiral stationary phases (CSPs) is considered one of the most effective methods to separate enantiomers because of the high separation efficiency and the general applicability [3, 4]. Therefore, the exploitation of new types of chiral selectors is still a research topic in the field of enantioseparation.

Gerhard K. E. Scriba (ed.), Chiral Separations: Methods and Protocols, Methods in Molecular Biology, vol. 1985, https://doi.org/10.1007/978-1-4939-9438-0_10, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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Emerging hybrid organic-inorganic materials (HOIM) with both organic and inorganic components have attracted a lot of attention because the integrative design, which is expected to provide excellent and unique properties [5–7]. Compared with conventional materials, HOIMs obtained by simultaneous reaction of organic molecules and inorganic components have abundant and homogeneously distributed organic functional groups within the whole framework instead of simply modifying the surface of inorganic oxides. Weak bonds still exist between organic and inorganic phases in such materials [8, 9]. HOIMs were endowed with new functions and features owing to the tunable functional organic groups in the pore walls or channels, which can achieve chromatographic separations not only on the surface of the materials but also at the inner part of the material. Moreover, the loading amount of chiral selectors in HOIMs and uniformly distributed organic functional groups can be tailored through controlling the ratio of chiral precursors over inorganic precursors in the preparation process. This makes HOIMs to have a great prospect as chiral stationary phases. In 2008, Okamoto and coworkers reported a novel method for synthesizing organic-inorganic hybrid materials using cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) and tetraethyl orthosilicate [10]. The proposed silica hybrid spheres exhibited similar chiral recognition and possessed a higher loading capacity compared to a commercial chromatographic column coated with the same chiral selector. Therefore, HOIMs are emerging in chiral analysis due to their superiority as separation materials. Important results of hybrid organic-inorganic materials used as chiral stationary phases have been achieved in the last 10 years. In 2007, Yang and coworkers synthesized bifunctionalized mesoporous organosilica spheres with trans-(1R,2R)-diaminocyclohexane in the pores and applied these as chiral stationary phase in highperformance liquid chromatography (HPLC). The column packed with the bifunctionalized mesoporous organosilica spheres exhibits higher selectivity and resolution for racemic amino acids than a column packed with trans-(1R,2R)-diaminocyclohexane (DACH)SiO2 prepared by the conventional post-synthesis grafting method [11]. In 2008, Yang and coworkers prepared a new mesoporous organic-inorganic sphere with trans-(1R,2R)-bis-(ureido)cyclohexane covalently bridged in the pore wall by co-condensation of N,N´-bis-[(triethoxysilyl)propyl]-trans-(1R,2R)-bis-(ureido)cyclohexane and 1,2-bis(trimethoxysilyl)ethane through a hierarchical double templating method. The hybrid material was employed as a novel kind of chiral stationary phase in HPLC. The column packed with the hybrid material efficiently separated the enantiomer of R/S1,10-bis-2-naphthol even at a high sample load and at high flow rates because of the high chiral selector loading and high surface area of the material [12]. In 2012, Di and coworkers synthesized

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mesoporous organosilicas with both R-(+)-1,10 -binaphthyl-2,20 diamine and ethane moieties bridging in the framework. They used C18TMACl as structural directing agent, one-step co-condensation of N,N0 -bis-[(triethoxysilyl)propyl]-(R)-bis-(ureido)-binaphthyl and 1,2-bis(triethoxysilyl)ethane [13]. The column packed with these organosilica spheres exhibited greater selectivity for R/ S-1,10 -bi-2,20 -naphthol. In 2013, Di and coworkers used a layerby-layer method for the synthesis of novel chiral core-shell silica microspheres with a DACH moiety bridged in the mesoporous shell. The functionalized core-shell silica microspheres were characterized and tested as chiral stationary phases in HPLC. R/S1,10 -bi-2,20 -naphthol, R/S-6,60 -dibromo-1,10 -bi-2-naphthol, and R/S-1,10 -bi-2,20 -phenanthrol were enantioseparated rapidly on the column packed with the DACH core-shell silica particles [14]. In 2014, Bao and coworkers synthesized a hybrid of CDMPC as chiral stationary phase (organic/inorganic: 70/30, w/w) via the sol-gel method [15]. Compared to a commercial Chiralpak IB column, better enantioseparation was achieved on this material for pindolol, metoprolol, propranolol, bisoprolol, and atenolol. In 2015, Zhao and coworkers fabricated a β-cyclodextrin-based periodic mesoporous organosilica (PMO) CSP via one-step copolymerization of silanized monochlorotriazinyl β-cyclodextrin and N-benzoyl-L-tyrosine ethyl ester (BTEE) in the presence of cetyltrimethylammonium bromide (CTAB) as template [16]. Functional groups such as β-cyclodextrin, triazinyl, and ethyl were introduced in the pore channels and pore walls of the hybrid material, respectively, which made this hybrid material a multifunctional stationary phase including groups for enantioresolution, anion exchange, and achiral separations. Among the various chiral separation materials, cellulose derivatives are the most commonly used chiral selectors for CSPs because of their ability to enantioseparate a large number of chiral compounds [17–20]. In 2007, Ikai and coworkers reported an efficient immobilization of polysaccharide derivatives onto silica gel via intermolecular polycondensation of triethoxysilyl groups, specifically using the 3-(triethoxysilyl)propyl group as cross-linker [21]. On this basis, the group reported the synthesis of organicinorganic hybrid materials using CDMPC bearing a small amount of 3-(triethoxysilyl)propyl residues and tetraethyl orthosilicate as a CSP for HPLC [10]. Nanocrystalline cellulose (NCC) not only retains the major properties of cellulose, but has also some unique characteristics such as a high surface area and optical properties. This makes NCC a promising material for the preparation of CSPs. Recent data show that NCC suspensions can form a chiral nematic liquid-crystalline phase, which consequently can be utilized as a template to prepare chiral ordered materials. These have been applied as new chiral separation materials [22, 23].

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Based on the merits of hybrid cellulose-based CSPs as well as the selective chiral discrimination properties, nanocellulose hybrid organic-inorganic core-shell CSPs for HPLC were designed by coating NCC derivatives on silica gel to further study the applications of nanocellulose in chiral separation. By using layer-by-layer and sol-gel methods, a nanocellulose derivative was introduced into the hybrid porous shell by the copolymerization reaction of organosilica precursors [24, 25]. These NCC-based CSPs showed better peak shape and higher column efficiency compared to a cellulose-based CSP, which indicated that NCC is an attractive material for CSPs. NCC derivative-based chiral selectors followed typical normal-phase HPLC behavior in hydrocarbon-alcohol mobile phases. Several examples of deviation from this behavior have been recently reported [26, 27]. In aqueous organic mobile phases, polysaccharide derivative-based CSPs are known to follow atypical reversed-phase behavior. In some cases even up to 30% water (v/v) could be used especially in combination with aprotic organic solvents such as acetonitrile [28–31]. This chapter describes the synthesis of a NCC-based CSP and its application to enantioseparations in the normal-phase and the reversed-phase elution mode. The synthesis of the material is outlined in Fig. 1. OH

a. triphenylchloromethane/pyridine b. 3,5-dimethylphenyl isocyanate c. conc. HCl/methanol

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Fig. 1 Scheme of the preparation of hybrid organic-inorganic materials as chiral selectors for HPLC enantioseparations

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Materials

2.1 Instrumentation and Materials

1. A commercial HPLC instrument with a UV detector: In the present study, a Waters HPLC System (Waters, Milford MA, USA) composed of a Waters 515 HPLC pump, a Waters 2487 UV detector, and a Rheodyne 7725i injector with a 20 μL sample loop. 2. A commercial column slurry packing apparatus: An Alltech 95551U HPLC slurry packing instrument (Alltech, Nicholasville, KY, USA) is suitable. 3. A commercial pH meter. 4. A commercial sonication bath for sonicating samples and degassing mobile phases. 5. A laboratory centrifuge capable of centrifuging a volume of 100–200 mL. 6. A commercial lyophilizer for freeze-drying samples. 7. A Soxhlet extraction apparatus for purification of the hybrid organic-inorganic material. 8. Dialysis tube membranes with a molecular weight cutoff of 7500.

2.2 Mobile Phases and Solutions

Use HPLC-grade organic solvents. Use ultrapure water (18.25 MΩ·cm at 25  C) prepared by a suitable water purification system. All reagents should be of analytical grade. 1. Sample solutions: Dissolve analytes at a concentration of 1 mg/ mL in the respective mobile phase. 2. Mobile phases for separations under normal-phase conditions: Mix the appropriate amounts of n-hexane and an alcohol. In the experiments described below, the following mobile phases were applied depending on the analyte: n-hexane-isopropanol (99.5/0.5, v/v), n-hexane-isopropanol (97/3, v/v), nhexane-ethanol (97/3, v/v), and n-hexane-isopropanol-chloroform (70/15/15). Filter through 0.45 μm filters and sonicate for 10 min before use. 3. Mobile phases for separations under reversed-phase conditions: Mix the appropriate volumes of acetonitrile and water. In the experiments described below, the following mobile phases were applied depending on the analyte: acetonitrile/water (15/85, v/v), acetonitrile/water (20/80, v/v), and acetonitrile/water (30/70, v/v). Filter through 0.45 μm filters and sonicate for 10 min before use.

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Methods Carry out all procedures at room temperature unless otherwise specified. Perform chemical reactions in a well-ventilated hood. Chemicals may be hazardous to human health; thus, observe safety precautions when handling chemicals and wear protective gear if applicable.

3.1

Synthesis of NCC

1. Disperse 5.0 g microcrystalline cellulose (see Note 1) in 50 mL sodium hypochlorite solution (see Note 2) for 12 h at room temperature. 2. Sonicate suspension of 30 min. 3. Dilute with 200 mL ultrapure water to stop the reaction. 4. After letting the suspension settle, decant supernatant and centrifuge for 10 min at 10,000  g. 5. Wash residue repeatedly with ultrapure water. A colloidal suspension of NCC is obtained. 6. Place colloidal suspension in 50 mL portions in dialysis membrane tubings (cutoff 7500) with the aid of a 100 mL graduated cylinder. Place five of these tubings in 10 L of deionized water. Replace 5 L of water every day for 3 days until the pH of the suspension becomes neutral (see Note 3). 7. Lyophilize the suspension to obtain dry NCC material.

3.2 Synthesis of NCC 3,5-Dimethylphenyl Carbamate Derivative

The synthesis of the material is outlined in Fig. 1. 1. Place 1.0 g freeze-dried NCC in a round-bottom flask equipped with a magnetic stir bar and a reflux condenser closed with a drying tube containing calcium hydroxide. Add 50 mL dry pyridine (see Note 4) and stir for 24 h at 80  C. 2. Add 3.5 g triphenylchloromethane (trityl chloride) and stir for 12 h at 80  C. 3. Ad 4.0 mL 3,5-dimethylphenyl isocyanate to the mixture and stir for 24 h at 80  C. 4. Let the reaction mixture cool to room temperature and pour the solution into 200 mL methanol while stirring. A white precipitate is formed. 5. Collect product by filtration and wash with methanol. 6. Suspend the solid in 2% (v/v) hydrochloric acid in methanol (see Note 5) and stir for 24 h at room temperature. 7. Collect the white solid by filtration and wash with 30 mL methanol. Dry at 60  C under vacuum for 24 h. 8. Dissolve 1.5 g of the dried solid in 60 mL pyridine (see Note 4) containing 1.5 g anhydrous lithium chloride in a round-

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bottom flask equipped with a magnetic stir bar and a reflux condenser closed with a drying tube containing calcium hydroxide. Stir for 2 h at room temperature. 9. Add 1.2 mL 3-(triethoxysilyl)propyl isocyanate and stir the mixture for 16 h at 80  C. 10. Let the mixture cool to room temperature and pour into 200 mL. Collect the precipitated NCC 3,5-dimethylphenyl carbamate derivative by filtration. Wash the product with methanol and dry at 60  C for 24 h under vacuum. 3.3 Preparation of Hybrid OrganicInorganic Material

1. Disperse 3.0 g activated 5 μm silica gel particles (see Note 6) in 100 mL of a 0.025 M cetyltrimethylammonium bromide (CTAB) solution (see Note 7) and sonicate for 30 min. Let stand for another 1 h. 2. Collect the silica gel particles by filtration, wash with 50 mL ultrapure water, and dry at 60  C for 12 h under vacuum. 3. Dissolve 0.05 g NCC 3,5-dimethylphenyl carbamate derivative obtained according to Subheading 3.2 in 25 mL pyridine (see Note 4) in a 100 mL round-bottom flask equipped with a condenser closed by a calcium hydroxide drying tube and a magnetic stir bar at room temperature. 4. Add 3 mL of tetraethyl orthosilicate (TEOS) and 2 mL of ethanol and continue stirring at room temperature. 5. In a separate flask, dissolve 0.1 g CTAB in 1.0 mL of 0.037 g/ mL aqueous hydrofluoric acid solution and add 0.5 mL concentrated hydrochloric acid. 6. After complete dissolution of CTAB, add this solution to the mixture prepared in step 4. 7. Cool the mixture to 15  C and stir the mixture for 4 h to form a stable hybrid silica sol. 8. Place the 3.0 g CTAB-silica obtained in steps 1–2 in the hybrid silica sol and let stand for 1.5 h. 9. Centrifuge the solution to collect the particles. Wash the silica particles repeatedly with ultrapure water. 10. Dry the particles at 60  C under vacuum for 12 h. 11. Repeat steps 3–10 five times if required and combine products to obtain sufficient quantities of the material. 12. Extract excessive CTAB by Soxhlet extraction using 50% aqueous ethanol to obtain the pure hybrid organic-inorganic material. Dry under vacuum for 12 h at 60  C.

3.4

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1. Connect the stainless steel column (150  4.6 mm) to the slurry packing apparatus. 2. Suspend 2.0 g hybrid organic-inorganic material in a mixture of 25 mL dioxane and 25 mL chloroform and sonicate for 2 min.

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3. Place the slurry in the slurry reservoir of the packing apparatus and pack into the column at a pressure of 50 MPa. 4. Use n-hexane as displacement solvent for the packing. 1. Install the hybrid organic-inorganic CSP column in the HPLC instrument.

3.5 Example 1: Enantioseparations in the Normal-Phase Mode

2. Place the respective mobile phase (see Note 8) in the solvent reservoir and equilibrate column at a flow rate of 1.0 mL/min until a stable baseline is obtained. 3. Set detection wavelength to 254 nm. 4. Inject sample solution (20 μL) and record chromatogram. Examples of enantioseparations in the normal-phase mode on the hybrid organic-inorganic CSP are shown in Fig. 2 (see Note 9). 1. Install the hybrid organic-inorganic CSP column in the HPLC instrument.

3.6 Example 2: Enantioseparations in the Reversed-Phase Mode

2. Place the respective mobile phase (see Note 10) in the solvent reservoir and equilibrate column at a flow rate of 1.0 mL/min until a stable baseline is obtained. 3. Set detection wavelength to 254 nm. 4. Inject sample solution (20 μL) and record chromatogram.

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Examples of enantioseparations in the reversed-phase mode on the hybrid organic-inorganic CSP are shown in Fig. 3 (see Note 9).

4

Notes 1. Microcrystalline cellulose from Merck KG (Darmstadt, Germany) gave best results in our hands but materials from other commercial sources may be suitable as well. 2. The sodium hypochlorite solution should contain an active chlorine content of not less than 10%. Wear protective gloves, protective clothing, and eye protection when handling the solution. 3. Dialysis is performed to remove the excess of acid. 4. Pyridine is toxic. Wear protective gear and operate in a wellventilated hood. 5. 1000 g of a 2% HCl solution in methanol can be prepared from 46.0 mL concentrated hydrochloric acid and 946 g methanol. 6. For silica gel activation, place 3 g silica gel (5 μm particle size) in 50 mL concentrated hydrochloric acid and leave at room temperature for 24 h. Filter and wash with ultrapure water until the pH of the filtered solution is neutral. Dry at 80  C under vacuum for 24 h. 7. A 0.025 M cetyltrimethylammonium bromide (CTAB) solution is prepared by dissolution of 0.92 g of CATB in 100.0 mL ultrapure water.

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8. The amount of the alcohol content in n-hexane increases the polarity of the mobile phase. An increase will result in shorter elution times due to the weakening of interactions such as hydrogen bonds between the analyte and the chiral selector. The concentration of the alcohol should be studied during method optimization. 9. HPLC instruments from different companies as well as different instruments from the same supplier may yield slightly different results even when using identical experimental conditions. Thus, the variables may require slight changes when transferring a certain analytical method from one instrument to another; instruments from different manufacturers may have different operation conditions. 10. Mobile-phase additives such as THF and CHCl3 can be used in enantioseparations on the hybrid organic-inorganic CSP to improve peak shape and separation performance. References 1. Lorenz H, Seidel-Morgenstern A (2014) Processes to separate enantiomers. Angew Chem Int Ed Engl 53:1218–1250 2. Ward TJ, Ward KD (2012) Chiral separations: a review of current topics and trends. Anal Chem 84:626–635 3. Okamoto Y, Ikai T (2008) Chiral HPLC for efficient resolution of enantiomers. Chem Soc Rev 37:2593–2608 4. Wang Z, Ouyang J, Banyans WRG (2008) Recent developments of enantioseparation techniques for adrenergic drugs using liquid chromatography and capillary electrophoresis: a review. J Chromatogr A 862:1–14 5. Guo Y, Hu C, Wang X et al (2001) Microporous decatungstates: synthesis and photochemical behavior. Chem Mater 13:4058–4064 6. Fukaya N, Haga H, Tsuchimoto T et al (2010) Organic functionalization of the surface of silica with arylsilanes. A new method for synthesizing organic–inorganic hybrid materials. J Organomet Chem 695:2540–2542 7. Kickelbick G (2007) Hybrid materials, synthesis, characterization and applications. WileyVCH, Weinheim 8. Sanchez C, Julia´n B, Belleville P, Popall M (2005) Applications of hybrid organic–inorganic nanocomposites. J Mater Chem 15:35–36 9. Wight AP, Davis ME (2002) Design and preparation of organic-inorganic hybrid catalysts. Chem Rev 102:3589–3614

10. Ikai T, Yamamoto C, Kamigaito M, Okamoto Y (2008) Organic–inorganic hybrid materials for efficient enantioseparation using cellulose 3,5–dimethylphenylcarbamate and tetraethyl Orthosilicate. Chem Asian J 3:1494–1499 11. Zhu G, Jiang D, Yang QH et al (2007) Trans(1R,2R)-diaminocyclohexane functionalized mesoporous organosilica spheres as chiral stationary phase. J Chromatogr A 1149:219–227 12. Zhu G, Zhong H, Yang QH, Li C (2008) Chiral mesoporous organosilica spheres: synthesis and chiral separation capacity. Microporous Mesoporous Mater 11:36–43 13. Ran RX, You LJ, al DB (2012) A novel chiral mesoporous binaphthyl–silicas: preparation, characterization and application in HPLC. J Sep Sci 35:1854–1862 14. Wu XB, You LJ, Di B (2013) Novel chiral core–shell silica microspheres with trans–(1R,2R)–diaminocyclohexane bridged in the mesoporous shell: synthesis, characterization and application in high performance liquid chromatography. J Chromatogr A 1299:78–84 15. Weng XL, Bao ZB, Xing HB et al (2013) Synthesis and characterization of cellulose 3,5–dimethylphenylcarbamate silica hybrid spheres for enantioseparation of chiral beta–blockers. J Chromatogr A 1321:38–47 16. Wang LT, Dong SQ, Han F et al (2015) Spherical beta-cyclodextrin silica hybrid materials for multifunctional chiral stationary phases. J Chromatogr A 1383:70–78

NCC Core-Shell Hybrid Materials 17. Shen J, Okamoto Y (2016) Efficient separation of enantiomers using stereoregular chiral polymers. Chem Rev 16:1094–1138 18. Shen J, Ikai T, Okamot Y (2014) Synthesis and application of immobilized polysaccharide -based chiral stationary phases for enantioseparation by high-performance liquid chromatography. J Chromatogr A 1363:51–61 19. Wang ZQ, Liu JD, Chen W, Bai ZW (2014) Enantioseparation characteristics of biselector chiral stationary phases based on derivatives of cellulose and amylose. J Chromatogr A 1346:57–68 20. Tang S, Mei XM, Chen W et al (2018) A highperformance chiral selector derived from chitosan(p-methylbenzylurea) for efficient enantiomer separation. Talanta 185:42–52 21. Ikai T, Yamamoto C, Kamigaito M, Okamoto Y (2007) Immobilization of polysaccharide derivatives onto silica gel: facile synthesis of chiral packing materials by means of intermolecular polycondensation of triethoxysilyl groups. J Chromatogr A 1157:151–158 22. Zhang JH, Xie SM, Zhang M et al (2014) Novel inorganic mesoporous material with chiral nematic structure derived from nanocrystalline cellulose for high-resolution gas chromatographic separations. Anal Chem 86:9595–9602 23. Zhang JH, Zhang M, Xie SM et al (2015) A novel inorganic mesoporous material with a nematic structure derived from nanocrystalline cellulose as the stationary phase for highperformance liquid chromatography. Anal Methods 7:3448–3453 24. Zhang XL, Wang LT, Dong SQ et al (2016) Nanocellulose derivative/silica hybrid coreshell chiral stationary phase: preparation and enantioseparation performance. Molecules 21:561–575 25. Zhang XL, Wang LT, Dong SQ et al (2016) Nanocellulose 3,5-dimethylphenylcarbamate

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derivative coated chiral stationary phase: preparation and enantioseparation performance. Chirality 28:376–381 26. Pierini M, Carradori S, Menta S et al (2017) C3-(Phenyl-4-oxy)-5-phenyl-4,5-dihydro-(1H) -pyrazole: a fascinating molecular framework to study the enantioseparation ability of the amylose (3, 5-dimethylphenylcarbamate) chiral stationary phase. Part II. Solvophobic effects in enantiorecognition. J Chromatogr A 1499:140–148 27. Matarashvili I, Ghughunishvili D, Chankvetadze L et al (2017) Separation of enantiomers of chiral weak acids with polysaccharide-based chiral columns and aqueous mobile phases in high-performance liquid chromatography: typical reversed-phase behavior. J Chromatogr A 1483:86–92 28. Chankvetadze B, Yamamoto C, Okamoto Y (2001) Enantioseparation of selected chiral sulfoxides using polysaccharide-type chiral stationary phases and polar organic, polar aqueous-organic and normal-phase eluents. J Chromatogr A 922:127–137 29. Jibuti G, Mskhiladze A, Takaishvili N et al (2012) HPLC separation of dihydropyridine derivatives enantiomers with emphasis on elution order using polysaccharide-based chiral columns. J Sep Sci 35:2529–2537 30. Gallinella B, Bucciarelli L, Zanitti L et al (2014) Direct separation of the enantiomers of oxaliplatin on a cellulose-based chiral stationary phase in hydrophilic interaction liquid chromatography mode. J Chromatogr A 1339:210–213 31. Shedania Z, Kakava R, Volonterio A et al (2018) Separation of enantiomers of chiral sulfoxides in high-performance liquid chromatography with cellulose-based chiral selectors using methanol and methanol-water mixtures as mobile phases. J Chromatogr A 1557:62–74

Chapter 11 Cyclofructans as Chiral Selectors: An Overview Garrett Hellinghausen and Daniel W. Armstrong Abstract Cyclofructans are cyclic oligosaccharides made of β-2,1-linked fructofuranose units. They have been utilized as chiral selectors, usually after derivatization, with high-performance liquid chromatography (HPLC), gas chromatography (GC), capillary electrophoresis (CE), and supercritical fluid chromatography (SFC). The focus herein will be directed to their development and applications as chiral selectors in various chiral separation techniques. Discussion of their use in hydrophilic liquid interaction chromatography (HILIC) will be limited. Their use in liquid chromatography, especially their improvements with the use of superficially porous particles (SPPs) will be emphasized. Method parameters and future directions are also discussed. Key words Derivatized cyclofructans, Primary amines, Enantiomeric separations, Crown ethers, Isopropyl-cyclofructan, Dimethylphenyl-cyclofructan, R-naphthylethyl-cyclofructan, Sulfated-cyclofructan, Superficially porous particles, Cycloinulohexaose, Cycloinuloheptaose

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Introduction to Cyclofructans Cyclofructans are cyclic oligosaccharides made of β-2,1-linked fructofuranose units. They were first discovered in 1989 by Kawamura and Uchiyama, who produced cyclofructan by fermentation of inulin from a strain of Bacillus circulans (OKUMZ31B) [1]. In 1994, Kushibe et al. reported a different strain of Bacillus circulans (MCI-2554), which led to more efficient cyclofructan production [2]. They have many common uses, such as ion trapping reagents [3–6]. A work on gas-phase chiral recognition of cyclofructans using permethylated-cyclofructan-6 (PM-CF6) and cyclofructan7 (PM-CF7) was reported in 1998 [7]. A direct FAB mass spectrometric approach enabled discrimination between enantiomers of several amino acid esters in a high vacuum. In 2009, the first use of cyclofructans as chiral selectors in LC was demonstrated [8]. The unique structure and synthesis of CF6-based chiral stationary phases (CSPs) as well as their chromatographic performance in terms of enantiomeric separations were demonstrated. Since then, a plethora of separations have been reported with cyclofructan-

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based chiral selectors, not only in LC, but also in GC, CE, SFC, and HILIC. In this chapter, the use of cyclofructan-based chiral selectors in all these chromatographic techniques is considered with an emphasis on their most useful applications.

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Cyclofructan-Based Chiral Stationary Phases for HPLC and SFC

2.1 Bonded Derivatized Cyclofructan Chiral Selectors

Different basic cyclofructans are distinguished by the number of linked fructose units, i.e., 6-linked fructose unit is cycloinulohexaose or cyclofructan-6 (CF6). CF6 has been the most studied cyclofructan chiral selector due to its availability in large amounts and good purity from crystallization [9, 10]. CF7 (cycloinuloheptaose) has become more obtainable due to effective preparative separations [11]. CF8 (cycloinulooctaose) is less accessible. The central macrocycle of CF6 is a natural 18-crown-6 core with the fructose units oriented alternatively around its center with most hydroxy groups aligned on the same side of the macrocycle (Fig. 1) [9–12]. Thus, CF6 has distinct surfaces with one side of the macrocycle being more hydrophilic than the other. Multiple hydroxy groups of the CF6 are close in proximity and form internal hydrogen bonds, folding the molecule and making the typical crownether inclusion complex difficult [12]. Also, cyclofructans do not have a hydrophobic inclusion complex capability like cyclodextrins [13]. Therefore, native cyclofructans had limited success for chiral separations when bonded to a silica support as CSPs [14]. However, they were useful for achiral separations in HILIC (see Subheading 3). Interestingly, derivatized cyclofructans proved to be exceptional for enantiomeric separations. Cyclofructan derivatization was performed at various hydroxy groups, which disrupts their internal hydrogen bonding and allows the molecule to unfold, promoting chiral interactions [8]. Derivatization of native cyclofructan can be performed after or partially

Fig. 1 General cyclofructan structure (cyclofructan-6; n ¼ 1, cyclofructan-7; n ¼ 2, cyclofructan-8; n ¼ 3)

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before bonding it to silica. A set of 14 cyclofructan CSPs were derivatized with various aliphatic or aromatic groups (Fig. 2) [8]. Perhaps, the most apparent result was that aliphatic derivatives had higher enantioselectivity for primary amines compared to the aromatic-functionalized CF6 CSPs [8]. Less enantioselectivity for chiral primary amines was observed with more hydroxy group substitution. These results suggested that the CF6 intramolecular hydrogen bonding was disrupted after partial derivatization, causing a “relaxation” of the molecular structure [8]. Overall, the most universal and highest enantiomeric selectivity for primary amines was with the isopropyl-CF6 (IP-CF6) CSP [8]. When the primary amine groups were farther away from the chiral center or sterically blocked, less enantioselectivity was observed. Further studies were performed to show the broad enantioselectivity from IP-CF6 for primary amines, which is illustrated in Fig. 3 with representative enantiomeric separations [15]. This shows that IP-CF6 has high enantioselectivity for primary amines with other groups present in the compound, such as alcohols, amides, and esters. Chiral analytes without primary amine moieties were far better separated by aromatic-derivatized CF6 CSPs [8]. This broader enantioselectivity was attributed to the increased π–π and dipole–dipole interactions, and steric and hydrogen bonding interactions offered by the aromatic-derivatized CF6 CSPs according to their geometry and size. The initial study tested ten aromatic functionalities that contained various electron-withdrawing groups, like chloro and nitro groups, as well as electron-donating groups such as methyl substituents [8]. Nitro groups were detrimental to enantiomeric separations, while the best selectors were the 3,4 and 4,3 chloro and phenyl moieties. Aromatic functionalities were also tested with CF7, and the most applicable chiral selectors from these studies were found to be R-naphthylethyl-cyclofructan-6 (RN-CF6) and dimethylphenyl-cyclofructan-7 (DMP-CF7) [8, 16]. A variety of chiral analytes were separated with these two CSPs and representative enantiomeric separations of acids, secondary amines, tertiary amines, alcohols, and others are shown in Fig. 4 [16]. Several other separations, including paclitaxel precursor phenylisoserine analogs, phytoalexins, metal complexes, spirobrassinins, illicit drugs, Betti base analogs, chiral catalyst auxiliaries, pharmaceuticals, pentahelicenes, biaryl atropisomers, methionine in supplements, and others, have also been reported [17–28]. Comparisons of cyclofructans to other HPLC CSPs, like cyclodextrins and polysaccharides, have shown that they have unique and enhanced enantioselectivities for many compounds, especially primary amines [29–31]. Reports of new derivatized cyclofructans with chlorinated aromatic functionalities as well as cationic functionalities have further expanded the usefulness of the cyclofructan class of chiral selectors [32, 33].

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Fig. 2 Scheme showing various chemically bonded cyclofructan-based stationary phases. Tested aliphatic derivatizing groups included methyl, ethyl, isopropyl, and tert-butyl isocyanates (ME, ET, IP, and TB). Tested aromatic derivatizing groups included 3,5-dimethylphenyl, 3,5-dichlorophenyl, p-tolyl, 4-chlorophenyl, 3,5-bis

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Fig. 3 Enantiomeric separations of various compounds with primary amine groups by aliphatic isopropylcyclofructan. (a) 2-Amino-1,2-diphenylethanol, (b) amlodipine, (c) tocainide, (d) 4-methyl-α-phenylphenethylamine, (e) 2,20 -diamino-1,10 -binaphthalene, and (f) endo-2-amino norbornane hydrochloride. Conditions for (a) acetonitrile(ACN):methanol(MeOH):acetic acid(HOAc):triethylamine(TEA) (30:70:0.3:0.2, v/v/v/v), 20  C, 1 mL/min; (b) ACN:MeOH:HOAc:TEA (60:40:0.3:0.2, v/v/v/v), 0  C, 1 mL/min; (c) ACN:MeOH:HOAc:TEA (75:25:0.3:0.2, v/v/v/v), 20  C, 1 mL/min; (d) ACN:MeOH:HOAc:TEA (30:70:0.3:0.5, v/v/v/v), 20  C, 1 mL/ min, (e) heptane:ethanol (80:20, v/v), 20  C, 1 mL/min; (f) ACN:MeOH:HOAc:TEA (30:70:0.3:0.2, v/v/v/v), 20  C, 0.5 mL/min (reproduced by permission of Elsevier from ref. 15 © 2010)

ä Fig. 2 (continued) (trifluoromethyl)phenyl, R-1(1-naphthyl)ethyl, S-1-(1-naphthyl)ethyl, and S-α-methylbenzyl isocyanates (DMP, DCP, DTP, MMP, MCP, RN, SN, SMP, DNP, and NTP). All derivatization groups were bonded to the cyclofructan via a carbamate or a thiocarbamate linkage, with the exception of dinitrophenyl and dinitrophenyl-trifluoromethyl groups, which were attached via an ether linkage (reproduced by permission of the American Chemical Society from ref. 8 © 2009)

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Fig. 4 Enantiomeric separations of various compounds by aromatic-derivatized cyclofructans: (a, c, f) 3,5-dimethylphenyl-cyclofructan-7 (DMP-CF7) and (b, d, e) R-1(1-naphthyl)ethyl-cyclofructan-6 (RN-CF6). (a) Dansyl-norleucine cyclohexylammonium salt, heptane(Hep):ethanol(EtOH):trifluoroacetic acid (TFA) (80:20:0.1, v/v/v), 20  C, 1 mL/min; (b) 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline hydrobromide, Hep:EtOH:TFA (60:40:0.1, v/v/v), 20  C, 1 mL/min; (c) Tro¨ger’s base, Hep:EtOH:TFA (80:20:0.1, v/v/v), 20  C, 1 mL/min; (d) benzoin, Hep:isopropyl alcohol:TFA (99:1:0.1, v/v/v), 0  C, 1 mL/min; (e) [Ru(phen)3]Cl2, methanol:acetonitrile:ammonium nitrate (60:40:0.2, v/v/w), 20  C, 1 mL/min; (f) ethyl 11-cyano-9,10-dihydro-endo-9,10-ethanoanthracene-11-carboxylate, Hep:EtOH:TFA (95:5:0.1, v/v/v), 20  C, 1 mL/min (reproduced by permission of the Royal Society of Chemistry from ref. 16 © 2011)

Typically, the most broadly useful class of CSPs for separating chiral primary amines are synthetic chiral crown ether-based stationary phases [34]. However, their applications are mainly restricted exclusively to primary amines and strong acidic, aqueous

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mobile phases are always necessary. All cyclofructan-based CSPs utilize common organic solvents and are highly stable. Column performance was maintained after more than 1000 injections for a primary amine in the polar organic mode (POM), which consisted of acetonitrile, methanol, and a mixture of acidic and basic additives [8]. These columns were also stable at lower temperatures, which increased enantioselectivity at the expense of efficiency. Thus, cyclofructan-based CSPs are also valuable for chiral SFC separation applications, like high-throughput preparative separations, and they have broader enantioselectivity than just primary amines. The high loading of cyclofructan-type phases was demonstrated in their initial report with a baseline separation of 4200 μg of N(3,5-dinitrobenzoyl)-phenylglycine on the RN-CF6 column in the POM [8]. Interactions contributing to retention via a linear free energy relationship were investigated to determine SFC applicability for the RN-CF6 and DMP-CF7 CSPs [35]. Screening and optimization protocols were developed based on previous studies for the enantiomeric separation of several compounds, including derivatized amino acids, other primary amines, and α-aryl ketones using the IP-CF6, RN-CF6, and DMP-CF7 CSPs [35–40]. For neutral and acidic compounds, better resolution was typically obtained in the normal-phase mode (NPM) with ethanol and isopropanol as alcohol modifiers. When testing additives for separation of basic compounds, fronting asymmetric wide peaks were observed with an acidic additive, due to strong interactions between basic analyte and weakly acidic silica-based stationary phase [8]. Basic additives decreased retention and selectivity of basic compounds, possibly due to the competition with basic analytes for the primary interaction sites on the CSP [8]. On-column racemization was also observed for α-aryl ketones when using basic additives, which could be controlled to study their rate of enantiomerization [37]. For the enantiomeric separation of amines in the POM, the optimal ratio and choice of additives was acetic acid and triethylamine (0.3:0.2, v/v) [8, 36]. Similarly, for the NPM, it was trifluoroacetic acid and triethylamine (0.3:0.2, v/v) [8, 36]. Recent studies focused on using these protocols with new column technologies to increase throughput as described in subsequent sections. 2.2 The Future of Superficially Porous Particle-Bonded Cyclofructans

Cyclofructans bonded to SPPs (superficially porous particles) have been shown to provide high-throughput and effective separations of a variety of chiral molecules [41–47]. SPPs decrease all contributions to band broadening (i.e., longitudinal diffusion, eddy dispersion, and resistance to mass transfer) [48–52]. First, the longitudinal diffusion of a solute band is partially dictated by an obstruction factor, which represents the blockage of flow paths by the packed bed. SPPs have an increased obstruction factor due to the solid core, resulting in a lower diffusional contribution

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[48]. The decreased eddy dispersion of a solute is mostly contributed to the better packing homogeneity across the SPP columns (i.e., from wall to center of the column) [49, 50]. Since SPPs have a shorter porous layer path length due to their shell thickness, there is decrease of mass transfer contributions to band broadening [41, 42, 51]. This is mostly seen for large molecules with small diffusion coefficients and smaller molecules that have slow adsorption-desorption kinetics. SPP columns can yield reduced plate heights of 1.3–1.5, compared to FPP columns, which generally have reduced plate heights >2.0 [52]. Native CF6 CSPs were utilized in the HILIC mode, indicating efficiency improvements of 25–65% with the 2.7 μm diameter SPP column (FRULIC-N column) compared to the 3 μm FPP column and efficiency values 2–4 higher than those obtained using the 5 μm FPP column (Fig. 5) [41]. Retention times of analytes on the SPP stationary phase were shorter compared to the FPPs due to the lower loading of the chiral selector, and the morphology of the particles [41]. High-throughput achiral separations, typically of

Fig. 5 Comparison of native CF6 chiral stationary phases bonded with superficially porous particles versus fully porous particles in the HILIC mode. The values on the top of the peaks correspond to the efficiency in terms of number of plates (N) on column. Column dimensions ¼ 150  4.6 mm (i.d.); flow ¼ 0.7 mL/min; temperature ¼ 30  C; injection volume ¼ 0.5 μL. (a) Acetonitrile(ACN):ammonium acetate(NH4OAc) (25 mM) (75:25, v/v); (b) ACN:NH4OAc (25 mM) (85:15, v/v), (c) ACN:NH4OAc (25 mM) (75:25, v/v); (d) ACN:NH4OAc (100 mM) (70:30, v/v). UV detection of 254, 210, 280, and 254 nm, respectively (a–d). The numbers (e.g., 1–4) represent the peaks in order of elution (1 and 4 correspond to the earliest and latest eluted analyte, respectively) (reproduced by permission of Elsevier from ref. 41 © 2014)

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polar analytes, using the HILIC mode with native and derivatized cyclofructan-based SPP CSPs have been reported [53–59]. The use of resins, instead of silica, to bond cyclofructans by “click” chemistry applications has also been noted [60]. Retention and selectivity of these analytes have proved different and unique compared to other HILIC stationary phases. These will not be discussed in detail, but the most prominent separations which have been observed include those of nucleic acids, peptides, xanthines, β-blockers, salicylic acid and its derivatives, and maltooligosaccharides [53–59]. One of the first chiral selectors bonded with SPPs was the isopropyl-cyclofructan-6 (SPP IP-CF6 which was commercialized as LarihcShell-P or LS-P). Enantiomeric separations of amlodipine, NOBIN, 1-(1-naphthyl)ethylamine, and fipronil were evaluated using 2.7 μm diameter SPPs, 5 μm FPP, and 3 μm FPPs bonded to IP-CF6 (Fig. 6) [43]. SPPs, compared to FPPs, provided similar selectivity, but much smaller retention factors and higher resolutions. Similarly, comparisons of 2,20 -bis(diphenylphosphinoamino)-1,10 -binaphthyl (BINAM) and α-aryl ketones have been made [37, 43]. LS-P was further utilized in a comprehensive enantiomeric separation report of 150 amines [47]. In this study, 95% of

Fig. 6 Enantiomeric separation comparisons of (a) amlodipine, (b) 1-(1-naphthyl)ethylamine, (c) 20 -amino1,10 -binaphthalen-2-ol (NOBIN), and (d) fipronil with isopropyl-cyclofructan-6 chiral stationary phases bonded with superficially porous particles (SPPs) versus fully porous particles (FPPs). Column dimensions: 150  4.6 mm (i.d.); column 1: 5 μm FPPs; column 2: 3 μm FPPs; column 3: 2.7 μm SPPs. Experimental conditions: (a) acetonitrile (ACN):methanol (MeOH):acetic acid (HOAc):trimethylamine (TEA) (80:20:0.3:0.2, v/v/v/v); (b) ACN:MeOH:HOAc:TEA (60:40:0.3:0.2, v/v/v/v); (c, d) heptane:ethanol (95:5, v/v) (reproduced by permission of Elsevier from ref. 42 © 2014)

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Fig. 7 Enantiomeric separations of catecholamines with primary amine groups: (a) octopamine, (b) normetanephrine, (c) norphenylephrine, and (d) norepinephrine using LarihcShell-P® (100  4.6 mm (i.d.), 2.7 μm particle size. Experimental conditions: (a–c) acetonitrile(ACN):methanol(MeOH):acetic acid(HOAc):triethylamine (TEA) (60:40:0.3:0.2, v/v/v/v), 25  C, 1 mL/min; (d) ACN:MeOH:HOAc:TEA (90:10:0.3:0.2, v/v/v/v), 45  C, 1 mL/min. UV detection at 254 nm (reproduced by permission of Elsevier from ref. 47 © 2018)

the primary amines, including pharmaceuticals, stimulants, reagents, and amino acids, were baseline separated by LS-P, most with a resolution of 2.0–2.5 [47]. Enantiomeric separations of catecholamines with primary amine groups are shown in Fig. 7 [47]. Most of these separations were achieved in 5 min. Ultrafast chiral separations of fluorinated active pharmaceutical ingredients and their desfluoro impurities have also been reported [45]. Further, ultrafast chiral separations in 10–20 s have been achieved for BINAM and 2-chloro-indan-1-ylamine using DMP-CF7 bonded to SPPs and LS-P (Fig. 8) [43]. Overall, high-throughput screening of compounds with primary amine groups using cyclofructanbased SPP CSPs has been highly successful. While the most universal application of the isopropyl-CF6 (LarihcShell-P) is for primary amines, it is well known that the other derivatized cyclofructans have different mechanisms that provide enantioselectivity for other neutral and acidic compounds. Separation reports of compounds other than primary amines for LS-P are fewer but do exist. For instance, Tro¨ger’s base, which has no primary amine moiety, was separated by the IP-CF6 column [8].

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Fig. 8 Ultrafast enantiomeric separations of (a) 2,20 -bis(diphenylphosphinoamino)-1,10 -binaphthyl (BINAM) and (b) 2-chloro-indan-1-ylamine using cyclofructan-based chiral selectors bonded to superficially porous particles (SPPs). Experimental conditions: (a) DMP-CF7 SPP (30  4.6 mm (i.d.)), heptane:ethanol 90:10 (v/v), 4.80 mL/min, 22  C; (b) SPP IP-CF6 (LarihcShell-P, LS-P) (100  4.6 mm (i.d.)), acetonitrile:methanol: trifluoroacetic acid:triethylamine 70:30:0.3:0.2 (v/v/v/v), 4.50 mL/min, 22  C (reproduced by permission of the American Chemical Society from ref. 43 © 2015)

Fig. 9 Enantiomeric separations of compounds without primary amine groups: (a) bicalutamide, (b) closantel, (c) famoxadone using LarihcShell-P (100  4.6 mm (i.d.), 2.7 μm particle size). Experimental conditions: (a) hexane (Hex):ethanol (EtOH):trifluoroacetic acid (TFA):trimethylamine (TEA) (80:20:0.3:0.2:, v/v/v/v), 0.8 mL/ min; (b) heptane(Hep):EtOH:TFA:TEA (80:20:0.3:0.2, v/v/v/v), 0.8 mL/min; (c) Hex:isopropyl alcohol(95:5, v/v), 1.0 mL/min. UV detection at 254 nm. Chromatograms (b) and (c) adapted from ref. 61

Recently, pesticides like closantel and famoxadone were baseline separated using LS-P (Fig. 9) [61]. The prostate cancer drug, bicalutamide, was also separated by LS-P (Fig. 9). These three compounds have no primary amine groups and were all separated with normal-phase solvents. These applications demonstrate that the isopropyl-derivatized cyclofructan-6 (LarihcShell-P) has broader enantioselectivity than just for primary amines. It seems that future studies should focus on identifying these mechanisms so that users can predict where they might apply. In the meantime, faster, more effective separations using other cyclofructans than the isopropyl-derivatized cyclofructan-6 will most likely be achieved with the use of superficially porous particles.

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CE and GC Applications of Cyclofructans Cyclofructans were first introduced as valuable chiral selectors for CE in 2009, exhibiting low UV absorption, high solubility in water, and minimal wall interactions [62]. Enantioselectivities of 110 primary, secondary, tertiary, and quaternary amines were compared between native cyclofructans 6 and 7 and their corresponding sulfated derivatives. 82% of these amines were separated, with 66 out of 110 amine baseline separated by one or both sulfated cyclofructans [62]. A representation of the enantiomeric separation of four basic pharmaceuticals is shown in Fig. 10 [63]. Minimal interactions were observed between native cyclofructans and amine compounds in aqueous buffers for CE, but sulfated cyclofructans bind basic, cationic analytes strongly. The strength of this electrostatic interaction was dependent on pH and was found to also contribute to the chiral separation of basic analytes, along with

Fig. 10 Enantiomeric separations of pharmaceutical amines (tamsulosin, tiropramide, bupivacaine, and norephedrine) with capillary electrophoresis (CE) using sulfated cyclofructan-6 (S-CF6). Capillary column dimensions: 50 μm i.d., 365 μm o.d., total length ¼ 48.5 cm (effective length ¼ 40 cm); running buffer: 100 mM phosphoric acid þ Tris þ 0.7% (w/v) S-CF6, pH 2.5; injection: 50 mbar for 5 s; temperature: 15  C; voltage: þ25 kV; UV detection: 200 nm (reproduced by permission of John Wiley & Sons from ref. 63 © 2013)

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hydrogen bonding interactions. Normal and reversed polarity modes can be used with sulfated cyclofructan, but the reversed polarity mode usually produced electropherograms with better peak shapes. Compared to conventional chiral selectors like crown ethers and sulfated cyclodextrins, sulfated derivatized cyclofructans had better enantioselectivity for cationic analytes, and not only primary amines [63–66]. Since it is well known that cyclofructans complex with metals, several studies have focused on investigating their binding patterns [67–71]. The IP-CF6 was studied as a CE additive and separated atropisomers of R,S-1,10 -binaphthalene2,20 -diyl hydrogen phosphate (R,S-BNP) with the addition of Ba2 + to the background electrolyte (BGE) (Fig. 11) [72]. Increased migration time and resolution were observed with higher concentrations of Ba2+, most likely due to increased electrostatic interactions between the cationic charged IP-CF6 complex with Ba2+ and the anionic phosphate groups of R,S-BNP [72]. Further studies with 1,10 -binaphthyl-2,20 -diyl hydrogen phosphate (BHP) confirmed this behavior and other ions were investigated, like Pb2+ [66]. Chiral separations of sulfonic and phosphoric acids with barium-complexed derivatized CF6 stationary phases in HPLC were also evaluated [73]. In these studies, the nature of the barium counteranion was examined and the elution strength was observed as acetate > methanesulfonate > trifluoroacetate > perchlorate [73]. Additionally, chiral ionic liquids, like D-alanine tert.-butyl ester lactate, have been mixed in the BGE with cyclofructan-based

Fig. 11 Effect of barium acetate addition to the background electrolyte (BGE) on enantiomeric separation of R, S-BNP (1,10 -binaphthalene-2,20 -diyl hydrogen phosphate). Capillary dimensions: 50 μm i.d., 365 μm o.d., total length ¼ 33 cm (24.5 cm to the detector); running buffer: 100 mM sodium borate, pH 10.0, with addition of 20 mM isopropyl-cyclofructan-6, voltage: þ15 kV, injection: 50 mbar for 5 s, temperature: 25  C; UV detection: 214 nm (reproduced by permission of Elsevier from ref. 72 © 2014)

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additives, which increased enantiomeric resolution in some cases [74]. Overall, cyclofructans have been evaluated as competitive CE additives, and further applications of their enhanced enantioselectivity compared to traditional additives remain to be investigated. In GC, limited chiral separation reports using cyclofructans have been made, since cyclodextrins dominate chiral GC [75]. The first use of cyclofructans in GC was reported in 2010, with the evaluations of per-O-methylated CF6 and CF7 as well as 4,6-di-O-pentyl CF6 [76]. Native cyclofructans have high melting points and are not soluble in other liquid stationary phases so they are not useful for GC. However, when derivatized, they can be dissolved into an achiral matrix and made suitable. Enantiomers of esters, β-lactams, alcohols, and amino acid derivatives were separated [76]. Chiral separations using 4,6-di-O-pentyl-3-O-trifluoroacetyl and 4,6-di-O-pentyl-3-O-propionyl CF6s were performed, which included 47 enantiomeric separations of derivatized amino acids, amino alcohols, amines, alcohols, tartrates, and lactones [77]. Significant advantages over traditional cyclodextrins have not been reported, but comprehensive knowledge concerning derivatized cyclofructans for chiral GC remains to be determined.

4

Conclusions Native cyclofructans are most effective in HILIC, but when derivatized they become powerful selectors for chiral separation techniques. The three cyclofructans: isopropyl-cyclofructan-6, Rnaphthylethyl-cyclofructan-6, and dimethylphenyl-cyclofructan-7 have been the most applicable of all tested derivatives in HPLC. They can operate in common organic solvents, overcoming limitations of synthetic crown-ether stationary phases for primary amine enantiomeric separations. Also, the cyclofructan-based CSPs are not exclusive to the separation of primary amines. The R-naphthylethyl-cyclofructan-6 and the dimethylphenyl-cyclofructan-7 have great enantioselective capabilities for metal complexes. Since they are most selective in normal-phase solvents, they are also useful for SFC. Sulfated cyclofructans have demonstrated the most success in CE sometimes with the use of additive salts. Research is more limited for cyclofructan-based chiral selectors in GC, but they do show differences in terms of enantioselectivity to conventional chiral stationary phases. Recent applications have focused on the use of isopropyl-cyclofructan-6 with new superficially porous particles, emphasizing its high and near universal enantioselectivity for chiral primary amines. However, new applications using cyclofructan-based chiral selectors remain to be discovered, and their continued success in chiral separations will likely expand.

Cyclofructans as Chiral Selectors

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References 1. Kawamura M, Uchiyama T, Kuramoto T, Tamura Y, Mizutani K (1989) Enzymic formation of a cycloinulo-oligosaccharide from inulin by an extracellular enzyme of Bacillus circulans OKUMZ 31B. Carbohydr Res 192:83–90 2. Kushibe S, Sashida R, Morimoto Y (1994) Production of cyclofructan from inulin by Bacillus circulans MCI-2554. Biosci Biotechnol Biochem 58:1136–1138 3. Yoshie N, Hamada H, Takada S, Inoue Y (1993) Complexation of cycloinulonexaose with some metal ions. Chem Lett 22:353–356 4. Shizuma M, Takai Y, Kawamura M, Takeda T, Sawada M (2001) Complexation characteristics of permethylated cycloinulohexaose, cycloinuloheptaose, and cycloinulooctaose with metal cations. J Chem Soc Perkin Trans 2:1306–1314 5. Takai Y, Okumura Y, Tanaka T, Sawada M, Takahashi S, Shiro M, Kawamura M, Uchiyama T (1994) Binding characteristics of a new host family of cyclic oligosaccharides from inulin: permethylated cycloinulohexoase and cycloinuloheptaose. J Org Chem 59:2967–2975 6. Uchiyama T, Kawamura M, Uragami T, Okuno H (1993) Complexing of cycloinulooligosaccharides with metal ions. Carbohydr Res 241:245–248 7. Sawada M, Takai Y, Shizuma M, Takeda T, Adachi H, Uchiyama T (1998) Measurement of chiral amino acid discrimination by cyclic oligosaccharides: a direct FAB mass spectrometric approach. Chem Commun 14:1453–1454 8. Sun P, Wang C, Breitbach ZS, Armstrong DW (2009) Development of new chiral stationary phases based on native and derivatized cyclofructans. Anal Chem 81:10215–10226 9. Sawada M, Tanaka T, Takai Y, Hanafusa T, Hirotsu K, Higuchi T, Kawamura M, Uchiyama T (1990) Crystal structure of cycloinulohexaose. Chem Lett 19:2011–2014 10. Sawada M, Tanaka T, Takai Y, Hanafusa T, Taniguchi T, Kawamura M, Uchiyama T (1991) The crystal structure of cycloinulohexaose produced from inulin by cycloinulooligosaccharide fructanotransferase. Carbohydr Res 217:7–17 11. Wang C, Breitbach ZS, Armstrong DW (2010) Separations of cycloinulooligosaccharides via hydrophilic interaction chromatography (HILIC) and ligand-exchange chromatography. Sep Sci Technol 45:447–452

12. Immel S, Schmitt GE, Lichtenthaler FW (1998) Cyclofructins with six to ten β-(1!2)linked fructo-furanose units: geometries, electrostatic profiles, lipophilicity patterns, and potential for inclusion complexation. Carbohydr Res 313:91–105 13. Armstrong DW, DeMond W (1984) Cyclodextrin bonded phases for the liquid chromatographic separation of optical geometrical, and structural isomers. J Chromatogr Sci 22:411–415 14. Wang C, Sun P, Armstrong DW (2010) Cyclofructans, a new class of chiral stationary phases. In: Berthod A (ed) Chiral recognition in separation methods. Springer, Heidelberg 15. Sun P, Armstrong DW (2010) Effective enantiomeric separations of racemic primary amines by the isopropyl carbamate-cyclofructan6 chiral stationary phase. J Chromatogr A 1217:4904–4918 16. Sun P, Wang C, Padivitage NLT, Nanayakkara YS, Perera S, Qiu H, Zhang Y, Armstrong DW (2011) Evaluation of aromatic-derivatized cyclofructans 6 and 7 as HPLC chiral selectors. Analyst 136:787–800 ´ , Ilisz I, Pataj Z, Fu¨lo¨p F, 17. Aranyi A, Bagi A Armstrong DW, Pe´ter A (2012) Highperformance liquid chromatographic enantioseparation of amino compounds on newly developed cyclofructan-based chiral stationary phases. J Sep Sci 35:617–624 18. Gondova´ T, Petrovaj J, Kutschy P, Armstrong DW (2013) Stereoselective separation of spiroindoline phytoalexins on R-naphthylethyl cyclofructan 6-based chiral stationary phase. J Chromatogr A 1272:100–105 19. Hrobonova K, Moravcik J, Lehotay J, Armstrong DW (2015) Determination of methionine enantiomers by HPLC on the cyclofructan chiral stationary phase. Anal Methods 7:4577–4582 20. Ilisz I, Grecso´ N, Forro´ E, Fu¨lo¨p F, Armstrong DW, Pe´ter A (2015) High-performance liquid chromatographic separation of paclitaxel intermediate phenylisoserine derivatives on macrocyclic glycopeptide and cyclofructan-based chiral stationary phases. J Pharm Biomed Anal 114:312–320 21. Majek P, Krupcik J, Breitbach ZS, Dissanayake MK, Kroll P, Ruch AA, Slaughter LM, Armstrong DW (2017) Determination of the interconversion energy barrier of three novel pentahelicene derivative enantiomers by dynamic high resolution liquid

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chromatography. J Chromatogr B Analyt Technol Biomed Life Sci 1051:60–67 22. Moravcˇ´ık J, Hrobonˇova´ K (2013) Highperformance liquid chromatographic method for enantioseparation of underivatized α-amino acids using cyclofructan-based chiral stationary phases. Nova Biotechnol Chim 12:108–119 23. Moskalˇova´ M, Kozlov O, Gondova´ T, Budovska´ M, Armstrong DW (2017) HPLC enantioseparation of novel spirobrassinin analogs on the cyclofructan chiral stationary phases. Chromatographia 80:53–62 24. Moskalˇova´ M, Petrovaj J, Gondova´ T, Budovska´ M, Armstrong DW (2016) Enantiomeric separation of new phytoalexin analogs with cyclofructan chiral stationary phases in normal-phase mode. J Sep Sci 39:3669–3676 25. Padivitage NLT, Dodbiba E, Breitbach ZS, Armstrong DW (2014) Enantiomeric separations of illicit drugs and controlled substances using cyclofructan-based (LARIHC) and cyclobond I 2000 RSP HPLC chiral stationary phases. Drug Test Anal 6:542–551 26. Woods RM, Patel DC, Lim Y, Breitbach ZS, Gao H, Keene C, Li G, La´szlo´ K, Armstrong DW (2014) Enantiomeric separation of biaryl atropisomers using cyclofructan based chiral stationary phases. J Chromatogr A 1357:172–181 27. Qiu H, Padivitage NLT, Frink LA, Armstrong DW (2013) Enantiomeric impurities in chiral catalysts, auxiliaries, and synthons used in enantioselective syntheses. Part 4. Tetrahedron Asymmetry 24:1134–1141 28. Shu Y, Breitbach ZS, Dissanayake MK, Perera S, Aslan JM, Alatrash N, MacDonnell FM, Armstrong DW (2015) Enantiomeric separations of ruthenium (II) polypyridyl complexes using HPLC with cyclofructan chiral stationary phases. Chirality 27:64–70 29. Kalı´kova´ K, Janecˇkova´ L, Armstrong DW, Tesarˇova´ E (2011) Characterization of new R-naphthylethyl cyclofructan 6 chiral stationary phase and its comparison with R-naphthylethyl β-cyclodextrin-based column. J Chromatogr A 1218:1393–1398 30. Lim Y, Breitbach ZS, Armstrong DW, Berthod A (2016) Screening primary racemic amines for enantioseparation by derivatized polysaccharide and cyclofructan columns. J Pharm Anal 6:345–355 31. Vozka J, Kalı´kova´ K, Janecˇkova´ L, Armstrong DW, Tesarˇova´ E (2012) Chiral HPLC separation on derivatized cyclofructan versus cyclodextrin stationary phases. Anal Lett 45:2344–2358

32. Khan MM, Breitbach ZS, Berthod A, Armstrong DW (2016) Chlorinated aromatic derivatives of cyclofructan 6 as HPLC chiral stationary phases. J Liq Chromatogr R T 39:497–503 33. Padivitage NL, Smuts JP, Breitbach ZS, Armstrong DW, Berthod A (2015) Preparation and evaluation of HPLC chiral stationary phases based on cationic/basic derivatives of cyclofructan 6. J Liq Chromatogr R T 38:550–560 34. Hilton M, Armstrong DW (1991) Evaluation of a crown etheric column for the separation of racemic amines. J Liq Chromatogr 14:9–28 35. Janecˇkova´ L, Kalı´kova´ K, Vozka J, Armstrong DW, Bosa´kova´ Z, Tesarˇova´ E (2011) Characterization of cyclofructan-based chiral stationary phases by linear free energy relationship. J Sep Sci 34:2639–2644 36. Woods RM, Breitbach ZS, Armstrong DW (2014) Comparison of enantiomeric separations and screening protocols for chiral primary amines by SFC and HPLC. LCGC N Am 32:742–745 37. Breitbach AS, Lim Y, Xu QL, Ku¨rti L, Armstrong DW, Breitbach ZS (2016) Enantiomeric separations of α-aryl ketones with cyclofructan chiral stationary phases via high performance liquid chromatography and supercritical fluid chromatography. J Chromatogr A 1427:45–54 38. Geryk R, Vozka J, Kalı´kova´ K, Tesarˇova´ E (2013) HPLC method for chiral separation and quantification of antidepressant citalopram and its precursor citadiol. Chromatographia 76:483–489 39. Kalı´kova´ K, Sˇlechtova´ T, Vozka J, Tesarˇova´ E (2014) Supercritical fluid chromatography as a tool for enantioselective separation; a review. Anal Chim Acta 821:1–33 40. Vozka J, Kalı´kova´ K, Roussel C, Armstrong DW, Tesarˇova´ E (2013) An insight into the use of dimethylphenyl carbamate cyclofructan 7 chiral stationary phase in supercritical fluid chromatography: the basic comparison with HPLC. J Sep Sci 36:1711–1719 41. Dolzan MD, Spudeit DA, Breitbach ZS, Barber WE, Micke GA, Armstrong DW (2014) Comparison of superficially porous and fully porous silica supports used for a cyclofructan 6 hydrophilic interaction liquid chromatographic stationary phase. J Chromatogr A 1365:124–130 42. Spudeit DA, Dolzan MD, Breitbach ZS, Barber WE, Micke GA, Armstrong DW (2014) Superficially porous particles vs. fully porous particles for bonded high performance liquid chromatographic chiral stationary phases:

Cyclofructans as Chiral Selectors isopropyl cyclofructan 6. J Chromatogr A 1363:89–95 43. Patel DC, Breitbach ZS, Wahab MF, Barhate CL, Armstrong DW (2015) Gone in seconds: praxis, performance, and peculiarities of ultrafast chiral liquid chromatography with superficially porous particles. Anal Chem 87:9137–9148 44. Patel DC, Wahab MF, Armstrong DW, Breitbach ZS (2016) Advances in high-throughput and high-efficiency chiral liquid chromatographic separations. J Chromatogr A 1467:2–18 45. Barhate CL, Breitbach ZS, Pinto EC, Regalado EL, Welch CJ, Armstrong DW (2015) Ultrafast separation of fluorinated and desfluorinated pharmaceuticals using highly efficient and selective chiral selectors bonded to superficially porous particles. J Chromatogr A 1426:241–247 46. Barhate CL, Joyce LA, Makarov AA, Zawatzky K, Bernardoni F, Schafer WA, Armstrong DW, Welch CJ, Regalado EL (2017) Ultrafast chiral separations for high throughput enantiopurity analysis. Chem Commun 53:509–512 47. Hellinghausen G, Roy D, Lee JT, Wang Y, Weatherly CA, Lopez DA, Nguyen KA, Armstrong JD, Armstrong DW (2018) Effective methodologies for enantiomeric separations of 150 pharmacology and toxicology related 1 , 2 , and 3 amines with core-shell chiral stationary phases. J Pharm Biomed Anal 155:70–81 48. Broeckhoven K, Cabooter D, Desmet G (2013) Kinetic performance comparison of fully and superficially porous particles with sizes ranging between 2.7 μm and 5 μm: intrinsic evaluation and application to a pharmaceutical test compound. J Pharm Anal 3:313–323 49. Bruns S, Stoeckel D, Smarsly BM, Tallarek UJ (2012) Influence of particle properties on the wall region in packed capillaries. J Chromatogr A 1268:53–63 50. Gritti F, Farkas T, Heng J, Guiochon G (2011) On the relationship between band broadening and the particle-size distribution of the packing material in liquid chromatography: theory and practice. J Chromatogr A 1218:8209–8221 51. Gritti F, Guiochon G (2012) Facts and legends about columns packed with sub-3-μm coreshell particles. LCGC N Am 30:586–595 52. DeStefano JJ, Langlois TJ, Kirkland JJ (2008) Characteristics of superficially-porous silica particles for fast HPLC: some performance comparisons with sub-2-microm particles. J Chromatogr Sci 46:254–260

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53. Qiu H, Loukotkova´ L, Sun P, Tesarˇova´ E, Bosa´kova´ Z, Armstrong DW (2011) Cyclofructan 6 based stationary phases for hydrophilic interaction liquid chromatography. J Chromatogr A 1218:270–279 54. Shu Y, Lang JC, Breitbach ZS, Qiu H, Smuts JP, Kiyono-Shimobe M, Yasuda M, Armstrong DW (2015) Separation of therapeutic peptides with cyclofructan and glycopeptide based columns in hydrophilic interaction liquid chromatography. J Chromatogr A 1390:50–61 55. Wang Y, Wahab MF, Breitbach ZS, Armstrong DW (2016) Carboxylated cyclofructan 6 as a hydrolytically stable high efficiency stationary phase for hydrophilic interaction liquid chromatography and mixed mode separations. Anal Methods 8:6038–6045 56. Padivitage NLT, Dissanayake MK, Armstrong DW (2013) Separation of nucleotides by hydrophilic interaction chromatography using the FRULIC-N column. Anal Bioanal Chem 405:8837–8848 57. Padivitage NLT, Armstrong DW (2011) Sulfonated cyclofructan 6 based stationary phase for hydrophilic interaction chromatography. J Sep Sci 34:1636–1647 58. Eastwood H, Xia F, Lo MC, Zhou J, Jordan JB, McCarter J, Barnhart WW, Gahm KH (2015) Development of a nucleotide sugar purification method using a mixed mode column & mass spectrometry detection. J Pharm Biomed Anal 115:402–409 59. Kozlı´k P, Sˇ´ımova´ V, Kalı´kova´ K, Bosa´kova´ Z, Armstrong DW, Tesarˇova´ E (2012) Effect of silica gel modification with cyclofructans on properties of hydrophilic interaction liquid chromatography stationary phases. J Chromatogr A 1257:58–65 60. Qiu H, Kiyono-Shimobe M, Armstrong DW (2014) Native/derivatized cyclofructan 6 bound to resins via “click” chemistry as stationary phases for achiral/chiral separations. J Liq Chromatogr R T 37:2302–2326 61. Hellinghausen G, Readel ER, Wahab MF, Lee JT, Lopez DA, Weatherly CA, Armstrong DW (2019) Mass spectrometry compatible enantiomeric separations of 100 pesticides using coreshell chiral stationary phases and evaluation of iterative curve fitting models for overlapping peaks. Chromatographia 82(1):221–233 62. Jiang C, Tong MY, Breitbach ZS, Armstrong DW (2009) Synthesis and examination of sulfated cyclofructans as a novel class of chiral selectors for CE. Electrophoresis 30:3897–3909 63. Zhang YJ, Huang MX, Zhang YP, Armstrong DW, Breitbach ZS, Ryoo JJ (2013) Use of

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sulfated cyclofructan 6 and sulfated cyclodextrins for the chiral separation of four basic pharmaceuticals by capillary electrophoresis. Chirality 25:735–742 64. Weatherly CA, Na YC, Nanayakkara YS, Woods ˆ , Armstrong DW RM, Sharma A, Lacour JO (2014) Reprint of: enantiomeric separation of functionalized ethano-bridged Tro¨ger bases using macrocyclic cyclofructan and cyclodextrin chiral selectors in high-performance liquid chromatography and capillary electrophoresis with application of principal component analysis. J Chromatogr B Analyt Technol Biomed Life Sci 968:40–48 65. Na YC, Berthod A, Armstrong DW (2015) Cation-enhanced capillary electrophoresis separation of atropoisomer anions. Electrophoresis 36:2859–2865 66. Prˇibylka A, Sˇvidrnoch M, Tesarˇova´ E, Armstrong DW, Maier V (2016) The empirical comparison of cyclofructans and cyclodextrins as chiral selectors in capillary electrophoretic separation of atropisomers of R,S-1,10 -binaphthalene-2,20 -diyl hydrogen phosphate. J Sep Sci 39:973–979 67. Reijenga JC, Verheggen TPEM, Chiari M (1999) Use of cyclofructan as a potential complexing agent in capillary electrophoresis. J Chromatogr A 838:111–119 68. Wang C, Yang SH, Wang J, Kroll P, Schug KA, Armstrong DW (2010) Study of complexation between cyclofructans and alkali metal cations by electrospray ionization mass spectrometry and density functional theory calculations. Int J Mass Spectrom 291:118–124 69. Wang L, Chai Y, Sun C, Armstrong DW (2012) Complexation of cyclofructans with transition metal ions studied by electrospray ionization mass spectrometry and collisioninduced dissociation. Int J Mass Spectrom 323–324:21–27

70. Wang L, Li C, Yin Q, Zeng S, Sun C, Pan Y, Armstrong DW (2015) Construction the switch binding pattern of cyclofructan 6. Tetrahedron 71:3447–3452 71. Wang L, Li Y, Yao L, Sun C, Zeng S, Pan Y (2014) Evaluation and determination of the cyclofructans-amino acid complex binding pattern by electrospray ionization mass spectrometry. J Mass Spectrom 49:1043–1049 72. Maier V, Kalı´kova´ K, Prˇibylka A, Vozka J, Smuts J, Sˇvidrnoch M, Sˇevcˇ´ık J, Armstrong DW, Tesarˇova´ E (2014) Isopropyl derivative of cyclofructan 6 as chiral selector in liquid chromatography and capillary electrophoresis. J Chromatogr A 1338:197–200 73. Smuts JP, Hao XQ, Han Z, Parpia C, Krische MJ, Armstrong DW (2014) Enantiomeric separations of chiral sulfonic and phosphoric acids with barium-doped cyclofructan selectors via an ion interaction mechanism. Anal Chem 86:1282–1290 74. Stavrou IJ, Breitbach ZS, KapnissiChristodoulou CP (2015) Combined use of cyclofructans and an amino acid ester-based ionic liquid for the enantioseparation of huperzine A and coumarin derivatives in CE. Electrophoresis 36:3061–3068 75. Xie SM, Yuan LM (2017) Recent progress of chiral stationary phases for separation of enantiomers in gas chromatography. J Sep Sci 40:124–137 76. Zhang Y, Breitbach ZS, Wang C, Armstrong DW (2010) The use of cyclofructans as novel chiral selectors for gas chromatography. Analyst 135:1076–1083 77. Zhang Y, Armstrong DW (2011) 4,6-Di-Opentyl-3-O- trifluoroacetyl/propionyl cyclofructan stationary phases for gas chromatographic enantiomeric separations. Analyst 136:2931–2940

Chapter 12 High-Performance Liquid Chromatography Enantioseparations Using Macrocyclic Glycopeptide-Based Chiral Stationary Phases: An Overview Istva´n Ilisz, Tı´mea Orosz, and Antal Pe´ter Abstract Since their introduction by Daniel W. Armstrong in 1994, antibiotic-based chiral stationary phases have proven their applicability for the chiral resolution of various types of racemates. The unique structure of macrocyclic glycopeptides and their large variety of interactive sites (e.g., hydrophobic pockets, hydroxy, amino and carboxyl groups, halogen atoms, aromatic moieties) are the reasons for their wide-ranging selectivity. The commercially available Chirobiotic™ phases, which display complementary characteristics, are capable of separating a broad variety of enantiomeric compounds with good efficiency, good column loadability, high reproducibility, and long-term stability. These are the major reasons for the frequent use of macrocyclic antibiotic-based stationary phases in HPLC enantioseparations. This overview chapter provides a brief summary of general aspects of antibiotic-based chiral stationary phases including their preparation and their application to direct enantioseparations of various racemates focusing on the literature published since 2004. Key words Chirality, Enantiomer, High-performance liquid chromatography, Chiral stationary phases, Direct separation, Macrocyclic glycopeptide antibiotics

1

Introduction Since their introduction by Armstrong and coworkers [1, 2] macrocyclic glycopeptides have been shown to be an exceptionally useful class of chiral selectors for the separation of enantiomers of biological and pharmacological importance by HPLC, thin-layer chromatography, capillary electrophoresis (CE), and capillary electrochromatography (CEC). Macrocyclic antibiotics possess several characteristics that allow them to interact with analytes and serve as chiral selectors. There are hundreds of these compounds and, unlike other classes of chiral selectors, they comprise a large variety of structural types. However, only a few appear to be effective as chiral stationary phases (CSPs). The glycopeptides teicoplanin, ristocetin A, and vancomycin have been extensively used as chiral

Gerhard K. E. Scriba (ed.), Chiral Separations: Methods and Protocols, Methods in Molecular Biology, vol. 1985, https://doi.org/10.1007/978-1-4939-9438-0_12, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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selectors in the form of chiral phases in HPLC. CSPs based on these glycopeptides have been commercialized under the trademark Chirobiotic™ by Astec and recently by Sigma-Aldrich. In the past two decades these CSPs have had a rapid and significant impact in the field of enantioseparations. This chapter presents HPLC applications and does not discuss analogous procedures and applications by chiral thin-layer chromatography, supercritical fluid chromatography, CE, or CEC. Focus is given to the literature published since 2004. For earlier publications the reader is referred to a comprehensive review published in the first edition of Chiral Separations, Methods and Protocols [3], as well as further numerous reviews, monographs, and book chapters on this topic [4–21].

2

General Issues of Macrocyclic Glycopeptides There are hundreds of macrocyclic antibiotics described in the literature which, unlike other classes of chiral selectors, comprise a large structural variety. In general, these compounds have molecular masses greater than 600 but less than 2200. There are acidic, basic, and neutral derivatives. The macrocyclic antibiotics used for chiral separations in HPLC include ansamycins (rifamycins), glycopeptides (avoparcin, teicoplanin, ristocetin A, vancomycin and their analogs), and the polypeptide antibiotic thiostrepton. Selected physicochemical properties of the most important macrocyclic antibiotics applied in HPLC enantioseparations are listed in Table 1, and their molecular structures are depicted in Figs. 1 and 2.

3

Chiral Recognition Mechanism Enantioseparation achieved by macrocyclic antibiotic-based CSPs may be possible via several different mechanisms, including inclusion into the hydrophobic pocket, π–π complexation, dipole stacking, hydrogen bonding, electrostatic and short-distance van der Waals interactions, steric effects, or combinations thereof. According to the generally accepted three-point model, chiral recognition requires a minimum of three simultaneous interactions between a selector and a selectand where at least one of the interactions is stereochemically dependent. The key step in chiral recognition is the ability of the selector to interact differently with the enantiomers. Transient diastereomeric complexes formed between selector and selectands with different physical and chemical properties result in an enantioseparation. There are probably several mechanisms depending on the nature of the analyte and the mode of chromatography. Obviously, solvent selection determines whether π–π interaction, H-bonding, hydrophobic interactions, etc.

0

0

1

0

1

1

Nocardia mediterranei

4

0

0

1

1

1

1

Nocardia mediterranei

[14]

Produced by

References

[14]

0

5

0

Own calculations

a

1 2

1 2

0

[14]

Streptomyces candidus

0

0

1

6

1

2

16

5

3 7

32

[14, 22]

Actinoplanes teichomycetius

0

0

1

8

0

1

14

3

4 7

23

1

Number of. . .

0

9

0

9

Hydrophobic tail

Asymmetric centers Macrocycles Aromatic rings Sugar moieties Hydroxy groups Primary amines Secondary amines Amido groups Carboxylic groups Methoxy groups Methyl esters

1877

α ¼ 1908 β ¼ 1943

755

Molecular weight

698

Teicoplanin A2–2

Avoparcin

Rifamycin B

Properties

Rifamycin SV

Glycopeptides

Ansamycins

[14]

Nonomuraea ATCC 39727

0

0

2

7

1

0

11

2

a

Synthetic compound

Synthetic compound a

0

0

1

8

1

0

11

2

4 7

18

2

1817

Dalbavancin

0

0

0

8

1

0

12

2

4 7

2 18

1

1789

Teicoplanin MDL 63,246

B0 ¼ 19 B1 ¼ 18 4 7

B0 ¼ 1732 B1 ¼ 1718

Teicoplanin A-40,926

[14]

Synthetic compound

0

0

1

6

0

1

7

0

4 7

8

0

1197

Teicoplanin aglycone

Table 1 Comparison of the physicochemical properties of macrocyclic antibiotics as potential chiral selectors

[14, 22]

Nocardia lurida

1

0

0

6

0

2

21

6

4 7

38

0

2066

Ristocetin A

[14, 22]

Streptomyces orientalis

0

0

1

7

1

1

9

2

3 5

18

0

1449

Vancomycin

[14]

Streptomyces orientalis

0

0

1

7

0

2

9

2

3 5

18

0

1435

[23, 24]

Amycolatopsis orientalis

0

0

1

7

0

3

9

3

3 5

22

0

1558

Norvancomycin Eremomycin

[25–27]

Amycolatopsis balhimycina

0

0

1

7

1

1

8

2

3 5

17

0

1446

Balhimycin

14

Streptomyces azureus

0

0

0

11

1

0

5

0

2 1

17

0

1665

Thiostrepton

Polypeptides

Istva´n Ilisz et al.

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OH

HO

OH CH3

O O



R=H



R = Cl

O

H2N

NH

O

O

OH O

HO HO

O

Cl

Cl

OH HN

NH

N H O

O

O

HN

O

O

OH

O

HO

Cl

NH

HN

OH

O

OH

O

NH

HO

OH O

HN

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NH O

O O

NH O

NH

O O

OH

O

NH

O

HO

OH

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HN O

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NH

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O

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O

O

O OH

O HOOC HO

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Cl

NH

NH HN

HO O

OH

OH

OH

NH O NH O Cl

HO

OH

O HO

OH HN

Teicoplanin A-40,926 B1

HO

OH

NH O

HO

OH

O

Teicoplanin A-40,926 B0

HO

OH

O HO

O

O

O

HO

Cl

O HOOC HO

HO

OH

NH O

O

O

HOOC HO

O

O

HN

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Cl

NH O

HO

OH

NH

OH

O

O

O

OH NH O

HO

OH

NH O

O

OH OH

HN

CH3 OH

OH

O

Cl

NH O

O

O

O

HO

OH

NH

O

O O

Teicoplanin A2-2

HO

O

NH

O

Avoparcin OH

NH O

Cl

HN

NH

NH2

CH3

OH

HO

HO

O

OH OH

O

OH

OH

O

HO

O

NH2

O

HO O

OH

NH O

O

HO

O O

O

O

O

HO

OH

NH O

O

O

OH

HO

HO

NH

O

NH O

NH

HO

OH

NH

O

OH

R

HO

OH

HO

OH

O

O

N

OH NH

NH O NH O Cl

OH

NH

O

HN O OH

O HO

NH O

O

Teicoplanin MDL 63,246

Dalbavancin

Fig. 1 Structure of avoparcin, teicoplanin A2–2, teicoplanin, teicoplanin A-40,926 B0, teicoplanin A-40,926 B1, teicoplanin MDL 63,246, and dalbavancin

Macrocyclic Glycopeptide-Based Chiral Stationary Phases HO

OH CH3

O H2N

OH O

H2N

O

HO

O

Cl

OH NH O

NH

O

HN

HO

O O

CH3 NH2

O OH

OH

O

O

OH OH

Teicoplanin aglycone

Ristocetin A

H3C HN

H2N

NH2

NH O

HO

O

NH

HO

O

HO

NH O

Cl O O HO

O

Cl

O

O

O

Cl O

OH

HO

O

NH

CH3

O

NH2

O

OH

O

O

HO

COOH

HN O

Cl O

HO

CH3

O

OH

NH O NH

OH

O

O HO

NH

O

HO

HO

O

NH

HO

COOH

HN

NH2

NH

O

OH

NH

HO

CH2OH OH OH

O

HO HO

O O

O

O

OH

NH

O

O

COOCH3

NH O

O

OH

O

HO

H3C HO

OH

NH O

O

O

CH2OH

OH

O NH

HO

O

O

O

OH

O

HO NH

O

HO

NH

OH

NH

Cl

NH

HO

NH

O

OH

O

205

OH

NH2

H3C

H3C

Vancomycin

Norvancomycin

H3C HN O

NH2

NH NH

HO

O

HO

O O

OH

NH O

O O

HO HO

O

CH3

O

H3C

NH2

Eremomycin

O

HO OH

NH O

NH2

Cl O

OH

Cl O

HO

O

O HO

COOH

HN

NH

HO

H3C

O

HO

O

NH

O CH3

OH

NH2

NH

HO

O

O

O

O HO

NH

O

COOH

HN

NH Cl O

H3C HN

HO

O

NH

O

O

NH2 O

O OH

Balhimycin

Fig. 2 Structure of teicoplanin aglycone, ristocetin A, vancomycin, norvancomycin, eremomycin, and balhimycin

predominate. On the one hand the structural diversity of macrocyclic glycopeptide-based CSPs provides almost all types of intermolecular interactions leading to chiral recognition. On the other

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hand, this variety makes it difficult to identify the exact mechanism that leads to chiral recognition. From the results relating to the possible chiral recognition mechanism, it can be concluded that there is no generally valid conception for chiral recognition of racemic compounds on macrocyclic glycopeptide-based CSPs. Detailed discussion related to mechanistic aspects of chiral separations on these CSPs can be found in recent publications [13, 15, 18, 21, 28] and Chapter 1.

