K. Hostettmann A. Marston M. Hostettmann Preparative Chromatography Techniques

K. Hostettmann A. Marston M. Hostettmann Preparative Chromatography Techniques

Springer-Verlag Berlin Heidelberg GmbH

K. Hostettmann A. Marston M. Hostettmann Preparative Chromatography Techniques Applications in Natural Product Isolation Second, Completely Revised and Enlarged Edition With 59 Figures ~Springer

Professor Dr. K. Hostettmann Dr. A. Marston Dr. M. Hostettmann University of Lausanne Institute of Pharmacognosy and Phytochemistry CH-1015 Lausanne - Dorigny Switzerland ISBN 978-3-642-08288-7 Die Deutsche Bibliothek - CIP-Einheitsaufnahme Hostettmann, Kurt: Preparative chromatography techniques : applications in natural product isolation 1 K. Hostettmann ; A. Marston ; M. Hostettmann. - 2., completely rev. and enl. ed. ISBN 978-3-642-08288-7 ISBN 978-3-662-03631-0 (ebook) DOI 10.1007/978-3-662-03631-0 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translations, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other ways, and storage in data banks. Duplication of this publication Of parts thereof is only permitted under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for under the German Copyright Law. Springer-Verlag Berlin Heidelberg 1998 Originally published by Springer-Verlag Berlin Heidelberg New York in 1998 Softcover reprint of the hardcover 2nd edition 1998 The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. production Editor: Christiane Messerschmidt, Rheinau Typesetting: Fotosatz-Service Kiihler OHG, Wiirzburg Cover design: Design & Production GmbH, Heidelberg SPIN: 10470948 52/3020-5 4 3 2 1 0- Printed on acid-free paper

Preface Over the past few years, increasing attention has been paid to the search for bioactive compounds from natural sources. The success of plant-derived products such as paclitaxel (Taxol) in tumor therapy or artemisinin in the treatment of malaria has provided the impetus for the introduction of numerous research programmes, especially in Industry. A great deal of effort is being expended in the generation of novel lead molecules of vegetable, marine and microbial origin by the use of high throughput screening protocols. When interesting hits are found, it is essential to have methods available for the rapid isolation of target compounds. For this reason, both industry and academia need efficient preparative chromatographic separation techniques and experience in their application. Purified natural products are required for complete spectroscopic identification and full characterization of new compounds, for biological testing and for the supply of pharmaceuticals, standards, and starting materials for synthetic work. Obtaining pure products from an extract can be a very long, tedious and expensive undertaking, involving many steps. Sometimes only minute amounts of the desired compounds are at hand and these entities may be labile. Thus it is an advantage to have access to as many different methods as possible in order to aid the isolation process. Although a certain amount of trial and error may be involved, nowadays there is the possibility of devising suitable rapid separation schemes by a judicious choice of the different techniques available. While a vast amount of literature is available about analytical procedures, very few monographs specifically treat preparative chromatography. The aim here is to fill this gap and provide a guide to isolation strategy. Since the first edition of this book in 1986, there have been important advances in the study of compounds from natural sources. The rapid development of spectroscopic techniques, including 2D-NMR methods, automated instrumentation and routine availability of X-ray crystallography, has greatly simplified the structure elucidation of natural products. Consequently, the

