hand book of affinity chromatography

1. Handbook of Affinity Chromatography Second Edition 2006 by Taylor & Francis Group, LLC

2. CHROMATOGRAPHIC SCIENCE SERIES A Series of Textbooks and Reference Books Editor: JACK CAZES 1. Dynamics of Chromatography: Principles and Theory, J. Calvin Giddings 2. Gas Chromatographic Analysis of Drugs and Pesticides, Benjamin J. Gudzinowicz 3. Principles of Adsorption Chromatography: The Separation of Nonionic Organic Compounds, Lloyd R. Snyder 4. Multicomponent Chromatography: Theory of Interference, Friedrich Helfferich and Gerhard Klein 5. Quantitative Analysis by Gas Chromatography, Josef Novk 6. High-Speed Liquid Chromatography, Peter M. Rajcsanyi and Elisabeth Rajcsanyi 7. Fundamentals of Integrated GC-MS (in three parts), Benjamin J. Gudzinowicz, Michael J. Gudzinowicz, and Horace F. Martin 8. Liquid Chromatography of Polymers and Related Materials, Jack Cazes 9. GLC and HPLC Determination of Therapeutic Agents (in three parts), Part 1 edited by Kiyoshi Tsuji and Walter Morozowich, Parts 2 and 3 edited by Kiyoshi Tsuji 10. Biological/Biomedical Applications of Liquid Chromatography, edited by Gerald L. Hawk 11. Chromatography in Petroleum Analysis, edited by Klaus H. Altgelt and T. H. Gouw 12. Biological/Biomedical Applications of Liquid Chromatography II, edited by Gerald L. Hawk 13. Liquid Chromatography of Polymers and Related Materials II, edited by Jack Cazes and Xavier Delamare 14. Introduction to Analytical Gas Chromatography: History, Principles, and Practice, John A. Perry 15. Applications of Glass Capillary Gas Chromatography, edited by Walter G. Jennings 16. Steroid Analysis by HPLC: Recent Applications, edited by Marie P. Kautsky 17. Thin-Layer Chromatography: Techniques and Applications, Bernard Fried and Joseph Sherma 18. Biological/Biomedical Applications of Liquid Chromatography III, edited by Gerald L. Hawk 19. Liquid Chromatography of Polymers and Related Materials III, edited by Jack Cazes 20. Biological/Biomedical Applications of Liquid Chromatography, edited by Gerald L. Hawk 2006 by Taylor & Francis Group, LLC

3. 21. Chromatographic Separation and Extraction with Foamed Plastics and Rubbers, G. J. Moody and J. D. R. Thomas 22. Analytical Pyrolysis: A Comprehensive Guide, William J. Irwin 23. Liquid Chromatography Detectors, edited by Thomas M. Vickrey 24. High-Performance Liquid Chromatography in Forensic Chemistry, edited by Ira S. Lurie and John D. Wittwer, Jr. 25. Steric Exclusion Liquid Chromatography of Polymers, edited by Josef Janca 26. HPLC Analysis of Biological Compounds: A Laboratory Guide, William S. Hancock and James T. Sparrow 27. Affinity Chromatography: Template Chromatography of Nucleic Acids and Proteins, Herbert Schott 28. HPLC in Nucleic Acid Research: Methods and Applications, edited by Phyllis R. Brown 29. Pyrolysis and GC in Polymer Analysis, edited by S. A. Liebman and E. J. Levy 30. Modern Chromatographic Analysis of the Vitamins, edited by Andr P. De Leenheer, Willy E. Lambert, and Marcel G. M. De Ruyter 31. Ion-Pair Chromatography, edited by Milton T. W. Hearn 32. Therapeutic Drug Monitoring and Toxicology by Liquid Chromatography, edited by Steven H. Y. Wong 33. Affinity Chromatography: Practical and Theoretical Aspects, Peter Mohr and Klaus Pommerening 34. Reaction Detection in Liquid Chromatography, edited by Ira S. Krull 35. Thin-Layer Chromatography: Techniques and Applications, Second Edition, Revised and Expanded, Bernard Fried and Joseph Sherma 36. Quantitative Thin-Layer Chromatography and Its Industrial Applications, edited by Laszlo R. Treiber 37. Ion Chromatography, edited by James G. Tarter 38. Chromatographic Theory and Basic Principles, edited by Jan ke Jnsson 39. Field-Flow Fractionation: Analysis of Macromolecules and Particles, Josef Janca 40. Chromatographic Chiral Separations, edited by Morris Zief and Laura J. Crane 41. Quantitative Analysis by Gas Chromatography, Second Edition, Revised and Expanded, Josef Novk 42. Flow Perturbation Gas Chromatography, N. A. Katsanos 43. Ion-Exchange Chromatography of Proteins, Shuichi Yamamoto, Kazuhiro Naka-nishi, and Ryuichi Matsuno 44. Countercurrent Chromatography: Theory and Practice, edited by N. Bhushan Man-dava and Yoichiro Ito 45. Microbore Column Chromatography: A Unified Approach to Chromatography, edited by Frank J. Yang 46. Preparative-Scale Chromatography, edited by Eli Grushka 47. Packings and Stationary Phases in Chromatographic Techniques, edited by Klaus K. Unger 48. Detection-Oriented Derivatization Techniques in Liquid Chromatography, edited by Henk Lingeman and Willy J. M. Underberg 49. Chromatographic Analysis of Pharmaceuticals, edited by John A. Adamovics 2006 by Taylor & Francis Group, LLC

4. 50. Multidimensional Chromatography: Techniques and Applications, edited by Hernan Cortes 51. HPLC of Biological Macromolecules: Methods and Applications, edited by Karen M. Gooding and Fred E. Regnier 52. Modern Thin-Layer Chromatography, edited by Nelu Grinberg 53. Chromatographic Analysis of Alkaloids, Milan Popl, Jan Fhnrich, and Vlastimil Tatar 54. HPLC in Clinical Chemistry, I. N. Papadoyannis 55. Handbook of Thin-Layer Chromatography, edited by Joseph Sherma and Bernard Fried 56. GasLiquidSolid Chromatography, V. G. Berezkin 57. Complexation Chromatography, edited by D. Cagniant 58. Liquid ChromatographyMass Spectrometry, W. M. A. Niessen and Jan van der Greef 59. Trace Analysis with Microcolumn Liquid Chromatography, Milos KrejcI 60. Modern Chromatographic Analysis of Vitamins: Second Edition, edited by Andr P. De Leenheer, Willy E. Lambert, and Hans J. Nelis 61. Preparative and Production Scale Chromatography, edited by G. Ganetsos and P. E. Barker 62. Diode Array Detection in HPLC, edited by Ludwig Huber and Stephan A. George 63. Handbook of Affinity Chromatography, edited by Toni Kline 64. Capillary Electrophoresis Technology, edited by Norberto A. Guzman 65. Lipid Chromatographic Analysis, edited by Takayuki Shibamoto 66. Thin-Layer Chromatography: Techniques and Applications: Third Edition, Revised and Expanded, Bernard Fried and Joseph Sherma 67. Liquid Chromatography for the Analyst, Raymond P. W. Scott 68. Centrifugal Partition Chromatography, edited by Alain P. Foucault 69. Handbook of Size Exclusion Chromatography, edited by Chi-San Wu 70. Techniques and Practice of Chromatography, Raymond P. W. Scott 71. Handbook of Thin-Layer Chromatography: Second Edition, Revised and Expanded, edited by Joseph Sherma and Bernard Fried 72. Liquid Chromatography of Oligomers, Constantin V. Uglea 73. Chromatographic Detectors: Design, Function, and Operation, Raymond P. W. Scott 74. Chromatographic Analysis of Pharmaceuticals: Second Edition, Revised and Expanded, edited by John A. Adamovics 75. Supercritical Fluid Chromatography with Packed Columns: Techniques and Applications, edited by Klaus Anton and Claire Berger 76. Introduction to Analytical Gas Chromatography: Second Edition, Revised and Expanded, Raymond P. W. Scott 77. Chromatographic Analysis of Environmental and Food Toxicants, edited by Takayuki Shibamoto 78. Handbook of HPLC, edited by Elena Katz, Roy Eksteen, Peter Schoenmakers, and Neil Miller 79. Liquid ChromatographyMass Spectrometry: Second Edition, Revised and Expanded, Wilfried Niessen 80. Capillary Electrophoresis of Proteins, Tim Wehr, Roberto Rodrguez-Daz, and Mingde Zhu 2006 by Taylor & Francis Group, LLC

5. 81. Thin-Layer Chromatography: Fourth Edition, Revised and Expanded, Bernard Fried and Joseph Sherma 82. Countercurrent Chromatography, edited by Jean-Michel Menet and Didier Thibaut 83. Micellar Liquid Chromatography, Alain Berthod and Celia Garca-Alvarez-Coque 84. Modern Chromatographic Analysis of Vitamins: Third Edition, Revised and Expanded, edited by Andr P. De Leenheer, Willy E. Lambert, and Jan F. Van Bocxlaer 85. Quantitative Chromatographic Analysis, Thomas E. Beesley, Benjamin Buglio, and Raymond P. W. Scott 86. Current Practice of Gas ChromatographyMass Spectrometry, edited by W. M. A. Niessen 87. HPLC of Biological Macromolecules: Second Edition, Revised and Expanded, edited by Karen M. Gooding and Fred E. Regnier 88. Scale-Up and Optimization in Preparative Chromatography: Principles and Bio-pharmaceutical Applications, edited by Anurag S. Rathore and Ajoy Velayudhan 89. Handbook of Thin-Layer Chromatography: Third Edition, Revised and Expanded, edited by Joseph Sherma and Bernard Fried 90. Chiral Separations by Liquid Chromatography and Related Technologies, Hassan Y. Aboul-Enein and Imran Ali 91. Handbook of Size Exclusion Chromatography and Related Techniques, Second Edition, edited by Chi-San Wu 92. Handbook of Affinity Chromatography, Second Edition, edited by David S. Hage 2006 by Taylor & Francis Group, LLC