4

Mobile-Phase Selection In all chromatographic modes, the selectivity and retention factors are mainly controlled by the nature and concentration of the mobile-phase components together with other variables such as the pH of the mobile phase. Because of the variety of the functionalities present within the macrocyclic glycopeptides, CSPs may be used in reversed-phase mode (RPM), normal-phase mode (NPM), polar ionic mode (PIM), or polar organic mode (POM). The possibility to operate in different modes is one of the major advantages of the glycopeptide CSPs, since different compounds separate best under different experimental conditions. Obviously, the solubility of the analyte in different solvents also affects the choice of the mobile-phase modes. The Chirobiotic™ columns have been found to be one of the most useful CSPs for the enantioseparation of drugs, pharmaceuticals, agrochemical toxins, amino acids, and their analogs. In order to find the best possible column and chromatographic conditions for the enantiomeric separation of amino acids a “decision tree” has been developed [29]. Protocols for the method development and optimization of chromatographic conditions using glycopeptide CSPs can be found in several publications [4, 29, 30]. Each type of interaction has different strengths in different mobile phases, so by going from one mobile phase type to another, on the same column, the mechanism changes, giving another opportunity for efficient separation.

4.1

Polar Ionic Mode

The PIM is a very effective mode for the chiral separation of ionizable racemates on macrocyclic glycopeptide-based CSPs. It is extensively used in the pharmaceutical industry mainly because of its speed and ability to be used easily with mass spectrometric detection. The dominant interactions between the analyte and the CSP usually involve π–π interactions, H-bonding, electrostatic, dipolar and steric interactions, or some combination thereof [31]. Equilibration of the column is fast and PIM offers a good alternative to normal-phase cellulose- or amylose-based applications when needed. Chiral selectivity in PIM is determined by the ratio of acid to base in the mobile phase. Once selectivity has been

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207

detected for a column during the screening mobile phases, the next step is to vary the acid-base ratio to determine the preferred ratio— a higher acid or a higher base content. In general, acidic molecules prefer a higher base content, while bases prefer a higher acid concentration. One should always keep in mind that in case of macrocyclic glycopeptide-based CSPs the selector is ionizable, so changes in the acid-to-base ratio will also affect the degree of ionization of the CSP itself. Acetic acid (AcOH) and triethylamine (TEA) are the most common additives in PIM; however, volatile salts may also be used. A typical starting mobile-phase composition can be MeOH/AcOH/TEA (100/0.1/0.1 v/v/v) or MeOH/ammonium formate (100/0.1 v/v). Decreasing the ratio of acid to base will lead to the change in selectivity. Varying the concentration with maintaining the same acid-to-base ratio usually has no significant effect on the selectivity but it will influence peak efficiency and retention. 4.2 ReversedPhase Mode

Ionic interaction is dominant not only in PIM but also in RPM. However, the additional possibility of the formation of inclusion complexes in RPM offers further opportunities for efficient chiral recognition. Inclusion occurs with the shallow pockets of the glycopeptide. Thus, for a RPM separation a different mechanism can be involved. The retention and selectivity in RPM are controlled mainly by pH, buffer (type and concentration), organic modifier (type and concentration), and to some extent flow rate. Typically, lowering the pH suppresses non-chiral retention mechanisms and silanol activity, which in turn enhances the chiral interactions. To achieve enantioseparation for most amino acids and small peptides non-buffered hydro-organic solvent mixtures as mobile phases are sufficient. However, for most other compounds an aqueous buffer is usually necessary to enhance resolution [13]. The type of the organic solvent used will greatly affect the separation; thus, it is advisable to test several different types. MeOH, ethanol (EtOH), acetonitrile (ACN), 2-propanol (2-PrOH), and tetrahydrofuran (THF) are the most common solvents that give good selectivities for various types of analytes. A typical starting composition of a mobile phase is ACN/buffer (pH 3.5–7.0) 10/90 (v/v) or alcohol/buffer 20/80 (v/v). Decreasing the flow rate may result in increased resolution with an acceptable increase in retention time.

4.3 Polar Organic Mode

For the resolution of neutral chiral analytes the NPM can be applied besides RPM or POM depending on the polarity of the racemate. More polar molecules are generally separated better in POM. In this mode a single polar organic solvent, e.g., MeOH, EtOH, 2-PrOH, or a mixture of these, is used as mobile phase. The dominant interactions between the analyte and the CSP usually

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involve hydrogen bonding, electrostatic, dipolar and steric interactions, or some combination thereof [31]. Retention is controlled by the polarity of the mobile phase. Increasing the polarity decreases retention. ACN, THF, or methylene chloride is a suitable mobile-phase additive to increase solubility of the analytes. Methyltert.-butyl ether (MtBE) and dimethylsulfoxide (DMSO) can also be added to reinforce steric effects and/or increase solubility. 4.4 NormalPhase Mode

NPM is well established and commonly used in drug discovery. In NPM using nonpolar mobile phases such as hexane or heptane in combination with a polar organic modifier such as EtOH or 2-PrOH, the CSP behaves as a polar stationary phase. The presence of polar functional groups and aromatic moieties of the glycopeptide may provide several interactions required for enantiorecognition, i.e., hydrogen bonding, π–π interactions, dipole stacking, and steric repulsion [3]. Optimization of a separation can be performed by adjusting the percentage of a polar organic modifier. Different combinations of polar and nonpolar solvents can affect the selectivity. The separation efficiencies with hexane/EtOH mobile-phase mixtures are usually higher than those obtained with hexane/2PrOH mixtures [3].

4.5 pH Considerations

In order to maintain a constant pH and reproducible retention times during any HPLC separation, buffering of the mobile phase is recommended. All macrocyclic glycopeptide-based selectors have ionizable groups; thus, their charge and perhaps their conformation can vary with the pH of the mobile phase [3]. Due to variations in their ionizable functional groups and, consequently, their pI values the pH of the mobile phase will have different effects on different macrocyclic antibiotics. Since in RPM and PIM ionic interactions play an important role in chiral recognition the pH of the mobile phase has a great impact on both retention and selectivity. Variation of the pH can alter the ionization of both the selector and the selectand. Therefore, the pH can affect the interaction mechanism even if the analyte is a neutral molecule. As a general rule, the starting pH of the mobile phase should be close to the pI value of the glycopeptide antibiotic used as chiral selector. Generally for screening experiments the pH around the pI of the antibiotics (pI  1) is a good starting point.

5 Preparation of Chiral Stationary Phases Based on Different Macrocyclic Antibiotics The concept of utilizing macrocyclic antibiotics as chiral selectors has been introduced by Armstrong et al. [1, 2]. In order to obtain an efficient CSP the following requirements must be fulfilled when macrocyclic antibiotics are bound to silica gel:

Macrocyclic Glycopeptide-Based Chiral Stationary Phases

209

(a) A stable linkage between the chiral selector and the silica gel matrix. (b) The chiral recognition properties of the glycopeptide are retained when bound to a solid support. (c) The geometrical arrangement of the chiral selector maximizes enantioselectivity. (d) The synthetic procedure for binding the selector to the silica gel can be scaled up. Organosilanes containing terminal carboxylic acid groups, i.e., [1-(carbomethoxy)ethyl]methyldichlorosilane and [2-(carbomethoxy)ethyl]trichlorosilane, were used to immobilize vancomycin and thiostrepton via their amino groups to the solid support, leading to the formation of stable amide bonds between the glycopeptides and the modified silica [2]. The likewise organosilanes containing terminal amino groups, i.e., (3-aminopropyl)triethoxysilane and (3-aminopropyl) dimethylethoxysilane, were applied for rifamycin B [2]. The same binding chemistry was applied for the coupling of the aglycone moiety of vancomycin via the carboxylic acid groups [32]. Methods described earlier for the preparation of cyclodextrinbased CSPs were also used to bind the macrocyclic antibiotics avoparcin, teicoplanin, ristocetin A, and vancomycin analogs to silica gel using epoxy groups containing organosilanes, including (3-glycidoxypropyl)trimethoxysilane, (3-glycidoxypropyl)dimethylethoxysilane, and (3-glycidoxypropyl)triethoxysilane [2, 33]. In the final dipropyl ether linkage structure, the glycopeptides are linked to the silica gel via a stable C–N bond. Recently, new CSPs were prepared by the binding of vancomycin [34] and eremomycin [35] to epoxy-activated silica gel and in the latter case eremomycin immobilized on silica was further modified with bovine serum albumin (BSA) yielding a mixed binary chiral sorbent [36]. The terminal diol functionality of silica gel was also being applied for the binding of macrocyclic molecules. The periodate oxidation of diol groups yields aldehyde functions [37]. Subsequently, macrocyclic glycopeptides bearing amino groups can be immobilized by reductive amination of aldehyde-functionalized silica in combination with sodium cyanoborohydride. This binding chemistry has been exploited for the immobilization of vancomycin [38–42], ristocetin A [43], and, recently, the glycopeptide MDL 63,246 [44, 45]. According to Svensson et al. [40] vancomycin was randomly linked to silica through one or both of its amino groups. Additionally 9-fluorenylmethoxycarbonyl (FMOC)-amino-protected vancomycin was immobilized, and vancomycin was then recovered by cleavage of the protecting group. No advantages were found for the use of a well-defined CSP as an alternative to the randomly linked vancomycin CSP.

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(3-Isocyanatopropyl)triethoxysilane and (3-isocyanatopropyl) dimethylchlorosilane as isocyanate-containing organosilanes can immobilize compounds in anhydrous dimethyl formamide. The isocyanate organosilanes possess different functionalities at opposite terminals: at one end they have a highly reactive isocyanate group, and at the other end they behave as trialkoxy- or dialkylmonochlorosilanes, respectively [46]. The teicoplanin analog A-40,929 and the teicoplanin aglycone were grafted covalently to a silica surface via a bifunctional aliphatic isocyanate (1,6-diisocyanatohexane) [47–49]. Norvancomycin was linked to silica gel via a spacer with different functionalities at the opposite terminal, e.g., isothiocyanate at one end and triethoxysilane at the other end [50]. D’Acquarica [46] investigated the influence of different spacers and the nature of the silica matrix on the chiral performance. The optimal synthetic strategy for the grafting of teicoplanin A2–2 included the formation of two ureidic functions on the CSP structure, spaced by a six-carbon-atom aliphatic chain. A new synthetic route for the stabilization of vancomycin was developed by Anan’eva et al. Mercaptosilica was modified by gold nanoparticles and then heated to react with 3-mercaptopropionic acid and vancomycin [51]. Vancomycin CSP was created via selfassembly and photochemical transformation of diazoresin. With UV treatment, the ionic bonding between silica particles and diazoresin, and diazoresin and vancomycin, was turned into covalent binding through a unique photochemical reaction of diazoresin [52].

6

Applications of Different Macrocyclic Antibiotic-Based CSPs Early information on the applications of macrocyclic antibiotics can be found in papers cited in numerous previous reviews and book chapters [3–8, 10–21, 29, 30, 53–56]. The recent progress covered in this overview concerns the period of time between 2004 and 2018. Applications of macrocyclic antibiotics as chiral selectors in CE, CEC, and thin-layer chromatography are not discussed here. Because of the structural differences, the macrocyclic glycopeptides are to some extent complementary to one another. Whenever a partial enantioresolution is obtained with one glycopeptide selector, there is a high probability that at least a baseline separation can be achieved with another glycopeptide. Each type of interaction has a different strength in different mobile phases. Thus, by exchanging one type of mobile phase with another type on the same column the recognition mechanism may change. This results in another opportunity for an efficient enantioseparation. Figure 3 illustrates examples of this complementary behavior.

Phenylalanine

3-Amino-2-benzylpropanoic acid O

O

Column

OH

OH

NH2

NH2

0.35

1.0

0.30

0.8

0.25 0.6

0.15

AU

AU

Chirobiotic T

0.20

0.4

0.10

0.2

0.05 0.0

0.00

-0.2

-0.05 0

5

10

15

20

0

5

10

Time (min)

20

25

30

0.4

0.8

0.3

0.6

0.2

AU

AU

0.4

Chirobiotic T2

15

Time (min)

0.1

0.2 0.0 0.0 -0.1 -0.2 0

5

10

15

0

20

5

10

15

20

25

Time (min)

Time (min)

0.8

0.3

0.6 0.2

AU

Chirobiotic TAG

AU

0.4 0.1

0.2 0.0

0.0

-0.1

-0.2 0

5

10

15

20

25

0

30

5

10

15

20

25

30

35

Time (min)

Time (min) 0.8

0.8 0.6

0.6 0.4

AU

AU

0.4

Chirobiotic R

0.2

0.2 0.0

0.0 -0.2

-0.2 -2

0

2

4

6

8

10

12

14

16

-2

18

0

2

4

6

8

10

12

14

16

Time (min)

Time (min) 1.0

1.4 1.2

0.8

1.0 0.6

0.6

AU

Chirobiotic V

AU

0.8

0.4

0.4

0.2

0.2 0.0

0.0 -0.2

-0.2 0

5

10

Time (min)

15

20

0

5

10

15

20

Time (min)

Fig. 3 Enantioseparations of phenylalanine and 3-amino-2-benzylpropanoic acid on macrocyclic glycopeptidebased CSPs. Experimental conditions: columns: Chirobiotic™ T, T2, TAG, R, and V; mobile phase: 0.1% triethylammonium acetate (pH 4.1)/MeOH 20/80 (v/v); flow rate: 0.5 mL min1; UV detection at 215 nm; ambient temperature

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6.1 Application of VancomycinBased CSPs

Vancomycin was the first macrocyclic antibiotic to be evaluated as a CSP in HPLC [2]. Vancomycin-based CSPs have been commercialized as Chirobiotic™ V and Chirobiotic™ V2. The columns differ in the chemistry for the binding of the macrocyclic glycopeptides to the silica gel support. Since 2005, numerous papers have appeared on the enantioseparations of different analytes on vancomycinbased CSP. Examples have been summarized in Table 2. El Deeb [57] developed and validated a HPLC method for the separation and enantiomeric impurity quantitation of the β1-receptor antagonist atenolol on a Chirobiotic™ V2 column. The direct enantioseparation of the serotonin-norepinephrine reuptake inhibitor duloxetine and its R enantiomer was achieved by HPLC on a Chirobiotic™ V CSP by Yang et al. [58]. The operational parameters (buffer pH, organic modifiers, temperature, and flow rate) were varied in order to achieve baseline resolution of the enantiomers. The limit of detection of this method was 0.06 μg mL1. The parasympathomimetic or cholinergic agent for the treatment of dementia of the Alzheimer type and dementia due to Parkinson’s disease rivastigmine (Exelon™) enantiomers were separated on a Chirobiotic™ V CSP by Xu et al. [59]. The effect of the temperature was investigated in the range of 5–30  C in order to determine the values of Δ(ΔH) and Δ(ΔS) from the van’t Hoff plot. Zuo et al. [60] developed a sensitive and specific HPLC–tandem mass spectrometry (MS/MS) method for the simultaneous detection of (S)-warfarin, (R)-warfarin, (S)-7-OH-warfarin, and (R)-7OH-warfarin in human plasma. The selectivity of 7-OH-warfarin from 4-, 6-, 8-, and 10-hydroxywarfarins for a Chirobiotic™ V column was addressed. The chiral separation of (R)-warfarin and (S)-warfarin in the hepatoma HepG2 cell line and the internal standard p-chlorowarfarin enantiomers was performed on a Chirobiotic™ V2 column by Malakova et al. [61]. The enantiomers were quantified with the aid of a fluorescence detector and the limit of detection was found to be 0.121 μmol L1 of (S)-warfarin and 0.109 μmol L1 of (R)-warfarin. Chiral chromatography and electrospray ionization mass spectrometry (HPLC-ESI-MS) of the enantiomers of the serotoninnorepinephrine reuptake inhibitor venlafaxine [62] and its major metabolite O-desmethylvenlafaxine in human plasma were performed on Chirobiotic™ V column [62]. The resolution of the enantiomers of arotinolol, a mixed α/β-blocker [84], and including the separation from the degradation products and other co-formulated compounds, was successfully achieved on a vancomycin CSP [63]. The method was highly selective without interference from the degradation products and co-formulated compounds. The detection limit of 20 ng mL1 was determined for each enantiomer. Enantiomeric resolution of the β-blocker bufuralol in plasma and pharmaceutical formulations was

Drugs

Drugs

Group of racemates

Vancomycin

Fluoxetine, norfluoxetine

Amphetamine, methamphetamine, methylenedioxyamphetamine, methorphan, methylenedioxymethamphetamine, ephedrine, pseudoephedrine Ketoprofen Aryloxyaminopropanol derivatives

Amphetamine, methamphetamine

Vancomycin

PIM

MeOH/0.04% NH4TFA

0.05 M KH2PO4 (pH 6.0)/2-propanol (50/50/ v/v) MeOH/ACN/AcOH/TEA (45/55/0.3/ 0.2 v/v/v/v)

Vancomycin Vancomycin, Teicoplanin

[76]

[75]

[74]

[73]

[71] [72]

[67] [68] [69] [70]

[61] [62] [63] [64] [65] [66]

[57] [58] [59] [60]

(continued)

RPM [77] PIM [78]

PIM

PIM

PIM

PIM PIM

PIM

PIM PIM

RPM RPM PIM PIM PIM PIM

RPM PIM PIM RPM

Mode Refs.

MeOH/AcOH/NH3 (100/0.1/0.02 v/v/v)

MeOH/AcOH/TEA (100/0.2/0.1 v/v/v) MeOH/ACN/AcOH/NH3 (80/20/0.02/0.01 or 60/40/ 0.02/0.01 v/v/v/v) MeOH/TFA/NH3 in different ratios 0.1% TEAA (pH 4.1)/ACN in different ratios MeOH/AcOH/TEA (100/0.025/0.75 v/v/v) EtOH/MeOH/aq. AcOH (pH 6.7)/TEA (50/50/0.225/ 0.075 v/v/v/v) EtOH/MeOH/aq. AcOH (pH 6.7)/TEA (50/50/0.225/ 0.075 v/v/v/v) EtOH/aq. NH4OAc (92.5/7.5 v/v)

0.5% TEAA (pH 4.5)/MeOH/ACN (5/45/50 v/v/v) MeOH/AcOH/TEA (100/0.04/0.01 v/v/v) MeOH/AcOH/TEA (100/0.02/0.01 v/v/v) 10 mM TEAA (pH 4.4)/ACN (v/v) Gradient elution: 90/10 (v/v) ! 10/90 (v/v) 10 mM TEAA (pH 4.1)/MeOH/ACN (64/5/31 v/v/v) 30 mM NH4OAc (pH 6.0)/MeOH (15/85 v/v) MeOH/AcOH/TEA (100/0.02/0.03 v/v/v) MeOH/AcOH/TEA (100/0.015/0.010 v/v/v) 2.5 mM NH4NO3 in EtOH (pHa 5.1) MeOH/AcOH/TEA (100/0.2/0.1 v/v/v)

The most effective mobile phases (v/v/v)

Vancomycin Vancomycin 2 Vancomycin 2

Vancomycin Vancomycin

Vancomycin

Vancomycin Vancomycin

Vancomycin Vancomycin Vancomycin Vancomycin Vancomycin Vancomycin

Vancomycin Vancomycin Vancomycin Vancomycin

Selectors

Propranolol, terbutaline, salbutamol, warfarin Atenolol Atenolol, metoprolol, fluoxetine Alprenolol, propranolol

Venlafaxine Arotinolol Bufuralol Terbutaline, salbutamol Mirtazapine, N-desmethyl mirtazapine Tartalolol, arotinolol Trantinterol

Atenolol Duloxetine Rivastigmine Warfarin

Analytes

Table 2 Enantioseparation of stereoisomers of different analytes on vancomycin-based CSPs

Macrocyclic Glycopeptide-Based Chiral Stationary Phases 213

Vancomycin degradation product Vancomycin immobilized on silica modified by gold nanoparticles Vancomycin

Amlodipin, atropine, baclofen, ibuprofen, mandelic acid, Phe

Alprenolol, oxprenolol, atenolol, pindolol, metoprolol, nadolol

Amino acids

N-Moc-α-amino acids Vancomycin Teicoplanin Ristocetin A

Crystalline degradation products—vancomycin

Vancomycin Vancomycin, teicoplanin, teicoplanin aglycone, ristocetin A

Cinacalcet Chiral xanthonic derivatives

Chlortrimeton, benzoin

Selectors

Analytes

Agrochemical Haloxyfop-methyl toxins Fenoxaprop-p-ethyl Indoxacarb

Group of racemates

Table 2 (continued)

NPM [82]

n-Hexane/MeOH or n-hexane/2-PrOH

15 mM NH4OAc (pH 4.1 or 5.9)/MeOH (80/20 (v/v) MeOH/ACN/AcOH/TEA (25/75/0.25/0.25 (v/v/v/v)

NPM [51] RPM

n-Hexane/2-propanol (80/20 v/v) 0.3% TEAA (pH 4.0)/ACN (30/70 v/v)

RPM [83] PIM

[50]

[81]

[79] [80]

PIM NPM RPM POM PIM PIM RPM RPM RPM

Mode Refs.

MeOH containing 2.5 mM NH4HCOO n-Hexane/EtOH or n-hexane/2-PrOH aq. TEAA (pH 4.2)/MeOH, NH4OAc (pH 6)/MeOH 100% MeOH, 100% EtOH or 100% 2-PrOH MeOH/AcOH/TEA 0.1% NH4TFA in MeOH aq. TEAA (pH 6.5)/MeOH (15/85 v/v) 20 mM Sodium citrate (pH 6.3)/THF (90/10 v/v) 30 mM NH4OAc (pH 6.0)/MeOH (15/85 v/v)

The most effective mobile phases (v/v/v)

214 Istva´n Ilisz et al.

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215

achieved on a Chirobiotic™ V CSP [64]. Co-formulated compounds did not interfere. The stability of the bufuralol enantiomers at different temperature was also studied. A rapid baseline enantioseparation for terbutaline and salbutamol was obtained in shorter than 10 min on Chirobiotic™ V using the polar ionic mode with mobile phase containing 2.5 mM NH4NO3 in EtOH (pHa 5.1) [65]. Mirtazapine and its metabolite N-desmethyl mirtazapine in rat plasma were determined on Chirobiotic™ V column. The repeatability of the HPLC-fluorescencepolarimetric detection was acceptable [66]. Enantioseparation of tartalolol and arotinolol in rat plasma was performed on Chirobiotic™ V using UV detection and PI mode. No enantiomeric separation was observed in the absence of TEA [67]. HPLC-MS/MS method was developed for the determination of trantinterol content in rat [68] and human [70] plasma. Separation of enantiomers was achieved on Chirobiotic™ V using MeOH/ACN/AcOH/ NH3 mobile phases. The best resolution was performed at the 2:1 acid-to-base ratio [69]. Chromatographic behavior was predicted for enantiomers of propranolol, terbutaline, salbutamol, and warfarin using DryLab HPLC method development software on Chirobiotic™ V [70]. It was concluded that the reversed-phase retention mechanism on Chirobiotic™ V follows the solvophobic theory. Enantioseparation for β-blockers, atenolol in mouse plasma [71], alprenolol, propranolol [72], fluoxetine, and norfluoxetine in a wastewater treatment plant [74] was optimized applying Chirobiotic™ V column. The effect of MeOH content [71] and EtOH content [72–74], buffer concentration, and pH on chromatographic parameters was systematically investigated. Optical isomers of amphetamine-type stimulants were separated on Chirobiotic™ V2 column applying HPLC-MS/MS technique in urine [76] and originated from clandestine laboratories [76]. The best separation was achieved in PI mode in mobile phases compatible with MS detection. For the separation of ketoprofen enantiomers vancomycin as chiral mobile-phase additive was applied in phosphate buffer/2-propanol eluent [77]. Physicochemical interactions of aryloxyaminopropanol derivatives on Chirobiotic™ V and Chirobiotic™ T CSPs were studied in PI mode. It was concluded that the interactions closest to stereogenic centers generally had a greater impact on enantioresolution [78]. Cinacalcet enantiomers in rat plasma were determined on Chirobiotic™ V CSP with MS/MS detection [79]. The PI mode has many advantages not only in terms of speed, but also in being beneficial for preparative separation. Enantioresolution of chiral xanthonic derivatives was performed on Chirobiotic™ V, Chirobiotic™ T, Chirobiotic™ TAG, and Chirobiotic™ R in NPM, RPM, POM, and PIM [80]. The effects of mobile-phase composition, percentage of organic modifier, pH, nature, and concentration of different additives were studied. Chirobiotic™ V and

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Chirobiotic™ T under RPM and NPM, respectively, presented the best chromatographic parameters. Two crystalline degradation products of vancomycin were immobilized on silica surface and applied for enantioseparation of acidic and basic drugs (amlodipin, atropine, baclofen, ibuprofen, mandelic acid, Phe) [81]. Both RPM and PIM were found to be applicable to all investigated molecules. Very recently, new synthetic routes were applied for the immobilization of vancomycin [51, 52]. Separation of chlortrimeton and benzoin enantiomers was achieved on a vancomycin CSP based on diazotized silica and prepared under UV light radiation. Enantioseparation was successfully carried out in NP and RP modes [52]. Anan’eva developed a silica-based CSP modified with gold nanoparticles and immobilized with vancomycin on the surface [51]. However, the enantioseparation of β-blockers in most cases failed, and RS values were lower than 1.0. Enantiomeric separations of three agrochemical toxins (haloxyfop-methyl, fenoxaprop-p-ethyl, and indoxacarb) were carried out by HPLC on crystalline degradation products of vancomycin as CSP by Aboul-Enein et al. [82]. Excellent stereoselectivity for the enantiomers of haloxyfop-methyl and fenoxaprop-p-ethyl and chiral recognition was achieved for indoxacarb in the NPM. The chromatographic results were compared with those on commercial vancomycin-based CSPs. The comparative enantioseparation of a series of unsaturated N-methoxycarbonyl-(N-Moc)-α-amino acids has been described for three types of glycopeptide phases, i.e., Chirobiotic™ V in comparison to Chirobiotic™ T and Chirobiotic™ R, containing vancomycin, teicoplanin, and ristocetin, respectively, as chiral selectors [83]. 6.2 Application of CSPs Based on Teicoplanin and Related Macrocyclic Glycopeptides

Teicoplanin and its analogs have been used successfully for the resolution of many types of racemic analytes, such as amino acids, drugs, toxins, small peptides, and peptidomimetics, as summarized in Tables 3 and 4. Teicoplanin-based CSPs have been commercialized under the trade names Chirobiotic™ T and Chirobiotic™ T2 differing in the binding chemistry. Furthermore, the teicoplanin aglycone has been bound to silica gel in Chirobiotic™ TAG, while ristocetin A has been immobilized on silica gel and commercialized as Chirobiotic™ R.

6.2.1 Separation of Amino Acid Enantiomers

The comparative enantioseparation of a series of unsaturated NMoc-α-amino acids showed that the best results in terms of enantioselectivity and resolution were obtained on the Chirobiotic™ R column in the PIM [83]. In order to gain a better understanding of the roles of the polar functional groups of the teicoplanin-based CSPs, the enantioseparations of a wide variety of racemic compounds, e.g., Trp-,

Amino acids, acids

Teicoplanin Teicoplanin Teicoplanin aglycone Ristocetin A Teicoplanin 2-Aminomono- and dihydroxycyclopentanecarboxylic and Teicoplanin aglycone 2-aminodihydroxycycloRistocetin A hexanecarboxylic acids Monoterpene-based β-amino acids Teicoplanin Teicoplanin aglycone Aliphatic and aromatic α-amino acids Teicoplanin aglycone Isoxazoline-fused Teicoplanin, 2-aminocyclopentanecarboxylic acids teicoplanin aglycone, vancomycin, vancomycin aglycone

Ala-, Tyr-, Trp-derivatives γ-Amino acids

Teicoplanin

Teicoplanin, teicoplanin aglycone, methylated teicoplanin aglycone Teicoplanin

Trp-, Phe-, Leu-, mandelic acidderivatives, profens, β-blockers

Chlorophenoxypropionic acids, branched-chain amino acids L,D-Threonine, L,D-methionine

Teicoplanin

Selector

N-Moc-α-amino acids

Group of racemates Racemates

RPM [91] PIM POM RPM [92] RPM [93] POM PIM

0.1% TEAA (pH 4.1)/MeOH (10/90 v/v) MeOH/AcOH/TEA (100/0.1/0.1 v/v/v) 100% MeOH 50 mM TEAA (pH 5.8)/MeOH (90/10 v/v) 0.01% TEAA (pH 4.1)/MeOH (60/40–80/20 v/v) 100% MeOH MeOH/AcOH/TEA (100/0.1/0.1 v/v/v)

(continued)

RPM [90]

RPM [88] RPM [89] PIM

RPM [86] PIM RPM [87]

RPM [83] PIM RPM [85] PIM

Mode Refs.

0.1% TEAA (pH 4.1–6.5)/MeOH (20/80 v/v) 0.1% TEAA (pH 4.1–6.5)/EtOH (20/80 (v/v)

0.1% TEAA/MeOH MeOH/AcOH/TEA (100/0.1/0.1 v/v/v) H2O/MeOH; H2O/EtOH H2O/2-PrOH/ACN 5.0 mM Phosphate buffer (pH 7.0)/MeOH 0.1% TEAA (pH 4.1)/MeOH 10:90 v/v) MeOH/AcOH/TEA (100/0.1/0.1 v/v/v)

15 mM TEAA (pH 4.1 or 5.9)/MeOH 80/20 (v/v) MeOH/ACN/AcOH/TEA (25/75/0.25/0.25 v/v/v/v) 1% TEAA/MeOH (60/40 v/v) MeOH/ACN/AcOH/TEA (55/45/0.3/0.2 v/v/v/v)

The most effective mobile phase (v/v/v)

Table 3 Enantioseparation of stereoisomers of amino acid and its analogs on teicoplanin, on its analogs, and on ristocetin A-based CSPs

Macrocyclic Glycopeptide-Based Chiral Stationary Phases 217

Selector

The most effective mobile phase (v/v/v)

Teicoplanin Teicoplanin aglycone Ristocetin A Teicoplanin Teicoplanin

Teicoplanin Teicoplanin aglycone, vancomycin

Fully constrained β-amino acid Met, Cys, homo-Cys

Teicoplanin Teicoplanin Teicoplanin aglycone

Methionine Unusual chlorinated and fluorinated amino acids Underivatized proteinogenic amino acid Teicoplanin in Arctic lakes aglycone

Amino acids, β-blockers, Amino oxazolidinones, mandelic acid, acids, coumachlor, proglumide, amino thalidomide, warfarin, mianserin acid analogs, drugs Carbocyclic β-amino acids possessing limonene skeleton

50 mM aq. NH4OAc (pH 6.0)/MeOH (90/10 v/v) MeOH/AcOH/TEA (100/0.5/0.1, 100/0.1/0.1 and 100/0.3/0.3 v/v/v) A: 0.1% formic acid, B: 0.1% formic acid in MeOH; gradient elution: 0–15 min 30% B, 15–20 min 100% B NH4HCOO(pHa 4.5)/MeOH 10/90 v/v) 25 mM aq. Phosphate buffer/1 mM aq. octanesulfonic acid/ ACN (94/3/3 v/v/v)

n-Heptane/EtOH (80/20 v/v) H2O/MeOH (40/60–20/80 v/v) 0.1% TEAA (pH 4.1)/ACN (80/20 v/v) 100% MeOH MeOH/ACN/AcOH/TEA (45/55/0.3/0.2 and 40/60/0.3/0.2 v/v/v/v) MeOH/AcOH/TEA (100/0.01/0.01 and 100/0.1/0.1 v/ v/v) 0.1% aq. TEAA/MeOH (90/10 v/v)

MeOH/ACN/AcOH/TEA (100/0/0.1/0.1 v/v/v/v) Bicyclo[2.2.2]octane-based 2-amino-3- Teicoplanin, MeOH/ACN/AcOH/TEA (50/50/0.1/0.1 v/v/v/v) carboxylic acid teicoplanin aglycone Ristocetin A 0.1% TEAA (pH 4.1)/MeOH (50/50 v/v) Phenylisoserine derivatives Teicoplanin Teicoplanin aglycone, vancomycin, vancomycin aglycone

Group of racemates Racemates

Table 3 (continued)

[94]

[100]

PIM [101] RPM [102–104]

PIM

RPM [98] PIM [99]

PIM [97] RPM

NPM [96] RPM POM PIM

RPM [95]

PIM

Mode Refs.

218 Istva´n Ilisz et al.

Phe, Tyr, Trp

Met, Val, Leu, Ala, Nval, Nleu

Underivatized Phe and Val N-protected amino acids, α-aryloxy acids, herbicides, anti-inflammatory agents

Ristocetin A Teicoplanin immobilized on sub-2 μm totally porous particles Teicoplanin immobilized on sub-2 μm totally porous particles Teicoplanin

RPM [107]

SFC

EtOH/H2O (80/20 and 90/10 v/v) MeOH/H2O (90/10 v/v)

CO2/(MeOH/H2O) [60/(90/10) v/v)]

[108]

RPM [105] RPM [106] PIM NPM

MeOH/H2O (60/40 or 40/60 v/v) 20 mM aq. NH4OAc/MeOH (15/85 v/v) 20 mM aq. NH4OAc/ACN (15/85 v/v) MeOH/ACN/AcOH/TEA (40/60/0.055/0.03 v/v/v/v) n-hexane/EtOH (70/30 v/v)

Macrocyclic Glycopeptide-Based Chiral Stationary Phases 219

Drugs

Group of racemates

[116] [117]

[118]

RPM NPM PIM

PIM

Carvedilol Ketoprofen

Teicoplanin Teicoplanin Teicoplanin

MeOH/AcOH/DEA (100/0.15/0.05 v/v/v) 1.0% aq. TEAA (pH 6.8)/MeOH (10/90 v/v) 10 mM aq. NH4OAc (pH 4.2)/MeOH (70/30 v/v)

[119] [120] [121]

[115]

RPM

Teicoplanin aglycone

PIM RPM RPM

[114]

RPM

[109] [110] [112114] [111] [112]

Refs.

[113]

PIM PIM

RPM RPM RPM

Mode

PIM

10 mM TEAA (pH 5.5)/EtOH (20/80 v/v) H2O/EtOH (80/20 v/v/v) H2O/MeOH (HPLC or μ-HPLC) (50/50 or 30/70 v/v) 0.1% aq. TEAA (pH 4.0)/MeOH (30/70 v/v) MeOH/AcOH/TEA (100/0.02/0.025 (v/v/v) MeOH/AcOH/TEA (100/17.5 mM/4.8 mM)

The most effective mobile phases (v/v/v)

MeOH/AcOH/TEA (100/0.025/0.017 v/v/v) Teicoplanin, teicoplanin aglycone, vancomycin Teicoplanin 20 mM TEAA (pH 6.4)/MeOH (10/90 v/v)

Teicoplanin Teicoplanin aglycone Teicoplanin Teicoplanin aglycone Teicoplanin Teicoplanin, teicoplanin aglycone, methylated teicoplanin aglycone

Selectors

H2O/ACN/HCOOH (85/15/0.02 v/v/v) H2O/ACN/HCOOH/NH4HCOO (78/22/0.02/10 mM v/v/v) Eflornithine Teicoplanin aglycone 10 mM TEAA (pH 4.5)/EtOH (75/25 v/v) n-Hexane/EtOH (80/20–50/50 v/v) Xanthones Teicoplanin, teicoplanin aglycone, MeOH/AcOH/TEA (100/0.5/0.5 v/v/v) vancomycin, ristocetin A Teicoplanin MeOH/TEA (100/0.1 v/v), MeOH/EtOH/TEA (90/10/ Propranolol, 0.1 v/v/v), MeOH/2-PrOH/TEA (90/10/0.1 v/v/v), and metoprolol, atenolol, MeOH/ACN/TEA (90/10/0.1 v/v/v) pindolol

Pregabalin Vigabatrin Triiodothyronine Thyroxine Bisoprolol 1-Methyl-2piperidinoethylesters of 2-, 3-, and 4alkoxyphenylcarbamic acid Aryloxyaminopropanol type potential β-blockers Bambuterol, terbutaline Molindone

Analytes

Table 4 Enantioseparation of stereoisomers of different analytes on teicoplanin and on its analog-based CSPs

220 Istva´n Ilisz et al.

Drugs

20 mM aq. TEAA (pH 6.0)/MeOH (75/25 v/v)

Teicoplanin

RPM RPM PIM HILIC RPM RPM

PIM POM RPM

10 mM aq. NH4OAc/MeOH (50/50 v/v) 10 mM aq. NH4OAc (pH 4.5)/MeOH (2/98 v/v) MeOH/TEA (100/0.05 v/v) 20 mM aq. NH4OAc /ACN (5/95 v/v) 0.1% aq. TEAA/MeOH (90/10 v/v) 2.5–50 mM NH4HCOO (pH 3.2)/ACN/ (65/35, 30/70 v/v) 50 mM NH4HCOO (pH 3.2)/MeOH (50/50 v/v), 50 mM NH4HCOO (pH 3.2)/THF (90/10 or 80/20 v/v)

MeOH/NH4HCOO (100/0.025, 100/0.5, 100/0.2 v/v); MeOH/AcOH/NH4OH (100/0.2/0.05 v/v/v); ACN/MeOH/AcOH/NH4OH (50/50/0.3/0.2 v/v/v/v); 100% MeOH; 16 mM aq. NH4HCOO/(pH 3.6)/MeOH (10/90 v/v)

RPM

NPM

n-Hexane with EtOH and 2-PrOH

Teicoplanin

Teicoplanin Teicoplanin Teicoplanin Teicoplanin, vancomycin Teicoplanin and Enkephalin, vancomycin bradykinin, immobilized on vasopressin, LHRH sub-2 μm peptides superficially porous Tryptic digest of equine particles apomyoglobin Modified macrocyclic Tobacco alkaloids, glycopeptide and nicotine metabolites teicoplanin and derivatives, immobilized on tobacco-specific sub-2 μm nitrosamines superficially porous particles

Cis- and transdiastereomers of 2-amino analogs of indole phytoalexins Fluorinated 2-(phenanthren-1yl)propionic acid 3-n-Butylphthalide Salbutamol Modafinil Therapeutic peptides

POM

MeOH/2-PrOH (40/60 v/v)

Ibuprofen, carboxyibuprofen, 2-hydroxy ibuprofen, chloramphenicol, ifosfamide, indoprofen, ketoprofen, naproxen, praziquantel Teicoplanin aglycone Ifosfamide and its Ndechloroethylated metabolites

(continued)

[130]

[129]

[125] [126] [127] [128]

[124]

[123]

[122]

Macrocyclic Glycopeptide-Based Chiral Stationary Phases 221

[131]

[132] [133]

TeicoShell: 0.1% TEAA/MeOH (50/50, 75/25, 70/30 v/v) and RPM 0.1% H3PO4/ACN (70/30 v/v) NicoShell: 1.0% TEAA/ACN (40/60 v/v)

RPM

RPM

MeOH/H2O þ borneol or fenchol-based ionic liquids

0.45% aq. TEAA (pH 3.6)/EtOH (80/20 v/v)

Teicoplanin Teicoplanin aglycone Vancomycin

H2O/ACN/NH4NO3 (50/50/80 mM v/v) MeOH/ACN/H2O/NH4NO3 (60/20/20/40 mM v/v/v/v)

[139]

[138]

RPM RPM

[135–137]

POM

[134]

Refs.

Mode

The most effective mobile phases (v/v/v)

Teicoplanin, 100% MeOH teicoplanin aglycone Teicoplanin aglycone H2O/EtOH/KPF6 (15/85/30 mM v/v/v)

Modified macrocyclic glycopeptide and teicoplanin immobilized on sub-2 μm superficially porous particles Teicoplanin þ borneol or fenchol-based ionic liquids Ristocetin A

Verubecestat and its intermediates

Mandelic acid, vanilmandelic acid, phenyllactic acid Ofloxacin, levofloxacin

Selectors

Analytes

Other Sulfoxides compounds Heterohelicenium cations Ruthenium (II) polypyridyl complexes

Group of racemates

Table 4 (continued)

222 Istva´n Ilisz et al.

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223

Phe-, and Leu-derivatives, mandelic acid analogs, profens, β-blockers, and organic acids, were evaluated on Chirobiotic™ T and TAG as well as a methylated teicoplanin aglycone CSP (Me-TAG; all the hydroxy groups of TAG were methylated by diazomethane) [85]. Improved separation efficiencies for many acidic analytes were obtained by methylating the H-bonding groups of TAG. Ionic and dipolar interactions between the carboxylate group of the analytes and the amino groups of the macrocyclic antibiotics as well as hydrophobic interactions were important for enantioseparations in RPM, while the H-bond interactions were relatively weak. Me-TAG offers higher hydrophobicity which can accentuate the interactions between analytes and hydrophobic moieties of the selector. However, these interactions are not necessarily stereoselective. In POM, electrostatic and dipolar interactions between polar functional groups are the major contributors in chiral recognition. Another important factor is the steric fit which can be changed with modifications of the teicoplanin structure. Three structurally diverse groups of analytes, i.e., branchedchain amino acids, amino alcohols (β-blockers), and chlorophenoxypropionic acids, were examined using various mobile-phase compositions and separation modes in combination with different teicoplanin coverage and distinct linkage chemistry [86]. The chlorophenoxypropionic acids, branched-chain amino acids, and β-blockers exhibited good separation on Chirobiotic™ T2. The adsorption behavior of L,D-threonine and L,D-methionine has been investigated on a column packed with teicoplanin bonded to a silica support in the RPM. The study was performed under nonlinear adsorption isotherm conditions [87]. The hydrophobic C11 acyl side chain attached to the D-glucosamine group of teicoplanin served as anchor moiety for the immobilization of this chiral selector on C8 and C18 apolar support material [88]. It was found that the enantiomer elution sequence of Ala, Tyr, and Trp derivatives on these modified C8 and C18 stationary phases was reversed (D < L) relative to that classically observed elution order with teicoplanin covalently immobilized to a silica gel support (L < D). Three underivatized cyclic γ-amino acids were successfully enantioseparated by Pe´ter et al. on macrocyclic glycopeptidebased CSPs (Chirobiotic™ T, T2, TAG, and R) [89]. An increase of the alcohol modifier in the mobile phase and a decrease of the column temperature generally increased the enantioseparation. Mechanistic aspects of chiral recognition were discussed with respect to the structures of the analytes. The enantioseparation of hydroxycycloalkane amino acid analogs and five monoterpene-based 2-aminocarboxylic acids was investigated by using Chirobiotic™ T, T2, TAG, and R columns [90, 91]. Of the four columns, Chirobiotic™ T and TAG appeared

224

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to be most suitable for the enantioseparations of 2-aminomono- or dihydroxycycloalkanecarboxylic acids and monoterpene-based 2-aminocarboxylic acids. The elution sequence was determined in most cases, but no general rule could be established correlating the elution sequence to the absolute configuration. On a Chirobiotic™ TAG column under RPM conditions, a reversible change in adsorption behavior was observed for five aliphatic and aromatic amino acids leading to a retention time shift when a preparative-scale column was treated under harsh preparative chromatographic conditions [92]. The enantioseparation of unusual isoxazoline-fused 2-aminocyclopentanecarboxylic acids was performed on Chirobiotic™ T, TAG, V, and VAG columns in the temperature range of 5–40  C [93]. The best separation of four enantiomers in one chromatographic run was achieved applying 0.1% TEAA (pH 4.1)/ MeOH, 100% MeOH, and MeOH/AcOH/TEA mobile phases. Enantiomers of bicyclo[2.2.2]octane-based 2-amino-3-carboxylic acid exhibited good resolution on Chirobiotic™ T, TAG, and R [94]. Thermodynamic studies revealed that enantioseparations were usually enthalpically driven, but on Chirobiotic™ R entropically driven separation was also registered. Separation of phenylisoserine derivatives was carried out on Chirobiotic™ T, TAG, and V CSPs mainly in RP mode [95]. Separation was influenced by the eluent pH and by the MeOH content of 0.1% TEAA (pH 4.1)/ MeOH mobile phase. Enantiomers of amino acids, β-blockers, oxazolidinones, mandelic acid, coumachlor, proglumide, thalidomide, warfarin, mianserin, etc. were successfully separated on newly developed teicoplanin, teicoplanin aglycone, and vancomycinbased selectors immobilized on 1.9 μm narrow particle size distribution (NPSD) silica [96]. Applications of the three liquid chromatographic modes yielded in separations with resolution values between 1.5 and 5.7 and retentions within 2 min. Best separation of carbocyclic β-amino acids possessing limonene skeleton was performed on Chirobiotic™ TAG applying PI mode. In RPM the effects of pH, MeOH content, and alcohol additives while in PIM the effects of co- and counterions were studied [97]. The loading effects for Met were investigated on Chirobiotic™ T. The high loading yielded in the activation of the column as indicated by the increased retention [98]. This effect could slowly be reversed by flushing the column. Chirobiotic™ T column was successfully applied to separate almost all unusual chlorinated and fluorinated Tyr and Phe analogs applying PI mode [99]. D- and L-proteinogenic amino acids in Arctic lakes were separated and identified on Chirobiotic™ TAG by application of HPLC-MS/ MS technique and PI mode [100].

Macrocyclic Glycopeptide-Based Chiral Stationary Phases

225

Enantioseparation of fully constrained β-amino acids was compared on Chirobiotic™ T and polysaccharide-based Lux Amylose2 CSPs [101]. It was concluded that the polysaccharide-based CSP was a better choice in NP mode. The imbalance in the Met or homo-Cys metabolism is connected to many disorders. The enantioseparation of Cys, homo-Cys, and Met as standards and in human plasma was carried out with 2D-HPLC technique with application of Purospher C18 in the first and Chirobiotic™ T or TAG in the second dimension using electrochemical detection [102, 103]. The effect of temperature for Cys, homo-Cys, and Met on chromatographic parameters was investigated and thermodynamic parameters were calculated [104]. The applied mobile phase in all cases was phosphate buffer with octane sulfonic acid. Underivatized Phe and Val were selected to predict chromatographic behavior using DryLab HPLC method development software on Chirobiotic™ R [105]. It was concluded that the chiral recognition mechanisms tend toward a hydrophilic interaction liquid chromatography (HILIC) rather than RP mode. Very recently, teicoplanin immobilized onto Titan-120 1.9 μm totally porous silica was applied under different eluent conditions, HILIC, PIM, RPM, and NPM [106]. N-protected amino acids, α-aryloxy acids, herbicides, and anti-inflammatory agents were baseline separated on a short (2 cm) and ultrashort (1 cm) columns with analysis time in order of 1 min. Min et al. immobilized teicoplanin on sub-2 μm superficially porous silica particles (SPPs) and applied for the enantioseparation of native amino acids. The best mobile-phase conditions were found employing EtOH/H2O or MeOH/H2O at 80/20 or 90/10 ratios [107]. Subcritical fluid chromatographic (SFC) method was applied on Chirobiotic™ T for the enantioseparation of Phe, Tyr, and Trp in a mobile-phase system containing 60% CO2 and 40% MeOH/ H2O in 90/10 ratio [108]. 6.2.2 Enantioseparations of Pharmaceutical Drugs

Pregabalin (Lyrica™) is the S enantiomer of a γ-amino acid analog used to treat neuropathic pain and as adjunct therapy for partial seizures with or without secondary generalization in adults. The drug is also effective against chronic pain in disorders such as fibromyalgia. The direct chiral separation of S enantiomer from its R enantiomer was developed through a serial coupling of Chirobiotic™ T and TAG stationary phases so that baseline separation of the enantiomers was achieved [109, 140]. A direct chiral HPLC method was developed and validated for the resolution and quantification of antiepileptic drug enantiomers (R)- and (S)-vigabatrin in pharmaceutical products on a teicoplanin aglycone CSP [110]. The stability of the vigabatrin enantiomers at different temperatures was studied.

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The hormones triiodothyronine (T-3) and thyroxine (T-4) are released from the thyroid gland upon stimulation by pituitary gland hormone thyroid-stimulating hormone (TSH). T-3 and T-4 affect almost every physiological process in the body including growth and development, metabolism, body temperature, and heart rate. HPLC separation of the T-3 and T-4 enantiomers was performed on Chirobiotic™ T and TAG CSPs, using H2O/MeOH mobilephase systems [141, 142]. In a new investigation the best conditions for the enantioseparation of T-4 were found on Chirobiotic™ T with MeOH and 0.1% aq. TEAA (pH 4.0) (70/30 v/v) mobile phases [143]. Bisoprolol is a selective β1-adrenergic receptor blocker [144]. An HPLC method has been developed and validated by Hefnawy et al. for the determination of (S)- and (R)-bisoprolol in human plasma [111]. Four macrocyclic antibiotic CSPs, Chirobiotic™ T, TAG, Me-TAG, and V, were compared with regard to the enantioseparation of 1-methyl-2-piperidinoethyl esters of 2-, 3-, and 4-alkoxyphenylcarbamic acid (potential local anesthetic drugs) [112]. The enantiomers were baseline separated in POM. The thermodynamic parameters revealed that the separation of the enantiomers on the Me-TAG CSP was enthalpy driven, while the separation on the vancomycin CSP was entropy driven. The highest resolution factors were achieved with a PIM system on Chirobiotic™ V, T, and TAG columns for the HPLC separation of enantiomers of potential β-blockers of the aryloxyaminopropanol type with a morpholino moiety in the hydrophilic part of the molecule [113]. The analysis of the enantiomers of the long-acting β2-adrenoceptor agonist terbutaline (used in the treatment of asthma) and its active metabolite bambuterol in rat plasma by an HPLC-MS/MS method was achieved using a Chirobiotic™ T column [114]. Another HPLC-MS/MS method employed a Chirobiotic™ TAG column for the enantioseparations of molindone used in the treatment of schizophrenia [115, 145]. The method was optimized and subsequently validated for analysis of patient plasma. Complete baseline separation was achieved under isocratic RPM conditions. The enantiomers of eflornithine used for the treatment of facial hirsutism [146] could be separated and analyzed in human plasma samples with a Chirobiotic™ TAG column using evaporative light scattering detection [116]. Four macrocyclic glycopeptide CSPs (Chirobiotic™ T, TAG, V, and R) have been investigated for the determination of the enantiomeric purity of 14 new chiral derivatives of xanthones under multimodal conditions (NPM, RPM, and PIM). The best enantioselectivity and resolution were achieved on Chirobiotic™ T and Chirobiotic™ R under NP condition [117].

Macrocyclic Glycopeptide-Based Chiral Stationary Phases

227

Enantiomers of β-blockers, propranolol, metoprolol, atenolol, pindolol [118], and carvedilol in plasma [119] were determined on Chirobiotic™ T with UV and MS detection, respectively. The best mobile-phase conditions were achieved using MeOH and TEA with different alcohol content. Enantioseparation of different profens was successfully carried out on Chirobiotic™ T and TAG [120–122]. For ketoprofen the best separation was achieved in RP mode [120]. Enantioseparation of pharmacologically active compounds in environmental samples such as ibuprofen, carboxyibuprofen, 2-hydroxyibuprofen, chloramphenicol, ifosfamide, indoprofen, ketoprofen, naproxen, and praziquantel was reported on Chirobiotic™ T by LC-MS technique in mobile phase 10 mM aq. NH4OAc(pH 4.2)/MeOH (70/30 v/v) [121]. PO mode containing MeOH and 2-PrOH was the best for the enantioresolution of ifosfamide and its metabolites [121]. Computational simulation indicated that the TAG basket provides rich environment with multiple charged centers, hydrophobic pockets, and functional groups that are available to participate in hydrophobic, H-bond, dipole-dipole, and cation-π interactions [122]. Chiral separation of novel cis- and trans-diastereomers of 2-amino analogs of indole phytoalexins was performed on Chirobiotic™ T containing n-hexane and EtOH or 2-PrOH as modifiers [123]. The thermodynamic study revealed that Δ(ΔH ) and Δ(ΔS ) were negative, meaning that the enantioseparation was enthalpically driven. Stereochemical characterization of fluorinated 2-(phenanthren-1-yl)propionic acid was carried out by enantioselective HPLC applying Chirobiotic™ T in RP mode and circular dichroism detection [124]. 3-n-Butylphthalide is administered for the clinical treatment of cerebral ischemia. Its enantioseparation was performed on Chirobiotic™ T at RP condition and MS detection [125]. β-Agonist salbutamol enantiomers were enantioresolved and determined in natural waters on Chirobiotic™ T with UV detection [126]. To maintain column performance additives such as TEA were avoided. The best mobile phase contained NH4OAc and MeOH. Separation of wake-promoting modafinil enantiomers was carried out on Chirobiotic™ T with MeOH/TEA mobile phases and UV detection [127]. The total analysis time was less than 6 min. The developed method was selective, precise, and robust. Separation of therapeutic peptides on Chirobiotic™ T and V CSPs showed that Chirobiotic™ T appears to function well in both HILIC and RP modes [128]. Teicoplanin and vancomycin antibiotic macrocyclic glycopeptide selectors were attached covalently to the surface of 2.7 μm superficially porous particles (SPPs) (TeicoShell and VancoShell, respectively). The core diameter and shell thickness of the SPPs were 1.7 μm and 0.5 μm, respectively. Ultrafast isocratic separations

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of enkephalin, bradykinin, vasopressin, LHRH peptides, and tryptic digest of equine apomyoglobin were achieved in acidic mobile phases containing ammonium formate as buffer and MS/MS detection techniques. Shorter retention times were obtained when THF was used as mobile-phase organic modifier, but higher efficiency was obtained when ACN was used in the eluent [129]. Chiral tobacco alkaloids, nicotine metabolites and derivatives, and tobacco-specific nitrosamines were successfully separated on newly developed modified macrocyclic glycopeptide selector covalently bonded to 2.7 μm SPPs (NicoShell) and TeicoShell [130]. Most frequently RP and PI mobile phases were applied, and all eluents were MS compatible. The study also provides optimal separation condition for the analyses of nicotine isomers [130]. Verubecestat is an inhibitor of β-site amyloid precursor protein-cleaving enzyme. Synthetic route developments involve diastereoselective transformations and determination of each intermediate and final active pharmaceutical ingredient (API) is basically important. TeicoShell allowed good enantioseparation of all verubecestat intermediates in mobile phases containing 0.1% TEAA/ MeOH (50/50, 75/25, 70/30 v/v) and 0.1% H3PO4/ACN (70/30 v/v) [131]. Enantioseparation and determination of verubecestat enantiomers (API) were easily performed on NicoShell with mobile phase 1.0% TEAA/ACN (40/60 v/v) [131]. (1S)-()-borneol- and (1R)-(þ)-fenchol-based chiral ionic liquids (CILs) as mobile-phase additives were applied to improve the enantioseparation of mandelic acid, vanilmandelic acid, and phenyllactic acid on Chirobiotic™ T CSP. The thermodynamic study indicated negative Δ(ΔH ) and Δ(ΔS ) values [132]. The same authors used new CILs based on (1R,2S,5R)-()-menthol as mobile-phase additive for the enantioseparation of acidic compounds mentioned above [133]. The conclusion was the same: enantioseparation was enthalpy driven. The enantiomeric biodegradation of ofloxacin and levofloxacin was monitored on Chirobiotic™ R with fluorescence and MS detection [134]. The best separation condition was obtained in the 0.45% aq. TEAA(pH 3.6)/EtOH (80/20 v/v) mobile-phase system. 6.2.3 Miscellaneous Compounds

The chiral analytes 2-, 3-, and 4-toluyl methyl sulfoxides with different 2-, 3-, and 4-halogen substituents on the aromatic ring were separated on Chirobiotic™ TAG and Chirobiotic™ T columns in the temperature range of 10–50  C [135, 136]. The method was also extended to the determination of chiral sulfoxides in human plasma [137]. The effects of different substituents and their positions in the aromatic ring of the sulfoxides on their enantioseparation were correlated with thermodynamic data. The sequence of elution of the sulfoxide enantiomers did not change in

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the temperature range investigated. The (S)-(þ) enantiomers always eluted first with the exception of the 4-(methylsulfinyl) biphenyl, where the (R)-() enantiomer was less retained. Heterohelicenium cations adopt a twisted helical structure that renders them chiral. The compounds are configurationally stable and their enantiomers were resolved for the first time by HPLC on Chirobiotic™ TAG CSP using water-based eluents containing potassium hexafluorophosphate (KPF6) as additive [138]. The effects of the mobile-phase composition and the analyte structure on the retention and enantioselectivity were investigated. The elution sequence of the analyte enantiomers was determined by online circular dichroism detection. Among the five different commercial macrocyclic antibioticbased CSPs (Chirobiotic™ T, T2, TAG, V, and R) the Chirobiotic™ T2 column was most effective in the enantioseparation of ruthenium(II) polypyridyl complexes [139]. All the complexes followed the same elution order. 6.3 Enantioseparations on Miscellaneous Macrocyclic Antibiotics

A recently introduced new CSP is based on the immobilization of the macrocyclic antibiotic eremomycin on epoxy-activated silica (Table 5). The application of the new CSP for the preparative enantioseparation of methionine using simulated moving-bed (SMB) chromatography was evaluated by Zhang et al. [147]. The column-to-column reproducibility was excellent and the long-term

Table 5 Enantioseparations on miscellaneous macrocyclic antibiotics Group of racemates Analytes Drugs

Selectors

The most effective mobile phases (v/v/v)

Mode Refs.

RPM [147] 100 mM NaH2PO4/MeOH (80/20 v/v) KH2PO4 (pH 4.5)/ Mixed Ketoprofen, fenoprofen, RPM [35] eremomycinindoprofen, ibuprofen, MeOH (50/50 v/v) bovine serum flurbiprofen albumin RPM [148] Metoprolol, pindolol, alprenolol, Eremomycin 0.1% aq. TEAA oxprenolol, labetalol, atenolol Vancomycin (pH 4.5)/MeOH/ Trp, Phe, DOPA, Met, Glu ACN (5/20/75 v/ v/v) ACN/AcOH (97/3 v/ v) α-Phenylcarboxylic acids Eremomycin EtOH/H2O RPM [149] (30/70–70/30 v/v) 0.1% NH4OAc/MeOH RPM [150] Heterocyclic compounds, acids, Dalbavancin NPM amines, alcohols, sulfoxides (50/50 v/v) and sulfilimines, amino acids n-Heptane/EtOH (80/20 v/v) Methionine

Eremomycin

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stability of the preparative stationary phase was satisfactory according to the results of perturbation experiments performed before and after long-term SMB runs. A mixed eremomycin-bovine serum albumin (BSA) selector was constructed by immobilization on silica surface [36]. This CSP was successfully operated for the enantioresolution of different profens. β-Blockers and amino acid enantiomers were resolved on eremomycin and vancomycin-based CSPs [148]. For β-blockers RP mode containing 0.1% TEAA/MeOH/ ACN was applied, while for amino acids ACN/AcOH mobilephase system ensured the best separation. Adsorption of enantiomers of α-phenylcarboxylic acids on silica gel with immobilized eremomycin was investigated in aq. EtOH mobile phase [149]. The experimental data showed that an increase in the content of organic modifier (EtOH) lowered the retention and resolution factor. The thermodynamic parameters ΔH and TxΔS were lowest at ca. 40–50% EtOH content. Dalbavancin is a new compound in the macrocyclic glycopeptide family which has been immobilized to silica gel. Approximately 250 racemates, including heterocyclic compounds, chiral acids, chiral amines, chiral alcohols, chiral sulfoxides and sulfilimines, amino acids, and amino acid derivatives were tested on the new CSPs [150]. As dalbavancin is structurally related to teicoplanin, the same set of chiral compounds was screened on two commercially available teicoplanin CSPs, Chirobiotic™ T and T2, for comparison. The dalbavancin CSPs were complementary to the teicoplanin CSPs. References 1. Armstrong DW (1994) A new class of chiral selectors for enantiomeric separations by LC, TLC, GC, CE and SFC. In: Pittsburg conference abstracts. p. 572. 2. Armstrong DW, Tang Y, Chen S, Zhou Y, Bagwill C, Chen JR (1994) Macrocyclic antibiotics as a new class of chiral selectors for liquid-chromatography. Anal Chem 66:1473–1484 3. Xiao TL, Armstrong DW (2004) Enantiomeric separation by HPLC using macrocyclic glycopeptide-based chiral stationary phases. In: Gu¨bitz G, Schmid MG (eds) Chiral separations. Methods and protocols. Humana Press, Totowa, pp 113–171 4. Beesley TE, Scott RPW (1998) Chapter 8. In: Beesley TE, Scott RPW (eds) Chiral chromatography. Wiley, Chichester, pp 221–263 5. Bojarski J (1999) Antibiotics as electrophoretic and chromatographic chiral selectors. Wiadom Chem 53:235–247 6. Dolezalova M, Tkaczykova M (2000) Control of enantiomeric purity of drugs. Chem Listy 94:994–1003

7. Ward TJ, Farris AB (2001) Chiral separations using the macrocyclic antibiotics: a review. J Chromatogr A 906:73–89 8. Gasparrini F, D’Acquarica I, Misiti D, Pierini M, Villani C (2003) Natural and totally synthetic receptors in the innovative design of HPLC chiral stationary phases. Pure Appl Chem 75:407–412 9. Dungelova M, Lehotay J, Rojkovicova T (2003) Chiral separations of drugs based on macrocyclic antibiotics in HPLC, SFC and CEC. Ceska Slov Farm 52:119–125 10. Dungelova J, Lehotay J, Rojkovicova T (2004) HPLC chiral separations utilising macrocyclic antibiotics—a review. Chem Anal 49:1–17 11. Ali I, Kumerer K, Aboul-Enein HY (2006) Mechanistic principles in chiral separations using liquid chromatography and capillary electrophoresis. Chromatographia 63:295–307 12. Beesley TE, Lee JT (2007) Chiral separation techniques, 3rd edn. Wiley-VCH, Weinheim 13. D’Acquarica I, Gasparrini F, Misiti D, Pierini M, Villani C (2008) HPLC chiral

Macrocyclic Glycopeptide-Based Chiral Stationary Phases stationary phases containing macrocyclic antibiotics: practical aspects and recognition mechanism. Adv Chromatogr 46:109–173 14. Ilisz I, Berkecz R, Pe´ter A (2006) HPLC separation of amino acid enantiomers and small peptides on macrocyclic antibioticbased chiral stationary phases: a review. J Sep Sci 29:1305–1321 15. Ilisz I, Berkecz R, Pe´ter A (2009) Retention mechanism of high-performance liquid chromatographic enantioseparation on macrocyclic glycopeptide-based chiral stationary phases. J Chromatogr A 1216:1845–1860 16. Ilisz I, Pataj Z, Pe´ter A (2010) Macrocyclic glycopeptide-based chiral stationary phases in high performance liquid chromatographic analysis of amino acid enantiomers and related analogs. In: Fitzpatrick DW, Ulrich HJ (eds) Macrocyclic chemistry: new research developments. Nova Science Publishers, Hauppauge, pp 129–157 17. Cavazzini A, Pasti L, Massi A, Marchetti N, Dondi F (2011) Recent applications in chiral high performance liquid chromatography: a review. Anal Chim Acta 706:205–222 18. Scriba GKE (2012) Chiral recognition mechanisms in analytical separation sciences. Chromatographia 75:815–838 19. Ali I, Al-Othman ZA, Al-Warthan A, Asnin L, Chudinov A (2014) Advances in chiral separations of small peptides by capillary electrophoresis and chromatography. J Sep Sci 37:2447–2466 20. Al-Othman ZA, Al-Warthan A, Ali I (2014) Advances in enantiomeric resolution on monolithic chiral stationary phases in liquid chromatography and electrochromatography. J Sep Sci 37:1033–1057 21. Scriba GKE (2016) Chiral recognition in separation science—an update. J Chromatogr A 1467:56–78 22. Gasper M, Berthod A, Nair UB, Armstrong DW (1996) Comparison and modeling study of vancomycin, ristocetin A, and teicoplanin. Anal Chem 68:2501–2514 23. Gause GF, Brazhnikova MG, Lomakina NN, Berdnikova TF, Fedorova GB, Tokareva N, Borisova VN, Batta GY (1989) Eremomycin—new glycopeptide antibiotic: chemical properties and structure. J Antibiot 42:1790–1799 24. Berdnikova TF, Shashkov AS, Katrukha GS, Lapchinskaya OA, Yurkevich NV, Grachev AA, Nifant’ev NE (2009) The structure of antibiotic eremomycin B. Russian J Bioorg Chem 35:497–503

231

25. Nadkarni SR, Patel MV, Chatterjee S, Vijayakumar EK, Desikan KR, Blumbach J, Ganguli BN, Limbert M (1994) Balhimycin, a new glycopeptide antibiotic produced by Amycolatopsis sp. Y-86,21022. Taxonomy, production, isolation and biological activity. J Antibiot 47:334–341 26. Chatterjee S, Vijayakumar EKS, Nadkarni SR, Patel MV, Blumbach J, Ganguli BN (1994) Balhimycin, a new glycopeptide antibiotic with an unusual hydrated 3-amino-4-oxoaldopyranose sugar moiety. J Org Chem 59:3480–3484 27. Pelzer S, Su¨ßmuth R, Heckmann D, Recktenwald J, Huber P, Jung G, Wohlleben W (1999) Identification and analysis of the balhimycin biosynthetic gene cluster and its use for manipulating glycopeptide biosynthesis in Amycolatopsis Mediterranei DSM5908. Antimicrob Agents Chemother 43:1565–1573 28. L€ammerhofer M (2010) Chiral recognition by enantioselective liquid chromatography: mechanisms and modern chiral stationary phases. J Chromatogr A 1217:814–856 29. Beesley TE, Lee JT (2009) Method development strategy and applications update for Chirobiotic chiral stationary phases. J Liquid Chrom & Related Tech 32:1733–1767 30. Aboul-Enein HY, Ali I (2003) Macrocyclic glycopeptide antibiotics-based chiral stationary phases. In: Aboul-Enein HY, Wainer IW (eds) Chiral separations by liquid chromatography and related technologies. Marcel Dekker, Inc., New York, pp 137–175 31. Hrobonova K, Lehotay J, Cizmarikova R, Armstrong DW (2001) Study of the mechanism of enantioseparation. I. Chiral analysis of alkylamino derivatives of aryloxypropanols by HPLC using macrocyclic antibiotics as chiral selectors. J Liquid Chrom & Related Tech 24:2225–2237 32. Ghassempour A, Abdollahpour A, TabarHeydar K, Nabid MR, Mansouri S, AboulEnein H (2005) Crystalline degradation products of vancomycin as a new chiral stationary phase for liquid chromatography. Chromatographia 61:151–155 33. Armstrong DW, DeMond W (1984) Cyclodextrin bonded phases for the liquid chromatographic separation of optical, geometrical, and structural isomers. J Chromatogr Sci 22:411–415 34. Petrusevska K, Kuznetsov MA, Gedicke K, Meshko V, Staroverov SM, SidelMorgenstern A (2006) Chromatographic enantioseparation of amino acids using a new chiral stationary phase based on a macrocyclic

232

Istva´n Ilisz et al.

glycopeptide antibiotic. J Sep Sci 29:1447–1457 35. Staroverov SM, Kuznetsov MA, Nesterenko PN, Vasiarov GG, Katrukha GS, Fedorova GB (2006) New chiral stationary phase with macrocyclic glycopeptide antibiotic eremomycin chemically bonded to silica. J Chromatogr A 1108:263–267 36. Fedorova IA, Shapovalova EN, Shpigun OA, Staroverov SM (2016) Bovine serum albumin adsorbed on eremomycin and grafted on silica as new mixed-binary chiral sorbent for improved enantioseparation of drugs. J Food Drug Anal 24:848–854 37. Ernst-Cabrera K, Wilchek M (1986) Silica containing primary hydroxyl groups for high performance affinity chromatography. Anal Biochem 159:267–272 38. Svensson LA, Karlsson KE, Karlsson A, Vessman J (1998) Immobilized vancomycin as chiral stationary phase in packed capillary liquid chromatography. Chirality 10:273–280 39. Do¨nnecke J, Svensson LA, Gyllenhaal O, Karlsson KE, Karlsson A, Vessman J (1999) Evaluation of a vancomycin chiral stationary phase in packed capillary supercritical fluid chromatography. J Microcol Sep 11:521–533 40. Svensson LA, Do¨nnecke J, Karlsson KE, Karlsson A, Vessman J (1999) Vancomycin based chiral stationary phases for micro column liquid chromatography. Chirality 11:121–128 41. Wikstro¨m H, Svensson LA, Torstensson A, Owens PK (2000) Immobilisation and evaluation of a vancomycin chiral stationary phase for capillary electrochromatography. J Chromatogr A 869:395–409 42. Desiderio C, Aturki Z, Fanali S (2001) Use of vancomycin silica stationary phase in packed capillary electrochromatography I. Enantiomer separation of basic compounds. Electrophoresis 22:535–543 43. Svensson LA, Owens PK (2000) Enantioselective supercritical fluid chromatography using ristocetin A chiral stationary phases. Analyst 125:1037–1039 44. Fanali S, Catarcini P, Presutti C, Stancanelli R, Quaglia MG (2003) Use of short-end injection capillary packed with a glycopeptide antibiotic stationary phase in electrochromatography and capillary liquid chromatography for the enantiomeric separation of hydroxy acids. J Chromatogr A 990:143–151 45. Fanali S, Catarcini P, Presutti C (2003) Enantiomeric separation of acidic compounds of pharmaceutical interest by capillary

electrochromatography employing glycopeptide antibiotic stationary phases. J Chromatogr A 994:227–232 46. D’Acquarica I (2000) New synthetic strategies for the preparation of novel chiral stationary phases for high-performance liquid chromatography containing natural pool selectors. J Pharm Biomed Anal 23:3–13 47. Chirobiotic Handbook, 5th ed. Guide to using macrocyclic glycopeptide bonded phases for chiral LC separations. Whippany, NJ: Astec, Advanced Separation Technologies Inc.; 2004. 48. Berthod A, Yu T, Kullman JP, Armstrong DW, Gasparrini F, D’Acquarica I, Misiti D, Carotti A (2000) Evaluation of the macrocyclic glycopeptide A-40,926 as a highperformance liquid chromatographic chiral selector and comparison with teicoplanin chiral stationary phase. J Chromatogr A 897:113–129 49. Berthod A, Chen X, Kullman JP, Armstrong DW, Gasparrini F, D’Acquarica I, Villani C, Carotti A (2000) Role of the carbohydrate moieties in chiral recognition on teicoplaninbased LC stationary phases. Anal Chem 72:1767–1780 50. Diana J, Visky D, Roets E, Hoogmartens J (2003) Development and validation of an improved method for the analysis of vancomycin by liquid chromatography. Selectivity of reversed-phase columns towards vancomycin components. J Chromatogr A 996:115–131 51. Anan’eva A, Polyakova Ya A, Shapovalova EN, Mazhuga AG, Shpigun OA (2018) Separation of β-blocker enantiomers on silica modified with gold nanoparticles with immobilized macrocyclic antibiotic vancomycin. J Anal Chem 73:152–115 52. Yu B, Zhang S, Li G, Cong H (2018) Lightassisted preparation of vancomycin chiral stationary phase based on diazotized silica and its enantioseparation evaluation by highperformance liquid chromatography. Talanta 182:171–177 53. Berthod A (2006) Chiral recognition mechanisms. Anal Chem 78:2093–2099 54. Berthod A (2009) Chiral recognition mechanisms with macrocyclic glycopeptide selectors. Chirality 21:167–175 55. Berthod A, Qiu HX, Staroverov S, Kuznestov MA, Armstrong DW (2010) Chiral recognition with macrocyclic glycopeptides: mechanisms and applications. In: Berthod A (ed) Chiral recognition in separation methods:

Macrocyclic Glycopeptide-Based Chiral Stationary Phases mechanisms and applications. Springer, Heidelberg, pp 203–222 56. Ilisz I, Pataj Z, Aranyi A, Pe´ter A (2012) Macrocyclic antibiotic selectors in direct HPLC enantioseparations. Sep Pur Rev 41:207–249 57. El Deeb S (2010) Evaluation of a Vancomycin-based LC column in enantiomeric separation of atenolol: method development, repeatability study and enantiomeric impurity determination. Chromatographia 71:783–787 58. Yang J, Lu XM, Bi YJ, Qin F, Li FM (2007) Chiral separation of duloxetine and its R-enantiomer by LC. Chromatographia 66:389–393 59. Xu Z, Zhou N, Xu X, Xu XX (2007) Enantioseparation of rivastigmine by high performance liquid chromatography using vancomycin chiral stationary phase. Chin J Anal Chem 35:1043–1046 60. Zuo Z, Wo SK, Lo CMY, Zhou L, Cheng G, You JHS (2010) Simultaneous measurement of S-warfarin, R-warfarin, S-7-hydroxywarfarin and R-7-hydroxywarfarin in human plasma by liquid chromatography–tandem mass spectrometry. J Pharm Biomed Anal 52:305–310 61. Malakova J, Pavek P, Svecova L, Jokesova I, Zivny P, Palicka V (2009) New highperformance liquid chromatography method for the determination of (R)-warfarin and (S)warfarin using chiral separation on a glycopeptide-based stationary phase. J Chromatogr B 877:3226–3230 62. Liu W, Wang F, Li H (2007) Simultaneous stereoselective analysis of venlafaxine and O-desmethylvenlafaxine enantiomers in human plasma by HPLC-ESI/MS using a vancomycin chiral column. J Chromatogr B 850:183–189 63. Hefnawy MM, Al-Shehri MM (2010) Chiral stability-indicating HPLC method for analysis of arotinolol in pharmaceutical formulation and human plasma. Arabian J Chem 3:147–153 64. Hefnawy MM, Sultan MA, Al-Shehri MM (2007) HPLC separation technique for analysis of bufuralol enantiomers in plasma and pharmaceutical formulations using a vancomycin chiral stationary phase and UV detection. J Chromatogr B 856:328–336 65. Hashem H, Tru¨ndelberg C, Attef O, Jira T (2011) Effect of chromatographic conditions on liquid chromatographic chiral separation of terbutaline and salbutamol on Chirobiotic V column. J Chromatogr A 1218:6727–6731

233

66. Rao RN, Kumar KN, Ramakrishna S (2011) Enantiomeric separation of mirtazapine and its metabolite in rat plasma by reverse polar ionic liquid chromatography using fluorescence and polarimetric detectors connected in series. J Chromatogr B-Anal Techn Biomed Life Sci 879:1911–1916 67. Hefnawy MM, Asiri AJ, Al-Zoman NZ, Mostafa GA, Aboul-Enein HY (2011) Stereoselective HPLC analysis of tertatolol in rat plasma using macrocyclic antibiotic chiral stationary phase. Chirality 23:333–338 68. Jing L, Li K, Qin F, Wang X, Pan L, Wang Y, Cheng M, Li F (2012) Determination of L-trantinterol in rat plasma by using chiral liquid chromatography-tandem mass spectrometry. J Sep Sci 35:2678–2684 69. Qin F, Wang Y, Wang L, Zhao L, Pan L, Cheng M, Li F (2015) Determination of trantinterol enantiomers in human plasma by high-performance liquid chromatography— tandem mass spectrometry using vancomycin chiral stationary phase and solid phase extraction and stereoselective pharmacokinetic application. Chirality 27:327–331 70. Wagdy HA, Hanafi RS, El-Nashar RM, Aboul-Enein HY (2013) Predictability of enantiomeric chromatographic behavior on various chiral stationary phases using typical reversed phase modeling software. Chirality 25:506–513 71. Hefnawy MM, Al-Shehri MM, Abounassif MA, Mostafa GAE (2013) Enantioselective quantification of atenolol in mouse plasma by high performance liquid chromatography using a chiral stationary phase: application to a pharmacokinetic study. J AOAC Int 96:976–980 72. Ribeiro AR, Afonso CM, Castro PML, Tiritan ME (2013) Enantioselective HPLC analysis and biodegradation of atenolol, metoprolol and fluoxetine. Environ Chem Lett 11:83–90 73. Ribeiro AR, Afonso CM, Castro PML, Tiritan ME (2013) Enantioselective biodegradation of pharmaceuticals, alprenolol and propranolol, by an activated sludge inoculum. Ecotox Environ Safe 87:108–114 74. Ribeiro AR, Maia AS, Moreira IS, Afonso CM, Castro PML, Tiritan ME (2014) Enantioselective quantification of fluoxetine and norfluoxetine by HPLC CrossMark in wastewater effluents. Chemosphere 95:589–596 75. Wang T, Shen B, Shi Y, Xiang P, Yu Z (2015) Chiral separation and determination of R/Smethamphetamine and its metabolite R/Samphetamine in urine using LC-MS/MS. Forensic Sci Int 246:72–78

234

Istva´n Ilisz et al.

