High performance mass spectrometry: Chemical applications

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

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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

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HIGH

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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 ]-<^ 3

2

CH

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

+ H

+

+ 3

+ H0 2

(4b)

The b r o a d m e t a s t a b l e peak a t t r i b u t e d t o r e a c t i o n (4a) i n d i c a t e s t h a t i t o c c u r s w i t h t h e r e l e a s e o f 0.93 eV o f t r a n s l a t i o n a l energy, i n e x c e l l e n t agreement w i t h a v a l u e o f 1.1 eV c a l c u l a t e d from r e s u l t s based on ICR s p e c t r o m e t r y and t h e q u a s i e q u i l i b r i u m t h e o r y (OET) o f mass s p e c t r a ( 7 ) . The m e t a s t a b l e peak a r i s i n g from r e a c t i o n (4b) Ts v e r y narrow and i s c o n s i s t e n t w i t h t h e r e l e a s e o f o n l y 0.0016 eV o f t r a n s l a t i o n a l energy. The e f f e c t o f t h e β-slitwidth on s e n s i t i v i t y and on t h e ease o f assignment o f p r e c u r s o r i o n masses has been s t u d i e d on an ΑΕΙ MS9 i n s t r u m e n t , o f N i e r - J o h n s o n geometry (81). The s t a n d a r d ^ - s l i t w i d t h i n such an i n s t r u m e n t i s 0.508 cm. which has an energy bandpass o f ± 0.661 o f t h e n o m i n a l energy o f t h e i o n beam. I f energy r e l e a s e i n a m e t a s t a b l e t r a n s i t i o n i s s u f f i ­ c i e n t l y l a r g e t o produce a beam w i t h a s p r e a d i n energy at t h e β-slit w h i c h i s g r e a t e r t h a n t h i s energy band­ p a s s , some o f t h e beam i s l o s t w i t h a consequent r e d u c ­ t i o n i n s i g n a l i n t e n s i t y . Any r e d u c t i o n i n t h e βs l i t w i d t h r e s u l t s i n a further loss of s i g n a l . I f on the o t h e r hand t h e energy s p r e a d w i t h i n t h e beam i s l e s s t h a n t h a t p a s s e d by t h e β-slit, t h e s l i t w i d t h may be r e d u c e d w i t h o u t l o s s o f s e n s i t i v i t y u n t i l t h e two become comparable a f t e r w h i c h any f u r t h e r r e d u c t i o n i n s l i t w i d t h leads to a f a l l i n s e n s i t i v i t y . Consequent­ l y , i f one w i s h e s t o compare t h e r e l a t i v e i n t e n s i t i e s of t h e peaks g i v e n by two m e t a s t a b l e t r a n s i t i o n s , i t i s u s u a l l y necessary to s p e c i f y the 3 - s l i t w i d t h at which the measurements a r e t a k e n . F o r example, i n F i g u r e 2, the two s i g n a l s a r e n o r m a l i z e d t o be e q u a l a t a s l i t ­ w i d t h o f 0.508 cm., b u t a t any l o w e r s l i t w i d t h t h e y a r e c l e a r l y unequal. S i m i l a r c o n s i d e r a t i o n s a p p l y when one r e d u c e s t h e β-slitwidth i n an a t t e m p t t o reduce t h e o v e r l a p p i n g o f m e t a s t a b l e peaks i n o r d e r t o a s s i g n masses o f p r e c u r s o r i o n s more r e a d i l y . F o r example, i f an i n t e n s e , broad peak i s o b s e r v e d f o r t h e t r a n s i t i o n i i m , i t may be d i f f i c u l t t o o b s e r v e a low i n t e n s i t y s i g n a l f o r t h e transition (mi-l) -* m . I f a m e t a s t a b l e peak i s i n t r i n s i c a l l y b r o a d owing t o t h e r e l e a s e o f energy i n the f r a g m e n t a t i o n , a r e d u c t i o n o f t h e β-slitwidth r e d u c e s t h e i n t e n s i t y o f t h e peak b u t n o t i t s w i d t h so t h a t a " p r e c u r s o r mass r e s o l v i n g power based on a 5% a d j a c e n t peak c o n t r i b u t i o n r i s e s v e r y l i t t l e as t h e s l i t w i d t h i s d e c r e a s e d . I f t h e energy r e l e a s e i s s m a l l +

+

2

+

+

2

11

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

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Organic Mass Spectrometry

Figure 1. Typical peak shape for transition C H *+ -» 7

Figure 2. Effect of β-slitwidth W on sensi­ tivity S for two metastable transitions; the points are experimental data, and the lines are calculated assuming a rectangular beam profile.

7

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

1.

9

Metastable Transitions

JENNINGS

so t h a t t h e energy s p r e a d i n t h e beam i s l e s s t h a n t h e energy bandpass o f t h e β-slit, a s u b s t a n t i a l improve­ ment i n " p r e c u r s o r mass r e s o l v i n g power" i s o b s e r v e d as the 3 - s l i t w i d t h i s reduced. R e s u l t s obtained f o r a number o f m e t a s t a b l e t r a n s i t i o n s a r e i l l u s t r a t e d i n F i g u r e 3. The main advantages o f t h e a c c e l e r a t i n g v o l t a g e scan method o f o b s e r v i n g m e t a s t a b l e t r a n s i t i o n s a r e i t s s i m p l i c i t y and s e n s i t i v i t y c o u p l e d w i t h i t s a b i l i t y t o g i v e d e t a i l e d i n f o r m a t i o n on energy r e l e a s e i n f r a g ­ mentations. I t s major d i s a d v a n t a g e s a r e t h a t t h e p r a c t i c a l range over w h i c h Vi/Vo may be v a r i e d i s l i m i t e d t o about f o u r and t h a t t h e s o u r c e t u n i n g c h a r ­ a c t e r i s t i c s may change d u r i n g t h e s c a n t h e r e b y a l t e r i n g t h e e f f i c i e n c y o f e x t r a c t i o n o f i o n s from t h e s o u r c e . The l a t t e r problem c a n be overcome t o a l a r g e e x t e n t by a d j u s t i n g t h e t u n i n g so t h a t i t i s r e l a t i v e l y i n s e n s i ­ t i v e t o V i a l t h o u g h t h i s u s u a l l y e n t a i l s some s a c r i f i c e of s e n s i t i v i t y . I n some a p p l i c a t i o n s , t h e b r o a d peaks produced by t h i s s c a n , even a t reduced β-slitwidths, may cause some u n c e r t a i n t y i n t h e assignment o f p r e c u r ­ sor i o n masses, p a r t i c u l a r l y i f t h e scan i s used t o study c o l l i s i o n induced decompositions. The t h i r d g e n e r a l method i n w h i c h t h e e l e c t r i c s e c t o r v o l t a g e Ε i s scanned a t c o n s t a n t V and Β s h a r e s many o f t h e advantages and d i s a d v a n t a g e s o f t h e p r e ­ v i o u s method, e s p e c i a l l y when used w i t h an i n s t r u m e n t of r e v e r s e d geometry ( 4 ) . I t makes use o f t h e f a c t that i f m i i i n tEe f i e l d - f r e e region preceding the e l e c t r i c s e c t o r , m a r e t r a n s m i t t e d when +

+

2

+

2

m /m 1

2

=

E /E 1

(5)

2

where E i and E a r e t h e e l e c t r i c s e c t o r v o l t a g e s r e ­ q u i r e d t o t r a n s m i t i o n s m i (main beam) and m . I n i n s t r u m e n t s o f N i e r - J o h n s o n geometry, t h e method has n o t been w i d e l y a p p l i e d s i n c e i t r e q u i r e s t h e u s e o f a s e n s i t i v e d e t e c t o r between t h e two s e c t o r s . Even t h e n , w i t h o u t mass a n a l y s i s o f t h e p r i m a r y i o n beam, peaks a r e o b s e r v e d f o r a l l t r a n s i t i o n s f o r which e q u a t i o n (5) i s s a t i s f i e d , i . e . , without s e l e c t i o n of either i i or m . A l t h o u g h assignment i s n o t d i f f i c u l t f o r s i m p l e compounds, i t becomes much more d i f f i c u l t as t h e com­ p l e x i t y o f t h e m o l e c u l e i n c r e a s e s . N e v e r t h e l e s s , such a d i s p l a y , known as an i o n k i n e t i c energy (IKE) s p e c t r u m , may have a p p l i c a t i o n s as a " f i n g e r p r i n t " spectrum. In a r e v e r s e geometry i n s t r u m e n t , however, t h e main beam i s m a s s - a n a l y z e d by p a s s i n g i t t h r o u g h a m a g n e t i c s e c t o r b e f o r e t h e e l e c t r i c s e c t o r so t h a t m i i s s e l e c t e d by a d j u s t i n g t h e m a g n e t i c f i e l d . Decompo2

+

+

2

+

+

2

+

10

HIGH

PERFORMANCE

MASS

SPECTROMETRY

+

s i t i o n s o f 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 two s e c t o r s g i v e r i s e to v a r i o u s d a u g h t e r i o n s m2 w h i c h are s e l e c t e d by s c a n n i n g the e l e c t r i c s e c t o r v o l t a g e E. The peak-shape c h a r a c t e r i s t i c s and o t h e r f e a t u r e s a r e v e r y s i m i l a r t o those o f the V s c a n method e x c e p t t h a t one o b t a i n s i n f o r m a t i o n on the p r o d u c t s g i v e n by the f r a g m e n t a t i o n o f a p a r t i c u l a r i o n m i r a t h e r t h a n on a l l p a r e n t i o n s o f a p a r t i c u l a r d a u g h t e r ion m . The r e s u l t i n g spectrum i s a m a s s - a n a l y z e d i o n k i n e t i c energy (MIKE) s p e c t r u m . Two advantages o f t h i s method o v e r the V s c a n method a r e t h a t the s o u r c e c o n d i t i o n s a r e c o n s t a n t t h r o u g h o u t the s c a n so t h a t i t can be t u n e d f o r maximum s e n s i t i v i t y a t the maximum p r a c t i c a l v a l u e o f V, and the range o f E i / E w h i c h i s p r a c t i c a b l e i s s u b s t a n t i a l l y g r e a t e r t h a n the range o f V i / V o i n the p r e v i o u s method. Problems a s s o c i a t e d w i t h d e c o m p o s i t i o n d u r i n g a c c e l e r a t i o n a r e absent and c o l ­ l i s i o n - i n d u c e d d e c o m p o s i t i o n s o f p r o d u c t s for«med i n i o n - m o l e c u l e r e a c t i o n s i n a h i g h p r e s s u r e source a r e l e s s l i k e l y t o mask peaks a r i s i n g from u n i m o l e c u l a r decompositions. Set a g a i n s t t h e s e a d v a n t a g e s , however, i s the f a c t t h a t s i n c e o b s e r v a t i o n s a r e made s u b s t a n ­ t i a l l y l a t e r i n the i o n f l i g h t p a t h t h a n i n the V s c a n method, a s m a l l e r number o f i o n s , i n g e n e r a l , w i l l decompose i n t h e f i e l d - f r e e r e g i o n b e f o r e the e l e c t r i c s e c t o r o f a r e v e r s e geometry i n s t r u m e n t t h a n i n the same r e g i o n o f 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 r e f o r e , the s e n s i t i v i t y i s l o w e r . In a d d i t i o n , a t h i g h v a l u e s o f E i / E , the energy o f the t r a n s m i t t e d m i o n s i s low, t h e r e b y l e a d i n g t o a r e d u c e d g a i n a t the e l e c t r o n m u l t i p l i e r so t h a t the w i d e r range i s o b t a i n e d t o some e x t e n t a t the expense o f s e n s i t i v i t y . A p p l i c a ­ t i o n s o f t h i s method a r e d i s c u s s e d by o t h e r a u t h o r s e l s e w h e r e i n t h i s volume. The l a s t two methods d i f f e r from those d e s c r i b e d above i n t h a t two o f the t h r e e p a r a m e t e r s V, Ε and Β a r e l i n k e d t o g e t h e r and scanned w h i l e the t h i r d i s h e l d constant. Each has the f e a t u r e o f g i v i n g narrow meta­ s t a b l e peaks comparable w i t h peaks g i v e n by i o n s formed i n t h e s o u r c e i n a normal mass spectrum so t h a t , i n e f f e c t , a h i g h r e s o l u t i o n spectrum o f m e t a s t a b l e peaks i s obtained. I n each s c a n , peaks a r e g i v e n by d a u g h t e r i o n s formed from a chosen p a r e n t i o n m i and the meta­ s t a b l e t r a n s i t i o n s a r e r e a d i l y a s s i g n e d . N e i t h e r method g i v e s any i n f o r m a t i o n on peak shape and hence no i n ­ f o r m a t i o n on the r e l e a s e o f t r a n s l a t i o n a l energy i n a f r a g m e n t a t i o n can be deduced. In the f i r s t o f the two l i n k e d scans Β i s h e l d c o n s t a n t and the a c c e l e r a t i n g v o l t a g e V and £he e l e c ­ t r i c s e c t o r v o l t a g e Ε a r e l i n k e d such t h a t V^/E i s +

+

+

2

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

2

+

2

2

+

1.

Metastable Transitions

JENNINGS

11

constant throughout the scan ( 9 ) . Since Ε i s d i r e c t l y p r o p o r t i o n a l t o V , t h e energy o f i o n s t r a n s m i t t e d by t}ie e l e c t r i c s e c t o r , V^/E i s e q u i v a l e n t t o m a i n t a i n i n g VW constant d u r i n g the scan. A t one p o i n t d u r i n g t h e s c a n , a t t h e c r o s s - o v e r p o i n t , V - V* as shown i n F i g u r e 4, t h e main beam i s t r a n s m i t t e d and t h e p a r e n t ion i i i s s e l e c t e d by a d j u s t i n g t h e m a g n e t i c f i e l d B. I f t h i s i o n fragments t o g i v e m i n the f i e l d - f r e e r e g i o n between t h e s o u r c e and e l e c t r i c s e c t o r , m ions are t r a n s m i t t e d by t h e e l e c t r i c s e c t o r when e

+

+

2

+

2

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

Vj

=

(m /m )V 2

1

(6)

1

where V i i s t h e a c c e l e r a t i n g v o l t a g e a c t u a l l y a p p l i e d t o t h e m i i o n s when m ions are transmitted. In order f o r these m i o n s t o be c o l l e c t e d a t t h e f i x e d magnetic f i e l d s t r e n g t h Β +

+

2

+

2

#

where vî = V a t t h e c r o s s o v e r p o i n t . tions are s a t i s f i e d Vj/Vj

%

=

(1/Vj)

=

kCl/v/)^

%

V /E 1

When b o t h equa(8)

2

In t h e s c a n , Ε and hence V* 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 t i m e t w h i c h has e l a p s e d s i n c e t h e i n i t i a t i o n o f the s c a n so t h a t m

2

=

m t /t 1

1

2

=

K/t

2

(9)

where t i and t a r e t h e t i m e s a f t e r i n i t i a t i o n o f t h e s c a n a t w h i c h m i and m are c o l l e c t e d . T h i s t y p e o f s c a n has been used on t h e ΑΕΙ MS50 i n s t r u m e n t w i t h VÎ = 2kV. When V r e a c h e s i t s i n s t r u m e n t a l maximum v a l u e o f 8kV, V = 4kV so t h a t t h e minimum v a l u e o f m /mi = 0 . 5 f o r a m e t a s t a b l e peak t o be o b s e r v a b l e . I f the crossover point i s s e t at a l o w e r v a l u e i n t h e r e g i o n o f l k V , t h e range c a n be e x t e n d e d so t h a t t h e minimum v a l u e o f m /mi i s app r o x i m a t e l y 0.33 b u t p r a c t i c a l problems may a r i s e i n m a i n t a i n i n g source s e n s i t i v i t y a t low a c c e l e r a t i n g voltages. T h i s i l l u s t r a t e s two o f t h e main d i s a d v a n t a g e s o f t h e method, l i m i t e d range and v a r i a t i o n o f s o u r c e t u n i n g c o n d i t i o n s d u r i n g t h e scan. In a d d i t i o n , a narrow β-slitwidth i s r e q u i r e d t o e l i m i n a t e peaks a r i s i n g from p a r e n t i o n s o f mass ( m i + l ) o r ( m i - l ) , 2

+

+

2

2

2

+

+

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

HIGH

•050"

PERFORMANCE

IOO* VV

,5

· °"

2

0

MASS

SPECTROMETRY

0

Figure 3. Effect of β-slitwidth W on precursor mass resolution R for various meta­ stable transitions

Scan time (arbj International Journal of Mass Spectrometry and Ion Physics

Figure 4. Plot of voltage vs. scan-time showing the cross-over point at V · (2kV) t

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

1.

Metastable Transitions

JENNINGS

13

but one cannot e l i m i n a t e low i n t e n s i t y s p u r i o u s peaks w h i c h o c c a s i o n a l l y a r i s e from d e c o m p o s i t i o n s w h i c h occur i n the a c c e l e r a t i n g r e g i o n . D e s p i t e these d i s a d v a n t a g e s , t h i s l i n k e d s c a n has p r o v e d v e r y u s e f u l i n t h e i n v e s t i g a t i o n o f t h e fragmen­ t a t i o n o f the m o l e c u l a r i o n s o f a s e r i e s o f s u b s t i t u t e d t e t r a f l u o r o c y c l o b u t a n e s ( 1 0 ) . The r e a c t i o n s o f f l u o r o e t h y l e n e and f l u o r o p r o p e n e m o l e c u l a r i o n s w i t h f l u o r o o l e f i n s s t u d i e d u s i n g ICR s p e c t r o s c o p y have been i n t e r ­ p r e t e d as p r o c e d i n g t h r o u g h a r e a c t i o n complex h a v i n g a c y c l o b u t a n e s t r u c t u r e and t h e f r a g m e n t a t i o n s o f t h e m o l e c u l a r i o n s o f t h e f l u o r o c y c l o b u t a n e s were shown t o be c o n s i s t e n t w i t h t h i s h y p o t h e s i s . Metastable t r a n s i ­ t i o n s o f d e u t e r a t e d m o l e c u l a r i o n s showed t h a t t h e s p e c i f i c i t y of the l a b e l l i n g i s retained i n t h e i r frag­ m e n t a t i o n so t h a t , f o r example, peaks were o b s e r v e d f o r the f r a g m e n t a t i o n s

F

D

In t h e second o f t h e l i n k e d s c a n s , t h e a c c e l e r a ­ t i n g v o l t a g e V i s h e l d c o n s t a n t and the m a g n e t i c f i e l d Β and e l e c t r i c s e c t o r v o l t a g e Ε a r e scanned such t h a t B/E i s c o n s t a n t t h r o u g h o u t t h e s c a n ( 1 1 ) . A p r o t o t y p e o f t h i s s c a n has r e c e n t l y been f i t t e d t o the Warwick MS50 i n s t r u m e n t , and a s c h e m a t i c o f t h e c i r c u i t i s shown i n F i g u r e 5. The m a g n e t i c f i e l d i s a d j u s t e d so t h a t t h e i o n m i i s c o l l e c t e d under normal o p e r a t i n g c o n d i t i o n s , and t h e e l e c t r i c s e c t o r v o l t a g e Ε i s mea­ s u r e d b y means o f a d i g i t a l v o l t m e t e r . The l i n k e d s c a n i s t h e n s w i t c h e d i n so t h a t t h e e l e c t r i c s e c t o r r e f e r ­ ence v o l t a g e i s c o n t r o l l e d by t h e o u t p u t o f t h e f i e l d monitor. The v a r i a b l e g a i n a m p l i f i e r i s a d j u s t e d so t h a t Ε i s i d e n t i c a l t o t h a t used i n normal o p e r a t i o n so that i i i o n s a r e a g a i n c o l l e c t e d . The s c a n i s t h e n i n i t i a t e d , and as Β and Ε f a l l , B/E remains c o n s t a n t as shown i n F i g u r e 6. Under normal o p e r a t i n g c o n d i t i o n s , i o n s mi sub­ j e c t e d t o an a c c e l e r a t i n g v o l t a g e V w i l l pass t h r o u g h an e l e c t r i c s e c t o r o f r a d i u s R w i t h v e l o c i t y v when the e l e c t r i c f i e l d E i s such t h a t +

+

+

x

0

m ν 1

/R = eE

Ί

or

2

+

2V =

REQ

(ID

s i n c e Ve = ^ m i V i . The m i o n s a r e c o l l e c t e d i f they pass t h r o u g h a m a g n e t i c f i e l d o f s t r e n g t h B i such t h a t x

HIGH

PRIMARY REFERENCE -20V

E S A SUPPLY

PERFORMANCE

MASS

ACCELERATING VOLTAGE POWER SUPPLY

-20V (MAIN BEAM) FIELD

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

MONITOR

B/E SCAN

Figure 5. Circuit used for B/E linked scan

Figure 6. Plot of B/E scan showing normal scan at E and Β and Ε values for collection of m * formed from various precursors 0

2

SPECTROMETRY

1.

2

m v /r 1

1

15

Metastable Transitions

JENNINGS

=

B ev 1

2

1

2

2

2

o r n^/e = r B / 2 V = r B / R E C I 2) 1

1

Q

where r i s t h e r a d i u s o f t h e m a g n e t i c s e c t o r . I f m i ·> m o c c u r s i n t h e f i e l d - f r e e r e g i o n be­ tween t h e s o u r c e and e l e c t r i c s e c t o r , m ions of v e l o c i t y v i a r e t r a n s m i t t e d by t h e e l e c t r i c s e c t o r o n l y if +

+

2

+

2

E

=

2

(m /m )E 2

1

(13)

0

and by t h e m a g n e t i c s e c t o r o n l y i f

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

m

v

2 l

/

r =

B

2

e

v

o

l

r

B

2

=

m

m

B

( 2/ l^ i

(

1 4

)

Hence b o t h Ε and Β need t o be r e d u c e d by t h e same fraction to (m /ii) of their o r i g i n a l value. Hence, the l i n k e d s c a n o f B/E h e l d c o n s t a n t a l l o w s a l l daugh­ ter ions m from t h e s e l e c t e d m i i o n t o be c o l l e c t e d i n t u r n . The s c a n has t h e advantage t h a t t h e s o u r c e e x t r a c t i o n e f f i c i e n c y i s constant since V i s not var­ i e d , and t h e range i s l i m i t e d o n l y by t h e range o v e r w h i c h t h e H a l l probe i s l i n e a r i n B. The s e n s i t i v i t y i s h i g h since products of decompositions i n the f i r s t f i e l d f r e e r e g i o n a r e c o l l e c t e d , b u t , as i n t h e s c a n o f Ε i n a r e v e r s e geometry i n s t r u m e n t , t h e r e s p o n s e o f t h e e l e c t r o n m u l t i p l i e r w i l l f a l l as t h e v a l u e o f Ε f a l l s . The r e l a t i v e i n t e n s i t i e s o f peaks w i t h i n a g i v e n scan w i l l depend on t h e energy r e l e a s e f o r each decomposi­ t i o n , t h e l i n e a r i t y o f t h e H a l l p r o b e , and t h e f a l l o f f i n s e n s i t i v i t y . N e v e r t h e l e s s , t h e narrow peaks and ease o f assignment make t h e scan i d e a l f o r a p p l i c a t i o n s i n w h i c h one m e r e l y wants t o a s c e r t a i n whether o r n o t there i s a metastable t r a n s i t i o n f o r the process m i m . I f one c o n s i d e r s t h e f o r m a t i o n o f m i o n s from a s e r i e s o f p a r e n t i o n s , t h e p a r t i c u l a r v a l u e s o f B/E w h i c h a r e r e q u i r e d f o r t h e i r c o l l e c t i o n a r e g i v e n by 2

+

+

2

+

+

2

+

2

m /e 2

=

2

(r B

2 2

)/E

(15)

2

and s o form a p a r a b o l a as shown i n F i g u r e 6. The v a l u e required f o r the c o l l e c t i o n of m from a p a r t i c u l a r m i i s found a t t h e i n t e r s e c t i o n o f t h e l i n e a r B/E p l o t and t h e p a r a b o l a . One c a n show t h a t t h e v a l u e o f Β i s t h a t a t w h i c h t h e m e t a s t a b l e peak would appear i n a normal mass s p e c t r u m , i . e . , a t an a p p a r e n t mass o f m / m i . When s c a n n i n g t o o b t a i n d a u g h t e r i o n s from a p a r t i c u l a r p a r e n t i o n m i , one may o b t a i n s p u r i o u s peaks o f low i n t e n s i t y i f t h e r e i s an i n t e n s e s i g n a l +

2

+

2

2

+

16

HIGH

+

PERFORMANCE

MASS

SPECTROMETRY

+

a r i s i n g from ( m i + l ) •> m or i f there i s s i g n i f i c a n t r e l e a s e o f i n t e r n a l energy. Such peaks can u s u a l l y be r e c o g n i z e d w i t h o u t d i f f i c u l t y and a r e , i n any c a s e , m i n i m i z e d by r e d u c i n g the $ - s l i t w i d t h . When used t o s t u d y c o l l i s i o n - i n d u c e d decomposi­ t i o n s , e i t h e r b y r a i s i n g the a n a l y z e r tube p r e s s u r e o r by u s i n g a c o l l i s i o n chamber between the s o u r c e and e l e c t r i c s e c t o r , t h i s scan s t i l l g i v e s sharp peaks and assignment i s t h e r e b y s i m p l i f i e d . For example, t h e c o l l i s i o n - i n d u c e d d e c o m p o s i t i o n o f the t o l u e n e molecu­ l a r i o n gave many sharp peaks down t o masses as low as m/e = 25, C H . I t has a l s o been used i n a s t u d y (12) o f t h e r e a c t i o n between the v i n y l m e t h y l e t h e r molecu­ l a r i o n and 1,3-butadiene i n which methanol i s e l i m i ­ n a t e d t o y i e l d what i s c o n s i d e r e d t o be the 1 , 3 - c y c l o hexadiene m o l e c u l a r i o n . The p r o d u c t s o b t a i n e d by t h e c o l l i s i o n - i n d u c e d d e c o m p o s i t i o n o f t h i s i o n produced by ion-molecule r e a c t i o n s i n a h i g h p r e s s u r e source a r e v e r y s i m i l a r t o those g i v e n by the 1 , 3 - c y c l o h e x a d i e n e m o l e c u l a r i o n when t h i s compound i s i n t r o d u c e d d i r e c t l y i n t o the s o u r c e , t h e r e b y s u p p o r t i n g t h i s s u g g e s t i o n . 2

+

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

2

Conclusion. In t h e above a c c o u n t , we have a t t e m p t e d t o p o i n t out t h e advantages and d i s a d v a n t a g e s o f the v a r i o u s methods o f o b s e r v i n g the p r o d u c t s o f m e t a s t a b l e t r a n s i ­ t i o n s and i t i s c l e a r t h a t a l l have t h e i r uses i n particular situations. For most a p p l i c a t i o n s , a V scan ( N i e r - J o h n s o n geometry) o r Ε s c a n ( r e v e r s e geometry) t o g e t h e r w i t h a l i n k e d s c a n , p r e f e r a b l y the B/E s c a n because o f i t s g r e a t e r r a n g e , w i l l g i v e the r e q u i r e d i n f o r m a t i o n . The methods s h o u l d be r e g a r d e d as comple­ mentary r a t h e r than i n c o m p e t i t i o n .

Literature Cited. 1. 2. 3. 4.

Cooks, R.G., Beynon, J.H., Caprioli, R.M. and Lester, G.R., "Metastable Ions", (Elsevier Scientific Publishing Co.), Amsterdam, 1973. Jennings, K.R. in "Mass Spectrometry, Techniques, and Applications", p. 419 (Ed. G. W. A. Milne, Wiley Interscience), New York, 1971. Holmes, J.L. and Benoit, F.M. in MTP Int. Rev. of Sci. Physical Chemistry Series 1 (1972) 5, 259. (Ed. A. Maccoll, Butterworths), London. Beynon, J.H. and Cooks, R.G. in TMP Int. Rev. of Sci. Physical Chemistry Series 2, (1975), 5, 159. (Ed. A. Maccoll, Butterworths), London.

1.

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

5.

JENNINGS

Metastable Transitions

17

Sen Sharma, D.K., Jennings, K.R. and Beynon, J.H., Organic Mass Spectrom., (1976), 11, 319. 6. Huntress, W.T., J r . , Sen Sharma, D.K., Jennings, K.R. and Bowers, M.T., Int. J. Mass Spectrom Ion Phys. (submitted for publication). 7. Bowers, M.T., Chesnavich, W.J. and Huntress, W.T., Jr., Int. J. Mass Spectrom Ion Phys., (1973), 12 357. 8. Jones, S., Elliott, R.M. and Jennings, K.R., unpublished work. 9. Weston, A.F., Jennings, K.R., Evans, S. and Elliott, R.M., Int. J. Mass Spectrom Ion Phys. (1976), 20, 317. 10. Derai, R., Ferrer-Correia, A.J.V., Mitchum, R.K., and Jennings, K.R., unpublished work. 11. Bruins, A.P., Jennings, K.R., Evans, S., Int. J. Mass Spectrom. Ion Phys. (in press). 12. van Doorn, R., Nibbering, N.M.M., Ferrer-Correia, A.J.V. and Jennings, K.R., unpublished work. Received December 30, 1977

2 Potential Energy Surfaces for Unimolecular Reactions of Organic Ions RICHARD D. BOWEN and DUDLEY H. WILLIAMS

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

University Chemical Laboratory, Cambridge, United Kingdom

In s t u d y i n g t h e r e a c t i o n s undergone by charged s p e c i e s i t i s o f t e n advantageous t o examine t h e chemis t r y u s i n g i o n beams. Such methods f a c i l i t a t e a t i g h t e r c o n t r o l o f the chemistry than i s p o s s i b l e i n c l a s s i c a l experiments i n s o l u t i o n . F o r i n s t a n c e , comp l i c a t i o n s caused by s o l v a t i o n a r e c o m p l e t e l y e l i m i n a t e d , and t h e o c c u r r e n c e o f r e a c t i o n s i s e a s i l y d e t e c t e d by t h e appearance o f m e t a s t a b l e peaks i n t h e mass spectrum. The i m p o r t a n c e o f m e t a s t a b l e peaks i n mass s p e c t r o m e t r y has been d i s c u s s e d i n d e t a i l ( 1 - 3 ) ; i n t h e p r e s e n t c o n t e x t t h i s i m p o r t a n c e a r i s e s because metast a b l e peaks a r e u s u a l l y produced by t h e s l o w , u n i molecular decompositions of ions having a w e l l - d e f i n e d range o f i n t e r n a l e n e r g y , j u s t above t h e t h r e s h o l d f o r r e a c t i o n . A l t h o u g h c l e a r l y d e f i n e d examples o f t h e i n t e r v e n t i o n o f i s o l a t e d s t a t e s a r e known ( 4 ) , such cases are probably the e x c e p t i o n r a t h e r than the r u l e . Indeed, t h e u n i m o l e c u l a r c h e m i s t r y o f e l e v e n members o f the C

H

n 2n l +

C

n = 2

1 2

- >

homologous s e r i e s o f i o n s c a n be e i t h e r r a t i o n a l i z e d o r p r e d i c t e d without invoking the i n t e r v e n t i o n o f i s o l a t e d s t a t e s ( 5 ) . A consequence o f t h e f a c t t h a t m e t a s t a b l e d i s s o c i a t i o n s u s u a l l y o c c u r w i t h l i t t l e e x c e s s energy i n the t r a n s i t i o n state i s that the a b i l i t y o f possible decay c h a n n e l s t o compete a g a i n s t one a n o t h e r i s c r i t i c a l l y dependent on t h e a c t i v a t i o n e n e r g i e s f o r t h e p r o c e s s e s concerned ( 6 ) . T h i s i s e l e g a n t l y demonstrat e d by t h e o c c u r r e n c e o f i s o t o p e e f f e c t s i n t h e decomp o s i t i o n o f s u i t a b l y l a b e l l e d i o n s . These i s o t o p e e f f e c t s span t h e e n t i r e range o f t h o s e known i n s o l u t i o n c h e m i s t r y , and i n some cases a r e s p e c t a c u l a r l y large. For instance, the metastable decompositions o f a l l t h e v a r i o u s D - l a b e l l e d methanes have been documen-©0-8412-0422-5/78/47-070-018$10.00/0

2.

BOWEN

Unimolecuhr

AND WILLIAMS

Reactions of Organic Ions

19

t e d ( 7 ) , and, a l t h o u g h c a l c u l a t i o n s i n d i c a t e t h a t t h e t h r e s h o l d f o r l o s s o f ϋ· from t h e m o l e c u l a r i o n s i s o n l y 0.08 eV above t h a t f o r Η· l o s s , t h e energy d i s ­ c r i m i n a t i o n i s so s t r o n g t h a t l o s s o f ϋ· i s n o t o b s e r v e d e x c e p t from CDi^. S t u d i e s o f l a b e l l e d ethane and propane have a l s o been r e p o r t e d ( 8 - 1 0 ) , and f o r CD CHt t h e r a t i o o f H :D l o s s i s ca."150:1 w h i l e t h a t f o r Η·:ϋ· l o s s from C D C H i s > 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

3

6

+

6

3

9

2

+

9

+

9

1 3

+

9

22

HIGH

PERFORMANCE

MASS

SPECTROMETRY

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

2

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

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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

<0.01

<3

HOCH2CH2Y

1.8±0.2

11.5±0.9

CH3CHYOH

1.9±0.1

10.1±0.5

CH3CH2OY

2.0±0.1

12.0±1.1

CH3OCH2Y

1

From t h e d a t a o f T a b l e I i t i s e v i d e n t t h a t two d i s t i n c t p o p u l a t i o n s o f i o n s , w h i c h do n o t i n t e r c o n v e r t a p p r e c i a b l y a t energies appropriate to metastable t r a n s i t i o n s , a r e b e i n g formed. Not o n l y do C H 0 i o n s formed from p r e c u r s o r s o f g e n e r a l f o r m u l a 29 undergo e x c l u s i v e methane l o s s , as opposed t o i o n s g e n e r a t e d from 30, 31 o r 32 w h i c h undergo l o s s o f methane and a c e t y l e n e , b u t a l s o t h e m e t a s t a b l e peak shapes f o r methane l o s s from t h e two t y p e s a r e d i f ferent. That f o r methane l o s s from i o n s p r o d u c e d from 29 i s g a u s s i a n , whereas methane l o s s from i o n s h a v i n g 30, 31 o r 32 t y p e p r e c u r s o r s i s f l a t - t o p p e d i n d i c a t i n g k i n e t i c energy r e l e a s e (52^5_3). Furthermore, the measured v a l u e s f o r t h e A P s f o r CHi* l o s s from t h e two +

2

f

5

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

2.

BOWEN

Unimolecular Reactions of Organic Ions

AND WILLIAMS

29

kinds o f ions, while only approximate, y i e l d values f o r the t r a n s i t i o n s t a t e e n e r g i e s w h i c h a r e d i f f e r e n t ( 5 4 ) . F i n a l l y , D l a b e l l i n g s t u d i e s (55) on i o n s p r o d u c e d from 29 (presumably s t r u c t u r e 7 J r e v e a l t h a t CH<* l o s s o c c u r s w i t h 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 hydro­ gen atoms w h i l e CFU l o s s from i o n s w i t h p r e c u r s o r s t r u c t u r e s 30 o r 31 (presumably s t r u c t u r e 6 i s t h e most s t a b l e form) i n v o l v e s s p e c i f i c e l i m i n a t i o n o f the h y d r o x y l h y d r o g e n , w h i c h does n o t l o s e i t s i d e n t i t y t o a s i g n i f i c a n t e x t e n t (56J ( i n t h e e n e r g y - r e l e a s i n g metastable t r a n s i t i o n ) . The above d a t a s e r v e t o i l l u s t r a t e t h e arguments i n f a v o r o f 6 and 7 r e a c t i n g o v e r p o t e n t i a l s u r f a c e s which a r e d i s t i n c t . The o v e r a l l case i s o v e r w h e l m i n g l y s t r o n g and f u r t h e r developments (57_) o f t h e p o t e n t i a l s u r f a c e a p p r o a c h c a n e x p l a i n much o f t h e d e t a i l e d c h e m i s t r y o f t h e C H 0 i o n . The e s t i m a t e d p o t e n t i a l surface i s depicted i n Figure 4, which s c h e m a t i c a l l y shows t h e d i f f e r e n t energy r e l e a s e p r o f i l e s (metastable peaks) f o r t h e two i n d e p e n d e n t d i s s o c i a t i o n s l e a d i n g t o HC=0 and CHi* (see a l s o T a b l e I ) . I t s h o u l d be n o t e d t h a t p r o t o n a t e d o x i r a n e (33) and p r o t o n a t e d a c e t a l d e h y d e (6) a r e e x p e c t e d t o i n t e r c o n v e r t p r i o r t o m e t a s t a b l e d i s s o c i a t i o n s . T h i s e x p l a i n s why l a b e l l i n g s t u d i e s r e v e a l t h a t b o t h t h e c a r b o n atoms o f i o n s o r i g i n a l l y g e n e r a t e d as 34 become e q u i v a l e n t p r i o r to metastable t r a n s i t i o n s (58)7 i a l s o why t h e deu­ t e r i u m atom a t t a c h e d t o oxygen i n 35 r e t a i n s i t s i d e n ­ t i t y (S6) w h i l e a l l carbon-bound hydrogens i n § become equivalent. +

2

5

+

a n <

*

+

+

CH CH=OH

CH CH=OD

34

35

3

33

3

Other i m p o r t a n t examples o f t h i s 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 a r e C H N (22), C H 0 (20i, 5_9-61) and 0 ι » Η Ο ( 6 2 ) , where t h e m e t a s t a b l e abundance c r i ­ t e r i o n c a n be used t o c l a s s i f y t h e i o n s i n t o 3 , 4 and 5 groups r e s p e c t i v e l y , w h i c h do n o t i n t e r c o n v e r t p r i o r t o 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 s . The C H N system i s p a r t i c u l a r l y i n s t r u c t i v e i n t h a t one i o n (16) i s u n i q u e i n u n d e r g o i n g s o l e l y C Hi* l o s s . Deuterium"labelling s t u d i e s r e v e a l that t h i s i s a s p e c i f i c process which may be r a t i o n a l i z e d i n terms o f a f o u r - c e n t e r " mech­ anism (Scheme 1 ) . The m e t a s t a b l e peak f o r t h i s reaction +

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A *-

CH =NHCH CH 2

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

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BOWEN

Unimolecular Reactions of Organic Ions

AND WILLIAMS

31

i s g a u s s i a n (22). Ions h a v i n g s t r u c t u r e s 14 and 15 a l s o undergo e t h y l e n e l o s s , b u t t h i s p r o c e s s has a~ h i g h e r a c t i v a t i o n energy t h a n i s o m e r i z a t i o n r e a c t i o n s l e a d i n g t o l o s s o f i d e n t i t y o f a l l t h e carbon-bound hydrogens o f 14 and 15 (22_) . The m e t a s t a b l e peak f o r t h i s reaction~ïs f l a t - t o p p e d t h u s i n d i c a t i n g t h a t 14 and 15 decompose o v e r a s e p a r a t e s u r f a c e from 16. T h i s ~ c o n c l u s i o n i s f u r t h e r s t r e n g t h e n e d by t h e ~ o c c u r r e n c e o f a new d i s s o c i a t i o n pathway, NH l o s s , f o r i o n s o f s t r u c t u r e 14 and 15. The t h i r d c l a s s o f i o n s , i n i t i a l l y g e n e r a t e d ~ a s 17~or 18, undergo b o t h 0 Ηι* and NH l o s s t o g e t h e r wïïh a t h i r d r e a c t i o n , H loss, in metastable t r a n s i t i o n s . F u r t h e r m o r e , t h e abundances of C Hit, NH and H l o s s e s a r e i n d e p e n d e n t , t o a f i r s t a p p r o x i m a t i o n , o f t h e p r e c u r s o r , thus i n d i c a t i n g d e c o m p o s i t i o n o v e r a common s u r f a c e . The m e t a s t a b l e peak shapes f o r t h e s e r e a c t i o n s a r e g a u s s i a n f o r C FU and NH l o s s and f l a t - t o p p e d f o r H l o s s . Clearly, i o n s o f i n i t i a l s t r u c t u r e 17 and.18 decompose o v e r a common s u r f a c e w h i c h i s d i s t i n c t "from t h o s e o v e r w h i c h 16 o r 14 and 15 d i s s o c i a t e ( 2 2 ) . The u n i m o l e c u l a r dëcompôsition~pathways and the" c o r r e s p o n d i n g m e t a s t a b l e peak shapes a r e summarized i n Scheme 1. A n o t h e r i n t e r e s t i n g example o f t h e g e n e r a l form of p o t e n t i a l energy s u r f a c e d e p i c t e d i n F i g u r e 3 i s the 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 s o f t h e C H N 0 ions i n t h e mass s p e c t r a o f some a l k y l - n i t r o b e n z e n e s ( 6 3 ) . S t a r t i n g from p r e c u r s o r s o f t h e g e n e r a l s t r u c t u r e OzNCeH^CHzR w i t h R=Br, CH o r C H , t h e m e t a s t a b l e dec o m p o s i t i o n s o f m/e 136 a r e found t o be l o s s o f NO f o r p a r a - s u b s t i t u t e d precursors while f o r the corresponding meta-isomers o n l y N 0 l o s s i s o b s e r v e d . T h i s e s t a b l i s h e s t h a t t h e i o n s formed by l o s s o f R from t h e m o l e c u l a r i o n s o f m- and £-0 NC HuCH R do n o t decompose o v e r t h e same p o t e n t i a l s u r f a c e . This i n turn leads t o the conclusion that e i t h e r or both o f the b e n z y l i c i o n s t r u c t u r e s (36 and 37) w h i c h might p l a u s i b l y be formed a t t h r e s h o l d do n o t undergo r e v e r s i b l e r i n g e x p a n s i o n t o t h e n i t r o t r o p y l i u m i o n 38. 3

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t I t i s p o s s i b l e t h a t 17 and 18 undergo 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 onto the~same p o t e n t i a l s u r f a c e as t h a t o v e r w h i c h 16 decomposes. However, t h e i n t e r n a l energy o f such i o n s w i l l be much h i g h e r t h a n t h a t of i o n s g e n e r a t e d as 16 and so t h e new d e c o m p o s i t i o n c h a n n e l s , f o r l o s s o f ~ H and NH a r e a c c e s s i b l e . Nevert h e l e s s , t h i s cannot be t h e case f o r 14 and 15 because the m e t a s t a b l e peak f o r C Hi l o s s i s â~differënt shape t h u s p r e c l u d i n g d e c o m p o s i t i o n o v e r t h e same s u r f a c e as 16 b u t w i t h g r e a t l y d i f f e r e n t i n t e r n a l e n e r g i e s . 2

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38 Rate-Determining Decomposition.

