Ion-Exchange Chromatography and Related Techniques 9780443153693

Table of contents :
Cover
Half Title
Handbooks in Separation Science Series
Ion-Exchange Chromatography and Related Techniques
Copyright
Contents
Contributors
1. Concepts and milestones in the development of ion-exchange chromatography
1. Introduction
2. Fundamentals
2.1. Retention mechanism for small ions
2.2. Retention mechanisms for polyelectrolytes
3. Column chromatography
3.1. Porous polymer ion exchangers for the separation of low-mass ions
3.2. Restricted access media
3.3. Ion exchangers for large-scale separations
4. Large-scale ion-exchange separations
5. Landmark developments in biotechnology for downstream processing using ion-exchange chromatography
References
2. Equilibria and kinetics of ion-exchange of biopolymers
1. Introduction
2. Dynamic models
2.1. Classification of the models
2.2. Formulation of mass balance equations in the GR model
2.3. Transport-dispersive model
2.3.1. Model formulation
2.4. Compatibility of GR and TD models
2.5. Reaction-dispersive model
3. Kinetic equations of adsorption-desorption rate
3.1. Kinetics of SMA formalism
3.2. Kinetics of cooperative adsorption
3.3. Kinetics of protein unfolding upon adsorption
4. Adsorption-desorption equilibria: Isotherm equations
4.1. SMA formalism
4.2. Cooperative adsorption isotherm
4.3. CPA isotherm
4.4. Determination of isotherm coefficients
4.4.1. SMA model
4.4.2. Cooperative adsorption isotherm
4.4.3. CPA isotherm
5. Causes of misinterpretation of the elution data
5.1. Effect of feed viscosity on the process kinetics
5.2. Effect of competitive adsorption
5.3. Effect of column void volumes
6. Procedure for design of IEX process
References
3. Stationary phases for ion separations
1. Ion-exchange terminology
2. Classification of ion-exchangers
2.1. Matrix or type of substrate material
2.1.1. Inorganic materials
2.1.2. Synthetic organic polymers
2.1.3. Hybrid matrices
2.2. Structure of ion-exchangers
2.2.1. Column packing morphology
2.2.2. Localization of fixed charges in ion-exchangers
Ionogenic groups distributed in a whole volume of particle
Controlled porosity particles or superficially porous ion-exchangers
Electrostatically agglomerated ion-exchangers
Immobilized ionogenic polymer layers
Encapsulated ion-exchangers
Ion-exchangers coated with an oppositely charged polymer
Covalent bonding or grafting of a polymer layer to an activated substrate surface
Isolated ionogenic groups on substrate surfaces, or chemically modified substrates
2.3. Types of functional groups
2.3.1. Positively charged functional groups (anion-exchangers)
2.3.2. Negatively charged groups (cation-exchangers)
2.3.3. Zwitterionic and polyampholyte ion-exchangers
2.3.4. Complexing ion-exchangers
2.4. Ion-exchange capacity
References
4. Stationary phases for the separation of biopolymers by ion-exchange chromatography
1. Introduction
2. Uniform agarose-based ion-exchange chromatographic media
3. Gigaporous ion-exchange chromatographic media
3.1. Gigaporous PSt-based ion-exchange chromatographic media
3.2. Gigaporous PGMA-based ion-exchange chromatographic media
3.3. DEAE macroporous agarose chromatographic media
3.4. CM macroporous agarose chromatographic media
4. Other ion-exchange stationary phases for bioseparations
4.1. Monolithic columns
4.2. Membrane chromatography
4.3. Cryogels
4.4. Mixed-mode chromatography
5. Summary and outlook
References
5. Ion-exchange separations of biomacromolecules on grafted and surface-modified polymers
1. Introduction
2. Stationary phases
2.1. Design of polymer-functionalized ion exchangers
2.2. Introduction of the surface polyelectrolytes and their modification
2.3. Typical commercial stationary phases
3. Adsorption and uptake theory
3.1. Three-dimensional adsorption
3.2. Facilitated mass transfer by chain delivery effect
4. Applications
4.1. Features of practical applications
4.2. Application examples
References
6. Extraction chromatography of actinides
1. Introduction
2. Extractants for actinide separation
3. Ligand impregnated resins for actinides
3.1. Monoamide impregnated resins
3.2. Malonamide impregnated resins
3.3. Diglycolamide impregnated resins
3.4. Multiple DGA impregnated resins
4. Room temperature ionic liquids in extraction chromatography
4.1. TODGA/RTIL resin
4.2. C4DGA and T-DGA/RTIL resins
5. Ligand grafted resins for actinides
5.1. Monoamide grafted resins
5.2. Malonamide grafted resins
5.3. Diglycolamide grafted resins
6. Composite beads for extraction chromatography
7. Perspectives
Abbreviations
References
7. Ion-exchange membrane chromatography
1. Introduction
2. Transport phenomena in membrane chromatography
3. Module design
4. Promising ion-exchange membranes for bioseparations
5. Conclusions
References
8. Ion-exclusion chromatography
1. Principle
2. Apparatus
3. Ion-exchange resin columns used in ICE
4. Eluent conditions
5. Detection methods
5.1. Conductivity detection
5.1.1. Direct detection
5.1.2. Enhancement of conductivity by postcolumn reaction
5.2. UV-VIS detection
5.2.1. Direct UV detection
5.2.2. Postcolumn derivatization
5.3. Mass spectrometry
5.4. Charged aerosol detector
6. Separations of nonionized substances
7. Separation of ammonium and amines
8. Vacancy ion-exclusion chromatography
9. Ion-exclusion/cation-exchange chromatography
10. Ion-exclusion/anion-exchange chromatography
Abbreviations
References
9. Chelation ion chromatography
1. Introduction
2. Theoretical aspects of complexation in liquid chromatography
2.1. Complexation in the mobile phase
2.2. Complexation in the stationary phase
3. Ion-exchange chromatography with the complex formation in the mobile phase
3.1. Cation-exchange chromatography with complexing eluents
3.1.1. Fixed-site cation-exchangers and complexing eluents
3.1.2. Dynamically modified cation-exchangers and impregnated adsorbents
3.1.3. Ion-pair chromatography of complexed metal ions
3.2. Anion-exchange chromatography
3.2.1. Fixed-site anion-exchangers and complexing eluents
3.2.2. Dynamically modified anion-exchangers and ion-pair mode
4. Chelating phases for ion-exchange chromatography
5. Application areas of chelating ion-exchangers
Abbreviations
References
10. Displacement chromatography with ion-exchangers
1. Principles of displacement chromatography
1.1. Basic concepts of displacement chromatography
1.2. Variant forms of displacement chromatography
1.2.1. Selective displacement chromatography
1.2.2. Sample displacement chromatography
1.2.3. Complex displacement chromatography
1.3. Theoretical models for displacement chromatography
2. Ion-exchange displacers
2.1. Displacers for ion-exchange chromatography
2.2. Approaches for displacer screening and design
3. Applications of ion-exchange displacement chromatography
3.1. Displacer chromatography process development and optimization
3.2. Applications
3.2.1. Displacement chromatography for the purification of recombinant proteins
3.2.2. Displacement chromatography for proteomic analysis
3.2.3. Applications of sample displacement chromatography
4. Prospects and outlook
References
11. Instrumentation for ion chromatography
1. Solvent delivery systems for IC applications
1.1. High-pressure piston pump
1.2. Eluent production modules
2. Detectors for IC
2.1. Conductivity detection
2.1.1. Suppressors for suppressed conductometry
Column-type suppressors
The membrane type suppressors
2.1.2. Charge detector
2.1.3. Direct conductometry (nonsuppressed conductometry)
2.2. Electrochemical detection
2.3. Photometric detection
2.4. Postcolumn reaction system
2.5. Mass spectrometry detection
2.6. Multiple detections
3. Injection system
3.1. Injection valve with sample loop
3.2. Preconcentration
4. Column oven
5. Column hardware
References
12. Instrument platforms for large-scale ion-exchange separations of biomolecules
1. Introduction
2. Chromatography columns
3. Ion exchange matrices
3.1. Process steps in ion-exchange chromatography
4. Chromatography equipment
5. Scale-up of ion-exchange processes
5.1. Understanding the product and resin selection
5.1.1. Column design and size
5.1.2. Process parameters
5.1.3. Validation and cleaning
5.1.4. Equipment and facility consideration
5.1.5. Mode of operations of IEC
5.2. Necessary calculations for IEC scale-up
5.3. Common problems associated with IEC scale-up from lab to manufacturing scale
5.3.1. Pressure drop
5.3.2. Buffer preparation at a manufacturing scale
5.3.3. Column packing and cleaning
5.3.4. Validation of a scaled-up process
References
13. Method development for large molecules IEX separations
1. Introduction
2. Column, stationary phase, and instrumentation considerations
2.1. Stationary phase characteristics
2.2. Column dimensions
2.3. Column hardware and instrumentation
2.4. Detection
3. Elution modes
3.1. Salt gradient mode
3.1.1. General considerations
3.1.2. Practical considerations for salt-gradient separations
Mobile phase pH and buffer
Salt additive in the mobile phase
3.2. pH gradient mode
3.2.1. General considerations
3.2.2. Practical considerations for pH gradient separations
Mobile phase buffers
The effect of the column on pH of the effluent
Empirical correction of nonlinear pH gradients
3.3. Salt-mediated pH gradient mode
4. IEX mode for nucleic acid separations
4.1. General considerations
4.2. Practical considerations
4.3. Denaturing elution conditions for nucleic acid analysis
5. On-off mechanism of retention
5.1. Multisegmented gradients (multiisocratic elution) vs linear gradients
6. Need for IEX platform methods
7. Systematic method development
7.1. Screening experiments
7.2. Method optimization in general
8. Practical advice for systematic method optimization
8.1. Method optimization in salt gradient mode
8.2. Method optimization in pH gradient mode
8.3. Method optimization in salt mediated pH gradient mode
8.4. Method optimization in ion-pairing IEX
9. Perspectives
Declaration of competing interest
References
14. Sample preparation for ion-exchange separations
1. Introduction
2. Liquid-phase extraction
2.1. Solid samples
2.2. Gas phase samples
2.3. Liquid-liquid extraction
3. Solid-phase extraction
3.1. Inorganic ions (matrix removal and concentration)
3.2. Inorganic ions (speciation)
3.3. Inorganic ions (extraction chromatography)
3.4. Organic ions
3.5. Biomacromolecules
3.6. Microextraction formats
4. Membrane-based extraction
4.1. Electrodialysis
4.2. Electromembrane extraction
References
15. Separation of ions by ion chromatography
1. Anion-exchange chromatography
1.1. Eluents
1.2. Stationary phases
1.3. Separation of anions
2. Cation-exchange chromatography
2.1. Stationary phases
2.2. Separation of cations
References
16. Applications of ion chromatography in environmental analysis
1. Introduction
2. Ion chromatography and related techniques
2.1. Water samples
2.2. Atmospheric samples
2.3. Solid samples
3. IC development perspectives
References
17. Applications of ion chromatography in food analysis
1. Introduction
2. Inorganic anions
3. Inorganic cations
4. Carbohydrates
5. Organic acids
6. Food safety issues
References
18. Separation of saccharides by ion-exchange chromatography
1. Introduction
2. Separation of saccharides by HPAEC
2.1. Monosaccharide and disaccharides analysis
2.2. Oligosaccharides and polysaccharides separation
2.3. Glycoconjugate separation
3. Separation of saccharides by ligand exchange chromatography
3.1. Monosaccharide and disaccharides separations
3.2. Oligosaccharides separation
4. Ion exclusion chromatography
5. Ion-pair chromatography
5.1. Monosaccharides and disaccharides separations
References
19. Separation of oligonucleotides by ion-exchange chromatography
1. Introduction
2. Column packings for the ion-exchange separations
3. Nucleotides and sugar nucleotides
3.1. Nucleotide sugars
4. Oligonucleotides
5. Phosphorothioated oligonucleotides
6. Mixed mode stationary phases
7. Multidimensional chromatography
8. Purification of oligonucleotides
References
20. Separation of oligonucleotides by ion-exchange and ion-pair chromatography
1. Introduction
2. Types of oligonucleotides
3. Endogenous RNAs
3.1. Messenger RNA
3.2. Ribosomal RNA
3.3. Transfer RNA
3.4. Small nuclear RNA
3.5. Small nucleolar RNA
3.6. Micro RNA
3.7. Long noncoding RNAs
4. Therapeutic oligonucleotides
4.1. Small interfering RNA
4.2. Antisense oligonucleotides
4.3. Aptamers
4.4. mRNA therapeutics
4.5. Single guide RNA
5. Charged-based separations of oligonucleotides
5.1. Ion-exchange chromatography
5.2. Ion-pair chromatography
5.3. Mixed-mode chromatography
6. Nonspecific adsorption
7. Sample preparation for oligonucleotides
8. Liquid chromatography-mass spectrometry of oligonucleotides
8.1. Two-dimensional liquid chromatography separations
9. Selected applications of ion-exchange and ion-pair chromatography to the determination of oligonucleotides
9.1. IEC of siRNAs
9.2. IEC of an aptamer
9.3. IPC of an antisense therapeutic
9.4. Two-dimensional chromatographic approaches for impurity determinations
9.5. Two-dimensional chromatographic separation of an mRNA digest
10. Future directions
References
21. Separation of proteins by ion-exchange chromatography
1. Introduction
2. Adsorption: Principles and models
3. Evolution of resins
4. Investigational approaches
5. Common modes of operation
6. Perspectives
References
22. Separation of proteins by mixed-mode chromatography
1. Introduction
2. Mixed mode ligands
3. Effect of salt and pH on mixed-mode chromatography
4. The third dimension
5. High-throughput screening of mixed-mode resins
6. Monoclonal antibody purification
7. Conclusion
References
23. Process modeling of protein separations by ion-exchange chromatography
1. Introduction
2. Mechanistic modeling of chromatography
2.1. Mechanistic model equations and zone movement in the column
3. Plate height and related variables in isocratic elution
4. Resolution Rs in isocratic elution
5. Ion-exchange equilibria
6. Linear gradient elution
6.1. Retention in linear gradient elution
6.2. Peak width, HETP, and Rs in linear gradient elution
6.3. Iso-resolution curves in linear gradient elution
7. Applications of the mechanistic models and the simplified methods
7.1. Stepwise-elution process design based on linear gradient elution data
7.2. Flow-through chromatography
7.2.1. Process design
7.3. Model simulations for flow-through chromatography
7.4. Summary
8. Capture process design
8.1. Multicolumn periodic counter current operation
8.2. Flow velocity gradient loading operation
8.3. Model simulations
8.4. Summary
References
24. Applications of ion-exchange chromatography for the purification of antibodies
1. Antibodies: Introduction, recombinant expression, manufacturing, and purification
1.1. Monoclonal antibodies and variant forms
1.2. Antibody expression, manufacturing, and purification
1.2.1. Recombinant expression and manufacturing
1.2.2. Purification workflow
Antibody purification workflow
2. Cation-exchange chromatography of antibodies
2.1. Principles and goal
2.2. Starting conditions and parameters for optimization
2.2.1. Resin screening
2.2.2. Elution program development
2.2.3. Optimization of loading condition
2.2.4. Determination of operating space
2.3. Case studies
3. Anion-exchange chromatography of antibodies
3.1. Principles and goal
3.2. Starting conditions and parameters for optimization
3.3. Case studies
4. Commercial ion-exchange resins
4.1. Ion-exchange ligands, linking chemistries, and backbone materials
4.2. Commercial ion-exchange resins
5. Scale-up and scale-down of ion-exchange chromatography
6. Tips and tricks
Acknowledgment
References
25. Conformational changes of biomolecules in ion-exchange chromatography
1. Introduction
2. Mechanisms
2.1. Interconversion between different conformational states
2.2. Unfolding and aggregation
3. Detection of protein conformational changes
3.1. Spectroscopic methods
3.2. Differential scanning fluorimetry (DSF) and differential scanning calorimetry (DSC)
3.3. Hydrogen/deuterium exchange (HDX)
4. Key contributing factors
4.1. Protein intrinsic stability
4.2. Stationary phase properties
4.2.1. Ligand
Ligand type
Ligand density
4.2.2. Particle morphology
Pore size distribution (PSD)
Polymer-grafted resins
4.2.3. Surface property (hydrophobicity)
4.3. Practical implications
4.3.1. Mass load
4.3.2. Contact time (hold step)
4.3.3. Operating flow rates
4.3.4. Operating temperature
4.3.5. Mobile phase pH
4.3.6. Salt and excipients
References
26. Continuous ion-exchange chromatography for protein polishing and enrichment
1. Introduction
2. Limits of batch chromatography
3. Continuous chromatography for protein purification
3.1. Rotating chromatography
3.1.1. Column switching chromatography
3.1.2. Annular chromatography
3.2. Simulated moving bed
3.2.1. Classical simulated moving bed
3.2.2. Gradient simulated moving bed
3.2.3. Simulated moving bed for ternary separation
3.3. Multicolumn continuous chromatography
3.3.1. Multicolumn counter-current solvent gradient purification
3.3.2. Two-column batch-to-batch recirculation process
3.3.3. N-rich
3.3.4. Flow2
3.3.5. Multicolumn self-displacement chromatography
3.4. Comparison of different continuous modes
4. Process design for continuous purification
4.1. Experiment-based design
4.2. Model-based design
5. Applications for protein polishing using ion-exchange
5.1. Monoclonal antibodies
5.2. PEGylated proteins
6. Conclusion
References
27. Separation of bio-particles by ion-exchange chromatography
1. Introduction
2. The principles of separating bio-particles by IEC
3. Challenges and coping strategies for separating bio-particles by IEC
3.1. Limitations of low binding capacity and coping strategies
3.2. Time-consuming issue and coping strategies
3.3. Low recovery and coping strategies
3.3.1. Effects of media pore size on recovery
3.3.2. Effects of ligand density on recovery
3.3.3. Effects of mobile phase on recovery
4. Design of IEC-based bio-particle separations
4.1. Special considerations
4.1.1. Expression systems
4.1.2. The source of impurities
4.1.3. Flow-through mode
4.1.4. Separation stages
4.1.5. Salts in the mobile phase
4.2. Representative examples of bio-particle separations by IEC
4.2.1. Separation of HBsAg-VLPs
4.2.2. Separation of the influenza virus
4.2.3. Separation of AAV Serotype 9
4.2.4. Separation of EVs
5. Future prospects
Abbreviations
Acknowledgment
References
28. Virus removal in bioprocessing using charged media
1. Introduction
2. Virus physicochemical properties
3. Protein interference
4. Chromatography format
5. Cation exchange and multimodal ligands
6. Conclusions
References
29. Role of temperature in ion-exchange processes of separation and purification
1. Influence of temperature on the stability of ion-exchange resins
2. Influence of temperature on the kinetics and dynamics of ion exchange
3. Influence of temperature on the ion-exchange equilibrium
4. The role of temperature in traditional ion-exchange processes using auxiliary reagents
4.1. Reagentless separation processes
References
Index
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Citation preview

ION-EXCHANGE CHROMATOGRAPHY AND RELATED TECHNIQUES

Series: Handbooks in Separation Science

ION-EXCHANGE CHROMATOGRAPHY AND RELATED TECHNIQUES Edited by

PAVEL N. NESTERENKO Department of Chemistry, Lomonosov Moscow State University, Moscow, Russian Federation

COLIN F. POOLE Department of Chemistry, Wayne State University, Detroit, MI, United States

YAN SUN Department of Biochemical Engineering, School of Chemical Engineering and Technology and Key Laboratory of Systems Bioengineering and Frontiers Science Center for Synthetic Biology (Ministry of Education), Tianjin University, Tianjin, China

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States Copyright © 2024 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-443-15369-3 For information on all Elsevier publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Candice Janco Acquisitions Editor: Charlotte Rowley Editorial Project Manager: Lindsay Lawrence Production Project Manager: Sruthi Satheesh Cover Designer: Matthew Limbert Typeset by STRAIVE, India

Contents

4. Stationary phases for the separation of biopolymers by ion-exchange chromatography

Contributors xi 1. Concepts and milestones in the development of ion-exchange chromatography

GUANGHUI MA

1. Introduction 69 2. Uniform agarose-based ion-exchange chromatographic media 71 3. Gigaporous ion-exchange chromatographic media 76 4. Other ion-exchange stationary phases for bioseparations 87 5. Summary and outlook 89 References 89

COLIN F. POOLE, LINLING YU, AND YAN SUN

1. 2. 3. 4. 5.

Introduction 1 Fundamentals 5 Column chromatography 10 Large-scale ion-exchange separations 16 Landmark developments in biotechnology for downstream processing using ion-exchange chromatography 18 References 19

5. Ion-exchange separations of biomacromolecules on grafted and surface-modified polymers

2. Equilibria and kinetics of ion-exchange of biopolymers

LINLING YU

1. Introduction 91 2. Stationary phases 93 3. Adsorption and uptake theory 103 4. Applications 106 References 108

DOROTA ANTOS AND WOJCIECH PIA ˛ TKOWSKI

1. 2. 3. 4.

Introduction 25 Dynamic models 27 Kinetic equations of adsorption-desorption rate 31 Adsorption-desorption equilibria: Isotherm equations 33 5. Causes of misinterpretation of the elution data 39 6. Procedure for design of IEX process 44 References 44

6. Extraction chromatography of actinides S.A. ANSARI AND P.K. MOHAPATRA

1. 2. 3. 4.

Introduction 113 Extractants for actinide separation 114 Ligand impregnated resins for actinides 115 Room temperature ionic liquids in extraction chromatography 125 5. Ligand grafted resins for actinides 129 6. Composite beads for extraction chromatography 136 7. Perspectives 138 References 139

3. Stationary phases for ion separations PAVEL N. NESTERENKO

1. Ion-exchange terminology 49 2. Classification of ion-exchangers 49 References 65

v

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Contents

7. Ion-exchange membrane chromatography

10. Displacement chromatography with ion-exchangers

RICCARDO ONESTI, SARA GIANCATERINO, MARCO ROSELLI, SERENA BANDINI, AND CRISTIANA BOI

GUOFENG ZHAO AND YAN SUN

1. Introduction 145 2. Transport phenomena in membrane chromatography 146 3. Module design 149 4. Promising ion-exchange membranes for bioseparations 156 5. Conclusions 157 References 157

8. Ion-exclusion chromatography MASANOBU MORI

1. 2. 3. 4. 5. 6. 7. 8. 9.

Principle 163 Apparatus 164 Ion-exchange resin columns used in ICE 165 Eluent conditions 166 Detection methods 167 Separations of nonionized substances 173 Separation of ammonium and amines 173 Vacancy ion-exclusion chromatography 173 Ion-exclusion/cation-exchange chromatography 174 10. Ion-exclusion/anion-exchange chromatography 175 Abbreviations 176 References 176

9. Chelation ion chromatography PAVEL N. NESTERENKO AND EKATERINA P. NESTERENKO

1. Introduction 181 2. Theoretical aspects of complexation in liquid chromatography 182 3. Ion-exchange chromatography with the complex formation in the mobile phase 185 4. Chelating phases for ion-exchange chromatography 200 5. Application areas of chelating ion-exchangers 203 References 204

1. Principles of displacement chromatography 211 2. Ion-exchange displacers 215 3. Applications of ion-exchange displacement chromatography 219 4. Prospects and outlook 221 References 223

11. Instrumentation for ion chromatography ELENA VENIAMINOVNA RYBAKOVA

1. Solvent delivery systems for IC applications 228 2. Detectors for IC 231 3. Injection system 239 4. Column oven 240 5. Column hardware 240 References 241

12. Instrument platforms for large-scale ion-exchange separations of biomolecules ANURAG S. RATHORE, ANUPA ANUPA, KANTI N. MIHOOLIYA, AND NITIKA NITIKA

1. Introduction 243 2. Chromatography columns 246 3. Ion exchange matrices 246 4. Chromatography equipment 248 5. Scale-up of ion-exchange processes 248 References 259

13. Method development for large molecules IEX separations MATEUSZ IMIOŁEK AND SZABOLCS FEKETE

1. Introduction 264 2. Column, stationary phase, and instrumentation considerations 264 3. Elution modes 266 4. IEX mode for nucleic acid separations 274 5. On-off mechanism of retention 275 6. Need for IEX platform methods 277

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Contents

7. Systematic method development 278 8. Practical advice for systematic method optimization 278 9. Perspectives 281 Declaration of competing interest 282 References 282

14. Sample preparation for ion-exchange separations COLIN F. POOLE

18. Separation of saccharides by ion-exchange chromatography YUAN ZHANG AND JIE LI

1. Introduction 371 2. Separation of saccharides by HPAEC 372 3. Separation of saccharides by ligand exchange chromatography 375 4. Ion exclusion chromatography 377 5. Ion-pair chromatography 379 References 383

1. Introduction 287 2. Liquid-phase extraction 288 3. Solid-phase extraction 293 4. Membrane-based extraction 304 References 307

15. Separation of ions by ion chromatography ANNA UZHEL AND OLEG SHPIGUN

1. Anion-exchange chromatography 316 2. Cation-exchange chromatography 326 References 331

16. Applications of ion chromatography in environmental analysis

19. Separation of oligonucleotides by ion-exchange chromatography COLIN F. POOLE

1. Introduction 387 2. Column packings for the ion-exchange separations 393 3. Nucleotides and sugar nucleotides 394 4. Oligonucleotides 398 5. Phosphorothioated oligonucleotides 400 6. Mixed mode stationary phases 401 7. Multidimensional chromatography 402 8. Purification of oligonucleotides 404 References 408

20. Separation of oligonucleotides by ion-exchange and ion-pair chromatography

RAJMUND MICHALSKI

1. Introduction 333 2. Ion chromatography and related techniques 333 3. IC development perspectives 340 References 346

17. Applications of ion chromatography in food analysis EDWARD MUNTEAN

1. Introduction 351 2. Inorganic anions 352 3. Inorganic cations 356 4. Carbohydrates 358 5. Organic acids 360 6. Food safety issues 362 References 365

MICHAEL G. BARTLETT

1. 2. 3. 4. 5. 6. 7. 8.

Introduction 414 Types of oligonucleotides 414 Endogenous RNAs 415 Therapeutic oligonucleotides 416 Charged-based separations of oligonucleotides 418 Nonspecific adsorption 421 Sample preparation for oligonucleotides 422 Liquid chromatography-mass spectrometry of oligonucleotides 423 9. Selected applications of ion-exchange and ion-pair chromatography to the determination of oligonucleotides 425 10. Future directions 429 References 430

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Contents

21. Separation of proteins by ion-exchange chromatography RAINER HAHN AND NICO LINGG

1. Introduction 435 2. Adsorption: Principles and models 436 3. Evolution of resins 438 4. Investigational approaches 446 5. Common modes of operation 450 6. Perspectives 454 References 455

22. Separation of proteins by mixed-mode chromatography XAVIER SANTARELLI AND CHARLOTTE CABANNE

1. Introduction 461 2. Mixed mode ligands 462 3. Effect of salt and pH on mixed-mode chromatography 465 4. The third dimension 468 5. High-throughput screening of mixed-mode resins 468 6. Monoclonal antibody purification 469 7. Conclusion 471 References 471

23. Process modeling of protein separations by ion-exchange chromatography SHUICHI YAMAMOTO

1. Introduction 473 2. Mechanistic modeling of chromatography 474 3. Plate height and related variables in isocratic elution 477 4. Resolution Rs in isocratic elution 480 5. Ion-exchange equilibria 481 6. Linear gradient elution 482 7. Applications of the mechanistic models and the simplified methods 487 8. Capture process design 496 References 502

24. Applications of ion-exchange chromatography for the purification of antibodies GUOFENG ZHAO, YONGBO LI, YUNTAO WU, SHANSHAN LI, XIAOMEI CHEN, WEI ZHANG, AND JIN XIE

1. Antibodies: Introduction, recombinant expression, manufacturing, and purification 506 2. Cation-exchange chromatography of antibodies 510 3. Anion-exchange chromatography of antibodies 514 4. Commercial ion-exchange resins 516 5. Scale-up and scale-down of ion-exchange chromatography 517 6. Tips and tricks 517 Acknowledgment 520 References 520

25. Conformational changes of biomolecules in ion-exchange chromatography JING GUO AND XUANKUO XU

1. Introduction 521 2. Mechanisms 522 3. Detection of protein conformational changes 525 4. Key contributing factors 527 References 532

26. Continuous ion-exchange chromatography for protein polishing and enrichment YU-CHENG CHEN, RUO-QUE MAO, SHAN-JING YAO, AND DONG-QIANG LIN

1. Introduction 535 2. Limits of batch chromatography 536 3. Continuous chromatography for protein purification 537 4. Process design for continuous purification 547 5. Applications for protein polishing using ion-exchange 548 6. Conclusion 550 References 550

ix

Contents

27. Separation of bio-particles by ion-exchange chromatography XUAN LIN, ZHIGUO SU, GUANGHUI MA, AND SONGPING ZHANG

1. Introduction 553 2. The principles of separating bio-particles by IEC 555 3. Challenges and coping strategies for separating bio-particles by IEC 555 4. Design of IEC-based bio-particle separations 564 5. Future prospects 570 Acknowledgment 573 References 573

28. Virus removal in bioprocessing using charged media CARYN L. HELDT

1. Introduction 579 2. Virus physicochemical properties 580 3. Protein interference 584

4. Chromatography format 585 5. Cation exchange and multimodal ligands 587 6. Conclusions 588 References 588

29. Role of temperature in ion-exchange processes of separation and purification V.A. IVANOV AND R.KH. KHAMIZOV

1. Influence of temperature on the stability of ion-exchange resins 592 2. Influence of temperature on the kinetics and dynamics of ion exchange 593 3. Influence of temperature on the ion-exchange equilibrium 594 4. The role of temperature in traditional ion-exchange processes using auxiliary reagents 600 References 609

Index 615

Contributors

Bhabha

Mateusz Imiołek Waters Corporation, Cell and Gene Therapy Consumables, Geneva, Switzerland

Dorota Antos Department of Chemical and Process Engineering, Rzeszo´w University of Technology, Rzeszo´w, Poland

V.A. Ivanov M. V. Lomonosov Moscow State University, Department of Chemistry, Moscow, Russian Federation

Anupa Anupa School of Interdisciplinary Research, Indian Institute of Technology Delhi, New Delhi, India

R.Kh. Khamizov Vernadsky Institute of Geochemistry and Analytical Chemistry of the Russian Academy of Sciences, Moscow, Russian Federation

S.A. Ansari Radiochemistry Division, Atomic Research Centre, Mumbai, India

Serena Bandini Department of Civil, Chemical, Environmental and Materials Engineering (DICAM), University of Bologna, Bologna, Italy

Jie Li

China Medical University, Shenyang, China

Shanshan Li Downstream Process Development, Shell Biotech, Shanghai, China

Michael G. Bartlett College of Pharmacy, University of Georgia, Athens, GA, United States

Yongbo Li Downstream Process Development, Shell Biotech, Shanghai, China

Cristiana Boi Department of Civil, Chemical, Environmental and Materials Engineering (DICAM), University of Bologna, Bologna, Italy

Dong-Qiang Lin College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, China

Charlotte Cabanne Bordeaux INP, CBMN, UMR 5248, Pessac, France

Xuan Lin State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, China

Xiaomei Chen Analytical and Pharmaceutical Sciences, Shell Biotech, Shanghai, China

Nico Lingg Department of Biotechnology, Institute of Bioprocess Science and Engineering, University of Natural Resources and Life Sciences, Vienna, Austria

Yu-Cheng Chen College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, China Szabolcs Fekete Waters Corporation, Cell and Gene Therapy Consumables, Geneva, Switzerland

Guanghui Ma State Key Laboratory of Biochemical Engineering, Institute of Process Engineering; Key Laboratory of Biopharmaceutical Preparation and Delivery, Chinese Academy of Sciences, Beijing, China

Sara Giancaterino Department of Civil, Chemical, Environmental and Materials Engineering (DICAM), University of Bologna, Bologna, Italy Jing Guo Biologics Development, Global Product Development & Supply, Bristol Myers Squibb, Devens, MA, United States

Ruo-Que Mao College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, China

Rainer Hahn Department of Biotechnology, Institute of Bioprocess Science and Engineering, University of Natural Resources and Life Sciences, Vienna, Austria

Rajmund Michalski Institute of Environmental Engineering of Polish Academy of Sciences, Zabrze, Poland Kanti N. Mihooliya Department of Chemical Engineering, Indian Institute of Technology Delhi, New Delhi, India

Caryn L. Heldt Department of Chemical Engineering; Health Research Institute, Michigan Technological University, Houghton, MI, United States

xi

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Contributors

P.K. Mohapatra Radiochemistry Division, Bhabha Atomic Research Centre, Mumbai, India Masanobu Mori Faculty of Science and Technology, Kochi University, Kochi, Japan Edward Muntean Department of Food Sciences, Faculty of Food Science and Technology, University of Agricultural Sciences and Veterinary Medicine, Cluj Napoca, Romania Ekaterina P. Nesterenko Department of Chemistry, Lomonosov Moscow State University, Moscow, Russian Federation Pavel N. Nesterenko Department of Chemistry, Lomonosov Moscow State University, Moscow, Russian Federation Nitika Nitika Department of Chemical Engineering, Indian Institute of Technology Delhi, New Delhi, India Riccardo Onesti Department of Civil, Chemical, Environmental and Materials Engineering (DICAM), University of Bologna, Bologna, Italy

Zhiguo Su State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, China Yan Sun Department of Biochemical Engineering, School of Chemical Engineering and Technology and Key Laboratory of Systems Bioengineering and Frontiers Science Center for Synthetic Biology (Ministry of Education), Tianjin University, Tianjin, China Anna Uzhel Department of Chemistry, Lomonosov Moscow State University, Moscow, Russia Yuntao Wu Bestchrom (Zhejiang) Biosciences Ltd, Zhejiang, China Jin Xie Downstream Process Development, Shell Biotech, Shanghai, China Xuankuo Xu Biologics Development, Global Product Development & Supply, Bristol Myers Squibb, Devens, MA, United States Shuichi Yamamoto Biomedical Engineering Center (YUBEC), Yamaguchi University, Ube, Japan

Wojciech Pia˛tkowski Department of Chemical and Process Engineering, Rzeszo´w University of Technology, Rzeszo´w, Poland

Shan-Jing Yao College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, China

Colin F. Poole Department of Chemistry, Wayne State University, Detroit, MI, United States

Linling Yu Department of Biochemical Engineering, School of Chemical Engineering and Technology and Key Laboratory of Systems Bioengineering and Frontiers Science Center for Synthetic Biology (Ministry of Education), Tianjin University, Tianjin, China

Anurag S. Rathore Department of Chemical Engineering, Indian Institute of Technology Delhi, New Delhi, India Marco Roselli Department of Civil, Chemical, Environmental and Materials Engineering (DICAM), University of Bologna, Bologna, Italy Elena Veniaminovna Rybakova Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, Moscow, Russia Xavier Santarelli Bordeaux INP, CBMN, UMR 5248, Pessac, France Oleg Shpigun Department of Chemistry, Lomonosov Moscow State University, Moscow, Russia

Songping Zhang State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, China Wei Zhang Downstream Process Development, Shell Biotech, Shanghai, China Yuan Zhang China Medical University, Shenyang, China Guofeng Zhao Analytical and Pharmaceutical Sciences, Shell Biotech, Shanghai, China

C H A P T E R

1 Concepts and milestones in the development of ion-exchange chromatography Colin F. Poolea, Linling Yub, and Yan Sunb a

Department of Chemistry, Wayne State University, Detroit, MI, United States Department of Biochemical Engineering, School of Chemical Engineering and Technology and Key Laboratory of Systems Bioengineering and Frontiers Science Center for Synthetic Biology (Ministry of Education), Tianjin University, Tianjin, China b

O U T L I N E 1. Introduction

1

2. Fundamentals 2.1 Retention mechanism for small ions 2.2 Retention mechanisms for polyelectrolytes

5 6

3. Column chromatography 3.1 Porous polymer ion exchangers for the separation of low-mass ions 3.2 Restricted access media

10

3.3 Ion exchangers for large-scale separations 4. Large-scale ion-exchange separations

16

5. Landmark developments in biotechnology for downstream processing using ion-exchange chromatography 18

8

References

12 14

1 Introduction

19

zeolites, sands, and clays with fixed charged sites in the soil [1–3]. These studies eventually led to the development and characterization of synthetic inorganic ion-exchange materials, such as aluminosilicates (zeolites), clays and other layered materials, metal phosphates,

Ion exchange as a stoichiometric process was first described in the early 1850s as a phenomenon in the interaction of soils with electrolyte solutions associated with the presence of

Ion-Exchange Chromatography and Related Techniques https://doi.org/10.1016/B978-0-443-15369-3.00027-4

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Copyright # 2024 Elsevier Inc. All rights reserved.

2

1. Concepts and milestones

heteropolyoxometalates, and hydrous oxides [1]. These materials found numerous industrial uses, such as catalysts and for water treatment, but generally lack a suitable morphology and mechanical properties for use in ion-exchange chromatography (IEC). The synthesis of highcapacity organic polymer resins in bead form, initially by polycondensation of phenols and amines with formaldehyde and later by polymerization of monomers, such as styrene, divinylbenzene, and acrylates between 1935 and 1945 was an important development that established a general matrix adaptable by surface functionalization for chromatographic applications [4–7]. Early materials often suffered from low compression resistance, poor dimensional stability in different solvents (swelling), and low kinetic performance. After a slow evolution and optimization of particle morphology, the descendants of these early materials remain at the forefront of chromatographic applications for the separation of ionic species [8]. The early 1950s also saw the development of liquid ion exchangers largely for the extraction of metal ions from water. Typically, these are high-molecular mass organic acids or bases, sparingly soluble in water, but readily soluble in water-immiscible organic solvents suitable for the extraction of ions from aqueous solution or their transport across a liquid membrane to achieve selective isolation, concentration, and matrix simplification. This led to the development of extraction chromatography using liquid ion exchangers supported on an inert support to process samples in a column configuration [9]. Ion-exchange membranes also appeared in the early 1950s but largely for nonchromatographic applications, such as electrodialysis. It was much later that they were adopted for the purification of biological macromolecules as an alternative to particle-packed column formats [10,11]. Ionexchange membranes are porous films or thin sheets of a surface functionalized polymer that can be stacked in series to increase capacity and performance. Their design allows for fast sample processing and ease of scalability. Later

they would play an important role in the development of eluent suppressors for ion chromatography using conductivity detection [12]. The mid-1950s saw the development of hydrophilic cellulose-, dextran-, and agarosebased resins in bead form for both analytical and preparative separations of biomolecules [6,13]. Porous organic polymer beads in use at that time were typically hydrophobic, denaturing, and strongly retentive of biomolecules. Carbohydrate-based polymers possess a hydrophilic surface and were easily functionalized opening opportunities for the separation of biomacromolecules by ion-exchange chromatography at moderate pressures with retention of their native structure. They were enormously impactful on early life-science applications of ion-exchange chromatography. Early materials were characterized by poor compression resistance, poor dimensional stability in different solvents (swelling), and low kinetic performance. After optimization of particle morphology, these disadvantages were minimized if not completely overcome. These later generation carbohydrate polymers are an important resource in biotechnology for the large-scale separations of biomolecules (see Section 3.3). In the mid-1960s and early 1970s surfacemodified porous silica particles revolutionized the practice of liquid chromatography laying the foundation for the explosive growth of high-performance liquid chromatography (HPLC) on the road to become one of the most important separation techniques of the 21st century [14,15]. Stationary phases suitable for ionexchange chromatography appeared early in the evolution of these chemically bonded phases but have not displaced porous organic polymer resins for many applications in IEC, in part due to their lower loading capacity and poor stability to aqueous solutions (2 < pH > 8), this being more limiting for ion-exchange separations than other forms of HPLC. Organic-inorganic composite substrates were developed later to address some of the pH limitations of silica-based substrates but had less impact on IEC and ion

1 Introduction

chromatography (IC) than for reversed-phase liquid chromatography (RPLC). A feature of this period was that it catalyzed developments in porous organic polymers resulting in particle designs of higher efficiency, smaller particle size, higher mechanical strength, and with biocompatible surfaces which allowed porous organic polymers to maintain their position as the main particle type for modern high-performance IEC, while other separation methods within the lexicon of HPLC techniques tended to migrate toward porous silica-based stationary phases. By the late 1970s ion-exchange resins had been supplemented by materials containing immobilized chelate groups for highly selective separations of ions leading to ion-chelation chromatography as an alternative to ion-exchange methods with an emphasis on higher selectivity for a smaller number of target ions [16]. Monolithic ion-exchange materials with a unitary biporous structure in rod form containing convective pores supporting high flow rates, lower pressure operation, and more favorable mass transfer as well as macropores to control retention appeared in the late 1990s [17]. Polymeric monoliths of low surface area have found use in the separation of biomacromolecules where a large surface area is not required. Ion-exchange chromatography as we know it today was built on a foundation of knowledge accumulated over many years punctuated by several outstanding achievements beginning in about 1947 with the preparative separation of the rare earth elements by displacement ion-exchange chromatography [3,18]. The rare earths have similar solution properties, and their isolation in a pure form is challenging. They were essential to the accelerated atomic weapons program under the umbrella of the Manhattan Project. Their separation by displacement ionexchange chromatography was achieved by loading a relatively large volume of sample on an ion-exchange column in the sulfonic acid form as a zone extending a significant length along the column. The column, or rather several columns connected in series were eluted continuously

3

with a solution containing citric acid and ammonium citrate. The rare earths relocate into bands on the resin bed, with each band having a specific citrate and hydrogen ion concentration and ionic strength associated with it. Each band undergoes an automatic sharpening of boundaries with each band riding on the backside of the proceeding band containing individual rare earths that move as a contiguous zone down the column when equilibrium has been achieved. At elution from the column, the individual rare earths are arranged within the sample zone in the order of their complexation constant with citrate, with the more strongly complexed rare earths eluting at the head of the reorganized sample zone. Within a few years, IEC was being used for isotope separations beginning with 15N and 14N as the ammonium ion [1,18]. Ions in solution are solvated diluting the mass difference between isotopes with separation factors that are extremely small. This led to specialized approaches such as counter-current ion-exchange separation in columns employing a balance between hydraulic flow and electromigration in opposing directions. This remains a specialized field with methods published for many elements. For more than 70 years, ion exchange has been used in clinical and biochemistry laboratories for the routine separation of amino acids, protein hydrolyzates, and other physiologically important amines to identify metabolic disorders and to sequence the structure of biopolymers [19,20]. Dedicated instruments, amino acid analyzers, have been in production for over half a century [20–22]. Separations are performed on strong cation-exchange resins with the amino acids in the protonated form using an optimized combination of pH, buffer composition, and temperature gradients. The amino acids are usually detected after postcolumn reaction with ninhydrin or o-phthalaldehyde. The separation methods are robust and more than forty physiologically important amine compounds can be separated in under 2 h. From early days commercially produced instruments operate in an automated environment and

4

1. Concepts and milestones

preceded developments in general HPLC instruments, which only attained similar features many years later. This traditional method of amino acid analysis is now challenged by new methods using precolumn derivatization and RPLC [19,23]. There is a balance of opinion as to which method is preferred, perhaps the newer methods for the fast separation of simpler mixtures and the amino acid analyzer for reliable quantification of complex mixtures. Another traditional application of ion exchange is the isolation, purification, and separation of peptides, proteins, nucleotides, and other biomacromolecules on either relatively soft nondenaturing gels for preparative chromatography or wide-pore, microparticle packings for analytical separations [24–27]. RPLC is now the preferred choice for the separation of peptides and nucleotides, but for biomacromolecules ion exchange offers complementary selectivity and a separation environment that is less denaturing. The latter is a critical consideration for the large-scale production of biopharmaceuticals and a significant contribution to the contemporary ion-exchange literature. Ion exchange has a long history for the separation and isolation of carbohydrates. Traditional methods are based on ligand exchange interactions employing cation exchangers loaded with metal ions, such as calcium (recommended for separating sugar alcohols), lead (more effective than calcium for separating isomeric mono- and disaccharides), and silver (preferred for determining oligomeric distributions) [28–30]. Water or aqueous organic solvent mixtures are used as the mobile phase. Retention is governed by a combination of size exclusion and electrostatic attraction between the electronegative sugar oxygen atoms and electropositive metal cations. Carbohydrates are also separated by anion-exchange chromatography of their negatively charged borate complexes. Since carbohydrates are weak acids (pKa
11.9–12.5), they can be separated by anion-exchange chromatography at high pH (>12) with sodium hydroxide

or sodium hydroxide/sodium acetate solutions as the mobile phase. The carbohydrates are expected to possess a single negative charge per saccharide unit (regardless of the degree of oligomerization) and elute in order of increasing size and degree of effective ionization. The need for a widely applicable and sensitive detector compatible with gradient elution techniques was solved with the development of the pulsed amperometric detector (PAD), which together with high-efficiency pellicular ion-exchange packings, is credited with revolutionizing the analysis of complex carbohydrate mixtures [31]. A major contemporary application of IEC is the separation of small inorganic and organic ions by ion chromatography [32,33]. The term ion chromatography is used here to describe a specific instrumental approach based on IEC that gave its name to a dedicated instrument, an ion chromatograph, but its contemporary use encompasses a wider range of separation methods in which it has become synonymous with a sample type rather than a specific technique. Ion chromatography evolved from studies in the mid-1970s that sought to combine the selectivity of ion exchange for the separation of inorganic ions with universal ion detection based on conductivity [12,34–36]. Despite limitations in early approaches, ion chromatography was quickly accepted in large part because it was able to replace many tedious wet chemical analyses with a simple instrumental approach capable of determining several ions in a single method. In its original form, ion chromatography benefited from the synergy between high efficiency pellicular column packings with a low exchange capacity; the use of low ionic strength mobile phases containing eluent ions with a high affinity for the stationary phase; and the use of a suppressor column combined with flow-through conductivity detection. Within a few years, nonsuppressed ion chromatography was developed using low-capacity ion-exchange resins with weak electrolyte eluents with a high affinity for the stationary phase

2 Fundamentals

[37]. Both approaches remain in use today. Modern ion chromatography allows greater flexibility in the choice of detection methods and utilizes a wide range of ion-exchange materials optimized for specific applications. Modern instruments are machined from polymeric parts and feature electrochemical generation of mobile phase compositions, and low volume suppressor systems that allow full automation of system operating processes. Although instrumentation for ion chromatography and HPLC may look similar, they have evolved separately, and to some extent synergistically. Conventional HPLC instruments are not generally employed for ion chromatography without significant modification due to the corrosive mobile phases necessary for ion-exchange separations.

2 Fundamentals IEC is used for the separation of ions and easily ionized substances (e.g., substances that form ions by pH manipulation or complex formation). One of the principal contributions to retention is the electrostatic attraction between mobile phase ions, both sample and eluent, for immobilized ion centers of opposite charge in the stationary phase. Separations depend on differences in the relative affinity of sample and mobile phase ions for the stationary phase ion centers in a dynamic exchange system, in which sample ions and eluent ions interact with multiple stationary phase ion centers as they migrate down the column. The charges that are bound to the resin phase are called fixed charges and provide a reference point for naming the other ions participating in the ion-exchange system [2,38]. Mobile phase ions with the same sign as the fixed charge ions are called co-ions, and those with an opposite charge are called counter-ions. The ion exchanger is a solid or liquid containing the fixed charge sites. It may be indicated as in the

5

salt form; in which case the counter-ions are neither hydrogen nor hydroxide or in the acid form (counter-ion is the hydrogen ion) or base form (counter-ion is the hydroxide ion). An anion exchanger is employed for the separation of negatively charged ions (anions) and a cation exchanger for the separation of positively charged ions (cations). Ion exchangers are broadly classified as strong or weak depending on the fixed charge site and the mobile phase pH. Strong anion and cation exchangers have sulfonic acid, phosphonic acid, and quaternary amine fixed charge sites that retain their ionic form independent of the mobile phase pH. Weak anion and cation exchangers have carboxylic acid or primary, secondary, or tertiary amine fixed charge sites whose charge state depends on the mobile phase pH and can be switched to neutral to aid elution. The capacity of an ion exchanger is determined by the number of fixed charged sites per unit mass of column packing with units of milliequivalents per gram of dry resin or milliequivalents per milliliter of wet resin. In the latter case, it is usual to state the type of counter-ion present in the resin since this affects the degree of swelling of the resin and hence its volume. For typical separations by IEC, column packings with an ion-exchange capacity of about 3–5 mEq/g are used and for IC about 0.1–0.5 mEq/g or less. Higher capacity resins generally require eluents of higher ionic strength for elution, and ionexchange capacity is a variable in method development for ion exchangers with the same fixed charge and in choosing detection options. Samples of high ionic strength may require suitable dilution before loading or the selection of column packings of high exchange capacity for effective chromatographic separation. Transport of sample ions in column chromatography occurs entirely in the mobile phase. Transport is an essential component of chromatographic systems since the common experimental arrangement for chromatographic separations employs a sample inlet and detector

6

1. Concepts and milestones

at opposite ends of the column with sample introduction and detection occurring in the mobile phase. IEC separations can be performed using elution, frontal analysis, or displacement transport modes. Elution chromatography is widely used for analytical separations. The mobile phase is continuously passed through the column after introduction of the sample which proceeds to migrate along the column to the exit where ions are detected as separated zones with a near Gaussian profile. Initially, the column packing is equilibrated with mobile phase counter-ions, the sample is introduced as a focused band that occupies a small fraction of the column length and sample ions separated under conditions in which their adsorption isotherms are linear (dilute solution), and their relative migration along the column results from the competition between sample ions and the higher concentration of counter-ions for the fixed charge sites of the column packing. Frontal analysis is rarely used for analytical separations, but as a method of measurement of physicochemical properties and in fundamental investigations. For frontal chromatography, the sample as a component of the mobile phase is passed continuously through the column and the breakthrough of sample ions monitored at the column exit. The least retained ion elutes first followed by the other ions in turn, each of which contains several components identical to the ions in the zone eluting before it. Ideally, if equilibrium is attained the detector output is comprised of a series of steps of increasing height. Frontal analysis is used to determine adsorption isotherms for single component or simple mixtures and to concentrate weakly retained ions from a concentrated sample matrix [39–41]. At the end of the separation, the column is contaminated with sample ions, and a lengthy column cleaning procedure between samples is required. Displacement chromatography is employed mainly for preparative-scale separations under nonlinear conditions [41–44]. The column is first equilibrated with mobile phase containing

counter-ions of low elution strength (sometimes referred to as the carrier). The sample typically dissolved in the carrier is introduced into the column as a contiguous zone that occupies a significant column length. Migration of sample ions commences by passing a mobile phase through the column containing a concentrated solution of a counter-ion (displacer) with a higher affinity for the fixed charge sites than any of the sample ions. Sample ions with a higher affinity for the fixed charge sites displace those from the stationary phase with a weaker affinity causing the sample ions to reorganize into adjacent zones containing identical sample ions within the sample application zone. The displacer ion moves the sample ions down the column, and provided that the column is long enough to attain a steady state, a succession of rectangular bands of separated sample ions (displacer train) exits the column. The advantages of displacement chromatography for preparative applications are that the separated ionic species are obtained at a higher concentration than for elution chromatography, mobile phase consumption is less, and band tailing is reduced because of the self-sharpening boundaries obtained. Separations are generally faster than for elution chromatography, but the cycle time is generally longer, due to the need to regenerate the column (rinse away the displacer and equilibrate with the carrier phase) between each sample injection. Moreover, careful collection of each eluted component is required because they leave the column as connected rectangular bands.

2.1 Retention mechanism for small ions General theories for the retention of small ions in IEC are based on either stoichiometric or electrostatic double-layer models [2,32,33,45]. Stoichiometric models provide a simple picture of the retention process with reasonable predictive capability. These useful features obscure physical deficiencies in the treatment of

7

2 Fundamentals

electrostatic interactions by the law of mass action. Stoichiometric models fail to consider the effect of long-range, multibody interactions on the equilibrium distribution constant. On the other hand, stoichiometric models provide a convenient starting point for a general picture of the ion-exchange process. If we assume that the sample and mobile phase ions are in equilibrium with the fixed charge sites on the stationary phase, an equilibrium expression can be written as yAM + xES $ yAS + xEM x

y

x

y

(1)

where Ax represents a sample ion of charge x, Ey a mobile phase counter-ion of charge y, and subscripts M and S refer to ions in the mobile and stationary phases, respectively. The equilibrium constant (selectivity coefficient) is given by. KA,E ¼ ½AS x y ½EM y x =½AM x y ½ES y x

(2)

To be correct the concentration terms should be expressed as activities, but stationary phase activities cannot be evaluated by formal methods. The selectivity coefficient is a measure of the preference of the stationary phase for one ion over another for a specific stationary phase but changes in order are usually minor for common ion exchangers. Polyvalent ions are typically retained more strongly than monovalent ions owing to their stronger electrostatic interactions with the fixed charge sites on the stationary phase. For ions of the same charge, retention tends to decrease with increasing hydrated ion radius. The mass distribution constant for the sample ion Ax is given by DA ¼ [ASx]/[AMx] and is related to the chromatographic retention factor for the sample ion by kA ¼ DAW/VM, where W is the mass of stationary phase and VM is the column hold-up volume. The ratio of W/VM is the phase ratio. Substituting these terms into Eq. (2) gives. KA,E ¼ ðkA V M =W Þy ð½EM y =½ES y Þx

(3)

For analytical separations, the concentration of sample ions is generally much less than the concentration of mobile phase counter-ions and [ESy] can be replaced by Q/y, where Q is the ion-exchange capacity of the stationary phase. Substituting for [ESy] in Eq. (3) gives. log kA ¼ log ðW=V M Þ + ð1=yÞ log ðKA,E Þ + ðx=yÞ log ðQ=yÞ  ðx=yÞ log ½EM y  (4) For a particular column, mobile phase composition, and sample ion KA,E, Q, W, and VM will be constant and Eq. (4) simplifies to. log kA ¼ C  ðx=yÞ log ½EM y 

(5)

where C is a system constant. In which case, the retention of sample ions is determined by the selectivity coefficient, the ion-exchange capacity of the stationary phase, the phase ratio, and the concentration of mobile phase counter-ion. Increasing the selectivity coefficient, ionexchange capacity of the stationary phase or phase ratio increases retention while increasing the concentration of the mobile phase counterion decreases retention. Increasing the charge on the mobile phase counter-ion (y) decreases retention, while increasing the charge of sample ions (x) increases retention. A plot of log kA vs log [EMy] should be linear with a negative slope given by the ratio of the charge on the sample and mobile phase counter-ions (x/y). Exceptions are known to Eq. (5), particularly for organic ions when nonionic interactions contribute to retention. For electrostatically agglomerated pellicular ion exchangers, deviations from Eq. (5) were attributed to electrostatic interactions with the oppositely charged core particle [46]. The above models assume the mobile phase contains a single mobile phase counter-ion. For mixtures of polyprotic acids, widely used as eluents in IC, modification is required [2,32,45]. The dominant equilibrium approach assumes that the most highly charged mobile phase counterion is solely responsible for elution and retention

8

1. Concepts and milestones

can be modeled assuming that the mobile phase contains only this counter-ion. The effective charge approach assumes that all charged competing ions are responsible for elution of sample ions in proportion to their charges. In Eq. (5), the charge y is replaced by the calculated effective charge for the multicomponent mobile phase counter-ions. The dual eluent species approach considers both the relative concentrations of the counter-ions and their different affinities for the stationary phase. The agreement with experimental data typically reflects the reliability of the assumptions with regard to the real system properties. Electrostatic ion-exchange models attempt to explain retention by the accumulation of ions in the double layer, which is typically a nonstoichiometric process, together with contributions from specific ion interactions with the charged surface as well as changes in ion solvation [2,47]. General models are formulated on the Gouy-Chapman diffuse double-layer theory combined with a mass action equation for specific adsorption of both co-ions and counterions to the charged surface. Ions are treated as point charges and the mobile phase as a continuum with constant polarizability. The stationary phase is regarded as a rigid plane with fixed charged sites per unit surface area. Sample ions are present at trace concentrations compared with mobile phase ions. The ion concentration profile is determined by the change in the electrostatic potential as a function of distance from the stationary phase surface, and a stoichiometric association constant is used to describe the concentration dependence of specific binding of counter-ions to the stationary phase surface. The retention factor of a sample ion depends on the total concentration of electrolyte in the mobile phase through its influence on the double layer, the surface potential, and the concentration of fixed charge sites on the stationary phase. Numerical solutions are required because of the integral form of the model and its interdependence on the

positions of ions with reference to the surface of the stationary phase, the surface density of fixed charge sites, and the bulk concentration of counter-ions. Analytical solutions are possible by imposing restrictions on the variables [47]. Electrostatic models can successfully explain observed experimental deviations from stoichiometric models but are difficult to evaluate.

2.2 Retention mechanisms for polyelectrolytes The retention of polyelectrolytes like proteins in IEC is a complex process that depends on many factors as well as the number and distribution of charge sites interacting with the surface of the stationary phase and on nonelectrostatic interactions [48–52]. Retention can be manipulated through changes in the mobile phase pH and ionic strength typically, which modify the electrostatic surface potential of polyelectrolytes. Also, the identity of mobile phase co-ions can significantly influence the retention of proteins through their effects on protein solubility and aggregation. Sizeexclusion effects can also affect retention by preventing access of the polyelectrolyte to the total pore volume of the stationary phase where the largest number of fixed charge sites are located. In addition, since Donnan membrane potentials are developed on charge-carrying supports, counter-ions may be excluded from the pore volume by ion repulsion [53]. As a consequence, the retention mechanism for a polyelectrolyte is never simple and needs to consider the purpose of the separation, whether for analytical purposes, which in most cases conform to a linear adsorption isotherm requiring estimation or control of fewer parameters, or preparative separations on overloaded columns, described by a nonlinear adsorption isotherm and requiring protein-protein interactions in the adsorbed and solution state to be considered.

2 Fundamentals

Models for the ion-exchange separation of polyelectrolytes can be broadly categorized as empirical models and mechanistic models [26,27,49]. Typical empirical models adopt a black box approach in which output responses are related to the input variables based on statistical evaluation of experimental data obtained from a design of experiments approach. It is likely that many experiments will be required to predict or optimize output responses, and often only a limited knowledge of the system properties is obtained. System information may not be transferrable to another system or polyelectrolyte. On the other hand, mechanistic models can provide insight into the retention mechanism and its relationship to the physicochemical properties of the system typically at the expense of considerable computational effort. Mechanistic models consist of sets of partial differential equations describing mass transport and thermodynamic adsorption phenomena in the column. Contemporary modeling of protein retention in IEC is dominated by stoichiometric adsorption models in which the protein is modeled as either a sphere interacting with a planar surface of opposite charge, as a planar surface (slab) interacting with a planar stationary phase of opposite charge, or as a colloidal particle interacting with a planar stationary phase [50,51]. The interaction of the protein with the ion exchanger is ascribed to a reversible stoichiometric displacement of counter-ions bound to the fixed charge sites on the ion exchanger by charged proteins contained in the mobile phase. For analytical separations, the stoichiometric displacement model (SDM) is the most straightforward approach and adequate in many cases [52]. The slab model predicts the often-observed dependence of the retention factor (log k) on the reciprocal of the square root of the mobile phase ionic strength. The SDM model has been extended to include both linear and nonlinear adsorption isotherms with the introduction of the steric mass action (SMA) model [50,51]. In

9

this case, nonlinear adsorption behavior is attributed to a reduction of the number of counter-ions due to displacement from the surface of the ion exchanger by the protein or from steric shielding due to its size. The total number of adsorption sites beneath the adsorbed protein on a charged surface is larger than the number of adsorption sites that interact directly with the protein, in which case a significant number of counter-ions are sterically hindered from participating in the ion-exchange equilibrium, Fig. 1 [54]. The SMA model accounts for the multipoint nature of the interaction of the protein with the ion exchanger through the value assigned to the characteristic charge, or a process called charge regulation [51,52,54,55]. The net charge for a polyelectrolyte is influenced by the electrostatic field at the surface of the ion exchanger. Although SMA models provide a conceptually simple description of protein adsorption, their ability to accurately reflect electrostatic interactions between proteins and ion exchangers has been questioned. Colloidal particle adsorption (CPA) models are nonstoichiometric models that provide a more realistic representation of electrostatic interactions [52,56]. In this case, charge regulation of a polyelectrolyte in the adsorbed state is represented as an electric double layer, and the PoissonBoltzmann equation is used to account for the electrostatic effect produced by the ion exchanger on the charge state of the protein. The lengthy calibration of mechanistic models involving many experimental and computational estimates for system properties for the specific protein studied is a disadvantage for general applications. Multiscale models attempt to understand the relationship between protein structure and their ionexchange properties such as retention or adsorption isotherm parameters [48,49]. Quantitative structure-property relationships are used to leverage machine learning algorithms and existing experimental data to predict target properties from protein structure. This

10

1. Concepts and milestones

FIG. 1 Representation of a protein with four negatively charged groups interacting with the fixed charged sites of a cation exchanger. The chloride counter-ions form undissociated ion pairs with the sites beneath the protein in the gray area, while the chloride ion is an exchangeable counter-ion outside the gray area. From H. Shen, D.D. Frey, Effect of charge regulation on steric mass-action equilibrium for the ion-exchange adsorption of proteins. J. Chromatogr. A 1079 (2005) 92–104 with permission.

starts by encoding the amino acid sequence of a series of reasonably homologous proteins with a group of global and patch-specific descriptors to train the model to reproduce the experimentally determined target property. If the model is sufficiently general, it can be used to predict the target property for other proteins in the same system, at least for proteins with structural features like the proteins in the training set and perhaps assist in understanding the change in the target property with variation of the system parameters. In one sense, multiscale models afford a deeper understanding of system properties and their relationship to protein structure, while on the other hand, the molecular descriptors may be too numerous and lack obvious chemical meaning for a general interpretation of the physicochemical properties of the system. In the latter case though, model development is largely a computational procedure potentially more efficient and time saving than models requiring experimental inputs.

3 Column chromatography In the period before HPLC, separations were performed at low pressures with totally porous inorganic oxide or porous polymer particles with an average particle size of 30–200 μm. These materials had a high sample capacity, useful for preparative chromatography, but for analytical separations on account of their large particle size, wide particle size distribution, and deep pores resulting in slow mass transfer, column efficiency was poor and separation times long. The introduction of pellicular particles (porous layer beads) in the 1960s could be considered the first column packing for HPLC. These materials had particle diameters of 30–55 μm consisting of a glass bead core onto which was fused a 1–3 μm porous silica layer. The rigid core permitted operation at high pressures and the thin, porous shell improved mass transfer properties. Reaction of the porous silica layer with organosilane reagents containing ionizable functional groups allowed the synthesis of the first

3 Column chromatography

high-performance ion-exchange packings for HPLC [57]. The importance of pellicular particles in the evolution of high-pressure liquid chromatography, also abbreviated to HPLC, was short-lived and declined rapidly in the early 1970s after the introduction of totally porous silica microparticles with a narrow size range and average particle diameter < 10 μm. These materials offered a more favorable compromise between mechanical stability and kinetic performance but for IEC applications were limited by their poor stability at high pH due to the solubility of the silica matrix. Compared with porous layer beads, totally porous microparticles provided faster separations and an increase of up to an order of magnitude in column efficiency and sample capacity. It was not until the 1990s that pellicular packings resurfaced in the form of nonporous microparticles of 0.5–5 μm diameter [58] and superficially porous particles of 535 cm/h, the column backpressure for commercially available stationary phases increased rapidly, indicating the collapse of the agarose microspheres. However, the maximum flow rate of the high-crosslinked agarose microspheres increased to as high as 1452 cm/h, without obvious collapse. The high cross-linked uniform agarose microspheres can be further modified by introducing ion-exchange ligands useful for fast-flow protein chromatography [3]. Generally, diethylaminoethyl (DEAE) weak anion-exchange media have a high binding capacity ideal for the purification of biomacromolecules. The ion-exchange capacity of the stationary phases has important effects on the static adsorption behavior (e.g., static adsorption capacity and binding constant) as well as the structure and activity of biomacromolecules such as hepatitis B surface antigen (HBsAg). This is because the structure of biomacromolecules is complex and easily altered during the binding process with the chromatographic media. Due to the multisite binding of a protein on the stationary phase, the protein conformation may change and be deactivated. Also, the multisite binding may be too strong for elution, resulting in irreversible adsorption. For biomacromolecules with multiple subunits, the selection of an ion-exchange media with a low ion-exchange capacity is beneficial for their structural stability and retention of bioactivity [4–6]. Preparation of DEAE chromatographic media with a controllable ion-exchange capacity and its effect on purification of HBsAg were studied. The important factors affecting the ion-exchange capacity of the stationary phases were the concentration of sodium hydroxide, DEAE
HCl concentration, reaction time, and temperature. It was confirmed that the highest ion-exchange capacity of 0.1135 mmol/mL was obtained with 5.0 M sodium hydroxide, 1.5 M DEAE
HCl, a reaction time of 4.5 h, and a temperature of 60°C. Also, DEAE media with different ion-exchange

2 Uniform agarose-based ion-exchange chromatographic media

capacities were obtained by varying the reaction time. With the increase of ion-exchange capacity, the adsorption capacity for BSA increased gradually and the dissociation constant decreased gradually. This is mainly due to the increase of protein adsorption sites on the stationary phases with the increase in ion-exchange capacity. Meanwhile, the decrease of the dissociation constants is mainly caused by the enhancement of the interactions between the protein and the stationary phase. However, the increase in adsorption capacity was not a simple linear relationship with the increase of ion-exchange capacity, indicating that the availability of DEAE ligands decreased with the increase of ion-exchange capacity [6]. Ion-exchange chromatography of HBsAg is illustrated in Fig. 5 [4]. The effect of ligand density on the purification efficiency was investigated, showing that a high ligand density resulted in HBsAg’s inactivation, which was caused by strong multisite binding [4]. Reducing the ligand density of the stationary phases appropriately was conducive to maintaining the HBsAg’s structure with an improved activity recovery. When HBsAg is 90

A280

A280 (mAU)

Conductivity 180

60

90

30

0 0

50

25

Conductivity (ms/cm)

270

0 75

Volume (ml)

FIG. 5 The elution profile of ion-exchange chromatography. Conditions: DEAE Sepharose FF column (ligand density: 0.041 mmol/mL, 75 mm  16 mm ID, CV ¼ 15 mL) was used, and 15 mL of desalted HIC active fraction was loaded and eluted stepwise with 9% buffer B (20 mM sodium phosphate, pH 7.0, added 1.0 M NaCl) and 100% buffer B in sequence [4].

