Table Of ContentModern Methods of Plant Analysis
New Series Volume 3
Editors
H.E Linskens, Nijmegen
J.E Jackson, Adelaide
Volumes Already Published in this Series:
Volume 1: Cell Components
1985, ISBN 3-540-15822-7
Volume 2: Nuclear Magnetic Resonance
1986, ISBN 3-540-1591O-X
Volume 3: Gas Chromatography/Mass Spectrometry
1986, ISBN 3-540-15911-8
Gas Chromatography/
Mass Spectrometry
Edited by
H.E Linskens and IE Jackson
Contributors
R.S. Bandurski G. Combaut A. Ehmann P. Hedden
B. Janistyn H. Kameoka H. Kodama D.V. Lynch
IK. MacLeod H. Nyberg L.M.S. Palni L. Rivier
R.R. Selvendran B.IH. Stevens S.A.B. Tay
G.A. Thompson Jr., L. Witte
With 98 Figures
Springer- Verlag
Berlin Heidelberg New York Tokyo
Professor Dr. HANS FERDINAND LINSKENS
Botanisch Laboratorium
Faculteit der Wiskunde en Natuurwetenschappen
Katholieke Universiteit
Toernooiveld
NL-6525 ED Nijmegen
The Netherlands
Professor Dr. JOHN F. JACKSON
Department of Biochemistry
Waite Agricultural Research Institute
University of Adelaide
Glen Osmond, S.A. 5064
Australia
ISBN-13 :978-3-642-82614-6 e-ISBN-13 :978-3-642-82612-2
DOl: 10.1 007/978-3-642-82612-2
Library of Congress Cataloging-in-Publication Data. Gas chromatography/mass spectrometry. (Modern methods
of plant analysis; new ser., v. 3) 1. Plants -Analysis. 2. Gas chromatography. 3. Mass spectrometry. I. Linskens, H. F.
(Hans F.), 1921-. II. Jackson, J. F. (John F.), 1935-. III. Series. QK865.G37 1986 58l.l9'285 86-3875
ISBN-13 :978-3-642-82614-6 (U .S.)
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© by Springer-Verlag Berlin Heidelberg 1986
Softcover reprint of the hardcover 1s t edition 1986
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2131/3130-543210
Introduction
Modern Methods of Plant Analysis
When the handbook Modern Methods of Plant Analysis was first introduced in
1954 the considerations were:
1. the dependence of scientific progress in biology on the improvement of existing
and the introduction of new methods;
2. the difficulty in finding many new analytical methods in specialized journals
which are normally not accessible to experimental plant biologists;
3. the fact that in the methods sections of papers the description of methods is
frequently so compact, or even sometimes so incomplete that it is difficult to
reproduce experiments.
These considerations still stand today.
The series was highly successful, seven volumes appearing between 1956 and
1964. Since there is still today a demand for the old series, the publisher has
decided to resume publication of Modern Methods of Plant Analysis. It is hoped
that the New Series will be just as acceptable to those working in plant sciences and
related fields as the early volumes undoubtedly were. It is difficult to single out the
major reasons for success of any publication, but we believe that the methods
published in the first series were up-to-date at the time and presented in a way that
made description, as applied to plant material, complete in itself with little need to
consult other publications.
Contributing authors have attempted to follow these guidelines in this New
Series of volumes.
Editorial
The earlier series Modern Methods of Plant Analysis was initiated by Michel
v. Tracey, at that time in Rothamsted, later in Sydney, and by the late Karl Paech
(1910-1955), at that time at Tiibingen. The New Series will be edited by Paech's
successor H. F. Linskens (Nijmegen, The Netherlands) and John F. Jackson
(Adelaide, South Australia). As were the earlier editors, we are convinced "that
there is a real need for a collection of reliable up-to-date methods for plant analysis
in large areas of applied biology ranging from agriculture and horticultural
experiment stations to pharmaceutical and technical institutes concerned with raw
material of plant origin". The recent developments in the fields of plant
biotechnology and genetic engineering make it even more important for workers
in the plant sciences to become acquainted with the more sophisticated methods,
which sometimes come from biochemistry and biophysics, but which also have
VI Introduction
been developed in commercial firms, space science laboratories, non-university
research institutes, and medical establishments.
Concept of the New Series
Many methods described in the biochemical, biophysical, and medical literature
cannot be applied directly to plant material because of the special cell structure,
surrounded by a tough cell wall, and the general lack of knowledge of the specific
behavior of plant raw material during extraction procedures. Therefore all
authors of this New Series have been chosen because of their special experience
with handling plant material, resulting in the adaptation of methods to problems
of plant metabolism. Nevertheless, each particular material from a plant species
may require some modification of described methods and usual techniques. The
methods are described critically, with hints as to their limitations. In general it will
be possible to adapt the methods described to the specific needs of the users of this
series, but nevertheless references have been made to the original papers and
authors. While the editors have worked to plan in this New Series and made efforts
to ensure that the aims and general layout of the contributions are within the
general guidelines indicated above, we have tried not to interfere too much with
the personal style of each author.