Isomerization P r i o r to Unimolecular

T h i s c o r r e s p o n d s t o the 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 5. Here the energy needed t o cause i s o m e r i z a t i o n o f s t r u c t u r e B to A i s more t h a n t h a t needed t o cause u n i m o l e c u l a r decompos i t i o n of B . Consequently, i o n B d i s s o c i a t e s i n preference to i s o m e r i z i n g to A . However, i o n A lies i n a p o t e n t i a l w e l l w h i c h i s so deep t h a t the l o w e s t a c t i v a t i o n energy p r o c e s s i s i s o m e r i z a t i o n t o B , w h i c h i s formed i n a h i g h l y e x c i t e d v i b r a t i o n a l l e v e l . The i o n B thus formed, t h e n d i s s o c i a t e s more r a p i d l y t h a n i t r e t u r n s t o s t r u c t u r e A , and does so w i t h a r a t e c o n s t a n t w h i c h i s much g r e a t e r t h a n t h a t (k-10 sec" ) normally a s s o c i a t e d w i t h metastable transitions. I t i s p o s s i b l e t h a t A and B may undergo t h e same r e a c t i o n s i n m e t a s t a b l e t r a n s i t i o n s , but i f t h e p o t e n t i a l s u r f a c e i s o f the g e n e r a l form d e p i c t e d i n F i g u r e 5, then f o u r c o n d i t i o n s must be s a t i s f i e d . ( i ) The m e t a s t a b l e peaks f o r any common p r o c e s s e s must be s i m i l a r . However, because o f t h e l a r g e r amount of i n t e r n a l energy p r e s e n t i n i o n s o f s t r u c t u r e B w h i c h have t h e i r o r i g i n as i s o m e r i z e d "A type i o n s , t h e m e t a s t a b l e peaks f o r d e c o m p o s i t i o n o f such i o n s a r e l i k e l y t o be broadened r e l a t i v e t o t h o s e f o r d i s s o c i a t i o n o f i o n s o r i g i n a l l y g e n e r a t e d as B . ( i i ) As d i s s o c i a t i o n o f the r e a r r a n g e d i o n s o f s t r u c t u r e B i s f a s t r e l a t i v e t o the normal time s c a l e of m e t a s t a b l e t r a n s i t i o n s , energy ceases t o be the dominant f a c t o r and " e n t r o p y e f f e c t s become i m p o r t a n t . Thus, f o r r e a r r a n g e d i o n s , s i n g l e bond-cleavage d i s s o +

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BOWEN

AND WILLIAMS

Unimolecuhr Reactions of Organic Ions

ÇH Ç H

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

2

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Accounts of Chemical Research

Figure 4. Potential energy surface and energy release profiles for slow unimolecuhr decomposition of the isomeric cations C H O* 2

s

Accounts of Chemical Research

Figure 5. Potential energy surface corresponding to rate-determining isomerization of ion A* to B* prior to unimolecular decomposition

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c i a t i o n s a r e f a v o r e d , whereas t h e r e i s d i s c r i m i n a t i o n a g a i n s t t h o s e d i s s o c i a t i o n s w h i c h have h i g h l y o r d e r e d transition states. Consequently, ions produced v i a the 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 a r e l i k e l y t o show a marked p r e f e r e n c e f o r s i n g l e b o n d - c l e a v a g e p r o c e s s e s ("source t y p e r e a c t i o n s ) even i f such p r o c e s s e s a r e not t h e most f a v o r a b l e e n e r g e t i c a l l y . (iii) AP measurements f o r t h e d e c o m p o s i t i o n s o f i o n s g e n e r a t e d as B , w i l l g e n e r a l l y r e v e a l d i f f e r e n t a c t i v a t i o n e n e r g i e s f o r competing p r o c e s s e s . However, s i n c e t h e r a t e - d e t e r m i n i n g s t e p s t a r t i n g from A i s the i s o m e r i z a t i o n t o B , t h e a c t i v a t i o n e n e r g i e s f o r a l l t h e e v e n t u a l d e c o m p o s i t i o n s must be t h e same and must be g r e a t e r t h a n any o f t h o s e s t a r t i n g from B . (iv) L a b e l l i n g s t u d i e s on i o n s s t a r t i n g from A may r e v e a l a lower degree o f l o s s o f i d e n t i t y o f t h e l a b e l than occurs i f the corresponding l a b e l l e d B i o n i s generated d i r e c t l y . T h i s i s because t h e r e i s l e s s time f o r i s o m e r i z a t i o n p r o c e s s e s t o cause l o s s o f i n t e g r i t y o f l a b e l l e d s p e c i e s o f h i g h i n t e r n a l energy ( r e a r r a n g e d "A type i o n s ) which d i s s o c i a t e r e l a t i v e l y rapidly. Although a l l u s i o n s t o the p o s s i b i l i t y o f ions r e a c t i n g over t h i s k i n d o f s u r f a c e were made f o r C 3 H 8 N " (22) and C H 0 (6_4) i o n s , i t was n o t u n t i l 1975 t h a t the f i r s t d e f i n i t e c a s e , i n t h e C 3 H 0 system ( 6 5 ) , was documented. Here i o n s o f m/e 59, generated~From the fragmentât i o n o f t h e m o l e c u l a r i o n s o f s u i t a b l e e t h e r s and a c e t a l s , may be a s s i g n e d s t r u c t u r e s 10 and 11 on t h e b a s i s o f α-cleavage. Ions o f s t r u c t u r e IQ undergo m a i n l y H 0 T o s s , t o g e t h e r w i t h some C ttk loss i n metastable t r a n s i t i o n s . AP measurements r e v e a l t h a t t h e t r a n s i t i o n s t a t e energy f o r H 0 l o s s i s some 5 k c a l mol" below t h a t f o r 0 Η^ l o s s . Loss o f CH =0 i s n o t o b s e r v e d i n m e t a s t a b l e t r a n s i t i o n s , even though t h e r e i s a complete CH =0 group p r e s e n t i n JQ, because the minimum energy needed t o p r o d u c e CH =0 + C Hs+ i s s i g n i f i c a n t l y g r e a t e r (9 k c a l m o l " ) t h a n t h e measured a c t i v a t i o n energy f o r C Hi* l o s s . However, i o n s gener­ a t e d i n i t i a l l y as H do undergo CH =0 l o s s , t o an a p p r e c i a b l e e x t e n t , i n m e t a s t a b l e t r a n s i t i o n s , and H 0 l o s s becomes a v e r y minor (ca 1% t o t a l m e t a s t a b l e i o n c u r r e n t from m/e 59) p r o c e s s . One r a t i o n a l i z a t i o n o f t h i s i s g i v e n Tn Scheme 2, where 11 i s c o n s i d e r e d to undergo 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 t o 10 p r i o r t o m e t a s t a b l e d i s s o c i a t i o n s . The r a t e - d e t e r m i n ­ i n g s t e p i s most s i m p l y f o r m u l a t e d as a symmetry-for­ b i d d e n (66) 1 , 3 - h y d r i d e s h i f t , though o t h e r mechanisms a r e p o s s i b l e . 11

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

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Unimolecular Reactions of Organic Ions

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Scheme 2 H en ζ +

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The r e s u l t i n g i o n 10, formed by 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 11 now'fias s u f f i c i e n t i n t e r n a l energy t o undergo CH =0 l o s s v i a s i m p l e bond c l e a v a g e o f t h e C-0 bond i n 10. 0 Ηι» l o s s i s a l s o p o s s i b l e , whereas H 0 l o s s , w h i c h r e q u i r e s c o n s i d e r a b l e rearrangement o f IQ p r i o r t o d i s s o c i a t i o n , i s g r e a t l y d i s f a v o r e d by "entropy" f a c t o r s . Further evidence i n favor o f the i s o m e r i z a t i o n i s found i n AP measurements f o r t h e d e c o m p o s i t i o n s o f i o n s i n i t i a l l y g e n e r a t e d as 1 1 , w h i c h a r e t h e same w i t h i n e x p e r i m e n t a l e r r o r , and c o r r e s p o n d t o an i n t e r n a l energy c o n s i d e r a b l y g r e a t e r t h a n t h a t needed t o cause d i s s o c i a t i o n o f 10. I n a d d i t i o n , t h e m e t a s t a b l e peaks f o r H 0 and C FU l o s s e s from i o n s g e n e r a t e d as 10 and 11, r e s p e c t i v e l y , a r e b r o a d e r a t h a l f h e i g h t i n ~ t h e l a t t e r case. T h i s o b s e r v a t i o n (65) t o g e t h e r w i t h Dl a b e l l i n g s t u d i e s , which r e v e a l a higher s p e c i f i c i t y f o r r e a c t i o n s s t a r t i n g from 11 t h a n J.0 (65) , a r e con­ s i s t e n t w i t h t h e h y p o t h e s i s t h a t 11 undergoes 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 t o 10 p r i o r t o m e t a s t a b l e transitions. The r e l e v a n t p o r 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 i s summarized i n F i g u r e 6. The second r e p o r t e d case o f r a t e - d e t e r m i n i n g isomerization p r i o r to unimolecular d i s s o c i a t i o n s i s a l s o i n t h e C3H70 s y s t e m , namely, p r o t o n a t e d p r o p i o n a l d e h y d e and p r o t o n a t e d acetone ( 6 7 ) . The e v i ­ dence p r e s e n t e d i s s i m i l a r t o t h a t f o r IQ and H above, and t h e r e l e v a n t p a r t o f t h e p o t e n t i a l s u r f a c e i s d e p i c t e d i n F i g u r e 7. I t i s p o s s i b l e t h a t t h i s type o f p o t e n t i a l sur­ f a c e w i l l p r o v e t o be more common t h a n h i t h e r t o i m a g i n e d as p r e v i o u s s t u d i e s o f t h e C 3 H 8 N " (22) and C3H 0 (6£) systems r e v e a l c a s e s where i s o m e r T z a t i o n may be tïïe r a t e - d e t e r m i n i n g s t e p i n d i s s o c i a t i o n o f some i o n i c s t r u c t u r e s . H a v i n g d i s c u s s e d t h e g e n e r a l shapes o f p o t e n t i a l s u r f a c e s o v e r w h i c h i o n s may be c o n s i d e r e d t o d i s s o c i a t e , i t i s i n s t r u c t i v e t o examine t h e s p e c i a l e f f e c t w h i c h t h e n a t u r e o f t h e f i n a l ( d i s s o c i a t i o n ) s t e p has upon t h e shape o f t h e m e t a s t a b l e peak f o r t h a t p r o c e s s .

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

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

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Figure 7. Potential energy surface for slow unimolecular decomposition of ions 8 and 9

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AND WILLIAMS

Unimolecuhr Reactions of Organic Ions

37

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

C o r r e l a t i o n o f M e t a s t a b l e Peak Shape w i t h P o t e n t i a l Energy S u r f a c e o f t h e D i s s o c i a t i o n StëpT C l e a r l y t h e r e a r e two b a s i c p o s s i b i l i t i e s ; namely that the reverse r e a c t i o n w i l l or w i l l not involve act i v a t i o n energy. These c o r r e s p o n d t o t h e p o t e n t i a l s u r f a c e s shown i n F i g u r e s 8(a) and 8 ( b ) , r e s p e c t i v e l y . In t h e former c a s e , i t i s c l e a r t h a t energy i s r e l e a s e d i n t h e d i s s o c i a t i o n ; and t h i s energy may be absorbed i n t o t h e v i b r a t i o n a l modes o f t h e p r o d u c t s o r may appear as t r a n s l a t i o n a l energy. T h i s k i n e t i c ene r g y r e l e a s e i s e v i d e n c e d by t h e b r o a d e n i n g o f t h e m e t a s t a b l e peak, w h i c h becomes f l a t - t o p p e d i n c a s e s where a r e l a t i v e l y s p e c i f i c and l a r g e amount o f energy i s i n v o l v e d . C o n v e r s e l y , i n c a s e s where t h e r e i s no r e v e r s e a c t i v a t i o n e n e r g y , l i t t l e o r no k i n e t i c energy r e l e a s e i s e x p e c t e d and t h e m e t a s t a b l e peak i s gauss i a n i n shape. Some c a s e s a r e n o t so c l e a r c u t as t o be b r a c k e t e d i n t o e i t h e r c a t e g o r y above; f o r i n s t a n c e , t h e t r a n s i t i o n s t a t e geometry may be f a i r l y f l e x i b l e , t h u s p e r m i t t i n g t h e r e l e a s e o f a range o f k i n e t i c e n e r g i e s w h i c h r e s u l t s i n t h e b r o a d e n i n g o f t h e metast a b l e peak f o r t h e p r o c e s s , a l t h o u g h i t f r e q u e n t l y remains g a u s s i a n i n shape. I n such c a s e s a v a l u e f o r ' f g k i n e t i c energy r e l e a s e may be computed from t h e peak w i d t h a t h a l f h e i g h t ( 6 8 ) . A l t e r n a t i v e l y , l o o k i n g a t the r e a c t i o n i n r e v e r s e , a g a u s s i a n m e t a s t a b l e peak c o n t a i n s t h e i n f o r m a t i o n t h a t t h e r e v e r s e r e a c t i o n can be made t o go by a l l o w i n g t h e p r o d u c t s (or p o s s i b l y t h e p r o d u c t s i n a suitably excited v i b r a t i o n a l level) to d r i f t together. A f l a t - t o p p e d peak c o r r e s p o n d s t o a d e f i n i t e energy b a r r i e r t o t h e r e v e r s e r e a c t i o n , w h i c h cannot be o v e r come by m e r e l y e x c i t i n g t h e p r o d u c t s i n t h e v i b r a t i o n al levels. R a t h e r , t h e p r o d u c t i o n and n e u t r a l must be g i v e n r e l a t i v e t r a n s l a t i o n a l energy i n o r d e r t o make the r e v e r s e r e a c t i o n go. C o n c e p t u a l l y , a f l a t topped m e t a s t a b l e peak f o r a g i v e n r e a c t i o n means t h a t t h e p r o d u c t s must be "pushed t o g e t h e r " i n o r d e r t o make t h e r e v e r s e r e a c t i o n go. One s i t u a t i o n i n w h i c h t h e p o t e n t i a l s u r f a c e i s o f t h e g e n e r a l form l i k e l y t o g i v e r i s e t o f l a t - t o p p e d m e t a s t a b l e peaks i s t h a t o f s y m m e t r y - f o r b i d d e n d i s s o c i a t i o n s (66J). Here a " c r o s s i n g " o f two m o l e c u l a r o r b i t a l energy l e v e l s o c c u r s , and upon a t t a i n i n g t h e t r a n s i t i o n s t a t e , w h i c h w i l l g e n e r a l l y be w e l l d e f i n e d g e o m e t r i c a l l y , d i s s o c i a t i o n occurs over a r e p u l s i v e potential surface. For i n s t a n c e , c o n s i d e r t h e c o n c e r t e d 1 , 2 - e l i m i n a t i o n o f H from i o n i z e d ethane (39) p r o c e e d i n g v i a a t

ie

a

v

r

a

e

2

38

HIGH

PERFORMANCE

MASS

SPECTROMETRY

planar t r a n s i t i o n state. The r e l e v a n t c o r r e l a t i o n diagram i s g i v e n i n F i g u r e 9 (69). A n a l y s i s o f t h i s d i a g r a m , l e a d s t o the c o n c l u s i o n t h a t the r e a c t i o n i s symmetry-forbidden. K i n e t i c energy r e l e a s e i s expect e d s i n c e the h i g h t r a n s i t i o n s t a t e energy i s p a r t i a l l y due t o the occupancy o f a m o l e c u l a r o r b i t a l c h a r a c t e r i z e d by mutual r e p u l s i o n between the i o n i z e d e t h y l e n e and H (see · i n F i g u r e 9 ) . E x p e r i m e n t a l l y , i o n i z e d ethane does i n d e e d l o s e H w i t h k i n e t i c energy r e l e a s e (70). A s i m i l a r a n a l y s i s f o r i o n i z e d methylamine ( 4 0 J 7 p r o t o n a t e d f o r m a l d e h y d e ( 4 1 ) , p r o t o n a t e d t h i o f o r m a l d e h y d e (42) and p r o t o n a t e d methylene i m i n e (43) l e a d s t o t h e c o n c l u s i o n t h a t t h e s e m o l e c u l e s , i f t h e y undergo c o n c e r t e d 1 , 2 - e l i m i n a t i o n s o f H , s h o u l d do so w i t h k i n e t i c energy r e l e a s e . E x p e r i m e n t a l l y , H l o s s e s are observed t o occur i n metastable t r a n s i t i o n s and i n each case the peak i s o f a f l a t - t o p p e d shape ( 7 0 ) , thus i n d i c a t i n g k i n e t i c energy r e l e a s e (52,53). THê f a c t t h a t a l l t h e s e d i s s o c i a t i o n s a r e 1,2-ëlimmat i o n s i s e s t a b l i s h e d by d e u t e r i u m l a b e l l i n g s t u d i e s (^,ΙΟ^,ΤιΟ,Τ^) , w h i c h show t h a t 2

2

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

2

2

+

CHaCHa*

CH =0H

40

41

3

39

+

CH NH Î 2

2

CH =SH 2

42

+

CH =NH 2

2

43

C H C D » l o s e s o n l y HD i n m e t a s t a b l e t r a n s i t i o n s (8,10^) and t h a t the h e t e r o a t o m d e u t e r a t e d a n a l o g u e s o f 4U t o 43 a l s o e l i m i n a t e o n l y HD i n u n i m o l e c u l a r decomposit i o n s (7_0,71) . F u r t h e r i n t e r e s t i n g a p p l i c a t i o n s o f o r b i t a l symmetry t o 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 s e x i s t (72,73) and one p a r t i c u l a r l y r e l e v a n t i n t h e p r e s e n t c o n t e x t i s t h e d e d u c t i o n t h a t H l o s s from i o n i z e d e t h y l e n e (44) o c c u r s v i a a 1 , 1 - e l i m i n a t i o n from the i o n i z e d cârbene 45. Such a p r o c e s s i s symmetry-allowed (660 and c o n s e q u e n t l y no k i n e t i c energy r e l e a s e i s o b s e r v e d to accompany H l o s s from C H i * . Were the r e a c t i o n t o o c c u r v i a a c o n c e r t e d 1 , 2 - e l i m i n a t i o n , i t would be symmetry-forbidden (66) and a f l a t - t o p p e d m e t a s t a b l e peak would be expectecT. A consequence o f the p r o p o s e d 3

3

2

2

1,2-H ^

+

CH

2

= CH * 2

2

V

ν

1,1-

*CH-CH

shift 44

f

3

•

CHECH

+ H

2

elimination 45

mechanism i s t h a t a l l t h e hydrogens i n l a b e l l e d i o n i z e d e t h y l e n e s h o u l d become e q u i v a l e n t p r i o r t o m e t a s t a b l e

Unimolecular Reactions of Organic Ions

WILLIAMS

(a)

(b)

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

transition state

reactant

reactant

Figure 8. Potential energy surfaces corresponding to dissociation step; (a) with reverse activation energy, (b) with no reverse activation energy

Figure 9. Correlation diagram for concerted, suprafacial 1,2-eUmination of H2 from ionized C H &

6

40

HIGH

PERFORMANCE

MASS

SPECTROMETRY

t r a n s i t i o n s ; hence, 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 forms o f 44 must be e x p l i c a b l e 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 any hydrogen 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 f a v o r i n g s e l e c t i o n o f H r a t h e r t h a n D. E x p e r i ­ m e n t a l l y i t i s found (74) t h a t t h e l a b e l l i n g r e s u l t s can be i n t e r p r e t e d i n tTTis way. Moreover, t h e i s o t o p e e f f e c t s o b s e r v e d a r e such as t o s u g g e s t s t r o n g l y t h a t t h e e l i m i n a t i o n o f H i s c o n c e r t e d . T h i s i s good e v i ­ dence t h a t t h e f o r m u l a t i o n o f H l o s s from C Hif* as a c o n c e r t e d 1 , 1 - e l i m i n a t i o n from t h e i o n i z e d carbene 45 is correct. The c o r r e l a t i o n o f t h e m e t a s t a b l e peak shape t o t h e o r b i t a l symmetry o f t h e r e a c t i o n c o n c e r n e d i s o n l y one p a r t i c u l a r example o f t h e p o t e n t i a l s u r f a c e ap­ proach. I t s h o u l d be n o t e d t h a t any d i s s o c i a t i o n w h i c h occurs through a channel i n which the products a r e formed on a m u t u a l l y r e p u l s i v e p o t e n t i a l s u r f a c e c a n g i v e r i s e t o k i n e t i c energy r e l e a s e . An e l e g a n t example o f t h i s s i t u a t i o n i s found i n t h e d e c o m p o s i t i o n , v i a C Hi l o s s , o f t h e C 3 H ? 0 and C3HeN s p e c i e s 8 and 14, w h i c h may be f o r m u l a t e d as o c c u r r i n g v i a t h e mechanism o f Scheme 3. 2

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

2

2

+

2

f

+

Scheme 3 +

1,2-H

CH CH CH= ΟΗχ 3

2

1,2-H

+

3

3

2

2

^

2

^

2

shift

CH CH CH=NH

+

^CH CHCH 0Ht==^CH CH CH OH 2

shift

«- CH CHCH NH 3

2

2

+

* CH =0H

2

2

~

CH CH CH NH 2

2

2

2

C 2 H k

+ CH =NH 2

2

-C Hif 2

14

46

U s i n g t h e i s o d e s m i c i n s e r t i o n method w i t h a c o r r e c t i o n o f 10 and 3 k c a l m o l " f o r t h e d e s t a b i l i z i n g e f f e c t o f a 3- and γ - Ο Η ( o r -NH ) group (41,43) y i e l d s t h e e s t i m a t e d p o t e n t i a l s u r f a c e s shown i n F i g u r e s 10 and 11. A l s o g i v e n i n t h e s e f i g u r e s a r e t h e m e t a s t a b l e peak shapes f o r 0 Ηι» l o s s from t h e i o n s i n q u e s t i o n . I t i s i m m e d i a t e l y a p p a r e n t t h a t i n t h e oxygen system ( F i g u r e 10) energy must be c o n t i n u o u s l y p u t i n t o t h e system t o a t t a i n and f i n a l l y d i s s o c i a t e t h e r e a c t i n g c o n f i g u r a t i o n 22. C o n s e q u e n t l y , t h e m e t a s t a b l e peak f o r t h i s p r o c e s s i s e x p e c t e d t o be g a u s s i a n and narrow, as i s o b s e r v e d ( 6 7 ) . I n t h e c o r r e s p o n d i n g n i t r o g e n system ( F i g u r e l T T , t h e r e a c t i n g c o n f i g u r a t i o n 46 now 1

2

2

BOWEN

AND WILLIAMS

Unimolecular Reactions of Organic Ions

182 CH2=CH +CH =OH 2

2

CH CH CH OH 2

2

2

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

3

2

Journal of the American Chemical Society

Figure 10. Potential energy surface and energy release profile for C H hss starting from 8 t

k

217 ^

CH CH CH=NH 3

2

ν

CH CH CH NH 2

2

2

2

2

Journal of the American Chemical Society

Figure 11. Potential energy surface and energy release profile for C U loss starting from 14 %

k

42

HIGH

PERFORMANCE

MASS

SPECTROMETRY

a p p r o x i m a t e s t h e t r a n s i t i o n s t a t e and d i s s o c i a t e s exothermically. T h e r e f o r e , k i n e t i c energy r e l e a s e i s p o s s i b l e , and i t i s found t h a t the m e t a s t a b l e peak f o r t h i s t r a n s i t i o n i s f l a t - t o p p e d corresponding t o a r e l e a s e o f ~9 k c a l m o l " o f t r a n s l a t i o n a l energy (22). As t h e e s t i m a t e d e x o t h e r m i c i t y o f t h e d i s s o c i a t i o n o f 46 t o p r o d u c t s i s -27 k c a l m o l " , i t c a n be seen t h a t a p p r o x i m a t e l y one t h i r d o f t h e energy r e l e a s e d i s p a r t i t i o n e d i n t o t h e t r a n s l a t i o n a l mode. U s i n g t h i s f i g u r e , and c o n s t r u c t i n g analogous p o t e n t i a l s u r f a c e s f o r t h e homologues o f 8 and 14 (47 and 48 r e s p e c t i v e l y ) i n w h i c h t h e h e t e r o a t o m [X-H] group i s r e p l a c e d by an [X-CH ] group, r e s u l t s i n t h e p r e d i c t i o n s t h a t ( i ) b o t h the oxygen and n i t r o g e n c a s e s ought t o e l i m i n a t e C Hk v i a 49 and 50 r e s p e c t i v e l y (Scheme 4 ) . ( i i ) The oxygen c a s e ~ ( 4 7 ) i s o n l y r e l a t i v e l y m i l d l y e x o t h e r m i c (^11 k c a l m o l ) and so o n l y a s m a l l q u a n t i t y o f energy (^4 k c a l m o l " ) i s e x p e c t e d t o be r e l e a s e d as t r a n s l a ­ tion, ( i i i ) The n i t r o g e n analogue (48) i s e s t i m a t e d t o d i s s o c i a t e v i a 50 w i t h t h e r e l e a s e o f ^34 k c a l mol ( i . e . , ^11 k c a l m o l " as t r a n s l a t i o n ) . 1

1

3

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

2

r T

1

1

1

Scheme 4 1,2-H-shifts

+

CH CH CH=OC:H 3

2

+

+

CH CH CH 0CH3

3

2

CH =0CH

2

2

2

3

-C2Hi*

47

49 +

+

+

CH CH CH=NHCH ^ CH2CH2CH2NHCH 3

2

3

3

_

>

c R

CH =NHCH 2

3

50

48

E x p e r i m e n t a l l y , i t i s observed that ( i ) both ions o r i g i n a l l y g e n e r a t e d as 47 and 48 undergo C H loss i n m e t a s t a b l e t r a n s i t i o n s (62^7_5). ( ϋ ) The peak f o r CH l o s s from 47 i s g a u s s i a n b u t b r o a d ; a v a l u e o f ^3 k c a l mol" may be computed as t h e a v e r a g e k i n e t i c energy r e l e a s e (7_5), measured from the peak w i d t h a t h a l f h e i g h t (68). ( i i i ) The peak f o r C H l o s s from 48 i s s i m i l a r t o t h a t f o r C H^ l o s s from 14; however, the k i n e t i c energy r e l e a s e i s somewhat g r e a t e r (^11 k c a l mol" (7J5)), i n good a c c o r d w i t h t h e e x p e c t e d value. 2

2

k

1

2

2

1

k

k

2.

BOWEN

AND WILLIAMS

Unimolecular Reactions of Organic Ions

43

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

Conclusion. The c o n c e p t t h a t t h e m e t a s t a b l e d i s s o c i a t i o n s o f o r g a n i c i o n s may be c o n s i d e r e d as o c c u r r i n g o v e r an appropriate p o t e n t i a l surface i s discussed. I t s use a s s i s t s i n u n d e r s t a n d i n g phenomena such as " s c r a m b l i n g " and has a w i d e r use i n t h e s y s t e m a t i c a n a l y s i s o f unimolecular i o n i c r e a c t i o n s . In p a r t i c u l a r , conside r a t i o n o f that p o r t i o n o f the p o t e n t i a l surface which governs t h e f i n a l d i s s o c i a t i o n s t e p o f t e n p e r m i t s a b e t t e r u n d e r s t a n d i n g o f t h e shape o f t h e m e t a s t a b l e peak f o r t h a t p r o c e s s . Conversely, c o n s i d e r a t i o n o f the shape o f t h e m e t a s t a b l e peak f o r a u n i m o l e c u l a r r e a c t i o n may y i e l d v a l u a b l e m e c h a n i s t i c i n f o r m a t i o n concerning the r e a c t i o n i n question.

Abstract. Results from many studies of metastable ion decompositions i n double focussing mass spectrometers are interpreted i n terms of the potential surfaces over which gas-phase organic ions decompose. The concept of the potential surface enables the chemistry of these ions to be clearly understood. Three general types of surfaces are proposed: (1) complete e q u i l i bration of isomers prior to unimolecular decomposition, (2) no equilibration of isomers prior to unimolecular decomposition, and (3) rate-determining isomerization prior to unimolecular decomposition. Examples of each are presented and discussed. Literature Cited. 1. 2. 3. 4. 5. 6.

Cooks, R . G . , Beynon, J.H., C a p r i o l i , R.M., and Lester, G.R., "Metastable Ions", Elsevier, Amsterdam, (1973). Williams, D.H. and Howe, I., "Principles of Organic Mass Spectrometry", McGraw-Hill, London, (1972). See the relevant chapters i n "Mass Spectrometry-Specialist Periodical Reports", Vols. 1-3, The Chemical Society, London. Simm, I.G., Danby, C.J., and Eland, J.H.D., J.C.S. Chem. Comm., (1973), 832. Bowen, R.D. and Williams, D . H . , J.C.S. Perkin I I , (1976), 1479. Rosenstock, H.M., Dibeler, V . H . , and Harlee, F.N., J. Chem. Phys., (1964), 40, 591.

44

7. 8. 9. 10. 11.

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12. 13. 14. 15. 16. 17.

18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

HIGH

PERFORMANCE

MASS

SPECTROMETRY

H i l l s , L.P., Vestal, M.L. and F u t r e l l , J.H., J. Chem. Phys., (1971), 54, 3834. Löhle, U . , and Ottinger, Ch., J. Chem. Phys., (1969), 51, 3097. Vestal, M. and F u t r e l l , J.H., J. Chem. Phys., (1970), 52, 978. L i f s h i t z , C. and Sternberg, L., Int. J. Mass Spectrometry Ion Phys., (1969) 2, 303. Williams, D.H., Cooks, R . G . , and Howe, I., J. Am. Chem. Soc., (1968), 90, 6759. Beynon, J.H., Saunders, R.A. and Williams, A.E., Ind. Chem. Belge., (1964), 29, 311. Polanyi, J.H., Accounts Chem. Res., (1972), 5, 161. Carrington, T., Accounts Chem. Res., (1974), 7, 20. Davis, B . , Williams, D.H., and Yeo, A.N.H., J. Chem. Soc. (Β), (1970), 81. Yeo, A.N.H. and Williams, D.H., J. Am. Chem. Soc., (1971), 93, 395. McLafferty, F.W., Kornfeld, R., Haddon, W.F., Levsen, K . , Sakai, I., Bente, P . F . , Tsai, S.-C. and Schuddemage, H.D.R., J. Am. Chem. Soc., (1973), 95, 3886. Reyes, B.G. and Harrison A.G., Org. Mass Spectrom., (1974), 9, 221. Beauchamp, J.L. and Dunbar, R . C . , J. Am. Chem. Soc., (1970), 92, 1477. McLafferty, F.W. and Sakai, I., Org. Mass Spectrom., (1973), 7, 971. Levsen, K. and McLafferty, F.W., J. Am. Chem. Soc., (1974), 96, 139. Uccella, N.A., Howe, I., and Williams, D.H., J. Chem. Soc. (Β), (1971), 1933. Winkler, J. and McLafferty, F.W., J. Am. Chem. Soc., (1973), 95, 7533. McLafferty, F.W. and Winkler, J., J. Am. Chem. Soc., (1974), 96, 5182. Dunbar, R . C . , J. Am. Chem. Soc., (1975) 97, 1382. Bentley, T.W. and Johnstone, R.A.W., Adv. Phys. Org. Chem., (1970), 8, 197. Siegel, Α., J. Am. Chem. Soc., (1974), 96, 1251. Davidson, R.A. and S k e l l , P . S . , J. Am. Chem. Soc., (1973), 95, 6843. Lossing, F.P. and Semeluk, G.P., Canad. J. Chem. (1970), 48, 955. Lossing, F.P. and Maccoll, Α., Canad. J. Chem. (1976), 54, 990. Lossing, F . P . , Canad. J. Chem., (1971), 49, 357. Lossing, F . P . , Canad. J. Chem., (1972), 50, 3973.

2;

BOWEN

33. 34. 35. 36. 37.

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

38.

39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52.

AND WILLIAMS

Unimolecuhr Reactions of Organic Ions

45

Lossing, F . P . , reported at the American Society for Mass Spectrometry Conference, Houston, Texas, U.S.A., (1975). Lossing, F.P. and Traeger, J.C., J. Am. Chem.Soc., (1975), 97, 1579. e.g., Wanatabe, Κ., Nakayama, J., and Mottl, J., J. Quant. Spect. Rad. Transfer, (1962), 2, 369. e.g. Refaey, K.M.A. and Chupka, W.A., J. Chem. Phys., (1968), 48, 5205. e.g., Haney, M.A. and Franklin, J.L., J. Phys. Chem., (1969), 73, 4328. For a compilation of ΔΗ values for various ions and neutrals see Franklin, J. L., D i l l a r d , J.G., Rosenstock, H.M., Herron, J.L., Draxl, Κ., and F i e l d , F . H . , "Ionization Potentials, Appearance Potentials, and Heats of Formation of Gaseous Positive Ions", National Bureau of Standards, Washington, D . C . , (1969). Beynon, J.H., Cooks, R . G . , Jennings, K.R. and Ferrer-Correia, A.J., Int. J. Mass Spectrometry Ion Phys., (1975), 18, 87. Rosenstock, H.M., Int. J. Mass Spectrometry Ion Phys., (1976), 20, 139. e.g., Radom, L., Pople, J.Α., and Schleyer, P.von R., J. Am. Chem.Soc., (1972), 94, 5935. Ref. 38, Appendix 2; see also Franklin, J.L., Ind. Eng. Chem., (1949), 41, 1070, and Franklin, J.L., J. Chem. Phys., (1953), 21, 2029. Bowen, R.D. and Williams, D . H . , Org. Mass Spectrom., 12, 475 (1977). Aue, D.H. Davidson, W.R. and Bowers, M.T., J. Am. Chem. Soc., (1976), 98, 6700. Howe, I. and McLafferty, F.W., J. Am. Chem. Soc., (1971), 93, 99. Beynon, J.H., Corn, J.E., Battinger, W.E., C a p r i o l i , R . M . , and Benkeser, R.A., Org. Mass Spectrom., (1970), 3, 1371. Holmes, J.L. and Terlouw, J.K., Org. Mass Spectrom., (1975), 10, 787. Smith, G.A. and Williams, D . H . , J. Chem. Soc. (Β), (1970), 1529. Bowen, R.C. and Williams, D . H . , Org. Mass Spectrom, 12, 453 (1977). Shaw, M.A., Westwood, R. and Williams, D . H . , J. Chem. Soc. (Β), (1970), 1773. Shannon, T.W. and McLafferty, F.W., J. Am. Chem. Soc., (1966), 88, 5021. Beynon, J.H. and Fontaine, A.E., Zeitschrift für Naturforschg, (1967), 22a, 334, and references cited therein. f

46

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53.

HIGH

PERFORMANCE

MASS

SPECTROMETRY

Beynon, J.H., Saunders, R . A . , and Williams, A.E., Zeitschrift für Naturforschg, (1965), 20a, 180. 54. Hvistendahl, G . , Bowen, R.D. and Williams, D . H . , J. Am. Chem. Soc., in press. 55. Hvistendahl, G. and Williams, D.H., J. Am. Chem. Soc., (1975) 97, 3097, (see pp. 3099 and 3100). 56. Van Raalte, D. and Harrison, A.G., Canad. J. Chem., (1963), 41, 3118. 57. Hvistendahl, G . , Bowen, R.D. and Williams, D . H . , unpublished results. 58. Harrison, A.G. and Keyes, B . G . , J. Am. Chem. Soc., (1968), 90, 5046. 59. Tsang, C.W. and Harrison, A.G., Org. Mass Spectrom., (1970), 3, 647. 60. Tsang, C.W. and Harrison, A.G., Org. Mass Spectrom., (1971), 5, 877. 61. Tsang, C.W., and Harrison, A.G., Org. Mass Spectrom., (1973), 7, 1377. 62. Mead, T . J . and Williams, D . H . , J.C.S. Perkin I I , (1972), 876. 63. Westwood, R., Williams, D.H. and Yeo, A.N.H., Org. Mass Spectrom., (1970), 3, 1485. 64. Mead, T.J. and Williams, D . H . , J. Chem. Soc. (Β), (1971), 1654. 65. Hvistendahl, G. and Williams, D . H . , J. Am. Chem. Soc., (1975), 97, 3097. 66. Woodward, R.B. and Hoffmann, R., "The Conserva­ tion of Orbital Symmetry", Verlag Chemie, Winheim/Bergstr., Germany, (1970). 67. Hvistendahl, G . , Bowen, R.D. and Williams, D. Η., J.C.S. Chem. Comm., (1976), 294. 68. T e r w i l l i g e r , D.T., Beynon, J.H. and Cooks, R . G . , Proc. Roy. Soc., (1974), 341, 135. 69. Smyth, K.C. and Shannon, T.W., J. Chem. Phys., (1969), 51, 4633. 70. Hvistendahl, G. and Williams, D . H . , J. Am. Chem. Soc., (1974), 96, 6753. 71. Beynon, J.H., Fontaine, A.E., and Lester, G.R., Int. J. Mass Spectrometry Ion Phys., (1968) 1, 1. 72. Hvistendahl, G. and Williams, D . H . , J.C.S. Perkin II, (1975), 881. 73. Williams, D. H. and Hvistendahl, G., J. Am. Chem. Soc., (1974), 96, 6755. 74. Hvistendahl, G. and Williams, D.H., J.C.S. Chem. Comm., (1975), 4. 75. Williams, D.H. and Bowen, R.D., J. Am. Chem. Soc., 99, 3192 (1977). RECEIVED December 30, 1977

3 Structures of Gas-Phase Ions from Collisional Activation Spectra* F. W. MC LAFFERTY

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch003

Department of Chemistry, Cornell University, Ithaca, NY 14853

"Double resonance" techniques have provided revolutionary new information for a variety of spectroscopic techniques in recent years. In mass spectrometry, double resonance has been utilized in the ion cyclotron resonance spectrometer to identify in a complex mixture of ions the precursor and product ions of a particular ion-molecule reaction. This paper concerns a technique in which a mass spectrum can be obtained of each peak in a mass spectrum; the ions representing that peak are activated by collision, causing their further decomposition to produce the "collisional activation" (CA) mass spectrum (1-3). The reverse-geometry double-focussing mass spectrometer (4) has special advantages for the measurement of such CA spectra. The sample is ionized and the resultant ions are mass analyzed in the magnetic analyzer; ions of a particular m/e value are brought into focus on a collision chamber containing a gas such as helium at ~10 torr pressure. In some ion-atom interactions part of the ion's kinetic energy is converted into internal energy without appreciable scattering of the ion. The resulting decompositions yield product ions which are separated in the electrostatic analyzer to yield the CA mass spectrum. In both formation and utility the CA spectrum resembles a normal mass spectrum; the decompositions of the collisionally activated ion follow the predictions of the quasiequilibrium theory, so that the resulting product ions are indicative of the precursor ion's structure. In addition, in most of the observed decomposition processes sufficient energy has been added by collision to make the resulting ion abundances virtually independent of the original internal energy ("temperature") of the precursor ion; the only ion energy-dependent processes are those which are involved in the decomposition of metastable ions (the MI spectrum, measured in the same manner except in the absence of the collision gas). Thus -4

Collisional Activation and Metastable Ion Characteristics. 55. For part 54, see reference 10. American Chemical © 0^12-042^^ 1155 16th St. N. W. Waehtogtoit, D. C. 20036

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch003

48

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both the masses and abundances of the CA spectrum, excluding peaks i n the MI spectrum, are characteristic of the precursor ion structure on a quantitative b a s i s , i n the same way that a normal mass spectrum i s a reproducible characteristic of the structure of the precursor molecule. Areas i n which CA-MS appears to be especially promising are fundamental studies of organic ion structures and reactions, MS structure determination of complex organic molecules through elucidation of the isomeric identity of fragment ions, and separation/identification of complex mixtures through MS separation of ionized molecular species which are then identified from their CA mass spectra. An excellent review of the field by Levsen and Schwarz has appeared (3), and Cooks reports on some related results (5); this article w i l l use as examples more recent work of our laboratory. CA-MS Instrumentation Most recent CA-MS studies have utilized "direct analysis of daughter ions (DADI) " (4) (also called "mass analyzed ion kinetic energy spectroscopy [MIKES]") (6) with a revers edgeometry double-focussing mass spectrometer (7) although Jennings and coworkers have recently shown there are advantages in using a normal geometry double-focussing instrument with a "linked" scan of the electrostatic and magnetic analyzers (8). Instrumentation aspects of importance include achieving purity of the precursor ion, resolution of the CA spectrum, data a c q u i s i tion and reduction, sensitivity, and quantitative reproducibility of spectra. As i s w e l l know i n normal mass spectrometry, the peaks in a unit resolution mass spectrum often contain isobaric multiplets; for example, C3H7+ and C 2 H 3 0 , both nominally m/e 43, actually differ i n mass by 37 millimass units. "Pure" precursor ions of course are desirable for CA spectra, and this can be achieved with sufficient resolving power (9); a CA-MS instrument with a double-focussing mass spectrometer before the c o l l i s i o n chamber i s under construction at Cornell as a solution to this problem (10). Even i f a l l the precursor ions have the same elemental composition, these s t i l l may contain isomeric impurities; formation of the ions using different ionizing electron energies, using different ionization methods (e.g., chemical ionization), or from different precursor molecules can be used to improve isomeric purity. A more serious problem i s the resolution achievable i n the CA spectrum. In the reversed-geometry instrument, the CA product ions are separated by the electrostatic analyzer, taking advantage of the fact that the loss i n mass on decomposition results i n a concommitant loss i n kinetic energy. However, i n the c o l l i s i o n process a small fraction of the ion's kinetic energy is converted into internal energy and, more seriously, internal +

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch003

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Ions from Collisional Activation Spectra

49

energy i n excess of that required for the decomposition can be released i n the reaction coordinate, appearing as a positive or negative velocity vector; this process has been extensively studied i n connection with the "flat-top" peaks produced i n metastable ion decompositions (6). This uncertainty i n the kinetic energy of a particular product ion broadens the peak, the extent of this broadening depending on the amount of energy variation relative to the total kinetic energy. Increasing the ion accelerating voltage decreases this fraction and therefore i n creases the resolution of the CA spectrum (11). A similar approach i s to use "post-acceleration" of the ions after the c o l l i s i o n chamber (12); an instrument under construction at the F O M Institute i n Amsterdam w i l l float the electrostatic analyzer and collector at as much as 40 kV, which should greatly enhance the resolution of the CA spectrum. Double-focussing mass analysis is employed i n instruments such as the spark source MS i n order to bring ions of widely varying kinetic energies but the same mass into coincident focus, and this principle w i l l be employed in the tandem double-focussing CA-MS instrument under construction at Cornell (10). The linked-scan, normal geometry double-focussing technique employed by Jennings and coworkers (8) apparently gives improved resolution for CA mass spectra; this arises because the ions produced by CA decomposition i n the field-free region before the electrostatic analyzer thus traverse most of the double-focussing path of the instrument. As already amply demonstrated i n normal MS, utilization of a dedicated computer for automated data acquisition and reduction also i s advantageous for CA-MS. The Cornell reversedgeometry CA-MS instrument was coupled to a DEC PDP-8 computer i n 1972 (7), and i n 1975 this was replaced by a PDP-11/10 dedicated computer which directs the multiple scanning of the CA spectrum through a D/A converter to the ESA (11). Special software and hardware give flexibility for selected mass scanning, averaging of the repeat scans, cathode ray tube display of the most recent and the averaged scans, comparison with previous spectra, multicomponent analysis of spectra, and so forth. Of special importance for ion structure characterization is the ±2% precision i n CA peak abundances which can be achieved with this new data system (11). In fact, this has been used to analyze ternary mixtures of isomeric C 3 H 7 S ions with ±10% accuracy based on the peak abundances of their CA spectra (13). This i s reminiscent of the quantitative analysis of light hydrocarbon mixtures using normal MS, the major incentive for the original development of the analytical mass spectrometer. In the Cornell tandem double-focussing CA-MS instrument under construction (10), we plan to have the PDP-11/45 computer also control the mass analysis of the precursor ion. The technique of "Signal Enhancement i n Real-Time" (14) w i l l be used to keep a particular precursor ion peak i n focus on the c o l l i s i o n chamber +

50

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during the continuous scan of the first magnet by an offsetting increase i n the ion accelerating voltage, during which time the CA-MS data (complete spectrum or selected ions) w i l l be obtained; the computer w i l l then direct the ion accelerating potential to focus the next precursor ion peak on the c o l l i s i o n chamber while its CA spectrum i s measured, and so forth (10), Such automated data acquisition and control for both MS sections in concert w i l l be especially advantageous for its use as a separation/identification system for complex mixtures.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch003

Structures of Gaseous Organic Ions The structures of a wide variety of ions have been determined by CA-MS. Table I l i s t s the elemental compositions of isomeric ions which have been studied by CA-MS at Cornell; additional studies by Levsen, Schwarz, Van de Sande, Beynon, Cooks, and their coworkers are listed i n the Levsen-Schwarz review (3). Table II shows proposed structures for 9 isomeric C3H50+ ions which give distinguishable CA spectra (there i s much better evidence for the first s i x than for the last three structures) (15). CA spectra, like normal mass spectra, can be used to characterize an ion's structure through rationalization of the decomposition products or through quantitative comparison with reference spectra of ions of established structures. This can be illustrated by studies of CgHg" isomers carried out by Dr. Claus Kôppel at Cornell recently (16). 1-

Table I. C o l l i s i o n a l Activation Studies CH

C

+ 4

, C H 3

H

+

C

+ 3

H

7 7 ' 7 8

C U 0*, 2

, C H 3

t #

C

H

+ 7

, C H *, C H 4

+

C

8

4

H

+

8 9 ' 9 11 ' +

C

H

, CgH^t, CgH^t

13 9

+

+

C H O , C H O , C H 0+, C H 0

4

2

s

3

s

3

+

6

6

n

+

6

3

+

C H 0t, C H O , C H O , C 6

+ g

g

n

+

+

l 2

1

H o \ C^H^ot g

4

C H N , C H N ; C H S , C^S" "; C ^ g C l " 2

6

3

Oligopeptides

8

2

5

+

?

