Table Of ContentLiquid
Chromatography Mass
Spectrometry
Techniques and Applications
MODERN ANALYTICAL CHEMISTRY
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Liquid
Chromatography Mass
Spectrometry
Techniques and Applications
Alfred L. Yergey
National Institutes of Health
Bethesda, Maryland
Charles G. Edmonds
Battelle Pacific Northwest Laboratories
Richland, Washington
lvor A. S. Lewis
King's College
London, United Kingdom
and
Marvin L. Vestal
Vestee Corporation
Houston, Texas
Springer Science+Business Media, LLC
Library of Congress Cataloging in Publication Data
Liquid chromatography/mass spectrometry: techniques and applications / Alfred
L. Yergey ... [et al.].
p. cm. - (Modern analytical chemistry)
Includes bibliographical references.
ISBN 978-1-4899-3607-3 ISBN 978-1-4899-3605-9 (eBook)
DOI 10.1007/978-1-4899-3605-9
1. Liquid chromatography. 2. Mass spectrometry. 1. Yergey, Alfred L. II. Series.
QD79.C454L5537 1989 89-35915
543'.0894-dc20 CIP
© Springer Science+Business Media New York 1990
Originally published by Plenum Press New York in 1990.
Softcover reprint of the hardcover I st edition 1990
AII rights reserved
No part of this book may be reproduced, stored in a retrieval system, or transmitted
in any form or by any means, electronic, mechanical, photocopying, microfilming,
recording, or otherwise, without written permis sion from the Publisher
Preface
This book is intended both to be an introduction to techniques and applications of
liquid chromatography/mass spectrometry and to serve as a reference for future
workers. When we undertook its writing, we chose not to cover the field,
particularly applications, exhaustively. Rather we wished to produce a book that
would be of use to people just beginning to use the technique as well as to more
advanced practitioners. In this regard, we have sought to highlight techniques
and applications that are of current importance, while not neglecting descriptions
of approaches that may be of significance in the future. We hope that we have
succeeded in this. At the same time we hope that the bibliography, with indexes
classified by author and title, will make this book of value to those who may
disagree with our emphasis.
ACKNOWLEDGMENTS. One of us (C.G.E.) wishes to acknowledge the en
couragment of Professor J. A. McCloskey in undertaking this project. All four of
us are grateful for the continuous and expert assistance of V. A. Edmonds in the
preparation of the Bibliography.
Alfred L. Ye rgey
Bethesda, Maryland
Charles G. Edmonds
Richland, Washington
lvor A.S. Lewis
London, England
Marvin L. Vestal
Houston, Texas
v
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2. Direct Liquid Introduction Interfaces . . . . . . . . . . . . . 5
2.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.2. Operating Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.3. Specific DLI Interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.3.1. Capillary Inlets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.3.2. Diaphragm Interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.3.3. Nebulizing Interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.4. Interfaces Using Unusual Fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.4.1. Continuous Flow FAB Interfaces . . . . . . . . . . . . . . . . . . . . . . . 14
2.4.2. SFC Interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Additional References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3. Mechanical Transport Devices ................... 19
3 .1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3.2. Continuous Transport Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.2.1. Moving-Wire Interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.2.2. Moving-Belt Interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.2.3. The Heated-Wire Concentrator . . . . . . . . . . . . . . . . . . . . . . . . . 29
3. 3. Mechanized Fraction Collectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3. 3. 1. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.3.2. Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
4. Thermospray ................................... 31
4. 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
4.2. Apparatus for Thermospray . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
vii
viii Contents
4.2.1. The Vaporizer Probe ................................. 34
4.2.2. The Thennospray Ion Source . . . . . . . . . . . . . . . . . . . . . . . . . . 34
4.2.3. Electron Gun . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
4.2.4. Discharge Electrode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
4.2.5. Vacuum System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
4.2.6. Thennospray Control System . . . . . . . . . . . . . . . . . . . . . . . . . . 36
4.2.7. Ion Optics and Mass Analyzer ......................... 36
4.3. Mechanisms Involved in Thennospray Vaporization and Ionization 37
4.3.1. Vaporization and Nebulization in the Thennospray
Capillary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
4.3.2. Free Jet Expansion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
4.4. Ionization Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
4.4.1. Production of Molecular Ions . . . . . . . . . . . . . . . . . . . . . . . . . . 62
4.4.2. Ion Evaporation from Charged Liquid Droplets . . . . . . . . . . . 64
4.4.3. Ion Production from Charged Liquid Droplets-
A Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
4.4.4. Ion Production from Charged Solid Particles . . . . . . . . . . . . . 72