76. Popovic A, McBriar T, He P, Beavis A (2017) Chiral determination and assay of optical isomers in clandestine drug laboratory samples using LC-MSMS. Anal-Methods UK 9:3380–3387 77. Gherdaoui D, Bekdouche H, Zerkout S, Fegas R, Righezza M (2016) Chiral separation of ketoprofen on an achiral NH2 column by HPLC using vancomycin as chiral mobile phase additive. J Iran Chem Soc 13:2319–2323 78. Boronova K, Lehotay J, Hrobonova K, Armstrong DW (2013) Study of physicochemical interaction of aryloxyaminopropanol derivatives with teicoplanin and vancomycin phases in view of quantitative structure-property relationship studies. J Chromatogr A 1301:38–47 79. Ramisetti NR, Bompelli S (2014) LC-MS/ MS determination of cinacalcet enantiomers in rat plasma on Chirobiotic V column in polar ionic mode: application to a pharmacokinetic study. Biomed Chromatogr 28:1846–1853 80. Phyo YZ, Cravo S, Palmeira A, Tiritan ME, Kijjoa A, Pinto MMM, Fernandes C (2018) Enantiomeric resolution and docking studies of chiral xanthonic derivatives on chirobiotic columns. Molecules 23(1). pii: E142). https://doi.org/10.3390/ molecules23010142 81. Abdollahpour A, Heydari R, Shamsipur M (2017) Two synthetic methods for preparation of chiral stationary phases using crystalline degradation products of vancomycin: column performance for enantioseparation of acidic and basic drugs. AAPS Pharm Sci Tech 18:1855–1862 82. Mojtahedi MM, Chaiavi S, Ghassempour A, Tabar-Heydar K, Sharif SJG, Malekzadeh M, Aboul-Enein HY (2007) Chiral separation of three agrochemical toxins enantiomers by high-performance liquid chromatography on a vancomycin crystalline degradation products-chiral stationary phase. Biomed Chromatogr 21:234–240 83. Boesten JMM, Berkheij M, Schoemaker HE, Hiemstra H, Duchateau ALL (2006) Enantioselective high-performance liquid chromatographic separation of Nmethyloxycarbonyl unsaturated amino acids on macrocyclic glycopeptide stationary phases. J Chromatogr A 1108:26–30 84. Zhao J, Golozoubova V, Cannon B, Nedergaard J (2001) Arotinolol is a weak partial agonist on beta 3-adrenergic receptors in brown adipocytes. Can J Physiol Pharmacol 79:585–593

85. Xiao TL, Tesarova E, Anderson JL, Egger M, Armstrong DW (2006) Evaluation and comparison of a methylated teicoplanin aglycone to teicoplanin aglycone and natural teicoplanin chiral stationary phases. J Sep Sci 29:429–445 86. Honetschlagerova VM, Srkalova S, Bosakova Z, Coufal P, Tesarova E (2009) Comparison of enantioselective HPLC separation of structurally diverse compounds on chiral stationary phases with different teicoplanin coverage and distinct linkage chemistry. J Sep Sci 32:1704–1711 87. Poplewska KR, Pitkowski W, SeidelMorgenstern A, Antos D (2007) Influence of preferential adsorption of mobile phase on retention behavior of amino acids on the teicoplanin chiral selector. J Chromatogr A 1173:58–70 88. Haroun M, Ravelet C, Grosset C, Ravel A, Villet A, Peyrin E (2006) Reversal of the enantiomeric elution order of some aromatic amino acids using reversed-phase chromatographic supports coated with the teicoplanin chiral selector. Talanta 68:1032–1036 89. Pataj Z, Ilisz I, Aranyi A, Forro E, Fu¨lo¨p F, Armstrong DW, Pe´ter A (2010) LC separation of γ-amino acid enantiomers. Chromatographia 71:13–19 90. Berkecz R, Ilisz I, Benedek G, Fu¨lo¨p F, Armstrong DW, Pe´ter A (2009) Highperformance liquid chromatographic enantioseparation of 2-aminomono- and dihydroxycyclopentanecarboxylic and 2-aminodihydroxycyclohexanecarboxylic acids on macrocyclic glycopeptide-based phases. J Chromatogr A 1216:927–932 91. Sipos L, Ilisz I, Pataj Z, Szakonyi Z, Fu¨lo¨p F, Armstrong DW, Pe´ter A (2010) Highperformance liquid chromatographic enantioseparation of monoterpene-based 2-amino carboxylic acids on macrocyclic glycopeptidebased phases. J Chromatogr A 1217:6956–6963 92. Bechtold M, Felinger A, Held M, Panke S (2007) Adsorption behavior of a teicoplanin aglycone bonded stationary phase under harsh overload conditions. J Chromatogr A 1154:277–286 93. Sipos L, Ilisz I, Nonn M, Fu¨lo¨p F, Pataj Z, Armstrong DW, Pe´ter A (2012) Highperformance liquid chromatographic enantioseparation of unusual isoxazoline-fused 2-aminocyclopentanecarboxylic acids on macrocyclic glycopeptide-based chiral stationary phases. J Chromatogr A 1232:142–151 94. Pataj Z, Ilisz I, Grecso´ N, Palko´ M, Fu¨lo¨p F, Armstrong DW, Pe´ter A (2014) Enantiomeric

Macrocyclic Glycopeptide-Based Chiral Stationary Phases separation of bicyclo[2.2.2]octane-based 2-amino-3-carboxylic acids on macrocyclic glycopeptide chiral stationary phases. Chirality 26:200–208 95. Ilisz I, Grecso´ N, Forro´ E, Fu¨lo¨p F, Armstrong DW (2015) High-performance liquid chromatographic separation of paclitaxel intermediate phenylisoserine derivatives on macrocyclic glycopeptide and cyclofructanbased chiral stationary phases. J Pharm Biomed Anal 114:312–320 96. Barhate CL, Wahab MF, Breitbach ZS, Bell DS, Armstrong DW (2015) High efficiency, narrow particle size distribution, sub-2 μm based macrocyclic glycopeptide chiral stationary phases in HPLC and SFC. Anal Chim Acta 898:128–137 97. Orosz T, Grecso N, Gy L, Zs S, Fu¨lo¨p F, Armstrong DW, Ilisz I, Pe´ter A (2017) Liquid chromatographic enantioseparation of carbocyclic beta-amino acids possessing limonene skeleton on macrocyclic glycopeptide-based chiral stationary phases. J Biomed Anal 145:119–126 98. Fuereder M, Panke S, Bechtold M (2012) Simulated moving bed enantioseparation of amino acids employing memory effectconstrained chromatography columns. J Chromatogr A 1236:123–131 99. Kucerova G, Vozka J, Kalikova K, Geryk R, Plecita D, Pajpanova T, Tesarova E (2013) Enantioselective separation of unusual amino acids by high performance liquid chromatography. Sep Purif Technol 119:123–128 100. Barbaro E, Zangrando R, Vecchiato M, Turetta C, Barbante C, Gambaro A (2014) D- and L-amino acids in Antarctic lakes: assessment of a very sensitive HPLC-MS method. Anal Bioanal Chem 406:5259–5270 101. Sardella R, Ianni F, Lisanti A, Scorzoni S, Marini F, Sternativo S, Natalini B (2014) Direct chromatographic enantioresolution of fully constrained beta-amino acids: exploring the use of high-molecular weight chiral selectors. Amino Acids 46:1235–1242 102. Deakova Z, Durackova Z, Armstrong DW, Lehotay J (2015) Separation of enantiomers of selected sulfur-containing amino acids by using serially coupled achiral-chiral columns. J Liquid Chromatogr Rel Technol 38:789–794 103. Deakova Z, Durackova Z, Armstrong DW, Lehotay J (2015) Two-dimensional high performance liquid chromatography for determination of homocysteine, methionine and cysteine enantiomers in human serum. J Chromatogr A 1408:118–124

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104. Bystricka Z, Bystricky R, Lehotay J (2016) Thermodynamic study of HPLC enantioseparations of some sulfur-containing amino acids on teicoplanin columns in ion-pairing reversed-phase mode. J Liquid Chromatogr Rel Technol 39:775–781 105. Wagdy HA, Hanafi RS, El-Nashar RM, Aboul-Enein HY (2014) Enantiomeric separation of underivatized amino acids: predictability of chiral recognition on ristocetin A chiral stationary phase. Chirality 26:132–135 106. Ismail OH, Ciogli A, Villani C, Martino MD, Pierini M, Cavazznini A, Bell DS, Gasparrini F (2016) Ultra-fast high-efficiency enantioseparations by means of a teicoplanin-based chiral stationary phase made on sub-2 μm totally porous silica particles of narrow size distribution. J Chromatogr A 1427:55–68 107. Min Y, Sui Z, Liang Z, Zhang L, Zhang Y (2015) Teicoplanin bonded sub-2-μm superficially porous particles for enantioseparation of native amino acids. J Pharm Biomed Anal 114:247–253 108. Sanchez-Hernandez L, Bernal JL, Jesus del Nozal M (2016) Chiral analysis of aromatic amino acids in food supplements using subcritical fluid chromatography and Chirobiotic T2 column. J Supercrit Fluid 107:519–525 109. Zhang YZ, Holliman C, Tang D, Fast D, Michael S (2008) Development and validation of a direct enantiomeric separation of pregabalin to support isolated perfused rat kidney studies. J Chromatogr B 875:148–153 110. Al-Majed AA (2009) A direct HPLC method for the resolution and quantitation of the R-()- and S-(þ)-enantiomers of vigabatrin (γ-vinyl-GABA) in pharmaceutical dosage forms using teicoplanin aglycone chiral stationary phase. J Pharm Biomed Anal 50:96–99 111. Hefnawy MM, Sultan MAA, Al-Shehri MM (2007) Enantioanalysis of bisoprolol in human plasma with a macrocyclic antibiotic HPLC chiral column using fluorescence detection and solid phase extraction. Chem Pharm Bull 55:227–230 112. Rojkovicova T, Lehotay J, Armstrong DW, Cizmarik J (2006) Study of the mechanism of enantioseparation. Part XII. Comparison study of thermodynamic parameters on separation of phenylcarbamic acid derivatives by HPLC using macrocyclic glycopeptide chiral stationary phases. J Liq Chrom Rel Techn 29:2615–2624 113. Hrobonova K, Lehotay J, Cizmarikova R (2005) HPLC separation of enantiomers of some potential beta-blockers of the aryloxyaminopropanol type using macrocyclic

236

Istva´n Ilisz et al.

antibiotic chiral stationary phases—studies of the mechanism of enantioseparation, Part XI. Pharmazie 60:888–891 114. Luo W, Zhu L, Deng J, Liu A, Guo B, Tan W, Dai R (2010) Simultaneous analysis of bambuterol and its active metabolite terbutaline enantiomers in rat plasma by chiral liquid chromatography–tandem mass spectrometry. J Pharm Biomed Anal 52:227–231 115. Aparasu RR, Jano E, Johnson ML, Chen H (2008) Hospitalization risk associated with typical and atypical antipsychotic use in community-dwelling elderly patients. Am J Geriatr Pharmacother 6:198–204 116. Malma M, Bergqvista Y (2007) Determination of eflornithine enantiomers in plasma, by solid-phase extraction and liquid chromatography with evaporative light-scattering detection. J Chromatogr B 846:98–104 117. Fernandes C, Tiritan ME, Cass Q, Kairys V, Fernandes XM, Pinto M (2012) Enantioseparation and chiral recognition mechanism of new chiral derivatives of xanthones on macrocyclic antibiotic stationary phases. J Chromatogr A 1241:60–68 118. Morante-Zarcero S, Sierra I (2012) Comparative HPLC methods for beta-blockers separation using different types of chiral stationary phases in normal phase and polar organic phase elution modes. Analysis of propranolol enantiomers in natural waters. J Pharm Biomed Anal 62:33–41 119. Poggi JC, Da Silva FG, Coelho EB, Marques PM, Bertucci C, Lanchote LV (2012) Analysis of carvedilol enantiomers in human plasma using chiral stationary phase column and liquid chromatography with tandem mass spectrometry. Chirality 24:209–214 120. He X, Lin R, He H, Sun M, Xiao D (2012) Chiral separation of ketoprofen on a chirobiotic T column and its chiral recognition mechanisms. Chromatographia 75:1355–1363 121. Camacho-Munoz D, Kasprzyk-Hordern B (2017) Simultaneous enantiomeric analysis of pharmacologically active compounds in environmental samples by chiral LC–MS/ MS with a macrocyclic antibiotic stationary phase. J Mass Spectrom 52:94–108 122. Ravichandran S, Collins JR, Singh N, Wainer IW (2012) A molecular model of the enantioselective liquid chromatographic separation of (R,S)-ifosfamide and its N-dechloroethylated metabolites on a teicoplanin aglycon chiral stationary phase. J Chromatogr A 1269:218–225

123. Gondova T, Petrovaj J, Kutschy P, Curillova Z, Salayova A, Fabian M, Armstrong DW (2011) Enantioseparation of novel amino analogs of indole phytoalexins on macrocyclic glycopeptide-based chiral stationary phase. Chromatographia 74:751–757 124. Bertucci C, Pistolozzi M, Tedesco D, Zanasi R, Ruzziconi R, Pietra DMA (2012) Stereochemical characterization of fluorinated 2-(phenanthren-1-yl)propionic acids by enantioselective high performance liquid chromatography analysis and electronic circular dichroism detection. J Chromatogr A 1232:128–133 125. Diao X, Ma Z, Lei P, Zhong D, Zhang Y, Chen X (2013) Enantioselective determination of 3-n-butylphthalide (NBP) in human plasma by liquid chromatography on a teicoplanin-based chiral column coupled with tandem mass spectrometry. J Chromatogr B 939:67–72 126. Rosales-Conrado N, Dell’Aica M, Eugenia de Leon-Gonzalez M, Perez-Arribas LV, PoloDiez LM (2013) Determination of salbutamol by direct chiral reversed-phase HPLC using teicoplanin as stationary phase and its application to natural water analysis. Biomed Chromatogr 27:1413–1422 127. Harvanova M, Gondova T (2017) New enantioselective LC method development and validation for the assay of modafinil. J Pharm Biomed Anal 138:267–271 128. Shu Y, Lang JC, Breitbach ZS, Qiu H, Smuts JP, Kiyono-Shimobe M, Yasuda M, Armstrong DW (2015) Separation of therapeutic peptides with cyclofructan and glycopeptide based columns in hydrophilic interaction liquid chromatography. J Chromatogr A 1390:50–61 129. Wimalasinghe RM, Breitbach ZS, Lee JT, Armstrong DW (2017) Separation of peptides on superficially porous particle based macrocyclic glycopeptide liquid chromatography stationary phases: consideration of fast separations. Anal Bioanal Chem 409:2437–2447 130. Hellinghausen G, Roy D, Wang Y, Lee JT, Lopez DA, Weatherly CA, Armstrong DW (2018) A comprehensive methodology for the chiral separation of 40 tobacco alkaloids and their carcinogenic E/Z-(R,S)-tobaccospecific nitrosamine metabolites. Talanta 181:132–141 131. Barhate CL, Lopez DA, Makarov AA, Bu X, Morris WJ, Lekhal A, Hartman R, Armstrong DW (2018) Macrocyclic glycopeptide chiral selectors bonded to core-shell particles enables enantiopurity analysis of the entire

Macrocyclic Glycopeptide-Based Chiral Stationary Phases verubecestat synthetic route. J Chromatogr A 1539:87–92 132. Feder-Kubis J, Flieger J, TatarczakMichalewska M, Plazinska A, Madejska A, Swatko-Ossor M (2017) Renewable sources from plants as the starting material for designing new terpene chiral ionic liquids used for the chromatographic separation of acidic enantiomers. RSC Adv 7:32344–32356 133. Flieger J, Feder-Kubis J, TatarczakMichalewska M, Plazinska A, Madejska A, Swatko-Ossor M (2017) Natural terpene derivatives as new structural task-specific ionic liquids to enhance the enantiorecognition of acidic enantiomers on teicoplaninbased stationary phase by high-performance liquid chromatography. J Sep Sci 40:2374–2381 134. Maia AS, Castro PML, Tiritan ME (2016) Integrated liquid chromatography method in enantioselective studies: Biodegradation of ofloxacin by an activated sludge consortium. J Chromatogr B 1029–1030:174–183 135. Mericko D, Lehotay J, Skacani I (2006) Effect of temperature on retention and enantiomeric separation of chiral sulfoxides using teicoplanin aglycone chiral stationary phase. J Liq Chrom Rel Techn 29:623–638 136. Mericko D, Lehotay J, Skacani I (2007) Separation and thermodynamic studies of chiral sulfoxides on teicoplanin-based stationary phase. J Liq Chrom Rel Techn 30:1401–1420 137. Mericko D, Lehotay J, Cizmarik J (2008) Enantioseparation of chiral sulfoxides using teicoplanin chiral stationary phases and kinetic study of decomposition in human plasma. Pharmazie 63:854–859 138. Villani C, Laleu B, Mobian P, Lacour J (2007) Effective HPLC resolution of [4] heterohelicenium dyes on chiral stationary phases using reversed-phase eluents. Chirality 19:601–606 139. Sun P, Krishnan A, Yadav A, MacDonnell FM, Armstrong DW (2008) Enantioseparations next term of chiral ruthenium (II) polypyridyl complexes using HPLC with macrocyclic glycopeptide chiral stationary phases (CSPs). J Mol Struct 890:75–80 140. Crofford LJ, Rowbotham MC, Mease PJ, Russell IJ, Dworkin RH, Corbin AE, Young JP, LaMoreaux LK, Martin SA, Sharma U (2005) Pregabalin for the treatment of fibromyalgia syndrome: results of a randomized, double-blind, placebo-controlled trial. Arthritis Rheum 52:1264–1273 141. Svanfelt J, Eriksson J, Kronberg L (2010) Analysis of thyroid hormones in raw and treated wastewater. J Chromatogr A 1217:6469–6474

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142. Koidl J, Hodl H, Schmid MG, Neubauer B, Konrad M, Petschauer S, Gubitz G (2008) Enantiorecognition of triiodothyronine and thyroxine enantiomers using different chiral selectors by HPLC and micro-HPLC. J Biochem Biophysical Meth 70:1254–1260 143. Gondova T, Petrovaj J, Sucha M, Armstrong DW (2011) Stereoselective HPLC determination of thyroxine enantiomers in pharmaceuticals. J Liquid Chromatogr Rel Technol 34:2304–2314 144. Bu¨hring KU, Sailer H, Faro HP, Leopold G, Pabst J, Garbe A (1986) Pharmacokinetics and metabolism of bisoprolol-14C in three animal species and in humans. J Cardiovasc Pharmacol 11:21–28 145. Jiang H, Li Y, Pelzer M, Cannon JM, Randlett C, Junga H, Jiang X, Qin C, Ji CQ (2008) Determination of molindone enantiomers in human plasma by high-performance liquid chromatography–tandem mass spectrometry using macrocyclic antibiotic chiral stationary phases. J Chromatogr A 1192:230–238 146. Wolf JE, Shander D, Huber F, Jackson J, Lin CS, Mathes BM, Schrode K (2007) Randomized, double-blind clinical evaluation of the efficacy and safety of topical eflornithine HCl 13.9% cream in the treatment of women with facial hair. Int J Dermatol 46:94–98 147. Zhang L, Gedicke K, Kuznetsov MA, Staroverov SM, Seidel-Morgenstern A (2007) Application of an eremomycin-chiral stationary phase for the separation of dl-methionine using simulated moving bed technology. J Chromatogr A 1162:90–96 148. Fedorova IA, Shapovalova EN, Shpigun OA (2017) Separation of β-blocker and amino acid enantiomers on a mixed chiral sorbent modified with macrocyclic antibiotics eremomycin and vancomycin. J Anal Chem 72:76–82 149. Blinov AS, Reshetova EN (2014) Effect of the concentration of organic modifier in an aqueous-ethanol mobile phase on the chromatographic retention and thermodynamic characteristics of the adsorption of enantiomers of α-phenylcarboxylic acids on silica gel with immobilized eremomycin antibiotic. Russ J Phys Chem A 88:1778–1784 150. Zhang XT, Bao Y, Huang K, BarnettRundlett KL, Armstrong DW (2010) Evaluation of dalbavancin as chiral selector for HPLC and comparison with teicoplaninbased chiral stationary phases. Chirality 22:495–513

Chapter 13 Application of Sub-2 Micron Particle Silica Hydride Derivatized with Vancomycin for Chiral Separations by Nano-Liquid Chromatography Chiara Fanali and Salvatore Fanali Abstract 1.8 μm Silica hydride particles have been derivatized with vancomycin and applied to the enantioseparation of some racemic herbicides and nonsteroidal anti-inflammatory drugs (NSAIDs) by nano-liquid chromatography. The chiral stationary phase (CSP) was packed for only 11 cm and the enantiomers were separated utilizing a laboratory-assembled instrumentation. The new CSP was very effective for the separation of the above mentioned acidic compounds, while poor resolutions were obtained for basic compounds. Mixtures of acetate buffer with methanol or acetonitrile allowed the chiral resolution of all compounds. Fast chiral separation of a NSAIDs-related compound can be achieved in less than 60 s. Key words Vancomycin, Enantiomers, High-performance liquid chromatography, Nano-liquid chromatography, Capillary electrochromatography, Chiral stationary phases

1

Introduction A large number of compounds, belonging to the environmental, pharmaceutical, agrochemical, and biochemical fields, exist as two or more stereoisomers because of their chemical structure (presence of stereogenic center/s). It is known that the enantiomers of chiral compounds possess identical physicochemical properties; however they can react differently in the presence of a chiral environment (see biological processes). Most of the novel drugs are commercialized as a single enantiomer because the two enantiomers can have different pharmacological activities. For example, ( )-epinephrine, a sympathomimetic drug, administered for cardiac stimulation is ten times more potent than its enantiomer. Naproxen, a nonsteroidal anti-inflammatory drug (NSAID), is another drug used for inflammation and stiffness diseases and the R-form is 28 times more potent than its antipode [1]. It is noteworthy to remark that in some cases one of the two enantiomers

Gerhard K. E. Scriba (ed.), Chiral Separations: Methods and Protocols, Methods in Molecular Biology, vol. 1985, https://doi.org/10.1007/978-1-4939-9438-0_13, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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could be even toxic and dangerous for human health. This is the case of S-naproxen, which is hepatotoxic in contrast to the R-enantiomer. Therefore chiral compounds represent a key issue in both research and application fields due to their involvement in biochemical processes with possible consequences for human health. As a result, reliable analytical and preparative methods are needed for their qualitative and quantitative determination. Enantiomer separations can be obtained utilizing the indirect and direct methods and both are based on the use of a chiral selector (CS). In the first case the two enantiomers react with the CS forming stable diastereoisomers, which exhibit different physicochemical properties so that they can be separated in conventional media. In the direct method, the two enantiomers form labile diastereoisomeric complexes during the separation process in the presence of a CS, which can be bonded or adsorbed onto the stationary phase or on the capillary wall. Analytical methods, so far employed for chiral separations, include capillary electrophoresis (CE), gas chromatography (GC), thin-layer chromatography (TLC), supercritical fluid chromatography (SFC), and high-performance liquid chromatography (HPLC), the latter in both conventional and microfluidic formats [2]. Among the large number of CS currently employed, macrocyclic antibiotics exhibit very high enantioselectivity toward a large number of compounds and, therefore, they have been widely used in analytical chemistry. Since their first use in HPLC by Armstrong’s group [3], the compounds have been widely applied for enantiomer separations utilizing other separation techniques, e.g., CE (also including capillary electrochromatography, CEC) [4–9] or nano-LC [10–13]. In CE the CS (vancomycin, teicoplanin, or other glycopeptide antibiotics) is usually added to the background electrolyte generating detection problems (lowering the sensitivity) due to the strong UV absorption. This is not a problem in CEC or nano-LC because the CS is bonded to silica particles packed into the capillary column. Vancomycin or teicoplanin containing CSPs utilized mainly in HPLC are commercially available, e.g., under the trade name Chirobiotic®. 1.1 Properties of Vancomycin and Enantioresolution Mechanism

Vancomycin belongs to the class of glycopeptide antibiotic with chemotherapeutic properties against bacteria. The compound is produced using microorganisms (Amycolatopsis orientalis). Vancomycin is composed of amino acids and saccharides (Fig. 1). The enantiomeric properties of vancomycin derive from the presence of 18 stereogenic centers. A large number of functional groups such as hydroxy, amino, amide, carboxylic, and aromatic strongly influence the interactions with analyte enantiomers. Considering the presence of one carboxylic and two amino groups, vancomycin exhibits zwitterionic properties and therefore the pH of the mobile phase strongly influences its charge.

Nano-Liquid Chromatography Chiral Separations

CH3 HO H3C

OH

H

NH2

O

HO HO HO

O

O

HN

O

O

NH

O

Cl

O

O O NH2

O

HN

O

NHCH3

H N

Cl

O

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H HO

NH H

H O

OH

N H HOOC OH

Fig. 1 Chemical structure of vancomycin

Vancomycin can be dissolved in water and to a lower extent in organic solvents with good stability at pH 3–6 [14]. Therefore, due to the presence of different functional groups in the chemical structure of vancomycin, this CS can be involved in several non-stereoselective as well as stereoselective interactions such as π–π interactions, hydrogen bonds, hydrophobic interactions, electrostatic interaction, or repulsion. In addition, inclusion complexation can also take place due to the presence of the sugar moiety [15]. Vancomycin has been widely studied as a CS utilizing different analytical techniques and achieving quite different results. For example, in CE this CS was simply added to the background electrolyte where (1) the two amino and the carboxylic groups could interact with the enantiomers and (2) the molecule was free to rotate in contrast to LC or CEC where one nitrogen is bonded to the stationary phase (typically silica gel) via a linker. As consequence, in CE very good enantiomer resolution of acidic compounds has been obtained, while in CEC or nano-LC mainly basic chiral compounds were separated [16–19]. However, it should be noted that the different behaviors of vancomycin toward the separation of basic or acidic enantiomers and the type of silica gel used in the synthesis of the CSP have to be considered as another parameter. In fact, in a recent paper of our group, ordinary silica with 5 μm particles and silica hydride (sub-2 micron particles) were derivatized with vancomycin and applied to the enantiomer resolution of both, basic and acidic compounds. The CSP based on silica hydride offered very high enantioresolution for acidic compounds and very poor resolution for basic analytes [11].

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1.2 Use of Sub2 Micron Particles as Stationary Phase

2

Most vancomycin-based CSPs currently employed in HPLC make use of particles of 5 μm diameter and these columns are commercially available. Considering the van Deemter equation, when using CSPs or other type of stationary phases, it can be concluded that a decrease of plate height can be obtained by reducing the particle size. In addition, since the second part of the van Deemter plot is rather flat, a higher flow rate could be used without sacrificing the efficiency, thus reducing the analysis time. This led to the production of small-diameter silica particles (1.7–1.8 μm) totally porous or core shell. Although excellent results were obtained with these columns, it must be remarked that a very high back pressure was observed. These columns have been applied in ultra high-performance liquid chromatography (UHPLC) where dedicated pumps (high pressure) have to be used [20]. 1.7 μm Porous silica particles modified with the Whelk-O1 selector have been used by Gasparrini’s group [20] in UHPLC for the fast enantioseparation of some drugs (e.g., flurbiprofen, naproxen). The column (50 mm  4.3 mm) offered very high efficiency using both normal-phase and reversed-phase mode with a number of theoretical plates in the range of 244,000–278,000 plates per meter. In a recent work, Armstrong’s group [21] reported an ultrafast (sub-second) chiral separation utilizing three CSs, including teicoplanin. The column (5 mm  4.6 mm) was packed with 2.7 μm core-shell particles. Our group used capillary columns of different inner diameters packed with 1.8 μm C18 porous silica particles for the separation of nonsteroidal anti-inflammatory drugs by nano-LC. This was done utilizing lengths of 50 mm and an ordinary HPLC pump with a split device to reduce the flow rate. Analysis time was less than 2 min [22]. Subsequently, the same type of hydride silica was derivatized with vancomycin and used for the enantioseparation of both acidic (herbicides, NSAIDs) and basic (β-blocker) compounds [11]. In this chapter, the preparation of a chiral capillary column (11 cm packed) is described, which allowed to obtain very good enantiomer separations of acidic compounds, while basic enantiomers were only partly separated. The different behaviors toward the studied compounds may be partly ascribed to the type of particles used.

Materials

2.1 Nano-LC Apparatus and Equipment

1. A laboratory-made apparatus allowing a flow rate of 10–1700 nL/min or a commercial nano-LC instrument can be used. The laboratory-made nano-LC system utilizing a commercial HPLC pump can be constructed as outlined in Fig. 2 using a passive split-flow system to reduce the flow rate to the nL/min levels and a suitable nano-LC six-port injection

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50 mm ID

split

130 mm ID

LC Pump

Loop 50 mm

valve

Packed capillary

UV detector

MeOH

Fig. 2 Scheme of a laboratory-assembled nano-LC instrumentation

valve or a valve modified according to [23]. The HPLC pump and the injection valve are connected to a stainless steel T-piece (Vici, Valco, Houston TX, USA) by 500 μm ID stainless steel tubes with lengths of 70 and 5 cm, respectively. The third port of the T-piece is connected to reservoir using a 20 cm long, 50 μm ID fused silica capillary. One end of the capillary column is directly connected to the valve, while the other end featuring the detection window is mounted to a commercial UV–vis on-column detector. Injections in this system are carried out by filling the loop with the sample solution and switching the valve for a defined period of time, which controls the injection volume. The loop is subsequently flushed with mobile phase to remove the sample solution (see Note 1). 2. A commercial ultrasonic bath for synthesis of the CSP and for degassing of solutions. 3. A commercial laboratory centrifuge for spinning down the particles during CSP synthesis. 4. A commercial pH meter for pH adjustment of buffer solutions. 5. A commercial water purification system for preparation of ultrapure water. 6. A stainless steel HPLC column (10 cm  4.6 mm ID). 7. Fused silica capillaries (75 μm ID  365 μm OD, polyimide coated). 2.2 Solutions for Synthesis of Vancomycin CSP

1. 60 mM Sodium periodate solution: Dissolve 0.64 g sodium periodate in 50 mL water:methanol (4:1, v/v). 2. 3 mM Vancomycin and 10 mM cyanoborohydride solution in 50 mM phosphate buffer, pH 7.04: In a 100 mL volumetric flask, dissolve 0.54 g dibasic sodium phosphate heptahydrate in

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90 mL water and adjust pH to 7.04 with 0.1 M phosphoric acid. Add water to the mark and mix. Dissolve 223 mg vancomycin hydrochloride (see Note 2) and 31.4 mg sodium cyanoborohydride in 50 mL of the buffer. 3. 10 mM Cyanoborohydride in 50 mM phosphate buffer, pH 3.1: In a 100 mL volumetric flask, dissolve 0.78 g monobasic sodium phosphate dihydrate in 90 mL water and adjust pH to 3.1 using 0.1 M sodium hydroxide. Add water to the mark and mix. Dissolve 31.4 mg sodium cyanoborohydride in 50 mL of the buffer. 2.3

Mobile Phases

1. 500 mM Ammonium acetate buffer, pH 4.5: In a 500 mL volumetric flask, mix 15 g glacial acetic acid in 300 mL water and adjust the pH to 4.5 using 1 M sodium hydroxide. Add water to the mark and mix. 2. Mobile phase A: Mix 5 mL of 500 mM ammonium acetate buffer, pH 4.5, with 10 mL of water and 85 mL methanol (HPLC grade). Sonicate for 5 min. 3. Mobile phase B: Mix 1 mL 500 mM ammonium acetate buffer, pH 4.5, with 9 mL water and 90 mL acetonitrile (HPLC grade). Sonicate for 5 min.

2.4

3

Sample Solutions

Prepare stock solutions of the compounds (chiral herbicides or NSAIDs) at a concentration of 1 mg/mL using acetonitrile as solvent. For sample solutions, dilute to a concentration of 100 or 50 μg/mL using acetonitrile.

Methods

3.1 Synthesis of Vancomycin CSP

The synthesis of the vancomycin CSP is outlined in Fig. 3. 1. Suspend 200 mg of diol silica hydride particles (particle size 1.8 μm, see Note 3) in 15 mL 60 mM sodium periodate solution and sonicate for 1 h. 2. Centrifuge at 2800  g for 5 min and discard supernatant. 3. Resuspend particles in 20 mL water and swirl gently. Centrifuge at 2800  g for 5 min and discard supernatant. Repeat this washing step three times. 4. Suspend the particles in 15 mL 3 mM vancomycin and 10 mM cyanoborohydride solution in 50 mM phosphate buffer, pH 7.04, and sonicate for 1 h. 5. Centrifuge at 2800  g for 5 min and discard supernatant. 6. Resuspend particles in 20 mL water and swirl gently. Centrifuge at 2800  g for 5 min and discard supernatant. 7. Suspend particles in 15 mL 10 mM cyanoborohydride in 50 mM phosphate buffer, pH 3.1, and sonicate for 1 h.

Nano-Liquid Chromatography Chiral Separations

Si

Si

O

OH

Si O

NaIO4

OH

Si

oxidaon

O

O Si O Si

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Vancomycin/NaCNBH3 pH 7.04

CHO CHO

Aminaon&reducon

O

Si O Si O Si O

CHO O

NaCNBH3

Si

pH 3.1

O

reducon

Vancomycin

Si O Si O

OH O

Vancomycin

Fig. 3 Scheme of the synthesis of the CSP containing vancomycin attached to silica hydride

8. Centrifuge at 2800  g for 5 min and discard supernatant. 9. Resuspend particles in 20 mL water and swirl gently. Centrifuge at 2800  g for 5 min and discard supernatant. Repeat this washing step three times. 10. Resuspend particles in 20 mL methanol and swirl gently. Centrifuge at 2800  g for 5 min and discard supernatant. Repeat this washing step three times. 11. Remove residual methanol at room temperature under vacuum in a rotary evaporator. 3.2 Packing of the Capillary Column

1. Cut a 50 cm long piece of the capillary. 2. Connect one end of the capillary to the stainless steel HPLC column (10 cm  4.6 mm ID). 3. Connect the opposite end of the capillary to the mechanical frit. 4. Suspend 2–3 mg RP18 silica gel with a particle size of 5 μm (see Note 4) in 1 mL of acetone or acetonitrile and sonicate for 10 min. 5. Place suspension in the HPLC column reservoir and connect to the pump. 6. Pack the capillary for about 10–11 cm. 7. Remove the RP18 stationary phase from the HPLC column reservoir and wash with water. 8. Flush the packed capillary with water for about 30 min. 9. Prepare the inlet frit with a heated wire at about 700  C for 6–7 s under a flow of water (30 MPa).

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10. Remove the mechanical frit. 11. Connect the capillary on the frit side and flush with acetonitrile to remove the excess of RP18 stationary phase. 12. Remove the capillary and connect to the HPLC column reservoir with the frit on the opposite side. 13. Suspend 2–3 mg of the silica hydride-vancomycin stationary phase in acetone:water (1:1, v/v) mixture. 14. Place suspension in the HPLC column reservoir and connect to the pump 15. Pack the capillary for 11 cm at 30 MPa. 16. Wash the HPLC column reservoir with water in order to remove the CSP. 17. Repeat steps 4–9 for the preparation of the outlet frit. 18. Connect the capillary with the inlet frit to the pump and flush with acetonitrile to remove the excess of RP18 phase. 19. Cut the capillary close to the inlet frit and at a distance of about 4 cm from the outlet frit (see Note 5). 20. Prepare a small window (width of about 0.5 cm) close to the outlet frit (about 2 cm) for online UV detection (see Note 6). 21. Gently clean the detection window with methanol. 3.3 Nano-LC Enantiomers Separation

1. Connect the capillary (inlet frit side) with the HPLC pump. 2. Flush system with the appropriate mobile phase at 30–35 MPa for 30 min (see Note 7). 3. Put the capillary with its window into the UV detector. 4. Connect the capillary (inlet side) to the injector valve (see Note 8). 5. Fill the valve loop with the mobile phase. 6. Equilibrate the column with the mobile phase (30 min). 7. Fill the loop with sample solutions with 50 μL syringe. 8. Inject for 4–5 s (see Note 9). 9. Flush the loop with the mobile phase (see Note 10). 10. Start the nano-LC separation and record chromatograms (see Tables 1 and 2 and Fig. 4) (see Note 11).

4

Notes 1. For further details concerning the nano-LC laboratoryassembled instrumentation see ref. 11. 2. Vancomycin is a potent antibiotic that can be harmful for human health. Therefore the compound should not be inhaled. In addition, skin contact should also be avoided.

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Table 1 Chromatographic data related to the nano-LC separation of herbicide enantiomers Compounds

tR1 (min)

tR2 (min)

k1

k2

α

RS

Dichlorprop

2.77

3.70

3.70

1.12

1.91

2.95

Diclofop

2.96

3.81

3.81

1.22

1.69

2.26

Fenoprop

2.91

3.70

3.70

1.04

1.72

2.60

Fluazifop

2.23

2.90

2.90

0.67

2.38

2.81

Haloxyfop

2.29

3.24

3.24

0.87

2.69

3.36

Mecoprop

2.63

3.35

3.35

0.89

1.83

2.50

Capillary column packed with 1.8 μm diol hydride-based silica particles derivatized with vancomycin. Experimental conditions: capillary column, 75 μm ID, Lpack: 11 cm, Leff: 13 cm; mobile phase, 500 mM ammonium acetate buffer pH 4.5/H2O/MeOH (5:10:85, v/v/v), elution in isocratic mode, flow rate 230 nL/min (to 1.8 min); samples, solutions 50 μg/mL diluted in ACN, 40 nL injected (reproduced with permission of Elsevier from ref. 11 © Elsevier 2015)

Table 2 Chromatographic data related to the nano-LC separation of NSAID enantiomers Compound

tR1 (min)

tR2 (min)

k1

k2

α

RS

Carprofen

4.76

5.63

1.70

2.19

1.29

1.72

Cicloprofen

3.31

4.10

0.88

1.33

1.50

2.38

Flurbiprofen

3.09

3.97

0.75

1.24

1.66

2.85

Ibuprofen

2.31

2.62

0.33

0.52

1.54

1.72

Indoprofen

6.38

7.30

2.62

3.14

1.20

1.27

Ketoprofen

3.60

4.45

1.16

1.66

1.44

2.29

Naproxen

3.22

4.01

0.80

1.25

1.55

2.52

Suprofen

4.24

5.16

1.45

1.98

1.37

2.19

Mobile phase: 500 mM ammonium acetate buffer pH 4.5/H2O/ACN (1:9:90, v/v/v), elution in isocratic mode, flow rate 360 nL/min (to 1.8 min); samples, solutions 50 g/mL in ACN, 70 nL injected. For other experimental conditions, see Table 1 (reproduced with permission of Elsevier from ref. 11 © Elsevier 2015)

´ 3. Diol silica hydride with 1.8 μm particle size and 100 A˚ pore size can be obtained from MicroSolv Technology Corp (Eatontown, NJ, USA). 4. Lichrospher particles have been employed; however any other RP18 material (not end-capped material) can be used. Because the presence of residual-free silanol groups, frit preparation is easier with this material as compared to the synthesized CSP.

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Fig. 4 Enantioseparations of selected herbicides (a) and NSAIDs (b) analyzed by nano-LC. Capillary column, 75 μm ID, with an effective length of 13 cm and a 11 cm packed bed of 1.8 μm diol silica hydride particles with covalently bound vancomycin. For other experimental conditions, see Tables 1 and 2 (reproduced by permission of Elsevier from [11] © 2015 Elsevier)

5. A straight cut of the capillary is required to avoid dead volumes. Therefore the use of commercial capillary cutter is advised. In addition, a careful control of the ends of the capillary under a microscope is advised. 6. The window is prepared removing the polyimide layer from the capillary using a razor. Special attention must be paid, because the capillary is now very fragile and can be easily broken. 7. The flushing procedure with the mobile phase is done outside the nano-LC instrument only when a new packed capillary is prepared. 8. The capillary is connected directly to the injection valve to avoid dead volumes that can lead to band broadening. 9. In this work, a modified injection valve was used. However the same approach can be undertaken utilizing commercial injector valves.

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10. The loop was flushed utilizing 100 μL syringe to completely remove the sample solution. 11. Very good enantiomer separations were obtained with the short capillary due to the high enantioselectivity of the column. The increase of the mobile-phase flow at 1700 nL/min allowed to achieve chiral resolutions of NSAID-related racemic compound in less than 60 s. References 1. De-Miao C, Qiang F, Na L, Song-Xian Z, Qian-Qian Z (2007) Enantiomeric separation of naproxen by high performance liquid chromatography using CHIRALCEL OD as stationary phase. Chin J Anal Chem 35:75–78 2. D’Orazio G, Fanali C, Asensio-Ramos M, Fanali S (2017) Chiral separations in food analysis. TrAC—Trends Anal Chem 96:151–171 3. Armstrong DW, Tang YB, Chen SS et al (1994) Macrocyclic antibiotics as a new class of chiral selectors for liquid chromatography. Anal Chem 66:1473–1484 4. Armstrong DW, Rundlett K, Reid GL III (1994) Use of a macrocyclic antibiotic, rifamycin b, and indirect detection for the resolution of racemic amino alcohols by CE. Anal Chem 66:1690–1695 5. Ward TJ, Oswald TM (1997) Enantioselectivity in capillary electrophoresis using the macrocyclic antibiotics. J Chromatogr A 792:309–325 6. Desiderio C, Fanali S (1998) Chiral analysis by capillary electrophoresis using antibiotics as chiral selector. J Chromatogr A 807:37–56 7. Aboul-Enein HY, Ali I (2001) Macrocyclic antibiotics as effective chiral selectors for enantiomeric resolution by liquid chromatography and capillary electrophoresis. Chromatographia 52:679–691 8. Tang A-N, Wang X-N, Ding G-S, Yan X-P (2009) On-line preconcentration and enantioseparation of thalidomide racemates by CEC with the hyphenation of octyl and norvancomycin monoliths. Electrophoresis 30:682–688 9. D’Orazio G, Fanali S (2010) Coupling capillary electrochromatography with mass spectrometry by using a liquid-junction nanospray interface. J Chromatogr A 1217:4079–4086 10. Rocchi S, Fanali C, Fanali S (2015) Use of a novel sub-2 μm silica hydride vancomycin stationary phase in nano-liquid chromatography. II. Separation of derivatized amino acid enantiomers. Chirality 27:767–772

11. Rocchi S, Rocco A, Pesek JJ, Matyska MT, Capitani D, Fanali S (2015) Enantiomers separation by nano-liquid chromatography: use of a novel sub-2μm vancomycin silica hydride stationary phase. J Chromatogr A 1381:149–159 12. D’Orazio G, Cifuentes A, Fanali S (2008) Chiral nano-liquid chromatography-mass spectrometry applied to amino acids analysis for orange juice profiling. Food Chem 108:1114–1121 13. D’Orazio G, Aturki Z, Cristalli M, Quaglia MG, Fanali S (2005) Use of vancomycin chiral stationary phase for the enantiomeric resolution of basic and acidic compounds by nanoliquid chromatography. J Chromatogr A 1081:105–113 14. Armstrong DW, Rundlett KL, Chen J-R (1994) Evaluation of the macrocyclic antibiotic vancomycin as a chiral selector for capillary electrophoresis. Chirality 6:496–509 15. Loukili B, Dufresne C, Jourdan E et al (2003) Study of tryptophan enantiomer binding to a teicoplanin-based stationary phase using the perturbation technique: investigation of the role of sodium perchlorate in solute retention and enantioselectivity. J Chromatogr A 986:45–53 16. Fanali S, Desiderio C (1996) Use of vancomycin as chiral selector in capillary electrophoresis. Optimization and quantitation. J High Resolut Chromatogr 19:322–326 17. Ward TJ, Dann C III, Brown AP (1996) Separation of enantiomers using vancomycin in a countercurrent process by suppression of electroosmosis. Chirality 8:77–83 18. Desiderio C, Aturki Z, Fanali S (2001) Use of vancomycin silica stationary phase in packed capillary electrochromatography I. Enantiomer separation of basic compounds. Electrophoresis 22:535–543 19. Fanali S, Rudaz S, Veuthey JL, Desiderio C (2001) Use of vancomycin silica stationary phase in packed capillary electrochromatography. II: Enantiomer separation of venlafaxine

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and O-desmethylvenlafaxine in human plasma. J Chromatogr A 919:195–203 20. Kotoni D, Ciogli A, Molinaro C et al (2012) Introducing enantioselective ultrahighpressure liquid chromatography (eUHPLC): theoretical inspections and ultrafast separations on a new sub-2-mm Whelk-O1 stationary phase. Anal Chem 84:6805–6813 21. Farooq Wahab M, Wimalasinghe RM, Wang Y et al (2016) Salient sub-second separations. Anal Chem 88:8821–8826

22. D’Orazio G, Rocco A, Fanali S (2012) Fastliquid chromatography using columns of different internal diameters packed with sub-2 μm silica particles. J Chromatogr A1228:213–220 23. Chankvetadze B, Bergenthal D, Wennemer H. Vorrichtung zur Trennung von Substanzgemischen mittels Flu¨ssigchromatographie. DE10260700. 2004.

Chapter 14 Cinchona Alkaloid-Based Zwitterionic Chiral Stationary Phases Applied for Liquid Chromatographic Enantiomer Separations: An Overview Istva´n Ilisz, Attila Bajtai, Antal Pe´ter, and Wolfgang Lindner Abstract For the early 2000s, chromatographic methods applying chiral stationary phases (CSPs) became the most effective techniques for the resolution of chiral compounds on both analytical and preparative scales. Highperformance liquid chromatography (HPLC) employing various types of chiral selectors covalently bonded to silica-based supports offers a state-of-the-art methodology for “chiral analysis.” Although a large number of CSPs are available nowadays, the design and development of new “chiral columns” are still needed since it is obvious that in practice one needs a good portfolio of different columns to face the challenging task of enantiomeric resolutions. The development of the unique chiral anion, cation, and zwitterion exchangers achieved by Lindner and his partners serves as an expansion of the range of the efficiently applicable CSPs. In this context this overview chapter discusses and summarizes direct enantiomer separations of chiral acids and ampholytes applying zwitterionic ion exchangers derived from Cinchona alkaloids. Our aim is to provide comprehensive information on practical solutions with focus on the molecular recognition and methodological variables. Key words Chirality, Enantiomer separation, High-performance liquid chromatography, Ion-exchangers, Zwitterionic chiral stationary phases, Cinchona alkaloids

1

Introduction The past three decades have seen an extraordinary growth in the field of so-called chiral technologies. Liquid chromatographic enantiomer separations—as part of this field—initially were carried out indirectly via derivatization using various enantiomerically pure reagents to form diastereomeric derivatives that could be separated on achiral, in particular on reversed-phase (RP) columns. However, by now, the so-called direct chromatographic enantiomer separation techniques applying chiral stationary phases (CSPs) became the most popular methods applied for analytical and preparative separations.

Gerhard K. E. Scriba (ed.), Chiral Separations: Methods and Protocols, Methods in Molecular Biology, vol. 1985, https://doi.org/10.1007/978-1-4939-9438-0_14, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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Since the first publication on a CSP utilized for gas chromatographic separation in 1966 [1], chiral separation techniques have become a very sophisticated field of analytical chemistry, where liquid chromatography-based methods dominate to date the field. Until now, a large number (>100) of CSPs prepared for enantioseparations based on LC and super- or subcritical fluid chromatography (SFC) have appeared on the market or have been described in the literature. However, the chiral selectors (SOs) of these CSPs tend to be derived from relatively few classes of chiral starting materials; amino acids (natural or unnatural), peptides, proteins, cyclodextrins, cyclofructans (mostly in the derivatized form), derivatized linear or branched polysaccharides, macrocyclic compounds such as macrocyclic antibiotics and chiral crown ethers, ion exchangers, and other fully synthetic chiral compounds can be utilized for this purpose. In this contribution, we will concentrate on Cinchona alkaloid-derived zwitterionic SOs and CSPs, applied as chiral ion exchangers. As extensively outlined in the paper by L€ammerhofer and Lindner [2], the first description of the application of Cinchona alkaloids for enantiomeric resolution by LC dates already back to the early 1950s [3, 4]. In the 1980s, Izumoto et al. [5, 6] and Petterson et al. [7–9] developed LC-based methods utilizing ion-pair formation between the basic quinuclidine residue of quinine and acidic analytes, while a silica-supported Cinchona alkaloidbased chromatographic material was prepared by Rosini et al. [10]. Later, CSPs based on Cinchona alkaloids were described by several groups. However, all these CSPs suffered from low enantioselectivities and a narrow application range. A conceptual structural modification developed by Lindner et al. resulted in a breakthrough in developing Cinchona carbamate-type CSPs through carbamoylation of the secondary hydroxy group of the native alkaloids [11]. In 2002, t-butyl carbamate derivatives of quinine (QN)- and quinidine (QD)-based CSPs were introduced into the market. These phases have been marketed by Chiral Technologies Europe with trade names Chiralpak® QN-AX and Chiralpak® QD-AX and have been extensively used since 2005. A few years later these Cinchona alkaloid-based chiral weak anion-exchanger (WAX) units have been conceptionally further modified by fusing the QN and QD moiety in a combinatorial synthesis approach with a strongly acidic aminosulfonic acid-based chiral synthon, a strong cation-exchanger (SCX) moiety, to generate this way a single zwitterionic (ampholytic) chiral selector motif [12]. The developed zwitterionic ion-exchange-type CSPs became recently also commercially available as Chiralpak ZWIX(þ)™ and ZWIX()™. These new ampholytic CSPs provided strong unequivocal evidence for synergistic effects of the two oppositely charged ionic subunits and sites of the chiral SOs as outlined in Fig. 1. They can act as chiral anion and cation exchangers but also as

Cinchona-Based Chiral Stationary Phases

253

Fig. 1 Structures of zwitterionic chiral stationary phases (numbers refer to the references, in which the CSPs have been described)

zwitterionic ion exchangers through double-electrostatic interactions occurring simultaneously with ampholytic analytes. With other words, zwitterionic CSPs have a much broader spectrum of selectivity since they will interact stereoselectively with basic or acidic analytes via single-electrostatic interactions in synergistic combination with additional intermolecular interactions triggered by stereochemical (configurational and conformational) parameters. Structures of zwitterionic chiral SOs and of the respective CSPs, which have been developed recently, are illustrated in Fig. 1.

2

Retention Mechanism of Cinchona Alkaloid-Based Ion Exchangers Over the last years, a number of variants of QN- and QD-based chiral SOs and CSPs have been developed as chiral anion exchangers. All of these brush-type CSPs have in common the bulky quinuclidine moiety with a tertiary amino group with a pKa about 9.8 [13], undergoing protonation under acidic conditions. The protonated nitrogen will function as the positively charged site of the chiral WAX-type SO which is able to form long-range electrostatic interactions with a negatively charged site of the acidic chiral

254

Istva´n Ilisz et al.

selectand (SA). Three of the four chiral centers of the quinuclidine moiety (1S, 3R, 4S) are identical (Fig. 1), while the configuration of chiral centers of the C8 and C9 atoms of the Cinchona backbone changes from (S)-C8 and (R)-C9 for QN to (R)-C8 and (S)-C9 for QD. The absolute configuration of the chiral C8 and C9 atoms turned out to be of crucial importance for the overall stereochemically driven molecular recognition, leading to the formulation of the term “pseudo-enantiomeric” behavior of QN and QD and their consequent derivatives. The chiral binding cavity around the carbamate group is characterized by a hydrogen-accepting and hydrogen-donating functionality and the C8 and C9 configuration of the SO in combination with the actively driven SO–SA interactions is essential in the regulation of the chromatographic enantioselectivity and elution order, which can be expressed by the differences of the stability constants of the intermediate (R)-SO–(R)-SA and (R)SO–(S)-SA associates. The same applies for the formally enantiomeric pair of (S)-SO–(R)-SA and (S)-SO–(S)-SA associates. Applied to the pseudo-enantiomeric SO moieties and respective CSPs (see Fig. 1), this leads to a reversal of the elution order of the enantiomers of chiral analytes, as often demonstrated. However, this concept may not necessarily apply for all applications of the pseudo-enantiomeric QN- and QD-type selectors as in reality they are diastereomers to each other. The zwitterionic SOs (Fig. 1) are based on the chemical fusion of a weak positively charged moiety related to the protonated quinuclidine-containing site with a moiety bearing a deprotonated, thus negatively charged, site related to a sulfonic acid residue. The molecular part of the anion-exchange site of the ZWIX(þ) selector is based on QN, while the cation-exchange site is based on (1S,2S)cyclohexyl-1-amino-2-sulfonic acid, and the two charged molecular parts are bridged via the well-established carbamoyl group. According to the pseudo-enantiomer concept outlined above, the ZWIX() selector is composed of QD and the (1R,2R)-cyclohexyl-1-amino-2-sulfonic acid. The two charged sites of these zwitterionic and ampholytic SOs are nine bonds apart from each other but cannot approximate each other very closely in space because of the rigidity of the multichiral center-based SO molecules. However, these rather bulky ampholytic selectors, in turn, are able to interact via long-range electrostatic interactions in a double-ion-pairing fashion with ampholytic SAs (Fig. 2). Such a concept, in combination with the steric arrangements of the substituents of the SO and SA molecules, fulfills the necessary requirement to generate an enantioselective molecular recognition scenario. Further intermolecular SO–SA interactions such as hydrogen bonding may come into play but are not a necessary prerequisite. As demonstrated by a large body of examples summarized in Table 1, this double-ion-pairing concept

Cinchona-Based Chiral Stationary Phases

255

Fig. 2 Scheme of possible interactions of a cinchona alkaloid-based zwitterionic chiral stationary phases with ampholytic analytes

worked successfully, among others, for free α-, β-, and γ-amino acids and very diverse structures of short peptides. Because of the ampholytic character of the ZWIX SOs and the somewhat independent property and spatial position of the positively and negatively charged sites, these ampholytic SOs can, in principle, also act individually as chiral anion exchanger or chiral cation exchanger (Table 1). However, a structural peculiarity exists in both cases, because of the intrinsically existing, stoichiometrically acting counterionic functional group within the ampholytic selector motif, which will affect the overall chromatographic retention behavior.

3

The Stoichiometric Displacement Model Adjustment of retention with organic bases and acids used as mobile-phase additives thus acting as displacers and counterions is a common practice in ion-exchange chromatography. In principle, it also applies to the described Cinchona alkaloid-based chiral ion exchangers. For a more detailed discussion of the underlying concept, including supporting investigations on the molecular recognition phenomena, we refer to the review of L€ammerhofer and Lindner [2]. In brief, the stoichiometric displacement model describes the ion-exchange process according to Eq. 1, where retention depends on the concentration of a counterion [X] in a commonly linear fashion according to Eq. 1, where Z is directly proportional to the ratio of the effective charge number of the solute and counterion [56–58]:



Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

α-, β-, and γ-aminophosphonic acids

MeOH containing 50 mM NH3 and 200 mM FA

Alkylsulfonic acid-, arylsulfonic acid-, Underivatized α-, β-, and γ-amino acids MeOH containing 25 mM DEA and and aminocyclohexanesulfonic 50 mM FA acid-carbamoylated quinine and quinidine

Acidic (N-blocked) amino acids; basic MeOH containing 25 mM DEA and analytes, zwitterionic analytes: amino 50 mM FA acids

Alkylsulfonic acid-, butanesulfonic acid-, and aminocyclohexanesulfonic acidcarbamoylated quinine and quinidine

MeOH, ACN, H2O/MeOH, H2O/ ACN containing TFA, FA, and AcOH as acids and NH3, DEA, and TEA as base in different ratios

Trp, α-MeTyr, α-Me-mTyr, α-MeDOPA, e-β-MeTic, β-Phe, N-protected-Phe, -Trp, and -Leu

HPLC, 15  C 0.4 mL min1 UV, 258 nm

HPLC, 25  C 0.4–0.7 mL min1 UV, 254 nm and CAD

HPLC 25  C 1.0 mL min1 UV, 254 nm and CAD

HPLC 25  C 1.0 mL min1

Trp, Phe, β-Phe, mefloquine, tocainide, MeOH containing different amounts of HPLC, 25  C NH3/AcOH in different ratios N-deisopropyl-disopyramide, 1.0 mL min1 N-protected-Phe, -Trp, and -Glu UV, 280 nm

4 Quinine-based zwitterionic CSPs

10 Quinine-based zwitterionic CSPs

HPLC, 25 C Chiral acids, bases, zwitterionic analytes MeOH containing 25 mM DEA and (amino acids and their analogs) 50 mM FA for acidic and zwitterionic 1.0 mL min1 analytes; MeOH/ACN (10/90 v/v) UV, 254 nm containing 25 mM DEA and 50 mM FA for basic analytes

Mode

Quinidine-based zwitterionic CSPs

The most effective mobile-phase compositions

Stereoisomers

Chiral selectors or columns

Table 1 Stereoisomer separations by zwitterionic CSPs based on Cinchona alkaloids

[18]

[17]

[16]

[15]

[14]

[12]

Ref.

256 Istva´n Ilisz et al.

MeOH/AcOH/NH4OAc (98/2/0.5 or 96/4/1 v/v/w); MeOH containing 5 mM NH3 and 40 mM FA v/v/w); MeOH/ACN (50/50 or 5/95 v/v) containing 15.5 or 2.5 mM FA, respectively

HPLC, 25  C MeOH/ACN/H2O (49.7/49.7/ 0.6 v/v/v) containing 2.5 mM DEA 0.1 mL min1 and 4.0 mM FA UV, 254 nm

2-Hydroxyglutaric acid

Aliphatic α- and β-hydroxycarboxylic acid

Nα-Boc-N4(hydroorotyl)-L-and D-4aminophenylalanine Cationic 1,2,3,4tetrahydroisoquinoline analogs

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

MeOH/ACN (25/75 v/v) containing 12.5 mM TEA and 25 mM AcOH; MeOH/ACN (75/25 v/v) containing 12.5 mM TEA and 25 mM AcOH

HPLC 10–50  C 0.6 mL min1 UV, 215–230 nm

HPLC 10–25  C 1.0 mL min1 CAD

A: MeOH/ACN (25/75 v/v), B: HPLC, 25  C 1.0 mL min1 MeOH/ACN (75/25 v/v) all containing 90 mM FA; gradient CAD profile: 0–100% B in 30 min; MeOH or EtOH or PrOH containing 60 mM NH3, DEA, or TEA

(continued)

[25]

[24]

[23]

[22]

[21]

ACN/MeOH/AcOH (95/5/0.05 v/ HPLC v/v) 10–40  C 0.3–1.0 mL min1 CAD

3-Hydroxybutyric-, 3-hydroxydecanoic-, and 3-hydroxymiristic acid

Chiralpak ZWIX(þ)™

[20]

MeOH/ACN/H2O (49/49/2 v/v/v) HPLC-ESI-MS 40  C containing 12.5 mM aq. NH4HCOO and 25 mM FA 0.5 mL min1

Mefloquine and its carboxy metabolite in blood

Chiralpak ZWIX()™

[19] HPLC, 25  C ACN/H2O (90/10 v/v), MeOH/ ACN/H2O (49/49/2 v/v/v), 0.4–1.0 mL min1 MeOH/THF/H2O (49/49/2 v/v/ UV, v), MeOH/H2O (98/2 v/v) all 230–254–270 nm and ELSD containing 25 mM DEA and 50 mM FA

Proteinogenic α-amino acids, DOPA, Kyn, dipeptides, N-protected amino acids, etc.

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

Cinchona-Based Chiral Stationary Phases 257

Trp and its methyl, methoxy and chloro-analogs and Trp metabolites D-

Chiralpak ZWIX(þ)™

Chiralpak ZWIX()™



MeOH/ACN/1.0 M aq. NH4HCOO/FA (500/500/25/ 2 v/v/v/v)

Peptide fragments from lipopeptide isolated from endophytic Pseudomonas poae strain RE*1-1-4 Calchinons: New designer drugs

Chiralpak ZWIX(þ)™

HPLC-ESI-MS 25  C 0.5 mL min1

UHPLC-ESIQTOF-MS 0.7 mL min1 MeOH/ACN volume ratio: 3/1; 1/1; HPLC, 25  C 1/3; 1/9 and MeOH/ACN/H2O 1.0 mL min1 (49/9/2 v/v/v) all containing UV, 254–280 nm 25 mM NH3 or DEA and 50 mM FA

MeOH/H2O (98/2 v/v) containing 9.4 mM NH4HCOO and 9.4 mM FA

Ref.

[31]

[30]

[29]

[28]

[27]

HPLC, 25 C [26] 0.5 mL min1 UV, 230–254–270 nm and ELSD

Mode

HPLC, 25  C MeOH/H2O (98/2 v/v) containing 20–50 mM DEA and 25–75 mM FA 0.5 mL min1 UV, 254 nm

MeOH/ACN (50/50 v/v), MeOH/ ACN/H2O (49/49/2 v/v/v), MeOH/THF/ H2O (49/49/2 v/v/v), MeOH/ H2O (98/2, 90/10 or 80/20 v/v) all containing 25 mM DEA and 50 mM FA

The most effective mobile-phase compositions

2-((2-(4-Propoxyphenyl)quinolin-4-yl) MeOH/THF/H2O (49/49/2 v/v/v) HPLC, 25  C oxy) alkylamines containing 12.5 mM DEA and 0.1 mL min1 25 mM FA UV, 200–350 nm

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

Proteinogenic amino acids and their derivatives

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

and L-Leu and D- and L-allo-Ile in animal plasma

Stereoisomers

Chiral selectors or columns

Table 1 (continued)

258 Istva´n Ilisz et al.

MeOH/H2O (96/4 v/v) containing 5.0 mM NH4HCOO and 5.0 mM FA MeOH/ACN containing NH3, DEA, TEA, and DIPEA as bases and FA, TFA, AcOH, and HFBA as acids in different amounts and ratios HPLC: H2O/MeOH (1/99 v/v) containing 30 mM TEA and 60 mM FA or MeOH/ACN (75/25 v/v) containing 30 mM TEA and 60 mM FA SFC: CO2/MeOH (60/40 or 70/30 v/v) containing 30 mM TEA and 60 mM FA HPLC: H2O/MeOH (1/99 v/v) containing 30 mM TEA and 60 mM FA SFC: CO2/MeOH (70/30 v/v) containing 30 mM TEA and 60 mM FA MeOH/ACN (50/50 v/v) containing 25 mM base (NH3, EA, DEA, TEA, or PA) and 50 mM acid (AcOH or FA) MeOH/ACN (80/20, 70/30, 60/40, 50/50 v/v) containing 25 mM base (NH3, DEA, TEA) and 50 mM acid (AcOH or FA); MeOH/H2O (98/2 v/v) containing 25 mM TEA and 50 mM FA

Pregabalin

Fmoc-Leu, Trp, salbutamol

N-Fmoc proteinogenic amino acids

N-Fmoc proteinogenic amino acids

Aliphatic and aromatic secondary amino acids

β2-Amino acids

Chiralpak ZWIX(þ)™

Chiralpak ZWIX(þ)™

Chiralpak ZWIX(þ)™

Chiralpak ZWIX()™

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

[37]

[35, 36]

[34, 35]

[33]

[32]

(continued)

HPLC [38] 5 to 55  C 0.6 mL min1 UV, 215 and 230 nm

HPLC 5 to 55  C 0.6 mL min1 UV, 230 nm CAD

HPLC, 25  C 0.6 mL min1 UV, 262 nm SFC, 40  C 2.0 mL min1 UV, 262 nm

SFC 20–50  C 2.0 mL min1 UV, 262 nm

HPLC 5–50  C 0.6 mL min1 UV, 262 nm

HPLC, 25  C 0.8 mL min1 UV, 230 nm

HPLC-ESI-MS 25  C 1.0 mL min1

Cinchona-Based Chiral Stationary Phases 259

[42]

[43]

[44]

[45]

Isoxazoline-fused MeOH/ACN (75/25 v/v) containing HPLC 2-aminocyclopentanecarboxylic acids 25 mM TEA and 50 mM AcOH 10–50  C (β-amino acids) 0.6 mL min1 UV, 215–230 nm CAD MeOH/ACN (50/50 v/v) containing HPLC 25 mM base (PA, TPA, BA, or TBA) 10–50  C and 50 mM AcOH 0.6 mL min1 UV, 215–230 nm MeOH/ACN (25/75 or 75/25 v/v) HPLC containing 25 mM NH3 or TEA and 10–50  C 50 mM AcOH 0.6 mL min1 CAD

β2- and β3-homoaminoacids

Cyclic β-amino acids

Unusual β3-aminoacids with alkyl, aryl, MeOH/ACN (50/50 v/v) containing HPLC and heteroaryl side chains 25 mM TEA and 50 mM AcOH 10–50  C 0.6 mL min1 UV, 230 nm CAD

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

[40, 41]

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

HPLC 10–50  C 0.6 mL min1 UV, 230 nm CAD

MeOH/ACN (50/50 or 25/75 v/v) containing 25 mM base (NH3, EA, DEA, TEA, or PA) and 50 mM acid (AcOH or FA)

Monoterpene-based β-amino acids

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

[39]

HPLC 10–50  C 0.6 mL min1 UV, 230 nm

MeOH/ACN (50/50 v/v) containing 25 mM base (NH3, EA, DEA, TEA, or PA) and 50 mM acid (AcOH or FA)

Bicycl0[2.2.2]octane-based 3-amino2-carboxylic acids

Ref.

Mode

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

The most effective mobile-phase compositions

Stereoisomers

Chiral selectors or columns

Table 1 (continued)

260 Istva´n Ilisz et al.

MeOH/ACN (75/25 v/v) containing HPLC 25 mM DEA and 50 mM AcOH 5–40  C 0.6 mL min1 CAD MeOH/ACN (60/40 v/v) containing HPLC 5–40  C 25 mM TEA and 50 mM FA or 6.25 mM TEA and 12.5 mM FA 0.6 mL min1 CAD MeOH/ACN (50/50 v/v) containing HPLC 25 mM DEA and 50 mM AcOH 5–50  C 0.6 mL min1 CAD

Limonene-based carbocyclic β-amino acids

N-methylated, guanidinated, and NFmoc cyclic β-amino acids

Cyclic β-amino acids and cyclic β-aminohydroxamic acids

Dipeptides: Ala-Val, Pro, Phe, Ala-Phe, MeOH containing 25 mM DEA and 50 mM FA Gly-Phe, Gly-Val, Gly-Leu, Gly-Trp, Gly-Pro, Pro-Gly, Gly-Thr, Gly-Asp

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™ ZWIX(þA) and ZWIX(A)

Alkylsulfonic acid-, butanesulfonic acid-, and aminocyclohexanesulfonic acidcarbamoylated quinine and quinidine

MeOH containing 0.5% AcOH Alkylsulfonic acid-, arylsulfonic acid-, Lys-Ala-Ala, and Ala-Ala tri- and dipeptides of variable stereochemical MeOH containing 100 μM NaOAC and aminocyclohexanesulfonic acidconfigurations carbamoylated quinine and quinidine

Alkylsulfonic acid-, arylsulfonic acid-, Ala, Val, Phe homo and heterochiral di-, MeOH containing 25 mM DEA and tri-, and tetrapeptides 50 mM FA and aminocyclohexanesulfonic acidcarbamoylated quinine and quinidine

MeOH/ACN (50/50 or 80/20 v/v) HPLC containing 25 m MDEA and 50 mM 5–40  C AcOH 0.6 mL min1 UV, 230 nm CAD

Cyclic β3-amino acids

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™ ZWIX(þA) and ZWIX(A)

HPLC-ESI-MS 25  C 5.0 μL min1

HPLC, 25  C 1.0 mL min1 UV, 254 nm and CAD

HPLC 25  C 1.0 mL min1 UV, 254 nm and CAD

MeOH/ACN (50/50 v/v) containing HPLC 25 mM TEA and 50 mM AcOH 5–50  C 0.6 mL min1 CAD

Cyclic β-aminohydroxamic acids

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

(continued)

[52]

[51]

[16]

[50]

[49]

[48]

[47]

[46]

Cinchona-Based Chiral Stationary Phases 261

HPLC 15 to þ45  C 0.4 mL min1 CAD

Mode

14 pairs of diastereomeric and MeOH/ACN/H2O (49/49/2 v/v/v) HPLC-ESI-MS 25  C enantiomeric Ser and Thr containing containing 12.5 mM NH3 and 25 mM FA 0.5 mL min1 di-, tri-, and tetrapeptides

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

HPLC, 25  C Homochiral and heterochiral peptides: MeOH; MeOH/ACN (98/2 v/v); MeOH/THF (98/2 v/v); MeOH/ 1.0 mL min1 Gly-Asn, Gly-Asp, Gly-Leu, Gly-Ser, H2O (98/2 v/v); MeOH/ACN CAD Gly-Val, Gly-Nva, Gly-Nle, Gly-Phe, (90/10 v/v), MeOH/THF Gly-Met, Gly-Gly-Leu, Ala-Val, (90/10 v/v), MeOH/H2O Pro-Phe, Leu-Leu, Leu-Leu-Leu (90/10 v/v) all containing 12.5 mM DEA and 25 mM FA

Di-, tripeptides: Leu-Val, Gly-Gly-Val

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

Chiralpak ZWIX(þ)™ and Chiralpak ZWIX()™

[53]

Ref.

[55]

[54]

HPLC, 25  C [19] ACN/H2O (90/10 v/v), MeOH/ ACN/H2O (49/49/2 v/v/v), 0.4–1.0 mL min1 MeOH/THF/H2O (49/49/2 v/v/ UV, v), MeOH/H2O (98/2 v/v) all 230–254–270 nm and ELSD containing 25 mM DEA and 50 mM FA

MeOH containing 25 mM DEA and Homo- or heterochiral dipeptides: 50 mM FA Ala-Pro, Gly-Pro, Leu-Pro, Phe-Pro and Pro-Pro

Ethanesulfonic acid-, arylsulfonic acid-, and aminocyclohexanesulfonic acidcarbamoylated quinine and quinidine

The most effective mobile-phase compositions

Stereoisomers

Chiral selectors or columns

Table 1 (continued)

262 Istva´n Ilisz et al.

Cinchona-Based Chiral Stationary Phases

263

logk ¼ log K Z  Z log ½X 

ð1Þ

In this equation KZ is a system-specific constant, which is related to (1) the ion-exchange equilibrium constant K (L/mol), (2) the specific surface area S of the adsorbent (m2/g), (3) the charge density on the surface qx (mol/m2), and (4) the mobilephase volume Vm in the column (L), as described by Eq. 2. (Vm is composed of the stagnant pore volume and the flowing interstitial volume between the particles.)
Z KS q x KZ ¼ ð2Þ Vm Thus, the amount of chiral selectors bound to the surface of the adsorbent plays a dominant role. K depends on the sum of the active SO–SA interactions of which the strongest is the electrostatic one. In other words, K will be different for the two entries of a resolvable pair of enantiomers because of the different magnitude of the sum of intermolecular interactions. As a result, the quotient of KR over KS (we assume KR > KS) will be directly related to the chromatographic selectivity, α. Based on this model, information can be derived on the charges involved in the overall ion-exchange process. The above phenomenologically described mechanism will become somewhat more complicated when discussing the mode of ampholytic ion exchange in context with ampholytic, or cationic or anionic, analytes. The intrinsic, stoichiometrically fixed intramolecular counterion effect is a special peculiarity of the ampholytic ZWIX selectors and needs to be discussed together with the counterion effect of the mobile-phase components depending on the applied experimental conditions. The related experimental facets of this concept are discussed in the forthcoming chapter.

4

Parameters to Be Optimized In the case of CSPs based on an ion-exchange process, retention depends on electrostatic interactions between an ionic (or ionizable) solute and the ionic (or ionizable) functional group (s) of the SO. In this context it should be noted that the ionic sites are always solvated, and the size of the solvation shell depends on the type of solvent components (see also later). In the case of Cinchona alkaloid-based SOs, the nitrogen in the quinuclidine ring is protonated under acidic conditions, applying any of the usual chromatographic modes (pKa ¼ 9.8) [13]. It is important to emphasize that eluent acidity will determine not only the ionic state of the quinuclidine ring, but also the ionization of the analyte. Consequently, pH values allowing attractive ionic interactions must

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be controlled via the mobile phase. However, we have to take into account that we are actually dealing with an apparent pH because of the primarily used nonaqueous mobile-phase conditions, which leads to a shift of pKa values compared to aqueous solutions. Obviously, a selector based on the quinuclidine ring will act as a positively charged anion-exchanger site. Accordingly, anions or acid additives are required to act as counterions to displace the solutes from this charged interaction site. In the case of the ZWIX phases, besides the protonated nitrogen of the quinuclidine ring, a SCX site (in most cases a deprotonated cyclohexane sulfonic acid moiety with a pKa of about 1) is also available for ionic interactions. Thus, with ampholytic SAs two ion pairs may be formed simultaneously via interactions with the anion- and the cation-exchanger sites of the SOs. Based on the spatial arrangement of the SO and SA molecules, an enantioselective resolution will occur. To displace the ampholytic SAs from both ionic sites, acidic and basic mobile-phase additives are generally required as counterions. The most important experimental variables by which elution can be adjusted are (1) the type and ratio of solvents of the polar organic bulk mobile phase, (2) the nature and concentration of acid and/or base additives, and (3) the temperature and the flow rate. 4.1 Effect of MobilePhase Composition

Relevant applications of the zwitterionic-type CSPs have been listed in Table 1 with information on the resolved analytes, chromatographic conditions, and detection methods applied. Nonaqueous polar organic solvents (methanol (MeOH) or acetonitrile (ACN)) in combination with acid and/or base modifiers (polar ionic (PI) mode) proved to be the preferential mobile phases when employing Cinchona alkaloid-based CSPs. The use of MeOH as a protic solvent (which can suppress H-bonding interactions) and ACN as a polar, but aprotic, solvent (which can strengthen ionic interactions, but interferes with π–π interactions) seems to be the best combination. It allows the reduction (suppression) of nonspecific hydrophobic interactions with the CSP, thereby enhancing enantioselectivity. Varying the nature and concentration of the organic solvents is the primary choice to tune the overall chromatographic performance. Zwitterionic Cinchona alkaloid-based selectors (similarly to the WAX QN-AX- and QD-AX-type columns) are mostly employed with MeOH- and/or ACN-based but acidic eluent systems using base and/or acid additives (Table 1). Other organic solvents like ethanol (EtOH) [21], 2-propanol (IPA) [21, 37], and tetrahydrofuran (THF) [34, 47, 55, 59] have been tested to improve the separation performance. On Chiralpak ZWIX(þ)™, the replacement of MeOH with 2 volume percent (vol%) EtOH or IPA resulted in slightly reduced retention factors. Less polar alcohols induced a decrease in retention [21], which suggests the partial involvement of hydrophobic SO–SA interactions. Enantioselectivity observed for

Cinchona-Based Chiral Stationary Phases

265

hydroxyalkanoic acids was highest with EtOH, but MeOH gave the best resolutions indicating the occurrence of some kinetic effects. Applying Chiralpak ZWIX(þ)™ and ZWIX()™ as well as mobile phases based on mixtures of IPA and ACN (using IPA instead of MeOH), retention of the studied secondary amino acids increased substantially with IPA present in the eluent system. This is probably due to the decreased ability of IPA to solvate the amino acids in the mobile phase. However, no significant improvement in enantioselectivity could be reached [37]. In cases when MeOH and ACN were applied as bulk solvents and only partial separation could be reached, replacement of MeOH or ACN with THF, in turn, may offer improved enantioselectivity and resolution [34, 47, 55, 59]. However, such improvements strongly depended on the amino acid involved in the experiment [47]. Separation of the basic trans-paroxetine enantiomers was investigated on Cinchona alkaloid-based zwitterionic CSPs [59]. Upon changing MeOH or ACN to THF, a significant improvement in enantioselectivity was observed. The MeOH/ THF or ACN/THF ratio exerted a strong effect on the chromatographic parameters. In MeOH/THF systems, increasing of the MeOH content resulted in decreasing retentions, while in ACN/THF systems increasing retentions were observed with an increasing ACN content. Increased retention factors were also obtained for underivatized oligopeptides on Chiralpak ZWIX (þ)™ employing mobile phases with increasing THF content in MeOH in the range of 0–10 vol%, while separation factors were not altered by THF addition [55]. In summary, zwitterionic phases are mostly employed with MeOH- and/or ACN-based mobile phases. All published data so far show that retention increases substantially with an increasing ACN content in eluents containing MeOH and ACN in different ratios. As concerns selectivity, diverse trends can be observed and the separation factor usually enhances with increasing ACN content for aliphatic analytes but an opposite trend was observed for aromatic analytes. This is most probably due to the influence of ACN on π–π interactions [37]. The observed chromatographic behavior can be summarized as follows: 1. An increase of retention factors with the increase of the content of the polar, but aprotic, ACN is probably due to decreased solvation of the charged sites resulting in a thinner solvation shell, thereby influencing the strength of the electrostatic interactions between the SA and the SO, which is affected by the third power of the distance of the charged sites. 2. The trend to a selectivity increase in the ACN-rich mobile phase can be explained by promotion of electrostatic and H-bonding interactions, which may enhance chiral

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recognition. In a solvent mixture with a higher MeOH content, the extent of SA solvation is more pronounced than in the aprotic ACN, resulting in shorter retention as a consequence of weaker electrostatic interactions. In addition, the protic MeOH can partially inhibit hydrogen bonding-type interactions influencing this way the chiral recognition. 3. The combination of long-range ionic, H-bonding (e.g., between the carbamate group of the SO and a suitable moiety of the SA), van der Waals, π–π, and steric interactions will determine the overall enantioselective separation performance. 4.2 Effect of Water Addition

RP conditions favor hydrophobic interactions, while mobile phases with lower dielectric constants may promote ionic, hydrogen bonding, and dipole-dipole interactions. After introducing the Cinchona alkaloid-based zwitterionic phases [12], it soon became clear that RP conditions generally led to lower separation performances. However, it is worth mentioning that adding water even at a low percentage might be advantageous for either sample solubility or MS detection compatibility reasons. Relatively few papers have been published describing the influence of water on the enantioseparations by zwitterionic phases. Mobile-phase systems to study the effects of water as bulk solvent component were employed for the enantioseparations of chiral zwitterionic SAs [15]. By enhancing the water content of the MeOH-based eluent up to 20 vol%, retention of amino acids slightly decreased. For the most hydrophobic Trp further enhancement of the water content (up to 80 vol %) led to marked increases of retention, while the effect on less hydrophobic solutes could be characterized with unchanged or slightly reduced retention times. It is important to note that both enantioselectivity and resolution were continuously decreasing with an increasing water content in this study. Depending on the hydrophobicity of the studied solutes, RP-type retention increments occurred only at high water contents, thus overwhelming the electrostatic forces. Caused by the strong solvation effects of water molecules in the water-rich eluent, interactions between the oppositely charged SO and SA moieties is considerably reduced. As a result, the formation of the SO–SA complex based on double ion pairing is weakened. To detect any subtle effects induced by water present in the eluent, enantioseparations of free amino acids were studied in MeOH- and ACN-based mobile phases with particular attention on low percentages of the water content [19]. Increasing the water content up to 20 vol% led to a drastic reduction in retention factors, while further increases up to 60 vol% led to diminished retention for most of the studied amino acids. With a water content in the range of 60–80 vol%, slightly increased retention factors were found. It was experimentally shown that a low percentage (2%) of

Cinchona-Based Chiral Stationary Phases

267

water in the mobile phase could be beneficial for certain separations resulting in better peak shapes and shorter analysis times. The influence of water in lower percentages (10 vol%) was also studied in additional cases [18, 32, 34, 45, 46, 55]. The presence of water in the eluent turned out to be favorable for the separation factor of peptides with lipophilic residues [55], while no beneficial effect of the water addition (1–5 vol%) was observed for the enantioseparation of highly polar aminophosphonic acids [18]. Reduced retention times and resolution, but an increased S/N ratio, were obtained with an increasing water content from 2 to 5 vol%, when the enantiomers of the γ-amino acid derivative pregabalin were separated using LC-MS determination [32]. For enantioseparations of 9-fluorenylmethyl-chloroformate (Fmoc)protected amino acids, applying an eluent containing 1–2 vol% water was found to be advantageous, while a further increase of the water content resulted in a decrease in the chromatographic performance [34]. Employing hydro-organic eluent systems (MeOH and/or ACN and water) for the enantioresolution of primary β-amino acids, the best separations were achieved with ACN as bulk solvent containing not more than 10 vol% of water [45]. In the case of β-aminohydroxamic acids, addition of water to the MeOH/ACN mobile phase system in increasing amounts led to a marked reduction in retention times followed by a continuous decrease in selectivity [46]. As a consequence of its solvation power, water may significantly influence the electrostatic interactions, thus playing a determining role in the ion-exchange process. As a general tendency, shorter retentions and lower selectivities and resolutions are obtained with increasing water content. It should be noted, however, that in some cases a low water content (typically 1–2 vol%) may still have a positive effect on the overall separation performance generating better peak shapes and shorter, but still sufficient, retention times. 4.3 Effect of the Nature and Concentration of Acid and Base Additives

To modulate the primary ionic interaction between SO and SA, nonaqueous polar organic solvents are generally modified by the addition of an acid and/or base. The additives greatly influence the ionization and ion-pair formation of both SO and SA, and the anions and cations of acid and base additives may therefore affect the chromatographic performance. For traditional ion exchangers, the retention can be adjusted conveniently by variation of the counterion concentration: the higher the counterion concentration the lower the retention, which is due to the competition between the SAs and the counterions for the ionic functional groups of the SO. Retention based on the abovementioned ion-exchange process in a nonaqueous environment can be described by the simple displacement model [56–58]: the plot of log k versus log c gives a linear relationship, where the slope of the straight line is

268

Istva´n Ilisz et al.

proportional to the effective charge involved in the ion-exchange process. Several articles discuss the validity of the simple displacement model for anion exchangers [14, 22, 60–64] and for zwitterionic phases [14, 21, 34–36, 44–50, 59]. The slope of log k versus log c generally varies in the range of 0.3 to 1.2 and 0.1 to 0.8 for anion exchangers and zwitterionic CSPs, respectively. The significantly lower absolute values of the slopes determined for the zwitterionic phases compared to, e.g., a genuine anion exchanger, can be attributed to a certain extent to the intrinsic and, thus, stoichiometrically fixed counterion effects of the zwitterionic SO moiety, which intramolecularly counterbalances the effects of the counterions present in the mobile phase. In these systems, counterions have a much lower effect on the retention times of the SAs than they have on a conventional single (mono)-ion chromatographic column. Consequently, it is important to emphasize that the retention times of SAs can only be adjusted in a limited range by the variation of the counterion concentration applying zwitterionic CSPs. It is important to note as well that enantioselectivity, in most cases, was not significantly affected by the concentration of the counterion employing either anion- or zwitterion-exchanger SO. Practically identical slopes were obtained for each enantiomer applying the same chromatographic conditions. Zwitterionic CSPs can be applied as cation, anion, and zwitterion exchangers. Comparing the slopes of the double-logarithmic curves determined for monoionic and zwitterionic modes, it can be stated that the zwitterionic mode can generally be characterized with markedly shallower slopes, indicating a definitive difference between the zwitterionic and monoionic modes. The variation of the nature of the acid and base added to the mobile phase is an additional parameter to obtain optimal separations with zwitterionic SOs. It is important to note that zwitterionic SOs act simultaneously as cation and anion exchangers. Therefore, acid and base additives play the role of co- and counterions concurrently. Moreover, one should keep in mind that the ion-exchange characteristics of a zwitterionic CSP will also be dependent on the structure of the analyte. Whether the analyte is present in anionic, cationic, or zwitterionic form, the type of the acid and base additive may have significantly different influences on the chromatographic behavior. Because of this special characteristic, the effects of the acid and base additives on retention, selectivity, and resolution are rather complex and difficult to deconvolute. To investigate the effects of the nature of the acid and base additives, separations are generally carried out with constant bulk solvent composition and an excess of the acid component in the mobile phase, which is applied to ensure that the bases are present in their protonated “ammonium” form. The results cited below were obtained on the commercially available Chiralpak ZWIX(þ)™ and ZWIX()™ columns.

