Pyrolysis mass spectrometry of recent and fossil biomaterials: compendium and atlas

TECHNIQUES AND INSTRUMENTATION IN ANALYTICAL CHEMISTRY - VOLUME 3 PYROLYSIS MASS SPECTROMETRY OF RECENT AND FOSSIL BI...

Author: Henk L.C. Meuzelaar | etc.

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TECHNIQUES AND INSTRUMENTATION IN ANALYTICAL CHEMISTRY

- VOLUME 3

PYROLYSIS MASS SPECTROMETRY OF RECENT AND FOSSIL BIOMATERIALS COMPENDIUM AND ATLAS

TECHNIQUES AND INSTRUMENTATION IN ANALYTICAL CHEMISTRY Volume 1 Evaluation and Optimization of Laboratory Methods and Analytical Procedures. A Suwey of Statistical and Mathematical Techniques by D.L. Massart, A. Dijkstra and L. Kaufman Volume 2 Handbook of Laboratory Distillation by E . Krell Volume 3 Pyrolysis Mass Spectrometry of Recent and Fossil Biomaterials. Compendium and Atlas by H.L.C. Meuzelaar, J. Haverkamp and F.D. Hileman

TECHNIQUES AND INSTRUMENTATION IN ANALYTICAL CHEMISTRY - VOLUME 3

PYROLYSIS MASS SPECTROMETRY OF RECENT AND FOSSIL BIOMATERIALS COMPENDIUM AND ATLAS

Henk L.C. Meuzelaar Biomaterials Profiling Center, University of Utah, Salt Lake City, UT, U.S.A.

Johan Haverkamp Department of Biomolecular Physics, F.O. M. Institute for Atomic and Molecular Physics, Amsterdam, The Netherlands

Fred D. Hileman Analytical Chemistry Division, Monsanto Research Corporation, Dayton, OH, U.S.A.

E LSEV IE R SCI ENTl F IC PUBLISHING COMPANY Amsterdam - Oxford - New York 1982

ELSEVIER SCIENTIFIC PUBLISHING COMPANY Molenwerf 1 P.O. Box 21 1,1000 AE Amsterdam, The Netherlands Distributors for the United States and Canada:

ELSEVIER SCIENCE PUBLISHING COMPANY INC. 52, Vanderbilt Avenue New York, NY 10017

ISBN 044442099-1 (Vol. 3) ISBN 044441 744-3 (Series)

0 Elsevier Scientific Publishing Company, 1982 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher, Elsevier Scientific Publishing Company, 1000 AH Amsterdam, The Netherlands Printed in The Netherlands

Dedicated to Professor Dr. Jaap Kistemaker in honour of his 65th birthday

“Is rational mass spectrometric analysis possible if molecular size is such that thermal dissociation . . . is unavoidable? Without a doubt the answer is yes. ’‘ J. Kistemaker (1 954)

This Page Intentionally Left Blank

V II

CONTENTS

............................ Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PART I COMPENDIUM OF BASIC PRINCIPLES AND APPLICATIONS . . . . . . . . . Abbreviations used

Chapter 1 . ORIGINS AND DEVELOPMENT OF PYROLYSIS MASS SPECTROMETRY OF BIOMATERIALS . . . . . . . . . . . . . . . . . . . . . 1.1. 1.2. 1.3. 1.4. Chapter 2 . 2.1. 2.2. 2.3. 2.4. 2.5. 2.6. 2.7. Chapter 3 . 3.1. 3.2. 3.3. 3.4. 3.5. 3.6. Chapter 4. 4.1. 4.2. 4.3.

4.4. 4.5. 4.6. 4.7. Chapter 5 . 5.1. 5.2. 5.3. 5.4.

.. ...................... ......................

The first reports Direct probe Py-MS Laser Py-MS . . . . Filament Py-MS . . .

..................... ..................... FROM FINGERPRINTING TO STRUCTURAL INVESTIGATION . . . . . . . Operational fingerprinting; a new concept . . . . . . . . . . Complexity of the sample . . . . . . . . . . . . . . . . . . . Additivity of component spectra . . . . . . . . . . . . . . . Availability of reference spectra . . . . . . . . . . . . . . Availability of standard reference materials . . . . . . . . . Knowledge of relevant pyrolysis mechanisms . . . . . . . . . . Availability of ancillary analytical methods . . . . . . . . . PYROLYSIS MECHANISMS IN BIOMATERIALS . . . . . . . . . . . . . General aspects . . . . . . . . . . . . . . . . . . . . . . . Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . Nucleic acids . . . . . . . . . . . . . . . . . . . . . . . . Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . Miscellaneous biopolymers . . . . . . . . . . . . . . . . . . THE TECHNIQUE OF CURIE-POINT PYROLYSIS MASS SPECTROMETRY . . . Sample preparation . . . . . . . . . . . . . . . . . . . . . . Pyrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . Pyrolysate transfer . . . . . . . . . . . . . . . . . . . . . Ionisation . . . . . . . . . . . . . . . . . . . . . . . . . . Mass analysis . . . . . . . . . . . . . . . . . . . . . . . . Ion detection . . . . . . . . . . . . . . . . . . . . . . . . Signal recording . . . . . . . . . . . . . . . . . . . . . . . REPRODUCIBILITY IN CURIE-POINT PYROLYSIS MASS SPECTROMETRY . . Qualitative reproducibility . . . . . . . . . . . . . . . . . Quantitative reproducibility . . . . . . . . . . . . . . . . . Factors influencing long-term reproducibility . . . . . . . . Prospects for inter-laboratory reproducibility . . . . . . . .

IX

XI XI11 1 3 3 4 5 6 9 9 9 10 10

11 13 13 15 15 16 19 21 24 25 29 29 33 37 41 42 45 45

47 47 49

50 52

.

.................. A v a i l a b l e computer programs . . . . . . . . . . . . . . . . . 6.1. Data pre-processing . . . . . . . . . . . . . . . . . . . . . 6.2. 6.2.1. Pattern scaling . . . . . . . . . . . . . . . . . . . 6.2.2. Feature s c a l i n g . . . . . . . . . . . . . . . . . . . Univariate s t a t i s t i c a l analysis . . . . . . . . . . . . . . . 6.3. 6.4. M u l t i v a r i a t e s t a t i s t i c a l analysis . . . . . . . . . . . . . . V i s u a l i s a t i o n techniques . . . . . . . . . . . . . . . . . . . 6.5. Computer-assisted chemical i n t e r p r e t a t i o n . . . . . . . . . . 6.6. 6.6.1. S u b t r a c t i o n techniques . . . . . . . . . . . . . . . . 6.6.2. Factor analysis . . . . . . . . . . . . . . . . . . . Computer-assisted q u a n t i t a t i v e a n a l y s i s o f m i x t u r e s . . . . . 6.7. Future developments . . . . . . . . . . . . . . . . . . . . . 6.8. Chapter 7 . SELECTED APPLICATIONS TO BIOMATERIALS . . . . . . . . . . . . 7.1. Scope o f a n a l y t i c a l p y r o l y s i s techniques . . . . . . . . . . . 7.2. Medical a p p l i c a t i o n s . . . . . . . . . . . . . . . . . . . . . 7.2.1. C l i n i c a l microbiology . . . . . . . . . . . . . . . . 7.2.2. Other c l i n i c a l a p p l i c a t i o n s . . . . . . . . . . . . . 7.2.3. Q u a l i t y control . . . . . . . . . . . . . . . . . . . 7.3. Biological applications . . . . . . . . . . . . . . . . . . . 7.4. Environmental a p p l i c a t i o n s . . . . . . . . . . . . . . . . . . 7.5. Biogeochemical a p p l i c a t i o n s . . . . . . . . . . . . . . . . . Chapter 6

DATA ANALYSIS PROCEDURES

............ Contents ................................ INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 1.1. General remarks . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Sample p r e p a r a t i o n . . . . . . . . . . . . . . . . . . . . . . 1.3. A n a l y t i c a l c o n d i t i o n s . . . . . . . . . . . . . . . . . . . . . Spectrum p r e s e n t a t i o n format . . . . . . . . . . . . . . . . . 1.4. 1.5. Spectrum c o n t r i b u t i o n s from i n o r g a n i c c o n s t i t u e n t s . . . . . . P r o t e i n s p e c t r a (group B) . . . . . . . . . . . . . . . . . . . 1.6. 1.7. N u c l e o t i d e and n u c l e i c a c i d spectra (group C ) . . . . . . . . . 1.8. L i p i d s p e c t r a (group D) . . . . . . . . . . . . . . . . . . . . 1.9. Spectra o f hurnic m a t e r i a l s and geopolymers (group F) . . . . . 1 . 1 0. Spectra o f o t h e r b i o c h e m i c a l l y i m p o r t a n t compounds (group G) . 2. PYROLYSIS MASS SPECTRA . . . . . . . . . . . . . . . . . . . . . . . Carbohydrates and Glycoconjugates . . . . . . . . . . . . . . . Group A . Peptides and P r o t e i n s . . . . . . . . . . . . . . . . . . . . . Group B . Nucleotides and N u c l e i c Acids . . . . . . . . . . . . . . . . . Group C . Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . Group D . N a t u r a l Products . . . . . . . . . . . . . . . . . . . . . . . Group E . Humic M a t e r i a l s and Geopolymers . . . . . . . . . . . . . . . . Group F . Group G . Other Biochemically Important Compounds . . . . . . . . . . . . Polymers of Non-Biological O r i g i n . . . . . . . . . . . . . . . Group H . REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SUBJECT I N D E X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PART I 1 ATLAS OF SELECTED PYROLYSIS MASS SPECTRA

55 55 56 56 58 60 62 66 68 68 69 70 73 77 77 77 77 83 85 86 89 92 99 101 105 105 105 106 106 107 109 110 110 110 111 113 113 151 167 179 195 213 245 259 275 287

IX

A b b r e v i a t i o n s used

amu

Atomic mass u n i t s

CI

Chemical i o n i s a t i o n

CID

Col1 i s i o n - i n d u c e d d i s s o c i a t i o n

EI

E l e c t r o n impact i o n i s a t i o n Energy o f i o n i s i n g e l e c t r o n s

FD

F i e l d desorption

FI

Field ionisation

GC/MS

Gas chromatography i n tandem w i t h mass s p e c t r o m e t r y

HRMS

High r e s o l u t i o n mass s p e c t r o m e t r y

MS

Mass s p e c t r o m e t r y

MS/MS

Mass s p e c t r o m e t r y i n tandem w i t h mass s p e c t r o m e t r y

MW

Mo7 e c u l a r we igh t

m lz Py-GC

The mass o f t h e i o n d i v i d e d by i t s charge ( u s u a l l y u n i t y )

Py-MS

Pyrolysis-mass s p e c t r o m e t r y

Py- T RMS

Time-resol ved p y r o l y s i s-mass s p e c t r o m e t r y

TC

Teq tT

P y r o l y s i s - g a s chromatography

C u r i e-poi n t temperature E q u i l i b r i u m temperature Total heating time Temperature r i s e t i m e


XI

Preface

I n recent years pyrolysis mass spectrometry (Py-MS) has played an increasingly important r o l e in analytical pyrolysis, which i s t r a d i t i o n a l l y dominated by pyrolysis gas chromatographic methods. This tendency f i r s t became evident during the I I I r d International Symposium on Analytical Pyrolysis held in July 1976 in Amsterdam, and has continued t o manifest i t s e l f during the 1978 conferences in Budapest (IVth International Symposium on Applied and Analytical Pyrolysis) and Plymouth, New Hampshire (Gordon Conference on Analytical Pyrolysis), and a l s o a t international mass spectrometry conferences and in the recently established Journal of A n a l y t i c a l and Applied P y r o l y s i s . Whereas s p e c i a l l y designed Py-MS systems using galvanically heated filament or d i r e c t probe pyrolysers have been q u i t e successful in s t r u c t u r a l investigations and k i n e t i c s t u d i e s involving synthetic polymers and model compounds, Curie-point Py-MS systems have proved t o be uniquely advantageous in applications t h a t require maximum reproducibility in fingerprinting and a l s o in applications t h a t require routine analysis o f hundreds or thousands of samples. This has made the Curie-point approach e s p e c i a l l y valuable f o r extremely complex samples, such as a r e encountered amon: materials of biological o r i g i n , ranging from biopolymers, c e l l s , microorganisms and t i s s u e s t o humic substances, sediments, p e a t s , coals and s h a l e s . Probably the most extensively used Py-MS system i s the f u l l y automated Curie-point Py-MS system a t the F.O.M. I n s t i t u t e f o r Atomic a n d Molecular Physics in Amsterdam. From January 1977 t o January 1980 t h i s system produced about 18,000 pyrolysis mass spectra f o r over 100 d i f f e r e n t users. One visitor t o t h i s f a c i l i t y , Dr. F. W. McLafferty, described h i s impressions as follows: "For biopolymers and other high molecular weight substances, automated pyrolysis-MS, developed by H . L. C . Meuzelaar, P. G. Kisternaker, W. Eshuis, M . A. Posthumus and A . J . H . Boerboom of t h e FOM

I n s t i t u t e i n Amsterdam, i s now an impressive r o u t i n e a n a l y t i c a l t o o l running l i t e r a l l y thousands o f samples per year, such as geo- and s y n t h e t i c polymers, c o a l , sewage, and u r i n e without sample workup.

The c a p a b i l i t y t o characterise b a c t e r i a i s p a r t i c u l a r l y

impressive and could r e v o l u t i o n i z e the c l a s s i c a l methods which have been used by c l i n i c a l laboratories since Pasteur."

In t h e course of t h e i r own involvement with the Py-MS f a c i l i t y a t the F . O . M . I n s t i t u t e , the authors* have sensed an increasing need f o r a compendium describing the

*

H . L . C . Meuzelaar was founder and Director o f t h e F.O.M. Pyrolysis Centre from 1970 t o 1978; J . Haverkamp i s the present Director of the F . O . M . Pyrolysis Centre; and F. Hileman was a guest s c i e n t i s t a t the Centre in 1978.

XI1 b a s i c p r i n c i p l e s , t e c h n i q u e s and a p p l i c a t i o n s of C u r i e - p o i n t Py-MS aimed a t p a s t , p r e s e n t and f u t u r e users o f t h e method.

The a u t h o r s f u r t h e r f e l t t h a t t h i s

compendium s h o u l d focus on r e c e n t and f o s s i l b i o m a t e r i a l s r a t h e r t h a n on s y n t h e t i c compounds because of t h e above-mentioned unique advantages o f C u r i e - p o i n t Py-MS f o r t h e a n a l y s i s of e x t r e m e l y complex samples. The need f o r a compendium became even more u r g e n t w i t h t h e ' a d v e n t o f c o m m e r c i a l l y a v a i l a b l e C u r i e - p o i n t Py-MS systems f r o m a t l e a s t two d i f f e r e n t m a n u f a c t u r e r s * i n 1979.

T h i s prompted t h e a u t h o r s t o i n c l u d e a s m a l l A t l a s o f r e f e r e n c e s p e c t r a o f

c a r e f u l l y s e l e c t e d b i o m a t e r i a l s which should h e l p new users o f C u r i e - p o i n t Py-MS systems t o " t u n e " t h e i r i n s t r u m e n t s t o t h e e x i s t i n g systems and t o e v a l u a t e unknown spectra

.

A few compounds o f n o n - b i o l o g i c a l o r i g i n , e.g. s y n t h e t i c polymers, a r e i n c l u d e d i n t h e A t l a s t o demonstrate t h e a p p l i c a b i l i t y o f t h e t e c h n i q u e t o t h e s e c l a s s e s o f compounds and t o p r o v i d e some r e l a t i v e l y s i m p l e s p e c t r a f o r " t u n i n g " purposes. Moreover, s y n t h e t i c compounds a r e o f t e n encountered as c o n s t i t u e n t s or contaminants i n biomaterials offered f o r analysis. In order t o avoid confusions and disappointment it should be stressed t h a t the A t l a s part of t h i s book w i l ? , primariZy a s s i s t in the qualitative interpretation of

pyroZysis mass spectra.

I n genera?,, the peak assigments given are tentative and have

not y e t been confirmed by high resoZution MS

GT

MS/MS.

Although a d e f i n i t e l e v e l o f

i n t e r l a b o r a t o r y r e p r o d u c i b i l i t y can be shown t o e x i s t between C u r i e - p o i n t Py-MS systems o f t h e same b a s i c design, as d i s c u s s e d i n Chapter 5, t h e e s t a b l i s h m e n t o f a l i b r a r y f o r q u a n t i t a t i v e comparison between s p e c t r a f r o m d i f f e r e n t i n s t r u m e n t s , e.g. f o r f i n e d i f f e r e n t i a t i o n between b a c t e r i a l p a t t e r n s , i s s t i l l beyond t h e p r e s e n t s t a t e o f the art. A l t h o u g h many a p p l i c a t i o n s o f t h i s t e c h n i q u e deal w i t h t h e c l a s s i f i c a t i o n and i d e n t i f i c a t i o n o f microorganisms, f u n g i and c e l l s , model s p e c t r a o f such m a t e r i a l s have n o t been i n c l u d e d i n t h e A t l a s as t h e i r " f i n g e r p r i n t s " depend s t r o n g l y on e x p e r i m e n t a l c o n d i t i o n s such as c u l t i v a t i o n and p r e p a r a t i o n methods. F i n a l l y , an i m p o r t a n t c a t e g o r y o f r e a d e r s t o whom t h i s Compendium and A t l a s s h o u l d p r o v e h e l p f u l a r e t h o s e c o n s i d e r i n g t h e a p p l i c a t i o n o f Py-MS techniques t o t h e i r own s p e c i f i c problems i n t h e a n a l y s i s o f b i o m a t e r i a l s o f w i d e l y d i f f e r e n t t y p e s .

The

a u t h o r s hope t h a t t h e broad range o f a p p l i c a t i o n s and s p e c t r a p r e s e n t e d w i l l e n a b l e a f a i r assessment o f t h e p r e s e n t c a p a b i l i t i e s and l i m i t a t i o n s o f C u r i e - p o i n t Py-MS i n t h e a n a l y s i s o f complex b i o m a t e r i a l s , w i t h r e g a r d b o t h t o known a p p l i c a t i o n s and t o more o r l e s s c l o s e l y r e l a t e d new a p p l i c a t i o n s n o t s p e c i f i c a l l y covered i n t h i s t e x t .

*

E x t r a n u c l e a r L a b o r a t o r i e s I n c . , P i t t s b u r g h , U.S.A., Winsford, U.K.

and V.G.

Micromass L t d . ,

XI11

Acknowledgements

The a u t h o r s g r a t e f u l l y acknowledge t h e g i f t o f samples by t h e N a t i o n a l Bureau o f Standards (Washington, U.S.A.), gezondheid (R.I.V.), B o r s t (R.I.V.,

B i l t h o v e n , The Netherlands; p o l y s a c c h a r i d e a n t i g e n s ) ,

D r . J.

B i l t h o v e n , The Netherlands; Neisseria gonnorrhaea s t r a i n s ) , D r .

Engel (R.I.V.,

H.M.B.

Drs. E. C . Beuvery ( R i j k s i n s t i t u u t v o o r de V o l k s -

B i l t h o v e n , The Netherlands; Mycobacterim s t r a i n s ) , D r . H.?1.

Greven (Organon, Oss, The Netherlands; s y n t h e t i c o l i g o p e p t i d e s ) , D r . K. H a i d e r and D r . C . Saiz-Jimenez ( F o r s c h u n g s a n s t a l t fiir L a n d w i r t s c h a f t ,

humic m a t e r i a l s ) , Dr. S.M.

Kunen ( U n i v e r s i t y o f Utah, U.S.A.;

D r . S. R. L a r t e r (Union O i l Company, U.S.A.; P.A.

Braunschweig, W. Germany; a i r particulates)

kerogens), D r . J. Id. de Leeuw and P r o f .

Schenck ( T e c h n i c a l U n i v e r s i t y o f D e l f t , The N e t h e r l a n d s ; o i l s h a l e s , c o a l s and

l i g n i t e ) , D r . T. Meindersma (Academisch Ziekenhuis D i j k z i g t , Rotterdam, The N e t h e r lands; KZebsieZZa s t r a i n s ) , D r . J . H. P e t a j a n ( U n i v e r s i t y o f Utah, U.S.A.;

muscle

samples), D r . G. Schutgens ( B i n n e n g a s t h u i s , Amsterdam, The N e t h e r l a n d s ; u r i n e samples), D r . G. S p o s i t o ( U n i v e r s i t y o f C a l i f o r n i a , R i v e r s i d e , U.S.A.; and Ir. A. L. van Wezel (R.I.V.,

sludge samples)

B i l t h o v e n , The Netherlands; p o l i o m y e l i t i s v i r u s

preparations). F u r t h e r , t h e a u t h o r s a r e i n d e b t e d f o r u n p u b l i s h e d d a t a t o D r . P. G. Kistemaker (F.O.M.

I n s t i t u t e f o r Atomic and M o l e c u l a r Physics, Amsterdam, The N e t h e r l a n d s ;

l a s e r Py-MS o f DNA),

D r . M. A. Posthumus ( A q r i c u l t u r a l U n i v e r s i t y , Wageningen, The

Netherlands; Py-MS o f n u c l e i c a c i d s ) , D r . H. -R.

S c h u l t e n ( U n i v e r s i t y o f Bonn, !i.

Germany; scheme o f p y r o l y t i c d e g r a d a t i o n o f g l y c o q e n ) , and D r . W. Windig (F.O.M. tute; f a c t o r analysis with rotation). B. B r a n d t (F.O.M.

Insti-

F i n a l l y , t h e a u t h o r s thank Ms. A. Tom and Ms.

I n s t i t u t e f o r Atomic and M o l e c u l a r P h y s i c s , Amsterdam) f o r p r e -

p a r i n g most o f t h e analyses, D r . 14. Eshuis f o r computer a n a l y s i s o f t h e d a t a , D r . P. G. Kistemaker, D r . M. A. Posthumus and D r . J. [I. de Leeuw f o r c r i t i c a l r e a d i n g o f t h e m a n u s c r i p t and Mrs. M. Van f o r t y p i n g and e d i t i n q t h e m a n u s c r i p t .

The r e s e a r c h

r e p o r t e d i n t h i s book was s u p p o r t e d by t h e Foundation f o r Fundamental Research on M a t t e r (F.O.M.)

and t h e M i n i s t r y o f H e a l t h and Environmental Hygiene i n The N e t h e r -

l a n d s and by t h e U n i t e d S t a t e s Department o f Energy.


PART I COMPENDIUM OF BASIC PRINCIPLES AND APPLICATIONS


3 Chapter 1

O R I G I N S AND DEVELOPMENT OF PYROLYSIS MASS SPECTROMETRY

1.1.

OF BIOMATERIALS

THE FIRST REPORTS

As e a r l y as 1952, Zemany ( r e f . 1 ) p i o n e e r e d t h e a p p l i c a t i o n o f p y r o l y s i s mass s p e c t r o m e t r y (Py-MS) t o t h e c h a r a c t e r i s a t i o n o f biopolymers such as albumin and pepsin.

A l t h o u g h he employed a r e l a t i v e l y p r i m i t i v e o f f - l i n e f i l a m e n t p y r o l y s i s

technique, h i s r e s u l t s showed t h a t c h a r a c t e r i s t i c and r e p r o d u c i b l e f i n g e r p r i n t s c o u l d be o b t a i n e d .

A f t e r Zemany's p u b l i c a t i o n , no f u r t h e r Py-MS s t u d i e s o f b i o -

m a t e r i a l s appear t o have been r e p o r t e d f o r more t h a n a decade.

I n s t e a d , two y e a r s

l a t e r Davison, Slaney and Wragg ( r e f . 2 ) p u b l i s h e d t h e f i r s t account o f a d i f f e r e n t a n a l y t i c a l p y r o l y s i s technique, namely p y r o l y s i s gas chromatography (Py-GC), which r e q u i r e d l e s s expensive and c o m p l i c a t e d i n s t r u m e n t a t i o n t h a n Py-MS and soon found a p p l i c a t i o n i n t h e s y n t h e t i c polymer f i e l d and a number o f r e l a t e d areas. e a r l y 1960's, t h e search f o r e x t r a t e r r e s t r i a l l i f e compounds

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-

I n the

o r , a t l e a s t , complex o r g a n i c

as p a r t o f t h e s c i e n t i f i c m i s s i o n o f planned space probes prompted t h e

a p p l i c a t i o n o f Py-GC t o biochemical problems, e.g.,

s t u d i e s o f b i o p o ymers and

microorganisms by W i l s o n e t aZ. ( r e f . 3 ) and Oyama ( r e f . 4 ) . gered an e x t e n s i v e s e r i e s o f s t u d i e s by R e i n e r e t aZ. ( r e f s . 5

This,

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n turn, t r i g -

7 ) , who Dioneered

t h e a p p l i c a t i o n o f Py-GC i n m i c r o b i o l o g y . Meanwhile, Zemany's r e p o r t on Py-MS seemed almost f o r g o t t e n , i n s p i t e o f t h e s t r o n g p o t e n t i a l advantages o f Py-MS o v e r Py-GC w i t h r e g a r d t o speed o f a n a l y s i s , l o n g - t e r m r e p r o d u c i b i l i t y and s u i t a b i l i t y o f t h e d a t a f o r computer p r o c e s s i n g . Towards t h e end o f t h e 196O's, however, s c a t t e r e d r e p o r t s on t h e use o f d i r e c t Py-MS f o r t h e c h a r a c t e r i s a t i o n o f complex o r g a n i c m a t e r i a l s appeared i n t h e l i t e r a t u r e . Hummel's group i n Cologne s t a r t e d an i m p r e s s i v e s e r i e s o f experiments ( r e f s . 8, 9 ) u s i n g b o t h d i r e c t probe and f i l a m e n t p y r o l y s i s i n combination w i t h f i e l d i o n i s a t i o n and e l e c t r o n impact i o n i z a t i o n mass s p e c t r o m e t r y f o r s t r u c t u r a l e l u c i d a t i o n o f s y n t h e t i c polymers.

A l s o , p u b l i c a t i o n s d e a l i n g w i t h b i o c h e m i c a l a p p l i c a t i o n s o f Py-

MS appeared, r e v e a l i n g t h e e x i s t e n c e o f a t l e a s t t h r e e d i f f e r e n t p y r o l y s i s approaches namely, d i r e c t probe p y r o l y s i s , l a s e r p y r o l y s i s and f i l a m e n t p y r o l y s i s .

