CRC handbook of chromatography. Volume I, Plant pigments [1st ed.] 9781351367158, 1351367153, 978-1-138-10504-1, 978-1-315-15056-7

Table of contents :
Content: Part 1:1.Chromatographic Methods for the Separation of Carotenoids. Tables. Part 2: 1.Chromatographic Methods for the Separation of Porphyrins and Metalloporphyrins. Tables. Part 3:1.Chromatographic Methods for the Separation of Chloro-phylls. Tables. Index.

Citation preview

CRC Series in Chromatography Editors-in-Chief

Gunter Z w eig, Ph.D . and Joseph Sherm a, Ph.D . General Data and Principles

Lipids

Gunter Zweig, Ph.D. and Joseph Sherma, Ph.D.

Helmut K. Mangold, Dr. rer. nat.

Hydrocarbons Walter L. Zielinski, Jr., Ph.D.

Carbohydrates Shirley C. Churms, Ph.D.

Inorganics M. Qureshi, Ph.D.

Drugs Ram Gupta, Ph.D.

Phenols and Organic Acids Toshihiko Hanai, Ph.D.

Terpenoids Carmine J. Coscia, Ph.D.

Amino Acids and Amines S.

Blackburn, Ph.D.

Steroids Polymers

)seph C. Touchstone, Ph.D.

Charles G. Smith, Norman E. Skelly, Ph.D., Carl D. Chow, and Richard A. Solomon

Pesticides and Related Organic Chemicals Plant Pigments

Joseph Sherma, Ph.D. and Joanne Follweiler, Ph.D.

Hans-Peter Kost, Ph.D.

Nucleic Acids and Related Compounds Ante M. Krstulovic, Ph.D.

CRC Handbook of Chromatography Plant Pigments Volume I Fat-Soluble Pigments Editor

Hans-Peter Kost, Dr. rer. nat. Botanic Institute University of Munich F.R.G.

Editors-in-Chief

Gunter Zweig, Ph.D.

Joseph Sherma, Ph.D.

President Zweig Associates Arlington, Virginia (Deceased)

P rofessor of Chemistry Lafayette College E aston, Pennsylvania

First published 1988 by CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 Reissued 2018 by CRC Press © 1988 by Taylor & Francis CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www. copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organiza-tion that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. A Library of Congress record exists under LC control number: 87021821 Publishers Note The publisher has gone to great lengths to ensure the quality of this reprint but points out that some imperfections in the original copies may be apparent. Disclaimer The publisher has made every effort to trace copyright holders and welcomes correspondence from those they have been unable to contact. ISBN 13: 978-1-138-10504-1 (hbk) ISBN 13: 978-1-315-15056-7 (ebk) Visit the Taylor & Lrancis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

CRC SERIES IN C H R O M A TO G RA PH Y SERIES PREFACE The fat-soluble photosynthetic pigments present in plants and algae, including chloro­ phylls, carotenoids, and related pigments, comprise an important class of compounds with an extensive literature. Dr. Kost and his co-authors have done an admirable job in searching out and organizing much of the critical chromatographic data and methodology in the present volume. Because of the chemical nature of these prenyllipid compounds, liquid chromatography is preferred for their isolation, separation, and determination. The most widely used methods include low pressure column LC, paper chromatography, TLC, and, most recently, HPLC. All of these methods are covered by Dr. Kost. Chromatography was “ invented” in the early 1900s by Michael Tswett, a Russian botanist and plant physiologist who first applied liquid-solid chromatography on a column of chalk to resolution of the complex natural mixture of yellow and green chloroplast pigments in the extracts of leaves he was studying. On a personal note, I was fortunate to work with Dr. Harold Strain for five summers at the Argonne National Laboratory when I first began to teach. Dr. Strain was one of the first important American chromatography experts and used all variations of liquid chromatography extensively in his studies of photosynthetic pigments. My experience with Dr. Strain set the foundation for my lifelong career of research and writing in chromatography. Readers of this Handbook are asked to contact the Series Editor if they find errors or omissions in coverage as well as with suggestions for future volumes and authors within the Handbook of Chromatography series. Joseph Sherm a

THE EDITORS-IN-CHIEF Gunter Zweig, Ph.D., received his undergraduate training at the University of Maryland, College Park, where he was awarded the Ph.D. in biochemistry in 1952. Two years following his graduation, Dr. Zweig was affiliated with the late R. J. Block, pioneer in paper chro­ matography of amino acids. Zweig, Block, and Le Strange wrote one of the first books on paper chromatography, which was published in 1952 by Academic Press and went into three editions, the last one authored by Gunter Zweig and Dr. Joe Sherma, the co-Editor-in-Chief of this series. Paper Chromatography (1952) was also translated into Russian. From 1953 to 1957, Dr. Zweig was research biochemist at the C. F. Kettering Foundation, Antioch College, Yellow Springs, Ohio, where he pursued research on the path of carbon and sulfur in plants, using the then newly developed techniques of autoradiography and paper chromatography. From 1957 to 1965, Dr. Zweig served as lecturer and chemist, University of California, Davis and worked on analytical methods for pesticide residues, mainly by chromatographic techniques. In 1965, Dr. Zweig became Director of Life Sci­ ences, Syracuse University Research Corporation, New York (research on environmental pollution), and in 1973 he became Chief, Environmental Fate Branch, Environmental Pro­ tection Agency (EPA) in Washington, D.C. From 1980 to 1984 Dr. Zweig was Visiting Research Chemist in the School of Public Health, University of California, Berkeley, where he was doing research on farmworker safety as related to pesticide exposure. During his government career, Dr. Zweig continued his scientific writing and editing. Among his works are (many in collaboration with Dr. Sherma) the now 11-volume series on Analytical Methods fo r Pesticides and Plant Growth Regulators (published by Academic Press); the pesticide book series for CRC Press; co-editor of Journal of Toxicology and Environmental Health; co-author of basic review on paper and thin-layer chromatography for Analytical Chemistry from 1968 to 1980; co-author of applied chromatography review on pesticide analysis for Analytical Chemistry, beginning in 1981. Among the scientific honors awarded to Dr. Zweig during his distinguished career were the Wiley Award in 1977, the Rothschild Fellowship to the Weizmann Institute in 1963/64; and the Bronze Medal by the EPA in 1980. Dr. Zweig authored or co-authored over 80 scientific papers on diverse subjects in chro­ matography and biochemistry, besides being the holder of three U.S. patents. In 1985, Dr. Zweig became president of Zweig Associates, Consultants in Arlington, Va. Following his death on January 27, 1987, the Agrochemicals Section of the American Chemical Society posthumously elected him a Fellow and established the Gunther Zweig Award for Young Chemists in his honor. Joseph Sherma, Ph.D., received a B.S. in Chemistry from Upsala College, East Orange, N.J., in 1955 and a Ph.D. in Analytical Chemistry from Rutgers University in 1958, carrying on his thesis research in ion exchange chromatography under the direction of the late William Rieman III. Dr. Sherma joined the faculty of Lafayette College in September, 1958, and is presently Charles A. Dana Professor and Head of the Chemistry Department. Dr. Sherma, independently and with others, has written over 300 research papers, chapters, books, and reviews involving chromatography and other analytical methodology. He is editor for residues and trace elements of the Journal of the Association of Official Analytical Chemists and a member of the advisory board of the Journal of Planar Chromatography. He is a consultant on analytical methodology for many companies and government agencies. Dr. Sherma has received two awards for superior teaching at Lafayette College and the 1979 Distinguished Alumnus Award from Upsala College for outstanding achievements as an educator, researcher, author, and editor. He is a member of the ACS, Sigma Xi, Phi Lambda Upsilon, SAS, AIC, and AO AC. Dr. Sherma’s current interests are in quantitative TLC, mainly applied to clinical analysis, pesticide residues, and food additives.

IN TROD UCTION While doing scientific work, many people regularly come across colored compounds that are either contained within plant or animal tissues or that perhaps represent an excreted component of the culture medium. Often pigments, because of their light-absorbing prop­ erties, have well-defined tasks, e.g ., protection from light or as sensor and antenna pigments. Needless to say, when involved with the analysis of these pigments, one should have suitable literature available. Not only in scientific research, but also in routine analysis the exact determination of plant pigments plays a more and more pronounced role. For example, carotenoids may be used as food dyes, enhancing an unappetizing color. Their exact ap­ plication necessitates exact analysis. For this purpose, the Handbook o f Chromatography: Plant Pigments, Volume I: FatSoluble Pigments has been compiled, in its essence an assemblage of tables where, besides data obtained by modem separation methods, older sources, often difficult to access, have also been included to give maximum possible information. It is a simple truth that if a pigment is unambiguously identified and described, it will keep the same chromatographic properties, the same absorption maxima, and the same molar extinction coefficient forever! Especially in older books, there are many valuable data that may easily be overlooked and “ buried” by the nearly logarithmically growing flood of data published today. On the other hand, scientific methods of analysis and identification at present are developing more rapidly than ever: for example, modem high performance liquid chromatography (HPLC). Often a sample with little or no prior preparation can be injected directly onto tiny columns. The setup often is so sensitive that literally traces of pigments, not noticeable to the naked eye, are sufficient to obtain qualitative as well as quantitative data. Unfortunately, however, the new methods are rather expensive in terms of apparatus, equipment, and supply of suitable chemicals and solvents. This creates a clear limit of availability, especially for small lab­ oratories and individual researchers. Most scientists, when dealing with a simple problem of separation and identification, just do not need sophisticated equipment, but reliable and cheap methods that are nevertheless reasonably quick and easy to handle. Also, even with the best possible instrumental equipment, it is indispensible to carry out some preliminary separation and identification steps before committing to the use of an expensive column that might easily be rendered inoperative by incompatible compounds. The present Handbook is intended to give information on not only the most recent but also the proven older techniques. In this sense, I wish the users of the book good success. The Editor

THE EDITOR Dr. Hans-Peter Kost, Dr. rer. nat. is at present Privatdozent at the Botanic Institute of the University of Munich, Munich, F.R.G. Dr. Kost received his chemistry diploma from the University of Saarbrucken in 1970 and subsequently was awarded the degree of Dr. rer. nat. in Natural Science, specializing in phytochemistry, from Munich University in 1974. From 1975 to 1977 he was a postdoctoral fellow at the University of California, Los Angeles. In 1981 he was promoted to Dr. rer. nat. habil. and was thereafter lecturer at the Munich Ludwigs-Maximilian-University. From 1984 to 1986 he substituted for different professors, including a period of one semester in Saarbrucken. Dr. Kost is a member of the German Chemical Society (Gesellschaft Deutscher Chemiker, GDCh) and the American Chemical Society. He is also a member of the International Society for the Study of the Origin Of Life and the International Electrophoresis Society, as well as an elected member of the New York Academy of Sciences. Dr. Kost has published about 50 articles in scientific journals, chiefly dealing with the chemistry and physiology of tetrapyrrolic pigments of plants and animals.

ACKNO W LEDGM EN TS H.-P. Kost wishes to thank all these many people who have helped with advice and practical support to finish the present volume: I was given very substantial assistance by my former technician G. Widerer, in industry. Dr. E. Schropp has helped a great deal in planning the conception of the carotenoid part of the volume. I would like to gratefully acknowledge the competent support of Dr. E. Benedikt in preparing the “ Porphyrins” part of the present volume and for much graphic work, especially concerning the “ Carotenoids” and the “ Porphyrins” sections of the book. My special thanks are devoted to all my friends and colleagues who helped me in all thinkable ways. I don’t want to fail to thank CRC press for help, advice, and often patience. My special thanks are devoted to Ms. Amy Skallerup. Finally, I want to emphasize the invaluable support in compiling data given by my wife, Dr. E. Kost-Reyes. Equally valuable, however, is the moral support she has given me over all these last years.

A DV ISORY BOARD

Bruce Burnham, Ph.D.

Eliana Kost-Reyes, Ph.D.

President Porphyrin Products Logan, Utah

Jesenwang Federal Republic of Germany

Brian H. Davies, Ph.D.

Hugo Scheer, Dr.habiI.rer.nat.

Professor Department of Biochemistry and Agricultural Biochemistry University College of Wales Aberystwyth Wales

Professor Botanical Institute University of Munich Munich Federal Republic of Germany

CON TRIBU TO RS

Eva Benedikt, Ph.D. Botanical Institute University of Munich Munich Federal Republic of Germany

Eliana Kost-Reyes, Ph.D. Jesenwang Federal Republic of Germany

Hugo Scheer, Dr.habil.rer.nat.

President Porphyrin Products Logan, Utah

Professor Botanical Institute University of Munich Munich Federal Republic of Germany

Brian H. Davies, Ph.D.

Eva Schropp, Ph.D.

Professor Department of Biochemistry and Agricultural Biochemistry University College of Wales Aberystwyth Wales

Botanical Institute University of Munich Munich Federal Republic of Germany

Bruce Burnham, Ph.D.

Hans-Peter Kost, Ph.D. Research and Development Serva-Technik, GmbH Heidelberg Federal Republic of Germany

TA BLE OF CONTENTS

Part I: CAROTENOIDS B. H. Davies and Hans-P. Kost CHROMATOGRAPHIC METHODS FOR THE SEPARATION OF CAROTENOIDS Introduction.................................................................................................................................... 3 Some Remarks on Carotenoid Formation and Sources........................................................... 3 Carotenoid Characterization.........................................................................................................4 Carotenoid Handling and Storage........................................................................................... 4 Carotenoid Crystallization and Melting P oints..................................................................... 4 Spectroscopic Methods.............................................................................................................. 4 UV-vis Spectroscopy of Carotenoids................................................................................. 4 Recording of Absorption S pectra................................................................................... 5 Quantitative Determination of Carotenoids....................................................................5 Infrared Spectroscopy of Carotenoids ................................................................................5 ‘H NMR Spectroscopy of Carotenoids..............................................................................6 Separation of Carotenoids by Chromatography — Introductory Rem arks........................ 6 Alternative Procedures for the Analysis of Carotenoids...................................................... 7 References........................................................................................................................................7 TABLES FOR THE ESTIMATION AND SEPARATION OF CAROTENOIDS General T ab le s..............................................................................................................................11 Table I. 1. Main Absorption Maxima of Lycopene in Various Aliphatic ^-Alcohols...........................................................................................................11 Table 1.2. Main Absorption Maxima of Lycopene in Various Solvents...................... 11 Table I. 3. Name List for Carotenoids (Tables 1.5, 1.6, I.PC, I. TLC, and I. L C )........................................................................................................... 12 Table I. 4. Name List for Carotenoids (HPLC and GC Tables) ..................................91 Table I. 5. Qualitative Spectroscopic Data: Absorption Maxima in Different Solvents.............................................................................................93 Table I. 6. Quantitative Spectroscopic Data: Molar Extinction Coefficients (cm “ 'M _l) of Carotenoids ............................................................................119 Paper Chromatography of Carotenoids...................................................................................131 Table N otes............................................................................................................................. 131 Table I. PC 1. Cellulose Papers and Impregnated Cellulose Papers— I ..................... 132 Table I. PC 2. Cellulose Papers and Impregnated Cellulose Papers—II..................... 138 Thin Layer Chromatography of Carotenoids.......................................................................... 141 Table N otes..............................................................................................................................141 Table I. TLC 1. TLC on Silica Gel G L ay ers................................................................142 Table I. TLC 2. TLC on Various One-Component Layers—I .................................... 145 Table I. TLC 3. TLC on Various One-Component Layers— II.................................... 147 Table I. TLC 4. TLC on Various One-Component Layers—III...................................150 Table I. TLC 5. TLC on Various Two-Component Layers (Containing Silica Gel)— I ....................................................................153 Table I. TLC 6. TLC on Various Two-Component Layers (Containing Silica Gel G)— I I ...............................................................154 Table I. TLC 7. TLC on Two- and Multicomponent Layers.......................................... 156 Table I. TLC 8. TLC on “ Thinlayer A” ...........................................................................158 Table I. TLC 9. TLC on Various Reversed-Phase L ay ers..............................................159

Liquid Chromatography of Carotenoids................................................................................. 161 N o tes........................................................................................................................................161 Table I. LC 1. Survey of Different Adsorbents and Solvents Used for Carotenoids.................................................................................................162 High Performance Liquid Chromatography of Carotenoids................................................. 171 Table Notes..............................................................................................................................171 Table I. HPLC 1. Carotenoids (Retention Times)............................................................. 172 Table I. HPLC 2. Carotenoids: Complementary Survey — I ..................................... 176 Table I. HPLC 3. Carotenoids: Complementary Survey — II..................................... 178 Gas-Liquid Chromatography of Carotenoids...........................................................................179 Table Notes..............................................................................................................................179 Table I. GC 1. GC of Carotenoids — 1.............................................................................. 180 Table I. GC 2. GC of Carotenoids — I I ............................................................................ 183

PART II: PORPHYRINS (EXCLUSIVE OF CHLOROPHYLLS) Bruce F. Burnham and Hans-P. Kost CHROMATOGRAPHIC METHODS FOR THE SEPARATION OF PORPHYRINS AND METALLOPORPHYRINS Structure, Function, Occurrence, and Biosynthesis of Porphyrins.................................. 189 Introductory Remarks ....................................................................................................... 189 Biosynthesis........................................................................................................................ 189 Characterization of Porphyrins............................................................................................. 191 Spectroscopic M ethods......................................................................................................191 UV-vis Spectroscopy......................................................................................................191 Infrared Spectroscopy.................................................................................................... 193 NMR Spectroscopy....................................................................................................... 193 Mass Spectroscopy......................................................................................................... 193 Melting Points..................................................................................................................... 194 Partition Behavior (HC1 N um bers)................................................................................. 194 Estimation and Separation of Porphyrins............................................................................ 194 Notes on Porphyrin Stability........................................................................................... 194 Prechromatography Purification and Sample Preparation............................................ 195 Sample Preparation from Basic Aqueous Solutions................................................. 195 Ethyl Acetate Extraction........................................................................................... 195 Absorption and Concentration on T a lc .................................................................. 195 Sample Preparation from Acidic Aqueous Solutions............................................... 195 Absorption and Concentration on DEAE-Cellulose..............................................197 Extraction of Porphyrins...............................................................................................197 Extraction from T a lc ................................................................................................ 197 Extraction from DEAE Cellulose............................................................................ 197 Preparation and Extraction of Porphyrin Methyl Esters....................................... 197 Chromatography............................................................................................................ 198 Paper Chromatography (P C )........................................................................................198 Thin-Layer Chromatography (TLC)............................................................................ 198 Liquid Chromatography (L C )...................................................................................... 198 High Performance Liquid Chromatography (H P L C )............................................... 198 Sources and Materials for H PLC ............................................................................ 199 Hyperpressure Gas Chromatography (HPGC)........................................................... 199 Paper Electrophoresis (P E L )........................................................................................ 199 References...............................................................................................................................200

TABLES FOR THE ESTIMATION AND SEPARATION OF PORPHYRINS AND METALLOPORPHYRINS General T ab les...........................................................................................................................205 Table II. 1. Trivial Names and Structures of Common Porphyrins............................ 205 Table II. 2. Quantitative Spectroscopic Data: Molar Extinction Coefficients...........206 Table II. 3. HC1 Numbers of Porphyrins and Porphyrin Esters...................................207 Paper Chromatography (PC) of Porphyrins.......................................................................... 208 Table II. PC 1. Free Acid Porphyrins................................................................................208 Table II. PC 2. Porphyrin Esters........................................................................................ 209 Table II. PC 3. Porphyrin Esters (Two-Solvent Systems).............................................. 211 Table II. PC 4. Porphyrin ‘‘Derivatives” ......................................................................... 212 Table II. PC 5. Porphyrins and Metalloporphyrins..........................................................213 Thin Layer Chromatography (TLC) of Porphyrins...............................................................215 Table II. TLC 1. Free Acid Porphyrins............................................................................ 215 Table II. TLC 2. Porphyrin Esters.....................................................................................217 Table II. TLC 3. Metalloprophyrins................................................................................. 219 High Performance Thin Layer Chromatography (HPTLC) of Porphyrins........................220 Table II. HPTLC L Porphyrin Esters............................................................................... 220 Liquid Chromatography (LC) of Porphyrins.........................................................................221 Table II. LC 1. Porphyrin E ste rs...................................................................................... 221 Table II. LC 2. Porphyrins, Hemins, and Esters (Silica G e l)....................................... 222 Table II. LC 3. Porphyrins and Porphyrin Esters (Sephadex)....................................... 223 High Performance Liquid Chromatography (HPLC) of Porphyrins.................................. 224 Table II. HPLC 1. Porphyrins and Porphyrin Esters...................................................... 224 Table II. HPLC 2. Porphyrins and Porphyrin Esters: Separation of Isom ers..............225 Table II. HPLC 3. Survey of Sample Workup for HPLC of Porphyrins (Condensed T ab le )...................................................................................................................................229 Hyperpressure Gas Chromatography (HPGC) of Porphyrins and Metalloporphyrins. ... 230 Table II. HPGC 1. Porphyrins and Metalloporphyrins................................................... 230 Paper Electrophoresis (PEL) of Porphyrins.......................................................................... 231 Table II. PEL 1. Porphyrins...............................................................................................231

PART Ills CHLOROPHYLLS Hugo Scheer CHROMATOGRAPHIC METHODS FOR THE SEPARATION OF CHLOROPHYLLS Introductory, Functional, and Biosynthetic Considerations............................................235 Introduction...................................................................................................................... 235 Structure, Function, Occurrence, Biosynthesis...........................................................235 Characterization of Chlorophylls: Spectroscopic Methods............................................. 239 UV-vis Spectra................................................................................................................. 239 Fluorescence Spectra.......................................................................................................241 Circular Dichroism .......................................................................................................... 242 NMR Spectroscopy.......................................................................................................... 242 Mass Spectroscopy.......................................................................................................... 243 Estimation and Separation of Chlorophylls.......................................................................244 Nonchromatographic Analytical Techniques................................................................ 244 Prechromatography Purification.................................................................................... 244

Chromatography.................................................................................................................245 Introductory Remarks................................................................................................... 245 Chlorophylls...................................................................................................................245 Preparative Chromatography.................................................................................... 243 Analytical Chromatography on TLC plates...........................................................247 Analytical Column Chromatography.......................................................................247 Continuous Liquid-Liquid Partition........................................................................ 248 Pheophytins.................................................................................................................... 248 Preparative Separations.............................................................................................248 Analytical Chromatography .................................................................................... 249 Special Considerations for Chlorophyll High Performance Liquid Chromatography (Appendix A ) .................................................................................249 Pum ps......................................................................................................................... 249 Columns...................................................................................................................... 249 Silylation of Reverse-Phase HPLC C olum ns....................................................... 249 E luents........................................................................................................................250 Detectors.................................................................................................................... 250 Derivatization Reactions and Formation of Artifacts (Appendix B ) ......................250 Pheophytinization of Chlorophylls.......................................................................... 250 Common Artifacts..................................................................................................... 251 Artifacts Involving the Central Magnesium Atom............................................251 Artifacts Involving the Isocyclic R in g .............................................................. 251 Artifacts Involving Carbon C -20........................................................................ 252 Artifacts Involving the Oxidation of the Macrocycle.......................................252 Artifacts Involving the 3-Vinyl G roup.............................................................. 252 Artifacts Involving Propionic Ester Side C hains............................................. 252 References...............................................................................................................................253 TABLES FOR THE ESTIMATION AND SEPARATION OF CHLOROPHYLLS General T ab les........................................................................................................................... 261 Table III. 1. Name List: Structure, Functions, Occurrence and Spectra of Chlorophylls..................................................................................................261 Table III. 2. Quantitative Spectroscopic Data: Molar Extinction Coefficients (cm -1 mol ') of Chlorophylls. Complementary Spectroscopic D ata.. 266 Table III. 3. Effects of Chemical Modifications on Chlorophyll Absorption............. 270 Table III. 4. Estimation of Chlorophylls by Spectroscopic Techniques......................277 Table III. 5. Discriminatory Detection Wavelengths for Chlorophylls....................... 279 Table III. 6. Degradation Products of Chlorophylls........................................................280 Paper Chromatography (PC)..................................................................................................... 282 Table III. PC. 1. Survey of Chromatography Systems.................................................... 282 Thin Layer Chromatography (T L C )........................................................................................283 Table III. TLC 1. Survey of Different Adsorbents Used for Chlorophylls..... 283 Table III. TLC 2. TLC of Total Pigment Extracts..............................................285 Table III. TLC 3. TLC of Nonesterified Pigments..............................................286 Liquid Chromatography (LC)....................................................................................................287 Table III. LC 1. Survey of Different Adsorbents Used for Chlorophylls..................... 287 Table III. LC 2. LC of Total Pigment Extracts................................................................288 Table III. LC 3. Preparative LC of Total Pigment Extracts........................................... 289 Table III. LC 4.LC of Reduction Products of Chlorophylls a and b .......................... 290 Table III. LC 5.LC of Bacteriochlorophyll e Derivatives............................................. 290 Table III. LC 6.Liquid-Liquid Partition of Chlorophyll Derivatives............................ 290

High Performance Liquid Chromatography (HPLC)........................................................... 292 Table III. HPLC 1. HPLC of Total Extracts...................................................................292 Table III. HPLC 2. HPLC of Chlorophylls and Derivatives........................................ 294 Table III. HPLC 3. HPLC of Chlorophyllous Pigments IncludingFree A cids........... 297 Table III. HPLC 4. HPLC of Bacteriopheophytins a ....................................................300 Table III. HPLC 5. HPLC of Chlorophylls a, b, c, and Some Unknowns................ 301 Table III. HPLC 6. HPLC of Derivatives of Bacteriochlorophylls c ........................ 302 Table III. HPLC 7. HPLC of Prime Chlorophylls.........................................................303 Table III. HPLC 8. HPLC of Chlorophyll Derivatives..................................................304 Table III. HPLC 9. HPLC of Bacteriomethylpheophorbides d .................................... 305 Table III. HPLC 10. HPLC of Nonesterified Chlorophyll Pigm ents........................... 306 INDEX........................................................................................................................................309

Part I: Carotenoids B. H. D avies and H ans-P. Kost

Chromatographic Methods for the Separation of Carotenoids

Volume I: Fat-Soluble Pigments

3

IN TRO D U CTIO N Many of the vividly red, orange, or yellow flowers and fruits, as well as a number of animals, owe their appearance to the presence of a class of more-or-less unsaturated tetraterpenoids called “ carotenoids” .1 The name comes from the carrot, Daucus carota, from which the prechromatographic “ carotene” was isolated (Wackenroder, 1831); only by chro­ matography could it be shown that there are a-, (3-, y-, and 8-carotenes in the carrot.13 Since then, the carotenoids have been extensively studied in many branches of natural science.13 5 The greater lability of the chlorophylls during autumnal necrosis reveals the carotenoids in the “ fall colors” of deciduous trees. Carotenoids are present in the thylakoid membranes of higher plants, algae, and photosynthetic bacteria; here one part of their function is to serve with lesser or greater efficiency as accessory pigments for light-harvesting in photosynthesis. They are not confined to the photosynthetic organelles; however, their presence and synthesis in so many fungi, yeasts, and bacteria which sometimes exhibit intense colors suggest another, wider function: the universal function of carotenoids as photoprotectants (compare References 3 to 5, 7 to 9). Carotenoids are also contained in the display apparatus of a variety of flowers;1011 here they help to attract insects. Many animals contain carotenoids, also, but only via their diet, for they cannot synthesize them as plants can. Chemically, carotenoids are hydrocarbons with numerous conjugated double bonds. The first carotenoid whose structure was elucidated (by Karrer in 1930) was lycopene, the red pigment of tomatoes and other fruits (for historic background, see References 1 and 12). SOM E R EM A R K S ON CA R O TEN O ID FO RM ATION AND SO U R C ES3 5 710 1315 The first “ typical” intermediate in carotenoid biosynthesis is isopentenyldiphosphate, which is formed via hydroxymethylglutaryl CoA and then mevalonic acid from the con­ densation of three molecules of acetyl CoA which arise from the intermediary metabolism of carbohydrates and lipids. By the action of an isomerase, isopentenyldiphosphate is con­ verted to dimethylallyldiphosphate, which easily splits off a diphosphate anion while leaving a carbonium ion. The carbonium ion may now condense head-to-tail with one molecule of isopentenyldiphosphate to form geranyldiphosphate. By the addition of a further molecule of isopentenyldiphosphate, famesyldiphosphate, a C-15 intermediate, is synthesized. After their formation from this compound, two tetraisoprenoid geranyl-geranyldiphosphates are condensed to molecules of the phytoene, a colorless compound which contains only three conjugated double bonds. Via stepwise dehydrogenation, phytoene is converted to phytofluene, ^-carotene, and neurosporene. Eventually, the intensely red-colored lycopene is formed. Lycopene may be converted via ring formation — the a- or (3-ionone rings of a- or (3-carotene, respectively. Animal pigments may have undergone considerable modifications; an example for such a modified pigment is the conversion of (3-carotene into canthaxanthin by the brine shrimp, Artemia salina L. (Crustacea, Branchiopoda).15 Some animals accumulate carotenoids in quantity (e.g., goldfish, flamingo), while others retain just as much as they require to convert via intestinal bisectioning of the molecule into retinal, which is the basis of all animal vision. Further conversion products are its primary alcohol retinol (vitamin A) and the corresponding acid (retinoic acid). Since all of these “ retinoids” are derived from dietary plant carotenoids, it is clear that the analysis of plant carotenoids has considerable nutritional significance. Today, another source of carotenoids is chemical synthesis; its route is often according to published procedures (compare References 1 and 16). A number of synthetic carotenoids

4

CRC Handbook of Chromatography: Plant Pigments

are used as food dyes. Since treating the principles of synthesis would exceed the scope of this book, the reader is referred to the respective literature. 16 Several hundreds of carotenoids are known, and their number is increasing logarithmically. 1 · 17 (See Table 1.3.)

CAROTENOID CHARACTERIZATION 1· 19 Carotenoid Handling and Storage Carotenoids, once extracted, are labile and require protection from heat, light, and oxygen. No more heat than absolutely necessary should be used in their manipulation. Low boiling solvents should be employed wherever possible and the concentration of carotenoid solutions should be carried out under reduced pressure using a rotatory evaporator. Carotenoids should not be exposed to bright light; the laboratory window(s) should not face the sun, and vessels, chromatography columns, and TLC tanks should be covered with black cloth, aluminum foil, etc., where appropriate. It is good practice to develop thin-layer chromatograms (TLC) in an inert atmosphere like nitrogen, although it may not always be necessary. As some carotenoids are acid labile (isomerization; furanoid epoxide formation), contact with acids must be avoided. Alkali is used for saponification, but it must be borne in mind that some carotenoids (e.g., astaxanthin) are also alkali labile. 1 Carotenoids should be stored in aliphatic or aromatic hydrocarbon solvents (under nitrogen or argon), in the dark, and in the deep freeze ( - 20°C). Carotenoid Crystallization and Melting Points Around 1950, when many carotenoid syntheses were carried out (for review see Reference 16), an important criterion of purity was the determination of melting points of the crystalline synthetic compounds; for that purpose enough crystalline material was available. Although chromatographic isolation procedures provide enough material for carrying out a variety of spectroscopic and chemical identification procedures, there is often not sufficient material for the production of crystals, and achievement of crystallization requires much experience. For example, carotenoids tend to form mixed crystals, thereby sometimes barring the possibility of purification by fractionated crystallization. Another drawback of carotenoid crystallization is the rapid decomposition of "crystalline" but semipure material, which participants of phytochemical courses can confirm. This decomposition is most pronounced if carotenoids are obtained as a liquid, noncrystalline film. Only where pure carotenoids are crystallized can they be conserved in the solid state and kept for a long time, especially when stored under an inert gas, like argon or nitrogen, and in the dark. A quick, general description of how to carry out carotenoid crystallization is given in the following: the method is based on the fact that many carotenoids dissolve poorly in methanol, but very well in aromatic solvents like benzene or toluene. In a centrifuge tube, the carotenoid to be crystallized (e.g., lycopene) is dissolved and hot methanol is added to begin crystallization. The sample is allowed to stand for 2 hr and then centrifuged. The crystals obtained are washed with boiling methanol and dried over phosphorus pentoxide. Spectroscopic Methods20 The spectroscopic methods usually applied comprise IR spectroscopy, UV-vis spectroscopy, nuclear magnetic resonance spectroscopy (NMR), and mass spectroscopy. The latter two methods are of especially great value in characterizing carotenoids. Unless the structure of a carotenoid has been confirmed by 1H NMR and mass spectroscopy (and, in the case of a carotenoid with one or more chiral centers, by CD), it cannot be considered as unambiguously identified. This does not, of course, apply to the known carotenoids of some plants. UV-Vis Spectroscopy of Carotenoids A key feature of carotenoids is their long system of conjugated double bonds on their

Volume I: Fat-Soluble Pigments

5

hydrocarbon “ backbone” . These conjugated double bonds give rise to the yellow or red carotenoid color. Mostly, carotenoids exhibit three distinct absorption maxima which have been listed in Table 1.5. Usually, the carotenoid double bonds are in all-trans positions (see figures for Table 1.5); however, cw-isomers of individual carotenoids are also known. The absorption spectra (= electron spectra) of the carotenoids are very much dependent on the solvent (solvent effect20). A few examples are given: the wavelength of absorption of some carotenoids (e.g., lycopene) exhibits a bathochromic shift when homologous, primary, al­ iphatic alcohols with increasing chain lengths are used as solvents. Table 1.1 gives the absorption maxima of lycopene in aliphatic Ai-alcohols. If we compare aliphatic and aromatic solvents, a bathochromic shift may be observed in aromatic solvents (Table 1.2).

Recording of Absorption Spectra Many spectrophotometers do not measure precisely in terms of reproducibility of wave­ length. It is, therefore, best to first scan the spectrum of the carotenoid to be measured and then to overlay the spectra with the spectrum of a standard dydimium or a holmium oxide filter in order to enable correlations to the correct wavelength (filter available, e.g., from E. Zeiss, Oberkochen, F.R.G.). Although it is advisable not to use too concentrated solutions, absorption values A of up to 0.8 are preferable in order to exploit fully the measuring range of the spectrophotometer. The cuvettes used (preferably quartz) should be freshly cleaned, matching pairs. Solvents have to be redistilled according to the procedures recommended; they should be free of contaminants, especially from those absorbing in the UV region. For quantitative determinations, the volume of the carotenoid solutions should be known. Since carotenoids tend to bleach, absorption measurements should be carried out immediately following pigment elution. It should always be borne in mind that the values measured might be too low because of pigment bleaching due to light, oxydation, etc. If the absorption coefficient is not known, it is a good practice to use the values combined in Table 1.6 as a guide. In most spectrophotometers 1-cm light-path standard cuvettes are used. Other cuvettes may be used as well in order to achieve a sufficient absorbance of very dilute solutions without the need to concentrate.

Quantitative Determination of Carotenoids After their separation by a chromatographic procedure, the amount of carotenoid(s) ob­ tained may be determined, for example, for further work or yield calculations of enzymic synthesis, etc. Usually, there will not be enough carotenoid to be determined by direct weighing. Therefore, quantitative determinations of carotenoids in most cases are carried out spectrophotometrically via the extinction coefficients A (1 %, 1 cm) or molar extinction (absorbance) coefficients e. Electronic spectra (= absorption spectra) may invariably be recorded, to enable the facile determination of concentration and/or absolute amount of carotenoid present. We have compiled data on molar extinction coefficients in Table 1.6 listing names in strictly alphabetical order. Infrared Spectroscopy o f Carotenoids20 The technique of infrared spectroscopy of carotenoids is mostly used in connection with synthetic work. IR spectra indicate qualitatively the functional groups present. So it is of value to detect acetylenic (2170 cm -1), allenic, or hydroxy groups and keto-functions, especially those inaccessible to chemical reagents in some pigments like capsanthin and fucoxanthin.20 Since a conjugated polyene system usually causes weak peaks only, the technique of infrared spectroscopy has not been considered as major in carotenoid chemistry. Infrared spectra of carotenoids may either be taken from a KBr-pellet, in solution (for example, chloroform), from another suitable solvent, or from thin films. Infrared spectro­ scopy has been considered as being useful for the detection of colorless contaminants. For more information, the reader is referred to the literature (for review, see Reference 20).

6

CRC Handbook of Chromatography: Plant Pigments

]H NMR Spectroscopy of Carotenoids20 One of the most prominent features of proton magnetic resonance spectroscopy is that the chemical shifts and coupling constants in a good approximation generally depend only on the immediate environment of the different protons. The interpretation of proton magnetic resonance spectra will, therefore, give detailed information on structural subunits that are present in an unknown molecule. This feature is termed additivity; it simplifies the inter­ pretation of spectra considerably. Coupling constants and chemical shifts of some carotenoids have been tabulated.20 'H NMR spectra usually are recorded in deuteriochloroform (purity >99.5% ) using tetramethylsilane (TMS) as an internal standard. Much of the information gained applies to the end groups of the carotenoid. Additional information can be obtained from the shifts of methylene and methine protons. For example, the appearance of CH signals between about 3 and 5.5 ppm indicates the presence of an -OR-substituted end group, whereas the presence o f-C H 2- multiplets at about 2 and 2.5 ppm indicates a neighboring double bond or carbonyl function; 2, 3, and 4-protons of a (3 end group usually give rise to characteristic multiplets that will help to identify the compound. For more information, the reader is referred to the literature.17-20

Separation of Carotenoids by Chromatography Introductory Remarks To achieve a satisfactory reproducibility of Rf values and retention volumes or retention times, a number of parameters have to be controlled. A few of these parameters are the following: 1. 2.

3. 4. 5. 6. 7. 8.

9.

A constant physical and chemical composition of the sorbents used, including particle size, water content, and respective degree of activation, is imperative. The solvent systems employed should be freshly prepared. Only analytical grade solvents should be used. Otherwise, impurities like peroxides, etc., may decompose sensitive compounds. Also, minute amounts of impurities acting as solvent system components (like water) may greatly influence Rf values. Constant temperature must be maintained. Rf values vary to some degree with the applied amount; overloading has to be strictly avoided, especially since it usually results in separations of poor quality. The degree of chamber saturation is very important. Always exclude light when colored compounds are being separated, especially carotenoids. When working with carotenoids it is good practice to work in an atmosphere of nitrogen. One has always to bear in mind that plants do not possess a time-constant pigment composition. For example, pigments of flowers are limited in their accessibility to the reproductive period of the plant. Drying in the oven should be avoided altogether, since it destroys many carotenoids and other pigments. A number of plants (e.g., yellow pansies) may be freeze-dried, however, without considerable losses.24 Gen­ erally, special care has to be taken in the handling of the individual plant material and the preparation of extracts. In order to achieve real reproducibility, the experiments should be repeated a number of times.

The goal of the present work is to give quick and precise information on the chromato­ graphic separation of plant pigments, including a certain number of related synthetic pigments or pigments from sources other than plants. More detailed information is given in the notes to the respective chromatographic tables.

Volume I: Fat-Soluble Pigments

7

Alternative Procedures for the Analysis of Carotenoids The rapid development of small computers in recent years (e.g., the IBM AT®, the Apple®, and others) facilitated extensive mathematical treatment of recorded absorption spectra. For carotenoids, a method has recently been proposed for the “ deconvolution” of spectra of biochemical mixtures into the amounts of its individual constituents.203 This method has been named “ multicomponent analysis” , and since it might circumvent the necessity of carrying out an actual chromatographic separation, a short description follows: the problem solved by multicomponent analysis (MCA) is the determination of the unknown relative concentrations c, in an overdetermined system of equations. Due to systematical or statistical errors, this system does not provide exact solutions. To determine solutions with the smallest possible errors, the criterion of least squares is chosen. The solution is calculated with the aid of a suitable program on a computer which is directly linked to a precise spectropho­ tometer. The computer will then determine the amount of each individual pigment. Con­ centrations or relative amounts are printed out together with the applicable standard deviation. One must, of course, bear in mind that only mixtures with known qualitative composition may be analyzed. Prior to the actual analysis, it is necessary to record a “ matrix” of absorption spectra of the components. There is no way to avoid the separation and identi­ fication of the individual components of mixtures of unknown qualitative composition. The main potential application of this relatively new method seems to lie in standard determi­ nations, e.g., in the fruit juice industry.

REFERENCES Isler, O., Ed., Carotenoids, Birkhauser-Verlag, Basel, 1971. Heftmann, E., Ed., Chromatography, 3rd ed., Van Nostrand, Reinhold, New York, 1975. Stahl, E., Dunnschichtschromatographie, Springer-Verlag, Berlin, 1967. Goodwin, T. W ., Ed., The Comparative Biochemistry o f Carotenoids, Chapman and Hall, London, 1952. 4. Goodwin, T. W., The Biochemistry o f the Carotenoids, Vol. 1, Plants, Chapman and Hall, London, 1980. 5. Britton, G., The Biochemistry o f Natural Pigments, Cambridge University Press, Cambridge, 1983. 6. Pfander, H., Ed., with Gerspacher, M., Rychinir, M., and Schwabe, R., Key to Carotenoids: Lists o f

1. 2. 2a. 3.

Natural Carotenoids, rev. ed., Birkhauser, Basel, 1987. 7. Goodwin, T. W., Ed., Chemistry and Biochemistry o f Plant Pigments, Vol. 2, 2nd ed., Academic Press, London, 1976. 8. Davies, B. H., Carotenoids, in Chemistry and Biochemistry o f Plant Pigments, Vol. 2, 2nd ed., Goodwin, T. W ., Ed., Academic Press, London, 1976, 38. 9. Britton, G., The Biochemistry o f Natural Pigments, Cambridge University Press, Cambridge, 1983. 10. Czygan, F.-C., Ed., Pigments in Plants, 2nd ed., G. Fischer Verlag, Stuttgart, 1980. 11. Sitte, P., Falk, H., and Liedvogel, B., Carotinoide, Springer-Verlag, Berlin, 1934, 117. 12. Zechmeister, L., Carotinoide, Springer, Berlin, 1934. 13. Davies, B. H., Pigments in Plants, 2nd ed., Czygan, F.-C., Ed., G. Fischer Verlag, Stuttgart, 1980, 31. 14. Davies, B. H., C-30 Carotenoids, in International Review o f Biochemistry, Vol. 14., Biochemistry o f Lipids II, Goodwin, T. W ., Ed., University Park Press, Baltimore, 1977, 51. 15. Davies, B. H., Hsu, W.-J., and Chichester, C. O., Comp. Biochem. Physiol., 33, 601, 1970. 16. Mayr, H. and Isler, O., in Carotenoids, Isler, O., Ed., Birkhauser-Verlag, Basel, 1971, 328. 17. 7th Int. Symp. on Carotenoids, Book of Abstracts, University of Munchen, Munich, 1984. 18. Weast, R. C. and Astle, M. J., Eds., Handbook o f Chemistry and Physics, 62nd ed., CRC Press, Boca Raton, Fla., 1981— 82. 19. Liaaen-Jensen, S., in Carotenoids, Iler, O., Ed., Birkhauser-Verlag, Basel, 1971, 61. 20. Vetter, W., Englert, G ., Rigassi, N., and Schwieter, U., in Carotenoids, Isler, O., Ed., BirkhauserVerlag, Basel, 1971, 189. 20a. Jochum, P. and Schrott, E. L., Anal. Chim. Acta, 157, 211, 1984. 21. Karrer, P. and Juncker, E., Carotinoide, Birkhauser-Verlag, Basel, 1948.

8

CRC Handbook of Chromatography: Plant Pigments 22. Frye, A. H ., J. Org. Chem., 16, 914, 1951. 23. Zweig, G. and Sherma, J ., Handbook Series in Chromatography, Vols. 1 and 2, 4th ed., CRC Press, Boca Raton, Fla., 1978. 24. Wanner, G. and Kost, H .-P., unpublished.

Tables for the Estimation and Separation of Carotenoids

All carotenoid tables have been compiled by G. Widerer, E. Schropp, and H.-P. Kost. Structures were drawn by E. Benedikt and H.-P. Kost.

Volume I: Fat-Soluble Pigments G EN ERA L TABLES Table L I

MAIN ABSORPTION MAXIMA OF LYCOPENE IN VARIOUS ALIPHATIC fi-ALCOHOLS Alcohol Methanol Ethanol n-Butanol /7-Octanol

Max 1 (nm) 443 446 449 451

Max 2 (nm) 470 473 475 479

Max 3 (nm) 501 503.5 507 511

Table 1.2

MAIN ABSORPTION MAXIMA OF LYCOPENE IN VARIOUS SOLVENTS Solvent Acetone /7-Hexane Methylene chloride Chloroform Carbon tetrachloride Cyclohexane Benzene Toluene Pyrrole

Max 1 (nm)

Max 2 (nm)

Max 3 (nm)

446 445 455

472 470 483

503 505 516

457 455

485 483

518 516

446 456 456 462

474 484 484 490

506 519 517 525

Table based on author’s data.

11

Where the name of a pigment is marked with an asterisk (*), different structures have been described in the literature. Second vertical row: names which are actually included in Tables 1.5 through I. LC 1. Third vertical row: completes list of names of each pigment; arrow indicates name under which further detailed information can be found in this table. Consult Reference 6 in the “ Methods” section for recent clarification of this subject.

C = 12.011 g m o l-1 H = 1.0079 g m ol-' O = 15.9994 g m ol"1

The present chapter of the Handbook deals with the chromatographic separation of a vast number of carotenoids. We, therefore, thought it practical to unify all carotenoids dealt with in Tables 1.5 through I. LC 1 in the form of a “ name list” . Table 1.3 contains names, synonyms, formulas, molecular weights, and structures of carotenoids mainly of plant origin; however, other naturally occurring carotenoids have been included as well, if advisable. First vertical row: this row contains alphabetically arranged pigment names appearing in Tables 1.5 through I. LC 1; also, synonyms, molecular (“ sum” ) formula CxHyOz, molecular weights, and structures.1,618 The molecular weights are rounded. The values are calculated from the molecular formula using a programmable desk-top computer employing the following molecular weights:18

NOTES

Table 1.3 NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC)

12 CRC Handbook of Chromatography: Plant Pigments

Cynthiaxanthin 7,8,7',8'-Dehydrozeaxanthin 3,3'-Dihydroxy-7,8,7',8'-dehydro-(3-carotene Pectenoxanthin (3/?,37?)-7,8,7\8'-Tetradehydro-(3, p-carotene-3,3' -diol 7,8,7', 8'Tetradehydrozeaxanthin

Alloxanthin

Cynthiaxanthin

Alloxanthin

Anhydrodeoxy-flexixanthin

Anchovyxanthin

Anhydrodeoxy-flexixanthin 4- Ketotorulene

Zeaxanthin

Cryptomonaxanthin

Aleuriaxanthin

C^H , •,()->

C^H ^O

C38H480 4

Sum formula

3',4'-Didehydro-(3,iJ/-caroten-4- C ^H ^O one 3',4'-Didehydro-4-keto-ycarotene 4-Ketotorulene

Zeaxanthin

(27?)-r,16'-D idehydro-l',2'dihydro-p,i|/-caroten-2'-ol

—►3-Hydroxy-4,4'-diketo-(3carotene -|3-

Aleuriaxanthin

Adonirubin

Actinioerythrin-bis-a-ketol Actinioerythrin Actinioerythrol 4,4'-Diketo-3-hydroxy-pcarotene 3Hydroxy-4,4'-diketo carotene

Actinioerythrin-bis-a-ketol Actinioerythrol (free diol)

Actinioerythrol (free diol) 3,3'-Dihydroxy-2,2'-dinor-p,pcarotene-4,4'-dione-3,3'diacylate — —> Actinioerythrin

All synonyms

Actinioerythrin Actinioerythrol (free diol)

Names mentioned in Tables 1.5 through I. LC

Actinioerythrin

Pigment

548.85

564.85

552.88

Mol wt

Structure

Volume I: Fat-Soluble Pigments 13

Anhydrorhodovibrin “ P-481”

Anhydrosaproxanthin Celaxanthin

Antheraxanthin

Aphanicin Canthaxanthin 4,4'-Diketo-(3-carotene Euglenanone 4,4'-Dihydroxy-(3-carotene Isozeaxanthin Aphanin Echinenone 4-Keto-(3-carotene Myxoxanthin

Anhydrorhodovibrin

Anhydrosaproxanthin

Antheraxanthin

Aphanicin

Aphanin

“ Aphanicol”

Anhydroeschscholtzxanthin

Names mentioned in Tables 1.5 through I. LC

Anhydroeschscholtzxanthin

Pigment

C41H580

C^H^o

Sum formula

—> Echinenone

—» 4,4'-Dihydroxy-(3-carotene

3-Hydroxy-3',4'-dehydro-ycarotene 3-hydroxytorulene 3,3'-Dihydroxy-5,6-epoxy-(3carotene 5.6Epoxy-5,6-dihydro-(3,(3carotene-3,3'-diol 5 .6- Epoxyzeaxanthin Zeaxanthin-5,6-epoxide —* Canthaxanthin

ol

1-Methoxy-l ,2-dihydro-3,4dehydrolycopene “ P-481” Celaxanthin C^H ^O 3',4'-Dehydrorubixanthin 3',4'-Didehydro-(3,i|/-caroten-3-

dihydro-i|/,ijj-carotene

1-Methoxy-3,4-didehydro-1,2-

Dianhydroeschscholtzxanthin 2,3,2',3',4',5'-H exadehydro4 ,5'-retro-(3, (3-carotene

All synonyms

584.88

550.87

566.91

530.84

Mol wt

0

Structure

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC)

14 CRC Handbook of Chromatography: Plant Pigments

4-Hydroxy-p-carotene Isocryptoxanthin “ Myxoxanthol” (3-Apo-8'-carotenal P-Apo-10'-carotenal p-Apo-4-carotenal (3-A po-12' -carotenal

“ Aphanol”

P-Apo-8'-carotenal

P-Apo-10'-carotenal

(3-Apo-4-carotenal P-Apo-12 '-carotenal Apo-2-lycopenal Apo-6'-lycopenal |3-Apo-8'-carotenal P-Apo-10'-carotenal p-Apo-4-carotenal p-A po-12' -carotenal Apo-3-lycopenal Apo-8'-lycopenal Crocetindialdehyde P-Apo-8'-carotenoic acid P-Apo-4'-carotenoic acid Neurosporaxanthin

P-Apo-8'-carotenal

P-Apo-10'-carotenal

(3-Apo-12'-carotenal

Apo-8,8'-carotendial P-Apo-2'-carotenoic acid P-Apo-4'-carotenoic acid

8'-Apo-i|i-caroten-8'-al

8'-Apo-(3-caroten-8'-al 10'-Apo-p-caroten-l0'-al 12'-Apo-p-caroten-12'-al

6'-Apo-i|/-caroten-6'-al

P-Apo-2'-carotenal

P-Apo-2'-carotenal

“ P-Apo-2-carotenal” “ P-Apo-3-carotenal” “ P-Apo-4-carotenal”

Aphanizophyll

Aphanizophyll

—» Crocetindialdehyde P-Apo-8'-carotenoic acid —> Neurosporaxanthin

—> Apo-3-lycopenal

—►(3-Apo-8' -carotenal —> (3-Apo-10'-carotenal —►P-Apo-12'-carotenal

“ P-Apo-4-carotenal” 12'-Apo-(3-caroten-12'-al —> Apo-6'-lycopenal

“ p-Apo-3-carotenal” 10'-Apo-(3-caroten-10'-al

“ P-Apo-2-carotenal” 8'-Apo-(3-caroten-8'-al “ p-Carotenal”

3',4'-Didehydro-2'-apo-P-caroten-2-al

—» p-Apo-8'-carotenal -» P-Apo-lO'-carotenal —» P-Apo-12'-carotenal

4-Hydroxymyxoxanthophyll 2'-(P,L-Rhamnopyranosyloxy)-3',4'-didehydro-l',2 'dihydro-p,i[/-carotene-3,4, 1'triol —> 4-Hydroxy-p-carotene

C25H340

C27H360

C30H40O

C,7H480

C46H66Os

350.54

376.58

416.65

508.79

747.02

Volume I: Fat-Soluble Pigments 15

—> (3-Apo-8'-carotenoic acid —» (3-Apo-10'-carotenoic acid —►(3-Apo-12'-carotenoic acid

(3-Apo-4'-carotenoic acid Neurosporaxanthin (3-Apo-8'-carotenoic acid (3-Apo-10'-carotenoic acid

(3-Apo-12'-carotenoic acid

4'-Apo-(3-caroten-4-oic acid

(3-Apo-2'-carotenol

(3-Apo-8'-carotenol

(3-Apo-10'-carotenol (3-Apo-8'-carotenol “ Apo-2-lycopenal” Apo-6'-lycopenal “ Lycopenal”

Apo-3-lycopenal Apo-8'-lycopenal

(3-Apo-2'-carotenol

(3-Apo-8'-carotenol

P-Apo-10'-carotenol 8'-Apo-[3-caroten-3-ol “ Apo-2-lycopenal”

Apo-3-lycopenal

8'-Apo-(3-caroten-8'-oic acid 10'-Apo-[3-caroten-10'-oic acid 12'-Apo-|3-caroten-12'-oic acid

(3-Apo-12'-carotenoic acid

(3-Apo-12'-carotenoic acid

Apo-8'-lycopenal 8'-Apo-tJ/-caroten-8'-al

8'-Apo-(3-caroten-3-ol (3-Citraurinene — —> (3-Apo-8'-carotenol —> Apo-6'-lycopenal

3 ',4 '-Didehydro-2'-apo-(3-caroten-2-ol

12'-Apo-(3-caroten-12'-oic acid Retinylidenetiglic acid —> Neurosporaxanthin

10'-Apo-(3-caroten-10'-oic acid

p-Apo-lO'-carotenoic acid

(3-Apo-10'-carotenoic acid

8'-Apo-(3-caroten-8'-oic acid “ (3-Apo-2'-carotenoic acid”

AH synonyms

(3-apo-8'-carotenoic acid

Names mentioned in Tables 1.5 through I. LC

(3-Apo-8'-carotenoic acid

Pigment

C30H42O

C37H50O

C,5H340-> ‘

C27H366 0 2

C30H40O2

Sum formula

418.66

510.80

366.54

392.58

432.64

Mol wt

C

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC) Structure

16 CRC Handbook of Chromatography: Plant Pigments

5,6-Epoxy-3-hydroxy-5,6dihydro-10'-apo-p-carotenlO'-al 5,6-Epoxy-3-hydroxy-5,6dihydro- 1 0 '-apo-p-carotenlO'-al {3-Citraurin 3-Hydroxy-10'-apo-P-caroten-lO'-al

Apo-lO'-violaxanthin

3-Hydroxy-12'-apo-p-caroten- 1 2 '-al

Astacene

Astaxanthin

Astaxanthindiacetate Astaxanthindiester Astaxanthinmonoester

“ Apo-4-zeaxanthinal”

Astacene

Astaxanthin

Astaxanthindiacetate Astaxanthindiester Astaxanthinmonoester

“ Apo-2-zeaxanthinal” “ Apo-3-zeaxanthinal”

“ Apoviolaxanthinal”

5,6-Epoxy-3-hydroxy-5,6dihydro-12'-apo-p-caroten-

“ Apo-2-lycopenal” Apo-6 '-lycopenal “ Lycopenal” Apo-3-lycopenal Apo-8 '-lycopenal

Apo-12'-violaxanthal

Apo-8 '-Lycopenal

Apo-6 '-lycopenal

“ Apo-2-lycopenal”

“ Euglenarhodon” “ Salmon acid” 3,4,3\4'-Tetraketo-P-carotene “ 4,5,4',5'-Tetraketo-pcarotene” 3,3'-Dihydroxycanthaxanthin (3S,3'5)-3,3'-Dihydroxy-p,pcarotene-4,4 '-dione 3,3'-Dihydroxy-4,4'-diketo-pcarotene “ Ovoester” — — —

3-Hydroxy-12'-apo-p-caroten1 2 '-al

—» 5,6-Epoxy-3-hydroxy-5,6dihydro-lO'-apo-p-carotenlO'-al —►5,6-Epoxy-3-hydroxy-5,6dihydro-1 0 '-apo-p-carotenlO'-al —*■ p-Citraurin —* 3-Hydroxy-10'-apo-p-caroten-lO'-al

5,6-Epoxy-3-hydroxy-5,6-dihydro-12'-apo-P-caroten-12'-al

“ Lycopenal” —►Apo-3-lycopenal

6 '-Apo-iJ/-caroten-6 '-al

C40H52O 4

CwH^C^

C 25H 340-,

C 32H420

596.85

592.82

382.54

442.68

Ho

366.

Volume I: Fat-Soluble Pigments 17

3'~Hydroxyechinenone 4-Keto-3 '-hydroxy-(3-carotene

Aurochrome

Auroxanthin

Azafrin

4,4 -Diapophytoene Rhodoviolascin Spirilloxanthin OH-Lycopene Rhodopin Rhodoviolascin Spirilloxanthin

Asteroidenone

Aurochrome

Auroxanthin

Azafrin

Bacterial phytoene Bacterioerythrin a

Bacteriopurpurin a

“ Bacterioerythrin (3”

Asterinic acid

Names mentioned in Tables 1.5 through I. LC

Asterinic acid

Pigment

—►Rhodoviolascin

-» OH-Lycopene

—> 4,4'-Diapophytoene Rhodoviolascin

(5/?,6/?)-5,6-Dihydroxy-5,6dihydro-10'-apo-(3-carotenlO'-oic acid

5,8,5',8'-D iepoxy-5,8,5',8'-tetrahydro-(3,(3-carotene-3,3'diol “ 5,8,5',8'-Diepoxyzeaxanthin” “ 3,3'-Dihydroxy-5,8,5',8'-diepoxy-(3-carotene”

5, 8 ,5 ', 8 '-Diepoxy-5,8 ,5 ', 8 '-tetrahydro-(3, (3-carotene

(3-Carotene-5,8,5',8'-diepoxide

“ ^-Carotene” *

M ixture of 7 ,8 ,7 ', 8 '-tetradehydroastaxanthin and 7 , 8 didehydroastaxanthin —> 3'-Hydroxyechinenone

All synonyms

C 27H 380 4

C H O

Sum formula

426.59

600.88

568.88

Mol wt

Structure

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC)

18 CRC Handbook of Chromatography: Plant Pigments

“ Bisdehydro-(3-carotene” “ Dehydrocarotene III” Retrobisdehydro-(3-carotene 3,4,3',4'-Tetradehydro-(3,(3carotene —» ” Dehydrolycopene” —» Sarcinaxanthin*

—> Sarcinaxanthin*

—►Bacterioruberin a

—> Oscillol-2,2'-di-((7-methylmethyl-pentoside)

Hydroxyspirilloxanthin OH-Spirilloxanthin 3,4,3',4'-Bisdehydro-(3carotene

3,4,3',4'-Bisdehydro-(3carotene

“ Dehydrolycopene” Sarcinaxanthin

Sarcinaxanthin

Bacterioruberin Bacterioruberin a

Oscillol-2,2'-di(0-methylmethyl-pentoside)

“ a-Bacterioruberin monomethyl ether” “ Bisdehydro-(3-carotene”

3,4,3',4'-Bisdehydro-(3carotene

Bisdehydrolycopene (2/?,6S,2'/?,6'S)-2,2'-Bis-(4hydroxy-3-methyl-2-butenyl)-y,y-carotene (2/?,6/?,27?,67?)-2,2'-Bis-(4hydroxy-3-methyl-2-butenyl)-€,e-carotene 2,2'-Bis-(3-hydroxy-3-methylbutyl)-3,4,3',4'-tetradehydro- 1 , 2 , 1 ', 2 ' -tetrahy dro­ ll*,i|j-carotene-l, 1 '-diol 2,2'-Bis(0-methyl-5-C-methylpentosyloxy)-3,4,3',4'-tetradehydro-1 , 2 , 1 ', 2 'tetrahy dro-ijj, i}/-carotene1 , 1 '-diol

—» 3,4,3\4'-Bisdehydro-(3carotene

—> OH-Spirilloxanthin

ten e-l,r-d io l “ Didemethylated spirilloxanthin”

Bacterioruberin 2,2'-Bis-(3-hydroxy-3-methylbutyl)-3,4,3',4'-tetradehydrol , 2 , l ', 2 '-tetrahydro-i}/,il;-caro-

Bacterioruberin Bacterioruberin a

Bacterioruberin a

—> Bacterioruberin a

Bacterioruberin Bacterioruberin a

Bacterioruberin

C 50H 76O 4

532.85

741.15

Volume I: Fat-Soluble Pigments 19

Oscillaxanthin

Bixin

Bixin Bixin

Caloxanthin

Aphanicin Canthaxanthin 4,4'-Diketo-(3-carotene Euglenanone

Capsanthin

Capsanthin-diester

Capsanthin-5,6 -epoxide

Bixin

“ a-Bixin” “ Bixin II”

Caloxanthin

Canthaxanthin

Capsanthin

Capsanthin-diester

Capsanthin-5,6 -epoxide

Names mentioned in Tables 1.5 through I. LC

2,2'-Bis-((3-L-rhamnopyranosyloxy)-3,4,3',4'-tetradehydro- 1 , 2 , T , 2 ' -tetrahydrovJ;,v|;-carotene-1,1 '-diol

Pigment

Capsanthin-monoepoxide 5,6-Epoxy-3,3'-dihydroxy-5,6dihydro-(3,K-caroten-6'-one

—

(3/?,3'S,5'/?)-3,3'-Dihydroxy(3,K-caroten-6'-one

Aphanicin (3,(3-Carotene-4,4'-dione Chlorellaxanthin 4,4'-Diketo-(3-carotene Euglenanone

(3,(3-carotene-3,3'-diol

6 ,7-Didehydro-5,6 -dihydro-

“ Bixin II” —> Bixin —» Bixin

"a-Bixin”

Bixin natural Bixin cis Bixin lower melting

—» Oscilloxanthin

All synonyms

C^H^CX

C^H^CX

C^H^CX

C^H^CX

C 2sH 30O 4

Sum formula

600.88

584.88

564.85

568.88

394.51

Mol wt

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC) Structure

20 CRC Handbook of Chromatography: Plant Pigments

P-Carotene p, P-Carotene a-Carotene p ,E-Carotene

p, P-Carotene -,. a-Carotene

C40Hs6

"Carotene" P, P-Carotene tj,,a-Carotene Neo-p-carotene Pseudo-a-carotene -,. P-Carotene

P-Carotene P, P-Carotene

P-Carotene

p,E-Carotene

CwH,o

P,E-Carotene (6' R)-P,E-Carotene

a-Carotene p ,E-Carotene

-,. P-Zeacarotene

-,. Flavacin

-,. P-Apo-8' -Carotenal -,. P-Carotene

-,. P-Cryptoxanthin

-,. Chrysanthemaxanthin

C"1H560 4

C40H,o04 C,,,H, 004

a-Carotene

"Carotene X"

Carotene oxide

'' P-Carotenal'' "Carotene"

Caricaxanthin

Chrysanthemaxanthin Flavoxanthin Cryptoxanthin P-Cryptoxanthin P-Apo-8 '-Carotenal P-Carotene p, P-Carotene Flavacin Mutatochrome P-Zeacarotene p ,-Zeacarotene

-,. Torularhodin

Capsorubin-diester Torularhodin

Capsorubin-diester 16' -Carboxyl-3' ,4' -dehydro"(-carotene "Carcinoxanthin"

Capsorubin

Capsanthin-5 ,6-epoxidediester Capsanthin-5 ,6-epoxide -,. Capsanthin-5 ,6-epoxide Capsanthinmonoester Capsochrome 5,8-Epoxy-3,3' -dihydroxy-5,8dihydro-P, K-caroten-6' -one (3S ,5R ,3' S,5'R)-3,3' -DihyCapsorubin droxy-K,K-caroten-6,6' -dione

Capsanthin-5 ,6-epoxidediester Capsanthinmonoepoxide Capsanthinmonoester Capsochrome

536.88

536.88

600.88

600.88

....N

"':::: 1:;

2!

0o·

~ "t,

p-Carotene-diepoxide p-Carotene-5,6,5',6'diepoxide 5,6,5', 6' -Diepoxy- P-carotene Aurochrome —> Aurochrome

Names mentioned in Tables 1.5 through I. LC

p-Carotene-5, 8,5', 8' diepoxide (3/?,3'/?)-p,p-Carotene-3,3'- Zeaxanthin diol p,p-Carotene-4,4'-diol 4,4'-Dihydroxy-p-carotene Isozeaxanthin (3/?, 3 7?, 6 7?)-p, e-Carotene3-Hydroxy-3'-hydroxy-a3,3'-diol carotene Lutein “ Xanthophyll” i|/,i|/-Carotene-16,16'-diol Lycophyll (3/?,37?)-p,p-Carotene-3,3'- Physalien diol dipalmitate (3R,37?,67?)-p,e-CaroteneHelenien 3,3'-diol dipalmitate Lutein dipalmitate P,p-Carotene-4,4'-dione Aphanicin Canthaxanthin 4,4' -Diketo- p-carotene Euglenanone P,K-Carotene-3',6'-dione Cryptocapsone p,K-Carotene-3',6'-dione Cryptocapsone

P-Carotene-5,6,5',6'diepoxide

P-Carotene-diepoxide

Pigment

Sum formula

568.88

Mol wt

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC) Structure

24 CRC Handbook of Chromatography: Plant Pigments

a-Carotene-5 ,6-epoxide "5,6-Monoepoxy-acarotene" 13-Carotene-monoepoxide 5 ,6-Monoepoxy-!3-carotene

a-Carotene-5,6-epoxide

Pyrenoxanthin "Troltein" Pyrenoxanthin Cryptoxanthin 13-Cryptoxanthin 4-Hydroxy-13-carotene Isocryptoxanthin Myxoxanthol a-Cryptoxanthin a-Cryptoxanthin Zeinoxanthin 4-Hydroxy-a-carotene Rubixanthin Gazaniaxanthin L ycoxanthin

13,E-Carotene-3, 19,3 '-trio!