Cinchona-Based Chiral Stationary Phases

269

When zwitterionic phases were operated in the zwitterionic mode for the resolution of free amino acid enantiomers and in the anion-exchange (AX) mode for acidic solutes, the type of basic additive, i.e., NH3, diethylamine (DEA), or tiethylamine (TEA), turned out to have negligible influence on the overall chromatographic performance [15]. It was concluded that, on the one hand, the effects of the base additive possibly balance each other. On the other hand, the intramolecular counterion within the zwitterionic SO is able to compensate the different elution strengths originating from the different types of base additives. The type of acid additive, i.e., acetic acid (AcOH), formic acid (FA), or trifluoroacetic acid (TFA), was found to have more pronounced influence on the chromatographic behavior suggesting that the AX-type interactions were more dominant in the retention mechanism than the cation-exchange-type interactions. Applying NH3 as a base additive, the influence of acids of different strengths and chain lengths (FA, AcOH, propionic acid) was studied in the enantioseparations of aminophosphonic acids [18]. The generally negative trend observed for separation efficiency with higher chain length of the acid was ascribed to its lower acidity and, therefore, to less pronounced counterion properties. Interestingly, opposite results were obtained for the separation of oligopeptides; that is, FA applied in the same concentration showed usually weaker elution strength than AcOH [55]. In the same study, three basic additives (NH3, DEA, TEA) were tested as counterions for the cation-exchange moiety. For most of the studied peptides, the elution strength enhanced in the sequence TEA < DEA < NH3. The effects of seven bases (ethylamine (EA), DEA, TEA, propylamine (PA), tripropylamine (TPA), butylamine (BA), and tributylamine (TBA)) and two acids (AcOH and FA) were investigated with zwitterionic phases applied in AX mode for the enantioseparation of proteinogenic Fmoc-amino acids [34, 36]. Neither the nature of the base nor the nature of the acid had a significant influence on the retention and enantioselectivity. Different bases (e.g., NH3, EA, DEA, TEA, PA, TPA, BA, TBA) and two acids (AcOH and FA) were also selected to study the influence of co- and counterions in the enantioseparations of β-amino acids [39, 43], isoxazoline-fused 2-aminocyclopentanecarboxylic acids [42], trans-paroxetine [59], and cyclic β-amino acids [44]. Increased retention factors were found in all cases as the degree of alkyl substitution of the nitrogen atom increased. Moreover, the retention factor also increased in several cases as the alkyl chain length in the amino group increased [42, 44, 59]. Retention factors, in most cases, decreased in the sequence TEA > DEA > EA  NH3. No significant variation of the chromatographic parameters could be observed with the change of the acid type (AcOH or FA). Regarding the influence of acid and base additives on the elution strength, in general, quite similar results were obtained for secondary amino acids [37],

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tetrahydroisoquinoline analogs [25], β2-amino acids [38], monoterpene-based 2-aminocarboxylic acids [40], and limonenebased carbocyclic β-amino acids [48]. NH3 exhibited the highest elution strength, while TEA the lowest elution strength. The acid applied (AcOH or FA) had only a slight effect on retention. As a general trend, the elution strength increases in the order TEA  DEA  EA  PA  NH3, but the competitive ability of the bases is not directly connected with their basicity. The larger the mobile-phase additive in size, the less effective it is in displacing a protonated SA from the SO–SA ion-pair complex under LC conditions. As a result, the elution strength decreases. 4.4

Flow Rate

The relatively slow mass transfer properties due to the presence of heterogeneous adsorption sites being characteristic of silica-based enantioselective ion-exchanger-type stationary phases is a fact to deal with [65]. Only a few articles discuss the effect of flow rate on the chromatographic performance applying a Cinchona alkaloidbased zwitterionic SO. Moderate gain in efficiency was realized when the eluent flow rate was reduced from 1 to 0.3 mL min1 on Chiralpak ZWIX(þ)™ used in the enantioseparations of aliphatic hydroxyalkanoic acids [21]. In the enantioresolution of aminophosphonic acids, increased plate heights were obtained with faster velocities in the range of 0.66–1.33 mm/s on Cinchona alkaloid-based zwitterionic CSP [18]. Because of the increased analysis time, a compromise of 0.93 mm/s velocity was selected as the optimum. More detailed studies by van Deemter analysis on H–u curves and the kinetics of the chromatographic process (effects of particle size, pore size, selector density, etc.) of the zwitterionic CSPs have not yet been published.

5 Analyte Separations in Biological Matrices on Cinchona Alkaloid-Based Ion-Exchanger CSPs Separation of amino acid enantiomers in biological samples (tissues, blood, physiological fluids, etc.) is an important task but, at the same time, it is also a big challenge. To increase the sensitivity of the methods, the amino group of amino acids gets most often derivatized with a UV or fluorescence active reagent before separation and analysis [66]. Generally, in a two-dimensional HPLC (2D HPLC) setup, implementing a “heart cut” concept, two columns are connected via a switching valve allowing the transfer of fractions of eluent of the “primary” column onto the second column [67]. Most commonly, the heart-cut 2D HPLC systems are implemented by connecting an RP column in the first dimension to a chiral column in the second dimension, including a QN- or QD-based anionic or zwitterionic stationary phase. The results obtained in this field are mainly connected to the work of Hamase

Cinchona-Based Chiral Stationary Phases

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and coworkers [68–74]. The enantiomers of the amino acids Val, allo-Ile, Ile, Leu, Pro, and 4-OH-Pro in mammals [68, 69]; of Val, allo-Ile, Ile, and Leu in mammals and physiological fluids [72]; of Ala, Asp, Glu, Ser, Leu, and allo-Ile in Japanese black vinegars [73]; as well as Asp and Glu in the tissues and physiological fluids of mice [74] were derivatized with 4-fluoro-7-nitro-3,1,3-benzoxadiazole (NBD-F) and separated successfully applying Chiralpak® QN-AX, QD-AX [68, 72], QN-AX, QD-AX, QN-2AX, QD-2AX [69], QN-AX [73], and QD-AX [74] columns in the second dimension using fluorescence detection (Table 1). For the 5-(dimethylamino) naphthalene-1-sulfonyl (DNS)-protected Ala, Arg, Asp, Glu, Ile, Leu, Phe, and Val in milk sample, t-butylcarbamoylated QN and QD CSP and UV detection were applied [67]. The enantioseparation of Ala, Asp, and Ser in rat plasma and tissues using 6-aminoquinilyl-N-hydroxysuccinimidyl carbamate (AccQ) and p-N,N,Ntrimethylammonioanilyl N0 -hydroxysuccinimidyl carbamate iodide (TAHS) as derivatization reagents applying zwitterionic ZWIX(þ) column in the second dimension and a highly sensitive MS/MS detection has also been described [70]. A new pre-column derivatization reagent [2,5-dioxopyrrolidin-1-yl(2-(6-methoxy-4-oxoquinolin-1(4H)-ylethyl carbonate] with a 6-methoxy-4-quinoline (6-MQD) moiety for analyses of proteinogenic amino acids has recently been designed and synthesized. The 6-MOQ derivatives of all proteinogenic amino acids were separated using the combination of three enantioselective columns, QN-AX, ZWIX(þ), and NBD-Ala enantiomer-based CSP [71]. This reagent would be very suitable for the two-dimensional HPLC system combining fluorescence detection (in the first dimension) and sensitive MS/MS detection (in the second dimension). Very recently AccQ derivatives of 19 proteinogenic amino acids have been directly separated on QN-based SO covalently bonded to superficially porous particles in an UHPLC system with MS/MS detection [75]. The method was suitable for the determination of all proteinogenic L- and D-amino acids in the brain of wild-type mice, mutant mice lacking D-amino acid oxidase activity, and heterozygous mice.

6 Separation of Homochiral and Heterochiral Peptide Epimers on Cinchona Alkaloid-Based Ion-Exchanger CSPs The Cinchona alkaloid-based ion exchanger CSPs possesses not only enantioselective but also diastereoselective properties. A typical example for enantioselective and diastereoselective discrimination on Cinchona alkaloid-based ion exchangers is the separation of homochiral and heterochiral peptide epimers. Examples are the separation of phosphinic pseudo-dipeptides (hPheψ[P(O)OH)

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CH2]Phe) on QD-AX CSP [76], 3,5-dinitrobenzoyl-, 3,5-dichlorobenzoyl-, and N-Fmoc-protected di- and tripeptides [77] as well as N-derivatized dipeptides of Ala-Ala, Ala-Leu, Leu-Ala, and Ala-Phe [78] on carbamoylated QD-based monolith column. Various zwitterionic phases based on alkylsulfonic acid-, arylsulfonic acid-, and aminocyclohexanesulfonic acid-carbamoylated QN and QD were successfully applied for the enantio- and diastereoseparation of underivatized homo- and heterochiral di-, tri-, and tetrapeptides applying the PI mode [16, 51–53]. ZWIX(þ) and ZWIX() were applied for the chiral discrimination of di- and tripeptides Leu-Val and Gly-Gly-Val [19]; di-, tri-, and tetrapeptides containing Ser and Thr [54]; as well as homo- and heterochiral peptides of Gly-Asn, Gly-Asp, Gly-Leu, Gly-Ser, Ala-Val, Pro-Phe, Leu-Leu, Leu-Leu-Leu, etc. [55]. It is noteworthy that the interconversion of cis and trans isomers of dipeptides containing C-terminal Pro could be observed by dynamic chromatography on zwitterionic CSPs at temperatures ranging from 15 to þ45  C. cis-trans Isomers could be separated below 0  C. Above 0–10  C, however, plateau formation and peak coalescence phenomena occurred, which is characteristic of a dynamic process at the timescale of partitioning. At and above room temperature, full coalescence was observed, which allowed separations of enantiomers without interference from interconversion effects [53].

7

Conclusions Although a rather large collection of diversely structured “chiral columns” are nowadays available on the market, the design of new chiral stationary phases offering more efficient separations under shorter analysis time with higher robustness is still a challenging task to reach and is valid for all types of CSPs, including the described chiral ion exchangers. This may include also the development of dedicated CSPs for certain families of chiral analytes of very high polarity, etc. In this context, diastereoselective aspects may also be of interest. Cinchona alkaloid-based CSPs are frequently applied either as weak anion exchangers for the resolution of diverse chiral acids or as zwitterions for the resolution of anionic, cationic, and ampholytic compounds. Besides traditional LC-based applications, these columns can also be efficiently utilized in SFC [34–36, 61, 79]. It is important to mention that recent developments in surface chemistry with advances in material sciences have enabled the development of highly efficient superficially porous particles [80] or separation-based sensors [81] based on Cinchona alkaloids. From the chromatographic point of view, new particle morphology, switching to, e.g., core-shell or sub-2 μm-type particles will

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probably further widen the application spectrum of Cinchona alkaloid-based selectors and CSPs toward highly efficient ultrafast enantiomer separations. Along this line, even comprehensive 2D UHPLC methodologies can be envisioned. References 1. Gil-Av E, Feibush B, Charles-Sigler R (1966) Separation of enantiomers by gas liquid chromatography with an optically active stationary phase. Tetrahedron Lett 7:1009–1015 2. L€ammerhofer M, Lindner W (2008) Liquid chromatographic enantiomer separation and chiral recognition by cinchona alkaloid-derived enantioselective separation materials. Adv Chromatogr 46:1–107 3. Grubhofer N, Schleith L (1953) Modified ion exchangers as specific adsorbents. Naturwissenschaften 40:508–512 4. Grubhofer N, Schleith L (1954) Splitting of racemic mandelic acid with a optically anion exchange. Hoppe Seylers Z Physiol Chem 296:262–266 5. Izumoto S, Sakaguchi U, Yoneda H (1983) Chromatographic study of optical resolution. 10. Stereochemical aspects of the optical resolution of cis(N)-[Co(N)2(O)4] complexes by reversed-phase ion-pair chromatography with cinchona alkaloid cations as the ion-pairing reagents. Bull Chem Soc Jpn 56:1646–1651 6. Miyoshi K, Natsubori M, Dohmoto N, Izumoto S, Yoneda H (1985) Chromatographic study of optical resolution. 12. Optical resolution of bis(oxalato)(1,10-phenanthroline)cobaltate(iii) and its related anion complexes with cinchona alkaloid cations as eluent. B Chem Soc Jpn 58:1529–1534 7. Pettersson C (1984) Chromatographicseparation of enantiomers of acids with quinine as chiral counter ion. J Chromatogr 316:553–567 8. Pettersson C, Schill G (1986) Separation of enantiomers in ion-pair chromatographic systems. J Liq Chromatogr 9:269–290 9. Pettersson C, Gioeli C (1988) Improved resolution of enantiomers of naproxen by the simultaneous use of a chiral stationary phase and a chiral additive in the mobile phase. J Chromatogr 435:225–228 10. Rosini C, Bertucci C, Pini D, Altemura P, Salvadori P (1985) Cinchona alkaloids for preparing new, easily accessible chiral stationary phases. 1. 11-(10,11-dihydro-60 -methoxycinchonan-9-ol)-tiopropylsilanized silica. Tetrahedron Lett 26:3361–3364

11. Lammerhofer M, Lindner W (1996) Quinine and quinidine derivatives as chiral selectors. 1. Brush type chiral stationary phases for high-performance liquid chromatography based on cinchonan carbamates and their application as chiral anion exchangers. J Chromatogr A 741:33–48 12. Hoffmann CV, Pell R, L€ammerhofer M, Lindner W (2008) Synergistic effects on enantioselectivity of zwitterionic chiral stationary phases for separations of chiral acids, bases, and amino acids by HPLC. Anal Chem 80:8780–8789 13. Kacprzak K, Gawronski J (2009) Resolution of racemates and enantioselective analytics by cinchona alkaloids and their derivatives. In: Song CE (ed) Cinchona alkaloids in synthesis and catalysis. Wiley-VCH, Weinheim, pp 421–463 14. Hoffmann CV, Reischl R, Maier NM, L€ammerhofer M, Lindner W (2009) Stationary phase-related investigations of quinine-based zwitterionic chiral stationary phases operated in anion-, cation-, and zwitterion-exchange modes. J Chromatogr A 1216:1147–1156 15. Hoffmann CV, Reischl R, Maier NM, L€ammerhofer M, Lindner W (2009) Investigations of mobile phase contributions to enantioselective anion- and zwitterion-exchange modes on quinine-based zwitterionic chiral stationary phases. J Chromatogr A 1216:1157–1166 16. Wernisch S, Pell R, Lindner W (2012) Increments to chiral recognition facilitating enantiomer separations of chiral acids, bases, and ampholytes using Cinchona-based zwitterion exchanger chiral stationary phases. J Sep Sci 35:1560–1572 17. Pell R, Sic´ S, Lindner W (2012) Mechanistic investigations of cinchona alkaloid-based zwitterionic chiral stationary phases. J Chromatogr A 1269:287–296 18. Gargano AFG, Kohout M, Macı´kova´ P, L€ammerhofer M, Lindner W (2013) Direct high-performance liquid chromatographic enantioseparation of free α-, β- and γ-aminophosphonic acids employing cinchona-based chiral zwitterionic ion exchangers. Anal Bioanal Chem 405:8027–8038

274

Istva´n Ilisz et al.

19. Zhang T, Holder E, Franco P, Lindner W (2014) Method development and optimization on cinchona and chiral sulfonic acid-based zwitterionic stationary phases for enantiomer separations of free amino acids by highperformance liquid chromatography. J Chromatogr A 1363:191–199 20. Geditz MCK, Lindner W, L€ammerhofer M, Heinkele G, Kerb R, Ramharter M, Schwab M, Hofmann U (2014) Simultaneous quantification of mefloquine (þ)- and ()enantiomers and the carboxy metabolite in dried blood spots by liquid chromatography/ tandem mass spectrometry. J Chromatogr B 968:32–39 21. Ianni F, Pataj Z, Gross H, Sardella R, Natalini B, Lindner W, L€ammerhofer M (2014) Direct enantioseparation of underivatized aliphatic 3-hydroxyalkanoic acids with a quinine-based zwitterionic chiral stationary phase. J Chromatogr A 1363:101–108 22. Caldero´n C, Horak J, L€ammerhofer M (2016) Chiral separation of 2-hydroxyglutaric acid on cinchonan carbamate based weak chiral anion exchangers by high-performance liquid chromatography. J Chromatogr A 1467:239–245 23. Caldero´n C, L€ammerhofer M (2017) Chiral separation of short chain aliphatic hydroxycarboxylic acids on cinchonan carbamate-based weak chiral anion exchangers and zwitterionic chiral ion exchangers. J Chromatogr A 1487:194–200 24. Ianni F, Carotti A, Marinozzi M, Marcelli G, Di Michele A, Sardella R, Lindner W, Natalini B (2015) Diastereo- and enantioseparation of a Nα-Boc amino acid with a zwitterionic quinine-based stationary phase: focus on the stereorecognition mechanism. Anal Chim Acta 885:174–182 25. Ilisz I, Grecso´ N, Fu¨lo¨p F, Lindner W, Pe´ter A (2015) High-performance liquid chromatographic enantioseparation of cationic 1,2,3,4-tetrahydroisoquinoline analogs on cinchona alkaloid-based zwitterionic chiral stationary phases. Anal Bioanal Chem 407:961–972 26. Zhang T, Holder E, Franco P, Lindner W (2014) Zwitterionic chiral stationary phases based on cinchona and chiral sulfonic acids for the direct stereoselective separation of amino acids and other amphoteric compounds. J Sep Sci 37:1237–1247 27. Fukushima T, Sugiura A, Furuta I, Iwasa S, Iizuka H, Ichiba H, Onozato M, Hikawa H, Yokoyama Y (2015) Enantiomeric separation of monosubstituted tryptophan derivatives and metabolites by HPLC with a Cinchona alkaloid-based zwitterionic chiral stationary

phase and its application to the evaluation of the optical purity of synthesized 6-chloro-Ltryptophan. Int J Tryptophan Res 8:1–5 28. Sugimoto H, Kakehi M, Jinno F (2015) Method development for the determination of d and l-isomers of leucine in human plasma by high-performance liquid chromatography tandem mass spectrometry and its application to animal plasma samples. Anal Bioanal Chem 407:7889–7898 29. Carotti A, Ianni F, Sabatini S, Di Michele A, Sardella R, Kaatz GW, Lindner W, Cecchetti V, Natalini B (2016) The “racemic approach” in the evaluation of the enantiomeric NorA efflux pump inhibition activity of 2-phenylquinoline derivatives. J Pharm Biomed Anal 129:182–189 30. Gerhardt H, Sievers-Engler A, Jahanshah G, Pataj Z, Ianni F, Gross H, Lindner W, L€ammerhofer M (2016) Methods for the comprehensive structural elucidation of constitution and stereochemistry of lipopeptides. J Chromatogr A 1428:280–291 31. Wolrab D, Fru¨hauf P, Moulisova´ A, Kucharˇ M, Gerner C, Lindner W, Kohout M (2016) Chiral separation of new designer drugs (Cathinones) on chiral ion-exchange type stationary phases. J Pharmaceut Biomed 120:306–315 32. Chennuru LN, Choppari T, Nandula RP, Zhang T, Franco P (2016) Direct separation of pregabalin enantiomers using a zwitterionic chiral selector by high performance liquid chromatography coupled to mass spectrometry and ultraviolet detection. Molecules 21. pii: E1578 33. Hanafi RS, L€ammerhofer M (2018) Response surface methodology for the determination of the design space of enantiomeric separations on cinchona-based zwitterionic chiral stationary phases by high performance liquid chromatography. J Chromatogr A 1534:55–63 34. Lajko´ G, Grecso´ N, To´th G, Fu¨lo¨p F, Lindner W, Pe´ter A, Ilisz I (2016) A comparative study of enantioseparations of Nα-Fmoc proteinogenic amino acids on Quinine-based zwitterionic and anion exchanger-type chiral stationary phases under hydro-organic liquid and subcritical fluid chromatographic conditions. Molecules 21. pii: E1579 35. Lajko´ G, Ilisz I, To´th G, Fu¨lo¨p F, Lindner W, Pe´ter A (2015) Application of Cinchona alkaloid-based zwitterionic chiral stationary phases in supercritical fluid chromatography for the enantioseparation of Nα-protected proteinogenic amino acids. J Chromatogr A 1415:134–145 36. Lajko´ G, Grecso´ N, To´th G, Fu¨lo¨p F, Lindner W, Ilisz I, Pe´ter A (2017) Liquid and

Cinchona-Based Chiral Stationary Phases subcritical fluid chromatographic enantioseparation of Nα-Fmoc proteinogenic amino acids on Quinidine-based zwitterionic and anion-exchanger type chiral stationary phases. A comparative study. Chirality 29:225–238 37. Ilisz I, Gecse Z, Pataj Z, Fu¨lo¨p F, To´th G, Lindner W, Pe´ter A (2014) Direct highperformance liquid chromatographic enantioseparation of secondary amino acids on Cinchona alkaloid-based chiral zwitterionic stationary phases. Unusual temperature behavior. J Chromatogr A 1363:169–177 38. Ilisz I, Grecso´ N, Aranyi A, Suchotin P, Tymecka D, Wilenska B, Misicka A, Fu¨lo¨p F, Lindner W, Pe´ter A (2014) Enantioseparation of β2-amino acids on cinchona alkaloid-based zwitterionic chiral stationary phases. Structural and temperature effects. J Chromatogr A 1334:44–54 39. Ilisz I, Grecso´ N, Palko´ M, Fu¨lo¨p F, Lindner W, Pe´ter A (2014) Structural and temperature effects on enantiomer separations of bicyclo [2.2.2]octane-based 3-amino-2-carboxylic acids on cinchona alkaloid-based zwitterionic chiral stationary phases. J Pharmaceut Biomed Anal 98:130–139 40. Pataj Z, Ilisz I, Gecse Z, Szakonyi Z, Fulop F, Lindner W, Peter A (2014) Effect of mobile phase composition on the liquid chromatographic enantioseparation of bulky monoterpene-based beta-amino acids by applying chiral stationary phases based on Cinchona alkaloid. J Sep Sci 37:1075–1082 41. Ilisz I, Pataj Z, Gecse Z, Szakonyi Z, Fu¨lo¨p F, Lindner W, Pe´ter A (2014) Unusual temperature-induced retention behavior of constrained β-amino acid enantiomers on the zwitterionic chiral stationary phases ZWIX(þ) and ZWIX(). Chirality 26:385–393 42. Ilisz I, Gecse Z, Lajko´ G, Nonn M, Fu¨lo¨p F, Lindner W, Pe´ter A (2015) Investigation of the structure–selectivity relationships and van’t Hoff analysis of chromatographic stereoisomer separations of unusual isoxazoline-fused 2-aminocyclopentanecarboxylic acids on Cinchona alkaloid-based chiral stationary phases. J Chromatogr A 1384:67–75 43. Ilisz I, Grecso N, Misicka A, Tymecka D, Lazar L, Lindner W, Peter A (2015) Comparison of the separation performances of Cinchona alkaloid-based zwitterionic stationary phases in the enantioseparation of beta(2)and beta(3)-amino acids. Molecules 20:70–87 44. Ilisz I, Gecse Z, Lajko G, Forro E, Fulop F, Lindner W, Peter A (2015) High-performance liquid chromatographic enantioseparation of cyclic beta-amino acids on zwitterionic chiral

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stationary phases based on Cinchona alkaloids. Chirality 27:563–570 45. Ilisz I, Grecso´ N, Papousˇek R, Pataj Z, Barta´k P, La´za´r L, Fu¨lo¨p F, Lindner W, Pe´ter A (2015) High-performance liquid chromatographic separation of unusual β3-amino acid enantiomers in different chromatographic modes on Cinchona alkaloid-based zwitterionic chiral stationary phases. Amino Acids 47:2279–2291 46. Lajko´ G, Orosz T, Grecso´ N, Fekete B, Palko´ M, Fu¨lo¨p F, Lindner W, Pe´ter A, Ilisz I (2016) High-performance liquid chromatographic enantioseparation of cyclic β-aminohydroxamic acids on zwitterionic chiral stationary phases based on Cinchona alkaloids. Anal Chim Acta 921:84–94 47. Grecso´ N, Forro´ E, Fu¨lo¨p F, Pe´ter A, Ilisz I, Lindner W (2016) Combinatorial effects of the configuration of the cationic and the anionic chiral subunits of four zwitterionic chiral stationary phases leading to reversal of elution order of cyclic β3-amino acid enantiomers as ampholytic model compounds. J Chromatogr A 1467:178–187 48. Lajko´ G, Orosz T, Ugrai I, Szakonyi Z, Fu¨lo¨p F, Lindner W, Pe´ter A, Ilisz I (2017) Liquid chromatographic enantioseparation of limonene-based carbocyclic β-amino acids on zwitterionic Cinchona alkaloid-based chiral stationary phases. J Sep Sci 40:3196–3204 49. Orosz T, Forro´ E, Fu¨lo¨p F, Lindner W, Ilisz I, Pe´ter A (2018) Effects of N-methylation and amidination of cyclic β-amino acids on enantioselectivity and retention characteristics using Cinchona alkaloid- and sulfonic acid-based chiral zwitterionic stationary phases. J Chromatogr A 1535:72–79 50. Bajtai A, Fekete B, Palko´ M, Fu¨lo¨p F, Lindner W, Kohout M, Ilisz I, Pe´ter A (2017) Comparative study on the liquid chromatographic enantioseparation of cyclic β-amino acids and the related cyclic β-aminohydroxamic acids on Cinchona alkaloid-based zwitterionic chiral stationary phases. J Sep Sci 41:1216–1223 51. Wernisch S, Lindner W (2012) Versatility of cinchona-based zwitterionic chiral stationary phases: enantiomer and diastereomer separations of non-protected oligopeptides utilizing a multi-modal chiral recognition mechanism. J Chromatogr A 1269:297–307 52. Bobbitt JM, Li L, Carlton DD, Yasin M, Bhawal S, Foss FW, Wernisch S, Pell R, Lindner W, Schug KA (2012) Diastereoselective discrimination of lysine–alanine–alanine peptides by zwitterionic cinchona alkaloidbased chiral selectors using electrospray

276

Istva´n Ilisz et al.

ionization mass spectrometry. J Chromatogr A 1269:308–315 53. Wernisch S, Trapp O, Lindner W (2013) Application of cinchona-sulfonate-based chiral zwitterionic ion exchangers for the separation of proline-containing dipeptide rotamers and determination of on-column isomerization parameters from dynamic elution profiles. Anal Chim Acta 795:88–98 54. Reischl RJ, Lindner W (2015) The stereoselective separation of serine containing peptides by zwitterionic ion exchanger type chiral stationary phases and the study of serine racemization mechanisms by isotope exchange and tandem mass spectrometry. J Pharm Biomed Anal 116:123–130 55. Ianni F, Sardella R, Carotti A, Natalini B, Lindner W, L€ammerhofer M (2016) Quininebased zwitterionic chiral stationary phase as a complementary tool for peptide analysis: Mobile phase effects on enantio- and stereoselectivity of underivatized oligopeptides. Chirality 28:5–16 56. Kopaciewicz W, Rounds MA, Fausnaugh J, Regnier FE (1983) Retention model for highperformance ion-exchange chromatography. J Chromatogr 266:3–21 57. Sellergren B, Shea KJ (1993) Chiral ion-exchange chromatography—correlation between solute retention and a theoretical ion-exchange model using imprinted polymers. J Chromatogr A 654:17–28 58. Stahlberg J (1999) Retention models for ions in chromatography. J Chromatogr A 855:3–55 59. Grecso´ N, Kohout M, Carotti A, Sardella R, Natalini B, Fu¨lo¨p F, Lindner W, Pe´ter A, Ilisz I (2016) Mechanistic considerations of enantiorecognition on novel Cinchona alkaloid-based zwitterionic chiral stationary phases from the aspect of the separation of trans-paroxetine enantiomers as model compounds. J Pharm Biomed Anal 124:164–173 60. Xiong X, Baeyens WRG, Aboul-Enein HY, Delanghe JR, Tu TT, Ouyang J (2007) Impact of amines as co-modifiers on the enantioseparation of various amino acid derivatives on a tertbutyl carbamoylated quinine-based chiral stationary phase. Talanta 71:573–581 61. Pell R, Schuster G, L€ammerhofer M, Lindner W (2012) Enantioseparation of chiral sulfonates by liquid chromatography and subcritical fluid chromatography. J Sep Sci 35:2521–2528 62. Gargano AFG, Lindner W, L€ammerhofer M (2013) Phosphopeptidomimetic substance libraries from multicomponent reaction: Enantioseparation on quinidine carbamate stationary phase. J Chromatogr A 1310:56–65

63. Woiwode U, Sievers-Engler A, ˜ oz Zimmermann A, Lindner W, Sa´nchez-Mun OL, L€ammerhofer M (2017) Surface-anchored counterions on weak chiral anion-exchangers accelerate separations and improve their compatibility for mass-spectrometry-hyphenation. J Chromatogr A 1503:21–31 64. Pe´ter A, Grecso´ N, To´th G, Fu¨lo¨p F, Lindner W, Ilisz I (2016) Ultra-trace analysis of enantiomeric impurities in proteinogenic NFmoc-amino acid samples on Cinchona alkaloid-based chiral stationary phases. Isr J Chem 56:1042–1051 65. Lammerhofer M, Gyllenhaal O, Lindner W (2004) HPLC enantiomer separation of a chiral 1,4-dihydropyridine monocarboxylic acid. J Pharm Biomed Anal 35:259–266 66. Ilisz I, Pe´ter A, Lindner W (2016) State-of-theart enantioseparations of natural and unnatural amino acids by high-performance liquid chromatography. TrAC-Trend Anal Chem 81:11–22 67. Ianni F, Sardella R, Lisanti A, Gioiello A, Cenci Goga BT, Lindner W, Natalini B (2015) Achiral–chiral two-dimensional chromatography of free amino acids in milk: a promising tool for detecting different levels of mastitis in cows. J Pharm Biomed Anal 116:40–46 68. Hamase K, Morikawa A, Ohgusu T, Lindner W, Zaitsu K (2007) Comprehensive analysis of branched aliphatic d-amino acids in mammals using an integrated multi-loop two-dimensional column-switching high-performance liquid chromatographic system combining reversed-phase and enantioselective columns. J Chromatogr A 1143:105–111 69. Tojo Y, Hamase K, Nakata M, Morikawa A, Mita M, Ashida Y, Lindner W, Zaitsu K (2008) Automated and simultaneous two-dimensional micro-high-performance liquid chromatographic determination of proline and hydroxyproline enantiomers in mammals. J Chromatogr B 875:174–179 70. Karakawa S, Shimbo K, Yamada N, Mizukoshi T, Miyano H, Mita M, Lindner W, Hamase K (2015) Simultaneous analysis of d-alanine, d-aspartic acid, and d-serine using chiral high-performance liquid chromatography-tandem mass spectrometry and its application to the rat plasma and tissues. J Pharm Biomed Anal 115:123–129 71. Oyama T, Negishi E, Onigahara H, Kusano N, Miyoshi Y, Mita M, Nakazono M, Ohtsuki S, Ojida A, Lindner W, Hamase K (2015) Design and synthesis of a novel pre-column derivatization reagent with a 6-methoxy-4-quinolone moiety for fluorescence and tandem mass spectrometric detection and its application to chiral

Cinchona-Based Chiral Stationary Phases amino acid analysis. J Pharm Biomed Anal 116:71–79 72. Han H, Miyoshi Y, Ueno K, Okamura C, Tojo Y, Mita M, Lindner W, Zaitsu K, Hamase K (2011) Simultaneous determination of d-aspartic acid and d-glutamic acid in rat tissues and physiological fluids using a multi-loop two-dimensional HPLC procedure. J Chromatogr B 879:3196–3202 73. Miyoshi Y, Nagano M, Ishigo S, Ito Y, Hashiguchi K, Hishida N, Mita M, Lindner W, Hamase K (2014) Chiral amino acid analysis of Japanese traditional Kurozu and the developmental changes during earthenware jar fermentation processes. J Chromatogr B 966:187–192 74. Han H, Miyoshi Y, Koga R, Mita M, Konno R, Hamase K (2015) Changes in d-aspartic acid and d-glutamic acid levels in the tissues and physiological fluids of mice with various d-aspartate oxidase activities. J Pharm Biomed Anal 116:47–52 75. Du S, Wang Y, Weatherly CA, Holden K, Armstrong DW (2018) Variations of L- and Damino acid levels in the brain of wild-type and mutant mice lacking D-amino acid oxidase activity. Anal Bioanal Chem 410:2971–2979 76. Mucha A, L€ammerhofer M, Lindner W, Pawełczak M, Kafarski P (2008) Individual

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stereoisomers of phosphinic dipeptide inhibitor of leucine aminopeptidase. Bioorg Med Chem Lett 18:1550–1554 77. Wang Q, Sa´nchez-Lo´pez E, Han H, Wu H, Zhu P, Crommen J, Marina ML, Jiang Z (2016) Separation of N-derivatized di- and tri-peptide stereoisomers by micro-liquid chromatography using a quinidine-based monolithic column—analysis of L-carnosine in dietary supplements. J Chromatogr A 1428:176–184 78. Wang Q, Zhu P, Ruan M, Wu H, Peng K, Han H, Somsen GW, Crommen J, Jiang Z (2016) Chiral separation of acidic compounds using an O-9-(tert-butylcarbamoyl)quinidine functionalized monolith in micro-liquid chromatography. J Chromatogr A 1444:64–73 79. Pell R, Lindner W (2012) Potential of chiral anion-exchangers operated in various subcritical fluid chromatography modes for resolution of chiral acids. J Chromatogr A 1245:175–182 80. Patel DC, Breitbach ZS, Yu J, Nguyen KA, Armstrong DW (2017) Quinine bonded to superficially porous particles for high-efficiency and ultrafast liquid and supercritical fluid chromatography. Anal Chim Acta 963:164–174 81. Patel DC, Wahab MF, O’Haver TC, Armstrong DW (2018) Separations at the speed of sensors. Anal Chem 90:3349–3356

Chapter 15 Enantioseparations by High-Performance Liquid Chromatography Based on Chiral Ligand Exchange Federica Ianni, Lucia Pucciarini, Andrea Carotti, Roccaldo Sardella, and Benedetto Natalini Abstract Although the first application of chiral ligand-exchange chromatography (CLEC) in HPLC dates back to late 1960s, this enantioselective strategy still represents the elective choice for the direct analysis of compounds endowed with chelating moieties. As a specific feature of the CLEC mechanism, the interaction between the chiral selector and the enantiomer does not take place in direct contact. Indeed, it is mediated by a central metal ion that, acting as a Lewis acid, simultaneously coordinates the two species, selector and analyte, through the activation of dative bonds. As a consequence, two diastereomeric mixed ternary complexes are generated in the column, ultimately leading to the stereoisomeric discrimination. CLEC applications can be carried out both with the chiral selector included in the mobile phase (chiral mobile phase, CMP), or as a part of the stationary phase. In the latter case, the chiral selector can be either covalently immobilized onto a solid support (bonded CSP, B-CSP) or physically adsorbed onto a conventional packing material, coated chiral stationary phase (C-CSP). In this chapter, a selection of CLEC applications with CMP- and C-CSP-based chiral systems is presented. Key words Chiral ligand-exchange chromatography, Chiral mobile phase, Coated chiral stationary phase, Dynamic coating

1

Introduction Since the beginning of chiral chromatography, it was self-evident that direct resolution of enantiomers assumes great importance, having an influent impact especially on the following steps involving, inter alia, the possibility to isolate increasing amounts of optical isomers. Indeed, the mechanism of ligand exchange applied to chiral chromatography electively allows the possibility to resolve a racemic mixture without enantiomer derivatization, thus disclosing its potential in the preparative process. Still today, chiral ligandexchange chromatography (CLEC) constitutes the method of choice for the enantioresolution of both racemates and

Gerhard K. E. Scriba (ed.), Chiral Separations: Methods and Protocols, Methods in Molecular Biology, vol. 1985, https://doi.org/10.1007/978-1-4939-9438-0_15, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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Fig. 1 Proposed models for the ternary complexes formed between the chiral additive and eluent N, N-dimethyl-L-phenylalanine, Cu(II), and a L- or D-amino acid

enantiomeric scalemic mixtures of compounds endowed with the ability to form complexes through dative bonds. As well known, a central metal ion (most commonly Cu(II)) is the Lewis acid acceptor on which hexadentate mixed diastereomeric complexes are formed (Fig. 1). As a consequence, this methodology can be applied to the separation and resolution of α- and β-amino acids, α-hydroxy acids, diols, diamines, amino alcohols, quinolones, and small peptides, all of them showing the complex-forming ability. Since its introduction by Gil-Av [1] in 1966 for GC and by Davankov in 1968 for LC [2], this methodology has been extensively applied. Two alternative and conceptually different approaches can be pursued to get compounds enantioseparated in CLEC systems. The first implies the use of a chiral mobile phase (CMP) obtained through the addition of a suitable chelating species to the eluent system. The second is based on the use of a chiral stationary phase (CSP) in which the chiral selector is either covalently immobilized onto a solid support (bonded CSP, B-CSP) or physically adsorbed onto a conventional packing material, usually C18 chains or porous graphitic carbon (coated CSP, C-CSP) [3, 4]. Besides the possibility to get underivatized amino acids enantioseparated [5–9], CLEC methodology offers a number of additional advantages: the possibility to detect UV transparent molecules through the generation of UV/vis-active metal complexes; the use of either commercially available and cost-effective or easy-to-synthesize chiral enantiodiscriminating agents (chiral selectors); for CMP and C-CSP systems, the employment of rather inexpensive achiral columns; and the easy analytical-to-semi-preparative scale-up. Moreover, due to the use of water as the main eluent component, CLEC methods can assume an “eco-friendly” character as well [5–9]. With CLEC-CMPs two distinct situations can occur: the first in which the enantiodiscriminating agent is exclusively present in the mobile phase and the second in which it partitions between the two chromatographic phases [3, 4]. In the more common, latter situation, the main complexation events taking place in the column can be realistically described by Scheme 1.

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+

+ Scheme 1 Complexation equilibria for a chiral mobile phase-based CLEC system

+ Scheme 2 Complexation equilibria for a chiral stationary phase-based CLEC system

In Scheme 1, A indicates the enantiomeric analyte, M the divalent metal cation, and C the chiral selector. The superscripts m and s refer to the location of the species in the mobile and stationary phases, respectively; K refers to the formation constant of the ternary complex (complexation constant). When C is dynamically coated onto a hydrophobic surface, the reversible complexation of the analyte into the ternary complex (AMCs) may happen by direct interaction of the analyte in the mobile phase, Am, with the immobilized chiral selector-metal ion complex, MCs. Alternatively, a two-step process may be described according to Scheme 2. In the two-step process, the analyte first transfers from the mobile to the stationary phase (Am $ As) where it is then coordinated by the binary MCs adduct (As + MCs $ AMCs). Equilibria described in Scheme 2 can take place both with C-CSP and B-CSP systems operating according to the CLEC concept. So far, both for CMP and C-CSP systems, a large number of physicochemically diverse chelating species have been evaluated as chiral selectors. Among these, a major role is played by amino alcohol derivatives [10–13] and amino acid derivatives [14–23]. Moreover, other less conventional compounds endowed with strong capacity to coordinate transition metal ions are fruitfully used as chiral selectors for CLEC applications [24–30]. Since the CLEC mechanism is based on complexation equilibria superimposed to hydrophobic interactions, relevant variations of the chromatographic performances (mostly in terms of retention, enantioselectivity, and resolution factors) might occur even upon slight modifications of the eluent composition, mostly in terms of pH, type and content of metal ion and salt, type and

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content of organic modifier, type and content of buffer systems; and type and content of additional ionic additive. Furthermore, the eluent flow rate as well as special features of the achiral column and its temperature should be carefully considered as well, with CMP and C-CSP systems. In the following sections, a selection of CLEC applications with CMP- and C-CSP-based chiral systems is presented. Accordingly, while examples 1 and 2 refer to CLEC applications in the CMP mode, examples 3–9 offer the opportunity to realize C-CSP separations.

2

Materials

2.1 Instrumentation and Materials

1. A commercial HPLC instrument, for example, a Shimadzu LC-20A Prominence system (Shimadzu, Kyoto, Japan) equipped with a CBM-20A communication bus module, two LC-20AD dual-piston pumps, and a SPD-M20A photodiode array detector. 2. Column ovens for thermostating the column, such as Grace® (Sedriano, Italy) heater/chiller (Model 7956R). 3. A commercial pH meter for pH adjustment of the mobile phase. 4. An ultrasonic bath for degassing mobile phases. 5. 0.22 μm Membrane filters for filtering mobile phases and sample solutions. 6. An OptimaPak C18 column (150 mm  4.6 mm i.d., 5 μm, ˚ pore size) (see Note 1). 100 A 7. A Nova-Pak C18 column (150 mm  4.0 mm i.d., 4 μm, 60 A˚ pore size) (see Note 1). 8. A GraceSmart C18 column (250 mm  4.6 mm i.d., 5 μm, ˚ pore size) (see Note 1). 120 A 9. A LiChrospher 100 C18 column (250 mm  4.0 mm i.d., 5 μm, 100 A˚ pore size) (see Note 1). 10. A μBondapak C18 column (300 mm  3.9 mm i.d., 10 μm, 125 A˚ pore size) (see Note 1). 11. A L-column ODS (150 mm  4.6 mm i.d., 5 μm, 120 A˚ pore size) (see Note 1). 12. A Sumipax ODS column (150 mm  4.6 mm i.d., 5 μm, 120 A˚ pore size) (see Note 1). 13. A Spherisorb ODS2 column (150 mm  4.6 mm i.d., 5 μm, ˚ pore size) (see Note 1). 80 A

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2.2 Solution and Mobile Phases

Use HPLC-grade organic solvents and ultrapure water obtained by a suitable water purification system. All chemicals should be of analytical grade.

2.2.1 Mobile Phases

1. Mobile phase for example 1 (10 mM L-leucine and 5 mM CuSO4  5H2O) (see Note 2). For 1.0 L of aqueous solution, dissolve 1.31 g of L-leucine in 700 mL of water and 1.25 g of CuSO4  5H2O in 300 mL of water. Mix both solutions (see Note 3). Mix this solution and methanol at a ratio of 88:12 (v/v) and adjust the pH to 4.8 with concentrated trifluoroacetic acid. Filter (0.22 μm) and sonicate the solution for 20 min (see Notes 4 and 5). 2. Mobile phase for example 2 (4.0 mM (S)-phenylalaninamide and 2.0 mM Cu(CH3COO)2 in sodium acetate pH 7.5) (see Notes 2, 3, and 6). Prepare 1.0 L of 0.3 M aqueous solution of sodium acetate by dissolution of 24.61 g in 1000 mL water. In 700 mL of the sodium acetate solution dissolve 0.66 g of and in 300 mL of the sodium acetate solution dissolve 0.36 g of Cu(CH3COO)2 H2O (see Note 4). Mix both solutions (see Note 3). Mix this solution with acetonitrile at a ratio of 72:28 (v/v). Adjust the pH of this solution to 7.5 with 5 N NaOH (for 1.0 L, dissolve 200 g of sodium hydroxide in water). Filter (0.22 μm) and sonicate the solution for 20 min (see Note 4). 3. Mobile phase for example 3 (1.0 mM CuSO4  5H2O) (see Note 2). Dissolve 249.68 mg of CuSO4  5H2O in 1000 mL water (see Note 2). Filter through 0.22 μm filters and sonicate for 20 min. 4. Mobile phase for example 4 (1.0 mM Cu(CH3COO)2): Dissolve 181.63 mg of Cu(CH3COO)2 in 1000 mL water. Filter through 0.22 μm filters and sonicate for 20 min. 5. Mobile phase for example 5 (0.1 mM CuSO4  5H2O) (see Note 2). Dissolve 24.97 mg CuSO4  5H2O in 1000 mL water: acetonitrile 90:10 (v/v). Filter through 0.22 μm filters and sonicate for 20 min. 6. Mobile phase for example 6 (0.5 mM Cu(CH3COO)2) (see Note 2). Dissolve 90.82 mg Cu(CH3COO)2 in 1000 mL water and adjust pH to 5.7 with 0.5 N NaOH (for 1.0 L, dissolve 20 g of sodium hydroxide in water). Filter through 0.22 μm filter and sonicate the eluent for 20 min (see Note 4). 7. Mobile phase for example 7 (0.5 mM Cu(CH3COO)2) (see Note 2). Dissolve 90.82 mg of Cu(CH3COO)2 in 1000 mL water. Filter the solution through 0.22 μm filters and sonicate the eluent for 20 min (see Note 7). 8. Mobile phase for example 8 (1.0 mM CuSO4  5H2O) (see Note 2). Dissolve 249.68 mg of CuSO4  5H2O in 1000 mL water.

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Filter the solution through 0.22 μm filters and sonicate the eluent for 20 min (see Note 7). 9. Mobile phase for example 9 (0.5 mM CuSO4  5H2O) (see Note 2). Dissolve 124.84 mg 0.5 mM CuSO4  5H2O in 1000 mL water and adjust the pH to 6.0 with 2.0 mM sodium acetate solution (for 1.0 L, dissolve 164.1 mg of sodium acetate in water). Filter through 0.22 μm filter and sonicate for 20 min (see Note 4). 2.2.2 Sample Solutions

3

Dissolve the analytes mentioned in the examples in the respective mobile phase at a concentration in the range of 0.5–1.0 mg/mL. In the case of amino acids of derivatives such as DNS-amino acids, either a mixture of the racemic acid derivatives or a single racemic amino acid may be used. Filter all sample solutions through 0.22 μm filters before injection.

Methods

3.1 Example 1: Enantioresolution of Ofloxacin Using Lleucine as Chiral Mobile-Phase Additive

1. Install the OptimaPak C18 column (150 mm  4.6 mm i.d., 5 μm, 100 A˚ pore size) in the HPLC system (see Note 1). 2. Equilibrate the column by recycling the mobile phase for experiment 1 for 12 h at a flow rate of 1.0 mL/min through the column (see Note 8). 3. Replace the mobile phase from the recycling with fresh mobile phase (see Notes 9 and 10). 4. Set the column oven at a temperature 25  C and equilibrate the column at a flow rate of 1.0 mL/min for another 15 min. 5. Set the detector to 293 nm and check if the baseline is stable (see Note 11). 6. Inject 20 μL of the ofloxacin sample solution and record the chromatogram. A representative chromatogram is shown in Fig. 2. Further examples can be found in ref. 15.

3.2 Example 2: Enantioresolution of DNS-Amino Acids Using (S)phenylalaninamide as Chiral Mobile-Phase Additive

1. Install the Nova-Pak C18 column (150 mm  4.0 mm i.d., ˚ pore size) in the HPLC system (see Note 1). 4 μm, 60 A 2. Equilibrate the column by recycling the mobile phase for experiment 2 for 12 h at a flow rate of 0.5 mL/min through the column (see Note 8). 3. Replace the mobile phase with fresh mobile phase (see Notes 9 and 10). 4. Set the column oven to 25  C and equilibrate the column at a flow rate of 0.5 mL/min for another 30 min.

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Fig. 2 Representative chromatogram of the enantioseparation of ofloxacin enantiomers (modified with permission of Elsevier from ref. 15 © 2007)

Fig. 3 Representative chromatogram of the simultaneous chemo- and enantioseparation of a mixture of D,LDns amino acids (modified with permission of Elsevier from ref. 17 © 1988

5. Set the detector at 254 nm and check if the baseline is stable. 6. Inject 20 μL of the sample solution and record the chromatogram. A representative chromatogram of a mixture of 11 DNS-amino acids is shown in Fig. 3. Further examples can be found in ref. 17. The separation of complex mixtures may be improved by using gradient elution (see Note 12).

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3.3 Example 3: N-decyl-S-trityl-(R)cysteine as Chiral Selector for the Enantioseparation of α-Amino Acids 3.3.1 Synthesis of N-decyl-S-trityl-(R)cysteine

All chemical reactions should be performed in a well-ventilated hood. All security measures for handling chemicals have to be observed. Wear protective clothing and safety goggles.

1. Dissolve 1.0 g (2.75 mmol) of S-trityl-(R)-cysteine in 15 mL methanol in a 100 mL round-bottom flask equipped with a magnetic stir bar at room temperature. 2. While stirring, add 0.19 g (3.0 mmol) of sodium cyanoborohydride and continue to stir for 20 min at room temperature. 3. Add 0.67 mL (3.50 mmol) decanal and continue to stir at room temperature for 12 h. 4. Remove the solvent under reduced pressure using a rotatory evaporator. 5. Purify the residue by flash chromatography with 120 g silica gel, by eluting with 3 L ethyl acetate/methanol as gradient from 0 to 20% (in volume) to obtain N-decyl-S-trityl-(R)cysteine as vitreous solid. The product can be further characterized using NMR spectroscopy.

3.3.2 Preparation of the N-decyl-S-trityl-(R)cysteine-Coated Chiral Stationary Phase

1. Install a GraceSmart C18 column (250 mm  4.6 mm i.d., 5 μm, 120 A˚ pore size) in the HPLC system (see Note 1). 2. Dissolve 0.9 g N-decyl-S-trityl-(R)-cysteine in 500 mL water: methanol (10:90, v/v) and pump the solution by recycling through the column at a flow rate of 0.3 mL/min for 48 h at room temperature (see Note 9). 3. Remove excessive N-decyl-S-trityl-(R)-cysteine by washing the column with 50 mL water:methanol (98:2, v/v) at a flow rate of 1.0 mL/min.

3.3.3 Enantioseparation of Amino Acids

1. Fill the mobile phase for experiment 3 in the solvent reservoir of the HPLC system (see Note 9). 2. Set the column oven to 25  C and equilibrate the column with the mobile phase for experiment 3 in open cycle at a flow rate of 0.4 mL/min for 24 h. 3. Set the detector wavelength to 254 nm and the flow rate at 1.0 mL/min and monitor until a stable baseline is obtained (see Note 13). 4. Inject 20 μL of an amino acid sample solution and record the chromatogram (see Notes 14, 15, and 16). 5. Monitor the column performance with periodical injections of rac-proline and perform the analysis of the first sample only when the separation and resolution factor values are close to 1.25 and 2.30, respectively.

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Fig. 4 Representative chromatogram of the enantioseparation of nor-leucine (modified with permission of Elsevier from ref. 19 © 2017)

A representative chromatogram of the separation of nor-leucine is shown in Fig. 4. Further examples can be found in ref. 19. 3.4 Example 4: (S)()-α,α-di(2naphthyl)-2pyrrolidinemethanolBased Coated Chiral Stationary Phase for the Enantioseparation of α-Amino Acids 3.4.1 Preparation of the (S)-()-α,α-di(2naphthyl)-2pyrrolidinemethanol-Based Coated Chiral Stationary Phase

1. Install a LiChrospher 100 C18 column (250 mm  4.0 mm i. d., 5 μm, 100 A˚ pore size) in the HPLC system (see Note 1). 2. Dissolve 0.5 g (S)-()-α,α-di(2-naphthyl)-2-pyrrolidinemethanol (for example, from Sigma-Aldrich) in 1.0 L methanol and pump this solution under recycling through the column at a flow rate of 0.5 mL/min for 4 days (see Notes 9 and 17). 3. Remove the excess of unbound (S)-()-α,α-di(2-naphthyl)-2pyrrolidinemethanol by washing the column with 50 mL water:methanol (98:2, v/v) at a flow rate of 1.0 mL/min.

3.4.2 Enantioseparation of α-Amino Acids

1. Fill the mobile phase for experiment 4 in the solvent reservoir of the HPLC system (see Note 9). 2. Set the column oven to 25  C and equilibrate the column by flowing the mobile phase in open cycle at a flow rate of 1.0 mL/min for 12 h. 3. Replace mobile phase with fresh mobile phase and set the detector wavelength to 254 nm. Monitor until a stable baseline is obtained. 4. Inject 20 μL of the sample solution and record the chromatogram.

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Fig. 5 Representative chromatogram of the enantioseparation of tyrosine (modified with permission of Wiley-VCH from ref. 10 © 2007)

5. Monitor the column performance with periodical injections of rac-tyrosine and perform the analysis of the first sample only when the separation and resolution factor values are close to 1.70 and 4.50, respectively. A representative chromatogram is shown in Fig. 5. Further examples can be found in ref. 10. 3.5 Example 5: (1S,2R)-N,Ncarboxymethyl Dodecylnorephedrine as Chiral Selector for the Enantioseparation of α-Amino Acids 3.5.1 Synthesis of (1S,2R)-N,Ncarboxymethyl Dodecylnorephedrine Monosodium Salt

All chemical reactions should be performed in a well-ventilated hood. All security measures for handling chemicals have to be observed. Wear protective clothing and safety goggles.

1. Dissolve 3.0 g (16 mmol) of (1S,2R)-norephedrine in 50 mL of dry methylene chloride in a 100 mL round-bottom flask equipped with a magnetic stir bar under a nitrogen atmosphere at room temperature. 2. While stirring under nitrogen, add dropwise 5 mL (36 mmol) of triethylamine and 3.8 mL (16 mmol) of lauroyl chloride previously dissolved in 10 mL of dry methylene chloride. 3. Continue to stir at room temperature under nitrogen for 30 min. 4. Transfer the organic solution to a separating funnel and wash with 60 mL 0.5 M HCl and subsequently with 60 mL 0.5 M NaOH. 5. Dry the organic layer with anhydrous MgSO4 and filter with paper filter in a Bu¨chner funnel. 6. Remove the organic solvent under reduced pressure using a rotatory evaporator and crystallize the resulting solid from methylene chloride/petroleum ether. Crystallization produces about 6.0 g of (1S,2R)-N-lauroyl norephedrine as a white crystalline solid (m.p. 73–75  C).

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7. Dissolve 5 g (15 mmol) of (1S,2R)-N-lauroyl norephedrine in 15 mL of dry THF into a 200 mL round-bottom flask and add a solution of 2.28 g (60 mmol) LiAlH4 in 50 mL of dry THF through a dropping funnel, over 30 min at 0  C. 8. Keep under reflux the whole mixture for 20 h, and then cool it to 0  C and quench by adding water. 9. Pass the whole mixture through a celite bed and then remove THF under reduced pressure using a rotatory evaporator. 10. Transfer the remaining aqueous solution into a separating funnel and extract the obtained (1S,2R)-N-dodecyl norephedrine with methylene chloride. 11. Dry the methylene chloride solution over anhydrous MgSO4 and then remove methylene chloride with a rotatory evaporator. 12. Crystallize the solid from a methylene chloride/petroleum ether solution. Crystallization produces about 4.0 g of (1S, 2R)-N-dodecyl norephedrine as a white crystalline solid (m.p. 52–54  C). 13. Dissolve 3.80 g (11.9 mmol) of (1S,2R)-N-dodecyl norephedrine in 30 mL of dry methylene chloride in a 100 mL roundbottom flask equipped with a magnetic stir bar under a nitrogen atmosphere. Add under stirring 1.46 mL (13.1 mmol) ethyl bromoacetate in 10 mL of dry methylene chloride at room temperature. Subsequently, add 1.83 mL (13.1 mmol) triethylamine at room temperature. 14. Continue to stir the solution for 24 h at room temperature. 15. Transfer the methylene chloride solution into a separating funnel and wash the reaction mixture with water. Then, dry the organic phase over anhydrous MgSO4, filter the solution on a sintered glass filter, and then concentrate it with a rotatory evaporator under reduced pressure. 16. Purify the oily residue by column chromatography on silica gel with an ethyl acetate/n-hexane 5/95 (v/v) eluent solution, thus obtaining about 1.5 g of the colorless oil (5R,6S)-4dodecyl-5-methyl-6-phenyl-2,3,5,6-tetrahydro-4H-1,4-oxazin-2-one. 17. Dissolve 1.4 g (3.90 mmol) of (5R,6S)-4-dodecyl-5-methyl-6phenyl-2,3,5,6-tetrahydro-4H-1,4-oxazin-2-one in 20 mL of methanol in a 50 mL round-bottom flask equipped with a magnetic stir bar at room temperature. 18. Add dropwise under stirring 3.45 mL of a 1 M NaOH methanolic solution and continue to stir the solution for 5 h at room temperature.

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19. Evaporate the solvent with a rotatory evaporator and then remove the solvent traces under high vacuum with a glass oven dry system for 10 h to obtain about 1.50 g of pure oily (1S,2R)-N,N-carboxymethyl dodecylnorephedrine monosodium salt. The product can be further characterized using NMR spectroscopy. 3.5.2 Preparation of the (1S,2R)-N,Ncarboxymethyl Dodecylnorephedrine Monosodium Salt-Coated Chiral Stationary Phase

1. Install a Waters μBondapak C18 column (250 mm  4.6 mm i. d., 10 μm, 125 A˚ pore size) in the HPLC system (see Note 1). 2. Dissolve 1.3 g of (1S,2R)-N,N-carboxymethyl dodecylnorephedrine monosodium salt in 15 mL methanol:water (1:1, v/v) and pump this solution through the column under recycling at a flow rate of 0.5 mL/min at room temperature (see Note 18). 3. Remove the unbound (1S,2R)-N,N-carboxymethyl dodecylnorephedrine monosodium salt by washing the column with a methanol:water (1:1, v/v) solution for 60 min at a flow rate of 0.3 mL/min.

3.5.3 Enantioseparation of α-Amino Acids

1. Fill the mobile phase for experiment 5 in the solvent reservoir of the HPLC system (see Note 9). 2. Set the column oven to 25  C and equilibrate the column with the mobile phase at a flow rate of 0.8 mL/min for 2 h. 3. Set the detector wavelength to 254 nm and monitor until a stable baseline is obtained. 4. Inject 20 μL of the sample solution and record the chromatogram. A representative chromatogram of the enantioseparation of a mixture of α-amino acids is shown in Fig. 6. Further examples can be found in ref. 13.

3.6 Example 6: 2-(2Hydroxy)hexadecyl(S)-1,2,3,4tetrahydro-3isoquinolinecarboxylic Acid as Chiral Selector for the Enantioseparation of α-Amino Acids

All chemical reactions should be performed in a well-ventilated hood. All security measures for handling chemicals have to be observed. Wear protective clothing and safety goggles.

3.6.1 Synthesis of the 2(2-Hydroxy)hexadecyl-(S)1,2,3,4-tetrahydro-3isoquinolinecarboxylic Acid

1. Dissolve 4.0 g (24.2 mmol) of L-phenylalanine in 10 mL of 37% (w/w) formaldehyde and 42 mL of 37% (w/w) hydrochloric acid in a 100 mL round-bottom flask equipped with a reflux condenser and a magnetic stir bar and stir at 60  C for 24 h.

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Fig. 6 Representative chromatogram of the simultaneous chemo- and enantioseparation of five racemic α-amino acids (modified with permission of Francis & Taylor from ref. 13 © 1993)

2. Cool the solution in an ice water bath to 0–5  C until a precipitate is formed. 3. Filter the reaction mixture using a paper filter in a Bu¨chner funnel to obtain a white precipitate. 4. Wash the precipitate on the Bu¨chner with 50 mL ice-cold water and dry the solid in a vacuum desiccator at room temperature for 12 h. 5. Redissolve the solid in 50 mL of boiling water in a 100 mL round-bottom flask and add dropwise a solution of 30% (m/v) ammonia until the pH is about 7. 6. Cool the aqueous solution with an ice bath to 0–5  C until a solid is formed. 7. Filter the solid using a paper filter in a Bu¨chner funnel and wash the obtained solid with 20 mL ice-cold water, 20 mL anhydrous ethanol, and 20 mL ethyl ether. 8. Dry the solid in a vacuum desiccator at room temperature for 24 h to obtain about 2.95 g (16.7 mmol) of (S)-1,2,3,4-tetrahydro-3-isoquinolinecarboxylic acid ((S)-THIQCA). 9. Dissolve 0.88 g (4.97 mmol) of (S)-THIQCA in a mixture of 6 mL of water and 54 mL of methanol in a 100 mL roundbottom flask equipped with a magnetic stir bar at room temperature.

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10. While stirring, add 0.21 g of NaOH and continue to stir at room temperature for 1 h. Add 1.50 g (6.25 mmol) of 1,2-epoxyhexadecane and keep stirring the mixture at room temperature for 24 h. 11. Neutralize the solution by adding 0.1 M HCl dropwise periodically checking the solution pH with pH paper. A white solid is formed. 12. Filter the solid using a paper filter in a Bu¨chner funnel and wash the solid with hot 50 mL methanol:water (90:10, v/v). 13. Dry the solid in a vacuum desiccator at room temperature for 24 h to get 1.87 g (4.48 mmol) of 2-(2-hydroxy)hexadecyl(S)-1,2,3,4-tetrahydro-3-isoquinoline carboxylic acid. The product can be further characterized using NMR spectroscopy. 3.6.2 Preparation of the 2-(2-Hydroxy)hexadecyl(S)-1,2,3,4-tetrahydro-3isoquinoline Carboxylic Acid-Coated Chiral Stationary Phase

1. Install a μBondapak C18 column (300 mm  3.9 mm i.d., 10 μm, 125 A˚ pore size) in the HPLC system (see Note 1). 2. Pump methanol at a flow rate of 1.0 mL/min through the column for 3 h. 3. Dissolve 1.85 g of 2-(2-hydroxy)hexadecyl-(S)-1,2,3,4-tetrahydro-3-isoquinoline carboxylic acid in 150 mL methanol: water (1:1, v/v) and pump this solution by recycling through the column at a flow rate of 0.5 mL/min for 2.5 h at room temperature (see Note 18). 4. Remove the unbound 2-(2-hydroxy)hexadecyl-(S)-1,2,3,4tetrahydro-3-isoquinoline carboxylic acid by washing the column with water at 0.4 mL/min for 2 h at room temperature (see Note 18).

3.6.3 Enantioseparation of α-Amino Acids

1. Fill the mobile phase for experiment 6 in the solvent reservoir of the HPLC system (see Note 9). 2. Set the column oven to 25  C and equilibrate the column with the mobile phase at a flow rate of 0.8 mL/min for 2 h (see Notes 9 and 14). 3. Set the detector wavelength to 254 nm and the flow rate at 1.0 mL/min and monitor until a stable baseline is obtained. 4. Inject 20 μL of the sample solution and record the chromatogram. Representative chromatograms of the enantioseparation of proline and methionine are shown in Fig. 7. Further examples can be found in ref. 24.

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Fig. 7 Representative chromatograms of the enantioseparation of (a) proline and (b) methionine (modified with permission of Elsevier from ref. 24 © 2006) 3.7 Example 7: L-Stearoylcarnitine as Chiral Selector for the Enantioseparation of α-Amino Acids and α-Hydroxycarboxylic Acids 3.7.1 Synthesis of L-Stearoylcarnitine Hydrochloride

All chemical reactions should be performed in a well-ventilated hood. All security measures for handling chemicals have to be observed. Wear protective clothing and safety goggles.

1. Dissolve 5.2 g of L-carnitine hydrochloride in 9.0 mL of trifluoroacetic acid in a 50 mL round-bottom flask equipped with a magnetic stir bar at room temperature. 2. While stirring, add 11.5 g stearoyl chloride as solid to one portion and continue to stir the mixture at 50  C for 2 h. 3. Let the reaction mixture stand at room temperature for 18 h and then pour the reaction mixture into 300 mL of diethyl ether so that a solid phase is formed. 4. Dissolve the obtained product in 80 mL methanol, filter, and recrystallize from 20 mL diethyl ether to obtain pure L-stearoylcarnitine hydrochloride. 5. Dry the solid final product in a vacuum desiccator at room temperature for 24 h to obtain about 9.9 g of L-stearoylcarnitine hydrochloride (m.p. 172  C; ½α25 D ¼ 13.7, c ¼ 1.0% in methanol). The product can be further characterized by NMR spectroscopy.

3.7.2 Preparation of the L-Stearoylcarnitine Hydrochloride-Coated Chiral Stationary Phase

1. Install a L-column ODS (150 mm  4.6 mm i.d., 5 μm, 120 A˚ pore size) into the HPLC system (see Note 1). 2. Dissolve 140 mg of L-stearoylcarnitine hydrochloride in 2 L of a 1:1 (v/v) mixture of 10 mM sodium diphosphate buffer, pH 6, and methanol. Pump the solution by recycling through

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the column at a flow rate of 1.0 mL/min for 40 h at room temperature (see Note 17). 3. Remove the unbound L-stearoylcarnitine by washing the column with water for 60 min at a flow rate of 1.0 mL/min. 3.7.3 Enantioseparation of α-Amino Acids and α-Hydroxycarboxylic Acids

1. Fill the mobile phase for experiment 7 in the solvent reservoir of the HPLC system (see Note 9). 2. Set the column oven to 25  C and equilibrate the column with the mobile phase at a flow rate of 1.0 mL/min for 2 h (see Notes 10 and 14). 3. Set the detector wavelength to 254 nm and monitor until a stable baseline is obtained. 4. Inject 20 μL of the sample solution and record the chromatogram (see Note 19). Representative chromatograms of the enantioseparation of isoleucine and lactic acid are shown in Fig. 8a, b, respectively. Further examples can be found in ref. 25.

Fig. 8 Representative chromatograms of the enantioseparation of (a) isoleucine and (b) lactic acid (modified with permission of Elsevier from ref. 25 © 2001)

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3.8 Example 8: (R,R)tartaric acid mono-(R)1-(α-naphthyl) ethylamide as Chiral Selector for the Enantioseparation of Carboxylic Acids and Amines with Another Polar Chelating Moiety in Alpha Position

All chemical reactions should be performed in a well-ventilated hood. All security measures for handling chemicals have to be observed. Wear protective clothing and safety goggles.

3.8.1 Synthesis of (R,R)tartaric acid mono-(R)-1(α-naphthyl)ethylamide

1. Dissolve 22.79 g (105 mmol) of (R,R)-O,O-diacetyl tartaric acid anhydride in 300 mL tetrahydrofuran in a 500 mL roundbottom flask equipped with a reflux condenser and a magnetic stir bar at room temperature. 2. While stirring, slowly add 18.84 g of (R)-1-(α-naphthyl)ethylamine dissolved in 100 mL tetrahydrofuran, and continue to stir the mixture at 60  C for 4 h. 3. Remove the solvent under reduced pressure using a rotatory evaporator. 4. Dissolve the solid residue in 200 mL methylene chloride and extract the organic solution with 200 mL of 1.0 M potassium hydroxide in a separating funnel. 5. Transfer the aqueous solution in a 500 mL round-bottom flask equipped with a magnetic stir bar and stir at room temperature for 3 h. 6. Afterwards, while stirring, add dropwise 6 M HCl until a colorless solid is obtained. 7. Filter the solid using paper filter in a Bu¨chner funnel and wash it with 100 mL water to obtain the colorless crystalline solid. 8. Dry the obtained (R,R)-tartaric acid mono-(R)-1-(α-naphthyl) ethylamide in a vacuum desiccator at room temperature for 24 h (m.p. 147–150  C; [α]D20 ¼ +51.2, c ¼ 0.2% in methanol). The product may be further characterized by NMR spectroscopy.

3.8.2 Preparation of the (R,R)-tartaric acid mono(R)-1-(α-naphthyl) ethylamide HydrochlorideCoated Chiral Stationary Phase

1. Install a Sumipax ODS column (150 mm  4.6 mm i.d., 5 μm, ˚ pore size) into the HPLC system (see Note 1). 120 A 2. Dissolve 250 mg (R,R)-tartaric acid mono-(R)-1-(α-naphthyl) ethylamide hydrochloride in 500 mL of a methanol:water (2:8, v/v) solution, in order to have a 0.05% (w/v) concentration of the chiral selector. Pump the solution of the selector by recycling through the column at a flow rate of 1.0 mL/min for 10 h at room temperature (see Notes 17, 18, and 20). 3. Remove the unbound (R,R)-tartaric acid mono-(R)-1-(α-naphthyl)ethylamide by washing the column with a water/ methanol (95/5; v/v) solution for 2 h at a flow rate of 0.5 mL/ min at room temperature.

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Fig. 9 Representative chromatogram of the enantioseparation of 1aminoethylphosphonic acid (modified with permission of Elsevier from ref. 27 © 1995) 3.8.3 Enantioseparation of Carboxylic Acids and Amines

1. Fill the mobile phase for experiment 8 in the solvent reservoir of the HPLC system (see Note 9). 2. Set the column oven to 25  C and equilibrate the column in open cycle with the mobile phase at a flow rate of 1.0 mL/min for 2 h (see Notes 9 and 14). 3. Set the detector wavelength to 254 nm and monitor until a stable baseline is obtained. 4. Inject 20 μL of the sample solution and record the chromatogram. A representative chromatogram of the enantioseparation of 1-aminoethylphosphonic acid is shown in Fig. 9. Further examples can be found in refs. 28 and 31.

3.9 Example 9: N2-noctyl-(S)phenylalaninamide as Chiral Selector for the Enantioseparation of Dipeptides

All chemical reactions should be performed in a well-ventilated hood. All security measures for handling chemicals have to be observed. Wear protective clothing and safety goggles.

3.9.1 Synthesis of N2-noctyl-(S)phenylalaninamide

1. Dissolve 8.2 g (0.05 mol) of (S)-phenylalaninamide in 200 mL of methanol at 40  C in a 500 mL three-neck round-bottom flask equipped with a reflux condenser and a magnetic stir bar. 2. While stirring, add 7.27 mL (0.05 mol) of n-octanal and 1.64 g of Pd/C (10%) (20% w/w) as catalyst under nitrogen. 3. Keep the solution under stirring at 40  C for 12 h under hydrogen flow at atmosphere pressure. 4. Remove the catalyst by filtration through a celite pad and remove the solvent under reduced pressure using a rotatory evaporator. 5. Dissolve the obtained crude product in 100 mL MeOH acidified with gaseous hydrochloric acid, until the hydrochloride salt is obtained as a precipitate.

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6. Filter the solid using a paper filter in a Bu¨chner funnel and wash it with 100 mL diethyl ether to remove residual aldehyde. Subsequently, continue washing the solid with a 5% (w/v) aqueous NaOH solution to remove the unreacted (S)-phenylalanine amide. 7. Dry the solid under glass oven and recrystallize it as the hydrochloride from MeOH/diethyl ether (70% yield, m.p. 212  C; ½α25 D ¼ +34.0, c ¼ 1 in ethanol 95%). The product can be further characterized using NMR spectroscopy. 3.9.2 Preparation of the N2-n-octyl-(S)phenylalaninamide Hydrochloride-Coated Chiral Stationary Phase

1. Install a Spherisorb ODS2 column (150 mm  4.6 mm i.d., ˚ pore size) into the HPLC system (see Note 1). 5 μm, 80 A 2. Dissolve 276.22 mg of N2-n-octyl-(S)-phenylalaninamide in 1.0 L of mobile phase for experiment 9 to give 1.0 mM solution (see Note 9). 3. Pump the solution through the column in open cycle at 1.0 mL/min for 15 h at room temperature (see Note 17). 4. Remove the unbound N2-n-octyl-(S)-phenylalaninamide by washing the column with water:methanol (95:5, v/v) for 2 h at a flow rate of 1.0 mL/min at room temperature (see Notes 20 and 21).

3.9.3 Enantioseparation of Dipeptides

1. Fill the mobile phase for experiment 9 in the solvent reservoir of the HPLC system. 2. Set the column oven to 25  C and equilibrate the column with the mobile phase at a flow rate of 1.0 mL/min for 2 h (see Notes 10 and 19). 3. Set the detector wavelength to 254 nm and monitor until a stable baseline is obtained. 4. Inject 20 μL of the sample solution and record the chromatogram. Representative chromatograms of enantioseparations of the dipeptides alanyl-alanine and alanyl-leucine are shown in Fig. 10a, b, respectively. Further examples can be found in ref. 21. The selector can also be used for the enantioseparation of α-amino acids, α-amino acid amides and esters, and α-hydroxy acids. Detailed information on the crystal structure of Bis(N2-n-octyl(S)-phenylalaninamide)Cu(II) complexes is reported in ref. 31.

4

Notes 1. The use of the specified column is recommended in order to obtain successful results. The use of other columns may generate different chromatographic performances.

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Fig. 10 Representative chromatogram of the enantioseparation of dipeptides (a) alanine-alanine and (b) alanine-leucine (modified with permission of Wiley from ref. 21 © 1996)

2. The use of a different Cu(II) salt as the Cu(II) source in the mobile phase can generate a substantially different chromatographic performance. Moreover, the ratio 2:1 is usually used for ligand:metal, respectively. 3. Solubilization of the binary metal/(chiral selector)2 assembly is strongly facilitated if the chiral selector and the Cu(II) salt are separately solubilized. 4. The pH of the mobile phase is the factor mostly influencing the chromatographic behavior in CLEC systems. Therefore, consider that quite different results can be generated by even slight changes in the mobile phase pH. 5. Enantioseparation is completely lost at pH values 3.5, while, when the pH of the eluent system exceeds 5.0, Cu(II) ions tend to precipitate and block the chromatographic system. 6. A chiral ratio of selector:Cu(II) of 2 mM:1 mM in the eluent system improves the resolution of polar amino acids. In contrast, a ratio chiral selector:Cu(II) of 4 mM:2 mM in the eluent system improves the resolution of apolar amino acids. 7. A low amount (from 5 up to 20%) of methanol or acetonitrile in the mobile phase could be required for the analysis of particularly hydrophobic compounds. Consider that the presence of an organic modifier in the mobile phase could result in a variation of the chromatographic performance. Moreover, excessive amounts of organic modifier in the mobile phase often result in the precipitation of the complex in the column and eluent system as well. Also, the progressive desorption of the selector could occur, gradually leading to phase deactivation. 8. Equilibration of the column takes at least 12 h. Thus, shorter equilibration times can result in decreased analytical performance.

