Process Scale Liquid Chromatography

Process Scale Liquid Chromatography Edited by G. Subramanian

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VCH W

Weinheim - New York Base1 Cambridge - Tokyo

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Process Scale Liquid Chromatography Edited by G. Subramanian

0VCH Verlagsgesellschaft mbH, D-69451 Weinheim (Federal Republic of Germany), 1995 Distribution: VCH, P. 0. Box 101161, D-69451 Weinheim (Federal Republic of Germany) Switzerland: VCH, P. 0. Box. CH-4020 Basel (Switzerland) United Kingdom and Ireland: VCH, 8 Wellington Court, Cambridge C B l 1 H Z (United Kingdom) USA and Canada: VCH, 220 East 23rd Street, New York, NY 100104606 (USA)

1

Japan: VCH, Eikow Building. 10-9 Hongo 1-chome, Bunkyo-ku. Tokyo 113 (Japan)

ISBN 3-527-28672-1

Process Scale Liquid Chromatography Edited by G. Subramanian

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VCH W

Weinheim - New York Base1 Cambridge - Tokyo

Ganapathy Subramanian 60 B Jubilee Road Littlebourne Canterbury Kent CT 3 1TP. UK

This book was carefully produced. Nevertheless, authors. editor and publisher d o not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statcmcnts. data, illustrations, procedural details or other items may inadvertently be inaccurate.

Published jointly by VCH Verlagsgcsellschaft. Weinheim (Federal Republic of Germany) VCH Publishers. New York, NY (USA)

Editorial Director: Dr. Don Emerson, Dr. Steffen Pauly Production Manager: Claudia Gross1

Library of Congress Card No. applied for

British Library Cataloguing-in-Publication Data: A catalogue record for this book is available from the British Library

Die Dcutsche Bibliothek - CIP-Einheitsaufnahme Process scale liquid chromatography / ed. by G . Subramanian. Weinheim ; New York ; BaSel ; Cambridge ;Tokyo : VCH, 1995 ISBN 3-527-28672-1 NE: Subramanian, Ganapathy [Hrsg.]

0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim (Federal Republic of Germany). 1995

Printed on acid-free and low-chlorine paper

All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form -by photoprinting, microfilm, or any other means - nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such. are not to be considered unprotected by law. Composition: K + V Fotosatz Gm b H, D-64743 Beerfelden Printing: strauss offsetdruck GmbH, D-69509 Morlenbach Bookbinding: Wilhelm Osswald Co., D-67433 Neustadt Printed in the Federal Republic of Germany

+

Preface

Preparative and process-scale liquid chromatography have gained considerable importance over the past two decades, not only as a research and development tool, but as a viable alternative to more traditional purification techniques in the production environment. In recent years, there have been several advances in the development of matrix, designs, and systems, and in the understanding of theory, which have enabled liquid chromatography to be applied successfully in large-scale separations of biological molecules, thus making process-scale liquid chromatography a subject in its own right, but with its own problems as well. Large-scale chromatography using different matrices for selective process separations is carried out routinely in many areas of the bioprocessing industry. However, relatively little data is available in the scientific literature, for two main reasons. Firstly, many commercial processes involve proprietary technology, which precludes any opportunity for publication, and secondly, the cost of carrying out large-scale separations of a non-proprietary feedstock solely for academic purposes is often too high. This book aims to provide a theoretical basis for the understanding and practical application of liquid chromatography in large-scale separations. I am indebted to the contributors, who have shared their practical knowledge and experience. Each chapter represents an overview of its chosen topic. Chapter 1 describes chromatography systems, designs and control systems for process-scale separations. The current state of theory in large-scale separation by liquid chromatography, for various applications, is discussed in Chapter 2, and alternative modes of operation of chromatographic columns in the process situation are presented in Chapter 3. The application of size-exclusion chromatography in process-scale purification of proteins is discussed in Chapter 4. Chapters 5 and 6 give an account of the application of polymeric media in process-scale separations and ion-exchange liquid chromatography in biochemical separations, respectively. Instrument design for industrial supercritical-fluid chromatography and its application in industrial separation, and the scaling up of supercritical-fluid chromatography to large-scale applications are described in Chapters 7 and 8. Affinity chromatography and its application in large-scale separations is reviewed in Chapter 9.

a It is my hope that this book will bring the accumulated knowledge of process separations to scientists in industry, and that it will stimulate further progress in the field of process-scale liquid chromatography. I wish to express my sincere thanks to Dr. Don Emerson and all his colleagues for their invaluable help. Canterbury, Kent October 1994

G. Subramanian

Contents

1

Chromatography Systems Fred Mann

1.1 1.2 1.2.1 1.2.2 1.2.3 1.2.4 1.2.5 1.2.6 1.2.7 1.2.8 1.3 1.4 1.4.1 1.4.2 1.4.3 1.4.4 1.4.5 1.4.6 1.4.7 1.5 1.5.1 1.5.2 1.5.3 1 S.4 1 S.5 1.5.6 1.6 1.6.1 1.6.2 1.6.3 1.7 1.8

Introduction

-

Design and Control

................................... ..................................... ................................... Material Compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electrical Standards .........................................

1 2 3 3

6

Reliability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Serviceability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . System Design . . . . . . . . . . . . . . . . . . . . . Component Selection Column ............................... Pumps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Valves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pipework . . . . . . . . . . . . . . . . . . . . . . . .

8 9 9 11 11 14

.................................. ........................................... Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Temperature . . UV/Visible Ads .............................. Refractive Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pH/Conductivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dedicated Controller . . . . . General Purpose Controller .................. Computer-Based Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

17 17 18 19 20 20 21 21 22

15

24 24 28 29

VIII

2

Contents

The Practical Application of Theory in Preparative Liquid Chromatography

Geoffrey B. Cox Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Why Theory? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . How much Theory? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Single Solutes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mass Overload . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Simple Model: Single Component which Follows a Langmuir Isotherm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1.2 Computer Simulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2 Volume Overload . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Multiple Solutes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.1 Computer Simulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.2 The Effects of Column Efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Optimisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.1 Production Rate Optimisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.2 Cost Optimisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.2.1 Laboratory Scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.2.2 Production Scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.3 Practical Optimisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

38 41 42 44 46 50 54 56 58 58 59 61 63

Appendix 1. Calculation of Column Saturation Capacity . . . . . . . . . . . . . . . . Appendix 2. Mathematical Models for Preparative Chromatography . . . . . . Mass-Balance Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Craig Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

64 65 65 67

2.1 2.2 2.3 2.4 2.4.1 2.4.1.1

3

33 33 34 34 35

Alternative Modes of Operation of Chromatography Columns in the Process Situation

Derek A. Hill 3.1 3.2 3.2.1 3.2.2 3.2.3 3.2.4 3.2.5 3.3 3.4

Process Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alternative Chromatographic Modes and Techniques . . . . . . . . . . . . . Elution Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Displacement Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Frontal Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Operating Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Miscellaneous Operating Techniques ............................ The Use of Alternative Modes and Techniques in Process Situations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

71 73 73 74 75 76 77 78 80

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4

Contents

IX

Process Scale Size Exclusion Chromatography

Jan-Christer Janson 4.1 4.2 4.3 4.3.1 4.3.2 4.3.3 4.3.4 4.4 4.5 4.6 4.6.1 4.6.2 4.6.3 4.7 4.8

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Separation Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Column Packing Materials for Process Scale SEC . . . . . . . . . . . . . . . Dextran Gels and Polyacrylamide Gels ......................... Agarose Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Composite Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Choice of Separation Medium ............................. Adsorption Effects of SEC Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Eluent in SEC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Practices of Process Scale SEC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Column Dimension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gel Preparation and Column Packing .......................... Feed Stock Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatographic Productivity in SEC .......................... Strategy for Scaling-up of SEC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5

Polymers and their Application in Liquid Chromatography

81 82 85 85 86 87 87 89 89 90 90 92 93 96

Linda L . Lloyd and John E Kennedy 5.1 5.2 5.3 5.4 5.4.1 5.4.1.1 5.4.1.2 5.4.1.3 5.4.1.4 5.4.2 5.4.2.1 5.4.2.2 5.4.2.3 5.4.3 5.4.3.1 5.4.3.2 5.4.3.3 5.4.3.4 5.4.3.5 5.5 5.5.1 5.5.2

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Polymer Network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manufacturing Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Types of Polymeric Matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Synthetic Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Polystyrene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Polyacrylamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Polymethacrylate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Miscellaneous Synthetic Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Natural Polymers ............................................ Dextran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Agarose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Composite Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................. Surface Coatings . . . . . . . . . . . . . . . Pellicular Supports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Core Shell Grafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pore Matrix Composites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interpenetrating Networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Polymer Physico-chemico Characteristics . . . . . . . . . . . . . . . . . . . . . . . Particle Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pore Size and Pore Size Distribution ...........................

99 100 101 101 103 103 103 104 104 104 104 105 106

108 108 108 109 109 109 110

X

Contents

5.5.3 5.5.4 5.5.5 5.5.6 5.5.7 5.5.7.1 5.5.7.2 5.5.7.3 5.5.7.4 5.6 5.6.1 5.6.2 5.6.3 5.6.4 5.6.5 5.6.6 5.6.7 5.6.8 5.7 5.7.1 5.7.2 5.7.3 5.7.4 5.8

Surface Area ......................... .................... Mechanical Rigidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Column Efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eluent Compatibility and Solvent Strength . . . . . . . . . . . . . . . . . . . . . . Activation and Functionalisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Polystyrene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Polyacrylamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Polymethacrylate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Size Exclusion Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reversed Phase Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hydrophobic Interaction Chromatography ...................... Ion Exchange Fractionations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Affinity Supports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chiral Separations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hydrophilic Interaction Chromatography ....................... High Speed Separations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Practical Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Choice of Adsorbent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemical Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fouling and Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recovery of Mass and Biological Activity ....................... Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6

Biochemical Applications of Process-Scale Ion-Exchange Liquid Chromatography

111 113 114 116 117 117 118 118 118 119 120 121 121 122 123 123 124 124 125 125 126 126 127 127

Peter R. Levison 6.1 6.2 6.3 6.4 6.4.1 6.4.2 6.4.3 6.4.4 6.5

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Principles of Ion-Exchange Chromatography .................... Throughput . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biochemical Applications of Process-Scale Ion-Exchange Liquid Chromatography ............................................. Chromatography of Hen Egg-White Proteins .................... Chromatography of Goat Serum Proteins ....................... Chromatography of a Monoclonal Antibody .................... Chromatography of DNA-Modifying Enzymes . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

131 132 134 236 137 142 146 247 150

Contents

.

7

XI

Instrumental Design and Separation in Large Scale Industrial Supercritical Fluid Chromatography

Pascal Jusforgues 7.1 7.2 7.3 7.3.1 7.3.2 7.3.3 7.3.4 7.4 7.4.1 7.4.2 7.4.3 7.5 7.6

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Principle. Advantages and Drawbacks . . . . . . . . . . . . . . . . . . . . . . . . . . Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pumping System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatographic Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fraction Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eluent Recycling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Separation Costs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Why PS-SFC is Expensive . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Why PS-SFC is Cheap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Purification Costs Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications: SFC vs HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8

Scaling-up of Supercritical Fluid Chromatography to Large-Scale Applications

153 153 157 157 157 158 158 159 159 159 160 161 161

Christopher D. Bevan and Christopher J: Mellish 8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9 8.10 8.11 8.12 8.13 8.14

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Supercritical Fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Choice of Supercritical Fluids ................................. The Scaling-up Process ....................................... The History of Preparative SFC ............................... Safety Considerations .The Column Shield Jacket . . . . . . . . . . . . . The Basic Chromatography ................................... Loading and Injection of Samples ............................. Design and Construction of the Sample Introduction Pressure Vessel (SIPV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Collection of Fractions from the Preparative Supercritical Fluid Chromatograph ............................................. High Pressure Trapping with Subsequent Recovery by Solidification of the Carbon Dioxide ....................................... Development of Large Scale Commercial Systems . . . . . . . . . . . . . . . . Detection of Solutes in Preparative SFC ........................ Recent Developments in SFC and SFE .........................

163 163 165 167 169 171 172 172 175 178 180 186 188 189

XI1 9

Contents

Affinity Chromatography and its Applications in Large-Scale Separations Christopher R. Goward

9.1 9.2 9.3 9.3.1 9.3.2 9.3.3 9.3.4 9.3.5 9.4 9.4.1 9.5 9.6 9.7 9.7.1 9.7.2 9.7.3 9.7.4 9.7.5 9.7.6 9.8 9.9 9.9.1 9.9.2 9.10 9.10.1 9.10.2 9.10.3 9.10.4 9.10.5

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Support Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Important Features of a Ligand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Coupling of a Ligand to the Support Matrix .................... Activation of the Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Capacity of the Adsorbent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ligand Leakage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Triazine Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Process Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Scale Up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . chromatography Column and Other Equipment . . . . . . . . . . . . . . . . . Process Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatography Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... Washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Selective Elution ............................................. Non-selective Elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Flow Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cleaning and Storage of Adsorbents ........................... Protein Engineering Applied to Protein Purification . . . . . . . . . . . . . Release of the Affinity Tail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Examples of the Use of Affinity Tails .......................... Examples of Some Large-Scale Affinity Methods . . . . . . . . . . . . . . . . Protein G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Streptavidin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucokinase and Glycerokinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Human Serum Albumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunoaffinity Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

193 194 195 197 198 198 200 200 201

202 202 203 203 203 204 205 205 205 206 207 208 208 209 210 210 211 211 212 212

215

Contributors

Christopher D. Bevan Glaxo Group Research Ltd Structural Chemistry Department Greenford Road Greenford Middlesex UB6 OHE United Kingdom (Chapter 8) Geoffrey B. Cox Prochrom Chemin des Blanches-Terres BP 9 F-54250 Champigneulles France (Chapter 2) Christopher R. Goward Camar Portondown Salisbury SP4 OJG United Kingdom (Chapter 9) Derek A. Hill The Wellcome Foundation Ltd Temple Hill Dartford DAI 5AH Kent United Kingdom (Chapter 3 )

Jan-Christer Janson Pharmacia Bioprocess Technology AB S-75 182 Uppsala Sweden (Chapter 4 ) Pascal Jusforgues Prochrom Chemin des BlanchesTerres BP 9 F-54250 Champigneulles France (Chapter 7) John F. Kennedy Birmingham Carbohydrate and Protein Technology Group Research Laboratory for the Chemistry of Bioactive Proteins and Carbohydrates School of Chemistry University of Birmingham Edgebaston Birmingham, B15 2TT United Kingdom (Chapter 5 )

XIV

Contributors

Peter R. Levison Whatman International Ltd Springfield Mill Maidstone ME14 2LE Kent United Kingdom (Chapter 6 )

Fred A. Mann Amicon Ltd Upper Mill Stonehouse Gloucestershire GLlO 2BJ United Kingdom (Chapter I )

Linda L. Lloyd Chembiotech Ltd Institute of Research and Development University of Birmingham Research Park Vincent Drive Birmingham B15 2SQ United Kingdom (Chapter 5 )

Christopher J. Mellish Glaxo Group Research Ltd Bioengineering Unit Greenford Road Green ford Middlesex UB6 OHE United Kingdom (Chapter 8)

List of Symbols and Abbreviations

A B C ci(k,I ) c m cm,i C S

cs, i

D m

dP

H K

Ki k' kb L

Lf N

Parameter in Knox Eqn (11); relates to packed bed Parameter in Knox Eqn; relates to diffusion in mobile phase Parameter in Knox Eqn; relates to mass transfer between phases Mobile phase concentration at the kth time step and Ith distance step Mobile phase concentration of solute Mobile phase concentration of solute i Stationary phase concentration of solute Stationary phase concentration of solute i Diffusion coefficient of solute in the mobile phase Particle diameter of packing material Height equivalent to a theoretical plate ( L / N ) Distribution coefficient between the mobile and stationary phases Distribution coefficient of solute i between the mobile and stationary phases Capacity factor in non-linear range of the isotherm Capacity factor in linear range of the isotherm Column length Loading factor (w,/ws) Column efficiency (no. of plates) for peak in non-linear range of the isotherm Column efficiency for peak in linear range of the isotherm Number of Craig stages Operating pressure Stationary phase concentration at the kth time step and Ith distance step Time Elapsed time per transfer in Craig simulation Elution time of non-retained peak Linear flow velocity Volume of mobile phase in the column (interstitial+ pore volume) Volume of stationary phase in column Mass of sample injected Column saturation capacity Longitudinal distance in column

XVI At Az @

@ rl V

List of Symbols and Abbreviations

Time step in finite difference equation (A2.3); [ = 2H(1 + k ’ ) / u ] Distance step in finite difference equation; [ = HI Phase ratio VJV, Column resistance parameter Solvent viscosity Reduced flow velocity (ud,/D,)

1 Chromatography Systems Design and Control

-

Fred Mann

1.1 Introduction ‘Anything formed of parts placed together’, ‘a set of things considered as a connected whole’ is the dictionary definition of a system. A liquid chromatography system is thus defined as parts or components that are connected together to allow the process of liquid chromatography to take place. All chromatography systems are basically the same (Fig. 1-1) whether they are used for analytical, small scale preparative or process use. The main components are common and consist of: -

the stationary phase or matrix

- the column to contain the matrix -

a pump to push mobile phase through the column means for selecting or mixing different solvents to produce gradients, either step or linear sample injection detection on the column outlet fraction collection control and/or data collection

The particular use, however, for which a chromatography system is required, will influence the relative importance and requirements of the individual components. In analytical systems for instance, the objective is to identify components present in a small sample volume; consequently these systems must be able to accommodate highly efficient columns containing very small particle diameter packings producing high column back pressures. Minimal volume in pipework, valves, and detector flow cells are required and a large emphasis is placed on data handling with automatic calculation of peak areas for concentration determination. There is no requirement for a fraction collector. In preparative systems, on the other hand, where the objective is to obtain purified components of the sample, fraction collection is a necessary requirement. However, preparative systems also differ depending on their use. In process development where the system may be used in the investigation and development of many different purification problems, flexibility is paramount, with the ability to operate with dif-

2

I Chromatography Systems

-

Design and Control Controller

Recorder

Soivent reservoirs

I

D

I

I

I

C

B

Detector

A

Fraction vessels Column

Fig. 1-1. Basic chromatograph components.

ferent media, solvents, columns, and detection requirements. In production where a system is dedicated to a single use, flexibility is no longer required and reliability will be the most important criteria. In terms of control even the simplest flash chromatography separation is usually in reality, under very sophisticated control; that is the direct manual control of the operator who performs and coordinates all the functions of: solvent selection, mixing, solvent and sample addition to the column, visual monitoring of the flow or movement of coloured bands and collection of the different separated components in suitable receptacles. However, by utilizing instrumentation to monitor the state of the system together with automation of valve switching and pump control, the direct operator involvement can be reduced, thus lowering labour cost, and increasing reproducibility and reliability.

1.2 System Requirements Before entering into the detailed design of systems it is first beneficial to consider the overall requirements the system must satisfy, namely:

1.2 System Requirements

-

3

functionality material compatibility pressure requirements electrical standards hygiene control/automation reliability serviceability

1.2.1 Functionality Chromatography separations are based not on the system but on the sample interaction with the stationary and mobile phases. If, however, the potential of this interaction is to be realized the system must not adversely affect the process. In reality the effect of the system must inevitably be to reduce the efficiency of the separation, for instance by sample dilution in the pipework. The challenge in designing a system is to reduce this negative impact to a minimum. In considering the functionality, therefore, it is necessary to consider not only the number of required solvent inlets, fraction outlets, pump and valve types, sensors, detectors, etc., but also the pipework size and configuration to ensure that they are optimal for the required flow and that dead legs and dilution zones are kept to a minimum.

1.2.2 Material Compatibility Selection of materials should be such that no problems can arise from adsorption to, or leaching from, components within the system. Materials must be compatible with all solutions used in the process, including those used for regeneration, cleanParticular attention must be paid to the use of plastics or ing, and storage [I]. elastomeric seals with organic solvents. The effect of solvents is dependent on concentration, temperature and contact time. Attack may result in softening and dissolving of the polymer and/or leaching of plasticizers or other components. For these reasons HPLC systems invariably use only stainless steel and PTFE as construction materials. It is particularly important to ensure that plastic or elastomeric materials selected for incorporation into systems destined for use in pharmaceutical production processes are acceptable and will satisfy any regulatory requirements (eg, US Food and Drug Administration (FDA) [2], US Pharmacopeia [3]). In the case of aqueous systems where halide ions are present, stainless steel of at least 3 16L grade is required, and in the case of high concentrations of chloride even more resistant grades may be specified. Alternatively, plastic may be used in place of steel.

4

I Chromatography Systems

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Design and Control

Attention should also be given not only to materials in direct contact with the process stream but also to external materials used for manufacture of frames and cabinets. Many chromatographs used for pilot or production purposes are located in environments promoting corrosion (eg, wet areas) and appropriate specification of these external materials is just as important as that applied to process pipework.

1.2.3 Pressure It is necessary to ensure that not only are all components within the system appropriately rated for the maximum operating pressure of the system, but that the design of the system adequately covers the pressure drop in the column and the associated pipework and valving at the maximum flowrate required. The major contribution to pressure drop in the system is invariably the packed column bed. Information on this can be derived from the early process development trials and from the matrix manufacturer. Caution should be exercised to ensure that the data relates to performance of the matrix in the column diameter proposed. With rigid particle packings (eg, Silica) data from small-diameter columns (25 - 50 mm diameter) can readily be extrapolated to larger diameter columns. In the case of soft or deformable matrices this may not be the case. With these matrices in small diameter columns, the bed is supported by the wall of the column, the effect of which is lost as the diameter of the column is increased (>200mm diameter). This leads to greater than expected pressure drops in larger columns. In some cases the matrix may be so deformable that required flow rates cannot be achieved in large-diameter columns due to the compressibility of the matrix. Pressure drop is related to flowrate and this relationship is particularly important when designing a system with a wide flowrate range. The pressure/flow relationship of a packed bed is linear, whereas with pipework it is a square factor where a doubling in flowrate will result in a four-fold increase in pressure drop. Consequently, although in order to minimize dilution within the system at low flowrate, it is desirable to use small bore tubing and valves, this may produce at high flowrates an undesirably high pressure drop requiring a compromise between minimum system volume and pressure drop. Pressure drop after the column should be given special care as the column inlet pressure, to which the column must be designed and built, will be the sum of both the packed bed/column pressure drop and that of the post column pipework and valving. Whereas increasing the pressure rating of a small analytical column is relatively inexpensive, increasing the pressure ratings of large scale production columns can be considerable, due to the need for greater material thicknesses and differing designs to cope with, in particular, the higher end loadings on the larger columns as the pressure increases. Implications of pressure vessel regulations also need to be considered, especially with respect to the column. Requirements differ between countries and is usually dependent on a combination of pressure and volume. For instance the A D Merkblatt regulations [4] in Germany stipulate more stringent requirements for design and

1.2 System Requirements

5

testing of a column if P (bar) x V (litres) is > 200. In the US many states classify larger chromatography columns as unfired pressure vessels, necessitating that they be designed, built and tested in accordance with the American Society of Mechanical Engineers (ASME) regulations [ 5 ] .

1.2.4 Electrical Standards Electrical design and manufacture also needs to conform to appropriate standards. In the past each country had its own standards and regulations but there are now unified European standards under the auspices of CENELEC (European Committee For Electrotechnical Standardization) which covers the following countries: Austria, Belgium, Denmark, Finland, France, Germany, Greece, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the UK. Each country still tends to maintain local standards classifications but these are directly equivalent to the European Norms. For instance an electrical design and manufacture applicable to process chromatographs is British Standard BS 2771 [6] (Electrical Equipment of Industrial Machines) which is equivalent to European Norm EN 6024. In addition the nature of the environment in which chromatographs are installed, and their use with the potential for liquid leaks or wash down will necessitate that enclosures meet the appropriate standards for dust and water protection (Table 1-1). Most installations will utilize IP54 protection. The use of organic solvents can mean that equipment needs to be designed and built to comply with standards for explosion proofing enabling it to operate in a hazardous area. Sometimes the chromatograph itself does not use flammable solvents but is installed in a hazardous area where flammable solvents are being used for other processes. In this case the chromatograph will still need to be built to comply with explosion proofing regulations. Individual country regulations have also Table 1-1. Summary of IP protection numbers. IP codes: ingress protection First numeral 0 1 2 3 4 5 6

No protection Objects >50mm Objects >12mm Objects >2.5 mm Objects >l.Omm Dust protected Dust tight

Protection against solid bodies

Second numeral 0 I 2 3 4 5 6 7 8

No protection Vertically dripping water 75 to 90" angled dripping water Splashed water Sprayed water Water jets Heavy seas Effects of immersion Indefinite immersion

Protection against liquids

6

1 Chromatography Systems - Design and Control

been standardized by CENELEC for European countries. In the US appropriate sections of the NEC (National Electrical Code) apply.

1.2.5 Hygiene The purification of sample feedstocks derived from micro-organisms or natural products, coupled with the use of mobile phases containing buffers and other components designed to maintain biological activity, can present ideal conditions for contamination of the system and proliferation by unwanted micro-organisms and the generation of pyrogens. In these circumstances, effective cleaning, and if necessary sterilization is required if a product of the required purity is to be obtained. This problem is encountered more in low or medium pressure systems than in HPLC. In the latter, solvents, matrices, and sample feedstocks are less conducive to microbial growth. In low and medium pressure chromatography, process hygiene is not only important in terms of preventing contamination of the product but also in prolonging the life of the stationary phase; the polymeric gel matrices commonly used being susceptible to bacterial degradation. In such cases the systems are cleaned in place (CIP) with strong alkaline or other appropriate cleaning or sanitizing solutions. For this to be effective the cleaning solution must be carried effectively throughout the entire system. Sanitary design aims to meet the above requirements by ensuring that there are no unswept volumes within the flow path that would provide opportunities for microorganisms to be harboured. No standards per se exist for sanitary designs of chromatographs but well established principles are utilized from other process applications, for instance the dairy industry (ie, the American 3-A standards developed by the US Dairy Industry). ‘Tri-clamp’ style pipework fittings are preferred in place of threaded or ferruletype connectors, as the face to face seal with flush fitting gasket does not provide crevices for microbial growth. In contrast, ferrule fittings invariably provide a ‘dead area’ between the tube and outer fitting in front of the ferrule. Similarly in the case of valves, diaphragm types are preferred as the design permits free flow across the whole internal surface, in contrast to ball valves where not only can there be a contaminating ‘plug’ within the ball itself, but the area between the ball and packing seal may form a crevice for microbial growth. In the design of sanitary systems attention has increasingly been focused, not only on the need for effectively flushed fittings and connections, but also on the surface of the pipework, valve or column itself, being non-conducive to bacterial or fungal attachment and growth. The smoother a surface the easier it is to clean and the less likely it is to harbour micro-organisms. Stainless steel is traditionally polished mechanically using an abrasive polishing pad or mop with an abrasive paste. A coarse abrasive is used first to take out major imperfections with successively finer grades being used to obtain a smoother surface. The surface finish is often specified in terms of the final abrasive grade used, for in-

1.2 System Requirements

7

stance, a 180 grit finish. The higher the grit number the smoother the finish. Although grit number is very often used to describe the finish, variation may occur between different sources of the same grit number and also be dependent on how worn the abrasive is. A more accurate comparison can be made by actual measurement of the surface texture of the steel. A stylus type instrument is used which is moved across the surface and produces a trace of the surface profile, together with a ‘Roughness Average’ (Ra) reading. Comparison of the Ra measurements still requires caution as correct setting of the measuring instrument (ie, cutoff) relative to the surface being measured is critical if representative results are to be achieved. Table 1-2 shows a comparison of typical Ra values for different grit finishes. Table 1-2. Surface finish.

Grit finish

120 Grit 180 Grit 240 Grit 320 Grit

Typical Ra“ Pm

Micro inch

0.8-1.2 0.4-0.8 0.3 -0.4 0.15-0.3

31-47 15-31 11-15 6-11

Ra is the roughness average. Ra is also known as the arithmetic average (AA) and centreline average (CLA). It is the arithmetic average of the absolute values of the measured profile height deviations taken within the sampling length and measured from the graphical centreline.

a

The surface of stainless steel, even if highly polished mechanically, is in fact not smooth but consists of a series of peaks and troughs. In fact the very act of polishing, being an abrasive process, actually increases the number of peaks and troughs and thus the actual surface area available for bacterial attachment. In addition the polishing action tends to ‘bend over’ the tops of the peaks thus trapping polishing abrasive or other dirt within the troughs which can make subsequent cleaning difficult. Increasingly electropolishing is being used as a final polishing step in addition to, or even as, a replacement for traditional mechanical polishing. Electropolishing is in effect the reverse of electroplating. It is an electrolytic process with the item to be polished made anodic in a strongly acidic electrolyte and positioned adjacent to a formed cathode plate. A high anode surface current progressively and preferentially dissolves the metal at the peaks of the surface. The peak to trough height is reduced and a smoother brighter surface with a lower total surface area results. The benefit of electropolishing in reducing the growth of bacteria on the steel surface has been demonstrated [7]. Stainless steel is utilized because of its high resistance to corrosion which is a result of the thin passive surface oxide film which will form naturally in the air. However, this passive film will only occur on clean surfaces. If areas of the surface are covered

8

1 Chromatography Systems - Design and Control

by oxides resulting from welding or heat treatment, or by oils from the polishing operation, the natural passive layer cannot form and corrosion can be initiated in these areas when they come into contact with corrosive media. For these reasons it is important that stainless steel pipework and other process fabrications are thoroughly cleaned, particularly at the welds to ensure the passive surface oxide film can form. This is normally achieved by washing the surface with an acidic ‘pickling’ paste or liquid (usually a mixture of chromic and nitric acids), to achieve a chemically clean surface on which the protective passive layer can form. Electropolished surfaces require no such treatment as the electrochemical action in itself results in a passivated surface. It would seem ideal for both maximum hygiene and corrosion protection that all process pipework and fittings be electropolished. However, this is often not possible as to be effective the electropolishing process requires a closely contouring cathode which is not feasible in many fabrications. In such cases electropolished pipe and valves may be utilized and either fitted together using clamps or directly welded using inert gas purged orbital welding techniques. The latter is an automated technique that provides an almost flush weld on the surface of the pipe with minimum oxide and crust formation. For maximum corrosion protection the pipework can be passivated after welding by recirculating the ‘pickling’ solution.

1.2.6 Control The main reasons for controlling a piece of equipment or process is to either reduce cost, by decreasing operator involvement and hence labour costs; increase throughput by, for instance, operating 24 h a day, which can in turn reduce capital investment, and/or to ensure greater reproducibility and reliability. In addition the need for full process recording and documentation for GMP and Regulatory compliance may more easily be met by automated systems. The level of automation required, and in particular the ability for the operator to influence the process, will depend on the use to which the system is put. A system used in development or for production of a variety of products will require more flexibility than one used in a dedicated single use production process. In the latter simplicity and reliability particularly in the user interface is more important.

1.2.7 Reliability All systems should be reliable but this is an absolute requirement for a production system. In development or research, reliability may be sacrificed for capability or flexibility. In production the need for reliability argues for keeping the system simple; that is only incorporate the components and features that are essential to the performance, control and recording of the process. In addition select components for their suitability for a production environment.

1.3 System Design

9

The location in which the system is being utilized needs to be considered. On the one hand the system needs to be designed or selected for reliable operation in such locations, eg, cold room; on the other hand the quality and stability of the services (power, air) need to be ensured. Particularly with automated systems in industrial situations, the susceptibility to, and implications of, power drop outs and other potential electrical problems, need to be considered. Be cautious of scaled up analytical components that may not be rugged enough.

1.2.8 Serviceability Systems should be designed with the aim of minimum service requirements but routine adjustments and calibration of sensors will be necessary. Layout of the various components should be such as to facilitate access to those items such as filters, pH probes, and detector lamps which will require periodic replacement. In selecting instrumentation, attention should be given to its stability, requiring less frequent recalibration and to its ease of recalibration when required. Certain pH and conductivity probes for instance can only be calibrated accurately under flow conditions which is not as easy to perform as static calibration. Design of the system should also address this need to facilitate recalibration. In production it is usual to implement routine preventative maintenance to reduce the likelihood of component failure during a run. The requirements and frequency of such servicing should be advised by the manufacturer or compiled from individual component manuals.

1.3 System Design Bearing in mind the general requirements, the next stage in designing a system is to produce a functional or block flow diagram of the process. This will usually also take into account not only the chromatography step but also the upstream and downstream requirements of the process. This identifies the number and volumes of solvents, sample, and fractions, together with column size. Production processes are invariably developed initially at the bench scale where stationary and mobile phases are selected and sample volumes determined. In the majority of cases the application is then simply scaled up by increasing the diameter of the column while maintaining the bed height constant, and increasing all volumes, including sample, elution, and regeneration solutions in direct proportion to the increase in column cross sectional area. Linear flow rate is maintained the same so that volumetric flow also increases in proportion to the increase in column cross sectional area, and process times remain constant. This stage will also identify the interdependence of the various stages of the overall process and indicate possible control interactions for instance, a fraction tank in the

Anicon

plant

+$

interlock

Valve normally open

Valve normally closed

0PC

0 In control panel

On control panel

0 On 8

Scope o f supply

L

Others

[TERMINATION

I TRlCLAMP

_ ._ _ ._ ._I

l

1.4 Component Selection

11

chromatography process may be utilized as a product feed tank, or recirculation tank in a following ultrafiltration step. Following the overall process flow diagram a more detailed piping and instrumentation drawing or ‘P & ID’ (Fig. 1-2) is produced which defines all pipework, valves, instrumentation, and their interconnection, together with process functions and control interlocks. This is now the basis for selection of the individual components.

1.4 Component Selection 1.4.1 Column As discussed earlier, the objective when constructing a Chromatography system is to minimize any deleterious effects the system introduces to the chromatography. Next to the media and solvent selection the most critical component is probably the column. The role of which is not only to contain the matrix but also to enable it to be packed and maintained homogeneously and provide uniform flow throughout the whole cross sectional area of the bed in order to ensure both maximum capacity and efficiency. At the same time as little dilution as possible is desirable, either within the bed itself, or as the sample is introduced to the column, or the separated fractions exit. Analytical columns utilize a single inlet and outlet port with the packed bed contained between two mesh or sintered frits. When the sample is injected onto the column through the single inlet port, some radial dispersion may occur in the frit, but usually the column will behave in the infinite diameter mode. As such the sample enters as a single point injection which as it flows through the packed bed disperses radially, but may not reach the wall of the column before exiting through the bottom frit. This effect can be beneficial in analytical applications where the failure of the sample to contact the walls negates any peak broadening, resulting from differences in flow profile between that existing between the bed and the column wall, and that within the bed itself. This results, therefore, in sharper peaks. For preparative applications, however, the infinite diameter mode is not so desirable as it reduces the effective capacity of the column. Here, complete utilization of the whole packed bed is required and so it is necessary to ensure uniform presentation of the sample across the whole cross section of the column, together with uniform flow. Although this will result in some peak broadening, due to the wall effect, this is reduced as the column diameter increases. Several approaches have been used to assist radial distribution. The simplest of these consists of a column end plate with a single inlet/outlet port, next to which is a coarse mesh, followed by a finer mesh to retain the matrix particles. The coarse Fig. 1-2. Typical piping and instrumentation drawing or P &ID of chromatograph. This drawing defines the hardware and instrumentation on the system.

12

1 Chromatography Systems

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Design and Control

mesh acts to provide channels for radial distribution. On larger columns this approach may be augmented by having several inlet/outlet ports manifolded together. An alternative approach is to machine distribution channels directly into the column end plate radiating out from the single inlet/outlet port. A bed support, consisting of a mesh or sinter material, is then placed between the end cell and the bed. Further refinements whereby the distribution channels are machined in the form of a cone with the apex at the inlet/outlet port, improves the flow profile by preventing the possibility of air locks forming which could cause uneven flow in part of the bed. A combination of radial, coned distribution channels, together with either a sinter or composite mesh permitting limited radial dispersion, can be considered as an ‘infinite multiport distribution system’, and as such gives an optimal flow profile (Fig. 1-3).

Fig. 1-3. Example of column end cell design providing uniform flow distribution.

As mentioned earlier, chromatography applications are usually scaled up by increasing the cross sectional area of the column consequently it is important that columns selected for both pilot studies, and final production, are directly scaleable. Selection of the column depends on the type of matrix used and the pressure and solvent requirements.

1.4 Component Selection

13

For low pressure chromatography ( I -7 bar) there are commercially available columns, constructed of plastic, glass, or stainless steel, ranging in size from laboratory scale up to 2 m diameter (glass maximum 1 m). Transparent plastic or glass columns are usually preferred as they permit visual inspection of the packed bed, which can facilitate packing and determination of fouling of the matrix. Glass columns are, however, not robust and pressure rating especially of the larger sizes is limited (1 m 1 bar rating). Glass can be protected and pressure rating increased by wrapping with glass reinforced plastic (GRP) but then it loses its transparency. Plastic columns, although being more robust, may not have the required solvent compatibility. Stainless steel, although robust and solvent resistant, is unsuitable for products that are susceptible to metal ions or where prolonged use of high concentrations of halide ions may cause corrosion. A new range of columns has recently been introduced to the market, made from TPX (tri-methyl-pentene polymer), providing transparency with good resistance to most commonly used chromatography solvents, and pressure rating of 7 bar. High pressure columns are invariably made of stainless steel because of the high pressure involved, and solvent resistance to all organic solvents is provided by the use of PTFE or other suitable fluoropolymer seals. Traditionally, low pressure columns have been packed by pouring the matrix gel slurry into the column and either letting it settle under gravity or by flowing mobile phase through the column to settle the bed. This may require the use of a column extension or filler tube which is removed once the bed is settled. The use of a column with an adjustable end cell greatly facilitates this operation as it means that accurate measurement of the gel volume is unnecessary, as is'the case with a fixed volume column. In addition if any further settling of the bed occurs during running, then adjustment can be made to take up any void. Simple gravity settling or flow packing do not suffice to pack efficiently the smaller particle size HPLC packings. In these cases the columns are usually associated with ancillary packinghnpacking equipment. The most widely used technique is that of axial compression, although alternative approaches such as radial compression have been used. In axial compression the slurry is introduced into the column and then compressed by moving the end cell, expelling excess solvent. In small diameter columns the bed may be stable for many runs but in larger diameter columns, particularly with spherical matrices, the bed may settle further, giving rise to voids and loss of efficiency. In these cases the use of equipment offering constant bed compression will alleviate this problem, as the end cell will automatically adjust to take up any voids formed during running. The continuing development to produce more rigid polymeric gel matrices for low pressure chromatography has now seen the adoption of axial compression in this area, enabling both faster packing, unpacking, and automatic compensation for void formation. As discussed earlier columns, in particular large or high pressure ones, may be classified as pressure vessels and be subject to regulations which differ from country to country. Besides the column needing to meet initial design and test requirements, regulations may require an annual inspection or retest.

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1 Chromatography Systems - Design and Control

1.4.2 Pumps The main requirements of the pump are that it provides stable and reliable flow rates over the range required. There are a number of pump types available, including: peristaltic, centrifugal, gear, lobe, plunger, and diaphragm. A number of issues relevent to selection of the most appropriate pump are considered below. As stated the most important criteria is the provision of a stable and reliable flow rate over the entire range required irrespective of changes in system backpressure. For this reason positive displacement pumps (piston, diaphragm) are preferred, and are the only option with high pressure systems. In such cases diaphragm pumps, although generally more expensive, offer greater reliability than piston pumps due to the lack of the piston seal, failure of which will cause leakage. On high-pressure diaphragm pumps the diaphragm is usually driven by hydraulic fluid. Possible contamination of the process stream, owing to rupture of this diaphragm, can be eliminated by selecting double-diaphragm versions. In this case two diaphragms are fitted together with a sensing element that detects if either of the diaphragms has ruptured. This enables an alarm to be signalled while still permitting completion of that process run. The even load distribution on the diaphragm, when driven hydraulically, results in very long service intervals between diaphragm replacements. Low pressure applications can also benefit from the use of diaphragm pumps, both with hydraulically driven, and low cost direct mechanically coupled diaphragms. However, on large flow rate sytems (10-20 L min-' and above), gear or lobe pumps offer significant cost advantages over diaphragm or piston pumps. These pumps, not being positive displacement, will produce a decreasing output as back pressure increases, but this can be compensated for by having automatic feedback control of the pump via a flow meter. Centrifugal pumps are not generally found on chromatography systems as they are not readily available in the flow rates required. Peristaltic pumps are commonly used in simple low pressure laboratory applications and have the advantage of offering, in principle, the 'cleanest' pump as the process stream only contacts the tubing. The disadvantage is that the tube may split, resulting in loss of valuable of product. Although this risk can be reduced by routinely replacing the tubing or utilizing special pump heads that can be stopped in such a position as to seal the ruptured tube if a cessation in flow is detected, peristaltic pumps are not usually selected for production systems. One disadvantage of positive displacement pumps is the pulsation of the pump outlet. It is undesirable that this reaches the column as it can lead to disturbance, and thus loss of efficiency of the packed bed. For low pressure systems, appropriately sized bubble traps placed on the outlet side of the pump can act as pulsation dampeners. On high pressure systems it is necessary to use specific pulsation dampeners. These consist of a gas chamber (air or nitrogen) separated from the process stream by a diaphragm or bellows. As the pump discharges the gas is compressed absorbing some of the pump energy which is then released during the pump inlet stroke thus smoothing the flow. For pulsation dampeners to be effective the initial gas pressure needs to be approximately 80% of the maximum pump discharge pressure. On systems that experience a range of back pressures, due to either changes

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in flow rate or columns, it is necessary to constantly tune the pulse dampener gas pressure to maintain efficient dampening. This can be removed by installing a back pressure regulator downstream of the pulse dampener. This will maintain the back pressure on the pump and pulse dampener constant, irrespective of changes downstream of the regulator, allowing the pulse dampener to operate at its optimal setting. Susceptibility of the sample to shear damage should be considered. Most proteins can be so damaged, and although the chromatography process usually employs a single pass, in contrast for example to ultrafiltration, it may nevertheless be desirable to select low-shear pumps. Lobe and peristaltic pumps have lower shear characteristics than gear or centrifugal pumps. Especially where sanitary concerns are important the interior of the pump must be easily flushed clean with no areas for residue entrapment that could promote microbial growth. Thorough cleaning of the system is facilitated if the pump head is easily removable. All materials of pump construction coming into fluid contact must be compatible with all process solutions, including those used for cleaning. High pressure diaphragm pumps usually feature a stainless steel pump head with PTFE diaphragm. Hastealloy pump heads are available, at extra cost, for use with high concentrations of halides. Medium and low pressure diaphragm and piston pumps are available with various plastic pump heads, as are gear pumps. Lobe pumps generally utilize stainless steel for both the casing and the rotors. If cleaning or sterilizing with either hot CIP solutions or steam, then the pump must be appropriately specified so that damage does not occur, either due to thermal expansion of components, or degradation of seals.

1.4.3 Valves Valves, whether manually or automatically operated, permit selection of solvents, isolation of the column, bypass of the bubble trap and filters, and the collection of individual fractions. Ideally multiport valves would be selected as they minimize dead volumes. Ball valves are readily available in multiport configuration at small sizes (3 - 12 mm), and can be used as three way valves for fraction collection and four way for filter and column bypass. Ball valves are, however, traditionally not considered sanitary, although there are recent additions to the market that are claimed to be. The concern with ball valves for sanitary applications is twofold, firstly the plug of solvent that is trapped within the ball when the valve is closed, and secondly that the area between the ball and the packing seal may form a crevice for microbial growth. In the case of three way or four way valves the plug within the ball may be of little consequence as the valve is always open in one position or the other, but the problem of the crevice remains. The most recent designs of ball valve aim to overcome this problem by closer tolerances between the packing and the ball with the aim of preventing any crevice. Traditionally diaphragm valves have always been preferred in sanitary situations, and these were only available in two way valves requiring manifolding of 3 or more

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valves in order to provide the same function as a 3 or 4 way ball valve. More recently 3 way sanitary diaphragm valves have also become available, thus enabling more compact arrangements to be utilized. In all cases careful attention should be paid to valve layout so as to ensure minimum hold up volume, deadlegs, and orientation of the valves to ensure free draining. Automation requirements also affect valve choice. In low pressure applications, either on the pump inlet side or post column on high pressure systems or throughout on low pressure systems, diaphragm valves can be either electrically or pneumatically actuated, simply and compactly. Ball valves conversely require an actuator that converts the linear motion into a rotary one which is, therefore, more bulky, and expensive. Another advantage of diaphragm valves, discussed in more detail later under automation, is simplified position sensing for feedback control. High pressure systems are restricted to ball valves in the high pressure parts of the circuit as diaphragms are limited in pressure capability (below 10 bar).

1.4.4 Pipework The pipework must meet the requirements for material compatibility, pressure rating, and as discussed earlier be sized to give the minimal pressure drop, while at the same time giving minimal internal volume. Pipework should be arranged so as to aid draining and minimize the possibility of air locks forming. Pressure and sanitary requirements will dictate type of connections. It can be beneficial to minimize these by welding valves directly into the line but enough connections should remain to enable the system to be dismantled for thorough cleaning if required.

1.4.5 Filters All solutions passed through the system must be free of particulate contamination. This is essential to protect the column, where accumulation of particulates will lead to clogging of the packed bed or column bed support, resulting in increased back pressure, non-uniform flow, and thus loss of efficiency. In addition to the column, particulate contamination can also result in damage to pumps, valves, and Pelton wheel or turbine flow meters. Filters can either be incorporated in the system or solutions can be prefiltered. If the latter approach is utilized care must be exercised to ensure that there is no risk of particle contamination subsequent to the filtering step. If filters are incorporated on the system it is common to install separate ones for the mobile phase and sample. In many cases the system includes a single filter for the mobile phase which is bypassed during loading of the prefiltered sample. For maximum hygiene, filtration should be down to 0.2 pm to prevent bacterial contamination. Filters, especially small porosity, may also act as air traps, as once wetted, the bubble point of the filter is such as to prevent air being passed. For this reason they

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should always be installed after the bubble trap. Pressure indication either side of the filter is required, either directly on the filter housing, or using sensors elsewhere in the system, so that the pressure drop across the filter can be monitored. An increase in such pressure drop indicates plugging of the filter and can be used to determine replacement. With production systems in particular, irrespective of whether filters are installed on the system, it is recommended that a filter is still installed prior to the pump during initial commissioning in order to trap any residual particulates originating from fabrication or installation upstream of the chromatograph.

1.4.6 Bubble Trap Bubble traps serve not only as pulsation dampeners, but also to protect the column from the introduction of air. This is only necessary in low pressure systems, with the ‘soft gel’ polymeric matrices. Small amounts of air entering a silica based matrix will have no deleterious effect on the matrix, although it may result in flow irregularities leading to loss of efficiency. The air, however, can easily be flushed out, especially if the column is operated bottom to top. ‘Soft gel’ matrices, however, will contract on drying out resulting in cracking of the bed and serious flow irregularities. Even if the air is subsequently flushed out of the column, the matrix will not recover properly. It is necessary to unpack the column and reslurry the gel to ensure thorough wetting, and then repack. The bubble trap acts to trap small amounts of air that may be introduced during connection and disconnection of the solvent inlet lines. If large amounts of air are introduced the bubble trap may not have sufficient volume to cope and air will be passed to the column. This can be avoided by either sensing the liquid level in the bubble trap and alarming the system if it falls too low, or by fitting an air sensor after the bubble trap also set to alarm the system if it senses air.

1.4.7 Gradient The chromatographic process may require that mobile phase composition is changed during the process, in either a step wise or linear fashion, necessitating the need to be able to mix two solvent-inlet streams in varying proportions. This mixing, to produce a ‘gradient’ of solvent composition, may be achieved in one of two ways. Either by switching a valve between the two solvent streams on the inlet or low pressure side of the pump, the duration of the valve position being in relation to the ratio of solvents required, or by the use of individual pumps for each solvent operating at different flow rates, again in proportion to the ratio required and mixing on the downstream or high pressure side of the pump. On process systems the two pump approach is usually achieved by using a double headed diaphragm or piston pump. The individual pump heads are fitted with

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automatic stroke length adjusters which are used to vary the flow output from each pump head in proportion to the ratio required. The pump heads are driven from the same motor shaft, the speed of which is varied, to alter the total flow rate. Although both pneumatic and electrical stroke length adjusters are available, pneumatic is normally preferred as it is less expensive and responds more rapidly. High pressure mixing, as described, has the advantage of being less susceptible to differences in solvent inlet tank levels than low pressure mixing but results in only half the installed pumping capacity being utilized. Low pressure mixing is usually less expensive but in order to ensure greatest accuracy it is important to ensure the supply pressures on both solvent inlets are similar. This can be achieved by either prepressurization of solvent tanks, or the use of small constant header tanks. Low pressure mixing also allows the full flow range of the pump to be utilized. However, with positive displacement pumps, which have a distinct inlet and outlet phase it is important that the valve cycle frequency does not coincide with the inlet/outlet cycle of the pump, as this would result in only one solvent being pumped. It is necessary to ensure, especially where the flow rate is being varied by altering the pump speed, that the valve cycle frequency is automatically varied to avoid resonance with the pump. The ability to mix solvents may have benefits other than just gradient generation. It can also be used to dilute concentrated buffers with water thus saving on tank and space requirements. In a production system gradient reproducibility is more essential than absolute accuracy.

1.5 Instrumentation The degree and type of instrumentation can vary on a system but in all cases the provision of instrumentation is to enable the operator to better monitor the status of the chromatographic separation, providing him with the opportunity to take actions or alter parameters as required or merely provide a record of the separation. The need for instrumentation can be considered in two categories; firstly to ensure that the system is performing as required, and secondly to monitor the actual chromatographic separation. In the first case this includes monitoring of such parameters as flow, pressure, temperature, air inclusion, and takes place before the column in order to ensure the conditions within that column itself are maintained, such as to effect the desired separation. In the second case monitoring is performed on the column outlet with the main objective of identifying when the individual fractions elute. Post column detection is a very important part of analytical chromatographs where sophisticated diode array detectors may be utilized, and base line correction and peak area calculations are used for concentration determination of individual fractions. In contrast, in process chromatography, post column detection tends to be used purely qualitatively and may even be absent. Although detection may be required during method development and at the pilot stage, a particular chromatographic separation may be so consistent that it may be

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sufficient to fractionate the column eluent on the basis of time or volume. On the other hand the utilization of a post column detector could alert the operator to a divergence from the expected performance earlier, in time to perform corrective action and save valuable product from loss or rework. In practice even though a particular separation may be well characterized by the time it is put into production there is still the likelihood of variation due to minor changes in concentration and impurities in the sample feedstock, changes in solvent composition, and variation in column performance. Consequently it is desirable to monitor the column eluent, not only to confirm at the earliest opportunity the correct operation of the process, but also enable modification of fraction collection to take account of these minor process variables.

1.5.1 Flow The solvent flow through the column is a critical parameter. Changes in flow rate will not only affect the efficiency of the separation but will also influence the elution time of fractions. Flow rate must, therefore, be maintained within well defined limits and deviations from this alerted to the operator. Analytical systems rarely utilize flow meters relying instead on highly accurate ‘dosing pumps’ where the flow rate may be accurately calculated from the pump speed. On preparative chromatographs it is usual to install flow meters even on those systems utilizing diaphragm or piston positive displacement pumps. The reason being that the accuracy of these pumps is less than their analytical counterparts, coupled with the fact they are often operated at their flow rate extremes, where the flow inaccuracy is even greater. A flow meter can be used, not only to monitor flow rate but by use of a feedback control circuit, also increase or decrease pump speed to maintain constant flow. For conducting solutions ‘Magflow’ meters are preferred. These work on the electromagnetic induction principle where there are no moving parts, the electrodes can be contained in an inert material and there is no pressure drop. Consequently they are the method of choice for sanitary systems but will not operate with deionized water or solvents where the conductivity is below 5 pS. They are also not available for small pipework systems (< 6 mm). In these cases flow meters utilizing a turbine or paddle wheel are utilized. The turbine or paddle wheel is located in a housing incorporated in the pipework. Imbedded in the turbine or paddle wheel are magnetic elements which, as it revolves, are sensed by a proximity sensor mounted outside the pipe. The speed of revolution being proportional to flow. It is important when selecting a sensor to take full account of flow range, solvent viscosity, and flow cell pressure drop. Preparative systems, particuarly development or pilot ones, may be required to operate over a wide dynamic flow rate range with different buffer compositions or solvents. In such cases it may be necessary to operate at the limits of flow meter performance where inaccuracy is greatest. In such cases sensors should be selected such that those flow rates which are most critical to the process (ie, elution) fall

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within the most accurate range, and those less critical (ie, cleaning) fall at the extremes.

1.5.2 Pressure Monitoring of pressure within the system serves not only to warn of a potential overpressurization, due for instance to a valve sticking, but also gradual increases in back pressure indicate column or filter plugging. Similarly a sudden drop in back pressure may indicate a failed connection. For maximum safety the system, including column, should be protected from overpressure by a mechanical pressure relief valve (spring seat or bursting disc type) set to relieve at the lowest individual component, maximum pressure rating. Columns particularly glass or plastic may have a pressure rating significantly lower than that of the pump and pipework and must be suitably protected. Bourdon tube type pressure gauges may be used for visual pressure indication and may be fitted with limit switches for alarming. Increasingly though pressure monitoring is achieved by the installation of strain gauge pressure transducers linked to a suitable controller. This enables not only a constant monitoring of pressure to a chart recorder or other data logger, but also gives the operator the ability to easily change pressure alarm set points, for instance, to protect an easily compressible matrix. Location of the pressure sensor will be in the high pressure circuit before the column, although occasionally it may be necessary to install a sensor after the column to protect pressure sensitive probes, for instance, pH or refractive index. Installation requires careful consideration to reduce increased hold up volume and dead legs, and so simple T pieces should be avoided. Bourdon type gauges should be fitted with diaphragm fluid separators, especially on sanitary systems. Pressure transducers are usually encased in a stainless steel diaphragm faced body that can be incorporated directly onto a pipework connection. On systems with positive displacement pumps, location after the pulse dampener or bubble trap is preferred to reduce the likelihood of erratic readings due to pulsation.

1.5.3 Temperature Active temperature control of preparative or process chromatographs is rarely used but temperature monitoring may be included in cases where temperature fluctuations may occur. These may arise from heat of mixing of feed solvents during gradient generation, from storage of feed solvents at different temperatures, or from failure of on line heat exchangers. Measurement will be by resistance transducer coupled to a suitable controller.

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1.5.4 UV/Visible Adsorption Monitors Most biologically active compounds, whether proteins or synthetic drugs, absorb light in the UV or visible spectrum and so can be detected by absorption monitors. During process development flexibility is required, so a detector with both variable wavelength and pathlength is most suitable; the latter to prevent saturation of the detector at high solute concentrations. The use of a variable wavelength detector on the process scale is, in contrast, not usually necessary and is probably best avoided. The UV absorption spectra are sufficiently wide to usually enable enough sensitivity to be achieved at one of the available spectral lines in a fixed wavelength detector. Fixed wavelength detectors have the advantage of lower cost, simplicity, and hence reliability. Most systems will use 254 nm for detection of small molecular weight synthetic components and 280 nm for proteins. In preparative chromatography, sensitivity is usually only a problem in terms of saturation of the detector due to high solute concentrations. Variable pathlength flow cells are available where the effective pathlength can be reduced down to 0.3 mm if required. Again in dedicated production units, where flexibility is not required, fixed pathlength flow cells are preferred, and these are available in a range of present pathlengths, enabling them to be matched to the absorption characteristics of the sample.