13,E-Caroten-4-ol (3R-)l3,lj,-Caroten-3-ol (3R-)5 '-cis-!3,lj,-Caroten-3-ol lj, ,lj,-Caroten- I 6-ol

13,E-Caroten-3' -ol (3R ,6' R- )13,E-Caroten-3-ol

13, 13-Caroten-4-ol

13,E-Carotene-3,20,3 '-trio! (3R-)13, 13-Caroten-3-ol

l3,l3-Carotene-2,3,3' -trio!

13,13-Carotene-2,3,3 '-trio!

13-Carotene-monoepoxide

a-Carotene-5 ,6-epoxide "5 ,6-Monoepoxy-acarotene"

a-Carotene-epoxide

-

-

-

-

-

4-Hydroxy-a-carotene Rubixanthin Gazaniaxanthin Lycoxanthin

a-Cryptoxanthin a-Cryptoxanthin

4-Hydroxy-13-carotene

Pyrenoxanthin 13-Cryptoxanthin

"Troltein"

13-Caroten-5,6-epoxide 13-Carotene-monoepoxide 5 ,6-Epox y-5 ,6-dihydro-13, 13carotene 5 ,6-Monoepoxy-!3-carotene

a-Carotene-5 ,6-epoxide 5 ,6-Epoxy-5 ,6-dihydro-13carotene ' '5, 6-Monoepox y-a-carotene' ' - a-Carotene-epoxide

552.88

584.88

C40 H56 0,

552.88

C40 H,6 0

C40 H56 0

ii:"

'-"

N

c:;-

;::

~

;:!

~-

"ti

ii:" Crocetindialdehyde

—> Crocetindimethylester —►Crocetin

—> Crocetin

—►Crocetin

Crocetin trans Crocetin stable “ a-Crocetin” “ Crocetin I” 8 , 8 '-Diapocarotene- 8 , 8 '-dioic acid

All synonyms

Crocetindialdehyde

Crocetindial

“ y-Crocetin” Crocetin trans

“ a-Crocetin’’

Crocetin Crocetin trans

Names mentioned in Tables 1.5 through I. LC

Crocetin

Pigment

C38H54O l9

C 22H280 4

C32H440 14

C 20H24O 2

C 20H 24O4

Sum formula

814.83

652.69

296.41

328.41

Mol wt

356.46

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC) Structure

28 CRC Handbook of Chromatography: Plant Pigments

Crocoxanthin

Cryptocapsin

Cryptocapsone

Cryptochrome

Cryptoflavin Cryptoxanthin-5,8 -epoxide

Alloxanthin Cynthiaxanthin

Cryptoxanthin P-Cryptoxanthin

Cryptoxanthin P-Cryptoxanthin

Crocoxanthin

Cryptocapsin

Cryptocapsone

Cryptochrome

Cryptoflavin

Cryptomonaxanthin

“ Cryptoxanthene”

Cryptoxanthin

,6

-dione

—> p-Cryptoxanthin

—> P-Cryptoxanthin

—►Alloxanthin

Cryptoxanthin-5,8 -epoxide 5,8-Epoxycryptoxanthin 5,8-Epoxy-5,8-dihydro-p,Pcaroten-3-ol 3-Hydroxy-5,8-epoxy-(3carotene

Cryptoxanthin-5,8 ,5 ', 8 'diepoxide 5,8,5',8'-Diepoxy-5,8,5',8'-tetrahydro-p,p-caroten-3-ol ” 3-Hydroxy-5,8,5',8'-diepoxy(3 -carotene”

p,K-Carotene-3

(3'5,5'/?)-3'-Hydroxy-{3,K-caroten-6 '-one

7,8-Dehydrozeinoxanthin (3/?,6'fl)-7,8-Didehydro-p,ecaroten-3-ol 3-Hydroxy-7,8-dehydro-acarotene

C4oH560

C4oH560

2

3

C^H^O;,

C ^H ^O

568.88

584.88

566.87

568.88

550.87

Volume I: Fat-Soluble Pigments 29

-

Cryptoxanthin diepoxide ~-Cryptoxanthin 5,6,5' ,6' diepoxide

Cryptochrome

Cryptoxanthin diepoxide ~-Cryptoxanthin 5,6,5' ,6' diepoxide Cryptoxanthinepoxide Cryptoxanthin 5 ,6-epoxide

Cryptoxanthin diepoxide

Cryptoxanthin 5,8,5' ,8' diepoxide ~-Cryptoxanthin 5,6,5' ,6' diepoxide

Cryptoxanthinepoxide

~-Cryptoxanthin-5,6,5' ,6' diepoxide 5,6,5' ,6' -Diepoxy-5,6,5' ,6' -tetrahydro-~,~-caroten-3-ol - Cryptochrome

Cryptoxanthin ~-Cryptoxanthin

Cryptoxanthin-5 ,6-epoxide ~-Cryptoxanthin-5 ,6monoepoxide 5, 6-Epox y-5 ,6-dihydro-~, ~caroten-3-ol

Cryptoxanthindiepoxide

''Caricaxanthin'' (3R)-~ .~-Caroten-3-ol •'Cryptoxanthene'' Cryptoxanthin •'Cryptoxanthol'' 3-Hydroxy-~-carotene Physoxanthin

3-H ydrox y-cx-carotene 3'-Hydroxy-cx-carotene Physoxanthin Zeinoxanthin

(3R ,6' R)-~,e-Caroten-3-ol

~-Cryptoxanthin

~,e-Caroten-3' -ol

All synonyms

cx-Cryptoxanthin Zeinoxanthin

Names mentioned in Tables 1.5 through I. LC

cx-Cryptoxanthin*

Pigment

C'"1H56 0,

C'",H, 6 0,

C4(,H, 6 0

C4(,H, 6 O

Sum formula

568.88

584.88

552.88

552.88

Mol wt

H

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC) Structure

"'::::0::,-

;:,:

o'.o'

~ ""ti

sS"

""ti

•-.,:

-§;::-

~

i:::,

:;:

~

9

Sarcinaxanthin —> Corynexanthin —> ‘T rollein” *

Lycopersene Sarcinaxanthin Corynexanthin

“ Trollein”

Dehydroadonirubin

Dehydroadonixanthin

3,4-Dehydro-p-carotene —» 3,4-Monodehydro-p3,4-Monodehydro-p-carotene carotene 3,4,3',4'-Bisdehydro-p—> 3,4,3',4'-Bisdehydro-Pcarotene carotene

Dehydroadonirubin

Dehydroadonixanthin

“ Dehydrocarotene II”

Dehydrocarotene III

P-Doradecin 3 '-Hydroxy-3,4-diketo-pcarotene “ 3'-Hydroxyeuglenanone”

3-Oxocanthaxanthin Phoeniconone Phoenicoxanthin* 3,4,4'-Triketo-p-carotene

—> Alloxanthin

—» Lutein

—> ^-Cryptoxanthin

—> Cryptoxanthinepoxide

—> Cryptoflavin

—> Phytofluene —> Lycopersene

Cynthiaxanthin

“ Cucurbitaxanthin”

p-Cryptoxanthin 5,6monoepoxide “ Cryptoxanthol”

Cryptoxanthin 5,8-epoxide

“ Decahydro-p-carotene” 7,8,11,12,15,7',8',11',12', 15' -Decahy dro-ij/, i|/-carotene Decahydrolycopene Decaprenoxanthin Decaprenoxanthin monoglucoside Deepoxyneoxanthin

—> Cryptoxanthinepoxide

Cryptoxanthinepoxide Cryptoxanthin 5,6-epoxide Cryptoxanthin 5,8-epoxide Cryptoflavin Cryptoxanthinepoxide Cryptoxanthin 5,6-epoxide Cryptoxanthin P-Cryptoxanthin 3-Hydroxy-3'-hydroxy-acarotene Lutein “ Xanthophyll” Alloxanthin Cynthiaxanthin Phytofluene Lycopersene

Cryptoxanthin 5,6-epoxide

C^H^O^

H0

C ^H ^O , 580.85

580.85

Volume I: Fat-Soluble Pigments 31

All synonyms

Bisdehydrolycopene 3,4,3',4'-Tetradehydro-i|/,v|/carotene 3,4,3',4'-Tetradehydrolycopene

“ Dehydrolycopene”

“ Dehydrolycopene”

3'-Dehydrolutein

3,4-Diketo-(3-carotene “ Euglenanone” 3-Hydroxy-4-oxo-2,3-dehydro(3-carotene 3-Oxoechinenone 3-Hydroxy-3'-keto-cx-carotene -» 3-Hydroxy-3'-keto-aPhilosamiaxanthin carotene

Dehydrohydroxyechinenone “ Euglenanone”

Dehydroretrocarotene Dehydroretro-(3-carotene 4,4'-Didehydro-(3-carotene 4,5'- Didehydro-4, 5 ' -retro- (3, (3carotene Isocarotene Retrodehydrocarotene Retrodehydro-(3-carotene 3,4-Dehydro-(3-carotene 3,4-Monodehydro-(33,4-Monodehydro-(3-carotene carotene Torulene Torulene Sarcinaxanthin -> Sarcinaxanthin Phytoene —►Phytoene Phytofluene Phytofluene

“ Dehydro-(3-carotene” Dehydroretrocarotene Dehydroretro-(3-carotene Retrodehydrocarotene Retrodehydro-(3-carotene

Names mentioned in Tables 1.5 through I. LC

Dehydrohydroxyechinenone

3',4'-Dehydro-y-carotene Dehydrogenans-P-439 Dehydrogenans-phytoene Dehydrogenans-phytofluene

3,4-Dehydro-iJt-carotene

“ Dehydro-0-carotene”

Pigment

Sum formula

532.85

564.85

534.87

Mol wt i

i

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC) Structure 'Y * ')

32 CRC Handbook of Chromatography: Plant Pigments

Dehydrorhodopin

7 , 8 -Dehydrozeinoxanthin

Dehydrosqualene 3.4Dehydrotorulene 7,8,7',8'-Dehydrozeaxanthin

3',4'-Dehydrorubixanthin

Dehydrorhodovibrin

3.4-

Dehydroretro-P-carotene

Hydroxyspirilloxanthin OH-Spirilloxanthin Anhydrosaproxanthin Celaxanthin 4,4'-Diapophytoene 3,4-Dehydrotorulene Alloxanthin Cynthiaxanthin Crocoxanthin

‘‘Dehydro-P-carotene” Dehydroretrocarotene Dehydroretro-p-carotene Retrodehydrocarotene Retrodehy dro- p-carotene ‘‘Dehydro-P-carotene” Dehydroretrocarotene Dehydroretro-P-carotene Retrodehydrocarotene Retrodehydro-p-carotene 3,4-Dehydrorhodopin ‘‘O H -P 481”

Phytofluene 2'-Dehydroplectaniaxanthin

carotene” 11,12-Dehydrophytoene 2'-Dehydroplectaniaxanthin

Dehydroretrocarotene

Torularhodin-aldehyde

carotene” “ 3,4-Dehydro-1 8 -0 x0 -7 -

3,4-Dehydrolycopene

Phytoene Torularhodin-aldehyde

Dehydrolycopene

15,15'-Dehydrolycopersene “ 3 ',4 '-Dehydro-17-oxo-7-

3.4-

—►Crocoxanthin

—> 4,4'-Diapophytoene — —> Alloxanthin

—> Anhydrosaproxanthin

3,4-Didehydro-1,2-dihydroi|/,iJj-caroten-l-ol 1,2-Dihydro-3,4-dehydro-1OH-lycopene “ OH-P 481” ‘‘OH-P 482” —> OH-Spirilloxanthin

‘‘Dehydro-P-carotene

—» Phytofluene 1'-Hydroxy-3',4'-didehydrol ', 2 '-dihydro-p,>|(-caroten-2 'one 1'-Hydroxy-2'-keto-1',2'dihydrotorulene —> ‘‘Dehydro-P-carotene

-> Torularhodin-aldehyde

~ > Phytoene -* Torularhodin-aldehyde

Monodehydrolycopene

3 ,4 -Didehydro-i|i,vJj-carotene

C4oH560

C^ H« 0 :

552.88 OH

566.87

534.87

Volume I: Fat-Soluble Pigments 33

7,8,7' ,8' -Tetrahydro-4,4' diapocarotene

4,4' -Diapo-~-carotene

4,4' -Diapo-Lycopen-4-al

4,4' -Diaponeurosporene

4,4' -Diapophytoene

4,4' -Diapo-~-carotene

4,4' -Diapo-lycopen-4-al

4,4' -Diaponeurosporene

4,4' -Diapo7,8, 11, 12,7' ,8' ,11 ', 12' -octahydro-lj,, lj,-carotene

-

4,4' -Diapophytoene

7,8-Dihydro-4,4' -diapocarotene C,0H42

C,0 H44

-

Anhydroeschscholtzxanthin Crocetindialdehyde Crocetin Crocetin trans

Dianhydroeschscholtzxanthin 8,8' -Diapocarotene-8,8' -dial 8,8' -Diapocarotene-8,8' -dioic acid

402.66

404.68

582.86

3,3' -Dihydroxy-7,8-dehydro-~- C40 H,40, caroten-5' ,6' -epoxide 5,6-Epoxy-7' ,8' -didehydro-5,6dihydro-~, ~-carotene, 3 ,3 '-diol

Diadinoxanthin

Diadinoxanthin

Anhydroeschscholtzxanthin Crocetindialdehyde Crocetin

556.87

Mol wt

C40 H, 40 2

Sum formula

I' ,2' -Dihydro-1 '-hydroxy-4ketotorulene I' -Hydroxy-3' ,4' -didehydro1',2' -dihydro-~.lj,-caroten-4one 4-Keto-l ',2' -dihydro-1 ' hydroxytorulene

All synonyms

Deoxyflexixanthin I' ,2' -Dihydro-1 '-hydroxy-4ketotorulene 4-Keto-l ',2' -dihydro-1 ' hydroxytorulene

Names mentioned in Tables 1.5 through I. LC

Deoxyflexixanthin

Pigment Structure

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC)

"'

~

~

;::

o'.o"

~ '"ti

s-

'"ti

';'.

;:,:-

.g

~...,

.:i

;:!

~

Q

->

"Dehydro-P-carotene" Dehydroretrocarotene Dehydroretro-P-carotene Retrodehydrocarotene Retrodehydro-P-carotene 3 ,4-Dehydro-P-carotene 3,4-Monodehydro-p-carotene

3 ,4-Didehydro-13, P-carotene

->

P-Apo-2' -carotenol

Asterinic acid C40 H500 4 3,3' -Dihydroxy-7 ,8-didehydro[3,[3-carotene-4,4' -dione

Torularhodin-aldehyde

->

P-Apo-2' -carotenol

3' ,4' -Didehydro-p,,j,-carotene-16' -al 4,4' -Didehydro-P-carotene

->

P-Apo-2' -carotenal

3' ,4' -Didehydro-2' -apo-Pcaroten-2' -al 3' ,4' -Didehydro-2' -apo-pcaroten-2' -ol ->

566.87

C40 H, 4 0,

(3R,3'R-)7,8-Didehydro-[3,[3caroten-3,3' -diol 7 ,8-Didehydrozeaxanthin

Diatoxanthin

Diatoxanthin

Part of asterinic acid

404.68

C,0 H44

7 ,8, 11, 12-Tetrahydro-4,4' diapocarotene

4,4' -Diapo-7 ,8, 11, 12tetrahydrolycopene

4,4'-Diapo-7,8,11,12tetrahydrolycopene

7 ,8-Didehydroastaxanthin

406.69

C,0 H••

7,8, 11, 12, 7' ,8' -Hexahydro4,4' -diapocarotene

4,4' -Diapophytofluene

4,4' -Diapophytofluene

P-Apo-2' -carotenal

408.71

C,0 H48

"Bacterial phytoene" "Compound X" C,0 -Phytoene Dehydrosqualene 4,4' -Diapo7,8, 11, 12, 7' ,8', 11 ', 12' -octahydro-,J,, ,Ji-carotene 7 ,8, 11, 12, 7' ,8', 11 ', 12' -Octahydro-4,4' -diapocarotene

4,4' -Diapophytoene

4,4' -Diapophytoene

~

~

w u,

~

;:! ~ ::::

D -> ->

Monadoxanthin

Torularhodin

Crocoxanthin

Crocoxanthin

Torularhodin

Monadoxanthin

Torulene 3,4-Dehydrolycopene Diatoxanthin

All synonyms

-> ->

3 ,4-Dehydrorhodopin "OH-P 481" Heteroxanthin ->

Heteroxanthin

->

Aleuriaxanthin

Deepoxyneoxanthin "Trollein"

3 ,4-Dehydrorhodopin

->

Saproxanthin

"Trollein"*

Aleuriaxanthin

Saproxanthin

->

Plectaniaxanthin

Plectaniaxanthin

Anhydrosaproxanthin -> Anhydrosaproxanthin Celaxanthin -> Anhydrodeoxyflexixanthin Anhydrodeoxyflexixanthin 4-Ketotorulene 3' ,4' -Didehydrochlorobactene Caloxanthin -> Caloxanthin

-> ->

->

Torulene 3,4-Dehydrolycopene Diatoxanthin

Names mentioned in Tables 1.5 through I. LC Sum formula

Mot wt

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC) Structure

t,,I

"'

~

~

3

0o·

"il

~

iS"

"il

~

°"-§

0

I::,

3

t3

9

~

a a ;,,;-

~

;::

I::,

::r::

Q n

O'I

5,6,5' ,6'-Diepoxy-5,6,5' ,6' tetrahydro-13, 13-carotene

5,6,5' ,6' -Diepoxy-[3-carotene

4' ,5' -Didehydro-4,5' -retro13,tlJ-carotene Didehydroretro--y-carotene 4' ,5' -Didehydro-4,5' -retrol3,l3-carotene-3,3' -diol 4' ,5' -Didehydro-4' ,5 '-retro13, 13-carotene-3 ,3 '-di one 3 ,4-Didehydrorhodopin 7 ,8-Didehydrozeaxanthin "Didemethylated spirilloxanthin'' ''Diepoxy-[3-carotene''

7' ,8' -Didehydro-5,6-dihydrol3,l3-carotene-3,5,3' -trio! 3' ,4' -Didehydro-1 ',2' -dihydro-13,tlJ-carotene-2, I' ,2' trio! 3' ,4' -Didehydro-4-keto--ycarotene 3 ,4-Didehydrolycopene 4' ,5' -Didehydro-4,5' -retro13, 13-carotene -+ 2-Hydroxyplectaniaxanthin

-+ Anhydrodeoxyflexixanthin

2-Hydroxyplectaniaxanthin

Anhydrodeoxyflexixanthin 4-Ketotorulene 3 ,4-Didehydrolycopene '' Dehydro-[3-carotene'' Dehydroretrocarotene Dehydroretro-[3-carotene Retrodehydrocarotene Retrodehydro-[3-carotene Retrodehydro--y-carotene

-+ 13-Carotene-diepoxide

-+ 13-Carotene-diepoxide

-+ 13-Carotene-diepoxide

-+ Bacterioruberin o:

-+ Diatoxanthin

-+ Rhodoxanthin

Rhodoxanthin

3,4-Didehydrorhodopin Diatoxanthin Bacterioruberin Bacterioruberin o: 13-Carotene-diepoxide 13-Carotene-5,6,5' ,6' diepoxide ''Diepoxy-[3-carotene'' 5,6,5' ,6' -Diepoxy-[3-carotene 13-Carotene-diepoxide 13-Carotene-5,6,5' ,6' diepoxide ''Diepoxy-[3-carotene'' 13-Carotene-diepoxide [3-Carotene-5,6,5' ,6' diepoxide "Diepoxy-[3-carotene'' 5,6,5' ,6' -Diepoxy-[3-carotene

-+ Eschscholtzxanthin

Retrodehydro--y-carotene Eschscholtzxanthin

-+ Retrodehydro--y-carotene

-+ Retrodehydro--y-carotene

-+ "Dehydro-[3-carotene"

-+ Heteroxanthin

Heteroxanthin

C40 H, 6 0

552.88

'-I

~

"';:,:c:;'

~

~-

~ "t,

1,2,7 ,8, I' ,2' ,7' ,8' -Octahydrolj, ,lj,-carotene-1, I' -diol

-. Part of asterinic acid 4,4' -Diketocynthiaxanthin Diketotetradehydrozeaxanthin 3,3' -Dihydroxy-7 ,8, 7' ,8' -tetradehydro-13,13-carotene-4,4' dione ---> Phillipsiaxanthin

Part of asterinic acid

C,0 H,,O,

C"'H 64 0,

Sum formula

-. Eschscholtzxanthin

All synonyms

Eschscholtzxanthin

Names mentioned in Tables 1.5 through I. LC

''3,3' -Dihydroxyretro-13carotene" 3,3' -Dihydroxy-7,8, 7' ,8' -tetradehydro-13, 13-carotene4,4' -dione

Pigment

564.85

576.94

Mol wt

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC) Structure

;:,: c:::,'

~

3

0o·

~ "'tl

s

"'tl

~

O Rhodoviolascin

Rhodoviolascin

,2, l',2 '-tetrahydro-3',4'-didehydro-il/,4>caroten-4-one

1 ,l'-Dimethoxy-l

—►Torulene

C 42H620 2

1,1 '-Dimethoxy-3,4,3',4'C42HS60 4 tetradehydro-l,2,l\2'-tetrahydro-i|Mji-carotene-2 ,2 '-dione ” 2-Ketospirilloxanthin” “ P-512” P-518 —> Part of asterinic acid —» 3,3'-Dihydroxy-7,8,7',8'-tetradehydro- (3, (3-carotene-4,4' dione —* Torulene

3-Hydroxy-4,4'-diketo-(3carotene Rhodoxanthin

2,2'-Diketospirilloxanthin P518

—» 3-Hydroxy-4,4'-diketo-(3carotene —►Rhodoxanthin

Rhodoxanthin

‘‘3,3'-Diketodehydro-(3carotene” 4,4'-Diketo-3-hydroxy-(3carotene “ 3,3'-Diketoretro-(3carotene”

2,2'-Diketospirilloxanthin

—» Part of asterinic acid 3,3'-Dihydroxy-7,8,7',8'tetradehydro-(3,p-carotene4,4'-dione —> Rhodoxanthin

Part of asterinic acid

4,4'-Diketocynthiaxanthin

598.95

624.90 O

Volume /: Fat-Soluble Pigments 45

C40HS4O

Phytofluene Dehydroadonixanthin Aphanin p,P-Caroten-4-one “ Echininone” 4-Keto-P-carotene Myxoxanthin

—> Phytofluene -> Dehydroadonixanthin

p-Carotenone

Aphanin Echinenone 4-Keto-p-carotene Myxoxanthin

—> p-Carotenone

(P)-Carotenonaldehyde

5,6-Dioxo-10'-apo-5,6-secop-caroten-lO'-al 5,6,5',6'-Diseco-p,p-carotene- 5,6,5', 6' -tetrone Dodecahydrolycopene P-Doradecin

Echinenone

(3S,5/?,6/?,3\S,57?,6'S)-5',6'C42H580 5 Epoxy-6,7-didehydro5,6,5',6'-tetrahydro-(3,P-carotene-3,5,3'-triol-3-acetate Neoxanthin-3-acetate —►((3)-Carotenonaldehyde

—> Violerythrin

Violerythrin

Dinoxanthin

—> Crocetindimethylester

Crocetindimethylester

C42H6O04

Sum formula

Dinoxanthin

—►1, r-Dimethoxy-3',4'-didehydro-1,2,1' ,2'-tetrahydroi|i,t(;-caroten-4-one —> Crocetindimethylester —» Methylbixin (trans)

1,l'-Dimethoxy-3',4'-didehydro-1,2,1' ,2'-tetrahydrovJ;,i|;-caroten-4-one Crocetindimethylester Methylbixin (trans)

l,l'-D im ethoxy-l,2,r,2'-tetrahydro-3' ,4'-didehydroi|/,v|/-caroten-4-one Dimethylcrocetin Dimethyl-6,6'-diapocarotene6,6'-dioate Dimethyl-8,8'-diapocarotene8,8'-dioate 2,2'-Dinor-P,(3-carotene3,4,3',4'-tetrone

All synonyms

1,r-Dim ethoxy-l ,2,l',2 '-te- — trahydro-i|Mjt-carotene-4,4'dione

Names mentioned in Tables 1.5 through I. LC

1,r-Dimethoxy-l ,2,1 \2'-tetrahydro-v|i,i|i-carotene-4,4'dione

Pigment

642.92

628.93

ch3c

9^

Mol wt

i

l

l

Structure

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC)

Q

46 CRC Handbook of Chromatography: Plant Pigments

(3S,5R ,6R, 3 'S,5 'R ,6' S, )5' ,6' ,Epoxy-6,7-didehydro5,6,5' ,6' -tetrahydro-f3,f3carotene-3 ,5,3 '-triol-3acetate 5 ,6-Epoxy-5 ,6-dihydro-f3carotene 5 ,6-Epoxy-5 ,6-dihydro-f3, f3carotene

5,6-Epoxy-7' ,8' -didehydro5 ,6-dihydro-f3, f3-carotene3,3' -diol 5' ,6' -Epoxy-6,7-didehydro5,6,5' ,6' -tetrahydro-f3,f3carotene-3,5, 19' ,3' -tetrol (3S,5R,6R ,3'S,5' R ,6'5)-5' ,6' Epoxy-6, 7-didehydro5 ,6,5' ,6' -tetrahydro-f3,f3carotene-3,5,3' -trio! 5' ,8' -Epoxy-6,7-didehydro5,6,5' ,8' -tetrahydr~-f3.f3carotene-3,5,3' -trio!

5,8-Epoxycryptoxanthin

''5,8-Epoxy-cx-carotene'' 5,8-Epoxy-[3-carotene

Eloxanthin

"Echininone"

---->

---->

---->

---->

Vaucheriaxanthin

Neoxanthin Trollixanthin

Neochrome Trollichrome

---->

----> ---->

Dinoxanthin

5 ,6-Monoepox y-cx-carotene

f3-Carotenemonoepoxide 5,6-Monoepoxy-[3-carotene

---->

---->

---->

---->

---->

Aphanin Echinenone 4-Keto-[3-carotene Myxoxanthin Isolutein Luteinepoxide Lutein-5 ,6-epoxide Taraxanthin Xanthophyllepoxide Flavochrome Flavacin Mutatochrome Cryptoflavin Cryptoxanthin-5,8-epoxide Diadinoxanthin

f3-Carotenemonoepoxide

cx-Carotene-epoxide

Dinoxanthin

Neochrome

Neoxanthin

Vaucheriaxanthin

Diadinoxanthin

Cryptotlavin

Flavochrome Flavacin

Luteinepoxide

Echinenone

-...J

""

""

:::.

~

O

Chrysanthemaxanthin

'"t,

~

5"

'"t,

~

~..., {;

i::,

3

cl

n;:-

Luteinepoxide

---->

Structure

Chrysanthemaxanthin Flavoxanthin Trollixanthin

Cryptoflavin

---->

Mol wt

0o·

Flavacin

---->

Sum formula

00

.

Flavochrome

Cryptoxanthinepoxide

Mutatoxanthin

Antheraxanthin

All synonyms

---->

---->

Mutatoxanthin

Cryptoxanthinepoxide Cryptoxanthin-5,6-epoxide Flavacin Mutatochrome Cryptoflavin Cryptoxanthin-5 ,8-epoxide Isolutein Luteinepoxide Lutein-5,6-epoxide Taraxanthin Xanthophyllepoxide Flavochrome

---->

Antheraxanthin

Names mentioned in Tables 1.5 through I. LC

5, 6-Epox y-5 ,6-dihydro-13, 13carotene-3 ,3 '-diol 5,8-Epoxy ,-5,8-dihydro-13,13carotene-3 ,3 '-diol 5 ,6-Epox y-5 ,6-dihydro-13, 13caroten-3-ol 5 ,8-Epoxy-5, 8-dihydro-13, 13carotene 5, 8-Epoxy-5, 8-dihydro-13, 13caroten-3-ol (3S,5R,6S,3' R,6' R)-5,6Epoxy-5,6-dihydro-!3,E-carotene-3,3 '-diol

Pigment

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC)

''5 ,8-Epoxy-rubixanthin'' (3S,5R,6S,3' S,5' R,6' R)-5,6Epoxy-3,3' ,5' -trihydroxy6', 7 '-didehydro5 ,6, 7 ,8,5' ,6' -hexahydro13, 13-caroten-8-one (3S,5R,6S,3' S,5' R,6' R)-5,6Epoxy-3,3' ,5' -trihydroxy6', 7' -didehydro5,6, 7 ,8,5' ,6' -hexahydro13, 13-caroten-8-one-3 '-acetate

5 ,6-Epoxy-3-hydroxy-5 ,6dihydro-1 O' -apo-!3-carotenlO' -al 5 ,6-Epoxy-3-hydroxy-5 ,6dihydro-12' -apo-!3-caroten12' -al 5,6-Epoxy-lutein ''5 ,8-Epoxy-lutein''

5' ,6' -Epoxy-3 ,3 '-dihydroxy7,8-didehydro-5' ,6' -dihydro10, l I ,20-trinor-i3,i3-carotenl 9', 11 '-olide 5' ,6' -Epoxy-3,3' -dihydroxy7,8-didehydro-5' ,6' -dihydro10, l l ,20-trinor-i3,i3-carotenl 9', 11 '-olide-3-acetate 5 ,6-Epoxy-3 ,3 '-dihydroxy5,6-dihydro-!3,K-caroten-6' one 5,8-Epoxy-3,3' -dihydroxy5 ,8-dihydro-13, K-caroten-6' one ''5 ,8-Epoxy-3-hydroxy--ycarotene" ---> Capsanthin-5,6-epoxide

---> Capsochrome

---> Rubichrome

Capsanthin-5 ,6-epoxide

Capsochrome

Rubichrome

Fucoxanthin "Fucoxanthol"

5,6-Monoepoxy-lutein Chrysanthemaxanthin

--->

--->

--->

Fucoxanthin

Fucoxanthinol

---> Rubichrome

--->

Apo-12' -violaxanthal

--->

'' Apoviolaxanthinal'' Apo-IO' -violaxanthal

---> Pyrroxanthin

Pyrroxanthin

5 ,6-Epoxy-3-hydroxy-5 ,6dihydro- l O' -apo-!3-carotenl O' -al 5 ,6-Epoxy-3-hydroxy-5 ,6dihydro-12' -apo-!3-caroten12' -al 5,6-Monoepoxy-lutein Chrysanthemaxanthin Flavoxanthin Rubichrome Fucoxanthinol

---> Pyrrhoxanthinol

Pyrrhoxanthinol

C,,H, 0 O, 408.58

1,0

,,.

;:: ~

~

()Q' ;:l

~ "t,

(::-

~ :::-

~

:---

~

~

:::;:l

Names mentioned in Tables 1.5 through I. LC

4-Keto-4'-ethoxy-(3-carotene 4-Keto-4'-ethoxy-(3-carotene Dehydrohydroxyechinenone Aphanicin Canthaxanthin 4,4'-Diketo-(3-carotene Dehydrohydroxyechinenone Astacene

4 '-Ethoxy-(},(3-caroten-4-one 4'-Ethoxy-4-keto-(3-carotene “ Euglenanone” Euglenanone

“ Euglenarhodon”

Eschscholtzxanthin

Eschscholtzxanthin

,6 -Epoxy-3,5,3 -trihyPeridininol droxy-6,7-didehydro5,6,5',6'-tetrahydro1 0 . 1 1 .20- trinor-(3,(3-caroten19', 11 '-olide 5',6'-Epoxy-3,5,3'-trihyPeridinin droxy-6,7-didehydro5,6,5' ,6 '-Tetrahydro1 0 . 1 1 .20- trinor-(3,(3-caroten19', 1 1 '-olide-3-acetate 5 ,6 -Epoxyzeaxanthin Antheraxanthin 5,8-Epoxyzeaxanthin Mutatoxanthin Eschscholtziaxanthin Eschscholtzxanthin

5

Pigment

-> Dehydrohydroxyechinenone —> Astacene

4',5'-Didehydro-4,5'-retro-(3,(3carotene-3,3 '-diol -3,3'-Dihydroxydehydro-pcarotene” ” 3,3'-Dihydroxyretro-(3carotene” Eschscholtziaxanthin Retrodehydrozeaxanthin ’’ -> 4-Keto-4'-ethoxy-(3-carotene -> 4-Keto-4'-ethoxy-(3-carotene -» Dehydrohydroxyechinenone -* Canthaxanthin

Antheraxanthin Mutatoxanthin -> Eschscholtzxanthin

—> Peridinin

—» Peridininol

All synonyms

C40H54O 2

Sum formula

566.87

Mol wt

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC) Structure

50 CRC Handbook of Chromatography: Plant Pigments

Flavochrome

Flavorhodin Neurosporene Proneurosporene · 'Protetrahydrolycopene'' •'Tetrahydrolycopene'' "5,6,5',6'Tetrahydrolycopene'' Chrysanthemaxanthin Flavoxanthin

Flavochrome

Flavorhodin

Neochrome Trollichrome Neoxanthin Trollixanthin Neochrome Trollichrome

Fucoxanthin "Fucoxanthol"

Foliachrome

Fucoxanthin

Fucochrome

Foliaxanthin

Flexixanthin

Flexixanthin

Flavoxanthin

Mutatochrome

Flavacin

Chrysanthemaxanthin

Neochrome

Neoxanthin

(3S,5R,6S,3' S,5' R,6' R)-5,6Epoxy-3,3' ,5' -trihydroxy6' ,7' -didehydro-5,6,7,8,5' ,6' hexahydro-P, P-caroten-8-one3' -acetate "Fucoxanthol"

->

->

3, I' -Dihydroxy-3' ,4' -didehydro-l ',2' -dihydro-P-lfi-caroten4-one 4-0xosaproxanthin -> Neochrome

->

•· 5 ,8-Epoxy-a-carotene'' 5 ,8-Epoxy-5 ,8-dihydro-P,tcarotene -> Neurosporene

P-Carotene-5 ,8-epoxide Carotene oxide Citroxanthin 5 ,8-Epoxy-p-carotene 5,8-Epoxy-5,8-dihydro-P,Pcarotene Mutatochrome

C.,H,,06

C'°H,40,

C 40 H, 6 0

6

c..,H, 0

658.92

582.86

552.88

552.88

HO

HO

-c

8

::l $::,

cS

:::-

~ r.i

;>:-

g

~

~ ::,

r.i

Q

N

7', 8 '-Didehydro-5,6 -dihydroC4oH560 4 (3,(3-carotene-3,5,6,3'-tetrol T , 8 '-Didehydro-5,6 -dihydroP,(3-carotene-3,5,3'-triol? “ 3,3 ',5 '-Trihydroxy-6 '-hydro7,8-dehydro-0-carotene” ‘‘Vawc/zer/fl-Heteroxanthin” 3,3',8-Trihydroxy-5,6-epoxy-(3carotene —> Phytoene —> Anhydroeschscholtzxanthin -* Phytofluene -* —> Phytofluene

Heteroxanthin

Phytoene Anhydroeschscholtzxanthin’

Phytofluene

4,4'-Diapophytofluene

Phytofluene

“ Hexadecahydrolycopene” 2,3,2',3',4',5'-Hexadehydro4,5'-retro-0,(3-carotene 15-d.s-7,8,l 1,12,7',8'Hexahydro-i|/,i|/-carotene 7,8,11,12,7', 8 '-Hexahydro4,4' -diapocarotene 7, 8 ,11,12,7', 8 'Hexahydrolycopene

C 72H n60 4

Heteroxanthin*

—> Myxobactone

—►(3-Citraurin —» 3 -hydroxy-10'-apo-(3-caroten-lO'-al

Myxobactone

(3-Citraurin

3-Hydroxy- 10'-apo-(3-caroten-lO'-al

0-Hexosyl-4-keto-l'-hydroxy-1 ', 2 ' -dihydro-ycarotene ” 3-Hydroxy-{3-apo-2carotenal” 3-Hydroxy-(3-apo-10'carotenal

l-Mannosyloxy-3,4-didehydro- C^H^C^ 1 , 2 -dihydro-8 '-apo-i|/-caroten8 '-ol

“ 1-Hexosyl-l ,2-dihydro-3,4didehydro-apo-8 'lycopenol”

'-Diapophytofluene

“ 1-Hexosyl-l,2-dihydro-3,4didehydro-apo-8 'lycopenol”

4 ,4

(3/?, 3,/?,6'/?)-(3, e-Carotene3,3'-diol dipalmitate Lutein dipalmitate Xanthophyll dipalmitate

Helenien Lutein dipalmitate

Helenien

—> Myxobactin

Myxobactin

l'-Glucosyloxy-3,4,3',4'-tetradehydro-1 ' ,2 '-dihydro(3,if*-carotene

596.80

600.88

H0^

H

Rubixanthin -> 3-Hydroxy-4,4' -diketo-13carotene

-->

->

carotene

4-Hydroxy-a-carotenc

o.-Citraurin

3-Hydroxy-4,4' -diketo-13-

~

'' Apo-3-zeaxanthinal'' 3-Hydroxy-13-apo-10' -carotenal ----. "Apo-4-zeaxanthinal"

->

All synonyms

->

3-Hydroxy- IO' -apo-l3-caroten- IO' -al 3-Hydroxy-12 '-apo-l3-caroten- I 2' -al a-Citraurin

13-Citraurin

Names mentioned in Tables 1.5 through I. LC

4,4' -Diketo-3-hydroxy-13carotene 3-Hydroxy-4,4' -diketo-13carotene 2-Hydroxy-a-carotene a-Cryptoxanthin Zeinoxanthin a-Cryptoxanthin

(3R)-3-Hydroxy-8' -apo-l3-caroten-8' -al 3-Hydroxy- lO' -apo-l3-caroten- l O' -al 3-Hydroxy-12' -apo-l3-caroten-l 2' -al 3-Hydroxy-8' -apo-e-caroten8' -al 3-Hydroxycanthaxanthin

Pigment

C 40 H'i 6 0

C40H,"O

C 07 H, 0 0,

Sum formula

552.88

552. 88

392.58

Mol wt

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC) Structure

Cc

cc ~

Si

~ "'1:l 0q"

i:S"

"'1:l

-~

-§::,-

~

F:.

Si

2

~ ~ r.i ::,-

Q

§:

e

::i:

r.i

Q

"'"

Ul

“ 3-Hydroxy-5,8,5',8'-diepoxy-(3-carotene” (3/?)-3-Hydroxy-5' ,6 '-dihydro-5'-apo-18'-nor-(3-caroten-6 '-one Hydroxydihydro-y-carotene

“ 4-Hydroxy-4,4'diaponeurosporene ’’ 1'-Hydroxy-3',4 '-didehydro1',2'-dihydro-(3-iJj-caroten2 '-one 1'-Hydroxy-3',4 '-didehydro1' ,2'-dihydro-(3,i|/-caroten-4one

1

\2'-D ihydro-l'-hydroxy-ycarotene

Reticulataxanthin

—> Deoxyflexixanthin

Deoxyflexixanthin 1' ,2'-D ihydro-1'-hydroxy-4keto-torulene 4-Keto-1', 2 '-dihydro-1'-hydroxy-torulene Cryptochrome

—> l',2'-D ihydro-l -hydroxy-ycarotene

Reticulataxanthin

-> Cryptochrome

4-Hydroxy-7'-8'-dihydro-4,4'diapo-carotene -► 2'-Dehydroplectaniaxanthin

“ 4-Hydroxy-4,4'diaponeurosporene ’’ 2'-Dehydroplectaniaxanthin

Hydroxyechinenone Hydroxyechinenone 4'-hydroxyechinenone 3'--Hydroxy-(3,(3-caroten-43 '-Hydroxyechinenone —> 3'-Hydroxyechinenone one 4-Keto-3'-hydroxy-(3-carotene 4'-Hydroxy-(3,(3-caroten-44-Hydroxy-4'-keto-(3-carotene —> 4-Hydroxy-4'-keto-(3one carotene 3-Hydroxy-(3,e-caroten-3'-one 3-Hydroxy-3'-keto-a-carotene —> 3-Hydroxy-3'-keto-aPhilosamiaxanthin carotene (3'S,57?)-3'-Hydroxy-0,KCryptocapsin Cryptocapsin caroten-6 '-one 3-Hydroxycitranaxanthin Reticulataxanthin —> Reticulataxanthin 3-Hydroxy-7, 8 -dehydro-aCrocoxanthin -> Crocoxanthin carotene 3-Hydroxy-3',4'-dehydro-y- Anhydrosaproxanthin —» Anhydrosaproxanthin carotene Celaxanthin

3-Hydroxy-(3,(3-caroten-4-one

C 30H42O

418.66

Volume I: Fat-Soluble Pigments 55

1'-Hydroxy-l'- 2 'dihydrospheroidenone 3'-Hydroxy-3,4-diketo-(3carotene

4-Hydroxy-7',8'-dihydro4,4'-diapocarotene 1-Hydroxy-1,2dihydrolycopene ‘41-Hydroxy-1 ,2dihydroneurosporene ’’ l'-H ydroxy-l', 2 'dihydroneurosporene 1-Hydroxy-1 ,2dihydrophytofluene 1'-Hydroxy - 1 ',2 'dihydrophytofluene 1'-Hydroxy-1'-2'dihydrospheroidene

r-H ydroxy-r,2'-dihydro-,ycarotene l'-Hydroxy-r,2'-dihydro-ycarotene 1'-Hydroxy-l',2'-dihydroP,iJ;-caroten-4-one

Pigment

l'-H ydroxy-r,2'-dihydro-ycarotene r,2'-D ihydro-l'-hydroxy-ycarotene l',2'-D ihydro-r-hydroxy-4keto-y-carotene 4-Keto-1' ,2'-dihydro-1'-hydroxy-y-carotene 4-K eto-l'-hydroxy-l',2'dihydro-y-carotene 4-Hydroxy-4,4'diaponeurosporene OH-Lycopene Rhodopin Chloroxanthin OH-Neurosporene Chloroxanthin OH-Neurosporene OH-Phytofluene Phytofluenol OH-Phytofluene Phytofluenol 7',8'-Dihydrorhodovibrin OH-Spheroidene OH-Y “ OH-R” OH-Spheroidenone Dehydroadonixanthin

AH synonyms

—> Dehydroadonixanthin

—» PH-Spheroidenone

—> OH-Y

—►OH-Phytofluene

-» OH-Phytofluene

-^Chloroxanthin

—* Chloroxanthin

—» 4-Hydroxy-4,4'diaponeurosporene —* OH-Lycopene

—►1 ',2'-Dihydro-r-hydroxy-ycarotene —> r,2'-D ihydro-l'-hydroxy-ycarotene —> 4-K eto-l'-hydroxy-l',2'dihydro-y-carotene

Names mentioned in Tables 1.5 through I. LC Sum formula

Mol wt

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC) Structure

56 CRC Handbook of Chromatography: Plant Pigments

—> Dehydroadonixanthin —> Lutein

—> 4-Hydroxy-4'-keto-(3carotene —►Cryptoflavin

3-Hydroxy-3'-keto-a-carotene 3'-Dehydrolutein Philosamiaxanthin 3-Hydroxy-(3,e-caroten-3'-one 3-Hydroxy-3'-keto-a-carotene Philosamiaxanthin 3-Hydroxy-4-keto-(3-carotene Hydroxyechinenone —> Hydroxyechinenone

3-Hydroxy-3'-keto-a-carotene

3-Hydroxy-5,8-epoxy-(3carotene “ 3' -Hydroxyeuglenanone ’’ 3-Hydroxy-3'-hydroxy-acarotene

4 '-Hydroxyechinenone 4-Hydroxy-4' -keto- (3-carotene Cryptoflavin Cryptoxanthin-5, 8 -epoxide Dehydroadonixanthin Lutein “ Xanthophyll”

3 '-Hydroxyechinenone Asteroidenone C^H^CL 4Keto-3'-hydroxy-(3-carotene 3'-Hydroxy-(3,(3-caroten-4-one 3'-Hydroxy-4-oxo-(3-carotene 4Keto-3'-hydroxy-(3-carotene

y -Hydroxyechinenone

4'-Hydroxyechinenone

Hydroxyechinenone

Hydroxyechinenone

C4QH5402

3-Hydroxy-(3,(3-caroten-4-one C^H^vjCL 3Hydroxy-4-keto-(3-carotene

4,4'-Diketo-3-hydroxy-(3Adonirubin carotene 4,4'-Diketo-3-hydroxy-03Hydroxy-4,4'-diketo-(3carotene carotene 3-Hydroxycanthaxanthin 3-Hydroxy-(3, ^-carotene-4,4' dione “ Metridene” Phoenicoxanthin

3' -Hydroxy-4,4' -diketo- (3carotene

566.87

566.87

566.87

580.85

o

H C

^

^

Volume I: Fat-Soluble Pigments 57

Names mentioned in Tables 1.5 through I. LC AH synonyms

4-Hydroxy-4'-keto-(3-carotene 4'-Hydroxyechinenone 4'-Hydroxy-p,(3-caroten-4-one 4-Hydroxy-4'-keto-(3-carotene 4'-Hydroxyechinenone r-H ydroxy-2'-keto-l',2'2'-Dehydroplectaniaxanthin —» 2'-Dehydroplectaniaxanthin dihydrotorulene 19-Hydroxylutein Pyrenoxanthin —> “ Trollein” * “ Trollein” 16-Hydroxylycopene Lycoxanthin —> Lycoxanthin 3Hydroxy-3'-methoxy-a3-Hydroxy-3'-methoxy-a— carotene carotene 1'-Hydroxy - 1 -methoxy-3,4“ OH-R” ->• OH-Spheroidenone didehydro-1,2, r ,2 ',7 ',8 'OH-Spheroidenone hexahy dro-ij;, i[/-caroten-2 one l'-Hydroxy-l-methoxy2-Ketorhodovibrin —» 2-Ketorhodovibrin 3,4,3' ,4' -tetradehydro1 , 2 , 1 ', 2 ' -tetrahydro-i|/, i|>caroten-2 -one l'-Hydroxy-l-methoxyl-methoxy-l'-hydroxy—» l-methoxy-l'-hydroxy1 , 2 , 1 ', 2 '-tetrahy dro-ij;, iji1 , 2 , l ', 2 -tetrahydro-i|/,i|/-car1 , 2 , l ', 2 '-tetrahydro-i|i,i|/-carocaroten-4-one oten-4-one ten-4-one Thiothece-OH-484 2'-(4-Hydroxy-3-methyl-2-bu- Sarcinaxanthin —> Sarcinaxanthin tenyl)-2-(3-methyl-2-butenyl)-e,6-caroten-18-ol 4Hydroxymyxoxanthophyll Aphanizophyll —> Aphanizophyll Hydroxyneurosporene Chloroxanthin —> Chloroxanthin OH-Neurosporene 3'-Hydroxy-4-oxo-(3-carotene 3'-Hydroxyechinenone —> 3'-Hydroxyechinenone 4-Keto-3 '-hydroxy-(3-carotene

Pigment C 40H,4O,

Sum formula

C4 lHS80 ,

566.87

Mol wt

582.91

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC) Structure

58 CRC Handbook of Chromatography: Plant Pigments

—> OH-Phytofluene

—» Dehydrohydroxyechinenone

3'-Hydroxy-5,6-seco-p,pcarotene-5,6 -dione 3Hydroxysemi-P-carotenone Triphasiaxanthin Hydroxyspheroidene 7',8'-Dihydrorhodovibrin OH-Spheroidene OH-Y “ Hydroxyspheroidenone” OH-R OH-Spheroidenone Hydroxyspirilloxanthin Hydroxyspirilloxanthin OH-Spheroidenone 1-Hydroxy-l ,2,7 ',8 'Chloroxanthin tetrahydrolycopene OH-Neurosporene 3-Hydroxytorulene Anhydrosaproxanthin Celaxanthin Isocarotene “ Dehydro-(3-carotene” Dehydro-retro-p-carotene Retrodehydro-carotene Retrodehydro-p-carotene Iso-^-carotene 7,8,11,12-Tetrahydro-v|iipcarotene 7,8,11,12Tetrahydrolycopene Isocrocetin Crocetin Isocryptoxanthin 4-Hydroxy-P-carotene Isocryptoxanthin “ Myxoxanthol”

“ OH-R” OH-Spheroidenone Triphasiaxanthin

—> Crocetin —> 4-Hydroxy-P-carotene

—> Asym. ^-carotene

—> “ Dehydro-P-carotene”

—> Anhydrosaproxanthin

—» Chloroxanthin

—> OH-Spirilloxanthin

—> OH-Spheroidenone

—►Triphasiaxanthin —> OH-Y

—» Triphasiaxanthin

—> OH-Spheroidenone

2-Hydroxyplectaniaxanthin 3\4'-Didehydro-l',2'-dihydro- C40H 56O^ p,il/-carotene-2 , r , 2 '-triol Rhodoauranxanthin

Dehydrohydroxyechinenone “ Euglenanone” OH-Phytofluene Phytofluenol

Hydroxyplectaniaxanthin

“ Hydroxy-R”

2-

3-Hydroxy-4-oxo-2,3-dehydro-P-carotene Hydroxyphytofluene

HOv.

Volume I: Fat-Soluble Pigments 59

OH

Isofucoxanthinol

Isolutein Luteinepoxide Lutein-5,6 -epoxide Taraxanthin Xanthophyllepoxide Methylbixin (trans)

Isofucozanthinol

Isolutein

Isorenieratene Leprotene

(3-Isorenieratene

4,4'-Dihydroxy-|3-carotene Isozeaxanthin

4-Keto-a-carotene

Aphanin Echinenone 4-Keto-(3-carotene Myxoxanthin

Isorenieratene

(3-Isorenieratene

Isozeaxanthin

4-Keto-a-carotene

4-Keto-(3-carotene

Isomethylbixin

Isofucoxanthin

Names mentioned in Tables 1.5 through I. LC

Isofucoxanthin

Pigment

—» Echinenone

(3,i|/-Caroten-4-one

—> 4,4'-Dihydroxy-(3-carotene

(3,(p-Carotene

Luteinepoxide

Pentaxanthin ? 3,5,3',5'-Tetrahydroxy-6',7'didehydro-5,8,5' 6 '-tetrahydro-(3,(3-caroten-8-one

3,5,3',5'-Tetrahydroxy-6',7'didehydro-5,8,5',6'-tetrahydro-(3, (3-caroten-8-one-3' acetate

All synonyms

C40HS4O

C 42H 5g0 6

Sum formula

550.87

532.85

528.82

616.88

658.92

Mol wt

1

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC) Structure

60 CRC Handbook of Chromatography: Plant Pigments

4-Keto-4' -ethoxy-(3-carotene

4-Keto-4'-ethoxy-(3-carotene

4'-Ethoxy-(3,(3-caroten-4-one 4'-Ethoxy-4-keto-(3-carotene

—> OH-Spheroidenone

—►Deoxyflexixanthin

(i-ijj-Carotene-4-one —» 4-Keto-l'-hydroxy-1',2'dihydro-7 -carotene

2

C 40H 54O 2

C 42H580

C^H ^O

l'-((3,D-Glucopyranosyloxy)-2'- C 46HM0 8 hydroxy-3',4'-didehydrol',2'-dihydro-p,i|/,caroten-4one

4-Ketophleixanthophyll

4-Ketophleixanthophyll

2-Keto-OH-spirilloxanthin

l',2'-Dihydro-l'-hydroxy-4keto-7 -carotene keto-7 -carotene 4-K eto-r,2'-dihydro-l'-hyl'-Hydroxy-l ',2'-dihydro-(3,iJ/droxy-y-carotene caroten-4-one 4-Keto-l '-hydroxy - 1 ',2'-dih- 4-Keto- l',2'-dihydro-1'-hyydro-y-carotene droxy-y-carotene 4-Keto-l'-hydroxy- 1 ' ,2'-dihydro-y-carotene 2-Ketorhodovibrin —> 2-Ketorhodovibrin

1 \2'-D ihydro-l'-hydroxy-4-

4-Keto-l'-hydroxy-1', 2'-dihydro-7 -carotene

4-Keto-3'-hydroxy-(3-carotene 3'-Hydroxyechinenone —» 3'-Hydroxyechinenone 4-Keto-3 '-hydroxy-(3-carotene 4-Keto-4'-hydroxy-(3-carotene 4-Keto-4'-hydroxy-(3-carotene —

“ 2-Keto-7',8'dihydrorhodovibrin ’’

4-Keto-l ',2'-dihydro-l'-hydroxy-torulene

4-Keto- 7 -carotene 1',2'-Dihydro-1'-hydroxy-4keto-y-carotene 4-K eto-l' ,2'-dihydro-1'-hydroxy-7 -carotene 4-Keto-1'-hydroxy-1' ,2'-dihydro-7 -carotene Deoxyflexixanthin 4-K eto-l' ,2 '-dihydro-1'-hydroxy-torulene “ OH-R” OH-Spheroidenone

4 -Keto-7 -carotene 4-K eto-l',2 '-dihydro-l'-hydroxy-7 -carotene

745.01

568.88

566.87

594.92

550.87

L J

L

Volume I: Fat-Soluble Pigments 61

2,2'-Diketospirilloxanthin P-518 Anhydro-deoxy-flexixanthin 4-Ketotorulene Isorenieratene Leprotene ‘T rollein” Pyrenoxanthin Torularhodin

3-Hydroxy-3'-hydroxy-acarotene Lutein ‘‘Xanthophy IP 1

Helenien Lutein dipalmitate

“ 2-Ketospirilloxanthin,,

Lutein

Lutein dipalmitate

“ Lusomycin”

Loroxanthin

Leprotene

4-Ketotorulene

2-Ketorhodovibrin

Names mentioned in Tables 1.5 through I. LC

2-Ketorhodovibrin

Pigment

Sum formula

(3/?,3'/?,6'/?)-(3,€-CaroteneC40H ^O 2 3,3'-diol “ Cucurbitaxanthin” 3,3 '-Dihydroxy-a-carotene 3Hydroxy-3'-hydroxy-acarotene “ Luteol” “ Xanthophyll” —> Helenien

—> Torularhodin

—> “ Trollein” *

—> Isorenieratene

—> Anhydro-deoxy-flexixanthin

l'-Hydroxy- 1-methoxyC4 ,H 5f)O x 3,4,3',4'-tetradehydro1 , 2 , 1 ', 2 '-letrahydro-i[i,i|)-caroten-2 -one 2Keto-OH-spirilloxanthin OH-P-511 —►2,2'-Diketospirilloxanthin

All synonyms

568.88

9^3

596.89

Mol wt

,

,

,

Structure

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC)

OH

62 CRC Handbook of Chromatography: Plant Pigments

Isolutein Luteinepoxide Lutein-5,6 -epoxide Taraxanthin Xanthophyllepoxide

Luteochrome

3-Hydroxy-3'-hydroxy-otcarotene Lutein “ Xanthophyll”

Luteoxanthin

Apo-2-lycopenal Apo-6 '-lycopenal

Lycopene Poly-cA-lycopene Prolycopene Rhodopurpurin Lycophyll Lycoxanthin

Lutein-5,6 -epoxide

Luteochrome

“ Luteol”

Luteoxanthin

“ Lycopenal”

Lycopene

Lycopene-16,16'-diol Lycopene-16-ol

Isolutein Luteinepoxide Lutein-5,6 -epoxide Taraxanthin Xanthophyllepoxide

Luteinepoxide

i|/,i|j-Carotene Poly-cA-lycopene Prolycopene Rhodopurpurin —> Lycophyll —» Lycoxanthin

5,6,5',8'-Diepoxy-5,6,5',8'-tetrahydro-(3,(3-carotene-3,3'diol 3,3 '-Dihydroxy luteochrome —> Apo-6 '-lycopenal

5,6,5',8'-Diepoxy-5,6,5',8'-tetrahydro-p, (3-carotene —> Lutein

3,3'-Dihydroxy-5,6 -epoxy-acarotene Eloxanthin (35,5^,65,3'/?,6'/?)-5,6-Epoxy5,6-dihydro-(3, e-carotene-3,3 'diol Lutein-5,6 -epoxide Taraxanthin Tareoxanthin —» Luteinepoxide

C40H S6

C4()H560 4

C4()H560 2

C 40H 56O 3

536.88

600.88

568.88

584.88

H0

I

Volume I: Fat-Soluble Pigments 63

Lycoxanthin

Lycoxanthin

l-Mannosyloxy-3,4-didehydro-1,2-dihydro-8'-apo-i|tcaroten-8 '-ol l-Methoxy-3,4-didehydro1,2-dihydro-i|i,i|i-carotene l-Methoxy-3,4-didehydro1,2,7',8',1 l ' , 1 2 '-hexahydro-i|/,v|/-carotene 1'-Methoxy-3 ', 4 '-didehydro1,2,7,8,1' ,2 '-hexahydrovjLvJj-caroten-l-ol 1-Methoxy-3 ,4-didehydro1,2,7', 8 ' -tetrahydro-i}/,4/carotene L-Methoxy-3 ', 4 ' -didehydro1 , 2 , L , 2 '-tetrahydro-i|/,i|icaroten- 1 -ol

Lycophyll

Lycophyll

4M|/-Carotene-16,16'-diol “ 3,3'-Dihydroxylycopene” Lycopene-16,16'-diol

7,8,11,12,15,7',8',11', 12', 15'-Decahydro-i|nJicarotene Decahydrolycopene Dihydrophytoene

All synonyms

Rhodovibrin

7',8'-Dihydrorhodovibrin OH-Spheroidene OH-Y “ P-450” Spheroidene —> Rhodovibrin

—» “ P-450”

—> OH-Y

iJ/,iJ/-Caroten-16-ol 16-Hydroxy-lycopene Lycopen-16-ol “ l-Hexosyl-l,2-dihydro-3,4- —> “ l-Hexosyl-l,2-dihydrodidehydro-apo-8 '3,4-didehydro-apo-8'lycopenol” lycopenol’’ Anhydrorhodovibrin —> Anhydrorhodovibrin “ P-481” “ P-412” —> “ P-412”

Lycopersene

Names mentioned in Tables 1.5 through I. LC

Lycopersene

Pigment

C40H^6O

C40H56O 2

C^H^,

Sum formula

552.88

568.88

546.96 I

Mol wt

I

I

I

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC) Structure

64 CRC Handbook of Chromatography: Plant Pigments

l-Methoxy-2-keto-7',8'-dihy- Pigment R dro-3,4-dehydrolycopene Spheroidenone l-Methoxy-4-oxo-l ,2-dihyThiothece-460 dro-8 '-apo-ij;-caroten-8 ' -al

“ 1-Methoxy-l '-hydroxyl , 2 , l ' , 2 '-tetrahydro-v|;,iJ/caroten-4-one” Thiothece-OH-484

“ 1-Methoxy-l'-hydroxyl , 2 , r , 2 '-tetrahydro>,i{/caroten-4-one”

—> Thiothece-460

', 2 '-tetrahydro-iJ/,i|/-caroten-4-one “ OH-Okenone” Thiothece-OH-484 —> Spheroidenone

1 ,2 , 1

1 '-Hydroxy-1-methoxy-

,2 ,7 ',8 ',1 1', 12'-----hexahydro-iJi,i}/-caroten-4one

—> “ P-450”

—►Anhydrorhodovibrin

1 -Methoxy-l

Anhydrorhodovibrin ‘‘P-481’ ’ “ P-450” Spheroidene

1-Methoxy-1,2-dihydro-3,4dehydrolycopene “ 4-Methoxy-5,6dihydrolycopene ’’

—

—> Thiothece-474

l-M ethoxy-1,2,7',8',11', 12'hexahydro-t|/,i|/-caroten-4one

1-Methoxy-1,2-dihydro-iJ/,ifjcaroten-4-one

Thiothece-474

Okenone

-» Thiothece 474

Thiothece 474

Okenone

Spheroidenone

Pigment R Spheroidenone

1-Methoxy-1,2-dihydro-i|/,vJicaroten-4-one

l-Methoxy-3,4-didehydro1,2,7' , 8 '-tetrahydro-i|/,i|/caroten-2 -one 1'-Methoxy-1' ,2'-dihydro(3,i|/-caroten-4'-one 1'-M ethoxy-l',2'-dihydroX,i|/-caroten-4'-one l'-M ethoxy-l',2 '-dihydro Neurosporaxanthinmethylester —» Methyl-apo-6 '-lycopenoate

C 16H „ 0 4 ‘

C „H 440 2

0 4 ,1-16002

Sum formula

“ Methyl- 1-hexosyl-l,2-dihy- “ Methyl-1-hexosyl-1 ,2-dihy- Methyl-l-mannosyloxy-3,4-di- C 37HS20 8 dro-3,4-didehydroapo-8'dro,3,4-didehydroapo-8'dehydro-1,2-dihydro-8'-apo-i|/lycopenoate” lycopenoate” caroten-8 '-oate Methyl-l-mannosyloxy-3,4- “ Methyl- 1 -hexosyl-l , 2 -dihy- —» “ Methyl-1-hexosyl-1 , 2 -dididehydro-l,2-dihydro-8'dro-3,4-didehydroapo-8'hydro-3,4-didehydroapo-8'apo-v|j-caroten-8 '-oate lycopenoate” lycopenoate” Methyl-l'-methoxy-4'-oxoThiothece-484 —> Thiothece-484 1 ', 2 ' -dihydro-x, vjt-caroten16(or 17 or 18)-oate

Methylbixin (trans)

oate

Methyl-6 '-apo-i|/-caroten-6 '-

Neurosporaxanthinmethylester Methyl-apo-6 '-lycopenoate

1-Methoxy-l ,2,7',8'-tetrahy- — dro-ijj,i|/-caroten-4-one “ P-450” —> “ P-450” Spheroidene Hydroxyspirilloxanthin —» OH-Spirilloxanthin OH-Spirilloxanthin

Names mentioned in Tables 1.5 through I. LC

Methylbixin (trans)

Methyl-apo-6 '-lycopenoate

l-Methoxy-l,2,7',8'-tetrahydro-ij/ ,i}t-caroten-4-one l-Methoxy-l,2,7',8'-tetrahydro-3,4-dehydrolycopene l'-Methoxy-3,4,3',4'-tetradehydro-1,2,1' ,2'-tetrahydrot|/,v|;-caroten-l-ol Methyl-4'-apo-p-caroten-4'oate Methyl-6 '-apo-i|;-caroten-6 'oate

Pigment

624.81

409.54

472.71

584.92

Mol wt

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC) Structure

ru

66 CRC Handbook of Chromatography: Plant Pigments

Myxol-2' -0-methyl-methylpentoside

3-Hydroxy-4,4' -diketo-13carotene

3-Hydroxy-4,4' -diketo-13carotene 4,4' -Diketo-3-hydroxy-13carotene Micronone Microxanthin

Mutatoxanthin

5,6-Monoepoxylutein Mutatochrome

Mutatoxanthin

3,3 '-Dihydroxy-5,8-epoxy-13carotene 5 ,8-Epoxy-5 ,8-dihydro-13, 13carotene-3,3' -diol 5 ,8-Epoxyzeaxanthin Zeaxanthin-5, 8-epoxide Zeaxanthinfuranoide

"Dehydrocarotene II" 3,4-Dehydro-13-carotene 3,4-Monodehydro-13-carotene 3 ,4- Dehydro-13-carotene 3 .4-Dehydro-13 .13-carotene 3 .4- Monodehydro-13-carotene -> 3.4-Dehydrolycopene 3 ,4-Dehydrolycopene -> OH-Spirilloxanthin Hydroxyspirilloxanthin OH-Spirilloxanthin -> a-Caroten-epoxide 5 ,6- Monoepoxy-a -carotene 13-Carotene-monoepoxide -> 13-Carotene-monoepoxide 5 ,6-Monoepox y-13-carotene 5,6-Epoxy-lutein -> Luteinepoxide -> Flavacin Flavacin Mutatochrome

3,4-Monodehydro-13-carotene

Monodehydrolycopene Monodemethylated spirilloxanthin 5 ,6-Monoepoxy-a -carotene 5 ,6-Monoepox y-13-carotene

Monadoxanthin 7 ,8-Didehydro-13.E-carotene3,3'-diol

->

->

Myxol-2' -0-methylmethylpentoside

Monadoxanthin

Micron one Microxanthin

2' -(0-Methyi-5-C-methylpentosyloxy)-3' ,4' -didehydro1',2' -dihydro-13,1)1-carotene3,1'-diol "Metridene"

C,,H, 0 0,

C4 oH.'i4

C40 H, 4 0,

584.88

534.87

566.87

0\ -...1

""

~

~

;:;:

"'\l c;Q'

~

~

::2.