6. edited by David S. Hage University of Nebraska Lincoln, Nebraska, U.S.A. Handbook of Affinity Chromatography Second Edition Boca Raton London New York Singapore A CRC title, part of the Taylor & Francis imprint, a member of the Taylor & Francis Group, the academic division of T&F Informa plc. 2006 by Taylor & Francis Group, LLC

7. Published in 2006 by CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 2006 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8247-4057-2 (Hardcover) International Standard Book Number-13: 978-0-8247-4057-3 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Catalog record is available from the Library of Congress Visit the Taylor & Francis Web site at and the CRC Press Web site atTaylor & Francis Group is the Academic Division of T&F Informa plc. 2006 by Taylor & Francis Group, LLC For permission to photocopy or use material electronically from this work, please access www.copyright.com http://www.taylorandfrancis.com http://www.crcpress.com organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA

8. ix Preface Welcome to the Handbook of Afnity Chromatography, Second Edition. The purpose of this book is to guide scientists, students, and laboratory workers in the theory, use, and applications of afnity chromatography. Since its original use almost 100 years ago, afnity chromatography has become an important tool in a large variety of areas. This is illustrated in this book through examples and topics that range from the elds of biology, biotechnology, biochemistry, and molecular biology to analytical chemistry, pharmaceutical science, environmental science, and clinical chemistry. overview of afnity chromatography and discusses important factors to consider in the development of afnity methods, including the choice of support material, immobilization reviews of common afnity methods, such as bioafnity chromatography, immunoafnity chromatography, DNA afnity chromatography, boronate afnity chromatography, dye- ligand and biomimetic afnity chromatography, and immobilized metal-ion afnity chromatography. respectively) explore the preparative, analytical, and biophysical applications of afnity methods in areas such as biochemistry, molecular biology, biotechnology, pharmaceutical capillary electrophoresis, mass spectrometry, microanalytical systems, chromatographic immunoassays, and molecularly imprinted polymers. This book is the result of a collaborative effort involving 48 scientists and students from 23 laboratories and organizations located throughout the world. I would like to thank each of these individuals for their contributions to this project. I would also like to thank my wife Jill and my sons Ben and Brian for their assistance in the preparation of this text. I hope that all who use this handbook will nd it a valuable addition to their work in afnity chromatography or related areas. David S. Hage 2006 by Taylor & Francis Group, LLC This handbook is divided into six sections. Section I (Chapters 1 to 4) provides an method, and application or elution conditions. Section II (Chapters 5 to 10) provides detailed Sections III, IV, and V of this book (Chapters 11 to 16, 17 to 21, and 22 to 25, analysis, proteomics, clinical testing, and environmental analysis. Section VI (Chapters 26 to 30) considers recent developments in this eld, including the use of afnity ligands in

9. xi Contents SECTION I Introduction and Basic Concepts Chapter 1 An Introduction to Afnity Chromatography .............................................. 3 David S. Hage and Peggy F. Ruhn Chapter 2 Support Materials for Afnity Chromatography........................................ 15 Chapter 3 Immobilization Methods for Afnity Chromatography............................................................................ 35 Chapter 4 Application and Elution in Afnity Chromatography ............................... 79 SECTION II General Afnity Ligands and Methods Chapter 5 Bioafnity Chromatography..................................................................... 101 Chapter 6 Immunoafnity Chromatography ............................................................. 127 Chapter 7 DNA Afnity Chromatography ................................................................ 173 Chapter 8 Boronate Afnity Chromatography.......................................................... 215 2006 by Taylor & Francis Group, LLC Per-Erik Gustavsson and Per-Olof Larsson Hee Seung Kim and David S. Hage David S. Hage, Hai Xuan, and Mary Anne Nelson David S. Hage, Min Bian, Raychelle Burks, Elizabeth Karle, Corey Ohnmacht, and Chunling Wa David S. Hage and Terry M. Phillips Robert A. Moxley, Shilpa Oak, Himanshu Gadgil, and Harry W. Jarrett Xiao-Chuan Liu and William H. Scouten

10. xii Contents Chapter 9 Dye-Ligand and Biomimetic Afnity Chromatography .......................... 231 Chapter 10 Immobilized Metal-Ion Afnity Chromatography................................. 257 SECTION III Preparative Applications Chapter 11 General Considerations in Preparative Afnity Chromatography......... 287 Chapter 12 Afnity Chromatography of Enzymes ................................................... 313 Chapter 13 Isolation of Recombinant Proteins by Afnity Chromatography................................................................... 347 Chapter 14 Afnity Chromatography in Antibody and Antigen Purication......................................................................... 367 Chapter 15 Afnity Chromatography of Regulatory and Signal-Transducing Proteins............................................................ 399 Chapter 16 Receptor-Afnity Chromatography........................................................ 435 SECTION IV Analytical and Semipreparative Applications Chapter 17 Afnity Methods in Clinical and Pharmaceutical Analysis................... 461 Chapter 18 Afnity Chromatography in Biotechnology........................................... 487 Chapter 19 Environmental Analysis by Afnity Chromatography........................... 517 Chapter 20 Afnity Chromatography in Molecular Biology ................................... 547 2006 by Taylor & Francis Group, LLC N.E. Labrou, K. Mazitsos, and Y.D. Clonis Daniela Todorova and Mookambeswaran A. Vijayalakshmi Anuradha Subramanian Felix Friedberg and Allen R. Rhoads Anuradha Subramanian Terry M. Phillips Allen R. Rhoads and Felix Friedberg Pascal Bailon, Michele Nachman-Clewner, Cheryl L. Spence, and George K. Ehrlich Carrie A.C. Wolfe, William Clarke, and David S. Hage Neil Jordan and Ira S. Krull Mary Anne Nelson and David S. Hage Luis A. Jurado, Shilpa Oak, Himanshu Gadgil, Robert A. Moxley, and Harry W. Jarrett

11. Contents xiii Chapter 21 Afnity-Based Chiral Stationary Phases................................................ 571 SECTION V Biophysical Applications Chapter 22 Quantitative Afnity Chromatography: Practical Aspects..................... 595 Chapter 23 Quantitative Afnity Chromatography: Recent Theoretical Developments.......................................................... 629 Chapter 24 Chromatographic Studies of Molecular Recognition and Solute Binding to Enzymes and Plasma Proteins................................................................................ 663 Chapter 25 Afnity-Based Optical Biosensors......................................................... 685 SECTION VI Recent Developments Chapter 26 Afnity Ligands in Capillary Electrophoresis ....................................... 699 Chapter 27 Afnity Mass Spectrometry.................................................................... 737 Chapter 28 Microanalytical Methods Based on Afnity Chromatography.............. 763 Chapter 29 Chromatographic Immunoassays............................................................ 789 Chapter 30 Molecularly Imprinted Polymers: Articial Receptors for Afnity Separations ......................................................... 837 2006 by Taylor & Francis Group, LLC Sharvil Patel, Irving W. Wainer, and W. John Lough David S. Hage and Jianzhong Chen Donald J. Winzor Sharvil Patel, Irving W. Wainer, and W. John Lough Sheree D. Long and David G. Myszka Niels H. H. Heegaard and Christian Schou Chad J. Briscoe, William Clarke, and David S. Hage Terry M. Phillips Annette C. Moser and David S. Hage Karsten Haupt

12. xv Editor David S. Hage is a professor of analytical and bioanalytical chemistry at the University of Nebraska. He received his B.S. in chemistry and biology from the University of WisconsinLa Crosse in 1983 and a Ph.D. in analytical chemistry from Iowa State Uni- versity in 1987. After nishing a postdoctoral position at the Mayo Clinic in 1989, he joined the faculty at the University of Nebraska. His interests include the study of biological interactions by afnity methods and analytical applications of afnity chromatography for biological and environmental agents. He is the author of over 120 research articles and reviews on the topic of afnity chromatography and received the 1995 Young Investigator Award from the American Association for Clinical Chemistry for his work. He has pre- sented numerous seminars and workshops on afnity methods and is on the editorial boards of several journals in the eld of chemical separation and analysis. 2006 by Taylor & Francis Group, LLC

13. xvii Contributors Pascal Bailon Hoffmann-LaRoche Inc. Nutley, New Jersey Min Bian Department of Chemistry University of Nebraska Lincoln, Nebraska Chad J. Briscoe MDS Pharma Services Lincoln, Nebraska Raychelle Burks Department of Chemistry University of Nebraska Lincoln, Nebraska Jianzhong Chen Department of Chemistry University of Nebraska Lincoln, Nebraska William Clarke Department of Pathology Johns Hopkins School of Medicine Baltimore, Maryland Y. D. Clonis Laboratory of Enzyme Technology Department of Agricultural Biotechnology Agricultural University of Athens Athens, Greece George K. Ehrlich Hoffmann-LaRoche Inc. Nutley, New Jersey Felix Friedberg Department of Biochemistry and Molecular Biology College of Medicine Howard University Washington, DC Himanshu Gadgil Department of Biochemistry University of Tennessee Health Science Center Memphis, Tennessee Per-Erik Gustavsson Department of Pure and Applied Biochemistry Center for Chemistry and Chemical Engineering University of Lund Lund, Sweden David S. Hage Department of Chemistry University of Nebraska Lincoln, Nebraska 2006 by Taylor & Francis Group, LLC