This

d i v e r s i f i c a t i o n r e f l e c t e d t h e e x i s t e n c e o f t h e same t h r e e approaches i n Py-GC s i n c e d i r e c t probe p y r o l y s i s , u s i n g a heated c a p i l l a r y , can be regarded as t h e e q u i v a l e n t o f "oven" o r " f u r n a c e " p y r o l y s i s i n Py-GC.

F o r a d e t a i l e d account o f t h e develop-

ment o f p y r o l y s i s t e c h n i q u e s up t o 1967, t h e r e a d e r i s r e f e r r e d t o t h e e x c e l l e n t r e v i e w b y Levy ( r e f . 10). d e t a i l e d and complete

Developments up t o 1979 a r e d e s c r i b e d i n two h i g h l y

r e v i e w s by I r w i n and S l a c k ( r e f . 11) and I r w i n ( r e f .

12),

4 which s h o u l d be c o n s u l t e d by e v e r y worker i n t h e f i e l d of a n a l y t i c a l p y r o l y s i s . S i n c e t h e above t h r e e p y r o l y s i s approaches s t i l l p e r s i s t i n Py-MS today, t h e f u r t h e r e v o l u t i o n o f t h e s e t e c h n i q u e s w i l l be d i s c u s s e d i n t h e f o l l o w i n g paragraphs. 1.2.

DIRECT PROBE PY-MS

The f i r s t a p p l i c a t i o n o f t h i s t e c h n i q u e t o b i o p o l y m e r s may w e l l have been t h e s t u d y of DNA p y r o l y s e d d i r e c t l y i n t h e source o f a h i g h r e s o l u t i o n magnetic s e c t o r i n s t r u m e n t r e p o r t e d by B o e t t g e r and K e l l y ( r e f . 1 3 ) i n 1969.

T h i s s t u d y was p r i m a r -

i l y m o t i v a t e d by t h e search f o r e x t r a t e r r e s t r i a l l i f e , and was p u b l i s h e d as an

extended a b s t r a c t o n l y .

Among t h e p y r o l y s i s p r o d u c t s i d e n t i f i e d were i n t a c t bases.

One y e a r l a t e r , Charnock and Loo ( r e f . 1 4 ) i n d e p e n d e n t l y r e p o r t e d an a l m o s t i d e n t i c a l

A curious aspect o f t h i s p u b l i c a t i o n i s t h a t t h e authors completely a v o i d e d t h e use o f t h e t e r m p y r o l y s i s - o r e q u i v a l e n t terms - t h u s c r e a t i n g t h e experiment.

m i s l e a d i n g i m p r e s s i o n t h a t t h e DNA sample was somehow evaporated f r o m t h e d i r e c t probe r a t h e r than pyrolysed.

T h i s a m b i g u i t y has p e r s i s t e d i n some o f t h e l a t e r

l i t e r a t u r e r e p o r t s on d i r e c t probe Py-MS by o t h e r a u t h o r s , r e f l e c t i n g a b a s i c d i f f i c u l t y i n a c c u r a t e l y d e f i n i n g t h e boundaries between e v a p o r a t i o n and p y r o l y s i s i n p r a c t i c a l experimental conditions.

As a r e s u l t , t h e l i t e r a t u r e on d i r e c t probe

mass s p e c t r o m e t r y abounds w i t h v a p o r i s a t i o n experiments where t h e l a r g e r , l e s s v o l a t i l e m o l e c u l e s were i n a d v e r t e n t l y p y r o l y s e d , and w i t h p y r o l y s i s experiments i n w h i c h t h e more v o l a t i l e molecules were u n d o u b t e d l y evaporated i n t a c t . A f t e r 1970, f u r t h e r d i r e c t probe Py-MS s t u d i e s on b i o m a t e r i a l s were r e p o r t e d by Wiebers et aZ. ( r e f s . 15

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17), A n h a l t and Fenselau ( r e f . 18), R i s b y and Yergey

( r e f s . 19, Z O ) , Buchhorn e t aZ. ( r e f . 21) and L i d e r w a l d ( r e f . 22).

The work o f

Wiebers e t aZ. on n u c l e i c a c i d s appears t o r e p r e s e n t a d i r e c t c o n t i n u a t i o n of Charnock and L o o ' s e x p e r i m e n t s .

The r e p o r t by A n h a l t and Fenselau d e a l s w i t h t h e

d i f f e r e n t i a t i o n o f microorganisms on t h e b a s i s o f l i p i d p a t t e r n s i n t h e h i g h e r mass range, i l l u s t r a t i n g t h e above-mentioned problems o f d e f i n i n g t h e boundaries between p y r o l y s i s and v a p o r i s a t i o n .

R i s b y and Yergey's s t u d i e s opened a new dimension i n

d i r e c t p r o b e Py-MS t h r o u g h t h e i n t r o d u c t i o n o f t i m e - r e s o l v e d r e c o r d i n g o f p y r o l y s i s p a t t e r n s r e f e r r e d t o by t h e a u t h o r s as l i n e a r programmed t h e r m a l d e g r a d a t i o n mass s p e c t r o m e t r y (LPTDMS) and a p p l i e d t o t h e a n a l y s i s o f microorganisms and leukaemic I n o r d e r t o d e t e c t l a r g e e v a p o r a t e d o r p y r o l y s e d molecules -

w h i t e blood c e l l s .

most p r o b a b l y o f a l i p i d n a t u r e impact i o n i s a t i o n techniques.

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t h e y used chemical i o n i s a t i o n r a t h e r t h a n e l e c t r o n

F i n a l l y , Luderwald and Buchhorn d e s c r i b e d t h e a n a l y s i s

o f c o n n e c t i v e t i s s u e samples f o r t h e presence o f poly(ethyleneterephtha1ate) p a r t i c l e s o r i g i n a t i n g from p r o s t h e t i c implant devices. C h a r a c t e r i s t i c f e a t u r e s o f t h e above d e s c r i b e d d i r e c t probe Py-MS experiments a r e t h e slow heating r a t e s ( t y p i c a l l y less than l"C/s),

t h e low p y r o l y s i s temperatures

( g e n e r a l l y below 400°C) and t h e r e l a t i v e l y l o n g r e s i d e n c e t i m e o f t h e p r o d u c t s i n t h e p y r o l y s i s zone.

The slow h e a t i n g r a t e t e c h n i q u e has t h e advantage of a l l o w i n g

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p y r o l y s i s t o o c c u r d i r e c t l y i n t h e i o n source, t h u s a v o i d i n g l o s s o f p r o d u c t s t h r o u g h a d s o r p t i o n on t r a n s f e r l i n e s .

T h i s enables t h e t e c h n i q u e t o be used w i t h

slow scanning magnetic s e c t o r i n s t r u m e n t s w h i l e s t i l l a f f o r d i n g t i m e - r e s o l v e d r e g i s t r a t i o n o f t h e p y r o l y s i s p a t t e r n s . Also, minimal s p e c i a l i s e d i n s t r u m e n t a t i o n i s r e q u i r e d s i n c e d i r e c t probe i n l e t s a r e a v a i l a b l e with most mass spectrometers. Disadvantages o f t h e approach a r e c o n s i d e r a b l e c o n t a m i n a t i o n o f t h e i o n source ( w h i c h may have an adverse i n f l u e n c e on l o n g - t e r m r e p r o d u c i b i l i t y ) , e x c e s s i v e c h a r r i n g because o f t h e slow h e a t i n g r a t e ( r e f . 23) and t h e occurrence of secondary p y r o l y s i s r e a c t i o n s because o f t h e l o n g r e s i d e n c e t i m e of t h e p r o d u c t s i n t h e p y r o l y s i s zone. 1.3.

LASER PY-MS

A second l i n e o f Py-MS s t u d i e s on b i o m a t e r i a l s which has i t s f o u n d a t i o n s i n t h e l a t e 1 9 6 0 ' s , i s l a s e r Py-MS.

Here p i o n e e r i n g s t u d i e s were r e p o r t e d i n 1966 by

V a s t o l a and P i r o n e ( r e f . 24), who used a r u b y l a s e r i n c o m b i n a t i o n w i t h a t i m e - o f f l i g h t mass spectrometer f o r t h e a n a l y s i s o f c o a l samples.

S i m i l a r experiments were

p u b l i s h e d by Joy e t aZ. ( r e f . 25) i n 1968, whereas i n 1970 Karn e t aZ. ( r e f . 26) d e s c r i b e d l a s e r Py-MS o f c o a l u s i n g an o f f - l i n e p y r o l y s i s t e c h n i q u e w i t h a r u b y and a C02 l a s e r and a n a l y s i n g t h e p y r o l y s i s p r o d u c t s by h i g h r e s o l u t i o n mass spectrometry.

L a s e r Py-MS c r e a t e s y e t a n o t h e r d e f i n i t i o n problem.

During intense l a s e r

r a d i a t i o n , f r a g m e n t a t i o n o f molecules may o c c u r by d i r e c t bond s c i s s i o n due t o e l e c t r o n i c e x c i t a t i o n ( p h o t o l y s i s ) r a t h e r t h a n by r e l a x a t i o n o f v i b r a t i o n a l e x c i t a t i o n r e s u l t i n g i n h e a t i n g o f t h e whole m o l e c u l e ( p y r o l y s i s o r t h e r m o l y s i s ) .

Some o f

t h e l a t e r Py-MS s t u d i e s t r i e d t o a v o i d t h i s a m b i g u i t y by u s i n g i n f r a r e d l a s e r s ( m a i n l y C02 l a s e r s ) a t beam i n t e n s i t i e s which p r e c l u d e d t h e o c c u r r e n c e o f m u l t i photon e x c i t a t i o n .

Examples o f such s t u d i e s i n c l u d e t h e work o f Vanderborgh and

F l e t c h e r on s y n t h e t i c polymers and geopolymers ( r e f . 27) and t h e s t u d i e s o f Kistemaker e t at. on s y n t h e t i c polymers ( r e f . 28) and n u c l e i c a c i d s ( r e f . 29).

To

confuse f u r t h e r t h e i s s u e o f process d e f i n i t i o n s i n l a s e r mass s p e c t r o m e t r y : even when u s i n g i n f r a r e d l a s e r s , spontaneous i o n i z a t i o n o f o r g a n i c m o l e c u l e s occurs t h r o u g h i o n - m o l e c u l e r e a c t i o n s between s m a l l i n o r g a n i c c a t i o n s o r a n i o n s and n e u t r a l o r g a n i c m o l e c u l e s ( r e f s . 30, 31).

A p a r t from t h e s e i n t a c t i o n - m o l e c u l e complexes,

o f t e n r e f e r r e d t o as " q u a s i - m o l e c u l a r i o n s " , fragment i o n s can a l s o be found, o b v i o u s l y formed by thermal f r a g m e n t a t i o n e i t h e r b e f o r e o r a f t e r t h e i o n - m o l e c u l e r e a c t i o n ( r e f . 30). The most r e c e n t development i n l a s e r Py-PIS appears t o be t h e l a s e r m i c r o p r o b e mass a n a l y s e r (LAMMA) developed by Kaufmann e t aZ. ( r e f . 32) and f u r t h e r improved by Wechsung e t aZ. ( r e f . 33).

O r i g i n a l l y developed f o r t h e a n a l y s i s o f i n t r a c e l l u l a r

e l e c t r o l y t e s and t r a c e metal c o n c e n t r a t i o n s , t h e LAMMA system has a l s o produced s p e c t r a e x h i b i t i n g c h a r a c t e r i s t i c fragment p a t t e r n s o f p o l y m e r i c o r g a n i c m a t e r i a l s ( r e f . 34) and c e l l s ( r e f s . 35, 36) as w e l l as t h e above d e s c r i b e d c l a s s of

6

i o n - m o l e c u l e complexes ( r e f . 3 7 ) .

The h i g h s p a t i a l r e s o l u t i o n ( b e t t e r t h a n 0.5 pm)

allows f i n g e r p r i n t i n g o f l e s s than 10-l'

g o f sample.

However, n e u t r a l fragments

a r e n o t r e g i s t e r e d by t h e LAMMA i n s t r u m e n t s i n c e t h e m i n u t e amounts o f p r o d u c t s produced p e r l a s e r s h o t p r e c l u d e t h e use o f f u r t h e r i o n i s a t i o n procedures.

There-

f o r e , most o f t h e d e t e c t e d i o n s a r e p r o b a b l y p h o t o l y s i s p r o d u c t s and/or i n t a c t i o n m o l e c u l e complexes and due c a u t i o n should be e x e r c i s e d i n r e f e r r i n g t o t h e s e s p e c t r a as

"

py r o 1y s is mas s s p e c t r a "

.

I n p r i n c i p l e , l a s e r Py-MS has tremendous p o t e n t i a l because i t a l l o w s r a p i d d i r e c t h e a t i n g o f t h e sample w h i l e h e a t i n g o f l a r g e r e a c t i v e s u b s t r a t e areas i s avoided.

Moreover, t h e h i g h s p a t i a l r e s o l u t i o n a c h i e v e a b l e opens u p a whole new

d i m e n s i o n i n t h e a n a l y s i s o f many d i f f e r e n t t y p e s o f samples.

Nevertheless, t h e r e

a r e s e r i o u s p r a c t i c a l problems i n c o n t r o l l i n g t h e amount o f energy d e p o s i t e d p e r u n i t sample volume, i n d e f i n i n g optimum parameters and i n c o n s t r u c t i n g s u i t a b l e mass s p e c t r o m e t e r s f o r r e c o r d i n g l a s e r p y r o l y s i s phenomena.

These problems may t a k e many

more y e a r s b e f o r e b e i n g a d e q u a t e l y r e s o l v e d .

A h i g h l y p r o m i s i n g approach towards t h e simultaneous d e t e c t i o n o f i o n s f r o m l a s e r processes, w i t h o u t r e s o r t i n g t o t i m e - o f - f l i g h t

systems o r p h o t o g r a p h i c p l a t e s , i s

t h e development o f e l e c t r o - o p t i c a l i o n d e t e c t o r s u s i n g channel e l e c t r o n m u l t i p l i e r a r r a y s i n c o m b i n a t i o n w i t h v i d i c o n o r p h o t o d i o d e a r r a y r e a d o u t systems ( r e f s . 38, 39).

T h i s approach, however, i s c o s t l y , t e c h n i c a l l y complex and s t i l l i n a r e l a t i v e l y

e a r l y s t a g e o f development. 1.4.

FILAMENT PY-MS

The t h i r d l i n e o f Py-MS s t i l l a c t i v e l y pursued t o d a y i s t h e f i l a m e n t p y r o l y s i s approach.

I n 1970, Simon's group i n Z u r i c h p u b l i s h e d t h e r e s u l t s o f p r e l i m i n a r y Py-

MS s t u d i e s on f a t t y a c i d s a l t s , pigment dyes and s u b s t i t u t e d benzoic a c i d s ( r e f . 40) u s i n g a C u r i e - p o i n t p y r o l y s e r connected t o a magnetic s e c t o r mass s p e c t r o m e t e r system t h r o u g h an empty c a p i l l a r y column and a m o l e c u l a r s e p a r a t o r .

Curie-point

p y r o l y s i s , as f i r s t d e s c r i b e d by Giacobbo and Simon ( r e f . 41), belongs t o t h e broad group o f f i l a m e n t p y r o l y s i s t e c h n i q u e s b u t d i f f e r s f r o m t h e o t h e r members of t h e g r o u p i n t h a t t h e f i l a m e n t i s i n d u c t i v e l y heated by a h i g h - f r e q u e n c y c o i l r a t h e r t h a n heated by a g a l v a n i c c u r r e n t , and t h e e q u i l i b r i u m t e m p e r a t u r e o f t h e f i l a m e n t

i s determined by t h e C u r i e - p o i n t o f t h e f e r r o m a g n e t i c f i l a m e n t r a t h e r t h a n by a s e r v o - c o n t r o l l e d power s u p p l y .

The i n d u c t i o n h e a t i n g p r i n c i p l e a l l o w s f o r b a t c h

p r o c e s s i n g o f samples s i n c e t h e f i l a m e n t s a r e c o m p l e t e l y i n t e r c h a n g e a b l e , p r o v i d e d t h a t t h e f i l a m e n t dimensions and t h e f e r r o m a g n e t i c a l l o y a r e k e p t c o n s t a n t . f a c t , because o f t h e l o w c o s t , C u r i e - p o i n t f i l a m e n t s a r e d i s p o s a b l e .

In

Moreover, t h e

c o n t a c t l e s s h e a t i n g p r i n c i p l e a l l o w s f o r easy a u t o m a t i o n o f sample exchange procedures, as demonstrated by t h e s u c c e s s f u l c o n s t r q c t i o n o f f u l l y automated Py-GC ( r e f . 42)

and Py-MS ( r e f . 43) systems.

A p a r t f r o m t h e s e t e c h n i c a l d i f f e r e n c e s , however,

7

C u r i e - p o i n t t e c h n i q u e s a r e f u l l y comparable t o o t h e r f i l a m e n t p y r o l y s i s t e c h n i q u e s employing s i m i l a r h e a t i n g r a t e s and e q u i l i b r i u m temperatures. I n 1973, Meuzelaar and Kistemaker a t t h e F.O.M.

I n s t i t u t e f o r Atomic and M o l e c u l a r

Physics i n Amsterdam r e p o r t e d t h e development of a C u r i e - p o i n t Py-MS t e c h n i q u e f o r f a s t d i f f e r e n t i a t i o n o f b a c t e r i a l s t r a i n s ( r e f . 44).

The Py-MS system d i f f e r e d f r o m

Simon's o r i g i n a l system i n t h a t t h e p y r o l y s i s r e a c t o r was enclosed i n t h e vacuum system and was connected t o t h e open e l e c t r o n impact i o n i s e r o f t h e quadrupole mass f i l t e r t h r o u g h a heated, g o l d - c o a t e d expansion chamber.

A l i q u i d nitrogen-cooled

screen surrounded t h e expansion chamber and a s i g n a l averager a l l o w e d r e c o r d i n g o f f a s t r e p e t i t i v e mass scans. group r e p o r t e d f u r t h e r t e c h n i c a l develop-

D u r i n g t h e n e x t few y e a r s , t h e F.O.M.

ments, such as t h e use of h i g h speed i o n c o u n t i n g ( r e f . 43), f u l l a u t o m a t i o n ( r e f . 43) and computerised d a t a p r o c e s s i n g techniques ( r e f . 45). t o b i o m a t e r i a l s were r e p o r t e d ( r e f s . 46

-

Also, new a p p l i c a t i o n s

49), i n c l u d i n g c h a r a c t e r i s a t i o n o f humic

m a t e r i a l s ( r e f . 50) and whole s o i l s ( r e f . 51), as w e l l as geochemical a p p l i c a t i o n s ( r e f . 52) and m e d i c a l a p p l i c a t i o n s ( d e s c r i b e d i n Chapter 7 ) .

The a n a l y s i s o f

b i o m a t e r i a l s b y f i l a m e n t p y r o l y s i s t e c h n i q u e s i n d i r e c t c o m b i n a t i o n w i t h mass s p e c t r o m e t r y was a l s o d e s c r i b e d b y o t h e r groups, e.g.

S c h u l t e n and G o r t z ( r e f . 53), employing C u r i e - p o i n t p y r o l y s i s i n t h e i o n source o f a h i g h - r e s o l u t i o n f i e l d i o n i s a t i o n mass spectrometer f o r t h e a n a l y s i s o f glycogen, and by Hileman ( r e f . 54) u s i n g a Pyroprobe (Chemical Data Systems, I n c . ) f i l a m e n t p y r o l y s e r i n t h e r e a g e n t gas i n l e t o f a chemical i o n i s a t i o n mass spectrometer f o r t h e c h a r a c t e r i s a t i o n o f muscie t i s s u e samples. R e c e n t l y , t h e d i s t i n c t i o n between f i l a m e n t and oven p y r o l y s i s t e c h n i q u e s has become l e s s marked t h r o u g h t h e i n t r o d u c t i o n o f a new C u r i e - p o i n t p y r o l y s i s t e c h n i q u e i n Py-MS ( r e f s . 55, 5 6 ) .

I n s t e a d o f employing a f e r r o m a g n e t i c f i l a m e n t , t h i s

t e c h n i q u e uses s m a l l , h o l l o w f e r r o m a g n e t i c c y l i n d e r s i n which t h e sample i s placed. The h e a t i n g c h a r a c t e r i s t i c s o f t h e s e c y l i n d e r s a r e s i m i l a r t o t h e h e a t i n g p r o f i l e s o f the wires.

The p y r o l y s i s process d i f f e r s f r o m f i l a m e n t p y r o l y s i s , however, i n

t h a t t h e p y r o l y s i s products experience m u l t i p l e c o l l i s i o n s w i t h t h e h o t c y l i n d e r wall.

I n t h i s r e s p e c t , t h e t e c h n i q u e resembles d i r e c t probe and o t h e r oven p y r o l y s i s

techniques.

I t d i f f e r s f r o m t h e l a t t e r , however, i n t h a t t h e h e a t i n g r a t e s a r e

o r d e r s o f magnitude h i g h e r t h a n i n c l a s s i c a l oven p y r o l y s e r s .

A p p l i c a t i o n s of t h i s

t e c h n i q u e i n Py-MS i n c l u d e t h e s t u d y of p y r o l y s i s mechanisms i n r e l a t i v e l y v o l a t i l e model compounds ( r e f s . 55, 57 (ref.

61).

-

5 9 ) , pigment dyes ( r e f s . 55, 60) and a n t i b i o t i c s

A p a r t f r o m i t s a p p l i c a t i o n t o t h e p y r o l y s i s of r e l a t i v e l y v o l a t i l e

compounds, however, i t remains t o be determined whether t h i s new approach o f f e r s any s i g n i f i c a n t advantage o v e r f i l a m e n t p y r o l y s i s i n t h e a n a l y s i s o f b i o m a t e r i a l s . Because of t h e t r a n s i e n t h i g h p r e s s u r e s i n t h e oven d u r i n g p y r o l y s i s secondary r e a c t i o n s may o c c u r i n t h e gas phase, e.g. p h e n y l b u t a n o i c a c i d ( r e f . 57).

f o r m a t i o n o f d i b e n z y l upon p y r o l y s i s o f

These r e a c t i o n s can be m i n i m i s e d by u s i n g

8 submicrogram amounts of sample ( r e f . 56).

A t t h e s e sample l e v e l s , 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 t o a v o i d s i g n i f i c a n t c o n t r i b u t i o n s f r o m i n s t r u m e n t background due t o r e s i d u a l c o n t a m i n a t i o n s o f oven and/or vacuum system.

I n our

experience, t h e above-mentioned problems have an adverse e f f e c t on r e p e a t a b i l i t y and r e p r o d u c i b i 1it y

.

F i n a l l y , a h i g h l y s p e c i a l i s e d Py-PIS t e c h n i q u e r e p o r t e d by S c h u l t e n e t aZ. ( r e f . 62) s h o u l d be mentioned.

I n t h i s experiment, a DNA sample was p y r o l y s e d d i r e c t l y on

t h e heated e m i t t e r o f an FD source and t h e i o n s were analysed by a h i g h r e s o l u t i o n mass spectrometer equipped w i t h a p h o t o p l a t e d e t e c t o r .

T h i s Py-FDIMS t e c h n i q u e

a l l o w e d t h e d e t e c t i o n o f u n u s u a l l y l a r g e p y r o l y s i s fragments of DNA, i n c l u d i n g some i n t a c t d i n u c l e o t i d e ions.

9 Chapter 2

FROM FINGERPRINTING TO STRUCTURAL INVESTIGATION

2.1.

OPERATIONAL FINGERPRINTING; A NEW CONCEPT

The c l a s s i c a l a p p l i c a t i o n s o f f i n g e r p r i n t i n g t e c h n i q u e s a r e c l a s s i f i c a t i o n and i d e n t i f i c a t i o n o f t h e o r i g i n a l m a t e r i a l , using a l i b r a r y o f reference f i n g e r p r i n t s . G e n e r a l l y i n t h e s e a p p l i c a t i o n s , l i t t l e o r no chemical i n t e r p r e t a t i o n o f t h e p a t t e r n Computer e v a l u a t i o n o f p y r o l y s i s f i n g e r p r i n t s

i s a t t e m p t e d o r even r e q u i r e d .

(pyrograms) i n c o m b i n a t i o n w i t h t h e g r e a t l y i n c r e a s e d speed o f a n a l y s i s a f f o r d e d by modern Py-MS t e c h n i q u e s has opened up i m p o r t a n t new areas o f a p p l i c a t i o n , namely s c r e e n i n g , q u a l i t y c o n t r o l and process m o n i t o r i n g ( r e f . 48, 49, 63).

These p r o c e -

dures can be regarded as " o p e r a t i o n a l f i n g e r p r i n t i n g " t e c h n i q u e s i n t h a t no 1 i b r a r y o f r e f e r e n c e s p e c t r a i s r e q u i r e d b u t r e f e r e n c e samples a r e u s u a l l y p r e s e n t i n t h e b a t c h o f samples s u b m i t t e d f o r a n a l y s i s .

I n most a p p l i c a t i o n s o f t h i s t y p e , a

s i n g l e c l a s s o f samples dominates (e.g. p u r e samples) and o n l y a m i n o r f r a c t i o n o f t h e samples belongs t o one o r more d i f f e r e n t c l a s s e s (e.g. contaminated samples). I n o u r experience, o p e r a t i o n a l f i n g e r p r i n t i n g a u t o m a t i c a l l y l e a d s t o t h e q u e s t i o n : what i s t h e b i o c h e m i c a l n a t u r e o f t h e observed v a r i a t i o n o r changes which make t h e o u t l i e r s d i f f e r e n t from t h e c e n t r a l c l u s t e r .

Thus, as shown i n s e v e r a l o f t h e above

c i t e d l i t e r a t u r e r e f e r e n c e s ( r e f . 49, 50, 52, 53), Py-MS t e c h n i q u e s a r e i n c r e a s i n g l y employed t o address d i r e c t l y problems c o n c e r n i n g t h e b i o c h e m i c a l n a t u r e , c o m p o s i t i o n and s t r u c t u r e o f t h e sample.