1.5.5 Refractive Index Although refractive index detectors are common on analytical systems due to their universal nature, they have a number of disadvantages for process use. Their advantage is their ability to detect compounds such as carbohydrates, lipids, and simple peptides, which possess no measurable UV absorption. Offset against this is their incompatibility with gradient operation and often their need for a flow splitter to cope with process scale flow rates. This latter is undesirable for any detector, due to the fact that the relative flow rate through the detector cell can vary depending on solvent viscosity and flow rate. The vast majority of refractive index detectors are differential detectors where the refractive index of the sample is measured relative to a reference liquid. This enables them to be used in a wide range of applications but requires a fairly delicate flow cell. Absolute refractive index detectors are available which although only covering a limited range and less sensitive, are more robust and have sensor probes which can be easily inserted in the process stream. These are especially suited to explosion proof applications.

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1.5.6 pH/Conductivity pH and conductivity sensors are used not only for monitoring conditions during the chromatography run, but also in the control of regeneration, cleaning and equilibration. pH probes are not ideally suited to in-line process use, due to their sensitivity to fouling, requiring cleaning and recalibration. They are also sensitive to temperature and have limited pressure capability. Although double reference-electrode self checking systems are available, they are not very compact and result in large internal volume flow cells. Consequently single electrode systems are commonly used but these must have provision for easy removal of the electrode for cleaning, calibration, and replacement. Storage of the system will require the electrode to be kept wet, either in situ, or removed from the unit. Similarly it may be necessary to protect the electrode during cleaning from, for instance, hot caustic solutions, in which case a self sealing fitting or other means of isolation is required. Conductivity sensors may be either the contacting type or the induction type. The contacting type may utilize either 2 or 4 electrodes. The four electrode cell permits measurement of higher ranges (up to 200 mS), and the correction of polarization effects due to deposits forming on the electrodes. Two electrode types are suitable for lower conductivity ranges and are easier to insert into a flow through cell but cannot compensate for polarization. Four electrode types are incorporated into the system in a similar manner to pH probes. The induction sensor works on the principle of the solution coupling the magnetic field of two magnetically isolated induction coils and has the advantage of not having electrodes; therefore there is no polarization and no decrease in performance owing to fouling. They are also capable of a much higher measurement range, up to 2000 mS. The disadvantages are that these units are currently available in sizes that require relatively large hold up volume fittings. If only one pH and/or conductivity sensor are utilized it is usual to locate them after the column to monitor the column eluent. They may, however, be additionally located prior to the column to act either in an alarm only mode to protect the column from extremes of pH due, for instance, to failure of a caustic dilution step, or to enable equilibration of a column to be continued until the pre and post column pH or conductivity measurements are equivalent.

1.6 Control The simplest chromatography system can consist of manually operated valves, a variable speed pump, detector and chart recorder to log the detector output. As already discussed, however, significant advantages can be obtained by automating the unit. This can cover just gradient generation or encompass full process control including sample injection, fraction collection, column regeneration, cleaning cycles, together with automatic alarm handling and documentation of process variables.

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The degree and type of automation will be dependent on the use to which the chromatograph is put. A development unit requires flexibility such that it can be used with a range of columns and chromatographic techniques. The operator needs to be able to easily change operating parameters such as solvent selection, flow, and pressure limits. The objective of such a development unit is to develop and test fully automated methods for later use in production. Consequently the ability to easily program and edit such methods is also of high priority. In contrast a dedicated production unit running only one separation process will require to have the method changed infrequently, if at all, and then only by authorized individuals. Consequently in this case the emphasis will be placed on simplicity, reliability, and security of the user interface. In both cases a record of the separation process will be required but again needs may differ. On a development unit it is usually sufficient to monitor and record process variables such as pressure, UV and the operating state as signalled by the control system. With production units the requirements of GMP compliance may dictate that not only is the intent of the control system monitored, but also the actual state of the hardware, this requiring the installation of valve position feedback. At first sight it may seem desirable to monitor all variables, including all valve positions. However, each component monitored will require at least one interface with the controller which will increase the cost, may result in a larger than really necessary controller being utilized, and may in itself lead to an error due to the greater complexity of the system. Only valves critical to the operation, therefore, need be monitored. In the case of two way diaphragm operated valves it is sufficient to use a single sensor to monitor the actuated position, but with multi-way valves or rotary valves it is necessary to sense both valve states, thus requiring two sensors, as the valve may fail in an intermediate position. Control automation can be achieved in essentially one of three ways, or any combination of these, namely: dedicated controller, general purpose controller, or computer based systems. All three are based on a microprocessor and as a result the distinctions and capabilities somewhat overlap.

1.6.1 Dedicated Controller Dedicated controllers are designed and built for a specific application and as a result tend to be used where there is a market or requirement for a significant number of identical units. In these cases the development cost can be offset by the lower unit cost through multiple manufacture. The dedicated controller has the advantage that it is usually easy to operate and ready for use. Its disadvantage is that it is usually programmed in assembly language, has a specific input/output configuration and thus its ability to be modified or enhanced is severely limited. Although a dedicated controller could be utilized for the complete chromatograph, and is used as such in analytical instruments, where unit numbers justify its use, its use in process chromatographs is usually restricted to specific instruments, for instance UV monitors, or gradient controllers, which are then integrated into the whole.

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1.6.2 General Purpose Controller These controllers are designed to perform a wide variety of tasks and as such are usually expandable often in modular form to enable the processing capability and input/output configuration to be matched to requirements. Examples of these types of controllers include programmable logic controllers (PLCs) and a large variety of industrial controllers. PLCs have long been used for industrial control purposes, being considered rugged and reliable for these environments. Although more flexible than dedicated controllers, with programing in high level languages including Ladder Logic, BASIC, C, etc., they still require skilled personnel to make program changes. For this reason PLCs tend to be used in such a way that the basic program does not require changing once commissioned. System parameters that need to be adjusted during the process or from process to process are dealt with by separate dedicated controllers that can easily be adjusted by the operator. PLCs are, therefore, more suited to production applications than development situations.

1.6.3 Computer-Based Systems In this case a computer, usually P C based, in conjunction with an I/O interface is used to control and monitor the system. The advantage of the P C is the power and flexibility of its operating system enabling it to accommodate a wide range of sophisticated software programs. The benefits of the computer-based system depends very much on the way in which this additional power is utilized. With dedicated controllers and PLC-based systems data logging is usually restricted to use of a chart recorder. The addition of the P C allows both the utilization of more extensive data logging, recording not only sensor variables, but also events such as valve switching and alarm conditions, together with the ability to replot data graphically, and incorporate into different report formats. Although the P C is powerful enough to perform all the control functions required of the chromatograph, extensive use of its capabilities by the user for data manipulation or method composition may result in undesired deterioration in its operating speed for the control functions. It is common, therefore, to link a dedicated or general purpose controller to the PC. This is used for direct control of the I/O, together with control of tasks such as gradient generation and peak detection for fraction collection. The P C is then used for data logging and user interface to the controller. This approach enables the system to perform all the functions of a PLC or dedicated controller, but with a much more user friendly interface allowing the operator, with no special programing skills, to readily reprogram methods and view and manipulate data. In order to control the chromatograph the user needs to be able to see the system status, control it either manually or by programed events, and to document what has occurred (Table 1-3). Several chromatographic control systems exist, providing essentially the above functions, but by way of example I have selected to describe that used on Amicon chromatographs with which I am most familiar.

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Table 1-3. Requirements of a chromatography controller. Control - Manual override of all output functions - Automatic switching of valves/events on basis of time, accumulated flow or dependent on sensor outputs Control of flow and pressure Gradient generation Peak detection

System status Process status display of valve position and sensor readings Alarm display-pressure, flow, air, pH, conductivity, valve position, tank level

Data logging Of all variables, events and alarms

Output

To printert’plotter, floppy disk, network, other software programs for report writing

Access Security of access to different levels of control limited to authorized personnel

Safety Safe power-up-shutdown sequencing

The Amicon approach is to use a general purpose industrial controller linked to a PC. The industrial controller, based on an enhanced Z80 processor is utilized for control of all I/O, both digital and analogue, and communicates to the PC via an RS232 interface. The PC provides the user interface via a software package running within the Microsoft Windows graphical environment. A series of display screens allows the user to view the system status, control it, and display logged data. Interaction by the operator is via either the keyboard or pointing device (trackball/mouse). Security of access is provided for, by the system initially booting up with a screen requiring the operator to enter a password. This can be set to allow different levels of access to the system, for instance, an operator may only run methods, whereas a supervisor also has the ability to edit method files if required. On correct entry of the password the system status display can be accessed. This takes the form of a picture display of the system showing all valves, pump and sensor outputs (Fig. 1-4). Colours are used to display the active flow path and windows are used for numerical display of all sensor outputs. The display is also interactive, allowing the operator to switch valves and pumps by moving the cursor to the appropriate ‘switch’ on the display and clicking the mouse. Thus this display is all that is required to both monitor and control the system manually. In addition, should an alarm condition occur a separate alarm panel is displayed, indicating which alarm has been triggered.

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Fig. 1-4. Example of control screen of Amicon K Prime chromatograph, showing interactive control picture and log display.

For automatic operation it is first necessary to ‘write a method’. This is achieved by selecting the method editor and literally building up a method. A method consists of a series of programed events that the controller will then execute. The events can either be programed on the basis of elapsed time or cumulative flow; the latter having the advantage that any fluctuations in flow, for whatever reason, will be accounted for. All possible events in the chromatograph are listed and can be selected from a look up table and the time at which they are to occur entered. Several events can be selected to occur at the same time if required. Events may include not only valve switching, but also changes to flow rate, gradient composition, alarm set points, peak detection parameters, and data logging criteria. The use of subtimers also allows events to be based conditional on certain detector outputs, exceeding either above or below user entered values. This is particularly useful for column regeneration or equilibration where, for example, a buffer wash is initiated and continues until the pH falls below a preset value. When completed the method can be stored to floppy disk or printed out as hard copy. To run automatically the appropriate method is selected and initiated. At this point it is downloaded to the controller which then assumes control, reporting data

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back to the PC as required. However, during an automatic run the operator may override any action by manually controlling the system via the interactive screen display. Data logging is performed in both the manual and automatic modes and comprises a graphical file of all analogue variables and a tabular file of all events, such as valve switching, pump on/off, changes in flow set points, and alarm conditions. All analogue data is collected to the file but only that selected need be displayed. In this way there is no danger of data being lost or not recorded, but to avoid undue confusion only that necessary at any time can be displayed. The tabular log will record all set points and any changes made to them during the run. All valve switching is recorded and if valve position feedback is utilized the log will record this also, together with any manual overrides executed during an automatic run. This provides, therefore, a full record for GMP purposes. The data can be saved either to floppy disk or printed out as required. All screens are interlinked so that a change to a valve position, whether manually or automatically, will be signalled simultaneously on the system schematic display, as well as in the tabular log. Chromatography separations may vary considerably in their duration, from minutes for HPLC to several hours for low pressure applications. Continuous recording of all variables can potentially result, therefore, in very large data files. The size of the data file will depend on the sampling rate, the slower this is the smaller the file for a given period, but the danger is that important data is then not recorded. One approach is to select different sampling rates for different variables, for instance, a fast sampling rate is necessary for UV absorbance if short peaks are to be detected, whereas a slower rate for pressure monitoring would be acceptable. An alternative approach taken on the Amicon system is to allow the user to program each variable so that only changes in value greater than a user set percentage of full scale are reported to the log file, thus greatly reducing the data file while ensuring significant changes are recorded. In addition for ease of analysing the process later, all variables can additionally be printed out at preset times (ie, every 15 min) for comparison. Fraction collection can either be on the basis of time or volume, or can utilize a peak detection routine, most commonly based on the UV signal, to trigger the start and finish of each fraction. Although sophisticated peak detection routines are utilized on analytical systems, they are not always applicable to preparative or production systems. Peak shape, under analytical conditions, is usually well defined over a short time period, in preparative applications this is often not the case. The simplest and most reliable method is timed collection and can be used for reproducible separations or where several fractions are to be taken and pooled later. Peak detection may either be based on the amplitude of the detector signal exceeding a preset threshold or its slope. Threshold detection is easy to set up but is affected by changes in base line level, caused by long term drift or zero calibration errors. Slope detection is less affected by base line changes but may be inadvertently triggered by a small peak, shoulder, or other signal noise, not present when the set up parameters were entered. The choice of method is very much dependent on the elution profile of the particular separation, but for the majority of process or production purposes, threshold

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Desixn and Control

detection, coupled with time collection, will be preferred. Unlike laboratory fraction collectors, where a large number of fractions are collected sequentially, process chromatographs will minimize the number of fraction ports required by effective use of the automation provided. For instance, the product may be collected in one main fraction with the front and back of the peak being collected in other fractions. All other eluent may be directed to a fraction waste tank that can be analysed for active product prior to disposal. Regeneration, cleaning and equilibration solutions would be routed direct to a waste tank, thus requiring only a total of 5 fraction ports. The controller and its interface may be located directly with the chromatograph, or separately. For development work an integrated unit is desirable as frequent interaction with the chromatograph is required. In contrast a dedicated production unit will require little interaction, but may need to be monitored from a central control room. As it is desirable to keep cable runs to a minimum it is usual, when providing remote control, to site the controller itself with the chromatograph for direct connection to the I/O, and provide only the user interface, usually a PC, remotely. This has the advantage of only requiring an RS232 or similar interface between the controller and PC. Industrial environments may dictate the need for industrialized PCs which are less susceptible to dust and have more robust power supplies compared with desktop equivalents, but in either case the use of UPS (uninterruptible power supplies) may be advisable to ensure continuous running of the chromatograph.

1.7 Validation Any manufacturing process aimed at producing a pharmaceutical product for either human or animal use must be approved by the appropriate regulatory authorities, for instance, the US FDA (Food and Drug Administration) or the UK DHSS (Department of Health and Social Security). In order to obtain approval the process must be validated [S]. This is defined by the FDA as follows [9]: “Process validation is establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its pre-determined specifications and quality characteristics”. Validation documentation needs to include not only evidence of the suitability of raw materials of a process, but also the suitability of materials of construction of process equipment, in terms of purity and traceability, and the performance and reliability of equipment and systems. Validation must begin right back at the design stage as it is at this point that the specifications and operational limits of the system are defined. This preliminary step ensures that the equipment will be suitable for its intended function, and will not be adversely affected by its operating environment. Once the system is manufactured and installed it is subject to an installation and operational qualification (IQIOQ), the objective of which is to determine that the equipment meets the defined specifications.

1.7 Validation

29

Equipment evaluation, at this stage, should be based on actual testing, and not solely on manufacturers specifications. The equipment configuration performance characteristics, maintenance procedures, repair and parts list, and calibration methods, must all be documented at this stage. Test procedures must test the equipment at the limits under which it will be required to operate. This approach is known as ‘worst case testing’. It does not imply testing to the point of system failure, but only to the outer ranges of the operational limits that are established by the user. ‘Worst case testing’ should not unduly stress the equipment if components have been correctly chosen with respect to the operational requirements. Validation applies to not only the hardware and instrumentation, but also to the control software [lo]. It needs to be demonstrated clearly that the software accurately and reliably does what it claims to do, and to this end it should have been tested and verified, both at the program level and the system level and be fully documented.

1.8 Conclusion It has only been possible in the space available to give a general overview of process chromatography systems, but in so doing I have attempted to identify those criteria that need to be considered in selecting or designing such a system. In particular the use of the system will determine the capability, flexibility, and reliability required; careful attention to these points will help to ensure the most effective process solution. Examples of systems are shown in Fig. 1-5.

References [I] Cowan, T.T., Thomas, C.R., Process Biochem, 1988, 23, 5-11. [2] Code of Federal Regulations: Food and Drugs 21, Parts 170- 199, Washington: Office of the Federal Register, National Archives and Records Administration; 1991. [3] The United States Pharmacopeia National Formulary X X I I , Rockville: United States Pharmacopeial Convention Inc.; 1990; pp. 1497- 1500. [4] A D Merkblatt N6: Glass; NI; Plastic, W2, W4, W7 and B8: Stainless Steel, Essen: TUV (Vereinigung der Technischen Uberwachungsvereine). [5] Boiler and Pressure Vessel Codes, Section 11 Material specifications Section Vlll: Stainless Steel, Fairfield, NJ: American Society of Mechanical Engineers; 1986. [6] BS 2771: Part 1: Electrical Equipment of Industrial Machines Specification for General Requirements, Milton Keynes: British Standards Institution; 1986. [7] White, P. E., Stainless Steel Industry, 1988, September, 1-4. [8] Chapman, K. G., Pharin Technol, 1991, October, 82-96. [9] Guideline on General Principles of Process Validation, Rockville; Centre for Drugs and Biologies and Centre for Devices and Radiological Health, Food and Drug Administration; May 1987. [lo] Bluhm, A.R., Pharm Technol, 1989, November, 33-40.

30

1 Chromafograpl?y Sysienis

-

Design and Control

Fig. 1-5. Examples of high pressure explosion proof (a) and sanitary low pressure chromatograph systems (b).

References

Fig. 1-5 b

31

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2 The Practical Application of Theory in Preparative Liquid Chromatography Geoffrey B. Cox

2.1 Introduction Until recently, preparative HPLC was carried out with little or no regard to theory. This is not surprising, since it was not possible to draw practical conclusions from the theories then available. In fact, the predominant view of preparative LC was that once the mass load exceeded the linear range, almost anything could happen, and probably would. Practical experience was that peak shapes were strange and unpredictable and that on occasion components would inexplicably appear as impurities in all components collected in the separation. Under such circumstances, it is not very surprising that preparative HPLC was regarded as a last resort for large scale purification. Following the work of two groups - those of Guiochon and Snyder - the situation has changed dramatically. Theories which have been verified practically are now in place and the use of preparative chromatography is established upon a sound knowledge base.

2.2 Why Theory? Even though the theories of preparative HPLC have now largely been developed, when the practising chromatographer attempts to apply them to a practical problem such as optimisation of a preparative separation, a number of major obstacles appears, some of which are often felt to be insurmountable. Not least of these is the problem of the determination of the isotherms, especially the competitive isotherms between several solutes, when the components are not available in a pure state. Indeed, the practising chromatographer frequently reaches this point and declares that it is quicker to solve the problem by trial and error rather than by making all of the (frankly) tedious and sometimes difficult measurements required to enable the necessary computer simulations for the solution of the problem to be carried out. In cases where all that is required is the small scale isolation of pure materials from a mixture this position can appear to be reasonable, since at least some preliminary preparative separation would be required to generate enough product for the study of the iso-

34

2 The Practical Application of Theory in Preparative Liquid Chromatography

therms, by which point the aim of the work would have been completed! Where a purification is to be scaled up for production, there is a much stronger case to be made for a proper study of the separation. Even here, there is frequently opposition to such a study on the grounds that the development of the method of purification has to be performed rapidly and that there is no time for the more ‘fundamental’ study of the separation. In such environments, what use, then, is theory? The aim of this chapter is to demonstrate how a knowledge of the theory of preparative HPLC is vital to the development of sound preparative methodologies in the shortest possible time. A further benefit is that the theoretical knowledge assists markedly even in those separations for which nothing is known (or is ever likely to be known) about the absorption isotherms of the solutes concerned. Finally, a sound knowledge of theory is essential for optimisation of methodologies for production scale operation and also assists in the recognition of those cases for which a deeper study of the separation is necessary in order to develop a reasonable procedure.

2.3 How Much Theory? Anyone who is involved in the scale-up of separations to preparative levels should know at least some of the theoretical background. This is because without such a knowledge, the odd behaviour of some solutes under conditions of mass overload can be misinterpreted and vital clues to the best mode of separation can be missed. This is not to imply that everyone needs to be a theoretical genius or must be involved in the measurement of isotherms or running computer simulations. In many cases, especially for separations at the laboratory scale, all that is necessary is an understanding of the major results of the simulations which have been done as well as a qualitative knowledge of how the separations can deviate from the predicted result. Once this is known, it is relatively simple, after the analysis of a few chromatograms, to understand the basic properties of the solutes and therefore the types of interactions and their related effects in the separation. From this knowledge other experiments to exploit this understanding can readily be devised. On the other hand, if it is desired to optimise a separation as a production process, much more needs to be done. As noted later in this chapter, an extensive series of experiments is essential in order to build a data base from which the optimal conditions can be calculated.

2.4 Single Solutes Although preparative HPLC is by definition a technique of separation (which implies that at least two components must be present in the mixture), it is useful to consider the situation when one component only is overloaded. This is not only the sim-

2.4 Single Solutes

35

plest case, but it can be also a good first approximation to the situation where a major component is to be purified which contains only small amounts of minor impurities. We will see later what happens to those impurities. Our first consideration will be the case in which a small injection size is used, and the mass of sample is increased. This situation is that of muss overloud. It requires a high sample solubility, although, as we will see later, larger volumes can be used without seriously compromising the results. The other extreme is where the sample is so poorly soluble that its volume is the limiting factor in that a large volume may be introduced without raising the mass injected to a level sufficient to cause significant mass overload effects. This latter situation is that of volume overload. This will be considered later.

2.4.1 Mass Overload The first symptom of mass overload is seen as a broadening of the chromatographic peak as the mass of sample is increased. This is measured as a lowering of the efficiency (reduction in the number of theoretical plates) and increase in peak asymmetry, but as mass load is increased it often results in triangular shaped peaks which show typically a peak maximum at a reduced retention time and a tail which extends to the retention time of a peak resulting from an analytical load. Other, much more bizarre peak shapes can also be found. These represent cases where special interactions between the solute molecules and the stationary phase, the mobile phase or each other occur. Theoretically, chromatography may be described as a combination of thermodynamic and kinetic processes. The thermodynamic aspects control the retention and shape of the peak whilst the kinetic aspects control the sharpness of the band. Together they define the resolution between components. The fundamental thermodynamic parameter is the distribution coefficient of the solute between the phases. This is given as the ratio between the concentrations of a solute in the stationary and mobile phases.

The changes in the distribution coefficient with changes in the solute load can be shown by a graph plotting the stationary phase concentration against that in the mobile phase. Such a plot is known as the Adsorption Isotherm for the system. The slope of the graph gives the distribution coefficient; it is a simple matter, once the ratio of the phases in a column are known (not in itself always a simple task to determine) to derive the capacity factor for the solute from the equation relating the two parameters

k'=K@ where @ is the phase ratio, K/Vm.

(2)

36

2 The Practical Application of Theory in Preparative Liquid Chromatography

At low loads, the relation between the concentrations is linear and the distribution coefficient is constant. This is the region in which analytical chromatography is (or should be) carried out since the retention times and peak widths are independent of the mass load. At higher mobile phase concentrations the plot deviates from linearity. It is clear that here the distribution coefficient - and therefore the capacity factor - does not remain constant. Changes in the capacity factor from its value for analytical scale sample sizes cause a distortion of the peak; those parts of the peak which contain high concentrations of the solute will have a different capacity factor and thus will move with a different velocity from those parts at low concentration. The form of this distortion will depend upon the shape of the isotherm at high load. This can be derived if one makes some simple assumptions. The retention of a species is supposed to occur through a 1 : 1 competition with mobile phase molecules for active sites on the surface of the packing material. As the concentration of solute molecules adsorbed on the surface of the packing material increases, there are fewer sites remaining for the competition to take place. This reduction in the number of sites changes the simple equation shown to above to:

This is the simplest non-linear relation which is exhibited by single solutes under mass-overloaded conditions. The relation in Eq. (3) is the Langmui; Adsorption Isotherm. Other isotherms relating the stationary and mobile phase concentrations are possible, depending upon the individual properties of the solutes, mobile phases and packing materials. Very many solutes follow the Langmuir isotherm, which is one

100 1 80

--

60

--

40

-~

0

7

5

,

I

10

15

20

cm Fig. 2-1. The Langmuir isotherm. Data for benzyl alcohol in 30% aqueous methanol using Zorbax ODS at 25°C.

2.4 Single Solutes

0

2

6

4

8

10

12

14

31

16

time (min) Fig. 2-2. Peak shape of a solute following the isotherm depicted in Fig. 2-1; loading at 5 % of saturation capacity.

reason for its general use in the derivation of equations and relations in preparative chromatography. The isotherm for benzyl alcohol in a reversed phase system is shown in Fig. 2-1. The peak shape of a solute which follows the Langmuir isotherm is shown in Fig. 2-2. Some solutes show quite different isotherms. A number of compounds, many substituted nitrophenyls for example, exhibit ‘s’shaped isotherms such as that shown in Fig. 2-3. This is because the solute molecules associate on the surface of the pack-

200

150 100

50 0 0

5

10

cm Fig. 2-3. An ‘S’ shaped isotherm.

15

20

38

2 The Practical Application of Theory in Preparative Liquid Chromatography

0

2

4

6

8

10

12

14

16

time (min) Fig. 2-4. Peak shape of a solute following the isotherm depicted in Fig. 2-3; loading at 5 % of saturation capacity.

ing as they are adsorbed. This self association enhances the adsorption, leading at first to a disproportionate increase in the stationary phase concentration. This, in turn, gives a larger capacity factor (and retention) at these higher concentrations. Ultimately, as the solute concentration becomes very high, the surface available for adsorption again becomes limiting, the curve turns downward and the capacity factor for the high concentration part of the peak decreases. A typical peak shape is produced by such behaviour, as shown in Fig. 2-4. The equations which describe the non-Langmuir isotherms are often complex and cannot be derived from simple principles but are often approximated by polynomials fitted to the experimental data. 2.4.1.1 A Simple Model: Single Component which Follows a Langmuir Isotherm

To introduce the idea of modelling preparative separations, we can first consider the simplest (useful) case. This is a solute which follows a Langmuir isotherm. Three approaches have been taken to render this case simple enough to develop equations which allow the calculation of the position and approximate shape of the band. The first, due to Knox and Pyper [l], used an approximation to the isotherm. To a first approximation and at low concentrations, the Langmuir isotherm follows an equation of the form:

c, = Cm(l+Cm)

(4)

This form of the isotherm results in a linear rate of change of the capacity factor with mobile phase concentration and gives rise to a truly triangular peak shape. Knox also concluded that the efficiency of a peak under mass overloaded conditions

2.4 Single Solutes

39

changed with the square root of retention, just as for analytical chromatography (true for his triangular peaks). From these relations, together with the assumption that the total peak variance was made up from the sum of the variances of the kinetic and thermodynamic contributions, he derived an equation for the plate number of a peak under conditions of mass overload. This, using the notation of Snyder et al. [2] is: N

1

where N is the efficiency measured at the mass load w,,No and k', are the efficiency and capacity factor of the peak in a chromatogram run at an analytical load and w, is the saturation capacity of the column, which corresponds to the mass of a monolayer of solute molecules on the surface of the packing material in the column. It should be noted that the derivation used by Knox makes some mutually contradictory assumptions - of light overload for the isotherm approximation and of heavy overload in that the bands were the shape of right angled triangles (ie the mass overload effects far outweighed the kinetic band broadening). The inaccuracies arising from the simplifying assumptions notwithstanding, this work was vital to the advances in understanding of preparative LC. The predictions from Eq. (5) were found by Snyder et al. [2] to fit experimental data more closely if the 114 factor in the denominator was changed to 3 / 8 . In fact, this fraction changes with the mass load and presumably compensates for the isotherm approximation. These workers also developed an equation to give the capacity factor of a mass overloaded band starting from the Langmuir isotherm itself, assuming only that the peaks were triangular. This gave a relation which is more accurate at higher loads than that derived from the approximate isotherm. The most useful aspect of this is that on rearrangement, it yields an equation for the saturation capacity (w,)of the column: k' kb W , = CNwx----1 + k' kb -k' This value is the load of the solute which would be adsorbed as a complete monolayer on the surface of the packing. It may be measured by two chromatographic experiments, one at analytical load and the other at preparative load. The The preparative run, analytical run yields the capacity factor at low load (Po). measured for the band by the usual equamade at load w,, gives the efficiency (N), tions and using the value of the retention of the peak maximum for the retention time of the band, and the capacity factor (k'),measured at the peak maximum. Due to the derivation of the equation, it applies only to solutes which follow the Langmuir isotherm. It can, therefore, be used to check Langmuir behaviour by measurement of w, at a variety of loads; a constant saturation capacity implies that the Langmuir isotherm is followed. The value of saturation capacity found here may be used in the isotherms employed for computer simulations (see below). This is an easy route to the determination of Langmuir isotherms.

40

2 The Practical Application of Theory in Preparative Liquid Chromatography

Guiochon et al. [3] also arrived at equations for retention and efficiency, based upon the solution of differential mass balance equations for chromatography using the ‘Ideal Model’ of chromatography. This makes the major assumption that the column efficiency is infinite, under which conditions it is possible to reach an analytical solution of the equations. Their equation for capacity factor converges with that of Snyder et al. at high values of efficiency and has the virtue of simplicity:

k’

-=

k:,

1/2 2 [I -Lf ]

(7)

Lfis the loading factor, equal to the ratio of sample load to column saturation capacity (w,/w, in Snyder’s terminology). This equation may be used when the column efficiency is high; above two thousand plates the errors are small. The equation for the loading factor is equally simple:

From a knowledge of the load, the analytical capacity factor and the capacity factor of the overloaded peak (calculated for the peak maximum), the saturation capacity can readily be obtained. By combining the value of capacity factor with the known kinetic band width (wb)of the column, a chromatogram may be constructed. To a first approximation, this is done by first fixing the points at retention times corresponding to the analytical and overload capacity factors. The baseline kinetic bandwidth (given by 4.t,./ is determined and half this value is subtracted from the retention time of the peak maximum to give the start of the peak and half is added to the analytical retention time to give the peak end. At first sight, it seems that the peak variances rather than the band widths should be added, but under mass overload the kinetic and thermodynamic band broadening processes are no longer completely independant. This interdependance means that the variance of the total process is no longer the sum of variances of the individual parts and the addition of band widths turns out to be as accurate a method as any other. If there is little interaction between the components or they are well separated, this approach can give a good approximation to the experimental chromatogram. The equations have the advantage of being rapid and simple, but the disadvantages of being semi-quantitative, susceptible to deviation from reality at higher loads, giving only an approximate peak shape and being unable to describe the interaction between solutes. The major use for such equations is to calculate the column saturation capacity for a solute. A typical example of such a calculation is given in Appendix 1 of this chapter. Another use for these simple equations, supposing that one does not have access to computer simulation programs (see below), lies in the fact that it is often useful early in the development of a preparative method to have an approximate idea of the maximum load which a set of chromatographic conditions can be likely to allow. This knowledge can be used to select the most promising line of approach to a preparative separation. Once the column saturation capacity for the major component

fi)

2.4 Single Solutes

41

has been determined from one of the equations, one may calculate the maximum load which will allow baseline separation between the solute and the first earlier eluting impurity using one of the above equations. Although at high loads displacement effects can be observed between the overlapping solute bands which may result in improved separations, it is rare that these will allow a load higher than 5 to 10 times this limit. From this approximate number it can often be determined if an approach to a separation is likely to be economic without performing an extensive study. Such calculations are useful in identifying grossly unacceptable methodologies; differentiation between the procedures which appear to have a higher likelihood of success still has to be based upon more extensive experiments.

2.4.1.2 Computer Simulations

When a better prediction of band shape is required, the simple equations can no longer be used. This is due to the assumptions inherent in their derivation. The equations of Knox and of Snyder both assume a triangular peak shape; those of Guiochon assume infinite efficiency. The problems lie in the fact that as the load reaches the non-linear area of the isotherms (this is said to be the region of nonlinear chromatography), one can no longer rely upon the additivity of variances to calculate the contributions of the different band spreading processes; the equations no longer have analytical solutions. Due to this, they have to be solved numerically. This is most readily done by the use of a computer to calculate the band profiles. This gives an accurate result for the band shape and retention for a given set of conditions, provided that the adsorption isotherm is known with some accuracy. The disadvantage is that the calculations can sometimes be lengthy and require a computer and the necessary software. Two approaches to the simulation of mass overloaded chromatography have been developed. One of these uses the Craig model of chromatography, which divides the column into sections, each approximating to a theoretical plate and following the physical distribution of the solute between the phases. This distribution is determined from the concentrations and the isotherm, as the solute is transported through the column. The other uses the differential mass balance equations of the system, integrating these over the time of the separation and the length of the column. Again, the solution of these relies upon the isotherm for the calculation of concentration of solutes between the phases. Although the two methods appear to be different, and reach the solution by somewhat different mathematical processes, their predictions are closely similar. The procedures are described in more detail in Appendix 2 for the chapter. These simulation processes are quite rapid, requiring only a few minutes processing time for moderate numbers of plates. The time required is a function of the square of the plate number; using a 80486 based 66 MHz personal computer a single solute simulation based on the mass balance model for 2000 plates takes a little over 30 s; the Craig calculation takes somewhat longer. The result of such a simulation is shown in Fig. 2-5; the results from the use of Eqs. (5 - 7) for the same conditions are also shown. This indicates that the simple equations are reasonably good at such predictions. Simulations are valuable since the results are very close to those which

42

2 The Practical Application of Theory in Preparative Liquid Chromatography

0

2

4

6

10

8

time (min) -~

-Simulation

~

~

Eq 5

Eq6

Eq 7

Fig. 2-5. Comparison of simulation data with the predictions of Eq. ( 5 - 7 ) Capacity factor 4.52, loading: 5% column saturation capacity, efficiency: 2000 plates.

are attainable experimentally and the computer has none of the less desirable characteristics of a liquid chromatograph. This means that the results of the calculations are reproducible and all parameters can be defined precisely and independently varied. Practical studies requiring a range of efficiencies, for example, require packing materials of different particle sizes but otherwise identical properties; this is difficult to attain experimentally. The real advantages of simulations accrue once more than one solute is considered; then there is no necessity to analyse multiple fractions in order to determine the individual concentration profiles and thus the purity and recovery of the solutes are easily calculated.

2.4.2 Volume Overload A frequent question which arises in preparative chromatography, largely due to work carried out prior to the advances in theory already mentioned, is one of the merits of volume overload versus mass overload. Volume overload occurs when the sample size is sufficiently large that its volume distorts the shape of the eluted peak. In practice, most chromatographic separations are affected to some extent by this, since the volume of the injected band does not have to be large in a modern high efficiency column to have some influence upon the efficiency measured for the eluted peak. This is especially true in preparative chromatography since most samples have limited solubility in the mobile phase and are injected in relatively large volume. Knox and Pyper demonstrated from their model [I] that the injection volume could be as

2.4 Single Solutes

43

large as half the final peak volume before any major effects could be discerned for a single solute. The width of a band eluting from a column in linear chromatography may be calculated from the sum of the variances of the different processes which influence it. For the present purposes we assume that the load is low and the predominant band spreading mechanism is due to the injection. The band variance may be considered as the sum of the variances due to the column and the injection volume. (It should be remembered that the variance is the square of the standard deviation of the band; the standard deviation can be measured as 1/4 of the baseline width, so variance is calculated as u2 = wb2/16.) If the column efficiency is high, and the column variance is therefore small compared with that of the injection, the latter will dominate and the peak will become rectangular, with a width equal to that of the injection. Under such circumstances, two peaks will be just resolved if their centres are separated by an elution volume equal to that of the injection. The band broadening due to mass overload in the absence of volume effects may be calculated from the equations given earlier. A load which will just allow resolution between two peaks may be calculated by setting the efficiency such that the band width of the second eluting peak is equal to the volume separating their centres. The two possibilities - of mass and volume overload - are shown in Fig. 2-6. Figure 2-6a shows the volume overload experiment whilst Fig. 2-6 b shows the mass overloaded run. Consider a separation of two components on an analytical column of 15 cmx4.6 mm in dimensions. This has approximately 1.5 mL volume, and we assume reasonable values of 5000 theoretical plates for the efficiency and a saturation capacity of 200 mg. The components have capacity factors under analytical conditions of 2 and 3 respectively. If volume overload is used, the sample volume corresponding to the peak separation in this case is 1.5 mL. In order to assess volume overload, this injection should not give rise to significant mass overload effects. Taking an arbitrary limit of 10% change in efficiency due to such mass overload effects, the maximum mass load turns out to be 0.41 1 mg. If the sample has sufficient solubility such that mass overload alone is employed without band spreading arising from the injection volume, a maximum load of 5.8 mg can be applied to the column. This result is typical of such calculations and indicates that mass overload is always the preferred mode. This calculation, incidentally, does not take into account the effects of displacement between the solutes. In fact the two bands under mass overload do not touch until a mass load somewhat in excess of 7 mg is reached. This illustrates that mass overload should always be used in preference to other options and a solute should be introduced in the highest possible concentration, consistent with its solubility in the mobile phase. The sample concentration which corresponds to the volume overload example above is approximately 0.3 mg/mL-'. Normally a sample solubility of between 10 and 100 mg/mL-' is desirable for preparative separations. A very poor solubility such as this usually is unacceptable and changes in temperature, mobile phase or the entire phase system are made in order to improve it to the level where mass effects predominate. There is a further need to reduce the volume overload effects other than those noted here. This is a consequence of the interactions between the solutes, which are considered in the following section.

I

a2

2 go004 m

A

2 0.002

~

1

0

0

(b)

2

1

6

4

,

8

10

time (min)

Fig. 2-6. (a) Volume overload. The effect of a 1.5 mL injection volume using a dilute sample. See text for details of the simulation conditions. (b) Mass overload. A mass overloaded separation under the same conditions as part (a). Sample loading: 3.5% saturation capacity.

2.5 Multiple Solutes As noted above, preparative chromatography is by definition carried out using samples which contain more than one component. In such cases, the complexity of the system increases. This is because in almost every case the solutes interact with each

2.5 Multiple Solutes

45

other. These interactions underlie the early belief that preparative chromatography was totally unpredictable. The results of the computer simulations have demonstrated that this branch of chromatography also follows rules, albeit rules more complicated than those which govern analytical chromatography. Solutes which interact with the same sites at the surface of the packing will compete for adsorption at these sites. If we assume for the moment that the solutes are in very high concentration and are the only species at the surface, we can begin to predict the likely effects of the interactions. We first consider the situation where the solutes use the same surface for adsorption - ie, have the same footprint on the surface of the packing. Since they are retained to a similar extent, they have more or less the same energy of adsorption at the surface. Thus the surface concentrations of the two species are governed by the law of mass action. Where a high concentration of one solute is present, it will compete more effectively for the sites and will thus exclude the solute present at lower concentration. If we recall from the single solute separation that the peaks will travel through the column approximately as triangles, then it can be seen that the front of the second solute, which is at high concentration, will displace the low concentration tail of the first eluted component. This will decrease the concentration of this component relative to the profile where there is no mutual interaction. The front of the second peak will be displaced relative to its position when injected alone by the low concentration tail of the first. This will be to a much lesser extent, but will lead to earlier elution of this part of the second eluting solute, distorting the peak shape from its original (approximately) triangular shape. When the two components have different footprints on the surface, the interactions will differ from those described above. Consider, for the moment, the effect of one solute requiring twice the surface area for adsorption. Again, since the solutes at this point co-elute, we can assume that their adsorption energies are approximately the same. In this case, one molecule of the larger solute will have to displace two of the smaller molecules; this requires twice the energy for the desorption as is gained from the subsequent adsorption and is less likely to occur. The displacement of the larger solute by the smaller is equi-energetic - although this also results in the liberation of surface area which can adsorb a second molecule of the smaller solute. This means that the solute with the larger footprint will be strongly displaced by the other. The actual result will depend upon which solute elutes first, as well as on their relative footprint size. Where the smaller solute elutes later, it will displace the tail of the first eluting component to a greater extent than seen in the case of equal footprints. The second component will not be so strongly displaced by the first, and will elute more closely to the peak shape found for the solute when injected alone. When the solute with smaller footprint elutes earlier, it will again displace the other solute. As the second eluting species is displaced, it will elute earlier, merging into the first peak. Again, since the earlier eluting solute is not so strongly displaced, this peak shape will be less effected by the interaction. The result of these interactions is that if the separation can be arranged such that the solute with smaller footprint (larger saturation capacity) elutes later, the strong displacements will enhance the separation, leading to higher recovery of pure material. Conversely, elution of this solute earlier than the other will lead to a very poor separation.

46

2 The Practical Application of Theory in Preparative Liquid Chromatography

2.5.1 Computer Simulations Due to the complex interactions, simple calculations as can be performed for single solutes are of no - or very little - value in multi-component preparative chromatography. All results which have led to our better understanding of mass overload have arisen from a combination of experiment and computer simulation. The key to

0

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(a)

6

8

10

6

8

10

time (min)

0

(b)

4

2

4

time (min)

Fig. 2-7. Computer simulation results from variation in relative load. Competitive Langmuir isotherms, k' (first component) 4.03, chromatographic selectivity: 1.12, N: SO00 plates. (a) Load: Solute 1: 1%; Solute 2: 9% of column saturation capacity. (b) Load: Solute 1: s%, Solute 2: 5% of column saturation capacity. (c) Load: Solute 1: 9%; Solute 2: 1% of column saturation capacity.

2.5 Multiple Solutes

0

Fig. 2-7 c

2

4

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47

10

time (min)

the simulation of multi-component mixtures is the competitive isotherm. This is an equation which describes the stationary phase concentration of one solute in terms of the mobile phase concentration of all of the components of the sample mixture. These competitive isotherms are more complex than those of single solutes, and only a few useful isotherm equations have been developed theoretically. One of these, which corresponds to the case of equal saturation capacities for the solutes, is the extension of the simple Langmuir isotherm - the competitive Langmuir isotherm:

When this relation is used in a computer simulation, it predicts some very strange peak profiles. Typical profiles for 1 : 9, 1: 1 and 9: 1 ratio mixtures are shown in Fig. 2-7. With a small concentration of the first eluted component, a strong displacement effect is seen, such that instead of being engulfed by the main peak, the minor component is forced to elute ahead of the major. When the minor component elutes later, it is pulled into the major peak. The 1 : 1 mixture shows evidence of both effects. These peak shapes correspond to the simple theory outlined above which may be used as a physical model to rationalise them. As expected from the physical model, the situation is further complicated if the saturation capacities of the solutes differ. The competitive Langmuir isotherm can no longer be used. An isotherm model has been theoretically derived from the Ideal Adsorbed Solution theory 141. This was calculated to allow use of individual isotherm parameters for solutes which follow the Langmuir isotherm but which have different saturation capacities. This allows the estimation of the changed displacements, although as could be expected, it certainly does not fit every case. Simulations using this isotherm are shown in Fig. 2-8. Two situations are depicted, one where the

48

2 The Practical Application of Theory in Preparative Liquid Chromatography 7

6 E 0 . 3

5

z4

U U

E

23

30

2 1

0

2

0

4

(a)

6

8

10

time (min)

--1

0

(b)

2

4

6

8

10

12

14

16

time (min)

Fig. 2-8. Computer simulation results using the competitive isotherms derived from the Ideal Adsorbed Solution Theory. (a) Conditions as Fig. 2-7 b except saturation capacities of components were 203 and 383 respectively. (b) Conditions as Fig. 2-7b except saturation capacities of components were 381 and 203 respectively.

first solute has a saturation capacity 50% smaller than the second, and the other where the elution order is reversed. The results of the simulations in comparison with those shown in Fig. 2-7 using the competitive Langmuir model illustrate the predictions of the simple physical model above. In the first case, as expected from the above discussion, an extremely strong displacement is observed, whilst in the second, there is extensive peak overlap. The peak shapes in the second example are not exactly in the form that one might expect, especially for the first eluted component and no re-

2.5 Multiple Solutes

0

2

4

6

8

10

49

12

time (min) Fig. 2-9. Separation of phenol and benzyl alcohol. Full line: detector wavelength 240 nm; dashed line: detector wavelength 290 nm. 3 mg phenol, 1 mg benzyl alcohol on a solumn 15 cm x 4.6 mm packed with Zorbax ODS ( 5 vm); flow rate: 30% aqueous methanol at I mL/rnin-'.

ports of such shapes have appeared in the literature. It is not currently clear if these strange shapes are real or if they result from the approximations involved in derivation of the isotherm. This isotherm has been used [5] to describe the chromatography of some steroids with success, but in this case the differences between the saturation capacities were not so extreme. Figure 2-9 shows an example of the displacements which can be observed experimentally. The separation of benzyl alcohol and phenol, which have saturation capacities of 381 and 203 mg respectively under the separation conditions [6]. Phenol is very strongly displaced by benzyl alcohol under these reversed phase conditions, although not quite as strongly as predicted by the Ideal Adsorbed Solution Theory isotherm as depicted in Fig. 2-8a. The true form of the competitive isotherm is not known in this case, even though the solutes individually follow the Langmuir isotherm. Preparative separations are rarely carried out between only two components. In practice one has many components. If one is to carry out computer simulations for such samples it is strictly necessary to have a competitive isotherm which describes the concentrations of all components. This can become a tall order, especially if all solutes do not fit the competitive Langmuir model which is one of the few available which can be used for more than two compounds. To a first approximation, one generally chooses the impurity peaks closest to the component of interest. It is not always a good assumption that the other components do not interact, especially at high loads when component bands overlap at high concentrations for much of their residence times in the column. Often experiments are performed to observe the peak shapes and position of impurities in order to assess their behaviour and to infer the type of isotherm which exists without necessarily determining it. Such experiments

50

2 The Practical Application of Theory in Preparative Liquid Chromatography

can identify the most important components in terms of the design of the separation.

2.5.2 The Effects of Column Efficiency The use of computer simulations allows rapid assessment of the effects of parameters on the separation. When the broad, strangely shaped peaks in a preparative separation are first seen, the intuitive reaction is that there is little point in using an efficient column for the separation; the mass overload effects mask totally the analytical performance of the column. Where there are interactions between solutes, however, the zones between the peaks and the purity and recovery of the components are functions of the column efficiency. Table 2-1 shows the recovery of two products from simulations of a separation of a solute containing 20% of an impurity in which only the plate number of the column is changed, from 500 to 5000. Two possible scenarios are shown here. The first is that with a competitive Langmuir isotherm; both solutes have identical saturation capacities. The second is an example where the saturation capacities are different; in this case the second eluting component has the higher Value. The benefit of increases in efficiency to the purity and recovery of the products is greatly evident. In addition, the great change in the recovery of the products depending upon the form of the competitive isotherm is also apparent; it is therefore vital in performing simulations to ensure that the competitive isotherms are accurate and in practice to ensure that the selectivity of the system is managed such that the most appropriate solute elutes last. Figure 2-10 shows a comparison of the chromatograms at 1000 and 5000 theoretical plates. Plate numbers in excess of 4000 do not significantly increase the recovery values for the second peak; above a certain number of plates the change in the profiles is very small for each additional increment in plate count since they begin to approach closely the profiles expected for an infinite efficiency. The recovery of the earlier eluting, minor component increases with efficiency even at plate numbers in excess of 5000. Figure2-11 shows a plot of recovery of this component plotted against plate number for various values of purity. Even at 8000 plates, the recovery of material at 98% purity remains a function of efficiency, and the maximum recovery at this purity - of around 60% - is reached only at an efficiency in excess of 10000 to 12000 plates. This illustrates the point made above concerning the injection volume. If the sample is loaded in a large volume, the effective efficiency of the column is much reduced from its analytical level and the separation is degraded with a concomitant loss of recovery of the product. Even though the peaks appear to be broad and eluted with low efficiency, it is the overlap zone between them which controls the resolution between the peaks and this can be critically dependent upon the column efficiency. It is clear that the best conditions for a separation depend upon the nature of the separation problem. In order to isolate the earlier eluting impurity one could operate at high load and accept the loss in recovery as a cost of attaining a high production rate of the product, or one could reduce the load and improve the recovery at the

0.003 0.1 1.I 10 31

0.04 44 84 96 100

0.02 8 29 78 100

2

For kb(l)=2; a = 1.1. Saturation capacity (Langmuir) = 100 mg. Saturation capacities (IAS theory): (1) = 80 mg; (2) = 120 mg. Loads: (1) = 1 mg; (2) = 5 mg.

100 98 95 90 80

Ideal adsorbed solution theory

0.0007 0.002 0.02 0.2 1.1

1

Solute: Purity

100 98 95 90 80

500

Number of plates:

Langmuir

Isotherm

0.01 13 34 53 72

0.0013 0.02 0.25 1.7 8

1

1000

Table 2-1. The influence of column efficiency upon recovery of products.

0.1 14 38 88

0.2 76 96 99 100

100

2

0.12 61 18 84 89

0.002 0.6 5.5 14 31

1

2000

1.1 95 99 99.99 100

0.55 19 44 94 100

2

24 87 90 92 93

0.008 18 31 40 48

1

4000

1 .I

3.2 99 99.99 100 100

23 41 98 100

2

0.015 28 40 46 52

1

5000

2.2 24 41 99

2

2 The Practical Application of Theory in Preparative Liquid Chromatography

2

4

6

8

10

12

time (min)

2

4

6

8

10

12

time (min) Fig. 2-10. Comparison of simulation results at (a) 1000 and (b) SO00 plates. Conditions as in Table 2-1.

expense of production. Changing the load will also change the relation between efficiency and recovery and this relation should also be understood in order to achieve the best result. In this particular example, isolation of the major component, which is usually the requirement in scale-up and production, cannot be achieved at high recovery and purity because of the tail of the first product which underlies it. Similar results in terms of recovery at a given purity are achieved for this component with column efficiencies above 2000 plates. In order to purify the major component, a significantly lower load must be used or some change in conditions is necessary to improve the selectivity, to change the elution order or even to change the iso-

2.5 Multiple Solutes

53

60 50

40 W

h

5

30

u

2 20 10 0

0

(4

2000

4000

6000

8000

Efficiency (Number of plates)

100 80 n

9

2. aJ

60

i+

$

40

ci 20 0 0

(b)

2000

4000

6000

8000

Efficiency (Number of plates)

Fig. 2-11. Plot of recovery versus plate number at various levels of purity. (a) First eluted component; (b) second eluted component.

therms. In order to proceed further with this separation, it would be necessary to know how the recovery of the product changes as a function of combinations of loading, the selectivity of the system, the column efficiency, changes in temperature, the nature of the packing material and (where relevant) changes in the mobile phase ionic strength and pH. For the majority of cases a moderate efficiency (in analytical terms) is necessary for preparative chromatography and plate counts in excess of 10000 are used only under special circumstances. One reason for this is that very high efficiencies can be

54

2 The Practical Application of Theory in Preparative Liquid Chromatography

expensive in terms of the use of small particle sizes and long columns (and therefore the associated high pressure of operation) and, as well shall see below, in most cases the cost of doing this more than offsets the costs of accepting a slightly lower recovery. The use of computer simulations gives two benefits. One lies in the quantitative results which are obtained when all of the parameters for the isotherms are known. The other is less tangible, but lies in the qualitative understanding of the processes involved. After a few basic scenarios have been mapped, most separations can be fitted reasonably easily into one of the general classes of separation. Thus, with relatively little data, one can begin to predict the possible behaviour of solutes and their impurities under mass overload; once one has the result of one or two runs under overloaded conditions, especially where the position and shape of all of the components have been obtained by collecting fractions through the chromatogram and analysing these by HPLC, the behaviour of the system can be at least qualitatively understood. From this understanding, other experiments can be designed to explore the limits of the effects observed and the results of these added to the earlier ones to produce a semi-quantitative picture of the separation. In many cases, for a laboratory scale separation, this is enough to design an adequate (but by no means optimal) separation. In other cases a simulation study can be carried out with approximate isotherms in order to approach a solution. This obviously depends upon the degree of success in finding a model which approximately fits experimental data to hand and would not be appropriate for a large scale separation. In order to go further and to begin to design preparative separations, it is necessary to understand what must be done in order to optimise them.

2.6 Optimisation If a separation is to be performed at large scale, it is usually necessary to carry out at least some level of optimisation. This may be simply to limit the time and cost of isolating a compound for further study, or may be to minimise the costs of production. It is important to remember that the reason for optimisation often influences the conditions chosen. This is because selection of conditions to give, for example, the maximum production rate (in kg h-') may not result in product which is isolat-

Table 2-2. Assumptions in deriving the production cost contributions of Fig. 2-12. Purity Recovery Efficiency required Pressure drop Particle diameter Capacity factor

99% 95% 3000 50 bar I0 pm 3

Solvent cost ($ L-') Solvent recovery ($ L-') Solvent losses Packing ($ g- ') Labour ($ h-') Amortisation (years)

5.0 0.1 5q o 4 30 5

2.6 Optimisation

55

ed at the lowest cost (in E, $ or FF kg-I). If we consider for the moment, optimisation of a production process, it is clear that the cost of the operation must be minimised in preference to any other strategy since this is the metric by which the process is judged. The cost of a typical process is made up from a number of contributions. These are: -

-

equipment cost cost of solvent: * cost of replacement * cost of recovery cost of packing material cost of power, utilities cost of labour cost of site cost of the loss of crude product

Ignoring the last item for the moment, one can derive for a particular separation the individual contribution of these costs to the total for a given annual production requirement. Such a breakdown is shown in Fig. 2-12; the assumptions made in deriving it are given in Table 2-2. Equipment costs were taken from Prochrom price lists. It is clear that at low production rates the cost of equipment and labour make up the vast proportion of the cost per kg of material purified. In these cases, the purification should be carried out under conditions which minimise these fixed costs, generally by operating the unit at the highest possible production rate. At very high procost / kg 300

I

TOTAL 200

-

I

0

....................................... solvent equipment -...--._ --.___

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56

2 The Practical Application of Theory in Preparative Liquid Chromatography 0.6

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Column Efficiency Fig. 2-13. The influence of efficiency on production rate and the efficiency of solvent use. Solid line: production rate; broken line: solvent efficiency.

duction requirements, the major costs shift to the solvent: the costs of recovery and the cost of solvent loss. Since it is generally cheaper to recover solvent than to replace it, the vast majority of large scale purifications use recovery of the solvent to reduce operating costs. In addition, because of the high cost of replacement, the efficiency of the solvent recovery process has to be maximised (it should be noted that the 5% loss of solvent used in this example is somewhat pessimistic). Even after all this is accomplished, the cost of solvent still predominates and the only solution is to consider operation under conditions of the maximum solvent efficiency - that is the isolation of the maximum quantity of product per unit volume of solvent. This is not the same as maximising the production rate of the process. Figure 2-13 shows the effect of efficiency on the production rate and on solvent efficiency for our hypothetical process. Although the curve for production rate passes through a maximum at a given value of efficiency, that for the solvent efficiency has no maximum. In fact, because the cost of increasing the column efficiency becomes much higher as high plate counts are achieved, there is usually a cost optimum which limits the efficiency chosen for the separation. It should be noted, however, that the column efficiency which gives the minimum cost at large annual production is always higher than that which gives the maximum production rate.