14. xviii Contributors Karsten Haupt Compigne University of Technology Centre de Recherches de Royallieu Compigne, France Niels Heegaard Department of Autoimmunology Statens Serum Institute Copenhagen, Denmark Harry W. Jarrett Department of Biochemistry University of Tennessee Health Science Center Memphis, Tennessee Neil Jordan Department of Chemistry and Chemical Biology Northeastern University Boston, Massachusetts Luis A. Jurado Department of Biochemistry University of Tennessee Health Science Center Memphis, Tennessee Elizabeth Karle Department of Chemistry University of Nebraska Lincoln, Nebraska Hee Seung Kim Department of Chemistry University of Nebraska Lincoln, Nebraska Ira S. Krull Department of Chemistry and Chemical Biology Northeastern University Boston, Massachusetts N. E. Labrou Laboratory of Enzyme Technology Department of Agricultural Biotechnology Agricultural University of Athens Athens, Greece Per-Olof Larsson Department of Pure and Applied Biochemistry Center for Chemistry and Chemical Engineering University of Lund Lund, Sweden Xiao-Chuan Liu Department of Chemistry California State Polytechnic University Pomona, California Sheree D. Long Biacore Inc. Piscataway, New Jersey W. John Lough Institute of Pharmacy and Chemistry University of Sunderland Sunderland, United Kingdom K. Mazitsos Laboratory of Enzyme Technology Department of Agricultural Biotechnology Agricultural University of Athens Athens, Greece Annette C. Moser Department of Chemistry University of Nebraska Lincoln, Nebraska Robert A. Moxley Department of Biochemistry University of Tennessee Health Science Center Memphis, Tennessee David G. Myszka Center for Biomolecular Interaction Analysis University of Utah Salt Lake City, Utah Michele Nachman-Clewner Hoffmann-LaRoche Inc. Nutley, New Jersey 2006 by Taylor & Francis Group, LLC

15. Contributors xix Mary Anne Nelson Department of Chemistry University of Nebraska Lincoln, Nebraska Shilpa Oak Department of Biochemistry University of Tennessee Health Science Center Memphis, Tennessee Corey Ohnmacht Department of Chemistry University of Nebraska Lincoln, Nebraska Sharvil Patel Bioanalytical and Drug Discovery Unit National Institute on Aging National Institutes of Health Baltimore, Maryland Terry M. Phillips Ultramicro Analytical Immunochemistry Resource Division of Bioengineering and Physical Sciences Ofce of Research Services National Institutes of Health Bethesda, Maryland Allen R. Rhoads Department of Biochemistry and Molecular Biology College of Medicine Howard University Washington, DC Peggy F. Ruhn MDS Pharma Services Lincoln, Nebraska William H. Scouten College of Sciences University of Texas at San Antonio San Antonio, Texas Christian Schou Department of Autoimmunology Statens Serum Institute Copenhagen, Denmark Cheryl L. Spence Hoffmann-LaRoche Inc. Nutley, New Jersey Anuradha Subramanian Department of Chemical Engineering University of Nebraska Lincoln, Nebraska Daniela Todorova Molecular Interaction & Separation Technology Labs Universit de Technologie de Compigne Compigne, France Mookambeswaran A. Vijayalakshmi Molecular Interaction & Separation Technology Labs Universit de Technologie de Compigne Compigne, France Chunling Wa Department of Chemistry University of Nebraska Lincoln, Nebraska Irving W. Wainer Bioanalytical and Drug Discovery Unit National Institute on Aging National Institutes of Health Baltimore, Maryland Donald J. Winzor Department of Biochemistry School of Molecular and Microbial Sciences University of Queensland Brisbane, Queensland, Australia Carrie A. C. Wolfe Division of Science and Mathematics Union College Lincoln, Nebraska Hai Xuan Department of Chemistry University of Nebraska Lincoln, Nebraska 2006 by Taylor & Francis Group, LLC

16. Section I Introduction and Basic Concepts 2006 by Taylor & Francis Group, LLC

17. 3 1 An Introduction to Afnity Chromatography David S. Hage Department of Chemistry, University of Nebraska, Lincoln, NE Peggy F. Ruhn MDS Pharma Services, Lincoln, NE CONTENTS 1.1 Introduction ............................................................................................................... 3 1.2 History of Afnity Chromatography......................................................................... 5 1.2.1 Origins of Afnity Chromatography............................................................. 5 1.2.2 Early Immobilization Methods ..................................................................... 6 1.2.3 Modern Era of Afnity Chromatography ..................................................... 7 1.3 Overview of Handbook........................................................................................... 11 References......................................................................................................................... 11 1.1 INTRODUCTION Chemical separation is an essential component of modern research and is widely used to process complex samples. Examples range from the trace analysis of a drug or hormone in blood to the large-scale isolation of a recombinant protein. The method of liquid chromatography has become particularly popular for these separations because of its ability to work with a wide range of substances. When combined with appropriate support materials, this technique can be used in either high-performance separations for chemical detection and measurement or in systems designed to purify a desired product. The wide range of stationary phases and mobile phases that can be employed in liquid chromatography also makes this method quite exible in terms of the types of chemical or physical properties that can be used as the basis for these separations. One of the most versatile forms of liquid chromatography is the technique known as afnity chromatography, which can generally be dened as a liquid chromatographic 2006 by Taylor & Francis Group, LLC

18. 4 Hage and Ruhn technique that uses a specic binding agent for the purication or analysis of sample components [16]. This technique makes use of the selective and reversible interactions that occur in many biological systems, such as the binding of an enzyme with a substrate or an antibody with an antigen. These interactions are used in afnity chromatography by immobilizing one of a pair of interacting molecules onto a solid support and placing it into a column. The immobilized molecule is referred to as the afnity ligand. This makes up the stationary phase of the afnity column. Figure 1.1 shows a typical scheme used to perform afnity chromatography. In this approach, a sample containing the compound of interest is injected onto the afnity Figure 1.1 Typical separation scheme for afnity chromatography. (Reproduced with permission from Hage, D.S., in Handbook of HPLC, Katz, E., Eksteen, R., Shoenmakers, P., and Miller, N., Eds., Marcel Dekker, New York, 1998, pp. 483498.) Response Apply Elute RegenerateApply Elute (a) (b) Time (or volume) Regenerate Wash 2006 by Taylor & Francis Group, LLC

19. An Introduction to Affinity Chromatography 5 column in the presence of a mobile phase that has the right pH, ionic strength, and solvent composition for solute-ligand binding. This solvent, which represents the weak mobile phase of an afnity column, is referred to as the application buffer. As the sample passes through the column under these conditions, compounds that are complementary to the afnity ligand will bind. However, due to the high selectivity of this interaction, other solutes in the sample will tend to wash or elute from the column as a nonretained peak. After all nonretained components have been washed from the column, the retained solutes are then eluted by applying a solvent that displaces them from the column or that promotes dissociation of the solute-ligand complex. This solvent, which represents the strong mobile phase for the column, is known as the elution buffer. As the solutes of interest elute from the column, they are either quantitated directly or collected for later use. The application buffer is then reapplied to the system and the column is allowed to regenerate prior to the next sample injection. Due to the strong and selective binding that characterizes many afnity ligands, solutes that are quantitated or puried by these ligands can often be separated with little or no interference from other sample components. In many cases the solute of interest can be isolated in only one or two steps, with purication yields of 100-fold to several thousand- fold being common [26]. In work with hormone receptors, purication yields approaching one million-fold have even been reported with afnity-based separations [5]. The wide range of ligands available for afnity chromatography makes this method a valuable tool for the purication and analysis of compounds present in complex samples. Areas in which afnity chromatography has been used include biochemistry, pharmaceutical science, clinical chemistry, and environmental testing. This book will examine the variety and types of afnity ligands used in these areas and the main factors to consider in the development of such a method. Several specic applications will also be considered, including the use of afnity chromatography in small- or large-scale purication, analyte detection, and the characterization of biological interactions. Finally, a number of new developments in this eld and in related areas of work will be discussed. 1.2 HISTORY OF AFFINITY CHROMATOGRAPHY 1.2.1 Origins of Afnity Chromatography Although some consider afnity chromatography to be a relatively new method, it is actually one of the oldest forms of liquid chromatography. For instance, the earliest use of this method was just seven years after Michael Tswett reported the rst known use of column liquid chromatography [7]. This occurred in 1910 when Emil Starkenstein examined the binding of insoluble starch to the enzyme -amylase [8]. This is also the rst known case in which liquid chromatography was used for a separation involving a protein. The original studies with afnity chromatography all made use of insoluble materials that acted as both the stationary phase and support material. This is not surprising, since this is the simplest form of afnity separation. For instance, the insoluble starch used by Starkenstein acted as both a support material and as a substrate for amylase, thus leading to this enzymes binding and retention. Similar work with starch and amylase was conducted in the 1920s through 1940s by other investigators [912], with a 300-fold purication being obtained in one of these studies [12]. Other examples include the use of polygalacturonase as a support and ligand for the adsorption of alginic acid [13], the purication of pepsin through the use of edestin, a crystalline protein [14], and the isolation of porcine elastase with powdered elastin [15]. 2006 by Taylor & Francis Group, LLC