Volume Three - Gas Chromatography/Mass Spectrometry
The New Series in Modern Methods of Plant Analysis was initiated in 1985
with a volume on Cell Components, and quickly followed by a second volume on
Nuclear Magnetic Resonance (NMR). Both included chapters contributed by
world experts on all aspects of the chosen topics. We have now followed the
principle adopted in the second volume, that of presenting the application of a
relative new and powerful analytical technique, this time devoting the third
volume to gas chromatography coupled to mass spectrometry (GCjMS). Here
the separation power of GC is combined with the selective detection properties of
MS, where compounds are ionized within the mass spectrometer and fragment
ions separated and analyzed on the basis of their mass-to-charge ratio.
Gas chromatography was one of the earliest techniques used to determine
plant hormones. When it is combined with mass spectrometry, a technique
capable of functioning as a highly sensitive and selective gas chromatography
detector, especially when it is focused on a particular ion, then we have a very
sensitive analytical tool indeed. As little as 10 pg may be enough for detection
with this technique. Little wonder, then, that GCjMS is often used to determine
plant hormones. This is reflected in the make-up of the present volume, where we
feature chapters on determination of cytokinins, auxins, gibberellins, cyclic
nucleotides, and other biologically active compounds. Other plant materials are
not neglected, so that chapters on the determination of tobacco constituents,
essential oils, phospholipids, fatty acids, volatile flower compounds, pectic
polysaccharides, and terpenoids can also be found in this volume.
Introduction VII
As in the previous volume on NMR, we hope that this collection of chapters
by world experts on plant analysis by GCjMS will encourage the further use of
this highly sensitive and discriminating technique amongst scientists, students,
and industrial analysts working with plant materials.
Acknowledgements. The editors express their thanks to all contributors for
their efforts in keeping to production schedules, and to Dr. Dieter Czeschlik,
Ms. K. G6del and Ms. E. Schuhmacher of Springer publishers for their
cooperation with this and other volumes in Modern Methods of Plant Analysis.
The constant help of Jose Broekmans is gratefully acknowledged.
Nijmegen and Adelaide, May 1986 H. F. LINSKENS
J. F. JACKSON
Contents
The Use of Combined Gas Chromatography-Mass Spectrometry
in the Analysis of Plant Growth Substances
P. HEDDEN (With 8 Figures)
1 Introduction. . . . . . 1
2 Identification by GC-MS 1
2.1 Derivatisation . . . 2
2.1.1 Methylation. . 2
2.1.2 Trimethylsilylation . 2
2.1.3 Permethylation . . 3
2.2 Gas Chromatography . . 3
2.3 Qualitative Mass Spectrometry 4
2.4 Quantitative Mass Spectrometry 5
3 Gibberellins . . . . . . . . . 9
3.1 Extraction and Purification 10
3.2 GC-MS ........ . 11
4 Abscisic Acid and Related Compounds 14
4.1 Qualitative GC-MS . 16
4.2 Quantitative GC-MS 17
5 Brassinosteroids 18
References. . . . . . . . 19
Applications of Mass Spectrometry for the Examination
of Pectic Polysaccharides
R. R. SELVENDRAN and B. J. H. STEVENS (With 6 Figures)
1 Introduction. . . . . . . . . . . . . . . . . . . 23
2 Structural Analysis of Pectic Polysaccharides. . . . . 23
3 Determination of the Nature of the Glycosidic Linkages . 24
4 Separation and Identification of Partially Methylated Alditol Acetates. 27
5 Extensions of Methylation Analysis. . . . . . 29
5.1 Controlled Partial Acid Hydrolysis Studies. . . . . . . . . . . 29
5.2 fJ-Eliminative Degradation Studies . . . . . . . . . . . . . . 30
5.2.1 Neutral Glycosyl Residues Linked to Galacturonosyl Residues 32
5.2.2 Glycosyl Residues Linked to 0-4 of 2,4-Linked
Rhamnopyranosyl Residues. . 32
6 Sequencing of Sugar Residues in Pectins. . . . . . . . 33
6.1 Partial Acid Hydrolysis . . . . . . . . . . . . . 33
6.1.1 Characterisation of Oligo saccharides as Permethylated
Derivatives . . . . . . . . . . . . . . . . . . . 34
Contents IX