2.6.1 Production Rate Optimisation For smaller scale purifications, where the major cost contributions tend to be the fixed charges of labour and equipment, the simple cost considerations suggest that the production rate should be maximised. Several papers have appeared on this sub-

2.6 Optimisation

57

ject. Although this is not generally appropriate for large scale separations and, as we shall see below, may not always be relevant for laboratory scale separations, it is worth spending a few moments in considering this option. The most detailed publication is that of Golshan-Shirazi and Guiochon [7]. This work combined the results from an ideal model of chromatography (ie, one which assumes an infinite column efficiency) with considerations of the kinetic band spreading present in real systems. The predictions and equations were confirmed by computer simulations. These authors produced an equation which gives the production rate for the second component in a pair of components. The equation itself is somewhat complex and incorporates terms very specific to a given separation and the available columns and equipment. In broad terms, the results of the work indicated that the highest production rates were found for the highest operating pressure (which maximises the flow rate possible for a given column efficiency). As a corollary, it was noted that the best preparative performance was obtained from high efficiency columns operated at high flow rates, with the initial high plate count being traded for speed. The work also demonstrated that an optimum value of the ratio of the square of the particle diameter to the column length (d :/L) exists. This last parameter is also a complex function of the operating conditions, such as the maximum operating pressure, the isotherm parameters, etc, and its value is not simple to obtain. Since it has a strong influence on the production rate of the separation and a value of d i / L significantly different from the optimum can result in a large reduction in production rate, it would be useful to be able to obtain this easily. In order to avoid the complex calculations and experimental measurements, especially where not all of the parameters are available, the optimum value of d:/L can be approached by noting that the efficiency required to perform the preparative separation is always less than that required for an analytical resolution of unity between the bands. Since the two values do not greatly differ when the required column efficiency is moderate or low, one may take this latter value, which is easy to calculate from the general resolution equation. Thus, the combinations of column length and particle size may be calculated which would give this desired efficiency at the maximum safe operating pressure of the system (for this one would use the Darcy Eq. (10) which relates the flow rate, pressure ( P ) , particle size (dp),column length ( L ) ,column resistance parameter (@; = 500 for spherical media, 1000 for irregular) and the solvent viscosity (y ) and the Knox Eq. (1 1) which relates the reduced flow velocity (v) to the reduced plate height (h)).

B h =-++Av0.33+CV V

Since the optima in chromatography are generally broad, this approach, while not giving an optimum column, would give one which approaches it. The error in this procedure is greatest for separations which need high efficiency. As noted above, the

58

2 The Practical Application of Theory in Preparative Liquid Chromatography

optimum separation for large scale operation is a function of costs rather than of production rate and it is rare that one would be involved in optimising production rate for a system which merits a great deal of time and effort to be spent in its optimisation. Thus, for many applications, the simple approach seems to result in an acceptable compromise, especially since, unlike the full optimisation of production rate, this does not require the isotherm parameters for the two components. It should be noted that the optimisation of production rate results in some surprising operating conditions, in that if an appreciable pressure is available very fast separations are found to be optimal. It is important that a realistic maximum operating pressure is chosen which reflects the capabilities of the equipment to operate for prolonged periods at the very high flow rates, not only in terms of not restricting the flow in the system such that much of the pressure is lost in the tubing but also in terms of the response time of detectors and fraction collection valves and the probability that the pressure will rise slowly with time. This can occur through partial blockage of filters and perhaps the frits with the unavoidable dust and debris found in the solvents and sample. Additionally it must be remembered that strongly adsorbed sample components can affect the permeability of the column at its inlet and fines which may be present in the packing material or which may be generated with time can slowly move through the bed. This results in a diminution of permeability at the column outlet. If the recommendations of the authors [5] are followed exactly, a separation with a selectivity of 1.5 and maximum operating pressure of 200 bar (20 Mpa) should be performed using a dG/L ratio of 17 and a plate count of 175. Using 40 pm particles in a 75 cmx 5 cm column would give this number of plates at the operating pressure, but would result in a flow rate close to 4 L min-’ and a retention time for a peak with a capacity factor of 3 of only 60 s. This could pose problems in accurate fraction collection.

2.6.2 Cost Optimisation The situation changes somewhat when the costs of operation and of the material being purified are taken into account. The reason is that unless a ‘touching band’ separation is being carried out, there is always a certain loss of crude material which is not recovered in the pure product fractions. Where the cost of this crude is low, this is of little consequence. In contrast, if the cost of the crude product is very high, the losses of crude can become overwhelmingly important. This can be illustrated by consideration of two scenarios - a laboratory scale purification and an industrial scale operation.

2.6.2.1 Laboratory Scale In this example we take a purification of 200g of material. If we assume some ‘typical’ parameters, this quantity of material may be purified in 27 runs at a load of 7.5 g per run using a column 8 cm in diameter. If each chromatogram takes

2.6 Optimisation

59

20 min, this results in 9 hours’ operation. If the equipment is amortised over a period of 5 years and is used for separations most of the time, the cost of equipment for this purification would be around $150. Assuming a direct cost of $ 3 0 per hour for labour, the total labour cost (including an average value for fully distributed overhead) would be $700. The solvent use (in a laboratory solvents are not often recovered) would be 270 L at a cost of around $800 if we assume that the separation used 60% aqueous methanol. The cost of packing material depends upon the way in which it is used; we assume here that a reasonably good material is used ($ 5000 kg-’) and that it is used for other separations after cleaning. If its lifetime is 6 months, then the incremental cost for the packing material for this purification is around $40 - ie, is close to negligible. This means that the total cost of purification of the 200 g of product is approximately $1700, a cost of $8500 kg-’. At this cost, clearly the separation should be carried out as quickly as possible, the equipment being operated at the maximum production rate. It should be noted that such separations should be performed in the largest size equipment possible consistent with its regular use to allow different projects to share the cost of purchase and the reasonable control of the packing material inventory. The consequence of operation at maximum production rate will usually result in a recovery of only around 60 to 70% of the product. This implies loss of 30 to 40% of the crude material. Estimation of the cost of the latter is sometimes difficult in the laboratory environment. In the present case, if the material to be purified is the result of only 4 days’ work in the laboratory, it bears a labour cost of $2400. (If it is a recombinant product the price could be very much higher - a monoclonal antibody may have a value of 1 million dollars per gramme). If the chromatographic separation recovers 67% of the product in the desired purity, there is then an additional cost of $800 from product loss - nearly 50% of the total purification cost. If the product is the result of several weeks’ work, for example the end result of a difficult synthetic route or an isolation from some natural product, or is based upon expensive intermediates, the cost would be much higher. Quite obviously, even in the laboratory, operation at the maximum production rate is not always the optimum procedure; where the cost of the crude material is very high, conditions leading to maximum recovery are indicated. In this example where only a day or two will be spent upon the purification, the determination of isotherm data is not cost effective, if only upon the grounds that it takes about the same length of time to perform either operation. The ability to model the separation taking data generated from an analytical LC to measure column saturation capacities, etc, and making gross assumptions about the isotherms can result in a better set of chromatographic parameters, or at least an understanding of the consequences of changing the separation conditions to give higher purity in terms of run time and column loading. For small scale separations, this is probably the best that can be attempted.

2.6.2.2 Production Scale At the other extreme, large scale purification processes are extremely cost sensitive. In this case the cost of all items which make up the total cost should be known with

60

2 The Practical Application of Theory in Preparative Liquid Chromafography

3.0

/A

5

-Et

' 5

$201kg crude

w

2.8

0

u

1.E

0.4

(4 3.0

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A

total

G

ii

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$100/kg crude

E

b

2.0

-

c m

0

*

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~~\"----------ic--------

product losses

-

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x.. 0.0

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some accuracy. The assumptions made for this analysis are based upon the data of Table 2-2 which were from a small scale purification which was then assumed to be required to be performed at an annual production of around 20 tomes. The effect of the cost of the crude material on the end product cost is shown in Fig. 2-14. The recovery of the process was varied to yield material of the desired purity. This action

61

2.6 Optimisation 6.

total

3

E *

1

v

c

8

$1000/kg crude

4.

2.

0.

90

Fig. 2-14c

95

100

product recovery

taken alone would result in a variation of the annual production. In order to maintain the production requirement the column diameter (and associated parameters of flow rate, equipment cost, etc) is varied. The cost per kilogramme of the product turns out to be very much lower than seen for the laboratory purification; if the cost of crude is discounted, the purification cost is around $120 kg-I. At a cost of $ 2 0 kg-' for the crude, there is little change of the optimum from the zero cost case and the system should be operated at a recovery of between 70 and 80% (the optima are quite shallow and a variation of several percentage points in recovery makes little difference to the overall cost here). At a crude cost of $ 1000 kg-', discarding 20 to 30% of the product becomes very expensive - so much so that the recovery required is driven to be close to 100%. This means operation at a much lighter load, using more solvent and a larger column to achieve the desired production. Obviously there is a balance between the cost of this operation and that of losing a part of the crude. At a cost of $100 kg-', there is an optimum final product cost at around 95% recovery.

2.6.3 Practical Optimisation The above considerations are only a part of optimisation. Most of the parameters interact. A change in the solvent will influence the selectivity and therefore the efficiency, loading and column dimensions required for the separation. The operating pressure at a given flow rate will change due to the viscosity change, and, because the system should be operated at a pressure close to its maximum, the flow rate, par-

62

2 The Practical Application of Theory in Preparative Liquid Chromatography

tide size and column dimensions will be influenced. The sample solubility, and therefore the injection volume will change; these will also change the operating parameters of column loading and efficiency. These will influence the column dimensions. The change in solvent will effect the cost, not only of the replacement of solvent losses but also of the recovery of the solvent. Almost every other parameter interacts with the remainder in a similar fashion. Due to the multiple interactions, there are many variables which require study in optimisation. It is beyond the scope of this chapter to discuss the techniques of experimental design which can be employed in order to determine which variables are most important for any given separation, which experiments are necessary and how to analyse the resulting data. Needless to say, there are a number of statistically based techniques for this, most of which require the results of a large number of experiments in order to be effective. As an example, a three variable system requires the results of 14 measurements in order to begin to map the experimental space adequately. As variables are added, the number of required experiments escalates rapidly, as does the complexity of the equation required to describe the response function in terms of the parameters. Data of this sort can be acquired either experimentally or by use of a computer. The advantages of the computer are many. Not only are the separations performed more rapidly and reliably, there is no need to spend time on collecting fractions and analysing them; a properly written computer program can calculate yields and purity in only a few seconds following a simulation. Experimental determination of the amount of data needed completely to optimise a separation requires so much effort that it typically is not performed. This results in separations being performed under sub-optimal conditions. Since the economics of such separations cannot always justify the use of chromatography whereas a properly optimised separation may be better than the alternatives, it is important to carry out the onerous task of accumulating the data. Given the costs involved in carrying out a full clinical trial of a pharmaceutical product, it is surprising that the optimisation of its purifcation process (which will cost a minute fraction of this figure) is often omitted. In those cases where the isotherms are not available, something can still be done to begin to approach the optimum. In this case, experimental data of reasonably good quality is needed. The separation is defined by the required annual production, the purity required for the product, the cost of the crude product (which is not always well known, but estimates are usually available) and the operating pressure of the equipment. As noted above, this pressure is not necessarily the maximum available from the pump since many other factors may be involved. In practice, the dimensions of available columns is also a factor which must be used as a constraint in the calculations. Initial experiments involve separations at analytical scale, to determine the chromatographic parameters of the separation such as the effect of flow rate on column efficiency, so that the parameters of the Knox equation may be estimated, the effects of retention and solvent on the selectivity (note that in general the solvent system which gives the maximum selectivity is used, unless there are strong cost factors involved), the effects of packing material on selectivity, etc. In the ideal case, a few packings of different particle sizes and properties will be identified to give reasonable

2.7 Conclusions

63

performance. This is useful for generating data with different column efficiencies. Runs at elevated load are carried out and the performance data for the major peak are determined. If this appears to follow the Langmuir isotherm, a value for its saturation capacity can be determined for each packing material using the equations noted earlier. This gives an indication of which packing material will allow the hightest loading. Again, this may not necessarily be that chosen, since other factors such as cost and operating pressure can be involved. A series of experiments at high load is carried out, determining the recovery of product at the required purity for various values of loading, column efficiency and retention. These values should be widely spaced to cover as much of the possible range of values of the parameters. It should also be noted that since later experiments will be carried out to refine the data set, the first experiments should not cover too many incremental values for each parameter; experimental design theory suggests that three points for each parameter are adequate, one at each likely extreme of its range, the other midway between them. The costs of operating under these conditions to produce the required annual production are calculated, varying the column dimensions to give the desired efficiency at the given operating pressure for the packing material sizes used. The data can be used to make plots of the operating cost against the various parameters of the experiments. These plots are used to design other experiments; it is rare for there to be an abvious optimum in the costs from an initial set of data. These next experiments either are aimed to find minima in the cost plots or to refine their positions. Finding a reasonably good value of the parameters from experimental measurements is difficult due to the interaction between them. One can assume many to be largely independent to a first approximation when looking for gross effects, but when attempting to refine the data to reach the optimum position, this assumption is no longer valid. Ultimately, (and this is why the data needs to be reasonably good) the optimum reached is tested by making small changes in parameters to determine its sensitivity and to ensure that it is at a minimum point. Care should be taken in the choice of the initial parameter values to ensure that a minimum in cost which is reached is not simply a small perturbation of the function and is the real minimum; a sufficiently wide initial range usually avoids this problem.

2.7 Conclusions Preparative chromatography involves theory of some sort in almost every aspect, whether it is for calculation of the performance of a column to determine if it is sufficiently well packed for prolonged use or for a full optimisation. Very often calculations are made to determine the column and particle dimensions which allow operation of a separation at the maximum operating pressure of the equipment to maximise the production from the column. Overloading usually is more controllable if one knows the saturation capacity of the column, since this can allow at least approximate calculation of the appropriate load to try in a separation. Once the sample

64

2 The Practical Application of Theory in Preparative Liquid Chromatography

has been run at overload, the recognition of the various types of interactions as predicted by the computer simulations can save a great deal of time, since one can, initailly qualitatively and later in a semi-quantitative fashion, understand, follow and predict the movement of peaks as load is changed, Once the competitive isotherms for solutes are known, the theories of chromatography now allow the rapid optimisation of production rate and, through computer simulations, also allow the rapid prediction of preparative chromatograms under any desired conditions. These facilitate .the full optimisation of a production scale method. Clearly, the extent to which the theories of preparative LC are used depend greatly upon both the scale at which it is operated and upon the operating environment. Operation at the laboratory scale generally requires a minimum knowledge, whilst as scale is increased, the extent to which the theories and tools are used should increase dramatically, especially when optimisation of production scale purifications are being carried out.

Appendix 1 Calculation of Column Saturation Capacity The column saturation capacity is calculated from two chromatograms. One of these is made at analytical loads, whilst the other is run at a preparative loading. It is important that the load is chosen such that the peak due to the component of interest is not deformed by detector overload and that a reasonably large change in capacity factor is seen. It is recommended that a relative change of more than 10 or 15% in k’ is used. A typical pair of chromatograms are shown superimposed in Fig. 2-A 1.1. This data was generated for benzyl alcohol in a reversed phase system, which is known to conform to the Langmuir isotherm. An injection of 2.5mg was made for the preparative run. In this as in the majority of cases, Eq. (8) may be used for the calculation since the initial efficiency of the column was high (8400 plates). The capacity factor of the analytical peak and of the preparative peak, taking the position of its peak maximum, were calculated. In this example, the void time (to) of the column is 1.49 min, and the retention times of the analytical and preparative peaks are 13.62 and 11.68 min, respectively. This gives k’ values of 8.15 and 6.85 and a ratio (k’/kh) of 0.84. From Eq. (8), the loading factor Lf becomes (1 - fi)’, ie, Lf= 0.00697. The saturation capacity is found by dividing the load by the loading factor. This gives a saturation capacity for this column of 359 mg. An alternative calculation uses Eq. (6). The efficiency for the overloaded peak was measured to be 303 plates. Substituting the values into Eq. (6) yields: 6.85 x 8.15 7.85 ~(8.15-6.85)

W, = 1 / Z x 3 0 3 ~ 2 . 5 ~

65

Appendix 2 t r = 11.68 k = 6.85 N = 303

to = 1.49

0

5

10

I5

Fig. 2-Al.1. Chromatograms arising from the injection of 1 Fg (dashed line) and 2.5 mg (solid line) of benzyl alcohol on a column (15 cmx4.6 mm) packed with Zorbax ODS (5 Fm), using 30% aqueous methanol at Z mL min-' as mobile phase.

This gives a value of 340mg for the column saturation capacity. As noted earlier, this method is used when column efficiencies are low and the approximation of an infinite efficiency is no longer possible. For most HPLC separations, Eq. (8) can be used with sufficient accuracy. It should be noted that the saturation capacity is determined for the column. Saturation capacities for other columns containing the same phase system can be calculated by multiplying this value by the ratio of the column volumes. If the weight of packing material in the column is known, the saturation capacity per gram of packing can be calculated. This can be used to compare different packing materials for a separation.

Appendix 2 Mathematical Models for Preparative Chromatography Mass-Balance Model The first model to be discussed is based upon consideration of the mass balance in the column. A small slice across the column is considered. The mass balance equation for a single component takes the form [S]:

(A 2.1)

66

2 The Practical Application of Theory in Preparative Liquid Chromatography

Here the first term is the change of concentrations of the solute in the slice with time, the second term is the change in concentration due to flow (velocity = u in the ‘z’ direction - ie, along the column) and the term on the right hand side arises from the molecular diffusion, also in the direction of flow. The first term contains terms for the concentration in the stationary phase (C,) and the mobile phase (12,).If we assume that the rate of mass transfer between the phases is very fast, these two values are related through the adsorption isotherm. In deriving the equation we make a number of assumptions - that the concentration across the radius of the column is constant, the partial molar volumes of solute and mobile phase are constant and equal to their values at infinite dilution of the solute and that the mobile phase is a single component (otherwise we have to write a second mass balance equation for it) or, if it is a binary eluent, the solute is much more strongly bound to the packing than is the strong component of the mobile phase. This last condition is usually true in reversed phase chromatography; adsorption chromatography on silica may not necessarily follow this. The first difficulty arises in that the kinetics of mass transfer between the mobile and stationary phases are not always rapid. If one tries to introduce a finite rate of mass transfer, however, except for the simplest of cases (which conditions are not at all relevant to preparative LC) the set of equations becomes intractable and no solution can be found. Hence we are forced to assume instantaneous mass transfer in order to proceed. A second difficulty arises from the diffusion term. This, too, renders the equations intractable even to current numerical methods of solution. The problem is avoided by the assumption of infinite column efficiency. This instantly introduces a difficulty in that the model is then far from representing real chromatographic systems. The equations, however, may now be solved numerically. In fact, for a single solute, there is an analytical solution of the equations resulting from these assumptions [3]. When two solutes are of interest, similar equations are written for both and the set of equations is solved numerically. The above assumptions reduce the differential Eq. (A2.1) to a simpler equation: (A2.2) The solution of the differential equations is possible by use of the right computer program. The calculation is performed by setting up a two dimensional grid of points, with time in one direction and distance travelled along the column in the other Fig. 2-A2.1). In fact, the differential equation is replaced by a finite difference equation 191. In order to calculate the mobile phase concentration at a distance, time point ( k , l ) on the grid, the equation takes the form [lo]:

At the beginning of the calculation, all concentrations are set to zero except for the column inlet, where a concentration-time profile corresponding to the injection

Appendix 2

67

______)

Distance Fig. 2-A2.1. Representation of the calculation grid for the mass balance model.

is imposed. The concentrations of the solutes at each point of the grid are calculated from those at the two preceding points. The concentrations in the stationary phase (4J are calculated from the mobile phase concentrations (ci)and the competitive isotherm. The column output can be determined by taking the concentration values at a distance corresponding to the column outlet at different times whilst the peak profile at any moment can be determined by taking values at a fixed time at different positions through the column. An interesting feature of this method of solution is that the interval between points of calculation in the grid introduces an apparent dispersion effect which operates on the data in the same way as would a chromatographic dispersion if it could be incorporated into the model. Thus, by choosing an appropriate value for the time and distance intervals in the calculation it is possible to model a column having a finite number of plates. In practice, Az and A, are assigned values equal to H (the HETP) and 2 H / u ( l +k') respectively [I]. This is necessary, since the assumption of infinite efficiency means that concentration discontinuities through the column should occur which would otherwise prevent the solution of the equations.

Craig Model A much simpler model in concept is the Craig model of chromatography. This is more a phenomenological model in that it tries to mimic the physical processes occurring in the column. As we will see, it uses most of the same assumptions as are necessary for the solution of the mass balance model and it also gives closely similar results. The model assumes that the column can be divided into slices, as shown in Fig. 2-A2.2. Each slice contains mobile and stationary phases. The model works by first allowing the solute in the mobile and stationary phases in each slice to reach

68

2 The Practical Appfication of Theory in Preparative Liquid Chromatography

Equilibration

+

+

Mobile Phase \stationary Phase

b

1 Translation Fig. 2-A2.2. Schematic of the Craig model.

equilibrium, after which the mobile phase is moved from one slice into the next. The solutes are again allowed to reach their equilibrium concentrations and the process is repeated. The concentrations of the solutes in the two phases are calculated from their competitive adsorption isotherms at each equilibration. This calculation requires a numerical solution by iteration if the isotherms are not linear, basing the initial value of the concentration upon the last value calculated. This, incidentally, is the main reason why the Craig model simulations are slower than those which use the mass balance model. This model clearly also assumes that the mass transfer between the phases is infinitely fast and band spreading is, just as for the mass balance model, introduced by the interval between the points of calculation. The column efficiency is a function of the number of slices chosen for the calculation. Although each slice does not correspond exactly to a theoretical plate, there is a simple relation between the number of slices (usually called Craig Stages) used and the number of theoretical plates for the column: n,=N--1

k'

1+k'

(A2.4)

n, is the number of stages, N is the number of plates and k' is the analytical capacity factor of the solute. It is a feature of the model that because of the interrelation between the number of stages, the efficiency and the capacity factor, the number of plates for any column modelled will be a function of retention. For small selectivity values, this is not very important. When the efficiency and the capacity factors are large, the number of Craig stages becomes equal to the number of theoretical plates in the column. The model operates by calculation of the concentration profile along the column after each transfer. This corresponds to calculation of the profile after an elapsed time given by the flow rate and the number of stages chosen for the column:

References

69

(A2.5)

Here t, is the elapsed time per transfer, L is the column length, u the linear velocity in the column and n, is the number of Craig stages in the column. The model is programmed by assuming first that all concentrations are zero. For a Langmuir isotherm, for example, the saturation capacity of each stage is calculated from the saturation capacity required for the column. A solute - or mixture of solutes - is added to the first stage in a desired quantity; a function of the overall load and the injection volume (in terms of the number of stages). The calculation of the concentrations in both the mobile and stationary phases is made from the isotherm and the total quantity of solute in the stage (there is no point, at this stage, in calculating the remainder of the column, which is still empty). The material remaining in the mobile phase is then moved into the next stage. Again, the calculation of the equilibrium concentrations in the phases is made from the isotherm, this time for the first two stages and the material remaining in the mobile phase in each stage is then moved to the next. Each time, the new concentrations in each phase in the stage are calculated from the total quantity of material which is found in each stage. The process continues until all of the solutes are eluted from the ‘column’. The chromatogram is obtained by taking the quantity transferred out of the final stage and plotting it against the number of transfers multiplied by the elapsed time per transfer. This method results in essentially the same output data as the mass balance model and it is not surprising that they have been found to result in identical predictions. There is one difference between the two models. This is that in the Craig model, materials with low retention have efficiencies which are much higher than they should be - a non-retained peak is eluted with infinite efficiency. The mass balance model is opposite. The peak of interest is eluted with the desired efficiency whilst the peaks eluting earlier have a lower plate number and later eluting peaks have a higher plate count. In this respect, the two models can give slightly different results, particularly when large selectivities or very high overloads are considered.

References [I] [2] [3] [4] [5] [6] [7] [8] [9] [lo]

Knox J.H., Pyper, H.M. J Chromatogr, 1986, 363, 1. Snyder, L.R., Cox, G.B., Antle P.E. Chromatographia, 1987, 24, 82. Golshan-Shirazi, S., Guiochon G. Anal Chem, 1988, 60, 2364. LeVan, M.D., Vermeulen, T. J Phys Chem, 1981, 85, 3247. Golshan-Shirazi, S., Huang, J.-X., Guiochon, G. Anal Chem, 1991, 63, 1147. Cox, G.B., Snyder, L.R. J Chromatogr, 1989, 483, 95. Golshan-Shirazi, S., Guiochon, G. Anal Chem, 1989, 61, 1368. Guiochon, G., Golshan-Shirazi, S., Jaulmes, A. Anal Chern, 1988, 60, 1856. Godounov S.K. Mat Sb, 1959, 47, 271. De Jong, G. J., De Brij, R. J., Hoogendoorn, J., Pauli, L. F., Zeeman, J., Poppe, H. Poster presented at the 17th International Symposium on Column Liquid Chromatography, Hamburg, 1993.

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3 Alternative Modes of Operation of Chromatography Columns in the Process Situation Derek A. Hill

3.1 Process Chromatography Preparative chromatography has, in recent years, gained considerable importance, not only as a research and development tool, but as a viable alternative to more traditional purification techniques in a production environment. Leaving aside those examples where no alternative purification technique is successful, the high recoveries of purified product will often more than compensate for the relatively high processing cost even compared with a low cost technique such as recrystallisation, provided that the value of the product is high enough [I]. In process chromatography, as distinct from preparative chromatography used in non-process situations, the following points must be taken into consideration: The process must be optimised in terms of cost. The product will be marketed, or be a constituent of, or an intermediate in, the production of a product which will be marketed. The cost of the product will, therefore, usually be critical. - The required quality of the purified material must be clearly defined at the outset. If, for example, the product of the chromatographic step is to be used as an intermediate in further processing operations, it may be possible to produce material of a lower quality than if the product of the chromatographic step is a final product. An understanding of the tolerances which the process will allow is essential, since a lower quality product will be cheaper to produce. - The chromatographic separation will usually only represent a small part of the total production process. Production of the feed stock for the chromatography, isolation of the product and recovery of solvents will, generally, require a greater proportion of resources. The chromatographic separation must be considered, not in isolation, but as an integral part of the overall process. For example, process changes during the synthesis of the feed stock may effect the quality of the feed stock and hence require changes to be made to the chromatography. The interrelationship of these factors must be understood in order to develop a reliable process. The selection of a particular chromatographic technique, where alternatives are available, may be based on the ease of product isolation, solvent recovery and safety rather than upon purely chromatographic considerations. -

72 -

-

-

-

-

3 Alternative Modes of Operation of Clzromatography Columns

The chromatographic separation will often be the rate limiting step in production. This is particularly true in the situation where a chromatograph is to be used for a single process and the size of instrument purchased will be determined on the basis of running the instrument, as near as is possible, continuously. Idle plant is expensive and this is particularly true where, in order to operate safely under pressure, doubling the cross-sectional area of a column, for example, may require the larger column to be engineered in such a way as to more than double its capital cost. It may on occasions be worth considering installing two smaller units rather than one large one, since the capital cost may not be too different, and there will be a strategic advantage in the event of equipment failure. Equipment failure is less likely with modern instrumentation which demonstrate a high level of reliability, but the implications should be considered. It may be worth considering the installation of several small units rather than one large one. These units can then be run out of phase so that the purified product is produced in a continuous stream, thus opening up the possibility of continuous down-stream processing which may be advantageous under certain circumstances. Other factors such as the stability of the feed stock may need to be considered. There is little point in preparing a large batch of feed stock, only to find that it has decomposed before chromatography has been carried out. This may dictate either the scale of synthesis of the feed stock or the scale of chromatographic equipment purchased. If an option exists to carry out the chromatographic purification at more than one point in a multistage synthesis, without compromising the quality of the final product, it is usually preferable to carry out this step as late in the synthesis as possible since, with yield losses at each step, a lower amount of material will require purification. This will have to be balanced against possible waste of expensive reagents in carrying out a reaction on a crude intermediate. The importance of carrying out proper costing on alternatives can not be overemphasised. Automation of the chromatographic separation is essential. Not only is it a waste of resource to utilise highly trained operators in this situation, but it avoids human error. Additionally, by automating operations it is far easier to validate the process. Controls can be incorporated so that any required adjustments to the separation conditions can only be made by an authorised person and any such adjustments will be properly logged automatically so that compliance with ‘good manufacturing practice’ can be assured. The chromatographic process must be robust. Automation will be simpler and consequently cheaper if the separation conditions remain constant. This will have implications in the areas associated with the chromatographic step such as solvent recovery and preparation of mobile phase. The quality of the feed stock will need to be, as near as possible, constant. This may also dictate how often the stationary phase needs to be changed or whether a guard column should be used in a particular situation in order to maintain column performance. In the selection of a chromatographic method, environmental considerations are becoming increasingly important. The discharge of toxic substances to the environment, either in the form of waste streams or atmospheric emissions is unacceptable. Moreover, the increasing involvement of Regulatory bodies such as the

3.2 Alternative Chromatographic Modes and Techniques

73

European Pollution Agency and Her Majesty’s Inspectorate of Pollution can result in a process being closed down. Within the pharmaceutical industry, the Federal Drugs Administration requires the submission of an Environmental Assessment before an application for a product licence is considered. As a consequence, the selection of the chromatographic system may no longer be made solely on the basis of the best chromatography. Whilst, for example, chlorinated solvents may still be used, adequate controls must be built into plant and buildings to prevent discharge. For example, bunding around an area prevents accidental spillage entering drainage systems. It must be borne in mind that what is acceptable today may not be acceptable tomorrow. As recently as 1991, the use of the virtually non-toxic, non-flammable, but nowadays environmentally sensitive, fluorocarbon113 (1,1,2-trichloro-I ,2,2-trifluoroethane) was recommended as a replacement for hexane [2]. Operator safety, and the safety of the general public are also of utmost importance in developing a process. In consideration of the above it is therefore essential to examine, as widely as possible within the time available, all of the alternatives which are available to the chromatographer, in order to develop the required safe, reliable and cost-effective process. Throughput must be maximised in order to minimise the use of often flammable, toxic and costly solvents and to minimise expenditure on plant, equipment, labour and operating costs.

3.2 Alternative Chromatographic Modes and Techniques Tiselius [3] defined three distinct modes in which a chromatographic column can be operated, elution, displacement and frontal. In addition to this there are ways of operating chromatographic columns which can be considered as intermediate between these modes and, in addition, there are various techniques which have been applied to chromatographic separations in order to improve throughput. These will be discussed in this Section.

3.2.1 Elution Chromatography Elution chromatography is a technique which is familiar to all chromatographers, whatever the scale of operation. The interaction between the analytes and the stationary phase is modified by the interaction of the mobile phase with the stationary phase in such a way as to establish a series of equilibria, so that the time which an analyte spends in the mobile phase compared with the time it spends on the stationary phase is different for different analytes. Separation is thus achieved, the analytes which interact less with the stationary phase being eluted from the column faster than those which interact more strongly with the stationary phase (Fig. 3-1). It

74

3 Alternative Modes of Operation of Chromatography Columns

+c

time

Fig. 3-1. Elution chromatography. The left-hand diagram shows the on-column situation during a run whilst that on the right shows the resulting chromatogram.

should be noted that elution chromatography is a dilution process, the amount of dilution depending upon the rate of elution, with slower running components being eluted as more dilute solutions than faster running components. It should also be noted that the contribution of solvent cost to the overall cost of a chromatographic separation, even allowing for recovery, is in the region of 60% of the total.

3.2.2 Displacement Chromatography In displacement chromatography, interaction of the mobile phase with the stationary phase is minimal. Competitive interaction with the stationary phase takes place between the analytes as they are loaded onto the column, those analytes having a greater affinity for the stationary phase displacing those analytes having a weaker affinity, resulting in a series of bands of analytes being formed on the column. Addition to the mobile phase of a substance having a greater affinity for the stationary phase than the most strongly adsorbed analyte results in the latter being displaced from the stationary phase. This analyte in turn displaces the analyte of next greatest affinity for the stationary phase and a displacement train is set up. The analytes are eluted from the column in a series of bands with sharp boundaries between adjacent analytes (Fig. 3-2). This process is concentrative. (For examples of the use of displacement chromatography in preparative applications see refs. [4] and [ 5 ] ) . It should be noted that one severe disadvantage of displacement chromatography is the need to regenerate the column before a subsequent injection is carried out. This can be costly in terms of time and materials, and may even outweigh the advantages which displacement normally displays over elution.

3.2 Alternative Chromatographic Modes and Techniques _ _

A+B+C

-

15

_

I

L

D+

conc.

D + 0-

- A

D+

-B

D--,

time

Fig. 3-2. Displacement chromatography. The left-hand diagram shows the on-column situation during a run whilst that on the right shows the resulting chromatogram.

3.2.3 Frontal Chromatography In common with displacement chromatography, in frontal chromatography the interaction of the mobile phase with the stationary phase is minimal. Loading of a solution of the analytes results in the formation of a series of bands of analytes being formed on the column, but instead of introducing a displacer, the loading is continued until, when all the adsorption sites on the column are occupied, the analyte which has the least affinity for the stationary phase starts to elute. As loading is continued further, the analyte with the second lowest affinity for the stationary phase starts to elute along with the lowest affinity analyte. As loading is continued further, a point is eventually reached where all the adsorption sites on the column are occupied by the analyte with the greatest affinity for the stationary phase, and the compo-

I

A+B+-

--*

A+B+C

time

Fig. 3-3. Frontal chromatography. The left-hand diagram shows the on-column situation during a run whilst that on the right shows the resulting chromatogram.

76

3 Alternative Modes of Operation of Chromatography Columns

sition of the column eluate is equal to that of the load solution (Fig. 3-3). Washing the column free of unadsorbed material can be followed by displacement of the remaining analyte with an appropriate substance having an even higher affinity for the stationary phase. The process is concentrative. (For an example of how frontal chromatography is used in process and preparative applications see ref. [ti].) It should be noted that frontal chromatography, as indicated by Guiochon and Katti 171, is not well suited to the production of high quality materials. Although this limits its general applicability, there are occasions where it is the method of choice.

3.2.4 Other Operating Modes The situation, as defined by Tiselius [3], is, in fact, a simplification. In practice, when a chromatographic column is operated under conditions of mass overload, as is commonly the case in process situations, these definitions become invalid. Under elution conditions, as the concentration of solute is increased, the equilibria are upset and the interaction of the mobile phase with the stationary phase is reduced, so that the dominant mechanism is displacement chromatography. This so-called self-displacement effect was first described in the literature by Newburger and Guiochon [S], although it is now obvious that some early published preparative separations, where the loading and resolution were far better than would have been predicted from the low efficiency stationary phases used, demonstrated this effect. Where the concentration of analytes is high on the column, the predominant rnechanism is the displacement mode. However, at the back of the band where the analyte concentration is lower, the predominant mechanism becomes elution. This moves the band forward, keeping the concentration high at the front of the band and so maintaining the displacement train and a sharp boundary between adjacent peaks (Fig. 3-4). However, at high concentrations, we can encounter non-linearity of the isotherms which can lead to situations such as that depicted in Fig. 3-5, where the slower eluting component always contains some of the faster eluting component, irrespective of where the cut is made. This may be acceptable if the front peak is the required material (although yield will be reduced) but not if high quality second peak material is required. Golshan-Shirasi and Guiochon [9] have studied these effects via computerised simulation, and an awareness of this work is to be recommended to anyone working in the field of process chromatography. When we consider gradient elution, at least with high concentrations of analytes, the same is sometimes true. Increasing solvent strength concentrates the band and induces a displacement mechanism, the increasing solvent strength maintaining the high concentrations required to maintain the displacement train. In other cases and at lower concentrations, gradient elution will work by an elution mechanism. Particularly where there is a large difference in the strength of interaction of the different analytes with the stationary phase, the gradient is used simply to speed up the rate of elution of the slower-running components in order to be able ro remove them from the column more quickly and hence to carry out more injections in a unit time. This is usually the case when stepped gradients are used as opposed to continuous gradi-

3.2 Alternative Chromatographic Modes and Techniques

77

Fig. 3-4. Displacement between adjacent peaks.

Fig. 3-5. Displacement between adjacent peaks with non-linear isotherms.

ents. When gradient elution is used equilibration of the column with the original mobile phase will be required before subsequent runs are carried out, thus reducing the number of runs which can be carried out in unit time.

3.2.5 Miscellaneous Operating Techniques Various techniques have occasionally been used to improve the throughput of a chromatographic process. In the early days of modern preparative chromatography, the technique of recycle with peak shaving [lo] was used when running in the elution mode, to compensate for the poor performance of the stationary phases which were then available. The overall effect is simply the same as using a much longer column, although without the increased back-pressures which this would generate and with lower consumption of mobile phase since it is continuously recycled through the column. The peak shaving is only necessary to prevent the faster running components catching up with the slower running components of the previous cycle. Although sometimes useful in one-off separations, it is not a good method for process operation, since it is slow and difficult to automate. A far better option is to use a higher efficiency column and, if there is an intermediate fraction of lower than the required purity, to recycle it off-line by combining this fraction from a number of chromatographic runs, isolating the material and rechromatographing it. The recycle/peak shaving technique has recently been revived for use in preparative enantiomeric separations on triacetyl cellulose [ I I , 121. Since the triacetyl cellulose used for preparative separations is a fairly low efficiency stationary phase, this may be a better ap-

78

3 Alternative Modes of Operation of Chromatography Columns

proach than using the extremely long columns and run times which have been reported elsewhere. It is often forgotten that sequential injections onto a column can be overlapped in such a way as to utilise the window before the fastest running peak starts to elute from the column to collect fractions from the previous injection. In this way the column is used at its maximum capacity and solvent usage is minimised. The use of the so-called ‘flip-flop’ technique [I31 in which the direction of flow in the column is reversed and the sample is injected alternately at either end of the column has been demonstrated to provide higher throughputs with reduced solvent consumption when used with elution chromatography. This approach requires the use of a column such as the Dynamic Axial Compression system manufactured by Prochrom, Champigneulles, France, where the stability of the packed bed is unaffected by the direction of flow. The technique of simulated moving bed chromatography [I41 has been around for a considerable time in industry, although for commercial reasons much of the work remains unpublished. Due to the problems associated with engineering this technique, its use has been largely restricted to simple separations where the quality requirements for the product are fairly low. Recently, however, developments in instrumentation and in particular in microprocessor-based control systems, have considerably improved the prospects for a wider acceptance of the method. Its main drawback is that it is only suitable for separation of binary mixtures unless a two-stage separation process is used. However, it would seem to be ideally suited to the field of preparative enantiomeric separations, and it is likely that a rapid growth in its use will occur in this area, particularly since the required instrumentation is now becoming commercially available.

3.3 The Use of Alternative Modes and Techniques in Process Situations The touching band approach for determining the maximum loadability of a column, as proposed by Knox [15], is fine in non-process situations, and is probably the best way of carrying out such separations. The development time is minimal in that, with modern column technology and the ready availability of bulk, high-performance stationary phases, it is a relatively simple matter to scale-up an analytical separation to a preparative column and to increase the loading to the maximum allowed by the touching bands approach. Separation of the required amount of material can be achieved in a fraction of the time that would have been required to develop a preparative separation a few years ago. However, when we examine the requirements of process, we must pose the question as to whether this approach will truly give us the maximum possible loadability and throughput. The answer in most cases is definitely not. The effect of loading beyond the point at which bands touch will differ from separation to separation. We must differentiate between a mass overload and a volume

3.3 The Use of Alternative Modes and Techniques in Process Situations

79

a c a'

conc

b b' time

1

Fig. 3-6. Volume overload.

increased loading on peak shape.

time overload. In the case of a volume overload the peak shape will remain approximately Gaussian but will broaden until adjacent peaks touch, then further broaden so the overlapping of the peaks occurs. In this case it is the required purity of the product which will determine how far the column can be loaded beyond the point at which adjacent peaks touch. Figure 3-6 shows a pair of overlapping peaks. If 99% material is required, the fraction from a to a' must be discarded. However, if only 95% material is required, only the portion from b to b' will be discarded and the cut point c will give 100% yield of 90% material. In the case of mass overload, the peak becomes approximately triangular (Fig. 3-7), with a steep front edge. Usually, in the volume overload situation, there is little which can be done to increase loading. Often the optimum conditions for a separation (at least with normal phase work) occur at a solvent strength where the analytes have low solubility. This is not a problem in analytical applications, but for preparative work it may prevent us increasing the strength of the load solution and hence getting a smaller loading band, to which the profile of the eluting band(s) will be related. Thus, in designing systems for preparative work it is essential to try to find solvent mixtures in which the analytes have good solubility, for it is only with good solubility that the vast potential of mass overload can be exploited. There is one major problem associated with working at very high loading which is the determination of cut points. Ultraviolet detectors become overloaded at relatively low concentrations and, whilst refractive index detectors are better in this respect, they suffer from other disadvantages such as an inability to cope with solvent gradients. Split flow to the detector may be helpful, but can cause problems. Where displacement is taking place, the boundary between adjacent peaks can be very sharp, and if the materials are closely related, may not show on a detector suffi-

80

3 Alternative Modes of Operation of Chromatography Columns

ciently to enable the cut to be made at the right point. It is of course possible to cut fractions simply by time or volume and analyse them off-line, but this is not to be recommended for process use where the analysis of hundreds of samples per day might be necessary. The best method of detection is undoubtedly an analysis of the eluent as it elutes from the column. This is becoming more feasible as fast HPLC systems are being developed, but analysis time is still rather long compared with the rate of elution of materials from the column. Additionally, it is often not easy to install an analytical HPLC instrument as a part of or close to a preparative instrument, because of intrinsic safety requirements associated with the use of large amounts of flammable solvents. The problem of detection is one which needs to be addressed by manufacturers of large scale equipment since development of detectors has not kept pace with developments in the rest of the instrumentation and developments in column technology.

3.4 Conclusion Unfortunately, there is no simple answer to the problem of developing a separation for process use, and there is no substitute for experimentation. What is important, is that we should be aware of the wide range of options which are open to us, and not be blinkered by experience gained in analytical and small-scale preparative work. Process chromatography is a subject in its own right, with its own problems, not simply an extension of other forms of chromatography.

References [I] Hill, D. A. in: Preparative and Process-scale Liquid Chromatography, Subrarnanian, G. (ed.), Chichester: Ellis Horwood, 1991, p. 98. [2] Kelly, M. in: Preparative and Process-scale Liquid Chromatography, Subrarnanian, G. (ed.), Chichester: Ellis Horwood, 1991, p. 108. [3] Tiselius, A. Ark Kemi Mineral Geol, 1943, 16A, I . [4] Horvath, C., Nahum, A., Frenz, J. J Chromatogr, 1981, 218, 365. [5] Verzele, M., Dewaele, C., VanHaver, D. J Chromatogr, 1982, 249, 231. [6] Hill, D. A., Mace, P., Moore, D. J Chromatogr, 1990, 523, 11. [7] Guiochon, G., Katti, A. Chromatographia, 1987, 24, 165. [S] Newburger, J., Guiochon, G. J Chromatogr, 1990, 523, 63. 191 Golshan-Shirazi, S., Guiochon, G. J Chromatogr, 1991, 545, 1 and references cited therein. [lo] Bidlingmeyer, B.A. in: A Better Way to Isolate and Purgy, Publication B24, Milford, Mass: Waters Associates, 1979, p. 14. [ I l l Schlogl, K., Widhalm, M. Monatsh Chem, 1984, 115, 1113. [I21 Juaristi, E., Qintana, D., Lamatsch B., Seebach, D. J Org Chem, 1991, 56, 2553. [I31 Colin, H., Hilaireau, P., Martin, M. J Chromatogr, 1991, 557, 137. [I41 This subject is discussed in detail in Preparative and Production Scale Chromatography, Ganestos, G., Barker, P.E. (ed.), New York, Marcel Dekker, 1993. [I51 Knox, J. Guidelines for Developing Preparative HPLC Separations. Presented at the 7th International Symposium on Preparative Chromatography, Gent 1990.

4 Process Scale Size Exclusion Chromatography Jan-Christer Janson

4.1 Introduction There are two main application areas for size exclusion chromatography of proteins: desalting or group separation and fractionation. Both areas are industrially of equal importance but the prerequisites for their ability of being scaled-up are quite different. Desalting operations are typically based on the use of rigid particles, made of highly cross-linked dextran, polyacrylamide or cellulose, that totally exclude proteins and other high molecular weight material while allowing free access of low molecular weight components of the sample into the stagnant liquid in the pores of the particles. The high diffusion coefficient of low molecular weight solutes enables the use of large particles and high operational flow-rates in desalting applications. The beneficial physical properties of the highly cross-linked polymer particles allow their packing in large columns without height or diameter restrictions and the separation principle allows the application of proportionally large sample volumes (in practice up to -25% of the total column volume). The column productivities in desalting operations are thus normally very high (in the order of 1 g protein per cm’ column cross sectional area per hour). The principle of fractionation by SEC, ie, the total separation of two or more proteins that differ sufficiently in size and/or shape, is in the majority of cases based on all the components diffusing into the interior of the particle gel matrix. With few exceptions, fractionation SEC is therefore a process with comparatively low productivity due to the low gel diffusivity of proteins, the requirement of long columns, low flowrates and, for optimum resolution, small sample volumes (0.005 - 0.05 Vc).In many industrial applications, scaling-up of fractionation SEC means stacking several short, large diameter columns connected by small bore tubing. The sample dry weight concentration as such is not a restriction in SEC, rather there is a linear relationship between this and the chromatographic productivity. What is usually the most serious sample load limiting factor, besides a small sample volume, is the relative viscosity of the sample that should not exceed a value of 1.5, which corresponds to a globular protein concentration of approximately 7%. There are several review articles published on SEC [l -41 which will provide a more comprehensive treatment than this chapter allows. (Further information on process scale SEC can be obtained from [ 5 ] and [6].)

82

4 Process Scale Size Exclusion Chrornutogruphy

4.2 Separation Principle The separation in SEC is, by definition, only dependent on differences in the sizes and shapes of the molecules to be separated, and the pore size distribution of the three dimensional network of the gel materials used for the chromatography. The molecules in the sample solution mixture, which is pumped into the packed bed as a narrow zone, distribute themselves practically momentarily between the stationary gel phase and the flowing aqueous buffer outside the gel particles. At any particular moment during this process, a certain mass fraction of each molecular species, the size of which depends on the fraction of the gel phase sterically available to that molecule, is moving down the column with the speed of the flowing liquid. The total mass transport of each molecular species down the column is effected at a speed which is inversely proportional to the fraction of the gel phase available to this particular molecule. It should be possible to make an analogy to isocratic partition chromatography in which molecules are separated according to their solubility in the liquid fraction of the gel phase. According to this principle, the separation in SEC is not dependent on differential diffusion rates; all molecules which enter the gel phase are supposed to distribute themselves through the whole gel particle cross-section while occupying only that fraction which is available to them by pure steric restriction. The classical equations describing SEC are the following: V, = V,+K,.

v,

(1)

and

v, = VO+K,; Vg

(2)

where: V, elution volume of the molecule (also called V,) V, void volume (elution of the totally excluded molecule) KO distribution coefficient Vp internal liquid pore volume of the gel phase (V,-V,-V, or, sometimes easier to measure, VR,H20-V,) K,, distribution coefficient Vg total volume of the gel phase V, total matrix volume V, geometrical column volume In practice, K,, has become the favoured distribution coefficient because of the ease with which it may be determined. The experimentally determined relationship between solute size (Stokes’ radius or molecular weight) and the distribution coefficient is a fundamental characteristic of any gel filtration medium known as the selectivity curve. Since SEC separates not only according to molecular size (mass) but also shape, two molecules with the same molecular weight but with different shapes will elute after different V,, with a rod-shaped molecule eluting earlier than a flexible coil which in turn will elute earlier than a spherically shaped, compact molecule

4.2 Separation Principle 0.8

83

Proteins: Chymotrypsinogen (25000) Cytochrome C (12600) Aprotinin (6500) Vitamin B12 (1400)

0.6.

Peptides:

5

Ac-(Gly-Leu-Gly-Ala-Lys -Gly-Ala-Gly-Val-Gly)n-amide (n=l 5, Mw=830-3900)

0.4-

-

PEG: s PEG 6000 PEG 1500 PED 600 PEG 150

0.2 -

L1000 .,-. 10000 100000

0.0

.

,

1

_ m l

100

Log molecular weight (Da)

Fig. 4-1. Selectivity curves of SuperdexB 30 prep grade for three categories of solutes: polyethylene glycols (PEG), peptides and proteins, respectively.

(Fig. 4-1). This explains why some polypeptides may fall outside calibration curves prepared with standard globular proteins. Theoretically, the steepest selectivity curve and thus the highest separation power is obtained with the idealized gel medium in which all the pores are of identical size, giving a separation range per pore volume of approximately one decade [7]. Such gels do not exist but are approached by gels made of point cross-linked linear polymers such as dextran and polyacrylamide. Since the proteins are eluted according to the available pore volume fraction, the total separation volume cannot be larger than the total volume of the liquid in the gel phase. The relative pore volumes vary between approximately 52% and 97% for different SEC media, with the lower value being typical for porous glass and some silica materials and the higher for certain polysaccharide-based media. The restricted separation volume in SEC reduces the maximum number of components that can be separated to (adapted from [7]): ,

nR, =

1f

5/ 5 + 6

*

(Nmax)‘’’/4Rs

(3)

where nR, is the number of peaks separated with a resolution factor of R,, V p is the internal liquid pore volume of the packed gel particles, V, is the void volume of the column and Nma,is the maximum number of theoretical plates in the column. The maximum number of components that can be separated in SEC is small compared with other liquid chromatography techniques. Even in HPLC mode of SEC not more

84

4 Process Scale Size Exclusion Chromatography

than approximately 12 peaks can be separated if a complete separation is required (ie, when R, = 1.5). In this context it should be remembered that the only parameter which governs the separation selectivity in SEC is the steepness and shape of the selectivity curve. The size of the chromatographic particles only shortens the time required for the molecules to achieve diffusive equilibrium in the gel medium. In other words, one can in principle get the same resolution in SEC irrespective of particle size merely by optimizing the operating flow-rate for the eluting buffer. Once applied to the top of the column, the sample zone in SEC is subjected to three dispersion factors: (1) Longitudinal axial diffusion which for proteins can be neglected at normal flow-rates. (2) Zone spreading due to flow irregularities in the chromatographic bed caused by an inferior packing procedure and/or a broad or uneven particle size distribution. (3) The most important cause of zone broadening, the restricted diffusion of the large protein molecules inside the sieving network of the gel particles which slows down the establishment of concentration equilibrium between the flowing and stationary liquid phases. The strong dependency of the resolution on the initial width of the sample zone is typical for SEC [8] and is especially significant for HPLC type SEC media where optimum resolution in analytical mode requires a sample volume of 0.2% of the bed volume or less. In preparative SEC, the optimum sample size (ie, sample volume) is a compromise between purity and chromatographic throughput and is unique for every separation situation. As was discussed above, the ability of an SEC medium to achieve a particular separation depends primarily on the relationship between the sample size and the total pore volume of the column [l].Gels which have pore volumes which are small compared with the overall gel volume, ie, those in which gel matrix occupies a large volume, such as silica and certain synthetic organic polymer gels, will therefore have a relatively low separation power (peak capacity). This is a factor which speaks in favour of media where the matrix is composed of single polymer chains such as dextran or polyacrylamide. In some respects it is misleading to refer to ‘pores’ in SEC media, since this often implies the existence of well defined spaces in a matrix composed of stationary elements. At the molecular level, the gel forming elements of many of the most useful media have a mobility not much different from the mobility they would have in free solution. Any individual space in such a gel is continually changing both its size and shape, just those properties which determine its ability to exclude other molecules by steric exclusion. The dynamic nature of the spaces in these gels means that the pore size distribution must be defined operationally in terms of its exclusion properties [9]. It almost certainly explains the observation that pore size distributions for gels where the exclusion properties are defined by flexible polymer chains are smoother than for those for gels where the excluding elements are expected to be stiffer.

4.3 Column Packing Materiuls for Process Scale SEC

85

4.3 Column Packing Materials for Process Scale SEC The ideal packing material for process scale SEC offers a range of narrow selectivities that satisfy the demand of optimal elution position for a range of protein molecular weights. It has got a satisfactory V , / K ratio that gives high peak capacity and high loadability. It is also complying with all the requirements of an industrial environment with regard to physical and chemical robustness as well as fulfilment of regulatory demands such as validability [ 101. Many different gel forming substances have been proposed in attempts to find the ideal SEC medium. These may be grouped into three main types based on the structure of the gel forming matrix: materials in which the matrix is composed of individual polymer chains; gels in which the matrix is built from aggregated polymer chains, and combinations of these. In these two cases, the same elements define the pore size distribution and provide the mechanical strength of the gel. The first type tends to be mechanically weak whereas the pore size distribution of the second kind may be uneven and the matrix volume may be high. In the third type, composite gels, aggregated matrix elements provide a mechanically strong supporting gel and the pore size distribution is given by linear polymer chains grafted to the supporting gel structure.

4.3.1 Dextran Gels and Polyacrylarnide Gels Gels based on cross-linked dextran Sephadex@ (Pharmacia Biotech AB, Uppsala, Sweden), or polyacrylamide, BioGel@ P (BioRad Laboratories, Richmond, CA, USA) were among the first to reach widespread use for SEC of proteins. The separation range, selectivity curve, is controlled by the degree of cross-linking of the polymer chains, and media are available with exclusion limits up to about 1x lo6 for globular proteins. The loosely cross-linked gels, with exclusion limits adequate for fractionation of protein mixtures, are mechanically weak and thus not suitable for process scale applications. However, the low content of gel matrix gives them a large separation volume and reduces the risk of adsorptive interactions. They are also characterized by relatively steep selectivity curves and give excellent separations and this is why they are preferred as constituents of composite gel media as shown below. The more tightly cross-linked kinds, eg, Sephadex G-25 and BioGel P-2, have high mechanical strength and separation ranges which make them ideal for desalting and buffer exchange operations. Both cross-linked dextran and polyacrylamide gels are usually supplied as dry powders which must be fully swollen before being packed in a column. The other kinds of SEC media are supplied as suspensions and little special treatment is needed.

86

4 Process Scale Size Exclusion Chromutogrup fiy

4.3.2 Agarose Gels Gels based on agarose alone [11] are examples of the second type of gel structure mentioned above. The individual polysaccharide chains are aggregated to form stiff fibres (Fig. 4-2). A gel containing 4% agarose, eg, Sepharose 4B, is thus much stronger than a gel containing 4 % dextran, eg, Sephadex G-200, at the same time that its exclusion limit is about two orders of magnitude higher. The separation range of agarose gels is controlled by the agarose content, gels containing as little as 2% agarose being used for special purposes.

Fig. 4-2. Scanning electron micrograph of an agarose gel (2%). The white bars represent 500 nm. In SuperdexO SEC media, the voids surrounding the agarose network skeleton are occupied by covalently linked dextran polymer chains. (Preparation and photo: A. Medin, Institute of Biochemistry, Uppsala University, Uppsala, Sweden.)

