High Performance Mass Spectrometry: Chemical Applications 9780841204225, 9780841205352, 0-8412-0422-5

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High Performance Mass Spectrometry: Chemical Applications Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.fw001

Michael L. Gross, EDITOR University of Nebraska

A symposium co-sponsored by the University of Nebraska—Lincoln, the National Science Foundation, ΑΕΙ Scientific, and INCOS Corp., Lincoln, November 3-5, 1976.

ACS

SYMPOSIUM

SERIES

AMERICAN CHEMICAL SOCIETY

WASHINGTON, D. C.

1978

70

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.fw001

Library of Congress Data Main entry under title: High performance mass spectrometry. (ACS symposium series; 70 ISSN 0097-6156) Includes bibliographies and index. 1. Mass spectrometry—Congresses. I. Gross, Michael L. II. University of Nebraska— Lincoln. III. Series: American Chemical Society. ACS symposium series; 70. QD96.M3H53 543'.085 78-789 ISBN 0-8412-0422-5 ASCMC8 70 1-358 1978

Copyright © 1978 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of thefirstpage of each article in this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective works, for resale, or for information storage and retrieval systems. The citation of trade names a n d / o rnamesof manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. PRINTED IN THE UNITED STATES OF AMERICA

ACS Symposium Series

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.fw001

Robert F . G o u l d , Editor

Advisory Board Kenneth B. Bischoff

Nina I. McClelland

Donald G. Crosby

John B. Pfeiffer

Jeremiah P. Freeman

Joseph V. Rodricks

E. Desmond Goddard

F. Sherwood Rowland

Jack Halpern

Alan C. Sartorelli

Robert A. Hofstader

Raymond B. Seymour

James P. Lodge

Roy L. Whistler

John L. Margrave

Aaron Wold

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.fw001

FOREWORD The ACS S Y M P O S I U M S E R I E S was founded in 1974 to provide a medium for publishing symposia quickly in book form. The format of the S E R I E S parallels that of the continuing A D V A N C E S I N C H E M I S T R Y S E R I E S except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. As a further means of saving time, the papers are not edited or reviewed except by the symposium chairman, who becomes editor of the book. Papers published in the ACS S Y M P O S I U M S E R I E S are original contributions not published elsewhere in whole or major part and include reports of research as well as reviews since symposia may embrace both types of presentation.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.pr001

PREFACE A4Tass spectrometry has emerged as a mature scientific discipline in chemistry. Investigators no longer are limited to slow scans at low resolution of volatile samples ionized by electron impact. In recent years, a number of significant developments have occurred that are considered examples of "High Performance Mass SpectrometryThese include chemical and field ionization, ultra-high resolution, new methods of defocused metastable scans, collisional activation spectrometry, field ionization kinetics, and new techniques in gas chromatography/mass spectrometry and sample introduction. To provide a forum for discussing the chemical applications of these techniques, a symposium was held by the Department of Chemistry, University of Nebraska, Lincoln, Nebraska, Nov. 3-5, 1976. The papers presented at that symposium have been updated during the past year (1977) and are collected in this volume. The primary objective of this volume is to survey current research in high performance mass spectrometry. Solution to problems in chemical analysis is one major theme. Complete identification and quantitation of trace amounts of materials in biological and environmental systems requires many of the methods of high performance mass spectrometry: GC/MS, exact mass measurements, and improved methods of sample vaporization and ionization. Another emphasis is the determination of structure and properties of gas-phase ions. This research is important in understanding the intrinsic properties of these species in the absence of solvent. Moreover, a thorough understanding of the mechanism of mass spectral fragmentations must rely on these fundamental studies. Accordingly, the volume has been divided into two sections. The first includes chapters describing methods for acquiring metastable ion data that relate to stable and low-energy decomposing ions and the application of these methods for determining ion structures, properties, and potential energy surfaces. By way of contrast, the capability to investigate extremely rapid ionic reactions using field ionization kinetics is described. The second section is a survey of analytical applications that can be explored with high performance mass spectrometry. General applications on qualitative and quantitative analyses using high resolution with vii

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.pr001

direct inlet and G L C methods of sample introduction are described. Furthermore, selective methods of chemical ionization and the development of simultaneous recording of positive and negative ion chemical ionization are also reviewed. The application of mass spectrometry to important problems in biochemistry and pharmaceutical chemistry are discussed in four of the chapters. In particular, methods of obtaining specificity in biological assays and the use of field desorption and an alternative to field desorption for handling nonvolatile materials are covered. Petroleum and solid state analysis and the use of ultra-high resolution, metastable methods, and spark source ionization are discussed for the analysis of complex mixtures in three chapters. The last two chapters are illustrative of two of the methods currently used in computer-assisted spectral interpretation. They are included because high performance techniques in mass spectrometry can produce tremendous quantities of data that place onerous interpretation demands on the user. This volume and the original symposium are intended to introduce the various aspects of high performance mass spectrometry and to provide a review of the current state of knowledge. Our hope is that the book will be useful to scientists with a modest background in mass spectrometry who wish to make use of the technique in their own research and to the expert who is interested in a critical review. At the opening remarks session of the symposium, G. G. Meisels, Chairman of the Chemistry Department at the University of Nebraska, offered an alternate definition of high performance mass spectrometry. He said that high performance mass spectrometry is a branch of research practiced by high performance scientists. Indeed, this volume is a collection of papers by many of the "highest performance mass spectroscopists at work today. I must acknowledge their cooperation in assembling this book. Moreover, I wish to thank Jan Deshayes for assisting in the organization of the symposium and overseeing the typing of the majority of manuscripts. The manuscripts were typed and assembled by Kathy Steen, Velaine Zbytniuk, and Sharon Coufal. Many of the conference details were handled by my graduate students: David Russell, David Hilker, and Stan Wojinski. I am also indebted to these persons for their efforts. F. H. Field and T. L. Isenhour presented plenary lectures at the symposium, but the press of other duties did not allow them to contribute to this volume. However, I am indebted to them for their participation. ,,

viii

Finally, the symposium was made possible because of generous support from the National Science Foundation, the University of Nebraska Research Council, Kratos-AEI Scientific Apparatus, and the INCOS Corp. Their contributions are gratefully acknowledged.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.pr001

University of Nebraska MICHAEL L. GROSS Lincoln, Nebraska December 30, 1977

ix

1 Observation of Metastable Transitions in a High Performance Mass Spectrometer K. R. JENNINGS

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch001

Department of Molecular Sciences, University of Warwick, Coventry, United Kingdom

A metastable transition is the unimolecular or collision induced decomposition of an ion after leaving the source exit s l i t and before reaching the collector of a mass spectrometer. The daughter ions produced in these decompositions give rise to metastable peaks in mass spectra produced in a magnetic deflection instrument. During the past ten years, various methods of observing metastable transitions in high performance double focussing instruments of various geometries have been introduced (1,2,3,4). This article aims to give a comparative account of these methods together with illustrations of the use of some of them in the author's laboratory. No single method has established i t s e l f as ideal for a l l applications, and before considering the methods in detail, one must be clear what information is required or may be obtained from a study of metastable transitions. If one is concerned with obtaining detailed information about the potential energy surface over which a particular decomposition occurs, the method must be capable of yielding information on peak shapes, and if possible, the energy resolution should be high enough to reveal any structure in the peak. On the other hand, if one's prime aim is to establish whether or not there is a metastable peak arising from the process m ->m or alternatively to assign as precisely as possible the mass of either m or m when one of them is known, a method which produces a very narrow peak, the position of which on the mass or voltage scale can be measured with high precision, is to be preferred. The choice of method may also be influenced by a consideration of whether one is particularly interested in collision-induced decompositions or whether one particularly wishes to avoid them. Other factors such as sensitivity, rapidity of scan, +

1

+

2

+

1

©0-8412-0422-5/78/47-070-003$05.00/0

+

2

4

HIGH

PERFORMANCE

MASS

SPECTROMETRY

d i s c r i m i n a t i o n e f f e c t s and the i n t e r p r e t a t i o n o f t i v e peak i n t e n s i t i e s must a l s o be c o n s i d e r e d .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch001

Methods o f O b s e r v i n g Applications.

Metastable

T r a n s i t i o n s and

rela­ their

I n a d o u b l e f o c u s s i n g i n s t r u m e n t , the t h r e e pos­ s i b l e v a r i a b l e s a r e V, the a c c e l e r a t i n g v o l t a g e , E, the e l e c t r i c s e c t o r v o l t a g e and B, the m a g n e t i c f i e l d s t r e n g t h . These may be scanned i n v a r i o u s ways t o a l l o w the c o l l e c t i o n o f d a u g h t e r i o n s formed i n metas­ t a b l e t r a n s i t i o n s w h i c h o c c u r i n the f i e l d f r e e r e g i o n s between the s o u r c e and the f i r s t s e c t o r and between the two s e c t o r s . The major c h a r a c t e r i s t i c s o f the f i v e methods w h i c h have so f a r been used a r e summarized i n T a b l e I . In d i s c u s s i n g these methods, i t w i l l be as­ sumed, u n l e s s o t h e r w i s e s t a t e d , t h a t the mass s p e c t r o ­ meter has a geometry i n w h i c h the e l e c t r i c s e c t o r p r e ­ cedes the m a g n e t i c s e c t o r . The f i r s t method s i m p l y makes use o f the o f t e n d i f f u s e peaks o f low i n t e n s i t y w h i c h a r e o b s e r v e d i n a normal mass spectrum i n w h i c h Β i s scanned w i t h V and Ε f i x e d . The m e t a s t a b l e peaks may be masked by i n t e n s e normal p e a k s , and assignment i s u s u a l l y not d i f f i c u l t f o r peaks g i v e n by a low m o l e c u l a r w e i g h t compound. However, i t becomes i n c r e a s i n g l y d i f f i c u l t to a s s i g n low i n t e n s i t y m e t a s t a b l e peaks i n the mass spectrum o f a compound o f h i g h m o l e c u l a r w e i g h t . I f o n l y the more i n t e n s e m e t a s t a b l e peaks are o f i n t e r e s t , few problems a r e e n c o u n t e r e d , and the method has the advantage t h a t the normal mass spectrum and m e t a s t a b l e peaks a r e ob­ t a i n e d i n a s i n g l e scan. One o f the most w i d e l y used methods i s t h a t i n w h i c h the a c c e l e r a t i n g v o l t a g e V i s scanned w i t h Ε and Β fixed. In t h i s method the d a u g h t e r i o n m i s selec­ t e d under normal o p e r a t i n g c o n d i t i o n s but w i t h V and Ε s e t a t t y p i c a l l y h a l f the maximum v a l u e . A s c a n o f V i s t h e n i n i t i a t e d and a peak i s o b t a i n e d whenever d i f f e r e n t p r e c u r s o r i o n s m i f u l f i l l the r e q u i r e m e n t that V /V„ = m /m (1) +

2

+

1

1

2

where V i s the a c c e l e r a t i n g v o l t a g e r e q u i r e d f o r the c o l l e c t i o n of m i o n s formed i n the s o u r c e and V i s the v a l u e r e q u i r e d t o t r a n s m i t m i o n s formed from m i i o n s i n the f i e l d - f r e e r e g i o n between the source and e l e c t r i c s e c t o r . T h e r e f o r e , the method i s w e l l s u i t e d to g i v e i n f o r m a t i o n on p r e c u r s o r i o n s o f a g i v e n daugh­ t e r i o n . The s i g n a l i s p r o d u c e d by the c o l l e c t i o n o f d a u g h t e r i o n s o f a f i x e d mass and energy, d e f i n e d by 0

+

2

x

+

2

+

Metastable Transitions

JENNINGS

Table I.

Scan

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch001

Β

C h a r a c t e r i s t i c s o f D i f f e r e n t Methods f o r Observation of Metastable Transitions.

Fixed

Ease o f KE Assignment R e l e a s e

V, Ε

Often D i f ­ ficult

Ε, Β

Usually to nearest nominal mass

Yes

i) D i f f i c u l t i i ) Usually to nearest nominal mass

Yes

V, Β

Yes

Feature 2

G i v e s m /mi r a t i o . Low i n t e n s i t y r e l a ­ t i v e t o normal peaks. O v e r l a p pro­ blems. 2

G i v e s a l l mi o f s e l e c t e d m . Range l i m i t e d by p r a c t i ­ c a l Vi/Vo r a t i o . Source c o n d i t i o n s vary during scan. +

2

i ) Ε p r e c e d e s B: g i v e s a l l mi/m r a t i o s w i t h o u t mass a n a l y s i s . Requires EM between s e c t o r s , i i ) Β p r e c e d e s E: gives a l l m from s e l e c t e d m i . Wide range; c o n s t a n t source c o n d i t i o n s . 2

+

2 +

V V E

Β

Good; peaks narrow

No

+

Gives a l l m from s e l e c t e d m i . Range l i m i t e d t o mi/m ~3. Source c o n d i t i o n s vary during scan. 2 +

2

Β/Ε

V

Good; narrow peaks

No

+

Gives a l l m from s e l e c t e d m i . Wide range; c o n s t a n t source c o n d i t i o n s . 2 +

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch001

6

HIGH

PERFORMANCE

MASS

SPECTROMETRY

the s e l e c t e d v a l u e s o f Β and E. The spectrum i s an energy spectrum o f p r e c u r s o r i o n s w h i c h fragment t o y i e l d t h e s e daughter i o n s , and t h i s i s r e a d i l y used t o g i v e t h e masses o f p r e c u r s o r i o n s by means o f e q u a t i o n (1). I f t h e f r a g m e n t a t i o n i s accompanied by t h e r e l e a s e o f t r a n s l a t i o n a l energy, t h e m e t a s t a b l e peak i s b r o a d ­ ened. I f t h e 3 - s l i t w i d t h i s r e d u c e d i n an i n s t r u m e n t o f N i e r - J o h n s o n geometry, t h e i n c r e a s e d d i s c r i m i n a t i o n a t t h e β-slit causes t h e peak-shape t o change and f a c i l i t a t e s t h e e v a l u a t i o n o f t h e energy r e l e a s e . Un­ der such c o n d i t i o n s , t h e i n t e n s i t y i s r e d u c e d b u t s i g n a l a v e r a g i n g may be used t o improve t h e s i g n a l - t o n o i s e r a t i o , and, i n c e r t a i n c a s e s , t h i s has r e v e a l e d f i n e s t r u c t u r e i n m e t a s t a b l e peaks. S e v e r a l fragmen­ t a t i o n s which l e a d t o the p r o d u c t i o n o f 0 Η ions y i e l d m e t a s t a b l e peaks w h i c h p o s s e s s f i n e s t r u c t u r e (5J). The s i g n a l g i v e n by t h e Ci»rU i o n s formed i n t h e process +

3

3

+

C

H

7 7

+ +

*

C

H

4 4

+

+

C

H

3 3

+

i s r e p r o d u c e d i n F i g u r e 1. T h i s peak s t r u c t u r e i s r a t i o n a l i z e d by assuming t h a t t h e two p r o c e s s e s y i e l d l i n e a r and c y c l i c forms o f t h e C H ions, the enthal­ p i e s o f f o r m a t i o n o f w h i c h d i f f e r by 1.09 eV. The d i f f e r e n c e i n e n e r g i e s r e l e a s e d i n t h e two p r o c e s s e s w h i c h g i v e r i s e t o t h e two components o f t h e peak i s 0.52 eV. T h i s i n d i c a t e s t h a t a p p r o x i m a t e l y h a l f o f t h e d i f f e r e n c e i n e n t h a l p i e s o f f o r m a t i o n appears as t h e difference i n energies released. A s i m i l a r conclusion i s r e a c h e d from t h e s t r u c t u r e i n t h e m e t a s t a b l e peak a r i s i n g from t h e p r o c e s s +

3

C

H

5 5

+

*

C

H

3 3

+

+

3

C

H

2 2

^

A f u r t h e r use o f t h i s type o f s c a n has been i n t h e i n v e s t i g a t i o n o f t h e energy r e l e a s e w h i c h accompanies the f r a g m e n t a t i o n o f an i o n formed i n an i o n - m o l e c u l e r e a c t i o n i n a h i g h p r e s s u r e source. For t h i s type o f e x p e r i m e n t , t h e c o n d i t i o n s r e q u i r e c a r e f u l adjustment s i n c e i f the pressure i s too low, the y i e l d o f f r a g ­ menting i o n i s low and i f t h e p r e s s u r e i s t o o h i g h , c o l l i s i o n - i n d u c e d decompositions occur o u t s i d e the s o u r c e and mask t h e peak g i v e n by t h e u n i m o l e c u l a r decomposition. I n a s t u d y (6) o f t h e f r a g m e n t a t i o n o f the C H 3 O H 2 " i o n , i t was founcT t h a t b e s t r e s u l t s were o b t a i n e d w i t h a s o u r c e p r e s s u r e o f hydrogen o f 0.05 Torr w i t h a t r a c e o f methanol. M e t a s t a b l e peaks a r i s i n g from two f r a g m e n t a t i o n s c o u l d be o b s e r v e d 4

1.

Metastable Transitions

JENNINGS

CH OH

+

2

H

+ 3

+ CH OH 3

-

H

2

+

2

(4a)

[CH OH ]- 6UÏÏ:1. I n a d d i t i o n t o c o n c l u s i o n s based on t h e r e l a t i v e abundances o f m e t a s t a b l e p e a k s , u s e f u l i n f o r m a t i o n c o n c e r n i n g t h e t r a n s i t i o n s t a t e s f o r r e a c t i o n s c a n somet i m e s be deduced from t h e shapes o f m e t a s t a b l e p e a k s . A n e c e s s a r y ( b u t n o t s u f f i c i e n t ) c o n d i t i o n f o r decomp o s i t i o n o f i s o m e r i c i o n s o v e r t h e same p o t e n t i a l surface, with c l o s e l y similar i n t e r n a l energies, i s t h a t t h e m e t a s t a b l e peaks f o r t h e d i s s o c i a t i o n ( s ) o f such i o n s must be t h e same shape. F o r i n s t a n c e e v i dence has b e e n p r e s e n t e d (11) w h i c h s t r o n g l y s u g g e s t s t h a t t h e C H 0 i o n s i n t h e mass s p e c t r a o f p h e n o l ( J ) and t r o p o l o n e ( 2 ) , w h i c h decompose by l o s s o f CO i n m e t a s t a b l e t r a n s i t i o n s , have t h e same s t r u c t u r e ( o r m i x t u r e o f s t r u c t u r e s ) . I n each c a s e , t h e m e t a s t a b l e peak f o r t h e r e a c t i o n +

3

2

2

t

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch002

3

3

+

6

6

C H 0Î 6

6

C H i 5

6

+

CO

i s t h e same shape ( 1 1 ) . OH

2 C o n v e r s e l y , i f t h e m e t a s t a b l e peaks f o r d i s s o c i a t i o n o f isomeric ions are d i f f e r e n t , t h i s c o n s t i t u t e s s t r o n g e v i d e n c e t h a t t h e decay c h a n n e l s , t h r o u g h w h i c h reaction i s occurring, are d i f f e r e n t . A c l a s s i c case i s the decomposition o f the molecular ions o f the i s o m e r i c n i t r o p h e n o l s v i a e l i m i n a t i o n o f NO. I n t h e c a s e s o f o- and £-nitrophenol m o l e c u l a r i o n s t h e met a s t a b l e peak f o r NO l o s s i s b r o a d and f l a t - t o p p e d , whereas f o r m - n i t r o p h e n o l t h e peak i s g a u s s i a n ( 1 2 ) .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch002

20

HIGH

3c

PERFORMANCE

4c

MASS

SPECTROMETRY

5c

C l e a r l y , t h e r e i s a fundamental d i f f e r e n c e i n t h e p o t e n t i a l s u r f a c e o v e r w h i c h t h e m o l e c u l a r i o n o f mn i t r o p h e n o l (3b) decomposes. C o n s i d e r a t i o n o f t h e p l a u s i b l e p r o d u c t s t r u c t u r e s ( 4 a , b, c) r e v e a l s t h a t the charge can be more e f f e c t i v e l y ~ d e I o c a l i z e d i n 4a and 4c v i a a " c l a s s i c a l resonance e f f e c t " . Furthermore, t h e d i f f e r e n t m e t a s t a b l e peaks f o r NO l o s s i n d i c a t e t h a t 3b does n o t e q u i l i b r a t e w i t h e i t h e r 3a o r 3c prior~£o u n i m o l e c u l a r decomposition. The Concept o f t h e P o t e n t i a l S u r f a c e . A r e l a t i v e l y s i m p l e and e l e g a n t method o f summ a r i z i n g t h e c h e m i s t r y o f a g i v e n system i s t h e cons t r u c t i o n o f t h e p o t e n t i a l s u r f a c e over w h i c h t h e i o n s decompose ( 1 3 ) . The method may be used when o n l y one e l e c t r o n i c s t a t e i s i n v o l v e d (an a d i a b a t i c s u r f a c e ) and can be extended t o i n c l u d e cases where more t h a n one e l e c t r o n i c s t a t e i s i n v o l v e d (a d i a b a t i c s u r f a c e ) . The l a t t e r must be employed when " c r o s s i n g s " o c c u r (14)·

The c o n s t r u c t i o n o f t h e p o t e n t i a l s u r f a c e f o r an i o n i c system a l s o e n a b l e s t h e c h e m i s t r y t o be c l e a r l y understood. For i n s t a n c e , consider t y o i s o m e r i c ions A+ and B+, w h i c h can decompose i n t o C +D and E +F,

2.

BOWEN

Unimolecular Reactions of Organic Ions

AND WILLIAMS

respectively. Clearly i f t h e a c t i v a t i o n energies f o r i s o m e r i z a t i o n o f A and Β a r e l e s s t h a n t h o s e needed to cause d i s s o c i a t i o n o f e i t h e r A o r B t h e n a t energies a p p r o p r i a t e t o slow r e a c t i o n s , r a p i d e q u i l i ­ b r a t i o n o f A and B w i l l o c c u r p r i o r t o m e t a s t a b l e t r a n s i t i o n s ( F i g . 1 ) . C o n s e q u e n t l y , t h e slow r e a c t i o n s undergone by i o n s on such a s u r f a c e w i l l r e f l e c t tlje r e l a t i v e a c t i v a t i o n e n e r g i e s f o r d i s s o c i a t i o n t o C +D and E +F, i r r e s p e c t i v e o f whether t h e i o n s a r e i n i ­ t i a l l y formed as A o r B . An example o f t h i s s i t u ­ a t i o n i s the p o t e n t i a l s u r f a c e f o r decomposition o f C H! i n t o C»H + C Hi» and C H + C H . Irres­ p e c t i v e o f t h e p r e c u r s o r used t o g e n e r a t e C 6 H , such i o n s always undergo d e c o m p o s i t i o n i n a p p r o x i m a t e l y t h e same r a t i o (1.6:1 f a v o r i n g C H l o s s ) (6) t h u s i n d i ­ c a t i n g that the a c t i v a t i o n energies f o r i n t e r c o n v e r s i o n of a l l t h e a c c e s s i b l e c o n f i g u r a t i o n s o f C H i a r e l e s s t h a n t h o s e f o r d e c o m p o s i t i o n . A s i m i l a r ca§e o c c u r s i n t h e u n i m o l e c u l a r d e c o m p o s i t i o n o f Ci*H where n e a r l y a l l t h e m e t a s t a b l e i o n c u r r e n t i s due t o CHi* l o s s (15) a l t h o u g h a s m a l l and a l m o s t c o n s t a n t p e r c e n t a g e i s due t o C Hi* l o s s (15J) . R a p i d e q u i l i b r i t i o n of a l l the p o s s i b l e structures of ^ Η ( F i g . 2) r e q u i r e s t h a t complete l o s s o f i d e n t i t y o f a l l c a r b o n and hydrogen atoms i n l a b e l l e d C»*H i o n s must precede u n i m o l e c u l a r d e c o m p o s i t i o n . T h i s i s borne o u t by D and C l a b e l l i n g s t u d i e s , which e s t a b l i s h s t a t i s t i c a l l o s s of CHi* i n m e t a s t a b l e t r a n s i t i o n s , i r r e s p e c t i v e o f t h e p r e c u r s o r s t r u c t u r e (15). T h i s l o s s o f i d e n t i t y o f t h e atoms o f l a b e l l e d i o n s i s sometimes r e f e r r e d t o as " s c r a m b l i n g " o r "randomization". Such e x p r e s s i o n s s h o u l d be used w i t h c a u t i o n , f o r t h e y may appear t o i m p l y t h a t t h e chemis­ t r y o f t h e system i s n o t u n d e r s t o o d , whereas i n f a c t , r e f e r e n c e t o a s u i t a b l e p o t e n t i a l s u r f a c e (e.g. F i g . 2 above) r e a d i l y e x p l a i n s t h e c h e m i s t r y . F u r t h e r m o r e , i t i s important t o grasp t h a t i t i s the r a p i d e q u i l i ­ b r a t i o n o f v a r i o u s i s o m e r i c s t r u c t u r e s w h i c h causes t h e l o s s o f i d e n t i t y o f atoms i n l a b e l l e d s p e c i e s and n o t the p r e s e n c e o f a s u i t a b l e s y m m e t r i c a l i o n on t h e potential surface. That t h e p r e s e n c e o f such a sym­ m e t r i c a l i o n i s n o t a p r e r e q u i s i t e i s e v i d e n t from t h e Ci*H case c o n s i d e r e d above, where l o s s o f i d e n t i t y o f a l l hydrogen and c a r b o n atoms o c c u r s p r i o r t o m e t a s t ­ a b l e t r a n s i t i o n s , even though no s t r u c t u r e f o r C^Hg* e x i s t s i n w h i c h a l l t h e hydrogen and c a r b o n atoms a r e equivalent. In a d d i t i o n t o t h e c r i t e r i o n o f m e t a s t a b l e abun­ dances (16), and l a b e l l i n g s t u d i e s , o t h e r i m p o r t a n t methods a r e a v a i l a b l e w h i c h a s s i s t i n t h e c o n s t r u c t i o n +

+

+

+

+

+

+

+

6

3

+

+

9

+

2

3

7

3

6

i 3

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch002

21

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6

3

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of p o t e n t i a l s u r f a c e s . These a r e C o l l i s i o n a l A c t i v a t i o n ( C A ) , I o n C y c l o t r o n Resonance (ICR) and energy measurements o f v a r i o u s k i n d s . The f i r s t two t e c h n i q u e s p e r m i t t h e d e t e c t i o n o f any i o n w h i c h e x i s t s i n a s i g n i f i c a n t w e l l on t h e p o t e n t i a l s u r f a c e . Thus, f o r i n s t a n c e , f o r C 2 H 0 , CA s t u d i e s i n d i c a t e o n l y two s t r u c t u r e s , p r e s u m a b l y 6 and 7, w h i c h e x i s t i n r e l a t i v e l y deep p o t e n t i a l w e l l s (17) ; t h i s i s i n agreement w i t h a s t u d y o f t h e m e t a s t a b l e d e c o m p o s i t i o n s o f the i o n (!L8) and w i t h a p r e v i o u s ICR s t u d y (19J) . + + CH CH=OH CH 0=CH +

5

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch002

3

3

2

S i m i l a r c o n c l u s i o n s have a l s o been r e a c h e d i n t h e C H 0 system (2£) ; and t h e C H N and C H N systems ( 2 1 ) , where once a g a i n t h e onium type i o n s (8-11; 14-18) appear t o e x i s t i n p o t e n t i a l w e l l s : + + • + CH CH CH=OH (CH ) C=OH CH CH 0=CH CH CH=OCH +

3

7

2

+

6

3

8

a n d

3

2

3

2

8

3

9 + CH CH=NH 3

2

11

+ CH NH=CH

2

3

3

2

13

+

+

CH CH CH=NH 2

3

10

12 3

2

(CH ) C=NH

2

3

14

2

+

CH CH NH=CH

2

3

2

15

+ CH CH=NHCH 3

3

2

16 + (CH ) N=CH 3

17

2

2

18

I t s h o u l d be n o t e d , however, t h a t t h e d i f f e r e n c e i n CA s p e c t r a o f two i o n s does n o t n e c e s s a r i l y p r e clude i s o m e r i z a t i o n p r i o r to metastable d i s s o c i a t i o n s . T h i s i s because t h e a c t i v a t i o n e n e r g i e s f o r i n t e r c o n v e r s i o n o f t h e i o n s may be l e s s t h a n t h o s e f o r d i s s o c i a t i o n b u t n e v e r t h e l e s s l a r g e enough t o cause c o n s i d e r a b l e d i f f e r e n c e s i n t h e CA s p e c t r a . An example o f t h i s s i t u a t i o n i s found i n t h e C H e N system where i o n s o f s t r u c t u r e s 14 and 15 have d i f f e r e n t CA s p e c t r a (21) a l t h o u g h an e a r l i e r m e t a s t a b l e i o n s t u d y r e v e a l s tïïât t h e s e i o n s decompose over t h e same s u r f a c e i n metastable t r a n s i t i o n s (22). +

3

2.

BOWEN

23

Unimolecular Reactions of Organic Ions

AND WILLIAMS

CA s p e c t r o s c o p y has a l s o been a p p l i e d t o t h e long-standing "benzyl versus t r o p y l i u m " problem (23^ 2£) ; s e v e r a l C H s p e c i e s w i t h d i f f e r e n t CA spect r a a r e o b s e r v e d ( 2 3 ) . E l e g a n t r e s u l t s stem from a d e u t e r i u m l a b e l l i n g s t u d y (24) where C H s D ions o f n o m i n a l s t r u c t u r e 1§ a r e o b s e r v e d t o l o s e :CD i n CA s p e c t r o s c o p y , thus p r o v i n g t h a t such low energy i o n s do n o t c o l l a p s e t o t r o p y l i u m s t r u c t u r e s . These s t u d i e s (23,24-), t o g e t h e r w i t h ICR work (2_5) w h i c h r e v e a l s tHât two d i s t i n c t C H p o p u l a t i o n s (presumab l y b e n z y l and t r o p y l i u m ) , w i t h d i f f e r e n t r e a c t i v i t i e s , e x i s t (25), c o n s t i t u t e strong evidence i n favor of b o t h t r o p y l i u m and b e n z y l c a t i o n s e x i s t i n g i n p o t e n t i a l w e l l s i n t h e gas phase. +

7

7

+

7

2

2

+

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch002

7

7

+

19 N e v e r t h e l e s s , s t u d i e s on h i g h e r energy i o n s (met a s t a b l e and s o u r c e r e a c t i o n s ) produced from 2 , 6 - C t o l u e n e (20) ( 2 6 ) , t h e t r o p y l i u m s a l t (21) (27) and d o u b l y l a b e l l e c T c y c l o h e p t a t r i e n e (28) s u g g e s t i s o m e r i z a t i o n o f benzyl to tropylium (or e q u i l i b r a t i o n of the two (2Ί5)) o c c u r s . T h i s i s because 20, f o r i n s t a n c e , fragments t o y i e l d some u n l a b e l l e d C s H and C H * . This i s not c o n s i s t e n t with simple c o l l a p s e to a 13

2

+

5

1 3

2

2

20 t r o p y l i u m i o n w h i c h t h e n r e a c t s w i t h o u t rearrangement s i n c e i o n s such as t h e c a t i o n o f 21 can o n l y l o s e C H or C C H without p r i o r rearrangement. The use o f energy measurements, w h i c h y i e l d h e a t s o f f o r m a t i o n (ΔΗ-f) o f s p e c i e s o f i n t e r e s t , i n con­ s t r u c t i n g p o t e n t i a l s u r f a c e s i s i n many ways s e l f evident. Where r e l i a b l e v a l u e s a r e a v a i l a b l e ( f o r i n s t a n c e : s e v e r a l s a t u r a t e d a l k y l i o n s ( 2 9 , 3 0 ) , some u n s a t u r a t e d carbonium i o n s (31-34) and v a r i o u s o t h e r s p e c i e s ( 3 5 - 3 8 ) ) , t h e r e l a t i v e l e v e l s o f r e a c t a n t s and p r o d u c t c o m b i n a t i o n s on a p o t e n t i a l s u r f a c e can be accurately assigned. A case where t h i s i s p o s s i b l e i s 2

1 3

2

2

24

HIGH

PERFORMANCE MASS

SPECTROMETRY

the (\Η + s u r f a c e r e f e r r e d t o abçve, where t h e h e a t s o f f o r m a t i o n o f t h e i s o m e r s o f Ci»H (29), product ions (29,31) and n e u t r a l s (38) a r e a l l a v a i l a b l e . In cases wïïëre t h e r e i s i n c o m p l e t e d a t a c o n c e r n i n g t h e h e a t s o f f o r m a t i o n o f t h e s p e c i e s o f i n t e r e s t , appearance pot e n t i a l (AP) measurements may be made. However, on conventional instruments there are systematic errors a s s o c i a t e d w i t h AP d e t e r m i n a t i o n s , and s i g n i f i c a n t e r r o r s a r e l i k e l y t o o c c u r . Many o f t h e d i f f i c u l t i e s a s s o c i a t e d w i t h AP measurements a r e e s s e n t i a l l y i n s t r u m e n t a l i n n a t u r e and have been d i s c u s s e d r e c e n t l y (39,40). AP measurements u s i n g c o n v e n t i o n a l mass spect r o m e t e r s may be u s e f u l i n d e t e r m i n i n g rough A H v a l u e s , but i n v i e w o f t h e d i f f i c u l t i e s a t t e n d i n g such measurements (3J9,40), t h e y s h o u l d n o t be r e g a r d e d a s b e i n g a c c u r a t e tô~T)etter t h a n ( s a y ) ±10 k c a l m o l " . Other ways around t h e p r o b l e m o f o b t a i n i n g ΔΗ^ v a l u e s f o r relevant species are calculations (41), estimation t e c h n i q u e s (5,42,£3), and p r o t o n a f f i n i t y (PA) mea­ surements (37,3T). The f i r s t two a r e e s p e c i a l l y use­ f u l i n c a s e s wKêre t h e s p e c i e s o f i n t e r e s t a r e unl i k e l y t o be a c c e s s i b l e v i a e x p e r i m e n t a l AP o r PA d e t e r m i n a t i o n , e.g., t h e 3 - h y d r o x y - l - p r o p y l c a t i o n ( 2 2 ) . The l a s t method i s o f t e n u s e f u l when t h e r e q u i s i t e c a t i o n cannot be g e n e r a t e d v i a d i r e c t i o n i z a t i o n o f t h e c o r r e s p o n d i n g r a d i c a l and i s c a p a b l e o f y i e l d i n g accurate r e s u l t s . An i n g e n i o u s a p p l i c a t i o n o f t h e t e c h n i q u e i s t h e d e t e r m i n a t i o n o f t h e h e a t o f format i o n o f t h e 2 - p r o p e n y l c a t i o n (23) by p r o t o n a t i o n o f propyne ( 4 4 ) . 9

9

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch002

f

1

+

+

CH CH CH 0H 2

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22

2

CH C=CH 3

2

23

When t h e methods o u t l i n e d above a r e a p p l i e d , t h r e e b a s i c t y p e s o f p o t e n t i a l s u r f a c e s a r e found t o occur. Complete E q u i l i b r a t i o n o f Isomers P r i o r t o Unimolecul a r Decomposition. T h i s c o r r e s p o n d s t o t h e g e n e r a l form o f p o t e n t i a l s u r f a c e shown i n F i g u r e 1. Here t h e a c t i v a t i o n energ i e s f o r i s o m e r i z a t i o n processes are l e s s than those f o r d i s s o c i a t i o n , and d e c o m p o s i t i o n o c c u r s o v e r t h e same s u r f a c e , from t h e same i o n ( o r i o n s ) i r r e s p e c t i v e of t h e o r i g i n o f t h e i o n under s t u d y . Examples o f t h i s g e n e r a l case a r e numerous, f o r i n s t a n c e t h e Ci»H +

9

2.

BOWEN

AND WILLIAMS

+

25

(15) and C H i (6) i s o m e r s r e f e r r e d t o above. Four g e n e r a l c r i t e r i a o f e x p e r i m e n t a l measurements must be s a t i s f i e d b e f o r e t h i s k i n d o f s u r f a c e may be s a f e l y assigned t o a given i o n . ( i ) The c r i t e r i o n o f m e t a s t a b l e abundances (ljS) must h o l d good, i . e . t h e d e c o m p o s i t i o n o f i o n s gener­ a t e d i n i t i a l l y as d i f f e r e n t s t r u c t u r e s must o c c u r t h r o u g h t h e same c h a n n e l ( s ) and i n s i m i l a r r a t i o s . However, some changes i n t h e r a t i o s o f i o n s decompo­ s i n g t h r o u g h t h e v a r i o u s c h a n n e l s may be o b s e r v e d ; t h e s e changes r e f l e c t t h e s l i g h t d i f f e r e n c e s i n i n t e r ­ n a l e n e r g i e s o f i o n s produced f r o m d i f f e r e n t p r e c u r s o r s (16) . (ii) The m e t a s t a b l e peak shapes must be t h e same f o r each d e c o m p o s i t i o n c h a n n e l , i r r e s p e c t i v e o f t h e precursor o f the ion. (iii) The AP's f o r each d e c o m p o s i t i o n channel must be t h e same ( a f t e r s u i t a b l e c o r r e c t i o n s have been made t o compensate f o r t h e d i f f e r e n t h e a t s o f formation of precursors) i r r e s p e c t i v e of the o r i g i n o f t h e i o n s under s t u d y . (iv) There must be l o s s o f i d e n t i t y o f some atoms i n l a b e l l e d i o n s , i . e . , t h e d e c o m p o s i t i o n o f l a b e l l e d i o n s must be s t a t i s t i c a l , o r must be c a p a b l e o f b e i n g i n t e r p r e t e d i n terms o f s t a t i s t i c a l s e l e c t i o n o f t h e n e c e s s a r y atoms t o g e t h e r w i t h an i s o t o p e e f f e c t . When i s o t o p e e f f e c t s a r e i n o p e r a t i o n , they must be the same f o r i o n s g e n e r a t e d from d i f f e r e n t p r e c u r s o r s . Whereas t h e t h i r d c r i t e r i o n i s l e s s r e l i a b l e when the AP measurements a r e made u s i n g c o n v e n t i o n a l mass s p e c t r o m e t e r s because o f t h e s y s t e m a t i c e r r o r s w h i c h may c o m p l i c a t e t h e a n a l y s i s ( 3 9 , 4 0 ) , t h e o t h e r t h r e e a r e s u i t a b l e f o r work u s i n g s t a n d a r d mass spectrome­ ters. An i n t e r e s t i n g example o f t h e i m p o r t a n c e o f con­ s i d e r i n g i s o t o p e e f f e c t s i n c o n j u n c t i o n w i t h complete e q u i l i b r a t i o n o f t h e atoms i n l a b e l l e d i o n s i s f u r ­ n i s h e d by two independent s t u d i e s (45,46) o f t h e c o m p e t i t i v e l o s s o f Η· and D« from t E e m o l e c u l a r i o n s of l a b e l l e d toluenes. For the three precursors 24-26, l o s s o f Η· and D» i n m e t a s t a b l e t r a n s i t i o n s from t h e m o l e c u l a r i o n s c a n be a c c o u n t e d f o r i f t h e l o s s o f hydrogen r a d i c a l i s assumed t o be s t a t i s t i c a l w i t h an i s o t o p e e f f e c t o f 2.8:1 (45) o r 3.5:1 (46) i n f a v o r o f Η· l o s s . The s l i g h t cTTscrepancy between the two v a l u e s found i n t h e two s t u d i e s i s presumably due t o a p o p u l a t i o n o f i o n s w i t h l o n g e r l i f e t i m e s (and hence lower i n t e r n a l e n e r g i e s ) i n t h e l a t t e r s t u d y ( 4 6 ) . S i m i l a r r e s u l t s a r e found (45) f o r t h e l a b e l l e c T ~ c y c l o h e p t a t r i e n e m o l e c u l a r i o n s T7 and 28; 6

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch002

Unimolecular Reactions of Organic Ions

3

26

HIGH

PERFORMANCE

MASS

SPECTROMETRY

t h i s i s c o n s i s t e n t w i t h e q u i l i b r a t i o n of the toluene and c y c l o h e p t a t r i e n e m o l e c u l a r i o n s p r i o r t o m e t a s t a ­ b l e d e c o m p o s i t i o n s (see c r i t e r i o n ( i v ) above).

24

25

26

27

28

Perhaps t h e b e s t example o f t h e j o i n t a p p l i c | t i o n of the c r i t e r i a i s a d e f i n i t i v e study o f the 0 Η · r a d i c a l c a t i o n s (47). In t h i s study the metastable abundances, peak sïïapes and t h e appearance p o t e n t i a l s f o r the v a r i o u s decomposition channels o f C H * ions g e n e r a t e d from propene and c y c l o p r o p a n e a r e shown t o be c o n s i s t e n t w i t h complete e q u i l i b r a t i o n o f t h e two p o s s i b l e structures o f the molecular i o n p r i o r to metastable t r a n s i t i o n s . Other members o f t h e H * homologous s e r i e s o f r a d i c a l c a t i o n s behave s i m i l a r l y (48,49). Other examples o f t h e a p p l i c a t i o n o f t h e c r i t e r i o n o f m e t a s t a b l e abundances (16) t o show complete e q u i l i bration of a l l accessible reactant configurations prior to m e t a s t a b l e d e c o m p o s i t i o n s i n c l u d e some C Η and C H 2 _ i o n s ( 5 0 ) . '

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch002

3

3

6

6

+

c

n

2 n

η

n

n

n

Δ η

λ

3

No E q u i l i b r a t i o n o f Isomers P r i o r t o U n i m o l e c u l a r Decomposition? T h i s c o r r e s p o n d s t o t h e g e n e r a l form o f p o t e n t i a l s u r f a c e i l l u s t r a t e d i n F i g u r e 3. Here t h e a c t i v a t i o n energies f o r i s o m e r i z a t i o n processes are g r e a t e r than t h o s e f o r d i s s o c i a t i o n , and d e c o m p o s i t i o n o c c u r s over two s e p a r a t e p o t e n t i a l s u r f a c e s . D e c o m p o s i t i o n o v e r t h i s g e n e r a l p o t e n t i a l s u r f a c e can be d e t e c t e d by t h e following observations: (1) The d e c o m p o s i t i o n ( s ) o f i o n s on t h e two s e p a r a t e " h a l v e s o f t h e s u r f a c e w i l l i n g e n e r a l be d i f f e r e n t b o t h i n t h e n a t u r e o f t h e r e a c t i o n ( s ) under­ gone and i n t h e e x t e n t t o w h i c h any common r e a c t i o n s occur. (ii) The shapes o f t h e m e t a s t a b l e peaks f o r any common r e a c t i o n s w i l l n o t , i n g e n e r a l , be t h e same. Indeed, i n some c a s e s (12^, 5 ^ ) , d r a s t i c d i f f e r e n c e s a r e observed. (iii) The AP's f o r any common d e c o m p o s i t i o n s w i l l be d i f f e r e n t i n g e n e r a l . 1 1

BOWEN

AND WILLIAMS

unimolecular Reactions of Organic Ions

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch002

Accounts of Chemical Research

Figure 1. Potential energy surface corresponding to complete equilibration to two ions, A* and B*, prior to slow unimolecular decomposition 231 CH CH 3

/ ^

208

2

+ CH =CH 2

2

+ CH,

Accounts of Chemical Research

Figure 2. Potential energy surface for slow unimolecular decomposition of the isomeric cations C H +

4

9

Accounts of Chemical Research

Figure 3. Potential energy surface corresponding to no interconversion of two ions, A* and B , prior to slow unimolecular decomposition +

28

HIGH

PERFORMANCE

MASS

SPECTROMETRY

( i v ) The b e h a v i o r o f l a b e l l e d i o n s on t h e two " h a l v e s " o f t h e s u r f a c e w i l l n o t u s u a l l y be t h e same. In p a r t i c u l a r , i s o t o p e e f f e c t s , when p r e s e n t , a r e u n l i k e l y t o be i d e n t i c a l . An e x c e l l e n t example o f t h i s k i n d o f p o t e n t i a l surface i s C H 0 . Ions g e n e r a t e d from p r e c u r s o r s h a v i n g t h e e t h e r m o i e t y ( s t r u c t u r e 29) behave i n a c o m p l e t e l y d i f f e r e n t way t o t h o s e g e n e r a t e d from 1a l k a n o l s ( 3 0 ) , 2 - a l k a n o l s (31) and compounds o f f o r m u l a ( 3 2 J , where Y i s a v a r i a b l e group. Two +

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch002

2

5

CH3OCH2Y

CH3CHYOH

YCH2CH2OH

CH3CH2OY

29

30

31

32

metastable decompositions o f C 2 H s O ions are observed ( 5 1 ) ; C H l o s s a t m/e 8.02 and CPU l o s s a t m/e 18.7. TEê" abundance d a t a , t o g e t h e r w i t h t h e k i n e t i c energy r e l e a s e w h i c h accompanies CHi* l o s s , a r e g i v e n i n Table I (SI). +

2

2

Table I . Precursor structure

Metastable

Decompositions o f C 2 H s O

+

ions.

m/e 8.02 m/e 18.7

k i n e t i c energy r e l e a s e ( k c a l m o l " ) o f m/e 18.7

m ++ , nu + + ity +e

+

Photo i o n i zat ion

ην

m^

Photo Dissociation

+

m-j* + Ν + m-j** + Ν

+Ν

Excitation

m^

+ Ν + e"

m^

+Ν

++

m^ + Ν + m^

2

+

+ e~

+ m

+ m..

+ Ν -•m + m + Ν

mi

m^

m«

Charge exchange

Stripping

+

m^

Dissociation

+

m^

Emission

The fast analyzed species i s underlined.

INDUCED PROCESSES

SPONTANEOUS PROCESSES

Metastable Fragmentation

Table I . Reactions Studied V i a Translational Energy Measurements

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch004

ο

w

s ο

ο χ

Ο

4. COOKS

61

Kinetic Energy Spectrometry

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch004

Analysis of Mixtures by MIKES/CID By choosing an appropriate configuration for a double focusing mass spectrometer and by studying secondary fragmentations, the analysis of mixtures can readily be achieved. Q-4) The mass-analyzed ion k i n e t i c energy (MIKE) arrangement allows tor ion selection as a prelude to dissociation. Kinetic energy analysis of the fragments provides a fragmentation pattern from which the structure of the reactant ion, and hence that of the corresponding neutral molecule, can be inferred. Figure 1 summarizes the principles of this method of mixture analysis. The method i s an alternative to gas chromatography/mass spectrometry and i t has the advantage that i t employs electromagnetic rather than chromatographic separation. D i f f i c u l t i e s of v o l a t i l i t y , lengthy development times and chemical contamination or degradation associated with chromatography are avoided. This i s accomplished at the expense of a loss i n sensit i v i t y associated with measurements on secondary rather than primary ions. Although both electron impact and chemical ionization have been employed i n these analyses, chemical ionization i s preferred because i t secures the structural relationship between the neutral molecule of interest and the corresponding ion which i s actually separated and analyzed. The secondary fragmentation reaction may be spontaneous or collision-induced. The l a t t e r are much preferred since ( i ) more reactions are observed, ( i i ) the t o t a l signal due to secondary ions i s higher, t y p i c a l l y by an order of magnitude, ( i i i ) there i s a greater tendency for simple bond cleavages i n CID than i n metastable ion reactions. To i l l u s t r a t e the s e n s i t i v i t y of t h i s technique consider the scan shown i n Figure 2. This peak i s a part of the MIKE spectrum taken i n the presence of c o l l i s i o n gas, of the (M + H ) ion of p-nitrophenol which was present i n a mixture of nitrobenzenes. Of special note i n these spectra (compare r e f . l c ) i s the cleanliness of the background as contrasted to gc/ms spectra. This i s a consequence of the double f i l t e r which operates i n obtaining these spectra. The signal to noise characteristics of the peak shown i n Figure 2 are consistent with a detectability l i m i t of ca. 10~Hg for t h i s experiment. Quantitation of the procedure has not been investigated i n d e t a i l but the general considerations should be similar to those i n volved i n quantitation of gc/ms data. For highest accuracy t h i s demands use of an internal standard, preferably an i s o t o p i c a l l y labeled variant of the compound of interest. An additional consideration, i f collision-induced dissociation i s employed, i s that the pressure of the target gas must be held constant. Figure 3 demonstrates the type of accuracy which i s readily achieved, variations i n c o l l i s i o n gas pressure being the major source of error i n t h i s experiment. One of the more attractive features of t h i s method i s i t s i n s e n s i t i v i t y to the presence of large amounts of impurities. Solvent +

HIGH PERFORMANCE MASS SPECTROMETRY

(M,*H)* Ionize (M +Hf Mass (M +H)

[(Μ +Η)**]—

3

M

3

CI

m

Excite

2

3

3i

m

Energy H

32

Analyze

(M +rtf Analyze 3

m 99 Characterization

Separation

Collision Spectroscopy

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch004

Figure 1. Principle of mixture analysis using the reversed sector massanalyzed ion kinetic energy (MIKE) spectrometer

5xl0" g/sec n

Figure 2. Illustration of sensitivity of the MIKES mixture analysis method. The peak shown, scanned in 3 min, is a characteris­ tic fragmentation (loss of OH) of the protonated molecular ion of p-nitrophenol. 86

88

90

%E

—X— 3-methyl-2-butonon« — 0 — 2-pentonont — o — 3-ptntanont

Mole

Fraction Analytical Letters

Figure 3. Analysis of ketones in mixtures of varying composition by electron impact ionization followed by MIKES

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch004

4. COOKS

Kinetic Energy Spectrometry

63

may constitute the major portion of the sample without deleterious results. Indeed, the solvent could probably be used to advantage as the CI reagent gas i n some cases. These considerations suggest that the method might f i n d appropriate application i n the continuous analysis of organic reaction mixtures or effluents. Cases i n which workup must be minimized, e.g., where chromatography a l t e r s the composition of the mixture are of special interest. Results on the analysis of the mixture resulting from the methylation of 2-butanone by methyl iodide i n the presence of base are given i n Table I J. Each compound was quantitated using c h a r a c t e r i s t i c peaks i n the MIKE spectrum of i t s molecular ion (electron impact). I t i s noteworthy that the mixture included isomers which are not separated i n the mass analysis stage of the procedure. The results given are for an early and a l a t e aliquot. They indicate the progress of methylation and the s t e r i c control to which t h i s reaction i s subject. The agreement between results employing metastable ion dissociations and CID i s of the order expected i n the absence of more sophisticated internal standards. Currently, mixture analysis by CI/MIKES i s being extended to more complex compounds including natural products and pharmaceuticals. Classes of conpounds which, because of v o l a t i l i t y or thermal i n s t a b i l i t y , cannot readily be separated by gas chromatography are of special interest. For t h i s reason the MIKE spectra of barbiturates have been examined. (2) There have been continued d i f f i c u l t i e s i n the analysis of these compounds, even using the newer mass spectrometric techniques. The ethyl- and a l l y l substituted barbiturates give most d i f f i c u l t y i n analysis and we therefore chose to examine several of the most refractory isomeric and homologous barbiturates. The MIKE spectra of the (M + H ) and (M + C2H5)* ions were found to c l e a r l y distinguish them. Figure 4 compares the MIKE spectra taken i n the presence of c o l l i s i o n gas of four protonated barbiturates. Two sets of isomers and two sets of homologs are represented. The sec-butyl group of butabarbital and the sec-amyl group of pentobarbital are associated with intense fragmentation by alkene elimination. The substituents which bear fewer hydrogens on the ot-carbon show t h i s reaction to a lesser extent. The s i m i l a r i t i e s i n the spectra shown are t y p i c a l of those observed for homologous barbiturates. The current s i t u a t i o n regarding mixture analysis by MIKES i s that the technique appears to be complementary to gc/ms, being applicable i n situations i n which gc i s d i f f i c u l t . The spectra are r e a d i l y interpreted and quantitation seems not to present any new problems. As occurred i n the development of gc/ ms, i t seems l i k e l y that automation of the instrumentation w i l l be required to achieve i t s f u l l potential which should include continuous monitoring c a p a b i l i t i e s . The somewhat lower sensit i v i t y compared with gc/ms should not often preclude i t s use, and the potential to analyze i n v o l a t i l e and thermally unstable b i o l o g i c a l compounds i s a most a t t r a c t i v e compensatory feature. +

HIGH PERFORMANCE MASS

SPECTROMETRY

Table I I . Analysis of Reaction Mixtures from the Methylation of Butanone

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch004

Early

Late

+

M*

Unimol

Unimol + CID

72

0.15

0.15

86

0.18

0.20

86

0.17

0.16

100

0.35

0.35

100

0.02

0.02

114

0.09

114

128

Unimol

Unimol + CID

0.04

0.04

0.10

0.88

0.92

0.03

0.02

0.08

0.02

—

-

0.01

0.01

4. COOKS

Kinetic Energy Spectrometry

65

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch004

Fragmentation Mechanisms and Metastable Ions i n Chemical Ionization Chemical ionization allows ready access to types o f ions which cannot be generated by electron impact. There has, however, been r e l a t i v e l y l i t t l e attention given the fragmentation mechanisms of these ions. This has been a consequence o f the emphasis accorded the determination of molecular weights by CI and the resulting efforts to minimize fragmentation. When secondary reactions are studied, fragmentations are naturally highlighted and i t has been observed that metastable ions i n CI often have i n t e n s i t i e s comparable to those observed i n EI. The study of metastable ions i n CI reveals the existence of a fragmentation mechanism i n which a neutral alkane molecule i s eliminated from onium type ions. For example, protonated t-butyl ethyl ether fragments by eliminating a butane molecule (1) while H CH.

Œ CH=ÔH + C H 3

4

CD

10

CH / I 3 CH. the trimethyloxonium ion losses methane. The reaction has also been observed i n halonium and ammonium ions. In a l l cases i t probably occurs v i a a four-centered mechanism (see below). I t i s sometimes observed i n competition with a l k y l r a d i c a l loss and alkene molecule elimination. The reaction i s quite general, since i t also occurs i n odd-electron ions. (3) Thus i t has been observed from the molecular ions of ketones and amines. Figure 5 compares the fragmentations of the molecular ion and the (M + H ) ion of diisopropyl ketone. The s i m i l a r i t y i n behavior o f the odd and even electron ions i n undergoing loss of an alkane molecule i s remarkable. Alkane loss has long been known i n the mass spectra of alkanes themselves, but, except for an isolated observation on ketones (4), the reaction has u n t i l recently gone unnoticed. I t now seems~that i t constitutes an important general fragmentat i o n mechanism which p a r a l l e l s the well-known alkene eliminat i o n (McLafferty rearrangement) i n the types of ions i n which i t occurs and i n i t s importance as a favored process at low internal energy. The four centered mechanism has been e x p l i c i t l y demonstrated f o r diisopropyl ketone by deuterium labeling. +

C H

C

3^ / \ ^ C

Œ

3" / H

C H

C

D

x

X

CH

3 3

>

H-C-D C^

+ 0=C=C Χ

3

(

Ή

X

3

3

66

HIGH PERFORMANCE MASS SPECTROMETRY

(M*H)

Butabarb (s-Bu)

+

' 212

Pentobarb (s-Am!

(M*H) 226

-132

-J

(M+H)

Butethal (n-Bu)

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch004

212

Amobarb (i-Am)

(M+H) 226

ι r Figure 4. MIKE spectra taken in presence of target gas of the proto­ nated barbiturates indicated. The stable ion beam, (M + H)* is recorded at an attenuation of JO . 3

|

M

>

o

M

o

Ο

Figure 10. CI (CH^) mass spectrum of a mixture of 3-aminopentane and 2-aminobutane showing formation of proton boundQ dimers

_JL_

175

2

NH + >-NH + CH

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch004

COOKS

Kinetic Energy Spectrometry

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch004

(tIOOO)

1 60

40 %E

Figure 11. MIKE spectrum (no collision gas) of the proton bound dimer formed from 2-aminobutane and 3-aminopentane. The stronger base gives the large metastable peak for formation of the BH+ ion.