20. 6 Hage and Ruhn As this suggests, much of the earliest work with afnity supports involved its use in the purication of enzymes. But research was also being conducted at this time in the selective purication of antibodies with biological ligands. This arose from the work by Landsteiner, who showed in 1920 that antibodies can recognize and bind substances with a specic structure, referred to as antigens [16]. This type of binding plus the ability of polyclonal antibodies to form insoluble complexes with antigens led to the growth in the 1930s of immunoprecipitation as an important technique for antibody purication [1719]. For instance, this approach was used by Kirk and Sumner to isolate antibodies against urease and to demonstrate that these antibodies were proteins [17]. This approach was similar to the work being performed in the purication of enzymes in that a ligand (i.e., the antigen) was used to create a specic biological interaction with the target of interest (the antibodies). However, rather than having the ligand be the same as the solid used for this isolation, this solid was formed as a result of the binding process. 1.2.2 Early Immobilization Methods Although the use of insoluble ligands and immunoprecipitation proved the potential for biological interactions as a means for chemical isolation, this approach was still limited to solutes for which an appropriate ligand and/or support was available. However, this began to change in the 1940s and 1950s as synthetic techniques became available for placing a broader range of ligands onto insoluble materials. These efforts began by employing solids that contained a noncovalently adsorbed layer of ligand. For instance, in 1935 DAlessandro and Soa used antigens coated on kaolin and charcoal for the isolation of antibodies associated with syphilis and tuberculosis [20]. A similar type of support was used by Meyer and Pic in 1936 [21]. However, it was soon realized that a more stable system would be obtained by chemically bonding the ligand to the support. This was rst used by Landsteiner and van der Scheer in 1936, when they adapted a diazo-coupling technique used to prepare hapten conjugates [22]. In this case, they attached a number of haptens to a solid material based on chicken erythrocyte stroma, with this material then being used for the isolation of antibodies for these haptens (see Figure 1.2). Work with other, more durable support materials also began to appear.A key development in this area occurred in 1951, when Campbell and coworkers used an activated form Figure 1.2 An early example of the use of immobilized antigens for antibody purication. These were used by Landsteiner and van der Scheer in 1936 for performing cross-reactivity studies of immune sera. (Reproduced with permission from Landsteiner, K. and van der Scheer, J., J. Exp. Med., 63, 325339, 1936.) 1 cc of suberanilic acid immune serum absorbed with azostromata made from p-Aminoadipanilic acid p-Aminosebacanilic acid Unabsorbed immune serum p-Aminoadipanilic acid p-Aminosuberanilic acid p-Aminosebacanilic acid Azoproteins made from casein and 0 0 + + +++ +++ ++ +++ +++ ++++ ++++ ++++ + ++ 0 0 +++ +++ 2006 by Taylor & Francis Group, LLC

21. An Introduction to Affinity Chromatography 7 of cellulose (p-aminobenzylcellulose) for immobilizing the protein serum albumin. This material was then used to isolate antialbumin antibodies from rabbit serum [23]. Similar work appeared by Lerman in the preparation of immobilized hapten supports [24] and in 1953 through the use of a covalently immobilized ligand for purifying the enzyme mush- room tyrosinase (see Figure 1.3) [25]. Other studies in immunopurication later began to appear with ligands attached to such supports as polyaminostyrene [26] and glass beads [27]. By 1966, several reviews on such work had appeared [2834]. After the report by Lerman [25], the use of immobilized ligands for enzyme isolation received only limited attention for some time, probably due to the rise of ion exchange with cellulose in the 1950s and 1960s as a separation tool in enzymology. However, this began to change in the mid-1960s, when Arsenis and McCormick began to use cellulose-based afnity supports for the isolation of avokinase and avin mononucleotide-dependent enzymes [35, 36]. 1.2.3 Modern Era of Afnity Chromatography The next major development came about in the late 1960s. There were three things that led to this event. The rst was the creation of beaded agarose supports by Hjerten in the mid-1960s [37]. This provided a more efcient and exible support than cellulose for use with biopolymers in liquid chromatography. The second development was the discovery of the cyanogen bromide immobilization method. This was reported in 1967 by Axen, Porath, and Ernback, providing a more convenient and general approach that could be used to attach proteins and peptides to polysaccharides [38]. The third development was a 1968 report by Cuatrecasas, Wilchek, and Annsen in which agarose and the cyanogen bromide method were used together to create immobilized nuclease inhibitor columns [39]. These columns were then used for purifying the enzymes staphylococcal nuclease, -chymotrypsin, Figure 1.3 Early examples of immobilized inhibitors that were used by Lerman in 1953 for the isolation of tyrosinase. (Reproduced with permission from Lerman, L.S., Proc. Natl. Acad. Sci. U.S.A., 39, 232236, 1953.) Cellulose O NN NN OH OH Cellulose O COOHNN NN OH COOH NN OH AsO3H2 Cellulose O NN OH HO OH Cellulose O NN OH Cellulose O NN OH NN CH2 CH3 NNCellulose O CH2 NNCellulose O CH2 2006 by Taylor & Francis Group, LLC

22. 8 Hage and Ruhn and carboxypeptidase A. An example of such a separation is shown in Figure 1.4. This was the same report in which the term afnity chromatography was rst used to describe this separation technique [39]. The techniques and applications that appeared in this last report led to a rapid increase in interest in the use of afnity ligands in liquid chromatography. This is illustrated and 2001 that have contained the phrase afnity chromatography. The number of such reports has grown from four in 1968 to approximately 1000 per year over most of the last decade. An even greater number of reports (i.e., roughly three times the number shown in Figure 1.5) have included the use of related phrases like immunoafnity chromatography. These results clearly demonstrate the widespread use of afnity chromatography and the impact it has had on modern separations. Following the reintroduction of afnity chromatography in 1968, a variety of ligands and applications for this method began to appear. For instance, the next six years saw the introduction of such methods as DNA-cellulose afnity chromatography [40], boronate afnity chromatography [41], dye-ligand afnity chromatography [42], and immobilized metal-ion afnity chromatography [43]. The ligands that are used in these techniques and other ligands and approaches that are now commonly used in afnity chromatography. Figure 1.4 One of the rst examples of modern afnity chromatography (Reproduced with permission from Cuatrecasas, P.,Wilchek, M., andAnnsen, C.B., Proc. Natl. Acad. Sci. U.S.A., 68, 636643, 1968.) Absorbancy,280m 0.2 0.2 2 4 6 8 10 12 14 16 1.0 0.6 0.2 0.6 0.6 Euent, ML Carboxypeptidase A (Unsubstituted Sepharose) Carboxypeptidase A (L-Tyrosyl-D-Tryptophan Sepharose) Carboxypeptidase B (L-Tyrosyl-D-Tryptophan Sepharose) 2006 by Taylor & Francis Group, LLC the types of targets they retain are summarized in Table 1.1. This same table also shows by Figure 1.5, which shows the number of publications that have appeared between 1968

23. AnIntroductiontoAfnityChromatography9 Figure 1.5 Number of articles per year that appeared from 1968 to 2001 and contained the phrase afnity chromatography. A total of 26,132 articles were identied in this search, which was performed using CAPLUS and MEDLINE. The inclusion of other phrases, such as afnity chromatographic, immunoafnity chromatography, and immunoafnity chromatographic led to the identication of 75,742 articles from this same period of time. NumberofArticlesPublished 1200 1000 800 600 400 200 0 1968 1970 1972 1974 1976 1978 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 Publication Year 2006 by Taylor & Francis Group, LLC

24. 10HageandRuhn Table 1.1 Common Ligands Used in Afnity Chromatography Method Ligand Retained Solutes Immunoafnity chromatography Antibodies Antigens (e.g., drugs, hormones, peptides, proteins, viruses, cell components) Antigens Antibodies against the antigens Afnity chromatography of enzymes Inhibitors, substrates, cofactors, coenzymes, and miscellaneous ligands Enzymes Lectin afnity chromatography Lectins Sugars, glycoproteins, and glycolipids Protein A/protein G afnity chromatography Protein A, protein G, and related ligands Antibodies and antibody fragments DNA afnity chromatography DNA or RNA DNA/RNA-binding proteins, complementary nucleotides Boronate afnity chromatography Boronates Carbohydrates, nucleosides, nucleotides, nucleic acids, glycoproteins, and catechols Dye-ligand afnity chromatography Synthetic dyes Proteins and enzymes Biomimetic afnity chromatography Ligands generated using combinatorial chemistry or from peptide, phage display, ribosome display, or aptamer libraries Various targets, including proteins and enzymes Immobilized metal-ion afnity chromatography Metal-ion chelates Metal-binding amino acids, peptides, proteins, and nucleotides 2006 by Taylor & Francis Group, LLC

25. An Introduction to Affinity Chromatography 11 1.3 OVERVIEW OF HANDBOOK This book is designed to introduce the reader to the topic of afnity chromatography and to the most common methods that are employed in this technique. The rst section of this handbook deals with the basic components of an afnity chromatographic system. This followed by a review of immobilization methods for attaching ligands to such supports under the topics of bioafnity, immunoafnity, and DNA afnity chromatography. Examples dyes and biomimetic compounds, and immobilized metal-ion chelates, respectively. isolation of targets. This was the original application of afnity chromatography and remains one of the largest uses for this technique. In this section, a review of general items to consider in the design of preparative chromatographic systems is provided in Chapter 11. ligands in the isolation of enzymes, recombinant proteins, antibodies and antigens, regulatory or signal-transducing proteins, and targets for receptors. Along with its traditional use in preparative work, afnity chromatography has also looks at some specic areas in which this method continues to play a signicant role in research and analysis, including clinical testing and pharmaceutical analysis, biotechnology, environmental analysis, molecular biology, and chiral separations, respectively. Another growing eld has been the use of afnity columns to characterize and study biological interactions. This approach is sometimes referred to as quantitative afnity chro- aspects of the experiments involved in such studies are considered (Chapter 22). An in-depth afnity-based biosensors for such work is considered (Chapter 25). to afnity chromatography or are special applications of this technique. The discussion in this nal section includes the use of afnity ligands in such areas as capillary electro- systems based on afnity chromatography and the use of afnity columns in chromatographic- ularly imprinted polymers. REFERENCES 1. Ettre, L.S., Nomenclature for chromatography, Pure Appl. Chem., 65, 819872, 1993. 2. Hage, D.S.,Afnity chromatography, inHandbook of HPLC, Katz, E., Eksteen, R., Shoenmakers, P., and Miller, N., Eds., Marcel Dekker, New York, 1998, pp. 483498. 3. Turkova, J., Afnity Chromatography, Elsevier, Amsterdam, 1978. 4. Scouten, W.H., Afnity Chromatography: Bioselective Adsorption on Inert Matrices, Wiley, New York, 1981. 2006 by Taylor & Francis Group, LLC begins with a discussion of support materials that are used in this method (Chapter 2), (Chapter 3). The selection of application and elution conditions are then examined in Chapter 4. Common ligands employed in afnity chromatography are examined in Section II More-detailed presentations are then made in Chapters 12 to 16 regarding the use of afnity been playing an increasing role in the analysis of chemicals. Section IV (Chapters 17 to 21) Section III (Chapters 11 to 16) discusses the use of afnity chromatography for the of synthetic or nonbiological ligands are then given in Chapters 8 to 10, including boronates, (Chapters 5 to 10). A variety of biological ligands are rst considered in Chapters 5 to 7 matography. This area is examined in Section V (Chapters 22 to 25). First, various practical look at the theory behind this method is then given (Chapter 23), and its use in the study of enzymes and plasma proteins is reviewed (Chapter 24). Finally, the use and development of Section VI (Chapters 26 to 30) presents several new methods that are closely related phoresis (Chapter 26) and mass spectrometry (Chapter 27). The creation of microanalytical based immunoassays are also described in this section (Chapters 28 and 29). The last chapter (Chapter 30) explores a new approach for creating afnity columns using molec-