3.

Ions from Collisional Activation Spectra

MCLAFFERTY

51

Table II. Possible Isomeric Structures of ΟβΕ^Ο* Ions

+ CH =CHCH=OH 2

HC=CCH OH 2

C H = C H O+= C H

+ CH CH C=0 3

2

2

2

A variety of molecules, including isotopically labeled derivatives, were used to form C 3 H g ions; their CA spectra showed that at least 13 isomers have lifetimes >10" seconds. Although many of the isomers do isomerize when formed with higher internal energies, these results contrast sharply with our previous conceptions of ready "scrambling" of hydrocarbon ions. Part of this misconception has arisen from the fact that most previous studies utilized the unimolecular decomposition products of an ion as evidence of its structure, and even the lowest a c t i ­ vation energy for ion decomposition is s t i l l higher than that for an isomerization. For example, the scrambling of isotopic labels on decomposition of the CyHj* ion from toluene indicated that this ion had the symmetrical tropylium, not the benzyl, structure (17, 18),but CA spectra show conclusively that both ions are formed and are stable for >10~ seconds (19). Similarly, the C 8 H g isomers m ethyl tropylium and a, o-, m-, and jj-methylbenzyl ions are found to be stable if formed with sufficiently low energy. However, as found for cycloheptatriene and toluene (19), isomerization does occur between the molecular ions of methylcycloheptatriene, ethylbenzene, and the xylenes (16). A number of recent studies have increased our basic knowledge of both solution and gas phase ionic reactions by direct comparison of particular reactivities (e.g., a c i d i t i e s , basicities) i n the two media (20 -22). A c l a s s i c NMR study by Olah and Porter (23) of the ionization of β-phenylethyl chloride (a, X = CI) i n superacid medium gives detailed NMR evidence for the formation of the ethylenebenzenium (ç) and oc-phenylethyl (d) cations, with the β-ethylphenyl ion (b) as the proposed intermediate (see Scheme I). Although this medium should mini­ mize solvent effects, we find that the unimolecular decomposition of a, with X as either CI or C H 3 , ions produces negligible amounts of either c or d as stable ionic products with the 10*"^ sec lifetime requirements of the CA technique. Instead, at low electron energies these precursors y i e l d ions whose CA spectra are identical to those formed by the protonation of benzocyclobutene, which thus presumably are formed originally as structure e. In contrast, ionization of β-phenylethyl bromide and iodide (a, X = Br or I) yields the ethylenebenzenium i o n , c, at low +

5

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch003

2

5

+

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch003

52 HIGH PERFORMANCE MASS SPECTROMETRY

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch003

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Ioris from Collisional Activation Spectra

53

electron energies . This appears to indicate that the formation of e i s favored energetically, possibly through an anchimericas sis ted reaction, but this pathway has more stringent steric requirements, so that the larger Br and I leaving groups cause the formation of c to be favored. Another problem which has been studied extensively on a theoretical as w e l l as experimental basis involves the relative energies of the possible C3H7"** isomers. Equilibrium constant measurements on ion-molecule reactions by Chong and Franklin (24) indicate the isopropyl structure to be 8 kcal/mol more stable than the protonated cyclopropane, but ion cyclotron resonance measurements (25) are consistent with complete isomerization of the latter to the former structure i n 10*" ^ seconds. Because the CA spectra of C3H7"*" ions formed by protonation of propene and of cyclopropane are identical (26), the isomerization also should be essentially complete i n 10~^seconds, indicating that the activation energy for the isomerization of protonated cyclopropane i s very low. If the transition state for this isomerization i s s i m i ­ lar to the η-propyl structure, this also indicates that its heat of formation i s surprisingly close to that of the protonated c y c l o ­ propane isomer (26). The differences i n the CA spectra of isomeric hydrocarbon ions are often relatively small, paralleling the behavior found for normal mass spectra of hydrocarbons. For example, the CA spec­ tra of sec- and tert-butyl ions are nearly the same (27). One method to increase these differences i s to form a derivative of the ions. In the case of the C 4 H g ions, simple derivatives can be formed by the reaction of the newly formed ions directly i n the MS ion source with acetyl chloride; the resulting ΟβΗ^Ο" " ions show substantial differences i n their CA spectra. The C4H9"*" ions formed from η-butyl bromide when derivatized i n this fashion give a CA spectrum which i s identical to that from sec-butyl ions, while isobutyl ions isomerize to give a 7:3 mixture of secand tert-butyl ions. Both gas phase and solution determinations show the heat of formation of tert-butyl ions to be 16 kcal/mol less than that of sec-butyl ions, so that conformational effects outweigh enthalpic factors. Obviously i t is important to the understanding of solution reactions involving organic ions to be able to separate out completely the effect of the solvent on the reaction. +

1

Applications of CA to Molecular Structure Determination Although such applications have been restricted i n the past because of instrumentation limitations, CA spectra should provide v i t a l additional information for the structure elucidation of complex molecules. The interpretation of a normal, unit resolution mass spectrum utilizes the masses of the pieces of the molecule to reconstruct the molecule's structure. Exact mass measurement of the fragments by high-resolution mass

54

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spectrometry can give the elemental compositions of these fragments, an obvious aid to putting the pieces of the molecular puzzle together correctly. As described above, the CA spectrum can provide the isomeric structure of the fragment i o n , obviously an even more valuable aid i n the interpretation process. For example, i f high-resolution measurements showed that an un­ known molecule produced a significant Οβ^Ο" " i o n , at least the nine isomeric structures of Table II should be considered as possible molecular fragments. Measuring the CA spectrum of the C 3 H 5 0 ions from the molecule should indicate the proper isomer. For example, a steroid containing a 17-α-hydroxyethyl group shows a significant C 2 H 5 C " peak i n its normal mass spectrum; the CA spectrum of this peak identifies i t as the C H 3 C H ( O H ) isomer eliminating the p o s s i b i l i t y , for example, that the steroid contains a - C H 2 O C H 3 group (2). Such applications of CA spectra have been especially useful for oligopeptide samples. The amino acid sequence i n fragment ions can be deduced from their CA spectra, and the presence of leucine be distinguished from that of isoleucine (28). 1

+

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch003

1

MS/CAMS As a Separation/Identification System Recognition of the usefulness of the gas chromatograph/ mass spectrometer/computer (GC/MS/COM) system for the identification and quantitation of complex unknown mixtures has led to an amazing growth i n its analytical applications i n this decade. GC/MS/COM involves separation of the mixture and identification of the individual components, with automated data reduction; other such separation/identification systems include liquid chromatography/MS/COM. Separation can also be done mass spectrometrically, characterizing the products from the decompositions of metastable ions (29) or, as we suggested i n a study on sequencing oligopeptides i n mixtures i n this fashion (30), the identification of the mass-separated ions can also be done by CAMS. Results from a surprising number of laboratories indicate a high potential for MS/CAMS as a separation/identifi­ cation system. For example, Levsen and Schulten (31) identified major components of a complex mixture produced by the pyrolysis of deoxyridonucleic a c i d , and Cooks and coworkers (5) achieved accuracies of ±20% on the quantitative analysis of a f i v e component ketone mixture. Of special note i s the 1 0 " gram sensitivity achieved i n the latter investigation. Recent studies at Cornell (9) have examined several aspects of MS/CAMS, including separation, sensitivity, mixture ionization techniques, computer identification of CA spectra, and automation. MS/CAMS appears to be especially promising i n application to complex mix­ tures for the quantitative analysis for specific components (similar to the "selected ion monitoring" i n GC/MS), and for the identification of individual components. 11

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch003

3.

MCLAFFERTY

55

Ions from Collisional Activation Spectra

As a separation technique, mass spectrometry has the advantage over GC of submillisecond time requirements, which would be especially advantageous for continuous analyzers. Of special importance i s the lower sample volatility requirement of MS, using direct probe introduction or special ionization methods such as field desorption or direct chemical ionization. Note that the lack of volatility restrictions on liquid chromato­ graphy i s the chief incentive for the proposed LC/MS separation/ identification systems. Resolution i n MS i s based on an entirely different principle than i n chromatography, so that it should obviously be a complementary separation technique, and a pos­ sible solution for problems i n which chromatographic resolution is inadequate. In normal mass spectrometry double-focussing instruments are employed to improve resolution, making possible the separation of isobaric multiplets. We have recently applied this (9) to improve the separation for MS/CAMS, obtaining characteristic CA spectra of each of the doublets C H 2 3 5 C l 2 and C5H12 (nominal mass 84), and also of C H C 1 C 1 and Ο Η Ο (nominal mass 86). For MS/CAMS the ionization of the sample should give a minimum number (preferably 1) of peaks for each component of a complex mixture introduced into the MS ionization chamber. Lowering the ionizing electron energy reduces the number of peaks per component and increases the relative abundance of the molecular ion, but lowers the overall sensitivity. "Soft" ionization methods such as chemical ionization often give only one peak (plus its isotopic peaks) per compound, but different reagent gases can be necessary to obtain peaks for a l l compo­ nents as the CI ionizing reagent gases (9). 3 5

2

3 7

5

1 0

Future Possibilities For C o l l i s i o n a l Activation Mass Spectra As with conventional mass spectrometry and GC/MS, the availability of CAMS systems which are convenient to use, have automated operation and data reduction, are reasonable i n price, and which show improved sensitivity and resolution, should provide, a powerful additional incentive to further applica­ tions i n a variety of areas. Improved instrumentation such as the "ZAB" reverse-geometry double-focussing mass spectrometer of VG Micromass, the "post-acceleration" reverse-geometry spec­ trometer at the FOM Institute (12), as w e l l as the tandem doublefocussing instrument under construction at Cornell (10) should aid this problem, but smaller inexpensive instruments must also become available commercially. In this regard, i t should be possible to u t i l i z e a quadrupole MS as the mass analyzer, accelerate the ions into a c o l l i s i o n region, possibly using a molecular beam of c o l l i s i o n gas as demonstrated by Moran and collaborators (32), followed by post-acceleration and separation of the CA spectrum i n an electrically isolated electrostatic analyzer (12). With no magnet, complete computer control with

56

HIGH PERFORMANCE MASS SPECTROMETRY

rapid mass response i n both the mass analysis steps should be possible. The analysis of highly complex mixtures such as biological fluids, pollutants, insect pheromones, food aromas and flavors, and pyrolysis products i s becoming of increasing importance. The sensitivity, specificity, and speed of analysis which should be possible with MS/CAMS should make it a p p l i cable to mixtures of organic compounds on a routine b a s i s , even to on-line control, for many important problems which can only be analyzed with difficulty at present.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch003

iterature Cited 1. McLafferty, F. W . , Bente, III, P. F., Kornfeld, R., Tsai, R . - C . , and Howe, I., J. Am. Chem. Soc., (1973), 95, 2120. 2. McLafferty, F. W . , Kornfeld, R., Haddon, W. F., Levsen, K., Sakai, I., Bente, III, P. F., Tsai, S . - C . , and Schuddemage, H. D. R., J. Am. Chem. Soc., (1973), 95, 3886. 3. Levsen, K. and Schwarz, H., Angew. Chem., Internat. Edit., (1976), 15, 509. 4. Maurer, J. H., Brunee, C., Kappus, G., Habfast, K . , Schröder, U., and Schulze, P., 19th Annual Conference on Mass Spectrometry, Atlanta, 1971, paper K-9. 5. Kruger, T. L., Litton, J. F., Kondrat, R. W., and Cooks, R. G., Anal. Chem., (1976), 48, 2113. 6. Cooks, R. G., Beynon, J. H., Caprioli, R. M., and Lester, G. R., "Metastable Ions", Elsevier, Amsterdam, 1973. 7. Wachs, T., Bente, III, P. F., and McLafferty, F. W., Int. J. Mass Spectrom. Ion Phys., (1972), 9, 333. 8. Weston, A. F., Jennings, K. R., Evans, S., and Elliot, R. M., Int. J. Mass Spectrom. Ion Phys., (1976), 20, 317. 9. McLafferty, F. W. an d Bockhoff, F. M., Anal. Chem., submitted. 10. McLafferty, F. W. in "Analytical Pyrolysis", H. L. C. Meuzelaar, Ed., Elsevier, Amsterdam, 1977. 11. Wachs, T., Van de Sande, C. C., Bente, III, P. F., Dymerski, P. P., and McLafferty, F. W., Int. J. Mass Spectrom. Ion Phys., accepted. 12. Tuithof, H. H., Int. J. Mass Spectrom. Ion Phys., submitted. 13. Van de Graaf, B. and McLafferty, F. W . , in preparation. 14. McLafferty, F. W . , Michnowicz, J. Α., Venkataraghavan, R., Rogerson, P., and Giessner, B. G., Anal. Chem., (1972), 44, 2282. 15. Dymerski, P. P. and McLafferty, F. W . , in preparation. 16. Köppel, C., Van de Sande, C. C., Nibbering, Ν. M. M., Nishishita, T., and McLafferty, F. W., J. Am. Chem. Soc., in press.

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch003

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Ioris from Collisional Activation Spectra

57

17. Rylander, P. N., Meyerson, S., Grubb, H. M., J. Am. Chem. Soc., (1957), 79, 842. 18. Bursey, J. T., Bursey, M. M., and Kingston, D. G. I., Chem. Rev., (1973), 73, 191. 19. McLafferty, F. W. and Winkler, J., J. Am. Chem. Soc., (1974), 96, 5182. 20. Arnett, Ε. M., Acc. Chem. Res., (1973), 6, 404. 21. Taagepera, M., Hehre, W. J., Topsom, R. D., and Taft, R. W., J. Am. Chem. Soc., (1976), 98, 7438. 22. Farneth, W. E. and Brauman, J. I., J. Am. Chem. Soc., (1976), 98, 7891. 23. Olah, G. A. and Porter, R. D., J. Am. Chem. Soc., (1971), 93, 6877. 24. Chong, S.-L. and Franklin, J. L., J. Am. Chem. Soc., (1972), 94, 6347. 25. McAdoo, D. J., McLafferty, F. W., and Bente, III, P. F . , J. Am. Chem. Soc., (1972), 94, 2027. 26. Dymerski, P. P., Prinstein, R. M., Bente, III, P. F . , and McLafferty, F. W . , J. Am. Chem. Soc., (1976), 98, 6834. 27. Dymerski, P. P. and McLafferty, F. W . , J. Am. Chem. Soc., (1976), 98, 6070. 28. Levsen, K . , Wipf, H.-K., and McLafferty, F. W., Org. Mass Spectrom., (1974), 8, 117. 29. McLafferty, F. W. and Bryce, T. Α., Chem. Commun., (1967), 1215. 30. Wipf, H.-K., Irving, P., McCamish, M., Venkataraghavan, R., and McLafferty, F. W., J. Am. Chem. Soc., (1973), 95, 3369. 31. Levsen, K. and Schulten, H.-R., Biomed. Mass Spectrom., (1976), 3, 137. 32. Moran, T. F . , Wilcox, J. B., and Abbey, L. Ε., J. Chem. Phys., (1976), 65, 4540. Acknowledgment We are indebted to the National Institutes of Health (grant 16609) and the Army Research Office, Durham, (grant 13136-C) for generous financial support of research described i n this review. RECEIVED December 30,1977

4 Ion Chemistry via Kinetic Energy Spectrometry R. G. COOKS

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

Department of Chemistry, Purdue University, W. Lafayette, IN 47907

This paper describes studies on ion structure, reaction mechanism and thermochemistry all of which rely on the measurement of ion kinetic energy. In all experiments the ion beam undergoes modification after i t leaves the source and it is subjected to sequential analysis by magnetic and electric fields. This ensures that a particular reaction occurring in the analyzer can be selected for study. The methodology is derived from that developed for the study of metastable ions, i.e., the spontaneous delayed fragmentations of ionized molecules. In this work, measurements on metastable ions are made in conjunction with ion/molecule interactions at high relative kinetic energy occurring after ion formation and acceleration. The reactions of interest are classified as secondary processes in contrast to primary processes which occur in the ion source. According to this view, primary processes may involve complex reaction sequences, and chemical and photochemical processes may follow the primary ionization steps, but the beam is not perturbed after extraction from the ion source. Simple mass spectra are recorded. This has been the traditional source of information on ion chemistry as well as the most important route to the applications of mass spectrometry. Secondary processes on the other hand include all those in which the ions issuing from the ion source subsequently undergo measurable alterations in charge state, mass, gross structure or electronic state. Adequate characterization of these processes usually requires that two independent measurements be performed on the ion beam after i t leaves the source. Secondary processes may be spontaneous and involve emission of a photon, an electron, an atom or a molecule. Of these processes, only the latter is well-characterized, and i t represents a metastable ion dissociation. Secondary processes which are not spontaneous may occur when the ion interacts with a gas atom or molecule, an electron, a surface, a photon beam or an electromagnetic field. Reactive collisions are of no importance in the kilovolt energy range so the inelastic reactions of interest are confined to electronic transitions, charge stripping, charge ©0-8412-0422-5/78/47-070-058$10.00/0

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

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59

exchange and photoemission and the dissociations which can follow these processes. Thus, our study of chemistry w i l l be based on measurements on the processes shown i n Table I . The structure of the reactant ion i s frequently of major interest when secondary processes are studied. This structural information i s often derived by monitoring the dissociations, either spontaneous or collision-induced, of the ion i n question. This often provides direct access to molecular structure and con­ stitutes a method of product analysis f o r ion source reactions. Other methods of characterizing an ion through i t s charge changing c o l l i s i o n s or v i a simple excitation reactions w i l l also be noted below. Thermochemical measurements accomplish t h i s purpose, and ion enthalpy measurements made i n the ion source by varying the energy deposited upon ionization represent the t r a ­ d i t i o n a l source of t h i s data. Using fast ion beams k i n e t i c energy loss measurements associated with ion/molecule reactions provide analogous data. I t i s a fortunate fact that for conditions commonly f u l f i l l e d i n mass spectrometers, the heat of reaction for ion/molecule reactions i s measured to a good approximation as the k i n e t i c energy change (loss i f endothermic, gain i f exothermic) of the fast species.* The value of k i n e t i c energy measurements i n studying secondary processes i s further enhanced by the k i n e t i c energy amplification associated with the disposal of internal energy of an excited species as r e l a t i v e translational energy of i t s fragments. The range of k i n e t i c energies of the fragment ions can be 1 0 the actual center of mass energy release. This makes possible sophisticated measurements on energy p a r t i t i o n i n g as well as the characterization of the reacting ion and i n favorable cases i t s fragmentation mechanism. This measurement i s applicable to both spontaneous and collision-induced dissociations. In summary, the chief concern of t h i s paper i s the use of secondary processes to characterize ions formed i n a chemical ionization source. To f u l l y exploit the r i c h chemistry associated with low energy, gas-phase ion/molecule reactions, direct methods for characterizing the structures of product ions are needed. For t h i s purpose, we use the conjunction of a chemical ionization source as reaction vessel with the methods of ion k i n e t i c energy spec­ trometry for analysis. 5

*The target r e c o i l energy i s given by j j| g- where m i s the mass of the ion,Ν that of the target, Q i s the heat of reaction and Ε i s the reactant ion translational energy.

CO

i

α·

CO CO

w

CO

Photon-induced

C o l l i s ion-induced (Surface-induced)

m.

Autoionization

+

++

3

n

m

3

Photoexcitation

m^ + hj •+ m^ *

r

ι -> 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*H)+|

Figure 5. MIKE spectra showing spontaneous alkane elimination from both the molecular ion and the protonated molecule for diisopropyl ketone

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

4.

COOKS

67

Kinetic Energy Spectrometry

Studies i n other cases are i n hand. A tentative generalization i s that the reaction i s f a c i l i t a t e d by positive charge density on the heteroatom or functionalized carbon. The study of metastable ions i n CI not only provides informat i o n on fragmentation mechanisms but i t i s a valuable source o f information on the internal energies of ions extracted from the source. I f c o l l i s i o n s relax the ions below the activation energy for the lowest energy fragmentation then no metastable ions w i l l be observed. Experiments i n which an inert bath gas i s admitted to the CI source and metastable ion abundances are followed are p a r t i c u l a r l y attractive as a new method of studying energy transfer i n ion/molecule c o l l i s i o n s . In a related type of experiment a correlation has been observed between the presence of metastable ions i n CI and the activation energy f o r the lowest energy fragmentation. (5) Thus, C3H5 " ions which require 2-3 eV of internal energy to undergo H loss show essentially no metastable peak even when generated by a variety of ion/molecule reactions. The process can, however, be induced by c o l l i s i o n and the k i n e t i c energy release i s then 0.47 eV i n good agreement with the value of 0.43 eV found f o r collision-induced dissociation of C3H5"" ions generated by electron impact. Figure 6 which i s adapted from ref. 6 shows these results as compared to the metastable dissociations of the same ions. The l a t t e r occur v i a two competitive reactions to give a composite metastable peak. On c o l l i s i o n , only the higher activation energy, higher frequency factçr reaction i s observed. In contrast to the behavior of C^Hr , the molecular ions of substituted nitrobenzenes which can undergo NO* loss a t considerably lower internal energies, show abundant metastable peaks. These reactions correspond e n t i r e l y to those observed for the same ions generated by electron impact, both sets of ions showing composite metastable peaks with the same k i n e t i c energy releases. Figure 7 i l l u s t r a t e s the composite metastable peaks associated with NO' loss from the molecular ions of pchloro and p-hydroxy nitrobenzene. The contrasting behavior o f the nitrocompounds and CJir can be interpreted i n terms of the e f f i c i e n c i e s of quenching of the states responsible f o r fragmentation on the time scale associated with metastable ions. 4

2

1

Isotope Labeling and the MIKE Spectrometer I t would be d i f f i c u l t to overestimate the importance that labeled compounds have had i n the elucidation of gas-phase ion chemistry. Frequently the synthesis of pure compounds, highly incorporated at s p e c i f i c s i t e s , has been the challenging and time consuming step i n such work. The MIKE spectrometer relaxes the requirements for both chemical and isotopic purity and so f a c i l i tates t h i s type of endeavor. (7) Quite simply, mass selection acts as a f i n a l stage of chemical and isotopic p u r i f i c a t i o n i n the synthesis. Subsequent to mass-analysis, either metastable or c o l l i s i o n -

68

HIGH PERFORMANCE MASS

SPECTROMETRY

ENERGY RELEASE

Threshold for (A)-

CID

m* E (A)"0.5eV rev

Thresholdfor(B)-

0 27eV EI CH=C-CH2+H

0 43eV;0-47eV EI CI

2

12.0 -

E (B)«l.2eV rtv

CALCULATED BARRIER _

I OOeV EI

not observed

PR0PENYL+

ο CH Ç«CH

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

2

2

Figure 6. Thermochemistry of the reactions C H * -» C H * + H (6). Also shown are the measured kinetic energy releases for the unimolecular (m*) and collision-induced dissociations (CID) when the ion is generated by electron impact (EI) and chemical ionization (CI). 3

5

3

S

2

a)pOH

80 %e

Figure 7. Composite metastable peaks for NO- loss from the molecular ions of the substituted nitrobenzenes indicated. The molecular ions were formed in the CI source by charge exchange with argon ions, were mass selected, and their energy spectra were plotted.

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

4.

COOKS

Kinetic Energy Spectrometry

69

induced dissociations may be studied depending upon whether react­ ing ions of higher or lower internal energy are of interest. These points are i l l u s t r a t e d by the elimination of propane from the diisopropyl ketone molecular ion. (Id) The acidic hydrogens on the α-carbons are readily exchanged with i n base. The p a r t i a l l y incorporated compound obtained by a single exchange step serves for analysis by the MIKES technique as i l l u s t r a t e d i n Figure 8. This shows part of the MIKE spectrum of the à^compound (401 of the ketone sample) compared to that of the unlabeled compound. Alkane elimination i s seen to involve loss of both labeled hydrogen atoms. Indeed, informative results are obtained on both the cL-ketone and the cL -ketone which can be selected independently for study. Experiments run i n the presence of target gas show, i n addition, collision-induced dissociation by loss of an a l k y l radical. The MIKE spectra of the labeled compounds confirm this and show that no hydrogen isomerization i n volving the methyl groups occurs. As a variation on the above approach i n which the MIKES i s used to purify a crude labeled compound, the synthesis i t s e l f can be carried out i n the CI source. Thus, using simple labeled reagents the ion of interest can be synthesized, purified and i t s reactions characterized, a l l within the mass spectrometer. Proton A f f i n i t i e s using Metastable Ions Metastable ions, because they refer to species which^have_^ just enough energy to react at a rate of approximately 10 sec , provide a source of thermochemical data on reaction thresholds. A long standing generalization i s that i f competitive fragmentations from the same ion both give r i s e to metastable peaks, the reactions must have similar activation energies. I f i n addition, the reactions are of similar type so that their frequency factors are comparable, then the relative magnitudes of their activation energies can be expected to determine the relative metastable peak heights. Thus, comparison of a kinetic quantity, the relative rates of two reactions from a given ion, allows the determination of a thermochemical quantity, the relative free energies of activation. In the cases of interest here, a direct correlation with enthalpy of activation i s expected because of the detailed s i m i l a r i t y i n the competitive reactions examined. Furthermore, this correlation should extend to the enthalpies of reaction provided reverse activation energies are negligible or similar. Thus, the r e l a t i v e metastable ion abundance should correlate with the relative product enthalpies. Figure 9 i l l u s t r a t e s the basis of the method. We have applied (8) these concepts to the competitive metastable ion dissociations of proton bound dimers, Β ΗΒ . These reactions Ί

+

B HB

2

B]HB

2

1

+

+ Β Η +B χ

+

+ Bi + B H 2

2

+

?

(2) (3)

70

HIGH PERFORMANCE MASS SPECTROMETRY

Analytical Chemistry

70 71 72 Mass

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

Figure 8. Partial MIKE spectra (no collision gas) of the molecular ions of unlabeled and labeled ketones mass selected from a partially incoroprated sample

Figure 9. (a) Rate constant vs. internal energy curves for competitive reactions of different activation energy but simihr frequency factor. When the internal energy is high enough for the higher energy process to occur fast enough to be observed as a metastable ion, the lower energy process is going faster still, and it dominates the spectrum at all internal energies; (b) the same situation illustrated by the very different state densities for the competitive fragmentations at an internal energy in excess of both activation energies.

4. COOKS

71

Kinetic Energy Spectrometry

are p a r t i c u l a r l y appropriate for t h i s treatment since they are simple bond cleavages, presumably involving steeply r i s i n g rate constant vs internal energy curves. Furthermore reverse activation energies are l i k e l y to be small. The difference i n product enthalpies for the competitive reactions (2) and (3) i s +

Δ(ΔΗ ) = AHfC^H*) + ΔΗ (Β ) - AH (B ) - ΔΗ (Β Η ) £

£

2

£

]L

£

2

(4)

but the proton a f f i n i t y PA i s the negative of the enthalpy change on addition of a proton, +

+

ΡΑ(Β ) = ΔΗ (Β ) + ΔΗ (Η ) - ΔΗ (Β^Η ) Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch004

χ

£

1

£

£

(5)

hence substituting i n (4) we have, Δ (AH ) - ΡΑ(Β ) - PA(B ) £

χ

(6)

2

Thus, the more abundant metastable peak for reactions (2) and (3) should indicate the base of greater proton a f f i n i t y . The order­ ing of proton a f f i n i t i e s should be independent of the internal energy d i s t r i b u t i o n of the reactant ion B^HB^"" although the meta­ stable ion abundance r a t i o w i l l depend on t h i s factor. We leave for elsewhere an attempt to develop t h i s quantitative r e l a t i o n ­ ship further; here we present results on the r e l a t i v e ordering of proton a f f i n i t i e s by t h i s new method. A mixture of 3-aminopentane and 2-aminobutane introduced into the CI source v i a a methane gas stream gave the mass spectrum shown as Figure 10. The ions at m/e 88 and 74 are due to pro­ tonated amines, those at 114, 128 and 142 appear to be immonium ions formed by transamination of the (M - H ) ions and those at 147, 161 and 175 are the proton bound dimers. The MIKE spectrum of the asymmetrical dimer m/e 161 i s shown i n Figure 11. As expected from data taken by equilibrium studies (9), 3-amino­ pentane has the larger proton a f f i n i t y (the difference i s 1 kcal mole-1). The method i s expected to be sensitive to small differences i n proton a f f i n i t y as the schematic i l l u s t r a t i o n of Figure 9 shows. This s e n s i t i v i t y i s i l l u s t r a t e d by a comparison of s-butylamine and pyridine which have been found by i c r (10) t o have v i r t u a l l y identical proton a f f i n i t i e s . Figure 12 i s a slow scan through part of the MIKE spectrum of the proton bound dimer of these two bases. Protonated pyridine i s formed i n higher abundance suggesting that pyridine has the greater proton a f f i n i t y , probably by 0.1 to 0.2 kcal mole-1. These results were obtained without correcting for c o l l e c t i o n and detector e f f i c i e n c i e s . This can be done by studying labeled ions i f isotope effects can be ignored. The dimer of pyridine and dr-pyridine provides a case i n point, results for i t being shown i n Figure 13. This type of experiment can also be designed for the determination of isotope effects on proton a f f i n i t i e s . 1

+

161

2

147

4

142

128 114

JUL

88

74

>

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.

NiBBERiNG

91

Field Ionization Kinetic Studies

" 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

6

5.

NiBBERiNG

Field Ionization Kinetic Studies

93

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.

94

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

3.

HIGH PERFORMANCE MASS

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

98

HIGH PERFORMANCE MASS SPECTROMETRY

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 .

6. HADDON

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

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

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

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3.6 3.4 -

y/ 293 299 / #

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#

3.3 -

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/

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0.8

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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

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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

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

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

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

HADDON

Organic Trace Analysis

200

240

210

320

360

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M/E

200

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M/E American Chemical Society

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

108

HIGH PERFORMANCE MASS SPECTROMETRY

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

(discard)

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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Organic Trace Analysis

, 104 MV

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

2800 MV

, 171 MV

65 MV

U 649 MV.

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5 6 1

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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

t

HIGH PERFORMANCE MASS SPECTROMETRY

512 MV

217 MV

157 MV

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

M,

- 5 MV

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299

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MASS Association of Official Analytical Chemists, Inc.

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

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

6. HADDON

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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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HIGH PERFORMANCE MASS SPECTROMETRY

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t

100 T

TIME, SEC. Association of Official Analytical Chemists, Inc.

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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American Chemical Society

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90

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

loi

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American Chemical Society

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

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%

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

R T

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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.

118

3. 4. 5. 6. 7. 8. Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch006

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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122

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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 ) .

124

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

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

7.

KIMBLE

Gas Chromatography/High R

125

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

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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

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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

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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.

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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

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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 (<10 ppm) c a n be made u s i n g GC-MS con­ d i t i o n s on q u a d r u p o l e s p e c t r o m e t e r s o p e r a t i n g i n t h e pulsed p o s i t i v e negative i o n c o n f i g u r a t i o n (10). Comparison o f E I and CI Methods: In order t o f u l l y a p p r e c i a t e the l i m i t a t i o n s o f EI method and the p o t e n t i a l o f CIMS b o t h i n a n a l y t i c a l c h e m i s t r y and i n t h e s t u d y o f fundamental p r o c e s s e s i n gas phase, a c o m p a r i s o n o f EI and CIMS i s g i v e n below. Under E I c o n d i t i o n s sample m o l e c u l e s a r e p l a c e d i n the i o n s o u r c e under h i g h vacuum ( 1 0 " t o 10 t o r r ) and a r e i o n i z e d by impact o f an e n e r g e t i c (>50eV) e l e c t r o n beam. S i n c e a 50eV e l e c t r o n t r a v e l s w i t h a v e l o c i t y o f 4.2 χ 10 cm/sec, i t t r a n s v e r s e s a m o l e c u l a r d i a m e t e r i n c a . 2.4 χ 1 0 " s e c . I o n i z a t i o n of the sample molecuTe o c c u r s on t h i s time s c a l e . S i n c e t h e f a s t e s t m o l e c u l a r v i b r a t i o n , a C-H s t r e t c h i n g v i b r a ­ t i o n , has a p e r i o d o f about 10~ s e c , a l l atoms c a n be c o n s i d e r e d t o be e f f e c t i v e l y a t r e s t d u r i n g t h i s p e r i o d (11). EI i o n i z a t i o n t h e r e f o r e i n v o l v e s e l e c t r o n i c e x c i t a t i o n by Frank-Condon type o f p r o c e s s . During 5

8

1 6

lk

6

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152

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PERFORMANCE

MASS

SPECTROMETRY

t h i s i o n i z a t i o n p r o c e s s , the i o n produced a c q u i r e s energy i n t h e range o f l - 8 e V , and, t h u s , f r e q u e n t l y undergoes e x t e n s i v e f r a g m e n t a t i o n . S i n c e a h i g h vacuum i s employed under EI c o n d i t i o n s , i o n - m o l e c u l e c o l l i s i o n s a r e e f f e c t i v e l y p r e c l u d e d . The i n t e r n a l energy of the i o n s , t h e r e f o r e , remains i n n o n - e q u i l i b r i u m d i s t r i b u t i o n from the i n s t a n t o f i o n i z a t i o n . Formation of fragment i o n s from the e x c i t e d p a r e n t i o n , i s exp l a i n e d by the Q u a s i - e q u i l i b r i u m t h e o r y (QET) (12) w h i c h assumes t h a t i n i t i a l e x c i t a t i o n energy i s r a n domized t h r o u g h o u t the m o l e c u l e a t a r a t e w h i c h i s f a s t r e l a t i v e t o the r a t e o f bond d i s s o c i a t i o n . QET p r e d i c t s t h a t fragment i o n s w i l l be formed by a s e r i e s o f competing c o n s e c u t i v 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 reactions. I t i s i m p o r t a n t t o r e a l i z e h e r e t h a t the EI p r o c e s s s t a n d s i n c o n t r a s t t o the u s u a l k i n e t i c s i t u a t i o n e n c o u n t e r e d b o t h i n s o l u t i o n and under CI conditions. I n t h e s e s i t u a t i o n s , m o l e c u l e s are c o n t i n u a l l y e n e r g i z e d and d e e n e r g i z e d by c o l l i s i o n s , and a M a x w e l l - B o l t z m a n type d i s t r i b u t i o n o f e n e r g i e s i s e i t h e r approached o r r e a l i z e d . In CIMS, a s e t o f r e a g e n t i o n s a r e f i r s t g e n e r a t e d by bombarding a s u i t a b l e r e a g e n t gas a t p r e s s u r e s between 0.5 and 1 t o r r , w i t h h i g h energy e l e c t r o n s (100 to 500eV). Sample m o l e c u l e s a r e i n t r o d u c e d i n the u s u a l manner but a t a cone, below 0.1% t h a t o f the r e a g e n t gas. Under t h e s e c o n d i t i o n s o n l y the r e a g e n t gas i s i o n i z e d by EI and sample m o l e c u l e s a r e i o n i z e d o n l y by i o n - m o l e c u l e r e a c t i o n s . In g e n e r a l , gas-phase i o n m o l e c u l e r e a c t i o n s a r e a p p r e c i a b l y f a s t e r t h a n r e a c t i o n s between n e u t r a l s p e c i e s . R e a c t i o n s p r o c e e d i n g a t or near d i f f u s i o n c o n t r o l l e d r a t e s a r e not uncommon under c h e m i c a l i o n i z a t i o n c o n d i t i o n s . An e x p l a n a t i o n f o r t h e s e l a r g e c r o s s - s e c t i o n s f o r r e a c t i o n can be found i n the t r e a t ment by L a n g e v i n ( 1 3 ) . Long range a t t r a c t i v e f o r c e s , w h i c h r e s u l t from p o l a r i z a t i o n o f the n e u t r a l m o l e c u l e by t h e a p p r o a c h i n g i o n , a r e produced. Depending upon t h e p r o x i m i t y and the r e l a t i v e v e l o c i t y o f the two s p e c i e s , t h e s e a t t r a c t i v e f o r c e s may cause the d i s t a n c e of c l o s e s t approach t o be s u f f i c i e n t l y s m a l l f o r a r e a c t i o n t o o c c u r . F u r t h e r m o r e , i f the i o n approaches the t a r g e t m o l e c u l e t o w i t h i n a c e r t a i n range o f d i s t a n c e s , the t r a j e c t o r y t a k e s on a s p i r a l o r o r b i t i n g n a t u r e around the m o l e c u l e . The o r b i t i n g b e h a v i o r o f the two s p e c i e s i n c r e a s e s the d u r a t i o n o f the i n t e r a c t i o n , i^.e. the l i f e t i m e o f the i o n - m o l e c u l e complex, p e r m i t s the i o n and m o l e c u l e t o p e r t u r b each o t h e r s e l e c t r o n i c s t r u c t u r e , and t o sample s e v e r a l p o s s i b l e a c t i v a t e d complexes. I o n i z a t i o n o f sample does not 1

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8.

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Positive and Negative Ion Chemical Ionization

A N D SETHI

153

o c c u r by a Frank-Condon p r o c e s s , s i n c e t h e l i f e t i m e o f the i o n - m o l e c u l e complex c a n be l o n g compared w i t h v i b r a t i o n a l time p e r i o d s . Under CI c o n d i t i o n s t h e amount o f energy i m p a r t e d to t h e sample i o n i s dependent i n p a r t on t h e e x o t h e r m i c i t y o f t h e i o n - m o l e c u l e r e a c t i o n employed. I n CI w i t h methane and i s o - b u t a n e , t h e most commonly used r e a g e n t g a s e s , t h e i o n i z a t i o n o c c u r s by e i t h e r p r o t o n t r a n s f e r t o , o r h y d r i d e a b s t r a c t i o n from, t h e sample. S i n c e t h e e x o t h e r m i c i t y o f gas-phase p r o t o n t r a n s f e r and h y d r i d e a b s t r a c t i o n r e a c t i o n s i s u s u a l l y low ( 0 3eV), t h e r e s u l t i n g e v e n - e l e c t r o n i o n s a r e r e l a t i v e l y s t a b l e towards f u r t h e r f r a g m e n t a t i o n . Those i o n s t h a t do fragment g e n e r a l l y do so by pathways d i f f e r e n t from those a v a i l a b l e t o t h e odd e l e c t r o n s p e c i e s g e n e r a t e d i n i t i a l l y under E I c o n d i t i o n s . A c c o r d i n g l y , t h e s t r u c ­ t u r a l i n f o r m a t i o n o b t a i n e d from E I and CI s p e c t r a o f the same sample i s u s u a l l y complementary. The op­ p o r t u n i t y f o r sample i o n t o undergo 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 under CI c o n d i t i o n s a l s o c o n t r i b u t e s t o t h e reduced fragmen­ t a t i o n o b s e r v e d i n t h e CI mode. A lower l i m i t f o r t h e number o f c o l l i s i o n s e x p e r i e n c e d by an i o n i n t h e CI s o u r c e c a n be e s t i m a t e d , by u s i n g t h e e x p r e s s i o n Z

c

= K.N

where Ζ i s t h e number o f c o l l i s i o n s , Κ i s t h e r a t e c o n s t a n t f o r t h e r e a c t i o n and Ν i s t h e number d e n s i t y of gas m o l e c u l e s . T y p i c a l values f o r Κ * 10~ cm . m o l e c u l e " • s e c " and Ν = 2 χ 1 0 (150°C, 1 t o r r ) y i e l d a v a l u e o f 2 χ 1 0 c o l l i s i o n s / s e c o r *1 c o l l i s i o n e v e r y 1 0 ~ s e c . (3b) The p r o t o n a f f i n i t y (P.A.) o f a m o l e c u l e i s de­ f i n e d as t h e heat l i b e r a t e d on p r o t o n a t i o n . The h i g h e r the P.A. o f a r e a g e n t m o l e c u l e , more s t a b l e i s i t s p r o ­ t o n a t e d form ( r e a g e n t i o n ) . The e x o t h e r m i c i t y o f p r o ­ t o n t r a n s f e r w i l l , o f c o u r s e , depend b o t h on t h e a c i d i ­ t y o f t h e r e a g e n t m o l e c u l e and t h e b a s i c i t y o f t h e sample m o l e c u l e . F o r a g i v e n sample, t h e p r o t o n a f ­ f i n i t y v a l u e s g i v e n below, 9

1

1

3

1 6

7

7

H

+ H

2

+

CHi* + H NH

3

+ H

+ HÎ +

+

+

ΔΗ = -101 K c a l / m o l e ; Ρ.Α.(Ηβ) - 101 CHt -> 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

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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 <

4b CI(NO) MW 68 NO

116 M-243Q

J 50| ω ^ 7lj

87j 20 m/e 40

100 4d

CJ(NO) MW84

REL . ABUND. a ι

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

100-

93

100

Û

ζ S

0 1

σ

4e CI (NO) MWI40

I39| 170 3-DECENE M~H M«NO|

UJ 0

40

m/e

80

30

"V*

140

I8Û

Figure 4. CI (NO) mass spectra of: (a) 1-pentanol, (b) 2-pentanol, (c) 2methyl-2-butanol, (d) cyclohexane, (e) 3-decene. The intensity of reagent ions is 50 to 100 times greater than as shown.

160

HIGH

NEGATIVE ION (NÏCÎMS)

PERFORMANCE

MASS

SPECTROMETRY

CHEMICAL IONIZATION MASS SPECTROMETRY

N e g a t i v e i o n s can be formed i n the gas phase by the f o l l o w i n g t h r e e mechanisms, depending upon the energy i n v o l v e d (22). AB + e(:0eV) AB + e(0-15eV)

*AB•A+B* B~+A"

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

AB + e(>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 <

1

46

50

197

31

+ I,2 3 2 Z

A

l,2

259

41

Z

I

H

I,2 2

PhCH=CHCOCH MW 146 M-l 145 3

I 0 0 J CH3O-

188 17 2 9

d

50*

1*-

ο:

100 •

CE

Kl

MW 2 2 6

J ce 501

tu

5 0 , 17 29

ω <

ι ι d

lu

50.

I47| M+|*

PERMETHYLATED TETRAPEPTIDE

H

1,2,3

MW 5 8 0

323>

'46_

N0

•385

2

31 CH 0"" 3

2171

288

433 A 1.2,3

M-l 579.

100. Analytical Chemistry

Figure 6. Pulsed positive negative ion CI mass spectra: (a) électron capture-impact type spectrum of amytal; (b) Bronsted acid-Brônsted base spectrum of traxis-4-phenyl3-buten-2-one; (c) Bronsted acid-Brônsted base electron capture spectrum of the Nacetyl-per-methylated-methyl ester of Met-Gly-Met-Met. The intensity of reagent ions is 50 to 100 times greater than as shown.