48

'

50

'

52

%F

Figure 12. Part of the MIKE spectrum of the s-butylamine/ pyridine proton bound dimer illustrating a case in which the metastable peaks for Reactions 2 and 3 differ in height by less than a factor of two. The proton affinity difference in this case is estimated to be 0.1 to 0.2 kcal mol' . 1

74

HIGH PERFORMANCE MASS SPECTROMETRY

As a f i n a l i l l u s t r a t i o n of the s e n s i t i v i t y of the MIKES approach i n investigating ion chemistry, Figure 14 shows a p a r t i a l MIKE spectrum of a C - labeled pyridine/s-BuNH2 dimer formed using only unenriched compounds. These species were selected from the complicated mixture of ions present i n the ion source. The doubly labeled ion represents only I I of the protonated dimer ion current. Nevertheless, i t s metastable reactions can r e a d i l y be studied the l a b e l pattern shown i n part (b) being that associated with pyridine and agreeing with the s t a t i s t i c a l l y required r a t i o of 3:10:5 for 80 :81 :82 . Work currently i n hand has shown that the method can be extended to bases belonging to other functional groups, and a ladder of b a s i c i t i e s i s being established. Collision-induced dissociation, although less sensitive to small differences i n proton a f f i n i t i e s than are metastable ion reactions, has been found to give corresponding r e s u l t s . The k i n e t i c energy releases for some of these reactions have been measured, and the small values recorded are consistent with the proposed n e g l i g i b l e effect of reverse a c t i v a t i o n energy. I 3

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch004

+

+

+

Ion Structure Determinations In keeping with the importance of determining ion structures, there has been a great deal of emphasis placed on t h i s subject i n recent years. Measurements of metastable ion characteristics, especially k i n e t i c energy release, ion enthalpies, and c o l l i s i o n a l activation (collision-induced dissociation) spectra have been most valuable i n t h i s regard. Our recent work i n t h i s area has f a l l e n into two categories, (a) the application of established methodology to investigate the structures of ions generated by chemical ionization, (b) the testing of new methods for characterizing ions. Examples from each topic are covered i n turn below. The combination of a chemical i o n i z a t i o n source with a MIKE spectrometer allows ions formed by CI to be independently characterized. This i s r e a d i l y done by mass-selection, followed by collision-induced dissociation. The r e s u l t i n g MIKE spectrum w i l l contain metastable peaks as w e l l as those due to CID. Figure 15 shows a set of these spectra for the ethylated nitrobenzenes indicated.(11) A s t r i k i n g feature of the spectr| i s the occurrence of major reactions common to a l l four (M + C-Hr) ions. The reactions correspond to loss of C H., C X O aild CJlrONO. The l a t t e r two processes can occur as simple Bond cleavages only i f the n i t r o group i s ethylated. ,As a further test of t h i s structure, the corresponding protonated molecules were examined i n the same way. Major reactions now observed were loss of HO" and MXJT, supporting the conclusion that the n i t r o group i s more basic than the r i n g or the para substituent. A characteristic of r i n g a l k y l a t i o n or protonation i s the loss of the substituent as a free r a d i c a l . Thus, one can contrast the absence of CI* loss i n the spectrum of ethylated 2

4. COOKS

Kinetic Energy Spectrometry

Py....Ht...Py (d ) e

W )PyH* 9

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch004

PyH

%Ε

Figure 13. Metastable peaks for the formation of labeled and un­ labeled pyridinium ions from a partially labeled dimer. Collection and detection efficiencies can be estimated from this data if second­ ary isotope affects are assumed negligible.

(Py...H ..NH Bu )-*C +

l

t

t

ι βι •

51

52

53

%E

Figure 14. Illustration of the selectivity and sensi­ tivity of the MIKES tech­ nique for studying ion thermochemistry. The doubly labeled ion stud­ ied was selected from the naturally occumng mix­ ture.

75

HIGH PERFORMANCE MASS SPECTROMETRY

o)pNO

.197* t

-28 -45

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch004

50

200 I77

150

100

+

b)pCN

J

L 150

100

50

186

c)pCI

50

150

100

186

d)pOH

50

100

150

Figure 15. MIKE spectra taken in the presence of collision gas of the (M + Ety ions of the substituted nitrobenzenes indicated. The abscissae of the spectra are calibrated in terms of ion mass. The stable ion beams have been divided by approximately 10 . s

4. COOKS

Kinetic Energy Spectrometry

77

£-chloronitrobenzene (Figure 15c) with the spectra of protonated p-chlorophenol, ethylated p-chlorobenzoic acid and ethylated pchlorobenzonitrile. Loss of CI* i n these compounds gives peaks which are 53%, 50%, and 3% of the most abundant fragment ions, respectively. In these and other cases there i s also evidence for a mixture of ring and substituent alkylated ions. We turn now to the testing of newer methods of characterizing ions. Those which employ the same instrumentation and methods as CID and metastable ion reactions are most attractive. Thus, charge stripping (7) provides a case i n point, the cross section

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch004

+ N+ m^* + Ν + e-

(7)

for the reaction being readily measured r e l a t i v e to that f o r an ion chosen to serve as an internal standard. This procedure was adopted i n comparing the isomeric C;$HgN ions generated from t butylamine and diethylamine. The l a t t e r showed a peak due to charge stripping which was more than an order of magnitude more intense than the former. I t seems probable that t h i s difference i s due less to differences i n the cross section f o r stripping (a process with a large energy loss which should be r e l a t i v e l y i n ­ sensitive to structural variation) than i t i s due to differences i n the s t a b i l i t y of the nascent doubly charged ions. Stripping at k i l o v o l t energies i s a v e r t i c a l process, the doubly charged ion being generated with the same structure as i t s singly charged precursor. Thus, the greater extent of fragmentation of the more highly branched C3HgN ion generated from t-butylamine i s the expected result. Although t h i s method of characterizing ions r e l i e s on a single parameter, i t i s of some interest as a means of sampling singly charged ions of lower average internal energy than i s the case f o r collision-induced dissociation.(12) A consequence i s that comparisons between stripping and CID sEould aid i n uncovering isomerizations occurring below the threshold for fragmentation. As a second example of newer methods of characterizing ions, we s h a l l consider measurements of k i n e t i c energy loss f o r collision-induced dissociations. Kinetic energy releases can be measured from the same data so we consider them together. As an example, H loss from a l k y l cations can be considered. Analogies i n structure and fragmentation mechanism of C2H3 , C?H C1 , C H3 , C H5 , and C H are inferred from the fact that these ions when generated from a variety of precursors give the energy losses and k i n e t i c energy releases shown i n Table I I I . The methyl cation shows contrasting behavior i n both i t s k i n e t i c energy loss and i t s energy release. +

++

2

+

2

+

+

3

+

3

+

4

3

78

HIGH PERFORMANCE MASS

SPECTROMETRY

Table III. Energy Releases and Losses for A l k y l Ions

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch004

Ion

Energy Loss (eV)

Energy Release (meV)

10.3±1.6

330±15

9.4±1.4

339t31

11.3±0.5

334±4

11.0

334

10±1

296±17

6.4±0.9

83±4

Conclusion Ion chemistry and i t s analytical applications have been treated here with a focus on particular instrumentation and types of measurements. The combination of chemical ionization with the mass-analyzed ion k i n e t i c energy (MIKE) technique i s valuable i n determining the structures of ions generated by low energy ion/molecule reactions, i n the analysis o f complex mixtures without p r i o r separation and i n mechanistic studies employing labeled compounds. The usefulness of k i l o v o l t energy ion/molecule reactions as a surprisingly subtle probe of ion properties i s shown. The compatibility of these and metastable ion reactions i n terms of instrumentation and the complementary information they supply i s noteworthy. Wider use of charge changing c o l l i s i o n s as well as collision-induced dissociation for organic ions seems appropriate. The range of inquiry which can be based on the rather simple measurement of ion k i n e t i c energy remains a constant source of pleasure t o t h i s research group. Acknowledgement This work has been supported by the National Science Foundation and done i n collaboration with T. L. Kruger, V. M. Franchetti, J . F. L i t t o n , R. W. Kondrat, C. S. Hsu, R. Flammang, E. Soltero-Rigau and D. Cameron.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch004

4. COOKS

Kinetic Energy Spectrometry

79

Abstract Secondary reactions including spontaneous and collision­ -induced dissociation and charge changing collisions, have been studied by the methods of ion kinetic energy spectrometry. Emphasis is on ions generated by chemical ionization which are mass-selected before reaction and energy analyzed after. Appli­ cations to the analysis of mixtures without chromatographic separation are demonstrated. Ion structure determinations are made on CI generated ions. Kinetic energy release and loss measurements are used to characterize ions. Studies on labeled compounds employing partially labeled material are shown to be possible so facilitating mechanistic investigations. Alkane elimination is shown to be a general low energy fragmentation occurring in both odd- and even-electron ions. The occurrence of metastable ion dissociations in chemical ionization experiments has been explored and these reactions form the basis for a new and sensitive method of ordering proton affinities in the gas phase. Literature Cited 1. Smith, D.H., Djerassi, C., Maurer, K.H., and Rapp, U . , J . Am. Chem. Soc., (1974), 96, 3482. 2. Schlunegger, U.P., Angew. Chem., Int. Ed. Engl., (1975), 14, 679. 3. Kruger, T.L., Litton, J.F., and Cooks, R.G., Anal. Chem., (1976), 9, 533. 4. Kruger, T.L., Litton, J.F., Kondrat, R.W., and Cooks, R.G., Anal. Chem., (1976), 48, 2113. 5. Levsen, Κ., and Schulten, H.R., Biomed. Mass Spectrom., (1976), 3, 137. 6. Soltero-Rigau, E., Kruger, T.L., and Cooks, R.G., Anal. Chem., (1977), 49, 435. 7. Litton, J.F., Kruger, T.L., and Cooks, R.G., J . Am. Chem. Soc., (1976), 98, 2011. 8. Cooks, R.G., Yeo, A.N.H., and Williams, D.H., Org. Mass Spectrom., (1969), 2, 985. 9. Cameron, D., Clark, J.E., Kruger, T.L., and Cooks, R.G., Org. Mass Spectrom., in press. 10. Holmes, J . L . , Osborne, A.D., and Weese, G.M., Org. Mass Spectrom., (1975), 10, 867. 11. Beynon, J.H., Brothers, D.F., and Cooks, R.G., Anal. Chem., (1974), 46, 1299. 12. Cooks, R.G., and Kruger, T.L., J. Am. Chem. Soc., (1977), 99, 1279. 13. Aue, D.H., Webb, H.M., and Bowers, M.T., J . Am. Chem. Soc., (1972), 94, 4726. 14. Kruger, T.L., Flammang, R., Litton, J.F., and Cooks, R.G., Tetrahedron Letters, (1976), 4555. 15. Cooks, R.G., Beynon, J.H., and Litton, J.F., Org. Mass Spectrom., (1975), 10, 503. RECEIVED December 30, 1977

5 Field Ionization Kinetic Studies of Gas-Phase Ion Chemistry N. M. M. NIBBERING

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch005

Laboratory for Organic Chemistry, University of Amsterdam, Amsterdam, The Netherlands

In the last few years the method of Field Ionization Kinetics (FIK) has been developed rapidly by several groups (1). The technique has proved to be successful in providing detailed information on the mechanisms of decompositions of ions in the gas phase (1,2). This information has been d i f f i c u l t or even impossible to obtain from conventional electron impact (EI) studies. Although the underlying theory of the FIK method has been described extensively i n detail (1,3), i t w i l l be repeated here i n a very simple way. If, for example, a 10 μm conditioned wire emitter (4) at potential V (- 8 kV) is positioned approximately 1.5 mm from a grounded and slotted cathode, gas phase molecules may be ionized in the strong e l e c t r i c f i e l d at a very narrow region close to the emitter. To a good approximation, the potential is equal to the emitter potential V at this point (Figure 1). Suppose that the ions, formed by either direct ionization or unimolecular decomposition close to the emitter, are stable for ≥ 1 0 sec. They w i l l acquire a kinetic energy eV (Turing acceleration from the emitter to the cathode and w i l l pass the e l e c t r i c sector of a double focussing mass spectrometer, set to transmit ions with a kinetic energy eV . Consequently, they w i l l be mass analyzed at their correct m/e-values. In this way a f i e l d ionization (FI) spectrum is obtained, containing peaks corresponding to the ions generated very near to the emitter, i . e . , wijhin approximately 1 0 sec. Fragment ions, m , generated by expulsion of (M-m) from M ions between the emitter and cathode at potent i a l V (Figure 1) are not transmitted through the e l e c t r i c sector, because they have insufficient kinetic 0

0

-5

0

0

- 1 1

+

+

x

©0-8412-0422-5/78/47-070-080$05.00/0

5.

81

Field Ionization Kinetic Studies

NiBBERiNG

energy (em/M ( V Q - V ^ ) + eVy) t o be f o c u s s e d by t h i s s e c t o r . However, t h e s e i o n s can be t r a n s m i t t e d t h r o u g h the e l e c t r i c s e c t o r by i n c r e a s i n g the e m i t t e r p o t e n ­ t i a l , V Q , by an amount A V , such t h a t the k i n e t i c energy of the fragment i o n s , m , i s g i v e n by e q u a t i o n (1) (3a). +

e V

0

=

ΤΓ

( V

0

+

Δ Υ

* V

+

e V

X

Thus i t i s p o s s i b l e t o m o n i t o r a t a f i x e d e l e c t r i c s e c ­ t o r v o l t a g e the abundance o f m i o n s as a f u n c t i o n o f the e m i t t e r p o t e n t i a l V Q + A V ( 3 a ) . A scan o f the e m i t t e r p o t e n t i a l , a c h i e v e d by i n c r e a s i n g A V , a l l o w s one t o v i e w the d e c o m p o s i t i o n s o f i o n s M o c c u r r i n g a t e v e r - i n c r e a s i n g d i s t a n c e s from the e m i t t e r . Of c o u r s e , these i n c r e a s e d d i s t a n c e s correspond to longer l i f e ­ t i m e s (5) o f the p a r e n t i o n , M . E x p r e s s i n g the abun­ dance o f m i o n s due t o l o s s o f (M-m) as a f u n c t i o n o f time f o l l o w i n g FI o f M, the FIK c u r v e o f m i s ob­ t a i n e d ( F i g u r e 2 ) . The time r a n g e , c o v e r e d c o n t i n u ­ o u s l y by the FIK method, i s a p p r o x i m a t e l y 1 0 " to 10" s e c . A d d i t i o n a l p o i n t s a t lçnger t i m e s can be o b t a i n e d from d e c o m p o s i t i o n s o f M i o n s i n the f i r s t and second f i e l d f r e e r e g i o n s , which a r e a l s o a c c e s s i b l e by the c o n v e n t i o n a l EI t e c h n i q u e ( F i g u r e 1 ) . The s t r e n g t h o f the FIK method i s shown t o f u l l advantage, i f i t i s used i n c o m b i n a t i o n w i t h a double f o c u s s i n g mass s p e c t r o m e t e r and w i t h i s o t o p i c l a b e l l i n g , f o r the e l u c i d a t i o n o f mechanisms o f gas-phase ion decomposition processes at 1 0 " t o 10' sec. (3a). The f i r s t and a l r e a d y c l a s s i c a l example o f such mecFP a n i s t i c s t u d i e s has been the FIK s t u d y on c y c l o h e x e n e 3,3,6,6-dit ( 6 ) . S i n c e then many m e c h a n i s t i c FIK s t u d i e s Kave "appeared i n the l i t e r a t u r e , t o w h i c h the reader i s r e f e r r e d ( l c , 2 , 7 ) . In our l a b o r a t o r y , tïïe FIK method i n c o m b i n a t i o n w i t h d e u t e r i u m l a b e l l i n g has been a p p l i e d t o gas phase d e c o m p o s i t i o n s o f the m o l e c u l a r i o n s o f 2-phenoxyethyl c h l o r i d e and o f 3 - p h e n y l p r o p a n a l , as w i l l be d i s c u s s e d below. In t h i s way, we i n t e n d t o show the a p p l i c a t i o n of t h i s p o w e r f u l t e c h n i q u e f o r i n v e s t i g a t i o n o f gasphase i o n i c d e c o m p o s i t i o n s .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch005

+

+

+

1 1

9

1 1

7

FIK Study on 2-Phenoxyethyl

Chloride (8).

A p r e v i o u s EI s t u d y o f 2-phenoxyethyl

chloride,

C 6 H 5 O C H 2 C H 2 C I , i n combination w i t h deuterium l a b e l l i n g has shown, t h a t the m o l e c u l a r i o n s l o s e i n a p p r o x i 1

m a t e l y e q u a l amounts «C^Cl and

2

•CH C1 ( 9 ) . 2

T h i s must

82

HIGH PERFORMANCE MASS SPECTROMETRY

slotted cathode emitter

/

J

electric sector set to transmit ions with a kinetic energy eV magnetic sector

v

x

Q

1 field free region

,'

detector

st

2 field free region

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch005

nd

Figure 1. Double-focusing field ionization mass spectrometer (not drawn to scale)

Figure 2. FIK curve of ions m*

(right) *Advances in Mass Spectrometry Figure 3. FI spectra of 2-phenoxyethyl chloride and of its 1,1-d«- and 2,2,-d analogs. The left ordinate scale refers to the intensities of ions with m/e < 130; the right ordinate scale to those of ions with m/e > 130. f

5.

83

Field Ionization Kinetic Studies

NiBBERiNG

94

63

.O-CH2-CH2-CI

COT

1

?

6

ri» 80 60

107

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch005

107

Lo

121

121 20

15 20

I lift 60

40

I ill.J

100

80

120

160

15 20

0-CD,-CH,-CI

65

158

I·/·

HOO 0.84 80 0.64

M 158

94

—95

60

0.44

M 158

—•'94 109

40

123 109

0.24

123

I-20 15 20

liiiji 40

60

-lilt— 80

100

ϊ ί ! 0 13θ/J\lI50

160

84

HIGH P E R F O R M A N C E MASS

SPECTROMETRY

a r i s e from a n e a r l y c o m p l e t e l y e q u i l i b r a t e d p o s i t i o n a l i n t e r c h a n g e o f t h e phenoxy group and t h e c h l o r i n e atom i n t h e m o l e c u l a r i o n s p r i o r t o l o s s o f a CH C1 r a d i c a l . We have a d d r e s s e d o u r s e l v e s t o t h e q u e s t i o n o f whether FIK c o u l d show t h e time dependence o f t h i s p o s i t i o n a l i n t e r c h a n g e . Of c o u r s e , normal e l e c t r o n - i m p a c t ( E I ) mass s p e c t r o m e t e r s p r e s e n t an i n t e g r a t e d view o f i o n c h e m i s t r y from t h e time o f f o r m a t i o n t o ^ 1 0 " s e c , and, t h e r e f o r e , t h i s i n f o r m a t i o n i s n o t o b t a i n a b l e on t h e s e instruments. The F I s p e c t r a o f 2-phenoxyethyl c h l o r i d e and o f the 1,1-d - and 2,2,-d analogues a r e g i v e n i n F i g u r e 3 without c o r r e c t i o n f o r c o n t r i b u t i o n s of n a t u r a l isotopes and o f i s o t o p i c i m p u r i t i e s . They show t h a t a t 2

6

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch005

2

2

1 1

10 s e c , t h e o r i g i n a l - C r ^ C l group i s e l i m i n a t e d from the m o l e c u l a r i o n s w i t h o u t s u b s t a n t i a l i n t e r f e r e n c e by the p o s i t i o n a l i n t e r c h a n g e p r o c e s s . To f o l l o w t h e p o s i t i o n a l i n t e r c h a n g e i n t h e m o l e c u l a r i o n s as a f u n c t i o n o f t j m e , F I K measurements were p e r f o r m e d on the (M-CH C1) and (M-CD C1) i o n s g e n e r a t e d from t h e 1 , l - d - and 2,2-d m o l e c u l a r i o n s . Thç measured abundances o f t h e (M-CH C1) and (M-CD C1) i o n s a r e e x p r e s s e d i n p e r c e n t a g e s o f t h e i r sum as a f u n c t i o n o f time and t h e s e r e s u l t s a r e g i v e n i n F i g u r e s 4a and 4b f o r t h e 1 , l - d - and 2,2-d compounds, r e s p e c t i v e l y . I t i s v e r y c l e a r from these f i g u r e s , t h a t a t s h o r t e r times t h e p o s i t i o n a l i n t e r c h a n g e o f t h e phenoxy group and t h e c h l o r i n e atom cannot compete w i t h t h e e l i m i n a t i o n o f -CD C1 (-CH C1) from t h e 1,1d2 - ( 2 , 2 - d ) m o l e c u l a r i o n s , b u t i t competes much more e f f e c t i v e l y a t l o n g e r times (~ 1 0 " sec) c o r r e s p o n d i n g t o m o l e c u l a r i o n s o f lower i n t e r n a l energy. This observation i n d i c a t e s that the p o s i t i o n a l interchange o c c u r s i n t h e gas phase m o l e c u l a r i o n s o f 2-phenoxye t h y l c h l o r i d e , and t h a t i t i s a p r o c e s s h a v i n g a lower f r e q u e n c y f a c t o r and lower a c t i v a t i o n energy than t h e e l i m i n a t i o n o f -CH C1 v i a a s i m p l e c l e a v a g e . Note a l s o from F i g u r e 4 t h a t t h e t i m e - r e s o l v e d p o s i t i o n a l i n t e r change reaches a t l e a s t a h i g h e r p e r c e n t a g e o f e q u i l i b r a t i o n (87-931) than can be deduced from p r e v i o u s E I r e s u l t s (851) ( 9 ) , w h i c h r e p r e s e n t o n l y an i n t e g r a t e d v a l u e o v e r t h e time range up t o ~ 10 s e c . 2

2

2

2

2

2

2

2

2

2

2

1 0

2

6

FIK Study on 3 - P h e n y l p r o p a n a l The

(10).

e l i m i n a t i o n s o f C H 0 and o f C r U 0 from t h e 3 2 1 m o l e c u l a r i o n s o f 3 - p h e n y l p r o p a n a l , C H CH CH CHO upon e l e c t r o n impact have been s t u d i e d p r e v i o u s l y by D- and C-13 l a b e l i n g (11,12). In s p i t e o f the extensive 2

2

3

6

5

2

2

NiBBERiNG

Field Ionization Kinetic Studies

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch005

mVe 107

n

-11.0

-11.0

/el09

-10.5

-10.0

-9.5

logt

-10.5

logt Advances in Mass Spectrometry

Figure 4. Intensities of m/e 107 and m/e 109 expressed in percentage of their sum as a function of time for the l,l-d - and 2,2-d -2-phenoxyethyl chlorides [(a) and (b), respectively]. The dashed lines refer to the percentage of equilibrated positional interchange of the phenoxy group and the chlorine atom. 2

2

86

HIGH PERFORMANCE MASS SPECTROMETRY

hydrogen r a n d o m i z a t i o n i n t h e m o l e c u l a r i o n s p r i o r t o o r d u r i n g f r a g m e n t a t i o n , i t has been s u g g e s t e d t h a t a hydrogen atom from p o s i t i o n 2 m i g r a t e s t o t h e p h e n y l r i n g d u r i n g t h e e l i m i n a t i o n s o f C H 0 and o f C 3 H 4 O ( 1 1 ) . T h i s h y p o t h e s i s c a n be t e s t e d w i t h t h e F I K method. I n F i g u r e 5 t h e E I spectrum and t h e F I s p e c t r a o f 3 - p h e n y l p r o p a n a l a t an unheated and h e a t e d e m i t t e r have been r e p r o d u c e d . I t i s s e e n , t h a t t h e i o n s m/e 92 and m/e 78, g e n e r a t e d by e l i m i n a t i o n s o f C H 0 and CaHitO from t h e m o l e c u l a r i o n s r e s p e c t i v e l y , a r e p r e s e n t i n t h e F I s p e c t r a . The F I s p e c t r a o f 3phenylpropanals, l a b e l l e d with deuterium i n the 1 p o s i t i o n ( 1 - d i ) , the 2 p o s i t i o n , (2,2-d ), the 3 p o s i t i o n ( 3 , 3 - d ) and i n t h e p h e n y l r i n g (0-ds) show t h a t t h e i o n s m/e 92 and m/e 78 r e t a i n t h e hydrogen atom from p o s i t i o n 1 t o tïïe e x t e n t o f a p p r o x i m a t e l y 90% (10.)· C o n t r a r y t o c o n c l u s i o n s from p r e v i o u s E I r e s u l t s (11) we now c o n c l u d e t h a t a hydrogen atom from p o s i t i o n 1 and n o t from p o s i t i o n 2 m i g r a t e s t o the p h e n y l r i n g d u r i n g t h e e l i m i n a t i o n s o f C H 0 and o f C r U 0 , a t l e a s t w i t h i n 1 0 " s e c . The e l i m i n a t i o n o f C H 0 o c c u r s by a 1,5 hydrogen m i g r a t i o n , i^.e. , t h e w e l l - k n o w n M c L a f f e r t y r e a r r a n g e m e n t , whereas the second e l i m i n a t i o n might p r o c e e d v i a a 1,5and/or a 1,4-hydrogen s h i f t (Scheme 1 ) . 2

2

2

2

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch005

2

2

2

2

1 1

3

2

2

Scheme 1

0

0 The e l i m i n a t i o n o f C3rU0 g i v e s an i s o l a t e d peak a t m/e 78 i n t h e F I spectrum ( F i g u r e 5 ) , so t h a t t h i s r e a c t i o n c a n be r e a d i l y s t u d i e d by F I K making u s e o f the v a r i o u s d e u t e r a t e d 3 - p h e n y l p r o p a n a l s . This i s not t r u e f o r t h e e l i m i n a t i o n o f C H 0 because t h i s r e a c t i o n i s s u b j e c t t o i n t e r f e r e n c e by t h e s i m p l e c l e a v a g e o f t h e C - C bond i n t h e d e u t e r a t e d 3phenylpropanals which occurs w i t h randomization o f hydrogen atoms (cf_. peaks a t m/e 91 and 92 i n t h e F I spectra, given i n Figure 5). F I K c u r v e s have been measured f o r t h e [M-C3H D 0 (x+y=4)] i o n s from t h e v a r i o u s d e u t e r a t e d ^ 2

2

+

3

2

5.

NiBBERiNG

87

Field Ionization Kinetic Studies

3 - p h e n y l p r o p a n a l s . E x p r e s s i n g t h e measured abundances o f t h e s e i o n s from d e u t e r a t e d 3 - p h e n y l p r o p a n a l i n p e r c e n t a g e s o f t h e i r sum as a f u n c t i o n o f time i n t h e g r a p h s , p r e s e n t e d i n F i g u r e 6, t h e f o l l o w i n g i n t e r e s t i n g r e s u l t s are obtained. i ) The hydrogen atom o r i g i n a l l y a t p o s i t i o n 1 i s p r e d o m i n a n t l y t r a n s f e r r e d t o g i v e CerU** ions at short i o n l i f e t i m e s . i i ) T r a n s f e r o f t h e hydrogen atoms from p o s i t i o n 3 t o y i e l d t h e C H * i o n s becomes more i m p o r t a n t a t l o n g e r t i m e s (note t h e i n c r e a s e o f m/e 80, i _ . e. C n^O * , with increasing time). 6

6

+

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch005

s

2

i i i ) The hydrogen atoms from p o s i t i o n 2 a r e n o t i n v o l v e d i n the p r o d u c t i o n o f C H ions i n t h e measured time range. + e

6

6

i v ) One o f t h e o r i g i n a l p h e n y l r i n g hydrogen atoms i s i n c r e a s i n g l y r e t a i n e d i n t h e n e u t r a l e l i m i n a t e d CatUO m o i e t y a t l o n g e r t i m e s , a l b e i t to a small extent. The f o l l o w i n g e x p l a n a t i o n i s proposed f o r t h e s e results. I n t h e measured time range o f 1 0 " to 1 0 " * s e c , two s p e c i f i c hydrogen exchange p r o c e s s e s i n the molecular ions o f 3-phenylpropanal occur p r i o r t o o r d u r i n g t h e f o r m a t i o n o f t h e C H * i o n s . The most i m p o r t a n t hydrogen exchange p r o c e s s t a k e s p l a c e between t h e hydrogen atoms from p o s i t i o n s 1 and 3, as d e p i c t e d i n Scheme 2. 1 0 , 3

9

5

+

6

6

Scheme 2

b

a

d

c

c KrÇ 6

etc.

HIGH PERFORMANCE MASS V. too

y. 100

PhCH CH CH0 2

SPECTROMETRY

2

El 78 (a)

50

50

65 0

Ί

1

Ί

115 _iL.

Τ

133

ο too

PhCH CH CHO

1.25

2

2

Fi (b)

OJ75

50 025

67

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch005

0

I

I

I

Γ

Ί

2

too

2

133

I high emitter temperature! (c)

3.0

1.0

0

Γ

syo PhCH CH CHO FI

50

615·(134*921

0

—ι )

I 110

100

90

70

130 m/e

120

Journal of the American Chemical Society

Figure 5. EI and FI spectra of 3-phenylpropanal [(b) unheated emitter, (c) heated emitter]. In the FI spectra the left ordinate scale refers to the intensities of ions with m/e < 134; the right ordinate scale to those of ions with m/e > 134. %

.

100

»

1

1

1

1

1

• 1 _

80

60

£3~ 2CH CDO CH

Q-CD CH2CHO

2

2

40

m/e 7$^ 20 — 1

1 I I

I

I

'

m/f80^-«-

I I

100 m/#78

"

~*~—*—--*^£n/f83

80

60

{3* 2 °2CH0 CH

C

^-CH CH CHO 2

2

AO m/e 82

20

-

m/e 79 • 103

, 1 , , , < 1 10.0 9.5 10.3

,1 10.0

9.5

Journal of the American Chemical Society

Figure 6. Intensities of the (M-C H D O fx + y = 4)Υ' ions expressed in percentage of their sum as a function of time for 3-phenylpropanals, deuterated in the 1, 2, and 3 positions ana in the phenyl ring as indicated s

x

y

5.

NiBBERiNG

Field Ionization Kinetic Studies

89

I t s h o u l d be n o t e d t h a t the hydrogen atom a b s t r a c t i o n from p o s i t i o n 3 by t h e r a d i c a l s i t e a t the oxygen atom i n t h e r e a c t i o n a b ( o r the e q u i v a l e n t p r o t o n a b s t r a c t i o n from p o s i t i o n 3 by the l o n e p a i r o f e l e c ­ t r o n s a t the oxygen atom, i f the charge r e s i d e s i n i t i a l l y i n the p h e n y l r i n g ) has been o b s e r v e d p r e v i o u s l y i n the m o l e c u l a r i o n s o f e t h y l 3-phenylpropionate (13). The o t h e r hydrogen exchange p r o c e s s a t s h o r t t i m e s i s b e s t e x p l a i n e d by an exchange between the hydrogen atoms from the p h e n y l r i n g and p o s i t i o n 1, as r a t i o n a l i z e d i n Scheme 3. Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch005

Scheme 3

A s i m i l a r exchange p r o c e s s , as p r e s e n t e d i n Scheme 3, has been o b s e r v e d i n the m o l e c u l a r i o n s o f 3-phenylp r o p y l bromide ( 1 4 ) . I t f u r t h e r s u p p o r t s the i d e a t h a t the e l i m i n a t i o n o f C2H2O v i a a M c L a f f e r t y r e a r ­ rangement ( v i d e supra) o c c u r s s t e p w i s e , w h i c h i s now w e l l a c c e p t e d (15). I n any e v e n t , the o p e r a t i o n o f b o t h schemes 2 and 3 a c c o u n t s f o r the i n c o r p o r a t i o n of the two hydrogen atoms from p o s i t i o n 3 i n the ΟβΗβ i o n s a t l o n g e r t i m e s ( v i d e s u p r a ) . T h i s FIK s t u d y does n o t show any a c t i v e p a r t i c i p a t i o n o f the hydrogen atoms from p o s i t i o n 2 i n the hydrogen exchange p r o c e s s e s p r i o r t o o r d u r i n g the f o r m a t i o n o f the 0βΗ ' i o n s . I t i s o f i n t e r e s t t o note t h a t such exchanges have been o b s e r v e d f o r m e t a s t a b l e decompo­ s i t i o n s i n the f i r s t f i e l d f r e e r e g i o n f o r i o n s g e n e r a t e d by E I (11_). T h i s s u g g e s t s t h a t an exchange between the hydrogen atoms a t p o s i t i o n 2 and, f o r example, t h e p h e n y l r i n g can o n l y compete w i t h the exchange p r o c e s s e s g i v e n i n schemes 2 and 3 f o r m o l e c u l a r i o n s o f l o w e r i n t e r n a l energy, i - e . t h o s e decomposing i n the f i r s t f i e l d f r e e r e g i o n . U n f o r ­ t u n a t e l y , t h i s p r o p o s a l c o u l d not be t e s t e d because of t h e absence o f a m e t a s t a b l e peak f o r the e l i ­ m i n a t i o n o f C3Hi»0 from the m o l e c u l a r i o n s o f 3p h e n y l p r o p a n a l under F I c o n d i t i o n s . The mechanism f o r the C H« 0 e l i m i n a t i o n r e q u i r e s f u r t h e r comment. The C H * i o n s , r e s u l t i n g from t h i s +

6

+

6

6

3

+

90

HIGH PERFORMANCE MASS SPECTROMETRY

e l i m i n a t i o n , appear t o o r i g i n a t e from the m o l e c u l a r i o n s and from the (M-28) * i o n s , as shown by appro­ p r i a t e m e t a s t a b l e d e c o m p o s i t i o n s i n the f i r s t f i e l d f r e e r e g i o n under EI c o n d i t i o n s . T h e r e f o r e , i t may be c o n c l u d e d t h a t t h e C H * i o n s are g e n e r a t e d by suc­ c e s s i v e hydrogen m i g r a t i o n from p o s i t i o n 1 t o the p h e n y l r i n g , c a r b o n monoxide l o s s i n a slow and r a t e d e t e r m i n i n g s t e p and a f a s t e l i m i n a t i o n o f e t h y l e n e , as i n d i c a t e d i n scheme 1. F i n a l l y , some comments s h o u l d be made on c i n n a m y l a l c o h o l , which has been s t u d i e d e x t e n s i v e l y by EI i n c o m b i n a t i o n w i t h D- and C - l a b e l l i n g (16>,17). This compound, an isomer o f 3 - p h e n y l p r o p a n a l , behaves q u i t e d i f f e r e n t l y upon E I , a l t h o u g h i t s m o l e c u l a r i o n s e l i m i ­ n a t e C H 0 and C 3 H 4 O as w e l l (16^,17). FI s p e c t r a o f c i n n a m y l a l c o h o l a t an unheated ancPheated e m i t t e r a r e g i v e n i n F i g u r e 7. C l e a r l y these s p e c t r a are d i f ­ f e r e n t from the FI s p e c t r a o f 3 - p h e n y l p r o p a n a l ( F i g u r e 5 ) . However, peaks a t m/e 92 and m/e 78 a r e o b s e r v e d , e s p e c i a l l y a t h i g h e r emTtter t e m p e r a t u r e , a l t h o u g h t h e i r abundances were too low t o o b t a i n r e l i a b l e FIK results. N e v e r t h e l e s s , i t may be c o n c l u d e d t h a t s m a l l f r a c t i o n o f the m o l e c u l a r i o n s o f c i n n a m y l a l c o h o l may have i s o m e r i s e d t o 3 - p h e n y l p r o p a n a l , p o s s i b l y as follows : +

6

6

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch005

1 3

2

2

C H CH=CHCH OH 6

5

2

ί

, +

*

+

C H CH-CH CH=OH*>C H CH CH CHo' * 6

5

2

£

6

5

2

2

h

Note, t h a t i o n g i s i d e n t i c a l t o i o n b i n Scheme 2. I t i s i n t e r e s t i n g Tn t h i s r e s p e c t t h a t tEe r a t i o o f i n t e n ­ s i t i e s o f the m e t a s t a b l e peaks i n the f i r s t f i e l d f r e e r e g i o n f o r t h e l o s s o f C 2 H 2 O and C3Hi*0 upon EI a r e ^150 f o r c i n n a m y l a l c o h o l and ^200 f o r 3 - p h e n y l p r o p a n a l . T h i s would mean t h a t i n c i n n a m y l a l c o h o l t h e e l i m i n a ­ t i o n o f C 3 H 4 O w h i c h r e q u i r e s a h i g h e r a c t i v a t i o n energy, can compete more e f f e c t i v e l y w i t h the l o s s o f C 2 H 2 O than i n 3-phenylpropanal, i f t h e i r decompositions i n t h e same t i m e window are c o n s i d e r e d . Ions h seem, t h e r e f o r e t o be s l i g h t l y more e x c i t e d t h a n t h o s e d i r e c t l y g e n e r a t e d from 3 - p h e n y l p r o p a n a l . T h i s can be e x p l a i n e d , i f the i s o m e r i z a t i o n of cinnamyl a l c o h o l p r o c e e d s t h r o u g h a s l o w s t e p , presumably f -> to e v e n t u a l l y g i v e a r e l a t i v e l y h i g h e r e x c i t e d 3-phenyl­ p r o p a n a l i o n . The e l i m i n a t i o n s o f C 2 H 2 O and o f CaH^O can t h e n compete more e f f e c t i v e l y w i t h each o t h e r i n the f i r s t f i e l d f r e e r e g i o n . A s i m i l a r phenomenon has been d e s c r i b e d r e c e n t l y . Fast unimolecular d i s -

5.

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" PhCH=CHCH OH 2

FI

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch005

- ία)

I

L

l

l

1

I

1

ι

I

1

- PhCH=CHCH OH 2

_

133

FI (high emitter temperature)

(b) 1I,

" . . . . ι . , 60

70

.ii 80

90

, .1 , 1 100

110

LJ 120

ι

130

I m/e

Journal of the American Chemical Society

Figure 7. FI spectra of cinnamylalcohol at an unheated (a) and heated (h) emitter. The left ordinate scale refers to the intensities of ions with m/e < 134; the right ordinate scale to those of ions with m/e > 134.

92

HIGH PERFORMANCE MASS SPECTROMETRY

s o c i a t i o n s o f the (M-methyl) i o n from m e t h y l i s o p r o p y l e t h e r i n t h e m e t a s t a b l e r e g i o n are a t t r i b u t e d t o a r a t e d e t e r m i n i n g i s o m e r i z a t i o n o f t h i s i o n t o the (M-methyl) s t r u c t u r e from d i e t h y l e t h e r (18J).

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch005

Conclusions. The r e s u l t s , o b t a i n e d from FIK f o r the systems d e s c r i b e d , show t h a t t h i s method i s e x t r e m e l y v a l u a b l e f o r o b t a i n i n g a deeper i n s i g h t i n t r u e gas-phase i o n d e c o m p o s i t i o n s and i n the l o n g - s t a n d i n g p r o b l e m o f s c r a m b l i n g o f hydrogen atoms. T h i s l a t t e r phenomenon appears t o c o n s i s t o f a s e r i e s o f s p e c i f i c hydrogen exchange p r o c e s s e s , as n o t e d p r e v i o u s l y by o t h e r a u t h o r s (2a). Experimental. E x p e r i m e n t a l d e t a i l s w i l l be d e s c r i b e d f u l l y e l s e where (8,10) and the most i m p o r t a n t c o n d i t i o n s are o n l y given here. The FIK e x p e r i m e n t s were p e r f o r m e d on a V a r i a n MAT 711 double f o c u s s i n g mass s p e c t r o m e t e r (Mattauch-Herzog geometry) equipped w i t h a c o m m e r c i a l l y combined EI/FI/FD s o u r c e . Data a c q u i s i t i o n and d a t a p r o c e s s i n g were a c h i e v e d w i t h the V a r i a n S p e c t r o System 100 on l i n e and s p e c i a l computer programmes, w r i t t e n f o r t h e FIK e x p e r i m e n t s , were used ( 1 9 ) . F u r t h e r exp e r i m e n t a l c o n d i t i o n s f o r 2-phenoxyetEyl c h l o r i d e were: I n l e t system: r e f e r e n c e i n l e t system a t 130°C Ion s o u r c e t e m p e r a t u r e : 100°C Emitter Current: S mA Analyzer pressure: 6x10" Torr Source p r e s s u r e : 5 x 1 0 " T o r r Mass r e s o l u t i o n (10% v a l l e y d i f i n i t i o n ) : 1200 Energy r e s o l u t i o n ( a t an a c c e l e r a t i n g v o l t a g e o f 8453 Volts): 1.4* V o l t a g e a t f o c u s s i n g e l e c t r o d e : ±6 kV. The e x p e r i m e n t a l c o n d i t i o n s employed f o r t h e 3p h e n y l p r o p a n a l s t u d i e s were: I n l e t system: c o o l e d d i r e c t i n s e r t i o n probe at -5 t o -10°C Ion s o u r c e t e m p e r a t u r e : 98°C E m i t t e r c u r r e n t : 0 mA Analyzer pressure: 6x10" Torr Source p r e s s u r e : 7x10" Torr Mass r e s o l u t i o n (101 v a l l e y d e f i n i t i o n ) : 500 Energy r e s o l u t i o n ( a t an a c c e l e r a t i n g v o l t a g e o f 8400 Volts): II V o l t a g e a t f o c u s s i n g e l e c t r o d e : ±6 kV. 9

6

9

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For b o t h s t u d i e s a c t i v a t e d t u n g s t e n w i r e e m i t t e r s o f 10 \xm w i t h an average n e e d l e l e n g t h o f 10 ym were used and p o s i t i o n e d 1.45 mm from t h e grounded s l o t t e d c a t h o d e .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch005

Acknowledgement. The a u t h o r i s e x t r e m e l y g r a t e f u l t o Drs. J. v a n der G r e e f and Mr. F. A. P i n k s e , who, w i t h g r e a t enthu­ s i a s m , have d e v e l o p e d t h e F I K method i n h i s l a b o r a t o r y . He f u r t h e r acknowledges t h e c o n t r i b u t i o n s o f Drs. C. B. T h e i s s l i n g and Dr. P. W o l k o f f C h r i s t e n s e n i n t h e c h e m i c a l f i e l d and t h e c o n t r i b u t i o n o f Dr. C. W. F. K o r t who d e v e l o p e d t h e s o f t w a r e f o r FIK. F i n a l l y , he w o u l d l i k e t o thank P r o f . H. D. Beckey and h i s group o f the U n i v e r s i t y o f Bonn f o r s t i m u l a t i n g d i s c u s s i o n s and the g i f t o f some w e l l - a c t i v a t e d e m i t t e r s .

Abstract. Field Ionization Kinetics has provided a clear i n ­ sight into the positional interchange of the phenoxy group and halogen atom in the molecular ions of 2-phen­ oxyethyl chloride and into the various hydrogen ex­ change processes i n the molecular ions of 3-phenyl­ propanal prior to or during fragmentation. This information could not be obtained from previous elec­ tron impact studies. Literature Cited. 1.

2.

a. Beckey, H. D . , Field Ionisation Mass Spectro­ etry, (1971), Akademie-Verlag, Berlin and Pergamon Press, Oxford, b. Robertson, A. J. B . , i n "Mass Spectrometry", Maccoll, A. (Ed.), Int. Rev. Sci., Physical Chemistry Ser. One, V o l . 5, Butterworths, London and University Park Press, Baltimore, (1972), p. 103. c) Derrick, P. J., i n "Mass Spectrometry", Maccoll, A. (Ed.), Int. Rev. Sci., Physical Chemistry Ser. Two, V o l . 5, Butterworths, London and Boston, Mass., (1975), p. 1. a. Derrick, P. J. and Burlingame, A. L., Acc. Chem. Res., (1974) 7, 328. b. McMaster, Β. N., Johnstone, R. A. W. (Ed.), i n Mass Spectrometry (Specialist Periodical Reports), The Chemical Society, London, (1975), V o l . 3, p. 33. c. Burlingame, A. L., Kimble, B. J., and Derrick, P. J., Anal. Chem., (1976) 48, 368R.

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SPECTROMETRY

a. Falick, Α. Μ., Derrick, P. J., and Burlingame, A. L., Int. J . Mass Spectrom. Ion Phys., (1973) 12, 101. b. Beckey, H. D. Hey, Η., Levsen, K. and Tenschert, G., Int. J . Mass Spectrom. Ion Phys., (1969) 2, 101. 4. Schulten, H.-R. and Beckey, H. D., Org. Mass Spectrom., (1972), 6, 885. 5. Falick, Α. Μ., Int. J . Mass Spectrom. Ion Phys., (1974) 14, 313. 6. Derrick, P. J., Falick, A. M. and Burlingame, A. L., J . Am. Chem. Soc., (1972) 94, 6794. 7. Borchers, F . , Levsen, K. and Beckey, H. D., Int. J. Mass Spectrom. Ion Phys., (1976) 21, 125. 8. Greef, J . van der, Theissling, C. B . , and Nibbering, Ν. Μ. Μ., 7th International Mass Spectrometry Conference, Florence, I t a l y , 1976, Paper No. 168; will appear in Advan. Mass Spectrom. 7 Daly, N. (Ed.). 9. Theissling, C. Β . , Nibbering, Ν. Μ. Μ., and Boer, Th.J. de, Advan. Mass Spectrom., (1971) 5, 642. 10. Christensen, P. Wolkoff, Greef, J . van der, and Nibbering, N. M. M. J. Am. Chem. Soc. (submitted) 11. Venema, Α., Nibbering, Ν. M. M . , and Boer, Th.J. de, Org. Mass Spectrom., (1970) 3, 583. 12. Venema, A. and Nibbering, Ν. Μ. Μ., Org. Mass Spectrom., (1974) 9, 628. 13. Resink, J . J., Venema, Α., and Nibbering, Ν. M. M. Org. Mass Spectrom., (1974) 9, 1055. 14. Nibbering, Ν. M. M. and Boer, Th.J. de, Tetrahedron, (1968) 24, 1427. 15. Kingston, D. G. I., Bursey, J. T., and Bursey, M. M . , Chem. Rev., (1974) 74, 215. 16. Schwarz, H . , Köppel, C. and Bohlmann, F . , Org. Mass Spectrom., (1973) 7, 881. 17. Köppel, C. and Schwarz, Η., Org. Mass Spectrom., (1976) 11, 101. 18. Hvistendahl, G. and Williams, D. H . , J. Am. Chem. Soc., (1975) 97, 3097. 19. Greef, J . van der, Pinkse, F. Α., Kort, C. W. F. and Nibbering, Ν. Μ. Μ., Int. J . Mass Spectrom. Ion Phys., i n press. RECEIVED December 30, 1977

6 Organic Trace Analysis Using Direct Probe Sample Introduction and High Resolution Mass Spectrometry WILLIAM F. HADDON

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch006

Western Regional Research Center, U.S. Department of Agriculture, Albany, CA 94710

Many interesting problems in organic trace analysis are inaccessible to combined gas chromatographymass spectrometry (GC/MS). For example, a recent treatise on the analysis of organic pollutants in water points out that between 80 and 90 percent of the extractable organic compounds in polluted water f a i l , even after derivitization, to pass through a gas chromatographic column (1). One approach for introducing less volatile compounds into the mass spectrometer in a highly purified state is the use of a liquid chromatograph coupled directly to the mass spectrometer (2,3,4). However, as an alternative to utilizing chromatographic separation prior to analysis, we can consider the mass spectrometer itself as an analytical instrument which is capable in itself of performing highly efficient separations, based, for example, on mass or energy differences of generated ions or on selective ionization, as well as precise identification and quantitation, based on reference mass spectra obtained separately on known compounds. This use of a mass spectrometer for combined separation-identification functions might be called "mass spectrometer-mass spectrometer" (MS/MS) analysis, with the direct probe serving as the vehicle for sample introduction. A number of laboratories have reported results using this approach, and the methods used to achieve selectivity in complex mixtures have included field ionization (5), chemical ionization (6,7), negative chemical ionization (8,9), collisional activation (10), mass-analyzed ion kinetic energy (11), and high resolution electron ionization (EI) (12-15) techniques. This report describes the use of the high resolution EI method in which particular ions are monitored as a function of temperature as the components of interest volatilize ©0-8412-0422-5/78/47-070-097$10.00/0

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch006

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from t h e d i r e c t i n t r o d u c t i o n p r o b e . The term h i g h r e s o l u t i o n s e l e c t e d i o n m o n i t o r i n g (SIM) i s used t o d e s c r i b e t h i s type o f experiment ( 1 6 ) , and the i o n abundance p r o f i l e r e c o r d e d as a f u n c t i o n o f t e m p e r a t u r e i s c a l l e d an e x a c t mass fragmentogram. In many o r g a n i c t r a c e a n a l y s i s p r o b l e m s , the p r e p a r a t i o n o f h i g h l y p u r i f i e d samples amenable t o low r e s o l u t i o n mass s p e c t r a l i d e n t i f i c a t i o n r e q u i r e s ex­ tensive e f f o r t . Employing i n c r e a s e d mass r e s o l u t i o n t o a c h i e v e h i g h e r s e l e c t i v i t y can reduce the e x t e n t o f sample p u r i f i c a t i o n s i g n i f i c a n t l y i n many c a s e s , and r e c e n t examples o f GC-high r e s o l u t i o n SIM f o r b o t h b i o f l u i d a n a l y s i s (17,18) and e n v i r o n m e n t a l m o n i t o r i n g (19) a r e i l l u s t r a t i v e . In many l a b o r a t o r i e s concerned w i t h a n a l y z i n g p a r t i c u l a r components o f complex m i x t u r e s , the s a v i n g s i n t h e c o s t o f sample p r e p a r a t i o n made p o s s i b l e by the a v a i l a b i l i t y o f h i g h r e s o l u t i o n SIM c a p a b i l i t y may j u s t i f y the h i g h e r i n i t i a l c o s t o f a s u i t a b l e h i g h r e s o l u t i o n mass s p e c t r o m e t e r . Furthermore, s m a l l r a d i u s ( 6 - i n c h o r l e s s ) m a g n e t i c s e c t o r mass s p e c t r o ­ meters h a v i n g b o t h the f a s t scan r a t e s r e q u i r e d by GC/MS a p p l i c a t i o n s and the c a p a b i l i t y f o r h i g h r e s o ­ l u t i o n (10-20,000) o p e r a t i o n w i t h good s e n s i t i v i t y a r e becoming a v a i l a b l e c o m m e r c i a l l y . On a t l e a s t one s m a l l r a d i u s i n s t r u m e n t o f t h i s t y p e , image c u r v a t u r e c o r ­ r e c t o r s have been employed t o enhance the h i g h r e s o ­ l u t i o n p e r f o r m a n c e , and f u r t h e r improvements i n r e s o ­ l u t i o n and s e n s i t i v i t y f o r these s m a l l r a d i u s i n s t r u ­ ments w i l l u n d o u b t e d l y be f o r t h c o m i n g . Two examples d e s c r i b e d below from the f i e l d s o f c l i n i c a l and f o o d c h e m i s t r y i l l u s t r a t e the u t i l i t y o f t h e h i g h r e s o l u t i o n d i r e c t probe method f o r t r a c e analysis. In a d d i t i o n , p e s t i c i d e s i n human t i s s u e have been measured a t h i g h r e s o l u t i o n on a p h o t o - p l a t e equipped mass s p e c t r o m e t e r (Γ5), and s i m i l a r h i g h r e s o l u t i o n methods have been employed t o measure p e s t i ­ c i d e s i n f o o d samples (9) and b i o f l u i d s (8) u s i n g n e g a t i v e c h e m i c a l i o n i z a t i o n , and t o q u a n t i t a t e drug m e t a b o l i t e s r a p i d l y i n b l o o d plasma (7.) u s i n g c h e m i c a l i o n i z a t i o n mass s p e c t r o m e t r y . These papers c o n t a i n e x c e l l e n t d e s c r i p t i o n s o f the p r a c t i c a l problems o f p r e p a r i n g samples and q u a n t i t a t i n g the r e s u l t s . Experimental H i g h r e s o l u t i o n SIM o p e r a t i o n can be a c h i e v e d on a m a g n e t i c s e c t o r mass s p e c t r o m e t e r by i n c o r p o r a t i n g a s w i t c h e d v o l t a g e d i v i d e r c i r c u i t , or by employing c o m p u t e r - d r i v e n power s u p p l i e s .

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99

Organic Trace Analysis

The o v e r - a l l e l e c t r o n i c s t a b i l i t y n e c e s s a r y f o r q u a n t i t a t i v e work depends on t h e p r e c i s i o n r e q u i r e d and on t h e mass r e s o l u t i o n s e l e c t e d f o r t h e a n a l y s i s . These s t a b i l i t y r e q u i r e m e n t s c a n be d e r i v e d from t h e G a u s s i a n c u r v e o f F i g u r e 1, w h i c h r e p r e s e n t s a mass s p e c t r a l peak. The 10 p e r c e n t v a l l e y d e f i n i t i o n o f mass s p e c t r o m e t e r r e s o l u t i o n c o r r e s p o n d s t o t h e o r d i ­ n a t e v a l u e a t a d i s t a n c e 2.44 σ from t h e c e n t e r o f t h e peak (5 p e r c e n t o f maximum h e i g h t f o r a s i n g l e p e a k ) , where σ i s t h e s t a n d a r d d e v i a t i o n o f t h e c u r v e . The o v e r - a l l i n s t r u m e n t s t a b i l i t y , S, i n p a r t s p e r m i l l i o n (ppm) f o r a p a r t i c u l a r a n a l y s i s i s 6

S = (a /2.44)(10 /R) U) where σ i s the number o f s t a n d a r d d e v i a t i o n s t o t h e o r d i n a t e a t t h e r e q u i r e d p e r c e n t a c c u r a c y , and R i s t h e mass r e s o l u t i o n (Μ/ΔΜ, 10 p e r c e n t v a l l e y d e f i n i t i o n )

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch006

x

T a b l e I . Mass S p e c t r o m e t e r S t a b i l i t y i n P a r t s p e r M i l l i o n Necessary f o r Q u a n t i t a t i v e High R e s o l u t i o n S e l e c t e d Ion M o n i t o r i n g . Resolution

Desired Q u a n t i t a t i v e Accuracy

(Percent):

99

95

90

80

16.

45.

61.

89.

9.8

27.

36.

53.

10,000

4.9

13.

18.

26.

15,000

3.3

9.0

12.

18.

20,000

2.4

6.7

9.2

2.7

3.6

3,000 5,000

50,000

.98

C a l c u l a t e d from Eq. 1. 'Μ/ΔΜ based on 10 p e r c e n t v a l l e y

13. 5.

definition.