26. 12 Hage and Ruhn 5. Parikh, I. and Cuatrecasas, P., Afnity chromatography, Chem. Eng. News, 63, 1732, 1985. 6. Walters, R.R., Afnity chromatography, Anal. Chem., 57, 1099A1114A, 1985. 7. Tswett, M., The chemistry of chlorophyll, phylloxanthin, phyllocyanin, and chlorophyllane, Biochem. Zeit., 5, 632, 1907. 8. Starkenstein, E., Ferment action and the inuence upon it of neutral salts, Biochem. Z., 24, 210218, 1910. 9. Ambard, L., Amylase: Its estimation and the mechanism of its action, Bull. Soc. Chim. Biol., 3, 5165, 1921. 10. Holmbergh, O., Adsorption of -amylase from malt by starch, Biochem. Z., 258, 134140, 1933. 11. Tokuoka, Y., Koji amylase, IX: Existence of -amylase, J. Agric. Chem. Soc. Japan, 13, 586594, 1937. 12. Hockenhull, D.J.D. and Herbert, D., The amylase and maltase of Clostridium acetobutylcium, Biochem. J., 39, 102106, 1945. 13. Lineweaver, H., Jang, R., and Jansen, E.F., Specicity and purication of polygalacturonase, Arch. Biochem., 20, 137152, 1949. 14. Northrup, J.H., Crystalline pepsin, VI: Inactivation by - and -rays from radium and by ultraviolet light, J. Gen. Physiol., 17, 359363, 1934. 15. Grant, N.H. and Robbins, K.C., Porcine elastase and proelastase, Arch. Biochem. Biophys., 66, 396403, 1957. 16. Landsteiner, K., Specic serum reactions induced by the addition of substances of known constitution (organic acids), XVI: Antigens and serological specicity, Biochem. Z., 104, 280299, 1920. 17. Kirk, J.S. and Sumner, J.B., The reaction between crystalline urease and antiurease, J. Immunol., 26, 495504, 1934. 18. Marrack, J.R. and Smith, F.C., Quantitative aspects of immunity reactions: The combination of antibodies with simple haptenes, Brit. J. Exp. Pathol., 13, 394402, 1932. 19. Heidelberger, M. and Kabat, E.A., Quantitative studies on antibody purication, II: The dissociation of antibody from pneumococcus-specic precipitates and specically aggluti- nated pneumococci, J. Exp. Med., 67, 181199, 1938. 20. dAlessandro, G. and Soa, F., The adsorption of antibodies from the sera of syphilitics and tuberculosis patients, Z. lmmunitats., 84, 237250, 1935. 21. Meyer, K. and Pic, A., Isolation of antibodies by xation on an adsorbent-antigen system with subsequent regeneration, Ann. Inst. Pasteur, 56, 401412, 1936. 22. Landsteiner, K. and van der Scheer, J., Cross reactions of immune sera to azoproteins, J. Exp. Med., 63, 325339, 1936. 23. Campbell, D.H., Luescher, E., and Lerman, L.S., Immunologic adsorbents, I: Isolation of antibody by means of a cellulose-protein antigen, Proc. Natl. Acad. Sci. U.S.A., 37, 575578, 1951. 24. Lerman, L.S., Antibody chromatography on an immunologically specic adsorbent, Nature, 172, 635636, 1953. 25. Lerman, L.S., A biochemically specic method for enzyme isolation, Proc. Natl. Acad. Sci. U.S.A., 39, 232236, 1953. 26. Manecke, G. and Gillert, K.E., Serologically specic adsorbents, Naturwissenschaften, 42, 212213, 1955. 27. Sutherland, G.B. and Campbell, D.H., The use of antigen-coated glass as a specic adsorbent for antibody, J. Immunol., 80, 294298, 1958. 28. Isliker, H.C., Chemical nature of antibodies, Adv. Prot. Chem., 12, 387463, 1957. 29. Kabat, E.A. and Mayer, M.M., Experimental Immunochemistry, 2nd ed., Charles C Thomas, Springeld, IL, 1961, pp. 781797. 30. Manecke, G., Reactive polymers and their use for the preparation of antibody and enzyme resins, Pure Appl. Chem., 4, 507520, 1962. 31. Sehon, A.H., Physicochemical and immunochemical methods for the isolation and characterization of antibodies, Brit. Med. Bull., 19, 183191, 1963. 2006 by Taylor & Francis Group, LLC

27. An Introduction to Affinity Chromatography 13 32. Weliky, N., Weetall, H.H., Gilden, R.V., and Campbell, D.H., Synthesis and use of some insoluble immunologically specic adsorbents, Immunochemistry, 1, 219229, 1964. 33. Weliky, N. and Weetall, H.H., Chemistry and use of cellulose derivatives for the study of biological systems, Immunochemistry, 2, 293322, 1965. 34. Silman, I.H. and Katchalski, E., Water-insoluble derivatives of enzymes, antigens, and antibodies, Annu. Rev. Biochem., 35, 873908, 1966. 35. Arsenis, C. and McCormick, D.B., Purication of liver avokinase by column chromatography on avine-cellulose compounds, J. Biol. Chem., 239, 30933097, 1964. 36. Arsenis, C. and McCormick, D.B., Purication of avin mononucleotide-dependent enzymes by column chromatography on avin phosphate cellulose compounds, J. Biol. Chem., 241, 330334, 1966. 37. Hjerten, S., The preparation of agarose spheres for chromatography of molecules and particles, Biochem. Biophys. Acta, 79, 393398, 1964. 38. Axen, R., Porath, J., and Ernback, S., Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides, Nature, 214, 13021304, 1967. 39. Cuatrecasas, P., Wilchek, M., and Annsen, C.B., Selective enzyme purication by afnity chromatography, Proc. Natl. Acad. Sci. U.S.A., 68, 636643, 1968. 40. Alberts, B.M., Amodio, F.J., Jenkins, M., Gutmann, E.D., and Ferris, F.L., Studies with DNA-cellulose chromatography, I: DNA-binding proteins from Escherichia coli, Cold Spring Harbor Symp. Quant. Biol., 33, 289305, 1968. 41. Weith, H.L., Wiebers, J.L., and Gilham, P.T., Synthesis of cellulose derivatives containing the dihydroxyboryl group and a study of their capacity to form specic complexes with sugars and nucleic acid components, Biochemistry, 9, 43964401, 1970. 42. Staal, G., Koster, J., Kamp, H., Van Milligen-Boersma, L., and Veeger, C., Human erythrocyte pyruvate kinase, its purication and some properties, Biochem. Biophys. Acta, 227, 8692, 1971. 43. Porath, J., Carlsson, J., Olsson, I., and Belfrage, B., Metal chelate afnity chromatography, a new approach to protein fraction, Nature, 258, 598599, 1975. 2006 by Taylor & Francis Group, LLC

28. 15 2 Support Materials for Afnity Chromatography Per-Erik Gustavsson and Per-Olof Larsson Department of Pure and Applied Biochemistry, Lund University, Lund, Sweden CONTENTS 2.1 Introduction ............................................................................................................. 16 2.2 Properties of Support Materials.............................................................................. 16 2.2.1 Chemical Inertness...................................................................................... 16 2.2.2 Chemical Stability....................................................................................... 20 2.2.3 Mechanical Stability.................................................................................... 21 2.2.4 Pore Size...................................................................................................... 21 2.2.5 Particle Size................................................................................................. 22 2.2.5.1 Zonal Elution Chromatography................................................... 23 2.2.5.2 Adsorption-Desorption Afnity Purication ............................... 24 2.2.5.3 Partial Loading in Adsorption-Desorption Afnity Purication ..................................................................... 24 2.2.5.4 Particulate Contaminants: Large Beads/Expanded Beds ....................................................... 25 2.2.6 Standard Commercially Available Afnity Supports.................................. 25 2.3 Afnity Supports with Special Properties .............................................................. 26 2.3.1 Nonporous Supports.................................................................................... 26 2.3.2 Membranes .................................................................................................. 26 2.3.3 Flow-Through Beads................................................................................... 28 2.3.4 Continuous Beds ......................................................................................... 29 2.3.5 Expanded-Bed Adsorbents.......................................................................... 30 2.4 Summary and Conclusions ..................................................................................... 30 Symbols and Abbreviations.............................................................................................. 31 References......................................................................................................................... 32 2006 by Taylor & Francis Group, LLC