VI Preface main challenge is now the efficient isolation and purification of bioactive components from different organisms (plants, animals, microorganisms etc.). These range from highly polar to very lipophilic compounds. Bioactivity can often be lost during chromatography and it is essential to have techniques available which provide gentle separation conditions. New and improved chromatographic processes are constantly being introduced and these have to be employed judiciously in an effort to solve the separation problems at hand. The aim of this volume is to build on the material covered in the first edition and to bring together the rather disperse applications of the new chromatographic techniques so that the reader has the means of selecting an approach for his own separation problem. For this reason, a critical look has been taken at the different methods available. This is supplemented by numerous examples of each chromatographic procedure, which can be used for reference purposes. No attempt has been made to provide an exhaustive literature survey but consultation of the bibliography given with each section will cover most of the ground necessary for an appreciation of the possibilities available. The first part of the book describes the important area of sample preparation. Much time can be saved by pre-treatment of a sample before chromatography. Liquid-solid chromatographic procedures are then covered, with emphasis on techniques which involve the application of pressure. All-liquid chromatography is introduced and some idea of given of its advantages in the separation of problematic labile compounds. Chapters on the separation of macromolecules and chiral substances are also provided. These two themes are of vital importance for the isolation of new pharmaceuticals, gene products and even primary metabolites. Finally, there is an insight into strategy when designing separation protocols. Lausanne, July 1997 K. Hostettmann A. Marston M. Hostettmann

Contents List of Symbols and Abbreviations...... 1 Introduction.............. 2 Sample Preparation and Purification 2.1 Extraction............... 2.2 Supercritical Fluid Extraction (SFE) 2.3 Solvent Partition 2.4 Filtration 2.5 Gel Filtration.. 2.6 Precipitation 2.7 Solid Introduction in Chromatography 2.8 Removal of Chlorophyll 2.9 Removal of Waxes.. 2.10 Removal of Tannins 2.11 Solid-Phase Extraction. 2.12 Preliminary Purification for Preparative High-Performance Liquid Chromatography 2.13 References.... 3 Planar Chromatography.......... 3.1 Preparative Thin-Layer Chromatography 3.1.1 Adsorbents................. 3.1.2 Sample Application.... 3.1.3 Choice of Mobile Phase and Development 3.1.4 3.1.5 3.1.6 3.2 3.2.1 3.2.2 3.2.3 3.3 4 4.1 4.1.1 4.2 of the PTLC Plate.............. Isolation of Separated Substances Impurities in Substances Separated by PTLC Overpressured Layer Chromatography (OPLC) Centrifugal Thin-Layer Chromatography Historical Development..... Apparatus.... Application of the Chromatotron References............. Special Column Chromatography Dry-Column Chromatography Applications............ Vacuum Liquid Chromatography XI 1 3 3 4 6 8 8 8 8 9 9 10 11 12 13 15 15 15 16 16 17 17 18 19 19 20 24 31 33 33 36 39

VIII Contents 4.2.1 4.3 5 5.1 5.1.1 5.1.2 5.1.3 5.1.4 5.1.5 5.1.6 5.1.7 5.1.8 5.1.9 5.1.10 5.1.11 5.1.12 5.1.13 5.2 5.2.1 5.2.2 5.2.3 5.2.4 5.3 5.3.1 5.3.2 5.3.3 5.3.4 5.3.5 5.4 6 6.1 6.2 6.3 6.4 6.5 7 7.1 7.1.1 7.1.2 7.1.3 7.1.4 7.1.5 7.2 7.2.1 7.2.2 Applications........................ Preparative Pressure Liquid Chromatography.... Basic Principles...................... Method Development and Optimisation........ Columns.... Stationary Phases..................... Column Packing Methods................ Sample Introduction................... Pumps.... Detectors.... Mobile Phases....................... Collection of Separated Material............ Shave and Recycle Chromatography.......... Column Overloading and Heart Cutting........ Column Switching.................... Peak Magnitude..................... Different Preparative Pressure Liquid Chromatographic Methods............... Flash Chromatography.................. Low-Pressure LC (LPLC)................. Medium-Pressure LC (MPLC).... High-Pressure LC (HPLC).... Supercritical Fluid Chromatography (SFC).... Supercritical Fluids.................... Load.... Special Considerations.................. Large-Scale Systems................... Separations by SFC.................... Preparative Gas Chromatography........... Columns.... Injection.... Sample Collection.................... Applications........................ Countercurrent Chromatography........... Droplet Countercurrent Chromatography....... Apparatus.... Choice of Solvents.................... Applications........................ Non-Aqueous Solvent Systems.... Perspectives........................ Rotation Locular Countercurrent Chromatography.. Description of the Method............... Solvent Selection..................... 41 48 50 50 53 54 56 61 63 65 65 66 67 67 68 70 71 71 72 81 88 99 117 118 118 118 119 119 121 128 128 129 129 130 134 135 136 137 138 142 153 154 154 155 156