T a b l e I l i s t s t y p i c a l c a l c u l a t e d v a l u e s o f S. These v a l u e s show t h a t q u a n t i t a t i v e p r e c i s i o n o f 90 p e r c e n t o r g r e a t e r s h o u l d be e a s i l y a c h i e v e d on most commercial mass s p e c t r o m e t e r s w h i c h have been d e s i g n e d f o r a c ­ c u r a t e mass measurement a p p l i c a t i o n s . When o n l y one o r two peaks need t o be m o n i t o r e d , t h e c i r c u i t r y o f an e x i s t i n g peak matcher c a n be modi­ f i e d t o m o n i t o r b o t h peaks by s w i t c h i n g between peak t o p s . (2,19) On one w i d e l y used commercial h i g h r e s o ­ l u t i o n i n s t r u m e n t , t h i s i s done by d i s c o n n e c t i n g t h e peak matcher scan c o i l s ( 2 ) . A l t e r n a t i v e l y , r e p e t i t i v e

100

HIGH PERFORMANCE MASS SPECTROMETRY

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch006

s c a n s o v e r narrow mass ranges can be u t i l i z e d , but a t reduced s e n s i t i v i t y . An advantage o f t h i s l a t t e r dynamic method o f r e c o r d i n g d a t a i s t h a t the peak p r o f i l e information i s continuously a v a i l a b l e during the e x p e r i m e n t . An i n c r e a s e beyond two c h a n n e l s can be a c h i e v e d w i t h o u t a computer system by i n c o r p o r a t i n g a s w i t c h a b l e v o l t a g e d i v i d e r c i r c u i t (13) (20) ( 2 1 ) . I t i s imp o r t a n t f o r a c i r c u i t o f t h i s type to employ samplea n d - h o l d a m p l i f i e r s f o r each c h a n n e l t o p r o v i d e cont i n u o u s o u t p u t s i g n a l s s u i t a b l e f o r d r i v i n g an o s c i l l o g r a p h i c or m u l t i p l e pen r e c o r d e r . Computer-Controlled Power S u p p l i e s f o r H i g h R e s o l u t i o n SIM. A d e d i c a t e d computer can p e r f o r m b o t h d a t a c o l l e c t i o n and i n s t r u m e n t c o n t r o l f u n c t i o n s f o r h i g h r e s o l u t i o n SIM when used i n c o n j u n c t i o n w i t h a programmable power s u p p l y f o r the mass s p e c t r o m e t e r a c c e l e r a t i n g and e l e c t r i c s e c t o r v o l t a g e s . The computer p r o v i d e s some a d d i t i o n a l b e n e f i t s over a h a r d - w i r e d system: The number o f mass c h a n n e l s can be l a r g e and can be changed d u r i n g the a n a l y s i s ; the f r a c t i o n o f t i m e a t a p a r t i c u l a r mass v a l u e can be v a r i e d a c c o r d i n g t o the e x p e c t e d abundance o f the peak; and the s y s t e m , when s u i t a b l y programmed, can sweep s h o r t mass r e g i o n s t o p r o v i d e peak p r o f i l e i n f o r m a t i o n w h i c h can be used b o t h t o a s c e r t a i n the degree o f i n t e r f e r e n c e from i s o b a r i c i o n s near the e l e m e n t a l c o m p o s i t i o n o f i n t e r e s t , and t o make a u t o m a t i c p e r i o d i c a d j u s t m e n t s o f the a c c e l e r a t i n g v o l t a g e t o compensate f o r d r i f t i n voltages. F i g u r e 2 shows the programmed power s u p p l y c i r c u i t r y u s e d i n our l a b o r a t o r y f o r h i g h r e s o l u t i o n SIM on a CEC 21-110A d o u b l e - f o c u s s i n g mass s p e c t r o m e t e r . Many o f the e l e c t r o n i c and programming d e t a i l s , i n c l u d i n g c h o i c e o f s u i t a b l e power s u p p l y u n i t s , a r e from the Washington U n i v e r s i t y system o f Holmes f221 f o r low r e s o l u t i o n SIM. In the USDA system a s i n g l e programmer (D/A c o n v e r t e r ) w i t h a 1 - v o l t o u t p u t d r i v e s a p a i r o f power s u p p l i e s i n s e r i e s , one w i t h a v o l t a g e g a i n o f 100 (KEPCO OPS-2000, Kepco, I n c . , F l u s h i n g , N.Y.) f o l l o w e d by a second w i t h a g a i n of -1 (KEPCO NTC2000). These power s u p p l i e s d i r e c t l y r e p l a c e the e l e c t r i c s e c t o r s u p p l y f o r m e r l y used w i t h the i n s t r u ment. The 21-110 r e q u i r e s p l u s and minus 375 v o l t s f o r n o m i n a l 8 kv. o p e r a t i o n . We d e r i v e a f i x e d p o r t i o n o f t h i s v o l t a g e from the i n t e r n a l r e f e r e n c e s u p p l y o f the OPS-2000 t h r o u g h R and R , and a v a r i a b l e p o r t i o n from the D/A c o n v e r t e r (DATEL DAC-169, 16 b i t p r e c i s i o n , D a t e l Systems, I n c . , Canton, Ma.) t h r o u g h R i . The 2

3

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6. HADDON

101

Organic Trace Analysis

KEPCO

NTC-2000

100 (EA+E.)

0 to +1000V

INV (IN) E (0 to +1V or 0 to +10V) A

SPC-16 D/A

CONVERTER INTERFACE 16-BIT LATCH

LOGIC SIGNALS

DATEL DAC-169 16 BIT D/A CONVERTER

-100 EA +EI] 0 to -1000V

Rl

NULL

WNr-

KEPCO OPS-2000

10KÛ

R2

10KÛ

+6.2V REF

Figure 2. Programmed power supply interface for CEC 21-110 mass spectrometer

102

HIGH PERFORMANCE MASS SPECTROMETRY

s e t t i n g o f R d e t e r m i n e s the f r a c t i o n o f f i x e d v o l t a g e a c c o r d i n g to the r e q u i r e m e n t s o f mass range and p r e c i s i o n . For a t e n p e r c e n t mass r a n g e , each d i g i t a l s t e p o f the programmed v o l t a g e c o r r e s p o n d s t o a 1.5 ppm change i n mass. The r e l a t i o n s h i p between mass and v o l t a g e o u t p u t f o r programmed a c c e l e r a t i n g v o l t a g e o p e r a t i o n i s 3

where m i s the mass i n f o c u s a t the c o l l e c t o r w i t h the D/A c o n v e r t e r a t 0 v o l t s , or d i g i t a l b i t s e t t i n g , r e q u i r e d t o ' ? o c u s mass m. ~Eq. 2 i s d e r i v e d from the mass s p e c t r o m e t e r e q u a t i o n by w r i t i n g a c c e l e r a t i n g v o l t a g e as a sum o f the a c c e l e r a t i n g v o l t a g e a t m p l u s the added v o l t a g e from the D/A c o n v e r t e r and a p p l i e s to b o t h s i n g l e and d o u b l e focussing sector instruments. F i g u r e 3 shows t h a t a l i n e a r r e l a t i o n s h i p i s o b t a i n e d f o r Eq. 2 f o r the 21110 mass s p e c t r o m e t e r m o d i f i e d as i n F i g u r e 2 over the mass range 293-331. V a l u e s of D/A c o n v e r t e r v o l t a g e w h i c h f o c u s p a r t i c u l a r e x a c t masses can be d e t e r m i n e d by l o c a t i n g i n t e r n a l r e f e r e n c e p e a k s , t y p i c a l l y d e r i v e d from p e r f l u o r o k e r o s e n e (PFK), and u t i l i z i n g them t o d e r i v e the s l o p e and i n t e r c e p t by l e a s t squares f i t to Eq. 2. A l t e r n a t i v e l y , the s p e c t r o m e t e r can be s e t to the c e n t e r o f a known peak, w h i c h g i v e s m directly, and k can be c a l c u l a t e d by m a n u a l l y l o c a t i n g one or more a d d i t i o n a l r e f e r e n c e peaks as V j j / a i s v a r i e d . The computer can be programmed to a d j u s t the app l i e d a c c e l e r a t i n g v o l t a g e d u r i n g an e x p e r i m e n t t o compensate f o r i n s t a b i l i t i e s i n the mass s p e c t r o m e t e r by sweeping a s i n g l e r e f e r e n c e peak p e r i o d i c a l l y , and c h a n g i n g the v o l t a g e a t each m/e a c o n s t a n t amount t o c o r r e c t f o r s h i f t s i n peak c e n t r o i d , a l t h o u g h t h e r e a r e s m a l l e r r o r s i n t h i s p r o c e d u r e because mass and v o l t a g e a r e not l i n e a r l y r e l a t e d (Eq. 2 ) . Feedback c o n t r o l o f t h i s type a l l o w s the use o f l e s s e x p e n s i v e power supp l i e s , w h i c h o f t e n have e x c e l l e n t s h o r t term s t a b i l i t y but p o o r e r l o n g term or t e m p e r a t u r e s t a b i l i t y (2_2) . M i c r o p r o c e s s o r - b a s e d SIM modules are becoming a v a i l a b l e , and one c o m m e r c i a l system s u i t a b l e f o r h i g h r e s o l u t i o n measurement i n c l u d e s a peak s t a b i l i z i n g c a p a b i l i t y and the f a c i l i t y f o r m o n i t o r i n g up to 8 m/ev a l u e s ( 2 3 ) . As w i t h a l l SIM c i r c u i t s l i m i t e d t o peak s w i t c h i n g , the one drawback r e l a t i v e to a computerbased system i s t h a t peak p r o f i l e i n f o r m a t i o n i s not e a s i l y o b t a i n e d . A s o l u t i o n to t h i s p r o b l e m i s to devote t h r e e c h a n n e l s o f the peak s e l e c t o r t o the same peak, one a t the c e n t e r , and two a t the 10 p e r c e n t

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch006

0

0

0

6. HADDON

Organic Trace Analysis

103

3.6 3.4 -

y/ 293 299 / #

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch006

#

3.3 -

0

/

0.2

0.4

0.6

# 3

0.8

°°

1.0

VD/A, VOLTS

Figure 3. Mass-voltage relationship for programmed accelerating voltage operation of 21110 mass spectrometer. M = m/e value focused at collector. V = voltage output of D/A converter. D/A

104

HIGH PERFORMANCE MASS SPECTROMETRY

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch006

i n t e n s i t y p o i n t s on the h i g h and low mass s i d e o f the peak. Three e x a c t mass fragmentograms c o i n c i d e n t i n t i m e ( t e m p e r a t u r e ) and h a v i n g 1:10:1 i n t e n s i t y r a t i o s c o n f i r m the absence o f i n t e r f e r i n g i s o b a r i c i o n s d u r i n g the a n a l y s i s ( 2 4 ) . Applications Aflatoxin Analysis. I n our l a b o r a t o r y we have measured s m a l l amounts o f a f l a t o x i n s i n foods and f e e d s , and i n the b i o f l u i d s o f e x p e r i m e n t a l a n i m a l s (25-27). A f l a t o x i n s are metab­ o l i t e s o f the mold A s p e r g i l l u s F l a v u s , and one o f the metabolites, a f l a t o x i n B i , i s of p a r t i c u l a r i n t e r e s t because i t i s a n a t u r a l l y o c c u r r i n g c a r c i n o g e n w h i c h i s sometimes p r e s e n t a t up to s e v e r a l p a r t s - p e r - b i l l i o n (ppb) i n some a g r i c u l t u r a l p r o d u c t s (28-30). The s t r u c t u r e s and p a r t i a l EI mass s p e c t r a o f s i x a f l a ­ t o x i n s are shown i n F i g u r e 4, and the mass s p e c t r a o f about 50 a d d i t i o n a l m y c o t o x i n s a r e a v a i l a b l e e l s e w h e r e (3JL). P u r i f i c a t i o n and subsequent t r a n s f e r o f a f l a ­ t o x i n s i n t o the mass s p e c t r o m e t e r i s d i f f i c u l t because the compounds a r e u n s t a b l e when h i g h l y p u r i f i e d , bond s t r o n g l y t o g l a s s s u r f a c e s , and cannot be s e p a r a t e d by gas chromatography. These f a c t o r s r e s t r i c t the use o f low r e s o l u t i o n mass s p e c t r a l a n a l y s i s f o r s t r u c t u r a l l y s p e c i f i c v e r i f i c a t i o n or q u a n t i t a t i o n of a f l a t o x i n s . The use o f h i g h r e s o l u t i o n SIM on p a r t i a l l y p u r i f i e d a f l a t o x i n c o n t a i n i n g m i x t u r e s i n t r o d u c e d v i a the d i r e c t probe s i g n i f i c a n t l y l o w e r s the l i m i t s o f d e t e c t i o n f o r mass s p e c t r a l a n a l y s i s o f t h e s e compounds, and has the a d d i t i o n a l advantage t h a t p r i o r i s o l a t i o n p r o c e d u r e s can be s i m p l i f i e d (12) . By u s i n g h i g h r e s o l u t i o n SIM we have d e t e c t e d a f l a t o x i n s B i and Mi i n e x p e r i m e n t a l m i l k samples a t 19 and 140 ppb, r e s p e c t i v e l y . An a n a l y t i c a l e x t r a c t i o n scheme f o r o b t a i n i n g p a r t i a l l y p u r i f i e d a f l a t o x i n s from f r e e z e - d r i e d m i l k and s u i t a b l e f o r use i n c o n j u n c t i o n w i t h h i g h r e s o l u t i o n mass s p e c t r a l a n a l y s i s i s shown i n F i g u r e 5. E x t r a c t A c o n t a i n s a l l o f the a f l a t o x i n s . In e x t r a c t s Β and C, w h i c h are more h i g h l y p u r i f i e d , a f l a t o x i n s B i and Mi a r e p a r t i a l l y s e p a r a t e d because of d i f f e r e n c e s i n p a r t i t i o n c o e f f i c i e n t s i n the e x t r a c t i o n s o l v e n t s (32). H i g h r e s o l u t i o n SIM d a t a f o r e x t r a c t s A and C a r e shown i n F i g u r e s 6 and 7. These d a t a o f i n t e n s i t y vvs. mass r e p r e s e n t scans o v e r 0.3 amu f o r each a p p r o p r i a t e i n t e g r a l m/e a t mass r e s o l u t i o n o f 7,000. The m i d d l e s c a n f o r e x t r a c t A ( F i g . 6 ) , w h i c h was r e c o r d e d a t the t e m p e r a t u r e o f maximum r a t e o f v o l a t i l i z a t i o n o f the a f l a t o x i n s , shows the Mi m o l e c u l a r i o n a t m/e 328, the

6. HADDON

Organic Trace Analysis

105

M-16 i o n o f Mi and m o l e c u l a r i o n o f B i (same e l e m e n t a l c o m p o s i t i o n , C i H i 0 ) a t m/e 312, and the M-29 i o n o f Mi a t m/e 299. The abundance r a t i o [M] ·/[M-29] agrees w i t h i n e x p e r i m e n t a l e r r o r w i t h t h e r a t i o o f t h e s e peaks i n t h e r e f e r e n c e low r e s o l u t i o n mass spectrum o f a f l a t o x i n Mi shown i n F i g u r e 4 and c o n f i r m s t h e absence o f i n t e r f e r e n c e a t a r e s o l u t i o n o f 7000 f o r this particular extract. Figure 7 gives equivalent d a t a f o r e x t r a c t C o b t a i n e d from t h e same sample o f f r e e z e - d r i e d m i l k . The a d d i t i o n a l peak a t m/e 314 i s from 12 ng a f l a t o x i n B , added as an i n t e r n a l s t a n d a r d . The abundance r a t i o s between a f l a t o x i n i o n s a t m/e 312 and 328 i n t h e s e scans r e f l e c t t h e d e c r e a s e d amount o f a f l a t o x i n B f o r e x t r a c t C w h i c h r e s u l t e d from s o l v e n t p a r t i t i o n i n g during extraction. Thus f o r e x t r a c t A ( F i g . 6) t h e i n t e n s i t y r a t i o o f C i H i 0 t o C i H i 0 i s 0.32, compared t o 0.06 f o r pure Mi (see F i g . 4 e ) , but f o r e x t r a c t C ( F i g . 7) t h e r a t i o i s 0.092, w h i c h i n d i c a t e s l e s s a f l a t o x i n B , as e x p e c t e d . The methodo l o g y t o q u a n t i t a t e t h e compounds i s c u r r e n t l y b e i n g developed. A n o t a b l e f e a t u r e o f t h e d i r e c t probe method f o r a f l a t o x i n a n a l y s i s i s an enhancement o f s e n s i t i v i t y w h i c h o c c u r s when p a r t i a l l y p u r i f i e d samples o f a f l a t o x i n a r e r u n on t h e mass s p e c t r o m e t e r , r e l a t i v e t o measurements on h i g h l y p u r i f i e d samples. Apparently, t h i s i s because b o n d i n g t o t h e s u r f a c e o f t h e sample c o n t a i n e r s i s s u b s t a n t i a l l y reduced i n a complex mixt u r e f o r t h e s e compounds. The d a t a o f F i g u r e 8 i l l u s t r a t e t h i s enhancement o f s e n s i t i v i t y f o r a f l a t o x i n B i added t o an e x t r a c t o f p o o l e d human u r i n e s . The c u r v e s o f F i g . 8 a r e e x a c t mass fragmentograms, r e c o r d e d d u r i n g r a p i d t e m p e r a t u r e programming o f t h e d i r e c t probe from about 50°C t o 250°C, w i t h the mass s p e c t r o m e t e r tuned t o the m o l e c u l a r i o n o f B , m/e 312.0635, a t 7000 r e s o l u t i o n . A c o m p a r i s o n o f t h e response o b t a i n e d i n c u r v e A o f F i g . 8 f o r 1.3 ng p u r e B i w i t h t h a t o f c u r v e D, o b t a i n e d from 0.03 ng B i added t o the u r i n e e x t r a c t , i l l u s t r a t e t h e 1 0 0 - f o l d enhancement o f s e n s i t i v i t y f o r d e t e c t i n g a f l a t o x i n s B i i n a complex m i x t u r e . The s e n s i t i v i t y enhancement i s somewhat g r e a t e r f o r a f l a t o x i n M i , f o r w h i c h n e g l i g i b l e response i s o b t a i n e d f o r 30 ng o f p u r i f i e d compound, compared t o a s i g n a l t o background r a t i o o f b e t t e r than 100 f o r 13 ng i n t r o d u c e d i n a m i x t u r e , as shown i n F i g s . 6 and 7. Q u a n t i t a t i v e H i g h R e s o l u t i o n SIM f o r A n a l y z i n g P u r i n e s i n B l o o d and T i s s u e . Q u a n t i t a t i v e d i r e c t probe measurements a t t h e 2100 ppm l e v e l f o r f i v e p u r i n e components o f human b l o o d 7

2

6

+

+

2

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch006

x

7

2

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American Chemical Society

Figure 4a, b, c. 70 eV mass spectra of aflatoxins

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch006

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Figure 4d, e, /. 70 eV mass spectra of aflatoxins

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FREEZE DRIED MILK

1:1 MeOH-H0 2

Residue (discard)

primary extract

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch006

3 vol. CHC1, Combined CHCI3 extracts (Extract A)

MeOH-H0 phase 2

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evaporate residue re-dissolve in 1:1 MeOH-H0 2

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(discard) Association of Official Analytical Chemists, Inc.

Figure 5. Analytical extraction of aflatoxins B and M from freeze-dried milk t

t

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, 104 MV

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Figure 6. High resolution SIM scans of intensity in millivolts (mV) vs. mass for 13 ng aflatoxin M and 1.7 ng aflatoxin B in 100fig dried milk extract A. Direct introduction probe temperature increases from A to C. t

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HIGH PERFORMANCE MASS SPECTROMETRY

512 MV

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Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch006

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Figure 7. High resolution SIM scans of intensity in millivolts (mV) vs. mass for extract C. 12.4 ng aflatoxin B (mass 314) added as internal standard. Direct probe temperature increases from A to B. 2

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111

and t i s s u e have been r e p o r t e d by Snedden and P a r k e r u s i n g h i g h r e s o l u t i o n SIM (13) ( 3 3 ) . The h i g h r e s o l u t i o n mass s p e c t r a l a n a l y s i s f o r t h e p u r i n e s p r o v i d e d q u a n t i t a t i v e measurement o f h y p o x a n t h i n e ( I ) , x a n t h i n e ( I I ) , u r i c a c i d ( I I I ) , a l l o p u r i n o l (IV) and p u r i n o l (V) (see F i g u r e 9 ) , w h i c h a r e i m p o r t a n t i n t h e s t u d y and t r e a t m e n t o f gout. The use o f h i g h r e s o l u t i o n mass s p e c t r o m e t r y made p o s s i b l e a c c u r a t e a n a l y s i s a t t h e s e l e v e l s w i t h o u t p r e - p u r i f i c a t i o n o f t h e samples. More r e c e n t l y t h e same group has used h i g h r e s o l u t i o n mass s p e c t r o m e t r y t o q u a n t i t a t e o e s t r o g e n and p r o g e s t e r o n e i n human o v a r i a n t i s s u e a t 10-50 ppm, a g a i n u s i n g p r o c e d u r e s t h a t r e q u i r e no c h e m i c a l s e p a r a t i o n o r d e r i v i t i z a t i o n p r i o r t o mass s p e c t r a l a n a l y s i s ( 1 4 ) . To i n s u r e t h a t t h e i r method i s v a l i d , t h e a u t h o r s r e c o r d e d a h i g h r e s o l u t i o n spectrum o f t h e m i x t u r e t o be a n a l y z e d a t t h e chosen r e s o l u t i o n , i n t h i s case 20,000, and compared t h e abundances o f c h a r a c t e r i s t i c mass peaks i n t h e e x p e r i m e n t a l sample w i t h those i n t h e h i g h r e s o l u t i o n r e f e r e n c e spectrum o f t h e pure compound. F i g u r e 10 i l l u s t r a t e s t h e e x c e l l e n t agreement o b t a i n e d a t f i v e o f t h e s i x masses examined f o r I I I i n human muscle t i s s u e . S i m i l a r experiments w i t h the o t h e r p u r i n e s o f F i g u r e 9 y i e l d e d a s e t o f masses a p p r o p r i a t e f o r a n a l y z i n g I-V t o g e t h e r . I n t e g r a t i n g t h e s e l e c t e d i o n p r o f i l e s ( e x a c t mass fragmentograms) a t f i v e masses as a f u n c t i o n o f temp e r a t u r e gave a s e t o f r e l a t i v e peak a r e a s . These peak a r e a s were r e l a t e d t o t h e r e l a t i v e amounts o f I-V u s i n g a s e t o f f i v e l i n e a r s i m u l t a n e o u s e q u a t i o n s and r e sponse c o e f f i c i e n t s d e v e l o p e d from runs on p u r i f i e d compounds, a c c o r d i n g t o e s t a b l i s h e d p r o c e d u r e s o f q u a n t i t a t i v e mass s p e c t r o m e t r y ( 3 4 ) . The i n t r o d u c t i o n o f a c c u r a t e l y weighed (mg amounts) samples f a c i l i t a t e d the c a l c u l a t i o n o f a b s o l u t e c o n c e n t r a t i o n s . An e v a l u a t i o n o f t h e s e n s i t i v i t y and measurement e r r o r f o r t h e a n a l y s i s r e v e a l s t h a t d i f f e r e n t methods o f sample p r e p a r a t i o n g i v e d i f f e r e n t q u a n t i t a t i v e p r e cision. F i g . 11 shows t h a t t h e b e s t r e s u l t s were o b t a i n e d u s i n g powdered samples o f d e s s i c a t e d b l o o d and muscle t i s s u e . D e p o s i t i n g t h e samples on t h e d i r e c t probe s u r f a c e ( g o l d ) by e v a p o r a t i o n o f an aqueous s o l u t i o n gave s i g n i f i c a n t l y lower p r e c i s i o n f o r a g i v e n sample amount, a p p a r e n t l y because o f i n t e r a c t i o n s between t h e sample and t h e s u r f a c e o f t h e probe f o r t h e more p o l a r compounds. When a l e s s p o l a r s u b s t a n c e , c a f f e i n e , was a n a l y z e d , t h e r e was no dependence o f p r e c i s i o n on t h e method o f p r e p a r i n g t h e sample, as shown i n F i g . 11.

112

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Figure 8. Exact mass fragmentogram for C H 0 (aflatoxin Bi molecular ion, m/e 312.0635): A, 1.65 ng B from reference solution; B, blank, extract B; C, urine extract plus 0.13 ng B ; D, urine extract plus 0.030 ng B . î7

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Figure 9. Structures of purines analyzed by high resolution SIM: I, hypoxanthine; II, xanthine; III, uric acid; IV, allopurinol; and V, oxipurinol.

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Figure 10. Partial high resolution mass spectrum of substances evaporated from human muscle, showing peaks attributable to uric acid. ( a) Relative intensities of uric acid peaks from muscle; (b) relative intensities of sam peaks from uric acid; (c) nominal mass of multiplet.

25

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Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch006

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Figure 11. Dependence of analytical precision of high resolution SIM results on concentration and diluent for caffeine, hypoxanthine, xanthine, and uric acid in solution and solid mixtures

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Discussion The mass s p e c t r o m e t e r i s w e l l s u i t e d t o t r a c e a n a l y s i s o f the t y p e d i s c u s s e d h e r e f o r two r e a s o n s : The p r o c e s s o f i o n p r o d u c t i o n by the mass s p e c t r o m e t e r i s g e n e r a l l y l i n e a r o v e r many o r d e r s o f magnitude o f sample c o n c e n t r a t i o n ; and the s e l e c t i v i t y o f the method can be e x t r e m e l y g r e a t when e i t h e r h i g h mass r e s o l u ­ t i o n , a s e l e c t i v e i o n i z a t i o n method, or a c o m b i n a t i o n of t h e s e a r e employed t o e f f e c t s e p a r a t i o n between d i f f e r e n t components o f m i x t u r e s . The wide dynamic range n e c e s s a r y f o r q u a n t i t a t i o n i s w e l l accomodated by electron multiplier detection. Photoplate detection w i l l be g e n e r a l l y l e s s s u i t a b l e f o r t r a c e a n a l y s i s problems o f t h i s n a t u r e because of extensive blackening of the p h o t o g r a p h i c p l a t e near i n t e n s e l i n e s , and nonl i n e a r i t y of response. The a f l a t o x i n s a r e f a v o r a b l y a n a l y z e d by h i g h r e s o l u t i o n SIM i n complex m i x t u r e s i n p a r t because o f t h e i r low r a t i o o f hydrogen t o c a r b o n , w h i c h l e a d s t o an e x a c t mass v a l u e below the masses o f o t h e r sub­ stances, p r i m a r i l y l i p i d s , t y p i c a l l y present i n food e x t r a c t s . Many o t h e r e n v i r o n m e n t a l p o l l u t a n t s i n ­ c l u d i n g h a l o g e n a t e d p e s t i c i d e s and many o f the common m y c o t o x i n s (35.) have comparable o r g r e a t e r mass d e f e c t s and s h o u l d be amenable t o a n a l y s i s by t h i s method. We have f r e q u e n t l y o b s e r v e d peaks below the e x a c t mass p o s i t i o n s o f the a f l a t o x i n s , as shown f o r the scans a t m/e 299 and 312 o f F i g u r e 6. These peaks may a r i s e from h a l o g e n a t e d p e s t i c i d e c o n t a m i n a n t s . High reso­ l u t i o n SIM may be a v a l u a b l e complement t o GC/MS and t o n e g a t i v e c h e m i c a l i o n i z a t i o n t e c h n i q u e s (8_,_9) f o r such compounds. Surface e f f e c t s . I n t e r a c t i o n s w i t h the s u r f a c e o f cont a i n e r s used t o i n t r o d u c e samples v i a the d i r e c t probe can a f f e c t d e t e c t i o n l i m i t s a d v e r s e l y , and an under­ s t a n d i n g o f s u r f a c e e f f e c t s appears to be i m p o r t a n t f o r f u l l y u t i l i z i n g the method i n t r a c e a n a l y s i s . In a d e t a i l e d s t u d y o f s u r f a c e i n t e r a c t i o n s f o r s m a l l pep­ t i d e s , Friedman (36-38) d e s c r i b e s the r a t e o f v o l a t i ­ l i z a t i o n (dn/dt) o f a pure compound from a s u r f a c e by the e q u a t i o n

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch006

%

H

= N Ae" o

E / R T

(3)

where N i s the number o f sample m o l e c u l e s i n i t i a l l y on the s u r f a c e , A i s a f r e q u e n c y f a c t o r , and the exponen­ t i a l term e ~ ' g i v e s the f r a c t i o n o f m o l e c u l e s a t t e m p e r a t u r e Τ w i t h s u f f i c i e n t energy t o overcome s u r ­ face bonding. Ε i s the a c t i v a t i o n energy f o r removing 0

E

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a m o l e c u l e from the s u r f a c e . A h i g h r a t e o f v o l a t i l i z a t i o n i s r e a l i z e d by i n c r e a s i n g N , f o r example by d i s p e r s i n g the sample by e v a p o r a t i o n from s o l u t i o n and by r e d u c i n g E, by u s i n g an i n e r t d i r e c t probe s u r f a c e . The enhanced s e n s i t i v i t y f o r a f l a t o x i n s i n mixt u r e s t h a t we o b s e r v e i s c o n s i s t e n t w i t h Friedman's model f o r s u r f a c e v o l a t i l i z a t i o n . Apparently other compounds c o d e p o s i t e d from s o l u t i o n a l o n g w i t h the a f l a t o x i n s e i t h e r occupy a c t i v e s i t e s on the sample tubes p r e f e r e n t i a l l y , o r s h i e l d the a f l a t o x i n s from them, r e s u l t i n g i n more e f f i c i e n t v o l a t i l i z a t i o n a r i s i n g from a r e d u c t i o n i n E. The c u r v e s o f measurement e r r o r f o r the p u r i n e s ( F i g . 1 1 ) , w h i c h i n d i c a t e lower p r e c i s i o n f o r more p o l a r compounds, a r e s u g g e s t i v e o f s u r f a c e i n t e r a c t i o n s ^ but the i n t e r p r e t a t i o n i s l e s s o b v i o u s , because d i s p e r s i n g the compounds over the g o l d s u r f a c e o f the sample probe a c t u a l l y l o w e r e d the p r e c i s i o n o f the a n a l y s i s . The p r e s e n c e o f h i g h concent r a t i o n s of i n o r g a n i c s a l t s probably accounts f o r t h i s e f f e c t f o r the p u r i n e s . I n Friedman's s t u d y o f s m a l l peptide v o l a t i l i z a t i o n , traces of i n o r g a n i c s a l t s l o w e r e d the r a t e o f v o l a t i l i z a t i o n s u b s t a n t i a l l y a t a given temperature (38). The s u r f a c e e f f e c t p r o b l e m i s an i m p o r t a n t a r e a f o r f u r t h e r study. I t has been s u g g e s t e d r e c e n t l y t h a t a r e d u c t i o n i n s u r f a c e b o n d i n g e x p l a i n s the s p e c t r a o b t a i n e d from " n o n - v o l a t i l e " compounds a b s o r b e d on a c t i v a t e d (surface-prepared) tungsten emitter wires u s i n g f i e l d d e s o r p t i o n mass s p e c t r o m e t r y (39,40.). M i n i m i z i n g s u r f a c e i n t e r a c t i o n s appears p r o m i s i n g as a g e n e r a l method t o enhance s e n s i t i v i t y f o r mass s p e c t r a l a n a l y s i s of l e s s v o l a t i l e substances. Of c o u r s e , i t i s i m p o r t a n t t o r e c o g n i z e t h a t the c h e m i c a l i n s t a b i l i t y o f a p a r t i c u l a r s u b s t a n c e may be s i g n i f i c a n t l y d e c r e a s e d r e l a t i v e t o i t s s t a b i l i t y i n a s o l u t i o n or s o l i d sample m a t r i x ; f o r example, a f l a t o x i n s d i s p e r s e d on a s u r f a c e show g r e a t l y enhanced r e a c t i v i t y to UV l i g h t . Ion Source C o n t a m i n a t i o n . C o n t a m i n a t i o n o f the i o n s o u r c e , w h i c h may o c c u r r a p i d l y i n some c a s e s , can a p p r e c i a b l y r e d u c e the s e n s i t i v i t y o f the method. T h i s f a c t o r must be weighed w i t h the advantages o f u s i n g the d i r e c t probe when a l t e r n a t i v e GC/MS p r o c e d u r e s a r e a p p l i c a b l e . The r a p i d i t y o f s o u r c e c o n t a m i n a t i o n v a r i e s w i d e l y i n the few examples r e p o r t e d . Snedden and P a r k e r e x p e r i e n c e d l e s s t h a n 20 per c e n t d e c l i n e i n s e n s i t i v i t y i n s i x months o f p u r i n e a n a l y s i s ( 1 3 J . C o n v e r s e l y , Dougherty (8.) found t h a t s e n s i t i v i t y f o r p o l y c h l o r i n a t e d b i p h e n y l (PCB) d e t e c t i o n was s e v e r e l y r e d u c e d a f t e r r u n n i n g t e n samples o f u n p u r i f i e d human u r i n e e x t r a c t . M i n i m a l s o l v e n t e x t r a c t i o n and column 0

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chromatography p r i o r t o d i r e c t probe a n a l y s i s s o l v e d t h i s p r o b l e m , as i t has i n our l a b o r a t o r y f o r a f l a t o x i n a n a l y s i s . Other approaches t o r e d u c i n g i o n source c o n t a m i n a t i o n may be a p p l i c a b l e i n some c a s e s . F o r example, i t s h o u l d be p o s s i b l e t o m e c h a n i c a l l y v e n t t h e v o l a t i l i z e d sample away from t h e i o n i z a t i o n chamber u n t i l t h e temperature o f v o l a t i l i z a t i o n f o r t h e compounds o f i n t e r e s t i s reached (see F i g . 8 ) , t h e r e b y reducing the q u a n t i t y o f m a t e r i a l which enters the i o n source. Chemical B i n d i n g E f f e c t s . The c h e m i c a l s t a t e o f t h e sample c a n a f f e c t i t s v o l a t i l i t y . A comparison o f the q u a n t i t a t i v e mass s p e c t r a l measurements f o r u r i c a c i d i n b l o o d plasma w i t h t h e c o n c e n t r a t i o n v a l u e s o b t a i n e d by e n z y m a t i c a n a l y s i s s u g g e s t s t h a t t h e mass s p e c t r o meter d e t e c t s o n l y t h e u r i c a c i d w h i c h i s bound l o o s e l y t o b l o o d p r o t e i n s , and n o t t h e u r i c a c i d i n s o l u t i o n , w h i c h may e x i s t p r i m a r i l y as t h e sodium s a l t . These d a t a , w h i c h must be c o n s i d e r e d p r e l i m i n a r y , suggest t h a t mass s p e c t r a l a n a l y s i s , when p e r f o r m e d w i t h o u t p r i o r sample t r e a t m e n t , may i n d i c a t e i n d i r e c t l y t h e e x t e n t o f c h e m i c a l b i n d i n g f o r s m a l l m o l e c u l e s i n some cases. Conclusions High r e s o l u t i o n s e l e c t e d i o n m o n i t o r i n g measurements on samples i n t r o d u c e d on t h e d i r e c t probe c a n p r o v i d e a c c u r a t e and h i g h l y s p e c i f i c q u a l i t a t i v e and q u a n t i t a t i v e i n f o r m a t i o n about compounds p r e s e n t i n t r a c e amounts. T h i s m i x t u r e a n a l y s i s method i s app l i c a b l e t o some compounds w h i c h cannot be s e p a r a t e d by gas chromatography because o f low v o l a t i l i t y o r chemical instability. The h i g h e s t s e n s i t i v i t i e s a r e obt a i n e d f o r s u b s t a n c e s h a v i n g l a r g e mass d e f e c t s and t h i s embraces a number o f c l a s s e s o f compounds, i n c l u d i n g h a l o g e n a t e d p e s t i c i d e s and m y c o t o x i n s , w h i c h a r e o f p a r t i c u l a r i n t e r e s t i n e n v i r o n m e n t a l and f o o d c h e m i s t r y . M a g n e t i c s e c t o r mass s p e c t r o m e t e r s h a v i n g s u f f i c i e n t e l e c t r o n i c s t a b i l i t y f o r a c c u r a t e mass measurement c a n be m o d i f i e d f o r a c c u r a t e q u a n t i t a t i v e h i g h r e s o l u t i o n s e l e c t e d i o n m o n i t o r i n g measurements.

References 1. Keith, L.H., in Identification and Analysis of Organic Pollutants in Water, L.H. Keith, ed. (Ann Arbor Science Publishers, Ann Arbor) 1976, p. iii. 2. McFadden, W.H., Schwartz, H.L. and Evans, S., J. Chromatogr. (1976) 122, 389.

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9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

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Arpino, P.J., Dawkins, B . J . , and McLafferty, F.W., J. Chromatogr. Sci. (1974) 12, 574. Carroll, D.I., Dzidic, I., Stillwell, R.N., Haegele, K.D. and Horning, E.C., Anal. Chem. (1975) 47, 2369. Sphon, J.Α., Dreifuss, P.A. and Schulten, H.R., J. Assoc. Official Anal. Chemists, (1977) 60, 73. Hunt, D.F. and Ryan III, J.F., Anal. Chem., (1972) 44, 1306. Weinkam, R.J., Rowland, M. and Meffin, P . J . , Biomed. Mass Spectrom. (1977) 4, 42. Dougherty, R.C. and Piotrowska, K., Proc. Nat'l. Acad. Sci. USA, (1976) 73, 1777. Dougherty, R.C. and Piotrowska, K., J. Assoc. Official Anal. Chemists, (1976) 59, 1023. Levson, K. and Schulten, H.R., Biomed. Mass Spec­ trom. (1976) 3, 137. Soltero-Rigau, E. Kruger, K.L. and Cooks, R.G., Anal. Chem. (1977) 49, 435. Haddon, W.F., Masri, M.S., Randall, V.G., Elsken, R.H., and Meneghelli, B . J . , J. Assoc. Official Anal. Chemists (1977) 60, 107. Snedden, W. and Parker, R.B., Anal. Chem. (1971) 43, 1651. Snedden, W. and Parker, R.B., Biomed. Mass Spec­ trom. (1976) 3, 275. Hutzinger, O., Jamieson, W.D., and Safe, S., Nature (London) (1974) 252, 698. Fenselau, C., Anal. Chem. (1977) 49, 563A. Millington, D.S., Buoy, M.E., Brooks, G., Harper, M.E. and Griffiths, Κ., Biomed. Mass Spectrom. (1975) 2, 219. Millington, D.S., J. Steroid Biochem. (1975) 6, 239. Evans, K.P., Mathias, Α., Mellor, Ν., Silvester, R. and Williams, A.E., Anal. Chem. (1975) 47, 821. Gallegos, E . J . , Anal. Chem. (1975) 47, 1150. Hammar, C.G., and Hessling, R., Anal. Chem. (1971) 43, 298. Holmes, W.F., Holland, W.H., Shore, B.L., Bier, D.M. and Sherman, W.R., Anal. Chem. (1973) 45, 2063. Vacuum Generators, Ltd., Altrincham, Cheshire, England. Millington, D.S., personal communication. Haddon, W.F., Wiley, M. and Waiss, A.C., Anal. Chem. (1971) 43, 268. Masri, M.S., J. Am. Oil Chem. Soc. (1970) 47, 61. Masri, M.S., Haddon, W.F., Lundin, R.E., and Hseih, D.P.H., J. Agric. Food Chem. (1974) 22, 512.

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28. Wilson, B.J. and Hayes, A.W. in Toxicants Oc­ curring in Foods, National Academy of Sciences, Washington, D.C., pp. 372-423 (1973). 29. Heildelberger, C., Annu. Rev. Biochem. (1975) 44, 79. 30. Ong, T.M., Mutat. Res. (1975) 32, 35. 31. Aflatoxin Mass Spectral Data Bank, Pohland, A.E. and Sphon, J.Α., ed., U.S. Food and Drug Adm., Bureau of Foods, Division of Chem. and Phys., Washington, D.C. 32. Masri, M.S., Page, J.R., and Garcia, V.G., J . Assoc. Official Anal. Chemists (1969) 52, 641. 33. Parker, R.B., Snedden, W., and Watts, R.W.E., Biochem. J . (1969) 115, 103. 34. Kiser, Introduction to Mass Spectrometry and Its Applications, Prentice-Hall, Inc., Englewood Cliffs (1965), p. 216. 35. Kingston, D.G.I., J. Assoc. Official Anal. Chemists (1976) 59, 1016. 36. Beuhler, R . J . , Flanigan, E., Greene, L . J . and Friedman, L . , Biochem. and Biophys. Res. Commun. (1972) 46, 1082. 37. Beuhler, R.J., Flanigan, E., Greene, L . J . and Friedman, L . , Biochem. (1974) 13, 5061. 38. Beuhler, R.J., Flanigan, Ε., Greene, L . J . and Friedman, L . , J . Am. Chem. Soc. (1974) 96, 3990. 39. Soltman, B., Sweeley, C.C. and Holland, J.F., Anal. Chem. (1977) 49, 1164. 40. Hunt, D.F., Shabanowitz, J . and Botz, F.K., Anal. Chem. (1977) 49, 1160. RECEIVED December 30, 1977

7 Introduction to Gas Chromatography/High Resolution Mass Spectrometry B. J. KIMBLE

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

University of California-Berkeley, Berkeley, CA 94720

The combination of a gas chromatograph (GC), a mass spectrometer (MS) and an on-line computer is now well-established as an exceptionally powerful and versatile tool for identifying and quantitating the components present in organic mixtures. As with any successful technique, many improvements have been incorporated in recent years in order to provide the maximum amount of information that can be derived from a GC/MS analysis. Significant developments include the use of: a) glass capillary columns (1-3), for minimized surface effects and increased chromatographic resolution; b) direct interfacing of capillary columns to the mass spectrometer ion source (4-7), to reduce sample losses in a molecular separator and to preserve chromatographic resolution by elimi nating large-volume connectors; c) high pressure ionization methods (e.g., chemical ionization, 8-14), to provide complementary in formation to assist in the interpretation of electron impact spectra; d) improved computer techniques, for automated data acquisition, data handling (15-17), spectrum recognition (18-23) and interpretation (24-26). An a d d i t i o n a l approach f o r t h e improved u t i l i t y o f t h e GC/MS t e c h n i q u e would be t o d e t e r m i n e t h e r e c o r d ed mass v a l u e s i n each spectrum w i t h s u f f i c i e n t a c c u r a c y t o be a b l e t o compute t h e e x a c t e l e m e n t a l comp o s i t i o n o f each o f t h e i o n s p e c i e s c o n t r i b u t i n g t o t h e spectrum (27) . Such an achievement would p r o v i d e t h e maximum amount o f s t r u c t u r a l i n f o r m a t i o n t h a t c a n be d i r e c t l y d e r i v e d from a mass spectrum — i . e . , a d a t a ©0-8412-0422-5/78/47-070-120$10.00/0

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s e t c o m p r i s e d o f e x a c t e l e m e n t a l c o m p o s i t i o n s and t h e i r r e l a t i v e abundances. The r o u t i n e a v a i l a b i l i t y o f e l e m e n t a l c o m p o s i t i o n s p e c t r a from a GC/MS a n a l y s i s would s u b s t a n t i a l l y a s s i s t t h e i n t e r p r e t e r / a n a l y s t i n s e v e r a l ways, a l l o f w h i c h d e r i v e from t h e i n c r e a s e d s t r u c t u r a l s p e c i f i c i t y i n h e r e n t i n such d a t a . I n t h e case o f mass s p e c t r a which a r e , i n f a c t , interprétable by a t r a i n e d spect r o m e t r i s t , exact elemental composition i n f o r m a t i o n would e l i m i n a t e t h e guesswork i n v o l v e d i n r e l a t i n g mass v a l u e s and mass d i f f e r e n c e s t o s t r u c t u r a l u n i t s , and t h u s , would r e s u l t i n more r a p i d and r e l i a b l e i n t e r pretations. T h i s i s p a r t i c u l a r l y t r u e when t h e r e c o r d e d spectrum r e s u l t s from t h e c o - e l u t i o n o f two o r more components, and t h e i n t e r p r e t i v e p r o c e s s has t o i n c l u d e t h e c o r r e c t assignment o f masses i n t o t h e i r appropriate "sub-spectra". Elemental composition i n f o r m a t i o n would a l s o f a c i l i t a t e t h e l o c a t i o n and q u a n t i t a t i o n o f s p e c i f i c components i n m i x t u r e s , s i n c e the i n t e n s i t i e s f o r a p a r t i c u l a r e l e m e n t a l c o m p o s i t i o n c o u l d be p l o t t e d as a f u n c t i o n o f scan number; such an e l e m e n t a l c o m p o s i t i o n chromatogram would c o n s t i t u t e a more s p e c i f i c s t r u c t u r a l i n d i c a t o r t h a n do n o m i n a l mass chromatograms. Of p a r t i c u l a r u t i l i t y , e l e m e n t a l c o m p o s i t i o n s p e c t r a would p r o v i d e d i r e c t s t r u c t u r a l i n f o r m a t i o n i n t h e case o f " h a r d - t o - i n t e r p r e t " s p e c t r a o f t r u l y unknown components — t h a t i s , when l i t t l e o r no c h e m i c a l information i s a v a i l a b l e to a s s i s t i n the i n t e r p r e t a t i o n . A l s o , unexpected compounds c a n a r i s e i n a v a r i e t y o f s i t u a t i o n s as a r t i f a c t s o r c o n t a m i n a n t s , o r due t o i n a d e q u a t e f r a c t i o n a t i o n and/or d e r i v a t i z a t i o n p r o c e d u r e s , e t c . ; and o f c o u r s e , " d i f f i c u l t " s p e c t r a occur p a r t i c u l a r l y i n analyses o f mixtures c o n t a i n i n g s e v e r a l hundred components. W i t h t o d a y s i n c r e a s i n g need t o s t u d y more and more c o m p l i c a t e d samples ( p a r t i c u l a r l y e n v i r o n m e n t a l m i x t u r e s such as sewage e f f l u e n t s , and samples f o r w h i c h m i n i m a l s e p a r a t i o n and f r a c t i o n a t i o n steps are d e s i r e d ) , i t i s t h i s aspect o f complex m i x t u r e a n a l y s i s t h a t would p a r t i c u l a r l y benef i t from t h e a v a i l a b i l i t y o f e l e m e n t a l c o m p o s i t i o n spectra. To d a t e , t h e concept o f e l e m e n t a l c o m p o s i t i o n assignments i n GC/MS a n a l y s e s has been e x p l o r e d i n a v a r i e t y o f ways, and a p p l i c a t i o n s o f t h e t e c h n i q u e seem l i k e l y t o i n c r e a s e as s u i t a b l e commercial i n s t r u m e n t a t i o n becomes more w i d e l y a v a i l a b l e . U n f o r t u n a t e l y , a somewhat ambiguous t e r m i n o l o g y has a l r e a d y e v o l v e d conc e r n i n g use o f t h e p h r a s e s " a c c u r a t e mass measurement" and, i n p a r t i c u l a r , " h i g h r e s o l u t i o n " . In general, 1

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t h e s e terms a r e used t o c o n t r a s t w i t h t h e measurement o f n o m i n a l masses ( e . g . , m/e 100 o r 101) and i n s t r u m e n t o p e r a t i o n a t n o m i n a l mass r e s o l u t i o n ( e . g . , Μ/ΔΜ < 1,000); however, i t s h o u l d be r e c o g n i z e d t h a t i n p r a c t i c e t h e s o - c a l l e d a c c u r a t e mass measurements may n o t be p a r t i c u l a r l y " a c c u r a t e " , n o r t h e mass r e s o l u t i o n p a r t i c u l a r l y " h i g h " . F o r s i m p l i c i t y , t h e acronyms GC/LRMS and GC/HRMS w i l l be used here t o denote GC/MS a n a l y s e s i n v o l v i n g t h e c o n t i n u o u s r e c o r d i n g o f complete mass s p e c t r a a t n o m i n a l (low) r e s o l u t i o n and h i g h e r mass r e s o l u t i o n s r e s p e c t i v e l y . I t s h o u l d a l s o be n o t e d t h a t t h e terms " a c c u r a t e mass measurement" and " h i g h r e s o l u t i o n " a r e n o t synonymous; t h i s may be i l l u s t r a t e d by summarizing t h e approaches t h a t have been r e p o r t e d i n the l i t e r a t u r e concerning the use o f elemental c o m p o s i t i o n i n f o r m a t i o n i n GC/MS a n a l y s e s . E l e m e n t a l c o m p o s i t i o n m o n i t o r i n g o f GC e f f l u e n t s . I n t h i s approach, t h e o b j e c t i v e i s t o u t i l i z e t h e mass s p e c t r o m e t e r as a h i g h l y s t r u c t u r a l l y s p e c i f i c GC d e t e c t o r f o r q u a n t i t a t i v e s t u d i e s , p a r t i c u l a r l y when the compound o f i n t e r e s t i s p r e s e n t i n a h i g h l y complex m i x t u r e (28-35). The mass s p e c t r o m e t e r i s o p e r a t e d a t some i n c r e a s e d mass r e s o l u t i o n ( e . g . , 10,000) w i t h a s i n g l e a c c u r a t e mass ( c h a r a c t e r i s t i c o f t h e components o f i n t e r e s t ) f o c u s s e d c o n t i n u o u s l y on t h e d e t e c t o r . The i n c r e a s e d mass r e s o l u t i o n aims t o reduce t h e i n t e r ­ f e r e n c e s from o t h e r i o n s h a v i n g t h e same n o m i n a l mass but o f a d i f f e r e n t e l e m e n t a l c o m p o s i t i o n , thus en­ h a n c i n g compound s p e c i f i c i t y w i t h r e s p e c t t o c o - e l u t i n g components from t h e gas chromatograph, i n s t r u m e n t back­ ground, column b l e e d , e t c . E l e m e n t a l c o m p o s i t i o n assignments from s p e c t r a r e ­ c o r d e d a t n o m i n a l mass r e s o l u t i o n . Here, t h e masses o f the r e c o r d e d peaks i n each spectrum a r e d e t e r m i n e d w i t h g r e a t e r t h a n n o m i n a l mass a c c u r a c y , and e l e m e n t a l c o m p o s i t i o n s a r e computed from t h e mass measurements (36; see a l s o 37, 3 8 ) . However, as t h e mass r e s o l u t i o n i s n o t i n c r e a s e ? , problems a r i s e i f t h e nominal mass r e s u l t s from i o n s o f more t h a n one e l e m e n t a l compo­ sition i . e . , i f t h e peak i s a c t u a l l y an u n r e s o l v e d doublet or m u l t i p l e t . This disadvantage represents a p a r t i c u l a r problem i n t h e a n a l y s i s o f complex m i x t u r e s when t h e components o f t h e m i x t u r e may n o t be chromat o g r a p h i c a l l y r e s o l v e d , and more c o m p l i c a t e d s p e c t r a a r e o f t e n produced. The u s e o f a double-beam mass s p e c t r o m e t e r f o r GC/MS a n a l y s e s (39^-41) i n which a mass c a l i b r a n t and t h e sample a r e a n a l y s e d s e p a r a t e l y but s i m u l t a n e o u s l y u s i n g two s e p a r a t e i o n s o u r c e s , a

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common mass a n a l y z e r and two s e p a r a t e d e t e c t o r s s h o u l d improve mass measurement a c c u r a c y b u t does n o t s o l v e the problem o f inadequate r e s o l u t i o n o f doublet o r m u l t i p l e t masses i n t h e sample s p e c t r a . A n o t h e r t e c h n i q u e , w h i c h u t i l i z e s a q u a d r u p o l e mass s p e c t r o ­ meter t o " s i m u l t a n e o u s l y p r o d u c e p o s i t i v e and n e g a t i v e i o n s p e c t r a , may a l s o be d e v e l o p e d f o r a c c u r a t e mass measurement i n GC/MS s t u d i e s (£2).

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

1 1

E l e m e n t a l c o m p o s i t i o n assignment from s p e c t r a r e c o r d e d a t i n c r e a s e d mass r e s o l u t i o n . I n t h i s c a s e , t h e aim i s t o c o n t i n u o u s l y r e c o r d complete mass s p e c t r a t h r o u g h o u t a c h r o m a t o g r a p h i c a n a l y s i s a t a s u f f i c i e n t l y h i g h mass r e s o l u t i o n t o e l i m i n a t e u n r e s o l v e d peaks and w i t h s u f ­ f i c i e n t mass measurement a c c u r a c y t o e x a c t l y d e t e r m i n e a l l o f t h e e l e m e n t a l c o m p o s i t i o n s p r e s e n t i n each spec­ trum. I n p r i n c i p l e , t h i s GC/HRMS method c a n be under­ t a k e n u s i n g mass s p e c t r o m e t e r s equipped f o r e i t h e r p h o t o p l a t e d e t e c t i o n (43-45) o r e l e c t r i c a l d e t e c t i o n (44-51, see a l s o 5 2 ) . I n p r a c t i c e , however, i t i s t h e l a t t e r method (which p a r a l l e l s n o m i n a l mass GC/MS o p e r a t i o n ) w h i c h has t h e p o t e n t i a l f o r p r o v i d i n g t h e maximum f l e x i b i l i t y and speed i n terms o f c o m p u t e r i z e d o p e r a t i o n , r e p e t i t i v e spectrum a c q u i s i t i o n , l a r g e spectrum c a p a c i t y , and t h e g e n e r a t i o n o f t o t a l i o n i z a ­ t i o n and mass chromatograms. I t i s t h i s GC/HRMS approach u t i l i z i n g magnetic s c a n n i n g and e l e c t r i c a l d e t e c t i o n w h i c h w i l l be d i s ­ c u s s e d more f u l l y h e r e . I t i s not intended t o describe d e t a i l s o f i n s t r u m e n t d e s i g n and s e t - u p p r o c e d u r e s , b u t r a t h e r t o present f o r the chemist/user the p r i n c i p a l p a r a m e t e r s i n v o l v e d , and t h e i r r e q u i r e m e n t s , l i m i t a ­ t i o n s and i n t e r - r e l a t i o n s h i p s ; i t i s t h i s knowledge t h a t becomes i m p o r t a n t i n p r a c t i c a l terms f o r t h e e f f e c t i v e and e f f i c i e n t i n t e r p r e t a t i o n o f GC/HRMS d a t a . Background The p r i m a r y parameters o f i n t e r e s t i n o b t a i n i n g e l e m e n t a l c o m p o s i t i o n s p e c t r a a r e mass measurement a c c u r a c y (a measure o f a b i l i t y t o a c c u r a t e l y d e t e r m i n e the t r u e mass) and dynamic mass r e s o l u t i o n ( t h e degree t o w h i c h masses c a n be r e s o l v e d under magnetic s c a n n i n g conditions). I n v e s t i g a t i o n o f t h e s e parameters de­ pends, o f c o u r s e , on t h e s p e c i f i c mode o f s p e c t r o m e t e r and computer o p e r a t i o n ; t h e d i s c u s s i o n h e r e i s based on d e v e l o p m e n t a l GC/HRMS s t u d i e s u n d e r t a k e n u s i n g a modi­ f i e d ΑΕΙ MS-902 d o u b l e - f o c u s s i n g mass s p e c t r o m e t e r t o g e t h e r w i t h o n - l i n e , r e a l - t i m e d a t a a c q u i s i t i o n and r e d u c t i o n (47-49, 5 3 ) .

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I n o u t l i n e , a spectrum i s produced by exponen­ t i a l l y s c a n n i n g over the mass range from h i g h mass t o low mass. The r e s u l t i n g a n a l o g s i g n a l from the de­ t e c t o r a m p l i f i e r i s d i g i t i z e d a t e q u a l time i n t e r v a l s and the d i g i t a l v a l u e s f o r each peak d e t e c t e d above a c o n s t a n t p r e s e t t h r e s h o l d v a l u e a r e saved f o r a n a l y s i s , t o g e t h e r w i t h the a b s o l u t e time o f o c c u r r e n c e o f t h e peak w i t h r e s p e c t t o the s t a r t o f the scan (54). The d i g i t a l v a l u e s f o r each peak d e f i n e the peak p r o f i l e , from w h i c h the peak i n t e n s i t y i s d e t e r m i n e d from i t s a r e a (5J5, the sum o f t h e d i g i t a l v a l u e s f o r t h a t peak) and the peak p o s i t i o n i n time i s d e t e r m i n e d from t h e t i m e computed f o r the peak c e n t e r - o f - g r a v i t y o r cent r o i d (54). Mass/time f u n c t i o n . In the i d e a l c a s e , t h e r e f o r e , t h e mass (M) o f any peak i s an e x p o n e n t i a l f u n c t i o n o f t h e e l a p s e d time ( t ) from the s t a r t o f the s c a n ( F i g u r e 1 ) . That i s , M « "

t / T

(1)

e

,f

where τ i s t h e t i m e c o n s t a n t " o f the e x p o n e n t i a l f u n c ­ t i o n , i . e . , t h e time t a k e n t o scan between any mass, M , and the mass M /e (where e=2.718). At the s t a r t o f the s c a n , t = 0 and M i s e q u a l t o the maximum mass value, M , so t h a t e q u a t i o n (1) becomes x

x

m a x

M = M

max

e"

t / x v

(2) J

S c a n r a t e . The s c a n r a t e i n t h e commonly quoted u n i t s o f seconds/mass decade (a mass decade c o v e r s the range Μ t o M /10) can be d e r i v e d from t h i s e q u a t i o n by con­ s i d e r i n g t h e t i m e d i f f e r e n c e ( t - t i ) f o r two masses Mi and Mi/10: χ

x

2

M

>

M

e

• max "

t l / T

a n d

M

'

-

M

max

β

'*

ΐ Λ

These e q u a t i o n s combine t o g i v e t -ti

= τ l o g 10 = τ (3) G 10 where τ i s the time t o scan one mass decade, i . e . , the s c a n r a t e i n seconds/decade. 2

1 0

R e s o l u t i o n . As n o r m a l l y d e f i n e d , the mass r e s o l u t i o n , R, a t 10% v a l l e y s e p a r a t i o n o f two e q u a l mass peaks i s g i v e n by

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Resolution Mass Spectrometry

= Μ/ΔΜ

(4)

where M i s t h e n o m i n a l mass and ΔΜ i s t h e a c c u r a t e mass d i f f e r e n c e f o r t h e two mass peaks ( F i g u r e 2 ) . The time d i f f e r e n c e , A t , between t h e maxima o f t h e s e two peaks can be d e t e r m i n e d from t h e r a t e o f change o f mass w i t h time (from e q u a t i o n 2) g i v i n g -M d

M

= —5**

. '

t / T

e

·dt

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

which approximates t o τ f o r s m a l l v a l u e s o f ΔΜ and A t . (2) g i v e s AM -At M ~T~

D i v i d i n g by e q u a t i o n

and u s i n g e q u a t i o n ( 4 ) , t h i s reduces t o At = " I

(5)

From symmetry c o n s i d e r a t i o n s , t h e time d i f f e r e n c e , A t , between t h e peak maxima i s e q u a l t o t h e time t a k e n t o scan a c r o s s one o f t h e s e peaks between t h e p o i n t s w h i c h a r e a t 5% o f t h e maximum peak h e i g h t ( F i g u r e 2 ) . Thus, i f τ and At a r e known, then t h e r e s o l u t i o n c a n be c a l c u l a t e d f o r each s i n g l e peak t h r o u g h o u t t h e mass range. A l s o , e q u a t i o n ( 5 ) shows t h a t f o r c o n s t a n t mass r e s o l u t i o n , R , and s c a n r a t e τ (=τ l o g 1 0 ) , t h e w i d t h s o f a l l s i n g l e t peaks measured a t 5 1 o f r h e i r maximum h e i g h t s h o u l d be c o n s t a n t . E q u a t i o n s 3 and 5 , combined t o g i v e 1 0

t =

- T

1

0

/ R

log

e

10 ,

(6)

p e r m i t v a r i o u s i n s t r u m e n t parameters t o be c a l c u l a t e d and compared w i t h e x p e r i m e n t a l r e s u l t s . F o r example, f o r a c o n s t a n t r e s o l u t i o n (10% v a l l e y ) o f 10,000 and a s c a n r a t e o f 6 seconds/decade, t h e p e a k w i d t h f o r a l l masses a t t h e i r 5% l e v e l s i s g i v e n by At =

s e c = 260.6 ysec ΙΟ* χ 2.3026

PERFORMANCE

MASS

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

HIGH

ρ _ M . M _ " M -M, ΔΜ ~ 2

Τ

At

Figure 2. Mass resolution (10% valley defi­ nition)

SPECTROMETRY

7.

