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Phytochemical ethods

GUIDE TO MODERN TECHNIQUES

OF PL NT

N LYSIS



Phytochemical

Methods

GUIDE TO MODERN TECHNIQUES

OF PL NT N LYSIS

J B Harborne

Professor o otany

University o Reading

Second dition

LON ON

NEW

YORK

CH PM N ND

H LL



First

published

1973

ky

Chapman

and

Hall Ltd

11 New Fetter Lane, London EC4P4EE

Reprinted

1976

Second edition 1984

Published

in

the

USA by

Chapman and Hall

733

Third

Avenue, New

York NY 10017

©

1973,

1984]

B Harborne

Enset (Photosetting)

,

Midsomer

Norton, Bath, Avon

Softcover reprint

of

the hardcover 1st edition 1984

by Richard

Clay

(The

Chaucer

Press) Ltd, Bungqy

ISBN-13: 978-94-010-8956-2 e-ISBN-13: 978-94-009-5570-7

DOl: 10.1007/978-94-009-5570-7

All

rights

reserved. No part oj

his

book may be reprinted, or reproduced

or utilized in a' )'Jorm or by any electronic, mechanical

or

other means,

now known or

hereafter

invented, including

photocopying

and

recording,

or

in

a' )'

information

storage and retrieval system,

without

permission

in

writingJrom

the

publisher.

British Library Cataloguing in

Publication

Data

Harborne,IB.

Phytochemical methods.-2nd ed.

1. Plants-Ana ysis 2 Bioorganic chemistry

3.

C h e m i s t ~ y Analytic

Qualitative

I Title

581.18 24 QK865

Library ojCongress Cataloging

in

Publication

Data

HarhorneJB. (Jeffrev B.)

Phytochemical

methodJ.

Includes

bibliographies and

index.

I Plants-Analysis. I Title.

QK865.H27

1984

581.19 285

84-7750



Contents

Priface to

First

dition

VII

Priface to Second

dition

IX

Glossary

XI

1

Methods

of

Plant Analysis

1

1 1

Introduction

1 2

Methods

of

extraction and isolation

4

1 3

Methods

of

separation

7

1 4

Methods

of

identification

16

1 5

Analysis

of

results

29

1 6

Applications 3

2

Phenolic Compounds

37

2 1

Introduction

37

2 2

Phenols and phenolic acids

39

2 3

Phenylpropanoids

46

2 4

Flavonoid pigments

55

2 5 Anthocyanins 6

2 6

Flavonols and flavones

69

2 7

Minor

flavonoids xanthones and stilbenes

76

2 8

Tannins

84

2 9 Quinone

pigments

89

3

he

Terpenoids

1

3 1

Introduction

1

3 2

Essential oils

103

3 3

Diterpenoids

and

gibberellins

116

3 4

Triterpenoids

and

steroids

120

3 5

Carotenoids

129

4 Organic Acids Lipids and Related Compounds

142

4 1

Plant acids

142

4 2

atty acids

and

lipids

lSI

V



VI Contents

4.3

Alkanes

and

related hydrocarbons

160

4.4 Polyacetylenes

164

4.5

Sulphur compounds

169

Nitrogen Compounds

76

5.1

Introduction

176

5.2

Amino acids

178

5.3 Amines

187

5.4

Alkaloids

192

5.5 Cyanogenic glycosides

202

5.6

Indoles

205

5.7

Purines pyrimidines and cytokinins

208

5.8

Chlorophylls

214

6

Sugars and their Derivatives

222

6.1

Introduction

222

6.2

Monosaccharides

223

6.3

Oligosaccharides

231

6.4

Sugar alcohols and cyclitols

236

7

Macromolecules

243

7.1

Introduction

243

7.2

Nucleic acids

244

7.3

Proteins

251

7.4

Polysaccharides

264

ppendix

277

A List ofRecommended TL Systems for All Major Classes of

Plant Chemical

ppendix B

28

Some Useful Addresses

ndex

282



Preface to First dition

While there are many books available on methods oforganic and biochemical

analysis, the majority are either primarily concerned with the application

of

a

particular

technique (e.g. paper chromatography) or have been written for an

audience of chemists

or

for biochemists working mainly with animal tissues.

Thus

no simple guide to modern methods

of

plant analysis exists and the

purpose of

the present volume is to fill this gap. t is primarily intended for

students in the plant sciences, who have a botanical or a general biological

background. t should also be of value to students in biochemistry, phar-

macognosy, food science

and

natural products organic chemistry.

Most books on chromatography, while admirably covering the needs of

research workers, tend to overwhelm the student with long lists of solvent

systems and spray reagents that can be applied to each class of organic

constituent.

The

intention here

is

to simplify the situation by listing only a

f w

specially recommended techniques

that

have wide currency in phytochemical

laboratories. Sufficient details are provided to allow the student to use the

techniques for themselves and most sections contain some introductory

practical experiments which can be used in classwork.

After a general introduction to phytochemical techniques, the book contains

individual chapters describing methods

of

identifying phenolic compounds,

terpenoids, fatty acids

and

related compounds, nitrogen compounds, sugars

and their derivatives and macromolecules. The attempt has been made to

cover practically every class of organic plant constituent, although in some

cases, the account is necessarily brief because

of

space limitations. Special

attention

has, however, been given to detection

of

endogeneous plant growth

regulators

and

to methods

of

screening plants for substances

of

pharmaco-

logical interest. Each chapter concludes with a general reference section,

which is a bibliographic guide to more advanced texts.