HIGH

164

PERFORMANCE

MASS

SPECTROMETRY

B r o n s t e d A c i d - B r o n s t e d Base Type S p e c t r a : S i m u l t a n e o u s p r o d u c t i o n o f B r o n s t e d a c i d and B r o n s t e d base s p e c t r a i s a c c o m p l i s h e d by u s i n g a r e agent gas composed o f methane and m e t h y l n i t r i t e . The q u a n t i t y o f m e t h y l n i t r i t e employed i s i n s u f f i c i e n t t o a l t e r t h e p o p u l a t i o n o f methane r e a g e n t i o n s . Accordi n g l y , a c o n v e n t i o n a l methane CI spectrum o f t h e sample i s o b t a i n e d on t h e p o s i t i v e i o n t r a c e . In the negative i o n mode m e t h y l n i t r i t e i s e f f i c i e n t l y c o n v e r t e d t o CH 0 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 r e a c t i o n shown below. 3

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

CH3ON0 — -

K;H O" + NO 3

Methoxide t h e n f u n c t i o n s as a s t r o n g B r o n s t e d base and a b s t r a c t s p r o t o n s from o r g a n i c m o l e c u l e s t o produce (M-l) i o n s . I n most cases t h e excess energy l i b e r a t e d i n t h e p r o t o n a b s t r a c t i o n r e a c t i o n remains i n t h e new bond t h a t i s formed (CH OH). C o n s e q u e n t l y , t h e sample ( M - l ) " i o n seldom p o s s e s s e s enough energy t o undergo e x t e n s i v e f r a g m e n t a t i o n . Ions c h a r a c t e r i s t i c o f sample m o l e c u l a r w e i g h t a r e almost always seen u s i n g t h i s methodology. The s p e c t r a o f trans-4-pheny1-3-buten-2one ( F i g u r e 6b) i l l u s t r a t e s t h e u t i l i t y o f methanem e t h y l n i t r i t e m i x t u r e as r e a g e n t - g a s under PPINICI conditions. I t s h o u l d be mentioned t h a t a wide v a r i e t y o f a n i o n i c n u c l e o p h i l e s and b a s e s , l e s s b a s i c t h a n CH 0 , can be g e n e r a t e d f o r s t u d y as n e g a t i v e i o n r e a g e n t s by s i m p l y a d d i n g a t h i r d component t o t h e methane-methyln i t r i t e m i x t u r e employed above. When t h e t h i r d compound r e a c h e s a r e l a t i v e c o n c e n t r a t i o n o f about 5% o f the t o t a l m i x t u r e , a l l o f t h e CH 0~ formed by e l e c t r o n c a p t u r e i s consumed by i o n m o l e c u l e r e a c t i o n s i n v o l v i n g p r o t o n t r a n s f e r t o CH 0~ from t h e t h i r d component. I n t h i s way ( M - l ) " i o n s from c y c l o p e n t a d i e n e , a c e t o n e , mercaptans, n i t r i l e s , e t c . c a n be g e n e r a t e d and employed as n e g a t i v e i o n r e a g e n t s . 3

3

3

3

E l e c t r o n Capture - B r o n s t e d A c i d - B r o n s t e d Base S p e c t r a : I f a m i x t u r e o f methane-methyl n i t r i t e i s employed as t h e CI r e a g e n t gas and t h e q u a n t i t y o f m e t h y l n i t r i t e i s i n s u f f i c i e n t t o consume t h e a v a i l a b l 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 , t h e r e a c t a n t s p e c i e s genera t e d i n t h e i o n s o u r c e c o n s i s t o f C H , CH 0 and t h e r m a l e l e c t r o n s . U s i n g t h e PPINICI t e c h n i q u e t h e r e a g e n t c o m b i n a t i o n produces s i m u l t a n e o u s l y B r o n s t e d a c i d CI s p e c t r a on t h e p o s i t i v e i o n t r a c e and a m i x t u r e +

5

3

8.

HUNT

Positive and Negative Ion Chemical Ionization

A N D SETHI

165

o f B r o n s t e d base and e l e c t r o n c a p t u r e CI s p e c t r a on t h e negative i o n trace. This combination of reagents i s p a r t i c u l a r l y u s e f u l when t h e problem a t hand r e q u i r e s t h e d e t e r m i n a t i o n o f b o t h sample m o l e c u l a r w e i g h t and d e t a i l e d s t r u c t u r a l i n f o r m a t i o n . Sequencing o f p o l y p e p t i d e s by MS i s a good example o f such a problem. F o r a der i v a t i z e d p o l y p e p t i d e , t h e PPINICI [CHi*-CH ONO ( t r a c e ) ] spectrum shows an ( M - l ) " i o n d e r i v e d from r e a c t i o n o f the sample w i t h CH 0", t h e p o s i t i v e i o n B r o n s t e d a c i d spectrum p r o v i d e s b o t h t h e N- and t h e C - t e r m i n a l s e quence i o n s , and t h 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 spectrum shows i o n s w h i c h o c c u r a t m/e v a l u e s 29 u n i t s (-N»CH -permethylation) h i g h e r t h a n t h e N - t e r m i n a l a c y l sequence i o n on t h e p o s i t i v e i o n t r a c e . Thus, Nt e r m i n a l sequence i o n s c a n be e a s i l y r e c o g n i z e d by t h e appearance o f d o u b l e t s s e p a r a t e d by 29 mass u n i t s on p o s i t i v e and n e g a t i v e i o n t r a c e s . Shown i n F i g u r e 6c i s t h e PPINICI (CH^-CH ONO) spectrum o f N - a c e t y l p e r m e t h y l a t e d - m e t h y l e s t e r o f Met-Gly-Met-Met. 3

3

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

3

3

Oxygen as a PPINICI

Reagent:

When oxygen a t 1 t o r r c o n t a i n i n g 10% hydrogen i s employed as t h e CI r e a g e n t gas, 0 , O 2 and a popul a t i o n o f t h e r m a l e l e c t r o n s f u n c t i o n as t h e r e a c t a n t s i n t h e p o s i t i v e and n e g a t i v e i o n modes, r e s p e c t i v e l y . +

1

2

0

2

0

2

+

—

K)

+ e"

—

K ) - 0-

2

2

0- + H — * H 0 + e" 2

2

We f i n d t h a t t h i s r e a g e n t gas m i x t u r e i s i d e a l l y s u i t e d f o r the a n a l y s i s o f t e t r a c h l o r o d i b e n z o d i o x i n (TCDD), p o l y a r o m a t i c h y d r o c a r b o n s , and a l c o h o l s (6). P o s i t i v e i o n CI ( 0 ) s p e c t r a o f a r o m a t i c m o l e c u l e s u s u a l l y consist of a s i n g l e i o n corresponding to M . T h i s i o n i s formed by e l e c t r o n t r a n s f e r from t h e sample t o 0 +. P o s i t i v e i o n C I ( 0 ) s p e c t r a a r e , t h e r e f o r e , analogous t o low v o l t a g e EI s p e c t r a e x c e p t t h a t under CI c o n d i t i o n s t h e r e i s no l o s s i n sample s e n s i t i v i t y . Under low v o l t a g e EI c o n d i t i o n s sample s e n s i t i v i t y may drop by 1 o r 2 o r d e r s o f magnitude. I n t h e n e g a t i v e i o n mode, p o l y a r o m a t i c h y d r o c a r bons e i t h e r r e a c t w i t h 0 o r c a p t u r e an e l e c t r o n and t h e n s u f f e r r e a c t i o n w i t h a d i r a d i c a l oxygen m o l e c u l e . Depending on t h e s t r u c t u r e o f t h e m o l e c u l e t h e r e s u l t i n g i o n s may c o r r e s p o n d t o M-, ( M - l ) , (M+14) , 2

+

2

2

1

2

166

HIGH

PERFORMANCE

MASS

SPECTROMETRY

(M+15)", (M+31)", o r (M+32)". PPINICI w i t h oxygen as t h e r e a g e n t gas i s p a r t i c u l a r l y s u i t e d f o r d i f f e r e n t i a t i o n o f i s o m e r i c p o l y a r o m a t i c s such as the C ^ H i 2 p a i r , b e n z o ( g h i ) p e r y l e n e and i n d e n o ( 1 , 2 , 3 - c d ) p y r e n e ( F i g u r e 7 ) , and m o l e c u l e s such as RC and RSHi* which have t h e same m o l e c u l a r w e i g h t but d i f f e r e n t e l e m e n t a l compositions. I n the case o f the C * H i p a i r , b o t h isomers e x h i b i t a s i n g l e i o n c o r r e s p o n d i n g to M i n the p o s i t i v e i o n mode. On the n e g a t i v e i o n t r a c e b e n z o p e r y l e n e shows i o n s c o r r e s p o n d i n g t o M and (M+15)" i n a 1/1 r a t i o . The l a t t e r s p e c i e s i s p r o b a b l y a p h e n o l i c a n i o n r e s u l t i n g from the r e a c t i o n o f M w i t h oxygen f o l l o w e d by the e l i m i n a t i o n o f OH*. The n e g a t i v e i o n spectrum o f indenopyrene a l s o e x h i b i t s the same two i o n s , M and (M+15)", but i n a r a t i o o f 10/1. F u r t h e r , the r a t i o o f the t o t a l p o s i t i v e t o t o t a l n e g a t i v e sample i o n c u r r e n t i s 0.5 f o r the benzoperyl e n e and 22 f o r the i n d e n o p y r e n e . The p r e s e n c e o f a five-membered r i n g i n the indenopyrene f a c i l i t a t e s f o r m a t i o n o f a s t a b l e n e g a t i v e i o n and easy i d e n t i f i c a t i o n o f t h i s compound even i n the p r e s e n c e o f the o t h e r C i»Hi2 i s o m e r s . A l t h o u g h RSHt* and RC compounds have the same mol. wt. and e x h i b i t a s i n g l e i o n a t the same m/e v a l u e on the p o s i t i v e i o n t r a c e , t h e s e two t y p e s oT m o l e c u l e s a r e e a s i l y d i f f e r e n t i a t e d i n the n e g a t i v e i o n mode. S u l f u r c o n t a i n i n g m o l e c u l e s undergo attachment o f oxygen t o M- and form (M+32)- i o n s whereas p o l y a r o m a t i c s c o n t a i n i n g o n l y c a r b o n and hydrogen form M and (M+15)"ions ( p h e n o l i c a n i o n s ) under C I ( 0 ) c o n d i t i o n s . I f b o t h compound t y p e s a r e p r e s e n t i n the same sample m i x t u r e , i o n s from each appear as an u n r e s o l v e d d o u b l e t a t M on the p o s i t i v e i o n t r a c e and as a d o u b l e t separ a t e d by 17 mass u n i t s (M+15 and M+32) on the n e g a t i v e ion trace. N I C I ( 0 ) i s a l s o o f v a l u e as a t e c h n i q u e f o r analyzing alcohols. Molecules containing alcohol groups form a hydrogen bond t o O 2 t o produce ( M + O 2 ) i o n and a l s o r e a c t w i t h 0 t o g i v e (M-17)" i o n s . 2

3

2

2

+

1

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

1

1

2

3

1

2

+

2

1

1

1

A n a l y s i s o f N o n v o l a t i l e Compounds: Most mass s p e c t r o m e t r i c t e c h n i q u e s r e q u i r e the sample m o l e c u l e s t o be i n the gaseous s t a t e p r i o r t o i o n i z a t i o n and t h u s a r e s e v e r e l y l i m i t e d i n t h e i r app l i c a t i o n t o the a n a l y s e s o f s a l t s and t h e r m a l l y l a b i l e compounds. Thermal energy i s the most common f o r c e used t o break the i n t e r m o l e c u l a r bonds and s u r f a c e m o l e c u l e bonds and t o f a c i l i t a t e i n t r o d u c t i o n o f sample m o l e c u l e s i n t o the gaseous s t a t e . Input o f energy i n t o

8.

HUNT

Positive and Negative Ion Chemical Ionization

A N D SETHI

100-

PPNICI(0 /H ) 2

2

Ν/ Ρ =

POS

22.2

276 0

50H Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch008

167

02

2

ΧΙΟ Ν/Ρ-0.5

ο ζ < ο ζ

3 ω < ,-

276 291

oc

(Μ + 15)50Η

NEG benzo(ghi) perylene I00

mw276

indeno(l,2,3-cd)pyrene mw 2 7 6

J

Figure 7. PPNICI (0 /H ) mass spectra of two isomers of C H : (a) benzo(ghi) per­ ylene; (b) indeno(l,2,3-cd-)pyrene. The intensity of reagent ions is 50 to 100 times greater than as shown. 2

2

22

J2

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

168

HIGH

PERFORMANCE

MASS

SPECTROMETRY

v i b r a t i o n a l l e v e l s o f the m o l e c u l e can e i t h e r r e s u l t i n d i s s o c i a t i o n o f i n t r a - m o l e c u l a r , i n t e r - m o l e c u l a r , or s u r f a c e - m o l e c u l e bonds. For most 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 , the former p r o c e s s becomes dominant, and e x t e n s i v e t h e r m a l d e g r a d a t i o n o f the sample o c c u r s d u r i n g the v a p o u r i z a t i o n s t e p . A v a r i e t y o f t e c h n i q u e s have been d e v e l o p e d t o overcome t h i s p r o b l e m . D e r i v a t i z a t i o n o f p o l a r group i n the m o l e c u l e t o e l i m i n a t e i n t e r m o l e c u l a r hydrogen b o n d i n g , i s perhaps the most common and s u c c e s s f u l method. U n f o r t u n a t e l y , d e r i v a t i z a t i o n adds an unwanted e x t r a s t e p i n sample a n a l y s i s . A t t e m p t s t o reduce s u r f a c e - m o l e c u l a r i n t e r a c t i o n s by v o l a t i l i z i n g the sample from r e l a t i v e l y i n e r t m a t e r i a l such as T e f l o n a l s o have shown some p r o m i s e ( 2 4 ) . Two t e c h n i q u e s , w h i c h i n c r e a s e the r a t e o f sample v a p o r i z a t i o n r e l a t i v e t o the r a t e o f p y r o l y s i s by p l a c i n g l a r g e amounts o f energy i n t o the m o l e c u l e on a t i m e s c a l e t h a t i s f a s t compared w i t h the v i b r a t i o n a l time p e r i o d ( 1 0 ~ to 1 0 " s e c ) , have a l s o shown considerable potential. The more p r o m i s i n g o f t h e s e t e c h n i q u e s , plasma d e s o r p t i o n mass s p e c t r o m e t r y ( 2 5 ) , i n v o l v e s impact o f h i g h energy (100 MeV) fission p r o d u c t s from C f onto the back s i d e o f m e t a l f o i l c o a t e d w i t h a m o l e c u l a r l a y e r o f sample. S i m u l t a n e o u s d e s o r p t i o n and i o n i z a t i o n o f sample m o l e c u l e s r e s u l t s . Many p o l a r and v e r y h i g h m o l e c u l a r w e i g h t compounds (e.£., V i t a m i n B ) a f f o r d i o n s c h a r a c t e r i s t i c o f sample m o l e c u l a r w e i g h t when a n a l y z e d by t h i s new methodology. The second h i g h energy t e c h n i q u e employs a p u l s e d l a s e r f o r b o t h d e s o r p t i o n and i o n i z a t i o n ( 2 6 ) . T h i s method has not been a p p l i e d t o the a n a l y s i s o f t h e r m a l l y l a b i l e m o l e c u l e s but has shown p r o m i s e f o r a n a l y s i s of s a l t s . The mass s p e c t r o m e t e r i c t e c h n i q u e w h i c h i s now most commonly used f o r the a n a l y s i s o f n o n - v o l a t i l e samples i s f i e l d d e s o r p t i o n ( 2 7 ) . T h i s t e c h n i q u e was i n t r o d u c e d by Beckey i n 1969, and employs a v e r y s t r o n g e l e c t r o s t a t i c f i e l d (1-4V/A ) t o i o n i z e m o l e c u l e s a b s o r b e d on s p e c i a l l y p r e p a r e d s u r f a c e , and a comb i n a t i o n o f t h i s f i e l d and t h e r m a l energy to desorb the i o n i z e d sample m o l e c u l e s . The a c t i v a t e d s u r f a c e cons i s t s o f dense, h i g h l y branched c a r b o n m i c r o n e e d l e s (30 \xm l o n g ) grown on 10 ]im t u n g s t e n w i r e . A c c o r d i n g t o M u l l e r (2*8), the s t r o n g f i e l d l o w e r s the b a r r i e r f o r e l e c t r o n t u n n e l l i n g from m o l e c u l e t o the w i r e s u r f a c e and i n c r e a s e s the r a t e o f i o n i c des o r p t i o n by l o w e r i n g the r e q u i r e d h e a t - o f - d e s o r p t i o n . 1 2

1 3

2 5 2

X 2

0

8. The

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Positive and Negative Ion Chemical Ionization

169

r a t e c o n s t a n t Κ f o r i o n i c d e s o r p t i o n i s g i v e n as Κ = ν · exp (-Q/kt) 1 3

1

where ν i s t h e v i b r a t i o n a l f r e q u e n c y ( 1 0 s e c " ) * and Q i s t h e heat o f i o n i c d e s o r p t i o n , r e d u c e d from t h e thermodynamic v a l u e Q° Q° - Ha + IP - φ 3

1

by a S c h o t t k y t e r m , 3. δ η ^ /

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

Q = Q° - 3 . 8 n

3 / 2

F

2

1 / 2

where Ha = Heat o f d e s o r p t i o n o f n e u t r a l

molecule

IP = I o n i z a t i o n p o t e n t i a l φ

= Work f u n c t i o n o f t h e s u r f a c e

F

= A p p l i e d f i e l d i n V/A°

η

= Charge o f t h e e v a p o r a t i n g i o n

The e f f e c t o f t h e f i e l d c a n be more c l e a r l y seen by i n s p e c t i n g t h e changes i n t h e energy l e v e l s o f t h e s u r f a c e - m o l e c u l e i n t e r a c t i o n on a p p l i c a t i o n o f t h e field. F i g u r e 8 shows such energy l e v e l s when ΙΡ-φ i s a large p o s i t i v e value. This i s u s u a l l y the s i t u a t i o n f o r o r g a n i c m o l e c u l e s a d s o r b e d on t h e s u r f a c e . F o r t h e s e m o l e c u l e s t h e IP i s between 9-12 ev and φ v a r i e s between 4-6 ev. F i g u r e 8b shows t h a t i n t h e p r e s e n c e o f a s t r o n g f i e l d t h e i o n i c c u r v e " c r o s s e s t h e atomic c u r v e a t Xc. ( I n f a c t t h e c u r v e s r e p e l r a t h e r than c r o s s because t h e r e a r e no symmetry o r s p i n d i f f e r e n c e s w h i c h p e r m i t degeneracy i n t h e two s t a t e s . ) On t h e r m a l e x c i t a t i o n t h e d e s o r p t i o n from t h e atomic ground s t a t e w i l l r e s u l t e i t h e r i n t h e e m i s s i o n o f an i o n by a d i a b a t i c t r a n s i t i o n , . i . e . , f i e l d i o n i z a t i o n a t o r beyond Xc, o r by t h e e m i s s i o n o f a n e u t r a l by n o n - a d i a b a t i c t r a n s i t i o n a l o n g t h e atomic c u r v e . A t v e r y h i g h f i e l d s ( F i g u r e 8 c ) , t h e i n t e r s e c t i o n p o i n t , X c , comes so c l o s e to t h e s u r f a c e t h a t Q i s g r e a t l y r e d u c e d . The r e ­ s u l t i n g s e p a r a t i o n o f t h e two s t a t e s becomes so l a r g e that d e s o r p t i o n o f the molecule can only occur i n i o n i c form. A l s o n o t e t h a t t h e p r o b a b i l i t y o f i o n i c de­ s o r p t i o n i n c r e a s e s as t h e p o l a r i z i b i l i t y (a) o f t h e m o l e c u l e i n c r e a s e s , due t o t h e l o w e r i n g o f energy o f i o n i c d e s o r p t i o n by 1/2 otF . T h i s l a t t e r term c o r ­ responds t o t h e p o l a r i z a t i o n energy o f t h e m o l e c u l e . 1 1

2

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PERFORMANCE

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

170

CO

X(Al

Journal of Chemical Physics

Figure 8. Potential energy diagrams for the field desorption process in the situation where: (a) the ionization potential (IP) is large when compared with the work func­ tion (φ); (b) in afieldof intermediate strength (200 MV/cm), field ionization can occur beyond critical distance Xc; (c) in the presence of highfield(400 MV/cm). Desorp­ tion of covalently bound molecules requires a reduced activation energy Q, followed byfieldionization beyond Xc.

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

8.

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A N D SETHI

Positive and Negative Ion Chemical Ionization

171

D e s p i t e t h e above t r e a t m e n t , t h e r e does n o t e x i s t a comprehensive t h e o r y w h i c h c a n a c c o u n t f o r a l l t h e r e s u l t s o b t a i n e d i n FD e x p e r i m e n t s w i t h l a r g e o r g a n i c molecules. Neither i s i t p o s s i b l e t o p i n p o i n t the magnitude o f f i e l d g e n e r a t e d near t h e s u r f a c e o f t h e e m i t t e r i n c o m m e r c i a l FD i n s t r u m e n t s . At a given applied p o t e n t i a l the strongest e l e c t r i c a l f i e l d s are g e n e r a t e d a t s u r f a c e s h a v i n g t h e s m a l l e s t r a d i i (i_.e. , sharpest p o i n t s ) . Therefore, f i e l d desorption i s thought t o o c c u r from t h e t i p o f t h e c a r b o n d e n d r i t e on the e m i t t e r , and m o l e c u l e s a r e thought t o m i g r a t e t o the d e n d r i t e t i p w i t h t h e a i d o f t h e r m a l e n e r g y , t h r o u g h some type o f f l u i d s t r u c t u r e formed on t h e surface. R e c e n t l y , H o l l a n d e_t al_. (2£) o b s e r v e d t h a t a normal f i e l d d e s o r p t i o n mass spectrum c a n be o b t a i n e d , at the usual emitter temperature, without the a p p l i c a t i o n o f h i g h (10KV) e x t e r n a l v o l t a g e ( t h e d e s o r p t i o n field). T h i s work i s r e v i e w e d i n a n o t h e r c h a p t e r i n t h i s volume. S i n c e t h e i o n a c c e l e r a t i n g v o l t a g e (3KV) was l e f t on i n t h e i r e x p e r i m e n t s , a f i e l d on t h e o r d e r o f 10 V/cm i s s t i l l p r e s e n t i n t h e v i c i n i t y o f t h e e m i t t e r . From t h e i r e x p e r i m e n t s , H o l l a n d et^ a l . , c o n c l u d e d t h a t t h e sample t r a n s p o r t and d e s o r p t i o n i s independent o f t h e a p p l i e d v o l t a g e o v e r t h e range 7KV to 12KV. To e x p l a i n t h e i r r e s u l t s , H o l l a n d e t a l . p o s t u l a t e d an i o n i z a t i o n model i n w h i c h t h e FTelcT, i f p r e s e n t , m e r e l y a c t s as a v e h i c l e t o remove i o n s once t h e y a r e formed by c h e m i c a l r e a c t i o n s i n a s e m i - f l u i d l a y e r o f sample m o l e c u l e s on t h e s u r f a c e o f t h e e m i t ters. I n o u r l a b o r a t o r y , we have r e c e n t l y employed FD e m i t t e r s as s o l i d probes under CI c o n d i t i o n s and agree w i t h H o l l a n d t h a t , f o r many m o l e c u l e s , t h e p r e s e n c e o f a h i g h e x t e r n a l f i e l d i s u n n e c e s s a r y . Our e x p e r i m e n t s a r e c o n d u c t e d on a F i n n i g a n q u a d r u p o l e mass s p e c t r o meter w i t h o u t a p p l i c a t i o n o f an e x t e r n a l f i e l d t o t h e emitter. I t i s i m p o r t a n t t o n o t e , however, t h a t o u r e x p e r i m e n t s were c o n d u c t e d under CI c o n d i t i o n s w h i l e H o l l a n d ert a l . c a r r i e d o u t t h e i r e x p e r i m e n t s under E I conditions. A l l o f o u r s t u d i e s have been p e r f o r m e d on F i n n i g a n model 3 200 o r 3300 q u a d r u p o l e i n s t r u m e n t s equipped w i t h CI i o n s o u r c e s and an INCOS Model 2300 d a t a system. Methane a t 0.5 t o r r p r e s s u r e was used as t h e r e a g e n t gas, w i t h t h e i o n s o u r c e t e m p e r a t u r e between 100-250°C. Normal f i e l d d e s o r p t i o n e m i t t e r s were u s e d . Sample p r e p a r a t i o n i n v o l v e d p l a c i n g a drop o f s o l u t i o n cont a i n i n g t h e sample i n a s u i t a b l e s o l v e n t on t h e s u r f a c e o f t h e e m i t t e r u s i n g a 10 \ii s y r i n g e . The sample was i n t r o d u c e d t o t h e i o n i z a t i o n chamber by removing t h e 3

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

172

HIGH

PERFORMANCE

MASS

SPECTROMETRY

r e p e l l e r assembly from the CI i o n s o u r c e and by p r e s s i n g t h e FD e m i t t e r s i n the h o l e thus v a c a t e d . In t h i s c o n f i g u r a t i o n the e m i t t e r w i r e i s s i t u a t e d d i r e c t l y on l i n e w i t h the e l e c t r o n e n t r a n c e h o l e , 3 mm back o f the ion-exit s l i t . The s p e c t r a were g e n e r a t e d by s i m p l y h e a t i n g the e m i t t e r w i r e r a p i d l y and s c a n n i n g the mass s p e c t r u m a t 4 sec/decade. Many s a l t s , e.g.., sodium and p o t a s s i u m b e n z o a t e s , c r e a t i n e and a r g i n i n e h y d r o c h l o r i d e s , c h o l i n e c h l o r i d e , and t h e r m a l l y l a b i l e compounds l i k e g u a n o s i n e , c y c l i c a d e n o s i n e monophosphate (C-AMP), s u g a r s , d i o x a t h a n (a p e s t i c i d e ) and a r g i n i n e - c o n t a i n i n g u n d e r v i t i z e d pept i d e s , a l l a f f o r d s p e c t r a c o n t a i n i n g ions which f a c i l i t a t e assignment o f sample m o l e c u l a r w e i g h t as w e l l as 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 . Some t y p i c a l s p e c t r a a r e shown i n F i g u r e 9 . None o f t h e s e compounds g i v e s an i o n c h a r a c t e r i s t i c o f molecu l a r w e i g h t under c o n v e n t i o n a l E I , C I , or F I ( f i e l d ionization) conditions. A t l e a s t t h r e e mechanisms f o r the o b s e r v e d desorpt i o n and i o n i z a t i o n o f samples on e m i t t e r s u r f a c e s under CI c o n d i t i o n s d e s e r v e c o n s i d e r a t i o n . Mechanism I .

Thermal d e s o r p t i o n o f sample "followed by c n e m i c a l i o n i z a t i o n "of the gaseous n e u t r a l m o l e c u l e .

As the e m i t t e r c u r r e n t ( t e m p e r a t u r e ) i s i n c r e a s e d , a p o i n t i s r e a c h e d where the c r y s t a l l a t t i c e o f the sample b r e a k s down and m i g r a t i o n o f sample m o l e c u l e s t o the t i p s o f the carbonaceous d e n d r i t e s o c c u r s i n the r e s u l t i n g s e m i - f l u i d s t a t e . D e s o r p t i o n from the e m i t t e r s u r f a c e o c c u r s a t a lower t e m p e r a t u r e t h a n t h a t r e q u i r e d f o r d e s o r p t i o n from c o n v e n t i o n a l s o l i d s p r o b e s because the s u r f a c e - s a m p l e b o n d i n g , and samplesample i n t e r a c t i o n s a r e m i n i m i z e d a t the t i p s o f the carbonaceous d e n d r i t e s . Mechanism I I .

F i e l d d e s o r p t i o n and i o n i z a t i o n m e d i a t e d by CI r e a g e n t i o n s

T h i s mechanism i s a n a l a g o u s to t h a t o p e r a t i n g under c o n v e n t i o n a l f i e l d d e s o r p t i o n c o n d i t i o n s e x c e p t the r e q u i r e d s t r o n g f i e l d i s p r o v i d e d by the i o n s i n the CI r e a g e n t gas r a t h e r t h a n by an e x t e r n a l 10KV p o t e n t i a l d i f f e r e n c e a p p l i e d t o the e m i t t e r and draw-out p l a t e . A c c o r d i n g t o Mechanism I I , sample m o l e c u l e s m i g r a t e to the t i p s o f the e m i t t e r d e n d r i t e s a t some c r i t i c a l t e m p e r a t u r e . There t h e y e x p e r i e n c e a f i e l d g e n e r a t e d by n e a r b y i o n s i n the r e a g e n t gas p l a s m a . T h i s f i e l d

8.

HUNT

1001

9α

166 I83|CKNH ) 30ma 2 8 8 3

ο

MW 4 5 2

Z,H+NH^

s

METHIONY^faGINYl^HENYLAi

0

m < tu« Τ Ι oc

1 199^

2

6

8

2B8|2*ëp 300

2D0 I0ÔT

9b

CI(NH )

29ma

3

180

400

MW504 R A F

F I N 0 S

E

HzOH

5Ό\ .198

lu

OC

(S,0HfNH?

(S 0H+NHt)

r**-

f

(M+NH*)

i l 200

100

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9e

l4e

400

500

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3 0 0 m/e

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2

6

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M A Y T A N S I N E

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

173

Positive and Negative Ion Chemical Ionization

A N D SETHI

486 . (631-AH;

0

ji. 2

. 0

0

z

400

*

692

w ii

_

,η/ ' β

600 Analytical Chemistry

Figure 9. Activated-emitter, solid-probe, NH (0.5 torr) or CH (0.5 torr) CI spectra of: (a) methionyl-arginyl-phenylafonine, (b) raffinose, (c) creatin, (d) potas­ sium benzoate, (e) maytansine—a large macrocyclic natural product with anti­ tumor properties. Ions designated by an asterisk contain a methane reagent ion, C H * or C H +, plus the sample molecule or a neutral fragment derived from the sample molecule. S

2

5

S

5

k

174

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PERFORMANCE

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l o w e r s t h e energy b a r r i e r f o r an e l e c t r o n t o t u n n e l from t h e sample i n t o t h e m e t a l and f a c i l i t a t e s i o n i z a t i o n o f t h e sample on t h e e m i t t e r s u r f a c e and d e s o r p t i o n o f t h e r e s u l t i n g i o n . In o r d e r f o r t h e i o n i z a t i o n t o o c c u r by t h i s mechanism, t h e f i e l d s g e n e r a t e d by t h e i o n i c plasma would have t o be comparable t o t h a t r e q u i r e d f o r c o n v e n t i o n a l FD e x p e r i m e n t s (ca.. IV/A°). We have a t t e m p t e d t o e s t i m a t e t h e magnitude o f t h e f i e l d i n d u c e d by an i o n i n t h e c l o s e v i c i n i t y (4-5A°) o f a m o l e c u l e a b s o r b e d on t h e s u r f a c e . F o r n o n p o l a r m o l e c u l e s , t h e l o n g range i n t e r a c t i o n p o t e n t i a l due t o the p o l a r i z a t i o n o f m o l e c u l e i s g i v e n as 2

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

V ( r ) = -âe /2y

lf

where

α = Average p o l a r i z i b i l i t y o f t h e m o l e c u l e γ = I n t e r n u c l e a r d i s t a n c e between t h e i o n and t h e m o l e c u l e e = U n i t e l e c t r i c charge For l a r g e o r g a n i c m o l e c u l e s , a t s m a l l d i s t a n c e s , t h e i n t e r a c t i o n energy c a n be between 0.1 t o 1 eV. F o r m o l e c u l e s w i t h a permanent d i p o l e moment, t h e i n t e r ­ a c t i o n i s i n c r e a s e d by up t o an o r d e r o f magnitude. These i n t e r a c t i o n s a r e e s t i m a t e d by " l o c k e d d i p o l e moment" o r ADO (average d i p o l e o r i e n t a t i o n ) t h e o r i e s (30) . Such an i n t e r a c t i o n , when i m p r e s s e d over v e r y s h o r t d i s t a n c e s (few A°) a t sharp p o i n t s , c a n g e n e r a t e strong e l e c t r i c a l f i e l d s . These f i e l d s can be e s t i ­ mated u s i n g t h e e q u a t i o n s d e v e l o p e d by E y r i n g e t a l . (31) w h i c h r e l a t e t h e p o t e n t i a l g r a d i e n t s , f i e l c T s , g e n e r a t e d a t t h e end o f a sharp m e t a l p o i n t s e p a r a t e d by a g i v e n d i s t a n c e from t h e c o u n t e r e l e c t r o d e , w i t h the a p p l i e d p o t e n t i a l and t h e c o o r d i n a t e s o f t h e m e t a l p o i n t . Under o u r c o n d i t i o n s t h e c o u n t e r e l e c t r o d e i s r e p l a c e d by i o n s . R e s u l t s o f these c a l c u l a t i o n s p o i n t t o f i e l d s on t h e o r d e r o f 0.05 t o 0.5V/A a t an i n t e r ­ n u c l e a r d i s t a n c e o f 4A°. 0

Mechanism I I I . C h e m i c a l i o n i z a t i o n o f t h e sample on t h e e m i t t e r s u r f a c e and t h e r m a l d e s o r p t i o n o f the r e s u l t i n g i o n . A c c o r d i n g t o t h i s mechanism, n e u t r a l sample m o l e c u l e s m i g r a t e t o t h e t i p s o f t h e e m i t t e r d e n d r i t e s where they a r e i o n i z e d by a c h e m i c a l r e a c t i o n w i t h a CI r e a g e n t ion. Energy t o desorb t h e r e s u l t i n g sample i o n i s p r o v i d e d by t h e t e m p e r a t u r e o f t h e e m i t t e r , t h e exo­ t h e r m i c i t y o f t h e i o n - m o l e c u l e r e a c t i o n o r p o s s i b l y by f i e l d s g e n e r a t e d by t h e i o n plasma a t t h e e m i t t e r surface.

8.

HUNT

A N D SETHI

Positive and Negative Ion Chemical Ionization

175

Work i s c u r r e n t l y i n p r o g r e s s i n o u r l a b o r a t o r y t o d e t e r m i n e w h i c h o f t h e above mechanisms i s r e s p o n s i b l e f o r t h e observed r e s u l t s .

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

A c c u r a t e Mass Measurement: D e t e r m i n a t i o n o f m o l e c u l a r c o m p o s i t i o n by a c c u r a t e mass measurements i s u s u a l l y a c c o m p l i s h e d w i t h a double f o c u s i n g 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 o p e r a t i n g a t h i g h r e s o l u t i o n (>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 (<10 ppm) under GC cond i t i o n s have been d e m o n s t r a t e d u s i n g t h i s methodology, but t h e c o s t o f t h e n e c e s s a r y i n s t r u m e n t a t i o n i s s t i l l quite high. I n an e f f o r t t o make e l e m e n t a l c o m p o s i t i o n d a t a a v a i l a b l e t o u s e r s o f q u a d r u p o l e mass s p e c t r o m e t e r s , we have r e c e n t l y examined the p o s s i b i l i t y o f o b t a i n i n g a c c u r a t e mass measurement d a t a u s i n g PPNICI m e t h o d o l ogy. R e s u l t s t o date i n d i c a t e t h a t a c c u r a t e mass measurements (<10 ppm) c a n be a c h i e v e d w h i l e o p e r a t i n g a t u n i t r e s o l u t i o n on a q u a d r u p o l e s p e c t r o m e t e r u s i n g s c a n speeds c o m p a t i b l e w i t h r o u t i n e GC-MS c o n d i t i o n s (5 sec s c a n c y c l e t i m e ) . The e x p e r i m e n t a l technique i n v o l v e s r e c o r d i n g s p e c t r a o f a reference substance (PFK) and sample s i m u l t a n e o u s l y i n t h e n e g a t i v e i o n mode and p o s i t i v e i o n mode, r e s p e c t i v e l y . T h i s i s p o s s i b l e because t h e PFK n e g a t i v e i o n c u r r e n t i s 600 times l a r g e r t h a n t h e c o r r e s p o n d i n g p o s i t i v e i o n c u r r e n t . Thus, by r e c o r d i n g s p e c t r a o f sample p l u s a

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

176

HIGH

PERFORMANCE

MASS

SPECTROMETRY

t r a c e q u a n t i t y o f PFK, i t i s p o s s i b l e t o o b t a i n s p e c t r a where r e f e r e n c e i o n s o n l y appear on the n e g a t i v e i o n t r a c e and o n l y sample i o n s appear on the p o s i t i v e i o n t r a c e . A c c u r a t e mass measurement o f p o s i t i v e l y c h a r g e d sample m o l e c u l e s i s t h e n a c c o m p l i s h e d by u s i n g an INCOS Model 2300 d a t a system t o scan the s p e c t r u m , t o a c q u i r e d a t a s i m u l t a n e o u s l y from b o t h p o s i t i v e and n e g a t i v e i o n m u l t i p l i e r s , t o d e t e r m i n e peak c e n t r o i d s , and t o c a l ­ c u l a t e the e x a c t mass o f the i o n s on t h e p o s i t i v e i o n t r a c e based on t h e i r p o s i t i o n s i n time r e l a t i v e t o PFK ions i n the n e g a t i v e i o n t r a c e . M u l t i p l e s p e c t r a o f sample + PFK ( t r a c e ) a r e r e ­ c o r d e d by r e p e t i t i v e l y s c a n n i n g the q u a d r u p o l e i n s t r u ­ ment o v e r a mass range 65-650 u s i n g a s c a n c y c l e t i m e o f 5 s e c . When c o c a i n e was a n a l y z e d by the above p r o c e d u r e and d a t a from any f i v e c o n s e c u t i v e scans were a v e r a g e d , measured v a l u e s f o r the (M+H) i o n a t m/e = 304.155 were found t o be w i t h i n 10 ppm (3 mmu) οΓ t h e o r e t i c a l v a l u e . I t i s i m p o r t a n t t o note t h a t no r e f e r e n c e compound s u p p r e s s i o n i s o b s e r v e d i n t h i s method, s i n c e o n l y a t r a c e q u a n t i t y o f PFK i s u s e d . +

Conclusion In t h i s c h a p t e r , we have d i s c u s s e d methods o f s e l e c t i v e i o n i z a t i o n u s i n g b o t h p o s i t i v e l y and nega­ t i v e l y c h a r g e d r e a g e n t gases. The c o m b i n a t i o n o f b o t h t e c h n i q u e s i n a s i n g l e mass s p e c t r o m e t e r has been de­ s c r i b e d and some a p p l i c a t i o n s p r e s e n t e d . A p a r t i c u ­ l a r l y u s e f u l a p p l i c a t i o n i s the c a p a b i l i t y t o o b t a i n e x a c t mass d a t a u s i n g a q u a d r u p o l e mass s p e c t r o m e t e r . I n a d d i t i o n , a new t e c h n i q u e f o r o b t a i n i n g mass s p e c t r a o f n o n v o l a t i l e samples has been o u t l i n e d . Literature 1. 2.

3.

4. 5.

Cited.

Dempster, A. J., P h i l . Mag., (1916), 31, 438. (a.) Tal'yoze, V. L. and Lyubimova, A. K . , Dokl. Akad. Nauk SSSR, (1952), 86, 909. (b.) Field, F. H.; Franklin, J. L.; and Lampe, F. W., J. Am. Chem. Soc., (1957), 79, 2419. Field, F. Η., Accounts Chem. Res., (1968), 1, 42. Some recent reviews are: (a) Munson, M.S.B., Anal, Chem., (1977), 49, 772A. (b) Field, F. H . , "Ion-Molecule Reactions", J. L. Franklin, E d . , Plenum Press, Ν. Y. (1972). Hunt, D. F. and Ryan, J. F., J.C.S. Chem. Comm., (1972), 620. Hunt, D. F.; McEwen, C. N . ; and Upham, R. Α . , Anal. Chem., (1972), 44, 1292.

8.

HUNT

6. 7. 8.

9. 10.

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

11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

21. 22.

23. 24. 25. 26.

A N D SETHI

Positive and Negative Ion Chemical Ionization

177

Hunt, D. F.; Stafford, G. C.; Crow, F. W.; and Russell, J. W., Anal. Chem., (1976), 48, 2098. Hunt, D. F., unpublished results. Fales, H. M. and Wright, G. J., Abstracts, Twenty­ -Fifth Annual Conference on Mass Spectrometry and Allied Topics, Washington, D . C . , May 1977, No. W­ -11. Hunt, D. F.; Shabanowitz, J.; and Botz, F. Κ., Anal. Chem., (1977), 49, 1160. Hunt, D. F.; Stafford, G. C.; Shabanowitz, J.; and Crow, F. C., Anal. Chem., in press. Cooks, R. G . ; Beynon, J. H . ; Caprioli, R. M.; and Lester, G. R., "Metastable Ions", Elsevier Com­ pany, Amsterdam, 1973. Rosenstock, H. M.; Wallenstein, M. B . ; Wahrhaftig, A. L.; and Eyring, E., Proc. Natl. Acad. Sci. U.S., (1952), 38, 667. Knewstubb, P. F., "Mass Spectrometry and Ion­ -Molecule Reactions", Cambridge University Press, 1969. Hunt, D. F., and Ryan, J. F. III, Anal. Chem. (1972), 44, 1306. Freiser, B. S.; Woodin, R. L.; and Beauchamp, J. L., J. Amer. Chem. Soc., (1975), 97, 6893. Dzidic, I . , J. Amer. Chem. Soc., (1972), 94, 8333. Wilson, M. S.; Dzidic, I.; and McCloskey, J. Α . , Biochim. Biophys. Acta, (1971), 240, 623. Dzidic, I. and McCloskey, J. Α . , Org. Mass Spec­ trum, (1972), 6, 939. Hunt, D. F., Adv. Mass Spectrometry, (1974), 1, 517. Hogg, A. M. and Nagabhushan, T. L., Abstracts, Twentieth Annual Conference on Mass Spectrometry and Allied Topics, Dallas, Texas, June 1972, No. P2. Beggs, D . , i b i d , No. N4. For recent reviews see: (a) Dillard, J. G . , Chem. Rev., (1973), 73, 589. (b) K. Jennings, Mass Spectrometry, Vol. 4, Specialist Periodical Reports, The Chemical Society, Burlington House, London, 1977. Warman, J. M. and Sauer, M. C., Jr., J. Chem. Phy., (1970), 52, 6428. Beuhler, R. J.; Flanigan, E.; Greene, L. J.; and Friedman, L., Biochem., (1974), 13, 5060. Macfarlane, R. D. and Torgerson, D. F., Science, (1976), 191, 920. Mumma, R. O. and Vastola, F. J., Org. Mass Spec­ trom., (1972), 6, 1373.

178

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27.