75

adsorbed, strong multisite binding occurs between the protein and the stationary phases, which breaks the balance of the forces maintaining the protein’s structure, not only causing the conformational change of the protein, but also promoting its inactivation. The strong multisite binding between HBsAg and the stationary phases is avoided using stationary phases with a low ion-exchange capacity, and HBsAg’s inactivation in the adsorption-desorption process is inhibited to a certain extent. Therefore, the ionexchange capacity of the stationary phases needs to be controlled according to the characteristics of both the target biomolecules and the biosystems to be purified. Strong cation-exchange media with different ion-exchange capacities (0.05–0.24 mmol/mL) based on agarose microspheres were used for purification of recombinant human lactoferrin (rHLF) expressed in breast bioreactor and the effects of ion-exchange capacity were studied [7]. Firstly, the static adsorption equilibrium was studied using lysozyme as the model protein, showing that with an increase in ionexchange capacity, the maximum adsorption capacity (qm) gradually increased, and the dissociation constant (Kd) gradually decreased. Also, the dynamic adsorption of lysozyme showed that the dynamic capacity increased and the breakthrough curve was steep, indicating that the stationary phases had good dynamic adsorption performance. Among them, the stationary phase with the highest ion-exchange capacity had a higher dynamic capacity than the commercially available stationary phases. Secondly, the purification performance for stationary phases with different ion-exchange capacities was investigated. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the rHLF was of high purity (>95% by high-performance size exclusion chromatography) and the recovery over 80%. In addition, with the increase of ionexchange capacity, the maximum purification capacity of rHLF per milliliter of stationary

76

4. Stationary phases for the separation of biopolymers

phases gradually increased, and 1 mL of stationary phases with a highest ion-exchange capacity (0.24 mmol/mL) could purify about 50 mL of milk, which was higher than that of commercially available stationary phases, thus minimizing the process time and improving the purification efficiency.

3 Gigaporous ion-exchange chromatographic media Biomacromolecules such as vaccines, nucleic acids, virus particles, have very high molecular weights, often reaching millions or even tens of millions of Da, and their structures are complex and easily changed. Due to the small pore size of traditional chromatographic media (e.g., several nanometers to tens of nanometers), the mass transfer of the solute is mainly controlled by diffusion during chromatography, which leads to a slow separation speed and easy inactivation of biomacromolecules, and is not conducive to large-scale applications of fast protein chromatography. Gigaporous media have through pores with good permeability, and convective mass transfer can be obtained for biomolecules, thus improving the purification efficiency and compatibility for fast protein chromatography.

3.1 Gigaporous PSt-based ion-exchange chromatographic media Professor Guanghui Ma and her team evaluated gigaporous poly(styrene-divinylbenzene) (PSt) microspheres prepared by a reverse micelles swelling method. The surfactant was added in the oil phase and then dispersed in the water phase to form oil droplets containing surfactant. When the surfactant in the oil phase reached a certain amount, the surfactant absorbed water and swelled, and a two-phase structure was formed in the oil droplets. Next, the suspension polymerization method was used to obtain gigaporous PSt microspheres

with an average pore size of 300–500 nm. The reverse micelles swelling method has a couple of advantages: the pore size of the microspheres is easily controlled, and the method consists of simple and reproducible steps easily scaled up. These gigaporous PSt microspheres have high rigidity for high pressure operation, stable properties, and favorable chromatographic properties. To solve the nonspecific adsorption caused by the strong hydrophobic interaction between the microspheres’ surface and proteins, the hydrophobic surface of gigaporous PSt microspheres should be coated with hydrophilic groups. Professor Guanghui Ma and her team modified gigaporous PSt microspheres using polyvinyl alcohol (PVA) and natural polysaccharide, respectively. The results showed that the reaction with PVA effectively covered the hydrophobic surface of the gigaporous particles. The hydrophilicity of the modified PSt microspheres was improved, and the amount of nonspecific adsorption of BSA decreased from 89.55 mg/g dried microspheres to 7.72 mg/g dried microspheres. To further reduce the nonspecific adsorption of gigaporous PSt microspheres, agarose was physically coated onto the gigaporous particles and subsequently chemically cross-linked. Agarose chains with a natural helical structure provided a more favorable coating of the hydrophobic surface of PSt microspheres than linear PVA molecules, and the nonspecific adsorption of BSA decreased from 89.55 mg/g dried microspheres to 1.18 mg/g dried microspheres. Also, the stability of the hydrophilic coating was improved using agarose modification. For PSt microspheres soaked in 1 M HCl or 1 M NaOH for 24 h at room temperature, no obvious leakage was detected [8]. Using agarose-coated gigaporous PSt microsphere as the matrix, DEAE groups were introduced to provide a weak anion-exchange chromatographic media. By optimizing the reaction conditions, the ion-exchange capacity was suitably controlled, and the maximum ionexchange capacity was obtained for 7.0 M

3 Gigaporous ion-exchange chromatographic media

sodium hydroxide, 2.5 M DEAE
HCl, a reaction time 2 h, and a temperature of 70°C. Scanning electron microscopy (SEM) images showed that the surface of the DEAE gigaporous PSt microspheres was rough, and some macropores with an average pore size over 500 nm were distributed on the surface, while the surface of DEAE agarose-based media was smooth with tiny pores, Fig. 6 [9]. Mercury intrusion porosimetry showed that the average pore size for DEAE gigaporous PSt microspheres was about 300 nm within a range from 100 to 600 nm, Fig. 7 [9]. The effective porosity of this gigaporous media for BSA is 22% higher than that of the commercially available agarose media. The pressure-flow velocity curves showed that the

FIG. 6

77

backpressure was much lower than the commercially available agarose media at the same flow rate, Fig. 8 [9]. For gigaporous PSt microspheres, a good linear relationship between the pressure and the flow rate is observed until the flow rate reached 3612 cm/h, while for the commercially available agarose media, at a flow rate of 1668 cm/h, the backpressure increased sharply, indicating collapse of the column bed. Also, the permeability coefficient of the gigaporous PSt microspheres was 3.34 times higher than that of the commercially available agarose media. Due to its high mechanical strength, low back pressure, and excellent permeability, gigaporous PSt microspheres have great potential for use in fast protein chromatography [9].

SEM images of DEAE-AP (A, 1900; B, 10,000) and DEAE-FF (C, 1900; D, 10,000) microspheres [9].

78

4. Stationary phases for the separation of biopolymers

0.35 Agap-co-PS DEAE-AP

1.5

Flow limit

0.30 Backpressure (MPa)

Log Differential Intrusion[ml/g]

2.0

1.0

0.5

0.0

0.25 0.20 0.15 0.10

Native PS DEAE-AP DEAE-FF

0.05 0.00

–0.5

10

100

0

1000

500

1000 1500 2000 2500 3000 3500

Pore Diameter (nm)

Flow velocity (cm/h)

Comparing the column efficiency of the two stationary phases at different flow rates, the greater plate height (HETP) indicates lower column efficiency. At the lowest flow rate (180 cm/h), the HETP of the DEAE-FF column was 1.24 times that of the DEAE-AP column (gigaporous media). With the increase of flow rate, the gap between the two stationary phases increased. For DEAE-FF column, HETP increased rapidly, indicating that the resistance to mass transfer mainly existed in the interior of the stationary phase, and the resolution

decreased at high flow rates. For the DEAE-AP column, the HETP increased slightly with flow rate, indicating that the convective mass transfer significantly improves the column efficiency. Using BSA as a model protein, the static adsorption of DEAE gigaporous PSt media is shown in Fig. 9A [9]. The equilibrium adsorption capacity of DEAE gigaporous PSt media for BSA was lower than that of DEAE-FF, probably because DEAE gigaporous PSt media had a lower surface area, thus reducing the concentration of binding sites. Fig. 9B illustrates the difference in dynamic

90

Dynamic capacity (mg BSA/ml bed)

FIG. 8 Relationship between the column backpressure and flow velocity. Column, 100 mm  4.6 mm ID; mobile phase, high-purity water [9].

Adsorbed amount of BSA (mg/ml resin)

FIG. 7 Pore size distribution curves of gigaporous Agapco-PS and DEAE-AP microspheres [9].

(A)

80 70 60 50 40 30 20

DEAE-AP DEAE-FF

10 0

0.2

0.4

0.6

0.8

1.0

1.2

Equilibrium of BSA concentration (mg/ml)

1.4

45

(B)

DEAE-AP DEAE-FF

40 35 30 25 20 15 250

500

750

1000 1250 1500 1750 2000

Flow velocity (cm/h)

FIG. 9 (A) The static adsorption isotherms of BSA to DEAE-FF and DEAE-AP microspheres; (B) dynamic binding capacity of BSA on the DEAE-AP and DEAE-FF columns at 10% breakthrough as a function of flow velocity. Column, 100 mm  4.6 mm ID; sample, 2 mg/mL BSA; mobile phase, 20 mM Tris-HCl buffer, pH 8.0 [9].

79

3 Gigaporous ion-exchange chromatographic media

binding capacity for the two stationary phases at different flow rates. The dynamic binding capacity of DEAE gigaporous PSt media for BSA was 1.8–2.1 times higher than that of DEAE-FF, indicating that DEAE gigaporous PSt media could greatly improve the mass transfer efficiency [9]. To investigate efficiency of DEAE gigaporous media in practical applications, the purification

(A1)100

(A2)

(A3) 70

80 TRA

TRA

181cm/h

MYO

80

of myoglobin (MYO), transferrin (TRA), and BSA on a column packed with DEAE gigaporous media at different flow rates was investigated and compared with a commercially available DEAE-FF stationary phase. The resolution of the three proteins on DEAE-FF decreased significantly with an increase in flow rate, Fig. 10 [9]. At a flow rate of 722 cm/h, the

TRA

361cm/h

BSA

40

BSA

40

BSA

40 30 20

20

20

MYO

50

UV signal (mAU)

UV signal (mAU)

UV signal (mAU)

60 60

722cm/h

60

MYO

10 0

0

10

20 30 T (min)

0

40

(B1)

0

20

(B2) MYO

120

361cm/h

MYO

100

722cm/h

UV signal (mAU)

TRA

60 40

TRA

80 60 BSA

40

BSA

UV signal (mAU)

100

80

0 0

10 T (min)

20

5 T (min)

10

0

2600cm/h

MYO

80 TRA

60 BSA

40

20

20

20

0

(B3) 120

100 UV signal (mAU)

10 T (min)

0

0 0

5 T (min)

10

0

1

2

3

T (min)

FIG. 10 Separation of modern proteins on (A) DEAE-FF column and (B) DEAE-AP column. Column, 100 mm  4.6 mm ID; 20 mM Tris-HCl buffer, pH 8.0; injection size, 100 μL; linear gradient, 100% buffer A (20 mM Tris-HCl, pH 8.0) to 50% buffer B (1.0 M NaCl in buffer A) in 30 mL eluate; protein concentrations: MYO 5 mg/mL, TRA 5 mg/mL, BSA 10 mg/mL [9].

80

4. Stationary phases for the separation of biopolymers

peak shape changed, indicating that the column bed had likely collapsed. However, the chromatographic performance of DEAE-AP was not affected by flow rate. With an increase in flow rate, resolution was basically unchanged. This gigaporous media achieved complete protein separation within 3 min at 2600 cm/h, indicating its potential for fast protein chromatography. The static adsorption capacity of ovalbumin, BSA, haptoglobin, thyroglobulin, and HBsAg virus-like particles (HB-VLPs) on DEAE gigaporous PSt media were investigated. For small- or medium-sized proteins (such as ovalbumin and BSA), both DEAE-FF and DEAECapto had high adsorption capacities. For large proteins (such as thyroglobulin and HB-VLPs), the adsorption capacity increased with the pore size, due to the greater accessibility provided by the gigaporous media [10]. As shown in Fig. 11, the adsorption of HB-VLPs to both DEAE-AP120nm and DEAE-AP-280nm was remarkably different to that of DEAE-FF and DEAE-Capto [11]. The adsorption capacity of DEAE-AP-280 nm was about 12.9 times higher than DEAE-FF and 4.5 times higher than DEAE-Capto. The adsorption capacity of DEAE-AP-120 nm

q (mg-protein/ml-media)

6 5 4 3 2

DEAE-FF DEAE-Capto DEAE-AP-120 nm DEAE-AP-280 nm

1 0 0.00

0.02

0.04 0.06 C (mg/ml)

0.08

0.10

FIG. 11 Adsorption isotherms of HB-VLPs on DEAE-FF, DEAE-Capto, DEAE-AP-120 nm, and DEAE-AP-280 nm media at 25°C [11].

was about nine times higher than DEAE-FF. This was probably due to the gigaporous structure of DEAE-AP improving the accessibility of HB-VLPs to binding sites [11]. Compared with DEAE-AP (120 and 280 nm), it took a longer time for HB-VLPs to reach adsorption equilibrium on DEAE-FF and DEAE-Capto. The best-fit values of De for HB-VLPs from the pore diffusion model increased with the pore size of the stationary phases, and DEAE-AP-280 nm had the highest De values, about 11.4 times higher than DEAE-FF, and 6.5 times higher than DEAE-Capto, respectively. CLSM images showed the distribution of FITC labeled HB-VLPs in the four anion-exchange media after a 6 h incubation, Fig. 12 [11]. It was obvious that the adsorption of HB-VLPs to both DEAE-FF and DEAE-Capto was basically limited to the outer surface of the stationary phases because the pore size of the stationary phases was similar to the particle size of HB-VLPs, and the adsorbed layer blocked their pores making the binding sites in the inner pores inaccessible to more HB-VLPs. However, HB-VLPs were evenly distributed over the gigaporous media, especially for DEAE-AP-280 nm, indicating that their pore sizes were large enough for efficient mass transfer [11]. The effect of loading amounts on antigen recovery of HB-VLPs eluted from four stationary phases is shown in Fig. 13 [11]. The recovery of HB-VLPs from DEAE-FF and DEAE-Capto decreased at higher loading. The recoveries from DEAE-AP-120 nm and DEAE-AP-280 nm increased gradually. The eluted fractions were analyzed by HPSEC as shown in Fig. 14 [11]. Two peaks were observed, i.e., peak A at 14 min and peak B at 22 min. The area of Peak A increased with an increase of the stationary phases’ pore size. HPSEC of the standard HB-VLPs had mainly a single peak at 14 min and a very small peak at 22 min. Transmission electron microscopy (TEM) showed that the image of peak A was similar to the standard for HB-VLPs, with a diameter of about

3 Gigaporous ion-exchange chromatographic media

81

FIG. 12 Representative equatorial CLSM images of the media (top) and the corresponding intraparticle fluorescence intensity profiles (bottom) for the four anion-exchange media after being incubated with 0.282 mg/mL FITC-HB-VLPs for 6 h. Media and particle diameters are as follows: (A) DEAE-FF, 97 μm, (B) DEAE-Capto, 105 μm, (C) DEAE-AP-120 nm, 107 μm, (D) DEAE-AP-280 nm, 92 μm [11].

80

Loading quantities (mg protein/ml-media):

Antigen recovery from ELISA (%)

70

0.5

1.0

1.5

2.0

60 50 40 30 20 10 0 D EA

E-FF

D EA

E-Ca

pto

D EA

E-AP

D EA

-120n

m

E-AP

-280n

m

FIG. 13 Effect of loading quantities (0.5, 1.0, 1.5, and 2.0 mg protein/mL-media) on ELISA-recovery of HB-VLPs eluted from four anion-exchange media. Conditions: a chromatographic column (50 mm  10 mm ID) packed with each of the anion-exchange media, including DEAE-FF, DEAECapto, DEAE-AP-120 nm, and DEAE-AP-280 nm, was loaded with 25, 50, 75, 100 mL HB-VLPs (0.08 mg/mL) solutions at a flow rate of 80 cm/h to yield a protein loading quantity of 0.5, 1.0, 1.5, and 2.0 mg protein/mL-media, respectively. Then the adsorbed HB-VLPs were eluted with 1.0 M NaCl in pH 7.0, 20 mM sodium phosphate buffer, the elution peak was polled, and the antigen was detected by ELISA [11].

20–30 nm, confirming that it was the normal assembly of HB-VLPs. Peak B was the disassembly between 5 and 10 nm. The percentage of HB-VLPs content in the elution was defined as the ratio of the area of peak A to the sum of the areas of peak A and peak B, and at higher loadings, the percentage of HB-VLPs normal assembly gradually increased for the gigaporous media, while it decreased for the conventional agarose media, consistent with the recovery of HB-VLPs. HB-VLPs adsorption and disassembly during the desorption process in agarose or gigaporous media are explained as follows. When HB-VLPs were adsorbed on DEAE-FF and DEAE-Capto, additional multisite binding occurred on the surface of the stationary phases and induced their irreversible disassembly, resulting in inactivation. On the contrary, when HB-VLPs were adsorbed on gigaporous media with a pore size was about 10 times larger, the multisite binding was reduced, and most of the disassembly avoided, leading to a higher recovery of the normal assembly.

82

4. Stationary phases for the separation of biopolymers

FIG. 14 (A) HPSEC assay of HB-VLPs eluted by 1.0 M NaCl in 20mM sodium phosphate buffer (pH 7.0) from DEAE-FF, DEAECapto, DEAE-AP-120 nm, and DEAE-AP-280 nm at the same loading quantity (2.0 mg/mL). The HPSEC profile of HB-VLPs standard was also compared. (B) TEM image of purified HB-VLPs standard. Bar, 100 nm; (C) TEM image of HP-VLPs in Peak A after HPSEC assay. Bar, 100 nm; (D) TEM image of HP-VLPs in Peak B after HPSEC assay. Bar, 50 nm [11].

3.2 Gigaporous PGMA-based ionexchange chromatographic media Polyacrylate is another common matrix for chromatographic media with good pressure resistance, good stability and high flow rate capability. After chemical modification, it is frequently employed for protein purification. Professor Guanghui Ma and her team prepared gigaporous PGMA microspheres using the

reverse micelles swelling technology to verify if this method was suitable for different monomer systems. Gigaporous PGMA microspheres with an average pore size of about 450 nm were prepared using Span 80 as the surfactant, a mixture of isooctane and 4-methyl-2-pentanol as the diluent, and DVB as the cross-linking agent, Figs. 15 and 16 [12,13]. Gigaporous PGMA microspheres were successfully modified with polyethylenimine

3 Gigaporous ion-exchange chromatographic media

83

FIG. 15 Effect of the co-operation of surfactant and diluents on the morphology of particles (cross-linking agent DVB, concentration of cross-linking agent 27.5%) [12].

Log Differential Intrusion / (mL/g)

2.2 2.0

IO:MP=3:2

1.8

180 nm 450 nm

IO:MP=1:1

1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 –0.2 1

10

100

1000

Pore Size Diameter/nm

FIG. 16

Effect of different ratio of the mixed solvents on the pore size distribution (the samples of Fig. 15C and D) [12].

84

4. Stationary phases for the separation of biopolymers

(PEI) to obtain anion-exchange media. PEI of various molecular weights and different branching agents was used for the modification. The ion-exchange capacity increased for the branched generations in the range of 0.11–0.32 mmol/mL. The dynamic binding capacity of BSA increased at first and then decreased, influenced by the PEI coating on the internal structure of the modified microspheres. The dynamic binding capacity of BSA on the PEI-modified gigaporous PGMA microspheres at a flow rate of 2800 cm/h (22 mg/mL) was slightly lower than at 361 cm/h (26 mg/mL), suggesting that it did not change significantly with the increase in flow rate. It showed good performance for the separation of three model proteins, even at a high flow rate of 1084 cm/h, Fig. 17 [14].

3.3 DEAE macroporous agarose chromatographic media Professor Guanghui Ma and her team prepared macroporous agarose microspheres using a micelles swelling method and double emulsification method, respectively. For FIG. 17 Separation of model proteins on PGMA-DVB-PEI600 three at different flow rates, the chromatographic peaks for lysozyme, BSA, and ovalbumin, in the order of left-to-right [14].

macroporous agarose microspheres prepared by micelles swelling method, the transparency of the microspheres was poor with numerous dark spots on the surface, and obvious macropores distributed on the surface [15]. According to inverse size exclusion chromatography, the pore size distribution was more than 40 nm, even close to 60 nm for macroporous agarose microspheres, while the pore size was no more than 40 nm for agarose microspheres prepared using conventional methods. The pore structures were basically the same before and after cross-linking [15]. For macroporous agarose microspheres prepared by double emulsification method, there was also many dark spots, and large pores up to several micrometers at the surface [16]. DEAE macroporous agarose media were prepared from two types of macroporous agarose microspheres. CLSM was used to study the distribution of FITC-labeled HBsAg in these microspheres during the static adsorption process, Fig. 18 [17]. Both conventional DEAE agarose media and commercial DEAE agarose media were used for comparison. A concentration of 4 wt% agarose solution was used for both the macroporous agarose

3 Gigaporous ion-exchange chromatographic media

85

FIG. 18 CLSM pattern of DEAE agarose-based microspheres incubated with HBsAg after 5 s (top) and 60 min (bottom), respectively. The matrix of media are as follows. (A) Conventional microspheres, (B) macroporous microspheres prepared by micelles swelling method, (C) macroporous microspheres prepared by double emulsion method, (D) commercial media [17].

media and conventional agarose microspheres, while a concentration of 6 wt% agarose solution was used for the commercial media. After 5 min, FITC-HBsAg was distributed on the outer surface of the microspheres, indicating that HBsAg was mainly bound to surface DEAE groups and had yet to enter the internal structure. After 60 min, FITC-HBsAg had almost completely entered the internal structure of the macroporous agarose microspheres, while for other microspheres, FITC-HBsAg was still only bound to the surface and could not enter the interior. The lower the agarose concentration the larger was the pore size. Compared with the commercial media, more HBsAg entered the conventional microspheres. The laser signal distribution of different microsphere profiles was compared. After 60 min, the signal intensity for FITC-HBsAg distributed in the inner part of the macroporous agarose microsphere was nearly 10 times higher than

for the commercially available stationary phases, indicating that its binding efficiency was significantly improved under these conditions. This was because the macroporous agarose microspheres had larger pores, and HBsAg was subjected to lower diffusion resistance when entering its interior, and hence bound faster and more fully.

3.4 CM macroporous agarose chromatographic media Professor Guanghui Ma and her team proposed a combined method of precross-linking and micelles swelling to prepare highly crosslinked macroporous agarose microspheres (HMA), followed by reaction with carboxymethyl (CM) groups to prepare cation-exchange media [18]. The maximum flow velocity of HMA was much higher than that of Sepharose 4FF and higher than that of other highly cross-linked

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agarose microspheres (HA). Moreover, the column pressure of HMA was lower than that of other stationary phases at the same mobile phase flow velocity, largely dependent on the macropores which reduced the flow resistance allowing the column to be operated at a high flow velocity. The plate height for the Sepharose 4FF column increased significantly at higher flow velocities, typical for interior diffusioncontrolled chromatography. Comparatively, the plate height for the HMA column increased only slightly with flow velocities up to 1200 cm/h, demonstrating accelerated intraparticle mass transfer leading to a high column efficiency. Static adsorption studies showed that the lysozyme adsorption capacity of 174 mg/mL for CM-HMA was 47% higher than that of CM-4FF, probably because the macropores in HMA provided more channels for protein transport and the available surface area for protein binding was enhanced. The adsorption kinetics of FITC-IgG in CM-HMA was studied by CLSM

FIG. 19

and compared CM-4FF, Fig. 19 [18]. CM-4FF fluorescence increased gradually from the outer surface to the interior. However, it was still concentrated on the particle surface even after adsorption of FITC-IgG for 60 min, mainly caused by the mass transfer limitation of IgG inside the media. Fluorescence for CM-HMA was visible within the whole particle after only 5 min and was evenly distributed from the outer layer to its core after 60 min, facilitating the mass transfer of biomacromolecules inside the media. The separation of a mixture of lysozyme and IgG on both CM-HMA and CM-4FF at different flow velocities is shown in Fig. 20 [18]. For CM-HMA, a satisfactory separation of the two proteins was obtained at flow velocities up to 1146 cm/h within 2 min, and the elution profile was not affected by the increase in the flow velocity, mainly due to its improved mechanical strength and enhanced mass transfer. However, for CM-4FF, protein resolution decreased significantly with an increase of the flow velocity. They

Dynamic adsorption process of IgG on (A) CM-4FF and (B) CM-HMA observed by CLSM [18].

4 Other ion-exchange stationary phases for bioseparations

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FIG. 20

The effect of flow velocity on the ion-exchange chromatographic performance of CM-HMA (A–C) and CM-4FF (D, E) columns. Conditions: column size, 160  10 mm ID (2 mL packed bed); equilibration buffer, 20 mmol/L. HAc-NaAc, pH 4.6; elution buffer, 20 mmol/L HAc-NaAc, 1.0 M NaCl, pH 4.6 [18].

cannot be separated completely at 382 cm/h, indicating that the column may have already collapsed at this flow velocity [18].

4 Other ion-exchange stationary phases for bioseparations 4.1 Monolithic columns Most monolithic columns used for bioseparation are obtained by polymerization of styrene or acrylate monomers. The monomer is polymerized in the column, forming porous polymer globules by suspension polymerization with a macroporous interconnected structure. Generally, in the presence of a porogen, monomers and cross-linkers are polymerized by heat or ultraviolet light. The amount of initiator, types and amounts of monomers, cross-linkers, and porogen, temperature, and reaction time all play

an important role in establishing the pore structure [19]. There are large differences in the structure of monolithic columns prepared by different techniques [19]. Macropores with a size of a few micrometers or more are formed in monolithic cryogels. Aligned macroporous channels with a size of approximately 10 μm are formed by freeze casting. There are several types of monolithic columns for ion-exchange chromatography such as PGMA monoliths. This type of stationary phase has advantages for fast bioseparations due to the presence of a high concentration of macropores, which improve the diffusion of the solutes within the column at high flow rates. The pore structure of the monolithic columns is optimized, such as porosity and the ratio of large and small pores. On the one hand, the effective surface area of the column is maintained to the extent possible while on the other hand, the high flow rate desired for the purification process is achieved. Therefore,

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its binding capacity and resolution are not affected by the flow rate, leading to a fast process. Ion-exchange monolithic columns are widely used for purification and analysis of various biosystems, e.g., peptides, antibodies, viruses, VLPs, oligonucleotides, and polynucleotides like plasmid DNA and RNA, indicating their potential for bioprocess control and quality control of final bioproducts [20].

4.2 Membrane chromatography The stationary phases for membrane chromatography are membranes whose physical and chemical properties play an important role. The key characteristics include the effective surface area, pore size and its distribution, and ligand type and charge, which not only influence the separation process but also column loading and elution properties of proteins. Polymer is the common membrane matrix, such as regenerated cellulose, nylon, polyethersulfone, polysulfone, polypropylene, and polyvinylidene fluoride. A series of key factors should be considered for the selection of the membrane matrix, e.g., pore structure, mechanical strength, lifetime and reusability, and nonspecific adsorption. As the binding of biomolecules to the membrane relates to the charge of the ligand, it is important for membrane modification to select appropriate systems. Common methods such as physical coating, chemical treatment, plasma treatment, self-assembly, layer-by-layer assembly, and ligand-grafting have been used for membrane modification. Membrane morphology include plate, folding, hollow fiber, and roll configurations. Based on the flow characteristics, they can be classified as: radial, axial, laterally-fed, and z2 laterally-fed membrane modules. Advantages of ion-exchange membrane chromatography include favorable diffusion properties, short diffusion times, reduced denaturation of biomolecules, and favorable economics, with obvious advantages for the fast purification of biomolecules, such as peptides,

monoclonal antibodies, serum proteins, DNA, and viruses [21,22].

4.3 Cryogels Cryogel is a porous polymer material prepared by freezing or low-temperature treatment. Both synthetic and natural polymers are used for preparing cryogels by various techniques, e.g., free-radical polymerization, free-radical cross-linking copolymerization via irradiation, graft polymerization with ionexchange groups, physical gel formation, and chemical gel formation, etc. The characteristics of cryogels are influenced by several factors including the polymer molecular weight, freezing rate, freezing-thawing cycles, and thawing rate, etc. Cryogels have several advantages such as an interconnected macroporous structure, good mechanical strength and stability, and biocompatibility. Macroporous cryogels with ionexchange sites have been used for the separation of enzymes, glycoproteins, antibodies, and DNA. They are suitable for rapid purification process displaying a high adsorption capacity with retention of bioactivity [23].

4.4 Mixed-mode chromatography Mixed-mode chromatography adopts a variety of interaction mechanisms to achieve the purification of biosystems, with ion-exchange and hydrophobic interactions the most common. The salt tolerance of these stationary phases has obvious advantages over traditional ion-exchange separations. They solve the problem of high conductivity loading that ionexchange chromatography cannot achieve, and thus can adapt to the high salt conditions of cell cultures. At the same time, multiple interactions between the stationary phase and target biomolecules provide additional possibilities in the design of purification methods. Mixed-mode stationary phases are

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References

also effective for the purification of complex samples both in the detection and removal of trace impurities, leading to a high recovery with an improved throughput. Typical stationary phases can be classified into four groups. Type I is obtained by mixing different types of particulate chromatographic media each with a single chemistry followed by packing them into a column. Type II media is obtained by modifying the surface with a mixture of ligands having different functionalities. Type III and Type IV are obtained using ligands that contain ion-exchange groups as part of the hydrophobic ligand. Type III media have charged functional groups close to the surface with hydrophobic chains extending into the mobile phase, while for Type IV media ionexchange groups are located at the free terminus of the hydrophobic chain. Both silica gel and polymers are used as the stationary phase matrix. Different structures are utilized in the design of mixed-mode stationary phases to provide more opportunities for interactions, e.g., functionalized nanomaterials, pyridinyl-based ligands, and co-polymers. Commercially available stationary phases include Capto MMC, Capto adhere, Capto Core from Cytiva; PPA Hypercel, HEA Hypercel and MEP Hypercel from Pall; Eshmuno HCX from Merck Millipore; and Toyopearl MX-Trp-650M from TOSOH. The ligand type and the pore structure of the matrix are key elements for understanding their chromatographic properties, e.g., binding capacity, adsorption kinetics, adsorption selectivity, etc. Mixed-mode chromatography is an excellent choice for overcoming difficulties in capture, intermediate steps, and polishing steps in the purification of biomolecules, e.g., monoclonal antibodies, antibody fragments, peptides, recombinant proteins, and oligonucleotides. These media have outstanding advantages including rich ligand types providing high selectivity, favorable cost and ligand stability, and a wide choice of operating conditions. With the development of

novel ligands and matrices, mixed-mode stationary phases will likely acquire more applications in both the purification and analysis of biomolecules [24,25].

5 Summary and outlook As one of the most effective methods for purification of biomolecules, ion-exchange chromatography is often selected for the downstream processing of biotechnology products due to its mild interactions, wide application, excellent processing capacity, and suitable resolution. Results are closely associated with the structures, functions, and properties of the ionexchange media. Therefore, the reasonable design and regulation of such key factors as particle size and its distribution, pore structure, ionexchange capacity, and pressure resistance are important factors for efficient bioseparations. Also, the stationary phase cost, loading capacity, and solvent consumption are of equal importance in large-scale purifications. For biomolecules with complex structures, it remains a challenge to simultaneously obtain a high accessible surface area, high resolution, and short process times in the search for new ion-exchange media.

References [1] Q.Z. Zhou, L.Y. Wang, G.H. Ma, Z.G. Su, Preparation of uniform-sized agarose beads by microporous membrane emulsification technique, J. Colloid Interface Sci. 311 (1) (2007) 118–127. [2] Q.Z. Zhou, Preparation and Application of UniformSized Controllable Agarose Gel Beads, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 2008. [3] X. Zhao, Preparation of High-Performance Agarose Microsphere for Potential Bioseparation Media, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 2014. [4] Y.D. Huang, J.X. Bi, L. Zhao, G.H. Ma, Z.G. Su, Regulation of protein multipoint adsorption on ion-exchange adsorbent and its application to the purification of macromolecules, Protein Expr. Purif. 74 (2) (2010) 257–263.

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[5] Y.D. Huang, J.X. Bi, Y. Zhang, W.B. Zhou, Y. Li, L. Zhao, Z.G. Su, A highly efficient integrated chromatographic procedure for the purification of recombinant hepatitis B surface antigen from Hansenula polymorpha, Protein Expr. Purif. 56 (2) (2007) 301–310. [6] Y.D. Huang, Protein Stability and Chromatography Process Optimization of Multi-Subunit Vaccines, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 2008. [7] Q. Bai, Y. Zhang, Y.J. Wang, J. Luo, Y. Li, Y.D. Huang, R.Y. Ma, Z.G. Su, Purification and characterization of recombinant human lactoferrin expressed in a cattle mammary bioreactor, Chin. J. Biotechnol. 26 (11) (2010) 1576–1583. [8] J.B. Qu, Modification of Gigaporous Polystyrene Microspheres for Potential Bioseparation Media, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 2009. [9] J.B. Qu, X.Z. Wan, Y.Q. Zhai, W.Q. Zhou, Z.G. Su, G.H. Ma, A novel stationary phase derivatized from hydrophilic gigaporous polystyrene-based microspheres for high-speed protein chromatography, J. Chromatogr. A 1216 (2009) 6511–6516. [10] M.R. Yu, Study of Gigaporous Chromatographic Process of Virus-like Particles Purification, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 2015. [11] M.R. Yu, Y. Li, S.P. Zhang, X.N. Li, Y.L. Yang, Y. Chen, G.H. Ma, Z.G. Su, Improving stability of viruslike particles by ion-exchange chromatographic supports with large pore size: advantages of gigaporous media beyond enhanced binding capacity, J. Chromatogr. A 1331 (2014) 69–79. [12] W.Q. Zhou, T.Y. Gu, Z.G. Su, G.H. Ma, Synthesis of macroporous poly(glycidyl methacrylate) microspheres by surfactant reverse micelles swelling method, Eur. Polym. J. 43 (2007) 4493–4502. [13] W.Q. Zhou, Study on Preparation and Characterization of Gigaporous Polymeric Microspheres by Novel Surfactant Reverse Micelles Swelling Method, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 2007. [14] R.Y. Zhang, Q. Li, Y. Gao, J. Li, Y.D. Huang, C. Song, W.Q. Zhou, G.H. Ma, Z.G. Su, Hydrophilic modification gigaporous resins with poly(ethylenimine) for highthroughput proteins ion-exchange chromatography, J. Chromatogr. A 1343 (2014) 109–118.

[15] L. Zhao, L. Huang, Y.D. Huang, K. Zhu, X.J. Che, Y.X. Du, J.W. Gao, D.X. Hao, R.Y. Zhang, Q.B. Wang, G.H. Ma, Preparation and structural regulation of macroporous agarose microspheres for highly efficient adsorption of giant biomolecules, Colloid Polym. Sci. 300 (2022) 691–705. [16] L. Huang, Y.D. Huang, L. Zhao, K. Zhu, X.X. Wu, D.N. Zhou, Z.G. Su, G.H. Ma, Q.B. Wang, Preparation of macroporous agarose-based chromatographic media using a double emulsification method and its binding with HBsAg, Chem Ind Eng 36 (4) (2019) 70–79. [17] L. Huang, Porosity Research and Performance Characterization of Macroporous Agarose Chromatography Medium, China University of Mining & Technology, Beijing, 2019. [18] X. Zhao, L. Huang, J. Wu, Y.D. Huang, L. Zhao, N. Wu, W.Q. Zhou, D.X. Hao, G.H. Ma, Z.G. Su, Fabrication of rigid and macroporous agarose microspheres by precross-linking and surfactant micelles swelling method, Colloids Surf. B: Biointerfaces 182 (2019) 110377. [19] S. Eeltink, S. Wouters, J.L. Dores-Sousa, F. Svec, Advances in organic polymer-based monolithic column technology for high-resolution liquid chromatographymass spectrometry profiling of antibodies, intact proteins, oligonucleotides, and peptides, J. Chromatogr. A 1498 (2017) 8–21. [20] A. Podgornik, S. Yamamoto, M. Peterka, N.L. Krajnc, Fast separation of large biomolecules using short monolithic columns, J. Chromatogr. B 927 (2013) 80–89. [21] A. Nath, M.M. Zin, M.A. Molna´r, S. Ba´nv€ olgyi, I. Ga´spa´r, G. Vatai, A. Koris, Membrane chromatography and fractionation of proteins from whey—a review, Processes 10 (2022) 1025. [22] C. Boi, S. Dimartino, Advances in membrane chromatography for the capture step of monoclonal antibodies, Curr. Org. Chem. 21 (2017) 1753–1759. [23] N. Babanejad, K. Mfoafo, E. Zhang, Y. Omidi, R. Razeghifard, H. Omidian, Applications of cryostructures in the chromatographic separation of biomacromolecules, J. Chromatogr. A 1683 (2022) 463546. [24] K. Zhang, X. Liu, Reprint of “mixed-mode chromatography in pharmaceutical and biopharmaceutical applications”, J. Pharm. Biomed. Anal. 130 (2016) 19–34. [25] V. Halan, S. Maity, R. Bhambure, A.S. Rathore, Multimodal chromatography for purification of biotherapeutics—a review, Curr. Protein Pept. Sci. 20 (2019) 4–13.

C H A P T E R

5 Ion-exchange separations of biomacromolecules on grafted and surface-modified polymers Linling Yu Department of Biochemical Engineering, School of Chemical Engineering and Technology and Key Laboratory of Systems Bioengineering and Frontiers Science Center for Synthetic Biology (Ministry of Education), Tianjin University, Tianjin, China O U T L I N E 1. Introduction

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2. Stationary phases 2.1 Design of polymer-functionalized ion exchangers 2.2 Introduction of the surface polyelectrolytes and their modification 2.3 Typical commercial stationary phases

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3. Adsorption and uptake theory

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4. Applications 4.1 Features of practical applications 4.2 Application examples

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1 Introduction

products, as is widely acknowledged in the biopharmaceutical industry [1,2]. Thus, to increase the productivity of IEC has long been a key issue for industrial-scale downstream processing of biomacromolecules, including recombinant therapeutic proteins, antibodies, and vaccines, which have become one of the largest markets for the pharmaceutical industry.

Although ion-exchange chromatography (IEC) has successfully dominated the industrialscale downstream processing of many biological products, the limited productivity of chromatography operation units remains the bottleneck in the large-scale production of biopharmaceutical

Ion-Exchange Chromatography and Related Techniques https://doi.org/10.1016/B978-0-443-15369-3.00004-3

3.1 Three-dimensional adsorption 3.2 Facilitated mass transfer by chain delivery effect

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Copyright # 2024 Elsevier Inc. All rights reserved.

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The stationary phase (ion exchanger) is the core of IEC technology and consists of two structural elements. The ion-exchange group is involved in the exchange process and interacts directly with the target biomacromolecules and the matrix is where the ion-exchange groups are attached. Both components affect the IEC performance and represent the key to understanding the productivity of the chromatographic process, which is reflected in the dynamic binding capacity (DBC) determined by both the equilibrium adsorption capacity and the mass transfer rate. Consequently, various strategies have been devised to improve the adsorption capacity and uptake kinetic properties of the stationary phase. For instance, a wide variety of matrix chemistry, including silica-based [3,4], cellulose-based [5–9], agarose-based [10,11], dextran-based [12,13], and composite-based [14] types have been developed. Additionally, a wide variety of matrix morphology, including nonporous beads [15], porous beads [5–9,16,17], membranes [18–21], and monoliths [22–24], have been fabricated. With regard to the ion-exchange groups, many variations have been evaluated for IEC separations of biomacromolecules as well [25].

Generally, ion-exchange groups are immobilized on the matrix surface by coupling with short spacers, common in traditional ion exchangers, Fig. 1A. Increasingly, ion exchangers with ionexchange groups on surface extenders (grafted and surface-modified polymer chains formed by grafting reactions) have attracted attention due to their greatly enhanced productivity for biomacromolecules compared with traditional ion exchangers. This new type of ion exchanger is called a polymer-functionalized ion exchanger, Fig. 1B. Thereby, ion-exchange groups directly located on the matrix surface are denoted as surface ion-exchange groups or surface ligand, and ion-exchange groups located on the surface extenders are denoted as polymeric ion-exchange groups or polymeric ligands. Since M€ uller reported the firstly polymerfunctionalized ion exchangers, the Fractogel series (Merck Millipore, Darmstadt, Germany) in 1990 [26] with a greatly improved adsorption performances, also called tentacle-type ion exchangers, an increasing number of IEC stationary phases with polymeric ion-exchange groups have been developed, including Sepharose XL, Streamline XL, and Capto series (Cytiva,

FIG. 1 Schematic illustration of the distribution of ion-exchange groups on IEC stationary phase. (A) Nongrafting IEC media and (B) polymer-functionalized IEC media. The quantity of polymer chains and ion-exchange groups in this figure do not represent their real densities on the pore surface. The size of the polymer chains, spacers, and ion-exchange groups do not represent their real sizes.

2 Stationary phases

formerly GE Healthcare Life Sciences and Pharmacia, Uppsala, Sweden), Bestarose XL and Mustang XL series (Bestchrom (Shanghai) Biosciences, Shanghai, China), Nuvia series (Bio-Rad, Hercules, CA, United States), Toyopearl GigaCap series (Tosoh Bioscience, Montgomeryville, PA, United States), and Eshmuno series (Merck Millipore, Darmstadt, Germany), as well as many lab-made polymer-functionalized ion exchangers [27–32] in the last 30 years. Thereafter, the polymerfunctionalized ion exchangers have dominated the market due to their more competitive chromatographic performance compared with traditional nongrafting ion exchangers, including high adsorption capacities [33–36], fast mass transfer rates [37–43], wide binding conditions [44–46], flexible elution conditions [47–50], etc., summarized in earlier reviews [51–53]. Therefore, this chapter will focus on ion exchangers with polymeric ion-exchange groups. Firstly, the design and fabrication methods will be summarized for typical commercial stationary phases. Then, the possible mechanisms of their competitive chromatographic performances will be discussed, especially their enhanced adsorption capacity and accelerated uptake kinetics. Finally, special considerations for their practical application in IEC separation process of biomacromolecules and some laboratory-level and industrial-level examples will be provided highlighting their advantages, including higher productivity and flexible operation capability, together with their challenges and coping strategies.

2 Stationary phases 2.1 Design of polymer-functionalized ion exchangers For an IEC stationary phase, both matrix and the coupled ion-exchange groups affect performance. The amount and nature (including polarity, electronegativity, and dissociation

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constant, etc.) of the ion-exchange groups determine the charge density (ionic capacity), and thus the specificity and binding strength of the biomacromolecules. Meanwhile, the physical and chemical nature of the matrix determines the attainable number of ion-exchange groups, stability, and flow characteristics of the ion exchanger. Especially, the three-dimensional (3D) structure of the matrix (particle porosity, pore size, and particle size distribution) determines the accessibility of ion-exchange groups to the biomacromolecules. In other words, the available binding sites of an IEC stationary phase are mainly dependent on the matrix structure and the ligand chemistry (properties of the ion-exchange groups). For traditional ion exchangers, the incorporation of ion-exchange groups onto the matrix surface has little impact on the 3D structure of the matrix nor the ion exchanger. However, the ion-exchange groups in polymer-functionalized ion exchangers are generated by the grafting/ modification of the surface extenders (polymer chains) with charged groups onto the matrix surface, and seriously affected the 3D structure of the ion exchangers, including the partial or total occupation of the pore volume and an increase in the particle size. Thus, the design of polymer-functionalized IEC media is more complicated than for traditional ion exchangers. The basic principle for their design is summarized as follows: (1) For the porous ion exchangers, the matrix should be macroporous, superporous, or gigaporous, allowing the polymer chains to enter the inner pores or to be in situ synthesized by the polymerization reaction. Then the polymer chains would form a grafting layer and occupy the pore space in the grafting reaction. Hence, the polymer chains should not be too big/long to enter the pores. More importantly, the decrease in the pore size of the matrix by the introduction of surface extenders (the

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thickness of the formed grafting layer) should be acceptable for the target biomacromolecules and blocking of the pores avoided. That is, the effective porosity of the porous ion exchangers for the target biomacromolecules should not be seriously affected after grafting, ensuring the number of available binding sites for the target biomacromolecules is preserved. Hence, the density of the surface extenders (grafting density) should be well controlled. Besides, surface extenders of extremely low grafting density are not sufficient to form an extended 3D structure for the grafting layer. Therefore, the thickness of the grafting layer should be adapted to the size of the target biomacromolecules, which is determined by the grafting density and the length of the polymer chains. (2) For nonporous ion exchangers, all the polymer chains are located on the outside surface of the matrix and the polymer chain length is not severely restricted. However, the grafting layer should not be too thick, since long polymer chains tend to collapse and have a strong impact on the flow characteristics of the ion exchangers. (3) Considering part or all the ion-exchange groups are located on the polymer chains, the charge density of the polymer chains is of great importance. It not only affects the 3D structure, including the thickness of the grafting layer, but also the ion-exchange behavior of the biomacromolecules, including the binding and elution steps. Additionally, the activity of the biomacromolecules is seriously influenced by their interactions with the charged polymer chains (polyelectrolytes). Polyelectrolytes of extremely high charge density should be avoided, as they generally form a rigid and extended grafting layer, block pores, and exhibit extremely strong binding of biomacromolecules. Thus, the recovery of target biomacromolecules (both

yield and activity) will be significantly reduced. In addition, polyelectrolytes of extremely low charge density should be avoided as they typically lead to collapse of the polymer layer and fail in the formation of a suitable 3D architecture. In short, both the matrix and the surface polyelectrolytes play important roles in the design of polymer-functionalized IEC media, and the two key structural elements and their complex interplay should be given sufficient consideration, as well as in the introduction process of the surface polyelectrolytes. Thus, the modification approach of the ready-coupling polymer chains onto the matrix surface and polymerization reactions for the in situ synthesis of polymer chains will be summarized in the following subsection.

2.2 Introduction of the surface polyelectrolytes and their modification There are two main approaches for the fabrication of polymer-functionalized IEC media. The surface-modification of the ready-coupling polymer chains, by which a ready-coupling polymer is directly attached to the preactivated matrix surface through a grafting reaction, called the “grafting to” approach (Fig. 2A and B). The second approach is the in situ synthesis and grafting of the polymer chains, in which the low-molecular-weight monomers “grow” into long polymer chains at single sites on the matrix surface, called a “grafting from” approach (Fig. 2C and D). For electroneutral polymer chains, a second modification is required to generate the ion-exchange groups, also called the functionalization procedure, to provide binding sites for the biomacromolecules (Fig. 2A and C). Besides, some modifications of these two approaches have been described to adjust the charge density and the distribution of ion-exchange groups (Fig. 2E–H). Thus, as shown in Fig. 2, different introduction routes for the surface

FIG. 2 Schematic illustration of different introduction routes for surface polyelectrolytes in the preparation of polymerfunctionalized ion exchangers. (A) “Grafting to” with electroneutral polymers; (B) “grafting to” with charged polymers; (C) “grafting from” with electroneutral monomers; (D) “grafting from” with charged monomers; (E) modification on “grafting to” polymers; (F) modification on “grafting from” polymers; (G) “grafting from” by copolymerization of different monomers; and (H) “grafting to” on the charged surface of a traditional ion exchanger. The size and quantity of polymer chains, spacers, and charged groups, as well the modified groups, do not represent their real sizes and densities. Reproduction from L. Yu, Y. Sun, Recent advances in protein chromatography with polymer-grafted media, J. Chromatogr. A 1638 (2021) 461865 with permission.

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polyelectrolytes result in different structure and charge characteristics for the polyelectrolyte chains, as well as in different types of ion exchangers, typically classified into three types (Type I, Type II, and Type III). For the “grafting to” approach, when a readycoupling electroneutral polymer is used, for instance, natural dextran [30,37–40,54–56], an activating treatment of the media (both the matrix and the grafted polymer) is usually needed for coupling the charged groups in the second modification procedure, such as the epoxidation [56] or allylation [54]. Hence, randomly distributed ion-exchange groups in both the grafted polymer chains and on the matrix surface are generated. This type of “grafting to” ion exchangers is denoted as Type I (Fig. 2A). When a ready-coupling polyelectrolyte is used in the “grafting to” approach, such as poly(ethylenimine) [57–60] or alginate [61], the fabrication procedure is much simpler, and only a grafting procedure is required. Consequently, all the ion-exchange groups are located in grafted polymer chains only, and this type of “grafting to” ion exchanger is denoted as Type II (Fig. 2B). For the “grafting from” approach, when an electroneutral monomer is used, for example, glycidyl methacrylate [62,63], the second modification for the introduction of charged groups follows the in situ polymerization reaction. There is generally no need for any activation procedure, due to the modifiable groups that typically exist in the electroneutral monomers, which can react with the epoxy groups in glycidyl methacrylate [62,63]. Thus, unlike the Type I ion exchangers, all the ion-exchange groups are in the grafted polymer chains, and this type of “grafting from” ion exchanger is denoted as Type III (Fig. 2C). When a charged monomer is used, it is like the Type II ion exchangers that only one in situ polymerization reaction (grafting procedure) is required. Thus, this type of “grafting from” ion exchangers also belongs to Type III.

The charge density of the polymer chains is of great importance in the polymer-functionalized IEC media, and a suitable charge density is required to obtain a high chromatographic performance. The charge density of the surface polyelectrolytes in Type I ion exchangers can be easily regulated by the activating treatment in the second modification procedure. However, the charge density of the surface polyelectrolytes in Type II and Type III ion exchangers is a fixed value, determined by the chemical nature of the ready-coupling polyelectrolyte and monomer, respectively. Therefore, in order to adjust the charge density of the polyelectrolyte extenders, further modification procedures including an activating treatment for the Type II and Type III ion exchangers are needed (Fig. 2E and F), and sometimes the conversion of the type of polymer-functionalized IEC media would happen by altering the distribution of ion-exchange groups (Fig. 2E, Type II turns to Type I). In addition, use of different monomers in the in situ polymerization reaction (Fig. 2G) and introduction of polyelectrolytes onto ion exchangers (charged surface, Fig. 2H) represent alternative routes to regulate the charge density and/or the distribution of ion-exchange groups. Moreover, more than one modification step and strategy can be used to adjust the structural and chemical properties of the surface polyelectrolytes. Therefore, charge reduction [64,65], charge increase [66,67], charge reversal [68–71], control of the charge distribution [72,73], and branching increase [74] of the polymer-functionalized IEC media can be realized. It is clear that the ready-coupling polymer chains in the “grafting to” approach (Type I and Type II ion exchangers) can be natural or synthetic. Furthermore, there are many reactive or activated groups in the ready-coupling polymer chains, so their grafting points (anchored sites) onto the matrix surface are usually random and multiple (Fig. 2A, B, E, and H). In addition to the random-multiple-anchored site, the branching degree and the molecular weights

2 Stationary phases

distribution (i.e., polymer dispersity index, PDI) of the ready-coupling polymer chains are commonly heterogeneous, leading to the heterogeneous status of the grafted chains on the matrix surface in the “grafting to” approach (Type I and Type II ion exchangers), Fig. 2A, B, E, and H. However, the polymer chains in the “grafting from” approach (Type III ion exchangers) are all synthetic, and the in situ synthesized polymer chains are grown from single-anchor sites as shown in Fig. 2C, D, F, and G, and are generally linear without branches (Fig. 2C, D, and F), except if a branching comonomer is used in the copolymerization route as illustrated in Fig. 2G and Ref [74]. Moreover, using the newly developed robust technology of atom transfer radical polymerization (ATRP) in the “grafting from” approach [15,75–77], homogeneous polymer chains in the grafting layer of well-controlled nanoscale architectures can be easily obtained, including a known chain length, well-defined chemical composition, single-anchor site, and stretching status of the linear polymer chains, as shown in Fig. 2C and D. Therefore, the various approaches and modification methods produce a diversity of polymer-functionalized ion exchangers with different structural and chemical properties and thus quite different chromatographic performances.

2.3 Typical commercial stationary phases There are many commercially available IEC media with grafted and surface-modified polymers, and their fabrication routes and structural characteristics provided by the manufacturers are listed in Table 1. Though some manufacturers also include multimodal ion exchangers in the scope of IEC, in this chapter, we focus on single-mode ion exchangers only. The commercially available polymerfunctionalized IEC media can be divided into three categories. Ion exchangers grafted by ready-coupling polymer chains, typically the

97

natural and electroneutral polymer—dextran, including the Sepharose XL, Streamline XL, and Capto series (Cytiva), and Bestarose XL and Mustang XL series (Bestchrom). They are representative of Type I media and are prepared by grafting dextran onto the surface of the matrix, which is a natural polymer such as agarose (Sepharose XL, Streamline XL, Bestarose XL, Mustang XL, and Capto series). Their fabrication routes are similar to Fig. 2A, with charge densities adjusted from 37 to 350 μmmol/mL. The main difference among these ion exchangers is their macroporous matrix, the grafting density, molecular weight of dextran chains, and ionic capacity (charge density). The second category is the tentacle-type ion exchangers, including the Fractogel and Eshmuno series from Merck Millipore. They are representative of Type III ion exchangers prepared by the in situ synthesis and grafting of the polymer chains onto the matrix surface. Fractogel is prepared by polymerizing vinyl monomers containing charged groups onto the surface of a polymethacrylate matrix. The ion-exchange groups are located only on the polyvinyl extenders (tentacles) of different lengths, as illustrated in Fig. 2D. The Eshmuno series was also synthesized by this tentacle technology with only minor modification. For instance, the four tentacular strong cation exchangers Eshmuno S, Eshmuno CP-FT, Eshmuno CPS, and Eshmuno CPX are based on similar rigid hydrophilic polyvinylether base matrixes (Eshmuno base matrixes) with sulfoisobutyl ion-exchange groups. However, their surface charge densities are different as a result of the different (co-)polymerization procedures, as shown Fig. 2D and G. Similarly, Capto S ImpAct (Cytiva) is fabricated using proprietary technology, formed by random grafting of pyrrolidone and sulfonate onto a high-flow agarose matrix, Fig. 2G. Thus, Capto S ImpAct is also a Type III ion exchanger. The third category is represented by the Capto ImpRes series (Cytiva), Nuvia series

LE 1 .

Summary of the fabrication routes, structure, and adsorption characteristics of the commercially available polymer-functionalized IEC

a

Cation/ anion

Matrix

Surface extender (polymer or monomer)

harose XL

Anion

Agarose

Dextran

I

Quaternary amine

180–260

160 mg/mL for BSA at 300 cm/h

pharose XL

Cation

Agarose

Dextran

I

Sulfopropyl

180–250

160 mg/mL for lysozyme Cy at 300 cm/h

mLine Q XL

Anion

Agarose

Dextran

I

Quaternary amine

230–330

>110 mg/mL for BSA at 300 cm/h

mLine SP XL

Cation

Agarose

Dextran

I

Sulfopropyl

180–240

>140 mg/mL for lysozyme Cy at 300 cm/h

Q

Anion

Agarose

Dextran

I

Quaternary amine

160–220

>100 mg/mL for BSA at 600 cm/h

DEAE

Anion

Agarose

Dextran

I

Diethylaminoethyl

290–350

>90 mg/mL for ovalbumin Cy at 600 cm/h

S

Cation

Agarose

Dextran

I

Sulfoethyl

110–140

>120 mg/mL for lysozyme Cy at 600 cm/h

S ImpAct

Cation

Agarose

Pyrrolidone III and sulfonate

Sulfoethyl

37–63

>85 mg/mL for BSA at 220 cm/h >90 mg/mL for lysozyme at 220 cm/h >100 mg/mL for IgG at 220 cm/h

Q ImpRes

Anion

Agarose

Proprietary surface extender

–

Quaternary amine

150–180

Cy >55 mg/mL for BSA at 150 cm/h >48 mg/mL for β-lactoglobulin at 150 cm/h

SP ImpRes

Cation

Agarose

Proprietary surface extender

–

Sulfonate

130–160

>95 mg/mL for BSA at 150 cm/h >70 mg/mL for lysozyme at 150 cm/h

Type Ion-exchange group

Ionic capacity (μmol/mL)

Dynamic binding capacity Co

Cy

Cy

Cy

Cy

Cy

arose XL

Anion

Agarose

Dextran

I

Quaternary amine

180–260

>160 mg/mL for BSA at 300 cm/h

starose XL

Cation

Agarose

Dextran

I

Sulfopropyl

180–250

160 mg/mL for lysozyme Be at 300 cm/h

ond Q Mustang

Anion

Agarose

Dextran

I

Quaternary amine

170–230

>100 mg/mL for BSA at 200 cm/h

ond SP Mustang Cation

Agarose

Dextran

I

Sulfopropyl

160–220

>100 mg/mL for lysozyme Be at 200 cm/h

gel EMD DMAE Anion

Crosslinked polymethacrylate

Vinyl monomers originally containing charged groups

III

Diethylaminoethyl

40–66

100 mg/mL for BSA at 150 cm/h

M M

gel EMD DEAE Anion

Crosslinked polymethacrylate

Vinyl monomers originally containing charged groups

III

Diethylaminoethyl

70–110

100 mg/mL for BSA at 150 cm/h

M M

gel EMD TMAE Anion

Crosslinked polymethacrylate

Vinyl monomers originally containing charged groups

III

Trimethylammoniumethyl 50–90

100 mg/mL for BSA

M M

gel EMD TMAE Anion

Crosslinked polymethacrylate

Vinyl monomers originally containing charged groups

III

Trimethylammoniumethyl 43–73

100 mg/mL for BSA at 200 cm/h

M M

gel EMD TMAE Anion (M)

Crosslinked polymethacrylate

Vinyl monomers originally containing charged groups

III

Trimethylammoniumethyl 140–190

150 mg/mL for BSA of gel at 150 cm/h

M M

Be

Be

C

LE 1 Summary of the fabrication routes, structure, and adsorption characteristics of the commercially available polymer-functionalized IEC —cont’d

a

Cation/ anion

Matrix

Surface extender (polymer or monomer)

Type Ion-exchange group

Ionic capacity (μmol/mL)

Dynamic binding capacity Co

Crosslinked polymethacrylate

Vinyl monomers originally containing charged groups

III

Trimethylammoniumethyl –

180 mg/mL for BSA at 150 cm/h

Cation

Crosslinked polymethacrylate

Vinyl monomers originally containing charged groups

III

Carboxyethyl

95–145

100 mg/mL for lysozyme M at 300 cm/h M

gel EMD SO3

Cation

Crosslinked polymethacrylate

Vinyl monomers originally containing charged groups

III

Sulfoisobutyl

95–140

150 mg/mL for lysozyme M at 100 cm/h M

gel EMD SO3

Cation

Crosslinked polymethacrylate

Vinyl monomers originally containing charged groups

III

Sulfoisobutyl

90–130

130 mg/mL for lysozyme M at 200 cm/h M

gel EMD SE (M)

Cation

Crosslinked polymethacrylate

Vinyl monomers originally containing charged groups

III

Sulfoisobutyl

85–125

160 mg/mL for lysozyme M at 220 cm/h M

uno Q

Anion

Hydrophilic polyvinylether polymer

Proprietary surface tentacles

III

Trimethylammoniumethyl 90–190

gel EMD TMAE Anion ap (M)

gel EMD d (M)

150 mg/mg for BSA at 600 cm/h

M M

M M

uno S

Cation

Hydrophilic polyvinylether polymer

Proprietary tentacles

III

Sulfoisobutyl

50–100

165 mg/mL for lysozyme M at 300 cm/h M

uno CP-FT

Cation

Hydrophilic polyvinylether polymer

Proprietary tentacles with negatively charged ligands and neutral spacers

III

Sulfoisobutyl

–

70 mg/mL for lysozyme at 300 cm/h

uno CPS

Cation

Hydrophilic polyvinylether polymer

Proprietary tentacles with negatively charged ligands and neutral spacers

III

Sulfoisobutyl

–

160 mg/mL for lysozyme M at 300 cm/h M

uno CPX

Cation

Hydrophilic polyvinylether polymer

Proprietary tentacles with negatively charged ligands and neutral spacers

III

Sulfoisobutyl

60

120 mg/mL for pIgG at 250 cm/h

M M

HP-Q Resin

Anion

UNOsphere epoxide

Proprietary surface extender

–

Quaternary amine

48–88

>50 mg/mL for thyroglobulin at 100 cm/h

Bio

HR-S Resin

Cation

UNOsphere epoxide

Proprietary surface extender

–

Sulfonate

100–180

>70 mg/mL for hIgG at 300 cm/h

Bio

Q Resin

Anion

UNOsphere epoxide

Proprietary surface extender

–

Quaternary amine

100–170

>170 mg/mL for BSA at 300 cm/h

Bio

M M

C

LE 1 Summary of the fabrication routes, structure, and adsorption characteristics of the commercially available polymer-functionalized IEC —cont’d

Cation/ anion

Matrix

S Resin

Cation

UNOsphere epoxide

PEARL ap Q-650M

Anion

Surface extender (polymer or monomer)

Type Ion-exchange group

Ionic capacity (μmol/mL)

Proprietary surface extender

–

Sulfonate

90–150

110 mg/mL for hIgG at 300 cm/h

Hydroxylated polymethacrylic polymer

Proprietary surface extender

–

Trimethylamine

200–300

105–155 mg/mL for BSA at To 212 cm/h Bio

PEARL Anion ap DEAE-650M

Hydroxylated polymethacrylic polymer

Proprietary surface extender

–

Diethylaminoethyl

150–250

>156 mg/mL for BSA at 212 cm/h

To Bio

PEARL ap S-650S

Cation

Hydroxylated polymethacrylic polymer

Proprietary surface extender

–

Sulfoisobutyl

140–240

>170 mg/mL for BSA at 220 cm/h

To Bio

PEARL ap CM-650

Cation

Hydroxylated polymethacrylic polymer

Proprietary surface extender

–

Carboxyethyl

180–280

>110 mg/mL for γ-globulin at 212 cm/h

To Bio

a

unavailable from the manufacturers. ovine serum albumin. om the product data sheets provided by the manufacturers.

Dynamic binding capacity Co

Bio

3 Adsorption and uptake theory

(Bio-Rad), Toyopearl GigaCap series (Tosoh Bioscience), whose grafting details are not provided by the manufacturers. The Nuvia series of ion exchangers are prepared by grafting surface extenders by proprietary technology onto a hydrophilic UNOsphere epoxide macroporous matrix with an optimized charge density. The Toyopearl GigaCap series media are built on a hydroxylated methacrylic polymer matrix with a polymeric functionalization. Since their structures are unknown, they cannot be classified as Type I/II/III. It should be noted that the commercially available polymer-functionalized IEC media display high chromatographic performance, especially high DBC values of 100 mg/mL for many proteins (Table 1). This is 2–5-fold higher than traditional nongrafting ion exchangers. Many lab-made polymerfunctionalized ion exchangers have been developed [51], among them ion exchangers with DBC values 200 mg/mL, including DEAE-modified and DEAE-dextran-grafted agarose (Type I, prepared by the route in Fig. 2H), sodium acetate partial neutralized poly(allylamine) grafted agarose (Type II, prepared by the route in Fig. 2E), and several ATPR Type III ion exchangers (Fig. 2D and G).

3 Adsorption and uptake theory A high DBC value requires both a high equilibrium adsorption capacity and a fast mass transfer rate. The 3D architecture of the polyelectrolyte layer offers more accessible binding space for the biomacromolecules than the two-dimensional (2D) binding surface of traditional ion exchangers, which is favorable for a high static binding capacity (equilibrium adsorption capacity), and the “chain delivery” of bound biomacromolecules on the polyelectrolyte chains facilitates mass transfer by flexible chain swings.

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3.1 Three-dimensional adsorption The binding sites (ion-exchange groups) for biomacromolecules on traditional nongrafting ion exchangers are located only on the matrix surface, so biomacromolecules can be adsorbed only on the matrix surface, presenting a 2D adsorption surface (Fig. 3A). Thus, the maximum adsorption density of the target biomacromolecules is limited by both the specific surface area (phase ratio) of the matrix and the charge density of the ion-exchange groups. More specifically, for an ion exchanger with a small specific surface area and high charge density, such as a nonporous, gigaporous, or monolithic matrix, the theoretical maximum adsorption density can be estimated by the available surface area of the matrix and the size of the biomacromolecules. By comparison, part or all of the binding sites for biomacromolecules on the polymerfunctionalized ion exchangers are located in the polyelectrolyte extenders (Fig. 1B), and biomacromolecules can be adsorbed in the grafting layer, presenting a 3D adsorption space (Fig. 3B). In other words, the polyelectrolyte grafting layer extends the 2D adsorption surface into the 3D adsorption space. Thus, the maximum adsorption density of the target biomacromolecules is not limited by the surface area of the matrix, but by the available pore volume (accessible binding sites) of the ion exchanger. Moreover, the long polyelectrolyte extenders provide more flexible ways for the biomacromolecules to interact with the ionexchange groups. Consequently, the increase of polymeric ligand of the polymer-functionalized ion exchangers, such as the increased grafting density, chain length, and charge density of polyelectrolyte extenders, increases the accessible binding sites in the grafting layer and in the flexible interaction with the binding sites. Whereas the increase in ionic capacity

104

5. Ion-exchange separations of biomacromolecules

FIG. 3 Schematic illustration of the adsorption of biomacromolecules onto an IEC stationary phase. (A) 2D adsorption surface in nongrafting IEC media and (B) 3D adsorption space in polymer-functionalized IEC media (represented by Type III media). The quantity of polymer chains and ion-exchange groups in this figure do not represent their real densities on the pore surface. The size of matrix, polymer chains, spacers, ion-exchange groups, and biomacromolecules do not represent their real sizes. Biomacromolecules are represented by bovine serum albumin (BSA) with PDB ID of 3V03.

(surface ion-exchange groups) of traditional nongrafted ion exchangers does not effectively increase the available binding sites for biomacromolecules, since many of the surface ionexchange groups are shield by the adsorbed biomacromolecules. Therefore, at the same value of the total ionic capacity (polymeric ion-exchange groups plus surface ion-exchange groups), the increase of only the polymeric ion-exchange groups can increase the number of accessible binding sites (Fig. 3B). That is, the increase in the density of polymeric ionexchange groups is more effective than the density of surface ion-exchange groups for enhancing the adsorption capacity of biomacromolecules [72,73]. Certainly, polymeric ionexchange groups of too high or too low a density are not suitable for adsorption, as detailed in Section 2.1.