The rigidity of agarose gels may be further increased and their chemical and thermal stability greatly improved by cross-linking [I21 such as in Sepharose@Fast Flow gel media. Since most of the cross-linking occurs between agarose chains in the same fibre, the stability of the fibre is increased, but its exclusion properties are essentially unchanged. Cross-linked agarose gels have separation ranges which are the same as the uncross-linked gels. The selectivity curves are rather flat and their selectivity is consequently low. Like dextran- or polyacrylamide-based gels, the media with the highest exclusion limits are mechanically weakest. The exclusion limits of agarose gels are so high that they find their biggest use in SEC fractionation of protein complexes and molecules with extended structures, eg, polysaccharides and also for the synthesis of composite gel media as described below.

4.3 Column Packing Materials .for Process Scale SEC

87

4.3.3 Composite Gels The most recently introduced SEC media are nearly all composite gels of the third type mentioned above. They differ in the chemical composition of both the supporting matrix which accounts for their mechanical properties, and the linear polymer chains which account for their exclusion properties. The first representatives of this type were UltrogelTMAcA, in which a polyacrylamide gel was trapped in the pores of an uncross-linked agarose gel [13], and SephacryP in which dextran chains are covalently grafted to macroporous, highly cross-linked bisacrylamide [ 141. A more recent development, Superdex@,is based on a highly cross-linked, rigid agarose supporting matrix which carries covalently grafted dextran chains [ 151. The special character of some of these gels is evident from a comparison of the maximum operating pressures for media with very different fractionation ranges but the same particle size distributions. For example, both Sephacryl S-100 HR (upper fractionation range for globular proteins ca. 1 x lo5) and Sephacryl S-500 HR (upper fractionation range for globular proteins ca. 8 x lo7) permit operating pressures up to 0.2MPa. The selectivity curves of composite media reflect the concentration and type of the excluding polymer chains. Thus, Superdex 75 and Superdex 200 have steep selectivity curves and exclusion properties closely similar to those of the corresponding dextran gels, Sephadex G-75 and Sephadex G-200, respectively, although they allow much higher flow rates to be used. Naturally, the smaller particle sizes give better resolution under otherwise comparable conditions.

4.3.4 The Choice of Separation Medium The SEC medium is chosen firstly on the basis of its fractionation range considered in relation to the sizes of the components to be fractionated, and secondly on the basis of its other physical and chemical properties. A good indication of the separation power of different media is given by their selectivity curves. The choice is made so that the target protein elutes with a K,, in the range 0.2 to 0.5, smaller values of K,, being favoured when critical contaminants are smaller than the target protein and larger values when the contaminants are larger. The choice between media with similar selectivity curves can only be made on the basis of trial runs since small details of gel structure made have effects on the resolution of closely eluting peaks which are difficult to foresee. In choosing between different media, questions of their chemical and physical stability become important since they govern the conditions which can be used for both running and cleaning the media. The newer composite media are generally to be preferred since their greater mechanical strength and smaller particle sizes allow higher flow rates to be used whilst maintaining resolution. A particle size range should be chosen that gives the desired degree of resolution for the required chromatographic productivity (sample load and flow-rate). Once again, a series of trial runs may be necessary to find the optimal gel. The relationship

88

4 Process Scale Size Exclusion Chromatography

between sample volume, resolution and particle size [2] is of significance in this context. For many preparative purposes the particle size which gives the optimum combination of resolution and separation time for a given sample volume may not necessarily be the smallest. For many high resolution process scale SEC applications, such as final polishing steps, a bead size of approximately 30 pm has proven adequate. In Table 4-1 are listed representative SEC media for process scale applications and their properties.

Table 4-1. Characteristics of some media for process scale SEC.

For desalting applications: Medium

Matrix

Dry particle sizea (pm)

Approximate separation range for proteins (kD)

Supplier

Sephadex G-25 Coarse Sephadex G-25 Medium Sephadex G-25 Fine

Dextran Dextran Dextran

100 - 300 50 - 150 20- 80

1 x lo3- 5 x lo3 1 x lo3- 5 x lo3 1 x l o 3- 5 x lo3

Pharmacia Pharmacia Pharmacia

BioGel P-6 Fine BioGel P-6 Medium

Polyacrylamide Polyacrylamide

45 - 90 90 - 180

1 x lo3- 6 x 1O3 1 x I O3 - 6 x 1O3

BioRad BioRad

For fractionation applications: Medium

Sephacryl S-100 HR

Matrix

Dextran/ bisacrylamide Sephacryl S-200 HR Dextranl bisacrylamide Sephacryl S-300 HR Dextran/ bisacrylamide Sephacryl S-400 HR Dextran/ bisacrylamide Sephacryl S-500 HR Dextran/ bisacrylamide Superdex 30 prep grade Agarose/ dextran Superdex 75 prep grade Agarose/ dextran Superdex 200 prep grade A g a r 0 4 dextran Toyopearl HW 40s Polymeric Toyopearl HW 50s Polymeric Toyopearl HW 55s Polymeric Toyopearl HW 65s Polymeric Toyopearl HW 75s Polymeric a

Particie size Approximate separation (w) range for proteins (kD)

Supplier

25 - 75

I x lo3- 1 x lo5

Pharmacia

25 - 75

5x103-2.5~10~

Pharmacia

25 - 75

1 x 1O4 - 1.5 x 1O6

Pharmacia

25 - 75

2 x lo4- 8 x lo6

Pharmacia

25 - 75

2 x lo4- 3 x 1O9

Pharmacia

24 - 44

1 x lo2- 1 x lo4

Pharmacia

24 - 44

3 x lo3- 7 x lo4

Pharmacia

24 - 44

1 x l o 4 - 6 x lo5

Pharmacia

25 - 40 25 - 40 25 - 40 25 - 40 25 - 40

1 x 102- I x 104 5 x 10’ - 8 x lo4 1 x lo3- 7 x 10’ 5 x lo4- 5 x 106 5 x 105- 5 x lo7

Tosoh Tosoh Tosoh Tosoh Tosoh

Bed volumes obtained are in the range 4 - 6 mL g-’ dry Sephadex G-25. Copolymer of oligoethylene glycol, glycidylmethacrylate and pentaerythreitoldimethacrylate.

4.5 The Eluent in SEC

89

4.4 Adsorption Effects of SEC Media Most SEC media expose either primary alcohol hydroxyls or amido groups on their polymer matrix surfaces. These groups are known to be non-interactive with most proteins and should thus create an ideal SEC environment. However, sometimes the presence of trace constituents in the polymer matrix raw material, or as a consequence of the manufacturing process, other groups, such as hydrophobic and/or ionic may appear in the final product. Thus, silica and agarose inherently contain low concentrations of negatively charged groups. These will cause adsorption of polycations such as basic proteins and exclusion of polyanions such as acidic proteins when low ionic strength buffers are used [16]. In order to increase their chemical and physical stability, covalent cross-links are introduced into modern agarose media during their manufacturing procedure. As a consequence, these become less hydrophilic and retardation of hydrophobic proteins can be observed at high ionic strengths [17]. As a rule of thumb, the ionic strength of buffers used in SEC, in order to prevent adsorption due to the presence of either ionic or hydrophobic groups, should preferably be in the range 0.1 -0.2. However, these effects are not always negative and may sometimes even contribute to the improvement of a separation.

4.5 The Eluent in SEC A characteristic feature of SEC is that the influence of the composition of the eluent on the separation can usually be completely neglected. Within wide limits set by the stability of the separation medium, the eluent can thus be chosen to suit the properties of the sample, in particular the stability of the biological activity of the target protein. Special components may be added almost without restriction to solve special problems of solubility or to meet other specific requirements. For example, detergents at concentrations below the critical micelle concentration or water miscible organic solvents, may be added to improve the solubility of hydrophobic proteins and peptides. Buffer composition, p H and ionic strength may be chosen to suit the requirements of a subsequent step like ion exchange chromatography or product formulation. It should be noted that a number of additives which improve solubility will also affect the shape of the protein molecules and thus their elution position.

90

4 Process Scale Size Exclusion Chromatography

4.6 Practices of Process Scale SEC 4.6.1 Column Dimension Resolution in SEC is proportional to the square root of the column length. Doubling the column length thus will only improve the resolution by a factor of approximately 1.4. Also, long columns are difficult to pack properly and give rise to high flow resistances. In addition, as the separation time is directly proportional to column length, a long column will require a long elution time. In many applications using small diameter (20- 50 pm) particles, a column length of 60 cm has proven adequate, providing column volumes of 4.7 L for a 100 mm diameter column and 18.8 L for a 200 mm diameter column. The usual column lengths for process scale applications, using particle sizes in the range 25 -75 pm, are 90- 100 cm. However, due to the flow resistances experienced with many modern semi-rigid gel media especially in columns with large diameters, and with media with small particle sizes, it is preferable to achieve the desired column length by stacking two to six shorter columns in series connected by small bore pipes. Shorter columns allow a much higher flow velocity during packing, resulting in beds with low V,-values and less zone dispersion. Minimum dispersion of the sample zone during application to the column bed is achieved

Fig. 4-3. Pharmacia Process Stack Column PS 370 in an insulin production facility (see Table 4-2).

4.6 Practices of Process Scale SEC

91

Fig. 4-4. Pharmacia BioProcess Stainless Steel Columns 800/300 in a human serum albumin production facility (see Table 4-3).

Table 4-2. Conditions for process scale SEC of insulin. Column Total bed height Column diameter Total bed volume Gel type Pump Flow-rate Sample volume Eluent Cycle time Column productivity

Pharmacia Procees Stack Column PS 370-1/4" 90cm ( 6 x l 5 c m ) 37 cm 96L (6xI6L) Sephadex '2-50 Superfine Special Grade Sera double membrane pump R 410L 1 4 L h - ' (13.1cmh-' linear flow) 2 L (28 g L- partially purified insulin) 1 M acetic acid 7h 56 g partially purified insulin per cycle

'

Table 4-3. Conditions for process scale SEC of HSAa on Sephacryl S-200 HR. Sample Sample volume Eluent Flow-rate Bed height Cycle time Column productivity a

HSA = human serum albumin

60-7OgL-' HSA solution in acetate p H 5.5 4% of the column volume 0.02 M sodium acetate p H 6 40cmh-' 90 cm (3 x 30 cm stainless steel columns) 40 min (sample application interval) 320 g HSA/I 000 cm2 h - '

92

4 Process Scale Size Exclusion Chromatography

by avoiding excessive volumes and excessive pressure drops in connecting pipes and in the sample distribution layer between the bed top and the column end piece (radial pressure drop in the sample distribution layer of the column end piece would cause radial irregularity in the shape of the sample zone and thus in zone broadening). The most successful example of using the concept of stacked columns in process scale SEC is in the insulin industry. Here the standard procedure involves the use of six column segments 15 cm bed height and 37 cm diameter giving a total column length of 90 cm and a total column volume of 96 L (Fig. 4-3). The conditions for this SEC application are listed in Table 4-2. For industrial applications with feed volumes in the range 20-25 L, such as in the plasma protein fractionation industry, see Table 4-3, normally three stainless steel columns, 30 cm bed height and 80 cm diameter, total volume 150 L, are connected in series (Fig. 4-4). The volume of the gel bed is chosen taking in account the volume of sample to be applied and peak separation volumes. Typical sample volumes for fractionation on Superdex 75 prep grade (average particle size 34 pm) are in the range 0.5 - 4% of bed volume.

4.6.2 Gel Preparation and Column Packing Modern SEC media require little preparation before packing except for those supplied as dry powders. These materials must be swollen fully, preferably in hot buffer solution, before packing. In all cases the gel suspension must be at the temperature at which the column will be packed and used. The quality of the column packing is crucially important to the success of size exclusion chromatography, and special care should be exercised when packing large diameter columns. Make sure the column is not damaged and that all parts are really clean. It is specially important that the nets and net fasteners are not damaged. See that the column tube is fixed or placed on a sturdy stand or fundament. Use a spirit level to assure a perfect vertical mount of the tube (for long columns) or a perfect horizontal mount of the bottom end plate (for short, wide columns). Pack the column at the temperature at which it will be used. Carefully remove air from underneath the bottom bed support net by passing eluent through it from underneath. Suck out any remaining air bubble. The gel suspension should be so thick that it will still pour easily and so thin that air bubbles will rise rapidly to the top. Pour the gel into the column down the column wall in one batch, avoiding the introduction of air bubbles as far as possible. A packing reservoir may be necessary to extend the column to make this possible. To assure a homogeneous gel slurry in wide diameter columns, stir the gel suspension slowly and manually with a paddle-like stirrer (never use motor-driven stirrers). By holding the paddle radially against the column inner wall, make sure that all movements within the slurry come to a rest. Then remove the paddle vertically. Pack the column using the flow rates or packing pressures recommended by the manufacturer for each individual gel type. Continue packing the column until the length of the packed bed is constant and equilibrate finally with two bed volumes of eluent buffer.

4.7 Chromatographic Productivity in SEC

93

A well packed and properly cared for column will give good results for many runs, repaying the time and trouble in its preparation; a poorly packed column will never give a good result.

4.6.3 Feed Stock Preparation The feed stock solution must be centrifuged and/or filtered to remove particles. It is not necessary to equilibrate it with the eluent as this equilibration will take place during the run. However, it may be necessary to dilute viscous samples or to remove polysaccharides or nucleic acids which can increase viscosity.

4.7 Chromatographic Productivity in SEC The chromatographic productivity in SEC is defined as the amount of adequately purified protein recovered per column cross-sectional area and chromatographic cycle time. The critical parameters in the optimization of SEC productivity are the sample size and flow-rate, respectively. The sample volume restriction in SEC, as discussed above, makes it mandatory to divide the feed stock into consecutively processed aliquots. The smaller the aliquots, the faster the possible flow-rate at constant resolution. The optimum conditions for processing Vfeed litres load volume per hour will correspond to [I]

vnj

where is the aliquot volume, Kinjis related to the shape of the applied sample plug. The optimal value is equal to 12 (the variance of a square-wave distribution) and is approached at larger sample volumes, such as those in desalting operations, are but for small sample volumes values around 5 are more common. V, and defined as above. D, is the solute diffusion coefficient in the gel particle and dp is the average particle diameter. The corresponding linear flow-rate, u, is given by

where L is the column bed length. The linear flow-rate is obtained by the volumetric flow-rate divided by the column cross-sectional area. These equations are useful for selecting optimum conditions and for scaling-up separation schemes. Governed by requirements of protein purity and column productivity, it is possible to find an optimum balance between flow-rate and sample volume. One way is to use simulations, correlated with data from experiments, to predict the optimum conditions. Such a study was performed by Arve [I81 for the separation of IgG and transferrin on Superdex 200. The recovery of IgG at different levels of final purity

94

4 Process Scale Size Exclusion Chromatography 5% Relative load volume

’/

, /

‘lyrecovery

10 20 30 40 50

60 Linear flow-rate (cmlh)

Fig. 4-5. Computer simulation of productivity and recovery of IgG in SEC on Superdexa 200 prep grade as a function of linear flow-rate and relative sample load (expressed as To of total bed volume). Simulations performed for an initial and final concentration of transferrin of 16.7 and 0.01%, respectively. In order to be able to increase the recovery at maximum productivity, both flow-rate and sample load will have to be decreased. Correlations from experimental work on a 2.6 cmx60 cm column. (Courtesy B. Arve [18].)

was determined as a function of flow-rate and sample volume at varying levels of transferrin contamination. Correlations with experimental data were made by computer simulation. The result of one set of conditions with initial and final IgG purities of 83.3% and 99.9%, respectively, are shown in Fig. 4-5. Of particular interest is the fact that the recovery curves go through a maximum in this operation range. A line drawn through the maxima of these curves should give the combination of sample volume and flow-rate at which one should operate to achieve the desired recovery at maximum productivity. When the optimal conditions for maximum productivity have been established, the cycle capacity is increased by proportionally increasing the column diameter and the sample volume. There seems to be only one way to further increase the column productivity in SEC, and that is to make maximum use of the available separation volume in the operating column, ie, not allowing any ‘empty’ spaces in the chromatogram. The processing time of an optimized procedure may thus be reduced by 33% by utilizing the dead time during elution of the void fraction to applying a new sample after only 2/3 of the total column volume is eluted. In this way, it is possible in favourable cases

4.7 Chromatographic Productivity in SEC

I

1

; -

;

95

:

Time (h)

Fig. 4-6. Elution diagram for process scale SEC of human serum albumin on Sephacryl S-200 HR. Sample application at an optimum interval of 40 min will make maximum use of the available column separation space. The albumin is collected in a volume of approximately 0.15 F.(Courtesy Berglof et al. [19].)

to apply three sample cycles per column bed volume. However, this can only be accomplished when the protein of interst and its accompanying impurities are eluted within a rather narrow volume window. Berglof et al. [I91 described such a favourable situation for the final step in the process purification of human serum albumin. The peaks to be separated were spaced in such a way that three cycles were performed per eluted column volume (Fig. 4-6) which means that the column productivity could be increased three fold as compared with the traditional way of operating SEC columns. Also, the cycle time can be further minimized by maximizing the flow-rate once the protein of interest has been eluted to rapidly rinse the column before the next sample is applied. There is a linear relationship between productivity and sample concentration in SEC. However, the restriction is related to the effect of increasing protein concentration on the viscosity of the sample relative to the eluent rather than the dry weight content as such. A relatively viscous sample zone is hydrodynamically unstable and viscous fingering soon develops with catastrophic effects on the resolution. As a rule of thumb, the concentration of a globular protein in SEC samples should not exceed 70 mg mL-' or a relative viscosity of 1.5. The most favourable type of SEC from a productivity point of view is desalting or buffer exchange using column packing materials such as Sephadex G-25 which totally excludes proteins but includes low molecular weight impurities and salts. Here sample volumes of approximately 20% of the column volume are routinely used and the flow-rates applied do not have to be adjusted to compensate for the slow diffusion rates of proteins in gels. One example is the de-ethanolization of human serum albumin obtained in the Cohn cold ethanol procedure. Introduced in 1972 by Friedli and Kistler [20], typically 12 L of albumin solution, containing 9% protein and 9% ethanol, is de-ethanolized in 13.5 min using a 40 cm diameter and 60 cm bed height column (total bed volume 75 L) of Sephadex G-25 Coarse operating at a linear flow-

96

4 Process Scale Size Exclusion Chroinuto~ruph~v

Fig. 4-7. Pharmacia BioProcess Stainless Steel Columns for desalting SEC operations.

rate of 240 cm h - ' . The corresponding buffer consumption is 55.5 L and the dilution factor is 1.5 - 1.9. Periodically, and during shut-downs, the column is rinsed with 2% NaOH, followed by 1 % formaldehyde. This procedure assures a high level of sanitation (bacterial counts usually < 10 per mL) and a media life length > 7000 cycles or 2 years' production. In Fig.4-7 are shown a variety of stainless steel columns used in industrial desalting operations and in Table 4-4 are listed column types and sizes, gel types, pumps and conditions normally used for desalting SEC and solvent removal in biotech industry.

4.8 Strategy for Scaling-up of SEC As has been discussed above, the resolution in SEC is more affected by increasing the sample zone width than increasing the operating flow-rate. This fact might be used for suggesting a simple strategy for the scaling up and optimization of the column productivity in SEC. Thus, in the first optimization step, one adjusts the column length and flow-rate to obtain a reasonable resolution, but primarily satisfying the desired chromatographic cycle time. In the next step, the desired resolution is obtained by adjusting the relative sample volume. Finally, the desired column productivity is achieved by adjusting the diameter of the gel filtration column keeping the linear flow-rate and the relative sample volume (ie, the sample zone width) constant. This apparently simple strategy requires that the columns are packed to the same

a

G-25 fine or G-25 fine or (3-25 fine G-25 fine or G-25 fine G-25 fine or G-25 coarse G-25 coarse G-25 coarse G-25 coarse G-25 coarse G-25 coarse G-25 coarse

Up to 0.3 0.3 - 1 0.4 - 3 0.4-4 1-6 1-9 Up to 15 Up to 25 u p to 35 up to 55 Up to 60 u p to 100 Up to 500 BPG 100/500 BPG 100/950 KS 370/15 BPG 200/950 KS 370/30 BPG 300/950 BPSS 400/600 BPSS 400/1000 BPSS 600/600 BPSS 600/1000 BPSS 800/600 BPSS 800/1000 BPSS 1800/1000

Recommended column type

0-30 45 - 64 15 45 - 64 30 45 - 64 60 100 60 100 60 100 100

Bed height (cm)

0-2.4 3.5-5.0 16 14.1 -20.1 32 3 1.8 - 45.0 75 125 170 280 300 500 2500

(L)

Bed volume

0 - 15 0- 15 0-50 0-50 0-50 0-50

R409 W R409 W R410Lb R410 L R410Lb R410 L

SSP-NDC

Flow-rate range (L h-‘)

Pump type

20 60 40 60 15 30 15 30 15 30 30

60

30

Approximate cycle time (min)

Maximum column loading depends on the application and requirement. Generally, sample size should not exceed 20% of column volume. A pump Sera 41 L (Seybert and Rahier, D-3524 Immenhausen, Germany) is required for packing. Stainless Steel Pumps Ltd., Eastbourne, Sussex, UK. Specifications vary according to application and flow-rate range. All BPSS columns will operate at flow-rate up to 5 mL cm-’h-’.

medium

medium

medium medium

Recommended Sephadex G-type

Sample volume to be processed/cyclea (L)

Table 4-4. Columns, gel types, pumps and conditions for desalting and solvent removal.

98

4 Process Scale Size Exclusion Chromatography

density and homogeneity irrespective of the column diameter. There are many examples in industry that this is possible, but it requires experience and skill in packing large diameter columns. In cases when this is not possible to achieve, one may compensate for less than optimal column packing quality by increasing the column length as discussed by Naveh 1211.

References Hagel, L., Janson, J.-C. in: Chromatography: Heftmann, E. (ed.), Amsterdam: Elsevier, 1992: 5th edn, pp. A267-A307. Hagel, L., in: Protein Purification, Principles, High Resolution Methods and Applications: Janson, J.-C., Ryden, L. (eds.), New York: VCH Publishers Inc., 1989; pp. 63- 106. Barth, H. G., J Chrom Sci, 1980, 18, 409-429. Yau, W. W., Kirkland, J. J., Bly, D. D., Modern Size Exclusion Liquid Chromatography: New York: Wiley, 1979. Kelley, T. T., Wang, T. G., Wang, H. Y., in ACS Symposium Series No. 3 14, Washington, DC: American Chemical Society, 1986, 193-207. Sofer, G. K., Nystrom, L.-E., Process Chromatography, A Practical Guide, London: Academic Press, 1989. Hagel, L. in: Aqueous Size-Exclusion Chromatography, Dubin, P. (ed.), Amsterdam: Elsevier, 1988, p. 146. Hagel, L., J Chromatogr, 1992, 591, 47-54. Hagel, L., J: Chromatogr, 1985, 324, 422-427. Sofer, G. K., Nystrom, L.-E., Process Chromatography, A Guide to Validation, London: Academic Press, 1991, pp. 1- 80. HjertCn, S., Biochim Biophys Acta, 1964, 79, 393 -398. Porath, J., Janson, J X . , Lags, T., J Chromatogr, 1971, 60, 167- 177. Uriel, J., Bull SOCChim Biol, 1966, 48, 969. Johansson, I., Unpublished report, Pharmacia Fine Chemicals AB, Uppsala, Sweden, 1976 IGgedal, L., Engstrom, B., Ellegren, H., Lieber, A.-K., Lundstrom, H., Skold, A., Scheming, M., J Chromatogr 1991, 537, 17. Edwards, S. L., Dubin, P. L., J Chromatogr, 1993, 648, 3-7. Dubin, P.L., Principi, J.M., Anal Chem, 1989, 61, 780-781. Arve, B., unpublished report, Pharmacia LKB Biotechnology AB, Uppsala, Sweden, 1989. Berglof, J.H., Eriksson, S., Anderson, I., Dev B i d Stand, 1987, 67, 25-29. Friedli, H., Kistler, P., Chimica, 1972, 26, 25. Naveh, D., BioPharm, 1990, 5, 28-36.

5 Polymers and their Application in Liquid Chromatography Linda L. Lloyd and John F. Kennedy

5.1 Introduction The use of polymers in process scale liquid chromatography has its origin in the work of Sober and Peterson [I] who in 1954 made the observation that proteins could be adsorbed onto a diethylaminoethyl (DEAE) derivatised cellulose and then selectivity desorbed by increasing the ionic strength of the eluent. This example of a derivatised polymer being used for preparative anion exchange chromatography of a biological macromolecule was quickly followed by the use of carboxymethyl (CM) cellulose for cation exchange chromatography and in 1959 Porath and Flodin [2] reported the use of a cross-linked polydextran gel, which when swollen by aqueous eluents, could be used to separate biological macromolecules based on differences in their solution size. This formed the basis of gel filtration chromatography. These materials are all microporous polymer networks and as such the porosity is determined by the degree of cross-linking, the lower the degree of cross-linking the larger the pores, but with increasing porosity being accompanied by a reduction in rigidity and hence mechanical strength. Therefore, this type of polymer matrix can only be operated under low/medium pressure conditions. It was in 1964 that Moore successfully synthesised a macroporous poly(styrene/ divinylbenzene) copolymer which enabled polymers to be utilised for liquid chromatography without the restrictions of operating pressure previously imposed [3]. The rigidity and controlled pore size and structure of these polymer matrices has enabled the development of high speed, high performance separations. The use of polymers, both synthetic and of natural origin, for liquid chromatography is a vast topic which has seen many significant advances in recent years. In order to exploit this technology to its fullest potential it is necessary to have some understanding of the different types of polymer which can be utilised and how the physicochemical properties of the various polymer networks can be manipulated according to the requirements of the individual fractionation. It is impossible within the confines of this chapter to provide a full survey of all the commercially available polymeric adsorbents for liquid chromatography but references has been made to some of the more commonly used packings to provide examples in relevent sections.

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5 Polymers and their Application in Liquid Chromatography

5.2 The Polymer Network Porous polymeric adsorbents for liquid chromatography can be categorised as being either microporous of macroporous depending upon the morphology of the individual particle. The type of pore structure is dependent upon the degree of crosslinking of the polymer chains within each particle. Microporous polymer particles are lightly cross-linked, typically less than 10% of the total monomer content is cross-linker, with the pore size being determined by the percentage of the cross-linking monomer used. The lower the degree of cross-linker the bigger are the pores in the presence of a good solvent but the swollen polymer network is less rigid. The swollen polymer network, being only lightly cross-linked, is semi-rigid and easily compressed and therefore can only be operated under low pressures. If the operating pressure exceeds the compressibility of the polymer network it will collapse so restricting the flow through the packed bed and hence column permeability. Microporous beads are therefore restricted in their use to low/ medium pressure applications. This often requires the use of large particles packed in large diameter short columns operated at low flow rates. Macroporous polymers are copolymers which are highly cross-linked having been synthesised with a high percentage of the cross-linking monomer. The suspension polymerisation is carried out in the presence of a porogen, a compound which is soluble in the monomer mixture but insoluble in the polymer, to produce rigid spherical particles which contain large voids or pores. These porous particles are rigid and even in the presence of a good solvent exhibit minimal swell. They are therefore mechanically much more stable and hence able to operate at higher pressures. It is therefore possible to use smaller particle sized adsorbents packed in smaller columns and operated at higher flow rates. Figure 5-1 shows a schematic of the morphology of microporous and macroporous polymer particles.

A

B

Fig. 5-1. Schematic representation of the morphology of (A) microporous and (B) macroporous polymer matrix. In the microporous structure there is a lower number of cross-links with the pores being located between the polymer chains. In the case of the macroporous structure the degree of cross-linking is much higher and hence the pores used for chromatographic separations are external to the polymer chains.

5.4 Dpes of Polymeric Mutrices

101

5.3 Manufacturing Process Particulate polymer supports suitable for liquid chromatography can be produced from a wide range of starting materials and using one of severe1 manufacturing technologies. Beaded polymers can be prepared from synthetic monomers such as styrene, acrylamide and methacrylate or gels from natural sources, polysaccharides including dextran, agarose and cellulose. Although the actual manufacturing process will vary according to the chemical nature of the monomers used and product required, the method of production of a porous particle is based on a two phase suspension system. The suspension polymerisation process used for the manufacture of porous particles involves the use of a water-organic two phase system [4]. The reaction mixture containing the monomer or monomers, cross-linking monomer, polymerisation initiator, porogen and droplet stabiliser is suspended in droplet from in an immiscible liquid. In the case of water-insoluble reactants, such as those used to produce polystyrene particles, the organic phase is dispersed by controlled stirring in water, and for water soluble monomers including acrylamide the aqueous reaction mixture is dispersed in an immiscible organic oil. The monomer mixture is stirred to give droplets of the required chromatographic adsorbent particle size. The polymerisation proceeds within the droplets with the growing polymer chains precipitating as they reach a critical size. The presence of the cross-linking monomer and the porogen produces a rigid three-dimensional structure with holes of a predetermined size, distribution and geometry. The choice of porogen enables the physical characteristics of the pores to be optimised for the separation of solutes of different size and chemical characteristics - from small pharmaceuticals to biological macromolecules. The polysaccharide based chromatographic media is normally produced using a suspension gelation and suspension cross-linking system. Again it is necessary to form a suspension of droplets but in this case they contain the polysaccharide, rather than a suspension of monomers. The conversion of the liquid droplet to a solid particle is effected by chelation, gelation, solvent extraction or chemical cross-linking depending upon the physico-chemical properties of the polysaccharide.

5.4 Types of Polymeric Matrices Polymeric matrices for liquid chromatography can be divided into two groups based on the origin of their component monomers. The synthetic polymers including polystyrene, polyacrylamide and polymethylmethacrylate and the natural polymers such as the polysaccharides dextran, agarose and cellulose. Although in general the synthetic polymers tend to be macroporous and the natural polymers microporous this is not always so. Polystyrene particles with less than 10% cross-linker have been utilised after derivatisation as ion exchangers for amino acid and carbohydrate sepa-

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5.4 v p e s of Polymeric Matrices

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rations and agarose when cross-linked for size separations of biological macromolecules. The molecular structures of the most common polymer systems are shown in Fig. 5-2.

5.4.1 Synthetic Polymers 5.4.1.1 Polystyrene

Polystyrene based particles are most commonly produced by a method involving the suspension of organic droplets in an aqueous media [3]. The matrix is a co-polymer of styrene and the divalent cross-linker divinylbenzene (Fig. 5-2A). The percentage of the cross-linker can be varied to produce either microporous of macroporous particles. For the production of macroporous particles the pore size, pore geometry and distribution are controlled by the choice of porogen, normally an organic which can be either a solvent or non-solvent for the polymer depending upon the particle morphology required. The particle size of the polymer beads will be dependent upon the formation and stabilisation of the droplets prior to polymerisation and therefore a distribution of particles is obtained. An alternative patented production method has been developed which produces monodispersed particles [5]. This process involves the formation of a latex particle onto which monomer droplets are absorbed and polymerised. This is the manufacturing process used for the commercially available Monobeads (Dyno). The hydrophobic macroporous polystyrene beads are exceptionally chemically stable and are used for organic phase gel permeation chromatography products, Styragel (Waters) and PLgel (Polymer Laboratories) and reversed phase HPLC separations, PRP (Hamilton) and PLRP-S (Polymer Laboratories). It is possible to surface modify the particle to mask the hydrophobicity of the base polymer and produce ion exchange and hydrophilic materials, PGSAX, PGGFC (Polymer Laboratories). In the microporous form after derivatisation to form strong cation exchangers these materials are commonly used for carbohydrate and amino acid separations, Aminex (BioRad).

5.4.1.2 Polyacrylamide

Polyacrylamide particles are formed by the polymerisation of a suspension in oil of the water soluble acrylamide monomer with methylenebisacrylamide as described by Flodin [6] (Fig. 5-2B). As in the case of the polystyrene matrix the porosity/permeability of the matrix can be controlled by the amount of the cross-linker used. How-

-

Fig. 5-2. Molecular structures of some of the most commonly used polymer systems. (A) Polystyrene, a copolymer of styrene and divinylbenzene, (B) polyacrylamide, a copolymer of acrylamide and methylenebisacrylamide, (C) HEMA, a copolymer of ethylene glycol methacrylate and bisethylene glycol methacrylate, (D) cross-linked dextran, (E) agarose, (F) cellulose.

104

5 Polymers and their Applicntion in Liquid Chromatograpliy

ever, increasing the amount of cross-linker reduces the pore size and due to the chemical structure of methylenebisacrylamide introduces both ionic and hydrophobic functionalities into the polymer structure which may result in the hydrophilic acrylamide matrix exhibiting non-specific interactions with certain types of solute. Polyacrylamide particles are most often used for size separations of small carbohydrates and peptides, Biogel P (BioRad).

5.4.1.3 Polymethacrylate A number of porous polymer matrices have been developed using derivatives of methacrylate as the monomer. Heitz et al. [7] described a method of preparation of a particulate polymethylmethacrylate which was cross-linked with ethanedimethacrylate. More recently the preparation of a commercially available material hydroxyethylmethacrylate, HEMA (Alltech) (Fig. 5-2 C), produced by the polymerisation of ethylene glycol methacrylate and bisethylene glycol methacrylate has been reported by Coupek et al. [8]. The production of polyglycidyl methacrylate, commercially available as Eupergit (Rohm Pharma), has also been described by the same group 191. The mechanical and chemical stability of these methacrylate based adsorbents makes them attractive alternatives for process chromatography where a robust column packing is essential. They have been used for size separations and hydrophobic interaction chromatography and after derivatisation as affinity matrices and for ion exchange chromatography.

5.4.1.4 Miscellaneous Synthetic Polymers A number of other synthetic polymer networks have been developed and commercialised for liquid chromatography including: polyvinylacetate cross-linked with butanediol divinyl ether, Merkogel GPC packings (Merck), polyvinylalcohol, Fractogel and Toyopearl (Toyo Soda), a hydroxylated acrylic monomer cross-linked with a bifunctional agent, Trisacryl (Sepracor) and a hydrophilic vinyl polymer, TSKgel PW (Toyo Soda).

5.4.2 Natural Polymers 5.4.2.1 Dextran Dextran is a homopolymer of 1,6-linked a-D-glucopyranose monomers (Fig. 5-2 D). The individual polymer chains do not associate on the molecular level to give an ordered three-dimensional structure. It therefore lacks the structural rigidity required for a chromatographic support. To improve the mechanical strength the individual polymer chains are cross-linked, for example, using epichlorohydrin in the case of

5.4 Types of Polymeric Matrices

105

Sephadex (Pharmacia). The degree of cross-linking controls both the porosity and the mechanical strength, the higher the degree of cross-linking the smaller the pores. The free hydroxyls in the dextran hydrophilic polymer enable derivatisation to produce affinity and ion exchange matrices. However, due to the cross-linking the permeability of these materials for biological macromolecules is reduced. 5.4.2.2 Agarose

Agarose is a linear polysaccharide consisting of alternate galactose-based monomers, 1,3-linked-~-galactoseand 2,4-linked 3,6-anhydro-~-galactose(Fig. 5-2E). It is obtained by extraction from seaweed and washed with phosphate buffer to remove the negatively charged agaropectin which is simultaneously extracted and would otherwise impart unwanted ion exchange functionalities side by side with the neutral polysaccharide. Unlike dextran, agarose is capable of exhibiting a crystalline microstructure due to hydrogen bonding between the individual polysaccharide chains; Fig. 5-3 shows a schematic of the two types of structure, dextran and agarose. The commercial adsorbent, Sepharose (Pharmacia), is produced by cooling a dispersion of agarose to produce microcrystalljne particles as described by Hjerten [lo].

Fig. 5-3. Schematic representation of the pore structure of (A) dextran and (B) agarose. The effect of the agarose crystalline microstructure, hydrogen bonding between the individual polymer chains, on the pore geometry and size is clearly evident.

A

B

The pores produced by the association of these polymer chains are sufficiently large to enable the diffusion of biological macromolecules into the matrix but because the chains are not covalently cross-linked the mechanical and chemical stabilities are not good and the particles degrade over a period of continuous use. To improve the mechanical and chemical stability of the matrix, individual polymer chains may be cross-linked by treatment of the non-cross-linked bead with epichlorohydrin, diepoxides or divinylsulphone [l I]. As the cross-linking is between associated polymer chains there is no reduction in the pore size or permeability of a bead when cross-linking occurs. This is the basis of the commercial product Sepharose CL (Pharmacia). Agarose being hydrophilic and highly permeable is ideally suited for size exclusion chromatography of biological macromolecules. Also the availability of numerous

106

5 Polymers and their Application in Liquid Chromatography

hydroxyl groups enables surface modification, to introduce for example ion exchange functionalities or affinity ligands, to be achieved with relative ease. The affinity material, Affi-Gel (BioRad) is an N-hydroxysuccinimide ester of a derivatised, crosslinked agarose bead which can be used for coupling in both organic and aqueous solvents.

5.4.2.3 Cellulose Cellulose is a long chain polymer consisting of ~-D-glUCOpyranOSe repeat units linked by 1,4-glycosidic bonds (Fig. 5-2F). It is an abundant natural polymer being present in plant material. It is most commonly obtained from wood and cotton or from the recycling of products containing derivatives of these materials. Cellulose is not homogeneous in that there are regions where the polymer chains are ordered to give an insoluble crystalline structure which is interspersed by water soluble amorphous material. Microcrystalline cellulose powders consist of irregularly shaped fibrous particles which are not suitable as chromatographic adsorbents. Regenerated cellulose in the form of regularly sized cellulose microspheres suitable for chromatographic applications is produced by dissolution of the cellulose followed by droplet dispersion and subsequent solvent extraction or covalent cross-linking. Determan and Weiland [ 121 described the preparation of non-cross-linked beads by solvent extraction, and Chitumbo and Brown [I31 the preparation of cross-linked cellulose beads.

A

B

D

C

E

Fig. 5-4. Schematic representation of composite polymeric particles. (A) Surface coating, (B) pellicular, (C) core shell graft, (D) pore matrix composite, (E) interpenetrating polymer networks.

5.4 Types of Polymeric Matrices

107

Celluloses in fibre, microgranular, bead and sheet form suitable for chromatographic applications are commercially available. The main use of this type of material is as a base matrix for ion exchange, CM and DEAE cellulose and affinity chromatography (Whatman).

5.4.3 Composite Materials As has been seen in the preceding sections, the polymeric liquid chromatography adsorbents cover a wide variety of chemical types, mechanical rigidity, thermal stability and solvent compatibility depending upon the nature of the component monomers and cross-linker. When the polymeric materials are compared with the inorganic materials such as silica and alumina there may be improved chemical stability but also limitations on the particle rigidity, as in the case of the microporous polysaccharide gels, or limited accessibility within the macroporous structures. Attempts to overcome these limitations have been made by the development of composite materials ie, adsorbents which are produced from more than one type of polymer or from polymer/inorganic combinations. Figure 5-4 represents schematically the type of composite materials discussed in this section. 5.4.3.1 Surface Coatings The surface chemistry of an adsorbent will determine its solvent compatibility and the type of interaction it is able to undertake with a given solute in a chromatographic system. Although the particle (organic polymer or inorganic) may be rigid and the pore structure ideal, as in the case of the controlled pore size and distribution which may be achieved with the macroporous materials, unless the surface chemistry is also acceptable the particle cannot be used for chromatographic separations. One means of utilising a support with the necessary pore morphology but unacceptable surface chemistry is by coating the surface with a suitable polymer so totally masking undesirable non-specific interactions. Anion exchange matrices for the separation of proteins have been produced by polyethyleneimine coating technology. In the case of inorganic silica a thin layer of the polyamine is adsorbed onto the surface through ion-pair formation between the silanols and the amine groups and the polymer coating then cross-linked in position to provide a stable coating [14]. The same principle has been used to surface coat negatively functionalised macroporous polystyrene [15]. These poIymeric coatings may then be further derivatised to introduce alternative functionalities if required. When the base matrix and the cross-linked coating are both chemically stable polymers an exceptionally robust material is produced. An example of such a material is the strong anion exchanger, PL-SAX (Polymer Laboratories), which is a macroporous polystyrene coated with cross-linked polyethyleneimine and subsequently derivatised to give a quaternary amine functionality. This type of structure with either an inorganic or polymeric base is widely used for both analytical and preparative separations.

108

5 Polymers and their Application in Liquid Chromatography

5.4.3.2 Pellicular Supports A pellicular chromatographic packing consists of a rigid core with a relatively thick outer shell. The outer shell is cross-linked by the inclusion of a difunctional monomer and for added stability the outer shell may be covalently attached to the core. An example of this type of packing is the material developed for carbohydrate analysis, CarboPac (Dionex), which is a pellicular anion exchange resin bead which has a 5 pm non-porous sulphonated polystyrene bead core with a 0.1 pm quaternary amine latex. These materials have gained widespread acceptance for analytical separations and small-scale fractionations. 5.4.3.3 Core Shell Grafts Core shell grafts are similar in concept to the pellicular supports being composed of a rigid core and relatively long flexible polymer chains which have been grafted to the bead surface. The outer shell is, however, normally thinner than that of a pellicular support. The rigid core may be porous, non-porous, inorganic or polymeric as long as the surface can be activated for attachment/grafting of linear polymer chains. This type of technology has been used to produce the tentacle type ion exchanges [ 161 which were developed for macromolecule separations. The core is a macroporous polyvinyl alcohol polymer or silica matrix with polymeric tentacles and is reported to exhibit improved efficiency due to non-hindered diffusion or steric limitations of the solute stationary phase interactions. 5.4.3.4 Pore Matrix Composites Macroporous adsorbents, although being mechanically stable, have less surface area than the microporous materials - under some conditions this can result in a decrease in sample capacity. However, unlike the macroporous particles, the microporous materials have limited mechanical rigidity so making them suitable only for operation under low and medium pressure conditions. In an attempt to combine the capacity of microporous materials with the rigidity of macroporous particles pore matrix composites have been developed. Within the pore of macroporous, inorganic or polymeric organic particle a microporous gel is produced. As reported by Kolla and Wilchek [I71 when a macroporous silica is soaked in a solution of an organic monomer containing cross-linker and initiator polymerisation results in the formation of a polymer gel network within the pore structure of the bead. Although a microporous network is required, limited cross-linking is necessary to ensure that the swollen polymer network is anchored within the pore during use and subsequent clean up procedures. However, under certain circumstances it may be possible with lightly cross-linked networks to link covalently the polymer chain to the internal pore surface of the particle. An ion exchange pore matrix composite has recently been produced from a macroporous polystyrene, HyperD media (Sepracor). It is described by the manufacturer as a ‘gel in a shell’ which combines the high diffusion and load

5.5 Polymer Physico-chemico Characteristics

109

capacity of soft gel with the mechanical rigidity of macroporous materials for biomolecule purification. 5.4.3.5 Interpenetrating Networks

It is possible to produce porous particles which are composed of two different types of polymer chains. Within the matrix of a polymer bead a second polymeric network is formed. It is essential for this type of material that an even distribution of the two polymer networks is obtained for good chromatographic performance. Arshady et al. have recently reported the production of an interpenetrating polymer network produced from a copolymer of dimethylacrylamide vinylferrocene polymerised within a preformed network of cross-linked polydimethylacrylamide [ 181. Such an organo-polymer system may be suitable for the fractionation of certain metal binding proteins.

5.5 Polymer Physico-chemico Characteristics The performance characteristics of a liquid chromatography adsorbent are one of the most important factors in achieving successful analytical separations and preparative fractionations. It is therefore of paramount importance that the relationships between polymer physico-chemico characteristics and chromatographic performance are fully understood. In size exclusion chromatography, where the separation is based on differences in solute hydrodynamic volume, the pore size, structure and distribution are of primary importance but for interactive chromatography, such as ion exchange and affinity, the available surface area play an important role as it determines solute capacity. In all cases the size of the particles, the chemical nature of the polymer backbone and/or surface coating, solvent compatibility and swelling characteristics must all be considered when assessing polymer suitability and likely performance.

5.5.1 Particle Size The two phase suspension process, with the exception of the method reported by Elingsen et al. [ 5 ] , produces spherical polymeric particles with a range of diameters. The particle size distribution is determined by the size of the microbeads which in turn is governed by the size of the monomer droplets. Monomer droplet size and stability are influenced by a wide range of parameters, including viscosity, concentration, reactor design, rate and type of mixing and presence of a droplet stabiliser. It has been shown [19] that given optimised polymerisation conditions it is possible to obtain, with a two phase suspension polymerisation system, particles which d o not

110

5 Polymers and their Application in Liquid Chromatography

deviate from the mean size by more than 100%. However, on a large production scale the deviation is often much larger than this, 5 - 50 or 10- 100 K r n distributions not being uncommon. For good permeability in liquid chromatography a narrow particle size distribution is required. This may be achieved in a number of ways according to the particle size range, particle fragility and polymer type. For large particles dry sieving may be suitable while for smaller particle size ranges air classification may be necessary. Sedimentation may be necessary to remove fine particles or fragments at the end of the sizing process which would otherwise significantly reduce the permeability so limiting the column throughput. In practice for analytical separations and smallscale fractionations particles as small as 3 pm are used but in general for larger scale purifications 10- 100 pm particles are used. The stability of the packing under pressure and flow rate must also be considered. For the less rigid microporous materials large particles tend to be used so keeping the operating pressure down and reducing the risk of packed column bed collapse.

5.5.2 Pore Size and Pore Size Distribution The pore size and geometry of a chromatographic packing are of paramount importance for efficient chromatography. In order to minimise the band broadening by limiting restricted diffusion it is necessary for the pore size and shape to be suffi-

Fig. 5-5. Size exclusion chromatography calibration curves, plots of elution volume versus molecular weight for a series of macroporous polystyrene materials designed for reversed phase Chromatography and as base matrices for subsequent, modification. Calibration performed using a 300 mm x 7.5 mm ID column and polystyrene standards with a tetrahydrofuran eluent. Curve (A) is a non-porous particle, (B) IOOA, (C) 300A, (I))IOOOA, and (F) 4000 A pore sized materials. VOLUME (MLI

5.5 Polymer Physico-chernico Characteristics

111

ciently large to allow free access to the molecules to be separated. In size exclusion chromatography (SEC) the separation mechanism is dependent upon the molecular size of a solute in solution and not on an interaction between the solute and the stationary phase. Therefore an SEC calibration curve, plot of elution volume versus molecular size, may be related to the pore size and pore size distribution of a chromatographic adsorbent [20]. Figure 5-5 illustrates typical SEC calibration curves for a series of polystyrene adsorbents which have been used for reversed phase chromatography or as a base matrix for further derivatisation. From these calibration curves it can be seen that with the smaller pore sizes there is a molecular size cut-off above which the molecules are unable to permeate the pore structure. There is also a rapid fall in the slope of the calibration curve due to the absence of small pores. Between these two points the calibration curve is relatively shallow as there is a distribution of pore sizes, the SEC resolving range. This optimised controlled distribution of pore sizes, with the sharp cut-offs at the top and the bottom of the curve, is similar to those obtained with the inorganic matrices, and enables materials to be chosen which have sufficiently large pores to allow free mobility of the macromolecule within the matrix so reducing band broadening due to restricted diffusion.

5.5.3 Surface Area As with inorganic matrices, the surface area, total pore volume and mean pore diameter of polymeric liquid chromatography packings are related. The methods for measurement of pore size commonly used are mercury porosimetry or nitrogen adsorption. The use of mercury porosimetry involves the use of pressure which is not compatible with obtaining surface area information about polymeric particles in the non-compressed state. However, when nitrogen adsorption measurements are performed for the determination of surface area and the results are compared with those of an inorganic support of the same pore size and distribution, as measured by SEC, the surface area is considerably higher for the organic polymer (Table 5-1). These high values are due to the biporous nature of the polymer matrix [21]. In addition

Table 5-1. The relationship between matrix pore size and surface area as determined by single point nitrogen adsorption for a range of organic, polystyrene, and inorganic, silica, liquid chromatography adsorbents. Pore size

Solid 100 300 1000 4000

(A)

Surface area (rn2g-') Polystyrene

Silica

3 414 384 267 139

300 200

-

-

112

5 Polymers and their Application in Liquid Chromatography

to the surface area located within the macropores the nitrogen adsorption isotherms show the presence of surface area within pores of diameter less than 20 These micropores located between the polymer chains and their surfaces will only be accessible to very small solutes. If the retentions obtained for a standard test mixture of peptides chromatographed using the series of polystyrene reversed phase adsorbents whose SEC calibration curves are shown in Fig. 5-5 and whose surface areas as determined by nitrogen adsorption given in Table 5-1, are compared (Fig. 5-6) then it can be clearly seen that as the pore size increases so retention decreases. This correlates with a reduction in the available surface area for solute stationary phase interaction as the pore size increases. If rather than this series of small solutes, proteins of increasing molecular weight are used to determine (by frontal loading) the capacity (in mg protein per mL packed column bed) of these same adsorbents after their conversion to strong anion exchangers the influence of available surface area on loadability can clearly be seen (Table 5-2). Although the ionic capacity decreases as the pore size increases the same

A.

A

B

20

3

VI

I= y1

P

E

A

i

'

MINUTES

3i

100

300

1000 PORE SIZE C AI

4obO

Fig. 5-6. (A) Separation of 5-C-terminal amide decapeptides (mixture RPS-PO010 [spi]) using a polystyrene, PLRP-S 100 A (Polymer Laboratories), reversed phase column with a linear gradient of 1 to 30% acetonitrile in water containing 0.1% TFA at a flow rate of 1.0 mLmin-'. Peak identification: (1) Ala3-Gly4(free amino), (2) Gly3-Gly4(N'-acetylated), ( 3 ) Ala3-Gly' (N'-acetylated), (4) Va13-Gly4(Na-acetylated), ( 5 ) Val3-Val4(Na-acetylated). The plot of retention time for these five synthetic amino acids as a function of pore size of the polystyrene HPLC reyersed phase material is shown in (B), (1) non-porous, (2) 4000A, (3) IOOOA, (4) 300A, ( 5 ) 100A pore size.

5.5 Polymer Physico-chemico Characteristics

1 13

Table 5-2. The relationship between pore size and specific ionic (In equivalents) or protein capacity (gmL-' column volume), as determined by frontal analysis for a range of strong anion exchangers based on a macroporous polystyrene matrix, PL-SAX. Molecular weight (kD) Ionic capacity Thyroglobulin y-Globulin BSA /I-Lactoglobulin

669 90 66 35

Pore size

(A)

100

300

I000

4000

0.51 6.0 14.0 9.0 22.0

0.26 10.0 31.0 20.0 108.0

0.23 17.0 38.0 36.0 46.0

0.21 23.0 34.0 16.0 20.0

trend is not observed with protein molecules. The optimum pore size for column capacity is dependent upon the size of the protein. For the smallest protein /3-lactoglobulin, molecular weight 35 kD, maximum capacity is achieved with the 300 A pore size material but as the size of the protein increases so its ability to diffuse fully into the smaller pores is limited and higher loadings are obtained with the larger pore size materials where there is more accessible surface area. With the largest protein, thyroglobulin which has a molecular weight of 669 kD, the highest capacity is achieved with the 4000 A pore size material.

5.5.4 Mechanical Rigidity Packed column bed homogeneity and stability are essential for high resolution separations. In large-scale separations the columns used are both wider and longer than those routinely applied to analytical and laboratory fractionations and the operating flow rates are higher. Increasing the column length increases the weight of the adsorbent and so will lead to an increase in the hydrostatic pressure. Within these columns there will be a continued abrasive action on the particles which may result in the formation of fines due to particle fragmentation which will collect at the column outlet so restricting flow. It is therefore essential that the column packings possess sufficient mechanical rigidity to be able to withstand such operating conditions. The microporous and macroporous polymer structures, as discussed earlier, differ in their pore structure. The microporous polymers even when cross-linked to improve mechanical rigidity are able to operate only at low pressure and therefore if the higher flow rates are to be used with larger columns then it is necessary to increase the particle size if the gels are not to be compressed. Alternatively it may be possible to use stacked columns [22] to limit the effect of large column operating conditions. The hydrophilic polysaccharide, agarose, is mechanically more stable than the other lightly cross-linked materials due to the hydrogen bonding between the individual polymer chains - this stability induces a crystalline microstructure. It is therefore possible, particularly after cross-linking the agarose chains, to use smaller par-

114

5 Polymers and their Application in

6000-

A

Liquid Chromatography

B

C E

0

M &

W

E

3

M M W re %

4000-

2000-

-I

I

I

Fig.5-7. Plot of flow rate versus column pressure for a series of polystyrene HPLC matrices (A) 100 A, 5 pm, (B) 300 A, 8 pm, (C) 1000 A, 8 prn, (D) 4000 A, 10 pm, and (E) non-porous, 8 pm particle. The eluent was acetonitrile/water (40 : 60 w/w) and the column dimensions were 250 mm x 4.6 mm ID.

ticle size adsorbents for high resolution separations even in the larger columns ~31. In the case of the adsorbents which have a macroporous polymer core or inorganic core with a microporous shell then the mechanical rigidity is higher than the microporous networks of the natural polysaccharide gels. With the highly crosslinked polymers, such as the polystyrene adsorbents, it has been shown that spherical particles may be produced which are mechanically stable to pressures in excess of 3000psi (21 MPa) [24]. Figure 5-7 shows the plot of flow rate versus operating pressure for the series of polystyrene adsorbents where deviation from linearity only occurs at operating pressures in excess of 4000 psi (28 MPa) when acetonitrilelwater, a poor solvent for the polystyrene matrix, is used. The difference in the slope of the plots is due to differences in the mean particle size and distribution and not to the pore size of the matrix.

5.5.5 Column Efficiency The performance characteristics of the stationary phase, whatever its type, are important factors in achieving successful separations. The efficiency of the column will be an indicator not only of the column performance, adsorbent and packed bed characteristics but also of system performance. In considering only the contribution of the stationary phase to column performance the factors which influence the sample band as it travels through the packed bed must be considered. There are five fac-

5.5 Polymer Physico-chemico Characteristics

1 15

tors which will contribute to the sample band broadening during its passage through the column: (1) the diffusion of molecules away from the centre of the band due to Brownian motion and the concentration gradient (longitudinal molecular diffusion); (2) differential paths through the column (eddy diffusion); (3) the velocity distribution for the mobile phase as it moves through the particles, the velocity being greatest at the centre of the channel (mobile phase mass transfer); (4) the different distances the molecules diffuse into the pools of stagnant mobile phase within the pores of the matrix (stagnant mobile phase mass transfer); (5) the different distances of migration into the stationary phase or residence time of the solute adsorbed onto the stationary phase (stationary phase mass transfer).

-

E

n

=

IY

0.02

Fig. 5-8. Plot of the dependence of HETP on flow velocity for the small pore size macroporous, polystyrene matrix, PLRP-S 200 A (Polymer Laboratories), using a small test probe, nitrobenzene. Eluent was acetonitrile/ water (90: 10 w/w) and the column dimensions were 200 mm x46 mm ID.