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PERFORMANCE

MASS

SPECTROMETRY

Beckey, H. D. and Schulten, H. R., Angew. Chem. Internat. E d i t . , (1975), 14, 403. 28. Muller, W. W., Phys. Rev., (1956), 102, 618. 29. Holland, J. F.; Soltman, B.; and Sweeley, C. C . , Biomed. Mass Spectrom., (1976), 3, 340. 30. Bowers, M. T. and Su, Timothy, "Interactions Be­ tween Ions and Molecules", P. Ausloos, E d . , Plenum Press, Ν. Y. (1974). 31. Eyring, C. F.; Mackeown, S. S.; and Millikan, R. Α., Phys. Rev., (1928), 31, 900. 32. Aspinal, M. L.; Compson, K. R.; Dowman, Α. Α . ; E l l i o t t , R. M.; and Haselby, D., Abstracts, Twenty Third Annual Conference on Mass Spectrometry and Allied Topics, Houston, Texas, May 1975, No. C-2. Received December 30, 1977

9 Investigations of Selective Reagent Ions in Chemical Ionization Mass Spectrometry K. R. JENNINGS

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

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

The great majority of reagent ions used in chemical ionization mass spectrometry (1) are either charge exchange reagents such as A r and N or ions which react as gas phase acids or bases such as H and C H or H and CH O . For each group of reagent ions, the amount of fragmentation depends primarily on the exothermicity of the electron or proton transfer reaction. There are, however, other types of reagent ions which undergo different reactions with specific classes of compounds and which, therefore, may give more detailed information about the presence or location of particular functional groups. within the molecule. This paper gives a brief account of work recently carried out in our laboratories in which the reactions of the vinyl methyl ether molecular ion, CHOCH=CH and the atomic oxygen negative ion, O' with a number of substrates were investigated. +

+

2

+

3

-

+

5

-

3

+

3

2

Reactions of the Vinyl Methyl Ether Molecular Ion. Previous work using ion cyclotron resonance (ICR) spectrometry on the reactions of olefinic molecular ions with various fluoro-olefins (2,3) showed that in each system, a new olefinic ion was Formed. Deuterium-labelling experiments suggested that these reactions occur by the formation of a substituted cyclobutane ion as an intermediate which fragments with the retention of the structural identity of the substituted methylene groups. A similar reaction was observed (4) in the vinyl methyl ether system in which the two secondary ions of m/e = 88 and 84 correspond to the elimination of ethylene and methanol, respectively. In each system, therefore, an olefinic ion attacks a neutral olefin molecule to yield an ion of even mass by the elimination of an olefin, and in the ©0-8412-0422-5/78/47-070-179$05.00/0

180

HIGH

PERFORMANCE

SPECTROMETRY

v i n y l m e t h y l e t h e r system a f u r t h e r i o n o f even mass i s formed by the l o s s o f methanol from the r e a c t i o n complex. The r e a c t i o n s o f i o n s o f t h i s t y p e w i t h o t h e r u n s a t u r a t e d s p e c i e s were t h e r e f o r e i n v e s t i g a t e d w i t h a v i e w t o u s i n g t h i s t y p e o f r e a c t i o n to l o c a t e the p o s i t i o n o f d o u b l e bonds i n m o l e c u l e s . No u s e f u l r e s u l t s were o b t a i n e d u s i n g C H C F * and C Fi»* as r e ­ agent i o n s s i n c e t h e y r e a c t e d m a i n l y by charge t r a n s ­ fer. But i n a number o f s y s t e m s , r e a c t i o n s o f the CH 0CHCH * i o n p r o v i d e d i n f o r m a t i o n w h i c h e n a b l e s one t o l o c a t e the p o s i t i o n o f a d o u b l e bond ( 5 ) . In s t u d ­ i e s i n t h e h i g h p r e s s u r e s o u r c e o f the d o u b l e f o c u s s i n g MS50 mass s p e c t r o m e t e r , i t was f o u n d t h a t a 9:1 m i x t u r e o f C 0 and CH 0CHCH gave a good y i e l d o f CH 0CHCH * and s u b s t a n t i a l l y r e d u c e d the r e a c t i o n o f t h i s i o n w i t h i t s parent molecule. The r e a c t i o n s o f i n t e r e s t a r e o f the g e n e r a l t y p e Η Η γ CH 0CH=CH t + X-CH=CH-Y 2

3

2

2

2

2

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

MASS

3

2

3

2

X

3

2

H XCH=CH

+

2

0CH

3

Η YCH=CHOCH Î 3

i n w h i c h , i n g e n e r a l , the s i m p l e r o f the two p o s s i b l e o l e f i n s i s e l i m i n a t e d as a n e u t r a l and a s u b s t i t u e n t c o n t a i n i n g a h e t e r o a t o m i s r e t a i n e d a l o n g w i t h the m e t h o x y l group as p a r t o f the o l e f i n i c i o n . Hence, f o r example, a l l t e r m i n a l o l e f i n s r e a c t by the f o r m a t i o n o f a complex f r o m w h i c h e t h y l e n e i s e l i m i n a t e d . The e x p e c t e d even-mass s e c o n d a r y i o n s were obs e r v e d i n the mass s p e c t r a g i v e n by the r e a c t i o n s o f CH 0CHCH * i o n s w i t h a number o f s i m p l e h y d r o c a r b o n olefins. In each c a s e , a f u r t h e r even-mass i o n was o b s e r v e d due t o the l o s s o f CH 0H from the r e a c t i o n complex, t o g e t h e r w i t h odd mass i o n s w h i c h a r i s e from the l o s s o f atoms o r r a d i c a l s from the complex. These r e a c t i o n s r e a d i l y a l l o w one t o d i s t i n g u i s h between i s o m e r i c s p e c i e s such as 1-butene and 2-butene. In F i g u r e I t h e v e r y d i f f e r e n t s p e c t r a g i v e n by 1-octene and t r a n s - 4 - o c t e n e a r e i l l u s t r a t e d . B o t h CH =CHCH CH= CH and CeH CH CH=CH r e a c t e d as t e r m i n a l m o n o - o l e f i n s and gave a c h a r a c t e r i s t i c peak due t o the l o s s o f C Hi» from the r e a c t i o n complex. S i m i l a r r e a c t i o n s were a l s o o b s e r v e d w i t h a l l y l f o r m a t e and w i t h CH =CH-CH C00H. When o l e i c a c i d was r u n as an "unknown , the p o s i t i o n o f i t s d o u b l e bond was c o r r e c t l y l o c a t e d by means o f the r e a c t i o n 3

2

3

2

2

5

2

2

2

2

2

11

2

9.

Selective Reagent Ions

JENNINGS

CH (CH ) CH=CH(CH )7COOH 3

2

7

+

2

181 CH 0CH=CH Î 3

2

+

CH (CH )7CH-CH-(CH )7C00H t 3

2

2

CH CH-0CH 2

CH (CH ) CH=CH 3

2

7

2

+

3

CH OCH=CH-(CH ) COOH ί 3

2

7

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

m/e = 200 The i m p o r t a n c e o f t h e r e a c t i o n i n w h i c h methanol i s e l i m i n a t e d i n c r e a s e s as t h e s i z e o f the m o l e c u l e i n c r e a s e s and i s t h e o n l y r e a c t i o n y i e l d i n g an even mass i o n w i t h u n s a t u r a t e d c y c l i c compounds and w i t h n o n - c o n j u g a t e d d i e n e s such as 1,4 and 2 , 6 - o c t a d i e n e s . U n s a t u r a t e d n i t r i l e s a r e s i m p l y p r o t o n a t e d . The con­ j u g a t e d s p e c i e s 1,3-butadiene undergoes a D i e l s - A l d e r r e a c t i o n i n w h i c h methanol i s e l i m i n a t e d . I t i s p l a n n e d t o e x t e n d t h i s s t u d y t o more c o m p l i ­ c a t e d u n s a t u r a t e d systems. I n v i e w o f t h e q u i t e gen­ e r a l n a t u r e o f t h e r e a c t i o n , i t i s hoped t h a t o t h e r o l e f i n i c i o n s w i l l be i d e n t i f i e d as u s e f u l r e a g e n t ions. I t s h o u l d t h e n be p o s s i b l e t o l o c a t e the po­ s i t i o n s o f c a r b o n - c a r b o n double bonds i n a wide v a r i e t y of compounds w i t h o u t p r i o r d e r i v a t i z a t i o n and i n f a v o r ­ a b l e c a s e s , as i n t h e 1 - f l u o r o p r o p e n e s ( 6 ) , i t may be p o s s i b l e t o d i s t i n g u i s h c i s - and t r a n s - i s o m e r s . Reactions o f the

A t o m i c Oxygen N e g a t i v e

Ion,

In r e c e n t y e a r s 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 mass s p e c t r o m e t r y (7) has become i n c r e a s i n g l y i m p o r t a n t . I n c e r t a i n a p p l i c a t i o n s , t h e t y p e o f spectrum produced and t h e s e n s i t i v i t y o f the method make i t s u p e r i o r t o p o s i t i v e c h e m i c a l i o n i z a t i o n , as w i l l be d e s c r i b e d e l s e w h e r e i n t h i s volume. Most n e g a t i v e r e a g e n t i o n s r e a c t p r i m a r i l y as s t r o n g gas phase bases b u t t h e r a d i c a l a n i o n , 0 · , has been found t o have some u n i q u e r e a c t i o n s which y i e l d i n t e r e s t i n g s t r u c t u r a l i n f o r ­ m a t i o n i n a number o f c a s e s . Most o f t h e e a r l y s t u d i e s (8_,9) were c a r r i e d o u t at l o w p r e s s u r e s i n a ICR s p e c t r o m e t e r . N 0 was found to be a c o n v e n i e n t s o u r c e o f 0· i o n s a l t h o u g h t h e f a c t t h a t t h e y a r e formed w i t h 0.38 eV o f t r a n s l a t i o n a l energy makes N 0 l e s s s a t i s f a c t o r y as a s o u r c e o f 0· i o n s f o r d e t e r m i n a t i o n o f r a t e c o n s t a n t s . I n t h e com­ b i n e d E I / C I s o u r c e o f the MS50, f u r t h e r r e a c t i o n s i n ­ v o l v i n g N 0 may o c c u r , b u t t h e s e were s u p p r e s s e d by u s i n g a 9:1 m i x t u r e o f N and N 0 a t a t o t a l p r e s s u r e 2

2

2

2

2

182

HIGH

PERFORMANCE

MASS

SPECTROMETRY

o f 0.5 T o r r . T h i s g a v e a h i g h y i e l d o f 0· i o n s w i t h o n l y a t r a c e o f N 0 ~ i o n s . The D a l y d e t e c t o r o f the MS50 was r e p l a c e d by an e l e c t r o n m u l t i p l i e r f o r n e g a t i v e i o n work, but m o d i f i c a t i o n s c u r r e n t l y b e i n g c a r r i e d out to the D a l y d e t e c t o r w i l l e n a b l e i t to o p e r a t e i n the n e g a t i v e i o n mode. E a r l y work (8) on the r e a c t i o n of 0· w i t h CH CD had shown t h a t o n l y CH C> and C D C i o n s are formed, i n d i c a t i n g t h a t the r e a c t i o n o c c u r s by H abstraction from a s i n g l e c a r b o n atom. T h i s was l a t e r shown (9) to bç a r e a c t i o n c h a r a c t e r i s t i c o f many 1 - o l e f i n s a l t K o u g h H a b s t r a c t i o n from a l k y l groups p r e d o m i n a t e u n l e s s the t e r m i n a l =CH group i s a c t i v a t e d by the p r e s e n c e o f an e l e c t r o n w i t h d r a w i n g group i n the m o l e c u l e , e.g., F o r CF . Other c l a s s e s o f compounds were found to undergo the H a b s t r a c t i o n r e a c t i o n . In the case o f a l i p h a t i c n i t r i l e s , the f a c t t h a t the r e a c t i o n o c c u r s w i t h nC 3 H 7 C N but not w i t h (CH ) CHCN p r o v i d e s s t r o n g e v i d e n c e that H a b s t r a c t i o n t a k e s p l a c e a t the α-C atom._ In an e x t e n s i v e s t u d y o f the r e a c t i o n s of 0· i o n s w i t h c a r b o n y l compounds ( 1 0 ) , the major r e a c t i o n s were f o u n d t o be o f the general~~type 2

2

:

2

2

+

2

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

2

3

+

2

3

2

+

2

(M-l)"

+

•OH

(1)

(M-2)·

+

H0

(2)

->·

(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

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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

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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

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Selectivity in Biomedical Applications

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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

f

175

1

200

ι

225

1

250

I

275

I

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TEMPERATURE (6VMIN)

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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

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195

.COOH

Figure 4. Cyclophosphamide I and its major human metabolites

,NH 0

1

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2

— * N

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

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108 0 N-P-OCH3

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92

212

JUL Γ 90

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<% Cancer Research

Figure 6. Electron impact mass spectrum of dimethyl phosphoramide mustard

122

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^N-P-OCH N(CH i

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3

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213 60

5 4oH 20 1 Irfll m 90

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177 1-J_ 170

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227 I 230

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Figure 7. Electron impact mass spectrum of trimethyl phosphoramide mustard

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

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A.

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m/e 213

m/e 215 160°

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Urine Extract

m/e 199

Λ-

m/e 201

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m/e2l5 160*

Γ70°

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190*

TEMPERATURE

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Figure 8. Selected ion records of urine extract from a patient receiv­ ing cyclophosphamide and of authentic derivatized phosphoramide mustard

Selectivity in Biomedical Applications

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

FENSELAU

163 Spectrum scanned urine extract 49

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.*? . ••

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Figure 10. Electron impact mass spectrum taken at ΐtin Figure 10

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of an e x t r a c t o f p a t i e n t plasma. I n t h i s c a s e , many l i p o p h i l i c endogenous c o n t a m i n a n t s fragment t o form i o n s o f the masses b e i n g m o n i t o r e d , and the i n t e r ­ f e r e n c e i s h i g h enough to r e n d e r the m e t a b o l i t e ' s i d e n t i f i c a t i o n ambiguous. A c c o r d i n g l y , the mass s p e c t r o m e t e r had t o be employed i n such a way as t o obtain additional s e l e c t i v i t y . In t h i s c a s e , c h e m i c a l i o n i z a t i o n was used i n p l a c e o f e l e c t r o n impact i o n i ­ zation. F i g u r e 12 shows the mass spectrum o f the monom e t h y l d e r i v a t i v e o f phosphoramide mustard o b t a i n e d with isobutane chemical i o n i z a t i o n . T h i s spectrum i s dominated by p r o t o n a t e d m o l e c u l a r i o n s p e c i e s (m/e 235, 237, 239) and t h e s e t h r e e i o n s were chosen f o r s e l e c t e d i o n m o n i t o r i n g . In t h i s c a s e , c h e m i c a l i o n i z a t i o n a l l o w s us t o m o n i t o r i o n s of the h i g h e s t mass p o s s i b l e , the p r o t o n a t e d m o l e c u l a r i o n . I t i s w e l l documented t h a t the h i g h e r the mass, the lower the p r o b a b i l i t y t h a t c o n t a m i n a n t s and o t h e r compounds w i l l fragment t o i o n s o f the same mass. C h e m i c a l i o n i z a t i o n a l s o i n ­ c r e a s e s s e l e c t i v i t y i n a n o t h e r way. F r a g m e n t a t i o n can be seen t o be g r e a t l y reduced i n the spectrum i n F i g u r e 12; s i m i l a r l y , f r a g m e n t a t i o n o f endogenous contaminants w i l l be g r e a t l y r e d u c e d , a g a i n l o w e r i n g the p r o b a b i l i t y of c o i n c i d e n c e w i t h the i o n s b e i n g m o n i t o r e d . Figure 13 p r e s e n t s p r o f i l e s o f the i o n s o f mass 235, 237 and 239, m o n i t o r e d as the plasma e x t r a c t i s e l u t e d from the gas chromatograph. On the b a s i s o f t h e s e i o n p r o f i l e s , i t was c o n c l u d e d t h a t phosphoramide mustard i s i n d e e d a c i r c u l a t i n g metabolite i n patients receiving cyclo­ phosphamide. T h i s i d e n t i f i c a t i o n was based on c o i n ­ c i d e n t r e t e n t i o n time w i t h a s t a n d a r d compound, on the f o r m a t i o n o f the i o n s c o n s i d e r e d c h a r a c t e r i s t i c o f the d e r i v a t i z e d m e t a b o l i t e , and on the f o r m a t i o n of t h e s e i o n s i n the a p p r o p r i a t e i n t e n s i t y r a t i o s o f about 9:6:1. The a n a l o g p r o f i l e s g e n e r a t e d by the s e l e c t e d i o n m o n i t o r a r e d i r e c t l y q u a n t i t a t a b l e , and once the meta­ b o l i t e had been i d e n t i f i e d i n p a t i e n t s ' b l o o d and u r i n e , the c l i n i c i a n s were v e r y keen t o assay i t s c o n c e n t r a t i o n as a f u n c t i o n o f t i m e . One p a r t i c u l a r advantage which mass s p e c t r o m e t r y b r i n g s t o a s s a y s o f t h i s s o r t i s t h a t i t p e r m i t s the use o f the i d e a l i n t e r n a l s t a n d a r d , t h a t i s the compound i t s e l f l a b e l l e d w i t h heavy i s o t o p e s , H , C , Ο , N . These i n t e r n a l s t a n d a r d s w i l l have c h e m i c a l c h a r a c t e r i s t i c s most n e a r l y a p p r o x i m a t i n g those o f the assay sample i t s e l f , and l o s s e s i n e x t r a c t i o n , d e c o m p o s i t i o n , d e r i v a t i ­ z a t i o n , column a b s o r p t i o n , e t c . a r e d i r e c t l y compen­ s a t e d by comparable l o s s e s i n the i n t e r n a l s t a n d a r d . 2

1 3

1 8

1 5

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

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201

Selectivity in Biomedical Applications

TEMPERATURE

Cancer Research

Figure 11. Selected ion records obtained by electron impact of phsma extract from a patient receiving cyclophosphamide

100

235

ISOBUTANE Cl 80

Cl

0 II

Cl

;; _p-N

60

1 NH

0CH 0

237

2

>

«5 LU ce

40

20 1239 100

120

140

160

180

200

220

240

260

Cancer Research

Figure 12. Chemical ionization mass spectrum of monomethyl phosphoramide mustard

202

PERFORMANCE

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I

I

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

HIGH

M/E 237

I

I

I

i

I

M/E 239

'

I 150·

I

^

I 160·

I 170·

I

—

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180* 190* TEMPERATURE

1

1

1

200*

2Κ>·

220·

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 <Ηphosphoramide mustard were a l s o used i n s t u d i e s o f m u s t a r d a l k y l a t i o n mechanisms ( 6 ) . F i g u r e 15 p r e s e n t s scans o f t h e m o l e c u l a r i o n r e g i o n s o f d - and dU -phosphoramide mustard t r i m e t h y l derivatives. Each compound c o n t a i n s two c h l o r i n e atoms, and t h u s t h r e e p r o t o n a t e d m o l e c u l a r i o n s a r e d e t e c t e d i n each spectrum. From t h e s e two s e t s o f [M + H] i o n s f o u r peaks were s e l e c t e d f o r m o n i t o r i n g . A l t h o u g h some s e n s i t i v i t y i s s a c r i f i c e d i n m o n i t o r i n g f o u r peaks r a t h e r than two, a d d i t i o n a l r e l i a b i l i t y i s g a i n e d s i n c e t h e i n t r a m o l e c u l a r i n t e n s i t y r a t i o c a n be checked as an a s s u r a n c e t h a t t h e c o r r e c t m e t a b o l i t e i s b e i n g d e t e c t e d , and t h a t i n t e r f e r e n c e i s m i n i m a l . Some f l e x i b i l i t y i s a l s o g a i n e d , because t h e i n t e r m o l e c u l a r r a t i o s o f m e t a b o l i t e t o i n t e r n a l s t a n d a r d may be c a l ­ culated i n s e v e r a l d i f f e r e n t combinations. F i g u r e 16 p r e s e n t s t h e s e l e c t e d i o n r e c o r d s from a plasma sample c o n t a i n i n g d - p h o s p h o r a m i d e m u s t a r d and di»^phosphor­ amide m u s t a r d . I t i s i m p o r t a n t t o know t h a t c r o s s t a l k i s m i n i m a l , t h a t t h e r e i s l i t t l e o r no s i g n a l con­ t r i b u t e d by t h e di*-compound t o masses m o n i t o r e d f o r t h e d -compound, and v i c e v e r s a . F i g u r e 17 p r e s e n t s a c a l i b r a t i o n c u r v e f o r phosphoramide m u s t a r d e x t r a c t e d from plasma. A l o g - l o g s c a l e i s used so t h a t t h e measurements may be p r e s e n t e d t h r o u g h two o r d e r s o f magnitude. A l l p o i n t s e x c e p t t h e l o w e s t were d e t e r ­ mined w i t h e x p e r i m e n t a l u n c e r t a i n t i e s between 1 and 5%. F i g u r e 18 p r e s e n t s t h e p r o f i l e s o f t h e concen­ t r a t i o n s i n p a t i e n t plasma o f n o r n i t r o g e n m u s t a r d , phosphoramide m u s t a r d and c y c l o p h o s p h a m i d e as a f u n c ­ t i o n o f time t h r o u g h 24 h o u r s . The s e l e c t i v i t y o f f e r e d by t h e mass s p e c t r a l a s s a y compares f a v o r a b l y w i t h t h e a l t e r n a t i v e n o n s p e c i f i c s p e c t r o p h o t o m e t r i c assay based on t h e f o r m a t i o n o f c o l o r e d p r o d u c t s from any a l k y l ­ a t i n g compounds p r e s e n t . I n a d d i t i o n , t h e mass spec4

h

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

203

0

0

0

204

HIGH

Η

Ρ^

2 Η 5

H (

\ CO2C2H5

PERFORMANCE

f-CD OH

SOC,

2

\

MASS

^

2

H

SPECTROMETRY

X^CD2a

CD OH

Ns^O^CI

2

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

Figure 14. Synthesis of d^-nornitrogen mustard

C I C H

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CICH^ 100

8/
3

CICDg-^

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r

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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

I

0.01

I

I

0.03

0.1

t

03

I

1.0

I

3.0

1

10.0

/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

HIGH

PERFORMANCE

MASS

SPECTROMETRY

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

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

500«

400

8

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

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

210

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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

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Field Desorption Mass Spectrometry

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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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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

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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 . +

+

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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. +

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I50
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50

Emitter Current (mA)

Figure 3. Temperature vs. the emitter current for the activated emitters used in this study (10 μ tung­ sten wire—30 μ average needle length). Region I obtained by melting of known samples and cross calibration to direct probe heated; Region II ob­ tained by resistance measurements on activated emitters; Region III obtained by resistance and optical pyrometer measurements.

I

50

100

1

I ' I ' I

150

1

m/e

I ' I • I ' I ' I

200

250

Analytical Chemistry

Figure 4. Field desorption spectrum of adenosine obtained by emitter current programming

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M

FD

80-

C

6 5 - J ^ Q^l^r^J_ H

N

H

607. AO-

M

W

4

7

C H

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3

S ^ N ^

20-

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150

200

500

A00

300

Figure 5. Comparison of field desorption and electron impact spectra of a synthetic nu­ cleoside analog. Sample furnished by E. C. Tayler and L. E. Crane of Princeton Univer­ sity, Princeton, ΝJ.

10080 F D BO 7. 40 20

M W I 4 7

çooH CHNHg CH

2

CH

2

88 M +l

COOH 1

1

IB

Figure 6. Comparison of field desorption and électron impact of glutamic acid

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FD s p e c t r a o f g a l a c t i t o l and s u c r o s e a r e shown i n F i g . 8. In n e i t h e r case was a m o l e c u l a r i o n o b s e r v e d i n the EI mode. The i n c r e a s e d f r a g m e n t a t i o n o f the d i s a c c h a r i d e i n d i c a t e s e i t h e r an i n c r e a s e d t h e r m a l l a b i l i t y o r the f o r m a t i o n o f i o n s w i t h h i g h e r i n t e r n a l energies. I n a l l o f the above examples, the s p e c t r a shown were t a k e n a t o r s l i g h t l y below the m i d - p o i n t o f the BAT. At s l i g h t l y h i g h e r t e m p e r a t u r e s , the p r e s e n c e o f i n o r g a n i c i m p u r i t i e s w i l l l e a d t o the p r o d u c t i o n o f c a t i o n i z e d m o l e c u l a r s p e c i e s as shown i n F i g . 9. F i g u r e 10 shows the FD s p e c t r u m o f the p o t a s s i u m s a l t of naphthyl s u l f a t e . T h i s i s an example o f a compound t h a t w i l l y i e l d o n l y c a t i o n i z e d s p e c i e s and i l l u s t r a t e s the u s e f u l n e s s o f c a t i o n i z a t i o n i n p r o d u c i n g i o n s from m o l e c u l e s t h a t c o u l d not o t h e r w i s e be a n a l y z e d by FD. The i n t r o d u c t i o n o f s m a l l amounts o f c a t i o n i m p u r i t y i s becoming a v i a b l e e x p e r i m e n t a l p a r a m e t e r i n the a n a l y s i s o f p o l a r compounds. W i t h c o n t i n u e d i n c r e a s e s i n t e m p e r a t u r e , t h e c a t i o n i z e d s p e c i e s become dominant. I f t h e r e a r e s i g n i f i c a n t l e v e l s o f i m p u r i t y , the i n o r g a n i c c a t i o n i t s e l f becomes the major s p e c i e s detected. The a n a l y s i s o f f i e l d d e s o r p t i o n s p e c t r a has i n d i c a t e d a u b i q u i t o u s dependence o f the n a t u r e o f the i o n s p r o d u c e d upon e m i t t e r t e m p e r a t u r e , the s o l v e n t u s e d , and t h e p r e s e n c e o f s m a l l c a t i o n s . These p a r a meters s t r o n g l y s u g g e s t a c h e m i c a l i n v o l v e m e n t i n the i o n i z a t i o n mechanism, and i n v e s t i g a t i o n s were c o n d u c t e d to d e t e r m i n e the e x a c t r o l e o f the s t r o n g e l e c t r i c f i e l d i n t h i s method. F i e l d image t h e o r y would p r e d i c t t h a n an i o n s h o u l d desorb i n the p r e s e n c e o f a s t r o n g f i e l d a t t e m p e r a t u r e s below those r e q u i r e d i n the absence o f t h e f i e l d . In f a c t , t h i s s i t u a t i o n has o f t e n been p o s t u l a t e d and was g e n e r a l l y a c c e p t e d as f a c t p r i o r to t h i s study. E l e c t r o n Impact I o n i z a t i o n o f F i e l d Desorbed Samples"! F i g u r e 11 shows the r e s u l t s o f an i n v e s t i g a t i o n w h i c h compares the BAT f o r u r i d i n e w i t h l a r g e v a r i a t i o n s o f the a p p l i e d v o l t a g e f i e l d . A d d i t i o n a l d a t a f o r t h i s s t u d y were o b t a i n e d u s i n g a n o v e l t e c h n i q u e c a l l e d EI/D ( 2 4 ) . T h i s t e c h n i q u e employs the t h e r m a l d e s o r p t i o n o f the sample from the a c t i v a t e d e m i t t e r w i t h subsequent i o n i z a t i o n by t h e e l e c t r o n beam. The g e o m e t r i c changes o f the s o u r c e n e c e s s a r y f o r t h i s t e c h n i q u e a r e shown i n F i g . 12. T h i s method g e n e r a t e s i o n s a f t e r the compound has l e f t the e m i t t e r and the t e m p e r a t u r e o f maximum i o n d e t e c t i o n i s c a l l e d b e s t e m i t t e r t e m p e r a t u r e (BET) i n a n o m e n c l a t u r e a n a l o gous t o BAT. For a l l the compounds t e s t e d t o d a t e , BET

11.

SWEELEY

100 80

ΗΟ-<ζ

80

^ - C H Ç H COOH 2

MW

1 '

15

M

FD

'

219

Field Desorption Mass Spectrometry

ET AL.

1 ' 1 .

181

, . , . , . , . , 1 , . J . J 1 , . , . ,

100

5 0

150

200

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

100

18

ι—'—ι—•—ι—•—ι— —r 1

150

200

Figure 7. Comparison of field desorption and electron impact spectra of tyrosine

100 80 7.

FD

M

W

'

8

87

M+!

ÇH OH

2

2

HCOH HOCH HOÇH HCOH OH OH

60 40

2

20 •

50

100

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60

KOH

AO

M-31

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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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HIGH

PERFORMANCE

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

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.

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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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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

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Field Desorption Mass Spectrometry

223

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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

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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+©)

+

<8$O (®) t

+

SMALL ION MOBILITY Μ+Φ—(M+®)

α

M (nM+®)*

+

+

+

(Μ-(ηΗ)Η+ηΦ)

+

+

T H E R M A L IONIZATION L A T T I C E ION MOBILITY

®* ®®®*

Biomedical Mass Spectrometry

Figure 14. A proposed ionization mechanism for temperature-dependent chemical generation of ions observed infielddesorption spectra

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

226

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to those o b t a i n e d by a c o n v e n t i o n a l Knudsen c e l l (27). The t e m p e r a t u r e a t t a i n a b l e by e m i t t e r h e a t i n g may exceed 1800°C and i n the f i n a l s t a g e o f the a n a l y s i s , t h e s e i n o r g a n i c s p e c i e s may be d r i v e n o f f w i t h the i o n s p r o d u c e d i n a c c o r d a n c e w i t h c l a s s i c a l Langmuir evaporation. The e s s e n t i a l p a r t s o f t h i s p r o p o s e d mechanism may be condensed as f o l l o w s : (1) A c t i v e t h e r m a l d e s o r p t i o n must procède o r be conc o m i t t a n t w i t h i o n f o r m a t i o n and d e t e c t i o n . (2) M o s t , i f not a l l , o f the i o n s o b s e r v e d a r e genera t e d by t h e r m a l l y - i n d u c e d c h e m i c a l r e a c t i o n s o c c u r r i n g i n the s e m i - f l u i d phase on o r near the emitter surface. (3) The r e l a t i v e o r g a n i c and i n o r g a n i c compound m o b i l i t i e s a r e paramount i n d e t e r m i n i n g the i o n i c species observed. (4) Most o f the o r g a n i c sample d e s o r b s as n e u t r a l molecules. Because o f the t h i n f i l m n a t u r e o f the sample on the e m i t t e r and the e f f i c i e n t h e a t i n g p r o c e s s , d e s o r p t i o n o c c u r s w i t h a minimum o f f r a g m e n t a t i o n and a t s l i g h t l y lower t e m p e r a t u r e s than would be a n t i c i p a t e d (18, 29^) . (5) A h i g h e l e c t r i c f i e l d i s needed t o e x t r a c t and c o l l e c t i o n s from the s e m i - f l u i d phase. (6) The h i g h f i e l d w i l l s h i f t the e q u i l i b r i a o f r a p i d t h e r m a l l y g e n e r a t e d c h e m i c a l r e a c t i o n s to f a v o r formation of p o s i t i v e i o n i c species. (7) I t i s p o s s i b l e t h a t the h i g h energy needed f o r the g e n e r a t i o n o f i o n s o f the type M may be a t t a i n e d i n the s u r f a c e c h e m i s t r y o f the s e m i - f l u i d phase by c l a s s i c a l thermodynamic p a r a m e t e r s w i t h l i t t l e i n f l u e n c e o f the a p p l i e d f i e l d . (8) I n summary, the e m i t t e r may be v i e w e d as a h i g h temperature microchemical r e a c t o r . Whether the model, p a r t i c u l a r l y items 2 and 7, i s e s s e n t i a l l y p r o v e n t o be c o r r e c t or n o t , i t has a l r e a d y been o f c o n s i d e r a b l e v a l u e . I t has f o c u s e d a t t e n t i o n upon the c h e m i c a l p a r a m e t e r s o f the a n a l y s i s and, t h e r e b y , has been i n s t r u m e n t a l i n the d e c r e a s e o f an o r i g i n a l f a i l u r e r a t e o f 50-70% t o a p r e s e n t f a i l u r e r a t e o f a p p r o x i m a t e l y 5%. Inorganic c a t i o n s , the p r e s e n c e o f w h i c h were o r i g i n a l l y a bane t o the method, a r e now s i m p l y r e a g e n t s t o be s e l e c t i v e l y a p p l i e d where t h e y may be o f b e n e f i t i n the p r o d u c t i o n o f c a t i o n i z e d species of molecules. I n a d d i t i o n , the model i n t r o duces a degree o f c h e m i c a l i n t u i t i o n i n t o t h e method and, i n so d o i n g , r e l i e v e s many o f the a n x i e t i e s engendered by attempts t o c o r r e l a t e e x p e r i m e n t a l r e s u l t s with c l a s s i c a l theory.

11.

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227

T h i s model s t r e n g t h e n s t h e u s e o f f i e l d d e s o r p t i o n as an a n a l y t i c a l method f o r p o l a r , n o n - v o l a t i l e com­ pounds and expands i t s p o t e n t i a l f o r t h e a n a l y s i s o f complex b i o l o g i c a l m o l e c u l e s . C l e a r l y , t h e r o l e o f t h e f i e l d must c o n t i n u e t o be a s u b j e c t o f i n v e s t i g a t i o n u n t i l t h e e x a c t n a t u r e o f i o n f o r m a t i o n can be d e f i n e d . Once t h i s o c c u r s , t h e method s h o u l d p a s s from the p r e ­ s e n t p r e d o m i n a n t l y a r t i s a n s t a g e t o t h a t o f an a p p l i e d science.

References

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

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Moor, J., and Waight, E. S., Org. Mass Spectrom., (1974), 9, 903. Krone, H . , and Beckey, H. D., Org. Mass Spectrom., (1969), 2, 427. Schulten, H. R., Beckey, H. D., B e l l e l , Ε. M . , Foster, A. B . , Jarman, Μ., and Westwood, J. H . , J. Chem. Soc. Chem. Commun., (1973), 13, 416. Schulten, H. R., and Games, D. Ε . , Biomed. Mass Spectrom., (1974), 1, 120. Schulten, H. R., and Beckey, H. D., Org. Mass Spectrom., (1973), 7, 861. Winkler, H. U . , and Beckey, H. D . , Biochem. Biophys. Res. Commun., (1972), 46, 391. Adlercreutz, H . , Soltmann, B . , and Tikkanen, M . , J. Steroid Biochem., (1974), 5, 163. Games, D. E., Jackson, A. H . , Millington, D. S., and Rossiter, M . , Biomed. Mass Spectrom., (1974), 1, 5. Maurer, K. H . , and Rapp, U . , Biomed. Mass Spec­ trom., (1975), 2, 307. Sammons, M. C . , Bursey, M. M . , and Brent, D. Α . , Biomed. Mass Spectrom., (1974), 169. Beckey, H. D., J. Mass Spectrom. and Ion Phys., (1969), 2, 500. Muller, E. W., Phys. Rev. (1956), 102, 618. Gomer, I . , J. Chem. Phys., (1959), 31, 341. Eyring, C. F., MacKcown, S. S., and Millikan, R. Α., Phys. Rev. (1928), 31, 900. Beckey, H. D., H i l t , E., Schulten, H. R., J. Phys. E., (1973), 6, 1043. Beckey, H. D., Heindricks, Α . , and Winkler, H. U . , Int. J. Mass Spectrom. and Ion Phys., (1970), 3, 9. Barofsky, D. F., and Barofsky, E., Int. J. Mass Spectrom. Ion Phys., (1974), 14, 3. Schulten, H. R., Beckey, H. D., Boerboom, A. J. H . , and Meuzelaar, H. I. C . , Anal. Chem., (1973), 45, 2358.

228

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19.

HIGH

PERFORMANCE

MASS

SPECTROMETRY

Olson, K. L., Cook, J. C . , Rinehart, K. L., Bio­ med. Mass Spectrom., (1974), 1, 358. 20. Soltmann, B . , 2nd Annual Field Desorption Work­ shop, Urbana, 1975. 21. Kummler, D . , and Schulten, H. R., Org. Mass Spec­ trom., (1975), 10, 813. 22. Maine, J. W., Soltmann, Β . , Holland, J. F., Young, N. D., Gerber, J. N . , and Sweeley, C. C . , Anal. Chem., (1976), 48, 427. 23. Moor, J., and Waight, E. S., Org. Mass Spectrom., (1974), 9, 903. 24. Soltman, B . , Sweeley, C. C. and Holland, J. F., Anal. Chem., (1977), 49, 1164. 25. Holland, J. F., Soltmann, B . , and Sweeley, C. C . , Biomed. Mass Spectrom., (1976), 3, 340. 26. Hiraoka, Κ., and Kebarle, P . , #48L 7th Int'l Conf. on Mass Spec., Florence, Italy, Sept. 1976. 27. Cherpha, W. Α . , and Ingham, U. G . , J. Phys. Chem., (1955), 59, 100. 28. Beuhler, R. J., Flanigan, E., Greene, L. J., and Friedman, L. Biochem., (1974), 13, 5060. 29. Beuhler, R. J., Flanigan, E., Greene, L. J., and Friedman, L., J. Am. Chem. Soc., (1974), 96, 3990. Received December 30, 1977

12 Mass Spectrometry Applications in a Pharmaceutical Laboratory D. A. BRENT, C. J. BUGGE, P. CUATRECASAS, B. S. HULBERT, D. J. NELSON, and N. SAHYOUN

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

Wellcome Research Laboratories, Burroughs Wellcome Co., Research Triangle Park, NC 27709

To discuss mass spectral research applications in the pharmaceutical area, we have selected a series of representative problems taken from work carried out at the Wellcome Research Laboratories. At this facility, the mass spectrometers are located in a central laboratory servicing all departments involved in pharmaceutical research and production. The instru­ ments used are the Varian MAT CH5-DF and 731 double focussing mass spectrometers. Both instruments are interfaced to a Varian SS100 data system and a Varian C-1024 time averaging computer. Each instrument can be coupled to a gas chromatο­ graph, has defocussed metastable ion capability, and can measure accurate masses. Analysis of Individual Components in A Complex Mixture By Selected Ion Monitoring Mass Spectrometry has been a powerful tool in the analysis of complex mixtures (1-3). When coupled to a gas chromatograph, a full mass spectrum can be taken of each peak eluting from the chromatograph. When these peaks contain more than one component, the mass spectrometer can be used as a specific detector to quantitate each component under a single peak (4). This is accomplished by sequentially monitoring ions that are characteristic of each component. The area of the envelope ©0-8412-0422-5/78/47-070-229$05.00/0

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

230

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MASS

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created by monitoring the intensity of an ion with time i s proportional to the concentration of the compound being measured. In addition, single or multiple ion dection i s more sensitive than scanning methods. This type of analysis can be applied to complex s o l i d s , e.g. tyramine i n freeze dried brain tissue (5). Further s p e c i f i c i t y i n the monitoring of complex mixtures can be accomplished by carrying out the measurement at high resolution (6). Allopurinol, oxipurinol, hypoxanthine, xanthine, and u r i c acid were d i r e c t l y quantitated i n freeze dried muscle tissue at the 10-100 ppm level with an accuracy of + 20% (6). A resolution of 20,000, 101 valley d e f i n i t i o n , was used to quantitate these purines and purine analogs. At t h i s resolution some integer masses were resolved into as many as f i v e fractional masses. One of the fractional masses at each integer mass selected i s s p e c i f i c f o r the compound to be measured. We have applied the technology of analyzing a mixture by single ion monitoring at high resolution to patent infringement cases (7). In Argentina, Austria, Czechoslovakia, Hungary, Mexico, Poland, Portugal, Spain, Switzerland, Uruguay, Venezuela, Yugoslavia, inter a l i a , newly developed drugs can only be protected by patents covering synthetic routes rather than by patents covering the compound i t s e l f or i t s usage. Therefore, i t i s valuable to have a method to determine the synthetic route used to monitor possible infringement cases. A compound cannot be manufactured absolutely pure. Some of the impurities present may characterize a single synthetic route or a particular class of routes. The samples are analyzed for the presence of characteristic impurities. The material to be analyzed (along with binding agents, excipients, and other drugs) i s ground by mortar and pestle to a fine powder. About 3 mg of a sample i s introduced into the mass spectrometer v i a the direct probe i n l e t . The sample i s examined under low resolution conditions at a variety of probe temperatures f o r ions characteristic of known impurities. I f these ions e x i s t , the same experiment i s carried out at 10,000 resolution, 101 v a l l e y d e f i n i t i o n . For example, one of the synthetic routes (8) to the drug trimethoprim, (1), involves an intermediate acetal, (2). Ions used f o r the i d e n t i f i c a t i o n of the impurity must be resolved from ions i n the spectrum of the drug, binder, or excipients. One of the ions choosen f o r (2) i s i t s molecular ion. One reason f o r t h i s choice i s that (1) cannot interfer because i t i s of lower molecular weight. Figure 1 i s an o s c i l l o graphic trace taken of the molecular ion of (2) at a concentrat i o n of .0005% i n trimethoprim. The sample was heated from ambient to 110° ( l e f t to right i n the figure). The m/e 293 and m/e 295 regions were alternately brought into focus using the peak matcher accessory on the mass spectrometer. Ions from the chemical mass marker perf luorokerosene (PFK) are used as reference peaks to determine the appropriate position f o r the

Figure J.

V

y

Sequential single-peak monitoring of the molecular ion of (2). The sample was heated from ambient to 110°C (left to right on the chart); (a) m/e 292.9824 from PFK, (b) m/e 294.9970 from PFK,(c) m/e 295.1420, the molecular ion of (2).

M c

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1

M.W.

290

PERFORMANCE

2.

M.W.

MASS

SPECTROMETRY

295

molecular ion of (2). The PFK i s introduced into the source v i a a heated i n l e t " a t the same time the direct insertion probe i s being used to vaporize the test sample. We have employed t h i s method of monitoring possible patent infringements f o r about f i v e years with a high degree of success i n defense of our patents covering synthetic routes to trimethoprim. A similar strategy was used to determine whether or not some of the pharmacological a c t i v i t y of a codeine, (3), sample was due to trace impurities of morphine, (4). w

Both morphine and codeine have intense molecular ions and small fragment ions. The molecular ion, m/e 299, of codeine fragments by the loss of a methyl group to give an ion at m/e 284. The C isotope of t h i s fragment has the same integer mass as the molecular ion of morphine. They d i f f e r i n compositions by a C vs CH. Therefore t h e i r accurate masses d i f f e r s l i g h t l y (285.1320, C isotope peak from the M-15 of codeine and 285.1365, the M of morphine. The resolution necessary to separate these ions with a 10% valley i s 63,775 (Μ/ΔΜ). This problem can be approached two ways using mass spectro­ metry. The f i r s t i s to calculate the intensity of the C 13

13

12

13

13

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12.

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ET AL.

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233

isotope peak expected and compare t h i s intensity with an averaged intensity observed experimentally. There are several problems involved with t h i s approach. Accurate measurement of peak heights i s d i f f i c u l t , and small variations exist i n abundances of naturally occurring isotopes. A low concentration of morphine w i l l lead to only a small perturbation of the isotopic r a t i o . Even i f a difference were observed, i t i s not certain that morphine i s the cause. A second approach i s to resolve the potential m/e 285 doublet at >60,000 resolution, obtain the percentage of peak height of m/e 285 due to an ion whose accurate mass corresponds to morphine, and calculate an approximate concentration of morphine i n codeine by a direct comparison of t h e i r molecular ions. The l a t t e r approach was selected. At f i r s t , we had d i f f i c u l t i e s i n tuning the resolution of our Varian MAT 731 to the level needed f o r t h i s experiment. We sent a sample of the codeine to Drs. Gross and Lyon at the university of Nebraska Lincoln to attempt the experiment on t h e i r A.E.I. MS-50. The m/e 285 peak i s 0.95% o f the base peak, m/e 299. The A.E.I. MS-50 set to a resolution of >70,000 showeï a doublet at m/e 285. The intensity of the ions was too weak to get a good peak height measurement. At the time Drs. Gross and Lyon had no method of integrating t h i s signal. After careful source cleaning and alignment we were able to obtain a resolution of 62,500 with the Varian MAT 731 and integrated the signal by taking 264 scans into a time averaging computer, Figure 2. The experiment was run over a twenty degree probe temperature range to l i m i t possible d i s t i l l a t i o n effects. In making the calculation the following assumptions were made: 1) No change i n concentration was observed due to fractional d i s t i l l a t i o n . 2) The ionization cross sections of morphine and codeine are the same. 3) The same amount of ion current i s contained i n molecular ions of morphine and codeine at 70 eV. 4) The molecular ion o f codeine does not fragment by the loss o f CH « It i s f e l t that these approximations are reasonable f o r the accuracy needed i n the pharmaceutical evaluation. The experiment indicated a l e v e l of 0.7 parts per thousand of morphine i n the codeine sample. This level was too small to account f o r the pharmacological effects observed. 2

Mixture Analysis By Metastable Ion Methods Metastable methods are discussed more f u l l y i n other sections of t h i s volume. Of the several choices to examine metastable ions, the defocused metastable ion techniques o f accelerating voltage scans keeping the magnetic and e l e c t r i c sector constant and mass analyzed ion k i n e t i c energy methods have been most profitable to our research. The defocused

234

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HIGH

Figure 2. The average of 264 scans over the m/e 285 doublet at 62,500 resolution

PERFORMANCE

MASS

SPECTROMETRY

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

12.

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ET AL.

235

Applications in a Pharmaceutical Lab

metastable work was carried out on the Varian MAT CH5-DF mass spectrometer. This instrument has a reverse geometry, .i.e. the source i s followed by the magnetic sector, e l e c t r i c sector, and electron m u l t i p l i e r . In t h i s configuration no metastable ions are seen i n the primary ion spectra. However, accelerating voltage scans keeping the magnetic and e l e c t r i c sectors constant can be used to observed metastables formed i n the f i r s t f i e l d free region. Mass analyzed ion k i n e t i c energy spectra (MIKES) or direct analysis of daughter ions (DADI) spectra can be used to observe metastable ions formed i n the second f i e l d free region (9,10). The accelerating voltage scan experiment i s carried out by focusing a daughter ion on the electron muljipliçr at 1 or 2 kV accelerating voltage. For the t r a n s i t i o n Mj -> M +(M -M ), the accelerating voltage i s increased u n t i l a daughter ion from a s p e c i f i c parent/daughter decomposition i s observed. The mass of the parent i s calculated by the equation M =M (Vm/Vo) where M^mass of the parent ion; M Emass of the daughter ion; Vo = accelerating voltage required to focus the primary ion daughter; and Vm i s the accelerating voltage required to focus the metastable ions daughter. Thus, a l l parents of a given daughter ion can be determined within a parent/daughter r a t i o that i s limited by the maximum accelerating voltage of the instrument, e.g. a Varian MAT CH-5DF has a maximum r a t i o of 3 and a Varian MAT 731 has a maximum r a t i o o f 5. In the MIKES or DADI mode, a parent i s focused on the electron m u l t i p l i e r of a reverse geomety instrument. The daughter ions from the decomposition i n the second f i e l d free region are brought into focus by decreasing the potential on the e l e c t r i c sector. The mass of the parent ion of these daughters has been selected by adjustment of the magnetic sector. Thus a l l daughters from a given parent over the complete mass range can be determined. The equation used i s M =M!(E/Eo); where Eo i s the e l e c t r i c sector's potential while focusing the parent and Ε i s the e l e c t r i c sector potential needed to observe the daughter ion. An example o f a problem that can be solved by defocused metastable techniques i s given below. In the reaction of (5) with an equivalent of base, i t i s not certain whether the f i r s t methylation occurs at the phenolic s i t e or on the pyrimidine section of the molecule (11). The crude reaction mixture was examined by low resolution electron impact mass spectrometry using 70 eV of ioning energy. The spectrum (Figure 3) appears to indicate a mixture. The masses at m/e 277, 291, 305 and 319 are molecular ions of (5) and mono-, d i - , and trimethylated (5). Reducing the electron voltage from 70 eV to 20 eV reduces the fragmentation. Therefore, most of the ion current i n the spectrum i s contained i n the molecular ions, Figure 4. From the 20 eV spectrum the following r a t i o of concentrations was 2

1

2

2

2

1

2

Figure 3. Mass spectrum of the products of the reaction of (5) with methyl iodide; 70 eV.