3.2 Facilitated mass transfer by chain delivery effect The surface transport of the bound biomacromolecules on the chromatographic media significantly contributes to the overall mass transfer. There are mainly four typical mechanisms of surface transport in IEC processes: activated jump, electrophoresis, electrostatic coupling effect, and the chain delivery effect [52]. The chain delivery effect is a passive transport mechanism for bound biomacromolecules that only exists for polymer-functionalized IEC media [51]. The accelerated uptake kinetics observed on polymer-functionalized IEC media can be explained by the chain delivery effect, Fig. 4. The direct visualization of protein uptake in DEAE- and DEAE-dextran-modified bare capillaries provides a microscopic view

3 Adsorption and uptake theory

FIG. 4 Schematic illustration of the chain delivery effect for bound biomacromolecules on polymer-functionalized ion exchangers. The dashed lines and dotted circles represent the grafted polymer chains before a swing. The dotted boxes containing a Y-type pattern highlight the sites for the delivery of bound biomacromolecules, represented by immunoglobulin G (IgG) in this case. The size and quantity of polymer chains, spacers, charged groups, and binding sites on IgG do not represent their real sizes and densities. Reproduction from L. Yu, Y. Sun, Recent advances in protein chromatography with polymer-grafted media, J. Chromatogr. A 1638 (2021) 461865 with permission.

of the chain delivery effect for bound biomacromolecules [78]. The grafted polyelectrolyte chains on the matrix surface are flexible and can change configuration to form a grafting layer of extended 3D architecture with a large amount of bound biomacromolecules (Fig. 3B). If the chains are close enough, the bound biomacromolecules on one polyelectrolyte chain can easily touch a neighboring chain by the flexible chain motion, in which the bound biomacromolecule can be transferred between neighboring chains from one binding sites to another, resulting in the facilitated mass transfer of the bound biomacromolecule (Fig. 4). In other words, the chain

105

delivery effect is driven by the chemical potential gradient as well as electrical potential gradient toward the center of the matrix, in addition to the interactions between the neighboring flexible chains mediated by the bound biomacromolecules. Furthermore, the chain delivery effect includes mass transfer between two neighboring chains, denoted as chain-chain delivery, and exists in the Type I, II, and III ion exchangers. The mass transfer between one polyelectrolyte chain and its adjacent surface ion-exchange group, denoted as the chain-surface delivery, can only exist in Type I ion exchangers (Fig. 4). Thus, the charge density of polyelectrolyte chains has a significant impact on the chain delivery effect. Firstly, the charge density directly determines the chain extension feature and the rigidity/flexibility which limits the possibility of chain swings. Secondly, the charge density affects the binding strength of the biomacromolecules to the polymeric ion-exchange group, which determines the availability of the bound biomacromolecules to be delivered between two polyelectrolyte chains (and/or one polyelectrolyte chain and its adjacent surface ion-exchange group), and thus limits the possibility of chain delivery by swings. Thirdly, the charge density of polyelectrolyte chains greatly affects the adsorption capacity of biomacromolecules and thus limits the amount of bound biomacromolecules that can participate in the chain delivery. Besides, the grafting density, which directly determines the distance of two neighboring chains (and/or one polyelectrolyte chain and its adjacent surface ionexchange group), together with the chain length and degree of branching, influences the possibility of the bound biomacromolecules to touch and interact with another binding site by chain swings, limiting the effectiveness of the swings. In general, an increase in the surface density of the ion-exchange groups corresponds to an increase in the probability of “chain-surface” delivery, whereas the increase of the polymeric

106

5. Ion-exchange separations of biomacromolecules

ion-exchange groups, including the increased grafting density, chain length, and charge density of polyelectrolyte extenders, favors an increase in the probability of “chain-chain” delivery. Given that the ionic capacity of a polyelectrolyte chain is equivalent to more than two orders of magnitude that of surface ionexchange groups, for the same total ionic capacity (polymeric ion-exchange groups plus surface ion-exchange groups), the increase in amount of surface ion-exchange groups is far more than that of the polymer chains, and the increased “chain-surface” delivery will compensate and even overcome the weakened “chain-chain” delivery. As detailed in Section 3.1, the increase in polymeric ion-exchange groups is more effective than the density of surface ion-exchange groups at enhancing the adsorption capacity, and thus more effective at increasing the number of biomacromolecule participating in chain delivery. Consequently, the ionic capacity of the polymer-functionalized IEC media plays a complicated role in its uptake kinetics of biomacromolecules, and a proper distribution of ionexchange groups on the matrix surface and/or in the polyelectrolyte extenders needs to be carefully designed.

4 Applications 4.1 Features of practical applications It is well known that the features of chromatographic processes are codetermined by the properties of the stationary and mobile phases, and that adjusting the mobile phase composition is a typical practical operation to control the chromatographic process. Especially, the pH, ionic strength, and counter-ions of the mobile phase are crucial factors for controlling the ion-exchange process, that is, the competitive displacement between counter-ions and biomacromolecules at the ion-exchange sites of the stationary phase. For the traditional nongrafting IEC media, there

are two electrostatic interactions that are important, namely the electrostatic interaction between biomacromolecules and ion-exchange groups and the electrostatic interaction between the counterions and the same ion-exchange groups. In contrast, for the polymer-functionalized IEC media, the electrostatic interaction between neighboring polymeric ion-exchange groups (and/or one polymeric ion-exchange group and its adjacent surface ion-exchange group) affects the 3D structure of the grafting layer, the rigidity/flexibility of the polyelectrolyte chains, and the accessibility of the binding sites, with a significant impact on the 3D adsorption space and chain delivery effect. Therefore, the pH, ionic strength, and mobile phases counter-ions have more complicated roles on the IEC separation of biomacromolecules on grafted and surface-modified polymers, providing more flexible options to control the binding and elution of biomacromolecules [61,65–67,79–83]. In detail, pH determines the net charge of biomacromolecules and degree of dissociation (net charge) of ion-exchange groups (including polymeric and surface ion-exchange groups). Thus, a more complicated and sensitive change in the ion-exchange separation of biomacromolecules on grafted and surface-modified polymers is generally observed by altering pH [84,85], providing a flexible elution option. Salt ions shield the electrostatic interaction. Hence, high ionic strength weakens electrostatic attraction between biomacromolecules and ion exchangers, which is unfavorable for binding but good for chain delivery. In addition, high ionic strength also weakens the electrostatic repulsion between polyelectrolyte chains, increasing chain flexibility and enhancing the accessibility of the ion-exchange groups, benefiting the binding and mass transfer of biomacromolecules. Therefore, most polymerfunctionalized ion exchangers display a better salt-tolerance behavior than traditional nongrafted ion exchangers [46,59,80,86,87]. That is, polymer-functionalized ion exchangers offer a wide mobile phase composition operating range

4 Applications

for ionic strength but require large elution volumes for gradient elution or high ionic strengths for isocratic elution. Besides, the salt-tolerance generally varies among biomacromolecules, which are eluted at different ionic strengths, giving rise to high-resolution separations. The type of counter-ion significantly affects the competitive displacement of counter-ions and biomacromolecules since the binding preference for counter-ions at an ion-exchange group only depends on their structure and not on the location of the ion-exchange groups (on polymers or on the matrix surface). The counter-ion preferences exhibit the same order on IEC media whether it is a polymerfunctionalized ion exchanger or a traditional nongrafted ion exchanger. However, counterions of high preference for the ion-exchange sites weaken electrostatic attraction between biomacromolecules and the ion exchanger, which is unfavorable for binding but good for chain delivery, like the effect of high ionic strength. Since the chain delivery mechanism contributes significantly to the total mass transfer for polymer-functionalized ion exchangers, a more sensitive change of binding and elution of biomacromolecules is commonly observed for polymer-functionalized IEC media by changing the counter-ion type [49,50]. Consequently, counter-ion selection provides a flexible option for optimizing binding and elution.

4.2 Application examples Many biomacromolecules, such as nucleic acids, polysaccharides, and proteins (including antibodies, enzymes, recombinant proteins, viruses, and virus-like particles), are separated by IEC during downstream processing. Herein, we focus on the practical applications of commercial polymer-functionalized ion exchangers for the laboratory-scale and industrial-scale separations of proteins and nucleic acids, as the lab-made ones have been summarized previously [51–53].

107

Monoclonal antibodies, consisting of a variety of isomers and aggregates, are regarded as the most important class of pharmaceutical biomacromolecules. The industrial-scale separation and purification platforms for antibodies typically include a cation-exchange chromatography unit for capture and a cation- or an anion-exchange chromatography unit for polishing [88]. The polymer-functionalized ion exchangers exhibit improved performances for the downstream processing of antibodies. Stein and Kiesewetter tested six cation exchangers for the binding of monoclonal antibody and removal of host cell protein on lab-scale columns and observed high step yields and reduced host cell protein contamination for two tentacle cation exchangers, Fractogel EMD SO3 (M) and Fractogel EMD SE Hicap (M), on a 200 mm  16 mm ID column [89]. Diedrich et al. conducted an industrial-scale separation preparative on a tentacle-type Fractogel EMD SO3 cation exchanger at loadings of 72.5, 82.8, 93.1, and 118.2 g/L from a preconditioned and filtered fermentation broth [90]. Additionally, the polymer-functionalized ion exchangers also work well for the separation and purification of other proteins, including β-lactoglobulin from whey by 260 mm  70 mm ID and 200 mm  80 mm ID columns packed with Nuvia Q [91], penicillin G acylase from E. coli homogenate and B. megaterium culture medium on packed-bed and expanded-bed columns with Streamline SP XL [92], recombinant human interleukin-11 from Escherichia coli culture medium by a 4.7 mm ID  100 mm prepacked column containing 4.7 mL Capto Q [93]. Recently, viruses and virus-like particles (VLPs) have attracted increasing attention due to their use in vaccines and cancer therapy. It is noteworthy that the viruses and VLPs are more complex than the soluble proteins in the ion-exchange process, because their surface usually contains carbohydrates, lipids, and proteins with different charges that vary with pH in different ways, unlike the net charge of soluble

108

5. Ion-exchange separations of biomacromolecules

proteins which tend to change according to their pI values. Nevertheless, the polymerfunctionalized ion exchangers also displayed enhanced IEC performance. For example, Eckhardt et al. compared eight anion-exchange, cation-exchange, and hybrid media in a 1-mL column for the purification of oncolytic measles virus, an emerging class of cancer therapeutics [94], and from a suspension in Vero cells feed [95]. The cation exchanger Eshmuno CPX was identified as the most promising resin, with a yield of 80.7% infectious MV particles and removal of 98.3% of total protein and 80.5% of host cell DNA [95]. Pereira Aguilar et al. achieved the direct capture and purification of the enveloped HIV-1 gag VLPs with a diameter of about 100–200 nm, from the CHO cells culture supernatant, with a 200 mm  16 mm ID column packed with 5.43 mL of Fractogel EMD TMAE Hicap (M) in a single IEC step [96]. The direct loading of cell culture supernatant onto the column resulted in a total particle recovery of about 35% together with a high purity in a single IEC unit, in which the VLPs only bind to the outer surface of the IEC media while the negatively charged low-mass impurities bind to the interior of the IEC beads and well separated by elution, while the noncharged and positively charged impurities were excluded from the column in the flow-through and wash steps [96]. Although the industrial platform process for the purification of nucleic acids is not well established, their reported gram-scale production generally involves at least one IEC unit [97,98]. For example, Chen et al. compared three anion exchangers for the separation of oligonucleotides, with GigaCap Q-650 M exhibiting narrower peak widths in a 2 mL column (55 mm  6.6 mm ID) compared to the nongrafted ion exchangers [99]. In addition, Matos et al. found that a 4.5 mL Capto Q ImpRes column of 100 mm  7.7 mm ID afforded a stronger interaction of doublestranded DNA (dsDNA) compared with single-stranded DNA (ssDNA), as well as

providing recognition for guanylate bases for samples of deoxynucleotides or poly(dG) which other media did not [100].

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C H A P T E R

6 Extraction chromatography of actinides S.A. Ansari and P.K. Mohapatra Radiochemistry Division, Bhabha Atomic Research Centre, Mumbai, India O U T L I N E 1. Introduction

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2. Extractants for actinide separation

114

3. Ligand impregnated resins for actinides 3.1 Monoamide impregnated resins 3.2 Malonamide impregnated resins 3.3 Diglycolamide impregnated resins 3.4 Multiple DGA impregnated resins

115 116 119 119 122

4. Room temperature ionic liquids in extraction chromatography 4.1 TODGA/RTIL resin 4.2 C4DGA and T-DGA/RTIL resins

125 125 127

1 Introduction Solvent extraction methods, being continuous in nature, are the workhorse of numerous separation industries. However, a major disadvantage associated with this technique is the generation of large volumes of secondary organic wastes. This aspect is particularly pronounced when the separation is realized from very large volumes of extremely dilute solutions. Under such a condition, the solvent

Ion-Exchange Chromatography and Related Techniques https://doi.org/10.1016/B978-0-443-15369-3.00018-3

5. Ligand grafted resins for actinides 5.1 Monoamide grafted resins 5.2 Malonamide grafted resins 5.3 Diglycolamide grafted resins

129 130 130 135

6. Composite beads for extraction chromatography

136

7. Perspectives

138

Abbreviations

138

References

139

extraction technique becomes cost prohibitive, and it is required to choose a technique where the organic solvent inventory is low. In this context, extraction chromatography (XC) has certain advantages, particularly when the separation involves very low concentrations of recoverable constituents from a large volume of solution [1,2]. The XC-based separation is sometimes also referred to as “green separation” technique as the extractant or organic solvent involved in the technique is extremely low

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Copyright # 2024 Elsevier Inc. All rights reserved.

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6. Extraction chromatography of actinides

compared with solvent extraction. The XC technique combines the selectivity and separation properties of solvent extraction with the ease of operation of chromatographic methods when performed in the column mode. Indeed, Horwitz et al. [3] have shown a similar correlation between the solvent extraction and XC for the extraction of lanthanides by D2EHPA (di-2ethylhexyl phosphoric acid). There is, however, a difference between the two techniques with respect to the activities of the ligand and the metal/ligand complex in solution, and on a solid support. In view of the fast-diminishing natural fossil fuels, nuclear energy is slowly emerging as a major alternative energy resource [4]. However, the future of nuclear energy programs depends upon the safe disposal of highly radioactive waste generated at the various stages of the nuclear fuel cycle. For example, large volumes of highly radioactive liquid waste, known as high-level waste (HLW), are generated during the reprocessing of the spent nuclear fuel. The HLW contains mainly unextracted U, Pu (from the PUREX losses), bulk of the minor actinides (Am, Np, Cm), and a host of elements like lanthanides, Tc, Pd, Zr, I, Cs, Sr, Ni, Sb, and Zr [5–8]. At present, the most accepted strategy for the management of liquid radioactive waste is to vitrify it as oxides in borosilicate glass matrices followed by its disposal in deep geological repositories [9–11]. However, due to the very long half-lives of the minor actinides, the surveillance of these vitrified blocks for such a long period will be a daunting task. An alternative proposed concept is the complete removal of minor actinides from the radioactive waste and their subsequent burning in the high flux reactors/accelerators in suitable chemical forms [12–16]. After removing the minor actinides, along with the long-lived fission product elements, the residual waste can be vitrified and buried in subsurface repositories at a muchreduced risk and cost.

Apart from HLW which is generated during the reprocessing of the spent nuclear fuel, large volumes of actinide bearing radioactive wastes are also generated in the laboratories involved either in the quality control of nuclear materials or the research laboratories handling actinides. These laboratory wastes are chemically and radiologically very complex in nature. Due to the toxic and hazardous nature of actinides, these wastes cannot be disposed like any other industrial waste. Instead, the removal of even trace amount of transuranium elements (particularly plutonium and americium) is essential for the safe disposal of these wastes [17,18]. Use of XC technique for the separation of actinides from laboratory waste is particularly encouraging looking at several advantages of the technique over traditional solvent extraction. A few obvious advantages are the low ligand invitatory, lower secondary waste generation, and ease of operation when separation is performed in a column. These merits of XC methods are advantageous while handling the radioactive solutions. The aim of this chapter is to provide a summary of the work on the separation of actinides by XC techniques. Work on ligand impregnated resins and chemically bonded resins will be highlighted. Although there are numerous studies on the XC separation of actinides and lanthanides using various extractants, the focus of this chapter will be limited to amidebased ligands like monoamides, diamides, and diglycolamides.

2 Extractants for actinide separation Though TBP is widely used in reprocessing industries for separation of tetra and hexavalent actinides, it has poor affinity for trivalent actinides, and therefore, it cannot be used for the separation of trivalent actinides. The ligand which can extract trivalent actinides is

115

3 Ligand impregnated resins for actinides

commonly referred to as “actinide partitioning” extractants. In the past few decades, separation scientists have been engaged in the development of new extractants for actinide partitioning [19–21]. In this context, several organophosphorus and amide-based extractants have been developed. Among the organophosphorus compounds, octyl-(phenyl)-N,N-diisobutyl carbamoyl methyl phosphine oxide (CMPO, I) has been extensively investigated [20]. The bifunctional nature of CMPO facilitates the extraction of trivalent actinides (Am and Cm) at a moderate acidity of 3–4 M nitric acid. However, stripping of the extracted actinides from the loaded organic phase with dilute acid solution is rather difficult and requires a complexing agent. Other phosphorus-based extractants, such as trialkyl phosphine oxide (TRPO) and diisodecyl phosphoric acid (DIDPA), have limitations as they extract trivalent actinides only at lower acidities (
1 M nitric acid), and are ineffective for HLW conditions of 3–4 M nitric acid [20]. Substituted malonamides, on the other hand, were a better choice for trivalent actinides under HLW conditions [20–22]. The fact that these extractants show poor extraction at lower acidities (
1 M nitric acid) makes them versatile for stripping of the loaded metal ions. In addition, malonamides are completely incinerable which implies that the amount of secondary waste generated can be reduced significantly by their incineration. Among malonamide-based extractants, N,N0 -dimethyl-N,N0 -dibutyl tetradecyl malonamide (DMDBTDMA, II) has been extensively studied for actinide partitioning [22]. The distribution ratios for trivalent actinides with DMDBTDMA are lower than those with CMPO, which necessitates the use of a higher concentrations of the former. Efforts to design more efficient extractants with a similar amide skeleton led to the development of diglycolamides (DGA, III), which display far superior affinities toward trivalent actinides compared with the malonamides and CMPO [23]. The affinity of DGA ligands toward the trivalent actinides has been further enhanced by

functionalizing multiple DGA moieties on a suitable scaffold, which provides preorganized DGA units for cooperative complexation [24–29]. O

O

i-butyl N

Ph

P

C C H2

i-butyl

C8H17

I

C4H9 N

O

O

C

C

CH3 N

C H

H3C

C4H9

C14H29

II O

O R

N C R

O C H2

C C H2

R N R

III 3 Ligand impregnated resins for actinides As the name indicates, these XC materials are prepared by physical impregnation of the ligands on an inert solid support, which may be an organic or inorganic porous polymeric material. Most used solid supports are Chromosorb-W, Chromosorb-102, XAD-4, XAD-7, Amberchrom CG-71, etc. Impregnation of ligands on the support is realized by dissolving them in a volatile solvent (such as acetone, methanol, toluene)

116

SCHEME 1

6. Extraction chromatography of actinides

Schematic of making impregnated resins.

followed by mixing the solid support to obtain a suitable composition (Scheme 1). After mixing the solid with the ligand solution, the solvent is evaporated to obtain the ligand impregnated resin. The composition of the ligand and the solid support is extremely important for obtaining the best-quality XC resin. In general, resins having a ligand composition of 10%–50% (w/w) on the support are prepared. Higher loadings are not favored due to loss of the free-flowing nature of the resin as the material may become sticky. Studies have shown that the extraction behavior of the resin is independent of the nature of the diluent used for impregnation of the ligand [30]. However, the performance of the XC resin is strongly dependent on the nature of the solid support [31]. While XAD-7 (a polyacrylic ester [–CH2–CH (COOR)–]n with a hydrophobic surface of moderate polarity) based resins were useful for certain cases [32], XAD-4 (polystyrene divinylbenzene) was effective for other cases [33]. ChromosorbW (dimethyldichlorosilane-treated acid-washed diatomaceous silica), on the other hand, was the best choice for DGA ligands compared with Chromosorb-102 (styrene divinylbenzene), XAD-4 and XAD-7 [34]. Amberchrom CG71, a macroporous polyacrylic ester, is the support for the commercial XC resins available from Eichrom Technologies, United States [35]. The selection of inert solid support is based on the particle size, porous nature, surface area, and hydrophobicity of the surface. Since most of the organic ligands impregnated on the resin are hydrophobic in nature (as compared to water), supports having a hydrophobic surface show better retention of

the ligand inside the pores due to hydrophobichydrophobic interactions. Table 1 lists a series of amide-based impregnated resins that have been used for the separation of actinides.

3.1 Monoamide impregnated resins Monoamides offer an alternative to TBP for the separation of tetra and hexavalent actinides [20–22]. The physicochemical properties of monoamides can be tuned by the judicious choice of their alkyl groups. For example, the linear alkyl chain N,N-dihexyloctanamide (DHOA, IV) has been studied as a PUREX solvent vis-a`-vis TBP [22]. On the other hand, one of the branched alkyl chain monoamides, viz. di-2-ethyhexyl-iso-butryamide (D2EHiBA, V), was found to be highly selective for uranyl ions over tertravalent thorium and was proposed as an alternative solvent for the THOREX process [22]. Although these monoamides have been studied extensively by liquid-liquid extraction, few studies have targeted their use in the XC technique. A systematic study was pursued with a series of monoamide-based XC resins for the separation of uranium from acid the solutions [31]. The monoamides used were di-2-ethylhexyl butyramide (D2EHBA), di-2-ethyhexyl-iso-butyramide (D2EHiBA) and di-2-ethyhexyl acetamide (D2EHAA). The extraction of UO2+ 2 ions by these ligands followed the order: D2EHAA> D2EHBA> D2EHiBA, which is consistent with the extraction behavior of these ligands observed in solvent extraction [22]. The effects of solid support on the extraction of uranium by these ligands

117

3 Ligand impregnated resins for actinides

TABLE 1

Summary of the amide-based extraction chromatography resins studied for the separation of actinides.

Ligand

Support

Studies performed/comments

References

D2EHAA

XAD-7

Separation of UO2+ 2 from acidic feed solution. Better than D2EHiBA resin

[31]

D2EHiBA

XAD-7

Separation of U(VI) from acidic feed solution. Inferior to D2EHAA resin

[31]

D2EHPrA

Chromosorb-W

4+ 4+ from acidic feed solution. Separation of UO2+ 2 , Pu , and Np The resin performance was better than the TBP resin

[36]

DMDBTDMA

Chromosorb-W

Separation of actinides and lanthanides under HLW conditions

[30]

3+

3+

4+

UO2+ 2

DMDOHEMA

Amberchrom

Sorption of Am , Cm , Th , and in its performance than TODGA resin

TODGA

Chromosorb-W

Separation of actinides and lanthanides under HLW conditions

[34]

TODGA

Magnetic particles

Minor actinides recovery under HLW conditions

[38]

TODGA

Silica/polymer

Minor actinides recovery by MAREC process

[39]

225

ions. The resin is inferior

225

[37]

TODGA

Amberchrom

(i) Ac/ Th separation for medical application (ii) Reprocessing of FBR-MOX fuel by ERIX process (iii) Distribution behavior of 60 elements

[40–42] [43] [44]

TODGA/TBP

Amberchrom

Separation of actinides and lanthanides under HLW conditions

[45]

T2EHDGA

Amberchrom

(i) Separation of Ra/Ac for medical application (ii) Separation of minor actinides and Ra/Ac

[46,47] [35]

T-DGA

Chromosorb-W

Separation of minor actinides from moderate acidic feed conditions

[48]

C4DGA

Chromosorb-W

(i) Separation of minor actinides from moderate acidic feed conditions (ii) Separation of Th4+ from moderate acidic feed conditions (iii) Separation of Np4+ from moderate acidic feed conditions

[48] [12] [49]

TAM-4-DGA

Chromosorb-W

Separation of minor actinides from moderate acidic feed conditions

[50,51]

HONTA

Chromosorb-W

Selective separation of Pu

were entirely different for XAD-4 and XAD-7. For any given amide, the maximum Kd for XAD-7 based resin was approximately three times higher than the corresponding Kd values for the XAD-4 based resin. XAD-4 had a styrene divinyl benzene ˚ backbone with an average pore diameter of 100 A 2 and a surface area of 750m /g, whereas, XAD-7 had a polyacrylic ester backbone with an average ˚ and a surface area of pore diameter of 300–400 A 2 380m /g. Since the amount of extractant loaded

4+

over trivalent and hexavalent actinides

[52]

on both supports was the same (40% w/w), it was difficult to understand the higher extraction behavior of the XAD-7 resins since its surface area was approximately half of the XAD-4. However, if one considers the pore diameter of the support, which was about three times larger for XAD-7, it can be concluded that the support with a larger pore diameter favors impregnation of the resin. An XC resin was also prepared by impregnating 45% w/w of di(2-ethylhexyl)-propionamide

118

6. Extraction chromatography of actinides 2

10

C6H13

O N

C

C6H13

C7H15

1

10

Kd (mL/g)

IV

0

10

N O

C -1

10

0

2

3

4

5

6

[HNO3], M

V

FIG. 1 Effect of aqueous feed acidity on Kd of uranyl ions with D2EHPrA resin. Reproduced with permission from R.B. Gujar, S.A. Ansari and P.K. Mohapatra, Actinide ion uptake from acidic radioactive feeds using an extraction chromatographic resin containing a branched dialkyl amide, J. Chromatogr. A 1635 (2021) 461728.

N O

1

C

VI (D2EHPrA, VI) on Chromosorb-W [36]. The resin was evaluated for the recovery of uranium from acidic feed solutions. As shown in Fig. 1, the Kd values of UO2+ 2 increased with the feed acidity up to 4 M nitric acid and became constant thereafter up to 6 M nitric acid. Such a behavior is typical for the extraction of metal ion by the “solvation mechanism,” described by the following equilibrium reaction: UO2 2+ ðaq:Þ + 2NO3  ðaq:Þ + xLðResinÞ   Ð UO2 ðNO3 Þ2 ðLÞx ðResinÞ

(1)

Here, x represents the number of ligand L units attached with the metal ion in the extracted complex. The above equilibrium signifies that an increase in the nitrate ion concentration at higher feed acidity favors the formation of UO2+ 2 /Lx complex on the resin. A further increase in the feed acidity (beyond 4 M nitric

acid) leads to competition for the ligand from the nitric acid and the metal ion. It is worth noting that the monoamide ligands do form complexes with acids and at higher acidities the acid-ligand complexation (adduct of the type: D2EHPrAHNO3) will be dominant leading to a decrease in the free ligand concentration on the resin, and hence acts as an antagonist. A combination of positive extraction effect due to increased nitrate ion concentration at higher acidities, and negative extraction effect due to acid-ligand adduct formation results in flattening of the extraction curve. Apart from uranium, the resin also showed good extraction capability for Pu4+ and Np4+ ions. The column studies with a 2.1 cm3 bed volume glass column (0.4 cm id) containing 0.35 g resin, exhibited breakthrough after a loading of 8.30 mg of uranium, which is quite encouraging for the recovery of uranium from a large volume of acid solution containing very low concentrations of actinides.

3 Ligand impregnated resins for actinides

3.2 Malonamide impregnated resins The monodentate ligands like TBP and monoamides can form complexes with tetra and hexavalent actinides, but not with trivalent actinides due to their lower ionic potential. To overcome these issues, multidentate ligands such as malonamides (bidentate) and diglycolamides (tridentate) ligands came into the picture to target trivalent actinides. Of several malonamides, DMDBTDMA was probably the first amide-based ligand proposed for the recovery of trivalent actinides from HLW in the DIAMEX process (diamide extraction process) proposed by the French researchers [53–56]. Although the major studies reported with this ligand were based on solvent extraction, including large scale demonstrations (10–50 L scale) utilizing mixer-settlers or centrifugal contactors, reports on the XC separation of actinides with this ligand are limited [30]. DMDBTDMA resin was prepared by impregnating 50% w/w ligand on Chromosorb-W. The distribution behavior of several actinide ions and fission product ele4+ 3+ 3+ + ments such as UO2+ 2 , Pu , Am , Eu , Cs , 2+ and Sr were investigated with this resin from a wide range of nitric acid solutions. As shown in Fig. 2, the resin showed a selective and

5

10

4

Kd (mL/g)

10

4+

Pu 2+ UO2

3

10

2

10

1

3+

10

Eu 3+ Am

0

10

1

2

3

4

5

6

[HNO3], M

FIG. 2 Distribution profiles of actinides and fission elements by DMDBTDMA resin [30].

119

4+ over efficient uptake of Am3+, UO2+ 2 , and Pu + fission product elements such as Cs and Sr2+ from moderate acidities. At 3 M nitric acid, the uptake of metal ions followed the order: 3+ 3+ 2+ + Pu4+ > UO2+ 2 ≫ Am  Eu ≫ Sr  Cs , which was similar to the ligand affinity observed in liquid-liquid extraction [22]. The possibility of using DMDBTDMA resin for the recovery of trace concentrations of Am3+ in the presence of relatively large amounts of Nd3+ and UO2+ 2 was also studied [30]. The results indicated that the presence of macro concentrations of Nd3+ and UO2+ 2 in the feed significantly affected the Kd values of Am3+ ion. The column performance of the resin for actinide ions was good. However, stripping of the loaded actinides (Am, U, and Pu) from the column was poor with dilute nitric acid solution (pH 2). Nonetheless, an efficient stripping was obtained with 0.05 M oxalic acid.

3.3 Diglycolamide impregnated resins Linear alkyl chain DGA ligands, such as N,N,N0 , N -tetra-n-octyl diglycolamide (TODGA), are promising for the extraction of trivalent actinides from acidic feed solution [23]. Solvent extraction properties of TODGA were also extended to the XC separation mode [34]. A comparison of TODGA, CMPO, and DMDBTDMA-impregnated resins (50% loading in each case) were made for the extraction of the Am3+ ion. As shown in Fig. 3, the Kd of Am3+ by the TODGA resin increased sharply up to 1 M nitric acid and remained steady thereafter up to 6 M nitric acid. This behavior was similar to the CMPO resin [57], but was in sharp contrast to the DMDBTDMA resin, where the Kd values increased gradually with nitric acid concentration and reached moderate values above 3 M nitric acid. The Kd values of Am3+ by different resins at 3 M nitric acid followed the order: 7200 (TODGA) > 2000 (CMPO) > 35 (DMDBTDMA). The performance of TODGA and DMDOHEMA resins, prepared by impregnating the two ligands on Amberchrom CG-71C, was also compared [37]. The uptake of Mo(VI), Pd2+ and Zr4+ was relatively higher for the DMDOHEMA resin compared with 0

120

Kd-Am

6. Extraction chromatography of actinides

10

4

10

3

10

2

10

1

10

0

TODGA CMPO DMDBTDMA

0

1

2

3

4

5

6

[HNO3], M

FIG. 3 Distribution coefficient (Kd) of Am3+ as a function of HNO3 concentration by different extractant impregnated on Chromosorb-W. Reproduced with permission from S.A. Ansari, P.N. Pathak, M. Husain, A.K. Prasad, V.S. Parmar, V.K. Manchanda, Extraction chromatographic studies of metal ions using N,N,N0 ,N0 -tetraoctyl diglycolamide (TODGA) as the stationary phase, Talanta 68 (2006) 1273–1280.

the TODGA resin. Nevertheless, the sorption of Zr4+ and Mo(VI) was successfully masked by addition of oxalic acid in the feed. Similarly, the extraction of Pd2+ was suppressed by its selective complexation with HEDTA in the feed solution without affecting the extraction of actinides. The sorption profiles of several metal ions such as 3+ 2+ + Am3+, Eu3+, Pu4+, UO2+ 2 , Fe , Sr , and Cs on TODGA resin were obtained from a series of nitric acid solutions [34]. The Kd values for actinides were similar to their distribution ratios observed in solvent extraction, i.e., An3+  An4+ > AnO2+ 2 . The Kd values for Cs+ and Fe3+ were less than 0.5mL/g for the full range of acidity investigated, suggesting insignificant sorption of these metal ions. An XC resin prepared by impregnating TODGA onto magnetic particles (as the inert support) indicated an identical results to those obtained by impregnating TODGA on Chromosorb-W [38], suggesting that the role of extractant is more dominant than that of the

support material. The advantages of such resins, however, include the fact that they can be added directly to the bulk solution and can be separated by a magnetic field after sorption of the metal ions, leaving behind the lean aqueous stream. To evaluate the applicability of TODGA resin, the effect of some selected metal ions, viz. UO2+ 2 and Nd3+ on the sorption of Am3+ was studied [34]. The sorption of Am3+ was not affected even in the presence of 20 g/L of uranium. However, the Kd value decreased sharply in the presence of Nd3+ suggesting a strong competition between Nd3+ and Am3+. In the column studies, 10mg of europium (used as a surrogate of Am3+) could be loaded without any breakthrough of 241Am. On the other hand, when a solution containing 20 g/L of U, spiked with 241Am tracer (at 3 M nitric acid), was passed through the column, no breakthrough for 241Am was observed even after passing 100 mL of the feed solution. This result 3+ indicated that UO2+ 2 did not compete with Am . In a similar study, an XC resin prepared by impregnating 30% TODGA + 10% TBP on Amberchrom CG-71C suggested that all the actinides could be separated from the other constituents of simulated HLW in the column operation [45]. Hoshi et al. [43] used TODGA impregnated resin for the development of a flow sheet for electrolytic reduction and ion exchange (ERIX) process for the reprocessing of FBR-MOX fuel dissolved in 3 M nitric acid. The ERIX process consists of three stages, viz. pretreatment, main process, and finally, the minor actinide separation with a combination of TODGA/BTP column for the recovery of actinides and lanthanides separately. Zhang et al. [39,58–62] proposed TODGA impregnated resin for the Minor Actinide Recovery by Extraction Chromatography (MAREC) process. They extensively studied the radiolytic and hydrolytic stability of the resin under HLW conditions. Horwitz et al. [63] demonstrated the synergistic enhancement in the uptake of trivalent actinides by TODGA resin in the presence of carrier trivalent metal ions from a hydrochloric acid medium (e.g., Fe, Ga, In, Tl which tend to

3 Ligand impregnated resins for actinides

form anionic species in hydrochloric acid medium). They observed that the synergistically extracted species contained a trivalent actinide or a lanthanide complexed by three TODGA molecules with three anionic metal chloride complex ions balancing the charge. They succeeded in the separation of Am3+ and Pu4+ in tracer quantities from a soil sample. After leaching the Am3+/Pu4+ from the soil sample by 3 M hydrochloric acid, the leached solution was passed through a TODGA column where all impurities were washed out, except Am and Pu. A significant amount of Fe was also retained by the column which was washed out with 3 M nitric acid. Finally, Am3+/Pu4+ were recovered with a mixture of 0.03 M oxalic acid and 0.25 M hydrochloric acid. More than 95% recovery of Am3+/Pu4+ was possible with a concentration

121

factor of 25 from a 450 mL feed solution. Horwitz et al. [40,41] also patented the procedure for the separation of nuclear medicine grade 225Ac from 229 Th target using a TODGA-coated resin column. Radchenko et al. [42] demonstrated the separation of Ac from irradiated Th target by a TODGA resin column. The irradiated Th target was dissolved in acid, and the entire amount of Th was converted into an anionic complex which was separated from Ac3+ on an ionexchange column. Finally, the Ac3+, contaminated by other cations such as Ra3+, Ba2+, and Ln3+, was separated by the TODGA column. Pourmand and Dauphas [44] determined the Kd values of sixty elements by TODGA resin (Fig. 4). They also reported the elution behavior of 32 elements with the TODGA resin column. TODGA resin was found to be highly versatile

FIG. 4 Distribution coefficients (Kd) of 58 elements on TODGA resin in logarithmic scale as a function of HCl concentration. Reproduced with permission from A. Pourmand, N. Dauphas, Distribution coefficients of 60 elements on TODGA resin: application to Ca, Lu, Hf, U and Th isotope geochemistry, Talanta 81 (2010) 741–753.

122

6. Extraction chromatography of actinides

with immense potential for matrix-analyte separation for high-precision elemental and isotope analysis of terrestrial, and extra-terrestrial materials. TODGA resins and columns are now available commercially from Eichrom Technologies Inc., United States. In contrast to the linear alkyl chain derivatives of DGA (e.g., TODGA), the XC studies with branched alkyl chain DGA, viz. T2EHDGA, are limited [35,46,47]. The performance of T2EHDGA resin is similar to that of TODGA resin for the separation of actinides and lanthanides under identical experimental conditions [35]. T2EHDGA resin was shown to offer a new possibility for the rapid, robust, and effective separation of 225Ra from 225Ac in targeted cancer therapy [46,47]. The resin showed a strong retention for Ac3+ from 6 to 7 M nitric acid and its efficient stripping with dilute nitric acid. The separated Ac3+ could be loaded on a cation-exchange column for production of a 225 Ac/213Bi generator [47]. Consequently, the use of T2EHDGA was implemented into the routine procedure used at ITU, Karlsruhe to produce 225Ac/213Bi radionuclide generators.

3.4 Multiple DGA impregnated resins During the past few decades, the DGA class of ligands has emerged as the most effective extractants for the separation of trivalent actinides from HLW in the proposed “Actinide Partitioning” program [23]. DGA ligands are TABLE 2

known to show a preference for trivalent actinides over the hexavalent uranyl cation, which is not the case for other ligands proposed for actinide partitioning such as CMPO and malonamides. The higher affinity of DGA for trivalent actinides was assigned to the formation of molecular aggregates through reverse micelle formation [64,65]. Several fundamental complexation studies, including crystal structures [66] and EXAFS studies [67], indicated the coordination of three DGA ligands with Eu3+/Am3+ via three donor oxygen atoms of these ligands. Taking the clue from DGA aggregate formation, it was predicted that preorganization of three to four DGA molecules on a molecular platform would enhance the affinity of these ligands. As predicted, it was realized that the extraction of actinides increased several folds upon functionalizing the multiple DGA moieties onto a suitable platform such as tripodal C-atom, tripodal N-atom or crown ether [24–29]. Such multiple-DGA functionalized ligands not only show higher extraction capability for trivalent actinides, but also show higher selectivity over uranium. Two such multiple-DGA functionalized ligands, viz. C4DGA (VII) and T-DGA (VIII), showed an order of magnitude higher metal ion extraction in XC compared with the conventional TODGA resin [48]. As shown in Table 2, several multiple DGA ligands impregnated resins have been prepared for the removal of hazardous actinides ions like Am3+, Pu4+, and UO2+ 2 from acidic feed solutions. At

Distribution coefficient (Kd) of metal ions on impregnated resins prepared by different DGA ligands. Kd at 3 M HNO3

Metal ions

TAM-4-DGA [50]

T-DGA [48]a

C4DGA [48]a

TODGA [34]b

Eu3+

(3.7  0.4)  105

8335  410

8330  380

9018

(4.3  0.4)  10

9939  340

6985  253

7212

Pu

(2.9  0.3)  10

4757  138

5305  168

5130

UO2+ 2

10.6  2.1

9.8  0.5

0.50  0.31

110

3+

Am

4+

a

5 5

Aqueous phase: 3 M HNO3; temperature: 25°C. Ligand loading: a10% (w/w), b47% (w/w).

123

3 Ligand impregnated resins for actinides

any acidity, the Kd values of Am3+ were higher with T-DGA resin compared to C4DGA resin. This behavior was explained by steriochemical effects where C4DGA with four DGA arms requires more energy for bringing the coordinating donor atoms together for a favorable complexation. Whereas the extraction of trivalent and tetravalent actinide ions was very high, the hexavalent uranyl ion was poorly extracted, providing a better separation factor for trivalent actinides over uranium when compared with conventional TODGA resin. C8H17 N C8H17 C8H17 N

C8H17 C8H17 C8H17 NC H O 8 17 N O O O O O O O O C8H17 O O O N C8H17 N N N C8H17 C8H17

C8H17

O

O

O

VII

VIII

O

O

O O R2N

NR2 N

O O

O

N

O

O O

N

NR2

N R = n-octyl O

NR2 O

O

IX

In the series of multiple DGA ligands, a unique ligand was studied where four DGA moieties were functionalized on a tetraaza-12-crown-4 scaffold (TAM-4-DGA, IX). Solvent extraction and complexation studies with TAM-4-DGA provided encouraging results for the separation of trivalent actinides [68]. In view of their excellent extraction properties for actinides, an XC resin was prepared by impregnating about 0.1 mmol of TAM-4-DGA on each gm of Chromosorb-W [50,51]. The Kd values of Am3+ and Pu4+ with this XC resin at moderate acidity of 3–4 M nitric acid were of the order of 105 mL/g. Whereas the extraction of trivalent and tetravalent actinides was very large, the hexavalent uranyl ion was poorly extracted as its Kd value was about 10 mL/g, thus providing a high separation factor for Am3+ and Pu4+ over UO2+ 2 . The Kd values for actinides with TAM-4-DGA resin were significantly larger than those for T-DGA and C4DGA ligands (Table 2). The resin capacity for Pu was 12.1  0.8 mg/g of resin, which corresponded to a 1:2 metal/ligand complex. A column study was performed to examine the possible use of this resin for the recovery/preconcentration of Pu. As shown in Fig. 5, a total of 17 mL of loading solution could be passed without breakthrough of Pu4+, which corresponded to 2.05 mg Pu on the column prepared with 200 mg of resin. The Pu4+ ion was eluted within 15 mL of eluent containing 0.5 M oxalic acid in 0.5 M nitric acid. The recovery of Pu was 97.2%. Oxalic acid was used as eluent due to its excellent complexing ability for Pu4+, and a saturated

124

6. Extraction chromatography of actinides

400 Loading curve

125

Elution curve

300

g / mL

g / mL

100

75

200

50 100 25

0

0 0

5

10

15

20

25

Volume of feed (mL)

0

4

8

12

16

Volume of eluent (mL)

Loading and elution curves for Pu4+ on TAM-4-DGA resin column. Feed solution: 0.128 mg/mL Pu4+ at 1 M HNO3; eluent: 0.5 M oxalic acid in 0.5 M HNO3. Reproduced with permission from P. Banerjee, S.A. Ansari, P.K. Mohapatra, R.J.M. Egberink, T.P. Valsala, D.B. Sathe, R.B. Bhatt, J. Huskens, W. Verboom, Highly efficient plutonium scavenging by an extraction chromatography resin containing a tetraaza-12-crown-4 ligand tethered with four diglycolamide (DGA) pendent arms, J. Chromatogr. A 1653 (2021) 462419.

FIG. 5

solution of oxalic acid is used for the final precipitation of Pu4+ for its conversion into PuO2. To develop new ligands for selective complexation of tetravalent actinides over their corresponding trivalent and hexavalent counterparts, a series of nitrilotriacetamide ligands were studied [69]. These ligands possess three amide moieties attached to a tripodal N atom (X) and show good selectivity for tetravalent actinides [70,71]. A nitrilotriacetamide ligand with n-octyl groups (HONTA, X with R ¼ n-octyl) exhibited high extraction capability and selectivity for Th4+ ion from a wide range of aqueous phase acidity (0.10–10.0M nitric acid) [72]. The separation factor between Th4+ and trivalent lanthanides was as high as 1000. Further studies confirmed that HONTA also had favorable extraction capability for Pu4+, but extremely poor extraction capability 3+ ions [26]. Looking at these for UO2+ 2 and Am features of HONTA, an XC resin was prepared using Chromosorb-W support and evaluated for the selective separation of Pu4+ for analytical applications [52].

R

R

R

N

R

N R

N

N O

O

O

R=

R C8H17

X Fig. 6 shows the nitric acid concentration dependent Kd values of Pu4+ on the HONTA resin. The results for Am3+ and UO2+ 2 are also included in the figure for comparison purpose. The Kd of Pu4+ was in the range of a few thousands in the entire range of acidity investigated (0.5–6 M nitric acid). In comparison to Pu4+, the Kd for the other actinides (Am3+ and UO2+ 2 ) was extremely low and never exceeded 5 mL/g over the entire range of feed acidity. The separation factors for Pu4+ over Am3+ or UO22+ were in the range of a few thousands. The largest separation factor was at the lowest feed acidity

4 Room temperature ionic liquids in extraction chromatography 5

10

4

x10

4

10

3

Kd (mL/g)

10

2

10

Separation factor Pu/Am Pu/U

1.2

0.8 1

10

Separation Factor

1.6 Kd values Pu(IV) Am(III) U(VI)

0.4

0

10

-1

0.0

10

0

1

2

3

4

5

6

[HNO3], M

FIG. 6 Influence of aqueous feed acidity on the distribution coefficient (Kd) and separation factor of actinides on HONTA resin. Reproduced with permission from R.B. Gujar, S.A. Ansari, P. K. Mohapatra, R.J.M. Egberink, J. Huskens, W. Verboom, Highly efficient extraction chromatography resin containing hexa-noctyl nitrilotriacetamide (HONTA) for selective recovery of plutonium from acidic feeds, Ind. Eng. Chem. Res. 62 (2023) 5954–5961.

(0.5 M nitric acid), primarily due to higher Kd value of Pu4+ as the Kd values of Am and U were insignificant. This feature illustrates the excellent selectivity of HONTA resin for the tetravalent actinide ion over the tri- and hexavalent actinide ions. The loading capacity for Pu was 58.2 mg/g resin corresponding to the formation of 1:1 metal/ligand complex. More than 20 mg of Pu4+ could be loaded onto the column prepared with 0.4 g of HONTA resin, and the efficient elution of Pu4+ was obtained with 0.5 M oxalic acid + 0.5 M nitric acid eluent. The column was also suitable for analytical separation of Pu from a mixture of actinides (Am, Pu, and U).

4 Room temperature ionic liquids in extraction chromatography Room temperature ionic liquids (RTILs) are being studied as alternative diluents to conventional solvents, and the metal ion extractions in

125

RTILs were shown to be extraordinary in a few cases [73–78]. Solvent extraction studies employing TODGA, C4DGA, and T-DGA for the extraction of Am3+ were highly encouraging due to manifold increase in its extraction capability in RTIL [79–82]. Similar results with RTIL were reported for XC resins [83,84]. In this section, the results with XC resins containing TODGA, T-DGA, and C4DGA in a RTIL, namely 1-butyl-3-methylimidazolium bis (trifluoromethanesulfonyl)imide (C4mimTf2N), will be discussed.

4.1 TODGA/RTIL resin A series of XC resins were prepared by varying the composition of TODGA and RTIL (C4mimNTF2) impregnated on Chromosorb-W [85]. In the first series, TODGA composition varied between 10% and 30% w/w at a fixed (10% w/w) RTIL concentration. In the second series, the RTIL composition was varied between 10% and 30% w/w at a fixed (10% w/w) TODGA concentration (Table 3). In further set of resins, both the TODGA and RTIL composition were varied simultaneously from 10% to 30% w/w. This exercise was performed to investigate the effect of ligand and RTIL on the extraction of actinides. Interestingly, no change in the extraction behavior of the resin was observed at any composition over a wide range of acidities (0.01–6 M nitric acid), and the Kd values of Am3+ were in the range 1  104 to 2.5  104 mL/g for all resins. The Kd value was distinctly different from those obtained with resin prepared from TODGA (without RTIL) impregnated on Chromosorb-W [34], where the Kd of Am3+ increased from 0.01 M nitric acid (Kd < 1) to 6 M nitric acid (Kd ¼ 7500). Additionally, the Kd of Am3+ was less with pure TODGA resin as compared with the TODGA/RTIL resin even with 47% w/w loading of ligand in the former vis-a`-vis 10% w/w loading in the later. High Kd value for Am3+ at lower acidity (0.01 M nitric acid) with TODGA/RTIL resin

126

6. Extraction chromatography of actinides

TABLE 3

Composition of TODGA-RTIL resins prepared in various composition [85].

Resin

Weight ratio (L: RTIL: solid)

TODGA (% w/w)

RTIL (% w/w)

Kd-Am at 0.01 M HNO3

Kd-Am at 3 M HNO3

XC1

0.2 g: 0.2 g: 1.6 g

10

10

20,770

15,785

XC2

0.4 g: 0.4 g: 1.2 g

20

20

14,310

17,170

XC3

0.75 g: 0.75 g: 1.0 g

30

30

15,870

19,780

XC4

0.2 g: 0.4 g: 1.4 g

10

20

17,315

11,540

XC5

0.2 g: 0.6 g: 1.2 g

10

30

15,930

17,465

XC6

0.4 g: 0.2 g: 1.4 g

20

10

12,285

11,085

XC7

0.6 g: 0.2 g: 1.2 g

30

10

12,895

17,850

was an indication of an ion-exchange extraction mechanism, and indicated difficulty in stripping the metal ions from the loaded resin. However, the sorbed metal ions was efficiently eluted with a complexing solution (EDTA), where the Kd of Am3+ was 3  104) and remained constant in the range of 0.01–4 M nitric acid. On the other hand, the Kd value for T-DGA resin decreased linearly with increasing nitric acid concentration. The distribution pattern for Am3+ ion on

128

6. Extraction chromatography of actinides 4

4x10

4

3x10

T-DGA 4

Kd, Am(III)

2x10

C4DGA 4

1x10

3

8x10

3

6x10

0

1

2

3

4

5

6

[HNO3], M

FIG. 9

Influence of nitric acid concentration on the distribution coefficient of Am3+ on T-DGA and C4DGA/RTIL resins. Reproduced with permission from R.B. Gujar, S.A. Ansari, W. Verboom, P.K. Mohapatra, Multi-podant diglycolamides and room temperature ionic liquid impregnated resins: an excellent combination for extraction chromatography of actinides, J. Chromatogr. A 1448 (2016) 58–66.

the two resins indicated different extraction mechanisms. Whereas an ion-exchange mechanism was likely for the T-DGA resin, a “solvation” extraction mechanism was more

TABLE 4

appropriate for the C4DGA resin between 0.1 and 4 M nitric acid. This behavior is in sharp contrast to the observations made with the same resins prepared in n-dodecane, where the Kd value of Am3+ increased gradually between 0.1 and 3 M nitric acid and remained constant thereafter [48]. As evident from Fig. 9, the Kd value of Am3+ at any acidity was higher with T-DGA resin as compared to C4DGA resin. The Kd of Am3+ for both resins in n-dodecane were several folds lower than for those prepared with RTIL (Table 4). A similar effect was observed in solvent extraction studies, where the distribution ratio (D) for the metal ions was many folds higher in RTIL than in a paraffinic solvent such as n-dodecane [79]. As the Kd of Am3+ at lower acidities was higher, stripping the column with dilute acid solution was not possible, and required a complexing reagent, such as guanidine carbonate and EDTA [87]. Reusability of the resin was evaluated by operating successive sorption and desorption cycles with Am3+ at 1 M nitric acid. The Kd with T-DGA resin remained constant, within the limits of experimental error. C4DGA resin exhibited poor reusability, where the Kd value decreased about 40% of the original value in the second cycle. This decrease in the Kd

Distribution coefficient of metal ions (Kd) by T-DGA and C4DGA resins. Kd at 3 M HNO3

Metal ions

T-DGA resin

C4DGA resin

TODGA/RTILa

TODGAb

Eu3+

50240  3510

37550  2070

18415  810

9018

3+

30910  1975

8940  1840

1575  735

7212

3+

47590  2880

9710  585

–

–

Pu

37210  1960

34680  1935

17840  815

5130

UO2+ 2

80  3.8

10  0.4

70.5  5.4

110

2+

5.5  0.3

BkIII) > Cm(III) > Am(III) (b) Sc3+ > U(VI) > Y3+ > Lu3+ > Yb3+ > Tm3+ > Er3+ > Bi3+ >Ho3+ > Tb3+ > Eu3+ > Gd3+ > Dy3+ > Sm3+ > Nd3+>Pr3+ >Ce3+ > La3+

[96,97]

TRU resin (octylphenyl-N,N-di-isobutyl carbamoylphosphine oxide (CMPO))

237

[98,99]

U/TEVA resin, diamyl amylphosphonate (DAAP)

237

[100]

Np, 241Am, 239Pu, 232Th, 238U

Np, 241Am, 239Pu, 232Th, 238U

4 Chelating phases for ion-exchange chromatography The previous discussion utilized complexation in the mobile phase to vary separation selectivity. Their apparent disadvantage is associated with a need to use rather strong and sometimes expensive complexing reagents as eluents. Also, separated metals are eluted in the form of stable complexes requiring

additional effort for further treatment of these complexes and complexing reagents. A possible solution is the use of adsorbents with chelating functional groups that provide a selective recognition of metals through the formation of coordination compounds or associates in the stationary phase. In this case, the eluent is composed of common mineral acids and electrolytes like nitrates, chlorides, and perchlorates of alkali metals.

4 Chelating phases for ion-exchange chromatography

A simple preparation of chelating ionexchangers is by impregnation and dynamic modification. Originally, these types of stationary phases were designed for extraction chromatography [101], where hydrophobic adsorbent, for example, PTFE, is coated with a solution of chelating reagent in the organic solvent, immiscible with aqueous eluents. The column efficiency of this liquid-liquid separation system was relatively low due to poor mass transfer in the surface liquid layer, and this type of stationary phase was replaced with impregnated and dynamically modified adsorbents. In the first case, the PS-DVB particles were saturated with a solution of a bulky aromatic reagent in an organic solvent, which provided swelling of the resin and a more efficient penetration of the reagent into the particle volume. Further washing with water caused the shrinking and entrapment of the reagent. The metallochromic indicators, such as xylenol orange, methyl thymol blue, phthalein purple, and others (Table 5) were used for impregnation due to the presence of aromatic rings in their structure along with chelating groups, which provide additional coordination of reagent on the aromatic backbone of the polymer resin. In many recent publications, column chromatography using impregnated and dynamically modified adsorbents is considered a variation of extraction chromatography. Efficient and selective separations of lanthanides and actinides were reported for adsorbents dynamically modified with diethyl-(2-ethylhexyl)phosphoric acid (DEHPA) [96,97] and with commercial resins TRU and UTEVA [98–100]. The structures for chelating extractants are shown in Table 5. More information on extraction chromatography can be found in Chapter 6. The separation selectivity of chelating ionexchangers depends on the type of chelating groups and their charges. All chelating groups contain polar atoms (O, N, S, P, etc.) which are responsible for the coordination of metals, and in many cases, chelating groups are charged.

201

The retention of metal ions due to the sole formation of surface complexes is possible for neutral ligands such as β-diketonates and crown ethers (see examples in Table 5). In this case, the contribution of electrostatic interactions to the retention of metal ions is minimal. Chelation is also a dominant retention mechanism for adsorbents with positively charged ligands, but the repulsion between protonated functional groups (e.g., 8-hydroxyquinolinol, polyamines, amidoxime, etc.) and metal cations can reduce retention in proportion to the charge and size of the ions. Undoubtedly, the most efficient separations in chelation ion chromatography have been reported for adsorbents possessing negatively charged groups (iminodiacetic acid, phosphonic acid, glyphosate—see Table 5). The reason is the long-distance electrostatic attraction of cations, which brings them into close proximity to the chelating groups for the subsequent formation of surface complexes. Due to the improved mass transfer, very efficient separations are possible as shown in Fig. 7. The relative contribution of electrostatic interactions in the total retention of metals depends on the stability constants of surface complexes and can be evaluated from the dependence of the distribution constant KD or retention factor k of metals on their stability constants β1. The linearity of the log k (or log KD)–log β1 plot indicates a prevalence of chelation in retention mechanisms. A corresponding plot for iminodiacetic acidfunctionalized silica is shown in Fig. 8. In this case, electrostatic interactions were suppressed by using an eluent containing 0.8 M potassium nitrate, as described earlier. It should be noted that the bonding chemistry can influence the chelating properties of an adsorbent. For example, IDA-functionalized adsorbents have been prepared by many different methods [66,102,103]. The most common method includes silica surface activation with 3-glycidoxypropyltriethoxysilane followed by a reaction with IDA [66]. This adsorbent

202

FIG. 7

9. Chelation ion chromatography

Separation of metal ions by using chelating adsorbents with negatively charged functional groups. Top: HEIDAsilica, 5 μm, 250  4.0 mm ID; eluent 25 mМ HNO3—0.75 М KNO3, 1 mL/min; 75°C; detection: PCR with Arsenazo III at 650 nm. Bottom: AMP-silica 3 μm, 150  4.0 mm ID; eluent 50 mМ HNO3—0.8 М KNO3, 1 mL/min; detection: PCR with PAR-ZnEDTA at 510 nm. Adapted from P.N. Nesterenko, P. Jones, Recent developments in the high-performance chelation ion chromatography of trace metals, J. Sep. Sci 30(11) (2007) 1773–1793.

5 Application areas of chelating ion-exchangers

203

FIG. 8 Correlation between retention of lanthanides on HEIDA-silica and stability constants of corresponding complexes with IDA and 2-hydroxyethyliminodiacetic acid (HEIDA). Chromatographic conditions are the same as in Fig. 7.

structure is shown in Table 5. The adsorbent contains a hydroxyl group in a β-position relative to nitrogen, and this group can coordinate metal ions too [61]. Due to this additional coordination, the stability of lanthanide complexes is much higher for 2-hydroxyethyliminodiacetic acid (HEIDA) compared to IDA (Fig. 8). Therefore, it is reasonable to consider a bonded ligand, such as HEIDA, rather than IDA. The log k  log β1 dependence of lanthanides separated using the earlier mentioned adsorbent show a stronger correlation with the HEIDA stability constants compared with IDA, as shown in Fig. 8. Competitive metal complexation between ligands in the eluent and surface-bonded chelating groups provides additional control over the retention order and separation selectivity of metal ions for specific applications [104].

5 Application areas of chelating ion-exchangers The complexing capability of cation ionexchangers is typical for various comprehensive IC columns. If diluted, noncomplexing mineral acids (methanesulfonic, perchloric, nitric) are used as eluents, complexation in the stationary phase is notable for carboxylicand phosphonic-type cation-exchange resins. These resins are also known as weak cationexchangers. Modern cation-exchange resins (Hamilton PRP-X800, IonPac CS19, Metrosep C1) contain covalently grafted poly(butadiene-maleic acid) or polyitaconic acid fragments, which have a superior selectivity for the separation of alkali- and alkaline-earth metals. A combination of phosphonic and carboxylic acid ion-exchange groups was used to increase the separation selectivity of

204

9. Chelation ion chromatography

manganese (II) from co-eluted alkaline-earth metal cations and the intragroup selectivity of lanthanides [105]. This design was used for the IonPac CS12 and IonPac CS12A (ThermoFisher, United States) chromatographic columns. It should be noted that carboxylic and phosphonic acid cation-exchangers can operate as chelating resins if electrostatic interactions are suppressed by the addition of concentrated potassium nitrate to the eluent [106]. Commercially available weak base anion-exchangers, for example, common aminopropylsilica can be also used in chelation ion chromatography as two adjacent amino groups can effectively coordinate metal ions [107]. Some complexing adsorbents saturated with transition metal ions are also produced for ligand-exchange chromatography [108] and related immobilized metal affinity chromatography (IMAC) [109]. Due to the high separation selectivity and elevated tolerance to the ionic strength of the samples, chelation ion chromatography can be used for the analysis of brines [9,110], seawater [111], mussels [78], and calcareous skeletons of marine plankton [112]. The application of CIC is especially effective for the direct determination of transition metals [113], alkaline-earth metals [61,114], aluminum [64], and other metals in seawater. Complexation assists in speciation analysis, for example, of iron(II) and iron(III), when the addition of complexing reagents stabilizes different oxidation states during the separation [115]. The determination of transition metals in fuel ethanol also has been accomplished by CIC [65]. This is a small part of reported applications and more can be found in monographs [1,26,116].

Abbreviations CIC CMPO CSA DAAP

chelation ion chromatography octylphenyl-N,N-di-isobutyl carbamoylphosphine oxide camphor-10-sulfonic acid diamyl, amylphosphonate

DAP DBSA DCTA DEHPA DNNS DTPA EDTA EGTA EHPA En HDS HIBA HMBP IDA NTA PAR PCR PTFE REE TBA UHPLC

1,2-diaminopropionic acid dodecylbenzenesulfonic acid 1,2-diaminocyclohexanetetraacetic acid di-(2-ethylhexyl)phosphoric acid di-nonylnaphtahenesulfonic acid diethylenetriaminepentaacetic acid ethylenediaminotetraacetic acid ethylene glycol-bis(2-aminoethyl ether)-N,N,N0 , N0 -tetraacetic acid 2-ethylhexylphosphonic acid mono-2-ethylhexyl ester ethylenediamine sodium hexadecanesulfonate α-hydroxyisobutyric acid bis(1,1,3,3-tetramethyl)phosphinic acid iminodiacetic acid nitrilotriacetic acid 4-(2-pyridylazo)resorcinol postcolumn reaction polytetrafluoroethylene rare-earth elements tetrabutylammonium ultra-high-performance liquid chromatography

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C H A P T E R

10 Displacement chromatography with ion-exchangers Guofeng Zhaoa and Yan Sunb a

Analytical and Pharmaceutical Sciences, Shell BioTech, Shanghai, China bDepartment of Biochemical Engineering, School of Chemical Engineering and Technology and Key Laboratory of Systems Bioengineering and Frontiers Science Center for Synthetic Biology (Ministry of Education), Tianjin University, Tianjin, China O U T L I N E 1. Principles of displacement chromatography 1.1 Basic concepts of displacement chromatography 1.2 Variant forms of displacement chromatography 1.3 Theoretical models for displacement chromatography 2. Ion-exchange displacers 2.1 Displacers for ion-exchange chromatography

211 212 213 215

3. Applications of ion-exchange displacement chromatography 3.1 Displacer chromatography process development and optimization 3.2 Applications

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4. Prospects and outlook

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References

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215

1 Principles of displacement chromatography 1.1 Basic concepts of displacement chromatography Displacement chromatography [1–4] is an operating mode that is different from elution chromatography more typically used for analytical

Ion-Exchange Chromatography and Related Techniques https://doi.org/10.1016/B978-0-443-15369-3.00003-1

2.2 Approaches for displacer screening and design

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separations. In displacement chromatography, most of the components in the feedstock bound to the stationary phase are displaced by the continuous input of a compound called a displacer. The displacer is selected/designed to have a higher affinity for the stationary phase than all or most of the components in the feedstock. As the displacer binds to the stationary phase, most of the

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FIG. 1

10. Displacement chromatography with ion-exchangers

Chromatographic profiles of (A) elution chromatography versus (B) displacement chromatography.

components in the feedstock are displaced by the displacer due to their lower binding affinity, moving ahead of the displacer flow (zone). As the components move through the column, any component of higher affinity for the stationary phase will in turn serve as the displacer of the components of a lower affinity, and the final distribution of the components will be a “displacement train” (Fig. 1B), in which the displaced components are separated but connected to each other. This leads to the separation of different displaced components with high concentration and high purity. After all the displaced components are discharged from the column, the displacer is eluted using a strong eluent, and the column is regenerated for the next operation. Displacement chromatography has several distinct differences from elution chromatography. In elution chromatography, target components are resolved into separate peaks, usually baseline separated. To achieve an acceptable separation, the loading amount must be controlled at a reasonably low level, and column capacity cannot be fully utilized. On the contrary, in displacement chromatography, a high loading amount is the prerequisite for the development of a displacement train, otherwise the components cannot reach the equilibrium concentration for displacement and will be mixed with adjacent component zones. Therefore, column utility is greatly improved, and preparative-scale samples can be processed on analytical-scale columns. Another

difference is that in a fully developed displacement train, the displacer moves after the displaced components train, while an eluent must penetrate through the target components to take effect. Displacement chromatography can be operated practically on any kind of chromatography column and target components, provided that a suitable displacer is available. Numerous examples have been reported for reversed-phase [5–7], normal-phase [8], hydrophobic interaction [9–13], and ion-exchange columns, with isotopes [14,15], small molecules [16], peptides [5], proteins, or nucleic acids [17,18] as target products. As the subject of this book is ion-exchange chromatography, only ion-exchange displacement chromatography will be discussed here, with a focus on biomolecules.

1.2 Variant forms of displacement chromatography 1.2.1 Selective displacement chromatography In a typical displacement chromatography process, the displacer should have a higher affinity for the stationary phase than all the sample components. This may not be possible for real samples with complicated compositions. In this case, selective displacement chromatography [19–21] is of value, where target components are displaced but strong-binding impurities are either retained on the column or eluted in the

1 Principles of displacement chromatography

213

FIG. 2 Variants of displacement chromatography process. (A) Selective displacement chromatography; (B) sample displacement chromatography.

displacer region (Fig. 2A). Also, there may be very weak-binding impurities eluted by either the mobile phase or the salts induced by the displacer in ion-exchange chromatography. Another case for complicated displacement systems is “spacer displacement chromatography” [22] with heterogenous displacers. In this case, some weaker-binding components in the displacer will appear in the middle of the displacement train and serve as the “spacer” for different target components. This may improve the recovery and purity of the products. 1.2.2 Sample displacement chromatography The choice of the displacer is the key to a successful displacement chromatography process. However, it is not always easy to find a displacer with good performance, low cost, and minimal safety concerns. Therefore, “sample displacement chromatography” [23–28], or elution-modified displacement chromatography [29,30], was proposed. As the name suggests, no displacer is used in sample displacement chromatography except the load sample itself. A stronger-binding component in the sample, which must also be of sufficient abundance, is eluted under traditional conditions and acts as the displacer for other components (Fig. 2B). Though not a typical displacement process in a strict manner, sample displacement chromatography can still adopt the advantages and theories of displacement chromatography.

1.2.3 Complex displacement chromatography In routine displacement chromatography, the displacer interacts with the stationary phase and displaces the target components into the solution. However, in complex displacement chromatography [31], the displacer competes with the stationary phase in binding to the targeted component. Once sufficient displacer is bound to the target component, the displacer-target complex is dissociated from the stationary phase and released into the mobile phase. This mode is also described as ligand-assisted displacement chromatography in the separation of metal ions [32].

1.3 Theoretical models for displacement chromatography Numerous efforts have been made to establish theoretical models for the displacement process to aid the development of displacement chromatography. A simple and straightforward model is the operating line plot [2,3,33] (Fig. 3). To design/predict a displacement process, the static binding isotherms of the target components and the displacer need to be determined. The operating line is a straight line drawn from the origin of the isotherms to a point on the displacer isotherm that corresponds to the displacer concentration

214

FIG. 3 Operating chromatography.

10. Displacement chromatography with ion-exchangers

line

plot

for

displacement

employed. The intersection of the operating line and the target component isotherm depicts the concentration of the component in an isotachic displacement train. The operating line plot is an ideal model for displacement chromatography where the binding characteristics of all components are independent from each other, and the mass transfer rates in the mobile and stationary phases are infinite. In real situations, however, there are several deviations. The presence of multiple components will

lead to a competitive binding phenomenon, and slow mass transfer rates will lead to a concentration gradient at zone boundaries, which are denoted as “shock layers” (Fig. 4). The components in the shock layer region are a mixture of two or multiple components and cannot be retrieved as a purified product. Moreover, for macromolecules in ion-exchange chromatography, multiple binding sites on the stationary can be either occupied or shielded by a single solute molecule. Once the macromolecule is displaced by small molecule displacers, the shielded binding sites will also be occupied by displacer molecules, releasing salt counter-ions into the mobile phase, and thus inducing a salt gradient. All these factors contribute to the outcome of the displacement process. Cramer et al. proposed a steric mass action (SMA) model [34–37] to describe the adsorption behavior of macromolecules in ionexchange systems. It is a three-variable equation (Eq. 1) where the solute concentration on the stationary phase, Q, is dependent on the mobile phase solute concentration C and the mobile phase salt concentration Cs. The SMA parameters ν (the characteristic charge), σ (the steric factor), and K (the equilibrium constant)

FIG. 4 Shock layers in displacement chromatography. (A) Shock layer of a single component; (B) shock layer between two components. Figure redrawn from J. Zhu, G. Guiochon, Production rate of an isotachic train in displacement chromatography, AIChE J. 41(1) (1995) 45–57.