KIMBLE

Gas Chromatography/High

Resolution Mass Spectrometry

127

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

For a d i g i t i z a t i o n r a t e o f 50 kHz, t h e d a t a p o i n t s a r e s e p a r a t e d i n time u n i t s o f 1/50,000 s e c o r 20 y s e c , so t h a t i n t h i s example, t h e w i d t h s o f a l l s i n g l e t peaks s h o u l d be d e f i n e d by 260/20 = 13 d a t a p o i n t s between t h e i r 5% l e v e l s . Mass measurement a c c u r a c y . I t i s t h e a b i l i t y t o mea­ sure masses i n a spectrum w i t h h i g h a c c u r a c y t h a t i s the b a s i s f o r d e t e r m i n i n g t h e c o r r e c t e l e m e n t a l com­ p o s i t i o n s . As p e r f e c t l y e x a c t mass measurements cannot be a c h i e v e d i n p r a c t i c e , t h e approach used t o g e n e r a t e a h i g h r e s o l u t i o n spectrum i s t o compute, f o r each measured mass, a l i s t o f p o s s i b l e e l e m e n t a l compo­ s i t i o n s (56) whose a c c u r a t e masses f a l l w i t h i n a s m a l l mass "wincfow" around each measured mass. I n o r d e r t o r e s t r i c t t h e e l e m e n t a l c o m p o s i t i o n l i s t i n g t o somewhat manageable p r o p o r t i o n s , t h e u s e r i s r e q u i r e d t o s e l e c t the t y p e s and maximum numbers o f each element t h a t he c o n s i d e r s r e l e v a n t on c h e m i c a l grounds. He i s a l s o r e q u i r e d t o i n p u t t h e " w i d t h " o f t h e mass window t o be used f o r t h e c a l c u l a t i o n s i . e . , t h e measured mass ± 6ppm, o r a mass window o f 26. The v a l u e o f 6 must be chosen c a r e f u l l y so t h a t the c o r r e c t e l e m e n t a l c o m p o s i t i o n f o r a p a r t i c u l a r measured mass i s i n c l u d e d i n t h e l i s t o f p o s s i b i l i t i e s . I f t h e chosen v a l u e o f 6 i s t o o s m a l l , t h e n t h e c o r r e c t c o m p o s i t i o n may n o t be l i s t e d ; however, i f t h e chosen v a l u e o f δ i s s i g n i f i c a n t l y l a r g e r than necessary, then the r e s u l t i n g l i s t o f c o m p o s i t i o n s r a p i d l y becomes t o o u n w i e l d y t o e v a l u a t e . The s e l e c t i o n o f t h e a p p r o p r i a t e v a l u e o f 6 f o r any p a r t i c u l a r s e t o f i n s t r u m e n t oper­ a t i n g parameters depends d i r e c t l y on a knowledge o f t h e mass measurement a c c u r a c y d e t e r m i n e d e x p e r i m e n t a l l y under t h o s e c o n d i t i o n s . The a t t a i n m e n t o f h i g h mass measurement a c c u r a c y i s dependent upon many f a c t o r s (57-60) such as adequate d e f i n i t i o n o f a c t u a l peak shapes, tTïë absence o f abnormal peakshapes, d e f i n i t i o n o f t h e a c t u a l mass/time f u n c t i o n , s o f t w a r e a l g o r i t h m s f o r mass c a l c u l a t i o n , and mass r e s o l u t i o n . The dependency on mass r e s o l u t i o n r e l a t e s t o t h e p r e s e n c e o f u n r e s o l v e d peak d o u b l e t s o r m u l t i p l e t s ( F i g u r e 3 ) ; f o r these peakshapes, t h e p o s i t i o n o f t h e c e n t e r - o f - g r a v i t y o f t h e o v e r a l l peak p r o f i l e i s i n a c c u r a t e , r e s u l t i n g i n i n a c c u r a t e mass measurements f o r t h e i o n s p e c i e s a c t u a l l y p r e s e n t . I n g e n e r a l , improved mass measurements c a n be o b t a i n e d by a v e r a g i n g t h e measurements from s e v e r a l s p e c t r a ( 6 1 , 6 2 ) . T h i s p r o c e s s r e d u c e s t h e e f f e c t o f t h e random e r r o r s a s s o c i a t e d w i t h a c c u r a t e mass d e t e r m i n a t i o n s , and, t h u s , a s m a l l e r mass window c a n be used i n t h e

128

HIGH

PERFORMANCE

MASS

SPECTROMETRY

generation o f p o s s i b l e elemental compositions f o r averaged mass measurements. S t a t i s t i c a l l y , the mass measurement a c c u r a c y would be e x p e c t e d t o be improved by a f a c t o r o f /n where η i s t h e number o f measurements averaged f o r a p a r t i c u l a r a c c u r a t e mass.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

P r a c t i c a l C o n s i d e r a t i o n s and t h e E v a l u a t i o n o f Instrument Performance? GC and f a s t - s c a n n i n g HRMS. I n p r a c t i c a l t e r m s , t h e a b i l i t y t o d e t e r m i n e a c c u r a t e mass measurements f o r s p e c t r a r e c o r d e d from c h r o m a t o g r a p h i c e f f l u e n t s c e n t e r s on the performance o f t h e f a s t - s c a n n i n g h i g h r e s o l u t i o n mass s p e c t r o m e t e r and i t s d a t a system (57, 63^-67). As f o r GC/LRMS, t h e gas chromatograph and GC/MS i n t e r f a c e may be o f v a r i o u s d e s i g n s p r o v i d e d t h a t : a) the i n c r e a s e d p r e s s u r e i n t h e i o n s o u r c e due t o the c a r r i e r gas p e r m i t s o p e r a t i o n o f the mass spectrometer ; b) each c h r o m a t o g r a p h i c peak i s o f s u f f i c i e n t d u r a t i o n so t h a t s p e c t r a w i l l be r e c o r d e d f o r each component; c) a s u f f i c i e n t amount o f each component i s i n t r o ­ duced v i a t h e GC i n l e t system i n t o t h e i o n s o u r c e so t h a t r e c o g n i z a b l e s p e c t r a c a n be recorded. As t h e s e d e s i g n c o n s i d e r a t i o n s have been t h e s u b j e c t o f many p u b l i c a t i o n s ( e . g . , 6 8 ) , o n l y the most i m p o r t a n t a s p e c t s f o r GC/HRMS w i l lïïe"d i s c u s s e d f u r t h e r h e r e . The main parameters f o r GC/HRMS o p e r a t i o n a r e summarized i n T a b l e 1; many o f t h e s e p a r a m e t e r s , o f c o u r s e , a l s o p e r t a i n t o GC/LRMS a n a l y s e s , b u t they become somewhat more c r i t i c a l f o r t h e c o n c o m i t a n t measurement o f a c c u r a t e masses under f a s t - s c a n n i n g h i g h r e s o l u t i o n c o n d i t i o n s . The t a b l e a l s o i n d i c a t e s t h e p r i m a r y optimum r e q u i r e m e n t s f o r each parameter. F o r s e v e r a l o f these, there are c o n f l i c t i n g requirements due t o t h e i n t e r - r e l a t i o n s h i p s t h a t e x i s t between t h e d i f f e r e n t p a r a m e t e r s , and t h e a c t u a l v a l u e s u t i l i z e d f o r a n a l y s e s w i l l n e c e s s a r i l y be a s e t o f v a l u e s t h a t a c h i e v e t h e optimum compromise. Scan c y c l e t i m e . The o v e r a l l c y c l e time f o r r e c o r d i n g a s i n g l e spectrum i s a f u n c t i o n o f the s c a n r a t e , t h e mass r a n g e , and t h e time r e q u i r e d t o r e s e t the magnetic f i e l d between t h e minimum and maximum mass v a l u e s . As d e s c r i b e d above, t h e t h e o r e t i c a l s c a n r a t e , τ , i s a c o n s t a n t and i s d i r e c t l y p r o p o r t i o n a l t o t h e time c o n s t a n t o f t h e e x p o n e n t i a l f u n c t i o n , τ. I n p r a c t i c e , 1 0

7.

KIMBLE

Gas Chromatography/High Resolution Mass Spectrometry

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

T a b l e 1,

129

P r i m a r y C o n s i d e r a t i o n s f o r GC/HRMS

Parameter

Requirement

mass measurement accuracy

maximize t o l i m i t number o f p o s s i b l e e l e m e n t a l comp o s i t i o n s generated

dynamic mass resolution

maximize t o reduce number o f u n r e s o l v e d p e a k s , hence i n c r e a s e mass measurement accuracy minimize t o increase sensitivity

sensitivity

maximize t o reduce sample quantity requirements

scan t i m e ( f u n c t i o n of mass range and s c a n r a t e )

m i n i m i z e scan time t o p r e s e r v e chromatographic r e s o l u t i o n i n t o t a l i o n i z a t i o n and mass chromatograms maximize mass range f o r u s e f u l information maximize s c a n r a t e (sec/decade) for increased s e n s i t i v i t y and mass measurement accuracy

magnet r e s e t t i m e

m i n i m i z e t o reduce "wasted" t i m e and p r e s e r v e chromatographic resolution i n t o t a l i o n i z a t i o n and mass chromatograms

GC peak e l u t i o n time

m i n i m i z e f o r h i g h e r chromatographic r e s o l u t i o n maximize w i t h r e s p e c t t o s c a n c y c l e time ( i . e . , s c a n time + magnet r e s e t time) to ensure s u f f i c i e n t time f o r r e c o r d i n g mass s p e c t r a

130

HIGH

PERFORMANCE

MASS SPECTROMETRY

the mass/time f u n c t i o n i s n o t p e r f e c t o v e r t h e whole mass range; t h a t i s , τ i s n o t c o n s t a n t b u t v a r i e s w i t h mass as i l l u s t r a t e d i n F i g u r e 4. The use o f an i n t e r ­ n a l mass s t a n d a r d such as p e r f l u o r o k e r o s i n e ( P F K ) , w h i c h c o n t r i b u t e s peaks o f known a c c u r a t e mass t h r o u g h ­ out t h e mass r a n g e , p r o v i d e s a means f o r d e t e r m i n i n g the a c t u a l mass/time f u n c t i o n over s m a l l s e c t i o n s o f the mass range. Thus, a v a l u e o f τ can be a s s i g n e d t o each o f t h e c a l i b r a t i o n masses, and from each o f t h e s e v a l u e s t h e measured s c a n r a t e , τ , i n seconds/decade can be c a l c u l a t e d from e q u a t i o n 3. The v a r i a t i o n o b s e r v e d f o r a n o m i n a l s c a n r a t e o f 6 seconds/decade f o l l o w s t h e p r o f i l e f o r τ shown i n F i g u r e 4, and ranges between about 6.5 seconds/decade a t m/e 580 and 5.5 seconds/decade a t m/e 80; t h e a c t u a l o v e r a l l s c a n t i m e between m/e 800 and m/e 65 i s about 7.2 seconds (48, 49). I t s h o u l d be n o t e d t h a t t h e upper s e c t i o n oF~~the mass range (~ m/e 800-650) i s n o t e a s i l y u s a b l e f o r ac­ c u r a t e mass a s s i g n m e n t s due t o t h e low i n t e n s i t y o f t h e PFK c a l i b r a t i o n masses i n t h i s r e g i o n , and t h e r a p i d r a t e o f change o f τ i n t h i s r e g i o n p r e c l u d e s a c c u r a t e e x t r a p o l a t i o n o f τ above t h e h i g h e s t r e l i a b l e c a l i ­ b r a t i o n mass. The t i m e t a k e n t o r e t u r n t o t h e i n i t i a l maximum mass v a l u e f o r t h e n e x t scan ( t h e magnet r e s e t t i m e ) s h o u l d , o f c o u r s e , be m i n i m i z e d as f a r as p o s s i b l e t o r e d u c e "wasted" t i m e and m i n i m i z e t h e o v e r a l l scan c y c l e t i m e . As r e p o r t e d p r e v i o u s l y (£8), magnet c i r ­ c u i t m o d i f i c a t i o n s u n d e r t a k e n on a MS-902 mass spec­ t r o m e t e r have p e r m i t t e d t h i s t i m e t o be r e d u c e d t o 2.4 seconds w h i l s t m a i n t a i n i n g mass c a l i b r a t i o n up t o - m/e 650 f o r t h e subsequent s c a n . W i t h t h e s e i n s t r u m e n t p a r a m e t e r s , t h e r e f o r e , t h e o v e r a l l scan c y c l e t i m e ( i . e . , s c a n t i m e + magnet r e s e t time) i s j u s t under 10 seconds.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

1 0

GC peak e i u t i o n t i m e . The v a l u e f o r t h e o v e r a l l scan c y c l e t i m e i s i m p o r t a n t f o r t h e a d j u s t m e n t o f t h e gas c h r o m a t o g r a p h i c o p e r a t i n g parameters ( e . g . , f l o w r a t e , column t e m p e r a t u r e and programming r a t e ) . In order to e n s u r e t h a t mass s p e c t r a can be r e c o r d e d f o r any GC peak, t h e t i m e f o r e i u t i o n o f a peak has t o be a d j u s t e d so t h a t i t i s a t l e a s t t w i c e t h e scan c y c l e t i m e . As i l l u s t r a t e d i n F i g u r e 5, t h e minimum f a c t o r o f two d e r i v e s from t h e unknown time r e l a t i o n s h i p between t h e s t a r t o f a s c a n and t h e commencement o f e i u t i o n o f a GC peak. I t i s n e c e s s a r y , t h e r e f o r e , i n t h i s c a s e , t o ensure t h a t a l l c h r o m a t o g r a p h i c peaks e l u t e i n t o t h e mass s p e c t r o m e t e r i o n s o u r c e over a time p e r i o d o f a t

KIMBLE

Gas Chromatography/High

Resolution Mass Spectrometry

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

Mass A

Centroid of Mass A

Figure 3. Effect of inadequate mass resolution on mass measurement ac­ curacy

Figure 4. The variation of τ as a function of mass

Figure 5. The extreme relationships between scan cycle time and GC peak eiution time; scan cycle time equals one-half of the GC peak eiution time

132

HIGH

PERFORMANCE

MASS

SPECTROMETRY

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

l e a s t 20 seconds so t h a t a t l e a s t one u s a b l e spectrum can be r e c o r d e d . The u s e o f a GC peak e i u t i o n time o f j u s t t w i c e the scan c y c l e time r e s u l t s i n mass chromatograms which show a v a r i e t y o f p r o f i l e s from t h e same GC peak ( s e e F i g u r e 6 ) . S i m i l a r f e a t u r e s may a l s o be seen w i t h t o t a l i o n i z a t i o n chromatograms, which c a n c o m p l i c a t e the matching o f such o u t p u t w i t h gas chromatograms r e c o r d e d by a flame i o n i z a t i o n d e t e c t o r ; q u a n t i t a t i v e comparisons a r e a l s o more d i f f i c u l t t o u n d e r t a k e w i t h such v a r i a t i o n s i n peak h e i g h t s . The scan c y c l e t i m e , t h e r e f o r e , d i r e c t l y l i m i t s t h e o v e r a l l gas chromato­ g r a p h i c performance t h a t c a n be u t i l i z e d e f f e c t i v e l y . Mass measurement a c c u r a c y . The a b i l i t y t o a c c u r a t e l y measure masses under GC/HRMS c o n d i t i o n s i s , o f c o u r s e , the o b j e c t o f t h e e x e r c i s e . However, i t i s t h e i n t r o ­ d u c t i o n o f t h e sample v i a a gas chromatograph t h a t p r e s e n t s major problems f o r d e t e r m i n i n g a c c u r a t e mas­ s e s . Because o f t h e s h o r t r e s i d e n c e time o f each component i n t h e i o n s o u r c e , a w e l l - d e f i n e d peak p r o ­ f i l e f o r each mass has t o be d e t e r m i n e d i n a v e r y s h o r t time (~ 260 psec a t a s c a n r a t e o f 6 seconds/decade and a r e s o l u t i o n o f 10,000). A l s o , o n l y one o r two r e a ­ s o n a b l y i n t e n s e s p e c t r a c a n be e x p e c t e d t o be r e c o r d e d from each component. T h i s v i r t u a l l y e l i m i n a t e s t h e a b i l i t y t o average the measured masses t o improve a c c u r a c y , and, t h e r e f o r e , i t i s n e c e s s a r y t h a t h i g h a c c u r a c y measurements s h o u l d be r e l i a b l y produced f o r each i n d i v i d u a l spectrum. The l a r g e volume o f d a t a g e n e r a t e d from a GC/HRMS a n a l y s i s a l s o demands a h i g h degree o f r e l i a b i l i t y as d e t a i l e d i n v e s t i g a t i o n s i n t o anomalous f e a t u r e s a r e e x t r e m e l y time-consuming. I n a d d i t i o n t o t h e g e n e r a t i o n o f e l e m e n t a l com­ p o s i t i o n l i s t i n g s f o r measured a c c u r a t e masses, a d e t a i l e d knowledge o f a t t a i n a b l e mass measurement a c c u r a c y i s a l s o i m p o r t a n t f o r GC/HRMS s t u d i e s i n o r d e r t o g e n e r a t e a c c u r a t e mass chromatograms ( o r e l e m e n t a l c o m p o s i t i o n chromatograms) t o l o c a t e t h e e i u t i o n times o f s p e c i f i c components o r compound c l a s s e s f o r f u r t h e r d e t a i l e d s t u d y (£7). I n t h i s c a s e , the u s e r s e l e c t s the e l e m e n t a l c o m p o s i t i o n o f i n t e r e s t and t h e c o r ­ r e s p o n d i n g a c c u r a t e mass i s c a l c u l a t e d . An a p p r o p r i a t e mass window (δ) i s s p e c i f i e d and, f o r each spectrum, the i n t e n s i t y o f t h e a c c u r a t e mass which i s l o c a t e d w i t h i n t h i s s p e c i f i e d mass window around t h e c a l c u l a t e d mass, i s saved. The i n t e n s i t i e s p l o t t e d as a f u n c t i o n o f s c a n number c o n s t i t u t e t h e e l e m e n t a l c o m p o s i t i o n o r a c c u r a t e mass chromatogram. Here a g a i n , s e l e c t i o n o f t h e s i z e o f the mass window depends on knowledge o f t h e

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

7.

KIMBLE

Gas Chromatography/High

Resolution Mass Spectrometry

133

mass measurement a c c u r a c y ; t h e c h o i c e i s c r i t i c a l t o ensure t h a t t h e c o r r e c t masses a r e l o c a t e d b u t t h a t " e x t r a n e o u s " masses a r e e x c l u d e d as f a r as p o s s i b l e . M e n t i o n s h o u l d a l s o be made o f t h e g e n e r a t i o n o f nomin a l mass s p e c t r a and chromatograms from a c c u r a t e mass data. Here t h e i n t e n s i t i e s o f a l l a c c u r a t e masses t h a t f a l l w i t h i n t h e mass range between (X.0000-0.4000) amu and (X.0000+0.6000) amu a r e summed t o g i v e t h e i n t e n s i t y o f n o m i n a l mass X. The mass measurement a c c u r a c y becomes i m p o r t a n t f o r t h e a c c u r a t e masses t h a t may l i e c l o s e t o t h e l i m i t s o f t h i s r a n g e , and cons i d e r a t i o n s h o u l d be g i v e n t o t h e c o n v e n t i o n s used f o r d e f i n i n g t h e n o m i n a l mass o f an i o n ( 6 9 ) . In v i e w o f a l l t h e s e b a s i c c o n s i d e r a t i o n s , i t i s e s s e n t i a l t h a t a d e t a i l e d e v a l u a t i o n o f a c c u r a t e mass measurement c a p a b i l i t y s h o u l d be u n d e r t a k e n f o r a g i v e n s e t o f o p e r a t i n g parameters t o ensure r e l i a b l e s i n g l e scan s p e c t r a and a c c u r a t e mass chromatograms. The p r a c t i c a l d e t e r m i n a t i o n o f mass measurement accuracy i s undertaken f o r a p a r t i c u l a r s e t o f i n s t r u ment o p e r a t i n g p a r a m e t e r s by r e c o r d i n g a number o f s p e c t r a o f a s e l e c t e d compound whose p r i n c i p a l masses are u n i q u e l y known. Because o f t h e c o n t i n u o u s v a r i a t i o n i n sample f l o w r a t e w h i c h r e s u l t s from samples i n t r o d u c e d v i a a gas chromatograph, t h e b a s i c e v a l u a t i o n i s made under GC/HRMS o p e r a t i n g c o n d i t i o n s ( i . e . , w i t h normal c a r r i e r gas f l o w r a t e , column temp e r a t u r e , e t c . ) , but w i t h i n t r o d u c t i o n o f the e v a l u a t i o n compound v i a a b a t c h i n l e t system a t c o n s t a n t sample f l o w r a t e . A c t u a l GC/HRMS e v a l u a t i o n c a n t h e n be checked by i n t r o d u c i n g a known compound v i a t h e gas c h r o m a t o g r a p h i c i n l e t and v e r i f y i n g t h a t t h e r e s u l t i n g mass measurements a r e c o n s i s t e n t w i t h t h e b a s i c e v a l u a t i o n f o r s i m i l a r masses o f s i m i l a r i n t e n s i t i e s . The compound s e l e c t e d f o r e v a l u a t i o n o f mass measurement a c c u r a c y s h o u l d produce i o n masses t h a t a r e s i n g l e t peaks w e l l - s e p a r a t e d from o t h e r masses a t t h e o p e r a t i n g mass r e s o l u t i o n . I d e a l l y , t h e y s h o u l d a l s o a d e q u a t e l y c o v e r t h e mass range o f i n t e r e s t , and s h o u l d be o f a p p r o x i m a t e l y t h e same r e l a t i v e i n t e n s i t i e s t o p e r m i t e v a l u a t i o n o f mass measurement a c c u r a c y as a f u n c t i o n o f b o t h mass and i n t e n s i t y . In p r a c t i c e , a compromise has t o be made w i t h r e s p e c t t o t h e s e l a t t e r r e q u i r e m e n t s , and a v a r i e t y o f compounds f o r t h i s purpose have been r e p o r t e d i n t h e l i t e r a t u r e ( e . g . , p e r c h l o r o b u t a d i e n e , £1, and h e p t a c o s a f l u o r o t r i b u t y l amine, 48,49). E v a l u a t i o n o f t h e measurements o b t a i n e d f o r t h e s e l e c t e d masses from s e v e r a l scans can be u n d e r t a k e n by a v a r i e t y o f r e l a t e d methods. One approach (61)

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described the production o f a histogram which p l o t s the p e r c e n t a g e o f t h e t o t a l number o f mass measurements as a f u n c t i o n o f t h e i r mass measurement e r r o r s (the d i f ­ f e r e n c e between t h e measured mass and t h e t r u e mass). T h i s method, however, s u f f e r s from t h e drawback t h a t o n l y a l i m i t e d number o f scans were e v a l u a t e d , and t h e r e s u l t i n g h i s t o g r a m was g e n e r a t e d from c o m p o s i t e e r r o r measurements from i o n s o f d i f f e r i n g masses and a l s o differing relative intensities. As GC/HRMS a n a l y s e s n e c e s s a r i l y i n v o l v e t h e p r o ­ d u c t i o n o f l a r g e numbers o f s p e c t r a and r e q u i r e a c o r r e s p o n d i n g computer c a p a b i l i t y , a more d e t a i l e d s t a t i s t i c a l e v a l u a t i o n c a n be u n d e r t a k e n i f a l a r g e number ( e . g . , > 100) o f s p e c t r a a r e r e c o r d e d and e v a l ­ uated automatically. I n t h i s approach (7£), f o r each s e l e c t e d a c c u r a t e mass, M , t o be e v a l u a t e d , t h e mea­ s u r e d mass i s l o c a t e d i n each spectrum w i t h i n an e s ­ t i m a t e d mass window around t h e t r u e mass, Mt. F o r each v a l u e o f M , s e v e r a l p a r a m e t e r s a r e c a l c u l a t e d from a l l of the recorded s p e c t r a : a) The number o f s p e c t r a (N) f o r w h i c h t h e measured mass was found w i t h i n t h e mass window. (The e s t i m a t e d mass window s h o u l d be chosen so t h a t i t r e p r e s e n t s t h e a b s o l u t e maximum mass measure ment e r r o r t h a n c a n be t o l e r a t e d f o r s p e c t r a l interpretation e.g., ±20 ppm. Thus, f o r a l l v a l u e s o f Μ^, Ν s h o u l d c o r r e s p o n d t o t h e number o f s p e c t r a r e c o r d e d ; i . e . , a l l o f t h e measure ments w i l l be w i t h i n 20 ppm o f t h e i r t r u e v a l ­ ue) . b) The mean mass e r r o r , E. ( T h i s may be p o s i t i v e or n e g a t i v e w i t h r e s p e c t t o M t ) . c) The s t a n d a r d d e v i a t i o n o f t h e mass measurement e r r o r s (σ ppm). d) The s t a n d a r d d e v i a t i o n o f t h e mean mass e r r o r (o = σ//Ν). e) Mean peak i n t e n s i t y . f ) S t a n d a r d d e v i a t i o n o f t h e i n t e n s i t y measure­ ments. (For a d d i t i o n a l e v a l u a t i o n o f i n s t r u m e n t p e r f o r m a n c e , mean and s t a n d a r d d e v i a t i o n v a l u e s a r e a l s o c a l c u l a t e d f o r a v a r i e t y o f o t h e r p a r a m e t e r s a s s o c i a t e d w i t h each s e l e c t e d mass; t h e s e i n c l u d e τ, W. , W., , R, as ex­ p l a i n e d below). * These v a l u e s p e r m i t a more d e t a i l e d e v a l u a t i o n o f the r e l a t i v e e f f e c t s o f t h e s y s t e m a t i c and random e r r o r s a s s o c i a t e d w i t h mass measurement a c c u r a c y . I n the absence o f s y s t e m a t i c e r r o r s , t h e mean e r r o r Ε s h o u l d approach zero ( i . e . , t h e mean measured mass s h o u l d e q u a l t h e t r u e mass), and t h e r e i s a 99.78% t

t

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0

b

z

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chance t h a t t h e mean mass w i l l be w i t h i n 3 o o f t h e t r u e v a l u e . A l s o , f o r a normal d i s t r i b u t i o n , 99.78% o f t h e measurements f o r each mass s h o u l d f a l l w i t h i n 3σ o f the mean measured v a l u e , i . e . , v i r t u a l l y a l l o f t h e measurements s h o u l d f a l l w i t h i n a s p r e a d o f 6σ ppm. A s u i t a b l e c h o i c e f o r d e t e r m i n i n g t h e mass window (2δ) f o r e l e m e n t a l c o m p o s i t i o n l i s t i n g s and chromatograms would r e s u l t , t h e r e f o r e , from u s i n g a δ v a l u e e q u a l t o the sum o f t h e maximum v a l u e o f 3σ f o r t h e masses c o n s i d e r e d , and t h e maximum a b s o l u t e v a l u e o f Ε f o r t h e same masses (see F i g u r e 7 ) . The use o f t h e 3σ v a l u e s ( c o r r e s p o n d i n g t o 99.78% of t h e measurements f o r a normal d i s t r i b u t i o n ) r a t h e r t h a n 2σ v a l u e s (95% o f t h e measurements) becomes im­ p o r t a n t f o r GC/HRMS a n a l y s e s because t h e l a r g e number of s p e c t r a ( s e v e r a l hundred, each p o s s i b l y c o n t a i n i n g s e v e r a l hundred a c c u r a t e m a s s e s ) , t h a t a r e g e n e r a l l y r e c o r d e d n e c e s s i t a t e s a h i g h degree o f r e l i a b i l i t y as w e l l as a c c u r a c y . I n g e n e r a l , t h e number o f e l e m e n t a l c o m p o s i t i o n s computed f o r a t y p i c a l measured mass (M) becomes unmanageable f o r a mass window l a r g e r t h a n about M ± 15 ppm, p a r t i c u l a r l y f o r more complex mole­ c u l e s t h a t may c o n t a i n many t y p e s o f atoms ( e . g . , C, C , H, N, 0, S i , F ) . Large mass measurement e r r o r s are a l s o p r o b l e m a t i c f o r the g e n e r a t i o n o f elemental c o m p o s i t i o n chromatograms s i n c e t h e mass measurement d i s t r i b u t i o n (spread) may o v e r l a p w i t h t h e d i s t r i b u t i o n of measured masses f o r a n o t h e r e l e m e n t a l c o m p o s i t i o n p r e s e n t i n some o f t h e s p e c t r a . Thus, i n t e n s i t i e s o f e x t r a n e o u s masses may be i n c l u d e d i n t h e f i n a l o u t p u t even though t h e " c o r r e c t " mass window has been s e l e c ­ ted, and t h i s would r e s u l t i n one o r more a r t i f a c t peaks i n t h e e l e m e n t a l c o m p o s i t i o n chromatogram. I n p r a c t i c a l terms f o r s p e c t r a l i n t e r p r e t a t i o n , i t i s a l s o i m p o r t a n t t o know t h e minimum a b s o l u t e mass i n t e n s i t y v a l u e f o r w h i c h t h e d e s i r e d mass measurement a c c u r a c y can be a s s u r e d ; d a t a o u t p u t c o n t a i n i n g l o w e r i n t e n s i t y v a l u e s c a n t h e n be e v a l u a t e d w i t h a p p r o p r i a t e c a u t i o n . F i n a l l y , i t i s i m p o r t a n t t o remember t h a t t h e c o n s i d e r a t i o n o f mass measurement a c c u r a c y r e l a t e s o n l y to w e l l - d e f i n e d normal peak shapes. Any s i g n i f i c a n t d i s t o r t i o n o f such peak shapes, such as u n r e s o l v e d d o u b l e t s , s h o u l d be r e c o g n i z e d and t h e e f f e c t on mass measurement a c c u r a c y t a k e n i n t o a c c o u n t .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

m

1 3

Sensitivity. I n GC/HRMS, an e v a l u a t i o n o f o v e r a l l s y s ­ tem s e n s i t i v i t y must c o n s i d e r n o t o n l y t h e d e t e c t i o n o f s p e c i f i e d masses from a g i v e n q u a n t i t y o f a component i n j e c t e d i n t o t h e gas chromatograph (as f o r GC/LRMS), but a l s o t h e mass measurement a c c u r a c y t h a t c a n be

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

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Mass Chromatoçram

30 NUMBER

Figure 6. Representation of an accurate mass chromatogram for equal GC peak shapes; scan cycle time equals one-half of the GC peak eiution time

Figure 7. The evaluation of mass measurement accuracy; M = true mass, M = mean measured mass, Ε = mean error, σ = standard deviation of the measured masses. t

m

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o b t a i n e d f o r t h e s e masses. T h i s more s t r i n g e n t c r i ­ t e r i o n r e s u l t s i n somewhat l e s s s e n s i t i v i t y compared t o d e t e c t i n g t h e p r e s e n c e o f a p a r t i c u l a r mass, s i n c e t h e peak p r o f i l e s must be s u f f i c i e n t l y w e l l - d e f i n e d t h a t the peak c e n t e r - o f - g r a v i t y o r c e n t r o i d can be a c c u ­ r a t e l y and r e l i a b l y d e t e r m i n e d . S e v e r a l d i s c u s s i o n s have appeared i n t h e l i t e r a ­ t u r e c o n c e r n i n g the t h e o r e t i c a l a s p e c t s o f s e n s i t i v i t y and mass measurement a c c u r a c y (57_, 7 1 ) . B a s i c a l l y , t h e l i m i t i n g f a c t o r concerns the s t a t i s t i c s a s s o c i a t e d w i t h the i o n a r r i v a l times a t the d e t e c t o r , and t h i s d e t e r ­ mines how w e l l the r e s u l t i n g d i g i t i z e d peakshape i s d e f i n e d ; t h e f r e q u e n c y o f d i g i t i z a t i o n o r the number o f d a t a p o i n t s / m a s s peak i s a l s o a c o n s i d e r a t i o n here ( 7 2 ) . I t has been p r e d i c t e d (71) t h a t f o r a mass r e s o l u t i o n o f 10,000, mass measurement a c c u r a c i e s w i t h i n 10 ppm ( = 3σ, o r 99.78% c o n f i d e n c e l e v e l ) s h o u l d be a c h i e v e d w i t h 30 i o n s / p e a k , and w i t h i n 17 ppm (=3σ) f o r 10 i o n s / p e a k . E s t i m a t e s made from a c t u a l mass measure­ ments (73) i n d i c a t e t h a t 10-15 i o n s / p e a k a r e r e q u i r e d t o g i v e a mass measurement a c c u r a c y o f 30 ppm (=3σ). The r e l a t i o n s h i p between t h e a b s o l u t e peak i n t e n s i t y , I (the sum o f t h e d i g i t a l v a l u e s f o r a peak p r o f i l e ) , and the number o f i o n s , N, c o m p r i s i n g t h a t peak may be d e t e r m i n e d (61) from the e q u a t i o n I . Ν "

r

ÎRef

where G i s t h e g a i n o f t h e m u l t i p l i e r the primary v a r i a b l e under normal o p e r a t i n g c o n d i t i o n s . (the other parameters a r e : R = input r e s i s t a n c e of the m u l t i p l i e r a m p l i f i e r i n ohms, e = charge on an e l e c t r o n i n cou­ lombs, f = f r e q u e n c y o f d i g i t i z a t i o n i n s e c " , ν = v o l t a g e i n c r e m e n t r e p r e s e n t e d by one b i n a r y " b i t " on the A/D c o n v e r t e r i n v o l t s ) . As w i t h GC/LRMS, i t i s i m p o r t a n t t h a t t h e o v e r a l l s e n s i t i v i t y i s d e t e r m i n e d f o r a s e l e c t e d component i n t r o d u c e d t h r o u g h t h e gas chromatograph, p a r t i c u l a r l y t o t a k e i n t o a c c o u n t any l o s s e s t h a t may o c c u r when using a molecular separator i n t e r f a c e . I t i s also n e c e s s a r y f o r GC/HRMS t h a t one o r more i o n masses ( e . g . , basepeak and/or m o l e c u l a r i o n , e t c . ) a r e s e ­ l e c t e d i n o r d e r t o s p e c i f y the mass measurement ac­ c u r a c y t h a t can be o b t a i n e d from an i n j e c t i o n o f a g i v e n amount o f the compound. In a d d i t i o n , the l i m i t e d number o f h i g h r e s o l u t i o n s p e c t r a t h a t can be r e c o r d e d f o r any p a r t i c u l a r GC peak under GC/HRMS c o n d i t i o n s a l s o has t o be c o n s i d e r e d . In t h e l i m i t i n g c a s e , when 1

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the scan c y c l e time ( e . g . , -10 seconds) i s about oneh a l f o f t h e GC peak e i u t i o n t i m e , t h e maximum r e c o r d e d i n t e n s i t y f o r any mass may be e x p e c t e d t o v a r y by a f a c t o r o f about two (see F i g u r e 6 ) , depending on t h e time o f s t a r t i n g a scan i n r e l a t i o n t o t h e time when the GC peak b e g i n s t o e l u t e a r e l a t i o n s h i p which i s unknown i n p r a c t i c e . F o r an i n j e c t i o n o f Ν ng o f t h e s e l e c t e d component, t h e maximum sample f l o w r a t e a t t h e top o f t h e GC peak c o r r e s p o n d s t o about N/10 ng/sec f o r a GC peak e i u t i o n time o f 20 seconds. Measured mass i n t e n s i t i e s , t h e r e f o r e , w i l l be p r o p o r t i o n a l t o sample f l o w r a t e s between about N/20 and N/10 ng/sec. I f the v a r i a t i o n o f mass measurement a c c u r a c y w i t h r e s p e c t t o a range o f measured i n t e n s i t i e s has been d e t e r m i n e d under t h e same c o n d i t i o n s , t h e n an assessment can be made f o r t h e mass measurement a c c u r a c y e x p e c t e d f o r s e l e c t e d masses r e c o r d e d from an i n j e c t i o n o f Ν ng under a g i v e n s e t o f e x p e r i m e n t a l c o n d i t i o n s ( i . e . , s c a n r a t e , mass r e s o l u t i o n , m u l t i p l i e r g a i n , e t c . ) . Dynamic mass r e s o l u t i o n . A knowledge o f t h e a c t u a l mass r e s o l u t i o n o f each r e c o r d e d peak i s an i m p o r t a n t measure o f t h e o v e r a l l p e r f o r m a n c e o f a f a s t - s c a n n i n g h i g h r e s o l u t i o n mass s p e c t r o m e t e r , and d i r e c t l y r e l a t e s t o t h e a b i l i t y t o a c c u r a t e l y measure masses. A l s o , i t i s f r e q u e n t l y n e c e s s a r y t o be a b l e t o d e t e r m i n e whether o r n o t c e r t a i n mass d o u b l e t s would be e x p e c t e d t o be r e s o l v e d under t h e i n s t r u m e n t c o n d i t i o n s used. As d i s c u s s e d i n t h e s e c t i o n above on mass r e ­ s o l u t i o n , t h e mass r e s o l u t i o n (10% v a l l e y d e f i n i t i o n ) o f any peak c a n be d e t e r m i n e d from i t s w i d t h a t 5% maximum peak h e i g h t (W51) and τ ( e q u a t i o n 6 ) , where b o t h W51 and τ a r e measured i n t h e same u n i t s ( e . g . , seconds o r number o f d a t a p o i n t s ) . The a c t u a l v a r i a ­ t i o n o f τ t h r o u g h o u t t h e mass range (see F i g u r e 4) means t h a t t h e r e s o l u t i o n c a l c u l a t i o n f o r a p a r t i c u l a r peak s h o u l d i n c o r p o r a t e t h e a p p r o p r i a t e v a l u e o f τ f o r t h e mass peak c o n s i d e r e d ; t h i s v a l u e i s i n t e r p o l a t e d f o r each peak d u r i n g t h e mass assignment p r o c e s s based on t h e v a l u e s c a l c u l a t e d f o r t h e s u r r o u n d i n g c a l i ­ b r a t i o n masses. The d e t e r m i n a t i o n o f t h e a c t u a l peak w i d t h ¥5% f o r any peak i s somewhat more i n v o l v e d as i t cannot be d i r e c t l y measured from t h e s e t o f d i g i t a l v a l u e s t h a t c o n s t i t u t e a peak p r o f i l e . F o r i d e a l (Gaussian) peakshapes, however, i t i s p o s s i b l e t o c a l c u l a t e in terms o f t h e peak a r e a , A ( t h e sum o f t h e d i g i t a l v a l u e s f o r t h a t peak p r o f i l e ) , and t h e maximum peak h e i g h t , Η ( t h e maximum d i g i t a l v a l u e f o r t h e peak profile). From t h e g e n e r a l e q u a t i o n f o r a G a u s s i a n

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f u n c t i o n , t h e w i d t h a t 5% o f t h e maximum peak ( i . e . , a t H/20) c a n be shown t o be g i v e n by W

height

= 2σ y/1 log 2Ô = 4.8955 σ

5%

139

(7)

e

where σ i s t h e s t a n d a r d d e v i a t i o n o f t h e f u n c t i o n . a r e a A under t h i s c u r v e i s g i v e n by

The

A = Ησ/27 where Η i s t h e maximum peak h e i g h t , so t h a t , e l i m i n a ­ t i n g σ gives Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

W

5I

ïïj

=

ï

l o

Se

2 0

1

- '

9 5 3

ff

( c f . W51 = 1.90 A/H from c o n s i d e r a t i o n o f a t r i a n g u l a r peakshape). Therefore, the r e s o l u t i o n ( i . e . , 10% v a l l e y d e f i n i t i o n ) i s g i v e n by R

5%

=

i t

=

=

? *

( 9 )

(where τ i s measured i n number o f d a t a p o i n t s ) . Preliminary evaluation of this c a l c u l a t i o n using a c t u a l d a t a (70) i n d i c a t e s t h a t f o r w e l l - d e f i n e d normal peak shapes, t F e v a l u e s c a l c u l a t e d f o r W51 a r e a p p r o x i ­ m a t e l y c o n s t a n t , as would be e x p e c t e d , and a l s o c o r r e s ­ pond t o v a l u e s measured d i r e c t l y from p l o t s o f i n d i v i ­ d u a l peak p r o f i l e s . In p r a c t i c e , t h e u t i l i t y o f t h e c a l c u l a t i o n o f R5^ i s l i m i t e d t o t h e e v a l u a t i o n o f peak p r o f i l e s . T h i s i s because each peak i s r e c o r d e d as a s e r i e s o f d i g i t a l v a l u e s t h a t exceed a c o n s t a n t p r e s e t t h r e s h o l d w h i c h i s chosen t o e l i m i n a t e " n o i s e " b u t t o maximize the o v e r a l l dynamic range. I f t h e d i g i t a l v a l u e s f o r two c l o s e mass peaks do n o t c u t t h e t h r e s h o l d v a l u e a t the v a l l e y ( F i g u r e 8 a ) , t h e n t h e p r o f i l e w i l l be r e c o g ­ n i z e d as t h e p r o f i l e o f a s i n g l e peak and t h e r e s u l t i n g c a l c u l a t i o n s w i l l be i n e r r o r . I n o r d e r t h a t t h e p r o f i l e be r e c o g n i s e d as two d i s t i n c t p e a k s , t h e maxima f o r e q u a l - s i z e d peaks must be s e p a r a t e d by W^h "the w i d t h o f each peak measured a t t h e t h r e s h o l d ( F i g u r e 8b). This t h r e s h o l d peakwidth i s e a s i l y determined from t h e number o f d a t a p o i n t s r e c o r d e d f o r each peak, and t h i s number c a n be used t o c a l c u l a t e a " t h r e s h o l d r e s o l u t i o n " , R , f o r each mass peak from t h e e q u a t i o n tV|

R = th R

τ

(10)

PERFORMANCE

MASS

SPECTROMETRY

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

HIGH

Figure 8. (a) Peak profile for doublet separated with resolution R = t/W (10% valley definition); (b) Peak profiles for peaks resolved at threshold, R< = T / W . 5 %

5%

A

Î A

7.

KIMBLE

Gas Chromatography/High

141

Resolution Mass Spectrometry

In terms o f t h e i n t e r p r e t a t i o n o f a c t u a l d a t a , t h e t h r e s h o l d o r " e f f e c t i v e " r e s o l u t i o n w i l l more a c c u ­ r a t e l y r e f l e c t the o v e r a l l instrumental a b i l i t y to r e s o l v e c l o s e l y p o s i t i o n e d mass peaks and thus t o s u b s e q u e n t l y d e t e r m i n e t h e i r a c c u r a t e masses and e l e ­ mental c o m p o s i t i o n s . The d e c r e a s e i n e f f e c t i v e r e s o l u t i o n due t o t h e use o f t h e t h r e s h o l d p e a k w i d t h f o r t h e c a l c u l a t i o n r a t h e r than Ws%, can be i l l u s t r a t e d by assuming t h a t the t h r e s h o l d p e a k w i d t h f o r a G a u s s i a n peakshape i s ap­ p r o x i m a t e d by

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

W

« 6σ

th

(11)

where 6σ i s t h e base w i d t h w h i c h d e f i n e s 99.781 o f t h e peak a r e a . T h e r e f o r e , from e q u a t i o n s (7) and ( 1 1 ) , W , i s g i v e n by W

th

• ;nw5T 5* - · * w

1

2

W

w

5%

C a l c u l a t e d v a l u e s f o r Rs%, W^-h and R^h a r e shown i n T a b l e 2 f o r t h e case o f a n o m i n a l s c a n r a t e o f 6 seconds/decade and a n o m i n a l r e s o l u t i o n (Rs%) o f 10,000; c o r r e s p o n d i n g v a l u e s a r e a l s o g i v e n f o r t h e range o f τ e n c o u n t e r e d f o r t h i s n o m i n a l scan r a t e . These c a l c u l a t i o n s demonstrate a s i g n i f i c a n t d e c r e a s e i n e f f e c t i v e r e s o l u t i o n f o r W^h 6σ, a d e c r e a s e o f 18.41 compared t o t h e r e s o l u t i o n a t 5% maximum peak h e i g h t , and a d e c r e a s e o f up t o 25% compared t o t h e supposed r e s o l u t i o n o f 10,000. F u r t h e r d e c r e a s e s i n e f f e c t i v e r e s o l u t i o n a r e t o be e x p e c t e d f o r l a r g e peaks s i n c e a p e a k w i d t h o f 6σ f o r a G a u s s i a n peakshape c o r ­ responds t o t h e w i d t h a t 1.11% o f t h e maximum peak h e i g h t ; f o r l a r g e peaks, t h e a c t u a l t h r e s h o l d w i l l be s i g n i f i c a n t l y below 1.11% o f maximum peak h e i g h t , and the t h r e s h o l d p e a k w i d t h w i l l thus be l a r g e r t h a n 6σ. =

T a b l e 2.

Scanrate, τ

1 0 >

Comparison o f r e s o l u t i o n v a l u e s c a l c u l a t e d from p e a k w i d t h a t 5% o f maximum p e a k h e i g h t (10% v a l l e y d e f i n i t i o n ) and p e a k w i d t h a t 1.11% o f maximum p e a k h e i g h t . τ, sec.

sec/decade

W

W

th 5% =4.8955σ, =6σ, psec ysec

R

5%

R

th

5.5

2. 389

260.6

319.4

9,167

7,479

6.0

2. 606 2. 823

260.6 260.6

319.4 319.4

10,000 10,833

8,159 8,838

6. 5

142

HIGH

PERFORMANCE

MASS

SPECTROMETRY

In a d d i t i o n t o t h e a c t u a l d e t e r m i n a t i o n o f t h e e f f e c t i v e r e s o l u t i o n f o r each r e c o r d e d peak, t h e s e c a l c u l a t i o n s c a n be u t i l i z e d t o i d e n t i f y "abnormal" peakshapes t h a t c a n o c c u r when o p e r a t i n g a h i g h r e s o l u t i o n mass s p e c t r o m e t e r under GC/HRMS c o n d i t i o n s (60, 7 4 ) . A v a r i e t y o f such peak shapes c a n be r a p i d l y i d e n t i f i e d by comparison o f t h e c a l c u l a t e d p e a k w i d t h a t 5% h e i g h t (W51) and t h e measured t h r e s h o l d p e a k w i d t h (W h); abnormal peakshapes i n c l u d e : s m a l l " n o i s y " peaks, f l a t - t o p p e d ( " s a t u r a t e d " ) p e a k s , t a i l i n g p e a k s , and u n r e s o l v e d d o u b l e t s o r m u l t i p l e t s . A l l o f t h e s e peakshapes w i l l a d v e r s e l y a f f e c t t h e a c c u r a t e d e t e r m i n a t i o n o f t h e peak c e n t e r - o f - g r a v i t y and hence the measurement o f a c c u r a t e mass. The e f f e c t o f abnormal peak shapes on t h e q u a l i t y o f a c t u a l d a t a o u t p u t i s i l l u s t r a t e d i n F i g u r e 9. The p l o t shows t h e a c c u r a t e mass chromatogram f o r m/e 119.0861 ( = C H ) from a GC/HRMS a n a l y s i s o f t h e neut r a l f r a c t i o n o f an e x t r a c t o f p e t r o l e u m r e f i n e r y w a s t e w a t e r ( 7 5 ) . The c o m p l e x i t y o f t h i s f r a c t i o n i s e v i d e n c e d by t h e u n r e s o l v e d "hump" w h i c h i s a l s o p r e s e n t i n the f l a m e i o n i z a t i o n gas chromatogram and many o f t h e o t h e r mass chromatograms g e n e r a t e d from t h e GC/HRMS d a t a . N o t a b l e i n t h e chromatogram shown a r e the two a b r u p t zero i n t e n s i t y v a l u e s l o c a t e d a t scan numbers 96 and 143, i n d i c a t i n g t h a t no a c c u r a t e masses were l o c a t e d i n t h e s e scans w i t h i n the mass range m/e 119.0861 ± 20ppm, i . e . , c o r r e s p o n d i n g t o t h e e l e m e n t a l composition C 9 H 1 1 . D e t a i l e d i n v e s t i g a t i o n o f t h e s e two scans r e v e a l e d t h a t C 9 H 1 1 i o n s were i n d e e d p r e s e n t i n the s p e c t r a and t h a t t h e a b s o l u t e i n t e n s i t i e s were c o n s i s t e n t w i t h the o v e r a l l p r o f i l e observed i n t h e a c c u r a t e mass chromatogram. P l o t s o f t h e a p p r o p r i a t e peak p r o f i l e s , a l s o shown i n F i g u r e 9, i l l u s t r a t e t h e abnormal peak p r o f i l e s t h a t were a c t u a l l y r e c o r d e d ; t h e v e r t i c a l l i n e s denote the p o s i t i o n s o f t h e c a l c u l a t e d c e n t e r s - o f - g r a v i t y f o r t h e two p r o f i l e s . I n b o t h cases the c a l c u l a t e d c e n t e r - o f - g r a v i t y was s h i f t e d s u f f i c i e n t l y so t h a t t h e c o r r e s p o n d i n g a c c u r a t e mass was o u t s i d e the mass window used t o g e n e r a t e t h e chromatogram. Both o f t h e s e peak, p r o f i l e s would be e a s i l y r e c o g n i z e d as b e i n g abnormal by comparison o f t h e i r area/maximum h e i g h t r a t i o s ( g i v i n g t h e c a l c u l a t e d W51 v a l u e s ) and the measured t h r e s h o l d p e a k w i d t h s , Wth. In b o t h c a s e s , t h e a v a i l a b i l i t y o f s o f t w a r e a l g o r i t h m s f o r d e t e c t i n g p r o f i l e minima and r e t h r e s h o l d i n g would e f f e c t i v e l y overcome t h e e r r o r s i n t r o d u c e d by t h e s e u n r e s o l v e d peak p r o f i l e s .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

t

9

n

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

7.

Gas Chromatography/High

KIMBLE

τι·

12993.

0

10

sun η/ε»

20

30

143

Resolution Mass Spectrometry

us.i

40

90

t0

70

80

90

100

110

120

130

140

190

190

C,H 0 T

C.H„â

2754B9.5 24591 113.06 817 60

10

275934.9 10231 119. OB 520 60

10

SCAN 143

SCAN 96

Identification and Analysis of Organic Pollutants in Water

Figure 9. Accurate mass chromatogram for m/e 119.0861 (=C H ). ted from scans 96 and 143. 9

tl

Peak profiles plot-

144

HIGH

PERFORMANCE

MASS

SPECTROMETRY

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

Conclusions I t has been t h e i n t e n t i o n o f t h i s c h a p t e r t o summarize f o r the chemist/user the primary aspects o f GC/HRMS which need t o be c o n s i d e r e d when u n d e r t a k i n g e v a l u a t i o n and i n t e r p r e t a t i o n o f such d a t a . The degree o f d a t a e v a l u a t i o n n e c e s s a r y i n h i g h r e s o l u t i o n mass s p e c t r o m e t r y i s s i g n i f i c a n t l y g r e a t e r than f o r l o w r e s o l u t i o n s t u d i e s . Any e v a l u a t i o n must i n c l u d e mass measurement a c c u r a c y and i t s dependency on mass and peak i n t e n s i t y , dynamic r e s o l u t i o n f o r each mass peak, and t h e many p o s s i b l e e l e m e n t a l c o m p o s i t i o n s g e n e r a t e d f o r each mass peak. The d i f f i c u l t y i s compounded i n GC/HRMS because o f t h e l a r g e amounts o f d a t a t h a t a r e g e n e r a t e d d u r i n g each a n a l y s i s ( e . g . , 15-25 d a t a p o i n t s / mass peak, 500-1500 mass peaks/spectrum, 300-600 spectra/analysis). F o r t h e s e r e a s o n s , the p r i m a r y u t i l i t y o f GC/HRMS i s f o r complex samples whose components cannot be d e t e r m i n e d by i n t e r p r e t a t i o n o f t h e i r nominal mass s p e c t r a . Even w i t h t h e p r o d u c t i o n o f guaranteed h i g h q u a l i t y d a t a , a f l e x i b l e i n t e r a c t i v e and u s e r - o r i e n t e d s o f t w a r e system i s e s s e n t i a l i n o r d e r t o f u l l y u t i l i z e the a v a i l a b l e i n f o r m a t i o n w i t h i n a r e a s o n a b l e t i m e frame. Such s o f t w a r e c o n s i d e r a t i o n s i n c l u d e t h e a b i l i ty to r a p i d l y review elemental composition l i s t i n g s t h a t a r e l i m i t e d by u s e r - s p e c i f i e d c r i t e r i a , such as l i s t i n g s f o r s i n g l e masses, mass d i f f e r e n c e s , l i m i t e d mass r a n g e s , and f o r masses h a v i n g i n t e n s i t i e s above a s p e c i f i e d a b s o l u t e o r r e l a t i v e v a l u e . The a b i l i t y t o i n c l u d e f u n c t i o n a l groups o f e l e m e n t a l c o m p o s i t i o n s ( e . g . , C H , C O 2 C H 3 , O S 1 C 3 H 9 , e t c . ) as i n p u t f o r generating elemental composition l i s t i n g s i s also a s i g n i f i c a n t a s s i s t a n c e i n data i n t e r p r e t a t i o n (76). In p a r t i c u l a r , s o f t w a r e a l g o r i t h m s which u t i l i z e c h e m i c a l c o n c e p t s t o l i m i t t h e number o f c o m p o s i t i o n a l poss i b i l i t i e s g e n e r a t e d f o r each mass (26, 77-79) a r e n e c e s s a r y t o speed-up t h e i n t e r p r e t a t i o n p r o c e s s and a s s i s t i n the generation of a s e l f - c o n s i s t e n t set o f e l e m e n t a l c o m p o s i t i o n s f o r each h i g h r e s o l u t i o n spectrum o f i n t e r e s t . The u s e o f e l e m e n t a l c o m p o s i t i o n s g e n e r a t e d f o r a c c u r a t e mass d i f f e r e n c e s i s a l s o u s e f u l i n t h i s context, s i n c e the elemental compositional p o s s i b i l i t i e s f o r a r e l a t i v e l y s m a l l mass d i f f e r e n c e a r e s u b s t a n t i a l l y fewer than f o r t h e a c c u r a t e masses themselves. As f o r GC/LRMS, t h e a u t o m a t i c i d e n t i f i c a t i o n o f t h e s p e c t r a o f i n t e r e s t would a l s o s i g n i f i c a n t l y speed-up t h e o v e r a l l d a t a i n t e r p r e t a t i o n p r o c e s s . A l g o r i t h m s such as t h a t d e s c r i b e d by B i l l e r and Biemann (15) s h o u l d be implementable f o r r e a s o n a b l y 6

5

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

7.