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

g

Ο

S3

C/Î

>

ι

>

s 1

*ΰ

Ο S

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12. BRENT ET AL.

Applications in a Pharmaceutical Lab 237

238

HIGH

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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

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MASS

SPECTROMETRY

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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

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ET AL.

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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

i

t

s

a

r

e

2

6

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

HIGH

0

,

II

·

— 0 - P - 0 f Ç H

,BASE

2

BASE

PERFORMANCE

MASS

-HCN or BASE • CO

SPECTROMETRY

BASE FRAGMENT

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

+ P0,

H

___0

ION d

ΙΟΝ α

0=

I

I

HO

BASE CH

CHj

2

· Ο ION b

^

CH

• INDICATES ATTACHMENT SITE NOT RESOLVED. METHYLFURAN LOSES I Η WHEN ATTACHED.

3

ION c

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

d-m C

5

d-hm C

dA

dC

dG

dT

135

111

151

126

125

141

a

215

191

231

206

205

221

b

295

271

311

286

285

301

c

375

351

391

366

365

381

d

186

162

202

176

192

Base + H

*

* Ions b e l o n g i n g t o t h e d c l a s s a r e d e r i v e d from P 0 a t t a c h m e n t t o e x o c y c l i c amino groups o f t h e bases and do n o t appear when such groups a r e e i t h e r absent or a l k y l a t e d . 3

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

140

135 Α

C

148,

f,

66

α

G 202 -L

TC

180 ' 200 ' 2 έ θ

A 172

1,

léO

c 162

A

1215

, C

240

G 231

m /e

285

200 ' 300

J I £ J

260

A ? 2

A 295

320

C

A .3^5

' 400

G 391

Nucleic Acids Research

' 3é0

G *W ί .351 365

' 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

α:20

ω

C40H

S 60

ω

80

ιοο-r

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

«j

Lu >

UJ

109

,00 '

20

40"

60

ΘΟΗ

100

άθ

'

125

140 '

1^

JL

162

léo

'

2ô6

1 ' 2^0 '

205

240

242

' ^

.256

Nucleic Acids Research

' 2à0" ' 300 ' 3έθ

,285

' 3^

,365

' 360 ' 380 ' 400

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

1

176

OH

HO-P-O-ÇHf

5-METHYLDEOXYCYTIDINE- 5-PH0SPHATE

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

IQO

20

^ 40

LU >

è 60

Lu

ω ζ

80

100 i

108

120

117

126

148

140

141

135

,176

_ULil 160 180

162

220

J

231

240

'

240

280

J285

308

300

295

Nucleic Acids Research

?60

252 256

,282

320

,311

WHEAT GERM DNA

Figure 4. Mass spectrum of wheat germ DNA

200 JlL

II

205 206

186 191 192

215

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

340

360

i 351

348 375

380

391

400

> ζ

M

o

M

o

1

H

0

X

2

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

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120

112411

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141

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160

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220

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Nucleic Acids Research

260

5 2

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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

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RELATIVE INTENSITY 8

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>L C H

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

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

HIGH

8

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8

§

A1ISN31NI 3AI±V13U

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8

MASS

Q ου

ω

s

ο

1 S

ε

SPECTROMETRY

ο:

Lu

!5 _ι

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ω

>-

100

201 ιοβ

40

60

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ΙΟΟ

120

114

129

126

140

140

135

162168 1 8 , J . ιφΠΒΒΒίΒΙ

6 ^ ,206|

215 ! , :|263!

309!.

300

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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

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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

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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 <0.1

0 Compounds 0.5 4.3 6.7 10.2 2.4 0.2

3.1

24.3

TABLE XIV. (16) P a r t i a l T a r b o n Number D i s t r i b u t i o n as Mole % i n a Crude and i n a C o a l L i q u i d C no. 9 10 11 12 13 14 15 16 17

Crude

Coal L i q u i d

0.74 1.00 2.51 6.28 9.18 9.65 10.64 8.06 7.45

2.18 20.76 9.55 12.15 8.52 15.41 5.36 8.61 3.78

Conclusions. The methods we have d e v e l o p e d a r e a p p l i c a b l e t o samples from a v a r i e t y o f s o u r c e s and w h i c h c o n s t i t u t e many t y p e s . Examples i n c l u d e c o a l l i q u e f a c t i o n p r o d u c t s , s y n c r u d e s , s h a l e o i l s , and c o n v e n t i o n a l p e t r o l e u m s t r e a m s . The sample c h a r a c t e r i z a t i o n i s f a s t and d e t a i l e d . M i n o r changes i n c a l i b r a t i o n d a t a and i n t h e p r e s e n t a t i o n o f t h e summary t a b l e s as r e q u i r e d by d i f f e r e n t sample s o u r c e s i s a c c o m p l i s h e d by coded o p t i o n s b u i l t i n t o t h e computer programs. Although p r i o r separation into saturate, aromatic, and p o l a r f r a c t i o n s i s d e s i r a b l e f o r p e t r o l e u m f r a c t i o n s , i t i s not r e q u i r e d f o r the mostly aromatic coal l i q u i d s . Sample q u a n t i t y i s o f t e n l i m i t e d i n some b e n c h - s c a l e e x p l o r a t o r y r e s e a r c h . W i t h t h i s t e c h n i q u e many i m p o r t a n t c h a r a c t e r i s t i c s such as e l e m e n t a l a n a l y s i s , d e n s i t y , and d i s t i l l a t i o n c u r v e s

14.

LUMPKIN

A N D ACZEL

Ultra-High Resolution Mass Spectrometry

273

can be c a l c u l a t e d w h i c h would n o t o t h e r w i s e be o b t a i n a b l e because o f t h e s m a l l sample s i z e .

Literature Cited

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

1.

Aczel, T . , Foster, J.Q. and Karchmer, J.H., presented at Division of Fuel Chemistry, 157th National Meeting of ACS, Minneapolis, Minnesota, April (1969). 2. Aczel, T. and Lumpkin, H.E., presented at 18th Annual Conference on Mass Spectrometry and Allied Topics, San Francisco, California, June 14-19, 1970. 3. Aczel, T . , Allan, D . E . , Harding, J.H. and Knipp, E.A., Anal. Chem., (1970), 42, 341. 4. Aczel, T. and Lumpkin, H.E., presented at 19th Annual Conference on Mass Spectrometry and Allied Topics, Atlanta, Georgia, May 2-7, 1971. 5. Beynon, J.H., Mass Spectrometry and Its Application to Organic Chemistry, Elsevier, Amsterdam, (1960). 6. Brown, R.A., Anal. Chem., (1951) 23, 430. 7. Field, F.H. and Hastings, S . H . , Anal. Chem., (1956), 28, 1248. 8. Johnson, B.H. and Aczel, T . , Anal. Chem., (1967), 39, 682. 9. Lumpkin, H.E. and Johnson, B . H . , Anal. Chem., (1954), 26, 1719. 10. Lumpkin, H.E., Anal. Chem., (1958), 30, 321. 11. Lumpkin, H.E. and Taylor, G.R., Anal. Chem., (1961), 33, 476. 12. Lumpkin, H.E. and Aczel, T . , Anal. Chem., (1964), 36, 181. 13. Lumpkin, H.E., Anal. Chem., (1964), 36, 2399. 14. Lumpkin, H.E., Wolstenholme, W.A., E l l i o t t , R.M., Evans, S., and Hazelby, D . , presented at 23rd Annual Conference on Mass Spectrometry and Allied Topics, Houston, Texas, May 25-30, (1975). 15. O'Neal, M . J . , Jr. and Weir, T . P . , Jr., Anal. Chem. (1951), 23, 830. 16. Reprinted with permission from Reviews of Anal. Chem., (1971), 1, 226. Received December 30, 1977

15 Organic Mixture Analysis by Metastable Ion Methods Using a Double-Focusing Mass Spectrometer E. J. GALLEGOS

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

Chevron Research Co., Richmond, CA 94802

This paper describes two ways in which metastable transition spectra may be used in the analysis of organic compounds or mixtures. The f i r s t section describes a technique from which single ion detection GC-MS type information is obtained from the metastable transition spectra without the benefit of a gas chromatograph. The second section describes a technique for obtaining mass ratio and intensity information without the use of magnet, quadrupole, or time of flight techniques. Theory Metastable transitions in mass spectrometry were f i r s t reported by Hipple and Condon (1) in 1945. The theories of metastable transitions and the techniques of obtaining these kinds of data have been summarized recently. (2,3) A brief review of how and where metastable ions occur in a double focusing mass spectrometer pertinent to this work w i l l be given here. The composition of ions reaching the collector of a mass spectrometer is determined by the rate constants, k , of many competing consecutive, unimolecular decompositions of excited molecular and fragment ions. The recorded ion current intensity versus the massto-charge ratio gives the mass spectrum. The residence time of an ion in source is about 10 seconds. There is an additional ~10 seconds elapsed time after acceleration to collection. Decompositions requiring less than 10 seconds occur in the source and w i l l be analyzed and collected to give a normal mass spectrum. n

-6

-5

-6

©0-8412-0422-5/78/47-070-274$10.00/0

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

15.

275

Double-Focusing Mass Spectrometer

GALLEGOS

M e t a s t a b l e t r a n s i t i o n s are due t o t h o s e decom­ p o s i t i o n s w h i c h t a k e p l a c e a f t e r the i o n l e a v e s the s o u r c e but b e f o r e i t r e a c h e s the d e t e c t o r . These d e c o m p o s i t i o n s can t a k e p l a c e i n Regions 1-6 o f F i g u r e 1. These r e g i o n s are d e f i n e d by t h e i r d i f f e r e n t i o n r e s i d e n c e time and f i e l d o r f i e l d f r e e c o n f i g u r a t i o n . The o n l y m e t a s t a b l e t r a n s i ­ t i o n s w h i c h are d e t e c t e d and used i n t h i s work are t h o s e w h i c h o c c u r i n R e g i o n 2. Ions w h i c h decompose i n R e g i o n 2 have r e c e i v e d f u l l a c c e l e r a t i o n but have n o t been energy a n a l y z e d i n the e l e c t r o s t a t i c s e c t o r . They, h a v i n g l o s t mass, w i l l not have the r e q u i r e d t r a n s l a t i o n a l energy t o pass t h r o u g h the m o n i t o r s l i t and w i l l impinge on the bottom e l e c t r o s t a t i c p l a t e a t some p o i n t ( i) a ( i ) b > e t c . ; see F i g u r e 1. They can be made t o pass t h r o u g h the m o n i t o r s l i t e i t h e r by i n c r e a s i n g the a c c e l e r a t i n g v o l t a g e o r by l o w e r i n g the e l e c t r o s t a t i c p l a t e v o l t a g e . There are two ways t o use t h i s f a c t . I f the magnet i s s e t t o f o c u s on some mass, say fragment M , and t h e e l e c t r o s t a t i c v o l t a g e i s h e l d c o n s t a n t w h i l e the a c c e l e r a t i n g v o l t a g e i s i n c r e a s e d , a l l m e t a s t a b l e peaks observed a t the m u l t i p l i e r d e t e c t o r w i l l be due t o p r e c u r s o r i o n s Μχ ( a , b , . . . , n ) , w h i c h decompose i n R e g i o n 2 t o g i v e M . The mass o f the p r e c u r s o r s M i (a,b,...,n) can be c a l c u l a t e d from the f o l l o w i n g equation M

+

o r

M

+

2

+

2

+

V

m

Μχ

- M

(1)

2

ο

where M • mass o f d a u g h t e r o r fragment i o n Μχ • mass o f the p r e c u r s o r i o n V a c c e l e r a t i n g voltage at which a m e t a s t a b l e i s observed V • a c c e l e r a t i n g v o l t a g e a t w h i c h the main beam i s i n f o c u s 2

s

m

Q

T h i s , i n e s s e n c e , p r o v i d e s the o p p o r t u n i t y t o o b s e r v e and i d e n t i f y a l l p a r t i c i p a n t s i n a s p e c i f i c r e a c t i o n process +

(M!) a,b,c...

M

+ 2

- [(MOa.b.c... - M ] 2

w i t h o u t i n t e r f e r e n c e from any o t h e r i o n s or p r o ­ c e s s e s w h i c h are o c c u r r i n g s i m u l t a n e o u s l y . In e f f e c t , t h i s p r o v i d e s an i s o l a t i o n t e c h n i q u e w i t h o u t p h y s i c a l s e p a r a t i o n but w i t h a r e s u l t i n g c a p a b i l i t y somewhat s i m i l a r t o GC-MS.

(2)

2 kv-8 kv

Figure 1. Metastable transition schematic

Chevron Research Company

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

15.

GALLEGOS

Double-Focusing Mass Spectrometer

277

T h i s method o f r e c o r d i n g and i n t e r p r e t i n g meta­ s t a b l e t r a n s i t i o n s p e c t r a i s c a l l e d the a c c e l e r a t i n g v o l t a g e scan method and was used t o o b t a i n the d a t a d e s c r i b e d i n S e c t i o n I under r e s u l t s . The a l t e r n a t e method i s t o u s e the t o t a l i o n m o n i t o r o f a double f o c u s i n g i n s t r u m e n t as a detector. I f t h e a c c e l e r a t i n g v o l t a g e i s scanned i n a manner d e s c r i b e d above a l l o f the m e t a s 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 R e g i o n 2 w i l l be d e t e c t e d . The s p e c t r a o b t a i n e d c a r r y mass r a t i o , Μ!/Μ , and i n t e n s i t y i n f o r m a t i o n . The a c c e l e r a t i n g v o l t a g e s V and V and the mass r a t i o Mi/M are r e l a t e d a c c o r d i n g t o E q u a t i o n 1. Only the e l e c t r o s t a t i c s e c t o r i s used i n t h i s method. T h i s second method o f r e c o r d i n g and i n t e r ­ p r e t i n g metastable t r a n s i t i o n s p e c t r a , c a l l e d the IKES method, was used t o o b t a i n the d a t a d e s c r i b e d i n S e c t i o n I I i n the R e s u l t s S e c t i o n . 2

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

0

2

m

Instrumentation T h i s work was done on an ΑΕΙ MS-9 d o u b l e f o c u s 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 . The i n s t r u m e n t was m o d i f i e d so t h a t the a c c e l e r a t i n g v o l t a g e c a n be scanned from 2-8 kV. The e l e c t r o s t a t i c s e c t o r v o l t a g e i s h e l d c o n s t a n t a t 135 v o l t s w h i c h , w i t h an a c c e l e r a t i n g v o l t a g e o f 2 kV, w i l l produce normal mass s p e c t r a . The sample i s i n t r o d u c e d t h r o u g h an a l l - g l a s s , h o t i n l e t (4) o r by u s i n g a d i r e c t i n s e r t i o n probe. I n the f i r s t method o f r e c o r d i n g m e t a s t a b l e t r a n s i t i o n s , a c c e l e r a t i n g v o l t a g e s c a n s , the magnet i s adjusted t o a current which w i l l b r i n g i n t o focus a daughter i o n M of interest. I n the second method o f r e c o r d i n g m e t a s t a b l e t r a n s i t i o n s p e c t r a , the t o t a l i o n m o n i t o r i s used as the d e t e c t o r w h i c h means t h e magnet i s not u s e d . For b o t h the a c c e l e r a t i n g v o l t a g e i s t h e n scanned from 2-8 kV t o produce t h e m e t a s t a b l e t r a n s i t i o n s p e c t r a shown i n t h i s s t u d y . T h i s arrangement w i l l a l l o w m e t a s t a b l e t r a n s i ­ t i o n s t o be o b s e r v e d due t o p r e c u r s o r i o n s Μχ"" w i t h a mass up t o f o u r times t h a t o f the d a u g h t e r i o n M . +

2

1

+

2

Results S e c t i o n I . A c c e l e r a t i n g v o l t a g e scan a n a l y s i s of metastable ions i s p o s s i b l e only i f a decomposition p r o c e s s can be found t h a t i s u n i q u e l y c h a r a c t e r i s t i c o f a compound o r compound t y p e . There are many systems w h i c h obey the r e q u i r e m e n t s f o r t h i s t e c h ­ n i q u e o f a n a l y s i s . Three examples o f t h i s are g i v e n .

278

HIGH

PERFORMANCE

MASS

SPECTROMETRY

Terpanes and S t e r a n e s Terpanes and s t e r a n e s a r e two t y p e s o f compounds t h a t f a l l w e l l i n s i d e t h e s e r e q u i r e m e n t s . Many t e r ­ panes, e i t h e r p e n t a c y c l i c - o r t r i c y c l i c - s a t u r a t e d h y d r o c a r b o n s found i n n a t u r e , fragment upon e l e c t r o n impact t o g i v e a base peak a t m/e 191 fragment i o n . (5_,6) F i g u r e 2 shows the m e t a s t a b l e spectrum o f a u t h e n t i c gammacerane, a C t e r p a n e and a proposed scheme f o r f r a g m e n t a t i o n o f t h e p a r e n t i o n t o t h e m/e 191 fragment i o n . S i m i l a r l y , s t e r a n e s w h i c h are s a t u r a t e d t e t r a c y c l i c h y d r o c a r b o n s fragment t o g i v e a base peak a t m/e 217 fragment i o n . F i g u r e 3 shows t h e m e t a s t a b l e spectrum o f a u t h e n t i c c h o l e s t a n e , a C 7 s t e r a n e and a p r o p o s e d scheme f o r f r a g m e n t a t i o n o f the p a r e n t i o n t o t h e m/e 217 fragment i o n . 3 0

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

2

Green R i v e r S h a l e The w e l l - a n a l y z e d , s a t u r a t e d f r a c t i o n o f the e x t r a c t a b l e p o r t i o n o f Green R i v e r S h a l e (7) i s used t o demonstrate t h e use o f the method. F i g u r e 4 shows the m e t a s t a b l e s c a n o f m/e 191 o f t h i s f r a c t i o n . The most i m p o r t a n t peaks a r e t h o s e due t o C χ , C , and C , f o l l o w e d by t h e C o and C terpanes. S i m i l a r l y , F i g u r e 5 p r e s e n t s t h e m e t a s t a b l e r e s u l t s f o r a scan o f m/e 217. These r e s u l t s show t h a t t h e l a r g e s t meta­ s t a b l e peak i s due t o a C s t e r a n e f o l l o w e d by t h e C e and, f i n a l l y , C 7 s t e r a n e s . F i g u r e 6 shows t h e mass chromatograms o f m/e 191 and 217 f o r t h i s f r a c t i o n . The normal mass spectrum was used t o c a l c u l a t e the t o t a l c o n c e n t r a t i o n o f t e r p a n e s and s t e r a n e s i n t h i s sample. T h i s i s done i n t h e f o l l o w i n g manner. The m/e 191 fragment i o n measured 2.33% o f the t o t a l i o n i ­ z a t i o n o f the s a t u r a t e f r a c t i o n o f Green R i v e r S h a l e . T h i s fragment i o n r e p r e s e n t s o n e - e i g h t h o f t h e t o t a l i o n i z a t i o n i n t h e mass spectrum o f the average authen­ t i c t e r p a n e i n t h i s sample so t h a t the t o t a l t e r p a n e c o n t r i b u t i o n t o t h e t o t a l i o n i z a t i o n o f t h e sample i s 18.6%. The m/e 217 fragment i o n measured 1.25% o f the t o t a l i o n i z a t i o n o f the s a t u r a t e f r a c t i o n o f Green R i v e r S h a l e . T h i s fragment i o n r e p r e s e n t s about onef i f t e e n t h o f the t o t a l i o n i z a t i o n i n the mass spectrum o f the average s t e r a n e i n t h i s sample so t h a t the t o t a l s t e r a n e c o n t r i b u t i o n t o t h e t o t a l i o n i z a t i o n o f the sample i s about 18.8%. The v a l u e s o f c o n t r i b u t i o n t o t o t a l i o n i z a t i o n a r e e q u i v a l e n t t o c o n c e n t r a t i o n s . (8) 3

2 9

2

2 1

2 9

2

2

3 0

15.

GALLEGOS

279

Double-Focusing Mass Spectrometer

m/e 191

Terpane

m/e 191

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

J.

JL

J

Vo Chevron Research Company

Figure 2. Metastable spectrum of m/e 191 of gammacerane

m/e 217

c

2

7

Ci.

Sterane

m/e 217

. A

^

Λ

«

\ V

0

V

n

Chevron Research Company

Figure 3.

Metastable spectrum of m/e 217 of cholestane

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

HIGH PERFORMANCE MASS SPECTROMETRY

191

m/e

1 Si Retention

Time

ι

Η π II

6. Green River shale saturate cut m/e 191 terpane and m/e

217

m/e

«1*14·

217 sterane mass chromatogram

Chevron Research Company

M)l2«

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

282

HIGH

PERFORMANCE

MASS

SPECTROMETRY

The a c c e l e r a t i n g v o l t a g e s c a n r e s u l t s were o b t a i n e d by m e a s u r i n g t h e m e t a s t a b l e peak h e i g h t and t h e n n o r m a l i z i n g a l l components t o g i v e 18.6% t e r p a n e s and 18.81 s t e r a n e s . A comparison o f t h e r e l a t i v e c o n c e n t r a t i o n s o f t e r p a n e s and s t e r a n e s i n the s a t u r a t e f r a c t i o n o f Green R i v e r S h a l e as d e t e r m i n e d b y GC-MS and t h e a c c e l e r a t i n g v o l t a g e scan method a r e g i v e n i n T a b l e I .

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

Table I Comparison o f GC-MS and A c c e l e r a t i n g V o l t a g e Scan (AVS) Method R e s u l t s o f Terpanes and S t e r a n e s i n the S a t u r a t e P o r t i o n o f Green R i v e r S h a l e

GC-MS

AVS Method

Terpanes C

C

C

C

3 1 3 0 29 2 1

C20

Total

1.0 13.6 2.3

1.1 13.2 2.2

0.4 1.4

0.4 1.7

18.7

18.6

10.3 7.6 1.9

10.1 6.3 1.8 0.6

19.8

18.8

Steranes C 9 2

C

28

C 7 2

C 2 2

Total

The a c c e l e r a t i n g v o l t a g e scan t e c h n i q u e s g i v e s t h e sum o f a l l t e r p a n e s o r s t e r a n e s a t a g i v e n molec u l a r w e i g h t ; whereas, isomers c a n be d i s t i n g u i s h e d by GC-MS. The t o t a l t e r p a n e and s t e r a n e c o n c e n t r a t i o n s c a l c u l a t e d a r e w i t h i n 1% o f t h o s e o b t a i n e d by GC-MS.

15.

GALLEGOS

283

Double-Focusing Mass Spectrometer

P y r o l y z e d Green R i v e r S h a l e A s i m i l a r a n a l y s i s was made on kerogen p y r o l y z a t e from Green R i v e r S h a l e . The comparison o f the r e l a t i v e c o n c e n t r a t i o n s o f t h e C 7 t o C χ terpanes determined by the a c c e l e r a t e d v o l t a g e scan method and GC-MS a r e g i v e n i n T a b l e I I . 2

3

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

Table I I Comparison o f GC-MS and A c c e l e r a t i n g V o l t a g e Scan (AVS) Method R e s u l t s o f Terpanes and S t e r a n e s i n t h e S a t u r a t e Cut from t h e P y r o l y s i s O i l from Green R i v e r S h a l e Percent AVS GC-MS Method Terpanes

28 C2 7

0.5 2.0 2.0 0.2 2.4

1.3 2.0 2.0 0.9 1.5

Total

7.1

7.7

C

28 C2 7

0.8 0.6 0.3

0.7 0.5 0.2

Total

1.7

1.4

C C

3 X 3 0

C

2 9

C

Sterane C

2 9

T o t a l t e r p a n e s f o r a c c e l e r a t e d v o l t a g e scan r e s u l t s were c a l c u l a t e d from t h e normal mass spectrum as b e f o r e . Here t h e a c c e l e r a t i n g v o l t a g e s c a n method gives a low v a l u e f o r the C 7 terpanes. This i s because one o f t h e C 7 t e r p a n e s does n o t fragment e x c l u s i v e l y t o g i v e m/e 191 as t h e base peak. (9J) 2

2

284

HIGH

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

Alkyl

PERFORMANCE

MASS

SPECTROMETRY

Phthalates

A l k y l p h t h a l a t e s , used g e n e r a l l y as p l a s t i c i z e r s i n p l a s t i c t u b i n g f a b r i c a t i o n , have become u b i q u i t o u s c o n t a m i n a n t s i n the e n v i r o n m e n t . An o r g a n i c concen­ t r a t e from a r e f i n e r y e f f l u e n t w a t e r sample c o n s i s t i n g m a i n l y o f n a p h t h e n i c a c i d s , was found t o c o n t a i n low c o n c e n t r a t i o n s o f d i - n - b u t y l p h t h a l a t e . The mass s p e c t r a o f n - d i b u t y l p h t h a l a t e shows a c h a r a c t e r i z i n g base peak a t m/e 149, w h i c h r e s u l t s from the decompo­ s i t i o n r o u t e s g i v e n i n F i g u r e 7. The m e t a s t a b l e spectrum i s shown i n F i g u r e 8, t o p , f o r pure d i - n - b u t y l p h t h a l a t e . F i g u r e 8, b o t t o m , shows the s e a r c h f o r p h t h a l a t e s i n the o r g a n i c concen­ t r a t e from the r e f i n e r y w a t e r sample. The p r e s e n c e o f p h t h a l a t e i s c l e a r l y shown by the b l a c k peaks. M e t a s t a b l e s p e c t r a l Peaks A and Β a r e due t o T r a n s i t i o n s A and Β shown i n the scheme i n F i g u r e 7. Even i n the p r e s e n c e o f o t h e r components w h i c h form an m/e 149 i o n on e l e c t r o n i m p a c t , the p r o g e n i t o r s are s u f f i c i e n t l y d i f f e r e n t i n mass so t h a t the p h t h a l ­ a t e m e t a s t a b l e peaks are c l e a r l y r e s o l v e d . Summary The a c c e l e r a t i n g v o l t a g e s c a n t e c h n i q u e o f a n a l y s i s may be a p p l i e d o n l y i f the d e c o m p o s i t i o n p r o c e s s e s o f the m o l e c u l a r t y p e s b e i n g s e a r c h e d are w e l l u n d e r s t o o d . Once the magnet i s s e t t o a c c e p t o n l y one p r e s e l e c t e d mass, say M , the o n l y time the m u l t i p l i e r w i l l produce a s i g n a l i s when the c o r r e c t p a r e n t i o n , Μ ^ , and a c c e l e r a t i n g v o l t a g e , V , are f o u n d . A l l t h i s happens i n the p r e s e n c e o f many o t h e r components and t r a n s i t i o n s w h i c h are i g n o r e d . T h i s t e c h n i q u e has many o f the b e n e f i t s o f GC-MS but d o e s n ' t r e q u i r e an a c t u a l s e p a r a t i o n o f m o l e c u l e s . +

2

m

Section II Mass r a t i o o f p r e c u r s o r mass t o f i n a l mass, M i / 2 > 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

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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.

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ETAL.

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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

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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.

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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.

MCLAFFERTY

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

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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.

MCLAFFERTY

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

HIGH

PERFORMANCE

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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

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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 <100

2A

Four even-mass, four odd-mass

6-88

2B

Eight

47-102

3A

Seven

61-116

3B

Seven

89-158

4A

Six

117-200

4B

Six

159-(M - 1)

5-6

+

Primary neutral losses

5A

Five

0-2,15-20, 26-53

5B

Five

34-75

6A

Five

59-109 (MW>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

<65

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It is important to emphasize that STIRS has been designed as an aid to, not as a replacement for, the human interpreter, its use making the interpretation more efficient and possibly more complete. The basic way i n which an interpreter uses STIRS i s to examine the best matching compounds found for each data class and for the overall match factors (Table I) i n an attempt to find common structural features. For example, if one data class showed a substantial proportion of compounds containing an isoxazole ring and another data class found mainly acetoxy compounds, these would be important postulates for the interpreter to test i n trying to assemble a molecular structure for the unknown. A l l of the reference compounds are coded i n Wiswesser Line Notation (WLN), so that the interpreter often finds it more convenient to scan these by eye to detect common structural features. There are two ways i n which the computer can help i n this task. Currently Dr. Michael M. Cone i n our laboratory i s developing a program for computer identification of "super substructures" which involves the comparison of decoded WLNs of the selected compounds to find the largest common structural features. A special system which has already been implemented by Dr. H. E. Dayringer i n our laboratory (13) predicts the presence of 179 common substructural fragments from the STIRS results. For t h i s , the 15 compounds selected i n each data class as providing the best match to the unknowns mass spectrum are examined by the computer for the presence of each of the preselected substructural fragments, and the statistical significance of the number found i s evaluated by a "random drawing" model. For example, because 28% of the compounds i n the reference f i l e contain phenyl, even if the unknown does not contain phenyl an average of ~4*phenyl-containing compounds would s t i l l be expected i n the 15 compounds of highest match factor values. However, if for a particular unknown there are 10 phenyl compounds i n the top 15, statistically this w i l l occur by coincidence only once i n 113 times; it follows that i n >99% of cases this w i l l be due to the presence of phenyl i n the unknown compound, i . e . , i n <1% of predictions based on such data w i l l the result be a false positive. To check t h i s , using the overall match factor (MF11) two runs were made of 373 "unknown" mass spectra (every 50th spectrum i n the Registry data base) (21); the substructures predicted at the 2.0% or less false positive level actually contained an average of 1.0% false positives, i n excellent agreement. The MF11 results (13) at predicted levels of <0.5%, <0.2%, and<0.1% gave average false positives of 0.7%, 0.55%, and 0.45%, respectively; these are not as low as expected s t a t i s t i c a l l y , but this appears to be mainly due to the fact that most of the falsely selected substructures have similar mass spectral behavior (e.g., pyridyl identified as phenyl). Typical results are shown i n Table II. In practice, the substructures selected are ranked i n order of their predicted values, reporting only those of ^ 2 % false positives. Not surprisingly, more reliable

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Table II. Substructure Identification by STIRS and K-Nearest Neighbors^

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

Substructure

% in STIRS f i l e <2% FPk <0.5% FP

KNN 3/5£ 3/3

FP

RC FP

RC

FP

RC FP

4.8 19 0.8

Ester, anhydride

19

55

2.1

50 1.4

29

Phenyl, sub. phenyl

28

75

5.0

67 3.4

71 11.7

8

74

0.3

74 0.0

54

0.7 47 0.5

Doubly-branched C

11

44

3.4

42 1.5

41

4.1

25 1.3

20 Substr.— average

14

42

1.9

36 1.2

43

7.9

27 2.1

49

1.9

44 0.7

Chlorine

179 Substr. average^

2.9

51 3.7

^References 13 and 26. -RC, % recall; FP, % false positives. % h r e e of the five nearest neighbors contain the substructure. -Others: a l k y l ^ C ^ > C , ^ C , carbonyl, -OH, - N H , -NH-, trisubst. N, S, - C H 2 O - , F, C I , -Ο-, double bond, singlebranch C, double-branch C, carbocyclic rings, and heterocyclic rings. 2

3

2

predictions are generally found for the overall match factors MF11.0, 11.1, and 11.2, which are derived from combinations of the values for the individual data c l a s s e s , as shown i n Table I. The 202 substructural features tested varied widely i n their occurrence i n the reference file; almost half of the com­ pounds contained the substructures carbonyl and methyl/ methylene, with only 6 each of compounds containing azulene and pyrene i n a total of 18,806 compounds. The substructures also varied widely i n their recall; trimethylsilyl was identified correctly i n a l l but one of the 30 compounds tested which con­ tained i t , while 24 of the substructures gave zero r e c a l l . As the experienced mass spectrometrist knows, functional group infor­ mation varies widely i n its " v i s i b i l i t y " i n the mass spectrum, not only between different groups, but also according to the environ­ ment of the group i n the molecule. In an infrared spectrum, a carbonyl group almost always gives a characteristic peak i n the carbonyl-stretching region. However, from a mass spectrum the presence of a carbonyl group often must be inferred from a com­ bination of peaks: the acetyl or benzoyl groups can give

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characteristic peaks, but not usually the uncombined carbonyl group. Further, i n the mass spectrum of acetone the acetyl group i s very v i s i b l e as the base peak at m/e 43, but i n the mass spectrum of dimethylaminoacetone this peak i s small (5%) and "hidden" next to a larger peak; the competing fragmentation directed by the amino group i s dominant. For the carbonyl group, STIRS-MF11 achieved only a 31% recall at the 98% confidence l e v e l . However this does not necessarily mean that STIRS has not been optimized properly for the carbonyl group; unless some other algorithm can show a superior performance, i t i s more l i k e l y that mass spectrometry i s not particularly sensitive for the carbonyl group per se. Despite this variation i n r e c a l l , even the l i s t of 179 substructures with non-zero recall on average givesa substantial amount of information concerning an unknown structure. M u l t i plying the recall for each substructure by its proportion i n the data base and summing these values gives a figure of 2.55, and improvements to STIRS ( H , 15) have raised this to approximately 3; this i s the number of correct substructure identifications which STIRS should give for the average unknown spectrum. Because 1% of the 179 substructures should be indicated as false p o s i t i v e s , this means that on average there w i l l also be 2 incorrect substructure identifications. However, i f the interpreter recognizes that a few false positives are possible, he can often d i s card these as unlikely candidates due to other mass spectral or separate evidence. We have recently put a substantial effort into the improvement of recall values for the substructure l i s t by optimization of the STIRS data c l a s s e s , i n particular, the "characteristic ions" (14) and the "primary neutral l o s s e s " (15); those changes were adopted which showed improvements i n the average r e c a l l values for 373 "unknown" spectra, without degradation of the reliability performance. Not surprisingly, the best recall values are found for those substructures which are known to have a strong directing influence on mass spectral fragmentations. We are now engaged in a revision of "Mass Spectral Correlations" (27) based on the reference mass spectral f i l e of 29,468 compounds for STIRS; the substructures found to give the best correlations here w i l l be tested with STIRS, and i t i s expected that this w i l l give a much larger l i s t of substructures with generally higher r e c a l l values. We have also made substantial progress on an algorithm to determine the molecular weight from the unknown mass spectrum, even i f a molecular ion i s not present. System Evaluation; Effect of F i l e Composition of Tested and Reference Spectra It i s obviously necessary for interested workers to reach a common understanding of evaluation terms such as reliability (used here as synonomous with "% correct" X .01), false

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

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positives, and recall (equations 1 - 3), and their applicability for comparison of alternative systems (6 - 15, 26, 29). In testing the efficiency of the STIRS system we based this on false positives and recall values; yet for PBM, as done by others for other retrieval systems, we used reliability as w e l l as recall values. We feel that past reports, including our own, have not fully recognized the effect of the proportion of the component sought in both the tested and reference spectral files (28). For retrieval systems such as PBM the proportion of false positives i s also characteristic of the system's performance; the number of incorrect matches produced should be proportional to the size of the reference file (assuming these spectra are of ran­ dom composition, and that the proportion of correct answers i n the file i s small). In fact, PBM i s designed to make the Κ value a direct measure of the probability of a false positive if the mass and abundance data of each reference spectrum are distributed i n the same manner as those of the whole f i l e . However, at a pre­ dicted FP value of 2 " (K = 50), the observed FP =-1/50,000; such values are greater than the prediction i n large part because mass values tend to be clustered (such as "ion series"); sup­ porting t h i s , class IV matches at Κ = 50 give FP =-1/500,000. Because the recall and false positives values are independent of occurrence probabilities (equation 4), i t i s better to use RC and FP i n evaluating the performance of retrieval as well as interpre­ tive systems. 5 0

RL = Ρ -RC/(P -RC + P--FP) C

C

(4)

X

Retrieval systems are commonly tested using a data base containing one reference spectrum of each unknown; equation 4 shows for such cases that using a larger data base must reduce the reliability value found, as the number of correct retrievals w i l l remain the same while the number incorrect increases. If a reference spectrum i s retrieved for a true unknown, the user would l i k e to know the probability that this answer i s correcthowever, this reliability depends not only on the system's recall and false positives performance, but also on the probability that the unknown compound i s represented i n the reference f i l e (thus even i f i t gave "perfect" RC and FP values, PBM could not match an unknown not represented i n the data base). Doubling the number of compounds i n the data base should increase the proba­ b i l i t y that the unknown w i l l be represented, but i f the new reference spectra are of "rarer" compounds the reliability [Ic/(Ic + If)l of answers w i l l decrease because the number of false positives (If) must double. This i s the basic reason for the use of specialized data bases for unknowns from restricted areas such as drugs or flavors. Thus the reliability of retrieval results depends on the proportion of the compounds tested which are represented i n the data base and on the proportion of compounds in the data base which correspond to the compound tested. On

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the latter, the reliability of PBM results i s improved by including in the file multiple reference spectra of many of the more common compounds . It may be helpful to examine the application of such per­ formance results to evaluate the reliability of the system's pre­ diction for a real unknown. To illustrate, at the 50% recall level (average ΔΚ = 30) PBM shows -1/50,000 false positives* (11); i f for an unknown PBM finds one match (ΔΚ = 30) i n a 25,000 spec­ trum f i l e there w i l l be a 50% chance (25,000/50,000) that this i s a false positive. However, the offsetting chance that this i s the right answer w i l l only correspond to the full 50% recall value i f there i s a high probability that one of the reference spectra i s of the same compound. Thus the user must combine (equation 4) the RC and FP values with his own estimates of the probability that the unknown i s present i n the f i l e . In making a similar examination of the use of such evalu­ ation terms for STIRS, i t should be noted that i n effect STIRS i s matching the unknown against the spectral data of each sub­ structure sought. Thus the present STIRS "substructure matching" system has only 179 entries i n its reference f i l e , and 1.0% false positives corresponds to an average of two incorrect identifica­ tions per search; for our present PBM spectral file of 42,000 com­ pounds, a false positives of only 1/21,000 would yield this num­ ber incorrect per search. Allowing this higher false positives level for STIRS makes i t able to achieve a higher recall; again, however, the utility to the user w i l l also depend on the proba­ bility that one or more of the 179 substructures w i l l be present in the unknown. In our data base collected from a wide variety of sources (21) the occurrence probabilities range from 46% (carbonyl) down to 0.03% (azulene), with an average of 2.9% for each of the 179 selected substructures. At the 1.0% false p o s i ­ tives level STIRS (MF 11) gave an average recall of 49% (13); thus (as explained above) for an unknown taken at random from this data base on average i t should predict 2.55 (2.9% X 179 X 49%) correct (and 2 incorrect) substructures of 179 per unknown [more recent (14, 15) STIRS improvements have raised the recall to give ~3 correct]. Consider the 20 common substructures of Table I I , which actually show poorer than average RC and FP performance; on average STIRS should predict 1.01 (14% X 20 X 36%) of these 20 substructures correctly for only 0.24 (1.2% X 20) incorrect. Although the carbonyl and azulene substructure have equivalent 1% chances of being found as false positives, the carbonyl w i l l The testing of PBM utilized a data base of -24,000 spectra i n which an average of ~2 were of the same compound as the un­ known tested. Thus at 50% recall on average one correct answer is found ( I = 1). For this recall (low molecular weight trial set) the reliability = 65% = 1/(1 + I ) , so I - 0 . 5 , and FP = 0.5/ 24,000 =-1/50,000. c

f

f

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

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be identified on average i n 14% (46% X 31% recall) of such unknowns, but the azulene w i l l only be found i n 1/1,000 of this frequency (0.03% X 50% recall). Obviously a STIRS identification of such low-frequency substructures as azulene i n most cases i s a false positive, but usually the interpreter can recognize this from other information on the sample as w e l l as from interpretation of the spectrum. Re-emphasizing, this low frequency refers only to the compounds i n our comprehensive data base; the true reliability of the results i s determined by the actual frequency i n the environment from which the unknown has been taken. If this frequency for azulene should be high, STIRS w i l l show a high reliability for the identification of this substructure. To summarize, an important difference between tests of compound identification systems such as PBM and substructure identification systems such as STIRS i s that generally in the former there i s no attempt to study the system's ability to identify different types of compounds. A particular compound i s used only once i n the retrieval test, and so its occurrence frequency i s the same as a l l other compounds tested; this would also be true of the reference set if i t i s of a l l different compounds. Thus i f the reference files used i n different tests are approximately the same with respect to size and average number of spectra per compound, the "reliability" values obtained w i l l be directly related to the "false positives" values at a particular recall l e v e l (equation 4). For evaluations of retrieval systems such as PBM either RL or FP values can be reported, but data necessary for their interconversion should be given. It i s better to report "false positives" for the evaluation of substructural identification systems such as STIRS, as the substructure occurrence frequency can vary by orders of magnitude. Also, following the common practice for studies of document retrieval systems (17) [and of PBM (11)3, recall should be evaluated as a function of the false positive l e v e l . In reports of particular improvements to STIRS (13-15) we used the value of RC/(RC + FP) as a modified " r e l i a b i l i t y " term, really a measure of the utility of the system; this combined recall and false positives i n a single term that would be independent of the substructure occurrence frequency. However, there has been considerable confusion over the use of this term, and i t now appears preferable to report RC and FP separately. Comparison of STIRS With Other Available Mass Spectral Systems. One advantage of STIRS i s that i t i s the only interpretive system suitable for most types of organic compounds which is generally available, although interpretive information can be obtained from the results of retrieval systems. The Artificial Intelligence system i s available for selected compound types through Stanford University, as discussed i n another paper i n this program. Very extensive research studies on pattern recognition or learning machine approaches to mass spectral interpretation have been reported over the last half-dozen years, as

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summarized i n Professor Isenhour's talk, although such systems apparently have not seen any appreciable use for real unknowns. Justice and Isenhour have shown that the K-Nearest Neighbor algorithm i s superior to 5 other common pattern recognition methods i n its ability to extract information from mass spectra (7, 26); to compare KNN and STIRS, Professor Isenhour and his coworkers undertook an evaluation of the ability of KNN to identify 20 of the substructures examined i n our STIRS study, using the same data base and substructure identifications. The results of 4 representative substructures and the averages for the 20 are shown i n Table II along with the STIRS results at the ^ 2 % and<0.5% false positives l e v e l s . Comparing identification requirements giving equivalent average recall values (3/5 KNN v s . >2% FP STIRS) shows a factor of 4 reduction i n false positives for STIRS with an increase from 64% to 84% correct. Thus the incorporation of mass spectral knowledge has aided the performance of STIRS, and a similar input into KNN should improve the c l a s s i f i e r performance. Note, however, that KNN can only make predictions for substructures for which it has been pretrained, while the self-training feature of STIRS makes possible predictions on any structural feature which i s present i n the reference data base. PBM and STIRS as Aids to the Interpreter For a particular unknown it i s possible that a retrieval system such as PBM can identify an unknown mass spectrum with sufficient confidence that no human intervention w i l l be necessary. However, i n general, systems such as PBM and STIRS should be employed as an aid to the interpreter; if the system cannot give an identification with sufficient confidence or propose a complete structure, i t i s s t i l l very possible that the interpreter may be able to complete the identification. Processing an unknown spectrum through PBM or STIRS requires less than 1 minute on either our laboratory DEC PDP-11/45 computer or the C o r n e l l / TYMNET computer system (16). Thus for a l l but the simplest unknowns the information provided by these systems should at least yield a substantial time saving to the interpreter. In the case of total unknowns, such as compounds commonly encountered i n pollution samples, insect pheromones, and forensic analyses, even the most experienced interpreter can hardly be familiar with the mass spectral behavior of a l l compound c l a s s e s , and here STIRS and PBM often provide the structural information that the interpreter had not been able to elucidate. In the future we expect to see such matching and interpretive systems implemented on the same dedicated computers which now acquire and reduce the data from GC/MS and high-resolution systems. In fact, the UMC microprocessor-driven quadrupole system automatically applies PBM to mass spectral data for routine analyses, giving quantities and confidence levels for the presence of 16

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preselected compounds every second (4). Because the further application of GC/MS and LC/MS to important problems i s s t i l l seriously limited by the ability to identify and interpret the resulting mass spectra, there is s t i l l a real need for research on optimization of such programs.