The success o f a t t e m p t e d b i o c h e m i c a l i n t e r p r e t a t i o n o f

i n t e r e s t i n g f e a t u r e s i n p y r o l y s i s mass s p e c t r a , whether d i s t i n g u i s h e d by eye o r w i t h t h e a i d o f n u m e r i c a l computer techniques, depends c r i t i c a l l y on t h e f o l l o w i n g f a c t o r s : c o m p l e x i t y o f t h e sample, a d d i t i v i t y o f component s p e c t r a , a v a i l a b i l i t y o f r e f e r e n c e s p e c t r a f r o m s t a n d a r d m a t e r i a l s , knowledge o f r e l e v a n t p y r o l y s i s mechanisms and a v a i l a b i l i t y o f a n c i l l a r y a n a l y t i c a l methods.

These f a c t o r s w i l l be d i s c u s s e d i n

t h e f o l l o w i n g paragraphs.

2.2.

COMPLEXITY

OF THE SAMPLE

O b v i o u s l y a multicomponent sample g e n e r a l l y p r o v i d e s a spectrum more d i f f i c u l t t o i n t e r p r e t t h a n t h a t o f a sample c o n s i s t i n g of a s i n g l e , pure component. multicomponent samples

-

However, w i t h

even e x t r e m e l y complex samples such as whole c e l l s

-

often

o n l y one o r two components w i l l s u f f i c e t o a d e q u a t e l y d e s c r i b e t h e a n a l y t i c a l problem. T h e r e f o r e , i f s u i t a b l e c o n t r o l samples a r e a v a i l a b l e , s u b t r a c t i o n o f p a t t e r n s may y i e l d a much s i m p l e r p a t t e r n , m a i n l y r e p r e s e n t a t i v e o f t h e component(s) of i n t e r e s t .

10 Even when comparing m u l t i p l e samples, e.g.,

b a c t e r i a l s t r a i n s d i f f e r i n g i n more

t h a n one component, f a c t o r a n a l y s i s t e c h n i q u e s may bq c a l l e d upon t o r e v e a l r e l a t i v e l y s i m p l e f a c t o r s p e c t r a r e p r e s e n t a t i v e o f t h e v a r i o u s components i n v o l v e d (see Chapter 6 ) . These t e c h n i q u e s can o n l y be used i f t h e component s p e c t r a a r e a d d i t i v e which, i n t u r n , r e q u i r e s t h a t t h e r e s p e c t i v e p y r o l y s i s pathways a r e m u t u a l l y n o n - i n t e r f e r i n g .

2.3.

ADDITIVITY OF COMPONENT SPECTRA

Only p y r o l y s i s t e c h n i q u e s which m i n i m i s e t h e o c c u r r e n c e o f b i m o l e c u l a r r e a c t i o n s and secondary r e a c t i o n s , e.g.

r e c o m b i n a t i o n s between p y r o l y s i s p r o d u c t s , can be

expected t o reasonably f u l f i l l t h e c o n d i t i o n o f a d d i t i v i t y .

I n s t u d i e s by Posthumus

and N i b b e r i n g ( r e f . 58) t h e C u r i e - p o i n t oven p y r o l y s i s o f microgram amounts o f amino a c i d s d i d n o t cause a p p r e c i a b l e r e c o m b i n a t i o n r e a c t i o n s .

T h i s was shown by

t h e absence of d i k e t o p i p e r a z i n e f o r m a t i o n , a r e a c t i o n t h a t r e a d i l y o c c u r s d u r i n g c l a s s i c a l oven p y r o l y s i s ( r e f . 6 4 ) .

I n t h e case o f 4 - p h e n y l b u t a n o i c a c i d (and n o t

f o r o t h e r w - p h e n y l a l k a n o i c a c i d s ) a r e c o m b i n a t i o n r e a c t i o n , i.e. t h e f o r m a t i o n o f d i b e n z y l , c o u l d be observed ( r e f . 57). However, t h i s r e a c t i o n c o u l d be e l i m i n a t e d b y f u r t h e r r e d u c i n g t h e amount o f sample t o submicrogram l e v e l s ( r e f . 5 6 ) . When u s i n g C u r i e - p o i n t f i l a m e n t p y r o l y s i s , a d d i t i v i t y o f s p e c t r a i s r e a d i l y observed f o r such c l a s s e s o f compounds as p o l y s a c c h a r i d e s ( r e f s . 65, 66) and l i g n i n s ( r e f . 67) and, l e s s w e l l e s t a b l i s h e d , a l s o f o r l i p i d s and p r o t e i n s ( r e f . 68).

The

a d d i t i v i t y o f "Component s u b s p e c t r a " i s an i m p o r t a n t r e q u i r e m e n t f o r (semi ) q u a n t i t a t i v e a p p l i c a t i o n s o f Py-MS, e.g.

i n m o n i t o r i n g ppm c o n c e n t r a t i o n s o f t h e t o x i c ,

t e c h n i c a l polymer DEAE-dextran i n p o l i o m y e l i t i s v i r u s v a c c i n e p r e p a r a t i o n s ( r e f . 63). The a d m i x t u r e o f i n o r g a n i c s a l t s , and a l s o changes i n pH o f t h e sample s o l u t i o n s o r suspensions markedly i n f l u e n c e t h e p y r o l y s a t e p a t t e r n s o f p o l a r o r g a n i c compounds, even i f no d i r e c t o r g a n i c s a l t s can be formed. i n S e c t i o n 4.1,

T h i s phenomenon, f u r t h e r d i s c u s s e d

and i n some o f t h e A t l a s s p e c t r a , should be k e p t i n mind when

e v a l u a t i n g s p e c t r a o f complex samples f o r t h e presence o f a p a r t i c u l a r o r g a n i c component.

H i g h l y r e a c t i v e o r g a n i c groups have been found t o r e a c t w i t h i n o r g a n i c

components, e.g.

t h e methyl groups o f t h e trimethylammonium f u n c t i o n of c h o l i n e

r e s i d u e s o r e s t e r methyl groups (see A t l a s ) can r e a c t w i t h c h l o r i d e i o n s t o f o r m methylchloride.

I n f a c t , t h e l a t t e r r e a c t i o n i s q u a n t i t a t i v e and can be used t o

measure t h e c o n c e n t r a t i o n o f a c e t y l c h o l i n e i n b r a i n t i s s u e , as d e s c r i b e d by S z i l a g y i

et d.( r e f . 6 9 ) .

S i m i l a r f o r m a t i o n o f m e t h y l c h l o r i d e can be observed d u r i n g

p y r o l y s i s o f c h o l i n e - c o n t a i n i n g p h o s p h o l i p i d s ( r e f . 70). 2.4.

AVAILABILITY OF REFERENCE SPECTRA

U n t i l now, r e f e r e n c e s p e c t r a have n o t been r e a d i l y a v a i l a b l e from l i t e r a t u r e sources s i n c e t h e Py-MS t e c h n i q u e s used v a r y t o o w i d e l y t o a l l o w c l o s e comparison o f specta.

However, f r o m b u i l d i n g f o u r C u r i e - p o i n t Py-MS systems o v e r t h e p a s t

t e n years,

we know t h a t r e p r o d u c i b i l i t y between i n s t r u m e n t s o f t h e same b a s i c

11 d e s i g n i s r e m a r k a b l y good.

Moreover, as demonstrated i n Chapter 5, even a d e f i n i t e

degree of i n t e r - l a b o r a t o r y r e p r o d u c i b i l i t y i s r e a d i l y achieved w i t h comparable instruments.

T h i s has encouraged t h e F.O.M.

Pyrolysis Centre t o compile a c o l l e c t i o n

A

of s e v e r a l hundred p y r o l y s i s mass s p e c t r a o f n a t u r a l and s y n t h e t i c compounds. s e l e c t i o n of t h e s e s p e c t r a i s i n c l u d e d i n P a r t I 1 o f t h i s volume.

The purpose o f

t h i s A t l a s i s p r i m a r i l y t o e n a b l e t h e r e a d e r t o e v a l u a t e t h e a p p l i c a b i l i t y of C u r i e - p o i n t Py-MS t o d i f f e r e n t problems i n t h e a n a l y s i s o f b i o m a t e r i a l s , geopolymers, humic compounds and, t o a l i m i t e d e x t e n t , drugs and t e c h n i c a l polymers.

A t the

same t i m e , t h i s c o l l e c t i o n o f Py-MS r e f e r e n c e s p e c t r a enables s c i e n t i s t s w o r k i n g w i t h comparable Py-MS t e c h n i q u e s t o e v a l u a t e t h e degree o f i n t e r - l a b o r a t o r y 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 b y a p p r o p r i a t e " t u n i n g " o f t h e i r i n s t r u m e n t s . However, as was p o i n t e d o u t i n t h e Preface, t h i s c o l l e c t i o n o f s p e c t r a s h o u l d p r i m a r i l y e n a b l e a q u a l i t a t i v e comparison o f p y r o l y s i s mass s p e c t r a .

The e s t a b l i s h m e n t o f a l i b r a r y

f o r q u a n t i t a t i v e comparisons between s p e c t r a f r o m d i f f e r e n t i n s t r u m e n t s , e.g.

for

f i n e d i f f e r e n t i a t i o n between b a c t e r i a l s t r a i n s , i s beyond t h e p r e s e n t s t a t e o f t h e art. I t s h o u l d f u r t h e r be n o t e d t h a t e x t e n s i v e chemical i n t e r p r e t a t i o n o f t h e v a r i o u s

mass peaks i n t h e A t l a s s p e c t r a was n o t p o s s i b l e ; o n l y t e n t a t i v e assignments c o u l d be made on t h e b a s i s o f comparison w i t h r e s u l t s of o t h e r t e c h n i q u e s such as Py-HRMS ( r e f s . 53, 71, 72) o r Py-GC-MS

( r e f s . 73, 74).

The use o f such d a t a f r o m t h e

l i t e r a t u r e may g i v e i n c o r r e c t r e s u l t s , however, as p y r o l y s i s and sample t r a n s f e r conditions f o r d i f f e r e n t techniques a r e o f t e n very d i f f e r e n t .

T h e r e f o r e , tandem

mass s p e c t r o m e t e r systems f o r c o l l i s i o n a l induced d i s s o c i a t i o n ( r e f s . 75

-

81)

s h o u l d p r o v e i n v a l u a b l e f o r e s t a b l i s h i n g t h e chemical i d e n t i t y o f peaks observed i n Py-MS, s i n c e t h i s approach a l l o w s p r e c i s e d u p l i c a t i o n o f p y r o l y s i s and sample

t r a n s f e r c o n d i t i o n s n o r m a l l y used i n Py-MS (see S e c t i o n 2.7). Regardless o f t h e s i z e o f any l i b r a r y o f p y r o l y s i s mass s p e c t r a , t h e v a r i e t y o f b i o m a t e r i a l s t h a t can be i n v e s t i g a t e d i s so immense t h a t o f t e n p y r o l y s i s p a t t e r n s n o t r e p r e s e n t e d i n t h e l i b r a r y w i l l be generated.

Even i n t h e s e cases, a l i m i t e d

amount o f chemical i n t e r p r e t a t i o n o f such p a t t e r n s may s t i l l be p o s s i b l e on t h e b a s i s o f i n s i g h t i n t o - o r e m p i r i c a l knowledge o f - r e l e v a n t p y r o l y s i s mechanisms (see S e c t i o n 2.6.).

2.5.

AVAILABILITY OF STANDARD REFERENCE MATERIALS A d i f f i c u l t problem i n t h e c o m p i l a t i o n o f a r e f e r e n c e c o l l e c t i o n o f p y r o l y s i s

mass s p e c t r a o f complex b i o m a t e r i a l s i s t h e l i m i t e d a v a i l a b i l i t y o f s u i t a b l e s t a n d a r d materials.

A l t h o u g h complex b i o m a t e r i a l s can be f o u n d among t h e l a r g e c o l l e c t i o n

o f Standard Reference M a t e r i a l s i s s u e d by t h e N a t i o n a l Bureau o f Standards ( r e f .

8 2 ) , t h e s e m a t e r i a l s have n o t been t h o r o u g h l y c h a r a c t e r i s e d and d e f i n e d w i t h r e s p e c t t o t h e i r o r g a n i c s t , r u c t u r e and c o m p o s i t i o n .

Moreover, a l t h o u g h NBS Standard

M a t e r i a l s o f b i o l o g i c a l o r i g i n a r e u s u a l l y a v a i l a b l e as a t h o r o u g h l y homogenised,

12 f i n e powder, t h e NBS does n o t guarantee ( r e f . 83) homogeneity o f t h e m a t e r i a l down O f course, t o sample s i z e s below 100 micrograms such as a r e n o r m a l l y used i n Py-MS. inhomogeneity problems may be d e t e c t e d by r e p e a t e d Py-MS analyses and, i f necessary,

can be c o r r e c t e d by a v e r a g i n g t h e r e s u l t s of m u l t i p l e d e t e r m i n a t i o n s . T a b l e 1 l i s t s NBS Standard Reference M a t e r i a l s which i n t h e o p i n i o n o f t h e a u t h o r s may have p o t e n t i a l v a l u e as complex b i o l o g i c a l r e f e r e n c e samples i n Py-MS, and s p e c t r a f r o m some o f which can be found i n t h i s book.

P r e l i m i n a r y s t u d i e s on

f o u r m a t e r i a l s i n d i c a t e d i n T a b l e 1 f a i l e d t o d e t e c t marked inhomogeneities a t t h e 10 microgram l e v e l ( r e f .

84).

Less s o p h i s t i c a t e d , b u t i n e x p e n s i v e and g e n e r a l l y

a v a i l a b l e r e f e r e n c e m a t e r i a l s f o r l e s s demanding a p p l i c a t i o n s i n Py-MS s t u d i e s on b i o m a t e r i a l s are, f o r i n s t a n c e , t h e p o l y s a c c h a r i d e s amylose and c e l l u l o s e , t h e p r o t e i n s albumin and k e r a t i n and complex m a t e r i a l s such as m i l k homogenate and s o f t wood powder (Douglas f i r ) .

Homogenised b o v i n e m i l k i s e s p e c i a l l y u s e f u l as d i f f e r -

e n t samples show r e l a t i v e l y l i t t l e v a r i a t i o n i n t h e i r p y r o l y s i s mass s p e c t r a . Moreover, i t i s composed o f a wide v a r i e t y o f b i o l o g i c a l compounds, needs l i t t l e sample p r e p a r a t i o n ( d i l u t i o n w i t h w a t e r ) and produces a t h i n , u n i f o r m c o a t i n g on t h e pyrolysis filament.

Convenient sources o f some h i g h p u r i t y s y n t h e t i c polymers a r e

s t a t i o n a r y phases f o r gas chromatography and i o n exchange r e s i n s .

However, 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 p y r o l y s i s p a t t e r n s may be found between p r o d u c t s f r o m d i f f e r e n t m a n u f a c t u r e r s o r even between d i f f e r e n t batches f r o m a s i n g l e commercial source. TABLE 1 NBS Standard Reference M a t e r i a l s o f r e c e n t and f o s s i l b i o l o g i c a l o r i g i n . of t h e m a t e r i a l s marked w i t h * see f u r t h e r i n t h i s Volume.

Nr

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

.

Ma t e r ia 1 B o v i n e serum albumin a-Cellulose Natural rubber Orchard l e a v e s Tomato leaves P i n e needles Soinach Rice f l o u r *Wheat f 1o u r *Brewer's y e a s t Oyster t i s s u e A1 bacore t u n a Freeze-dried u r i n e *Urban p a r t i c u l a t e * R i v e r sediment E s t u a r i n e sediment Subbituminous c o a l Bituminous c o a l

NBS c a t . n r .

SRM 926 SRM 1006a SRM SRM SRM SRM SRM SRM SRM SRM SRM RM SRM SRM SRM SRM SRM SRM

385b 1571 1573 1575 1570 1568 1567 1569 1566 50 2671 1648 1645 1646 1635 1632a

For s p e c t r a

13

In conclusion, it can be stated that satisfactory standard reference materials for Py-MS analysis of complex biomaterials do not yet seem to exist. An ideal biomaterial for calibration ("tuning") of the Py-PIS systems should exhibit long-term chemical stability and should preferably be completely soluble in a simple solvent with good coating characteristics. Further, it should be sensitive to changes in analytical conditions and it should provide a pyrolysate with components representative of a wide range of chemical classes and molecular sizes. An example of a potentially good standard reference material for Py-MS is lignin. Some lignins are completely soluble in methanol or ethanol (ref. 85); lignins are chemically very stable and provide a wide range of pyrolysis products. Nitrogen-containing products are usually absent, however, and lignin is not highly sensitive to changes in pyrolysis or mass spectrometry conditions. Instant milk powder might be another interesting candidate, as it also is readily soluble and contains a wide range of biological compounds, e.g. nitrogen-containing compounds and lipids. However, the chemical stability of milk powder might prove to be inadequate. There is no doubt that the field of analytical pyrolysis would benefit greatly if NBS or other internationally recognised organisations would develop a set of carefully homogenised and stored biomaterials, 10 or 100 mg aliquots of which could be made available to the scientific community as standards for analytical pyrolysis. 2.6. KNOWLEDGE OF RELEVANT PYROLYSIS MECHANISMS Our present knowledge of pyrolysis mechanisms in biomaterials is at best very sketchy and does not compare with the level of knowledge of and insight into fragmentation mechanisms occurring during electron impact ionisation. Although Posthumus e t aZ. (refs. 57, 58) have presented strong evidence for the basic simplicity and straightforwardness of pyrolysis mechanisms in selected organic model compounds, which compare favourably with electron impact fragmentation mechanisms in these molecules, the extreme complexity of many biomaterials will undoubtedly prevent the achievement of a satisfactory degree of insight into the precise pyrolysis mechanisms involved. A more detailed overview of our present knowledge of pyrolysis mechanisms in biomaterials is presented in Chapter 3.

2.7. AVAILABILITY OF ANCILLARY ANALYTICAL METHODS A very difficult task is the qualitative interpretation of a single significant mass peak in a pyrolysis mass spectrum. With the exception of a few peaks in the lower mass range, e.g. at m/z 34 (hydrogen sulphide) or m/z 17 (ammonia), the chemical identity of such a peak cannot be established with certainty, let alone its molecular origin. High-resolution mass spectrometry can be used to establish the elemental composition of a peak in a pyrolysis mass spectrum (refs. 53, 71), provided that the pyrolysis experiment can be repeated closely under these conditions. Photoplate registration may be the method o f choice if the original spectrum was

14 o b t a i n e d w i t h a f a s t - s c a n n i n g mass spectrometer.

A l t e r n a t i v e l y , a few s e l e c t e d

m u l t i p l e t s may be r a p i d l y scanned by v a r y i n g t h e e l e c t r i c a l p o t e n t i a l s on a h i g h r e s o l u t i o n m a g n e t i c s e c t o r i n s t r u m e n t and by r e c o r d i n g t h e s i g n a l s w i t h a s i g n a l averager, as d e s c r i b e d by Freudenthal and Gramberg ( r e f . 86).

High r e s o l u t i o n mass

s p e c t r o m e t r y , o f course, does n o t d i s t i n g u i s h between i s o m e r i c s t r u c t u r e s .

Applica-

t i o n of Py-GC/MS t e c h n i q u e s t o e s t a b l i s h t h e chemical i d e n t i t y o f a Py-MS peak i s s e v e r e l y handicapped by d i f f e r e n c e s i n p y r o l y s i s c o n d i t i o n s and t r a n s m i s s i o n c h a r a c t e r i s t i c s o f t h e systems.

A p r o m i s i n g approach, though r e q u i r i n g e x t r e m e l y s p e c i a l i s e d i n s t r u m e n t a t i o n , i s t h e use o f MS/MS f o r t h e i d e n t i f i c a t i o n o f s e l e c t e d nominal mass peaks.

Using a

c o l l i s i o n a l induced d i s s o c i a t i o n (CID) t e c h n i q u e Levsen and S c h u l t e n ( r e f . 75) were a b l e t o i d e n t i f y s e v e r a l m o l e c u l a r i o n s i n t h e low-energy E I mass spectrum o f a DNA pyrolysate.

Another MS/MS system, e s p e c i a l l y designed f o r measuring v e r y s h o r t -

l a s t i n g p y r o l y s i s processes has been d e s c r i b e d by T u i t h o f e t aZ. ( r e f . 7 6 ) , u s i n g simultaneous e l e c t r o - o p t i c i o n d e t e c t i o n ,

With t h i s instrument, a p r e l i m i n a r y study

o f a component i n t h e p y r o l y s a t e s o f Mycobacteriwn c e l l s , i m p o r t a n t f o r sub-species i d e n t i f i c a t i o n has been d e s c r i b e d ( r e f , 8 0 ) .

I n t h i s s t u d y i t was demonstrated t h a t i s o m e r i c m o l e c u l a r i o n s such as t h o s e o f t r i m e t h y l a m i n e and propylamine show s i g n i f i -

c a n t l y d i f f e r e n t c o l l i s i o n - i n d u c e d fragment p a t t e r n s . M c L a f f e r t y ( r e f . 77) and Todd ( r e f . 8 1 ) c o n s t r u c t e d a h i g h r e s o l u t i o n i n s t r u m e n t c o n s i s t i n g o f two d o u b l e - f o c u s s i n g mass spectrometers i n tandem f o r t h e a n a l y s i s o f mu1 ticomponent m i x t u r e s .

A h i g h l y i n t e r e s t i n g development f o r s t r u c t u r e a n a l y s i s i s

t h e tandem mass s p e c t r o m e t e r c o n s i s t i n g o f t h r e e quadrupole assemblies r e c e n t l y d e s c r i b e d by Yost and Enke ( r e f . 8 7 ) , and of a d o u b l e quadrupole system, r e p o r t e d by Siege1 ( r e f . 8 8 ) .

These developments may b r i n g t h e use o f MS/MS t e c h n i q u e s w i t h i n t h e

r e a c h o f a much l a r g e r number o f l a b o r a t o r i e s d u r i n g t h e n e x t few y e a r s s i n c e commerc i a l v e r s i o n s a r e a l r e a d y a v a i l a b l e ( r e f s . 88, 89). O t h e r p r o m i s i n g approaches t o a more d e t a i l e d q u a l i t a t i v e a n a l y s i s o f m i x t u r e s o f p y r o l y s i s p r o d u c t s a r e t h e use o f s e l e c t i v e r e a g e n t gases i n C I - M S systems o r t h e d i f f e r e n t i a t i o n o f p y r o l y s i s p r o d u c t s w i t h t h e same nominal mass b u t d e r i v e d f r o m d i f f e r e n t p a r e n t compounds by comparing t i m e - r e s o l v e d p y r o l y s i s p a t t e r n s such as t h o s e o b t a i n e d by R i s b y and Yergey ( r e f s . 19, 20).

15 Chapter 3

PYROLYSIS MECHANISMS IN BIOMATERIALS

3.1.

GENERAL ASPECTS

Knowledge o f vacuum pyrolysis mechanisms in biomaterials i s most advanced f o r some c l a s s e s of r e l a t i v e l y simple compounds, such as amino acids ( r e f . 59). However, r e l a t i v e l y l i t t l e i s known about these mechanisms for most biopolymers, with t h e exception of t h e thermal degradation of polysaccharides such as c e l l u l o s e ( r e f . 90)

or glycogen ( r e f . 53).

Uhereas polytetrafluoroethylene produce t h a t s o f t i o n i s a t i o n techniques of biopolymers a r e usually much

some s y n t h e t i c polymers such as polystyrene o r extremely simp1 e pyrolysis mass s p e c t r a , provided a r e used (see Figure l ) , t h e pyrolysis mass spectra more complex.

To some e x t e n t , t h i s i s caused by a general d i f f e r e n c e in pyrolysis mechanisms. In polystyrene and polytetrafluoroethylene the major degradation reaction i s a straightforward depolymerisation by 6-bond s c i s s i o n ( i n i t i a t i o n followed by unzipping) ( r e f . 9 ) . This y i e l d s styrene (MW 104) and t e t r a f l u o r o e t h y l e n e ( M W l o o ) , respectively, and thus t h e low voltage electron impact spectra show almost exclusively t h e molecular ion of the monomers and, a t a much lower i n t e n s i t y , the molecular ions of t h e dimers and trimers provided t h a t the higher mass range i s scanned. None of the more common biopolymers possesses a s t r u c t u r e wherein each of t h e monomeric building blocks contributes a two-carbon segment t o the polymer backbone which could lead t o simple depolymerisation by p-scission.

Natural rubber, a poly-

isoprene, i s perhaps t h e only biopolymer t o y i e l d marked amounts of monomer under a n a l y t i c a l pyrolysis conditions ( s e e Section 3.5). Instead, most biopolymers decompose by a v a r i e t y of mechanisms often characterised by t h e elimination of s t a b l e neutral molecules, such as H20, HCN, CH20, Cti30H, H2S, C O , C02, C2H4 and H 2 , accompanied by t h e break-up of t h e polymer chain i n t o l a r g e r fragments. Fortunately, these fragments often r e t a i n c h a r a c t e r i s t i c s of t h e original monomeric building blocks. I t should be noted t h a t since the i n i t i a l pyrolytic reactions a r e h e t e r o l y t i c , t h e course of t h e thermal degradation and the r e l a t i v e y i e l d s of the various pyrolysis products can be s i g n i f i c a n t l y influenced by t h e presence of a c i d i c or a l k a l i n e c a t a l y s t s or o f s a l t s ( r e f . 91). In the following paragraphs the present s t a t e of knowledge of vacuum pyrolysis mechanisms f o r t h e major groups o f biopolymers will be b r i e f l y discussed.

16

D4 40

iH* CH

14 eV

d

20

I

.

70 eV

4

5

20

40

60

80

100

120 mlz

140

160

Figure 1. Pyrolysis mass spectra o f polystyrene obtained at low voltage (14 eV) and normal (70 eV) E I ionisation energies. Conditions: sample 2 ug; Tc 510°C. 3.2.