Contents IX 7.2.3 7.2.4 7.3 7.3.1 7.3.2 7.3.3 7.3.4 7.3.5 7.4 8 8.1 8.2 8.3 8.4 8.4.1 8.5 8.6 8.7 8.8 8.9 9 9.1 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 9.2 9.3 9.4 9.4.1 9.4.2 9.4.3 9.5 10 10.1 10.1.1 10.1.2 10.1.3 10.1.4 Applications........................ Perspectives........................ Centrifugal Partition Chromatography......... Instruments........................ Choice of Solvent..................... Operating Techniques.................. Applications........................ Perspectives........................ Separation of Macromolecules............. Size Exclusion Chromatography............ Ion Exchange Chromatography............. Hydrophobic Interaction Chromatography...... Reversed-Phase Chromatography.... Ion-Pair RPC.... Affinity Chromatography................ Metal Interaction Chromatography........... Tentacle Supports..................... The Influence of Buffers................. Separation of Chiral Molecules............. Chiral Separations by Medium- or High-Pressure Liquid Chromatography................. Cellulose Derivatives................... Cyclodextrin Phases................... Poly(meth)acrylamides.... rr-acidic and rr-basic Phases.... Ligand-Exchange Chromatography.... Chiral Separations by Flash Chromatography..... Chiral Separations by Gas-Liquid Chromatography..................... Chiral Separations by Countercurrent Chromatography..................... Droplet Countercurrent Chromatography....... Rotation Locular Countercurrent Chromatography.. Centrifugal Partition Chromatography......... Separation Strategy and Combination of Methods.. Hydrophilic Compounds................ Combinations Involving Liquid-Liquid Partition and Liquid Chromatography.............. Combinations Involving Liquid-Liquid Partition and Size Exclusion Chromatography.......... Combinations Involving Size Exclusion Chromatography and Liquid Chromatography.... Combinations Involving Polymeric Supports..... 157 160 161 163 168 171 172 194 195 202 203 206 209 210 212 213 214 214 215 216 217 217 221 222 222 223 224 224 225 226 226 226 227 228 230 231 232 232 234 235

X Contents 10.1.5 10.2 10.2.1 10.2.2 10.3 10.4 11 Combinations Involving Different Liquid Chromatographic Steps................. Lipophilic Compounds.................. Combinations Involving Liquid Chromatography and Planar Chromatography.............. Combinations Involving Different Liquid Chromatographic Techniques.............. Conclusion........................ Subject Index....................... 236 236 236 237 239 240 241

List of Symbols and Abbreviations CCC ecce CPC CSP CTLC DCCC DMSO DNP GC HIC HPLC HSCCC IR LPLC MPLC PTLC RLCC RPC SEC SFC SFE TBME TFA THF TLC VLC countercurrent chromatography centrifugal countercurrent chromatography centrifugal partition chromatography chiral stationary phase centrifugal thin-layer chromatography droplet countercurrent chromatography dimethylsulphoxide dinitrophenyl gas chromatography hydrophobic interaction chromatography high-pressure liquid chromatography high-speed countercurrent chromatography infra-red low-pressure liquid chromatography medium-pressure liquid chromatography preparative thin-layer chromatography rotation locular countercurrent chromatography reversed-phase chromatography size exclusion chromatography supercritical fluid chromatography supercritical fluid extraction tertiary butyl methyl ether trifluoroacetic acid tetrahydrofuran thin-layer chromatography vacuum liquid chromatography