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9. Before performing the loading of the chiral selector or pumping the mobile phase through the column, carefully filter the solution through a 0.22 μm membrane filter and degas by sonication for 20 min. 10. The chiral selector-Cu(II) complex is supposed to be partially adsorbed to the C18 column. Therefore, recycling the mobile phase contains less Cu(II) ions and chiral selector compared to the initial concentrations. Thus, in order to obtain reproducible enantioseparations, analyses should be performed with a freshly prepared mobile phase which replaces the mobile phase used for coating. 11. Under the present conditions, the enantiomer elution order is (S)-ofloxacin < (R)-ofloxacin. 12. The chemo- and enantioseparation of complex mixtures can be improved by means of the following gradient program: Time (min)

Buffer A (%)

Buffer B (%)

0

100

0

15

80

20

50

10

90

70

0

100

Buffer A: chiral selector:Cu(II)-acetonitrile 76:24 (v/v), pH at 7.5; Buffer B: chiral selector:Cu(II)-acetonitrile 69:31 (v/v), pH at 7.5. Column temperature, 25  C; 0.5 mL/min; UV wavelength at 254 nm. 13. For the detection of other analytes, a different wavelength may be applied for higher sensitivity. 14. In CLEC systems, a variation of the flow rate of the mobile phase and/or the column temperature can markedly affect the overall chromatographic performance. Generally, reduced flow rates do not affect the α values but can have a positive effect on column efficiency. The increase of column temperature accelerates the ligand-exchange rate and the mass transfer process, thus shortening the analyte analysis. Up to a certain temperature, a reduced retention can be accompanied by an improved column efficiency. However, the resolution and the column efficiency could also be negatively affected by the column temperature. 15. For the calculation of the chromatographic performance (that is, the retention, separation, and resolution factor values as well

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as the column efficiency) the time of the dead volume (t0) is obtained by injecting a sample solution containing sodium nitrate in the mobile phase as unretained marker. 16. After use, wash the column with 500 mL of water with trifluoroacetic acid 0.1% (v/v) to remove the majority of Cu(II) ions complexed to the chiral selector. Then, wash the column with pure MeOH (200 mL) to remove the chiral selector still adsorbed onto the C18 stationary phase. 17. Shorter recycling periods can generate low-performance C-CSPs. 18. Different coating rates (i.e., eluent velocity through the column during the loading process) can generate C-CSPs with different analytical performance. 19. The hold-up volume of the column is estimated from the retention time of methanol. 20. When the efficiency results are unsatisfactory, wash the column consecutively with 100 mL of MeOH, 100 mL of dichloromethane, and again 100 mL of MeOH. The chiral ligand is easily recovered by using the following procedure: (1) evaporate the solution under vacuum to eliminate chloroform; (2) add dropwise to the methanol solution containing the recovered chiral selector 6 M HCl to reach pH 2–3; (3) bubble H2S through the solution for 10 min, to precipitate CuS, and then filter the solution; (4) add 1.0 M NaOH to basic pH (>8) to precipitate the ligand; and (5) dissolve the precipitate in MeOH, add dropwise 2 M HCl in order to acidify the solution, and recrystallize from MeOH/diethyl ether (yield: 95%). 21. The adsorption of the ligand-Cu(II) complexes follows the Langmuir rule: the amount of the adsorbed selector is directly proportional to its concentration in the mobile phase. At the usual concentration, i.e., 1.0 mM ligand and 0.5 mM Cu(II), after saturation had occurred, 200 mg (0.33 mmol) of the Cu (II) complex of N2-n-octyl-(S)-phenylalaninamide hydrochloride is adsorbed, consistent with about 8% (w/w) of the column loading. References 1. Gil-Av E, Feibush R, Charles-Sigler R (1966) Separation of enantiomers by gas liquid chromatography with an optically active stationary phase. Tetrahedron Lett 7:1009–1015 2. Rogozhin SV, Davankov VA (1968) Chromatographic resolution of racemates on dissymmetric sorbents. Russ Chem Rev 37:565–575

3. Davankov VA, Kurganov AA, Ponomareva TM (1988) Enantioselectivity of complex formation in ligand-exchange chromatographic systems with chiral stationary and/or chiral mobile phases. J Chromatogr 452:309–316 4. Davankov VA (1994) Chiral selectors with chelating properties in liquid chromatography: fundamental reflections and selective review of

HPLC Enantioseparations by CLEC recent developments. J Chromatogr A 666:55–76 5. Natalini B, Sardella R, Giacche` N et al (2010) Chiral ligand-exchange separation and resolution of extremely rigid glutamate analogs: 1-aminospiro[2.2]pentyl-1,4-dicarboxylic acids. Anal Bioanal Chem 397:1997–2011 6. Natalini B, Sardella R, Macchiarulo A et al (2008) S-trityl-(R)-cysteine, a powerful chiral selector for the analytical and preparative ligand-exchange chromatography of amino acids. J Sep Sci 31:696–704 7. Natalini B, Sardella R, Pellicciari R (2005) O-benzyl-(S)-serine, a new chiral selector for ligand-exchange chromatography of amino acids. Curr Anal Chem 1:85–92 8. Sardella R, Macchiarulo A, Carotti A et al (2012) Chiral mobile phase in ligand-exchange chromatography of amino acids: exploring the copper(II) salt anion effect with a computational approach. J Chromatogr A 1269:316–324 9. Sardella R, Ianni F, Giacche` N et al (2012) Ligand-exchange enantioresolution of dihydroisoxazole amino acid derivatives acting as glutamatergic modulators. Tr Chromatogr 7:43–56 10. Natalini B, Sardella R, Macchiarulo A et al (2007) (S)-()-α,α-di(2-naphthyl)-2-pyrrolidinemethanol, a useful tool to study the recognition mechanism in chiral ligand-exchange chromatography. J Sep Sci 30:21–27 11. Sliwka M, S´lebioda M, Kołodziejczyk AM (1998) Dynamic ligand-exchange chiral stationary phases derived from N-substituted (S)-phenylglycinol selectors. J Chromatogr A 824:7–14 12. Hyun MH, Yang DH, Kim HJ et al (1994) Mechanistic evaluation of the resolution of a-amino acids on dynamic chiral stationary phases derived from amino alcohols by ligandexchange chromatography. J Chromatogr A 684:189–200 13. Hyun MH, Ryoo J-J, Lim NE (1993) Optical resolution of racemic α-amino acids on a dynamic chiral stationary phase by ligand exchange chromatography. J Liq Chromatogr 16:3249–3261 14. Gil-Av E, Tishbee A, Hare PE (1980) Resolution of underivatized amino acids by reversedphase chromatography. J Am Chem Soc 102:5115–5117 15. Yan H, Row KH (2007) Rapid chiral separation and impurity determination of levofloxacin by ligand-exchange chromatography. Anal Chim Acta 584:160–165

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16. Galaverna G, Panto` F, Dossena A et al (1995) Chiral separation of unmodified α-hydroxy acids by ligand exchange HPLC using chiral copper(II) complexes of (S)phenylalaninamide as additives to the eluent. Chirality 7:331–336 17. Armani E, Barazzoni L, Dossena A et al (1988) Bis(L-amino acid amidato) copper (II) complexes as chiral eluents in the enantiomeric separation of D,L-dansylamino acids by reversed-phase high-performance liquid chromatography. J Chromatogr 441:278–298 18. Lee SH, Oh TS, Lee HW (1992) Enantiomeric separation of free amino acids using N-alkyl-Lproline copper(II) complex as chiral mobile phase additive in reversed phase liquid chromatography. Bull Kor Chem Soc 13:280–285 19. Carotti A, Ianni F, Camaioni E et al (2017) Ndecyl-S-trityl-(R)-cysteine, a new chiral selector for “green” ligand-exchange chromatography applications. J Pharm Biomed Anal 144:31–40 20. Remelli M, Fornasari P, Dondi F et al (1993) Dynamic column-coating procedure for chiral ligand-exchange chromatography. Chromatographia 37:23–30 21. Galaverna G, Corradini R, Dossena A et al (1996) Copper(II) complexes of N2-alkyl-(S)amino acid amides as chiral selectors for dynamically coated chiral stationary phases in RP-HPLC. Chirality 8:189–196 22. Knox JH, Wan QH (1995) Chiral chromatography of amino- and hydroxy-acids on surface modified porous graphite. Chromatographia 40:9–14 23. Wan QH, Shaw PN, Davies MC et al (1997) Role of alkyl and aryl substituents in chiral ligand exchange chromatography of amino acids study using porous graphitic carbon coated with N-substituted-L-proline selectors. J Chromatogr A 786:249–257 24. Qinghua M, Shengqing W, Ying G et al (2006) Preparation and application of Isoquinolinecarboxylic acid derivative as chiral stationary phase for ligand exchange chromatography. Chin J Anal Chem 34:311–315 25. Kamimori H, Konishi M (2001) Evaluation and application of liquid chromatographic columns coated with ‘intelligent’ ligands: (I) acylcarnitine column. J Chromatogr A 929:1–12 26. Zaher M, Baussanne I, Ravelet C et al (2008) Copper(II) complexes of lipophilic aminoglycoside derivatives for the amino acid enantiomeric separation by ligand-exchange liquid chromatography. J Chromatogr A 1185:291–295

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ˆ i N, Kitahara H, Aoki F (1995) Direct sepa27. O ration of carboxylic acid and amine enantiomers by high-performance liquid chromatography on reversed-phase silica gels coated with chiral copper(II) complexes. J Chromatogr A 707:380–383 28. Fukuhara T, Yuasa S (1990) Novel ligandexchange chromatographic resolution of DL-amino acids using nucleotides and coenzymes. J Chromatogr Sci 28:114–117 29. Zaher M, Baussanne I, Ravelet C et al (2009) Chiral ligand-exchange chromatography of amino acids using porous graphitic carbon

coated with a dinaphthyl derivative of neamine. Anal Bioanal Chem 393:655–660 ˆ i N, Kitahara H, Aoki F (1993) Enantiomer 30. O separation by HPLC on reversed phase silica gel coated with copper(II) complexes of (R, R)-tartaric acid mono-amide derivatives. J Liq Chromatogr 16:893–901 31. Galaverna G, Pelosi G, Gasparri Fava G et al (1994) Chiral molecular laminates: crystal structures of bis(N2-n-alkyl-(S)-phenylalaninamidato)copper(II) complexes. Tetahedron Asymm 5:1233–1240

Chapter 16 Applications of Chiral Supercritical Fluid Chromatography Emmanuelle Lipka Abstract Nowadays polysaccharide chiral stationary phases are very popular and this originates for many reasons: (1) their wide applications window, (2) numerous different chemistry availability either in coated or in immobilized versions, and (3) large loading capability useful for preparative scale. Indeed chiral separations remain a hot topic (particularly in the pharmaceutical market) and in this field supercritical fluid chromatography is emerging rapidly. However, its use is more complex than high-performance liquid chromatography. The presented example illustrates analytical scale chiral separation method development and the effect of each operating parameter, i.e., flow rate, outlet pressure, and temperature variations on a chiral separation. Key words Supercritical fluid chromatography, Enantioseparation, Chiral stationary phase, Polysaccharide-based stationary phase

1

Introduction Methodologies offering the two enantiomers of chiral compounds are needed for biological testing following the FDA’s policy statement. Two approaches can be implemented to obtain enantiomers: the first consists of designing an enantioselective synthesis of the desired enantiomer. The second approach implies the synthesis of a racemic mixture which is subsequently resolved into the corresponding enantiomers. Therefore, the resolution giving simultaneously both enantiomers can be obtained by chromatographic methods. At the discovery stage of drug development, when a large number of molecules are required in milligram amounts for initial testing, stereoselective syntheses are neither time nor cost efficient, whereas upscaling to preparative chromatography is possible for both high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC). A recent review focuses on preparative SFC issues [1]. However only analytical scale is considered in this chapter.

Gerhard K. E. Scriba (ed.), Chiral Separations: Methods and Protocols, Methods in Molecular Biology, vol. 1985, https://doi.org/10.1007/978-1-4939-9438-0_16, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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Table 1 The density, ρ, and viscosity, η, of carbon dioxide and the diffusion coefficient, D, for naphthalene in carbon dioxide, D, under gas, supercritical, and liquid conditions ρ (kg m3) Gas, 313 K, 1 bar Supercritical, 313 K, 100 bar Liquid, 300 K, 500 bar

η (μPa s)

D (m2 s1)

2

16

5.1  10–6

632

17

1.4  10–8

1029

133

8.7  10–9

1.1 Characteristics and Properties of Supercritical Fluids

Supercritical fluid has properties intermediate between those of gases and liquids. The fluid can be adjusted for compounds to be sufficiently soluble to be eluted, while at the same time the viscosity and diffusion coefficients can be high enough to bring about relatively rapid mass transport. Table 1 shows typical values for the density and viscosity of a gas, supercritical fluid, and liquid, taking carbon dioxide as an example. Density is more than half that of the liquid, giving rise to reasonable solubility. In contrast, however, the viscosity of a supercritical fluid is much closer to that of a gas than that of a liquid. Thus, the pressure drop through a packed column in SFC is less than that for HPLC. Diffusion coefficients also shown in Table 1 for naphthalene in carbon dioxide are higher in a supercritical fluid than in a liquid. This has the advantage of faster transport. The supercritical fluid is the main constituent of the mobile phase used in SFC. So the consequences of these particular properties (i.e., low viscosity, high diffusivity of the mobile phase, and low pressure drop) permit high flow rates (i.e., short run times) with similar peak efficiency. Carbon dioxide (CO2) is the most often used supercritical fluid because its critical point (Pc ¼ 73 bar, Tc ¼ 31.1  C) is easily accessible and compatible with the stability of the compounds to be separated. It is also miscible with many organic solvents because the polarity of the CO2 is too low to elute quite polar compounds. Therefore a cosolvent (also called modifier) is usually added to the mobile phase up to 50% and can be either an alcohol or acetonitrile most of the time. It is noteworthy that CO2 is a nontoxic solvent and cheap and can be considered as “green” compared to the organic solvent. This feature fully answers the new guidelines leading to an environmentally friendly chromatography [2]. Subsequently, SFC started to carve out its niche [3].

1.2 Development of a Chiral SFC Method

Among the different methodologies developed to supply optically pure isomers, chromatographic resolution of racemic mixture has been recognized as a useful technology. Two methodologies can be pursued, called indirect and direct ones. The first, the “indirect” one, forms true diastereoisomers through derivatization of the compound with a chiral agent (CDA); subsequently those diastereoisomers can be separated in an achiral environment. The second,

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the “direct” one, requires a chiral stationary phase (CSP) to form transient diastereoisomers between the solute and the stationary phase. The direct approach is the most often used. 1.2.1 Chiral Stationary Phases

In a chiral development method, the type of stationary phase is a key choice in HPLC but in SFC as well. Polysaccharide-based CSPs are by far the most dominant and widely used CSPs over these last years and currently applied for more than 80% of the analytical [4] and more than 90% of the preparative separation of enantiomers due to their remarkable stability and loading capacity [5]. Among them, CSPs containing the coated selectors amylose tris (3,5-dimethylphenylcarbamate), cellulose tris(3,5-dimethylphenylcarbamate), amylose tris((S)-1-phenylethylcarbamate), and cellulose tris(methylbenzoate) showed broad enantioselectivity. The immobilized versions of the coated polysaccharide CSPs are also commercially available but they do not seem to display higher separation capability over the coated versions. However, they allow the use of stronger solvents such as chlorinated solvents, tetrahydrofuran, or ethyl acetate.

1.2.2 Mobile Phase: Type of Modifier (Cosolvent)

The Purnell equation defines the resolution Rs ¼

1 pffiffiffiffiffiffiffi k2 α1 N2   4 α 1 þ k2

ð1Þ

based on the retention, selectivity, and efficiency terms of the separation. In Eq. 1 the resolution factor was calculated using Rs ¼ 2 (tR2  tR1)/(ω1 + ω2), where tR1 and tR2 are the retention times of the peaks of interest and ω1 and ω2 are the peak widths measured at the baseline between tangents drawn to the peak sides. The retention (or capacity) factor (k) is a means of measuring the retention of an analyte on the chromatographic column for each second eluted enantiomer, and k2 was calculated by k ¼ (tR2  t0)/t0 where t0 was measured using 3,5-tri-tert.-butylbenzene (TTBB). N, the number of theoretical plates (or efficiency), is the factor used to determine the performance and effectiveness of columns, and was calculated for the second eluted peak (N2), using this equation: N ¼ 16(tR2/ω2)2. In the following section we will study the effect of the type and the percentage of the modifier in order to improve the resolution. In SFC, there are two main modifier effects: (1) the organic solvent changes the polarity of the mobile phase, modifying its solubilizing properties; (2) it changes the density of the mobile phase, particularly when pressure and temperature conditions are such that fluid compressibility is high (close to the critical point and when the fluid is more gas-like) [6]. However, this effect should be minor under the operating conditions used in most current applications of chiral SFC, where the fluid is denser, and thus more liquid-like.

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The type of modifier strongly influences the retention and the enantioselectivity but varies in an unpredictable way. For this reason, several mobile-phase compositions are usually employed in systematic screening strategies. Methanol is most of the time the first choice, followed by ethanol, and isopropanol. Acetonitrile is also tested. Retention has been seen to increase when modifier polarity decreased upon moving from methanol to ethanol and isopropanol, making it possible to consider chiral SFC as a normal-phase chromatographic mode. However, other studies have shown totally different retention variations which are in contradiction with the normal-phase theory. Thus, the effect of the nature of the modifier on retention seems to be both, compound and CSP dependent. On the contrary, acetonitrile as a cosolvent often results in poor efficiency and deteriorated peak shapes because of the non-protic character of this solvent, resulting in a very low recovery rate of silanol groups. Compared to methanol, an increase of retention is usually observed. Column efficiency also varies in an unpredictable way. Indeed, band broadening partly arises from slow diffusion between mobile and stationary phases. Thus, in theory, efficiency should increase when the mobile-phase viscosity decreases (as diffusion rates increase in less viscous solvents). So efficiency would be expected to increase from higher alcohols to methanol (MeOH). However, this trend is not systematically observed. 1.2.3 Mobile Phase: Percentage of Modifier (Cosolvent)

In chiral SFC, by increasing the percentage of cosolvents (the most polar constituent of the mobile phase), the retention times decrease up to a certain point where retention might increase again. Relatively large variations in retention are observed at small percentages of the modifier, while the relative variations are smaller at higher percentages of the modifier. The selectivity factor usually remains constant or slightly decreases under increased modifier proportions, while the separation efficiency can follow two trends: Usually the efficiency increases with the first few percent of the modifier because of mobile -phase absorption on the CSP and subsequently decreases when solvent proportion further increases because the diffusion rates of the solutes in the mobile phase decrease with the addition of a viscous solvent.

1.2.4 Mobile Phase: Use of Additives

For the separation of basic or acidic compounds, a third component, called additive, can be added to the mobile phase in order to improve the peak shape or even to initiate the elution of the compounds. The impact of the nature of the additive [7] or the effect of its concentration in the modifier, on the enantioselectivity, is a controversial topic. As a general rule, basic additives (like triethylamine (TEA), diethylamine (DEA), or isopropylamine (IPA)) are employed for the separation of basic compounds and

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acidic additives, e.g., trifluoroacetic acid (TFA) or formic acid (FA), for acidic compounds. However, the opposite can also generate good resolution, i.e., a basic additive for an acidic compound or vice versa. For a comprehensive study on this topic the reader is referred to ref. 8. 1.2.5 Operating Parameter: Flow Rate

The flow rate is rarely optimized in order to obtain a separation. Most of the time its variation is implemented to reduce analysis time when a separation has already been established. Nowadays, flow rates in analytical SFC are between 2 and 6 mL/min. Those high flow rates are possible because of the low viscosity of the fluids. When increasing the flow rate, the retention times decrease. Because as the fluid is compressible, changing its linear velocity in the column affects its density and, therefore, its eluting strength. Thus, considering the Purnell equation (Eq. 1) resolution decreases with increasing flow rates (retention times decrease and efficiency remains unchanged in the column due to the high diffusivity of the solutes).

1.2.6 Operating Parameter: Outlet Pressure (Back Pressure)

The stated back pressure values usually range from 80 to 200–250 bar. As previously said, mobile-phase density influences selectivity. When the pressure increases, the density of the fluid, and thus the elution strength, increases causing a decrease of retention times while separation factors decrease to a lesser extent or remain constant. The range of retention variation depends on mobile phase composition. Indeed the effect of pressure on resolution is significant when the fluid is more gas-like, i.e., when pure carbon dioxide or small percentages of modifier (below 5%) are used as a mobile phase. For liquid-like fluids (above 10% of cosolvent), the pressure effect is less important. In addition, as diffusivity is reduced under high-pressure conditions, column efficiency may be altered. The effect on selectivity and column efficiency is generally less significant than the effect on retention, and thus resolution is less affected by a change in pressure.

1.2.7 Operating Parameter: Temperature

Last but not least, temperature has the most complex effect. Indeed, it can act in two opposite ways on the retention. At constant pressure drop, an increase of the temperature enhances the coefficient of diffusion and the volatility of the solutes and then decreases their retention. Simultaneously, the density of the carbon dioxide (thus elution strength) is reduced, leading to increased retention [9]. On the one hand, polysaccharide CSPs should not be operated above 50  C. On the other hand, if the oven is unable to cool the temperature below room temperature, the available range of temperature variation is quite low (between 15–20 and 50  C in practice).

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The relationship between the temperature and the retention factor can be expressed in terms of the Van’t Hoff equation:     ΔH ΔS þ þ ln β ð2Þ ln ðkÞ ¼  RT R where T is the absolute temperature, R is the ideal gas constant, β is the phase ratio, and ΔH and ΔS are the standard molar enthalpy and standard molar entropy of the solute transfer (from mobile to stationary phase), respectively. Therefore, ln(k) plotted versus 1/T should be linear. Two main behaviors of the Naperian logarithm of the retention factor versus reversed temperature are reported: – The curve is crescent indicating that retention decreases with an augmentation of the temperature. This HPLC-close trend is explained by high proportion of cosolvent used and high back pressure (150 bar) applied causing an increase of the mobilephase density and resulting in a more liquid-like fluid. Therefore increasing temperature leads to an improved solute solubility in the mobile phase, decreasing retention times. – The curve is decreasing, meaning that retention increases with an increased temperature. This trend is classically observed in SFC (with low proportion of modifier), as the fluid density decreases (and thus the solvating power) when temperature increases, resulting in a reduction of the elution strength of mobile phase. Besides these two models, authors reported a “U-shaped” curve, deviation between the two previous extreme trends, explained by differential influence of the stationary phase on the retention mechanism [10]. An increase in column efficiency is usually expected from high temperature through increased mobile-phase diffusivity, most of the time in SFC but mainly in HPLC. Besides, temperature may lead to changes in the rigidity of the stationary phase and analytes, thus affecting the analyte entrance into the chiral cavities. In addition, temperature effect is also related to back pressure. Authors indicate that the use of pressures well above the critical pressure limits the effect of temperature on column efficiency [11]. A comprehensive review on SFC has demonstrated many reasons to work with subcritical rather than supercritical conditions [12] and proposed a starting point: moderate temperature of 25–30  C and pressure of 150 bar. Those complex and sometimes antagonistic effects do not allow to predict the temperature influence. Ideally, as pressure, temperature is not an influential parameter on retention and resolution.

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As in HPLC, the main parameter to obtain a chiral separation is the stationary phase, then the type and the percentage of cosolvent in the CO2-containing mobile phase. Flow rate and temperature should also be optimized. The main difference in comparison with HPLC is that in SFC a back pressure is applied after the column to ensure a constant pressure drop along the column so this parameter could also be optimized. In this work, we chose trans-stilbene oxide (TSO) as the probe to develop a chiral separation method through the stationary and mobile phase optimization and to explore the flow rate, temperature, and back pressure variation effects.

2

Materials

2.1 Instrumentation and Materials

1. A commercial SFC instrument, for example, a SFC-PICLAB hybrid 10–20 apparatus (PIC Solution, Avignon, France) equipped with a cryostat (e.g., Minichiller, Huber, Offenburg, Germany) for cooling the pump head used for pumping the CO2 to – 8  C; a thermostated suitable 10 column switching device (i.e., a column oven), a diode array detector (e.g., Smartline 2600 diode array detector, Knauer, Berlin, Germany), and an injection valve. 2. An ultrasonic bath for sample dissolution. 3. A suitable filtering device such as 0.45 μ PTFE syringe filters (15 mm diameter) for filtration of the solutions into the sample vials (see Note 1). 4. A 250 mm  4.6 mm i.d. Chiralpak AD-H with 5 μm particle size (Chiral Technologies Europe, Illkirch, France or Chiral Technologies, Inc., West Chester, PA, USA)

2.2 Chemicals and Solutions

Use HPLC-grade methanol (MeOH), ethanol (EtOH), propan-2ol (IPA), and acetonitrile (ACN). Use carbon dioxide (CO2) with a purity of 99.995%. All other chemicals should be of analytical grade. 1. trans-Stilbene oxide sample solution (1 mg/mL): Weigh 10 mg trans-stilbene oxide (TSO) into a 10 mL volumetric flask. Add 5 mL EtOH and sonicate until complete dissolution of the compound. Make up to the volume with EtOH.

3

Methods

3.1 General Installation of the System

1. Install the Chiralpak AD-H column into a thermostated suitable column-switching device according to the instructions of the manufacturer.

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2. Pressurize the system and check for leaks (see Note 2). 3. Place IPA in the cosolvent reservoir and set percentage of IPA at 100% and percentage of carbon dioxide at 0%; set the flow rate at 0.25 mL/min and flush the column for 45 min with this mobile phase. 4. Set the percentage of modifier (MeOH firstly) at 20% and the percentage of carbon dioxide at 80%; set the flow rate at 0.25 mL/min and flush the column for 45 min (see Note 3). 5. Set the flow rate at 1.0 mL/min and equilibrate for 1 min. 6. Set the flow rate at 4.0 mL/min and equilibrate the system for 2 min. 7. Set the outlet pressure at 150 bar. 8. Set the column oven temperature at 40  C. 9. Set the detection wavelength at 220 nm. 3.2

Applications

3.2.1 Example 1

The example illustrates the results of enantioseparation of TSO obtained when MeOH, EtOH, IPA, and ACN are used at 20% in CO2 on AD-H. 1. Place MeOH in the cosolvent reservoir and set percentage of MeOH at 20% (see Note 4) and percentage of CO2 at 80%. 2. Set flow rate at 4.0 mL/min. 3. Equilibrate the system for 2 min. 4. Inject about 20 μL TSO sample solution into the system and start recording the chromatogram for about 5 min. A chromatogram of the separation using 20% MeOH as cosolvent is shown in Fig. 1. 5. Place EtOH in the cosolvent reservoir. 6. Set percentage of EtOH at 20% and percentage of CO2 at 80%. 7. Repeat steps 2–4. A chromatogram of the separation using 20% EtOH as cosolvent is shown in Fig. 1. 8. Place IPA in the cosolvent reservoir. 9. Set percentage of IPA at 20% and percentage of CO2 at 80%. 10. Repeat steps 2–4. A chromatogram of the separation using 20% IPA as cosolvent is shown in Fig. 1. 11. Place ACN in the cosolvent reservoir. 12. Set percentage of ACN at 20% and percentage of CO2 at 80%. 13. Repeat steps 2–4. A chromatogram of the separation using 20% ACN as cosolvent is shown in Fig. 1 It can be seen in Fig. 1 that best resolution is obtained with EtOH as a modifier. ACN leads to the shorter retention times and lower resolution values.

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20% MeOH 20% EtOH

2500

20% IPA 20% ACN

2000

AU

1500 1000 500 0 –500

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Time (min)

Fig. 1 Overlaid chromatograms for TSO enantioseparations with a carbon dioxide mobile phase containing 20% of different modifiers MeOH or EtOH or IPA (λ ¼ 254 nm) or ACN on AD-H CSP, at 40  C; 150 bar back pressure; 4 mL min1; λ ¼ 220 nm

This comparison between different types of modifier can be run also on the OD-H column (see Note 5). In this case the general installation of the system (steps 1–11) should be implemented with the OD-H column or with any type of column used. 3.2.2 Example 2

This example illustrates the impact of the variation of the percentage of the cosolvent EtOH on retention and resolution in the range of 5–30%. 1. Place EtOH in the cosolvent reservoir and set percentage of EtOH at 5% and percentage of CO2 at 95%. 2. Set flow rate at 4.0 mL/min. 3. Equilibrate the system for 2 min. 4. Inject about 20 μL TSO sample solution into the system and start recording the chromatogram for about 10 min. A chromatogram of the separation using 5% EtOH is shown in Fig. 2. 5. Set percentage of EtOH at 10% and percentage of CO2 at 90%. 6. Repeat steps 2–4. A chromatogram of the separation using 10% EtOH as cosolvent is shown in Fig. 2. 7. Set percentage of EtOH at 15% and percentage of CO2 at 85%. 8. Repeat steps 2–4. A chromatogram of the separation using 15% EtOH as cosolvent is shown in Fig. 2. 9. Set percentage of EtOH at 20% and percentage of CO2 at 80%. 10. Repeat steps 2–4. A chromatogram of the separation using 20% EtOH as cosolvent is shown in Fig. 2.

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30% EtOH

1600

25% EtOH

1400

20% EtOH

AU

1200

15% EtOH

1000

10% EtOH

800

5% EtOH

600 400 200 0 0 -200

2

4

6

8

10

t (min)

Fig. 2 Overlaid chromatograms for TSO enantioseparations with a carbon dioxide mobile phase containing different percentages of EtOH on AD-H CSP; 150 bar back pressure; 4 mL/min; 40  C; λ ¼ 220 nm

11. Set percentage of EtOH at 25% and percentage of CO2 at 75%. 12. Repeat steps 2–4. A chromatogram of the separation using 25% EtOH as cosolvent is shown in Fig. 2. 13. Set percentage of EtOH at 30% and percentage of CO2 at 70%. 14. Repeat steps 2–4. A chromatogram of the separation using 30% EtOH as cosolvent is shown in Fig. 2. Figure 2 illustrates that as the percentage of cosolvent increases, retention times of the enantiomers decrease from 3.41 to 1.49 min and from 9.37 to 2.58 min for tr1 and tr2, respectively. As the percentage of cosolvent increases, resolution decreases from 11.41 to 4.11 while efficiency is seen to increase from 5 to 30%, respectively. 3.2.3 Example 3

This example illustrates the impact of the flow rate variation on retention and resolution in the range of 2.0–6.0 mL/min. 1. Place EtOH in the cosolvent reservoir and set percentage of EtOH at 10% and percentage of CO2 at 90%. 2. Set flow rate at 2.0 mL/min. 3. Equilibrate the system for 2 min. 4. Inject about 20 μL TSO sample solution into the system and start recording the chromatogram for about 14 min. A chromatogram of the separation using 2.0 mL/min is shown in Fig. 3. 5. Set flow rate at 3.0 mL/min. 6. Repeat steps 3 and 4. A chromatogram of the separation using 3.0 mL/min is shown in Fig. 3.

Enantioseparation by SFC 1400

10% EtOH 6mL/min

1200

10% EtOH 5mL/min

1000

10% EtOH 4mL/min

800

10% EtOH 3mL/min

AU

313

10% EtOH 2mL/min

600 400 200 0 -200

0

2

4

6

8

10

12

14

t (min)

Fig. 3 Overlaid chromatograms for TSO enantioseparations with a carbon dioxide mobile phase containing 10% of EtOH on AD-H CSP; at different flow rates; 150 bar back pressure; 40  C; λ ¼ 220 nm

7. Set flow rate at 4.0 mL/min. 8. Repeat steps 3 and 4. A chromatogram of the separation using 4.0 mL/min is shown in Fig. 3. 9. Set flow rate at 5.0 mL/min. 10. Repeat steps 3 and 4. A chromatogram of the separation using 5.0 mL/min is shown in Fig. 3. 11. Set flow rate at 6.0 mL/min. 12. Repeat steps 3 and 4. A chromatogram of the separation using 6.0 mL/min is shown in Fig. 3. Figure 3 illustrates that when the flow rate varies from 2.0 to 6.0 mL min1, the retention times strongly decrease whereas resolution only slightly decreases. Resolution values were equal to 9.53, 8.97, 8.75, 7.82, and 7.53, respectively, for TSO on AD-H CSP, with 10% of EtOH, 40  C at 150 bar. Indeed, column efficiency is not so much affected by an increase in flow rate, as the slope of van Deemter curve in SFC is much more flat than in HPLC (see Note 6). For instance lowering the flow rate ensures an improved resolution (see Note 7). 3.2.4 Example 4

This example illustrates the impact of the outlet pressure variation with 5% of EtOH as organic modifier on retention and resolution in the range of 80–200 bar. 1. Place EtOH in the cosolvent reservoir and set percentage of EtOH at 5% and percentage of CO2 at 95%. 2. Set flow rate at 4.0 mL/min. 3. Equilibrate the system for 2 min. 4. Set the outlet pressure at 80 bar.

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5. Inject about 20 μL TSO sample solution into the system and start recording the chromatogram for about 18 min. A chromatogram of the separation under 80 bar outlet pressure is shown in Fig. 4a. 6. Set the outlet pressure at 120 bar. 7. Repeat step 5. A chromatogram of the separation under 120 bar is shown in Fig. 4a. 8. Set the outlet pressure at 150 bar. 9. Repeat step 5. A chromatogram of the separation under 150 bar is shown in Fig. 4a. 10. Set the outlet pressure at 200 bar. 11. Repeat step 5. A chromatogram of the separation under 200 bar is shown in Fig. 4a. Figure 4a illustrates that at a concentration of 5% EtOH, the pressure increase strongly diminishes the retention times (from 5.00 to 3.01 min and from 15.27 to 7.93 min for tr1 and tr2, respectively), while the resolution slightly decreases (9.90, 11.62, 11.40, and 10.15) between 80 and 200 bar, respectively. 3.2.5 Example 5

This example illustrates the impact of the outlet pressure variation with 10% of EtOH as organic modifier on retention and resolution in the range of 80 to 200 bar. 1. Place EtOH in the cosolvent reservoir and set percentage of EtOH at 10% and percentage of CO2 at 90%. 2. Set flow rate at 4.0 mL/min. 3. Equilibrate the system for 2 min. 4. Set the outlet pressure at 80 bar. 5. Inject about 20 μL TSO sample solution into the system and start recording the chromatogram for about 10 min. A chromatogram of the separation under 80 bar outlet pressure is shown in Fig. 4b. 6. Set the outlet pressure at 120 bar. 7. Repeat step 5. A chromatogram of the separation under 120 bar is shown in Fig. 4b. 8. Set the outlet pressure at 150 bar. 9. Repeat step 5. A chromatogram of the separation under 150 bar is shown in Fig. 4b. 10. Set the outlet pressure at 200 bar. 11. Repeat step 5. A chromatogram of the separation under 200 bar is shown in Fig. 4b.

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Fig. 4 Overlaid chromatograms for TSO enantioseparations with a carbon dioxide mobile phase containing (a) 5% of EtOH, (b) 10% EtOH, and (c) 40% EtOH at different bar back pressure on AD-H CSP. Other experimental parameters: 40  C; 4 mL/min; λ ¼ 220 nm

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Figure 4b illustrates that at a concentration of 10% EtOH in the mobile phase, the pressure increase slightly reduces the retention times (from 2.93 to 2.16 min and from 7.45 to 4.80 min for tr1 and tr2, respectively), while the resolution remains quite constant (10.52, 10.22, 10.01, and 9.48) between 80 and 200 bar, respectively. 3.2.6 Example 6

This example illustrates the impact of the outlet pressure variation with 40% of EtOH as organic modifier on retention and resolution in the range of 80–200 bar. 1. Place EtOH in the cosolvent reservoir and set percentage of EtOH at 40% and percentage of CO2 at 60%. 2. Set flow rate at 4.0 mL/min. 3. Equilibrate the system for 2 min. 4. Set the outlet pressure at 80 bar. 5. Inject about 20 μL TSO sample solution into the system and start recording the chromatogram for about 5 min. A chromatogram of the separation under 80 bar outlet pressure is shown in Fig. 4c. 6. Set the outlet pressure at 120 bar. 7. Repeat step 5. A chromatogram of the separation under 120 bar is shown in Fig. 4c. 8. Set the outlet pressure at 150 bar. 9. Repeat step 5. A chromatogram of the separation under 150 bar is shown in Fig. 4c. 10. Set the outlet pressure at 200 bar. 11. Repeat step 5. A chromatogram of the separation under 200 bar is shown in Fig. 4c. Figure 4c illustrates that at a concentration of 40% EtOH in the mobile phase the pressure increase has no significant effect on retention or on resolution (varying from 5.59 to 5.37) (see Note 8).

3.2.7 Example 7

This example illustrates the impact of the temperature variation with 10% of EtOH as organic modifier on retention and resolution in the range of 25–45  C. 1. Place EtOH in the cosolvent reservoir and set percentage of EtOH at 10% and percentage of CO2 at 90%. 2. Set flow rate at 4.0 mL/min. 3. Equilibrate the system for 2 min. 4. Set the outlet pressure at 150 bar. 5. Set the temperature oven at 25  C. 6. Equilibrate the system for 5 min.

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10% EtOH 45°C 10% EtOH 40°C

1000

10% EtOH 35°C

800

10% EtOH 30°C 10% EtOH 25°C

AU

600 400 200 0 -200

0

1

2

3

4

5

6

7

8

t (min)

Fig. 5 Overlaid chromatograms for TSO enantioseparations with a carbon dioxide mobile phase containing 10% of EtOH on AD-H CSP at different column temperatures; 150 bar back pressure; 4 mL min1; λ ¼ 220 nm

7. Inject about 20 μL TSO sample solution into the system and start recording the chromatogram for about 8 min. A chromatogram of the separation at 25  C is shown in Fig. 5. 8. Set the temperature oven at 30  C. 9. Equilibrate the system for 5 min. 10. Repeat step 7. A chromatogram of the separation at 30  C is shown in Fig. 5. 11. Set the temperature oven at 35  C. 12. Equilibrate the system for 5 min. 13. Repeat step 7. A chromatogram of the separation at 35  C is shown in Fig. 5. 14. Set the temperature oven at 40  C. 15. Equilibrate the system for 5 min. 16. Repeat step 7. A chromatogram of the separation at 40  C is shown in Fig. 5. A high proportion of organic modifier (i.e., 10%) used and high back pressure (150 bar) applied cause an increase of the mobilephase density and result in a more liquid-like fluid. Therefore increasing temperature leads to an improved solute solubility in the mobile phase, decreasing retention times. This phenomenon is observed in Fig. 5 which illustrates the temperature variation for TSO between 25 and 45  C with 10% of EtOH in a CO2-containing mobile phase.

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3.3 General Uninstallation of the System

This procedure should be followed at the end of a working series, before to switch off the device. 1. Decrease the outlet pressure from 150 to 110 bar (see Note 9). 2. Decrease the outlet pressure from 110 to 80 bar. 3. Decrease the outlet pressure from 80 to 40 bar. 4. Decrease the flow rate from 4 to 2 mL/min. 5. Stop the flow rate. In case the column has to be removed from the oven, flush the column with MeOH or IPA (see Note 10).

4

Notes 1. Filtration is highly recommended (even mandatory) to avoid damaging the injection or selection rotors. 2. Leaks can be detected by frozen condensation at the place of the leak. 3. The columns are shipped with solvent (n-hexane:alcohol 90:10, v/v) for analytical column dimensions (4.6 mm ID). To avoid any damages it is recommended to flush them with 100% IPA before the first use in SFC mode. 4. Above 16% of a modifier at 50  C (according to T. Berger [13]) and due to the modifier dissolved in the CO2, the mobile phase is more often in subcritical state than in supercritical state however, without any substantial negative impact on the physical properties of the fluid. 5. If the analogous experiments would be performed the OD-H column peak shapes, retention times, and resolution could be different (as amylose and cellulose of similar chiral selectors differ in enantioseparation mechanism). 6. The following parameter values should be chosen as typical starting conditions: 20% of cosolvent; between 30 and 40  C and 150 bar back pressure. In addition, a flow rate of 3 or 4 mL/min will allow a rapid elution of the compounds. 7. Increasing flow rate by 1 or 2 mL/min leads to shorter analysis times while keeping resolution. 8. Low-pressure values should be avoided when using large amount of modifier as a two-phase system can be formed; in addition larger pressure avoids a noisy baseline detection. 9. A decrease by step of 40 bar is recommended to protect your outlet pressure regulator. 10. For a storage period exceeding 2–3 days flush the column with 100% IPA or MeOH modifier at 0.25 mL/min flow rate for

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45 min. Then the columns can be stored capped to avoid solvent evaporation and stored at room temperature. References 1. Speybrouck D, Lipka E (2016) Preparative supercritical fluid chromatography: a powerful tool for chiral separations. J Chromatogr A 1467:33–55 2. Galyan K, Reilly J (2018) Green chemistry approaches for the purification of pharmaceuticals. Curr Opin Green Sustain Chem 11:76–80 3. Tarafder A (2016) Metamorphosis of supercritical fluid chromatography to SFC: an overview. Trends Anal Chem 81:3–10 4. Chankvetadze B (2013) Enantioseparations by high-performance liquid chromatography using polysaccharide-base chiral stationary phases—an overview. In: Scriba GKE (ed) Chiral separations. Methods in molecular biology, vol. 970. pp 81–111 5. Francotte E (2001) Enantioselective chromatography as a powerful alternative for the preparation of drug enantiomers. J Chromatogr A 906:379–397 6. West C (2014) Enantioselective separations with supercritical fluids. Curr Anal Chem 10:99–120 7. Ye YK, Lynam KG, Stringham RW (2004) Effect of amine mobile phase additives on chiral subcritical fluid chromatography using polysaccharide stationary phases. J Chromatogr A 1041:211–217

8. Speybrouck D, Doublet C, Cardinael P, FiolPetit C, Corens D (2017) The effect of high concentration additive on chiral separations in supercritical fluid chromatography. J Chromatogr A 1510:89–99 9. Zehani Y, Lemaire L, Ghinet A, Millet R, Chavatte P, Vaccher C, Lipka E (2016) Exploring chiral separation of 3-carboxamido-5-aryl isoxazole derivatives by supercritical fluid chromatography on amylose and cellulose tris dimethyl- and chloromethyl phenylcarbamate polysaccharide based stationary phases. J Chromatogr A 1467:473–481 10. Yaku K, Aoe K, Nishimura N, Marishita F (1999) Retention mechanisms in super/subcritical fluid chromatography on packed columns. J Chromatogr A 848:337–345 11. Blackwell JA, Stringham RW (1997) Temperature effects on selectivity using carbon-dioxide based mobile phases on silica-based packed columns near the mixture critical point. Chromatographia 44:521–528 12. Lesellier E, West C (2015) The many faces of packed column supercritical fluid chromatography—a critical review. J Chromatogr A 1382:2–46 13. Berger T (1995) Packed column SFC. In: Roger M, Smith RM (eds) RSC chromatography monographs. Royal Society of Chemistry, London, pp 67–68

Chapter 17 Chiral Separations by Countercurrent Chromatography Sheng-Qiang Tong Abstract Chiral separations by countercurrent chromatography are mainly divided into two types: homogeneous chiral selector addition and interfacial chiral ligand exchange. In this chapter, we describe two methods for the enantioseparation of phenylsuccinic acid and α-hydroxy acids by high-speed countercurrent chromatography using hydroxypropyl-β-cyclodextrin and N-n-dodecyl-L-proline as chiral selectors for both above mentioned modes. Key words Biphasic solvent system, Countercurrent chromatography, Chiral ligand exchange, Chiral separation, Homogeneous chiral selector

1

Introduction Countercurrent chromatography is a kind of liquid-liquid partition chromatography, which does not use a solid support for the stationary phase. The separation depends on the partitioning performance of solutes between two immiscible liquid phases which can be composed of two or more solvents. Compared with conventional liquid chromatography, the main advantages of countercurrent chromatography are of the following two aspects: solid-free stationary phase and large capacity for sample injection. Shortcomings resulting from the stationary phase of conventional liquid chromatography, such as irreversible adsorption, contamination, and pH limitation, do not play a role in countercurrent chromatography [1–3]. Modern countercurrent chromatographic techniques, i.e., high-speed countercurrent chromatography and highperformance centrifugal partition chromatography, have been widely used in the preparative separation and purification of chemical components from natural products and synthetic mixtures [4, 5]. They are efficient alternative methods for the preparative separation of chemical components. The retention of stationary phase can reach up to 80%, which is much higher than that in conventional liquid chromatography. The analyte to be separated

Gerhard K. E. Scriba (ed.), Chiral Separations: Methods and Protocols, Methods in Molecular Biology, vol. 1985, https://doi.org/10.1007/978-1-4939-9438-0_17, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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can be partitioned into any space in the stationary phase. Meanwhile, a lower solvent consumption can be achieved for separations by countercurrent chromatography. The typical amount of sample that could be injected into the separation column of a typical semipreparative countercurrent chromatography can reach several hundreds of milligrams. Furthermore, no complex pretreatment of the sample is needed for a separation, which is much more convenient than that of conventional chromatographic technique [6]. The main disadvantage of countercurrent chromatography lies in its low separation efficiency; that is, the efficiency of the typical theoretical plates for a separation column in countercurrent chromatography is less than 2000, which is much lower than that of conventional liquid chromatography or capillary electrophoresis. Therefore, a high separation factor (α
1.4) is generally required for complete separation of analytes by countercurrent chromatography. Compared with conventional liquid chromatography, only a small number of publications are available with regard to chiral separations by countercurrent chromatography [7–9]. It is still a big challenge to obtain a complete enantioseparation of a racemate. A chiral selector added to the chromatographic system is necessary to introduce a chiral environment in the separation system. The solubility of the chiral selectors should be restricted to one phase of the solvent system only, while the racemate should partition freely in both phases. Thus, it is difficult to find suitable enantioseparation conditions for successful chiral separation by countercurrent chromatography. Since the chiral selector dissolved in one of the two phases is not immobilized to the stationary phase, the stereospecific molecular interaction between the chiral selector and the enantiomer would not be so efficient and sufficient because both of them are freely dispersed in the liquid phase. Although this is applied in chiral capillary electrophoresis, the high theoretical plates of separation systems of capillary electrophoresis greatly make up this disadvantage. Most chiral selectors reported in chiral separations by countercurrent chromatography come from separation methods such as liquid chromatography, capillary electrophoresis, enantioselective liquid-liquid extraction, diastereoisomeric crystallization, dynamic resolution, or chiral ligands in asymmetric synthesis [10–15]. So far, the following chiral selectors have been successfully applied in countercurrent chromatography and centrifugal partition chromatography: cinchona alkaloid derivatives, L-proline derivatives, β-cyclodextrin derivatives, vancomycin, cellulose and amylose derivatives, (þ)-(18-crown-6)-2,3,11,12-tetracarboxylic acid, tartaric acid derivatives, (S)-naproxen derivatives, and fluorinated chiral selectors [16, 17]. Chiral separations by countercurrent chromatography can be divided into two types: homogeneous chiral selector addition and

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Fig. 1 Schematic diagram of equilibrium between the enantiomers (EnH) and chiral selector (CS) in the separation column for homogeneous chiral selector addition

interfacial chiral ligand exchange. The separation mechanism for the two modes can be explained by a simplified mathematic model with the hypothesis that the chiral selector and the complexes formed between the chiral selector and the enantiomers are completely restricted to one of the two phases. 1.1 Homogeneous Chiral Selector Addition

The quadratic scheme of the equilibrium between a chiral selector and the enantiomers in countercurrent chromatography originally used by Oliveros et al. [18] can be simplified in the conventional chiral separation with monophasic recognition described by Ma et al. [19] based on the assumption that both, chiral selector and chiral selector–enantiomer complex remain only in the stationary phase (Fig. 1). In the case of 1:1 stoichiometry, the following equations apply: K D ¼ D ¼

½EnH org ½EnH aq

½EnH org þ ½CS‐EnH org

kf ¼

½EnH aq ½CS‐EnH org ½EnH org ½CSorg

ð1Þ ð2Þ ð3Þ

where KD is the partition coefficient as the ratio of the concentration of a substance in a single definite form in one phase to that in the other phase at equilibrium. D is the distribution ratio as the ratio of the concentration of a substance in various forms in one phase to that in the other phase at equilibrium. The distribution ratio D governs the retention of a solute, while the partition coefficient KD is useful for analytical calculations. kf is the complex formation constant of [CS-EnH+]org in the organic phase. Combined with Eqs. 1–3, the distribution ratio D of enantiomer (En) is expressed as follows: n o ð4Þ D  ¼ K D 1 þ k f  ½CSorg

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And the separation factor, α, is expressed as (supposing (þ)enantiomer more retained than ()-enantiomer in the organic phase) α¼

D þ 1 þ k f þ ½CSorg ¼ D  1 þ k f  ½CSorg

ð5Þ

Equation 5 indicates that the enantioseparation factor increases with the concentration of the chiral selector and with the magnitude of the kf+/kf ratio (monotonous increasing function). 1.2 Interfacial Chiral Ligand Exchange

Formation of diastereomeric ternary coordination complexes between the enantiomers, a transitional metal or nonmetallic ion, and chiral ligand is one of the most powerful chiral recognition methods because coordination bonds generally display much higher stereoselective binding force than other intermolecular forces, such as hydrogen bonds, van der Waals, or dipole-dipole interactions. Chiral ligand-exchange countercurrent chromatography is based on the ability of a chiral ligand (L-LiH) solubilized in the organic stationary phase through complexation with a transition metal ion (e.g., Cu2+) to preferentially extract one of the enantiomers (EnH) through the formation of a ternary L-Li: Cu2+:En electroneutral complex [20]. Highly alkylated ligands usually partition exclusively into the organic phase of a two-phase solvent system, and its solubilization requires that the free ligand (L-LiH) should be in the neutral form. This separation mechanism can be divided into the following two stages. As shown in Fig. 2, stage one (upper diagram) schematically illustrates the chemodynamic mechanisms taking place in a separatory funnel: the organic stationary phase containing the chiral ligand (L-LiH) in the upper portion and the aqueous mobile phase added with transition metal ion (Cu2+) in the lower portion. In this first stage, the transition metal ion Cu2+ is extracted into the organic phase to form a bisbinary complex L-Li:Cu2+:L-Li by releasing two protons into the aqueous phase. At this stage, the formation of a binary complex in the organic phase is critically affected by the pH of the aqueous phase since protonation and deprotonation take place at the interface. Stage two (lower diagram) illustrates the chiral separation mechanism occurring in the separation column where two phases are arbitrarily separated. In this stage, chiral ligand exchange takes place between the binary complex L-Li:Cu2+:L-Li and the enantiomers En of a racemate in the aqueous phase, resulting in the formation of neutral ternary complex L-Li:Cu2+:En in the organic phase. In this stage, the diastereoisomeric complex is formed as a homo-chiral ternary complex, i.e., L-Li:Cu2+:L-En, and a hetero-chiral ternary complex (L-Li:Cu2+:D-En), in which the enantioselectivity (α) was achieved if the stabilities of these two diastereoisomers differ.

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Fig. 2 Schematic diagram of speciation reactions in a chiral ligand-exchange two-phase solvent system (stage one) and equilibrium between racemates (EnH) and chiral ligand (LiH) in the separation column (stage two) (reproduced by permission of Elsevier from ref. 22 © 2014)

For the first stage (enantioselective liquid-liquid extraction), the formation constant kf1 for the binary complex is expressed as kf 1 ¼

½Li2 Cuorg ½Hþ 2aq ½LiH2org ½Cu2þ aq

ð6Þ

And the distribution ratio for transition metal ion is ½Li2 Cuorg D Cu ¼  2þ  Cu aq

ð7Þ

Combining Eqs. 6 and 7 establishes the following equation for stage one:   ð8Þ logD Cu ¼ logk f 1 þ 2 log½LiHorg þ pHaq Equation 8 allows to deduce log kf1 from the intercept of a plot of log DCu versus log[LiH]org þ pHaq. As for the second stage (chemodynamic equilibrium inside of the separation column), the partition coefficient KD for enantiomers is K D ¼

½EnH org ½EnH aq

The dissociation constant ka for enantiomers is

ð9Þ

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 ka ¼

En 

 aq

½Hþ aq

½EnH aq

ð10Þ

The formation constant kf2 for ternary complex is k f 2 ¼

½Li‐Cu‐En org ½LiHorg ½Li2 Cuorg ½EnH org

ð11Þ

The distribution ratio D for enantiomers can be expressed as D ¼

½EnH org þ ½Li‐Cu‐En org   ½EnH aq þ En  aq

ð12Þ

Combining Eqs. 6 and 9–12, the distribution ratio D for enantiomer can be expressed as    2þ  K D þ 2 D ¼ k k Cu ½ LiH  þ ½ H  f 1 f 2 aq org 2 aq ½Hþ aq þ ka ½Hþ aq ð13Þ Finally, the enantioseparation factor α by chiral ligandexchange countercurrent chromatography is expressed as   2 k f 1 k f 2þ Cu2þ aq ½LiHorg þ ½Hþ aq α¼ ð14Þ   2 k f 1 k f 2 Cu2þ aq ½LiHorg þ ½Hþ aq As shown in Eqs. 13 and 14, for a given two-phase solvent system at a constant separation temperature, the distribution ratio for enantiomers would be largely determined by the partition coefficient KD, the pH value of the aqueous phase, the binary and ternary complex formation constants, the concentration of the transition metal ion, and the concentration of the chiral ligand in the organic phase. The enantioseparation factor is critically dependent on several parameters such as the difference between the two ternary complex formation constants kf2+ and kf2, the concentration of the transition metal ion in the aqueous phase, the concentration of the chiral ligand in the organic phase, and the pH of the aqueous phase. β-Cyclodextrin (β-CD) derivatives are typical chiral selectors used in homogeneous chiral selector addition countercurrent chromatography for enantioseparation of racemic aromatic acids, while L-proline derivatives are typical chiral ligands used in interfacial chiral ligand-exchange countercurrent chromatography for enantioseparation of α-hydroxy acids. In this chapter, we describe in detail two established methods for chiral separations by countercurrent chromatography under the two typical modes [21, 22].

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Materials

2.1 Instrumentation and Materials

1. A commercial countercurrent chromatography instrument (see Note 1). 2. A commercial HPLC instrument equipped with a C18 column for chiral analysis (see Note 2). 3. A commercial pH meter with a glass electrode for pH adjustment of solutions (see Note 3). 4. A glass column (25 cm  30 mm ID) for purification of the chiral ligand. 5. Silica gel (200–300 μm). 6. Thin-layer chromatography GF254 plates (see Note 4).

2.2 Reagents and Solutions

Use HPLC-grade solvents for mobile phases and ultrapure water purified by a suitable water purification system. All other chemicals should be of analytical grade. Most chemicals are toxic. Handle with care taking the required safety precaution measures. Handle toxic solvents and chemicals in a ventilated hood. Also perform synthetic procedures of cleanup procedures by column chromatography in a ventilated hood. 1. 0.1 M Phosphate buffer, pH 2.51: Dissolve 11.04 g of sodium dihydrogen phosphate monohydrate in 800 mL of water. Adjust to pH 2.51 by addition of 0.1 M phosphoric acid (see Note 5). 2. 0.05 M Hydroxypropyl-β-CD solution: Dissolve 60.5 g of hydroxypropyl-β-CD in 800 mL of the 0.1 M phosphate buffer, pH 2.51, under stirring (see Note 6). 3. Mix 200 mL of n-hexane, 600 mL of methyl tert.-butyl ether, and 800 mL of 0.05 M hydroxypropyl-β-CD solution in a 2000 mL separatory funnel (see Note 7) and shake vigorously for 5 min at room temperature (see Note 8). Let stand and equilibrate at room temperature for 30 min (see Note 9). 4. Organic phase 1 and aqueous phase 1: Separate upper organic phase and lower aqueous phase and degas both phases by sonication before use. Use phases for the experiment described in Subheading 3.1. 5. Add 200 mL of n-butanol and 200 mL of water in a 500 mL separatory funnel and shake vigorously for 5 min at room temperature (see Note 8). Let stand and equilibrate at room temperature for 30 min (see Note 9). Separate upper organic phase and lower aqueous phase. 6. Aqueous phase A: Dissolve 4.0 mg of cupric acetate monohydrate in 100 mL of the aqueous phase obtained under step 5 to obtain a 0.2 mM cupric acetate solution.

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7. Organic phase A: Dissolve 1.417 g of N-n-dodecyl-L-proline (see Note 10) in 100 mL of the organic phase obtained under step 5. Dissolve 0.499 g of cupric acetate monohydrate in 100 mL of the aqueous phase obtained under step 5 (see Note 11). Transfer both solutions to a 500 mL separatory funnel and shake vigorously for 5 min. Let stand and equilibrate for at least 30 min. Discard the lower aqueous phase. 8. Mix the aqueous phase A prepared according to step 6 and the organic phase A described under step 7 in a 500 mL separatory funnel. Shake vigorously for 5 min at room temperature (see Note 8). Let stand and equilibrate for 30 min at room temperature (see Note 9). 9. Organic phase 2 and aqueous phase 2: Separate upper organic phase and lower aqueous phase and degas both phases by sonication before use. Use phases for the experiment described in Subheading 3.2.2. 10. Phenylsuccinic acid sample solution: Dissolve 712 mg of racemic phenylsuccinic acid in 20 mL of the organic phase obtained under step 4 (see Note 12). 11. α-Hydroxy acid sample solutions: Dissolve 2 mg of a racemic α-hydroxy acid (mandelic acid, 2-chloromandelic acid, or 4-methoxymandelic acid) in 1.0 mL of the aqueous phase obtained from step 9 (see Note 13). 12. HPLC mobile phase: Mix 400 mL of a 10 mM aqueous solution of hydroxypropyl-β-CD, 100 mL of acetonitrile, and 250 μL of trifluoroacetic acid. Adjust pH to 2.0 by addition of triethylamine. Filter (0.45 μm) and degas by sonication before use (see Note 14).

3

Methods

3.1 Example 1: Chiral Separation of Phenylsuccinic Acid by Homogeneous Chiral Selector Addition Using Hydroxypropyl-βcyclodextrin as Chiral Selector

1. Set temperature of the separation column of the preparative countercurrent chromatography instrument at 5  C (see Note 15). 2. Pump the organic phase 1 as obtained in Subheading 2.2, step 4, into the separation column at a flow rate of 30 mL/min until the column is completely filled with the organic phase (see Note 16). Do not rotate the column during the filling procedure. 3. Pump the aqueous phase 1 as obtained in Subheading 2.2, step 4, into the inlet of the separation column with head-to-tail elution mode at a flow rate of 2.0 mL/min while rotating the separation column at 850 rpm (see Note 17). 4. Equilibrate column. When the equilibrium is reached, the mobile phase eluting from the outlet is clear.

Chiral Countercurrent Chromatography 500

329

(-)-enantiomer

450 (+)-enantiomer

400 350

[mV]

300 250 200 150 100 50 0 0

50

100

150

200

250 300 [min]

350

400

450

500

550

Fig. 3 Chromatogram of enantioseparation of racemic phenylsuccinic acid by preparative high-speed countercurrent chromatography. Biphasic solvent system: n-hexane:methyl tert-butyl ether:0.1 M phosphate buffer with pH 2.51 containing 0.05 M hydroxypropyl-β-CD (0.5:1.5:2, v/v/v); sample: 712 mg of racemate dissolved in 20 mL of the organic phase; flow rate: 2.0 mL/min; elution mode: head to tail; revolution: 850 rpm; stationary-phase retention: 62.9% (reproduced by permission of Elsevier from ref. 21 © 2011)

5. Set detection wavelength to 254 nm. 6. Fill sample loop with the sample solution of phenylsuccinic acid as obtained in Subheading 2.2, step 10, and inject into the separation system. Record chromatogram. A typical chromatogram is shown in Fig. 3 (see Note 18). 7. For preparative isolation of the enantiomers, collect eluting fractions manually according to the elution profile, e.g., between 160 and 380 min and between 400 and 520 min. 8. Acidify fractions by addition of concentrated hydrochloric acid to pH 2 and extract three times with 450 mL methyl tert-butyl ether (see Note 19). Dry the combined organic layers with anhydrous sodium sulfate and filter, and evaporate the solvent under reduced pressure using a rotatory evaporator. 9. Subject each residue of the two fractions to the silica gel column chromatography with isocratic elution (chloroform:methanol:glacial acetic acid 10:1:0.05, v/v/v). Analyze fractions by TLC with the mobile phase: chloroform:methanol:glacial acetic acid (10:1:0.05, v/v/v) to collect the fractions showing spots with a Rf value of about 0.34. Combine fractions and evaporate the organic solvent under reduced pressure to yield about 285 mg of the (þ)-enantiomer and 290 mg of the ()enantiomer (see Note 20).

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10. The purity of the phenylsuccinic acid enantiomers can be analyzed by reversed-phase HPLC using the mobile phase prepared according to Subheading 2.2, step 12, and the following conditions: flow rate 0.6 mL/min, column temperature 30  C, and detection at 225 nm. Typical chromatograms are shown in Fig. 4. The HPLC purity for each enantiomer should be at least 98.5%. 3.2 Example 2: Chiral Separation of α-Hydroxy Acids by Chiral LigandExchange Countercurrent Chromatography 3.2.1 Synthesis of N-ndodecyl-L-proline

1. 5.75 g (50 mmol) L-Proline and 2.2 g (35 mmol) sodium cyanoborohydride in a 250 mL one-neck round-bottom flask equipped with a magnetic stir bar and a drying tube filled with anhydrous calcium chloride and add 75 mL methanol (see Note 21). 2. Add 10.15 g (55 mmol) n-dodecanal over a period of 20 min to the reaction mixture at room temperature. 3. Stir reaction mixture for 18 h at room temperature. 4. Evaporate solvent under reduced pressure using a rotatory evaporator. 5. Purify the residue by silica gel column chromatography using chloroform:methanol (8:0, v/v) to remove small amount of ndodecanal and using chloroform:methanol (8:2, v/v) to elute the target component N-n-dodecyl-L-proline (see Note 22). 6. Analyze fractions by TLC using chloroform:methanol (1:1, v/v) as mobile phase. Detect spots by iodine vapor. The Rf values of L-proline and N-n-dodecyl-L-proline are 0.31 and 0.65, respectively. 7. Combine fractions containing the target compound and remove the organic solvents under reduced pressure in a rotatory evaporator. 8. 8 Obtain N-n-dodecyl-L-proline as a white waxy solid. The yield is about 12 g.

3.2.2 Countercurrent Chromatography Enantioseparation

1. Set column temperature of the analytical countercurrent chromatography instrument at 10  C (see Note 23). 2. Pump the organic phase 2 as obtained in Subheading 2.2, step 9, into the separation column of the instrument apparatus with a flow rate of 5 mL/min until the entire column is filled with the organic phase (see Note 16). Do not rotate the column during the filling procedure. 3. Pump the aqueous phase 2 as obtained in Subheading 2.2, step 9, into the inlet of the separation column with head-to-tail elution mode at a flow rate 0.3 mL/min while rotating the separation column at 145.98  g (see Note 24). 4. Equilibrate column. When the equilibrium is reached, the mobile phase eluting from the outlet is clear.

Chiral Countercurrent Chromatography

Absorbance (225 nm) mAU

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25 (-)-enantiomer

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10

15

20

25

Time (minutes)

Fig. 4 Chromatogram of HPLC analyses of racemic phenylsuccinic acid and phenylsuccinic acid enantiomer purified from countercurrent chromatography. (a) Racemic phenylsuccinic acid; (b): (þ)-phenylsuccinic acid; (c): ()-phenylsuccinic acid (reproduced by permission of Elsevier from ref. 21 © 2011)

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5. Set detection wavelength to 254 nm. 6. Fill sample loop with a sample solutions of an α-hydroxy acid (mandelic acid, 2-chloromandelic acid, or 4-methoxymandelic acid) and inject into the separation system individually. Record chromatogram. Typical chromatograms of the α-hydroxy acids are shown in Fig. 5 (see Note 25).

4

Notes 1. Any commercial apparatus of high-speed countercurrent chromatography and high-performance centrifugal partition chromatography, which can provide suitable retention of stationary phase, can be used for the present chiral separations. In the present study, a TBE-20A analytical instrument and a TBE-300A preparative instrument (Shanghai Tauto Biotechnique, Shanghai, China) was used. Both instruments are equipped with a set of three multilayer coils. The TBE-20A analytical column consisted of 0.8 mm ID PTFE tubing with a total capacity of 20 mL while the TBE-300A preparative column consisted of 1.6 mm ID PTFE tubing with a total capacity of 270 mL. The β values of the analytical and preparative columns ranged from 0.60 to 0.78 and 0.46 to 0.73, respectively (β ¼ r/R, R ¼ 4.5 cm for analytical columns and 6.5 cm for preparative ones, where r is the distance from the coil to the holder shaft, and R, the revolution radius or the distance between the holder shaft and central axis of the centrifuge). The revolution speed of the column coils can be regulated with a speed controller in the range from 0 to 2000 rpm for the analytical centrifuge, and from 0 to 1000 rpm for the preparative centrifuge. Both separation columns are installed in a vessel that maintains column temperature at 5  C by a constanttemperature controller. Manual sample injection valves with a 1.0 mL loop for analytical apparatus and a 20.0 mL loop for preparative one are used to introduce the sample into the column. The solvents are pumped into the column with a constant-flow pump. Continuous monitoring of the effluent is achieved with a model UVD-200 detector (Shanghai Jinda Biotechnology Co., Ltd., Shanghai, China) and SEPU3000 workstation (Hangzhou Puhui Technology, Hangzhou, China) was employed to record the chromatogram. 2. In the present study a MS-20160509 system (Shimadzu, Japan) composed of a Shimadzu SPD-20Avp UV detector, a Shimadzu LC-20ATvp Multisolvent Delivery System, a Shimadzu SCL-10ASvp controller, a Shimadzu LC pump, and a LabSolutions MS-20160509 workstation were used. The

(mV)

Chiral Countercurrent Chromatography

24 22 20 18 16 14 12 10 8 6 4 2 0

333

(+)-mandelic acid (-)-mandelic acid 0

20

40

60

80 (min)

100

120

140

160

8 7 6 5

(mV)

4 3 2 1 0 (+)-2-chloromandelic acid

–1

(-)-2-chloromandelic acid

–2 –3

(mV)

0

50

100

150

200

250

300

(min)

22 20 18 16 14 12 10 8 6 4 2 0 –2 –4

(+)-4-methoxymandelic acid (-)-4-methoxymandelic acid 0

50

100

150

200

(min)

Fig. 5 Chromatograms of enantioseparation of three racemic α-hydroxy acids by analytical chiral ligandexchange high-speed countercurrent chromatography. Solvent system: n-butanol:water (1:1, v/v), in which 0.050 M of N-n-dodecyl-L-proline was added in the organic phase as chiral ligand and 0.025 M of cupric

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column was an YMC-Pack ODS-A (250 mm  4.6 mm ID, 5 μm) (YMC Co., Ltd., Kyoto, Japan). 3. Any commercial pH meter can be used. In the present study, the pH value for the test is determined with a Delta 320-s pH meter (Mettler-Toledo, Greifensee, Switzerland). 4. In the present study, commercial thin-layer chromatography plate GF254 (Qingdao Haiyang Chemical, Qingdao, China) was used. Any commercial normal-phase thin-layer chromatography plate can be used. 5. The pH value of aqueous phase could greatly affect enantiorecognition between enantiomers and hydroxypropyl-β-cyclodextrin because the enantiorecognition for enantiomers by hydroxypropyl-β-CD is only achieved when the free molecular enantiomer is present in the aqueous phase instead of ionic ones. pH value should not be more than 3.0. 6. Commercial hydroxypropyl-β-CD with a degree of substitution being 6.5–7.0 is used and the average molecular weight is around 1507 g/mol. Higher degree of substitution for hydroxypropyl-β-CD leads to a higher enantioseparation factor. Thus, peak resolution could be improved with hydroxypropyl-β-CD with higher degree of substitution. Also, the chiral selector should have high solubility in the selected solvent system but it should be dissolved in only one phase of the biphasic solvent system. 7. The biphasic solvent system is composed of n-hexane:methyl tert.-butyl ether:0.05 M hydroxypropyl-β-CD solution (0.5:1.5:2, v/v/v). The distribution ratio of racemate should be arranged within a suitable value (0.2–5) as for the selected biphasic solvent system added with the chiral selector. The organic solvent ethyl acetate and n-butyl acetate are two alternative solvents for methyl tert.-butyl ether in the organic phase. Higher enantiorecognition could be obtained when an organic solvent with large hydrophobicity is used. Enantiorecognition of hydroxypropyl-β-CD in the aqueous phase would be greatly disrupted by adding water-soluble organic solvent in the aqueous phase, such as methanol, ethanol, and acetonitrile. 8. Be cautious of the gas produced when shaking vigorously for preparation of biphasic solvent system in the separatory funnel.

ä Fig. 5 (continued) acetate was added in the aqueous phase as a transition metal ion; stationary phase: upper organic phase; mobile phase: lower aqueous phase; sample solution: 2 mg of racemates dissolved in 1 mL of the mobile aqueous phase; flow rate: 0.3 mL/min; revolution: 145.98  g column temperature: 10  C; stationary-phase retention: 15–20% (reproduced by permission of Elsevier from ref. 22 © 2014)

Chiral Countercurrent Chromatography

335

9. 30 min is sufficient for equilibration. No additional equilibration time is needed. 10. N-n-dodecyl-L-proline is not commercially available. The synthesis for N-n-dodecyl-L-proline is described in Subheading 3.2.1. 11. The pH value of the aqueous phase should be closely around 5.6, which is achieved by adding cupric acetate in the aqueous phase. The pH value can have a profound effect on the formation of binary and ternary complexes among chiral ligand, transitional metal ion, and enantiomer. No enantioseparation could be achieved if the pH value is below 5.6. Large amount of precipitation would be observed if the pH value of aqueous phase reaches 6.0. 12. It is very important to make sure that the amount of racemate to be injected is set to avoid saturation of the chiral selector. A maximum molar ratio of chiral selector/analyte (1:1) is determined to the limit capacity in chiral separation, in which chiral selector forms 1:1 complexes with enantiomers. 13. A maximum molar ratio of chiral ligand:transitional metal ion: analyte (1:1:1) is determined to the limit capacity in chiral separation, in which chiral ligand:Cu2+:enantiomer forms diastereoisomeric complexes with the molar ratio of 1:1:1. 14. The mobile phase was 10 mM hydroxypropyl-β-CD aqueous solution:acetonitrile:trifluoroacetic acid (80:20:0.05, v/v/v) (pH 2.5, adjusted with triethylamine). 15. Low separation temperature is favorable for improving peak resolutions during the enantioseparation by countercurrent chromatography. Thus, 0–10  C is preferred. 16. A large flow rate is preferred for pumping the organic phase into the column, which is used as the stationary phase. 17. Different rotation speed of the separation column might be used when different commercial preparative high-speed countercurrent chromatography apparatus is used, which is mainly dependent on the retention of the stationary phase. Generally, stationary-phase retention should be around 55–65% for the present separation. 18. The recommended sample volume in the standard separation using a commercial high-speed countercurrent chromatography unit with a partition efficiency of about 600–800 theoretical plates may be less than 5% of the total column capacity. 19. Extract three times with 450 mL methyl tert.-butyl ether, in which 150 mL methyl tert.-butyl ether is used each time. Ethyl acetate is an alternative solvent for the extractions.

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20. The collected fractions are subjected to silica gel column chromatography to remove slight amount of hydroxypropyl-β-cyclodextrin in the purified enantiomer. The recovery of the enantiomers should be about 80%. 21. It is not necessary to dry methanol and nitrogen is not necessary. 22. Chloroform is toxic. Handle with care taking the required safety precaution measures. Always use a ventilated hood. Dichloromethane could be used if chloroform is forbidden in the lab. 23. Low separation temperature is favorable for improving peak resolution during enantioseparation by countercurrent chromatography. However, stationary phase might be easily carried over during the separation if a very low column temperature is used because n-butanol is highly hydrophilic. 24. Different rotation speed of the separation column might be used when a different commercial analytical high-speed countercurrent chromatography apparatus is used, which is mainly dependent on the retention of the stationary phase. Generally, stationary-phase retention should be at least 20–25% for the present separation. 25. Recovery of enantiomer from the enantioseparation by analytical countercurrent chromatography could not be investigated since only 2 mg of racemate is injected for each separation.

Acknowledgments The financial support by the National Natural Science Foundation (No. 21105090), Zhejiang Province Natural Science Foundation (No. Y4100472), and Zhaohui Program of Zhejiang University of Technology (2013) is gratefully acknowledged. References 1. Ito Y (1981) Efficient preparative countercurrent chromatography with a coil planet centrifuge. J Chromatogr 214:122–125 2. Ito Y, Sandlin J, Bowers WG (1982) Highspeed preparative counter-current chromatography with a coil planet centrifuge. J Chromatogr 244:247–258 3. Ito Y, Conway WD (1984) Development of countercurrent chromatography. Anal Chem 56:534A–554A 4. Friesen JB, McAlpine JB, Chen SN, Pauli GF (2015) Countercurrent separation of natural

products: an update. J Nat Prod 78:1765–1796 5. Hu RL, Pan YJ (2012) Recent trends in counter-current chromatography. Trends Anal Chem 40:15–27 6. Ito Y (2005) Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography. J Chromatogr A 1065:145–168 7. Huang XY, Di DL (2015) Chiral separation by counter-current chromatography. Trends Anal Chem 67:128–133

Chiral Countercurrent Chromatography 8. Ward TJ, Ward KD (2012) Chiral separations: a review of current topics and trends. Anal Chem 84:626–635 9. Ma Y, Ito Y (2010) Chiral CCC. In: Cazes J (ed) Encyclopedia of chromatography, vol 1, 3rd edn. pp 413–415 10. Han C, Wang W, Xue G, Xu D, Zhu T, Wang S, Cai P, Luo J, Kong L (2018) Metal ion-improved complexation countercurrent chromatography for enantioseparation of dihydroflavone enantiomers. J Chromatogr A 1532:1–9 11. Xu W, Wang S, Xie X, Zhang P, Tang K (2017) Enantioseparation of pheniramine enantiomers by high-speed countercurrent chromatography using β-cyclodextrin derivatives as a chiral selector. J Sep Sci 40:3801–3807 12. Tong S, Wang X, Shen M, Lv L, Lu M, Bu Z, Yan J (2017) Enantioseparation of 3-phenyllactic acid by chiral ligand exchange countercurrent chromatography. J Sep Sci 40:1834–1842 13. Tong S, Shen M, Xiong Q, Wang X, Lu M, Yan J (2016) Chiral ligand exchange countercurrent chromatography: equilibrium model study on enantioseparation of mandelic acid. J Chromatogr A 1447:115–121 14. Lv L, Bu Z, Lu M, Wang X, Yan J, Tong S (2017) Stereoselective separation of β-adrenergic blocking agents containing two chiral centers by countercurrent chromatography. J Chromatogr A 1513:235–244 15. Wang S, Han C, Wang S, Bai L, Li S, Luo J, Kong L (2016) Development of a high speed countercurrent chromatography system with Cu(II)-chiral ionic liquid complexes and hydroxypropyl-β-cyclodextrin as dual chiral

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selectors for enantioseparation of naringenin. J Chromatogr A 1471:155–163 16. Huang X-Y, Pei D, Liu J-F, Di D-L (2018) A review on chiral separation by counter-current chromatography: development, applications and future outlook. J Chromatogr A 1531:1–12 17. Pe´rez E, Minguillo´n C (2007) Countercurrent chromatography in the separation of enantiomers. In: Subramanian G (ed) Chiral separation techniques, 3rd edn. pp 369–397 18. Oliveros L, Puertolas PF, Minguillon C, Camacho-Frias E, Foucault A, Goffic FL (1994) Donor-acceptor chiral centrifugal partition chromatography: complete resolution of two pairs of amino-acid derivatives with a chiral II donor selector. J Liq Chromatogr 17:2301–2318 19. Ma Y, Ito Y, Foucault A (1995) Resolution of gram quantities of racemates by high-speed counter-current chromatography. J Chromatogr A 704:75–81 20. Koska J, Haynes CA (2001) Modelling multiple chemical equilibria in chiral partition systems. Chem Eng Sci 56:5853–5864 21. Tong SQ, Yan JZ, Guan YX, Lu YM (2011) Enantioseparation of phenylsuccinic acid by high speed counter-current chromatography using hydroxypropyl-β-cyclodextrin as chiral selector. J Chromatogr A 1218:5602–5608 22. Tong SQ, Shen MM, Cheng DP, Zhang YM, Ito Y, Yan JZ (2014) Chiral ligand exchange high-speed countercurrent chromatography: mechanism and application in enantioseparation of aromatic α-hydroxy acids. J Chromatogr A 1360:110–118

Chapter 18 Cyclodextrins as Chiral Selectors in Capillary Electrophoresis Enantioseparations Gerhard K. E. Scriba and Pavel Ja´cˇ Abstract Due to their structural variability and their commercial availability, cyclodextrins are the most frequently used chiral selectors in capillary electrophoresis. A variety of migration modes can be realized depending on the characteristics of the cyclodextrins and the analytes. The basic considerations regarding the development of a chiral CE method employing cyclodextrins as chiral selectors are briefly discussed. The presented examples illustrate the separation modes of an acidic and a basic analyte with native and charged cyclodextrin derivatives as a function of the pH of the background electrolyte and the concentration of the cyclodextrin. Key words Capillary electrophoresis, Chiral separation, Enantioseparation, Cyclodextrin, Enantiomer migration order

1

Introduction Cyclodextrins (CDs) are cyclic oligosaccharides consisting of α(1!4)-linked D-glucopyranose units. They are produced by digestion of starch by the enzyme cyclodextrin glycosyltransferase initially isolated from various Bacillus strains [1, 2]. The most important industrially produced CDs differ in the number of glucopyranose units; that is, α-CD is composed of 6 units, β-CD of 7 units, and γ-CD of 8 units (Fig. 1). CDs are shaped like toroids with a lipophilic cavity and a hydrophilic outside surface. The wider rim contains the secondary 2- and 3-hydroxy groups, while the narrower rim is formed by the primary 6-hydroxy groups. Some properties of the native CDs are summarized in Table 1. The hydroxy groups can be derivatized yielding a large variety of CD derivatives containing uncharged or charged substituents (Table 2). CDs can be obtained from many companies including Merck, CDT Inc., or CyDex Inc. The most complete selection of CDs including variations in the degree of substitution and isomeric purity is supplied by Cyclolab and Cyclodextrin-Shop.