POLYSACCHARIDES

The general pathways for pyrolytic degradation o f polysaccharides, mainly derived by Shafizadeh e t aZ. (refs. 91, 92) from model studies on cellulose [a R(l + 4) glucan], involve splitting of the polymer chain by three basic chemical reaction mechanisms, viz. dehydration, retroaldolisation and decarboxylation. Using these basic mechanisms, Schulten and Gortz (ref. 53) were able to explain the pyrolytic degradation of glycogen [a branched glucan with a(l + 4) and a(l 6 ) linkages]. The hexose degradation pathways illustrated for glycogen in Figure 2 result in the formation of furan and pyran-type fragments and smal ler acyclic aldehyde and ketone -f

17

F i g u r e 2 . Some p y r o l y t i c d e g r a d a t i o n pathways proposed f o r hexose palymers. T h i s scheme i s m a i n l y based on a c o m p i l a t i o n o f p y r o l y s i s p r o d u c t s o f glycogen as observed by S c h u l t e n e t aZ. ( r e f s . 53, 94) u s i n g C u r i e - p o i n t pyrolysis-FI-HRMS a t v a r i o u s p y r o l y s i s temperatures. The m/z v a l u e s a r e i n d i c a t e d i n i t a l i c numerals. I t s h o u l d be n o t e d t h a t some fragments a r e h a r d l y p r e s e n t i n t h e C u r i e - p o i n t l o w v o l t a g e - E I s p e c t r a o f hexose polymers such as t h o s e i n c l u d e d i n t h e A t l a s . Moreover, some m o l e c u l a r i o n peaks, r e g u l a r l y observed i n t h e s p e c t r a , e.g. a t m/z 32, 60, 110, 112 and 124, a r e n o t e x p l a i n e d by t h i s d e g r a d a t i o n scheme. fragments.

As an example, t h e l a r g e s t commonly found c e l l u l o s e fragment i s l e v o g l u c o -

san (m/z 162), whereas a peak a t m/z 126 can be a t t r i b u t e d t o h y d r o x y m e t h y l f u r f u r a l (ref.

90) o r levoglucosenone ( r e f . 53) (see A t l a s ) .

A l t h o u g h as y e t r e l a t i v e l y l i t t l e work has been c a r r i e d o u t t o e l u c i d a t e r e a c t i o n pathways i n non-hexosyl p o l y s a c c h a r i d e s , e.g. N - a c e t y l hexosaminyl polymers ( r e f . 9 3 ) , t h e p y r o l y s i s mass s p e c t r a l p a t t e r n s shown f o r c e l l u l o s e and i t s N-acetylglucosamine analogue c h i t i n ( F i g u r e 3 ) i n d i c a t e a marked degree of correspondence i n b a s i c p y r o l y s i s fragments.

As t h i s may a l s o h o l d f o r o t h e r t y p e s of c a r b o h y d r a t e s , t h e hexose

model s t u d i e s s h o u l d a i d c o n s i d e r a b l y i n t h e q u a l i t a t i v e i n t e r p r e t a t i o n o f nonhexosyl p o l y s a c c h a r i d e s . N o t w i t h s t a n d i n g t h e s e a p p a r e n t b a s i c s i m i l a r i t i e s i n p y r o l y s i s pathways, d i f f e r e n t t y p e s o f sugar m o i e t i e s , e.g. pentoses, amino sugars, N - a c e t y l aminosugars, h e x u r o n i c a c i d s and deoxy- and anhydro sugars, o f t e n c o n t r i b u t e c h a r a c t e r i s t i c fragment s e r i e s

3

A CELLULOSE

60 98

3

J

J

-4

3

43

30 I

i/ 8(

YY

160

F i g u r e 3. P y r o l y s i s mass s p e c t r a o f c e l l u l o s e ( a ) and c h i t i n ( b ) . Note t h e predomi n a n c e o f even masses i n t h e c e l l u l o s e spectrum i n d i c a t i n g a l m o s t complete absence o f fragment i o n s . F o r t h e chemical i d e n t i t y o f t h e main i o n s i g n a l s see r e f . 53. The c h a r a c t e r i s t i c i o n s e r i e s i n t h e c h i t i n spectrum appears t o be s h i f t e d by 1 o r 41 amu, r e s p e c t i v e l y , r e l a t i v e t o t h e c e l l u l o s e p a t t e r n , i n d i c a t i n g t h e presence o f NH2 o r Na c e t y l f u n c t i o n a l groups i n s t e a d o f an OH group i n t h e fragments. C o n d i t i o n s : samples 1 0 pg; Tc 510°C; Eel 14 eV (see A t l a s ) . t o t h e p y r o l y s i s mass spectrum by v i r t u e o f t h e i r d i f f e r e n t s t r u c t u r e s .

For t y p i c a l

f r a g m e n t s e r i e s o b t a i n e d from these c a r b o h y d r a t e b u i l d i n g b l o c k s we r e f e r t o t h e Atlas. An example o f t h e s e n s i t i v i t y o f Py-PIS f o r s t r u c t u r a l d e t a i l s was a l s o g i v e n by Haverkamp e t u Z . (sialopolymers).

( r e f . 49) i n t h e a n a l y s i s o f N - a c e t y l n e u r a m i n i c a c i d polymers T h i s s t u d y shows t h a t t h e presence o f 0 - a c e t y l s u b s t i t u e n t s as

19 w e l l as o f n e u t r a l hexosyl m o i e t i e s i n t h e n a t i v e polymers i s r e a d i l y d e t e c t e d by Py-MS a n a l y s i s as s i m p l e cleavage r e a c t i o n s g i v e r i s e t o a c e t i c a c i d f r o m t h e f o r m e r

and i n t a c t r i n g fragments such as t h a t a t m/z 126 ( p r o b a b l y levoglucosenone) f r o m the latter.

F u r t h e r , d i f f e r e n t i a t i o n o f a(2

+

8) and a ( 2

-+

9 ) l i n k a g e t y p e s between

t h e monomeric u n i t s seems t o be p o s s i b l e as t h e p r i m a r y a l c o h o l i c f u n c t i o n p r e s e n t i n each o f t h e monomeric m o i e t i e s i n t h e a ( 2

+

8 ) c h a i n g i v e s r i s e t o an i n c r e a s e d

amount o f methanol as a s i m p l e p y r o l y t i c cleavage p r o d u c t . The n o t e d s c a r c i t y o f a l t e r n a t i v e a n a l y t i c a l t e c h n i q u e s f o r t h e r a p i d chemical c h a r a c t e r i s a t i o n o f microgram samples o f c a r b o h y d r a t e s makes Py-MS a p o w e r f u l t o o l , e s p e c i a l l y f o r t h e analysis o f i n s o l u b l e polysaccharides. 3.3.

NUCLEIC A C I D S I n t h e f i e l d o f n u c l e i c a c i d a n a l y s i s , p y r o l y s i s mass s p e c t r o m e t r y t e c h n i q u e s

have produced i n t r i g u i n g , sometimes p a r a d o x i c a l r e s u l t s . ( r e f s . 15

-

The work o f Wiebers e t aZ.

1 7 ) shows t h a t d i r e c t probe p y r o l y s i s produces l a r g e c h a r a c t e r i s t i c

fragments, a p p a r e n t l y r e p r e s e n t i n g i n t a c t bases o r even more o r l e s s s e v e r e l y dehydrated n u c l e o s i d e s .

A s a r e s u l t , they successfully applied t h i s technique t o

t h e c h a r a c t e r i s a t i o n o f unusual base m o i e t i e s i n n u c l e i c a c i d s .

However, when

a p p l y i n g C u r i e - p o i n t p y r o l y s i s t e c h n i q u e s t o t h e a n a l y s i s o f n u c l e i c a c i d s , Meuzelaar

e t aZ. ( r e f . 48) o b t a i n e d p y r o l y s i s mass s p e c t r a showing o n l y r i b o s e a n d / o r deoxyr i b o s e fragments (see F i g u r e 4).

Although t h i s enabled a c l e a r d i f f e r e n t i a t i o n t o

be made between RNA and DNA samples, t h e complete l a c k o f s i g n a l s d e r i v e d f r o m t h e base m o i e t i e s was b o t h d i s a p p o i n t i n g and p u z z l i n g .

L a t e r s t u d i e s by Posthumus e t

aZ. ( r e f . 72) and S c h u l t e n e t a2. ( r e f . 62), employing h i g h - r e s o l u t i o n f i e l d i o n i s a -

t i o n and f i e l d d e s o r p t i o n techniques, r e s p e c t i v e l y , as w e l l as l a s e r Py-MS s t u d i e s by Kistemaker e t aZ. ( r e f . 29) (see F i g u r e 5 ) and d i r e c t probe C I D measurements by Levsen and S c h u l t e n ( r e f . 75) succeeded i n d e t a i l i n g t h e d e g r a d a t i o n b e h a v i o u r o f n u c l e i c acids. The main mechanism t a k i n g p l a c e a t temperatures as low as 180°C appears t o be t h e e x p u l s i o n o f t h e sugar m o i e t y w i t h t h e simultaneous f o r m a t i o n o f base-phosphate condensates ( r e f . 72).

Under s t a n d a r d C u r i e - p o i n t Py-MS c o n d i t i o n s o n l y t h e sugar

m o i e t y i s d e t e c t e d , s i n c e t h e base-phosphate complex i s t r a p p e d i n t a c t on t h e r e l a t i v e l y c o l d w a l l o f t h e r e a c t i o n chamber.

I n d i r e c t probe p y r o l y s i s , however,

t h e base-phosphate complex i s a p p a r e n t l y f u r t h e r p y r o l y s e d , y i e l d i n g t h e base fragments.

A l t e r n a t i v e l y , some o f t h e base-phosphate complexes may c o n c e i v a b l y

r e a c h t h e i o n source w i t h o u t f u r t h e r w a l l c o l l i s i o n s and t h e n may fragment d u r i n g e l e c t r o n impact i o n i s a t i o n , a g a i n p r o d u c i n g i o n s c o r r e s p o n d i n g t o t h e base m o i e t i e s . A second i m p o r t a n t r e a c t i o n mechanism appears t o be t h e f o r m a t i o n o f polyphosphates ( r e f . 62) w i t h t h e e x p u l s i o n o f i n t a c t n u c l e o s i d e s .

Again, t h e s e n u c l e o s i d e s can

o n l y c o n t r i b u t e t o t h e mass spectrum by secondary p y r o l y s i s i n t o more v o l a t i l e fragments, o r b y d i r e c t d i f f u s i o n i n t o t h e i o n s o u r c e w i t h o u t f u r t h e r w a l l c o l l i s i o n s .

20

A

198

DNA

B

961 I

R NA

84

58

* jell4

F i g u r e 4. P y r o l y s i s mass s p e c t r a of DNA and RNA. Note t h e absence o f s i g n a l s f r o m t h e n u c l e i c a c i d bases and t h e dominance o f w a t e r - l o s s fragments f r o m t h e d e o x y r i b o s e and r i b o s e m o i e t i e s , r e s p e c t i v e l y . Peaks marked w i t h * i n t h e RNA spectrum denote p o s s i b l e c o n t a m i n a t i o n w i t h hexose-type compounds. C o n d i t i o n s : sample 10 119; Tc 510°C; Eel 14 eV.

A h i g h l y i n t e r e s t i n g t h i r d d e g r a d a t i o n pathway appears t o be d i r e c t s c i s s i o n o f t h e polymer c h a i n i n t o n u c l e o t i d e s (mono- o r diphosphates) o r even d i n u c l e o t i d e s , as observed by S c h u l t e n e t aZ. ( r e f .

62).

These l a r g e p h o s p h a t e - c o n t a i n i n g p r o d u c t s

c a n n o t be expected t o produce s i g n i f i c a n t m o l e c u l a r i o n s i g n a l s u s i n g e l e c t r o n i m p a c t i o n i s a t i o n t e c h n i q u e s , even i f t h e y somehow m i g h t r e a c h t h e i o n source.

- -

21

NUCLEOSIDES-2 H20

BASES

_;,,, 205 200

I,

II.,.

,,~, !.,

231

miz

250

F i g u r e 5. Laser p y r o l y s i s mass spectrum o f h e r r i n g DNA showing p r o m i n e n t s i g n a l s d e r i v e d f r o m t h e base and n u c l e o s i d e m o i e t i e s . C o n d i t i o n s : cw CO2 l a s e r , 50 W/cmz, p u l s e t i m e 0.1 s; Eel 14 eV. F o r e x p e r i m e n t a l s e t - u p see r e f e r e n c e 48. Compared w i t h p o l y s a c c h a r i d e s , t h e a p p l i c a t i o n o f Py-MS t e c h n i q u e s t o t h e a n a l y s i s o f n u c l e i c acids i s s t i l l i n i t s infancy.

However, t a k i n g i n t o account t h e i m p r e s s i v e

successes o b t a i n e d w i t h t h e mass s p e c t r a l a n a l y s i s o f i n t a c t n u c l e o s i d e s and n u c l e o t i d e s u s i n g some o f t h e newer d e s o r p t i o n t e c h n i q u e s , s i g n i f i c a n t breakthroughs i n t h e Py-MS a n a l y s i s o f n u c l e i c a c i d s , u s i n g combinations o f p y r o l y s i s and d e s o r p t i o n t e c h -

niques, as p i o n e e r e d by S c h u l t e n e t

3.4.

uZ. ( r e f . 62), may w e l l o c c u r i n t h e n e a r f u t u r e .

PROTEINS

As mentioned i n t h e f i r s t paragraph o f t h i s c h a p t e r , t h e a n a l y s i s of p r o t e i n s i s one o f t h e o l d e s t a p p l i c a t i o n s o f Py-MS ( r e f . 1 ) . Rather t h a n break-up o f t h e polymer backbone i n t o l a r g e fragments c h a r a c t e r i s t i c o f t h e o r i g i n a l b u i l d i n g b l o c k s , t h e dominant mechanism appears t o be t h e s p l i t t i n g o f f of appendages ( r e f .

95).

As a r u l e , h i g h l y c h a r a c t e r i s t i c s i g n a l s a r e f o u n d f o r t h e a r o m a t i c - and

s u l p h u r - c o n t a i n i n g amino a c i d m o i e t i e s , e.g.

hydrogen s u l p h i d e f o r c y s t ( e ) i n e and i n

c o m b i n a t i o n w i t h m e t h a n e t h i o l a l s o f o r m e t h i o n i n e ; p y r r o l e , p y r r o l i d i n e and m e t h y l p y r r o l e f o r ( h y d r o x y ) p r o l i n e ; phenol and c r e s o l f o r t y r o s i n e ; t o l u e n e , s t y r e n e and p h e n y l a c e t o n i t r i l e f o r p h e n y l a l a n i n e ; and i n d o l e and methyl i n d o l e f o r t r y p t o p h a n (see F i g u r e 6 ) . e.g.

As i s e v i d e n t from t h i s f a r f r o m e x t e n s i v e l i s t , some fragments,

p y r r o l e s and p h e n y l a c e t o n i t r i l e , must i n v o l v e s c i s s i o n o f t h e polymer c h a i n .

I n f a c t , some o f t h e a l i p h a t i c amino a c i d m o i e t i e s a l s o produce c o r r e s p o n d i n g nitriles.

N e v e r t h e l e s s , s i g n i f i c a n t n i t r i l e f o r m a t i o n i s n o t observed f o r t y r o s i n e o r

tryptophan. There seem t o be two p o s s i b l e e x p l a n a t i o n s f o r t h e l i m i t e d amount o f 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 t h u s f a r f r o m t h e Py-MS a n a l y s i s o f p r o t e i n s .

First, proteins

a r e composed o f a g r e a t e r v a r i e t y o f b u i l d i n g b l o c k s t h a n any o t h e r c l a s s o f b i o polymers.

T h i s i n t u r n p r o v i d e s f o r a g r e a t v a r i e t y o f p y r o l y s i s p r o d u c t s , many o f

w h i c h possess t h e same nominal mass.

Nominal r e s o l u t i o n mass s p e c t r a o b v i o u s l y

A 1

117

I

ACTH 4-10

Met-Glu-His-Phe-Arg-Trp-Gly

B 8 LPH 61-69

94

I Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys

--

L1

I

I

83

60

80

100

120 m/z 140

r

160

Figure 6. Curie-point pyrolysis mass spectra of synthetic oligopeptides representing sequences from adrenocorticotropin and p-lipotropin. Note characteristic ion signals of methionine (m/z 34, 48), phenylalanine (m/z 92, 104, 117), tyrosine (m/z 94, 108, 120, 122) and tryptophan (m/z 117, 131). Conditions: samples 10 ug; Tc 510°C; E el 14 eV. provide very 1 imited information on such an extremely complex pyrolysate. Secondly, there may well be a fundamental problem in the pyrolysis reaction mechanisms of proteins, namely a pronounced tendency for charring (ref. 95) involving the polymer backbone. This prevents the release of more or less complete amino acid moieties and instead results in the splitting off of appendages such as the phenol and indole fragments. As charring during pyrolysis appears to be inversely proportional t o the rate of heating o f the sample (ref. 23) - providing an important argument for

23 A

108 Tyr-Phe

117

120 m,z 140

160

B

I8 Phe-Tyr

1!7

0

43

78?9

J 100

(LL

L 3 m,z 140

160

Figure 7. Curie-point pyrolysis mass spectra of two closely related dipeptides, differing only in the amino acid sequence. Note changes in relative intensities at m/z 79, 92, 94, 104, 108, 117 and 120. The difference in intensity at m/z 60 may be unrelated to structural features and probably represents acetic acid derived from the acetate counter-ion of the terminal amino group. Conditions: sample 10 pg; Tc 510°C; Eel 14 eV. preferring faster filament pyrolysis techniques over slow furnace pyrolysis techniques such as direct probe pyrolysis - fast laser pyrolysis might help to produce larger building blocks in protein pyrolysis. Preliminary experiments by Kistemaker e t al. (ref. 2 9 ) , however, failed to demonstrate these larger fragments. In summing up the present situation, we have the distinct impression that, because of the limited success obtained and the availability of powerful alternative techniques, Py-MS techniques may not gain as much momentum in the chemical

c h a r a c t e r i s a t i o n o f p r o t e i n s as f o r some o t h e r biopolymers. However, i t s h o u l d be k e p t i n mind t h a t some i m p o r t a n t c l a s s e s o f p r o t e i y s , such as g l y c o p r o t e i n s , a r e l e s s amenable t o c o n v e n t i o n a l t e c h n i q u e s and may t h e r e f o r e c o n s t i t u t e an i m p o r t a n t potential f i e l d o f application.

A l s o , Py-MS may p r o v e t o be o f v a l u e i n t h e r a p i d

c h a r a c t e r i s a t i o n o f p r o t e i n p r e p a r a t i o n s , when o n l y a l i m i t e d amount o f chemical i n t e r p r e t a t i o n i s d e s i r e d ( r e f . 96).

A l t e r n a t i v e l y , t h e s e n s i t i v i t y o f Py-MS m i g h t

perhaps a l l o w i t s use i n tandem w i t h c o n v e n t i o n a l p r o t e i n c h a r a c t e r i s a t i o n t e c h n i q u e s , e.g.

f o r t h e a n a l y s i s o f e l e c t r o p h o r e t i c bands o r s p o t s .

F i n a l l y , Py-MS has proved

t o be o f v a l u e i n t h e c h a r a c t e r i s a t i o n o f unusual o l i g o p e p t i d e s t r u c t u r e s such as t h o s e encountered i n some a n t i b i o t i c s ( r e f . 61).

An example o f t h e Py-MS s p e c t r a o f

t w o c l o s e l y r e l a t e d d i p e p t i d e s i s shown i n F i g u r e 7, d e m o n s t r a t i n g t h e s e n s i t i v i t y o f t h e C u r i e - p o i n t Py-MS t e c h n i q u e t o m i n o r s t r u c t u r a l d e t a i l s .

3.5.

LIPIDS L i p i d s c o n s t i t u t e a n o t h e r i m p o r t a n t group o f b i o m a t e r i a l s which o f t e n have i n -

s u f f i c i e n t v o l a t i 1 i t y t o be analysed by d i r e c t chromatographic o r mass s p e c t r o m e t r i c techniques.

W i t h t h e e x c e p t i o n o f t h e t e r p e n e s , which c o u l d be regarded as o l i g o m e r s

o r polymers o f i s o p r e n e , l i p i d s u s u a l l y do n o t possess t h e r e p e a t i n g s u b u n i t s which a r e t h e hallmark o f polymeric materials.

F i g u r e 8 shows t h e p y r o l y s i s mass spectrum

o f n a t u r a l rubber. a polyterpene (cis-l,4-polyisoprene).

The dominant p y r o l y s i s

p r o d u c t s appear t o be t h e i s o p r e n e monomer a t m/z 68 and t h e dirner a t m/z 136.

Most

o t h e r p r o m i n e n t mass s i g n a l s appear t o be r e s i d u a l E I fragments i n s p i t e o f t h e l o w e l e c t r o n energy c o n d i t i o n s .

The s t r o n g E I f r a g m e n t a t i o n tendency i s e x p l a i n e d by

t h e a l i p h a t i c , branched hydrocarbon n a t u r e o f t h e p y r o l y s i s p r o d u c t s .

Natural

r u b b e r i s perhaps one o f t h e few biopolymers t h a t show a more o r l e s s r e g u l a r d e p o l y m e r i s a t i o n b e h a v i o u r under p y r o l y s i s c o n d i t i o n s .

I n o t h e r biopolymers thermal

d e g r a d a t i o n p r e v a i l s o v e r d e p o l y m e r i s a t i o n , as d i s c u s s e d i n S e c t i o n 3.1. C u r i e - p o i n t Py-MS has had o n l y l i m i t e d success i n t h e s t u d y of n o n - p o l y m e r i c l i p i d s because t h e r a p i d h e a t i n g and s h o r t r e s i d e n c e t i m e o f t h e p r o d u c t s i n t h e r e a c t i o n zone r e s u l t s i n t h e escape o f i n t a c t o r a t most m i n i m a l l y fragmented l i p i d m o i e t i e s , e.g.

complete f a t t y a c i d s , which a r e e a s i l y l o s t by condensation on t h e

w a l l s o f t h e r e a c t i o n chamber.

Thus, l i p i d m o i e t i e s t e n d t o be s t r o n g l y under-

r e p r e s e n t e d i n t h e p y r o l y s i s mass s p e c t r a o f complex l i p i d - c o n t a i n i n g samples.

By

u s i n g heated r e a c t i o n chamber w a l l s and p y r o l y s i n g d i r e c t l y i n f r o n t o f t h e i o n source, however, l i p i d molecules may p r o v i d e i n t e n s e s i g n a l s , as shown by t h e work o f A n h a l t and Fenselau ( r e f . 1 8 ) .

Even then, t h e use o f e l e c t r o n impact i o n i s a t i o n ,

e s p e c i a l l y a t t h e 70 eV e l e c t r o n energy used by t h e s e a u t h o r s , r e s u l t s i n severe f u r t h e r fragmentation o f t h e l i p i d products.

I n t h i s r e s p e c t , t h e use of chemical

i o n i s a t i o n t e c h n i q u e s f o r t h e a n a l y s i s o f l i p i d p r o d u c t s f r o m d i r e c t probe p y r o l y s i s , as r e p o r t e d by Risby and Yergey ( r e f . 19, 20), may be regarded as an improvement. From t h e p o i n t o f view o f p y r o l y s i s c o n d i t i o n s , however, i t would seem t o be

25

i8

:sHs monomer

f

f

53

ClaHls dimer

L 80

F i g u r e 8. C u r i e - p o i n t p y r o l y s i s mass spectrum o f n a t u r a l r u b b e r ( c i s - l , 4 - p o l y i s o p r e n e ) . Note prominent i o n s i g n a l s a t m/z 68 and 136, a p p a r e n t l y r e p r e s e n t i n g t h e molecu l a r i o n s o f monomeric and d i m e r i c u n i t s . ( f ) d e s i g n a t e s E I fragment i o n s . Condi t ions:sample 10 pg; Eel 14 eV (see A t l a s ) .

p r e f e r a b l e t o use r a p i d f i l a m e n t p y r o l y s i s r a t h e r t h a n d i r e c t probe p y r o l y . s i s t e c h niques, i n o r d e r t o a v o i d unnecessary secondary p y r o l y s i s r e a c t i o n s r e s u l t i n g i n t h e p r o d u c t i o n o f m u l t i p l e u n s a t u r a t e d o r even a r o m a t i c p y r o l y s i s p r o d u c t s . I n t e r e s t i n g r e s u l t s have a l s o been o b t a i n e d w i t h p h o s p h o l i p i d s owing t o t h e Weijman ( r e f . 70)

presence o f h i g h l y c h a r a c t e r i s t i c m o i e t i e s such as c h o l i n e .

r e p o r t e d t h e occurrence o f a c h a r a c t e r i s t i c i o n s e r i e s a t m/z 59, 71 and 89, presumed t o be t r i m e t h y l a m i n e , v i n y l d i m e t h y l a m i n e and N,N-dimethylaminoethanol (see F i g u r e 9 ) , i n t h e p y r o l y s a t e of fungal p h o s p h o l i p i d s .

, respectively

The abovementioned

i d e n t i t y o f t h e m/z 59 fragment m o l e c u l e i n t h e p y r o l y s a t e o f l e c i t h i n ( p h o s p h a t i d y c h o l i n e ) was proved by L o u t e r e t aZ. ( r e f . 80) u s i n g Py-MS/MS.

A c u r i o u s phenomenon

i s r e g u l a r l y observed i n t h e p y r o l y s i s o f phosphol p i d p r e p a r a t i o n s , namely t h e m e t h y l a t i o n o f c h l o r i d e i o n s r e s u l t i n g i n m e t h y l c h o r i d e (m/z 50, 52 i n F i g u r e 9

I

(see a l s o S e c t i o n 2 . 3 . ) .

3.6.

MISCELLANEOUS BIOPOLYMERS

An i m p o r t a n t b i o p o l y m e r which l e n d s i t s e l f e x t r e m e l y w e l l t o Py-MS a n a l y s i s i s lignin.

Upon p y r o l y s i s , whether r a p i d o r slow, l i g n i n degrades i n t o i t s p h e n o l i c

b u i l d i n g b l o c k s w i t h r e l a t i v e l y l i t t l e f o r m a t i o n o f s m a l l e r p y r o l y s i s p r o d u c t s (see F i g u r e 1 0 ) . Depending upon t h e t y p e o f l i g n i n analysed, v a r i o u s c h a r a c t e r i s t i c phenyl , methoxyphenyl ( g u a i a c y l ) , and dimethoxyphenyl ( s y r i n g y l ) s e r i e s a r e r e a d i l y

59

1.111

..I,,l.

.",

I

Figure 9. Pyrolysis mass spectrum of a fungal lipid fraction showing the characteristic ion series of choline-containing phospholipids namely at m/z 58 (C3H8N), 59 (trimethylamine), 71 (N,N-dimethylvinylamine) and 89 (N,N-dimethylaminoethanol ; sometimes more pronounced, depending on the sample matrix), Note also typical methylchloride ion signals at m/z 50 and 52. The peaks at m/z 107 and 109 should probably be assigned to N,N-dimethylvinylammoniumchloride. As discussed in the text, the lipid part of the molecule is strongly under-represented in the spectrum. Conditions: sample 10 pg; Tc 510°C; Eel 14 eV.

