Laboratory Handbook for the

Fractionation of Natural Extracts

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 Laboratory
Handbook for
the Fractionation of
Natural Extracts
Peter J. Houghton

and

Amala Raman
Pharmacognosy Research Laboratories,
Department of Pharmacy, King's College London

SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.

First edition 1998

© 1998 Peter J. Houghton and Amala Raman

Originally published by Chapman & H a l l i n 1998
Softcover reprint of the hardcover 1st edition 1998
Thomson Science is a division of International Thomson Publishing I ® P ®

Typeset in 10/12pt Palatino by Columns Design Ltd, Reading

ISBN 978-1-4613-7662-0 ISBN 978-1-4615-5809-5 (eBook)

DOI 10.1007/978-1-4615-5809-5
A l l rights reserved. No part of this publication may be reproduced, stored in a retrieval sys-
tem or transmitted in any form or by any means, electronic, mechanical, photocopying,
recording or otherwise, without the prior written permission of the publishers.
Applications for permission should be addressed to the rights manager at the London
address of the publisher.
The publisher makes no representation, express or implied, with regard to the accuracy of
the information contained in this book and cannot accept any legal responsibility or liability
for any errors or omissions that may be made.

A catalogue record for this book is available from the British Library
Library of Congress Catalog Card Number: 97-77954
 Contents

1 Introduction 1
1.1 The need for this book - an active extract, what happens
next? 1
1.2 Why is it important to find out the nature of the active
components? 2
1.3 Green chemicals - the renewed interest in natural
products as sources of bioactive compounds 4
1.4 What are we doing? 7
1.5 Bibliography 13

2 Important concepts 14
2.1 Introduction 14
2.2 Polarity 14
2.3 Dilution and concentration factors 14
2.4 Synergy 16
2.5 Deterioration, decomposition and storage 17
2.6 Biological variation 18
2.7 Authentication of material 20
2.8 Dereplication 21

3 Methods for extraction and sample clean-up 22

3.1 Introduction 22
3.2 Factors to be considered in selecting an extraction method 22
3.3 General extraction methods 27
3.4 Extraction methods for specific phytochemical groups 39
3.5 Clean-up techniques to remove unwanted substances 45
3.6 Bibliography 52

4 Crude fractionation procedures 54

4.1 Introduction 54
4.2 Choice of fractionation method 55
4.3 Common fractionation methods 56
4.4 Bibliography 65
vi Contents
5 Chromatographic fractionation procedures 66
5.1 Introduction 66
5.2 The principles of chromatography 66
5.3 Physico-chemical processes taking place in chromatography 71
5.4 Bibliography 81

6 Chromatographic procedures using columns and liquid-liquid

systems 82
6.1 Column chromatography 82
6.2 Liquid-liquid partition schemes 90
6.3 Bibliography 93

7 Layer chromatography 94
7.1 Layer chromatography 94
7.2 Preparative thin-layer chromatography and paper
chromatography 102
7.3 Bibliography 112

8 Analysis of crude extracts, fractions and isolated compounds 113

8.1 Introduction 113
8.2 Qualitative analysis of mixtures and isolates 114
8.3 Quantitative analytical methods 127
8.4 Biological assays 135
8.5 Bibliography 138

9 General methods 139

9.1 Basic techniques in handling extracts 139
9.2 Isolation of active compounds 145
9.3 Chromatographic procedures 146
9.4 Basic techniques in thin-layer chromatography 150
9.5 Bibliography 153

10 Practical exercises in chemical screening and fractionation of

crude extracts from plant materials 154
10.1 Chemical screening 154
10.2 Exercises in extraction, clean-up, fractionation and
chromatographic methods 162

Appendix A Recipes for chemical test reagents 184

Index 187