~

~

~

-

~

::::

s: ;:;:

Myxoxanthophyll Aphanin Echinenone 4-Keto-(3-carotene Myxoxanthin 4-Hydroxy-(3-carotene Isocryptoxanthin “ Myxoxanthol”

Myxol-2'-rhamnoside Myxoxanthin

“ Myxoxanthol”

Myxol-2'-0-methylpentoside P-476

Myxobactone

Myxobactone

Myxol-2'-O-methylmethylpentoside

Myxobactin

Names mentioned in Tables 1.5 through I. LC

Myxobactin

Pigment

—» 4-Hydroxy-(3-carotene

—» Myxoxanthophyll —*Echinenone

P'476

2'-(0-Methyl-5-C-methylpentosyloxy-3',4'-didehydro-l',2'dihydro-(3,iJ/-carotene-3, l'-diol

r,2'-D ihydro-l'-glucosyl-4ketotorulene 1 ' ,2'-Dehydro - 1 '-hydroxy-4-ketotorulene-glucoside 1'-Glucosyloxy-3',4'-didehyd r o - r , 2 '-dihydro-p,ifi-caroten4-one O-Hexosyl-4-keto - 1 '-hydroxyT , 2 ' -dihydro-y-carotene

l',2'-D ihydro-l'-glucosyl-3,4dehydrotorulene l',2'-D ihydro-r-hydroxy-3,4dehydrotorulene-glucoside 1 '-Glucosyloxy-3,4,3',4'-tetradehydro-1',2'-dihydro-(3,ij;carotene

All synonyms

C47H680 7

C j(,HmO,,

Sum formula

745.05

729.01

713.01

Mol wt

0

LJ

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC) Structure

68 CRC Handbook of Chromatography: Plant Pigments

Myxoxanthophyll

p-Carotene P,P-Carotene

Neochrome Trollichrome

Neofucoxanthin A,B Neoluteoxanthin U

Neoxanthin

Dinoxanthin

Neurosporaxanthin

Neurosporaxanthinmethylester

Myxoxanthophyll

Neo-p-carotene

Neochrome

Neofucoxanthin A,B Neoluteoxanthin U

Neoxanthin

Neoxanthin-3-acetate

Neurosporaxanthin

Neurosporaxanthinmethylester

Methyl-4'-apo-P-caroten-4'oate

p-Apo-4'-carotenoic acid 4'-Apo-p-caroten-4-oic acid

{3S,5R.6R,3'S,5'R,6'S)-5' ,6 'Epoxy-6 ,7-didehydro5,6,5\6'-tetrahydro-p,p-carotene-3,5,3'-triol Foliaxanthin “ 3,3',5'-Trihydroxy-5',6'-dihydro-5' ,6 '-epoxy-P-carotene’’ Trolliflor —> Dinoxanthin

5 ', 8 '-Epoxy-6 ,7-didehydro5,6,5',8'-tetrahydro-p,p-carotene-3,5,3'-triol Foliachrome Fucochrome Trollichrome* Trolliflavin — —

r,2'-D ihydro-3\4'-didehydro3,1 '-dihydroxy-y-caroten-2'yl-rhamnoside Myxol-2'-rhamnoside 2' -((3 ,L-Rhamnopyranosy loxy )3',4'-didehydro-l',2'-dihydroP,iJ/-carotene-3,1'-diol 2'-0-Rhamnosylmyxol —> P-Carotene

C 36H480 2

C 35H460 2

C 46H660 7

ho' '

512.77

498.75

600.88 H0 ‘-

600.88

731.02

h

Volume I: Fat-Soluble Pigments 69

7 ,8 , 1 1 , 1 2 ,7 ',8 \1 1 ', 1 2 'Octahydrolycopene

—» 1 , l'-Dihydroxy- 1 ,2, l',2'-tetrahydro-£-carotene OH-Phytofluene

1,1 '-Dihydroxy- 1 ,2, 1 ', 2'tetrahydro-£-carotene OH-Phytofluene Phytofluenol OH-Phytofluene Phytofluenol 4,4'-Diapophytoene

Phytofluene ^-Carotene 7, 8 ,7', 8 ' -Tetrahy dro-i|t, t];carotene Phytoene

—> Phytoene

Phytoene

15-c/.y-7,8,11,1 '1,1', 8 f, 11', 12' -Octahydro-t}/,ijicarotene 1,2,7,8, l',2',7 ',8 '-O ctahydro-i|/,ij;-carotene-1 , 1 '-diol 1,2,7, 8 ,7 ', 8 ', 1 1 ', 12'-Octahydro-i|i,iJ/-caroten- 1 -ol 1,2,7,8,11,12,7 ,8 -Octahydro-v|/,4/-caroten- 1 -ol 7,8,11,12,7',8',1 l',12'-O ctahy dro-4,4' -diapocarotene “ Octahydrolycopene” 5, 6 ,7, 8 ,5 ', 6 ',7 ', 8 'Octahydrolycopene

Sum formula

—> Phytoene

—> Phytofluene ^-Carotene

4,4'-Diapophytoene

—> OH-Phytofluene

3,3'-Dihydroxy-5,5'-dihydroC ^H ^O , 7,7'-didehydro-(3-carotene 6,7,6',7'-Tetradehydro5,6,5',6'-tetrahydro-(3,(3-carotene-3,3'-diol

Nostoxanthin

Nostoxanthin

(p-Carotene ” ij/-Carotene” 7, 8 -Dihydro-iJ/,^-carotene 7,8-Dihydrolycopene Flavorhodin “ Poly-c/s-iji-carotene” Proneurosporene Protetrahydrolycopene ’’ 4‘Tetrahydrolycopene” ” 5,6,5', 6 'Tetrahydrolycopene”

All synonyms

Flavorhodin Neurosporene Proneurosporene “ Protetrahydrolycopene” “ Tetrahydrolycopene” ‘‘5,6,5' ,6 'Tetrahydrolycopene”

Names mentioned in Tables 1.5 through I. LC

Neurosporene

Pigment

568.88

538.90

Mol wt

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC) Structure

70 CRC Handbook of Chromatography: Plant Pigments

OH-Lycopene Rhodopin

Chloroxanthin OH-Neurosporene “ l-M ethoxy-l'-hydroxy1 , 2 , 1 ' , 2 '-tetrahydro-i|j,i|;caroten-4-one ’’ Thiothece-OH-484 3,4 -Dehydrorhodopin Rhodovibrin OHP-481 3,4-Dehydrorhodopin 2-Ketorhodovibrin

OH-Phytofluene Phytofluenol

', 8 '-Dihydrorhodovibrin OH-Spheroidene OH-Y “ OH-R” OH-Spheroidenone “ Dihydroxylycopene” 1,2,1' ,2'-Tetrahydro-1,1'dihydroxy-lycopene 7 ', 8 '-Dihydrorhodovibrin OH-Spheroidene OH-Y

OH-Lycopene

OH-Neurosporene

OH-Phytofluene

OH-Pigment Y

OH-Spheroidene

OH-Rhodopin

“ OH-R”

“ OH P-482” OH P-511

“ OH P-481” *

7

OH-Chlorobactene

OH-Chlorobactene

“ OH-Okenone”

OH-£-Carotene

OH-£-Carotene C 40H54O

—> OH-Y

-» 1 ,2 ,l',2'-Tetrahydro-l ,1'dihydroxy-lycopene

dihydrophytofluene r-H ydroxy-1',2'dihydrophytofluene Hydroxyphytofluene 1,2 ,7 ,8 ,7 ',8 ', 11', 12'-Octahydro-if/^-caroten-1-ol -* OH-Y —> OH-Spheroidenone

1 -Hydroxy-1,2-

-» 3,4-Dehydrorhodopin —» 2 -Ketorhodovibrin

—» 3,4-Dehydrorhodopin —» Rhodovibrin

-> “ 1-Methoxy-l '-hydroxy1 , 2 , 1 ' , 2 '-tetrahydro-ijj,i}j-caroten-4-one

C 40HmO

“ Bacterioerythrin (3” C^H ^O 1 ,2-Dihydro-ili,il/-caroten-l-ol 1,2-Dihydro-1-OH-lycopene 1-Hydroxy-1,2-dihydrolycopene Rhodopin —* Chloroxanthin

1',2'-D ihydro-l'hydroxychlorobactene

Ol

r , 2 '-Dihydro-caroten-4'-one —►Myxol-2'-0-methylmethylpentoside

12

2

C41 H620

Q 4H80O ,2

C 52H 760

C4 ,H 540

|

^

I

O-CrjH^p^

570.94 0^3

Nl

oh

921.22

893.17

578.88

,

Volume I: Fat-Soluble Pigments 73

Peridinin

Pentaxanthin

Pectenoxanthin

5 \6'-E poxy-3,5,3 '-trihydroxy6,7-didehydro-5,6,5',6'-tetrahydro- 1 0 , 1 l , 2 0 -trinor-(i,pcaroten-19',1 T-olide-3-acetate Sulcatoxanthin

—> Isofucoxanthinol

—> Alloxanthin

—> 2,2'-Diketospirilloxanthin

-> Oscillol-2,2'-di-(0-methylmethylpentoside) —> 2,2'-Diketospirilloxanthin

—> Anhydrorhodovibrin

l-Methoxy-3,4-didehydro1,2,7',8'-tetrahydro-i|>,i|icarotene 4-Methoxy-5,6dihydrolycopene” 1-Methoxy-l ,2,7',8'-tetrahydro-3,4-dehydrolycopene “ Pigment Y” Spheroidene —> Myxol-2'-0-methylmethylpentoside

All synonyms

C,t,H50O 7

C 4 ,HA()0

Sum formula

OCH3

568.92

Mol wt

Structure

W

^

H

CRC Handbook of Chromatography: Plant Pigments

Peridinin

2,2'-Diketospirilloxanthin P-518 2,2'-Diketospirilloxanthin P-518 Alloxanthin Cynthiaxanthin Isofucoxanthinol

“ P-512“

P-518

P-496

P-496

“ P-481”

Myxol-2'-0-methylmethylpentoside P-476 Anhydrorhodovibrin

P-476

“ P-481”

“ P-450” Spheroidene

Names mentioned in Tables 1.5 through I. LC

“ P-450”

Pigment

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC)

74

Phleixanthophyll

Dehydroadonirubin 4-Keto-a-carotene Dehydroadonirubin 4,4'-Diketo-3-hydroxy-(3carotene 3-Hydroxy-4,4'-diketo-(3carotene

Physalien

a-Cryptoxanthin Cryptoxanthin (3-Cryptoxanthin

Phleixanthophyll

Phoeniconone Phoenicopterone Phoenicoxanthin*

Physalien

Physoxanthin*

Philosamiaxanthin

-» a-Cryptoxanthin* —> (3-Cryptoxanthin

(3/?,3'fl)-(3,0-Carotene-3,3’diol dipalmitate Zeaxanthin dipalmitate

r-((3,D-Glucopyranosyloxy)3' ,4 '-didehydro-1' ,2'-dihydro(3,i|/-caroten-2'-ol -» Dehydroadonirubin —» 4-Keto-a-carotene —» Dehydroadonirubin —> 3-Hydroxy-4,4'-diketo-pcarotene

“ 2,2'-Diketobacterioruberin” 1, l'-Dihydroxy-2,2'-diketoPhillipsiaxanthin 1,2, l',2'-tetrahydro-3,4,3',4'tetradehydrolycopene 1,1 -Dihydroxy-3,4,3 ,4'-tetradehydro-1 , 2 , 1 ', 2 '-tetrahydroiJ;,i|j-carotene-2,2'-dione “ 2,2' -Diketobacterioruberin ’’ 3-Hydroxy-3'-keto-a-carotene —> 3-Hydroxy-3'-keto-aPhilosamiaxanthin carotene

Phillipsiaxanthin

5',6'-Epoxy-3,5,3'-trihydroxy6,7-didehydro-5,6,5',6'-tetrahydro-10,1 l,20-trinor-(3,(3caroten-19', 1 l'-olide

Peridininol

Peridininol

C 7;Hll60 4

C46H660 7

C40H52O4

C,vH480 6

ho

C H jjC H ^co

1045.71

ft

OH

L

731.02

n

596.85

588.78

/

Volume I: Fat-Soluble Pigments 75

OH-Phytofluene Phytofluenol Pigment R Spheroidenone (3-Zeacarotene (3,-Zeacarotene “ P-450” Spheroidene Plectaniaxanthin

Phytofluenol

Poly-cis-y-carotene

Plectaniaxanthin

“ Pigment Y”

“ Pigment X”

(3,iJ/-Carotene y-Carotene Pro-y-carotene

Phytofluene

Phytofluene

Pigment R

Phytoene

Names mentioned in Tables 1.5 through I. LC

Phytoene

Pigment

y-Carotene

3',4'-didehydro-l',2'-dihydro(3, i|/-carotene-l', 2'-dihydroxyl ', 2 '-dihydrotorulene

“ P-450”

—> (3-Zeacarotene

—> Spheroidenone

Dehydrogenans-phytoene 15,15'-Dehydrolycopersene “ Hexadecahydrolycopene” 15-ds-7,8,l 1,12,7',8',11',12'Octahydro-v|/,i|/-carotene 7,8,11,12,7',8',I T , 12'Octahydrolycopene “ Decahydro-(3-carotene” Dehydrogenans-Phytofluene 1 1 , 1 2-Dehydrophytoene Dodecahydrolycopene 15-cis-7,8,l l,12,7',8'-hexahydro-ilM(;-carotene 7,8,11,12,7',8'hexahydrolycopene “ Octahydrolycopene” —» OH-Phytofluene

All synonyms

Sum formula

568.88

542.93

544.95

Mol wt

'

\

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC)

/= \

/

Structure

76 CRC Handbook of Chromatography: Plant Pigments

Pyrenoxanthin*

Pseudo-a-carotene

“ Protetrahydrolycopene”

Proneurosporene

Prolycopene

“ Pro-y-carotene”

Polycislycopene

“ Poly-cis-i|/-carotene”

“ Trollein”

“ Flavorhodin” Neurosporene Proneurosporene “ Protetrahydroly copene ” ‘‘Tetrahydrolycopene” “ 5,6,5', 6 'tetrahydrolycopene ’’ Lycopene Poly-cislycopene Pro lycopene Rhodopurpurin p, ^-Carotene y-Carotene Pro-y-carotene Lycopene Poly-c/s-lycopene Prolycopene Rhodopurpurin “ Flavorhodin” Neurosporene Proneurosporene “ Protetrahydrolycopene’ ’ ‘ ‘Tetrahydrolycopene” “ 5, 6 ,5 ', 6 'Tetrahydrolycopene” “ Flavorhodin” Neurosporene Proneurosporene “ Protetrahydrolycopene” ‘‘Tetrahydrolycopene” “ 5, 6 ,5 ', 6 'Tetrahydrolycopene” (3-Carotene (3, (3-Carotene —>“ Trollein” * (3,e-Carotene-3,20,3'-triol

—>(3-Carotene

—>Neurosporene

—►Neurosporene

—» Lycopene

—►y-Carotene

—►Lycopene

—» Neurosporene

HO

C40H56O,584.88

Volume I: Fat-Soluble Pigments 77

Pyrrhoxanthinol

Renierapurpurin

Renieratene

Reticulataxanthin

(3-Apo-12'-carotenoic acid 3,4,3',4'-Bisdehydro-pcarotene “ Dehydro-(3-carotene” Dehydro-retro-(3-carotene Retrodehydrocarotene Retrodehydro-(3-carotene “ Dehydro-(3-carotene” Dehydro-retro-p-carotene Retrodehydrocarotene Retrodehydro-P-carotene

Pyrrhoxanthinol

Renierapurpurin

Renieratene

Reticulataxanthin

Retinylidenetiglic acid Retrobisdehydro-p-carotene

Retrodehydro-(3-carotene

Retrodehydrocarotene

Pyrrhoxanthin

Names mentioned in Tables 1.5 through I. LC

Pyrrhoxanthin

Pigment

Sum formula

—> “ Dehydro-p-carotene”

3-Hydroxycitranaxanthin (3/?)-3-Hydroxy-5',6'-dihydro5'-apo-18'-nor-P-caroten-6'one —> P-Apo-12'-carotenoic acid —> 3,4,3',4'-Bisdehydro-(3carotene —> “ Dehydro-(3-carotene”

Aphanizophyll

Eschscholtzxanthin Myxoxanthophyll

“ Retrodehydrozeaxanthin” 2'-((3,L-Rhamnopyranosyloxy-)3' ,4'-didehydro- 1 ' , 2 'dihydro-(3,i|/-carotene-3, 1 'diol 2'-((3,L-Rhamnopyranosyloxy-)3 ' ,4' -didehydro-1', 2' ■dihydro-(3 ,vJ/-carotene3,4,l'-triol 2'-0-Rhamnosylmyxol Rhodoauranxanthin Rhodopin

Rhodopurpurin

Didehydroretro-y-carotene 4 ',5 '-Didehydro-4,5 '-retro-(3, iJjcarotene —> Eschscholtzxanthin —> Myxoxanthophyll

Retrodehydro-y-carotene

Retrodehydro-y-carotene

C42H60O 2

C4,H(S Semi-p-carotenone

Semi-P-carotenone

5,6-Seco-(3,(3-carotene-5,6dione (6'/?)-5,6-Seco-p,e-carotene5,6-dione

(2/?,6S,2'/?,6'S)-2,2'-Bis-(4-hydroxy-3-methyl-2-butenyl)7,7-CarOtene 2'-(4-Hydroxy-3-methyl-2-butenyl-)2-(3-methyl-2-butenyl)t.t-caroten-18-ol (2/?,6ft,2'/?,6'7?)-2,2'-Bis-(4hydroxy-3-methyl-2-butenyl)e, e-carotene Decaprenoxanthin Dehydrogenans-P-439

Sarcinaxanthin

Sarcinaxanthin*

C „H 420

C40H56O2

C40H56O2

C50H72O2

1

1

430.67

568.88

568.88

ho^ c^

^

705.12 X I

^

ll J

^ ^

Volume I: Fat-Soluble Pigments 81

All synonyms

Pigment R Spheroidenone

Rhodoviolascin Spirilloxanthin

“ Spirillotoxin”

“ 5,6-Dihydro-4-methoxy-lycopen-6-one“ l-Methoxy-3,4-didehydro1,2,7',8'-tetrahydro-if/,i|/-caroten-2-one “ l-Methoxy-2-keto-7',8'-dihydro-3,4-dehydrolycopene ’’ Pigment R —> Rhodoviolascin

3 ,19,3'-Trihydroxy-7,8-dihydro-(3,e-caroten-8-one 3,3', 19-Trihydroxy-7,8-dihydro-8-oxo-a-carotene “ Xanthophyll K,S“ Siphonaxanthin-monolaurate —> Siphonein Siphonein “ Xanthophyll K ,” Siphonaxanthin-monolaurate Siphonaxanthin-monolaurate “ Xanthophyll K ,” 3 ,19,3'-Trihydroxy-7,8-dihySiphonein dro-(3,e-caroten-8-one-19laurate “ Xanthophyll K ,“ (3,ijj-Carotene —> y-Carotene y-Carotene Pro-y-carotene “ P-450” —> “ P-450” Spheroidene

Siphonaxanthin “ Xanthophyll K,S”

Names mentioned in Tables 1.5 through I. LC

Spheroidenone

Spheroidene

“ Sphaerobolin”

Siphonein

Siphonaxanthin-monolaurate

Siphonaxanthin

Pigment

C4lH,80 2

C52H7KOs

C4()Hv,0 4

Sum formula

582.91 OCHj

783.19

600.88

Mol wt

I

I

Ct-^OH

I

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC) Structure

82 CRC Handbook of Chromatography: Plant Pigments

—> Alloxanthin

Alloxanthin Cynthiaxanthin 7 ',8 ', 11', 12'-Tetrahydro-ycarotene

—> 7 ',8 ',1 1 \ 12'-Tetrahydro-ycarotene

—> Nostoxanthin

Nostoxanthin

—» Alloxanthin —» ‘‘Dehydrolycopene”

3,4,3',4'-Bisdehydro-(3carotene ‘‘Dehydrolycopene”

3,4,3',4'-Tetradehydro-(3,(3carotene 3,4,3',4'-Tetradehydro-i|M(jcarotene (3/?,3'/?-)7,8,7',8'-Tetradehydro-(3,(3-carotene-3,3'-diol 3,4,3',4'Tetradehydrolycopene 6,7,6',7'-Tetradehydro5,6,5',6'-tetrahydro-(3,(3carotene-3,3'-diol 7,8,7',8'Tetradehydrozeaxanthin 7 \8 \1 1', 12'-Tetrahydro-(3,iJ/carotene

—> Asterinic acid 3,3'-Dihydroxy-7,8,7',8'tetradehydro-(3,(3-carotene4,4'-dione 4,4'-Diketocynthiaxanthin Diketotetradehydrozeaxanthin -» 3,4,3',4'-Bisdehydro-(3carotene —> ” Dehydrolycopene”

—> Luteinepoxide

—» Peridinin —» Luteinepoxide

—» Rhodoviolascin

Alloxanthin Cynthiaxanthin ‘‘Dehydrolycopene”

Part of asterinic acid

Rhodoviolascin Spirilloxanthin Peridinin Isolutein Luteinepoxide Lutein-5,6-epoxide Taraxanthin Xanthophyllepoxide Isolutein Luteinepoxide Lutein-5,6-epoxide Taraxanthin Xanthophyllepoxide

7,8,7',8'Tetradehydroastaxanthin

Tareoxanthin

Sulcatoxanthin Taraxanthin

Spirilloxanthin

C40H48O4

592.88

Volume I: Fat-Soluble Pigments 83

l,2 ,r,2 '-T e tra h y d ro -l,l'dihydroxy-lycopene

“ Dihydroxylycopene” 1,2, l',2'-T etrahydro-l, 1'dihydroxy-lycopene

Asym. ^-carotene 7,8,11,12-Tetrahydro-i|/,ijicarotene 7,8,11,12Tetrahydrolycopene 7,8,7',8'-Tetrahydro-i|i,v|t^-Carotene carotene 7,8,7', 8' -T etrahy dro-vjt,v|/carotene 1,2,l',2'-Tetrahydro-i|M};-car- “ Dihydroxy lycopene” otene-1 ,l'-diol 1,2, l',2'-T etrahydro-l, 1'dihydroxylycopene 1,2,7',8'-Tetrahydro-4,4-car- Chloroxanthin oten-l-ol OH-Neurosporene 7,8,7',8'-Tetrahydro-4,4'4,4'-Diapo-£-carotene diapocarotene 7.8.11.12- Tetrahydro-4,4'4,4'-D iapo-7,8,11,12diapocarotene tetrahydrolycopene

7 ',8 ',1 1 \ 12'-Tetrahydro-ycarotene

7',8',1 r,12'-Tetrahydro--ycarotene

7.8.11.12- Tetrahydro-if/,iftcarotene

Names mentioned in Tables 1.5 through I. LC

Pigment

“ Dihydroxylycopene” l,l'-D ih y d ro x y -l,2 ,r,2 'tetrahydrolycopene OH-Rhodopin 1,2, r,2'-Tetrahydro-iji,il;-carotene-1,1 '-diol 1,2,1' ,2'-Tetrahydrolycopene1,1 '-diol

—> 4,4'-D iapo-7,8,11,12tetrahydrolycopene

—» 4,4'-Diapo-£-carotene

—» Chloroxanthin

—» 1,2, l',2'-T etrahydro-l, 1'dihydroxylycopene

—> ^-Carotene

—» Asym.^-carotene

7 ',8 ',1 1', 12'-TetrahydroB,^-carotene

All synonyms

C^H ^O ,

C^H*,

Sum formula

572.91

540.91

Mol wt

Structure

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC)

.

84 CRC Handbook of Chromatography: Plant Pigments

“ Flavorhodin” Neurosporene Proneurosporene “ Protetrahydrolycopene ” ‘‘T etrahy droly copene ’’ “ 5 ,6 ,5 ',6 'Tetrahydrolycopene’’ “ 5,6,5',6'-Tetra“ Flavorhodin” hydrolycopene” Neurosporene Proneurosporene ‘ ‘Protetrahydrolycopene “ Tetrahydrolycopene’ ’ “ 5 ,6 ,5 ',6 'Tetrahy droly copene ” 7,8,7', 8 '-Tetrahydrolycopene ^-Carotene 7,8,7',8'-Tetrahydro-iK4>carotene 7,8,11,12-TetraAsym. ^-carotene hydrolycopene 7 ,8 ,1 1 ,12-Tetrahydro-i}j,il/carotene 7.8.11.12Tetrahydrolycopene “ 7 ',8 ',1 l',1 2 'Asym. ^-carotene Tetrahydrolycopene” 7 ,8 ,1 1 ,12-Tetrahydro-i|i,i};carotene 7.8.11.12Tetrahydrolycopene 1,2, l',2'-Tetrahydrolyco“ Dihydroxylycopene” pene-1,1 '-diol 1,2, l',2'-T etrahydro-l, 1'dihydroxylycopene 3,5,3',5'-Tetrahydroxy-6',7'- Isofucoxanthinol didehydro-5,8,5', 6 ' -tetrahydro-(3, p-caroten-8-one 3,5,3', 5' -Tetrahydroxy-6', 7' - Isofucoxanthin didehydro-5,8 ,5 ',6 '-tetrahydro-f3, (3-caroten-8-one-3acetate 3,4,3',4'-Tetraketo-(3Astacene carotene “ 4,5,4',5'-Tetraketo-(3Astacene carotene”

“ Tetrahydrolycopene”

—* Astacene

—►Astacene

-> Isofucoxanthin

—>Isofucoxanthinol

—>l,2 ,r,2 '-T etrah y d ro -l,T dihydroxylycopene

Asym. ^-carotene

-> Asym. ^-carotene

—►^-Carotene

Neurosporene

—> Neurosporene

Volume I: Fat-Soluble Pigments 85

Thiothece-460

Thiothece-414

Thiothece-414

Thiothece-4$4

1-Methoxy-l'-hydroxy1,2, r,2'-tetrahydro-i|i,i|/caroten-4-one Thiothece-OH-484

Torularhodin

Torularhodin-aldehyde

Thiothece-414

“ Thiothece-41%"

Thiothece-4%4

Thiothece-OH-4M

Torularhodin

Torularhodin-aldehyde

Names mentioned in Tables 1.5 through I. LC

Thiothece-460

Pigment

"3',4'-Dehydro-17'-oxo-ycarotene" "3,4-Dehydro-1 8 -oxo-ycarotene"

“ 16'-Carboxyl-3',4'-dehydroy-carotene" 3',4'-Didehydro-(3,iJj-caroten16'-oic acid "Lusom ycin"? *Torulene-carboxylic( 16'-)acid"

—» 1-Methoxy-l'-hydroxy1,2, l',2'-tetrahydro-ijMj/-caroten-4-one

Methyl-l'-methoxy-4'-oxor,2'-dihydro-x,^-caroten-16 (or 17 or 18)-oate

r-M ethoxy-l',2'-dihydro-p,i|icaroten-4'-one l'-Methoxy-l',2'-dihydro-tp,i|icaroten-4'-one " Thiothece-41%" —» Thiothece-414

l-Methoxy-4-oxo-l ,2-dihydro8'-apo->|/-caro(en-8'-al

All synonyms

C4()Hv O

C40H52O,

582.91

462.67

Mol wt

548.85

564.85

C42HS40 4

C4IH sX0 2

C,,H420 ,

Sum formula

R.

622.8

OCH3 \l

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC) Structure

3

86 CRC Handbook of Chromatography: Plant Pigments

Torulene

Torularhodin

Torulene

“ Torulene-carboxylic(16'-)acid Trihydroxy-a-carotene 3,19,3'-Trihydroxy-7,8-dihydro-(3,e-caroten-8-one 3,19,3'-Trihydroxy-7,8-dihydro-(3,€-caroten-8-one-19laurate 3 ,3 ',5 '-Trihydroxy-5', 6 '-dihydro-5 ',6'-epoxy-(3carotene 3,19,3'-Trihydroxy-7,8-dihydro-8 -oxo-a-carotene 3 ,3 ', 6 ' -Trihydroxy-5, 6 epoxy-a-carotene 3,3',6'-Trihydroxy-5,8epoxy-a-carotene 3, 8 ,3'-Trihydroxy-5,6 -epoxy(3-carotene “ 3,3',5'-Trihydroxy-6'-hydro-7,8 -dehydro-(3carotene” 1,l',2'-T rihydroxy-3,4,3',4'tetradehydro-1 , 2 , 1 ' , 2 '-tetrahydro-i|;,iJ/-caroten-2-one 3,4,4'-Triketo-(3-carotene Trollichrome Heteroxanthin —» Heteroxanthin

—> 2'-Dihydrophillipsiaxanthin —> Dehydroadonirubin

Heteroxanthin

Heteroxanthin

2'-Dihydrophillipsiaxanthin

Dehydroadonirubin

-> Trollixanthin

-^Siphonaxanthin

-► Neoxanthin

—> Siphonein

— -^Siphonaxanthin

', 4 '-Dehydro-y-carotene 3',4'-Didehydro-p, ^-carotene “ 3 , 3 '-Dimethoxy-7 -carotene” “ 3,3'-Dimethoxy-3',4'-dehydro-7 -carotene” —> Torularhodin 3

Trollichrome

Siphonaxanthin “ Xanthophyll K ,S“ Trollixanthin

Trihydroxy-a-carotene Siphonaxanthin “ Xanthophyll K ,S” Siphonaxanthin-monolaurate Siphonein “ Xanthophyll K ,“ Neoxanthin Trollixanthin

Torularhodin-methylester

Torularhodin-methylester

3',4'-Didehydro-p,i|j-caroten16'-al 16'-Oxotorulene Torulenal Methyl-3',4'-didehydro-(3,ii/caroten-16' -oate C40H54

C 41H 540 2

534.87

578.88 ^

Volume /: Fat-Soluble Pigments 87

Deepoxyneoxanthin

Trollichrome

Neochrome Trollichrome Neoxanthin Trollixanthin

Trollixanthin

(3-Zeacarotene (3,-Zeacarotene Heteroxanthin

“ Trollein” *

Trollichrome*

Trolliflavin

Trollixanthin

“ Unidentified II”

“ Vaucheria-Heteroxanthin”

Trolliflor

Triphasiaxanthin

Names mentioned in Tables 1.5 through I. LC

Triphasiaxanthin

Pigment

Sum formula

—» Heteroxanthin

5,6-Epoxy-5,6-dihydro-(3,e-carotene-3,3',6'-triol —» (3-Zeacarotene

a -c a r o te n e

3 ,3 ',6 /-Trihydroxy-5,6-epoxy-

C40H56O4

6'-Oxychrysanthemaxanthin C40H56O4 3,3',6'-Trihydroxy-5,8-epoxya-carotene 5.8Epoxy-5,8-dihydro-(3,e-carotene-3,3',6'-triol —> Neochrome —■» Neochrome —» Neoxanthin

(3, e-Carotene-3,19,3'-triol C40H56O3 Deepoxyneoxanthin 6.7Didehydro-5,6-dihydro(3,(3-carotene-3,5,3'-triol 19-Hydroxy-lutein Loroxanthin Pyrenoxanthin*

3'--Hydroxy-5,6-seco-(3,(3-carotene-5,6-dione 3-Hydroxy-semi-p-carotenone

All synonyms

600.88

600.88

584.88

584.88

Mol wt

HO

ho-

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC) Structure

88 CRC Handbook of Chromatography: Plant Pigments

Violaxanthin 9-ds-Violaxanthin Violeoxanthin

Violaxanthin Violeoxanthin 9-ds-Violaxanthin 13-ds-Violaxanthin Violaxanthin 9-ds-Violaxanthin Violeoxanthin

Violaxanthin

9-ds-Violaxanthin

3-Hydroxy-3'-hydroxy-acarotene Lutein “ Xanthophyll” Siphonaxanthinmonolaurate Siphonein Siphonaxanthin Helenien Lutein dipalmitate Isolutein Luteinepoxide Lutein-5,6-epoxide Taraxanthin Xanthophyllepoxide

“ Xanthophyll”

Xanthophyllepoxide

“ Xanthophyl K,S” Xanthophyll dipalmitate

“ Xanthophyll K ,”

Violerythrin

Violerythrin

13-ds-Violaxanthin Violeoxanthin

Vaucheriaxanthin

Vaucheriaxanthin

—» Siphonaxanthin —►Helenien

—» Siphonein

—» Lutein

2,2'-Dinor-(3,(3-carotene3,4,3\4'-tetrone

—» Violaxanthin

9-ds-Violaxanthin Violeoxanthin —> Violaxanthin

(3S,5R,6S,3'S,5'R,6'S)5,6,5',6'-Diepoxy-5,6,5',6'tetrahydro-(3,(3-carotene-3,3'-

5', 6'-Epoxy-6,7-didehydro5,6,5',6'-tetrahydro-(3,(3-carotene-3,5,19',3'-tetrol

C4oH560 4

C^H^Os

564.76

600.88

616.88

o h

ho

Volume I: Fat-Soluble Pigments 89

a-Zeacarotene

(3-Zeacarotene (3,-Zeacarotene

(3-Zeacarotene (3,-Zeacarotene

Zeaxanthin Zeaxanthin

Physalien Antheraxanthin Mutatoxanthin Mutatoxanthin Zeaxanthin a-Cryptoxanthin Zeinoxanthin

(3-Zeacarotene

(3,-Zeacarotene

Zeaxanthene Zeaxanthin

Zeaxanthin dipalmitate Zeaxanthin-5,6-epoxide Zeaxanthin-5,8-epoxide Zeaxanthinfuranoide “ Zeaxanthol” Zeinoxanthin

Names mentioned in Tables 1.5 through L. LC

a-Zeacarotene

Pigment

—> Zeaxanthin Anchovyxanthin (3/?,3'/?)-(3,(3-Carotene-3,3'dioi “ Zeaxanthene” “ Zeaxanthol” -» Physalien -> Antheraxanthin —» Mutatoxanthin -> Mutatoxanthin —> Zeaxanthin —> a-Cryptoxanthin

“ Carotene X ” ? “ Pigment X” ? -7I Ql .. .. , Q 7 ,8 -Dihydro-(3,iJ/-carotene 7',8'-Dihydro-y-carotene (3,-Zeacarotene (3-Zeacarotene

7',8'-Dihydro-8-carotene (6/?)-7',8'-Dihydro-e,i|;carotene

All synonyms

C40H56O,

C40H58

C4IJHW

Sum formula

568.88

538.90

538.90

I

I

Mol wt

I

v

^

Table 1.3 (continued) NAME LIST FOR CAROTENOIDS (TABLES 1.5, 1.6, I. PC, I. TLC, I. LC) Structure

H

T

90 CRC Handbook of Chromatography: Plant Pigments

Volume I: Fat-Soluble Pigments Table 1.4 NAME LIST FOR CAROTENOIDS (HPLC AND GC TABLES) Antheraxanthin Auroxanthin epimer 1 Auroxanthin epimer 2 Bacterioruberin Bacterioruberin neo A Bacterioruberin neo U Bacterioruberin neo V Bacterioruberin neo W 6-But-2-enylidene-l ,5,5-trimethyl-cyclo-hex-1-ene Canthaxanthin a-Carotene (3-Carotene (3-Carotene, cis isomer 1 (3-Carotene, cis isomer 2 (Z-)(3-Carotene (3, (3-Carotene (3,e-Carotene (3,i|/-Carotene (Z)-(3,iJ/-Carotene y-Carotene e,v|;-Carotene (Z-)e,iJ/-Carotene ^-Carotene (Z-)£-Carotene (3-Citraurin Cryptoxanthin all-(E-)(3-Cryptoxanthin (Z-)(3-Cryptoxanthin Cryptoxanthinester 3,4-Dehydrorhodopin Diadinoxanthin Diatoxanthin Dinoxanthin 2,2'-Diol Echinenone Fucoxanthin (3-Ionone Lutein Lutein neo A Lutein neo B Lutein neo U Lutein neo V Lutein epoxide Lutein-5,6-epoxide Lutein-3'-ether epimer 1 Lutein-3'-ether epimer 2 Lycopene

all-(E-)Lycopene Lycopersene (8/?-)Mutatoxanthin (85- )Mutatoxanthin Neochrome Neochrome epimer 1 Neochrome epimer 2 Neofucoxanthin A Neotucoxanthin B Neolutein A Neolutein B Neoperidinin Neoxanthin Neoxanthin neo A Neoxanthin X Neurosporene all-(£-)Neurosporene (5Z-)Neurosporene Okenone Peridinin Phytoene Phytofluene all-(£-)Phytofluene Phytol Rhodopin all-(£-)rubixanthin (5'Z-)Rubixanthin (Gazaniaxanthin) (9'Z-)Rubixanthin (13Z-)Rubixanthin ( 13'Z-)Rubixanthin (5'Z, 13Z-) or (5'Z, 13'Z-)Rubixanthin Spheroidenone Spirilloxanthin Squalene Tetrahydrospirilloxanthin 3,5,6,3'-Tetrol Tetrofuranoxyd Torulene Triacontane Violaxanthin Violacanthin-9-m Violaxanthin-13-m Violeoxanthin Xanthophyllester Zeaxanthin (9Z-)Zeaxanthin (13Z-)Zeaxanthin

91

92

CRC Handbook of Chromatography: Plant Pigments Table 1.4 (continued) NAME LIST FOR CAROTENOIDS (HPLC AND GC TABLES) Hydrogenated Carotenoids

H2-(3-apo-4'-carotenal H2-(3-apo- lO'-carotenal H2-(3-apo-8'-carotenoic acid H2-(3-apo-8'-carotenoic acid ethyl ester H2-(3-apo-8'-carotenoic acid methyl ester H2-(3-apo-8'-carotenal H2-astacene H2-azafrin H2-bixin H:-canthaxanthin H2-capsanthin H2-a-carotene H2-P-carotene H:-y-carotene H2-£-carotene H2-P-carotenone H2-carotinin H2-crocetin H2-cryptoxanthin H2-cryptoxanthin, Ac H2-cryptoxanthin, TMS H2-decapreno-P-carotene H2-3,4-dehydro-P-apo-8'-carotenal H2-dehydro-(3-carotene H:-4,4'-diapo-£-carotene H2-4,4'-diaponeurosporene H2-4,4'-diaponeurosporen-4-oate methylester H2-4,4'-diaponeurosporen-4-oic acid H2-4,4'-diapophytoene H2-4,4'-diapophytofluene H:-diethylcrocetin H2-dihydrosqualene H2-dimethoxyisozeaxanthin

H2-dimethoxyzeaxanthin H2-dimethylcrocetin H2-echinenone H2-fucoxanthin H2-4-hydroxy-4,4'-diaponeurosporene H2-isocryptoxanthin H2-isocryptoxanthin, Ac H2-isocryptoxanthin, TMS H2-isozeaxanthin H2-isozeaxanthin, diAc H2-isozeaxanthin, diTMS H2-lycopene H2-lycopersene H2-methylazafrin H2-methylbixin H2-neo-a-carotene H2-neo-P-carotene H2-neurosporene H2-physalien; C4()-fragment H2-phytoene H2-phytofluene H2-phytol H2-retinaldehyde H2-retinol H2-rubixanthin H2-squalene H2-tetrahydrosqualene H2-torularhodin H2-(3-zeacarotene H2-zeaxanthin H2-zeaxanthin, diAc H2-zeaxanthin, diTMS

Volume I: Fat-Soluble Pigments

93

Table 1.5 QUALITATIVE SPECTROSCOPIC DATA: ABSORPTION MAXIMA IN DIFFERENT SOLVENTS

NOTES In Table 1.5, the pigment names have been listed alphabetically (first vertical row). Since a number of carotenoids have been named differently by different authors, the same pigment may appear under two or even more names. If the desired pigment is not found, refer to row 1 (vertical) of the name list (Table 1.3). Second and following rows: literature references (superscripted) refer to absorption maxima (usually three) given in each row. Absorption maxima in parentheses indicate shoulders. “ Benzine” (solvent 3) is a mixture of hydrocarbons similar to petroleum ether (solvent 10), but in the literature (Karrer and Juckerla and Frye14) is nonspecified. Row 10, “ petroleum ether” , combines solvents with a boiling range of approximately 40 to 80°C (that is, it combines values recorded in light petroleum, bp 60—70 and 70— 80°C).

—

—

—

—

444, 482, 5169

—

493, 522.5s

(480), 500, (431)6 460, 485, 5208

Alloxanthin (trans)

Anhydrodeoxyflexixanthin (trans) Anhydroeschscholtzxanthin Anhydrorhodovibrin

365, 450, 476,

Aphanizophyll (trans)

50721

—

Aphanicin Aphanizophyll

—

—

—

—

Antheraxanthin (trans)

Antheraxanthin (cis)

460, 488, 52012 — 433, 457, 48813 — 457, 48716

Anhydrosaproxanthin Antheraxanthin

434, 464, 496la —

_

—

—

—

—

—

—

—

—

—

—

—

Alloxanthin

Actinioerythrol

—

Solvent 3 “ Benzine”

(480), 508, (538) —

Solvent 2 Benzene

Actinioerythrin

Pigment

Solvent 1 Acetone

494, 53316 454, 484, 51816 —

—

445, 478, 510 14 376, 50616

503, 539, 5787 —

—

454, 488, 518la —

495,533, 574* —

Solvent 4 Carbon disulfide

474, 50416 453, 482, 51020 —

—

430, 456, 48415 —

484, 516, 5497 —

—

(490), 518, (550)* (436), 460, 4892 —

518,(550)*

Solvent 5 Chloroform

— —

—

—

474, 503, 5387 358, 374, 455,483, 51710 — —

—

—

—

—

—

Solvent 6 Cyclohexane

—

— —

—

—

—

—

_

—

__

375, 455, 483, 5 17*1

__ __ __

__

__ __ __

__

350, 352, 446 . 472, 50221

—

__ —

462, 49416 —

__

__ —

— 424, 442, 47019 __

—

__

__

__

474, 496, 5296

—

__ __

__

__

__ __

__

489.5, 519.5s 490, (516)s

__

__

—

—

__

__

—

—

Solvent 12 Pyridine

—

Solvent 11 Methanol

4804 __

458, 488la

__

(470), 496, 529>

Solvent 10 Petroleum ether

451,(467),

—

__

__ —

_

Solvent 9 Hexane

_

Solvent 8 Ether

46917 4 2 1 , 4 4 3 , — 47318 — __ 445, 472, — 50220 _

— 422, 444, 4721S 419 , 443,

—

—

—

(427), 450, 4782 451, 4803

—

—

Solvent 7 Ethanol

Table 1.5 QUALITATIVE SPECTROSCOPIC DATA: ABSORPTION MAXIMA IN DIFFERENT SOLVENTS

94 CRC Handbook of Chromatography: Plant Pigments

—

—

—

—

—

—

Astaxanthin-monoester (cis) ’

Astaxanthin-monoester (trans)

Asterinic acid

Aurochrome

Auroxanthin

Auroxanthin (cis)

48822

(448), 47326

Apo-6'-lycopenal

47026

—

—

Apo-3-lycopenal

— — — — —

— — — —

— — — —

Apo-8'-lycopenal

—

—

Astacene Astaxanthin Astaxanthin-diacetate Astaxanthin-diester (cis) Astaxanthin-diester (trans)

—

—

—

—

44014

—

—

—

— 48519 47831 — —

488, 518la

— — — — — —

— — — — — 473, (496)25

p-Apo-4-carotenal (3-Apo-2'-carotenal (J-Apo-8' -carotenal P-Apo-lO'-carotenal (3-Apo-12'-carotenal (3-Apo-4'-carotenoic acid (3-Apo-8'-carotenoic acid P-Apo-lO'-carotenoic acid P-Apo-2'-carotenol Apo-8' -carotenol p-Apo-lO'-carotenol Apo-2-lycopenal

—

_

—

—

—

—

— — — — —

—

—

— — — 455.5, 490.5, 525.5 la 474, 502la

—

—

— — — — — —

__ __

__ — 47230 __ __ __

__

__

510(ca.)la 50330

__ __

335,385, 41033 __

45133 __

42814

54129 428, 45714 401,432,

—

(480), 518,

__ __ __

__

__

__

—

__

—

42518

42814 375 400

382,403,

__

—

__

—

__

42333

__

__

379,399, 425.5a>34

379.5,401,

__

43714

__

—

__

__ __

__ __

47 832

__

__

__

__

—

__

52229

(478), 495,

__

__

__ __ __ __ __ __ __ __

47132

__

__ __

__ 500 (broad)13 49329

__ __ __ __

__ __ __ __ __ __ __ __

47026

__

__

__ __ __ __

__

— 47230

__

—

(515)a-26 46826 46728

__

47726

(462), 479,

__

—

47827 __ 47431 47132 47 832

__

__

— __ __

__

__ __

__

__

__

280, 449,

__ 475, 50510

__

__ __ __ __

__ __ __ __

47322 42622 40322

__ __ __ __

__ __ __ __

__

42524

__

__

45024

__

__

470, 49525

__

—

__ __ __

__ __

__ __

—

__

454, 484la 4 3 5 23 41024 477, 50525

__ __

__ __

__ __ __ __

__ __ __ 469, 493.5 , 528.5 la 478, 508, 545la __

__

ca. 442la

__

__ __ __

_

98la

447^ __ __ __

49822 45722 43 522

__

__ __ __ __

__

__

__

__ __ __

—

__ __

__ __

__

__

__

490, 525la — __ —

__ __

__

__ __ — __ __ __

460 (ca.)la

__

Volume I: Fat-Soluble Pigments 95

485, 51840

483, 514la —

—

—

Capsanthin-5,6-epoxide — Capsanthin-5,6-epoxide----diester

—

— —

—

424, 448, 47641 —

Capsanthin-diester

Capsanthin-monoester

Capsochrome Capsorubin

Capsombin-diester

a-Carotene

464, 496,a 455, 486, 520la 457, 488, 52240

— — 486, 520la

— — —

Canthaxanthin Canthaxanthin (cis) Capsanthin

— 378,398, 481,511, 54935 —

—

— 374,389,466, 499, 533.535

Azafrin Bacterioruberin a

—

Solvent 2 Benzene

3,4,3',4'-Bisdehydro-p ----carotene Caloxanthin —

—

Auroxanthin (trans)

Pigment

Solvent 1 Acetone

447.5, 47814

— 444, 474, 506,a —

—

— —

—

— — 475,505la

—

—

— —

—

Solvent 3 “ Benzine”

450,475, 50537

482, 515la 468, 503, 541.5 la —

—

499, 534la —

—

50037 — 503, 542la

—

—

— 418,500.5, 533.5, 57235

__

Solvent 4 Carbon disulfide

432,457, 48537

—

462, 492la —

—

481, 511la —

—

(432), 458, 48420 48320 — —

428, 458,a 380,397, 475,506, 54435 —

__

Solvent 5 Chloroform

—

—

— —

—

— —

423,444, 4732

48540

— —

(420), 443, 47242

—

__

—

422,445, 47537

50440 — 444, 474? 50616 452, 477, 5 iq 40

__

—

__

__

421,445, 47443

—

__

—

__

__

—

__

__

_

__ __

47440

(450), 474,

_

__

__ 473 50240

__

—

__

__

_

__

—

_

__

— 47340

__

_

__

—

_

__

428, 458la

__

__

—

Solvent 12 Pyridine

Solvent 11 M ethanol

50340 —

__ __

47440

__

50440 (450), 474,

—

47240

(450), 474,

__

__

—

369,385, 461,494, 52835 4 7 123

__

Solvent 10 Petroleum ether

4 6 5 -^ 6 7 23 355^ 46539 — ’

46237

45621

__

462, 490, 52236 —

_

__

Solvent 9 Hexane

46738 — —

—

__

— _

__

__

42818 — —

Solvent 8 E ther

3g0 402

Solvent 7 Ethanol

(426), 449, 47520 47732

—

__

— —

__

Solvent 6 Cyclohexane

Table 1.5 (continued) QUALITATIVE SPECTROSCOPIC DATA: ABSORPTION MAXIMA IN DIFFERENT SOLVENTS

96 CRC Handbook of Chromatography: Plant Pigments

—

e-Carotene —

—

—

^-Carotene

^-Carotene (equilibrium mixture)

—

0-Carotene-diepoxide

—

—

triol Carotenonaldehyde

0-Carotenone

a-Carotene-5,6-epoxide — 0-Carotene— monoepoxide P, p-Carotene-2,3,3'-

—

■ 8 -Carotene

—

—

€ -Carotene

£-Carotene (trans)

—

—

€-Carotene

8-Carotene (trans)

,

(428), 452, 47744 (432), 453, 47948

7Carotene (trans) 8-Carotene —

7 -Carotene

O,

0 ,(3-Carotene

0-Carotene

431,462, 495la

—

426,452, 483.514 —

—

—

420, 446, 476Ia 453, 486 , 522la

455, 484la —

456, 48514

—

—

—

-

-

406, 432, 461la 440, 468 , 502la

—

— —

—

—

—

—

-

(414), 434, — 458, 488.554 — —

—

440, 465, 49553 — — —

447,47 7, 5 1 9la

—

—

463, 49245

430, 459, 491la 466, 499 , 538la

—

471, 503Ia 479, 51114

472, 50214

—

—

—

470, 50114

—

—

— 457,490, 52614

463,496, 533.514

—

450,485, 52014 —

—

—

423, 450, 482la 454, 489, 527la

454, 483la 459, 49214

456, 48414

—

—

—

-

452, 48314

—

—

440,470, 50314

446,475, 508.5 14

—

_

463, 49333

—

—

—

—

— —

—

—

—

—

419,440, 47523 -

—

—

—

—

_

457, 48523

—

—

—

450, 47850

— —

—

—

—

—

-

—

—

—

— —

—

—

_

—

(442),(473)la —

—

— —

—

—

—

—

417, 440, 4692 —

—

440,460, 48951

—

426,451, 47846 _

_ 405, 431, 458la 436, 466, 500Ia

— —

—

—

378, 400, 42552

—

—

— 428,458, 49014

431,462, 49414

425,450, 47833 421,449, 477a-47

_

—

404, 430, 457la

442, 4711a (420), 442, 4704-

417, 439, 47042

377, 382, 398, 410, 42255

296, 361, 379.5, 400.5, 424.556

285, 296, 359, 377, 398, 42352

380, 387, 400, 412, 42655

267, 417, 4 4 1 ,4 7 1 54 439, 47014

431,456, 487 m

281,431, 456, 48954

(410), 435. 460, 48952

421,443, 47 349

426,451, 47733 (427), 448, 47449

—

—

-

—

—

—

—

—

—

—

-

—

—

—

—

—

—

Volume I: Fat-Soluble Pigments 97

440, 465, 49553 — — —

—

—

—

—

— — —

—

—

—

—

—

Chloroxanthin (trans)

Chrysanthemaxanthin Citranaxanthin a-Citraurin

(3-Citraurin

Corynexanthin

Crocetin (stable trans)

Crocetindialdehyde

Crocetin-di-( (3-D-gluco- — syl)-ester

—

Chlorobactene (trans) Chloroxanthin

Crocetindimethylester

Crocetin-O-D-gentiobiosyl)-(p-D-glucosyl) ester

—

—

-

467, 497la

— — —

425, 452, 482y

_

—

Chlorobactene

—

—

Solvent 2 Benzene

Celaxanthin

Pigment

Solvent 1 Acetone

_

— —

—

45 1 .4 8 0 .5 16 — 449, 480, 5141a 457,490, 525la 435,466, 49516 426,453, 482la —

—

— —

450.5'la —

_

487.521. 56214 _

Solvent 4 C arbon disulfide

424.5,

—

424.5, 450.5 la —

459, 488la

— — —

—

— —

—

Solvent 3 “ Benzine”

_

—

434.5, 463la

—

—

423.447, 47816 434.5, 463la

—

430, 459la — _

—

— __

—

_

_

__

__

—

_

—

—

—

—

—

—

—

—

_

_

__

—

400, 422, 4502>

_

__

458™

40g 43Q

__ __ __

_

__

4 2 1,443, 47,60

__

4 2 1 , 443, 47,60

__

411 436 464 la

_

_

__

—

__

__ __

—

—

Solvent 12 Pyridine

—

_

_

—

— __ _

__

__ __

—

—

Solvent 11 M ethanol

_

—

458, 487la

4 2 1 ,4 5 0 la 453^ 495-™ 438, 4771a

__

470"

4 ,7 44Q

__

491”

456,486.5, 520la 435,461,

Solvent 10 Petroleum ether

—

_

421, 45016 __

__

__ __

456.486.5, 52016 —

Solvent 9 Hexane

—

—

—

— __

__

__

491s7 __

435,461,

—

Solvent 8 Ether

415,437, 46716 —

Broadla

4 2 1 ,4 4 8 la 475, (489)™ _

_

_ —

__

_

455.488, 520.5la

Solvent 7 Ethanol

__ __

—

Solvent 5 Solvent 6 Chloroform Cyclohexane

Table 1.5 (continued) QUALITATIVE SPECTROSCOPIC DATA: ABSORPTION MAXIMA IN DIFFERENT SOLVENTS

98 CRC Handbook of Chromatography: Plant Pigments

—

—

461, 4942

—

—

463, 49231 — — —

—

—

—

—

—

—

—

— — — —

Cryptoxanthindiepoxide P-Cryptoxanthin-5,65 ',6'-diepoxide Cryptoxanthin-5,6epoxide P-Cryptoxanthin-5',6'monoepoxide Cryptoxanthin-5',6'monoepoxide Cryptoxanthin-5,8epoxide P-Cryptoxanthinmonoester Cynthiaxanthin Dehydroadonirubin Dehydroadonixanthin Dehydro-(3-carotene

—

—

—

P-Cryptoxanthin

Dehydro-P-carotene (trans)

455, 486la

—

a-Cryptoxanthin

—

—

277, 308, 339, 354, 433,439 , 457, 475, 4 88^

439, 47014 —

— —

Cryptoflavin Cryptoxanthin

4 9 162 —

486, 51962

— —

—

Cryptocapsin

—

Cryptocapsone Cryptochrome

—

Crocoxanthin

—

— — — —

—

—

—

—

—

—

—

—

— 424,452, 485.5 14 —

— —

—

—

— — — 472, 504, 543la — 50267

—

—

479, 5122

—

—

—

473. 503la

453,483, 51865

459, 49014 452,483, 51914 —

— 424, 45614

—

—

— — — 455, 485, 518la —

—

—

(434), 456, 48315 456, 4882

432, 452, 48015 —

453, 482la

433,463, 49765

— 388, 409, 43915 438, 46814 433,463, 49714 —

—

(430), 454, 4822

—

— — — —

—

—

—

—

—

—

—

—

—

— —

—

—

—

— A l l 21 46627 —

—

424, 445, 47715 424,445, 477—

423, 442, 47215 —

442, 473la

(428), 449, 47 32

424,452, 486la —

— —

—

(421), 443, 4722

—

— — — —

—

—

—

—

—

—

—

—

—

—

— —

—

—

445,471,

424, 445, 47740 — 46 628 45528 —

428, 45466

—

422. 443. 47262 —

—

423.451. 484la 227, 267, 299, 333. 347,421, 427. 446. 462, 47564 446, 471a-u 423,451, 4846S 425, 451, 476a-34 —

— —

422, (427), 445, (462). 47 561 (445), 470, 49740

— — — 447, 475, 504la —

—

—

—

—

—

—

—

420,452, 485.56S

425,447, 48114 420, 446, 477M

— —

—

—

—

— — — —

—

—

—

—

—

—

—

—

—

—

— —

—

—

—

— — — —

—

—

—

—

—

—

—

—

—

— —

— —

—

—

Volume I: Fat-Soluble Pigments 99

—

—

—

4,4'-Diapolycopen-4-al

4,4'-Diapophytoene

4,4'-Diapophytofluene

4,4'-Diapo-£-carotene

Diadinoxanthin (trans)

480.5, 5085 (428), 449, 47949 340, 426, 447.5, 478^ —

—

—

—

—

—

— —

—

—

—

—

—

493,531, 570la

—

—

Solvent 2 Benzene

495, (522)69

Dehydrorhodopin

Deoxyflexixanthin Diadinoxanthin

3.4-

2'-Dehydroplectaniaxanthin Dehydro-retrocarotene

Dehydrolycopene

—

Dehydrolycopene

3.4-

—

Dehydro-(3-carotene —

Dehydro-hydroxyechinenone

3.4-

Pigment

Solvent 1 Acetone

—

—

—

—

—

— —

—

—

—

—

—

—

—

Solvent 3 “ Benzine”

—

—

—

—

—

— - 449, 474, 50 672 —

—

—

—

520,557, 60114

—

—

Solvent 4 Carbon disulfide

—

—

—

—

48 874 —

—

—

— —

—

—

—

—

— 432,455, 4 8215 —

—

—

—

—

—

__

__ 493,528, 567la —

__

Solvent 6 Cyclohexane

__

Solvent 5 Chloroform

—

—

—

47 774

— 424,446. 4 7672 425, 446, 4773 —

__

__ __

346.5, 36673

__ 297.573 (315.5), 330,

__

__ —

__ 50674 275, 285.5,

__ __

__

__

__

__

__ (358), 378,

__

338, 444.5 , 474-“

__

__ —

477.5 , (500)5 —

__

455, 483, 51771 476.5, 5035 —

__________

(358), (374),

__ 50270

445 472,

498.534, 574la __________

__

__

Solvent 12 Pyridine

_

421,445, 4 7 572 __

__

__

__

_

400, 42573 454, 476,

—

—

— —

__

__

_

—

__

__

Solvent 11 Methanol

486, 520hK _ _

__________

—

__ 476.504. 542la

__

49136 __

—

__

425, 462.

Solvent 10 Petroleum ether

__

_

—

Solvent 9 Hexane

__________

Solvent 8 Ether

—

_______

—

46627

__

Solvent 7 Ethanol

Table 1.5 (continued) QUALITATIVE SPECTROSCOPIC DATA: ABSORPTION MAXIMA IN DIFFERENT SOLVENTS

100 ___

(430), 452,

CRC Handbook of Chromatography: Plant Pigments

_

—

—

-

2,2'-Diketo-

48082

-

—

—

bacterioruberin 3.4Diketo-a-carotene 4,4'-Diketo-0-carotene —

—

—

tene (trans) 4,4'-Dihydroxy-0carotene Dihydroxy-£-carotene

—

—

—

—

—

—

—

439, 467, 501"

—

—

—

—

—

—

—

—

—

—

—

-

—

—

—

469, (489)78

—

471, (491)78

3,4'-Dihydroxy-0-caro- — tene (cis) 3,4'-Dihydroxy-(3-caro- —

droxy-4-keto-ycarotene r,2'-D ihydro-r-hydroxy-4-keto-y-carotene (trans) l',2'-D ihydro-l'-hydroxy-4-keto-torulene 7',8'-Dihydrorhodovibrin

—

—

—

1'2 ,'-Dihydro-1'-hy-

droxy-y-carotene l',2'-D ihydro-l'-hydroxy-y-carotene (trans) r,2'-D ihydro-l'-hy-

—

7,7'-Dihydro-0-carotene —

-

-

—

(434), 454, 4g249 -

—

3',4'-Didehydrochloro- (465), 491, bactene (trans) 52476 3.4Didehydrolycopene —

Diatoxanthin (trans)

tetrahydrolycopene Diatoxanthin

4,4'-D iapo-7,8,ll,12-

—

-

—

— —

—

—

—

—

457, 486, 522"

—

—

—

—

—

—

—

-

50081 —

361, 54380

—

—

439, 465.5, 498"

—

—

—

—

—

—

—

(433), 458, 48615 -

—

47681

-

(427), 450, 47838

—

—

—

—

-

—

-

47882

460, 480, (504)

—

—

—

0

40951

(440), 460,

(425), 449, 47515 428,452, 4793

—

-

—

—

—

—

-

-

—

-

466

-

451,480

~

—

—

—

450,(469), 4794

-

—

462 ^

-

378,400, 42579

429, 454, 486"

_

465,(490)

35°- (438); 459' 488™

—

-

-

—

—

—

—

—

458,491, 38a. 4C5, 42923

52477

430, 452, 483” -

(354), 374, 395 4.1Q7

—

—

—

—

-

-

Volume I: Fat-Soluble Pigments 101

—

—

— —

—

Eschscholtzxanthin

Flavacin

Flavochrome Flavorhodin

Flavoxanthin

5.6Epoxy-3-hydroxy5.6dihydro-12'-apo-Pcaroten-12 '-al (trans)

—

_

_

— 439, (468)86

_

—

432, 481lh

434. 462la —

459,486, 5207 —

421, 45016

— —

—

—

_

—

414, (440)86 392, (412) 86

_

Solvent 3 “ Benzine”

442, (470)86 419, (442) 86

437 86

„ 472-’

-

47044 Echinenone — 5.6Epoxy-3-hydroxy— 5.6dihydro-10'-apo-Pcaroten- lO'-al (cis) 5.6Epoxy-3-hydroxy— 5.6dihydro-10'-apo-Pcaroten-lO'-al (trans)

418.442,

Dinoxanthin

_

—

_

—

Solvent 2 Benzene

U '-D im ethoxy-I.21 \ 2 '-tetrahydro-i|i,(jjcarotene-4,4 '-dione l.r-D im ethoxy-1,2,- I '. 2 '-tetrahydro-3',4 'didehydro-i|Mj/-caroten-4-one

2,2'-Diketospirilloxanthin

Pigment

Solvent 1 Acetone

_

449, 47988

451, 48214 472, 50287

474,507. 5427 —

—

_

—

_

—

433, 46114 __ 430, 459la

451,478, 5107 —

—

_

_

-

_

_

_

Solvent 6 Cyclohexane

_ (470).

456,488, 5207 —

—

__

47316

-

_

Solvent 5 Chloroform

_____

49872 488— 49416 —

441,467,

_

530,561, 60383

Solvent 4 C arbon disulfide

44884

400,423.

_

__

448,476. 5057 —

—

_

_

470’-’ 458. 459"’

419.441.

_

_

Solvent 7 Ethanol

—

415. 428, 452: 1 __ __

—

422, 45016 __

438,464, 49573 —

__________

____

469” 45310

416.439.

_

—

Solvent 10 Petroleum ether

421 45()ia

422, 4 5 0 '4

442,472. 5()27 _

_

_

467”

416 438

Solvent 11 M ethanol

_

—

_

—

__________

452— 456*5

_

,370). 187 461,489. 52^ 584 ,368)7 387. 494.5 . 52784

Solvent 9 Hexane

_____

455-’ 1 __

_

Solvent 8 Ether

Table 1.5 (continued) QUALITATIVE SPECTROSCOPIC DATA: ABSORPTION MAXIMA IN DIFFERENT SOLVENTS

_

—

__

489 5 ->l6

__________

464

_

_

Solvent 12 Pyridine

102 CRC Handbook of Chromatography: Plant Pigments

________

—

—

Fucoxanthol

Gazaniaxanthin

447.5,476 , 5091a

—

— —

— —

— —

—

—

P-carotene Hydroxyechinenone 3'-Hydroxyechinenone

— —

—

-

—

-

—

—

—

—

1-Hydroxy-1,2-dihydro- — •y-carotene 3-Hydroxy-4,4'-diketo- —

-

—

4-Hydroxy-4,4'diaponeurosporene

—

—

caroten-12 '-al (trans) 4Hydroxy-f3-carotene

—

—

caroten-lO'-al {trans) 3-Hydroxy-12' apo-P*

—

—

—

caroten-lO'-al (cis) 3-Hydroxy-10'-apo-P-

caroten-12 '-al (cis) 3Hydroxy-12'-apo-P-

—

—

1-Hexosyl-l ,2-dihydro- — 3,4-didehydro-apo-8'lycopenol 3-Hydroxy- 10'-apo-(3- —

—

—

—

-

Heteroxanthin

278, 306, 342,356, 433, 439, 457,474, 48764

-

— —

—

—

— —

diaponeurosporene Helenien

Gazaniaxanthin (trans) 435,462,49291 — 4-((3-D-Glucopyrano— — syl)-oxy-4,4'-

483, 5105 (427), 449, 47148

Flexixanthin Fucoxanthin

— —

49481

—

-

—

—

—

—

—

—

—

-

— —

461,494.5, 5 3 1' a

—

— 445,477, 510la

474— 48185 471J)

48381

—

423.449. 47774

—

—

—

—

—

—

434, 456. 48515

-

— 400,424, 450, 47973

-

—

— 457, 492la

-

—

—

47938

—

—

-

-

-

46285^ (462)J)

~

415.438. 46774

(427), 451,

—

426, 448, 47713

-

391,416, 439, 46873

398, 423, 4482 434.5,462, 494.5 la

(330), (426), 449, (465)2

-

—

427,452. 482 -

—

-

390,414, 437, 46673

-

-

467 1

-

404, (425)

433, (458)

427, (452 )86

226, 267, 298,331, 348, 420, 427,445, 462, 475w

—

-

434.5,462.5, 494.5 la

(425). 447.5, 47590

460

494^3

'

(386). 413, 435, 465

408, (428)

434.5,462.2, 494.5 16

—

-

—

-

-

—

—

-

-

—

Volume I: Fat-Soluble Pigments 103

(430), 453, 480" —

—

—

0-Isorenieratene {trans)

4-Keto-a-carotene

4-Keto-p-carotene

4-Keto-y-carotene — 4-Keto-y-carotene {cis) — 4-Keto-y-carotene 471,(491)™ (trans)

Isozeaxanthin

—

—

Isorenieratene

(3-Isorenieratene

—

Isofucoxanthinol

— — —

—

—

—

—

424, 456, 487la 430, 463, 4929s —

—

— — —

—

—

—

—

—

—

—

— — —

—

—

—

444, 474, 506la 452, 484, 52098 456. 487, 508100 —

—

—

—

Isofucoxanthin

—

—

Solvent 4 Carbon disulfide

—

__

—

—

—

Solvent 3 “ Benzine”

—

(425), 449,

,

—

Hydroxy-3'-keto-a47794

Solvent 2 Benzene

4Hydroxy-4'-keto-(3— carotene 4-Hydroxy-4'-keto-(3460, (480)95 — carotene (trans) Isocryptoxanthin — —

3carotene

Pigment

Solvent 1 Acetone

— — —

—

—

—

—

—

—

—

—

—

—

— 47 020

—

Solvents Chloroform

— 467. (483)51 _

_ _

483™

__

(474)™ 432. 458.5,

451,4782s

_

_

(460)20

_____

Solvent 7 Ethanol

(428), 451, 478.5™ (425), 451.