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

Literature Cited 1. Morrison, J. D. and Dromey, R. G., to be published. 2. Waller, G. R., "Biochemical Applications of Mass Spec­ trometry", Wiley-Interscience, New York, 1972. 3. Hewlett-Packard Corp., Palo Alto, California 94304. 4. Universal Monitor Corp., a division of McDonnell-Douglas Corp., 430 North Halstead Street, Pasadena, California 91107. 5. Burlingame, A. L., Kimble, B. J., and Derrick, P. J., Anal. Chem., (1976), 48, 368R. 6. Pesyna, G. M. and McLafferty, F. W . , in "Determination of Organic Structures by Physical Methods", Nachocl, F. C., Zuckerman, J. J., and Randall, E. W . , Eds., Vol. 6, pp 91-155, Academic Press, New York, 1976. 7. Isenhour, T. L., Kowalski, B. R., and Jurs, P. C., Critical Review Anal. Chem., (1974), 4, 1. 8. Soltzberg, L. J., Wilkins, C. L., Kaberline, S.L.,Lam, T. F . , and Brunner, T. R., J. Am. Chem. Soc., (1976), 98, 7139. 9. Smith, D. H., Buchanan, B. G., Engelmore, R. S., Adlercreutz, H., and Djerassi, C., J. Am. Chem. Soc., (1973), 95, 6087. 10. McLafferty, F. W . , Hertel, R. H., and Villwock, R. D . , Org. Mass Spectrom., (1974), 9, 690. 11. Pesyna, G. M., Venkataraghavan, R., Dayringer, Η.E.,and McLafferty, F. W . , Anal. Chem., (1976), 48, 1362. 12. Kwok, K.-S., Venkataraghavan, R., and McLafferty, F. W . , J. Am. Chem. Soc., (1973), 95, 4185. 13. Dayringer, H. E., Pesyna, G. Μ . , Venkataraghavan, R., and McLafferty, F. W . , Org. Mass Spectrom., (1976), 11, 529. 14. Dayringer, H. E. and McLafferty, F. W . , Org. Mass Spectrom., (1976), 11, 543. 15. Dayringer, H. E., McLafferty, F. W . , and Venkataraghavan, R., Org. Mass Spectrom., (1976), 11, 895. 16. Office of Computer Services, Cornell University, Ithaca, New York 14853. 17. Salton, G., "Automatic Information Organization and Retrieval", McGraw-Hill, New York, 1968. 18. Grotch, S. L., 17th Annual Conference on Mass Spectrometry, Dallas, May, 1969, p 459. 19. Pesyna, G. M., McLafferty, F. W . , Venkataraghavan, R., and Dayringer, Η. E., Anal. Chem., (1975), 47, 1161.

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20. Abramson, F. P., Anal. Chem., (1975), 47, 45. 21. Stenhagen, E., Abrahamsson, S., and McLafferty, F. W . , "Registry of Mass Spectral Data", Wiley-Interscience, New York, 1974. 22. Pesyna, G. M., Ph.D. Thesis, Cornell University, 1975. 23. Hertz, H. S., Hites, R. Α., and Biemann, K., Anal. Chem., (1971), 43, 681. 24. Hites, R. A. and Biemann, Κ., Adv. Mass Spectrom., (1968), 4, 37. 25. Speck, D. D . , McLafferty, F. W . , and Venkataraghavan, R., in preparation. 26. Isenhour, T. L., Lowry, S. R., Justice, J. B., Jr., McLafferty, F. W . , Dayringer, Η. E., and Venkataraghavan, R., Anal. Chem., submitted. 27. McLafferty, F. W . , "Mass Spectral Correlations", Advances in Chemistry Series No. 40, American Chemical Society, Washington, 1963. 28. McLafferty, F. W., Anal. Chem., submitted. 29. Rotter, H. and Varmuza, K., Org. Mass Spectrom., (1975), 10, 874. Acknowledgment We are grateful to the National Institutes of Health (grant 16609) and to the Environmental Protection Agency (grant R804509) for financial support of this work, and to the National Science Foundation for partial funding of the DEC PDP-11/45 computer used in this research. Received December 30, 1977

18 Structure Elucidation Based on Computer Analysis of High and Low Resolution Mass Spectral Data DENNIS H. SMITH and RAYMOND E. CARHART

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch018

Departments of Chemistry, Genetics, and Computer Science, Stanford University, Stanford, CA 94305

A tremendous effort has been directed toward development of advanced instrumentation for mass spectrometric analysis. Advancements include ever-increasing sensitivities and resolving powers, new ionization techniques, metastable ion probes of ion decomposition and structure and computer systems for rapid acquisition and reduction of data. We sometimes lose sight of the fact that these developments are designed to provide information about chemical and biochemical structures at greater depth and in greater detail than previously available. The ultimate goal in most research in mass spectrometry is to provide powerful tools for molecular structure elucidation, either directly, by exploitation of existing techniques, or indirectly by development of new techniques. Concurrently, several computer-based techniques designed to assist chemists in the analysis and interpretation of mass spectral data have been developed. Analytical procedures for treatment of combined gas chromatographic/mass spectrometric data obtained at low resolving powers (1) (gc/1rms) ©0-8412-0422-5/78/47-070-325$10.00/0

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p r o v i d e mass s p e c t r a o f h i g h q u a l i t y f o r subsequent e x a m i n a t i o n by manual or computer methods. Library s e a r c h p r o c e d u r e s (2) and t h e i r e x t e n s i o n s (30 o r p a t t e r n r e c o g n i t i o n programs (4J) may p r o v i d e c l u e s t o the i d e n t i t y o f the s t r u c t u r e or be used t o d e t e r m i n e the s t r u c t u r e u n i q u e l y . A computer program f o r a n a l y s i s o f s p e c t r a based on c l a s s s p e c i f i c f r a g m e n t a t i o n r u l e s i s a v a i l a b l e (5) . These t e c h n i q u e s have o b v i o u s l i m i t a t i o n s (3,4,5) ; i n f a c t , the a b i l i t y t o i n t e r p r e t mass s p e c t r a l d a t a i n terms o f m o l e c u l a r s t r u c t u r e l a g s f a r b e h i n d the c a p a b i l i t i e s o f modern s p e c t r o m e t e r s t o produce h i g h q u a l i t y d a t a . There are s e v e r a l r e a s o n s f o r t h i s l a g : 1. There i s no f o r m a l t h e o r y r e l a t i n g molecular s t r u c t u r e s to t h e i r r e s p e c t i v e mass s p e c t r a w h i c h has p r e d i c t i v e power o f use t o the s t r u c t u r a l c h e m i s t . 2. (a c o r o l l a r y o f 1) Mass s p e c t r o m e t r y r a r e l y p r o v i d e s d e t a i l e d s u b s t r u c t u r a l i n f o r m a t i o n to a s s i s t i n e l u c i d a t i n g a s t r u c t u r e e x c e p t i n known c h e m i c a l c o n t e x t s where p r e v i o u s l y d e v e l o p e d r u l e s may be a p p l i e d r e t r o s p e c tively. 3. C u r r e n t methods do not make adequate use o f o t h e r knowledge about a p a r t i c u l a r compound, and; 4. The the c o m b i n a t o r i a l c o m p l e x i t y o f d e a l i n g w i t h the a c t u a l i n f o r m a t i o n c o n t e n t o f a mass spectrum has n o t , u n t i l now, been a d d r e s s e d . P o i n t s (3) and (4) w i l l be d i s c u s s e d i n some d e t a i l subsequently. In our l a b o r a t o r i e s we have been t r y i n g t o b r i n g n e w l y - d e v e l o p e d c o m p u t a t i o n a l t o o l s t o bear on g e n e r a l approaches t o a s s i s t i n g s t r u c t u r a l c h e m i s t s i n i n t e r p r e t a t i o n o f mass s p e c t r a . In t h i s paper we w i l l d i s c u s s the s t r e n g t h s and l i m i t a t i o n s o f t h e s e new t o o l s w h i l e assuming t h a t the r e q u i s i t e mass s p e c t r a l d a t a are a v a i l a b l e . We are engaged i n r e s e a r c h w h i c h i n v o l v e s the gc/ms a n a l y s i s o f complex m i x t u r e s t o g e t h e r w i t h subsequent a n a l y s i s of these data to e x t r a c t s p e c t r a of i n d i v i d u a l components ( l b ) and s e a r c h f o r the s p e c t r a i n l i b r a r i e s o f mass s p e c t r a l d a t a . The gc/lrms a n a l y s e s p r o v i d e an i m p o r t a n t p r e - s c r e e n i n g o f m i x t u r e s . Combined gc/ms d a t a o b t a i n e d a t h i g h mass s p e c t r o m e t e r r e s o l v i n g powers (gc/hrms) (6) y i e l d e l e m e n t a l c o m p o s i t i o n d a t a f o r n o v e l components. In any case the computer programs d e s c r i b e d i n subsequent s e c t i o n s a c c e p t e i t h e r low or h i g h r e s o l v i n g power d a t a (nominal masses or e l e m e n t a l compositions, r e s p e c t i v e l y ) . Computer-Assisted

S t r u c t u r e E l u c i d a t i o n Based

18.

Computer Analysis of High and Low Resolution 327

SMITH AND CARHART

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch018

P r i m a r i l y on Mass S p e c t r a l Data. Assume t h a t an i m p o r t a n t unknown component has been o b s e r v e d i n d a t a from a gc/ms a n a l y s i s o f a m i x t u r e ( e . g . , F i g u r e 1 ) . Assume f u r t h e r t h a t t h e spectrum o f t h e component ( F i g u r e 2) was n o t found in existing libraries. T h i s problem becomes a c l a s s i c problem o f s t r u c t u r e e l u c i d a t i o n . One, f o r example, might attempt t o i s o l a t e l a r g e r q u a n t i t i e s and o b t a i n a d d i t i o n a l s p e c t r a l d a t a . F o r many problems t h i s i s time-consuming and d i f f i c u l t . R e a l i z i n g t h a t h i g h r e s o l v i n g power d a t a a r e l e s s ambiguous t h a n d a t a p r o v i d e d by t h e low r e s o l u t i o n spectrum ( F i g u r e 2 ) , one might o b t a i n a gc/hrms spectrum and d e t e r m i n e e l e m e n t a l c o m p o s i t i o n s f o r the o b s e r v e d i o n s . The d a t a o b t a i n e d i n t h e example a r e p r e s e n t e d i n T a b l e I f o r major i o n s i n t h e spectrum. These d a t a may be t h e o n l y d a t a one c a n TABLE I . Elemental Compositions f o r S i g n i f i c a n t Fragment Ions i n t h e Mass Spectrum G i v e n i n F i g u r e 2. m/e

Composition

293

C

1 5

H

1 9

N0

5

261

C

1 4

H

1 5

N0

4

234

C

13 16

202

C

8 12

174

C H N0

142

C H N0

116

C H NO

91

H

H

7

6

5

C H ?

N 0

N 0

5

1 2

8

1 0

3

4

3

2

7

e a s i l y o b t a i n t o determine s t r u c t u r a l i n f o r m a t i o n about t h e unknown. Many c u r r e n t programs o p e r a t e under t h e assumption t h a t t h e s e a r e t h e o n l y d a t a . But, o f course, t h i s i s not t r u e . I n almost e v e r y instance a great deal of a d d i t i o n a l information about t h e unknown i s a v a i l a b l e . Some o f t h i s i n f o r m a t i o n i s f a c t u a l , f o r example, t h e p h y s i c a l and c h e m i c a l p r o p e r t i e s and t h e s o u r c e o f t h e m a t e r i a l s ,

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch018

328

HIGH

PERFORMANCE

Figure 1. Total ion current plot, scans 470-565, of the GCfirms analysis of the ether/ethyl acetate extracts of an acidi­ fied human urine subsequent to a 1 hr IN NaOH hydrolysis. Numbers and names associated with component spectra de­ tected by the CLEANUP program (lb) refer to the match scores and names of close library matches. Tetracosane, scan 508, is an internal standard for relative retention index cahuhtions.

MASS

5 0 0

SPECTROMETRY

5 5 0

91

547

116

142 174 202

234 261

,

Ί T'H"i"T Γ 150

200

250

1^293 300

Figure 2. 70 eV low resolution mass spectrum obtained for the component eluting at scan 547 (see total ion current plot, Figure 1)

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch018

18.

SMITH AND

CARHART

Computer Analysis of high and

Low

Resolution 329

and i s o l a t i o n and d e r i v a t i z a t i o n p r o c e d u r e s . Other i n f o r m a t i o n i s j u d g m e n t a l ; f o r example, knowledge o f o t h e r compounds p r e s e n t i n the same m i x t u r e , p l a u s i b i l i t y o f c h e m i c a l or b i o c h e m i c a l p r o c e s s e s w h i c h may have y i e l d e d the compound, and good intuitions. I f t h i s i n f o r m a t i o n can be b r o u g h t t o b e a r on the p r o b l e m , i t s h o u l d be e a s i e r t o s o l v e . We have d e v e l o p e d the CONGEN program f o r c o m p u t e r - a s s i s t e d s t r u c t u r e e l u c i d a t i o n . (7_) This program has a f l e x i b l e mechanism f o r e x p r e s s i o n and use o f c o n s t r a i n t s on the p l a u s i b i l i t y o f c e r t a i n chemical features (substructures, r i n g systems). (8J) A l t h o u g h d e s i g n e d f o r the g e n e r a l p r o b l e m o f i n c o r p o r a t i n g s t r u c t u r a l i n f e r e n c e s from d i f f e r e n t s p e c t r o s c o p i c techniques or other sources of chemical i n f o r m a t i o n , we are i n t r o d u c i n g c a p a b i l i t i e s f o r more t h o r o u g h use o f mass s p e c t r a l d a t a . The c a p a b i l i t i e s were d e v e l o p e d i n i t i a l l y i n e a r l i e r DENDRAL work. (5,9) The i m p o r t a n c e o f CONGEN i n problems such as t h a t o u t l i n e d above i s t h a t i t g i v e s the c h e m i s t a mechanism f o r e x p l o r i n g s t r u c t u r a l p o s s i b i l i t i e s under c o n s t r a i n t s e x p r e s s i n g h i s / h e r f a c t u a l o r j u d g m e n t a l knowledge. T h i s knowledge can be d e f i n e d , a p p l i e d t o a c u r r e n t p r o b l e m , and saved f o r f u t u r e use i n r e l a t e d p r o b l e m s , as i l l u s t r a t e d below. R e t u r n i n g t o the example, i t i s f o o l h a r d y t o c o n s i d e r s t r u c t u r a l possibilités i n the l i g h t o f the presumed m o l e c u l a r i o n , C 1 5 H 1 9 N O 5 . Without c o n s t r a i n t s , the number o f p o s s i b l e s t r u c t u r e s i s huge. However, knowledge t h a t the compound i s a component o f a m i x t u r e o f o r g a n i c compounds i s o l a t e d from human u r i n e , and t h a t the u r i n e was s u b j e c t e d to b a s i c h y d r o l y s i s p r i o r to e x t r a c t i o n provides a d d i t i o n a l i n f o r m a t i o n w h i c h can t o some e x t e n t be e x p r e s s e d as s t r u c t u r a l c o n s t r a i n t s (see b e l o w ) . More s p e c i f i c s t r u c t u r a l i n f o r m a t i o n i s a v a i l a b l e from the f a c t t h a t the f r a c t i o n c o n s i s t s o f e t h e r / e t h y l a c e t a t e e x t r a c t a b l e o r g a n i c a c i d s , w h i c h were s u b s e q u e n t l y e s t e r i f i e d u s i n g diazomethane p r i o r t o gc/ms a n a l y s i s . T h i s " h i s t o r y " of the sample p r o v i d e s a tremendous r e d u c t i o n i n the scope o f p o s s i b l e s t r u c t u r e s . Chemists use t h i s r e a s o n i n g a u t o m a t i c a l l y i n manual examination of s t r u c t u r a l p o s s i b i l i t i e s . To be t r u l y e f f e c t i v e , a program must somehow p r o v i d e the same c a p a b i l i t i e s when c o n f r o n t e d w i t h a mass spectrum. To a s s i s t c h e m i s t s i n making use o f such r e a s o n i n g , we are e x t e n d i n g CONGEN t o a l l o w e x p l o r a t i o n o f s t r u c t u r a l p o s s i b i l i t i e s f o r an unknown

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch018

330

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PERFORMANCE

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w i t h i n c o n s t r a i n t s p r o v i d e d by the mass spectrum and by f a c t u a l and j u d g m e n t a l knowledge s u p p l i e d by t h e c h e m i s t . We a r e d e v e l o p i n g two g e n e r a l approaches to t h e use o f mass s p e c t r a l d a t a . These two a p p r o a c h e s , MSPRUNE and Mass D i s t r i b u t i o n Graphs (MDG's), m i r r o r t h e c l a s s i c i n t e r p l a y between maximum use o f i n f o r m a t i o n i n r e t r o s p e c t i v e t e s t i n g v s . p r o s p e c t i v e g u i d a n c e ( p l a n n i n g ) toward hypot h e t i c a l s o l u t i o n s i n the p r o b l e m - s o l v i n g paradigm of h e u r i s t i c s e a r c h . (5^7J) i n p r i n c i p l e , the approches a r e complementary. They w i l l y i e l d the same answers by w o r k i n g on a p r o b l e m from two d i f f e r e n t d i r e c t i o n s . In p r a c t i c e we have made more p r o g r e s s t o date on t h e former (see b e l o w ) . We w i l l i l l u s t r a t e b o t h w i t h examples. I.

MSPRUNE - R e t r o s p e c t i v e T e s t i n g o f S t r u c t u r a l Candidates.

If i t i s p o s s i b l e to a r r i v e at a set of s t r u c t u r a l c a n d i d a t e s f o r an unknown based on c o n s t r a i n t s d e r i v e d from c h e m i c a l c o n s i d e r a t i o n s , o t h e r s p e c t r o s c o p i c d a t a and/or c h a r a c t e r i s t i c i o n s i n a mass s p e c t r u m , i t s h o u l d be p o s s i b l e t o t e s t each c a n d i d a t e i n some d e t a i l t o d e t e r m i n e i f i t i s c a p a b l e of p r o d u c i n g t h e o b s e r v e d spectrum. MSPRUNE makes such t e s t s and w i l l "prune", o r r e j e c t those s t r u c t u r e s w h i c h c o u l d not have y i e l d e d o b s e r v e d i o n s . In many p r o b l e m s , i n c l u d i n g the example ( F i g u r e 2, T a b l e I ) , i t i s p o s s i b l e t o a r r i v e a t a r e a s o n a b l e set o f c a n d i d a t e s t r u c t u r e s f o r an unknown u s i n g a v a i l a b l e d a t a and the f o l l o w i n g g e n e r a l p r o c e d u r e . A. Determine the m o l e c u l a r w e i g h t and f o r m u l a . I n t h e example, a c a n d i d a t e m o l e c u l a r i o n i s found a t m/e 293, o f c o m p o s i t i o n C i H i N 0 . T h i s i o n i s s e l e c t e d by MOLION ( 1 0 ) , our m o l e c u l a r i o n d e t e r m i n 5

9

5

TABLE I I . M o l e c u l a r Ion C a n d i d a t e s and Rankings f o r t h e Spectrum G i v e n i n F i g u r e 2. Candidate m/e

293 294 325 352 308

Ranking 100 71 50 46 43

18.

SMITH AND

CARHART

Computer Analysis of High and Low

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch018

a t i o n program, and makes sense c h e m i c a l l y . f i v e b e s t c a n d i d a t e s and t h e i r r a n k i n g s are i n Table I I .

Resolution 331

The given

B. D e r i v e superatoms (7) and c o n s t r a i n t s from a v a i l a b l e data" In the example, knowledge o f the source o f the sample t e l l s us t h a t i t i s an o r g a n i c a c i d from human u r i n e and was e s t e r i f i e d t o form m e t h y l e s t e r s p r i o r t o gc/ms a n a l y s i s . T h i s f r a c t i o n c o n t a i n s a r o m a t i c and a l i p h a t i c a c i d s i n a d d i t i o n t o c o n j u g a t e s o f these a c i d s w i t h b a s i c n i t r o g e n s , such as i n amino a c i d s . We can d e f i n e a s e t o f superatoms, or b u i l d i n g b l o c k s , w h i c h can be used to c o n s t r u c t s t r u c t u r e s and can be saved on a computer f i l e f o r f u t u r e use i n r e l a t e d p r o b l e m s . Such a s e t o f superatoms w i t h t h e i r a s s o c i a t e d names i s shown i n F i g u r e 3, where the bonds t o u n s p e c i f i e d atoms a r e f r e e v a l e n c e s w h i c h w i l l subs e q u e n t l y be bonded t o o t h e r atoms i n c l u d i n g h y d r o gen. In the example, the abundant m/e 91 i o n s u g g e s t s the superatom BZ ( F i g u r e 3 ) , w i t h no o t h e r s u b s t i t u e n t s a t t a c h e o f t o the a r o m a t i c r i n g . The number o f oxygen atoms and degree o f u n s a t u r a t i o n s u g g e s t two m e t h y l e s t e r f u n c t i o n a l i t i e s (EST, F i g u r e 3 ) . The s i n g l e n i t r o g e n s u g g e s t s a t l e a s t the p a r t s t r u c t u r e AMI, a r i s i n g from an a c i d c o n j u g a t e w i t h a b a s i c n i t r o g e n . There are perhaps o t h e r ways t o p h r a s e t h i s p r o b l e m , and a l t e r n a t i v e a s s u m p t i o n s , but t h e s e a s s u m p t i o n s w i l l s u f f i c e f o r i l l u s t r a t i v e purposes. C. Generate s t r u c t u r e s under a p p r o p r i a t e c o n s t r a i n t s from the c o m p o s i t i o n o f superatoms and r e m a i n i n g atoms. In our example, the c o m p o s i t i o n i s B Z i E S T AIM1C3H5. W i t h o u t c o n s t r a i n t s t h e r e are 78 s t r u c tural possibilities. T h i s l i s t c o n t a i n s many i m p l a u s i b l e s t r u c t u r e s . For example, i f we assume t h a t the compound i s an amino a c i d c o n j u g a t e , t h e n a p a r t s t r u c t u r e s i m i l a r to ACI must be p r e s e n t , i n fact -NHCH _2^~ 3Implementation of t h i s c o n s t r a i n t ^ l e a v e s 16 s t r u c t u r e s . 2

C 0 0 C H

f l

D. Use MSPRUNE t o t e s t the r e m a i n i n g s t r u c t u r a l c a n d i d a t e s t o d e t e r m i n e w h i c h c o u l d y i e l d key i o n s i n the o b s e r v e d s p e c t r u m ? MSPRUNE i s an e x t e n s i o n t o CONGEN w h i c h a l l o w s i n t e r a c t i o n w i t h the program t o c a r r y out the t e s t s . MSPRUNE o p e r a t e s u s i n g the f o l l o w i n g sequence o f s t e p s : D.l Obtain fragmentation r u l e s : A s e r i e s of q u e s t i o n s t o the u s e r o f MSPRUNE/CONGEN e l i c i t s the

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch018

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mass s p e c t r o m e t r i c f r a g m e n t a t i o n r u l e s t o be used i n i n t e r p r e t a t i o n o f the d a t a . We are c u r r e n t l y r e s t r i c t e d t o r u l e s used p r e v i o u s l y i n the MetaDENDRAL program INTSUM ( 9 ) . These r u l e s i n c l u d e c o n s t r a i n t s on c l e a v a g e o f a r o m a t i c r i n g s , m u l t i p l e bonds, more t h a n one bond t o t h e same atom, number o f s t e p s i n a f r a g m e n t a t i o n p r o c e s s , hydrogen t r a n s f e r s and l o s s (or t r a n s f e r ) o f o t h e r n e u t r a l s p e c i e s such as w a t e r o r c a r b o n monoxide. For t h e example, the c o n s t r a i n t s summarized i n T a b l e I I I were used. This set of c o n s t r a i n t s i s p a r t i c u l a r l y restrictive. The o n l y danger i n t h i s l e v e l o f r e s t r i c t i o n i s t h a t an i n c o r r e c t s t r u c t u r e may y i e l d a s i m p l e r e x p l a n a t i o n o f the spectrum t h a n the c o r r e c t s t r u c t u r e . The c o r r e c t s t r u c t u r e i n such a case may be m i s s e d . D.2 Input mass s p e c t r a l i o n s t o be e x p l a i n e d . The u s e r i s t h e n asked t o i n p u t the i o n s he/she w i s h e s t o be e x p l a i n e d . These i o n s may be e n t e r e d TABLE I I I . F r a g m e n t a t i o n P r o c e s s C o n s t r a i n t s Used i n t h e A n a l y s i s o f the Mass Spectrum G i v e n i n F i g u r e 2. Constraints Allow Adjacent Breaks:

a

User Response No

Allow Aromatic Breaks: A l l o w Breaks o f Double o r T r i p l e Bonds: Max Bonds t o Break i n a S i n g l e S t e p : Max Steps Per P r o c e s s : Max Bonds t o Break i n a P r o c e s s Allowed H Transfers: Allowed Neutral Transfer:

No^ No 1 2 2 -2-1012

Cleavage o f more t h a n one, non-hydrogen bond t o t h e same atom. I . e . , do not c l e a v e the a r o m a t i c r i n g . There a r e no a r o m a t i c r i n g s o r m u l t i p l e bonds a l l o w e d t o c l e a v e ; any number >1 i s m e a n i n g l e s s because t h e r e a r e no o t h e r degrees o f u n s a t u r a t i o n ( r i n g s ) ; thus e v e r y c l e a v a g e y i e l d s a fragment i o n . (9)

18.

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Computer Analysis of High and Low Resolution 333

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch018

as e i t h e r n o m i n a l masses o r e l e m e n t a l c o m p o s i t i o n s . O b v i o u s l y t h e l a t t e r form i s much more e f f e c t i v e t h a n n o m i n a l masses o f p o s s i b l e c o m p o s i t i o n a l a m b i g u i t y . The method o f s e l e c t i n g t h e i o n s t o be used i n t h e a n a l y s i s i s up t o t h e u s e r . I n t h e example, we chose i o n s on t h e b a s i s o f a) h i g h mass ( i n t u i t i v e l y o f g r e a t e r s t r u c t u r a l u t i l i t y ) , and, b) h i g h abundance. Ions o f low mass and low abundance have a g r e a t e r chance o f r e s u l t i n g from e i t h e r s e v e r a l d i f f e r e n t p l a c e s i n t h e m o l e c u l e o r from complex p r o c e s s e s beyond t h e a b i l i t y o f t h e s i m p l e r u l e s (Table I I I ) t o explain. D. 3 T e s t each c a n d i d a t e s t r u c t u r e t o d e t e r m i n e i f i t ~ c o u l d y i e l d i o n s i n p u t . A l l p o s s i b l e fragment a t i o n s a l l o w e d by t h e c o n s t r a i n t s a r e d e t e r m i n e d f o r each s t r u c t u r e , u s i n g an a l g o r i t h m s i m i l a r t o t h a t d e v e l o p e d f o r INTSUM ( 9 ) . For each f r a g m e n t a t i o n t h e mass and c o m p o s i t i o n oF t h e r e s u l t i n g i o n i s d e t e r mined, i n c l u d i n g a l l o w e d hydrogen and/or n e u t r a l t r a n s f e r s . A simple comparison o f these ions w i t h the i o n s i n p u t r e v e a l s whether o r n o t t h e s t r u c t u r e could y i e l d a l l ions input. I f not, the s t r u c t u r e i s rejected. I f so, the s t r u c t u r e i s r e t a i n e d . This e x p e r i m e n t a l v e r s i o n o f MSPRUNE t a k e s no c o g n i z a n c e o f i o n abundances i n t h i s c o m p a r i s o n . I t makes a simple e x i s t e n c e t e s t only. Given the ions of Table I and t h e c o n s t r a i n t s o f T a b l e I I , t h e l i s t o f 16 b e s t c a n d i d a t e s i s trimmed t o f i v e 1 - 5 , (see F i g u r e 4 ) . I f t h e o r i g i n a l s e t o f 78 p o s s i b i l i t i e s i s t e s t e d under t h e above c o n d i t i o n s * f o r MSPRUNE, 15 s t r u c t u r e s r e m a i n ; t h e spectrum i t s e l f i s a p o w e r f u l c o n s t r a i n t on p o s s i b l e s t r u c t u r e s . I f o n l y n o m i n a l masses a r e i n p u t , t h e s e t o f 16 s t r u c t u r e s i s n o t r e d u c e d by MSPRUNE; 16 s t r u c t u r e s remain. T h i s k i n d o f c o m p a r i son can q u a n t i t a t e t h e i n f o r m a t i o n c o n t e n t o f low v s . high r e s o l u t i o n spectra. E. E v a l u a t e Remaining S t r u c t u r e s . The s t r u e t u r e s w h i c h r e s u l t can be examined w i t h t h e h e l p o f CONGEN t o d e t e r m i n e a d d i t i o n a l c o n s t r a i n t s o r t o d e s i g n e x p e r i m e n t s t o d i f f e r e n t i a t e among t h e possibilities. W i t h knowledge o f human m e t a b o l i c p r o c e s s e s and t h e c h e m i s t r y o f t h e i s o l a t i o n p r o c e d u r e , i t i s easy t o a s s i g n 1, (see F i g u r e 4) p h e n y l a c e t y l g l u t a m i c a c i d d i m e t h y l e s t e r ( 6 ) , as t h e c o r r e c t s t r u c t u r e . Phenylacetic a c i d i s normally c o n j u g a t e d w i t h g l u t a m i n e and e x c r e t e d as phenyl a c e t y l g l u t a m i n e . The base c a t a l y z e d h y d r o l y s i s c o n v e r t e d t h e p r i m a r y amide f u n c t i o n a l i t y i n t o t h e o b s e r v e d c a r b o x y l i c a c i d ( 6 , see F i g u r e 4 ) : t h e

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch018

334

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d i m e t h y l e s t e r was formed on subsequent d e r i v a t i zation. In l a r g e r p r o b l e m s , i t i s p o s s i b l e t o use o t h e r f e a t u r e s o f CONGEN t o t e s t i n t u i t i o n s on possible structures. For example, i n t h i s problem where an amino a c i d c o n j u g a t e was s u s p e c t e d , i t was p o s s i b l e t o t e s t a u t o m a t i c a l l y every s t r u c t u r e f o r the p r e s e n c e o f one o f t h e known amino a c i d s k e l e t o n s . Of t h e f i v e f i n a l s t r u c t u r e s , ( 1 - 5 , see F i g u r e 4) o n l y one, 1, p o s s e s s e s a known amino a c i d s k e l e t o n . Of t h e 16 assumed c o n j u g a t e s , f o u r f o r m a l l y p o s s e s s a g l y c i n e , two a p h e n y l a l a n i n e , one an a s p a r t i c and one 1, a g l u t a m i c s k e l e t o n . Possible o r i g i n s of i m p o r t a n t f r a g m e n t a t i o n s i n t h e spectrum o f 1 a r e i l l u s t r a t e d i n Scheme 1. We have no i s o t o p i c l a b e l l i n g data t o support these suggestions. An A p p l i c a t i o n o f MSPRUNE. The p r o c e d u r e o u t l i n e d above p r o v e d e x t r e m e l y h e l p f u l i n a n a l y s i s o f unknown compounds o b s e r v e d i n a gc low r e s o l u t i o n ms e x p e r i m e n t . A p a t i e n t e x h i b i t i n g s i g n s o f m e n t a l r e t a r d a t i o n was r e f e r r e d t o S t a n f o r d . We examined o r g a n i c compounds i n t h i s p a t i e n t ' s u r i n e u s i n g p r o c e d u r e s d e s c r i b e d above f o r t h e example o f s t r u c t u r e 1. In f a c t , t h e s e compounds were o b s e r v e d i n t h e same o r g a n i c a c i d f r a c t i o n , b u t t h i s t i m e p r i o r t o any a l k a l i n e hydrolysis. The p o r t i o n o f t h e t o t a l i o n c u r r e n t v s . scan number p l o t where t h e unknowns were o b s e r v e d i s shown i n F i g u r e 5. The components i n q u e s t i o n , A^ C, were d e t e c t e d by t h e program CLEANUP ( l b ) a t scans 382, 402, and 406 ( F i g u r e 4 ) . R e s o l v e d s p e c t r a ( l b ) a r e shown i n F i g u r e s 6a and 6b. The spectra bear obvious s i m i l a r i t i e s ; i n f a c t the s p e c t r a a t scans 402 and 406 a r e n e a r l y superi m p o s a b l e . Gc/hrms (6) a n a l y s i s o f t h i s f r a c t i o n l e n t f u r t h e r e v i d e n c e "to s u p p o r t t h e r e l a t i o n s h i p # o f t h e unknown s t r u c t u r e s ; a l l s i g n i f i c a n t i o n s common t o t h e s p e c t r a p o s s e s s t h e same e l e m e n t a l c o m p o s i t i o n s (shown i n F i g u r e 6a f o r t h e s i g n i f i c a n t , h i g h e r mass i o n s ) . The MOLION program f i n d s m/e 207, C 1 1 H 1 3 N O 3 , t h e h i g h e s t r a n k i n g m o l e c u l a r i o n c a n d i d a t e f o r a l l t h r e e components. The unknowns a r e a p p a r e n t l y s t r u c t u r a l i s o m e r s . Thus, t h e y can be i n v e s t i g a t e d by CONGEN and MSPRUNE i n a s i n g l e r u n . F o r t h i s a p p l i c a t i o n , we assumed t h e p r e s e n c e o f an a r o m a t i c r i n g , a m e t h y l e s t e r f u n c t i o n a l i t y and an amide ( c o n j u g a t e )

SMITH AND CARHART

CH3O-?-

HO-

EST

OH

CH3O-

>=0

MEO

CO

Computer Analysis of High and Low

J?-NHAMI

—HNACI

Figure S. A set of superatoms useful for considering structural candidates in the context of solvent extractable organic acids from human urine (derivatized to methyl esters)

,JL^ '\\f ^^Α^μ

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch018

B2

Resolution 335

*

Figure 4. Candidate structures for unknown repre­ sented by the mass spectrum in Figure 2

Scheme 1

(—202 -CH^OHx* 142 174.

M16(-C H 02) 2

HDCH-234

261

2

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch018

336

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Figure 5. Total ion current plot, scans 357-426, of the solvent extractable organic acids from the urine of a patient exhibiting signs of mental retardation

119

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382 118 00

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50

150

τ

Ι

|

τ

|

Ι

200

Figure 6α. 70 eV low resolution mass spectrum of component A, scan 382, of the total ion current plot of Figure 5. Elemental compositions were determined by a subsequent GC/hrms experiment

SPECTROMETRY

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch018

18.

SMITH AND

CARHART

Computer Analysis of High and Low Resolution 337

l i n k a g e (superatoms PH, EST, and AMI r e s p e c t i v e l y ) . C o n s t r a i n t s f o r b i d d i r e c t c o n n e c t i o n o f t h e amide and e s t e r f u n c t i o n s , and a l k y l c h a i n s o f two o r more c a r ­ bons c o n n e c t e d t o t h e a r o m a t i c r i n g . G e n e r a t i o n o f s t r u c t u r e s under these c o n s t r a i n t s y i e l d e d 52 p o s s i b i l i t i e s . A number o f methods were used t o examine t h e s e s t r u c t u r e s . F o r example, auto­ m a t i c s u r v e y o f t h e s t r u c t u r a l p o s s i b i l i t i e s showed 6 s t r u c t u r e s w h i c h f o r m a l l y p o s s e s s known amino a c i d s k e l e t o n s . Use o f MSPRUNE under c o n s t r a i n t s used f o r the p r e v i o u s example (Table I I I ) , and w i t h t h e i o n s shown i n F i g u r e 5a was n o t t o o h e l p f u l ; 36 c a n d i d a t e s remained a f t e r t h i s t e s t . More s e v e r e c o n s t r a i n t s were then u s e d , s p e c i f i c a l l y c o n s i d e r i n g o n l y s i n g l e r a t h e r t h a n two s t e p p r o c e s s e s . This e f f e c t e d a dramatic r e d u c t i o n , l e a v i n g o n l y f o u r s t r u c t u r e s , 7 (see F i g u r e 7) and t h e t h r e e isomers r e p r e s e n t e d by 87 A p o s s i b l e source o f each i o n i s shown i n Scheme 2 f o r 8. L i t ­ e r a t u r e s u r v e y s r e v e a l e d t h a t 7 has been o b s e r v e d i n the dog b u t n e v e r i n man. S t r u c t u r e 8 i s p a r t i c u l a r l y a t t r a c t i v e because t h e r e a r e t h r e e s u b s t i t u t i o n isomers p o s s i b l e . These compounds a r e f o r m a l l y c o n j u g a t e s o f t o l u i c a c i d s w i t h g l y c i n e . We s u b s t a n t i a t e d t h i s h y p o t h e s i s and proved t h e s t r u c t u r e s by s y n t h e s i s o f the t h r e e i s o m e r s . The r e t e n t i o n i n d i c e s ( T a b l e IV) and mass s p e c t r a agree c o m p l e t e l y . Further i n v e s t i ­ g a t i o n s i n d i c a t e d t h a t x y l e n e s a r e e x c r e t e d by t h e body by f i r s t , o x i d a t i o n o f one o f t h e m e t h y l groups, and second, c o n j u g a t i o n w i t h g l y c i n e . Further, the r e l a ­ t i v e c o n c e n t r a t i o n s o f t h e t h r e e compounds c l o s e l y a p p r o x i m a t e t h e r e l a t i v e amount o f o r t h o , meta and p a r a isomers i n c o m m e r c i a l m i x t u r e s o f x y l e n e . The p a t i e n t had somehow been exposed t o q u a n t i t i e s o f x y l e n e . TABLE IV. R e l a t i v e R e t e n t i o n Indexes (R.R.I.) f o r Unknowns A-C and S y n t h e t i c O r t h o , Meta and P a r a - t o l u y l g l y c i n e s , as Determined by CLEANUP ( l b ) . Unknown

Scan#

R.R.I.

A

382

2060

Β

402

2128

C

406

2141

S y n t h e t i c Compound ortho-toluylglycine methyl e s t e r meta-toluylglycine methyl e s t e r para-toluylglycine methyl e s t e r

R.R.I. 2058 2128 2143

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119

MASS

Β 402

75-^

50 A

91

25 H

148 175

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch018

50

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T T H

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207

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100

1, Ι

Ι

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1

150

200

Figure 6b. 70 eV low resolution mass spectra of components Β and C (Figure 5). Elemental com­ positions of major ions are those given in Figure 6a.

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8

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Figure 7. Candidate struc­ tures for unknowns repre­ sented by spectra in Figures 6a and 6b Scheme 2

2

3

3

SPECTROMETRY

18.

SMITH AND

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch018

II.

CARHART

Computer Analysis of High and Low Resolution 339

Prospective Generation i t i e s b y MDGGEN.

of Structural

Possibil-

I t i s a common i n t u i t i o n t h a t mass s p e c t r a p o s s e s s a tremendous amount o f s t r u c t u r a l i n f o r m a t i o n , most o f w h i c h goes unused. Every i o n i s a p i e c e o f the o r i g i n a l s t r u c t u r e . There are many such i o n s r e p r e s e n t i n g p i e c e s w h i c h may o v e r l a p t o an unknown e x t e n t . I d e a l l y , one would l i k e a comput a t i o n a l method t o p e r f o r m a l l p o s s i b l e o v e r l a p s o f p i e c e s o f s t r u c t u r e r e p r e s e n t e d by t h e i o n s , t h e r e b y c o n s t r u c t i n g t h e s e t o f c a n d i d a t e s t r u c t u r e s . We a r e e x p l o r i n g an approach w h i c h c a n i n p r i n c i p l e c a r r y out t h i s g e n e r a l a n a l y s i s o f mass s p e c t r a p r o s p e c t i v e l y , by g e n e r a t i o n o f s t r u c t u r a l p o s s i b i l i t i e s d i r e c t l y from the mass s p e c t r a l d a t a . Note t h a t t h i s approach d i f f e r s from MSPRUNE, w h i c h u t i l i z e s r e t r o s p e c t i v e t e s t i n g o f p r e v i o u s l y generated candidate structures. Our approach i n t r o d u c e s t h e c o n c e p t o f a "mass d i s t r i b u t i o n g r a p h " (an "MDG"). An MDG i s a graph whose nodes p o s s e s s the p r o p e r t i e s o f mass and/or elemental composition. Edges between nodes p o s s e s s the p r o p e r t y o f m u l t i p l i c i t y , i . e . s i n g l e , d o u b l e , e t c . MDG s a r e i n c o m p l e t e l y s p e c i f i e d c h e m i c a l s t r u c t u r e s i n t h a t nodes o f MDG s c o n t a i n o n l y mass o r numbers o f atoms w i t h o u t s p e c i f i c a t i o n o f how such mass o r atoms a r e i n t e r c o n n e c t e d w i t h i n each node. I n a d d i t i o n , the edges w h i c h c o n n e c t nodes a r e n o t p r e c i s e c h e m i c a l bonds i n t h a t m u l t i p l e edges may o r may n o t become m u l t i p l e bonds i n complete s t r u c t u r e s (see b e l o w ) . We have d e v e l o p e d a method ("MDGGEN") t o c o n s t r u c t MDG's, and s u b s e q u e n t l y , complete s t r u c t u r e s , d i r e c t l y from the mass s p e c t r a l d a t a . We i l l u s t r a t e the u s e o f t h e c o n c e p t o f MDG's w i t h a s i m p l e example, the mass spectrum ( F i g u r e 8) and s t r u c t u r e o f h e x a n a l 9. The s p e c t r u m , F i g u r e 8, shows t h e e l e m e n t a l c o m p o s i t i o n s o f i o n s used i n t h e subsequent d i s c u s s i o n . A l t h o u g h MDG s and t h e s e c t i o n o f CONGEN, MDGGEN, w h i c h g e n e r a t e s them c a n use n o m i n a l masses, r e s u l t s are more p r e c i s e u s i n g e l e m e n t a l c o m p o s i t i o n s , i f known (see a l s o MSPRUNE, above). At the most g e n e r a l l e v e l , the MDG f o r a s t r u c t u r e i s a s i n g l e node p o s s e s s i n g a l l atoms and degrees o f u n s a t u r a t i o n ( r i n g s p l u s m u l t i p l e b o n d s ) , o r C H i 0 (one degree o f u n s a t u r a t i o n ) f o r t h i s 1

1

1

6

2

20

C2H5 CHO 4

40

s»

C3H7I

C2H40

m/e

60

C4H9

C4H8

C4H8O

80

111,1,

C6H1O

Tetrahedron

100

M* C6H12O

Figure 8. 70 eV low resolution mass spectrum of hexanal. Elemental compositions were determined by accurate mass measurements in a subsequent experiment at high resolving power.

ο d oc

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50H

c ο Ό C D Ώ <

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Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ch018

CO

18.