10 PHENOLS

I

I

I

z

P

~

20

Figure 10. Pyrolysis mass spectrum of a BjPrkmann lignin, obtained from straw, showing the presence of all three characteristic lignin building blocks in grass lignins. Conditions: sample 10 pg; Tc 610°C; Eel 15 eV. distinguishable, as reported by Meuzelaar e t aZ. (ref. 50). The chemical identities of the main pyrolysis products of lignin have been established by several authors, e.g. using Py-GC-PIS techniques (refs. 52, 97).

27 S o i l polymers, e.g.

humic a c i d s , as w e l l as geopolymers, e.g.

c o a l s and kerogens

a l t h o u g h n o t s t r i c t l y polymers i n t h e sense o f h a v i n g w e l l d e f i n e d r e p e a t i n g s u b u n i t s , a r e a n o t h e r c l a s s of macromolecular substances amenable t o Py-MS a n a l y s i s ( r e f . 52). The inore random, h i g h l y complex c o n f i g u r a t i o n s o f t h e s e substances however, have so f a r eluded precise s t r u c t u r a l e l u c i d a t i o n .

With regard t o coal s t r u c t u r e , a r e c e n t

r e p o r t by t h e Committee of Chemical Sciences o f t h e N a t i o n a l Research C o u n c i l , The Department of Energy,

USA ( r e f . 98), n o t e s t h a t :

" I t probably w i l l not be possible i n the foreseeabZe future actualZy t o

determine the "structure" of coal absoZutely i n the way we can with, say, organic compounds. Coal i s heterogeneous by nature and it varies i n structure no l e s s from millimeter t o millimeter i n the same seam than between coals from d i f f e r e n t rank formed from d i f f e r e n t species of pZant matter i n swamps hundreds or thousands miZes apart. The committee believes, hmever, t h a t it i s possible t o determine i n coals the key structural features t h a t a f f e c t u t i l i t y and r e a c t i v i t y If. As a r e s u l t , u n d e r s t a n d i n g o f p y r o l y s i s mechanisms i n t h e s e m a t e r i a l s i s h i g h l y s p e c u l a t i v e a t most, and chemical i n t e r p r e t a t i o n o f t h e i r p y r o l y s i s - m a s s s p e c t r a i s g e n e r a l l y c o n f i n e d t o t h e l a b e l l i n g o f c h a r a c t e r i s t i c i o n s e r i e s as " a l k e n e s " , "benzenes",

"naphthalenes",

"phenols",

" p y r r o l e s " , e t c . (see S e c t i o n 7.5).

Never-

t h e l e s s , t h i s may p r o v i d e u s e f u l i n s i g h t i n t o t h e degree o f a r o m a t i c i t y o r unsaturation,

i n t o t h e presence o f h e t e r o a t o m i c compounds, e.g.

and i n t o r e a c t i v i t y , e.g. c o a l c o n v e r s i o n b e h a v i o u r ( r e f . 9 9 ) . o f t e n n o t a v a i l a b l e as e a s i l y f r o m c o n v e n t i o n a l t e c h n i q u e s .

s u l p h u r compounds, This information i s


29

Chapter 4

THE TECHNIQUE OF CURIE-POINT PYROLYSIS MASS SPECTROMETRY

4.1. SAMPLE PREPARATION In filament pyrolysis techniques, ideally the sample is coated on the filament by applying one or more drops of a solution followed by drying in air, in an inert gas or in a vacuum chamber. Although readily soluble samples may be found among both synthetic and natural polymers, more often than not the sample is insoluble in most chemically inert solvents. Generally, insoluble samples can be handled by mechanical grinding or milling to form a fine powder (with admixture o f quartz sand to aid homogenisation), followed by ultrasonic suspension in a suitable solvent. Among the refractory samples which have been successfully processed in this manner are cells, tissues, whole soils, coals and shale rocks. Sampling and homogenisation of sticky or elastic materials, e.g. certain microorganisms and natural rubber, can be aided by grinding with cooling in liquid nitrogen. Some samples, e.g. bacterial colonies, sludges or urine, can be applied directly to the filament. Suitable solvents or suspending media are methanol, carbon disulphide and water. Carbon disulphide is most easily dried and produces relatively stable suspensions by virtue of its high specific density, However, it is toxic and stored samples tend to dry out rapidly. Water, although often the most natural choice for biomaterials, dries very slowly and therefore requires vacuum drying if time is an important factor. Methanol has been the preferred solvent or suspending medium in most of our recent studies. Further important factors in sample preparation are the pH and ionic strength of the sample solution or suspension, as these factors may strongly influence the pyrolytic fragmentation of (especially polar) organic compounds. This is demonstrated in Figure 1 1 , where pyrolysis mass spectra of polygalacturonic acid sampled from different suspension or solution media are compared. Such "matrix effects" may lead to artifacts on comparative analysis of samples by Py-PIS. A suitable means of preventing such artifacts is to "standardise" solutions or suspensions throughout the series of samples to be compared by using buffered saline, e.g. aqueous PBS buffer (0.01 11 phosphate, 0.145 F1 C1-, 0.17 M ;'aN pH 7.2). Even when disregarding any signals directly derived from residual solvent, a change o f solvent, e.g. from methanol to water or carbon disulphide may appreciably influence the pyrolysis pattern (refs. 100, 101). Careful attention should be paid to the cleanliness o f the pyrolysis wire and of the glass or quartz reaction tube surrounding the filament. The use of ferromagnetic wires made of a fast rusting alloy and in particular pure iron wires (Tc = 770°C),

30

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31 F i g u r e 11. P y r o l y s i s mass s p e c t r a o f p o l y g a l a c t u r o n i c a c i d , u s i n g d i f f e r e n t sample s o l v e n t s . ( a ) Suspension i n methanol; ( b ) s u s p e n s i o n / s o l u t i o n i n water, pH 3 . 8 ; ( c ) s o l u t i o n i n aqueous sodium h y d r o x i d e , pH 9.5;(d) s o l u t i o n i n phosphate b u f f e r e d s a l i n e (PBS), pH 6.5. Conditions:sample c o n c e n t r a t i o n s 1 mg/ml; samples 10 ug; Tc 510°C; E e l 14 eV. Note t h a t t h e a c i d i c aqueous and m e t h a n o l i c suspensions (a, b ) g i v e s i m i l a r s p e c t r a . Use o f t h e a l k a l i n e medium ( c ) r e s u l t s i n marked i n t e n s i t y changes, e.g. a t m/z 31, 32, 60, 68, 74, 85, 96, 102 and 114. The f o r m a t i o n o f f u r a n o i c components seems t o be reduced under t h e i n f l u e n c e o f a l k a l i . The n e u t r a l b u f f e r e d s o l u t i o n g i v e s an i n t e r m e d i a t e spectrum;the h i g h c o n c e n t r a t i o n o f c h l o r i d e i o n s g i v e s r i s e t o t h e i n t e n s e peaks a t m/z 36 and 38 (HCl+'). i n c o m b i n a t i o n w i t h aqueous s o l u t i o n s o r suspensions s h o u l d be avoided. p o i n t w i r e s a r e v e r y i n e x p e n s i v e , t h e y may be d i s c a r d e d a f t e r use.

As C u r i e -

Used g l a s s o r

q u a r t z r e a c t i o n tubes, however, can be c l e a n e d by b o i l i n g i n a c i d s (e.g. chromic a c i d ) , r i n s i n g i n d e - i o n i s e d w a t e r and subsequent oven d r y i n g .

The c h o i c e o f t h e

f i l a m e n t c l e a n i n g method, e.g. p r o l o n g e d h e a t i n g i n a w a t e r - s a t u r a t e d hydrogen atmosphere, u l t r a s o n i c c l e a n i n g i n o r g a n i c s o l v e n t s o r p r e - p y r o l y s i n g i n a vacuum environment, a l s o n o t i c e a b l y i n f l u e n c e s t h e p y r o l y s i s p a t t e r n ( r e f s . 100, 1 0 1 ) .

We

most f r e q u e n t l y use t h e r e d u c t i v e hydrogen c l e a n i n g t e c h n i q u e because i t removes o x i d e l a y e r s which may cause c a t a l y t i c r e a c t i o n s and/or change t h e e m i s s i v i t y o f t h e f i l a m e n t surface.

However, i t s h o u l d be n o t e d t h a t r e d u c t i v e c l e a n i n g t e c h n i q u e s

can cause severe hydrogen a b s o r p t i o n by t h e m e t a l .

T h i s may i n f l u e n c e t h e p y r o l y t i c

r e a c t i o n s , as demonstrated by K u t t e r e t aZ. ( r e f . 102) u s i n g s m a l l f e r r o m a g n e t i c c y l i n d e r s f o r t h e p y r o l y s i s o f n i t r o and azo compounds.

Hydrogen can a l s o be formed

p y r o l y t i c a l l y f r o m r e s i d u a l s o l v e n t s (methanol, w a t e r ) . To a p p l y t h e sample f r o m a s o l u t i o n o r suspension, a m i c r o p i p e t t e i s used t o d e p o s i t a 5 - m i c r o l i t r e drop c l o s e t o t h e t i p o f t h e f e r r o m a g n e t i c w i r e .

F i g u r e 12

shows a b a t c h o f 12 w i r e s b e i n g c o a t e d w h i l e p r o t r u d i n g f r o m t h e g l a s s r e a c t i o n t u b e s and s l o w l y r o t a t i n g t o ensure u n i f o r m d i s t r i b u t i o n o f t h e sample.

I f necessary

t h e whole assembly can be pumped o f f i n a vacuum e n c l o s u r e f o r f a s t d r y i n g .

After

d r y i n g , t h e w i r e s a r e r e t r a c t e d t o t h e p r o p e r p o s i t i o n i n t h e g l a s s r e a c t i o n tube, w h i c h a l s o serves as a p r o t e c t i v e c o v e r and even a l l o w s s h i p p i n g o f c o a t e d w i r e s by m a i l ( s e e F i g u r e 13).

An a l t e r n a t i v e c o a t i n g t e c h n i q u e may be used w i t h s l u r r i e s ,

sludges o r pastes which a r e t o o t h i c k f o r m i c r o p i p e t t i n g .

Such samples can be

s i m p l y smeared on t h e w i r e u s i n g a p l a t i n u m sample l o o p o r by s t i c k i n g t h e w i r e d i r e c t l y i n t o t h e sample.

With b a c t e r i a l c o l o n i e s , t h i s t e c h n i q u e , a l t h o u g h p r o -

v i d i n g l e s s u n i f o r m sample c o a t i n g s t h a n t h e drop technique, has c o n s i d e r a b l e advantages o v e r s e q u e n t i a l washing, f r e e z e - d r y i n g and re-suspending procedures w h i c h may cause i r r e p r o d u c i b l e changes i n t h e chemical c o m p o s i t i o n o f t h e sample and l o s s of c h a r a c t e r i s t i c m e t a b o l i t e s ( r e f . 103).

P r e f e r r e d amounts o f sample i n C u r i e -

p o i n t Py-MS v a r y between 1 and 20 micrograms.

Below 1 microgram, background s i g n a l s

may s i g n i f i c a n t l y c o n t r i b u t e t o t h e p a t t e r n s .

As r e p o r t e d by Meuzelaar ( r e f . 100)

and Windig e t aZ. ( r e f . 101) changing t h e amounts o f sample f r o m 2 t o 20 micrograms causes minimal changes i n t h e r e l a t i v e peak h e i g h t s o f t h e p a t t e r n s o f glycogen and

32

Figure 1 2 . Apparatus f o r the batch coating and drying of Curie-point wires. Note t h a t the wires a r e revolved by magnetic a t t r a c t i o n t o the base p l a t e assembly ( r i g h t ) which makes a slow c i r c u l a r movement. All wires can be withdrawn simultaneously i n t o the reaction tubes by s l i d i n g the manifold away from t h e base-plate. albumin.

Above 20 micrograms, however, the increase in overall peak height i s no longer proportional t o sample weight, indicating t h a t p a r t s of t h e material a r e blown off the wire before being pyrolysed. Occasionally, t h e use of amounts of samples u p t o 60 - 70 u g cannot be avoided, e . g . when pyrolysing whole s o i l samples containing l e s s than 1 % of organic material ( r e f . 51). I n t h i s case, however, sample blow-off i s n o t as severe, as the inorganic p a r t of the sample usually does not produce l a r g e amounts of gaseous pyrolysis products.

33

Figure 13. Coated Curie-point wires a n d reaction tubes i n shippinq c onta ine r which can hold u p t o 96 tubes. Typical concentrations of s o l u t i o n s or suspensions used f o r sample coating a r e 1 - 2 mg/ml. When applying 5 u l drops, t h i s amounts t o 5 - 10 micrograms per d r o p . The normal r e p e a t a b i l i t y of sample amounts applied to the filaments i s approximately ? lo % when using t h e d r o p technique a n d i50% when using the smear technique.

4.2. PYROLYSIS The Curie-point technique involves placing a thin ferromagnetic wire in a n induction c o i l connected t o a high frequency power supply (Figure 1 4 ) . Idhen t h e h . f . f i e l d i s switched o n , the ferromagnetic wire inductively heats t o i t s Curie-point temperature, a t which t h e wire l o s es i t s ferromagneticity a n d becomes paramagnetic. This causes a d r a s t i c l o s s in energy absorption from the h . f . f i e l d in the c o i l . Provided t h a t t h e wire dimensions and h . f . f i e l d parameters a r e properly matched,

) of t h e wire s t a b i l i z e s c lose t o the Curie-point eq a p o i n t where t h e residual energy absorption by Eddy c urre nts ( " s k i n a t temperature, th e equilibrium temperature ( T

34

glass reaction tube

I

F i g u r e 14. Schematic diagram o f t h e C u r i e - p o i n t w i r e / r e a c t i o n tube assembly l o c a t e d w i t h i n the h i g h frequency c o i l . e f f e c t " ) i s balanced by the loss o f heat through r a d i a t i o n and conduction ( r e f .

104).

Temperature/time p r o f i l e s f o r wires o f d i f f e r e n t ferromagnetic m a t e r i a l s ,

when u s i n g a Fischer Labortechnik 1.5 kW, 1.1 MHz h . f . power supply, a r e shown i n F i g u r e 15.

Intermediate curves can be obtained by using various a l l o y s o f these

A t 600"C, t y p i c a l degradation r e a c t i o n s i n polymers a r e Therefore, even w i t h temperature-

ferromagnetic m a t e r i a l s .

completed i n l e s s than m i 11iseconds ( r e f . 105). r i s e times (t,)

i n the m i l l i s e c o n d range, p y r o l y s i s w i l l be complete a t temperatures

w e l l below 600°C.t As a consequence, i t i s n o t s u r p r i s i n g t h a t changing T

from 510 eq t o 610°C does n o t d r a s t i c a l l y i n f l u e n c e the p y r o l y s i s p a t t e r n s o f biopolymers such

as glycogen and albumin ( r e f s . 100, 101).

For the same reason, changing the t o t a l

h e a t i n g time ( t i ) from 0.3 t o 1.2 s a l s o does n o t cause appreciable changes i n these p a t t e r n s ( r e f s . 100, 101).

However, a very low T

d r a s t i c changes i n p y r o l y s i s p a t t e r n s .

I I

I

I

I

i

4010-

Ni(T, =358

e.g. below 400"C, may cause eq ' This i s explained by t h e f a c t t h a t a t these

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I

1

0.2

0.4

0.6 -t

0.8

1.0

1.2

(s)

F i g u r e 15. Temperature/time p r o f i l e s and C u r i e - p o i n t temperatures f o r pure N i , Fe, and Co wires (diameter 0.5 mm) when using a 1.5 kW, 1.1 Mhz h . f . power supply.

temperatures t h e h a l f - l i f e o f a d e g r a d a t i o n r e a c t i o n may become l o n g compared w i t h t h e temperature-rise time.

I n t h i s s i t u a t i o n , p y r o l y s i s may be i n c o m p l e t e when

i s reached and t h u s changing t o t h e r hand, a h i g h T

e.g.

c

strongly influences the pyrolysis pattern.

T

eq On t h e

above 700 o r 800"C, may a l s o cause changes i n t h e

eq ' p y r o l y s i s p a t t e r n s owing t o t h e s t r o n g r a d i a n t h e a t i n g o f t h e r e a c t i o n t u b e , and may l e a d t o e i t h e r e v a p o r a t i o n o r secondary p y r o l y s i s o f compounds condensed on t h e

w a l l s o f t h e t u b e d u r i n g t h e i n i t i a l p y r o l y s i s stages o f t h e sample. It s h o u l d be n o t e d t h a t v a r y i n g T may be used t o enhance o r reduce t h e r e l a t i v e eq c o n t r i b u t i o n o f a p a r t i c u l a r component o r m o i e t y i n t h e p y r o l y s i s mass spectrum o f a

complex m i x t u r e o r conjugate.

For i n s t a n c e , because o f t h e r e l a t i v e t h e r m o l a b i l i t y

o f c a r b o h y d r a t e s i n comparison w i t h p r o t e i n s , c a r b o h y d r a t e s i g n a l s may be s p e c i f i c a l l y enhanced i n s p e c t r a o f c a r b o h y d r a t e - p r o t e i n m i x t u r e s o r g l y c o p r o t e i n s by s e l e c t i n g l o w e r p y r o l y s i s temperatures. oligopeptide mixture.

T h i s i s shown i n F i g u r e 16 f o r an o l i g o s a c c h a r i d e -

I n t h e 358°C spectrum ( a ) t h e p e p t i d e c o n t r i b u t e s a c h a r a c t e r -

i s t i c s e r i e s o f fragments a t m/z 48 (CH3SH, from t h e M e t - r e s i d u e s ) , 56 (C4H8 isomers, and a c r o l e i n ) , 94 (phenol, f r o m T y r ) , 108 ( c r e s o l , f r o m T y r ) and 117 ( i n d o l e , f r o m T r p ) , whereas c o n t r i b u t i o n s o f t h e Phe-residue (m/z 92, t o l u e n e , and 104, s t y r e n e ) a r e r e l a t i v e l y low.

Most o f t h e fragment peaks i n t h e spectrum o r i g i n a t e f r o m t h e

carbohydrate constituent.

I n t h e 610°C spectrum ( b ) t h e p e p t i d e s u b - p a t t e r n ( a r r o w s )

i s much more pronounced w i t h r e s p e c t t o t h e c a r b o h y d r a t e s u b - p a t t e r n .

Moreover,

a d d i t i o n a l i n f o r m a t i o n about t h e p e p t i d e c o n s t i t u e n t i s o b t a i n e d by t h e i n c r e a s e i n r e l a t i v e i n t e n s i t i e s a t m/z 34 (H2S, an a d d i t i o n a l fragment f r o m t h e Met r e s i d u e s ) , 92, 104, 120 ( h y d r o x y s t y r e n e , a d d i t i o n a l fragment f r o m Tyr) and 131 ( m e t h y l i n d o l e , from Trp).

The r e l a t i v e i n t e n s i t i e s o f h i g h mass range c a r b o h y d r a t e fragments (m/z

112, 114, 124, 126, 144) have decreased. The tT i s determined by t h e diameter o f t h e w i r e , t h e c o m p o s i t i o n o f t h e a l l o y , t h e s t r e n g t h o f t h e h i g h frequency f i e l d and t h e f i e l d f r e q u e n c y ( r e f . 1 0 4 ) .

Since

under p r a c t i c a l Py-MS c o n d i t i o n s p y r o l y s i s t a k e s p l a c e b e f o r e t h e w i r e reaches T

eq ' t h e h e a t i n g r a t e i s g e n e r a l l y t h o u g h t t o have a c r i t i c a l i n f l u e n c e on t h e p y r o l y s i s p a t t e r n s ( r e f . 105).

Unexpectedly, however, v a r y i n g t h e h e a t i n g r a t e by a f a c t o r o f

10 (changing tT o f a 510°C w i r e froin 0.1s t o 1.0s) does n o t a p p r e c i a b l y change t h e Py-MS p a t t e r n s o f glycogen and albumin ( r e f s . 100, 101).

Therefore, t h e emphasis

p l a c e d on a c c u r a t e r e p r o d u c t i o n o f t h e t e m p e r a t u r e / t i m e p r o f i l e i n t h e p y r o l y s i s gas chromatography l i t e r a t u r e (e.g. r e f s . 10, 11, 106, 107) does n o t seem t o be as

T i s n e i t h e r too low eq Other f a c t o r s , such as t h e c h o i c e o f t h e f i l a m e n t c l e a n i n g t e c h n i q u e

s t r i n g e n t under vacuum p y r o l y s i s c o n d i t i o n s , p r o v i d e d t h a t nor too high.

and o f t h e s o l v e n t , and f a c t o r s t h a t govern t r a n s f e r o f t h e p y r o l y s a t e t o t h e i o n s o u r c e o r i n f l u e n c e i o n i s a t i o n c o n d i t i o n s , appear t o d e t e r m i n e t h e r e p r o d u c i b i l i t y o f Py-MS more s t r o n g l y t h a n t h e t e m p e r a t u r e / t i m e p r o f i l e , e s p e c i a l l y w i t h r e s p e c t t o

1ong-term r e p r o d u c i b i 1 it y ( r e f s . 100, 101 ) .

36 6

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t

60

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20

40

60

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80

94

100

120

140

160

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Figure 16. Pyrolysis mass spectra of an oligosaccharide-oligopeptide mixture (ma1topentaose and Phe-Asp-Met-Trp-Gly-Met-Tyr, 1 :1 w/w ratio) showing the differences in fragmentation behaviour of both molecular components when different equilibrium temperatures are used.

In contrast to the relatively mild influence of changes in temperature profile, substitution of a ferromagnetic tube ("oven" pyrolysis) for the ferromagnetic wire produces pronounced changes in the pyrolysis mass spectra of most compounds as shown in Figure 17. As can be seen, oven pyrolysis of carbohydrate and proteinaceous materials produces a range of products due t o secondary pyrolysis reactions, e.g. unsaturated and aromatic hydrocarbons. Moreover, spectra of chemically related materials produced by oven pyrolysis tend to show fewer characteristic differences

37 t h a n t h e c o r r e s p o n d i n g f i l a m e n t s p e c t r a , whereas f o r o b t a i n i n g r e p r o d u c i b l e ovens p e c t r a s m a l l e r t o l e r a n c e s i n p y r o l y s i s and sample c o n d i t i o n s e x i s t .

I n conclusion,

t h e use o f f e r r o m a g n e t i c tubes i s recommended o n l y f o r r e l a t i v e l y v o l a t i l e samples which cannot be p y r o l y s e d e f f e c t i v e l y by t h e f i l a m e n t t e c h n i q u e , o r i n s p e c i a l cases where more r i g o r o u s thermal d e g r a d a t i o n i s d e s i r e d . 4.3.

PYROLYSATE TRANSFER I n t h e p r e v i o u s s t e p s , sample p r e p a r a t i o n and sample p y r o l y s i s , t h e o r i g i n a l

sample was t r a n s f o r m e d i n t o a multicomponent m i x t u r e o f p y r o l y s i s p r o d u c t s ( t h e pyrolysate).

R e p r o d u c i b l e mass s p e c t r o m e t r i c a n a l y s i s of t h i s mu1 ticomponent

mixture requires c a r e f u l c o n t r o l o f several f a c t o r s , i n c l u d i n g t h e t r a n s f e r condit i o n s between t h e p y r o l y s i s zone and t h e i o n source, t h e i o n i s a t i o n c o n d i t i o n s and t h e subsequent mass a n a l y s i s and i o n d e t e c t i o n c o n d i t i o n s . I d e a l p y r o l y s a t e t r a n s f e r c o n d i t i o n s s h o u l d a1 l o w t h e p y r o l y s i s p r o d u c t s t o r e a c h t h e i o n i s a t i o n zone w i t h o u t any l o s s , d e g r a d a t i o n o r r e c o m b i n a t i o n o f p r o d u c t s d u r i n g t r a n s f e r ; i n p r a c t i c e these conditions are almost never f u l f i l l e d .

First,

some p y r o l y s i s p r o d u c t s t e n d t o remain on t h e f i l a m e n t i n t h e f o r m o f n o n v o l a t i l e c h a r s and t h u s a r e c o m p l e t e l y i n a c c e s s i b l e t o f u r t h e r a n a l y s i s .

Presently available

e v i d e n c e i n d i c a t e s t h a t t h e amount o f c h a r formed i s i n v e r s e l y p r o p o r t i o n a l t o t h e h e a t i n g r a t e ( r e f . 23) and t h u s can be m i n i m i s e d by a v o i d i n g e x c e s s i v e l y s l o w heating rates.

Some p r o d u c t s v o l a t i l e enough t o escape f r o m t h e p y r o l y s i s zone may

be d i f f i c u l t t o t r a n s f e r t o t h e i o n i s a t i o n zone.

These p r o d u c t s may have a tendency

t o condense on t h e w a l l s o f t h e r e a c t i o n chamber a n d / o r subsequent t r a n s f e r l i n e s . H e a t i n g t h e s e w a l l s t o a h i g h enough t e m p e r a t u r e t o a v o i d c o n d e n s a t i o n may r e s u l t i n f u r t h e r thermal d e g r a d a t i o n o f t h e p r o d u c t s .

O b v i o u s l y , t h e i d e a l s o l u t i o n would be

t o a c h i e v e a w a l l - l e s s t r a n s f e r o f p y r o l y s i s p r o d u c t s , t h a t i s , by p y r o l y s i n g d i r e c t l y i n f r o n t o f t h e i o n source, o r by a n a l y s i n g o n l y t h o s e m o l e c u l e s which reach the i o n i s a t i o n region w i t h o u t previous w a l l c o l l i s i o n s .

However, i n p r a c t i c e

i t i s d i f f i c u l t t o a v o i d s t r o n g c o n t a m i n a t i o n o f t h e i o n source under t h e s e c o n d i -

tions.

I f t h e beam o f p y r o l y s i s p r o d u c t s i s s u f f i c i e n t l y c o l l i m a t e d t o a v o i d w a l l

c o n t a c t i n t h e i o n source, t h e n c o n t a m i n a t i o n may be m i n i m i s e d b u t t h e s i g n a l i n t e n s i t y w i l l be s t r o n g l y reduced.