Gerhard K. E. Scriba (ed.), Chiral Separations: Methods and Protocols, Methods in Molecular Biology, vol. 1985, https://doi.org/10.1007/978-1-4939-9438-0_18, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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340

OR RO

OR OR

O OR

OR

OR

RO

5

4

RO

OR OR RO O

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3

OR

RO

O RO

OR

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O

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a-CD

RO RO O

b-CD

g -CD

Fig. 1 Structures of α-CD, β-CD, and γ-CD Table 1 Properties of native CDs α-CD

β-CD

γ-CD

6

7

8

Molecular formula (anhydrous)

C36H60O30

C42H70O35

C48H80O40

Molecular weight (g/mol)

972

1135

1297

˚) Approximate inner diameter (A

4.7–5.3

6.0–6.5

7.5–8.3

˚) Approximate outer diameter (A

14.6

15.4

17.5

Approximate height (A˚)

7.9

7.9

7.9

Approximate volume of cavity (A˚3)

174

262

427

Solubility in water (g/100 mL)

14.5

1.85

23.2

Water molecules in cavity

6

11

17

D-Glucopyranose

units

Due to their ability to form complexes with a variety of compounds, CDs have found numerous applications in the pharmaceutical, cosmetic, food, textile, chemical, and agrochemical industries [3–5]. In separation sciences, CDs have been used as chiral selectors in GC, HPLC, as well as capillary electrophoresis (CE) techniques including electrokinetic chromatography (EKC), micellar electrokinetic chromatography (MEKC), microemulsion electrokinetic chromatography (MEEKC), and capillary electrochromatography (CEC). In fact, CDs are the most widely used chiral selectors in CE. Advantages are the UV transparency as well as the fact that they can be used in aqueous and nonaqueous background electrolytes. This has been documented in countless publications, which have been summarized in reviews, e.g., [6–14], book chapters [15, 16], or a monograph [17]. General aspects of

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Table 2 Examples of commercially available CDs Derivative

Substituents

Native CDs α-CD

H

β-CD

H

γ-CD

H

Neutral CDs Methyl-α-CD

CH3, randomly substituted

Methyl-β-CD

CH3, randomly substituted

Methyl-γ-CD

CH3, randomly substituted

Heptakis(2,6-di-O-methyl)-β-CD

CH3 in positions 2 and 6

Heptakis(2,3,6-tri-O-methyl)-β-CD

CH3 in positions 2, 3, and 6

Hydroxypropyl-α-CD

CH2–CH2–CH2–OH, randomly substituted

Hydroxypropyl-β-CD

CH2–CH2–CH2–OH, randomly substituted

Hydroxypropyl-γ-CD

CH2–CH2–CH2–OH, randomly substituted

Negatively charged CDs Carboxymethyl-β-CD

CH2-COONa, randomly substituted

Sulfated α-CD

SO3Na, randomly substituted

Sulfated β-CD

SO3Na, randomly substituted

Sulfated γ-CD

SO3Na, randomly substituted

Sulfobutylether-β-CD

CH2–CH2–CH2–CH2–SO3Na, randomly substituted

Succinyl-β-CD

CO–CH2–CH2–COOH, randomly substituted

Heptakis(6-O-sulfo)-β-CD

SO3Na in position 6

Heptakis(2,3-di-O-acetyl-6-O-sulfo)-β-CD

CH3CO in positions 2 and 3, SO3Na in position 6

Hexakis(2,3-di-O-methyl-6-O-sulfo)-α-CD

CH3 in positions 2 and 3, SO3Na in position 6

Heptakis(2,3-di-O-methyl-6-O-sulfo)-β-CD

CH3 in positions 2 and 3, SO3Na in position 6

Octakis(2,3-di-O-methyl-6-O-sulfo)-γ-CD

CH3 in positions 2 and 3, SO3Na in position 6

Positively charged CDs (2-Hydroxy-3trimethylammoniopropyl)-α-CD

CH2–CH(OH)–CH2–N(CH3)3Cl, randomly substituted

(2-Hydroxy-3trimethylammoniopropyl)-β-CD

CH2–CH(OH)–CH2–N(CH3)3Cl, randomly substituted

(2-Hydroxy-3trimethylammoniopropyl)-γ-CD

CH2–CH(OH)–CH2–N(CH3)3Cl, randomly substituted

6-Monodeoxy-6-monoamino-β-CD

NH2 instead of one 6-OH group

Heptakis(6-deoxy-6-amino)-β-CD

NH2 in position 6

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CE enantioseparations can be found, for example, in review papers [18–22] as well as monographs [23, 24]. The chiral recognition mechanism of CDs is believed to occur in most cases via the inclusion of lipophilic moieties of the analyte into the hydrophobic cavity of the CDs displacing solvent molecules (typically water) from inside the cavity [25]. However, it has also been shown that inclusion complex formation is not a prerequisite for a successful enantioseparation. Secondary interactions between CD and the analytes may include hydrogen bonding or dipole-dipole interactions with the hydroxy groups or with other polar substituents of the CDs [26]. In the case of charged CDs, ionic interactions will also contribute or may even dominate the complexation mechanism. The increased interactions between oppositely charged analytes and CDs often allow the use of very low selector concentrations in enantioseparations. 1.1 Migration Modes of CE Enantioseparations

As in chromatographic techniques, CD-mediated enantioseparations in CE are based on the reversible formation of diastereomeric complexes between the enantiomers of a solute and the CDs. However, in contrast to chromatography, the selector is not fixed to a support and may even possess an electrophoretic mobility itself. As a consequence, the transient diastereomeric complexes between a CD and the solute enantiomers may differ in their association constants and/or their electrophoretic mobilities leading to enantioseparations. The mobilities of the CDs as well as the CD-analyte complexes offer a variety of different modes and flexible analytical systems. The experimental conditions can even be selected in a suitable way to reverse the migration order of the analyte enantiomers [27]. Figure 2 illustrates some popular migration modes in chiral CE. In the case of a basic analyte in acidic media in the presence of neutral CDs (Fig. 2a) the protonated analyte migrates to the detector at the cathodic end of the capillary. The CD does not possess an electrophoretic mobility but may be transported by the electroosmotic flow (EOF). Subsequently, the stronger complexed enantiomer migrates second as it is complexed for a longer period of time compared to the weaker bound enantiomer and the complex itself has a lower mobility compared to the free analyte. In the case of an acidic (negatively charged) analyte in moderately acidic or alkaline background electrolytes the analyte migrates to the anode but is eventually transported to the detector at the cathodic end of the capillary by the strong EOF (Fig. 2b). Under these circumstances the stronger complexed enantiomer migrates first because the mobility in the opposite direction to the anode is slowed down. If the pH is lowered so that the EOF is reduced in such a way that the anodic mobility of the analytes exceeds the EOF the analytes can be detected at the anode upon reversing the polarity of the applied voltage (Fig. 2c). Thus, the weaker bound enantiomer will

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Fig. 2 Scheme of selected separation modes in CD-mediated chiral CE

migrate first resulting in a reversal of the enantiomer migration order compared to the situation with higher pH buffers described in Fig. 2b. For scenario 2c, the analyte must possess a negative charge to ensure migration toward the detector at the anode. The electrophoretic mobility of charged CDs can also lead to interesting applications as illustrated for negatively charged CDs. In a low-pH-background electrolyte a protonated analyte migrates to the cathode while the CD migrates toward the anode (Fig. 2d). In this scenario, the weaker bound enantiomer is detected first. A general advantage of selectors with the opposite charge to the analytes is their counter-directed mobility, which allows the use of low concentrations of the chiral selector. In the case of high CD concentrations or strong binding of the enantiomers to the selector, the compounds may not reach the detector at the cathode

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because the solutes are transported by the negatively charged CD to the anode. In this case, the polarity of the applied voltage can be reversed and detection is carried out at the anodic end of the capillary (Fig. 2e). The enantiomer which forms the stronger complex with the CD is detected first in this scenario as it is accelerated toward the anode by the negatively charged CD. Compared to the situation discussed in Fig. 2d, a reversal of the enantiomer migration order is observed. These conditions can also be applied to the analysis of uncharged compounds. Provided that the EOF is strong enough, neutral analytes can be detected at the cathodic end of the capillary in the presence of negatively charged CDs analogous to the situation illustrated in Fig. 2d. However, in most cases the carrier mode of the charged selectors will be exploited for enantioseparations of neutral analytes in analogy to Fig. 2e. Comparable scenarios can also be envisaged for positively charged CDs and negatively charged compounds. Moreover, many further migration schemes have been described also depending on differences in the complexation strength of charged and uncharged species as well as considering the mobility of the complexes [20, 22, 27, 28]. Furthermore, the combination of charged and uncharged CDs has been a successful strategy using the uncharged CD for the separation of the analyte enantiomers and the charged CD for their mobilization [29]. 1.2 Development and Optimization of a Chiral CE Method

The aim of method development of any analytical separation technique is to obtain an assay that allows the separation of the analytes in a short analysis time. Besides the physicochemical characteristics of the analyte, experimental factors including type and concentration of the CD, pH, type, and concentration of the background electrolyte, additives such as organic solvents or surfactants, applied voltage, or capillary temperature affect enantioseparations in CE. In case of water-insoluble, lipophilic, or neutral compounds MEKC or MEEKC should be considered. A summary of method development can be found in [30]. Generally, water-soluble charged compounds are analyzed by EKC. Typical method development starts with the selection of an appropriate buffer pH and a CD. At present the selection of the CD cannot be rationalized and depends largely on the experience of the analyst. In many cases a reversal of the enantiomer migration order can be observed when switching from one native CD to another or when using different CD derivatives of the same native CD. Pure single isomer CDs (e.g., heptakis(6-O-sulfo)-β-CD) are not necessarily required for successful enantioseparations. In fact, many reported separations have been achieved using randomly substituted derivatives. However, randomly substituted CDs are a mixture of isomers differing in their degree of substitution, i.e., the number and the position of the substituents. Therefore, randomly substituted CDs from various suppliers may differ in this respect

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and differences may even be observed from batch to batch for a given CD from the same supplier. It has been demonstrated that the source of the CD and the degree of substitution may affect the enantioseparation of one compound while this may have no effect for another analyte. Moreover, it cannot be predicted if a higher or lower degree of substitution of a given CD results in a better enantioseparation; examples for both scenarios have been reported [31, 32]. Combinations of CDs especially the combination of charged and neutral CDs have been a very successful strategy for CE enantioseparations [29]. Screening approaches have been described in an attempt to find more or less generalized starting conditions without excessive testing of CDs. Many users prefer negatively charged CDs as they can be used for uncharged compounds as well [33–35]. At low pH basic compounds are protonated and migrate to the cathode while the negatively charged CDs migrate to the anode. Neutral compounds interacting with the negatively charged CDs are transported to the anode and can be detected upon reversing the polarity of the applied voltage. Most acidic analytes are protonated at low pH and behave as neutral compounds. A screening strategy using sulfated CDs is outlined in Fig. 3. According to the charged resolving agent migration (CHARM) model developed by Vigh and coworkers [36] screening should be performed in a low pH

50 mm fused silica capillary, 20 cm effective length 300 V/cm, reversed polarity (anodic detection) 50 mM phosphate buffer, pH 2.5 5% (w/v) S-a-CD, S-b-CD, or S-g -CD

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Try other CDs or other selectors Final method

Fig. 3 Scheme of method development using negatively charged CDs. S-α-CD, S-β-CD, and S-γ-CD refer either to randomly sulfated CDs or to single-isomer sulfated CD derivatives [33–35]

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buffer (pH 2.2–2.5) and a high pH buffer (pH 9.5) depending on whether the analytes are ionized or neutral. Further strategies including both neutral and charged CDs have also been developed [37–40]. Upon selection of the suitable CD further method optimization should be performed. Besides optimization of the CD concentration, proper adjustment of the pH of the background electrolyte may critically affect an enantioseparation especially in the case of ionizable analytes. Working in the pH range close to the pKa values of the compounds can maximize the separation selectivity, especially of structurally closely related substances, due to an increasing contribution of the complex mobility. Other factors to be optimized include the type and concentration of the buffer, applied voltage, temperature of the capillary, and buffer additives such as organic solvents or surfactants. In addition, adjustment, suppression, or reversal of the EOF by dynamic or permanent coating of the capillary wall may be considered. In the case of MEKC methods, the nature and concentration of the surfactant have to be considered, and in the case of MEEKC the composition of the microemulsion, i.e., the type of the organic phase and type and concentration of the surfactant as well as the co-surfactant. Factors affecting CD-mediated enantioseparations have been summarized in [41]. One has to keep in mind that the desired resolution may also depend on the intended purpose. For example, when racemates have to be resolved a resolution value of 1.5 may be sufficient. However, when one enantiomer has to be quantified in the presence of a large excess of the other enantiomer, i.e., for the determination of the stereoisomeric purity of a compound, a much larger RS value has to be achieved in order to avoid overlapping of the small peak of the minor stereoisomer by the very large peak of the major stereoisomer. In practice it is common to use a variety of potential operating conditions to assess which operating conditions may prove useful. Standard sets of buffers and CDs may be tested in an overnight sequence to discover which conditions provide some enantioseparation. The initial conditions are subsequently optimized. Nowadays, multivariate optimization is preferred over univariate methods using quality by design and design of experiments methodology [42–45]. This reduces the number of experiments required to determine the best experimental conditions and, more importantly, allows the identification of the experimental parameters, which significantly influence the method. Optimization and method robustness can be concluded on a rational basis. Overall, this methodology results in the scientific understanding of the analytical process.

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Materials

2.1 CE Apparatus and Equipment

1. A commercial CE instrument with a high-voltage source (up to 30 kV) and a photodiode array detector. A P/ACE MDQ CE System (Sciex, Framingham, MA, USA) is suitable (see Note 1). 2. Uncoated fused silica capillaries with an internal diameter of 50 μm, an effective length of 30 cm, and a total length of 40.2 cm (see Note 2). Install the capillary into the capillary cartridge according to the manufacturer’s instructions. 3. A commercial pH-meter for pH adjustment of the background electrolytes. 4. An ultrasonic bath for sample and CD dissolution as well as for degassing of the solutions. 5. Syringe filters containing polyester or nylon filter membranes with a pore size of 0.20 μm. The use of 0.45 μm filters is also possible. The membrane material must be chemically inert to the solutions to be filtered. 6. A water purification system for preparation of ultrapure water (e.g., a Milli-Q Direct 8 system, Millipore, Billerica, MA, USA).

2.2 Background Electrolytes (See Notes 3 and 4)

1. BGE 1: 50 mM Phosphate buffer, pH 6.5, 2.5 mg/mL of β-CD. Dissolve 690 mg of NaH2PO4·H2O in approx. 50 mL of Milli-Q water and adjust pH to 6.5 using 1 M NaOH. Adjust the volume of the solution to 100.0 mL with Milli-Q water. Dissolve 25 mg of β-CD in approx. 5 mL of the buffer (see Note 5) under sonication (15 min) and adjust the volume to 10.0 mL with buffer. 2. BGE 2: 50 mM Phosphate buffer, pH 3.0, 2.5 mg/mL of β-CD. Dissolve 340 μL of 85% H3PO4 in approx. 50 mL of MilliQ water and adjust pH to 3.0 using 1 M NaOH. Adjust the volume of the solution to 100.0 mL with Milli-Q water. Dissolve 25 mg of β-CD in approx. 5 mL of the buffer (see Note 5) under sonication (15 min) and adjust the volume to 10.0 mL with buffer. 3. BGE 3: 50 mM Phosphate buffer, pH 2.5, 2 mg/mL of sulfated β-CD. Dissolve 340 μL of 85% H3PO4 in approx. 50 mL of MilliQ water and adjust pH to 2.5 using 1 M NaOH. Adjust the volume of the solution to 100.0 mL with Milli-Q water. Dissolve 20 mg of sulfated β-CD sodium salt (degree of

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substitution 12–15; see Note 6) in approx. 5 mL of the buffer and adjust the volume to 10.0 mL with buffer. 4. BGE 4: 50 mM Phosphate buffer, pH 2.5, 30 mg/mL of sulfated β-CD. Dissolve 340 μL of 85% H3PO4 in approx. 50 mL of MilliQ water and adjust pH to 2.5 using 1 M NaOH. Adjust the volume of the solution to 100.0 mL with Milli-Q water. Dissolve 300 mg of sulfated β-CD sodium salt (degree of substitution 12–15; see Note 6) in approx. 5 mL of the buffer and adjust the volume to 10.0 mL with buffer. Filter all buffer solutions through a 0.20 μm polyester or nylon membrane syringe filter into the buffer vials and degas by sonication for 5 min prior to use. 2.3

Sample Solutions

1. 1,10 -Binaphthyl-2,20 -diyl hydrogen phosphate solution (see Note 7): Prepare stock solutions (1 mg/mL) of each enantiomer of 1,10 -binaphthyl-2,20 -diyl hydrogen phosphate by dissolving 10 mg of each compound in approx. 5 mL of methanol and adjust the volume to 10.0 mL with methanol. Mix 200 μL of (S)-(þ)-1,10 -binaphthyl-2,20 -diyl hydrogen phosphate stock solution with 100 μL of (R)-()-1,10 -binaphthyl-2,20 -diyl hydrogen phosphate stock solution and adjust the volume to 10.0 mL with 10% aqueous methanol. Transfer the solution to the sample vial. 2. Ofloxacin solution (see Note 7): Prepare stock solutions (1 mg/mL) of ofloxacin and levofloxacin by dissolving 10 mg of each compound in approx. 5 mL of methanol and adjust the volume to 10.0 mL with methanol. Mix 400 μL of ofloxacin stock solution and 200 μL of levofloxacin stock solution and dilute to 10.0 mL with MilliQ water. Transfer the solution to the sample vial.

3

Methods

3.1 Conditioning and Rinsing Procedures for the Fused Silica Capillary ( See Note 8)

Filter all rinsing solutions through a 0.20 μm polyester or nylon membrane syringe filter.

3.1.1 Preconditioning of a New Capillary

Rinse the new capillary at a pressure of 138 kPa (20 p.s.i.) subsequently with 1. 0.1 M Phosphoric acid for 10 min.

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2. 1 M Sodium hydroxide for 20 min. 3. 0.1 M Sodium hydroxide for 20 min. 4. Milli-Q water for 10 min. 5. The appropriate background electrolyte for 10 min. 3.1.2 Conditioning of the Capillary Between Analyses

Rinse subsequently with filtered (0.2 μm) solutions at a pressure of 138 kPa (20 p.s.i.) with 1. 0.1 M Phosphoric acid for 2 min. 2. 0.1 M Sodium hydroxide for 2 min. 3. Milli-Q water for 2 min. 4. The appropriate background electrolyte for 4 min.

3.1.3 Rinsing of the Capillary for Storage

Rinse subsequently with filtered (0.2 μm) solutions at a pressure of 138 kPa (20 p.s.i.) with 1. 0.1 M Phosphoric acid for 10 min. 2. 0.1 M Sodium hydroxide for 10 min. 3. Milli-Q water for 10 min. For short-term (overnight) storage place capillary ends into vials containing Milli-Q water. For long-term storage dry capillary by purging with air at a pressure of 34.5 kPa (5 p.s.i.) for 5 min. After the overnight storage of the capillary rinse it next day with steps 1–3 as described in Subheading 3.1.3. Thereafter rinse it at 138 kPa (20 p.s.i.) for 10 min with the appropriate background electrolyte. Apply the same procedure when changing the background electrolyte. After the long-term storage condition the capillary as described in Subheading 3.1.1.

3.2

CE Analysis

3.2.1 Example 1

After conditioning of the capillary (see Note 8) select the appropriate background electrolyte and fill into buffer vials (see Note 9). Carry out CE measurements at the specified parameters including UV detection wavelength and applied high voltage. Set the temperature of the capillary to 20  C. Introduce sample solutions hydrodynamically at a pressure of 3.4 kPa (0.5 p.s.i.) for 6 s (see Note 10). The example illustrates the separation of negatively charged analytes using a neutral CD in the presence of a significant EOF as illustrated schematically in Fig. 2b. Use BGE 1 as run buffer and 1,10 -binaphthyl-2,20 -diyl hydrogen phosphate as analyte. Introduce the sample at the anodic end of the capillary, and carry out the detection at the cathodic end. Set data sampling rate to 4 Hz (see Note 11) and autozero time of the detector to 1.0 min.

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c

d

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3

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Fig. 4 Enantioseparation of 1,10 -binaphthyl-2,20 -diyl hydrogen phosphate using β-CD as chiral selector at (a) pH 6.5 and (b) pH 3.0

Applied voltage: 30 kV (ramp time 0.17 min). Detection wavelength (see Note 12): 210 nm (bandwidth 10 nm). Detector reference wavelength (see Note 13): 340 nm (bandwidth 50 nm). Generated current under the experimental conditions: approx. 80 μA. A typical electropherogram is shown in Fig. 4a. The stronger complexed enantiomer (S)-(þ)-1,10 -binaphthyl-2,20 -diyl hydrogen phosphate is detected first. The separation has been reported in [46]. 3.2.2 Example 2

The example illustrates the separation of negatively charged analytes using a neutral CD in the absence of a significant EOF under reversed polarity of the applied voltage as illustrated schematically in Fig. 2c. Use BGE 2 as run buffer and 1,10 -binaphthyl-2,20 -diyl hydrogen phosphate as analyte. Introduce the sample at the cathodic end of the capillary, and carry out the detection at the anodic end. Set data sampling rate to 4 Hz (see Note 11) and autozero time of the detector to 1.0 min. Applied voltage: 30 kV (ramp time 0.17 min). Detection wavelength (see Note 12): 210 nm (bandwidth 10 nm). Detector reference wavelength (see Note 13): 340 nm (bandwidth 50 nm). Generated current under the experimental conditions: approx. –50 μA.

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d

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Fig. 5 Enantioseparation of ofloxacin using sulfated β-CD as chiral selector at pH 2.5. (a) Low selector concentration (2 mg/mL) under normal polarity of the applied voltage and (b) exploiting the carrier ability of the selector at high concentrations (30 mg/mL) under reversed polarity of the applied voltage

A typical electropherogram is shown in Fig. 4b. The weaker complexed enantiomer (R)-()-1,10 -binaphthyl-2,20 -diyl hydrogen phosphate is detected first. The separation has been reported in [46]. 3.2.3 Example 3

The example illustrates the separation of positively charged analytes using a negatively charged CD in the absence of a significant EOF under normal polarity conditions as illustrated schematically in Fig. 2d. Use BGE 3 as run buffer and ofloxacin as analyte. Introduce the sample at the anodic end of the capillary, and carry out the detection at the cathodic end. Set data sampling rate to 4 Hz (see Note 11) and autozero time of the detector to 1.0 min. Applied voltage: 30 kV (ramp time 0.17 min). Detection wavelength (see Note 12): 291 nm (bandwidth 10 nm). Detector reference wavelength (see Note 13): 450 nm (bandwidth 50 nm). Generated current under the experimental conditions: approx. 70 μA. A typical electropherogram is shown in Fig. 5a. The weaker complexed (R)-enantiomer migrates first.

3.2.4 Example 4

The example illustrates the separation of positively charged analytes using a negatively charged CD in the absence of a significant EOF exploiting the carrier ability of the selector under reversed polarity of the applied voltage as illustrated schematically in Fig. 2e.

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Use BGE 4 as run buffer and ofloxacin as analyte. Introduce the sample at the cathodic end of the capillary, and carry out the detection at the anodic end. Set data sampling rate to 4 Hz (see Note 11) and autozero time of the detector to 6.0 min. Applied voltage: 20 kV (ramp time 0.17 min). Detection wavelength (see Note 12): 291 nm (bandwidth 10 nm). Detector reference wavelength (see Note 13): 450 nm (bandwidth 50 nm). Generated current under the experimental conditions: approx. –95 μA. A typical electropherogram is shown in Fig. 5b. The stronger complexed (S)-enantiomer, levofloxacin, migrates first.

4

Notes 1. CE instruments from different companies as well as different instruments from the same supplier may yield slightly different results even when using identical experimental conditions. Thus, the variables may require slight changes when transferring a certain analytical method from one instrument to another so that fine-tuning of the parameters of a published method can be necessary. Moreover, instruments from different manufacturers may have different operation conditions such as the maximum of pressure that can be applied. 2. Capillaries from different suppliers may lead to slightly different separation performances. Even capillaries from the same supplier may vary to a certain extent. Thus, the purchase of larger quantities of capillaries is recommended especially if a method is intended for validated routine analysis in an industrial environment. 3. Preparation of buffers according to different procedures results in buffers differing in ionic strength which may affect the separation selectivity. For example, a 50 mM phosphate buffer, pH 2.5, may be prepared (1) by mixing 50 mM sodium dihydrogen phosphate (monobasic sodium phosphate, NaH2PO4) and 50 mM disodium hydrogen phosphate (dibasic sodium phosphate, Na2HPO4) in appropriate proportions to obtain the desired pH; (2) by dissolving the appropriate amount of 85% phosphoric acid in a certain amount of water and adjusting to pH 2.5 by addition of sodium hydroxide solution before making up the final volume by addition of water; (3) by adjusting 50 mM phosphoric acid to pH 2.5 by addition of a sodium hydroxide solution; and (4) by adjusting 50 mM sodium dihydrogen phosphate to pH 2.5 by addition of diluted phosphoric

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acid. In the first and second cases the buffer concentration is 50 mM with respect to phosphate, in the case (3) the molarity of phosphate is below 50 mM, and in the case (4) phosphate molarity is higher than 50 mM. The deviation from the desired molarity will depend on the concentration of the sodium hydroxide solution and phosphoric acid used for pH adjustment. In addition, when using different salts, e.g., the potassium or lithium phosphate salts, or different bases, e.g., potassium hydroxide or lithium hydroxide, for the preparation, the resulting buffers differ in the counterions which may also affect a separation. Thus, careful characterization of the buffer is required for reproducible results. In addition, buffers can only be stored for a limited period of time even at low temperatures. 4. Due to the temperature dependence of dissociation equilibria buffer pH should be adjusted at the temperature that is used during the electrophoretic run. Specifically, the change of the pKa per Kelvin (or degree Celsius) of organic zwitterionic buffers is significant. 5. Due to the limited aqueous solubility of β-CD (max. 18 mg/ mL in water) urea at a concentration of 1–2 M is typically added when higher β-CD concentrations are required for an enantioseparation. It has been shown that urea can also affect separation selectivity. 6. Randomly substituted CDs are a mixture of isomers with varying degrees of substitution and substitution patterns (i.e., the number and positions of the substituents are different). Therefore, CDs from different sources and even different batches from the same supplier may vary in this respect which may lead to varying separation selectivity or resolution depending on the batch of selector used. In most cases the separation can be optimized by variation of the concentration of the chiral selector. Chemically defined single-isomer CDs are also available such as heptakis(6-O-sulfo)-β-CD or heptakis(2,3-di-Omethyl-6-O-sulfo)-β-CD. However, the use of randomly substituted CDs may also result in higher enantioresolutions compared to single-isomer CDs. In the presented examples, sulfated β-CD with a degree of substitution of 12–15 was used. 7. Nonracemic mixtures are used in order to easily detect the enantiomer migration order. Preparation of such solutions is only possible if at least one of the enantiomers is available in the pure form. 8. Conditioning of the capillary is important in order to obtain reproducible conditions of the inner wall of the capillary. Therefore, careful preconditioning of the capillary is required. Moreover, it is necessary to include all rinsing steps into

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validation procedures when developing CE procedures for quality control. 9. Different vials containing the background electrolyte should be used for rinsing of the capillary and for the analytical separation. Buffer levels should be the same in the analysis vials in order to avoid a hydrodynamic flow due to differences in hydrostatic pressure between the vials. Buffer should be replaced after a number of injections (typically between 2 and 10 injections) because of buffer depletion. In the present examples the buffer was replaced after six analyses. 10. When applying hydrodynamic injection, the actually injected amount of the sample may vary depending on the temperature or the viscosity of the solution. Thus, adjustment of the injection time and/or pressure may be required. In the present examples the samples were injected at ambient temperature. Typical injection plug length in CE corresponds to approx. 1–2% of capillary length. 11. The data sampling rate or data collection rate is a parameter that should be optimized during the method development and/or transfer. Ideally, the peak should have a Gaussian shape and should be defined by at least 20 data points. With an increased data sampling rate, the baseline noise will increase simultaneously. 12. The detection wavelength is typically set at the maximum absorbance of an analyte. The bandwidth is usually narrow (typically 5–10 nm) representing the number of diode responses, which are used to obtain a signal at the selected wavelength. In the presented examples, the signal is collected at 210  5 nm (Examples 1 and 2) and at 291  5 nm (Examples 3 and 4). The resulting sensitivity of the method can also differ between CE instruments of different generations or different manufacturers. 13. The reference wavelength compensates the fluctuations of lamp intensity as well as the changes in the absorption of background electrolyte. A reference wavelength should be chosen in the range with no or very low absorbance. The bandwidth is typically wide, i.e., 25–100 nm. Proper settings of the reference wavelength can improve the signal-to-noise ratio and reduce the drift of the baseline. When the reference wavelength range is set too close to the absorbance of the respective analytes, the sensitivity of the method can be significantly reduced. In the presented examples, the reference wavelength was set at 340  25 nm (Examples 1 and 2) and at 450  25 nm (Examples 3 and 4).

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References 1. Biwer A, Antranikian G, Heinzle E (2002) Enzymatic production of cyclodextrins. Appl Microbiol Biotechnol 59:609–617 2. Jin Z (2013) Cyclodextrin chemistry. World Scientific Publishing, Singapore 3. Bilensoy E (ed) (2011) Cyclodextrins in pharmaceutics, cosmetics and biomedicine. Current and future industrial applications. Wiley, Hoboken 4. Dodziuk H (ed) (2006) Cyclodextrins and their complexes: chemistry, analytical methods, applications. Wiley-VCH, Weinheim 5. Iacovino R, Caso JV, Di Donato C et al (2017) Cyclodextrins as complexing agents: preparation and applications. Curr Org Chem 21:162–176 6. Zhu Q, Scriba GKE (2016) Advances in the use of cyclodextrins as chiral selectors in capillary electrokinetic chromatography: fundamentals and applications. Chromatographia 79:1403–1435 7. Saz JM, Marina ML (2016) Recent advances on the use of cyclodextrins in the chiral analysis of drugs by capillary electrophoresis. J Chromatogr A 1467:79–94 8. Cucinotta V, Contino A, Giuffrida A et al (2010) Application of charged single isomer derivatives of cyclodextrins in capillary electrophoresis for chiral analysis. J Chromatogr A 1217:953–967 ˇ ezanka P, Navra´tilova´ K, R ˇ ezanka M et al 9. R (2014) Application of cyclodextrins in chiral capillary electrophoresis. Electrophoresis 35:2701–2721 10. Escuder-Gilabert L, Martı´n-Biosca Y, MedinaHerna´ndez MJ et al (2014) Cyclodextrins in capillary electrophoresis: recent developments and new trends. J Chromatogr A 1357:2–23 11. Zhou J, Tang J, Tang W (2015) Recent development of cationic cyclodextrins for chiral separation. Trends Anal Chem 65:22–29 12. Fanali S (2009) Chiral separations by CE employing CDs. Electrophoresis 30: S203–S210 13. Chankvetadze B (2009) Separation of enantiomers with charged chiral selectors in CE. Electrophoresis 30:S211–S221 14. Scriba GKE (2008) Cyclodextrins in capillary electrophoresis enantioseparations—recent developments and applications. J Sep Sci 31:1991–2011 15. Gu¨bitz G, Schmid MG (2010) Cyclodextrinmediated chiral separations. In: Van Eeckhaut A, Michotte Y (eds) Chiral

separations by capillary electrophoresis. Chromatogr Science Series, vol. 100. CRC Press, Boca Raton, pp 47–85 16. Chankvetadze B (2006) The application of cyclodextrins for enantioseparations. In: Dodziuk H (ed) Cyclodextrins and their complexes: chemistry, analytical methods, applications. Wiley-VCH, Weinheim, pp 119–146 17. Tang W, Ng SC, Sun D (2013) Modified cyclodextrins for chiral separation. Springer, New York 18. Sa´nchez-Lo´pez E, Marina ML, Crego AL (2016) Improving the sensitivity in chiral capillary electrophoresis. Electrophoresis 37:19–34 19. Ja´cˇ P, Scriba GKE (2013) Recent advances in electrodriven enantioseparations. J Sep Sci 36:52–74 20. Scriba GKE (2013) Differentiation of enantiomers by capillary electrophoresis. Top Curr Chem 340:209–276 21. Scriba GKE (2011) Fundamental aspects of chiral electromigration techniques and application in pharmaceutical and biomedical analysis. J Pharm Biomed Anal 55:688–701 22. Chankvetadze B (2007) Enantioseparations by using capillary electrophoretic techniques. The story of 20 and a few more years. J Chromatogr A 1168:45–70 23. Chankvetadze B (1997) Capillary electrophoresis in chiral analysis. Wiley, Chichester 24. Van Eeckhaut A, Michotte Y (eds) (2010) Chiral separations by capillary electrophoresis. Chromatogr Science Series, vol. 100. CRC Press, Boca Raton 25. Biedermann F, Nau WM, Schneider HJ (2014) The hydrophobic effect revisited—studies with supramolecular complexes imply high-energy water as noncovalent driving force. Angew Chem Int Ed 53:11158–11171 26. Schneider HJ (2009) Binding mechanisms in supramolecular complexes. Angew Chem Int Ed 48:3924–3977 27. Chankvetadze B (2002) Enantiomer migration order in chiral capillary electrophoresis. Electrophoresis 23:4022–4035 28. Hammitzsch-Wiedemann M, Scriba GKE (2009) Mathematical approach by a selectivity model for rationalization of pH- and selector concentration-dependent reversal of the enantiomer migration order in capillary electrophoresis. Anal Chem 81:8765–8773 29. Fillet M, Hubert P, Crommen J (2000) Enantiomeric separations of drugs using mixtures of

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charged and neutral cyclodextrins. J Chromatogr A 875:123–134 30. W€atzig H, Degenhardt M, Kunkel A (1998) Strategies for capillary electrophoresis. Method development and validation for pharmaceutical and biological applications. Electrophoresis 19:2695–2752 31. Rocheleau MJ (2005) Generic capillary electrophoresis conditions for chiral assay in early pharmaceutical development. Electrophoresis 26:2320–2329 32. Dubsky´ P, Svobodova´ J, Tesarˇova´ E, Gasˇ B (2010) Enhanced selectivity in CZE multichiral selector enantioseparation systems: proposed separation mechanism. Electrophoresis 31:1435–1441 33. Evans CE, Stalcup AM (2003) Comprehensive strategy for chiral separations using sulfated cyclodextrins in capillary electrophoresis. Chirality 15:709–723 34. Ates H, Mangelings D, Vander Heyden Y (2008) Fast generic chiral separation strategies using electrophoretic and liquid chromatographic techniques. J Pharm Biomed Anal 48:288–294 35. Zhou L, Thompson R, Song S et al (2002) A strategic approach to the development of capillary electrophoresis chiral methods for pharmaceutical basic compounds using sulfated cyclodextrins. J Pharm Biomed Anal 27:541–553 36. Williams BA, Vigh G (1997) Dry look at the CHARM (charged resolving agent migration) model of enantiomer separations by capillary electrophoresis. J Chromatogr A 777:295–309 37. Liu L, Nussbaum MA (1999) Systematic screening approach for chiral separations of basic compounds by capillary electrophoresis with modified cyclodextrins. J Pharm Biomed Anal 19:679–694

38. Jimidar MI, Van Ael W, Van Nyen P et al (2004) A screening strategy for the development of enantiomeric separation methods in capillary electrophoresis. Electrophoresis 25:2772–2785 39. Souverain S, Geiser L, Rudaz S, Veuthey JL (2006) Strategies for rapid chiral analysis by capillary electrophoresis. J Pharm Biomed Anal 40:235–241 40. Deeb SE, Hasemann P, W€atzig H (2008) Strategies in method development to quantify enantiomeric impurities using CE. Electrophoresis 29:3552–3562 41. Servais AC, Crommen J, Fillet M (2010) Factors influencing cyclodextrin-mediated chiral separations. In: van Eeckhaut A, Michotte Y (eds) Chiral separations by capillary electrophoresis. Chromatogr science series, vol. 100. CRC Press, Boca Raton, pp 87–107 42. Sentellas S, Saurina J (2003) Chemometrics in capillary electrophoresis. Part A: method for optimization. J Sep Sci 26:875–885 43. Dejaegher B, Mangelings D, Vander Heyden Y (2012) Experimental design methodologies in the optimization of chiral CE or CEC separations: an overview. Methods Mol Biol 970:409–427 44. Orlandini S, Gotti R, Furlanetto S (2014) Multivariate optimization of capillary electrophoresis methods: a critical review. J Pharm Biomed Anal 87:290–307 45. Orlandini S, Pinzauti S, Furlanetto S (2013) Application of quality by design to the development of analytical separation methods. Anal Bioanal Chem 405:443–450 46. Chankvetadze B, Schulte G, Blaschke G (1996) Reversal of enantiomer elution order in capillary electrophoresis using charged and neutral cyclodextrins. J Chromatogr A 732:183–187

Chapter 19 Application of Dual-Cyclodextrin Systems in Capillary Electrophoresis Enantioseparations Anne-Catherine Servais and Marianne Fillet Abstract The enantioseparation of acidic and neutral compounds can be successfully achieved in capillary electrophoresis (CE) using dual-cyclodextrin (CD) systems. This chapter describes how to separate the enantiomers of acidic or neutral substances using dual-CD systems made up of a negatively charged CD derivative, i.e., sulfobutyl-β-CD or carboxymethyl-β-CD, in combination with a neutral one, namely heptakis(2,3,6tri-O-methyl)-β-CD. An acidic compound (carprofen) and a weakly acidic drug (pentobarbital) were selected as model compounds. Key words Capillary electrophoresis, Dual-cyclodextrin system, Acidic compounds, Neutral compounds

1

Introduction Efficient stereoselective separation is of utmost importance for comparative pharmacodynamic and pharmacokinetic studies on each enantiomer, toxicological investigations, as well as quality control of chiral drugs. Liquid chromatography, capillary electrophoresis (CE), and more recently supercritical fluid chromatography are the most widely used techniques for chiral separations of nonvolatile compounds. The success of CE for enantioseparations comes from its very high efficiency, low reagent consumption, and high flexibility [1]. In most CE enantioseparations, cyclodextrins (CDs) are added into the background electrolyte (BGE) [1–3]. As a complete enantioseparation cannot always be achieved using a single CD (especially for uncharged analytes), dual-CD systems were developed to increase both selectivity and resolution. These systems are often favorable due to the differences in the complexation mechanisms of the two CDs with the enantiomers (i.e., complexation stability, chiral recognition pattern, and influence on analyte mobility) [4–6]. Overviews of dual-CD systems can be found in specialized

Gerhard K. E. Scriba (ed.), Chiral Separations: Methods and Protocols, Methods in Molecular Biology, vol. 1985, https://doi.org/10.1007/978-1-4939-9438-0_19, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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review articles [7, 8] and in a book chapter [9]. Several types of combinations can be used: a neutral with a charged CD [6, 10–12], two charged CD derivatives [12], or two neutral CDs [13]. In the case of a separation only based on complexation constant differences (KR 6¼ KS), an enantiomeric resolution can be observed provided that the mobilities of the free and complexed forms of the analytes are different (μf 6¼ μc), as described in the following equation [14, 15]: Δμ ¼

ðμ f  μc ÞðK R  K S Þ½C  1 þ ðK R þ K S Þ½C  þ K R K S ½C 2

where [C] is the concentration of the chiral selector. In dual-CD systems, both chiral selectors can act in a synergistic way as well as counteract each other. Crommen and coworkers developed mathematical models in order to predict the enantioselectivity in systems containing two CDs and therefore to rationalize the optimization of such systems in terms of resolution and migration times [8, 16, 17]. It is worth noting that these equations are valid provided that only 1:1 complexation occurs and that the two CDs lead to independent complexation (no mixed complexes). In addition, both chiral selectors, mostly CD derivatives, are assumed to be pure, well-characterized compounds, which is rarely the case in practice. The model presented in ref. 8 is more general than previous ones since it is valid for charged and neutral analyte enantiomers and for ionic and uncharged CDs. The development of a dual-separation system may include the optimization of the affinity pattern via the selection of the suitable CD, or the mobility terms by choosing the appropriate concentration of an anionic, cationic, or neutral CD. A selectivity improvement in dual-CD systems can be obtained if one CD accelerates the analyte and the other one decelerates it or has no influence on its mobility and if the affinity pattern of the enantiomers for each CD is opposite. It is worth noting that multiselector models should be used when dealing with CD derivatives that are not single isomers but mixtures of CDs with different degrees of substitution and various positions of substituents. Multiselector models as well as particular features of mixtures of selectors can be found in a review article [18]. The dual systems proposed in this chapter, consisting of a highly selective neutral CD, namely heptakis(2,3,6-tri-Omethyl)-β-CD (TM-β-CD) in combination with a nonselective or poorly selective negatively charged CD, i.e., sulfobutyl-β-CD (SB-β-CD) or carboxymethyl-β-CD (CM-β-CD), are especially useful for neutral compounds or ionizable analytes present in uncharged form [6, 10, 11, 19].

Dual CD systems

2 2.1

359

Materials Equipment

1. CE system equipped with a UV detector and a temperature control system (15–60  C  0.1  C): A HP3D CE System (Agilent Technologies, Waldbronn, Germany) is suitable. 2. Uncoated 50 μm i.d. fused silica capillaries (e.g., from Polymicro Technologies, Phoenix, AZ, USA) with a total length of 44 cm and an effective length of 37 cm to the detector. 3. Cellulose-based membrane filters (0.2 μm). 4. A commercial pH meter for pH adjustment.

2.2 Background Electrolytes

Prepare all solutions using Milli-Q water and analytical grade reagents. Filter them through a cellulose-based membrane filter (0.2 μm) before use. 1. BGE 1: 100 mM Phosphoric acid adjusted to pH 3 with triethanolamine. To prepare 100 mL of BGE 1, introduce 80 mL of water and 1.15 g of phosphoric acid (85%) in a volumetric flask of 100.0 mL, bring to volume with water, and mix. Adjust this solution to pH 3 with triethanolamine. 2. BGE-CD 1: 5 mM SB-β-CD (e.g., from CyDex Pharmaceuticals, Lenexa, KS, USA, or Cyclolab, Budapest, Hungary) and 15 mM TM-β-CD (e.g., Cyclolab, Budapest, Hungary, or Sigma-Aldrich, Saint-Louis, MO, USA) in BGE 1 (see Notes 1, 2, and 3). To prepare 10 mL of BGE-CD 1, introduce 88 mg of SB-β-CD (having an average degree of substitution of 4) and 214 mg of TM-β-CD in a volumetric flask of 10.0 mL. Dissolve and bring to volume with BGE 1. 3. BGE 2: 100 mM Phosphoric acid adjusted to pH 5 with triethanolamine. To prepare 100 mL of BGE 2, introduce 80 mL of water and 1.15 g of phosphoric acid (85%) in a volumetric flask of 100.0 mL, bring to volume with water, and mix. Adjust this solution to pH 5 with triethanolamine. 4. BGE-CD 2: 10 mM CM-β-CD (e.g., from Cyclolab, Budapest, Hungary) and 50 mM TM-β-CD in BGE 2 (see Notes 5–8). To prepare 10 mL of BGE-CD 2, introduce 142 mg of CM-β-CD (having an average degree of substitution of 3.5) and 715 mg of TM-β-CD in a volumetric flask of 10.0 mL. Dissolve and bring to volume with BGE 1.

2.3

Sample Solutions

1. Carprofen sample solution (50 μM): Prepare a solution at a concentration of about 14 μg/mL in a mixture of water:methanol (9:1) (see Note 4). 2. Pentobarbital sample solution (50 μM): Prepare a solution at a concentration of about 11 μg/mL in a mixture of water:methanol (7:3) (see Note 4).

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Methods 1. At the beginning of the working day, wash the capillary with 1 M NaOH, 0.1 M NaOH, water, and then BGE 1, each for 10 min at a pressure of approximately 1 bar.

3.1 Example 1: Enantioseparation of Acidic Drugs

2. Before each injection wash the capillary with BGE-CD 1 for 3 min at a pressure of approximately 1 bar. 3. Inject the sample solution hydrodynamically by applying a pressure of 50 mbar for 5 s. 4. Separate the enantiomers of acidic analytes using the following parameters (see Note 9): – Applied voltage: 25 kV (negative polarity). – Capillary temperature: 25  C. – UV detection at 230 nm for carprofen. A typical electropherogram of the enantioseparation of carprofen is shown in Fig. 1. 5. At the end of each working day wash the capillary with water for 10 min.

0,0025

0,0020

mAU 0,0015

0,0010

0,0005

0,0000 0

2

4

6

8

min.

10

12

14

16

Fig. 1 Enantioseparation of carprofen in dual-CD system. BGE-CD: 5 mM SB-β-CD and 15 mM TM-β-CD in 100 mM phosphoric acid-triethanolamine (pH 3) (reproduced by permission of John Wiley & Sons from ref. 11 © 1997)

Dual CD systems 0,004

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pH 5 pH 3

0,003

mAu 0,002

b 0,001

a 0,000

2

4

6

8

10

12

14

16

18

min.

Fig. 2 Enantioseparation of pentobarbital in dual-CD systems. BGE-CD: (a) 5 mM SB-β-CD and 30 mM TM-β-CD in 100 mM phosphoric acid-triethanolamine (pH 3) or (b) 10 mM CM-β-CD and 50 mM TM-β-CD in 100 mM phosphoric acid-triethanolamine (pH 5) (reproduced by permission of Elsevier from ref. [6] © 1998) 3.2 Example 2: Enantioseparation of Weakly Acidic or Neutral Drugs

1. At the beginning of the working day, wash the capillary with 1 M NaOH, 0.1 M NaOH, water, and then BGE 2, each for 10 min at a pressure of approximately 1 bar. 2. Before each injection wash the capillary with BGE-CD 2 for 3 min at a pressure of approximately 1 bar. 3. Inject the sample solution hydrodynamically by applying a pressure of 50 mbar for 5 s. 4. Separate the enantiomers of weakly acidic or neutral analytes using the following parameters (see Note 9): – Applied voltage: 25 kV (negative polarity). – Capillary temperature: 25  C. – UV detection at 210 nm for pentobarbital. Enantioseparations of pentobarbital are displayed in Fig. 2. 5. At the end of each working day wash the capillary with water for 10 min.

4

Notes 1. SB-β-CD used in the described experiments possesses an average degree of substitution of four sulfobutyl groups. As a result, this CD exhibits a strong negative charge at any

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commonly used pH in CE. Various SB-β-CD qualities differing in the degree of substitution are commercially available. All of them are able to separate enantiomers but SB-β-CDs with different degrees of substitution may provide different migration times due to the different charges and, more importantly, enantioseparations of the same analyte may differ considerably depending on the degree of substitution of the CD. Examples have been reported where SB-β-CD with a certain degree of substitution could resolve the enantiomers of a given drug while it was not possible to separate the analyte enantiomers with another SB-β-CD possessing a different degree of substitution. Single-isomer, well-characterized CD derivatives are always recommended. 2. No enantioseparation or very poor resolution can be observed for most acidic drugs when they are in the deprotonated anionic form [10, 11]. At pH 3, carprofen (pKa: 4.3) is mainly present in the uncharged form. As the phosphoric acid/triethanolamine BGE results in a low anodic EOF due to the coating of the capillary with triethanolamine [10], carprofen migrates to the anodic side of the capillary. The addition of a neutral CD alone cannot lead to a chiral separation as the electrophoretic mobilities of the free and the complexed forms of the analytes are not significantly different. It is worth noting that no chiral separation could be observed when SB-β-CD alone was added to the BGE, although the migration time of carprofen decreased due to analyte interaction with SB-β-CD [11]. In the proposed dual system, the neutral CD derivative provides enantioselectivity and the negatively charged CD derivative acts as a carrier. 3. After optimization, 15 mM TM-β-CD was found to lead to the maximum resolution for the carprofen enantiomers. For the optimization of a chiral separation of acidic compounds the concentration of the neutral CD has to be optimized in the 10–50 mM range. Regarding the anionic CD derivative, it was demonstrated that the concentration of SB-β-CD has only a limited impact on the enantioseparation and 5 mM SB-β-CD was found to be optimal in terms of analysis time [10, 11]. 4. This dissolution medium for the analytes was found to be optimal in terms of analyte stacking. 5. CM-β-CD used in these experiments has an average degree of substitution of 3.5. The dissociation of the carboxylic acid groups of the CD depends on the pH of the BGE. 6. At pH 5, very weakly acidic analytes are still neutral. As can be seen in Fig. 2, the use of a dual-CD system at pH 5 instead of pH 3 increases the enantioresolution of very weakly acidic

Dual CD systems

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drugs such as pentobarbital. This can be explained by the significant increase of the negative charge of CM-β-CD in this pH range leading to a higher mobility difference between the free and complexed forms of analyte enantiomers at pH 5. 7. After optimization, 50 mM TM-β-CD concentration was found to lead to the maximum resolution for the pentobarbital enantiomers. The neutral CD concentration resulting in the highest enantioresolution value for your compound of interest has to be optimized in the 10–50 mM range. Regarding the anionic CD derivative, it was demonstrated that the concentration of CM-β-CD has only a limited impact on the enantioseparation and 10 mM CM-β-CD was found to be optimal in terms of analysis time [10, 11]. 8. In some cases, a higher enantioseparation can be obtained by replacing TM-β-CD by heptakis(2,6-di-O-methyl)-β-CD (Cyclolab, Budapest, Hungary) in the dual-CD system [6]. 9. The CD-containing BGE, i.e., BGE-CD 1 or BGE-CD 2, has to be renewed every 60 min of analysis time in order to avoid the depletion which impairs method performance and robustness. References 1. Jac P, Scriba GK (2013) Recent advances in electrodriven enantioseparations. J Sep Sci 36 (1):52–74 2. Chankvetadze B, Fillet M, Burjanadze N, Bergenthal D, Bergander C, Luftmann H, Crommen J, Blaschke G (2000) Enantioseparation of aminoglutethimide with cyclodextrins in capillary electrophoresis and studies of selector-selectand interactions using NMR spectroscopy and electrospray ionization mass spectrometry. Enantiomer 5(3–4):313–322 3. Servais AC, Rousseau A, Fillet M, Lomsadze K, Salgado A, Crommen J, Chankvetadze B (2010) Capillary electrophoretic and nuclear magnetic resonance studies on the opposite affinity pattern of propranolol enantiomers towards various cyclodextrins. J Sep Sci 33 (11):1617–1624 4. Lurie IS, Klein RFX, Dalcason TA, Lebelle MJ, Brenneisen R, Weinberger RE (1994) Chiral resolution of cationic drugs of forensic interest by capillary electrophoresis with mixtures of neutral and anionic cyclodextrins. Anal Chem 66(22):4019–4026 5. Lelievre F, Gareil P, Bahaddi Y, Galons H (1997) Intrinsic selectivity in capillary electrophoresis for chiral separations with dual cyclodextrin systems. Anal Chem 69(3):393–401

6. Fillet M, Fotsing L, Crommen J (1998) Enantioseparation of uncharged compounds by capillary electrophoresis using mixtures of anionic and neutral beta-cyclodextrin derivatives. J Chromatogr A 817(1–2):113–119 7. Lurie IS (1997) Separation selectivity in chiral and achiral capillary electrophoresis with mixed cyclodextrins. J Chromatogr A 792 (1–2):297–307 8. Fillet M, Hubert P, Crommen J (2000) Enantiomeric separations of drugs using mixtures of charged and neutral cyclodextrins. J Chromatogr A 875(1–2):123–134 9. Servais AC, Crommen J, Fillet M (2009) Factors influencing cyclodextrin-mediated chiral separations. In: Van Eeckhaut A, Michotte Y (eds) Chiral separations by capillary electrophoresis, Chromatographic sciences, vol 100. CRC Press, pp 87–107 10. Fillet M, Bechet I, Schomburg G, Hubert P, Crommen J (1996) Enantiomeric separation of acidic drugs by capillary electrophoresis using a combination of charged and uncharged betacyclodextrins as chiral selectors. HRC—J High Res Chrom 19(12):669–673 11. Fillet M, Hubert P, Crommen J (1997) Enantioseparation of nonsteroidal antiinflammatory drugs by capillary electrophoresis

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using mixtures of anionic and uncharged betacyclodextrins as chiral additives. Electrophoresis 18(6):1013–1018 12. Delplanques T, Boulahjar R, Charton J, Houze C, Howsam M, Servais AC, Fillet M, Lipka E (2017) Single and dual cyclodextrins systems for the enantiomeric and diastereoisomeric separations of structurally related dihydropyridone analogues. Electrophoresis 38 (15):1922–1931 13. Novakova Z, Pejchal V, Fischer J, Cesla P (2017) Chiral separation of benzothiazole derivatives of amino acids using capillary zone electrophoresis. J Sep Sci 40(3):798–803 14. Chankvetadze B (1997) Separation selectivity in chiral capillary electrophoresis with charged selectors. J Chromatogr A 792(1–2):269–295 15. Chankvetadze B, Lindner W, Scriba GKE (2004) Enantionmer separations in capillary electrophoresis in the case of equal binding constants of the enantiomers with a chiral selector: commentary on the feasibility of the concept. Anal Chem 76(14):4256–4260

16. Abushoffa AM, Fillet M, Hubert P, Crommen J (2002) Prediction of selectivity for enantiomeric separations of uncharged compounds by capillary electrophoresis involving dual cyclodextrin systems. J Chromatogr A 948 (1–2):321–329 17. Abushoffa AM, Fillet M, Servais AC, Hubert P, Crommen J (2003) Enhancement of selectivity and resolution in the enantioseparation of uncharged compounds using mixtures of oppositely charged cyclodextrins in capillary electrophoresis. Electrophoresis 24 (3):343–350 18. Mullerova L, Dubsky P, Gas B (2014) Twenty years of development of dual and multi-selector models in capillary electrophoresis: a review. Electrophoresis 35(19):2688–2700 19. Crommen J, Fillet M, Hubert P (1998) Method development strategies for the enantioseparation of drugs by capillary electrophoresis using cyclodextrins as chiral additives. Electrophoresis 19(16–17):2834–2840

Chapter 20 Enantioseparation by Capillary Electrophoresis Using Cyclodextrins in an Amino Acid-Based Ionic Liquid Running Buffer Joachim Wahl and Ulrike Holzgrabe Abstract For enantioseparations of chiral drugs in capillary electrophoresis, chiral ionic liquids (CIL) can be employed instead of traditional running buffer containing a chiral selector. CILs can be applied solely or in addition to the often used cyclodextrin derivatives. Here the separation of phenethylamines, especially of ephedrine, is described using tetrabutylammonium L-argininate (125 mM) in phosphate buffer (75 mM, pH 1.5) in addition to β-cyclodextrin (30 mM). Using this dual-chiral running buffer system ephedrine, pseudoephedrine and methylephedrine, but not norephedrine, could be easily resolved. Key words Amino acid-based chiral ionic liquids, Cyclodextrins, Phenethylamines, Capillary electrophoresis

1

Introduction In capillary zone electrophoresis, ionic analytes are separated in open tubular capillaries due to their differential migration which is caused by their respective charge-to-size/mass ratios. In order to move an ionic analyte through the capillary, high voltage and a running buffer (RB) are needed. Often used RBs are phosphate, borate, acetate, and formate buffer of different molarity and pH [1]. More recently, ionic liquids (ILs) have been applied in CE [2]. ILs are solvents composed of an organic or inorganic anion and an organic cation and are characterized by a melting point mostly lower than 100
C which is caused by the large size of the ions. Typical cations are tetraalkylammonium and -phosphonium as well as alkylated imidazolinium, pyridinium, piperidinium, and pyrrolidinium cations, and often used anions are tetrafluoroborate, hexafluorophosphate, bis(trifluoromethylsulfonyl)imide, and trifluoromethanesulfonate. ILs can be used as a sole BGE or in combination with the traditional buffers mentioned above. The

Gerhard K. E. Scriba (ed.), Chiral Separations: Methods and Protocols, Methods in Molecular Biology, vol. 1985, https://doi.org/10.1007/978-1-4939-9438-0_20, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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complexity of those systems, especially the influence of IL molarity, pH, and alkyl chain length of the IL on the electroosmotic flow (EOF), has extensively been discussed by Wahl and Holzgrabe [2]. ILs also play a role in the enantioseparation of chiral substances. Either the IL can be chiral, i.e., a chiral ionic liquid (CIL), such as amino acid-based CILs, or a chiral selector can be added to the RB, e.g., a cyclodextrin (CD) derivative. A combination of both may also be applied. Such systems have been reviewed by KapnissiChristodoulou et al. [3] and Greno et al. [4]. Dual-separation systems, consisting of CILs and CDs, might result in an even better enantioseparation than obtained by a sole chiral selector. At present, it is difficult to predict a resolution of enantiomers because a plethora of interactions between analytes and chiral selectors can take place. The enantiomeric analytes form diastereomeric complexes with the CDs, but the ILs may also complex with the CDs as well as (C)IL-analyte complexes may interact with the CD. In addition, CILs can form diastereomeric complexes with the chiral analytes and, last but not least, the CILs may interact either with the analyte-CD or with the analyte-CIL-CD complex. All these chiral recognition processes may synergistically add to the enantioseparation, but not necessarily. They may also neutralize or even thwart a resolution. Over the past years, we have systematically studied the enantioand diastereoseparation of phenethylamine derivatives, i.e., ephedrine, pseudoephedrine, norephedrine, and methylephedrine, by means of CD-modified CE using neutral and negatively charged CDs [5, 6], of sulfated CD-modified microemulsion electrokinetic chromatography [7], and by the application of the IL tetrabutylammonium chloride (TBAC) in combination with neutral CDs [8] as well as the amino acid-based CILs with β-CD. The optimization of the latter system, especially with regard to the TBA-amino acid IL, can be found in [9]. The best resolutions were obtained when using basic amino acids, especially arginine, in the dual-RB system. A comparison of the data of all separation systems is summarized in Table 1. The only RB system which is able to separate the enantiomers of all phenethylamines and all compounds from each other is the negatively charged heptakis(2,3-di-O-acetyl-6-O-sulfo)-β-CD (HDAS-β-CD) [5, 6, 10, 11]. In all other systems it is difficult to resolve the ephedrine, methylephedrine, and norephedrine enantiomers. In contrast, the pseudoephedrine isomers can be baseline separated with every system. The enhancement of chiral separations of phenethylamines was already shown using TBA+-based ILs combined with various cyclodextrins as background electrolyte additive [2, 8], but an even better enantioseparation of ephedrine and the methylephedrine enantiomers was achieved by adding the amino acid-based salt tetrabutylammonium L-argininate (TBA-L-Arg) to a 75 mmol/L

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Table 1 Comparison of the enantioseparations revealed with the different RB separation systems Phosphate buffer (50 mM pH 3.0)a

Phosphate buffer (75 mM pH 2.5), 125 mM TBACb 35 mM β-CD

50 mM HP-β-CD

60 mM Me-β-CD

Phosphate buffer (75 mM pH 2.5), 125 mM TBA-L-Argc 30 mM β-CD

12 mM Phenethyl amines β-CD

12 mM HDAS

Ephedrine

1.3

16.9

3.3

2.7

1.3

3.6

Pseudoephedrine

3.1

12.6

11.0

17.5

19.4

12.2

Methylephedrine

0.9

10.0

2.7

2.6

1.8

3.3

Norephedrine

–

4.7

1.0

100,000). 5. Samples from commercial vendors can be used. Certified standards of tioconazole, isoconazole nitrate, and fenticonazole nitrate can be obtained from the European Directorate for the Quality of Medicines and Healthcare (EDQM), Strasbourg, France. 6. To identify any system peak, preferably start run with sample solvent (methanol). 7. Flushing the capillary between consecutive run is crucial to avoid carryover and to ensure reproducible inner wall condition. If changing buffer shifts the migration time, extend the conditioning time to 10–15 min. 8. It is suggested to run individual enantiomers in triplicates in order to identify their migration order before performing simultaneous enantioseparation.

Acknowledgments This work was supported by the Fundamental Research Grant Scheme from the Ministry of Higher Education (Malaysia) under vote number R.J130000.7826.3F262 (78314) and the National Science Foundation awarded by the Ministry of Science, Technology and Innovation (Malaysia) to S.M. Abdul Wahib is much appreciated. References 1. Saz JM, Marina ML (2016) Recent advances in the use of cyclodextrins in the chiral analysis of drugs by capillary electrophoresis. J Chromatogr A 1467:79–94 2. Rezanka P, Navratilova K, Rezanka M, Kral V, Sykora D (2014) Application of cyclodextrins in chiral capillary electrophoresis. Electrophoresis 35:2701–2721 3. Zhu Q, Scriba GKE (2016) Advances in the use of cyclodextrins as chiral selectors in capillary electrokinetic chromatography: fundamentals and applications. Chromatographia 79:1403–1435 4. Huang L, Lin J, Xu L, Chen G (2007) Non aqueous and aqueous-organic media for the enantiomeric separations of neutral organophosphorus pesticides by CE. Electrophoresis 28:2758–2764 5. Li W, Zhao L, Zhang H, Chen X, Chen S, Zhu Z, Hong Z, Chai Y (2014) Enantioseparation of new triadimenol antifungal active

compounds by electrokinetic chromatography and molecular modeling study of chiral recognition mechanisms. Electrophoresis 35:2855–2862 6. Terabe S, Otsuka K, Ichikawa K, Tsuchiya A, Ando T (1984) Electrokinetic separations with micellar solutions and open-tubular capillaries. Anal Chem 56:111–113 7. Terabe S (2008) Micellar electrokinetic chromatography for high-performance analytical separation. Chem Rec 8:291–301. The Japan Chemical Journal Forum and Wiley Periodicals, Inc. 8. Hu S-Q, Guo X-M, Shi H-J, Luo R-J (2015) Separation mechanisms for palonosetron stereoisomers at different chiral selector concentrations in MEKC. Electrophoresis 36:825–829 9. Van Zomeren PV, Hilhorst MJ, Coenegracht PMJ, De Jong GJ (2000) Resolution optimization in micellar electrokinetic chromatography

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using empirical models. J Chromatogr A 867:247–259 10. Deeb SE, Iriban MA, Gust R (2011) MEKC as a powerful growing analytical technique. Electrophoresis 32:166–183 11. Lin X, Hou W, Zhou C (2003) Enantiomer separation of miconazole by capillary electrophoresis with dual cyclodextrin systems. Anal Sci 19:1509–1512 ˇ esla P, Blomberg L, Hamberg M, Jandera P 12. C (2006) Characterization of anacardic acids by micellar electrokinetic chromatography and mass spectrometry. J Chromatogr A 1115:253–259 13. Wan Ibrahim WA, Warno SA, Aboul-Enein HY, Hermawan D, Sanagi MM (2009) Simultaneous enantioseparation of cyproconazole, bromuconazole, and diniconazole enantiomers by CD-modified MEKC. Electrophoresis 30:1976–1982 ˜a-Traverso J, 14. Mene´ndez-Lo´pez N, Valiman Castro-Puyana M, Salgado A, Garcı´a MA, Marina ML (2017) Enantiomeric separation of the antiuremic drug colchicine by electrokinetic chromatography: method development and quantitative analysis. J Pharm Biomed Anal 138:189–196 15. Hu S-Q, Wang G-X, Guo W-B, Guo X-M, Zhao M (2014) Effect of low concentration sodium dodecyl sulfate on the electromigration of palonosetron hydrochloride stereoisomers in micellar electrokinetic chromatography. J Chromatogr A 1342:86–91 16. Ibrahim WAW, Arsad SR, Maarof H, Sanagi MM, Aboul-Enein HY (2015) Chiral separation of four stereoisomers of ketoconazole drugs using capillary electrophoresis. Chirality 27:223–227 17. Pe´rez-Ferna´ndez V, Garcı´a MA, Marina ML (2010) Enantiomeric separation of cis-bifenthrin by CD-MEKC: quantitative analysis in a commercial insecticides formulation. Electrophoresis 31:1533–1539 18. Yu T, Du Y, Chen B (2011) Evaluation of clarithromycin lactobionate as a novel chiral selector for enantiomeric separation of basic drugs in capillary electrophoresis. Electrophoresis 32:1898–1905 ´ , Mene´ndez-Lo´pez N, Boltes K, 19. Garcı´a MA Castro-Puyana M, Marina ML (2017) A capillary micellar electrokinetic chromatography method for the stereoselective quantitation of bioallethrin in biotic and abiotic samples. J Chromatogr A 1510:108–116 20. Kodama S, Nakajima S, Ozaki H, Takemoto R, Itabashi Y, Kuksis A (2016) Enantioseparation of hydroxyeicosatetraenoic acids by

hydroxypropylγ-cyclodextrin-modified micellar electrokinetic chromatography. Electrophoresis 37:3196–3205 21. Orlandini S, Pasquini B, Caprini C, Del Bubba M, Dousˇa M, Pinzauti S (2016) Enantioseparation and impurity determination of ambrisentan using cyclodextrin-modified micellar electrokinetic chromatography: visualizing the design space within quality by design framework. J Chromatogr A 1467:363–371 22. Mikuma T, Iwata YT, Miyaguchi H, Kuwayama K, Tsujikawa K, Kanamori T, Kana H, Inoue H (2016) Approaching over 10 000-fold sensitivity increase in chiral capillary electrophoresis: cation-selective exhaustive injection and sweeping cyclodextrin-modified micellar electrokinetic chromatography. Electrophoresis 37:2970–2976 23. Lin E-P, Lin K-C, Chang C-W, Hsieh M-M (2013) On-line sample preconcentration by sweeping and poly(ethylene oxide)-mediated stacking for simultaneous analysis of nine pairs of amino acid enantiomers in capillary electrophoresis. Talanta 114:297–303 24. Petr J, Ginterova P, Znaleziona J, Knob R, Losˇta´kova´ M, Maier V, Sˇevcˇik J (2013) Separation of ketoprofen enantiomers at nanomolar concentration levels by micellar electrokinetic chromatography with on-line electrokinetic preconcentration. Cent Eur J Chem 11:335–340 25. Cheng H, Zhang Q, Tu Y (2012) Separation of fat-soluble isoquinoline enantiomers using β-cyclodextrin modified micellar capillary electrokinetic chromatography. Curr Pharm Anal 8:37–43 26. Ibrahim WAW, Wahib SMA, Hermawan D, Sanagi MM, Aboul-Enein HY (2012) Chiral separation of vinpocetine using cyclodextrinmodified micellar electrokinetic chromatography. Chirality 24:252–254 27. Hermawan D, Wan Ibrahim WA, Sanagi MM, Aboul-Enein HY (2010) Chiral separation of econazole using micellar electrokinetic chromatography with hydroxypropyl-γ-cyclodextrin. J Pharm Biomed Anal 53:1244–1249 28. Wan Ibrahim WA, Hermawan D, Sanagi MM, Aboul-Enein HY (2010) Stacking and sweeping in cyclodextrin-modified MEKC for chiral separation of hexaconazole, penconazole, myclobutanil. Chromatographia 71:305–309 29. Wan Ibrahim WA, Wahib SMA, Hermawan D, Sanagi MM, Aboul-Enein HY (2013) Separation of selected imidazole enantiomers using dual cyclodextrin system. Chirality 25:328–335

Chapter 25 Carbohydrate-Based Polymeric Surfactants for Chiral Micellar Electrokinetic Chromatography (CMEKC) Coupled to Mass Spectrometry Vijay Patel and Shahab A. Shamsi Abstract Polymeric surfactants (molecular micelles, MoMs) with a variety of chiral head groups and chain lengths may be the most promising chiral selectors when used for sensitive detection of chiral compounds in micellar electrokinetic chromatography-mass spectrometry (MEKC-MS). Various carbohydrate-based MS compatible surfactants with phosphate and sulfate head groups have been recently synthesized in our laboratory for its application in CMEKC-MS. In this chapter, we illustrate that the synthesized glucopyranoside-based MoMs are fully compatible with electrospray ionization MS and can be successfully used as a chiral selector for high-throughput screening of multiple chiral compounds using MRM mode in CMEKC-MS/MS experiments. This chapter describes in detail synthesis and utility of α- and β-glucopyranoside-based polymeric surfactant with two different chain lengths and head groups. The presented examples optimize the effect of appropriate millimolar concentration of monomer sugar surfactants required for polymerization as it affects the separations of acidic and basic compounds. Under the optimized concentration of the monomer needed for polymerization (i.e., an equivalent monomer concentration of MoMs), the superiority of MEKC-MS over MEKC-UV is evident. Structurally similar basic drugs with the difference in hydrophobicity are first tested in MEKC-MS to find the optimum head group and optimum chain length with the aim for developing a widely applicable polymeric glucopyranoside-based surfactant. The partial enantioresolution of several structurally similar basic compounds is significantly improved when switching from one head group to another head group of the glucopyranoside MoMs. Thus, complementary separations using poly-N-β-D-SUGP versus poly-N-β-D-SUGS were seen. This phenomenon also exists when comparing the MoMs, which differ in an anomeric configuration such as poly-N-α-D-SUGP and poly-N-β-D-SUGP. Key words Capillary electrophoresis, Chiral micellar electrokinetic chromatography (CMEKC-MS), Polymeric glucopyranoside surfactants, Molecular micelles (MoMs), Poly-N-α-D-SUGP, Poly-N-α-DSUGS, Poly-N-β-D-SUGP, Poly-N-β-D-SUGS

1

Introduction Polymeric surfactants (molecular micelles, MoMs) prepared from amino acid-based surfactants and peptide-based surfactants having anionic head groups are abundantly applied to the separation of enantiomers of racemic compounds containing positively

Gerhard K. E. Scriba (ed.), Chiral Separations: Methods and Protocols, Methods in Molecular Biology, vol. 1985, https://doi.org/10.1007/978-1-4939-9438-0_25, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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charged secondary or tertiary amino group [1–5]. In addition, enantioseparations of neutral, negatively and positively charged atropisomers [6, 7] and derivatized amino acids [8, 9] can be achieved by micellar electrokinetic chromatography (MEKC). Recently, several classes of cationic [10] and anionic drugs [11] along with their metabolites are simultaneously enantioseparated using polymeric amino acid and dipeptide surfactants by MEKCmass spectrometry (MS) by Shamsi and coworkers. Thus, the continuing development of new and improved polymeric chiral surfactants with exceptional discrimination abilities is needed for the development of both fast and sensitive MEKC-MS. A new generation of polymeric surfactants, based on two sugar types, N-alkenyl α-D- and β-D-glucopyranoside, was recently developed in our laboratory (Fig.1). Several key advantages of this new generation of glucopyranoside-based polymeric surfactants are noted: (a) multiple stereogenic centers favor enantioseparation of diverse group of analytes; (b) compatibility with electrospray ionization mass spectrometry (ESI-MS) and significantly lower ion-suppression compared to unpolymerizable monomers; (c) zero critical micelle concentration (CMC) allowing its use at significantly lower concentration resulting in lower operating current for CE-MS; (d) environmentally friendly and biodegradable; (e) separation of multichiral center compounds using only a single chiral selector is possible. In particular, the advantages (b) and (c) noted above enables this class of polymeric surfactants to be fully compatible with ESI-MS. In this chapter, we describe in detail the preparation and application of N-alkenyl α-D- and β-D-glucopyranoside with different chain lengths and head groups as chiral selectors for MEKCMS/MS. The first type of glucose based, i.e., polymeric α-D-glucopyranoside-based surfactants with different chain lengths and head groups is successfully synthesized and characterized as chiral selectors in MEKC-MS/MS [12]. First, the effect of polymerization concentration of the monomer surfactant, N-undecenyl-α-Dglucopyranoside-4,6-hydrogen phosphate sodium salt (α-DSUGP), was evaluated by enantioseparation of an anionic compound (1,10 -binaphthyl-2,20 -diyl-hydrogen phosphate, BNP) and a zwitterionic compound (dansylated phenylalanine, Dns-Phe) by MEKC-UV to find the optimum molar concentration for polymerization. Next, MEKC-UV and MEKC-MS were compared for the enantioseparation of BNP. The influence of polymeric glucopyranoside-based surfactant containing phosphate and sulfate head groups as well as C8 and C11 chain lengths on chiral resolution was evaluated for two classes of cationic drugs (ephedrine alkaloids and β-blockers). Finally, enantioselective MEKC-MS of ephedrine alkaloids and β-blockers were profiled at their optimum pH 5.0 and 7.0, respectively using ammonium acetate buffer and optimum polymeric N-α-D-glucopyranoside surfactant. The LOD for most

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Fig. 1 Chemical structures of sugar-based polymeric surfactants. (a) Polysodium N-undecenyl-β-D-glucopyranoside-4,6-hydrogen phosphate (poly-β-D-SUGP); (b) polysodium N-undecenyl-α-D-glucopyranoside-4,6hydrogen phosphate (poly-α-D-SUGP); (c) polysodium N-octenyl-β-D-glucopyranoside-4,6-hydrogen phosphate (poly-β-D-SOGP); (d) polysodium N-octenyl-α-D-glucopyranoside-4,6-hydrogen phosphate (poly-α-D-SOGP); (e) polysodium N-undecenyl-β-D-glucopyranoside-6-hydrogen sulfate (poly-β-D-SUGS); (f) polysodium N-undecenyl-α-D-glucopyranoside-6-hydrogen sulfate (poly-α-D-SOGS); (g) polysodium Noctenyl-β-D-SOGS; (h) polysodium N-octenyl-α-D-SOGS

of the enantiomers ranges from 10 to 100 ng/mL with S/N of at least 3.0. In a recent study, the second type, i.e., polymeric β-D-glucopyranoside-based surfactants with phosphate and sulfate head groups

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containing C8 and C11 hydrocarbon tails was compared at the optimum pH 5.0 for MEKC-MS/MS [13]. It has been shown that the selectivity of poly-β-D-SUGS and poly-β-D-SUGP is complementary to one another. Therefore, if a chiral compound is not separated using one polymeric glucopyranoside, it has a high probability of being enantioresolved using the other glucopyranoside surfactant. In addition, the enantioresolution of two isomeric surfactants, which differ in the anomeric orientation (containing phosphate or sulfate head group), shows that the β-form of poly-DSUGS or poly-D-SUGP is a preferred orientation with a high success rate for enantioseparation.

2

Materials

2.1 Instrumentation and Materials

1. A commercial CE instrument hyphenated to a mass spectrometer. In the present study, an Agilent 7100 series CE instrument interfaced to a 6410 series triple quadrupole mass spectrometer (Agilent Technologies, Palo Alto, CA) is suitable. The Agilent Mass Hunter Workstation Data Acquisition software to acquire data and the Mass Hunter Qualitative Analysis software (B.07.00 version) for chromatographic data analysis. The Agilent Optimizer software to optimize multiple reaction monitoring (MRM) parameters (e.g., fragmentor voltage, collision energy, product ion m/z) for each analyte through flow injection analysis experiments. 2. A commercial HPLC pump equipped with a 1:100 splitter to deliver sheath liquid. 3. A Cobalt 60 panoramic irradiator for polymerization of the surfactants (e.g., from Phoenix Memorial Laboratory, University of Michigan, Ann Arbor, MI, USA). 4. A flash chromatography fritted column (1800 length, 1.800 ID) for surfactant purification. 5. A commercial ultrasonic bath for mobile phase vacuum degassing. 6. A commercial pH meter for the pH adjustment of the mobile phases and the sheath liquid. 7. Fused silica capillaries with an internal diameter of 50 μm, outer diameter of 360 μm, cut to a total length of 90 cm (e.g., available from Polymicron Technologies, Phoenix, AZ, USA). 8. 0.45 μm nylon syringe filters for filtration of the polymeric surfactant solution. 9. 0.2 μm filters for filtering the sheath liquid.

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10. A 1000 MW cut-off cellulose ester membrane for polymer dialysis (e.g., 1000 MW cut-off Spectra/Por, from Rancho Dominguez, CA, USA). 11. Silica gel for flash chromatography (pore size 60 A˚). 2.2 Solutions and Background Electrolytes

Use HPLC grade organic solvents and ultrapure water (18.8 mΩ) obtained from a suitable water purification system. 1. Binary solvent 1 (methanol/ethyl acetate, 0.05:10, v/v): Add 1000 mL of ethyl acetate in a graduated cylinder and add 50 mL of methanol into it. Sonicate the solution for 10 min. 2. Binary solvent 2 (ethyl acetate/n-hexane 2:1, v/v): Add 600 mL of ethyl acetate in a graduated cylinder and add 300 mL of nhexane into it. Sonicate the solution for 10 min. 3. Ternary solvent (10:2:1 ethyl acetate/methanol/water): Add 1000 mL of HPLC ethyl acetate to 200 mL of methanol and 100 mL of water. Sonicate for 10 min. 4. TLC visualization solution: Add 2.5 mL concentrated sulfuric acid to 50 mL methanol. 5. Stock solutions: Weigh out 0.0010 or 0.0020 g of chiral analytes (1,10 -binaphthyl-2,20 -dihydrogen phosphate (BNP), dansylated phenylalanine (Dns-Phe), atropine, homatropine, pseudoephedrine, ephedrine, norephedrine, methylephedrine, atenolol, metoprolol, carteolol, and talinolol, dissolve in 1.0 or 2.0 mL of methanol, and store at 20  C. Prepare working solutions of the analytes at 0.1 or 1 mg/mL by dilution of the stock solutions with water/methanol (50:50, v/v). Prepare fresh working solutions on a daily basis. 6. Ammonium acetate solution (7.5 M): Dissolve 57.81 g ammonium acetate in 100.0 mL water in a volumetric flask. A commercial ready-made solution can also be used. 7. Electrolyte 1 (12.5 mM NaH2PO4 and 12.5 mM Na2HPO4, pH 7.0, 45 mM poly-α-D-SUGP): Weigh out 0.1500 g of NaH2PO4 and 0.1774 g of Na2HPO4 and dissolve in 80 mL of ultrapure water. Adjust pH to 7.0 (see Note 1). Transfer the solution to 100 mL volumetric flask and bring to volume with ultrapure water. Prepare final MEKC running buffer by adding 45 mM poly-α-D-SUGP in background electrolyte at polymerization concentrations (20, 50, 75, and 100 mM). Filter the electrolyte with 0.45 μm nylon membrane filter prior to its use. 8. Electrolyte 2 (20 mM NH4OAc, pH 10.8, 15 mM poly-α-DSUGP): Add 80 mL of ultrapure water to a beaker and add 267 μL of 7.5 M ammonium acetate solution. Adjust the pH to 10.8 by adding 13.4 M ammonium hydroxide. Transfer the solution to 100 mL volumetric flask and bring to volume with ultrapure water. Prepare final MEKC running buffer by adding

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15 mM poly-α-D-SUGP to the background electrolyte. Filter the electrolyte with 0.45 μm nylon membrane filter prior to its use. 9. Electrolyte 3 (25 mM NH4OAc, pH 7.0, 30 mM poly-α-DSUGP): Add 80 mL of ultrapure water to a beaker and add 333 μL of 7.5 M NH4OAc solution. Adjust the pH to 7.0 by adding ammonium hydroxide. Transfer the solution to 100 mL volumetric flask and bring to volume with ultrapure water. Prepare final MEKC running buffer by adding 30 mM poly-α-D-SUGP to the background electrolyte. Filter the electrolyte with 0.45 μm nylon membrane filter prior to its use. 10. Electrolyte 4 (25 mM NH4OAc, pH 5.0,15 mM poly-β-DSUGS): Add 80 mL of ultrapure water to a beaker and add 333 μL of 7.5 M sol NH4OAc solution. Adjust the pH to 5.0 by adding acetic acid. Transfer the solution to 100 mL volumetric flask and bring to volume with ultrapure water. Prepare final MEKC running buffer by adding 15 mM poly-β-D-SUGS to the background electrolyte. Filter the electrolyte with 0.45 μm nylon membrane filter prior to its use. 11. Electrolyte 5 (25 mM NH4OAc, pH 5.0, 15 mM poly-β-DSUGP): Add 80 mL of ultrapure water to a beaker and add 333 μL of 7.5 M sol NH4OAc solution. Adjust the pH to 5.0 by adding acetic acid. Transfer the solution to 100 mL volumetric flask and bring to volume with ultrapure water. Prepare final MEKC running buffer by adding 15 mM poly-β-D-SUGP in background electrolyte. Filter the electrolyte with 0.45 μm nylon membrane filter prior to its use. 12. Electrolyte 6 (25 mM NH4OAc, pH 7.0, 15 mM poly-β-DSUGP): Add 80 mL of ultrapure water to a beaker and add 333 μL of 7.5 M sol NH4OAc solution. Adjust the pH to 5.0 by adding ammonium hydroxide. Transfer the solution to 100 mL volumetric flask and bring to volume with ultrapure water. Prepare final MEKC running buffer by adding 15 mM poly-β-D-SUGP to the background electrolyte. Filter the electrolyte with 0.45 μm nylon membrane filter prior to its use. 2.3 Sheath Liquid Solution

1. Mix HPLC grade methanol and ultrapure water in the ratio of 80/20 (v/v). Add 160 mL of methanol and 40 mL of ultrapure water in a graduated cylinder. 2. To this mixture, add 7.5 M NH4OAc solution and dilute to a final concentration of 5 mM NH4OAc. Sonicate the mixture for 10 min and vacuum degas thoroughly prior to its use.

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Methods

3.1 Synthesis of Polymeric Surfactants

The details of synthetic procedures for various derivatives of carbohydrate-based chiral polymeric surfactant with D-optical configuration are provided below: The polymeric surfactant included are four α- and β-forms of phosphorylated D-glucose head groups with C8 and C11 hydrocarbon chains (Fig. 1a–d) and four α- and β-isomers of sulfated D-sugars with the same two aforementioned chain lengths (Fig. 1e–h). All steps are carried out at room temperature unless noted otherwise. Perform all synthetic steps in a wellventilated hood. Observe appropriate safety regulations for all chemicals.

3.1.1 Synthesis of Carbohydrate-Based Surfactant Containing Phosphated Glucose as Head Group and Nundecenyl and N-octenyl Hydrocarbon Chain with αand/or β-Configuration

The steps of the synthesis are outlined in Schemes 1, 2, 3, 4, and 5. The numbers in the text refer to the structures in the Schemes. 1. To synthesize β-configuration of phosphated sugar surfactants of eight and eleven hydrocarbon chain length, dissolve equimolar amounts (5.0 g, 0.0128 mol) of β-D-glucose pentaacetate (1) and (1.6 mL, 0.0128 mol) boron trifluoride diethyl etherate in 50 mL of anhydrous dichloromethane under nitrogen in a 250 mL round-bottom flask. Add 3.9 mL (0.0192 mol) of 10-undecen-1-ol or 2.9 mL (0.0192 mol) of 7-octene-1-ol and stir overnight (~18 h) to yield N-octenyl or N-undecenyl-β-D-glucopyranoside pentaacetate (2 or 3, Scheme 1, Glycosylation). Identify the product by 1H NMR spectroscopy (see Note 2) 2. Prepare a saturated solution of sodium bicarbonate by stirring ~30 g (0.36 mol) sodium bicarbonate in 250 mL of ultrapure water in a 500 mL beaker. Add the saturated sodium bicarbonate solution dropwise to the product (obtained in step 1) to obtain a pH ~7 to neutralize the excess boron trifluoride (see Note 3).