KIMBLE

Gas Chromatography/ High Resolution Mass Spectrometry

145

i n t e n s e , a c c u r a t e l y measured masses; however, more complex a l g o r i t h m s (17) would r e q u i r e t h e a b i l i t y t o r e c o r d a s u b s t a n t i a l number o f s p e c t r a t h r o u g h o u t t h e t i m e o f e i u t i o n o f a gas c h r o m a t o g r a p h i c peak. In e s s e n c e , t h e r e f o r e , t h e r o u t i n e u t i l i t y o f t h e GC/HRMS t e c h n i q u e demands an e x t e n s i v e s o f t w a r e capa­ b i l i t y f o r the r a p i d e v a l u a t i o n of instrument p e r f o r ­ mance, f o r t h e p r o d u c t i o n o f r e l i a b l e h i g h - q u a l i t y d a t a , and f o r t h e e f f i c i e n t h a n d l i n g o f t h e d e t a i l e d information contained i n the elemental composition s p e c t r a r e c o r d e d o f gas c h r o m a t o g r a p h i c e f f l u e n t s . I n p a r t i c u l a r , t h e r o u t i n e use o f such s o f t w a r e f o r i n ­ strument e v a l u a t i o n and performance m o n i t o r i n g i s v i t a l i f t h e p o t e n t i a l o f t h e GC/HRMS t e c h n i q u e i s t o be e x p l o i t e d i n a m e a n i n g f u l way.

REFERENCES 1.

Novotny, Μ., and Zlatkis, Α., Chromatographic Reviews, (1971), 14, 1. 2. Grob, Κ., and Grob, K., J r . , J . Chromatog., (1974), 94, 53, and references therein. 3. Grob, K., and Grob, G., p. 75 in "Identification and Analysis of Organic Pollutants in Water", ed. L.H. Keith; Ann Arbor Science Publishers, Ann Arbor, Michigan, 1976. 4. Henderson, W., and Steel, G., Anal. Chem., (1972), 44, 2302. 5. Grob, K., and Jaeggi, Η., Anal. Chem., (1973), 45, 1788. 6. Leferink, J. G., and Leclercq, P. Α., J. Chroma­ tog., (1974), 91, 385. 7. Henneberg, D., Henrichs, U . , and Schomburg, G., Chromatographia, (1975), 8, 449. 8. Schoengold, D. Μ., and Munson, B., Anal. Chem., (1970), 42, 1811. 9. Arsenault, G. P., Dolhun, J. J., and Biemann, K., Anal. Chem., (1971), 43, 1720. 10. Arsenault, G. P., p. 817 in "Biochemical Appli­ cations of Mass Spectrometry", ed. G. R. Waller; Wiley-Interscience, 1972. 11. Blum, W., and Richter, W. J., Tetrahedron Letters, (1973), 835. 12. Fentiman, A. F., Foltz, R. L . , and Kinzer, G. W., Anal. Chem., (1973), 45, 580. 13. Jelus, B., Munson, Β., and Fenselau, C., Anal. Chem., (1974), 46, 729. 14. Ryhage, R., Anal. Chem. (1976), 48, 1829. 15. Biller, J. E., and Biemann, Κ., Anal. Letters, (1974), 7, 515.

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16. Nau, Η., and Biemann, Κ., Anal. Chem., (1974), 46, 426. 17. Dromey, R. G., Stefik, M. J., Rindfleisch, T. C., and Duffield, Α. Μ., Anal. Chem., (1976), 48, 1368. 18. Mathews, R. J., and Morrison, J . D., Aust. J. Chem., (1974), 27, 2167, and references therein. 19. Sweeley, C. C., Young, N. D., Holland, J. F., and Gates, S. C., J . Chromatog., (1974), 99, 507. 20. Abramson, F. P., Anal. Chem., (1975), 47, 45. 21. Mellon, F. Α., p. 117 in "Mass Spectrometry, Volume 3", ed. R. A. W. Johnstone; Specialist Periodical Reports, The Chemical Society, London, 1975. 22. Pesyna, G. M., Venkataraghavan, R., Dayringer, H. Ε., and McLafferty, F. W., Anal. Chem., (1976), 48, 1362. 23. Smith, D. H., Yeager, W. J., Anderson, P. J., Fitch, W. L . , Rindfleisch, T. C., and Achenbach, M., Anal. Chem., (1977), 49, 1623. 24. Lederberg, J., p. 193 in "Biochemical Applications of Mass Spectrometry", ed. G. R. Waller; Wiley­ -Inter science, 1972. 25. Kwok, K.-S., Venkataraghavan, R., and McLafferty, F. W., J . Am. Chem. Soc., (1973), 95, 4185. 26. Dromey, R. G., Buchanan, B. G., Smith, D. H., Lederberg, J., and Djerassi, C., J. Org. Chem., (1975), 40, 770. 27. Beynon, J . Η., in Adv. Mass Spectrom., Volume 1, ed. J . D. Waldron; Pergamon Press, NY, 1959. 28. Millington, D. S., J. Steroid Biochem., (1975), 6, 239. 29. Millington, D. S., Buoy, Μ. Ε., Brooks, G., Harper, M. E., and Griffiths, Κ., Biomed. Mass Spectrom., (1975), 2,219. 30. Telling, G. M., Bruce, Τ. Α., and Althorpe, J., J. Agr. Food Chem., (1971), 19, 937. 31. Gough, Τ. Α., and Webb, K. S., J. Chromatog., (1972), 64, 201, and (1973), 79, 57. 32. Crathorne, B., Edwards, M. W., Jones, N. R., Walters, C. L . , and Woolford, G., J . Chromatog., (1975), 115, 213. 33. Sen, N. P., Miles, W. F., Seaman, S., and Law­ rence, J. F., J . Chromatog., (1976), 128, 169. 34. Gallegos, E. J., Anal. Chem., (1975), 47, 1150. 35. Evans, K. P., Mathias, Α., Mellor, Ν., Silvester, R., and Williams, Α. Ε., Anal. Chem., (1975), 47, 821.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

7.

KIMBLE

Gas Chromatography/High

Resolution Mass Spectrometry

147

36. Prater, T. J., Harvey, Τ. Μ., and Schuetzle, D., Twenty-Fourth Annual Conference on Mass Spec­ trometry and Allied Topics, San Diego, 1976, p. 625. 37. Hammar, C.-G., and Hessling, R., Anal. Chem., (1971), 43, 299. 38. Haddon, W. F., and Lukens, H. C., Twenty-Second Annual Conference on Mass Spectrometry and Allied Topics, Philadelphia, 1974, p. 436. 39. Barber, Μ., Chapman, J. R., Green, Β. N . , Merren, T. O., and Riddoch, Α., Eighteenth Annual Con­ ference on Mass Spectrometry and Allied Topics, San Francisco, 1970, p. B299. 40. Chapman, J . R., Merren, T. O., and Limming, H. J., Nineteenth Annual Conference on Mass Spectrometry and Allied Topics, Atlanta, 1971, p. 218. 41. Wolstenholme, W. Α., and E l l i o t t , R. Μ., American Laboratory, (1975), Nov., 68. 42. Hunt, D. F., Stafford G. C., Shabanowitz, J., and Crow, F. W., Anal. Chem., (1977), 49, 1884; see also chapter by D. F. Hunt, this volume. 43. Watson, J. D., and Biemann, Κ., Anal. Chem., (1965), 37, 844. 44. Habfast, Κ., Maurer, Κ. Η., and Hoffmann, G., Twentieth Annual Conference on Mass Spectrometry and Allied Topics, Dallas, 1972, p. 414. 45. Habfast, K., Maurer, Κ. H., and Hoffmann, G., p. 408 in "Techniques of Combined Gas Chromatogra­ phy/Mass Spectrometry," ed. W. H. McFadden; John Wiley and Sons, N.Y., 1973. 46. McMurray, W. J., Green, Β. Ν., and Lipsky, S. R., Anal. Chem., (1966), 38, 1194. 47. Kimble, B. J., Cox, R. E., McPherron, R. V., Olsen, R. W., Roitman, Ε., Walls, F. C., and Burlingame, A. L . , J . Chromatog. S c i . , (1974), 12, 647. 48. Kimble, B. J., Walls, F. C., Olsen, R. W., and Burlingame, A. L . , Twenty-Third Annual Conference on Mass Spectrometry and Allied Topics, Houston, 1975, p. 503. 49. Kimble, B. J., McPherron, R. V., Olsen, R. W., Walls, F. C., and Burlingame, A. L . , Adv. Mass Spectrom., Volume 7, in press; Proceedings of the Seventh International Mass Spectrometry Confer­ ence, Florence, Italy, August 1976. 50. Rapp, U . , Schröder, U . , Meier, S., and Elmenhorst, H., Chromatographia, (1975), 8, 474.

148

51. 52. 53. 54.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

55. 56. 57. 58. 59. 60. 61.

62. 63. 64. 65. 66.

HIGH PERFORMANCE MASS SPECTROMETRY

Dobberstein, P., Zune, Α., and Wolstenholme, W. A., Twenty-Fourth Annual Conference on Mass Spec­ trometry and Allied Topics, San Diego, 1976, p. 234. Chapman, J. R., Evans, S., and Holmes, J. M., Sixteenth Annual Conference on Mass Spectrometry and Allied Topics, Pittsburgh, 1968, p. 313. Burlingame, A. L . , Olsen, R. W., and McPherron, R. V., p. 1053 in Adv. Mass Spectrom., Volume 6, ed. A. R. West; Applied Science, 1974. Smith, D. H., Olsen, R. W., Walls, F. C., and Burlingame, A. L . , Anal. Chem., (1971), 43, 1796. Burlingame, A. L . , Smith, D. Η., and Olsen, R. W., Anal. Chem., (1968), 40, 13. Burlingame, A. L . , p. 15 in Adv. Mass Spectrom., Volume 4, ed. E. Kendrick; The Institute of Petro­ leum, London, 1968. McMurray, M. J., p. 43 in "Mass Spectrometry: Techniques and Applications", ed. G. W. Milne; Wiley-Interscience, 1971. Klimowski, R. J., Venkataraghavan, R., and McLaf­ ferty, F. W., Org. Mass Spectrom., (1970), 4, 17. Boettger, H. G., and Kelly, Α. Μ., Seventeenth Annual Conference on Mass Spectrometry and Allied Topics, Dallas, 1969, p. 337. Banner, Α. Ε., Thirteenth Annual Conference on Mass Spectrometry and Allied Topics, St. Louis, 1965, p. 193. Burlingame, A. L . , Smith, D. H., Merren, T. O., and Olsen, R. W., p. 17 in "Computers in Analy­ tical Chemistry", ed. C. H. Orr and J. A. Norris; Progress in Analytical Chemistry, Vol. 4, Plenum Press, N.Y., 1969. Venkataraghavan, R., Klimowski, R. J., and McLaf­ ferty, F. W., Accounts Chem. Res., (1970), 3, 158. Merritt, C., Issenberg, P., Bazinet, M. L . , Green, Β. N . , Merren, T. O., and Murray, J. G., Anal. Chem., (1965), 37, 1037. Habfast, K., p. 3 in Adv. Mass Spectrom., Volume 4, ed. E. Kendrick; The Institute of Petroleum, London, 1968. McMurray, M. J., Lipsky, S. R., and Green, Β. N . , p. 77 in Adv. Mass Spectrom., Volume 4, ed. E. Kendrick; The Institute of Petroleum, London, 1968. Bowen, H. C., Clayton, E., Shields, D. J., and Stanier, Η. Μ., p. 257 in Adv. Mass Spectrom., Volume 4, ed. E. Kendrick; The Institute of Petro­ leum, London, 1968.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch007

7.

KIMBLE

Gas Chromatography/ High Resolution Mass Spectrometry 149

67. Hilmer, R. Μ., and Taylor, J. W., Anal. Chem., (1973), 45, 1031. 68. McFadden, W. Η., "Techniques of Combined Gas Chromatography/Mass Spectrometry"; Wiley-Inter­ -science, Ν.Υ., 1973. 69. Fales, Η. Μ., Heller, S. R., Milne, G. W. Α., and Sun, T., Biomed. Mass Spectrom., (1974), 1, 295. 70. Kimble, B. J., and McPherron, R. V., unpublished work. 71. Campbell, A. J., and Halliday, J. S., Thirteenth Annual Conference on Mass Spectrometry and Allied Topics, St. Louis, 1965, p. 200. 72. Halliday, J . S., p. 239 in Adv. Mass Spectrom., Volume 4, ed. E. Kendrick; The Institute of Petro­ leum, London, 1968. 73. Green, Β. Ν., Merren, T. O., and Murray, J. G., Thirteenth Annual Conference on Mass Spectrometry and Allied Topics, St. Louis, 1965, p. 204. 74. Barber, M., Green, Β. Ν., McMurray, W. J., and Lipsky, S. R . , Sixteenth Annual Conference on Mass Spectrometry and Allied Topics, Pittsburgh, 1968, p. 91. 75. Burlingame, A. L . , Kimble, B. J., Scott, E. S., Wilson, D. M., and Stasch, M. J., p. 587 in "Iden­ tification and Analysis of Organic Pollutants in Water", ed. L. H. Keith; Ann Arbor Science Pub­ lishers, Ann Arbor, Michigan, 1976. 76. Kundred, Α., Spencer, R. B., and Budde, W. L . , Anal. Chem., (1971), 43, 1086. 77. Biemann, K., and McMurray, W., Tetrahedron Let­ ters, (1965), 647. 78. Hilmer, R. Μ., and Taylor, J . W., Anal. Chem., (1974), 46, 1038. 79. Venkataraghavan, R . , McLafferty, F. W., and Van Lear, G. E., Org. Mass Spectrom., (1969), 2, 1. RECEIVED December 30, 1977

8 Analytical Applications of Postive and Negative Ion Chemical Ionization Mass Spectrometry DONALD F. HUNT and SATINDER K. SETHI

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch008

Department of Chemistry, University of Virginia, Charlottesville, VA 22901

As early as 1916 Dempster (1) observed an ion at m/e=3, which was correctly identified as H3 . By 1925 it was well established that this ion was produced by a secondary process resulting from c o l l i s i o n between ion (H+2) and neutral species ( H ) in the mass spectrometer ion source. Studies of such ion molecule c o l l i s i o n s were largely neglected u n t i l 1952, when interest was revived by the observation of the ion, CH+5 formed by the reaction, (2) +

2

.

CH+4 + C H —-> CH+5 + CH.3. 4

The b i r t h of Chemical Ionization Mass Spectrometry (CIMS) took place when Field (3) and Munson (3a) realized that an ion such asCH+5could ionize sample molecules by transferring a proton to them in the gas-phase. Such an ionization process is t o t a l l y different from ionization of a molecule by removal of an electron, as is done in most other mass spectrometric methods. Here it is a chemical reaction between the primary ion (reagent ion) and the sample molecule which is responsible for ionization of the sample. It is possible to control both the energetics of sample ion formation as well as the type of structural information obtained in the resulting mass spectrum. Different CI reagents undergo different ion-molecule reactions with the same sample molecule, and each ion-molecule reaction affords different structural information about the sample in question. Ion molecule reactions have been developed to identify different organic functional groups and to differentiate, primary, secondary and t e r t i a r y alcohols (4), 1 ° , 2 ° . and 3° amines (5), c y c l i c alkanes from ©0-8412-0422-5/78/47-070-150$10.00/0

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch008

8.

HUNT

A N D SETHI

Positive and Negative Ion Chemical Ionization

151

o l e f i n s ( 4 ) , s u l p h u r c o n t a i n i n g a r o m a t i c s from nons u l p h u r c o n t a i n i n g a r o m a t i c s , (6) and even i n some cases the o x i d a t i o n s t a t e o f the heteroatom i n polya r o m a t i c h y d r o c a r b o n s (7) . R e c e n t l y t h e r e have been a t t e m p t s t o d i s t i n g u i s h s t e r e o i s o m e r s i n t h e gas-phase u s i n g o p t i c a l l y a c t i v e r e a g e n t gases ( 8 ) . In a d d i t i o n t o an e f f i c i e n t t e c h n i q u e f o r t h e p r o d u c t i o n o f a wide v a r i e t y o f p o s i t i v e i o n s , CI i s a l s o an e x c e l l e n t method f o r g e n e r a t i n g n e g a t i v e l y charged sample i o n s . Due t o t h e l a r g e c o n c e n t r a t i o n o f t h e r m a l o r near t h e r m a l energy e l e c t r o n s , produced d u r i n g i o n i z a t i o n o f t h e CI r e a g e n t gas, t h e r e s o n a n c e e l e c t r o n c a p t u r e mechanism o p e r a t e s e f f i c i e n t l y t o produce l a r g e c o n c e n t r a t i o n s o f n e g a t i v e sample i o n s under CI c o n d i t i o n s . A d e s c r i p t i o n o f o u r i n i t i a l r e s e a r c h e f f o r t i n n e g a t i v e i o n CI w i l l be p r e s e n t e d l a t e r i n the chapter. P a r t i c u l a r l y noteworthy i s the development o f methodology w h i c h f a c i l i t a t e s s i m u l ­ taneous d e t e c t i o n o f b o t h p o s i t i v e and n e g a t i v e i o n s on a q u a d r u p o l e mass s p e c t r o m e t e r (6). In a d d i t i o n t o t h e above work we have a l s o r e ­ c e n t l y d e v e l o p e d methodology f o r o b t a i n i n g CI mass s p e c t r a o f n o n v o l a t i l e s a l t s and t h e r m a l l y l a b i l e m o l e c u l e s , under CI c o n d i t i o n s u s i n g q u a d r u p o l e i n ­ s t r u m e n t s w i t h f i e l d d e s o r p t i o n e m i t t e r s as s o l i d probes b u t i n t h e absence o f an e x t e r n a l l y a p p l i e d f i e l d (9). We have a l s o demonstrated t h a t a c c u r a t e mass measurements ( NHÎ

ΔΗ = -127 K c a l / m o l e ; P.A.(Crit) - 127 ΔΗ = -207 K c a l / m o l e ;

show t h a t r e a c t i o n w i t h Ht w i l l

P.A. (NHt) = 207

produce p r o t o n a t e d

154

HIGH

PERFORMANCE

MASS

SPECTROMETRY

sample i o n , [ M + H ] , h a v i n g 26 Kcal/mole more energy than those g e n e r a t e d by p r o t o n t r a n s f e r from CH+. Due t o v e r y h i g h p r o t o n a f f i n i t y o f ammonia, the NHi* w i l l o n l y t r a n s f e r a p r o t o n t o m o l e c u l e s w h i c h a r e more b a s i c t h a n ammonia. A c c o r d i n g l y , the NH^ i o n f i n d s u t i l i t y as a r e a g e n t f o r s e l e c t i v e l y i o n i z i n g b a s i c components i n a m i x t u r e o f o r g a n i c compounds. In many c a s e s e x t e n s i v e f r a g m e n t a t i o n o f the sample i s d e s i r a b l e i n o r d e r t o o b t a i n as much s t r u c t u r a l i n f o r m a t i o n as p o s s i b l e . F r a g m e n t a t i o n under EI i s due t o h i g h i n t e r n a l energy o f the m o l e c u l a r i o n a l t h o u g h the f r e e r a d i c a l c h a r a c t e r o f Mt lowers a c t i v a t i o n energy f o r many o t h e r w i s e i n a c c e s s i b l e decomp o s i t i o n pathways. F o r t u n a t e l y E l - t y p e s p e c t r a can be o b t a i n e d under CI c o n d i t i o n s by u s i n g p o w e r f u l one e l e c t r o n o x i d i z i n g agents l i k e N j * . The n i t r o g e n r a d i c a l c a t i o n formed by e l e c t r o n impact on N2 gas a t 1 torr

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch008

+

N* + e(80eV)

-> N *

+ N* + e

AB + NÎ'

•+ A B '

AB + N*

+ AB

2

+

+ e

+ N

2

+ N

2

(1) ΔΗ - -(2-8)eV

+ e

(2)

ΔΗ - -(0-3)eV

(3)

can t r a n s f e r 2-8eV o f energy t o the sample m o l e c u l e (AB) d u r i n g the i o n i z a t i o n s t e p ( E q - 2 ) . Extensive f r a g m e n t a t i o n o f the r e s u l t i n g Mt i o n r e s u l t s and a spectrum i d e n t i c a l t o t h a t produced by EI methodology i s o b t a i n e d . M e t a s t a b l e (N*) can a l s o i o n i z e the sample as shown i n ( E q - 3 ) . S e l e c t i v e Reagent Gases f o r P o s i t i v e Ion CIMS Argon-Water : When an argon-water m i x t u r e i s employed as the CI r e a g e n t gas, the s p e c t r a o b t a i n e d e x h i b i t f e a t u r e s c h a r a c t e r i s t i c o f b o t h c o n v e n t i o n a l EI and B r o n s t e d a c i d CI s p e c t r a (T4) . Use o f t h i s reagent gas m i x t u r e i s p a r t i c u l a r l y v a l u a b l e when an i o n c h a r a c ­ t e r i s t i c o f the sample m o l e c u l a r weight and abundant fragment i o n s c h a r a c t e r i s t i c o f m o l e c u l a r s t r u c t u r e a r e b o t h r e q u i r e d t o s o l v e the a n a l y t i c a l problem a t hand. E l e c t r o n bombardment o f A r / H 0 (20/1) a t 1 t o r r produces i o n s a t m/e 4 0 ( A r ) , 8 0 ( A r J ) , and 1 9 ( H 0 ) as w e l l as a p o p u l a t i o n o f m e t a s t a b l e argon n e u t r a l s (Ar*). P r o t o n t r a n s f e r from H 0 t o a sample m o l e c u l e i s u s u a l l y o n l y s l i g h t l y e x o t h e r m i c and seldom r e s u l t s i n e x t e n s i v e f r a g m e n t a t i o n o f the r e s u l t i n g M+l i o n . In c o n t r a s t e l e c t r o n t r a n s f e r from sample t o A r i s h i g h l y e x o t h e r m i c (4-6eV) and p r o d u c e s from the sample 2

+

+

3

+

3

+

8.

HUNT

Positive and Negative Ion Chemical Ionization

A N D SETHI

155

an energy r i c h r a d i c a l c a t i o n w h i c h s u f f e r s fragment a t i o n t o produce a E l - t y p e s p e c t r u m . The a b i l i t y t o r e c o r d b o t h E I - and C l - t y p e s p e c t r a i n a s i n g l e s c a n i s p a r t i c u l a r l y u s e f u l when t h e maximum s t r u c t u r a l i n f o r m a t i o n p o s s i b l e i s d e s i r e d and t h e q u a n t i t y o f sample a v a i l a b l e f o r analysis i s only s u f f i c i e n t f o r a s i n g l e e x p e r i m e n t . A m i x t u r e o f n i t r o g e n and w a t e r a f f o r d s s p e c t r a i d e n t i c a l t o t h o s e o b t a i n e d w i t h argon and w a t e r as t h e CI r e a g e n t . F o r t h e purpose o f c o m p a r i son, c o n v e n t i o n a l EI and C I ( A r - H 0 ) s p e c t r a o f d i - n p e n t y l a m i n e a r e shown i n F i g u r e 1. R e a c t i o n o f t h e amine w i t h H 0 a f f o r d s a s i n g l e i o n [ M + l ] . I n cont r a s t t h e EI spectrum d i s p l a y s a r e l a t i v e l y weak molecular ion. 2

+

+

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch008

3

Deuterium o x i d e : When D 0 i s employed as t h e CI r e a g e n t , a l l a c t i v e hydrogens a t t a c h e d t o N,S, o r 0 atoms i n an o r g a n i c sample undergo exchange d u r i n g t h e l i f e time o f t h e sample i n t h e i o n s o u r c e o f t h e mas s spectrometer. A r o m a t i c hydrogens have a l s o been shown t o undergo exchange {IS). I f t h e mol. wt. o f t h e sample i s a l r e a d y known from p r e v i o u s C l f C H O s p e c t r a , t h e number o f a c t i v e hydrogens i n t h e m o l e c u l e can be c o u n t e d by i n s p e c t i o n o f t h e mol. wt. r e g i o n o f t h e CI (D 0) spectrum. D i f f e r e n t i a t i o n o f 1°, 2°, and 3° amines i s e a s i l y a c c o m p l i s h e d i n t h i s manner (5>) . I n the CI ( D 0 ) spectrum o f 6 - k e t o e s t r a d i o l ( F i g u r e 2 ) , the M+l peak o b s e r v e d i n t h e w a t e r CI s p e c t r a a t m/e=287 i s s h i f t e d t o m/e=290. T h i s l a t t e r i o n c o r responds t o d - k e t o e s t r a c T i o l + D and r e s u l t s from exchange o f t h e two a c t i v e hydrogen atoms i n t h e d i o l f o l l o w e d by d e u t e r a t i o n . 2

#

2

2

+

2

Ammonia E l e c t r o n bombardment o f ammonia g e n e r a t e s NHÎ a l o n g w i t h ( N H ) H and (NH ) H+. These i o n s f u n c t i o n as weak B r o n s t e d a c i d s and w i l l o n l y p r o t o n a t e s t r o n g l y b a s i c s u b s t a n c e s l i k e amides ( 1 6 ) , amines (17) , and some a , 3 - u n s a t u r a t e d k e t o n e s (Γ8Γ) . The r e s u l t i n g sample i o n s seldom undergo f r a g m e n t a t i o n because o f t h e low e x o t h e r m i c i t y a s s o c i a t e d w i t h p r o t o n t r a n s f e r r e a c t i o n s ( F i g u r e 3 a ) . A l d e h y d e s , k e t o n e s , e s t e r s , and a c i d s , w h i c h a r e n o t s u f f i c i e n t l y b a s i c tô a c c e p t a p r o t o n from NH+, show i o n s i n C I ( N H ) s p e c t r a r e s u l t i n g from t h e e l e c t r o p h i l i c attachment o f NH^ t o t h e molec u l e CFigure 3b) ( 1 9 ) . A n o t h e r i n t e r e s t i n g a s p e c t o f CI (NH ) r e s e a r c h i s the f i n d i n g t h a t ammonia can be employed as a r e a g e n t gas f o r t h e d i r e c t a n a l y s i s o f o r g a n i c s i n w a t e r . The +

3

2

3

3

3

3

HIGH

100*

1 El

sort 30,

44

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch008

SPECTROMETRY

43. 58

70.

IOO-f

it!

MASS

1UDT

MWI57

ο CI

PERFORMANCE

,157 *f 158

43

50i

44 301

1561

158, 20

60

"V·

100

—ι— 140

170

Figure 1. EI and CI (N +H 0) mass spectra of di-n-pentylamine. The intensity of reagent ions is 50 to 100 times greater than as shown. 2

2

IOC

Analytical Chemistry

Figure 2. CI (H 0) and CI (D 0) mass spectra of 6-ketoestradiol. The inten­ sity of reagent ions is 50 to 100 times greater than as shown. 2

2

8.

HUNT

A N D SETHI

157

Positive and Negative Ion Chemical Ionization

ammonium i o n i s n o t s u f f i c i e n t l y a c i d i c t o p r o t o n a t e w a t e r . A c c o r d i n g l y , o r g a n i c s i n water c a n be s e l e c t i v e l y i o n i z e d when ammonia i s employed as t h e CI reagent gas. The NH+ i o n c a n a l s o be used as s t e r e o c h e m i c a l probe o f o r g a n i c s t r u c t u r e s . S i m p l e a l c o h o l s a r e n o t i o n i z e d under CI (NH ) c o n d i t i o n s . I n c o n t r a s t , d i o l s i n w h i c h t h e two h y d r o x y l groups c a n s i m u l t a n e o u s l y form i n t r a m o l e c u l a r hydrogen bonds t o t h e NH+ i o n a r e i o n i z e d (20). D i f f e r e n t i a t i o n o f t r a n s d i a x i a l d i o l s from t h e 3 T e q u a t o r i a l o r a x i a l - e q u a t o r i a l isomers i s e a s i l y a c c o m p l i s h e d by CI (NH ) mass s p e c t r o m e t r y (7) (Figure 3c). L i k e ammonia, methylamine, i s a l s o a u s e f u l r e agent gas. Aldehydes and k e t o n e s r e a c t w i t h CH NH i n t h e CI s o u r c e t o form p r o t o n a t e d S c h i f f bases ( 2 1 ) . The r e a c t i o n i s q u i t e s e n s i t i v e t o t h e s t e r i c e n v i r o n ment o f t h e c a r b o n y l group ( 7 ) . 3

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch008

3

3

3

N i t r i c O x i d e : N i t r i c o x i d e i s one o f t h e most v e r s a t i l e r e a g e n t gases f o r p o s i t i v e i o n CIMS. E l e c t r o n bombardment o f n i t r i c o x i d e a f f o r d s NO w h i c h f u n c t i o n s as an e l e c t r o p h i l e , h y d r i d e a b s t r a c t o r , and one e l e c t r o n o x i d i z i n g agent toward o r g a n i c samples. Depending on t h e type o f o r g a n i c f u n c t i o n a l groups p r e s e n t , any o r a l l o f t h e above r e a c t i o n s may be o b s e r v e d . We f i n d t h a t n i t r i c o x i d e CI s p e c t r a a r e p a r t i c u l a r l y u s e f u l f o r i d e n t i f y i n g organic f u n c t i o n a l groups i n sample m o l e c u l e s , f o r d i f f e r e n t i a t i n g o l e f i n s from c y c l o a l k a n e s , and f o r f i n g e r p r i n t i n g h y d r o c a r b o n mixtures. Of p a r t i c u l a r i n t e r e s t i s t h e f i n d i n g t h a t CI (NO) s p e c t r a c a n be employed t o d i f f e r e n t i a t e p r i mary, s e c o n d a r y , and t e r t i a r y a l c o h o l s . N i t r i c o x i d e CI s p e c t r a o f t e r t i a r y a l c o h o l s c o n t a i n o n l y ( M - 1 7 ) i o n s formed by a b s t r a c t i o n o f t h e h y d r o x y l group t o form n i t r o u s a c i d . S p e c t r a o f s e c o n d a r y a l c o h o l s e x h i b i t three i o n s ; ( M - l ) , which corresponds t o a p r o t o n a t e d k e t o n e ; ( M - 1 7 ) ; and (M-2+30) . The l a t t e r i o n i s g e n e r a t e d by t h e o x i d a t i o n o f t h e a l c o h o l f o l lowed by a d d i t i o n o f N 0 t o t h e r e s u l t i n g k e t o n e . Spectra o f primary alcohols also e x h i b i t ions corr e s p o n d i n g t o ( M - l ) and (M-2+30) . I n a d d i t i o n , however, an i o n , ( M - l ) , u n i q u e f o r p r i m a r y a l c o h o l s i s o b s e r v e d . T h i s i o n i s p r o d u c e d by h y d r i d e a b s t r a c t i o n from C i o f t h e a l d e h y d e formed on o x i d a t i o n o f t h e p r i m a r y a l c o h o l . F i g u r e s 4 a , b, c show CI(NO) s p e c t r a of three isomers o f pentanol. +

+

+

+

+

+

+

+

+

158

HIGH

Scheme:

PERFORMANCE

MASS

SPECTROMETRY

NO* as a S e l e c t i v e CI Reagent.

TERTIARY ALCOHOLS: +

(R) C-OH

R C (M-17)

3

3

SECONDARY ALCOHOLS: +

(R) CH-0H - ^ - * ( R ) C - 0 H — K R ) aC=0 i i ^ ( R ) C 0 * N 0 (M-l) (M-2+30) +

2

2

2

+

-*(R) CH

+

2

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch008

(M-17)

+

PRIMARY ALCOHOLS: +

RCH -0H —

^RC H-OH

2

(M-l)

^RCH=0 —

^ΗΟ···ΝΟ

+

(M-2+30)

+

+

+

->RC =0 (M-3)

+

D i f f e r e n t i a t i o n o f o l e f i n s and c y c l o a l k a n e s h a v i n g the same M. W. i s a l s o e a s i l y a c c o m p l i s h e d u s i n g n i t r i c o x i d e as r e a g e n t . Spectra of cycloalkanes e x h i b i t only an [ M - l ] i o n s whereas those o f o l e f i n s c o n t a i n b o t h [M-l]+ and [M+30] i o n s ( 1 9 ) . The l a t t e r s p e c i e s r e s u l t s from e l e c t r o p h i l i c a d d i t i o n o f N 0 t o t h e double bond ( F i g u r e 4d, e ) . CI(NO) s p e c t r a o f h y d r o ­ carbons c l o s e l y resemble those o b t a i n e d under f i e l d i o n i z a t i o n c o n d i t i o n s . Over 80% o f t h e i o n c u r r e n t i n CI(NO) s p e c t r a o f most h y d r o c a r b o n s i s c a r r i e d by t h e [ M - l ] i o n . T h i s s i t u a t i o n stands i n sharp c o n t r a s t t o t h a t o b t a i n e d under e i t h e r EI o r C I ( C H O c o n d i t i o n s , where e x t e n s i v e f r a g m e n t a t i o n o f h y d r o c a r b o n m o l e c u l e i s observed. In a d d i t i o n t o t h e above r e s u l t s , i t i s p o s s i b l e to use CI(NO) s p e c t r a t o i d e n t i f y many f u n c t i o n a l groups i n o r g a n i c m o l e c u l e s . Spectra of a c i d s , alde­ hydes and k e t o n e s show M+30, M-17; M+30, M - l ; and M+30 i o n s r e s p e c t i v e l y . One drawback t o t h e use o f n i t r i c o x i d e as a CI r e a g e n t i s t h a t i t i s a s t r o n g o x i d i z i n g agent and t h e r e f o r e r a p i d l y d e s t r o y s h o t m e t a l f i l a ­ ments used t o produce t h e beam o f i o n i z i n g e l e c t r o n s . To overcome t h i s p r o b l e m we have d e v e l o p e d a Townsend e l e c t r i c discharge ( f i l a m e n t l e s s ) source f o r producing a beam o f i o n i z i n g e l e c t r o n s o r i o n s ( 6 ) . +

+

+

+

+

8.

HUNT

159

Positive and Negative Ion Chemical Ionization

A N D SETHI

160 180 Figure 3. CI (NH ) mass spectra of: (a) triethylamine, (b) cyclohexanone, (c) Ό-(-) ribose. The intensity of reagent ions is 50 to 100 times greater than as shown. S

locr 8
10eV)

Resonance e l e c t r o n capture Dissociative electron capture

+

•A~+B +e

Ion p a i r p r o d u c t i o n

+

A +B"+e W i t h the e x c e p t i o n o f a s m a l l p o p u l a t i o n o f low energy secondary e l e c t r o n s produced under EI c o n d i t i o n s d u r i n g p o s i t i v e sample i o n f o r m a t i o n , most of the e l e c t r o n s a v a i l a b l e under EI c o n d i t i o n s p o s s e s s energy i n e x c e s s o f lOeV. A c c o r d i n g l y , most n e g a t i v e sample i o n s are produced by e i t h e r i o n - p a i r f o r m a t i o n o r by d i s s o c i a t i v e e l e c t r o n c a p t u r e mechanisms, and most o f the sample i o n c u r r e n t i s c a r r i e d by low mass f r a g m e n t s , s p e c i e s l i k e O , HO", C l " and CN~, e t c . Ions of t h i s type p r o v i d e l i t t l e s t r u c t u r a l i n f o r m a t i o n about the sample m o l e c u l e i n q u e s t i o n . In c o n t r a s t t o the above s i t u a t i o n , Wurman and Sauer (23») showed t h a t the t h e r m a l i z a t i o n o f e l e c t r o n s can o c c u r i n a f r a c t i o n o f m i c r o - s e c o n d i n the p r e s e n c e o f gases l i k e methane and iso-butane. The r e s u l t i n g l a r g e p o p u l a t i o n o f t h e r m a l e l e c t r o n s makes r e s o n a n c e e l e c t r o n c a p t u r e the dominant mechanism f o r f o r m a t i o n o f n e g a t i v e i o n s under CI c o n d i t i o n s . Once formed the n e g a t i v e l y charged sample i o n s s u f f e r up t o s e v e r a l hundred s t a b i l i z i n g c o l l i s i o n s w i t h n e u t r a l r e a g e n t gas m o l e c u l e s b e f o r e they e x i t the i o n i z a t i o n chamber. U n l i k e the r e s u l t s o b t a i n e d by h i g h energy e l e c t r o n i m p a c t , s p e c t r a r e c o r d e d under CI c o n d i t i o n s e x h i b i t abundant m o l e c u l a r anions (M-) f o r many t y p e s of molecules. F u r t h e r , t h o s e m o l e c u l e s t h a t fragment under n e g a t i v e i o n CI c o n d i t i o n s g e n e r a l l y do so by e l i m i n a t i o n o f s m a l l m o i e t i e s from the p a r e n t a n i o n . S i n c e the s t r u c t u r a l f e a t u r e s w h i c h s t a b i l i z e a negat i v e charge on an o r g a n i c m o l e c u l e are not u s u a l l y the same as t h o s e t h a t s t a b i l i z e a p o s i t i v e c h a r g e , e l e c t r o n c a p t u r e n e g a t i v e i o n CI s p e c t r a t e n d t o p r o v i d e s t r u c t u r a l i n f o r m a t i o n complementary t o t h a t a v a i l a b l e i n the p o s i t i v e i o n mode. 1

8.

HUNT

Positive and Negative Ion Chemical Ionization

A N D SETHI

Perhaps t h e most e x c i t i n g f e a t u r e o f n e g a t i v e i o n CIMS i s t h e f i n d i n g t h a t t h e s e n s i t i v i t y a s s o c i a t e d w i t h i o n f o r m a t i o n by e l e c t r o n c a p t u r e i n t h e CI s o u r c e can be 100-1.000 t i m e s g r e a t e r t h a n t h a t a v a i l a b l e by any p o s i t i v e i o n methodology. T h i s r e s u l t s u g g e s t s t h a t n e g a t i v e i o n CIMS w i l l soon become t h e method o f c h o i c e f o r t h e q u a n t i t a t i o n o f many o r g a n i c s i n complex m i x t u r e s by GCMS. Key t o t h e s u c c e s s o f t h e n e g a t i v e i o n t e c h n i q u e i s t h e development o f c h e m i c a l d e r i v a t i z a t i o n procedures which f a c i l i t a t e i n t r o d u c t i o n o f groups i n t o t h e sample under a n a l y s i s t h a t enhance b o t h f o r m a t i o n o f m o l e c u l a r a n i o n s , M , by e l e c t r o n c a p t u r e and s t a b i l i z a t i o n o f t h e r e s u l t i n g M toward unde­ s i r a b l e fragmentation. Preliminary studies indicate t h a t p e n t a f l u o r o b e n z a l d e h y d e and p e n t a f l u o r o b e n z o y l c h l o r i d e a r e e x c e l l e n t reagents f o r t h i s purpose. R e a c t i o n o f t h e s e two r e a g e n t s w i t h p r i m a r y amines and phenols f a c i l i t a t e s d e t e c t i o n o f these c l a s s e s o f compounds a t t h e femtogram ( 1 0 ~ g) l e v e l by n e g a t i v e i o n GC-CIMS methodology ( T a b l e I ) . 1

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch008

1

1 5

TABLE J_.

Detection L i m i t s f o r D e r i v a t i v e s of Primary Amines and Phenols

Compound

GCMS D e t e c t i o n L i m i t 10 χ 1 0 "

Signal/Noise

1 5

g

4/1

5

g

4/1

P e n t a f l u o r o b e n z o y l amphetamine

>)TMS

Κ*-

Pentafluorobenzylidene dopamine-bis-trimethyl s i l y l CO-C F 6

1

6

5

Δ ' -Tetrahydrocannabinol pentafluorobenzoate

ether

20 χ 10

1 5

g

4/1

161

HIGH

162

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch008

P u l s e d P o s i t i v e and N e g a t i v e

PERFORMANCE

Ion CI

MASS

SPECTROMETRY

(PPINICI):

S i m u l t a n e o u s r e c o r d i n g o f p o s i t i v e and n e g a t i v e i o n CI mass s p e c t r a on F i n n i g a n Model 3200 and Model 3300 q u a d r u p o l e mass s p e c t r o m e t e r s i s a c c o m p l i s h e d by p u l s i n g the p o l a r i t y o f the i o n source p o t e n t i a l (±110V) and f o c u s i n g l e n s p o t e n t i a l (±10-20V) a t a r a t e o f 10 kHz as i l l u s t r a t e d i n F i g u r e 5. Under t h e s e con­ d i t i o n s , p a c k e t s o f p o s i t i v e and n e g a t i v e i o n s a r e e j e c t e d from the i o n - s o u r c e i n r a p i d s u c c e s s i o n and e n t e r the q u a d r u p l e mass f i l t e r . U n l i k e the magnetic i n s t r u m e n t s , i o n s o f i d e n t i c a l m/e, but d i f f e r e n t p o l a r i t y , t r a v e r s e the quadrupoTe f i e l d w i t h e q u a l f a c i l i t y and e x i t the r o d s a t the same p o i n t . Detec­ t i o n o f i o n s i s a c c o m p l i s h e d s i m u l t a n e o u s l y by two continuous diode m u l t i p l i e r s operating w i t h f i r s t dynode p o t e n t i a l s o f o p p o s i t e p o l a r i t y . The r e s u l t i s t h a t p o s i t i v e and n e g a t i v e i o n s a r e r e c o r d e d s i m u l ­ t a n e o u s l y as d e f l e c t i o n s i n o p p o s i t e d i r e c t i o n on a c o n v e n t i o n a l l i g h t beam o s c i l l o g r a p h . E l e c t r o n C a p t u r e - EI Type S p e c t r a : As n o t e d e a r l i e r when N or argon i s used as r e a g e n t gas, the p o s i t i v e i o n CI s p e c t r a are essen­ t i a l l y i d e n t i c a l to t h a t o b t a i n e d under EI c o n d i t i o n s . In the n e g a t i v e i o n mode, sample i o n s a r e formed by e l e c t r o n capture. N e g a t i v e i o n s are not produced from n i t r o g e n or a r g o n under CI c o n d i t i o n s . U s i n g PPINICI t e c h n i q u e w i t h N as a r e a g e n t gas we can s i m u l t a n e o u s ­ l y d e t e c t and r e c o r d EI type s p e c t r a on the p o s i t i v e i o n t r a c e and i o n s produced by resonance e l e c t r o n c a p t u r e on the n e g a t i v e i o n t r a c e . S i n c e the s t r u c ­ t u r a l f e a t u r e s t h a t s t a b i l i z e p o s i t i v e and n e g a t i v e fragment i o n s a r e not u s u a l l y the same, the above methodology p e r m i t s one t o s i m u l t a n e o u s l y r e c o r d spec­ t r a w h i c h c o n t a i n complementary s t r u c t u r a l i n f o r m a t i o n . An example o f t h i s case ( F i g u r e 6a) i s the ΕΙ-EC spec­ trum o f a m y t a l . 2

2

E l e c t r o n Capture - Bronsted

A c i d Type S p e c t r a :

I f methane o r i s o b u t a n e i s used as r e a g e n t gas f o r PPINICIMS, B r o n s t e d a c i d type CI s p e c t r a a r e produced on the p o s i t i v e i o n t r a c e and e l e c t r o n c a p t u r e s p e c t r a a r e g e n e r a t e d on the n e g a t i v e i o n t r a c e . N e g a t i v e i o n s d e r i v e d from methane o r i s o b u t a n e are not o b s e r v e d i n t h i s mode o f o p e r a t i o n .

8.

HUNT

163

Positive and Negative Ion Chemical Ionization

A N D SETHI

5

PPN1-C1

POS EM

RODS RODS

U

ooooo*

-•1600V I600V

H ~I

- - -++ + - -

^

^ „ „ u ,i LB 0

NE G EM •V 1 6 0 0 V

2 ION "^LENS SOURCE +I0V + 4-10 V P U L S E RATE

|0 KHz

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch008

Analytical Chemistry

Figure 5. Pulsed positive negative ion chemical ionization (PPNICI) mass spectrometer: FIL-filament, EM-electron mul­ tiplier, and LBO-Hght beam oscillograph

1001

28.

χ 50

-

50j

6J> 100

Φ Ώ

d ζ 3

ιοω —

ο ζ 3

ι

ω
10,000) i n p a r t t o r e s o l v e sample i o n s and r e f e r e n c e i o n s produced s i m u l t a n e o u s l y . The a c c u r a t e mass o f t h e sample i o n i s t h e n c a l c u l a t e d by e x t r a p o l a t i o n from the measured mass o f a nearby r e f erence i o n . Drawbacks o f t h e above methodology i n c l u d e the h i g h c o s t o f the n e c e s s a r y i n s t r u m e n t a t i o n , l o w s e n s i t i v i t y w h i c h always accompanies o p e r a t i o n a t h i g h r e s o l u t i o n , sample i o n s u p p r e s s i o n due t o t h e l a r g e q u a n t i t i e s o f r e f e r e n c e compound r e q u i r e d t o produce abundant r e f e r e n c e i o n s a t h i g h mass, and t h e d i f f i c u l t y i n u s i n g the method f o r GC-MS a n a l y s i s due t o t h e s l o w s c a n speeds u s u a l l y r e q u i r e d t o m a i n t a i n adequate ion current a t high r e s o l u t i o n . To overcome t h i s p r o b l e m , A s p i n a l eit a l . ( 3 2 ) , i n 1975, d e v e l o p e d a t e c h n i q u e f o r o b t a i n i n g a c c u r a t e mass measurements a t low r e s o l u t i o n u s i n g a d o u b l e beam m a g n e t i c s e c t o r mass s p e c t r o m e t e r . I n t h i s method two p o s i t i v e i o n beams, one c o n t a i n i n g i o n s from t h e i n t e r n a l s t a n d a r d and one beam c o n t a i n i n g i o n s from t h e sample a r e p r o d u c e d i n two s e p a r a t e i o n s o u r c e s , p a s s e d t h r o u g h the same m a g n e t i c f i e l d s i m u l t a n e o u s l y , and c o l l e c t e d s e p a r a t e l y a t two e l e c t r o n m i l t i p l i e r s . A c c u r a t e mass measurements (·

(M+0-R)"

+

R-

(3)

->

(M-2-R)"

+

R- +

(M-l)·

+

OH"

2

H0

(4)

2

(5)

F u r t h e r r e a c t i o n o f OH" l e a d s to the p r o d u c t i o n o f (Μ­ Ι ) " so t h a t the i n t e n s i t y r a t i o (M-l) /(Μ-2)· r i s e s as the p r e s s u r e r i s e s . In the h i g h p r e s s u r e s o u r c e , the t h r e e i s o m e r i c pentanones g i v e q u i t e d i f f e r e n t s p e c t r a as shown i n F i g u r e 2, each o f w h i c h can be r a t i o n a l i z e d i n terms o f the above r e a c t i o n s . The v a r y i n g y i e l d s o f (Μ-2)· i o n s a r e a t t r i b u t e d to the r e l a t i v e ease o f a b s t r a c t i o n of H from the c a r b o n atoms a d j a c e n t to the c a r b o n y l group s i n c e d e u t e r i u m - l a b e l l i n g e x p e r i ­ ments i n d i c a t e d t h a t H a b s t r a c t i o n o c c u r r e d o n l y from t h e s e p o s i t i o n s . I n the case o f ( C H ) C H C 0 C H ( C H ) 2 , no (Μ-2)· i o n s are formed, c o n s i s t e n t w i t h t h i s i n t e r ­ p r e t a t i o n ^ Hence i n compounds o f t h i s type^, the r e a c ­ t i o n s o f 0· i o n s can be used to d e t e c t the p r e s e n c e o f a c t i v a t e d CH groups. W i t h a r o m a t i c compounds (11) , r e a c t i o n s analogous to ( l ) - ( 3 ) o c c u r but a f u r t h e r r e a c t i o n c h a r a c t e r i s t i c +

2

+

2

3

2

2

3

9.

Selective Reagent Ions

JENNINGS

183

o f a r o m a t i c compounds i s : M

+

0·

+

(M-H+0)-

+

Η·

(6)

I n c e r t a i n c a s e s , l o w i n t e n s i t y peaks were o b s e r v e d due to t h e occurrence o f the r e a c t i o n Μ

+

0·

(M-H+O-HX)"

+

Η·

+

HX (7)

Benzene g i v e s a v e r y s i m p l e s p e c t r u m c o n s i s t i n g o f a p p r o x i m a t e l y e q u a l i n t e n s i t y peaks a t (Μ-2)· and (MH+0) o n l y . W i t h 1 , 3 , 5 - b e n z e n e - d 3 , the major low mass peak i s (Μ-3)· s u g g e s t i n g 1,2 e l i m i n a t i o n o f H a l ­ though 1,4 e l i m i n a t i o n cannot be r u l e d o u t . The two h i g h e r mass peaks were o f v e r y s i m i l a r i n t e n s i t i e s , i n d i c a t i n g a n e g l i g i b l e H/D i s o t o p e e f f e c t . Pyridine y i e l d s a s p e c t r u m c l o s e l y r e s e m b l i n g t h a t o f benzene, but t h e r e i s i n a d d i t i o n a low i n t e n s i t y (M-l)"" peak. S i n c e t h e hydrogen atoms a r e no l o n g e r e q u i v a l e n t , i t is of interest to investigate positional s e l e c t i v i t y i n the displacement r e a c t i o n . I n t h e case o f 4-deuterop y r i d i n e , t h e peak a r i s i n g from d e u t e r i u m d i s p l a c e m e n t i s o n l y s l i g h t l y l e s s i n t e n s e t h a n t h a t due t o hydrogen d i s p l a c e m e n t a l t h o u g h s t a t i s t i c a l l y , an i n t e n s i t y r a t i o o f 1:4 would be p r e d i c t e d . T h i s i n d i c a t e s a marked p r e f e r e n c e f o r t h e d i s p l a c e m e n t o f the hydrogen atom i n the 4 - p o s i t i o n . The r e s u l t s o b t a i n e d w i t h 1,3,5benzene-d.3 show t h a t t h i s cannot be a t t r i b u t e d t o an isotope effect. However, i t was w i t h t h e a l k y l a r o m a t i c compounds t h a t t h e most i n t e r e s t i n g s p e c t r a were o b t a i n e d . Ob­ s e r v a t i o n s on s p e c t r a g i v e n by d e u t e r o - t o l u e n e s showed t h a t t h e two major peaks a r i s e from t h e a b s t r a c t i o n o f H from the m e t h y l group t o g i v e t h e ( M - l ) " i o n and t h e d i s p l a c e m e n t o f a r i n g hydrogen atom t o form the (MH+0)~ i o n s . The t h r e e i s o m e r i c x y l e n e s g i v e t h e spec­ t r a shown i n F i g u r e 3 and i t i s seen t h a t the metaisomer g i v e s a spectrum q u i t e d i f f e r e n t from t h o s e o f the o t h e r two i s o m e r s . The prominence o f t h e (Μ-2)· i o n appears t o a r i s e from the p r e s e n c e o f t h e two m e t h y l groups meta t o each o t h e r s i n c e when one o f t h e m e t h y l groups i s f u l l y d e u t e r a t e d , t h e (Μ-3)· i s formed by l o s s o f HOD, i n d i c a t i n g t h a t one hydrogen atom i s removed from each m e t h y l group i n f o r m i n g t h e (Μ-2)· ion. This i s also consistent with the observation that the (Μ-2)· i o n i s t h e base peak i n t h e s p e c t r u m g i v e n by m e s i t y l e n e . In t h e s p e c t r a o f a l l t h e a l k y l benzenes d i s c u s s e d above, t h e r a t i o I ( M - H + 0 ) " / I ( M - l ) " l i e s i n t h e range 0.07-0.19. However, i n t h e case o f t - b u t y l b e n z e n e ,

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch009

2

+

HIGH

PERFORMANCE

MASS

SPECTROMETRY

ΛΛΛ/ (b) / S / V ^

[MW-OCK,]*

[MM'-OCKJ]* 155

142138

ΊΪ2

ÏOÔ~~ "Ve

155

p

138

112

100

Organic Mass Spectrometry

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch009

Figure 1. Spectra given by 1-octene and trans-4-octene on reaction with CHsOCHCH,*

(a)

(b)

es, «Ι ι 15 Figure 2. Ο ·" NCI spectra of three isomeric pentanones, (a) C H COC H (b) CH CO-n-C H (c) CH COCH(CH ) 2

2

5>

s

s

7>

5

3

s 2

(a)

ι. 104

.21

151 I

105 (b) 1 .

121 1

Τ

105

Μ Figure 3. O" NCI spectra of the three isomeric xylenes, (a) o-xylene, (b) m-xylene, (c) p-xylene

•

1.

121 1

151

1

9.

185

Selective Reagent Ions

JENNINGS

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch009

+

t h i s r a t i o r i s e s t o about 5, s u g g e s t i n g t h a t t h e H a b s t r a c t i o n t o form t h e ( M - l ) ~ i o n i s l a r g e l y from t h e c a r b o n atom α t o t h e a r o m a t i c r i n g . The a b i l i t y t o d i s t i n g u i s h p o s i t i o n a l isomers i l l u s t r a t e d by t h e s p e c t r a o f t h e x y l e n e s i s a l s o i l ­ l u s t r a t e d by t h e s p e c t r a g i v e n by t h e t h r e e m e t h y l p y r i d i n e s . The r e l a t i v e i n t e n s i t i e s o f t h e major peaks a r e g i v e n i n t h e T a b l e I . I t i s seen t h a t t h e base peak o f t h e 2 - m e t h y l p y r i d i n e i s t h e (M-H+O)" i o n where­ as when t h e 4 - p o s i t i o n i s b l o c k e d , as i n 4-methylp y r i d i n e , t h i s i s a peak o f low i n t e n s i t y , c o n s i s t e n t w i t h t h e o b s e r v a t i o n s made on 4 - d e u t e r o p y r i d i n e . This was n o t always t h e c a s e , however, and t h e s p e c t r a g i v e n by t h e t h r e e f l u o r o t o l u e n e s on r e a c t i o n w i t h 0· a r e very s i m i l a r .

Table I .

R e l a t i v e I n t e n s i t i e s o f t h e M a j o r Peaks i n the 0" NCI S p e c t r a o f M e t h y l p y r i d i n e s

Ion m/e

2-Methylpyridine

(M-H+O)" 108

3-Methylpyridine

4-Methylpyridine

100

77

26

(M-3H+0)" 106

13

40

15

(M-CH^+0)" 94^

13

13

9

(M-H)~ 92

68

100

100

95

89

57

10

36

9

(M-2H) 91

7

(M-3H)" 90

186

high performance mass spectrometry

Conclusion. This brief account of the reactions of CH OCHCH * and 0 · indicates the possibilities of the use of ionmolecule reactions in probing structural features of certain classes of compounds. Undoubtedly, other ions w i l l be found which w i l l undergo different types of reactions and one can look forward to the establishing of a series of selective reagent ions which w i l l allow one greatly to extend the type of structural informa­ tion obtainable. 3

2

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch009

Experimental. The work was carried out in part using a Varian V5900 ion cyclotron resonance (ICR) spectrometer for preliminary work at low pressure and in part in an MS50 double focussing mass spectrometer (ΑΕΙ Scientific Apparatus Ltd) equipped with a dual purpose electronimpact/chemical ionization (EI/CI) source. The source pressure was measured by means of an MKS "Baratron" (Model 77H-10) the output of which fed the control unit of a Granville-Phillips automatic pressure regulator (series 216) which controlled the flow rate of the major component in the high pressure source. The instrument was modified to allow i t to operate in the negative ion mode. Literature Cited. 1. 2. 3. 4. 5. 6. 7. 8.

Field, F . H . , MTP Int. Rev. of S i c . , Series One, Physical Chemistry, Vol. 5 (Ed. A. Maccoll), Ch. 5, Butterworths, London, 1972. Ferrer-Correia, A.J.V. and Jennings, K.R., Int. J. Mass Spectrom Ion Phys., (1973), 11, 111. Drewery, C.J., Goode, G. C., and Jennings, K.R., Int. J. Mass Spectrom Ion Phys., (1976), 22, 211. Drewery, C.J. and Jennings, K.R., Int. J. Mass Spectrom. Ion Phys., (1976), 19, 287. Ferrer-Correia, A . J . V . , Sen Sharma, D.K. and Jennings, K.R., Org. Mass Spectrom., (1976), 11, 867. Drewery, C.J., Goode, G . C . , and Jennings, K.R., Int. J. Mass Spectrom Ion Phys., (1976), 20, 403. Jennings, K.R., Mass Spectrometry, Vol. 4, Specialist Periodical Report, Chemical Society, London, Ed. R.A.W. Johnston, 1977, p. 209. Goode, G.C. and Jennings, K.R., Adv. in Mass Spectrom., (1974), 6, 797.