T h e r e f o r e , t h e g l a s s r e a c t i o n t u b e shown i n F i g u r e

14 serves two purposes, n a l f e l y t o t r a p t h e r e l a t i v e l y i n v o l a t i l e p y r o l y s i s p r o d u c t s which m i g h t contaminate t h e i o n source and t o o b t a i n maximum s i g n a l i n t e n s i t y by p r o d u c i n g a f o r w a r d o r i e n t e d beam o f v o l a t i l e p y r o l y s i s p r o d u c t s w h i c h may d i r e c t l y e n t e r t h e expansion chamber o r t h e i o n source. The expansion chamber shown i n F i g u r e 18 s e r v e s t o broaden t h e p r e s s u r e l t i m e p r o f i l e i n t h e i o n source, i n o r d e r t o e n a b l e a s u f f i c i e n t number o f mass scans t o be made t o o b t a i n a r e p r e s e n t a t i v e averaged mass spectrum o f t h e p y r o l y s a t e . expansion chamber s h o u l d have c h e m i c a l l y i n e r t w a l l s , e.g. order t o avoid degradation o f p y r o l y s i s products.

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39 F i g u r e 17. Comparison o f p y r o l y s i s mass s p e c t r a o f biopolymers o b t a i n e d by t h e f i l a ment t e c h n i q u e (Fe w i r e , Tc 770°C; s p e c t r a a, b ) and t h e oven t e c h n i q u e (Fe tube, Tc 770°C; s p e c t r a c, d ) . The oven s p e c t r a a r e c o r r e c t e d f o r background, i n t h e f i l a m e n t s p e c t r a background c o n t r i b u t i o n s a r e n e g l i g i b l e . Note t h e c o n s i d e r a b l e i n c r e a s e i n r e l a t i v e peak i n t e n s i t i e s i n t h e oven s p e c t r a ( a r r o w s ) when compared t o t h e corresponding filament spectra.

electronmultiplier

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hf coil

bu fer volume

cold screen

CURIE-POINT PYROLYSIS MS SYSTEM

F i g u r e 18. Schematic diagram o f a C u r i e - p o i n t Py-MS system. T y p i c a l o p e r a t i n g c o n d i t i o n s : b u f f e r volume (expansion chamber) temperature 150-175°C; E e l 13-15 eV; i o n energy 5-10 eV; mass range m/z 15 t o between m/z 130 and m/z 200; scanning speed 8-10 s p e c t r a / s ; t o t a l scanning t i m e 15-30 s.

heated t o a temperature h i g h enough t o p r e v e n t condensation o f p y r o l y s i s p r o d u c t s f r o m l e a v i n g t h e r e a c t i o n tube, y e t l o w enough t o a v o i d secondary p y r o l y s i s r e a c t i o n s .

A good compromise temperature range appears t o be 150-200°C.

The t e m p e r a t u r e s h o u l d

be k e p t f a i r l y c o n s t a n t i n o r d e r t o a v o i d v a r i a t i o n s i n i o n i s a t i o n f r a g m e n t a t i o n p a t t e r n s due t o d i f f e r e n t i n i t i a l temperatures o f t h e molecules e n t e r i n g t h e i o n i s a t i o n r e g i o n ( r e f . 101, 108, 109).

I n t h e c o n f i g u r a t i o n shown i n F i g u r e 18, t h e

w a l l s o f t h e r e a c t i o n tube a r e heated by r a d i a t i o n f r o m t h e p y r o l y s i s f i l a m e n t o n l y and a r e t h e c o l d e s t l o c a t i o n i n t h e t r a n s f e r l i n e .

As a consequence, most p y r o l y s i s

p r o d u c t s w i t h low v o l a t i l i t y and a l s o l a r g e evaporated compounds, e.g. l i p i d s , w i l l condense on t h e w a l l o f t h e r e a c t i o n t u b e d i r e c t l y around t h e p y r o l y s i s zone. A l t h o u g h t h e l o s s o f such substances l e a d s t o a l o s s of v a l u a b l e i n f o r m a t i o n , t h e advantage o f t h i s s i t u a t i o n i s t h a t c o n t a m i n a t i o n o f expansion chamber and i o n source i s minimal.

In f a c t , a system such as t h a t shown i n F i g u r e 18 can be used

f o r o v e r 1 y e a r , a n a l y s i n g more than ( r e f . 100, 101).

1,000 samples, w i t h o u t c l e a n i n g t h e i o n source

40

L mlz 256

I

mlz 64

2

4 t(s)6

8

10

Figure 19. Selected ion p r o f i l e s from time-resolved Py-PIS analysis of an o i l - s h a l e kerogen sample. The p r o f i l e s were obtained by removing t h e modular expansion chamber, prolonging t T t o approximately 6 s . The p r o f i l e s a t m/z 56, believed t o represent mainly Cq-alkenes, shows a t l e a s t four overlapping k i n e t i c events. The ion p r o f i l e s a t m/z 34 ( H 2 S t ‘ ) and m/z 64 (S02” and/or S 2 ” ) a r e even more complex. Especially the m/z 64 p r o f i l e shows many overlapping events, the f i r s t of which appears t o represent e a r l y d i s t i l l a t i o n of elemental sulphur from the sample, thus producing an Sg+’ molecular ion signal a t m/z 256 a s well as a s u i t e of fragment ions, among which the S2+’ ion contributes t o the ion p r o f i l e a t m/z 64. Conditions: Sample 10 pg (from methanol suspension); Tc 610°C; t T 6 s (estimated); t z 10 s; Eel 20 eV. A newly designed version of the Py-MS i n l e t system ( r e f . 110) incorporates a

modular design which allows easy removal of the expansion chamber, thus positioning t h e reaction tube d i r e c t l y in f r o n t of the ion source. I n a d d i t i o n , the reaction tube i s designed in such a way t h a t the walls around the pyrolysis zone may be preheated t o several hundred degrees without heating the sample, which i s temporarily kept in a cool portion of the reaction tube. The p o t e n t i a l l y stronger contamination of the ion source can be controlled by operating the ion source a t a s l i g h t l y higher temperature than the reaction tube, e . g . a t 200 and 1 7 5 ” C , respectively. The pressure/time p r o f i l e of pyrolysis products entering the ion source can be broadened s u f f i c i e n t l y by slowing the heating r a t e of the wire t o 100°C/s. As demonstrated in Figure 19 t h i s allows f o r e f f i c i e n t scanning of the pressure/time p r o f i l e by the

41 quadrupole, b u t i s s t i l l o r d e r s o f magnitude f a s t e r t h a n t h e h e a t i n g r a t e s o f d i r e c t p r o b e systems, t h u s m i n i m i z i n g e x c e s s i v e c h a r format'ion.

In p r i n c i p l e , t h i s con-

f i g u r a t i o n combines t h e advantages o f C u r i e - p o i n t p y r o l y s i s ( b a t c h p r o c e s s i n g , easy a u t o m a t i o n ) and f a s t f i l a m e n t h e a t i n g (minimal c h a r f o r m a t i o n , m i n i m a l secondary r e a c t i o n s ) w i t h t h o s e o f d i r e c t probe t e c h n i q u e s (minimal l o s s o f l o w - v o l a t i l i t y p y r o l y s i s products, p o s s i b i l i t y o f o b t a i n i n g t i m e - r e s o l v e d p y r o l y s i s p r o f i l e s ) .

4.4.

IONISATION I n s e l e c t i n g t h e i o n i s a t i o n technique, two c r i t e r i a a r e c o n s i d e r e d e s s e n t i a l .

F i r s t , minimal i o n i s a t i o n f r a g m e n t a t i o n o f p y r o l y s i s p r o d u c t s s h o u l d o c c u r w i t h p r i n c i p a l l y m o l e c u l a r i o n s b e i n g formed.

Secondly, t h e i o n i s a t i o n process must be

s t a b l e enough t o p r o v i d e a h i g h degree o f l o n g - t e r m r e p r o d u c i b i l i t y .

While no

i o n i s a t i o n t e c h n i q u e i s i d e a l i n b o t h r e s p e c t s , t h e advantages and disadvantages o f t h e t h r e e m a j o r " s o f t " i o n i s a t i o n techniques, i . e .

l o w v o l t a g e E I , C I and F I , w i l l be

b r i e f l y discussed. Low v o l t a g e E I has been t h e p r i n c i p a l t e c h n i q u e used t h r o u g h o u t t h e C u r i e - p o i n t Py-

MS s t u d i e s r e p o r t e d so f a r .

T r i a l s t u d i e s u s i n g compounds t y p i c a l o f p y r o l y s a t e s

i n d i c a t e t h a t e l e c t r o n e n e r g i e s between 13 and 15 eV appear t o be a good compromise between minimum y i e l d o f fragment i o n s and maximum y i e l d o f moTecular i o n s ( r e f . 4 6 ) . Once t h e e l e c t r o n energy has been chosen, e v e r y e f f o r t s h o u l d be made t o keep t h i s energy c o n s t a n t t o w i t h i n 0.1 eV.

I n r e p r o d u c i b i l i t y studies, a variation i n electron

energy f r o m 13.9 t o 14.1 eV r e s u l t e d i n a 3.4% and 4.0% mean r e l a t i v e d e v i a t i o n i n t h e i n t e n s i t i e s o f t h e 40 most i n t e n s e peaks i n t h e s p e c t r a o f albumin and glycogen, r e s p e c t i v e l y ( r e f s . 100, 101). I n s p i t e o f t h i s s e n s i t i v i t y t o m i n o r changes i n o p e r a t i n g conditions,

low v o l t a g e E I has t h e unique advantage o f b e i n g a b l e t o i o n i s e T h i s a l l o w s f o r a v e r y open d e s i g n o f t h e i o n source and

m o l e c u l a r beams e f f i c i e n t l y .

I n c o n t r a s t , b o t h CI and F I i n v o l v e c o n s i d e r a b l e i n t e r -

m i n i m i s e s memory e f f e c t s .

a c t i o n o f p y r o l y s i s p r o d u c t s w i t h s u r f a c e s d u r i n g t h e i o n i z a t i o n procedure ( F I o n l y f o r p o l a r products).

F i n a l l y , l o w v o l t a g e E I i s r e a d i l y o b t a i n a b l e on most mass

spectrometers whereas t h e C I and F I techniques may r e q u i r e e l a b o r a t e equipment modif ic a t i on. The C I t e c h n i q u e i s e x t r e m e l y e f f i c i e n t i n g e n e r a t i n g ( q u a s i - ) m o l e c u l a r i o n s t h e r e b y a l l o w i n g t h e d e t e c t i o n of t h e s e i o n s even when t h e sample i s p r e s e n t i n v e r y low c o n c e n t r a t i o n s ( r e f . 111).

An a d d i t i o n a l f e a t u r e o f C I i s t h e degree o f s e l e c t i -

v i t y as t o which p a r t i c u l a r chemical m o i e t y can be i o n i s e d , depending on t h e s e l e c t i o n

of t h e r e a g e n t i o n ( r e f . 1 1 2 ) .

As an example o f how t h i s c o u l d be used i n Py-MS

s t u d i e s , one m i g h t c o n c e i v a b l y enhance t h e c o n t r i b u t i o n o f n i t r o g e n - c o n t a i n i n g p y r o l y s i s p r o d u c t s , e.g.

d e r i v e d from p r o t e i n s i n t h e sample, by u s i n g a r e a g e n t i o n

w i t h a high proton a f f i n i t y ,

such as C4H5

+.

However, s e v e r a l i n h e r e n t f e a t u r e s o f C I

i o n source d e s i g n p o t e n t i a l l y l i m i t i t s u s e f u l n e s s in Py-MS s t u d i e s .

First, the

42

p r o l o n g e d r e s i d e n c e t i m e i n t h e t i g h t i o n source may cause d r a s t i c changes i n t h e chemical c o m p o s i t i o n o f t h e p y r o l y s a t e .

Secondly, u n l e s s t h e sample i s p y r o l y s e d

s l o w l y and t h e p r o d u c t s a r e d i l u t e d w i t h r e a g e n t gas, p y r o l y s i s compounds formed i n h i g h c o n c e n t r a t i o n s ( e . g . H20, NH3) c o u l d m o d i f y t h e i o n i s a t i o n process by s u b s t i t u t i n g f o r t h e o r i g i n a l r e a g e n t gas ( r e f . 5 4 ) . be determined by sample-dependent f a c t o r s .

Thus t h e i o n i s a t i o n process may then

A l t h o u g h C I s t u d i e s o f multicomponent

hydrocarbon m i x t u r e s have been r e p o r t e d ( r e f . 113), so f a r no l o n g - t e r m r e p r o d u c i b i l i t y s t u d i e s on multicomponent m i x t u r e s c o n t a i n i n g h i g h l y p o l a r compounds appear t o have been c a r r i e d o u t .

U n t i l such d a t a become a v a i l a b l e t h e v a l u e o f C I i n

q u a n t i t a t i v e Py-MS s t u d i e s ( " f i n g e r p r i n t i n g " ) cannot be e s t a b l i s h e d w i t h c e r t a i n t y . As d i s c u s s e d on page 14,

however, C I i s p o t e n t i a l l y a v e r y v a l u a b l e a n c i l l a r y

t e c h n i q u e f o r q u a l i t a t i v e s t u d i e s o f t h e p y r o l y s a t e , u s i n g s e l e c t i v e r e a g e n t gases. I n F I t h e i o n i s a t i o n process i s dependent t o some e x t e n t on t h e c o n d i t i o n o f t h e e m i t t e r s u r f a c e , e s p e c i a l l y w i t h p o l a r compounds.

T h i s i s shown by t h e f r e q u e n t

occurrence o f protonated molecular ions, i n d i c a t i n g the r o l e o f surface i o n i s a t i o n processes i n F I o f t h e s e compounds ( r e f . 114).

Changes i n t h e c o n d i t i o n o f t h e

e m i t t e r s u r f a c e may t h u s change t h e c h a r a c t e r and d i s t r i b u t i o n o f t h e i o n s produced f r o m multicomponent m i x t u r e s c o n t a i n i n g r e l a t i v e l y p o l a r compounds. n o t e x i s t w i t h m i x t u r e s o f a p o l a r substances.

T h i s problem does

I n f a c t , F I has been shown t o be

s u p e r i o r t o low v o l t a g e E I i n t h e q u a n t i t a t i v e a n a l y s i s o f hydrocarbon m i x t u r e s ( r e f . 115).

Except f o r some s e l e c t e d geopolymers and s y n t h e t i c polymers, however, most

complex o r g a n i c m a t e r i a l s w i l l y i e l d p y r o l y s a t e s c o n t a i n i n g v a r y i n g amounts o f p o l a r components.

T h e r e f o r e , F I i s u n l i k e l y t o become a r o u t i n e i o n i s a t i o n t e c h n i q u e i n

f i n g e r p r i n t i n g o f b i o m a t e r i a l s by Py-MS.

However, F I can be a v e r y v a l u a b l e t o o l f o r

d e t e r m i n i n g t h e elemental c o m p o s i t i o n o f i n t a c t m o l e c u l a r i o n s i n p y r o l y s a t e s e s p e c i a l l y i n c o m b i n a 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 o m e t r y ( r e f s . 53, 71, 116). A l t h o u g h l o w - v o l t a g e E I may have t h e drawbacks o f r e s i d u a l f r a g m e n t a t i o n and p o o r s e n s i t i v i t y i n comparison w i t h C I o r F I , t h e i n h e r e n t advantages o f s i m p l i c i t y and a b i l i t y t o o p e r a t e i n a beam mode, t h u s r e d u c i n g c o n t a m i n a t i o n , have made i t t h e method of c h o i c e i n most C u r i e - p o i n t Py-MS s t u d i e s r e p o r t e d so f a r .

Further, i t i s

w o r t h m e n t i o n i n g t h a t t h e problem o f r e s i d u a l f r a g m e n t a t i o n d u r i n g l o w v o l t a g e E I i s t o some e x t e n t compensated f o r by t h e f a c t t h a t p y r o l y s i s p r o d u c t s r e p r e s e n t an unusual c o l l e c t i o n o f r e l a t i v e l y s t a b l e compounds w h i c h o f t e n produce s t a b l e m o l e c u l a r i o n s d u r i n g l o w - v o l t a g e E I ( r e f . 100).

Among t h e more common b i o m a t e r i a l s , l i p i d s are

t h e o n l y c l a s s o f compounds w h i c h c o n s i s t e n t l y prsduce l o w v o l t a g e

EI Py-MS p a t t e r n s

s t r o n g l y dominated by fragment i o n s .

4.5.

MASS ANALYSIS

The c h o i c e o f t h e mass s p e c t r o m e t e r i n Py-MS i s d i c t a t e d by t h e p y r o l y s i s c o n d i t i o n s , t h e a n a l y s i s speed r e q u i r e d , t h e mass range and r e s o l u t i o n d e s i r e d , t h e

43 p r e f e r r e d i o n i s a t i o n mode, t h e c o s t o f t h e i n s t r u m e n t and t h e need f o r computerisation.

F a s t h e a t i n g r a t e s g e n e r a l l y demand e i t h e r i n s t r u m e n t s w i t h simultaneous i o n

detection capability, i.e.

incorporating photoplate o r channelplate detectors, o r f a s t

s c a n n i n g i n s t r u m e n t s such as quadrupoles, t i m e - o f - f l i g h t s p e c t r o m e t e r s o r v o l t a g e scanned magnetic s e c t o r i n s t r u m e n t s .

However, t h e r e q u i r e m e n t s o f h i g h a n a l y s i s speed

and sample t h r o u g h p u t may r u l e o u t t h e use o f p h o t o p l a t e systems. may weigh a g a i n s t t h e use o f p h o t o p l a t e i n s t r u m e n t s . with time-of-flight

instruments, e s p e c i a l l y i f coupled w i t h t h e requirement o f u n i t

r e s o l u t i o n a t h i g h e r mass ranges. q u i r e d , e.g.

Also, c o s t factors

Mass range may become a problem

I f i o n i s a t i o n techniques o t h e r than E I are r e -

F I o r C I , o n l y magnetic s e c t o r i n s t r u m e n t s and quadrupoles come i n t o

F i n a l l y , ease o f c o m p u t e r i s a t i o n p r o v i d e s a s t r o n g argument i n f a v o u r

consideration. o f quadrupoles.

Most o f t h e C u r i e - p o i n t Py-MS work r e p o r t e d has been performed w i t h quadrupole instruments.

The o n l y r e a l disadvantages o f quadrupoles f o r Py-MS s t u d i e s a r e t h e

l i m i t e d i o n t r a n s m i s s i o n a t h i g h e r mass ranges and t h e i n a b i l i t y t o p e r f o r m h i g h r e s o l u t i o n studies.

I f f u r t h e r m a j o r breakthroughs were t o be made i n t h e development

o f F o u r i e r t r a n s f o r m i o n c y c l o t r o n resonance mass s p e c t r o m e t r y f o r r o u t i n e a n a l y t i c a l a p p l i c a t i o n s ( r e f . 117), t h i s t e c h n i q u e m i g h t w e l l become t h e p r e f e r r e d approach f o r Py-MS s t u d i e s .

A l t e r n a t i v e l y , t h e r e c e n t i n t r o d u c t i o n o f commercial m a g n e t i c s e c t o r

i n s t r u m e n t s f e a t u r i n g f a s t v o l t a g e scanning o v e r s e v e r a l mass decades ( r e f . 118) m i g h t a l s o become an a t t r a c t i v e a l t e r n a t i v e t o quadrupole systems, e s p e c i a l l y i f h i g h r e s o l u t i o n v e r s i o n s o f t h e s e magnetic s e c t o r i n s t r u m e n t s c o u l d be developed. When u s i n g quadrupoles i n a C u r i e - p o i n t Py-MS c o n f i g u r a t i o n , as shown i n F i g u r e 18, t y p i c a l o p e r a t i n g c o n d i t i o n s a r e : e l e c t r o n energy 14 eV (open cross-beam i o n i s e r ) , i o n energy 5-10 eV, mass range m/z 16 ( l o w e s t o r g a n i c m o l e c u l a r i o n i s CH4) t o m/z 200, scanning speed 8-10 s p e c t r a / s and t o t a l scanning t i m e 10-30 s. chamber i s heated t o 150-ZOO'C,

Whereas t h e expansion

t h e i o n source i s n o t s p e c i a l l y heated and t h e mass

s p e c t r o m e t e r housing i s u s u a l l y a t room temperature.

The l i q u i d n i t r o g e n - c o o l e d

shroud around t h e i o n source a c t s as a v e r y e f f i c i e n t pump f o r o r g a n i c m o l e c u l e s , w h i c h t r a p s most p y r o l y s i s p r o d u c t s escaping i o n i s a t i o n .

T h i s p r e v e n t s them f r o m r e -

e n t e r i n g t h e i o n source a f t e r m u l t i p l e c o l l i s i o n s w i t h t h e p o o r l y d e f i n e d s u r f a c e s o f t h e vacuum envelope o r q u a d r u p o l e head.

A p a r t f r o m b e i n g a v e r y e f f i c i e n t pump, t h e

l i q u i d n i t r o g e n - c o o l e d screen i s a l s o a pump w i t h a h i g h l y c o n s t a n t performance, m a i n l y d e f i n e d by elementary parameters such as t e m p e r a t u r e and s u r f a c e area.

The

c o n s t a n t pumping c h a r a c t e r i s t i c s o f t h e c o l d screen a r e an i m p o r t a n t f a c t o r i n t h e achievement o f long-tei-m r e p r o d u c i b i l i t y i n t h e a n a l y s i s o f multicomponent samples. The o t h e r s o l u t i o n t o t h i s problem, d i f f u s i o n - l i m i t e d pumping ( r e f . 119), does n o t a c h i e v e t h e same pumping e f f i c i e n c y as t h e c o l d screen by f a r .

To p r e v e n t t h e accum-

u l a t i o n o f e x c e s s i v e amounts o f t r a p p e d p r o d u c t s , t h e screen i s n o t permanently c o o l e d b u t i s a l l o w e d t o h e a t up t o room temperature a t t h e end o f each day.

The m o l e c u l e s

r e l e a s e d d u r i n g t h e h e a t i n g up p e r i o d a r e t h e n pumped o f f by t h e main vacuum pump o f

44

Display Terminal

Q Recorder

Mini- cornp. (32k core rn.)

F i g u r e 20. Schematic diagram o f an automated C u r i e - p o i n t Py-MS system. F o r a d e t a i l e d d e s c r i p t i o n , see r e f e r e n c e 43. Note t h e 3 6 - p o s i t i o n t u r n t a b l e ; t h e 1 i q u i d n i t r o g e n - c o o l e d screen c o m p l e t e l y s u r r o u n d i n g t h e i o n source and t h e h i g h speed i o n c o u n t i n g channei. T y p i c a l t i m e needed t o a n a l y s e one b a t c h o f 36 samples i s 1 hour. t h e system, which may e i t h e r be a d i f f u s i o n pump o r a t u r b o m o l e c u l a r pump.

Finally, t h e c o l d screen, by v i r t u e o f i t s h i g h pumping speed ( a p p r o x i m a t e l y 10 l / s . c m 2 ) p r o v i d e s f o r e x t r e m e l y l o w o r g a n i c background s i g n a l s and f a s t r e c o v e r y o f t h e system

a f t e r each a n a l y s i s . F i g u r e 20 shows a c o m p l e t e l y automated C u r i e - p o i n t Py-MS system. system was b u i l t a t t h e F.O.M.

T h i s automated

I n s t i t u t e f o r Atomic and M o l e c u l a r P h y s i c s i n Amsterdam

( r e f . 43) and has been i n o p e r a t i o n s i n c e 1976.

Manual v e r s i o n s based on t h e p r i n c i -

p l e s shown i n F i g u r e 18 o r f o l l o w i n g a somewhat d i f f e r e n t approach a r e c o m m e r c i a l l y a v a i l a b l e ( r e f . 120).

Removal of t h e expansion chamber and p r e - h e a t i n g o f t h e r e -

a c t i o n t u b e w a l l s r e q u i r e s mass ranges up t o m/z 800 o r h i g h e r i f f u l l use i s t o be made of t h e i n f o r m a t i o n a v a i l a b l e f r o m l a r g e e v a p o r a t e d o r p y r o l y s e d m o l e c u l a r s p e c i e s , e.g.

lipids.

The i n c r e a s e d scanning t i m e p e r spectrum caused b y t h i s l a r g e

mass range has t o be compensated f o r by s l o w e r h e a t i n g r a t e s o f t h e sample o r l o n g e r r e s i d e n c e t i m e s o f t h e p y r o l y s a t e i n t h e expansion chamber, i f used.

45 I O N DETECTION

4.6.

I n p r i n c i p l e , i o n c o u n t i n g has c o n s i d e r a b l e advantages o v e r analogue i o n c u r r e n t monitoring.

The l a r g e bandwidth of p u l s e a m p l i f i e r s used i n i o n c o u n t i n g systems

a l l o w s t h e r e c o r d i n g of e x t r e m e l y small s i g n a l s a t maximum scanning speed o f t h e quadrupole mass s p e c t r o m e t e r . C u r r e n t a m p l i f i e r s , i n c o n t r a s t , have l o w e r bandwidths a t high amplifications,

t h u s l i m i t i n g t h e scanning speed o r s p o i l i n g peak r e s o l u t i o n .

Moreover, t h e use of p u l s e a m p l i f i e r s g r e a t l y f a c i l i t a t e s t h e a u t o m a t i o n o f Py-MS systems s i n c e samplesproducing unexpectedly s m a l l o r l a r g e s i g n a l s do n o t r e q u i r e a change i n a m p l i f i c a t i o n s e t t i n g by t h e o p e r a t o r .