SMITH AND

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Computer Analysis of High and Low Resolution 341

example. MDGGEN r e q u i r e s i n p u t s i m i l a r t o t h a t o f MSPRUNE. I t r e q u i r e s mass s p e c t r o m e t r i c fragmentat i o n r u l e s . The s u i t e o f r u l e s i s t h e same as t h a t mentioned p r e v i o u s l y (Table I I I ) w i t h an a d d i t i o n a l parameter f o r the number o f i o n s w h i c h are a l l o w e d to go u n e x p l a i n e d . I t a l s o r e q u i r e s mass s p e c t r a l d a t a , i n p u t by t h e u s e r , who a g a i n s e l e c t s whatever peaks a r e deemed r e l e v a n t . MDGGEN o p e r a t e s by s e l e c t i n g i n p u t i o n s one a t a time. Each i o n s e l e c t e d r e s u l t s i n an attempt by the program t o a p p o r t i o n atoms ( o r mass) i n nodes o f the c u r r e n t MDG among nodes o f new MDG s such t h a t f r a g m e n t a t i o n o f t h e new MDG s under i n p u t r u l e s would y i e l d t h e i o n . The r e s u l t s f o r h e x a n a l b e g i n n i n g w i t h the m o l e c u l a r i o n MDG ( C H i 0 ) and u s i n g t h e o b s e r v e d i o n C H as t h e f i r s t s e l e c t e d i o n a r e shown i n F i g u r e 9. The number o f MDG s r e s u l t i n g a f t e r i n c o r p o r a t i o n o f a new i o n depends on t h e f r a g m e n t a t i o n t h e o r y i n use. Simple bond c l e a v a g e o f the f i r s t MDG ( F i g u r e 9) w i t h o u t hydrogen t r a n s f e r y i e l d s C H d i r e c t l y . However, i f hydrogen t r a n s f e r s (0, 1 o r 2) a r e a l l o w e d , t h e second MDG i s a l s o a p o s s i b i l i t y . Cleavage o f t h e s i n g l e bond w i t h t r a n s f e r o f two hydrogens i n t o t h e c h a r g e d fragment would y i e l d C H . I f two s t e p p r o c e s s e s a r e a l l o w e d , t o g e t h e r w i t h hydrogen t r a n s f e r s , t h e n t h e r e a r e f i v e o t h e r MDG s g e n e r a t e d a t t h i s l e v e l ( F i g u r e 9) each o f w h i c h would y i e l d C H w i t h t h e i n d i c a t e d c l e a v a g e s and hydrogen transfers. MDGGEN uses some s i m p l e p a r i t y c o n s i d e r a t i o n s to a v o i d g e n e r a t i n g nonsense MDG s w h i c h have unspecified connections. These c o n s i d e r a t i o n s a r e the same as those used by mass s p e c t r o s c o p i s t s . An odd mass i o n c o n t a i n i n g an even number o f n i t r o g e n s ( e . g . , 0) c a n a r i s e from c l e a v a g e o f an odd number o f bonds ( e . g . , a s i n g l e bond) accompanied by t r a n s f e r o f an even number o f hydrogen atoms ( e . g . , 0). Or t h e same i o n c a n a r i s e from c l e a v a g e o f an even number o f bonds ( e . g . , 2) accompanied by a t r a n s f e r o f an odd number o f h y d r o g e n s , and so f o r t h . Thus the MDG C H - C H 0 as a s i n g l e s t e p , one hydrogen t r a n s f e r p r o c e s s t o y i e l d C H i s nonsense -- o n l y i o n i c s t r u c t u r e s o r f r e e r a d i c a l s can be c o n s t r u c t e d from t h a t MDG. Other c o n s t r a i n t s are a p p l i e d t o the MDG s i f s e l e c t e d by the u s e r , and some examples i n c l u d e f o r b i d d i n g c l e a v a g e o f more t h a n one bond t o t h e same c a r b o n atom o r f o r b i d d i n g c l e a v a g e o f m u l t i p l e bonds. Note t h a t the t h i r d MDG o f F i g u r e 9 cannot be r e j e c t e d on 1

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Figure 9. Mass distribution graphs illustrating the various ways of obtaining a C H ion from a C H O molecular ion under different assumptions concerning numbers of bonds cleaved, number of steps, and numbers of hydrogen atoms transferred in the fragmentation process S

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Computer Analysis of High and Low Resolution 343

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the l a t t e r c o n s t r a i n t . The two i n t e r c o n n e c t i n g bonds may o r may not be a double bond i n f i n a l s t r u c t u r e s depending on how i n t e r c o n n e c t i o n s a r e made among atoms i n v a r i o u s nodes o f the MDG. Each MDG r e s u l t i n g from a p p l i c a t i o n o f t h e f i r s t i o n i s t h e n f u r t h e r e l a b o r a t e d by s e l e c t i n g a n o t h e r i o n and a s k i n g how, under t h e e x i s t i n g f r a g m e n t a t i o n t h e o r y , the n e x t i o n c o u l d be o b t a i n e d from each MDG. T h i s g e n e r a l l y r e s u l t s i n e x p a n s i o n o f t h e MDG s i n t o more complex g r a p h s , accompanied however by a g r e a t e r s p e c i f i c i t y o f each node. T h i s e x p a n s i o n i s under t h e c o n t r o l o f an o p e r a t o r w h i c h d e t e r m i n e s l e g a l o v e r l a p s o f e x i s t i n g MDG s w i t h new MDG s i m p l i e d by t h e n e x t i o n . An example i s shown i n F i g u r e 10. The f i r s t MDG r e p r e s e n t s one way o f o b t a i n i n g the o b s e r v e d i o n ( F i g u r e 8) Ci*H& from h e x a n a l , by c l e a v a g e o f two bonds w i t h no accompanying hydrogen t r a n s f e r . Assume t h a t t h e n e x t i o n a p p l i e d was C2H5, w h i c h c a n be o b t a i n e d from the MDG 0 Η -0ι,Η Ο. There i s o n l y one way t o p e r f o r m the o v e r l a p (the o p e r a t o r i s d e s i g n a t e d (+), F i g u r e 1 0 ) , y i e l d i n g the MDG 0 Η - 0 Η = 0 Ηι»0. The e l a b o r a t e d MDG c a n y i e l d b o t h Ci*H and C H . However, i t i s s t r u c t u r a l l y more s p e c i f i c because the assembly o f atoms C^He i s a p p o r t i o n e d i n t o more p r e c i s e u n i t s , C2H5-C2H3. Assume t h a t the n e x t i o n chosen was C H . One o f the p o s s i b l e MDG s f o r o b t a i n i n g C H , c l e a v a g e o f t h e two bonds i n t e r ­ c o n n e c t i n g C H 6 = C H 0 w i t h one hydrogen t r a n s f e r , was shown i n F i g u r e 9. The o v e r l a p o p e r a t o r expands the p r e v i o u s MDG i n t o f i v e more e l a b o r a t e , b u t more s p e c i f i c MDG s ( F i g u r e 1 0 ) , from w h i c h a l l i o n s t o t h a t p o i n t (0ι*Η , C H and C H ) c a n be o b t a i n e d . Note t h a t MDG;s 1, 2, 4 and 5 a t t h i s l e v e l would be e l i m i n a t e d a t t h i s p o i n t g i v e n t h e c o n s t r a i n t f o r b i d d i n g c l e a v a g e o f more t h a n one bond t o t h e same c a r b o n atom; t h e r e i s no o t h e r way o f o b t a i n i n g the i o n C H from t h e s e MDG s g i v e n t h e assumed origin of C H (above). T h i s p r o c e d u r e (under t h e c o n s t r a i n t s o f s i n g l e step processes, f o r b i d d i n g cleavage o f m u l t i p l e bonds, f o r b i d d i n g c l e a v a g e o f more t h a n one bond t o t h e same c a r b o n atom, t r a n s f e r o f 0 o r 1 hydrogen atoms and a l l o w i n g a t most one i o n t o go u n e x p l a i n e d ) r e s u l t s i n two p o s s i b l e MDG s f o r hexanal, using the ions l a b e l l e d with elemental c o m p o s i t i o n s i n F i g u r e 8. These a r e shown i n Scheme 3. The f i r s t MDG i s s t r u c t u r a l l y v e r y s p e c i f i c and y i e l d s o n l y the c o r r e c t s t r u c t u r e o f h e x a n a l 9. The second MDG i n d i c a t e s a more common 1

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Figure 10. A sequence illustrating the élaboration of MDG's as explanations are determined for new data points (ions of specific elemental composition; see text for details)

Scheme 3

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o c c u r r e n c e , where one o r more nodes o f the MDG c a n y i e l d d i f f e r e n t p a r t s t r u c t u r e s . I n t h i s example, the node - C H - y i e l d s e i t h e r t h e 1,1- o r 1,2d i s u b s t i t u t e d C=C, thus p r o d u c i n g two a l t e r n a t i v e s t r u c t u r e s f o r t h e second MDG, 10 and 11, Scheme 3, both enols. As a d e v e l o p i n g , e x p e r i m e n t a l p r o c e d u r e , MDGGEN has s e v e r a l l i m i t a t i o n s . The f o r e m o s t i s the i n h e r e n t c o m b i n a t o r i a l c o m p l e x i t y o f even s i m p l e p r o b l e m s . Even w i t h severe c o n s t r a i n t s t h e r e are u s u a l l y s e v e r a l MDG s r e s u l t i n g from t h e a p p l i c a t i o n o f a s i n g l e i o n t o each MDG a t t h e p r e v i o u s s t e p . The t a s k o f d e t e r m i n i n g a l l t h e o v e r l a p s f o r s e v e r a l i o n s i n t h e spectrum o f a m o l e c u l e o f s i g n i f i c a n t s i z e i s time-consuming. T h i s i s due i n l a r g e p a r t t o o u r c u r r e n t i n a b i l i t y t o add c h e m i c a l i n t e l l i g e n c e t o t h e p r o c e d u r e . As we d i s c u s s e d above, any p r o b l e m i s s i m p l i f i e d i f t h e r e i s a way o f r e p r e s e n t i n g and u t i l i z i n g cons t r a i n t s based on knowledge o f t h e p r o b l e m . We a r e p u r s u i n g t h i s p r o b l e m i n MDGGEN by d e v i s i n g ways t o i n c o r p o r a t e known superatoms i n t h e p r o c e d u r e . As i n the p r o b l e m o f s t r u c t u r e g e n e r a t i o n , problems a r e s i m p l i f i e d when c o l l e c t i o n s o f atoms a r e a g g r e g a t e d i n t o known s u b s t r u c t u r e s . A n o t h e r l i m i t a t i o n i s the extent o f d u p l i c a t i o n inherent i n the p r o c e d u r e . The same s t r u c t u r e i n many cases can be c o n s t r u c t e d from two o r more MDG's. F o r example, t h e s t r u c t u r e o f h e x a n a l c a n be o b t a i n e d from MDG s 1, 5 and 7, F i g u r e 9. T h i s r e s u l t s because MDG s a r e i n a sense o n l y e x p l a n a t i o n s o f i o n s . A g i v e n s t r u c t u r e may account f o r a g i v e n i o n i n s e v e r a l d i f f e r e n t ways. We o b t a i n a s e p a r a t e MDG f o r each o f t h e s e e x p l a n a t i o n s , each o f w h i c h w i l l y i e l d t h e same s t r u c t u r e . A n o t h e r l i m i t a t i o n i s i n the f r a g m e n t a t i o n t h e o r y i t s e l f (see Conclusions). I f i o n s a r i s e from p r o c e s s e s more complex t h a n those a l l o w e d by t h e t h e o r y , then t h e c o r r e c t s t r u c t u r e may w e l l be m i s s e d o r no s t r u c t u r e s obtained a t a l l . F o r t h i s r e a s o n , we c u r r e n t l y a l l o w some number o f i o n s t o go u n e x p l a i n e d . I n the h e x a n a l example, t h i s number was one. Under the t h e o r y u s e d , s t r u c t u r e 9 cannot l o s e t h e elements o f H 0 observed i n t h e s p e c t r u m , and 10 and 11 cannot y i e l d HC0+, a l s o observed i n t h e s p e c t r u m . But w i t h one i o n a l l o w e d t o go u n e x p l a i n e d , 9-11 result. 2

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We have p r e s e n t e d some p r e l i m i n a r y r e s u l t s o f our e f f o r t s toward programs t o a s s i s t i n the g e n e r a l a n a l y s i s o f mass s p e c t r a . We have p o i n t e d out the need t o i n c o r p o r a t e as much f a c t u a l and j u d g m e n t a l knowledge a s i s r e a s o n a b l e . Only i n t h i s way can mass s p e c t r a l d a t a , i n c o n j u n c t i o n w i t h a g e n e r a t o r o f c h e m i c a l s t r u c t u r e s , p r o v i d e s i g n i f i c a n t cons t r a i n t s on s t r u c t u r a l p o s s i b i l i t i e s . Indeed, we f e e l t h a t s u c c e s s f u l systems o f the f u t u r e must make use o f such knowledge. Chemists use i t i n t h e i r r e a s o n i n g about m o l e c u l a r s t r u c t u r e s and mass s p e c t r a ; program a s s i s t a n t s must do the same. We a r e p r o g r e s s i n g a l o n g s e v e r a l l i n e s t o make the MSPRUNE and MDGGEN e x t e n s i o n s t o CONGEN more u s e f u l . We a r e c u r r e n t l y i n v e s t i g a t i n g ways t o make much more d e t a i l e d s t a t e m e n t s o f f r a g m e n t a t i o n t h e o r y t o CONGEN, f o r use e i t h e r by MSPRUNE o r MDGGEN. We a r e u s i n g a subgraph r e p r e s e n t a t i o n o f r u l e s f o r i n p u t by a u s e r (the r e p r e s e n t a t i o n used i n Meta-DENDRAL [ 1 1 ] ) . Such r u l e s may r e f e r t o a l p h a - c l e a v a g e s , âTlylic c l e a v a g e s , e t c . , or be d e t a i l e d , c l a s s - s p e c i f i c r u l e s i n i n s t a n c e s where t h a t i n f o r m a t i o n about a s t r u c t u r e i s a v a i l a b l e . We are e x t e n d i n g MSPRUNE t o a l l o w i n v e s t i g a t i o n o f a complete spectrum and a r a n k i n g o f c a n d i d a t e s t r u c t u r e s based on the agreement o f p r e d i c t e d v s . o b s e r v e d s p e c t r a . These are a l l f e a t u r e s 0 7 e a r l i e r DENDRAL work (5) now b r o u g h t t o bear on c h e m i c a l problems i n the g e n e r a l framework o f CONGEN t o provide a powerful t o o l for computer-assisted structure elucidation. Acknowledgment. We w i s h t o thank the N a t i o n a l A e r o n a u t i c s and Space A d m i n i s t r a t i o n (NGR-05-020-004), and the N a t i o n a l I n s t i t u t e s o f H e a l t h (RR 00612 and GM 20832) f o r t h e i r s u p p o r t o f t h i s r e s e a r c h and f o r the NIH s u p p o r t o f the SUMEX computer f a c i l i t y (RR 00785) on w h i c h the CONGEN program i s d e v e l o p e d , m a i n t a i n e d and made a v a i l a b l e t o a n a t i o n w i d e ' community o f u s e r s .

Literature Cited. 1.

2.

a) B i l l e r , J . E . and Biemann, Κ., Anal. Lett., (1974), 7, 515; b) Dromey, R.G., Stefik, M.J., Rindfleisch, T.C. and Duffield, A.M., Anal. Chem., (1976), 48, 1368. See review by R.G. Ridley in "Biochemical Applications of Mass Spectrometry", G.R.

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Waller, E d . , Wiley-Interscience, New York, NY, (1972), p. 177. 3. Kwok, K . - S . , Venkataraghavan, R. and McLafferty, F.W., J. Amer. Chem. Soc., (1973), 95, 4185. 4. Jurs, P.C. and Isenhour, T.L., "Chemical Applica­ tions of Pattern Recognition", Wiley-Inter­ science, New York, NY (1975). 5. Smith, D.H., Buchanan, B . G . , Engelmore, R.S., Duffield, A . M . , Yeo, Α . , Feigenbaum, E . A . , Lederberg, J. and Djerassi, C . , J. Amer. Chem. Soc., (1972), 94, 5962. 6. Gc/hrms data were obtained using a Varian-Mat 711 mass spectrometer connected to a Digital Equipment Corp. PDP 11/45 computer system of our own design. 70 ev mass spectra were obtained at a resolving power of 5000 (extended by software doublet resolution routines) at 8 or 10 sec/decade scans. 7. Carhart, R . E . , Smith, D.H., Brown, H. and Djerassi, C . , J. Amer. Chem. Soc., (1975), 97, 5755. 8. Carhart, R . E . , and Smith, D.H., Computers in Chemistry, 1, 79 (1976). 9. Smith, D.H., Buchanan, B . G . , White, W.C., Feigenbaum, E . A . , Lederberg, J. and Djerassi, C., Tetrahedron, (1973), 29, 3117. 10. Dromey, R . G . , Buchanan, B . G . , Smith, D.H., Lederbert, J. and Djerassi, C . , J. Org. Chem., (1975), 40, 770. 11. Buchanan, B . G . , Smith, D.H., White, W.C., Gritter, R . J . , Feigenbaum, E . A . , Lederbert, J., and Djerassi, C . , J. Amer. Chem. Soc., (1976), 98, 6168. Received December 30, 1977

INDEX

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ix001

A Accelerating voltage scan experiment 235 Accelerating voltage scan method .277,282 Accurate mass measurement 175 1- Actene 184 Activation studies, collisional 50,97 Adenosine,fielddesorption spectrum of 216 Adenylate cyclase inhibitor, field desorption mass spectrum of the 244 Aflatoxins 115 analysis 104 analytical extraction of 108 mass spectra of 106 Alcohol(s) 150,158 cinnamyl 90 Aliphatic nitriles 182 Alkanes 291 C 287 C 287,292 elimination, spontaneous 66 Alkenes, C 290 Alkene isomers, C 287 Alkyl aromatic compounds 183 ions, energy releases and losses for .. 78 phthalates 284 Alkylbenzenes 263,292 Alkylnaphthalenes 263 Allopurinol 111,230 Amines 150 and phenols, detection limits for derivatives of primary 161 quantitation of bis-(2-chloroethyl) 188 2- Aminobutane 71-73 3-Aminopentane 71-73 Ammonia 155 Ammonium ions 65 Anthracenes, phenanthrenes 263 Appearance potential (AP) measurements 24 Argon-water 154 Aromatic(s) 151 compounds 182 distribution in a heavy coal liquid 272 high sulfur petroleum 268 Arsenic 308 Aspergillus flavus 104 6

8

5

6

Atomic oxygen negative ion, reactions of the 181 Autoionization 60 Β Bacteriophage DNAs, mass spectra of 255-259 Barbiturates 63,66 Benzocyclobutene 51 Benzodithiophenes 263 Benzo ( ghi ) perylene 167 Benzonitrile 212 Benzyl vs. tropylium problem 23 Best anode temperature (BAT) 209 on electricfield,dependence on .... 221 Best emitter temperature (BET) 218 Binding effects, chemical 117 Biofluids 98 Biomedical application, prognosis forfielddesorption mass spectrometry in 209 Biomedical application, selectivity in 188 Blood Ill and tissue, quantitative high resolution SIM for analyzing purines in 105 Boron 302 Brain tissue, freeze dried 230 Bronsted acid-Bronsted base type spectra 162-164 1,3-Butadiene 16 Butane 65 Butanone, analysis of reaction mixtures from the methylation of 64 2-Butaiione by methyl iodide, methylation of 63 s-Butylamine 71 -pyridine proton-bound dimer 73 ieri-Butylethyl ether 65 C C H loss C H , 1,2-elimination of hydrogen from ionized C H 0 ions, possible isomeric structures of C H . radical cations

349

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39

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51 26

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Ci Hi Oe, exact mass fragmentogram for 112 CH OH ion, fragmentation of the ... 6 Cations C H t radical 26 α-phenylethyl 51 slow unimolecular decomposition of the isomeric 27,33 Cadmium 304 Caffeine 111-114 Carbohydrates 209 Carbon number distribution as a mole % 272 Carbonyl compounds 182 Cell, Knudsen 226 Centroid 124-137 Charge exchange 60 Chemical ionization 97 mass spectrometry, analytical applications of positive and negative ion 150 mass spectrometry, investigations of selective reagent ions in 179 mass spectra 72,156,159,173, 201 methods, comparison of EI and 151 pulsed positive and negative ion (PPINICI) 162 reagent, NO* as a selective 158 CID, analysis of mixtures by MIKE61 CIMS, selective reagent gases for positive ion 154 p-Chlorobenzoic acid 77 p-Chlorobenzonitrile 77 p-Chloronitrobenzene 67,77 p-Chlorophenol 77 Cholestane 279 Chromatogram, accurate mass 143 Chromatogram for equal GC peak shapes, mass 136 Chromatography high pressure liquid (hplc) 243 /mass spectrometer/computer (GC/MS/COM) system, gas .. 54 /mass spectrometry (GC/MS), gas 97,120 Cinnamyl alcohol 90 FI spectra of 91 Circuit, B/E linked scan 14 Circuit, voltage divider 100 Coal extract 267 Hiawatha 295 liquid 272 liquefaction product 268 products, ultra-high resolution mass spectrometry analysis of petroleum and 261 Cobalt 308 7

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Collisional Activation (CA) 22 mass spectra ( MS ) 47,55 -MS instrumentation 48 -MS as a separation/identifica­ tion system, MS/ 54 to molecular structure determina­ tion, applications of 52 Collision-induced dissociation 68 Composition monitoring of GC effluents 122 Compositions for significant fragment ions in the mass spectrum 327 Compounds, analysis of nonvolatile .. 166 Computer analysis of high and low resolution mass speceral data 325 applications in mass spectrometry .. 310 -assisted structure elucidation based primarily on mass spectral data 326,327 -controlled power supplies for high resolution SIM 100 identification of unknown mass spectra 311 printout for cadmium by isotope dilution spark source mass spectrometry 306 programs, data acquisition and 266 system, gas chromatograph/mass spectrometer/ (GC/MS/COM) 54 Condon Process, Frank153 Contamination, ion source 116 Creatin 173 Crude liquid, partial carbon number distribution as mole % in a 272 Crude oil 271 Cyclic alkalines 150 Cycloalkanes 158,292 Cycloheptatriene 25,51 1,3-Cyclohexadiene 16 Cyclohexane 159,289 Cyclohexanone 159 Cyclohexene-3,3,6,6-d, FIK study on 81 Cyclophosphamide 194,198, 202 (I) and its major human metabolites 195 nornitrogen mustard 206 Cyclopropane 52 4

D Data acquisition and computer programs 266 Data presentation, quantitative analysis and 269 3-Decene 159 Decomopsition, slow unimolecular ... 24-36

INDEX

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ix001

Deoxyribonucleic acids ( DNA ) detection of modified nucleotide in 251

351

Electron impact (continued) mass spectrum 197,199 E. coli 258 analysis of DNA, pyrolysis 249 comparison offielddesorption mass spectra of bacteriophage .255-259 pyrolysis electron impact mass and 217-219 spectral analysis of 248, 249 electron capture162 salmon sperm 252 of 3-phenylpropanal 86,88 wheat germ 254 of a synthetic nucleoside 215 Dependence of BAT on electric field 221 of polydeoxyribonucleotides 250 Derivative of phosphoramide mustard, 1,2-Elimination of hydrogen 37-39 mass spectrum of the trimethyl 196 Elimination, spontaneous alkane 66 Derivatives of primary amines and Emission 60 phenols, detection limits for 161 Emitter current programmer Desorption and ionization of samples, (ECP) 214,215 mechanisms for the observed 172 Emitter current, temperature vs 216 Deuterium labeling 81 Energy Deuterium oxide 155 curves, rate constant vs. internal .... 70 Diazomethane 195 diagrams, potential 170 Dibenzofurans 263 kinetic 97 Diethyl ether 92 releases 68 Diisopropyl ketone 65,69 measurements, reactions studied via Dilution techniques for trace analysis 299 translational 60 Dimer, proton-bound 72,73 releases and losses for alkyl ions .... 78 Dimethyl phosphoramide mustard .... 197 release profiles 33,41 Dipping 212 reverse activation 39 Direct analysis of daughter ions spectra (MIKES), mass analyzed (DADI) 48,235 ion kinetic 48, 235 spectrum 240 spectrometry, ion chemistry via Direct probe sample introduction high kinetic 58 resolution mass spectrometry, surface, potential 18,27,33,36-41 organic trace analysis using 97 Equilibration to two ions 27 Dissociation 60 Equilibration of isomers, complete ... 24 collision-induced 68 Equilibrium theory (QUET), quasi- 152 photo 60 Ethane 37 step 37-39 Ethylbenzene 51 symmetry-forbidden 37 Ethylene 38 unimolecular 68 Ethylenebenzenium 51 Distribution as a mole % 272 0-Ethylphenyl ion 51 Double-focusing mass Exchange, charge 60 spectrometer 47,82, 274,301 Excitation 60 Doublet, peak profile for 139 Extraction of aflatoxins, analytical .... 108 Drug metabolites 98,209 analysis of 194 F Dry spike isotope technique, trace elements by a 306 Field desorption (FD) 168 analysis 224 and electron impact spectra, £ comparison of 217-219 E. coli DNA 258 mass spectrometry, analysis of Electricfield,dependence of BAT on 221 natural products using 243 Electron capture—Bronsted acidmass spectrometry in biomedical Brônsted base spectra 162,164 applications, prognosis for 209 Electron capture-EI type spectra 162 samples, electron impact ionization Electron impact 68 of 218 and CI mass spectra of spectra di-2-pentylamine 156 of adenosine 216 and CI methods, comparison of 151 of the adenylate cyclase inhibitor 244 ionization 62,218 of galactitol and sucrose 218, 219

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ix001

352

HIGH

PERFORMANCE

MASS

SPECTROMETRY

spectra (continued) Gas-phase ion chemistry, field of naphthyl sulfate 218 ionization kinetic studies of 80 of the potassium salt of naphthyl Gas-phase ions from collisional sulfate 220 activation spectra, structures of .. 47 of sodium barbiturate 220 Gases for positive ion SIMS, selective reagent 154 Field image theory 210 Field ionization (FI) 97 Gaussian model for mass spectral peak 101 Glutamic acid 217 kinetic 337 curve of ions 82 Glycine Glycosides 209 study on cyclohexene-3,3,6,6-^4 81 Gout Ill 278-283 of gas-phase ion chemistry 80 Green River shale on 2-phenoxyethyl chloride 81 on 3-phenylpropanal 83 H mass spectrometer, double-focusing 82 Halonium ions 65 spectra 83 133 of cinnamyl alcohol 91 Heptacosafluorotributylamine 300 of 2-phenoxyethyl chloride 83 Herzog geometry, Mattauch34(W43 of 3-phenylpropanal 86,88,90 Hexanal 289 theory 210 Hexane isomers 296 Fluoro olefins 13 l-Hexyl-n-C 295 Fluoroethylene 13 Hiawatha coal Fluoropropene 13 High pressure liquid chromatography (hlpc) 243 Formaldehyde 38 High resolution 122 Fragmentation -low voltage technique, advantages of the CH OH ion 6 and disadvantages of 262 mechanisms in chemical ionization 65 nomenclature and 262 metastable 60 MS, GC, and fast-scanning 128 paths of phthalate 285 MS, primary considerations for pattern 249 GC/ 129 process constraints 332 209 rules 326 Hormones 151,270 theory 341 Hydrocarbons mixture of polychlorinated 241 Fragmentogram for Ci Hi 0 , exact pure 287 mass 112 Frank-Condon process 153 Hydrogen deficiency nomenclature .... 263 Hydrogen, 1,2-elimination of 37 p-Hydroxy nitrobenzene 67 G Hypoxanthine 111-114, 230 20

+

3

2

7

2

6

Galactitol and sucrose, FD spectra of 218,219 Gammacerane 279 Gas chromatogram of urine extract 189,199 Gas chromatography (GC) detectors, sensitivity of 192 effluents, elemental composition monitoring of 122 /high resolution mass spectrometry (HRMS) 120 and accelerating voltage scan ( AVS ) method results, comparison of 282 fast-scanning HRMS 128 primary considerations for 129 peak elution time 129-131 peak shapes, mass chromatogram for equal 136

I ICR spectroscopy 13 Ideno (l,2,2-ed-)pyrene 167 Instrumentation 277 Instrument performance 128 Intensities of the major peaks in the O-NCI spectra of methylpyridines 185 Intensity vs. mass 104,109,110 INTSUM 332 Ionization 68 chemical 68,97 detector, flame 132 electron impact 62 field 97 offield-desorbedsamples, electron impact 218 fragmentation mechanisms in chemical 65

353

INDEX

Ionization (continued) mechanism, proposed 225 metastable ions in chemical 65 negative chemical 97 of -phenylethyl chloride 51 of samples, mechanisms for the observed desorption and 172 Ion(s) ammonium 65 CH 342 chemical ionization mass spectrometry 150,179 chemistry,fieldionization kinetic studies of gas-phase 80 chemistry via kinetic energy spectrometry 58 from collisional activation spectra, structures of gas-phase 47 current plot 328,336 cycloheptatriene molecular 25 cyclotron resonance ( ICR ) 22 in defining structure, use of defocused metastable 239 (DADI), direct analysis of daughter 48,235 energy releases and losses for alkyl 78 equilibration to two 27 β-ethylphenyl 51 fragmentation of the CH OH 6 halonium 65 kinetic energy (IKE) method 277 kinetic energy spectra .9,48,97,284,290 labeled and unlabeled pyridinium .. 75 in the mass spectrum 327 metastable 47 methods using a double-focusing mass spectrometer 274 methods, mixture analysis by metastable 233 monitoring, analysis of individual components in a complex mixture by selected 229 monitoring (SIM), high-resolution selected 98 possible isomeric structures of CsH 0 51 potential energy surfaces for uni­ molecular reactions of organic .... 18 reactions of the atomic oxygen negative 181 reactions of the vinyl methyl ether molecule 179 records 202-205 of plasma extract 201 of the urine extract 193,198

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ix001

3

7

+

3

+

5

6

Ion(s) (continued) source contamination 116 geometries 221 pulsed radiofrequency (RF) 299 structure determinations 74 structures of gaseous organic 50 trimethyloxonium 65 Isomerization, rate-determining 32,33 Isomers, hexane 289 Isotope deposited onto pure silver powder, separated 307 dilution spark source mass spectrometry 300,306 dilution techniques for trace analysis, multielement 299 labeling 67 ratio measurements 190 technique, trace elements by a dry spike 306 J

Johnson geometry, Nier-

6-10

Κ Kerogen pyrolyzate 283 6-Ketostradiol, CI mass spectra of .... 156 Ketones, analysis of 62 Ketones, MIKE spectra of 70 Knudsen cell 226 L Lipids Liquefaction product, coal

115 268

M Magnet reset time 129 Magnetic analyzer 300 Magnetic scanning 123 Manganese 308 Mass-analyzed ion 97 Mass-analyzed ion kinetic energy spectroscopy (MIKES) 48,235 /CID, analysis of mixtures by 61 sensitivity of 62 spectra 66,73,76 of ketones 70 spectrometer, reversed sector 62 technique, selectivity and sensitivity of the 75 Mass chromatogram, accurate 143 chromatogram for equal GC peak shapes 136

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ix001

354 Mass (continued) distribution graphs 330,342 doublets and resolving power 264 fragmentograms, exact 105,112 intensity vs 104 measurement accuracy 123-132 evaluation of 136 measurements, resolution and 267 resolution 126 dynamic 129, 138 elemental composition assignment 122,123 on mass measurement accuracy, effect of inadequate 131 resolving power, precursor 7-9 SIMS scans of intensity vs 109,110 spectra 288-290 of aflatoxins 106,107 of bacteriophage DNAs 255-259 computer identification of unknown 311 future possibilities for collisional activities 55 isotope dilution spark source 305 spectral analysis of DNA, pyrolysis electron impact 249 data classes used in STIRS 315 computer-assisted structure elucidation based primarily on 326,327 structure elucidation based on computer analysis of high and low resoltuion 325 peak, Gaussian model 101 systems, comparisons of STIRS with other available 321 spectrometer /computer (GC/MS/COM) system, gas chromatograph/ 54 double-focusing field ionization .. 82 double-focusing spark source 301 -mass spectrometer (MS/MS) analysis 97 MS-702 301 organic mixture analysis by metastable ion methods using a double-focusing 274 PPINICI 163 reverse-geometry, double-focusing 47 stability 99 spectrometry analytical applications of positive and negative ion chemical ionization 150

HIGH

PERFORMANCE

MASS

SPECTROMETRY

spectrometry ( continued ) analysis of natural products using field desorption 243 analysis of petroleum and coal products, ultra-high resolution 261 applications in a pharmaceutical laboratory 229 in biomedical application, prognosis forfielddesorption 209 /CAMS as a separation/identification system 54 computer applications in 310 computer printout for cadmium by isotope dilution spark source 306 data, acquisition and reduction of 310 detection and identification of minor nucleotides in intact deoxyribonucleic acids by pyrolysis electron impact ... 248 gas chromatography- (GC/MS), 97 gas chromatography /high resolution 120 instrumentation, CA48 investigations of selective reagent ions in chemical ionization .. 179 isotope dilution spark, source 300 metabolite analysis using 242 negative ion chemical ionization (NICIMS) 160 organic trace analysis using direct probe sample introduction and high resolution 97 plasma desorption 168 spark source (SSMS) 299 spectrum CI 72,201 collisional activation (CA) 47 electron impact 197-199 elemental compositions for significant fragment ions in the 327 of the trimethyl derivative of phosphoramide mustard 196 -time function 124-126 transfer 223 -voltage relationship 103 Mattauch-Herzog geometry 300 Maytansine 173 McLafferty rearrangement 86-89 MDGs 344 MDGGEN, prospective generation of structural possibilities by 339 Measurement accuracy, mass 123-136 Mechanism, four-centered 29,65 Mechanisms for the observed desorption and ionization of samples ... 172

355

INDEX

Metabolites analysis using mass spectrometry .... 242 cyclophosphamide I) and its major human 195 drug 194,209 of trimethoprim 242 Metastable decompositions of C H 0 ions 28 fragmentation 60 ions 47 in chemical ionization 65 in defining structure, use of defocused 239 methods, mixture analysis by .233,274 proton affinities using 69 peaks 35,75 spectrum 279,280 transitions 276 different methods for observation of 5 in a high performance mass spectrometer 3 methods of observing 4 Methane 65 Methanol 16 Methionyl-arginyl-phenylalamine 173 Method, accelerating voltage scan 9 Methodology 249 Methods of observing meastable transitions 3-5 Methyl-alkyl phthalate 196 Methylamine 38 Methylated products, mixture of 240 Methylation of butanone 63,64 2-Methyl-2-butanol 150 Methylcycloheptatriene 51 5-Methyldeoxycytidine-5'-phosphate .. 253 Methylene amine 38 Methyl iodide 236,237 methylation of 2-butanone by 63 Methylisopropyl ether 92 Methylpyridines, relative intensities of the major peaks in the O'NCI spectra of 185 Microsyringe extrusion 212 Microsyringe sample addition 213 Milk, freeze-dried 108,109 Mixture analysis, principle of 62 Mixture by selected ion monitoring, analysis of individual com­ ponents in a complex 229 Mole % in a coal liquid 272 Mole % in a crude liquid 272 Molybdenum 304 Monomethyl derivative of phosphoramide 200 Monomethyl phosphoramide mustard 201 MSPRUNE, application of 334 +

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ix001

2

5

Muscle tissue 111,230 Muscles, human 113 Mustard, derivatives of phos­ phoramide 195 Mustard, mass spectrum of the trimethyl derivative of phos­ phoramide 196 Mycotoxins 115

Ν Naphthenes 263 Naphthenic acids 284 Naphthyl sulfate, FD spectrum of 218-220 Natural products using field desorption mass spectrometry, analysis of 243 Negative ion chemical ionization mass spectrometry (NICIMS) .... 160 Nier-Johnson geometry 6-10 Nitric oxide 157 NO as a selective CI reagent 158 Nitrobenzenes 67,68,76 Nitrogen 42,270 Nitrophenols 19 Nomenclature and high resolution .... 262 Nomenclature, hydrogen deficiency .. 263 Nonvolatile compounds, analysis of .... 166 d -Nornitrogen mustard 204 Nornitrogen mustard, cyclophos­ phamide 206 Nuclear magnetic resonance (NMR) spectroscopy 243 Nucleic acids 248 Nucleoside(s) 209,245 analog, synthetic 217 cyclophosphates from the ribodinucleotides, formation of 249 electron impact (EI) spectrum of a synthetic 215 in intact deoxyribonucleic acids 248 in DNAs, detection of modified 251 +

4

Ο

frûns-4-Octene 184 ONCI spectra 184 Olefins 151,158 Oligonucleotides 249 Organic ions, structures of gaseous .... 50 Organic mixture analysis by metastable ion methods 274 Ovarian tissue, human Ill Oxipurinol 112,230 Oxygen 42,270 as a PPINICI reagent 165

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ix001

356

HIGH

Paraffins 263 Peakwidth, threshold 139-141 Pentafluorobenzoyl amphetamine 161 Pentafluorobenzylidene dopaminebis-trimethyl sylyl ether 161 1-Pentanol 159 2-Pentanol 159 Pentanones 184 di-2-Pentylamine, EI and CI mass spectra of 156 Peptiae volatilization 116 Perchlorobutadiene 133 Perfluorokerosene (PFK) 102,230 Pesticides 98,115 Petroleum 293 aromatics, high sulfur 268 and coal products, ultra-high reso­ lution mass spectrometry, analysis of 261 Pharmaceutical laboratory, mass spectrometry applications in a ... 229 Phenanthrenes/anthracenes 263 Phenols, detection limits for deriva­ tives of primary amines and 161 2-Phenoxyethyl chlorides 81-85 l-Phenyl-n-C o 296 a-Phenylethyl cations 51 β-Phenylethyl chloride, ionization of 51 3-Phenylpropanal 81-90 Phosphoramidate linkage 251 Phosphoramide monomethyl derivative of 200 mustard 195-198,201-205 Photo dissociation 60 Photoexcitation 60 Photographic plate 299 Photoionization 60 Phthalates 286 fragmentation paths of 285 Plasma desorption mass spectrometry 168 extract 202 ion records of 201 Plot, B/E scan 14 Polychlorinated hydrocarbons, mixture of 241 Polydeoxyribonucleotides 250 Polynucleotide poly (dA · dt), synthetic 257 Polypeptides 209 Potassium benzoate 173 Potassium salt of naphthyl sulfate, FD spectrum of the 220 Potential surface, concept of the 20 Power supplied for high resolution SIM, computer-controlled 100 2

PERFORMANCE

MASS

SPECTROMETRY

Power supply interface, programmed 101 Pulsed positive and negative ion CI (PPINICI) 162 mass spectra 167 mass spectrometer 163 reagent, oxygen as a 165 Probability based matching (PBM) .. 311 and STIRS as aids to the interpreter 322 Profiles, energy release 33,41 Profiles, peak 139,143 Propene 52 Propoxyphene 193 Proton affinities using meastable ions 69 Proton-bound dimer 73 s-butylamine/pyridine 73 formation of 72 Purines 112 in blood and tissue, quantitative high resolution SIM for analyzing 105 Purinol Ill Pyridine 71 proton-bound dimer, s-butylamine 73 Pyridinium ions, labeled and unlabeled 75 Pyrolysis electron impact mass spectra analysis of DNA 248,249 Pyrolysis oil 283 Pyrolytic fragmentation of poly­ deoxyribonucleotides 250 Pyrolyzate, kerogen 283

Quantitative analysis and data presentation Quasi-equilibrium theory (QUET)

152

R Radiofrequency (RF) ion source, pulsed 299 Raffinose „ 173 "Randomization* 21 Rate constant vs. internal energy curves 70 Rate-determining isomerization 32,33 Reaction(s) mixtures from the methylation of butanone 64 of organic ions 18 studied via translational energy measurements 60 Reagent ions in chemical ionization mass spectrometry, investigations of selective 179 Reduction, data acquisition and 266 Reliability plots, recall/ 312

357

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ix001

INDEX

Resolution 124 dynamic mass 138 effect of β-slitwidth on precursor mass 12 elemental composition assignment 122,123 high 122 mass 126 and mass measurements 267 on mass measurement accuracy, effect of inadequate mass 131 mass spectrometry, organic trace analysis 97 values, comparison of 141 Resolving power, mass doublets and .. 264 Resolving power, precursor mass 7-9 Resonance effect, classical 20 Resonance techniques, double 47 Retention indexes, relative 337 Reverse-geometry double-focusing mass spectrometer 47 Ribodinucleotides, formation of nucleoside cyclophosphates from the 249 D-(-)Ribose 159

Sensitivity 129,135 effect of 0-slitwidth on 8 of GC detectors 192 of the MIKES technique, selectivity and 75 Shale, Green River 278-283 Silver powder, separated isotopes deposited onto pure 307 0-slitwidth 8,12 Sodium barbiturate, FD spectrum of 220 Spark source mass spectrometry (SSMS) 299 computer printout for cadmium by isotope dilution 306 double-focusing 301 isotope dilution 300,305 Spectra of adenosine, field desorption 216 Bronsted acid-Bronsted base type 162,164 CI mass 159,173 of cinnamyl alcohol, FI 91 comparison offielddesorption and electron impact 217,219 DADI 240 electron capture-EI type 162 FI 83 ion kinetic energy ( IKE ) 9 S of 6-ketoestradiol, CI mass 156 Saccharides 209 MIKE 66,76 Salmon sperm DNA 252 structures of gas-phase ions from collisional activation 47 Salts 172 O-NCI 184 Saturate, metastable spectrum 280 of di-2-pentylamine, EI and CI mass 156 Scan of 3-phenylpropanal, EI 88 coils 99 PPINICI mass 167 cycle time 128-131,138 recorded at increased mass method, accelerating voltage 9,277 resolution 123 rate 124 recorded at nominal mass resolution 122 time 129 of a synthetic nucleoside, electron Scanning, magnetic 123 impact (EI) 215 "Scrambling" 21 system evaluation 318 Selected ion monitoring ( SIM ) Spectrometer, metastable transitions for analyzing purines in blood in a high performance mass 3 and tissue 105 Spectrometer, reversed sector MIKE 62 computer-controlled power supplied for high resolution .... 100 Spectroscopy ICR 13 high resolution 98 MIKES 48 scans of intensity vs. mass 109,110 nuclear magnetic resonance ( NMR ) 243 Selectivity, approaches for enhancing 190 Stability, mass spectrometer 99 Selectivity and sensitivity of the Steranes 278-282 MIKES technique 75 Stripping 60 Self-training interpretive and Structural candidates, retrospective retrieval system (STIRS) 311 testing of 330 as aids to the interpreter, PBM and 322 Structural possibilities by MDGGEN 339 with other available mass spectral Structure(s) systems, comparison of 321 of C H 0 ions, possible isomeric .... 51 mass spectral data classes used in .. 315 determination, applications of CA substructure identification by 317 to molecular 52 +

3

5

358

HIGH

Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0070.ix001

Structure ( s ) ( continued ) determinations, ion 74 elucidation based on computer analysis of high and low reso­ lution mass spectral data ... 325-327 Studies, collisional activation 50 Substructure identification by STIRS 317 Sucrose, FD spectra of galactitol and 218,219 Sulfur 270 petroleum aromatics, high 268 Superatoms 331,335 Surface volatilization 116 Synthetic polynucleotide poly (dA · dt) 257

PERFORMANCE

MASS

Transitions, methods of observing metastable Trichlorobenzenes Triethylamine Trimethoprim metabolites of Trimethyl derivative of phos­ phoramide mustard, mass spectrum of the Trimethyl phosphoramide mustard .... Trimethyloxonium ion Tropylium problem, benzyl vs Tungsten emitter wires Tyramine Tyrosine

4 241 159 230 242 196 197 65 23 116 230 219

U

Τ Temperature, best annode (BAT) ... 209 Temperature, best emitter (BET) ... 218 Temperature vs. emitter current 216 Terpanes 278-282 Tetracosane 328 Tetrafluorocyclobutanes 13 Tetrahydrocannabinol pentafluorobenzoate 161 Thermal diffusion cuts 294 Thioformaldehyde 38 Threshold peakwidth 139-141 Time function, mass124-126 GC peak eiution 129-131 magnet reset 129 scan cycle 131,138 Tissue freeze-dried brain 230 freeze-dried muscle 230 human ovarian Ill muscle Ill quantitative high resolution SIM for analyzing purines in blood and 105 Toluene 51 Trace analysis using direct probe sample introduction, high resolution mass spectrometry 97 analysis, multielement isotope dilution techniques for 299 elements by a dry spike isotope technique 306 metal concentrations 304

SPECTROMETRY

Ultra-high resolution mass spec­ trometry analysis of petroleum and coal products 261 Unimolecular decomposition 24,32-36 of the isomeric cations, slow 27,33 Unimolecular dissociation 68 Uric acid 111-114, 230 Urine 112,336 extract 196 gas chromatogram of 189,199 ion records of the 193,198 human 328,335 V Vinyl methyl ether 16 molecular ion, reactions of the 179 Volatilization, peptide 116 Volatilization, surface 116 Voltage(s) 223 divider circuit 100 relationship, mass103 scan experiment, accelerating 235 scan method, accelerating 9,277, 282 technique, advantages and disad­ vantages of high resolution-low 262 W Water, argonWheat germ, DNA

154 254 X

Xanthine Xylenes

111-114,230 51,184,337