1 I

I

The longitudinal molecular diffusion will not contribute significantly to the overall column performance and by using spherical particles of uniform diameter and shape the eddy diffusion can be minimised. By using small particles the mobile phase mass transfer will also be reduced. The stagnant mobile phase and stationary phase mass transfer will be very much influenced by the geometry of the pore structure as they are dependent upon the molecules diffusing into the pore of the matrix. It is therefore essential that whatever the size of the molecule this diffusion is not hindered if high performance separations are to be achieved. Figure 5-8 shows the plot of plate height (HETP) versus linear flow velocity for a high performance, small molecule, polystyrene reversed phase matrix, PLRP-S 100 A (Polymer Laboratories). For the small molecule nitrobenzene it is evident that there is an optimum linear velocity for maximum efficiency and that high performance separations can be achieved with this polymer adsorbent. When the size of the molecule is increased (Fig. 5-9) there is no minimum in the curve. When the mechanically stable polystyrene matrices are used as the core and a strong anion exchange coating is applied it can be seen that the pore size does influence the efficiency of the separation. The 4000 A material is more efficient in all cases but with the protein ferritin which has a molecular weight of 470 kD the improvement is most dramatic. This is due to there being less hindered diffusion of the large biomolecule with the increased pore size.

116

5 Polymers and their Application in Liquid Chromatography

I

I

2 FLOW RATE (MLIMINI

1

Fig. 5-9. Plot of reciprocal column efficiency versus flow rate for the test probes. (A) Adenosine 5-monophosphate, (B) myoglobin, (C) ferritin using the strong anion exchanger, PLSAX (Polymer LFboratories) or 4000 A (----) pore with 1000 A (-) size. Data obtained under non-interactive conditions, ie, with a high salt eluent.

k

5.5.6 Eluent Compatibility and Solvent Strength The eluent compatibility of a polymeric adsorbent will be dependent upon the chemical structure of the polymer backbone, chemical type of the cross-linking agent, degree of cross-linking, and any subsequent covalent or dynamic modifications carried out. The natural polysaccharide polymers in their native state are hydrophilic and are therefore compatible with aqueous eluents whereas the synthetic polymers can be hydrophobic, as in the case of polystyrene, and hence compatible with organic eluents, or hydrophilic, as in the case of polyacrylamide, and so be compatible with aqueous mobile phases. It is of course possible to modify the eluent compatibility of a polymeric matrix by surface coating or derivatisation. For example, the very hydrophobic macroporous polystyrene matrices may be coated with a hydrophilic polymer to make ion exchange adsorbents or materials suitable for aqueous size separations [25]. With polymeric packings the solvent strength appears to have a major influence on peak symmetry for small molecules. A study correlating the peak symmetry with the column void volume for a macroporous polystyrene reversed phase adsorbent for a series of eluents of different strengths showed that the void volume was significantly smaller for a good solvent compared with a poor solvent [ 2 6 ] .With good solvents the peak symmetry is significantly improved. This decrease in void volume with good solvents may be attributed to the ability of the solvent to swell the polymer matrix and hence to ‘wet’ the surface of the bead. Indeed, one of the major differences

5.5 Polymer Physico-chemico Characteristics

1 17

between macroporous polymers and microporous gels is the extent to which the volume of the bead increases when wet. With macroporous resins the swell is typically less than 15% from the dry state to the fully swollen form whereas for microporous gels the swell may be several hundred per cent. Indeed, it is this ability to swell, ie, solvation of the polymer chains, in a good solvent which imparts the porous nature to the microporous gels.

5.5.7 Activation and Functionalisation Activation and functionalisation of polymeric particles can be carried out to produce liquid chromatography adsorbents with a range of functionalities. The type of chemistries employed will depend upon the chemical nature of the polymer and the required final product functionalities. Covalent attachment may be carried out, as is required for core shell grafts or coatings applied which may or may not be further derivatised. In all cases it is essential that the derivative is stable to the chromatographic conditions employed and any clean up procedures used.

5.5.7.1 Polystyrene

Porous polystyrene matrices have been derivatised by chloromethylation to produce both cation and anion exchangers [27, 281. This chemistry, however, involves the reaction of the monosubstituted aromatic ring with chlorosulphonic acid. With lightly cross-linked materials the amount of the difunctional cross-linker, divenylbenzene, is small (less than 12%) and therefore high capacity ion exchangers can be obtained. However, with the macroporous highly cross-linked adsorbents the difunctional monomer is in vast excess and the derivatisation reaction is therefore limited by the availability of the monosubstituted ring. Only low capacity ion exchangers are produced which retain some of the hydrophobicity of the polystyrene backbone so making them unsuitable for many applications, particularly the separation of biopolymers which requires a very hydrophilic matrix if non-specific interactions are to be avoided. Yang et al. [29] reported the production of a weak cation exchanger by oxidation of macroporous poly(methy1styrene-divinylbenzene)or of poly(ch1oromethylmethylstyrene-divinylbenzene) with an ionic capacity of 4 meq 8-l. The separation mechanism was reported to be a combination of ion exchange and hydrophobic interaction with the solutes investigated, heterocyclic bases, nucleotides, nucleosides and amino acids. In order to produce a hydrophilic adsorbent from a polystyrene core it is necessary to shield the hydrophobic polymer backbone. One approach reported by Rounds et al. [15] involves electrostatic adsorption of a polyamine onto the surface of sulphonated, microparticulate polystyrene. This adsorbed layer is cross-linked into position and subsequently quaternised. The performance of this material was comparable with that of commercially available packings for the separation of biological macromolecules. The adsorption and subsequent cross-linking of a neutral, hydro-

1 18

5 Polymers and their Application in Liquid Chromatography

philic polymer onto a polystyrene particle has also been demonstrated to produce aqueous SEC adsorbents [25].

5.5.7.2 Polyacrylamide

The two most common acrylamide based gels used for liquid chromatography are Bio-Gel P (BioRad), a polyacrylamide, and Trisacryl (Sepracor), a poly[(trishydroxymethyl)methylacrylamide]. Inman and Dintzis [30] reported the activation of BioGel P material using carbodiimide or glutaraldehyde for immobilisation of biological macromolecules including antibodies and enzymes. A macroporous acrylamide containing a high percentage of the cross-linker methylenebisacrylamide was produced and subsequently derivatised to produce on octadecyl functionality for reversed phase separations [24]. This material was shown to have good permeability and mechanical rigidity when compared with a macroporous polystyrene and silica based octadecyl materials. The Trisacryl polyacrylamide-based gels are very hydrophilic with many hydroxyl groups located on the main polymer structure. This enables activation to be achieved using those chemistries developed for derivatisation of carbohydrate hydroxyl groups.

5.5.7.3 Polymethacrylate

The most commonly used methacrylate-based liquid chromatography adsorbents are the hydrophilic polymers, HEMA (Alltech) a polyhydroxyethylmethacrylate and Eupergit (Rohm Pharma) a polyglycidylmethacrylate. In addition to its use as a size exclusion chromatography adsorbent, HEMA (Alltech), has also been derivatised for use in ion exchange chromatography and as an activated support is available for coupling affinity ligands. Eupergit (Rohm Pharma) is mainly activated for coupling biological ligands for affinity chromatography. In both cases there are available hydroxyl groups which can be derivatised using similar chemistries to the methods used for polysaccharide gels. An adsorbent for the rapid resolution of proteins has been reported by Burke et al. [31] based on a polyethyleneimine covalent coating of a non-porous polymethacrylate bead. Quantitative recovery of protein is achieved when individual proteins are in the sub-microgramme range.

5.5.7.4 Polysaccharides

Polysaccharide gels are produced from dextran (Sephadex, [Pharmacia]), agarose (Sepharose, [Pharmacia]) and cellulose (Whatman) with cross-linking in some cases to improve the mechanical stability. Although some of these adsorbents are used for size exclusion chromatography they are also used for ion exchange and affinity chromatography. The structure of the polysaccharide and degree of cross-linking will

5.6 Applications

119

determine the type and number of groups available for derivatisation. Any available primary hydroxyls in the polysaccharide backbone are the normal route for activation, being more reactive than secondary ones. The chemically modified polysaccharide gels can be divided into groups based on their application: the ion exchange adsorbents where the final product should be unreactive and stable and the activated intermediates that are used for immobilisation of affinity ligands and therefore the activated group must be available for chemical coupling with the resultant product being stable. Since the first work of Sober and Peterson [I] using derivatised cellulose for ion exchange chromatography there has been continued interest in the development of improved adsorbents. Weak ion exchangers, carboxymethyl (CM) and a range of alkylaminoalkyl derivatives such as diethylaminoethyl (DEAE), cation and anion respectively, are produced from the polysaccharide gels by the formation of alkyl ethers. For the preparation of a strong anion exchange material quaternary amine derivatives are required such as the diethyl-(2-hydroxypropyl)-aminoethylfunctionality [32].If the polysaccharide matrix is to be used as a support for affinity chromatography then an ‘activated’ gel must first be produced to which can be coupled the ligand, often a protein, via OH, NH2 or SH groups present in its structure. There are a number of different chemistries which can be used for activation/coupling in acidic, basic or organic media. The choice of chemistry will be partly dependent upon the solvent compatibility and stability of the matrix and ligand as well as of the stability of the covalent linkage produced. Activation using carbonyldiimidazole, periodate and sulphonyl chloride can be used as can pre-formed cyclic carbonates and cyclic imidocarbonates. The products of activation may, however, be different according to the polysaccharide which is used, for example, the action of cyanogen bromide on dextran or cellulose produces a trans-2,3-imidocarbonate but with agarose which does not contain vicinal hydroxyl groups a cyclic imidocarbonate is not energetically favoured and therefore a cyanate ester is formed [33]. Both products will couple ligands containing free amino groups - the cyclic imidocarbonate via nucleophilic attack and the cyanate ester to form an isourea derivative.

5.6 Applications Liquid chromatography is normally used in a purification scheme after some initial separation processes. It is therefore necessary to have a chromatographic support which has high resolution, selectivity and capacity for the solute of interest if the desired product purity is to be achieved at an economical cost. The choice of separation mechanism will be partly determined by the chemical and physical characteristics of the solute of interest compared with those of the contaminants. Separation mechanisms based on size/shape, charge, hydrophobicity and biological recognition can be used with specialist chiral phases being available should a racement need to be separated. In many processes it is necessary to use a combination of techniques, each with a different selectivity to enable product of the required purity to be produced.

120

5 Polymers and their Application in Liquid Chroinatography

5.6.1 Size Exclusion Chromatography Size exclusion chromatography, also called gel filtration and gel permeation chromatography, is the only form of liquid chromatography which is non-interactive. The separation is based upon the ability of a molecule to diffuse into a porous matrix; the degree of penetration and hence residence time within the particle will be governed by the size of the molecule in solution. The larger the molecule the less it diffuses into the porous structure and therefore the earlier it will elute from the column. Small molecules will, however, be able to diffuse fully into the structure and therefore will have a longer residence time in the column. It is clear that the requirements/ restrictions placed on a packing material for size exclusion chromatography will be very demanding. For use in an aqueous system the packing must be hydrophilic with no hydrophobic or charged patches which could result in interactions between the packing material and the sample molecules. The packing must be sufficiently porous with a good pore size distribution to be able to differentiate between molecules of different sizes. The selectivity and resolution of this technique is lower than with the interactive forms of chromatography but if the packing material and elution conditions are optimised is should be possible to fractionate molecules if the difference in their hydrodynamic volume is greater than 10%. The most commonly used materials for size exclusion chromatography of water soluble macromolecules are the polysaccharide based gels, cellulose, dextran and agarose. Polyacrylamide Biogel P (BioRad) has been widely used for the separation of small molecules and oligosaccharides. Rigid polymers such as the hydrophilic polyhydroxyethylmethacrylate,HEMA (Alltech), and the vinyl polymer TSKgel PW (Toyo Soda) are used for analytical separations or small scale preparative fractionations. Polystyrene based particles (PLgel [Polymer Laboratories] and Stryragel [Waters]) are routinely used for the separation of synthetic polymers but are too hydrophobic for the separation of water soluble species. However, it has recently been

I

I

I

2

I

SALT CONCENTRATION

M

Fig. 5-10. Plot of retention versus salt concentration for the proteins (A) a-chymotrypsinogen A, (B) lysozyme, (C) ovalbumin, (D) myoglobin, using the size exclusion matrix PL-GFC 300A (Polymer Laboratories).

5.6 Applications

121

shown that it is possible to coat a polystyrene matrix with a hydrophilic polymer and to achieve size separations of proteins and natural polymers [25]. Figure 5-10 shows a plot of retention versus salt concentration for proteins on a coated polystyrene packing, PL-GFC (Polymer Laboratories). It is clear that there is a wide range of salt concentrations over which the elution volume does not change, ie, a size separation is taking place.

5.6.2 Reversed Phase Chromatography Reversed phase chromatography is commonly used for the separation of small molecules but the strength of the interaction with proteins is such that the conditions required for elution often result in loss of biological activity. The most commonly used reversed phase adsorbents are based on a silica matrix with a bonded hydrocarbon phase but chemically these materials are not very stable and can have residual silanol groups which may interact particularly with basic solutes. Attempts have been made to overcome this problem by coating them with a polymer layer [34] or by using mechanically stable polymers which are able to operate under HPLC conditions. Macroporous polystyrene has been used in its unmodified form for the separation of small molecules [35] and protein separations [36] and after derivatisation with a hydrocarbon phase (ACT- I [Interaction]). Indeed it has been demonstrated by Burton et al. [37] that better resolution of complex protein mixtures may be achieved with polymeric materials. Peptide purification is most commonly carried out using reversed phase columns where excellent selectivity and resolution is achieved. Figure 5-11 illustrates the purification of a crude synthetic peptide using a small pore size polymeric reversed phase material.

5.6.3 Hydrophobic Interaction Chromatography Hydrophobic interaction chromatography, as reversed phase chromatography, uses a non-polar stationary phase and a polar mobile phase. However, the adsorbents used for hydrophobic chromatography have a lower density of shorter hydrocarbon chains. These materials are therefore less hydrophobic with the interaction between solute and stationary phase being promoted by the use of salt. It is a technique widely used for the purification of proteins as the mild elution conditions are unlikely to cause denaturation. Many of the polymer matrices, for example HEMA (Alltech) which has an aliphatic backbone with a large number of hydroxyl groups, could be used directly for hydrophobic interaction chromatography as they have both hydrophobic and hydrophilic character. Adsorbents based on the hydrophilic vinyl polymer - TSKgel PW, TSKgel Phenyl PW and TSKgel Ether PW (Toyo Soda) - have been compared for the separation of proteins [38]. The Ether support was shown to be more hydrophilic and therefore less likely to lead to denaturation of the proteins.

122

5 Polymers and their Application in Liquid Chromatography

\

A

1

0

I

I

I

9

I

0

1

9

MINUTES

Fig. 5-11. Isocratic purification of the crude synthetic peptide, ACP (65-74), from its truncated failure sequences using the polystyrene reversed phase matrix, PLRP-S 100 A (Polymer Laboratories). Column dimensions, 150 mmx4.6 mm ID and the eluent was acetonitrWwater (20: 80 v/v) containing 0.1% TFA. (A) Shows the separation of approximately 5 pg of peptide, (B) shows the separation of approximately 1 mg of peptide.

5.6.4 Ion Exchange Fractionations Polysaccharide based ion exchangers have been used for several decades for the separation of biomolecules. They have become the primary adsorbents used in the purification of recombinant protein products as the mild elution conditions do not lead to conformational or structural changes, denaturation, even of labile biomolecules. The first materials to be produced were based on cellulose but these were quickly followed by cross-linked dextran and agarose based supports. More recently we have seen the development of ion exchangers based on rigid polymers which are able to operate at higher pressure and flow rates. Ion exchange can be divided into two categories: anion exchange, where the functional group has a positive charge, and cation exchange where the functional group has a negative charge. These categories can be further divided into weak exchangers, where the nett charge is dependent upon the pH, and strong ion exchangers, where the net charge is independent of the pH environment. With the polysaccharide gels the most commonly used functional groups are the DEAE weak anion exchangers and the CM weak cation exchangers. The adsorbents are available in a range of pore sizes for the fractionation of different

5.6 Appiications

123

sized solutes. Recently macroporous chemically stable polymers have also been used for the production of ion exchangers and in this case strong ion exchange functional groups are attached as in: Mono Q and Mono S (Pharmacia), modified TSKgel (Toyo Soda) and PL-SAX and PL-SCX (Polymer Laboratories).

5.6.5 Affinity Supports Affinity chromatography is possibly the most powerful of all the liquid chromatography techniques for the purification of biological macromolecules. The technique, often described as ‘bioselective’ chromatography, utilises the natural interaction and selectivity of many biological macromolecules such as enzymes, antibodies and antigens for their complementary molecule. The use of the technique for purification requires the formation of a complex between the molecule of interest and a ligand attached to a stationary phase. The association constant for this complex should be in the range IO3-1O8 for the reversible formation of a complex under chromatographic conditions [39]. The choice of the support matrix and immobilisation method is critical to the success of an affinity purification. The matrix must not be involved in nonspecific interactions with the molecule of interest and the ligand must be immobilised in the correct orientation for interaction and maintain its biological activity after coupling. It is necessary for the production of high capacity adsorbents that the available surface area is optimised. Agarose has a high surface to volume ratio and has been frequently used as an affinity support matrix. The rigid macroporous polymers have recently received much attention as potential support matrices since affinity purifications can take advantage of the rapid equilibrium of these HPLC materials so leading to reduced cycle times and hence higher throughputs. An alternative to the immobilisation of a biological ligand has been reported which utilises a synthetic ligand, typically a modified dye structure, which will be more stable to the chromatographic conditions and exhibit an affinity for a particular class of compound [40]. This type of ligand has been termed ‘biomimetic’ as it mimics the biospecific interactions of the biomolecules. These ligands could be of great potential in the future of process affinity chromatography as, unlike biological molecules, they do not have antigenic activity, should any of the ligand leach into a human pharmaceutical preparation, are stable and can be sterilised without the loss of activity.

5.6.6 Chiral Separations The resolution of optical isomers and subsequent fractionation of the biologically active isomer from its enantiomer, which may be inactive, inhibit biological activity or indeed in some cases even be toxic, is essential for a pharmaceutical grade preparation. A number of polymeric liquid chromatography adsorbents have been used for

124

5 Polymers and their Application in Liquid Chromatography

the enantiomeric separations, including triacetyl cellulose, which has been used to resolve the enantiomers of methaqualone [41]. Polyacrylamide and polymethacrylate supports, after attachment of a chiral molecule, have been used successfully. One of the more recent materials to be commercialised is Cyclobond (Advanced Separation Technologies) which is a cyclodextrin (an oligoglucose molecule with a cyclic structure) attached to an inorganic, silica support. The cyclodextrin is formed enzymically with a toroid, open barrel, structure where the glucose molecules around the larger end are orientated so that the secondary hydroxyls rotate to the right and the smaller end of the barrel is rimmed by the more polar primary hydroxyls. The internal surface of the barrel is hydrophobic so that the hydrophobic proportions of a molecule are attracted to the inner surface forming an inclusion complex. The formation of the inclusion complex will be dependent upon the ability of a molecule or portion of a molecule to fit into the cavity of the cyclodextrin. There will therefore be an element of structural selectivity which has been utilised for resolving racemate mixtures including the D, L-mixtures of certain dansyl or beta-naphthylamino acids ~421.

5.6.7 Hydrophilic Interaction Chromatography Hydrophilic interaction chromatography is a variant of normal phase chromatography where a hydrophilic adsorbent is eluted with a hydrophobic mobile phase and solute retention increases with increasing hydrophilicity. The elution order will therefore be the opposite to that achieved with reversed phase chromatography. A neutral hydrophilic packing, PolyHydroxyethyl Aspartamide (PolyLC) based on an inorganic silica matrix with a coating in which ethanolamine is incorporated into a polysuccinimide layer has been reported to show promise for the separation of histones, membrane proteins, phosphorylated amino acids and peptides [43].

5.6.8 High Speed Separations A serious limitation to the high speed fractionation of large molecules is intraparticle diffusion. Many attempts have been made to reduce this factor and so increase the throughput of a chromatographic process. Three options have been explored: (1) the use of non porous packings coated with an adsorbed layer; (2) reducing the particle size; (3) increasing the size of the pores. When non-porous packings are used the available surface area is low and therefore the capacity is low. Whilst this may be acceptable for analytical work or for small-scale separations it is a serious limitation for preparative and process chromatography. Reducing the particle size reduces the time constant for diffusion as the migration distance into the pore is reduced, but when small particles are used the column permeability is decreased so increasing operating pressures and the column becomes more susceptable to fouling. One of the more recent innovations in polymeric adsorbents which could lead to significant ad-

5.7 Pructical Considerations

125

vances in the area of preparative and process chromatography has been the development of large pore matrices. It had been observed that by increasing the pore size of an adsorbent to 4000 A the speed of analysis of biological macromolecules could be increased without a decrease in resolution [44]. This has certain implications for preparative and process chromatography as it would mean a significant increase in the throughput of a chromatographic fractionation. This type of material was examined by Rodrigues et al. [45] who concluded that with very large pore materials the improvement in performance of the chromatographic process was due to convection enhancing the effective diffusion within the matrix. This philosophy has lead to the commercialisation of a range of polymeric materials for what has been termed ‘perfusion chromatography’ (Poros [PerSeptive Biosystems]). The flow rates are several times higher than with conventional smaller pore HPLC materials so increasing the throughput of the system [46]. An alternative novel technology for bioprocess chromatography has been reported by DePalma [47] and termed Hyper Diffusion Chromatography. This material is reported to combine the high diffusion and capacity of a microporous gel with the mechanical rigidity at high flow rates of the macroporous polymers. The HyperD media (Sepracor) is a composite of a rigid polystyrene matrix with a hydrogel pore infill, termed by the manufacturers as ‘a gel in a shell’. The rigidity of the polystyrene pore framework prevents the hydrogel from collapsing at high flow rates. This rigid shell format combined with rapid diffusion through the hydrophilic gel, which is reported to exhibit minimal non-specific binding and high recovery of proteins, enables high binding capacities to be achieved at high flow rates. The rapid kinetics of interaction between protein and gel enables high resolution fractionations to be achieved at high flow rates. It would be expected that these characteristics would lead to high throughput and hence good productivity of fractionation on both preparative and process scales.

5.7 Practical Considerations 5.7.1 Choice of Adsorbent When choosing an adsorbent for a liquid chromatography separation, whether analytical, preparative or process scale, there are a number of practicalities which need to be considered. The type of packing material must be selected with due consideration to the type of molecule being separated: size, shape, hydrophobicity and electrostatic characteristics are all important as are the contaminants from which it is to be separated. None of the adsorbents used in liquid chromatography are completely inert in their behaviour towards the sample and mobile phase and therefore the physical and chemical characteristics of the adsorbent must also be related to the sample type for optimised separations with maximum throughput and column lifetime. The required purity of the final product must be considered when devising a

126

5 Polymers and their Application in Liquid Chromatography

purification protocol. For certain applications, such as the preparation of human pharmaceuticals, very high purity is required by the legislators and therefore the number of stages in the process is likely to be large with highly selective adsorbents being used to achieve good resolution. However, when a final product is, for example, going to be used as an additive in a biological washing powder then it is unnecessary to purify it to such a high level. Indeed the economics of the process, as dictated by final product cost, do not permit complex multistage protocols to be used. Within the vast array of commercially available polymeric adsorbents there are many different surface chemistries with a range of hydrophobicities and electrostatic charges.

5.7.2 Chemical Stability When considering the stability of a chromatographic packing it is essential to take into account the base matrix, functional groups, leakage of monomers or derivatisation agents and residual chemically active groups all of which may contribute to the ageing of the chromatographic support. Even though a functional group may be covalently attached the bond may in some instances be acid or alkali labile and therefore mobile phases or regeneration solutions must be chosen which are compatible with the polymer, base matrix and or surface coating. Matrices such as the polystyrene based packings are generally stable within the pH range 1 - 13 but some of the polymethacrylates and polysaccharides are restricted to use within the pH range 2- 12. With samples that contain lipids and pigments it may be necessary to regenerate the adsorbent using polar organics such as ethanol or acetic acid and therefore the differential sweWshrinkage of the polymer in the mobile phase and cleaning solution must be considered. If the swell differential is too large then there will either be a build up in pressure or voiding which will disrupt the packed bed homogeneity leading to chanelling and a loss in column performance. The macroporous polymers have very low swell and as such can easily withstand changes in eluent polarity but in the case of the microporous gels, which are only lightly cross-linked, changes in polarity may cause a sufficient change in the size of the polymer bead to disrupt the packed bed. Any decomposition or bleed from the column packing will contaminate the product which under certain circumstances can result in additional expensive cleanup procedures having to be implemented. This obviously will have a detrimental effect on process economy.

5.7.3 Fouling and Regeneration The chromatographic adsorbent may become fouled due to strong irreversible binding or the reaction of active components of a sample, not necessarily the solute of interest. This type of fouling would result in a loss in sample capacity and a change in the selectivity of the packing material. Alternatively, colloidal fines or particulate matter introduced in the eluent or sample feed streams may be deposited at the head

5.8 Summary

127

of the column or within the interstitial or pore volume so reducing the permeability of the packed column. With the separation of recombinant products, biologically active substances, fouling due to microbial growth can also occur. Even when the column packing and the sample have been well matched fouling will inevitably occur and when, after a period of time the selectivity and capacity decrease, regeneration must be carried out. With the polymeric adsorbents which have exceptional chemical stability a wide array of cleaning options are available including acid, alkali, organic solvents, detergents and protein solubilising agents. Often a series of cleaning/ solubilising agents are applied to desorb the contaminants and flush to waste. Regeneration time will depend upon the number of stages involved and the physical/chemical characteristics of the packing material. The diffusion of the cleaning solvent and desorbed contaminants will be dependent upon the pore size/geometry and particle size of the packing with larger, open pores and small particles having better diffusion rates and shorter equilibration times.

5.7.4 Recovery of Mass and Biological Activity In the isolation of biologically active compounds the aim is to achieve purification without loss of activity. With small molecule pharmaceuticals the loss in therapeutic activity is small even when aggressive mobile phases are used and the interactions between stationary phase and solute is strong. However, with large biomolecules, proteins, which are structurally very complex, it is necessary to maintain the threedimensional structure if biological activity is to be preserved. Partial unfolding or denaturation of a biopolymer is known to occur during a chromatographic process and has been attributed to eluent and/or surface induced effects [48]. The residence time in the chromatographic column has also been shown to be related to the extent of conformational changes. Polymeric adsorbents are available which have very strong solute stationary phase interactions, such as in the case of the polystyrene reversed phase materials, and would therefore be expected to result in denaturation of biomolecules but more gentle packings are also available, such as the ion exchangers and hydrophobic interation adsorbents.

5.8 Summary The primary concerns of the process chromatographer are final product purity and process economy. This can be interpreted as selectivity and productivity as defined by capacity and speed. The choice of the chromatographic media has a dramatic effect on the overall performance of the process separation and is therefore critical if these objectives are to be achieved. There are now available many different types of polymeric packings (Table 5-3) which enable options to be considered but which only a few years ago where not available.

128

5 Polymers and their Application in Liquid Chromatography

Table 5-3. Examples of commercially available polymeric packing materials for liquid chromatography. This is not an exhaustive list but is meant simply to illustrate the number of suppliers and type of polymers currently available.

Supplier

Polymer base

Trade name

Advance Separations Technology

Cyclodextrin

Cyclobond

Alltech

Hydroxyethylmethacr ylate

HEMA

BioRad

Agarose Polyacr ylamide Polystyrene

Affi-Gel Biogel P Aminex

Dionex

Polystyrene

CarboPac

Dyno

Polystyrene

Monobeads

Hamilton

Polystyrene

PRP

Interaction Chemicals

Polystyrene

ACT-I

Merck

Polyvinyl acetate

Merkogel

Pharmacia

Agarose Dextran

Sepharose Sephadex

PolyLC

Polysuccinimide

Polyhydroxethyl Aspartamide

Polymer Laboratories

Polyethyleneimine Polyhydroxyl polymer Polystyrene

PL-SAX PL-GFC PLgel, PLRP-S

Rohm Pharma

Polyglycidylmethacrylate

Eupergit

Sepracor

Polystyrene Trishydroxymethyl methylacrylamide

HyerD media Trisacryl

Toyo Soda

Hydrophilic vinyl polymer Polyvinyl alcohol

TSKgel P W Fractogel, Toyopearl

Waters

Polystyrene

Styragel

Whatman

Cellulose

For separations based on molecular size in solution, ie, size exclusion chromatography, the semi-rigid hydrophilic gels, dextran, agarose or cellulose based, are normally the materials of choice. In order to improve mechanical stability some cross-linking has been carried out which has increased the throughput of the fractionation process. The highly cross-linked synthetic polymers are not yet widely used on a process scale. In interactive chromatography, such as ion exchange, the productivity of the separation depends upon a number of related variables. The binding capacity of the media and the maximum operational flow rate are the most important. With the natural polymers, such as dextran, agarose and cellulose, which have high available surface areas and hence capacities, the compressibilities of the gels limits the operational flow rates which can be used so restricting the amount of product which can

References

129

be fractionated in a given period of time. Cross-linking of these natural polysaccharide gels does improve the mechanical rigidity but also by introducing cross-linking points restricts the free diffusion of molecules within the pore structure which results in a reduction of usable capacity with increasing flow rate. The introduction of the macroporous synthetic polymer supports which have increased mechanical stability does enable higher flow rates to be used, although the available surface area and hence sample capacity are lower than the soft gels. Throughput can be increased with these materials under certain circumstances. With the recent activity in the development of polymeric packings specifically designed for preparative and process chromatography, there have been a number of materials commercialised which are likely to lead to significant increases in selectivity and productivity. The wide pore adsorbents which are able to harness convective enhanced diffusion to maintain good mass transport within the porous structure of the particles as the flow rate increases and the macroporous shell with a hydrophilic gel pore infill which combines the mechanical rigidity of the shell with the high capacity of the gel infill both look promising.

References Sober, H.A., Peterson E.A., J A m Chem SOC1954, 76, 1711. Porath, J., Flodin, P., Nature 1959, 183, 1657. Moore, J. C., J Polym Sci I, Part A 1964, 2, 835. Sherrington, D. C., in: Polymer Supported Reactions in Organic Synthesis: Hodge, P., Sherrington, D. C. (eds.) Chichester, Wiley 1980; chapter 1. [5] Elingsen, T., Aune, O., Ugelstad, J., Hogan, S., J Chromatogr 1990, 535, 147. [6] Flodin, P., French Patent 1363978 1964 [7] Heitz, W., Ulliner, H., Hoeker, H., Makromol Chem 1966, 98, 42. [8] Coupek, J., Krivakova, M., Pokorny, S., J Polym Sci 1973, 42, 185. [9] Svec, F., Hradil, J., Coupek, J., Kalal, J., Angew Makromol Chem 1975, 48, 109. [lo] Hjerten, S., Biochim Biophys Acta 1964, 79, 393. [ I l l Porath, J., Janson, J. C., Laas, T., d Chromatogr Sci 1971, 60, 167. [I21 Determan, H., Weiland, T., Makromol Chem 1968, 114, 263. [I31 Chitumbo, K., Brown, W., J Polym Sci 1971, 36, 279. [I41 Alpert, A. J., Regnier, F.E., J Chromatogr 1979, 185, 375. [I51 Rounds, M.A., Rounds, W. D., Regnier, EE., J Chromatogr 1987, 397, 25. [I61 Muller, W., J Chromatogr 1990, 510, 133. [I71 Kolla, P., Koehler, J., Schomburg, G., Chromatographia 1987, 23, 465. [I81 Arshady, R., Corain, B., Lora, S., Palma, G., Rosso, U., Okan, F., Zecca, M., Adv Muter 1990, 2, 412. [I91 Arshady, R., Ledwith, A., React Polym 1983, 1, 159. [20] Dawkins, J.V., Heming, M., Makromol Chem 1975, 176, 1795. [21] Tanaka, N., Hashiznme, K., Araki, M., Tsuchiya, H., Okuno, A., Iwaguchi, K., Ohnishi, S., Takai, N., J Chromatogr 1988, 448, 95. [22] Janson, J. C., in: Advances in Biochemical Engineering, Vo/.25 : Fiechter, A. (ed.) Heidelberg. Springer-Verlag, 1982, p. 44. [23] Hjerten, S., Trends in Anal Chem 1984, 3(3), 87. [l] [2] [3] [4]

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5 Polymers and their Application in Liquid Chromatography

Dawkins, J.V., Lloyd, L.L., Warner, F.P., J Chromatogr 1986, 352, 157. Lloyd, L.L., J Chromatogr 1991, 544, 201. Bowers, L. D., Pendigo, S., J Chromatogr 1986, 371, 243. Hajos, P., Inczedy, J., J Chromatogr 1980, 201, 253. Lee, D., J Chromatogr Sci 1984, 22, 327. Yang, Y.B., Nevejans, F., Verzele, M., Chromatographia 1985, 20, 735. Inman, J. K., Dintzis, H.M., Biochem 1969, 8, 4047. Burke, D. J., Duncan, J. K., Dunn, L. C., Cummings, L., Siebert, C. J., Ott, G. S., J Chromatogr 1986, 353, 425. Kennedy, J. F., White, C. A., in: Bioacfive Carbohydrates: in Chemistry, Biochemistry and Biology : Chichester: Ellis Horwood 1983, p. 288. Kohn, J., Wilchek, M., Enzyme Microb Techno1 1982, 4 , 161. Davankov, V. A., Kurganoc, A. A,, Unger, K. K., J Chromatogr 1990, 500, 5 19. Cope, M. J., Davidson, I.E., Analyst 1987, 112, 417. Tweeton, K., Tweeton, T.N., J Chromatogr 1986, 359, 111. Burton, W. G., Nugent, K. D., Slattery, T.K., Summers, B. R., J Chromatogr 1988, 443, 363. Kato, Y., Kitamura, T., Hashimoto, T., J Chromatogr 1986, 360, 260. Janson, J.C., Trends in Biotech 1984, 2, 31. Jones, K., LC GC 1991, 4(9), 32. Isaksson, R., Erlandsson, P., Hansson, L., Holmberg, A., Berner, S., J Chromatogr 1990,498, 257. Armstrong, D. W., J Chromatogr Sci 1984, 22, 411. Alpert, A. J., J Chromatogr 1990, 499, 177. Lloyd, L. L., Warner, F.P., J Chromatogr 1990, 512, 365. Rodrigues, A., Lopes, J., Lu, Z.P., Loureiro, J., Dias, M., J Chromatogr 1992, 590, 93. Fulton, S.P., Shahadi, A. J., Gordon, N.F., Afeyan, N. B., Biotechnology 1992, 10, 635. DePalma, A., Genetic Engineering News 1993, 13(1), 18. Hearn, M. T. W., Hodder, A.N., Aguilar, M. I. Chromatogr, 1985, 327, 47.

6 Biochemical Applications of Process-Scale Ion-Exchange Liquid Chromatography Peter R. Levison

6.1 Introduction As a technique in liquid chromatography ion-exchange can be carried out at high pressure, medium pressure or low pressure. The scope of this article is restricted to the use of ion-exchange in low pressure chromatography. As a process-scale technique, ion-exchange chromatography is extensively used in the industrial treatment of water both for waste minimisation and also as a treatment for potable water, condensates and diluents for many industrial processes. The use of ion-exchange in these areas are well documented and representative applications can be found in several texts [I- 31. Ion-exchange processes occur in living systems influenced by the charged properties of many of the constituents of the cell, eg, cell membrane lipids or cell wall proteins and the constituents of individual organelles, eg, nucleic acids, proteins, etc. [4]. These ion-exchange properties of biological molecules, whilst facilitating their physiological function, can be exploited in liquid chromatography as a means for their removal from the biological system in which they are present. Downstream processing is the generic term given to a defined series of unit processes resulting in the isolation of a target biopolymer from a crude feedstock which could be a tissue extract, biological fluid, tissue culture, fermentation, etc. Downstream processing is routinely carried out in the bioprocessing industry and the number and sequence of these unit processes will depend on several factors including the nature of the feedstock, the degree of purity required for the target, the yield requirements and general economic considerations. Low pressure liquid chromatography is an essential part of many downstream processes and will typically consist of a sequence of discrete procedures, each relying on a different chemical or physical interaction between the feedstock components and the chromatographic stationary phase. Dependant on the nature of the target material the chromatographic steps referred to as process-scale may be carried out in laboratory columns where only low levels of target may be present, eg, mg quantities, or using large contactors where kilogram quantities of target are being separated. When developing a process, initial studies will be carried out at laboratory-scale. Here, the selection of chromatographic techniques will be made such that a series of steps starting from crude feedstock and resulting in purified product will be iden-

132

6 Biochemical Applications of Process-Scale Ion-Exchange Liquid Chromatography

tified. Furthermore, the conditions would be developed to ensure that process efficiency and hence process economics were optimal. Selection of the chromatographic techniques, while dictated by the chemical and physical properties of the target protein and other feedstock components, will be influenced by several external factors which include media costs, availability and consistency of supply of bulk media, regulatory requirements, environmental issues, incorporation of additional unit operations including filtration, desalting, etc., to support the procedure. There are several techniques available and routinely used in low pressure liquid chromatography and these have been adequately described elsewhere [5 -71. Of the chromatographic techniques available those typically scaled-up are ion-exchange, hydrophobic interaction and size exclusion with the first being the most widely used.

6.2 Principles of Ion-Exchange Chromatography As the name implies, ion-exchange is simply an exchange of solute ions of like charge, facilitated by some external factor. A biochemical example of an ionexchange process being the transmission of an action potential along a nerve where Na+ ions initially enter the nerve cell and subsequently K + ions leave the nerve cell thereby propagating transmission of the nerve impulse [S]. Ion-exchange chromatography is simply an adsorption/desorption process utilizing this principle of ion-exchange and is therefore reliant on the chemical properties of the solute molecules to be separated. During the adsorption stage, a charged solute ion binds to a stationary phase bearing the opposite charge, while solute molecules of either neutral charge or similar charge to the stationary phase are unretained. Desorption is simply effected by exchanging the bound solute ion with a counter-ion of similar charge, typically sodium or chloride ions. Ion-exchange stationary phases bear either a positive or negative charge for adsorption of either anions or cations respectively. The principle of anion and cation exchange is represented in Fig. 6-1. Low pressure ion-exchange media are available in bulk from several suppliers and in the bioprocessing industry, are traditionally based on polysaccharide supports including cellulose, agarose and dextran [9, 101. For anion-exchange chromatography the support matrix is derivatized with positively charged functional groups, typically amines, and for cation exchangers the matrix would be derivatized with negatively charged functional groups, typically acids. Whatman International Ltd. manufacture a range of anion- and cation-exchange celluloses which have either weak or strong charged groups attached to the cellulose molecules. For anion-exchange celluloses the functional groups present are the tertiary amine, diethylaminoethyl (DEAE): CH2 - CH,

-CH2 -CH2

-

NH'

6.2 Principles of Ion-Exchange Clwomatography

Anion-Exchange

133

Ca tion-Exchange

I 1 \

ADSORPTION

WASH

i

-000 -000 DESORPTION (NaCI)

Fig. 6-1. Principles of anion and cation exchange.

and the quaternary amine N,N,N-trimethyl-2-hydroxypropylamine(QA):

OH CH3 I I -CH,-CH-CH,-N+-CH3 I CH3 The functional groups used for cation-exchange celluloses are the carboxylic acid, carboxymethyl (CM): - CH,

-CO;H+

the sulfonic acid, sulfoxyethyl (SE): - C H ~- C H -SOTH+ ~

and orthophosphate (P):

134

6 Biochemical Applications of Process-Scale Ion-Exchange Liquid Chromatography

0 /I

-0- P -O-H+ I

0-NH,' The selection of either anion- or cation-exchange media is entirely dependent on the nature of the solute ions to be separated although for protein separations anionexchange tends to be the more widely used technique. In an aqueous environment proteins can be regarded as polyions and have an overall electrical charge dependent primarily on the secondary structure of the protein and post-translational factors such as glycosylation [ I l l . The isoelectric point, PI of a protein is the pH at which it bears no net charge and this is dependent on structural aspects of the protein molecule. For p H > PI the protein bears an overall negative charge and thus binds to an anion-exchanger and for pH < PI the protein bears an overall positive charge and thus binds to a cation-exchanger. Factors including the p H and ionic strength of the mobile phase, the pKa of the functional group and the surface charge density of the protein will influence the choice of the most suitable functional group with which to effect the separation. Unlike other techniques including size exclusion, ion-exchange is an adsorptive process and therefore isocratic elution is not recommended and often not practical. Selective desorption is simply carried out by increasing the ionic strength and/or adjustment of the p H closer to the PI of the bound protein or the pKa of the functional group on the exchanger.

6.3 Throughput If an ion-exchange step is to be used in an industrial process then the economics will dictate that a certain throughput must be obtained. For a process step throughput is the amount of product purified per unit time at the desired level of purity. In order for the unit process to be commercially sound it is generally accepted that the added value of the resulting product exceeds the costs of its manufacture, ie, media costs, mobile phase costs, labour, capital equipment costs, utilities, depreciation, etc. Throughput is influenced by several factors which include media selection, capacity of the medium, adsorption/desorption kinetics of the medium and process time, ie, flow rate. These influences have been described in detail elsewhere [12]. Having selected the most appropriate ion-exchange medium based upon the considerations mentioned above, the mode of use of the exchanger needs to be determined. Generally, a process-scale separation would have been carried out at laboratory-scale during the preliminary stages of development. In these developmental stages the separation would often be carried out using small column-based chromatography systems, if for no other reason than that they are widely available, simple to use and relatively inexpensive. However, when the process is scaled-up several external factors may

6.3 Throughput

135

Table 6-1. Factors influencing the choice of batch versus column techniques. 1 . Volume and concentration of feedstock

Scale, ie, amount of media required Sanitary requirements Media losses Process time Labor requirements I. Centrifugation capability 8. Shear constraints 9. Automation 2. 3. 4. 5. 6.

influence mode of use of the ion-exchanger. These can include the factors listed in Table 6- 1. The two generalized techniques used in process-scale ion-exchange liquid chromatography are either batch stirred tank or column. A batch stirred tank is a simple equilibrium process in which the ion-exchange medium is stirred as a slurry with the feedstock in an open system. The media is then collected by a filtrationkentrifugation process prior to the washing and elution stages. A column process is part of a closed system in which the ion-exchange medium is contained within a vessel fitted with porous end supports, and through which the feedstock, wash buffers and elution buffer all pass. Columns are available in a range of sizes and designs from several manufacturers and can use either axial or radial flow technologies. We have evaluated several axial and radial flow columns and compared their performance in separations using Whatman ion-exchange celluloses 113- 161. Of those factors listed in Table 6-1, the major feature which determines the requirement for a batch procedure will be scale of the process. In separations where large volumes of a dilute feedstock are to be treated with a small volume of medium, then the process time using a column may be so long as to preclude its use. For example, adsorption of protein onto 100 kg of ion-exchanger in a column would take 4 h for 800 L of feedstock operating at a flow rate of 2 bed volumes h-I, ie, 200 L h-I. If the feedstock volume were increased to 8000 L then this adsorption process would take 40 h under similar operating conditions which likely would be undesirable and economically impractical. In these circumstances a batch adsorption may be the process technique of choice. A major factor which determines the requirement for a column process is that of sanitary operating conditions. In order to operate a process under these conditions a closed system is ideal and this may preclude the use of batch process unless a suitable self-contained plant operating area is available. A more detailed discussion of the factors listed in Table 6-1 has been reported elsewhere [12].

136

6 Biochemical Applications of Process-Scale Ion-Exchange Liquid Chromatography

6.4 Biochemical Applications of Process-Scale Ion-Exchange Liquid Chromatography Large-scale ion-exchange chromatography is carried out routinely in many areas of the bioprocessing industry. However relatively little data is available in the scientific literature presumably due to two primary reasons. Firstly, many commercial processes involve proprietary technology which precludes any opportunity for publications of detailed experimental results. Secondly, the cost of carrying out a large-scale separation of a non-proprietary feedstock for academic purposes is often too high to warrant such a study being carried out for the purpose of publication of such data. Biochemical applications of process-scale ion-exchange liquid chromatography which have been reported in the literature include the isolation of uridine phosphorylase from Escherichia coli [ 171, prochymosin from Escherichia coli [18], L-asparaginase from Erwinia spp. [19, 201, monoclonal antibodies [21, 221 and albumin from human plasma [23,24]. We have carried out several process-scale evaluations using Whatman ion-exchange celluloses and these will be discussed in more detail. Dependent on the pH of the feedstock and the PI of both the target protein and the contaminants within Is target a cation? ie, pH < p i

I s target an anion? ie, pH > p l

Is target to be retained and con tam in ants unretained?

Yes

Use anionexchange ie, Positive Step

No

Use cationexchange ie, Negative Step

Is target to b e retained and contaminants unretained?

Yes

Use cationexchange ie, Positive Step

Fig. 6-2. Approaches to ion-exchange chromatography.

No

Use anionexchange ie, Negative Step

3 37

6.4 Biochemical Applications of Process-Scale Ion-Exchange Liquid Chromatography

the feedstream, the selection of either anion or cation-exchange will be made. As described above, for p H > p I a protein would bind to an anion exchanger and for pH
6.4.1 Chromatography of Hen Egg-White Proteins Hen egg-white is a complex mixture of solutes comprising 10.5% (w/v) protein [25]. Although hen egg-white is reported to contain in excess of 40 proteins [25] the most abundant basic protein is lysozyme [PI 10.51 and the most abundant acidic protein is ovalbumin [PI 4.51 [25]. Consequently hen egg-white is a useful mixture for both anion- and cation-exchange chromatography, and is used commercially in the food processing and enzyme-manufacturing industries. The laboratory-scale chromatography of diluted, clarified hen egg-white at pH 7.5 on the Whatman anion-exchange cellulose DE 52 and cation-exchange cellulose CM52 is represented in Figs. 6-3a and 6-4a respectively. As would be expected the basic protein lysozyme is unretained on DE52 while being the most strongly retained on CM52. Conversely the ovalbumin component is strongly retained on DE52 whereas it is eluted rapidly from CM52. In each case the loading of egg-white proteins is 70 mg, ie, 7 mL of a 10 mg mL-' solution which accounts for 2% of the effective binding capacity of the column of either DE 52 and CM 52. In such analytical separations, the chromatography is optimal since protein :protein interactions and the various factors influencing throughput will be minimal. In order to demonstrate scaleability both separations were scaled-up 1000-fold by increasing column diameter from 1.5 cm to 45 cm whilst maintaining bed depth at - 16 cm. The separations of hen egg-white proteins on DE 52 and CM 52 are represented in Figs. 6-3 b and 6-4 b respectively. The chromatograms demonstrate that both separations directly scale-up from analytical to process columns. Under process-scale loadings several factors including protein : protein displacement, kinetics, etc., will affect the chromatography such that the analytical profile will change as loading increases. If ovalbumin is to be isolated from egg-white then use of a positive anion-exchange step is desirable. Since ovalbumin is the most strongly bound component on DE52 (Fig. 6-3) then as loading progresses it is reasonable to assume that the ovalbumin will displace the less acidic proteins. This is analogous to frontal elution/displacement chromatography reported for preparative HPLC separations [26]. In order to assess the effects of loading on the separation of egg-white proteins a column of DE52 (1.5 cm

-

-

138

6.4 Biochemical Applications of Process-Scale Ion-Exchange Liquid Chromatography

1 Load sample Buffer wash

Volume passed ImL)

Gradient s t a r t

0.50

0.50

A 0.40

0.4 0

0.30

0.30

-

E

I

j 0

E

W

c u

W

rn n

73

;0.20 n

0.20 $ u

a

0.1 0

0.1 0

0

vt

2o Load sample Buffer wash

40

!

60

80

100

120 Volume passed (Ll

140

160

180

0 200

Gradient s t a r t

Fig. 6-3. Column chromatography of hen egg-white proteins on DE52 using 0.025 M-Tris/HCl buf-

139

6.4 Biochemical Applications of Process-Scale Ion-Exchange Liquid Chromatography

0.80 ~-

5

3.80

(a)

0.70-

0.70

0.60 -

3.60

0.50

-

0.50

0.40

-

0.40

m

=I

E

%

.-

m

n

n

0

N

L

-5 'D

0.30 -

0.30 IF;

4

0.20 o 0 0.10

.

0 0.10

2

~

0

0I

40

60

80

2o Load sample

100

120

140

160

180

200 220

240

Volume passed (mL)

Gradient s t a r t

1

0.80

-

0.40

0.60

m N

Z 0.40

0

~-

m

n

0.30

~-

- 0.30

n

.TI

Q

0.20 -

01

20

Load sample Gradient s t a r t

40

60

80

100 120 140 160 Volume passed (L)

180

200

220

-

0.20

--

0.10

240

0

Fig. 6-4. Column chromatography of hen egg-white proteins on CM52 using 0.01 M-sodium acetate buffer, pH 4.5 at (a) laboratory scale (1.5 cm i.d. x 15.5 cm) at a flow rate of 2 mL min-' and (b) process scale (45 cm i.d.xl6cm) at a flow rate of 1000mLmin-l.

140

6 Biochemical Applications of Process-Scale Ion-Exchange Liquid Chromatography

1.20 1.00

E 0.80

0 N co OI

: 0.60 m 5

o

L

I I

< 0.40 ul

0.20

0 80

U

90

Volume passed (mL)

Fig. 6-5. Absorbance profile of column eluate at 280 nm during a saturation loading of DE52 with 10 mg mL-' hen egg-white proteins using 0.025 M-TridHCl buffer, pH 7.5 at laboratory scale (1.5cm i.d.xI5.5 cm) at a flow rate of 1 mLmin-'.

i. d. x 15.5 cm) was loaded with 8 mg mL-' egg-white solution (875 mL) by which time the absorbance at 280nm of the column effluent was similar to that of the feedstock. The breakthrough curve for this study is represented in Fig. 6-5. During this study a total of 13 fractions were collected and samples were rechromatographed at analytical scale on a column of DE52. These chromatograms are represented in Fig. 6-6. Fraction 1 (Fig. 6-6 a) contains lysozyme, with all acidic material adsorbing to the DE 52. The inflection in Fig. 6-5 (fraction 2) at a loading of - 720 mg (90 mL) represents the displacement of ovotransferrin by the more acidic components (Fig. 6-6 b). A second breakthrough occurs around fraction 8 where the ovalbumin starts to elute from the column (Fig. 6-6c). Following a buffer wash and salt elution the protein bound to the DE 52 was predominately ovalbumin. These data demonstrate that anion-exchange chromatography can be used effectively to purify ovalbumin using a displacement process. If ovotransferrin were the desired target then it would be reasonable to collect and pool fractions 2 - 6 which contain lysozyme and ovotransferrin and rechromatograph them on a second column of DE 52 to eliminate the lysozyme contaminant. The molecular weight of ovalbumin is 45 000 compared with 76600 for ovotransferrin [25]. In order to facilitate the displacement of ovotransferrin by ovalbumin, hen egg-white can be chromatographed on the Whatman anion-exchange cellulose DE 92. This exchanger is based on fibrous cellulose and has less accessible pores then DE52 which results in size exclusion of proteins typically larger than - 80000 Da. The analytical chromatography of hen egg-white protein on DE92 is represented in Fig. 6-7 a and compared with DE 52 Fig. 6-7 b. The ovotransferrin peak is reduced on

142

6.4 Biochemical Amlicafions of Process-Scule Ion-Exchange Liquid Chromatography

0.50

10.50

0.40

- 0.40

5

-

g 0.30

-

0.30 I€

N

01

W u

P-

c m n

5 O

-

;; 0.20

0.20

VI

n Q

0.10

Jo 180

0

Vt

A

20

40

60

t

B

80

100 120 Volume passed (mL1

140

160

200

C

A = L o a d sample

B = B u f f e r wash

C =Gradient s t a r t

Fig. 6-6 a

0.50

0.50

0.40

0.40

0.30

0.30 !-

-

E N

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D W

.-

m n

L

0

5 0.20

0.20 5

n a

0.10

0.10

n

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20

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A 1 3 A = Load sample

Fig. 6-6 b

60

1

80

100 120 Volume passed ( m L )

140

C B = Buffer wash

C =Gradient s t a r t

160

180

200

142

6 Biochemical Applications of Process-Scale Ion-Exchange Liquid Chromatography

0.50

0.40 I

s

cg 0.30

0

(I/i

al u m t

n

::0.20 n L

Q

0.10

A

B

A = Load sample

0.30

-2 E TW II

.-L

0

0.20 ;=

0.1 0

C B = Buffer wash

C =Gradient s t a r t

Fig. 6-6c Fig. 6-6. Rechromatography using DE 52 of individual fractions eluting during the saturation loading of a laboratory column (1.5 cm i.d.xl5.5 cm) of DE52 with IOmg mL-’ hen egg-white proteins. Samples of Fraction 1 (a), Fraction 2 (b) and Fraction 8 (c) were chromatographed at laboratory scale (1.5 cm i.d.x 15.5 cm) using 0.025 M-Tris/HCI buffer at a flow rate of 1 mL min-’.

DE92 compared with DE52 due to size exclusion a feature which is advantageous in the isolation of ovalbumin. We have reported the large-scale separation of ovalbumin from diluted egg-white using DE52 127, 281 and DE92 [29] for both batch stirred tanks and column operation. In each study 200L of 10mgmL-’ egg-white was contacted with 25 kg of exchanger. The results demonstrated effective isolation of ovalbumin from the eggwhite for the reasons described above at high binding capacity and good product recovery 127 - 291.

6.4.2 Chromatography of Goat Serum Proteins Blood proteins are fractionated by various procedures to give a range of purified components which may be used for a number of therapeutic or diagnostics purposes. Procedures for the fractionation of plasma proteins are adequately reviewed elsewhere 1301. Fractionation of blood proteins from a range of hosts is carried out at large-scale and in these processes ion-exchange chromatography is routinely used. In

143

6.4 Biochemical Applications of Process-Scale Zon-Exchnnze Liquid Chromatography

10.50 0.40-

~

0.40

0.30N

al u

c rn

!

A B6 A z L o a d samDle

80 100 120 Volume passed ImL)

140

160

180

200

C B = B u f f e r wash

[=Gradient s t a r t

0.50

0.40

0.30

I

-d

E al

2

0

0.20 5

0.10

0 60 A B A = L o a d sample

100 120 Volume passed ImL)

80

140

160

180

200

c B:Buffer

wash

[=Gradient s t a r t

Fig.6-7. Column chromatography of hen egg-white proteins on (a) DE92 and (b) DE52 using 0.025 M-Tris/HCl buffer, pH 7.5 at laboratory scale (1.5 cm i.d. x 15.5 cm) at flow rates of 2 mL min-'.