29. 16 Gustavsson and Larsson 2.1 INTRODUCTION A key item in the selection and design of an afnity chromatographic method is the choice of support material. The supports used in afnity chromatography must meet several requirements. Ideally, such a support should be inexpensive and allow solutes to have rapid, unhindered access to the immobilized afnity ligand. In addition, the support should play a completely passive role during the separation while also being able to couple the desired afnity ligand. These are not trivial requirements. For example, quick access of solutes to the ligand will require small support particles. But if these solutes are large biomolecules, the support must also have large pores. For the support to play a passive role, it must be chemically inert toward the chromatographic solvents and solutes, have no ionic or hydrophobic groups, and be resistant toward the mechanical strains exerted during the chromatographic process. And yet, for the support to be used for ligand attachment, it must be able to undergo chemical modication or somehow adsorb the ligand. In reality, no true ideal support exists for afnity chromatography. This is because many of these requirements are in direct conict with each other. As a result, all current afnity supports involve some compromise in these properties and are generally geared toward a particular application. Some extreme examples of this are micrometer-sized nonporous particles, which are optimized for rapid analytical or micropreparative separations, and 0.2-mm expanded-bed particles or 0.4-mm Big Beads, which are optimized for separating crude extracts in the early stages of a purication process. This chapter examines the various properties that should be considered when selecting a support for afnity chromatography. By being familiar with these properties, one can make an informed choice in selecting a support for a given application. 2.2 PROPERTIES OF SUPPORT MATERIALS 2.2.1 Chemical Inertness The rst important property for a successful afnity column is that it should rmly and specically bind the desired solute while leaving all other molecules in the sample or process stream untouched. This requires that the support within the column contain an afnity ligand that is capable of forming a suitably strong complex with the solute of interest. Second, the support material must be inert to other solutes to avoid the simulta- neous binding of nondesired sample components. This requires that the support have a chemical character that is very similar to that of the medium in which it is operating. Since almost all afnity separations occur in aqueous solutions, the support should thus be as hydrophilic as possible. As a rule, the mobile phase used in afnity separations has a low ionic strength. The support should therefore contain as few charges as possible to prevent ionic interactions. Many supports that are available today fulll these requirements. This is because either their basic structure has the desired properties or because they are provided with a hydrophilic coating that gives them such properties. A well-known example is the polysaccharide agarose, which was used in the rst modern application of afnity chromatography [1]. This is sold under several trade names, like Sepharose Fast Flow from Amersham Bio- polymeric chains of the disaccharide agarobiose, which in turn is made up of D-galactose in agarose are clustered together in bundles. These form a porous and hydrophilic network, 2006 by Taylor & Francis Group, LLC sciences or Af-Gel from Bio-Rad Laboratories (see Table 2.1). Agarose consists of and 3,6-anhydro-L-galactose, as shown in Figure 2.1a. The individual polymeric chains

30. SupportMaterialsforAffinityChromatography17 Table 2.1 Examples of Traditional Afnity Supports Trade Name Material Average Particle Diameter (m) Examples of Available Ligands Preactivated Form Available? Manufacturer/Supplier Sepharose HP Sepharose FF Agarose Agarose 34 90 Protein A, heparin, Cibacron Blue Protein A, heparin, Cibacron Blue Yes Yes Amersham Biosciences Amersham Biosciences Mimetic series Agarose 105 Synthetic ligands No Prometic Biosciences/ACL Af-Gel Agarose 150300, 75150 Protein A, heparin, Cibacron Blue Yes Bio-Rad Laboratories Sepharose Big Beads Agarose 200 IMACa No Amersham Biosciences Cellthru Big Beads Agarose 400 Cibacron Blue, heparin, IMACa Yes Sterogene TSK-Gel Polymethacrylate 10 Boronate, IMACa , heparin Yes TosoHaas/Supelco Af-Prep Polymethacrylate 10, 50 Protein A, polymyxin No Bio-Rad Laboratories SigmaChrom AF Polymethacrylate 20 Protein A, IMACa , Cibacron Blue No Supelco Fractogel Polymethacrylate 30, 65 IMACa , heparin Yes Merck ProteinPak Silica 40 No Yes Waters Bakerbond Silica 40 No Yes J.T. Baker Trisacryl Polyacrylamide derivative 60, 200 Cibacron Blue, Basiline Blue No BioSepra/Pall Af-Gel 601 Polyacrylamide Not specied Boronate No Bio-Rad Laboratories Cellune Cellulose 85, 90, 170 Heparin, IMACa , gelatin Yes Chisso/Amicon/Millipore a IMAC, immobilized metal-ion afnity chromatography.. 2006 by Taylor & Francis Group, LLC

31. 18 Gustavsson and Larsson where the groups facing the solvent have a minimum tendency to attract sample components. Cellulose is another example of a polysaccharide support that is used in afnity chromatography. Another example of a support used in afnity chromatography is silica. This is used in the method of high-performance liquid afnity chromatography (HPLAC) or high- performance afnity chromatography (HPAC), which was rst reported in 1978 [2]. Although silica-based materials are certainly hydrophilic, they are unsuitable for afnity chromatography unless they have rst been modied at their surface. This is the case because the native surface of silica is primarily covered with silanol groups. These groups are weak acids that give silicas surface a strong negative charge at neutral pH. These charges, in combination with other binding forces, often result in the irreversible adsorption of solutes like proteins to native silica. However, several schemes can be used to render this surface inert toward such solutes, including polymer coating techniques and reactions between silica and alcohols or trialkoxysilanes [3, 4]. An example of such a scheme is the reaction of silica with -glycidoxypropyltrimethoxysilane [5], followed by acid hydrolysis of the resulting epoxy groups; this leaves the modied silica with a hydrophilic, noncharged surface (shown in Figure 2.1b) that has little or no binding to many biological compounds. The polymeric support polystyrene (illustrated in Figure 2.1c) is also unsuitable in its original form for afnity separations due to the highly hydrophobic character of this material. Native polystyrene, which is often used as a reversed-phase material, must rst Figure 2.1 Common materials used in the preparation of afnity supports. The structure in (a) shows the repeating unit of agarose: D-galactose and 3,6-anhydro-L-galactose. The structure in (b) shows diol-bonded silica, and (c) is the structure of polystyrene/divinylbenzene. CH2OH OH OH CH2 O H H O H HO H H H HO O OH HO H O Si CH2 CH2CH2 OCH2 CH2CH OH OH O O (a) (b) (c) 2006 by Taylor & Francis Group, LLC

32. Support Materials for Affinity Chromatography 19 be rendered hydrophilic by one of various surface-coating techniques before it can be used in other chromatographic methods [6, 7]. Polymeric supports based on polymethacrylate [8, 9] are more hydrophilic than polystyrene supports and can be used directly in afnity chromatography. Examples of these and other polymeric afnity supports, such as those A support material should be inert toward solutes, but to make it suitable for afnity chromatography, it should also be easy to couple to a ligand. Since most support materials are rich in hydroxyl groups, the chemistries developed for the attachment of ligands have focused mainly on using these regions as anchoring points. Many such methods are available [10, 11], A support material with a tendency to adsorb solutes in a nonspecic manner may still be useful, provided that the surrounding medium is modied accordingly. For instance, if one changes the buffer in an afnity column so that the buffers chemical character (e.g., hydrophilicity and ion strength) matches that of the support, there will be a minimum tendency toward nonspecic adsorption. As an example, when a support has charged groups present that can create undesired ionic interactions, a mobile phase buffer with an ionic strength of about 0.15 M can be used to suppress such binding [12, 13]. Even if the support is essentially free of nonspecic interactions with sample components, the nal afnity adsorbent obtained with this support could have a dramatically different character. This occurs because the procedure used to couple the ligand to the support may introduce undesired groups that might then act as a new source of nonspecic binding. A well-known example occurs during the activation of agarose and other polysaccharide supports with cyanogen bromide. In one standard protocol for this method, a large excess of cyanogen bromide is used. This excess is necessary because the alkaline conditions used for the reaction lead to a substantial breakdown of the active cyanate ester groups (see Figure 2.2a). Several breakdown products (including charged ones) may form when this occurs, which later leads to nonspecic interactions between the support and injected solutes. To avoid this problem, a modied protocol can be utilized that involves activating the support at a low temperature in the presence of a cyanogen transfer agent (e.g., triethylamine), which results in a less-altered support [14]. The cyanogen transfer agent in this alternative technique increases the electrophilicity of the cyanogen bromide by complex formation (as shown in Figure 2.2b). This allows the activation to be carried out at a neutral pH, which, in turn, minimizes hydrolysis of the cyanate ester groups and increases the overall reaction yield. Figure 2.2 Reaction schemes showing (a) the classical cyanogen bromide (CNBr) activation technique and (b) the modied cyanogen bromide activation technique using a cyano transfer agent (CTA). OH OH CTA-CN+ O OCN CNBr Overall reaction yield 20% of added CNBrBr + pH 7-8 (a) (b) 2006 by Taylor & Francis Group, LLC based on polyacrylamide, are listed in Table 2.1. with these being described in greater detail in Chapter 3.