2 Ion-exchange displacers

can be experimentally determined for a given combination of solute and stationary phase. Using the SMA binding isotherm, the displacement profile of proteins can be accurately predicted with induced salt gradients considered.   ν Q Cs C¼ K Λ  ðν + σ ÞQ

(1)

where C and Q are the solute concentrations in the mobile and stationary phases, respectively; Cs is the background salt concentration; Λ is the total ionic bed capacity; ν, σ, and K are the SMA parameters. Guiochon et al. [38–40] used a competitive Langmuir model and a constant pattern approach to model the shock layer thickness in two-component displacement chromatography. The shock layer thickness is dependent on mobile phase velocity, the concentration and retention factor of the displacer, and the separation factor of the two components. The rationale was further developed by Natarajan and Cramer [40] using the steric mass action formalism and solid film linear driving force model to accurately describe the effects of flow rate, particle diameter, and degree of difficulty for the separation on shock layer pattern and the yields obtainable in ion-exchange chromatography. The simulated displacement chromatography profile matches the experimental results and can potentially be used for the in silico optimization of ion-exchange displacement parameters.

2 Ion-exchange displacers 2.1 Displacers for ion-exchange chromatography The displacer is an indispensable component of displacement chromatography technology. Several factors make a suitable displacer.

215

A displacer candidate should have a high affinity to common stationary phases for use with diverse target molecules; an effective means of column regeneration is also a prerequisite; and it should preferably be of high purity, inexpensive, nontoxic, and easy to separate from the target product. Various existing molecules, either natural or synthetic, have been utilized as ion-exchange displacers. These are summarized for highmolecular-weight and low-molecular-weight ion-exchange displacers in Tables 1 and 2, respectively. In 2007, Sachem, Inc. launched a set of commercial ion-exchange displacers for protein separation, i.e., Expell, Isolis, and Propell displacers, both in SP (cation-exchange) and Q (anion-exchange) forms. These were the first set of dedicated commercial displacer products for industrial applications. The chemical compositions of these displacers are proprietary, but from related patents, it can be surmised that the cation displacers are linear oligomers of organic amines while the anionic displacers are polysulfonates with aromatic rings (Fig. 5). A regeneration solution, Regenerate, was also provided by Sachem, Inc. to facilitate column regeneration after displacement operation.

2.2 Approaches for displacer screening and design The selection and design of ion-exchange displacers are relatively straightforward—to screen or design positive or negative polyions. The candidate molecules can be evaluated by column experiments or batch adsorption. As the most important aspect for displacers is the affinity to the stationary phase, the common approaches for compound screening in drug discovery can be readily adopted. Vutukuru and Kane [47] used surface plasmon resonance (SPR) spectroscopy for the high-throughput screening of displacers. The candidate molecule library is immobilized, as self-assembled monolayers, and the affinity of two model

TABLE 1

High-molecular-weight displacers employed for the ion-exchange displacement chromatography of proteins.

Stationary phase

Displacer

Proteins

References

DEAE Sephadex A 50 DEAE Sephacel DEAE cellulose

Carboxymethyl dextrans

Ovalbumin β-lactoglobulins A and B Serum proteins RNA polymerase II

[1]a

CM cellulose

Ampholine (pH 4–6)

Human serum albumin α-Fetoprotein

[1]a

TSK DEAE 5PW

Chondroitin sulfate Polygalactouronic acid

β-lactoglobulins A and B

[1]a

TSK DEAE 5PW

Carboxymethyl dextrans

Alkaline phosphatase

[1]a

TSK DEAE 5PW

Chondroitin sulfate

β-lactoglobulins A and B

[1]a

RG Bio PSM 300

Nalcolyte 7105 PEI 600

Cytochrome c and lysozyme RNAse, α-chymotrypsinogen A, and lysozyme

[1]a

TSK DEAE 5PW

Chondroitin sulfate

β-galactosidase

[1]a

Matrex PAE-300

Polyvinyl sulfonic acid

Ovalbumin, conalbumin

[1]a

Tris Acryl DEAE M

Carboxymethyl starch

Lactate dehydrogenase

[1]a

Matrex PAE-300

Dextran sulfate Polyvinyl sulfonic acid

β-lactoglobulins A and B

[1]a

Fractogel DEAE 650S DEAE cellulose

Carboxymethyl dextrans

Guinea pig serum proteins, mouse liver cytosol proteins

[1]a

Protein-Pak SP-8HR

Protamine sulfate

α-chymotrypsinogen A, cytochrome c, and lysozyme

[1]a

Protein-Pak Q-8HR

Heparin

β-lactoglobulins

[1]a

Waters SP-8HR

DEAE-dextrans

α-chymotrypsinogen A, cytochrome c

[1]a

Waters Q-8HR Waters DEAE-8HR

Pentosan polysulfate Dextran sulfate

β-lactoglobulins A and B, α-lactalbumin, soybean trypsin inhibitor

[1]a

Protein-Pak Q-8HR

Block methacrylic polyampholytes

β-lactoglobulins A and B

[1]a

POROS 10HQ POROS HQ/M

Heparin

β-lactoglobulins A and B

[1]a

POROS HS/M POROS HS50

Protamine sulfate

rHu-BDNF

[1]a

UNO Q1 and Q6

Polyacrylic acid

α- and β-lactalbumins

[1]a

Q2 strong anionexchanger

Copolymer of N,N-dimethylacrylamide and 7-hydroxy-3-methyl-4-vinylindanone

α-lactalbumin and β-lactoglobulin

[41]

Bio-Scale S2 strong anion-exchange column UNO S1 column (monolith)

Poly(diallyl dimethyl ammonium chloride)

Lysozyme and cytochrome c

[42,43]

UNO Q column (monolith)

Polyacrylic acid

Plasmid, protein, and lipopolysaccharides

[44]

a

Reference [1] is a book chapter that summarized the ion-exchange displacers reported before 2000.

TABLE 2 Low-molecular-weight displacers ( λE (e.g., positive peak with potassium acid phthalate eluent) and “indirection detection” ¼ λS < λE (e.g., negative peak with nitric acid eluent). In both cases, these IC system configurations use a relatively low-capacity ionexchange column (0.01–0.05 meq/column). Nonsuppressed conductometry is generally less sensitive than suppressed detection while quite

sufficient for some applications. The presence of a system peak (negative or positive) in the chromatogram may interfere with the detection of some ions.

2.2 Electrochemical detection Electrochemical detection can be used for electroactive species, specifically those readily amenable to reduction or oxidation. There are different approaches. Amperometry measures the cell current at a constant potential; pulsed amperometry measures the cell current at the plateau of a pulsed potential; and coulometry measures the current at a constant potential with full conversion of the analyte. Amperometric detectors are some of the most selective and sensitive detectors for IC separations [7,8]. Electrochemical detector cells typically contain three electrodes: a working electrode (WE), where the electrochemical reaction occurs; a reference electrode (RE) for current potential measurement; and an auxiliary electrode (AE)

236

11. Instrumentation for ion chromatography

of opposite pole to the WE. The operation of an electrochemical detector is based on continuous amperometry. A certain potential is applied between the working and reference electrode, where solutes passing over the working electrode are reduced or oxidized. The instantaneous current is monitored and provides the detector signal (Fig. 10). Amperometric detection requires close control of eluent temperature, pH, and eluent flow (eluent flow through the reactor should be continuous and pulse free) to obtain stable and reproducible results. Pulsed amperometric detection (PAD) is the most common mode for IC applications, utilizing a measuring potential and two cleaning potentials to provide constant electrochemical regeneration of the electrode surface. In PAD, the detection is performed using a continuous series of cyclic potentials applied to the working electrode. The measured potential is applied first, and the current is measured after a suitable equilibration time. Afterwards, a large positive potential is applied to the electrode causing the oxidative removal of reaction products, and a subsequent negative potential to return the working electrode to its original state. The whole process is repeated with each cycle lasting typically less than 1 s, with each rapid measurement taken with what is essentially a freshly

FIG. 10

prepared electrode surface. The selection of the working electrode is an important consideration. Gold for carbohydrates, amino acids, amines, aminoglycosides, and thio-organics; platinum for sulfite, cyanide, alcohols, and hydrazine; silver for cyanide, sulfide, iodide, and other inorganic species; and glassy carbon for phenols catecholamines, and other organics. PAD has been used for the detection of anions, such as nitrite, nitrate, thiosulfate, and certain halide ions, following IC separation. However, the most significant application is for the determination of carbohydrates and amino acids.

2.3 Photometric detection Photometric detection includes direct photometry, indirect photometry, and fluorescence detection. A schematic diagram of a UV/Vis absorbance detector is shown in Fig. 11 and for a fluorescence detector in Fig. 12. Direct photometric detection in IC is useful for ionic species that exhibit absorbance in the UV/Vis region: organic acids, halides (such as bromide and iodide), oxyanions, including nitrate, nitrite, bromate, iodate, chlorate, and perchlorate [7,9]. Most inorganic cations do not absorb significantly in the UV/Vis region.

Schematic design of a pulsed amperometric detector cell.

2 Detectors for IC

FIG. 11

Schematic diagram of a UV/Vis absorbance detector.

FIG. 12

Schematic diagram of a fluorescence detector.

A combination of a postcolumn reactor and visible detection is commonly used to detect transition and heavy metal ions. Indirect UV/Vis absorption in IC requires the use of an absorbing eluent ion. For anion detection, benzoate- and phthalate-based eluents have been used for indirect UV absorbance detection [8]. This approach is able to accurately and sensitively detect “transparent ions” that are not visible to a photometric detector when the ions of the eluent absorb light. For example, the phthalate ion absorbs in the UV region (high background signal). The phthalate ion removes the target anions from the column, and their passage through the detector cell results in a drop in

237

optical absorbance and a negative peak. This approach has many advantages over nonsuppressed conductometry detection. A disadvantage is that it cannot be applied to eluent gradient separations. Direct fluorescence detection is rather uncommon for inorganic ions as only a few species exhibit significant fluorescence without pre- or postcolumn derivatization. The significant issues of background interference and quenching effects, which often hinder fluorescence detection, have reduced its utility in IC to a small number of applications, like the determination of pesticides and some pharmaceutical drugs.

238

11. Instrumentation for ion chromatography

2.4 Postcolumn reaction system Postcolumn reaction (PCR) is performed by mixing the column effluent and reagents at the column exit forming new products suitable for UV/Vis, fluorescence, or electrochemical detection. The main application of PCR is either to enhance detection sensitivity for a broad range of analytes or to provide selective detection of target analytes. Within the PCR system (Fig. 13), a reagent is supplied by a pump or by overpressure of a reagent bottle. The flow of reagent is usually mixed with the column effluent by means of a T-connector inside a heated reactor coil or mixer where the analytes are converted to new products with favorable detection properties. A typical example is the detection of amino acids after a reaction with ninhydrin in the case of the classical amino acid analysis. Further examples include the trace analysis of iodide, bromate, hexavalent chromium, other

FIG. 13

transition and heavy metals, and lanthanides after IC separation.

2.5 Mass spectrometry detection The suppressed conductivity detection feature of IC is what makes it one of the most compatible and useful forms of liquid chromatography as an inlet for mass spectrometry (MS). The IC suppressor not only eliminates the accumulation of salts from the ion source but also assists in reducing or eliminate the ion signal suppression. For coupling an IC to MS, an electrospray (ESI), heated electrospray (HESI), or inductively coupled plasma (ICP) ion source is typically used. IC-ICP-MS is used exclusively for speciation in trace elemental analysis (TEA). The ionization efficiency of the ICP is typically significantly higher than for an ESI source, and IC-ICP-MS is the technique of choice for speciation of elements

Apparatus for postchromatographic reaction detection.

239

3 Injection system

such as arsenic, selenium, mercury, chromium, sulfur, etc. IC-ESI-MS is typically used for the analysis of molecular species—polar anionic and cationic pesticides, disinfection byproducts, as well as major metabolites such as sugar phosphates. For analytes not readily ionized in solution, ESI-MS can present an attractive detection option and can provide high sensitivity. If the solute is cationic in solution, positive mode ESI is chosen and conversely, when the ions are anionic, negative ion mode ESI is more suitable. Weak acid solutes, such as carboxylic acids, are typically monitored in the negative ion mode. Neutral molecules poorly ionized by ESI (for example, sugars) can be detected by the formation of adduct ions with alkali metal salts.

2.6 Multiple detections For some complex samples, it may be helpful to use several detectors in serial. For example, the determination of a trace amount of nitrite in the presence of large amounts of chloride is possible with series coupled UV and CD detectors. For the simultaneous detection of sulfide and cations in water, it is possible to separate ions on a cation-exchange column and detect sulfide with a UV detector and ammonia and other cations by a CD detector with a suppressor. For the

FIG. 14

Flow paths for a six-port injection valve.

detection of all ions in a single sample it is important that the UV detector is placed in front of the suppressor. In case of multiple detections using a destructive detector, like electrochemical detectors, the destructive detector must be the last in the series.

3 Injection system 3.1 Injection valve with sample loop The injection system may be manual or automated, but both are based on the injection valve. The injection valve is typically a six-port, electrically actuated valve designed to introduce a precise sample amount into the column, usually by displacement from a sample loop. IC systems typically use 10–100 μL sample loops made of PEEK capillary tubing. The injection valve has two switchable operating positions: load and inject. Eluent flows through either the load or inject path, depending on the valve position (Fig. 14). In the load position, the sample is loaded into the sample loop, where it is held until injection. Eluent flows from the pump, through the valve, and to the column, bypassing the sample loop. The sample flows from the syringe or autosampler line (if installed), through the valve, and into the sample loop. The excess sample

240

11. Instrumentation for ion chromatography

flows out to waste. In the inject position, the sample is swept onto the column for separation. Eluent flows from the pump, through the sample loop, and onto the column, carrying the contents of the sample loop with it. The same flow scheme is used for loading and injecting samples with an autosampler by fullloop injection mode. The loop may be partially filled (partial loop injection). In this case, the loop must not be filled to more than 50% of the total loop volume otherwise the injection may not be precise.

3.2 Preconcentration Ion chromatography is frequently used to determine ions at low concentration levels, perhaps in the low ng/L (ppb) range. One way to extend IC methods to lower detection limits is to increase the loop volume (up to 2 mL, depending on column size and capacity). An alternative is to replace the sample loop with a concentrator column. A concentrator column is a short column (typically 30–50 mm in length) containing ionexchange resin. The function of the concentrator column is to strip ions from a relatively large volume of sample (from several mL up to several liters). This may require an auxiliary sample pump to flush the sample through the concentrator column and then to waste. The valve is subsequently switched to the inject position and the ions adsorbed by the concentrator column are eluted into the analytical column by the eluent for separation. The advantage of this system is the ability to perform routine analyses for ions up to pg/L (ppt) levels. This is a typical demand in the electric power and semiconductor industries.

4 Column oven A column oven is a useful option for trace analysis. The conductivity detector is sensitive to changes in eluent temperature and a column

oven can facilitate maintaining a constant eluent temperature. This will help to minimize detector noise and lower detection limits. The column temperature will also affect ion retention times and can be used as a variable in method development.

5 Column hardware The column consists of a column body and frits and is filled with the stationary phase. Guard columns have lengths from 30 to 50 mm and separation columns from 100 to 300 mm with inside diameters from 1 to 9 mm. IC column body, fittings, frits, and tubing are typically made of PEEK. The frits at both ends of the column hold the column packing in place. A replaceable frit at the top of the column acts as a filter to protect the column from particles. Guard columns generally contain the same material as the separation column and are located directly in front of the separation column. Guard columns are used for protection over the lifetime of the separation column from particles and dissolved contaminants. Guard columns are changed when the separation of a standard is no longer acceptable, or column pressure is beyond the normal range. A possible reason for a column pressure jump after several sample injections could be the clogging of the column frit with particles. In this case, a change of the frit at the top of the column can be helpful. The construction of a column body and type of fitting used to connect tubing to the column are shown in Fig. 15. Reusable PEEK fittings with ferrules are used almost exclusively to connect tubing to IC columns and other instrument components. During connection of the fitting, the tubing should be bottomed out or pushed completely into the column end before tightening, to ensure that there is no unnecessary dead volume in the connection. The same principle is used for the connection of any other instrument components.

References

FIG. 15

241

The construction of a column body and fittings used to connect tubing to the column.

References [1] B. Evans, The history of ion chromatography: the engineering perspective, J. Chem. Educ. 81 (2004) 1285–1292. [2] H. Small, T.S. Stevens, W.C. Baumann, Novel ion exchange chromatographic method using conductometric detection, Anal. Chem. 47 (1975) 1801–1809. [3] T.S. Stevens, J.C. Davis, H. Small, Hollow fiber ionexchange suppressor for ion chromatography, Anal. Chem. 53 (1981) 1488–1492. [4] H. Small, J.M. Riviello, C.A. Pohl, Ion Chromatography Using Frequent Regeneration of Batch-type Suppressor, 1997. Application filed by Dionex Corp in 1995, granted in 1997, US Patent 5,597,734.

[5] Thermo Scientific, Product Manual for Capillary Charge Detector Cell (QDC-300), 2014. [6] J.S. Fritz, D.T. Gjerde, Discovery and early development of non-suppressed ion chromatography, J. Chromatogr. Sci. 48 (2010) 525–532. [7] J. Weiss, Handbook of Ion Chromatography, WileyVCH, Weisbaden, 2016. [8] Н. Small, T.E. Miller, Indirect photometric chromatography, Anal. Chem. 54 (1982) 462–469. [9] P.R. Haddad, P.E. Jackson, Ion Chromatography: Principles and Applications, Elsevier, Amsterdam, 1990.

C H A P T E R

12 Instrument platforms for large-scale ion-exchange separations of biomolecules Anurag S. Rathorea, Anupa Anupab, Kanti N. Mihooliyaa, and Nitika Nitikaa a

Department of Chemical Engineering, Indian Institute of Technology Delhi, New Delhi, India School of Interdisciplinary Research, Indian Institute of Technology Delhi, New Delhi, India

b

O U T L I N E 1. Introduction

243

2. Chromatography columns

246

3. Ion exchange matrices 3.1 Process steps in ion-exchange chromatography

246

4. Chromatography equipment

248

5. Scale-up of ion-exchange processes

248

246

1 Introduction Ion-exchange (IEX) is a well-established technique for the separation of charged molecules [1,2]. It is based on ionic interactions between charged molecules and charged ligands on the chromatographic resin, thereby enabling the separation of biomolecules that differ by only one amino acid owing to the charge properties of biomolecules and their interaction with the

Ion-Exchange Chromatography and Related Techniques https://doi.org/10.1016/B978-0-443-15369-3.00025-0

5.1 Understanding the product and resin selection 248 5.2 Necessary calculations for IEC scale-up 254 5.3 Common problems associated with IEC scale-up from lab to manufacturing scale 258 References

259

chromatographic media [3,4]. This technique has been utilized for purifying enzymes [5–7], peptides [8], proteins [5,9], antibodies [9,10], as well as amino acids and nucleic acids [11,12]. The nature of ionic interactions can be binding or repulsive based on the net charge present on the biomolecule and the charged ligand. Fig. 1 illustrates the interactions between the oppositely charged molecules and ligands on the resin beads.

243

Copyright # 2024 Elsevier Inc. All rights reserved.

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12. Instrumental platforms for bioseparations

Sample flow

Anion exchange chromatography

Cation exchange chromatography

Sample injection (Column equilibrium)

Sample adsorption

Sample desorption

FIG. 1 Schematic representation of IEX showing the adsorption and desorption of the sample by counterions in anion exchange (left side) and cation exchange (right side).

In preparative chromatography, the resin is usually packed into glass or steel columns. The pressure resistance of the resin beads depends on the resin matrix which in turn determines its suitability for different applications. Resins with bead diameters >40 μm have low pressure resistance and are limited to purification in preparative scale chromatography. Beads of a smaller particle size, typically 3–10 μm, with a narrow size range are mostly used for analytical applications in high-performance liquid chromatography (HPLC). Based on the separation mechanism and properties of the stationary phase, the separation of biomolecules can be categorized by size exclusion, ion-exchange, hydrophobic interactions, reversed-phase, and

affinity chromatography. For size-exclusion chromatography (SEC) the stationary phase is typically composed of agarose beads of various pore sizes and isocratic mobile phases are used for the separation. Target compounds are eluted in decreasing order of molecular size. For hydrophobic interaction chromatography (HIC) separations result from both hydrophobic and charge-based interactions as proteins contain both hydrophobic and hydrophilic amino acids. The stationary phase contains long carbon chains that can interact with the hydrophobic patches of the protein. The sample is loaded on the HIC column at high ionic strength to minimize charge-based interactions so that the hydrophobic interactions dominate. Separations

1 Introduction

are typically performed with a decreasing ionic strength gradient. This results in a strengthening of the charge-based interactions and as a result proteins elute based on both their net charge and hydrophobicity. Due to this duality of interactions, HIC often offers unique selectivity compared to other modes of chromatography. HIC is extensively used in preparative scale chromatography as it is nondestructive and maintains the biological activity of the protein. Reversed-phase liquid chromatography (RPLC) uses an aqueous-organic solvent mixture as a mobile phase to control adsorption of the target compounds onto a hydrophobic stationary phase. This mode of chromatography is destructive for many proteins and is rarely used in for preparative scale applications where recovery of product with retention of its biological activity is important. Affinity chromatography is based on the molecular recognition of target molecule by purpose-designed ligands of the stationary phase. For example, for monoclonal antibody (mAb) products, affinity chromatography is often used as the first step for capturing the mAb product. The Fc region of a monoclonal antibody has an affinity toward Protein A and as a result for the isolation of monoclonal antibodies from other host cell proteins, Protein A chromatography is preferred. The Fc region of a monoclonal antibody binds to the Protein A resin and the remaining unbound impurities pass through the column. For elution of a monoclonal antibody, a low pH wash is

245

used to reverse the affinity of the monoclonal antibody to the Protein A resin [13]. Separations by ion-exchange chromatography (IEC) are categorized as anion-exchange chromatography (AEX) or cation-exchange chromatography (CEX) depending on the nature of the bound ligand of the stationary phase. In CEX, the bound ligand or resin matrix is negatively charged and retains positively charged molecules while negative and neutral molecules pass through the column. In AEX, the bound ligand or resin matrix is positively charged retains negatively charged compounds. To elute the resin adsorbed biomolecules one of two strategies are commonly employed. A mobile phase of higher ionic strength than the sample solvent allows the elution of the protein from the stationary phase [14]. Since the charge on a protein is pH dependent, a change in the pH of the mobile phase can be used to neutralize its charge state and elute it from the stationary phase. If the pH of the mobile phase is less than the isoelectric point of the protein (pI, the pH when the net charge of protein is zero), the net protein charge is positive and CEX can be used [15]. In CEX, the less positively charged proteins elute first followed by more positively charged proteins. If the pH of the mobile phase is higher than the protein pI, the protein is negatively charged and AEX can be used for the separation from other positively charged impurities. In AEX, the less negatively charged proteins elute first followed by the more negatively charged proteins.

246

12. Instrumental platforms for bioseparations

2 Chromatography columns Various types of columns are commercially available and dominate current practices. Column design impacts the separation process in terms of the ease of column packing, resin quality, available bed height, and scalability for large scale manufacturing [16]. Prepacked columns are available to facilitate rapid screening during early process development. Column manufacturers are making available larger prepacked columns, suitable for pilot and even commercial production in larger numbers. The column hardware varies with the internal diameter (I.D.) and column height, as well as among different vendors. Selection of a particular column depends on a number of factors including process scale, feed titer, and retention time needed for the molecule to bind efficiently. These factors as well as the impact of scale are discussed in the following sections.

3 Ion exchange matrices The versatile IEX unit operation allows a wide range of matrices to be employed for the purification of target biomolecules. The resin media is selected depending on whether IEX is used for capturing and concentrating the sample, for intermediate purification, or for final polishing. The resin porosity and bead size determine the

TABLE 1 Type of exchanger

resolving power and binding capacity of the resin. The type of resin charged functional group determines whether it is categorized as a weak or strong ion exchanger [17,18]. For example, the diethylaminoethyl (DEAE) functional group is a weak anion exchanger while a quaternary ammonium functional group is a strong anion exchanger. Likewise, for cation exchangers, a carboxymethyl (CM) functional group is a weak cation exchanger, and sulfopropyl (SP) functional group is a strong cation exchanger. Table 1 categorizes the different types of IEX resins based on charge and strength.

3.1 Process steps in ion-exchange chromatography The following process steps are typically required when performing ion-exchange chromatography: equilibration; sample loading; washing; elution; cleaning in place; and re-equilibration. The equilibration step is performed to acclimatize the chromatography resin to the binding buffer (pH, conductivity). These conditions are chosen according to the pI of the protein and the type of IEX chromatography to be performed (AEX/CEX). Typically, the conductivity of the binding buffer is kept low, preferably below 5 mS to minimize the effect of the counter-ions on the separation and to maximize product binding. Once the column is

Some commercially available IEX resins and their characteristics. pH range

Commercial name

Cl
, HCOO
, CH3COO
, SO42


2–12

Q Sepharose, Capto Q

Diethylaminoethyl (DEAE), O(CH2)2 N+H(C2H5)2

Cl
, HCOO
, CH3COO
, SO42


2–9

DEAE-Sepharose, Capto DEAE

Sulfopropyl (SP), (CH2)3SO3


Na+, H+

4–13

SP Sepharose, Capto S

6–10

CM Sepharose, CM Cellulose

Functional group

Counterion

Strong anion

Quaternary ammonium (Q), CH2N+(CH3)3

Weak anion Strong cation Weak cation




Carboxymethyl (CM), (OCH2COO )

+

+

Na , H

3 Ion exchange matrices

247

equilibrated, it can be loaded with the sample. Most commonly, the target protein is bound to the column resin (bind and elute mode). Alternatively, conditions are chosen such that the target protein flows through the column while the impurities bind to it (flow-through mode). For sample loading, the dynamic binding capacity (DBC) of the resin must be estimated. This is defined as the maximum amount of a target protein that can be loaded onto the column without loss of protein [19]. A higher DBC is desirable as it implies that a smaller amount of resin (smaller column) will be adequate for processing a fixed amount of sample. A series of binding experiments with varying amounts of the target protein is conducted to estimate the DBC. The results are plotted to determine the maximum amount of product that can be bound by a given amount of resin before reaching saturation. The DBC is an important factor in the development

of a biopharmaceutical manufacturing processes since it directly impacts the yield and column size required. To maximize DBC, several factors need to be optimized, such as pH, ionic strength, flow rate, and column capacity. Accurate determination of DBC is critical for successful design and eventual scale-up of the IEC step. Once sample loading is complete, the column is washed with the equilibration buffer to remove nonspecifically bound proteins or impurities that may be present in the void spaces of the column. This helps to enhance the purity of the bound target protein. After the washing step, the target protein needs to be eluted based on a change in pH or conductivity. For the latter, ions with a higher lyotropic property can be used to replace those with lower lyotropic properties based on the Hofmeister series [20].

For elution, different gradients can be utilized to change the concentration of the binding buffer compared with the elution buffer. When the target protein is bound to the column without any impurities bound to the column, a step gradient, that is, a sharp change in concentration of the elution buffer may be optimal. However, if the bound proteins contain both the target protein and other impurities, a gradual increase in elution buffer strength may be better as this will allow the weakly bound proteins to elute first followed by the release of strongly bound proteins. The simplest approach is a linear increase in the percentage of elution buffer. For those cases where impurities are closely related to

the product, a sigmoidal gradient may improve the separation [21,22]. Proteins eluting with different percentages of elution buffer are collected and analyzed for purity. Even with 100% elution buffer, some impurities may be tightly bound to the resin and require removal to regenerate the charged sites before the next sample can be applied. Depending on the resin matrix, cleaning can be achieved by extreme changes in pH, extreme changes in conductivity, or both. The most common cleaning buffer for high pH is NaOH, which offers high conductivity as well as high pH, thereby removing otherwise tightly bound impurities. In cases where such extreme pH changes cannot be used, a high

248

12. Instrumental platforms for bioseparations

concentration of salt (such as NaCl) is used [23]. In most cases, a mixture of NaOH and NaCl has proven to be efficient for resin cleaning [23]. Leaving the column in such denaturing conditions is likely to adversely affect the resin and thus a final equilibration is required to remove the cleaning buffer. This is achieved by flushing the column with a binding buffer. If the column is to be used again, the process can be continued from step two, if not, the column can be stored in a storage buffer as per the column manufacturer’s instructions. The most common storage solutions are 10 mM NaOH and 20% ethanol [24]. These help with the efficient long-term storage of columns without affecting their integrity.

4 Chromatography equipment For the purification of target proteins, the equipment used plays a critical role in designing the process steps. A variety of equipment is commercially available from different sources, for example, the AKTA systems from Cytiva are among the most popular automated chromatography equipment for lab-scale purification. Other notable manufacturers include MerckMillipore, Repligen, Phenomenex, and Pall Corporation. When choosing equipment, it is important to consider the scale, the type of separation, and the specific needs of the application. Typical chromatographic hardware consists of multiple pumps, valves, sensors, and the control panel which displays the status of the system and allows the user to pause or continue operation. Most vendors offer modular systems that can be customized to the application. Process chromatographs are complex equipment and require sophisticated software for process monitoring and control. For example, the UNICORN software from Cytiva is used to run the € AKTA chromatography systems. This software is quite user-friendly and allows multiple experiments to be performed simultaneously with real-time monitoring of chromatographic

parameters such as pH, pressure, conductivity, and other process variables. Most software is compatible with other data analysis platforms, such as Microsoft Excel, facilitating data capturing and processing during experimental runs for easy and efficient reporting. Depending on the scale, whether pilot or commercial, the equipment available varies in size, automation, and cost [25]. Key features of some pilot and commercial scale process chromatographs are presented in Table 2.

5 Scale-up of ion-exchange processes The widespread use of IEC for protein purification is due to its robustness and efficiency in a large-scale manufacturing environment [26–28]. Large scale operations are expensive as well as time consuming. As a result, it is common practice to perform key activities such as process development, process optimization, and process characterization at the lab scale (Fig. 2). The final process is then scaled-up to pilot or commercial scale as desired, with the scaling procedure based on factors such as the amount of resin, column size, column design, process parameters, and the desired process monitoring and control strategy [29–33].

5.1 Understanding the product and resin selection When designing an IEC step, it is important to understand the physicochemical properties of the product (like its isoelectric point), the resin (like retention, capacity, pore size, specificity), as well as, the impact of process variables (such as pH, particles size, solvents, types of salts and their concentrations, flow rate, gradient or isocratic, gradient shape and length, temperature, types of additives) on the performance of the separation [26,32,34]. The properties of the product are utilized for screening chromatographic resins (Fig. 3).

249

5 Scale-up of ion-exchange processes

TABLE 2

Chromatographic instrument for pilot and commercial scale operations.

Equipment

Manufacturer

Scale

Key features

AKTA Pilot 600

Cytiva

• 26–200 mm column ID • 0.1–1200 mL/min flow rate

Bridge transition to GMP

AKTA Process DCS

Cytiva

• 1–2000 L/h flow rate

Customizable with distributed control system

NGC Medium Pressure Chromatography System

Biorad

• Purification capacity from mg to g • Up to 100 mL/min flow rate

Preconfigured and customization provided and automated through ChromLab software

CoPrime Biochromatography

Merck Millipore

• Pilot and production scale • 180–1200 L/h

Fully automated and configurable system which provides gradient precision throughout the operating flow rate range

Resolute Flowdrive MU

Sartorius

• From clinical to commercial manufacturing

In compliance with CFR 21 which ensures data integrity

FIG. 2

Illustration of the various tasks that are performed before the commercial chromatography process is finalized. (1) High throughput and conventional methods are used for efficient screening of chromatography resins. (2) Process development, optimization, and characterization activities are performed using lab scale equipment. (3) Once the final commercial process has been defined, it is scaled up to be performed in the manufacturing plant.

250

12. Instrumental platforms for bioseparations

FIG. 3 The schematic view of screening of IEC resins. Pack resins in gravity columns or high-throughput microplates and check the binding of molecule of interest. Remove nonbinding resins and select the resin which has appropriate binding. Then, optimize process parameters with resin chosen. If selectivity is low, repeat the initial screening. At last, finalize the elution mode, analyze yield and purity, and scale up to manufacturing. Check reusability and perform economic analysis [35,36].

(a) High-throughput screening (HTS) of the chromatographic resins HTS of IEC resins using automated, platforms are becoming increasingly common in the biopharma industry. Automated stations enable handling of solutions and suspensions to determine the dynamic capacity of resins and the elution profiles of protein mixtures at the microliter scale [26,37–40]. Various highthroughput platforms such as microplates and prepacked column kits are commercially available for high-throughput screening of IEC resins. Table 3 describes a few representative products for high-throughput resin screening. Furthermore, robotic platforms can offer information about static capacity and uptake

kinetics, traditionally derived from otherwise tedious batch experiments. Not only are automated platform rapid and efficient, but they are also significantly more robust than manual operations [38]. (b) Critical components of resin screening Typically, conventional screening of chromatographic resins involves using small gravity columns with volumes ranging from 1 to 10 mL [26]. Column experiments include isocratic and gradient elution experiments with frontal analysis, while batch experiments measure static capacity and uptake curves [42]. The screening of more than 20 anion-exchange resins and 25 weak and strong cation-exchange resins for

251

5 Scale-up of ion-exchange processes

TABLE 3

High-throughput platforms for resin screening.

S. no.

Platform or product

Applications and characteristics

References

1

AcroPrep filter plates from Pall Corporation

• 24-, 96-, and 384-well microplates with the accurate dispense of nanoliters of liquid • Very low hold-up volume • Withstand high flow rate and allow the rapid screening • High-throughput resin screening

[41]

2

Predictor 96-well filter plates from Cytiva

• 96-well plates are prepacked with commercial chromatography media • It can be operated in manual or automated mode • It also determines adsorption isotherms

[40]

3

MediaScout minicolumn from ATOLL GmbH

• It includes a base plate with a standard 96-well microtiter plate and up to 96 prepacked mini columns filled with resins • Customization can be implemented as per the requirement of the process

[40]

purification of proteins, for example, lipolase (pI of 4.3), BSA (pI of 5–5.2), insulin precursor (pI of 5.3), anti-FVII mAb (pI of 6–7), myoglobin (pI of 7–8), aprotinin (pI around 10.5), and lysozyme (pI around 11) have been reported [43]. Apart from resin screening, a vital attribute to keep under consideration is the selectivity or binding strength of the resin [32]. This refers to the ability of the resin to preferentially bind or interact with target proteins in a mixture over others. The selectivity of a resin depends on several factors, including the functional groups on the resin, the buffer pH, and the ionic strength of the eluent [32,44]. A classical isocratic retention measurement assesses the binding strength, where equilibration and protein elution are carried out at equal ionic strength. The ratio of the retention volume to the column volume is a measure of the binding strength. In cases where the target protein binds weakly due to a high-conductivity loading solution, resins with the strongest retention should be selected for further testing [26]. However, if a protein binds strongly to all resins, a resin with strong binding at low salt concentrations should be selected to minimize salt consumption, especially if a pH

gradient cannot be used for elution [26]. Apart from selectivity, other considerations that are likely to impact the choice of resin include resin cost, the physical and chemical stability of the resin, bed height, lifetime and reusability of the resins, availability with respect to desired manufacturing-scale, leaching of ligands, and batch-to-batch variation in resin quality [32]. 5.1.1 Column design and size Column design plays a crucial role in the scale up to large scale operations. Column diameter, bed height, column material and strength, and packing density should be optimized to achieve maximum efficiency and productivity. For IEC, scale-up is typically performed linearly keeping the product loading constant (Fig. 4). First, a scale-up factor is calculated by dividing the amount of the product in the load at large scale with that at a small scale. Second, column volume is estimated by multiplying the column volume at a small scale with the scale-up factor. Typically, bed height is maintained constant as one scales up the process, to keep the plate number and column efficiency constant [26,32,45,46]. Third, since we know the bed height and the

252

FIG. 4

12. Instrumental platforms for bioseparations

Scale-up of column size from laboratory to manufacturing scale.

desired column volume, the required column diameter can be calculated. Since columns are available in discrete sizes (with respect to diameter), the chosen column should have an equal or greater diameter than the calculated value. This is done so that the maximum loading capacity of the resin is not exceeded. Column packing is another key issue for large scale operations. The packing density should be optimized to ensure optimal mass transfer, and the bed should be packed uniformly to avoid channeling. 5.1.2 Process parameters During the development of IEC-based processes, it is crucial to carefully monitor and optimize the process parameters to achieve the desired selectivity and yield. One important parameter to control is the buffer system and

its pH, salt concentration, operating temperature, and flow rate [26,31,32]. These factors are crucial in determining the retention time and selectivity of the resin, and hence, must be optimized to achieve an efficient separation. The flow rate is another essential factor affecting the resin’s capacity, retention time, and resolution. Furthermore, it is essential to ensure that the buffer system is compatible with the resin and does not lead to degradation or product loss. The buffer system also affects the retention time and selectivity of the resin. Hence, it is necessary to choose the buffer system and optimize its concentration and pH to achieve the desired separation. 5.1.3 Validation and cleaning Once the scale-up process has been completed, validating the IEC process and the resin cleaning in place (CIP) at the industrial scale is

5 Scale-up of ion-exchange processes

essential. It is important to ensure that product quality is maintained and the process is reproducible and scalable. CIP of ion-exchange resins is critical for successful scale-up of the process. CIP procedures should be established to ensure that the column is thoroughly cleaned between batches to prevent cross-contamination and maintain product purity. CIP validation should be performed to ensure that the cleaning procedures are effective and do not leave any residue on the column. It demonstrates that cleaning eliminates any leftover product, process, or environmental residue, allowing the safe production of the following items with the equipment or system. There are several necessary considerations for the implementation of cleaning validation [47,48], which are also comprehensively explained in different technical reports of the PDA on biotech cleaning validation, such as: (a) Designing and developing a process for cleaning The cleaning process design is essential for a comprehensive cleaning validation program before its implementation in a manufacturing facility. Understanding the critical quality attributes (CQAs) and critical process parameters (CPPs) of the cleaning process is crucial for effective cleaning process design. (b) Degradation effects and analytical methods In bioprocessing, the active ingredient is typically vulnerable to degradation from hot, alkaline, aqueous cleaning solutions, which is a necessary part of the cleaning process to eliminate the denatured proteins. However, this degradation can impact the cleaning validation process as well. Therefore, several analytical methods can be used to identify the removal of active ingredients, including HPLC, ELISA, Bradford assay, and conductivity measurements. (c) Sampling methods For biotech manufacturing, liquid rinse samples are often used for sampling as the

253

equipment is usually glass columns or hardpiped. Additionally, mock or blank runs are sometimes conducted to provide a comprehensive overview of total carryover during active bulk manufacturing. (d) Sustaining the validated condition Maintaining the validated state of a cleaning process is crucial for ensuring product quality, safety, and purity. Methods for validation maintenance include change control, periodic monitoring based on risk assessment, and data trending review. Biotech manufacturing industries design their cleaning processes with a reasonable margin of safety to ensure that actual residue values obtained during qualification protocols or routine maintenance remain well below the acceptance criterion limit. Apart from these parameters, several other factors also influence the efficacy of cleaning validation, including implementation of cleaning validation protocols, planning and management for cleaning validation, and evaluation and mitigation of risks [48]. 5.1.4 Equipment and facility consideration Scaling up IEC requires consideration of equipment and facility requirements. This includes selecting appropriate chromatographic systems, pumps, and other equipment. Ensuring that the equipment is appropriate for the specific process and product is being purified is important. The facility should also be designed to accommodate the scale-up process and ensure that it meets regulatory requirements. 5.1.5 Mode of operations of IEC In IEC, elution is primarily achieved by varying the salt concentration and pH of the eluent [49,50]. This process involves binding the protein of interest to ion exchange resins based on their charges, either positive or negative. During elution, the eluent conditions are adjusted to disrupt the ionic interactions between the protein

254

12. Instrumental platforms for bioseparations

and the resin, releasing the protein from the column. This can be achieved by increasing the salt concentration or changing the eluent’s pH. Doing so reduces the ionic strength and electrostatic interactions between the protein and resin, allowing the protein to be eluted from the column. Isocratic and gradient elution are the commonly used modes of operation in IEC [51–53] (Fig. 5), with displacement chromatography also used in niche applications [54–56]. However, other modes of operation can be employed at a large scale, such as frontal chromatography [32,57], which has traditionally been used in the chemical industry. While each mode of operation in IEC has practical considerations, there are also practical issues that are specific to each mode of interaction. For example, in isocratic elution, buffer preparation, column size and packing, and flow rate must be carefully considered to

FIG. 5 Mode of elution during IEC process optimization.

separate the product of interest effectively. In gradient elution, gradient preparation, column size, and packing, and gradient profile must be optimized to ensure adequate separation. Similarly, in frontal chromatography, the sample size and the stationary phase properties must be carefully selected to ensure efficient separation of the target component from the sample mixture. Moreover, the choice of mode of operation will depend on multiple factors, such as the sample complexity, the required purity of the target protein, the economics of the process, and the availability of equipment and facilities. Overall, selecting the appropriate mode of interaction for the IEC scale-up from lab to manufacturing scale will depend on carefully considering the practical issues specific to each mode and the factors relevant to the purification process.

5.2 Necessary calculations for IEC scale-up Scale-up calculations are crucial in IEC because they help to determine the optimal operating conditions for large-scale processes [26,32,58]. Determining optimal operating conditions at the lab scale is relatively easy but becomes complicated at the manufacturing scale. Therefore, scale-up calculations are used to predict the behavior of the IEC process at a larger scale. The calculations consider factors such as the resin capacity, bed height, flow rate, column size and diameter, packing efficiency, and the pressure drop across the column to determine the optimal conditions for a largescale process. The objectives are to achieve improved separation efficiency, increased productivity, and reduced production costs. The process of packing a column with ion exchange resins is critical for the successful operation of IEC at a manufacturing level which allows handling the increased sample volume by increasing the column diameter while maintaining the proportional column volume [5,26,32,46]. Although volumetric scale-up is

255

5 Scale-up of ion-exchange processes

not always appropriate or complex, some mathematical considerations are frequently used for IEC scale-up processes, Table 4. These include: (a) Porosity

The porosity of the resin bed can affect the flow rate, mass transfer, and pressure drop in the column, and can be calculated using the following equation:

In IEC, the porosity of the resin bed is a critical parameter to consider during scale-up [59].

TABLE 4

ε¼

ðV b
V r Þ Vb

Various equations used for scale-up of IEC processes.

S. no.

Process stage

Attribute

Equation

1

Column packing

Porosity

rÞ ε ¼ ðVbV
V b

2

Column packing

Column’s total volume

Vb ¼ A  H

3

Column packing

Resin volume

Vr ¼ ε  Vb

4

Column packing

Linear velocity

Q V ¼ ðAε Þ

5

Column packing

Bed height

H ¼ VAb

6

Column packing

Plate height

HETP ¼ NL

7

Column packing

Plate number

N¼K

8

Column packing

Pressure drop

ΔP ¼

9

Column packing

Void volume

Vv ¼ Vt
Vb

10

IEC process

Retention time

0Þ tR ¼ ðV r
V F

11

IEC process

Retention factor

0Þ k ¼ ðtRV
V 0

12

IEC process

Separation factor

α ¼ kk1

13

CIP

Flow rate

Q¼AV

14

CIP

Contact time

t ¼ VQb

15

IEC process

Mass balance

F  Cin + R  Rin ¼ F  Cout + R  Rout

16

IEC process

Gradient elution

Cout ¼ (Kd  Cin)/{1 + (Kd  (Veff + Vr)/Vr)} {exp(
b  D)
1}


Vr 2 w KμV

ðε3 D2p Þ

2

Abbreviations: ε, porosity; Vb, total volume of the column; Vr, volume of the resin in the column; A, cross-sectional area of column; H, height of the resin bed; V, linear velocity; Q, flow rate of mobile phase; HETP, plate height; L, column length; N, plate number; w, peak width at half height; K, proportionality constant; μ, viscosity of the mobile phase; Dp, diameter of the resin beads; Vv, void volume of the resin bed; tr, retention time; F, flow rate of the mobile phase; k, retention factor; α, separation factor; k2, retention factor for the second compound; k1, retention factor for the first compound; t, contact time; F, feed flow rate; Cin, concentration of the target protein in the feed; R, resin flow rate; Rin, concentration of the target protein in the resin; Cout, concentration of the target protein in the effluent; Rout, concentration of the target protein in the resin after the exchange; Kd, dissociation constant of the protein-resin complex; Veff, effective volume of the resin; b, salt gradient; D, diffusion coefficient of the protein.

(1)

256

12. Instrumental platforms for bioseparations

where ε is the porosity of the resin bed, Vb is the total volume of the column, and Vr is the volume of the resin in the column. The total volume of the column (Vb) can be calculated by multiplying the cross-sectional area of column (A) by the height of the resin bed (H), as follows: Vb ¼ A  H

(2)

The volume of the resin (Vr) can be calculated by multiplying the porosity of the resin (ε) by the total volume of the column (Vb), that is, Vr ¼ ε  Vb

(3)

Once the porosity of the resin bed and its other parameters are calculated, it can be used to determine the appropriate flow rate and bed height for the scaled-up column. In general, a higher porosity allows for a higher flow rate and results in a lower binding capacity. Hence, it is critical to balance these factors when optimizing the IEC process. (b) Linear velocity The linear velocity is the velocity at which the mobile phase (buffer or eluent) flows through the resin bed. It affects the column’s mass transfer, resolution, and binding capacity [5,26,32,36,60]. The linear velocity is a vital parameter to consider during scale-up, and it is calculated by Eq. (4): V¼

Q ð A  εÞ

(4)

where V is the linear velocity, Q is the flow rate of the mobile phase, A is the column’s crosssectional area, and ε is the porosity of the resin bed. During scale-up, it is critical to maintain the same linear velocity constant to ensure that the mass transfer and resolution are not affected. This is commonly achieved by maintaining the bed height and the linear flow velocity constant during scale-up. The bed volume is proportionately increased to accommodate the larger amount of product in the feed.

(c) Bed height The bed height in IEC relates the height of the resin bed to the volume of the mobile phase [32,36,46,61]. Generally, a higher bed height leads to a higher separation resolution and pressure drop across the column, which can affect the separation efficiency [36,61]. Hence, the bed height equation is used to optimize the bed height for a given separation, considering the flow rate, column dimensions, and other operating parameters. The bed height can be calculated using Eq. (5): H¼

Vb A

(5)

where H is the height of the resin bed, Vb is the volume of the resin bed in the column, and A is the column’s cross-sectional area. (d) Plate number The plate number is a measure of column efficiency [32,36,61,62]. The plate number represents the number of equilibriums stages a solute travers as it travels down the column. The higher the plate number, the better separation a column can achieve. The plate number can be calculated from the plate height (HETP), which relates the column efficiency to the physical properties of the column and the mobile phase as follows: HETP ¼

L N

(6)

where HETP is the plate height, L is the column length, and N the plate number. The plate number can be determined by:  2 Vr N¼K w

(7)

where N is the plate number, Vr is the retention volume of the solute, w is the peak width at half height, and K is the proportionality constant

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5 Scale-up of ion-exchange processes

which includes geometrical and thermodynamic factors (such as the phase ratio and retention factor). In IEC, the retention volume is defined as the volume of mobile phase required to elute a solute from the column. The peak width at half height measures the width of the eluted peak. (e) Pressure drop The pressure drop across the column can be calculated from Darcy’s law for fluid flow in a porous medium [36,63] as follows: KμV  ΔP ¼  ε3  D2p

where Vv is the void volume of the resin bed, Vt is the total volume of the column, and Vb is the volume of the resin in the column. (g) Retention time The retention time of a solute in IEC is a function of several variables, including the properties of the resin, the properties of the protein or compound being purified, and the operating conditions of the column [26,36]. It can be calculated as follows: tR ¼

(8)

where ΔP is the pressure drop, K is the packing constant, μ is the viscosity of the mobile phase, V is the linear velocity of the mobile phase, ε is the porosity of the resin bed, and Dp is the diameter of the resin beads. To scale up the process, it is critical to consider the effect of these factors, such as bed height, particle size, and flow rate, on the pressure drop and to ensure that the pressure drop in the larger column does not exceed the operating limits of the system (the column).

ðV r
V 0 Þ F

(10)

where tR is the retention time, Vr is the elution volume of the solute, V0 is the void volume, and F is the flow rate of the mobile phase. (h) Retention factor The retention factor is a measure of how well a solute binds to the resin in the column, and is determined by: k¼

ðtR
V 0 Þ V0

(11)

where k is the retention factor. (i) Separation factor

(f ) Void volume The void volume refers to the volume of the empty space within the column not occupied by the resin or the sample being purified [64,65]. It is an important parameter when designing and optimizing a chromatographic process and can be determined using a noninteracting solute, such as salt, that does not bind to the ion exchange resin. The solute is added to the column and allowed to flow through, and the time it takes to pass through the column is measured. This time is called the “dead time” or “void time.” The void volume can be determined using the following equation: Vv ¼ Vt
Vb

(9)

The separation factor during purification describes the degree of separation between two compounds loaded onto the column [66]. It can be calculated as: α¼

k2 k1

(12)

where α is the separation factor, k2 is the retention factor of the second compound, and k1 the retention factor of the first compound for two peaks eluting in the order first and second. (j) Flow rate The flow rate of the cleaning solution can be calculated as: Q¼AV

(13)

258

12. Instrumental platforms for bioseparations

where Q is the flow rate, A is the column’s crosssectional area, and V is the velocity of the cleaning solution. (k) Contact time Optimizing the contact time of a cleaning solution in a column is crucial as it can significantly impact contaminant removal efficiency without damaging the resin [47,48]. A contact time that is too short may result in ineffective cleaning, while a contact time that is too long can cause damage to the column’s properties. Therefore, careful consideration should be given to optimizing the contact time of the cleaning solution to achieve the desired level of cleaning without compromising the integrity of the column. The contact time of the cleaning solution with the resin can be determined using the following equation: t¼

Vb Q

(14)

where t is the contact time, Vb is the volume of the resin in the column, and Q is the flow rate of the cleaning solution. In addition to flow rate and contact time, other factors that need to be considered during CIP include residence time, Reynolds number, and shear rate. This is important to ensure the cleaning process’s effectiveness while maintaining the equipment’s integrity. By carefully considering and optimizing these parameters, CIP procedures can be carried out safely and efficiently, improving product quality and reducing downtime.

5.3 Common problems associated with IEC scale-up from lab to manufacturing scale In this section, we briefly discuss some issues that could arise during scale-up and large-scale operations of IEC separations.

5.3.1 Pressure drop A uniform pressure is critical for effective and robust column operation. While this is easy to achieve in lab scale operations, as the column size increases, the cross-sectional area and the volumetric flow rate increase as well to maintain a constant residence time. Since the permeability of the resin remains constant, the pressure drop increases proportionally to the square of the column diameter [67,68]. If the pressure drop exceeds the limits of the column hardware or column resin, it could result in multiple undesirable outcomes including reduced flow rate, column compression, and ultimately column failure. While it is best to mitigate this risk during process development and design, other strategies that can be used include using resins with a larger particle size and higher permeability and reducing the bed height. 5.3.2 Buffer preparation at a manufacturing scale Buffer preparation for large-scale separations can be challenging and time-consuming. Preparing larger volumes of buffer results in several challenges, such as the need for expensive large-scale storage and equipment [69,70]. The time-consuming process of mixing often results in the degradation of buffer ingredients, the need for large-scale filters or sterilizing facility, and problems in validating the buffer preparation. Increasingly the industry is moving toward implementing automated buffer systems which significantly reduce labor, save time, and offer favorable process economics [70]. Use of single-use systems can further reduce the risk of contamination and eliminate the need for cleaning and sterilizing large storage equipment. 5.3.3 Column packing and cleaning Packing large scale columns (particularly >50 cm diameter) is nontrivial. Manufacturers spend considerable time and resources to create optimal packing protocols that can account for

References

packing parameters, including the slurry concentration, packing flow rate, and packing pressure. Regular monitoring of the packed bed during the packing process and after use can also help identify issues and ensure that the column performance is optimized. Additionally, packing materials with an optimum particle size distribution can help minimize bed compression and ensure uniform packing. Choosing the appropriate column size and bed height can also help achieve a consistently packed bed with high separation performance. Possible issues that may arise include: (a) Uneven packing: This can result in variations in flow rate and binding capacity, leading to poor separation [61]. (b) Bed compression: Excessive pressure during column packing can cause bed compaction, reducing the column’s void volume and binding capacity [65]. (c) Channeling: Uneven packing or bed compression can lead to the formation of preferential flow paths, resulting in channeling and reduced separation performance. (d) Resin bed expansion: The ion exchange resin can expand due to temperature or buffer composition changes, thereby resulting in uneven packing. (e) Large-scale column cleaning: Operating large columns requires significant volumes of cleaning and regeneration solutions, which can be expensive and time-consuming to prepare, and require specialized equipment. In addition, these create a major challenge with respect to waste disposal. 5.3.4 Validation of a scaled-up process Validating performance at a manufacturing scale can be challenging. The increased complexity of the process and the need for larger sample sizes can make obtaining accurate and reproducible results difficult [71]. The validation of the process at a large scale is usually

259

associated with several problems, such as variability in resin performance causing variation in the process, which is challenging to validate. Ion-exchange separation is a highly effective technique for separating and purifying products based on ionic strength. However, implementing IEC at manufacturing scale presents various challenges, such as pressure drop, buffer preparation, column packing, column cleaning and regeneration, buffer recycling, and validation. To overcome these challenges and enable successful implementation at a large scale, several strategies are used, including process design, resin selection, regeneration, scale-up process, process control, and waste disposal. These strategies are crucial when designing and implementing large-scale ion exchange processes to ensure optimal performance and process economics. Careful planning and meticulous attention to detail are necessary to ensure successful operation of IEC at a manufacturing scale.

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C H A P T E R

13 Method development for large molecules IEX separations Mateusz Imiołek and Szabolcs Fekete Waters Corporation, Cell and Gene Therapy Consumables, Geneva, Switzerland O U T L I N E 1. Introduction

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2. Column, stationary phase, and instrumentation considerations 2.1 Stationary phase characteristics 2.2 Column dimensions 2.3 Column hardware and instrumentation 2.4 Detection

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3. Elution modes 3.1 Salt gradient mode 3.2 pH gradient mode 3.3 Salt-mediated pH gradient mode

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4. IEX mode for nucleic acid separations 4.1 General considerations 4.2 Practical considerations 4.3 Denaturing elution conditions for nucleic acid analysis 5. On-off mechanism of retention 5.1. Multisegmented gradients (multiisocratic elution) vs linear gradients

Ion-Exchange Chromatography and Related Techniques https://doi.org/10.1016/B978-0-443-15369-3.00030-4

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7. Systematic method development 7.1 Screening experiments 7.2 Method optimization in general

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8. Practical advice for systematic method optimization 278 8.1 Method optimization in salt gradient mode 278 8.2 Method optimization in pH gradient mode 280 8.3 Method optimization in salt mediated pH gradient mode 280 8.4 Method optimization in ion-pairing IEX 280 9. Perspectives

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Declaration of competing interest

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References

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Copyright # 2024 Elsevier Inc. All rights reserved.

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1 Introduction Liquid chromatography (LC) method development is defined by several key steps followed to end up with a suitable analytical process. Generally, one determines the goal of the procedure, sets out the approach and, after the practical development, moves onto prevalidation and validation of the experiments, while documenting all the actions. The case of ionexchange chromatography (IEX) separations is no different; however, this chapter focuses on practical aspects of the method development specifically related to two class of large molecule biologics: proteins (i.e., antibodies and related products) and nucleic acids (i.e., mRNAs). Such a focus stems from the current momentum behind the development of these types of therapeutics in the biopharmaceutical industry and is aligned with our experience. For more general, universally applicable considerations the reader is invited to consult other publications, for example instructive texts by Snyder et al. [1]. Owing to their intrinsically charged nature, biopolymers are ideal substrates for IEX. Sequence and/or posttranslational modification caused differences in overall (effective or accessible) net charge and its distribution allows for efficient fractionation of the biomolecules based on their electrostatic interactions with a charged stationary phase. Due to its usually mild and nondenaturing conditions, IEX can routinely be used in many stages of the biomolecule lifecycle, starting from capture, and intermediate purification to the final polishing steps. Such at scale processes are discussed in detail in further chapters, while here we focus on elaborating general principles regarding the analytical scale method development. IEX can finely resolve differences in surface charge, which makes it an ideal tool for monitoring charge variants of therapeutic antibodies (monoclonal antibody (mAb) and antibodydrug-conjugate (ADC)) or shortmer impurities

of therapeutic oligonucleotides. As such, it has become an essential tool offering unique selectivity in modern biopharmaceutical analysis, indispensable for the quality control. However, its potential is not fully harnessed due to the challenging method development, typically characterized by laborious trial-and-error approaches investigating multiple variables impacting the separation. The analyst has to consider column and stationary phase characteristics (weak/strong anionic/cationic resin, etc.), decide on an elution mechanism (pH, salt gradients, or combination thereof, i.e., salt-mediatedpH gradients), investigate key factors (buffer agent, ionic strength, temperature, etc.) and multiparametrically optimize them all in a timely manner. Therefore, it is not surprising that emerging method development strategies are often aided by automation and in silico simulations. According to our broad experience in the subject we will discuss those essential variables and their influence on separations, together with new approaches in the field further extending the potential of IEX and facilitating the method development process.

2 Column, stationary phase, and instrumentation considerations 2.1 Stationary phase characteristics Decision of the mode of chromatography is typically dictated by the properties of the sample; as such, the separation of nucleotides is performed on anion exchange (AEX) columns, while cation exchange (CEX) is the suggested starting point for peptides and proteins unless they are negatively charged below pH 7. Secondly, polymeric based strong exchangers are recommended as the first choice, as they allow screening of a wide pH range owing to their superior stability, pH independent ion exchanging capacity (stationary phase ligand: sulfonic acid pKa at
2.0–2.5 or quaternary amines, with charge independent of pH) and

2 Column, stationary phase, and instrumentation considerations

typically higher selectivity [2]. The type of stationary phase will typically have the determining effect on peak widths, but the overall efficiency of the separation will be highly dependent on the elution conditions. To evaluate orthogonal conditions, the use of weak exchangers might be beneficial as selectivity differs according to changes in resin structure, the strength of secondary interactions, and the ionexchange ligands and their capacity. This is recognized in some more specific applications to separate closely related biomolecules and their impurities, where mixed mode chromatography (IEX/RP) was applied to analyze small interfering ribonucleic acids [3]. It is important to consider the column technical parameters; nonporous particles, despite their more limited binding capacity, are preferred for high-resolution analyses due to the low diffusivity of biomolecules and the nature of strong electrostatic interactions (so to avoid insufficient mass transfer related band broadening). Although not typically used for analytical applications, organic monolithic stationary phases are a suitable medium when the main goal of the separations is its high-capacity/ throughput character and column longevity [4]. As the material, most of the state-of-theart columns use stable, highly cross-linked poly(styrene-divinylbenzene) (PS/DVB) that can be operated at high pressures, extreme pH (2–12) and high temperature (60°C) extending the accessible method development space. Regarding the particle size, columns packed with 3–5 μm particles universally provide higher resolution and peak capacity than those with larger ones, still commonly available commercially [5]. However, the expected increase in the separation power was not observed by decreasing the particle size, possibly due to the on-off interaction mechanism of biomolecules with the stationary phase. In fact, for most typical application like CEX in mAb separations, it was found that columns packed with nonporous polymeric particles smaller than 2.5 μm, do

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not provide increased efficiency, only affecting the permeability, and therefore its use is of limited benefit and can possibly lead to very high pressures with certain, low ionic strength mobile phases [6].

2.2 Column dimensions It becomes increasingly clear that the use of short, narrow bore columns is of advantage when it comes to the separation of large molecules. In general, biomolecules starting from moderately sized solutes (>5 kDa), exhibit an on-off (bind and elute) type retention mechanism which causes only the first segment of the column to effectively contribute to the separation (1–2 cm), making the overall column length not important [7]; As such, efficient IEX analysis can be obtained in a short time (50 nt) can exist as several conformers due to their propensity to form short intramolecular interactions and a wide array of accessible base-stacked conformations [70]. Denaturing conditions, like high pH, elevated temperature, and the presence of organic cosolvent, can disrupt base-pairing and basestacking interactions. Under these conditions, the intact RNA can be linearized and eluted as sharper peaks.

4.2 Practical considerations Most IEX methods apply a linear or multisegmented salt gradient in a buffered aqueous mobile phase. Typical mobile phase buffers are based on tris at pH 7–9, HEPES at pH 6.8–8.2, sodium carbonate at pH 10–11, or sodium phosphate at pH 11–12. Most often, the mobile phase buffer concentration is 20–30 mM. For traditional salt gradient separations, it is common to use 1–2 M NaCl, KCl, MgCl2, NaBr, or NaClO4 as additives in the mobile phase [71,72]. Recently, an alternative method based on the use of an ion pairing agent and relatively mild elution conditions was proposed [71,73]. A gradient formed of weak ion-pairing cations (e.g., tetramethylammonium chloride, TMAC) can produce intriguing AEX separations of mRNAs. This approach was referred to as ion pairing anion exchange “IPAX” and appears to provide different recovery and selectivity as compared with classical salt gradients and opens up an exciting, new avenue in research for nucleic acid AEX method development [71].

4.3 Denaturing elution conditions for nucleic acid analysis The use of elevated temperature to achieve fully or partially denaturing conditions is a common technique that can additionally be used to increase the retention of oligonucleotides. The

5 On-off mechanism of retention

separation can also be modulated by changing the eluent pH [74,75]. However, the degradation of RNA molecules is a concern at elevated pH [68]. In reality, pH induced hydrolysis is a very slow process, typically negligible on the time scale of a chromatographic separation [69,75]. Another effect of high pH is the elimination of inter- and intrastrand hydrogen bonding, which can typically be overcome at a pH higher than 10. High pH separations are often combined with low temperature (T ¼ 10–15°C) conditions to maintain acceptable retention. At both high pH and high temperature, elution of mRNA species might be challenging due to undesirably strong interactions and high retention. Organic solvents such as acetonitrile or methanol in small amounts (e.g., 10%–15% in the mobile phase) might also aid the partial denaturation and in some cases can be used to adjust selectivity [69]. The addition of 5–6 M urea or 10% formamide is also used for denaturing AEX [69]. Short RNAs elute as sharp peaks in nondenaturing conditions simply because they exhibit more homogenous adsorption/desorption behavior and take on a minimal secondary structure. On the contrary, RNA molecules longer than 100 nt often elute as broad, tailing peaks in AEX if nondenaturing conditions are used. Cernigoj et al. showed that gradient elution from a weak anion exchanger, in the presence of guanidine hydrochloride (Gdn) in the mobile phase, roughly doubles the resolution between open-circular and supercoiled plasmid isomers [76]. It also improves resolution among linear, and multimeric/aggregated forms. Less tailing and sharper elution peaks led to an increase in sensitivity by about 30%.

5 On-off mechanism of retention Large solutes follow a particular type of elution behavior, which is often referred to as an “on-off” or “bind and elute” mechanism [77–79].

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This process includes only a few adsorptiondesorption steps, instead of the common multiple-step partitioning process [80]. Therefore, large solutes are very sensitive to mobile phase strength and composition. A slight change in mobile phase composition can result in total binding of the analyte at the column inlet or, conversely, its complete release from the column [8,81]. Thus, at some point during peak elution, band broadening and compression effects of large solute bands equilibrate with each other. This balance leads to a quasisteady state that minimizes any further broadening of the migrating peak. Upon reaching this state, the solute velocity (migration) equals the migration speed of the mobile phase. Consequently, beyond this point, the protein zones will travel with the same velocity, and the selectivity will not change. Therefore, when separating large solutes displaying the on-off elution mechanism, multisegmented gradients are more practical compared with linear gradients [82]. Up to this date, all molecules of MW 15–20 kDa (including: antibody fragments, intact antibodies, fusion proteins, antibody-drug-conjugates, oligonucleotides, and intact mRNAs) separated in all IEX elution modes (salt gradient, pH gradient, salt mediated pH gradient and ion-pairing IEX) were found to follow the on-off mechanism [7].

5.1 Multisegmented gradients (multiisocratic elution) vs linear gradients One consequence of the on-off mechanism is that the elution distance between peaks can be—nearly—arbitrarily set by combining gradient segments of different steepness [82]. The recently developed “multiisocratic elution mode” approaches the selectivity limits of multilinear gradients as it combines multiisocratic (binding) steps and very short steep (eluting) gradient segments at solute elution. Such an elution program allows the desired selectivity to be set, while maintaining sharp peaks due to significant band compression effects resulting from

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the use of very steep gradient steps. A uniform peak distribution (equidistant band spacing) can be achieved with the multiisocratic approach. The elution distance between the peaks can be freely programmed by adjusting the length of the isocratic segments. In the end, both the sensitivity and resolution are better than those observed using common linear gradients. Fig. 2 shows a comparison of linear and multisegmented gradient CEX separations for a partially digested mAb sample. When applying a

linear gradient (salt-mediated pH gradient), the critical peak pair was the second and third peak. They were separated with a resolution of Rs ¼ 1.79. After optimizing the gradient and adding an isocratic hold between the critical peak pair, the resolution was improved, up to Rs ¼ 5.34. Moreover, the multisegmented gradient was set to achieve an equidistant peak distribution. Such multisegmented gradients are often applied for the separation of empty and full AAV

FIG. 2 CEX separation of daratumumab subunits (partially digested sample) using a 100  4.6 mm column, and a generic linear gradient (A) or an optimized multisegmented gradient (B). Elution mode: salt-mediated pH gradient. Unpublished data from authors’ laboratory.