While the enormous chemical variation

of

secondary metabolism in plants

has long been appreciated, variation in primary metabolism (e.g. in the

enzymes

of

respiration and

of

photosynthetic pathways) has only become

apparent quite

recently. With the realization

of

such chemical variation in

both

the small

and

large molecules

of

the plant kingdom, systematists have

ll



VIll

Preface to First dition

become interested in phytochemistry

for

shedding new light on plant relation

ships. A new discipline

of

biochemical systematics has developed and since

the present book has been written with some emphasis on comparative

aspects it should be a useful implement to research workers

in

this field.

In

preparing this book for publication the author has been given advice and

suggestions from many colleagues. He would particularly like to thank Dr

E

C. Bate-Smith

r

T. Swain r T

A

Smith and Miss Christine Williams

for

their valuable assistance. He

is

also grateful to the staff

of

Chapman and Hall

for expeditiously seeing the book through the press.

Reading

June

1973

J

B H.



Preface to Second dition

Since the preparation of the first edition there have been several major

developments in phytochemical techniques. The introduction

of

carbon-13

NMR spectroscopy now provides much more detailed structural information

on

complex molecules while

HPLC

adds a powerful and highly sensitive

analytical tool to the armoury of the chromatographer. With HPLC it is

possible to achieve for involatile compounds the type ofseparations that GLC

produces for volatile substances. Spectacular developments have also occur

red in the techniques of mass spectrometry; for example the availability

of

a

fast-atom

bombardment

source makes it possible in FAB MS to determine

the molecular weight

of

both very labile and involatile plant compounds.

These

techniques

are

described briefly in the appropriate sections

of

this new

edition.

During

the last decade the number

of

new structures reported from plant

sources has increased enormously and among some classes

of

natural

constituent the number of known substances has doubled within this short

time-span. The problems ofkeeping up with the phytochemical literature are

as a result of all this activity quite considerable although computerized

searches through Chemical bstracts have eased the burden for those scientists

able to afford these facilities.

In

order to aid at least the student reader

literature

references in this second edition have been extensively updated to

take into account the most recent developments. Additionally some new

practical experiments have been added to aid the student in developing

expertise in studying for example phytoalexin induction in plants and

allelopathic interactions between plants.

Since the first edition phytochemical techniques have become of increasing

value in ecological research following the realization that secondary con

stituents have a significant role in determining the

food

choice of hose animals

that

feed on plants. Much effort has been expended on analysing plant

populations for their toxins

or

feeding deterrents. Most such compounds were

included in the first edition except for the plant tannins. A new section has

therefore been included on tannin analysis in Chapter 2

IX



Preface to Second dition

With

this new edition the opportunity has been taken to add two

appendices - a checklist ofTL procedures for all classes

of

plant substance

and a list ofuseful addresses for phytochemists. Some errors in the first edition

have been corrected

but

others may remain and the author would welcome

suggestions for further improvements.

As with the first edition the author has benefited considerably from help

and advice of many colleagues. He would particularly like to thank his

co-workers in the phytochemical unit and his students who have been willing

guinea

pigs in the development

of

new phytochemical procedures.

Reading

December 1983

Jeffrey

B

Harborne



lossary

GENERAL

ABBREVIATIONS

TLC

GLC

PC

UV

= thin layer chromatography

gas liquid chromatography

paper chromatography

ultraviolet

nm

=

mass spectroscopy

mobility relative to front

= relative retention time

= nanometres

IR

=

infrared

mol

wt

=

molecular weight

NMR nuclear magnetic resonance M molar

HPLC high performance liquid chromatography

CHEMICALS

BSA N,O-bis trimethylsilyl)acetamide

PVP Polyvinylpyrrolidone for removing phenols)

BHT butylated hydroxy toluene anti-oxidant)

EDT

A ethylenediaminetetracetic acid chelating agent)

Tris tris hydroxymethyl)methylamine buffer)

PMSF phenyl methane sulphonyl fluoride

SDS sodium dodecyl sulphate

CHROMATOGRAPHIC

SUPPORTS

Kieselguhr diatomaceous earth

Decalso sodium aluminium silicate

DEAE-cellulose diethylaminoethyl-treated

PEl -cellulose polyethyleneimine-treated

ECTEOLA cellulose = epichlorohydrin-triethanolamine-alkali treated

PPE

= polyphenyl ether

OV

= methyl siloxane polymer

TXP

=

trixylenylphosphate SE

=

silicone oil

DEGS

diethyleneglycol succinate XE nitrile silicone

Apiezon L stop-cock grease Embacel acid-washed celite

Carbowax polyethylene glycol support

Chromosorb = firebrick support Poropak = styrene polymer support

ODS octadecylsilane

Xl



ll

lossary

CHROMATOGRAPHIC SOLVENTS

MeOH

=

methanol HC

2

H

=

formic acid

EtOH

=

ethanol HOAc

=

acetic acid

iso PrOH

=

iso propanol CHCl

3

=

chloroform

n BuOH

=

n butanol

CH

2

Cl

2

=

methylene dichloride

iso BuOH

=

iso butanol EtOAc

=

ethyl acetate

PhOH

= phenol

Me2CO

= acetone

NHEt2

= diethylamine MeCOEt = methyl ethyl ketone

Et

2

0 = diethyl ether

C

6

H

6

= benzene

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