144

6 Biochemical Applications of Process-Scale Ion-Exchange Liquid Chromatography

order to assess the effectiveness of ion-exchange chromatography we investigated the isolation of immunoglobulin G (IgG) from goat serum [31, 321. The immunoglobulin G content of a 10% (v/v) diluted goat serum was 0.95 mg mL-' reflecting a concentration of 11070 (w/w) total protein. Existing procedures for isolation of IgG involved several steps including salt precipitation, anion-exchange chromatography and size-exclusion chromatography [31]. Salt precipitation is a useful step at laboratory-scale and used carefully can effect a good degree of purification. However, in order for it to be effective the protein precipitate is usually collected as a pellet by centrifugation, redissolved in a minimum volume of buffer, and then desalted by either dialysis or size-exclusion chromatography. The protein solution would then typically be microfiltered to eliminate any particulates prior to subsequent chromatography. At small-scale such an approach is tolerable but at large-scale the additional unit processes, described above - which render the fractionated protein in a suitable form for subsequent ion-exchange chromatography - are cumbersome, time consuming and costly. An attractive proposition for large-scale chromatography would be to eliminate the salt-precipitation step completely and replace it with a more scaleable procedure. To this end we reported the use of the fibrous weak anion-exchange cellulose Whatman CDR as a guard column for treatment of diluted goat serum. Using 0.02 M-Tris/HCl buffer, pH 7.5, the IgG fraction (PI 6.9- 8.6) passed unretained through the column of CDR while some of the more acidic components were retarded by this medium [32]. The post-CDR goat serum was then chromatographed on the quaternary amine anion-exchange cellulose Whatman QA 52. A typical chromatogram is illustrated in Fig. 6-8. This QA52 procedure is an example of a negative step whereby the IgG is collected in the unretained fraction while the contaminants adsorb to the QA52. The purpose of our work was to isolate the IgG fraction at high purity with less emphasis on overall yield. QA52 partially retained

-

-

0.5

0.4

z

0

", 0.3 A

aJ

1

Y

m t

n

0'VI 0.2

n

z l

1

/ I

0

50

I I

I

I

I

I

100 150 200 250 300 350 400 Elution volume (mL1

Fig. 6-8. Column chromatography of post-CDR goat serum on QA52 using 0.02 M-Tris/HCl buffer, pH 7.5 at laboratory scale (1.5 cm i.d. X 12 cm). A denotes a buffer wash and B denotes a wash using buffer containing 0.5 M-NaCI.

6.4 Biochemical Applications of Process-Scale Ion-Exchange Liquid Chromatography

145

IgG under our experimental conditions and increasing either the ionic strength of the buffer, or reducing the pH, were both effective at improving yield but also decreased the capture efficiency of non-IgG proteins thereby reducing product purity. Under optimized mobile phase conditions of 0.02 M-Tris/HCl buffer, pH 7.5, we were able to recover 55% (w/w) of applied IgG at a purity of 100% as determined by immunoelectrophoresis [32]. Having developed and optimized the process at laboratory scale we reported effective scale-up of the QA52 step 415-fold [32]. In this study we wished the QA52 step to be the final stage of the purification so use of the negative step has two advantages. Firstly, the separation was engineered such that the IgG component eluted in a pure state and secondly the IgG fraction did not require a desalting step or buffer change for subsequent use. These two features may be economically attractive were this to be a commercial application of process-scale ion-exchange liquid chromatography. In order to subfractionate the IgG pool further (if required) a sample of the unretained material from the QA 52 step was chromatographed on the cation-exchange cellulose Whatman SE52 [31]. A typical chromatogram is represented in Fig. 6-9. The data demonstrates 2 peaks of protein, the first which was unretained by the SE52 and the second which adsorbed to the exchanger. Both components had similar molecular weights and were immunologically identical to goat IgG but had slightly different isoelectric points [311. Further analysis using a protein A affinity column demonstrated that the unretained peak from the SE 52 column comprised principally the subclass IgG, whereas the component which bound to SE52 was principally the subclass IgG2 [31].

-

Fig. 6-9. Column chromatography of post-QA52 goat serum IgG fraction on SE52 using 0.01 M-Tris/HCI buffer, pH 7.5 at laboratory scale (1.5cm i.d.xl1.5cm) at a flow rate of 2mLmin-'. A denotes a buffer wash.

Elution volume (rnL1

146

6 Biochemical Applications of Process-Scale Ion-Exchange Liquid Chromatography

6.4.3 Chromatography of a Monoclonal Antibody In the previous section we describe a purification of an immunoglobulin G pool from goat serum. These immunoglobulins are polyclonal in nature containing antibodies raised by the host in support of their own immune system together with any specific antibodies which may have been raised following a specific challenge with a foreign antigen. The use of polyclonal antisera is widespread in various immunoassays including ELISA, immunodiffusion, etc., and a wide range of antisera are available commercially at various levels of purity. In order to facilitate much higher selectivity in, for example, immunoassays, immunodiagnostics and immunoaffinity chromatography the use of monoclonal antibodies is becoming widespread. Monoclonal antibodies are typically produced by either mammalian cell culture or from the ascites fluid of a host, typically mouse. Further background information on monoclonal antibodies is available in the literature [33]. In order to investigate the role of ion-exchange chromatography in the purification of a monoclonal antibody we used hybridoma L243 which produces an IgG2, antibody against a non-polymorphic determinant on human I a-like molecules [34]. This antibody is cytotoxic for human peripheral blood lymphocytes and recognises a 28 000- 34000 Da HLA-DR molecule which has been identified on the surface of Bcells, monocytes, macrophages and activated T-cells. Hybridoma L 243 (ATCC HB55) was derived by fusion of NS-1 myeloma cells and spleen cells of a BALB/c mouse previously inoculated with the B lymphoblastoid cell line RPMI 8866 [34]. The hybridoma cells were cultured in an Opticell 5200R bioreactor at Charles River Biotechnical Services (Margate, Kent, UK) and the culture fluid containing 91 '70 DMEM and 5% fetal bovine serum used as feedstock for ion-exchange chromatography. The culture fluid contained 0.05-0.2 mg IgG,, mL-l in the presence of a large excess of fetal bovine serum albumin. Consequently it was decided to perform a positive ion-exchange step using cation-exchange. Under optimized conditions the acidic albumin contaminant should be unretained by the exchanger while the monoclonal antibody should adsorb. A typical chromatogram of the culture fluid on the cationexchange cellulose Whatman SE53 is represented in Fig. 6-10. The bulk protein was unretained on the SE53 as anticipated with all IgG,, activity remaining bound to the exchanger. The IgGza eluted as a major peak (1 130- 1200 mL) ahead of some colored components present in the culture medium. The IgG,, fraction collected from the SE53 column was further purified by size exclusion chromatography using Sephacryl S-300 (Pharmacia LKB Biotechnology, Milton Keynes, UK). The elution profile of this fraction is represented in Fig. 6-1 1 a. The results demonstrate a major peak of MW- 160000 Da (83.9 mL) which contained IgG2, as determined by immunodiffusion tests. By comparison the elution profile of the culture medium on Sephacryl S-300 is shown in Fig. 6-1 1b. Clearly the IgG,, component (81.7 mL) identified by immunoassay is a minor component of a highly heterogeneous protein pool. The results of this study demonstrate that use of a positive cation-exchange chromatographic step can be used to significantly enrich the content of a monoclonal antibody from a tissue culture medium in a single step. The monoclonal antibody could

147

6.4 Biochemical Applications of Process-Scale Ion-Exchange Liquid Chromatography

0.30

0.80 0.70

0.25 0.60

E 0.20

.-.

0.50 !-,

-

W 0 N

0

W

2 0.15

0.40

n

2 ._ D

L

v) 0

0.30

2 0.10

2

0.20 0.05 0.1 0

/ 0 0

LI

200

400

600

0

800

1000

1200

1400

1600

Volume passed (mL)

Fig. 6-10. Column chromatography of hybridoma L243 tissue culture fluid on SE 53 using 0.02 M-sodium acetate buffer, pH 5.5 at laboratory scale (1.5 cm id. x 15.5 cm) at a flow-raie of 2 mL min-’.

be further purified by either size-exclusion chromatography as reported here or by affinity chromatography using either the immobilised antigen against the monoclonal antibody or a group specific adsorbent such as immobilised protein A. In any event the ultimate product application and criteria for purity would determine what, if any, further purification was required.

6.4.4 Chromatography of DNA-Modifying Enzymes Molecular biology has emerged as a routine technique in the development and manufacture of a range of recombinant DNA-derived products in several areas including therapeutics and diagnostics. In order for the molecular biologist to perform manipulations on recombinant DNA it is necessary to have available a range of DNA-modifying enzymes. These fall into three major categories. Firstly, endonucleases (restriction enzymes) which selectively cleave the DNA molecule at defined nucleotide sequences, secondly, ligases to join chains of nucleotides together and thirdly polymerases to facilitate in-vitro replication of the DNA template. These three groups of DNA-modifying enzymes are essential in the development of the ‘genetically engineered’ expression system for the target molecule and consequently are commercially available as tools for the molecular biologist. Unlike some of the other biochemical applications of process-scale ion-exchange liquid chromatography

6 Biochemical Applications of Process-Scale Ion-Exchange Liquid Chromatography

0.03

0.02 0

m

N W

u c m

n L

VI 0

2 0.01

0

100

120

Volume passed (mL) 8:

0.03

146.0

-

0.02 0 N W

al LJ c m n L

0

wl

n

a 0.01

-

0-

I

0

20

172.9

Fig. 6-11. Column chromatography of (a) post-SE53 IgG,, peak and (b) L243 tissue culture fluid on sephacryl S-300 using 0.5 M-NaCl at laboratory scale (1.5 cm i.d.x90cm) at a flow rate of I mLmin-'.

6.4 Biochemical Applications of Process-Scale Ion-Exchange Liquid Chromatography

149

discusssed above the scale of manufacture of DNA-modifying enzymes is frequently smaller than that of a bulk protein such that a laboratory column of chromatography medium should suffice. Furthermore, the criteria for purity are perhaps less stringent than for other proteins since, provided that there is no other contaminating enzyme activity in the preparation, protein homogeneity may not be a prerequisite. The catalytic domain of all three groups of DNA-modifying enzymes has a requirement for a phosphate group which is either hydrolyzed (endonucleases) or condensed (ligases, polymerases). The cation-exchange cellulose Whatman P 11 has application not only as a cation-exchanger but also as a pseudoaffinity medium for phosphate-dependent enzymes by virtue of the phosphate functional group. Biochemical applications of cellulose phosphate include the purification of various kinases and phosphatases [35]. We have investigated the chromatography of over 20 DNA-modifying enzymes using P 11 [36]. These proteins can all be isolated from the host bacterial cell lysate preparation by chromatography on P 11 to which DNAmodifying enzymes bind. A representative chromatogram is shown in Fig. 6-12. A benefit of using a cation-exchanger is that nucleic acids which could mask or hamper the activity of the DNA-modifying enzymes are unretained by P 11. The salt-eluted material from the P 11 column (fractions 9 - 11) were found to contain endonuclease activity and were also free of any other contaminating enzyme activity [36]. This elution profile is typical for all DNA-modifying enzymes we have studied [36] and activity can be detected by either cleavage of lambda phage DNA or selected plasmids; fusion of Hind 111 digested lambda DNA; or incorporation of radioactive labelled deoxynucleotides into replicated DNA molecules, for endonucleases, ligases or polymerases, respectively [36]. 1.0 0.9

0.8

0.7

z

0.6 -L

0 OD

N

al v

0.5

m c n L

U

m

v) 0

0.4

n

4

0.3 0.2

0.1 0

-

I

1

I

I

I

I

10 20 Fractions

I

0

Fig. 6-12. Column chromatography of Providencia sfuartii cell lysate on P 11 using 0.01 M-potassium phosphate buffer, pH 7.5 containing 0.2 M-NaCI and 0.007 M-2-merCaptOethanol at laboratory scale (1 cm i.d. x 20 cm) at a flow rate of 0.5 mL min-‘.

150

6 Biochemical Applications of Process-Scale Ion-Exchange Liquid Chromatography

This protocol gives a rapid and efficient single step purification of a group of commercially important bacterial enzymes. If further fractionation of these enzymes should be required then chromatography by anion-exchange may facilitate further purification, although in terms of the functional role of these enzymes may in fact be an unnecessary luxury.

6.5 Conclusion Throughput is the key to economic success of commercially important biochemical applications of process-scale ion-exchange liquid chromatography. We have described the principles of ion-exchange and how it may be used to replace existing more cumbersome process stages. Examples have been given to the application of both anion- and cation-exchangers in either positive or negative adsorption processes dependent on the nature of the feedstock and the requirements of the process. Used carefully ion-exchange chromatography proves an extremely useful step in processscale liquid chromatography and can give rise to significant purification factors with excellent recovery of applied protein. Acknowledgements The author wishes to acknowledge the contributions of his colleagues within Whatman International Ltd. who carried out the work described in this review: Stephen E. Badger, Brian N. Brook, Jayne A. Cox, Navin D. Pathirana, Michael Streater and David W. Toome based in Maidstone, UK, and Edward T. Butts, Mark L. Koscielny and Linda Lane of Whatman Inc. based in Fairfield, NJ, USA. Acknowledgement is made to John M. Ward and co-workers in the Department of Biochemistry, University College London, UK for collaboration in the studies of DNA-modifying enzymes.

References [l] Streat, M. Ion-Exchange for Industry, Chichester: Ellis Horwood Ltd., 1988.

[2] Williams, P.A., Hudson, M. J. (eds.) Recent Developments in Ion-Exchange 2, London: Elsevier Applied Science, 1990. [3] Abe, M., Kataoka, T., Suzuki, T. New Developments in Ion-Exchange, Tokyo: Kodansha, 1991. [4] Williams, R. J. P., in: Recent Developments in Ion-Exchange 2, Williams, P. A., Hudson, M. J. (eds.), London: Elsevier Applied Science, 1990, pp. 3- 15. [ 5 ] Hammond, P.M., Scawen, M.D. J Biotechnol, 1989, 11, 119- 134. [6] Scawen, M.D., Hammond, P.M. J Chem Tech Biotechnol, 1989, 46, 8 5 - 103.

References

151

Lowe, C. R. A n Introduction to Affinity Chromatography, Amsterdam: Elsevier Biomedical Press, 1979; pp. 276-292. Ganong, W. F. Review of Medical Physiology, Los Altos: Lange Medical Publications, 1981, 10th edn; pp. 37-39. Friefelder, D. Physical Biochemistry, San Francisco: W. H. Freeman and Company, 1989; 2nd edn; p. 249. Rossomando, E. F., in: Methods in Enzymology: Deutscher, M. P. (ed.) San Diego: Academic Press, 1990; Vol. 182, p. 309. Stryer, L. Biochemistry, San Francisco: W.H. Freeman und Company, 1981, 2nd edn; p. 11. Levison, P. R., in: Preparative and Process-Scale Liquid Chromatography: Subramanian, G. (ed.) Chichester: Ellis Horwood Ltd., 1991; pp. 146-161. Lane, L., Koscielny, M. L., Butts, E. T., Levison, P. R., in: Pittsburgh Conference Abstract Book, New York, March 1990; Pittsburgh: Pittsburgh Conference, 1990; p. 1078. Lane, L., Koscielny, M. L., Levison, P. R., Toome, D. W., Butts, E.T., Bioseparation 1990, 1, 141 - 147. Levison, P. R., in: Pharmaceutical Manufacturing International, Barber, M. S., Barnacal, P. A. (eds), London: Sterling Publications International Ltd., 1991; pp. 147 - 149. Levison, P. R., Badger, S.E., Toome, D. W., Butts, E.T., Koscielny, M. L., in: Upstream and Downstream Processing in Biotechnology III, Huyghebaert, A., Vandamme, E. (eds.), Antwerp: The Royal Flemish Society of Engineers (k.viv.) 1991; pp. 3-21 -3-28. Weaver, K., Chen, D., Walton, L., Elwell, L., Ray, P., Biopharm 1990, July/August, pp. 25-28. Marston, F. A. O., Angal, S., Lowe, P. A., Chan, M., Hill, C. R., Biochem SOCTrans 1988, 16, 112- 115. Goward, C. R., Stevens, G. B., Collins, I. J., Wilkinson, I. R., Scawen, M. D., Enzyme Microb Technol 1989, 12, 229-247. Lee, S.-M., Ross, J.T., Gustafson, M.E., Wroble, M.H., Muschik, G.H., Appl Biochem Biotechnol 1986, 12, 229-247. Duffy, S.A., Moellering, B. J., Prior, G. M., Poyle, K. R., Prior, C. P., Biopharm 1989, June, pp. 34-47. Jungbauer, A., Unterluggauer, F., Uhl, K., Buchacher, A., Steindl, F., Pettauer, D., Wenisch, E., Biotechnol Bioeng 1988, 32, 326-333. Sofer, G. K., Nystrom, L. E., Process Chromatography A Practical Guide, San Diego: Academic Press, 1989; pp. 61 -63. Stoltz, J. F., Rivat, C., Geschier, C., Colosetti, P., Dumont, L., Pharm Technol Int 1991, June, pp. 60-65. Stevens, L., Comp Biochem Physioll991, lOOB, 1-9. Herbert, N. R., in: Preparative and Process-Scale Liquid Chromatography, Subramanian, G. (ed.), Chichester: Ellis Harwood Ltd., 1991; pp. 9-38. Levison, P. R., Badger, S. E., Toome, D. W., Carcary, D., Butts, E. T., in: Advances in Separation Processes, Inst Chem Eng Symp Series, 1990; No. 118, pp. 6-1-6-11. Levison, P.R., Badger, S.E., Toome, D. W., Carcary, D., Butts, E.T., in: Separations for Biotechnology 2, Pyle, D. L. (ed.), London: Elsevier Applied Science, 1990; pp. 381 -389. Levison, P. R., Badger, S.E., Toome, D. W., Koscielny, M. L., Lane, L., Butts, E. T., J Chromatogr 1992, 590, 49-58. Curling, J. M., Methods in Plasma Protein Fractionation, London: Acadeic Press, 1980. Schwartz, W.E., Clark, F.M., Sabran, I.B., LC-GC Magazine 1986, 4, 442-448. Levison, P. R., Koscielny, M. L., Butts, E. T., Bioseparation 1990, 1, 59-67. Goding, J. W., Monoclonal Antibodies: Principles and Practice, New York: Academic Press, 1983. Lampson, L., J Immunol 1980, 125, 293-299. Kinyanjui, P. W., Pearlman, R. E., Eur J Biochem 1991, 195, 55 - 63. Ward, J. M., Wallace, L. J., Cowan, D., Shadbolt, P., Levison, P. R., Anal Chim Acta 1991,249, 195-200.

References

Fig. 1-5 b

31

7 Instrumental Design and Separation in Large Scale Industrial Supercritical Fluid Chromatography Pascal Jusforgues

7.1 Introduction Preparative chromatography can be practised on two different scales: laboratory (recovery of grams of sample) and production (recovery of kilograms of sample). Preparative Scale Supercritical Fluid Chromatography (PS-SFC) has been practised on the laboratory scale from the very beginning of SFC in 1962 [I]. More information on these historical experiments has been gathered by Berger et al. [2] and Bevan [3]. This chapter is devoted to production PS-SFC which appears to be the crossbreeding of three successful techniques: analytical SFC, preparative HPLC and industrial Supercritical Fluid Extraction (SFE). It was created in 1982 when Professor Perrut patented the PS-SFC process with total recycling of the eluent 141. Ever since, it has been developed and made reliable in a university laboratory on a pilot scale [5 - 71 before becoming commercially available in 1990 [8]. During this work some technological choices have been made. Thus the developed process is only one of the multiple possibilities that could be imagined initially (for example, the pressurization of the fluid is carried out by condensing it and pumping it with a high pressure liquid pump; another choice would have been to compress it with a gas compressor). The process described here is the result of the work begun on 1982 since it is the only process available today.

7.2 Principle, Advantages and Drawbacks PS-SFC can be described as a modification of preparative HPLC. Their principles differ on four major points: -

The liquid solvent is replaced by a supercritical fluid, that is to say a fluid whose pressure and temperature are maintained at values higher than its critical pressure and temperature (Fig, 7-1). The supercritical fluid can be pure or a mixture. In most cases, carbon dioxide is used pure or mixed with a few percent of a “classical” solvent, called a modifier. The supercritical fluid has a high density (close

154

7 Instrumental Design and Separation

to liquid densities) that is correlated with high solubilities of the products to be purified. - The cycle time is shorter in PS-SFC (typically between 4 and 15 min). - At the column outlet, the purified products are instantaneously separated from the supercritical eluent by depressurizing it (the gas phase has no solvent power) while, in HPLC, the products are recovered diluted in the liquid eluent. - After separation of the product and the eluent, the latter is instantly and completely recycled. Figures 7-1 and 7-2 give more details on the PS-SFC process principle by describing the eluent cycle. For a better understanding of the thermodynamic cycle, typical pressure and temperature values are given here below when carbon dioxide is used. Transposition to other supercritical fluids is easily done by taking into account their respective critical pressure and temperature.

I C r

PRESSURE

1

I

SUPERCRITICAL FLUID

SOLID

TEMPERATURE

Fig. 7-1. Phase diagram of a pure substance. Dotted lines are the solid-liquid and gas-liquid equilibrium curves. Solid line is the eluent cycle of PS-SFC. Further explanations see text.

Eluent Cycle: 1. The eluent is stored in a small buffer reservoir in a liquid state (more precisely under gas-liquid equilibrium).

P 45 bar, T 10°C. 2. The liquid phase is pressurized by a pump to working pressure (typically between 100 and 300 bar).

P 200 bar, T 10°C.

7.2 Principle, Advantages and Drawbacks

155

CONDENSER

tI) VY

PRESSURE CONTROL

PURE PRODUCTS RECOVERY

Fig. 7-2. Flow sheet of a PS-SFC process. Further explanations see text.

3. The pressurized liquid is heated to working temperature. P 200 bar, T 45 "C. The eluent is now supercritical. Injection is made through a loop in which the sample has been previously dissolved in the eluent. 4. The supercritical eluent brings the sample through the chromatographic column (the pressure drop is amplified on the diagram for the sake of clarity). The separation then takes place.

5. The mixture (eluent + separated products) is depressurized in an adiabatic, thus isoenthalpic, way. P 45 bar, T 10°C. After depressurization, the eluent is under gas-liquid equilibrium.

156

7 Instrumental Design and Separation

6. The fluid is heated up to become gaseous. P 45 bar, T 25 " C .

In this phase, the eluent solvent power drastically decreases and the pure products condense into small droplets. Physical separation of the gaseous eluent and the separated products occurs in a cyclone, under dynamically enhanced gravity. 7. The gaseous eluent is recycled. It is cleaned on an adsorbent bed and condensed before being returned to the buffer reservoir. P 45 bar, T 10°C.

The cycle is complete. When a modifier is used with the supercritical eluent, the gas phase cannot be passed through the adsorbent bed. In this case, the gas phase is directed through a patented separator, which both cleans the eluent and adjusts the modifier concentration to the desired value. Periodic operations (injection, fraction collection, etc.) and continuous operations (regulation of temperature, pressure, flowrate, etc.) are completely automated. PS-SFC has several advantages over preparative HPLC: - The chemical properties of the new eluents offer a new range of selectivities (though it has been often said that carbon dioxide is close to liquid n-hexane, the opposite allegation has been demonstrated as often). - Retention times and selectivities can be continuously modified by changes of the operating temperature and pressure. The transport properties important for chromatography (viscosity, diffusion coefficients) are at least one order of magnitude better than in HPLC, allowing improved efficiencies at higher eluent speed and, thus, shorter cycle times. - One of the major drawbacks of HPLC is eliminated: storage, manipulation and treatment of cubic meters of solvents is replaced by storage of a few kilograms of eluent. - Purified product dilution is generally reduced to 1 : 1 (no dilution) or, in case of use of a modifier, to 1 :2 (worst case being 1 :10) (note that the modifier used will be a pure and harmless solvent like ethanol). On the other hand, PS-SFC has several drawbacks that limit its applications: - To maintain the eluent over its critical point, high pressure technology must be used (typically 200 bars). - Solvent capacity of supercritical fluids does not cover such a broad range as liquid solvents capacity (eg, proteins cannot be processed by PS-SFC).

7.3 Technology

157

7.3 Technology As can be seen in Fig. 7-2, the process is packed with chemical engineering. We will review here the key points of the process which determine its successful operation.

7.3.1 Pumping System The pump is a membrane type metering pump. It is the pressure source of the process, so that it is closely related to the safety concerns. In the process described here, four different safety levels are used: The first level is electronic: the computer checks the pressure through pressure gauges and reacts to any unscheduled pressure variation. Second, an electro-mechanical circuit breaker controlled by the pressure is placed at the outlet of the pump. Third, a relief valve on the hydraulic oil circuit of the pump prevents any over pressure generation by the pump. Fourth, several relief valves protect the process volumes against over pressure risks. Pumping the liquid eluent (for example carbon dioxide) is not as easy as pumping a normal liquid because the “cavitation” risk is much greater than usually. Indeed the pumped liquid is, on the suction side of the pump, a boiling liquid in equilibrium with its saturated vapor pressure, thus the slightest depression due to pump suction makes the liquid boil, bubbles appear and pumping is inefficient. That is the reason why the pump heads must be cooled and maintained at a temperature level lower than the eluent buffer volume. Even with such precautions, the pump efficiency is still dependent on small temperature and pressure variations on the suction side. Moreover, the carbon dioxide compressibility under these conditions is more important than that of classical liquids. Thus it is not possible to use the pump as a perfect metering pump and one can only consider the delivered flow rate as an approximation of the required flow rate. For these reasons, the flow rate regulation is made through an indirect method: the pump is permanently delivering an excessive flow rate and a part of this flow (the excess) is sent to a derivation line which is a bypass between the outlet and the inlet of the pump. The amount of bypass flow is regulated permanently by the computer.

7.3.2 Chromatographic Column Many negative experiments were made to obtain good pre-packed columns, but the lifetime of a pre-packed column in PS-SFC is only a few hours: quick rearrangement of particles leaves unacceptable void volumes and the maximum efficiencies ob-

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7 Instrumental Design and Separation

tained with a prepacked column (60 mm ID) were of a few hundred theoretical plates per meter. It is now well accepted that, for liquid chromatography, the obtention of large diameter, very efficient and stable columns requires the dynamic axial compression technology. The similarity between the HPLC and SFC columns and stationary phases suggest that the dynamic axial compression is the good solution and indeed efficiencies of 63000 plates per meter were obtained with a good stationary phase. Time stability of these columns is good and life time has not yet been evaluated (because it is too long). Moreover these columns support pressurization and depressurization of the eluent well, as long as the compression is maintained.

7.3.3 Fraction Collection After the column the eluent and purified product mixture is depressurized violently and heated to about 20 "C to vaporize the eluent. Theoretically it is easy to separate two phases by simple decantation. However, the practice is much different: the liquid (or solid) pure product forms a mist (or a dust) that is taken away from the traps by the gas flow and special devices must be used to stop the liquid (or solid) in the traps. The most efficient devices used are a patented model of cyclonic separators. The eluent turns rapidly around the axis of a cylindric tube. The heavy phase carried by the gas is centrifugated and agglomerates on the walls of the tube before falling to the bottom of the tube. The gas phase is evacuated by the top of the tube. These separators have several advantages: - First of all they are efficient (almost 100% of the condensable part of the product is stopped). - They have no moving mechanical parts. - They have a low retention volume of eluent.

7.3.4 Eluent Recycling Recycling of the gaseous eluent is easy when it is a pure fluid (CO,); the gas is passed through classical adsorbents columns to be cleaned then it is condensed. However, the use of a modifier mixed with the eluent makes things more difficult: the modifier (generally ethanol) will be partly recycled with the eluent (because its vapor pressure is not negligible). If we try to clean the gas on adsorbents, they will be very rapidly saturated. On the other hand, the modifier concentration in the column must be perfectly constant (not to change retention times) but the concentration of modifier at the outlet of the traps is not known because it is the result of complex physico-chemical phenomena which occur under non-equilibrium conditions. The solution of these two problems has been found and patented: the gas is cleaned by passing it through a liquid (the modifier) and the modifier is distributed between the liquid and gas

7.4 Separation Costs

159

phase to reach equilibrium. The concentration of modifier in the gas phase is controlled by a simple temperature control.

7.4 Separation Costs 7.4.1 Why PS-SFC is Expensive The main part of investment cost comes from the high pressure devices: the lowest pressure is in the recycling part of the process and it is still 45 bar. Thus, the process must include more than 50 high pressure valves, 15 high pressure volumes, 3 or 4 high pressure pumps, more than 200 m of high pressure tubes (and the corresponding high pressure fittings or weldings), 10 high pressure indicators and so on. The physico-chemical properties of the supercritical fluid being strongly dependent on pressure and temperature, means that these two parameters must be closely controlled all along the process, which must include 6 closed-loop regulation systems. As can be seen on the eluent cycle (Fig. 7-I), the PS-SFC process requires 3 or 4 different temperature levels and as many heating or cooling systems which must be added to the investment cost. Finally, safety systems related to the manipulation of high pressure compressible fluids are necessary all along the process loop. Strict parameter control, safety control and periodic valve actuation requires an automation system: the operator controls the process through a computer. All these points justify that the investment cost represents typically 51% of the total purification cost (see example below).

7.4.2 Why PS-SFC is Cheap Once the investment is paid, operating costs are much reduced. Eluent is cheap and completely recycled. The price of the packing material depends on the purification case but it is important to note that no special packing is required (all the HPLC packings are currently used in SFC). The energy cost is due to the heating and cooling devices required. Thanks to the automation system (included in the investment cost), labor cost is much reduced: one operator is required on the basis of 2 or 3 h per day for 24 h per day operations. The “miscellaneous” part of the cost includes environment, buildings, insurance, administration, etc.

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7 Instrumental Design and Separation

7.4.3 Purification Costs Range Typical Example. Figure 7-3 represents a typical example of cost share-out for the treatment of a natural crude mixture. These results are extrapolated from experiments made on a 60 mm ID to a real industrial production of 3 tonnes per year on a 200mm ID column.

One will notice the specific conditions of the example given in Fig. 7-3: - Favorable conditions: required purity is not very high. - Unfavorable conditions: 2 products must be recovered and many impurities are eluted before, in-between and after the two products. Ideal Case. If, in the previous example, the two products alone were present in the crude and had to be separated, cycle time would be reduced from 9 to 6 min, injected quantities would be increased up to 100 g per injection and yield would be as high as 95%. Production rate would then reach 6.8 tonnes per year on a 200 mm ID column. All other expenses being unchanged (investment, labor, energy, etc.), the purification cost would then be about US $ 92 per kg.

Worst Case. One of the worst possible cases (from an economics point of view) has been studied too. To make comparison easier with the above case, figures have been projected for a 200mm ID column. A racemic mixture of enantiomers has to be separated in two fractions of 99% purity. Due to the low capacity of the packing material used, very small quantities can 51%Investment

Eluent Fig. 7-3. Purification costs for the treatment of a natural crude. Conditions: 200 mm ID column; pure carbon dioxide eluent; 400 kg h-l flow rate; 180 bar; 50°C; packing is raw silica replaced every month; 2 compounds were collected and 5 main impurities removed; 1st compound purity increased from 50 to 95%; 2nd compound purity increased from 30 to 90%; injected quantities are 85 g every 9 min; collected quantities are 62.5 g per injection; work time is 24 h per day during 300 days per year; production rate is 3 tonnes per year. On this basis, with a US $ 1.6 million investment and a 5 year investment depreciation, the purification cost is US $ 209 per kilogram of purified product. Note: this cost does not include the crude price (part of the crude is lost since the yield is 62.5/85 = 73.5% of the injected crude material).

7.6 Conclusion

161

be injected (1.1 g every 5 min) (a little more than “touching bands” injections). Ethanol (8%) is added to carbon dioxide. Eluent flow rate is quite low (80 kg h-’). As a result of these conditions, investment cost is increased up to US $ 1.8 million (modifier addition to the eluent and removal from the pure products). Energy requirements are slightly decreased (because of lower flow rates). Manpower is increased (modifier manipulation). Packing price is difficult to evaluate since it is not commercially available: we evaluate it to twice the price of the above example. Other expenses are identical. Under these conditions 86.4 kg are produced each year at a price of US $ 8250 per kilogram!

7.5 Applications: SFC vs HPLC We have seen three examples of PS-SFC purification representing almost the full range of purification costs that can be achieved: ideal case at US $ 100 per kilogram, medium case at US $200, and high cost case at US $ 8000 per kilogram. These costs are comparable with the range of purification costs that can be achieved by HPLC. However, in the medium cost case studied above, HPLC purification cost was evaluated to US $ 350 per kilogram where SFC cost is only US $ 200 per kilogram. These figures induce a general rule for future evaluations: when a separation will be technically possible by both HPLC and SFC, the result of the economical comparison will depend upon the features of the separation. The main parameters will be: Solubilities of samples in supercritical fluid. SFC eluent: pure CO, or C 0 2 plus modifier. - HPLC solvent: pure, binary or ternary mixture; cheap or expensive; easy to recycle or not; level of residual traces allowed in product. - Respective selectivities, loadabilities and cycle times, etc. -

The main condition to use PS-SFC for a separation is the possibility to dissolve the sample in the supercritical fluid. This solubility can be evaluated from experiments or from literature on the applications of the supercritical fluids (extraction or analytical chromatography) (eg, [9,10]).

7.6 Conclusion PS-SFC appears to be a complementary technique to preparative HPLC. Actually, it can be presented as one of the multiple alternatives to HPLC that aims at reducing the cost and solvent consumption while maintaining the efficiency of separation: Simulated Moving Bed, Flip-Flop, Back-Flush, PS-SFC, Multi-Dimensional Chromatography, etc. On this basis, the potential applications of PS-SFC can be evaluat-

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7 Instrumental Design and Separation

ed to about 10% of HPLC applications. This potential is far from being realized yet because PS-SFC was only introduced commercially in 1990 and users must still be educated about its potential.

References Klesper, E., Corwin A.H., Turner, D.A., J Org Chem 1962, 27, 700. Berger, C., Perrut, M., J Chromatogr 1990, 505, 37-43. Bevan, C., this book, Chapter 8. Perrut, M., French Patent 8209649, 1982. Jusforgues, P., Dissertation, I.N.P.L. Nancy, France, 1988. Berger, C., Dissertation, I.N.P.L. Nancy, France, 1989. Doguet, L., Dissertation, I.N.P.L. Nancy, France, 1992. Prochrom, BP 9, 54250 Champigneulles, France (unpublished results). SFC Applications, The 1988 Workshop on Supercritical Fluid Chromatography, Park City, Utah, 12-14 January 1988. Markides, K.E., Lee, M.L. (eds.), Brigham Young University Press, 1988. SFC Applications, The 1989 Workshop on Supercritical Fluid Chromatography, Snow Bird, Utah, 13-15 June 1989. Markides, K.E., Lee, M.L. (eds.), Brigham Young University Press, 1989.

8 Scaling-up of Supercritical Fluid Chromatography to Large-Scale Applications Christopher D. Bevan and Christopher J. Mellish

8.1 Introduction Supercritical fluid chromatography (SFC) has been employed at the analytical scale by a number of workers for several years [I]. Scaling-up the technique for preparative use has been investigated and developed by relatively few and still presents a number of challenges. This Chapter relates the problems associated with scaling-up the technique for use at the laboratory preparative level and above, and pays special attention to the technical demands on materials and apparatus to ensure complete safety in the operation of the chromatograph. The ideas and designs of others will be reviewed and compared with the authors' own approach. The advantages and disadvantages offered by the various systems will be explained in sufficient detail for the reader to adopt the system most suited to hidher needs. The non-specialist reader may not be familiar with the use of 'supercritical fluids as mobile phases in chromatography so the Chapter is introduced with a brief explanation of the nature of the supercritical state.

8.2 Supercritical Fluids Supercritical fluid chromatography (SFC) is a form of liquid chromatography where the usual liquid solvent mobile phase has been replaced with a supercritical fluid. A supercritical fluid is a substance that is above its critical point. The boiling point of a liquid is the temperature at which the maximum vapour pressure of the liquid is equal to the external pressure or the temperature at which the liquid boils freely under that pressure [2]. For water the boiling point at atmospheric pressure is 100"C. However, if the pressure above the water is increased the boiling point is raised. The elevation of the boiling point of a substance with increase in pressure is shown in Fig. 8-1. The curve continues increasing to a point called the critical point. The temperature and pressure at this point are called the critical temperature and pressure respectively, which are characteristic of the particular substance. The curve

164

.,

8 Scaling-up of Supercritical Fluid Chromatography to Large-Scale Applications

Liquid

Pressure

The boiling point curve

Fig. 8-1. The boiling point curve.

Temperature

Pressure

bar

Liquid I

Supercritical fluid

--- - - - - - - - - - -

73 --

Gas

Fig. 8-2. The phase diagram of carbon dioxide. -56

31

Temp"C

for carbon dioxide is shown in Fig. 8-2 with the supercritical region indicated. A fluid in this region is neither a true liquid nor a true gas and has some of the properties of each. As indicated by the boundaries shown in Fig. 8-2 the fluid will not condense to a liquid irrespective of how much pressure is applied and equally will not boil if the temperature is increased. The supercritical fluids have some very interesting and potentially useful properties for the chromatographer. The solvation strength of many supercritical fluids approach those of liquid solvents, hence many substances will dissolve in them. Supercritical fluids have diffusion coefficients which are close to those of gases, hence they are able to transport dissolved solutes through materials very quickly. The low viscosity of the supercritical fluid also allows them to flow easily through small openings. Perhaps the most dramatic property of the supercritical fluid state is the absence of a surface meniscus. The supercritical fluid exhibits no surface tension properties. This combination of properties means that a supercritical fluid can penetrate a material as though it was a gas under high pressure but with the very important difference that it has solvation properties which approach those of a liquid. Hence these fluids may be very useful for use as phases in extraction and chromatography. The extractive properties of supercritical carbon dioxide have been exploited for many years on an industrial scale for the decaffeination of coffee and the removal

. -

E

ul

w

0.928

n

0 ._ U c 0

2

0

0.464

0

n v

.

7.38

8.3 Choice of Supercritical Fluids

73.8

738

165

Fig. 8-3. The phase diagram of supercritical and near critical CO,.

Pressure in bar

of nicotine from tobacco [3]. Their use in preparative chromatography however is more recent and limited. The solvating power of a supercritical fluid generally increases as its density increases. The mobile phase elution strength can be increased by increasing the applied pressure on the fluid to increase its density. The relationship between density and pressure of supercritical and subcritical carbon dioxide is shown by Fig. 8-3. Although supercritical carbon dioxide is a solvent of relatively low polarity, its solvation power varies considerably with density. The curves are particularly steep close to the critical point. For example, just above the critical temperature (31 .I " C ) a fivefold increase in pressure changes the density from 0.4 to 0.9 g mL-'. If the pressure is raised further, from 350 to 700 Bar the increase in density only changes from 0.9 to 0.98 g mL-'. Pressure can be an effective way to control solvent elution strength. In many cases, however, the range of solvation power available through density variation is not great enough. For these cases the addition of a few percent of a polar solvent such as methanol or acetonitrile to the supercritical carbon dioxide can modify its properties. The modifier solvent may render the carbon dioxide subcritical but it now combines the solvating power of the high density carbon dioxide and the polar nature of the added modifier.

8.3 Choice of Supercritical Fluids Many fluids have been investigated for use in supercritical fluid chromatography (SFC) and extraction (SFE). Table 8-1 lists the critical points of temperature and pressure for some of them. The physical and chemical properties of a supercritical fluid depend upon temperature and pressure and are of paramount importance when designing a supercritical fluid chromatograph or extractor. Excessively high pressure or temperature require-

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8 Scaling-up of Supercritical Fluid Chromatography to Large-Scale Applications

Table 8-1. Critical points for a selection of fluids.

Fluid

Critical temperature ("C)

Critical pressure (Bar)

Ethylene Carbon dioxide Ethane Nitrous oxide Propylene Propane Ammonia Hexane Water

9.3 31.1 32.3 36.5 91.9 96.7 132.5 234.2 374.2

50.4 73.8 48.8 12.1 46.2 42.5 112.8 30.3 220.5

ments could make the apparatus too expensive to be practical. Choice of fluid is particularly important. The most familiar solvent, water, is a poor choice for SFC or SFE from an engineering and safety standpoint. The critical parameters are among the highest for common solvents. Ammonia is particularly unpleasant to work with. A fume cupboard or other ventilation precautions are needed to keep it out of the laboratory atmosphere. It is also extremely corrosive and requires that certain parts of the chromatograph be gold plated to resist dissolution. Nitrous oxide has been used fairly extensively because it is quite polar and has reasonably accessible values of critical temperature and pressure. However, there is evidence from Sievers et al. [4] of violent explosive reactions between nitrous oxide and modifiers. The alkanes and alkenes pose fire hazards and are of low polarity. The most popular and safe supercritical fluid is undoubtedly carbon dioxide for use in SFC and SFE. Thermally labile materials chromatographed in it are unlikely to be affected because its critical temperature is only slightly above room temperature. The critical pressure of carbon dioxide (73.8 Bar) is not difficult to maintain with modern high performance liquid chromatography (HPLC) solvent delivery systems. Carbon dioxide has the additional advantages of being non-flammable, chemically inert, odour free, available in a state of high purity at a low cost and does not present a solvent disposal problem. It is beyond the scope of this chapter to review the extensive literature available for the use of analytical supercritical fluid chromatography and extraction. However it is important for the reader to appreciate the problems posed by scaling-up the analytical technique for preparative and process scale applications. The studies explored in this chapter will present the reader with a number of different approaches from a selection of key workers in this field to the solution of the scaling-up process. A choice can then be made by the reader of the approach that best suits his or her problem and resources.

8.4 The Scaling-up Process

161

8.4 The Scaling-up Process Analytical SFC employs much of the apparatus used in high performance liquid chromatography. The packed columns that are used are typically 4.6 mm internal diameter and from 150 to 250 mm in length. The usual micro-particulate packing materials such as 5 pm diameter spherical silicas and bonded silicas as well as polystyrene divinylbenzene copolymeric packings can be employed for analytical SFC. The quantity of packing inside an analytical column is typically a couple of grams. The loading of analyte for analysis purposes is usually very low indeed; typically a microgram per gram of packing. Small scale preparative work could be carried out on analytical columns up to a maximum loading of a few milligrams per gram of packing. Above this loading the resolution of the chromatograph can suffer and gross distortion of peak shape and variation in peak retention times will occur as the loading is progressively increased. Clearly a production rate of a few milligrams per run would be of interest to characterise a substance only, using other analytical techniques such as nuclear magnetic resonance spectroscopy (NMR), infra-red spectroscopy (IR) and mass spectrometry (MS) or to produce a limited quantity of a primary analytical standard. Isolation of larger quantities of material by preparative SFC demands the use of larger columns than those used in analytical SFC. Columns of 150 to 250mm in length packed with 5 pm diameter micro-particulate silicas can generate 10000 theoretical plates which is adequate for the majority of separations. There is not much to be gained therefore, by increasing the length of the column unduly. The loading of the column is increased by employing more packing in the column, thus it is the column diameter which must be increased for preparative purposes. A doubling of the column diameter in a fixed length column would increase the column volume by a factor of four. Hence by doubling the diameter the quantity of packing that can be accommodated can be increased by four and consequently the loading can be increased by a factor of four. Standardised columns supplied principally for HPLC are commercially available in a number of dimensions (Table 8-2). High quality chromatographic packing materials for HPLC and SFC are expensive. The cost of filling a process scale column with a micro-particulate-bonded silica could be over fi 15000. Table 8-2 shows columns whose diameters have increased from 4.6 to 150 mm, a factor of over thirty times. The loading capacity has increased from a few milligrams per run to several grams per run on the process scale column. The actual loading that can be achieved on a particular size of column will be highly dependant upon the extent of separation of the peak of interest from the peaks of undesired components. If this separation is large the column may be loaded at considerably higher levels than those indicated in Table 8-2. There are examples in the literature where columns are loaded to the extent of greater than 10 mg loading to 1 g of packing. In supercritical fluid chromatography the limit of loadability is more likely to be a limit imposed by the solubility of the sample in the supercritical fluid rather than the capacity of the column for the quantity of substance applied.

168

8 Scaling-up of Supercritical Fluid Chromatography to Large-Scale Applications

Table 8-2. Column dimensions and packing requirements for scale-up of SFC from analytical to process scale operation. ~

Column

Standard analytical Semi-preparative Preparative Large preparative Pilot scale Process scale

Diameter (mm) 4.6 10

22 44 80 150

~

---

Nominal imperial equivalent (inches)

Column volume (mL)

Packing required (dry weight, g)

1/4 1/2 1 2 4 6

4.2 19.6 95 380 1250 4400

3 14 61 266 815 3080

--

Column volume calculated by assuming a standard length of 250 mm. A packing density of 0.7 g m t - ' has been assumed for the caIculation of the weight of dry packing required to fill these columns. Naturally, this density will vary from packing type to type and must be calculated individually for the particular column desired.

In order to achieve a comparable retention time when scaling up from analytical or semi-preparative to larger systems the mobile phase flow rate must be increased in proportion to the increase in column area or column diameter squared. If an analytical system of 4.6 mm column diameter is operated at a mobile phase flow rate of 1 mL min-', a 22 mm diameter column would need to be run at a flow rate of about 23 mL min-l in order to maintain the linear flow velocity constant. This preparative flow rate was calculated by applying Eq. (1).

where

D, = preparative column diameter DA = analytical column diameter A = analytical flow rate P = preparative flow rate Application of Eq. (1) indicates that the predicted flow rates for process scale columns of say 150 mm diameter will be very large indeed. Scale-up from an analytical column at 1 mL min-' to a 150 mm diameter process column requires a flow rate of over 1 L min-l to maintain a constant linear flow velocity. The pumping of supercritical fluids at high flow rates and high pressures requires the use of specialised solvent delivery systems, whereas for the analytical and preparative scales a modified HPLC solvent delivery system will prove adequate. Fortunately the back pressure (resistance to flow presented by the packed column) is inversely proportional to the square of the column diameter. Hence, by increasing the column diameter from 4.6 to 150mm, a pressure reduction factor of over one thousand times would be expected. However, in order to maintain a proportional linear velocity of mobile phase through the larger column, the back pressure will remain

8.5 The History of Preparative SFC

169

more or less constant. It is very important to increase the diameter of all the connecting tubing in the system when scaling up from analytical levels. Failure to do this will result in large pressure drops across lengths of relatively narrow tubing and it may be found that the major source of back pressure is from the connecting tubing and fittings rather than from the chromatographic column packing. Typical inner diameter of tubing on analytical systems range from 0.18 to 0.25 mm, which are too narrow for large scale preparative systems. Supercritical fluids have flow properties which are similar to gases under high pressure and hence they can flow fairly easily at high velocities through relatively narrow tubing. The pressure drop across a packed chromatographic column is considerably less for a supercritical fluid than for a liquid. For example, a typical pressure drop across an analytical column (4.6 mm i.d. x 250 mm) filled with micro-particulate silica when operated with supercritical carbon dioxide at 266 Bar (4000 psi), 50 "C, at a flow rate of 5 mL min-' is about 20 Bar. If these conditions were adopted using an HPLC mobile phase such as methanol/water the pressure drop would be ten times the SFC figure. It is advantageous to have a low pressure drop across an SFC column because the solvating power of the fluid, and consequently its eluting strength, is proportional to its density which in turn is proportional to the applied pressure. To obtain reproducible chromatography the elution strength should remain constant along the length of the column. If the pressure differential is large the solutes may precipitate from solution as the pressure and hence density of the fluid falls. With packed columns of the usual 250mm length this is not often a problem. It is prudent to use columns of a length that just satisfies the requirements of the separation and no more in process scale SFC because the micro-particulate packings are so expensive.

8.5 The History of Preparative SFC The possibility of preparative separation was suggested at the very beginning of SFC development by Klesper and coworkers [5] who stated in their first report that porphyrins could be recovered at the outlet valve. Klesper uses the terms high pressure gas chromatography above the critical temperature to describe their investigation, the term supercritical fluid chromatography had not been coined at this time. Ten years later Jentoft and Gouw [6] reported a sophisticated SFC system that allowed fractionation of components. A turntable holding six glass collection tubes was enclosed inside a high pressure vessel which could be pressurised with nitrogen gas. The high pressure maintained the carbon dioxide containing solutes in liquid or liquid like states depending upon the temperature. The column effluent containing a peak component was collected in a tube under high pressure. After collection, the pressure in the vessel was reduced slowly to atmospheric pressure, leaving only the solutes in the glass tubes. In 1977 Hartman and Klesper [7] described an SFC system with an open air fraction collector that was designed to be used with a low boiling point organic solvent. The authors were attempting to separate styrene oligomers preparatively by SFC.

170

8 Scaling-up of Supercritical Fluid Chromatography to Large-Scale Applications

In 1986 Perrut and Jusforgues [8] reported a preparative SFC system equipped with a 60 mm i.d. column and high pressure collection vessels which were selected by computer controlled valves placed between the separation column and the vessels. In order to reduce fluid consumption the mobile phase fluid was recondensed back to the delivery pump after separation of the solutes. Saito et al. [9] have developed specialised apparatus for preparative SFC. They describe a novel pressure regulating system for constant mass flow SFC and carry out a physico-chemical analysis of mass flow reduction in pressure programming by using an analogous circuit model. These Japanese workers have published many applications of the SFC technique to complex separation problems. For example Yamauchi and Saito [ 101 used semi-preparative supercritical fluid chromatography to fractionate lemon-peel oil and Saito and Yamauchi [11] isolated tocopherols from wheat germ oil by recycle semi-preparative SFC. Supercritical fluids have been used to extract and then chromatograph eicosapentanoic acid and docosahexaenoic acid esters from esterified fish oil by employing programmed extraction-elution with supercritical carbon dioxide [ 121. In spite of the significance of the publications cited the technique of preparative SFC has received relatively little attention when compared with analytical SFC. The method has the potential capability of replacing normal phase preparative HPLC because when coupled with supercritical fluid extraction (SFE) it carries out the extraction, preconcentration and chromatographic fractionation in a single run according to Saito et al. [13]. The use of supercritical carbon dioxide allows easy separation of solutes at low temperature in an oxygen free environment, which is essential for the separation of labile compounds such as tocopherols [131. In additions a non-flammable, non-polluting and inexpensive mobile phase is very helpful for safety and economy in the laboratory and in a production process. At Glaxo the authors have found that analytical SFC is about five times faster than HPLC to achieve the same separation efficiency and chromatographic peaks are very sharp [14]. Re-equilibration of the column following changes in mobile phase composition is also more rapid than for HPLC which allows for very rapid method development. It is perhaps surprising therefore that given this catalogue of advantages the technique has not been adopted widely and developed more intensively and comprehensively. This is probably explained by the success of HPLC is being able to perform the majority of separations adequately, rather than any serious limitations of SFC. Commercial HPLC instrumentation is widely available and highly developed whereas SFC instrumentation is in its relative infancy. At Glaxo a number of chromatographic techniques are employed to characterise and purify novel drug candidate compounds from our synthesis laboratories. Occasionally a separation is required that is not satisfactorily achieved by HPLC or GLC and it is here that SFC has found a niche. Analytical SFC has been in use at Glaxo since 1986 and has become indispensable as a sample introduction technique for mass spectrometry (SFCMS). The development of preparative SFC started in 1988 with specialised apparatus constructed on site to build a preparative chromatograph that incorporates some novel solutions to sample introduction and collection problems. The construction of the preparative supercritical fluid chromatograph designed and built by the authors

8.6 Safety Considerations

-

The Coluinn Shield Jacket

I7 1

will be described in some detail in this chapter and its features compared and contrasted with other published systems. The ongoing development of preparative SFC at Glaxo has been reported elsewhere by the first author [14- 161, but these have been as lectures or poster presentations. This chapter reports all of the development work to date in a detailed form that can be compared alongside the developments of others.

8.6 Safety Considerations - The Column Shield Jacket Preparative HPLC columns are designed to operate routinely at hydraulic pressures of several hundred Bar. If a failure occurs in the tubing or the fittings at either end of the column during HPLC operation, the result would be an undramatic leak of mobile phase. However, if these columns are used with supercritical fluids at high pressures, the loss of the column end fittings or a fracture of the tubing would result in an explosive discharge of gas and column packing material. An enormous amount of energy is stored in the supercritical fluid which must be contained safely. For this reason the preparative chromatographic column is contained within a shield jacket. The jacket is constructed entirely from stainless steel and has been hydraulically tested to withstand an internal pressure of 600 Bar (9000 psi) applied almost instantaneously. The shield jacket shown in Fig. 8-4 can comfortably accommodate a

Fig. 8-4. Column shield jacket.

112

8 Scaling-up of Supercritical Fluid Chromatography to Large-Scale Applications

22 mm x 250 mm preparative HPLC column and was designed by the authors in consultation with Dr. Swales of the Mechanical Engineering Department of University of Leeds (UK) and built by Lancashire Fittings Ltd, Harrogate, Yorkshire (UK). In the event of a column failure the presence of pressure inside the shield jacket can be checked by unscrewing a small orifice vent valve at the top of the jacket before dismantling the jacket. The simple vent valve was chosen in preference to a pressure gauge, as an indicator of internal pressure because the tolerance of a pressure gauge to an explosive build up of pressure was unknown. Unfortunately the heavy mass of steel jacket does mean that it takes at least 1 h for the column ro reach operating temperature. The large thermal mass of the jacket ensures that short term temperature fluctuation is damped out.

8.7 The Basic Chromatograph The basic chromatograph around which the preparative scale-up modifications have been made is manufactured by Gilson. An hydraulic diagram of the system is shown in Fig. 8-5. The original system employed a TescomT” needle valve as a back pressure regulator to maintain high pressure within the system. If was found that such a valve proved to be unreliable if it was necessary to change the pressure frequently in the system by adjustment of this valve. The internal volume of this valve is large which made post-valve sample collection very difficult as the valve’s volume is a significant fraction of the column volume and contributes to extra column band broadening. It is essential to have a pressure regulating valve which has a small internal volume and has the capability of controlling the pressure independently of mobile phase mass flow rate. In addition to these features, it is desirable to heat the effluent stream to prevent blockages by solid particles of carbon dioxide and precipitated solutes caused by the self cooling of the fluid as it expands after depressurisation. The back pressure regulation on the latest system is by means of an electronically controlled valve described by Saito et al. [9] and manufactured by the Jasco Corporation of Japan. This device has proved to be much more reliable than the manually operated valve and has a very small internal volume.

8.8 Loading and Injection of Samples One area of preparative SFC that would benefit from further investigation is the sample injection technique. With the exception of on-line extraction/chromatography, the sample is usually introduced as a solution in an organic solvent and injected onto the column by means of a loop rotary valve. The sample loop is then flushed with a high density liquid that eventually becomes the supercritical fluid mobile phase on entering the heated column.

8.8 Loading and In.jection of Samples

113

Mixer

v2

1-1 [cooler

1

Restrictor

'I '

(1 of 12)

I

UV detector

Fig. 8-5. Hydraulic diagram of the preparative SFC system built at Glaxo.