F u r t h e r advantages o f p u l s e

c o u n t i n g systems a r e t h e d i r e c t i n s i g h t i n t o t h e s i g n a l sampling s t a t i s t i c s f o r each peak, t h e l a c k o f immediate s i g n a l r e d u c t i o n w i t h decreases i n m u l t i p l i e r g a i n ( p r o v i d e d t h a t t h e g a i n remains above a c e r t a i n minimum l e v e l ) , and t h e absence o f s i g n a l f l u c t u a t i o n s caused by mechanical v i b r a t i o n s i n m u l t i - s t a g e dynode m u l t i -

A s e r i o u s disadvantage o f i o n c o u n t i n g , Typical maximum c o u n t i n g r a t e s a r e o f t h e o r d e r o f 106 i o n s / s . Assuming a scan r a t e o f 103 amu/s, t h e maximum number o f i o n s a c c u r a t e l y counted f o r any g i v e n mass w i l l be l e s s 3 t h a n 10 / s . I n t h e Py-MS c o n f i g u r a t i o n s shown i n F i g u r e s 18 and 20, t h e p r e s s u r e r i s e i n t h e i o n source a f t e r each p y r o l y s i s l a s t s f o r about 5-10 s. Thus t h e t o t a l maximum number o f i o n s measured p e r peak i s w e l l below 5.10 3 S i n c e a t l e a s t 102 p l i e r s (e.g. caused by mechanical pumps).

however, is t h e l i m i t e d c o u n t i n g r a t e o f c o m m e r c i a l l y a v a i l a b l e systems.

.

i o n s a r e r e q u i r e d f o r a r e a s o n a b l y w e l l d e f i n e d (10% l e v e l ) peak a m p l i t u d e measurement, t h e u s e f u l dynamic range ( r a t i o between s m a l l e s t and l a r g e s t a c c u r a t e l y measured peak i n a spectrum) i s l e s s t h a n 50:1!. F o r t h i s reason, Meuzelaar e t aZ. used a s p e c i a l l y b u i l t high-speed i o n c o u n t i n g 7 system, c a p a b l e o f c o u n t i n g up t o 2.10 i o n s / s ( r e f . 43), t h u s o b t a i n i n g a 2 0 - f o l d improvement i n t h e u s e f u l dynamic range,

C o n s t r u c t i o n of t h e high-speed i o n c o u n t i n g

system r e q u i r e d s p e c i a l p r e c a u t i o n s t o be t a k e n a g a i n s t i n t e r f e r e n c e f r o m o u t s i d e n o i s e s i g n a l s , e.g. amplifier.

t r i a x i a l f e e d t h r o u g h and c a b l e c o n n e c t i o n s f r o m m u l t i p l i e r t o

The m u l t i p l i e r used was a Bendix 4700 c h a n n e l t r o n m u l t i p l i e r .

When

u s i n g amounts o f sample below 20 ug and i o n i z i n g a t 14 eV, o n l y a r e l a t i v e l y s m a l l number o f mass s i g n a l s encountered i n b i o m a t e r i a l s exceeded t h e maximum c o u n t r a t e i n t h i s system. 4.7.

SIGNAL RECORQING

Recording o f p y r o l y s i s mass s p e c t r a o b t a i n e d by quadrupole i n s t r u m e n t s can be done e i t h e r by a s i g n a l a v e r a g e r o r by computer.

S i g n a l averagers a r e o f t e n s u i t a b l e

f o r i n t e r f a c i n g w i t h quadrupole mass s p e c t r o m e t e r s ( r e f . 44).

Most s i g n a l a v e r a g e r s

w i l l s u p p l y a v o l t a g e ramp o u t p u t which can be used t o d i r e c t t h e mass scan o f t h e quadrupole.

A l t e r n a t i v e l y , t h e quadrupole may use i t s i n t e r n a l scan and t h e s i g n a l

a v e r a g e r may f o l l o w t h e quadrupole scan i n a t r i g g e r mode.

Some s i g n a l a v e r a g e r s

a r e even equipped w i t h p u l s e c o u n t i n p u t s a l l o w i n g d i r e c t r e c o r d i n g o f s i g n a l s

46 g e n e r a t e d by i o n c o u n t i n g equipment.

However, as w i t h c o m m e r c i a l l y a v a i l a b l e i o n -

c o u n t i n g equipment, t h e maximum c o u n t r a t e of p u l s e i n p u t s on s i g n a l averagers 6 g e n e r a l l y does n o t exceed 2 - 3.10 p u l s e s / s . The advantages o f spectrum r e c o r d i n g by s i g n a l averagers a r e s i m p l i c i t y of o p e r a t i o n , ease o f d a t a i n s p e c t i o n and h i g h scan speed.

Disadvantages, i n comparison w i t h s i g n a l r e c o r d i n g by computer, a r e l i m i t e d

memory s i z e , u n s u i t a b i l i t y f o r f u r t h e r d a t a p r o c e s s i n g and i n a b i l i t y t o p e r f o r m more complex scanning r o u t i n e s , e.g.

jumping t o s e l e c t e d peaks.

The l i m i t e d memory s i z e

w i l l generally preclude t h e recording o f time-resolved p y r o l y s i s patterns.

To a l l o w

f u r t h e r d a t a p r o c e s s i n g , on- o r o f f - l i n e t r a n s f e r of s p e c t r a l d a t a t o a s u i t a b l e computer system w i l l be necessary. Various computerized quadrupole systems a r e now c o m m e r c i a l l y a v a i l a b l e .

However,

when s e l e c t i n g a computerized system f o r Py-MS a p p l i c a t i o n s , a t t e n t i o n has t o be p a i d t o some s p e c i a l requirements n o t f u l f i l l e d by e v e r y system.

I n f a c t , none o f

t h e p r e s e n t l y a v a i l a b l e mass spectrometer/computer systems i s i d e a l l y s u i t e d f o r PyMS a p p l i c a t i o n s .

F i r s t , t h e computer s h o u l d be r e a d i l y programmable t o p e r f o r m

s i g n a l a v e r a g i n g t a s k s f o r t h e purpose o f o b t a i n i n g a s i n g l e i n t e g r a t e d mass spectrum, e s p e c i a l l y i f t h e a v a i l a b l e memory s i z e does n o t p e r m i t s e q u e n t i a l s t o r a g e o f a l l s p e c t r a f o r l a t e r summation.

Secondly, if t i m e - r e s o l v e d p y r o l y s i s p a t t e r n s a r e t o

be recorded, t h e memory s i z e o f t h e computer s h o u l d be l a r g e enough t o accomodate s e q u e n t i a l l y r e c o r d e d s p e c t r a o r , a l t e r n a t i v e l y , f a s t dumping o f r e c o r d e d s p e c t r a t o d i s k s h o u l d be p o s s i b l e d u r i n g t h e measurements.

T h i r d l y , t h e computer/mass spec-

t r o m e t e r i n t e r f a c e s h o u l d be capable o f h a n d l i n g i o n c o u n t i n g s i g n a l s i n s t e a d o f analogue s i g n a l s .

A t p r e s e n t , t h i s c a p a b i l i t y i s a v a i l a b l e on one o f t h e commercial

Py-MSsystems ( r e f . 120a) b u t o n l y f o r c o u n t r a t e s up t o 2.106 ions/second.

Finally,

t h e computer system s h o u l d be e v a l u a t e d w i t h r e g a r d t o i t s c a p a b i l i t y f o r n u m e r i c a l e v a l u a t i o n o f t h e p y r o l y s i s mass s p e c t r a .

None of t h e c o m m e r c i a l l y a v a i l a b l e mass

spectrometer/computer systems p r o v i d e s s o f t w a r e packages f o r m u l t i v a r i a t e s t a t i s t i c a l a n a l y s i s such as d e s c r i b e d i n Chapter 6.

Therefore, t h e u s e r e i t h e r has t o develop

h i s own s o f t w a r e by t r a n s l a t i n g a v a i l a b l e software packages f r o m l a r g e r computer systems, o r may s i m p l y want t o make an on- o r o f f - l i n e c o n n e c t i o n t o a l a r g e system.

A minimum "minicomputer" c o n f i g u r a t i o n s u i t a b l e f o r d e v e l o p i n g m u l t i v a r i a t e s t a t i s t i c a l a n a l y s i s programs w i l l p r o b a b l y have a t l e a s t 64K c o r e memory, a f l o a t i n g p o i n t processor, two d i s k s ( o r one d i s k and one magnetic t a p e ) , a p r i n t e r and a g r a p h i c d i s p l a y t e r m i n a l w i t h h a r d copy u n i t o r X / Y p l o t t e r .

47

Chapter 5

REPRODUCIBILITY I N CURIE-POINT PYROLYSIS MASS SPECTROMETRY

QUALITATIVE REPRODUCIBILITY A high degree of qualitative reproducibility, that is the continuous presence of the same features in analysis patterns obtained from the same sample, is a prime requirement for any fingerprinting technique (ref. 107). In our experience, Curiepoint Py-MS techniques show excellent qualitative reproducibility for all kinds of complex biomaterials analysed so far. Similar conclusions were drawn by Hickman and Jane from a comparative study of three different Py-#S techniques in the analysis of various polymeric materials (ref. 121). Considering the large number of different, possibly even competing, pyrolysis reactions that occur in highly complex biomaterials, the stability of the pyrolysis patterns of such materials, a s also observed in Py-GC (ref. 122), is an intriguing phenomenon. In fact, many deliberate changes in pyrolysis conditions, e.g. a 15-fold increase (from 0.1 -1.5 s ) in tT (refs. 100, 101), fail to change the qualitative composition of the pyrolysate, provided that the occurrence of secondary reactions is minimized and T is high enough to ensure eq complete pyrolysis within tT. Marked qualitative changes in pyrograms, e.g. due to formation of recombination products, may occur, however, when samples become excessively large, or when pyrolysis is carried out inside a capillary tube rather than on the surface of a filament. Drastic changes in pyrograms can also be expectec to occur if the selected T i s s o low that the half-lives of degradation reactions eq become longer than tT, allowing isothermal pyrolysis processes to take place at, or close to, T of the filament. Windig e t aZ. (ref. l o g ) , who applied factor analysis eq (see Section 6.6.2) to the qualitative evaluation of the influence of changes in a number of pyrolytic and mass spectrometric conditions, demonstrated that an increase in T (358 - 770°C) for polysaccharide materials results in a stronger degradation eq of the compounds to small, less characteristic fragment molecules, whereas the effect for a typical protein is completely different, viz. the formation of more strongly dehydrogenated pyrolysis products. Since qua1 itative changes in the pyrolysates are brought about only by such gross, deliberate changes in pyrolysis conditions, such changes hardly ever pose a significant problem under controlled analytical conditions. A problem with regard to qualitative reproducibility may be encountered when a high instrument background is present due to insufficient trapping efficiency of the liquid nitrogen-cooled screen or unusually heavy contamination of the expansion 5.1.

48 chamber o r i o n source.

However, C u r i e - p o i n t Py-MS systems such as t h a t shown i n

F i g u r e 20 a r e s p e c i f i c a l l y designed t o e l i m i n a t e such problems as r i g o r o u s l y as possible.

N a t u r a l l y , even a l o w i n s t r u m e n t a l background may s t r o n g l y i n f l u e n c e t h e

p y r o l y s i s p a t t e r n s i f t h e amount o f sample p y r o l y s e d i s t o o low t o p r o v i d e an adequate signal-to-background r a t i o .

For t h e t y p e o f i n s t r u m e n t shown i n F i g u r e s 18 and 20,

t h e minimum sample s i z e f o r o b t a i n i n g s a t i s f a c t o r y s i g n a l - t o - b a c k g r o u n d r a t i o s i s a p p r o x i m a t e l y 1 microgram f o r substances y i e l d i n g complex p y r o l y s a t e s , e.g.

bio-

polymers (see F i g u r e 21), as compared w i t h 10 ng o r l e s s f o r compounds which y i e l d a s i n g l e dominant component, e.g.

polystyrene (see Figure 1).

Some t y p e s o f sample

t e n d t o g i v e low s i g n a l y i e l d s , e s p e c i a l l y samples c o n t a i n i n g r e l a t i v e l y l a r g e amounts o f i n o r g a n i c c o n s t i t u e n t s , e.g. whole s o i l samples, compounds f o r m i n g l a r g e

v)

z

20

10

20

40

60

80

100

120

140

160

F i g u r e 21. P y r o l y s i s mass s p e c t r a o f ( a ) background produced by h e a t i n g a c l e a n w i r e ( b j and ( c ) 1 and 5 ug, r e s p e c t i v e l y , o f a galacto-gluco-glucurono-araban. Note t h a t t h e spectrum o f t h e 1 ug sample s t i l l shows an o v e r a l l s i g n a l - t o - b a c k g r o u n d r a t i o o f b e t t e r t h a n 1 O : l . F u r t h e r n o t e t h a t t h e i n t e n s i t y o f t h e 5 Ug spectrum i s o n l y about 4 t i m e s t h a t o f t h e 1 ug spectrum, i n d i c a t i n g p o s s i b l e i n a c c u r a c y o f a c t u a l sample s i z e .

49

amounts o f c h a r , e.g.

condensed p o l y n u c l e a r s t r u c t u r e s such as a r e found i n h i g h

r a n k c o a l s , o r compounds e v a p o r a t i n g i n t a c t and condensing on t h e r e l a t i v e l y c o l d r e a c t i o n t u b e w a l l , such as c h o l e s t e r o l and o t h e r r e l a t i v e l y v o l a t i l e compounds. W i t h such samples, l o w s i g n a l - t o - b a c k g r o u n d r a t i o s may be o b t a i n e d i n s p i t e o f a seemingly adequate sample s i z e . 5.2.

QUANTITATIVE REPRODUCIBILITY

Most p y r o l y s i s mass s p e c t r o m e t r y a p p l i c a t i o n s a l s o r e q u i r e a d e f i n i t e degree o f q u a n t i t a t i v e r e p r o d u c i b i l i t y , t h a t i s t h e c o n t i n u o u s presence o f f e a t u r e s w i t h s i m i l a r r e l a t i v e i n t e n s i t i e s i n a n a l y s i s p a t t e r n s o b t a i n e d f r o m t h e same sample. O b v i o u s l y , s h o r t - t e r m q u a n t i t a t i v e r e p r o d u c i b i l i t y , b e t t e r termed " r e p e a t a b i l i t y " , much more r e a d i l y achieved t h a n l o n g - t e r m q u a n t i t a t i v e r e p r o d u c i b i l i t y .

is

Typical

f i g u r e s f o r s h o r t - t e r m ( l e s s t h a n 1 day) and l o n g - t e r m (more t h a n 1 month) r e p r o d u c i b i l i t y as determined i n C u r i e - p o i n t Py-MS s t u d i e s on s e l e c t e d biopolymers ( r e f . 100) a r e i n t h e 1-3% and 8-11% ranges (average d i f f e r e n c e observed i n 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 40 most i n t e n s e peaks), r e s p e c t i v e l y . l o n g - t e r m r e p r o d u c i b i 1it y observed f o r glycogen.

F i g u r e 22 shows t h e l e v e l o f

The above-mentioned l e v e l s o f

q u a n t i t a t i v e r e p r o d u c i b i l i t y can be achieved o n l y a f t e r some b a s i c c o n s i d e r a t i o n s r e g a r d i n g i o n s i g n a l s t a t i s t i c s and dynamic range o f t h e a m p l i f i e r a r e c a r e f u l l y observed.

F o r example, an average r e p r o d u c i b i l i t y l e v e l o f +.5% c a n n o t be o b t a i n e d i f '

t h e average peak s i g n a l i n t h e mass s p e c t r a i s produced by l e s s t h a n 400 i o n s ( f o r 400 d e t e c t e d i o n s t h e u n c e r t a i n t y i n t h e s i g n a l magnitude measured i s r o u g h l y

+. 400"2

= +. 20, o r i 5%).

On t h e o t h e r hand, i f t h e s i g n a l s become t o o l a r g e , b o t h

p u l s e and analogue a m p l i f i e r s may s u f f e r s i g n a l l o s s , l e a d i n g t o a n o n - l i n e a r response.

T h i s may a d v e r s e l y a f f e c t t h e q u a n t i t a t i v e r e p r o d u c i b i l i t y , e s p e c i a l l y i f

t h e sample s i z e s v a r y c o n s i d e r a b l y between d u p l i c a t e analyses.

F o r a more compre-

h e n s i v e d i s c u s s i o n o f dynamic range problems i n p u l s e a m p l i f i e r s , t h e r e a d e r i s r e f e r r e d t o S e c t i o n 4.6.

.

I n v i e w o f t h e i n h e r e n t v a r i a b i l i t y o f many samples o f b i o l o g i c a l o r i g i n , a 5 -

10% l e v e l o f i n s t r u m e n t i r r e p r o d u c i b i l i t y i s s a t i s f a c t o r y f o r most Py-MS a p p l i c a t i o n s . F u r t h e r , some a p p l i c a t i o n s do n o t r e q u i r e a h i g h degree o f l o n g - t e r m r e p r o d u c i b i l i t y s i n c e r e f e r e n c e samples a r e co-analysed w i t h t h e unknown samples, as d i s c u s s e d i n S e c t i o n 2.1 ( " o p e r a t i o n a l f i n g e r p r i n t i n g " ) .

T y p i c a l examples o f o p e r a t i o n a l f i n g e r -

p r i n t i n g , where t h e need f o r l i b r a r y s p e c t r a i s o b v i a t e d , a r e p r o v i d e d by s t u d i e s on t h e subspecies i d e n t i f i c a t i o n o f m y c o b a c t e r i a r e p o r t e d b y Wieten et a Z . ( r e f s .

123

-

125), as w e l l as by t h e analyses o f t i s s u e and body f l u i d samples d e s c r i b e d i n Chapters 6 and 7, r e s p e c t i v e l y .

However, even i f n o t r e q u i r e d i n some a p p l i c a t i o n s ,

a h i g h l e v e l o f l o n g - t e r m q u a n t i t a t i v e r e p r o d u c i b i l i t y i s an i n d i s p e n s a b l e i n g r e d i e n t f o r a c h i e v i n g any degree o f i n t e r - l a b o r a t o r y r e p r o d u c i b i l i t y .

50 A

GLYCOGEN

I6O

Po

8

AFTER 26 DAYS

C

AFTER 3 4 DAYS

20

40

60

80

100

120

140

160

mn F i g u r e 22. Long-term r e p r o d u c i b i l i t y o f glycogen p y r o l y s i s mass s p e c t r a . C o n d i t i o n s : sample 5 pg; Tc 610°C; t T 0.1 s ; t c 0 . 9 s; i n l e t t e m p e r a t u r e 150OC; Eel 14 eV. ( a ) Glycogen, suspended i n methanol; ( b ) t h e same suspension, analysed a f t e r 26 days o f s t o r a g e a t -20°C; ( c ) f r e s h suspension o f glycogen p r e p a r e d froni t h e same batch, a n a l y s e d 34 days a f t e r t h e a n a l y s i s o f ( a ) . 5.3.

FACTORS INFLUENCING LONG-TERM REPRODUCIBILITY

F a c t o r s i n f l u e n c i n g l o n g - t e r m r e p r o d u c i b i l i t y i n C u r i e - p o i n t Py-MS have been s t u d i e d i n d e t a i l by Meuzelaar ( r e f . 100) and Windig e t aZ. ( r e f . 101).

Table 2

l i s t s p o s s i b l e f a c t o r s i n f l u e n c i n g v a r i o u s s t e p s o f t h e a n a l y t i c a l p r o c e d u r e i n Py-MS.

A d e t a i l e d e x a m i n a t i o n o f f a c t o r s i n v o l v e d i n sample p r e p a r a t i o n and p y r o l y s i s steps showed t h a t some d e l i b e r a t e changes i n e x p e r i m e n t a l c o n d i t i o n s caused changes i n average peak h e i g h t ( 4 0 most i n t e n s e peaks) o f o v e r 20%, as l i s t e d i n T a b l e 3.

In

p r a c t i c e , however, t h e e x p e r i m e n t a l parameters i n v o l v e d a r e c o n t r o l l e d much more closely.

T h e r e f o r e , t h e e s t i m a t e d combined c o n t r i b u t i o n o f t h e s e f a c t o r s t o l o n g -

t e r m v a r i a t i o n s observed i n t h e s p e c t r a i s l e s s t h a n 3

-

4%. T h i s 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 m a j o r causes o f i m p e r f e c t l o n g - t e r m r e p r o d u c i b i l i t y must be sought i n f a c t o r s i n f l u e n c i n g sample t r a n s f e r and/or mass s p e c t r o m e t r i c a n a l y s i s r a t h e r t h a n i n f a c t o r s i n f l u e n c i n g sample p r e p a r a t i o n and p y r o l y s i s .

51 TABLE 2 Factors possibly influencing long-term r e p r o d u c i b i l i t y Sampl e Preparation

F i 1ament Heating

Product Transfer

Mass Analysis

cleaning o f sample c a r r i e r

temperature r i s e time

inlet temperature

ionisation

solvent/suspending liquid

equilibrium temperature

residence time

extraction

sample s i z e

t o t a l heating time

surface activity

transmission

TABLE 3 I n f l u e n c e o f sample p r e p a r a t i o n and p y r o l y s i s parameters on pyrogram v a r i a b i l i t y o f b o v i n e serum albumin and glycogen

Average d i f f e r e n c e i n 40 most i n t e n s e peaks ( X )

Parameter V a r i e d

A1 bumi n

Glycogen

a . W i r e c l e a n i n g method ( 3 techniques )*

6.0 - 17.4

14.8

-

22.7

b . Suspending l i q u i d (H20, MeOH, CS2)

7.7

-

15.4

72.0

-

22.9

c . Sample s i z e ( 5 , 10, 20

5.7

-

8.6

5.2

-

12.8

7.1

-

9.1

5.2

-

10.3

?a)

d. Temperature r i s e t i m e (0.1, 0.5, 1.0 s )

e. E q u i l i b r i u m t e m p e r a t u r e (510, 610°C) f. Total heating time (0.3, 0.6, 1.2 s )

Estimated c o n t r i b u t i o n o f a - f t o long-term v a r i a b i l i t y

12.0 3.2

-

14.9 4.5

3.2

-

6.5

2 - 4

2 - 3

Actual short-term v a r i a b i l i t y (. 1 day)

0.9

2.4

1.0

-

2.9

Actual long-term v a r i a b i l i t y ( 3 4 days)

9.9 - 11 .o

8.3

-

9.7

*

-

C l e a n i n g t e c h n i q u e s used: i n d u c t i v e h e a t i n g , s o l v e n t c l e a n i n g , and h e a t i n g i n a r e d u c i n g Bunsen b u r n e r f l a m e ( r e f s , 130, 1 0 1 ) .

52 E l e c t r o n energy i n p a r t i c u l a r proved t o b e a h i g h l y c r i t i c a l parameter.

Changing

t h e e l e c t r o n energy s e t t i n g s f r o m 14.0 t o 14.1 eV caused more t h a n a 10% d i f f e r e n c e

i n average r e l a t i v e peak i n t e n s i t i e s .

Since actual e l e c t r o n impact energies a r e n o t

r e a d i l y d e f i n e d t o w i t h i n a few t e n t h s o f an e l e c t r o n v o l t and changes o f t h a t magnitude m i g h t e a s i l y be caused by c o n t a m i n a t i o n o f t h e i o n s o u r c e o r by e l e c t r o n i c i n s t a b i l i t i e s d u r i n g p y r o l y s i s , f l u c t u a t i o n i n e l e c t r o n energy may be a m a j o r f a c t o r l i m i t i n g long-term r e p r o d u c i b i l i t y .

O t h e r p o s s i b l e troublesome f a c t o r s a r e a d s o r p t i o n

phenomena on t h e w a l l s o f r e a c t i o n t u b e and expansion chamber, as w e l l as changes i n i o n e n e r g i e s and mass r e s o l u t i o n by c o n t a m i n a t i o n o f i o n source and quadrupole r o d s . C o n t r o l l e d h e a t i n g o f t h e i o n source c o u l d reduce t h e s e problems.

I n view o f a l l

t h e s e p o s s i b l e o r a c t u a l sources o f v a r i a t i o n , t h e degree o f i n t e r - l a b o r a t o r y r e p r o d u c i b i l i t y a c h i e v e d w i t h i n s t r u m e n t s showing 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 i o n - s o u r c e and quadrupole d e s i g n i s remarkable, as w i l l b e d i s c u s s e d i n t h e f o l l o w i n g paragraphs. 5.4.

PROSPECTS FOR INTER-LABORATORY REPRODUCIBILITY

As y e t , no s y s t e m a t i c s t u d y has been undertaken t o d e t e r m i n e t h e l e v e l o f i n t e r l a b o r a t o r y r e p r o d u c i b i l i t y o b t a i n a b l e w i t h C u r i e - p o i n t Py-PIS.

Preliminary data,

however, suggest t h a t t h e p r o s p e c t s f o r i n t e r - l a b o r a t o r y r e p r o d u c i b i 1 i t y between i n s t r u m e n t s o f t h e same b a s i c d e s i g n as shown i n F i g u r e 18 a r e p r o m i s i n g .

Figures

23 and 24 show s p e c t r a o f Douglas f i r wood and urban p a r t i c u l a t e (NBS, SRM 1648), r e s p e c t i v e l y , o b t a i n e d on d i f f e r e n t Py-MS i n s t r u m e n t s i n two d i f f e r e n t l a b o r a t o r i e s .

I n s p i t e o f a s i m i l a r b a s i c design, t h e two i n s t r u m e n t s , a f u l l y automated system b u i l t a t t h e F.O.M.

I n s t i t u t e f o r Atomic and M o l e c u l a r P h y s i c s i n Amsterdam (see

F i g u r e 20) around a R i b e r QM17 mass f i l t e r , and a Py-MS system a t t h e B i o m a t e r i a l s P r o f i l i n g Center, U n i v e r s i t y o f Utah, S a l t Lake City, b u i l t by E x t r a n u c l e a r Labs. I n c . ( P i t t s b u r g h ) around a Spectre1 275 quadrupole mass f i l t e r , were s i g n i f i c a n t l y d i f f e r e n t w i t h r e g a r d t o t h e dimensions o f t h e expansion chamber, i o n s o u r c e and mass f i l t e r .

A l s o t h e r e were n o t a b l e d i f f e r e n c e s i n t h e o p e r a t i n g c o n d i t i o n s o f t h e

p y r o l y s e r , i o n s o u r c e and quadrupole mass f i l t e r . were o b t a i n e d f r o m d i f f e r e n t batches.

Moreover, t h e Douglas fir samples

Hence, i n many ways t h e s p e c t r a i n F i g u r e s 23

and 24 p r e s e n t " w o r s t case" examples o f i n t e r - i n s t r u m e n t a l and i n t e r - l a b o r a t o r y r e p r o d u c i b i l i t y and t h e presence o f some obvious d i f f e r e n c e s i n t h e s e s p e c t r a i s therefore not surprising.