_

_

__

—

Solvent 6 Cyclohexane

____

— — —

—

—

—

—

—

—

—

—

—

—

—

__________

Solvent 8 Ether

—

(425), 452. 48099 —

(424), 449, 47839 (430), 453. 48297 —

454, (474)"

454, (473)"

_________

Solvent 10 Petroleum ether

— — —

45882

465. (490)™ 350, 461102 462102

—

(428), 451, 47 9 " 453.5, 470l()l —

—

—

—

—

—

—

4 5 1 ,47996

—

—

Solvent 9 Hexane

—

—

Solvent 11 Methanol

— — —

—

—

—

—

—

(445), 475, (505)la —

—

—

—

Table 1.5 (continued) QUALITATIVE SPECTROSCOPIC DATA: ABSORPTION MAXIMA IN DIFFERENT SOLVENTS

— —

—

—

—

—

—

—

—

—

—

—

—

—

Solvent 12 Pyridine

104 —

CRC Handbook of Chromatography: Plant Pigments

—

—

— —

44 8,4 74, 505.5 107

Lutein-5,6-epoxide

Luteochrome Luteoxanthin

Lycopene

— —

480, (507)6 48 5,512, (540)104 465 , 490 , 5236

Luteinepoxide

—

—

—

—

—

Lutein (trans)

365, 474102

—

—

—

—

Lutein

—

—

—

_

—

— 407 ,43 2, 46062 455,487, 52214

453, 48216

—

_

429 ,45 6, 48562

—

—

_

—

Leprotene

4-Ketotorulene

4-Keto-l',2'-dihydro1 '-hydroxy-y-carotene 4-K eto-l',2'-dihydro1 '-hydroxytorulene 4-Keto-4'-ethoxy-3carotene 4-Keto-3'-hydroxy-3carotene 4-Keto-l'-hydroxy1 ', 2'-dihydro-y-carotene (cis) 4-Keto-l'-hydroxy1 ', 2 '-dihydro-y-carotene (trans) 4-Keto-4'-hydroxy-3carotene 4-Ketophleixantophyll 2-Ketorhodovibrin

_

_

_

_

447,475.5, 506*4

— —

—

—

—

—

—

— —

—

—

_

—

—

_

_

_

_

477,507.5, 54814

451, 48214 —

440, 470, 50189 472, 50216

_

477, 499, 51716 446,475, 50533

—

— . —

—

—

—

—

_

_

_

453,480, 51714

— —

—

—

_

428, 460, 49516 428,454, 48333

—

— —

—

—

—

—

_

_

_

—

— —

—

—

—

—

—

(431), 457.5, 48138 — —

—

—

—

420, 442, 46989 (417), 440, 46946 — 396,420, 4462 443,472, 50314

(423), 446, 474106

421,445, 47346

470, 490, (516)51 —

— 520104

—

—

—

—

480.(504 )M

4 6 7 ,(483)M

—

— —

—

—

—

422,445, 474105

—

—

— —

—

—

—

—

—

—

— 395,421, 448x 34 448,472, 50336

442, 47116

—

425, 452, 48416 420,(425), 445, (460), 4744 421, 446, 4743,47 421,445, 474x 34

—

445,471, 502107

— —

—

—

—

420,444, 47433

— 485,51 1, 5471()4 456 , 483, 5186 —

—

45 882 — 50222

462102

45 2102

—

—

—

—

—

45882

—

—

—

—

—

—

—

—

—

—

—

—

—

461,490, 526107

—

—

—

—

— —

—

—

—

—

—

—

—

Volume I: Fat-Soluble Pigments 105

Methyl-1-hexosyl-1 ,2- 469, 49792 dihydro-3,4-didehydroapo-8'-lycopenoate Monadoxanthin —

1-Methoxy-l,2— 7 ',8 ',1 1',12'-hexahydro-ift ,vJi-caroten-4-one 1-Methoxy-l'-hydroxy- — 1,2,l',2'-tetrahydroi|Mj/-caroten-4-one 1-Methoxy-l,2-7',8'-te---trahydro-vJ/,vJ/-caroten4-one Methyl-apo-6'445,469, lycopenoate (495)26 Methyl-apo-6'-lycopen- — oate (trans) Methylbixin (trans) —

1-Methoxy-l,2-dihydroijMjj-caroten-4-one

Lycoxanthin

—

Lycophyll

—

Solvent 2 Benzene

—

—

—

—

—

—

—

—

_

—

—

_

—

_

444,473, 50414 444, 473, 50414 —

—

Solvent 3 “ Benzine”

—

456,487, 52114 448,474, 505109 456, 487, 52114 — —

446.472, 50426

Lycopene (trans)

Pigment

Solvent 1 Acetone

50426

—

—

—

_

—

—

—

—

—

—

—

—

—

—

—

Solvent 5 Chloroform

—

—

—

472,506, 54614 473, 507, 54714 —

—

Solvent 4 Carbon disulfide

_

—

—

— 50326 —

—

—

—

—

—

—

—

Solvent 6 Cyclohexane

_

—

—

—

—

—

—

—

_

444,474, 50514 444. 474, 50514 —

—

Solvent 7 Ethanol

—

—

—

—

—

—

—

—

—

—

—

_

Solvent 8 Ether

422, (427), 445, (461), 47561

—

—

(360), 377, (463), 487, 51984 (330), 345, (436.5), 460.5, 49084 —

(360), 375, (462), 488. 52084 398.5, 420, 446K4

286. 295. 425. 448, 476, 50754 447,473, 50416 —

Solvent 9 Hexane

Table 1.5 (continued) QUALITATIVE SPECTROSCOPIC DATA: ABSORPTION MAXIMA IN DIFFERENT SOLVENTS

4 /2 ,

—

432, 456, 49023 _ _

446,

(448), 471.

—

—

—

441,468. 5011,)K 443. 469, 500109 —

445. 474. 505*

Solvent 10 Petroleum ether

—

—

—

—

—

—

—

—

—

—

—

Solvent 11 Methanol

—

_

—

—

—

—

—

—

—

—

—

—

Solvent 12 Pyridine

106 CRC Handbook of Chromatography: Plant Pigments

47762

— —

(450), 478, 5 1076 (350), (363), (450), 474, 50576

—

— —

413,438,

46649

Myxoxanthin Myxoxanthol

Myxoxanthophyll

Neochrome

Neofucoxanthin A Neofucoxanthin A and B

Neoxanthm

Neurosporaxanthin

471.5, (496)25

420,441,

—

Myxobacton

Myxoxanthophyll (trans)

—

Myxobactin

-

— —

-

462. 488, 5 2276 —

— —

—

—

439, 468la

—

Mutatoxanthin

-

—

-

466, 49733

__ __

__ __

__

488110 464,494, 529110 —

—

431,459, 48888 —

—

—

—

—

-

466”

422,449,

__ _

-

473110 441,474, 508110 (460). 488, 52276 _ _

—

__

437, 468la

—

—

—

—

— _

50651 460, 480, (504)s 1 470110 —

-

—

__ __

_

—

— —

_

-

467”

_

46611)5

414,437,

__

4 2 1 ,44968 __ 447105 417,438,

-

(375). 398.

__

—

—

454^ 477

__

448, 475, 505111 __

__

_

427, 45788

467”

415,439,

__

436, 465a-34 472"3 477.5, 50523

466” 414,

416,437,

44516

_

_

__

__

—

465110 431,465, 495110 ’ —

—

__

426, 45614

__

4 64113 471, 50523

470"3

440, 470" -

_

__

_

— —

_

__

_______

-

_

_

(465 ) 493 528™

— —

_

__

443, 4731a

__

__ —

__

—

__________

420,445,

_______

__

__

—

41 ^ 439

45 242 3 j q (402) 423 44942 __

409,428,

__________

__________

46123

— _

—

426, 45619

45442 —

—

__

(407) 428

__

45062 __

__

397,422,

43642 __

_

(418), 428,

417 442,

______

__________ 46762

__

—

____

__

_

___

—

__

—

—

—

—

—

459, 492la

435, 46916

479, 511la 47218 — 47118

459, 489.516

—

-

—

—

—

-

—

—

— —

—

—

—

—

—

—

—

440, 47016

—

460, 492la

—

—

—

—

—

—

—

Mutatochrome (equilib- — rium mixture) Mutatchrome (trans) —

Mutatochrome

3,4-Mono-dehydro-(3- — carotene 5.6Mono-epoxy-(3carotene 5.6Mono-epoxy-0-car- — otene (trans) 5.6Mono-epoxylutein (trans)

__________

__________

Volume I: Fat-Soluble Pigments 107

— —

—

—

—

—

—

—

—

—

(350), (440), 464 , 49453 —

—

—

—

-

—

Nostoxanthin

OH-C-Carotene

OH-Chlorobactene

OH-Chlorobactene (trans) r,u i OH-Lycopene ^ nuKI OH-Neurosporene

OH-P-481

OH-Phytofluene

oh -r

OH-Spheroidene

440.5, 467.5, 501.583

-

—

—

—

—

—

—

—

-

— _

—

—

—

Solvent 3 “ Benzine”

486113

Solvent 2 Benzene

Neurosporaxanthin — (equilibrium mixture) Neurosporaxanthin— methylester Neurosporaxanthin— methylester (trans) Neurosporene —

Pigment

Solvent 1 Acetone

-

_

—

—

—

—

—

—

Solvent 4 Carbon disulfide

-

_

—

(432) 457, 48520

—

—

—

—

-

—

~

—

-

Solvent 5 Solvent 6 Chloroform Cyclohexane

—

—

~

(426), 448, 47520 —

Solvent 7 Ethanol

-

_

—

—

—

50425

Solvent 8 Ether

_

-

—

—

—

416, 440, 47023

Solvent 9 Hexane

472, (495)25

(460)' f 2' 515115

455' 5®2' 515114 ^'367 ^ '

445, 474, 506114 414, 437, 4661'4

376 396, 41533 435 461, 49153

(392), 413, 438, 46952

-

—

—

— —

—

— —

—

—

—

Solvent 11 Solvent 12 Methanol Pyridine

(360), 473.5, 47325

474» 50525

Solvent 10 Petroleum ether

Table 1.5 (continued) QUALITATIVE SPECTROSCOPIC DATA: ABSORPTION MAXIMA IN DIFFERENT SOLVENTS

108 CRC Handbook of Chromatography: Plant Pigments

466, 490, 522103 390, 470, 499, 532103 — —

—

495, 528, 559115 510, 539, 575115 47148 465, 502117

Oscillaxanthin (cis) Oscillaxanthin (trans)

P-481

P-518

—

— —

— —

_

— —

—

Phytoene (trans) Phytofluene

Phytofluene (cis)

— _ ‘ —

— _

— —

— 338,355, 37416 —

—

—

— _

—

—

_

_ _

— —

—

— —

_ —

—

—

446^ — (495), 528, — 559118 454, 478, 5096 —

Phleixanthophyll (trans) (368), (428), 456, 478, 509119 Physalien — Phytoene —

Phleixanthophyll

Peridininol Phillipsiaxanthin

Peridinin

P-412 P-450

Oscillaxanthin

— (460), 484, 51222 _

Okenone Okenone (trans)

— —

— — 373, 389, 467116 —

OH-Spirilloxanthin OH-Spirilloxanthin (trans)

501, 53083

484, (505)104

OH-Spheroidenone

_

_ _

494,528, 56816 — —

— —

_ 529, 565114

—

— _

— _

—

—

528,562, 601115 454,480, 5 1272 — _ _

_

_ _

_

— _

_

—

— —

— _

(465), 489, 5226 —

—

—

360, 54380

_

_ _

476,501, 53420 — —

— —

—

—

—

— —

— _

—

—

— _

—

—

_

_ _

— —

—

— —

_ —

—

— 276, 286, 29651 — 332,348, 36651 —

—

—

—

47272

522115

468,492, 52620 — —

— —

—

487104

—

— —

— —

—

—

— _

—

—

_

_ _

— —

_

— —

_ —

—

_

_

_ _

_

— 275, 286, 298120 286, 29856 331,347, 36762 —

—

554 —

431,454, 48472 —

—

— —

_

48422 —

—

—

118

—

—

— —

46772

—

—

— —

— —

_

— —

— —

—

452, 48023 — (264), 275, — 286, 29652 — — (317), 331, — 346, 36852 249, 257, — 304, 318, 331.5, 347.5, 36756

—

—

— 488.518,

412, 438114 423, 450, 480114 455 , 482, 514114 487.5,518. 555115 455, 485122

— —

_

460, 483, 516104 489, 523114 369,385, 462, 494, 528'16 — —

—

— —

— —

(465). 493, 526h —

— —

_

—

_

—

— —

_

— —

_ _

—

Volume I: Fat-Soluble Pigments

109

— —

— 457.5M 457.5U —

—

— (380), (462), 487, 5 18119

—

445, 472, 5048 — 363,3 78,46 0, 488, 522125

Prolycopene (cis) Proneurosporene

Protetrahydro-lycopene Pyrrhoxanthin Pyrrhoxanthinol Renierapurpurin

Renieratene

Reticulataxanthin Retro-dehydro-y-carotene (trans)

Rhodopin

Rhodopin (trans) Rhodopurpurin Rhodovibrin

— — 372,388, 473, 503, 535123

—

457, 476, 50 798 — —

— — — 464, 487, 5191cxi

— —

455.5, 485la

—

Prolycopene

—

Pigment R

—

Plectaniaxanthin Pro-y-carotene

—

Phytofluenol

—

Solvent 2 Benzene

475,499, 530121 454, 478, 50869 — — 447.5, 477la

—

Phytofluene (trans)

Pigment

Solvent 1 Acetone

— — —

—

— —

—

— — — —

— —

—

— —

—

—

—

Solvent 3 “ Benzine”

478, 508, 547la — 511, 5 5087 (408), 491, 522, 559125

463, 496, 5 3 298 — —

— 5 12 , 5 8672 — 477, 504, 544100

495,519.5, 553121 — 460.5, 493.5 la 469.5, 500.5la — —

—

—

Solvent 4 Carbon disulfide

453, 486, 5211a — — 370,385, 469, 498, 532123

— —

—

— — — —

— —

453.5, 484la

— (444), 473la

499121

—

—

Solvent 5 Chloroform

296, 447, 474,507 — — —

— —

—

— — — —

— —

—

— —

—

—

—

Solvent 6 Cyclohexane

(445), 474, 505la - — —

— —

—

— 47 172 __ —

— —

445, (471 ),a

— (437), (465)la

488121

—

—

Solvent 7 Ethanol

332, 348,

__

_ — —

—

— —

—

447, 470, 501la _

__ 358,374, 455, 483, 516123

440, 470, 50116 _

__ 48322

—

__

_

__ —

—

__ __

__ __ 463, 490124 __

__ __

—

__

__ __

__ __ —

__ __

__ __

__ __ __ __

463123

_

—

__ __ __ __ 407, 43016 45 9 , 48 7 72

__ — __ —

__ __

__ __

(414), 436, (463)123 440, 46818 (404), 436,

__ __ __ __

—

__ —

__ —

__ (435), 464la

__ —

__

__

Solvent 12 Pyridine

—

__

__

Solvent 11 Methanol

—

__

36836

332, 348,

Solvent 10 Petroleum ether

460,482, 513121

—

__ —

—

__

__

36816 —

Solvent 9 Hexane

Solvent 8 Ether

Table 1.5 (continued) QUALITATIVE SPECTROSCOPIC DATA: ABSORPTION MAXIMA IN DIFFERENT SOLVENTS

110 CRC Handbook of Chromatography: Plant Pigments

— —

—

(350), (440), 465, 49553 —

Rubichrome Rubixanthin

Rubixanthin (m )

Rubixanthin (trans)

—

_

—

—

—

—

—

—

484, (505)128

— (455), 484, (505)115

Sintaxanthin

Siphonaxanthin

Siphonaxanthinmonolaurate Siphonein

Spheroidene

Spheroidene (trans)

Spheroidenone

Spheroidenone (cis) Spheroidenone (tram)

440, 468, 5009 438, 467, 4999 (475), 502, 530128 378, 492.59 (475), 501, 530"5

—

— —

424 ,45 1, 481la — 45 8,486, 518la 422126

—

—

—

—

—

482,511, 5489 474,503.5, 542la — —

Semi-a-carotenone Semi-p-carotenone

Sarcinaxanthin

Saproxanthin {trans)

Saproxanthin

451,478.5, 509107 (350), (363), 450, 473, 50376 —

—

Rhodoxanthin

Rubixanthinepoxide

—

Rhodoviolascin

— —

—

—

—

_

—

—

(415). 440, 469la — 446,470, 501la _

—

—

—

—

458,489, 524la — 432,463, 495.5 14 —

—

_

(490), 520, 553128 — (490), 520, 553115

—

—

_

—

—

436,466.5. 499ld 499, 533la 499, 538la

—

461,491, 52614 —

—

496,534, 573.5 la 491,525, 564la 476, 50614 461,494, 53314 —

_

—

—

—

—

—

— —

—

—

— —

_

—

—

46690

—

46690

_

— —

—

—

—

—

—

—

423.451. — 480la — — (487),(519)la —

(370). 460, 4 86 . 5 18107 —

474. 50414

—

476,507. 544la 482,510, 546la — 439,474, 50914 —

_

— 488115

—

—

—

452— 46490

455127

445—45190

_

(415). 441. 469.5la — —

—

—

—

—

448, 48014 433,463, 49614 —

(465), 491, 526la 438, 495la

— —

—

—

—

—

—

—

— —

—

448.472.5, 503107 —

—

—

—

— —

—

—

— — 515115

— 460,482.5,

—

48 3 129

—

—

—

—

—

—

—

—

— —

—

—

—

—

—

—

— —

—

_

— —

48 8130

(427), 450. 47890 450, (473)127

—

— —

—

(360.5). 445. 4 70 , 500107 —

—

456,487, 5 21la— — 432,463, 495.565 430, 455, 48518 —

—

(427), 450. 47890 405. 427. 453. 48474 —

—

—

415,440. 46916 — 443,469. 500la (425), 448. 475126 —

—

—

—

—

522,489, 45 836 — 432,462, 49465 —

—

— —

—

—

—

—

—

—

_

— _

_

(372). 490, 55 2 107 —

—

—

—

— —

_

Volume I: Fat-Soluble Pigments 111

373, 389, 468, 498, 533' 16

—

—

Spirilloxanthin (trans)

Taraxanthin

Taraxanthin (trans)

—

—

— —

— —

—

458,485, 5208

—

—

428.5,455, 485131 —

378,395. 479,510, 548.535 —

Solvent 2 Benzene

365,448,474, 506.58

7.8.11.12- Tetrahydroly- — copene (equilibrium mixture) 5,6,5',6'-Tetra— hydrolycopene Thiothece-460 (trans) (445), 471, 506132

l,2,l',2'-Tetrahydro1,1'-dihydroxylycopene (trans) Tetrahydrolycopene 7.8.11.12- Tetrahydrolycopene

7,8,7',8'-Tetrahydro— tKi|/-carotene 7.8.11.12- Tetrahydro- — iK4>-carotene 1,2,1',2'-Tetrahydro— 1 , 1 '-dihydroxy lycopene

—

Spirilloxanthin

Pigment

Solvent 1 Acetone

—

— —

—

—

—

—

—

—

443, 472la

—

—

Solvent 3 “ Benzine”

—

—

— —

—

479,506, 5438

—

—

441,469, 501la —

496.5, 534, 573.5 16

50051

—

— —

—

458,485, 518s

—

—

—

— —

—

__

______

—

__

__

__ —

—

—

—

Solvent 6 Cyclohexane

—

476, 507, 54416

(418), 495, (475), 505, 532, 571.55433S

Solvent 4 Carbon Solvents disulfide Chloroform

_

—

__

__

__

_

__ __

__

__

__________

418.552 285,296, __ 350, 372, 391,415s2 413.5, 437.5, __ 467.523 (412), 435, (445), 473.5 , 460.5, (500)132 494.5 132

__ 354 ( 374 394.5 ,

4 10 , 43316

__

__ __ __

__

363,446, 473, 5048

__

__

41984 __

__

__

__ __

_

—

—

Solvent 11 M ethanol

_

528116

368.384, 461,493. 528369, 385, 462,494,

Solvent 10 Petroleum ether

—

__

__________

375.5 , 397, 42152

__

443, 4721(1

__

49335

Solvent 9 Hexane

—

—

__

__

_

—

463,493. 528s7

Solvent 8 Ether

__________ 445 459

_

469106

420, 441,

—

_

—

Solvent 7 Ethanol

Table 1.5 (continued) QUALITATIVE SPECTROSCOPIC DATA: ABSORPTION MAXIMA IN DIFFERENT SOLVENTS

—-

__

__

__ __

—

__

__

__

_

—

—

374.5 , 395,

Solvent 12 Pyridine

112 _______

CRC Handbook of Chromatography: Plant Pigments

(380), (463)5

—

— — —

—

—

—

—

—

—

556136 —

—

-

Torulene

Triphasiaxanthin

Trollichrome Trollixanthin Vaucheriaxanthin

Violaxanthin

Violaxanthin (cis)

Violaxanthin (trans)

Violaxanthin (9-cis)

Violaxanthin ( 13-d.v)

Violeoxanthin

Violerythrin Xanthophyll

Xanthophyll K1

Xanthophyll K1S

—

a-Zeacarotene

_

—

Xanthophyllepoxide

Thiothece-OH-484 (trans) Torularhodin

Thiothece-484 (trans)

(450), 477, (504)132 (465), 478.5, 518132 460, 484.5, 515132 —

Thiothece-474 (trans)

_

—

430,456, 482.5131

-

_

— 420,447.5, 4 7 7 5 1a

—

_

—

—

—

426,453, 48262

432, 459la 457, 483la —

485 , 5 19, 55716 470,503. 541la —

—

—

—

—

-

_

— —

—

—

—

—

_

'

417.5,443, 47214

— — —

—

—

—

—

—

_

_

—

472, 501.5 ld

-

_

— 447,474, 50537

—

—

—

—

440,469, 500.5 14

450. 479la 473. 501la —

500 . 541, 58216 491,525, 565la —

—

—

—

—

458-468™

4671*'

580136 —

—

—

—

—

424,451.5, 48214

430, 458la 455, 482la —

483 , 515, 55416 469,501, 539lu 480, 510134

—

—

—

-

—

— —

—

423. 448. 47945 422,448. 47845

—

—

430,453, 48345

— — —

—

—

—

—

—

424.446, 47343 __

4 4 ^ -4 5 2 * '

454-^66™

— 420,446.5, 476la

414, 436, 46546

_

—

(328), 417. 440, 471106

309, 408, 430, 45717

417.5,442.5, 471.5 14

424, 451la 447, 474la 419, 443, 471,3S

456,486, 520lu —

—

—

—

_

__

-

—

~ —

—

—

—

421,441, 47111)5

— —

—

—

—

—

—

_

— —

(378), 457.5, 483,516 s —

474. 488, 500123 458, 484, 513.5132 459, 484, 515132 5 15 133

-

’ • 449137

300 A 1 I

—

420,440. 46737

_

417.5, 440, 470a-34

327, 415, 438. 46817

_

__

442, 471K

(428). 450. 4781*'

(428), 450, 477*'

—

—

—

417,440, 420,443, 4 7017 4 17 . 47 2 33 441, 469a 47

— — —

440. 467, 495134

468 , 502 , 53936 —

—

—

—

__

-

—

418,444, 473.5la

—

—

—

415,440. 46914

— — —

—

—

(462), 486, (51 1)132 (460), 483. (510)132 —

477132

-

__

—

—

_

—

—

—

—

— — —

475,508. 545la —

—

—

—

—

Volume I: Fat-Soluble Pigments 113

Zeinoxanthin

Containing 30% acetone.

—

Zeaxanthin (trans)

a

—

(430), 453, 479103 —

Pi-Zeacarotene

Zeaxanthin

Pigment

Solvent 1 Acetone

—

432,459, 48862 —

—

Solvent 2 Benzene

—

423,451.5. 483.5 14 —

—

Solvent 3 “ Benzine”

449, 474, 505139

450,482, 5 I7 14 —

—

Solvent 4 C arbon disulfide

434, 456, 485139

429,462, 49514 —

__

Solvent 5 Chloroform

—

__

—

__

Solvent 6 Cyclohexane

__ _

4783 —

_

__

Solvent 8 Ether

423.5,451, 48314 428,452,

__

Solvent 7 Ethanol 2

430,451, 480138 __

4

Solvent 10 Petroleum ether

478x 34 422 (428), _ 445, (463), 4744 446.5, 472x 34

(426), 449.5, 476.5X34 429,451,

__

Solvent 9 Hexane 7

—

—

__

__

__

Solvent 12 Pyridine

—

__

Solvent 11 M ethanol

421.5,449.5, 480.5la

Table 1.5 (continued) QUALITATIVE SPECTROSCOPIC DATA: ABSORPTION MAXIMA IN DIFFERENT SOLVENTS

114 CRC Handbook of Chromatography: Plant Pigments

Volume I: Fat-Soluble Pigments

115

REFERENCES

1. Hertzberg, S., Liaaen-Jensen, S., Enzell, C. R., and Francis, G. W., Acta Chem. Scand., 23, 3290, 1969. la. Karrer, P. and Jucker, E., Carotinoide, Verlag Birkhauser, Basel, 1948. 2. Hager, A. and Stransky, H., Arch. Mikrobiol., 73, 77, 1970. 3. Egger, K., Nitsche, H., and Kleinig, H., Phytochemistry, 8, 1583, 1969. 4. Chapman, D. J., Phytochemistry, 5, 1331, 1966. 5. Aasen, A. J. and Liaaen-Jensen, S., Acta Chem. Scand., 20, 1970, 1966. 6. Hertzberg, S. and Liaaen-Jensen, S., Acta Chem. Scand., 21, 15, 1967. 7. Karrer, P. and Leumann, E., Helv. Chim. Acta, 51 (34), 445, 1951. 8. Ryvarden, L. and Liaaen-Jensen, S., Acta Chem. Scand., 18, 643, 1964. 9. Barber, M. S., Jackman, L. M., Manchand, P. S., and Weedon, B. C. L., J. Chem. Soc., C, 2166, 1966. 10. Surmatis, J. O., Acta Chem. Scand., 31, 186, 1966. 11. Liaaen-Jensen, S., Acta Chem. Scand., 17, 500, 1963. 12. Francis, G. W., Hertzberg, S., Andersen, K., and Liaaen-Jensen, S., Phytochemistry, 9, 629, 1970 13. Toth, G. and Szabolcs, J., Phytochemistry, 19, 629, 1980. 14. Frye, A. H., J. Org. Chem., 16, 914, 1951. 15. Stransky, H. and Hager, A., Arch. Mikrobiol., 71, 164, 1970. 16. Goodwin, T. W., Carotenoids, in Modern Methods o f Plant Analysis, Vol. 3, Paech, K. and Tracey, M. V., Eds., Springer-Verlag, Berlin, 1955, 272.

17. 18. 19. 20. 21. 22.

Stewart, J. and Wheaton, T. A., J. Chromatogr., 55, 325, 1971. Jungalwala, F. B. and Cama, H. R., Biochem. J., 85, 1, 1962 Czeczuga, B., Comp. Biochem. Physiol., 48B, 349, 1974. Stransky, H. and Hager, A., Arch. Mikrobiol., 72, 84, 1970. Hertzberg, S. and Liaaen-Jensen, S., Phytochemistry, 5, 565, 1966. Aasen, A. J. and Liaaen-Jensen, S., Acta Chem. Scand., 21, 970. 1967. 23. Isler, O. and Schudel, P., Carotine und Carotinoide, in Wiss. Veroff. dt. Ges. Erncihr., Vol. 9, Steinkopff, Darmstadt, 1963, 54.

24. 25. 26. 27. 28. 29. 30. 31 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53.

Singh, H., John, J., and Cama, H. R., J. Chromatogr., 75, 146, 1973 Aasen, A. J. and Liaaen-Jensen, S., Acta Chem. Stand., 19, 1843, 1965. Kjpsen, H. and Liaaen-Jensen, S., Phytochemistry,, 8, 483, 1969. Egger, K. and Kleinig, H., Phytochemistry, 6, 903, 1967. Egger, K., Phytochemistry, 4, 609, 1965. Sprensen, N. A., Liaaen-Jensen, S., Bprdalen, B., Haug, A., Enzell, C., and Francis, G., Acta Chem. Scand., 22, 344, 1968. Leftwick, A. P. and Weedon, B. C. L., Chem. Commun., 1, 49, 1967. Campbell, S. A., Mallams, A. K., Waight, E. S., and Weedon, B. C. L., Chem. Commun., p 941, 1967. Kleinig, H. and Egger, K., Phytochemistry, 6, 611, 1967. Stobart, A. K., McLaren, J., and Thomas, D. R., Phytochemistry, 6, 1467, 1967. Knowles, R. E. and Livingston, A. L., J. Chromatogr., 61, 133, 1971. Liaaen-Jensen, S., Acta Chem. Scand., 14, 950, 1960. Foppen, F. H. and Gribanovski-Sassu, O., Biochim. Biophys. Acta, 176, 357, 1969. Merlini, L. and Cardillo, G., Gazz. Chim. Ital., 93, 949, 1963. Grob, E. C. and Pflugshaupt, R. P., Helv. Chim. Acta, 48, 930, 1965. Hsieh, L. K., Lee, T.-C., Chichester, C. O., and Simpson, K. L., J. Bacterial., 118, 385, 1974. Camara, B. and Moneger, R., Phytochemistry, 17, 91, 1978. Hiyama, T., Nishimura, M., and Chance, B., Anal. Biochem., 29, 339, 1969. Hertzberg, S. and Liaaen-Jensen, S., Phytochemistry, 6, 1119, 1967. Eichenberger, W. and Grob, E. C., Helv. Chim. Acta, 181, 1556, 1962. Johansen, J. E., Svec, W. A., and Liaaen-Jensen, S., Phytochemistry, 13, 2261, 1974. Toth, G. and Szabolcs, J., Phytochemistry, 19, 629, 1980. Braumann, T. and Grimme, H. L., J. Chromatogr., 170, 264, 1979. Nelson, J. W. and Livingston, A. L., J. Chromatogr., 28, 465, 1967. Bjdrnland, T., J. P hycoi, 15, 457, 1979. Bjdrnland, T., Phytochemistry, 21, 1715, 1982. Smallidge, R. L., Phytochemistry, 12, 2481, 1973. Kleinig, H. and Reichenbach, H., Arch. Mikrobiol., 68, 210, 1969. Davies, B. H., Hallett, C. J., London, R. A., and Rees, A. F., Phytochemistry, 13, 1209, 1974. Liaaen-Jensen, S., Acta Chem. Scand., 18, 1703, 1964.

116

CRC Handbook of Chromatography: Plant Pigments

54. Manchand, P. S., Riiegg, R., Schwieter, U., Siddons, P. T., and Weedon, B. C. L., J. Chem. Soc., p. 2019, 1965. 55. Nakayama, T. O. M., Arch. Biochem. Biophys., 75, 356, 1958. 56. Davis, J. B., Jackman, L. ML, Siddons, P. T., and Weedon, B. C. L., J. Chem. Soc., C, 2154, 1966. 57. Sherma, J., J. Chromatogr., 52, 177, 1970. 58. Yokoyama, H. and White, M. J., J. Org. Chem., 30, 2481, 1965. 59. Yamaguchi, M., Bull. Chem. Soc. Jpn., 30, 979, 1957. 60. Pfander, H. and Wittwer, F., Helv. Chim. Acta, 58, 1608, 1979. 61. Chapman, D. J., Phytochemistry, 5, 1331, 1966. 62. Buckle, K. A. and Rahman, F. M. ML, J. Chromatogr., 171, 385, 1979. 63 Cholnoky, L., Szabolcs, J., Cooper, R. D. G., and Weedon, B. C. L., Tetrahedron Lett., 19, 1257, 1963. 64. Cholnoky, L., Szabolcs, J., and Nagy, E., Liebigs Ann. Chem., 616, 207, 1958. 65. Hida, M. and Ida, K., Bot. Mag. Tokyo, 77, 458, 1964. 66. Bodea, C. and Tamas, V., Ann. Chem., 671, 57, 1964. 67. Zechmeister, L. and Wallcave, L., J. Am. Chem. Soc., 75, 5341, 1953. 68. Davies, B. H., Hallett, C. J., London, R. A., and Rees, A. F., Phytochemistry, 13, 1209, 1974. 69. Arpin, N. and Liaaen-Jensen, S., Phytochemistry, 6, 995, 1967. 70 Isler, O., Montavon, M., Riiegg, R., and Zeller, P., Helv. Chim. Acta, 39, 454, 1956. 71. Jackman, L. M. and Liaaen-Jensen, S., Acta Chem. Scand., 15, 2058, 1961. 72. Loeblich, A. R. and Smith, V. E., Lipids, 3, 5, 1967. 73. Taylor, R. F. and Davies, B. H., Biochem. J., 139, 751, 1974. 74. Taylor, R. F. and Davies, B. H., Biochem. J., 153, 233, 1976. 75. Allen, M. B., Fries, L., Goodwin, T. W., and Thomas, O., J. Gen. Microbiol., 34, 259, 1964. 76. Hertzberg, S. and Liaaen-Jensen, S., Phytochemistry, 8, 1259, 1969. 77. Singh, R. K., Britton, G., and Goodwin, T. W., Biochem. J., 136, 413, 1973. 78. Hertzberg, S. and Liaaen-Jensen, S., Acta Chem. Scand., 20, 1187, 1966. 79. Fiasson, J.-L. and Arpin, N., Bull. Soc. Chim. Biol., 49, 537, 1967. 80. Schwieter, U., Riiegg, R., and Isler, O., Helv. Chim. Acta, 49, 992, 1966. 81. Ungers, G. E. and Cocney, J. J., J. Bacterial., 96, 234, 1968. 82. Petracek, F. J, and Zechmeister, L., J. Am. Chem. Soc., 78, 1427, 1956. 83. Jackman, L. M. and Liaaen-Jensen, S., Acta Chem. Scand., 18, 1403, 1964. 84. Schmidt, K. and Liaaen-Jensen, S., Acta Chem. Scand., 27, 3040, 1973. 85. Krinsky, N. I. and Goldsmith, T. H., Arch. Biochem. Biophys., 91, 271, 1960. 86. Szabolcs, J., Pure Appl. Chem., 47, 147, 1976. 87. Karrer, P. and Solmssen, U., Helv. Chim. Acta, 19, 1019, 1936. 88. Karrer, P. and Jucker, E., Helv. Chim. Acta, 28, 300, 1945. 89. Eskins, K. and Harris, L., Photochem. Photobiol., 33, 131, 1981. 90. Ricketts, T. R., Phytochemistry, 6, 1375, 1967. 91. Arpin, N. and Liaaen-Jensen, S., Phytochemistry, 8, 185, 1969. 92. Aasen, A. J., Francis, G. W., and Liaaen-Jensen, S., Acta Chem. Scand., 23, 2605, 1969. 93. Bonnett, R., Spark, A. A., and Weedon, B. C. L., Acta Chem. Scand., 18, 1739, 1964. 94. Liaaen-Jensen, S. and Hertzberg, S., Acta Chem. Scand., 20, 1703, 1966. 95. Liaaen-Jensen, S., Acta Chem. Scand., 19, 1166, 1965. 96. Wallcave, L. and Zechmeister, L., J. Am. Chem. Soc., 75, 4495, 1953. 97. Jensen, A., Acta Chem. Scand., 20, 1728, 1966. 98. Yamaguchi, M., Bull. Chem. Soc. Jpn., 31, 739, 1958. 99. Liaaen-Jensen, S., Acta Chem. Scand., 19, 1025, 1965. 100. Cooper, R. D. G., Davis, J. B., and Weedon, B. C. L., J. Chem. Soc., 10, 5637, 1963 101. Entschel, R. and Karrer, P., Helv. Chim. Acta, 112, 983, 1958. 102. Leftwick, A. P. and Weedon, B. C. L., Acta Chem. Scand., 20, 1195, 1966. 103. Hertzberg, S. and Liaaen-Jensen, S., Phytochemistry, 5, 557, 1966. 104. Liaaen-Jensen, S., Acta Chem. Scand., 17, 555, 1963. 105. Jeffrey, S. W., Biochem. J., 80, 336, 1961. 106. Nitsche, H. and Egger, K., Phytochemistry, 8, 1577, 1969. 107. Aasen, A. J. and Liaaen-Jensen, S., Acta Chem. Scand., 20, 811, 1966. 108. Conti, S. F. and Benedict, C. R., J. Bacteriol., 83, 929, 1962. 109. Kj0sen, H. and Liaaen-Jensen, S., Acta Chem. Scand., 25, 1500, 1971. 110. Heilbron, I. M. and Lythgoe, B., J. Chem. Soc., p. 1376, 1936. 111. Sherma, J., Anal. Lett. , 3, 35, 1970. 112. Sherma, J. and Latta, M., J. Chromatogr., 154, 73, 1978.

Volume I: Fat-Soluble Pigments

117

113. Zalokar, M., Arch. Biochem. Biophys ., 70, 568, 1957. 114. Liaaen-Jensen, S., Cohen-Bazire, G., Nakayama, T. O. M., and Stanier, R. K., Biochim. Biophys. Acta , 29, 477, 1958. 115. Liaaen-Jensen, S., Acta Chem. Scand., 17, 303, 1963. 116. Liaaen-Jensen, S., Acta Chem. Scand., 14, 953, 1960. 117. Pinckard, J. H., Kittredge, J. S., Fox, D. L., Haxo, F. T., and Zechmeister, L., Arch. Biochem. Biophys., 44, 189, 1953. 118. Arpin, N. and Liaaen-Jensen, S., Bull. Soc. Chim. Biol., 49, 527, 1967. 119. Hertzberg, S. and Liaaen-Jensen, S., Acta Chem. Scand., 20, 1187, 1966. 120. Kushwaha, S. C., Pugh, E. L., Kramer, I. K. G., and Kates, M., Biochim. Biophys. Acta, 260, 492, 1972. 121. Goodwin, T. W., Land, D. G., and Sissins, M. E., Biochem. J., 64, 486, 1956. 122 Strain, H. H., Manning, W. M., and Hardin, G., Biol. Bull., 86, 169, 1944 123. Quereshi, A. A., Kim, M., Quereshi, N., and Porter, J. W., Arch. Biochem. Biophys., 162, 108, 1974. 124. Yokoyama, H., White, M. J., and Vandercook, C. E., J. Org. Chem., 30, 2482, 1965. 125. Liaaen-Jensen, S., Acta Chem. Scand., 13, 2143, 1959. 126. Yokoyama, H. and White, M. J., J. Org. Chem., 30, 3994, 1965. 127. Kleinig, H. and Egger, K., Phytochemistry, 6, 1681, 1967. 128. Liaaen-Jensen, S., Acta Chem. Scand., 17, 489, 1963. 129. Aasen, A. J. and Liaaen-Jensen, S., Acta Chem. Scand., 21, 970, 1967. 130. Liaaen-Jensen, S., Acta Chem. Scand., 17, 500, 1963. 131. Eugster, C. H. and Karrer, P., Helv. Chim. Acta, 40, 69, 1957. 132. Andrewes, A. G. and Liaaen-Jensen, S., Acta Chem. Scand., 26, 2194, 1972. 133. Simpson, K. L., Nakayama, T. O. M., and Chichester, C. O., J. Bacterial., 88, 1688, 1964. 134. Yokoyama, H. and Guerrero, H. C ., J. Org. Chem., 35, 2080, 1970. 135. Kleinig, H. and Egger, K., Z. Naturforsch., 22, 868, 1967. 136. Hertzberg, S. and Liaaen-Jensen, S., Acta Chem. Scand., 23, 3290, 1969. 137. Davies, B. H., Chemistry and Biochemistry o f Plant Pigments, Goodwin, T. W., Eds., Academic Press, London, 1965, 489. 138. Allen, M. B., Fries, L., Goodwin, T. W., and Thomas, D. M., J. Gen. Microbiol., 34, 259, 1934. 139. Livingston, A. L. and Knowles, R. E., Phytochemistry, 8, 1511, 1969.

Volume I: Fat-Soluble Pigments

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Table I. 6 Q U A N T IT A T IV E SP E C T R O SC O P IC DATA: M O L A R E X T IN C T IO N C O E F F IC IE N T S ( c m O F C A R O T E N O ID S

NOTES Since a number of presumably identical carotenoids have been named differently by different authors, the same pigment may appear under two or even more names. If the desired pigment is not found, refer to row 1 (vertical) of the name list (Table 1.3). The first vertical row contains the pigment names. In the following vertical rows, the first number of each pair gives the wavelength and the second the molar extinction coefficient (with the reference number superscripted to it). If, in the original literature, the A (1%, 1 cm) value is published instead of the molar extinction coefficient, the corresponding e-value is calculated according to the following formula: e = 0.1 x A (1%, 1 cm) x mol. wt. where e = molar extinction coefficient and A (1%, 1 cm) = absorbance of a 1% solution of the carotenoid in a cuvette with a path length of 1 cm. The molecular weight is deduced from Table 1.3. With the values given, it is easily possible to calculate the carotenoid concentration as well as the absolute milligram amounts by using the molecular weight. For the preparation of the present table, the utmost care has been exercised. The available literature has been followed, especially where reviews have been used. Compound names are in strictly al­ phabetical order. The amount of carotenoid may be calculated using the formula: mmol of carotenoid = volume (in m€) x absorbance x e _1 The amount of carotenoid in milligrams is calculated by multiplication by the molecular weight: amount of carotenoid (in mg) = mmol x mol. wt. x e _1

120

CRC Handbook of Chromatography: Plant Pigments Table I. 6 (continued) QUANTITATIVE SPECTROSCOPIC DATA: MOLAR EXTINCTION COEFFICIENTS (cm 'M-') OF CAROTENOIDS Solvent: Acetone Pigment

e

Aleunaxanthin 463 Arhydroeschscholtzxanthin — Antheraxanthin — (3-Apo-2'-carotenal — (3-Apo-8'-carotenal — (3-Apo- lO'-carotenal — [3-Apo-12'-carotenal (trans) — (3-Apo-4'-carotenoic acid (trans) 473 (3-Apo-8'-carotenoic acid (trans) — (3-Apo-lO'-carotenoic acid (trans) — Apo-3-lycopenal —

134.9* — — — — — — 122.7-' — — —

— — — — — — — — — — 488

— — — — — — — — — — U6.06

— — — — — — — — — — —

— — — — — — — — — — —

— — — — — — — — — — —

— — — — — — — — — — —

— — — — — — — — — — 475

— — — — — — — — — — I18.37

Astacene Astaxanthin-diacetate Aurochrome

— — —

— — —

— 482 387 409 434

— I06.0X 73.59 II6.09 115.09

— — —

— — —

— — —

— — —

— —

—

Auroxanthin A/afrin Bactenorubenn a 3.4,3',4'-bisdehydro-(3-carotene Bixin Canthaxanthin

— — 499 — — —

— — 194 2*** — — —

— — — — — 480

— — — — — 118.2*

— — — — — —

— — — — — —

— — — — — —

— — — — — —

— — — — — 469

Capsanthin

—

—

121 (h 95.02

—

—

—

—

—

Capsorubin a-Carotene

— 448

— I45.0*3

318 483 483 489 —

132.2* -

— 477

— 117.0*

— 456

— .129.93

— -

—

(3-Carotene

454

134 4 *3

465

125.5*

484

107 8*

465

128 6*

457 485

134.54 I 18 34

—

—

426 451 481

90 0 136 0 127.0

—

—

—

(3, (3-Carotene -y-Carotene

— —

— —

— —

— —

— —

—

8-Carotene

—

—

—

—

—

8-Carotene (trans)

—

—

—

—

e-Carotene

—

—

—

„ , e.-Carotene *

—

—

—

_

e

_ 444

Amax

€

Cyclohexane

Amax

_

Amax

Chloroform

e

(3-Carotene-di-epoxide

Amax

Carbon disulfide

Benzene

Amax

—

—

—

—

—

—

(3-Carotenone Chlorobactene

— —

— —

— —

— —

— —

—

— — — 124 3*

~

_ 155 2

—

419 475

^-Carotene

€

49**

97.7*

99 94 . 4 I51.44

Volume I: Fat-Soluble Pigments

121

Table I. 6 (continued) Q U A N T IT A T IV E SPE C T R O SC O P IC D ATA: M O L A R E X T IN C T IO N C O E F F IC IE N T S (c m ~' M~X) O F C A R O T E N O ID S

Ethanol Xmax

Ether e

Xmax

— — 446 — — — —

— — — — 137 43 _ — — — — — — — —

— —

— —

—

Petroleum ether

Hexane e

Xmax

— —

e

Xmax

Xmax

— — — —

— —

— —

— —

— —

448 430

108.84 87.74

— _

— —

— —

— —

—

—

—

—

—

—

—

—

—

498

100.2 1

402 —

111.21 —

— —

— —

— —

— —

_ 409

_

— — —

— — —

— — —

— — —

471 — —

127.0 11 — —

471 456 463 465—467

—

—

—

422 427 446 463 475 451 436

98.215 96.115 145.715 98.515 133.715 134 5 13 105.2x l6

—

426

118.814 —

—

— —

— —

— —

— —

— 462

— 148.21

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

400

—

—

—

—

—

_ 93.9 1

—

e

— — — —

_

143.81 — _ _ _ _ 138.91 — 110.0 1 — 82.5 1 — 75.74 —

e

— — — —

_

— 160.02

Xmax

Pyridine

460 — _ 498 457 435 414

_

— 499

e

Methanol

—

— — — —

— _ _ — — — —

_ _

_ _

_ _

_

— — — —

_

_

_

—

127.94 — 165.71 — 107.312 — 124.34

— — —

— — —

— — —

422 444 473

102.04 150.34 135.34

—

—

—

—

451 448

134.217 — 139.218

—

—

—

— —

— —

— —

— —

—

—

—

—

—

—

—

—

—

—

—

—

451 433 459 490 280 431 456 488 281 431 456 489 266 416 440 470

134.5 ly 119.020 171 020 150.020 40.020 150.020 174.020 165.020 44.020 lll.O 20 174.020 159.020 36.020 104.020 167.020 168.020

138 2*

396 399

108.221 135.222

—

—

_

—

—

435 461 491

116.06 161 06 143.06

—

—

_

—

122

CRC Handbook of Chromatography: Plant Pigments Table I. 6 (continued) QUANTITATIVE SPECTROSCOPIC DATA: MOLAR EXTINCTION COEFFICIENTS ( c n i ' A / 1) OF CAROTENOIDS Solvent: Acetone Pigment

Carbon disulfide

Benzene

Chloroform

Cyclohexane

Xmax

e

Xmax

e

Xmax

e

Xmax

e

Xmax

e

Chlorobactene (trans) Chloroxanthm

— —

— —

— —

— —

— —

— —

— —

— —

— —

— —

Chrysanthemaxanthin Citranaxanthm Crocetin Crocetin-dialdehyde

— — — —

— — — —

— — — —

— — — --

— — — —

— — — —

— — — —

— — — —

— — — —

— — — —

Crocetin-dimethylester

—

—

—

—

—

—

—

—

—

—

Cryptocapsin

—

—

—

486 520 491 277 433 439 457 475 488 —

112.026 87.026 108.026 22.0 15 86 .515 84.2 15 130.015 86.515 116.915 —

—

—

—

—

—

—

Cryptocapsone a-Cryptoxanthin

— —

— -

— —

— —

— —

— —

— —

— —

(3-Cryptoxanthin

—

—

—

—

—

—

—

Deepoxyneoxanthin Dehydro-3 -carotene Dehydro-(3-carotene (trans) 3.4Dehydro-(3-carotene 3.4Dehydro-lycopene Dehydroretrocarotene

— — — — — —

— — — — — —

— — — — — —

— — — — — —

— — — — — —

— — — — — —

— — — — — —

— — — — — —

— — — — — —

— — — — — —

3.4Dehydrorhodopin Deoxyflexixanthin 7,7'- Di hydro- (3-carotene

— — —

— — —

— — —

— — —

— ——

— — —

— — —

— — —

— — —

— — —

2,2'-Di hydroxy-(3-carotene 4,4'-Dihydroxy-(3-carotene 2,2'-Diketobacterioruberin

452 — —

117.21 — —

— — —

— — —

— — —

— — —

— — 361 543

— — — — 32.232 — 145.032

— — —

4,4'-Diketo-(3-carotene

—

—

—

—

—

—

—

—

—

—

Echinenone Eschscholtzxanthin

— —

— —

472 —

115.2* —

— —

— —

— —

— —

461 —

116.2 1 —

Flavoxanthin Fucoxanthinol Gazaniaxanthin Helenien

— — 462 —

— — 142.6 1 —

— — — —

— — — —

— — — —

—

447 452 —

128.8* 126.61 —

149.11 — — — — — 20.713 — 87.8 15 85.8 15 130.9 15 88.0 15 118.I 15 — — — — — —

— — — —

2-Hydroxy-a-carotene 2-Hydroxy-(3-carotene 4-Hydroxy-(3-carotene

432 — — 278 433 439 457 474 487 — — —

— — —

— — —

— — —

— — 457 485

— — 138.54 121.94

— —

Volume I: Fat-Soluble Pigments Table I. 6 (continued) Q U A N T IT A T IV E SP E C T R O SC O P IC DATA: M O L A R E X T IN C T IO N C O E F F IC IE N T S (cm ' M 1) O F C A R O T E N O ID S Ethanol

Ether

Petroleum ether

Hexane

im»T

(

Amax

e

— —

— —

— —

— —

— —

— —

421 — —

I22.81

_

—

—

—

— —

— —

— —

— —

— —

_

__

_

__

—

_

_

_

—

—

__

—

—

—

—

—

445

137 4 1

—

—

—

— — — —

— — — —

— — — —

— — — —

— 471 471 — — —

166.028 — — —

— — —

— — —

— — —

— — —

— — —

— — —

—

—

—

—

450

134.0*1

—

—

—

—

—

— —

— —

— —

— —

466 468 — 438 464 495

114.0*1 111 O23 — 136 04 185.94 147.44

452

89.6*

—

—

—

—

—

—

—

—

Amax

267 420 427 445 462 475

e

Amax

Methanol e

Amax

e

Pyridine Amax

c

460 415 439 469 — 463 450 408 430 458

159.923 91 224 137.824 137.824

— —

— —

— —

— —

98.025 141 9 1 86.94 155.34 172.54

— — —

— — —

— — —

— — —

—

400 422 450

83.44 138.I4 141 94

_

—

—

—

—

267 421 427 446 462 475 452 480

25.615 — 97.9 15 94.615 145.515 96.8 15 134.515 131.04 — 109.04

—

—

—

26 5 n 98.115 95.615 145.5*5 99.0 ' 5 133 9 *5

— 166.027 — 461 492 446 472 502 483 476.5 382 405 429

—

—

—

—

— — — —

—— — — — —

— — — — —

— — — — —

— — —

— — —

— — —

— — —

—

—

—

—

—

—

—

—

—

—

—

458 —

118.9* —

— —

— —

— —

— —

—

—

—

—

—

—

—

-

— 124.21 160.51 124.129 171.429 139.629 171.430 150.231 105.44 165.94 161 54

—

123

124

CRC Handbook of Chromatography: Plant Pigments Table I. 6 (continued) QUANTITATIVE SPECTROSCOPIC DATA: MOLAR EXTINCTION COEFFICIENTS ( c m - ' M 1) OF CAROTENOIDS Solvent: Acetone Pigment

Xmax

c

Xmax

€

Xmax —

1-Hydroxy-1,2-dihydro-y-carotene—

—

—

—

4-Hydroxyechinenone — 3Hydroxy-3-keto-a-carotene 449 4Hydroxy-4-keto-(3-carotene — (trans) 2Hydroxyplectaniaxanthin 476 Isocryptoxanthin — Isofucoxantin — Isorenieratene —

— 137.534 —

— — —

— — —

143.0 * — — —

(3-lsorenieratene

—

— — — 443 465 493 —

—

Carbon disulfide

Benzene

Chloroform c

Xmax

—

Cyclohexane

e

Xmax

c

—

—

—

—

— — —

— — —

— — —

— — —

— — —

— — — — — — 95. 036 — 123 036 106.036 — 456 487 508

— — — —

— — — —

— — — —

— — — —

— — — —

88.036 — 118.036 107.036

—

—

—

— — —

Isozeaxanthin

—

—

—

—

—

—

—

—

—

—

4-Keto-a-carotene

—

—

—

—

—

—

—

—

—

—

4-Keto-y-carotene (trans) 4-Keto-4-ethoxy (3-carotene 4-Keto-4'-hydroxy-(3-carotene 4-Keto-phleixanthophyll Lutein Lutein-5,6-epoxide Lycopene

471 — — 480 — — 474

131 l 38 — — — — — 142.039 — — 458 — — 185 041 487

— — — — 127.2 1 — 180.91

— — — — 475 — —

— — — — 122.9 1 — —

— — — — — — —

— — — — — — —

— — — — — — —

— — — — — — —

Lycopene (trans) Lycophyll Lycoxanthin Lycoxanthin (trans)

— 508 474 476

— — — —

— — — —

— — — —

— — — —

— — — —

— — — —

— — — —

— — — —

Methyl-apo-6-lycopenoate Methylbixin (trans)

— —

— 184.3* 170.343 186.9^ 193.5 — —

— —

— —

— —

— —

— —

— —

— —

— —

Methyl- 1-hexosyl-l ,2-dihydro-3,4- 469 didehydro-apo-8' -lycopenoate 3,4-Monodehydro-(3-carotene — Mutatochrome —

122.346 —

—

—

—

—

—

—

—

— —

— —

— —

— —

— —

— —

157.948 —

— 77.09 113.09 101.09 — —

— —

Myxoxanthophyll Neochrome

— 416 437 463 — —

— —

— —

— —

— —

— —

— —

878 —

Neoxanthin

—

—

—

—

—

—

—

—

—

—

Neurosporaxanthin

—

—

486

110.249

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

Neurosporaxanthin-methylester (trans) Neurospore ne

— —

—

—

—

—

—

—

—

—

—

OH-Chlorobactene

—

—

—

—

—

—

—

—

—

—

OH-R

_

_

OH-Spheroidene OH-Speroidenone OH-Y (trans)

— — —

— — —

_ 467.5 501 467.5

_ 163.552 124.452 163.553

_

_ — — —

_ — — —

_ — — —

_ — — —

_ — — —

— — —

Volume I: Fat-Soluble Pigments

125

Table I. 6 (continued) Q U A N T IT A T IV E SPE C T R O SC O P IC DA TA : M O L A R E X T IN C T IO N C O E F F IC IE N T S (cm ' M 1) O F C A R O T E N O ID S Ethanol

Ether

Amax

e

—

—

—

e

Amax

—

—

—

—

—

—

—

—

—

—

—

—

— — —

— — —

— — —

—

—

—

— — 445 440 —

Petroleum ether

Hexane

Amax

e

Amax

Methanol c

Pyridine

Amax

e

A.max

e

128.0° 156.06 143.06 127.51

—

—

—

—

—

444 462 494 454

—

—

—

—

—

—

454

127.512

—

—

—

—

— — —

451 — —

134.027 — —

— 453 452

105.435I37.523

— —

— —

— —

— —

—

—

—

—

—

—

—

—

—

—

452 453.5

451 478 —

— —

— —

— —

459 458

145.11 — 140.440 _ — —

— _

126 O33 122.0 11

— _

126.037 160.94

— _

—

—

—

— — — — 446 472 505 474

136.54 — 121 74 _ _

— —

_

_

— —

_

_ _ _ 120.84 — 185.24 169.14 157.842 —

_

— — _

— — _

_

— — _

_

_

_

—

—

—

—

—

—

—

—

—

—

—

— —

— —

— —

— —

— —

— —

471 432 456 490

122.91 — 108.323 — 165.523 165.523

— —

— —

— —

—

—

—

—

—

—

461

124.623

—

—

—

—

—

424

136.41

428

—

12547

— —

—

— -

—

— —

_

—

— —

-

_

_

438

136.419

_

—

—

—

—

472

122.249

477.5

85.55

—

—

—

—

—

—

473.5

143.65

—

—

440

157.31

—

—

—

—

—

—

—

—

—

—

—

—

—

—

413 437.5 468 435 461 491 482

—

—

—

—

—

—

456

_ _

_ _

_ _

_

464 470 —

133 7X-49 — 114.7y*49 — —

— —

95.050 147.050 147.050 100.06 135.06 115.06 120.251

—

—

—

—

—

—

—

—

—

—

—

—

146.753

—

—

—

—

CRC Handbook of Chromatography: Plant Pigments

126

Table I. 6 (continued) QUANTITATIVE SPECTROSCOPIC DATA: MOLAR EXTINCTION COEFFICIENTS ( c m O F CAROTENOIDS Solvent: Acetone Pigment Oscillaxanthin P-412 P-450 P-481 P-518

Xmax — _ _ _

e — _ _ _

e

— _

Xmax

— _

— _

— _

_ _ _

_ _

_ _

_ _

— _

_ _

— _ _ _

_ _

— — —

— — —

— — —

Phytoene

—

—

—

—

—

—

—

—

—

—

Phytoene ( 15-m) Phytoene (trans) Phytofluene Phytofluene (trans)

— — — —

— — — —

— — — —

— — — —

— — — —

— — — —

— — — —

— — — —

— — — —

— — — —

Plectanixanthin Pyrenoxanthin Renierapurpunn

474 — —

142.5* — —

— — —

— — —

— 103.1* —

— — —

— — —

—

—

—

—

—

—

—

—

Retro-dehydro-(3-Carotene Rhodopin Rhodopin (trans) Rhodovibrin Rhodoxanthin

— — All — —

— — — — —

— — — — —

— — 83.036 109.036 88.036 84.036 113.036 103.036 — — — — —

— 454 —

Renieratene

— — 477 504 544 467 497 532 — — — — —

— — — — —

— — — — —

473 474 — — 491

167 4* 165.97 — — 134 3*

— — — — 111.5*

— — — — —

— — — — —

— — 500 — —

— — 81 4* — —

— — — — —

— — — — —

104.024 157.024 147.024 93.424 99.024 127.024 — 91.756 138.656 132.356 — _

—

—

—

—

_

_

—

—

—

—

_

_

— — —

— — —

— — —

— — —

— — —

— _ —

—

—

Spheroidenone

—

—

— — —

— — —

109.054

— _

«

— — —

Spheroidene

561

Xmax

— — —

160.8* — 162.04 ’ — — — — — — 462

—

e

— — —

— — 165.442 — —

—

e

— _

_ _

Xmax

Cyclohexane

Philosamiaxanthin Phleixanthophyll Physalien

462 478.5 — — —

—

Xmax

Chloroform

360 543 — — —

Rubixanthin Saproxanthin Semi-a-carotenone Semi-(3-carotenone Sintaxanthin

—

Carbon disulfide

Benzene

30.632 138.132 — — —

— — — —

— — —

7' ,8 , 1 1 ', 12 -tetrahydro-y-carotene— 1,2,1',2'-Tetrahydro-l, 1'474 dihydroxylycopene 5,6,5',6'-Tetrahydrolycopene —

— 90.842

438 465 497 472 533 497 — 428.5 455 485 — _

—

—

—

—

—

—

—

7,8,11,12-Tetrahydrolycopene Torularhodin ToruIarhodin-aJdehyde

— — —

— — —

— — —

— — —

— — —

— 515 —

— — 109.157 _ — —

Spheroidenone (cis) Spirilloxanthin Taraxanthin

— — —

— _

— _

— _

— _

—

— _

— _

—

—

— _ —

Volume I: Fat-Soluble Pigments

127

Table I. 6 (continued) Q U A N T IT A T IV E SP E C T R O SC O P IC DATA: M O L A R E X T IN C T IO N C O E F F IC IE N T S (crn ' M 1) O F C A R O T E N O ID S Ethanol

Ether

Xmax

c

— — — — —

— — — — —

Xmax — — — — —

Petroleum ether

Hexane e

Xmax

— — — — —

— — — — —

e

Methanol

Xmax

— — — — —

— 412 450 482 518

— 452 480 276 286 297 — — 347 317 332 348 367

e

Xmax

— 135.321 176.421 141.721 118.751

e

490 — — _ —

144 81

— —

— —

— —

— —

— —

— —

—

—

—

—

—

—

— — — —

— — — —

— — — —

— — — —

286 286 347 —

4131 49 9* 85.6 1 —

— —

— —

— —

— —

— —

— —

— —

— —

— —

— —

— 490 516

— 140.74 104.74

483 —

181330 — _ _

_

—

—

—

—

467

105.2*

—

_

_

—

—

—

—

—

482.5

122.451

— 442

— 168.2*

— _

151.6 10

491 _

140.321 _ _

—

—

—

—

—

—

378

—

—

—

—

—

—

— — —

— — —

— — —

— — —

395 — —

136.3* — —

413.5 437.5 467.5 — 497 514

_

_

—

— _

_

493 _

_

_

472 473

_

_

_

— — 140.14 — 124.44 32.455 — 41.255 27.855 _ _ _ _ 81.421 _ 27.055 — 56.055 88.555 85.O55

17141 164.56

97.4*

_

— — — _ _ _

493 _

_

_

_

_

_ _

127039

_ _

_ _

_ —

_ _

_ —

_ —

_ _

— _

_

_

_

_

—

—

c

— _

_ _

— —

_

Xmax

67.02-1 — — — — — _ _ _ — _

446

_

Pyridine

_

_

_

_

_

_

—

_

—

—

95.94 _ 150.44 153.04 _ _ 166.64 — 155.9* —

_

—

_

_

_ _ _

_ _ _

_ _

128

CRC Handbook of Chromatography: Plant Pigments Table I. 6 (continued) QUANTITATIVE SPECTROSCOPIC DATA: MOLAR EXTINCTION COEFFICIENTS (cm 'A/ ') OF CAROTENOIDS Solvent: Acetone Pigment Torularhodin-methylester Torulene

Xmax — —

Carbon disulfide

Benzene c

Amax

— —

Tnphastaxanthin

—

—

Violaxanthm

—

—

Xanthophyllepoxide

—

—

a-Zeacarotene (3-Zeacarotene (31 -Zeacarotene Zeaxai#.jin

— — — 452

— — — 133.1*

— —

— 428 453.5 483 430 456 482.5 — — — —

t

Xmax

Chloroform c

Xmax

— —

— —

— —

— —

—

—

—

480 510

88.556 — 134.456 128.456 105.156 — 133 056 100.856 — — — — — — — —

—

—

Cyclohexane

€

Amax — —

— —

96.658 — 84.958

€ — —

—

—

—

—

—

—

—

—

—

— — — —

— — — —

— — — —

— — — —

— — — —

REFERENCES 1. Davies, B. H., Chemistry and Biochemistry o f Plant Pigments, Vol. 2, Goodwin, T. W., Eds., Academic Press, London, 1976, 150. 2. Warren, C. K. and Weedon, B. C. L., J. Chem. Soc., p. 3972, 1958a. 3. Hager, A. and Meyer-Bertenrath, T., Planta, 69, 198, 1966. 4. Isler, O. and Schudel, P., Carotine und Carotinoide, in Wiss. Veroff. dt. Ges. Ernahr., Vol. 9, Steinkopff, Darmstadt, 1963, 54. 5. Aasen, A. J. and Liaaen-Jensen, S., Acta Chem. Scand., 19, 1843, 1965. 6. Bonnett, R., Spark, A. A., and Weedon, B. C. L., Acta Chem. Scand., 18, 1739, 1964. 7. Surmatis, J. D., Acta Chem. Scand., 31, 186, 1966. 8. Campbell, S. A., Mallams, A. K., Waight, E. S., Weedon, B. C. L., Barbier, M., Lederer, E., and Salaque, A., Chem. Commun., p. 941, 1967. 9. Barber, M. S., Davis, J. B., Jackman, L. M., and Weedon, B. C. L., J. Chem. Soc., p. 2870, 1960. 10. Liaaen-Jensen, S., Acta Chem. Scand., 14, 950, 1960. 11. Petracek, F. J. and Zechmeister, L., J. Am. Chem. Soc., 78, 1427, 1956. 12. Liaaen-Jensen, S., Acta Chem. Scand., 19, 116, 1965. 13. Hiyama, T., Nishimura, M., and Chance, B., Anal. Biochem., 29, 339, 1969. 14. Braumann, T. and Grimme, H. L., Biochim. Biophys. Acta, 637, 8, 1981. 15. ChoinoKy, L., Szabolcs, J., and Nagy, E., Liebigs Ann. Chem., 616, 207, 1958. 16. Nelson, J. W. and Livingston, A. L., J. Chromatogr., 28, 465, 1967. 17. Simpson, K. L., Nakayama, T. O. M., and Chichester, C. O., J. Bacteriol., 88, 1688, 1964. 18. Schwieter, U., Bolliger, H. R., Chopard-dit-Jean, L. H., Englert, G., Kofler, M., Konig, A., von Planta, C., Rtiegg, R., Vetter, W., and Isler, O., Chimia (Switz.), 19, 294, 1965. 19. Banthorpe, D. V., Doonan, H. J., and Wirz-Justice, A., J. Chem. Soc. Perkin Trans. I, p. 1764, 1972. 20. Manchand, P. S., Riiegg, R., Schwieter, U., Siddons, P. T., and Weedon, B. C. L., J. Chem. Soc., p. 2019, 1965. 21. Liaaen-Jensen, S., Cohen-Bazire, G., Nakayama, T. O. M., and Stanier, R. K., Biochim. Biophys. Acta, 29, 477, 1958. 22. Nash, H. A., Quackenbush, F. W., and Porter, J. W., J. Am. Chem. Soc., 70, 3613, 1948. 23. Liaaen-Jensen, S., Acta Chem. Scand., 19, 1025, 1965. 24. Barber, M. S., Jackman, L. M., Manchand, P. S., and Weedon, B. C. L., J. Chem. Soc., C, 2166, 1966. 25. Yokoyama, H. and White, M. J., J. Org. Chem., 30, 2481, 1965. 26. Cholnoky, L., Szabolcs, J., Cooper, R. D. G., and Weedon, B. C. L., Tetrahedron Lett., 19, 1257, 1963. 27. Wallcave, L. and Zechmeister, L., J. Am. Chem. Soc., 75, 4495, 1953.