6.2 Partial Acetolysis. . . . . . . . . . . . . . . . . . . . 37
6.3 Enzymatic Hydrolysis . . . . . . . . . . . . . . . . . . 38
7 Sequencing of Pectic Polysaccharides by Partial Depolymerisation
of Permethylated Derivatives. 38
8 Experimental 40
References. . . . . . . . . . 43
GC-MS Methods for Cyclic Nucleotides in Higher Plants
and for Free High Unsaturated Fatty Acids in Oils
B. JANISTYN (With 10 Figures)
1 Introduction . . . . . . . . . . . . . . . . . . . . . 47
2 Methods of GC-MS . . . . . . . . . . . . . . . . . . 48
3 Adenosine-3': 5'-Monophosphate (cAMP) in Maize Seedlings
(Zea mays). . . . . . . . . . . . . . . . . . . . . . 48
4 An Isotope Dilution GC-MS Spectrometric Assay for cAMP
in Cultured Tobacco Tissue. . . . . . . . . . . . . 51
5 Stability of Cyclic Purine Nucleotides in the Presence of
Hydrochloric Acid During Extraction . . . . . . . . 55
6 Guanosine-3': 5'-Monophosphate (cGMP) in Maize Seedlings
(Zea mays). . . . . . . . . . . . . . . . . . . . . . 56
7 GC-Separation of Synthetic cAMP and cGMP in a Mixture. 59
8 Cyclic Pyrimidine Nucleotides in Plants? . 60
9 Free High Unsaturated Fatty Acids in Oils 60
10 Conclusions 64
References. . . . . . . . . . . . . . . . 65
GC-MS Methods for Lower Plant Glycolipid Fatty Acids
H. NYBERG (With 4 Figures)
1 Introduction . . . . . . . . . 67
2 Extraction of the Plant Material. 67
2.1 Handling and Storage . . . 67
2.2 Extraction with Organic Solvents 68
2.3 Purification of the Extract . . . 69
3 Separation of Glycolipids from the Total Lipid Extract. 70
3.1 Column Chromatography. . . . . 70
3.2 Thin-Layer Chromatography (TLC) 72
3.3 Other Applications. . . . . 73
4 Isolation of the Glycolipids. . . . . . 74
4.1 Thin-Layer Chromatography . . . 74
4.2 Localization of the Glycolipids on TLC. 75
4.3 Removing the Spots from the TLC Plates . 76
5 Derivatization of the Glycolipid Fatty Acids for GC-MS 76
5.1 General Features . . . . . 76
5.2 Formation of Methyl Esters. 77
5.3 Silylation and Other Methods 77
x
Contents
6 GLC Instrumentation for Fatty Acid Analysis. 78
6.1 General Features . . 78
6.2 Carrier Gas System . 79
6.3 Injection and Injectors 79
6.4 The Detector . . . . 82
7 Column Selection for Fatty Acid GLC Analysis 83
7.1 Column Types . . . . . . . . . . . . 83
7.2 Supports, Liquid Phases, and Their Characteristics . 84
8 Interpretation of GC Data and Calculation of Results . 87
8.1 Identification of Peaks Using Standard Compounds 87
8.2 Quantitation of Results. . . . . . . . . . . . 88
8.3 External and Internal Standardization . . . . . 89
9 Fatty Acid Ester Structure Determination by GC-MS 90
9.1 Equivalent Chain Lengths (ECL) . . . . . . . 90
9.2 Semilogarithmic Correlations . . . . . . . . . 91
9.3 Mass Spectrometers and Their Function Principles 91
9.4 Interpretation of Mass Spectra of Fatty Acid Esters 92
10 Abbreviations. 94
References. . . . . . . . . . . . . . . . . . . . . . 95
Analysis of Phospholipid Molecular Species by Gas Chromatography
and Coupled Gas Chromatography-Mass Spectrometry
D. V. LYNCH and G. A. THOMPSON, Jr. (With 6 Figures)
1 Introduction. . . . 100
2 Lipid Preparation . . . . . . . 101
2.1 Lipid Extraction . . . . . . 10 1
2.2 Purification of Phospholipids. 102
2.2.1 Column Chromatography. 102
2.2.2 Thin Layer Chromatography 103
3 Formation of Derivatives for GC or GC-MS . 104
3.1 Phospholipase C Treatment . . . . . . 104
3.2 Conversion of Diacylglycerols to Silyl Derivatives. 106
3.2.1 Formation of Trimethylsilyl Derivatives. . . 107
3.2.2 Formation of tert-Butyldimethylsilyl Derivatives 107
4 Gas Chromatography. 108
5 Mass Spectrometry. . . 110
5.1 Instrumentation . . III
5.2 Operating Conditions 111
5.3 Identification of Molecular Species 111
5.4 Quantitation of Molecular Species by GC-MS 113
5.5 Quantitation of Molecular Species by GC-MS Following Reduction
of Double Bonds Using Deuterium . . . . . . . . 115
5.6 Direct MS Analysis of Underivatized Phospholipids . 117
6 Determination of Positional Distribution of Acyl Chains
Using Phospholipase A2 . 117
7 Conclusion 118
References. . . . . . . . 119