4.4.5. Ionic Equilibria in a Vaporizing Droplet . . . . . . . . . . . . . . . . . 73
4.4.6. Gas Phase Ion-Molecule Reactions . . . . . . . . . . . . . . . . . . . . . 75
4.4.7. Repeller Effects ..................................... 78
4.5. Optimizing Thennospray LC/MS for Particular Applications . . . . . . 79
4.5.1. Selection and Modification of LC Conditions . . . . . . . . . . . . . 79
4.5.2. Selection of Ionization Technique . . . . . . . . . . . . . . . . . . . . . . 80
4.5.3. Operating Temperatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
4.5.4. Vaporizer Tip Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
4.6. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Additional References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
5. Particle Beam Interfaces ........................ 87
6. LC/MS of Nucleic Acid Constituents ............. 89
6.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
6.2. LC/MS of Nucleobases and Related Compounds . . . . . . . . . . . . . . . . 91
6.2.1. Nucleobases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
6.2.2. Methylxanthine Bases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
6.3. LC/MS of Nucleosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
6.3.1. Liquid Chromatography of Nucleosides . . . . . . . . . . . . . . . . . . 99
6.3.2. Methods Employed for Nucleosides . . . . . . . . . . . . . . . . . . . . . 99
6.3.3. Performance Comparisons for Nucleosides ............... 100
6.3.4. Application of LC/MS to Nucleoside Analysis ............ 105
6.4. LC/MS of Nucleotides ..................................... 117
Contents ix
6.4.1. Methods Employed for Nucleotides and Dinucleotides ...... 117
6.4.2. Applications of LC/MS to Nucleotides and Dinucleotides ... 117
6.5. Future Prospects .......................................... 123
Additional References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
7. LC/MS of Conjugated Molecules ................ 127
7 .1. Introduction .............................................. 127
7.2. Glucuronides ............................................. 128
7. 3. Acy lcarnitines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 3
7 .4. Sulfates .................................................. 134
Additional References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
8. Applications of LC/MS to Amino Acids, Peptides,
and Proteins .................................. 137
8.1. Background .............................................. 137
8.2. Applications Using Immobilized Enzyme Digestions ............. 143
8.3. Limitations and Future Work ................................ 145
9. A Bibliography of Combined Liquid
Chromatography/Mass Spectrometry ........... 149
9.1. Introduction .............................................. 149
9.1.1. Methods ........................................... 149
9.1.2. Organization of the Bibliography ....................... 150
Additional References ...................................... 151
9.2. Bibliography ............................................. 151
9. 3. Author Index ............................................. 255
9.4. Subject Index ............................................. 278
Index ........................................................ 305
Chapter 1
Introduction
Despite the power and usefulness of gas chromatography/mass spectrometry
(GC/MS), there are many compounds that are intractable to analysis by this
technique because of the difficulty or impossibility of forming satisfactory ther
mally stable derivatives. Certain classes of biological compounds that are pre
charged, phospholipids, for example, or conjugates such as glucuronides or glu
tathione adducts are particularly subject to this limitation. While mass spectra of
such materials can be obtained using many of the newer direct sample introduc
tion techniques such as direct chemical ionization (DCI), fast atom bombardment
(FAB), and 252Cf desorption, on-line chromatographic introduction of these
kinds of compounds was not possible until the development of a practical liquid
chromatographic/mass spectrometric interface.
The development of such a practical interface has occurred relatively re
cently, and in fact there are no citations in Chemical Abstracts under the subject
heading "Liquid Chromatography and Mass Spectrometry" before 1971, and the
number of such citations remained at a very low level for the next several years.
However, it should be noted that pioneering work demonstrating the potential for
such an interface was done by Tal'roze and his colleagues in 1968 [68TA28]. In
the past few years, the field has grown substantially, leading to the publication of
about 40 original papers a year for the last five years. To put this number in
perspective, however, the recent annual publication rates for LC/MS are equal to
about 25% of the papers published in GC/MS and about 4% of the total publica
tions in mass spectrometry.
A major characteristic of any of the interfaces between a liquid chromato
graph and a mass spectrometer is the elimination of the need for chemical
modification of the sample by derivatization. This elimination becomes a sub
stantial practical advantage that permits the mass spectrometric analysis of non
volatile, thermally labile, and/or precharged molecules. On the other hand, these
interfaces must be able to accommodate the greatly increased molecular flow
rates associated with liquid chromatography relative to gas chromatography.
Microbore liquid chromatographs operating at flow rates of about 10 f.JJ/min