Volume I: Fat-Soluble Pigments

129

Table I. 6 (continued) QUANTITATIVE SPECTROSCOPIC DATA: MOLAR EXTINCTION COEFFICIENTS (cm' ' M~' ) OF CAROTENOIDS Ethanol

Ether c

441

150.23

__ _ _ 450

__ _

lm i«

Petroleum ether

Hexane «

Xmax

e

Xmax

Methanol e

Xmax

e

497 460 484 518

170.8* 123 84 173.34 143.34

— —

— —

— —

— —

—

—

—

—

— — — —

— — — —

— — — —

—

—

—

441

153.259

__ _ _ _ _ 144 5 1 —

__ —

421 426 — —

99.760 104.560 — —

421 428 427 452 480

132.0 1 — 135.81 — 97.060 — 133.74 — 116.64

—

Xmax

— —

—

_

e

Pyridine

REFERENCES 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60.

Zechmeister, L. and Wallcave, L., J. Am. Chem. Soc., 75, 5341, 1953. Isler, O., Montavon, M., Rtiegg, R., and Zeller, P., Helv. Chim. Acta, 39, 454, 1956. Jackman, L. M. and Liaaen-Jensen, S., Acta Chem. Scand., 15, 2058, 1961. Aasen, A. J. and Liaaen-Jensen, S., Acta Chem. Scand., 20, 1970, 1966. Schwieter, U., Riiegg, R., and Isler, O., Helv. Chim. Acta, 49, 992, 1966. Entschel, R. and Karrer, P., Helv. Chim. Acta, 41, 402, 1958. Liaaen-Jensen, S. and Hertzberg, S., Acta Chem. Scand., 20, 1703, 1966. Jensen, A., Acta Chem. Scand., 20, 1728, 1966. Cooper, R. D. G., Davis, J. B., and Weedon, B. C. L., J. Chem. Soc., p. 5637, 1963. Bush, W. V. and Zechmeister, L., J. Am. Chem. Soc., 80, 2991, 1958. Hertzberg, S. and Liaaen-Jensen, S., Acta Chem. Scand., 20, 1187, 1966. Hertzberg, S. and Liaaen-Jensen, S., Acta Chem. Scand., 21, 15, 1967. Goodwin, T. W., Carotenoids, in Modern Methods o f Plant Analysis, Vol. 3, Paech, K. and Tracey, M. V., Eds., Springer-Verlag, Berlin, 1955, 272. Aasen, A. J. and Liaaen-Jensen, S., Acta Chem. Scand., 20, 811, 1966. Ryvarden, L. and Liaaen-Jensen, S., Acta Chem. Scand., 18, 643, 1964. Kjdsen, H. and Liaaen-Jensen, S., Acta Chem. Scand., 25, 1500, 1971. Markham, M. C. and Liaaen-Jensen, S., Phytochemistry, 7, 839, 1968. Kjdsen, H. and Liaaen-Jensen, S., Phytochemistry, 8, 483, 1969. Aasen, A. J., Francis, G. W., and Liaaen-Jensen, S., Acta Chem. Scand., 23, 2605, 1969. Hertzberg, S. and Liaaen-Jensen, S., Phytochemistry, 6, 1119, 1967. Hertzberg, S. and Liaaen-Jensen, S., Phytochemistry, 8, 1259, 1969. Zalokar, M., Arch. Biochem. Biophys., 70, 568, 1957. Nakayama, T. O. M., Arch. Biochem. Biophys., 75, 356, 1958. Liaaen-Jensen, S., Acta Chem. Scand., 17, 303, 1963. Jackman, L. M. and Liaaen-Jensen, S., Acta Chem. Scand., 18, 1403, 1964. Liaaen-Jensen, S., Acta Chem. Scand., 17, 500, 1963. Liaaen-Jensen, S., Acta Chem. Scand., 17, 489, 1963. Davis, J. B., Jackman, L. M., Siddons, P. T., and Weedon, B. C. L., J. Chem. Soc., C, 2154, 1966. Eugster, C. H. and Karrer, P., Helv. Chim. Acta, 40, 69, 1957. Simpson, K. L., Nakayama, T. O. M., and Chichester, C. O., J. Bacteriol., 88, 1688, 1964. Yokoyama, H. and Guerrero, H. C., J. Org. Chem., 35, 2080, 1970. Karrer, P. and Jucker, E., Helv. Chim. Acta, 26, 626, 1943. Petzold, E. N., Quackenbush, F. W., and McQuistan, M., Arch. Biochem. Biophys., 82, 117, 1959.

Volume I: Fat-Soluble Pigments

131

PA PER C H R O M A TO G RA PH Y OF C A R OTENOIDS TA BLE NOTES The chromatography of carotenoids has a long tradition dating back to the beginning of the 20th century. (See References 1 and 12 in Methods section.) After PC had been invented, the introduction of adsorbent-loaded paper(s) led to a rapid development in the area of carotenoid chemistry, since a better resolution compared to the columns used previously was possible. Especially useful was the development of circular PC, which even today can be considered as being superior to other methods in some aspects: its advantages are the excellent resolution, the speed of separation, and the minimal apparatus requirements (glass Petri dishes, appropriate solvents, papers, wicks, and — if possible — a nitrogen tank or source). The carotenoid mixture is dissolved (e.g., in acetone) and 10 to 100 (xg of carotenoid is applied to the center of the paper; the diameter of the spot should not exceed 1 cm. After the application of each portion of the solution, the solvent is evaporated in a stream of nitrogen. After application, a small drop of acetone is added at the center of the paper to concentrate the carotenoids in the form of a ring (= origin). A wick is inserted at the center and the paper, with the wick protruding from the lower surface, is set horizontally in the lower half of a Petri dish with solvent. When the wick is in contact with the solvent, the apparatus is closed, using the upper Petri dish. Chromatography should be carried out in the dark, but nonitrogen atmosphere is required. Different solvent systems have been applied by different authors and can be looked up in Table I. PC 1, together with the applicable literature which is, therefore, not explicitly listed at this place. When carried out with paper with a diameter of 12.5 to 15 cm, a separation will take approximately 15 to 20 min and is, therefore, often more rapid than the development of TLC plates. In the laboratory, papers may be impregnated with a number of adsorbents; however, this poses a certain problem in terms of standardization. Papers have been loaded with the carbonates of calcium, zinc, or magnesium, with magnesium hydroxide or sucrose. Com­ mercially available sorbent-loaded papers seem to give better resolution and reproducibility. For these tables, references dealing exclusively with papers still commercially available have been included. Due to the considerable amount of data, two tables (I. PC 1 and I. PC 2) have been composed. Table I. PC 1 deals with papers from Schleicher and Schiill (Diiren, F. R. G.) and Table I. PC 2 with Whatman papers. The papers mentioned in Tables I. PC 1 and I. PC 2 are either untreated, reverse phase, or sorbent filled. In many cases, simple cellulose paper is not sufficient for separation. Here, the application of adsorbent-loaded papers has led to excellent results. The papers most often used are those filled with kieselguhr (20%, Schleicher & Schiill No. 287), aluminum oxide (Schleicher & Schiill No. 288), aluminum hydroxide (equ. 7.5% A 1203, Whatman AH 21), and silica gel (equ. 22% Si02, Whatman SG 81). Activation of the papers by heat is possible, but the additional activity (up to a strength equivalent to Brockmann grade II, Schleicher & Schiill paper No. 667) is quickly lost due to the uptake of moisture. Since some papers are no longer available, they are not included in the respective tables. The most versatile adsorbent-loaded paper is kieselguhr paper, which is considered to be superior for the separation of cis-trans isomeric xanthophylls. Alumina-impregnated papers separate “ less polar carotenes” (a- from (3-carotene, for example) and give improved resolution of xanthophylls. Aluminum hydroxide and silica gel-loaded papers have been applied in a number of cases. It has to be noted that adsorbent-loaded papers should be prewashed with the appropriate solvent system prior to use when a further analysis of pigments is planned. Even then, eluates from kieselguhr paper are unsuitable for mass spectrometric examination.

— — — —

— — — —

— — — —

— — — —

— — — — — — 1 0 8 — — __ — — — — — — — — — __

— — — —

(3,(3-Carotene-2,3,3'-triol Chlorobactene (trans)

— — — —

— — — —

(3-Carotene (cis) (3-Carotene (trans) y-Carotene y-Carotene (trans)

62"

__

83"

6 8 '°

63 13

8 8 12

8 8 12

95 13

2 4 ' --------------------------------------------------- — — — — — — — __ __ __ __ __ — — — — — — — — __ __ __ — — — — — — — — __ 709 __ — — — — — — — — — 709 __ — — — — — — — — — — __ — — — — — — — — 509 — — 35 1 — — — — — — — — — __ — — — — — — — — __ __ 9510

Astacene Astaxanthin (trans) Asterinic acid Auroxanthin epimer 1 Auroxanthin epimer 2 Bacterioruberin (trans) a-Bacterioruberin (trans) Canthaxanthin (3-Carotene — — — — — — — — — __

__ __ __ __

— — — — __ __ __ __ __ __ — — — — — — — — __ __ — — — — — — — __ __ __ — — — — — — — — __ __

——

Aphanizophyll Apo-4'-carotenoic acid Apo-6 '-lycopenal Apo-8 '-lycopenal

— —

__

— —

__ __

__

4(F

__ __

__ __

__

__

__ __

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__ 2829 __ __ __ __ __ __ __ 2 10 __ __ __ __ __

__

532' 571 0

—

—

264

__

__ __ __ __ __ __ __ __

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9810 __

__

__ __ __ __ __ __ __ __

__ __

7321

__

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98l(i 9810 __

__

__ __ __ __ __ __ __ __

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—

—

55 14 771 4

3914

__ __

6515

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__

4514

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351 0

__ __ __ __ __ __ __ __

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4410

__ __ __

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__ __

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__ __ __ __

9^34

—

__ __ __ __ __ __ __

—

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334

__

__ __ __

P2 P2 P2 P2 P2 P2 P2 P3 P3 SI4 S15 S I 6 SI7 S18 S19 S20 S3 SI4 T2,3 T2,3 T2,3 T2,3 T2,3 T2.3 T2,3 T2,3 T2.3 D1 Dl D1 D1 Dl Dl D1 DI D1

Rf x [oq

P2 SI3 T2,3 Dl

__ __ 241X 6513 56IS __

__ __

P2 P2 SU SI2 T2.3 T2 D1 Dl

__ __ __

P2 P2 P2 P2 P2 S6 S7 S 8 S9 S10 T2 T1 T2 T2 T2 D1 D1 Dl Dl D1

—

P2 S5 T2 D1

Anhydrorhodovibrin (trans)

P2 S4 T2 D1

__ __ __

P2 P2 S2 S3 T2 T2 Dl D1

— — — — __ __ __ __ __ __ — — — — — — __ __ __ __ — — — — __ __ __ __ __ __

PI SI Tl D1

Actinioerythrin Actinioerythrin-bis-a-ketol Aleuriaxanthin

Compound

PaPer Solvent Technique Detection

Table I. PC 1 CELLULOSE PAPERS AND IMPREGNATED CELLULOSE PAPERS — I

_____ _____

_____

__ __

__ __ __ __

__

4 2i

__ __ __ __ __ __

46>

__ __

__ __ __

P4 S2I T2.3 D1

132

CRC Handbook of Chromatography: Plant Pigments

—

—

— —

— — — — — — — — — — — — — — — — — —

— — — — — —

— — — — — — — —

—

—

—

—

—

25“

39

__ __

794

— — — — — — — — — — — — — — — __ __ __ — — — — — — — — — — — _ _ _ — — — — — — — — — — — — —

__ __

8 16

-

-

68

34“

20 (

^

80 7 2 22

90 91"'

63'

59 26

—

64 —

__ __

__ __

23 40“

—

80

01

O 1 H)

____

__ 401° 6

—

—

—

— —

— —

—

—

— — — — — — — — —

—

— —

— — — — 8310 — 5721 — — — 8710 —

—

—

— —

— —

—

—

— 367 — — 3539 — 72l() — 8336

—

— —

—

— 3634 — — — —

— — —

— —

— —

—

—

— — — — — — — — —

—

—

—

— — — — — —

—

—

— —

— —

—

—

— — — — — — — — —

—

— —

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— — — — — —

—

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— —

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— — — — — — — — —

—

— —

—

— — — — 661 —

—

—

— —

— —

—

—

— — — — — — 431 — —

—

— —

—

—

Volume I: Fat-Soluble Pigments 135

PI P2 P3 P4 SI 52 53 54 55 56 57 58 59 510 511 512

= = = = = = = = = = = = = = = =

cellulose Schleicher & Schiill No. 287 (silica impregnated) Schleicher & Schiill No. 597 cellulose, impregnated withtriglyceride petroleum ether-carbontetrachloride = 3:2 acetone-methanol = 49:1 benzene-acetone = 7:3 benzene-acetone = 1:1 benzene-methanol = 99:1 benzene-methanol = 49:1 hexane-acetone = 1 9 : 1 hexane-acetone = 9:1 hexane-acetone = 7:3 hexane-acetone = 4:1 petroleum ether petroleum ether-acetone = 99:1

8

(Part 2), Holman, R. T., Ed., Pergamon Press, New York, 1965, 133.

1964.

11. Liaaen-Jensen, S., Hegge, E., and Jackman, L. J., Acta Chem. Scand., 18, 1703,

Lipids, Vol.

S 13 = petroleum ether-acetone = 49:1 S14 - petroleum ether-acetone = 19:1 S15 = petroleum ether-acetone = 17:3 S16 = petroleum ether-acetone = 9:1 S17 = petroleum ether-acetone = 7:3 S18 = petroleum ether-acetone = 4:1 S19 = petroleum ether-acetone = 1:1 S20 = petroleum ether-isopropanol = 197:3 S21 = methanol-acetone = 5:1 Technique T1 = ambient temperature, chamber saturation, ascending T2 = circular paper chromatography T3 = for co-chromatograms, the 3-divided paper technique was used (Jensen, A., Aasmuntrud, O., and Eimhjellen, K. E., Biochim. Biophys. Acta, 8 8 , 466, 1964) Detection D1 = visual observation

21. Hertzberg, S., Liaaen-Jensen, S., Enzell, C. R., and Francis, G. W., Acta Chem. Stand., 23, 3290, 1969. 22. Aasen, A. J. and Liaaen-Jensen, S., Acta Chem. Stand., 20, 1970, 1966. 23. Arpin, N. and Liaaen-Jensen, S., Phytochemistry, 6 , 995, 1967. 24. Hertzberg, S. and Liaaen-Jensen, S., Acta Chem. Scand., 20, 1187, 1966. 25. Liaaen-Jensen, S., Acta Chem. Stand., 17, 489, 1963.

12, 2751, 1973. 19. Arpin, N. and Liaaen-Jensen, S., Phytochemistry, 8 , 185, 1969. 20. Hertzberg, S. and Liaaen-Jensen, S., Phytochemistry, 5 , 5 5 7 , 1966.

12. Liaaen-Jensen, S., Acta Chem. Scand., 19, 1025, 1965. 13. Liaaen-Jensen, S., Phytochemistry, 4, 925, 1965. 14. Kj0sen, H. and Liaaen-Jensen, S., Phytochemistry, 8, 483, 1969. 15. Liaaen-Jensen, S., Acta Chem. Stand., 17, 500, 1963. 16. Fiasson, J. L. and Arpin, N., Bull. Soc. Chim. Biol., 49, 537, 1967. 17. Jackman, L. M. and Liaaen-Jensen, S., Acta Chem. Stand., 18, 1403, 1964. 18. Arpin, N., Kjosen, H., Francis, G. W., and Liaaen-Jensen, S., Phytochemistry,

REFERENCES

1 Egger, K., Phytochemistry\ 4, 609, 1965. 2. Francis, G. W., Hertzberg, S., Andersen, K., and Liaaen-Jensen, S., Phytochemistry, 9, 629, 1970. 3. Hertzberg, S. and Liaaen-Jensen, S., Acta Chem. Scand., 21, 15, 1967. 4. Aasen, A. J. and Liaaen-Jensen, S., Acta Chem. Stand., 19, 1843, 1965. 5. Aasen, A. J., Francis, G. W., and Liaaen-Jensen, S., Acta Chem. Stand., 23, 2605, 1969. 6 . Hertzberg, S. and Liaaen-Jensen, S. Acta Chem. Stand., 20, 1187, 1966. 7. Arpin, N. and Liaaen-Jensen, S., Bull. Soc. Chim. Biol., 49, 527, 1967. 8 . Smallidge, R. L. and Quackenbush, F. W., Phytochemistry, 12, 2481, 1973. 9. Fiksdahl, A., Mortensen, J. T., and Liaaen-Jensen, S., J. Chromatogr., 157, Il l , 1978. 10. Liaaen-Jensen, S. and Jensen, A., Progress in the Chemistry' o f Fats or Other

Solvent

Paper

Table I. PC 1 (continued) CELLULOSE PAPERS AND IMPREGNATED CELLULOSE PAPERS — I

136

CRC Handbook of Chromatography: Plant Pigments

30. 31. 32. 33.

26. 27. 28. 29.

Liaaen-Jensen, S. and Hertzberg, S., Acta Chem. Scand., 20, 1703, 1966. Markham, M. C. and Liaaen-Jensen, S., Phytochemistry\ 7, 839, 1968. Aasen, A. J. and Liaaen-Jensen, S., Acta Chem. Scand., 21, 970, 1967. Sdrensen, N. A., Liaaen-Jensen, S., Bordalen, B., and Hang, A., Acta Chem. Scand., 22, 344, 1968. Jackman, L. M. and Liaaen-Jensen, S., Acta Chem. Scand., 15, 2058, 1961 Liaaen-Jensen, S., Acta Chem. Scand., 17, 555, 1963. Liaaen-Jensen, S., Acta Chem. Scand., 14, 953, 1960. Jensen, A., Acta Chem. Scand., 20, 1728, 1966.

34. Jensen, A. and Liaaen-Jensen, S., Acta Chem. Scand., 13, 1863, 1959. 35. Liaaen-Jensen, S., Acta Chem. Scand., 13, 2142, 1959. 36. Hertzberg, S. and Liaaen-Jensen, S., Phytochemistry, 8, 1259, 1969. 37. Aasen, A. J. and Liaaen-Jensen, S., Acta Chem. Scand., 20, 811, 1966. 38. Hertzberg, S. and Liaaen-Jensen, S., Phytochemistry, 5, 565, 1966. 39. Surmatis, J. D., Ofner, A., Gibas, J., and Thommen, R., J. Org. Chem., 31, 186, 1966. 40. Liaaen-Jensen, S., Acta Chem. Scand., 19, 1166, 1965. 41. Davies, B. H., Chemistry and Biochemistry o f Plant Pigments, Goodwin, T. W., Eds., Academic Press, London, 1965, 489.

Volume I: Fat-Soluble Pigments 137

— — — — — — _ — — — — — 60 — — — — 00 — — 62 — 81 — 32 64

100

PI S5 T1 D1 1

— — — — — — 10 — — — — — 40 — 00 — — — —

_

— — —

20

— 45 35

P2 SI T1 D1 1

_ 27 — 00 — — — 42 25 38 00 — — — — 22 — 32 — —

— — 100 47 100 29 —

1

P2 S2 T1 D1

_

1

P2 S3 T1 D1 1

P2 S4 T1 D1

— — 65 — — — — — — 61 _ — — — — — — — —

_ — — — 62 — 28 — — — — _ — — — — 71 — — —

60 — — — — — —

20 — — — — — —

PI S4 T1 D1

— 21 100 — — — 100 — — 8 50 — — — 100 — — — 64 — — — — — — _ _ _ _ _ — 60 — — — — — — — 00 64 — — — — 50 — — — — — — _ 25 11 37 — — — 31 — — — 46 — — — 00 61 — _ _ _ _ — — — — 78 — — — — — — — 8 41 — — — — — 66 — 26 — — — — — — — — — —

— 73 50

1

PI S3 T1 D1 1

Canthaxanthin a-Carotene (3-Carotene 7 -Carotene ^-Carotene Cryptoxanthin Diadinoxanthin Diatoxanthin 5,6,5\6'-Diepoxy-(3-carotene Dinoxanthin Flavochrome Flavoxanthin Fucoxanthin Lutein Lycopene 5.6Monoepoxy-a-carotene 5. 6 Monoepoxy-[3-carotene Mutatochrome Neoxanthin Peridinin and Neoperidinin Phytofluene Rhodoxanthin Rubixanthin Taraxanthin Torulene Violaxanthin Zeaxanthin

1

PI S2 T1 D1

Rf x 100

PI SI T1 D1 1

Compound

Paper Solvent Technique Detection Literature

_

1

— — — — — 53 — — — — 7 — — 60 — 80 — 35 85

100 — — — — — —

2

_

P2 S5 T1 D1

— — — — — — — — — — — — — — 49 — — — —

— — — — — — —

P2 S6 T2 D1 3

— — — — — — 43 59 — — — — 27 70 — — — — 5 — — — — — — 48 —

P3 S7 T1 D1 4

__

44

— — 28 73 — — — — 5 23 — — — — — 48

__ __

—

57

54

P3 S9 T3a D1 5

— — 49 74 — — — — 32 51 — — — — — 65

— — — — — — 44 60 —

P3 S8 T3b D1 5

— — 96 — — — 61 62 — — — — 60 — — — — — — — — — — — — —

— — — — — — 54

P3 S7 T3a D1 4

Table I. PC 2 CELLULOSE PAPERS AND IMPREGNATED CELLULOSE PAPERS — II

__

— — 96 — — — 23 33 — — — — 10 — — — — — — — — — — — — —

P3 S10 T3b D1

138

CRC Handbook of Chromatography: Plant Pigments

Paper

REFERENCES

Detection

D1

T3

T2

= circular paper chromatography; for co-chromatograms the 3-divided paper technique was used (Jensen, A., Aasmuntrud, O., and Eimhjellen, K. E., Biochim. Biophys. Acta, 8 8 , 466, 1964) = two-dimensional chromatography: a, first dimension; b, second dimension. = visual observation

= petroleum ether (bp 60— 80°C)-chloroform = 7 : 3 = petroleum ether (bp 60— 110°C)-/z-propanol = 24:1 S10 = petroleum ether (bp 60— 1 10°C)-chloroform = 7 : 3 Technique T 1 = ambient temperature, chamber saturation, ascending

S8 S9

2

1. Valadon, L. R. G. and Mummery, R. S., Phytochemistry , 11, 413, 1972. . Arpin, N. and Liaaen-Jensen, S., Phytochemistry, 8 , 185, 1969. 3. Eskins, K., Sc hoi field, C. R., and Dutton, H. J., J. Chromatogr., 135, 217, 1977. 4. Jeffrey, S. W., Biochem. J . , 80, 336, 1961. 5. Jeffrey, S. W. and Allen, M. B., J. Gen. Microbiol., 36, 277, 1964.

n-hexane n-hexane-acetone = 99:1 n-hexane-acetone = 95:5 n-hexane-acetone = 85:15 n-hexane-acetone = 8:2 petroleum ether-acetone = 19:1 petroleum ether (bp 60— 80°C)-n-propanol = 24:1

— = = = = = =

P3

51 52 53 54 55 56 57

P2

= Whatman paper Chromedia AH 81 (impregnated with aluminum hydroxide equivalent to 7.5% A120 3) - Whatman paper Chromedia SG 81 (impregnated with 22% S i0 2) = Whatman 3 MM chromatography paper

PI

Volume I: Fat-Soluble Pigments 139

Volume I: Fat-Soluble Pigments

141

THIN LAYER CHROMATOGRAPHY TABLES

TABLE NOTES Thin-layer chromatography (TLC) of carotenoids is one of the most modem methods of carotenoid separation. Since the basic principles of TLC of carotenoids are already contained in Volumes I and II of the CRC Handbook Series on Chromatography (edited by Zweig and Sherma23) and other literature (see “ Methods” References 1 to 24 and Tables I. TLC 1 through I. TLC 9), and special precautions have already been discussed in “ Carotenoid Handling and Storage” of the Section “ Carotenoids” of this handbook, only a few remarks are given here. As already pointed out, carotenoids are extremely sensitive to light and oxygen. It is therefore necessary to chromatograph under subdued light or, better, in the dark. Since carotenoids on TLC plates are much more readily oxidized than on paper, the zones obtained should be rapidly eluted once chromatography has been finished. TLC has been performed on a variety of layers using different developing systems. Tables I. TLC 1 through I. TLC 4 contain Rf values from diverse one-component layers. Table I. TLC 5 and I. TLC 6 give Rf data obtained on two-component layers containing silica gel G. Tables I. TLC 7 and I. TLC 8 list Rf values from two- and multicomponent layers not containing silica gel G. Table I. TLC 9 contains values from reverse-phase plates.

— — —

—

—

—

— — — — —

00 — — — —

— —

2 — — 00

Astacene Azafrin Bixin

Canthaxanthin

Capsanthin

Capsorubin

a-Carotene (3-Carotene y-Carotene 5-Carotene e-Carotene

^-Carotene 0-Carotene (3-Citraurin Cryptocapsin Cryptoxanthin

Dehydro-retro-(3-carotene Diadinoxanthin

4,4'-Diapo-£-carotene 4,4'-Diapolycopen-4-al 4,4'-Diaponeurosporen-4-al 4,4'-Diaponeurosporene

9 7

—

4,4'-Diapophytoene 4,4'D iapophytofluene

—

(3-Apo-8'-carotenal

LI SI T1 D l,3 ,4 1

Antheraxanthin

Compound

Layer Solvent Technique Detection Literature

— —

— — — —

— —

— — — — _

— — _ — —

—

—

—

— — _

—

—

LI SI T1 D l,3 2

—

—

— —

— —

— — — —

— —

— — — — _

_

— 1

—

—

—

— — _

LI SI T1 D1 3

_

_

_

—

—

—

— —

—

—

LI S2 T1 D1 7

_

—

—

—

— —

—

—

LI S2 T1 D1 8

_

—

—

—

— —

—

—

LI S3 T1 D1 9

_

— —

— — — —

— —

— —

— — — —

— —

— —

— 52 59 —

— —

— —

— — — —

— —

— —

— — — —

— —

— — — — — 00 — — — — — — — — — — — — — 47 _ _ _ _ _ _

_

—

—

—

— —

—

—

LI SI T1 D l,2 6

_ _ _ _ _ _ _ _ _ _ _ _ _ _ — — — — — — — — — —

— _

—

—

—

_

— —

—

—

LI SI T1 D1 4,5X

_

— —

— 30 36 —

— —

— — — — _

_ — —

_

—

—

—

— —

—

—

LI S4 T1 D1 7

— —

— — — —

— 41

— — — —

— —

96

—

—

—

— —

—

—

LI S5 T1 D1 10

34 25

19 — — 14

55 45

35 — — 27

— —

28 — — — _

11 — — — _ — —

_ _ _ — —

—

—

—

— — _

—

—

LI S7 T1 D l,3 ,4 1

_ _ _ — —

—

—

—

— — _

—

—

LI S6 T1 D l,3 ,4 1

LI S9 T1 D1 12

_

_

_

— —

— — — —

— —

— — 53 —

89 87 80 — —

—

_

—

_

— —

—

—

_

— —

— — — —

85 —

— — — —

— 76 — — —

—

_

—

—

— —

—

—

Rf x 100

LI S8 T1 D5 11

— —

— — — —

— —

— — — —

— — — — —

—

—

50

42 —

—

—

LI S10 T1 D1 13

__

46 35

25 — — 22

— —

23 — — — _

— —

— — — —

— —

— — — — _

— 55 — — —

__ — — — — —

—

_

— — _

—

__

LI S12 T1 D1 3

—

—

— — _

—

—

LI S ll T1 D l,3 ,4 1

Table I. TLC 1 TLC ON SILICA GEL G LAYERS

— __

_

— —

— — — —

— —

— — — — —

__

_

— —

— — — —

— —

— —

— — — —

— —

— 24 — — 47 — — — _ _

91 87 70 — —

__

—

_

—

— _

__

__

_

LI S15 T1 D1 4

__ __ __

__

LI S14 T1 D5 11

— — — — _ _

— 87 _ — —

_

— — _

—

__

LI S13 T1 D1 14

_

_

_

— — — — —

__

_

—

— —

— — — —

— —

— —

— — — —

— —

1

— —

— — — —

— —

__ __ __

LI S18 T1 D1 14

__ __

__ __

— —

— — — —

— —

— —

— — — —

— —

— —

— — — —

— —

— — 75 —

—

100 — —

0 7 0 __ 7

_

0 7

_

0— 7

__ __ 0__ 7

0__ 7

LI S19 T1 D1 3 \ 15

— —

— — — —

__

—

0— 7

__ __ __

— 73 — 73a, 75 — 65 100 __ 100 __

__

_

—

__

__ __

100 100 100

13

15

90

2 __ 5 —

__ __

32

LI S18 T1 D1 15

—

__

__

__ — — — — —

— — —

__

11

___

__ __ __

LI S18 T1 D1 3

— 82 —

__

_

00

_

__ __ _

___

__

LI S17 T1 D1 15

15

__

LI S17 T1 D1 14

84 — — — — — — — _ _ 6

— — — — —

__

_

__ __ __

LI S16 T1 D1 4

142

CRC Handbook of Chromatography: Plant Pigments

—

— — — — —

— — — 00 — _

— — 4 1 — — — — —

Isolutein Isozeaxanthin 4-Keto-a-carotene Lutein Lycopene

Lycopersene Methylbixin Neoxanthin Neurosporene Neurosporene (cis) P-457

Peridinin Pendininol Phytoene Phytofluene Torularhodin-methylester Torulene Viol axanthin P-Zeacarotene Zeaxanthin

diaponeurosporene

4-Hydroxy-4-keto-pcarotene

— — — —

—

tetrahydrolycopene 1,1 '-Di hydroxy-1,2,1' ,2'-

tetrahydro-£-carotene Dinoxanthin Echinenone 4-Hydroxy-p-carotene 4-Hydroxy-4,4'-

—

4,4'-D iapo-7,8, 11,12-

— — 21 12 — — — — —

— — — — — _

— — — — —

—

— — — —

_

_

_

_

_

— — — 10 — — — — —

30 — — — —

— — — — —

—

— — — —

_

_

_

— — — 100 — — — — —

— — — — —

— — — — —

—

— — — —

_

_

_

— — 20 — — — — — —

— — — — — _

— — — — —

—

— — — —

_

_

— — — — — — — — —

— — — — — _

— — — — —

—

— — — 25

_

_

— — — — — — — — —

— — — — — _

— — 34 — —

—

— — — —

_

_

— — — — — — — — —

— — — — — _

— — — — —

—

— — — —

_

_

— — — — — — — — —

— — — — —

— — — — —

—

— — — 12

_

_

33 7 — — — — — — —

— — — — — 00

— — — — —

—

47 — — —

— — 23 17 — — — — —

— — — 9 — —

— — — — —

—

— — — —

—

19

— — 47 34 — — — — —

— — — 21 — _

— — — — —

—

— — — —

_

36 _

_

_

— — — — — — — — —

— — — — —

— — — — 67

—

— — — —

— _

— — — — — — — — —

— — — — — _

— 16 — — —

24

— 55 35 —

—

— — — — — — — — —

— — — — — _

— — — — —

—

— 82 — —

—

_

— — 35 28 — — — — —

— — — 18 —

— — — — —

—

— — — —

_

26

_

— — — — — — — — —

— — — — — _

— — — — —

—

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_ _

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— — — — — — 37 — —

— — 23 — — _

— — — 42 —

—

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— — — — — — — — —

— — — — —

— — — — 52

—

— — — —

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— — — 32 — — — — —

— — — 18 25 _

— — — — —

—

— — — —

—

_

_

— — — — — — — — —

— — — 80 84

— — — — 72

—

— — — —

00

_

_

_

— — — — — — — — —

— — — — —

— — — — —

—

— 10 — —

—

_

_

— — — — — — — — —

— — — — —

— — — — 74

—

— — — —

—

_

_

— — — — — — 19 — —

— — — — —

12 — — 41 —

—

— — — —

—

_

_

21 100 24

100

— — 100 100 100

100 97 — — —

— — — 35 100

—

— — — —

—

_

_

— 0— 7 — — —

— — — 0— 7 56

—

— — — —

—

— — — — — — — — — 0—7 — — — 0—7 — — — 0—7

— — — — —

— — — — —

—

— — — —

—

_

Volume I: Fat-Soluble Pigments 143

LI SI 52 53 54 55 56 57 58 59 510 511 512 513

= = = = = = = = = = = = = =

Lett., 5, 1257, 1963.

7. Taylor, R. F. and Davies, B. H., Biochem. J., 153, 233, 1976. 8 . Fox, D. L. and Hopkins, T. S., Comp. Biochem. Physiol., 19, 267, 1968. 9. Cholnoky, L., Szabolcs, J., Cooper, R. D. G., and Weedon, B. C. L., Tetrahedron

1974

Technique Detection

D3 D4 D5

S14 S 15 S 16 S 17 S 18 S19 T1 D1 D2

petroleum ether-methylene dichloride = 9:1 petroleum ether (bp 100 120°C) petroleum ether (bp 100— 1 20°C)-acetone = 49:1 petroleum ether (bp 90 1 10 C)-benzene = 1.1 methylene chloride-ethyl acetate = 4:1 undecane-methylene chloride = 4 :1 ambient temperature, chamber saturation, ascending visual observation spraying with Rhodamin 6 G in acetone (1% w/v) and observation under UV light = brown spots after exposure to J2 vapor = observation of fluorescence under UV light = spraying with KM n0 4 (0.5% in 50% sulfonic acid)

= = = = = = = = =

ger-Verlag, Berlin, 1967, 259. 15. Davies, B. H., Chemistry and Biochemistry o f Plant Pigments, Goodwin, T. W ., Ed., Academic Press, London, 1965, 514

istry, 13, 2261, 1974. 11 Parihar, D. B., Prahash, O. M., Bajaj, J., Tripathi, R., and Verma, K. K., J. Chromatogr., 59, 457, 1971. 12 Grob, E. C. and Pflugshaupt, R. P., Helv. Chim. Acta, 48, 930, 1965 13. Egger, K. and Kleinig, H., Phytochemistry, 6 , 903, 1967. 14. Bolliger, H. R. and Konig, A., Diinnschichtchromatographie, Stahl, E., Ed., Sprin-

10 Johansen, J. E., Svec, W. A., Liaaen-Jensen, S., and Haxon, F. T., Phytochem-

REFERENCES

Silica gel G (Merck) petroleum ether petroleum ether-acetone = 85:15 petroleum ether-acetone = 49:1 petroleum ether-acetone = 9:1 petroleum ether-acetone = 7:3 petroleum ether-benzene = 95:5 petroleum ether-benzene = 9:1 petroleum ether-benzene = 3:2 petroleum ether-benzene-ethanol = 25:25:2 petroleum ether-benzene-methanol = 4 9 :4 9 : 2 petroleum ether-ether = 99:1 petroleum ether-ether = 19:1 petroleum ether-ethyl acetate-diethyl amine= 58:30:12

1. Taylor, R. F. and Davies, B. H., Biochem. J., 139, 751, 1974 2. Davies, B. H., Goodwin, T. W., and Mercer, E. J., Biochem. J., 81, 40P, 1961. 3. Stobart, A. K., McLaren, J., and Thomas, D. R., Phytochemistry, 6 , 1467, 1967 4. Fiasson, J. L. and Arpin, N., Bull. Soc. Chim. Biol., 49, 537, 1967. 5. Davies, B. H., Jones, O., and Goodwin, T. W., Biochem. J., 87, 326, 1963 6 . Bramley, P. M., Davies, B. H., and Rees, A. F., Liq. Scintill. Counting, 3, 76,

Layer Solvent

Table I. TLC 1 (continued) TLC ON SILICA GEL G LAYERS

144

CRC Handbook of Chromatography: Plant Pigments

Volume I: Fat-Soluble Pigments Table I. TLC 2 TLC ON VARIOUS ONE-COMPONENT LAYERS — I Layer Solvent Technique Detection Literature

LI SI T1 D1 1

L2 S2 T1 D1 2

L3 S3 T1 D1 3

Compound Antheraxanthin Auroxanthin Azafrin Bixin Canthaxanthin Capsanthin Capsorubin a-Carotene (3-Carotene 7 -Carotene ^-Carotene Cryptoxanthin Cryptoxanthinmonoepoxide 4,4'-Diapolycopen-4-al 4,4'-Diaponeurosporen4-al Fucoxanthin 4-Hydroxy-4,4'diaponeurosporene Lutein Luteinepoxide Lycopene Lycopersene Methylbixin Micronone Microxanthin Neoxanthin Phytoene Phytofluene Siphonaxanthin Siphonaxanthinmonolaurate Siphonein Torularhodinmethylester Torulene Trollein Vaucheriaxanthin Violaxanthin Xanthophyll K Xanthophyll K1 Xanthophyll K1S (3-Zeacarotene Zeaxanthin

L3 S4 T1 Dl , 2 4

L4 S2 T1 D1 5

L5 L6 SI S3 T2 T1 D1 D1 1 6

L7 S5 T1 D1 7

10— 11 — 10— 11 10— 11 63 10— 11 10— 11 100 100 100 100 54 —

— — — — — — — — — — — — —

Rf x 100 — 36 — — — — — — — — — — —

— — — — — — — — — — — — —

— — — — — — — — — — — — —

— — — — — — — — — — — — —

16 — — — — — — — 100 — — — 68

— 52 — — — — — — — — — — —

— —

— —

— —

72 72

— —

— —

— —

— —

— —

15 —

— —

— 67

— —

— —

— —

95 —

— — — — — — — — — — — —

22 — — — — — — 11 — — 9 18

— — — — — — — — — — — —

— — — — — — — — — — — —

27 17 — — — — — 7 — — — —

— — — — — — — — — — — —

10— 11 — 100 100 81 — — — 100 100 — —

68 77 — — — 74 79 87 — — 87 —

— —

18 —

— —

— —

— —

— —

— 94

73 —

— — — — — _ _ — —

— 94 — — — — — — — — — 3 — — — 14 — — — — _ _ _ _ _ _ _ _ _ _ — — — — — — — 27

— — — — —

— —

98 — — 76 — — 10— 11 86 — 78 _ 7 2 87 100 — 10— 11 59

145

146

CRC Handbook of Chromatography: Plant Pigments Table I. TLC 2 (continued) TLC ON VARIOUS ONE-COMPONENT LAYERS — I Layer

Solvent

Technique Detection

LI L2 L3 L4 L5 L6 L7 SI 52 53 54 55 T1 T2 D1 D2

= = = = = = = = = = = = = = =

alumina silica gel silica gel G (Merck) MN silica gel N (Machery and Nagel) Kieselgur G (Merck) Mg3(P 0 4)2 polyamide benzene-methanol = 19:1 benzene-methanol = 50:2.5 benzene benzene-methanol-acetic acid = 87:11:2 butan-2-one-methanol-water = 5:5:1 ambient temperature, chamber saturation, ascending development of chromatogram under nitrogen visual observation brown spots after exposure to J2 vapor

REFERENCES 1. Stobart, A. K., McLaren, J., and Thomas, D. R., Phytochemistry, 6, 1467, 1967. 2. Kleinig, H. and Egger, K., Phytochemistry, 6, 1681, 1967. 3. Cardani, C ., Merlini, L., and Mondelli, R., Gazz. Chim. Ital., 92, 41, 1962. 4. Taylor, R. F. and Davies, B. H., Biochem. J., 153, 233, 1976. 5. Kleinig, H. and Egger, K., Z. Naturforsch., 22b, 868, 1967. 6. Davies, B. H., Chemistry and Biochemistry o f Plant Pigments, Goodwin, T. W., Ed., Academic Press, London, 1965, 514. 7. Ricketts, T. R., Phytochemistry, 6, 1375, 1967.

— — — — — — 52 — — — — — — — — — — — — — —

Azafnn B,x,n Canthaxanthin Capsanthin Capsorubin “ ■Carotene 0-Carotene 7Carotene 8-Carotene e-Carotene ^-Carotene Cryptocapsin Cryptoxanthin Cryptoxanthin-5,6-epoxide 4,4'-Diapo-£-carotene 4,4'-Diapolycopen-4-al 4,4'-Diaponeurosporen-4-al 4,4'-Diaponeurosporene 4,4'-Diapophytoene 4,4'-Diapophytofluene 4,4'-Diapo-7,8,l 1,12tetrahydrolycopene

— _ — — — — — — — — — — — — — — — — — — —

— — — — — — — —

— — — — — — — — 35 — — — 37 — — 21 67 58 37

— — — —

— — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — _ — — — — — — — — — — —

— — — — — — — — — — _ — _ 3 — — — 7 — — 00 45 19 7

— — — — — — — — 5 3 _ _ _ — — — — — — — — — — —

— — — —

LI L2 L2 L2 L2 L2 L2 SI S2 S3 S4 S5 S5 S6 Tl T2 Tl T2 T2pr. Tl T2pr. D1 D l,4 D1,2,4DI,4 D I,2,4Dl,2,4DI-3 1 2 3 2 4 3 4

3-Apo-8'-carotenal (3-Apo- lO'-carotenal (3-Apo-12'-carotenal Antheraxanthin

Compound

Layer Solvent Technique Detection Literature

— — — — — — _ _ _ 10 — — — 16 — — 4 61 38 18

— — — — — — — — 16 8 _ — _ — — — — — — — — — — —

— — — —

L2 L2 S6 S7 Tl T2pr. DI.2.4DI-3 3 4

_

__

— —

_ _

_

_ _ __ __ _ _ _ _ _ __ __ _ _ __ _ __ __ _ _ _ _

_

—

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_

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__

35

qq

_

_

_

_ _ _

_ _ _

_ _ _

_

_

_

^ 53 __ 7q 1000

_

__

L9 S22 Tl Dl 1

00 00 00 00 63 00 3 qq 66 - 5 0 0 100 97 100 97 100 97 100 97 100 97 _ __ __ 54 00 __ ______ __ __ __ __ __ __ __ __ _ _

qq

_

q q

L9 S2I Tl Dl I I

_ 00 — — 00 __ 3 1 97 — _ — — — 80 — — 74 — — 41 — — 55 _ _ 84 _ _ _ _ 9 97 g3 __ g3 __ __ __ __ __ __ __ __ __ __ __ __ __ __ __

__ __ __ __ __ _ _

_ _ _ _

— — — — _ _

_

— __

__

_ _ __ __ _ _ _ _ __

L5 L5 1.6 L7 L7 L8 S6 SI6 SI7 SI8 SI9 S20 T2 T2pr, T3 T1 Tl Tl D1.4D1-3 D1 D1 . 2 DI . 2 DI 2 4 9 I 0 I 0 I I I

__ __

_

__ _ 10 10 _ _ _ _ __

— —

__ __ __ _

L4 L5 L5 S15 S2 S5 Tl T2 T2pr Dl D 1.4D I-3 2 4

_ _ _ _ __ _ __ _ _ _ _ __ _ _ _ _ __ _ __

_

_

__ __ __ __

L4 L4 L4 L4 SI2 SI3 S13 S14 Tl Tl Tl Tl Dl D1 D1 Dl 7 7 8 7 7

Rr x 100

L4 L4 L4 S10 SI I SI I Tl Tl Tl D1.4DI Dl 6 7 8

— — — — — — — — — — — — — — — — — — — — — — __ __ __ 26 __ — — — — __ __ __ __ — — — — — — — 87 — — — — — — — 71 — 87 85 91 _ _ _ _ _ _ _ _ _ _ _ — — — — — — — — — — _ _ _ _ _ _ _ _ _ _ _ — — — — — __ __ __ __ __ __ — — — — — __ __ __ __ — — — — — __ __ __ __ — — — — — — — __ __ — — 70 — __ __ — — 70 — — — __ __ __ __ — — 31 — — — __ __ __ — — — — — — __ __ — — — — — — — — __

— — — —

L3 L3 S8 S9 Tl Tl Dl D1 5 5

Table I. TLC 3 TLC ON VARIOUS ONE-COMPONENT LAYERS — II

Volume I: Fat-Soluble Pigments 147

— — _

— — _

_

— _

-

— — — — _ _

Torulene Violaxanthin Xanthophyll (3-Zeacarotene Zeaxanthin

— — _

— — _ __ — 00 35 11 _ _ —

__

-----------------14 98 — 34 — — 98 — 88 — ----------_ _ _ — — — — — — — — — — 18 — — — — 62 — 19 — — 51 — — _ _ _ _ _ — — _ _ _ — — — — —

LyCOpenal Lycopene Lycopersene Methylbixin Mutatoxanthin Neoxanthin Neurosporene Phytoene Phytofluene Phytofluene (cis) Phytofluene (trans) Torularhodin-methylester

—

— —

__ __ __ __

__ __

__ — __ __ —

__ __ _

— — _ _ — — 46 — j7 M — __ __ _

— _ _ _ — 00 56 32 _ _ __

— __ __

— __ __ -

__ __ __ __ __

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-

— 67 — 40 32 __

_ _ _ _

-

— __ __

__ __

_

__ __

L2 L2 S6 S7 T1 T2pr. D l,2,4D l-3 3 4

__ __ __ __ __

L2 L2 L2 S5 S5 S6 T2Pr T2Pr D1,2,4D1,2,4Dl-3 4 3 4

__ __

— — — — — — — — — — — — — — — — —

Dihydroxy-a-carotene 4,4'-Diketo-3-hydroxy-(3-carotene Echinenone Hydroxy-a-carotene 4-Hydroxy-4,4'-diaponeurosporene Isozeaxanthin Lutein , Luteoxanthin a Luteoxanthin b

__ — __ — —

L2 S4 T2 D1,2,4D1,4 3 2

L2 S3

__

L1 L2 S1 S2 T2 D1 D l,4 1 2

__ __ __ __ —

Compound

^ _,°yent m^Ue tection L,teratUre

_

_

_

_

_

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_ _ _

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_

_ _ _

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__ __

_ _

L4 S15 T1 D1 7

_ _ _ _ _ _ _ — — _ _ _ _ _ _ _ _ _ _ _ _ __ __ __

__ __

__

__ __ ___

L4 L4 S13 S14 T1 T1 D1 D1 8 7

__

_ _

_

_

_ _

_

_ _

— — — — 64 57 — --------- ------------------------------------------------------------ -----30 37 __ __ __ __ __ __ 34 __ __ _ _ -----------------------------------------_

__

_ —

_ — _

_

__

__ __

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L4 S13 T1 D1 7

L5 L5 S6 S16 T2 T2pr. D l,4 Dl-3 2 4

q

_

_

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i

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I 23 63 _ _

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—

L6 L7 L7 L8 S17 S18 S19 S20 T3 T1 T1 T1 D1 D1 2 D1 2 D1 9 10 ’ 10 ’ 11

L9 S22 T1 D1 11

10 00

L9 S21 T1 D1 11

__ _

_ —

__ _

__ 19

_ _ _ — —

__ 97

__ _

_ _ 38 —

__ __ 500

98 _ 500

I I I _ to I 43 _ _ _ _ _ _ 97 _ _ _ _ _ _ _ _ _ _ _ g| , 3 __ — 81 82 __ — — ™ 69 20 _ _ _ _ _ _ _ 40 _ _ _ _ _ _ _ _ _ _ _ _ _ 4o _ _ _ _ _ _ 40 _ _ _ _ _ _ __ __ __ __ 00 94 83

—

__ __ ________ __ __ __ __ __

L5 S5 T2pr. Dl-3 4

I _ _ _ _

__ __

_

__ __ _ __ __

m

L4 S12 T1 D1 7

------------------------------------------------------ -----_ _ _ _ _ _ _ _ _ 84 _ _ _ _ _ _ _ _ _ 83 _ _ ----------------------------------------_ _ _ _ _ _ _ _ _ _ _ —

— ---__ __ _

L4 S ll T1 D1 8

Rf x

L4 S ll T1 D1 7

_ __ __

__ __

__ __ __ __ __

L4 S10 T1 Dl,4 6

__ __

— __ __

__ 100 __ __ __

L3 S9 T1 D1 5

—

_ _ _ _

— __ __

20 __ __ __ __

L3 S8 12T1 D1 5

Table I. TLC 3 (continued) TLC ON VARIOUS ONE-COMPONENT LAYERS — II

148

CRC Handbook of Chromatography: Plant Pigments

Solvent

Layer

LI L2 L3 L4 L5 L6 L7 L8 L9 SI 52 53 54 55 56 57 58 59 510 511

= = = = = = = = = = = = = = = = = = = =

REFERENCES

Detection

Technique

= = = = = = = = = = = = =

= = = = =

S12 S13 S14 S15 S16 S17 S18 S19 S2 0 S21 S22 T1 T2

T3 D1 D2 D3 D4

hexane-benzene = 4:1 hexane-ether = 1:1 hexane-ethyl acetate = 9:1 hexane-ethyl acetate = 2:1 hexane-ether = 99.5:0.5 hexane-ethyl acetate = 19:1 hexane-«-propanol = 99.9:0.1 hexane-rt-propanol = 99:1 benzene-petroleum ether = 9 :1 benzene carbon tetrachloride ambient temperature, chamber saturation, ascending previous to the actual use, the plates are washed with chloroformmethanol = 1:1, then with acetone; pr. indicates layer thickness of 1 mm development of chromatograms under nitrogen visual observation observation under UV light spraying with 0.5% aqueous KM n0 4 iodine vapor

1. David, H. L., J. Bacteriol., 119, 527, 1974. 2. Kushwaha, S. C. and Kates, M., Biochim. Biophys. Acta, 316, 235, 1973. 3. Taylor, R. F. and Davies, B. H., Biochem. J., 139, 751, 1974. 4. Kushwaha, S. C., Pugh, E. L., Kramer, J. K. G., and Kates, M., Biochim. Biophys. Acta, 260, 492, 1972. 5. Ungers, G. E. and Cooney, J. J., J. Bacteriol., 96, 234, 1968. 6 . Taylor, R. F. and Davies, B. H., Biochem. J., 153, 233, 1976. 7. Cardani, C., Merlini, L., and Mondelli, R., Gazz. Chim. Ital., 92, 41, 1962. 8 . Merlini, L. and Cardillo, G., Gazz. Chim. Ital., 93, 949, 1963. 9. Stobart, A. K., McLaren, I., and Thomas, D. R., Phytochemistry, 6, 1467, 1967. 10. Buckle, K. A. and Rahman, F. M. M., J. Chromatogr., 171, 385, 1979. 11. Bolliger, H. R. and Konig, A., Diinnschichtchromatographie, Stahl, E., Ed., Springer-Verlag, Berlin, 1967, 253.

Alox 25-22 (Brinkman Instruments) Alumina G (type E, Merck) CaCO, (specified as low in alkalinity) Silica gel G (Merck) Silica gel H (Merck) Kieselguhr G (Merck) Cellulose MN 300 (Machery and Nagel) MgO Mg 3(P 0 4)2 hexane-acetone = 99:1 chloroform-ether = 99:1 hexane-benzene = 95:5 hexane-benzene = 1 : 1 hexane-ether = 99.75:0.25 hexane-ether = 99:1 hexane-ether = 97:3 ether ether-petroleum ether = 1 : 1 chloroform ether-hexane = 9:1

Volume I: Fat-Soluble Pigments 149

— — — — — — — — —

10 0

— — — — — — — — —

—

|3-Carotene y-Carotene 5-Carotene e-Carotene Cryptoxanthin Dehydroadonirubin Diadinoxanthin Diatoxanthin Dihydroxy-excarotene Dihydroxy-3,4-dehydro-a-carotene

— — — — — — — — — — — — —

Tl Dl 2

S2

L2

— — — — — — — — — — — — —

1

LI Sl Tl Dl

Alloxanthin (trans) Antheraxanthin (3-Apo-8'-carotenal P-Apo-10'-carotenal (3-Apo-12'-carotenal Astacene Auroxanthin Azafrin Bixin Canthaxanthin Capsanthin Capsorubin a-Carotene

Compound

La>er Solvem Technique Detection Literature

__

84

— — — —

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—

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— 00 — — — — — 00

L3 S3 Tl Dl 3

__

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__

97 __ __

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— — — — —

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_

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L5 S9 Tl Dl 7

__

_ __ __ __ 99 __ __ __ _ __ __ __ __

__ __ __ __ __ __ __ __

L5 SIO Tl D1 5

__

__ __ __ __

_

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25— 30

__ __ __

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Rf x 100

8

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72 65

L7 SI3 Tl Dl 3

_

__

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45

—

__

__ __ __

— 58

__ __

32

__ __ __

__

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L8 SI4 Tl Dl 9

_

__

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—

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_

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L8 L8 L8 L8 SI4 SI4 SI4 S14 Tl Tl Tl Tl D1 Dl Dl Dl 10 11 12 13

Table I. TLC 4 TLC ON VARIOUS ONE-COMPONENT LAYERS — III

__

_

_

__

—

__ __ __ __ __ __ __ __

L8 S2 Tl Dl 2

_

100

__

—

__

__ __ __ __

95

__

L8 SI5 Tl Dl 14

_

—

—

__

__ __ __

__

__

L9 S I6 Tl Dl 15

150

CRC Handbook of Chromatography: Plant Pigments

Lutein-5,6-epoxide Lycopenal Lycopene Methylbixin Micronone Microxanthin Neoxanthin Prolycopene Renierapurpurin Renieratene Rhodoxanthin Siphonaxanthin Siphonaxanthinmonolaurate Siphonein Spheroidene Torularhodindimethylester Trollein Vaucheriaxanthin Violaxanthin Xanthophyll K Xanthophyll K1 Xanthophyll K1S Zeaxanthin

3,4-Diketo-acarotene Echinenone Fucoxanthin Isorenieratene Lutein

— — — — — — — — — — — — —

— — —

— — —

— —

— — —

— — —

— —

— 00

— — 00

— — 00

— — 00 00 — — — — — — — — —

— — — 00

— — — — 78 — _ _

— — — — — — — — 50 69 — — —

—

20

—

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— — —

— — — — — — 20 — — — — — —

75

— — —

—

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— — —

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— — — — — — — — — — 33 — —

— —

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— — —

— — — — — — 35 — — — — — —

90 — — — —■ — — 76

—

— —

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— — — 83

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— — — — — — —

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—

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16 — 33 25 34 22 32

34 — —

— — — 23 —

— — — — — — — —

— — 44 27 25

33

35

— — — — —

—

28

— — — — — — —

— — —

—

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87

— — — — — — 29

— — —

—

98

— — — — —

—

—

27

10 27

26

24

— — — — —

—

10 28 — —

1322

35

— — —— 31—

33

30

—

Volume /: Fat-Soluble Pigments 151

152

CRC Handbook of Chromatography: Plant Pigments

Table I. TLC 4 (continued) TLC ON VARIOUS ONE-COMPONENT LAYERS Layer

Solvent

Technique Detection

L1 L2 L3 L4 L5 L6 L7 L8 L9 Sl S2 S3 S4 SS S6 S7 S8 S9 S 10 S II Sl2 Sl3 Sl4 SIS Sl6 Tl T2 Dl D2

III

= Alumina G = CaC03 = Ca(OH), = Cellulose MN 300 = kieselguhr =MgO = Mg(OH),·MgC0 3 = polyamide = Silica gel H =petroleum ether-acetone = 199:1 = petroleum ether = methylene chloride-petroleum ether = 95:5 =petroleum ether-acetone-n-propanol = 90:10:0.25 = petroleum ether-carbon tetrachloride = 6:4 = petroleum ether-benzene-chloroform-acetone = 50:35: 10:5 =petroleum ether-acetone-n-propanol = 90:10:0.45 = petroleum ether-acetone = 200: I = petroleum ether-benzene = I: I = petroleum ether-n-propanol = 99: I = petroleum ether-benzene = 3: I =petroleum ether (bp 100-140°C)-benzene = 8:1:1 =petroleum ether-acetone = 25:3 =petroleum ether-methanol-butanone-2 = 8:1:1 =petroleum ether (bp IOO-I40°C)-methanol-butanone-2 = 8:1:1 =petroleum ether-benzene = 9:1 = ambient temperature, chamber saturation, ascending = development of chromatogram under nitrogen =visual observation = observation under UV light (A. = 366 nm)

REFERENCES I. Cooper, R. D. G., Davis, J, B., and Weedon, B. C. L., J. Chem. Soc., p. 5637, 1963. 2. Ungers, G. E. and Cooney, J, J., J. Bacterial., 96, 234, 1968. 3. Davies, B. H., Chemistry and Biochemistry of Plant Pigments, Goodwin, T. W., Ed., Academic Press, London, 1965, 489 4. Buckle, K. A. and Rahman, F. M. M., J. Chromatogr., 171, 385, 1979. 5. Bolliger, H. R. and Konig, A., Diinnschichtchromatographie, Stahl, E., Ed., Springer-Verlag, Berlin, 1967, 253. 6. Stobart, A. K., McLaren, J,, and Thomas, D. R., Phytochemistry, 6, 1467, 1967. 7. Barber, M. X., Jackman, L. M., Manchand, P. S., and Weedon, B. C. L., J. Chem. Soc., p. 2166, 1966. 8. Bramley, P. M. and Davies, B. H., Phytochemistry, 14, 463, 1974. 9. Egger, K., Phytochemistry, 4, 609, 1965. 10. Ricketts, T. R., Phytochemistry, 6, 1375, 1967. II. Egger, K. and Kleinig, H., Phytochemistry, 6, 903, 1967. 12. Kleinig, H. and Egger, K., Phytochemistry, 6, 611, 1967. 13. Kleinig, H. and Egger, K., Phytochemistry, 6, 1681, 1967. 14. Kleinig, H. and Egger, K., Z. Naturforsch. B, 22, 868, 1967. 15. Qureshi, A. A., Manok, K., Qureshi, N., and Porter, J, W., Arch. Biochem. Biophys., 162, 108, 1974.

Volume I: Fat-Soluble Pigments Table I. TLC 5 TLC ON VARIOUS TWO-COMPONENT LAYERS (CONTAINING SILICA GEL G) — I Layer Solvent Technique Detection Literature

LI SI T1 D1 1

L2 S2 T1 D1 2

L3 S2 T1 D1 2

Compound

Solvents

Technique Detection

L4 S4 T1 D1 3

L5 S5 T1 D1 4

L6 S6 T1 D1 5

L7 S7 T1 D1 3

— — — — — — — — — 90 — — — — — —

48 36 50 00 00 00 — — — 100 — — — — — — — — — —

— — — — — — 51 38 — 96 — — — — 63 — — — 17 —

Rf x 100

(3-Apo-8'-carotenal (3-Apo-10'-carotenal [3-Apo-12'-carotenal (3-Apo-8'-carotenoic acid (3-Apo-10'-carotenoic acid (3-Apo-12'-carotenoic acid Bixin Canthaxanthin Canthaxanthin (cis) (3-Carotene Cryptoxanthin Echinenone 4-Hydroxy-4'-keto-(3-carotene Isocryptoxanthin Isozeaxanthin Lutein Lycopene Rhodoxanthin Zeaxanthin Zeinoxanthin Layer

L4 S3 T1 D1 3

LI L2 L3 L4 L5 L6 L7 SI 52 53 54 55 56 57 T1 D1

= = = = = = = = = = = = = = = =

— — — — — — — 50 48 — — 72 40 55 26 — — — — —

— — — — — — — — — — — — — — — — — — — 48

— — — — — — — — — — — — — — — — — — — 65

— — — — — — — 43 — — 34 82 — — — — — 16 — —

— — — — — — — — — — 74 — — — — 55 — 94 57 —

6

— — —

Silica gel G with fluorescence indicator (Eastman chromagram sheets 6060) Silica gel G-lime = 1:4 Silica gel G-lime = 1:6 Silica gel G-Ca(OH )2 = 1:6 Silica gel G-MgO = 1 : 1 Silica gel G-plaster of Paris = 8:1 Silica gel G-rice starch = 98:2 benzene-methanol = 97:3 benzene-Ai-butanol = 49:1 benzene benzene-methanol = 49:1 benzene-petroleum ether = 1:1 cyclohexane-acetone = 47:3 ether-/i-hexane = 7:3 ambient temperature, chamber saturation, ascending visual observation

REFERENCES 1. Hsieh, L. K., Lee, T. Ch., Chichester, C. O., and Simpson, K. L., J. Bacteriol., 118, 385, 1974. 2. Livingstone, A. L. and Knowles, R. E ., Phytochemistry, 8 , 1311, 1969. 3. Bollinger, H. R. and Konig, A., Dunnschichtchromatographie, Stahl, E., Ed., SpringerVerlag, Berlin, 1967, 253. 4. Bramley, P. M. and Davies, B. H., Phytochemistry, 14, 463, 1974. 5. Singh, H., John, H., and Cama, H. R., J. Chromatogr., 75, 146, 1973.

153

__

10

— — 60 — — — — — — — — — —

86

Lycopene

— — — — — — — — — —

1

LI SI T1 D1

Alloxanthin (3-Apo-8'-carotenal (3-Apo-10'-carotenal (3-Apo-12'-carotenal (3-Apo-8'-carotenoic acid (3-Apo-10'-carotenoic acid (3-Apo-12'-carotinoic acid Astaxanthin Canthaxanthin “ -Carotene fi-Carotene (3, (3-Carotene (3,e-Carotene 7 -Carotene ^-Carotene (3-Citraurin Crocoxanthin a-Cryptoxanthin (3-Cryptoxanthin Diadinoxanthin Diatoxanthin Echinenone 4-Hydroxy-a-carotene Isorenieratene Lutein

Compound

Layer Solvent Technique Detection Literature

_

__ __

__ __

61 __ __

57

__ __

__ __

77

_

_

— — — — — — — — — — — — —

L3 S3 T1 D1 3

— — — — __ __ __ —

__

— — — — — — — — — — — — — _

L2 S2 T2 D2,3 2

_

_

_

_

4

j

__ __ __ __ __ __ __ __ __ __

__ __

__ __ __ __ __ __ __ __ __ __ __ __ __

_

_

_

5

__ __

__ __

38

__ __ __

67 96

__ __ __

6

__ __

82 59

__ __

_

_

__

71

__ __ __ __ __ __ __ __ __

__ __

l0 _ 20

__ __ __ __ __ __ __ __ __ __

40_50 __

__ __

84

88

__ __ __ __ __ __

Rf x 100

6

L6 S9 T1 D1

__ __ __ __ __ __

L5 S8 T1 D1

_ _ — —

__ __

— — — — — —

__ __ __ __

_

_

_

4

L4 S7 T1 D1

__ __ __ __

10 0

10 0

—

82

66

__ __ _ —

— — — — — — —

L4 S6 T1 D1 4

— — — — — — —

L3 S5 T1 D1 3

— —

__

42 — — — — — — —

L3 S4 T1 D1 3

57

52

__ __ __ __ __ __ __ __

58

__ __ __ __ __ __ __ __ __ __

__

75

__ __

__ __ 83

96 92

__ __

__ __ __ __ __ __

L7 S ll T1 D1 7

92 92

__ __

__ __ __ __ __ __

L7 S10 T1 D1 7

I

__

__ __ __ __ __ __

—

__ __

I

__ __ __ __ __

I

__ __ __ __

—

I

_

__ __

__ __ __ __ —

— —

__ __

22 12

__ __ __ __

— 81

__ __

__ __ __ __ __ __

L ll S14 T1 D1

_ —

__ __

__ __ _ —

__ __ __ __ __ __

L9 L10 S12 S13 T1 T1 D1 D1 8 6 9

__ __ __ __ __ __

L8 S12 T1 D1 8

Table I. TLC 6 TLC ON VARIOUS TWO-COMPONENT LAYERS (CONTAINING SILICA GEL G) — II

9

53

I

_

I

_

__ __

__ __

__ __

_ 100

00

_ 100

00

00

^3

00

71

__ 57

57

9

L ll S16 T1 D1

__

LI1 S15 T1 D1

280 0

00

154

CRC Handbook of Chromatography: Plant Pigments

1. 2. 3. 4. 5. 6. 7. 8. 9.