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FIG. 3 A step-gradient AEX separation of empty- (first eluting group of peaks) and full AAV capsids (second eluting group of peaks). From R.J. Whitfield, et al., Rapid high-performance liquid chromatographic analysis of adenovirus type 5 particles with a prototype anion-exchange analytical monolith column, J. Chromatogr. A 1216(13) (2009) 2725–2729, with permission.

capsids in the anion exchange mode. These gradients are referred to as a “modular discontinuous gradient,” “step gradient” or “two-step gradient.” Very often, the empty and the full particles are eluted at different linear holds, with a steep gradient segment between them [83–85]. Fig. 3 shows an example of such a step gradient separation. Hejmowski et al. developed an interesting approach based on a multiple step gradient with small conductivity increases of about 1 mS/cm which provided a high separation efficiency of empty and full AAV serotype 5 [86]. In some cases, the best overall separation is achieved with a nonlinear gradient. Joshi and coworkers investigated a fast, sigmoidal shape, salt gradient CEX method for efficient mAb separations [20].

6 Need for IEX platform methods With a keen emphasis on Quality by Design (QbD), and driven by a focus on patient safety, regulatory agencies (FDA and EMA) impose

tight mandates on understanding and monitoring of therapeutic macromolecules’ critical quality attributes (CQAs). A key aspect of biopharmaceutical QbD, which is yet to be truly leveraged, is the use of so-called “platform” strategies for CQA determination. The charge variant analysis—which is currently the main application of IEX for biopharmaceuticals—with a salt gradient elution, although widely used, has never been regarded as a platform method. This is partly due to the need for a careful and lengthy method optimization for each compound (as separations are correlated with the analyte’s pI and can span a wide range). However, the introduction of pH gradient elution has changed this perception permitting a single method to be used as a global starting point for any product. A broad range pH gradient can be set up so that, at some point, any target solute and its associated charged variants will reach their isoelectric points, that is, lose their net charge and elute from the column. This is facilitated by the availability of commercial buffer cocktails that offer exceptional linear

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control of a pH gradient. Often short columns and fast gradients are applied together for platform methods and are especially beneficial for high throughput screening campaigns.

7 Systematic method development 7.1 Screening experiments If the sample components are unknown, then an automated screening procedure can be helpful. A multicolumn IEX screening approach was proposed that includes 12 different columns and 24 mobile phases that are sequentially applied in an automated fashion for both cation and anion exchange modes [87]. This screening approach was applied to oligonucleotide, peptide, and protein separations. Blending various concentrated stock solutions allows screening of both pH and ionic strength in IEX methods with minimal user intervention and without the need for the preparation of separate mobile phases [88]. Fully automated scouting systems are also available (e.g., ChromSwordAuto). The screening module generates sequences automatically (considering column equilibration and washing steps) and runs them to scout various gradients, columns, solvents, buffers, and temperatures [89].

Among these three variables, they found that mobile phase pH was the most important. The mobile phase linear velocity also has a strong influence on the separation quality of large biomolecules [91,92]. Indeed, longitudinal diffusion is negligible for large molecules, while band broadening is mostly determined by the mass transfer resistance. Therefore, a low flow rate is preferred for high resolution separations, but a compromise must be found between the separation time and resolution. Computer-assisted method development and optimization, while common in RPLC protein separations, is now also becoming more popular in IEX. Thiemo et al. developed specific software for the parameter estimation, chromatogram simulation, and process optimization [93]. It provides numerical tools for solving various types of chromatography models, including the model combining the transport dispersive model (TDM) and SMA. Both non-LSS and LSS type computer-assisted method development procedures were recently reported for both salt- and pH-gradient modes in agreement with the quality by design (QbD) concept [25,94].

8 Practical advice for systematic method optimization

7.2 Method optimization in general

8.1 Method optimization in salt gradient mode

The method development in IEX was mostly based on trial-and-error or one factor-at-a-time (OFAT) approaches. Nowadays, thanks to the development of statistical tools and chromatographic modeling software, once lengthy optimization can be substantially sped up. However, it is critical to appropriately select the method variables. With the proper variables, the models can be calibrated with just a few initial experiments. Bai et al. showed that retention of IgG antibodies and selectivity of their separation strongly depend on mobile phase pH, stationary phase type, and salt-gradient steepness [90].

Based on our experience, the two most important variables for a salt gradient separation are the mobile phase pH and the salt gradient steepness. Column length has little effect on the quality of IEX separations (due to the on-off mechanism) thus short columns are preferred (i.e., 5 cm long). The flow rate should be set at a relatively low value to avoid band broadening (originating from mass transfer resistance). Temperature should be kept close to ambient to maintain nondenaturing conditions. Fig. 4 shows a proposed DoE for the separation of basic proteins in the salt gradient cation exchange mode.

8 Practical advice for systematic method optimization

279

FIG. 4 Recommended experimental design for the optimization of cation exchange salt gradient separation of antibody charge variants, based on six initial experiments to calibrate the model.

The gradient steepness needs to be studied at two levels while mobile pH should be studied at three levels (because of nonlinear effects). If modeling software is available [95], then fundamental chromatographic models and the corresponding resolution maps can be easily produced [89] and the working point identified within the entire, extrapolated design space.

Peak recovery (area) is often an important target of the optimization procedure when adequate peak resolution is achieved. Solute recovery of large biomolecules in IEX might be poor in some conditions, which makes peak tracking challenging and hence complicates modeling efforts. Fig. 5 shows an example of the selection of a working point based on resolution map

FIG. 5 Finding the working point on a two-dimensional resolution map. Sample: a partially digested monoclonal antibody. Elution mode: salt gradient CEX, 100  4.6 mm column operated at F ¼ 0.6 mL/min.

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optimization. A gradient time of tG ¼ 25 min and mobile phase pH of 5.6 resulted in the highest achievable resolution and most robust conditions. If no chromatographic modeling software is available, then “blind” factorial (full or fractional) designs can also be used to estimate the ideal working point.

8.2 Method optimization in pH gradient mode An important criterion in the pH gradient mode is that a linear gradient of A to B buffers should provide a linear pH response, otherwise, retention modeling becomes challenging. In the pH gradient mode, the two most important variables are the gradient steepness and mobile phase temperature since they both have a significant impact on selectivity and resolution. For most solutes, the relationship of retention time (or its transformation, i.e., logarithmic retention factor) with pH, gradient steepness, and temperature can be described by linear models [89]. This observation suggests that the method optimization with gradient steepness and temperature as model variables requires measurement at two levels only (Fig. 6). This design and modeling approach was applied for the optimization of a COVID-19 antibody therapeutics separation in the pH gradient cation exchange mode [9].

8.3 Method optimization in salt mediated pH gradient mode In theory, a salt mediated pH gradient broadens the experimental design space that can be mapped to tune selectivity and resolution. The probability of finding an appropriate separation, should significantly increase compared to a single salt- or pH gradient separation [58]. Another benefit of this combined elution mode is a gain in peak compression effects, especially for late eluting peaks. In the salt-mediated pH gradient mode, the most significant factors are the salt gradient steepness and the mobile phase salt concentration. The effect of the first variable can be described by linear models, thus two-factor levels should be studied. The latter variable should be investigated at three levels. Fig. 7 provides some recommendations for initial experiments.

8.4 Method optimization in ion-pairing IEX The concept of ion pairing in AEX can also be used in combination with a sodium chloride gradient. TMAC can be used as a mobile phase additive to form partially ion-paired mRNA molecules (thus decreasing the number of accessible charges). IPAX has thus far been performed with 25 mM TRIS and HEPES buffer (pH 7.5–8)

FIG. 6 Recommended experimental design for the optimization of cation exchange pH gradient separation. Only four initial experiments are required to calibrate the model.

9 Perspectives

281

FIG. 7 Recommended experimental design for the optimization of cation exchange salt mediated pH gradient separation. Six initial experiments are required to calibrate the model.

and 1–3 M TMAC gradients. In a classical salt gradient separation, relatively low temperatures (T ¼ 30–40°C) result in better recoveries while in the IPAX separation, higher temperatures up to 60°C were more beneficial. Therefore, mobile phase temperature needs to be studied carefully. Besides temperature, gradient steepness and salt concentration can be studied as important method variables. Due to the on-off elution behavior of large biomolecules, model parameters can be derived, and the mobile phase composition (c*) to elute a nucleic acid peak with retention factor of k ¼ 1 determined from two linear gradient experiments. A symmetrical %B window around the %B composition obtained for the c* value can then be set. With that, one can expect the solute to elute in the middle of the gradient window (a symmetrical elution window scaling approach) [71,72]. The gradient time and the elution window (symmetry range) can then be arbitrarily set such that either fast or long separations can be performed as desired.

9 Perspectives Future developments in the field could be geared toward the use of new types of columns with continuous stationary phase gradients. It

was shown that they may be beneficial for large molecule separations in the mobile phase gradient elution mode, especially when more than one type of interaction strength is modulated along the column [96]. First steps to the practical realization of this concept were made by the generation of charge density gradients on anion exchange latex particles but it has only been applied to the separation of common anions so far [97]. Ion-exchange capacity gradients were studied in more detail for the separation of ions of different valence, again confirming the potential selectivity gain for differently charged ions [98]. These results suggest that the technical challenge of column preparation might be worth pursuing, especially in the context of large molecules separated using mixed mode chromatography. The other possible advance in the field can utilize pressure as a parameter for tuning the selectivity, known as pressure-enhanced liquid chromatography [99]. Its application was demonstrated for reverse-phase large molecule separations, with the potential to significantly improve resolution, but the effect has not been observed for ion-exchange separations [100]. First studies shed light on how pressure modulation might affect the retention of biomolecules interacting with different IEX stationary phases [101,102]. Further

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research is needed to explore the impact of pressure on biomolecule separation selectivity and its possible application for the method development.

Declaration of competing interest BioResolve and IonHance are trademarks of Waters Technologies Corporation.

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C H A P T E R

14 Sample preparation for ion-exchange separations Colin F. Poole Department of Chemistry, Wayne State University, Detroit, MI, United States O U T L I N E 1. Introduction

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2. Liquid-phase extraction 2.1 Solid samples 2.2 Gas phase samples 2.3 Liquid-liquid extraction

288 288 290 291

3. Solid-phase extraction 3.1 Inorganic ions (matrix removal and concentration) 3.2 Inorganic ions (speciation)

293 293 297

1 Introduction As separation methods have become faster and typically fully automated the bottleneck in the sample workflow is generally sample preparation. The latter process exists only because many samples cannot be analyzed in their native form. After all, matrix components interfere in the separation or detection of target analytes, contaminate the separation system, or contain target analytes at concentrations too low for

Ion-Exchange Chromatography and Related Techniques https://doi.org/10.1016/B978-0-443-15369-3.00009-2

3.3 Inorganic ions (extraction chromatography) 3.4 Organic ions 3.5 Biomacromolecules 3.6 Microextraction formats

298 298 301 302

4. Membrane-based extraction 4.1 Electrodialysis 4.2 Electromembrane extraction

304 304 306

References

307

their detection. The process of sample preparation typically consists of a series of discrete and possibly manual operations required to ensure the compatibility of the sample with the separation system and the goals of the analysis [1–3]. For ion-exchange separations, an ideal sample is an aqueous solution free from interfering matrix components in which the target analytes are at an appropriate concentration for their detection. Real samples tend to deviate from an ideal sample in one or more respects. Insoluble material in the form of particles is a

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general problem causing system failure due to the restriction of narrow passageways in instrument and column components. Thus, the most universal sample preparation technique is filtration accompanied by dilution, if necessary, to minimize matrix problems. Filtration of small sample sizes is easily performed using syringe-based porous polymeric membrane disks of various dimensions and pore sizes [1]. Membrane filters with a mean pore size of 0.45 μm are suitable for general applications and 0.2 μm when removal of bacteria (sterilization) is required. Alternatively, or in addition, for a greater level of automation in-line filters before the injection valve are a common component of autosamplers, and postinjection in-line filters on the high-pressure side of the injection valve are a further possibility. Syringe filters are single-use devices while in-line filters are used for multiple samples processed sequentially with a lifetime limited by carryover and clogging, properties that depend on the sample matrix. Particle filters are available in different materials to ensure matrix compatibility and stability to solutions of extreme pH. It is not always possible to minimize matrix interference by dilution, especially for samples that contain target compounds already at low concentrations. In this case, sample preparation needs to incorporate a simultaneous or sequential preconcentration step. Since only aqueous solutions are typically analyzed by ion-exchange chromatography gaseous samples need to be transferred to the liquid phase and solids or liquids not readily soluble in an aqueous solution need to be extracted or digested before analysis. In general, nonaqueous samples represent a potential problem, for example, fats, oils, and organic solvent extracts since the matrix (solvent) is not generally compatible with the retention mechanism or mobile phase. A further general problem for the analysis of low-mass ions is caused by high-mass matrix components co-extracted from environmental or biological samples, such as proteins, lipids, humic acid

substances, lignans, and surfactants. Related problems arise for samples with a high concentration of a matrix ion outside the range that can be handled by the column capacity and selectivity. Samples of extreme pH may not be compatible with the separation mechanism and/or compatible with instrument components. While solutions of varying complexity will be identified for most of the above issues, few are fully automated and integrated with the sample separation. In addition, suitable approaches are frequently sample-type dependent. The lack of universal methods in many instances, adds to the complexity and variety of sample preparation choices and can be confusing in the absence of curated experiences. Also, the availability of equipment, method robustness, cost, time, and capability of automation impact method choices. Sample preparation approaches need to consider the dominant feature of samples suitable for separation by ion-exchange chromatography. Electrostatic forces underpin the separation mechanism of ion-exchange chromatography and to participate in the separation process, samples must be ions or neutral compounds easily ionized by manipulating the sample or mobile phase pH. In addition, electrostatic interactions facilitate the isolation of ions from neutral compounds, and electric fields the isolation of ions in ways that are inappropriate for neutral compounds. These approaches afford a unique capability for the dedicated isolation and preconcentration of ions not available to other sample types [1,4–7].

2 Liquid-phase extraction 2.1 Solid samples General sample processing steps for solid samples include drying (optional), grinding, homogenization, and dissolution followed by filtration and (optional) dilution or volume reduction. Target ions in low-solubility matrices

2 Liquid-phase extraction

may be isolated by leaching followed by decantation or centrifugation before filtration. Leaching results in the transfer of the target ions to a solvent in intermate contact with the sample in which the sample matrix has limited solubility. The rate and extent of extraction depend on the characteristic properties of the matrix, the properties of the target ions, and the distribution of the target ions within the matrix [8–10]. Solid and semisolid samples are initially treated by such processes as drying, grinding, chopping, homogenizing, and/or sieving to enhance the rate of extraction. These processes are used to reduce the particle size and to increase the sample surface area in contact with the leaching solution. Air-drying is suitable for inorganic and plant materials while freeze-drying is typically preferred for tissue and food commodities. The rate of extraction, recovery of the target ions, and the concentration of the soluble portion of the matrix determine the success of leaching for ion-exchange separations. Although many ions can be extracted using simple shaking and rocking devices faster extraction and usually higher recoveries are obtained using ultrasonic- and microwave-assisted apparatuses [11–13]. When matrix interference is not mitigated by typical matrix-target ion separation methods, then matrix destruction using digestion or combustion techniques provides an alternative option mainly for elemental analysis [2,10,14–16]. Dry ashing and wet acid digestion are the most important methods for the determination of metals. For dry ashing, the dried and homogenized sample is placed in a crucible and mineralized by heating in a muffle furnace for several hours or overnight at temperatures typically above 500°C [2,10,16]. The resulting inorganic ash is dissolved in dilute acid or water for ion exchange separations. In all open vessel methods, like dry ashing, the risk of target ion loss or contamination needs to be controlled. Wet acid digestion in open, or preferably closed, vessels is generally preferred [2,10–13,16,17].

289

Solid samples are mixed with a strong acid, for example, nitric or hydrochloric acid, and/ or oxidizing agents such as hydrogen peroxide, and heated at moderate temperatures to accelerate matrix decomposition. Several devices from simple hot plate and block heaters to more advanced microwave- and ultrasound-assisted extraction instruments are typically used. The latter facilitates accurate temperature, pressure (closed vessels) and time control, batch sample processing, and automated cool down, in a programmable environment. For wet acid digestion, microwave-assisted methods typically allow the use of less concentrated acids, shorter digestion times, and higher sample throughput due to batch sample processing, together with improved repeatability due to their integrated control and monitoring features. Alkali fusion provides an alternative approach to wet acid digestion mainly for glasses and geological samples [2,10]. The solid sample is mixed with a suitable fluxing agent (e.g., sodium hydroxide or sodium carbonate) and heated in a crucible to melt. After cooling, the fused mass is solvent extracted and diluted for ion exchange separation. Both wet acid digestion and fusion methods result in extracts containing a high concentration of ions associated with the digestion reagent, which without additional treatment may interfere with ion-exchange separations [3]. For samples with a high-carbon content, wet acid digestion may be incomplete due to the poor solubility of carbon particles favoring the use of combustion or dry ashing techniques. The most common are the Schoeninger flask, combustion bomb, and microwave-induced combustion techniques [11,15–21]. These approaches have several features in common but differ in sample size and throughput. The Schoeninger flask method uses an electrical discharge to initiate the combustion of a sample ( isoelectric point of the oxide/hydroxide) and anions under acidic conditions (sample pH < the isoelectric point of the oxide/hydroxide). In addition, metal oxides can be selected to exploit Lewis acid/base and ligand exchange interactions. Activated carbons are nonspecific adsorbents with ion-exchange sites associated with oxygen-containing impurities that can establish ionic interactions at an appropriate sample pH. As a general strategy, a single sample is aliquoted and processed on several different sorbents, each typically selected to retain an

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14. Sample preparation for ion-exchange separations

individual target species. Alternatively, all target species are retained on a single sorbent and selectively eluted in separate fractions by stepwise changes in the eluting solvent. In a further variation, the sample is initially complexed with a chelating reagent that preferentially reacts with a single chemical species and is isolated by a sorbent with a high affinity for the chelate. Each isolated fraction is assayed independently, generally by an elemental analysis technique, and the total elemental composition of the sample before fractionation may also be determined. These values are then summed to provide information on the distribution of the chemical species for the element in the sample. Although most reports are based on the general strategies outlined above [74–80], the selection of suitable sorbents and reagents is element and matrix dependent [74–76]. Column and cartridge methods predominate but batch processes have been described as well [76]. Most reports describe a single protocol and comparison studies are rare, even in review articles for specific elements [74,75,79,81]. All are multistep procedures with a low sample throughput. The highest literature coverage is for the environmentally or biomedically important elements (Cr, As, Fe, Hg, Sn, and Se).

3.3 Inorganic ions (extraction chromatography) Extraction chromatography utilizes an ion extraction reagent or solution of an ion extraction reagent in a water-immiscible solvent impregnated, embedded, or coated on an inert and porous polymer, silica, or diatomaceous earth support [82–84]. The extraction reagent is typically a liquid ion exchanger or chelating agent with high selectivity for the target ions. The solvents are generally polar organic solvents or room-temperature ionic liquids of low water solubility. These traditional sorbents are being re-imagined as covalently bonded or encapsulated sorbents with higher efficiency

and sample loading capacity [85,86]. Contemporary applications of extraction chromatography are focused on actinide and lanthanide elements, especially radionucleotides encountered in the processing of nuclear waste and recycling of nuclear fuels [85–89]. For the latter application, systems developed at an analytical scale are optimized with a view to subsequent use for large-scale separations using either column or batch processes with a focus on reagents that exhibit high selectivity, recyclability, and radiochemical stability. Typical reagents for actinides and lanthanides are represented by organophosphorus (e.g., trialkyl phosphates, dialkylphosphoric acids, trialkylphosphine oxides, etc.) [87,90] and amide (e.g., dialkylamide, diglycolamide, etc.) [85,86,89] complexing reagents and cryptand-containing ligands for group (II) metals (e.g., 90Sr) [88]. For coated and impregnated supports stability, sample capacity, and low efficiency are issues of varying importance that depend on the operating conditions (e.g., acidity, ionic strength, matrix composition, and pore network structure) [87,89]. See Chapter 6 for further details.

3.4 Organic ions The retention of organic ions by solid sorbents exhibits greater variation than typical inorganic ions due to their variable, and in many cases, manipulatable charge status and more favorable participation in general intermolecular interactions with the sorbent in addition to electrostatic attraction. Modern ion-exchange sorbents aim to exploit both types of interactions simultaneously using dual-phase or mixedmode sorbents to achieve improved selectivity and higher capacity [9,49,51,91,92]. Typical ion-exchange sorbents have either a silica or porous polymer skeleton decorated with immobilized ionic or ionizable functional groups. They are broadly classified as strong (SCX) or weak (WCX) cation exchangers with sulfonic acid or carboxylic acid functional groups,

3 Solid-phase extraction

respectively, or strong (SAX) or weak (WAX) anion exchangers with quaternary amine or primary, secondary, or tertiary amine functional groups, respectively. The distinguishing feature is that strong ion exchangers possess functional groups with a formal charge that is independent of the sample pH while weak ion exchangers have a variable charge dependent on the conditions employed during sample processing. For silica-based sorbents, their stability outside the pH range of 2–8 is poor except for silica-organic hybrid materials, but these are not widely used in SPE. Silica-based materials also generally have a greater capacity for nonelectrostatic interactions compared with polymer-based sorbents, which are typically stable over the full pH range. Commercially available polymer-based ionexchange sorbents typically have a poly(styrene-divinylbenzene), poly(divinylbenzene), or poly(vinylpyrrolidone-divinylbenzene) skeleton with functional groups incorporated into the polymer by either surface modification or by incorporation of monomers containing the target functional group or a suitable precursor subsequently converted to the target functional group [91–95]. Hypercross-linking of porous polymers is a common approach used to increase their microporosity increasing their specific surface area (>1000 m2/g) compared with conventional macroporous polymers (30 nm) and hydrophilic surfaces compatible with weak and reversible protein-sorbent interactions [100–102]. Sample complexity typically dictates that higher efficiency and capacity column formats are adopted for fractionation using several columns with different retention mechanisms (e.g., affinity, ion exchange, size exclusion, reversed phase, and hydrophobic interaction) and sequential fractionation and further separation on each column. Both low- and highresolution chromatographic systems can be employed in this process. Sample throughput is low and typically only semiautomated. For ion-exchange fractionation, pH or ionic strength gradients are typically used to distribute protein mixtures over a reasonable number of fractions for further separation. Weak ion exchangers are more flexible and afford higher selectivity for the fractionation of proteins and are the usual choice for proteins that retain their functionality over the pH range of 6–9, as well as for labile proteins that may require mild elution conditions. See Chapters 4 and 21 for further details.

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Classical SPE techniques are suitable for the extraction of peptides [101,103–106]. Preliminary solvent precipitation of proteins may be required to avoid cartridge contamination and blockage if the loss of peptides from co-precipitation is tolerable. Peptides contain a terminal carboxylic acid group and can be extracted from weakly basic solutions by anion-exchange sorbents when a nonselective extraction method is desired. Neutral and polar contaminants can be removed in the wash step by a selection of appropriate solvent systems. A limited fractionation of peptides is possible using stepwise changes in the pH or ionic strength of the elution solvent. A modern approach to proteomics is the use of integrated sample preparation systems performing several unit operations in a single device before separation by liquid chromatography [107–109]. These systems are designed for diagnostic purposes in clinical chemistry using only a few microliters of plasma. A typical workflow includes such operations as cell lysis, buffer exchange, reduction/ alkylation modification, digestion, and desalting in an on-chip or in-capillary tube format. A popular option incorporates a small column containing strong cation-exchange beads to capture proteins for on-bead sample manipulation and finally protein digestion. The resulting polypeptides are then analyzed by liquid chromatography-mass spectrometry. More than 290 proteins can be profiled in less than 2 h for a 1 μL plasma sample by this approach [107]. Devices that combine reversedphase clean-up with ion exchange fractionation allow the identification of 862 proteins in 12h (separation time for liquid chromatography) from 1 μL of depleted plasma [108]. Oligonucleotides of therapeutic interest are typically 7 that are not detected by suppressed conductivity due to their low dissociation. Conductivity detection does not always successfully handle the determination of anions at low concentrations in saline solution, especially when these ions are eluted on the tail of the chloride peak. However, a high concentration of chloride does not interfere with using a UV absorbance detector placed after the conductivity cell. The simultaneous application of two detection systems in series is a convenient solution for determining the anion composition of wastewater, seawater, body fluids, and extracts from meat products. MS detection is mostly implemented for trace and ultra-trace analysis to achieve lower detection limits. Each detection system determines a range of eluents, which are suitable for the separation of the target anions.

1.1 Eluents For many years sodium carbonate, sodium bicarbonate, or their mixture were widely used as eluents for the separation of anions with conductivity detection. After suppression, these

317

eluents convert to weakly dissociated H2CO3 with a very low background conductivity. However, the suppression of carbonate buffers results in a significant water dip as the conductivity of water from the sample is considerably lower than the eluent background conductivity. This negative dip can cause difficulties in the determination of early eluting anions. Depending on the concentration and the composition of the carbonate/bicarbonate buffer eluents of medium or strong eluent strength can be prepared. The choice of eluent is determined by the affinity of the eluent counter-ion(s) and target analyte ions for the stationary phase. Thus, sodium bicarbonate is suitable for the separation of monovalent anions, while the retention of divalent species is too strong. However, sodium carbonate facilitates the elution of divalent anions in a reasonable time, although there is a possibility of insufficient separation or coelution. A carbonate/bicarbonate mixture often provides optimum eluting conditions for ions of different valency. Nevertheless, such eluents do not allow the analysis of some polarizable and polyvalent anions. An absence of the possibility of increasing the eluent concentration during analysis makes carbonate/bicarbonate buffers unsuitable for the determination of strongly retained analytes. Utilizing a hydroxide eluent helps to achieve lower detectability for target ions, as hydroxide converts to water after suppression. Hydroxide ions possess a weak affinity for typical stationary phases and to elute polyvalent anions a relatively high concentration (up to 100 mmol/L) of hydroxide eluent is required. The initial concentration doesn’t influence the background signal, so hydroxide eluents are suitable for gradient elution. Increasing the hydroxide ion concentration leads to a decrease in anion retention and shorter separation times, explaining why gradient elution for multicomponent mixtures of anions of different valency is so frequently used. The pH of the eluent, which is closely connected to the eluent concentration,

318

15. Separation of ions by ion chromatography

primarily influences the retention of multivalent ions, because their valency is determined by the eluent pH. For example, in a carbonate/bicarbonate eluent orthophosphoric acid is present mainly as the divalent hydrophosphate ion, which elutes before sulfate. In a hydroxide eluent, the equilibrium shifts to trivalent phosphate, which elutes after sulfate. Manually prepared hydroxide eluents are always contaminated with carbonate that can result in irreproducible retention of anions. The use of electrolytic eluent generation helps to eliminate all the uncertainties of manual eluent preparation and to obtain carbonate-free hydroxide that, in turn, decreases background conductivity and baseline drift under gradient conditions. An alternative to suppressed conductivity detection is to connect the separator column directly to the conductivity cell (direct detection). This method was first demonstrated by Fritz et al. [5] for the determination of inorganic anions. To monitor anions with reasonable sensitivity, eluents with low conductivity and reasonable affinity for the stationary phase are required. Benzoates, phthalates, and osulfobenzoates meet this requirement and are typically used for nonsuppressed IC. To achieve appropriate retention of organic acids, the pH value of the eluent is varied between 4 and 7 to control the degree of acid dissociation. However, the background conductivity of aromatic eluents exceeds that of carbonate/bicarbonate eluents after suppression by 4–10 times, which reduces the detection limits and the linear range of the detector. Eluent choice is much broader for IC with spectrophotometric or amperometric detection. Due to good UV transmittance, alkali salts of phosphoric, sulfuric, and perchloric acids can be used with direct photometric detection. For UV-transparent anions indirect UV detection with a UV-absorbing eluent containing, for example, aromatic carboxylic acids can be used [6,7]. A wider range of eluents (alkali chlorides, chlorates, perchlorates, hydroxides, and carbonates) are compatible with amperometric detection. For MS detection, a

micromembrane suppressor placed between the separation column and ion spray interface is used for desalting the eluent [8]. Using a suppressor limits the choice of eluents to strong bases; however, in contemporary practice, many stationary phases for the separation of anions using a hydroxide eluent have been developed.

1.2 Stationary phases The matrix of ion exchangers is characterized by two main parameters—chemical and mechanical stability, which determine their field of application. For example, polymethacrylatebased resins find limited use due to their poor stability at pH values between 1 and 12. Consequently, such stationary phases are used in the nonsuppressed conductivity mode or with suppression using carbonate/bicarbonate eluents for the separation of seven inorganic anions, usually named “standard anions.” At the same time, polyvinyl alcohols are stable over the pH range of 0–14 and can be used with hydroxide eluents. However, due to poor mechanical stability, such columns are operated at lower flow rates compared with column packings prepared from other polymer substrates, and so separations take longer. Nowadays, poly(styrenedivinylbenzene) (PS-DVB) and ethylvinylbenzene/divinylbenzene (EVB/DVB) are the most widely used matrices for the preparation of ion exchangers due to their stability over the pH range of 0–14 and favorable mechanical strength as well as compatibility with organic solvents provided by high cross-linking or a high percentage of divinylbenzene in the resin. The high mechanical and chemical stability of PS-DVB or EVB/DVB ion exchangers allow the simultaneous separation of neutral compounds and ions in a reasonable time. Ion retention in general is determined by the surface concentration of fixed-charged sites on the ion exchanger. The ion-exchange capacity is defined as the number of ion-exchange sites per weight equivalent of the column packing [9]. Most anion exchangers have a capacity from

319

1 Anion-exchange chromatography

20 to 200 μequiv/g. An increase in ion exchange capacity is associated with an increase in retention. Nevertheless, the high capacity of an anion exchanger can be compensated to some extent by using a stronger counter-ion or by increasing the eluent concentration. The selectivity of the stationary phase depends mainly on its surface structure and method of attachment of the fixed charge sites to the matrix surface. The covalent attachment of single quaternary ammonium groups to the polymer matrix is the simplest route to prepare an anion exchanger. However, in the case of chemical derivatization ion-exchange sites can be unevenly distributed on the matrix surface or penetrate the pores of polymer particles. This results in ineffective shielding of the matrix and nonionic interactions of the anions with the matrix can cause significant retention of polarizable anions and decrease peak symmetry. The presence of ion-exchange sites in the pores increases the thickness of the ion-exchange layer and a decrease in mass transfer velocity is observed as well as an increase in band broadening. To minimize this effect spatially distancing of the functional groups from the matrix surface

is often utilized. However, the selectivity of chemically modified stationary phases is limited typically to the separation of standard anions (Fig. 2). Early in the development of IC nanobeadagglomerated anion exchangers were introduced [1]. These phases are typically based on a surface sulfonated PS-DVB or EVB/DVB matrix with a particle size of 5–10 μm to which is electrostatically attached a layer of fully aminated porous polymethacrylate or polyvinyl benzyl chloride beads of 0.1 μm diameter. This layer of latex particles is responsible for anion retention. The thin functional layer leads to fast ion-exchange processes and higher column efficiency. The selectivity of nanobeadagglomerated resin can be easily manipulated by changing the degree of latex cross-linking and the structure of functional groups. Nanobead-agglomerated anion exchangers with different selectivity are available for specific applications, for example, for the analysis of oxohalides and standard anions using a carbonate/bicarbonate eluent (Fig. 3). The elution of bromate before chloride allows the determination of the small amount of bromate in the

SO42-

15 μS

PO43-

NO3-

ClBr-

F-

NO2-

min 0

2

4

6

8

10

12

FIG. 2 Separation of standard anions obtained on a chemically modified anion exchanger. Eluent KOH, gradient elution. Flow rate: 1.0 mL/min. Detection: suppressed conductivity.

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15. Separation of ions by ion chromatography

FIG. 3

Separation of standard anions and oxohalides on an IonPac AS12A column. Eluent: 0.3 mmol/L sodium bicarbonate + 2.7 mmol/L sodium carbonate. Flow rate: 1.5 mL/min. Detection: suppressed conductivity. 1—fluoride, 2—chlorite, 3—bromate, 4—chloride, 5—nitrite, 6—bromide, 7—chlorate, 8—nitrate, 9—orthophosphate, 10—sulfate [10].

presence of much higher concentrations of chloride and sulfate in groundwater and wastewater. Among EVB/DVB-based stationary phases grafted ion exchangers containing two or more separately synthesized polymers are an important resource. Grafting an anion-exchange polymer to the surface of a highly cross-linked ethylvinylbenzene substrate can be achieved by different approaches, for example, using a base polymer with functional groups that can react with a second monomer. The thickness of the ion-exchange polymer layer can be controlled to achieve high chromatographic efficiency. Grafted anion exchangers allow the separation of some organic anions (glycolate, acetate, formate, oxalate) and standard inorganic anions

using carbonate/bicarbonate eluents or a gradient elution with hydroxide solution. Hyperbranched anion exchangers are used for different applications including the separation of haloacetic acids formed during the disinfection of drinking water via chlorination. Originally introduced by Pohl and Saini [11], the hyperbranched stationary phases were obtained by the electrostatic attachment of the linear positively charged polymer to the sulfonated polymer surface and further repeating modification cycles with diepoxide and methylamine. However, since that time other amino compounds, especially amino acids, have been used for the formation of a hyperbranched layer and its covalent attachment to the polymer matrix, leading to the development of anion exchangers with unique selectivity

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1 Anion-exchange chromatography

4 μS

Response

1

3

5 4

6

2

20

12

10 11

14 16 13

9

15

19

21

22

18 17

7 8

min 0

5

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85

FIG. 4

Separation of a multicomponent anion mixture on a hyperbranched anion exchanger. Eluent KOH, gradient elution. Flow rate: 1.0 mL/min. Detection: suppressed conductivity. 1—fluoride, 2—quinate, 3—glycolate, 4—acetate, 5—lactate, 6—formate, 7—galacturonate, 8—propionate, 9—chlorite, 10—chloride, 11—bromate, 12—nitrite, 13—sulfate, 14—malate, 15—glutarate, 16—oxalate, 17—phosphate, 18—nitrate, 19—fumarate, 20—citrate, 21—isocitrate, 22—trans-aconitate [12].

(Fig. 4). Such phases allow the determination of organic acids, including glycolic, acetic, formic, and lactic, as well as inorganic anions in samples with complex matrices, such as orange juice, in a single separation [12]. IC separations should provide satisfactory resolution of sample ions in as short time as possible. For faster separations, the flow rate can be increased but is limited by the maximum column operating pressure. Most columns in IC are operated at a flow rate from 1.0 to 1.5 mL/min. In recent years, the development of polymer monolithic columns for IC was started [13]. The main advantage is their higher flow permeability allowing a relatively low column back pressure at higher flow rates. Such phases are irreplaceable in high-speed and high-resolution separations of inorganic anions and organic acids using hydroxide eluent.

1.3 Separation of anions Many inorganic anions can be determined by IC, Table 1. Today, the determination of the

TABLE 1 Inorganic anions determined by ion chromatography. Standard anions

Fluoride, chloride, bromide, nitrite, nitrate, sulfate, phosphate

Polarizable anions

Iodide, thiocyanate, thiosulfate, perchlorate, chromate, molybdate, tungstate, arsenate, selenate

Oxyhalides

Bromate, hypochlorite, chlorite, chlorate, iodate, periodate

Sulfur compounds

Sulfide, sulfite

Polyphosphates

Pyrophosphate, tripolyphosphate, trimethaphosphate, tetrapolyphosphate

Nitrogen compounds

Cyanide, azide, cyanate, hyponitrite

Other anions

Borate, tetrafluoroborate, silicate, arsenite, selenite

standard anions in environmental samples by IC is a routine task. The separation of fluoride from the void volume and its determination in the presence of weakly retained organic acids was a problem

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for older stationary phases. New stationary phase architectures allow the fluoride to be determined simultaneously with other standard anions in different samples using carbonate/ bicarbonate or hydroxide eluents and conductivity detection. The selectivity of the stationary phase determines not only the possibility to separate different anions but also to determine target anions in samples with complex matrices. The concentration differences between neighboring ions are of great importance for the analysis of oxohalides, since they are typically present in small amounts in real-world samples. In this case, columns that allow the elution of the target ions before compounds at a high concentration are necessary. In some cases, the analysis of oxohalides in the submicrogram/liter range is required. The determination of weakly retained organic acids, such as glycolate, formate, and acetate, in a power station steam at a trace level is of great importance. Another example is the analysis of disinfection by-products, such as chlorite and chlorate in swimming pool water after chlorination. Detection of oxyhalides that can be formed after ozonation in drinking water at sub-μg/L level can be achieved by utilizing preconcentration techniques. However, it requires additional hardware, namely, a concentrator column and a second pump, and takes additional time for the preconcentration step. Nowadays, the determination of anions at sub-μg/L level is often realized via direct injection of a large sample volume. For this purpose, special high-capacity columns with 2 mm internal diameter are used, since they provide a fourfold increase in mass sensitivity compared to 4 mm diameter columns. The technique of large-volume injection can be significantly improved by using hydroxide eluent generation through a minimization of baseline drift during the gradient separation. The combination of a high-capacity anion exchanger with a 250 μL injection loop and electrolytically generated KOH as an eluent allows sub-μg/L detection limits for chlorite, bromate, chlorate,

and bromide using suppressed conductivity detection (Fig. 5). Polarizable anions (see Table 1) exhibit a high affinity for anion exchangers, and consequently, the simultaneous determination of polarizable and nonpolarizable anions was a challenging task for a long time. Modern stationary phases and gradient elution techniques allow the simultaneous separation of standard and polarizable anions (see Fig. 1). When conductivity detection is inadequate more specific detection systems can solve the problem. For instance, direct UV detection and amperometric detection are useful for the determination of iodide in the presence of high chloride concentrations. Amperometric detection is also utilized for the analysis of other halides, sulfide, cyanide, thiosulfate, nitrite, and oxyhalide anions at the microgram/liter level [15]. Some anions, such as orthosilicate, borate, and organic acids are difficult to detect by suppressed conductivity because they are only partially ionized after suppression. In this case, a charge detector is used, for example, for the analysis of organic acids typically found in fruit juices. A wide range of organic anions can be determined by IC—carboxylic, phosphonic, and sulfonic acids, organophosphates and organosulfates, carbohydrates, and amino acids. IC is used mainly when organic anions are to be simultaneously analyzed with inorganic anions. For a long time, the separation of some monovalent weakly retained carboxylic acids, such as glycolic, acetic, formic, and lactic, was considered difficult by IC because of the low selectivity of existing stationary phases. The addition of methanol to eluents led to an improved separation [16]. Nevertheless, the resolution of carboxylic acids in realworld samples is often lower than in model mixtures, especially in complex samples such as fruit juices, wine, and other beverages. Nowadays, hyperbranched anion exchangers allow the separation of these organic acids together with other di- and trivalent carboxylic acids and inorganic anions with hydroxide eluents in fruit juices and other beverages, as shown in Fig. 6. Despite the long separation time, IC with highly selective

1 Anion-exchange chromatography

323

FIG. 5 Analysis of bottled drinking water. Column: IonPac AS19. Eluent: 10 mM KOH 0–10 min, 10–45 mM 10–25 min. Column temperature: 30°C. Flow rate: 1.0 mL/min. Detection: suppressed conductivity. Sample: (A) Bottled water and (B) spiked bottled water. Analytes: (A) 2—Bromate (0.00098 mg/L), 3—chlorate (0.0042 mg/L); (B) 1—chlorite (0.022 mg/L), 2—bromate (0.0049 mg/L), 3—chlorate (0.022 mg/L), 4—bromide (0.022 mg/L) [14].

columns and conductivity detection has replaced several methods still applied for the determination of organic acids in fruit juices since they complement each other. Using hyperbranched anion exchangers with increased selectivity toward organic acids meets the requirements of “green chemistry” and makes it possible to define organic acid profiles in samples with complex matrices without using additional electrochemical or enzymatic methods for the determination of single organic compounds.

Sometimes, IC is the preferred choice because of its simplicity and absence of additional pretreatment steps. The conventional method for determining haloacetic acids, the disinfection products that need to be measured at trace levels in water, is gas chromatography with extraction and derivatization. However, modern anion exchangers allow the determination of these compounds at an acceptable level using MS detection without time-consuming sample preparation. IC can also be used to determine azide in

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16

2 1

7 μS

8

6 7 4 5

12 11

7

12

17

22

27

A

32

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9

5

4

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6

9 μS

7

16

B 10

15

20

25

30

35

9 2 3 μS

16

3 5

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15

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7 6

4 10

15

13

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C

9 20

25

30

35

10

14

13

min 0

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30

40

50

60

70

80

90

100

110

120

130

FIG. 6 Chromatograms of fruit juices diluted five times on a M4-DEA 250  4 mm i.d. column. Flow rate: 1.0 mL/min. Eluent: KOH, gradient elution. Detection: suppressed conductivity. Samples: A—grape juice, B—blueberry juice, C—mango juice. Analytes: 1—gluconic acid, 2—quinic acid, 3—shikimic acid, 4—glycolic acid, 5—acetic acid, 6—lactic acid, 7—formic acid, 8—galacturonic acid, 9—chloride, 10—sulfate, 11—malic acid, 12—tartaric acid, 13—oxalic acid, 14—phosphate, 15— nitrate, 16—citric acid [17].

pharmaceuticals, body fluids, and in buffers used for long-term storage of immunoaffinity columns. As a highly toxic compound, azide needs to be monitored in industries using it as a reagent. Azide content can be determined by gas chromatography or reversed-phase high-performance liquid chromatography (RP HPLC) with derivatization, however, IC with suppressed conductivity detection is preferable due to the absence of timeconsuming sample preparation steps [18]. Modern anion exchangers allow the determination of azide in samples containing a high concentration of chloride, phosphate, and citrate (Fig. 7). Short-chain organosulfates are typically separated by IC while long-chain organosulfates are

FIG. 7 Chromatogram of protein sample spiked with azide eluted from an immunoaffinity column. Azide concentration is equal to the concentration limit (0.06 mg/L). Column IonPac AS11 250 mm  4 mm i.d. Flow rate: 1.0 mL/min. Eluent: NaOH, gradient elution. Detection: suppressed conductivity. 1—chloride, 2—azide, 3—bromide, 4—overlapping peaks of phosphate and citrate [18].

325

1 Anion-exchange chromatography

product of the nerve agent sarin and needs to be monitored at low concentrations. The necessary detection limits can be achieved by a combination of IC with ICP-MS. In the case of bisphosphonates, which are active pharmaceutical ingredients for the treatment of osteoporosis and hypercalcemia, such high sensitivity is not required. Bisphosphonates can be analyzed by gas chromatography or RP HPLC with derivatization, IC with suppressed or nonsuppressed conductivity detection, as well as postcolumn derivatization with subsequent UV detection [20]. Among these methods, IC with suppressed conductivity detection is the most straightforward because it doesn’t require a derivatization step and provides simultaneous separation of ionic impurities (Fig. 9). The introduction of pulsed amperometric detection considerably enlarged the scope of IC by expanding its range of applications to include compounds such as carbohydrates and amino acids [22]. Since pKa values for carbohydrates lie between 12 and 14, they can be separated on anion exchangers with a hydroxide

usually analyzed by ion-pair chromatography. For short-chain alkyl sulfates high-performance liquid chromatography (HPLC) with ion-pair reagents can also used, however, the choice of ion-pair reagents and buffers is limited in the case of MS detection. IC with a hydroxide eluent offers an alternative approach to HPLC-MS. An important organosulfate that can be determined by IC is sulfamate. Sulfamate as well as sulfate are the degradation products of the antiepileptic drug topiramate. For monitoring topiramate degradation sulfamate and sulfate are easily determined by IC with conductivity detection. Since the retention of sulfamate is similar to weakly retained carboxylic acids, an anion exchanger with high selectivity toward these compounds, such as hyperbranched anion exchanger GM4 [19], is required. Using gradient allows the elution of sulfate in a reasonable time (Fig. 8). The determination of phosphorus-containing organic compounds like alkyl phosphonic acids and bisphosphonates is an important task. Methylphosphonic acid is the degradation

SO42-

Response

10 µS

С O32NH2SO30

2

4

6

8

10

12

14

16

18

20

22

min FIG. 8 Analysis of antiepileptic drug topiramate. Column GM4 250  4 mm i.d. Flow rate: 1.0 mL/min. Eluent: KOH, gradient elution. Detection: suppressed conductivity.

326

15. Separation of ions by ion chromatography

be classified as strong acid phases containing sulfonate groups and weak acid phases containing carboxylic groups. The ion-exchange process on a strong cation exchanger can be described in the following way: Resin
SO3
H+ + NaCl ¼ Resin
SO3
Na+ + HCl

FIG. 9 Separation of clodronate and potential ionic impurities. Column: IonPac AS5. Column temperature: 45°C. Eluent: NaOH, gradient: 20–100 mmol/L in 20 min. Flow rate: 1 mL/min. Detection: suppressed conductivity. 1—chloride, 2—nitrate, 3—diester of dichloromethylene bisphosphonic acid, 4—sulfate, 5—orthophosphate, 6—monoester of dichloromethylene bisphosphonic acid, 7—clodronate, 8— monochloromethylene bisphosphonic acid, 9— methylenebisphosphonic acid, 10—carbonyl bisphosphonic acid [21].

eluent. Amino acids are traditionally determined by cation exchanger chromatography; however, anion exchangers can be used as well for their analysis with integrated amperometric detection. The simultaneous determination of amino acids and carbohydrates by anionexchange chromatography with pulsed amperometric detection is illustrated in Fig. 10. Amino acids with primary amine functional groups can be determined at low picomole concentrations using fluorescence detection with postcolumn derivatization.

2 Cation-exchange chromatography For the separation of cations stationary phases with negatively charged groups and acidic eluents are used. Cation exchangers can

Cation retention is also determined by the size and charge of the analyte. Smaller cations elute faster than larger ones, while monovalent species elute before divalent cations. However, the introduction of complexing agents in the eluent can influence the retention behavior of cations, for example, reversing the elution of magnesium and calcium. Conductivity detection with or without suppression is applicable for cation detection. The suppressor in cation exchange chromatography is also used to decrease the eluent background conductivity and to convert the sample cations into a more conductive form. The high suppression capacity and low noise of self-regenerating membrane suppressors make them the most suitable for this application. The principle of suppressor systems in cation-exchange chromatography is illustrated below: Resin
NR3 + OH
+ HCl ¼ Resin
NR3 + Cl
+ H2 O Resin
NR3 + OH
+ NaCl ¼ Resin
NR3 + Cl
+ NaOH For the separation of alkali metals, ammonium, and small aliphatic amines on strong acid cation exchangers mineral acids, such as hydrochloric, sulfuric, or nitric acid, are typically used as eluents. The simultaneous analysis of alkali metals, alkaline-earth metals, and ammonium on weak acid cation exchangers is possible using eluents containing methanesulfonic or sulfuric acid. A mixture of ethylenediamine and dicarboxylic acids (tartaric or oxalic) is used as an eluent for the determination of

2 Cation-exchange chromatography

327

FIG. 10 Separation of 17 amino acids and nine carbohydrates. Column AminoPac PA 10. Eluent: NaOH + NaAc, gradient elution. Flow rate: 0.25 mL/min. Detection: integrated pulsed amperometry. 1—arginine, 2—fucose, 3—lysine, 4—arabinose, 5—glucose, 6—alanine, 7—threonine, 8—fructose, 9—glycine, 10—ribose, 11—valine, 12—serine, 13—proline, 14—sucrose, 15—lactose, 16—isoleucine, 17—leucine, 18—methionine, 19—raffinose, 20—maltose, 21—histidine, 22—phenylalanine, 23—glutamic acid, 24—aspartic acid, 25—cystine, 26—tyrosine [23].

alkaline-earth metals with nonsuppressed conductivity detection.

2.1 Stationary phases All polymer substrates used for the preparation of anion exchangers are utilized for cation exchangers. The ion exchange capacity is varied typically from 5 to 100 μequiv/g. Surfacesulfonated PS-DVB-based cation exchangers are mainly used for the separation of alkali metals with mineral acids as eluents and conductivity detection. Strong acid sulfonate groups do not provide the possibility of simultaneous separation of mono- and divalent cations due to the

high affinity of divalent cations for the stationary phase. Typically, alkali and alkaline-earth metals are analyzed sequentially with different eluents on strong acid cation exchangers. The exception is nanobead-agglomerated cation exchangers with an EVB/DVB support, a covalently bonded monolayer of aminated polymer particles, and an electrostatically attached second layer of sulfonated nanobeads. Due to the low density of sulfonate groups at the surface of the cation exchanger, the retention of divalent cations is close to that of monovalent cations allowing faster elution of alkaline-earth metals. More often weak acid cation exchangers with carboxylic functional groups are used for the

328

15. Separation of ions by ion chromatography

Implementing silica as a matrix for cation exchangers is attractive due to higher efficiency. Sulfonated silica-based cation exchangers and polymer-coated silica with a polymer layer obtained from butadiene and maleic acid are widely used. Polymer-coated silica with weak carboxylic groups is suitable for the simultaneous analysis of alkali and alkaline-earth metals with or without suppression.

2.2 Separation of cations FIG. 11 Gradient elution of Group I and II cations, ammonium, and alkylamines. Column: IonPac CS17 (250 mm  2 mm i.d.). Column temperature: 40°C. Eluent: MSA, gradient elution. Flow rate: 0.40 mL/min. Detection: suppressed conductivity. 1—lithium, 2—sodium, 3—ammonium 4—potassium, 5—ethylamine, 6—propylamine, 7— tert-butylamine, 8—sec.-butylamine, 9—isobutylamine, 10—n-butylamine, 11—1,2-dimethylpropylamine, 12—din-propylamine, 13—magnesium, 14—calcium [24].

simultaneous analysis of alkali and alkaline-earth metals. An example is chemically derivatized poly(vinyl alcohol)-based cation exchangers with carboxylic groups. However, cation exchangers consisting of a highly cross-linked mesoporous EVB/DVB with a grafted ion-exchange polymer are more common. The functional layer of these phases contains weak acid groups (carboxylic or carboxylic together with phosphonate) and facilitates the separation of alkali and alkaline-earth metals as well as ammonium and organic amines (Fig. 11). In contrast to nanobead-agglomerated cation exchangers mono- and divalent cations are eluted with methanesulfonic acid. Using gradient elution allows the reduction of the separation time with improved resolution and efficiency in some cases. For higher reproducibility electrolytically generated methanesulfonic acid is often used, especially for gradient separations. Silica is limited as a matrix for anion exchangers by its poor hydrolytic stability at basic pH, however, for the separation of cations with weak acidic eluents this is not a problem.

Cation-exchange chromatography is used for the separation of alkali and alkaline-earth metals, ammonium, aliphatic and aromatic amines, and transition metals. The advantage of IC compared with conventional instrumental methods is the simultaneous determination of ions. While in the past, strong sulfonate cation exchangers allowed only the consecutive determination of alkali and alkaline-earth metals, their simultaneous separation and ammonium are now a routine task for IC. Typically, polymer-based weak carboxylic cation exchangers with methanesulfonic acid as an eluent are used for the separation. The retention of alkali and alkaline-earth metals increases with increasing ionic radius. Ammonium, difficult to detect by other methods, usually elutes between sodium and potassium and can be determined even at low concentrations compared with alkali metals. Primary, secondary, and tertiary alkylamines are determined on carboxylic cation exchangers with suppressed or nonsuppressed conductivity detection. The selectivity of weak acid cation exchangers can be influenced by adding aprotic solvents, such as acetonitrile, to the eluent. The lower dissociation of carboxylic acids in the presence of acetonitrile results in a retention decrease of cations, especially divalent species. In the past, the addition of organic solvents to the eluent was often used for the rapid analysis of organic amines, while now organic solvents are required only in special cases, such as the separation of aromatic amines. The

2 Cation-exchange chromatography

FIG. 12 Gradient elution of Group I and II cations, ammonium, and biogenic amines. Column: IonPac CS17 (250 mm  2 mm i.d.). Column temperature: 40°C. Eluent: MSA, gradient elution. Flow rate: 0.40 mL/min. Detection: suppressed conductivity. 1—lithium, 2—sodium, 3—ammonium, 4—potassium, 5—magnesium, 6—calcium, 7—putrescine, 8—cadaverine, 9—histamine, 10—spermidine, 11— spermine [24].

development of weak acid cation exchangers with hydrophilic surfaces allows the determination of biogenic amines together with alkali and alkaline-earth metals using purely aqueous eluents with suppressed conductivity detection (Fig. 12). Biogenic amines are used as an indicator of food spoilage, and the necessary specificity of the analysis in complex food matrices can be provided by integrated pulsed amperometric detection. This detection method is extremely important for the specific determination of oxidizable amines in the presence of high concentrations of inorganic cations that are not detected. Primary amines and polyamines can also be determined by fluorescence detection after derivatization of primary amine functional groups by reaction with o-phthaldialdehyde in the presence of 2-mercaptoethanol. IC allows the determination of transition metals for multielement analysis at the nanogram/Liter range after preconcentration. To reduce the effective charge of the transition metals complex-forming reagents are added to the eluent. Weak organic acids such as citric, oxalic, tartaric, or pyridine-2,6-dicarboxylic acid

329

are typically used for this purpose. Transition metals form anionic or neutral complexes with these organic acids suitable for separation using anion or cation exchangers. Nowadays, for the analysis of transition metals nanobead-agglomerated stationary phases with anion- and cation-exchange functionalities are commonly used. Suppressed conductivity detection is impossible due to the formation of undissociated metal hydroxides during the suppressor reaction. Nonsuppressed conductivity detection can be used for the simultaneous determination of alkali, alkaline-earth, and transition metals (Fig. 13). However, the specificity of nonsuppressed conductivity detection is limited by high amounts of matrix cations, and therefore photometric detection with postcolumn derivatization is typically utilized (Fig. 14). The ultra-trace determination of transition metals is necessary for the power plant industry. The analytes can be concentrated on a small concentrator column, thus improving detection limits down to the single-digit nanogram/liter levels. Using high-capacity ion exchangers allows detection limits in the mid nanogram/ liter level by direct injection of up to 2000 μL samples. IC in the capillary format became available during the last two decades [27]. Using 0.4 mm internal diameter columns allows a decrease in flow rates and injection volumes to 10 μL/min and 0.4 μL, respectively. Capillary IC provides such advantages as prolonged system operation and increased mass sensitivity. 20 μL injection on a capillary column is equivalent to 2000 μL injection on a 4 mm internal diameter column, making capillary IC useful for ultra-trace analysis. Another advantage of IC is its capability to determine transition metals in different oxidation states. Thus, the simultaneous analysis of chromium (III)/(VI), which was a problem for a long time, is possible by ion-exchange chromatography. For chromium speciation, an anion exchanger and potassium hydrogen phthalate eluent are used to retain chromium (VI).

FIG. 13 Separation of alkali, alkaline-earth, and transition metals. Column: IonPac SCS1 (250 mm  4 mm i.d.). Eluent: 2.5 mM MSA and 0.8 mM oxalic acid. Flow rate: 1.0 mL/min. Detection: nonsuppressed conductivity. 1—copper, 2—lithium, 3—sodium, 4—ammonium, 5—potassium, 6—magnesium, 7—zinc, 8—cobalt, 9—nickel, 10—calcium, 11—strontium [25].

FIG. 14 Separation of transition metals. Column: PRP-1 (300  4.6 mm i.d.). Eluent: 1 M potassium nitrate, 0.25 mM chlorodipicolinic acid and 6.25 mM nitric acid (pH 2.2). Detection: spectral array [26].

References

Chromium (III) elutes in the column void volume and undergoes postchromatographic oxidation to chromium (VI). Then, both chromium species are determined by indirect photometric detection. Higher sensitivity is possible by combining a bifunctional ion exchanger with ICP-MS detection [28].

[13]

[14]

References [1] H. Small, T.S. Stevens, W.C. Bauman, Novel ion exchange chromatographic method using conductimetric detection, Anal. Chem. 47 (1975) 1801–1809. [2] O.I. Shchukina, A.V. Zatirakha, A.S. Uzhel, A.D. Smolenkov, O.A. Shpigun, Novel polymer-based anion-exchangers with covalently-bonded functional layers of quaternized polyethyleneimine for ion chromatography, Anal. Chim. Acta 964 (2017) 187–194. [3] C. Dengler, M. Kolb, M. L€aubli, Methodenvergleich der Ionenchromatographie mit und ohne chemische Suppression, GIT Fachz. Lab. 6 (1996) 609–614. [4] D.L. Strong, P.K. Dasgupta, Electrodialytic membrane suppressor for ion chromatography, Anal. Chem. 61 (1989) 939–945. [5] D.T. Gjerde, J.S. Fritz, G. Schmuckler, Anion chromatography with low-conductivity eluents, J. Chromatogr. 186 (1979) 509–519. [6] H. Small, T.E. Miller, Indirect photometric chromatography, Anal. Chem. 54 (1982) 462–469. [7] R.A. Cochrane, D.E. Hillman, Analysis of anions by ion chromatography using ultraviolet detection, J. Chromatogr. 241 (1982) 392–394. [8] R.C. Simpson, C.C. Fenselau, M.R. Hardy, R.R. Townsend, Y.C. Lee, R.J. Cotter, Adaptation of a thermospray liquid chromatography/mass spectrometry interface for use with alkaline anion exchange liquid chromatography of carbohydrates, Anal. Chem. 62 (1990) 248–252. [9] J. Weiss, Handbook of Ion Chromatography, fourth ed., WILEY-VCH Verlag GmbH & Co., KGaA, Weinheim, 2016. [10] J. Weiss, S. Reinhard, C. Pohl, C. Saini, L. Narayaran, Stationary phase for the determination of fluoride and other inorganic anions, J. Chromatogr. A 706 (1995) 81–92. [11] C. Pohl, C. Saini, New developments in the preparation of anion exchange media based on hyperbranched condensation polymers, J. Chromatogr. A 1213 (2008) 37–44. [12] A.S. Uzhel, A.V. Zatirakha, A.D. Smolenkov, O.A. Shpigun, Quantification of inorganic anions and organic

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

331 acids in apple and orange juices using novel covalently-bonded hyperbranched anion exchanger with improved selectivity, J. Chromatogr. A 1567 (2018) 130–135. L. Geiser, S. Eeltink, F. Svec, J.M.J. Frechet, Stability and repeatability of capillary columns based on porous monoliths of poly(butyl methacrylate-co-ethylene dimethacrylate), J. Chromatogr. A 1140 (2007) 140–146. B.M. De Borba, J.S. Rohrer, C.A. Pohl, C. Saini, Determination of trace concentrations of bromate in municipal and bottled drinking waters using a hydroxide-selective column with ion chromatography, J. Chromatogr. A 1085 (2005) 23–32. R.D. Rocklin, E.L. Johnson, Determination of cyanide, sulfide, iodide, and bromide by ion chromatography with electrochemical detection, Anal. Chem. 55 (1983) 4–7. J. Stillian, C. Pohl, New latex-bonded pellicular anion exchangers with multi-phase selectivity for highperformance chromatographic separations, J. Chromatogr. 499 (1990) 249–266. A.S. Uzhel, A.N. Borodina, A.V. Gorbovskaya, O.A. Shpigun, A.V. Zatirakha, Determination of full organic acid profiles in fruit juices and alcoholic beverages using novel chemically derivatized hyperbranched anion exchanger, J. Food Compos. Anal. 95 (2021) 103674. K. Vinkovic, V. Drevenkar, Ion chromatography of azide in pharmaceutical protein samples with high chloride concentration using suppressed conductivity detection, J. Chromatogr. B 864 (2007) 102–108. A.S. Uzhel, A.V. Zatirakha, K.N. Smirnov, A.D. Smolenkov, O.A. Shpigun, Anion exchangers with negatively charged functionalities in hyperbranched ionexchange layers for ion chromatography, J. Chromatogr. A 1482 (2017) 57–64. C.K. Zacharis, P.D. Tzanavaras, Determination of bisphosphonate active pharmaceutical ingredients in pharmaceuticals and biological material: a review of analytical methods, J. Pharm. Biomed. Anal. 48 (2008) 483–496. G.E. Taylor, Determination of impurities in clodronic acid by anion-exchange chromatography, J. Chromatogr. A 770 (1997) 261–271. R.D. Rocklin, C.A. Pohl, Determination of carbohydrates by anion exchange chromatography with pulsed amperometric detection, J. Liq. Chromatogr. 6 (1983) 1577–1590. H. Yu, Y.-S. Ding, S.-F. Mou, P. Jandik, J. Cheng, Simultaneous determination of amino acids and carbohydrates by anion-exchange chromatography with integrated pulsed amperometric detection, J. Chromatogr. A 966 (2002) 89–97.

332

15. Separation of ions by ion chromatography

[24] M. Rey, C. Pohl, Novel cation-exchange column for the separation of hydrophobic and/or polyvalent amines, J. Chromatogr. A 997 (2003) 199–206. [25] W. Zeng, Y. Chen, H. Cui, F. Wu, Y. Zhu, J.S. Fritz, Single-column method of ion chromatography for the determination of common cations and some transition metals, J. Chromatogr. A 1118 (2006) 68–72. [26] M.J. Shaw, P. Jones, P.N. Nesterenko, Dynamic chelation ion chromatography of transition and heavy metal ions using a mobile phase containing

4-chlorodipicolinic acid, J. Chromatogr. A 953 (2002) 141–150. [27] P. Kuban, P.K. Dasgupta, Capillary ion chromatography, J. Sep. Sci. 27 (2004) 1441–1457. [28] H. Hagendorfer, W. Goessler, Separation of chromium(III) and chromium(VI) by ion chromatography and an inductively coupled plasma mass spectrometer as element-selective detector, Talanta 76 (2008) 656–661.

C H A P T E R

16 Applications of ion chromatography in environmental analysis Rajmund Michalski Institute of Environmental Engineering of Polish Academy of Sciences, Zabrze, Poland O U T L I N E 1. Introduction

333

2. Ion chromatography and related techniques 2.1 Water samples 2.2 Atmospheric samples

333 334 338

1 Introduction Substances analyzed in environmental samples are of natural and anthropogenic origin. They are present in all compartments of the environment and have a huge impact on the quality of life [1]. They may be classified as chemical (inorganic and organic), biological, and physical [2]. An important part of environmental analytical chemistry is the determination of inorganic anions (e.g., F
, Cl
, NO2
, Br
, SO32
, NO3
, PO43
, SO42
), and cations (e.g., Li+, Na+, K+, Mg2+, Ca2+), as well as some organic ions (e.g., carboxylic acids, amines). Substances occurring in ionic forms are believed to exhibit biological activity and toxicity toward living organisms. Their determination was traditionally carried

Ion-Exchange Chromatography and Related Techniques https://doi.org/10.1016/B978-0-443-15369-3.00023-7

2.3 Solid samples

338

3. IC development perspectives

340

References

346

out using wet chemical methods. Unfortunately, many of these suffer from interferences and limited sensitivity and can be labor-intensive and difficult to automate [3]. Consequently, they are being replaced by instrumental methods, particularly ion chromatography (IC) and related techniques [4].

2 Ion chromatography and related techniques IC as a commercially available analytical method was reported in 1975 by Small, Stevens, and Bauman [5]. There are two main types of IC: Suppressed IC and nonsuppressed IC [6]. Nonsuppressed IC is less sensitive for common

333

Copyright # 2024 Elsevier Inc. All rights reserved.

334

16. Applications of ion chromatography in environmental analysis

inorganic anions than suppressed conductivity methods but still is useful for higherconcentration matrices. Ion exchange remains the primary separation mode in IC and can be subdivided into ion-pairing chromatography (IPC) [7], ion-exclusion chromatography [8], and ion chelation chromatography [9]. The separation and detection modes used in environmental analysis, as well as a selection of ionic species analyzed by IC and related techniques, are summarized in Table 1 (anions) and Table 2 (cations), respectively. The advantages of IC are fast and simultaneous separation and determination of several ions ( 8), aromatic sulfonates

Ion-pair, anionexchange, reversedphase

Conductivity

Phenols

Anion-exchange

UV, amperometry

Aliphatic alcohols

Ion-exclusion, reversed-phase, ion pair

Amperometry

Carboxylic acids

Sulfonic acids

Alcohols and phenols

336 TABLE 2

16. Applications of ion chromatography in environmental analysis

Typical cations determined by IC methods in environmental samples.

Analyte groups

Example analytes

Separation mechanism

Detection mode

Alkali and alkaline earth metals, ammonia

Li+, Na+, K+, Rb+, Cs+, Mg2+, Ca2+, Sr2+, Ba2+

Cation-exchange

Conductivity

Metals and metalloids

Cu2+, Ni2+, Zn2+, Co2+, Cd2+, Pb2+, Mn2+, Fe2+, Fe3+, Sn2+, Sn4+, Cr3+, Cr6+, V4+, V5+, As3+, As5+, Sb3+, Sb5+

Ion-exclusion, anion-exchange

Conductivity, UV/Vis

Lanthanides and actinides

La3+, Ce3+, Pr3+, Nd3+, Sm3+, Eu3+, Gd3+, Tb3+, Dy3+, Ho3+ Er3+, Tm3+, Yb3+, Lu3+

Anion-exchange, cation-exchange, cation-exchange, ion-pair

UV/Vis

Organic cations

Low molecular amines-alkylamines, mono-, di-, tri-, tetramethylamine, alkanolamines, monoethanolamine, diethanolamine

Cation-exchange, ion-pair

Conductivity

High molecular amines-alkylamines, aromatic amines, cyclohexylamines, quaternary ammonium ions, polyamines

Cation-exchange, ion-pair

Conductivity, UV

cations (e.g., Na+, K+, NH4+, Mg2+, Ca2+, Sr2+, Ba2+, organic amines). While the analysis of samples with simple matrices is relatively straightforward, problems arise when dealing with samples with complex matrices. In this context, some IC methods have been developed that can tolerate a high salt content, such as column switching [23–26] and multidimensional IC [24] in samples with difficult matrices such as seawater [25] or wastewater [26]. The determination of nontypical, but important ions such as cyanide and sulfite is important, because of their large-scale industrial use [27]. The next examples are polyphosphates widely used in everyday products and industrial water treatment applications [16], and chelating agents such as nitrilotriacetic acid (NTA) and EDTA [28]. There are a significant number of IC applications for metal and metalloids in a wide variety of environmental samples [13]. While alkali metal and alkaline earth cations are detected routinely using conductivity detection, for transition metals and lanthanides often postcolumn reaction (PCR) is used [29,30].