9.

Selective Reagent Ions

Dawson, J.H.J., and Jennings, K.R., J. Chem. Soc. Faraday Trans. II, (1976), 72, 700. 10. Harrison, A.G. and Jennings, K.R., J. Chem. Soc. Faraday Trans. I, (1976), 77, 1601. 11. Bruins, A . P . , Ferer-Correia, A . J . V . , Harrison, A . G . , Jennings, K.R. and Mitchum, R.K., Adv. in Mass Spectrom. 7, (in press). Received December 30, 1977

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch009

9.

JENNINGS

10 Selectivity in Biomedical Applications CATHERINE FENSELAU

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch010

Department of Pharmacology and Experimental Therapeutics, Johns Hopkins University School of Medicine, Baltimore, MD 21205

Often in pharmacology, c l i n i c a l studies, toxicology and studies of environmental residues, the sample is a minor component of a very complex mixture. Figure 1 shows an example of a typical mixture extracted from urine. An analogously obtained extract from blood, sweat or cerebral spinal fluid would contain even more compounds, because these physiologic fluids have a higher proportion of l i p o p h i l l i c endogenous components. If one is performing qualitative or quantitative assays of a specific component of such a mixture, i t is easy to see how interference from other compounds may lessen the r e l i a b i l i t y of the assay, degrade the sensitivity of the assay, and interfere with quantitation. Documentation of the loss in sensitivity and also in precision of mass spectral analyses of samples from physiologic fluids is presented in Tables 1 and 2. Table 1 indicates that, while 100 picograms of bis-2chloroethylamine (a metabolite of the antitumor drug cyclophosphamide) can be quantitatively detected as Table 1.

Quantitation of Bis-(2-chloroethyl)amine Pure from urine from plasma Signal/Noise = 30/1

1 X 10"-10 g 3 X 10"-8 g 9 X 10"-8 g

the trifluoroacetyl derivative in a particular gas chromatograph-mass spectrometer system, samples which had been extracted from urine and derivatized could only be assayed reliably down to about 30 nanograms ©0-8412-0422-5/78/47-070-188$10.00/0

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch010

10. FENSELAU

Selectivity in Biomedical Applications

189

ε •S .S:

ε-

ο

s s s Ο

s I 8

&

190

HIGH

PERFORMANCE

MASS

SPECTROMETRY

u s i n g the same p r o t o c o l . In moving from u r i n e to p l a s m a , a n o t h e r t h r e e - f o l d l o s s i n s e n s i t i v i t y was experienced. In Table 2, s t a n d a r d d e v i a t i o n ^ a r e p r e s e n t e d of i s o t o p e r a t i o measurements of C -cont a i n i n g carbon d i o x i d e from two s o u r c e s . Here meaT a b l e 2.

I s o t o p e R a t i o Measurements Sample

C0

2

from

Standard D e v i a t i o n

limestone

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch010

from u r i n e

± 0. 05 % ± 0. 15 %

surements performed on u r i n e samples were found to have a t h r e e - f o l d l a r g e r s t a n d a r d d e v i a t i o n than those made on samples o b t a i n e d from (homogeneous) l i m e s t o n e ( 1 ) . This i l l u s t r a t e s a degradation i n p r e c i s i o n . Enhancement o f S e l e c t i v i t y The e a s i e s t s o l u t i o n to the c o n t a m i n a t i o n problems e n c o u n t e r e d i n b i o m e d i c a l samples would be, o f c o u r s e , to p u r i f y samples b e f o r e one c a r r i e s out the mass s p e c t r a l a n a l y s i s . However, b i o m e d i c a l samples are u s u a l l y a v a i l a b l e i n o n l y l i m i t e d amounts, and i n p r a c t i c e , i t i s o f t e n e a s i e r t o employ s e l e c t i v e methods o f a n a l y s i s , t h a t i s , t o u t i l i z e mass s p e c t r a l t e c h n i q u e s t h a t s e l e c t as u n i q u e l y as p o s s i b l e f o r the sample o f i n t e r e s t . Table 3 i n d i c a t e s f i v e areas i n w h i c h one may enhance the s e l e c t i v i t y o f a n a l y s e s by mass s p e c t r o m e t r y . These approaches can be used i n v a r i o u s c o m b i n a t i o n s t o p r o v i d e enhanced r e l i a b i l i t y , p r e c i s i o n , and/or s e n s i t i v i t y . T a b l e 3.

Approaches f o r Enhancing

D 2) 3) 4) 5)

Selectivity

Sample p r e p a r a t i o n Ion p r o d u c t i o n Ion s e p a r a t i o n Ion d e t e c t i o n Data p r o c e s s i n g

The most i m p o r t a n t a s p e c t o f s e l e c t i v e sample p r e p a r a t i o n i s , of c o u r s e , i s o l a t i o n and p u r i f i c a t i o n . T h i s i s an a r t form which w i l l not be a d d r e s s e d i n t h i s e s s a y , except t o p o i n t out t h a t gas chromatography p r o v i d e s a v e r y p o w e r f u l t e c h n i q u e f o r p u r i f i c a t i o n or s e p a r a t i o n o f the compound o f i n t e r e s t . A l l of the

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch010

10.

FENSELAU

Selectivity in Biomedical

Applications

191

subsequent i l l u s t r a t i o n s w i l l be measurements made by combined gas chromatography-mass s p e c t r o m e t r y . S e l e c t i v e d e r i v a t i z a t i o n p r o c e d u r e s s h o u l d a l s o be e v a l u a t e d , f o r example t h e f o r m a t i o n o f b o r o n a t e d i e s t e r s from c i s d i o l s . However, many compounds i n a m i x t u r e a r e u s u a l l y d e r i v a t i z e d by any c h e m i c a l r e a c t i o n , and d e r i v a t i z a t i o n r e a c t i o n s u s u a l l y c o n t a m i n a t e a sample f u r t h e r , r a t h e r than s e l e c t i n g f o r t h e compound o f i n t e r e s t . One u s e f u l f u n c t i o n o f d e r i v a t i z a t i o n i s t o i n c r e a s e t h e m o l e c u l a r weight o f t h e sample beyond t h e mass range o f most i n t e r f e r i n g i o n s . A n o t h e r example o f s e l e c t i v e sample p r e p a r a t i o n might be t h e g e n e r a t i o n o f i s o t o p e c l u s t e r s . In Figure 2 a r e shown t h e s t r u c t u r e s o f propoxyphene and i t s d.7 a n a l o g . The major mode o f f r a g m e n t a t i o n i s a l s o i n d i c a t e d . A 1:1 m i x t u r e o f t h e s e two compounds was, . a d m i n i s t e r e d t o an a n i m a l , and u r i n e was c o l l e c t e d and e x t r a c t e d . The drug r e l a t e d m e t a b o l i t e s were p i c k e d out o f t h e many components d e t e c t e d by gas chromatography-mass s p e c t r o m e t r y on t h e b a s i s o f i s o t o p i c c l u s t e r s a t m/e 91 and m/e 98. These peaks r e p r e s e n t e d t h e b e n z y l and d - b e n z y l i o n s i n d i c a t e d i n t h e F i g u r e . T h i s approach p e r m i t t e d a t l e a s t 8 m e t a b o l i t e s t o be l o c a t e d and s u b s e q u e n t l y i d e n t i f i e d ( 2 J . A l t h o u g h s e l e c t i v e i o n i z a t i o n , t h e second c a t e g o r y i n T a b l e 3, has h i s t o r i c a l l y denoted p h o t o i o n i z a t i o n and l o w energy e l e c t r o n impact i o n i z a t i o n , t h e low s e n s i t i v i t y o f t h e s e t e c h n i q u e s has p r e v e n t e d t h e i r widespread a p p l i c a t i o n . I o n i z a t i o n techniques which c u r r e n t l y appear t o o f f e r t h e g r e a t e s t p o t e n t i a l f o r enhancing t h e s e l e c t i v i t y o f a n a l y s e s i n c l u d e p o s i t i v e and n e g a t i v e c h e m i c a l i o n i z a t i o n (3) and f i e l d i o n i z a t i o n and d e s o r p t i o n , a l l a d d r e s s e d by o t h e r a u t h o r s i n t h i s volume. The u t i l i t y o f c h e m i c a l i o n i z a t i o n i s i l l u s t r a t e d below. The t h i r d c a t e g o r y i s s e l e c t i v i t y i n i o n s e p a r a t i o n i n the ion analyser. C e r t a i n l y high r e s o l u t i o n a n a l y s i s comes t o mind as a method f o r r e d u c i n g t h e a m b i g u i t y i n an a n a l y s i s . T h i s was i l l u s t r a t e d q u i t e e a r l y i n a p h a r m a c o l o g i c a p p l i c a t i o n o f Bommer and Vane (4). C o l l i s i o n a l a c t i v a t i o n w i t h two s t a g e s ' o f i o n s e p a r a t i o n , and some o f t h e d e f o c u s s e d m e t a s t a b l e t e c h n i q u e s a r e a l s o examples o f s e l e c t i v i t y i n i o n separation. I n a l l t h e s e c a s e s , however, r e l i a b i l i t y of t h e a n a l y s i s i s i n c r e a s e d a t t h e expense o f sensitivity. The f o u r t h c a t e g o r y i n which one c a n i n c r e a s e t h e s e l e c t i v i t y o f a mass s p e c t r a l a n a l y s i s i s by s e l e c t i v e d e t e c t i o n . Here t h e preeminent example i s s e l e c t e d i o n monitoring. I n t h i s t e c h n i q u e , one m o n i t o r s t h e i n 7

192

HIGH

PERFORMANCE

MASS

SPECTROMETRY

t e n s i t i e s o f p r e s e l e c t e d , c h a r a c t e r i s t i c i o n s as a f u n c t i o n o f t i m e . As an example, F i g u r e 1 c o n t a i n s the gas chromatogram d e t e c t e d by f l a m e i o n i z a t i o n o f an e x t r a c t o f u r i n e from a morphine-dosed r a b b i t . In t h i s i n v e s t i g a t i o n i n the a u t h o r s l a b o r a t o r y , the g l u c u r o n i d e m e t a b o l i t e o f morphine was sought. I t s a p p r o x i m a t e e i u t i o n t i m e i s i n d i c a t e d by the arrow. F i g u r e 3 shows the s e l e c t e d i o n r e c o r d s o f s e v e r a l i o n s c h a r a c t e r i s t i c o f morphine g l u c u r o n i d e . A l l o f these p r o f i l e s a r e s i m p l e r t h a n the gas chromatogram i n F i g u r e 1. I n a d d i t i o n , i t may be o b s e r v e d t h a t when h i g h e r masses a r e m o n i t o r e d , fewer components o f the u r i n e e x t r a c t are detected. N e e d l e s s t o say, s e l e c t e d i o n m o n i t o r i n g can be used i n c o m b i n a t i o n w i t h o t h e r s e l e c t i v e t e c h n i q u e s , most n o t a b l y gas chromatography and c h e m i c a l i o n i z a t i o n . Ion p r o f i l e s a n a l o g o u s to those p r o v i d e d by s e l e c t e d i o n m o n i t o r i n g can a l s o be r e c o n s t r u c t e d from r e p e t i t i v e l y scanned c o n v e n t i o n a l s p e c t r a , b e s t done w i t h a computer. I n t h i s c a s e , a l l o f the i o n s det e c t e d i n each scan a r e s t o r e d i n the computer so t h a t any o f them may be c a l l e d out and p r o f i l e d . However, the o v e r a l l t e c h n i q u e i s l e s s s e n s i t i v e t h a n s e l e c t e d i o n m o n i t o r i n g by a f a c t o r as g r e a t as 10 *. This r e f l e c t s p r i m a r i l y the g r e a t e r sampling t i m e f o r the s e l e c t e d i o n t e c h n i q u e and a l s o the f a c t t h a t sample i s not wasted w h i l e u n i n f o r m a t i v e a r e a s o f the s p e c t r a a r e b e i n g scanned and d u r i n g a n a l y z e r r e s e t p e r i o d s . I n T a b l e 4, the p r a c t i c a l lower d e t e c t i o n l i m i t s a r e p r e s e n t e d f o r v a r i o u s k i n d s o f gas c h r o m a t o g r a p h i c d e t e c t o r s . T o t a l i o n c u r r e n t m o n i t o r i n g w i t h the mass s p e c t r o m e t e r has a s e n s i t i v i t y r o u g h l y comparable t o

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch010

1

1

T a b l e 4.

S e n s i t i v i t y o f GC

Detectors —g

T o t a l Ion C u r r e n t M o n i t o r i n g

10

Flame I o n i z a t i o n

10

•—— g

-9 g -12 E l e c t r o n Capture

10

g -12

S e l e c t e d Ion M o n i t o r i n g 10 g f l a m e i o n i z a t i o n d e t e c t i o n , w h i l e s e l e-c8t e d i o n moniMass t o r i n gChromatograms w i t h the mass s p e c t r o m e t e r i 10 s c l e ag r l y compet i t i v e w i t h the b e s t e l e c t r o n c a p t u r e d e t e c t o r s . S e l e c t e d i o n m o n i t o r i n g i s o f c o u r s e much more g e n e r a l l y a p p l i c a b l e t h a n e l e c t r o n c a p t u r e d e t e c t i o n . Thus, s e l e c t e d i o n m o n i t o r i n g has been r e p o r t e d t o be used w i t h s e n s i t i v i t y i n the p i c o g r a m range w i t h e l e c t r o n

10.

Selectivity in Biomedical Applications

FENSELAU

Propoxyphene

193

d -Propoxyphene 7

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch010

Figure 2. Structures and major fragmentation of propoxyphene and d -propoxyphene 7

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Figure 3. Selected ion records of the urine extract chromatographed in Figure 1 (re­ injected)

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impact i o n i z a t i o n , w i t h p o s i t i v e i o n c h e m i c a l i o n i z a t i o n , and a l s o w i t h n e g a t i v e i o n c h e m i c a l i o n i z a t i o n . The a u t h o r submits t h a t t h i s i s i n d e e d a c a t e g o r y o f h i g h p e r f o r m a n c e mass s p e c t r o m e t r y . A n a l y s i s o f Drug M e t a b o l i t e s The d i s c u s s i o n o f s e l e c t e d i o n m o n i t o r i n g above s e r v e s as an i n t r o d u c t i o n t o an account o f work a t Hopkins i n w h i c h mass s p e c t r o m e t r y has been used i n the s e l e c t e d i o n m o n i t o r i n g mode t o p r o v i d e enhanced s e n s i t i v i t y and r e l i a b i l i t y i n the a n a l y s i s o f m e t a b o l i t e s o f cyclophosphamide i n u r i n e and b l o o d . F i g u r e 4 shows the s t r u c t u r e o f c y c l o p h o s p h a m i d e ( I ) and o f i t s major m e t a b o l i t e s as they a r e now u n d e r s t o o d . Cyclophosphamide i s a w i d e l y used a n t i t u m o r d r u g , w h i c h i s not i t s e l f biologically active. R a t h e r , i t has been known f o r some y e a r s t h a t c y c l o p h o s p h a m i d e i s c o n v e r t e d t o c y t o t o x i c a c t i v e m e t a b o l i t e s by o x i d a t i v e m e t a b o l i s m i n the h e p a t i c microsomes. A l t h o u g h t h i s was u n d e r s t o o d e a r l y on, n o n e t h e l e s s , the drug was used c l i n i c a l l y f o r more t h a n 10 y e a r s b e f o r e the s t r u c t u r e s o f any o f i t s h e p a t i c m e t a b o l i t e s were e l u c i d a t e d , and i n f a c t , s u c c e s s f u l i d e n t i f i c a t i o n o f the m e t a b o l i t e s o c c u r r e d o n l y a f t e r mass s p e c t r o m e t r y was a p p l i e d t o the problem i n s e v e r a l l a b o r a t o r i e s , i n c l u d i n g our own. In F i g u r e 5, the s t r u c t u r e i s p r e s e n t e d f o r the c y t o t o x i c a c t i v e m e t a b o l i t e phosphoramide m u s t a r d , w h i c h was f i r s t i s o l a t e d and c h a r a c t e r i z e d from an i n v i t r o i n c u b a t i o n of r a d i o i s o t o p e l a b e l l e d cyclophosphamide w i t h mouse l i v e r microsomes (5J). The metab o l i t e was t r e a t e d w i t h diazomethane, c o n v e r t i n g i t t o a m i x t u r e o f mono-, d i - and t r i m e t h y l d e r i v a t i v e s as i s i n d i c a t e d i n F i g u r e 5. The major f r a g m e n t a t i o n p a t h o f t h e s e d e r i v a t i v e s a r e a l s o i n d i c a t e d i n the F i g u r e . A l t h o u g h the group a t Hopkins was v e r y p l e a s e d a t the time t o have i d e n t i f i e d the f i r s t a c t i v e m e t a b o l i t e o f c y c l o p h o s p h a m i d e , n o n e t h e l e s s t h i s m e t a b o l i t e had been o b t a i n e d from an i n v i t r o e x p e r i m e n t , and i t was deemed n e c e s s a r y t o examine b l o o d and u r i n e o f p a t i e n t s who were r e c e i v i n g the drug t o c o n f i r m t h a t such a metab o l i t e was formed i n v i v o i n humans. Because much s m a l l e r samples would be a v a i l a b l e from the p a t i e n t s and because t h e s e would be much more h i g h l y c o n t a m i n a t e d t h a n was the sample from the i n v i t r o microsomal experiment, s e l e c t e d i o n monitoring was employed t o l o o k f o r t h i s m e t a b o l i t e . In o r d e r t o use t h i s h i g h l y s e l e c t i v e d e t e c t i o n t e c h n i q u e , one must know what the c o n v e n t i o n a l l y scanned mass spectrum o f the compound l o o k s l i k e . In F i g u r e 6 i s the mass spectrum o f the d i m e t h y l d e r i v a t i v e o f phosphoramide m u s t a r d . Ions were chosen from

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch010

10.

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195

.COOH

Figure 4. Cyclophosphamide I and its major human metabolites

,NH 0

1

CI-

2

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L

«5.187 199,201 213,215

R-R'-H R«CH ,R'«H R=R'=CH 3

3

Figure 5. Structures and fragmentation of the derivatives of phosphoramide mustard treated with diazomethane

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t h i s spectrum f o r s e l e c t e d i o n m o n i t o r i n g w h i c h a r e , on the one hand, h i g h l y c h a r a c t e r i s t i c o f the substance b e i n g s o u g h t , and on the o t h e r , s u f f i c i e n t l y i n t e n s e t o p r o v i d e good s e n s i t i v i t y . From t h i s spectrum m/e v a l u e s 199 and 201 were chosen f o r monitoring."Tëcause t h e s e peaks r e p r e s e n t i o n s c o n t a i n i n g c h l o r i n e i s o t o p e s , one o f t h e i r c h a r a c t e r i s t i c f e a t u r e s i s t h e i r r e l a t i v e i n t e n s i t y r a t i o o f 3:1. F i g u r e 7 c o n t a i n s the mass spectrum o f the t r i m e t h y l d e r i v a t i v e o f phosphoramide m u s t a r d . From t h i s spectrum the p a i r o f peaks a t m/e 213 and 215 was s e l e c t e d f o r m o n i t o r i n g . Here a g a i n t h e s e peaks o c c u r i n the spectrum w i t h an i n t e n s i t y r a t i o o f a p p r o x i m a t e l y 3:1, r e f l e c t i n g the p r e s e n c e a t n a t u r a l abundance o f C I i n the lower mass i o n s and C I i n the h e a v i e r i o n s . F i g u r e 8 shows the s e l e c t e d i o n r e c o r d s o f t h e s e f o u r i o n s , m o n i t o r e d when a s t a n d a r d sample i s der i v a t i z e d and i n j e c t e d i n t o the gas chromatograph and when a u r i n e e x t r a c t i s d e r i v a t i z e d and i n j e c t e d i n t o the gas chromatograph mass s p e c t o m e t e r . From t h i s e x p e r i m e n t i t was c o n c l u d e d t h a t the p a t i e n t * s u r i n e d i d i n d e e d c o n t a i n the m e t a b o l i t e phosphoramide mustard. T h i s i d e n t i f i c a t i o n was based on the comparable r e t e n t i o n t i m e s o f m a t e r i a l e x t r a c t e d from t h e u r i n e and the s t a n d a r d compound, on the f o r m a t i o n o f the c h a r a c t e r i s t i c fragment i o n s w i t h t h e i r a p p r o p r i a t e r e l a t i v e i n t e n s i t i e s , and on the c h a r a c t e r i z a t i o n o f a l l t h r e e d e r i v a t i v e s o f the m e t a b o l i t e . (The monom e t h y l d e r i v a t i v e was d e t e c t e d i n a s e p a r a t e i n j e c tion. ) In o r d e r t o p r o v i d e a frame o f r e f e r e n c e f o r the s e l e c t i v i t y o f t h i s d e t e c t i o n method, the t o t a l i o n c u r r e n t gas chromatogram o f the same u r i n e e x t r a c t i s shown i n F i g u r e 9. The arrows on the a x i s i n d i c a t e the p o i n t s a t w h i c h the d i - and t r i m e t h y l d e r i v a t i v e s o f the m e t a b o l i t e were d e t e c t e d by s e l e c t e d i o n monitoring. One o f these arrows f a l l s on the t r a i l i n g edge o f the l a r g e r peaks i n the m i x t u r e . A conventional scanned s p e c t r u m was o b t a i n e d a t t h i s p o i n t , w h i c h i s shown i n F i g u r e 10. T h i s i s the s p e c t r u m o f a m e t h y l a l k y l p h t h a l a t e , a member o f a f a m i l y o f c o n t a m i n a n t s o f t e n e n c o u n t e r e d i n c l i n i c a l samples. A l l peaks c o n t r i b u t e d t o the spectrum by the m e t a b o l i t e o f c y c l o p h o s p h a m i d e had i n t e n s i t i e s l e s s t h a n 1% r e l a t i v e t o the i n t e n s i t y o f the base peak o f the c o n t a m i n a n t . N o n e t h e l e s s , the s e l e c t i v e d e t e c t i o n method c o m p l e t e l y i g n o r e s t h i s g r o s s c o n t a m i n a n t , because i t does not form i o n s o f the masses b e i n g m o n i t o r e d . In F i g u r e 11, s e l e c t e d i o n p r o f i l e s a r e p r e s e n t e d , o b t a i n e d by m o n i t o r i n g the c h r o m a t o g r a p h i c s e p a r a t i o n 3 5

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch010

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Figure 12. Chemical ionization mass spectrum of monomethyl phosphoramide mustard

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^

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Cancer Research

Figure 13. Selected ion records obtained by chemical ionization of: (A) authentic derivatized phosphoramide mustard, (B) plasma extract from a patient receiving cyclo­ phosphamide, (C) control plasma extract.

10.

Selectivity in Biomedical Applications

FENSELAU

The f i r s t s t e p i n t h e development o f an assay t e c h n i q u e f o r phosphoramide mustard was t h e s y n t h e s i s o f a du a n a l o g t o be used as an i n t e r n a l s t a n d a r d . Part of the s y n t h e t i c scheme i s i n d i c a t e d i n F i g u r e 14. L i t h i u m aluminum d e u t e r i d e r e d u c t i o n i s an i d e a l s t e p f o r i n t r o d u c i n g q u a n t i t a t i v e amounts o f d e u t e r i u m i n t o s p e c i f i c l o c a t i o n s i n the molecule. The p r o d u c t o f t h e r e a c t i o n w i t h t h i o n y l c h l o r i d e , n o r n i t r o g e n mustard, i s a general b u i l d i n g block present i n a l l the metabolites o f c y c l o p h o s p h a m i d e and a l s o a number o f o t h e r a n t i ­ tumor a l k y l a t i n g d r u g s . d - N o r n i t r o g e n mustard was e l a b o r a t e d t o d -phosphoramide m u s t a r d and d U - c y c l o ­ phosphamide by l i t e r a t u r e methods, p r e p a r a t o r y t o u n d e r t a k i n g c l i n i c a l a s s a y s o f t h e c o r r e s p o n d i n g unl a b e l l e d compounds. c U - N o r n i t r o g e n mustard and ο

fJ N(CH ) N-P

20

20h

r

250 2 è 0 2*0 2 Ô 0

m/e

271 70 2 Ô 0 250 2 é 0 2"

m/e

Figure 15. Protonated molecular ion region (chemical ionization) of phosphoramide mustard and d^phosphoramide mustard

Selectivity in Biomedical Applications

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch010

FENSELAU

Figure 16. Selected ion records of an extract of plasma containing phosphoramide mustard and à -phosphoramide mustard h

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/JUQ Phosphoramide Mustard/ml 10 fiQ Phosphoramide Mustard-D Cyclohexylamine Salt/ml 4

Cancer Research

Figure 17. Ratio of intensities of peaks at m/e 263 and 267 as a function of the amount of phosphoramide mustard added to 10 fig à -phosphoramide mustard cyclohexylamine salt in one mh plasma h

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600 Patient *2 • Cyclophosphamide ο Nornitrogen Mustard κ Phosphoramide Mustard

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch010

500«

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3001-

Ο Ε 200

lOOh

Hours Cancer Research

Figure 18. Concentration of cyclophosphamide nornitrogen mus­ tard as a function of time in plasma of a patient receiving 75 mg/kg inalhr infusion

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch010

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207

t r a l v a l u e s a r e u n i f o r m l y h i g h e r t h a n t h o s e o b t a i n e d by spectrophotometry. I n p a r t t h i s i s because decomp o s i t i o n o f these u n s t a b l e compounds d u r i n g workup and d e r i v a t i z a t i o n i s compensated by the s t a b l e i s o t o p e l a b e l l e d i n t e r n a l standard. The mass s p e c t r o m e t r i c assay i s h i g h l y r e l i a b l e , h i g h l y s p e c i f i c , r e l a t i v e l y e x p e n s i v e and t e d i o u s . The a s s a y whose development and a p p l i c a t i o n i s d e s c r i b e d above employs s e l e c t i v i t y from a number o f the c a t e g o r i e s i n T a b l e 3. S e l e c t i v i t y i s used i n sample p r e p a r a t i o n . F i r s t o f a l l the gas chromatog r a p h i c i n l e t system was u s e d . S e c o n d l y heavy r e l i a n c e was p l a c e d on i s o t o p e c l u s t e r s , b o t h from c h l o r i n e and from d e u t e r i u m / h y d r o g e n . S e l e c t i v i t y i n i o n p r o d u c t i o n has been i n c o r p o r a t e d b y the u s e o f i s o b u t a n e c h e m i c a l ionization. S e l e c t i v i t y i n i o n d e t e c t i o n was i n t r o duced by the u s e o f s e l e c t e d i o n m o n i t o r i n g . Some s e n s i t i v i t y was t r a d e d f o r r e l i a b i l i t y and f o r capab i l i t y t o do q u a n t i t a t i o n by m o n i t o r i n g f o u r i o n s i n s t e a d o f o n l y one. At i n t e r v a l s o f i n c r e a s i n g frequency, c l a i m s a r e p r e s e n t e d i n the l i t e r a t u r e f o r i n c r e a s e d s e n s i t i v i t y , e i t h e r a s s o c i a t e d w i t h a new t e c h n i q u e o r a p a r t i c u l a r instrument. Can i n c r e a s e d s e n s i t i v i t y be used i n b i o m e d i c a l a p p l i c a t i o n s ? Yes I Methodology o r i n s t r u m e n t s w i t h improved s e n s i t i v i t y can make i m p o r t a n t c o n t r i b u t i o n s . However, i t must be remembered t h a t even w o r k i n g w i t h samples p r e s e n t i n nanogram amounts s u f f i c i e n t t o be d e t e c t e d by c o n v e n t i o n a l s c a n n i n g , a n a l y s e s a r e s t y m i e d as the sample i s l o s t i n the normal n o i s e o r i n t e r f e r e n c e o f the o t h e r components o f a physiologic fluid. T h i s i s f u r t h e r compounded by i o n s c o n t r i b u t e d from column b l e e d and i n s t r u m e n t background. F o r i n c r e a s e d s e n s i t i v i t y t o be u t i l i z a b l e and u s e f u l , i t must be a p p l i e d i n c o n j u n c t i o n w i t h methodology w h i c h p r o v i d e s a p p r o p r i a t e s e l e c t i v i t y as well.

Literature Cited 1.

2.

G. E. Von Unruh, D. J. Hauber, D. A. Schoeller and J . M. Hayes. Detection Limits of Carbon-13 Labelled Drugs and Metabolites. Biomed. Mass Spectrom (1974) 1 345-349. S. L. Due, H. R. Sullivan and R. E. McMahon. Propoxyphene: Pathways of Metabolism in Man and Laboratory Animals. Biomed. Mass Spectrom (1976) 3 217-225.

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H. P. Tannenbaum, J . D. Roberts and R. C. Dougherty. Negative Chemical Ionization Mass Spectrometry - Chloride Attachment Spectra. Anal. Chem. (1975) 42 49-54 and references therein. 4. P. Bommer and F. Vane. The Use of Fragmentation Patterns of 1,4-benzodiazepines for the Structure Determination of Their Metabolites by High Resolution Mass Spectrometry. Fourteenth Annual Conference on Mass Spectrometry and Allied Topics, Dallas, Texas, 1966; M. A. Schwartz P. Bommer and F. Vane. Diazepam Metabolites in the rat: Characterization by High Resolution Mass Spectrometry and Nuclear Magnetic Resonance. Arch. Biochem. (1967) 121 508-516. 5. M. Colvin, C. A. Padgett and C. Fenselau. A Biologically Active Metabolite of Cyclophosphamide. Cancer Res. (1973) 33 915-918. 6. M. Colvin, R. B. Brundrett, M. N. Kan, I. Jardine and C. Fenselau. Alkylating Properties of Phosphoramide Mustard. Cancer Res. (1976) 36 11211126. 7. C. Fenselau, M. N. Kan, S. Billets and M. Colvin Identification of Phosphorodiamidic Acid Mustard as a Human Metabolite of Cyclophosphamide. Cancer Res. (1975) 35 1453-1457. (Reprinted by permission of Cancer Research, Inc.) 8. I. Jardine, C. Fenselau, M. Appler, M. N. Kan, R. B. Brundrett and M. Colvin. The Quantitation by Gas Chromatography Chemical Ionization Mass Spectrometry of Cyclophosphamide, Phosphoramide Mustard and Nornitrogen Mustard in the Plasma and Urine of Patients Receiving Cyclophosphamide Therapy. Cancer Res. Reprinted by permission of Cancer Research, Inc. Received December 30, 1977

11 Prognosis for Field Desorption Mass Spectrometry in Biomedical Application CHARLES C. SWEELEY, BERND SOLTMANN, and JOHN F. HOLLAND*

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch011

Department of Biochemistry, Michigan State University, East Lansing, MI 48824

Field Desorption Field desorption mass spectrometry is a method of increasing importance to the biological and medical sciences. It is reported by many investigators to be a panacea for the analysis of polar, non-volatile compounds, compounds that are simply not amenable to other types of mass spectrometry. Conversely, i t is claimed by others to be a black art that is more mystical than scientific, with successful analyses resulting more from good fortune than from clever experimental design. The truth is obviously somewhere between these divergent views. But the potential of the method for revealing definitive information on molecular species makes a better understanding of its processes a concern of a l l scientists who are engaged in efforts to determine the structures of molecules of biological importance. Some typical successes of the method to date include carbohydrates and saccharides (1-3), glycosides (6), nucleosides and nucleotides (5), polypeptides (6), hormones (2) and drugs and drug metabolites (8-10). In spite of this impressive l i s t , the potential of the method is just beginning to be realized. A vexing problem persists, however, in that the number of failures remains disturbingly high in many investigations. In field desorption mass spectrometry, the sample to be analyzed is physically placed upon the ion source anode when i t is external to the vacuum chamber. Subsequently, the anode is placed in the ion source and the vacuum restored. The sample is heated in the presence of a very large voltage f i e l d . At a specific temperature, called the best anode temperature (BAT), ions will be formed, many of which are accelerated and focused into the mass spectrometer for dispersion and *Author to whom correspondence should be addressed. ©0-8412-0422-5/78/47-070-209$10.00/0

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d e t e c t i o n . T h i s method o f i o n i z a t i o n , from i t s i n c e p t i o n by Beckey ( 1 1 ) , has been s u c c e s s f u l l y a p p l i e d t o a number o f compounds t h a t a r e p o l a r and n o n - v o l a t i l e and, i n g e n e r a l , not amenable t o a n a l y s i s u s i n g o t h e r types or i o n i z a t i o n . The i o n s p r o d u c e d by FD have v e r y low i n t e r n a l e n e r g y , and the r e s u l t i n g mass s p e c t r a n o r m a l l y have l a r g e peaks r e l a t e d t o t h e i r m o l e c u l a r i o n s , w i t h few or no fragment i o n s p r e s e n t . Cluster i o n s a r e o f t e n o b s e r v e d , but the s p e c t r a p r o d u c e d a r e much l e s s complex t h a n those o b t a i n e d by e l e c t r o n impact i o n i z a t i o n . For example, r i b o f l a v i n g i v e s a comp l e x e l e c t r o n impact sçectrum t h a t c o n t a i n s a v e r y s i p a l l m o l e c u l a r i o n , M . However, when o b s e r v e d by FD, M i s dominant and the o n l y major i o n i n the s p e c t r u m . I t i s c l e a r t h a t the i o n i z a t i o n p r o c e s s e s i n FD, whate v e r t h e y may be, y i e l d i o n s w i t h l i t t l e i n t e r n a l energy and f r a g m e n t a t i o n i s m i n i m i z e d . L e t us examine ways i n w h i c h t h i s may o c c u r . A t h e o r e t i c a l model f o r f i e l d d e s o r p t i o n was o r i g i n a l l y d e v e l o p e d from f i e l d i o n i z a t i o n t h e o r y , based upon m a t h e m a t i c a l d e r i v a t i o n s by M u e l l e r (12) and Gomer (13). As shown i n F i g . 1, t h i s model p r o p o s e s t h a t the p o t e n t i a l energy c u r v e s f o r the e l e c t r o n s o f a m o l e c u l e on the s u r f a c e o f a c o n d u c t o r a r e g r e a t l y a l t e r e d i n the p r e s e n c e o f s t r o n g e l e c t r i c f i e l d s (~ 1 0 V/cm). V o l t a g e g r a d i e n t s o f t h i s magnitude are o b t a i n a b l e i n the v i c i n i t y o f v e r y s h a r p p o i n t s s i n c e a p p l i e d v o l t ages e x h i b i t l a r g e , n o n - l i n e a r g r a d i e n t d i s t o r t i o n s i n the v i c i n i t y o f s m a l l r a d i i o f c u r v a t u r e s , w i t h the f i e l d r i s i n g a s y m p t o t i c a l l y as the s u r f a c e s a r e approached (14). F i e l d image t h e o r y p r e d i c t s t h a t i o n s may be formed i n one o f two manners i n t h e s e v e r y l a r g e f i e l d s . F i r s t l y , as seen from F i g . 1, the energy o f i o n i z a t i o n has been g r e a t l y r e d u c e d . The p o s s i b i l i t y o f t h e r m a l energy p r o v i d i n g the s t i m u l u s f o r the e l e c t r o n t o escape i n c r e a s e s as the energy w e l l becomes s h a l l o w e r . S e c o n d l y , i f the p o r t i o n o f the energy l i n e t h a t l i e s below t h e l e v e l o f t h e energy i n the w e l l becomes c l o s e to t h e w e l l i t s e l f , the p r o b a b i l i t y f o r e l e c t r o n t u n n e l i n g w i l l i n c r e a s e and i o n s can be g e n e r a t e d by t h i s quantum m e c h a n i c a l mechanism. Indeed, t h i s i s cons i d e r e d t o be t h e dominant mode i n f i e l d i o n i z a t i o n mechanisms. U n f o r t u n a t e l y , i n p r a c t i c e , the f i e l d image t h e o r y has p r o v i d e d the a n a l y s t w i t h l i t t l e i n t u i t i v e f e e l i n g f o r the p r o c e s s e s i n v o l v e d as they r e l a t e to experimental observations. As u s e r s , we, l i k e many o t h e r s , were v e r y f r u s t r a t e d by the l a r g e number o f s e e m i n g l y random f a i l u r e s . On the o t h e r hand, s u c c e s s f u l a n a l y s e s were o f t e n 8

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch011

SWEELEY

E T AL.

Field Desorption Mass Spectrometry

Analytical Chemistry

Figure 1. The effect of a strong field on a potential energy curve. The ordinate is energy and the abcissa is distance from the emitter surface.

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o b t a i n e d w i t h no knowledge as t o what was done r i g h t o r , f o r t h a t m a t t e r , o f t e n w i t h no knowledge o f any­ t h i n g b e i n g done d i f f e r e n t l y . The o n l y m e a n i n g f u l o b s e r v a t i o n was t h a t c h e m i c a l parameters were, i n a l l c a s e s , i n f l u e n t i a l t o the r e s u l t s o f a n a l y s e s . I t was t h i s dilemma t h a t encouraged our i n v e s t i g a t i o n i n t o f i e l d d e s o r p t i o n . T h i s i n v e s t i g a t i o n has c e n t e r e d on two major o b j e c t i v e s : t o c o n t r o l the v a r i a b l e s o f a n a l y s i s i n order to gain a n a l y t i c a l r e p r o d u c i b i l i t y ; and t o s t u d y the n a t u r e o f the mechanism i n v o l v e d i n the i o n i z a t i o n p r o c e s s e s i n o r d e r t o more i n t e l l i g e n t l y apply the technique. The i n i t i a l problem i n v o l v e d the anode i t s e l f . T h i s d e v i c e f u n c t i o n s as the i o n e m i t t e r and i s com­ p r i s e d o f a t h i n c o n d u c t i n g m e t a l upon w h i c h sharp n e e d l e s or p o i n t s have been e t c h e d or d e p o s i t e d i n a process c a l l e d a c t i v a t i o n . In t h i s study, a c t i v a t i o n of a 10y t u n g s t u n w i r e was a c c o m p l i s h e d by the de­ p o s i t i o n o f m i c r o - n e e d l e s o f amorphous c a r b o n i n a method s i m i l a r t o t h a t proposed by Beckey ert a l . (15) . The p r o c e s s o f a c t i v a t i o n i s c r i t i c a l b o t h to the q u a l i t y o f s p e c t r a produced and the r e p r o d u c i b i l i t y o f the a n a l y s i s . An a u t o m a t i c d e v i c e was d e s i g n e d and f a b r i c a t e d t o f a c i l i t a t e the programming o f the temper­ a t u r e o f the w i r e as i t was h e a t e d i n a low p r e s s u r e environment o f b e n z o n i t r i l e . C a r e f u l c o n t r o l and d u p l i c a t i o n o f the t i m e , t e m p e r a t u r e and p r e s s u r e p a r a ­ meters have r e s u l t e d i n the p r o d u c t i o n o f u n i f o r m a c t i v a t e d e m i t t e r s w i t h a t y p i c a l average n e e d l e l e n g t h of 30μ. The two most commonly used methods o f sample ad­ d i t i o n t o the a c t i v a t e d e m i t t e r s a r e d i p p i n g and m i c r o s y r i n g e e x t r u s i o n (16-18). In the d i p p i n g method, the e m i t t e r i s i n s e r t e d i n t o a s o l u t i o n o f the sample and the amount a d h e r i n g t o the m i c r o - n e e d l e s a f t e r removal i s allowed to dry p r i o r to a n a l y s i s . A v a r i a t i o n of t h i s method i n v o l v e s the f r e e z i n g o f the sample on the e m i t t e r s u r f a c e a f t e r i n s e r t i o n i n t o the sample s o l u ­ t i o n ( 1 9 ) . T h i s p r o c e s s can be r e p e a t e d , a l l o w i n g the a c c u m u l a t i o n o f l a r g e r amounts o f sample on the e m i t t e r . D i p p i n g methods a r e c o n v e n i e n t , but a more r e p r o d u c i b l e q u a n t i t y o f sample can be a p p l i e d u s i n g a microsyringe. In t h i s method, a known volume o f sample s o l u t i o n i s e x t r u d e d from a c a l i b r a t e d s y r i n g e ( u s u a l l y i n the range o f 200 n a n o l i t e r t o one m i c r o l i t e r ) and a l l o w e d t o adhere t o the m i c r o - n e e d l e s on the e m i t t e r s u r f a c e . A c o n v e n i e n t v a r i a t i o n o f t h i s method p l a c e s the e x t r u d e d drop i n t o the r e g i o n between two p a r a l l e l a c t i v a t e d e m i t t e r s as shown i n F i g . 2. A f t e r a d d i t i o n , the e m i t t e r s a r e s l o w l y s e p a r a t e d , d u r i n g which time

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ETAL.

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Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch011

11.

Figure 2. Microsyringe sample addition using adjacent parallel emitters. Separation will result in the entire sample being sorbed to either one of the emitters.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch011

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the t o t a l drop w i l l c l i n g t o one or the o t h e r e m i t t e r . T h i s approach g r e a t l y i n c r e a s e s the p o s s i b i l i t y o f a drop b e i n g a p p l i e d t o and h e l d by an e m i t t e r ( 2 0 ) . The g r e a t e s t u n c e r t a i n t y i n the i n i t i a l s t u d i e s i n f i e l d d e s o r p t i o n mass s p e c t r o m e t r y was the absence o f an a c c u r a t e knowledge o f the t e m p e r a t u r e d u r i n g a n a l y sis. Heat i s a p p l i e d t o the sample by p a s s i n g a c u r r e n t t h r o u g h the e m i t t e r w i r e ; however, a l l o f the o r i g i n a l methods i n v o l v e d the manual s e t t i n g o f an uncalibrated dial. Even under t h e s e p r i m i t i v e cond i t i o n s , the need f o r e x a c t t e m p e r a t u r e c o n t r o l was not c r i t i c a l u n t i l r e p r o d u c i b l e e m i t t e r s were a v a i l a b l e . At t h a t t i m e an a p p e a l i n g a l t e r n a t i v e p r e s e n t e d i t s e l f : namely, t o c a l i b r a t e the c u r r e n t p a s s i n g t h r o u g h the e m i t t e r a g a i n s t t e m p e r a t u r e . The e m i t t e r c u r r e n t can t h e n be used as the means o f t e m p e r a t u r e measurement and c o n t r o l (21-23). T h i s was a c c o m p l i s h e d i n our l a b o r a t o r y by the d e s i g n and f a b r i c a t i o n o f an e m i t t e r c u r r e n t programmer (ECP) ( 2 2 ) . T h i s s o l i d s t a t e d e v i c e e n a b l e s the r e g u l a t i o n o f c u r r e n t a t any v a l u e between 0 and 85 ma t o the n e a r e s t 0.1 ma and makes p o s s i b l e a l i n e a r program between any two c u r r e n t v a l u e s a t a p r e s e l e c t e d r a t e . The a c c u r a t e c o n v e r s i o n o f t h e s e c u r r e n t v a l u e s i n t o t e m p e r a t u r e was a d i f f i c u l t and time consuming t a s k . F i g u r e 3 i l l u s t r a t e s the n a t u r e o f t h e c u r r e n t - t e m p e r a t u r e r e l a t i o n s h i p f o r the u n i f o r m a c t i v a t e d e m i t t e r s used i n t h i s s t u d y . The upper v a l u e s on t h i s c u r v e were o b t a i n e d v i a an o p t i c a l p y r o m e t e r w h i c h v i e w e d the e m i t t e r s t h r u a s p e c i a l s a l t window mounted i n an e x p e r i m e n t a l i o n s o u r c e c o n s t r u c t e d f o r r e s e a r c h o b j e c t i v e s . The m i d d l e p a r t o f the c u r v e was d e t e r m i n e d by u s i n g r e s i s t a n c e measurements o f the e m i t t e r w i r e t o e x t r a p o l a t e between the upper and l o w e r r e g i o n s . The lower p a r t o f the c u r v e was o b t a i n e d by m e l t i n g a s e r i e s o f known compounds upon the e m i t t e r s u r f a c e . T a b l e 1 i l l u s t r a t e s the r e p r o d u c i b i l i t y a c h i e v a b l e w i t h t h e ECP on m u l t i p l e a n a l y s i s u s i n g the same emitt e r and on s u c c e s s i v e a n a l y s e s u s i n g d i f f e r e n t e m i t t e r s . T h i s d e v i c e , f o r the f i r s t t i m e , a l l o w e d c o n t r o l o f the t e m p e r a t u r e o f the sample d u r i n g a n a l y s i s and provided a reasonable estimate of i t s a c t u a l value. An a d d i t i o n a l b e n e f i t o f the ECP i s r e a l i z e d when a l i n e a r t e m p e r a t u r e s c a n i s p e r f o r m e d w i t h the mass s p e c t r o meter b e i n g c y c l i c a l l y scanned by the d a t a system. The r e s u l t i n g s t o r e d d a t a f i l e s can o u t p u t mass chromatograms i n f o r m a t s s i m i l a r t o those o f gas chromatography-mass s p e c t r o m e t r y w i t h the e x c e p t i o n t h a t the a b s c i s s a i s t e m p e r a t u r e and not e i u t i o n t i m e . Mixtures whose components desorb a t d i f f e r e n t t e m p e r a t u r e s can

SWEELEY E T A L .

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Field Desorption Mass Spectrometry

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be r e s o l v e d i n t i m e , and c o - d e s o r b i n g compounds c a n be r e s o l v e d by mass d i f f e r e n t i a t i o n . Figure 4 i l l u s t r a t e s the t y p e o f o u t p u t o b t a i n e d from a l i n e a r t h e r m a l s c a n o f t h e n u c l e o s i d e , a d e n o s i n e . The p o i n t s on t h e c u r rent V £ . i n t e n s i t y curve o f the t o t a l i o n i n t e n s i t y ( T i l ) a r e c r e a t e d by summing a l l o f t h e i o n i n t e n s i t i e s f o r each scan. I n t h e i n s e r t i n F i g . 4, t h i s q u a n t i t y i s p l o t t e d against the current a x i s along w i t h the i o n i n t e n s i t i e s o f m/e 267 (M ) and m/e 268 ( M + l ) i n a format s i m i l a r t o t h a t o f mass cKromatograms. Although we have no p r e f e r e n c e , some a u t h o r s r e f e r t o t h i s type o f o u t p u t as a thermogram ( 2 1 2 3 ) . The mass spectrum shown i n F i g . 4 was t a k e n a t t h e BAT, s c a n number 23. N o t i c e t h e dominance o f t h e m o l e c u l a r i o n c l u s t e r i n the spectrum. T h i s i s v e r y t y p i c a l o f FD s p e c t r a . +

+

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch011

1

T a b l e 1.

Wire#

REPRODUCIBILITY OF EMITTER CURRENT PROGRAMMER (ECP). R e p r i n t e d w i t h p e r m i s s i o n from final. Chem., 48, 428 (1976). Copyright American ChemicaT~~Society. C u r r e n t (mA)

# o f Measurements

1

19.2 ± 0.4

5

2

21.3 ± 0.4

5

3

20.3

1

4

21.0

1

5

20.2

1

F i g u r e 5 c o n t r a s t s t h e e l e c t r o n impact ( E I ) spectrum o f a s y n t h e t i c n u c l e o s i d e w i t h i t s FD spectrum. A g a i n t h e dominance o f t h e m o l e c u l a r i o n s p e c i e s i n t h e FD spectrum i s o b s e r v e d w h i l e t h e same s p e c i e s i s h a r d l y d e t e c t a b l e i n t h e E I spectrum. F i g u r e 6 comp a r e s t h e E I and FD s p e c t r a o f g l u t a m i c a c i d . The m a j o r FD peak f o r t h i s compound i s t h e M+l i o n , a s i t u a t i o n n o t uncommon w i t h o t h e r amino a c i d s . T y r o s i n e , however, as shown i n F i g . 7, e x h i b i t s no molecu l a r i o n s i n t h e E I spectrum and a v e r y l a r g e M i o n i n t h e FD spectrum. P r o t o n a t i o n t e n d e n c i e s would n o t p r e d i c t t h i s o b s e r v a t i o n . Q u i t e p o s s i b l y t h e nonb o n d i n g e l e c t r o n p a i r on t h e p h e n o l i c h y d r o x y l i s involved i n the i o n i z a t i o n of t h i s species v i a a t r u l y f i e l d dependent quantum m e c h a n i c a l mechanism. +

216

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I50 Λ

OH

1

; MW

20H

;a

OH 342

' I ' I ' I ' f ' I ' I '

50

100

150

200

250

300

Figure 8. The field desorption spectra of galactitol and sucrose

-37

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100

m/e Figure 9. The field desorption spectrum of sodium barbiturate at a temperature above the BAT showing cationization

lOO-i

30

mA

(M

+2K)

301

80604020' I ' I ' I

50

1

I

1

I

1

I ' I

100

\ • I ' I ' I ' \ ' I ' I ' I ' I ' I ' I ' I ' I

150

m/e

200

250

1

300

Figure 10. Field desorption spectrum of the potassium salt of naphthyl sulfate. Sample furnished by V. Rheinhold of Arthur D. Little Co., Cambridge, MA.

11.

SWEELEY E T AL.

EMITTER CURRENT (mA) ^

Field Desorption Mass Spectrometry

221

ANODE TEMP.

30 3 2 0 °

25^

.250 201.240*

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch011

Î220* 15

APPLIED VOLTAGE(KV) 10 Biomedical Mass Spectrometry

Figure 11. The dependence of the BAT on the applied electric field

Figure 12. Ion source geometries for field desorption (FD) and electron impact ionization using afielddesorption emitter (EI/D)

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MASS

SPECTROMETRY

has been t h e same as BAT, and t h e y b o t h a r e independent of t h e a p p l i e d f i e l d s t h r o u g h o u t the range from 14KV to 3KV. The 3KV a c t s as a minimum f o r t h e s e e x p e r i m e n t s s i n c e t h i s i s t h e a c c e l e r a t i n g v o l t a g e f o r the mass s p e c t r o m e t e r b e i n g u s e d , a V a r i a n CH-5-DF. T h i s p r e ­ s e n t s a dilemma, o f c o u r s e , s i n c e t h e v o l t a g e f i e l d s t h a t a r e needed t o c o l l e c t , a c c e l e r a t e and f o c u s t h e i o n s i n a m a g n e t i c s e c t o r mass s p e c t r o m e t e r may a l s o be involved i n ion production. This i s a s i t u a t i o n akin to a p e r s o n a t t e m p t i n g t o f i n d out what goes on i n the dark. I n o r d e r t o see a n y t h i n g , he must somehow i l ­ l u m i n a t e t h e r e g i o n d u r i n g w h i c h time i t i s no l o n g e r dark. Ion g e n e r a t i o n i n t h e much l o w e r f i e l d s o f the s o u r c e o f a q u a d r u p o l e mass s p e c t r o m e t e r would be an i d e a l way t o e x t e n d t h e s e s t u d i e s . Below t h e t h r e s h o l d o f t h e BAT, measurable amounts of d e s o r p t i o n were not found t o o c c u r even f o r p r o ­ l o n g e d p e r i o d s o f t i m e and w i t h h i g h a p p l i e d f i e l d s . These r e s u l t s s t r o n g l y i n d i c a t e t h a t the f i e l d i s not of major s i g n i f i c a n c e i n the d e t e r m i n a t i o n o f t h e temperature at which d e s o r p t i o n o c c u r s . A s e r i e s of e x p e r i m e n t s was c o n d u c t e d t h e n t o r e s o l v e the e f f e c t o f t h e f i e l d on t h e r a t e o f d e s o r p t i o n . I n t h e s e ex­ p e r i m e n t s , t h e r a t e o f d e s o r p t i o n , w i t h the FD f i e l d on, was compared w i t h t h e r a t e o f d e s o r p t i o n w i t h t h e f i e l d s o f t h e mass s p e c t r o m e t e r o f f . F i g u r e 13 i l ­ l u s t r a t e s one t y p e o f experiment from t h i s s e r i e s . F i g u r e 13A shows t h e normal t h e r m a l d e s o r p t i o n c u r v e o b t a i n e d by a l i n e a r t e m p e r a t u r e scan under s t a n d a r d FD conditions. In t h e r e m a i n i n g c u r v e s , the t h e r m a l scan was r e p e a t e d w i t h the same amount o f sample on t h e e m i t t e r ; however, a l l o f the v o l t a g e f i e l d s o f the mass s p e c t r o m e t e r were l e f t o f f u n t i l the p o i n t s marked were r e a c h e d , I f o r Β, I I f o r C, and I I I f o r D. The scans were t h e n completed as i n c u r v e A. T h i s experiment i n d i c a t e s t h a t t h e sample i s l e a v i n g a t a r a t e t h a t i s independent o f t h e a p p l i e d f i e l d s . Within experimental e r r o r , no e f f e c t o f t h e f i e l d was o b s e r v e d f o r any o f the compounds examined. I n summary, n e i t h e r the tem­ p e r a t u r e a t w h i c h d e s o r p t i o n o c c u r r e d nor t h e r a t e o f mass t r a n s f e r from the e m i t t e r w i r e i n t o t h e s p e c t r o ­ meter vacuum were i n f l u e n c e d by t h e h i g h e l e c t r i c field. C l e a r l y the f i e l d image t h e o r y does not ac­ commodate t h e s e o b s e r v a t i o n s . In a n o t h e r s e r i e s o f e x p e r i m e n t s c o n d u c t e d a t a c o n s t a n t emitter tempera£ure, the r a t i o o f i o n i n t e n ­ s i t i e s between M and MH remained i n v a r i e n t o v e r the e n t i r e a p p l i e d f i e l d range; and t h e i o n p a t t e r n s o f the s p e c t r a produced a l s o remained unchanged w i t h i n the r e p r o d u c i b i l i t y o f t h e system. On the o t h e r hand, +

SWEELEY

E T AL.

Field Desorption Mass Spectrometry

223

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch011

MASS TRANSFER AS A FUNCTION OF EMITTER CURRENT (TEMPERATURE)

Biomedical Mass Spectrometry

Figure 13. Measurement of mass transfer in the presence of and the absence of applied voltages

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch011

224

HIGH

PERFORMANCE

MASS

SPECTROMETRY

changes i n e m i t t e r t e m p e r a t u r e , s o l v e n t used i n p r e ­ p a r a t i o n , and t h e p r e s e n c e o f s m a l l amounts o f i n ­ organic c a t i o n s s i g n i f i c a n t l y a l t e r e d the nature o f the i o n s p r o d u c e d . Throughout t h e s e s t u d i e s , changes i n chemical parameters a l t e r e d the r e s u l t s o f the a n a l y s i s w h i l e changes i n t h e a p p l i e d f i e l d o n l y v a r i e d t h e absolute i n t e n s i t i e s of the ions detected but not t h e i r ratios. Based upon t h e a n a l y s e s o f our e x p e r i m e n t a t i o n and the o b s e r v a t i o n s o f many o t h e r s , we have p r o p o s e d an a l t e r n a t e mechanism f o r t h e g e n e r a t i o n o f many o f t h e ions that a r e observed i n f i e l d d e s o r p t i o n a n a l y s i s ( 2 5 ) . F o r c o n v e n i e n c e , t h e s i t u a t i o n on t h e e m i t t e r f o r a s i n g l e o r g a n i c compound o f t y p i c a l p u r i t y c a n be c l a s s i f i e d i n t o f o u r s t a g e s d u r i n g a n a l y s e s , as shown i n F i g . 14. The f i r s t s t a g e o c c u r s a t t e m p e r a t u r e s below t h e BAT. No i o n s a r e o b s e r v e d even a t t h e h i g h ­ e s t f i e l d s a v a i l a b l e by t h e i n s t r u m e n t . The second stage o c c u r s a t t h e BAT. A t t h i s t e m p e r a t u r e t h e o r g a n i c compound b e g i n s t o m e l t , c h a n g i n g i n t o a l i q u i d m o b i l e phase. Exposed t o t h e h i g h vacuum o f t h e mass spectrometer, thermal d e s o r p t i o n begins. In this expanding mixed phase, o f s o l i d - l i q u i d - g a s (semi­ f l u i d ) , i o n s a r e g e n e r a t e d by t h e r m a l l y induced chemi­ c a l r e a c t i o n s . At these temperatures, the m o b i l i t y o f the o r g a n i c component may g e n e r a t e m o l e c u l a r i o n s and c l u s t e r s o f m o l e c u l a r s p e c i e s . I f s m a l l amounts o f i n o r g a n i c c a t i o n s a r e p r e s e n t , which i s o f t e n t h e c a s e , i o n s o f t h e type Μ Θ and Μ θ , where θ i s t h e i n o r g a n i c c a t i o n , w i l l be formed by e l e c t r o p h i l i c a t t a c k o f t h e c a t i o n s upon t h e n e u t r a l s p e c i e s . A t these e l e v a t e d t e m p e r a t u r e s , e l e c t r o p h i l i c attachment i s n o t a d i a b a t i c but e n d o t h e r m i c ( 2 6 ) , a c o n d i t i o n t h a t c o i n c i d e s w i t h the o b s e r v a t i o n o~F^.ncreased c a t i o n i z a t i o n a t h i g h e r emitter currents. During s t a t e I I I , the temperature i s e l e v a t e d t o the p o i n t where t h e c a t i o n m o b i l i t y i s s u f f i c i e n t t o a l l o w t h e d e t e c t i o n o f o n l y c a t i o n i z e d m o l e c u l a r spe­ cies. Ion c l u s t e r s , c o n t a i n i n g two o r more c a t i o n s , b e g i n t o appear. A t t h i s h i g h e r t e m p e r a t u r e , f r a g ­ m e n t a t i o n o f t e n i n c r e a s e s and t h e appearance o f a l a r g e r number o f fragment i o n s o c c u r s . I f t h e r e a r e a p p r e c i a b l e amounts o f i n o r g a n i c s a l t s , t h e spectrum may become dominated by t h e h i g h l y mobile c a t i o n . This stage occurs a t the h i g h s i d e o f the BAT and t h e o r g a n i c s p e c i e s i s r a p i d l y t h e r m a l l y desorbed i n t o t h e vacuum. I n t h e f i n a l s t a g e , encompassing t h e h i g h e s t t e m p e r a t u r e s , i o n s and c l u s t e r i o n s a r i s i n g from t h e i n o r g a n i c l a t t i c e appear. These i o n s a r e v e r y s i m i l a r +

2

+

SWEELEY

Field Desorption Mass Spectrometry

E T AL.