L3 L4 L5 L6 L7 L8 L9 L10 L ll SI 52 53 54 55

LI L2

— — 35— 40 — — —

— — — — — —

— — — — 48 —

38 — — — — —

— 25 — — — —

— — — — — —

— — — — — — S6

— — — — — —

— — — — — —

— — — — — 27

— — — — — 47

— — — 75 — —

— — — — — —

— — — — — —

— — — — — —

= petroleum ether-acetone-benzene-isopropanol = 69.5:25:4:1.5 S7 = petroleum ether-acetone = 12:1 S8 = petroleum ether-benzene = 4:1 S9 = petroleum ether-benzene = 49:1 S10 = petroleum ether-benzene = 3:2 Sl l = petroleum ether-methylene chloride = 9:1 S12 = hexane-acetone = 49:1 S13 = petroleum ether-benzene = 9:1 S14 = petroleum ether-acetone = 75:25 S15 = petroleum ether-acetone = 9:1 S16 = petroleum ether-diethyl ether = 75:25 Technique T1 = ambient temperature, chamber saturation, ascending Detection D1 = visual observation D2 = spraying with a solution of Rhodamin 6G in acetone (1% w/v) D3 = observation under UV light

— — — — — —

REFERENCES

= Silica gel G with fluorescence indicator (Merck) = Silica gel G (Merck) impregnated with 3% AgN 03 (w/w) = Silica gel G (Merck)-aluminum oxide = 1 : 1 = Silica gel G (Merck)-calcium carbonate = 1 : 1 = Silica gel G (Merck)-calcium hydroxide = 1 : 4 = Silica gel G (Merck)-calcium hydroxide = 1 : 6 = Silica gel G (Merck)-Kieselguhr = 3:1 = Silica gel G (Merck)-lime = 1 : 4 = Silica gel G (Merck)-lime = 1 : 6 = Silica gel G (Merck)-magnesium oxide = 1 : 1 = Silica gel G (Merck)-plaster of Paris = 8:1 = petroleum ether-acetone = 85:15 = petroleum ether-ether = 7:3 = hexane-acetone = 3:1 = hexane-acetone = 1 3 : 7 = hexane-acetone = 7:3

— — — — — —

Hsieh, L. K ., Lee, T. C ., C hichester, C. O ., and Simpson, K. L ., J. BacterioL, 118, 385, 1974. Bramley, P. M. and Davies, B. H ., Phytochemistry, 14, 463, 1974. C hapm an, D. J ., Phytochemistry, 5, 1331, 1966. B jornland, T ., Phytochemistry, 21, 1715, 1982. Cooper, R. D. G ., Davis, J . B ., and W eedon, B. C. L ., J. Chem. Soc. London, p. 5637, 1963. Davies, B. H ., Chemistry and Biochemistry o f Plant Pigments, Goodwin, T. W., Ed., Academic Press, London, 1965, 489. P a rih ar, D. B., P rahash, D. M., B ajaj, J ., T ripathi, R. P., and Verma, K. K., J. Chromatogr., 59, 457, 1971. Livingstone, A. L. and Knowles, R. E ., Phytochemistry, 8, 1311, 1969. Singh, H ., Jo h n , J ., and C am a, H. R ., J. Chromatogr., 75, 146, 1973.

Solvent

Layer

Monadoxanthin Neoxanthin Phytoene (3-Zeacarotene Zeaxanthin Zeinoxanthin

Volume I: Fat-Soluble Pigments 155

Alloxanthin Anhydroeschscholtzxanthin Antheraxanthin Aphanizophyll (3-Apo-(3-carotenoic acid Astacene Auroxanthin Caloxanthin Canthaxanthin Capsanthin Capsorubin a-Carotene (3-Carotene y-Carotene e-Carotene 3-Carotene-5, 6 ,5 ', 6 '-diepoxide Crocoxanthin Cryptoxanthin Cryptoxanthin-5,6,5 ', 6 '-diepoxide Cryptoxanthin-5', 6 '-monoepoxide Diadinoxanthin Diatoxanthin Echinenone Eschscholtzxanthin Euglenanone Fucoxanthin Heteroxanthin 3'-Hydroxyechinenone

Compound

Layer Solvent Technique Detection Literature

_ _ — —

6

— 76 52 — — _ — 20 83 _ _ — — — — — — — — — 41 28 —

LI SI T1 D1 1

— _ 72 — —

88

20 — — — — _ — — — _ _ 97 92 — 96 — 75 77 — 79 — —

LI SI T1 D1 2

— — 52 — — _ — — — _ _ — — 71 — — — — — — — — — — _ — — —

LI SI T1 D1 3

_

_ _

_

4

— — 80

—

88

— — — — — — 77 — — — —

— 24 83

— — — 5 —

LI SI T1 D1

_

_ _

_

5

— 12 —

— — 82 79 41 28 — —

86

— 92 — —

— — —

— — 52 — —

LI SI T1 D1

_

_ _

_

1

— — —

— — — — — — — — — — 48 — —

— 32 —

21 — — — —

LI S2 T1 D1

_

_ _

_

1

— — —

— 92 — — — — — — — — — — —

13 — 77

— — — — —

LI S3 T1 D1

_

_ _

_

6

L3 S5 T1 D1 7

— — —

— — — — — — — — — — — — —

— — —

— — — — —

37

27 — —

52

— 100 — — — — 70 — — — — 92 22 68 44 — —

19

— 100 — — — — 62 — — — — 91 12

— — 65 42

50

— — — — 38

L3 S6 T1 D1 7

— — 58 24

34

— — — — 28

Rf x 100

L2 S4 T1 D1

Table I. TLC 7 TLC ON TWO- AND MULTICOMPONENT LAYERS

__ __

__ __ __ __

__ __ —

__

—

__ __ __

__ __

— — — — — — — — — — — — — — — — — — —

L4 S12 T2 D1

__

L3 S ll T1 D1 7 8

— —

7

L3 S10 T1 D1

— — — — — — — — — — — — 5 0 0 0 0 42 25 7 — — — — — — 55 37 20 62 42 15 60 42 15 — — — 25 10 00 — — — — — — — — — — — — 39 21 4 — — — — — __ — — __ — — __ 35 18 2 8 1 00 54 34 9 95 85 55

7

L3 S9 T1 D1

— — — — 15 72 — — 80 81 76 — 80 — — — — 74 — — — — 72 22 80 98

L3 S8 T1 D1 7

— — — — 30 69 — — 79 79 74 — 100 — — — — 76 — — — — 90 25 81 84

L3 S7 T1 D1 7

156

CRC Handbook of Chromatography: Plant Pigments

LI L2 L3 L4

SI 52 53 54 55

Layer

Solvent

= = = = =

= = = =

— — — — 65 — — — — _ _ — — — — — 61 _ _ — — — — 74 39

— — — — _ — — — — _ _ — — 49 — — — _ _ — — — — _ —

— — — — _ — — 75 — _ _ — — _ 10 — — _ _ — 36 — — _ 39 _ _ — — — — — _

—

6

— 63 — — _ — — — — _ _ — 3 _ — — — 49 — — —

— — — — _ _

_ — — — —

— — — 54

_ _

_

— — — — 63 _

— — 35 — — —

44 — — 22

50 52 67 31

_

_ _

S6

— — — — — _

— — _ — — — 56

— — — — _ — — — — 20 37 — 42 — — — 42 60 10 — — — 52 54 — — — 30 30

6

45

29 — 22 — — — 28

90

g

— — — 56 57 — 100 — —

95

— — — 34 35 — 100 — —

— — — 92 93 — 95 — — 75 22 40 — 82 — — — 43 64 9 — — — 76 78

— — — 91 95 — 30 — — 60 20 32 — 96 — — — 40 45 2 — — — 93 82

— — — — — — 36 10 68 45 14 — — — 00 00 00 — — — — — — 5 00 00 7 00 00 8 0 0 0 0 — — — 90 78 40 — — — — — — — — — 14 7 1 15 4 00 1 0 0 0 0 — — — — — — — — — 88 73 35 55 35 10

— — — 57

— — — — 70 — — — — — — — — 83 — — — — — — — — — 83 —

= petroleum ether (bp. 100— 120°C)-methanol-butan-2-one =4 : 1 : 1 S7 = petroleum ether (bp. 100— 1 20°C)-methanol-butan-2-one =2 : 1 : 1 S8 = methanol-butan-2-one = 1:1 S9 = water-methanol-butan-2-one = 1:5:5 S10 = water-methanol-butan-2-one = 1:3:3 S ll = water-methanol-butan-2-one = 2:3:3 S12 = methanol-water-benzene = 90:12:4 Technique T 1 = ambient temperature, chamber saturation, ascending T2 = two runs: the plate is developed first with chloroform, then with solvent system indicated Detection D1 = visual observation

— — _ 12 — — _ _ _ _ _ _ — — — — 19 — 69 — 74 _ 39 59

_ _

_

— — — —

C aC 0 3-M g0-C a(0H )2 = 15:3:2 MgO-Kieselguhr G (Merck) = 1 : 1 polyamide-Cellulose MN 300 = 85:15 powdered polyethylene-Cellulose MN 300 = 23:2; 25 g of the mixture are suspended in 75 m€ methanolchloroform = 4:1 “ benzine’’-acetone-chloroform-methanol = 50:50:40:1 “ benzine’ ’-acetone-chloroform-methanol = 50:50:40:8 “ benzine’’-chloroform-acetone = 50:40:30 benzene-acetone = 3:2 petroleum ether (bp. 100— 120°C)-methanol-butan-2-one = 8 :1:1

3Hydroxy-3'-keto-a-carotene 4Hydroxy-4'-keto-(3-carotene Isocryptoxanthin Isozeaxanthin Lutein Lutein (trans) Luteindipalmitate Lutein-5,6 -epoxide Luteoxanthin Lycopene Lycophyll Lycoxanthin Myxoxanthophyll Neoxanthin Nostoxanthin Oscillaxanthin Retroanhydro-(3-cryptoxanthin Rhodoxanthin Rubixanthin Torularhodin Trihydroxy-a-carotene Vaucheriaxanthin Vaucheriaxanthinester Violaxanthin Zeaxanthin

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CRC Handbook of Chromatography : Plant Pigments Table I. TLC 7 (continued) TLC ON TWO- AND MULTICOMPONENT LAYERS REFERENCES

1. 2. 3. 4. 5. 6.

Hager, A. and Stransky, H., Arch. Mikrobiol., 71, 132, 1979. Hager, A. and Stransky, H., Arch. Mikrobiol., 73, 77, 1970. Hager, A. and Stransky, H., Arch. Mikrobiol., 72, 68, 1970. Stransky, H. and Hager, A., Arch. Mikrobiol., 72, 84, 1970. Stransky, H. and Hager, A., Arch. Mikrobiol., 71, 164, 1970. Cyronak, M. J., Britton, G., and Simpson, K. L., Phytochemistry, 16, 612, 1977. 7. Bolliger, H. R. and Konig, A., Diinnschichtchromatographie, Stahl, E., Ed., Springer-Verlag, Berlin, 1967, 253. 8. Schenk, J. and Dussler, H. G., Pharmazie, 24, 116, 1969.

Table I. TLC 8 TLC ON “ THINLAYER A” Layer Solvent Technique Literature

LI SI T1 1

LI S2 T1 1

LI S2 T1 1

LI S3 T1 1

Compound* Antheraxanthin a-Carotene (3-Carotene y-Carotene ^-Carotene Lutein Lutein-5,6 -epoxide Lycopene Neoxanthin Rhodoxanthin Violaxanthin Zeaxanthin Layer

LI

Solvent

SI 52 53

Technique

a

T1

3 1 1 — — 2 3 — 5 — 4 2

5 — 1 — — 4 2 — 6

— 3 7

4 — — — — 3 1 — 5 7 2 6

— 1 2 3 4 — — 5 — — — —

= “ thinlayer A ” : 12 g Kieselguhr G (Merck 8129), 3 g silica gel “ unter 0.08 mm” for chromatography (Merck 7729), 3 g C aC 0 3 p. A. (Merck 2066), 0.02 g Ca(OH )2 p.A. (Merck 2047), 50 m t ascorbic acid (8 x 1 0 3 mol x €"' ) = “ benzine” (bp 100— 140°)-isopropanol-dist. water = 100:12:0.25 = “ benzine” (bp 100— 140°)-acetone-chloroform = 50:50:40 = “ benzine” (bp 100— 140°)-benzene-acetone = 40:10:1 = layer thickness 0.125 mm, ascending; the plates are dried for 1.5 hr at 50—60°C; good ventilation is necessary; the plates are to be used immediately after preparation

Order of separation; no Rf values. 1 = top pigment.

REFERENCE 1.

Hager, A. and Meyer-Bertenrath, T., Planta (Berlin), 69, 198, 1966.

Volume I: Fat-Soluble Pigments

159

Table I. TLC 9 TLC ON VARIOUS REVERSED-PHASE LAYERS Layer Solvent Technique Detection Literature

LI SI T1 D1 1

LI S2 T1 D1 2

LI S3 T1 D1 3

LI S3 T1 D1 4

LI S4 T1 D1 5

L2 S5 T1 D1 6

6

Compound

Solvent

7

L4 S5 T1 D1 7

L4 S6 T1 D1 7

L5 S5 T1 D1

— — — — — — — — — — — — — — — — — 5 — — 89 — — — — — — — —

— — — — — — — 8 — 94 — — — — — — — — — — — — — — — — — — —

L5 S7 T2 D1

L5 S8 T2 D1

8

8

L6 S9 T3 D1 8

Rf x 100

Alloxanthin (trans) (3-Apo-8'-carotenal Antheraxanthin Antheraxanthin (trans) Astacene Capsanthin Canthaxanthin (3-Carotene y-Carotene Cryptoxanthin Cryptoxanthinepoxide Dehydroadonirubin Diadinoxanthin (trans) Diatoxanthin (trans) Echinenone Fucoxanthin Isozeaxanthin Lutein Lutein-dipalmitate Luteinepoxide Neoxanthin Rhodoxanthin Siphonaxanthin Siphonein Torularhodin-methyl-ester Vaucheriaxanthin Violaxanthin Zeaxanthin Zeaxanthin (trans) Layer

L3 S6 T1 D1

LI = L2 = L3 = L4 = L5 = L6 = SI = 52 = 53 = 54 = 55 = 56 = 57 = 58 = 59 =

— — — — — — 38 — — — — — — — 23 — — — — — — — — — — — — 53 —

62 — — 66 — — — — — — — — 70 59 — — — — — — — — — — — — — — 56

— — 45 — — — — 00 — — 14 — — — — — — 36 — — 67 — — — — 85 52 37 —

— — — — — — — — — — — — — — — 62 — 39 — — 68 — 72 36 — — — — —

— — — — 45 — 38 — — — — 43 — — 23 — — — — — — — — — — — — — —

— — — — — — — 10 15 90 — — — — 61 — — — 2 — — — — — 48 — — — —

— — — — — 74 — 00 00 7 — — — — — — 49 56 — 72 95 26 — — — — 84 54 —

— — — — — — — — — 11 — — — — — — — — — — — — — — — — — — —

cellulose, impregnated with triglyceride kieselguhr, impregnated with paraffin kieselguhr, impregnated with vegetable oil Kieselguhr G (Merck), impregnated with coconut butter Kieselguhr G (Merck), impregnated with paraffin Kieselguhr G (Merck), impregnated with triglyceride acetone-methanol-water = 15:5:2 methanol-acetone-water = 40:10:3 methanol-acetone-water = 30:10:3 methanol-acetone-water = 15:5:2 methanol-acetone = 5:2 methanol-acetone-water = 20:4:3 acetone-methanol-water = 50:47:3 methanol-acetone-water = 76:20:4 methanol-acetone-water = 75:15:10

— 80 — — — — 94 22 — 91 — — — — 69 — — 100 11 — — — — — 57 — — — —

— 65 — — — — 86 3 — 80 — — — — 42 — — 97 00 — — — — — 25 — — — —

— — — — — — — 00 — 7 — — — — — — — 56 00 72 95 26 — — — — 84 55 —

CRC Handbook of Chromatography: Plant Pigments

160

Table I. TLC 9 (continued) TLC ON VARIOUS REVERSED-PHASE LAYERS Technique

Detection

T1 = ambient temperature, chamber saturation, ascending T2 = a slurry is prepared from Kieselguhr G (Merck) and dioxane-water = 3 : 1 and distributed equally onto glass plates (10 x 20 cm, 20 x 20 cm); the layer is dried for 4 hr at 100°C and thereafter partially impregnated with paraffin oil-petroleum ether (bp 100— 140°C) = 8:92 to about 3— 4 cm from the upper rim (ascending technique); for solvent removal, the plates are kept at 70°C for 1 hr T3 = the layer is prepared as described under T2; partial impregnation with an 8 % solution of a triglyceride with low acid content (for example, olive oil [e.g., Livio®]) D1 = visual observation

REFERENCES 1. 2. 3. 4. 5.

Kleinig, H. and Egger, K., Phytochemistry, 6, 611, 1967. Egger, K., Nitsche, H., and Kleinig, H., Phytochemistry, 8, 1583, 1969. Kleinig, H. and Egger, K., Z. Naturforsch. B, 22, 868, 1967. Kleinig, H. and Egger, K., Phytochemistry, 6, 1681, 1967. Egger, K. and Kleinig, H., Phytochemistry, 6, 903, 1967. 6 . Davies, B. H., in Chemistry and Biochemistry o f Plant Pigments, Goodwin, T. W., Ed., Academic Press, London, 1965, 489.

7. Stobart, A. K., McLaren, J., and Thomas, D. R., Phytochemistry, 6, 1467, 1967. 8 . Bolliger, H. R. and Konig, A., Diinnschichtchromatographie, Stahl, E., Ed., Springer-Verlag, Berlin, 1967, 253.

Volume I: Fat-Soluble Pigments

161

LIQUID CHROMATOGRAPHY (LC) TABLES

NOTES ON TABLE I. LC 1 A more “ classical” way to separate fat-soluble chloroplast pigments is column chro­ matography. A diversity of stationary phases has been used. Much of the early work (see Reference 12 in “ Methods” ) and a number of recent publications still focus on liquid chromatography. Table I. LC 1 deals with the separation of carotenoids on columns filled with various sorbents. Since often in the original literature small figures (drawings) instead of retention volumes, or other data have been published, no numbers are given. Also, the resolution of zones and order of zones eluted is strongly dependent on the experimental conditions. The user is therefore advised to consult the original literature, when pigment name, sorbent, and solvent (= eluent) have been found. The amount of pigment present in a band can be roughly estimated by judging from the width of this band and the depth of color. As stationary phases, mixtures often are used. One example for such a mixture is Ca(OH)2Hyflo-Super-Gel with toluene-petrol ether in various compositions as the mobile phase. The stationary phase is more polar than the mobile phase. Prior to the application to the column, the plant extract is evaporated to a small volume or brought to dryness and taken up in a small volume of hexane. In this way, a number of interfering pigments, such as flavins, are separated. For working on a semipreparative scale, a variety of column sizes is suitable. A more general rule from the chemical point of view is that the larger the molecule of the carotenoid applied, the more firm is the linkage to the column material. The distance between bands should be such that the bands appear well separated, especially the main bands: for example, for Neurospora crassa, y-carotene and neurosporene. Sterols tend to interfere with carotenoid separation; they also absorb in the UV region. For their removal they may be frozen out or digitonized and removed by subsequent centrifugation. For literature sources, the reader is referred to Table I. LC 1.

Aphanicin Aphanin Bacterioruberin Canthaxanthin “ ’Carotene p-Carotene

Neoxanthin P"481 Spirilloxanthin Violaxanthin Alumina (act. grade 0 — II) Aleuriaxanthin Anhydrorhodovibrin

7

-Carotene Cryptoxanthin Lycopene Lycophyll Lycoxanthin Mutatoxanthin

(3-Carotene

Alumina3 Antheraxanthin Astaxanthin

Pigment

3 4 5

S 15 S7 57 S8 S 13 S16 S 17 S18 S5 S6 815 S15 S15 815 815 819 7b

12

10

9b

6

3

11

9b 5

8

7b

6

6

la la la la 2 la la 2 2 2 la la la 2 2 la

Ref.

SI S2 S3 S5 56 S7 S8 S6 S9 S10 S9 S ll S12 S13 S 13 S 14

Solvent

Chloroxanthin Cryptoxanthin 3,4-Dehydrolycopene 2'-Dehydroplectaniaxanthin 2'-Dehydroplectaniaxanthin- l'-ester 3,4-Dehydrotorulene Deoxyflexixanthin 1 \2'-Dihydro-l'-hydroxy-y-carotene 1\2'-Dihydro-l'-hydroxy-4-keto-y-carotene Dihydroxy-£-carotene Dihydroxylycopene 1,1 '-Dihydroxy- 1 ,2 ,1 \2'-tetrahydro-£-carotene Echinenone Gazaniaxanthin 3-Hydroxy-3'-hydroxy-a-carotene 3-Hydroxy-3'-keto-a-carotene 4-Hydroxy-4'-keto-p-carotene

^-Carotene (cis) ^-Carotene (trans) Chlorobactene

^-Carotene

e-Carotene

8 -Carotene

7

p-Carotene (trans) -Carotene

Pigment

Table I. LC 1 SURVEY OF DIFFERENT ADSORBENTS AND SOLVENTS USED FOR CAROTENOIDS

S15 S7 S 10 S13 S15 S20 S20 S21 S5 S15 S15 S15 S15 S15 S15 S7 S19 S 17 SI S35 S30 S17 S7 S22 S8 S8 S23 S24 S25 S17 SI S9 S ll S8

Solvent

8

22 23 23

11

3 12 27 19 20 14 14 21 21 17

11

16 16 10 18d 4

21

13 14 33 3 10 15 15 15 21 3 10 17

Ref.

162

CRC Handbook of Chromatography: Plant Pigments

OH-Chlorobactene OH-Lycopene OH-Neurosporene

Neurosporene (cis) OH-£-Carotene

Lycophyll Lycoxanthin Methyl-apo-6 '-lycopenoate Myxoxanthin Neurosporaxanthin-methylester Neurosporene

Lycopersene

Lycopene

Isorenieratene (trans) (3-Isorenieratene (trans) Isozeaxanthin 4 -Keto-7 -carotene 4-Keto-3'-hydroxy-(3-carotene 4-Ketotorulene Lutein Lycopene S I3 S 15 S 11 S 17 S ll S 17 S9 S7 S7 S7 S7 S10 S13 S 15 S15 S19 S26 S35 S8 S19 S29 S27 S28 S 13 S17 S 13 S7 S7 S13 S13 S15 S5 S 17 S17 S8 S17 S 17 14 11 19 23 5 10 17 27 3 21 24 25 26 7a 33 7d 7b 7a 25 25 24 11 28 4 29 3 17 21 17 10 21 10 21 21

8

13 13

S.g. 434 S.g. 460

Rubixanthin Rubixanthin (cis) Rubixanthin (trans) Saproxanthin

Rhodovibrin

Phytofluene (cis) Phytofluene (trans) Plectaniaxanthin Rhodopin

Phytofluene

OH-Spirilloxanthin OH-Y P-412 P-450 P-481 P-518 Phytoene

OH-R

OH-P-481 OH-Phytofluene

S8 S17 S 17 S8 S8 S8 S17 S15 S13 S7 S17 S5 S5 S19 S29 S5 S5 S5 S15 S19 S29 S29 S29 S30 S8 S8 S8 S8 SI S8 SI S9 S23 S31 S32 S7 S33

21 10 21 4 29 21 4 21 21 21 29 10 21 7b 9h 10 17 21 3 T 9 9b 9b 12 5 10 4 5 22 10 10 32 32 30 31 32 32

Volume I: Fat-Soluble Pigments 163

y-Carotene (trans) 8-Carotene (trans) ^-Carotene ^-Carotene (trans) ^-Carotene

(3-Carotene (trans) y-Carotene

Apo-3-lycopenal Auroxanthin (3-Carotene

Alumina (act. grade III — IV) Antheraxanthin

1,2,1', 2 '-Tetrahydro-1,1'-dihydroxy lycopene 7.8.11.12- Tetrahydrolycopene (cis) 7.8.11.12- Tetrahydrolycopene (all trans) Torulene Zeaxanthin

S.g. 500 Spheroidenone Spirilloxanthin

Pigment

S9 S ll S19 S9 S5 S5 55 56 S6 56 S6 S6 57 S19 S15 S15 S6 S15 S6

S8 S7 S17 S17 S17 S ll S15 S15 S10 S14 S34

Solvent

35e 34g W 34g 16s 35e 39m 331 37h 38 34f 16' 35e 18J 34f 34f 37h 34f 16'

32 29 4 5 21 5 16 16 3 10 11

Ref.

Neurosporene

Lycopene (trans) 5,6-Monoepoxy-p-carotene (trans) 5,6-Monoepoxy lutein Mutatochrome Mutatochrome (trans) Neoxanthin

Lycopene

1-Hydroxy-1,2-dihydro-y-carotene Lutein

5,6-Diepoxy-(3-carotene {trans) 7,8-Dihydrosarcinaxanthin Echinenone Euglenanone Flavochrome Flavoxanthin

Crocetindial Cryptoxanthin Cryptoxanthin (trans) 3.4-Dehydrolycopene

Chrysanthemaxanthin

Pigment

Table I. LC 1 (continued) SURVEY OF DIFFERENT ADSORBENTS AND SOLVENTS USED FOR CAROTENOIDS

S8 S35 S10 S7 S8 S15 S15 S15 S35 S7 S8 S15 S8 S35 S19 S8 S10 S5 S6 S10 S15 S15 S 19 S13 S15 S8 S13 S15 S36 S37 S6 S6

Solvent

34g 33k 41 35e 34e 16* 37h 34e 40* 35e 35e 33k 34g 33k 18^ 34g 33k 40’ 16’ 331 37h 38 18s 34f 34e 34g 33k 34e 34g 35e I61 37h

Ref.

164 CRC Handbook of Chromatography: Plant Pigments

Rhodopin Rubixanthin (cis) Rubixanthin (trans) Sarcinaxanthin Torulene Violaxanthin P-Zeacarotene Zeaxanthin CaC0 3 a-Carotene P-Carotene Cryptoxanthin Lutein Neoxanthin Violaxanthin Zeaxanthin C aC0 3-Ca(OH)2-Diatomaceous earth = Alloxanthin {trans) Crocoxanthin a-Cryptoxanthin Diatoxanthin 4-Hydroxy-a-carotene Lutein Monadoxanthin Zeinoxanthin Ca(OH )2 a-Carotene

Phytofluene

OH-Chlorobactene Phytoene

2:2:1 (w/w) 43 43 43 43 43 43 43 43 45 44

S5 57

42 42 42 42 42 42 42

S8 S8 S8 S8 S8 S8 S8 S38 S38 S38 S38 S38 S38 S38 S38

18* 16' 34f 37h 38 39m 16' 37h 39m 34f 18J 34e 34e 40' 37h 34* 161 34g

S19 S5 S5 S5 S5 S5 S5 S5 55 56 S19 S13 S10 S13 S6 SI 1 S6 S8

Neurosporaxanthin Oscillaxanthin P-476 P-496

,2-dihydro-3,4-didehydro-apo-8'lycopenol 4-Ketophleixanthophyll Lycopene Methyl- 1 -hexosyl- 1 ,2-dihydro-3,4-didehydro-apo8 '-lycopenoate Myxoxanthophyll

1 -Hexosyl-l

Flexixanthin

P-Cryptoxanthin {cis) Lycopene Phytofluene Phytofluenol Zeaxanthin Ca(OH)2-Celite 535 = 4:1 P-Carotene 4 -Keto-a-carotene Retrodehydrocarotene Cellulose Deoxyflexixanthin

Cryptoxanthin a-Cryptoxanthin a-Cryptoxanthin (cis) P-Cryptoxanthin

P-Carotene-monoepoxide y-Carotene

(3-Carotene

7

SI S i SI S5 S43 S8 S44

31 28 11 30 30

19 40 47

SI S5 S9

20 20 20 20 47

46 46 46

S21 S39 S39 S41 S42 S35 S42 S8

45 44 44 44 45

44

S8

S5 S8 S7 S8 S8

44 45 45 45

S8 S5 S5 S5

l

44

S8

l

45 44

45 44

S5 $7 S5 S7

Volume I: Fat-Soluble Pigments 165

MgO-Celite 503 = 2:1 (w/w) 0-Carotene y-Carotene 3,4,3',4'-Bisdehydro-0-carotene 3 ,4-Dehydro-0-carotene Lycopene Rodoxanthin

0-Carotene (cis) Isocryptoxanthin Isocryptoxanthin (cis) MgO Fucoxanthin MgO-Celite = 3:2 Anhydroeschscholtzxanthin Antheraxanthin Auroxanthin Chrysanthemaxanthin Eschscholtzxanthin Flavoxanthin Lutein 5,6-Monoepoxylutein (trans) Neoxanthin Violaxanthin Xanthophyll Zeaxanthin

Sarcinaxanthin Lime Superfine (Sierrahydrated)-Celite 545 = 2:1 0-Carotene

Phyleixanthophyll

Pigment

50 51 34 34 34 51 34 34 34 34 34 51 34 51

S17 S19 SI S9 S ll S19 S ll S8 S17 S ll S8 S 19 SI S19 52 52 52 52 52 52

48 48 48 48 48

S45 S47 S46 S47 S47

S45 S46 S48 S47 S46 S47

15 19 40

Ref.

SI SI S8

Solvent

0-Carotene

Saproxanthin Microcel C Alloxanthin (trans) Crocoxanthin a-Cryptoxanthin Diatoxanthin 4-Hydroxy-a-carotene Lutein Monadoxanthin Zeinoxanthin Polyamide (0.2 — 0.8 mesh) 0-Carotene Sea Sorb 43 Magnesia-Celite 545 = 1:2 a-Carotene

MgO-Celite 545 = 1:1 0-Carotene ^-Carotene Echinenone Euglenanone Phytofluene Magnesium silicate Neurosporaxanthin (cis) Neurosporaxanthin (trans) S.g. 434 S.g. 460

Pigment

Table I. LC 1 (continued) SURVEY OF DIFFERENT ADSORBENTS AND SOLVENTS USED FOR CAROTENOIDS

53

S45

42 42 42 42 42 42

43 43 43 43 43 43 43 43

S53 S53 S53 S53 S53 S53 S53 S53

S54 S55 S56 S54 S55 S56

28 28 32 32 32 32

38 38 35 35 38

Ref.

S51 S52 S15 S9 S23 S10

S49 S49 S17 S8 S49

Solvent

166 CRC Handbook of Chromatography: Plant Pigments

a b c d e f

55 36 36 36 55 55 36 36 36 36 42 42 42 42 42

S58 S58 S58 S58 S58

42 42 42 42 42 42 42 42 42 42 42 42 42 42 42

S4 S57 S57 S57 S4 S4 557 S57 S57 S57

S54 555 556 S54 555 556 S54 555 556 S54 555 556 S54 555 556

Activity grade not available. 3% (v/w) water-deactivated alumina (i.e., Brockmann grade II). Alumina (Peter Spence, Type H); Brockmann grade II. 2% (v/w) water-deactivated alumina. Alumina, deactivated by treatment with 5% of water. Partially deactivated by treatment with 1% (v/w) of water.

Lycopene Phytoene Phytofluene Starch a-Carotene (3-Carotene Cryptoxanthin Lutein Neoxanthin

Sephadex-LH-20 Auroxanthin a-Carotene (3-Carotene ^-Carotene Chrysanthemaxanthin Lutein

Zeaxanthin

Violaxanthin

Neoxanthin

Lutein

Cryptoxanthin

S49 S49 S49 S49 S49 S49 S49 S49

S59 S59 S59 S59 S59

S19 S58 S19 S58 S19 S58 S19 S58 S19 S58 S19 S58 S19 S58

S58 S58

42 42

43 43 43 43 43 43 43 43

54 54 54 54 54

42 42 42 42 42 42 42 42 42 42 42 42 42

g Methanol-deactivated alumina. h Deactivated neutral alumina (80 — 200 mesh), 6 % water. 1 Woelm neutral (Brockmann grade III). J Alumina (Peter Spence, Type H); Brockmann grade IV. k Deactivated alumina made by treating grade “ O ” with methanol. 1 3:2 Mixture of activated and deactivated (Brockmann grade III) alumina.

ZnCOr Celite = 3:1 (w/w) a-Carotene (3-Carotene Diatoxanthin Lutein Zeaxanthin Z nC 02-Diatomaceous earth = 3:1 (w/w) Alloxanthin Crocoxanthin a-Cryptoxanthin Diatoxanthin 4-Hydroxy-a-carotene Lutein Monadoxanthin Zeinoxanthin

Zeaxanthin

Violaxanthin

Neoxanthin

Lutein

Cryptoxanthin

(3-Carotene

Violaxanthin Zeaxanthin Sugar a-Carotene

Volume I: Fat-Soluble Pigments 167

Solvent SI = 20— 30% acetone in petroleum ether 52 = ethyl ether-cold acetic acid = 20:1 53 = 15% KOH in 90% methanol 54 = chloroform 55 = petroleum ether 5 6 = 0—5% ether in petroleum ether 57 = 0 5% acetone in petroleum ether 5 8 = 10—25% acetone in petroleum ether 59 = 40—60% acetone in petroleum ether S10 = 30—50% ether in petroleum ether SI 1 “ 25 40% acetone in petroleum ether 512 = acetone-n-propanol = 9:1 513 = 15 25% ether in petroleum ether 514 = acetone 515 = 5— 15% ethyl ether in petroleum ether 516 = benzene-methanol = 9:1 517 = 5— 10% acetone in petroleum ether 518 = 7— 10% ethyl ether in hexane 519 = benzene 520 = petroleum ether-benzene = 85:15 521 = petroleum ether-benzene = 1:1 522 = 99% ether-1% methanol 523 = 1— 5% methanol in petroleum ether 524 = petroleum ether-methanol = 75:25 525 = benzene-methanol = 95:5 526 = benzene-ethyl ether = 75:25 527 = ether-methanol = 98:2 528 = ether 529 = 0 4% ethyl ether in hexane 530 = 1 2% methanol in benzene

benzene-acetone = 6 : 4 0.5— 1% methanol in ethyl ether 100% methanol from 5 0 % acetone in petroleum ether to 2% methanol in petroleum ether $ 3 5 = 50% ether in petroleum ether $ 3 5 — 60% acetone in petroleum ether $ 3 7 - ethanol $ 3 8 = 0 _ 2 0 % acetone in benzene $ 3 9 = 20— 30% benzene in petroleum ether $40 = petroleum ether-benzene = 95:5 S41 = 0— 30% ether in petroleum ether $42 = 1 0 — 50% ether in petroleum ether $ 4 3 — pyridine $ 4 4 = 30 % acetone in benzene $ 4 5 = hexane $ 4 5 = i _ 5% acetone in hexane $ 4 7 = 6— 15% acetone in hexane $ 4 8 = hexane-acetone = 75:25 $ 4 9 - q— 25% acetone in petroleum ether $50 = benzene-ether-ethanol = 2:3:1 $ 5 1 = methanol $ 5 2 = \% acetic acid in methanol $ 5 3 — 1 0 — 80% ethyl ether in petroleum ether $ 5 4 = l ,2-dichlorethane $ 5 5 = petroleum ether -I- 10— 50% acetone $ 5 5 = petroleum ether + 6 — 10% ^-propanol $ 5 7 - chloroform-methanol-Ai-hexane = 65:5:30 $ 5 8 = petroleum either + 5% /i-propanol $ 5 9 = petroleum ether -I- ethyl ether (cone unspecified)

S31 = $32 = $33 = $34 =

Table I. LC 1 (continued) SURVEY OF DIFFERENT ADSORBENTS AND SOLVENTS USED FOR CAROTENOIDS

168

CRC Handbook of Chromatography: Plant Pigments

Volume I: Fat-Soluble Pigments

169

REFERENCES 1. Czeczuga, B., Comp. Biochem. Physiol., 48B, 349, 1974. la. Czeczuga, B., Comp. Biochem. Physiol., 39B, 945, 1971; J. Insect. Physiol., 17, 2017, 1971. 2. Conti, S. F. and Benedict, C. R., J. Bacteriol., 83, 929, 1962. 3. Liaaen-Jensen, S., Phytochemistry, 4, 925, 1965. 4. Liaaen-Jensen, S., Acta Chem. Scand., 17, 500, 1963. 5. Ryvarden, L. and Liaaen-Jensen, S., Acta Chem. Scand., 18, 643, 1964. 6. Hertzberg, S. and Liaaen-Jensen, S., Phytochemistry, 5, 565, 1966. 7. Kushwaha, S. C. and Kates, M., Biochim. Biophys. Acta, 316, 235, 1973. 8. Liaaen-Jensen, S., Acta Chem. Scand., 19, 1166, 1965. 9. Kushwaha, S. C., Pugh, E. L., Kramer, J. K. G., and Kates, M., Biochim. Biophys. Acta, 260, 492, 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55.

1972. Liaaen-Jensen, S., Hegge, E., and Jackman, L. M., Acta Chem. Scand., 18, 1703, 1964. Hertzberg, S. and Liaaen-Jensen, S., Phytochemistry, 5, 557, 1966. Arpin, N. and Liaaen-Jensen, S., Phytochemistry, 6, 995, 1967. Liaaen-Jensen, S., Acta Chem. Scand., 19, 1025, 1965. Hertzberg, S. and Liaaen-Jensen, S., Acta Chem. Scand., 20, 1187, 1966. Manchand, P. S., Rtiegg, R., Schwieter, U., Siddons, P. T., and Weedon, B. C. L., J. Chem. Soc., p. 2019, 1965. Davies, B. H., Hallett, C. J., London, R. A., and Rees, A. F., Phytochemistry, 13, 1209, 1974. Fiasson, J. L. and Arpin, N., Bull. Soc. Chim. Biol., 49, 537, 1967. Bonnett, R., Spark, A. A., and Weedon, B. C. L., Acta Chem. Scand., 18, 1739, 1964. Hertzberg, S. and Liaaen-Jensen, S., Acta Chem. Scand., 21, 15, 1967. Aasen, A. J. and Liaaen-Jensen, S., Acta Chem. Scand., 20, 1970, 1966. Liaaen-Jensen, S., Cohen-Bazire, G., Nakayama, T. O. M., and Stanier, R. Y., Biochim. Biophys. Acta, 29, 477, 1958. Arpin, N. and Liaaen-Jensen, S., Phytochemistry, 8, 185, 1969. Liaaen-Jensen, S. and Hertzberg, S., Acta Chem. Scand., 20, 1703, 1966. Kjpsen, H. and Liaaen-Jensen, S., Phytochemistry, 8, 483, 1969. Markham, M. C. and Liaaen-Jensen, S., Phytochemistry, 7, 839, 1968. Surmatis, J. D., Ofner, A., Gibas, J., and Thommen, R., J. Org. Chem., 31, 186, 1966. Arpin, N. and Liaaen-Jensen, S., Bull. Soc. Chim. Biol., 49(5), 527, 1967. Aasen, A. J. and Liaaen-Jensen, S., Acta Chem. Scand., 19, 1843, 1965. Liaaen-Jensen, S., Acta Chem. Scand., 17, 489, 1963. Francis, G. W., Hertzberg, S., Andersen, K., and Liaaen-Jensen, S., Phytochemistry, 9, 629, 1970. Hertzberg, S. and Liaaen-Jensen, S., Phytochemistry, 8, 1259, 1969. Aasen, A. J. and Liaaen-Jensen, S., Acta Chem. Scand., 20, 811, 1966. Goodwin, T. W., Biochem. J., 58, 90, 1954. Jungalwala, F. B. and Cama, H. R., Biochem. J., 85, 1, 1962. Krinsky, N. I. and Goldsmith, T. H., Arch. Biochem. Biophys., 91, 271, 1960. Hasegawa, K., Methods Enzymol., 67, 261, 1980. Goldie, A. H. and Subden, R. E., Biochem. Genet., 10(3), 275, 1973. Bramley, P. M. and Davies, B. H., Phytochemistry, 14, 463, 1975. Mitzka-Schnabel, U., dissertation University of Munich, Munich, 1978. Arpin, N., Norgard, S., Francis, G. W., and Liaaen-Jensen, S., Acta Chem. Scand., 27, 2321, 1973. Aasen, A. J. and Liaaen-Jensen, S., Acta Chem. Scand., 21, 970, 1967. Strain, H. H. and Svec, W. A., Adv. Chromatogr., 8, 119, 1969. Chapman, D. J., Phytochemistry, 5, 1331, 1966. Zechmeister, L. and Pinckard, J. H., Experientia, 4(12), 474, 1948. Cholnoky, L., Szabolcs, J., and Nagy, E., Justus Liebigs Ann. Chem., 616, 207, 1958. Entschel, R. and Karrer, P., Helv. Chim. Acta, 41, 112, 1958. Aasen, A. J., Francis, G. W., and Liaaen-Jensen, S., Acta Chem. Scand., 23, 2605, 1969. Wallcave, L. and Zechmeister, L., J. Am. Chem. Soc., 75, 4495, 1953. Zechmeister, L. and Wallcave, L., J. Am. Chem. Soc., 75, 5341, 1953. Jensen, A., Acta Chem. Scand., 20, 1728, 1966. Karrer, P. and Leumann, E., Helv. Chim. Acta, 50/51, 445, 1951 Foppen, F. H. and Gribanovski-Sassu, Biochim. Biophys. Acta, 176, 357, 1969. Fric, F. and Haspel-Horvatovic, E., J. Chromatogr., 68, 264, 1972. Allen, M. B., Fries, L., Goodwin, T. W., and Thomas, D. M., J. Gen. Microbiol., 34, 259, 1964. Shimizu, S., J. Chromatogr., 59, 440, 1971.

Volume I: Fat-Soluble Pigments

171

HIGH PER FO R M A N C E LIQ UID C H R O M A TO G RA PH Y (HPLC) TABLES

TABLE NOTES In the last few years, the application of high-performance liquid chromatography has greatly increased. We, therefore, have included tables on HPLC which is basically a special application of liquid chromatography. Numbers for retention times (rR) are given only in relatively few publications. From other publications, however, it is possible to deduce approximate values from figures. The data thus obtained are compiled in Table I.HPLC 1, together with the applicable chromatographic parameters. The other relevant literature is combined in Tables I.HLPC 2 and I.HLPC 3, which are a complementary survey of the separation of carotenoids in HPLC systems.

length (mm) diameter (mm) form material

/R(min)

— — — — — — — — — — 4.2h 4.2h 4.6h — — — — — —

Antheraxanthin Antheraxanthin(d.v) Auroxanthin epimer 1 Auroxanthin epimer 2 Bacterioruberin all trans Bacterioruberin neo A Bacterioruberin neo U Bacterioruberin neo V Bacterioruberin neo W (3-Carotene (3, (3-Carotene (3,e-Carotene (3,i|/-Carotene (3-Citraurin Cryptoxanthin Diadinoxanthin Diatoxanthin Dinoxanthin 2,2'-Diol*

PI 250 4.6 st n.a. SI 1.4 300 n.a. D1 T1 1

Compound

Solvent Flowrate (m€/min) Pressure (psi) Temperature (°C) Detection Technique Literature

Packing Column

1

— — — — — — — — — — — — — — — — — — 8.0*

fR(min)

PI 250 4.6 st n.a. S2 1.4 300 n.a. D1 T1 1 1

— — 25.5f 25.9f — — — — — — — — — — — — — — _

fR(min)

PI 250 4.6 st n.a. S3 1.4 300 n.a. D1 T1

— — — — 14.6s 14. lg 15.18 16.08 16.38 — — — — — — — — — _

fR(min)

PI 250 4.6 st n.a. S4 1.4 300 n.a. D1 T1 2

— — — — — — — — — — — — — — — — — — _ _

fR(min)a

P2 135 6.35(OD) st SS S5 0.5 -C H 0 > -C H 2CH3> -C H 2CH2C 0 2M e>-C H 2C H X H 3> -C H 2C 0 2M e> -C H 2CH2N H C 0M e> -C 02M e> A )C 0 M e> -C 0 -C H 2C 0 2Me The differences in mass spectra of porphyrin “ type” isomers (e.g., type I and type III) are inconclusive, however. For more detailed information, the reader is referred to the literature.136a

Melting Points Historically, the use of melting points of porphyrin esters has been important. It has now been recognized, however, that many of the porphyrin esters crystallize with different structures,30’31 i.e., they are polymorphic and, therefore, melt a different temperatures.

Partition Behavior (HC1 Numbers) Many porphyrins are extractable from aqueous solutions into diethyl ether or ethyl actate after the pH is adjusted to values around 3 to 4. Once in either of these organic solvents, it is possible to extract the porphyrins into basic or acidic aqueous solutions (see below); this makes possible an important clean-up step prior to chromatography. The so-called HC1 number of porphyrins is mostly of historical interest. Nevertheless, it is of some practical use in the laboratory. The HC1 number32 is defined as the concentration of HC1 in that percent (w/v) which extracts two thirds of the porphyrin from an equal volume of an ether solution. The HC1 numbers depend jointly upon the dissociation of the porphyrin as a base and its ether-water partition coefficient. Representative values of HC1 numbers are given in Table II.3 (compare Reference 33).

ESTIMATION AND SEPARATION OF PORPHYRINS Notes on Porphyrin Stability The term “ stability’’ must be defined carefully. We use it to indicate that a given compound retains its structure and individual character. It need not decompose in the usual sense to be considered “ unstable” . Thus, any porphyrin in any solution except acidic ones can be seen as unstable because of the tendency to incorporate trace metal ions. As mentioned, metalloporphyrins have spectral characteristics quite different from the metal-free com­ pounds. Similarly, the metalloporphyrins have considerably different chromatographic be­ havior in all of the systems mentioned in this section. All solvents used for chromatographic purposes should be carefully washed with water to remove acids and alcohols. They should be dried with sodium sulfate, etc., passed over a silica gel column, and finally “ glass distilled” to ensure the removal of trace metals. Those solvents, e.g., lutidine, water, etc., for which the above obviously does not apply, should have EDTA added to give a final concentration of 1 mM. As a general rule, mixtures of solvents should be prepared fresh each day. This is imperative when the components react with each other: e.g., acetic acid and methanol react to form methyl acetate. While most porphyrins are quite stable in acidic solution, some are not stable under almost any conditions. Protoporphyrin is one of the best examples of an unstable porphyrin. In acid the vinyl groups at positions 2 and 4 tend to hydrate to produce the corresponding secondary alcohols, i.e., hydroxyethyl groups. Protoporphyrin is very unstable in the light — the photoreaction yields one of the two isomers of photoprotoporphyrin,37 39 a green compound

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to be seen on most chromatograms of porphyrins. All porphyrins should, therefore, be kept in dim light. Porphyrins in basic solution tend to be less stable. Whenever possible, they should not be stored under such conditions. It is best to store porphyrins as the dry crystalline esters or the dry dihydrochloride salts. Even the dry material should be stored in the dark.

Prechromatography Purification and Sample Preparation A discussion of sample preparation is difficult since there exist a multitude of starting materials. Some investigators start with leaves, some with sedimentary rocks, some with microbial cultures (or supernatants), and others with urine. The first problem is to solubilize the porphyrin, when present in insoluble form. At this point it is impossible to generalize. When appropriate, i.e., with leaves and rocks, it is necessary to work with finely divided material. Once the porphyrin is in soluble form, it is necessary to perform some preliminary clean-up and concentrating. The methods vary so widely that examples of each must be considered separately (Figure 4 and Table 1).

Sample Preparation from Basic Aqueous Solutions Basic aqueous solutions (pH 8) are prepared from leaves, rocks, liver, feces, culture supernatants, etc. Three techniques are practical for the extraction of porphyrins from these solutions:

Ethyl Acetate Extraction Add a volume of ethyl acetate equal to V2 the volume of the extract and shake it in a separatory funnel. Put the entire mixture into a beaker with a magnetic stirring bar and mix it vigorously. Adjust the pH with glacial acetic acid to about pH 3.2. Return the sample into the separatory funnel and mix the two phases thoroughly. After phase separation, remove the lower (aqueous) phase for reextraction. Place the ethyl acetate phase (containing por­ phyrins and metalloporphyrins) in a beaker. Extract the aqueous phase again, this time using only V2 of the volume of the ethyl acetate used in the first extraction. Monitor the results of each extraction with the aid of a long-wavelength UV light in a dark room. Continue the extraction process as long as the ethyl acetate fraction continues to show a pink-red fluo­ rescence. Take the combined ethyl acetate fractions and wash them several times with water. Next, extract the metal-free porphyrins out of the ethyl acetate with 3 M HC1 using a minimum volume of the latter. The solution of the porphyrins in hydrochloric acid is now ready to be chromatographed or esterified, as the case may be (see below). If the ethyl acetate phase retains significant color, we have to suspect that stable metalloporphyrins remained. There­ fore, wash the ethyl acetate with water at least twice. Then extract it in a solution of 1 M ammonia in water (minimum volume). Repeat the extraction as long as pigment continues to move from the organic to the aqueous phase. The aqueous phase now will contain metalloporphyrins. They are ready for chromatography or derivatization.

Absorption and Concentration on Talc Add talc (C aC 03, ca. 1 g/100 m€) to the basic solution and mix it well on a magnetic stirrer. Adjust the pH to 3.2 with glacial acetic acid. After thorough mixing and equilibration, filter off the talc on a Buchner funnel and wash with water. All the porphyrins and metal­ loporphyrins should be absorbed on the talc, which should fluoresce under long-wavelength UV light. The preparation is now ready for chromatography or derivatization.

Sample Preparation from Acidic Aqueous Solutions Acidic extract of tissue etc.: extracts made 1.5 M with HC1 should be protein-free and will not contain any metalloporphyrins. They should fluoresce salmon red under UV light.

FIGURE 4. Example for the workup of porphyrin-containing samples. Further procedures are given in citations to tables dealing with ‘‘porphyrins” and in Table II HPLC 3, this Section.

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Add 1 g talc per 100 m€ and adjust the pH to 3.2 with 3 M NaOH (after adding some saturated sodium acetate as buffer). Filter off the talc on a Buchner funnel and wash with water. The talc should fluoresce under long wavelength UV light.

Absorption and Concentration on DEAE Cellulose Add dry DEAE cellulose to the basic solution of the porphyrins. Mix thoroughly on a magnetic stirrer. Filter off the DEAE cellulose on a Buchner funnel and wash with water. All the porphyrins and metalloporphyrins should be on the DEAE cellulose which should fluoresce under long-wavelength UV light.

Extraction of Porphyrins Extraction from Talc Porphyrins may be extracted from talc with either strong mineral acids such as 3 M HC1 or bases such as 1 M NH4OH. They can, thus, be chromatographed directly. It is necessary, however, to pay attention to the solvent composition to be employed. For example, if one intends to use paper chromatography in lutidine water with an NH3 atmosphere or reversephase high-performance liquid chromatography (HPLC), one must be careful about the ionic state of the applied material. A HC1 extract of porphyrins will, of course, render di-cations. They will not migrate unless they are “ neutralized” . The same holds true for porphyrins in NH4OH. Metalloporphyrins may be extracted from talc by eluting them with 1 M NH4OH.

Extraction from DEAE Cellulose Porphyrins may be eluted from DEAE with 3 M HC1. Again, the fact that they are present in the form of a strongly acidic solution must be taken into account prior to chromatography.

Preparation and Extraction of Porphyrin Methyl Esters The most practical method for examining almost any unknown porphyrin mixture by chromatography is to prepare the methyl ester first. Two methods are commonly used (next two paragraphs). Esterification with methanol/mineral acid — Suspend the dry talc or DEAE cellulose to which the porphyrin is bound in methanol containing 5% concentrated sulfuric acid. After about 1 hr, filter on a Buchner funnel. Wash the talc or DEAE cellulose several times with methanol. Place the combined acidic methanol extracts in a closed container (screw-capped test tube) and place it into a boiling water bath for about 1 hr. Caution should be observed because of the developing internal pressure during heating. Alternatively, the acidic sus­ pension can be incubated at room temperature in the dark overnight. Esterification with boron trifluoride/methanol — Alternatively, porphyrins may be eluted from talc or DEAE cellulose with boron trifluoride (10 to 20% in methanol). Again, the esterification can be accelerated by heating as above, or the mixture may be allowed to stand overnight. If the sample of talc or DEAE cellulose is not thoroughly dry, it is practical to add a water-scavenging compound such as 2% or/Zio-trimethylformate. Extraction of porphyrin esters — To the reaction mixture in acidified methanol con­ taining the prophyrin esters, either chloroform or (better) methylene chloride is added (sep­ aratory funnel). After the contents of the funnel have been shaken, water is added to give two phases. The porphyrin ester will move quickly into the organic (lower) phase. The extraction should be repeated and monitored by observing the fluorescence under longwavelength UV light. Further, this extraction should be carried out as quickly as possible to avoid acidic hydrolysis of the porphyrin esters. The chloroform (or methylene chloride) extract of porphyrin esters should be washed several times in the separatory funnel with distilled water. This washing is generally accompanied by a change in color from purple to

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a brownish red, as the porphyrin di-cation ester is neutralized. The porphyrin ester can then be crystallized by adding methanol (1:1) and allowing the chloroform to evaporate in a gentle stream of warm air. The porphyrin ester is then ready for chromatography. Extraction from sedimentary rock — In some cases it is necessary or appropriate to extract a sample directly with organic solvents. Such is the case with sedimentary rocks.Toluene-methanol = 1 : 1 seems to be an efficient solvent. The filtered solution is then passed over an alumina column which removes and concentrates the porphyrins. The por­ phyrins are extracted from the alumina using one of the methods given above.

Chromatography Paper Chromatography The most practical method for preliminary studies involving porphyrins is undoubtedly chromatography. The technique is simple and does not involve complicated instrumentation. Large pickle jars are quite suitable as containers for the development of paper chromatograms. Detection of porphyrins is best done by visual observation of red fluorescent spots when the chromatogram is examined under long-wavelength UV irradiation in a dark room. Heme compounds are best visualized after spraying the chromatogram with an appropriate dye. (See, for example, Table II. PC 5.) The major limiting factor of paper chromatography is one of capacity. For clear resolution, the porphyrin should be hardly visible in the white light, but should fluoresce clearly under long-wavelength UV light.

Thin-Layer Chromatography (TLC) TLC of porphyrins is a very useful technique which provides several advantages. First, it is possible to buy thin-layer plates with a wide variety of adsorbants. Most commonly used are silica gel, polyamide (nylon), and talc (CaC03). The latter coating is not com­ mercially available. TLC on the analytical scale can, as a general rule, be adapted, by slight variations in the solvent mixture, to thick-layer or preparatory chromatography. Further, within limits, results obtained by TLC can be directly adapted (with modifications as they become apparent) to both large-scale liquid chromatography with the same adsorbant andJ or high-performance liquid chromatography column materials. Porphyrins on developed thinlayer chromatograms tend to be considerably less stable than usual. This is, no doubt, caused by the large surface area upon which they are exposed. If such chromatograms must be stored, they should be wrapped in plastic film (Saran® wrap) or aluminum foil and stored in the freezer. TLC plates that contain a fluorescent indicator frequently cause problems when used for porphyrin chromatography. The indicator is a zinc complex and there is enough “ free” zinc so that some of this metal is incorporated by the porphyrins during chromatography. This, being a continuous process, results in a streaking of each porphyrin as it is converted into the slower running zinc complex.

Liquid Chromatography (LC) This technique is of some use in large-scale preparatory work. It is of little or no use for analytical work. The obvious advantage of LC is the potential capacity of the system, i.e., one is limited only by the size of the column. It is, however, the most difficult system with which to obtain reproducible results.

High-Performance Liquid Chromatography (HPLC) In the last few years, the analysis of porphyrins has been revolutionized by application of HPLC. As is customary for porphyrins, medical applications have preceeded its application in plant biochemistry, which is now a rapidly developing area. HPLC is without doubt the most powerful chromatographic technique available at the present time. Separations that were impossible a few years ago are now routine laboratory procedures and take only a few

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minutes. The speed factor should be emphasized by the following example: in a trial run (15 min), an unknown mixture may exhibit several fast-running peaks in a solvent system such as ethyl acetate-hexane = 55:45. Greater resolution may be achieved quickly by simple adjustment of the solvent ratio, e.g., 50:50. This “ fine tuning” is only practical with HPLC. In addition to the advantage of speed afforded by HPLC, there is the particular feature that quantification is implicitly automatic. It should be noted that as with all chromatographic techniques discussed, the use of standard porphyrin mixtures is imperative for definitive work. A survey of a sample workup is given in the table (Table II. HPLC 3).

Sources and Materials for HPLC HPLC columns are available from many commercial sources. (For sources and literature, see HPLC tables.) Even though they may bear the same description, e.g., 5 pm silica gel, 5 pm C I8, etc., columns are not always interchangeable. Each column, even from a single manufacturer, must be “ fine tuned” with the use of standard porphyrin mixtures. Hyperpressure Gas Chromatography (HPGC) Porphyrins are a class of very slightly volatile compounds, although in a number of cases a purification could be achieved by sublimation.40 It is, therefore, not surprising that although the different forms of paper-, thin-layer, and column liquid chromatography have been widely used for the separation and analysis of porphyrins, conventional gas chromatography has remained of no importance. A few authors, however,41 43 were successful with a special form of gas chromatography, the so-called hyperpressure gas chromatography (HPGC). Carrier gases employed were dichlorodifluoromethane (c.t. 111.5°C) or monochlorodifluoromethane (c.t. 96°C) above their critical temperatures (c.t.) at pressures of 1000 to 1400 psi. Under these conditions, the amount of solid (e.g., porphyrin or metalloporphyrin) dissolved in the gas is much larger than expected from the normal increase of vapor pressure due to external pressure. The hyperpressure gas chromatograph and the necessary modifications have been described.41 For porphyrins, HPGC offers the only method for gas chromatography of these compounds as such. On the other hand, since metals may be separated in the form of their porphyrin metal chelates, the method offers a possibility for the separation of Cu(II), Ni(II), V(IV), and Sn(IV) in the form of their etioporphyrin metal chelates. Also, Ag(II) could be thus determined.43

Paper Electrophoresis In modem clinical chemistry, electrophoretic methods are increasingly used, especially for the analysis of serum proteins. From time to time, it might happen that urine from porphyric patients has to be analyzed. We, therefore, give a brief reference on paper elec­ trophoretic separations of porphyrins.44’45 For analysis, free porphyrins are used. As could be expected, the speed of migration in the electric field increases with the number of carboxyl groups. To obtain a complete dissociation of the carboxylic acid groups present, an alkaline buffer has to be used (e.g., 1/20 M barbiturate, pH 8.6). Compared to modem HPLC, paper electrophoresis is somewhat slow, but within 1 to 3 hr a satisfactory separation can be achieved.44 The method might be recommended if no other equipment than an electrophoretic setup is available and electrophoresis is routinely carried out. One also must consider that routine electrophoresis today is mostly carried out on cellulose acetate strips, whereas the literature refers to paper. For further information, the reader is referred to Table II. PEL 1.

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CRC Handbook of Chromatography: Plant Pigments REFERENCES

1. Smith, K.M., Ed., Porphyrins and Metalloporphyrins, Elsevier, Amsterdam, 1975. 2. Dolphin, D., Ed., The Porphyrins, Vol. 1 to 3 and 4 to 7, Academic Press, New York, 1978 and 1979. 3. Cox, M. T., Jackson, A. H., and Kenner, G. W., J. Chem. Soc. C., p. 1974, 1971. 4 DiNello, R. K. and Chang, C. K., The Porphyrins, Vol. 1, Dolphin, D., Ed., Academic Press, New York, 1978, 290. 5. Drabkin, D. L., The Porphyrins Vol. 1, Dolphin, D., Ed., Academic Press, New York, 1978, 31. 6 . Dolphin, D., Ed., The Porphyrins, Vol. 6 and 7, Academic Press, New York, 1979. 7. Chlorophylls, Scheer, H., in CRC Handbook o f Chromatography, Plant Pigments, Vol. 1 , Fat-Soluble Pigments, Kost, H.-P., Ed., CRC Press, Boca Raton, 1988. 8. Baker, E. W. and Palmer, S. E., The Porphyrins, Vol. 1, Dolphin, D., Ed., Academic Press, New York, 1978. 9. Hodgson, G. W., Ann. N.Y. Acad. Sci., 206, 670, 1973. 10. Bonnet, R., Ann. N.Y. Acad. Sci., 206, 722, 1973.

11. Pfaltz, A., Jaun, B., Fassler, A., Eschenmoser, A., Jaenchen, R., Gilles, H. H., Diekert, G., and Thauer, R. K., Helv. Chim. Acta, 65, 828, 1982. 12. Langhof, H., Muller, H., and Rietschel, L., Arch. Klin. Exp. Dermatol., 212, 506, 1961. 13. Andreoni, A. and Cubeddo, R., Eds., Porphyrin in Tumor Phototherapx, Plenum Press, New York, 1984. 14. Sano, S., The Porphyrins, Vol. 7, Dolphin, D., Ed., Academic Press, New York, 1979, 378. 15. Hewson, W. D. and Hager, L. P., The Porphyrins, Vol. 7, Dolphin, D., Ed., Academic Press, New York, 1979, 295. 16. Berk, P. D. and Berlin, N. I., Eds., Chemistry and Physiology of Bile Pigments, Fogarty Int. Center Proc. No. 35, DHEW Publ. No. (NIH) 77-1100, National Institutes of Health, Bethesda, Md., 1977. 17. Rudiger, W., Fortschr. Chem. Org. Naturst., 29, 61, 1971. 18. Scheer, H., Angew, Chem. Int. Ed. Eng., 20, 241, 1981. 19. Scheer, H., Light Reaction Path o f Photosynthesis, Vol. 35, Fong, F. K., Ed., Springer-Verlag, Berlin, 1982. 20. Bennet, A. and Siegelman, H. W., The Porphyrins, Vol. 6 , Dolphin, D., Ed., Academic Press, New York, 1979, 493. 21. Brumm, P. J., Fried, J., and Friedmann, H. C ., Proc. Natl. Acad. Sci. U.S.A., 80, 3943, 1983. 22. Benedikt, E. and Kost, H.-P., Z. Naturforsch., 38c, 753, 1983. 23. Lascelles, J., Tetrapyrrole Biosynthesis and Its Regulation, Benjamin, New York, 1964. 24. Battersby, A. R. and McDonagh, A. E., Porphyrins and Metalloporphyrins, Smith, K. M., Ed., Elsevier, Amsterdam, 1975, 61. 25. Frydman, R. B., Frydman, B., and Valasinas, A., The Porphyrins, Vol. 6 , Dolphin, D., Ed., Academic Press, New York, 1979, 3. 26. Jacobs, N. J., Jacobs, J. M., Bloomer, J. R., and Morton, K. O., Enzyme, 28, 206, 1982. 27. White, W. I., Bachmann, R. C ., and Burnham, B. F., The Porphyrins, Vol. 1, Dolphin, D., Ed., Academic Press, New York, 1978, 553. 28. Soret, J. L., Comptes Rendues, 97, 1267, 1883. 29. Gamgee, A., Z. Biol. Munich, 34, 505, 1897. 30. MacDonald, S. F. and Michl, K. H., Can. J. Chem., 34, 1768, 1956. 31. Morsingh, F. and MacDonald, S. F., J. Am. Chem. Soc., 82, 4377, 1960. 32. Willstatter, R. and Mieg, W., Ann. Chem., 350, 1, 1906. 33. Fischer, H. and Orth, H., Die Chemie des Pyrrols, Vol. 1 and 2, Akademische Verlagsgesellschaft, Leipzig, 1934 and 1937; reprinted by Johnson Reprint, New York, 1968. 34. Janson, T. R. and Katz, J. J., The Porphyrins, Dolphin, D., Ed., Academic Press, New York,

1979, 1. 35. LaMar, G. N. and Walker, F. A., The Porphyrins, Dolphin, D., Ed., Academic Press, New York,

1979, 61. 36. Scheer, H. and Katz, J. J., Porphyrins andMetalloprophyrins, Smith, K. M., Ed., Elsevier, Amsterdam, 1975, 399. 16a. Budzikiewicz, H., The Porphyrins, Vol. 3, Dolphin, D., Ed., Academic Press, New York, 1978, 395. 16b. Dougherty, R. C ., Biochemical Applications o f Mass Spectrometry, Waller, G. E., Ed., John Wiley & Sons, New York, 1972, 591. 37. Fischer, H. and Bock, H., Hoppe-Seyler s Z. Physiol. Chem., 255, 1, 1938. 38. Inhoffen, H. H., Brockmann, H., Jr., and Bliesener, K.-M., Ann. Chem., 730, 173, 1969. 39. Barret, J., Nature (London), 183, 1185, 1959. 40. Smith, K. M., Porphyrins and Metalloporphyrins, Smith, K. M., Ed., Elsevier, Amsterdam, 1975, 15.

Volume I: Fat-Soluble Pigments 41. Karayannis, N. M., Corwin, A. H., Baker, E. W., Klesper, E., and Walter, J. A., 1736, 1968. 42. Klesper, E., Corwin, A. H., and Turner, D. A., J. O r g . C h e m ., 27, 700, 1962. 43. Karayannis, N. M. and Corwin, A. H., A n a l. B io c h e m ., 26, 34, 1968. 44. With, T. K., S c a n d . J. C lin . L a b . I n v e s t., 8, 113, 1956. 45. Lockwood, W. H. and Davis, J. L., C lin . C h im . A c ta , 7, 301, 1962.

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GENERAL TABLES Table II. 1 TRIVIAL NAMES AND STRUCTURES OF COMMON PORPHYRINS Substituents3

Porphyrin Etioporphyrin-I Octaethylporphyrin Deuteroporphyrin-IX Mesoporphyrin-IX Hematoporphyrin-IX Protoporphyrin-IX Coproporphyrin-I Coproporphyrin-III Uroporphyrin-I Uroporphyrin-III Chlorocruoroporphyrin Pemptoporphyrin Deuteroporphyrin-IX 2,4di-acrylic acid 2.4- Diformyldeuteroporphyrin-IX 2.4- Diacetyldeuteroporphyrin-IX Deuteroporphyrin-IX 2,4disulfonic acid Phylloporphyrin-XVb Pyrroporphyrin-XV Rhodoporphyrin-XV Phylloerythrin Deoxophylloerythrin Pheoporphyrin-a5

1 Me Et Me Me Me Me Me Me H H Me Me Me

2

3 Et Et H Et -CH(OH)CH 3 V P P

4

5

7

8

CHO H Acr

Me Et Me Me Me Me Me Me H H Me Me Me

Me

CHO

Me

CHO

Me

P

P

Me

Me

Ac

Me

Ac

Me

P

P

Me

Me

S 0 3H

Me

S 0 3H

Me

P

P

Me

Me Me Me Me Me Me

Et Et Et Et Et Et

Me Me Me Me Me Me

Et Et Et Et Et Et

Me Me Me Me Me Me

H H - C 0 2H -CO— CH2C -C H 2—CH2c -CO— CHc

P P P P P P

Me Me Me Me Me Me

P P

Et Et H Et -CH(OH)CH 3 V P P P P V V Acr

6

Me Et Et Et Me P Me P Me P Me P Me P Me P H P H P Me P Me P Me P

Me Et P P P P Me P H P P H P P P

Et Et Me Me Me Me P Me

Me Me Me

I

C 0 2Me Note: Side-chain abbreviations — Me = methyl; Et = ethyl; V = vinyl; R = H or substituent; P = CH 2CH 2C 0 2R; A = CH 2C 0 2R; Acr = CH=CHCOOH. a b c

Substituents (1 to 8 ) are arranged clockwise at peripheral positions of porphyrin nucleus. Me-substituent at 7 bridge. Forms ring by linkage to 7 bridge.