Sample preparation is typically the most laborious part of the analysis and the main source of errors. Environmental water samples are collected usually into plastic containers made of polytetrafluoroethylene (PTFE), polypropylene (PP), polystyrene (PS), or high-density polyethylene (HDPE). Various processes can occur in the sample between sampling and analysis (e.g., oxidation, reduction, complexation, precipitation, biochemical and photochemical processes). Therefore, they should be analyzed as soon as possible after sampling. Sample preparation encompasses several operations, including filtration, dilution, pH change, standard addition, derivatization, liquid-liquid extraction, solid-phase extraction, distillation, microdiffusion, and membrane techniques [31]. It is important for reliable results that even routine processes (dilution, pH changes through sample preservation, pressure, and temperature changes) may affect the primary analyte forms [32]. In many cases, water samples require only filtration through a 0.45 μm filter (to remove solid

2 Ion chromatography and related techniques

TABLE 3

337

IC standard methods for environmental applications.

Standard number (year of publication)

Standard title

Water samples ISO 10304-1 (1992)

Water quality—Determination of dissolved fluoride, chloride, nitrite, orthophosphate, bromide, nitrate, and sulfate ions using liquid chromatography of ions. Part 1: Method for water with low contamination

Replaced by PN-ISO 10304-1 (2009)

Water quality—Determination of dissolved anions by using liquid chromatography of ions. Part 1: Determination of bromide, chloride, fluoride, nitrite, nitrate, orthophosphate, and sulfate

ISO 10304-2 (1995)

Water quality—Determination of dissolved anions by liquid chromatography of ions. Part 2: Determination of bromide, chloride, nitrate, nitrite, orthophosphate, and sulfate in wastewaters

ISO 10304-3 (1997)

Water quality—Determination of dissolved anions by liquid chromatography of ions—Part 3: Determination of chromate, iodide, sulfite, thiocyanate, and thiosulfate

ISO 10304-4 (1997)

Water quality—Determination of dissolved anions by liquid chromatography of ions. Part 4: Determination of chlorate, chloride, and chlorite in water with low contamination

ISO 14911 (1998)

Water quality—Determination of dissolved Li+, Na+, NH+4 , K+, Mn2+, Ca2+, Mg2+, Sr2+, and Ba2+ using ion chromatography—Method for water and waste water

ISO 15061 (2001)

Water quality—Determination of dissolved bromate—Method by liquid chromatography of ions

ISO 10304-1 (2009)

Walter quality—Determination of dissolved anions by liquid chromatography of ions. Part 1: Determination of bromide, chloride, fluoride, nitrate, nitrite, phosphate, and sulfate

ISO 11206 (2011)

Water quality—Determination of dissolved bromate—Method using ion chromatography (IC) and postcolumn reaction (PCR)

ISO 19340 (2017)

Water quality—Determination of dissolved perchlorate—Method using ion chromatography (IC)

ISO/WD 18127:2022 (Draft 2023)

Water quality—Determination of adsorbable organically bound fluorine, chlorine, bromine, and iodine (AOF, AOCl, AOBr, AOI)—Method using combustion and subsequent ion chromatographic measurement

US EPA Method 300.0 (1993)

The determination of inorganic anions in water by ion chromatography

US EPA Method 300.1 (1997)

The determination of inorganic anions in water by ion chromatography

US EPA Method 218.6 (1994)

Determination of dissolved hexavalent chromium in drinking water, groundwater, and industrial wastewater effluents by ion chromatography

Atmospheric samples ISO 11632 (1998)

Stationary source emission—Determination of mass concentration of sulfur dioxide—Ion chromatographic method

ISO 264125 (2010)

Ambient air quality—Guide for the measurement of anions and cations in PM 2.5

US EPA Method 300.7 (1995)

Dissolved sodium, ammonium, potassium, magnesium, and calcium in wet deposition by chemically suppressed ion chromatography

Solid samples EN 15192 (2006)

Characterization of waste and soil. Determination of chromium (VI) in solid material by alkaline digestion and ion chromatography with spectrophotometric detection

338

16. Applications of ion chromatography in environmental analysis

particles) or dilution with deionized water to obtain a suitable analyte concentration range. Usually, this concerns samples with an excess of major ions (e.g., Cl
, SO42
, Na+). Examples of chromatograms of such samples are shown in Fig. 1 (anions) and Fig. 2 (cations), respectively.

2.2 Atmospheric samples Atmospheric samples provide invaluable data on wet deposition of contaminants with a possible impact on certain environmental compartments. Emissions of anthropogenic pollutants are caused mainly by energy production, the large variety of industrial plants, traffic, agricultural activities, and other sources [33]. Samples occur in a variety of forms mainly fumes, dust, gases, vapors, mists, rain, and aerosols. IC applications in atmospheric samples can be divided into two parts. First is gas analysis after trapping of the gaseous analyte in a liquid or on a solid sorbent, and next conversion of the gaseous compounds into ionic species. The second is the characterization of the chemical composition of aerosols, that is, analysis of the particulate matter and wet precipitations. Airborne particulate matter is partially composed of water-soluble salts, and wet precipitates may contain a considerable concentration of different ionic species. The main air pollutants are inorganic compounds (e.g., nitrogen and sulfur oxides, metals, metalloids, respirable particulate matter) [34] and organic compounds (e.g., amines, carboxylic acids) that can be analyzed by IC methods after suitable sample preparation. The sampling methods can be divided into three groups [35,36]: 1. Passive methods (diffusion, permeation). 2. Denudation methods (absorption in solution, chemisorption, permeation). 3. Dynamic methods (freezing, absorption in solution, chemisorption). The most popular is absorption in a suitable solution. Its efficiency depends on solution

composition. For example, HF, NOx, SO2, and SO3 are usually absorbed in deionized water, sometimes with the addition of H2O2. For NH3 acidic solutions (e.g., a diluted sulfuric acid solution) are used. Wet denuder systems are used for the collection of soluble ionogenic gases and soluble ionic species adsorbed onto atmospheric particles, especially for online continuous monitoring purposes. Their main advantages are the simultaneous measurements of gas or aerosol analytes and measurement in the 24/7 mode. Moreover, since external energy is not required, the samplers are simple to construct and relatively inexpensive, can generally be re-used, are small size, and can be easily mounted at almost any site. Disadvantages include relatively long exposure time required to obtain adequate detection limits for trace gases, and only moderate precision due to the variability of the collection rate caused by atmospheric conditions. The first paper on gaseous sample analysis by IC was published in 1979 [37]. Among the many analytical methods available for air pollution monitoring and atmospheric research, the role of IC is still rather small but should not be underestimated. Easy sample preparation, avoidance of hazardous chemicals, and the option to operate a fully automated system are some of the reasons for the acceptance of IC as a reference method [38].

2.3 Solid samples A typical example of a solid environmental sample is soil, which is the vehicle by which substances are driven from the land surface to underground water, plants, and finally into food sources. IC is used for the determination of common anions and cations in soil extracts. This provides information on the fate of nutrient ions resulting from agricultural practices [39]. The analysis of the total nitrogen, phosphorous, sulfur, and their corresponding oxidized anions, for example, nitrite, nitrate, phosphate, and sulfate are of importance when assessing soil

339

2 Ion chromatography and related techniques

mV

Cl

SO42

1800 1600 1400 1200

F

1000

Sample diluted 1:100 800

Sample diluted 1:1000

600 

Br

400

NO3

Standard sample

PO43

200 0

2

4

6

8

10

12

14

16

18

20

22

24

26

min

FIG. 1 Example chromatograms of inorganic anions in brine sample. Separation conditions: Anions—F
, Cl
, NO3
, PO43
, SO42
Column—Metrohm Metrosep A Supp 3 Eluent—1.7 mM Na2CO3 + 1.6 mM NaHCO3 Eluent flow rate—0.85 mL/min Injection volume—20 L Detection—Suppressed conductivity

conditions and fertility. Both anion IC and ionexchange chromatography (IEC) have been used extensively for the determination of lowmolecular-weight organic acids in soil extracts [40]. To separate complex mixtures of inorganic and organic acids, two-dimensional chromatographic techniques can be used [24]. Another important application is the control of landfills, especially hazardous waste landfills [41]. The determination of anions and cations in sludge, leachates, and similar solid wastes by IC is similar, in practice, to the analysis of soil samples. Samples are usually leached under aqueous conditions, then filtered and pretreated using solidphase extraction (SPE), if necessary, before separation. Solid samples can be prepared by many methods, but finally, they must be converted into liquid form for sample introduction into the separation column. Sample dissolution or extraction is usually carried out at room

temperature. However, sample heating is sometimes necessary. The selected extraction method depends on the sample matrix and characteristics of the analyte ions. Water is the preferred solvent because not all the separation columns and suppressors can handle organic solvents. Sometimes water with small amounts of organic solvents (e.g., methanol), water with acid or base addition, or the eluent solution is applied. If none of these methods is effective, it may be necessary to use alkaline (e.g., NaOH, KOH, Na2CO3, or K2CO3) or acidic (e.g., KHSO4 or K2S2O7) fusion; combustion in an atmosphere of oxygen; oxygen or calorimetric bomb; chemical digestion in open containers; pressure digestion; or UV pyrolysis [42]. The combustion IC (CIC) methods are particularly useful for samples containing organic substances with heteroatoms (e.g., chlorine, sulfur, nitrogen, etc.), which can be converted into suitable ions for chromatographic analysis [43,44].

340

16. Applications of ion chromatography in environmental analysis

mV

Na+

900 800

Ca2+ K

+

700 600

Na+

500

K+

400

Sample diluted 1:100

Ca2+

300

Sample diluted 1:1000

200

Li+

Na+

NH4+

K+

Ca2+

Mg2+

100

Standard 0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32 min

FIG. 2 Example chromatograms of inorganic cations in brine sample. Cations—Li+, Na+, NH+4 , K+, Mg2+, Ca2+ Column—Metrohm Metrosep C3 Eluent—5 mM HNO3 Eluent flow rate—1.0 mL/min Injection volume—100 L Detection—Suppressed conductivity

Examples of IC and related techniques for environmental analyzes with details of the separation column, eluent, detection mode, and sample type are summarized in Tables 4–7.

3 IC development perspectives Since its introduction in 1975 IC has been a useful and reliable method for environmental research [97]. It has become a versatile and mature separation technique for the analysis of a vast number of ions and ionogenic substances. This results from many IC advantages such as good accuracy and precision; a wide range of applications; many detection modes; high selectivity, speed, and separation efficiency; welldeveloped hardware, low cost of consumables,

and the development of portable, fully automatic IC systems [98]. Miniaturized systems that require fewer consumables, generate less waste and have lower energy consumption compared to full-scale laboratory systems. Advances that have greatly accelerated the development of IC in recent years include the introduction of gradient elution and highperformance suppressors, dedicated stationary phases, capillary and multidimensional IC, and IC-based hyphenated techniques [99,100]. Recently, an important point in comparison of the utility of different analytical methods is green analytical chemistry aspects [101]. In the case of IC, eluents are relatively inexpensive, safe to use, environmentally friendly, and only a small amount of waste is generated (typically nontoxic). Like any other analytical method, IC and

341

3 IC development perspectives

TABLE 4

Examples of IC for the determination of inorganic anions in environmental samples.

Analytes

Column

Eluent

Detection mode

Sample matrix

Reference

F
, Cl
, NO3
, PO43
, SO42


Dionex Fast Run Anion

2.3 mM Na2CO3 + 4.8 mM NaHCO3

Suppressed conductivity

Surface water, rainwater

[45]

NO
2

Dionex IonPac AS4A

H3BO4 + Na2CO3, NaCl, Na2CO3 + NaHCO3

Suppressed conductivity, UV/ Vis, amperometric

Waters with a large excess of chloride

[46]

Cl
, NO2
, Br
, NO
3, HPO42
, SO42


Dionex IonPac AS4A

1.8 mM Na2CO3 + 1.7 mM NaHCO3

Suppressed conductivity

Waters from peatlands

[47]

Cl
, Br
, NO2
, NO3
, PO43
, SO42


Dionex IonPac AS4A

1.7 mM Na2CO3 + 1.8 mM NaHCO3

Suppressed conductivity

Rainwater

[48]

F
, Cl
, Br
, NO2
, NO3
, PO43
, SO42


Dionex IonPac AS5A

NaOH

Cl
, NO3
, SO42
, Br


Dionex IonPac AS4A-HC

Na2CO3 + NaHCO3

Suppressed conductivity, UV (λ ¼ 210 nm)

Seawater

[25]

ClO4


Dionex IonPac AS11

100 mM NaOH

Suppressed conductivity

Groundwater

[49]

CN


Dionex IonPac AS11

5 mM NaOH

Suppressed conductivity

Wastewater

[50]

F
, Cl
, Br
, PO43
, 2

NO
2 , SO4 , NO3

Dionex IonPac AS11

21 mM NaOH

Suppressed conductivity

Groundwater

[51]

NO2
, NO3
, Cl
, PO43
, SO42


Dionex IonPac AS9-HC

9.0 mM Na2CO3

Suppressed conductivity, UV (λ ¼ 225 nm)

Seawater, wastewater

[23]

TSKgel IC Anion SW + TSKgel IC Cation

5 mM Na2SO4

UV (λ ¼ 206 nm) or fluorescence (410/470 nm)

Rainwater

[52]

IO3
, I


Agilent G3154A/102

20 mM NH4NO3

ICP-MS

Seawater, groundwater

[53]

Cl
, ClO2
, ClO3
, ClO4
, BrO3
, Br
, IO3
, I


Waters C-Pak A

KNO3 + HNO3

ICP-MS

Surface water

[54]


+ NO
2 , NO3 , NH4

342 TABLE 5

16. Applications of ion chromatography in environmental analysis

Examples of IC for the determination of inorganic cations in environmental samples.

Analytes

Column

Eluent

Detection mode

Sample matrix

Reference

Li+, Na+, K+, Rb+, Cs+, NH4+, Mg2+, Ca2+, Sr2+, Ba2+

Dionex Fast Cation I, Fast Cation II

17 mM HCl + 0.26 mM 2,3-diaminopropionic acid (DPA)

Suppressed conductivity

Wastewater, surface water

[55]

Na+, K+, NH4+, Mg2+, Ca2+

Dionex CS 1

0.028 mM Ce(NO3)3

Indirect fluorescence

Rainwater, fog, clouds, aerosols

[56]

NH4+

Metrohm Metrosep C4

1.7 mM HNO3 + 0.7 mM DPA + 0.05 mM 18-crown-6

Suppressed conductivity

Saline waters

[57]

Na+, K+, NH4+, Mg2+, Ca2+

Dionex IonPac CS10

40 mM HCl + 12 mM DPA

Suppressed conductivity

Organic-rich natural water from peatlands

[58]

Na+, K+, Mg2+, Ca2+

Dionex IonPac CS12

20 mM CH3SO3H

Suppressed conductivity

Rainwater, snow

[59]

Na+, K+, NH4+, Mg2+, Ca2+

Sykam LCA K01

4.5 mM HNO3, 1 mM histidine + 1 mM dipicolinic acid + 12 mM HCl

Suppressed conductivity

Fog

[60]

K+, Na+, NH4+, Mg2+, Ca2+

Dionex IonPac Cation Fast I, IonPac CS10

50 mM HCl + 5.1 mM DPA

Suppressed conductivity

Ice cores

[61]

Na+, NH4+, K+

Metrohm Metrosep C2

4 mM tartaric acid + 0.75 mM DPA

Nonsuppressed conductivity

Atmospheric particulate matter

[62]

Na+, K+, Mg2+, Ca2+

Dionex IonPac CS12

20 mM MSA

Suppressed conductivity

Rainwater

[63]

Li+, Na+, NH4+, K+, Mg2+, Ca2+

Dionex IonPac CS16

26 mM MSA

Suppressed conductivity

Surface water, wastewater

[64]

Na+, K+, Mg2+, Ca2+

Metrohm Cation 1–2

4 mM tartaric acid + 0.75 mM DPA

Suppressed conductivity

Ice, snow

[65]

Na+, NH4+, K+, Mg2+, Ca2+

Metrohm Metrosepp Cation 1–2

10 mM tartaric acid

Nonsuppressed conductivity

Rainwater

[66]

NH4+

Metrohm Metrosep C4

1.7 mM HNO3 + 0.7 mM DPA

Nonsuppressed conductivity

Urban and marine aerosols

[67]

343

3 IC development perspectives

TABLE 6

Examples of IC for the determination of metals and metalloids in environmental samples.

Analytes

Column

Eluent

Fe , Fe , Ni , Cu2+, Zn2+, Co2+, Pb2+, Mn2+

Dionex IonPac CS5A

7.0 mM 2,6-pyrridine dicarboxylic acid (PDCA) + 66 mM KOH + 74 mM formic acid

Fe3+, Fe2+, Ni2+, Cu2+, Zn2+, Co2+, Pb2+

Dionex IonPac CS2

10 mM oxalic acid + 7.5 mM citric acid or 40 mM tartaric acid + 12 mM citric acid

Dionex IonPac CS5

6 mM PDCA or 50 mM oxalic acid + 95 mM LiOH

Cd2+, Pb2+, Zn2+, Cu2+

Dionex AS9-HC

Pd2+, Pt2+

Detection mode

Sample matrix

References

PCR with PAR UV (λ ¼ 530 nm)

Industrial wastewater

[68]

UV (λ ¼ 520 nm)

Groundwater

[69]

Na2CO3

Suppressed conductivity

Water

[70]

Dionex IonPac AG4-SC, IonPac AG10, IonPac AG11, IonPac AG15, IonPac AG16

Oxalic acid, HCl, HNO3, HClO4

ICP-MS

Urban road dust, atmospheric particulates

[71]

Cu2+, Ni2+, Zn2+, Co2+, Fe2+, Mn2+, Cd2+, Fe3+, Pb2+

Dionex IonPac CS5A

28 mM oxalic acid + 250 mM NaNO3

UV/Vis (λ ¼ 530 nm)

Atmospheric particulates

[34]

Cu2+, Ni2+, Zn2+, Co2+, Pb2+, Fe2+

Dionex IonPac CS2

20 mM oxalic acid + 20 mM citric acid

UV/Vis (λ ¼ 520 nm)

Airborne particulates

[72]

Cu2+, Ni2+, Co2+, Zn2+, Cd2+, Pb2+, Fe2+, Fe3+

Dionex HPIC-CSS

Oxalic acid

UV/Vis (λ ¼ 520 nm)

Groundwater, surface water

[73]

Cr3+, CrO42


Home-made iminodiacetate resin

0.70 M HNO3

ICP-MS

Seawater

[74]

Sb3+, Sb5+

Hamilton PRP-X100

Phthalic acid, tartaric acid, 4-hydroxybenzoic acid, benzoic acid, citric acid

ICP-AES

Surface water, solid extracts

[75]

AsO43
, AsO33


Dionex IonPac AS-9

NaOH, Na2CO3 + NaHCO3

ICP-MS

Soil

[76]

Pu3+, Am3+, U3+

Dionex IonPac CS10

HNO3 + DPA

ICP-MS

Fuel leaching solutions

[77]

As3+, As5+

Hamilton PRP-X100, Dionex AS7, AG7

75 mM Na3PO4, 2.5–50 mM HNO3

ICP-MS

Surface water, groundwater

[78]

Hg2+, methyl-Hg+, phenyl-Hg+

Discovery C18

35% methanol + 40% acetonitrile + 25% water + 0.1 Mm DCTA

ICP-MS

Seawater

[79]

3+

2+

2+

Continued

344

16. Applications of ion chromatography in environmental analysis

TABLE 6

Examples of IC for the determination of metals and metalloids in environmental samples—cont’d Detection mode

Sample matrix

References

NH4OH + (COOH)2 + PDCA

ICP-MS

Seawater

[80]

Self-made—G3154A101, G3154A102

5 mM CH3COONH4

ICP-MS, ESI-MS

Water, soils

[81]

Al complexes

Dionex IonPac CG2

0.2 M ammonium formate

ICP-MS

Surface water, groundwater

[82]

Fe2+, Fe3+, Cu2+, Ni2+, Zn2+, Co2+, Pb2+, Cd2+

Bio-Res 50W-X8, Dionex IonPac AS11

50 mM NH4NO3, NH4OH

ICP-MS

Environmental water

[83]

Analytes

Column

Eluent

La , Ce , Pr , Nd3+, Sm3+, Eu3+, Gd3+, Tb3+, Dy3+, Ho3+, Er3+, Tm3+, Yb3+, Lu3+

Dionex IonPac CS5A

V3+, V5+

3+

3+

TABLE 7

3+

Examples of IC for the determination of organic and other substances in environmental samples. Sample matrix

References

Fluorescence detection

River water

[84,85]

10 mM KCl, 10 mM HCL

ICP-MS

River water

[86]

Dionex IonPac CS17

2–10 mM methanesulphonic acid (MSA)

Suppressed conductivity

Atmospheric gas and particulate matter

[87]

Methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, trimethylamine, propylamine, butylamine

Metrohm Metrosep C4–150

6 mM HNO3 + 1.0% (v/v) acetone

Nonsuppressed conductivity

Particulate matter (PM2.5)

[88]

Glyphosate, AMPA

Metrohm Metrosep A Supp 4

22.5–45 mM CH3COONH4 + 10–30 mM NH4OH

MS/MS

Surface water (creeks, rivers, stormwater wetlands)

[89]

Methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, trimethylamine, propylamine, butylamine

Dionex IonPac CS19

1–10 mM MSA

Suppressed conductivity

Atmospheric samples

[90]

Analytes

Columns

Eluent

Detection mode

Oxalate, ascorbic acid

Dionex IonPac AS4A

Na2CO3 + NaHCO3

Glyphosate, aminomethylphosphonic acid (AMPA)

Laboratory prepared

Methylamine, ethylamine, dimethylamine, diethylamine, trimethylamine, triethylamine

345

3 IC development perspectives

TABLE 7

Examples of IC for the determination of organic and other substances in environmental samples—cont’d Sample matrix

Analytes

Columns

Eluent

Detection mode

Guanidine compounds

Dionex IonPac CS17

1–25 mM MSA (1 mM NaOH as postcolumn reagent)

Suppressed conductivity, amperometric detection

River, lake, marsh water

[91]

Glycolate, formate, acetate, pyruvate, oxalate, malate, malonate, maleate, fumarate

Metrohm Metrosep A Supp 15

5 mM Na2CO3 + 0.3 mM NaOH

Suppressed conductivity

Secondary organic aerosol

[85]

Formate, acetate, propanoate, glyoxylate, pyruvate, lactate, oxalate, valerate, malonate, furancarboxylate, fumarate, maleate, succinate, methylmaleate, glutarate, malate

Dionex IonPac AS11

KOH

Suppressed conductivity, MS

Gas and aerosol

[92]

Quinate, lactate, levulinate, glycolate, propanoate, butanoate, dipicolinate, malate, malonate, tartarate, maleate, oxalate, fumarate, citrate

Dionex IonPac AS17

0.6–80 mM KOH

ICP-MS

Atmospheric aerosols

[93]

Lactate, acetate, propionate, formate, succinate, malonate, oxalate

Dionex IonPac AS11-HC

1–25 mM KOH

Suppressed conductivity

Rainwater

[94]

Acetate, formate

Dionex IonPac AS14

3.2 mM Na2CO3 + 1.0 mM NaHCO3

Suppressed conductivity

Snow, hail, rainwater

[95]

Alltech Allsep A-2

2.0 mM Na2B4O7

Shodex IC SI-50 4E

1.5 mM NaHCO3 + 1.2 mM Na2CO3

Dionex IonPac AS4A

750 μM NaOH (A), 200 mM NaOH (B)

UV-VIS (254 nm)

Soil extracts

[96]

Acetate, gluconate, formate, pyruvate, glutarate, succinate, malate, oxalate, fumarate, citrate, trans-aconitate

related techniques have some limitations, typical of all separation methods. Very well-equipped IC systems (e.g., hyphenated with MS or ICP-MS) are relatively expensive and require experienced operators. The maximum system pressure is currently limited owing to the use of PEEK for hardware components and tubing, and the

References

application of membrane technology in eluent generators and suppressors. It takes time for sample preparation, as great accuracy is required, for example, in dilutions, often requiring attention to detail and good laboratory practice. However, the advantages outweigh the limitations of IC. The importance of IC and related techniques in

346

16. Applications of ion chromatography in environmental analysis

environmental research are unquestionable. IC will continue to evolve as more ionic contaminants become regulated, and not only in environmental research.

[15]

[16]

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C H A P T E R

17 Applications of ion chromatography in food analysis Edward Muntean Department of Food Sciences, Faculty of Food Science and Technology, University of Agricultural Sciences and Veterinary Medicine, Cluj Napoca, Romania O U T L I N E 1. Introduction

351

5. Organic acids

360

2. Inorganic anions

352

6. Food safety issues

362

3. Inorganic cations

356

References

365

4. Carbohydrates

358

1 Introduction Food analysis can be defined as the scientific study of the composition and properties of food products, which uses various techniques and methods to determine the physical, chemical, biological, and sensory characteristics of both foodstuffs and the raw materials used in their production. The data acquired through food analysis play a decisive role in ensuring food quality and food authenticity, in developing and improving food products, as well as in safety issues—by detecting contaminants, certain additives, and other substances that may be harmful to human health.

Ion-Exchange Chromatography and Related Techniques https://doi.org/10.1016/B978-0-443-15369-3.00013-4

The chemical analysis of food products entails identifying, detecting, and determining a wide range of substances from food matrices, providing valid and reliable information about the food’s composition. Among the many techniques currently used in food analysis, ion chromatography (IC) stands out as a valuable method, and in some cases, even the preferred method of analysis for certain substances in specific matrices, being recognized for its versatility, sensitivity, and accuracy. Nowadays, IC can be considered one of the most powerful analytical tools for numerous analytes including inorganic ions, organic acids, carbohydrates, sugar alcohols, amines, amino acids, aminoglycosides, proteins, peptides,

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Copyright # 2024 Elsevier Inc. All rights reserved.

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17. Applications of ion chromatography in food analysis

glycoproteins, pesticides, and many others. In comparison with other techniques, IC offers the benefit of simultaneously determining multiple ionic components from samples; its greatest strength at the very beginnings was its ability to simultaneously analyze inorganic anions, for which there were no other efficient analytical methods [1,2]. From the perspective of food analysts, IC is recognized as a versatile and adaptable technique, known for its minimal sample requirements, robustness, high sensitivity, costeffectiveness, versatility, and efficiency [3]. It can support various system configurations and settings, column types, eluents, as well as in-line sample preparation techniques [4,5]; most IC systems are user-friendly, while some of them are highly automated and capable of handling many samples [6]. IC analyses can be performed with high precision and accuracy in a relatively short time, in some cases allowing for direct sample analysis even without the need for sample preparation—such as in the case of combustion IC. Moreover, IC is a safe and eco-friendly method as it commonly employs dilute solutions of acids, alkalis, or salts as mobile phases and does not entail the purchase of costly and hazardous materials or the disposal of waste eluents [7,8]. Overall, numerous arguments can support IC as a valuable tool, to demonstrate foodstuffs are safe, nutritious, and desirable for consumers; it can be used for quality control and characterization of raw materials and ingredients, monitoring technological processes streams and the quality of end-products, waste streams, cleaning solutions, process water, ensuring food safety and determining the authenticity of certain foods and more. Its broad dynamic range makes it suitable for both trace contaminants and major components of food, providing accurate results for each scenario, while the sample preparation procedure is simple and fast for numerous categories of food products, which are usually diluted only with deionized water and filtrated before injection. However, despite IC being a mature and well-recognized technique, research

is ongoing toward increasing speed, sensitivity, efficiency, and resolution by using smaller particles in columns of smaller internal diameters (such as nano and capillary columns) and by utilizing hyphenated techniques like ion chromatography-mass spectrometry (IC-MS) [2,9,10]. The purpose of this chapter is to highlight several relevant applications of IC in food analysis and to direct the reader toward a more detailed approach, since there are numerous works dedicated to this subject either as books or chapters devoted to food analysis [2,3,11,12] as well as many research papers and application notes, some of them being referenced in this context.

2 Inorganic anions Inorganic anions are important analytes in analyzed food products, to ensure safety, quality, nutritional value, and in some cases even authenticity. Chloride is the most common anion in foodstuffs (many items contain sodium chloride) and its concentration is targeted routinely in food quality monitoring, while nitrate and nitrite can be harmful in high concentrations and, therefore, are related to regulatory compliance issues (see Section 5); meanwhile, the presence of certain inorganic anions in food products can be used to trace their origin and ensure food authenticity [13]. Inorganic anions analysis was the first application in that IC showed its greatest utility since for the simultaneous analysis of anions there is no other rapid analytical method. Water was among the first matrices for which IC analysis of inorganic anions was successfully applied, eventually becoming a reference method in accredited laboratories, where it replaced the classical “wet” chemical methods [14–16]. Water is a critical component for the food industry, where it plays vital roles in various processes such as dissolving, diluting, dispersing, heating, steam

2 Inorganic anions

generation, cooling, cleaning, etc.; it is used in various forms, including as a standalone beverage and as an ingredient in many foodstuffs. It is imperative to monitor water quality regardless of its type, as it can cause food safety problems and have a decisive impact on the properties of food products; additionally, poor water quality in industrial processes can negatively affect equipment and the entire manufacturing process, for example, hard water can lead to deposits on equipment surfaces, pipes, valves and decreases the effectiveness of cleaning, etc. Water samples can be easily analyzed by IC (in some cases even without a preliminary sample preparation stage), and most applications using either isocratic separations with carbonate/hydrogencarbonate mobile phases, low conductivity eluents (containing salts of phthalic, p-hydroxybenzoic, or salicylic acids) or electrolytically generated hydroxide eluents; the latter option, also known as reagent-free IC (RFIC), allows for both isocratic and gradient elution. Detection can be accomplished by either direct conductivity or by suppressed conductivity, the second one significantly improving sensitivity; baseline separations of both major inorganic anions and minor ones can be accomplished in from 5 to 25 min (Table 1). Beer production is another beneficiary of the advantages of IC, starting with the analysis of water used in brewing and ending with the quality control of beer. Water is a main ingredient in beer production and its composition is decisive for determining beer quality, thus thorough monitoring of its anion composition is required. Previous research has shown that excessive amounts of certain anions can negatively affect fermentation and the overall taste of beer: High levels of chloride can enhance the sweetness of the beer and inhibit yeast flocculation, and nitrate can cause problems if it is converted to nitrite which can lead to weak or incomplete fermentation [28]. Therefore, quality control targeting inorganic anions in beer is important for maintaining its quality and ion-exchange chromatography with either suppressed or nonsuppressed

353

conductivity detection is typically used for this [29], in certain cases being accomplished with the simultaneous separation of both inorganic and organic anions (Table 1). The analysis of inorganic anions in winemaking is also a notable issue since some anions have a significant impact on the taste and overall sensory experience of wine. Hence, sulfate can increase the perception of bitterness and dryness while chloride can contribute to the perception of saltiness. In addition, inorganic anions can harm the stability and preservation of wine or even the health of consumers (see Section 5). IC analysis of inorganic anions in wine can ensure the quality and safety of wines, allowing winemakers to achieve the desired taste profile and stability; most applications use suppressed conductivity, the simplest methods allowing only the separation of major inorganic anions (Table 1), while others using high-capacity anion exchangers enable both inorganic and organic anions to be separated (up to 30 analytes) in 45min [23]. In recent years, the consumption of fruit juices has increased as individuals become more cognizant of the impact of their dietary choices on their overall health and well-being. In this context, the analysis of inorganic anions can provide valuable information about the quality and safety of juices, since certain anions can be indicators of contamination from agricultural or industrial sources, or poor quality or processing practices, while anions such as nitrite and nitrate can have health implications if consumed in large quantities. As the demand for fruit juice grows, so have fraudulent practices, leading to an abundance of counterfeit juice on the market. IC is an appropriate tool for all these issues, enabling convenient chromatographic profiling of both inorganic and organic anions using suppressed conductivity, the most advanced systems using RFIC [22,24]. For milk and dairy products, the inorganic anions typically analyzed include chloride, iodide, nitrate, phosphate, thiocyanate, and sulfate, most of which occur as mineral compounds

354 TABLE 1

17. Applications of ion chromatography in food analysis

Representative IC applications for anions’ analysis. Separation time (min)

References

Suppressed conductivity

14

[17]

IonPac AG22 Fast, AS22-Fast (150  2 mm) • 4.5 mM Na2CO3/1.4 mM NaHCO3 • 0.5 mL/min

Suppressed conductivity

5

[18]

Beer (ale)—fluoride. chloride, nitrate, sulfate, phosphate, and six organic anions

IonPac ICE-AS6 (250  4 mm) • 0.8 mM heptafluorobutyric acid • 1 mL/min

Suppressed conductivity

20

[19]

Wine—chloride, phosphate, sulfite, sulfate, and four organic anions (fast screening)

Metrosep A Supp 10 (100  4 mm) • 5 mM Na2CO3/5 mM NaHCO3/5 μM HClO4 • 1 mL/min

Suppressed conductivity

18

[20,21]

Wine and fruit juices—fluoride, chloride, nitrite, bromide, nitrate, sulfate, phosphate, and nine organic anions

OmniPac PAX-100 (250  4 mm) • Water/12% methanol/16% ethanol/0.1 … 1 M NaOH (gradient) • 1 mL/min

Suppressed conductivity

35

[22]

Wine and fruit juices—fluoride, bromate, chloride, bromide, nitrate, sulfate, phosphate, and 21 organic anions

IonPac AS11-HC-4 μm (250  0.4 mm), IonPac AS11HC-9 μm (250  4 mm) • KOH (EG) • 0.4 mL/min

Suppressed conductivity

45

[23]

Fruit juices—fluoride, bromate, chloride, bromide, nitrate, sulfate, phosphate, arsenate, and 20 organic anions

IonPac AS11-HC-4 μm (250  0.4 mm) • 1.5 … 65 mM KOH-EG (gradient) • 0.015 mL/min

Suppressed conductivity

40

[24]

Milk products—iodide

IonPac AG11, AS11 Analytical (250  4 mm) • 50 mM HNO3 • 1.5 mL/min

Pulsed amperometry

4

[25]

Milk—fluoride, hydrogencarbonate, chloride, nitrite, bromide, nitrate, hydrogenophosphate, sulfate

Shodex IC I-524A (100  4.6 mm) • 1.5 mM p-hydoxyben-zoic acid/1.7 mM N,N-diethylethanolamine/ 10% CH3OH • 1.5 mL/min

Direct conductivity

20

[26]

Matrix/analytes

Column and mobile phase

Detection

Water—fluoride, chloride, nitrate, sulfate

Metrosep A Supp 5 (250  4 mm) • 3.2 mM Na2CO3/1 mM NaHCO3 • 0.7 mL/min

Water—fluoride, chloride, nitrite, bromide, nitrate, phosphate, sulfate

355

2 Inorganic anions

TABLE 1

Representative IC applications for anions’ analysis—cont’d Separation time (min)

References

Suppressed conductivity

15

[27]

Suppressed conductivity

7

[13]

Matrix/analytes

Column and mobile phase

Detection

Milk whey—chloride, sulfate, bromide, phosphate and citrate, isocitrate

IonPac AS11-HC (250  0.4 mm) • 20 mM NaOH • 0.38 mL/min

Rum, vodka—chloride, nitrate, and sulfate

IonPac AS4A (250  4 mm) • 17 mM Na2CO3/18 mM NaHCO3 • 2 mL/min

natural to milk as salts of magnesium, calcium, iron, copper, or manganese; these salts are also involved in milk coagulation, which ensures the production of high-quality curd for cheese making [30]. The concentration of these anions are relevant indicators of milk quality; hence their analysis is important to ensure that the milk and the dairy products made from it meet the necessary regulatory standards. The IC methods for analyzing anions in milk and dairy products are based on a quite laborious sample preparation involving such techniques as deproteinization, centrifugation, membrane filtration, dialysis, or solid-phase extraction; some representative applications are presented in Table 1. IC can be used to analyze inorganic anions in spirits, such as whiskey, vodka, and rum to establish authenticity; the presence of chloride, sulfate, and nitrate can be an indicator that a certain beverage is genuine, which is relevant for protecting consumers from counterfeit or adulterated products. IC analysis can be accomplished using isocratic separations, with carbonate/hydrogencarbonate eluents and suppressed or nonsuppressed conductivity detection [13,31]. A promising development in anion analysis is combustion IC, which combines sample preparation and analysis in one automated process, allowing for the simultaneous detection of fluoride, bromide, chloride, iodide, and sulfur [32].

Combustion IC is an efficient approach, integrating sample preparation and chromatographic analysis: a solid or liquid sample is placed in an autosampler and loaded into a combustion oven where pyrolysis and oxidation take place in an oxygen and water vapor stream at temperatures above 900°C. The high temperature leads to the release of sulfur oxides and hydrogen halides or halogens, which are then transported to an absorption unit where they react with oxygenated water producing sulfate and halide ions. A portion of the resulting solution is subsequently analyzed by IC, where the separation process involves an anion-exchange column, a suppressor, and a conductivity detector. A typical analysis takes around 15 min, with the slow step of the IC separation; for overcoming this drawback, in automatic systems, while one sample is being separated, the next sample is prepared for analysis, making the whole procedure time efficient. Combustion IC is a promising development due to its ability to accurately determine the halogens and sulfur content in complex matrices—difficult to achieve with traditional sample preparation methods. By eliminating tedious sample preparation steps and utilizing a highly sensitive, automated method that is easy to use, this approach saves time and produces fewer environmental contaminants compared to other

356

17. Applications of ion chromatography in food analysis

methods. Its convenience makes it suitable for many applications beyond food analysis, for example, industries such as petroleum, coal, plastic, semiconductor, pharmaceutical, environmental, extractive, and power generation [33–36]. However, as a relatively new technique, there is no current official method for combustion IC in food analysis.

3 Inorganic cations Inorganic cations originate both from raw materials and ingredients used in food production, some of them having important roles in human nutrition: potassium is indispensable for proper nerve and muscle function, calcium is essential for strong bones, magnesium in numerous enzymatic reactions and for proper muscle and nerve function, iron to produce hemoglobin, etc. At the other extreme, cations such as lead, cadmium, chromium (VI), and mercury are toxic to humans, hence their determination in foodstuffs is important for food safety. The analysis of cations is imperative for assessing the safety and quality of drinking water; in this area, IC enables the simultaneous determination of alkali and alkaline-earth metals, this approach being particularly notable especially for mineral waters, which are significant sources of microelements and macroelements [37]. Since the composition of water is affected by both geochemical and anthropogenic factors as well as processing [38], IC provides a fast and reliable quality control approach. The separation of alkali and alkaline earth cations is typically accomplished with high-capacity cationexchange columns, strong eluents (dilute acid such as nitric acid or methanesulfonic acid in the mM range) with direct conductivity detection, within a reasonable time involving minimal sample preparation (Table 2). Even more benefits can be obtained by performing simultaneous anion and cation determinations using specially-designed systems, which allows for comprehensive analysis in a shorter time frame [47,48].

In brewing, monitoring the quality of water for inorganic cations is a major issue since they have an impact on the taste of the final product. To ensure product quality and meet consumer expectations, it is essential to monitor cation profiles in beer. IC with conductivity detection is utilized to determine cations in both water and beer (Table 2); two-channel systems [40] allow the simultaneous determination of cations and anions in beer. Winemaking requires monitoring of alkali and alkaline-earth metals using conductivity detection as well as certain transition metals, typically with UV/VIS detection after postcolumn derivatization, while more timedemanding applications allow the determination of biogenic amines (Table 2). Fruit juices have significant amounts of cations essential for human health, such as calcium, potassium, and magnesium; in addition, different fruit species and varieties can be distinguished by their cation fingerprints, which can be used to identify a specific fruit or blending practices used in the juice production. The analysis of cations from fruit juices is quite simple, most applications requiring only a strong eluent and conductivity detection, sample preparation consisting typically of dilution and filtration [44,45]. Milk and dairy products are more challenging for cations analysis due to the more complex sample preparation required, but otherwise, IC is a convenient method for the determination of calcium, magnesium, and alkali metal cations [49]; also using an IonPac CS12A column and suppressed conductivity detection [50], it is possible to simultaneously determine choline—a vital micronutrient for several physiological processes such as acetylcholine synthesis, cellmembrane signaling, or lipid transport. A valuable by-product of the cheese-making process is milk whey, the liquid remaining after the separation of the curd; despite being a by-product, it is a valuable raw material with a wide range of commercial applications. The quality control of milk whey and its derivatives by IC is a useful method for the determination of cations, anions, and carbohydrates [27,51].

357

3 Inorganic cations

TABLE 2

Representative IC applications in cations’ analysis.

Matrix/analytes

Column and mobile phase

Detection

Separation time (min)

References

Water—lithium, sodium, ammonium, potassium, calcium, magnesium, strontium, barium

Metrosep C4 (150  4 mm) • 2 mM HNO3/ 2 mM dipicolinic acid • 0.9 mL/min

Direct conductivity

20

[39]

Beer—sodium, potassium, magnesium, calcium

Metrosep C6 (150  4 mm) • 2.3 mM HNO3/ 1.7 mM dipicolinic acid • 0.9 mL/min

Direct conductivity

20

[40]

Lager—sodium, ammonium, potassium, magnesium, calcium

IonPac CS12 (250  2 mm) • Water/1 … 0.1 mM NaOH (grad.) • 1 mL/min

Suppressed conductivity

11

[19]

Wine—sodium, ammonium, potassium, magnesium, and calcium

IonPac CS12A (250  2 mm) • 20 mM MSA • 1 mL/min

Direct conductivity

10

[41]

Wine—copper, zinc, iron(II), manganese

Metrosep C2 (150  4 mm) • 1.75 mM oxalic acid/2 mM ascorbic acid • 1 mL/min

UV/VIS, after postcolumn derivatization

12

[42]

Wine—sodium, potassium, calcium, magnesium, putrescine, and cadaverine

Metrosep C1 (100  4 mm) • 2.5 mM HNO3/ 10% acetone • 1 mL/min

Direct conductivity

26

[43]

Fruit juice—sodium, ammonium, potassium, magnesium, and calcium

IonPac CS12A (250  2 mm) • 20 mM MSA • 1 mL/min

Suppressed conductivity

5

[44]

Apple and orange juices—sodium, potassium, ammonium, magnesium, and calcium

Universal Cation 7u column (100  4.6 mm) • 3 mM HNO3 • 0.5 mL/min

Direct conductivity

15

[45]

Continued

358 TABLE 2

17. Applications of ion chromatography in food analysis

Representative IC applications in cations’ analysis—cont’d Column and mobile phase

Matrix/analytes

Detection

Separation time (min)

References

Milk powder—sodium, ammonium, potassium, calcium, magnesium + choline

Metrosep C Supp1 (250  4 mm) • 4 mM HNO3 50 μg/L Rb • 1 mL/min

Suppressed conductivity

30

[46]

Milk whey—lithium, sodium, ammonium, potassium, calcium, magnesium

IonPac CS12A (250  2 mm) • 20 mM MSA • 1 mL/min

Suppressed conductivity

10

[27]

Vodka—potassium, sodium, ammonium, and lithium

Akvilain C1 (250  4.6 mm) • 4 mM HNO3 • 2 mL/min

Direct conductivity

10

[31]

Last but not least, cation analysis can be used to authenticate spirits; potassium, sodium, ammonium, and lithium from vodka can be determined with a nitric acid eluent and nonsuppressed conductivity detection [31].

4 Carbohydrates Carbohydrates are major components of many foods and play a key role in the food industry, being used as sweeteners and preservatives in various processed foods such as candies, soft drinks, and baked goods. They are the preferred source of energy for the human body and are used in many medicinal and functional food applications. The use of IC for the analysis of carbohydrates provides accurate and precise results: common applications include the analysis of soft drinks, fruit juices, baked goods, and fermented products (e.g., beer, wine, yogurt) as well as the determination of the sugar content of fruits, vegetables, or plant extracts [52]. It can also be used

to establish the authenticity of certain foodstuffs such as honey, maple syrup, or fruit juices. Due to its high specificity, most applications use amperometric detection, but there are also a few instances in which conductivity or UV/VIS detection proved their usefulness (Table 3). Carbohydrate analysis is critical in enology; wines contain a significant amount of carbohydrates, with glucose and fructose being the major ones found in grapes. These sugars are used by yeast during fermentation to produce ethanol, but some remain unfermented; the amount remaining in a wine determines its classification as dry, semidry, semisweet, or sweet. IC allows the determination of carbohydrates in wine without a preliminary extraction [59]. In the brewing industry, fermentable carbohydrates are of primary importance and proper knowledge of the beer wort carbohydrate profile is important for controlling the brewing process and providing the desired beer quality [60]. Beer is a complex matrix that contains a variety of substances, including proteins, lipids, minerals, carbohydrates, and flavor compounds;

359

4 Carbohydrates

TABLE 3

Representative IC applications in carbohydrates analysis. Separation time (min)

References

Conductivity and pulsed amperometry

12

[53]

CarboPac PA 100 (250  4 mm) • Water/0.5 M NaOH/1 M CH3COONa (grad.) • 1 mL/min

Pulsed amperometry

25

[19]

Milk and dairy products— sucrose, galactose, glucose, lactose, and lactulose

CarboPac PA 20 (150  3 mm) • 10 mM KOH • 0.008 mL/min

Pulsed amperometry

12

[54]

Dairy products—arabinose, galactose, glucose, sucrose, fructose, lactose, and maltose

CarboPac PA 1 (250  4 mm) • 1 M CH3COONa/0.2 M NaOH/H2O/25 mM CH3COONa (grad.) • 0.25 mL/min

Pulsed amperometry

35

[55]

Milk whey—galactose, glucose, N-acetylgalactosamine, lactose, lactulose, and epilactose

CarboPac PA 1 (250  4 mm) • 10 mM NaOH/2 mM Ba(OAc)2 • 1 mL/min

Pulsed amperometry

20

[27]

Lactic acid beverages and fermented milk—lactose, glucose, fructose, and lactic acid

Shodex sugar SH-G(50  6 mm) + SH1011 (300  8 mm) • 5 mM H2SO4 • 1 mL/min

Refractive index + UV (210 nm)

10

[56]

Lactose-free milk and dairy products—residual lactose, galactose, fructose, sucrose, and lactulose

CarboPac PA 20 Fast 4 μm (30  2 mm) • Water/0.2 M NaOH/0.1 M CH3COONa/1 M CH3COONa (grad.) • 0.25 mL/min

Pulsed amperometry

15

[57]

Fruit juices—mannitol, sorbitol, glucose, fructose, and sucrose

Carbopac PA 1 (250  4 mm) • 0.15 NaOH/0.15 NaOH/ 0.4 mM CH3COONa (grad.) • 0.4 mL/min

Pulsed amperometry

8

[58]

Matrix/analytes

Column and mobile phase

Detection

Wine, beer—mannitol, arabinose, glucose, fructose, lactose; sucrose, raffinose, maltose, acetate, glycolate, formate

HPIC-AS6 (250  4 mm) • 80 mM NaOH • 1 mL/min

Beer—glucose, maltose, maltotriose, maltotetraose, maltopentaose, malto-hexaoze, maltoheptaose, maltooctaose, maltodecaose

the key sugars for brewing are maltose and maltotriose, which are converted to ethanol by yeast during fermentation and IC can separate these compounds allowing their accurate determination [29].

The analysis of soluble carbohydrates in dairy products is relevant for quality control, fermentation control, and establishing their nutritional value [61]. Lactose is the main soluble carbohydrate in milk, being followed by minor amounts

360

17. Applications of ion chromatography in food analysis

of galactose, glucose, fructose, sucrose, and maltose. Accurate monitoring of carbohydrates in milk, milk whey, and dairy products is required to guarantee product formulation and product quality, and for reporting ingredients for those sensitive to allergens or with a compromised immune system; it can be accomplished with high specificity using pulsed amperometry detection (Table 3). Evaluating the residual amount of lactose is imperative for the quality control of products claimed to be lactose-free [62] and IC is one of the most advantageous methods for this purpose [57]. The fruit juice industry finds IC invaluable for the precise and effective determination of soluble carbohydrates, ensuring that the juices meet specific quality standards; by monitoring their carbohydrates content, IC can help to determine their nutritional value, identify microbiologicalinduced changes during storage, and assist in their authentication [45]. The soluble carbohydrate content has a significant impact on the sensory properties and nutritional values of fruit products and is critical for individuals with diabetes. The main concerns with authenticity are related to the use of low-quality and less expensive ingredients in their production, instead of those stated on the label. Since fruit juices are primarily water and carbohydrates such as fructose, glucose, and sucrose, their adulteration is achieved by dilution with water, and by using less expensive ingredients such as different combinations of sugar solutions and syrups, adding food additives to mask poor quality, blending cheaper juices with more expensive juices, using a different type of fruit, partially replacing one type of fruit juice with a cheaper one, or adding peel and/or pulp wash [63]. Such practices are used to lower production costs and improve the appearance of a juice without disclosing these manipulations on the label. The most common method used of adulteration for fruit juices is to dilute a concentrated juice with water, then add a sweetener, and finally adjust the flavor and color as needed; inexpensive sweeteners like inverted sugar beet or cane syrup, high fructose

corn syrup, hydrolyzed inulin syrup, or sucrose, are commonly utilized in such cases [64]. IC can help to uncover these practices by revealing the chromatographic profiles of carbohydrates in genuine juices as well as those of suspicious samples, enabling their comparison; oligosaccharide fingerprinting is an effective method to identify adulteration by sugar syrups based on carbohydrate profiles [58,65].

5 Organic acids Organic acids are food components that arise from either natural raw materials or emerge during processing, while others may be used as food additives; they play major roles in sensorial properties and stability of foodstuffs, while some can be considered biomarkers to establish authenticity. Organic acids, such as malic and tartaric, are naturally present in plant-origin raw materials and contribute to the taste of food products; organic acids like citric and acetic are used as sourness agents to provide a tangy taste (e.g., in candies, soft drinks, sauces). Certain organic acids such as citric, lactic, and acetic can inhibit the growth of harmful microorganisms, preventing spoilage and ensuring that food products remain safe for consumption; this is particularly important for products such as pickles and sauerkraut, where they are produced during fermentation. In the beer industry, organic acid profiles are used to monitor the fermentation process and play an important role in determining the quality of the final product. The analysis of organic acids in beer is possible by IC using different detection methods (Table 4), for either organic acids alone or together with inorganic anions [70] or carbohydrates [53]. In winemaking, organic acids are useful indicators of wine quality, sensory characteristics, and stability, also for monitoring changes in composition and establishing authenticity; some of them originate from grapes (such as tartaric, malic, and citric), and others are formed during

361

5 Organic acids

TABLE 4

Representative IC applications in organic acids analysis. Separation time (min)

References

Suppressed conductivity

20

[19]

Shodex RSpak KC-G 6B (50  6 mm) + KC-811 (300  8 mm) • 4.8 mM HClO4 • 1 mL/min

VIS (430 nm, postcolumn derivatization)

30

[66]

Beer—acetate, succinate, pyroglutamate, lactate, pyruvate, malate, oxalate, citrate, and five inorganic anions

Shodex IC I-I524 A (100  4.6 mm) • 1.5 mM phthalic acid/ 1.38 mM tris(hydroxymethyl) aminomethane/0.3 M H3BO3 • 1.2 mL/min

Nonsuppressed conductivity

25

[67]

Wine—acetate, malate, tartrate, oxalate, and four inorganic anions (fast screening)

Metrosep A Supp 10 (100  4.0 mm) • 5 mM Na2CO3/5 mM NaHCO3/5 μM HClO4 • 1 mL/min

Suppressed conductivity

18

[20,21]

Wine and fruit juices—21 organic acids and 8 inorganic anions

IonPac AS11-HC (250  2 mm) • Water/methanol/KOH (EG) • 0.4 mL/min

Suppressed conductivity

30

[23]

Fruit juices and wine—19 organic acids + 8 inorganic anions

IonPac AS11-HC-4 μm (250  0.4 mm) • KOH-EG (grad.) • 0.015 mL/min

Suppressed conductivity

40

[24]

Beverages—gluconate, lactate, malate, tartrate, citrate, glycerol, sucrose, glucose, and fructose

IonPAC AS15 (25  2 mm) • 10 … 90 mM KOH (grad.) • 0.3 mL min

Suppressed conductivity, mass spectrometry

40

[68]

Fruit juices—oxalate, citrate, galacturonate, dehydroascorbate, malate, quinate, ascorbate, succinate, shikimate, fumarate, and three carbohydrates

Aminex HPX 87H (300  7.8 mm) • 0.005 N H3PO4 • 0.4 mL/min

UV and refractive index detection

20

[69]

Orange juice—19 organic acids and 10 inorganic anions

IonPac AS11-HC (250  0.4 mm) • KOH-EG (grad.) • 0.015 mL/min

Suppressed conductivity

36

[70]

Matrix/analytes

Column and mobile phase

Detection

Ale—lactate, acetate, pyruvate, succinate, malate, citrate, and five inorganic anions

IonPac ICE-AS6 (250  4 mm) • 0.8 mM heptafluorobutyric acid • 1 mL/min

Beer—citrate, pyruvate, gluconate, malate, succinate, lactate, fumarate, acetate, pyroglutamate

362

17. Applications of ion chromatography in food analysis

alcoholic fermentation (such as succinic, lactic, and acetic). Tartaric and malic acids are the most significant and largely responsible for the acidity of wines. During the aging process, changes in the organic acid composition occur, for example, the concentration of free tartaric acid decreases as it reacts and precipitates with other wine components, and malic acid is converted to lactic acid by bacteria during malolactic fermentation, resulting in a wine with lower acidity and distinct taste. Organic acids have an impact on the tartness of the wine, for instance, malic acid can give a green apple flavor while an excessive amount of acetic acid can result in an undesirable vinegar taste; spoiled wines may contain higher concentrations of acids such as acetic, propionic or lactic [71]. Organic acids can be determined by IC using suppressed conductivity detection with gradient elution on highcapacity anion-exchange columns, this being the favored approach since it allows the simultaneous determination of a wide range of organic and inorganic anions [23,24]. The assessment of organic acids in fruit juices is imperative since they greatly impact the overall quality of the product (e.g., citric acid, malic acid, and lactic acid are responsible for the distinctive sourness in many fruit juices), but they also act as preservatives, helping to extend the shelf life of the product. In addition, certain organic anions can serve as markers for authenticity, being useful indicators of adulteration with less expensive juices [68]. In this context, IC is an invaluable resource for quality and authenticity-related concerns in the juice industry, particularly when using capillary columns and systems with suppressed conductivity detectors (Table 4). A comparison of organic acid profiles for authentic juice and suspicious products provides evidence of blending since the organic acid composition is distinct for each juice type [72].

6 Food safety issues IC is the dominant technique in food safety for the quantification of various ionic species

such as nitrate, nitrite, sulfite, chromate, arsenate, perchlorate, biogenic amines, pesticides, and many others. Regulations set by food safety agencies require that the levels of such analytes stay below certain limits in food products and their use must be carefully controlled and monitored so that they can be considered safe for human consumption. Nitrites and nitrates are commonly used additives in food preservation and curing (E249–E252), particularly in the production of meats, where they help to prevent the growth of harmful bacteria while contributing to the characteristic color of cured meats. However, there are health concerns associated with their consumption, given their toxicity; furthermore, when exposed to high heat, nitrites can form carcinogenic nitrosamines. Moreover, they can cause methemoglobinemia—a condition in which the blood’s ability to carry oxygen is reduced [73]. It’s worth noting that nitrates are also found in foods of plant origin such as leafy greens, beetroot, and other vegetables (with relatively high levels in products harvested from fertilized fields) as well as in drinking water (because of anthropic pollution). IC allows the simultaneous determination of these anions together with other anions using suppressed conductivity (Tables 1 and 5); the content of nitrite and nitrate can be determined in a more specific and convenient way using UV detection [84], especially when the chloride concentrations exceed by thousands-fold the concentrations of these anions. Sulfite is an inorganic anion originating from sulfites used as preservatives and antioxidants (E221–E228) in certain foodstuffs and drinks in which they are added to prevent the growth of microorganisms, to enhance or preserve color and for stabilization purposes; the use of sulfites is an old and widely established practice in winemaking [85]. Consequently, the concentration of sulfite is regulated in food products because high levels can cause allergic reactions in some individuals [86,87]. The determination of sulfite is accomplished using either anion exchange or ion exclusion chromatography

363

6 Food safety issues

TABLE 5

Representative IC applications in the food safety analysis. Separation time (min)

References

Suppressed conductivity

20

[74]

IonPac AS1 (250  4 mm) • 5 mM Na2CO3/1 mM NaHCO3 • 1 mL/min

Suppressed conductivity

14

[75]

Meat products—nitrite and nitrate

IonPac AS11-HC (250  0.4 mm), 9 μm • KOH-EG (grad.) • 0.015 mL/min

Suppressed conductivity

20

[76]

Beer—nitrate, sulfite and chloride, phosphate, bromide, and sulfate

Metrosep A Supp 10 (100  4 mm) • 6 mM Na2CO3/4 mM NaHCO3/5 μM NaClO4 • 0.7 mL/min

Suppressed conductivity

22

[77]

Wine—nitrate, sulfite, lactate, chloride, and phosphate

Metrosep Anion Dual 2 (75  4.6 mm) • 2 mM NaHCO3/1.8 mM Na2CO3/15% acetone • 0.8 mL/min

Suppressed conductivity

18

[78]

Wine—nitrate, sulfite, fluoride, chloride, bromide, hydrogenophosphate, and sulfate

Shodex IC SI-90 4E (250  4 mm) • 1 mM Na2CO3/4 mM NaHCO3/5% acetone • 1.5 mL/min

Suppressed conductivity

18

[79]

Milk—perchlorate, iodide, and thiocyanate

Metrosep A Supp 15 (50  4 mm) • 4 mM Na2CO3/6 mM NaHCO3, 10% MeOH • 0.8 mL/min

Suppressed conductivity

28

[80]

Water—fluoride, bromate, chloride, bromide nitrate, and sulfate

IonPac AS23 (250  4 mm) • 4.2 mM Na2CO3/1 mM NaHCO3 • 1.1 mL/min

Conductivity

25

[81]

Wine—dopamine, tyramine, putrescine, cadaverine, histamine, serotonin agmatine, phenylethylamine, spermidine, and spermine

IonPac CS18 (250  2 mm) • 3 … 45 mM MSA (grad.) • 0.3 mL/min

Pulsed amperometry, suppressed conductivity

42

[82]

Matrix/analytes

Column and mobile phase

Detection

Water—nitrite, nitrate, selenate, arsenate, perchlorate, chromate and fluoride, chloride

Metrosep A Supp 7 (250  4 mm) • 10.8 mM Na2CO3/35% acetonitrile • 0.8 mL/min

Drinking water—nitrate, chromate, fluoride, chloride, bromide, phosphate, and sulfate

Continued

364 TABLE 5

17. Applications of ion chromatography in food analysis

Representative IC applications in the food safety analysis—cont’d

Matrix/analytes

Column and mobile phase

Detection

Wine—monomethylamine, trimethylamine, 2-phenylethylamine, putrescine, cadaverine, histamine, serotonin, ammonium, calcium, and magnesium

Metrosep C Supp 1 (150  4 mm) • 2.5 mM HNO3/0.1 mg/L Rb/25 mM HNO3/ 0.1 mg/L Rb (grad.) • 1 mL/min

Suppressed conductivity

[79,88,89] with typically suppressed conductivity or pulsed amperometric detection. Caution is necessary during the whole analysis to prevent the oxidation of sulfite to sulfate. Bromates are used as flour improvers and oxidizing agents in the baking industry; besides, the bromate anion can enter the food chain through water, as a by-product of the ozone disinfection treatment process (when the raw water contains bromide). Since bromate can be toxic to humans and long-term exposure to high levels can increase the risk of cancer [90], the use of potassium bromate as a food additive is banned in several countries. Table 5 provides some representative applications that utilize suppressed conductivity to measure bromate concentrations in food, particularly in bread and baked goods, to ensure that it does not pose a health risk; most of them use suppressed conductivity, but postcolumn derivatization with spectrophotometric detection has also been used for this purpose [91], as has RFIC for bromate determination in mineral waters [92]. Furthermore, IC is a standard method in several applications for the determination of bromate [14,93,94]. Biogenic amines are naturally occurring compounds that are produced by the breakdown of proteins and by the decarboxylation of amino acids by certain microorganisms. They are commonly found in fermented foods and aged products such as cheese, wine, and cured meats. These compounds can have both beneficial and negative effects on human health and their

Separation time (min)

References

30

[83]

content in food products is a relevant issue for the quality and safety of foodstuffs; high levels of biogenic amines may indicate spoilage, poor hygiene, or inadequate manufacturing practices [95]. Elevated levels in food products can cause nausea, headaches, sweating, heart palpitations, and hypertension, while some biogenic amines such as histamine may lead to severe allergic reactions and even food poisoning present in high concentrations [96]. Biogenic amines can also affect the organoleptic properties of food products; for example, high levels of histamine and tyramine can cause a bitter taste, while putrescine and cadaverine can give rise to an unpleasant smell, affecting consumer’s perception of the product quality and safety. It is possible to separate these compounds simultaneously with several inorganic cations [83]; suppressed conductivity detection enables the determination of most biogenic amines, but pulsed amperometry provides higher specificity [82], which is important when inorganic cations are present at high concentrations (they are not detected so that they do not interfere). Perchlorate originates mainly from the utilization of rocket fuels, fireworks, and fertilizers and can contaminate food products through pathways such as irrigation water, contaminated soil, or industrial pollution [97]. Assessing the concentration of perchlorate in foodstuffs is notable because it can inhibit the uptake of iodine by the thyroid gland, leading to thyroid disorders. In addition, it is known to be a

References

developmental toxicant and can affect the cognitive and behavioral development of fetuses and children, while long-term exposure to high levels of perchlorate can increase the risk of cancer [98]. The analysis of perchlorate in foodstuffs can be done using suppressed conductivity or mass spectrometric detection (Table 5); standard methods are available [99,100]. Arsenic in foodstuffs can have potential health effects including cancer, cardiovascular disease, and developmental disorders; long-term exposure to high levels of inorganic arsenic can cause skin lesions, cancer, and lung damage. Ensuring that arsenic is not present at levels that could pose a risk to human health is particularly relevant for food products that are known to contain higher levels of arsenic such as rice and seafood; IC provides a reliable method for these [24,74]. Chromate is an anion that contains chromium in the +6-oxidation state, which is toxic, mutagenic, and probably carcinogenic [101]; this is why US EPA and the EU have specified maximum admissible chromium concentrations for their drinking water directives. To determine whether chromate levels in food products pose a health risk, IC with suppressed conductivity detection is a suitable technique with the advantage that it simultaneously provides information for other anions of likely interest [74,75]; alternatively, UV-VIS spectroscopy after postcolumn derivatization [102] can be used for more specific detection.

References [1] R.J. McGorrin, One hundred years of progress in food analysis, J. Agric. Food Chem. 57 (18) (2009) 8076–8088. [2] J. Weiss, Handbook of Ion Chromatography, John Wiley & Sons, Hoboken, NJ, 2016. [3] R. Michalski, Food analysis: ion chromatography, in: J. Cazes (Ed.), Encyclopedia of Chromatography, third ed., vol. II, Taylor & Francis, CRC Press, 2010, pp. 909–912. [4] Y. Wang, Determination of nitrite and nitrate in fruit juice by in-line dialysis-ion chromatography, Chin. J. Health Lab. Technol. 12 (2010) 058–065.

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[5] Z. Xiong, Y. Dong, H. Zhou, H. Wang, Y. Zhao, Simultaneous determination of 16 organic acids in food by online enrichment ion chromatography – mass spectrometry, Food Anal. Methods 7 (9) (2014) 1908–1916. [6] J.M. Liu, C.C. Liu, G.Z. Fang, S. Wang, Advanced analytical methods and sample preparation for IC techniques, RSC Adv. 5 (72) (2015) 58713–58726. [7] T. D’Amore, A. Di Taranto, G. Berardi, V. Vita, M. Iammarino, Going green in food analysis: a rapid and accurate method for the determination of sorbic acid and benzoic acid in foods by capillary ion chromatography with conductivity detection, LWT 141 (2021) 110841. [8] R. Michalski, P. Pecyna-Utylska, Green aspects of IC versus other methods in the analysis of common inorganic ions, Separations 8 (12) (2021) 235–244. [9] R. Michalski, Principles and applications of ion chromatography, in: R. Michalski (Ed.), Application of IC-MS and IC-ICP-MS in Environmental Research, John Wiley & Sons Inc., Hoboken, NJ, 2016, pp. 1–46. [10] B. Paull, P.N. Nesterenko, Ion chromatography: fundamentals and instrumentation, in: S. Fanali, P.R. Haddad, C.F. Poole, P. Schoenmakers, D. Lloyd (Eds.), Liquid Chromatography, Elsevier, Amsterdam, 2013, pp. 157–191. [11] W.R. LaCourse, Ion chromatography in food analysis, in: S. Otles (Ed.), Handbook of Food Analysis Instruments, CRC Press, 2008, pp. 161–196. [12] E. Muntean, Food Analysis Using IC, Walter de Gruyter GmbH & Co KG, 2022. [13] D.W. Lachenmeier, R. Attig, W. Frank, C. Athanasakis, The use of ion chromatography to detect adulteration of vodka and rum, Eur. Food Res. Technol. 218 (1) (2003) 105–110. [14] ASTM D6581-18, Standard Test Methods for Bromate, Bromide, Chlorate and Chlorite in Drinking Water by Suppressed Ion Chromatography, ASTM International, 2018. https://www.astm.org/Standards/ D6581.htm. [15] ISO 10304-1, Water Quality. Determination of Dissolved Anions by Liquid Chromatography of Ions—Part 1: Determination of Bromide, Chloride, Fluoride, Nitrate, Nitrite, Phosphate and Sulfate, ISO, Geneva, Switzerland, 2007. https://www.iso.org/standard/46004.html. [16] US EPA Method 9056A, Determination of Inorganic Anions by IC, United States Environmental Protection Agency. Office of Groundwater and Drinking Water, USEPA, Cincinnati, 2007. [17] Application note S-287, Tap Water Analysis for Anions and Cations Using Metrohm Intelligent Partial Loop Technique (MiPT), Metrohm AG, Herisau, Switzerland, https://www.metrohm.com/content/dam/ metrohm/shared/application-files/AN-S-287.pdf.