AcO

OAc OAc

O

OAc

BF3

AcO HO

18 h at RT OAc

(1) ß-D-glucose pentaacetate Ac =

O

O CH 3

( )n

O

O

( )n

OAc OAc

OAc

(2) n=5, N-octenyl- ß-Dglucopyranoside pentaacetate (3) n=8, N-undecenyl- ß-Dglucopyranoside pentaacetate

Scheme 1 Reaction scheme for glycosylation of β-D-glucose pentaacetate (1) to yield N-alkenyl-β-Dglucopyranoside pentaacetates (2, 3)

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Vijay Patel and Shahab A. Shamsi AcO

HO ( )n

O

O

MeOH, CH 3ONa

OAc OAc

O

O

( )n

OH

3 h at 0 0C

OH

OH

OAc

(4) n=5, N-octenyl-ß-Dglucopyranoside (5) n=8, N-undecenyl-ß-D glucopyranoside

(2) n=5, N-octenyl-ß-Dglucopyranoside pentaacetate (3) n=8, N-undecenyl-ß-D glucopyranoside pentaacetate

Scheme 2 Reaction scheme for deacetylation of N-alkylenyl-β-D-glucopyranoside pentaacetates (2, 3) to Nalkylenyl-β-D-glucopyranosides (4, 5)

HO

Cl O

O

( )n

TEA

Cl

OH

P O Ph O

O

O P O

O O

( )n

O

24 h at RT

OH

OH

OH

OH

(6) n=5, N-octenyl-ß-Dglucopyranoside-4,6-phenyl phosphate (7) n=8, N-undecenyl-ß-Dglucopyranoside-4,6-phenyl phosphate

(4) n=5, N-octenyl-ß-D glucopyranoside (5) n=8, N-undecenyl-ß-D glucopyranoside

Scheme 3 Reaction scheme for phosphorylation of N-alkylenyl-β-D-glucopyranosides (4, 5) to yield N-alkylenyl-β-D-glucopyranoside-4,6-phenyl phosphates (6, 7)

O

O P O

OH

O

O

( )n

NaOH 1, 4-Dioxane

O OH

(6) n=5, N-octenyl-ß-D glucopyranoside-4,6-phenyl phosphate (7) n=8, N-undecenyl-ß-D glucopyranoside-4,6-phenyl phosphate

Na + -O

O

P O

OH

O

O

( )n

O OH

(8) n=5, Sodium-N-octenyl-ß-D glucopyranoside-4,6-hydrogen phosphate (9) n=8, Sodium-N-undecenyl-ß-Dglucopyranoside-4,6-hydrogen phosphate

Scheme 4 Reaction scheme for hydrolysis of phenyl group of N-alkylenyl-β-D-glucopyranoside-4,6-phenyl phosphates (6, 7) to yield sodium-N-alkylenyl-β-D-glucopyranoside-4,6-hydrogen phosphates (8, 9)

3. Transfer the resulting solution to a 1000 mL separatory funnel and remove excess of boron trifluoride diethyl etherate by shaking the funnel vigorously with 200 mL of ultrapure water. Collect the lower organic layer in a clean 1000 mL beaker. Dry the organic layer using a small amount of

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Scheme 5 Reaction scheme for synthesis of N-alkylenyl-α-D-glucopyranoside pentaacetates (10, 11) and sodium-N-alkylenyl-β-D-glucopyranoside phenyl-4,6-phosphates (12, 13). (II, III, and IV refer to reaction schemes)

anhydrous sodium sulfate while slowly swirling the beaker (see Note 4). Filter the organic solution into a clean 250 mL round-bottom flask and remove solvent at 60  C in a rotary evaporator under vacuum to obtain the product as yellow viscous liquid. 4. Dissolve the entire glycosylated product N-undecenyl-β-D-glucopyranoside acetate (2) or N-octenyl-β-D-glucopyranoside acetate (3) in a 250 mL round-bottom flask in 20 mL of anhydrous methanol. Add a catalytic amount (~1 mL) of sodium methoxide (25% solution in methanol) to the reaction mixture at 0  C using a disposable syringe and stir the mixture for 3 h on the ice bath (0  C) to yield the crude deacetylated product (Scheme 2, Deacetylation). 5. Add about 5 g of Amberlyst 15 hydrogen form of cation exchanger to the round-bottom flask containing the deacetylated product until a pH ~7 is obtained to remove the excess sodium methoxide. Filter the resulting mixture using Whatman #42 filter paper (or a similar filter paper) to remove the cation exchange resin. Transfer the filtrate to a 1000 mL round-bottom flask and add ~25 g silica gel before removing the solvent in a rotary evaporator at 60  C under vacuum. 6. Load a flash chromatography column up to 12 in. with silica gel and then load the dried deacetylated product mixed with silica gel from step 5 onto the top of the silica packed column (see Note 5). 7. Add about 1000 mL of the binary solvent 1 (methanol/ethyl acetate, 0.05:10 v/v) to elute pure deacetylated sugar product. First, pass the binary solvent through packed silica column containing dry loaded deacetylated product and collect 30 fractions of 15 mL eluate in each 15 mL clean falcon tubes.

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8. Spot each fraction on a TLC plate and develop using a binary solvent 1 (methanol/ethyl acetate, 0.05:10 v/v) as the mobile phase. After development of dry plate at room temperature visualize the spots by dipping the TLC plate into the visualization solution or by spraying the plate with the visualization solution and heat TLC plate on a hot plate. The spots appear as dark brown or black spot. 9. Combine fractions containing pure deacetylated product as indicated by the TLC in a round-bottom flask and evaporate solvents in a rotary evaporator at 60  C under vacuum to yield N-octenyl-β-D-glucopyranoside (4) or N-undecenyl-β-D-glucopyranoside (5) as colorless viscous liquids. The dried product can be stored in a round-bottom flask capped with rubber septum at room temperature until further use. Identify the product by 1H NMR spectroscopy (see Note 6). 10. Dissolve nearly 0.8 g (0.0027 mol) N-octenyl-β-D-glucopyranoside (4) or 1.18 g (0.0036 mol) N-undecenyl-β-D-glucopyranoside (5) in 50 mL of anhydrous dichloromethane in a 250 mL round-bottom flask under nitrogen. Add 648.5 μL (0.0046 mol) of triethylamine using a syringe and stir for 10 min while keeping the flask on ice bath to reduce heat generated by addition of triethylamine. Add 636.5 μL (0.0043 mol) of phenyl dichlorophosphate and stir the reaction mixture at room temperature for 3 h. Check the reaction by TLC after 3 h to confirm formation of the phosphorylated product. Spot the reaction mixture on TLC plate and develop using a binary solvent (ethyl acetate/n-hexane 2:1, v/v) as mobile phase. Multiple spots on TLC plate are an indication of the product formation (Scheme 3, Phosphorylation). 11. After stirring the reaction mixture for 24 h at room temperature, add about 25 g silica gel and evaporate the solvent at 60  C using a rotary vacuum evaporator. Load the silica gel-containing phosphorylated product on a flash chromatography column and carry out purification as described in step 6. Use a total of 1000 mL of binary solvent 2 (ethyl acetate/nhexane 2:1, v/v) for elution. Collect ~15 mL fractions into each falcon tube. Spot each fraction on a TLC plate and develop using a binary solvent 2 (ethyl acetate/n-hexane 2:1, v/v) as mobile phase. Develop plate and visualize spots as described in step 8. 12. Combine fractions containing the pure product of either (6) or (7) (about 5 fractions) in a 100 mL round-bottom flask and evaporate solvents using a rotary vacuum evaporator at 60  C to yield a sticky white solid product (octenyl- or undecenyl-βD-glucopyranoside-4,6-phenyl phosphate, 6, 7). Identify the

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product by NMR spectroscopy (see Note 7 for both chain lengths). 13. Dissolve the entire product of either octenyl- or undecenyl-β-D-glucopyranoside phenyl phosphate (6, 7) in 20 mL of 1,4-dioxane in a 250 mL round-bottom flask. Add 110 μL of sodium hydroxide (50% in water) and stir the reaction mixture for 20 h. After the first 3 h, check the reaction mixture by TLC for multiple spots on TLC plate indicating the product formation. Use ethyl acetate/n-hexane (2:1, v/v) as mobile phase (Scheme 4, Hydrolysis). 14. After stirring for 20 h, evaporate solvent 1,4-dioxane in a rotary vacuum evaporator to obtain the product as a powder. Neutralize the resulting product by dissolving it in 25 mL of ultrapure water for 30 min and add 1 M hydrochloric acid until a pH of about 7 is reached (pH paper should turn yellow). 15. After neutralization, transfer the solution of octenyl- or undecenyl phosphated surfactants (8, 9) to a separatory funnel and extract with 200 mL of ethyl acetate to remove organic impurities. Swirl the separatory funnels and allow the two layers to separate and clear. Collect the bottom aqueous layer in a beaker and stir overnight to remove residual ethyl acetate. Lyophilize the aqueous layer to yield solid salt form of the monomeric surfactant (i.e., either β-D-SOGP or β-D-SUGP). Identify the product of both chain lengths by 1H NMR spectroscopy (see Note 8). 16. For the synthesis of α-D-configured phosphated sugar surfactants with octenyl- or undecenyl carbon chains (i.e., compounds 12 and 13), proceed as described above for the respective β-D-configured derivatives (steps 1–15) except for using a different molar ratio of the reactants and longer reaction time in step 1. Specifically, in step 1, dissolve 5 g (0.0128 mol) β-D-glucose pentaacetate (1) and 1.6 mL (0.0129 mol) boron trifluoride diethyl etherate in 50 mL of anhydrous dichloromethane under nitrogen in a 250 mL round-bottom flask. Add 2.5 g (0.0192 mol) 7-octen-1-ol or 3.3 g (0.0192 mol) 10-undecen-1-ol and stir for 72 h (Scheme 5) to allow complete inversion of the configuration at the anomeric carbon. Identify the product of both chain lengths by NMR spectroscopy (see Note 9). 17. Follow the subsequent steps 2–15 as described with no further modifications.

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3.1.2 Synthesis of Carbohydrate-Based Surfactant Containing Sulfated Glucose as Head Group and N-undecenyl and Octenyl Hydrocarbon Chain with α- and/or βConfiguration

1. To synthesize β-D-configured sulfated sugar surfactants of eight and eleven hydrocarbon chain length, dissolve equimolar amounts (5.0 g, 0.0128 mol) of β-D-glucose pentaacetate (1) and (1.6 mL, 0.0128 mol) boron trifluoride diethyl etherate in 50 mL of anhydrous dichloromethane under nitrogen in a 250 mL round-bottom flask. Add 3.9 mL (0.0192 mol) of 10-undecen-1-ol or 2.9 mL (0.0192 mol) or 7-octene-1-ol and stir overnight (~18 h) to yield N-octenyl or N-undecenyl-β-D-glucopyranoside pentaacetate (2 or 3). To yield Noctenyl or N-undecenyl-α-D-glucopyranoside pentaacetate (10 or 11), carry out the above reaction for 72 h. 2. Prepare a saturated sodium bicarbonate solution by stirring ~30 g (~1.4 mol) sodium bicarbonate in 250 mL ultrapure water in a 500 mL beaker. Add the saturated sodium bicarbonate solution dropwise to the product (obtained in step 1) to obtain a pH ~7 to neutralize the excess boron trifluoride (see Note 10). 3. Transfer the resulting solution to a 1000 mL separatory funnel and remove excess boron trifluoride diethyl etherate by shaking the funnel vigorously with 200 mL of water. Collect the lower organic layer in a clean 1000 mL beaker. Dry the organic layer using a small amount of anhydrous sodium sulfate while slowly swirling the beaker (see Note 4). Filter the organic solution into a clean 250 mL round-bottom flask and remove solvent at 60  C in a rotary vacuum evaporator to obtain the product as yellow viscous liquid. 4. Dissolve all of the intermediates of N-octenyl-α- or β-D-glucopyranoside (e.g., 1.45 g or 0.005 mol) or N-undecenyl-α- or β-D-glucopyranoside (e.g., 1.65 g 0.005 mol) in 80 mL of anhydrous pyridine under nitrogen blanket. Add an equimolar amount (0.8 g, 0.005 mol) of sulfur trioxide pyridine complex and stir for 10 min while keeping round-bottom flask on ice bath to reduce heat generated by addition of sulfur trioxide pyridine complex. Continue the reaction under nitrogen blanket for 24 h (Scheme 6, Sulfonation). Check the reaction with TLC after 3 h to confirm formation of the product. Develop the TLC plate using 10:2:1 for ethyl acetate/methanol/water to check for product formation (see Note 11). 5. After 24 h of stirring, transfer the resulting product(s) in 1000 mL round-bottom flask using minimal amount of pyridine. Add ~25 g silica gel to the round-bottom flask and dry in rotary vacuum evaporator at 70  C to yield intermediate Nundecenyl- or N-octenyl-α-D-glucopyranoside-6-hydrogen sulfate (14, 15) or N-undecenyl- or N-octenyl-β-D-glucopyranoside-6-hydrogen sulfate (18, 19). 6. Purify the intermediate as described in steps 6 and 7 (Subheading 3.1.1) using a 10:2:1 (v/v/v) ethyl acetate/methanol/ water solvent mixture.

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Scheme 6 Reaction scheme for synthesis of N-alkylenyl-α-D-glucopyranoside-6-hydrogen sulfates (18, 19) and N-alkenyl-β-D-glucopyranoside-6-hydrogen sulfates (14, 15)

7. Collect eluate from the falcon tubes containing pure product in a 250 mL round-bottom flask and dry in rotary vacuum evaporator at 60  C to yield pure intermediate(s). 8. Dissolve the pure intermediate(s) in 50 mL of ultrapure water and add 1 mL of sodium hydroxide (10%) solution and stir the mixture (Scheme 7, formation of sulfated sodium salt). 9. Lyophilize the resulting sodium salt solution at 50  C collector temperature and 0.05 mbar pressure for 2 days to yield final products (20, 21, 22, and 23) sodium N-undecenyl- and octenyl-β-D-glucopyranoside-6-hydrogen sulfate (β-D-SUGS and β-D-SOGS), as well as sodium N-undecenyl and sodium N-octenyl-α-D-glucopyranoside-6-hydrogen sulfate (α-DSUGS and α-D-SOGS) monosodium salts. Identify the product of both chain lengths by 1H NMR spectroscopy of α- and β-D sulfated products (see Note 12). 3.2 Polymerization of Surfactant Monomers

1. Dissolve 20, 50, 75, and 100 mM amounts of α-D-SUGP, equivalent to 5-, 12.5-, 18.75-, and 20-times the critical micelle concentration in ultrapure water [see also 14] in four 50 mL clear borosilicate glass bottles and sonicate for 10–15 min to obtain a clear solution.

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Scheme 7 Reaction scheme for synthesis of sodium salts of N-alkylenyl-α-D-glucopyranoside-6-hydrogen sulfates (22, 23) and N-alkylenyl-β-D-glucopyranoside-6-hydrogen sulfates (20, 21)

2. Polymerize all four aforementioned amounts of α-D-SUGP surfactant solutions in each 20 mL size glass vial at the same time and temperature using a cobalt-60 gamma radiation source at a total dose of 20 MRad (Phoenix Laboratory, University of Michigan, Ann Arbor, MI). The polymerized bottles attained an amber color with similar intensity suggesting the polymerization occurred homogenously. 3. Transfer the polymerized solution from each amber bottle to a cutoff dialysis membrane (see Note 13) and stir in a beaker of water for 24 h to remove unreacted monomers of α-D-SUGP. Make sure to change the water solutions in the beaker every 8 h to promote efficient dialysis and to remove molecular impurities of less than 2000 Daltons. 4. Filter the dialyzed solutions and lyophilize at 50  C collector temperature and 0.05 mbar until a dry powder of polymer is obtained (see Note 14). Lyophilize poly-α-D-SUGP and obtain the 1H NMR to illustrate peak broadening and disappearance of vinyl protons as shown in Fig. 2. 5. Polymerize all remaining α- and β-vinyl phosphorylated and sulfated sugar monomers (i.e., octenyl and undecenyl phosphated and sulfated sugar surfactants) in a similar fashion (described in steps 1–4 in this section) except they are all polymerized at 100 mM equivalent monomer concentrations (EMCs).

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Fig. 2 400 MHz 1H NMR spectra of monomeric N-undecenyl-α-D-glucopyranoside-4,6-hydrogen phosphate (α-D-SUGP, top) and polymeric N-undecenyl-α-D-glucopyranoside-4,6-hydrogen phosphate (poly-α-D-SUGP, bottom) in D2O 3.2.1 Method to Test the Enantioresolution Capability of Poly-α-DSUGP

1. Install a fused silica capillary of a total length of 64.5 cm (360 μm OD, 50 μm ID) and an effective length of 56.0 cm in the CE-UV cassette of the Agilent CE system. 2. Flush the capillary with 1 M NaOH for 30 min followed by ultrapure water for 20 min. Next, flush the electrolyte 1 containing α-D-SUGP (polymerized at 20 mM EMC) for 4 min at 950 mbar. 3. Set up diode array detector at 210, 213, 214, and 254 nm. 4. Place electrolyte 1 in a set of four inlet and outlet buffer vials. 5. Inject sample solution of BNP at a concentration of 1 mg/mL and perform two duplicate runs at 5 mbar for 10 s. 6. Apply a separation voltage of +20 kV (positive polarity, detection at cathodic end of the capillary) and record electropherograms. After the two runs, repeat steps 2, 3, and 4 and inject Dns-Phe at a concentration of 1 mg/mL at a pressure of 5 mbar for 10 s. Apply a voltage of +20 kV.

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Fig. 3 Effect of polymerization concentrations (mM) of sodium N-undecylenyl-α-D-glucopyranoside-4,6hydrogen phosphate (α-D-SUGP) surfactant monomers on chiral resolution of the zwitterionic compound dansylated phenylalanine (Dns-Phe, a) and the anionic compound (1,10 -binaphthyl-2,20 diylhydrogen-phosphate) (BNP, b) in MEKC. (Reproduced by permission of Wiley-VCH from ref. 12 © 2016)

7. Repeat steps 1–6 for the other three EMCs (50, 75, and 100 mM) of poly-α-D-SUGP. 8. Examples of electropherograms obtained for the chiral separation of Dns-Phe and BNP at the optimum EMC of 100 mM and 20 mM poly-α-D-SUGP, respectively, are shown in Fig. 3a, b. 3.3 General Procedures and Conditions for CMEKCUV and CMEKC-MS/MS Experiments 3.3.1 CMEKC-UV Procedure

1. Use a fused silica capillary with a total length of 64.5 cm and an effective length of 56.0 cm. 2. Prepare a detection window at 8.5 cm from the detector end by removing 3 mm section of polyimide coating. 3. Install the fused silica capillary in CE-UV cassette according to the instructions of the manufacturer (see Note 15) and install the cassette in the CE instrument. 4. Transfer about 200 μL of analyte solution to the cone-shaped vials (250 μL size) and 350 μL of polymeric surfactant dissolved in background electrolytes to the buffer vials (500 μL size). 5. Flush the new fused silica capillary for 30 min with 1 M NaOH followed by 20 min with ultrapure water. 6. Pre-condition the capillary by flushing with background electrolyte containing polymeric surfactant of various EMC concentrations (5–100 mM) for 2–5 min before each run.

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7. Post-condition the capillary by flushing sequentially with ultrapure water for 2 min, 1 M NaOH for 4 min, and ultrapure water for 2 min. 8. Set up the capillary temperature and the voltage polarity according to the nature of analyte. 9. Keep the analyte solutions at 10–15  C in the carousel using a circulator water bath. Perform hydrodynamic injection at a pressure of 5–10 mbar for varying times in seconds as needed for each experiment. 3.3.2 CMEKC-MS/MS Procedure

1. Use a fused silica capillary with a total length of 60–120 cm (see Note 16). 2. Install a fused silica capillary in the CE-MS cassette by inserting capillary inlet to the injector side of the CE-MS cassette and the outlet into the nebulizer according to the instructions of the manufacturer. 3. To install capillary into the nebulizer, first slide the capillary from the top of the nebulizer body until the capillary emerges from the sprayer tip. Align the capillary end flat with the sprayer tip using a fingernail and secure the fitting screw to hold the capillary in position (see Note 17). 4. Connect the sheath flow and the nebulizing gas tubings to the nebulizer. Fill the sheath liquid bottle with sheath liquid and prime the HPLC pump to remove any air bubbles from the sheath flow tubing. An example of sheath liquid used in CMEKC-ESI-MS is as follows: 80/20 (v/v) MeOH/H2O containing 5 mM NH4OAc (pH 6.8) at a flow rate of 5 μL/ min. 5. Before the start of each MEKC-MS/MS experiment, rinse prepunctures and electrodes with 2-propanol. For maximum sensitivity, clean the spray shield and spray chamber with 2-propanol using a special cloth (see Note 18). 6. Pre-condition the capillary by flushing with background electrolyte containing surfactant of various EMC concentrations (5–50 mM) for 2–5 min before each runs. 7. Post-condition the capillary by flushing sequentially with water for 2 min, 1 M NH4OH for 4 min, and ultrapure water for 2 min. 8. Set the capillary temperature and the voltage polarity according to the nature of analyte. 9. Keep analyte solutions at 10–15  C in the vial handler using circulator water bath. Perform hydrodynamic injection at a pressure of 5–10 mbar for varying times in seconds.

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10. Before setting the spray chamber parameters for on-line MEKC-MS/MS, optimize the MS signal by performing multiple reaction monitoring (MRM) scan. Determine the fragmentor voltage and collision energy for each chiral analyte from flow injection HPLC. 3.3.3 Example 1

CMEKC-UV Experiment

The example illustrates the chiral separation of a model test analyte (BNP) using poly-α-D-SUGP by CMEKC-UV and CMEKC-MS/ MS. Use electrolyte 2 (see Subheading 2.2) as run buffer. 1. Use a 56.0 cm effective length (375 μm OD, 50.0 μm ID) capillary. 2. Set the detector detection wavelengths at 210, 213, 214, and 254 nm (bandwidth 4 nm) and the reference wavelength at 360 nm (bandwidth 100 nm). 3. Inject BNP sample (1 mg/mL) at the anodic end of the capillary at a pressure of 5 mbar for 10 s. 4. Apply a separation voltage of +20 kV (ramp time 0.17 min) and record electropherogram. A representative electropherogram is shown in Fig. 4a.

CMEKC-MS/MS Experiment

1. Use a 60.0 cm effective length (375 μm OD, 50.0 μm ID) capillary. 2. Set the following conditions for the mass spectrometer:

Fig. 4 Chiral separation of BNP by MEKC-UV (a), and MEKC-MS/MS (b). Peak identification: 1 ¼ R-BNP, 10 ¼ S-BNP (Reproduced by permission of Wiley-VCH from ref. 12 © 2016)

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(a) Spray chamber parameters: nebulizer pressure: 3 psi. (b) Drying gas temperature: 250  C. (c) Drying gas flow rate: 6 L/min. (d) Capillary voltage: 3000 V. (e) Fragmentor voltage: 200 V. (f) Collision energy: 41 eV. (g) MRM transition: 347.1 ! 79.1 (h) Sheath liquid: MeOH/H2O (80/20, v/v), 5 mM NH4OAc, pH 6.8 with a flow rate of 5 μL/min. 3. Inject BNP sample solution (0.1 mg/mL) at the anodic end of the capillary at a pressure of 5 mbar for 10 s. 4. Apply separation voltage of +20 kV (ramp time 0.17 min) and record electropherogram. A representative electropherograms of the CMEKC-MS/MS separation is shown in Fig. 4b. 3.3.4 Example 2

The example illustrates a simultaneous enantioseparation of norephedrine, pseudoephedrine, ephedrine, and N-methylephedrine by CMEKC-MS/MS using poly-α-D-SUGP as a chiral pseudophase. Use electrolyte 3 (see Subheading 2.2) as a run buffer and a sample solution containing 10 μg/mL of norephedrine, pseudoephedrine, ephedrine, and N-methylephedrine in MeOH/H2O (10/90, v/v). 1. Use a 60.0 cm effective length (375 μm OD, 50.0 μm ID) capillary. 2. Set the following conditions for the mass spectrometer: (a) Spray chamber parameters: nebulizer pressure: 3 psi. (b) Drying gas temperature: 250  C. (c) Drying gas flow rate: 6 L/min. (d) Capillary voltage: 3000 V. (e) Fragmentor voltages: 88, 64, and 98 V. (f) Collision energies: 25, 17, and 21 eV. (g) MRM transitions: 166.1 ! 115.1, 152.2 ! 117, 180.2 ! 147.2 for pseudoephedrine/ephedrine, norephedrine, and methylephedrine, respectively. (h) Sheath liquid: MeOH/H2O (80/20, v/v), 5 mM NH4OAc, pH 6.8 with a flow rate of 5 μL/min. 3. Inject sample solution containing the four alkaloids at a concentration of 10 μg/mL each at a pressure of 5 mbar for 10 s. 4. Apply separation voltage of +20 kV (ramp time 0.17 min) and record electropherogram.

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Fig. 5 Electropherograms for enantioseparation of ephedrine alkaloids using sugar surfactant with optimum head group and chain length at optimum pH 5.0 in MEKC-MS/MS. Peak identification: 1 ¼ (1R,2S)-()norephedrine, 10 ¼ (1S,2R)-(+)-norephedrine; 2 ¼ (1R, 2R)-()-pseudoephedrine, 20 ¼ (1S,2S)-(+)-pseudoephedrine; 3 ¼ (1R,2S)-()-ephedrine, 30 ¼ (1S,2R)-(+)-ephedrine; 4 ¼ (1R,2S)-()-N-methylephedrine, 40 ¼ (1S,2R)-(+)-N-methylephedrine (Reproduced by permission of Wiley-VCH from ref. 12 © 2016)

Representative electropherograms resulting from the experiment are shown in Fig. 5. The enantioresolution and enantioselectivity of the four alkaloids are in the following decreasing order: Nmethylephedrine > ephedrine > norephedrine > pseudoephedrine. The enantiomers of pseudoephedrine and ephedrine are separated simultaneously since both share the same MRM transition (166.1 ! 115) and cannot be distinguished by either accurate mass or MS/MS. The separation has been reported in [12]. 3.3.5 Example 3

The example illustrates the separation of four β-blockers by CMEKC-MS/MS using poly-α-D-SUGP. Use electrolyte 3 (see Subheading 2.2) as a run buffer. The sample solution consists of a mixture of atenolol, metoprolol, carteolol, and talinolol at a concentration of 10 μg/mL each in MeOH/H2O (10/90, v/v). 1. Use experimental settings (capillary and MS conditions) described for Example 2 with the following exceptions: (a) Fragmentor voltages: 137, 107, 83, and 98 V. (b) Collision energies: 25, 17, 17, and 13 eV. (c) MRM transitions 267.2 ! 145.2, 268.2 ! 116.2, 293.2 ! 237.2, and 364.3 ! 308.3 for atenolol, metoprolol, carteolol, and talinolol, respectively. 2. Inject sample solution containing the four β-blockers at a concentration of 10 μg/mL each at a pressure of 5 mbar for 10 s 3. Apply separation voltage of +20 kV (ramp time 0.17 min) and record electropherograms. Representative electropherograms resulting from the experiment are shown in Fig. 6. The elution order of four β-blockers (atenolol, metoprolol, carteolol, and talinolol) is based on the

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Fig. 6 Electropherograms of the enantioseparation of β-blockers. Peak identification: 1, 10 ¼ atenolol; 2, 20 ¼ carteolol; 3, 30 ¼ metoprolol; 4, 40 ¼ talinolol. For each β-blocker the R-enantiomer eluted earlier than the S-enantiomer. (Reproduced by permission of Wiley-VCH from ref. 12 © 2016)

increase in their hydrophobicity (i.e., log P values). The separation has been reported in [12]. 3.3.6 Example 4

The example illustrates the enantioseparation of two tropane alkaloids atropine and homatropine by CMEKC-MS/MS using poly-β-D-SUGS and poly-β-D-SUGP as chiral pseudostationary phases. Use electrolyte 4 containing poly-β-D-SUGS (see Subheading 2.2) and electrolyte 5 containing poly-β-D-SUGP as run buffers, respectively. Use a sample solution containing 0.1 mg/mL of atropine and homatropine in MeOH/H2O (10/90, v/v). 1. Use experimental settings (capillary and MS conditions) described for Example 2 with the following exceptions: (a) Fragmentor voltages: 41 and 76 V (b) Collision energies: 33 and 35 eV (c) MRM transitions: 290.2 ! 124.2 and 276.2 ! 125.2 for homatropine and atropine, respectively. 2. Inject sample solution containing the tropane alkaloids at a concentration of 0.1 mg/mL each at a pressure of 5 mbar for 10 s 3. Apply separation voltage of +20 kV (ramp time 0.17 min) and record electropherograms. Representative electropherograms resulting from the use of either poly-β-D-SUGS or poly-β-D-SUGP are shown in Fig. 7a (poly-β-D-SUGS) and Fig. 7b, (poly-β-D-SUGP), respectively. Poly-β-D-SUGS enantioseparated both atropine and homatropine, but run time for atropine was significantly longer. Poly-β-D-SUGP showed no enantioselectivity for atropine.

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Fig. 7 Electropherograms comparing the effect of the head group of the polymeric sugar-derived surfactants (poly-β-D-SUGS (a) and poly-β-D-SUGP (b)) on the chiral separation of atropine and homatropine in MEKC-MS/ MS. 3.3.7 Example 5

The example illustrates the enantioselectivity of poly-α-D-SUGP and poly-β-D-SUGP for pseudoephedrine and metoprolol. Use electrolyte 3 containing poly-α-D-SUGP (see Subheading 2.2) and electrolyte 6 containing poly-β-D-SUGP (see Subheading 2.2) as run buffers, respectively. 1. For the enantioseparation of pseudoephedrine use electrolyte 3 containing poly-α-D-SUGP as run buffer first and then the experimental settings (capillary and MS conditions) described for Example 2 with the following exceptions are mentioned below: (a) Fragmentor voltage: 88 V. (b) Collision energy: 25 eV. (c) MRM transition: 166.1 ! 115 m/z. 2. Inject sample solution containing 0.1 mg/mL pseudoephedrine in MeOH/H2O (10/90, v/v) at the anodic end of the capillary at a pressure of 5 mbar for 10 s.

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Fig. 8 Electropherograms comparing the chiral selectivity of polymeric surfactants in the α- and β-configuration (poly-α-D-SUGP (a, c) and poly-β-D-SUGP (b, d)) in MEKC-MS/MS for the enantioseparation of pseudoephedrine (a, b) and metoprolol (c, d)

3. Apply separation voltage of +20 kV (ramp time 0.17 min) and record electropherogram. A representative electropherogram is shown in Fig. 8a. 4. Repeat experiment using electrolyte 6 containing poly-β-DSUGP following steps 1–3. A representative electropherogram is shown in Fig. 8b.

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5. For the enantioseparation of metoprolol use electrolyte 3 containing poly-α-D-SUGP first and the experimental settings (capillary and MS conditions) described for Example 2 with the following exceptions: (a) Fragmentor voltage: 107 V. (b) Collision energy: 17 eV. (c) MRM transition: 268.2 ! 116.2 m/z. 6. Inject sample solution containing 0.1 mg/mL metoprolol in MeOH/H2O (10/90, v/v) at the anodic end of the capillary at a pressure of 5 mbar for 10 s. 7. Apply separation voltage of +20 kV (ramp time 0.17 min) and record electropherogram. A representative electropherogram is shown in Fig. 8c. 8. Repeat experiment using electrolyte 6 containing poly-β-DSUGP following steps 5–7. A representative electropherogram is shown in Fig. 8d. Pseudoephedrine is baseline enantioseparated with a longer run time using poly-β-D-SUGP whereas poly-α-D-SUGP showed a shorter run time and a lower chiral selectivity. On the contrary, poly-α-D-SUGP showed better chiral resolution for metoprolol (Fig. 8c, RS ¼ 1.0) compared to poly-β-D-SUGP which gave poor chiral separation (Fig. 8d, Rs ¼ 0.4). The bar plots shown in Fig. 8e provide a comparison of the enantioseparation of chiral compounds between poly-α-D-SUGP and poly-β-D-SUGP (unpublished data). More chiral compounds (37) were screened with poly-β-D-SUGP but this polymeric surfactant exhibited an overall higher success rate of 65% compared to success rate of 47% observed with poly-α-D-SUGP.

4

Notes 1. The pH of the MEKC buffer is always adjusted before the addition of polymeric surfactant. 2. N-undecenyl-β-D-glucopyranoside pentaacetate: 1H NMR (MeOD, 400 MHz) δ 1.3 (12H, m), 1.6 (2H, t), 2.1 (2H, q), 3.2 (1H, t), 3.3 (1H, t), 3.5 (1H, m), 3.7 (1H, m), 3.9 (2 H, m), 5.0 (2H, t), 5.8 (1H, m). N-octenyl-β-D-glucopyranoside pentaacetate: 1H NMR (MeOD, 400 MHz): δ 1.26 (4 H, m), 1.6 (2 H, m), 3.30–3.49 (2 H, m), 3.6 (2 H, m), 3.9 (3H, m), 4.09–4.14 (3H, m), 4.2 (1 H, s), 4.9 (1H, d), 5.0 (1H, d), 5.8 (1H, t). 3. The pH of the solution is checked by placing a few drops of the product on a pH paper and comparing the color of pH paper with color indicator chart.

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4. Organic solvents were dried by adding a small amount of anhydrous sodium sulfate and shaking the beaker. When sodium sulfate is added to the organic solvent, it absorbs water and clumps up. If no water is left in the organic solvent to absorb, additional sodium sulfate will no longer clump and usually floats freely indicating complete removal of residual water. 5. The chromatography column is packed by pouring dry silica gel using a plastic funnel while applying a slight vacuum at the bottom end. Tap the column gently while packing to allow the silica gel to settle down and pack tightly. Load the silica gel-containing dry product over the silica bed in the column and tap the column gently to make an even layer of the product. The solvent is poured slowly on the top of column bed without disturbing the product layer. The product layer may be covered with a layer of silica before adding the solvent to prevent uneven spreading of the product. 6. The 1H NMR spectrum of the deacetylated product is same as for the glycosylated product (see Note 2). The acetyl group protons in glycosylated product and hydroxy protons in the deacetylated product do not have adjacent protons for coupling resulting in lack of resonance from these protons. Thus, the spectra of the abovementioned products are same. 7. N-undecenyl-β-D-glucopyranoside-4,6-phenyl phosphate: 1 H NMR (CDCl3, 400 MHz) δ 7.40–7.22 (5H, m), 5.83 (1H, m), 4.98 (2H, m), 4.47–4.21 (4H, d), 3.9–3.47 (6H, m), 2.06 (2H, m), 1.64 (3H, m), 1.38 (13H, m). Noctenyl-β-D-glucopyranoside-4,6-phenyl phosphate: 1H NMR (CDCl3, 400 MHz) δ 7.41–7.23 (5H, m), 5.83 (1H, m), 5.03–4.94 (2H, m), 4.46–4.25 (4H, m), 3.90–3.78 (3H, m), 3.59–3.49 (1H, m), 2.05 (6H, s), 1.64 (2H, t), 1.30 (9H, m). 8. Sodium N-undecenyl-β-D-glucopyranoside-4,6-hydrogen phosphate: 1H NMR (D2O, 400 MHz) δ 5.80 (1H, m), 5.0–4.8 (2H, m), 4.40 (1H, d), 4.30–3.10 (8H, m), 1.96 (2H, q), 1.53 (2H, m), 1.21 (13H, m). Sodium Noctenyl-β-D-glucopyranoside-4,6-hydrogen phosphate: 1H NMR (D2O, 400 MHz) δ 5.8 (1H, m), 5.0–4.8 (2H, m), 4.40 (1H, d), 4.20–4.10 (1H, m), 4.0 (1H, m), 3.90–3.70 (2H, m), 3.70–3.50 (4H, m), 1.94 (2H, q), 1.50 (2H, m), 1.71 (7H, m). 9. N-undecenyl-α-D-glucopyranoside pentaacetate: 1H NMR (MeOD, 400 MHz): δ 5.87 (1H, m), 5.52 (2H, m), 5.13 (1Hanomeric, d), 4.85–5.10 (3H, m), 4.01–4.30 (5H, m), 3.71 (2H, m), 3.45 (2H, t), 2.05 (29H, m), 1.67 (8H, m), 1.39 (25H, m). N-octenyl-α-D-glucopyranoside

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pentaacetate: 1H NMR (MeOD, 400 MHz): δ 5.87 (1H, m), 5.52 (2H, m), 5.13 (1Hanomeric, d), 4.85–5.10 (3H, m), 4.01–4.30 (5H, m), 3.71 (2H, m), 3.45 (2H, t), 2.05 (23H, m), 1.67 (6H, m), 1.39 (17H, m). 10. Add 1 mL of saturated NaHCO3 at a time while stirring the reaction mixture. Check the pH by placing a drop of the mixture on a pH paper. Keep adding saturated NaHCO3 until the pH is about 7. 11. Reaction progress is monitored by spotting reaction mixture along with starting material on a TLC plate and developing with suitable solvent system. Multiple spots and spot other than starting material on developed TLC plate suggest formation of α and β-form of the sulfated products. 12. N-undecenyl-β-D-glucopyranoside-6-hydrogen sulfate monosodium salt: 1H NMR (D2O, 400 MHz): δ 5.80 (1H, m), 4.96–4.84 (2H, m), 4.34 (1Hanomeric, d), 4.20 (1H, d), 4.11 (2H, t), 3.79 (1H, t), 3.55 (2H, m), 3.37 (2H, m), 3.16 (1H, t), 1.95 (2H, q), 1.52 (2H, t), 1.24 (13H, m). Nundecenyl-β-D-glucopyranoside-6-hydrogen sulfate monosodium salt: 1H NMR (D2O, 400 MHz): δ 5.80 (1H, m), 4.96–4.84 (2H, m), 4.34 (1Hanomeric, d), 4.20 (1H, d), 4.11 (2H, t), 3.79 (1H, t), 3.55 (2H, m), 3.37 (2H, m), 3.16 (1H, t), 1.95 (2H, q), 1.52 (2H, t), 1.24 (13H, m); N-octenyl-β-Dglucopyranoside-6-hydrogen sulfate monosodium salt: 1H NMR (D2O, 400 MHz): δ 5.80 (1H, m), 4.97–4.79 (2H, m), 4.36 (1Hanomeric, d), 4.22 (1H, d), 4.12–4.08 (1H, m), 3.8 (1H, m), 3.56 (2H, m), 3.37 (2H, m), 3.16 (1H, m), 1.95 (2H, m), 1.52 (2H, m), 1.29 (6H, m); N-octenyl-α-D-glucopyranoside-6-hydrogen sulfate monosodium salt: 1H NMR (D2O, 400 MHz): δ 5.87 (1H, m), 5.00 (2H, m), 4.85 (1Hanomeric, d), 4.21 (2H, m), 3.87 (2H, m), 3.65 (3H, m), 3.51 (2H, t), 2.01 (4H, m), 1.51 (6H, m), 1.25 (10H, m). Nundecenyl-α-D-glucopyranoside-6-hydrogen sulfate monosodium salt: 1H-NMR (D2O, 400 MHz): δ 5.87 (1H, m), 4.98 (2H, m), 4.85 (1Hanomeric, d), 4.21 (2H, m), 3.87 (2H, m), 3.50–3.70 (3H, m), 3.49 (2H, t), 2.01 (2H, m), 1.51 (4H, m), 1.25 (18H, m). 13. The dialysis membrane must be soaked for at least 3 h in a beaker filled with ultrapure water to remove traces of unreacted sodium counterions and unpolymerized monomers. Rinse the membrane several times. Construct a dialysis bag from the membrane using gentle sealing ridges universal closure (clips) to fill the polymerized solution for dialysis. 14. Almost all molecular micelles are hygroscopic and should be stored in a desiccator for better run time repeatability and longer shelf life.

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15. Install the capillary in the alignment interface by first pushing alignment interface against the capillary insertion tool and sliding the capillary. Once the UV detection window is aligned with the alignment window, release the interface and place it in the interface holder of an empty cassette. Wind the capillary around the reel of the cassette if the capillary is too long. Avoid any contact between the capillary windings to prevent any heating due to high voltage. Finally, close the cassette cover and make sure that both the capillary ends are of same length as the cassette guiding pins. Install the cassette in the CE instrument carefully while guiding capillary ends to the electrodes. 16. For CMEKC-MS/MS experiments, a 50 cm or a longer fused silica capillary should be used because of instrument constraints. 17. The distance between the capillary tip and the spray tip can be adjusted if necessary by turning the adjustment screw on the nebulizer. For turning adjustment screw clockwise will retreat the capillary inside the nebulizer and turning anti-clockwise will protrude the capillary tip outside the nebulizer. The position of the capillary tip relative to the spray tip needs to be optimized for CMEKC-MS/MS analysis. Several experiments may be needed to optimize the position of the capillary tip relative to sprayer tip for a stable current without compromising the sensitivity. 18. The MS spray chamber should be cleaned with a lint-free cloth (Agilent part # 05980-60051). If the spray shield is too dirty, an abrasive paper (8000 grit, Agilent part # 8660-0852) can be used to remove stains.

Acknowledgment This work was supported by NIH grant (5-R21MH107985-02). References 1. Wang J, Warner IM (1994) Chiral separations using micellar electrokinetic capillary chromatography and a polymerized chiral micelle. Anal Chem 66:3773–3776 2. Billiot EJ, Thibodeaux SJ, Shamsi SA, Warner IM (2000) Evaluating chiral separation interactions by use of diastereomeric polymeric dipeptide surfactants. Anal Chem 71:4044–4049 3. Rizvi SA, Zheng J, Apkarian RP, Shamsi SA (2006) Polymeric sulfated amino acid surfactants: a class of versatile chiral selectors for micellar electrokinetic chromatography

(MEKC) and MEKC-MS. Anal Chem 79:879–898 4. Wang B, He J, Shamsi SA (2011) A high throughput multivariate optimization for the simultaneous enantioseparation and detection of barbiturates in micellar electrokinetic chromatography-mass spectrometry. J Chromatogr Sci 48:572–583 5. He J, Shamsi SA (2013) Chiral separations, methods and protocols (second edition). In: Scriba GKE (ed) CMEKC-MS with polymeric surfactants. Humana Press, New York, pp 319–348

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6. He J, Shamsi SA (2009) Multivariate approach for the enantioselective analysis in MEEKCMS: II. Optimization of 1,10 -binapthyl-2,20 -diamine in positive ion mode. J Sep Sci 32:1916–1926 7. Billiot EJ, Warner IM (2000) Examination of structural changes of polymeric amino acidbased surfactants on enantioselectivity: effect of amino acid order, steric factors, and number and position of chiral centers. Anal Chem 72:1740–1748 8. Valle BC, Morris KF, Fletcher KA, Fernand V, Sword DM, Eldridge S, Larive CK, Warner IM (2006) Understanding chiral micellar separations using steady state fluorescence anisotropy, capillary electrophoresis and NMR. Langmuir 23:425–435 9. Agnew-Heard KA, Shamsi SA, Warner IM (2000) Optimizing enantioseparation of phenylthiohydantoin amino acids with polymerized sodium undecanoyl-L-valinate in chiral electrokinetic chromatography. J Liq Chromatogr Relat Technol 239:1301–1317 10. Liu Y, Shamsi SA (2015) Development of novel micellar electrokinetic chromatography mass spectrometry for simultaneous enantioseparation of venlafaxine and dimethylvenlafaxine: application to analysis of drug-

drug interactions. J Chromatogr A 1420:119–128 11. Wang X, Hou J, Shamsi SA (2013) Development of a novel chiral micellar electrokinetic chromatography-tandem mass spectrometry assay for simultaneous analysis of warfarin and hydroxywarfarin metabolites: application to the analysis of serum samples of patients undergoing warfarin therapy. J Chromatogr A 1271:207–216 12. Liu Y, Lin B, Wang P, Shamsi SA (2016) Synthesis, characterization and application of polymeric α-D-glucopyranoside based surfactant: application for enantioseparation of chiral pharmaceuticals in micellar electrokinetic chromatography-tandem mass spectrometry. Electrophoresis 37:913–923 13. Liu Y (2016) Chiral capillary electrophoresismass spectrometry: Developments and applications of novel glucopyranoside molecular micelles. PhD dissertation, Georgia State University, Atlanta, GA 14. Liu Y, Wu B, Wang P, Shamsi SA (2016) Synthesis, characterization and application of polysodium N-alkylenyl α-D-glucopyranoside surfactants for micellar electrokinetic chromatography-tandem mass spectrometry. Electrophoresis 37:913–923

Chapter 26 Application of an (18-Crown-6)-2,3,11,12-Tetracarboxylic Acid-Based Chiral Stationary Phase in Capillary Electrochromatography Wonjae Lee, Kyung Tae Kim, and Jong Seong Kang Abstract Capillary electrochromatography is employed for the enantioseparation of α-amino acids and their derivatives. The synthesis and application of a covalently bonded chiral stationary phase containing ( )-(18-crown-6)-2,3,11,12-tetracarboxylic acid as chiral selector is described. Enantioseparations are performed using methanol/Tris-citric acid (20 mM, pH 3.0–4.5) (20:80, v/v) as mobile phase. Key words Amino acid, Capillary electrochromatography, Chiral stationary phase, (18-Crown-6)2,3,11,12-tetracarboxylic acid, Enantioseparation

1

Introduction Capillary electrochromatography (CEC) is considered a promising approach for analytical scale enantioseparations. CEC is a technique combining the advantages of both, capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). The electroosmotic flow (EOF) is used to propel the mobile phase through a capillary with typical inner diameters of 50–150 μm packed with a suitable chiral stationary phase. (18-Crown-6)-2,3,11,12-tetracarboxylic acid is a chiral crown ether, which has shown good enantioselectivity towards a wide spectrum of protonated primary chiral amines and amino acids as well as some secondary amine compounds in several analytical techniques [1, 2]. For example, (18-crown-6)-2,3,11,12-tetracarboxylic acid not only has been widely utilized as a chiral selector for the resolution of racemic amino compounds in CE and in NMR [2–9], but also has been used successfully as a chiral stationary phase for HPLC [10–19]. Both enantiomers, i.e., (+)- and ( )(18-crown-6)-2,3,11,12-tetracarboxylic acid, are commercially available.

Gerhard K. E. Scriba (ed.), Chiral Separations: Methods and Protocols, Methods in Molecular Biology, vol. 1985, https://doi.org/10.1007/978-1-4939-9438-0_26, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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In this chapter, the preparation of a chiral stationary phase with ( )-18-crown-6-2,3,11,12-tetracarboxylic acid covalently bound to aminopropyl silica gel is described. The application to the separation of amino acids and their derivatives by CEC is reported.

2

Materials

2.1 CE Apparatus and Equipment

1. A commercial CE instrument, allowing the application of pressure on both buffer reservoirs (for example, a HP3DCE system from Agilent, Waldbronn, Germany, is suitable). 2. A commercial ultrasonic bath for sample dissolution, degassing of the mobile phase, and preparation of the slurry of the stationary phase. 3. A suitable device for packing and fusing the capillary columns. 4. A commercial pH meter. 5. 0.2 μm pore size membrane filters. 6. 100 μm i.d., 365 μm o.d. polyimide-coated fused silica capillaries. 7. A capillary column cutter with diamond blade (e.g., a Shortix® cutter). 8. A suitable device for packing of the capillary, composed, for example, of a HPLC pump and a stainless steel slurry reservoir containing a 0.5 μm frit at the inlet end.

2.2 Chemicals for Synthesis of CEC Column

1. ( )-(18-Crown-6)-2,3,11,12-tetracarboxylic acid. ˚ , 5 μm AkzoNobel, 2. Aminopropyl silica gel (Kromasil 100 A Sundsvall, Sweden). 3. Tris(1,3-dichloro-2-propyl)phosphate.

2.3 Mobile Phase and Sample Solutions

Prepare all solutions using ultrapure water prepared by a suitable purification system to reach a resistivity of 18.2 MΩ cm at 25  C. Use HPLC-grade methanol and analytical grade reagents.

2.3.1 Mobile Phase

Dissolve 0.86 g Tris in 80 mL water. Adjust the pH to 4.0 using a solution of 3.84 g citric acid in 100 mL water (see Note 1). Transfer to a 100 mL volumetric flask and make up to the volume with water. Mix 10 mL of methanol with 40 mL of the Tris–citric acid buffer. Filter through 0.2 μm membrane filters and degas by sonication before use (see Note 2).

2.3.2 Sample Solutions (1 mg/mL)

Weigh 10 mg of the respective racemic amino acid or amino acid derivative or alternatively 5 mg of each amino acid enantiomer in a 10 mL volumetric flask. Add about 5–6 mL mobile phase and dissolve compounds. Sonicate in case of slow dissolution. Make up to the mark with mobile phase and store in a refrigerator.

CEC Using Chiral Crown Ether-Based Chiral Stationary Phase

3

447

Methods Carry out all procedures at room temperature unless otherwise specified. Carry out all synthetic procedures in a ventilated hood. Adhere to the appropriate safety instructions when handling the chemicals (see Note 3).

3.1 Synthesis of ( )(18-Crown-6)tetracarboxylic AcidBonded Silica

1. Weigh 300 mg of ( )-(18-crown-6)-2,3,11,12-tetracarboxylic acid into a 100 mL round bottom flask equipped with a condenser and a magnetic stir bar. Add 30 mL freshly distilled acetyl anhydride and reflux for 24 h. Remove acetyl chloride under reduced pressure using a rotary evaporator. ( )(18-crown-6)-2,3,11,12-tetracarboxylic dianhydride is obtained as a white, crystalline solid (yield approximately 275 mg) (see Note 4). 2. Add 2.5 g aminopropyl silica gel and 50 mL benzene (see Note 5) to a 100 mL round bottom flask equipped with a Dean-Stark trap, a condenser and a magnetic stir bar. Reflux until all water is removed by azeotropic distillation. Remove excess of benzene under vacuum in a rotary evaporator to afford dried aminopropyl silica gel (see Note 6). 3. Suspend the dried aminopropyl silica gel and 20 mL dried methylene chloride in a flask and add 0.24 mL triethylamine. 4. Dissolve 275 mg ( )-(18-crown-6)-2,3,11,12-tetracarboxylic dianhydride prepared in step 1 in 5 mL dry methylene chloride. Add the solution dropwise to the stirred aminopropyl silica gel suspension at 0  C (see Note 7). 5. Upon complete addition of the crown ether solution, stir at 0  C for 2 h. Let the suspension warm to room temperature and stir for 2 days at room temperature to obtain the crown ether-bonded silica gel. 6. Successively wash (about 30 mL for each solvent) the chiral crown ether-bonded silica gel with methanol, 1 M HCl solution, water, methanol, methylene chloride, and hexane. 7. Dry the (18-crown-6) chiral ether-bonded silica gel under vacuum (at 1 torr).

3.2 Capillary Column Packing

1. Suspend 10 mg of the ( )-18-crown-6-2,3,11,12-tetracarboxylic acid-bonded silica gel in 10 mL methanol and sonicate for 30 min. 2. Cut a 35 cm long piece of the capillary (see Note 8) and attach to a stainless steel slurry reservoir (30 mm  4.6 mm i.d.). 3. Place the slurry into a slurry reservoir containing a stainless steel 0.5 μm frit at the inlet end.

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4. Close the outlet end of capillary with stainless steel 0.5 μm frit (see Note 9). 5. Connect the reservoir to an HPLC pump and pump the slurry into the capillary at 8000 psi (see Note 10). 6. When the packed bed is about 20 cm long, remove the reservoir and connect the packed capillary to the same pump. Flush distilled water through the capillary. 7. After washing the capillary for about 30 min with water, prepare the inlet frit at a distance of 2 cm from the end of the packed bed by fusing the silica gel using a tungsten filament (see Note 11). 8. Prepare the outlet frit in the same way at a distance of about 19 cm from the end of the packed bed. 9. Remove the residual unwanted packing material by pumping methanol in the reversed direction. Use a lower pressure for this procedure. 10. Prepare the detection window immediately after the outlet frit by removing the polyimide coating using a tungsten filament (see Note 12). 11. Remove the extra part of the capillary by cutting 8 cm away from detection window (see Note 13). 12. Mount the packed capillary into the capillary holder of the instrument and condition the capillary by flushing with the mobile phase for 12~24 h before the chromatographic measurements. 3.3

CEC Analysis

1. Install capillary in the CE instrument according to the instructions of the manufacturer. 2. Add mobile phase to the inlet reservoir and place an empty reservoir at the outlet end. Apply a pressure at about 12 bar at the inlet end for several minutes and check for liquid drops in the outlet reservoir. 3. Fill both mobile phase reservoirs with mobile phase and apply a voltage of 20 kV for 5 min. 4. Inject the sample of the racemic amino acid or of the derivatives at 15 kV for 5 s (see Note 14). 5. Apply an external pressure of 12 bar for both buffer reservoirs. 6. Set detector to 210 nm and column temperature to 25  C. 7. Apply separation voltage of 20 kV and perform the CEC analysis (see Note 15). Examples of chromatograms of CEC enantioresolutions of tryptophan and 4-bromophenylalanine are shown in Fig. 1. Chromatographic data for other amino acids including variation of the

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Fig. 1 Typical electropherograms for the separation pattern of (a) tryptophan and (b) 4-bromophenylalanine at pH 4.0. CEC conditions: injection, 15 kV for 5 s; applied voltage 20 kV; applied pressures, 12 bar on both vials; detection, 210 nm; mobile phase 20:80 (v/v %) methanol/Tris–citric acid buffer (20 mM) (Reproduced by permission of Springer from ref. 20 © 2015)

pH of the mobile phase are summarized in Table 1. The separations are carried out in an analogous way as described above using the respective mobile phase listed in Table 1. Preparation of the buffers is performed according to Subheading 2.3 but with the pH adjusted to the desired pH in the range 3.0–4.5. Further examples can also be found in ref. 20.

4

Notes 1. Mobile phases with various pH values (pH 3.0, 3.5, and 4.5) can be prepared by the same method as the case of pH 4.0. Concentrated citric acid (0.5 M) can be used at first to narrow the gap from the starting pH to the required pH. After getting close to the desired pH, it is recommended to use diluted citric acid (0.05 M) to avoid a sudden drop below the required pH. 2. Solvents can be degassed by sonication under light vacuum for 5–10 min. If the solvents have been exposed to the atmosphere for a long time, they should be degassed again. Degassing can also be achieved by sonication. 3. All used chemicals described here should be considered hazardous to human health and should be handled with care in a

t1

–

14.53

13.58

11.50

11.90

11.62

10.08

13.85

–

11.41

12.85

13.44

13.78

14.59

14.76

–

Analyte

Phenylalanine

4-Bromophenylalanine

4-Chlorophenylalanine

4-Fluorophenylalanine

3-Fluorophenylalanine

2-Fluorophenylalanine

4-Amino-phenylalanine

4-Nitro-phenylalanine

4-Hydroxyphenylalanine

3-Hydorxyphenylalanine

Phenylglycine

4-Hydorxyphenylglycin

4-Fluorophenylglycine

Tryptophan

5-Hydroxytryptophan

α-Methyltryptamine

pH 3.0

–

18.46

18.42

18.41

17.06

16.71

13.67

–

18.57

12.69

16.82

15.74

14.84

19.27

21.65

–

t2

–

1.25

1.26

1.34

1.27

1.30

1.20

–

1.34

1.26

1.45

1.32

1.29

1.42

1.49

–

α

–

3.05

2.73

3.25

3.08

2.88

2.30

–

3.13

1.63

3.50

3.15

3.06

3.30

3.35

–

Rs

–

9.93

9.93

8.11

8.16

7.72

7.87

7.24

8.73

9.76

7.48

7.57

7.35

8.49

9.04

7.15

t1

pH 3.5

–

11.91

12.23

10.26

9.93

9.59

9.30

8.32

10.93

10.82

10.03

9.50

8.89

11.59

13.02

8.84

t2

–

1.20

1.23

1.27

1.22

1.24

1.18

1.15

1.25

1.11

1.34

1.25

1.21

1.36

1.44

1.24

α

–

1.90

1.99

2.63

2.26

2.34

1.92

2.01

2.41

1.04

2.71

2.14

1.71

2.60

2.59

2.11

Rs

9.54

7.37

7.42

5.97

6.10

5.86

6.15

6.11

6.61

8.88

5.67

5.76

5.65

6.40

6.76

5.26

t1

pH 4.0

9.85

8.52

8.79

7.33

7.23

7.04

7.11

6.92

7.87

9.93

6.99

6.76

6.47

7.76

8.41

6.06

t2

1.03

1.16

1.18

1.23

1.19

1.20

1.16

1.13

1.19

1.12

1.23

1.17

1.15

1.21

1.24

1.15

α

0.16

1.60

1.75

2.30

1.87

2.00

1.75

1.52

1.75

0.68

1.95

1.76

1.42

2.00

2.43

1.25

Rs

12.35

5.85

5.89

4.65

4.74

4.55

4.58

4.79

5.00

10.67

4.39

4.49

4.40

5.10

5.50

4.38

t1

pH 4.5

13.01

6.84

7.05

5.82

5.70

5.57

5.40

5.53

6.16

12.62

5.66

5.47

5.21

6.53

7.31

5.25

t2

1.05

1.17

1.20

1.25

1.20

1.22

1.18

1.16

1.23

1.18

1.29

1.22

1.18

1.28

1.33

1.20

α

0.16

1.79

1.90

1.84

1.73

1.95

1.72

1.35

1.82

0.95

1.94

1.71

1.68

2.44

2.37

1.75

Rs

Table 1 Chromatographic parameters of the enantiomeric resolution of α-amino acids and their derivatives by CEC at various pH values (Reproduced by permission of Springer from ref. 20 © 2015)

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ventilated hood. Wear protective clothing, gloves as well as safety glasses. 4. Freshly distilled acetic acid anhydride should be used for a fast and quantitative reaction. 5. Benzene is carcinogenic and should be handled with specific precautions. Do not breathe vapor or fumes. Use process enclosures, local exhaust ventilation. Do not get in your eyes, on skin, or on clothing. Wash thoroughly after handling. Wear protective goggles and impervious protective clothing to prevent skin contact. 6. When the excess benzene is removed using a rotary evaporator, a slow spinning rate is required to dry the aminopropyl silica gel. 7. The reaction should be performed under an argon atmosphere. 8. The capillary should be flushed with water and methanol successively for 10 min before attaching it to the reservoir. The washed capillary should be placed under vacuum to completely remove the solvent before packing. 9. The stainless steel frit serves as a temporary inlet frit and will avoid the effort of making a first frit, which is later cut off. 10. Hit the stainless steel reservoir intermittently with a rubber hammer to avoid precipitation of the silica gel. 11. The frit can be fused with a heated filament. The fusing device is prepared easily in-house from a tungsten wire and a variable AC autotransformer. The fusing will be performed with increased temperatures controlled by the variable AC autotransformer. Distilled water should flow through the capillary during fusing. 12. The capillary becomes fragile when the polyimide coating is removed. Handle with care to avoid breaking the capillary. 13. The capillary must be cut in square with a diamond blade. The cut end of capillary can be verified with magnifier or microscope. 14. Other injection voltages or injection times may apply for other samples. Adjust voltage and time for the injections according to the instructions of the instrument manufacturer. 15. Consult the instruction manual of the instrument manufacturer to perform efficient instrument control and data acquisition.

Acknowledgment This work was supported by Chungnam National University.

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References 1. Hyun MH (2006) Preparation and application of HPLC chiral stationary phases based on (+)(18-crown-6)-2,3,11,12-tetracarboxylic acid. J Sep Sci 29:750–761 2. Paik MJ, Kang JS, Huang BS, Carey JR, Lee W (2013) Development and application of chiral crown ethers as selectors for chiral separation in high-performance liquid chromatography and nuclear magnetic resonance spectroscopy. J Chromatogr A 1274:1–5 3. Bang E, Jung JW, Lee W, Lee DW, Lee W (2001) Chiral recognition of (18-crown-6)tetracarboxylic acid as a chiral selector determined by NMR spectroscopy. J Chem Soc Perkin Trans 2:1685–1692 4. Lee W, La S, Choi Y, Kim KR (2003) Chiral discrimination of aromatic amino acids by capillary electrophoresis in (+)- and ( )(18-crown-6)-2,3,11,12-tetracarboxylic acid selector modes. Bull Kor Chem Soc 24:1232–1234 5. Lee W, Bang E, Lee W (2003b) Chiral resolution of diphenylalanine by high-performance liquid chromatography on a crown-etherbased chiral stationary phase and by NMR spectroscopy. Chromatographia 57:457–461 6. Cho SI, Shim J, Kim MS, Kim YK, Chung DS (2004) Online sample cleanup and chiral separation of gemifloxacin in a urinary solution using chiral crown ether as a chiral selector in microchip electrophoresis. J Chromatogr A 1055:241–245 7. Xiao YG, Peter CH (2006) Enantiomeric separation of underivatized small amines in conventional and on-chip capillary electrophoresis with contactless conductivity detection. Electrophoresis 27:4375–4382 8. Lili Z, Lin Z, Reamer RA, Bing M, Zhinong G (2007) Stereoisomeric separation of pharmaceutical compounds using CE with a chiral crown ether. Electrophoresis 28:2658–2666 9. Abdalla AE, Fakhreldin OS (2011) Computational modeling of capillary electrophoretic behavior of primary amines using dual system of 18-crown-6 and -cyclodextrin. J Chromatogr A 1218:344–5351 10. Lee W, Jin JY, Baek CS (2005) Comparison of enantiomer separation on two chiral stationary phases derived from (+)-18-crown-62,3,11,12-tetracarboxylic acid of the same chiral selector. Microchem J 80:213–217 11. Hyun MH, Kim DH, Cho YJ, Jin JS (2005) Preparation and evaluation of a doubly

tethered chiral stationary phase based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid. J Sep Sci 28:421–427 12. Berkecz R, Sztojkov-Ivanov A, Llisz I (2006) High-performance liquid chromatographic enantioseparation of β-amino acid stereoisomers on a (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid-based chiral stationary phase. J Chromatogr A 1125:138–143 13. Jin JS, Lee W, Hyun MH (2006) Development of the antipode of the covalently bonded crown ether type chiral stationary phase for the advantage of the reversal of elution order. J Liq Chromatogr Relat Technol 29:841–848 14. Manolescu C, Grinberg M, Field C, Ma S, Shen S, Lee H, Wang Y, Granger A, Chen Q, McCaffrey J, Norwood D, Grinberg N (2008) Studies of the interactions of amino alcohols using high performance liquid chromatography with crown ether stationary phases. J Liq Chromatogr Relat Technol 31:2219–2234 15. Zhang C, Wei XH, Chen Z, Rustum AM (2010) Separation of chiral primary amino compounds by forming a sandwiched complex in reversed-phase high performance liquid chromatography. J Chromatogr A 1217:4965–4970 16. Lee A, Choi HJ, Jin KB, Hyun MH (2011) Liquid chromatographic resolution of 1-aryl1,2,3,4-tetrahydroisoquinolines on a chiral stationary phase based on (+)-(18-crown-6)2,3,11,12-tetracarboxylic acid. J Chromatogr A 1218:4071–4076 17. Asnin L, Sharma K, Park SW (2011) Chromatographic retention and thermodynamics of adsorption of dipeptides on a chiral crown ether stationary phase. J Sep Sci 34:3136–3144 18. Llisz I, Aranyi A, Pataj Z, Peter A (2012) Enantiomeric separation of nonproteinogenic amino acids by high-performance liquid chromatography. J Chromatogr A 1269:94–121 19. Kim KH, Seo SH, Kim HJ, Jeun EY, Kang JS, Mar W, Youm JR (2003) Determination of terbutaline enantiomers in human urine by capillary electrophoresis using hydroxypropyl-β-cyclodextrin as a chiral selector. Arch Pharm Res 26:120–123 20. Wu W, Kim KT, Adidi SK, Lee YK, Cho JW, Lee W, Kang JS (2015) Enantioseparation and chiral recognition of a-amino acids and their derivatives on ( )-18-crown-6-tetracarboxylic acid bonded silica by capillary electrochromatography. Arch Pharm Res 38:1499–1505

Chapter 27 Experimental Design Methodologies for the Optimization of Chiral Separations: An Overview Luiz Carlos Klein-Ju´nior, Debby Mangelings, and Yvan Vander Heyden Abstract In this chapter, the application of design of experiments (DoE) for chiral separation optimization using supercritical fluid chromatography (SFC), liquid chromatography (LC), capillary electrophoresis (CE), and capillary electrochromatography (CEC) methods is reviewed. Both screening and optimization steps are covered, including a discussion of each aspect, such as factor-, level-, and response selection. Different designs are also presented, highlighting their applications. Key words Method development, Design of experiments, Chiral separation, Supercritical fluid chromatography, Liquid chromatography, Capillary electrophoresis, Capillary electrochromatography

1

Introduction Chirality is a recurrent topic in drug design. From 1997, when chiral issues were raised at the Conference on Pharmaceutical Ingredients, until today, chiral drugs remain an important matter when considering drug development [1]. Whether drugs should be synthesized as racemic mixtures or as a single enantiomer, what are the pharmacological and toxicological activities of each enantiomer and how enantiomeric purity should be assayed are persistent inquiries [1–3]. In fact, this resulted in the term “chiral switch,” which is allusive to the development of single enantiomers based on existing drugs, marketed as racemates [1, 3]. Gu¨bitz and Schmid [4] stated that about half of the drugs are chiral and about 25% are used as pure enantiomers. However, pure enantiomer drugs are growing in proportion. In fact, Agranat [5] observed that in 1992, 40% of newly approved drugs were enantiomerically pure. In 2010, this fraction reached 70%. In 2015, all chiral drugs approved by the FDA were single enantiomers, with the exception of lesinurad [1]. In this sense, the development of technologies which are able to provide chiral information is essential in drug discovery [1, 6].

Gerhard K. E. Scriba (ed.), Chiral Separations: Methods and Protocols, Methods in Molecular Biology, vol. 1985, https://doi.org/10.1007/978-1-4939-9438-0_27, © Springer Science+Business Media, LLC, part of Springer Nature 2019

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Among these technologies, those related to chiral separations are indispensable for the study of drugs. The reason is that industry sometimes prefers to synthesize racemates and separates them into enantiomers afterwards rather than investing in enantioselective synthesis of a single enantiomer. In addition, regulatory authorities impose that enantioselective identification and quantification methods must be developed for each active pharmaceutical ingredient with chiral properties. They also demand that pharmacokinetic and toxicological assays are performed with both pure enantiomers and the racemate. Therefore, enantioselective separations are important in pharmaceutical industry. However, since physicochemical properties are identical for enantiomers, standard achiral chromatographic or electromigration techniques are unable to provide a separation [7]. It is imperative that a chiral environment is present to separate enantiomers. In chiral separations, either indirect or direct approaches can be used. In the direct approach, the separation is based on the use of a chiral selector, such as polysaccharides or cyclodextrins, which can be fixed to a support, forming a chiral stationary phase (CSP), or added to the mobile phase, as chiral mobile phase additive (CMPA) [6]. In this approach, separation of the enantiomers is based on the formation of transient diastereoisomeric complexes between the selector and enantiomers. The indirect approach, less used nowadays, involves the formation of diastereomeric derivatives by chemical reactions and subsequent separation in an achiral environment [1]. Taking into account that the direct approach is the most frequently used, the complexity level of the optimization process to achieve a good resolution between the analytes is higher than for an achiral separation. In addition to standard factors to be optimized (e.g., mobile phase composition, temperature, flow rate), the chiral selector, its concentration, and occasionally the stationary phase are additional factors to be added to the list. The factors to be varied depend on the approach that is used to obtain the separations, i.e., by means of a CSP or CMPA. In this sense, the optimization process is ideally performed by a design of experiments (DoE). DoE is a statistical methodology used for planning experiments and analyzing results. To achieve the objectives, multivariate methods are used, occasionally giving valuable interactions information, with a minimum number of experiments. The most used DoE approach is Response Surface Methodology (RSM). In this approach, the response y is dependent on a given number of factors k, varied at l levels. This usually results in building a second-order polynomial model, where the final aim is to determine suitable levels of the k factors resulting in the best or an acceptable response y [8–10]. Traditionally, a DoE approach can be divided into two parts: the screening of factors and the optimization of the levels of the most important factors. Therefore, in the first step, several factors

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(and occasionally their interactions) will be evaluated regarding their significant influence on the response(s). For the optimization, the significant factors previously selected will be optimized to achieve the best combination affording an optimal response. In the end, the theoretically predicted conditions should be validated experimentally [11]. In this book chapter, the application of DoE in the development of analytical chiral separation methods using chromatographic or electromigration techniques will be reviewed and discussed, covering the last 5 years. It is divided into two general topics: screening designs and response surface designs. Each topic will be further subdivided according to the separation technique.

2

Screening Designs Screening designs are used as a first step in an experimental optimization when examining a (relatively) large number of factors.1 Through a relatively small number of experiments, it aims at selecting important factors to be further optimized, using RSM. Therefore, it is imperative to properly select which factors will be evaluated at which levels.2 Factors can affect the response3 and are divided into controlled or uncontrolled. The latter cannot be included in a screening design. However, their influence must be reduced maximally between experiments. On the other hand, the controlled factors, all of which can somehow affect the response, should be evaluated in the screening design. In addition, factors can be also divided as qualitative (levels situated on a discrete scale; e.g., stationary phase type), quantitative (varying on a continuous scale; e.g., pH) and mixture-related (for mixtures composition, e.g., % organic modifier in mobile phase) [8, 9]. Some thought must also be given when selecting factor levels. Usually, two levels are evaluated in a screening: one at a low value and another at a high value. This will define the experimental domain.4 These maximum and minimum limits must be carefully chosen. Close limits may not allow enough variation in the response, leading to an inappropriate elimination of an important factor for the optimization step. On the other hand, large variations between the levels may result in a too large experimental domain in which some factor levels combinations (experiments) cannot be

1

Factors are variables that can be varied in an independent manner from each other, e.g., pH, temperature, concentration [9, 12]. 2 Levels are the values of a specific factor. Therefore, temperature (a factor), for example, can be evaluated at 30, 40, and 50
C (levels) [9, 12]. 3 Response is the measurable output from experiments, e.g., migration time, resolution [12]. 4 Experimental domain is defined by the ranges of the factors levels [12].

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performed anymore. Frequently, the levels are reported as codes, i.e., 1 (low level) and þ1 (high level) [9]. Finally, when executing a screening design, randomization and replication are also important issues. An experimental design should be ideally randomized, in order to minimize systematic influences in the response. Moreover, replication may give random variance estimation, avoiding misinterpretation of the results [8, 9]. However, it should be mentioned that when a large uncontrolled effect (e.g., a time effect) occurs during the execution of the design, randomization does not eliminate its influence from all factor-effect estimates. Concerning replication, it should be mentioned that some replicates (e.g., when measured under repeatability conditions) may underestimate the experimental error on a nonsignificant effect. Summarizing, factors, levels, and error estimation will impact on the screening design selection and its outcome. The screening design will be used to determine which factors have a significant impact on the response. For this purpose, usually models with linear or second-order interaction terms are built. In the first model, only the influence of the main effects is estimated. In the second model, the main effects are determined, as well as interactions, usually between two factors [11]. A Full Factorial Design, 2k, is occasionally used as screening design. This design allows estimating the effects of all factors, as well as their interaction effects. In this design, k factors are evaluated at two levels [low (1) and high (þ1)], giving 2k experiments [9, 11, 13]. Examples of Full Factorial Designs 22 and 23, giving a unique combination of levels and factors for each run (experiment), are shown in Table 1. In Fig. 1, a graphical representation of these Full Factorial Designs is given. However, practical execution of a 22 design is not recommended, as the low number of replicates at a given factor level may lead to poor effect estimates. The number of experiments of a Full Factorial Design increases exponentially with the number of factors, and becomes too high quickly to be feasible. A variation of the Full Factorial Design might then be used, i.e., when several factors (usually more than four) must be included in the screening design. Those designs are called Fractional Factorial Designs and they allow evaluating the effect of a larger number of factors on a given response, but with a smaller number of experiments. The Fractional Factorial Design is a fraction of the Full Factorial Design for a given number of factors. A Fractional Factorial Design allows estimating the main effects and occasionally some interaction effects. However, in a Fractional Factorial Design at least two effects are confounded, i.e., are estimated together. The design is represented as 2k-p, where k is the number of factors and p represents the fraction of the Full Factorial Design considered. For example, a 25 Full Factorial Design would require 32 runs. A half-fraction Factorial Design 25-1 contains

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Table 1 Full factorial designs Two factors (22)

Three factors (23) Factors

Factors

Run

x1

x2

Run

x1

x2

x3

1

1

1

1

1

1

1

2

+1

1

2

+1

1

1

3

1

+1

3

1

+1

1

4

+1

+1

4

+1

+1

1

5

1

1

+1

6

+1

1

+1

7

1

+1

+1

8

+1

+1

+1

Fig. 1 (a) Two-factor two-level Full Factorial Design (22), and (b) three-factor two-level Full Factorial Design (23)

16 runs, still affording to estimate the five main as well as nine interaction effects [8, 11]. Notice that each estimated effect here is a confounding of two effects. For five factors also a quarterFraction Factorial Design (25-2) may be considered, with 8 experiments to be executed. A specific type of factorial design is the Plackett-Burman Design, developed in 1946 [14]. It assumes, as in the case for

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Table 2 Two-level Plackett-Burman Design to evaluate 11 factors in 12 runs Factors Run

x1

x2

x3

x4

x5

x6

x7

x8

x9

x10

x11

1

+1

+1

1

+1

+1

+1

1

1

1

+1

1

2

1

+1

+1

1

+1

+1

+1

1

1

1

+1

3

+1

1

+1

+1

1

+1

+1

+1

1

1

1

4

1

+1

1

+1

+1

1

+1

+1

+1

1

1

5

1

1

+1

1

+1

+1

1

+1

+1

+1

1

6

1

1

1

+1

1

+1

+1

1

+1

+1

+1

7

+1

1

1

1

+1

1

+1

+1

1

+1

+1

8

+1

+1

1

1

1

+1

1

+1

+1

1

+1

9

+1

+1

+1

1

1

1

+1

1

+1

+1

1

10

1

+1

+1

+1

1

1

1

+1

1

+1

+1

11

+1

1

+1

+1

+1

1

1

1

+1

1

+1

12

1

1

1

1

1

1

1

1

1

1

1

most Fractional Factorial Designs, that interactions can be neglected. Therefore the main effects are estimated from a low number of experiments. A Plackett-Burman Design contains a number of experiments that is a multiple of four and the number of factors that can be evaluated at the most is one less than the number of experiments. Therefore, 11 factors can be evaluated with only 12 experiments (Table 2) [9, 11]. Thus, this design is frequently used when a larger number of factors must be evaluated. When the factors are selected, levels are established, and the experimental setup is built, the experiments must be executed as planned in order to obtain the responses for each run. Finally, the results must be analyzed in order to estimate the significance of the effects on the response. Traditionally, the influence of factors is determined either by the estimation of regression coefficients or effects. Coefficients for the factors can be estimated according to Eq. 1 as follows: y ¼ β0 þ

k X

βi x i þ ε

ð1Þ

i¼1

where y is the response, k the number factors, β0 the intercept or the constant term, βi the real coefficients, xi the factors, and ε the error. However, βi coefficients are most often estimated as b after

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obtaining the experimental data. Using least square regression, it is possible to establish a mathematical equation to describe the response variation as a function of the different factors. The coefficients, b, can be estimated as:  T 1  T  ð2Þ Xm:n Xn:m ym:i bn:1 ¼ Xn:m where X is the design matrix, XT the transposed matrix, y the response vector, and m and n are the number of lines and columns of the matrices [12]. Another option is to estimate the effects (Ex) on each response y: P P y ðþ1Þ  y ð1Þ ð3Þ Ex ¼ N =2 where ∑y(þ1) and ∑y(1) are the sums of the responses of factor x at high (þ1) and low (1) levels, and N is the number of design experiments. For interpretation and determination of the most important coefficients/effects, usually graphical and/or statistical analysis is performed. Both Pareto charts (Fig. 2) and normal probability or half-normal probability plots (Fig. 3) can be drawn. A Pareto chart is a bar plot where effects (or t values from a t-test) are plotted from the most to the least important. The bar length represents the effect (or t value) of each factor and a line, crossing all bars, represents the critical t value. Therefore, bars which exceed this line are considered significant. For the normal or half-normal probability plots, those effects which deviate from the line formed by the nonsignificant

Fig. 2 Pareto chart of a Fractional Factorial Design 25-1, where the red line represents the critical effect (α ¼ 0.05)

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Fig. 3 Half-normal probability plot for 11 effects (Reproduced with permission of Elsevier from ref. 15 © 2011)

ones are considered significant. Those which are on the straight line are unimportant. However, in addition to graphical evaluation, statistical analysis should ideally also be performed. This is often performed by a ttest: tx ¼

jE x j , t critical ðSEÞe

ð4Þ

where |Ex| is the absolute effect of factor x and (SE)e is the standard error. tx is compared to the tabulated critical value of t, which is defined based on the number of degrees of freedom and at a given significance level (most often α ¼ 0.05). When tx of an effect is equal to or larger than tcritical, it is significant. Alternatively, |Ex| can be compared to Ecritical, estimated as: E critical ¼ t critical  ðSEÞe , jE x j

ð5Þ

Those effects (in absolute values) that are equal or larger than the critical effect can be considered significant. The standard error can be estimated in several ways, such as from the variance of replicated experiments, or from a priori or a posteriori considered negligible effects. In the first case, (SE)e is estimated as: rffiffiffiffiffiffiffi 2s 2 ðSEÞe ¼ ð6Þ n where s2 is the variance of n replicated experiments. For the second approach, (SE)e is estimated from a priori considered negligible effects EN, as follows: sffiffiffiffiffiffiffiffiffiffiffiffi P 2ffi EN ð7Þ ðSEÞe ¼ nN

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Table 3 Factors examined in the screening designs of some case studies using liquid chromatography (a) or capillary electrophoresis/capillary electrochromatography (b) Factors

Type of factor

Number of levels

Ref.