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CRC Handbook of Chromatography: Plant Pigments Table II. 2 QUANTITATIVE SPECTROSCOPIC DATA: MOLAR EXTINCTION COEFFICIENTS UV-VIS Spectral Data of Porphyrin Esters (nm) — €m Compound Uroporphyrin octamethyl ester3 Coproporphyrin tetramethyl ester3 Protoporphyrin IX dimethyl ester Deuteroporphyrin IX dimethyl ester Mesoporphyrin-IX dimethyl ester Hematoporphyrin-IX dimethyl ester 3

Soret X 406 215 X 400 e 180 X 407 e 171 X 399.5 e 175 X 400 e 166 X 402 e 193

e

IV

III

II

I

502 15.8 498 14.3 505 14.2 497 13.4 499 13.6 499 15.0

536 9.3 532 9.9 541 11.6 530 10.1 533 9.6 534 9.5

572

627 4.2 621 5.0 630 5.4 621 5.0 621 4.9 622 4.3

6 .8

566 7.1 575 7.4 566 8.2 567 6.5 569 6.9

Ref. 1 2 2 2 2 3

Spectra identical for all isomers.

REFERENCES 1. Mauzerall, D., J. Am. Chem. Soc., 82, 2601, 1960. 2. Smith, K. M., Porphyrins and Metalloporphyrins, 1st ed., Elsevier, Amsterdam, 1975. 3. Caughey, W. S., Fujimoto, W. Y., and Johnson, B. D., Biochemistry, 5, 3830, 1966.

Volume l: Fat-Soluble Pigments Table II. 3 HCI-NUMBERS OF PORPHYRINS AND PORPHYRIN ESTERS Compound

HCI Number32,3

Uroporphyrin III Octamethyl ester Coproporphyrin III Tetramethyl ester Protoporphyrin IX Dimethyl ester Mesoporphyrin IX Dimethyl ester Hematoporphyrin IX Deuteroporphyrin IX Dimethyl ester Chlorocruoroporphyrin Rhodoporphyrin XV Dimethyl ester Pyrroporphyrin XV Dimethyl ester Phylloporphyrin XV Dimethyl ester Pheoporphyrin a 5 Phylloerythrin a

5.0 0.9 1.7 2.5 5.5 0.5 2.5 0.1 0.3 2.0 4.6 4.0 7.5 1.3 2.5 0.35 0.9 9.0 7.5

The HCI number is defined as the concentration of HCI in percent (w/v) which extracts two thirds of the porphyrin from an equal volume of an ether solution. The HCI numbers depend jointly upon the dissociation of the porphyrin as a base and its ether-water partition coefficient. 1

REFERENCES 1. Willstatter, R. and Mieg, W., Ann. Chem., 350, 1, 1906. 2. Fischer, H. and Orth, H., Die Chemie des Pyrrols, Vol. 1 and 2, Akademische Verlagsgesellschaft, Leipzig, 1934 and 1937. 3. Mauzerall, D .,7. Am. Chem. Soc., 82, 2601, 1960.

207

208

CRC Handbook of Chromatography: Plant Pigments PAPER CHROMATOGRAPHY OF PORPHYRINS Table II. PC 1 FREE ACID PORPHYRINS Paper Solvent Technique Detection Literature

PI SI T1 D1 1

PI S2 T2b D1 1 1

PI S3 T2b D1

Compound

a Sheets 20 x 23 cm. b 20°C. c 0.5— 7 |xg per spot.

Solvent

PI P2 P3 SI

= = = =

52 53 54 55 56

= = = = =

57 = Technique T1 = T2 =

Detection

P la S5 T lc D1 2

P2 S6 T2d D1 1

1

P2 S2 T2C D1 3

P3 S7 T3 D1

26 — — 54 — 84 86 87 88 — — 100 100

— — — — — — — — — — — 63 —

Rf x 100

Uroporphyrin Hexacarboxlic porphyrin Pentacarboxylic porphyrin Coproporphyrin Tricarboxylic porphyrin Protoporphyrin Mesoporphyrin Hematoporphyrin Deuteroporphyrin Pemptoporphyrin Phylloerythrin Etioporphyrin All porphyrin esters

Paper

PI S4 T2 D1 2

T3 = D1 =

3 — — 6 — 8 8 8 8 — 8 — 100

6 — 42 56 68 83 — — — — — — —

85—95 45— 55 — 20— 30 — — — 5— 9 — — — — —

— — — — — 10f 36f 100f 62f 36f — — —

— — — — — 9 21 53 33 21 — — —

19 — — 47 — 75 81 77 79 — — — 100

d 15°C. c 25°C. f Rf position of spot relative to hematoporphyrin.

Whatman No. l a Whatman No. 3 Schleicher and Schiill paper 2034 b 2,4-2,5-lutidine-water; upper phase as saturated solution; atmosphere satu­ rated with NH 3 vapor 2,6-lutidine-water = 5:3; atmosphere saturated with NH 3 vapor 0.1 M LiCl, 20°C; atmosphere saturated with NH 3 vapor ethanol-2,6 -lutidine-water = 30:3:67 pyridine-0.2 M sodium borate buffer pH 8 .6 = 1:9 2,4-lutidine-water; upper phase as saturated solution; atmosphere saturated with NH 3 vapor CCl4-isooctane = 7:3 descending, NH 3 vapof ascending, NH 3 vapor15 de Equilibration: cylindrical glass tanks (13 x 28 cm) were allowed to equili­ brate for 30 min before use with 20 m € of 0.88 M ammonia placed at the bottom; the tanks were also lined with filter paper soaked in concentrated ammonia Developing: 20 m€ of developing mixture was placed in a suitable-sized Petri dish placed at the bottom of the tanks horizontal in saturated atmosphere of the solvent vapor visual detection under UV light

REFERENCES 1. Falk, J. E., J . C h r o m a to g r ., 5, 277, 1961. 2. Belcher, R. V., Smith, S. G., Mahler, R., and Campbell, J., 1970. 3. Blumer, M., A n a l. C h e m ., 28, 1640, 1956.

J . C h r o m a to g r .,

53, 279,

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209

T able II. PC 2 P O R P H Y R IN EST E R S Paper Solvent Technique Detection Literature

PI SI Tl + D1 1

P2 S2 T1 D1 2

PI S3 T1 D1 3

Compound* Uroporphyrin I octamethyl ester Uroporphyrin III octamethyl ester Heptacarboxylic porphyrin III heptamethyl ester Hexacarboxylic porphyrin III hexamethyl ester Pentacarboxylic porphyrin III pentamethyl ester Coproporphyrin I tetramethyl ester Coproporphyrin III tetramethyl ester Protoporphyrin IX Monomethyl ester Dimethyl ester Mesoporphyrin IX dimethyl ester Hematoporphyrin IX Dimethyl ester Dimethyl ether Deuteroporphyrin IX dimethyl ester M onovinyl m onohydroxyethyl deuteroporphyrin dimethyl ester a b c d e f

P3 S4 T1 D1 4, 5

P3 S5 Tl + + D1 4, 5

P3 S6 Tl + + D1 4, 5

P4 S7 T1 D1

7 10

6

Rf x 100 00b f 23b' 40°

— — —

— — —

— — —

50c

—

—

—

58c

—

—

—

04 — — 74b —

— — — — 80d 60d

— — S3e 95 100 —

— — — —

0041 — 4041 —

— — — —

66b

—

—

—

51 59

23 23

24 23

3 59 59 20

80 63 45 74

71 46 46 61

Methyl esters of porphyrins. Standard reference. Enzymic. Porphyrin dimethyl esters on iron-impregnated paper; Rf values estimated from areas of greatest density as spots showed long diffuse fronts. Lutidine solution of porphyrins. Caution: separation of I and III isomers does not work completely reliably.

Paper

Solvent

PI = Whatman No. 1 P2 = Whatman No. 1 paper impregnated with 1.8% (w/v) ferric chloride calculated as FeCl3*6 H20 P3 = Whatman No. 3 SI = kerosene-dioxane = 4.0:1.0 52 = benzene-methanol = 100:5 53 = lutidine-water = 5:3.5 in ammonia atmosphere 54 = kerosene-tetrahydropyran-methylbenzoate = 5:1.4:0.35 55 = water-acetonitrile-n-propanol-pyridine = 3.8:1:2:0.5 (with silicone as stationary phase) 5 6 = water-acetonitrile-dioxane = 2 .3:2.8:0 .8 (with silicone as stationary phase) 57 = carbon tetrachloride-i-octane = 7:4

CRC Handbook of Chromatography: Plant Pigments

210

Table II. PC 2 (continued) PORPHYRIN ESTERS Technique

Detection

T1 = ascending " = Chloroform solutions of porphyrin esters (1 p.g/10 |xf) were applied by means of a micropipette graduated in 5 p i along a baseline 2 cm from one edge of a 21 cm square of Whatman No. 1 paper, in such a manner that they would run with the grain of the paper. Chromatography was carried out at 22 to 26°C. " ' = Second development by reversed phase paper chromatography. The partially de­ veloped paper chromatogram (Solvent Syl) was treated with a petrol ether (bp. 56 to 110°C) solution of Dow-Coming silicone No. 550 fluid (w/v 12.5/100) by dipping the paper into the solution or by pulling it through. After drying at 105 to 110°C for 3 min, the paper cylinder, made with the new basal line at the bottom, was placed in the second solvent mixture (either S5 or S6) which was used in an atmosphere saturated with water. The developing time was 1.5 hr for the solvent front (S5) resp. 50 min (S6) to ascend to about 8 cm. During the second development, the members in the unresolved spot from the first devel­ opment were separated. D1 = visual detection under UV light (366 mm) REFERENCES

1. 2. 3. 4. 5. 6.

Cornford, P. A. D. and Benson, A., J. C h r o m a to g r ., 10, 141, 1963. Henderson, R. W. and Morton, T. C., J. C h r o m a to g r ., 27, 180, 1967. Ellsworth, R. K., A n a l. B io c h e m ., 32, 377, 1969. Falk, J. E., J. C h r o m a to g r ., 5, 277, 1961. Chu, T. C. and Chu, E. J., J. B io l. C h e m ., 208, 537, 1954. Blumer, M., A n a l. C h e m ., 28, 1640, 1956.

Volume I: Fat-Soluble Pigments

211

Table II. PC 3 P O R P H Y R IN E ST E R S (T W O -SO L V E N T SY ST E M S) Paper Solvent Technique Detection Literature

PI S1/S3 T1 D1 1

PI S2/S4 T1 D1 1 1

PI S2/S5 T1 D1

PI S2/S6 T1 D1 1

PI S2/S7 T1 D1 1

Second run /i-propanol with Compound8

Rf x 100

Uroporphyrin I methyl ester Coproporphyrin I methyl ester Coproporphyrin III methyl ester Protoporphyrin IX methyl ester Mesoporphyrin IX methyl ester a

Kerosene /i-Decane w-Dodecane 17 47 67 84 89

14 42 70 86 92

w-Tetradecane n-Hexane

20 52 76 92 96

13 45 74 92 95

15 47 66 89 93

Porphyrin-methylesters, Chu et al.2

Paper Solvent

PI = Whatman No. 1 paper, 24°C SI = chloroform-kerosene = 2.6:4.0 first run S2 = chloroform first run; second run propanol-alkane = 1:5 Technique T1 = ascending; the paper was dried at 105— 110°C for about 4 min after completion of the first run; the atmosphere was saturated with the same solvents as used for development Detection D1 = visual observation under UV light; spraying the completed chromatograms with isooctane before observation under UV light increased markedly the sensitivity of the fluorescence, 0.005 |±g being observable; for spots of nonfluorescent porphyrin metal complexes, it was found that on spraying with a solution of fluoranthene in n-pentane, and then illuminating with light at 366 nm, the porphyrins and their metal complexes showed as dark spots against a fluorescent background, about 0.04 |xg being observable

REFERENCES 1. Falk, J. E., J. Chromatogr., 5, 277, 1961. 2 Chu, T. C., Green, A. A., and Chu, E. J.,

J.

Biol. Chem., 190, 643, 1951.

212

CRC Handbook of Chromatography: Plant Pigments Table II. PC 4 PORPHYRIN “ DERIVATIVES” Paper Solvent Technique Detection Literature

PI SI T1 D1 1

Compound*

b

1

PI SI T1 D1 1 1

PI S2 T1 D1

Rf x 100

Deuteroporphyrin Monohydroxyethyl Monovinyl-mono hydroxyethyl Monohydroxymethyl Monohydroxymethylmonovinyl Dihydroxymethyl 2-Formy 1-4-hydroxyethyl 2-Ethylene-glycol Hematoporphyrin Porphyrin a Chlorin a2 Mesorhodochlorin 2-a-Hydroxy Mesochlorin P6 2-a-Hydroxy Mesopheophorbide a 2-a-Hydroxy 3

PI S2 T1 D1

34 29

38 34

64b 60b

68b 66b

19 22

24 31

54b 54b

57b 56b

1 16

14 26

56b 58b

56b 64b

30 3 10 30 31

32 18 26 40 34

64b 56b 56b 65b 80b

61b 57b 62b 60b 78b

33

35

82b

80b

10

34

78b

72b

Hydroxylated porphyrins and chlorins and their acetylated products. Acetylated compound.

Paper Solvent

PI SI S2 Technique T1 Detection D1

= = = =

Whatman No. 1 chloroform-kerosene = 2.6:42 propanol-kerosene = 1:52 ascending, 22°C visual REFERENCES

1. Barret, J., Nature 183, 1185, 1959. 2. Chu, T.C., Green, A. G., and Chu, E. J., J. Biol. Chem., 190, 643, 1951.

Volume I: Fat-Soluble Pigments T able II. PC 5 P O R P H Y R IN S A N D M E T A L L O P O R P H Y R IN S Paper Solvent Technique Detection Literature

PI SI T1 D '/2 1

P2 S2 T2 D1 2

Compoundd

P3 S4 T 3++ D1 2

P? S5 T4 D3 2

P? S6 T4 D3 2

96 88

20 56

34

76

45

77

Rf x

Uroporphyrin-Fe-complex (urohemin) Coproporphyrin-Fe-complex (coprohemin) Protoporphyrin-dimethyl ester Cu-complex Fe-complex (protohemin, hemin) Ni-complex Mesoporphyrin-dimethyl ester Fe-complex Ni-complex Hematoporphyrin-Fe-complex (hematohemin) Deuteroporphyrin-Fe-complex (deuterohemin) Deoxophyllerythrin-monomethyl ester V-complex Ni-complex Deoxophyllerythrin-etioporphyrin V-complex Ni-complex Etioporphyrin III a

P3 S3 T 3+ D1 2

— —

— —

— —

7 13 —

— — 68

— — 63

10 10 — 12 —

— — — — —

—

72

77

—

—

—

62

76

15 8 23 47 20 54 63

—

—

— — — — —

7

—

Rf x 100 values of free and esterified porphyrins and metalloporphyrins.

Paper

Solvent

PI P2 P3 SI 52

= = = = =

53 = 54 = 55 = 56 Technique T1 T2 T3 Detection

= = = =

Whatman No. 3 Whatman No. 1 Schleicher and Schiill paper 2043 b carbon tetrachloride-i-octane = 7:3 2,4-2,5-lutidine-water (upper phase, saturated solution, atmosphere sat­ urated with NH3 vapor) 2,4-lutidine-water (upper phase, saturated solution, atmosphere saturated with NH3 vapor) 2,6-lutidine-water = 5:3, atmosphere saturated with NH3 vapor water-n-propanol-pyridine = 5.5:0.1:0.4, atmosphere saturated with water and pyridine vapor 2,6-lutidine-water = 3.3:2.7, atmosphere saturated with water vapor horizontal in saturated atmosphere of the solvent vapor descending, NH3 vapor, 19°C ascending, NH3 vapor, 15° C + , 25°C++ ( + , ++; see Table II. PC2)

D1 = visual D2 = porphyrin metal complexes: quenching of fluorescence in the case of metal complexes by spraying the completed and dried chromatogram with or dipping it in a saturated solution of fluoroanthene in n-pentane; it is then rapidly air-dried and observed under UV radiation at 366 nm D3 = formation of blue spots upon spraying with benzidine reagent; by the use of this special spray, Connelly et al.3 were able to detect as little as 3 x 10“4 |xg of hemine. Important note: since benzidine is carcinogenic, it is better to use sub­ stitutes, for example, tetramethyl-benzidine (see table and text)4 5

213

214

CRC Handbook of Chromatography: Plant Pigments Table II. PC 5 (continued) PORPHYRINS AND METALLOPORPHYRINS REFERENCES 1. 2. 3. 4. 5.

Blumer, M ., Anal. Chem., 28, 1640, 1956. Falk, J. E ., J. Chromatogr., 5, 277, 1961. Connelly, J. L., Morrison, M., and Stotz, E., J. Biol. Chem., 233, 743, 1958. White, W. I., J. Chromatogr., 138, 220, 1977. For applications, see (e.g.) Kost, H.-P. and Benedikt, E .,Z . Naturforsch. , 37c, 1057, 1982.

Volume I: Fat-Soluble Pigments THIN-LAYER CHROMATOGRAPHY OF PORPHYRINS Table II. TLC 1 FREE ACID PORPHYRINS Layer Solvent Technique Detection Literature

LI SI T1 D1 1

L2 S2 T2 D1 2

Compound

L4 S4 T4 D1 4

L5 S5 T5 D1 5

Rf x 100

Uroporphyrin Coproporphyrin Protoporphyrin Mesoporphyrin Hematoporphyrin Deuteroporphyrin Deuteroporphyrin monohydroxyethylmonovinyl Pemptoporphyrin a

L3 S3 T3 D1 3

60— 80 30— 50 10 10 10 10 —

76 40 — — — 2 —

— — 19 25 35 32 —

— — 5 — — — —

— — 33a 25a 3a 28a 17a

10

—

28

—

—

Approximate values.

Porphyrins were prepared from human and bovine excreta. Layer

Solvent

LI = talc, pharmacopeia quality;6 talc suspensions were prepared from 50 g of talc, 50 g of methanol, and 2 g of gypsum, thoroughly mixed in a shaker; the mixture was immediately poured into the applicator and placed in position on a row of clean plates of plain window glass lying on a smooth firm support; immediately after pouring the sus­ pension into the applicator, it was moved slowly forward over the plates to spread an 0.30-mm-thick talc layer; after drying for ‘/2 hr at room temperature, the plates were ready for use; they can be stored for several weeks at room temperature if required L2 = talc on glass plates (20 x 20 cm2); the talc is of pharmacopeia quality and is passed through a 0.2-mm sieve prior to use; equal weights of talc and methanol (analytical) are mixed and applied on the plates; layer thickness: 0.25 mm; the plates are dried in air at room temperature L3 = talc plates (20 x 20 cm2); mixture: 40 g of talc and 70 m€ of methanol, thickness 0.25 mm; dry at room temperature (five plates) L4 = 20 x 20 cm2 silica gel Polygram® (Brinkman Instruments, Inc., Westbury, N.Y.) without fluorescent indicator, layer thickness 0.25 mm L5 = silica gel G, layer thickness 0.25 mm SI = acetone-0.5 N HC1 = 7:3 (v/v) 52 = acetone-0.5 N HC1 = 6:4 (v/v) 53 = ethanol-lutidine-water = 30:3:67 54 = 2,6-lutidine, (practical 95%) 55 = benzene-methanol/formic acid = 8.5:1.5 (v/v)/0.3 M

215

216

CRC Handbook of Chromatography: Plant Pigments Table II. TLC 1 (continued) FREE ACID PORPHYRINS Technique T1 = ascending; porphyrins were applied as solutions in HC1, ammonia, or HCl-acetone T2 = ascending, 1 hr; about 5— 10 p€ solution containing 0.1— 20.0 pg of porphyrin is employed, diameter of the spots is below 5 mm T3 = ascending; develop in the tank for at least 12 hr at a temperature range of 20— 25°C, plates can be left to develop overnight T4 = ascending T5 = ascending, 21— 23°C, 30— 45 min, solvent front 10 cm from start Detection D1 = visual; spots of free porphyrins are detected by their red fluorescence in UV light in a dark room

REFERENCES 1. With, T. K., J. Chromatogr.,42, 389, 1969. 2. With, T. K., Clin. Biochem., 1, 30, 1967. 3. Belcher, R. V., Smith, S. G., Mahler, R., and Campbell, J., J. Chromatogr., 53, 279, 1970. 4. Ellsworth, R. K., Anal. Biochem., 32, 377, 1969. 5. Ellfolk, N. and Sievers, G., J. Chromatogr., 25, 373, 1966. 6. Pharmacopeia Nordica, Vol. 2, Busch, Copenhagen, 1963, 594.

Compound

a

Approximate values.

Uroporphyrin octamethyl ester Heptacarboxylic porphyrin heptamethyl ester Hexacarboxylic porphyrin hexamethyl ester Pentacarboxylic porphyrin pentamethyl ester Coproporphyrin-tetramethyl ester Tricarboxylic porphyrin trimethyl ester Protoporphyrin IX monomethyl ester Protoporphyrin IX dimethyl ester Mesoporphyrindimethyl ester Hematoporphyrin-dimethyl ester Acetyl derivative Deuteroporphyrin-dimethyl ester Tetramethyl-tetrapropylporphyrin Meso-tetrapheny 1-porphyrin

Layer Solvent Technique Detection Literature

— 55 — — — — — — — — —

—

30 — — — — — — — — —

—

2+ — — — — — — — — —

—

—

90 —

—

1

LI S3 T1 D1 2

8 —

1

LI S2 T1 D1

6 —

LI SI T1 D1 1

— —

10+ — — — — — —

10+ — — — — — —

30+

—

—

80a —

L2 S2 T2 D1 2

— —

20+

—

—

60d —

L2 SI T2 D1

—

10+

—

55 +

—

90d —

L2 S3 T2 D1 2

Table II. TLC 2 PORPHYRIN ESTERS

9

55

33

3

L4 S4 T3 D1

88

4

L3 S6 T4 D1

24 3

98

15

x 1®®

4

L3 S5 T1 D1

92 92 14

—

—

L3 S4 T3 D1 3 3

21

26 19

L5 S4 T3 D1 5

84

54

L6 S7 T1 D1 6

^5

20

L7 S8 T1 D1 7

70d

^5d

49a

36d

25d

14d 19d

L8 S9 T5 D1

Volume I: Fat-Soluble Pigments 217

1. 2. 3. 4. 5. 6. 7.

With, T. K., Thin layer chromatography of porphyrins and their esters on talc plates, private communication to Burnham, B. F. With, T. K., J. Chromatogr., 42, 389, 1969. Henderson, R. W. and Morton, T. C ., J. Chromatogr., 27, 180, 1967. Jackson, A. H., Semin. Hematol., 14, 193, 1977. Ellsworth, R. K ., Anal. Biochem., 32, 377, 1969. Doss, M. and Burger, H., Z. Physiol. Chem., 348, 936, 1967. Doss, M., Z. Klin. Biochem., 8, 197, 1970.

REFERENCES

LI = talc plates; 50 g of sieved talc and 3— 5 g of burned gypsum in 50 g of methanol L2 = 50 g of talc, 50 g of methanol, and 2 g of gypsum, thoroughly mixed in a shaker; the mixture was immediately poured into the applicator; the layer was dried for 30 min at room temperature, thickness: 0.30 mm L3 = 0.25 mm silica gel G slurried in water L4 = 0.25 mm silica gel G slurried in 3%(w/v)FeS04*7 H20 L5 = 0.25 mm silica gel G slurried in 0.3% (v/v) sulfuric acid L2 = 20 x 20 cm silica gel Polygram® without fluorescent indicator L4 = silica gel H L8 = silica gel F254 Solvent SI = chloroform-methanol = 1:1 52 = ethanol-ethyl acetate = 3:2 53 = acetone-glacial acetic acid-pyridine = 1:1:1 54 = benzene-methanol = 100:5 55 = acetone-rt-hexane = 3:7 56 = benzene-acetone = 99:1 57 = 2.6 lutidine (practical 95%) 58 = benzene-ethylacetate-methanol-butanol = 82:14:3:1 59 = benzene-ethyl acetate-methanol = 85:13.5:1.5 Technique T1 = ascending T2 = ascending; porphyrin esters were applied as chloroform solutions T2 = ascending; 1— 5 jxg of porphyrin was applied to the plate in 10 fi€ benzene from a capillary pipette; the plates were devel­ oped in cylindrical glass tanks 11 0 x 22 cm for about 70 min at 20°C; this allowed the solvent front to travel about 17 cm T4 = ascending; 2 hr in a tank that has been equilibrated with the developing solvent for at least 1 hr T5 = ascending, 40 min at 21°C Detection D1 = visual; spots are detected by their red fluorescence in UV light in a dark room

Layer

Table II. TLC 2 (continued) PORPHYRIN ESTERS

218

CRC Handbook of Chromatography: Plant Pigments

Volume I: Fat-Soluble Pigments Table II. TLC 3 METALLOPORPHYRINS Layer Solvent Technique Detection Literature

LI SI T1 D1 1 1

LI S2 T1 D1 2

Compound

L2/, S3 T2 D1

L*A S4 T2 D1 2

L4 S5 T3 D1 3

Rf x 100

Protoporphyrin-Fe-complex (protochemin) Monomethyl ester Dimethyl ester Mesoporphyrin-Fe-complex (mesohemin) Hematoporphyrin-Fe-complex (hematohemin) Deuteroporphyrin-Fe-complex (deuterohemin) Diacetyl derivative

5

1

47

28

31

45 95 —

35 55 —

— — 51

— — 32

49

—

—

—

13

81

—

—

40

20

43

—

—

12

8

Layer

LI = Eastman chromatogram sheets (silica gel 6061) L2/3 = 0.25 mm silica gel thin-layer plates/2 mm silica gel thick-layer plates L4 = polyamide poly-e-caprolactam coated on ethylene terephthalate Solvent SI = 2,6-lutidine-water = 20:1 52 = hexane-chloroform-methanol = 1:1:0.2 53 = H-butanol-water-acetic acid = 50:1.5:1.4 54 = hexane-A7-propanol-acetic acid = 10:5:1.5 55 = methanol-acetic acid = 97.5:2.5 Technique T1 = ascending, 20°C T2 = ascending; samples of the hemins = 1 p f of ap­ proximately 2 x 10 ■M pyridine solutions T3 = ascending; samples of the hemins (up to 0.25 pT of 1 x 10 3 M pyridine solutions were applied 1 cm from the end of 4 x 8-cm polyamide plates to give an initial spot diameter of 1— 2 mm Detection D1 = visual

REFERENCES 1. Asakura, T. and Lamson, D. W .,A/ia/. Biochem., 53, 448, 1973. 2. Dinello, R. K. and Dolphin, D. W ., Anal. Biochem., 64, 444, 1975. 3. Lamson, D. W., Coulson, A. F. W., and Yonetani, T., Anal. Chem., 45, 2273, 1970.

219

220

CRC Handbook of Chromatography: Plant Pigments HIGH PERFORMANCE THIN-LAYER CHROMATOGRAPHY OF PORPHYRINS Table II. HPTLC 1 PORPHYRIN ESTERS Layer Solvent Technique Detection Literature

LI SI T1 D1 1

Compound

LI S2 T1 D1 1

LI S3 T1 D1 1

Rf x 100

Uroporphyrin I-octamethyl ester Heptacarboxylporphyrin I-heptamethyl ester Hexacarboxylporphyrin I-hexamethyl ester Pentacarboxylporphyrin I-pentamethyl ester Coproporphyrin I tetramethyl ester Mesoporphyrin IX dimethyl ester

36 41

28 30

12 17

46

46

25

53

55

32

60

65

40

75

77

59

Layer

LI = HPTLC-Kieselgel 60 (E. Merck) without flu­ orescence indicator Solvent SI = benzene-petrol ether (40— 60°C b.p.)-methanol-ethyl acetate = 48.5:40.0:10.5:9.0 52 = carbon tetrachloride-ethyl acetate = 1:1 53 = hexane-butanone-2-acetic acid = 15:7.5:1.5 Technique T1 = ascending on HPTLC plates for Nano-DC (10 x 10 cm), E. Merck Detection D1 = visual

REFERENCE 1. Benedikt, E. and Kost, H.-P., unpublished.

Volume I: Fat-Soluble Pigments

221

LIQUID CHROMATOGRAPHY OF PORPHYRINS Table II. LC 1 PORPHYRIN ESTERS Packing Column

length (cm diameter (cm) material

Solvent3, Solvent Solvent, Temperature Detection Literature

PI 10 1.5 G Sl-1 S I-2 S I-3 Ambient D1 1

PI 10 1.5 G S2-1 S2-2 S2-3 Ambient D1 1 1

P2 10 1.5 G S3-1 S3-2 S3-3 Ambient D1 1

P3 10 1.5 G S4-1 S4-2 S4-3 Ambient D1 1

P3 10 1.5 G S5-1 S5-2 S5-3 Ambient D1 1

P4 10 1.5 G S6-1 S6-2 S6-3 Ambient D1 1

P4 10 1.5 G S7-1 S7-2 S7-3 Ambient D1

Order of elution of components (1 = first, 2 = second, 3 = third) Compound Uroporphyrin-octamethyl esters Coproporphyrin-tetramethyl esters Dicarboxylic porphyrin-dimethyl esters a

3

3

3

3

3

1

1

2

2

2

2

2

2

2

1

1

1

1

1

3

3

Stepwise elution with three consecutive solvents. Each solvent system elutes one component of the system of porphyrins given above in the order of elution given. Intermediate compounds, e.g., heptacarboxylic porphyrin heptamethyl ester, etc., will elute in-between uroporphyrin octamethyl ester and coproporphyrin tetramethyl ester.

Packing

PI P2 P3 P4 Solvent Sl-1 S I-2 5152 S2-2 5253 S3-2 5354 S4-2 5455 S5-2 5556 S6-2 5657 S7-2 S7-3 Detection D1

= aluminum oxide grade IV = aluminum oxide grade II = calcium carbonate grade V = magnesium oxide grade III = benzene-chloroform = 10:1 = benzene-chloroform = 1:1 3 = chloroform-methanol - 100:1 1 = petroleum ether (b.p. 40— 60°C)-chloroform = 1:1 = petroleum ether (b.p. 40— 60°C)-chloroform = 1:6 3 = chloroform-methanol = 100:1 1 = petroleum ether (b.p. 40— 60 °C)-chloroform = petroleum ether (b.p. 40— 60°C)-chloroform 3 = petroleum ether (b.p. 40— 60°C)-chlorodorm 1 = benzene = benzene-chloroform = 10:6 3 = chloroform-methanol = 100:1 1 = petroleum ether (b.p. 40— 60°C)-chloroform = 3:1 = petroleum ether (b.p. 40— 60°C)-chloroform = 1:1 3 = petroleum ether (b.p. 40— 60°C)-chloroform = 1:7 1 = chloroform-methanol = 100:0.5 = chloroform-methanol = 100:1 2 = chloroform-methanol = 100:2 1 = benzene-methanol = 100:4 = benzene-methanol = 100:8 = benzene-methanol = 100:10 = visual REFEREN CE

1. Nicholas, R. E ., Biochem. J .f 48, 309, 1951.

= 6:1 = 4:1 = 1:1

222

CRC Handbook of Chromatography: Plant Pigments Table II. LC 2 PORPHYRINS, HEMINS, AND ESTERS (SILICA GEL) Packing Column

length (cm) diameter (cm) material

Solvent Temperature Detection Literature

PI 60 2.5 G SI Ambient D1 1

Compound

PI 15 n.a. G S3 Ambient D1 2

Ve (m€)

Protoporphyrin IX Fe-complex Monomethyl ester Monomethyl esterFe-complex Dimethyl ester Dimethyl esterFe-complex Note:

PI 60 2.5 G S2 Ambient D1 1

— — — 300

— 575 — —

+ + + — + + —

— 230

— —

+ —

+ elutes first, + + elutes second, + + + elutes last

Packing Solvent

Detection

PI SI SI S3 D1

= = = = =

silica gel hexane-chloroform-methanol = 1:1:0.3 chloroform-methanol = 1:1 2,6-lutidine-water = 12:1 visual

REFERENCES 1. Asakura, T. and Lamson, D. W., Anal. Biochem., 53, 448, 1973. 2. Ellsworth, R. K., Anal. Biochem., 32, 377, 1969.

Volume I: Fat-Soluble Pigments Table II. LC 3 PORPHYRINS AND PORPHYRIN ESTERS (SEPHADEX) Packing Column

length (cm) diameter (mm) material

Solvent Temperature Detection Technique Literature

PI 60 8 G SI Ambient D1 T1 1

PI 60 8 G S2 Ambient D1 T1 1

1

Compound

PI 60 8 G S4 Ambient D1 T1 2

P2 87 25 G S5 Ambient D2 T2

— — — 32 — — — — 109 — — — 65

— 203 163 — 225 176 246 215 — — 271 238 —

Ve (m€)

Uroporphyrin Octamethyl ester. Octa-H-butyl ester. Coproporphyrin Tetramethyl ester Tetra-n-butyl ester Mesoporphyrin dimethyl ester Di-n-butyl ester Hematoporphyrin Deuteroporphyrin Dimethyl ester Di-n-butyl ester Porphyrin C Packing Packing Solvent

PI 60 8 G S3 Ambient D1 T1 1

PI P2 SI SI 53 54 55 Detection D1 D2 Technique T1

18 — — 78 — — — — — 570 — — —

— — — 25 — — — — — 38 — — —

23 — — 34 — — — — — — — — —

= = = = = = = = = =

Sephadex G-25 (Dextran gel) Sephadex LH-20 0.2 M borate buffer pH 8.6 0.002 M borate buffer pH 8.6 0.01 M borate buffer pH 8.6 0.05 M borate buffer pH 8.6 chloroform-methanol = 1:1, containing 1 g Tris base per liter optical absorption at the Soret peak (cells of 1-cm light path) visual for application to the column, porphyrins were dissolved in a minimum quantity (0.5— 1 m f) of 0.2 M sodium borate buffer pH 8.6; to each 2-m€ fraction, 2 m€ of 3 N HC1 were added and the mixture was further diluted with 1.5 M HC1, if necessary T2 = The elution profile was taken from porphyrin ester run separately on a 2.5 x 87cm column of Sephadex LH-20. Solvent, chloroform-methanol containing 1 g Tris base per liter.

REFERENCES 1. Rimington, C. and Belcher, R. V., J. Chromatogr., 28, 112, 1967. 2. Bachmann, R. C. and Burnham, B. F., J. Chromatogr., 41, 344, 1969.

223

Uroporphyrin Octamethyl ester Heptacarboxylic porphyrin Heptamethyl ester Hexacarboxylic porphyrin Hexamethyl ester Pentacarboxylic porphyrin Pentamethyl ester Coproporphyrin Tetramethyl ester Protoporphyrin IX-dimethyl ester Mesoporphyrin IX Dimethyl ester Harderioporphyrin-trimethyl ester Isoharderioporphyrin-trimethyl ester

Compound

Packing Temperature Solvent Flow rate (m€ x m i n 1) Column length (cm) diameter(mm) (ID) form material Detector Literature

— 8.6 — 5.2 — 3.2 — 2.0 — 1.2 — — — — —

12.2be — —

—

P2 Ambient S2 0.8— 1.3 25 20 Straight SS D2 2

1.8bc — 2.2bc — 3.4bc — 5.0bc — 7.4b,c — —

PI Ambient SI 0.5 25 26 Straight SS D1 1 3

—

— — —

— 5.8 — — — — — — — — —

P3 Ambient S3 1 183 n.a. n.a. SS 316 D3 3

11.2

— — 13.0

— — — — — — — — — — —

P3 Ambient S4 1 183 n.a. n.a. SS 316 D3 3

—

— 6.3 —

— — — — — — — — — — 7.1

/R (min)

P3 Ambient S5 1 183 n.a. n.a. SS 316 D3 4

—

— — —

— 7 — 4.5 — 3.5 — 2.1 — 1.8 —

P4 Ambient S6 1.5 20 4 Straight SS D3 5

Table II. HPLC 1 PORPHYRINS AND PORPHYRIN ESTERS

—

— — —

— 6.4 — 4.1 — 2.8 — 2.0 — 1.4 —

P5 Ambient S7 2— 3a n.a. n.a. Straight SS D4 6

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF PORPHYRINS

—

— 1.8d —

— 6.2d — 4.6d — 3.5d — 2.8d — 2.2d —

P5 Ambient S8 2 30 3.9 Straight SS D5 6

—

— 2.0C —

— 8.6e — 6.2C — 4.5C — 3.4C — 2.7C —

P5 Ambient S9 2 30 3.9 Straight SS D5

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CRC Handbook of Chromatography: Plant Pigments

Flow rate at 2 m€ x m in-1 until the pentacarboxyl porphyrin has passed, then at 3 m f x m i n 1. Standard free acid porphyrins (Porphyrin Products, Logan, Utah) in acetone-0.1 N HC1 = 10:1, v/v; 0.04 nmol of each compound. Approximate values. k' = 0.62 /r-0.96. k' = 0.66 fR-0.92.

Solvent

Packing

52 53 54 55 56 SI 58 59

PI P2 P3 P4 P5 SI

= Perkin Elmer Silica A 10 fim = Micro Pak CN, 10 p,m (Varian) = Corasil II = 5 (xm Partisil = 5 |xm Porasil = elution was performed for 25 min with a linear gradient of acetone-dilute acetic acid (2—90%A) as follows: acetone-0.23 M acetic acid = 70:30, v/v delivered from pump B in the reverse-pump exchange mode and 10% acetic acid delivered from pump A in the reversed-pump exchange mode = ethyl acetate-n-heptane-isopropanol = 40:60:0.5 = ethyl acetate-petrol ether (b.p. 60— 80°C) = 2.5:97.5 = ethyl acetate-petrol ether (b.p. 60— 80°C) = 25:75 = ethyl acetate-petrol ether (b.p. 60— 80°C) = 30:70 = ethyl acetate-cyclohexane = 60:40 = benzene-ethyl acetate-chloroform = 70:10:20 = benzene-ethyl acetate-methanol = 85:13.5:0.75 = ethyl acetate-heptane = 55:45

Vj = elution volume of the i,h component of mixture VQ = void volume of column

K = (V,-V0) x v 0->

d,c The capacity factor k' is defined as:

c d e

b

a

Volume I: Fat-Soluble Pigments 225

D5

D4

D2 D3

D1

REFERENCES

= Perkin Elmer series 3 liquid chromatograph equipped with an LC-55 UV-Vis digital spectrophotometer; porphyrins were de­ tected by their absorbance at 403 nm and by fluorescence in a Perkin Elmer fluorescence spectrophotometer model 240 A = spectrophotometric at 400— 402 nm (Variscan) = Cecil variable wavelength detector set at 400 nm and fitted with a 10-p,€ flow cell = Beckman model 25 spectrophotometer set at 400 nm and fitted with a microflow cell = Waters 44 monitor set at 403 nm

6. Straka, J. G., Kushner, J. P., and Burnham, B. F., High-performance liquid chromatography of porphyrin esters. Identification of mixed esters generated in sample preparation, Anal. Biochem., I l l , 269, 1981.

5. Petryka, Z. J. and Watson, C. J., A new rapid method for isolation of naturally occurring porphyrins and their quantitation after high performance liquid chromatography, Anal. Biochem., 84, 173, 1978.

1. Longas, M. O. and Pols-Fitzpatrick, M. B., High-pressure liquid chromatography of plasma free acid porphyrins, Anal. Biochem., 104, 268, 1980. 2. Miller, V. and Malina, L., High-performance liquid chromatographic analysis of biologically important porphyrins, J. Chromatogr., 145, 290, 1978. 3. Evans, N., Games, D. E., Jackson, A. H., and Matlin, S. A., Applications of high pressure liquid chromatography and field desorption mass spectrometry in studies of natural porphyrins and chlorophyll derivatives, J. Chromatogr., 115, 325, 1975. 4. Evans, N., Jackson, A. H., Matlin, S. A., and Towill, R., High performance liquid chromatographic analysis of porphyrins in clinical materials, J. Chromatogr., 125, 345, 1976.

Detector

Table II. HPLC 1 (continued) PORPHYRINS AND PORPHYRIN ESTERS

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CRC Handbook of Chromatography: Plant Pigments

Volume I: Fat-Soluble Pigments Table II. HPLC 2 PORPHYRINS AND PORPHYRIN ESTERS: SEPARATION OF ISOMERS Column packing Temperature Solvent Flow rate (m t x m i n 1) Column length (cm) diameter (mm, (I.D.) form material Detector Literature

PI Ambient SI 1.5ab 30 3.9 st SS D1 1 1

PI Ambient S2 1.5b 30 3.9 st SS D1 2

Compound Uroporphyrin I- isomer II- isomer III- isomer IV- isomer Uroporphyrin-octamethyl ester I-isomer Ill-isomer Heptacarboxylic porphyrin I-isomer I-isomer heptamethyl ester Ill-isomer Hexacarboxylic porphyrin I-isomer I-isomer hexamethyl ester Ill-isomer Pentacarboxylic porphyrin I-isomer I-isomer pentamethyl ester Ill-isomer Coproporphyrin I-isomer I-isomer tetramethyl ester Ill-isomer a b

P2 Ambient S3 0.9 30 4.0 st SS D2

tR

P2 Ambient S4 1.0 30 3.9 st SS D2 2

P3 Ambient S5~ 1.5 30 3.9 st SS D1 3

(min)

— — — —

— — — —

22 38 29 31.5

11 — 14 —

9.5 — 12 —

98 102

— —

— —

— —

— —

— — —

— 5 —

— — —

— — —

3.8 — 5.3

— — —

— 6 —

— — —

— — —

3.2 — 5.3

— — —

— 7 —

— — —

— — —

2.8 — 4.7

— — —

— 10 —

— — —

— — —

3.6 — 9.5

Two columns in series. Recycling mode.

Packing

Solvent

PI P2

= p-Porasil (Waters column model A, LC 202) = jx-Bondapak C 18 (Waters); column equipped with a Whatman 50 x 4.6-mm CO:PEL (ODS) 37- to 50-pm precolumn P3 = p-Bondapak (Waters) SI = n-heptane-glacial acetic acid-acetone-water = 90:60:30:0.5 52 = n-heptane-glacial acetic acid-acetone-water = 90:60:90:0.5 53 = acetonitrile-10 2 M phosphate buffer pH 6.95 = 4:96 54 = acetonitrile-10“ 2 M phosphate buffer pH 6.95 = 5:95 S5+ = separation of n-carboxylic porphyrins, n = 8...4

227

228

CRC Handbook of Chromatography: Plant Pigments Table II. HPLC 2 (continued) PORPHYRINS AND PORPHYRIN ESTERS: SEPARATION OF ISOMERS

Detector

No. of carboxyl groups

Percent acetonitrile in phosphate buffer (10-2 A/, pH 6.85; 5 x 10 4 M EDTA

8 7

2.5 5.0

6

10.0

5 4

12.5 15.0

D1 = Waters 440 detector, compounds monitored at 405 nm D2 = Schoeffel FS 970 fluorometer

REFERENCES 1. Bornmer, J. C., Burnham, B. F., Carlson, R. E ., and Dolphin, D., The chromatographic separation of uroporphyrin I and III octamethyl esters, Anal. Biochem., 95, 444, 1979. 2. Wayne, A. W., Straight, R. C ., Wales, E. E., and Englert, E., Jr., Isomers of uroporphyrin free acids separated by HPLC, J. HRC and CC, 2, 621, 1979. 3. Prior, M. et al., personal communication.

Volume ¡: Fat-Soluble Pigments

229

Table II. HPLC 3 SURVEY OF SAMPLE WORKUP FOR HPLC OF PORPHYRINS (CONDENSED TABLE) Procedure Preadsorption on silica gel Separation of uroporphyrin isomers Direct injection after addition of mesoporphyrin standard Solvent extraction method 4 Direct injection of urine preserved with sodium carbonate/tetrasodium EDTA; external standard solutions Isolation of uroporphyrins from urine and feces; conversion into methyl esters (methanol/sulfuric acid) Coprecipitation of uroporphyrin and coproporphyrin with calcium hydroxide; redissolution in hydrochloric acid; final pH chlorphyll b > chlorophyll a), which can lead (and also be useful) to selective demetalation. There is recent evidence that demetalation is also an enzymatic process in chlorophyll degradation. A second effect of Mg is the change in the reduction potential of the macrocycle towards a more ready oxidation. Many chlorophyll oxidations proceed via ir-cation radicals and have recently been shown to be also dependent on the coordination state of the central Mg. This metal ion is coordinatively unsaturated and bears either one or two extra ligands, depending mainly on the solvent system. The macrocycle of bacteriochlorophyll a is more readily oxidized with the Mg-bearing one (e.g., in acetone solution) rather than two “ extra” ligands (e.g., in methanolic solution). Last but not least are the spectroscopic properties of chlorophylls dependent on the sol­ vation — and by consequence aggregation — of chlorophylls. Any identification by spec­ troscopic comparison with authentic material must, therefore, be done under identical conditions. Artifacts Involving the Isocyclic Ring

The isocyclic ring E bears the enolizable (3-ketoester system, which is responsible for most of the observed reactions. The susceptible C-13 is further activated by being in a quasibenzylic position and by the strain of the ring. The ready enolization of C-132 is responsible for its epimerization to the so-called “ prime” pigments, and to the production of “ alio-

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merization” products (13-alkoxylated, -hydroxylated, or -acetoxylated pigments). In all allomerization products, C-132 is no longer enolizable. This is the basis of the negative “ phase test” for these compounds. Important with respect to artifacts is the inevitable formation of two quite stable epimers, thus increasing the number of separable fractions. Another well-characterized allomerization product is the 13' a-oxa-132-methoxy-chlorophyll (“ 10-methoxylactone” ). Heat treatment leads to the loss of the 132-COOCH3-substituent to yield the pyrochlorophylls. The situation has become more complicated by the implication of chlorophyll a , e.g., the 132-epimer of chlorophyll a as a constituent of photosystem I and by the finding of 132-hydroxychlorophylls, 132-hydroxypheophorbides, and pyropheophorbides in degreening Euglena and in aged bacterial and plant cell cultures. Here enzymatic reactions may occur besides the ready chemical reactions. Artifacts Involving Carbon C-20

The 20-methine bridge of the chlorin macrocycle is susceptible to electrophilic attack. Although no common alteration product, the 2 0 -0 derivative has recently been reported to be produced during the washing of chlorophyll solutions with tap water (which is chlorinated in most parts of the world). Again, these compounds are also implicated as natural products of important function. The occurrence of 2 0 -0 chlorophyll(s) has been demonstrated in photosystem I in stoichiometric amounts relative to P-700. Artifacts Involving Oxidation of the Macrocycle

This problem is important in bacteriochlorophylls a and b, which are readily oxidized to chlorophyll-type pigments. 3-Devinyl-3-acetylchlorophyll a and its derivatives are common contaminants of bacteriochlorphyll a and especially b preparations. Acetone is notorious as solvent, which has been related to the ready oxidation of solvent. Since methanol is, on the other hand, prone to induce allomerization, mixed systems have been found most safe. Chlorophylls of the chlorin-type (plant chlorophylls and bacteriochlorophylls c, d, and e) are stable towards oxidation under the common extraction conditions, but care should be taken in the presence of oxidants. Quinones of high redox potential (e.g., tetrachloroquinones) have been used as selective oxidants during chemical structure correlation studies for both the conversion of bacteriochlorins to chlorins and of chlorins to porphyrins. Artifacts Involving the 3-Vinyl Group

Although generally quite reactive, alterations of this substituent are generally much slower than at the aforementioned sites. Pigments of this type have been found as by-products during quinone oxidation. Some of the less common chlorophylls (e.g., chlorophyll d) are derived from (probably biosynthetic?) modifications of the 3-vinyl group, and the bacter­ iochlorophylls c,d, and e contain the 3-hydroxyethyl substituent. Artifacts Involving Propionic Ester Side Chains

The 172-ester group is attacked by chlorophyllase to produce transesterified pigments (e.g., methyl esters in methanolic solution) and/or the free acid. Chlorophyllase is active in the common extraction media, and its activity varies greatly with the biological material. The 132-carbomethoxy group is stable to transesterification and hydrolysis under all common extraction conditions.

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REFEREN CES 1. Tswett, M., Adsorptionsanalyse und Chromatographische Methode. Anwendung auf die Chemie des Chlo­ rophylls, Ber. Dtsch. Bot. Ges., 24, 384, 1906. 2a. Svec, W. A., The isolation, preparation, characterization, and estimation of the chlorophylls and the bacteriochlorophylls, in The Porphyrins, Vol. 5, Dolphin, D., Ed., Academic Press, New York, 1978, 341. 2b. Oelze, J., Analysis of bacteriochlorophylls, in Methods in Microbiology, Vol. 18, Academic Press, London, 1985, 252. 2c. Cavaleiro, J. A. S. and Smith, K. M., Chromatography of chlorophylls and bacteriochlorophylls, Talanta, 33, 963, 1986. 3a. Falk, H., Moornaert, G., Isenring, H. P., and Eschenmoser, A., Über Enolderivate der Chlorophyllreihe. Darstellung von 132, 173-Cyclophäophorbide Enolen, Helv. Chim. Acta, 58, 2347, 1975. 3b. Hynninen, P., Application of elution analysis to the study of chlorophyll transformations by column chromatography on sucrose, J. Chromatogr., 175, 75, 1979. 3c. Inhoffen, H. H., Jäger, P., and Mählhop, R., Partialsynthese von Rhoidin-g7-trimethylester aus Chlorine6-trimethylester, zugleich Vollendung der Harvard-Synthese des Chlorophylls a zum Chlorophyll b, JustusLiebigs Ann. Chem., 749, 109, 1971. 3d. Risch, N., Brockmann, H., Jr., and Gloe, A., Strukturaufklärung von neuartigen Bakteriochlorophyllen aus Chloroflexus aurianticus, Justus Liebigs Ann. Chem., p. 408, 1979. 3e. Smith, K. M., Partial synthesis of chlorophyll-A from rhodochlorin, Tetrahedron, 37, 399, 1981. 3f. Wasielewski, M. R. and Thompson, J. F., 9-Desoxo-9, 10-dehydrochlorophyll a, Tetrahedron Lett., p. 1043, 1978. 3g. Wolf, H. and Scheer, H., Photochemische Hydrierung von Phäophyrinen: 7,8-cis Phäophorbide, Justus Liebigs Ann. Chem., p. 1710, 1973. 4a. Thornber, J. P., Markwell, J. P., and Reinman, S., Plant chlorophyll protein complexes: recent advances, Photochem. Photobiol., 29, 1205, 1979. 4b. Cogdell, R. J. and Thornber, J. P., Light-harvesting pigment-protein complexes of purple photosynthetic bacteria, FEBS Lett., 122, 1, 1980. 4c. Gingras, G., Comparative review of photochemical reaction center preparations from photosynthetic bac­ teria, in The Photosynthetic Bacteria, Clayton, R. K. and Sistrom, W. R., Eds., Plenum Press, New York, 1978, chap. 6. 4d. Thornber, J. P., Trosper, T. L., and Strouse, C. E., Bacteriochlorophyll in vivo: relationship of spectral forms to specific membrane components, in The Photosynthetic Bacteria, Clayton, R. K. and Sistrom, W. R., Eds., Plenum Press, New York, 1978, chap. 7. 4e. Olson, J. M., Bacteriochlorophyll a-proteins from green bacteria, in The Photosynthetic Bacteria, Clayton, R. K. and Sistrom, W. R., Eds., Plenum Press, New York, 1978, chap. 8. 4f. Anderson, J. M. and Barrett, J., Light-harvesting pigment-protein complexes of algae, in Encyclopedia of Plant Physiology, n.s., Vol. 19, Photosynthesis III, Staehelin, L. A. and Amtzen, C. J., Eds., Springer Verlag, Berlin, 1986, 269. 4g. Anderson, B. and Anderson, J. M., The chloroplast thylakoid membrane — isolation, subfractionation, and purification of its supramolecular complexes, in Modern Methods of Plant Analysis, n.s., Vol. 1, Cell Components, Linskens, H. F. and Jackson, J. F., Eds., Springer Verlag, Berlin, 1985, 231. 4h. Thornber, J. P., in Encyclopedia of Plant Physiology, n.s., Vol. 19, Photosynthesis III, Staehelin, L. A. and Amtzen, C. J., Eds., Springer Verlag, Berlin, 1986, chap.3. 5. IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN), Nomenclature of tetrapyrroles, Pure Appl. Chem., 51, 2251, 1979. 6. Bonnett, R., Nomenclature, in The Porphyrins, Vol. 1, Dolphin, D., Ed., Academic Press, New York, 1978, 1. 7. Fischer, H. and Orth, H., Die Chemie des Pyrrols, Vol. 2, 2nd half, Akademische Verlagsgesellschaft, Leipzig, 1940; reprinted by Johnson Reprint Corp., New York, 1968. 8. Baker, E. W. and Palmer, S. E., Geochemistry of porphyrins, in The Porphyrins, Vol. 1, Dolphin, D., Ed., Academic Press, New York, 1978, 485. 9. Schoch, S., Scheer, H., Schiff, J. A., Siegelman, H. W., and Rtidiger, W., Pyropheophytin accompanies pheophytin in darkened light grown cells of Euglena, Z. Naturforsch., 36c, 827, 1981. 10. Feher, G. and Okamura, M.Y., Chemical composition and properties of reaction centers, in The Pho­ tosynthetic Bacteria, Clayton, R. K. and Sistrom, W. R., Eds., Plenum Press, New York, 1978, chap. 19. 11. Klimov, V. V., Dolan, E., Shaw, E. R., and Ke, B., Interaction between the intermediary electron acceptor (pheophytin) and a possible Plastoquinone-iron complex, Photosystem II, 77, 7227, 1980.

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12a. Scott, A. I., Irwin, A. J., Siegel, L., and Shoolery, J. S., Sirohydrochlorin. Prosthetic group of sulfite and nitrite reductase in its role in the biosynthesis of vitamin Bl2, J. Am. Chem. Soc., 100, 7987, 1978. 12b. Deeg, R., Kriemler, H. P., Bergmann, K.-H., and Müller, G., Neuartige, methylierte Hydroporphyvine und deren Bedeutung bei der Cobyrinsäure-Bildung, Z. Physiol. Chem., 358, 339, 1977. 12c. Imfeld, M., Arigoni, D., Deeg, R., and Mueller, G., Factor I ex Clostridium tetanomorphum: proof of structure and relationship to vitamin B12 synthesis, in Vitamin B12 and Intrinsic Factor, 3rd. Eur. Symp., de Gruyter, Berlin, 1979, 315. 12d. Battersby, A. R. and McDonald, E., Origin of the pigments of life: the type-III problem in porphyrin biosynthesis, Acc. Chem. Res., 12, 14, 1979. 13a. Siegel, L. M., Murphy, M. J., and Kamin, H., Reduced nicotinamide adenine dinucleotide phosphatesulfite reductase of enterobacteria. I, J. Biol. Chem., 248, 251, 1973. 13b. Vega, J. M. and Kamin, H., Spinach nitrite reductase, J. Biol. Chem., 252, 896, 1977. 14a. Agins, L., Ballantine, J. A., Ferrito, V., Jaccarini, J., Murray-Rust, P., Pelter, A., Psaila, A. F., and Schembri, P. J., Bonellin, Pure Appl. Chem., 51, 1847, 1979. 14b. Matthews, J. I., Braslavsky, S. E., and Camilleri, P., The photophysics of bonellin: a chlorin found in marine animals, Photochem. Photobiol., 32, 733, 1980. 15. Vernon, L. P. and Seely, G. R., Eds., The Chlorophylls, Academic Press, New York, 1966. 16a. Katz, J. J., Norris, J. R., Shipmann, L. S., Thurnauer, M. C., and Wasielewski, M. R., Chlorophyll functions in the photosynthetic reaction center, Annu. Rev. Biophys. Bioeng., 7, 393, 1978. 16b. Battersby, A. R. and McDonald, E., Biosynthesis of porphyrins, chlorins and corrins, in Porphyrins and Metalloporphyrins, Smith, K. M., Ed., Elsevier, Amsterdam, 1975, chap. 3. 16c. Katz, J. J., Chlorophyll, in Inorganic Biochemistry, Eichhorn, G., Ed., Elsevier, Amsterdam, 1973, 1022. 16d. Scheer, H. and Inhoffen, H. H., Hydroporphyrins: reactivity, spectroscopy, and hydroporphyrin analogues, in The Porphyrins, Dolphin, D., Ed., Vol. 2 (Part B), Academic Press, New York, 1978. 16e. Jones, O. T. G., Chlorophyll biosynthesis, in The Porphyrins, Vol. 3, Dolphin, D., Ed., Academic Press, New York, 1978, chap. 3. 16f. Weiss, C., Optical spectra of chlorophylls, in The Porphyrins, Vol. 3, Dolphin, D., Ed., Academic Press, New York, 1978, chap. 3. 16g. Brockmann, H., Jr., Stereochemistry and absolute configuration of chlorophylls and linear tetrapytrroles, in The Porphyrins, Vol. 2, Dolphin, D., Ed., Academic Press, New York, 1978, chap. 9. 16h. Sauer, K., Primary events and the trapping of energy, in Bioenergetics of Photosynthesis, Govindjee, Ed., Academic Press, New York, 1978, chap. 3. 16i. Papageorgiou, Chlorophyll fluorescence: an intrinsic probe of photosynthesis, in Bioenergetics of Photo­ synthesis, Govindjee, Ed., Academic Press, New York, 1975, chap. 6. 16j. Rüdiger, W. and Schoch, S., Chlorophylls, in Chemistry and Biochemistry of Plant Pigments, Vol. 2, Goodwin, Ed., in press. 16k. Bogorad, L., in Chemistry and Biochemistry of Plant Pigments, Vol. 1, Goodwin, T. W., Ed., Academic Press, London, 1976, 64. 161. Holden, M., in Chemistry and Biochemistry of Plant Pigments, Vol. 2, Goodwin, T. W., Ed., in press. 16m. Jackson, A. H., in Chemistry and Biochemistry of Plant Pigments, Vol. 1, Goodwin, T. W., Ed., Academic Press, London, 1976, 1. 16n. Schneider, H. A. W., in Pigments in Plants, Czygan, F. C., Ed., Gustav Fischer Verlag, Stuttgart, 1980, 237. 16o. Castelfranco, P. A. and Beale, S. I., in The Biochemistry of Plants, Vol. 8, Stumpf, P. K. and Conn, E. E., Eds., Academic Press, New York, 1981. 16p. Castelfranco, P. A. and Beale, S. I., Annu. Rev. Plant Physiol., 34, 241, 1983. 16q. Porra, R. J. and Meisch, H.-U., TIBS, 9, 99, 1983. 16r. Leeper, F. J., Nat. Prod. Rep., 2, 19 and 561, 1985. 16s. Larkum, A. W. D. and Barrett, J., Adv. Bot. Res., 10, 1, 1983. 17. Jones, M. S. and Jones, O. T. G., Ferrochelatase of Rhodopseudomonas spheroides, Biochem. J., 119, 453, 1970. 18. Csatorday, K., MacColl, R., and Berns, D. S., Accumulation of protoporphyrin IX and zinc protopor­ phyrin IX in Cyanidium caldarium, Proc. Natl. Acad. Sei. U.S.A., 78, 1700, 1981. 19. Schoch, S., Lempert, U., and Rüdiger, W., Über die letzten Stufen der Chlorophyllbiosynthese: Zwis­ chenprodukte zwischen Chlorophyllid und phytohaltigem Chlorophyll, Z. Pßanzenphysiol., 83, 427, 1977. 20. Scheer, H., Svec, W. A., Cope, B. T., Studier, M. H., Scott, R. G., and Katz, J. J., Structure of bacteriochlorophyll b, J. Am. Chem. Soc., 96, 3714, 1974. 21a. Shlyk, A. A., Fradkin, L. I., Rudoi, A. B., Prudnikova, I. V., and Savchenko, G. E., Group mechanism of pigment assembly in centers of chlorophyll biosynthesis, in Chloroplast Development, Developments in Plant Biology, Vol. 2, Akoyonoglu, G. and Akoyonoglu, J. H., Eds., Elsevier, New York, 1978, 119. 21b. Bednarik, D. P. and Hoober, K. J., Science, 230, 450, 1985.

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22. Dornemann, D. and Senger, H., Isolation and partial characterization of a new chlorophyll associated with the reaction centre of photosystem I of scenedesmus, FEBS Lett., 126, 323, 1981. 23. Rebeiz, C. A., Balanger, F. C., Freyssinet, G., and Saab, D. S., Chloroplast biogenesis. XXIX. The occurrence of several novel chlorophyll a and b chromophores in higher plants, Biochim. Biophys. Acta, 50, 234, 1980. 24. Scholz, B. and Ballschmiter, K., Do all 8 diastereomeric bacteriochlorophylls exist in nature, Angew. Chem., 20, 956, 1981. 25. Bazzaz, M. B., New chlorophyll chromophores isolated from a chlorophyll-deficient mutant of maize, Photobiochemistry, 2, 199, 1981. 26. Smith, K. M., Bisset, G. M. F., and Bushell, M. J., Partial synthesis of optically pure methyl bacteriopheophorbides c and d from methyl pheophorbide a, J. Org. Chem., 45, 2218, 1980. 27. Brockmann, H., Jr., Bacteriochlorophyll e: structure and stereochemistry of a new type of chlorophyll from Chlorobiaceae, Philos. Trans. R. Soc. London B, 273, 277, 1976. 28. Caple, M. B., Chow, H. C., and Strouse, C. E., Photosynthetic pigments of green sulfur bacteria (the esterifying alkohols of bacteriochlorophylls c from Chlorobium limicola, J. Biol. Chem., 253, 6730, 1978. 29. Steiner, R., Schafer, W., Bios, I., Wieschhoff, H., and Scheer, H., A2,10-Phytadienol as esterifying alcohol of bacteriochlorophyll b from Ectothiorhodospira halochloris, Z. Naturforsch., 36C, 417, 1981. 30. Katz, J. J., Strain, H. H., Harkness, A. L., Studier, M. H., Svec, W. A., Janson, T. R., and Cope, B. T., Esterifying alcohols in the chlorophylls of purple photosynthetic bacteria. A new chlorophyll, bacteriochlorophyll (gg), all-trans geranylgeranyl bacteriochlorophyllide a, J. Am. Soc., 94, 7938, 1972. 31a. Holt, A. S., Recently characterized chlorophylls, in The Chlorophylls, Vernon, L. P. and Seely, G. R., Eds., Academic Press, New York, 1966, 111. 31b. Gloe, A., Pfennig, N., Brockmann, H., Jr., and Trowitzsch, W., A new bacteriochlorophyll from brown-colored Chlorobiaceae, Arch. Mikrobiol., 102, 103, 1975. 32. Walter, E., Schreiber, J., Zass, E., and Eschenmoser, A., Bchl a ^ und Bphe ap in den photosynthetischen Reaktionszentren von R. rubrum G 9, Helv. Chim. Acta, 62, 899, 1979. 33a. Egle, K., Biologischer Chlorophyllabbau, in Handbuch der Pflamenphysiologie, Vol. 5, Part 1, Ruhland, W., Ed., Springer-Verlag, Berlin, 1960, 354. 33b. Yentsch, CH. S., The relationship between chlorophyll and photosynthetic carbon production with reference to the measurements of decomposition products of chloroplastic pigments, Mem. 1st Ital. Idrobiol., 18 (Suppl.), 322, 1965. 34. Morris, M. M., Park, K., and Mackinney, G., On the photodecomposition of chlorophyll in vitro, J. Agric. Food Chem., 21, 277, 1973. 35a. Katz, J. J. and Janson, T. R., Chlorophyll-chlorophyll interactions from 'H and l3C nuclear magnetic resonance spectroscopy, Ann. N.Y. Acad. Sci., 206, 579, 1973. 35b. Steiner, R., Wieschhoff, H., and Scheer, H., HPLC of bacteriochlorophyll b and its derivatives as an aid for structure analysis, J. Chromatogr., 242, 127, 1982. 35c. Gottstein, J. and Scheer, H., unpublished. 36a. Gottstein, J. and Scheer, H., Long-wavelength absorbing forms of bacteriochlorophyll a in solutions of Triton-X 100, Proc. Natl. Acad. Sci. U.S.A., 80, 2231, 1981. 36b. Scherz, A. and Parson, W. W., Oligomers of bacteriochlorophyll and bacteriopheophytin with spectro­ scopic properties resembling those found in photosynthetic bacteria, Biochim. Biophys. Acta, 766, 653, 1984; Exciton interactions in dimers of bacteriochlorophyll and related molecules, Biochim. Biophys. Acta, 766, 666, 1984. 36c. Scheer, H., Paulke, B., and Gottstein, J., Long-wavelength absorbing forms of bacteriochlorophylls, in Optical Properties and Structure ofTetrapyrroles, Blaur, G. and Sund, H., Eds., de Gruyter, Berlin, 1985, 507. 36d. Scherz, A., Rosenbach, V., and Malkin, S., Biochim. Biophys. Acta, in press. 37. Brockmann, H., Jr., and Kleber, I., Bacteriochlorophyll b, Tetrahedron Lett., p. 2195, 1970. 38. Davis, M. S., Forman, A., Hanson, L. K., Thornber, J. P., and Fajer, J., Anion and cation radicals of bacteriochlorophyll and bacteriopheophytin b. Their role in the primary charge separation of Rhodopseudomonas viridis, J. Phys. Chem., 83, 3325, 1979. 39. Dougherty, R. C., Strain, H. H., Svec, W. A., Uphans, R. A., and Katz, J. J., The structures, properties and distribution of Chlorophyll c, J. Am. Chem. Soc., 92, 2826, 1970. 40. Budzikiewicz, H. and Taraz, K., Chlorophyll c, Tetrahedron, 27, 1447, 1971. 41. Smith, K. M., General features of the structure and chemistry of porphyrin compounds, in Porphyrins and Metalloporphyrins, Smith, K. M., Ed., Elsevier, New York, 1975, chap. 1. 42. Goedheer, J. C., Visible absorption and fluorescence of chlorophyll and its aggregates in solution, in The Chlorophylls, Vernon, L. P. and Seely, G. R., Eds., Academic Press, New York. 43. Wolf, H. and Scheer, H., Stereochemistry and chiroptic properties of pheophorbides and related com­ pounds, Ann. N.Y. Acad. Sci., 206, 549, 1973.