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17. Applications of ion chromatography in food analysis

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[80] Application note S-297, Iodide, Thiocyanate and Perchlorate in Milk Applying Inline Dialysis, Metrohm AG, Herisau, Switzerland, https://www.metrohm.com/ content/dam/metrohm/shared/application-files/ANS-297.pdf. [81] S. Djam, M. Najafi, S.H. Ahmadi, S. Shoeibi, Determination of bromate in bottled water marketed in Iran by ion chromatography, J. Chem. Metrol. 13 (2) (2019) 47–52. [82] B.M. De Borba, J.S. Rohrer, Determination of biogenic amines in alcoholic beverages by IC with suppressed conductivity detection and integrated pulsed amperometric detection, J. Chromatogr. A 1155 (1) (2007) 22–30. [83] Application note CS-014, Biogenic Amines Besides Other Cations in Red Wine Applying a High-Pressure Gradient, Metrohm AG, Herisau, Switzerland, 2018. https://www.metrohm.com/content/dam/ metrohm/shared/application-files/AN-cs014.pdf. [84] Application note 132, Determination of Nitrite and Nitrate in Drinking Water Using Ion Chromatography With Direct UV Detection, Thermo Fisher Scientific, Sunnyvale, CA, USA, 1991. https://assets. thermofisher.com/TFSAssets/CMD/ApplicationNotes/4189-AU132_Apr91_LPN034527.pdf. [85] C.D. Di Mattia, A. Piva, M. Martuscelli, D. Mastrocola, G. Sacchetti, Effect of sulfites on the in vitro antioxidant activity of wines, Ital. J. Food Sci. 27 (4) (2015) 505–512. [86] K.W. Lien, D.P. Hsieh, H.Y. Huang, C.H. Wu, S.P. Ni, M.P. Ling, Food safety risk assessment for estimating dietary intake of sulfites in the Taiwanese population, Toxicol. Rep. 3 (2016) 544–551. [87] B. Timbo, K.M. Koehler, C. Wolyniak, K.C. Klontz, Sulfites—a food and drug administration review of recalls and reported adverse events, J. Food Prot. 67 (8) (2004) 1806–1811. [88] L. Chen, B. De Borba, J. Rohrer, Determination of Total and Free Sulfite in Foods and Beverages, Application note 54, Thermo Fisher Scientific, Sunnyvale, CA, USA, 2016. https://tools.thermofisher.com/content/ sfs/brochures/AN-54-IEX-Sulfite-Food-BeverageAN70379-EN.pdf. [89] A.L.M. Espinosa, A simplified method to determine total sulphite content in food and beverages via ion chromatography, The Column 16 (2) (2020) 12–16. [90] V. Shanmugavel, K.K. Santhi, A.H. Kurup, S. Kalakandan, A. Anandharaj, A. Rawson, Potassium bromate: effects on bread components, health, environment and method of analysis: a review, Food Chem. 311 (2020) 125964. [91] R. Michalski, A. Łyko, Determination of bromate in water samples using post column derivatization method with triiodide, J. Environ. Sci. Health Part A 45 (10) (2010) 1275–1280.

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C H A P T E R

18 Separation of saccharides by ion-exchange chromatography Yuan Zhang and Jie Li China Medical University, Shenyang, China O U T L I N E 1. Introduction

371

2. Separation of saccharides by HPAEC 2.1 Monosaccharide and disaccharides analysis 2.2 Oligosaccharides and polysaccharides separation 2.3 Glycoconjugate separation

372

3. Separation of saccharides by ligand exchange chromatography

377 377

372

4. Ion exclusion chromatography

377

373 375

5. Ion-pair chromatography 5.1 Monosaccharides and disaccharides separations

379

References

383

375

1 Introduction Saccharides are important functional materials. According to their degree of polymerization (DP), they can be classified as monosaccharides (DP 1), oligosaccharides (DP 2–10), polysaccharides (DP >10), and glycans. Saccharides in food and related samples play vital roles in diverse biological functions. Besides, they also serve as structural materials, such as components of membranes, and they participate in cellular recognition.

Ion-Exchange Chromatography and Related Techniques https://doi.org/10.1016/B978-0-443-15369-3.00016-X

3.1 Monosaccharide and disaccharides separations 3.2 Oligosaccharides separation

379

Various methods for the separation of saccharides by liquid chromatography (LC), gas chromatography (GC), and capillary electrophoresis have developed over many years. Therein, the chromatographic analysis of carbohydrates is frequently performed on ion-exchange stationary phases. Ion chromatography (IC) is wellestablished for the separation of ionic species in solution. It is based on the reversible exchange of ions between target ions and dissociable ions on the ion exchange resin. There are mainly four

371

Copyright # 2024 Elsevier Inc. All rights reserved.

372

18. Separation of saccharides by ion-exchange chromatography

types of IC for carbohydrate separations, highperformance anion-exchange chromatography (HPAEC), ligand-exchange chromatography, ion exclusion chromatography, and ion pair chromatography. This chapter provides an overview of ion chromatography methods for the separation of saccharides including insights into the separation mechanisms.

2 Separation of saccharides by HPAEC HPAEC is a liquid chromatographic technique first described in 1983. HPAEC is based on the principle that saccharides dissociate to anions in a medium with high pH (>12), and ion-exchange chromatography on an agglomerated thin-shell anion exchange resin column is possible. HPAEC is a powerful tool for saccharide separations due to its high resolution [1,2]. The most common columns employed for HPAEC are manufactured by Dionex including Carbopac PA1, Carbopac PA10, Carbopac PA20, and CarboPac PA100 [1–4], Table 1. These columns are stable over the pH range of 0–14 and provide rapid separations of saccharides. The mobile phase for HPAEC is relatively simple. Generally, sodium hydroxide or potassium hydroxide solutions are usually chosen to create an alkaline environment for saccharide ionization. Acetate or other salts may be added to the mobile phase to improve resolution and maintain acceptable peak shapes. Saccharides contain multiple hydroxyl groups, which are electrochemically active. Neutral and acidic monosaccharides and oligosaccharides can be dissociated into weak acid radical ions in strong alkali solutions of pH range of 12–14. Therefore, in eluents of high pH, they exist partially or completely in the anionic form and can be separated on an anion exchange column. Schmid et al. [4] employed HPAEC for the separation of glucose, fructose, and sucrose in beverages. A CarboPac PA100 column was used with a

mobile phase of 85mM KOH and 0.5 mM KCl. A fast baseline separation of the three monosaccharides within less than 6 min was obtained, Fig. 1. Moreover, this method avoided the slow equilibrium processes associated with gradient elution. Polysaccharides with similar degrees of polymerization are similar to each other with the same molecular structures and are difficult to separate by conventional liquid chromatographic techniques. In HPAEC, the pH of the mobile phase can be varied by gradient elution to control the degree of dissociation of the polysaccharides facilitating their separation.

2.1 Monosaccharide and disaccharides analysis The separation of monosaccharides is the most common application of HPAEC. Samples containing free monosaccharides or monosaccharides released from glycoconjugates by acid or enzyme hydrolysis can be separated. In recent years, this application has been improved using new anion-exchange columns and eluent generation techniques. Unlike the poor separation of disaccharides, the CarboPac PA20 column allows the separation of monosaccharides with good resolution, even for monosaccharides with close structures, with short separation times and high sensitivity. Monti et al. [1] separated lactose, galactose, and glucose from “lactose-free” hard cheese, using CarboPac PA20 and a mobile phase of 8 mM sodium hydroxide in under 15 min. HPAEC can be used to determine the monosaccharide constitutes glycoprotein. Sample preparation consists of hydrolysis before direct injection into the HPAEC system. This method achieves satisfactory sensitivity without derivatization. Harazono et al. [9] compared different conventional liquid chromatographic and HPAEC for the determination of the monosaccharide composition of biopharmaceuticals. The HPAEC method was the most convenient

373

2 Separation of saccharides by HPAEC

TABLE 1

Summary of carbohydrates detected by IC.

Matrix

Analytes

Determination methods

Cheese

Monosaccharides

Phellinus igniarius

Analytical columns

Mobile phase

References

HPAEC-PAD

CarboPac PA20 anionexchange column (150 mm
3 mm)

Gradient elution: A: ultrapure deionized water; B: 200 mM NaOH

[1]

Monosaccharides

HPAEC-DC

CarboPac PA10 (250 mm
4 mm)

Gradient elution: 15 mM NaOH

[2]

Beverages

Monosaccharides and disaccharides

HPAEC-UV

CarboPac PA100 analytical column (250 mm
4 mm)

Isocratic elution: 85 mM KOH with 0.5 mM KCl

[4]

Urine

Sugar alcohol

HPAEC-PAD

Thermo Scientific Dionex CarboPac PA20 (150 mm
3 mm) and Dionex CarboPac MA1 column (250 mm
4 mm)

Gradient elution: KOH and NaOH

[5]

Honeydew and nectar

Monosaccharide and disaccharides

Ion chromatography (IC)-PAD

IonPac PA10 (250 mm
4 mm)

Gradient elution: 35 mmol/L NaOH

[6]

Wood sample

Monosaccharides and disaccharides

HPAEC-PAD

Dionex CarboPac SA10 Guard (50 mm
4 mm) Dionex CarboPac SA10 Analytical (250 mm
4 mm) Dionex CarboPac SA10-4μm Analytical (250 mm
4 mm)

Isocratic elution: 1 mM KOH

[7]

Fermentation broths

Monosaccharides and disaccharides

HPAEC-PAD

Dionex CarboPac MA1 Analytical (250 mm
4 mm) and Dionex CarboPac PA1 Analytical (250 mm
4 mm)

Gradient elution: NaOH

[8]

as it avoided the need for derivatization and separated monosaccharides after hydrolysis with little interference. Notably, washing of the column after the separation to remove amino acids and small peptides is time-consuming. This problem was solved using electrolytic eluent generation and shorter columns. Pairing eluent generation with a 2
100 mm CarboPac PA20 column, 4 μm particle size, achieved a glycoprotein monosaccharide analysis separation in a shorter time.

2.2 Oligosaccharides and polysaccharides separation HPAEC can provide highly selective separations of oligosaccharides using a strong anionexchange stationary phase, under high-pH conditions with a hydroxide-based eluent. HPAEC is suitable for the determination of the degree of polymerization (DP 2–12) of xylooligosaccharides and effectively separates branched xylooligosaccharides in complex mixtures.

374

18. Separation of saccharides by ion-exchange chromatography

FIG. 1 (A) Separation of glucose, fructose, and sucrose under optimized conditions in a standard mixture containing 1.7 mg L1 of each analyte, and (B) apple juice (labeled organic) diluted 10,000-fold. Mobile phase: 85 mM KOH, 0.5 mM KCl; flow rate: 1 mL min1; detection at 266 nm (Agilent 1260 HPLC with 60 mm path length). Reproduced with permission from T. Schmid, et al., Analysis of saccharides in beverages by HPLC with direct UV detection, Anal. Bioanal. Chem. 408(7) (2016) 1871–1878.

Sialic acid is a negatively charged saccharide widely distributed in animal tissues and microorganisms and is a component of many polysaccharides, mucins, glycoproteins, and some lipids. Sialic acid plays an important role in the generation and development of the brain and nervous system. Moreover, the abnormal expression of sialic acid in cell membranes is associated with tumor metastases and proliferation.

X9MeGluA2

X8MeGluA2

X7MeGluA2

X12MeGluA

X10MeGluA

X8MeGluA

X11MeGluA

30

25

35

X1

X5

20

X9MeGluA

X9 + X6MeGluA

X5MeGluA

X4MeGluA

X8 X2MeGluA

X7 + X3MeGluA

X6

X2

X4

EIC (counts)

15

X10 + X7MeGluA

1.7e8

X3

FIG. 2 Overlaid extracted ion chromatograms 5.5e8 (EICs) of [M + Li]+ adducts for 25 compounds separated using HPAEC-QqQ-MS. Reproduced with permission from E.S. Rodrı´guez, et al., Determination of xylooligosaccharides produced from enzymatic hydrolysis of beechwood xylan using high-performance anion-exchange chromatography tandem mass spectrometry, J. Chromatogr. A 1666 (2022) 462836.

X1MeGluA

Rodrı´guez et al. [10] used HPAEC for the separation of xylooligosaccharides derived from enzymatically hydrolyzed commercial xylan from beechwood. A CarboPac PA200 was used for the separation with a mobile phase containing 150 mM NaOH and 150 mM NaOAc in the gradient elution mode. Twenty-five xylooligosaccharides of different DPs were characterized in a single chromatogram, Fig. 2.

0

10

20 Time (min)

30

40

3 Separation of saccharides by ligand exchange chromatography

Therefore, it is important to monitor the levels of sialic acid in humans. HPAEC is a wellestablished method for the separation of sialic acid even in complex matrices. Hurum and Rohrer [11] compared HPAEC and a conventional liquid chromatographic method for the determination of sialic acid in infant formula. Although both methods exhibited sufficient sensitivity for the determination of sialic acids in infant formula the HPAEC method was faster. For samples with a difficult to eliminate matrix, high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was preferred. HPAEC-PAD was used to determine sialic acid in human serum, proving its convenience and simplicity [12]. Polysialic acids are found on the glycoproteins of neural cells and elsewhere in nature. The introduction of the CarboPac PA200 column improved the separation of polysialic acids compared to CarboPac PA100 columns [13]. Basumallick and Rohrer [13] developed an HPAEC method for the separation of a homologous series of polysialic acids. Compared with existing methods, HPAE-PAD provides higher resolution of polysialic acids. The CarboPac PA200 column resolved homologs of polysialic acid in colominic acid with a DP up to 100 within 70 min. HPACE is a suitable method for other polysaccharides separations, for example, pneumococcal capsular polysaccharides (PnPS), a key virulence factor with more than 90 immunologically distinct serotypes associated with variations of the capsular polysaccharides. The separation of antigenically distinct polysaccharides in the conjugate vaccine is extremely challenging. The HPAEC method is a powerful tool to solve the problem [14]. Talaga et al. [15] established an HPAEC method for the separation of pneumococcal polysaccharides and their conjugates after hydrolysis in vaccines without additional sample cleanup. The polysaccharides, conjugates and hydrolysis products were separated within 35min. Also, this method is very sensitive, requiring less than 10 μg of polysaccharide.

375

2.3 Glycoconjugate separation For the separation of glycoconjugates, HPAEC is mostly used in vaccine production and quality control. Conjugated saccharides are the active vaccine ingredient while free (unconjugated) saccharides are impurities surviving the purification process. By directly determining the conjugated saccharides, the efficacy and quality of the vaccine can be established. However, the direct determination of conjugated saccharides is difficult. HPAEC can solve this problem. Rech et al. [16] employed HPAEC for the determination of conjugated saccharides directly in vaccines. This method is both faster and more convenient than conventional approaches based on total saccharide determination.

3 Separation of saccharides by ligand exchange chromatography The stationary phase for ligand exchange chromatography consists of an ion-exchange resin containing metal ions (such as Cu2+, Co2+, Ni2+, Fe2+, etc.), which can form coordination complexes with saccharides. The stability of the coordination complexes determines the relative retention of the saccharides. Based on this principle, the separation of mono, di, and trisaccharides is relatively simple using only water as the mobile phase at elevated temperatures to achieve acceptable resolution and peak widths. As well as saccharide compounds that can be separated include amines, polyols, organic acids, amino acids, alkenes, and alkyne derivatives. The use of cation-exchange resins saturated with a metal ion to resolve neutral sugars was first demonstrated in 1960 [17], but it was not until the 1980s that high-resolution ligand exchange stationary phases became commercially available. Ligand exchange chromatography achieves rapid and effective separation of saccharides, even for mixtures

376

18. Separation of saccharides by ion-exchange chromatography

of monosaccharides, disaccharides, and polysaccharides. Brereton and Green [18] developed ligand exchange chromatography for the separation of saccharides in dairy and soy products. The monosaccharides (glucose, galactose, and fructose), disaccharides (lactose and sucrose), and polysaccharides (raffinose and stachyose) were all separated an isolated with recoveries ranging from 88% to 110%, Fig. 3. New stationary phases were developed for the separation of saccharides. Ers€ oz et al. [19] used molecularly imprinted polymers and a quartz crystal microbalance as a detector for the determination of glucose. Methylacrylaminohistidine copper (II) chelate was used to create the template for the selective separation of glucose. The molecularly imprinted polymer had a high binding affinity for glucose in the presence of other saccharides. Ion-exchange resins containing metal ions (such as Ca2+, Pb2+, and Ag+) are most useful for the separation of saccharides. Ion-exchange

FIG. 3

resins loaded with Ca2+ or Pb2+ are preferred for the separation of mono and oligosaccharides and Ag+ for the separation of oligosaccharides [20]. Ul’yanovskii et al. [21] employed ligandexchange chromatography for the separation of apiose from other monosaccharides and sugar alcohols. A strong cation exchange polymeric stationary phase modified with Pb2+ was used with water as the mobile phase and compared with the Ca2+-modified stationary phase. Both metal-loaded stationary phases provided good separation of apiose from matrix components, however, the most complete separation of apiose and monosaccharides was obtained on the Ca2+-containing stationary phase. For the separation of the apiose-mannitol pair, the Pb2 + -containing stationary phase was preferable. In the case of pectins, many of which contain large amounts of mannitol, the Pb2+-containing stationary phase ensures baseline separation of apiose from other sugars.

Ligand-exchange chromatograms of seven saccharides. (a) Control solution, (b) control solution after extraction by ion-exchange SPE (extract-salt), and (c) solution identical to control containing NaCl and KCl after SPE ion-exchange extraction (extract + salt). Peak identities: (1) raffinose, (2) sucrose, (3) glucose, (4) xylose, (5) fructose, (6) mannitol, and (7) xylitol. Reproduced with permission from K.R. Brereton, D.B. Green, Isolation of saccharides in dairy and soy products by solid-phase extraction coupled with analysis by ligand-exchange chromatography, Talanta 100 (2012) 384–390.

4 Ion exclusion chromatography

Many factors influence separation performance by ligand-exchange chromatography. Moravc´ık et al. [22] explored the influence of the stationary phase ionic form on the separation of galactooligosaccharides from mixtures containing lactose, glucose, and galactose. They found that the hydrogen ion form was superior to other cationic forms. A much higher yield of galactooligosaccharides with the required purity was obtained for the hydrogen ion form owing to its better kinetic properties. These results were in accord with those of Rabelo et al. [23].

3.1 Monosaccharide and disaccharides separations Ligand-exchange chromatography is a wellestablished method for the separation of monosaccharides typically with Ca2+- and Pb2+-loaded cation exchangers. The commonly use eluent is water. Qian et al. [24] used ligand-exchange chromatography with a Ca2+-containing stationary phase for the separation of mono and disaccharides in bee pollen and propolis. There was some overlap between galactose and xylose but otherwise, all common hexoses and sucrose expected to be present in the samples were well separated from each other and matrix components. Mass spectrometry was used to examine the separation mechanism. The saccharides were detected as their doubly charged Ca2+-adduct ions.

3.2 Oligosaccharides separation Previous research indicated that Ag+-loaded cation-exchange resins retain oligosaccharides to a greater extent than the Ca2+ form for the same resins, resulting in the separation of a higher number of oligosaccharides. It is shown that increased retention and enhanced resolution are achieved by the formation of strong silver monodentate complexes with oligosaccharides, which are much stronger than the bidentate complexes formed between calcium and oligosaccharides. Additionally, to improve separations and peak

377

shapes, Dong’s research group evaluated a weak cation-exchange column loaded with Cu2+ and optimized related factors such as buffer concentration and organic solvent for the separation of chitooligosaccharides. A low concentration of organic solvent was beneficial for improving the solubility of chitooligosccharides and provided a higher resolution of chitooligosaccharides with different degrees of polymerization. Recently, a glutathione-based column for hydrophilic interaction/cationexchange mixed-mode chromatography was developed for the separation of oligosaccharides. The separation of neutral fructosan with a high degree of polymerization, basic chitooligosaccharides, and strongly acidic carrageenan oligosaccharides was achieved. Wang et al. [25] separated cycloinulooligosaccharides (cycloinulohexaose [CF6], cycloinuloheptaose [CF7], and cycloinulooctaose [CF8]) on a metal-loaded, strong cation-exchange column with higher selectivity than for hydrophilic interaction liquid chromatography. Ligand-exchange chromatography was utilized for the isolation and enrichment of saccharides by solid-phase extraction (SPE) [26]. Schemeth et al. [26] immobilized different lanthanides (La3+, Ho3+, Eu3+, Er3+, or Tb3+) on a cation-exchange resin for the extraction of saccharides in honey and a Cynara scolymus extract. They observed different binding affinities for saccharides by the lanthanide ions, although this had only a minor impact on their selection for SPE, it might have more impact on the separation of saccharides by ligand-exchange chromatography.

4 Ion exclusion chromatography Ion exclusion chromatography finds application in the separation of a wide range of small, neutral, or partially ionized molecules. The stationary phase is usually a high-capacity cation exchange resin combined with aqueous solutions of sulfuric acid as eluent. The size exclusion

378

18. Separation of saccharides by ion-exchange chromatography

mechanism is based on the physical exclusion of molecules unable to penetrate the pore structure of the resin. This property depends on the degree of cross-linking of the polymer matrix. Chinnici et al. [27] employed ion exclusion chromatography for the separation of three saccharides (sucrose, glucose, and fructose) in fruit juice with an Aminex HPX 87H column. Three saccharides were baseline separated with good peak shapes. Ion exclusion chromatography is also useful for isolating saccharides from a complex matrix. For example, Takeoka et al. [28] used ion exclusion chromatography to isolate saccharides from a fermentation broth containing many other potential interfering substances such as organic acids and ethanol. A low-molecular size exclusion column modified with a strong cationexchanger was used as the stationary phase with an eluent containing pyroglutamic acid. Saccharides belonging to eight different species in rice wine were characterized within 45 min. The production of monosaccharides such as glucose and xylose from lignocellulosic biomass is an important topic for the sustainable production of food ingredients. However, the exaction of monosaccharides from a complex matrix as hydrolyzed biomass is the limiting step. Lodi et al. [29] used ion exclusion chromatography for the separation of monosaccharides from a hemicellulose hydrolysate. They explored the separation mechanism between monosaccharides and interferents as well as among three monosaccharides. The separation is based on the ion exclusion principle, by which strong electrolytes are separated from nonelectrolytes and weak electrolytes using a strong ionexchange resin as a stationary phase. The strong electrolytes are excluded from the resin due to electrostatic repulsion with the fixed groups, while the monosaccharides are partitioned between the mobile phase and the stagnant liquid inside the particles and eventually adsorbed onto the resin. Monosaccharides

were eluted with symmetrical peaks before the total column porosity, being sterically excluded from the resin pores to a certain extent while the interferents were strongly adsorbed on the resin and eluted later and well separated from all the other components. Similar results were obtained by Mai et al. [30] using a column packed with Dowex 50WX4-400 cation exchanger resin (H+ form exchanged with 1-ethyl-3-methylimidazolium cation) to separate glucose and xylose from a biomass hydrolysate. Isotherm measurements indicated that xylose was more strongly bound than glucose. The ion exclusion column exhibited good performance for the separation of glucose from xylose. Horna´k and Pernthaler [31] employed ion exclusion chromatography for the separation of 18 saccharides including monosaccharides and amino sugars in freshwater samples of distinct matrix complexity. The separation was conducted on a Supelcogel C-610H column (H+ form). Even though at low concentrations (as low as nM concentrations) they were determined using high throughput conditions, Fig. 4. However, the attempted separation of cellobiose, glucose, xylose, galactose, mannose, and arabinose and the internal standard xylitol on a strong cation-exchanger (H+ form) with deionized water as the mobile phase was less satisfactory with only poor resolution of mannose, xylose, and galactose [32,33]. To solve this problem, Cheng and Chang [33] developed a column-switching high-performance liquid chromatography method for the separation of cellobiose, glucose, xylose, galactose, mannose, and arabinose in a salt solution. In this system, the H+ ion-exclusion column serves to separate the salt from the saccharides and to initially separate the saccharides and the internal standard. The first five sugars, cellobiose, glucose, xylose, galactose, and mannose, were separated on a Pb2+-containing cation-exchange column while arabinose and the internal standard xylitol

5 Ion-pair chromatography

379

FIG. 4 LC-MS extracted ion chromatograms of a mixture (100 nM each) of hexoses and deoxyhexoses (A), disaccharides and pentoses (B), and amino sugars (C) using multiple reaction monitoring (MRM) in the scan mode. Identical transitions were applied for isobaric analytes. Glc, glucose; Man, mannose; Gal, galactose; Fru, fructose; Fuc, fucose; Rha, rhamnose; Scl, sucralose; Cel, cellobiose; Mal, maltose; Suc, sucrose; Xyl, xylose; Lyx, lyxose; Ara, arabinose; Rib, ribose; MurNAc, N-acetylmuramic acid; (GlcNAc)2, N,N0 -diacetylglucosamine; ManNAc, N-acetyl-D-mannosamine; GlcNAc, N-acetyl-D-glucosamine; GalNAc, N-acetyl-D-galactosamine. Reproduced with permission from K. Hornˇa´k, J. Pernthaler, A novel ion-exclusion chromatography-mass spectrometry method to measure concentrations and cycling rates of carbohydrates and amino sugars in freshwaters, J. Chromatogr. A 1365 (2014) 115–123.

bypassed the Pb2+-containing column and detected. In the meantime, the five sugars retained on the Pb2+-containing column were eluted for detection arriving after arabinose and xylitol. All saccharides were baseline separated.

5 Ion-pair chromatography Ion-pair chromatography (IPC) is an increasingly popular method for the separation of saccharides, in which ion pair reagents (IPRs) are added to the mobile phase to aid the retention of the ionic analyte on a hydrophobic stationary phase. The technique utilizes the same types of stationary phases and mobile phases as reversed-phase liquid chromatography. The ion pair reagent is usually an alkylsulfonate, an alkylsulfate, or an alkylammonium salt. The high efficiency of reversed-phase chromatographic columns compared with columns used for ion exchange or ion chromatography makes IPC a useful alternative to these techniques.

5.1 Monosaccharides and disaccharides separations Kochanowski et al. [34] studied the separation of nucleotide sugars in cultured cells by IPC using tetrabutylammonium hydrogensulfate as the ion pair reagent. Thirteen compounds including five nucleotide sugars were baseline separated within 30 min, Fig. 5. However, the separation of nucleotide sugar isomers such as uridine diphosphate (UDP) glucose and UDP galactose remains a problem because of their structural similarity. Sha et al. [35] improved the ion-pair separation of nucleotide sugars using core-shell columns and connecting multiple columns in tandem to increase the separation capacity and ultimately enable high-resolution detection of nucleotide sugars from cell extracts. Under optimum conditions, all major nucleotide sugars including their isomers were separated by ion-pair chromatography. For heparin-derived disaccharides, Jones et al. [36] observed that the retention of the

380

18. Separation of saccharides by ion-exchange chromatography

FIG. 5 Ion-pair RP-HPLC separation of 40-μL PCA CHO cell extracts (4
106 cells, 48 hold). Briefly, the cellular pellet was submitted to PCA extraction. After a neutralization step, soluble nucleotides and nucleotide sugars were analyzed by ion-pair RP-HPLC. Reproduced with permission from N. Kochanowski, et al., Intracellular nucleotide and nucleotide sugar contents of cultured CHO cells determined by a fast, sensitive, and high-resolution ion-pair RP-HPLC, Anal. Biochem. 348 (2006) 243–251.

disaccharide was determined by the hydrophobicity of the stationary phase, the concentration of organic modifier in the mobile phase, the charge on the disaccharides at the mobile phase pH, as well as charge, concentration, and hydrophobicity of the ion-pair. After optimization of the influencing factors, 12 disaccharides were separated within 10 min, as shown in Fig. 6. Kemmei et al. [37] used ion-pair chromatography for the separation of six sugar alcohols (erythritol, xylitol, arabitol, sorbitol, mannitol, and dulcitol) in drink, eyedrops, contact lens wetting solutions, and mouthwashes. Three different ion-pairing reagents (tetramethylammonium chloride, tetraethylammonium chloride, and tetrapropylammonium chloride) were compared, Fig. 7. The tetrabutylammonium chloride ion-pairing reagent exhibited the best peak shapes and separated all six sugar alcohols within 40 min.

Doneanu et al. [38] employed ion-pair chromatography for the separation of heparin oligosaccharides with a degree of polymerization of six to ten. All oligosaccharides were separated (in addition seven isomeric hexasaccharides were baseline separated as well). Ion-pair chromatography is not commonly used for the separation of polysaccharides but can provide complementary information for chain mapping profiles and facilitates their detection at low concentrations by mass spectrometry. Li et al. [39] employed ion-pair chromatography-mass spectrometry for the separation of the heparins enoxaparin and nadroparin using pentylamine as the ion-pair reagent. Enoxaparin and nadroparin as well as their fragments were well separated and exhibited favorable properties for mass spectrometric detection. Ion-pair chromatography can be used

381

5 Ion-pair chromatography

IA

100

IS IIIS IIA

IIIA

%

IIS

IH IVH

IVA IIH

* IIIH

IH

0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 Time (min)

FIG. 6 Ion-pair separation of commercially available heparin-derived disaccharides. The mobile phase contains 20 mM tributylamine and 2.5 mM ammonium acetate. The peak marked with an asterisk is an impurity. IS, IIS, IIIS, IVS, IA, IIA, IIIA, IVA, IH, IIH, IIIH, and IVH indicate the 12 heparin disaccharides. Reproduced with permission from C.J. Jones, N. Membreno, C.K. Larive, Insights into the mechanism of separation of heparin and heparan sulfate disaccharides by reverse-phase ion-pair chromatography, J. Chromatogr. A 1217 (2010) 479–488.

for the characterization of lipopolysaccharides and related compounds. Kojima et al. [40] used tributylamine as an ion-pair reagent for the separation of lipopolysaccharides which interact with the ion-pair reagent through their charged phosphate groups. The separation was based on the difference in the number of charged

phosphate and ethanolamine groups, as nonstoichiometric substituents, on the polysaccharide backbone. This method accomplished retention of the O,N-deacylated derivative (deON), and polysaccharide portion (PS), Fig. 8, under conditions favorable for their detection at low concentrations by mass spectrometry.

382

18. Separation of saccharides by ion-exchange chromatography

FIG. 7 Chromatograms of standard solution of six sugar alcohols (E; erythritol, X; xylitol, A; arabitol, S; sorbitol, M; mannitol, D; dulcitol). The sugar alcohol concentrations were 1 mM for erythritol and xylitol, 0.1 mM for arabitol, sorbitol, mannitol and dulcitol. Chromatograms were obtained with an InertSustain C18 column and thermostated at 50 °C using 0.1 mM disodium molybdate and 1 mM hydrochloric acid with added (A) 4 mM tetrapropylammonium chloride and 1% v/v methanol, (B) 0.4 mM tetrabutylammonium chloride and 10% v/v methanol and (C) 0.1 mM tetrapentylammonium chloride and 20% v/v methanol as the mobile phase. The detector wavelength was set at 247 nm. Reproduced with permission from T. Kemmei, et al., Reversed phase ion-pair chromatographic separation of sugar alcohols by complexation with molybdate ion, J. Chromatogr. A 1547 (2018) 71–76.

References

383

FIG. 8 Separation of the O,N-deacylated LPS (A and C) and polysaccharide portion of LPS (B and D) by strong anionexchange chromatography with postcolumn fluorometric derivatization (SAX-FLD) (upper) and reversed-phase ion-pair chromatography with postcolumn fluorometric derivatization (RPIP-FLD) (lower). The HPLC conditions for SAX were column, HiTrap Q HP (1.0 mL); flow rate, 1.0 mL/min; eluent, 10 mM sodium phosphate (pH 7.0) with programmed liner gradient 5–25 min with sodium chloride 0–250 mM; postcolumn reagent, 100 mM taurine and 6 mM sodium phosphate (pH 7.0); postcolumn reagent flow rate, 0.3 mL/min, fluorescence detection, λex 350 nm and λem 430 nm. The HPLC conditions for RPIP were column, Waters Symmetry C18 (particle size 3.5 μm, 2.1 mm ID
50 mm); flow rate, 0.25 mL/min; eluent, 15 mM acetic acid containing 15 mM tributylamine adjusted at pH 7.0 with 45% KOH solution; programmed acetonitrile gradient, 5%–30% at 0–30 min and 30%–60% at 30–40 min for O,N-deacylated LPS (C) and 5%–20% at 0–30 min and 20%–40% at 30–40 min for polysaccharide portion of LPS (D); postcolumn reagent, 100 mM taurine and 6 mM sodium phosphate (pH 7.0); postcolumn reagent flow rate, 0.25 mL/min, fluorescence detection, λex 350 nm and λem 430 nm. Reproduced with permission from H. Kojima, et al., Improved separation and characterization of lipopolysaccharide related compounds by reverse phase ion pairing-HPLC/electrospray ionization-quadrupole-mass spectrometry (RPIP-HPLC/ESI-Q-MS), J. Chromatogr. B 878 (2010) 442–448.

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Nose, D. Kozaki, Multi-functional separation mode-ion chromatography using L-pyroglutamic acid eluent for simultaneous determination of sugars, organic acids, and ethanol during multiple parallel fermentation of rice wine, J. Am. Soc. Brew. Chem. (2023) 1–7, https://doi.org/10.1080/03610470.2022.2158437. G. Lodi, L.A. Pellegrini, A. Aliverti, B. Rivas Torres, M. Bernardi, M. Morbidelli, G. Storti, Recovery of monosaccharides from lignocellulosic hydrolysates by ion exclusion chromatography, J. Chromatogr. A 1496 (2017) 25–36. N.L. Mai, N.T. Nguyen, J.-I. Kim, H.-M. Park, S.-K. Lee, Y.-M. Koo, Recovery of ionic liquid and sugars from hydrolyzed biomass using ion exclusion simulated moving bed chromatography, J. Chromatogr. A 1227 (2012) 67–72. K. Hornˇa´k, J. Pernthaler, A novel ion-exclusion chromatography–mass spectrometry method to measure concentrations and cycling rates of carbohydrates and amino sugars in freshwaters, J. Chromatogr. A 1365 (2014) 115–123. C.-C. Wu, C. Cheng, A study of the hydrolysis of waste paper cellulose with a vertically hanging immobilized cellulase reactor and the reuse of the immobilized cellulase, J. Chin. Chem. Soc. 52 (1) (2005) 85–95. C. Cheng, K.C. Chang, Sampling, dilution, and loading device-coupled high-performance liquid chromatography method for successive on-line analyses of major carbohydrate products in immobilized cellulase hydrolysate of paper cellulose, Anal. Sci. 23 (3) (2007) 305–310.

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[34] N. Kochanowski, F. Blanchard, R. Cacan, F. Chirat, E. Guedon, A. Marc, J.L. Goergen, Intracellular nucleotide and nucleotide sugar contents of cultured CHO cells determined by a fast, sensitive, and high-resolution ionpair RP-HPLC, Anal. Biochem. 348 (2) (2006) 243–251. [35] S. Sha, G. Handelman, C. Agarabi, S. Yoon, A highresolution measurement of nucleotide sugars by using ion-pair reverse chromatography and tandem columns, Anal. Bioanal. Chem. 412 (15) (2020) 3683–3693. [36] C.J. Jones, N. Membreno, C.K. Larive, Insights into the mechanism of separation of heparin and heparan sulfate disaccharides by reverse-phase ion-pair chromatography, J. Chromatogr. A 1217 (4) (2010) 479–488. [37] T. Kemmei, S. Kodama, A. Yamamoto, Y. Inoue, K. Hayakawa, Reversed phase ion-pair chromatographic separation of sugar alcohols by complexation with molybdate ion, J. Chromatogr. A 1547 (2018) 71–76. [38] C.E. Doneanu, W. Chen, J.C. Gebler, Analysis of oligosaccharides derived from heparin by ion-pair reversedphase chromatography/mass spectrometry, Anal. Chem. 81 (9) (2009) 3485–3499. [39] D. Li, L. Chi, L. Jin, X. Xu, X. Du, S. Ji, L. Chi, Mapping of low molecular weight heparins using reversed phase ion pair liquid chromatography–mass spectrometry, Carbohydr. Polym. 99 (2014) 339–344. [40] H. Kojima, M. Inagaki, T. Tomita, T. Watanabe, S. Uchida, Improved separation and characterization of lipopolysaccharide related compounds by reverse phase ion pairing-HPLC/electrospray ionizationquadrupole-mass spectrometry (RPIP-HPLC/ESI-QMS), J. Chromatogr. B 878 (3) (2010) 442–448.

C H A P T E R

19 Separation of oligonucleotides by ion-exchange chromatography Colin F. Poole Department of Chemistry, Wayne State University, Detroit, MI, United States O U T L I N E 1. Introduction

387

2. Column packings for the ion-exchange separations

393

3. Nucleotides and sugar nucleotides 3.1 Nucleotide sugars

394 396

4. Oligonucleotides

398

1 Introduction Nucleotides are the natural building blocks of DNA and RNA. They are composed of three units: a nitrogen base (pyrimidine or purine), a pentose sugar, and a phosphate group at the 50 sugar position (Fig. 1) [1]. In an RNA the pentose sugar is ribose and for DNA deoxyribose (Fig. 2). Deoxyribose has a hydrogen atom substitution of the hydroxyl group at the 20 position of ribose. The nitrogen bases are cytosine, uracil (RNA only), thymine (DNA only), adenine, and guanine (Fig. 3). Oligonucleotides are single-strand linear polymers of nucleotide monomers connected by

Ion-Exchange Chromatography and Related Techniques https://doi.org/10.1016/B978-0-443-15369-3.00019-5

5. Phosphorothioated oligonucleotides

400

6. Mixed mode stationary phases

401

7. Multidimensional chromatography

402

8. Purification of oligonucleotides

404

References

408

phosphodiester bonds. They differ in their length (number of nucleotide monomers), sequence (order of nucleotide monomers), and whether they form secondary structures (single stranded or double stranded). Polynucleotides are longer strands of oligonucleotides although opinions vary as to the number of nucleotide monomers required to differentiate an oligonucleotide from a polynucleotide. This is sometimes defined by a specific application while biopolymers are generally described as polynucleotides. For example, in DNA hybridization experiments oligonucleotides are typically defined as containing 10–25 nucleotide monomers.

387

Copyright # 2024 Elsevier Inc. All rights reserved.

Nucleoside Triphosphate Nucleoside Diphosphate Nucleoside Monophosphate Nucleoside

O

O HO

O

OH

OH

N

O

P

O

P

NH2

P

O

N

O

OH

N

N

Nitrogenous Base Phosphate OH

OH

Pentose Sugar FIG. 1 General structure of a nucleotide. Reproduced from N. Gautam, J.A. Alamoudi, S. Kumar, Y. Alnouti, Direct and indirect quantification of phosphate metabolites of nucleoside analogs in biological samples, J. Pharm. Biomed. Anal. 178 (2020) 112902 with permission.

5

5

HOCH2 4 C

H

O H 3

H 2

C

C

OH

OH

Ribose

OH

HOCH2

C 1

4 C

H

H

OH

O H 3

H 2

C

C

OH

H

C 1 H

Deoxyribose

FIG. 2

Structure or ribose and deoxyribose sugars.

FIG. 3

Structure of pyrimidine and purine bases of natural nucleotides.

1 Introduction

Current interest in the analysis of oligonucleotides is largely related to the rapid growth of synthetic oligonucleotides as diagnostic and therapeutic agents as well as the biological significance of natural nucleotides and oligonucleotides [1–8]. Common applications as diagnostic agents include their use as primers in polymerase chain reactions and as probes for DNA sequencing. Therapeutic oligonucleotides are medium sized active pharmaceutical ingredients used in antisense and gene therapy to suppress the synthesis of target proteins. These include antisense oligonucleotides (ASOs), small interfering RNA (siRNA), microRNA (miRNA), aptamers, and catalytic DNA. ASOs are small single-stranded oligonucleotides typically containing 16–20 nucleotide monomers (7–8 kDa). siRNAs are double-stranded oligonucleotides with typically 20 or more base pairs consisting of an antisense strand (pharmacologically active) and a complementary sense strand that activate the RNA interference silencing pathway, thereby achieving catalytic degradation of the target messenger RNA. miRNAs are single-stranded, noncoding oligonucleotides (RNAs) of about 18–25 nucleotide monomers with a significant role in the posttranscriptional regulation of gene expression. Nucleic acid aptamers are single-stranded oligonucleotides containing 20–100 nucleotide monomers (6–30 kDa) with a high target specificity. Oligonucleotides are synthesized in a stepwise process proceeding from the 30 -end to the 50 -end of the oligonucleotide sequence by automated solid-phase synthesis typically using phosphoramidite chemistry [9]. Each cycle involves the addition of a single monomer unit in several chemical reactions separated by rinse steps to remove excess reagents. Typical reactions include functional group protection, activation and coupling, oxidation, capping, cleavage, and deprotection steps using various reagents and reaction conditions. Because of the accumulation of many small errors at each step, the final oligonucleotide product typically

389

contains a variety of closely related impurities not easily removed during final product purification. As the oligonucleotide chain length increases, so does the number of synthetic steps and the number and total amount of impurities. Some of the more common impurities include sequence deletions where one or more nucleotides fail to attach to the sequence during synthesis (shortmers) and sequence additions where one or more nucleotides add to the sequence in a single cycle during the synthesis (longmers). For an oligonucleotide of length nt (i.e., containing nt nucleotide monomers) shortmers are oligonucleotides of length (nt  x) and longmers of length (nt + x) where x is a whole number of nucleotide monomers (1, 2, 3, etc.) Other impurities occur by depurination, deamination, thermal stress, adduct formation, and degradation of the nucleotide bases (Fig. 4) [5]. Double-stranded siRNA impurities include mismatched sequences and noncomplementary single-stranded sequences. As well as solidphase synthesis, in vitro transcription, a template-directed method, typically provides high-quality material in amounts up to milligrams for RNAs up to several kilobases [6]. The transcription mixture contains premature transcripts and other by-products that require removal at the purification stage. Synthetic oligonucleotides for therapeutic purposes may have additional chemical modifications to enhance biostability or specificity. Typical modifications include conjugation, sugar modification, base modification, and backbone modification (Fig. 5) [5]. The replacement of one or more of the nonbridging oxygen atoms of the phosphate linker with sulfur (phosphorothioate) to enhance biostability toward nuclease enzymes is a common backbone modification [4,5]. This results in further impurities, for example, unreacted phosphodiester (PO) impurities in the phosphorothioated (PS) oligonucleotide. The hydroxyl group of the ribose sugar at the 20 position may be modified by O-methyl or

390

19. Separation of oligonucleotides by ion-exchange chromatography

FIG. 4 Structures of typical impurities from the solid-phase synthesis of ASO and siRNA oligonucleotides. Reproduced from A. Goyon, P. Yehl, K. Zhang, Characterization of therapeutic oligonucleotides by liquid chromatography, J. Pharm. Biomed. Anal. 182 (2020) 113105 with permission.

O-methoxyethyl groups. The type and number of possible modifications are quite wide reflecting specific therapeutic needs rather than general processes. Chemical modifications tend to add to the complexity of impurity profiles by increasing the number of closely related impurities. Since oligonucleotides for therapeutic purposes are regulated active pharmaceutical ingredients, the identity and number of impurities are of concern and contribute to drug

quality, efficacy, and manufacturing yield. Their removal during drug purification is often a challenge. Both natural and chemically modified oligonucleotides need to be purified for most applications. Chromatographic methods are typically used for monitoring impurities and for smallto large-scale purification [5–10]. The common methods for oligonucleotides and their typical impurities are ion-pair reversed-phase liquid

1 Introduction

391

FIG. 5 Chemical modification of therapeutic antisense oligonucleotides. Reproduced from A. Goyon, P. Yehl, K. Zhang, Characterization of therapeutic oligonucleotides by liquid chromatography, J. Pharm. Biomed. Anal. 182 (2020) 113105 with permission.

392

19. Separation of oligonucleotides by ion-exchange chromatography

chromatography (IP-RPLC), ion-exchange chromatography (IEX), hydrophilic interaction liquid chromatography (HILIC), and ureapoly(acrylamide) gel electrophoresis (ureaPAGE). Gel electrophoresis is the oldest approach and is still widely used but has several disadvantages [10]. The most important are: it is labor-intensive and slow; oligonucleotide recovery is poor; the method is not easily scalable; and isolated oligonucleotides are contaminated with acrylamide and salts that generally require removal. Also, the separation of sugar-modified (20 -position) oligonucleotides from unmodified oligonucleotides is generally impossible. The current gold standard for the analysis of oligonucleotides is IP-RPLC [5–8]. This approach benefits from the high peak capacity and fast separations possible using totally porous particles pl

2. Decrease of pH < pl

Bead

3. EluƟon by ionic repulsion

Mult imodal cat ion exchangers

Bead

1. Protein binding by hydrophobic interacƟon and/or ionic interact ion pH < pl

2. Increase of pH > pl

Bead

3. Elut ion by ionic repulsion

FIG. 3

Diagram of how mixed mode resins work (in green (light gray in print version): resin bead and ligand, in pink (gray in print version): protein, in blue (dark gray in print version): hydrophobic effect, in yellow (light gray in print version): ionic interaction or repulsion).

467

3 Effect of salt and pH on mixed-mode chromatography

Cat ion exchange

+ pH

–

Net charge

Net charge

Anion exchange

pl

+

IEX resin pH

Mixed mode resin

–

pl

FIG. 4

Comparison of the possible pH range for binding depending on the type of chromatography used: ion exchange (IEX) or mixed mode. Modified from Multimodal chromatography Handbook Cytiva.

Experiments at the “limits” of interaction between proteins and chromatographic media were performed to observe the influence of environmental properties on protein adsorption. The electrostatic attraction between the ligand and the surface negative charge areas of the protein could occur, but also a repulsion between the ligand and the positively charged areas of the protein. This may explain the partial retention of the proteins. As above, the addition of salt promotes adsorption. The interactions between proteins and mixed-mode ligands can be described as a succession of electrostatic and hydrophobic interactions guided by ionic strength, depending on the type of salt and protein. (c) pI > pH A basic protein (lysozyme) with a pI higher than the pH of the mobile phase was also studied. For a buffer without added salt; the protein is neither absorbed nor retained on the resins and passes through the column without interaction. At pH 8, the protein net charge is about +6.3, which can explain the charge repulsion between the two positive surfaces. However, even if the pH is increased to 9, giving a net charge close to 0 (0.4), retention does not change. Thus, a charge in repulsion occurs even at a pH where the net overall charge is favorable for binding and suggests that the effect of salt is

more beneficial for overcoming charge repulsion than promoting hydrophobic interactions. A retention curve as a function of salt concentration is shown in Fig. 5. This type of U curve is typical of the involvement of multimodal interactions [1,13]. Hydrophobic interactions take precedence over electrostatic interactions as the conductivity increases. The interactions of proteins and ligands in mixed-mode separations can be described as a succession of electrostatic and hydrophobic interactions guided by ionic strength according to the type of salt and protein.

15 Retention Volume (CV)

(b) pI  pH

10

5

Electrostatic

Hydrophobic

0 0

20 40 Conductivity (mS/cm)

60

FIG. 5 Influence of conductivity on retention of αchymotrypsinogen-A on resin HEA HyperCel, along with proposed effects involved.

468

22. Separation of proteins by mixed-mode chromatography

4 The third dimension Each of the hydrophobic and ionic interactions (attraction and/or repulsion) guides the separation in mixed-mode chromatography. The balance between interactions is the keystone of selectivity in mixed-mode separations. Moreover, the variation of these interactions is dependent on the physicochemical environment in which the chromatographic supports are used. The pH and salt conditions are the main variables for initial optimization: 1. The pH modifies the distribution of charges on the surface of the proteins. 2. Conductivity changes the balance between electrostatic and hydrophobic interactions. In addition, selectivity in mixed-mode chromatography can be modulated by a 3rd dimension. Compounds described as “modulators” or “additives” can be added to the mobile phase to alter protein/ligand interactions, Table 3. They improve selectivity and thus purity after introduction during loading, washing, or elution. Modulators can also be combined for a synergistic effect. Guanidine is extremely efficient for the elution of proteins adsorbed on mixed-mode resins without affecting the pH. Guanidine is particularly useful for cleaning columns postseparation. Guanidine is effective at disrupting hydrophobic interactions, hydrogen bonds, and

TABLE 3

Typical modulators and their effects.

Modulator

Effect

Urea, arginine, guanidine

Decrease of hydrophobic effect Decrease of hydrogen-bonding interactions

Alcohols

Decrease of hydrophobic effect

Glycerol

Decrease of hydrophobic effect

Ethylene glycol

Decrease of hydrophobic effect

Ammonium sulfate

Increase of hydrophobic effect

electrostatic interactions for mixed-mode chromatographic media. Nevertheless, the structural disturbances associated with high concentrations of guanidine can be a major drawback to maintaining protein function during purification. Arginine has a guanidium group and therefore properties similar to guanidine. Methylene and hydroxyl groups have also been explored in the solubilization mechanism through hydrophobic interactions and hydrogen bonds with proteins [14]. Arginine has shown its potential to reduce the binding of proteins on mixed-mode chromatographic media, in particular the MEP HyperCel resin [15].

5 High-throughput screening of mixed-mode resins Method development is more complex for mixed-mode chromatography resins than conventional resins. Several ligands and multiple binding and elution conditions must be screened. Each resin for mixed-mode chromatography contains ligands with different structures. Moreover, the interactions involved are complex. High-throughput techniques make it possible to test several experimental parameters efficiently [3,11]. Column experiments using “One Factor at a Time” (OFAT) are timeconsuming and may fail to identify the overall optimum conditions. High-throughput screening of resins and/or conditions allows many parameters to be tested simultaneously. The method development time is reduced, and a large amount of information is obtained. Microplates or miniaturized columns are the tools of choice for high-throughput screening [16–18]. It is necessary to plan the design of experiments adapted to the parameters to be tested to define the “input parameters.” The goals can be of a different nature: yield, purity, aggregates … These are the output parameters. For robust purification, it is necessary to identify the important parameters and their effects. The key points

6 Monoclonal antibody purification

are column packing, sampling, washing, elution, and cleaning (“Cleaning in Place”). There are multiple parameters: pH, conductivity, flow rate, buffers, additives, etc. The final objective is to optimize yield and purity while preserving product quality. These conditions must then be validated in the column (dynamic approach) after which the change of scale can be implemented, Fig. 6. In addition to method development, the above approach can be used for the evaluation of process robustness. Regulatory agencies (ICH, FDA) strongly recommended this approach for processes development: A more systematic approach (also defined as quality by design) can include, for example, incorporation of prior knowledge, results of studies using design of experiments, use of quality risk management, and use of knowledge management (from guideline annex of ICH Q8 Pharmaceutical Development). With the introduction of QbD pharmaceutical process development is moving toward better knowledge of the interactions between products and the manufacturing processes. QbD is defined as a systematic approach to drug development, which aims to better explore the characteristics of molecules, to better understand the manufacturing process by exploring its limits, and identifying critical

FIG. 6

469

parameters. With one purpose: that quality is no longer ensured by reaching a target value, but by a value range, called Design Space, in which the production parameters can vary without altering the quality of the final product. All the parameters studied constitute the design space. It can also be named knowledge space. The operating space corresponds to the optimal conditions chosen, Fig. 7. To go further, the modeling of the chromatographic interactions of a ligand with a protein can be explored. Different models based on the fundamental thermodynamic equations have been used [19–22]. More recently, mechanistic models have been used to predict a wide range of column properties for both multimodal cation-exchanger and anion-exchanger chromatographic media [23]. These approaches decrease the complexity of process development and increase the knowledge of the properties of mixed-mode resins.

6 Monoclonal antibody purification Mixed mode chromatography has been used for the efficient purification of many proteins, and in particular, as an alternative to conventional techniques for the purification of

Progress of process development from the DoE to the change of scale.

470

FIG. 7

22. Separation of proteins by mixed-mode chromatography

Spaces of the Quality by Design concept.

monoclonal antibodies (mAbs) [24–29]. Our team has also published several articles in this field [3,30–33]. Due to its ligand charge density, mixed-mode chromatographic media can be

used as a mAb capture step without any treatment of the culture medium. It affords a good yield and purity and the whole process meets regulatory requirements (Table 4).

TABLE 4 mAb, HCPs, DNA and aggregate values during the purification process of recombinant antibody from CHODP12 cell culture supernatant (for 1 L of culture) [31]. Step yield (%) Supernatant (1 L)

Purity (%)

HCPs (ppm)

45

24,417

DNA clearance (log)a

Aggregates (%) 6.5

Capture

HEA HyperCel

94

98.7

270

2

0.5

Intermediate

Capto MMC

94

99.3

132

0

0.4

Polishing

Sartobind Q

99

99.9

15

1

0.3

a

DNA clearance is expressed in log10 reduction.

References

7 Conclusion Although the potential of mixed-mode chromatography for protein purification has been largely demonstrated their use remains limited compared to traditional methods like ion exchange, hydrophobic interaction, or affinity chromatography. Specific tools such as high-throughput screening, modeling, and in silico simulations were developed to facilitate process method development for mixed-mode resins. New perspectives for their use are coming from the growing demand for nucleic acids (DNA and RNA) products in the therapeutic field. In a continuous process, mixed-mode chromatography could make it possible to simplify the process by reducing the number of necessary steps [34]. A digital twin of the mRNA-based SARS-COVID-19 vaccine was proposed that included a mixed-mode chromatography step. This aimed to improve manufacturing process capabilities [35]. Mixed-mode chromatography is a powerful technique with high selectivity for adsorption and elution at different conditions. This allows a wide variety of applications and reduces the number of steps in a purification process.

References [1] L.A. Kennedy, W. Kopaciewicz, F.E. Regnier, Multimodal liquid chromatography columns for the separation of proteins in either the anion-exchange or hydrophobic-interaction mode, J. Chromatogr. 359 (1986) 73–84. [2] J. Pezzini, C. Cabanne, R. Gantier, V.N. Janakiraman, X. Santarelli, A comprehensive evaluation of mixed mode interactions of HEA and PPA HyperCel™ chromatographic media, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 976–977 (2015) 68–77. [3] J. Pezzini, G. Joucla, R. Gantier, M. Toueille, B. Garbay, X. Santarelli, C. Cabanne, Antibody capture by mixed-mode chromatography: a comprehensive study from determination of optimal purification conditions to identification of contaminating host cell proteins, J. Chromatogr. A 1218 (2011) 8197–8208. [4] J.A. Woo, H. Chen, M.A. Snyder, Y. Chai, R.G. Frost, S.M. Cramer, Defining the property space for chromatographic ligands from a homologous series of mixedmode ligands, J. Chromatogr. A 1407 (2015) 58–68.

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C H A P T E R

23 Process modeling of protein separations by ion-exchange chromatography Shuichi Yamamoto Biomedical Engineering Center (YUBEC), Yamaguchi University, Ube, Japan O U T L I N E 1. Introduction

473

2. Mechanistic modeling of chromatography 474 2.1 Mechanistic model equations and zone movement in the column 474 3. Plate height and related variables in isocratic elution

477

4. Resolution Rs in isocratic elution

480

5. Ion-exchange equilibria

481

6. Linear gradient elution 6.1 Retention in linear gradient elution 6.2 Peak width, HETP, and Rs in linear gradient elution 6.3 Iso-resolution curves in linear gradient elution

482 482 484 485

1 Introduction Ion-exchange chromatography (IEC) is widely used for preparative and process scale separation of proteins and other biological

Ion-Exchange Chromatography and Related Techniques https://doi.org/10.1016/B978-0-443-15369-3.00006-7

7. Applications of the mechanistic models and the simplified methods 487 7.1 Stepwise-elution process design based on linear gradient elution data 487 7.2 Flow-through chromatography 489 7.3 Model simulations for flow-through chromatography 493 7.4 Summary 495 8. Capture process design 8.1 Multicolumn periodic counter current operation 8.2 Flow velocity gradient loading operation 8.3 Model simulations 8.4 Summary

496

References

502

497 498 500 501

products such as DNAs, viruses, and virus-like particles. Among such biological products, monoclonal antibody (mAb)-based drugs are currently produced in large quantities as major biopharmaceutical products. Downstream

473

Copyright # 2024 Elsevier Inc. All rights reserved.

474

23. Process modeling of protein separations

processing of mAb is very important and involves several chromatographic steps including IEC. Columns of 1–200 L are used for process-scale IEC. The reader can find information on the characteristics of the binding and elution mechanism (ion-exchange equilibria) and applications of IEC to proteins elsewhere in this book. However, designing a process-scale protein separation by IEC needs additional information such as mechanistic models and scale-up procedures. As a time to the clinic is crucial, rapid, and reliable, procedures (methods) for design, operation, and validation of the purification process are needed. Various high-throughput process design (HTPD) systems have been developed. Most systems use a standard 96-well micro-plate format or its modified version as a high-throughput system (HTS). HTPD may reduce the time and the sample volume with the aid of the design of experiments (DOE). Mechanistic (phenomenological) chromatographic models offer an alternative and powerful approach to HTPD. Although not new, several research groups are currently actively pursuing this approach for the reasons outlined as follows. Compared with DOE, which is commonly employed with HTS, the mechanistic model approach has several advantages: • It provides a better understanding of the factors affecting the separation • It allows for extrapolation if the theories/ models are valid • It enables the number of experiments to be reduced It is also useful for characterizing the new separation media/formats and gaining an understanding of the separation (retention or binding) mechanism. Since several (commercial and open source) mechanistic model simulation software packages are now available, this approach is easier to adopt than in earlier times. However,

laborious procedures are still needed to determine the parameters for the simulation. Simplified versions of the model are useful for obtaining such data quickly from a small number of experiments. In this chapter, the modeling of process-scale IEC of proteins is explained for various elution/ operation methods. Accelerated methods for determining or estimating parameters needed for model simulations are also described.

2 Mechanistic modeling of chromatography 2.1 Mechanistic model equations and zone movement in the column Several elution methods are employed for the IEC of proteins as shown in Fig. 1. For isocratic elution, the mobile phase composition is the same during elution, whereas a modifier (modulator) concentration in the mobile phase is changed during nonisocratic methods, such as linear gradient elution or stepwise elution. Usually, a salt is used as a modifier at a fixed (constant) pH for the IEC of proteins. Flow-through chromatography (FTC) is similar to isocratic elution except that a continuous sample feed, a large sample volume, is applied. Let us consider isocratic elution for understanding mechanistic chromatographic models. As shown in Fig. 2, porous particles (particle diameter dp) are packed into a cylindrical column of the column diameter dc and the packed bed height (length) Z [1]. The packed column is considered to consist of two phases: the mobile phase and the stationary phase. The total volume of the mobile phase (interparticle space) is called the void volume Vo, which is related to the total bed volume Vt by Vo ¼ Vt ε ¼ AcZε, where Ac ¼ πd2c /4 is the cross-sectional area of the column. The void fraction ε is the ratio Vo to Vt. Vo can be determined by the retention volume of a large

475

2 Mechanistic modeling of chromatography

(B) Linear gradient eluon (LGE)

concentraon

(A) Isocrac eluon (IE) contaminant Target contaminant

contaminant Target

contaminant

(D) Flow-through chromatography (FTC)

(C) Stepwise eluon (SE) Modifier(modulator) Breakthrough volume or me

contaminant

Target Target

contaminant

Modifier (modulator)

me or volume FIG. 1

Modifier (modulator)

me or volume

Elution methods (operation modes) of chromatography.

FIG. 2 Schematic representation of the separation of two components in chromatography (upper) and transport phenomena in the chromatography column (lower).

molecule completely excluded from the pore volume of the stationary phase (packing material). The equation describing the solute (sample) concentration in the mobile phase of the column as a function of time t and distance from the column inlet z, C(t, z), is described by Eq. (1), which considers axial dispersion.

DL is the axial dispersion coefficient (mixing in the mobile phase due to channeling and the unequal path length of the interstitial spaces), H ¼ (1  ε)/ε is the volumetric phase ratio, and u is the mobile phase velocity, which is related to the superficial velocity u0 or the volumetric flow rate Fv by

∂C ∂CS ∂ C ∂C +H ¼ DL 2  u ∂t ∂z ∂z ∂t

CS is the average solute concentration in the stationary phase. Various definitions are possible

2

(1)

u ¼ Fv =ðAc εÞ ¼ u0 =ε

(2)

476

23. Process modeling of protein separations

for the stationary phase concentration. Here, Cs is defined as Cs ¼ εP CP + ð1  εP ÞCq ¼ εP CP + ρP q

(3)

where εP is the particle porosity, CP is the concentration in the pore, Cq is the concentration on the solid surface, ρP is the particle density, and q is the concentration in kg-protein/ kg-adsorbent. CP, Cq, and C are related to the distribution coefficients KP and Kq. CP ¼ KP C

(4)

Cq ¼ Kq CP

(5)

Then, Cs is related to C with the (overall) distribution coefficient K. 
  Cs ¼ εP + ð1  εP ÞKpq KP C
 ¼ ðεP KP Þ + ð1  εP ÞKpq KP ¼ KC (6)
 K ¼ εP + ð1  εP ÞKpq KP ¼ ðεP KP Þ + ð1  εP ÞKpq KP ¼ KSEC + Kads (7) KSEC is the distribution coefficient due to size exclusion and Kads the distribution coefficient for adsorption (or interaction). In most experimental investigations, KP is considered to be unity. However, this may cause another contradiction for size exclusion [2]. The particle porosity εP must be constant. KP reflects the ratio of the pore volume accessible to a solute. The stationary phase concentration in the particle is described by the diffusion equation for a sphere with the radial coordinate r.    2 ∂CS ∂ CS 2 ∂CS ¼ DS + (8) r ∂r ∂t ∂r2 DS is the stationary phase diffusion coefficient. Mass transfer through the stagnant film around the particle (sphere) is described by the film mass transfer coefficient kF.    ∂CS 6kF CS,i ¼ C (9) ∂t dp K

CS,i ¼ KCi is the stationary phase concentration at the surface (r ¼ Rp ¼ dp/2). The above set of equations represents the most rigorous mechanistic model, sometimes called the rate model (Model 1, Table 1). This model adequately describes the spreading of the sample zone along the column due to several mass transfer mechanisms such as pore diffusion, film diffusion, and axial dispersion. Various models can be derived from this model by neglecting term(s) or parameter(s). If we neglect all the parameters affecting mass transfer, the equilibrium model is given by ∂C ∂CS ∂C +H ¼ u ∂t ∂z ∂t

(10)

Chromatographic models, including equilibrium models, are explained in several books and book chapters [1,3–8]. The simplified equilibrium models are important to indicate how the distribution coefficient (or isotherm) affects retention. The movement of a solute applied to a chromatographic column is described by the following equation from the equilibrium model Eq. (10). dz=dt ¼ u=ð1 + HKÞ

(11)

Here, z ¼ distance from the top of the column. Other models are summarized in Table 1. Model 2 is often referred to as the equilibrium-dispersive model, in which the effective or overall dispersion coefficient DL,e is responsible for the total zone spreading. The diffusion equation is replaced with the linear driving force (LDF) equation in Model 3. Since the axial dispersion is omitted in the mobile phase equation, the zone spreading is described only by the overall mass transfer coefficient Ks. This model is widely used for studying fixed-bed adsorption processes with relatively large particles [6]. In addition to rate models, alternative models referred to as the plate model or the tank-inseries model are available. The well-known Martin-Synge plate theory (model) regards a

3 Plate height and related variables in isocratic elution

477

TABLE 1 Basic and plate number equations for mechanistic chromatographic models. Model

Basic and plate number N equation

1

∂C ∂CS ∂2 C ∂C +H ¼ DL 2  u ∂t ∂z ∂z ∂t    2 ∂CS ∂ CS 2 ∂CS ¼ DS + 2 r ∂r ∂t ∂r    ∂CS 6kF CS,i ¼ C ∂t dp K

Mobile phase Stationary phase Film mass transfer

1 2ðDL =uÞ d2P Ra ð1  Ra Þu dP Ra ð1  Ra ÞKu ¼ + + N Z 30DS Z 3kF Z 2

∂C ∂CS ∂2 C ∂C +H ¼ DL,e 2  u ∂t ∂z ∂z ∂t

Mobile phase

1 2ðDL,e =uÞ ¼ N Z 3

Plate model

∂C ∂CS ∂C +H ¼ u ∂t ∂z ∂t

Mobile phase

 ∂CS ¼ KS KC  CS ∂t

Stationary phase (linear driving force)

1 2ð1  Ra Þ2 u 2ð1  Ra Þ2 ¼ ¼ N HKKS Z NTU  ∂CðiÞ ∂CSðiÞ u

Cði1Þ  CðiÞ +H ¼N Z ∂t ∂t 1/N ¼ 1/N

Models 1–3 are sometimes referred to as rate theory or rate models. Ra ¼ 1/(1 + HK) N equations for a linear isotherm (K ¼ constant). NTU (the number of transfer units) NTU ¼ 2N(1  Ra)2.

chromatography column as a series of plates. In each plate, the equilibrium is instantaneous. The zone spreading is described by the plate number N. Several versions of the plate model are available based on ordinary differential equations. One is the discontinuous flow model, or CraigCraig model, which is similar to liquid-liquid distribution operations. Both models have a single zone-spreading parameter. When the above models are solved numerically with appropriate isotherm data and initial and boundary conditions, elution curves for isocratic elution, linear gradient elution, stepwise elution (bind/elute), and FTC can be obtained.

Although numerical solutions (and analytical solutions in special cases) can predict elution curves, it is useful to have simplified methods for predicting elution behavior for the model simulations.

3 Plate height and related variables in isocratic elution For linear isocratic elution (K ¼ constant), HETP (height equivalent to a theoretical plate or plate height) is a commonly employed measure of column efficiency based on the peak

478

23. Process modeling of protein separations

tR

Cmax

HETP = Z(V/tR)2 w2 =8V2 Cmaxe-1

the peak maximum retention time [8–11]. Namely, μ10  tR, and μ2  σ 2 or by fitting the exponentially modified Gaussian model equation to the peak profile [3]. The retention time tR at a constant K is given by the following equation, which can be derived by integrating Eq. (11) from z ¼ 0 to z ¼ Z (t ¼ 0 to t ¼ tR) tR ¼ ðZ=u0 Þ½ε + ð1  εÞΚ  ¼ ðZ=uÞð1 + HΚ Þ (15) The retention volume is

W=4V

time FIG. 3

Chromatographic elution curves (chromatogram, elution profile). The peak width at C ¼ 0.5Cmax (half-width) becomes 2.35σ.

V R ¼ FtR ¼ V o + ðV t  V o ÞK ¼ V o ð1 + HKÞ (16) The retention factor k is also used instead of K and can be determined from VR. V R ¼ V 0o ð1 + kÞ

(17)

0

width (σ ¼ standard deviation of the elution curve) and the retention time tR, Fig. 3 HETP ¼ Z=N ¼ Zðσ=tR Þ2

(12)

tR and σ are precisely determined by the first and the second moment defined by the following equations. ð∞ ð∞ ð∞ 0 Ct dt= C dt ¼ Ct dt=μ0 (13) μ1 ¼ 2

0

0

μ2 ¼

ð∞ 0

Here, Vo is the peak elution volume of a nonretained (nonadsorbed) but fully permeable solute. Namely, a small solute that can use all the pore volume and does not interact with the ligand or polymer surface is employed to determine Vo0 . k is related to K as follows. V 0o ¼ V o + V p ¼ V t ε + V s εP ¼ V t ε + V t ð1  εÞεP ¼ V t ½ε + ð1  εÞεP  (18)

0

2 C t  μ01 dt=μ0

(14)

where μ0 ¼ 0th moment (area of the curve). Note that for low plate numbers (N < 30), curves are skewed with tailing and the assumptions μ10  tR, and μ2  σ 2 are no longer valid. Numerical integration based on Eqs. (13) and (14) is required. Therefore, it is better to measure symmetrical curves by decreasing the flow velocity. However, errors are always included in the leading part, the tailing part, and baseline drifts. It is often better to calculate HETP directly by simply measuring the peak width at C ¼ e1Cmax and

V R  V o ¼ V t ½ð1  εÞ εP  + V t ½ε + ð1  εÞ εP k ¼ V t ð 1  εÞ K (19) Therefore, K ¼ εP + ½ε=ð1  εÞ + εP k

(20)

As pointed out by Hearn [12], the determination of εP must be carefully examined. If the sample probe is not small enough, the value is smaller than the true εP value due to size exclusion. If there is some interaction between the probe and the stationary phase, the value may be larger than the true k value.