33. 20 Gustavsson and Larsson The ligand itself may be responsible for unwanted interactions on the nal support. For instance, suppose that some agarose beads contained an immobilized derivative of adenosine triphosphate (ATP) and that the nal support had a ligand concentration of 10 to 15 mol ATP per milliliter of packed-bed volume. This is a normal degree of substitution and would be suitable for retaining ATP-dependent proteins. However, this modication has also made the agarose into a cation-exchange resin through the presence of the phosphate groups on the ATP. At pH 7, the charge concentration on this gel would be about 50 mol per milliliter of packed bed. Taking into account the multivalency of the charged triphosphate groups, the mobile phase used with this support would need to contain a 0.1 to 0.2 M buffer to cancel the ion-exchange properties imposed by the ATP afnity ligand. Such effects should be considered whenever a new afnity adsorbent is being designed. To avoid these nonspecic effects and thereby improve selectivity, it may also be wise to avoid working at unnecessarily high ligand concentrations. A related source of unwanted binding can occur via the ligand spacer. The spacer molecule serves to make an immobilized ligand more accessible to an injected macromol- ecule. Hexamethylenediamine is often used as spacer for this purpose [10, 11], but this introduces both charged groups and a patch of hydrophobicity to the supports surface. Sometimes these extra functionalities may act with the ligand to give even stronger binding for the desired sample components. However, in most cases they will lower the selectivity of the adsorbent, due to its retention of undesired sample components. This phenomenon was observed early in the development of afnity chromatography and eventually led to the development of a separation mode known as hydrophobic interaction chromatography (HIC) [15]. 2.2.2 Chemical Stability Obviously, the afnity adsorbent should be chemically stable under the operating conditions that will be used in the column. This includes stability toward enzymes and microbes that might be present in the process stream. This also means that the support must be stable in the presence of the elution buffers, regenerating solvents, and cleaning agents that will be used with the column. Agarose-based supports are almost ideal in this respect, especially when they are in their cross-linked form (e.g., Sepharose Fast Flow from Amersham Biosciences). Such supports are not attacked by enzymes, can be used between pH 3 and 12, and can withstand all commonly used water-based eluants without shrinking or swelling [11]. An additional attractive feature of cross-linked agarose is that it easily withstands sanitation with 0.5 M sodium hydroxide. This last feature is especially important in industry, where regular sanitation is used to achieve reliable performance and good product quality. A preferred way of doing this is cleaning in place (CIP) with a strong sodium hydroxide solution. This strong alkaline solution is an active bactericide and removes otherwise irreversibly deposited material from the column, such as particles, denatured proteins, lipids, and other compounds. If not removed, such deposits can contaminate the desired product and lead to column clogging. The fact that cross-linked agarose is stable at high temperatures is also important, since this allows it to be sterilized by autoclaving at 121C. Inorganic materials like porous glass and silica are vulnerable to hydrolytic damage. These supports should not be used above pH 8, and preferably not above pH 7, for any prolonged period of time, since these conditions can lead to breakdown of their silica structure [11]. However, silica materials are rarely used without the presence of a coating to make them more inert toward solutes, as discussed in the previous section. To a certain extent, these coatings also shield and protect the silica from hydrolysis. Furthermore, certain brands of silica supports are treated to incorporate zirconium or aluminum into 2006 by Taylor & Francis Group, LLC

34. Support Materials for Affinity Chromatography 21 their surfaces, which can considerably improve the stability of these supports in an alkaline environment [16, 17]. Up to this point, it may appear that afnity supports based on agarose will be satis- factorily stable under all situations encountered. But this is not the case, since often the weak point is not the matrix itself but rather the ligand or the attachment between a ligand and the support. For example, derivatives of adenosine monophosphate (AMP) and nicotinamide adenine dinucleotide (NAD+ ) are both efcient ligands for purifying NAD+ -dependent dehy- drogenases. But these are also delicate molecules prone to breakdown, either spontaneously or in the presence of hydrolytic enzymes. In this case, less efcient but more stable ligands, such as the dye Cibacron Blue, may be preferred, especially in large-scale applications where the cost of frequently replacing the separation material may otherwise be prohibitive. An example where the anchoring between a ligand and matrix is the weak point on a support can be observed in the cyanogen bromide method. Although this method is convenient to use, it does lead to an isourea bridge between the support and ligand, which will be slowly hydrolyzed at an alkaline pH. This problem can be overcome by alternative immobilization techniques that give a more stable product. Examples include methods that couple through ether linkages using bisepoxides [10, 11] or various other approaches, 2.2.3 Mechanical Stability The mechanical stability of an afnity chromatographic support should be sufcient to withstand the pressure drop across a column when the column is run at an optimum speed and ow rate for a separation. Most packing materials meet this requirement in well-behaved systems. However, in preparative-scale work, the sample or feed stream can contain many substances that may foul the separation bed. Deposits of lipids, denatured proteins, and particulate contaminants may restrict ow and quickly raise the column backpressure to unacceptable levels. In the case of soft gels like standard agarose beads, high backpressures will compress the column bed, which will increase the pressure even further and ultimately cause a collapse of this bed. Stronger supports such as silica or heavily cross-linked polymers will not collapse at such pressures, but even here very high backpressures are undesirable. Analytical-scale chromatography is often performed using short- to medium-sized columns with small- sized beads. In this situation, supports like silica and polystyrene are preferred, since the pressure drops in these columns can be high, often extending up to several hundred bars [18, 19]. The higher pressures in these applications are due, in part, to the desire for good mass-transfer properties and fast analysis times, which are generally obtained through the use of fast ow rates and small-diameter supports. 2.2.4 Pore Size It was stated earlier that the ideal afnity support should allow unhindered access of a solute to the immobilized ligand. For a macromolecular solute, this requires a support that has large pores. But just how large must these pores be? An answer to this question is given by the Renkin equation [20], which allows one to estimate the effective diffusion coefcient (Deff) of a solute in a porous material. (2.1) In this equation, Rs/Rp is the ratio of the solutes radius (Rs) to the pore radius (Rp), p is the particle porosity, is the tortuosity factor, KD is the distribution coefcient for the solute, and D is the diffusion coefcient for the solute in free solution. By inserting different D DK R R R R RD p s p s peff = +1 2 10 2 09 0 953 . ( / ) . ( / ) . ( ss pR/ )5 / 2006 by Taylor & Francis Group, LLC as outlined in Chapter 3.

35. 22 Gustavsson and Larsson values for the ratio Rs/Rp, one nds that the pore diameter should be at least ve times the diameter of the solute to avoid severely restricted rates of diffusion. For a protein of normal size (i.e., a diameter around 60 ), a ratio of ve for Rp/Rs means that the support pores should be in the range of 300 . Several common supports are available with such pore sizes. For example, supports based on 4 and 6% agarose have pore sizes of about 700 and 300 , respectively. Silica particles are available with pores in the range of 40 to 4000 . Polymethacrylate particles can be obtained with pores that are 100, 200, 500, or 1000 . Support materials with very large pores give essentially unhindered diffusion for most solutes, but they also have a smaller surface area per milliliter of bed volume than supports with smaller pores. This reduced surface area leads to a diminished binding capacity. As a rule, a pore size of 300 to 700 is usually a good compromise in most situations encountered in afnity chromatography, since this gives fairly unrestricted diffusion for most biomolecules while also providing a relatively large surface area for retention. 2.2.5 Particle Size Afnity supports are available in a wide variety of particle diameters. These range from HPLC-type materials with diameters of 10 m or less [18, 19] to large particles for preparative work that have diameters of 400 m. But which particle size is preferred for a given application? The answer will depend on the purpose of the separation, the mechanical properties of the support, and the characteristics of the sample. From a theoretical viewpoint, it is always advantageous to have a small particle size, since this will promote fast mass transfer of a solute between the outer ow stream and interior of a support particle. Figure 2.3 gives a simplied view of the various steps that are involved in this process. In this model, sample molecules are transported down through the column by the ow of the mobile phase in the spaces between the support particles. To reach the afnity ligands, these molecules must diffuse through the stagnant mobile- phase layer surrounding the particles (i.e., the lm model) and proceed to the inside pore network. It is here that the sample molecules will nally bind to the afnity ligand. When the retained molecules are eluted, the same steps occur but in a reversed order. In this model, smaller support particles mean shorter diffusion distances, since they have shorter pores and a thinner stagnant mobile phase layer around and in the support. This, in turn, results in shorter times being needed for diffusion [21]. According to the Einstein equation [21], the diffusion time (td) that is required for a molecule to travel a given mean Figure 2.3 Transport processes that occur in a chromatographic column. FLOW 2006 by Taylor & Francis Group, LLC

36. Support Materials for Affinity Chromatography 23 distance (d) will be proportional to the square of the molecules diffusion distance, or td = d2 /2Deff (2.2) where Deff is the effective diffusion coefcient of the molecule in its surrounding medium. In preparative afnity chromatography, relatively large support particles are often used, making intraparticle diffusion the main factor limiting efciency. In this case, dimin- ishing the particle size will increase the rate of movement of solutes between the support and surrounding ow stream, giving an improved column performance. It is this effect that was the original driving force behind the use of smaller supports in afnity columns, thus giving rise to the technique of HPLAC [2, 5]. Under such conditions, a decrease in particle size by a factor of ve can make it possible to increase the ow rate by up to 25-fold and still retain good chromatographic performance. This results in a dramatic improvement in the productivity of the system. However, a point is eventually reached when a decrease in particle diameter no longer gives a proportional improvement in an afnity columns performance. This has been observed in many analytical-scale systems that use HPLC-type supports with particle sizes less than 10 m in diameter. Under these conditions, diffusion in the particle is now relatively fast, and it is the adsorption/desorption of sample molecules to and from the afnity ligand that becomes the limiting factor in speed and efciency [19]. Although better efciency is always obtained with small support particles, using a small particle size is not without difculties. One problem is the much higher ow resistance of these smaller particles. According to the Kozeny-Carman relationship, the pressure drop over a column (p) is inversely proportional to the square of the supports particle size (dp), p/L = Cu/(dp)2 (2.3) where L is the bed height, is the mobile phase viscosity, u is the linear ow velocity, and C is a constant that depends on the bed porosity. This equation indicates that decreasing a supports particle size by a factor of ve will increase the pressure drop across this support by a factor of 25. This increased ow resistance may lead to bed collapse when using soft gels such as agarose. And, although supports like silica can tolerate the higher pressures that result, these will require the use of more expensive pumps to work at such pressure, as is generally done in HPLC. Another route that could be taken with small afnity supports is to use a short and wide column instead of a long and narrow one. The advantages of this are that the shorter, wider column can be run at higher ow rates without creating high-pressure drops. Another drawback with small particle sizes, especially in preparative work, is the increased danger of fouling that exists when particulate contaminants are in the feed stream or sample. This occurs because the interstitial spaces in a bed of small particles can be too narrow for such agents to pass through. Such fouling will increase the ow resistance and may lead to bed collapse if the support material does not have sufcient mechanical strength, as discussed earlier in Section 2.2.3. As a result of these various requirements, the particle size to pick when designing a new afnity adsorbent will be a compromise between the desired chromatographic performance, properties of the feed stream, and the mechanical strength of the support. Some common selections made in specic cases will be described in the next few sections. 2.2.5.1 Zonal Elution Chromatography Zonal elution chromatography (also known as weak or dynamic afnity chromatography) is mainly used in analytical work and in basic studies of afnity phenomena. It is seldom 2006 by Taylor & Francis Group, LLC