256

CRC Handbook o f Chromatography : Plant Pigments

44a. Scheer, H. and Katz, J. J., Nuclear magnetic resonance spectroscopy of porphyrins and metalloporphyrins, in Porphyrins and Metalloporphyrins, 2nd ed., Smith, K. M., Ed., Elsevier, New York, 1975. 44b. Janson, T. R. and Katz, J. J., NMR spectra of diamagnetic porphyrins, in The Porphyrins, Vol. 4, Dolphin, D., Ed., Academic Press, New York, 1978, chap. 1. 45. Scheer, H., Darstellung und absolute Konfiguration von 7,8-cis Phäophorbiden und 9-Hydroxy-phäphorbiden, Ph.D. thesis, Technical Braunschweig, University, West Germany, 1971. 46. Norris, J. R., Scheer, H., and Katz, J. J., ENDOR spectroscopy of chlorophylls and the photosynthetic light conversion apparatus, in The Porphyrins, Vol. 4, Dolphin, D., Ed., Academic Press, New York, 1978, chap. 3. 47. Boxer, S. G., CIoss, G. L., and Katz, J. J., The effect of magnesium coordination on the nC and l5N magnetic resonance spectra of chlorophyll a. The relative energies of nitrogen Ntt* states as deduced from a complete assignment of chemical shifts, J. Am. Chem. Soc., 96, 7058, 1974. 48. Budzikiewicz, H., Mass spectra of porphyrins and related compounds, in The Porphyrins, Vol. 3, Dolphin, D., Ed., Academic Press, New York, 1978, chap. 9. 49a. Constantin, E., Nakatani, Y., Teller, G., Hueber, R., and Ourisson, G., Electron-impact and chemical ionization mass-spectrometry of chlorophylls, phaeophytins and phaeophorbides by fast desorption on a gold support, Bull. Soc. Chim. Fr., p. 303, 1981. 49b. Grotemeyer, J., Bosel, U., Walter, K., and Schlag, E. W., Multiphoton-ionization mass spectroscopy of native chlorophylls, J. Am. Chem. Soc., 108, 4233, 1986. 49c. Dougherty, R. C., Dreifuss, P. A., Sphon, J., and Katz, J. J., Hydration behavior of chlorophyll a: a field desorption mass spectral study, J. Am. Chem. Soc., 102, 416, 1980. 49d. Tabet, J. C., Jablonski, M., Cotter, R. J., and Hunt, J. E., Time resolved laser desorption. III. The metastable decomposition of chlorophyll a and some derivatives, Int. J. Mass Spectrom. Ion Phys., 65, 105, 1985. 50. Hunt, J, E., MacFarlane, R. D., Katz, J. J., and Dougherty, R. C., High-energy fragmentation of chlorophyll a and its fully deuterated analogue by 2S2Cf plasma desorption mass spectrometry, J. Am. Chem. Soc., 103, 6775, 1981. 51a. Scholz, B. and Ballschmitter, K., Preparation and reversed-phase high-performance liquid-chromatography of chlorophylls (technical note), J. Chromatogr., 208, 148, 1981. 51b. Iriyama, K., Shiraki, M., and Yoshiura, M., An improved method for extraction, partial purification, separation and isolation of chlorophyll from spinach, J. Liq. Chromatogr., 2, 255, 1979. 51c. Gleixner, G., Karg, V., and Kis, P., Rapid preparation of pure chlorophyll a, Experientia, 38, 303, 1982. 52. Whitlock, H. W., Jr., Hanauer, R., Oester, M. Y., and Bower, B. K., Diimide reduction of porphyrins, J. Am. Chem. Soc., 91, 7485, 1969. 53. Risch, N., Reich, H., Schormann, A., and Brockmann, H., Jr., Note on a simple method for the separation of chlorophyll derivatives of the A-series and B-series, Justus Liebigs Ann. Chem., p. 1519, 1981. 54. Scheer, H., Katz, J. J., and Norris, J. R., Proton-electron hyperfine coupling constants of the chlorophyll a cation radical by ENDOR spectroscopy, J. Am. Chem. Soc., 99, 1372, 1977. 55. Baum, S. J., Burnham, B. F., and Plane, R. A., Studies on the biosynthesis of chlorophyll: chemical incorporation of magnesium into porphyrins, Proc. Natl. Acad. Sei. U.S.A., 52, 1439, 1964. 56. Isenring, H. P., Zass, E., Smith, K., Falk, H., Le Luisier, J., and Eschenmoser, A., Enolisierte Derivate der Chlorophyllreihe: 132-Desmethoxycarbonyl-173-desoxy-cyclochlorophyllid a-enol und eine Methode zur Einführung von Mg unter milden Bedingungen, Helv. Chim. Acta, 58, 2357, 1975. 57. Wasielewski, M. R., A mild method for the introduction of magnesium into bacteriopheophytin a, Tet­ rahedron Lett., p. 1373, 1977. 58. Bucks, R. R. and Boxer, S. G., Synthesis and spectroscopic properties of a novel cofacial chlorophyllbased dimer, J. Am. Chem. Soc., 104, 340, 1982. 59. Hynninen, P. H., Application of elution analysis to the study of chlorophyll transformations by column chromatography on sucrose, J. Chromatogr., 175, 75, 1979. 60. Jeffrey, S. W ., Properties of two spectrally different components in chlorophyll c preparations, Biochim. Biophys. Acta, 177, 456, 1969. 61. Iriyama, K. and Yoshiura, M., Separation of chlorophyll a and chlorophyll b by column chromatography with sephadex LH-20 or powdered sugar, J. Chromatogr., 177, 154, 1979. 62. Sato, N. and Murata, N., Preparations of chlorophyll a, chlorophyll b and bacteriochlorophyll a by means of column chromatography with diethylaminoethylcellulose, Biochim. Biophys. Acta, 501, 103, 1981. 63. Braumann, I. and Grimme, L. H., Reversed-phase high-performance liquid-chromatography of chloro­ phylls and carotenoids, Biochim. Biophys. Acta, 637, 8, 1981. 64. Risch, N., Kemmer, T., and Brockmann, H., Jr., Chromatographische Trennung von Behl e, Justus Liebigs Ann. Chem., 1978, 585, 1978.

Volume I: Fat-Soluble Pigments

257

65. Brereton, R. G., Rajananda, V., Blake, T. J., Sanders, J. K., and Williams, D. H., “ In beam” electron impact mass spectrometry: the structure of a bacteriochlorophyll allomer, Tetrahedron Lett., p. 1671, 1980. 66. Ellsworth, R. K., Tsuk, R. M., and St. Pierre, L. A., Attribution of hydrolytic and esterifying “ chlorophylase” activities observed in vitro to two enzymes, Photosynthetica, 10, 312, 1970 Aiga, I. and Sasa, T., Formation of atypical chlorophyllide a, Plant Cell. Physiol., 11, 161, 1970. 67. Hynninen, P., Isolation of chlorophylls a and b using an improved two-phase extraction method followed by precipitation and a separation on a sucrose column, Acta Chem. Scand. B, 31, 829, 1977. 68. Goyens, L., Post, E., Dehairs, F., Vandenhout, A., and Bayens, W., The use of HPLC with fluorimetric detection for chlorophyll a determination in natural extracts of chloropigments and their degradation products, Int. J. Environ. Anal. Chem., 12, 51, 1982. 69. Brown, L. M., Hargrave, B. T., and MacKinnon, M. D., Analysis of chlorophyll a in sediments by HPLC, Can. J. Fish. Aquat. Sei., 38, 205, 1981. 70. Bessiere, J. and Montel, A., Methode rapide de dosage selectif des chlorophylls a et b: utilisation de la separation par HPLC, Water Res., 16, 987, 1982. 71. Scheer, H. and Rauscher, G., Empfindliche und flexible Kopplung von HPLC und AS, Labor Praxis, 4— 7, 24, 1980. 72. Brereton, and Sanders, private communication. 73. Watanabe, T., Nakazato, M., Mazaki, H., Hongu, A., Konno, M., Saitoh, S., and Honda, K., Biochim. Biophys. Acta, 807, 110, 1985.

Tables for the Estimation and Separation of Chlorophylls

Structure

Chlorophyll b C5 5 H7 0 N4 0 6Mg = ^06

MW = 892

Chlorophyll a R, = H, R2 = C2 H5, R3 = COOCH3 , R4 = H C5 5 H7 2 N4 0 5Mg

Pigment

A2

A2‘

R‘

Green plants Algaee Prochloro

All oxygenic photosynthetic organisms

Occurrence

A

A + RCd

Function1*

644, 430

662, 430'

Chlorophyll

655, 525, 412

667, 535, 505,' 408

Pheophytin

Table III.l NAME LIST: STRUCTURES, FUNCTIONS, OCCURRENCE, AND SPECTRA OF CHLOROPHYLLS

GENERAL TABLES

Volume I: Fat-Soluble Pigments 261

Structure

H

A2

Chlorophylls c,, c2 C3 5 H3 0 N4 O5Mg MW = 610 (Chi c2 has 2H less)

Chlorophyll d C 54 HTON4 0 6Mg

MW = 894

Ra

Pigment

Rhodophyta Chlorella (?)

Pheophyta Cryptophyta Pyrrophyta Bacillariophyta Chrysophyta Prasynophyta

Occurrence

A1

Af

Function0

688,447

626, 576, 444g (627, 578, 448)

Chlorophyll

692,547,516, 421

650, 592, 579, 532, 433h

Pheophytin

Table III.l (continued) NAME LIST: STRUCTURES, FUNCTIONS, OCCURRENCE, AND SPECTRA OF CHLOROPHYLLS

262 CRC Handbook o f Chromatography: Plant Pigments

Few species of photosynthetic bacteria"1 Heliobacterium Chl°mm

A2; A2,10 A2,6,10,14

Bacteriochlorophyll b (R, = COCH3) QsH 72N40 6Mg MW = 908 (R = A2) Bacteriochlorophyll g 0r

Rs

Ethyl or Methyl Phtyl, famesyl, cetyl, others

CHO

R4

R,

CH

i ___________ l_ ____________I_________

CH Ethyl, A 7 -propyl, /-butyl or neopentyl

R,

e

R,

d

c

Bchl

m Rhodopseudomonas viridis, Rp. sulfoviridis, Thiocapsa pfennigii, Ectothiorhodospira halochloris, Et. abdelmalekii contain bacteriochlorophyll b, and Helio­ bacterium chlorum, bacteriochlorophyll g.

Volume I: Fat-Soluble Pigments 265

Chlorophyll^

Chlorophyll c,

Methylpheophorbide/? Chlorophylle

Pheophytin b

430 (56.8)

430 (117.3) 429 (111.6) 430 433 (90.5) 432 471 (4.44) ------472 (5.48) -------

X max(nm) (e)

455 (158.4) 453 (158.8) Acetone 455 Acetone, 80% a q u e o u s ------460 (184.1) Ethanol, 96% 464 Diethylether 412 (73.4) 434 (190.9) Dioxane ------------Acetone, 80% a q u e o u s -----436 (160.0) Dioxane ------------Diethylether 447 ( 1 3 8 . 9 ) ------Diethylether 447 (159.9) Acetone 442 (70.7) Acetone 446 (212.3) Pyridine 461(211.1) Acetone 445 (195.8) Pyride 466 (280)

Ether

Pheophytina

Methylpheophorbide a 8 -Deethyl 8 -vinylchlorophyll a Chlorophyll b

Acetone Acetone, 80% a q u e o u s ------Ethanol, 96% Diethyl ether 408 (114.8) Dioxane ------Acetone, 80% aqueous 409 (113.8) Dioxane -------

410 (76.0)

X max(nm) (c)

Diethyl ether

Solvent

Chlorophyll a

Compound1* 578 (8.27)

X max(nm) (t)

-

599 (8.40) ------600 (9.46) 600 (7.8) 628 ( 1 3

-

600 (13.0)

-

-

-

Ref.

662 (90.0) 1 661(86.2) 15 663 (82.6)16 665 (81.0) 3 665 (74.4)17 667 (55.4) 2 667 (43.0) 5 667 (49.2) 3 6 6 6 (52.8) 6

X max(nm) (t)

-

644 (56.2) 2 642 (56.0) 15 645 (46.9)16 649 (47.6) 3 649 (40.0) 1 655 (37.2) 2 ------655 (81.6) 3 652 (30.6) 7 . 5 ) ------2 627 (18.1) 18 628 (9.6) 19 629 (23.9) 20 640 (21.4) 20 630 (22.7) 20 642 (19.4) 20

609.5 (8.53) 609 (6.98) 610 (10.4) 610 (7.77)

-

618 (17.5)

-

615 (14.5)

X max(nm) (e)

595 ( 1 1 . 5 ) -------

( 1 3 3 . 1 ) -----------536 (5.77) 558 (7.17) ( 1 0 7 . 2 ) -----------525 (12.6) 555 (7.69) ------------527 (13.2) 558 (8.38) 525 (11.8) 552 (7.72) ------579 (12.6)

549 (6.40)

( 9 4 . 6 ) -----------536 (4.26) 582 (10.3) ( 7 4 . 2 ) -----------505 (12.7) 534 (11.0) 506 (10.1) 535 (8.65) 505 (13.1) 536 (11.4) 506 (11.4) 535 (9.27)

533 (3.76)

X max(nm) (e)

(e) = Molar Extinction Coefficient x 10 3

Table III.2 QUANTITATIVE SPECTROSCOPIC DATA: MOLAR EXTINCTION COEFFICIENTS ( c m '1 m o l 1) OF CHLOROPHYLLS. COMPLEMENTARY SPECTROSCOPIC DATA»

266 CRC Handbook of Chromatography: Plant Pigments

Acetone6 Diethyl ether"

Acetone6 Diethyl ether

Acetone6 Diethyl etherf

Acetone6 Diethyl etherf

Bacteriochlorophyll c

Bacteriopheophytin c

Bacteriochlorophyll d

Bacteriopheophytin d

bteA Q

Bacteriochlorophyll bA210 Bacteriopheophytin bp or

ap floe ap fl'cc Bacteriochlorophyll bp

Bacteriopheophytin a

Diethyl ether Diethyl ether

Bacteriochlorophyll ap

Acetone Dioxane Carbon tetrachloride Acetone/methanol = 7:2 Methanol Diethyl ether Chloroform Diethyl ether Diethyl ether Diethyl ether Diethyl ether Diethyl ether" Dioxane“ Acetone Diethyl ether" Diethyl etheri

Diethyl etheri

Chlorophyll d

407 (82) 408 (78) 398 (237)

368 (94) 368 (81) 368 (226)

390 (Sh)

330 (45) —

379 (Sh) —

356 (56)

384 (62.7) 390 (59.4) 385 (61.0) 386 (58.9) 384 (61.0) 385 (58.9)

406 (100) 403 (84.7)

408 (87) 405 (68.7)

408 (100) 406 (86.2)

384 (65) 419 (55.4)

391(48.0)

447 (87.4)

392 (52.7) 445 (87.4) 358 (73.3) — 358 (85.3) 358 (40.2) — — — 365 (53.9) 357 (113.6) 363 (99.4) 357 (105.9) 358 (109.6) 357 (113.0) 358 (109.6)

424 (100) 448 (117) 425 (113.6) 505 (14) 501(8.8)

431(100) 429 (112) 431(113.2) 515 (17) 512 (8.5)

525 (28.3) 533 (26.2) 525 (28.2) 526 (27.5) 525 (28.3) 526 (27.5)

530 (2.73)

548 (3.6)

533 (14) 531(9.4)

— 602 (10)

547 (22) 544 (13)

574 (11) —

580 (27) 578 (25) 528 (50)

625 (3.64) 630 (3.91) 623 (3.10) 622 (3.24) 622 (3.0) 624 (3.09)

577 (20.8)

595 (8.47)

548 (16) 602 (65)

608 (17) 618 (11)

604 (15) 607 (5)

624 (14) 624 (13)

676 (18)d 678 (25)u

608 (15.4) 680 (10.7) 687 (11.5) 678 (9.51) 680 (8.32) 678 (9.51) 680 (9.55)

697 (9.11)

643 (12.8)

654 (61) 651(88.3) 650 (88.3) 658 (55) 658 (44.1)

668 (64) 659 (73) 660 (75.6) 664 (65) 663 (46)

688 (98.7) 686 (105) 773 (91.0) 770 (96.0) 772 (95.6) 775 (20.1) 772 (115) 781 (88.0) 767 (76.0) 772 (42.0) 749 (67.5) 757 (63.4) 749 (71.4) 751(67.9) 749 (71.6) 751(67.6) 792 (100) 795 (100) 794 (100) 794 (100) 776 (100)

11 14 18 11 14

11 14 18 11 14

21 21 2 9 18 22 9 9 2 9 2 2 10 10 10 10 9 9 12 13 13

Volume I: Fat-Soluble Pigments

267

Acetonebg Acetonebg Diethyl ether Acetone

Solvent 337 (48.5) 378 (19.5)

A. max(nm) (e) 458 439 432 432

(100) (100) (187) (165.6)

A max(nm) (e)

534 (11)

A max(nm) (e) — 571(8)

A max(nm) (e) 592 (19) 598 (9)

A max(nm) (*) 647 654 623 623

(34) (24) (22.6) (21.4)

A max(nm) («)

11 11 2 2

Ref.

1. 2. 3. 4. 5. 6 . 7. 8 . 9. 10.

REFERENCES Anderson, A. F. H. and Calvin, M., Nature, 194, 285, 1962. Smith, J. H. C. and Benitez, A., Modern Methods of Plant Analysis, Vol. 4, Paech, K. and Tracey, M., Eds., Springer, Berlin, 1955, 142. Vernon, L. P., Anal. Chem., 32, 1144, 1960. Jeffrey, S. W., Nature, 194, 600, 1962. Stern, A. and Wenderlein, H.,Hoppe-Seyler’s Z. Physiol. Chem., 175,405, 1936. Stern, A. and Wenderlein, H.,Hoppe-Seyler’s Z. Physiol. Chem., 174, 81, 1935. Stern, A. and Wenderlein, H.,Hoppe-Seyler’s Z. Physiol. Chem., 174, 32, 1935. Weigl, J. W., J. Am. Chem. Soc., 75, 999, 1953. Clayton, R. K., Photochem. Photobiol., 5, 669, 1966. Walter, E., Schreiber, J., Zass, E., and Eschemoser, A., Helv. Chim. Acta, 62, 899, 1979.

All extinctions have been given in molar units. For the often-used weight units (cm ‘-g '•€), these values have to be multiplied by the molecular weight. The latter is given in Table III. 1. b The subscripts GG, P, and A 2,10 refer to the esterifying alcohols geranylgeranol, phytol, and 2,10-phytadienol, respectively. c Arbitrary units. d In part due to the absorption of oxidation/isomerization product(s). c Mixture of several homologues; e calculated from the data of Reference 14, assuming the substituents R, = R3 = R5 -CH3, R2 = C 2 H5, R4 = famesyl. f Mixture of several homologues; e calculated from the data of Reference 14, assuming the substituents R, = R3 = CH3, R2 = C 2 H5, R4 = famesyl, R5 = H (Table 1). g Mean of bchl e-fractions isolated from six different species; the peak position varies by ± 2 nm, the relative intensities up to 50%.

a

Bacteriochlorophyll e Bacteriopheophytin e Protochlorophyll

Compound1*

(e) = Molar Extinction Coefficient x 10“ 3

Table III.2 (continued) QUANTITATIVE SPECTROSCOPIC DATA: MOLAR EXTINCTION COEFFICIENTS (c m " 1 m o l 1) OF CHLOROPHYLLS. COMPLEMENTARY SPECTROSCOPIC DATA3

268 CRC Handbook of Chromatography: Plant Pigments

11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Gioe, A., Dissertation, Universität Göttingen, Göttingen, 1977. Baumgarten, D. (with Sauer, K.), M.S. thesis, University of California, Berkeley, 1970. Steiner, R., Zulassungsarbeit, University of Munich, Munich, 1981. Holt, A. S., Chemistry and Biochemistry of Plant Pigments, Goodwin, T. W., Ed., Academic Press, London, 1965, 3. Strain, H. H., Thomas, M. R., and Katz, J. J., Biochim. Biophys. Acta, 75, 306, 1963. Hoffmann, P. and Werner, D., Jena Rev., 11, 1114, 1960. Wintermans, J.F.M .C., Photosynthetica, 3, 11, 199. Strain, H. H. and Svec, W., The Chlorophylls, Vernon, L. P. and Seely, G. R., Eds., Academic Press, New York, 1966. Jeffrey, S. W., Biochim. Biophys. Acta, 177, 456, 1969. Jeffrey, S. W., Biochim. Biophys. Acta, 279, 15, 1972. Holt, A. S. and Morley, H. V., Can. J. Chem., 37, 507, 1959. Kim, W. S., Biochim. Biophys. Acta, 112, 392, 1966.

Volume l: Fat-Soluble Pigments 269

Pheophytin s

Chlorophylls

Chlorophyll s Pheophytin s Chlorophylls Pheophytin s Bacteriopheophytin c

Pyromethylpheophorbide s

Methylpheophorbide a

Methylpheophorbide a Pheoporphyrin s s

Chlorin e()-tme

Isochlorin e4-dme

Reference compound

CH:NH: CH2NH, CHO ( = Chi b) CHO ( = Phe b)

H COCH( H C:HS C:PÇ CH(OCOCH,)CH, CHOH-CH, CHOH-CH, CH(OCOCH JCH, COCH, CHOHCH, COCH, COCH, C:HS CHO( = Chic/) CHO( = Chic/) 3-5-Ethylene

Change

- 16 1 7 5 - 6 1 1 6 5 — 8 1 — -4 1 7 +13 -4

Soret

- 6 + 5 - 1 0 + 5 - 19 +25 —1 2 + 2 6

Changes at C-7

- 12 + 3 + - 1 4 - 1 6 - 8 + 1 + + 2 + - 6 - 4 + 10 - 1 1 + 1 1 -1 0 -1 0 + 2 0 + +23 -9

Changes at C-3

Red

d f a (twofold) a

a a d c c d d b a. d b a d b b

a

Remarks

Table 111.3 EFFECTS OF CHEMICAL MODIFICATIONS ON CHLOROPHYLL ABSORPTION

7 7 7 8

1 1 1 1 2 2 2 2 2 2 3 3 4 2 5 ,6 5 ,6

Ref.

270 CRC Handbook of Chromatography: Plant Pigments

132-Epimer 131-Silylated enol

13'-Silylated enol

Cyclochlorophyll enol Pyro (= 132-H2) 132-Methoxyl 132-Acetoxy j 132-Methoxy-pyro Peripheral Mg-complex

Peripheral Mg-complex

Peripheral Mg-complex

Peripheral Mg-complex

Chlorophylls

Pheophytin s

Chlorophylls Methylpheophorbide b

Methylpheophorbide/?

Bacteriomethylpheophorbide s

Bacteriopheophytin/?

Methylpheophorbide s

Alkyl

Bacteriochlorophyll a

+20 -40' +30 - 70'

+3

+30 0

+25

+8

a, h, j, m

a, h, m

a (threefold), h. j

c, g c, g a (threefold), h, j

0 0 +20 -50 +75

0 0 +15

15 15

d, h a(sevenfold), n, j, k

18

18

18

17 17 18

16 17

^

10 ^

^

3 3 3 ^ 10 6, 11 14

c

a

±0 +6 -6 6 + 12 -58

c

100% B in 20 min, then isocratic A: methanol; B: water; C: methanol-ethanol = 1:1, step gradient; A-B = 85:15 for 17.5 min, A-B = 95:5 for 9.5 min, 100% A for 6 min, then C isopropanol in hexane, step gradient: 1 % for 20 min, 2% for 30 min, 5% for 12 min, then 1 0 % step gradient: methanol-water = 98:2 for 77 min, then methanol-water = 1:1 step gradient: A: methanol; B: water; C: ether; A-B = 80:20 for 20 min, 90:10 for 45 min, 95:5 for 45 min, 97.5:2.5 for 65 min, 100% A for 40 min; A-C = 90:10 for 30 min, 50:50 for 25 min, then 25:75 iso-Octane-98% ethanol = 9:1. REFERENCES

1. 2. 3. 4. 5.

Braumann, T. and Grimme, L. H., Biochim. Biophys. Acta, 637, 8, 1981. Braumann, T. and Grimme, L. H., J. Chromatogr., 170, 264, 1979. Iriyama, K., Yoshiura, M., and Shiraki, M., J. Chromatogr., 154, 302, 1978. Eskins, K., ScholField, C. R., and Dutton, H. J., J. Chromatogr., 135, 217, 1977. Stransky, H., Z. Naturforsch., 33c, 836, 1978.

Pheophytin a(KPheophytin aDHGG Pheophytin aTHGG, Pheophytin aP Pheophytin a P Pheophytin b(KPheophytin bnHGG Pheophytin bTHGG Pheophytin bP 3-Acetyl-3-devinylpheophytin aGG Pyropheophytin aGG Pyropheophytin ap Methylpheophorbide a Pyromethylpheophorbide a Chlorophyll aP Chlorophyll aP Chlorophyll bP Chlorophyll bP Pyrochlorophyll aP Pheophytin %

Compound“1*

Packing Column length (mm diameter (mm) (OD) material Solvent Flow rate (m€/min) Temperature (°C) Detection Literature

7.4 9.4 11.5 13.6 15.2 — — — — — 7.7d 8.0C 1.3C 2.1c 7.2 — — — 8.3 4.5*

— — — — — — — — — —

250 4.6 SS S2 1.5 Ambient D1 2

P2

4.6 5.6 6.7 8.0 9.4 — — — — —

250 4.6 SS SI 1.5 Ambient Dl 1

PI

2

— — — — — — — — — —

— — — — — 5.0 6.1 8.2 10.2 3.9

250 4.6 SS S2 1.5 Ambient D2

P2

3

— — — — — — — — — —

— — — — — — — — — —

250 4.6 SS S3 1.5 Ambient D3

P2

3

250 4.6 SS S2 1.5 Ambient D4 5

P2

— — — — — — — — — —

— — — — — — — — — —

— — — — — — — — —

_ _

— — — — — — — — —

/R' (corrected)* (min)

250 4.6 SS S4 1.5 Ambient D4 4

P2

— — — — — — — — —

_ _

— — — — — — — — —

250 4.6 SS S2 1.5 Ambient D3 7

P2

Table III. HPLC 2 HPLC OF CHLOROPHYLLS AND DERIVATIVES

__ — — — 34 44 19 27 — —

— — — — — — — — — __

120 4.5 SS S5 3 Ambient D5 8

P3

__ — — — 22 — 10 — — —

— — — — — — — — — __

250 4.5 SS S2 4 Ambient D5 9

P4

— — — — — — — — — —

— 5.5 — — — 10 __

— —

1800 3 SS S6 0.7 Ambient D6

P5

294 CRC Handbook of Chromatography : Plant Pigments

— — — —

— —

—

—

—

—

— — — — — — — — —

— — — — — — — — —

—

—

—

—

— — — — — — — — —

14.7g

19.5g 11.5g

15.3e

10. lg

12.1 — — — 22.5 17.1 — — 13.3g

a The subscripts GG, DHGG, THGG-I, THGG-II, P, and F stand for the pigments esterified with geranyl-geraniol ( = A2,6,10,14-phytatetraenol), dihydrogeranyl geraniol (= A2,10,14-phytatrienol), tetrahydrogeranyl geraniol I ( = A2,14-phytadienol), tetrahydrogeranyl geraniol II ( = A2,10phytadienol), phytol (= A2-phytaenol), and famesol, respectively. See Formula in Table III. 1 for phytanol. b For the structure of these compounds see Formula . Packing PI = |iBondapak C18, 10 (xm (Waters, Kònigstein) P2 = Lichrosorb C8, 10 |xm (Knauer, Oberursel)h P3 = Lichrosorb RP-18, 5 p.m (Knauer, Oberursel)h P4 = Partisil PX S 1025 ODS 2 (Whatman, U.S.A.) P5 = Corasil II

Oxidation products of bacteriochlorophyll bP (Structures unknown) Oxidation products of bacteriochlorophyll bTHGG.„ (Structures unknown)

a THGG II

Bacteriochlorophyll aP Bacteriochlorophyll aCK] Bacteriopheophytin aP Bacteriopheophytin ac^ Bacteriochlorophyll bP Bacteriochlorophyll bTHGGU Bacteriopheophytin bP Bacteriopheophytin £THGGI1 3-Acetyl-3-devinyl-8hydroxyethylchlorophyll aP 3-Acetyl-3-devinyl-8-hydroxyethyl-8deethylchlorophyll

—

— —

—

—

— — 6.0 3.9 — — — — —

—

— —

—

—

3.0 1.5 — — — — — — —

—

— —

—

—

— — — — — — — — —

—

— —

—

—

— — — — — — — — —

—

— —

—

—

— — — — — — — — —

SI S2 S3 S4 S5 S6

= = = = = =

methanol-acetone (90:10 v/v) methanol-water (95:5 v/v) methanol-1% sodium ascorbate in water (89:11 v/v) methanol-1% sodium ascorbate in water (95:5v/v) acetonitrile-water (94:6) ethylacetate-petroleum ether (20:80)

tR' = retention time corrected for dead time, Shoulder of pheophytin . Reference 5. Reference 6. Best detection wavelength for degradation products of bchl b is 680 nm. Columns filled with material from Merck, Darmstadt, F.R.G.

Solvent

c d e f 8 h

—

—

—

—

— — — — — — 4.7 3.5 —

Volume I: Fat-Soluble Pigments

295

1. 2. 3. 4. 5. 6 . 7. 8 . 9.

D1 = absorption at 667 nm D2 = absorption at 655 nm D3 = absorption at 600 nm REFERENCES

D4 = absorption at 525 nm D5 = absorption at 436 nm D6 = absorption at 412 and 434 nm

Schoch, S., Lempert, U., Wieschhofî, H., and Scheer, H., J. Chromatogr., 157, 357, 1978. Benz, J. and Rüdiger, W., Z. Naturforsch., 36C, 51, 1981. Steiner, R., Zulassung, University of Munich, Munich, 1980. Scheer, H., unpublished. Schoch, S., Wieschhofî, H., and Scheer, H., unpublished. Benz, J., Dissertation, University of Munich, Munich, 1980. Scholz, B. and Ballschmiter, K., J. Chromatogr., 208, 148, 1981. Shoaf, W. T., J. Chromatogr., 152, 247, 1978. Evans, N., Games, D. E., Jackson, A. H., and Matlin, S. A., J. Chromatogr., 115, 325, 1975.

Detection

Table III. HPLC 2 (continued) HPLC OF CHLOROPHYLLS AND DERIVATIVES

296 C R C H a n d b o o k o f C h r o m a to g r a p h y : P la n t P ig m e n ts

22.5 8.7 24

2 2

7.6 19.5 — — — — — — — — — —

6 .6

16 16.5

Chlorophyll ap Chlorophyll a'p Chlorophyllide a Pheophorbide b Pyropheophytin b Chlorophyll^ Chlorophyll aXHGG Chlorophyll aDHGG Chlorophyll Chlorophyll aF Chlorophyll aG Ethyl chlorophyllide a Methyl chlorophyllide a 132-Hydroxychlorophyll ap Pyrochlorophyll ap Pheophytin ap Pheophytin a p Pheophorbide a Pyropheophytin ap 2.52 2.46

3 .7

9.3 8.0 5.3

1 1 .0

13.0 13.8 — — — —

— — —

5 3 .9

17.8

1 0 .6

250 4.6 SS st S2 1.5 40 D2

n.a. n.a. SS st SI 2.0 Ambient D1 1 2

b

PI

PI

Compound11

Packing Column length (mm) diameter (mm) material form Solvent Flow rate (m€/min) Temperature (°C) Detection Literature

— — — — — — — — — 14.7 — 3.0 —

1 .5

7.1 — 2.2 — —

— — — — — — — — — — — — — — __ — — — __

250 4.6 SS st S4 1.5 Ambient D3 4 4

P3

tR (min)

150 n.a. SS st S3 1.5 Ambient D2 3

P2

__

7 .0

__

3 3 .7

__ — __

_ _

30.8 — 3.0 — — — — — — —

250 4.6 SS st S5 1.5 Ambient D4 5

P3

7

— — — __ __ __ __ __ __ _ __ _ __

8 .6

— — — — __ __ __ — __ _ __ — __

18.5 — — — —

bc

250 4.6 SS st S7 2.0 Ambient D5 6

P3

18.2 — 5.5 — —

bc

250 4.6 SS st S6 2.0 Ambient D5 5

P3

_

b

__ 3 3 —3 5 ° __

— — 16.5-18C 28 — — — — __ __ __ __ __ __ __

n.a. n.a. n.a. n.a. S8 4.0 29 D6

P4

Table III. HPLC 3 HPLC OF CHLOROPHYLLOUS PIGMENTS INCLUDING FREE ACIDS

_

6

b — — — 8.5,13 __ __ — __ __ __ __ __ __ __ __ _ __ 13— 15° __

n.a. n.a. n.a. n.a. S9 4.0 25 D6

P4

__ — __

__ __ __ — __ __ __ __ __ __ __ __

8

29.4 —

bcd

300 4 SS st S10 1.0 Ambient D7 7

P4

Volume I: Fat-Soluble Pigments 297

Chlorophyll bp Chlorophyll b'p Chlorophyllide b Pheophytin bp Pheophytin b'p Protochlorophyllpgk ProtochlorophylITHGGg ProtochlorophyllDPHGC;g Protochlorophyllcc8 Protochlorophyll(..I8gh ProtochlorophyllFg Protochlorophyllc, / Protochlorophyll x Protochlorphyllide Protopheophytinp Bacteriochlorophyll ap Bacteriochlorophyll a p Bacteriochlorophyll öTHGCli Bacteriochlorophyll ö1)Hgg' Bacteriochlorophyll aGG Bacteriomethyl chlorophyllide a Bacteriochlorophyllide a Bacteriopheophytin a Bacteriomethylpheophorbide a Bacteriopheophorbide a Bacteriopyropheophytin a Bacteriopyromethalpheophorbide a Bacteriopyropheophorbide a 5.6 4.9 — — — — — — — — —

14 14.5 3.8 18 19 — — — — — — — — — — 5.8 2.5— 1 1 . 2 — 58.0 6.4 7.3 — — 4.4 — — 27.5 — — — — —

7 .0

— 34.8 — 19.7 16.6 14.1 12.1 10.0

8 .1

7.2 — — 10.8 — — — — — — — — — — — — — — — — — — — — — — — —

5 .6

— — — — — — — — — — — — — — 30.5 — — — — — — — 34.3 22.0' 3.5 35.4 25.6 12.2

27.2 — — — — — — — — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — — — — — — — — —

Table III. HPLC 3 (continued) HPLC OF CHLOROPHYLLOUS PIGMENTS INCLUDING FREE ACIDS — — 6 , 1 0 .5 C — — — — — — — — — — 17 — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — —

— — 26.3 — — 30.8 — — — — — — — 12.7 — — —

298 CRC Handbook of Chromatography: Plant Pigments

1

= gradient: MeOH-H20 = 85:15 (0—25 min), 95:5 (25— 60 min) = MeOH-H20 - 95:5 = gradient: A: methanol-water = 8:2, B: ethyl acetate, 0—50% B in 20 min = absorption at 654 nm = fluorescence with selected excitation and emission wavelengths = absorption at 525 nm (605 for bacteriochlorophyll) = absorption at 667 nm = absorption at 440 nm = absorption at 445 nm = absorption at 436 nm

4. Schoch, S., private communication. 5. Davies, D. and Holdsworth, E. S., J. Liq. Chromatogr., 3, 123, 1980. 6 . Burke, S. and Aronoff, S., Anal. Biochem., 114, 367, 1981. 7. Eskins, K. and Harris, L., Photochem. Photobiol., 33, 131,1981.

D3 D4 D5 D6 D7

Detection D1 D2

S9 S10

S8

' No separation of C, and C2. g No difference in retention times forprotochlorophyll and 8 -desethyl-8 vinylprotochlorophyll (= “ mono-” and “ divinyl-protochlorophyll” , respectively.) Differentiation is possible from fluorescence excitation spectra. h Alcohol chain length estimated from /R-diagram. 1 Alcohols not rigorously identified with respect to the position of double bonds; pigments isolated from Rhodopseudomonas palustris. J Accompanying peak at 23.6 (= a'?). k 10 Further protochlorophylls probably differingin their esterifying alcohols have been identified in Cucumis moschato seedcoats.

REFERENCES

= Sorbax ODS (C1H, DuPont) = C8 — Rp - Partisil 10 ODS (Waters) = P x S 1025 ODO-2 (Whatman) = A: methanol-water = 75:25; B: ethyl acetate; convex gradient (Waters No. 7) 0—50% B in 10 min, the n isocratic 52 = methanol 53 = aqueous methanol, step graident: 4 min 90%, 12 min 98% methanol 54 = A: methanol-water = 80:20; B: ethyl acetate, conves gradient (Waters No. 8 ) 0—50% B in 30 min 55 = same as S4, except A: methanol-water = 91:9 56 = A: methanol; B: water, 70—95% B 57 = same as S6 , but water containing 5 mM tetrabutyl am­ monium phosphate (Waters Pic A); this improves the resolution of accompanying carotenoids and retards free acids

PI P2 P3 P4 SI

. Schwartz, S. J., Woo,S. L.,and vonElbe, J. H., J. Agric. Food Chem., 29, 533, 1981. 2. Shioi, Y., Fukae, R., and Sasa, T., Biochim. Biophys. Acta, 722, 72, 1983; Shioi, Y. andSasa, I., Plant Cell Physiol., 23, 24, 835, 1983. 3. Falkowski, P. G. and Sucher, J., J. Chromatogr., 213, 349, 1981.

Solvent

Packing

a The index indicates the esterifying alcohol: P = A2-phytaenol (phytol); THGG = A2,14-phytatrienol (tetrahydrogeranyl-geraniol); DHGG = A2,10,14-phytatrienol (dihydrogeranyl-geraniol); GG = A2,6,10,14-phytatrienol (geranyl-geraniol); F = A2,6,10-famatrienol (famesol); G = A2,6-geraniadienol (geraniol). The prime pigments (e.g., a') are the 132epimers. b Retention times estimated from figures. c The system has been used for the simultaneous separation of carotenoids. d Some peaks show evidence of containing more than one compound. c More than one peak; smaller peak probably due to the “ prime” prigments.

V olu m e I: F a t-S o lu b le P ig m e n ts

299

300

C R C H a n d b o o k o f C h r o m a to g r a p h y : P la n t P ig m e n ts

Table III. HPLC 4 HPLC OF BACTERIOPHEOPHYTINS Packing Column length diameter material Solvent Flow rate Temperature Detection Literature Compound**

b 1

d

PI

PI

PI

P2

240 7 Glass SI FI T1 D1

240 7 Glass S2 F2 T1 D1

240 7 Glass S3 F3 T1 D1

240 9 Glass S4 F4 T1 D1

1

1

1

1

tR (min)

6*

Bacteriopheophytin Bacteriopheophytin Bacteriopheophytin Bacteriopheophytin

a ’p ap a \ ]Cl aCK-

a

18.2 15.8 21.5 18.4

2 1 .8

20.3 26.2 24.2

18.8 16.0 24.0 19.7

17.9 17.0 — —

Formulas, see Table III. 1. Dimethyl ether Hexamethyl phosphor triamide. Methylcyclohexane.

Packing Solvent

Flow rate

Detection

PI P2 SI S2 S3 S4 FI F2 F3 F4

Partisil 5 (Whatman) Lichrosorb S; 60 5 p.m (Merck) pentane-ether-water saturated with ether = 16:15:5 pentane-benzene-DMEb = 40:40:3 pentane-MCc-DMEb-HMPAc = 100:20:4:0.3 pentane-MCd-acetonitrile = 50:10:3 1.5 m€/min 1.42 m6 /min 1.08 m^/min 2.67 m6 /min absorption 254, 260 nm REFERENCE

1. Walter, E., Schreiber, J., Zass, E., and Eschenmoser, A., Helv. Chim. Acta, 62, 899, 1979.

Volume I: Fat-Soluble Pigments

T able III. H PL C 5 H PLC O F C H L O R O PH Y LL S a, b, c, AN D SO M E U N K N O W N S Packing Column length diameter material Solvent Flow rate Temperature Detection Literature

PI

P2

P3

P4

65 0.5 PTFE SI FI Ambient D1 1

250 n.a. SS S2 F2 24°C D2 2

250 4.6 SS S3 F3 n.a. D3 3

250 4.6 SS S4 F3 n.a. D3 3

Compound

/R

tR

tR

fR(min)

Chlorophyll« Chlorophyllide a Chlorophyll b Chlorophyll c, + c2 Pheophytins a of unknown structure 1 2 3 4 Chlorophyll a (E 446, F 674)a Chlorophyll a (E 443, E 672)a Chlorophyll a (E 432, F 662)a Chlorophyll a (E 436, F 670)a

15.2 — 38.5 —

— — 48.6 —

18.3 5.2 — 8.3

21.4 — — 8.1

— — — — — — — —

45.8 — 46.2 — 46.6 — 47.2 — 47.7 — 51.2 — 61.8— 73.2 —

— — — — — — — —

a Mixture of several compounds of unknown structure; indicated are the maximum wavelengths for fluorescence excitation (E) and emission (F) of the major com­ ponent as identified by fluorescence analysis. Packing Solvent

PI P2 P3 SI 52

Flow rate Detection

53 54 FI F2 F3 D1 D2 D3

silica gel SS-05 (Japan Spectroscopic) Spherisorb (5 |xm) Partisil 10 ODS step gradient of isopropanolin hexane (0—20 min: 1%; 20—50 min: 2%; 50—60 min: 5%; 60—75 min: 10% step gradient: 0—39 min, benzene-hexane = 1:1; then acetone-benzene: 39—53 min, 6:94; 53—65 min, 8:92, then 16:84 gradient methanol-water = 70:30—95:5 S3 containing 5 mM tetrabutyl ammonium phosphate 16 |x€/min n.a. 2 m€/min absorption 380 nm fluorescence excitedat 425 nm absorption 440 or 650 nm REFERENCES

1. Iriyama, K., Shiraki, M., and Yoshikura, M., J. Liq. Chromatogr., 2, 255, 1979. 2. Rebeiz, C. A., Belanger, F. C., Freyssinet, G., and Saab, D. S., Biochim. Biophys. Acta, 590, 234, 1980. 3. Davies, D. and Holdsworth, E. S., J. Liq. Chromatogr., 3, 123, 1980.

301

302

CRC Handbook of Chromatography : Plant Pigments

Table III. HPLC 61 HPLC OF DERIVATIVES OF BACTERIOCHLOROPHYLLS c Packing Column length diameter material Solvent Flow rate Temperature Detection Literature

PI

PI

PI

300 7.8 SS S2 FI T1 D1

300 7.8 SS S3 F2 Tl D1

1

2

300 7.8 SS SI FI Tl D1 1, 3

Compound9

tR (min)

Bacteriomethylpheophorbide c Fraction 1(2'-R) Fraction 1(2‘-S) Fraction 2(2'-R) Fraction 2(2'-S) Fraction 3(2'-R) Fraction 3(2‘-S) Fraction 4(2‘-R) Fraction 4(2’-S) Bacteriomesomethylpheophorbide Fraction 1 Fraction 2 Fraction 3 Fraction 4 a

18 —

23 —

31 33 —

43 c

46 50 58 63 75 79 91 98 12.7 14.5 17.6 20.7

For formulas, see Table III. 1.

Packing Solvent

Flow rate Detection Temperature

PI SI S2 S3 FI F2 D1 Tl

= = = = = = = =

|x-Bondapak C-18 acetonitrile-water = 90:10 acetonitrile-water = 67:33 methanol-water = 85:15 2 mfVmin 3 m€/min absorption 405 nm ambient REFERENCES

1. Smith, K. M., Bushell, M. J., and Rimmer, J., J. Am. Chem. Soc., 102, 2437, 1980. 2. Smith, K M., Kehres, L. A., and Tabba, H. D., J. Am. Chem. Soc., 102, 7149, 1980. 3. Smith, K. M., Bisset, G. M. F., and Bushell, M. J., J. Org. Chem., 45, 2218, 1980.

V o lu m e I: F a t-S o lu b le P ig m e n ts

Table III. HPLC 7 HPLC OF PRIME-CHLOROPHYLLS1 Packing Column length diameter material Solvent Flow rate (m€/min) Temperature Detection Literature

PI

PI

P2

250 30 SS SI

150 4.6 SS S2

150 4.6 SS S3

10

1

1

Ambient D1

Ambient D2

7°C D2

1

1

2

tR (min)

Compound3 Chlorophyll a Chlorophyll a' Chlorophyll b Chlorophyll b' Pheophytin a Pheophytin a' Pheophytin b Pheophytin b' a b

21

16 37 26 ___ b ___ b ___ b ___ b

9.6 5.6 16.4 15.0 5.8 4.5 1 2 .6

10.7

6 .0

3.8 2 1 .1

n.a. 4.1 3.4 n.a. n.a.

Retention times measured from figures. Data not available, but separation achieved under similar conditions.

Packing

PI P2

= Nucleosil 50-5 Macherey & Nagel = Silica Senshupack 50-5

Solvent

51 52 53

= hexane/2-propanol = 97:3 = hexane/2-propanol = 98.6: 1.4 = hexane/2-propanol = 98.4: 1 . 6

Detection

D 1 = absorption D2 = absorption, 430 nm REFERENCES

1. Watanabe, T., Hongu, A., Honda, K., Nakazato, M., Konno, M., and Saitoh, S,,Anal. Chem., 56, 251, 1984. 2. Watanabe, T., Nakazato, M., Mazaki, H., Hongu, A., Konno, M., Saitoh, S., and Honda, K., Biochim. Biophys. Acta, 807, 110, 1985.

303

304

C R C H a n d b o o k o f C h r o m a to g r a p h y : P la n t P ig m e n ts

Table III. HPLC 8 HPLC OF CHLOROPHYLL DERIVATES Packing Column length diameter material Solvent Flow rate Temperature Detection Literature

PI

PI

250 4.6 SS SI 1.5 40°C D1

250 4.6 SS SI 1.5 40°C D1

1

2

Compounda,b

tR (min)

Methylchlorophyllide a Ethylchlorophyllide a Chlorophyll ac Chlorophyll aF Chlorophyll aGG Chlorophyll aDHGG Chlorophyll aTHGG Chlorophyll aP Chlorophyll a 'P Pheophytin aP 10-Hydroxychlorophyll aP Pyrochlorophyll aP Chlorophyll bP Chlorophyll b 'P Pheophytin bP ProtochlorophyllGG Protochlorophyll DHGG Protochlorophyll THGG ProtochlorophyllP ProtopheophytinP Bacteriochlorophyll a ^ Bacteriochlorophyll aDHGG Bacteriochlorophyll aTHGG Bacteriochlorophyll aP Bacteriochlorophyll a 'P Bacteriopheophytin aP Chlorophyll b ^ Chlorophyll ¿?DHGG Chlorophyll bTHGG Chlorophyll bp a

b

2.46 2.52 3.74 5.27 8.04 9.29 11.03 13.03 15.80 53.87 10.62 17.85 7.25 8.14 34.85 1 2 .1 2

14.10 16.62 19.69 58.05 4.36 4.93 5.61 6.45 7.28 27.52 — — — —

—

— — — —

— — — — — — — — —

— — — — — — —

— — — — — 4.72 5.36 6.19 7.22

Subscripts denote the esterifying alcohol. P = phytol, GG = geranyl-geraniol, DHGG = dihydrogeranylgeraniol, THGG = tetrahydrogeranylgeraniol, F = famesol. Sensitivity enhancement can be achieved by proper excitation and emission wave­ lengths. See original reference for details.

Packing Solvent Detection

PI = Cl 8 -silica, Sorbax-ODS (DuPont) SI = Methanol D1 = Fluorimetry + Integrator REFERENCES

1. Shioi, Y., Fukae, R., and Sasa, T., Biochim. Biophys. Acta, 722, 72, 1983. 2. Shioi, Y. and Sasa, T., Biochim. Biophys. Acta, 756, 127, 1983.

V o lu m e I: F a t-S o lu b le P ig m e n ts

Table III. HPLC 9 HPLC OF BACTERIOMETHYLPHEOPHORBIDES d PI

Packing Column length diameter material Solvent Flow rate Temperature Detection Literature

250 7.8 SS SI 5m€/min Ambient D1 1

Compoundabc’ R R S S R R S S a

b c

Et Prn Bu1 NP Et Prn Bu1 NP

Retention volume (m€) 74 124 182 232 49 85 136 177

Et Et Et Et Me Me Me Me

The isomers are characterized (in this order) by the stereochemistry at C-3] (R or S), the substituent at C- 8 (Et = ethyl, Prn = n-propyl, Bu1 = wo-butyl, NP = neopentyl), and the substituent at C-12 (Me or Et). Elution volumes are given in m€. R and S isomers with identical substituents at C- 8 and C-12 can be separated.

Packing Solvent Detection

PI = fx-Bondapak C-18 (Waters) SI = methanol-water = 85:15 D1 = absorption REFERENCE

1. Smith, K. M. and Goff, J. J. Chem. Soc. Perkin I, p. 1099 1985.

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Table III. HPLC 10 HPLC OF NONESTERIFIED CHLOROPHYLL PIGMENTS Packing Column length diameter material Solvent Flow rate Temperature Detection Literature

PI a a a SI

P2 2 0 10

Amb. D1

Glass S2 n.a. Amb. Visual

1

2

1

P3

P4

P4

P5

150 4 SS S3

250 4.6 SS S4

250 4.6 SS S5

250 4.6 n.a. S6

1

0 .2

1

1

25°C D2 3

Compound Protochlorophyllide a 8 -Deethyl-8 -vinyl-protochlorophyllide a Protopheophorbide a Protochlorophyll ap Chlorophyllide a Chlorophyllide a' 8 -Deethyl-8 -vinyl-chlorophyllide a Pheophorbide a Pheophorbide a' Methylchlorophyllide a Methylpheophorbide a Chlorophyll a Chlorophyll a' Pheophytin a Pheophytin a' Chlorophyllide b Chlorophyllide b' Pheophorbide b Pheophorbide b' Methylchlorophyllide b Methylpheophorbide b Chlorophyll b Chlorophyll b' Pheophytin b Pheophytin b' Chlorophyll c, Chlorophyll c2 Methylchlorophyllide c, Methylchlorophyllide c2

40°C D3 4d

40°C D3 4

20°C D4 5e

(min) 12.3 — — — 7.9 — — — — — —

— — — — — — — — — — — — — — — — — —

— — — 33b — — 21 4b — —

— 3.8b

— — — 5.6 — — 1 1 .8

— 9.7 16.5 20.3

6.4 — 14.4 — 4.4 — — 13.5 16.6

— — — — — — — —

—

—

— —

—

—

—

18(80)

—

2 0 .8

—

3.2b — — — — — — — — — —

27.7 29.0

— — — — — — — — — — — — 4.7 7.3 — —

—

— — — —

2 .6

— 9.1 — 5.1 13.9 18.9 19.4 24.4 25.2 8 .6 8 .6 11 11

22(65) 34(65) — — 17(50) — 21(50) — — —

—

— — 7.9 9.1 30.6 35.8 — — —

—

41(80) — — — — — — — 14.4 (80) —

—

— — — — — —

30 (80) — 16.6 (67) 24.5 (67) — —

a Analytical column, no dimensions given. b Elution volumes. c AA = ammonium acetate. d This system also separates chlorophylls esterified with different alcohols. e This system also separates chlorophylls and bacteriochlorophylls esterified with different alcohols.

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307

Table III. H PLC 10 (continued) H PL C O F N O N E ST E R IF IE D C H L O R O P H Y L L P IG M E N T S Packing

Solvents

Detection

PI P2 P3 P4 P5 SI S2C

= = = = = = =

p.-Bondapak C-18 (Waters) DEAE-Sepharose CL-6B (Pharmacia) 5 p, Spherisorb ODS-2 (Phase Sep) Partisil-10 ODS-2 (Whatman) or Sorbax-ODS (DuPont) Polyethylene, RP-HPLC grade (Polysciences) A: methanol-water = 80:20; B: ethyl-acetate, gradient linear A to 50% B. A: acetone; B: acetone-methanol = 10:1; C: acetone-methanol-AA = 70:30:0.3; D: acetone-water-AA = 80:20:1, step gradient. S3C = A: methanol-AA (1 M) in water = 8:2; B: methanol-acetone = 8:2; linear gradient A to 100% B in 15 min, then isocratic B 54 = methanol-water = 95:5, containing 13 mA/ acetic acid (pH = 4.2) 55 = methanol-water = 85:5, containing 13 mA/ acetic acid (pH = 4.2) 56 = acetone-water mixtures. The acetone concentration (v/v) is given in brackets with the retention times. D1 = absorption 436 nm D2 = absorption 650 nm D3 = fluorimetry and densitometry D4 = fluorimetry at optimum wavelengths. REFERENCES

1. 2. 3. 4. 5.

Eskins, K. and Harris, L., Photochem. Photobiol., 33, 131, 1986. Araki, S., Oohusa, T., Owata, T. and Murata, N., Plant Cell Physiol., 25, 841, 1984. Zapata, M., Ayala, A. M., Franco, J. M., and Garrido, J. L., Chrornatographia, 23, 26, 1987. Shioi, Y., Doi, M. and Sasa, T., J. Chromatogr., 238, 141, 1984. Shioi, Y. and Beale, S. I., Anal. Biochem., 162, 493, 1987.

Index

Volume I: Fat-Soluble Pigments INDEX A Absorbents, chlorophylls, 283—287 Absorption carotenoids, 5, 93— 115 deconvolution, 7 lycopene absorption maxima, 11 chlorophylls, chemically-modified, 270—275 Acetone, 11 Acetyl derivatives, TLC, 217 3-Acetyl-3-devinyl-8-hydroxyethylchlorophyll, 295 3-Acetyl-3-devinyl-8-hydroxyethyl-8-de-ethylchlorophyll, 295 3-Acetyl-3-devinylpheophytin a, 294 Actinioerythrin, 13, 43, 94, 132 Actinioerythrin-frA-a-ketol, 13, 132 Actinioerythrol, 13, 43, 94 Adonirubin, 13, 57 Adsorbents, 162— 168, 283 Ag (II) complex, HPGC, 230 Alcohols, 11 Aleuriaxanthin, 13, 36, 120— 121, 132, 162 Algal chlorophylls, 277 Aliphatic alcohols, 11 10-Alkoxy-methyIpheophorbide a, 272 Alloxanthin(s), 42 LC, adsorbents and solvents, 165— 167 spectroscopy, 94 synonyms, 13, 29, 31, 33, 74, 83 TLC, 150, 154, 156, 159 Alloxanthin (trans) LC, 165, 166 spectroscopy, 94 TLC, 150, 159 Alumina, LC, 162— 165 A-Aminolevulate, 237, 238 Anchovyxanthin, 13, 90 Anhydrodeoxyflexixanthin, 13, 36, 37, 62, 94 Anhydrodeoxyflexixanthin (trans), 94 Anhydroeschscholtzxanthin, 14, 34, 53 LC, 166 spectroscopy, 94, 120— 121 TLC, 156 Anhydrorhodovibrin LC, 162 PC cellulose papers, 132 spectroscopy, 94 synonyms, 14, 64, 65, 74 Anhydrorhodovibrin (trans), 132 Anhydrosaproxanthin, 14, 26, 36, 55, 59, 94 Anhydrospirilloxanthin, 33 Antheraxanthin(s), 91 HPLC, 172, 176, 292 LC, 162, 164, 166 spectroscopy, 94, 120— 121 synonyms, 14, 50, 43, 48, 90 TLC, 142, 145, 147, 150, 156, 158, 159 Antheraxanthin (trans), 159 Aphanicin

LC, 162 spectroscopy, 94 synonyms, 14, 20, 24, 26, 44, 50 “Aphanicol”, 42 Aphanin, 14, 26, 46, 47, 60, 68, 162 Aphanizophyll PC cellulose papers, 132 spectroscopy, 94 synonyms, 15, 58, 79 TLC, 156 Aphanizophyll (trans), 94 Aphanol, 15, 54 p-Apo-4'-carotenal, 180 P-Apo-8'-carotenal, 21, 142, 159, 1 8 0 , 184 p-Apo-10'-carotenal, 180 Apocarotenal(s) GC, 180, 184 hydrogenated, 92, 180, 184 spectroscopy, 95, 120— 121 synonyms, 15— 17, 21, 35 TLC, 142, 147, 150, 153, 154, 159 Apo-8,8'-carotenedial, 28 p-Apo-p-carotenoic acid, 156 Apo-4'-carotenoic acid, 132 p-Apo-8'-carotenoic acid, 92, 180 p-Apo-8'-carotenoic acid esters, 92, 180 p-Apo-8'-carotenoic acid ethyl ester, 180 p-Apo-8'-carotenoic acid methyl ester, 180 Apocarotenoic acids, 92 GC, 180 hydrogenated, 92, 180 PC cellulose papers, 132 spectroscopy, 95, 120— 121 synonyms, 15— 16, 69, 78 TLC, 153, 154, 156 p-Apo-8'-carotenol, 27 Apocarotenols, 16, 27, 35, 63 Apolycopenals LC, 164 PC cellulose papers, 132 spectroscopy, 95, 120— 121 synonyms, 15— 17, 63 Apo-violaxanthals, 49 Apo-violaxanthin, 17 Apoviolaxanthinals, 17, 49 Apozeaxanthinals, 17, 27, 54 Artemia salina, 3 Astacene GC, 180 hydrogenated, 92, 180 PC cellulose papers, 132 spectroscopy, 95, 120— 121 synonyms, 17, 50, 80, 85 TLC, 142, 150, 159 Astaxanthin(s), 4 LC, 162 PC cellulose papers, 132 spectroscopy, 95 synonyms, 17, 41—43, 73

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TLC, 154 Astaxanthin diacetate, 17,95, 120— 121 Astaxanthin diesters, 17, 95 Astaxanthin monoesters, 17, 95 Asterinic acid, 42, 45 PC cellulose papers, 132 spectroscopy, 95 synonyms, 18, 35, 44, 83 Asteroidenone, 18, 50 Asym.8-carotene, 59, 84, 85 Aurochrome, 18, 23, 24, 38, 95, 120— 121 Auroxanthin(s) LC, 164, 166, 167 spectroscopy, 95—96, 120— 121 synonyms, 18, 38, 42 TLC, 145, 150, 156 Auroxanthin epimers, 91, 132, 172 Azafrin GC, 180 hydrogenated, 92, 180 spectroscopy, 96, 120— 121 synonyms, 18, 42 TLC, 142, 145, 147, 150

B Bacterial phytoene, 18, 35, see also Phytoenes Bacteriochlorins, 240 Bacteriochlorophyllide a, 298 13-OH-Bacteriochlorophyll a, 280 Bacteriochlorophylls, 267, 268 biosynthesis, 239 chemical modifications, effects on absorption, 271, 272, 274—275 HPLC, 295, 304 porphyrin biosynthesis, 190 spectroscopy, 240, 277, 279 structures, functions, occurrence, and spectra, 263 Bacteriochlorophylls a biosynthesis, 238 chemical modifications, effects on absorption, 271 HPLC, 298, 304 LC, total pigment extracts, 289 spectroscopy, 279 Bacteriochlorophylls /?, 279 Bacteriochlorophylls c, 271 Bacteriochlorophylls d, 273 Bacteriochlorophylls e, 271, 291 Bacterioerythrins, 18, 71, 79 Bacteriomesomethylpheophorbide c, 302 Bacteriomethyl chlorophyllide a, 298 Bacteriomethylpheophorbides a, 271, 298 Bacteriomethylpheophorbides c, 302 Bacteriomethylpheophorbides e, 291 Bacteriopheophorbides a, 279, 298 Bacteriopheophorbides b, 279 Bacteriopheophytins, 267, 268 biosynthesis, 239 chemical modifications, effects on absorption, 271, 275 HPLC, 295, 298, 300 Bacteriopheophytins a, 271, 298, 300

Bacteriopheophytins /?, 271 Bacteriopheophytins c, 270 Bacteriopheophytins d, 273 Bacterioprotochlorophyllide, 263 Bacteriopurpurin, 18,79 Bacteriopyromethylpheophorbide a, 298 Bacteriopyropheopheophorbide a, 298 Bacteriopyropheophytin a, 298 a-Bacterioruberin monomethyl ether, 19, 72 Bacterioruberins, 91 HPLC retention times, 172 LC, 162 PC cellulose papers, 132 spectroscopy, 96, 120— 121 synonyms, 19, 37 a-Bacterioruberins (trans), 132, 172 Bactobilin, 189 Benzene, 11 Bisdehydro-p-carotene LC, 166 spectroscopy, 96, 120— 121 synonyms, 19, 31, 78, 83 Bisdehydrolycopenes, 19, 32 Bis(4-hydroxy-3-methyl-2-butenyl)carotenes, 19, 81 2,2,-Bis(3-hydroxy-3-methylbutyl)-3,4,3',4'tetradehydro-1,2,1 ',2'-tetrahydro-i}/,\|icarotene-l,l'-diol, 19 2,2'-Bis(0-methyl-5-C-methylpentosyloxy)3,4,3',4'tetradehydro-1,2,1 ',2'-tetrahydro\j/,\|/-carotene-l,l'-diol, 19, 73 2,2'-Bis(p,L-rhamnopyranosyloxy)-3,4,3/,4'tetradehydro-1,2,1 '^'-tetrahydro-v^Xj/carotene-l,l'-diol, 20, 73 Bixins GC, 180 hydrogenated, 92, 180 spectroscopy, 120— 121 synonyms, 20 TLC, 142, 145, 147, 150, 153 Boron-trifluoride, 197 Brine shrimp, 3 /i-Butanol, 11 6-But-2-enylidene-1,5,5-trimethyl-cyclohex-1-ene, 91, 184

c C30-Phytoene, 27, 35 Calcium carbonate, 165 Calcium hydroxide, 165 Caloxanthin, 20, 36, 96, 156 Canthaxanthin(s), 3, 44 GC, 180, 184 HPLC separation, 176 hydrogenated, 92, 180, 184 LC, 162 PC cellulose papers, 132, 138 spectroscopy, 96, 120— 121 synonyms, 14, 20, 24, 26, 50, 91 TLC, 142, 145, 147, 150, 153, 154, 156, 159 Canthaxanthin (cis), 153 Capsanthin

Volume I: Fat-Soluble Pigments GC, 180 hydrogenated, 92, 180 spectroscopy, 96, 120— 121 synonyms, 20, 42 TLC, 142, 145, 147, 150, 156, 159 Capsanthin-diester, 20, 96 Capsanthin-5,6-epoxide, 20, 21, 49, 96, 97 Capsanthin-5,6-epoxide-diester, 21, 96, 97 Capsanthin monoepoxide, 20, 21 Capsanthin monoester, 21, 96 Capsochrome, 21,49, 96 Capsorubin spectroscopy, 96, 120— 121 synonyms, 21, 42 TLC, 142, 145, 147, 150, 156 Capsorubin diester, 21, 96 Carbon tetrachloride, 11 Carboxyl-3',4'-dehydro-Y-carotene, 86 16'-Carboxy l-3',4'-dehydro-y-carotene, 21 Carcinoxanthin, 21, 27 Caricaxanthin, 21, 30 p-Carotenal, 21 a-Carotene, 3, 91 HPLC separation, 176, 292 hydrogenated, 92 LC adsorbents and solvents, 162, 165— 167 PC cellulose papers, 138 spectroscopy, 96, 120— 121 synonyms, 18, 22 TLC, 142, 145, 147, 150, 154, 156, 158 a-Carotene, cis- isomer, 197, 292 a-Carotene-5,6-epoxide, 97 ds-a-Carotene, 292 (3-Carotene, 3, 91 GC, 180, 184 HPLC, 172, 176, 178, 292 hydrogenated, 92, 180, 184 LC, 162, 164— 167, 291 PC cellulose papers, 132, 138 spectroscopy, 97, 120— 121 synonyms, 21, 23, 69, 77 TLC, 142, 145, 147, 150, 153, 154, 156, 158, 159 p-Carotene, cis- isomers, 91, 176, 292 p,p-Carotene HPLC retention times, 172 spectroscopy, 97, 120— 121 synonyms, 21, 23, 69, 77, 91 TLC, 154 P,e-Carotene, 21, 22, 91, 97, 154, 172 p,