The peak band broadening that arises from the transition from mixed solvent liquid to supercritical mobile phase is similar to the effect observed in HPLC when the solvent used to dissolve the sample has a higher elutropic strength than the mobile phase [17]. One part of the solute moves through the column along with the injection solvent that is only retained slightly and peak distortions may be observed such as leading edged peaks or double peaks. The volume of sample that can be introduced without flooding the column and thereby causing peak distortion is limited. For example, Cretier et al. [18] quote 1OpL as a typical injection maximum on a 4 mm x 150 mm packed column. The use of SFC on a preparative scale requires the introduction of relatively large volumes. It is desirable to minimise the quantity of foreign solvent present before sample injection. A number of investigators have sought novel solutions to this problem. The methods which should be appropriate for application to preparative SFC comprise the following. Cretier et al. [I81 describe an injection technique whereby the sample solution is passed from the sample injection loop to a packed pre-column. The solvent is then

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8 Scaling-up of Supercritical Fluid Chromatography to Large-Scale Applications

removed from the pre-column by passage of warm helium gas. Finally the carbon dioxide mobile phase is introduced and dissolves the solutes which have been deposited on the pre-column and focuses them onto the top of the preparative column. In this way the solvent can be removed completely and the amount of sample injected significantly increased. In practice the volume and consequently the amount of sample that can be injected are limited by the volume of the pre-column. However, the loading and venting cycle can be repeated a number of times before carrying out the elution with liquid carbon dioxide and the final preparative separation. When the volume of injection solvent is small, the dilution with the supercritical fluid mobile phase leads to conditions of elution for the solutes which are almost identical to the conditions of elution with the supercritical fluid itself. As the volume of injection is increased there is less dilution of the bolus of solvent by the mobile phase and the solvent acts as a second eluting mobile phase for part of the solute, giving rise to an adjacent earlier eluted peak. The amplitude of the band broadening depends on the nature of the sample solvent. Cretier et al. [18] show that the helium flow can be optimised for complete removal of the injection solvent and minimisation of sample loss. They also demonstrate that above an injection volume of 50 pL there is a loss of the sample during the precolumn loading step. In order to increase the injected amount without exceeding the volume capacity of the pre-column it is possible to load several plugs in succession with each plug kept smaller than 50 pL, the solvent being removed after introduction of each plug. Although the work of Cretier et al. [18] demonstrates convincingly the need to remove the foreign injection solvent, their experimental work was performed on an analytical scale chromatograph using a 2 m m x 3 0 m m pre-column and 4 mm x 150 mm main column. Extrapolation of these findings to the dimensions of a conventional preparative chromatograph (22 m m x 250 mm and larger columns) would need to be confirmed by further experimental work. The solutes used for the tests were dilute solutions of anthracene and pyrene. The solubility of the solute in the supercritical fluid could present a problem which would ultimately limit the amount of compound which could be chromatographed. The pre-column concentration technique whilst overcoming the problem of solvent removal does not guarantee that the solute of interest will redissolve rapidly and completely in the supercritical mobile phase. For good chromatography this is a pre-requisite. In practical terms of course, if the chromatographic separation of a desired compound from other compounds is large (a large a separation factor) the effect of peak broadening is not so important, so that provided the peak of interest can be isolated from other peaks its shape is immaterial. Unfortunately in many real life situations SFC is applied where other chromatographic methods have failed to provide sufficient resolution. Separation of complex mixtures into their components requires that the full potential of the separation technique be utilised. For these situations judicious choice of sample introduction conditions, solvent, solubility, etc. must be made. One particular advantage of using SFC is that it can generate a higher theoretical plate count per unit time than HPLC. This means that multiple chromatographic runs using small injections for each run could be used to separate complex mixtures in a small space of time.

8.9 Design and Contruction of the Sample Introduction Pressure Vessel (SIPV)

115

8.9 Design and Construction of the Sample Introduction Pressure Vessel (SIPV) The problem of injecting large volumes of solvent onto the chromatograph was recognised by the authors early in their development of the preparative supercritical chromatograph at Glaxo. Many of the problems have been overcome by the design of a sample introduction pressure vessel (SIPV) which replaces the loop valve injector in the chromatograph. It is a generally accepted rule in HPLC that samples should be dissolved in the mobile phase prior to injection. This is to ensure that the sample remains in solution at the head of the column and that the process of partitioning of the sample between mobile and stationary phases is left undisturbed by the presence of a solvent that may be of different polarity or elutropic strength to that of the mobile phase. As Cretier and other workers [I81 have found, failure to observe this practice may often result in distortion of peak shape resulting in degradation of resolution and reproducibility. At worst it can result in a complete precipitation of the compound in the tubing and frits at the head of the column with consequent blockage of the system. In HPLC it is a simple matter to ensure that the solubility of the sample in the chosen mobile phase is adequate. In analytical SFC the injection of small amounts of foreign solvents can often be tolerated provided that the solvent chosen is of a similar polarity to the mobile phase. Furthermore, in analytical SFC high solubility of the analyte in the injection solvent and mobile phase is not so important because much lower concentrations are employed, whereas in preparative SFC this loading can represent a primary limitation on the production rate of the purified compound. The compounds that require purification at Glaxo are novel and therefore their solubility in supercritical mobile phases is unknown. Indeed the data on solubility of compounds in supercritical carbon dioxide and other less popular fluids is very sparse indeed. The choice of polarity modifying solvents that can be used with supercritical carbon dioxide will also depend on the miscibility of such compounds with the fluid at the various temperatures and pressures employed in the chromatographic process. Clearly, immiscibility of mobile phase solvents must be avoided at all costs during a separation. The best way to ensure that the sample has dissolved completely in the chosen mobile phase and that the resulting solution is a single phase, is to observe the process directly before chromatography. In order to do this the authors designed and built a pressure vessel with observation ports incorporated into it so that the sample dissolution process in the mobile phase could be carried out under identical conditions to those employed for the chromatographic separation. The operational requirements for such a vessel place severe demands on the properties of the materials employed in its construction. The vessel has to withstand very high pressures and elevated temperatures and successfully contain supercritical carbon dioxide with modifiers such as methanol, ethanol or acetonitrile. The fluid must be easy to see and its pressure, temperature and composition easy to measure and control. Solid and liquid samples should be easy to introduce into the vessel and their mixing with the mobile phase fluid be facilitated. Above all the vessel must be designed and con-

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8 Scaling-up of Supercritical Fluid Chromatography to Large-Scale Applications

Fig. 8-6. Sample introduction pressure vessel (SIPV) and its associated heater.

structed so that it can be used safely in a laboratory environment. The sample introduction pressure vessel (SIPV) is pictured in Fig. 8-6 and the elements of its construction shown by Fig. 8-7. The volume of the chamber is 10 cm3 which should be adequate for the dissolution of most samples that require chromatography on a conventional 22 mm x 250 mm preparative HPLC column. The observation port hole windows are constructed from 32 mm thick toughened Pyrex@glass discs. Each window comprises two discs each 16 mm thick. This laminated construction was chosen in preference to a single 32 mm thick disc to reduce the risk of fracture propagation through the glass. The tensile strength of glass is dependant upon its history. Surface scratches can lower the strength of the supercooled fluid dramatically even with toughened glass. Lexan@polycarbonate blast shields have been fitted above the windows to prevent accidental scratching of the glass and to contain glass splinters being ejected into the laboratory in the unlikely event of an explosion. The sides of the vessel are protected by enclosure in a PVC pipe. The 10mL chamber is fabricated from stainless steel (304 grade) with the glass port hole windows sealed onto the chamber ring with ‘0’rings made from Kalrez@ high tensile strength fluoropolymer. Kalrez@ was selected after trying Teflon@and gold as ‘0’ring gaskets. A gold ring burst at 200 Bar under test and Teflon@leaked badly at about 20 Bar pressure. The inlet and outlet ports have been set at right angles to each other to induce a swirling flow in the chamber. The inlet and outlet severe service bonnet valves were chosen from the Whitey range (SS3NBS4) to withstand carbon dioxide at up to 400 Bar at up to 100°C. The design of these valves using a ball and seat prevents them from being damaged by over tightening. The fluids contained within the vessel can be heated by means of a 25 W cartridge heater with temperature control via a type T thermocouple.

8.9 Design and Construction of the Sample Introduction Pressure k s e l (SIPV)

177

Fig. 8-7. Sample introduction pressure vessel (SIPV), elements of its construction.

The in-line window arrangement ensures that the fluids in the chamber can be seen very clearly and can be illuminated with normal or polarised light if desired. The disappearance of the meniscus at the surface of the liquid carbon dioxide as it becomes a supercritical state is very easy to see with this instrument. In spite of its rugged construction, the vessel is pressurised and depressurised slowly in use to prevent undue stress on the components. Pressure testing to 400 Bar (6000 psi) with water and with carbon dioxide is performed on a regular basis to satisfy laboratory safety requirements and to minimise the danger of unexpected failure at lower pressures. The M 12 full nuts are tightened in the order shown from 1 to 10 using a torque wrench to 13.5 Nm (10 lbf ft) so that the load is distributed equally on the steel studs. This design ensures that samples come into contact only with fairly inert materials such as stainless steel, glass and Kalrez@. Other important safety precautions adopted comprise the following. The vessel is not left at an elevated pressure for excessive periods of time and certainly not for longer than 8 h. This allows the glass sufficient recovery time during the night to prevent stresses building up, and reduces the destructive effects of explosive decompression from within the Kalrez@‘0’rings. The safe working pressure of 400 Bar is relevant only at room temperature. A derating factor must be applied for operation at 50 “ C so that the safe working pressure becomes 370 Bar (5400 psi). When viewing directly through the glass, a full face shield should be worn. Fingers and other parts of the body should be kept well away from the area around the

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8 Scaling-up of Supercritical Fluid Chromatography to Large-Scale Applications

‘spider’s’ arms where they emerge from the PVC shield to avoid pressure blast in the event of the glass fracturing. If the apparatus is dismantled, new Kalrez@‘0’rings are fitted, together with new silicone rubber gaskets. The tightening torque on the nuts are never guessed, they are always set with a torque wrench, by increasing the torque gradually in a number of stages up to the present limit. The SIPV replaces the Rheodyne 7125 loop injection valve in the chromatograph shown in Fig. 8-5 when it is desired to dissolve samples in carbon dioxide mobile phases. The SIPV overcomes most of the problems encountered when trying to chromatograph relatively large volumes of sample in supercritical carbon dioxide. This facility allows the chromatograph to be used in a very flexible manner at the preparative scale.

8.10 Collection of Fractions from the Preparative Supercritical Fluid Chromatograph The collection of eluent fractions from a conventional preparative HPLC is a trivial matter. Provision of automated fraction collectors suitable for HPLC is no commonplace and instrumentation is highly developed and widely commercially available. However, collection of eluent from a preparative SFC is not trivial, particularly if complete collection of a fairly volatile solute is required. In a supercritical fluid chromatograph using carbon dioxide the expansion of the fluid at depressurisation is very large indeed. If carbon dioxide is depressurised from 300 Bar (4500 psi) to atmospheric pressure is expands to 355 times its original volume if its temperature is maintained constant. Hence at a flow rate of 10 cm3 min-l of supercritical carbon dioxide at 300 Bar, which corresponds to about 7 g carbon dioxide per minute, expansion after depressurisation yields 3.5 L of carbon dioxide gas per minute at NTP. On expansion the gas cools rapidly (Joule-Thompson effect) and tends to block the back pressure restrictor valve (particularly when the simple manual valves are used) with solid carbon dioxide and precipitated solutes. A number of workers have attempted to collect solutes from carbon dioxide solution after the restrictor valve and some have reported the difficulties they experienced and the poor quantitative recovery of solutes, in particular volatile solutes [19-211. Saito and Yamauchi [I91 attempted to collect tocopherols from wheat germ by preparative SFC and reported 30 to 50% recoveries which is unsatisfactory. They concluded that the compounds were probably removed by the mist of ethanol, used as modifier solvent, in the vent line and suggested that use of a cold trap might help. The system used by these Japanese workers incorporated the Jasco electronic back pressure regulator described by Saito et al. [9] which allows for collection at atmospheric pressure but does not solve the problem of non-quantitative recovery. Although there have been useful reports on preparative SFC by Jentoft and Gouw [22], Hartman and Klesper [7], and Perrut and Jusforgues [8] the technique has still

8.10 Collection of Fractions .from the Preparative Supercritical Fluid Chromatograph

179

MeOH

1

Flushed restrictor

380/o

acetophenone recovered

Heated system and sinter

20.5'10

acetophenone recovered

Heated system and flushed sinter

41 .50/o

acetophenone recovered

Fig. 8-8. The recovery of acetophenone from a carbon dioxide/methanol mobile phase by dissolution into methanol.

not been widely accepted mainly because fractions are collected in high pressure vessels when a fluid such as carbon dioxide is used. In order to obtain the fractions, the operator needs to wait until the last fraction has been collected and then the pressure in the vessel must be reduced to atmospheric pressure. Fractions cannot be dealt with as easily as those produced by preparative HPLC. In addition the safety requirements of collection at high pressure must be considered thoroughly. The simplest method for collecting solutes is to bubble the carbon dioxide eluent through a trapping solvent. The authors have investigated this method using acetophenone as a suitable test probe. Examples of the various solvent trapping configurations are displayed in Fig. 8-8 together with the results which demonstrate unequivocally that this approach is far from quantitative. In the examples given, the eluent from the preparative column is passed through a Rheodyne@7037 pressure restriction valve to maintain supercritical conditions. The carbon dioxide-methanol eluent is then allowed to expand to atmospheric pressure post restrictor. Recovery of the acetophenone from the carbon dioxide-methanol stream was attempted by simply flushing the Rheodyne@valve with methanol and collecting the compound in a 100 mL volumetric flask. This method showed very poor recovery,

1 80

8 Scaling-up of Supercritical Fluid Chromatography to Large-Scale Applications

38% at best. A second attempt at recovery by introducing a stainless steel sinter immersed in methanol in a 100 mL volumetric flask with and without heating of the lines gave even lower recoveries of the acetophenone. The highest recovery of acetophenone from the 5 mL min- carbon dioxidemethanol stream was achieved by trapping it in methanol using a flushed sinter with heated lines, yet this amounted to only 41.5%. The mass transfer of the acetophenone from the eluent to the trapping solvent is the limiting step since at mobile phase flow rates of less than 1 mL min-' recoveries are much higher. These simple experiments show that to quantitatively and reproducibly recover acetophenone from a fast flowing carbon dioxide-methanol eluent a more sophisticated method is required.

'

8.11 High Pressure Trapping with Subsequent Recovery by Solidification of the Carbon Dioxide Clearly the major problem in isolating solutes from supercritical carbon dioxide is the large increase in volume which occurs at depressurisation. A potentially attractive but technically more complex solution to this problem which avoids the rapid depressurisation is to isolate the solute under supercritical or almost supercritical conditions. In the author's system, shown by Fig. 8-5, the peak of interest is diverted from the main steam into a pressurised collection vessel where the mobile phase fraction can be isolated. Carbon dioxide undergoes a phase change from the liquid to the solid state at -56°C (see phase diagram for COz, Fig. 8-2). Hence, by cooling the pressurised collection vessel to below - 56 "C the carbon dioxide is solidified and the high pressure reduced to atmospheric pressure. The solid carbon dioxide may then be removed from the vessel together with the entrapped solute. The solid carbon dioxide may then be allowed to sublime away to leave the desired solute behind. The advantages of this approach are that loss of solute should be minimal and precipitation and clogging of the pressure restrictor is overcome completely. The internal volume of the restrictor is also of no consequence. The disadvantage is that a high pressure collection vessel must be designed and engineered. Initial collection of acetophenone and of various coloured dye tracers indicated that when using a stainless steel cylinder (10mmx250mm, 20mL volume) as the collection vessel the total volume of the cylinder was not available for the solute. The dye experiments indicated that about half the total volume was available for collection of the solute. The reasons for this are as follows. The Hagen-Poiseuille equation, which describes the velocity profile of a liquid flowing through a tube under laminar flow conditions predicts that the maximum velocity of the head of the parabolic flow profile will be twice the mean velocity of the liquid [23]. Breakthrough of the dye tracer would therefore be expected in about half the volume of the tube. Fluid mechanics predicts that if the flow is turbulent the difference between the mean and maximum flow velocities is much less than in laminar flow [23]. This means that un-

8.11 High Pressure Trapping with Subsequent Recovery

181

the positions shown

MI0 nut (lock) optional

1 turn increases/decreases volume bv l m l

Fig. vessel.

Experimental sample collection

der turbulent flow conditions more of the volume of the tube would be available for collection of the desired eluent than under laminar flow conditions. It is obviously desirable to utilise the volume of the collection vessel efficiently so methods for inducing turbulent flow were investigated. An experimental collection vessel was designed and built to investigate the effect of incorporating baffles into the collection vessel. This vessel is shown in Figs. 8-9 and 8-10. The size and spacing of the baffle discs could be optimised using this apparatus by employing a coloured dye in a stream of methanol to simulate the flow conditions experienced in the chromatograph. The flow patterns generated by the dye tracer allowed for an optimum design of collection vessel to be deduced. The series of photographs reproduced in Fig. 8-11 show the passage of a slug of black dye passing into and out of the experimental collection vessel. The effect of the baffle discs on the flow profiles can be seen clearly in this series. These experiments were conducted at a flow rate of 5 mL min-I. It is obvious from these photographs that much of the available volume of the collection vessel (60 mL) is being occupied by the dye.

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8 Scaling-up of Supercritical Fluid Chromatography fo Large-Scale Applications

Fig. 8-10. Experimental sample collection vessel and trial baffle discs.

A version of the baffled collection vessel was fabricated from stainless steel for use at high pressure. This vessel is pictured in Fig. 8-12 and its construction detailed by Fig. 8-13. The sample collection vessel has been constructed to withstand a working pressure of up to 400 Bar (6000 psi) and contain up to 60 cm3 of fluid. The internal baffle discs allow the vessel to fill up from the bottom without too much mixing in order that much of the available volume is used. Cooling of the vessel by immersion in liquid nitrogen is facilitated by locating each vessel in its individual PVC case. The base of each vessel is slotted to fit over a key bar at the base of the PVC case. The arrangement detailed in Fig. 8-14 allows for the top of the vessel to be removed without touching the very cold vessel’s body. The solidified carbon dioxide slug can then be drawn out of the vessel using the inlet tube and disc assembly. The carbon dioxide can then be allowed to sublime off to leave the solute as an uncontaminated and, if required, dry state by use of a dissicator. Up to twelve of these specially designed vessels can be connected in parallel to each other by switching valves as shown in Fig. 8-5. Thus in the authors’ system individual fractions of eluent may be trapped in the appropriate vessel and complete recovery of the solutes assured. Even volatile solutes such as acetophenone can be recovered in very high yield. Portions of the eluent that do not contain peaks of interest can be diverted via the Rheodyne 7000 valve (Fig. 8-5) to pass through the back pressure restrictor to waste. This system ensures that the pressure in the whole system can be controlled by the back pressure restrictor and the fractions can be isolated individually without having to wait for the last fraction to be collected. The absence of pressure changes on collection ensures that the density and therefore the solvating

Fig. 8-11. Collection of dyestuff in the experimental collection vessel.

Fig. 8-12. The sample collection vessel (open).

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8 Scaling-up of Supercrifical Fluid Chromatography to Large-Scale Applications

0INPUT

L K e y bar

Fig. 8-13. The SFC sample collection vessel.

power of the carbon dioxide mobile phase remains constant. In this system solutes do not precipitate from solvent when fractions are collected and pressure control is precise. Cooling of the collection vessels minimises the dangers of venting high pressure liquids and completely obviates the need for adsorption columns to trap solutes. Veisserik et al. 121, 241 report on the possibilities of using conventional preparative HPLC instruments for preparative SFC and have arrived at similar conclusions to those of the authors. The Estonian’s designs do not have collection vessels that are cooled however, and we have found that for complete recovery of volatile solute this solidification of the carbon dioxide is an important step.

-

8.11 High Pressure Trapping with Subsequent Recovery

MI2 Fullnut

a

SFC SAMPLE COLLECTION VESSEL

INLET 0

0

6-Off each M I 2 was her

20 40 60 80 100 120

Scale rnrn

0OUTLET

P.V.C. locating box

socket head CA

stainless steel grade 304 U

Fig. 8-14. Sample collection vessel and PVC locating box.

185

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8 Scaling-up of Supercritical Fluid Chromatography to Large-Scale Applications

8.12 Development of Large Scale Commercial Systems Preparative SFC would appear to be a promising method of producing fractions free from solvent contamination and at first sight if may appear surprising that there have been so few reports on semi-industrial scale SFC separations. In practice it turns out to be very difficult to overcome many of the problems outlined so far in this chapter. Eluent re-cycling, eluent-product separation, periodic injection of feed stock, fraction collection and column technology each present difficulties which although manageable at the laboratory scale become much more crucial at the process scale. Commercial systems have to operate reliably and regularly at a cost which is economically attractive to the user. These severe constraints may not apply quite so rigorously to a laboratory based research instrument. It is worth mentioning the work of Khosah [25] at the laboratory scale on elution with a supercritical fluid of compounds adsorbed on a porous material. Khosah discussed the technical and economic feasibility of the process at the pilot plant scale. Alkio et al. [26] published some results obtained with a preparative SFC unit built by conversion of a supercritical fluid extractor. Columns of 0.3 to 2 L could be mounted in this unit and be swept at flow-rates of up to 8 kg h-' of liquid carbon dioxide. However, both these devices suffer from a lack of automation and operating experience. Industrial development of these systems in the near future is unlikely. Berger and Perrut [27] report the promising results obtained in their laboratory since 1982. They have built a fully automated pilot unit, which can accommodate columns of internal diameter up to 60 mm and lengths of up to 1 m. The eluent can be totally recycled and its flow rate through the column can reach 50 kg h-I. These developments have been patented for commercial realisation in France, the United States and Europe respectively by Perrut [28]. A general scheme outlining the principles of the separation process is shown in the publication by Berger and Perrut [27]. Their initial studies were carried out with a synthetic mixture of naphthalene derivatives to demonstrate the feasibility of the process, its reliability and the stability of the hydrodynamics. Some technical difficulties regarding eluent-product separation and column efficiency were encountered and led to the design of high performance separators [29], and the adaptation of dynamic axial compression (DAC) to preparative SFC [30]. In order to widen the field of preparative SFC to more polar molecules an investigation of the addition of modifier solvents was carried out [31]. Several industrial scale separations were conducted by Berger and Perrut which demonstrated that preparative SFC could be a serious competitor to HPLC in the future for certain types of separation [32]. Commercial development of the Nancy industrial scale prototype has been carried out by Prochrom SA [33] and launched at the 7th International Symposium on Preparative Chromatography (Prep-90) at Ghent, Belgium in April 1990. Since its launch in 1990 at Ghent a number of units have been sold and the technology has raised a degree of excitement and interest from several companies. To the authors' knowledge the Prochrom system is the only fully automated industrial scale supercritical fluid chromatograph available for installation. The principle of operation of the Pro-

5.12 Developiient oj Large Scale Comiiercial S y s t e m

1 87

Fig. 8-15. The Prochrom process scale supercritical fluid chromatograph. (Photographed at the Prep-90 Exhibition, Gent, Belgium.)

chrom Super C 100 supercritical fluid chromatograph (pictured in Fig. 8-15) is explained by Jusforgues [34] in detail as part of Chapter7 of this book. Development of the Prochrom Process Scale SFC series, from the initial Super C 100 has involved the company in considerable investment. Customers who wish to apply this technology will also have to invest heavily in plant, training and a suitable serviced room for housing the unit. This investment couId well exceed three quarters of a million dollars and would be a major investment even for a large company. This high capital outlay would need to be offset against factors such as lower operating costs, higher quality product, ease of operation perhaps by virtue of computer control with the inherent saving in labour, and a long reliable lifetime so that the cost could be spread over a long period. We anticipate that the use of process scale SFC for the production of multi-kilogram quantities of product will be restricted primarily to high value products such as highly potent (low dose) pharmaceuticals whose annual production would be measured in kilograms rather than in tonnes. The extraction and isolation of specific highly active natural products could be another area where exploitation of the technique at the process scale could prove cost effective. Prochrom have compared the cost of isolating components from mixtures by process scale SFC and by HPLC. The recycling of carbon dioxide in the SFC instrument is a major cost saving factor in the comparison and coupled with the virtu-

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8 Scaling-up of Supercritical Fluid Chromatography to Large-Scale Applications

ally zero environmental impact of the process (no noxious solvents to dispose of) make the supercritical fluid option an attractive one for certain applications. Unlike preparative GLC, involatile thermally labile compounds can be treated, and unlike preparative HPLC, the products can be recovered free from solvent and thus be directly usable for tests, reactions or final use. Coupling of supercritical fluid extraction and preparative SFC make it possible to produce highly purified substances without any contact with conventional organic solvents, which could be of great interest to some parts of the pharmaceutical industry. Sometimes of course the addition of a modifier (co-solvent) is required for the elution of polar compounds and this may limit the use of preparative SFC for fractionating these compounds although solvent removal is usually easier and cheaper than it is in HPLC. Experience with analytical scale SFC has shown that a number of diverse modifiers, from the simple alcohols to ion-pairing and reverse micellar reagents, may be added to supercritical carbon dioxide in order to widen the scope of substances that can be separated by SFC. Many of these techniques could be scaled-up for preparative use. At the laboratory scale the use of preparative SFC will probably be restricted to separations that cannot be conveniently carried out by preparative HPLC. However, there are reports of chiral separations for example, where the selectivity obtainable from SFC is superior to that of HPLC [35]. The impact of legislation to control and limit the use of solvents that cause ozone depletion and the rising cost of solvent disposal could well help to tip the balance in favour of the choice of carbon dioxide but equally could limit the use of supercritical freons [36].

8.13 Detection of Solutes in Preparative SFC There are a variety of detectors that will be found suitable for the detection of organic compounds in supercritical fluid chromatography. Carbon dioxide is the most widely used fluid and has the advantage of being transparent in the ultraviolet part of the spectrum to 190 nm. Variable wavelength UV absorption detectors used for liquid chromatography may be employed for SFC but they must incorporate a cell that can withstand the very high pressures employed in the technique. A number of UV detector manufacturers produce suitably modified instruments for use with SFC. Sensitivity for the eluted solute peak is often much larger than with analytical SFC. In fact, as often happens in preparative HPLC, the variable wavelength UV detector must be de-tuned away from the wavelength of maximum absorption of the eluting species to prevent overload of absorption. For the vast majority of cases when using modified carbon dioxide mobile phases there is no need to look further than the simple UV detector. However, there are other detectors which have been demonstrated as having some specific advantages over UV absorption in certain cases. Detectors that have been

8.14 Recent Deveioprnents in SFC and SFE

189

studied include the infrared (IR) detector which has found particular application with xenon mobile phases since xenon has no IR absorption bands. Xenon is very expensive however, and its very high cost would prohibit its use in large scale SFC. The flame ionisation detector (FID) has been used in analytical SFC and could be used in preparative SFC by by-passing a very small fraction of the mobile phase flow through to the detector. Obviously the FID could not operate in a largely carbon dioxide atmosphere and would respond to many modifiers. The photoionisation detector (PID) has been used in analytical SFC but is better suited to highly sensitive detection of specific types of compound. The refractive index (RI) detector as used for analytical and preparative HPLC does suffer from being quite sensitive to the refractive index changes which take place in the superciritical fluid as its pressure and hence density fluctuate. It often takes some time for such detectors to ‘settle down’ in chromatographic systems. The authors have noticed that when using the Jasco@back pressure regulator the emergence of the solute peak(s) through the regulator is accompanied by a change in frequency of the solenoid valve oscillations. This is presumably caused by the differential compressibility of the solute in the mobile phase and the mobile phase itself as they each pass through the regulator. The emergence of solute can be perceived aurally. The diode array UV detector (DAD) has been employed for preparative SFC [13] and this has the advantage that the eluting materials may be partially characterised by acquisition of their UV absorption spectrum directly in the flowing stream. The light scattering detector (LSD) is a recent detector development that has found useful application in SFC. For non-UV-absorbing compounds, the first coupling of SFC on packed columns using carbon dioxide with light scattering detection was reported by Carraud et al. [37] and later by Nizery et al. [38]. This constitutes an almost universal detection method using SFC with packed columns and polar modifiers. The instrument employed in these studies and in further work reported by Herbreteau et al. [39] on the analysis of sugars is the Sedex 45 evaporative light-scattering detector (Sedere, Vitry sur Seine, France).

8.14 Recent Developments in SFC and SFE Recent developments in the preparative supercritical fluid chromatography and extraction area include the following commercially available instruments and innovations. Suprex have introduced the Prepmaster and Accutrap, two new products for automated quantitative sample preparation. The Prepmaster is a dedicated supercritical fluid extraction system designed for analytical and semi-preparative extractions. It can be used on-line, directly interfaced to a gas chromatograph or SFC; or off-line with the Accutrap cryogenic collection module. The Prepmaster can accommodate extraction vessels with capacities from 0.5 to 8 mL.

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8 Scaling-up of Supercritical Fluid Chromatography to Large-Scale Applications

The Accutrap SFE collection module is a stand-alone unit for off-line SFE. The extracted analytes from the Prepmaster pass through a heated restrictor into a cryogenic trap where they are focussed, ballistically heated to 40°C and desorbed with a suitable liquid solvent into an autosampler vial. Gilson have recently launched their 'SF3' SFC system which incorporates a pressure controller which is independent of flow rate and modifier concentration. They have addressed the problems of automating injection, fraction collection and extraction. The design and rationale behind the laboratory scale preparative SFC system described by the authors and built at Glaxo has been reported elsewhere on a number of occasions at specialist meetings on SFC and large scale HPLC [14- 161. Many of the technological innovations from this system were shared with Bartle and Clifford (University of Leeds). Bartle and Clifford have set up a system similar to that developed at Glaxo to investigate the relationships between the rate of production and (a) purity, (b) flow rate, and (c) the amount injected. Their aim is to develop rules for the optimisation of preparative SFC. Although intended for eventual use for high value materials such as pharmaceuticals the system is being studied using a model 50% mixture of fluorene and phenanthrene. 90% of each compound can be recovered by high pressure trapping with freeze out, with purities better than 95% and at a rate of 360mg h-'. These experiments were carried out using COz at a flow rate of 20mLmin-' (liquid) and 100 mg injected amounts of mixture. Their results and conclusions were presented at Keele University in October 1991 [40]. The development of preparative SFC is now at a stage where the technique can be used in the laboratory by the interested scientist. Individual components and complete systems are commercially available. At the process scale the technique can be applied using developed proven industrial systems although at present these represent a considerable financial investment for the user. Acknowledgements We would like to express our thanks to Miss Gillian Hope for her typing. Special thanks must go to my (CDB) family who put up without a Dad for several weekends whilst this chapter was in preparation.

References 11J Smith, R. M. (ed.) Supercritical Fluid Chromatography, RSC Chromatography Monographs, London: Royal Society of Chemistry; 1988, pp. 250. [2] Uvarov, E. B., Chapman, D. R., Isaacs, A., A Dictionary of Science, London: Penguin; 1971. [3] Randall, L.G., Sep Sci Tech 1982, 17, I- 118. [4] Sievers, R E . , Hansen, B., Chem Eng News 1991, 69(29), 2. [5] Klesper, E., Corwin, A.H., Turner, D.A., J Org Chem 1962, 27, 700-701. [6] Jentoft, R.E., Gouw, T. H., Anal Chem 1972, 44, 681 -686. [7] Hartmann, W., Klespes, E., J Polyrn Sci Lett Ed 1977, 15, 713-719.

References

3 91

Perrut, M., Jusforgues, P., Entropie 1986, 132, 107- 116. Saito, M., Yamauchi, Y., Kashiwazaki, H., Sugawara, M., Chromatographia 1988,25,9,801-804. Yamauchi, Y., Saito, M., J Chromatogr 1990, 505, 237-246. Saito M., Yamauchi, Y., J Chromatogr 1990, 505, 257-271. Higashidate, S., Yamauchi, Y., Saito, M., J Chromatogr 1990, 515, 295-303. Saito, M., Yamauchi, Y., Inomata, K., Kottkamp, W., J Chromatographic Sci 1989, 27, 84. Bevan, C. D., 2nd International Seminar, Developments on Preparative Chromatography, University of Cambridge, Cambridge, 8 December 1989. [15] Bevan, C. D., Preparative and Process-Scale Liquid Chromatography, Loughborough University of Technology, Loughborough, 11-14 December 1990. [I61 Bevan, C. D., CS Syn?posium on Extraction and Chromatography with Supercritical Fluids, University of Keele, Keele, 2-3 October 1991. [I71 Hoffman, N.E., Pan, S.L., Rustum, A.M., J Chroniatogr 1989, 465, 189. [I 81 Cretier, G., Majdalani, R., Rocca, J. L., Chromatographia 1990, 30, 11- 12. [19] Saito, M., Yamauchi, Y., J Chromatogr 1990, 505, 266, 270. [20] Campbell, R. M., Lee, M. L., A m Chem Soc Div Fuel Chem Pre 1985, 30, 189- 194. [21] Veisserik, J., Helbre, A., Kuuskmae, E., Poster 34, 7th International Symposiumon Preparative Chromatography, Ghent, 8-11 April 1990. [22] Jentoft, R.E., Gouw, T. H., Anal Chem 1972, 44,681-686. [23] Technology, a 2nd Level Course. Thermofluid mechanics and Energy - 5 Modelling Fluids, Milton Keynes: Open University Press 1982, 39-44. 1241 Veisserik, J., Somer, T., Poster presented at the International Symposium on Column Liquid Chromatography, Stockholm, 1989. [25] Khosah, R. P., Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, New Orleans, 1988. [26] Alkio, M., Harvala, T., Kompa, V., in: Proc. of 1st International Symposiumon Supercritical Fluids, Nice, Oct 1988; Perrut, M. (ed.) Nancy: Inst. National Polytechnique de Lorraine, 1988, pp. 389-396. [27] Berger, C., Perrut, M., J Chromatogr 1990, 505, 1, 37-43. [28] Perrut, M., French Patent 8209649, 1982; CA 100 (14): 105823: Method and Apparatus for fractionating mixtures by chromatography with elution by a fluid in the supercritical state. SocietC Nationale Elf Aquitaine SA, FR 82-9649, 3 June 1982; FR 2527934 A 1, 9 Dec 1983. [29] Perrut, M., French Patent 8510468, 1985; CA 107 (14): 117444s: Device for extraction - separation - fractionation by supercritical fluid. SocietC Elf Aquitaine (SNEA); FR 85-10468, 9 Jul 1985; FR2584618 A I , 16 Jan 1987. [30] Berger, C., Douget, L., Perrut, M., French Patent Request 1989. [31] Jusforgues, P., Perrut, M., French Patent 8610609, 1986. Process and apparatus for separating mixtures by extraction with the aid of supercritical fluid. SocietC Elf Aquitaine (SNEA) Eur. Pat. Appl., I l p p ; EP 87-40465, 25 Jun 1987; EP 254610 A l , 27 Jan 1988. [32] Berger, C., PhD Thesis, Institute National Polytechnique de Lorraine, Nancy, 1989. [33] Prochrom SA, BP9 54250, Champingneulles, France, 7th International Symposium on Preparative Chromatography, Prep-90, Ghent, Belgium, 8 -11 April 1990. [34] Jusforgues, P., this book, Chapter 7. [35] Macaudiere, P., Caude, M., Rosset, R., Tambute, A., J Chromatogr Sci 1989,27(7), 383 -384. [36] Jordan, J. W., Taylor, L. T., J Chromatogr Sci 1986, 24(3), 82- 88. [37] Carraud, P., Thiebaut, D., Caude, M., Rosset, R., Lafosse, M., Dreux, M., J Chromatogr Sci 1987, 25, 395. [38] Nizery, D., Thiebaut, D., Caude, M., Rosset, R., Lafosse, M., Dreux, M., JChromatogr 1989, 467, 49. [39] Herbreteau, B., Lafosse, M., Morin-Allory, L., Dreux, M., J Chromatogr 1990,505,299-305. [40] Bartle, K. D., Clifford, A. A., Fang, R., Jafar, S. A., Symposium on Extraction and Chromatography with Supercritical Fluids, Chromatographic Society and University of Keele, Keele, 2 - 3 October 1991. [8] [9] [lo] [I 11 [I21 [13] [I 41

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9 Affinity Chromatography and its Applications in Large-Scale Separations Christopher R. Goward

9.1 Introduction Proteins have binding sites for various molecules to enable them to carry out their biological function. Binding to these molecules is determined by the size and shape of the molecule bound, its hydrophobic and charge moieties and hydrogen bonding. The affinity for a particular molecule can be used to separate a protein from a mixture of substances by a process termed affinity chromatography. Affinity chromatography provides a simple procedure for separation of a protein from a complex mixture. It has been widely used in the laboratory and is beginning to be accepted on the industrial scale. In this process a target molecule specifically and reversibly inter21. A ligand is acts with a ligand which has been immobilised on to a matrix [I, covalently attached to a matrix and retains the ability to bind the target molecule. Unbound protein is washed from the affinity adsorbent and the target protein is eluted using the mildest conditions available. The principles of affinity chromatography are shown in Fig. 9-1. The bound target molecule is released intact after addition of an elution agent. Elution may be selective by addition of a biospecific agent or it may be non-selective by altering the ionic strength, the pH, polarity or addition of a denaturing agent or a chaotropic agent. Affinity chromatography may be a very powerful method once a procedure has been determined but the development of a suitable affinity adsorbent may be very slow. The ligand may be expensive, unstable, and may leach from the matrix resulting in a contaminated product. However, affinity chromatography is particularly useful for purification of large amounts of valuable proteins from high expression systems and particularly from secretory systems. Affinity chromatography may be particularly powerful when the target protein is a minor component of a complex mixture. The technique is likely to become more exploited on the large scale as very high resolution can be obtained, at least an order of magnitude higher than that obtained by other methods of chromatography; the average purification step achieves about 5 - 6 fold purification whereas the average for affinity chromatography is about 1OOfold [3]. Interferon has been purified on an immobilised antibody column with a 5000fold increase in specific activity [4]. The advantages and disadvantages of affinity chromatography are summarised in Table 9-1.

2. Target protein

1. Sample is applied to the adsorbent

IS

bound to the ligand

P rotein /

'

/ gand

'1

' Protein1

3. Other proteins are washed away

4. Target protein is eluted from the adsorbent

/

gand

Protein

I

Fig. 9-1. The principles of affinity chromatography.

Table 9-1. Advantages and disadvantages of affinity chromatography. Advantages

Disadvantages

Overall process time may be rapid Overall number of steps in a process are often reduced Capacity of the adsorbent can be high or low Process is not limited by volume of sample Resolution can be very high

Ligand may be expensive Development of an adsorbent may be very slow Regeneration of an adsorbent can be difficult

9.2 Support Matrix The selection of a support matrix to which the ligand is attached is very important. The support matrix should be hydrophilic for water-based solvent. It should be macroporous to cope with the high hydrodynamic pressure and thus avoid compression, and mechanically rigid to allow sufficient ligand to be coupled and allow even large molecules access to within the beads. The particles should ideally be small to improve the kinetics of binding and elution since the interfacial surface area de-

9.3 Important Features of a Ligand

195

creases exponentially with decreasing particle size and the distance a molecule has to migrate decreases with decreasing particle size 151. They should be spherical for uniform packing in a column. The matrix should be easily derivatised with various functional groups by stable covalent bonds to keep ligand leakage low, to be resistant to chemical and biological degradation, inert for minimum non-specific adsorption, inexpensive and reusable. Although the matrix should ideally be composed of small beads, there is greater compression with smaller particles so smaller beads have to be made from more rigid materials. Support media currently available for affinity chromatography and their performance and properties have been reviewed, for example, by Narayanan and Crane [6]. The ideal matrix is not available but there are many choices. Sepharose FF, Trisacryl and Fractogel ranges, which are in the 40 Fm range, give higher resolution than conventional gel beads of 9 0 y m or more and Sepharose FF, a highly cross-linked agarose, has been used at flow rates in excess of 300 cm h-' in for example purification of L-asparaginase from Erwinia chrysanthemi by cation-exchange chromatography 17, 81. Sepharose FF is produced by Pharmacia-LKB, IBF produce Trisacryl, a synthetic poly-N-acryloyl-2-amino-2-hydroxymethyl-1,3-propane diol [9] and Merck manufacture Fractogel TSK, a series of synthetic hydrophilic vinyl polymers. The hydrodynamic properties of these matrices make them suitable for large-scale work where high flow rates are desirable. Inorganic materials such as porous silica are rigid but these tend to bind proteins irreversibly even after derivatisation and this has limited their use [lo]. An added problem with silica is non-specific adsorption of substances due to a residual surface charge and the matrix is also soluble at alkaline pH. The newer generation of matrices are based on the early polymers but are cross-linked or made of composites. They have high rigidity and are less compressible and more uniform with smaller particle size such that these matrices are more suitable for large-scale separations. Polytetrafluoroethylene may prove to be an important matrix as it is mechanically rigid and chemically inert [ 1I] and polymer-coated polytetrafluoroethylene beads have been produced for immobilisation of ligands on to the fluorocarbon surface [12]. Another recent advancement is the introduction of perfusion chromatography 1131 in which the particles are interconnected with large pores through the particle in addition to the usual diffusive pores (Fig. 9-2). The advantage of this system is rapid access of molecules to the diffusive pores which makes separation and capacity unusually independent of the flow rate. The matrix is commercially available under the trade name POROSTM(Perseptive Biosystems, Cambridge, Massachusetts).

9.3 Important Features of a Ligand A ligand should ideally recognise only the target protein. Examples of affinity ligands are shown in Table 9-2. However, many group-specific adsorbents are commercially available and it may be unnecessary to invest in a specific adsorbent. The size of the ligand is important for if is too small the target protein may have insuffi-

196

9 Affinify Chromatography and its Applications in Large-Scale Separations

cient access to the ligand. If this is the case it may be necessary to use a spacer arm. If the ligand is too large it may be denatured and non-specific binding may be increased. As mentioned above, immobilised ligand (L) is covalently bound to the stationary phase and interacts specifically with the target protein (P).

(a) Conventional particle

Diffusive pore

(b) Perfusion particle

Throughpore

/

Diffusive pore

Fig. 9-2. Conventional and perfusion chromatography particles. (a) In a conventional chromatography particle there are no throughpores so convective flow is around the particle and the inner sites for adsorption are reached by molecular diffusion. (b) In a perfusion chromatography particle there are large throughpores which can be used for connective flow and so the inner site for adsorption are reached without molecular diffusion.

Table 9-2. Examples of affinity ligands for proteins. Ligand

Target protein

Protein G Protein A Protein L Antigen Antibody Hormone Nucleotides Protease inhibitors Cofactor Substrate Triazine dyes Streptavididavidin Phenyl boronate Lectins Heparin

Immunoglobulins Immunoglobulins Immunoglobulins Antibodies Antigen Receptor Nucleic acid binding proteins Proteases Enzyme Enzyme Dehydrogenases, kinases, polymerases Biotin-labelled proteins Glycoproteins Glycoproteins Fibronectin, fibrinogen

9.3 Important Features of a Ligand

197

Factors affecting retention are the equilibrium dissociation constant (Kd)for the ligand-protein complex and the number of available binding sites on the ligand. The value of Kd may be defined by

The interaction should be effectively reversible so that the target protein may be eluted without denaturation of protein or ligand. Affinity chromatography works well when the Kd is in the range lo4- lo8 M-'; if the Kd is lower it may prove impossible to elute undamaged target protein, if higher the interaction may be too weak. Note that the Kd for free ligand may be different to that for immobilised ligand. The ligand should be stable to the immobilisation and chromatography conditions including resistance to proteases, elution agents and cleaning agents.

9.3.1 Coupling of a Ligand to the Support Matrix The ligand may be coupled either directly to the matrix or to a spacer arm which itself is attached to the matrix. A spacer arm is used to reduce steric hindrance between the target molecule and the ligand (Fig. 9-3). In either case immobilisation should be through a region which will not interfere with binding to the target mole-

Fig. 9-3. Importance of the spacer arm. (a) In the absence of a spacer arm there may be insufficient space to allow contact with the binding site on the target protein. (b) Addition of a spacer arm may allow the crucial contacts between the ligand and the target protein to be made.

198

9 Affinity Chromatography and its Applications in Large-Scale Separations

cule. This may require a trial and error approach to determine the best means of immobilisation and it may prove necessary to chemically modify the ligand to achieve the immobilisation. The ligand should be as pure as economically possible before coupling to ensure maximum purity of the product after chromatography. The spacer may be hydrophobic or hydrophilic which may introduce non-specific hydrophobic or electrostatic attractions respectively and so it is usually a short linear hydrocarbon chain. Some commonly used spacer arms are 6-aminohexanoic acid, 1,6-diaminohexane and 3,3'-diaminodipropylamine.

9.3.2 Activation of the Matrix The matrix requires chemical activation before derivatisation with ligand. There are a large number of activating agents and methods available [14]. Activation is usually achieved by introduction of an electrophilic group into the matrix which can then react with nucleophilic groups (eg, amino, thiol or hydroxyl) of the ligand, though the reverse may be performed. The most effective methods of immobilisation are use of tresyl chloride and 1,l'-carbonyldiimidazole but these are too expensive for largescale work [I51 and so the cyanogen bromide method is often used. Many activated matrices can be obtained from suppliers of chromatography media.

9.3.3 Capacity of the Adsorbent The capacity of an adsorbent depends on the amount of ligand immobilised and its accessibility to the target protein. With high ligand concentrations the dissociation of the ligand may be limited yet a low ligand concentration may make the chromatography inefficient. Also, with a high ligand concentration there may be non-specific binding and steric hindrance. The optimum concentration of ligand is best found by experiment. A highly immobilised ligand does not necessarily mean a high capacity for the protein. For example, agarose lightly substituted with Cibacron Blue F-3GA binds more serum albumin than heavily substituted cellulose [I61 and a lightly substituted antibody matrix proved more efficient than a highly substituted matrix [17]. The reason for this may be explained by steric hindrance (Fig. 9-4). The amount of ligand coupled to a known amount of matrix may be determined from the difference in the amount added to the matrix and that amount remaining unbound after copious washing, provided that the ligand can be accurately measured. Other methods may be used such as direct spectroscopy of the gel or after solubilisation of the gel. The degree of substitution is important because if it is too high many non-specific adsorption sites are available and porosity is reduced. This is particularly true when the ligand is a large molecule. The capacity of the adsorbent for target protein can be determined from breakthrough curves [18]. A simple method for this is shown in Fig. 9-5.

Fig. 9-4. Steric occlusion of the ligand. (a) I f the matrix is heavily substituted with ligand, binding of the target protein to the ligand may be blocked. (b) At a lower concentration of ligand the target protein can freely interact with the ligand.

C

.-C0

20

60

40

80

100

Volume Fig. 9-5. Breakthrough curve. The useful capacity of the adsorbent should be determined before a large-scale operation is undertaken. This may be determined by a simple frontal analysis. The sample is applied to a known amount of adsorbent and the effluent is monitored for emergence of the protein of interest. The useful capacity may be defined as the amount applied at which there is 10% breakthrough in the effluent, as indicated by the broken line.

200

9 Affinity Chromatography and its Applications in Large-Scale Separations

9.3.4 Ligand Leakage To be cost effective an affinity adsorbent should be reusable and so stability of the matrix-ligand linkage is very important [6]. Ligand leakage and subsequent contamination of the product is a serious problem in affinity chromatography and is largely a result of binding ligands via a spacer to cyanogen bromide-activated agarose which is then attacked by nucleophiles. Leakage can be exacerbated by attack of the hydroxyl groups of agarose and carboxyl and carboxamide groups of polyacrylamide gels. Ligand leakage may greatly reduced by using N-hydroxysuccinimide-activatedester gel, linkages similar to those from bisoxirane coupling. Ligand leaked can be removed from the eluate by gel-filtration or ion-exchange chromatography. Ligand leakage is probably the worst drawback of immunoaffinity chromatography. Detachment from the adsorbent occurs mostly during elution of antigen. However, antibody dissociation from the adsorbent does not occur when antibodies are immobilised by amine or amide attachment. Antibodies may become attached by multiple links, which may reduce leakage, but there may be loss of binding ability by antibody.

9.3.5 Triazine Dyes Coupling the often labile ligand to a matrix is difficult and the coupled ligand is often unstable and open to biological attack. Reactive dyes offer an alternative ligand. These dyes are mainly produced by ICI (Procion) and CIBA (Cibacron) for use in the textile and printing industries. They consist of a polysulphonated anthroquinone, diazo or copper phthalocyanine chromophore linked to a reactive triazinyl group by a secondary amino bridge. An example of a triazine dye is shown in Fig. 9-6. The immobilised dye is stable, though prone to leakage from the matrix, and binds a wide range of proteins under specific conditions [19]. Dyes often leach from agarose due to hydrolysis of the dye-matrix linkage. Dye may also be released by breakdown of the matrix itself and dye-agarose conjugate leaks. Free dye may also be released which has been aggregated and adsorbed onto immobilised dye [20]. Traces of dyes can be removed by anion-exchange or gel filtration chromatography below pH 6.5. 0

N H,

Fig. 9-6. Example of a triazine dye: Procion Blue MX-3 G.

9.4 Process Design

201

Leakage of dyes from a matrix is a problem for the preparation of protein for diagnostic, therapeutic and some analytical purposes but the ether linkage is stable enough for most chromatography at neutral pH. Cross-linked agarose tends not to breakdown and synthesis of dyes solely for application in affinity chromatography allows improved matrix ligand conjugates [21]. There have recently been other advancements in the field of biomimetic dyes [22]. Techniques for binding dye to agarose are based on conventional textile dyeing methods. This reactive triazine group is coupled directly to the matrix at mildly alkaline pH and room temperature, although the dye has been immobilised via the chromophore with very encouraging results 1231. Dye may also be attached with a spacer arm inserted between the dye and the support. Methods for immobilisation of triazine dyes by ether linkage between the positively charged triazine moiety of reactive dyes and the hydroxyl groups of agarose by nucleophilic substitution have been described [24]. The dye solution is added to agarose containing 1-270 w/v NaCl to promote physical adsorption of the dye to the matrix and then NaOH to raise the pH to 10.5 and thereby generate the necessary nucleophiles for the conjugation. The temperature and time of incubation determines the degree of immobilisation. Complete coupling of dichlorotriazinyl dyes requires only 2 to 4 h at ambient temperature but the less reactive monochlorotriazinyl dyes require 72 h at ambient temperature or 16 h at 60°C [24]. The covalent attachment of dyes to matrices other than agarose for large-scale application follows similar if not identical protocols 1251.

9.4 Process Design An affinity chromatography technique may be sufficient to provide a one step process but it is often beneficial to precede it with a precipitation step or ion-exchange chromatography to reduce contamination and the amount of extract to be processed, to possibly improve resolution and remove proteolytic enzymes. Affinity chromatography media may be thus susceptible to fouling if used at too early a stage in a process and may be difficult to regenerate, which is of particular note due to their high cost. However, the method may be useful at an early stage to extract extracellular proteins or recombinant proteins produced in high concentrations by the cell. On a laboratory scale affinity chromatography has proved a very powerful method for purification of proteins 1261. It is undoubtedly used on the preparative scale but the literature contains relatively few examples which is in part due to problems with associated expensive and labile ligands. Affinity ligands may have only a limited application to large-scale work if the cost of preparing a suitable matrix is greater than the value of the product. Affinity chromatography requires appreciable chemical and biological knowledge to produce an adsorbent which may not then behave as expected. The use of group-specific media such as an immobilised coenzyme or nucleotide reduce this problem [27, 281 but conventional affinity matrices are difficult and expensive to synthesise. Immobilised triazine dyes have advantages over group-specific

202

9 Affinity Chromatography and its Applications in Large-Scale Separations

ligands in their commercial availability, low cost, resistance to chemicals and enzymes, they can be repeatedly used [29] and can be easily and directly attached to the matrix to a wide range of supports via the reactive triazine group without using toxic reagents such as cyanogen bromide.

9.4.1 Scale Up Any restrictions on large-scale work should be taken into account when the protocol is being developed. The process should be fully optimised in the laboratory and the sample volume may then be increased by 10 or 20 times. The column surface area is increased proportionally with the bed height constant; column diameter has little effect on resolution so the amount of adsorbent used can be increased by increasing the diameter of the column. However, the cost involved increases when the diameter of the column is increased and it becomes increasingly difficult to apply an even spread of the sample over the surface of the adsorbent. The affinity adsorbent, ionic strength, pH, linear flow rate and temperature should remain the same, though the flow rate may be increased if greater throughput is required. Large scale cell extracts often contain higher protein concentrations than small scale extracts due to different methods used for cell breakage, so care has to be taken not to overload the column. The scale up process is repeated until the desired scale is achieved. The intended use of the product has to be taken into account during the scale up and design of large-scale processes. For example, it may be necessary to show that any components of the adsorbent are not present in the final product if this is to be for human use. The affinity adsorbent, and other adsorbents used in the process, has to be controlled strictly to ensure that the product is sufficiently pure. This may preclude use of a particular adsorbent, particularly if the ligand leaches, due to the activation chemistry used in its manufacture. Alternatively it may be necessary to demonstrate that the ligand leakage into the product is of an acceptable level.

9.5 Chromatography Column and Other Equipment The column chosen for a large-scale chromatography step should have the lowest dead volumes possible and a distribution system which allows the sample to be applied over the surface of the adsorbent. Suppliers of columns for large-scale chromatography include Pharmacia-LKB (Milton Keynes, UK), Amicon Ltd (Stonehouse, Gloucestershire, UK) and Whatman Ltd (Maidstone, Kent, UK). These columns may be made of either glass, plastic or stainless steel. Large columns should be packed with adsorbent by filling the column with a 40% slurry of cross-linked adsorbent in 0.5 M NaCl through an extension to the top of the column. Excess fluid is pumped away, the outlet is closed and the top fitting is attached to the column. The pumps and other fittings should perform their task without contamination of the product,

9.7 Chromatography Conditions

203

particularly for therapeutic proteins. The pump of choice is a lobe rotor type which can have sanitary components and be operated over a wide range of flow rates. The total column size used depends on the capacity of the adsorbent for protein. The materials used for the column construction should be compatible with solvents and solutions used. Janson and Hedman [30] have detailed desirable features of large-scale chromatography columns. Dimensions of the column become more critical with process chromatography compared with laboratory chromatography because of problems due to back pressure which limits reduction in process time. Short fat columns are generally best to minimise the pressure drop across the bed. Proteins with low affinity for a ligand can be purified without a strong interaction with the ligand; the protein is delayed in its transition down the column and true chromatography can occur. In this case a longer column relative to the diameter may be used to good advantage in terms of resolution. Affinity chromatography is usually performed in a column, but batch binding and elution may be used.

9.6 Process Control It is often desirable to automate a process, particularly when a process is repeated for a number of cycles. This can be achieved with either microprocessor-based controllers or via computer programs to operate valves [26]. The process control system can also be used to monitor, record and change the separation conditions such as pressure, pH, conductivity, flow rate and temperature. Use of sensors allows feedback control of the process. These can be used to control flow rate and pressure and to detect the presence of air in the system. The emergence of protein from the column can be monitored by measuring the absorbance of the eluate and this can be used to initiate isolation of the product [26].