THIN

STATE

I

FILM IONIZATION M E C H A N I S M

REACTION

SOLID

TYPE

IONS DETECTED

STATE

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch011

NONE

I

SEMI-FLUID

SOLUTE

NONE

MOBILITY

M+M-*2M 2M — M*+M~ + 2Μ+Θ-Μ2Μ+Θ)

IDC SEMI-FLUID

(Μ+®) +θ-Η—(Μ+2θ)

^α®ύ*

M+H — (M+H)

Ε Γ FLUID LATTICE r

+

+

(M4-H) (M+©)

+

ι

>

s 1

*ΰ

Ο S

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch012

12. BRENT ET AL.

Applications in a Pharmaceutical Lab 237

238

HIGH

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch012

5.

M.W.

PERFORMANCE

MASS

SPECTROMETRY

277

determined from peak heights of the molecular ions: tri2.8

di10.0

mono-methylated (5) 8.2 ~

(5) l77

The accuracy of t h i s measurement i s dependent on the following assumptions: a l l the ion current i s contained i n the M ions; no d i s t i l l a t i o n effect has occurred, i^.e. the vapor phase concentrations are the same as those i n the s o l i d state; and the ionization cross sections of the molecules analyzed are the same. The fragmentation of (1) was studied as a model for methyl­ ated (5). The mass spectrum of (1) has prominent peaks at m/e 290 (Μ), 275 (M-CH ); 259 (M-CH 0), and 123. The accurate mass measurement of m/e 123 showed the ion composition to be 05Η7Ν . This ion contains the pyrimidine and benzylic methylene portions of (1). Using accelerating voltage scans with the magnetic and e l e c t r i c sector constant, i t was determined that m/e 290, 275, and 259 a l l fragmented to y i e l d m/e 123. To answer the o r i g i n a l question as to where the f i r s t methylation occurs on (5), the monomethylated species were examined without p u r i f i c a t i o n using defocused metastable ions, (the DADI or MIKES method). A l l monomethylated products have a molecular ion at m/e 291. Since these ions are known to fragment losing the benzene portion of the molecule, those molecular ions that fragment yielding m/e 138 are methylated on the pyrimidine portion of the molecule and those molecular ions that y i e l d m/e 124 are methylated on the benzene portion of the molecule. The compositions of m/e 291, 138 and 124 were confirmed by accurate mass measurement. One cannot make a conclusion about the o r i g i n of m/e 124 and 138 without the use defocused metastable ions or by examining only p u r i f i e d mono­ methylated species. For example m/e 124 would be generated from the molecular ion of (5) as well as monomethylated (5), and m/e 138 can be generated from the molecular ion of either 3

4

3

12.

BRENT

239

Applications in a Pharmaceutical Lab

ET AL.

CH 0 3

C

CH3O

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch012

m/e

H

2 y ^ N H

m/e 124

291

OCH3 C H

2V^NH

Y^NH \N^NHCH m/e

^N^NHCH

3

3

m/e 138

291

d i - or monomethylated (5). The DADI spectrum i s given i n Figure 5. The DADI spectrum of m/e 291 shows a t r a n s i t i o n , m/e 291 -> m/e 138 but not m/e 291 -* 124. Within the l i m i t s of detection of"the technique, Tt appears that of the monomethylated portion of the crude mixture, methylation occurs only on the pyrimidine section of the molecule. The Use Of Defocused Metastable Ions In Defining Structure The use of metastable ions to define a parent/daughter relationship can also be useful i n structure problems. For example, we were asked i f we could distinguish (6) and (7) by mass spectrometry. The electron impact mass spectrum of the compound analyzed gave a molecular ion and fragmentation pattern which would f i t either (6) or (7) with the expection of Μ- (^Η Ν0 . The elemental composition of t h i s ion was confirmed by accurate mass measurement. Structure (7) could lose N=C-C0 Et d i r e c t l y to y i e l d M- C4H5NO2. However, (6) could give the same ion by sequential loss o f CH =CH2 by a McLafferty rearrangement yielding a -C0 H which loses C0 followed by the loss of HCN. Although there are some exceptions, i t i s f e l t that metastable transitions are one step losses and usually occur i n one section of the molecule (12,13). The molecular ion was examined by DADI and shown toTragment d i r e c t l y to Μ- 0 Η Ν0 , thus favoring sturcture (7). However, other than the possible errors already 5

2

2

2

2

2

4

5

2

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch012

240 HIGH PERFORMANCE MASS SPECTROMETRY

•3

!

1

1 «**

g

5 ε

ι

δ

ίο

!

12.

BRENT

E T AL.

Applications in a Pharmaceutical Lab

241

0 H

OH

^-OCHgCr^

N

0

c

H

L

H N 2

*

ι CH

;

CH

3

H-0-CH CH

Τ

2

N~~ CH

3

0

3

3

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch012

7.

mentioned, molecules may rearrange thermally during v o l a t i l i ­ zation or a f t e r i o n i z a t i o n to a new structure from which the metastable ion i s formed. So although t h i s data was an early indication f o r structure (7), other physical and chemical evidence were required. Subsequent physical and chemical evidence supported the mass spectrometeric results. Analysis Of A Mixture V i a GC-MS A protocol f o r inducing l i v e r microsomal a c t i v i t y was established using Aroclor® 1254 l o t RCS9999 as the inducing agent. When a new l o t of t h i s material, RCS088, was purchased i t was noted that the physical properties were different. RCS9999 was yellow and s l i g h t l y viscous; RCS088 was clear and very viscous. The l a t t e r being more t y p i c a l of Aroclor® 1254. The problem was to determine the difference between these two l o t s of material and i f possible recreate l o t RCS9999. The b i o l o g i c a l a c t i v i t y data could not be used conveniently because these tests were too time consuming. I t was feared that the two materials would d i f f e r i n t h e i r capacity to induce l i v e r microsomal a c t i v i t y . Therefore a physical method was sought to distinguish any differences i n the two l o t s of material. Aroclor® i s a mixture of polychlorinated hydrocarbons. The f i r s t two d i g i t s of the numbers following the name refer to the numbers of carbons i n the system which are chlorinated and the l a s t d i g i t s refer to the percentage of chlorine i n the mixture (14). A l i t e r a t u r e procedure was followed to separate Aroclor® into i t s components by gc (15). The more highly chlorinated components elute l a s t . Tïïe column used was 6 f t χ 2 mm 3% OV-1 on Gas Chrom. Q 100/120 mesh. The column was held at 100° f o r 8 min and brought to 200° at 4°/min and held. The chromatogram of RCS 088 was clean f o r the f i r s t few minutes. At higher temperatures, at least 15 components eluted. RCS9999 had the same high temperature p r o f i l e but had over 10 components i n the low temp section of the chromatogram. These peaks could arise from trichlorobenzenes (a common diluent

242

HIGH

PERFORMANCE

MASS

SPECTROMETRY

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch012

of Aroclor®), less highly chlorinated biphenyls, or other materials. RCS9999 was examined by gc-ms and a combustion analysis was carried out f o r percent chlorine. Via gc-ms, the peaks which eluted f i r s t were i d e n t i f i e d as mono-, d i - , t r i - , and tetra-chlorinated biphenyls. The combustion analysis indicated 46.55% chlorine. The gas chromatograms of Aroclor® 1254, 1248 and 1242 were examined. A blend of 2 parts of 1242 and 1 part of 1254 was most l i k e RCS9999. These results were returned to the researcher i n i t i a t i n g the problem. The blend and l o t RCS088 are currently being tested to compare t h e i r a c t i v i t y i n inducing l i v e r microsomes to the a c t i v i t y of lot RCS9999. Metabolite Analysis Using Mass Spectrometry Mass spectrometry plays an important role i n structure elucidation of drug metabolites. The small amounts of material isolated i n these studies are s t i l l usually within the capabili t i e s of a mass spectrometer. The metabolites are often monitored by some tag throughout t h e i r i s o l a t i o n and p u r i f i c a t i o n , radio l a b e l , stable isotope l a b e l , s p e c i f i c chromaphore, etc. Drug biotransformation and biochemical conjugations are normally known processes (16). The compound being examined i s usually known to be a metabolite, and often the gross structural features of the parent drug are s t i l l present. The mass spectrum of the parent drug i s studied carefully by low and high resolut i o n mass spectrometry and defocused metastable ion techniques. The fragmentation of the parent i s used for a model for the metabolite. The reinvestigation of a t i c system used to isolate the metabolites of trimethoprim (1) led to the discovery of a new metabolite (17). The compound had a fluorescence on t i c similar to trimethoprim 1-oxide, a known metabolite. The mass spectrum of the material indicated a molecular weight 16 Daltons greater than the parent drug. The elemental composition of parent plus oxygen was further supported by accurate mass measurement of

OCH

8.

3

12.

BRENT

ET AL.

243

Applications in a Pharmaceutical Lab

m/e 306. The molecular ion fragmented by losing oxygen, and the remaining spectrum looked l i k e that of the parent drug. The loss of oxygen from the molecular ion was indicative of the oxygen residing on Ν and not on carbon. The lack of an M-2 appeared consistent with N-K) and not -NH-OH. Since the 1-oxide was a known metabolite which had a different R on t i c than t h i s product, the metabolite was l i k e l y the 3-oxiae, (8). The i r , uv, mass spectrum, and chromatographic properties of the metabolite were i d e n t i c a l with synthetic (8).

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch012

£

Analysis Of Natural Products Using F i e l d Desorption Mass Spectrometry F i e l d desorption mass spectrometry (fdms) i s a gentle method of ionizing molecules from the s o l i d state (18). A solution or suspension of the compound to be analyzed i s coated on an emitter. The emitter i s a 10 yM tungsten wire on which carbon dendrites are grown. The emitter i s introduced into the source on the end o f a direct probe. Most of the solvent evaporates during the insertion of the direct probe into the source. Several thousand v o l t s are placed on the emitter. In addition a small heating current can be applied. Under the influence of the high f i e l d and gentle heating, molecules are ionized by the loss of an electron to the emitter surface or by attachment of a cation (19). This technique has extended the range of compounds which can be examined by mass spectrometric methods to include thermally l a b i l e and/or non- v o l a t i l e molecules (18). For many samples, fdms eliminates the need f o r derivatizing the target molecule. F i e l d desorption mass spectrometry, nuclear magnetic resonance (nmr) spectroscopy, and high pressure l i q u i d chromato­ graphy (hplc) were used to determine the structure of a compound isolated from toad erythrocyte ghosts which inhibited adenylate cyclase. This product gave one uv absorbing peak on a DEAEcellulose ion-exchange column (20). Its molecular weight was estimated to be approximately 500 Daltons by Bio-Gel P-4 chromatography. Digestion with RNA-ase, DNA-ase, phospholipase A and C, papain, typsin, chymotrypsin, 5 -nucleotidase, spleen and snake venom phosphodiesterases, beef and heart c y c l i c nucleotide phosphodiesterase did not i n h i b i t the a c t i v i t y of t h i s compound. Some peptidases and alkaline phosphatase produced diminished a c t i v i t y . Therefore, nucleotides and peptides were suspected to be possible subunits of the b i o l o g i c a l l y active material. An early experiment with electron impact mass spec­ trometry had produced no information. An hydrolysis followed by amino acid analysis indicated the presence of a-aminoadipic acid. Since electron impact mass spectrometry had not worked, f i e l d desorption mass spectrometry was attempted. A summary of the ions seen i s given below. Only integer mass values were obtained, and the isotope peaks were too weak or complicated by 1

244

HIGH P E R F O R M A N C E MASS S P E C T R O M E T R Y

[M+H] ions to use to determine elemental composition. Also when t h i s work was carried out we had no means of adding spectra to improve the signal to noise r a t i o . Therefore we manually counted the number of times an ion appeared i n spectra taken at one milliamp increments of emitter heating current above the f i r s t evidence of ions as observed on the t o t a l ion current monitor. (For a more detailed explanation of the mechanics involved i n obtaining an f i e l d desorption mass spectrum, the authors suggest reading reference 18.) Compound assignment was based on integer mass values f i t t i n g known amino acid or nucleotide molecular weights.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch012

Table I

F i e l d Desorption Mass Spectrum of the Adenylate Cyclase Inhibitor

m/e

No. o f times the ion was seen.

Comment

90

(6)

Alanine + H

98/99

(3)

[H P0J /[H P0t ]

122

(8)

Cysteine + H

135

(4)

Adenine

144

(1)

251, 252, 253

(4)

?

268

CD

Adenosine

303/304

(2)

314/315

(2)

332

(2)

348

(2)

354

(4)

+

3

lt

+

f

?

? ? ?

Adenosinemonophosphate ?

12.

BRENT

Applications in a Pharmaceutical Lab

ET AL.

245

No α-amino adipic acid was seen. A repeat of the hydrolysis/ amino acid analyzer experiment showed an error; alanine was misassigned as α-aminoadipic acid. The p o s s i b i l i t y of the presence of cysteine was not confirmed by the hydrolysis experi­ ment. The presence of nucleosides and-tides was supported by the f i e l d desorption experiment. A fourier transform proton nmr was run on the material. Two different substituted adenines yielding a single proton at 68.14 were seen. A t r i p l e t at 66.48 and a doublet at 66.02 were observed. These signals were thought to be due to the l CH of a 2 -deoxysugar and the l C H of a 2 -oxysugar. Looking back at the f i e l d desorption results, some unassigned peaks could f i t 2 -deoxy AMP, i ^ e . m/e 251, 252, 253 and 332. The nmr signals at 66.48 and 66.02 were approximately of equal intensity. Since the unknown contained a mixture of nucleotides, the sample was subjected to hplc using a Partisil-10-SAX anion exchange column. Elut ion with phosphate buffer was known to separate nucleoside monophosphates with good resolution. The hplc results indicated two large 254 nm absorbing peaks of equal intensity eluting i n the monophosphate region (both of which had u.v. spectra i d e n t i c a l to 5 -AMP) and a small peak eluting at the region of ATP. Mixing experiments showed the f i r s t peak to be AMP. This component was destroyed by reaction with periodate. The second peak cochromatographed with 2 -deoxy AMP and was not affected by periodate oxidation. These peaks were collected and tested f o r b i o l o g i c a l a c t i v i t y . The inhibitory a c t i v i t y remained with the "2 -deoxy AMP" peak. At t h i s point synthetic 2 -deoxy-5 -AMP was tested and found to be inactive. A 5 -nucleotide would be contrary to the undimin­ ished a c t i v i t y after treatment with 5-nucleotidase. However, the reason that the 5 -nucleotidase experiment may have been i n error i s that the enzyme might have special requirements such as the presence of a 2 -hydroxy group. Work then branched i n two directions. Enzyme data s t i l l pointed to the p o s s i b i l i t y of amino acids or peptides linked to the active molecule. The nucleotide was examined f o r amino acids by hydrolysis followed by chromatography on an amino acid analyzer or t r i m e t h y l s i l y l a t i o n , then gc or gc-ms. In addition more enzymes were tested on the peak isolated by hplc. I t was found that rye grass 3 -nucleotidase diminished the peak height on hplc yielding deoxyadenosine. Therefore, synthetic 2 -deoxy3 -AMP was tested. I t had the same inhibitory a c t i v i t y against adenylate cyclase and the same chromatographic properties as the material isolated from toad erythrocyte ghosts. The compound i n question i s 2 -deoxy- 3 -AMP. The only piece of structural uncertainty i s i t s r e a c t i v i t y with peptidase. This piece of evidence, l i k e l y caused by impure enzyme, delayed the structure assignment by giving a false lead. However, i f i t weren t for the other enzyme data, the problem would be orders of magnitude more d i f f i c u l t . f

1

f

f

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch012

f

f

f

f

f

f

1

1

1

1

f

f

f

f

f

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch012

246

high performance mass spectrometry

Summary We have n o t attempted t o survey the e n t i r e scope o f mass spectrometry as a p p l i e d t o the pharmaceutical i n d u s t r y . Rather we have taken a c r o s s s e c t i o n o f problems t o i l l u s t r a t e the u t i l i t y o f h i g h performace mass spectrometry t o problems found i n the i n d u s t r y . The use o f h i g h r e s o l u t i o n mass spectrometry, defocussed metastable i o n techniques, coupled gas chromatographymass spectrometry, and the new i o n i z a t i o n technique o f f i e l d d e s o r p t i o n have been h i g h l i g h t e d . I n a l l cases s u c c e s s f u l s o l u t i o n t o a problem depended on good communication between researchers t o e s t a b l i s h the h i s t o r y o f the problem, d e f i n e the g o a l s , and t o p l a n the experiments which might l e a d t o a solution. In a l l cases, although mass spectrometry was emphas i z e d , other p h y s i c a l methods as w e l l as wet chemistry p l a y e d v i t a l i n t e r a c t i v e r o l e s i n the problem's s o l u t i o n . The h i g h performance mass spectrometer may have been circumvented i n many o f these problems, but t h i s would be a t the expense o f sample and time. The savings o f time and the a b i l i t y t o work w i t h extremely s m a l l q u a n t i t i e s o f sample are the keys t o the impact o f modern instrumentation. Acknowledgement Îhe authors wish t o acknowledge the h e l p f u l a s s i s t a n c e on s e v e r a l problems r e f e r r e d t o i n the t e x t by Drs. R.W. Morrison, D. C l i v e , and J.W. F i n d l a y and Mr. R.F. Butz.

References 1. Biemann, Κ., "Mass Spectromety", McGraw Hill Book Co., Inc., New York, 1962. 2. Waller, G.R., "Biomedical Applications of Mass Spectrometry", Wiley-Interscience, New York, 1972. 3. Junk, G.A., Int. J . Mass Spectrum. Ion Phys. (1972) 8, 1. 4. Sweeley, C.C., Elliot, W.H., Fries, I. and Ryhage, R., Anal. Chem., (1966) 38, 1549. 5. Boulton, A.A. and Majer, J.R., Can. J . Biochem. (1971) 49, 993. 6. Snedden, W. and Parker, R.B., Anal. Chem. (1971), 43, 1651. 7. Brent, D.A. and Yeowell, D.A., Chem. Ind. (1973), 190. 8. Cresswell, R.M. and Mentha, J., U.S. Patent 3513185, 19 May, 1970; Hoffer, Μ., U.S. Patent 3341541, 12 September, 1967. 9. Beynon, J.H., Cooks, R.G., Amy, J.W., Baitinger, W.E. and Ridley, T.Y., Anal. Chem., (1973), 45, 1023A. 10. Cooks, R.G., Beynon, J.H., Caprioli, R.M. and Lester, G.R., "Metastable Ions", Elsevier Scientific Pub. Co., Amsterdam, New York, 1973. 11. Brent, D.A. and Rouse, D.J., Varian CH5DF/MAT 311 Application Note No. 12, (1973).

12.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch012

12. 13.

BRENT

E T AL.

Applications in a Pharmaceutical Lab

247

Jennings, K.R., Chem. Commun., (1966), 283. Letcher, R.M. and Eggers, S.H., Tetrahedron Lett. (1967), 3541. 14. Armour, J.Α., J. Chromatogr. (1972), 72, 275. 15. Hirwe, S.N., Borchard, R.E., Hansen, L.G. and Metcalf, R.L., Bull. Environ. Contam. Toxicol. (1974), 12, 138. 16. LaDu, B.N., Mandel, H.G. and Way, E.L., eds., "Fundamentals of Drug Metabolism and Drug Disposition", The Williams and Wilkins Co., Baltimore, Md., 1971. 17. Sigel, C.W. and Brent, D.A., J . Pharm. Sci., (1973) 62, 674. 18. Brent, D.A., J . Assoc. Off. Anal. Chem. (1976) 59, 1006. 19. Holland, J . F . , Soltmann, B. and Sweeley, C.C., Biomed. Mass Spectrom. (1976) 3, 340. 20. Sahyoun, N., Schmitges, C.J., Siegel, M.I. and Cuatrecasas, P., Life Sci. (1976) 19, 1961. Received December 30, 1977

13 Detection and Identification of Minor Nucleotides in Intact Deoxyribonucleic Acids by Pyrolysis Electron Impact Mass Spectrometry J. L. WIEBERS Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch013

Department of Biological Sciences, Purdue University, West Lafayette, IN 47907

It has been demonstrated previously that intact DNA or polydeoxyribonucleotides can be subjected to mass spectrometric analysis without prior derivatization or chemical or enzymatic hydrolysis (1,2). When a polydeoxyribonucleotide is exposed to pyrolysis electron impact mass spectrometry (Py-EI-MS), the primary fragmentation process involves cleavage at the phosphodiester bonds linking the nucleotide residues. Subsequent fragmentations result in ion products which are diagnostic for the common nucleotide components of the polynucleotide (3). Ribooligonucleotides are not susceptible to Py-EI-MS and remain nearly inert under such conditions. Presumably, this different behavior is due to the presence of the 2'-hydroxyl group in the ribo compounds. In the original scheme (1) for the cleavage of deoxyribopolynucleotides, i t is proposed that the fragmentation involves extrusion of the 3'and 5'-phosphodiester entities from the polynucleotide, and that extrusion of the oxygen functions in the 3'- and 5'-positions is more energetically favored in deoxyribo compounds than in ribo compounds. This appears to be a reasonable i f not a complete explanation of the process. Similar types of fragmentations of nucleic acids have been observed in pyrolysis field desorption mass spectrometry (Py-FD-MS) (4,5,6). These studies demonstrated that DNA and RNA can be analyzed by Py-FD-MS, and reaction mechanisms involved in the pyrolysis process were proposed. High resolution analysis permitted the assignment of probable structures to the pyrolysis products of the sugar and phosphate moieties as well as the nucleic acid components. In more recent investigations (7,8), high resolution (HR) Py-FD-MS yielded structural information on the ion ©0-8412-0422-5/78/47-070-248$05.00/0

WIEBERS

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch013

13.

249

Minor Nucleotides in Intact DNA

p r o d u c t s from u n p r o t e c t e d r i b o - d i n u c l e o t i d e s . E v i ­ dence f o r the f o r m a t i o n o f n u c l e o s i d e c y c l o p h o s p h a t e s from the r i b o d i n u c l e o t i d e s p e r m i t t e d t h e d i f f e r e n t i a ­ t i o n o f d i n u c l e o t i d e sequence i s o m e r s . The a p p l i c a ­ t i o n o f the HR-Py-FD-MS method t o t h e base sequence a n a l y s i s o f o l i g o n u c l e o t i d e s has been p r o p o s e d ( 7 , 8 ) . Low r e s o l u t i o n p y r o l y s i s e l e c t r o n impact mass s p e c t ­ r o m e t r y (LR-Py-EI-MS) has been s u c c e s s f u l l y employed f o r t h e sequence d e t e r m i n a t i o n o f d i - , t r i - , and t e t r a d e o x y r i b o - o l i g o n u c l e o t i d e s by a p p l y i n g c o m p u t e r i z e d pattern r e c o g n i t i o n a n a l y s i s to the s p e c t r a l r e s u l t s ( 3 , 9 ) . An i n t r i g u i n g s t u d y on t h e a n a l y s i s o f t h e p y r o l y s i s p r o d u c t s o f DNA by combined low energy e l e c t r o n impact and c o l l i s i o n a l a c t i v a t i o n s p e c t r o m e t r y a l l o w e d t h e assignment o f t h e s t r u c t u r e s o f t h e d i f ­ f e r e n t components i n t h e complex m i x t u r e from t h e p y r o l y s i s without p r i o r separation (10). The i n v e s t i g a t i o n s c i t e d above i n d i c a t e t h a t b o t h s t r u c t u r a l and sequence i n f o r m a t i o n about n u c l e i c a c i d s can be o b t a i n e d v i a p y r o l y s i s mass s p e c t r o m e t r y . One a s p e c t o f such i n f o r m a t i o n i s t h e d e t e c t i o n o f m o d i f i e d n u c l e o t i d e s i n DNA and i s t h e s u b j e c t o f t h i s p a p e r . M o d i f i e d components a r e thought t o have f u n c t i o n a l s i g n i f i c a n c e f o r t h e DNA m o l e c u l e , and, t h e d e t e c t i o n and l o c a t i o n o f such components i n v i r a l genomes i s expected t o c o n t r i b u t e t o a b e t t e r understanding o f host-viral relationships. P y r o l y s i s E l e c t r o n Impact Mass S p e c t r a l A n a l y s i s o f DNA. Methodology. S o l u t i o n s o f DNA o r p o l y d e o x y r i b o n u c l e o t i d e s c o n t a i n i n g 0.01 t o 1.0 A 60nm i n t r o d u c e d i n t o c a p i l l a r y sample tubes and t a k e n t o d r y n e s s i n vacuo i n a d e s i c c a t o r c o n t a i n i n g P2O5. Samples a r e i n t r o d u c e d i n t o t h e s p e c t r o m e t e r (DuPont 21-490 B) by d i r e c t p r o b e , s l o w l y h e a t e d t o about 250° and the a n a l y s i s i s c a r r i e d o u t a t 70 eV, 3 χ 10' T o r r , s o u r c e t e m p e r a t u r e = 200°. S p e c t r a a r e r e c o r d e d a t the p o i n t a t w h i c h the maximum number o f i o n s a r e g e n e r a t e d as i n d i c a t e d by t h e i o n m o n i t o r . u

n

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F r a g m e n t a t i o n P a t t e r n . The g e n e r a l scheme p r o ­ pos ed~TôrtnTlïinis^ f r a g m e n t a t i o n o f DNA t o g e t h e r w i t h the s u g g e s t e d s t r u c t u r e s o f t h e i o n p r o d u c t s are o u t l i n e d i n F i g u r e 1. S p e c i e s a i s c o n s i d e r e d t o be t h e f i r s t v o l a t i l e p r o d u c t t h a t i s r e l e a s e d from the p o l y n u c l e o t i d e c h a i n and, t h u s , i s submitted t o electron-impact i o n i z a t i o n , which f r a g ments the m o l e c u l e t o t h e p u r i n e o r p y r i m i d i n e base and t o m e t h y l f u r a n . S u b s e q u e n t l y , i o n s b and c a r e

250

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Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch013

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3

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Nucleic Acids Research

Figure 1. Proposed scheme for the electron impact and pyrolytic fragmentation of polydeoxyribonucleotides

13.

wiEBERS

251

Minor Nucleotides in Intact DNA

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch013

formed t h r o u g h t h e attachment o f a PO3 m o i e t y t o t h e e x o c y c l i c amino group o f t h e base (presumably t h r o u g h a phosphoramidate l i n k a g e . T h i s i o n o n l y appears i n spectra of nucleotides that contain a free exocyclic amino group ( 3 ) . The p u r i n e o r p y r i m i d i n e base fragment and t h e i o n t y p e s a, b , c , and d, a r e s p e c i f i c f o r each o f t h e common deoxyriïïonucleoticTe r e s i d u e s found i n DNA. The mass v a l u e s o f t h e s e i o n t y p e s f o r t h e common r e s i d u e s a r e l i s t e d i n T a b l e I . T h e i r appearance i n the mass spectrum o f salmon sperm DNA i s shown i n F i g u r e 2. T a b l e I . Mass V a l u e s (m/e) o f D i a g n o s t i c Ions D e r i v e d from R e s i d u e s i n DNA and P o l y d e o x y r i b o n u c l e otides. N u c l e o s i d e Residue Ion

Type 5

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D e t e c t i o n o f M o d i f i e d N u c l e o t i d e s i n DNAs. The same t y p e s o f i o n p r o d u c t s documented above can be used t o d e t e c t m o d i f i e d n u c l e o t i d e r e s i d u e s p r e s e n t i n DNAs. F o r example, 5 - m e t h y l d e o x y c y t i d i n e appears i n s m a l l amounts i n many DNAs. A mass spectrum o f 5 - m e t h y l d e o x y c y t i d i n e - 5 - p h o s p h a t e ( F i g u r e 3) shows i o n s a t m/e 176, 205, 285, and 365. These v a l u e s a r e a n a l o g o u s t o t h e v a l u e s o f t h e i o n t y p e s a , b , c and d, f o r common DNA components ( T a b l e I ) ancT a r e cliagn o s t i c f o r the presence o f 5 - m e t h y l c y t i d i n e residues i n DNA. A spectrum ( F i g u r e 4) o f wheat germ DNA 1

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Nucleic Acids Research

' 3é0

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' 340 ' 3éO

SALMON SPERM DNA

Figure 2. Mass spectrum of salmon sperm DNA. A, G, C, and T, indicate the ions that arise from deoxyadenosine, deo sine, deoxycytidine, and thymidine residues, respectively. In Figures 2-8, peaks indicated by broken lines are drawn their actual relative intensity.

100

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Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch013

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Nucleic Acids Research

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Figure 3. Mass spectrum of 5-methyldeoxycytidine-5'-phosphate

1

176

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Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch013

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280

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Nucleic Acids Research

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,311

WHEAT GERM DNA

Figure 4. Mass spectrum of wheat germ DNA

200 JlL

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205 206

186 191 192

215

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch013

340

360

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348 375

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Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch013

13.

WIEBERS

Minor Nucleotides in Intact DNA

255

[which i s known t o c o n t a i n 5.6 moles o f 5-methyldeoxyc y t i d i n e p e r 100 gram atoms o f DNA phosphorus ( 1 1 ) ] c l e a r l y shows t h e e x p e c t e d i o n t y p e s a t m/e 176, 205, 285, and 365 f o r t h e 5 - m e t h y l d e o x y c y t i d i n e e n t i t y . Some DNAs have one o f t h e common components comp l e t e l y r e p l a c e d by m o d i f i e d r e s i d u e s . F o r example, b a c t e r i o p h a g e T2 DNA i s known t o have a l l o f i t s d e o x y c y t i d i n e r e s i d u e s h y d r o x y m e t h y l a t e d ( 1 2 ) . The spectrum o f T2 DNA ( F i g u r e 5) e x h i b i t s none o f t h e d i a g n o s t i c i o n s t h a t o r i g i n a t e from d e o x y c y t i d i n e r e s i d u e s ( T a b l e I ) , b u t r a t h e r , shows i o n s w i t h t h e e x p e c t e d v a l u e s f o r p r o d u c t s d e r i v i n g from 5h y d r o x y m e t h y l d e o x y c y t i d i n e r e s i d u e s ( T a b l e I ) . These r e s u l t s from t h i s p a r t i c u l a r DNA emphasize t h e v a l i d i t y o f t h e mass s p e c t r a l d e t e c t i o n method s i n c e t h e y q u a l i t a t i v e l y confirm the f i n d i n g s o f the chemical c o m p o s i t i o n s t u d i e s on t h i s DNA. As a f u r t h e r cont r o l f o r t h e method, mass s p e c t r a l a n a l y s i s o f t h e s y n t h e t i c p o l y n u c l e o t i d e p o l y ( d A - d T ) ( F i g u r e 6) y i e l d e d o n l y t h e e x p e c t e d i o n p r o d u c t s d e r i v i n g from dA and dT, and no e x t r a n e o u s i o n s were o b s e r v e d . Mass s p e c t r a o f b a c t e r i o p h a g e DNAs a r e more comp l e x than those o f the b a c t e r i a l hosts t h a t they i n fect. F o r example, t h e spectrum o f E. c o l i DNA ( F i g u r e 7) d i s p l a y s d i a g n o s t i c i o n s T o r 5-methyldeoxyc y t i d i n e , whereas, t h e spectrum ( F i g u r e 8) o f t h e DNA from b a c t e r i o p h a g e 0X-174 (which i n f e c t s E ^ c o l i ) i n d i c a t e s t h e p r e s e n c e o f 5 - m e t h y l d e o x y c y t i d i n e r e s i d u e s as w e l l as s e v e r a l d i f f e r e n t minor p u r i n e and p y r i m i d i n e components. A r e c e n t s t u d y (13) o f f o u r b a c t e r i o p h a g e DNAs y i e l d e d s p e c t r a w h i c h showed s e v e r a l s e r i e s o f i o n p r o d u c t s t h a t c o r r e s p o n d t o t h e i o n t y p e s a, b , c, and d and have mass v a l u e s w h i c h c o u l d be i n d i c a t i v e of t h e p r e s e n c e o f m e t h y l a t e d deoxyadenosines and o f substituted deoxyuridines. A l t h o u g h t h e f u n c t i o n ( s ) o f m o d i f i e d components i n DNAs has n o t been r e s o l v e d , i t i s o f g r e a t i n t e r e s t to m o l e c u l a r b i o l o g i s t s t o d e t e r m i n e n o t o n l y w h i c h modified n u c l e o t i d e s occur i n bacteriophages, but a l s o , t o d e t e r m i n e t h e i r l o c a t i o n i n t h e whole DNA molecule. The s t r a t e g y t h a t we a r e u s i n g i n o u r l a b o r a t o r y t o h e l p t o answer t h e s e q u e s t i o n s i s (a) to d e t e c t and i d e n t i f y t h e m o d i f i e d c o n s t i t u e n t s i n t h e i n t a c t DNA m o l e c u l e by mass s p e c t r a l a n a l y s i s , and (b) to t r e a t t h e DNA w i t h a p p r o p r i a t e endonuclease r e s t r i c t i o n enzymes. T h i s t e c h n i q u e p e r m i t s t h e c l e a v a g e o f the m o l e c u l e a t s p e c i f i c s i t e s y i e l d i n g s p e c i f i c f r a g ments whose s e q u e n t i a l o r d e r c a n be r e c o n s t r u c t e d . The fragments a r e s e p a r a t e d and c o l l e c t e d by g e l e l e c t r o p h o r e s i s and, s u b s e q u e n t l y , a r e a n a l y z e d by

100

ι,ΙΙ2

ιΙΟΘ

120

112411

|ΙΙ9

,117

|Ι26

|1 140

141

135

02

[Ι, 160

160

180

all

183

200

225»» 221 :

220

213 202 Si**, ,266 263ί

240

280

™

300

ι

3,1 501

295 282, •2Β5

Nucleic Acids Research

260

5 2

J250? j !. 11256 i •268

242

Τ 2 DNA

320

Figure 5. Mass spectrum of bacteriophage T2 DNA

«7°

186

215

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch013

340

3θβ

380 4 0 0

3 7 5

ί 365 j , 381 ^

360

343 j . .348

8

RELATIVE INTENSITY 8

8

8

8

>L C H

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch013

8

8

;r-ï

H 3 Q.

SI »

f — β

3

VMQ V>vWl w* sapifo&ioriN xomyi

LSZ

saaaaiAv

"gj

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch013

HIGH

8

8

8

§

A1ISN31NI 3AI±V13U

PERFORMANCE

8

MASS

Q ου

ω

s

ο

1 S

ε

SPECTROMETRY

ο:

Lu

!5 _ι

£

ω

>-

100

201 ιοβ

40

60

80Η

ΙΟΟ

120

114

129

126

140

140

135

162168 1 8 , J . ιφΠΒΒΒίΒΙ

6 ^ ,206|

215 ! , :|263!

309!.

300

,295

Nucleic Acids Research

280

280 285!

320

Figure 8. Mass spectrum of bacteriophage 0X-174 DNA

240 260 m/e

23Ι„ 228 236

1 1160 I 1 1 180 IIιΐι ι ,ΐιΙ,Ι.ί il 200 N 220

Ψ L

1

250 ,.237

311 313

ΦΧ-Ι74 DNA

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch013

340

343

360

365!:

368

380

.389! 382'

400

393

CO

Ci

8·

ο

ο

to

260

HIGH

PERFORMANCE

MASS

SPECTROMETRY

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch013

mass s p e c t r o m e t r y f o r t h e e x i s t e n c e o f m o d i f i e d r e s i ­ dues. T h i s p r o c e s s i s r e p e a t e d w i t h o t h e r endonuc l e a s e r e s t r i c t i o n enzymes t h a t c l e a v e t h e DNA m o l e c u l e a t d i f f e r e n t s i t e s , y i e l d i n g s m a l l e r and d i f f e r e n t f r a g m e n t s , w h i c h t h e n a r e s u b m i t t e d t o mass s p e c t r a l a n a l y s i s . U l t i m a t e l y , t h i s procedure i s expected t o p e r m i t t h e l o c a l i z a t i o n o f t h e u n u s u a l r e s i d u e s i n spe­ c i f i c r e g i o n s o f t h e m o l e c u l e . A t t h e p r e s e n t time t h e r e i s no o t h e r method f o r l o c a t i n g v e r y s m a l l amounts o f minor n u c l e o t i d e s i n DNA. Thus, t h e s e n s i ­ t i v i t y and t h e d e f i n i t i v e n e s s o f t h e mass s p e c t r o m e t e r i s being e x p l o i t e d i n the s o l u t i o n o f a c u r r e n t l y s i g n i f i c a n t problem i n m o l e c u l a r b i o c h e m i s t r y .

Literature Cited. 1.

Charnock, G.A. and Loo, J.L. Anal. Biochem., (1970), 37, 81. 2. Wiebers, J.L., Anal. Biochem., (1973), 51, 542. 3. Wiebers, J.L. and Shapiro, J.A., Biochemistry, (1977), 16, 1044. 4. Schulten, H.R., Beckey, H.D., Boerboom, A . J . H . , and Meuzelaar, H . L . C . , Anal. Chem., (1973), 45, 2358. 5. Meuzelaar, H . L . C . , Kistemaker, P . G . , and Posthumus, Μ.Α., Biomed. Mass Spectrom., (1974), 1, 312. 6. Posthumus, Μ.Α., Nibbering, N.M.M., Boerboom, A . J . H . , and Schulten, H.-R. Biomed. Mass Spectrom. (1974), 1, 352. 7. Schulten, H.-R. and Schiebel, H.M., Z. Anal. Chem. (1976), 280, 139. 8. Schulten, H.-R. and Schiebel, H.M., Nucleic Acids Res., (1976), 3, 2027. 9. Burgard, D.R., Perone, S.P., and Wiebers, J.L., Biochemistry, (1977), 16, 1051. 10. Levsen, K. and Schulten, H.R. Biomed. Mass Spectrom., (1976), 3, 137. 11. Spencer, J.H. and Chargaff, E. Biochim. Biophys. Acta, (1963), 68, 18. 12. Wyatt, G.R. and Cohen, S.S. Biochem. J., (1953), 55, 774. 13. Wiebers, J.L. Nucleic Acids Res., (1976), 3, 2959. Received December 30, 1977

14 Ultra-High Resolution Mass Spectrometry Analysis of Petroleum and Coal Products H. E. LUMPKIN and THOMAS ACZEL

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch014

Exxon Research and Engineering Co., Analytical Research Laboratory, Baytown, TX

The detailed characterization of petroleum in terms of molecular composition and advances in mass spectrometry have proceeded abreast for about 30 years. From the analysis of gases and low boiling liquids in the 1940's and gasoline type analysis in the early 1950's (6), the frontiers moved toward higher boiling fractions as heated inlet systems evolved (9,11,15). But inlet systems mutated and evolved so rapidly that the mass separation or resolving power of the basic instruments could not keep apace. Components of the very complex material we c a l l "rock o i l " were vaporized into MS ion sources and the resulting spectra were partially or wrongly interpreted. D i s t i l l a t i o n , thermal diffusion, and solid-liquid elution chromatography were used to separate petroleum into fractions, but the fractions were still very complex. Meanwhile, work in gas chromatography was beginning to show excellent separations in the lower boiling ranges and for specific mixtures of components. However, GC has not been of major value in separations of the higher boiling ranges because of its lack of resolution. In the 1960's high resolution (5,13) and in the mid-1970's ultra high resolution (14) mass spectrometers became available and were used in the characterization of petroleum and, more recently, coal liquids (1). When ion production methods which simplify the spectra such as low ionizing voltage (7), field ionization, and chemical ionization, are used along with the inherent power of these instruments, even such intransigent mixtures as petroleum and coal products begin to yield. This paper reviews the work and summarizes the results of using the low ionizing voltage electron impact technique with high and ultra high resolution mass spectrometers for the ©0-8412-0422-5/78/47-070-261$05.00/0

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch014

262

HIGH

PERFORMANCE

MASS

SPECTROMETRY

q u a n t i t a t i v e a n a l y s i s o f p e t r o l e u m f r a c t i o n s and coal products. The i n s t r u m e n t s used were t h e ΑΕΙ MS9 and MS50 mass s p e c t r o m e t e r s . For a method t o be q u a n t i t a t i v e i t must be based on s p e c t r a w h i c h a r e r e p r o d u c i b l e . I n a d d i t i o n , t h e r e s h o u l d be a l i n e a r r e s p o n s e t o c o n c e n t r a t i o n , and t h e r e l a t i v e s e n s i t i v i t i e s among a l l t h e compo­ n e n t s t o be d e t e r m i n e d s h o u l d be known. We have used c a l i b r a t i o n d a t a ( r e l a t i v e o r a b s o l u t e i n s t r u m e n t r e s p o n s e t o a g i v e n amount o f a component) d e r i v e d from p u r e compounds and s e p a r a t e d c o n c e n t r a t e s . E x t r a p o l a t i o n o f c a l i b r a t i o n d a t a i n t o r e g i o n s where d i r e c t i n s t r u m e n t a l measurements on c a l i b r a n t s a r e not a v a i l a b l e have been made by c o n s i d e r i n g aromat i c i t y , s t r u c t u r e , and m o l e c u l a r w e i g h t (4,10,12). The low i o n i z i n g v o l t a g e method has Fotïï advantages and d i s a d v a n t a g e s w i t h r e s p e c t t o methods u s i n g h i g h e r i o n i z i n g v o l t a g e s (Table I ) . O b v i o u s l y we b e l i e v e t h a t f o r a r o m a t i c and p o l a r compound m i x t u r e s , t h e low v o l t a g e t e c h n i q u e i s s u p e r i o r . The major advantage i s t h e s i m p l i f i c a t i o n o f the s p e c t r a t o the p o i n t that only molecular ions are p r o d u c e d . Moreover, t h e r e i s no i n t e r f e r e n c e among components, and c a l i b r a t i o n d a t a a r e more s i m p l y d e r i v e d . The major d i s a d v a n t a g e i s t h e l o s s i n s e n s i t i v i t y - a f a c t o r t o 5 t o 20. TABLE I, Advantages and D i s a d v a n t a g e s o f H i g h R e s o l u t i o n - Low V o l t a g e T e c h n i q u e . Advantages M o l e c u l a r Ion S p e c t r a . General A p p l i c a b i l i t y r e g a r d l e s s o f sample o r i g i n , processing, b o i l i n g r a n g e . Data on i n d i v i d u a l homologs. Extrapolated c a l i b r a t i o n data.

Disadvantages L i m i t a t i o n t o Double Bond Compounds - O l e f i n s , Aromatics, P o l a r s . Relat i v e l y low s e n s i t i v i t y and r e p r o d u c i b i l i t y . *

Nomenclature and H i g h R e s o l u t i o n . In t h e f o l l o w i n g d i s c u s s i o n , t h e c h e m i c a l names o f t h e compounds and compound t y p e s w i l l be used i n t e r c h a n g e a b l y w i t h t h e m o l e c u l a r i o n s e r i e s , where

14.

LUMPKIN

263

Ultra-High Resolution Mass Spectrometry

A N D ACZEL

the s e r i e s i s d e r i v e d from the e m p i r i c a l f o r m u l a expression C H The z-number i s a measure o f hydrogen d e f i c i e n c y ( T a b l e I I ) . Thus b u t y l b e n z e n e , C i o H i i f , i s t h e C i member o f t h e C H series of a l k y l b e n z e n e s . D i b e n z o f u r a n i s t h e f i r s t member and n u c l e u s o f the ?n-16° s e r i e s . The n u c l e u s d e t e r m i n e s t h e s e r i e s , and a l k y l s u b s t i ­ t u t i o n extends t h e c a r b o n number b u t does n o t a f f e c t the e m p i r i c a l f o r m u l a . U n f o r t u n a t e l y f o r t h e a n a l y ­ t i c a l c h e m i s t , t h e carbon number o f almost a l l s e r i e s c o n t i n u e on and on i n b o t h p e t r o l e u m and c o a l e x t r a c t s t o as h i g h m o l e c u l a r w e i g h t compounds as can be v a p o r i z e d . Of c o u r s e , as the b o i l i n g p o i n t of t h e f r a c t i o n i s i n c r e a s e d , more and more compound t y p e s and s e r i e s t y p e s a r e found. 0

c

H

o

r

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch014

n

TABLE I I . Hydrogen D e f i c i e n c y Nomenclature Ζin Compound Type

Example

Formula

Paraffins

n-Hexadecane

C 1 6H3

Naphthenes

MeCyclohexane

C7H1

Alkylbenzenes

Butylbenzene

C10H1k

A l k y l n a p h t h a l e n e s Naphthalene

ι»

k

C

H

n 2n+z +2 0 -6

Ci οΗβ

-12

Phenanthrenes/ Anthracenes

DiMePhenanthrene

C I G H H

-18

Dibenzofurans

Dibenzofuran

Ci H 0

-16 0

C1iHeS2

-14 S

B e n z o d i t h i o p h e n e s MeBenzodithiophene

2

8

2

I f the m a t e r i a l s t o be examined were o n l y h y d r o c a r b o n s , a r e s o l v i n g power o f about 7000 would be adequate because t h i s i s s u f f i c i e n t t o s e p a r a t e the h y d r o c a r b o n s e r i e s up t o a mass o f about 650. But b o t h p e t r o l e u m and c o a l - d e r i v e d m a t e r i a l s a r e made up o f compounds c o n t a i n i n g t h e elements 0, N, and S as w e l l as h y d r o c a r b o n s . These h e t e r o a t o m components l e a d t o i n c r e a s e d c o m p l e x i t y because now many compound t y p e s have m o l e c u l a r i o n s d i f f e r i n g s l i g h t l y i n mass a t t h e same n o m i n a l m o l e c u l a r weight. Thus m u l t i p l e t s a r e found t h r o u g h o u t t h e s p e c t r a , and r e s o l v i n g power r e q u i r e m e n t s a r e d i c t a t e d by the heteroatoms e n c o u n t e r e d and the m o l e c u l a r weight. Of c o u r s e , t h e more r e s o l u t i o n one has a v a i l a b l e , t h e h i g h e r the m o l e c u l a r w e i g h t a t w h i c h

264

HIGH

PERFORMANCE

MASS

SPECTROMETRY

TABLE I I I .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch014

Mass D o u b l e t s and R e s o l v i n g Power

Doublet

AMass

H12-C

,094

Max. Mass a t RP a 10K 80K 150K

Example

C1 i*H2 2

C15H10

190.1721

190.0782

940

7520

14000

910

7280

13500

364

2910

5460

2 6

1010

C i*Hg

Ô C H -S 2

8

O r

.091 C1oH1^

C βΗβ S

134.1095

CHu-0

CH -N 2

SH^-Ca

134.0190

.0364

.0126

C13H12

C12ΗβΟ

168.0939

168.0575

D O

2 V

ΟΠΟ

< ^ N - ^ s x ^ Η

Ci3H11

Ci2H9N

167.0861 C if H g

167.0735

1

34

.0034 Ci

2H1

ifS

190.0816

Ci

5H10

190.0782

270

1890

500

14.

LUMPKIN

A N D ACZEL

Ultra-High Resolution Mass Spectrometry

265

TABLE IV. R e s o l v i n g Power R e q u i r e d t o S e p a r a t e Members o f P o s s i b l e M u l t i p l e t s a t m/e 300 and 301

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch014

Formula

Mass

AMass

RP R e q u i r e d

H

300.2817

H

300.2453

0.0364

8,000

H

300.2089

0.0364

8,000

300.1912

0.0177

17,000

300.1878

0.0034

88,000

C H SO

300.1548

0.0330

9,000

C

22 20°

H

300.1514

0.0034

88,000

C

24 12

H

300.0939

0.0575

5,000

301.1830

0.0082

37,000

301.1548

0.0282

11,000

301.1466

0.0082

37,000

C

22 36

C

21 32°

C

20 28°2

C

20 28

C

23 24

H

S

H

1 9

2 4

13

301.1912 C C

C

H

22 24 H

22 23 C C

N

H

21 20°

C H NO 2 1

1 9

266

HIGH

PERFORMANCE

MASS

SPECTROMETRY

one c a n s e p a r a t e a d o u b l e t ( T a b l e I I I ) . The c l o s e s t l y i n g doublet r e q u i r i n g the greatest r e s o l u t i o n i n the samples we a n a l y z e i s t h e SH^-Ca d o u b l e t ( T a b l e IV).

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch014

Data A c q u i s i t i o n and Computer Programs. H i g h r e s o l u t i o n a n a l y s e s o f p e t r o l e u m and c o a l p r o d u c t s produce such a volume o f d a t a t h a t an onl i n e d a t a a c q u i s i t i o n system i s r e q u i r e d . We c u r r e n t l y use a Columbia S c i e n t i f i c I n d u s t r i e s MS r e a d o u t system t o sense peak t o p s and s l o p e changes and t o f u r n i s h d i g i t a l d a t a on peak h e i g h t and occurence t i m e t o a magnetic tape r e c o r d e r . Data from t h e magnetic tape a r e r e a d and h a n d l e d by computer programs w h i c h p e r f o r m a number o f s o p h i s t i c a t e d f u n c t i o n s (3^8>) as o u t l i n e d i n T a b l e V. A major f u n c t i o n i s mass measurement. From knowledge o f t h e o c c u r r e n c e t i m e s and masses o f peaks from r e f e r e n c e compounds i n a t i m e - i n t e n s i t y spectrum and t h e times o f t h e sample p e a k s , t h e masses o f t h e sample peaks TABLE V. Data A c q u i s i t i o n and R e d u c t i o n Purpose:

Determine f o r m u l a s and i n t e n s i t i e s o f 100-5000 peaks and c a l c u l a t e p e r c e n t abundance.

Functions:

Convert analog t o d i g i t a l s i g n a l s . Measure peak h e i g h t s o r a r e a s . Measure peak p o s i t i o n s i n time u n i t s . Separate p a r t i a l l y r e s o l v e d m u l t i p l e t s . Reject noise spikes. R e c o g n i z e s t a n d a r d r e f e r e n c e peaks. C a l c u l a t e masses and a s s i g n f o r m u l a e . C a l c u l a t e q u a n t i t a t i v e a n a l y s i s and p r i n t o u t i n p u t , d e t a i l e d d a t a , and summaries.

can be c a l c u l a t e d . The a c c u r a c y o f t h e mass measurements i s a f u n c t i o n o f t h e e l e c t r o n i c and m e c h a n i c a l s t a b i l i t y o f t h e mass s p e c t r o m e t e r . The MS50 i s a v e r y s t a b l e i n s t r u m e n t l e a d i n g t o a c c u r a t e mass measurements. When masses a r e known t o s u f f i c i e n t a c c u r a c y , t h e computer a s s i g n s unique e m p i r i c a l f o r m u l a e t o peaks and u l t i m a t e l y t h e a p p r o p r i a t e s e r i e s i n t h e g e n e r a l C H~ . n o m e n c l a t u r e . The q u a n t i t a t i v e a n a l y s i s i s c a l c u l a t e d i n a separate program u s i n g t h e measured peak h e i g h t s , t h e s e r i e s

14.

LUMPKIN

AND ACZEL

267

Ultra-High Resolution Mass Spectrometry

a s s i g n m e n t s , and an e x t e n s i v e s e t o f c a l i b r a t i o n d a t a . Examples o f the form o f d a t a g i v e n i n t h e q u a n t i t a t i v e program w i l l be d i s c u s s e d l a t e r . R e s o l u t i o n and Mass Measurements, Good mass measurement even a t a r e s o l v i n g power o f 20,000 a r e i n s u f f i c i e n t t o s e p a r a t e p o s s i b l e m u l t i p l e t s i n complex p e t r o l e u m and c o a l m i x t u r e s . For t h e d a t a i n T a b l e V I , t h e computer has o f f e r e d two o r t h r e e p o s s i b l e f o r m u l a e f o r s e v e r a l o f t h e p e a k s , t o g e t h e r w i t h the r e s p e c t i v e e r r o r i n mass measurement. The low l y i n g peak a t mass 296 i s C i e H i S 2 , based on mass measurement, w h i l e t h e mass 296.157 peak i s p r o b a b l y composed o f u n r e s o l v e d i o n s from t h e i n d i c a t e d h y d r o c a r b o n and s u l f u r compounds. A t mass 298 the mass measurements a r e p o o r e r , b u t one would s e l e c t t h e -28/S and -16/S0 r a t h e r t h a n t h e -38 and -26/0 t y p e s when t h e i r mass measurements a r e compared t o t h a t f o r t h e -24 s e r i e s .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch014

6

TABLE V I . C o a l E x t r a c t (~20,000 R e s o l u t i o n ) Intensity

Meas m/e

E r r o r MMU

Formula

(Series)

2,138

296.070

7.7

C2 itHe

2,138

296.070

4.3

C21H12S

(_30S)

2,138

296.070

0.9

CieHi

(20S2)

25,136

295.121

0.5

C22H16Û

(.280)

17,744

296.157

1.0

C2 3H2 0

(.26)

17,744

296.157

-2.4

C2 0H2 "»S

(_ieS)

6,868

298.084

6.0

C2 ι»Ηι 0

( . 3 8 )

6,868

298.084

2.6

C2 1H1 i»S

(_2 S) 8

5,708

298.142

6.1

C2 2H1eO

(.260)

5,708

298.142

2.7

Ci9H22SO

(_1 S0)

15,832

298.175

2.9

Cz3H2 2

(.2O

6

Coal l i q u e f a c t i o n products c o n t a i n r e l a t i v e l y l e s s s u l f u r t y p e s and more n i t r o g e n and oxygen t y p e s t h a n some p e t r o l e u m f r a c t i o n s . Less r e s o l u t i o n i s r e q u i r e d f o r t h e s e l a t t e r heteroatoms t h a n f o r s u l f u r , and R=40,000 appears adequate t o r e s o l v e many c o n s t i t u e n t s w i t h o u t r e s o r t i n g t o any p r i o r s e p a r a t i v e s t e p s . F o r example, 464 components i n

268

HIGH

PERFORMANCE MASS

SPECTROMETRY

106 d i f f e r e n t compound t y p e s were found f o r t h e c o a l product r e p o r t e d i n Table V I I . U l t r a h i g h r e s o l u t i o n (80,000) dynamic s c a n n i n g o f a h i g h s u l f u r p e t r o l e u m f r a c t i o n t a x e s t h e c a p a b i l i t i e s o f t h e mass s p e c t r o ­ meter, t h e d a t a a c q u i s i t i o n system, and t h e computer TABLE V I I . Unseparated Coal L i q u e f a c t i o n Product ~40,000 R e s o l u t i o n

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch014

Intensity

Meas. m/e

E r r o r , MMU

Formula

(Series)

C l gH ι 6 SO

(.2 2 S O )

-1. 5

C2

(. 3 0 )

292. 1470

-0. 7

C 2 0 H2 0 O2

349

292. 1812

-1. 5

C2 1H2 l»0

(.2 0 / O 2 ) (. i e / 0 )

245

292. 2206

1. 5

C2 2H2 8

(. 16)

293

292. 0924

0. 2

758

292. 1237

589

121

293. 0962

0. 5

392

293. 1223

1. 9

237

293. 1490

1. 3

173

293. 1764

-1. 5

246

293. 2151

0. 8

3H16

I s o t o p e from C

2 2

H

1 5

N

I s o t o p e from C20H23NO C21H27N

(.22/SO) (.2 9 / N ) (. 2 0 / O 2 ) (. 17/NO)

(. / N ) programs because o f t h e l a r g e number o f peaks as shown i n T a b l e V I I I . We had t o i n c r e a s e t h e d i m e n s i o n s o f o u r a r r a y s from 3500 t o 5000 f o r scans o f t h i s nature. I n T a b l e IX i s shown t h e d a t a a t s e l e c t e d masses f o r o n l y t h e h y d r o c a r b o n and s u l f u r compound 15

TABLE V I I I . High S u l f u r Petroleum Aromatics ~80,000 R e s o l u t i o n Intensity

Meas. m/e

E r r o r , MMU

Formula

(Series)

C2 2H10

423

274.0776

-0. 5

1,904

274.0820

0. 5

C 1 gHi t» S

(_2%/S)

601

274.0842

-0. 5

C1εΗι8S2

1,608

274.1348

-0. 7

C 2 0H1eO

(_iu/S ) (.22/0)

438

274.1362

-2. 7

C 1 7 H 2 2 so

(.12/SO)

4,206

274.1719

-0. 2

C21H2 2

5,104

274.1746

-0. 7

C l 8 H 2 6S

(.20) L10/S)

3,148

274.2656

-0. 2

C2 0H3 ^

(.6)

2

14.