479

3 Plate height and related variables in isocratic elution

When the HETP is plotted against the flow velocity (linear mobile phase velocity) u, a so-called Van Deemter curve is obtained. HETP ¼ Z=N ¼ A + B =u + C u + D u o

o

o

o

¼ 2DL =u + 2γ m Dm =u h i + HKd2p u= 30Ds ð1 + HKÞ2 h i + HK2 dp u= ð3kF Þð1 + HKÞ2

(21)

Here, γ m is the obstruction factor in the mobile phase. The first two terms (Ao + Bo/u) represent the dispersion (zone spreading) in the mobile phase. The third term Cou represents dispersion due to stationary phase diffusion and the fourth term for dispersion due to the stagnant film mass transfer around the particle. The above form of the HETP equation was first derived by Van Deemter et al. [13]. Later, a more exact form was proposed by Kubin [14] and Kucera [15] with the aid of moment equations from the solution in the Laplace domain of the partial differential equations of Model 1 in Table 1. Their final form in our notation is given by Eq. (21). In fixed-bed adsorption process design and operation, different parameters such as NTU (the number of transfer units), HTU (height of transfer units), and Kfav (overall volumetric mass transfer coefficient) are employed. For linear isocratic elution chromatography, these parameters are easily related to HETP as HETP ¼ 2HTU or NTU ¼ 2N. The dimensionless form of the HETP equation is useful as the results can be compared over a wide range of conditions on the same scale. The HETP equation, Eq. (21), can be rewritten with the dimensionless HETP, h ¼ HETP/dp, and the dimensionless velocity ν ¼ udp/Dm. h ¼ A∗ + B∗ =ν + C∗ ν + D∗ ν

(22)

h ¼ HETP=dp

(23)

ν ¼ udp =Dm ¼ Re Sc

(24)

A∗ ¼ 2ðDL =uÞ=dp ¼ 2=Pe

(25)

B∗ ¼ 2γ m h i C∗ ¼ HK= 30γ s ð1 + HKÞ2

(26) (27)

h i D∗ ¼ HK2 = 3Shð1 + HKÞ2

(28)

γ s ¼ Ds =Dm

(29)

Sh (¼ kfdP/Dm) is the Sherwood number and Re (¼ ρudP/η) is the Reynolds number based on the particle diameter and the linear mobile phase velocity, Sc (¼ η/ρDm) is the Schmidt number, and Pe (¼ udp/DL) is the Peclet number based on the particle diameter and the axial dispersion coefficient. ρ is the density of the solution and η is the viscosity of the solution. A* is constant with a value from 2 to 10 for typical velocity ranges. Namely, Pe is 0.2–0.5. γ s is an obstruction factor for stationary phase diffusion, implying a lower value for the molecular diffusion coefficient in the stationary phase (in most cases pore diffusion). The second term B*/ν is negligible for protein chromatography due to low Dm. The contribution of the fourth term D*ν is also low in most cases. A typical h-ν curve is shown in Fig. 4 along with the contribution of each term. This type of plot is usually called a “Van Deemter Plot” based on their original equation [13]. Eq. (21) has the same form as the original Van Deemter equation although the C and D terms are different from Eq. (21). Another empirical expression for h is given by h ¼ E∗ ν n

(30)

This equation can describe the experimental data over a certain velocity range. In fixed-bed adsorption processes, the stationary phase diffusion is dominant because of very large adsorbent particle size (packing materials). So the assumption of h ¼ C*ν is commonly employed. This approximation is also valid for chromatography at very high dimensionless velocities (ν > 200).

480

23. Process modeling of protein separations 10

h= E*Qn

6

A*+C*Q

4

2

0

h= HETP/dp

h= HETP/dp

8

C*Q A*

B*/Q

0

D*Q 0

20

Perfusion effect

40

60

80

Q = udp /Dm

0

100

Q= udp /Dm Left-hand side, the relationship between h (dimensionless HETP) and ν (dimensionless velocity), and right-hand side the perfusion effect.

FIG. 4

At very high dimensionless velocities and high pore-diameter/particle-diameter ratio values, convection through pores may enhance mass transfer. As a consequence, h does not increase with ν significantly. This is called a “perfusion” mechanism, Fig. 4. A modified, empirical expression for HETP considering the perfusion mechanism is given by [3]. h ¼ A∗ + F∗ ν=ð1 + aνÞ

(31)

(a)

Rs  1

(b)

Rs | 1

(c)

Rs | 2

(d)

t R1

W1

W2

me

tR 2

FIG. 5 Resolution, Rs.

4 Resolution Rs in isocratic elution In addition to HETP (a measure of the column efficiency), another measure of separation performance is needed, which considers the distance between the two adjacent peaks. The resolution Rs is the most employed measure for the separation efficiency of two adjacent peaks. Rs ¼ ðtR2  tR1 Þ=½ð1=2ÞðW 1 + W 2 Þ

(32)

When the two peaks have similar peak height areas, baseline separation is achieved when Rs > 1.3, Fig. 5. By assuming that W1 ¼ W2 ¼ 4σ 1 and inserting Eqs. (12) and (15) into the Rs equation, we obtain

Rs ¼ fHðK2  K1 Þg=f4ð1 + HK1 Þg N 1=2 ¼ fHðK2  K1 Þg=f4ð1 + HK1 ÞgZ1=2 HETP1=2 (33) This equation shows that Rs is proportional to the square root of the column length, Z. Although the HETP equation, Eq. (21) can be inserted into the resolution equation to relate Rs to various chromatography variables, the use of Eq. (30) is more convenient in order to understand the effect of individual parameters. Rs ¼ fHðK2 K1 Þg=f4ð1 + HK1 ÞgZ1=2 un dp1n Dm n (34) By using this equation, we can predict how Rs varies with such parameters as u and dp. As Dm

481

5 Ion-exchange equilibria

decreases with increasing molecular weight, the separation of larger molecules becomes more difficult. Based on the above discussion, it is easily understood why smaller particles result in higher resolution at high velocities. However, at the same time, the pressure drop, Δp, increases with decreasing particle diameter according to the Kozeny-Carman equation [3,5,16,17]. Δp ¼

κηZ ð1  εÞ2 u0 ε3 d2p

(35)

Here, κ is the constant (150180). Δp is proportional to u0 (¼ u/ε), 1/d2p, Z and η. For a packed bed with compressible or semirigid gels, the linear range Δp – u is smaller when the column size (diameter and/or height) is increased [3,5,17–20]. This is an important issue when the process is scaled up because the upper limit of the flow velocity is caused by bed compression, the optimum condition must be carefully sought by changing the column geometry (height to diameter ratio) even for the same bed volume.

5 Ion-exchange equilibria The ion-exchange or electrostatic interaction between a protein having Zp charged sites and the ionized (ion-exchange) group in the presence of a counter-ion (salt) is described as follows [1,8,21]: Ke

P + ZP S , P + ZP S

(36)

where P is the protein and S is the counter-ion (salt). The over bar indicates the concentration in the stationary phase. The equilibrium coefficient Ke is given by
 ZP P ½S Ke ¼
ZP (37) ½P S The application of the law of mass action to IEC of proteins was first presented by Boardman and

Partridge [22]. Later, a more elaborate treatment was developed by Regnier and coworkers [23,24] as the stoichiometric displacement model (SDM). Assuming that the activity coefficient is constant and close to unity, the following equation is derived [8].   Cq I ZP (38) Ke ¼ C Iq Cq is the protein concentration in the stationary phase (or the protein concentration bound to the ion-exchange group) and C is the protein concentration in the liquid phase (the mobile phase in chromatography). I is the salt (ion) concentration in the liquid phase (the mobile phase in chromatography), and Iq is the salt ion concentration in the stationary phase (or the salt (ion) concentration bound to the ion-exchange group). The total concentration in the stationary phase is governed by the ion-exchange capacity Λ, which is related to Iq and Cq as follows: Λ ¼ I q + Zp Cq

(39)

As proteins are large molecules, it is likely that the protein in the stationary phase may hide or shield some ion-exchange groups, which are not involved in protein binding. Based on this consideration, a concept of shielded charge or the steric factor (SF) was introduced by Cramer’s group [25] as the steric mass action (SMA) model. Here, SF (in their papers, the symbol σ is commonly used) is the average number of sites, which are sterically shielded by the protein. Then, the total capacity becomes:

 (40) Λ ¼ I q + SF + Zp Cq Ke ¼

  ZP Cq I C Λ  ðSF + ZP ÞCq

(41)

When the protein concentration is low, Λ ¼ Iq. The distribution coefficient Kq ¼ Cq/Cp is given by

23. Process modeling of protein separations

 Kq ¼ Cq =Cp ¼ Ke ΛZp I Zp

(42)

The number of binding sites, Zp, is also referred to as the characteristic charge in the SMA model (the symbol ν is used in their papers). Now, Eq. (7) is rewritten by inserting Eq. (42)

 K ¼ Kads + KSEC ¼ Ke ΛZp I Zp + KSEC ¼ AI B + KSEC (43) Here, B ¼ Zp is the number of binding sites (effective charge) and A ¼ KeΛB contains the ion-exchange equilibrium coefficient Ke and the ion-exchange capacity Λ and B.

6 Linear gradient elution When a certain substance in the mobile phase (modulator) is changed continuously with time, good resolution may be achieved that is not easily obtained with isocratic elution. Major advantages of this method (linear gradient elution, LGE) are as follows [8]. A linear increase of salt is used for LGE in IEC of proteins. (1) Resolution can be changed easily by modifying the gradient slope as well as the flow velocity. (2) Multiple solutes can be eluted in a single run. (3) False peaks due to a discontinuous change in the mobile phase do not exist. (4) Self-sharpening effect due to slower migrating velocities of the front part and faster velocities of the rear part of the peak exists. This can improve the elution peak shape automatically even when the zone is distorted due to a poor sample injection device and/or poorly packed bed.

6.1 Retention in linear gradient elution Compared with isocratic elution, LGE is complicated. At the beginning, the sample is tightly bound to the stationary phase. Usually, the salt

concentration is set to be low for IEC. Then, a linear increase in the salt concentration at a fixed pH is introduced into the column. The sample zone does not move as it is tightly bound to the column. As the salt concentration I increases, the isotherm shape changes as shown in Fig. 6. Eventually, the sample zone starts to move along the column. The zone trajectory is shown in Fig. 7. The zone movement can be described by Eq. (11) when the distribution coefficient K as a function of I is known. Since K depends strongly on I, which will be shown later, the sample zone moves down the column for a very narrow range of I. The linear increase of the modulator concentration (salt or counter-ion) for IEC is described by Eq. (44), where I0 is the initial salt concentration and g is the slope of the gradient [M/mL]. In an ideal case, the concentration at the column outlet is given by Eq. (45). Based on the equilibrium model, it is possible to calculate the zone movement numerically with Eq. (11), K(I), and Eq. (46), which describes I as a function of time t and distance from the column inlet z. Column inlet I ¼ I 0 + gFv t ¼ I 0 + gV

(44)

Column outlet I ¼ I 0 + gðFv t  V 0 Þ ¼ I 0 + gð V  V 0 Þ

(45)

In the column I ¼ I 0 + g½V  V 0 ðz=ZÞ ¼ I 0 + g½Fv tV 0 ð1 + HK0 Þðz=ZÞ (46) Staonary phase concentraon,_Cs

482

FIG. 6

Increasing salt concentraon

Mobile phase concentraon,_C

Adsorption isotherms as a function of salt concentration.

483

6 Linear gradient elution

distance from the column inlet outlet

outlet gradient

isocra c isoionic strength line peak trajectory

inlet

FIG. 7

Zone movement during isocratic and gradient elution.

V0 is the volume needed to elute the salt. K0 is the distribution coefficient (constant) for the salt. After mathematical manipulations, the following equation is obtained. dI=dt ¼ G½ðu=ZÞ  ð1 + HK0 Þ=Zðdz=dtÞ dI=dt ¼ Gðu=ZÞ½1  ð1 + HK0 Þ=ð1 + HKÞ (47) ¼ Gðu=ZÞHðK  K0 Þ=ð1 + HKÞ dI=dz ¼ GHðK  K0 Þ=Z

(48)

This equation is integrated to give IðR

GH ¼ I0

where

inlet

me or volume

dI K  K0

 GH ¼ gV o H ¼ gðV  V o Þ

(49)

(50)

The differential form of Eq. (49) is dðGHÞ=dI ¼ 1=½KðI Þ  K0 

(51)

when K is approximated to K ¼ AI B + K0

(52)

and following simple equation is obtained. GH ¼ I R B+1 =½AðB + 1Þ

(53)

Note that Eq. (52) is almost the same as Eq. (43) when KSEC  K0 . Eq. (53) indicates that the peak

shifts to larger elution volumes with a decreasing gradient slope in LGE, Fig. 8. The salt concentration at the peak position (peak retention volume, VR), IR, also decreases with a decreasing gradient slope. The IR values for different gradient slopes are plotted against the normalized gradient slope GH in Fig. 8. Once GH-IR plots are prepared, the data are usually fit to Eq. (53) as shown in Fig. 8. The B value is the number of binding sites (effective charges) involved in electrostatic interaction, whereas A includes the equilibrium coefficient, the binding site, and the ion-exchange capacity as shown in Eq. (43). The above-mentioned equations do not include the effect of the solute concentration C. This is because up to a certain sample loading the elution curve shows a geometrically similar profile. Then, in LGE, K varies with time during elution as the linear increase of I is introduced to the column. The tR therefore changes with the initial salt concentration I0 even when the same gradient slope g is used. However, we found that the peak salt concentration IR values do not change with I0. Similarly, the IR value remains constant when the sample volume is increased although VR changes. GH-IR plots can be employed for other chromatographic separations such as hydrophobic interaction liquid chromatography or reversed-phase liquid chromatography [27–29].

484

23. Process modeling of protein separations

0.1

Peak salt concentration IR

Normalized gradient slope GH [M]

Resource Q pH5.2

E - lactoglobulin B

0.01

3.1 cm/min 25 cm/min

E -lactoglobulin A

volume

Retention volume,VR

0.001

0.1

0.2

peak salt concentration, IR [M]

FIG. 8

GH-IR plots and LGE experiments [1,21,26].

6.2 Peak width, HETP, and Rs in linear gradient elution As explained previously, the distribution coefficient at IR (KR) in LGE can be determined if we define the apparent peak elution volume based on KR as V R app ¼ V o ð1 + HKR Þ

(54)

L ¼ M0.5 was derived from an asymptotic solution. pffiffiffiffiffi L¼ M for M  0:25 3:22M (56) L¼ for 0:25 < M < 12 1 + 3:13M L¼1 for M  12

The peak width is also independent of I0 or VF. However, we also have to consider the zonesharpening effect due to the linear gradient, Fig. 9. We define the zone-sharpening factor L as L ¼ W G =W I ¼ σ G =σ I

(55)

where W is the peak width at the baseline and the subscript I is for isocratic elution and G for LGE. L is given as a function of M as follows. These correlations were obtained based on the experimental and numerical LGE curves.

1 + HKR 2GH  J  ð1 + HK0 Þ

M¼

KR

KKR

(57)

zone spreading

Zone moving velocity dz/dt= u/(1+HK) zone sharpening

zone sharpening

KR= K at I=IR

time

FIG. 9 Linear gradient elution chromatography (zonespreading and zone-sharpening effects).

485

6 Linear gradient elution

where J ¼ jdK/dIj and M is a dimensionless group. L approaches 1.0 with increasing M. This means that the zone-sharpening effect is very weak and W is almost the same as for a constant modulator concentration I ¼ IE (isocratic elution). When M is less than 0.25, the relationship L2 ¼ M holds. Based on this relationship, the resolution is proportional to a single dimensionless parameter h

i0:5 Rs ∝ Ym ¼ ðZDa I a Þ= GH u dp 2 (58) Ia representing a dummy variable having a numerical value of 1 so that Ym becomes dimensionless. A similar Rs equation was derived by Snyder et al. [30] using a completely different approach. Eq. (58) was experimentally verified for both IEC and hydrophobic interaction liquid chromatography of proteins [31] and was successfully employed for scaling-up IEC columns from 15 L bench-scale to 370 L process scale [32]. Examples of calculation are shown in the book by Harison et al. [17]. However, it is still desirable to have more accurate methods, which can cover a wide range of flow rates. Because GHJ ¼ B/(B + 1),

Elution curves as a function of flow-velocity u at a fixed GH The above experimental data set as a function of GH W-u as a function of GH

IR

Vv-u as a function of GH

GH-IR curve L

HETP=(Z/L2)(Vv/VRapp)2 and u curve at a fixed GH for different GH values

L-M

M

VRapp=VR (1+HKR)

K-I J-I or B KR

FIG. 10 Flow diagram for determining HETP from linear gradient elution (LGE) curves [33].

determination of the column performance for the LGE-IEC of proteins. This method was verified by several groups [34–37].

6.3 Iso-resolution curves in linear gradient elution

Finally, the HETP from the LGE curve (HETP)LGE is determined as !2 Z σV HETPLGE ¼ 2 (60) app L VR

Because we have one more tunable operating parameter gradient slope for LGE compared with isocratic elution, the same resolution can be obtained at different combinations of flow velocity and gradient slope even for the same column. To predict resolution in LGE, the following parameters were proposed where (HETP)LGE is HETP for LGE [38]. Note that by assuming that HETP is proportional to u, a useful dimensionless group Ym ¼ [(ZDaIa)/(GHud2p)]0.5 Eq. (58) was derived from Eq. (61)
 (61) O ¼ ðZI a Þ= G ðHETPÞLGE

A flow diagram for the determination of (HETP)LGE-u is given in Fig. 10. Based on this method, the HETP can be determined from LGE curves as a function of u. It was confirmed that the HETP-u curve is independent of Z. This is one of the fundamental assumptions of the HETP concept. The curve is also unaffected by the initial salt concentration I0 and by the sample loading. These results confirm that the method is applicable to the

Ia is a dummy variable with a numerical value of 1 (unit is M). The elution curves for various combinations of experimental variables such as Z, G, and u are similar when the O values are set to be equal. Although the same resolution can be obtained with different combinations of operating/column variables, Fig. 11, there may be some optimum condition since the retention time tR and retention volume VR are a function of G and u.

M

ð1 + HKR Þ ðB + 1Þ 2B ð1 + HK0 Þ

(59)

486

Absorbance at 280 nm

4

Separation time [min]

30 25 U

20

6

time [min]

GH=0.018 u=9 cm/min

8

A

17

19 B

0.3

IR

8

12 10 volume [mL]

14

16

7

8

VR

9

0

=

FtR

B

15

Iso-resolution curve

Z= 5 cm

20 cm

10

10 cm

A

5 0

15

13

GH=0.049 u=2.3 cm/min

NaCl concentration [M]

23. Process modeling of protein separations

0

2

4

L

6

8

10

Relative elution volume V/Vt FIG. 11

Separation time and relative elution volume relationships as a function of column length [35,38].

For conventional porous bead columns, the peak broadens with increasing flow velocity due to diffusion mass transfer in the pores. Therefore, when the flow velocity u is increased, the gradient slope g must become shallower to obtain the same value for O. Likewise, when u is decreased, g must be increased. With the aid of the (HETP)LGE-u curve, this calculation can be done. The method for determining (HETP)LGEu relationships was described in Section 6.2. Once the u and g values are determined, the separation time tS and the relative buffer consumption VR/Vt are calculated as follows. tS  tR  ðI R  I o Þ=g + V 0

(62)

V R =V t ¼ FtR =V t ¼ tR Z=ðuεÞ

(63)

Here, V0 is the elution volume for the salt. The tR  VR/Vt curve at a constant O value is called the “iso-resolution curve.” The same resolution with different separation time and buffer consumption can be obtained with this curve.

The relative elution volume is often referred to as CV (column volume) and implies the relative buffer consumption. For the column length Z ¼ 5 cm, the upper limit (symbol U) and lower limit (symbol L) are shown in Fig. 11. The symbol A and B are the data from the elution curves shown in the inset (A) and (B), respectively. The open circles A and B in Fig. 11 are the data from the inset A and B, respectively. These data are in good agreement with the calculated isoresolution curve. As shown, the separation time becomes longer as the elution volume decreases. It is especially important to know where your separation conditions are located. For example, if your separation is carried out at point B in Fig. 11, it is not wise to decrease u to decrease buffer consumption. Likewise, if your separation is performed at point A, it is not advantageous to increase u as a large buffer consumption is required. For the calculation of the iso-resolution curves, there are the upper (U) and lower limits

7 Applications of the mechanistic models and the simplified methods

(L). The upper limit is determined by the gradient slope. The lower limit is determined by u or the pressure drop. When Z is increased, the curve shifts to the left and the region between the limit U and L becomes smaller. This means that buffer consumption decreases with increasing Z, but at the same time, fast separations become difficult. This is especially true for large-scale or process-scale separations where the maximum flow velocity due to the pressure drop limit is significantly affected by the column bed height. Fig. 12 illustrates how to adjust the operating conditions according to the iso-resolution curve. Although the effect of sample loading is not included, the resolution is constant up to a certain sample loading. A further increase of the sample loading results in lower resolution. Nevertheless, it is still important to determine the optimum conditions at low sample loadings, from which we can examine the effect of sample overloading.

Separation time [min]

Starting point

0

7 Applications of the mechanistic models and the simplified methods

Desired Desired separation separation time time

2

4

4

6

8

Starting point

2 Desired buffer consumption

00

Another important topic is the estimation of the (HETP)LGE-u curve with a small amount of experimental data. The axial dispersion term Ao can be estimated as discussed in Section 3. Two or three LGE curves for different GH and u values may provide sufficient data for the prediction of resolution and retention. The gradient slope g is one of the most important variables in LGE because it can be varied over a wide range compared with Z and u. Such a drastic increase in resolution cannot be achieved in isocratic elution even when u is decreased and/or Z is increased. However, the above-mentioned three variables in LGE must be carefully tuned to obtain the highest productivity and the desired resolution together with other constraints and/or requirements such as buffer consumption and separation time.

So far mechanistic models have been explained, which can be used for process modeling, process understanding, and process optimization. Several examples of the application of these models are presented later to facilitate an understanding of their use.

4

2

487

2

4

6

8

VR/Vt [-] FIG. 12 Illustration on how to adjust the flow velocity and the gradient slope to reduce separation time (upper) and buffer consumption (lower) [38].

7.1 Stepwise-elution process design based on linear gradient elution data Although LGE is an efficient elution method, for industrial processes stepwise-elution (SE) is preferred on account of its simpler operation procedure. Basically, a SE process can be designed by using the mathematical model when K(I) and mass transfer properties are known. As illustrated by Fig. 13, stepwise elution can be grouped into two mechanisms. In type I elution, a target protein is completely desorbed with an elution buffer, IE. Namely, the distribution coefficient of the protein in the elution

488

23. Process modeling of protein separations

(A)

(B)

Salt concentration Type I elution Type I elution K 20 and the isotherm is linear (K ¼ constant), single zone-spreading parameter models provide the same elution profiles [1,3]. In this study, the following equilibrium-dispersive model (Model 2 in Table 1) was chosen for the numerical simulations.

1 2ðDL,e =uÞ ¼ N Z 0

100

200 300 400 Eluon volume, V [mL]

(74)

DL,e is the effective dispersion coefficient, which considers the dispersion due to the stationary phase diffusion as well as axial dispersion. Instantaneous equilibration Cs ¼ KC is assumed in this model. DL,e is related to the plate number N by the following equation.

2

1000

chromatograms (C1)–(C15) in Fig. 24 indicate that dimer and trimer are removed until V ¼ 190 mL compared with the sample [sizeexclusion chromatogram (A) in Fig. 24]. The desorbed fraction by the buffer containing 1.0 M NaCl contained highly concentrated dimer and trimer [size-exclusion chromatogram (B) in Fig. 24].

∂C ∂Cs ∂2 C ∂C +H ¼ DL,e 2  u ∂t ∂z ∂z ∂t

3

493

500

FIG. 23 Flow-through chromatography experimental results (A) dimer/monomer ratio (B) UV absorbance signals at 280 nm; after the sample loading (400 mL), the mobile phase was applied to the column until V ¼ 500 mL; then, the mobile phase containing 1.0 M NaCl was pumped to the column to desorb the adsorbed proteins; eluted peak protein concentration was so high that the UV signal was flattened [44,45].

(75)

The analytical solution to Eq. (74) for constant K is given by the following equations.



 C=C0 ¼ f N, T∗  f N, T ∗  T∗F (76) C0 is the sample feed concentration. f (x,y), T⁎ and T∗F are given by Eq. (77).

pffiffiffi pffiffiffi 1 f ðx, yÞ ¼ erfc x  y 2

(77)

494

23. Process modeling of protein separations

Monomer

(A) Sample Dimer

110-130 mL

310-330 mL

(C1)

(C6)

130-150 mL

330-350 mL

Trimer 0

5 10 Eluon Volume [mL]

410-430 mL

(C11) 430-450 mL

(C2)

(C7)

150-170 mL

350-370mL

(C3)

(C8)

(C12) 450-470 mL

(C13)

Desorbed fracon with 1 M NaCl

Dimer

170-190 mL

(C4)

(B) Trimer

(C9)

Monomer 190-210 mL

(C5) 0

5 10 Eluon Volume [mL]

390-410mL

(C10) 5

0

370-390mL

470-490 mL

(C14) 490-510 mL

(C15)

10 0 5 10 0 Eluon Volume [mL]

10

5

FIG. 24 Size-exclusion chromatography (SEC) chromatograms of FTC experiment fractions. (A) Sample, (B) desorbed fraction by 1 M NaCl (V ¼ 520–530 mL in Fig. 23); (C1)–(C15) are SEC chromatograms of the fractions from V ¼ 110–510 mL in Fig. 23 (fraction volume ¼ 20 mL) [44,45].

TABLE 2 Calculated and experimental breakthrough volumes of monomer and dimer [44,45]. Calc. K [2]

Calc. V∗a [2]

Exp. V∗a [2]

Calc. Va [mL]

Exp. Va [mL]

Monomer

3.5

2.2

3.0

29.0

20

Dimer

44.5

22.7

21.0

216

202

T⁎ is the dimensionless time and T∗F is the dimensionless sample injection time (tF is the sample injection time). T∗ ¼ t=½ðZ=uÞð1 + HKÞ

(78)

T ∗F ¼ tF =½ðZ=uÞð1 + HKÞ

(79)

The error function complement erfc(x) is defined by Eq. (80)

2 erfcðxÞ ¼ pffiffiffi π

ð∞

et dt ¼ 1  erfðxÞ 2

x

2 ¼ 1  pffiffiffi π

ðx

et dt 2

(80)

0

For the simulation, C01 ¼ 9 kg/m3 and C02 ¼ 1 kg/m3 were used. Figs. 25 and 26 show calculated FTC profiles as a function of mobile phase salt concentration, I. It is clear that V∗F dramatically increases with decreasing I from 0.2 to 0.18 M. However, as mentioned previously, further decrease in I increases V∗a1. Aggregate removal (in this study dimer removal) is a difficult separation. Even using LGE baseline separation (complete separation) is not easy unless a long separation time is accepted. In LGE, it is not ΔK ¼ K2  K1 but

495

7 Applications of the mechanistic models and the simplified methods

10

I = 0.18 K1 = 3.5 K2 = 44.5 VF* = 18.1 Va1*= 2.1 RT = 240 s

Monomer (1)

Concentraon [kg/m3]

Concentraon [kg/m3]

10

5

0

0 20 CV = V/Vt [-]

I = 0.175 K1 = 4.0 K2 = 60.1 VF* = 24.9 Va1*= 2.3 RT = 240 s

0 10

Producvity, P [g/(mL· min)]

5

Concentraon [kg/m3]

Dimer (2)

Calculated flow-through chromatography elution profiles for two different salt concentrations [44,45].

Monomer (1)

Dimer (2)

I = 0.18 K1 = 3.5 K2 = 44.5 VF* = 18.1 Va1*= 2.1 RT = 240 s

Monomer (1)

5

Dimer (2)

0 10

0.5 0.4 dp = 34 μm RT = 2 min RT = 4 min RT = 8 min

0.3 0.2

dp = 60 μm RT = 4 min RT = 8 min

0.1 0 0

0.02 0.04 0.06 0.08 0.1 0.12 0.14 1/VF* [-]

FIG. 27

Productivity as a function of sample load volume

[44,45]. Monomer (1)

5 Dimer (2)

0

5

0

80

40 60 CV = V/Vt [-]

20

10

0

I = 0.2 K1 = 2.1 K2 = 14.8 VF *= 5.3 Va1* = 1.4 RT = 240 s

Dimer (2)

0

FIG. 25

Monomer (1)

20

40 60 CV = V/Vt [-]

I = 0.185 K1 = 3.0 K2 = 33.3 VF* = 13.2 Va1 *= 1.8 RT = 240 s

80

TABLE 3 Linear gradient elution separation performance at GH ¼ 0.002 [44,45]. pH

IR1

IR2

ΔIR

K1

K2

6

0.121

0.164

0.0437

10

10.4

7

0.148

0.217

0.0693

10.6

9.9

FIG. 26

Calculated flow-through chromatography elution profiles for I ¼ 0.175, 0.180, and 0.185 [44,45].

ΔIR, which determines the distance between two peaks. Table 3 shows calculated IR1 (monomer), IR2 (dimer), ΔIR, K1, and K2 values for LGE at GH ¼ 0.002. Unlike FTC processes, K1 and K2 values are similar and much larger than those for FTC processes. In LGE, the separation

performance (ΔIR) at pH 7 is much higher than that at pH 6, whereas the same separation performance can be obtained at pH 6 and 7 for FTC.

7.4 Summary With the aid of LGE experiments, FTC processes can be designed and optimized. Basically, small particles give higher productivity

496

23. Process modeling of protein separations

provided that the pressure drop is acceptable. Higher separation performance compared with LGE because a proper mobile phase salt concentration can be found at which the difference of the distribution coefficients is sufficiently high. On the other hand, the separation is sensitive to small changes in the mobile phase salt concentration and pH. Precise buffer preparation and monitoring of FTC processes are key factors for successful operations. One practical problem using FTC is that the UV signal does not provide useful information for the separation because the concentration of impurities during polishing steps is much lower than the target protein. Therefore, numerical simulations are quite useful for understanding the process. Various chromatographic models can be used for simulations. Several software packages including various mechanistic models are available [48–50]. However, these software approaches do not indicate how to obtain parameter values required for the simulations. It is also difficult to choose the correct model for a simulation. Fundamental knowledge of the chromatography of proteins in the elution mode is essential to use numerical simulations. Although the inverse method seems promising for parameter estimation, the Yamamoto method [33] can reduce the computational effort and provide reliable parameter values [51,52]. In addition to numerical simulations, advanced process analytical technology (PAT) for monitoring various impurities must be developed [53]. FTC is useful also for large bioparticles such as viruses [54,55]. However, since such bioparticles are much larger than proteins, their diffusion rates in the pores of conventional porous chromatography particles are very slow. Consequently, both the dynamic and static binding capacities become extremely low. Using convective media, such as membranes and monoliths can solve the mass transfer problems [56,57], although tuning of the proper convective pore size is needed [58].

For polishing steps, both anion exchange and cation exchange chromatography columns are commonly used as orthogonal separation methods [59–61]. However, other modes, such as hydrophobic interaction liquid chromatography (HIC), may be effective. GH-IR curves can be prepared for HIC as well [28]. Another interesting approach is to connect anion and cation IEC columns in series [60]. Optimization of FTC of proteins IEC is possible using model simulations of FTC profiles with the data obtained from LGE experiments. By choosing the proper mobile phase salt concentration, efficient FTC separation performance at high productivities can be obtained at pH 6, 7, and 8 although separations are improved with increasing pH for LGE.

8 Capture process design Since current IEC media have a high protein adsorption capacity (>100 g-protein/mL-gel), IEC can be effectively employed for the capture of proteins. The adsorption capacity can be assessed by measuring breakthrough curves (BTCs) at different flow velocities and/or sample protein concentrations, Fig. 28. Although BTCs can be described by the models in Table 1 with adsorption isotherms, several parameters are used for characterizing BTCs. Dynamic binding capacity (DBC) defined by Eq. (81) is used to describe the adsorption capacity [3,62]. DBC ¼ C0 V B =V t

(81)

Herein, VB is the breakthrough volume at C ¼ CB (XB ¼ CB/C0), which is often defined as the volume where XB ¼ 0.1 (10% breakthrough volume) (see Fig. 28). Similarly, the static binding capacity (SBC) also called the equilibrium binding capacity (EBC) is calculated by Eq. (82). SBC ¼ C0 V C =V t

(82)

497

8 Capture process design

X= C/C0

1

Breakthrough curve XC

Increasing velocity

XB 0

FIG. 28

VB VB

0

VC

Volume, V

Breakthrough curves (C0 ¼ sample protein concentration).

Herein, VC is defined as the volume where Xc ¼ 0.55–0.60 (see Fig. 28). DBC decreases with increasing flow velocity, u or decreasing residence time RT. There is a good correlation between DBC and u with the following dimensionless groups [63,64] E∗ ¼ DBC=SBC ¼ a1  a2 F∗

(83)

F* is derived from the irreversible isotherm pore-diffusion control constant-pattern breakthrough curve equation [3,6]. F∗ ¼ d2p =½Ds ðZ=uÞ

α is the dimensionless volume for the nonloading periods. Namely, αVt is the volume for tNL. RT ¼ Z/u0 ¼ Vt/Fv is the residence time based on the total CV. By using the E*-F* curve, DBC values can be calculated with Eq. (87). h

i DBC ¼ SBC a1  a2 d2p =Ds =ðεRTÞ (87) In addition to P, the buffer (solvent) consumption per recovered product (loaded amount) is an important criterion for the process efficiency [63].

(84)

V ∗NL ¼ V NL =ðC0 V F Þ ¼ V NL =ðDBC V t Þ ¼ α=DBC

As already mentioned, the stationary phase (pore) diffusion coefficient Ds can be determined from a HETP-u curve measured by an isocratic elution (pulse-response) experiment with nonbinding conditions. The efficiency of the capture process can be assessed by the productivity P defined by Eq. (85) as a function of u [62].

(88)

P ¼ ðC0 V F Þ=ðtC V t Þ ¼ DBC=tC

(85)

VF is the sample volume loaded during the loading period t1. The cycle time tC is the sum of the loading period (t1) and the nonloading period tNL (¼ t2 + t3 + t4 + t5) nonloading periods as shown in Fig. 29. tC ¼ t1 + t2 + t3 + t4 + t5 ¼ ðV B + αV t Þ=Fv ¼ ðV B + V NL Þ=Fv ¼ ½ðDBC=CF Þ + αRT (86)

A modified productivity considering V∗NL is given by Pc ¼ P=V ∗NL

(89)

As illustrated by Fig. 30, P increases with u until it reaches a maximum value [62]. However, V∗NL also increases as DBC decreases with u. The maximum Pc is obtained at a lower flow velocity than the value for the maximum P.

8.1 Multicolumn periodic counter current operation To improve process efficiency several approaches are possible. One is to use twoconnected columns for sample loading. After

498

23. Process modeling of protein separations

FIG. 29 Capture process (bind/elute or stepwise elution).

t2

t1

t3

t5

t4 I

impuries

me, t Sample loading

wash

total cycle me

tC = t1+ t2+ t3+ t4+ t5

P 0.5

DBC [g/L]

V*NL [L/g] or E* [–]

V*NL E*

0

regeneraon

1

Pc

Producvity P [g/(L·h)] Pc=P/V*NL [g/{(L·h)(L/g)} ] or DBC [g/L]

CIP(cleandesorpon in-place)

eluon

0

Flow velocity u [cm/min] or 1/tr [min-1]

Productivity, DBC, Pc, E*, V∗NL as a function of flow velocity or 1/tr. tr ¼ Z/u ¼ Vo/Fv ¼εVt/Fv ¼ εRT is the residence time based on the void volume Vo.

FIG. 30

the loading the columns are separated and the top column used for the nonloading protocol and the bottom column for sample loading. This continuous operation is called periodic counter current operation [65]. As is clear from Fig. 31, a much higher sample loading is possible compared with a single column loading based on DBC. In addition, this operation can be carried out continuously with a continuous sample feed and intermittent (periodic) elution.

normal constant flow (CF) velocity mode as shown in Fig. 32. This method is called a flow velocity gradient (LFG) loading [66]. A similar loading protocol using a step change of the flow velocity has been reported [67,68]. A linear flow velocity gradient (Fv1 at t ¼ 0 and Fv2 at t ¼ tg) is defined by Column 1

Fv A) C 0

M1

Column 2

M2

Column 3

Vt

8.2 Flow velocity gradient loading operation

B) F v

It is common to use a constant flow velocity for sample loading. When the flow velocity is decreased over time during loading, a much higher amount of the target protein can be loaded on the column compared with the

FIG. 31 Multicolumn periodic counter current (PCCC) operation. (A) The sample is loaded on the two-connected columns whereas Column 3 is used for the nonloading protocol. (B) Column 1 is separated and used for the nonloading protocol. Column 3 is connected to Column 2 for the loading.

Wash,Eluon

499

8 Capture process design

Fv ¼

ðFv2  Fv1 Þ t + Fv1 tg

(90)

By using DBC as a function of tr, LFG (Fv1, Fv2, and tg) can be designed to either shorten the loading time tload or increase DBC. The productivity P in LFG is calculated by Eq. (91), using Mload or the volume of the sample loaded Vload and the gradient time tg. ð tg C0 Fv dt C0 V F C0 ðFv1 + Fv2 Þ 0 P¼ ¼ ¼ V t tg  2V t V t tg C0 1 1 ¼ + (91) tr0,2 2 tr0,1

FIG. 32 Comparison of flow rates (A1), breakthrough curves (A2), and loading amounts (A3) for loading of IgG on a SP Sepharose FF column (0.2 mL OPUS MiniChrom column, 1 cm long with 0.5 cm in inner diameter, particle diameter 90 μm). Constant flow velocity, CF (tr ¼ 0.5 and 1.0 min), 4-step change (tr1 ¼ 0.1, tr2 ¼ 0.4, tr3 ¼ 0.7, tr4 ¼ 1.0 min) and LFG with (tr1, tr2) ¼ (0.5, 1 min) [66].

X=C/C0 [-]

0.8 0.6 0.4

(B)

(A)

LFG (tr = 0.5 – 2 min) CF (tr = 1.04 min) CF (tr = 2.09 min)

LFG (tr = 0.5 – 2 min) CF (tr = 1.04 min) CF (tr = 2.09 min)

tB

tB

0.2 0 0

FIG. 33

P is now independent of the gradient time tg and only related to the sum of Fv1 and Fv2 or 1/tr0,1 and 1/tr0,2. When a faster flow rate is applied, higher P can be achieved because of the shorter tg. However, the loaded amount C0VF is smaller as DBC decreases with increasing Fv. For the same Fv1 and Fv2, P remains constant and independent of the gradient slope. Fig. 32 shows the change of FV with time (A1), BTCs (A2) and loaded amount on the column (A3) for constant, 4-step change, and LFG. To illustrate the difference between the constant and LFG velocity loading, BTCs are shown for both the time axis and the volume axis in Fig. 33. LFG (tr ¼ 0.5–2 min) reduces the loading time

100 200

300 400 500 Time, t [min]

0

VB VB 20 10 Volume , V [mL]

Process efficiency improvement by LFG for Sepharose Fast Flow (SPFF) of IgG (A) X vs time t, and (B) X vs volume V. For tr ¼ 1.04 min, RT ¼ 2.89 min, DBC10% ¼ 77 mg/mL and tB,10% ¼ 222 min. For tr ¼ 2.09 min, RT ¼ 5.81 min, DBC10% ¼ 87 mg/mL and tB,10% ¼ 508 min. For tr ¼ 0.5  2.0 (LFG), RT ¼ 1.4–5.6 min, DBC10% ¼ 86 mg/mL and tB,10% ¼ 222 min. A 0.2 mL OPUS MiniChrom column packed with SPFF (90 μm) The column was 1 cm long with 0.5 cm inner diameter [66].

500

23. Process modeling of protein separations

tload by 56% while maintaining the same DBC (86 mg/mL) as that for CF (tr ¼ 2.09 min). This means that an approximately two-fold increase in P is possible by LFG. The mechanism is easily understood using numerical simulations with model 3 in Table 1. Fig. 34 shows calculated BTCs for (A) a constant high flow velocity at u ¼ u1, (B) a constant low flow velocity at u ¼ u2, (C) flow programming from u ¼ u1 to u2 (u2 at t* > 80), and (D) flow programming from u ¼ u1 to u2 (u2 at t* > 50). For BTC (C), the concentration X drops sharply at t* ¼ 80 when the flow velocity is decreased to u2. After that, the BTC (C) is superimposed on BTC (B). This behavior was experimentally confirmed as shown in Fig. 32. The flow velocity programmed curve BTC (D) is hardly distinguishable from BTC (B). As u1/u2 ¼ 0.7, the breakthrough time tB at X ¼ 0.1 is shorter for BTC (D) by ca. 15%. This behavior is due to the favorable isotherm used in the calculation (large KLC0 values). The zone self-sharpening effect in the column exists due to the migration velocity of the protein governed by the concentration. Because of this effect, a partially broadened zone in the column at the high velocity can

1

be compressed again with a lower velocity. The constant-pattern breakthrough curve is also due to this effect [6,7].

8.3 Model simulations It is not difficult to numerically simulate chromatograms or elution curves using a personal computer. However, it is still preferred to have a single zone-spreading parameter related to the plate number N. Different models containing a single zone-spreading parameter give identical elution curves in LGE provided they use the same value for N. Fig. 35 shows LGE curves numerically calculated by several models. A single lumped (or overall) zone-spreading parameter (N) was determined from the experimental (HETP)LGE-u and GH-IR curves. The calculated elution curves show favorable agreement with the experimental results. It is also promising that the calculated curve using a simple model, which does not need a numerical calculation is also in good agreement with the experimental results. Recently, several commercial and opensource simulation software packages have become available [48,49]. Fig. 36 shows simulated LGE curves using the open-source software CADET [69]. Model 1 in Table 1 along

Favorable isotherm

0.9 0.8

0.5

0.6

KL=15, Q=80, H = 1.857

0.4 0.2 0.1

0

100

200

300

400

SP Sepharose HP lysozyme

C0 = 1.0 mg/mL VF = 0.7 mL dc = 0.9 cm dp = 34 Pm Z = 20 cm u = 4.6 cm/min GH= 0.114 M I0 = 0.03 M

0.45

(A) Ks* = 0.02 (B) Ks* = 0.014 (C) Ks* = 0.02 (080) (D) Ks* = 0.02 (050)

0.3

0

KLQC 1  KLC

0.3

‫ ە ۑ‬experimental = calculated 1 = calculated 

Numerically calculated breakthrough curves for the constant and flow-programmed velocity mode for a favorable isotherm described by the Langmuir isotherm Cs ¼ KLQC/(1 + KLC) ¼ KC, As K∗s ¼ Ks (Z/u), the velocity u2 for BTC (B) is 0.7  u2 for BTC (A). The sample feed concentration C0 ¼ 1 is used for these calculations [66].

0.5

0.4

N ca.200

0.15

0.3

Dimensionless me, t*

FIG. 34

0.6

0

30

32

34

36

salt concentration, I [M]

Cs

C/C0 [-]

X = C/C0

0.7 0.6

38

time [min]

FIG. 35 Numerically calculated linear gradient elution curve. Modified from S. Yamamoto, Plate height determination for gradient elution chromatography of proteins. Biotechnol. Bioeng. 48 (1995) 444–451.

8 Capture process design

8.4 Summary

0.2 0.15 0.1 0.05 0

Salt concentraon I [M]

Protein concentraon C [mol/m3]

0.25

0.01

0 0

50 100 150 Eluon volume [mL]

200

FIG. 36 Numerically calculated linear gradient elution curves [69]. Sample: mAb, column: Eshmuno CPX column (20 cm  0.5 cm I.D.) Symbols are experimental data (C). Solid curves are simulation results for the mAb. Dotted curves represent salt concentrations, I. GH ¼ 0.006M, u ¼ 5.1 cm/min; GH ¼ 0.004 M, u ¼ 2.6cm/min; GH ¼ 0.003 M, u ¼ 2.0cm/min. CADET was used for the simulation.

with the following extended Langmuir type equation was used for the simulation.

 ∂Cq ¼ Ka I ka Cp Q  Cq  Kd Cq ∂t

501

(92)

Ka and ka are related to A and B in Eq. (53) as Ka ¼ A=½Qð1  εÞ

(93)

ka ¼ B

(94)

Namely, Ka and ka can be determined using values of A and B obtained from GH-IR curves. Other parameters are determined experimentally or estimated from literature sources. The model simulations shown in Figs. 35 and 36 are for relatively low sample loadings. Model simulations for high sample loadings need additional parameters for the ion-exchange equilibrium equations such as the shielded charge values and the total adsorption capacity. These parameters must be obtained by separate experiments. Several methods have been proposed to determine these parameters systematically and quickly [51,70,71].

Process modeling of protein separations by IEC was explained in this chapter by using simplified methods based on the mechanistic models. Some methods are useful for understanding the binding mechanism of proteins to ion-exchange ligands. For example, the separation of protein variants [26,72], retention of PEGylated proteins [73], retention of protein aggregates [74]; retention of oligo-DNAs [75,76] were analyzed. The retention of various mAbs was correlated with the surface charge distributions [77]. Recently, the effect of an amino acid substitution in a monoclonal antibody on retention was investigated by quantitative structure-property relationship (QSPR) models [78]. This type of study will be helpful for not only understanding the retention and separation behavior of proteins in IEC but also for estimating the model simulation parameters. Although several simulators are available, standardization of models and model parameters are needed along with a standard and efficient workflow for determining or estimating model parameters. LGE experiments can provide important data for parameter determinations. However, a proper choice of experimental conditions is essential. Such conditions are usually determined from a priori knowledge in most cases. Otherwise, a trialand-error approach is needed and is timeconsuming. Methods (procedures) for designing proper experimental conditions need to be developed, which can reduce the time required to obtain the required data. Finally, it is important to establish a proper column dimension, packing protocols, and packing quality inspection methods to obtain reliable data for scale-up. In some cases, small short columns do not provide reliable data, and such columns cannot be checked in advance by routine inspection methods [79].

502

23. Process modeling of protein separations

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C H A P T E R

24 Applications of ion-exchange chromatography for the purification of antibodies Guofeng Zhaoa, Yongbo Lib, Yuntao Wuc, Shanshan Lib, Xiaomei Chena, Wei Zhangb, and Jin Xieb a

Analytical and Pharmaceutical Sciences, Shell Biotech, Shanghai, China bDownstream Process Development, Shell Biotech, Shanghai, China cBestchrom (Zhejiang) Biosciences Ltd, Zhejiang, China O U T L I N E 1. Antibodies: Introduction, recombinant expression, manufacturing, and purification 506 1.1 Monoclonal antibodies and variant forms 506 1.2 Antibody expression, manufacturing, and purification 507 2. Cation-exchange chromatography of antibodies 2.1 Principles and goal 2.2 Starting conditions and parameters for optimization 2.3 Case studies 3. Anion-exchange chromatography of antibodies

Ion-Exchange Chromatography and Related Techniques https://doi.org/10.1016/B978-0-443-15369-3.00028-6

510 510 510 512 514

3.1 Principles and goal 3.2 Starting conditions and parameters for optimization 3.3 Case studies 4. Commercial ion-exchange resins 4.1 Ion-exchange ligands, linking chemistries, and backbone materials 4.2 Commercial ion-exchange resins

514 514 514 516 516 517

5. Scale-up and scale-down of ion-exchange chromatography 517 6. Tips and tricks

517

Acknowledgment

520

References

520

505

Copyright # 2024 Elsevier Inc. All rights reserved.

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1 Antibodies: Introduction, recombinant expression, manufacturing, and purification 1.1 Monoclonal antibodies and variant forms Antibodies, also known as immunoglobins (Ig), are Y-shaped protein molecules generated by the immune system that can bind specific target molecules (antigens) and initiate subsequent biological effects. There are different classes of immunoglobins, but the most studied and utilized is immunoglobin G (IgG). The targeting and binding capability has made IgG a versatile tool in research, therapeutic, and diagnostic applications. This chapter will primarily focus on the IEX purification of therapeutic IgGs, of which the principles can be readily adopted for the IgGs of other applications. Traditionally, antibodies are purified from serum or ascites of immunized animals. Such preparations are a mixture of antibodies with different sequences and binding affinities, and the productivity is low. The recombinant DNA technology has enabled the expression of antibodies with uniform sequences (“monoclonal antibodies,” or mAbs) in host cell lines, which is a great leap for consistent and large-scale manufacturing. Meanwhile, the technologies for antibody discovery have also undergone a series of revolutions, from the traditional animal immunization—screening—humanization approach to the current transgenic animals and single-B cell sequencing, enabling fast discovery of fully human mAbs. These technologies have enabled a boost in antibody therapeutics in the past 30 years. By 2022, there are 175 antibody therapeutics approved or in regulatory review, and almost 1200 more in clinical studies [1]. The therapeutic areas for antibodies include oncology, immunology, and infectious diseases. The IgG molecule consists of three distinct structural modules, i.e., two identical antigenbinding fragments (Fab) and one fragment

FIG. 1 IgG structure.

called fragment crystallizable (Fc), Fig. 1. The Fc portion does not bind to the target but has several important biological functions. Once the Fab part binds to a receptor on the cancer cell surface, the Fc portion initiates a series of cellkilling mechanisms such as antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell phagocytosis (ADCP). The Fc fragment can also bind to the neonatal Fc receptor (FcRn) by cell internalization, which protects the antibody from degradation and releases it back into circulation, extending its half-life [2]. As the structure of the Fc fragment is constant among different antibody molecules, it is also the most utilized binding target for affinity purification of antibodies and antibody-like molecules. Different variant forms of antibodies (Fig. 2) have emerged as therapeutics with several already flourishing due to an ever-increasing clinical and market demand. Unlike natural IgGs, which have identical binding structures in each arm, bispecific antibodies (bsAbs) adopt either a hybrid asymmetric structure with two different target-binding sites, or a fused structure of additional binding site(s) linked to a parent IgG molecule. The purpose of bispecific antibodies is usually to bring the two targets together, e.g., one arm binds to the cancer cell

1 Antibodies: Introduction, recombinant expression, manufacturing, and purification

507

FIG. 2 Therapeutic antibodies and variant forms. (A) mAbs; (B) Fc fusion proteins; (C) bispecific antibodies; (D) ADCs; (E) antibody fragments.

and the other arm binds to the immune cell (e.g., T cell) to initiate cancer cell-killing. Antibodydrug conjugates (ADCs) are antibodies conjugated with a cytotoxic drug by chemical reactions. For applications where binding is the only mechanism of action, antibody fragments (e.g., Fab or part of Fab) are also developed for the purpose of easy expression and better permeability. The Fc fragment of antibodies is also widely used to construct drug molecules that would otherwise be unstable or short acting, e.g., fusion proteins of Fc fragment with cell-surface receptors or hormones. Though the therapeutic mechanisms of these variant forms may vary, the purification workflow for antibodies and antibody-like molecules are much the same due to their structure

similarity. Antibody fragments do not have the IgG-like structure, and their purification is no different from any other non-IgG proteins. In this chapter, the applications of ion-exchange chromatography are discussed in the context of antibodies and antibody-like therapeutics.

1.2 Antibody expression, manufacturing, and purification 1.2.1 Recombinant expression and manufacturing Recombinant DNA expression technologies are widely used for the large-scale manufacturing of antibodies. The host cells for recombinant protein expression span different organisms,

508

24. Ion-exchange chromatography for antibody purification

and appropriate host cells should be selected according to the structural characteristics of the antibody molecules. Microbials like E. coli cannot produce glycosylated proteins and are thus only suitable for antibody fragments, as the Fc fragment must be glycosylated to be fully functional. The glycosylation pattern of yeast is significantly different from mammalian cells, and although there are reports of glycoengineered yeast to express proteins with mammalian glycoform, the overall application of yeast expression system is still mainly restricted to antibody fragments [3–5]. Murine cell lines are the most extensively studied mammalian cell lines with desirable glycosylation patterns for therapeutic antibodies. Until now, the most widely used cell system for antibody expression is Chinese Hamster Ovary (CHO) cells. Several earlier therapeutic antibodies also used NS0 or Sp2/0, both of which are murine lymphoid cells [6,7]. Baby Hamster Kidney (BHK) cells have also been reported for the expression of antibody-cytokine fusion proteins [8,9], but so far not for commercial antibody therapeutics. Although murine cell lines have been used to express proteins with similar glycosylation patterns to humans, they are still able to produce nonhuman glycosylations like α-1,3-galactose and N-glycolylneuraminic acid. Human cell lines [10,11], such as human embryonic kidney 293 (HEK293) and human embryonic retina cells (PER.C6), have also been used for the expression or antibodies and other recombinant protein therapeutics. These cell lines also feature high growth rates and high productivity. However, more experiences in viral risk control and clinical safety of human host cell lines are needed before wider application is possible. While the explorations of numerous products in different host cells flourish, the choice for a specific product depends on both the characteristic properties of the product, technology maturity, and regulatory acceptance. As a platform technology for the development of antibody

therapeutics, CHO cell lines are, and will continue to be, the prevailing technology for therapeutic antibodies in years to come. Numerous cases have demonstrated the quality and efficacy of antibody molecules expressed in CHO cells, and strategies for the mitigation of safety risks like virus and adventitious agents are well established. Several efforts have been made to improve the productivity of CHO cell culture, such as cell line development and culture media optimization. Today, the titer for antibody products in CHO cell culture is typically 5–10 g/L with reactors of 200–10,000 L. 1.2.2 Purification workflow The antibody-expressing cell lines are grown in a suspension cell culture from which the antibodies are secreted into the culture media. The cell culture harvest may contain both productrelated impurities and process-related impurities, as listed in Table 1. Antibody purification workflow A typical workflow for antibody purification is shown in Fig. 3 and typically includes three chromatographic steps, virus inactivation, and several filtration steps. The first chromatography step is usually Protein A affinity chromatography, which effectively captures the antibody molecules from the cell culture harvest as an intermediate of high purity. As the product in Protein A chromatography is eluted at low pH, a lowTABLE 1 Impurities in antibody manufacturing. Product-related impurities

Process-related impurities

Aggregates Fragments Charge variants Mismatched variants (for bsAbs)

Host cell protein Host cell DNA Protein A Cell culture media; antifoams Surfactant/detergents Cell banking reagents Virus contamination

1 Antibodies: Introduction, recombinant expression, manufacturing, and purification

509

FIG. 3 Antibody purification workflow. (A) General workflow; (B) and (C) common examples of the chromatographic steps.

pH viral inactivation and neutralization step typically follows. If low pH is not suitable for a specific product (e.g., product is unstable at the virus inactivation pH), a surfactant/ detergent (S/D) inactivation step is used instead, either before or after Protein A chromatography. The surfactant/detergent is removed in subsequent chromatography steps. There are options for the choice and order of the two polishing steps, but an almost indispensable step is cation-exchange chromatography (CEX) in the bind-and-elution mode. CEX can effectively remove product-related impurities like aggregates, fragments, and mismatched variants. Most antibodies have an isoelectric point (pI) of 7–9 and are positively charged at typical pH ranges. The material is loaded on the cation-exchange column at an appropriate pH and ionic strength. The target antibody molecule and product-related impurities are bound to the column and resolved by salt or pH elution.

Another important polishing step for antibody purification is anion-exchange chromatography (AEX) in the flow-through mode. AEX is useful for removing process-related impurities, such as host cell DNA, host cell protein, and potential virus contamination, which all have an acidic pI, Table 2. At a moderately high pH, the positively charged antibody molecule flows through the column, while negatively charged impurities are retained. In recent years, hydrophobic-ion-exchange mixed-mode adsorbents have been widely used for antibody purification. These adsorbents operate in a similar fashion to single-mode ion-exchange resins, but with improved selectivity. Mixed-mode adsorbents can be used instead of the corresponding CEX or AEX steps, respectively. For challenging cases where the CEX step does not effectively remove all product-related impurities, other modes need to be evaluated, e.g., hydroxyapatite or hydrophobic interaction

510

24. Ion-exchange chromatography for antibody purification

TABLE 2 Isoelectric points of antibodies and impurities. Molecule

pI

mAbs and bsAbs

7–9

Sialylated proteins

5–6

Host cell proteins

4.8–9.4 [12]

DNA

4.35 [13]

Protein A

5.1 [14]

Virus [15]

MVM: 6.2 REO3: 3.9 xMuLV: 5.8 PrV: 7.6

Note: MVM, minute virus of mice; REO3, reovirus type 3; PrV, pseudorabies virus; xMuLV, xenotropic murine leukemia virus.

chromatography (HIC). These polishing steps can be used either as an additional polishing step, or used in place of AEX, provided that process-related impurities can be removed effectively as well. The order of the polishing steps needs some strategic consideration. For some existing processes, CEX is the first polishing step after Protein A purification. However, as the product is usually eluted with a salt, a high level of which is unfavorable for the following AEX step. If the AEX step cannot tolerate such salt concentrations, an additional dilution or ultrafiltration step is needed to prepare the CEX eluate for AEX loading. Thus, increasingly AEX is being used as the first and CEX as the second polishing step.

2 Cation-exchange chromatography of antibodies 2.1 Principles and goal Cation-exchange chromatography is often the first polishing step to be developed, regardless of its position, or order, in the process workflow. Since it is operated in the bind-and-elution

mode, CEX is a critical step for the control of product-related impurities. The impurities, which differ in binding affinity to the resin, can be removed by elution with a moderate salt concentration before eluting the product. Also, high pH or dual pH-salt elution can be tried to optimize selectivity.

2.2 Starting conditions and parameters for optimization A typical procedure for bind-and-elution chromatography processes includes the following steps: (1) resin screening; (2) elution program development; (3) loading condition optimization; and (4) determination of the operating space. Key parameters of CEX process development are listed in Table 3. 2.2.1 Resin screening The first step is resin screening, which includes part of the elution program development as well. There are many commercially available ion-exchange resins to choose from. Weak ion-exchangers, strong ion-exchangers and mixed-mode adsorbents offer different selectivity and can be evaluated for the removal of productrelated impurities (e.g., aggregates or fragments) or any other difficult-to-remove impurities. Ligand coupling density and coupling chemistry will impact the loading capacity for different molecules, and the resin matrix may affect both selectivity and recovery. Bead size and pore structure can impact resolution. For process developers, there are usually a (set of ) platform chromatographic processes in place, but once specific needs for a new product are identified, either different from those of typical products (e.g., different structure, presence of specific impurities), or greater demands for yield or quality, additional resin screening is highly recommended. A list of commercially available ionexchange resins commonly used for antibody purification is provided in Section 4.

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TABLE 3

Typical operating parameters of antibody cation-exchange chromatography.

Parameter

Starting point

Range for optimization

Range for operating space

Loading pH

5.0

5.0 – (pI

Target  0.2

Loading conductivity

5 mS/cm

20–50 mM buffer

5 mS/cm

Loading amount

60 g/L

DBC  80%

As determined by optimization results

Linear flow rate

150–250 cm/h

No optimization needed

Target  50 cm/h

Elution pH

5.0

Same as loading pH of this step or the next step

Target  0.2

Gradient elution program

0%–100% of 1 M NaCl in 20 CV

Switch to stepwise elution

Target  2 mS/cm

0.5)

Note: DBC, dynamic binding capacity; CV, column volume.

Loading conditions will be initially established during resin screening and further optimized once the resin to be used is identified. To ensure stable performance, the loading (which is typically also the eluting) pH should be at least 0.5 pH unit below the isoelectric point (pI) of the target product. Most of the time a theoretically calculated pI from the amino acid sequence is sufficient to start process development. On the other hand, to minimize product aggregation and binding of acidic processrelated impurities, an operating pH 5.0 is recommended. However, once glycosylation or other posttranslation modifications influence the isoelectric point (e.g., sialyation, which is common for Fc fusion proteins), the situation becomes more complicated. Sialyation typically results in a broad pI range of 5–6, with different charge variants corresponding to a different number of sialic acids per molecule. Since loading such molecules at a lower pI is impractical, it is necessary to evaluate whether the molecule will bind to the CEX resin in the normal pH range, or AEX should be used instead.

TABLE 4 Buffer systems used for the ionexchange chromatography of antibodies. Buffer system

pH range

Glycine-HCl

2.2–3.6

Acetate

3.6–5.8

Citrate

3.0–6.6

Phosphate

5.8–8.0

Tris-HCl

7.1–8.9

Commonly used buffer systems for antibody ion-exchange chromatography are listed in Table 4. Once the starting pH is established, the material can be loaded at the recommended capacity of the resin as an initial test. The loading conductivity should usually be 5 mS/cm. A linear NaCl gradient from 0 to 1 M NaCl in 20 CVs is run to elute the protein, and fractions are collected. The fractions are analyzed for protein concentration and impurities, and the optimal resin is selected based on yield and product quality.

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2.2.2 Elution program development

2.2.3 Optimization of loading condition

A linear elution program should have been established as part of the resin screening step. If required, some tuning of the elution pH can be done. The loading pH can be either simultaneously changed with the elution pH or kept unchanged. For the latter case, the resulting program would be a pH/salt dual gradient. A further consideration is to match the loading pH in subsequent chromatography steps, to minimize a loss of separation performance in subsequent operations. Since stepwise elution is typically employed in large-scale manufacturing environments, the gradient elution program should be converted to a stepwise gradient. The salt concentration for the elution of the product is easily determined as it corresponds to the salt concentration at the peak maxima in gradient elution. One or more washing steps with lower salt concentrations (Table 5) may be added prior to elution to remove loosely bound impurities. The product from stepwise elution is analyzed and compared with the results from gradient elution, and fine tuning can be performed if needed.

Since the loading pH may have been optimized simultaneously during the elution program development, the major task is the determination of the loading capacity. The dynamic binding capacity (DBC) of the resin should be determined first and corresponds to the loading amount at 10% breakthrough. A safety factor of 80% is applied to calculate the loading capacity, which is experimentally verified for yield, purity, and impurities. If these results are unacceptable, lower loading amounts are evaluated. 2.2.4 Determination of operating space The operating space is the collection of acceptable ranges for critical process parameters to maintain process performance. The operating space of ion-exchange chromatography can be determined by either single-factor experiments or design-of-experiment (DOE) approaches. A typical set of operating ranges was provided in Table 3.

2.3 Case studies

TABLE 5 Wash buffers used for ion-exchange chromatography (bind-and-elution mode) of antibodies. Wash buffer

Purpose

Equilibration buffer + 0–50 mM NaCl

To remove weak-binding impurities, e.g., fragments

Buffers with pH values higher than equilibration

To remove weak-binding impurities; another wash step is usually needed to bring the pH back to target value

Equilibration buffer + 100–500 mM arginine

To remove hydrophobically bond impurities; more applicable to mixed-mode IEX

E x a m p l e 1: Cat i o n- e x c h ang e chromatography of a mAb The product is a monoclonal antibody with a pI of 8.7. A moderate pH/salt dual elution is employed. The CEX polishing step removes aggregates resulting in a purity of 99% by sizeexclusion chromatography, Tables 6 and 7. Charge heterogeneity remains unchanged. HCP is not removed at this step. Since this is the first step after Protein A chromatography, a Protein A spiking study was also performed, indicating that the CEX process reduced the Protein A level from 1826 to 305 ppm.

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TABLE 6 CEX process parameters for a mAb purification.

Example 2: Mixed-mode catione x cha n g e chr o m a t o g r a p hy o f a b sAb [16]

Parameter

Value

Resin

Capto S

Linear velocity

160 cm/h for load, wash, and elution

Equilibration buffer

50 mM sodium acetate, pH 5.0

Load pH

5.0

Load conductivity

8.5 mS/cm

Load amount

30–55 g/L

Wash 1 buffer

50 mM sodium acetate, pH 5.0

Wash 1 volume

4 CV

Wash 2 buffer

50 mM sodium acetate, pH 5.5

Wash 2 volume

4 CV

Elution buffer

50 mM sodium acetate, 86 mM NaCl, pH 5.5

Eluate collection start

Ascending to 100 mAU/mm

Eluate collection stop

Descending to 400 mAU/mm

Yield

85%

The product is an asymmetric bispecific antibody in IgG format with high levels of product-related impurities like half antibodies, homodimers, and aggregates. Capto MMC ImpRes, a mixed-mode cation-exchange resin, was used for its higher selectivity, Table 8. Variations were made to the washing conditions for the removal of impurities. The half antibody was removed during the washing steps and the homodimer is retained by column and stripped after elution. These steps increase the purity from 73.3% to 99.0% by size-exclusion chromatography with some sacrifice for the yield.

TABLE 8 MMCEX process parameters of a bsAb purification. Parameter

Value

Resin

Capto MMC ImpRes

Linear velocity

180 cm/h

Equilibration buffer

50 mM sodium acetate, pH 5.5

Load pH

5.5

TABLE 7 Impurity clearance after CEX for a mAb purification.

Load conductivity

Not specified

Quality attribute

Before CEX

After CEX

Load amount

20–30 g/L

Wash 1 buffer

50 mM sodium acetate, pH 5.5

Protein concentration (mg/mL)

9.1

4.7

Wash 1 volume

5 CV

Purity (SEC, %)

98.1

99.1

Wash 2 buffer

Purity (CE-SDS NR, %)

97.6

96.9

20 mM phosphate buffer, 21 mM NaCl, pH 7.4

Purity (CE-SDS R, %)

98.1

96.6

Wash 2 volume

5 CV

Charge heterogeneity (main peak %, cIEF)

71.2%

67.3%

Wash 3 buffer

50 mM sodium acetate buffer, 95 mM NaCl, pH 5.5

Host cell protein (ppm)

37

74

Wash 3 volume

5 CV

Host cell DNA (pg/mg)