(a)

Type of acid Type of base % organic modifier (MeOH) in mobile phase Concentration of acid Concentration of base

Qualitative Qualitative Mixture Quantitative Quantitative

4 4 2 2 2

[16] [16] [16] [16] [16]

(b)

Concentration of chiral selector Temperature Background electrolyte (BGE) concentration Background electrolyte (BGE) pH Voltage Type of cyclodextrin Capillary length

Quantitative Quantitative Quantitative Quantitative Quantitative Qualitative Quantitative

2 or 3 2 or 3 2 or 3 2 or 3 2 or 3 2 2

[17–20] [17–20] [17–20] [17–20] [17–20] [17] [18]

Finally, the third approach determines standard error from a posteriori considered negligible effects using the algorithm of Dong [15]. It estimates the (SE)e based on unimportant effects Ek, as follows: sffiffiffiffiffiffiffiffiffiffiffiffi P 2 Ek ðSEÞe ¼ ð8Þ m where m is the number of Ek. It also represents the degrees of freedom for tcritical. The unimportant factors are selected as: jE k j  2:5  s 0

ð9Þ

s 0 ¼ 1:5  medianjE x j

ð10Þ

where s0 is estimated as:

with median |Ex| the median of the absolute effects [15]. Notice that instead of a t-test also an equivalent ANOVA table can be used to determine the significance of effects. The important effects selected based on graphical and statistical analysis can be further optimized by RSMs, giving ultimately an optimal chiral separation. In the next sections, the application of screening designs for both chromatographic and electrophoretic chiral separations will be reviewed. Table 3 contains factors and numbers of levels evaluated in the different studies. 2.1 Chromatographic Methods

Chromatographic methods, such as liquid chromatography (LC) and supercritical fluid chromatography (SFC), are frequently used for the separation of chiral compounds, either at analytical or preparative level. In the last years, several technological

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improvements were made to these techniques, such as the development of Ultra-High-Performance Liquid Chromatography (UHPLC) in LC and the introduction of modern and more robust instruments for SFC, mainly with the aim of reducing analysis time and increasing resolution. Besides these improvements, the development of new chiral selectors can be considered a milestone for chiral drug separation and quality control. From the use of chiral crown ethers in the 1970s, until nowadays, about four decades have passed [21] and many other selectors are used nowadays. The most used CSP contains polysaccharide, macrocyclic antibiotic, cyclodextrin, or Pirkle-type selectors. The application of DoE can be a powerful approach to optimize enantiomeric separation. Using such approach, Hanafi and L€ammerhofer [16] applied a screening design to select important factors for the chiral separation of N-[(9H-fluoren-9-yl-methoxy)carbonyl]-L-leucine (Fmoc leucine), tryptophan, and salbutamol. A Cinchona-based zwitterionic stationary phase, obtained by the fusion of quinine carbamate and (S,S)-trans-2-aminocyclohexanesulfonic acid, was used on HPLC. The authors used a 2m4n Taguchi orthogonal array design with mixed levels, more specifically an L16 (2242) design, where m represents the number of factors at two levels (percentage of methanol in acetonitrile-based mobile phase, molarity of acid and molarity of base) and n the number of factors at four levels (type of acid and type of base) (see Table 4). Resolution was calculated as response. The authors observed that the best results were obtained for run 7 (Table 4), with acetic acid and diethylamine additives. Therefore, their molarities (ranging from 5 to 50 mM) and the percentage of methanol (from 5 to 40%) were selected for an optimization study. 2.2 Electromigration Techniques

Capillary electrophoresis (CE) and capillary electrochromatography (CEC) are electromigration techniques that are largely applied to environmental analysis and drug quality control. For CE, the separation is typically performed in a background electrolyte with a high electric potential. For CEC, an electrical field is also applied, similar to CE, but it is applied on a capillary filled with a stationary phase. Both acidic and basic compounds can be separated in CE and CEC, while the latter technique only allows the separation of neutral species [22–24]. CE and CEC are largely applied for enantiomeric separations. Linked to DoE, a powerful approach for the separation optimization of chiral compounds is created. Such approach was performed by Orlandini et al. [17, 18], Krait et al. [19], and Meng et al. [20]. Orlandini et al. [17, 18] used a similar strategy to achieve the chiral separation of levosulpiride using CE and ambrisentan using MEKC, respectively. Enantioresolution was used as response. In both studies, an asymmetric screening design was applied (see Table 5). Orlandini et al. [17] evaluated the type of neutral

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Table 4 2m4n screening design for the separation of Fmoc leucine, tryptophan, and salbutamol (Reproduced by permission of Elsevier from ref. 16 © 2018) Run

Type of acid

Type of base

1

TFA

NH3

2

FA

3

% methanol

Molarity of acid (mM)

Molarity of base (mM)

30

25

25

NH3

100

25

50

HOAc

NH3

100

50

25

4

HFBA

NH3

30

50

50

5

TFA

DEA

100

50

50

6

FA

DEA

30

50

25

7

HOAc

DEA

30

25

50

8

HFBA

DEA

100

25

25

9

TFA

TEA

30

50

50

10

FA

TEA

100

50

25

11

HOAc

TEA

100

25

50

12

HFBA

TEA

30

25

25

13

TFA

DIPEA

100

25

25

14

FA

DIPEA

30

25

50

15

HOAc

DIPEA

30

50

25

16

HFBA

DIPEA

100

50

50

TFA trifluoroacetic acid, FA formic acid, HOAc acetic acid, HFBA heptafluorobutyric acid, DEA diethylamine, TEA trimethylamine, DIPEA N,N-diisopropylethylamine

cyclodextrin (as qualitative factor) at two levels, while Britton–Robinson buffer concentration, buffer pH, sulfated-β-cyclodextrin concentration, and neutral cyclodextrin concentration were evaluated at three levels. The design can be specified as L16 (2135). In Orlandini et al. [18], capillary length and temperature were evaluated at two levels: borate concentration, pH, γ-cyclodextrin concentration, sodium dodecyl sulfate concentration, and voltage at three levels. Following the screening design, RSM was used for further optimization of the most important factors, being pH (9.2–10.2), cyclodextrin concentration (36–50 mM), and voltage (24–30 kV). In Krait’s study [19], a Fractional Factorial Design 25-1 was also applied for the enantioseparation of ambrisentan using CE. Alternatively to the study of Orlandini et al. [18], voltage, capillary temperature, pH of the background electrolyte, acetate concentration, and γ-cyclodextrin concentration were all evaluated at three levels. Enantioresolution was used as response. As

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Table 5 Asymmetric screening design for the chiral separation of levosulpiride (Reproduced by permission of Elsevier from ref. 17 © 2015) Britton-Robinson Sulfated-β-cyclodextrin Neutral buffer sodium sulfate Run cyclodextrin concentration (mM) pH concentration (mM)

Neutral cyclodextrin concentration

Voltage (kV)

1

MβCD

10

3.0 15

20

18

2

MβCD

5

3.0 11

30

13

3

MβCD

15

2.5 11

20

18

4

HEβCD

15

3.5

7

20

13

5

MβCD

10

3.5 15

10

13

6

HEβCD

15

3.0 15

30

8

7

HEβCD

10

3.5 11

30

18

8

HEβCD

10

3.0 11

20

13

9

MβCD

10

3.0

7

20

8

10

MβCD

15

3.0 11

10

13

11

MβCD

5

3.5 11

20

8

12

HEβCD

5

2.5 15

20

13

13

MβCD

10

2.5

7

30

13

14

HEβCD

5

3.0

7

10

18

15

HEβCD

10

2.5 11

10

8

16

HEβCD

10

3.0 11

20

13

MβCD Methyl-β-cyclodextrin, HeβCD (2-hydroxyethyl)-β-cyclodextrin

outcome, the authors detected that voltage, capillary temperature, and acetate concentration influenced significantly the response. Meng et al. [20] used CE for the separation of three stereoisomeric impurities of sitafloxacin, including enantiomers and diastereoisomers. Initially, the authors evaluated several types of cyclodextrins [α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, methyl-β-cyclodextrin, dimethyl-β-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, highly sulfated β-cyclodextrin, (2-hydroxypropyl)-γ-cyclodextrin, highly sulfated γ-cyclodextrin], as well as a ligand-exchange chiral selector composed of Cu2+ and D-phenylalanine (D-Phe). The best result was obtained for highly sulfated γ-cyclodextrin. However, the elution order was not satisfactory, since sitafloxacin eluted before the impurities, which is not desirable when the sample must be overloaded for the determination of minor impurities. In this sense, combinations of a cyclodextrin and the chiral ligand-exchange selector were

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Fig. 4 Electropherograms obtained for the separation of stereoisomeric impurities (1–3) and sitafloxacin (4) using (a) highly sulfonated γ-cyclodextrin, (b) γ-cyclodextrin, (c) Cu-phenylalanine ligand-exchange chiral selector, or a combination of γ-cyclodextrin and (d) the Cu-phenylalanine ligand-exchange selector (Reproduced with permission of Elsevier from ref. 20 © 2017)

evaluated. The best result was obtained when Cu-D-Phe was combined with γ-cyclodextrin. Interestingly, no separation was achieved neither for the ligand-exchange selector nor for γ-cyclodextrin when applied alone as selector (Fig. 4). Taking into account the complex method needed to perform the separation of sitafloxacin and its impurities, a Fractional Factorial Design 27-3 was used in order to evaluate the importance of each factor: γ-cyclodextrin concentration, Cu2+ concentration, DPhe concentration, pH, BGE concentration, voltage, and capillary temperature. As response, the resolution factor between the adjacent peak was used. Based on the regression coefficient plots, γ-cyclodextrin concentration, Cu2+ concentration, D-Phe concentration, and pH were selected as most important factors for further optimization. It should be noticed that using resolution as response is not without danger. It can only be used as response when the order of peaks does not change, which is not evident during optimization. The elution order can be determined using either the individual enantiomers or a scalemic mixture (i.e., a mixture of enantiomers in a ratio other than 1:1). However, in both cases it is necessary that the individual enantiomers are commercially available. The problem

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with resolution is that when peaks 3 and 4, for instance, elute once as 3-4 and once as 4-3 and are equally separated. Both resolutions are then equal (i.e., represented by the same value), but they represent different situations. Thus estimating effects or coefficients in such situation leads to wrong results and conclusions.

3

Response Surface Methodology After executing a screening design, a response surface design may be performed with the factors highlighted as important in order to determine an optimal condition to achieve the best response. Alternatively, important factors can also be selected based on historical knowledge [11]. Level ranges are also selected in accordance with results already obtained. However, the number of levels will be determined by the selected design. Several response surface designs are described. However, threelevel Full Factorial Designs, Box-Behnken Designs (BBD), and Central Composite Designs (CCD) are the most often used ones [12]. The graphical representation of each design is given in Fig. 5. A Full Factorial Design is often applied when only two factors need to be optimized. When three or more factors must be included, a Full Factorial Design is not a good choice because of the high number of experiments required (27 experiments for 3 factors, calculated as 3k, where k is the number of factors) [12]. BBD is a kind of subset of the factorial design 3k. In this sense, it is more efficient and economical than a Full Factorial Design. The experimental points are equidistant from a central point and situated on a hypersphere. It is a three-level design (1, 0, þ1) with equally spaced intervals, requiring 2 k(k  1) þ Cp experiments, where k is the number of factors and Cp, the number of replicates of the central point [8, 12, 25].

Fig. 5 Graphical representation of response surface experimental designs for the study of three factors: (a) three-level Full Factorial Design; (b) Box-Behnken Design; (c) Central Composite Design (Reproduced with permission of Elsevier from ref. 12 © 2008)

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Finally, CCD can be divided in three parts: (1) a Full or Fractional Factorial Design; (2) a star design, with axial points at a distance α from the center; and (3) a central point. All factors are evaluated at five levels (α, 1, 0, þ1, þα), requiring k2 þ 2 k þ Cp experiments, where k is the number of factors and Cp is the number of replicates of the central point. The distances α are often calculated using Eq. 11, resulting in 1.41 for two factors, 1.68 for three, and 2.00 for four factors [8, 12, 25]. k=

α¼24

ð11Þ

One design that is not always orthogonal is the D-optimal design. It is frequently used when some regions of the factor space cannot be explored, such as, for instance, some solvent combinations (Fig. 6a, b). For this reason, it is called an asymmetric design. This design is built firstly by defining the type of model to

Fig. 6 (a) A 32 Full Factorial Design in a rectangular symmetrical domain, (b) a restricted 32 Full Factorial Design in an asymmetrical domain, (c) the candidate points of the grid in the asymmetrical domain, and (d) the selected points constructing an 8-experiments D-optimal design (possible or selected experiments (l)) (Reproduced with permission of Elsevier from ref. 15 © 2011)

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model the response. This will require a minimal number of experiments (Nmin) to estimate the coefficients. Then, the number of experiments N that the analyst will execute is defined (Nmin  N). In a third step, the experimental space is then represented by a grid of potentially possible experiments (Ngrid) (Fig. 6c). Finally, the N experiments to be performed are the selection from Ngrid for which the determinant of X XT is maximal (Fig. 6d), where XT is the transposed matrix X [12, 15]. After response selection, the experimental setup is executed. In order to model the response as a function of the factors, a polynomial model is built: y ¼ β0 þ

k X i¼1

βi x i þ

k X i¼1

βii x 2i þ

k X

βij x i x j þ ε

ð12Þ

1ij

where y is the response, β0 the intercept, k the number of factors, βi the real coefficient of the linear terms, xi and xj represent the factors i and j, βii is a real coefficient of quadratic terms, βij a real coefficient of the interaction terms, and ε represents the error associated with the model [8]. The coefficients are estimated by least squares regression, as in Eq. 2. For the model evaluation, both graphical and statistical evaluations can be performed. Graphically, the model is usually visualized as contour (2D) or response surface (3D) plots (Fig. 7). These graphs show the correlation between the response and the factors, facilitating the interpretation of the model [25]. For the statistical evaluation, analysis of variance (ANOVA) may be performed. It aims to make a comparison between the regression variation with the random variation due to experimental error, allowing to detect the significance of the regression. For that, usually a table is built, as in Table 6 [12].

Fig. 7 Response surface (left) and contour (right) plots (Reproduced with permission of Springer from ref. 25 © 2017)

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Table 6 Analysis of variance Variation source

Sum of squares

Regression

SSreg ¼

Residuals

SSres ¼

Lack-of-fit

SSlof ¼

Pure error

SSpe ¼

Total

SSpe ¼

P m Pn1  j

i

P m Pn1  i

j j

P m Pni  i

j

P m Pni  i

j

ybi  yi

y ij  yi

Degrees of freedom

Mean squares

p1

MSreg ¼

SSreg p1

²

np

res MSres ¼ SS np

²

mp

lof MSlof ¼ SQ mp

²

nm

y ij  ybi

P m Pn1  i

² ybi  y

² y ij  y

SS

pe MSpe ¼ nm

n1

The significance of regression is estimated by: MSreg  F vreg , vres MSres

ð13Þ

where the ratio of the mean squares of regression (MSreg) and the mean squares of residuals (MSres) is compared with an F-value, considering the degrees of freedom of the regression (vreg) and of the residuals (vres). Therefore, a ratio higher than the tabulated Fvalue is considered significant, indicating that the variance due to regression is significantly higher than that of the residuals. In practice, this test is always highly significant for the considered model [12]. Another test is the lack-of-fit test, which is estimated as: MSlof  F vlof , vpe MSpe

ð14Þ

where the ratio of the mean squares of lack-of-fit (MSlof) and the mean squares of pure error (MSpe) is compared with an F-value, considering the degrees of freedom of the lack-of-fit variance (vlof) and of the pure error (vpe). In this case, no significant difference between MSlof and MSpe is expected, indicating that the model fits the data significantly well [12]. Finally, the coefficient of determination (R2) from test set experiments is also used to evaluate the predictive capacity of the model [8, 25]. However, all above statistical parameters are only useful when the quadratic model is used for calibration purposes, i.e., to predict the response for future samples. Here, in method optimization, the model is only used to indicate a suitable region and the interpretation of the above parameters is far less important. Accurate prediction is also of low importance here (see case study further). Most important is identifying a suitable region, which is then confirmed experimentally.

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For the determination of the optimal conditions, visual inspection of the contour or surface plots can be used. However, it is also possible to calculate the maximum and minimum based on the first derivate of mathematical Eq. 16: y ¼ β0 þ β1 x 1 þ β2 x 2 þ β11 x 21 þ β22 x 22 þ β12 x 1 x 2

ð15Þ

The critical point is calculated by: Δy ¼ β1 þ 2β11 x 1 þ β12 x 2 ¼ 0 ΔX 1

ð16Þ

Δy ¼ β2 þ 2β22 x 2 þ β12 x 1 ¼ 0 ΔX 2

ð17Þ

and

By solving both equations, it is possible to determine x1 and x2 values, giving the coordinates of the optimum point [12, 25]. As a last step, the optimal experimental conditions are defined and they must be experimentally confirmed. The robustness of the optimum can  occasionally be evaluated either from the equation by testing Δy around the optimum (should be small) or experimentally by Δxi means of a robustness test around the optimum [15]. When several responses are optimized simultaneously, the Derringer desirability function can be used. In this methodology, a desirability function is built for each response, transforming the response to a desirability scale between 0 (undesirable response) and 1 (a desirable response). This allows combining all responses to a global overall desirability value (D), defined as the geometric mean of individual desirability values [12]. In the next sections, an overview of the use of RSM for the optimization of the separation of chiral molecules will be presented. The separations were developed in SFC, LC, CE, and CEC. In Table 7, the factors and number of levels evaluated for several studies are presented. 3.1 Supercritical Fluid Chromatography

Both A˚sberg et al. [26] and Forss et al. [28] used a similar approach to separate different analytes. In both cases, a 33 design was used to evaluate the effects of temperature, backpressure, and percentage of organic modifier in mobile phase on the retention factor and the selectivity. In A˚sberg [26], in addition to analytical scale, preparative scale was also evaluated. Therefore, productivity was also determined. Productivity is defined as the amount of purified product per unit time, estimated as: Pr ¼

V inj C 0 Y Δt c

ð18Þ

where Vinj is the injection volume, C0 the sample concentration, Y is the recovery yield, and Δtc the cycle time, defined as the time between the complete elution of both isomers. In both studies,

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Table 7 Factors examined in the optimization designs of the case studies discussed using supercritical fluid chromatography (a), liquid chromatography (b), or capillary electrophoresis/capillary electrochromatography (c) Type of factors

Number of levels

Ref.

(a) Temperature Pressure % organic modifier in mobile phase Flow rate

Quantitative Quantitative Mixture Quantitative

3 or 5 3 or 5 3 or 5 5

[26–28] [26–30] [26–30] [27, 29, 30]

(b) % organic modifier (Hex, ACN, or MeOH) in mobile phase Concentration of acid Concentration of base Flow rate % water in mobile phase Temperature Buffer concentration pH Stationary phase

Mixture

3, 4 or 5

[16, 31–39]

Quantitative Quantitative Quantitative Mixture Quantitative Quantitative Quantitative Qualitative

5 5 3, 4 or 5 5 3, 4 or 5 3 3 or 5 3

[16, 33] [16, 31] [31, 37–39] [32] [33, 37, 38] [34–36] [35, 36] [37]

Factors

(c) Background electrolyte (BGE) pH Concentration of chiral selector (cyclodextrin) % organic modifier (MeOH or ACN) in mobile phase Temperature Background electrolyte (BGE) concentration Voltage Injection parameters Concentration of amino acid-based ionic liquid (AAIL)

Quantitative 2, 3 or 5 Quantitative 2, 3 or 5 Mixture

3

Quantitative 2 or 3

[17, 18, 20, 40–48] [17, 18, 20, 40, 42, 44, 46, 48] [41, 46]

Quantitative 2, 3 or 5

[19, 41, 42, 44, 46, 48, 49] [19, 41, 43–46, 48, 49]

Quantitative 2 or 3 Quantitative 3 Quantitative 3

[17–19, 44–46, 48, 49] [44, 48] [47]

ANOVA and R2 were used to evaluate the mathematical model. For Forss et al. [28], the response were mostly influenced by the pressure and temperature. For A˚sberg et al. [26], temperature was also significant, and methanol content. In Langaraday et al. [30] and Ghinet et al. [29], the optimization was focused on the semi-preparative scale enantioseparation and isolation of two synthetic bioactive compounds. Their approach was similar, using CCD to optimize outlet pressure, flow rate, and percentage of organic modifier effect on resolution. ANOVA and lack-of-fit were tested for model evaluation. In both cases, after optimization, it was possible to isolate each isomer with about 97% yield. In the study of Chen et al. [27], the enantioseparation of (þ)dinotefuran and ()-dinotefuran, a neonicotinoid insecticide, and its metabolites, (þ)1-methyl-3-(tetrahydro-3-furylmethyl) urea

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and ()1-methyl-3-(tetrahydro-3-furylmethyl) urea were determined in pollen, honey, water, and soil. In a first step, the authors evaluated using a one-variable-at-a-time (OVAT) approach the effect of different chiral columns and different co-solvents (methanol, ethanol, isopropanol, n-butanol, acetonitrile, and n-hexane) on the resolution, retention factor, and signal-to-noise ratio. This allowed the authors to select the best column and the percentage formic acid-methanol modifier. For the optimization step, a CCD was used to evaluate the influence of methanol percentage, mobile phase flow rate, automated backpressure regulator pressure, and column temperature on the resolutions, retention factors, and signal-to-noise ratios. ANOVA was used to determine the significant factors, while regression and lack-of-fit were tested for model evaluation. The optimal condition was estimated by visual inspection of the response surface plots and by the calculation of Derringer’s desirability function. Finally, the authors applied their methodology to analyze pollen, honey, water, and soil samples. Neither enantiomers were detected in pollen or honey. In water, two samples were contaminated. 3.2 Liquid Chromatography

For LC optimization, most studies used CCDs [16, 31, 33, 35, 36, 39]. It was mainly applied to evaluate the effect of mobile phase factors, such as fraction of organic phase and concentration of acidic or basic additives. Alternatively, some authors used a D-optimal mixture design [32, 37]. The D-optimal mixture design is an asymmetric design when applied in an asymmetric domain. It is frequently used, also for non-mixture variables, instead of symmetric designs, such as CCD, because otherwise impossible combinations between the factors would be proposed or only a part of the domain would be examined [9]. However, when applied in a symmetric domain, the D-optimal design can be considered as a symmetric design. Kannappan and co-workers [32] used such design to evaluate the effect of organic modifier (75–85%) and of water (15–25%) for the separation of ondansetron enantiomers. Since the factors are components of the mobile phase, their levels are not independent. Therefore, the mixture factors are expressed as fractions. The latter facts also mean that a D-optimal design, or whatever other design, was not needed to optimize the modifier/water composition. The experimental domain can be represented by a line and optimizing the composition means finding the appropriate water or modifier fraction. Saleh et al. [37], on the other hand, used this design to determine the optimal conditions evaluating chiral column (qualitative factor), mobile phase composition (mixture factor), flow rate, and temperature (quantitative factors). In total, nine experiments were performed to evaluate four factors at three levels.

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Fig. 8 Chromatograms for the separation of (a) Fmoc leucine, (b) tryptophan, and (c) salbutamol enantiomers, using optimal conditions derived from CCD (a, b) or 3-level Full Factorial Design (c). * co-eluting impurity (Reproduced with permission of Elsevier from ref. 16 © 2018)

An interesting approach was used by Hanafi et al. [16]. Aiming to perform the enantioseparation of Fmoc leucine, tryptophan, and salbutamol, the authors used a 2m4n screening design, as described in 2.1, identifying percentage of methanol, and concentration of diethylamine and acetic acid as important factors. For the optimization step, the authors evaluated each factor at five levels, using a CCD with 6 replicates of the central point (20 experiments). As responses, resolution, retention time, and retention factor were selected. For Fmoc leucine and tryptophan, the approach led to satisfactory results, affording baseline separation for both enantiomers (Fig. 8a, b). However, for salbutamol, poor enantiomeric separation was obtained. In fact, resolutions were below 1.5 in all experiments. From the analysis of the contour plot (Fig. 9a), it was decided that optimal conditions could be out of the examined experimental region, in the upper left corner when considering the concentrations of diethylamine and of acetic acid. However, a concentration of acetic acid below 0 is not possible. To further evaluate the experimental domain, the authors performed a Full Factorial Design 32, mainly to explore higher levels of diethylamine, keeping the percentage of methanol at 63%, as in the CCD. Higher levels of acetic acid were also considered in the second design. A new contour plot was obtained (Fig. 9b), giving higher resolutions, as shown in Fig. 8c (resolution 2.62). Notice, firstly that the predicted (contour plot) and the experimental resolution are quite different. From Fig. 9b, it can be deduced that the highest achievable resolution in the domain will be somewhat higher than 1.25, while the experimental value was 2.62. This item was already discussed above in Subheading 3. Notice also from both contour plots in Fig. 9 that optima in a

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Fig. 9 Contour plots for the separation (resolution) of salbutamol enantiomers derived from (a) CCD and (b) Full Factorial Design experiments Arrows indicate where optimum domain should be. Blue square: initially investigated domain of Fig. 9a (Adapted with permission of Elsevier from ref. 16 © 2018)

majority of cases are found in a corner of the domain and that it looks as if the real optimum is outside the examined domain as mentioned above. This conclusion is the consequence of the behavior of the response as a function of the factor levels and is found in a majority of optimizations. 3.3 Electromigration Techniques

As indicated in Table 5, most chiral separations covered in this chapter were performed using electromigration techniques. Taking into account DoE strategies, most studies applied CCD [18–20, 43, 49], as for SFC and LC. In addition, 3-level Full Factorial Design [40, 41, 47] and other orthogonal designs [44, 45] were largely applied. It can be noticed that the term orthogonal design is not very informative in relation to the design applied. Besides the D-optimal designs, all designs discussed in this chapter are orthogonal. An orthogonal design is any design in which the main effects, i.e., the effects of the fraction, can be estimated independently from each other. Thus, the estimation of the main effects is unconfounded. Therefore, it can be recommended to specify the design instead of using the term orthogonal design. However, for CE and CEC, in addition to mobile phase factor evaluation, such as pH and buffer concentration, additional factors were also evaluated, such as concentration of the chiral selector, temperature, and voltage. These parameters also play a pivotal role

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Fig. 10 Electropherogram for the separation of sitafloxacin (4) and stereoisomers (1–3) at optimal conditions (Reproduced with permission of Elsevier from ref. 20 © 2017)

in the enantioseparation, as reinforced by screening designs (see Subheading 2.2). In a study performed by Meng et al. [20], γ-cyclodextrin concentration, Cu2+ and D-Phe concentrations (ligand-exchange type chiral selector), and pH were selected as most important factors by a Fractional Factorial Design prior to the optimization step for the separation of the stereoisomers of sitafloxacin (see Subheading 2.2). Using a CCD, resolution and analysis time were measured as responses. By the analysis of response surface plots and using the Derringer’s desirability function, pH and D-Phe concentration were selected at intermediate level; γ-cyclodextrin concentration at low level; and Cu2+ concentration at high level as optimum. This allowed to have a high resolution value within an analysis time below 30 min (Fig. 10). Notice that if 4 factors are optimized, only a very small fraction of the entire response surface can be visualized, because 2 factors need to be fixed. One thus risks not to visualize the global optimum of the method.

4

Conclusions In this book chapter, both screening and optimization designs are discussed for the optimization of chiral separations, in SFC, LC, or CE/CEC. The different steps are discussed, including factors and level selection, the designs applied in the different situations, and the building and evaluation of the model. Most case studies found in the literature did not apply a screening step. This might be related to previous knowledge on the

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problem, allowing to perform directly the optimization step. Most frequently CCD are used for this optimization. Although the use of DoE for chiral separations is not a novelty, it is applied only to a limited extent, given the large number of chiral compounds analyzed. In these studies which used a DoE, most factors examined in the design are related to the mobile phase, such as pH, modifiers (type and concentration), and concentration of chiral selectors. No study was found using gas chromatography where a method was optimized by a DoE approach. The explanation may simply be that GC is much less used for chiral separations. The DoE approach is a powerful methodology that can be used for procedures optimization, including the optimization of chiral separations. Because of the complexity of this topic, evaluating the influence of several factors in a limited number of experiments is primordial, thus avoiding the unnecessary waste of time and reagents. References 1. Calcaterra A, D’Acquarica I (2018) The market of chiral drugs: chiral switches versus de novo enantiomerically pure compounds. J Pharm Biomed Anal 147:323–340 2. Stinson SC (1997) Chiral drug market shows signs of maturity. Chem Eng News 75:38–70 3. Agranat I, Caner H (1999) Intellectual property and chirality of drugs. Drug Discov Today 4:313–321 4. Gu¨bitz G, Schmid MG (2008) Chiral separation by capillary electromigration techniques. J Chromatogr A 1204:140–156 5. Agranat I, Wainschtein SR, Zusman EZ (2012) The predicated demise of racemic new molecular entities is an exaggeration. Nat Rev Drug Discov 11:972–973 6. Scriba GKE (2016) Chiral recognition in separation science—an update. J Chromatogr A 1467:56–78 7. Płotka JM, Biziuk M, Morrison C, Namies´nik J (2014) Pharmaceutical and forensic drug applications of chiral supercritical fluid chromatography. Trends Anal Chem 56:74–89 8. Candioti LV, De Zan MM, Ca´mara MS, Goicoechea HC (2014) Experimental design and multiple response optimization. Using the desirability function in analytical methods development. Talanta 124:123–138 9. Brynn Hibbert D (2012) Experimental design in chromatography: a tutorial review. J Chromatogr B 910:2–13 10. Nguyen NK, Lin DKJ (2011) A note on small composite designs for sequential experimentation. J Stat Theory Pract 5:109–117

11. Das AK, Mandal V, Mandal SC (2014) A brief understanding of process optimization in microwave-assisted extraction of botanical materials: options and opportunities with chemometric tools. Phytochem Anal 25:1–12 12. Bezerra MA, Santelli RE, Oliveira EP, Villar LS, Escaleira LA (2008) Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta 76:965–977 13. Hanrahan G, Montes R, Gomez FA (2008) Chemometric experimental design based optimization techniques in capillary electrophoresis: a critical review of modern application. Anal Bioanal Chem 390:169–179 14. Plackett RL, Burman JP (1946) The design of optimum multifactorial experiments. Biometrika 33:305–325 15. Dejaegher B, Vander Heyden Y (2011) Experimental designs and their recent advances in set-up, data interpretation, and analytical applications. J Pharm Biomed Anal 56:141–158 16. Hanafi RS, L€ammerhofer M (2018) Response surface methodology for the determination of the design space of enantiomeric separations on cinchona-based zwitterionic chiral stationary phases by high performance liquid chromatography. J Chromatogr A 1534:55–63 17. Orlandini S, Pasquini B, Del Bubba M, Pinzauti S, Furlanetto S (2015) Quality by design in the chiral separation strategy for the determination of enantiomeric impurities: development of a capillary electrophoresis method based on dual cyclodextrin systems

Design of Experiments in Method Optimization for the analysis of levosulpiride. J Chromatogr A 1380:177–185 18. Orlandini S, Pasquini B, Caprini C, Del Bubba M, Dousˇa M, Pinzauti S, Furlanetto S (2016) Enantioseparation and impurity determination of ambrisentan using cyclodextrinmodified micellar electrokinetic chromatography: visualizing the design space within quality by design framework. J Chromatogr A 1467:363–371 19. Krait S, Dousˇa M, Scriba GKE (2016) Quality by design-guided development of a capillary electrophoresis method for the chiral purity determination of ambrisentan. Chromatographia 79:1343–1350 20. Meng R, Kang J (2017) Determination of the stereoisomeric impurities of sitafloxacin by capillary electrophoresis with dual chiral additives. J Chromatogr A 1506:120–127 21. Patel DC, Wahab MF, Armstrong DW, Breitbach ZS (2016) Advances in high-throughput and high-efficiency chiral liquid chromatographic separations. J Chromatogr A 1467:2–18 22. Declerck S, Vander Heyden Y, Mangelings D (2016) Enantioseparations of pharmaceuticals with capillary electrochromatography: a review. J Pharm Biomed Anal 130:81–99 23. Zhu Q, Scriba GKE (2018) Analysis of small molecule drugs, excipients and counter ions in pharmaceuticals by capillary electromigration methods–recent developments. J Pharm Biomed Anal 147:425–438 ˜ a-Gonzalez JA, Ferna´n24. Ramos-Paya´n M, Ocan ´ dez-Torres RM, Llobera A, Bello-Lo´pez MA (2018) Recent trends in capillary electrophoresis for complex samples analysis: a review. Electrophoresis 39:111–125 25. Yolmeh M, Jafari SM (2017) Applications of response surface methodology in the food industry processes. Food Bioproc Tech 10:413–433 ˚ sberg D, Enmark M, Samuelsson J, Fornstedt 26. A T (2014) Evaluation of co-solvent fraction, pressure and temperature effects in analytical and preparative supercritical fluid chromatography. J Chromatogr A 1374:254–260 27. Chen Z, Dong F, Li S, Zheng Z, Xu Y, Xu J, Liu X, Zheng Y (2015) Response surface methodology for the enantioseparation of dinotefuran and its chiral metabolite in bee products and environmental samples by supercritical fluid chromatography/tandem mass spectrometry. J Chromatogr A 1410:181–189 28. Forss E, Haupt D, Sta˚lberg O, Enmark M, Samuelsson J, Fornstedt T (2017) Chemometric evaluation of the combined effect of

477

temperature, pressure, and co-solvent fractions on the chiral separation of basic pharmaceuticals using actual vs set operational conditions. J Chromatogr A 1499:165–173 29. Ghinet A, Zehani Y, Lipka E (2017) Supercritical fluid chromatography approach for a sustainable manufacture of new stereoisomeric anticancer agent. J Pharm Biomed Anal 145:845–853 30. Landagaray E, Vaccher C, Yous S, Lipka E (2016) Design of experiments for enantiomeric separation in supercritical fluid chromatography. J Pharm Biomed Anal 120:297–305 31. Kannappan V, Mannemala SS (2014) Multiple response optimization of a HPLC method for the determination of enantiomeric purity of S-ofloxacin. Chromatographia 77:1203–1211 32. Kannappan V, Kanthiah S (2017) Enantiopurity assessment of chiral switch of ondansetron by direct chiral HPLC. Chromatographia 80:229–236 33. Nistor I, Lebrun P, Ceccato A, Lecomte F, Slama I, Oprean R, Badarau E, Dufour F, Dossou KSS, Fillet M, Lie´geois J-F, Hubert P, Rozet E (2013) Implementation of a design space approach for enantiomeric separations in polar organic solvent chromatography. J Pharm Biomed Anal 74:273–283 34. Rosales-Conrado N, Guille´n-Casla V, Pe´rezArribas LV, Leo´n-Gonza´les ME, Polo-Dı´ez LM (2013) Simultaneous enantiomeric determination of acidic herbicides in apple juice samples by liquid chromatography on a teicoplanin chiral stationary phase. Food Anal Methods 6:535–547 35. Rosales-Conrado N, Dell’Aica M, Leo´n-Gonza´les ME, Pe´rez-Arribas LV, Polo-Dı´ez LM (2013) Determination of salbutamol by direct chiral reversed-phase HPLC using teicoplanin as stationary phase and its application to natural water analysis. Biomed Chromatogr 27:1413–1422 36. Rosales-Conrado N, Leo´n-Gonza´les ME, Polo-Dı´ez LM (2015) Development and validation of analytical method for clenbuterol chiral determination in animal feed by direct liquid chromatography. Food Anal Methods 8:2647–2659 37. Saleh OA, Yehia AM, El-Azzouny AS, AboulEnein HY (2015) A validated chromatographic method for simultaneous determination of guaifenesin enantiomers and ambroxol HCl in pharmaceutical formulation. RSC Adv 5:93749–93756 ˝cs L, 38. Szabo´ Z-I, Mohammadhassan F, Szo Nagy J, Komja´ti B, Nosza´l B, To´th G (2016) Stereoselective interactions and liquid

478

Luiz Carlos Klein-Ju´nior et al.

chromatographic enantioseparation of thalidomide on cyclodextrin-bonded stationary phases. J Incl Phenom Macrocycl Chem 85:227–236 39. Valliappan K, Vaithiyanathan SJ, Palanivel V (2013) Direct chiral HPLC method for the simultaneous determination of warfarin enantiomers and its impurities in raw material and pharmaceutical formulation: application of chemometric protocol. Chromatographia 76:287–292 40. Abdel-Megied AM, Hanafi RS, Aboul Enein HY (2018) A chiral enantioseparation generic strategy for anti-Alzheimer and antifungal drugs by short end injection capillary electrophoresis using an experimental design approach. Chirality 30:165–176 41. Albals D, Hendrickx A, Clincke L, Chankvetadze B, Vander Heyden Y, Mangelings D (2014) A chiral separation strategy for acidic drugs in capillary electrochromatography using both chlorinated and nonchlorinated polysaccharide-based selectors. Electrophoresis 35:2807–2818 42. Escuder-Gilabert L, Martı´n-Biosca Y, Sagrado S, Medina-Herna´ndez MJ (2014) Fast-multivariate optimization of chiral separations in capillary electrophoresis: anticipative strategies. J Chromatogr A 1363:331–337 43. Guo PW, Rong ZB, Li YH, Fung YS, Gao GQ, Cai ZM (2013) Microfluidic chip capillary electrophoresis coupled with electrochemiluminescence for enantioseparation of racemic drugs using central composite design optimization. Electrophoresis 34:2962–2969

˝s I, Sohajda T, Zhou W, Hu W, 44. Kazsoki A, Fejo Szente L, Be´ni S (2016) Development and validation of a cyclodextrin-modified capillary electrophoresis method for the enantiomeric separation of vildagliptin enantiomers. Electrophoresis 37:1318–1325 ˝cs MD-L, Nosza´l B, To´th G 45. Szabo´ Z-I, Szo (2016) Chiral separation of uncharged pomalidomide enantiomers using carboxymethyl-β-cyclodextrin: a validated capillary electrophoretic method. Chirality 28:199–203 46. Szabo´ Z-I, To´th G, Vo¨lgyi G, Komja´ti B, Hancu G, Szente L, Sohajda T, Be´ni S, Muntean D-L, Nosza´l B (2016) Chiral separation of asenapine enantiomers by capillary electrophoresis and characterization of cyclodextrin complexes by NMR spectroscopy, mass spectrometry and molecular modeling. J Pharm Biomed Anal 117:398–404 47. Wahl J, Holzgrabe U (2018) Capillary electrophoresis separation of phenethylamine enantiomers using amino acid based ionic liquids. J Pharm Biomed Anal 148:245–250 48. Caˆrcu-Dobrin M, Buda˘u M, Hancu G, Gagyi L, Rusu A, Kelemen H (2017) Enantioselective analysis of fluoxetine in pharmaceutical formulations by capillary zone electrophoresis. Saudi Pharm J 25:397–403 49. Zhang Q, Du Y (2013) Evaluation of the enantioselectivity of glycogen-based synergistic system with amino acid chiral ionic liquids as additives in capillary electrophoresis. J Chromatogr A 1306:97–103

INDEX A π-Acceptor ....................................................................... 15 α1-Acid glycoprotein (AGP)........................................... 13 Acidic compounds............................................... 189, 192, 228, 241, 306, 307, 362, 375, 379 Additives ....................................................... 6, 17, 22, 36, 39, 81–91, 96, 128, 138, 143, 149, 153, 159, 180, 189, 195, 196, 207, 208, 215, 224, 227–229, 255, 264, 267–270, 280, 282, 284, 306, 307, 343, 346, 366, 367, 407, 462, 471 Adrenaline........................................................................ 61 β-Adrenergic drugs ......................................................... 41 β-Agonists............................................................. 227, 387 AGP, see α1-Acid glycoprotein (AGP) Alkaloids ....................................................... 23, 161, 228, 251–272, 322, 418, 435–437 Alkynyl-functionalized silica ................................ 148, 151 Alprenolol ...................................214, 215, 229, 375, 376 Ambrisentan ......................................................... 411, 462 Amino acid ................................................... 9, 36, 45, 61, 82, 128, 172, 183, 206, 240, 252, 280, 366, 393, 417, 445, 471 β-Amino acids...................................................... 217, 224, 225, 260, 267, 269, 270 Amino alcohols........................................................ 17, 46, 82, 86, 196, 223, 280, 281 Aminopropyl silica....................................... 446, 447, 451 3-Aminopropyltriethoxysilane...................................... 150 Amlodipine ........................ 118, 120, 131, 133, 187, 191 Amphetamine ....................................................... 213, 215 Ampholiyte, see Ampholytic Ampholytic ................................. 252–255, 263, 264, 272 Amylose amylose tris(3-chloro-5-methylphenylcarbamate) (ACMPC) ................................................. 110, 135 amylose tris[(S)-α-methylbenzylcarbamate].............. 4 phenylcarbamate................................ 98, 99, 101, 116 tris(3,5-dimethylphenylcarbamate)....................... 3–5, 98, 132, 305 Anionic micelles ............................................................ 408 Aptamers.......................................................................... 25 Aqueous-organic ...............................................3, 82, 111, 118, 133, 137, 174 α-Aryl ketones ...................................................... 189, 191 AstecTM® ........................................................... 7, 20, 202

Atenolol .............................................................23, 37, 41, 43, 173, 212–214, 220, 227, 386, 421, 436, 437 Atropisomers ............................................... 185, 195, 418 α,α´-Azobisisobutyronitrile (AIBN)............................. 108

B Background electrolyte (BGE)....................................2, 3, 9, 10, 22, 195, 240, 241, 340, 342, 343, 346–352, 354, 357, 359–363, 365, 366, 368, 369, 373–379, 384, 385, 392, 394, 396, 398–403, 407, 408, 410, 412–414, 421–422, 432, 433, 461–463, 465, 471 Back pressure ................................................ 72, 104, 241, 307–309, 311, 312, 315, 317, 318, 470, 472 Basic analytes ................................................ 15, 189, 194, 241, 256, 342, 375, 377, 379, 383–387 Basic compounds ................................189, 306, 345, 462 Benzoin........................................................ 106, 188, 214 BGE, see Background electrolyte (BGE) BINAM, see 2,20 -Bis(diphenylphosphinoamino)1,10 -binaphthyl (BINAM) 4,4’-Bipyridine .................................................................. 4 2,20 -Bis(diphenylphosphinoamino)-1,10 binaphthyl (BINAM) ............................... 191, 193 β-Blockers ............................................................ 191, 212, 216–218, 223, 226, 227, 230, 418, 436, 437 Boric acid complexes............................................ 383–388 Bovine serum albumin (BSA)...............13, 160, 209, 230 Box-Behnken Design (BBD)............................... 463, 466 4-Bromophenylalanine ........................................ 448–450 Brush-type phase ..............................................13, 15, 253 BSA, see Bovine serum albumin (BSA) Bupranolol ............................................................ 375, 376

C Calixarenes............................................................ 2, 23, 25 Camphor sulfonic acid .................................................... 85 Capillary electrochromatography (CEC) open tubular ............................................................ 112 packed ...................................................................... 240 Capillary electrophoresis (CE) .............................ix, 1, 83, 112, 159, 194, 201, 240, 241, 322, 339–354, 357–363, 373–379, 407, 445, 462, 471 Capillary electrophoresis-mass spectrometry (CE-MS) ..................................391–404, 418, 433

Gerhard K. E. Scriba (ed.), Chiral Separations: Methods and Protocols, Methods in Molecular Biology, vol. 1985, https://doi.org/10.1007/978-1-4939-9438-0, © Springer Science+Business Media, LLC, part of Springer Nature 2019

479

CHIRAL SEPARATIONS: METHODS

480 Index

AND

PROTOCOLS

Capillary zone electrophoresis (CZE) ................ 365, 407 Carbohydrate................................ 3, 4, 11, 128, 417–443 Carbon dioxide (CO2)........................................ 225, 304, 307, 308, 310–318 Carprofen....................................131, 247, 359, 360, 362 Cavity .............................................................2, 4, 7–9, 23, 25, 46, 47, 84, 160, 254, 308, 339, 340, 342, 367, 408, 409 CCD, see Central composite design (CCD) CD, see Cyclodextrin (CD) CD-mediated enantioseparations....................9, 342, 346 CDShell® RSP ................................................................... 7 CE, see Capillary electrophoresis (CE) CEC, see Capillary electrochromatography (CEC) Cellobiohydrolase ........................................................... 13 CelluCoat® ...................................................................... 98 Cellulose 4-methylbenzoate ..................................................... 96 microcrystalline .............................................. 176, 179 triacetate (CTA) ............................................... 94, 111 tris(3,5-dichlorophenylcarbamate) .........99, 111, 132 tris(3,5-dimethylphenylcarbamate)....................... 3, 6, 98, 99, 104, 132, 172, 305 tris(4-chlorophenylcarbamate) ................................... 4 tris(4-methylbenzoate) ............................................... 4 tris(4-methylphenylcarbamate) .................................. 4 tris(phenylcarbamate) ...........................................4, 96 CE-MS, see Capillary electrophoresis-mass spectrometry (CE-MS) Central composite design (CCD) ...............................463, 466, 467, 471–476 Cerebrospinal fluid (CSF) ...........................393, 396–400 Cetyltrimethylammonium bromide (CTAB) .............173, 177, 179 Charged CDs.............................................. 341, 343–346, 351, 358, 362, 366, 373, 410 Chelate............................................................................. 17 Chemically bonded phases ........................................... 186 ChiraDex® ......................................................................... 7 Chiral additives.................................................43, 85, 280 Chiral ART Amylose-C................................................... 98 Chiral ART Amylose-SA ............................................... 109 Chiral ART Cellulose-C ................................................. 98 Chiral ART Cellulose-SB.............................................. 109 Chiral ART Cellulose-SJ................................................. 96 Chiralcel® OD .............................................................3, 98 Chiralcel® OD-H .............................................................. 3 Chiralcel® OJ................................................................... 96 Chiralcel® OZ ................................................................. 99 Chiral crown ether ...................................................19–20, 188, 252, 445, 447, 462 Chiral derivatization reagents................................. 58, 61, 69, 71, 72, 76, 393

Chiral discrimination ............................................ 20, 136, 159, 162, 174, 373, 408, 410 Chiral ion-exchange stationary phases........................... 15 Chiral ionic liquids (CILs)...................................... 17, 21, 22, 195, 228, 366, 368 Chiral ligand-exchange ...................................17, 41, 279, 323–326, 329–333, 464 Chiral ligand-exchange chromatography (CLEC) .......................................... 17, 86, 87, 279 Chiral micelles ...........................................................22–24 Chiral mobile phase (CMP) ................... 81, 90, 280–282 Chiral mobile phase additive (CMPA).......................... 22, 36, 81–91, 215, 284, 454 Chiralpak® AD .......................................... 3, 98, 130, 131 Chiralpak® AD-H ......................................................... 308 Chiralpak® AGP .............................................................. 13 Chiralpak® AY ................................................................. 99 Chiralpak® HSA .............................................................. 13 Chiralpak® IA ............................................................3, 109 Chiralpak® IB ...................................................3, 109, 173 Chiralpak® IC................................................................ 109 Chiralpak® IC-3 ..................................131–135, 142, 143 Chiralpak® ID-3 ................................................... 131–134 Chiralpak® IE-3 ................................................... 131–134 Chiralpak® IF-3.................................................... 132, 134 Chiralpak® ZWIX(+)..............15, 17, 252, 257–262, 264 Chiralpak® ZWIX(-) ............................................. 15, 252, 256–262, 265, 268, 272 Chiral polyol .................................................................. 383 Chiral recognition ............................................... 1–25, 48, 81, 83–85, 95–99, 108–111, 116, 120, 129, 133, 160–162, 172, 183, 202–209, 216, 223, 266, 324, 357, 366, 377, 408, 409 Chiral recognition mechanisms .................................1–25, 119, 202–206, 342, 374, 383 Chiral selector ............................................................2, 35, 57, 81, 130, 147, 159, 171, 183, 201, 240, 252, 280, 318, 322, 340, 358, 366, 373, 383, 392, 407, 418, 445 Chiral stationary phase (CSP) ...................................3, 35, 45, 57, 81, 94, 128, 147, 159, 171, 183, 201, 240, 251, 280, 305, 392, 445 Chiral sulfoxides .........................130, 134, 135, 226, 230 Chirasil-DEX® ................................................................... 7 ChiraSpher®..................................................................... 20 Chirex® (D)-penicillamine.........................................13, 17 CHIROBIOTIC® ........................................................... 14 ChiroSil®.......................................................................... 20 Chlorthalidone ................................................................ 23 Cinchona alkaloid ......................... 15, 161, 251–272, 322 Clenbuterol ................................................. 149, 153, 386 Click chemistry.................................................... 148–150, 161–163, 166, 191

CHIRAL SEPARATIONS: METHODS Clicked CSPs ....................................................... 148, 149, 152, 153, 161, 162 CMP, see Chiral mobile phase (CMP) CMPA, see Chiral mobile phase additive (CMPA) CO2, see Carbon dioxide (CO2) Coated capillary.................................................. 47, 52, 53 Coefficients ................................................. 190, 304, 307, 323, 325, 326, 458, 459, 465, 466, 468, 469 Column efficiency ........................................ 53, 105, 174, 299, 300, 306–308, 313 Complexation ........................................... 7, 9, 13, 20, 24, 25, 46, 82, 147, 202, 280, 281, 324, 344, 357, 358, 409, 410 Complexation constant........................................ 281, 358 Copolymerization ....................................... 108, 173, 174 Copper (Cu) .................................................................... 37 Core shell....................................................................7, 14, 104–106, 173, 174, 241, 272 Countercurrent chromatography ................... ix, 321–336 Counterions......................................................... 7, 84, 85, 224, 255, 263, 264, 267–269, 383 Counter migration technique (CMT) ......................... 392 (18-Crown-6)-2,3,11,12-tetracarboxylic acid ............. 19, 20, 322, 445–451 Crown ether ........................................................ 7, 19–20, 184, 188, 195, 196, 445, 447, 462 Crownpak® ...................................................................... 20 CSP, see Chiral stationary phase (CSP) Cu(II) ...................................................................... 41, 43, 86, 90, 280, 297–300 See also Copper (Cu) Cu(II)-complex .............................. 41, 43, 297, 299, 300 CuI(PPh3) ............................................................ 152, 166 Cyclobond® ................................................................... 7, 9 Cyclodextrin (CD) α-cyclodextrin (α-CD) ................4, 84, 339–341, 408 amino β-CD.................................................... 374, 378 β-cyclodextrin (β-CD) .................................... 8, 9, 84, 148, 322, 326–329, 334–336, 339–341, 347, 348, 350, 353, 366, 367, 369, 370, 408, 409, 411, 464 carboxymethyl-β-CD (CM-β-CD).....................86, 88 dual-cyclodextrin........................... 357–363, 407–415 γ-cyclodextrin (γ-CD)..................................... 4, 7, 84, 339–341, 345, 408, 410, 411, 463, 465, 475 heptakis(2,3-di-O-acetyl-6-O-sulfo)-β-CD (HDAS-β-CD).......................9, 10, 366, 374–377 heptakis(2,3-di-O-methyl-6-O-sulfo)-β-CD (HDMS-β-CD) ...................................... 9, 10, 374 heptakis(2,3,6-tri-O-methyl)-β-CD............... 341, 358 heptakis(2,6-di-O-methyl)-β-CD................... 341, 363 heptakis(6-O-sulfo)-β-CD (HS-β-CD).................. 8, 9, 341, 344, 353, 374, 409 hydroxyethyl-β-CD ................................328–330, 464

AND

PROTOCOLS Index 481

hydroxypropyl-α-CD .............................................. 341 hydroxypropyl-β-CD............................................... 341 hydroxypropyl-γ-CD............................................... 341 mono-6A-azido-β-CD........................... 151, 164, 165 6-monodeoxy-6-mono(3-hydroxy) propylamino-β-CD (PA-β-CD) ....................... 374, 375, 377–379 mono-6A-tosyl-β-CD .............................................. 165 mono-6-toluenesulfonyl-β-CD .............................. 163 octakis(6-O-methyl-2,3-di-O-pentyl)-g-CD.......... 374 phenylcarbamate CD ....................147, 148, 151, 152 single isomer CD............................................ 343, 353 succinyl-β-CD.......................................................... 341 sulfated β-CD ................................................ 341, 347, 348, 351, 353, 374, 376, 463, 464 sulfobutyl-β-CD ...................................................... 358 sulfobutylether-β-CD.............................................. 341 Cyclodextrin-based CSPs.............................159–168, 209 Cyclodextrin-mediated ........................................ 339–354 Cyclodextrin-modified ......................................... 407–415 Cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) ................ 407–415 Cyclofructan dimethylphenyl-cyclofructan ......................... 188, 196 isopropyl-cyclofructan...................187, 191, 195, 196 naphthylethyl-cyclofructan ............................ 185, 196 permethylated-cyclofructan .................................... 183 sulfated cyclofructan ...................................... 194–196 Cyclofructan-based CSPs.............................184–193, 196 Cyclohexyl-1-amino-2-sulfonic acid ..................... 15, 254 Cycloinuloheptaose (CF7) ......................... 184, 185, 196 Cycloinulohexaose (CF6) ..........................................7, 11, 183–185, 189, 190, 192–196 Cycloinulooctaose (CF8).............................................. 184 CZE, see Capillary zone electrophoresis (CZE)

D Dalbavancin ................................................. 203, 229, 230 Dansyl (Dns) ....................................................... 160, 162, 163, 166, 167, 285, 430, 432 Dansyl-amino acids ..................................... 160, 162, 163 Degree of substitution ........................................ 334, 339, 343, 345, 347, 353, 359, 361, 362, 402 Derivatization ...................................................... 4, 21, 47, 58–61, 69, 70, 75, 99, 184, 185, 187, 251, 271, 279, 304, 394, 398, 399, 403 Derringer desirability function ..................................... 470 Design of experiments (DoE) ............................ 346, 454, 462, 473, 476 D-gluconic acid δ-lactone.............................................. 383 α-D-Glucopyranoside .......................................... 418, 424, 425, 429, 432, 441, 442 β-D-Glucopyranoside........................................... 418, 419, 423–426, 429, 439, 441, 442

CHIRAL SEPARATIONS: METHODS

482 Index

AND

PROTOCOLS

Diacetone-D-mannitol .................................383–385, 387 Diastereomeric complexes .................................. 2, 17, 36, 57, 82, 202, 280, 342, 366, 367, 408 Diltiazem ....................................................................... 115 5-(Dimethylamino)naphthalene-1-sulfonyl (DNS) .............................................. 271, 284, 285 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC) .............................................. 61, 64, 73, 74 (S)-()-α,α-di(2-naphthyl)-2pyrrolidinemethanol.......................................... 287 Diols....................................... 17, 47, 209, 244, 247, 248 Dipeptides .......................................................23, 82, 166, 257, 261, 262, 271, 272, 296, 298, 418 2,5-Dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1, 3,5-triazin-2-yl)pyrrolidine-2-carboxylate (DMT-Pro-OSu) ...........................................59, 60 2,5-Dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1, 3,5-triazin-2-yl)pyrrolidine-2-carboxylate (DMT-(S)-Pro-OSu)........................61, 65–68, 74 (S)-1-(4,6-Dimethoxy-1,3,5-triazin-2-yl)pyrrolidin3-amine (DMT-3(S)-Apy) .......................... 61, 64, 66, 68, 74, 76 (3,3’-Diphenyl-1,1’-binaphthyl)-20-crown-6............... 20 Direct enantioseparation............................................... 212 Displacement model ....................................255–263, 267 DL-selenomethionine (SeMet) .....................36, 37, 39–42 DMT-3(R)-Apy.....................................61, 67, 68, 74, 76 DMT-Pro-OSu, see 2,5-Dioxopyrrolidin-1-yl-1(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidine2-carboxylate (DMT-Pro-OSu) DNS, see 5-(Dimethylamino)naphthalene1-sulfonyl (DNS) Dns-Phe ............................................................... 163, 166, 167, 418, 421, 430, 432 DoE, see Design of experiments (DoE) π-π Donor ..................................................................15, 16 Donor-acceptor .........................................................13–16 D-optimal design ........................................ 467, 471, 473 D-penicillamine ................................................................ 17 Drugs ............................................................ 9, 13, 23, 35, 41, 45, 70, 94, 115, 118, 133, 134, 140, 143, 185, 193, 206, 208, 213, 216, 218, 220–228, 239, 241, 258, 303, 357, 360–362, 374, 375, 377, 386, 407–415, 418, 453, 462 Dual-cyclodextrin................................ 357–363, 407–415 D-(+)-xylose .......................................................... 383–386 Dynamic coating .................................................. 281, 346

E EKC, see Electrokinetic chromatography (EKC) Electrokinetic chromatography (EKC).......................1, 7, 340, 343, 392, 393, 395–396, 407–415 Electron-donating ...............................82, 95–97, 99, 185 Electron-donating groups ..................................... 17, 185

Electron-withdrawing groups ...................................... 185 Electroosmotic flow (EOF) ................................ 342, 344, 346, 349–351, 366, 367, 369, 371, 408–410, 445 Electropherograms........................................................ 436 Electrophoretic mobilities .........342, 343, 362, 409, 410 Electrospray ionization (ESI) ............... 62, 393, 395–403 Electrospray ionization-mass spectrometry (ESI-MS) ......................................................57–76, 128, 257, 259, 262, 418, 433 Elution order ..................................................... 3–6, 9, 13, 15, 17, 58, 71, 75, 76, 90, 109, 110, 114–120, 134, 161, 223, 229, 254, 299, 436, 464, 465 Enantiodiscriminating................................................... 280 Enantiodiscrimination................................................... 135 Enantiomeric separations..............................45, 183–185, 187–189, 191–196, 206, 215, 216, 374, 391, 393, 462, 473 Enantiomer migration order .................. 9, 343, 344, 353 Enantiorecognition ................................................. 15, 81, 162, 208, 334, 408, 409 Enantioresolutions .....................................................9, 90, 173, 210, 215, 227, 230, 240–241, 267, 268, 279, 284, 353, 362, 363, 402, 410, 420, 430, 432, 448, 462 Enantioselective interactions ....................................96, 97 Enantioselectivity .................................................... 25, 46, 47, 96, 108, 111, 114, 115, 119, 129, 132, 133, 135, 136, 138, 139, 147, 162, 167, 185, 189, 192–196, 209, 213, 226, 229, 240, 249, 252, 254, 264, 265, 268, 269, 281, 306, 324, 358, 362, 383, 409, 410, 436, 437, 445 Enantioseparations ................................................ 7, 9, 11, 13, 17, 20, 23–25, 35–53, 70, 81–91, 93–121, 127–143, 147–156, 159–168, 171, 173, 174, 178–180, 201–230, 241, 248, 252, 265, 267–269, 271, 279, 310–313, 315, 317, 318, 322, 324, 326, 329–336, 339–354, 357–363, 365–371, 373–379, 385–387, 393, 407–415, 418, 420, 435–440, 445, 463, 471, 473 Enilconazole ...................................................................... 9 Ephedrine .............................................................. 23, 213, 366–368, 370, 418, 421, 435, 436 Eremomycin ........................................203, 209, 229, 230 ESI, see Electrospray ionization (ESI) ESI-MS, see Electrospray ionization-mass spectrometry (ESI-MS) Experimental design............................................. 453–476 External complexes ........................................................... 9

F Factorial Design .................................................. 456, 457, 459, 463, 465–467, 473–475 Fenticonazole ...............................................410, 413–415 Flavanone.............................................5, 9, 149, 153, 154

CHIRAL SEPARATIONS: METHODS Flavonoids ............................................................ 160, 162 (+)-1-(9-Fluorenyl)ethyl chloroformate ...................... 394 9-Fluorenylmethoxycarbonyl (FMOC) ............. 117–120, 209, 259, 261, 269, 403, 462, 463, 473 9-Fluorenylmethoxycarbonyl choride (Fmoc-Cl) ........................................ 394, 398, 402 Flurbiprofen ........................................................ 131, 241, 247, 375, 378 FMOC, see 9-Fluorenylmethoxycarbonyl (FMOC) Fmoc-amino acids ......................................................... 269 Fractional Factorial Design................................. 456, 459, 463, 465, 467, 475 Frit ....................................................................... 152, 166, 245–247, 446–448, 451 Full Factorial Design........................................... 456, 457, 463, 466, 467, 473, 474 Functionalized cyclodextrins ........................................ 147

G Gas chromatography (GC) .....................................ix, 1, 7, 25, 45–53, 57, 159, 171, 184, 194–196, 240, 340, 476 GC, see Gas chromatography (GC) Glucopyranoside based polymeric surfactants............. 418 Glycopeptides ...................................................... 7, 11–14, 83, 89, 90, 115, 201–230, 240 Guest-host complexes ....................................................... 7

H Halogen bonds....................................................... 2, 4, 13 H-bond, see Hydrogen bond (H-bond) HDAS-β-CD, see Heptakis(2,3-di-O-acetyl6-O-sulfo)-β-CD (HDAS-β-CD) HDMS-β-CD, see Heptakis(2,6-di-O-methyl6-O-sulfo)-β-CD (HDMS-β-CD) Helix ....................................................................... 3, 4, 20 Heptakis(2,6-di-O-methyl-6-O-sulfo)-β-CD (HDMS-β-CD)...................................................... 9 Herbicides............................................................ 219, 225, 241, 244, 247, 248 High-performance liquid chromatography (HPLC)................................ix, 3, 4, 7, 14, 17, 20, 21, 25, 37, 57, 59, 69–73, 75, 81–91, 93–121, 127–143, 147–156, 159–168, 171–175, 178, 180, 184–193, 195, 196, 201–230, 240, 241, 243–246, 256–259, 261, 262, 268, 271, 279, 303–305, 308, 309, 313, 327, 328, 330, 331, 340, 374, 375, 384, 412, 420–422, 433, 434, 445, 446, 448, 462 HILIC, see Hydrophilic interaction liquid chromatography (HILIC) Homochiral .......................................................20, 21, 47, 50–53, 262, 271–272

AND

PROTOCOLS Index 483

Homochiral pentyl cage..................................... 47, 50–53 Homogeneous............................................ 172, 322–324, 326, 328–330 Human serum albumin................................................... 13 Hybrid organic-inorganic materials .................... 171–180 Hydrobenzoin ............................................................... 160 Hydrodynamic injection ............................. 354, 385, 433 Hydrogen bond (H-bond) .......................................... 4–6, 15, 18, 23, 45, 202, 206, 223, 227, 264–266 Hydro-organic solvent .................................................. 207 Hydrophilic interaction liquid chromatography (HILIC) ............................................................118, 127–143, 184, 190, 191, 196 Hydroxy acids..................................................17, 82, 280, 297, 326, 328, 329, 332, 333 Hydroxycarboxylic acids ............................. 257, 293, 294 2-(2-Hydroxy)hexadecyl-(S)-1,2,3,4tetrahydro-3-isoquinolinecarboxylic acid ..................................................................... 289 Hydroxyproline .........................................................17, 86

I Ibuprofen (IBP) ...................................................... 61, 62, 64, 131, 160, 214, 227, 247 Imidazole drugs ................................................... 407–415 Immobilization.................................................... 9, 15, 94, 105, 107–109, 147, 161–164, 173, 209, 216, 223, 229, 230 Inclusion complexations ....................................... 82, 147, 241, 408–410 Inclusion complexes...................................................9, 46, 160, 184, 207, 342–344 Indanone ...................................................................86, 88 Indirect enantioseparations ............................................ 81 Indirect resolution .......................................................... 58 Induced fit ...................................................................4, 25 Interaction π–π............................................................2, 4–6, 8, 13, 15, 17, 20, 21, 23, 25, 82, 162, 185, 202, 206, 208, 241, 266, 408 dipole–dipole .............................................2, 7, 13, 25, 82, 128, 147, 162, 185, 227, 265, 324, 408 electrostatic.........................................................25, 82, 128, 147, 162, 194, 195, 202, 206, 223, 241, 253, 254, 263, 265–267, 373, 377, 379 ion-dipole ................................................................ 2, 7 ionic ...........................................................2, 7, 13, 15, 18, 25, 85, 207, 208, 223, 263, 264, 267 van der Waals ......................................................2, 4, 7, 25, 202, 266, 324 Interfacial......................................................322, 324–326 Ion-exchanges ........................15–18, 255, 263, 267, 268 Ionic liquids........................................... 17, 160, 365–371 Isoconazole.................................................. 410, 413, 415

CHIRAL SEPARATIONS: METHODS

484 Index

AND

PROTOCOLS

K Ketoprofen.......................................................86, 87, 110, 131, 160, 213, 215, 220, 227, 229, 247, 411 Kromasil®...................................... 20, 148, 149, 163, 446

L Labeling reaction...................................58, 59, 71, 73, 74 Labeling reagents ......................................................57–76 Lactobionic acid ...........................................383–386, 388 Larihc® ............................................................................... 7 LarihcShell-P® ...................................................... 191–193 L-carnitine ...................................................................... 293 LEC, see Ligand-exchange chromatography (LEC) Levofloxacin ........................................222, 228, 348, 352 Levosulpiride ........................................................ 462, 464 Ligand-exchange ..................................................... 17, 36, 37, 41, 87, 89, 279, 299 Ligand-exchange chromatography (LEC)........ 82, 86, 90 Lipodex .............................................................................. 7 L-leucine...............................................283, 284, 287, 401 Lorazepam ....................................................................... 23 Loxoprofen ...................................................................... 61 L-phenylalanine....................................................... 86, 289 L-proline.......................................... 17, 86, 322, 326, 329 L-sorbose........................................................................ 383 L-stearoylcarnitine ................................................ 293, 294 Lux® Amylose-2............................... 99, 115, 131, 132, 225 Cellulose-1.................................. 3, 98, 115, 121, 131 Cellulose-2................................................99, 131, 132 Cellulose-3........................................................ 96, 131 Cellulose-4.......................................99, 106, 116, 131 i-Amylose-1 .........................................................3, 109 i-Cellulose-5 ................................................... 132, 143

M Macrocyclic antibiotics............................................ 13, 82, 83, 89, 90, 201, 202, 208–230 Macrocyclic glycopeptides .......................................11–14, 83, 87, 115, 201–230 Marfey’s reagent.............................................................. 58 Mass spectrometry (MS)..................................... 7, 57–76, 128, 133, 140, 212, 215, 227, 228, 265, 391–403, 418–443 Medetomidine ................................................................... 8 MEEKC, see Microemulsion electrokinetic chromatography (MEEKC) MEKC, see Micellar electrokinetic chromatography (MEKC) MEKC-MS, see Micellar electrokinetic chromatographymass spectrometry (MEKC-MS) Metal ions ............................................................ 7, 17, 20, 82, 90, 280, 281, 324–326, 334, 335

Metal-organic frameworks (MOFs) ............20–22, 25, 46 Method development ............................................. 85, 90, 113, 118, 132–141, 206, 215, 225, 343, 345, 354, 373 Method optimization..........................180, 346, 453–476 See also Optimization 7-Methoxyflavanone ................................... 149, 153, 154 Metoprolol .......................................................... 173, 214, 386, 421, 436–440 Micellar electrokinetic chromatography (MEKC) ............................................................1, 7, 22, 25, 340, 343, 346, 399–400, 408–415, 418, 421, 422, 432, 439, 462 Micellar electrokinetic chromatography-mass spectrometry (MEKC-MS) ..............................399, 417–443 Microemulsion electrokinetic chromatography (MEEKC) ............................................1, 7, 22, 25, 340, 343, 346, 366 Microporous organic polymers (MOPs) ....................... 46 Migration modes.................................................. 342–344 Migration order...................................343, 344, 353, 415 MIPs, see Molecularly imprinted polymers (MIPs) Mobile phase selection......................................... 206–208 Models ........................................................................6, 21, 162, 202, 255–263, 267, 280, 308, 332, 345, 358, 454, 456, 467–469, 471, 472, 475 Modifiers.............................................................. 6, 17, 89, 112, 114, 117, 119, 128, 133, 189, 207, 208, 212, 215, 223, 227, 228, 230, 264, 282, 298, 304–306, 308, 310, 311, 313, 314, 316–318, 410, 455, 461, 470–472, 476 MOFs, see Metal-organic frameworks (MOFs) Molecular cages ............................................................... 25 Molecular dynamics .................................... 3, 4, 9, 15, 23 Molecularly imprinted polymers (MIPs) ....................... 21 Molecular micelles............................................ 22–24, 417 Molecular modeling..................................................... 3–5, 7–9, 11, 13, 15, 17, 19, 21, 22, 24 Monolithic column .............................................. 104, 105 Monoliths ...................................104, 105, 112, 114, 272 MOPs, see Microporous organic polymers (MOPs) MS, see Mass spectrometry (MS) Multivariate ............................................. 72, 76, 374, 454 Multivariate optimization ............................................. 346

N NACE, see Nonaqueous capillary electrophoresis (NACE) N-Alkylenyl-α-D-glucopyranoside-6-hydrogen sulfate ................................................................. 429 Nanocellulose ....................................................... 171–180 Nanocrystalline cellulose (NCC) .......173, 174, 176–177 Nano-liquid chromatography.............................. 239–249 Naphthyl ethylamine............................... 20, 61, 191, 295

CHIRAL SEPARATIONS: METHODS Naproxen ............................................................. 131, 221, 227, 239, 241, 247 NCC, see Nanocrystalline cellulose (NCC) N-decyl-S-trityl-(R)-cysteine........................................ 286 N-α-D-glucopyranoside ................................................ 418 Neutral CDs ........................................................ 341, 342, 345, 349, 350, 358, 362, 363, 366, 409, 410 Ninhydrin .................................................... 38, 40, 41, 43 (1S,2R)-N,N-carboxymethyl dodecylnorephedrine......................................... 288 N,N’-dicyclohexylcarbodiimide (DCC) ............. 150, 155 N,N-dioctyl-L-alanine ..................................................... 17 N-n-dodecyl-L-proline ........................328, 329, 333, 335 N2-n-octyl-(S)-phenylalaninamide............. 296, 297, 300 N-octenyl-α-D-glucopyranoside-6-hydrogen sulfate ........................................................ 429, 442 N-octenyl-β-D-glucopyranoside ................................... 426 N-octenyl-β-D-glucopyranoside-6-hydrogen sulfate ................................................................. 442 Nonaqueous ............................................ 9, 111, 264, 267 Nonaqueous background electrolyte .......................9, 340 Nonaqueous capillary electrophoresis (NACE).................................... 373–379, 383–388 Nonsteroidal anti-inflammatory drug (NSAID) .................................................... 83, 131, 160, 239, 241, 244, 247–249, 374 Noradrenaline......................................................... 61, 162 Norephedrine ..................................................5, 194, 288, 289, 366, 367, 370, 421, 435, 436 Normal phase ........................................................ 3, 6, 99, 110, 130, 135, 149, 153, 162, 174, 175, 178, 193, 196, 206, 306, 334 Normal-phase mode (NPM) .................................. 13, 83, 84, 111, 128, 156, 162, 178, 189, 206–208, 214, 216, 219–221, 225, 226, 229, 241 Normal probability plot................................................ 459 Norvancomycin ............................................................. 203 NPM, see Normal-phase mode (NPM) NSAID, see Nonsteroidal anti-inflammatory drug (NSAID) Nucleosil® Chiral-1......................................................... 17 N-undecenyl-α-D-glucopyranoside-4, 6-hydrogen phosphate ............................. 418, 432 N-undecenyl-α-D-glucopyranoside6-hydrogen sulfate ............................................ 442 N-undecenyl-β-D-glucopyranoside .............................. 426 N-undecenyl-β-D-glucopyranoside-4, 6-hydrogen phosphate ...................................... 441 N-undecenyl-β-D-glucopyranoside6-hydrogen sulfate ................................... 429, 442

O Ofloxacin ................................................86, 87, 222, 228, 284, 285, 299, 348, 351, 352

AND

PROTOCOLS Index 485

Optimization ........................................................... 53, 85, 94–103, 105–112, 120, 132, 138, 189, 206, 208, 309, 343–346, 358, 362, 363, 366, 453–476 Ovomucoid...................................................................... 13 Oxaliplatin ...........................................131, 133, 142, 143 Oxfendazole ......................................................... 131, 134

P Packed capillary ...................................112, 245, 248, 448 Pareto charts.................................................................. 459 Partial filling .................................................................. 397 Partition coefficient..................................... 323, 325, 326 Pentobarbital ............................................... 359, 361, 363 Peptides ...........................................................11, 83, 128, 191, 207, 213, 227, 252, 255, 258, 262, 267, 269, 271–272, 280 Pharmaceuticals .............................................. ix, 7, 35, 45, 57, 61, 70, 72, 74, 75, 111, 114, 128, 133–135, 141, 147, 159, 185, 192, 194, 206, 212, 221–228, 239, 359, 391, 453, 454 (S)-Phenylalaninamide ........................283, 284, 296, 297 Phenylalanine..................................................72, 160, 450 Phenylcarbamate ........................................................3, 95, 97–99, 110, 116, 147, 148, 151–152 Phenylethylamine ............................................................ 21 Phenylglycine................................................................. 450 Phenylsuccinic acid .............................................. 328–331 Pirkle-type selectors ............................................... 13, 462 Plackett-Burman Design...................................... 457, 458 Plasma .................................................................... 66, 161, 212, 215, 225–227, 258, 271, 395–397 POC, see Porous organic cage (POC) Polar ionic mode (PIM) .....................206–207, 215, 226 Polar organic mode (POM) ........................................3, 6, 13, 83, 84, 111, 130, 153, 161, 162, 189, 206–208, 214, 215, 217, 221–223, 226 Poly(sodium N-undecylenyl-α-D-glucopyranoside4,6-hydrogen phosphate) (Poly-N-α-DSUGP)...................................................... 419, 421, 430–432, 434–436, 438–440 Poly(sodium N-undecylenyl-α-D-glucopyranoside-6hydrogen sulfate) (Poly-N-α-D-SUGS) ........... 417 Poly(sodium N-undecylenyl-β-D-glucopyranoside4,6-hydrogen phosphate) (Poly-Nβ-D-SUGP)...................... 419, 420, 422, 437–440 Poly(sodium N-undecylenyl-β-D-glucopyranoside6-hydrogen sulfate) (PolyN-β-D-SUGS) .................419, 420, 422, 437, 438 Poly-(sodium undecyl-L-leucyl-L-valinate) (polySULV) ...................................................23, 24 Poly-(sodium undecyl-L-valyl-L-leucinate) (polySUVL) ......................................................... 23 Polycondensation ................................................. 109, 173 Polymeric surfactant ............................................ 417–443

CHIRAL SEPARATIONS: METHODS

486 Index

AND

PROTOCOLS

Polymerization ..................................................20, 21, 23, 418, 420, 421, 429–432 Poly-N-α-D-SUGP, see Poly(sodium N-undecylenyl-α-D-glucopyranoside-4,6hydrogen phosphate) (Poly-N-α-D-SUGP) Poly-N-α-D-SUGS, see Poly(sodium N-undecylenyl-α-D-glucopyranoside-6-hydrogen sulfate) (Poly-N-α-D-SUGS) Poly-N-β-D-SUGP, see Poly(sodium Nundecylenyl-β-D-glucopyranoside-4,6-hydrogen phosphate) (Poly-N-β-D-SUGP) Poly-N-β-D-SUGS, see Poly(sodium Nundecylenyl-β-D-glucopyranoside-6-hydrogen sulfate) ( Poly-N-β-d-SUGS) Polyol .................................................................... 383–388 Polysaccharide .............................................................. 3–6, 14, 93–121, 127–143, 173, 174, 185, 252, 307, 454, 462 Polysaccharide-based....................................................3, 6, 93–121, 127–143, 225, 305 Polysaccharide phenylcarbamates........... 95–99, 110, 111 POM, see Polar organic mode (POM) Porous organic cage (POC) .....................................45–53 Pregabalin ............................................220, 221, 259, 267 Primary amines.............................................................185, 187–189, 192, 193, 195, 196, 203 Propranolol.......................................................... 9, 23, 24, 37, 41, 43, 161, 162, 173, 213, 215, 220, 227, 386, 388 Proteins......................................................... 13, 115, 128, 228, 252, 393, 395, 396, 399 Proton pump inhibitors (PPI).................... 131, 133, 161 Pseudoephedrine ................................................. 213, 366, 367, 370, 421, 435, 436, 438–440 Pseudostationary phase..............393, 399, 408, 410, 437

Q Quinidine..................................15, 17, 85, 256, 261, 262 Quinine..............................................................15, 17, 23, 37, 39–42, 85, 252, 256, 261, 262, 462 Quinoline......................................................................... 15 Quinuclidine.................... 15, 17, 18, 252–254, 263, 264

R Randomization .............................................................. 456 Randomly substituted CDs ........................ 343, 353, 374 Razoxane .............................................................. 132, 134 Recognition mechanisms ...........................................1–25, 81, 119, 202–206, 210, 225, 342, 374, 383 Resolution ........................................................... 9, 20, 21, 35, 40–43, 45–47, 57–59, 73, 75, 82, 86–89, 91, 94, 96, 129, 134, 135, 138–140, 147, 153, 162, 167, 172, 189, 191, 195, 196, 207, 212, 213,

215, 221, 224, 226, 230, 241, 249, 252, 264, 265, 267–269, 272, 279–281, 286, 288, 298, 299, 303–305, 307, 308, 310–314, 316, 318, 322, 334–336, 346, 353, 357, 358, 362, 363, 366, 367, 371, 379, 386, 388, 395, 402, 410, 415, 418, 432, 440, 445, 450, 454, 455, 462, 465, 466, 471–475 Resolvosil® BSA .............................................................. 13 Response surface designs ..................................... 455, 463 Response surface plots ......................................... 472, 475 Retention factors ..................................................... 38, 40, 43, 129, 133, 135–137, 153, 167, 191, 206, 264, 265, 269, 308, 410, 470, 472, 473 Retention mechanism ................................ 128, 133, 134, 207, 215, 253–255, 269, 308 Reversed-phase mode (RPM)........................................ 13, 82–84, 111, 156, 161, 162, 167, 178, 179, 206–208, 213–218, 220–226, 241 Ricobendazole (RBZ) .........................131, 134–141, 143 Ristocetin A .......................................................11, 13, 87, 89, 201–203, 209, 213, 214, 222 Rivastigmine ......................................................... 212, 213 Robustness........................................................... 118, 119, 272, 346, 363, 470 ROESY NMR.................................................................... 8 RPM, see Reversed-phase mode (RPM)

S Salbutamol ............................................................... 37, 41, 43, 213, 215, 221, 227, 259, 385, 386, 462, 463, 473, 474 Screening ...........................................................70, 94, 99, 111, 114, 189, 192, 207, 208, 306, 345, 454, 455, 461–464, 470–473, 475 Screening designs ................................455–466, 473, 475 SDS, see Sodium dodecyl sulfate (SDS) Sepapak™-2 .................................................................... 99 SepapakTM-3.................................................................... 99 Sepapak™-4 ...................................................99, 113, 121 SFC, see Supercritical fluid chromatography (SFC) Single isomer CDs ............................................... 343, 353 Sodium dodecyl sulfate (SDS)................ 9, 408–412, 414 (S,S)-trans-2-aminocyclohexanesulfonic acid.............. 462 Stereorecognition.......................................................... 2, 4 Stereoregular polymers ................................................... 20 Sulfoxides............................. 47, 130, 132, 134, 226, 230 Supercritical fluid ....................... 147, 202, 304, 470–472 Supercritical fluid chromatography (SFC)................. ix, 1, 3, 7, 57, 111, 119, 147, 159, 184–193, 196, 225, 240, 252, 259, 272, 303–319, 357, 461, 470, 471, 473, 475 Superficially porous particles (SPPs) .........................7, 14, 185, 190–193, 196, 221, 225, 227, 228, 271, 272

CHIRAL SEPARATIONS: METHODS Suprofen ..............................................131, 247, 375, 378 Surfactants ................................................ 9, 23, 343, 346, 392, 393, 399, 408–410, 417, 418, 420, 424–433, 436, 438, 440

T Talinolol............................................ 9, 10, 421, 436, 437 Tartaric acid ...............................................................19, 85 (R,R)-tartaric acid mono-(R)-1-(α-naphthyl) ethylamide.......................................................... 295 Teicoplanin ...........................................11, 13, 14, 83, 87, 89, 201–203, 209, 210, 213–230, 240, 241 Teicoplanin aglycone ........................................11, 13, 14, 203, 205, 210, 213, 214, 217, 218, 220–224 TeicoShell® ............................................................. 14, 228 Terbutaline .......................................................... 213, 215, 226, 375, 376, 386 Tetrabutylammonium L‑argininate (TBA‑L‑Arg)..................................... 366, 368, 370 Thin-layer chromatography (TLC).........................35–44, 201, 202, 210, 240, 327, 329, 334, 421, 424, 426, 427, 442 Thioether ....................................................................... 162 Thiostrepton................................................ 202, 203, 209 Timolol .......................................................................... 162 Tioconazole ..................................................410, 413–415 TLC, see Thin-layer chromatography (TLC) Trans-(1R,2R)-diaminocyclohexane ........................... 172 Trans-stilbene oxide (TSO)................................. 308–317 Triazine labeling reagent ..........................................57–76 Triazole .......................................................................... 162 Triclabendazole ............................................................. 134 Triethoxysilyl .......................................109, 172, 173, 177 3H-1,2,3-Triazolo[4,5-b]pyridine-3-ol (HOAt) ............................................ 61, 64, 73, 74 2,2,2-Trifluoro-1-(9-anthryl)ethanol ................. 104, 114 Tro¨ger’s base ........................................... 93, 94, 188, 192 Tryptophan........................ 160, 448–450, 462, 463, 473

AND

PROTOCOLS Index 487

U UHPLC, see Ultra high-performance liquid chromatography (UHPLC) Ultra high-performance liquid chromatography (UHPLC) ................................................ 1, 59, 73, 241, 271, 462 Ultron ES-CD® ................................................................. 7

V Validated method .......................134, 212, 221, 226, 352 Validation....................................................................... 353 van Deemter equation ......................................... 105, 241 Van’t Hoff ...........................................129, 139, 212, 308 Vancomycin ............................................................. 11, 13, 14, 83, 86, 87, 89, 201–203, 209, 210, 212, 213, 215, 216, 218, 221, 224, 226, 227, 230, 239–249, 322 VancoShell® ..................................................................... 14 Vigabatrin ............................................................. 220, 221

W Warfarin ............................................................... 132, 212, 213, 215, 218, 224 Whelk-O1 ..................................................................13, 15

X Xanthones ........................................................................ 13 X-ray crystallography .......................................3, 7, 15, 19

Z Zwitterionic ................................................ 7, 11, 15, 240, 251–272, 353, 418, 432, 462 Zwitterionic ion-exchange..................................... 15, 252 ZWIX® .................................................... 15, 17, 254–265, 268, 271, 272