LUMPKIN

AND ACZEL

269

Ultra-High Resolution Mass Spectrometry

t y p e s from t h e same s e r i e s shown i n T a b l e V I I I . These components a r e r e a l ; they form a r e g u l a r s e r i e s a p p e a r i n g a t e v e r y 14 mass u n i t s b e g i n n i n g a t the r i g h t n u c l e a r m o l e c u l a r w e i g h t and a d d i t i o n a l compound t y p e s appear w i t h i n c r e a s i n g mass. B u t even t h e u l t r a h i g h r e s o l u t i o n MS50 mass s p e c t r o ­ meter runs o u t o f r e s o l v i n g power f o r t h e s e c l o s e lying multiplets. TABLE I X . High S u l f u r P e t r o l e u m A r o m a t i c s -80,000 R e s o l u t i o n Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch014

I n t e n s i t i e s A t S e r i e s Shown Mass CN 162 190 246 288 372

-6

12 10256 14 6880 18 3622 21 3230 27 772

-10/S

-20 -14/S

8332 8700 1851 6124 5263 5120 4202 -2668 UR-

2

-24/S -34-28/S

695 410 -2081 UR- 903 ---1562 UR---

1108

2

-38/S

540

CN - Carbon Number; UR - U n r e s o l v e d ; Avg. E r r o r 0.8 MMU A l i s t o f t h e compound t y p e s i d e n t i f i e d i n v a r i o u s p e t r o l e u m and c o a l l i q u i d samples i s g i v e n i n T a b l e X. A t o t a l o f 173 C, Η, Ν, 0 and S c o n t a i n i n g types are i n c l u d e d , n o t counting the m i s c e l l a n e o u s l i s t o f t y p e s w i t h t h e same e m p i r i c a l f o r m u l a e b u t different nuclear structures. Fortunately, a l l of t h e s e t y p e s a r e n o t o b s e r v e d i n a s i n g l e sample. Q u a n t i t a t i v e A n a l y s i s and Data P r e s e n t a t i o n . Our q u a n t i t a t i v e a n a l y s i s programs y i e l d a r r a y s o f w e i g h t p e r c e n t o f compound t y p e by c a r b o n number as t h e prime d a t a and c o v e r s many computer o u t p u t pages. From such a w e a l t h o f d e t a i l e d d a t a one c a n c a l c u l a t e many a d d i t i o n a l paramenters. Some o f t h e s e a r e g i v e n i n T a b l e XI and i n c l u d e q u i t e s t r a i g h t ­ f o r w a r d items such as average m o l e c u l a r w e i g h t , e l e ­ mental a n a l y s i s , and carbon number d i s t r i b u t i o n . W i t h some d a t a on b o i l i n g p o i n t s , l e s s o b v i o u s i n f o r m a t i o n such as d i s t i l l a t i o n c u r v e s and composi­ t i o n s o f any s e l e c t e d d i s t i l l a t e f r a c t i o n s a r e c a l c u l a b l e (2,) . One seldom needs t h e d e t a i l e d compound t y p e by c a r b o n number d a t a . T h e r e f o r e , we p r e p a r e summary t a b l e s p r e s e n t i n g d i f f e r e n t a s p e c t s o f t h e analysis. Example d a t a from such summary t a b l e s a r e

270

HIGH

PERFORMANCE

MASS

SPECTROMETRY

TABLE X. Compound Types i n V a r i o u s Samples Range i n G e n e r a l Formulas

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch014

Class Hydrocarbons

C

n 2n

S u l f u r Compounds

C

n 2n- 2

S u l f u r Compounds

C

n 2 n - 10 2

Oxygen Compounds

C

n 2 n - 2°

Oxygen Compounds

C

n 2 n - 6°2

t 0

C

n 2n-40°2

Oxygen Compounds

C

n 2 n - 6°3

t 0

C

n 2n-32°3

Oxygen Compounds

C

n 2 n - 10°4

N i t r o g e n Compounds

C

n 2n- 3

S u l f u r - O x y g e n Compounds

C

n 2n-

N i t r o g e n - O x y g e n Compounds

C

n 2n-

Miscellaneous N , S0 , 2

and N 0 2

2

H

t 0

H

C

S

H

n 2n-50

t 0

S

H

H

H

H

H

10

H

7

N

t o

SO

N0

H

C

S

n 2n-44

t o

t 0

H

H

C

C

H

S

n 2n-30 2

H

n 2n-48°

t 0

C

H

H

C

H

n 2n-18°4

H

n 2n-49

N

to C H . SO n

2 n

3 0

to C H . NO n

2 n

4 1

SO^,

Compounds

TABLE X I . Factors Calculated i n Quantitative Analysis. Data c a l c u l a t e d f o r up t o 3000 components, 100 homologous s e r i e s . Output i n c l u d e s p a r a m e t e r s l i s t e d below: 1. 2. 3.

Weight p e r c e n t f o r each component and s e r i e s . Average MW, c a r b o n atoms, c a r b o n atoms i n s i d e c h a i n s f o r each s e r i e s and f o r t o t a l aromatics. Elemental a n a l y s i s , molecular weight d i s t r i b u t i o n , c a r b o n number d i s t r i b u t i o n , d i s t i l l a t i o n , p r e d i c t i o n o f c o m p o s i t i o n o f narrow fractions.

C a l c u l a t i o n on IBM 370 computer y i e l d s 18 t a b l e s , t a k e s 20 seconds.

14.

LUMPKIN AND ACZEL

shown i n T a b l e s X I I , X I I I , and XIV. The compound t y p e summary t a b l e , e x c e r p t e d i n T a b l e X I I , a l s o g i v e s t h e average m o l e c u l a r w e i g h t and average c a r b o n number f o r each t y p e . The a r o m a t i c r i n g d i s t r i b u t i o n i n a heavy c o a l l i q u i d (i'able X I I I ) shows t h a t b o t h h y d r o c a r b o n s and oxygenated t y p e s a t t a i n maxima a t 4 r i n g s / m o l e c u l e . T h i s sample c o n t a i n e d an u n u s u a l l y h i g h amount o f oxygenated t y p e s , w h i c h a r e p r i m a r i l y hydroxy a r o m a t i c s . Those from p e t r o l e u m a r e m o s t l y a r o m a t i c f u r a n s . A p a r t i a l c a r b o n number d i s t r i b u t i o n i n a crude o i l and i n a c o a l l i q u i d ( T a b l e XIV) shows t h a t w h i l e t h e p e t r o leum has a smooth d i s t r i b u t i o n , t h e c o a l l i q u i d d i s p l a y s maxima a t C i , C i , C m , and C i . These c a r b o n number maxima a r e due t o t h e n a p h t h a l e n e , acenaphthene, p h e n a n t h r e n e , and pyrene a r o m a t i c and hydroaromatic n u c l e i . The p e t r o l e u m tends t o have l o n g a l k y l s u b s t i t u e n t s on t h e a r o m a t i c n u c l e i w h i l e much o f t h e c o a l l i q u i d i s c o n c e n t r a t e d a t t h e nuclear molecular weight. 0

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch014

271

Ultra-High Resolution Mass Spectrometry

2

6

TABLE X I I . P a r t i a l Compound Type Summary (Crude O i l ) Wt. P e t .

Type

Avg.

MW

Avg. C. No. 11.7

H

11.6

158.2

H

14.9

192.3

14.6

H

5.5

256.4

19.6

H

2.7

307.0

23.5

H

0.5

327.7

25.4

C

n 2n-6

C

n 2n-12

C

n 2n-18

C

n 2n-22

C

n 2n-28

C

n 2n-10

C

n 2n-16

C

n 2n-16°

H

S

4.1

215.4

13.8

H

S

7.3

250.0

16.7

0.2

179.6

12.8

H

272

HIGH P E R F O R M A N C E MASS

SPECTROMETRY

TABLE X I I I . A r o m a t i c D i s t r i b u t i o n i n a Heavy C o a l L i q u i d Aromatic Rings 1 2 3 4 5 6 7+

Hydrocarbons 2.5 15.7 19.5 26.8 5.0 2.6 0.5

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch014

72.6

S Compounds 0.4 1.4 0.8 0.4 s p e c t r a as d e s c r i b e d e a r l i e r c a r r y mass r a t i o and i n t e n s i t y i n f o r m a t i o n o n l y . The work t o be d e s c r i b e d h e r e was done t o a s s e s s the v a l u e o f t h e s e k i n d s o f d a t a f o r a n a l y s i s o f v o l a t i l e components. We used a l k a n e , a l k e n e , and c y c l o a l k a n e i s o m e r s , and h y d r o c a r b o n m i x t u r e s as examples. IKES s p e c t r a are compared t o mass s p e c t r a i n some i n s t a n c e s . A l l o f the IKES s p e c t r a were t a k e n a t low r e s o l u t i o n , i . e . , the m o n i t o r s l i t assembly o f the MS-9 mass s p e c t r o m e t e r was used as the d e t e c t o r . S i n c e the m o n i t o r s l i t assembly has an ^0.5 cm hole M

2

GALLEGOS

Double-Focusing Mass Spectrometer

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch015

0

0 m/e 149 Chevron Research Company

Figure 7. Main fragmentation paths of phthalate

286

PERFORMANCE

MASS

SPECTROMETRY

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch015

HIGH

Figure 8.

Metastable scan of m/e 149 of pure n-dibutyl phthahte and phthalates in organic from a water sample

15.

Double-Focusing Mass Spectrometer

GALLEGOS

287

i n i t , a l l o f t h e IKES s p e c t r a peaks w i l l appear as p a r t i a l l y r e s o l v e d d o u b l e t s o r as f l a t - t o p p e d p e a k s , w h i c h r e f l e c t t h e h o l e i n t h e d e t e c t o r . The masses o r c a r b o n number o f t h e m o l e c u l e s i n v o l v e d i n any p a r ­ t i c u l a r t r a n s i t i o n can be d e t e r m i n e d from t h e c a l c u l ­ a b l e mass r a t i o . Pure Hydrocarbon F i g u r e 9 shows t h e IKES and mass s p e c t r a o f f i v e C alkene isomers. Note t h a t a l l o f t h e IKES s p e c t r a o f t h e b r a n c h e d a l k e n e s have a base peak c o r r e s p o n d i n g to the C -*· C transition. The mass s p e c t r a does n o t show t h i s phenomenon. However, F i g u r e 10 g i v e s t h e IKES s p e c t r a o f c i s - 3 - h e x e n e w h i c h shows a base peak for the C + C transition. I n f a c t , t h e IKES s p e c t r a match v e r y c l o s e l y t h a t o f 2-methyl-1-pentene. T h e i r mass s p e c t r a a r e a l s o p r a c t i c a l l y i d e n t i c a l . F i g u r e 10, i n a d d i t i o n , c o n t r a s t s t h e IKES s p e c t r a o f c y c l o h e x a n e and t h r e e hexene i s o m e r s . The mass s p e c t r a f o r a l l f o u r a r e q u i t e s i m i l a r . The IKES s p e c t r a show much g r e a t e r c o n t r a s t . F i g u r e 11 shows t h e IKES and mass spectra, o f t h r e e C a l k e n e s . A l l show a IKES s p e c t r a base peak for the C + C transition. The base peak i n t h e i r mass s p e c t r a i s t h e ( M - 15) fragment i o n . T h i s w o u l d be due t o C s •+ C * t r a n s i t i o n . This implies a b a s i c d i f f e r e n c e i n the decomposition k i n e t i c s a t t i m e s o f 1 0 " t o 1 0 " seconds. F i g u r e 12 shows IKES and mass s p e c t r a o f some C -C alkanes. Note t h a t a l l t h e n o n s y m m e t r i c a l b r a n c h e d a l k a n e s have a IKES s p e c t r a base peak c o r ­ responding t o the C + C transition. F i g u r e 13 shows t h e IKES s p e c t r a o f m i x t u r e o f C a l k a n e s , C c y c l o a l k a n e s , and C alkylbenzenes. There i s a d e f i n i t e c o n t r a s t between t h e a l k a n e s and the a r o m a t i c a l k y l b e n z e n e s IKES s p e c t r a . The c e n t r o i d o f t h e IKES s p e c t r a i s g r e a t e s t f o r the a l k a n e s f o l l o w e d by t h e c y c l i c a l k a n e s and, f i n a l l y , the alkylbenzenes. T h i s j u s t means t h a t t h e a l k a n e s on t h e average j u s t b r e a k about i n h a l f and, i f n o t , t h e p o s i t i v e charge can s t a y w i t h t h e s m a l l e r fragment. The c y c l i c s c l e a v e about t h e same w i t h some p r e f e r e n c e t o l e a v e t h e charge on t h e r i n g system. The. a l k y l b e n z e n e s a l m o s t e x c l u s i v e l y l e a v e t h e p o s i t i v e charge on t h e π bonded system w h i c h , i n t h i s c a s e , i s always o f g r e a t e r mass. The same may be s a i d o f t h e d a t a shown i n F i g u r e 14. These samples a r e S i 0 - s e p a r a t e d c u t s from 6

+

+

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch015

5

3

+

+

5

3

5

+

+

5

2

+

+

k

5

8

6

6

+

5

8

+

3

8

8

2

HIGH

IKES Spectra

PERFORMANCE

MASS

SPECTROMETRY

Mass Spectra

• V

2-€thyl-l-Butene

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch015

m/e

1-Hexene

C

30

6

4-

90

m/e

4-Methyl-2-Pentene

c

6

·

30

I'M

m/e

90

2-Methyl-l-Pentene V 30

m/e

Chevron Research Company

Figure 9.

Mass and ikes spectra of some C alkanes 6

Figure 10. Mass and ikes spectra of cyclohexane and the hexane isomers

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch015

HIGH

PERFORMANCE

MASS

SPECTROMETRY

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch015

2-Pentene

30

70 m/e

2-Methyl-l-Butene

s

/L

30

70 m/e

ο f 2-Methyl-2-Butene

ο It

JU 30

m/e

Figure 11.

Mass and ikes spectra of some C alkenes s

70

GALLEGOS

Double-Focusing Mass Spectrometer

-V-

2,3,4-Trimethyl Pentane

ι II if

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch015

U/JI c, A A 2,4-Di methyl Pentane

~1

L

2,2-Dimethyl Butane

C

6

2-Methyl Pentane

Chevron Research Company

Figure 12. Ikes spectra of some alkanes

292

HIGH

PERFORMANCE

MASS

C Alkanes

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch015

8

C Cycloalkanes 8

C Alkylbenzenes s

Chevron Research Company

Figure 13. Ikes spectra of C alkanes, cycloalkanes, and alkylbenzenes 8

SPECTROMETRY

Double-Focusing Mass Spectrometer

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch015

GALLEGOS

Chevron Research Company

Figure 14. Ikes spectra of the saturate and aromatic fraction of a petroleum sample ASL 3084

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch015

294

HIGH

PERFORMANCE

MASS

SPECTROMETRY

Figure 15. Ikes spectra and Grou-type results of five thermal diffusion cuts of the saturate fraction of a petroleum sample

GALLEGOS

295

Double-Focusing Mass Spectrometer

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch015

15.

Chevron Research Company

Figure 16. Ikes spectra of the aromatic and saturate fractions of Hiawatha coal

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch015

296 HIGH PERFORMANCE MASS SPECTROMETRY

15.

297

Double-Focusing Mass Spectrometer

GALLEGOS

a p e t r o l e u m crude o i l . The main peaks o f the a r o m a t i c IKES s p e c t r a appear a t a c c e l e r a t i n g v o l t a g e s l e s s t h a n 3 kV, w h i c h means M /M r a t i o s a r e a l l l e s s t h a n 3/2. T h i s i m p l i e s two t h i n g s : f i r s t , t h e charge p r e f e r s t o s t a y on t h e l a r g e r p i e c e s u g g e s t i n g a π-bonded o r a r o m a t i c n u c l e u s on most o f the m o l e c u l e s and, second, t h e r e i s no l o n g - c h a i n b r a n c h i n g on any o f the m o l e c u l e s o f t h i s f r a c t i o n ; o t h e r w i s e , we would see peaks beyond 3 kV even i f an a r o m a t i c system is involved. The s a t u r a t e c u t shows most o f i t s peaks between about 3 and 5 kV, w h i c h i s a r a t i o Mj/M - 3/2 t o 5/2 averaged a t about 4/2. T h i s means t h a t on the average the m o l e c u l e s i n t h i s f r a c t i o n b r e a k i n h a l f . T h i s i s c o n s i s t e n t w i t h what i s known o f s a t u r a t e - t y p e molecules. F i g u r e 15 shows t h e IKES s p e c t r a o f f i v e t h e r m a l d i f f u s i o n c u t s o f the ASL 3084 s a t u r a t e f r a c t i o n d i s ­ c u s s e d above. The g r o u p - t y p e a n a l y s i s o f t h e s e c u t s i s shown below each s p e c t r a . The peaks t o n o t e a r e the h i g h r a t i o M /M > 4/2 f i l l e d peaks and t h e low r a t i o Μχ/Μ < 3/2 c r o s s - h a t c h e d peaks. The h i g h r a t i o peaks d i m i n i s h i n i n t e n s i t y a l o n g w i t h the p a r a f f i n s and o n e - r i n g components; whereas, the l o w r a t i o peak i n c r e a s e s w i t h i n c r e a s e d c o n c e n t r a ­ t i o n o f the m u l t i r i n g components. x

2

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch015

2

x

2

2

Miscellaneous

Samples

F i g u r e 16 shows t h e IKES s p e c t r a o f t h e S i 0 s a t u r a t e and a r o m a t i c c u t from a U t a h Hiawatha c o a l . The a r o m a t i c c u t shows two peaks marked A and Β w h i c h have Μ / Μ r a t i o s > 3/2 s u g g e s t i n g some l o n g s t r a i g h t c h a i n attachment t o a π-bonded n u c l e u s . GC-MS a n a l y s i s o f t h i s f r a c t i o n r e v e a l s the p r e s e n c e o f a homologous s e r i e s o f n - a l k y l b e n z e n e s . F i g u r e 17 shows t h e IKES s p e c t r a o f 1-phenyl-nC and l - h e x y l - n - C . The base peak i n t h e IKES spectra of l-phenyl-n-C i s due t o t h e C $ -> C t r a n s i t i o n , i . e . , the M /M r a t i o i s h i g h . The l-hexyl-n-C shows o n l y a s m a l l peak r e p r e s e n t i n g the e q u i v a l e n t t r a n s i t i o n . The base peak i s due t o the molecule breaking i n h a l f . 2

χ

2

2 0

2 0

+

2 0 x

2 0

2

2

7

298

HIGH

PERFORMANCE MASS

SPECTROMETRY

Conclusions T h i s s e c t i o n has shown how IKES s p e c t r a may be u s e d , somewhat l i k e IR o r UV, i n t h e i d e n t i f i c a t i o n o f gaseous m a t e r i a l s i n b o t h t h e q u a l i t a t i v e and q u a n t i ­ t a t i v e sense. C e r t a i n isomers n o t d i s t i n g u i s h a b l e by mass s p e c t r a a l o n e may w e l l be u n r a v e l e d u s i n g IKES spectra. T h i s s o r t o f approach may w e l l be u s e f u l i n GC-IKES a n a l y s i s s i m i l a r t o GC-MS.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch015

Literature Cited 1.

Hipple, J. A. and Condon, E. U . , Phys. Rev. 68,

2.

Cooks, R. G . , Beynon, J. H . , Copriol, R. Μ., and Lester, G. R., "Metastable Ions," Elsevier Scientific Publishing Company, New York, New York, 1973. Jennings, K. R., "Some Aspects of Metastable Transitions, Mass Spectrometry Techniques and Application," G. W. H. Milne, E d . , Wiley­ -Interscience, New York, New York, 1971. Teeter, R. M. and Doty, W. R., Res. Sci. Inst. 37,

54

3.

4.

(1945).

p 792

5.

(1966).

Budzikiewicz, H . , Wilson, J. Μ., and Djerassi, C, J. Am. Chem. Soc. 85, p 3688, 1963. 6. Tokes, L., Jones, G . , and Djerassi, C . , J. Am. Chem. Sci. 90, p 5465, 1968. 7. Gallegos, E. J., Anal. Chem. 43, p 1151, 1971. 8. Otvos, J. W. and Stevens, D. P . , J. Am. Chem. Soc. 78, p 546, 1956. 9. Gallegos, Ε. J., Anal. Chem. 47, p 1524, 1975. Received December 30, 1977

16 Multielement Isotope Dilution Techniques for Trace Analysis* J. A. CARTER, J. C. FRANKLIN, and D. L. DONOHUE

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch016

Oak Ridge National Laboratory, Oak Ridge, TN 37830

Instrumental mass spectrometry includes a variety of techniques for measuring the mass to charge (m/e) ratio of gaseous ions. For analytical purposes, positive ions produced by a suitable source in a vacuum are subsequently separated according to mass-to-charge ratio. The most often used source for trace analysis is the pulsed radiofrequency (RF) ion source. The early pioneering work in spark source mass spectrometry was conducted prior to the late 1950's (1-3), while commercial instrumentation became available in the early 1960's. The commercial designs employed the Mattauch-Herzog double focusing geometry (4) in which ions of a l l masses are simultaneously focused in one plane. The field of application of the technique has widened considerably, and with the advent of multielement isotope dilution techniques (5-7), spark source mass spectrometry (SSMS) should produce even better quantitative data. Basic SSMS Review. Figure 1 is a schematic diagram of a typical spark source mass spectrometer. The source and analyzer portions are normally operated in the low 10 torr region while the photographic plate region for prepumping is at a higher pressure, approximately 10 torr. Ions representative of the sample material are produced by the rf spark source. The spark between the conducting sample electrodes is produced by applying a pulsed radiofrequency potential of 30 -8

-8

* Research sponsored by the Energy Research and Development Administration under contract with the Union Carbide Corporation. ©0-8412-0422-5/78/47-070-299$05.00/0

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch016

300

HIGH

PERFORMANCE

MASS

SPECTROMETRY

t o 50 kV. Sample e l e c t r o d e s may be made d i r e c t l y from t h e sample m a t e r i a l i f i t i s a c o n d u c t o r o r s e m i c o n d u c t o r , but n o n - c o n d u c t i n g m a t e r i a l s must be mixed w i t h pure c o n d u c t o r s such as Ag o r g r a p h i t e and compressed. Ions produced i n t h e source a r e a c c e l e r a t e d i n t o the a n a l y z e r r e g i o n o f t h e mass s p e c t r o m e t e r by a p o t e n t i a l o f about 25 kV. S i n c e t h e i o n s have a wide k i n e t i c energy s p r e a d , t h e e l e c t r o s t a t i c a n a l y z e r p r o v i d e s t h e n e c e s s a r y energy f o c u s i n g p r i o r t o t h e mass s e p a r a t i o n i n t h e m a g n e t i c s t a g e . Since the M a t t a u c h - H e r z o g geometry b r i n g s i o n s o f d i f f e r e n t mass t o charge r a t i o t o f o c u s a t d i f f e r e n t p o i n t s i n a s i n g l e p l a n e , a p h o t o g r a p h i c p l a t e p l a c e d on t h e f o c a l plane o f the magnetic a n a l y z e r simultaneously d e t e c t s a l l i s o t o p e s w i t h m/e between 7 and 250. A t y p i c a l p h o t o g r a p h i c p l a t e has 15 graded e x p o s u r e s f o r a s i n g l e specimen so t h a t t h e c o n c e n t r a t i o n range from 0.01 t o o v e r 1000 ppm i s c o v e r e d . F i g u r e 2 i s a p h o t o g r a p h o f an ΑΕΙ MS702 spark s o u r c e i n s t r u m e n t used a t ORNL. The source g l o v e box was i n s t a l l e d t o p e r m i t use o f an i n e r t atmosphere and t o s e r v e as c o n t a i n m e n t f o r c e r t a i n r a d i o a c t i v e samples. The t e c h n i q u e o f SSMS, b e s i d e s p o s s e s s i n g h i g h s e n s i t i v i t y and comprehensive e l e m e n t a l c o v e r a g e , shows l i n e a r r e s p o n s e f o r many e l e m e n t s ; i . e . , t h e i o n i n t e n s i t y o f any s i n g l e i m p u r i t y s p e c i e s i s proportional t o the impurity concentration i n the b u l k sample, p r o v i d i n g t h e i m p u r i t y i s homogeneously d i s t r i b u t e d . T h i s p r o p e r t y i s d e m o n s t r a t e d by t h e l i n e a r i t y p l o t i n F i g u r e 3 f o r boron c o n c e n t r a t i o n s i n NBS s t e e l s t a n d a r d s i n w h i c h t h e l i n e a r r e s p o n s e e x t e n d s o v e r s e v e r a l o r d e r s o f magnitude. I s o t o p e D i l u t i o n Spark Source Mass S p e c t r o m e t r y . A n a l y s e s by SSMS i n a r e a s i n w h i c h s t a n d a r d s a r e not a v a i l a b l e (as i s t h e case o f e n v i r o n m e n t a l samples and r e l a t e d energy s o u r c e samples) r e q u i r e use o f t h e isotope d i l u t i o n technique. I s o t o p e d i l u t i o n , des­ c r i b e d by H i n t e n b e r g e r ( 8 J , i n v o l v e s a d d i n g t o t h e sample an e n r i c h e d minor i s o t o p e o f t h e element o f i n t e r e s t , f o l l o w e d by mass s p e c t r o m e t r i c measurement of the a l t e r e d isotope r a t i o s . The c o n c e n t r a t i o n o f t h e element b e i n g measured i s o b t a i n e d from e q u a t i o n ( 1 ) .

16.

CARTER

ET AL.

Multielement Isotope Dilution Techniques

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch016

ORNL-DWG. 73-3117

1. SAMPLE ELECTRODES 2. ACCELERATOR SLITS 3. ELECTROSTATIC ANALYZER 4. BEAM MONITOR 5. MAGNETIC ANALYZER 6. PHOTO PLATE ΑΕΙ Scientific Apparatus

Figure 1. Double-focusing spark source mass spectrometer

Figure 2. MS-702 mass spectrometer with the special glove box arrangement

301

Figure 3. Analytical calibration curve for boron in steel over six orders of magnitude (0.01-10,000 ppm)

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch016

16.

CARTER

ETAL.

303

Multielement Isotope Dilution Techniques

(D where χ

=

w e i g h t o f element i n t h e sample.

y

=

w e i g h t o f element i n t h e t r a c e r s p i k e .

=

isotope r a t i o of the spike,

R

t

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch016

reference isotope tracer isotope R

isotope r a t i o of the mixture,

m

reference isotope tracer isotope * R

s

=

i s o t o p e r a t i o o f t h e sample, reference isotope tracer isotope '

A

=

a t o m i c w e i g h t o f t h e sample element.

=

atomic weight o f the t r a c e r .

Τ

=

atom % o f t h e s p i k e i s o t o p e i n t h e t r a c e r .

S

=

atom % o f t h e s p i k e i s o t o p e i n t h e sample.

A

t

The terms: A , A , T, S, R , and y a r e u s u a l l y known i n advance, and S i n c e R c a n be v e r y a c c u r a t e l y measured, i t i s n o t u n u s u a l to o b t a i n p r e c i s i o n and a c c u r a c y on t h e o r d e r o f 1% f o r t r a c e element d e t e r ­ mination. A f t e r e q u i l i b r i u m between s p i k e i s o t o p e and sample r e f e r e n c e i s o t o p e i s o b t a i n e d , q u a n t i t a ­ t i v e r e s u l t s a r e a s s u r e d by i s o t o p e d i l u t i o n even when a l o w y i e l d i n g c h e m i c a l p u r i f i c a t i o n i s r e q u i r e d . Thermal i o n i z a t i o n s o u r c e s a r e used f o r s o l i d s and e l e c t r o n bombardment s o u r c e s f o r g a s e s , g i v i n g a c c u r a t e r e s u l t s w i t h s n l a l l samples. However, i s o t o p e d i l u t i o n has n o t been used u n t i l r e c e n t l y f o r a n a l y z i n g e n e r g y - r e l a t e d samples w i t h SSMS. Even though t h e method i s l i m i t e d t o elements h a v i n g two o r more n a t u r a l l y o c c u r r i n g o r l o n g - l i v e d i s o t o p e s , i t i s s e n s i t i v e and c a n be v e r y a c c u r a t e . S i n c e spark s o u r c e mass s p e c t r o m e t e r s have s i m i l a r t

1

304

HIGH

MASS

SPECTROMETRY

s e n s i t i v i t i e s f o r a l l e l e m e n t s , SSMS can be used w i t h o u t d e l e t e r i o u s e f f e c t s f o r many m a t r i c e s . For example, i n o r d e r t o o b t a i n the cadmium c o n c e n t r a t i o n i n a sample by i s o t o p e d i l u t i o n SSMS, cadmium e n r i c h e d in C d i s e q u i l i b r a t e d w i t h the normal cadmium so t h a t c h e m i c a l e q u i l i b r i u m i s e s t a b l i s h e d between the e n r i c h e d and n a t u r a l i s o t o p e s . T h e r e a f t e r , any t e c h n i q u e may be used t h a t p e r m i t s the t r a n s f e r o f 0.1-3 ng o f cadmium from the s o l u t i o n t o the s u r f a c e o f a s u i t a b l e e l e c t r o d e s u b s t r a t e such as machined graphite rods. For t r a c e m e t a l c o n c e n t r a t i o n s , any s u i t a b l e e x t r a c t a n t may be used t o c o n c e n t r a t e the m e t a l a f t e r e q u i l i b r a t i o n w i t h the e n r i c h e d s p i k e . H i g h e x t r a c t i o n e f f i c i e n c i e s a r e not r e q u i r e d s i n c e a t t h i s p o i n t the a n a l y s i s depends on e s t a b l i s h i n g a r a t i o between the e n r i c h e d s p i k e i s o t o p e and one o f the major i s o t o p e s of the metal being sought. The mass s p e c t r a o f cadmium and molybdenum spiked with enriched C d and M o are shown i n F i g u r e 4. C o n c e n t r a t i o n s o f the i n d i v i d u a l elements can be c a l c u l a t e d from the a l t e r e d i s o t o p e r a t i o s . The r e s u l t s o f a t y p i c a l i s o t o p e d i l u t i o n o f cadmium a r e shown i n T a b l e I , w h i c h i s a r e p r o d u c t i o n o f t h e format produced by the computer. The d a t a r e d u c t i o n c o m p u t e r - d e n s i t o m e t e r system w h i c h we use has been t h o r o u g h l y d e s c r i b e d e l s e w h e r e ( 9 ) . The f i r s t e n t r y on the t a b l e shows the e n r i c h m e n t o f the Cd s p i k e (88.4% Cd). The second e n t r y i s a r e p o r t o f the abundances o f the cadmium i n the sample, u s u a l l y the n a t u r a l abundance. Sample 318R was a n a l y z e d by m i x i n g 1.00 ml o f the s p i k e ( c o n t a i n i n g 1.00 ppm e n r i c h e d cadmium) w i t h a 1.00 ml a l i q u o t o f the unknown. Two d i f f e r e n t p h o t o p l a t e i o n exposures were measured t o y i e l d the % t r a n s m i t t a n c e v a l u e a t m/e 106 and 114 as l i s t e d i n the t a b l e . From the e m u l s i o n c a l i b r a t i o n and the o b s e r v e d % T - v a l u e s , the q u a n t i t y o f cadmium i n the unknown i s c a l c u l a t e d by the computer system and r e p o r t e d i n the "Nano-gm" column. Even though the p e r c e n t t r a n s m i t t a n c e s are a t o p p o s i t e ends o f the e m u l s i o n c a l i b r a t i o n c u r v e , the agreement i s v e r y good. The e x p e c t e d p r e c i s i o n f o r t h e s e a n a l y s e s i s ±3-5%. The d a t a r e d u c t i o n programs f o r t h i s i s o t o p e d i l u t i o n method a r e s u f f i c i e n t l y f l e x i b l e to accomodate a v a r i e t y o f e x p e r i m e n t a l a p p r o a c h e s . For example, any c o m b i n a t i o n o f volume or w e i g h t , c o n c e n t r a t i o n , i s o t o p i c abundance, and % t r a n s m i t t a n c e f o r b o t h the s p i k e and sample can be accommodated by the d a t a system. 1 0 6

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch016

PERFORMANCE

1 0 6

1 0 6

97

16.

CARTER

Multielement Isotope Dilution Techniques

ET AL.

ORNL-DWG. 76-2795

TYPICAL MASS SPECTRA FOR IDSSMS Cd

Mo

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch016

97

LMP_ « 0.375 Mo

SAMPLE Normal Isotopes

liiii

llllli

95

100 105

110 115

95

100 105

110 115

,06

Cd

- « 0.043

SPIKE Separated Isotopes

SAMPLE SPIKE

I ill 11 95

100

„ IlllL

105

110

115

^.o.s3 114

Cd

Figure 4. Isotope dilution spark source mass spectra for cadmium and molybdenum

305

306

HIGH

PERFORMANCE

MASS

TABLE I . Computer P r i n t o u t f o r Cadmium by I s o t o p e Spark Source Mass S p e c t r o m e t r y Cd

106

Spike Sample

88.400 1.220

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch016

Sample 318R 318R

SPECTROMETRY

Dilution

114

Vol

Conck

2.500 28.900

1.000 1.000

1.000

Nano-gm

106

114

4005.000 3933.000

19.3 64.6

16.2 60.0

Trace Elements by a "Dry S p i k e " I s o t o p e T e c h n i q u e . Due t o d i f f i c u l t y o f d i s s o l v i n g many c o a l and f l y a s h samples, a method was employed i n w h i c h d r y sample powders were mixed w i t h a c o n d u c t i n g m a t r i x m a t e r i a l w h i c h c o n t a i n e d numerous i s o t o p i c " s p i k e s " . (10) The m a t r i x was 99.9999% p u r e s i l v e r powder. The e n r i c h e d i s o t o p e s shown i n T a b l e I I were then d e p o s i t e d onto t h e m a t r i x from s o l u t i o n . A p p r o x i m a t e l y 50 yg/g o f i s o t o p i c a l l y normal erbium was added as a s i n g l e i n t e r n a l s t a n d a r d . Erbium was chosen because o f t h e l a r g e dynamic range i n i s o t o p i c c o m p o s i t i o n . The s p i k e d s i l v e r m a t r i x was p r e s s e d i n t o e l e c t r o d e s and s p a r k e d d i r e c t l y t o measure the l e v e l s o f t h e elements t o be a n a l y z e d (see T a b l e I I ) . B l a n k l e v e l s o f each element p r e s e n t i n t h e s i l v e r were d e t e r m i n e d i n a p r e v i o u s experiment. In o r d e r t o s t a n d a r d i z e t h e amounts o f each i s o t o p i c s p i k e p r e s e n t i n t h e m a t r i x m a t e r i a l , i t was n e c e s s a r y t o d e p o s i t a s o l u t i o n c o n t a i n i n g known amounts o f t h e n a t u r a l elements onto a weighed a l i q u o t o f t h e d r y m a t r i x . T h i s m i x t u r e was d r i e d and homogenized, f o l l o w e d by SSMS a n a l y s i s and i s o t o p e d i l u t i o n measurements t o y i e l d t h e apparent c o n c e n t r a t i o n s o f t h e n a t u r a l elements. I t was p o s s i b l e t o b a c k - c a l c u l a t e the a c t u a l c o n c e n t r a t i o n s of the s p i k e i s o t o p e s i n t h e m a t r i x . The a c c u r a c y o f any measurement s h o u l d be checked by t h e use o f s t a n d a r d s . F o r t h e a n a l y s i s o f c o a l , f l y ash and r e l a t e d g e o l o g i c a l samples, NBS S t a n d a r d Reference M a t e r i a l s such as NBS SRM 1632 ( C o a l ) and 1633 ( F l y Ash) a r e u s e f u l . From the a n a l y s i s o f t h e s e s t a n d a r d s , r e l a t i v e s e n s i t i v i t y f a c t o r s o r i n s t r u m e n t a l b i a s e s were d e t e r m i n e d .

16.

CARTER

ETAL.

Multielement Isotope Dilution Techniques

307

Homogenization o f the d e p o s i t e d i s o t o p e s was ensured by e x t e n s i v e s h a k i n g and g r i n i n d i n a m i x e r m i l l u s i n g a p l a s t i c c o n t a i n e r and p l a s t i c b a l l p e s t l e s . W i t h 30 minutes o f such s h a k i n g , the p r e c i ­ s i o n o b t a i n e d f o r d u p l i c a t e a n a l y s e s was 5-101, i n d i c a t i n g t h a t no g r o s s inhomogeneity e x i s t e d .

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch016

TABLE I I Separated

I s o t o p e s D e p o s i t e d Onto Pure Ag Powder

Element

Isotope

Pb Tl Ba Te In Cd Mo Sr Se Zn Cu Ni Fe Cr Κ

204 203 135 125 113 111 97 86 77 67 65 61 57 53 41

%

Enrichment 99. 73 94.96 93.6 91.23 96.36 91.33 94.25 95.73 94.38 89.68 99.70 92.11 93.63 96.4 99.35

Approx. Cone. i n Ag f j i g / g ) 10 5 50 10 5 5 5 100 10 30 30 30 100 30 100

In the a n a l y s i s o f t r a c e elements i n c o a l by t h i s method, the c o a l was f i r s t ashed (550°C) and an e x a c t amount o f ash mixed w i t h a known q u a n t i t y o f s i l v e r s p i k e . The m i x t u r e was homogenized, p r e s s e d , and s p a r k e d i n the s p a r k s o u r c e mass s p e c t r o m e t e r w i t h the u s u a l i o n d e t e c t i o n by I l f o r d Q-2 photop l a t e s . Data were r e a d on a m i c r o d e n s i t o m e t e r computer system d e s c r i b e d e l s e w h e r e (9). For the elements l i s t e d i n T a b l e I I , a r a t i o was measured between the s e p a r a t e d i s o t o p e and a n a t u r a l i s o t o p e o f each element. These r a t i o s were used t o c a l c u l a t e the c o n c e n t r a t i o n s o f each element i n the sample a c c o r d i n g t o the i n t e r n a l s t a n d a r d approach t o SSMS analyses (10). This technique i s s i m i l a r t o , but not the same a s , i s o t o p e d i l u t i o n , s i n c e the i s o t o p e s have not r e a c h e d c h e m i c a l e q u i l i b r i u m i n s o l u t i o n . A p p a r e n t l y i s o t o p i c e q u i l i b r i u m i s approached i n t h e i o n plasma. Once the amounts o f the elements i n T a b l e I I a r e

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch016

308

HIGH

PERFORMANCE

MASS

SPECTROMETRY

measured a s i m i l a r p r o c e d u r e a l l o w e d the o t h e r elements o f i n t e r e s t such as Co, Mn, As, e t c . , t o be measured v e r s u s the a p p r o p r i a t e i s o t o p i c s p i k e s . A g a i n , a s e n s i t i v i t y f a c t o r i s needed and can be o b t a i n e d from NBS o r o t h e r s t a n d a r d s . T a b l e I I I shows average r e s u l t s f o r NBS SRM 1632 ( C o a l u s i n g SRM 1633 ( F l y Ash) as a s t a n d a r d . These d a t a i n d i c a t e t h a t good p r e c i s i o n and a c c u r a c y a r e a v a i l a b l e a t l e v e l s l e s s t h a n one p a r t p e r m i l l i o n . The t e c h n i q u e d e s c r i b e d above f o r c o a l ash a n a l y s i s has a l s o been s u c c e s s ­ f u l l y a p p l i e d t o o t h e r ash samples and some g e o l o g i ­ c a l m a t e r i a l s w h i c h are d i f f i c u l t t o d i s s o l v e . As f o r o t h e r a n a l y t i c a l e n d e a v o r s , the a v a i l a b i l i t y o f s t a n d a r d s i s i m p o r t a n t t o ensure a c c u r a c y o f the r e s u l t s by the d r y i s o t o p e s p i k e technique. M

TABLE I I I . A n a l y s i s o f SRM Element Tl Pb Cd Zn Cu Ni Cr

1 1

1632

U s i n g SRM

Cone. + 0.6 33 0.4 32 15 15 19

± ± ± ± ± ± ±

RSD 0.2 3 0.2 8 3 3 3

1633

as

NBS 0.59 30 0.19 37 18 15 20.2

Standard

Certified ±

0.03 ±9 ± 0.03 +4 ±2 ±1 ± 0.5

Conclusions. M u l t i e l e m e n t i s o t o p e d i l u t i o n has added a capa­ b i l i t y f o r p r e c i s i o n and a c c u r a c y t o the h i g h s e n s i ­ t i v i t y and comprehensive e l e m e n t a l range o f spark s o u r c e mass s p e c t r o m e t r y . E n r i c h e d i s o t o p e s are a v a i l a b l e i n h i g h p u r i t y and h i g h enrichment f o r a l a r g e number o f elements so t h a t the number o f e l e ­ ments t h a t may be d e t e r m i n e d by IDSSMS i s q u i t e l a r g e . We have shown t h a t these i s o t o p e s can be used as i n t e r n a l s t a n d a r d s f o r m o n o - n u c l i d i c e l e m e n t s . We have a l s o shown t h a t d r i e d m i x t u r e s o f i s o t o p e s on c o n d u c t i n g powders can be added t o s o l i d samples as i n t e r n a l s t a n d a r d s w i t h a c c e p t a b l e p r e c i s i o n and accuracy. The p r e c i s i o n and a c c u r a c y o f the d r y s t a n d a r d method i s between 10 and 20%, and f o r the s o l u t i o n t e c h n i q u e the p r e c i s i o n and a c c u r a c y can a p p r o a c h 5-10% w i t h 1 χ 1 0 " g o f an element p r e s e n t on t h e e l e c t r o d e s . 9

16.

CARTER

ET AL.

Multielement Isotope Dilution Techniques

309

Literature Cited.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch016

1. 2.

Dempster, A.J., Nature, (1935) 135, 452. Gordon, J.G., Jones E.J., and Hippie, J.A., Anal. Chem., (1951) 23, 438. 3. Hannay, N.B., and Ahearn, A.J., Anal. Chem., (1954) 26, 1056. 4. Mattauch J. and Herzog, R., Z. Physik., (1934) 89, 786. 5. Leipziger, F.D., Anal. Chem., (1965) 37, 171. 6. Alvarez, R., Paulsen, P . J . , and Kelleher, D . E . , Anal. Chem., (1969) 41, 955. 7. Carter, J.A., Donohue, D . L . , Franklin, J.C., and Stelzner, R.W., Trace Substances in Environmental Health-IX, Univ. of Missouri, 1975. 8. Hintenberger, H . , "A Survey of the Use of Stable Isotopes in Dilution Analysis", Electromagnetically Enriched Isotopes and Mass Spectrometry, ed. by M. L. Smith, Academic Press, Chap. 21, pp. 177-189. (1956). 9. Stelzner, R.W., McKown, H . S . , and Sites, J.R., ACS Spring Meeting, 1975. 10. Jaworski, J.F., and Morrison, G.H., Anal. Chem., (1975) 47, 1173. 11. Ahearn, A.J., ed. "Mass Spectrometric Analysis of Solids", Elsevier, New York, 98 (1966). Received December 30, 1977

17 Computer Applications in Mass Spectrometry F. W. MC LAFFERTY and R. VENKATARAGHAVAN

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch017

Department of Chemistry, Cornell University, Ithaca, NY 14853

The information content of the mass spectrum of an average organic compound is unusually high, containing at least 50 bits of information (1); further, with modern instrumentation a mass spectrum can be obtained on a nanogram of compound in one second. Obviously, to utilize this tremendous quantity of data it must be acquired, reduced, and interpreted in a very rapid and efficient manner. The modern digital computer (COM) has proved to be ideal for all three of these duties, and its rapid increase in capabilities, and the concomitant reduction in cost, in the last decade has thus led to a similar revolution in mass spectrometry (MS). Acquisition and Reduction of MS Data The book of Waller (2) was very timely in showing the surprising advances in MS/COM systems that had occurred in the few years before 1971. This book contained reports from many leading MS research laboratories concerning the automated data acquisition and reduction systems then in use. There were a wide variety of these, the majority of which had been developed to a substantial extent in the reporting laboratory. In the intervening six years the field has changed dramatically; literally hundreds of mass spectrometry laboratories now have computerized data acquisition and reduction systems, and most of these are manufacturer supplied. Only a small fraction of these acquire data on, for example, magnetic tape for later processing on a central computer; the on-line minicomputer is the rule, but it has increasing competition from microprocessors and even sophisticated calculators. A computer-controlled GC/MS is now available for less than $50,000 (3), and a microprocessor-driven MS is available which gives the confidence of identification as well as quantitative analyses for 16 preselected compounds every second (4).

©0-8412-0422-5/78/47-070-310$05.00/0

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch017

17.

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AND

VENKATARAGHAVAN

Computer Applications

311

There are s t i l l challenging areas of high potential for further applications of on-line computers i n MS data acquisition and reduction, such as high-resolution MS, simplification of MS operation, maintenance and record keeping, c o l l i s i o n a l activation MS, and continuous analyzers (e.g., patient monitoring, process control) (5). However, just as mass spectrometry evolved so that most laboratories no longer constructed their own instruments, the field has suddenly progressed to where computer data a c q u i s i tion and reduction equipment which is superior for most a p p l i c a tions i s now available commercially. This i s not true, however, for a highly promising and challenging area, that of the computer identification of unknown mass spectra (6), and this w i l l be emphasized i n the remainder of our discussion. If efficient mass spectrometer systems with totally automated data acquisition, reduction, and identification could be made available at reasonable cost, this would surely open many important new areas of application for MS. Computer Identification of Unknown Mass Spectra The identification of unknown compounds from their mass spectra, whether as pure compounds or i n mixtures, i s a problem for which we feel i t i s better to use two approaches, retrieval and interpretation, with these applied sequentially. The unknown mass spectrum i s first matched against a library of a l l available reference spectra; if no spectrum i s retrieved which matches sufficiently w e l l , the unknown spectrum i s then interpreted to obtain as much structural information as possible. A variety of systems for both retrieval and interpretation have been proposed (6); interpretation using pattern recognition systems have been described at this meeting by Isenhour (7) and by Wilkins (8) and the Artificial Intelligence system (9) by Smith; thus this report w i l l emphasize the "Probability Based Matching" (PBM) (10, 11) and the "Self-Training Interpretive and Retrieval System" (STIRS) (12-15) developed at Cornell for these purposes. Both of these systems are actually available internationally to outside users over the TYMNET computer networking system (16). Probability Based Matching For document retrieval i n libraries (17). it i s w e l l known that optimization requires "weighting" of the descriptors or requirements sought; some are more important than others to the person conducting the search. Low resolution mass spectra contain two principal types of data, masses and abundances; the PBM system (10,, 11) employs a probability weighting of these. As first proposed by Grotch (18), the probability of occurrence of particular abundances (based on 100% for the most abundant peak) should follow a log normal distribution. This was shown to be true for a data base of 18,806 different compounds (19); abundance

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch017

312

HIGH

PERFORMANCE

MASS

SPECTROMETRY

ranges differing by a factor of two in their occurrence probability are>0.24%, >1.0%, >3.4%, >9.0%, >19%, >38%, and>73%. The probability of occurrence of the different mass values also varies widely. Because larger molecular fragments tend to decompose to give smaller fragments, higher m/e values are less common in mass spectra, the probability decreasing by a factor of 2 approximately every 130 mass units. Although this is a surprisingly smooth function at high m/e values, lower values show rather large differences in their probability of occurrence, with these variations repeated every 14 mass units. Thus, although m/e 39, 41, and 43 ions of 1% or greater abundance are each found in more than two-thirds of all reference spectra, peaks m/e 33, 34, and 35 each occur less than 10% as frequently (19). Thus in an unknown mass spectrum if abundant ions at m/e 34, 241, and 343 are matched by comparably abundant ions in a reference spectrum, this is far more significant in indicating that a correct retrieval has been made than if the unknown's m/e 39, 41, and 43 peaks were matched in a reference spectrum. These abundance and mass uniqueness weightings are used to calculate the probability that the match occurred by chance and is thus a "false positive"; the reciprocal of this probability (log base 2 "confidence index, K") is used to rate the degree of match. A second unique feature of PBM, which has been proposed independently by Abramson (20), is "reverse searching", and is valuable for the identification of components in mixtures. In this, PBM ascertains whether the peaks of the reference spectrum are present in the unknown spectrum, not whether the unknown's peaks are in the reference. Thus the reverse search in effect ignores peaks in the unknown which are not in the reference spectrum, as they could be due to other components of the mixture. Although reverse searching should thus reduce the capabilities of PBM for matching unknown spectra of pure compounds, this apparently is more than offset by the increased capabilities resulting from the data weighting. The system has been tested with over 800 "unknown" mass spectra taken from a large c o l l e c tion of mass spectra from diverse sources (21), and its performance has shown to be generally superior (22) to the widely accepted Biemann-MIT system (23) which matches the two largest peaks in each 14 mass unit region. Performance has been measured utilizing "recall/reliability" plots, methodology developed for library retrieval systems (17) in which recall (RC) is defined as the proportion of relevant spectra actually retrieved, and reliability (RL, or "% correct" X 0.01) is the proportion of retrieved spectra which are actually

17.

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Computer Applications

A N D VENKATARAGHAVAN

313 (1)

tt- -

( I

V c

FP = I / P f

+

(2)

¥

(3)

f

relevant; FP = proportion of false positives, I = number of cor­ rect identifications, If = number of false identifications, P = number of possible correct identifications, and Pf = number of possible false identifications (P + Pf = total unknowns examined). The "recall/reliability" plot is then constructed by determining the pair of these values achieved for particular Κ (or "ΔΚ") value thresholds (11); the higher the Κ value demanded by the user for an identification, the higher the probability that it is correct (RL), but the lower the chance of making an identification for a particular unknown (RC). Note that if the evaluation is made considering only the highest Κ selection as the match, the number of compounds tested will be equal to the number of possible cor­ rect identifications (P ), to Pf, and also to (I + If), so that for this evaluation RC = RL = (1 - FP). Two sets of "unknown" spectra of over 400 each were selected at random from the molecular weight (MW) ranges 144-160 and 232-312; at 15% recall these showed reliability values of 83% and 85%, respectively; at 25% R C , RL = 76% and 62%; and at 50% R C , RL = 65% and 42%. As discussed later, the 65% RL value corresponds to a false positives of ~ l / 5 0 , 0 0 0 . The lower results for the high M W set are mainly due to the use of only 15 peaks per spectrum, and this has been increased, de­ pending on M W , to as many as 26. Unknowns present as 30% and 10% components in mixtures gave reliabilities of 60% and 20%, respectively, at the 25% recall level; however, these results were far superior to those from the forward search system on the same unknowns. To repeat, for unknowns giving matches of unsatisfactory reliability, STIRS should be used also. Because retrieval of the spectrum of a compound whose structure is similar to, but not exactly the same a s , that of the unknown can be helpful to the user, four "classes of match" have been defined: I, identical compound or stereoisomer; II, class I or ring position isomer; III, class II or homolog; and IV, class III or an isomer of class III compound formed by moving only one carbon atom. The recall/reliability performance of PBM was evaluated separately for these four matching criteria; at the 50% recall level 95% of the compounds (low M W set) selected matched within the class IV criteria. As has been suggested previously (24), the subtraction of the reference spectrum of an identified compound from the mixture spectrum should produce a residual unknown spectrum which is easier to identify. This approach has been implemented so that at the end of the PBM run the computer automatically subtracts the best matching reference spectrum (or, at user command, some c

c

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch017

c

c

c

314

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PERFORMANCE

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SPECTROMETRY

other reference spectrum) from the unknown spectrum and notifies the user of any significant residual peaks. Although PBM is a reverse search system, its recall is lower for components in lower proportion of the mixture; thus subtracting out the contribution of an abundant component improves the PBM recall for other mixture components. Recently In Ki Mun in our laboratory has applied the PBM principles to the identification of the number of chlorine and bromine atoms present in a fragment ion from the abundances of the isotopic peaks. In a test of more than 1,000 "unknown" spectra taken from our large data base, the predictions of this PBM program were correct 90% of the time, and most of these incorrect predictions were due to inaccurate data.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch017

Self-Training Interpretive and Retrieval System In some interpretive methods such as Artificial Intelligence (9) the computer is programmed to recognize the mass spectral behavior of particular types of compounds or substructures . In pattern recognition or learning machine approaches, the computer trains itself for such recognition based on reference spectra which do and do not contain a particular structural feature. In contrast, there is no pretraining of STIRS (12) for the mass spectral behavior of particular structural moieties; rather, 15 classes of mass spectral data (Table I) have been selected which indicative of different compound types or substructures, but without designating what these types or substructures are. STIRS then, in effect, trains itself to interpret the unknown mass spectrum by matching the unknown data for each of these classes against the corresponding data for all of the reference spectra; particular substructures or types of compounds which are found in a substantial proportion of the best matching reference compounds have a correspondingly high probability of being present in the unknown. Thus STIRS does not have to be pretrained to recognize the presence of any particular structural feature; however, it cannot identify any feature which is not present in some compounds of the reference file. In further contrast to some applications of pattern recognition methods, we recommend that STIRS be used for positive information only; the presence of a structural feature in many of the selected reference compounds indicates the presence of that substructure in the unknown, but the absence of a substructure in the selections is not a reliable indication of its absence in the unknown. The present Cornell PBM system employs a data base of 41,429 different spectra of 32,403 different compounds. To increase the selectivity of STIRS, it uses a data base of only the best spectrum (25) of each compound, limiting this further to the 29,468 compounds which contain only the common elements H , C , N , O , F , S i , P, S, C I , Br, and/or I. A number of improvements made recently to the system will be described below. a r e

17.

MCLAFFERTY AND VENKATARAGHAVAN

315

Computer Applications

Table I. Mass Spectral Data Classes Used i n STIRS

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch017

Data Class

Description, maximum number of peaks

1

Ion Series (14 amu

2-4

Characteristic ions

separation)

Range of mass or mass loss 175)

6B

Five

76-149 (MW>250)

5C

Five

16-20, 30-38, 44-! 59-65, 72-76

6C

Five

26-28, 39-42,5266-70, 80-84

7, 8

11

Secondary neutral losses from most abundant odd-mass (MF7) and even-mass (MF8) loss Overall match factors

11.0

MF11.1 + MF11.2

11.1

2A + 2B + 3A + 3B + 4A + 4B

11.2

5A + 5B + 6A

99% of cases this w i l l be due to the presence of phenyl i n the unknown compound, i . e . , i n