37. 24 Gustavsson and Larsson used for preparative purposes, due to its relatively low capacity compared with other methods. The sample molecules in this case are injected as a narrow plug. These are then monitored as they travel through the bed at speeds that are dependent on the strengths of their respective interactions with the ligand. To obtain a good separation in this type of system, it is important to have a column with a good efciency, as is true in HPLC-based afnity methods. Ideally, this requires that the support be based on particles that are as small as reasonably possible. The use of small particles will reduce diffusion times, as discussed earlier, which also promotes the formation of narrow solute peaks. This can be performed by using HPLC-grade silica that has been modied for use in afnity chromatography, as discussed in Section 2.2.1.Another alternative is to use ultrasmall, nonporous particles based on micron-sized silica or polystyrene [18]; however, these materials are not yet commercially available in the afnity mode. More information on zonal elution chromatography and its applications in afnity 2.2.5.2 Adsorption-Desorption Afnity Purication The most common way of performing afnity chromatography for preparative-scale work is to use this as an adsorption-desorption process. In this strategy, the target molecule alone is bound tightly by the ligand, and the adsorbent has a high binding capacity. After performing a suitable wash to release weakly bound substances from the sample, the conditions on the column are drastically changed (e.g., by using a substantial pH shift) so that the target molecule loses its afnity for the ligand and is eluted. Such a protocol works well with large support particles, since there is no absolute need for high chromatographic efciency. Still, small support particles can certainly help speed up the adsorption step, make the washing step more efcient, and permit the target molecule to be eluted in a smaller volume (i.e., as a narrower peak). This is especially valuable in analytical applications, where sharper peaks allow for more convenient quan- titation, better limits of detection, and faster analysis times. In preparative-scale work, the greater ease of handling larger particles is often preferred in practice, with particle sizes in the range 30 to 100 m being common in such work. 2.2.5.3 Partial Loading in Adsorption-Desorption Afnity Purication Partial loading is a method for improving the performance of large particles in preparative- scale afnity chromatography. The trick in this approach is to utilize only the outer shell of the support particle and thus diminish the distances required for solute diffusion. Binding to a thin outer layer of the support takes only a fraction of the time needed to saturate the entire particle. However, this outer shell still represents a substantial fraction of the supports total binding capacity. determined based on the following equation [21], tshell/ttotal = 1 3(1 a/r)2 + 2(1 a/r)3 (2.4) where ttotal is the time needed to saturate the whole particle, tshell is the time needed to saturate a layer with thickness of a in the particle, and r is the particle radius. According to Figure 2.4, an outer shell with a depth equal to one fth the total support-particle radius (a/r = 0.2) will contain half the particle volume (Vshell/Vtotal = 0.5). But this gure also shows that such a shell will take only one tenth the time needed to ll the whole particle (tshell/ttotal = 0.1). 2006 by Taylor & Francis Group, LLC Figure 2.4 illustrates results for the partial loading technique. These results were separations can be found in Chapter 4 and in Chapters 18 to 24.

38. Support Materials for Affinity Chromatography 25 Admittedly, special arrangements have to be made to make full use of this principle. For instance, fast recirculation through the bed is needed to ensure that all the support particles experience the same loading concentration. Also, the washing and elution steps are not substantially improved by this approach, since the released substances (impurities or the desired compound) will still have access to the entire particle. 2.2.5.4 Particulate Contaminants: Large Beads/Expanded Beds In large-scale purication schemes, particulate contaminants such as cells and cell debris are a problem, since they tend to clog afnity columns. This problem becomes worse as the size of the packing materials decreases, as noted earlier. One solution to this problem is to use extra-large particles. This strategy has been adopted in products like Sepharose Big Beads from Amersham Biosciences and Cellthru Big Beads from Sterogene, which have particle sizes of 100 to 300 m and 300 to 500 m, respectively. These very large particles should be run in the partial-loading mode described in the previous section. If this is not done, their performance will suffer from their long diffusion distances. Another solution to particulate contaminants is to use the expanded-bed principle. In this technique, the distance between the adsorbent particles is increased to allow contaminants to pass through freely. The adsorbents necessary for this mode of operation will be described later in Section 2.3. 2.2.6 Standard Commercially Available Afnity Supports The previous sections of this chapter described the properties that an afnity support should have, such as a hydrophilic character, good chemical and physical stability, and an appropriate pore size for the target solute. Most of these criteria are presently met by Figure 2.4 The shrinking-core model for protein adsorption. It is assumed in this model that the protein binds strongly to the adsorbent (step adsorption) and that the binding kinetics are fast compared with diffusion (i.e., the normal situation in preparative afnity chromatography operated according to the adsorption-desorption principle). The radius of the entire support particle is given by r, and the thickness of the outer absorbed layer or shell is given by a. The relative volume of the shell is given by the ratio Vshell/Vtotal, where Vtotal is the total volume of the support particle and Vshell is the volume occupied by the outer layer occupied by the adsorbed molecule. The relative time needed to ll the exterior shell is tshell/ttotal, where tshell is the time need to ll the outer layer and ttotal is the time needed to completely saturate the support particle. 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 1.0 1.0 1.0 a/r ttotal tshellVshell Vtotal r a 2006 by Taylor & Francis Group, LLC

39. 26 Gustavsson and Larsson commercial adsorbents based on porous beads. Several examples of these supports are The adsorbents shown in Table 2.1 are available from a number of manufacturers and are supplied either in bulk or as prepacked columns. In addition, some of these materials can be obtained in forms that already contain one of several common afnity ligands, such as protein A, Cibacron Blue, heparin, or metal chelating groups. If other ligands are required, it may be necessary to rst nd a suitable activation and coupling procedure for the ligand (see preactivated supports, which greatly simplies the preparation of new afnity columns. 2.3 AFFINITY SUPPORTS WITH SPECIAL PROPERTIES Porous supports like agarose, polymethacrylate, or silica beads are the main workhorses in most current applications of afnity chromatography. However, in the past several years described for use in afnity chromatography. Many of these newer materials have properties that give them superior performance in certain applications. Materials that fall in this category include nonporous supports, membranes, ow-through beads, continuous beds, and expanded-bed particles. 2.3.1 Nonporous Supports Nonporous beads with diameters of 1 to 3 m can be an optimum choice for fast analytical or micropreparative separations, since the limiting factor of pore diffusion is virtually eliminated in these materials. Such beads may also be the best choice for fundamental or quantitative studies of afnity interactions [18], since the binding and dissociation behavior seen with these materials should be more directly linked with the interactions occurring between solutes and the afnity ligand. However, there is a substantial loss of surface area and binding capacity that occurs through the elimination of internal pores. For instance, 1.0-m nonporous particles have a surface area of about 5 m2 per milliliter of packed bed, but the corresponding value for porous silica with 300- pores is about ten times higher. This difference becomes even more accentuated when comparing larger beads. In addition, as was discussed earlier, a smaller particle size leads to larger column backpressures. Thus, micron-sized particles are usually used in shallow beds to avoid high system pressures. Difculties with backpressure can also be minimized by using monodisperse particles, which will create fewer problems than polydisperse supports [23]. Nonporous bers are another category of materials that have been used as supports for afnity chromatography. These can have very high dynamic capacities [24]. Another advantage of these supports is that, in spite of the fact that the bers are submicron in diameter, their backpressures are low due to the low packing density of the overall ber bed. 2.3.2 Membranes Membranes have been used for afnity chromatography in various formats, such as stacked sheets, in rolled geometries, or as hollow bers [25, 26]. Materials that are commonly used for these membranes are cellulose, polysulfone, and polyamide [25, 26]. Because of their lack of diffusion pores, the surface area in these materials is as low as it is in nonporous beads. However, the at geometry and shallow bed depth of membranes keep the pressure drop across them to a minimum. This means that high ow rates can be used, which makes 2006 by Taylor & Francis Group, LLC shown in Table 2.1. For more information on the preparation of such support materials, other types of supports have also become available commercially (Table 2.2) or have been see the reviews in the literature [4, 22]. Chapter 3 or the literature [10, 11]). Many of the commercial suppliers in Table 2.1 also offer

40. SupportMaterialsforAffinityChromatography27 Table 2.2 Examples of Commercial Afnity Supports with Special Properties Trade Name Format Material Examples of Available Ligands Preactivated Form Available? Manufacturer/Supplier Sartobind Stacked membranes Regenerated cellulose Protein A, IMAC Yes Sartorius Poros Flow-through beads (20- or 50-m diameter) Polystyrene/Divinylbenzene Protein A, protein G, heparin, IMACa Yes Applied Biosystems CIM Monolithic disk or tubes Polymethacrylate Protein A, protein G Yes BiaSeparations Streamline Composite beads (120- or 200-m diameter) Agarose plus quartz or stainless s

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