9.7 Chromatography Conditions 9.7.1 Adsorption A protein is bound to the affinity adsorbent under conditions sufficiently mild to bind as few contaminating proteins as possible. The sample is usually in equilibration buffer and may be either a crude cell extract, culture supernatant fluid or obtained after a precipitation or ion-exchange step. Adsorption is better from more concentrated samples and higher flow rates can be applied, but the concentration is limited by the viscosity of the sample. It is acceptable to apply dilute samples as these are concentrated on the adsorbent. Overall, the volume of sample is not crucial provided that it is not so large that time becomes a limiting factor, unless the protein is weakly

204

9 Affinity Chromatography and its Applications in Large-Scale Separations

bound in which case a small volume should be used (5% of the column volume) to prevent elution of non-bound protein. The flow rate used is limited in part by the mechanical properties of the matrix but in practice may be more dependent upon the concentration of immobilised ligand, the dissociation constant of target protein and ligand, and the concentration of target protein in the sample. Retardation on the column can be raised by increasing the column, length, lowering the flow rate and reducing the sample volume. Increasing the sample size can weaken binding but if the interaction is sufficiently specific, competition for the ligand may be low enough to allow column overloading. Binding to an affinity adsorbent involves a mixture of hydrophobic and electrostatic interactions, hydrogen bonding and protein conformation. Binding is affected by a number of conditions (Table 9-3). Increasing the ionic strength can be used to elute some proteins so electrostatic interactions are important for binding. The electrostatic interactions involve cation-exchange so the binding is stronger at low pH values. The ionic strength is often raised to about 250-500 mM to reduce non-specific binding to the adsorbent but it must not be so high that interaction of the protein with free ligand is prevented. Increasing the ionic strength promotes binding of some proteins which indicates the importance of hydrophobic interactions particularly when hydrophobic spacer arms are used. Hydrophobic interactions are lowered when the temperature is lowered so the temperature can be varied to suit the purification process. Table 9-3. Factors which affect binding to the affinity adsorbent Factor Immobilisation method used Attachment of ligand to the matrix Concentration of immobilised ligand Affinity of the target protein for the immobilised ligand Steric hindrance affecting availability of the ligand PH Ionic strength Temperature Flow rate

9.7.2 Washing After application of the sample, the adsorbent is washed to remove non-bound and non-specifically bound substances. It may be necessary to wash with several different buffers but care has to be exercised when the washing buffer is chosen not to remove the target protein. Non-specific binding occurs by ionic interactions between proteins and the ligand or charged groups present due to the nature of the coupling, or by hydrophobic interactions with the ligand. A suitable concentration of NaCl is often used in the buffer to aid the washing process.

9.7 Chromatography Conditions

205

9.7.3 Elution A dynamic equilibrium exists between bound and free target protein on the adsorbent. The environment in the column has to be changed to disrupt the interaction and so to reduce the affinity. As the complex is mostly maintained by hydrophobic interactions, hydrogen bonding and electrostatic attractions, elution is effected by weakening these forces by stepwise or continuous addition of a suitable agent. The mildest elution method is ideal but cost may be a consideration, particularly on the large scale, since some of the elution agents may be prohibitively expensive. Elution of protein from the adsorbent may be by specific cofactors or substrates or by nonspecific agents such as a pH change or an ionic gradient. If these methods fail, then chaotropes such as thiocyanate, ethylene glycol or urea are introduced but a disadvantage is that the protein may be denatured. Proteins are very often eluted using ionic gradients produced by increasing concentrations of buffer or salt. It is usually a matter of trial and error to find the most suitable eluent.

9.7.4 Selective Elution Selective elution is usually used with non-specific ligands. A substance is added which competes with ligand or target protein and may be a substrate, cofactor or inhibitor. The molecules which interact at the ligand binding site or elsewhere make the binding site no longer available due to a conformational change or by steric occlusion or they remove an agent essential for binding. In immunoaffinity chromatography, if the ligand is a hapten or if the determinants are known, these can be used to elute the protein. Competition for the ligand provides the ideal method but may prove too expensive, particularly on the large scale. The amount of free ligand necessary can be reduced by lowering the affinity, for example, by changing the buffer pH or ionic strength. Increasing the ionic strength may, however, increase hydrophobic interactions and have the opposite effect. The expense can also be limited by optimising the concentrations of free and bound ligand. The optimum concentration of free ligand can be determined from a linear gradient of ligand applied to the column; the concentration at which the target protein is released can be determined. It may prove difficult to elute the protein with free ligand if the bound ligand concentration is too high. The most selective elution conditions may be obtained by use of an expensive biospecific eluent, but the cost of an elution agent is an important consideration particularly in the overall economy of a large-scale process.

9.7.5 Non-selective Elution Non-selective elution may be more appropriate particularly in high-affinity systems where few contaminants have been adsorbed. Non-selective elution is usually used with specific ligands. Non-selective eluants change the conformation of the target

206

9 Affinity Chromatography and its Applications in Large-Scale Separations

protein and/or ligand and disrupt the interaction between them. They are usually introduced in gradients, which may be used to resolve several proteins, but use of gradients may not be practical on the large scale. Elution with NaCl or KCI, usually up to 1 M, is a very economical method. Nonhydrophobic interactions between the immobilised ligand and protein, including biospecific ionic or other polar forces, are disrupted. Hydrophobic interactions are increased when the ionic strength is raised so non-specific interactions may become stronger. Elution can be achieved by lowering the pH; the ionisation of charged groups is altered so that salt bridges are not maintained. This method is often used when immuno-binding is to be disrupted; the pH is reduced to below 3 usually with acetic acid, propionic acid or glycine/HCl buffer. The pH used for elution is limited by stability of the target protein, the ligand and the matrix. Increasing the temperature can be used to elute tightly bound proteins, in combination with other eluants. Temperature gradients can be used to elute weakly bound proteins. The ligand and target protein have to be stable to the temperature used. Electrophoretic elution can be used for charged proteins which are tightly bound. Elution can be effected by lowering the polarity of the buffer with surface tension reducing agents particularly at low temperature. These agents lower hydrophobic and van der Waals interactions and include detergents such as Triton X-100 (up to 1070 v/v), ethylene glycol (up to 50% v/v), ethanol (up to 2070 v/v) and dioxane (up to 10% v/v), though it should be noted that ethanol is of no use if the ligand is charged. Chaotropic agents are used when there is high affinity between the protein and the adsorbent and all else fails. The structure of water is disrupted to reduce the interaction, for example with either 3 M potassium thiocyanate, 2 M potassium iodide or 4 M magnesium chloride. Chaotropes are often used to deform and hence elute protein in immunoaffinity chromatography and other systems where there is strong affinity. It is often necessary to remove the chaotrope immediately after elution. They are used at neutral p H when antigen is unstable at acid pH, thus precluding elution at low pH. Conformational changes of the target protein and/or ligand are induced by denaturing agents such as 8 M urea or 6 M guanidine hydrochloride. They can be useful when the pH change required to elute a protein also denatures it. Presence of divalent metal ions, such as Cu, Ni, Co, Mn, or Zn, may make binding tighter. The adsorbed protein can be eluted with a chelating agent or simply by omitting the metal cation. After elution the target protein often needs to be moved to a gentle environment particularly after harsh elution conditions. This can be achieved by dilution, desalting columns, dialysis, diafiltration or, if the pH has been drastically altered, Tris base can be added to bring the pH to neutral.

9.7.6 Flow Rate The flow rate used is an important factor in affinity elution. The lowest flow rate acceptable to the chromatography conditions is used to obtain maximum recovery and resolution with a minimum dilution of the sample. This is particularly important if several proteins have bound to the affinity adsorbent. The direction of the flow

9.8 Cleaning and Storage of Adsorbents

207

is often best reversed for elution to avoid dilution and retardation as the protein travels down the column. However, if a gradient of non-selective eluent is used true chromatography may be desirable in which case the direction of flow should not be altered. Even if true chromatography is not necessary, it may be desirable to use a gradient for its zone-sharpening effect where the protein furthest down the column is in contact with a high concentration of eluent and so is eluted more rapidly than protein at the top of the column, which allows it to join the main group of proteins faster than it would if frontal elution were used. If the protein is tightly bound the flow may be stopped after addition of eluant to allow dissociation. However, this may not be a good idea if the protein is damaged by prolonged exposure to the eluant.

9.8 Cleaning and Storage of Adsorbents If the elution conditions have not irreversibly damaged the ligand, effective cleaning is required to ensure longevity of the adsorbent and its continued correct function in order to reduce the risk of contaminating the product with endotoxins or other unwanted bacterial products. This is particularly important on the large-scale since investment in the adsorbent may be considerable. Fouling of the column may be from particulate matter, non-specifically adsorbed substances or from microbial contamination [3 I]. Particulate matter including microorganisms may be removed from buffers and cell extracts with 2 - 10 wm pore size filters upstream of the column. The adsorbent should be cleaned as soon as possible after use to avoid bacterial growth and ideally affinity chromatography should not be used to separate a protein from a crude cell extract until after a precipitation step, using heat, pH or salts for example, or until another chromatography step has been completed. NaOH is the best choice, where applicable such as with dye ligands, because it sterilises the entire system, has good cleaning effect, cannot contaminate the product and it destroys pyrogens but does require careful handling on the large scale. Affinity adsorbents present a problem for cleaning if the ligand is labile. Specific methods may be used for a particular affinity gel, but a general method of washing may be used to disrupt ionic and hydrophobic interactions by washing the adsorbent with buffer at pH 8.5 followed by buffer at pH 4.5, both containing 0.5 M NaC1. The storage conditions chosen should prevent microbial growth. If the protein is to be used for therapeutic purposes 20% (v/v) ethanol or 0.1Yo (w/v) NaOH (if appropriate) may be most suitable. For non-therapeutic proteins, 0.2% (w/v) thimerosal, 0.02% (w/v) chlorhexadine, 0.02% (w/v) sodium azide or 0.5% (w/v) chloretone can be used but these bacteriostatic agents cannot be used with all affinity media, they have no cleaning effect and there is a low risk of bacterial contamination of the product. Storage conditions also depend on stability of the adsorbent, ligand and the nature of the covalent bond of ligand to matrix. Dye ligand affinity matrices can be stored over many months with little loss in protein binding capacity and matrices are re-usable. They are often far more stable than conventional affinity systems which

208

9 Affinity Chromatography and its Applications in Large-Scale Separations

may be susceptible to fouling by extraneous lipid and protein and to degradation by proteases, phosphatases, nucleotidases and so on which are found in most cell extracts. In contrast, protein ligands may change conformation and lose activity at extreme pH, high temperature or in the presence of denaturing agents.

9.9 Protein Engineering Applied to Protein Purification Molecular genetics has produced recombinant systems so that bacteria produce heterologous proteins in high concentration with high yields [32]. This increased concentration of the protein of interest is particularly important as the volume of bacterial culture required for a particular yield can be reduced. The purification process may require fewer steps as the protein is relatively pure at the start [32]. The gene can also be manipulated to produce proteins with more desirable properties including fusion of the genes producing peptides to the target protein to aid protein purification [33]. The aim is to make the purification process more simple and predictable. DNA sequence which codes for a polypeptide containing desirable properties to aid purification of the target molecule may be introduced upstream or downstream of the portion coding for the product. The target molecule with the fused affinity tail can be separated by adsorption to an appropriate ligand. Nonbound protein is washed through the column and the target protein eluted by changing conditions of pH, ionic strength or addition of a competing substance. An early example was the fusion of a polyarginine tail to the C-terminus of urogastrone which was subsequently removed from the purified product with immobilised carboxypeptidase A [34]. The use of tails on the large scale may be restricted to ion exchange whereas on the small scale affinity can be more applicable. Fusions have also been designed to aid correct folding and to protect small molecules from proteolytic enzymes as described for a protein A-DNase I fusion [35].

9.9.1 Release of the Affinity Tail The polypeptide tail may be released after chromatography by chemical or enzymic means. The released tail, if intact, may be captured by passing the mixture through the same chromatography adsorbent or separated by size or charge, but removal of the affinity tail, if necessary, is often the most difficult problem and methods used in the laboratory may not be suitable for use on the large scale [36]. The methods used for cleavage of tails has recently been reviewed [37] but note that removal of the tail is not always necessary [36]. The big advantage of the method is that, rather than screening matrices and optimising the process, the protein can be tailored to suit a purification method and hence process development time is reduced.

9.9 Protein Engineering Applied to Protein Purification

209

9.9.2 Examples of the Use of Affinity Tails Example of the variety of tails which have been used are shown in Table 9-4. Some examples of the use of affinity tails demonstrates the versatility of the technique. Mouse dihydrofolate reductase with a polyhistidine peptide tail was purified by using metal chelate chromatography followed by removal of the affinity peptide with carboxypeptidase A [38]. A short hydrophilic antigenic peptide of eight residues engineered on to the N-terminus of recombinant lymphokines allows them to be purified by using an immobilised monoclonal antibody specific for the first four amino acids of the peptide [39].

Table 9-4. Examples of fusion protein systems for protein purification. Tail

Ligand

C-terminal arginine Polyarginine Polyaspartic acid Polycysteine Polyhistidine Polyphenylalanine Histidine-tryptophan dipeptide Protein A Protein G Glutathione S-transferase Chloramphenicol acyl transferase FlagTMantigenic peptide /?-Galactosidase

Anhydrotrypsin Cation exchanger Anion exchanger Covalent Immobilised metal ion Phenyl Iminodiacetate IgC Ig G/albumin Glutathione p-Aminochloramphenicol Anti-Flag peptide Anti-/3-galactosidase

A fusion of two IgG-binding domains of staphylococcal protein A to recombinant human insulin-like growth factor I (Igf-I) [40], expressed in Escherichia coli or Staphylococcus aureus, can be separated on IgG-Sepharose. The fusion protein was secreted from Escherichia coli into 1000 L of culture medium. The culture medium was cleaned by cross-flow filtration and bound to 2 L IgG-Sepharose in portions of 35 L per cycle. The column was equilibrated with 50 mM Tris-HC1, pH 7.6, containing 150 mM NaCl and 0.05% (v/v) Tween 20 and eluted with 500 mM acetic acid titrated to pH 3.4 with ammonium acetate [41]. Eluates from 21 cycles were combined and 2 g of fusion protein were treated with hydroxylamine at pH 9.0 for 4 h to release the protein by cleaving an Asn-Gly bond. The solution was exchanged into 150 mM ammonium acetate, pH 6.0, and passed through IgG-Sepharose to capture released IgG-binding domains. The Igf-I was passed through the column and recovered. The secretion system ensures a high initial purification factor and the culture medium contains little or no proteolytic enzymes to damage the product. Disadvantages of such protein A fusions are the extreme conditions of pH of high concentrations of chaotropes required to elute them which may also denature the de-

2 10

9 Affinity Chromatography and its Applications in Large-Scale Separations

sired protein. There may also be difficulty in subsequent removal of the protein A moiety without damaging the desired protein. The second point is common to other fusions. An acid labile bond may be introduced at the junction between the species of protein or a protease sensitive site introduced in the same region. The target protein obviously has to be stable under such acid conditions and if proteases are used the protein has to be free of the particular cleavage point. For example, introduce Asn-Gly bonds (as in the example above) which are rare and can be broken with hydroxylamine [42]. Any Asn-Gly bonds in the required protein product may be removed by protein engineering if desirable properties of the protein are not affected by this action. A dual affinity system has been reported where tails are fused to the N-terminus and to the C-terminus which aids stability of the target molecule and allows two purification methods to be exploited. An example is the fusion of the albumin-binding domains of protein G and IgG-binding domains of protein A around a recombinant human insulin-like growth factor 11 [43]. This dual affinity fusion protein was more stable than a single N-terminal fusion protein. It is likely that these types of methods will be incorporated in the separation of proteins in the future. Dual affinity systems are useful for expression of unstable proteins as only full-length protein is obtained after purification [43,44].

9.10 Examples of Some Large-Scale Affinity Methods 9.10.1 Protein G Protein G is naturally found on the surface of some streptococci and binds to immunoglobulin G (IgG). An example of the simplicity and power of affinity chromatography is illustrated by the purification of recombinant protein G expressed by Escherichia coli. Cell extract is applied to IgG-Sepharose equilibrated with 50 mM Hepes-NaOH, pH 8.0 containing 250 mM NaC1. Unbound protein is washed from the column with the same buffer and pure protein G eluted with 100mM glycineHC1, pH 2.0. Any proteolytic fragments of the protein G molecule can be removed, if necessary, by subsequent anion-exchange chromatography on Q-Sepharose FF [45] as shown in Fig. 9-7. This method has proved useful but for large-scale work it has the disadvantage of initial cost and a relatively short useful lifetime due to irreversible fouling and loss of functional ligand from the matrix [46]. The affinity adsorbent was avoided and scale up of the process allowed by introduction of a new process. The protein G was shown to be stable to heating to 100°C [47] so a heat treatment at 80 "C to precipitate less stable protein was followed by anion-exchange chromatography on Q-Sepharose FF and resulted in pure protein [46). The new process has the potential to be scaled up considerably in contrast to the process using IgGSepharose.

Fig. 9-7. Electrophoresis of protein G samples. Samples of (A) marker proteins, (B) IgG-Sepharose eluate and (C) Q-Sepharose eluate were subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The relative molecular mass ( M , )markers are indicated to the left of the photograph.

9.10.2 Streptavidin Streptavidin is a biotin binding protein from Streptomyces avidinii with an extraordinary Kd of I O l 5 M - ' . This extreme affinity means that elution from immobilised biotin required 6 M guanidine hydrochloride at pH 1.5. By using 2-iminobiotin as a ligand rather than biotin the protein can be purified in a single chromatography step [48]. This illustrates that it may be worthwhile to consider an alternative ligand to what may at first appear to be the obvious natural choice.

9.10.3 Glucokinase and Glycerokinase Glucokinase may be used for assay of glucose and glycerokinase for assay of serum triglycerides. Both enzymes could be purified by immobilised nucleoside phosphates but this is prohibitively expensive on the large scale. Both proteins bind to Procion Blue MX-3G while Procion Brown H-3R could be used to separate glycerokinase

2 12

9 Affinity Chromatography and its Applications in Large-Scale Separations

and glucokinase which had proved difficult by anion-exchange chromatography [49, 501. A tandem column method was used with the first column containing Procion Brown H-3R to bind to glucokinase. The unbound glycerokinase was captured on the second column in the series containing Procion Blue MX-3 G. A method involving hydrophobic interaction chromatography was also developed to separate glucokinase from glycerokinase and this avoided use of triazine dyes which had resulted in contamination of the product with dye [51].

9.10.4 Human Serum Albumin Albumin was purified from human plasma by separation on Trisacryl-Cibacron Blue F-3 GA [52]. About 250 g was bound to a 55-L column over a total of 500 cycles. To allow the adsorbent to be used this number of times, the column was subjected to a vigorous regeneration procedure involving a wash with 6~ urea and then 3 M NaCl every 15 cycles and with proteolytic enzymes every 30 cycles. However, the protein was already about 68% pure as the triazine step was the last step in the process. Whole plasma (15 L) was treated with 10% (v/v) ethanol and then 6 M acetic acid. All chromatography steps were carried out at p H 8.6. The buffer was rapidly exchanged to 25 mM Tris-HC1, containing 1.5 mM NaCl, on a 48-L column of Trisacryl GF-05. Anion exchange chromatography was performed on a 48-L column of DEAE Trisacryl M, equilibrated with 25 mM Tris-HC1 containing 15 mM NaCl and eluted with 50 mM Tris-HC1 containing 600 mM NaC1, to remove IgG. Triazine dye affinity chromatography on a 55-L column of Blue Trisacryl (Cibacron Blue, F-3 GA), equilibrated with 50 mM Tris-HC1 containing 600 mM NaCl and eluted with 50 mM Tris-HC1 containing 3 M NaCl, gave pure albumin.

9.10.5 Immunoaffinity Chromatography Immobilised antibody specifically interacts with a single surface feature of protein, not necessary a ligand binding site, and therefore may not be regarded as a true affinity adsorbent. Production of an immunoaffinity adsorbent, particularly when monoclonal antibodies are used, is expensive so immunoaffinity chromatography will probably only be used on the large scale for purification of a high value product which cannot be purified by using cheaper methods. Immunoaffinity has been increasing in popularity as monoclonal antibodies [53] are becoming more available. A major advantage has been that low concentrations of antigen can be separated though this is becoming less of an advantage as genes are cloned and highly expressed when they can be adequately purified by other techniques. Single-step purification factors of several thousand fold are possible. A disadvantage of immunoaffinity adsorbents is their high cost so relatively small columns are used with repetitive operation. There is also a high risk of fouling and chemical denaturation, notably proteolysis. A further drawback of immunoaffinity chromatography is that the

References

2 13

method is based on recognition of antigenic determinants so modified or degraded target proteins, even if inactive, will be co-purified. The use of immunoaffinity chromatography for preparation of clinical products has been reviewed by Jack and Wade [54]. Human thyroid stimulating hormone (hTSH), human follicle stimulating hormone (hFSH) and human luteinising hormone (hLH) were purified from a side fraction of human pituitary growth hormone production by immunoaffinity chromatography on immobilised hormone-specific monoclonal antibodies using a cascade system [ 5 5 ] . In the cascade system, unbound fractions were passed to another column. Monoclonal antibodies were used to selectively purify these proteins despite similar molecular mass, isoelectric point and amino acid sequence. Monoclonal antibodies are very expensive so a small automated column was considered preferable with repeated cycles rather than large columns for single preparation of a batch. This limits the loss if the column becomes contaminated or degrated. The process was easily automated which reduces labour costs and extends the working day. The use of sterile buffers and clean room conditions extends the working life of the columns. Acknowledgements

Thanks to Prof. T. Atkinson and Dr. M. D. Scawen for their comments on the manuscript.

References Lowe, C. R., in: Laboratory Techniques in Biochemistry and Molecular Biology: Work, T. S., Work, E. (eds.) Amsterdam: North-Holland, 1979; pp. 274- 522. Scouten, W. H. Affinity Chromatography: Bioselective Adsorption on Inert Matrices, New York: Wiley, 1981. Bonnerjea, J., Oh, S., Hoare, M., Dunnill, P. Bio/Technology, 1986, 4, 954-958. Secher, D.S., Burke, D. C. Nature, 1980, 285, 445-450. Janson, J. C. Trends Biotechnol, 1984, 2, 31 -38. Narayanan, S. R., Crane, L. J. Trends Biofechnol, 1990, 8, 12- 16. Goward, C. R., Stevens, G. B., Collins, I. J., Wilkinson, I. R., Scawen, M. D. Enzyme Microb Echnol, 1989, 11, 810-814. Goward, C.R., Stevens, G.B., Tattersall, R., Atkinson, T. Bioseparation, 1992, 2, 335-341. Miron, T., Wilchek, M. Appl Biochem Biotechnol, 1985, 11, 445-456. Berkowitz, S. Bio/Technology, 1987, 5, 61 -62. Kobos, R.K., Eveleigh, J. W., Arentzen, R. Trends Biotechnol, 1989, 7, 101- 105. Stewart, D. J., Purvis, D. R., Lowe, C. R. J Chromatogr, 1990, 510, 177- 187. Afeyan, N. B., Fulton, S. P., Gordon, N. E, Mazsaroff, I., Varady, L., Regnier, F.E. Bio/Technology, 1990, 8, 203-206. Dean, P. D. G., Johnson, W. S., Middle, F. A. (eds.) Affinity Chromatography - A Practical Approach, Oxford: IRL Press, 1985. Clonis, Y.D. Bio/Technology, 1987, 5, 1290- 1293. Angal, S., Dean, P.D.G. Biochem J, 1977, 167, 301-303. Fowell, S.L., Chase, H.A. J Biotechnol, 1986, 4, 1- 13. Chase, H.A. J Chromatogr, 1984, 297, 179-202.

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[I91 Scawen, M.D., Atkinson, T., in: Reactive Dyes in Protein and Enzyme Technology: Clonis, Y., Atkinson, T., Bruton, C. J., Lowe, D. R. (eds.), Basingstoke: Macmillan, 1987, pp. 51 -85. [20] Stead, C.V. Bioseparation, 1991, 2, 129- 136. [21] Burton, S. J., Pearson, J. C., Stewart, D. L. Separationsfor Biotechnology, 1990, 2, 265-275. [22] Lowe, C.R., Burton, S. J., Burton, N.P., Alderton, W.K., Pitts, J.M., Thomas, J.A. Trends Biotechnol, 1992, 10, 442-448. [23] Burton, S. J., Stead, C. V., Lowe, C. R. J Chromatogr, 1990, 508, 109- 125. [24] Atkinson, T., Hamrnond, P.M., Hartwell, D.R., Hughes, P., Scawen, M.D., Sherwood, R.F., Small, D.A.P., Bruton, C. J., Harvey, M. J., Lowe, C. R. Biochem SOCTrans, 1981, 9, 290-293. [25] Qadri, F. Trends Biotechnol, 1985, 3, 7 - 12. [26] Atkinson, T., Scawen, M. D., Hammond, P. M., in: Biotechnology: Rehm, H.-J., Reed, G. (eds.) Weinheim: VCH Verlagsgesellschaft, 1987; Vol. 7A, pp. 279 - 323. [27] Lowe, C. R., Dean, P. D. G. FEBS Lett, 1979, 14, 313-316. [28] Mosbach, K., Guilford, H., Ohlsson, R., Scott, M. Biochem J. [29] Lamkin, G., King, E. Biochem Biophys Res Commun, 1976, 72, 560-565. [30] Janson, J.C., Hedrnan, G. Adv Biochem Eng, 1982, 25, 43-99. [31] Hammond, P.M., Scawen, M.D. J Biotechnol, 1989, 11, 119-134. [32] Scawen, M. D., Atkinson, T., Hammond, P. M., Sherwood, R. E, in: Molecular Biology and Biotechnology: Walker, J. M., Gingold, E. B. (eds.) Cambridge: The Royal Society of Chemistry, 1993, pp. 327-355. [33] Sherwood, R. F. Trends Biotechnol, 1991, 9, 1-3. [34] Sassenfeld, H.M., Brewer, S. J. Bio/Technology, 1984, 2, 76-81. [35] Popplewell, A. G., Gore, M. G., Scawen, M., Atkinson, T. Protein Eng, 1991, 4 , 963 -970. [36] Sassenfeld, H.M. Trends Biotechnol, 1990, 8, 88-93. [37] UhlCn, M., Moks T. Methods Enzynol, 1990, 185, 129.- 143. [38] Hochuli, E., Bannwarth, W., Dobeli, H., Gentz, R., Stuber, D. Bio/Technology, 1988, 6, 1321 - 1325. [39] Hopp, T.P., Prickett, K. S., Price, V.L., Libby, R. L., March, C. J., Ceretti, P.T., Urdal, D. L., Conlon, P. J. Bio/Technology, 1988, 6, 1204-1210. [40] Moks, T., Abrahmsen. L., Holmgren, E., Billich, M., Olsson, A., UhlCn, M., Pohl, G., Sterky, C., Hultberg, H., Josephson, S., Holmgren, A., Jorvall, H., Nilsson B. Biochemistry, 1987, 26, 5239-5244. [41] Moks, T., AbrahmsCn, L., Osterloff, B., Josephson, S., Ostling, M., Enfors, S.-O., Persson, I., Nilsson, B., U h l h , M. Bio/Technology, 1987, 5, 379-382. [42] Bornstein, P., Balian, G. Methods Enzymol, 1977, 47, 132-145. [43] Hammarburg, B., Nygren, P.-A., Holmgren, E., Elrnblad, A., Tally, M., Hellman, U., Moks, T., UhlCn M. Proc Nut1 Acad Sci USA, 1989, 86, 4367-4371. [44] Jansson, B., Palmcrantz, C., UhlCn, M., Nilsson, B. Protein Eng, 1990, 2, 555-561. [45] Goward, C. R., Murphy, J.P., Atkinson, T., Barstow, D.A. Biochem J, 1990, 267, 171 - 177. [46] Murphy, J.P., Atkinson, T., Hartwell, R., Stevens, G.B., Hinton, R. J., Goward, C.R. Bioseparation, 1992, 3, 63 -71. [47] Goward, C.R., Irons, L. I., Murphy, J. P., Atkinson, T. Biochem J, 1991, 274, 503 -507. [48] Bayer, E. A., Ben-Hur, H., Gitlin, G., Wilchek, M. J Biochem Biophys Methods, 1986, 13, 103 - 112. [49] Scawen, M.D., Harnrnond, P.M., Comer, M. J., Atkinson, T. Anal Biochem, 1983, 132, 413 -417. [50] Goward, C.R., Hartwell, R., Atkinson, T., Scawen, M.D. Biochem J, 1988, 237, 415-420. [51] Goward, C.R., Atkinson, T., Scawen, M.D. J Chromatogr, 1986, 369, 235-239. [52] Allary, M., Saint-Blancard, J., Boschetti, E., Girot, P. Bioseparation, 1991, 2, 167- 175. [53] Kohler, G., Milstein, C. Nature, 1975, 256, 495-497. [54] Jack, G. W., Wade, H.E. Trends Biotechnol, 1987, 5, 91 -95. [55] Jack, G. W., Blazek, R., James, K., Boyd, J.E., Micklem, L. R. J Chem Tech Biotechnol, 1987, 39, 45-58.

Index

a gel in shell 125 absolute refractive index detectors 21 absorption monitors 21 Accutrap 189 acrylamide monomer 101, 103 activation 117f. - of Eupergit 118 - of HEMA 118 - of polymers 117 adsorbents - fouling 126 - polymeric 100 - surface coating 107 adsorption effects of SEC media 89 adsorption isotherm 35 adsorption on an affinity adsorbent 203 Affi-Gel 106 affinity 109, 193 affinity adsorbents 193 - cleaning and storage 207 affinity chromatography 104, 123, 193ff., 197 ff., 210ff. - activating agents 198 - activation of the matrix 198 - advantages and disadvantages 194 - binding to an affinity adsorbent 204 - capacity of the adsorbent 198f. - cleaning and storage of adsorbents 207 - column for large-scale chromatography 202 - coupling of a ligand to the support matrix 197 - elution 205 - equilibrium dissociation constant 197 - examples of some large-scale methods 210 - flow rate in affinity elution 206 - fouling of adsorbent 207 - important features of a ligand 195

large-scale purification of human serum albumin 212 - ligand leakage 200 - methods of immobilisation 198 - non-selective elution 193, 205 - process control 203 - process design 201 - purification of protein G 210 - purification of streptavidin 211 - scaling up 202 - selective elution 193, 205 - spacer arms 198 - steric hindrance 198 - steric occlusion of the ligand 198f. - support matrix 194 - use of triazine dyes as ligands 200 - washing after application of the sample 204 affinity chromatography media 201 - fouling 201 affinity ligands 195 - important features 195 affinity ligands for proteins - examples 196 - resistance to proteases, elution agents and cleaning agents 197 affinity supports 123 affinity tails - examples of use 209 - release 208 agaropectin 105 agarose 101, 103ff., 118, 120, 122f., 132, 200 - affinity chromatography 106 - coupling of triazine dyes 200, 201 - crystalline microstructure 105 - ion exchange chromatography 105 - mechanical rigidity 113 - molecular structure 103 - schematic representation of the pore structure 105 -

216

Index

- separations of biological macromolecules 103 - size exclusion chromatography 105 agarose gel 86 - scanning electron micrograph 86 alumina 107 Aminex 103 amino acids, separation by hydrophilic interaction chromatography 124 ammonia, critical points for fluids 166 analytical chromatography 36 - diode array detectors 18 - distribution coefficient 36 - dosing pumps 19 - mass load 36 - post column detection 18 analytical column 11 analytical SFC 153, 175, 189 - flame ionisation detector (FID) 189 - injection 175 - photoionisation detector (PID) 189 anion exchange 122 - principles 133 - relationship between pore size and specific ionic or protein capacity 113 anion exchanger 134, 137 anion-exchange chromatography 99, 132, 144 - of goat serum proteins 144 antibodies, purification by affinity chromatography 123 antigens 212 - purification by affinity chromatography 123 automation 22 axial compression 13

bacteriostatic agents in affinity chromatography 207 ball valves 15 - sanitary applications 15 baseline separation 41 B-cells 146 benzyl alcohol, separation from phenol 49 Bio-Gel activation I18 BioGeF 85 Biogel P 104, 120 BioGel P-2 85 biological macromolecules, purification by affinity chromatography 123 biomimetic 123 bioprocess chromatography 125 bioselective 123 bisethylene glycol methacrylate 104 blood proteins, fractionation 142

bubble trap 17 butanediol divinyl ether 104 capacity factor 35, 39f., 64, 68 - analytical 40 - at low load 39 - of the overloaded peak 40 Carbo Pac 108 carbohydrates, separation by polyacrylamide 104 carbon dioxide 153f., 156f., 178 - critical points for fluids 166 - expansion 178 - phase diagram 164 - subcritical 165 - supercritical 164f. - supercritical fluid 153 - use in SFC 166 - use in SFE 166 carboxymethyl (CM) cellulose 99, 107, 119, 122 cation exchange 122, 132 - principles 133 cation exchanger 132, 134, 137 cation-exchange chromatography 99, 132 cellulose 81, 101, 118, 120, 122, 132 - base matrix 106 - microcrystalline 106 - molecular structure 103 - regenerated 106 chaotropic agents, use in affinity chromatography 193, 206 chemical stability 126 - of polymeric adsorbents 126 chiral separations 123 chromatograph 2, lOf., 26 - components 2 - example of control screen 26 - piping and instrumentation drawing (P&ID) 11 chromatographic productivity in SEC 93 chromatography 35 - combination of thermodynamic and kinetic processes 35 - Craig model 41 - goat serum proteins 142 - ‘ideal model’ 40 - of a monoclonal antibody 146 - rate limiting step in production 72 - simulation of mass overloaded chromatography 41 chromatography controller - computer based systems 23f.

Index dedicated controller 23 general purpose controller 23f. - requirements 25 chromatography system 1ff., 22 - analytical 1 - automation 22 - bubble trap 17 - cleaned in place (CIP) 6 - column selection 11 - component selection 11 - contamination 6 - control 1 ff., 8, 22 - design l f f . , 9 - electrical standards 5 - filters 16 - flow diagram 9 - flow rate 19 - functionality 3 - gradient 17 - hygiene 6 - instrumentation 18 - main components 1 - material compatibility 3 - monitoring of pressure 20 - overpressurization 20 - pipework 16 - preparative 1 - pressure 4, 20 - protection from overpressure 20 - pumps 14 - refractive index detectors 21 - reliability 8 - requirements 2 - sanitary design 6 - servicability 9 - solvent flow 19 - temperature monitoring 20 - valves 15 a-chymotrypsinogen, purification by size exclusion chromatography 120 CM-cellulose see carboxymethyl (CM) cellulose coffee, decaffeination 164 column efficiency 50ff., 56, 114 - influence on production rate 56 - influence on solvent efficiency 56 - influence upon recovery of products 51 column resistance parameter 57 column saturation capacity 64 column shield jacket 171 columns 11, 13 - analytical 11 - calculation of saturation capacity 64 -

217

- dimension in process scale SEC 90 - for large-scale affinity chromatography 202 - for PS-SFC 157 - high pressure chromatography 13 - low pressure chromatography 13 - maximum capacity 11 - maximum efficiency 11 - packing by axial compression 13 - packing in low-pressure chromatography 13 - performance 114 - preparative 11 - radial distribution 11 - saturation capacity 39f. - selection 11 competitive isotherms 47, 67 composite gels 87 - size exclusion chromatography 87 composite polymers, schematic representation 106 computer based systems 23 f. computer simulations 41, 46 - in multi-component preparative chromatography 46 - of mass overloaded chromatography 41 - using competitive isotherms derived from the ideal adsorbed solution theory 48 - variation in relative load 46 concentration profile 68 conductivity sensor 22 - four electrode types 22 - two electrode types 22 contamination of chromatography systems 6 core shell grafts 108 cost of solvent 54 cost optimisation - on laboratory scale 58 - on production scale 59 Craig model 41, 67f. - scheme 68 - simulations 68 critical point 163 critical points for fluids 166 critical temperature 163, 165 cross-linked agarose 86 cross-linked dextran 81, 85 - molecular structure 103 cross-linker 100 5-C-terminal amide decapeptides, separation using a polystyrene reversed phase column 112 cyclobond 124 cyclodextrin 124

218

Index

DEAE-cellulose see diethylaminoethyl (DEAE) cellulose decanation 158 dedicated controller 23 de-ethanolization of human serum albumin by SEC 95 deformable matrices, influence of pressure 4 denaturing agent, use in affinity chromatography 193 derivatization 118 desalting by size exclusion chromatography 81 detector 21 - fixed wavelength detector 21 - refractive index detector 21 - variable wavelength detector 21 dextran 101, 104f., 118, 120, 122, 132 - homopolymer 104 - schematic representation of the pore structure 105 dextran gels 85 - for size exclusion chromatography 85 diaphragm pumps 14 diaphragm valves 15f. dichlorotriazinyl dyes, ligands in affinity chromatography 201 diethylaminoethyl (DEAE) cellulose 99, 107, 119, 122, 132 differential mass balance equations 40 diffusion coefficients of supercritical fluids 164 diode array UV detector (DAD) 189 dispersion effect 67 displacement 73, 76f. - between adjacent peaks 76 - between adjacent peaks with non-linear isotherms 77 displacement chromatography 74, 76 distribution coefficient 35 divinylbenzene, divalent cross-linker 103 DNA-modifying enzymes 147 - chromatography 147 dosing pumps 19 droplet stabiliser 101 eddy diffusion 115 electrical standards 5 electrophoresis of protein G 211 electrophoretic elution in affinity chromatography 206 electropolishing 7 f. - of stainless steel 7

eluent - in size exclusion chromatography 89 - supercritical 156 eluent compatibility, of polymers 116 eluent cycle of PS-SFC 154 eluent recycling 158 - in PS-SFC 158 eluting buffer 84 elution 73 - in affinity chromatography 205 elution chromatography 73 f. - chromatogram 74 elution time 19 enzymes, purification by affinity chromatography 123 epichlorohydrin, cross-linker 104 ethane, critical points for fluids 166 ethylene, critical points for fluids 166 ethylene glycol methacrylate 104 Eupergit 104, 118 - activation 118 extracellular proteins, extraction by affinity chromatography 201 feed stock preparation, for size exclusion chromatography 93 filters 16 - air traps 16 fixed wavelength detector 21 flame ionisation detector (FID) 189 ‘flip-flop’ technique 78 flow 19 - uniform 11 flow diagram 9 flow meters 19 flow rate 19 - in affinity elution 206 fouling 124, 126 fraction collection 158 - in PS-SFC 158 fractionation - by size exclusion chromatography 81 - of blood proteins 142 Fractogel 104, 195 freons, supercritical 188 frontal 73 frontal chromatography, chromatogram 75 functionalisation of polymers 117 gel filtration 120, 200 - non-interactiv 120 gel filtration chromatography 99

Index gel medium 83 gel permeation chromatography 120 gel preparation in size exclusion chromatography 92 gels 85f. - agarose 86 - dextran 85 - polyacrylamide 85 general purpose controller 23 f. glucokinase 211 f. - purification by affinity chromatography 211. 212 glycerokinase 211 f. - purification by affinity chromatography 211, 212 gradient generation 20 - temperature fluctuations 20 gradients 1, 17 - formation 17 Hagen-Poiseuille equation 180 HEMA 104, 118, 120f. - derivatization 118 - molecular structure 103 - use for hydrophobic interaction chromatography 121 hen egg-white proteins 137 - separation by ion exchange chromatography 137f. hexane, critical points for fluids 166 high load 63 high pressure chromatography 13f. - columns 13 - pulsation dampeners 14 - pumps 14 high speed separations 124 - of large molecules 124 highly cross-linked polymers 81 histones, separation by hydrophilic interaction chromatography 124 homopolymer 104 HPLC 161 - comparison of applications to PS-SFC 161 - purification cost 161 HPLC system - material compatibility 3 HSA human serum albumin 91 human follicle stimulating hormone (hFSM), purification by immunoaffinity chromatography 213 human luteinising hormone (hLH), purification by immunoaffinity chromatography 213

219

human pituitary growth hormone 213 human serum albumin - conditions for process scale SEC 91 - de-ethanolization by SEC 95 - elution diagram for process scale SEC 94 - production facility 91 - purification by large-scale affinity chromatography 212 human thyroid stimulating hormone (hTSH), purification by immunoaffinity chromatography 213 hybridoma L 243 146f. hydrophilic interaction chromatography I24 hydrophilic vinyl polymer 104 hydrophobic interaction chromatography 104, 121 - separation of glucokinase from glycerokinase 212 hydroxyethylmethacrylate see HEMA hygiene 6 - in chromatography systems 6 Hyper D media 108 hyper diffusion chromatography 125 ideal adsorbed solution theory 47, 49 ideal model of chromatography 40 immobilisation in affinity chromatography 198 immobilised antibody column 193 immunoaffinity adsorbent 212 irnmunoaffinity chromatography 146, 200, 205, 212f. - ligand leakage 200 - selective elution 205 immunoglobulin G (IgG) 144, 146 - computer simulation of productivity and recovery in SEC on Superdex@200 prep grade 94 - isoIation 144 induction sensor 22 infinite column efficiency 66 infinite multiport distribution system 12 infrared (IR) detector 189 injection volume 42, 69 insulin - conditions for process scale SEC 91 - production facility 90 interactive chromatography 109 interferon, purification by affinity chromatography 193 interpenetrating polymer network 109

220

Index

ion exchange 109, 122, 131f. - functional groups 132ff. - in low pressure chromatography 131 ion exchanger 122 - polysaccharide based 122 - strong 122 - weak 122 ion-exchange celluloses 136 ion-exchange chromatographic 137 - hen egg-white proteins 137 ion-exchange chromatography 99, 104, 200f. - approaches 136 - of a monoclonal antibody 146 - principles 132 ion-exchange fractionations 122 ion-exchange liquid chromatography 131 - biochemical applications of process-scale 131 ionic strength 99 isotherms 36ff. - competitive 47 - Langmuir 36 - non-Langmuir 38 - ‘S’ shaped 37 Joule-Thompson effect 178

Knox Equation 57 Langmuir isotherm 36f., 63 f., 69 - competitive 47 - determination 39 - peak shape of the solute 37 ligands 8f., 195 - biomimetic 123 - important features 195 - leakage 200 - steric occlusion 198f. light scattering detector (LSD) 189 linear flow velocity 168 linear velocity 69 liquid chromatography 120 - application of polymers 99ff. - applications 119 - chemical stabiltiy of adsorbent 126 - choice of adsorbent 125 - examples of commericially available polymeric adsorbents 128 - inorganic materials 107 - permeability 110 - recovery of biological activity 127 - regeneration of the adsorbent 126 loading factor 40, 64

longitudinal molecular diffusion 115 low molecular weight solutes, diffusion coefficient 81 low pressure chromatography - hygiene 6 - pumps 14 - sanitary 29 lymphocytes 146 lysozyme 137 - purification by size exclusion chromatography 120 macrophages 146 macroporous polymers 99f. - schematic representation of the morphology 100 ‘Magflow’ meters 19 mass balance model 65ff. - representation of the calculation grid 67 mass overload 35 - increase in peak asymmetry 35 - triangular shaped peaks 35 material compability 3 - in chromatography systems 3 matrix 1 - fouling 13 maximum capacity 11 maximum production rate 54 maximum solvent efficiency 56 mechanical rigidity 113 - of macroporous polymers 113 medium pressure chromatography, hygiene 6 membrane proteins, separation by hydrophilic interaction chromatography 124 mercury porosimetry for measurement of pore size 111 Merkogel GPC 104 methacrylate - monomer I01 - use of derivatives as the monomer 104 methylenebisacrylamide 103f. microporous polymers 99 f. - schematic representation of the morphology 100 microspheres 106 mobile phase, gradient formation 17 mobile phase mass transfer 115 modifier 165, 188 - in PS-SFC 153, 156, 158 - preparative SFC 188 Mono Q 123 Mono S 123 Monobeads 103

Index monoclonal antibodies 146, 212 f. - chromatography 146 monocytes 146 monomers, synthetic 101 multi-component preparative chromatography 46 multiple solutes 44ff. - computer simulations 46 multiport valves 15 myoglobin, purification by size exclusion chromatography 120 natural polymers 101, 104, 106 nicotine 165 - removal from tobacco 165 nitrogen adsorption for measurement of pore size 111 nitrous oxide, critical points for fluids 166 non uniform flow 16 non-selective eluants in affinity chromatography 205 non-selective elution in affinity chromatography 205 optical isomers, resolution 123 optimisation - in preparative chromatography 54 - of cost on laboratory scale 58 - of cost on production scale 59 - of production rate 56 - practical 61 ovalbumin 137 - large-scale separation by ion-exchange chromatography 142 - purification by ion-exchange chromatography 140 - purification by size exclusion chromatography 120 overpressurization 20 ovotransferin, purification by ion-exchange chromatography 140 packing materials 85 particle size of polymers 109 peak broadening 11 peaks, triangular shaped 35 pellicular supports 108 peptides - selectivity curves of SuperdexB 30 prep grade 83 - separation by hydrophilic interaction chromatography 124 - separation by polyacrylamide 104

221

perfusion chromatography 125, 195f. - particles 196 pH probes 22 pH sensor 22 pharmaceutical products, validation 29 phase diagram 154 - near critical carbon dioxide 165 - supercritical carbon dioxide 165 phenol, separation from benzyl alcohol 49 phosphate buffer 105 photoionisation detector (PID) 189 pipework 16 piston pumps 14 plate height (HETP), dependence on flow velocity 115 PLCs 24 PLgel 103 PGGFC 103 PLRP-S 103 PGSAX 103, 107, 123 PLSCX 123 - synthetic 104 polyacrylamide 81, 101ff., 118 - activation 118 - molecular structure 102f. - monomer 103 - use in size exclusion chromatography 120 polyacrylamide gels 85 - for size exclusion chromatography 85 polyclonal 146 polyethylene glycols, selectivity curves of Superdex@30 prep grade 83 polyglycidyl methacrylate 104 polymer network 100 polymeric gel matrices, bacterial degradation 6 polymeric matrices 101 - types for liquid chromatography 101 polymerisation, manufacture of polymeric adsorbents 101 polymers 99 ff. - activation 117 - application in liquid chromatography 99 ff. - chemical stability 126 - composite materials 107 - core shell grafts 108 - eluent compatibility 116 - examples of commercially available polymeric adsorbents 128 - fouling 126 - functionalisation 117 - interpenetrating networks 109

222

Index

macroporous 100 manufacturing process 101 - mechanical rigidity 113 - microporous 100 - molecular structures 102 - natural 101, 104 - particle size 109 - pellicular supports 108 - pore matrix composites 108 - pore size 110 - pore size distribution 110 - regeneration 126 - solvent strength 116 - surface area 111 - surface coating 107 - synthetic 101, 103 polymethacrylate 104, 118 polymethylmehtacrylate 101 polypeptide tail 208 polypeptides 83 polysaccharides 118f. - as ion exchangers 119 - fractionation by size exclusion chromatography 86 - natural polymers 101 - use for affinity chromatography 119 polystyrene lolff., 111, 117 - derivatization by chloromethylation 117 - ion exchanger for amino acid and carbohydrate separations 101 - molecular structure 102f. polystyrene HPLC matrices 114 114 - flow rate versus column pressure polyvinylacetate 104 polyvinylalcohol 104 pore matrix composite 108 pore size 110f. - measurement by mercury porosimetry 111 - measurement by nitrogen adsorption 111 - of polymers 110 pore size distribution of polymers 110 porogen loof., 103 POROSTM 195 positive displacement pumps 14 post column detection, process chromato graphy 18 preparative chromatography 44 ff. - Craig model 67 - enantiomeric separation on triacetyl cellulose 77 - flow meters 19 - mass-balance model 65 - mathematical models 65 ff. -

-

- multiple solutes 44 - optimisation 54 - recyclelpeak shaving technique 77 - volume overload 42 preparative column 11 preparative enantiomeric separations 78 preparative high pressure liquid chromatography see preparative HPLC preparative HPLC 33 ff., 153 - comparison to PS-SFC 156 - mass overload 35 - overload of single solute 34 - practical application of theory 33ff. - volume overload 35, 42 preparative liquid chromatography 33 ff. - practical application of theory 33 ff. - volume overload 42 preparative scale supercritical fluid chromatography see PS-SFC preparative SFC 169ff., 188f. - basic chromatograph 172 - chiral separations 188 - collection of dyestuff in the experimental collection vessel 183 - column shield jacket 171 - detection of solutes 188 - diode array UV detector (DAD) 189 - experimental sample collection vessel 181f. - flame ionisation detector (FID) 189 - high pressure trapping of the carbon dioxide 180 - history 169 - hydraulic diagram 173 - loading and injection of samples 172 - recovery of acetophenone 179, 180 - safety considerations 171 - sample collection vessel 183ff. - sample introduction pressure vessel (SIPV) 175 - variable wavelength UV absorption detectors 188 preparative SFC see also PS-SFC (preparative scale supercritical fluid chromatography) preparative supercritical fluid chromatograph 178 - fraction collection 178 preparative supercritical fluid chromatography see preparative SFC; see also PS-SFC (preparative scale supercritical fluid chromatography) Prepmaster 189 pressure 20 pressure drop in chromatography systems 4

Index pressure gauges 20 - bourdon tube type 20 pressure transducers 20 pressurization 158 process chromatography 71 - general considerations 71 - mass overload 76 - post column detection 18 - use of alternative modes and techniques 78 process-scale ion-exchange liquid chromatography 134ff. - biochemical applications 136 Procion Blue MX-3G 211 f. Procion Brown H-3R 211 f. production - cost 54 - maximum production rate 54 production rate 56 - influence of column efficiency 56 - optimisation 56 programmable logic controllers (PLCs) 24 propane, critical points for fluids 166 propylene, critical points for fluids 166 protein complexes, fractionation by size exclusion chromatography 86 protein engineering, applied to protein purification 208 protein G - electrophoresis 211 - purification 210 proteins - isoelectric point 134 - purification by hydrophobic interaction chromatography 121 - selectivity curves of Superdex@30 prep grade 83 - separation by size exclusion chromatography 81, 120 PRP 103 PS-SFC 153ff. - chromatographic column 157 - comparison of applications to HPLC 161 - comparison to preparative HPLC 156 - drawbacks 156 - eluent cycle 154 - eluent recycling 158 - flow sheet fo the process 155 - fraction collection 158 - instrumental design and separation on a large scale 153ff. - modifier 153, 156

223

pumping system 157 - purification cost 160 - safety control 159 - separation cost 159 - separator 156, 158 - technology 157 PS-SFC see also preparative SFC pulsation dampeners 14f., 17 pumping system 157 - in PS-SFC 157 pumps 1, 14 - double-diaphragm versions 14 - pulsation damperners 14 - types 14 -

radial compression 13 radial dispersion 12 radial distribution 11f. recombinant proteins, extraction by affinity chromatography 201 recovery of biological activity 127 recycle/peak shaving technique 77 refractive index 21 refractive index (RI) detectors 21, 189 regeneration 126 - or the chromatographic adsorbent 126 restricted diffusion 110 reversed phase adsorbents 121 reversed phase chromatography 121 RheodynC 7037 pressure restriction valve 179 Rheodyne 7000 valve 182 Roughness Average (Ra) 7 ‘S’ shaped isotherm 37 ‘S’ unshaped isotherm 38 - peak shape of the solute 38 sample band broadening factors 115 sample introduction pressure vessel (SIPV) 175ff. - elements of its construction 177 saturation capacity 39, 64 f., 69 - calculation 64 scaling up 134 - of an ion-exchange step 134f. - size exclusion chromatography 96 scaling-up process 167 - for supercritical fluid chromatography 167 SEC see size exclusion chromatography selective elution 205 - in affinity chromatography 205 separator, in PS-SFC 156, 158 SephacryP 87

224

Index

Sephadex@ 85, 105 Sephadex G-25 85 Sepharose 105 Sepharose CL 105 Sepharose@Fast Flow 86 Sepharose FF 195 SFC see supercritical fluid chromatography SFE see supercritical fluid extraction silica 107 simulated moving bed chromatography 78 - preparative enantiomeric separations 78 simulation 76 single solutes 34 - calibration curves 110 size exclusion chromatography (SEC) 81 ff., 109, 111 - adsorption effects of media 89 - agarose 105 - agarose gels 86 - characteristics of media for desalting applications 88 - characteristics of media for fractionation applications 88 - choice of separation medium 87 - chromatographic productivity 93 - column dimension in process scale 90 - column packing 92 - column packing materials for process scale SEC 85 - composite gels 87 - computer simulation of productivity and recovery 94 - conditions for desalting and solvent removal 96, 97 - desalting 81 - dextran gels 85 - eluent 89 - elution diagram for process scale SEC of human serum albumin 95 - equations describing SEC 82 - feed stock preparation 93 - for separation of proteins 120 - fractionation 81 - fractionation of polysaccharides 86 - fractionation of protein complexes 86 - gel preparation 92 - of goat serum proteins 144 - polyacrylamide gels 85 - pore size distribution 82 - process scale SEC of human serum albumin 91 - process scale SEC of insulin 91 - selectivity curves 87

- separation power of different media 87 - separation principle 82 - separation volume 83 - stainless steel columns for desalting operations 96 - strategy for scaling-up 96 - viscosity of the sample 81 ‘soft gel’ polymeric matrices 17 solute - capacity factor 35 - distribution coefficient 35 solvation strength of supercritical fluids 164 solvent 56 - cost 54 - maximum efficiency 56 - recovery process 56 solvent efficiency 56 - influence of column efficiency 56 solvent flow 19 solvent strength of polymers 116 solvent viscosity 57 spacer arms in affinity chromatography 197, 201 stagnant mobile phase mass transfer 115 stainless steel 7f. - cleaning 8 - electropolishing 7 f. - passive surface oxide film 7 - Roughness Average (Ra) 7 stationary phase 1 stationary phase mass transfer 115 steric hindrance in affinity chromatography 197f. steric occlusion 199 streptavidin 211 - purification by affinity chromatography 211 Styragel 103, 120 styrene 103 - monomer 101 supercritical eluent 156 - solvent capacity 156 supercritical fluid 156 supercritical fluid chromatography (SFC) 163, 167f., 189 - choice of supercritical fluids 165 - coupling with supercritical fluid extraction 170 - development of large scale commercial systems 186 - light scattering detector (LSD) 189 - Prochrom process scale supercritical fluid chromatograph 187

Index - recent developments 189 - scaling-up process 167f. - scaling-up to large-scale applications 163ff. supercritical fluid extraction (SFE) 153, 189 - Accutrap 189, 190 - choice of supercritical fluids 165 - Prepmaster 189 supercritical fluid extractor, conversion to a preparative SFC unit 186 supercritical fluids 153, 163ff., 169 - choice 165 - diffusion coefficients 164 - flow properties 169 - solvation strength 164 supercritical state 163 Superdex@ 87 support matrix 194 - in affinity chromatography 194 surface area - measurement by nitrogen adsorption 111 - of polymers 111 - polystyrene 111 - silica 111 surface coatings 107 suspension polymerisation process 101 synthetic polymers 103

T-cells, activated 146 temperature control 20 temperature monitoring 20 thermal stability of agarose gels 86 throughput 134 tobacco, removal of nicotine 164 Toyopearl 104 TPX (tri-methyl-pentene polymer) 13 triacetyl cellulose 77, 124 triangular peak shape 38 triazine dyes 200, 212 triazine group 200, 201 Trisacryl 104, 118, 195 TSKgel 120 - modified 123 TSKgel Ether PW 121 TSKgel Phenyl PW 121 TSKgel PW 104, 121 - ball valves 15 - diaphragm valves 15 - multiport valves 15 variable wavelength detector 21 volume overload 35, 42, 44 water, critical points for fluids 166 xenon 189

225