C a r e f u l examination, however, r e v e a l s t h e most s e v e r e

d i f f e r e n c e s t o be a t t r i b u t a b l e t o d i f f e r e n t i o n t r a n s m i s s i o n e f f i c i e n c i e s i n t h e low and h i g h mass ranges o f t h e s p e c t r a . Note e s p e c i a l l y t h e h i g h t r a n s m i s s i v i t y i n t h e h i g h mass range shown b y t h e E x t r a n u c l e a r Labs. system, which employs a quadrupole r o d system o f l a r g e r dimensions t h a n t h e F.O.M.

system.

I t s h o u l d be p o i n t e d o u t t h a t o p t i m a l correspondence between s p e c t r a o b t a i n e d on

d i f f e r e n t quadrupole systems can be achieved o n l y by t r i a l and e r r o r , s i n c e i n s t r u ment s e t t i n g s such as

" e l e c t r o n energy" and " i o n energy" p r o v i d e o n l y a r o u g h

53

A

z

e

d -1

F

9

B

s 9

6

3

20

40

60

80

100

120

m/z

140

160

Figure 23. Comparison of pyrolysis mass spectra of Douglas fir wood obtained on different instruments: (a) Automated Py-MS apparatus; F.O.M. Institute, Amsterdam; (b) Extranuclear Labs. Py-MS instrument, Biomaterials Profiling Center, University of Utah. Difference in peak intensities in the low mass range of the spectra probably represent residual solvents (water, methanol) and/or different ion transmissivities of the quadrupole filters. Conditions: sample 10 p g ; Tc 510°C; Eel (a) 14 eV and (b) 1 1 eV. indication of the actual electron and ion energies involved. The spectra in Figures 23 and 24 were obtained with minimal effort to obtain optimum correspondence between the systems and thus do not represent the ultimate degree of inter-laboratory reproducibility obtainable. Since differences in relative ion transmission are a widely recognized problem in quadrupole instruments, magnetic instruments would appear to offer considerable advantages with regard to inter-laboratory reproducibility in Py-MS. Inherent disadvantages, however, such as slower scanning rates, lower sensitivity in low and medium mass ranges and higher vulnerability to ion source contamination problems,

I/I

6-

A

4-

>

2-

t m z Iw

z

z

B

20

40

60

80

100

120

m/z

140

160

F i g u r e 24. Comparison o f p y r o l y s i s mass s p e c t r a o f urban p a r t i c u l a t e (NBS, SRM 1648) o b t a i n e d on d i f f e r e n t i n s t r u m e n t . ( a ) Automated Py-MS apparatus, F.O.M. I n s t i t u t e , Amsterdam; ( b ) E x t r a n u c l e a r Labs, Py-MS i n s t r u m e n t , B i o m a t e r i a l s P r o f i l i n g Center, U n i v e r s i t y o f Utah. F o r d i f f e r e n c e s i n t h e low mass range, see F i g u r e 23. Condit i o n s : sample 10 ug; Tc 510°C; E e l ( a ) 14 eV and ( b ) 11 eV. have s o f a r p r e v e n t e d t h e development o f C u r i e - p o i n t Py-MS systems based on magnetic instruments f o r r o u t i n e a n a l y t i c a l applications.

F o r t u n a t e l y , modern computer

t e c h n i q u e s a l l o w some degree o f compensation f o r t r a n s m i s s i v i t y d i f f e r e n c e s between quadrupoles.

A s i m p l e way o f c o r r e c t i n g f o r t r a n s m i s s i v i t y d i f f e r e n c e s i s t o d i v i d e

t h e s p e c t r a i n t o s e c t i o n s o f 14 mass u n i t s and t o make comparisons between peak i n t e n s i t i e s w i t h i n these s e c t i o n s o n l y .

Since such c o r r e c t i o n methods a r e f a r f r o m

p e r f e c t , however, t h e l e v e l of i n t e r - l a b o r a t o r y 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 s h o u l d n o t be expected t o a l l o w d i r e c t comparison o f m i n u t e q u a n t i t a t i v e d i f f e r e n c e s ir, f i r , g e r p r i n t s w i t h i n the foreseeable f u t u r e .

Rather, t h e degree o f i n t e r - l a b o r a t o r y

r e p r o d u c i b i l i t y o b t a i n a b l e s h o u l d enable r e s e a r c h workers t o exchange d a t a r e g a r d i n g t h e presence and i n t e r p r e t a t i o n o f c h a r a c t e r i s t i c s i g n a l s i n d i f f e r e n t t y p e s o f m a t e r i a l s b e i n g analysed.

55

Chapter 6

DATA ANALYSIS PROCEDURES

6.1.

AVAILABLE COMPUTER PROGRAMS

E v a l u a t i o n of p y r o l y s i s mass s p e c t r a may e i t h e r be p u r e l y q u a n t i t a t i v e , u s i n g t h e s p e c t r a as f i n g e r p r i n t s o n l y , o r may i n v o l v e some degree o f q u a l i t a t i v e i n t e r p r e t a t i o n as t o t h e b i o c h e m i c a l n a t u r e , c o m p o s i t i o n o r s t r u c t u r e o f t h e sample.

Although i n

s e l e c t e d cases d i r e c t v i s u a l e v a l u a t i o n o f q u a l i t a t i v e aspects by an e x p e r i e n c e d o p e r a t o r i s p o s s i b l e , c o m p u t e r - a s s i s t e d e v a l u a t i o n t e c h n i q u e s have n o t o n l y g r e a t l y r e f i n e d q u a n t i t a t i v e comparisons between t h e s p e c t r a b u t a r e a l s o becoming i n d i s p e n s i b l e t o o l s f o r q u a l i t a t i v e i n t e r p r e t a t i o n , as shown by Burgard e t uZ. ( r e f s . 126, 127) i n t h e i n t e r p r e t a t i o n o f d i r e c t probe Py-FIS s p e c t r a f r o m n u c l e i c a c i d s . Computer-assisted t e c h n i q u e s used for t h e e v a l u a t i o n o f p y r o l y s i s mass s p e c t r a i n c l u d e data pre-processing techniques, u n i v a r i a t e s t a t i s t i c a l analysis techniques, m u l t i v a r i a t e s t a t i s t i c a l a n a l y s i s t e c h n i q u e s , f e a t u r e s e l e c t i o n and d a t a r e d u c t i o n t e c h n i q u e s , and d i s p l a y t e c h n i q u e s .

Nost o f t h e s e procedures a r e i n c l u d e d i n t h e

s p e c i a l Py-MS s o f t w a r e package developed by Eshuis e t uZ. ( r e f . 45) a t t h e F.O.M. I n s t i t u t e f o r Atomic and M o l e c u l a r Physics, Amsterdam, and a r e a v a i l a b l e t o i n t e r ested s c i e n t i s t s .

P r i n c i p a l component, d i s c r i m i n a n t and f a c t o r a n a l y s i s procedures

a r e a v a i l a b l e i n t h e w i d e l y used S t a t i s t i c a l Package f o r t h e S o c i a l Sciences (SPSS) ( r e f . 128) and i n t h e BMDP package ( r e f . 1 2 9 ) .

Other useful, widely a v a i l a b l e

m u l t i v a r i a t e s t a t i s t i c a l a n a l y s i s programs a r e t h e ARTHUR program package developed b y Kowalski ( r e f . 130) and t h e CLUSTAN package ( r e f . 131).

The l a t t e r programs a r e

s p e c i f i c a l l y o r i e n t e d towards t h e c l u s t e r a n a l y s i s approach i n c l a s s i f i c a t i o n problems. A f u l l d i s c u s s i o n o f computer a n a l y s i s t e c h n i q u e s s u i t a b l e f o r p y r o l y s i s mass s p e c t r a i s beyond t h e scope o f t h i s Chapter.

T h e r e f o r e , we s h a l l d i s c u s s o n l y t h e

most e s s e n t i a l d a t a p r o c e s s i n g s t e p s when a p p l y i n g t h e F.0.M program package t o a s e l e c t e d m e d i c a l problem, namely t h e comparative a n a l y s i s o f muscle b i o p s y samples from p a t i e n t s w i t h Duchenne muscular d y s t r o p h y (DMD) as w e l l as f r o m n o n - d y s t r o p h i c c o n t r o l s . T a b l e 4 shows t h e c l i n i c a l d a t a f o r b i o p s i e d muscle t i s s u e samples f r o m t h r e e DMD p a t i e n t s and t h r e e n o n - d y s t r o p h i c c o n t r o l s . I t s h o u l d be p o i n t e d o u t t h a t DMD i s an i n h e r i t e d , r e l e n t l e s s l y p r o g r e s s i v e d i s e a s e .

DMD, which o n l y m a n i f e s t s

i t s e l f c l i n i c a l l y i n boys, i s u s u a l l y diagnosed a t t h e age o f f o u r o r f i v e and l e a d s t o t e r m i n a l i l l n e s s a t t h e age o f t h i r t e e n o r f o u r t e e n . T h e r e f o r e i n c r e a s e d i n v o l v e ment was n o t e d f o r t h e 5, 7 and 8 y e a r o l d boys ( p a t i e n t s D , E and F ) .

Preparation

56

TABLE 4 Clinical data f o r t h e muscle biopsy samples from dystrophic p a t i e n t s and controls Patient

Age

Sex

Diagnosis

Muscle Type

A

26 2 36

M

M

control control control dystrophic dystrophic dystrophic

biceps abdomi ni s vastus (med. ) gastroc (med.) biceps biceps

B

C D E F

5

F M

7 8

M M

of t h e muscle t i s s u e samples f o r Py-MS a n a l y s i s subsequently involved freeze-drying, grinding t o a f i n e powder and suspending i n methanol.

DATA PRE-PROCESSING Each of t h e s i x muscle t i s s u e samples was analysed in quadruplicate, thus genera t i n g 24 spectra in t o t a l . Representative spectra obtained by pyrolysing 10 p g of muscle t i s s u e on filaments with a Curie-point temperature of 610°C and using electron impact i o n i s a t i o n a t 14 eV electron energy a r e shown i n Figure 25 f o r control C and p a t i e n t F. Visual examination of the spectra shows obvious s i m i l a r i t i e s (note t h e characteri s t i c ion c l u s t e r a t m/z 91 t o m/z 100) and differences (compare m/z 67) between the two spectra. The f i r s t question t o be addressed i s whether these differences a r e c h a r a c t e r i s t i c of t h e samples o r due t o variations introduced by t h e analytical procedure. Assuming t h a t these differences indeed prove t o be c h a r a c t e r i s t i c of the samples, t h e next question then becomes whether these differences in composition are c o r r e l a t e d with t h e involvement in the disease or a r e due t o i n t r a - and i n t e r individual v a r i a t i o n s in the samples. Before such questions can be addressed by making numerical comparisons between t h e s p e c t r a , a l l ion i n t e n s i t y measurements have t o be properly scaled. Two types of scaling operations a r e normally performed, namely pattern (= spectrum) s c a l i n g , more often referred t o as "normalisation", and f e a t u r e ( = mass peak) scaling. Both types of scaling will be discussed in some d e t a i l since previous t e x t s have paid l i t t l e a t t e n t i o n t o these procedures, whicti a r e highly crucial t o the outcome of t h e numerical evaluation procedure. 6.2.

6.2.1. Pattern s c a l i n g Pattern scaling (normalisation) i s performed t o compensate f o r variations i n the overall ion i n t e n s i t y caused by phenomena n o t relevant t o the analytical problem, such as differences in sample s i z e or changes in instrument s e n s i t i v i t y . The most d i r e c t , often s a t i s f a c t o r y way t o normalise mass spectra f o r numerical purposes i s t o express peak heights as percentage t o t a l ion i n t e n s i t y . This procedure works

57 8 CONTROL (C)

6

4

> c ln

2

z

z

0 PATIENT ( F )

83

20

'

40

'

60

80

100

120mlz 140

160

Figure 25. Pyro l y s i s mass spectra o f muscle t i s s u e from a control subject (C) and a dystrophic pat i e n t ( F ) . For c l i n i c a l data see Table 4. Conditions: sample 10 u g ; Tc G1O"C; Eel 1 4 eV. Arrows i n d i c a t e increased i n t e n s i t i e s as compared t o t h e o t h e r spectrum. b e t t e r a s t h e number of peaks increases and as the v a r i a t i o n in individual peak heights decreases. The main problem in using t h i s procedure i s t h e occurrence of very l a r g e peaks, e s p e c i a l l y when these peaks e x h i b i t a high degree of i n t r a - and/or inter-sample deviation. I f such a peak happens t o be unusually high in a given spectrum, then a l l other peaks w i l l be given low r e l a t i v e i n t e n s i t y values, which may considerably confuse f u r t h e r q u a n t i t a t i v e and q u a l i t a t i v e comparisons between t h e spectra. A simple solution t o t h i s problem i s t o exempt a l l peaks l a r g e r t h a n a c e r t a i n percentage of t o t a l signal i n t e n s i t y , in one or more o f t h e s p e c t r a compared, from t h e normalisation procedure. Once t h e remaining peaks have been normalised t o 100% o f t h e residual t o t a l ion i n t e n s i t y , the i n i t i a l l y omitted peaks can be scaled accordingly, thus bringing t h e sum of the r e l a t i v e i o n i n t e n s i t i e s t o a value g r e a t e r than 100%. The obvious shortcoming o f t h i s procedure i s , however, t h a t elimination of t h e l a r g e s t peaks i s a t best a very rough way o f eliminating potential sources of strong v a r i a t i o n . Some l a r g e peaks may be very s t a b l e and c o n t r i b u t e l i t t l e t o intra-sample ( i n n e r ) or inter-sample ( o u t e r ) d e v i a t i o n , whereas a r e l a t i v e l y small peak may be responsible f o r most o f t h e t o t a l (pooled) deviation in t h e system. An

58

alternative

procedure would t h e r e f o r e be t o exempt a l l peaks e x h i b i t i n g more than a

c e r t a i n amount of inner and/or outer deviation, depending on t h e a n a l y t i c a l problem. However, t h i s requires a knowledge of t h e s e deviations, which can be calculated accurately only a f t e r adequate normalisation. Therefore, t h i s approach requires i t e r a t i v e c a l c u l a t i o n of normalisation c o e f f i c i e n t s and variances while removing peaks with high deviation values u n t i l no more peaks can be found with deviation values above a c e r t a i n l e v e l . Apart from being demanding on computer time, t h i s i t e r a t i v e procedure needs t o be used j u d i c i o u s l y when high l e v e l s of inner o r outer deviation e x i s t in t h e d a t a , otherwise a l l b u t a few small peaks might be removed, and t h e normalisation might be based on poor signal s t a t i s t i c s . For t h e muscle biopsy data a compromise solution was adopted. Preliminary pattern s c a l i n g was performed on t h e basis of percentage ion i n t e n s i t y using a l l peaks. Subsequently, inner and outer deviation values were calculated f o r a l l peaks. Those peaks showing more than 5% r e l a t i v e ion i n t e n s i t y and/or an inner o r o u t e r deviation l a r g e r than 1% of t o t a l deviation were removed from t h e second and f i n a l pattern scaling operation. This r e s u l t e d i n t h e temporary removal of m/z 1 7 , 34, 43, and 67. I t should be noted t h a t t h e choice of t h e normalisation procedure and of most of t h e following numerical procedures depends l a r g e l y on t h e o p e r a t o r ' s a n a l y t i c a l philosophy. I f , f o r example, t h e pyrolysis mass spectra of polystyrene and polytetrafluoroethylene, which show peaks only a t m/z 104 and 100, r e s p e c t i v e l y , ( s e e Figure 1 ) a r e t o be compared numerically, then t h e i t e r a t i v e normalisation procedure will f a i l completely as both peaks a r e removed and no group of s t a b l e peaks common t o both s p e c t r a i s l e f t t o provide an i n t e r n a l reference f o r pattern s c a l i n g . Even i f t h i s problem i s ignored by using sample weight o r t o t a l ion c u r r e n t a s a normalisation reference, f u r t h e r problems may be encountered when a c t u a l l y comparing the spectra numerically, depending on t h e type of s i m i l a r i t y c o e f f i c i e n t used. In general i t can be s t a t e d t h a t t h e more s i m i l a r t h e s p e c t r a a r e t h a t a r e being compared, the more straightforward a numerical comparison i s l i k e l y t o be. The more d i s s i m i l a r t h e s p e c t r a , t h e g r e a t e r i s t h e d i f f i c u l t y in deciding which numerical procedures t o use. 6.2.2. Feature s c a l i n q Feature s c a l i n g , i . e . scaling t h e r e l a t i v e i n t e n s i t y values of d i f f e r e n t mass peaks, i s performed t o compensate f o r d i f f e r e n c e s i n r e l a t i v e peak i n t e n s i t i e s caused by phenomena not relevant t o t h e a n a l y t i c a l problem. For example, when t r y i n g t o d i f f e r e n t i a t e muscle biopsy samples by comparing pyrolysis mass s p e c t r a ,

the l a r g e s t peaks do not necessarily contain t h e most information r e l e v a n t t o t h e problem. However, i f no f e a t u r e scaling i s performed these l a r g e peak values will dominate t h e numerical comparison between spectra,wjth most types of algorithms.

59 Because i t i s p r e f e r a b l e t o base a c l a s s i f i c a t i o n o r d i f f e r e n t i a t i o n o f muscle biopsy samples on t h e most r e l i a b l e and c h a r a c t e r i s t i c f e a t u r e s r a t h e r than on t h e l a r g e s t , some form o f f e a t u r e s c a l i n g i s i n d i c a t e d .

Since m u l t i p l e analyses o f each

sample a r e made, intra-sample standard d e v i a t i o n s f o r each peak can be c a l c u l a t e d and peak i n t e n s i t i e s expressed i n u n i t s o f standard d e v i a t i o n .

The more d u p l i c a t e

analyses a r e a v a i l a b l e t h e more e f f e c t i v e l y t h i s procedure removes i n t r a - s a m p l e "noise" due t o i n s t r u m e n t a l sources.

I f m u l t i p l e samples had been a v a i l a b l e from

each i n d i v i d u a l , "noise" from i n t r a - i n d i v i d u a l

v a r i a t i o n s i n t h e composition o f t h e

sample c o u l d have been e l i m i n a t e d i n t h e same way.

An example o f t h i s s c a l i n g f o r

t h e DMD data i s g i v e n i n Table 5 i n which column ( a ) g i v e s t h e t o t a l i o n i n t e n s i t y f o r v a r i o u s peaks and column (b) t h e intra-sample d e v i a t i o n .

D i v i s i o n o f ( a ) by ( b )

g i v e s t h e peak i n t e n s i t i e s i n terms o f standard d e v i a t i o n u n i t s .

While t h i s v e r y

common procedure b r i n g s o u t t h e most r e l i a b l e f e a t u r e s , these f e a t u r e s a r e n o t n e c e s s a r i l y t h e most c h a r a c t e r i s t i c f e a t u r e s f o r o b t a i n i n g a c l e a r d i f f e r e n t i a t i o n between t h e samples. TABLE 5 I o n i n t e n s i t y parameters f o r t h e 15 masses w i t h g r e a t e s t c h a r a c t e r i s t i c i t y values i n t h e a n a l y s i s o f muscle samples ~~

~

~

~

(a)

(b) Intra-sample Ion I n t e n s i t y Deviation

% Total

Mass 67 a1 32 101 31 41 40 a4 68

ao

48 102 74 42 34

3.45 1.61 1.40 0.09 1.33 1.42 0.11 1.41 2.03 0.83 4.34 0. oa 0.37 3.00 4.07

0.19 0.06 0.09 0.01 0.08 0.06 0.01 0.05 0.07 0.03 0.19 0.01 0.02 0.07 0.14

(c) Inter-sample Deviation 4.39 0.77 1.09 0. oa 0.75 0.54 0.05 0.40 0.57 0.25 1.44 0.05 0.13 0.40 0.73

(a)/(b) Reproducibility Value

(c)/(b) "Characteristicity" Value

ia,i6 26. a3 15.56 9.00 16.63 23.67 11 .oo 28.20 29.00 27.67 22.84 8.00 18.50 42.86 29.07

23.60 13.24 11.50 10.15 9.53 8.78 8.56 8-30 8.24 7.97 7.54 6.14 5.71 5.63 5.29

To s o l v e t h i s problem, Eshuis e t aZ. ( r e f . 45) suggested t h e use o f a f e a t u r e s c a l i n g f a c t o r based on t h e r a t i o between o u t e r and mean i n n e r d e v i a t i o n , i n o r d e r t o b r i n g o u t those f e a t u r e s which a r e b o t h h i g h l y d i f f e r e n t between d i f f e r e n t classes o f p a t t e r n s ( h i g h o u t e r d e v i a t i o n ) and h i g h l y s t a b l e w i t h i n each g i v e n c l a s s ( l o w mean i n n e r d e v i a t i o n ) . w e l l known F i s h e r r a t i o .

This " c h a r a c t e r i s t i c i t y " f a c t o r i s closely r e l a t e d t o the Eshuis e t aZ. have shown t h a t t h e use o f t h i s f a c t o r

60

dramatically improves the separation of two classes of Listeria without making previous assumptions about the existence of these two classes (ref. 45). If used in this manner, the feature scaling acts as a contrast-enhancing procedure not entirely dissimilar to the computer procedures used for contrast enhancement in digital image processing operations. A distinct disadvantage of feature scaling techniques incorporating outer deviation values in the scaling factor, however, is the strong dependence of this factor on the particular set of samples used. Addition of a different sample to the set may appreciably change the scaling factors, sometimes making direct comparison between results obtained with different sets of samples difficult, if not impossible. This problem does not necessarily exist when mean inner deviation is used in the scaling factors only, since the sources of biological and instrumental "noise" variations may often be assumed to be similar for different sets of samples. In fact, this condition is almost a prerequisite for further statistical processing of combined data from different sets of samples. Referring again to Table 5, the column labelled "characteristicity" value lists the ratio of the outer to inner sample deviations for the fifteen masses with the largest "characteristicity" values. 6.3. UNIVARIATE STATISTICAL ANALYSIS

Feature scaling factors are primarily calculated for subsequent multivariate statistical analysis of the data, However, these factors also provide a primitive, but often effective, means of data reduction and/or feature selection. Data reduction for further numerical analysis procedures may be achieved by only considering the n mass peaks with the highest "characteristicity" values or highest reproducibility values, where n may have any value from unity to the total number of mass peaks measured. Feature selection on the basis of scaling factors can be helpful in selecting features to be displayed as one-, two- or three-dimensional plots. Alternatively, selected features may be used for further univariate statistical analysis, e.g. T-tests for the significance of observed trends in the intensity distributions. As an example of two-dimensional feature plots, Figure 26 shows the scatter plot o f the intensity distributions of the two masses with highest "characteristicity" values, namely m/z 67 and 81, respectively, for the DMD data. These masses both show a progressive increase in intensity with progressive involvement of the disease. In fact, T-test values show a significant separation between patient and control samples in spite of the small number of samples involved. At the same time, however, this two-dimensional plot showsthe basic shortcoming of univariate statistical analysis techniques in that the strong correlation between the two mass signals is completely ignored. The existence of this correlation is confirmed by the high value of the calculated correlation coefficient. In contrast, the scatter plot o f m/z 32 versus m/z 101 shown in Figure 27 reveals no strong correlation either with the degree o f involvement in the disease or between both masses. The intensity distribution of

61

-4-

D t

i

i O h

TOTAL

E

6

i

ION INTENSITY m/z 67

Figure 26. Scatter plot of the ion intensities at m/z 67 versus m/z 81 for muscle biopsy samples (Table 4), showing a strong increase in the dystrophic muscle (D,E,F) and a high degree of correlation (correlation coefficient 0.91) between m/z 67 and 81. T-tests for probability of chance occurrence of separation between normals and dystrophic case F yield p values less than 0.01 for both m/z 67 and m/z 81.

. N

C

E 0.2>

c fn z W

-0-

-

&-

z

z 0.10 -I

a &-

e

+

o

-

A

-

% TOTAL

1 2 ION INTENSITY m/z 32

Figure 27. Scatter plot of the ion intensities at m/z 32 versus m/z 101, showing absence of correlation with the disease as well as between both ion intensity distributions. Note high value for sample C (female control) at m/z 101. m/z 32 appears to be dominated by strong individual differences between all three controls (compare this finding with the pronounced differences in clinical data for these samples in Table 4), whereas m/z 101 shows an exceptionally high value for the only female sample (control C). Additional analyses of different samples will be required in order to confirm the significance of these observations.

S i m i l a r two-dimensional p l o t s c o u l d be c o n s t r u t t e d , o f course, f o r many o t h e r c o m b i n a t i o n s o f masses w i t h h i g h " c h a r a c t e r i s t i c i t y " values.

However, a f a r more

comprehensive approach i s t o use m u l t i v a r i a t e s t a t i s t i c a l a n a l y s i s t e c h n i q u e s which e n a b l e a comparison o f i n t e n s i t y d i s t r i b u t i o n s o f a much l a r g e r c o l l e c t i o n o f masses t o be made s i m u l t a n e o u s l y .

A l s o , t h i s approach a v o i d s t h e danger o f m i s j u d g i n g t h e

s i g n i f i c a n c e of seemingly non-random d i s t r i b u t i o n s when examining i s o l a t e d f e a t u r e s f o r an expected d i f f e r e n t i a t i o n i n i n t e n s i t y d i s t r i b u t i o n s . l e v e l o f 1% ( p

Obviously, a significance

5 0.01) does n o t p r o v e d i f f e r e n t i a t i o n i f o n l y observed a few t i m e s

among a hundred o r so d i f f e r e n t f e a t u r e s i n s p e c t e d . 6.4.

MULTIVARIATE STATISTICAL ANALYSIS I n s t e a d of c o n s i d e r i n g t h e i n t e n s i t y d i s t r i b u t i o n a t each i n d i v i d u a l mass v a l u e

s e p a r a t e l y , as i s t h e case i n u n i v a r i a t e s t a t i s t i c a l a n a l y s i s , m u l t i v a r i a t e s t a t i s t i c a l a n a l y s i s t e c h n i q u e s a l l o w c o n s i d e r a t i o n o f n mass d i s t r i b u t i o n s i n n-dimensional space s i m u l t a n e o u s l y .

Whereas i t i s r e l a t i v e l y easy t o v i s u a l i z e two- o r even

t h r e e - d i m e n s i o n a l s c a t t e r p l o t s o b t a i n e d by p l o t t i n g t h e i n t e n s i t y d i s t r i b u t i o n s a t t w o o r t h r e e mass v a l u e s s i m u l t a n e o u s l y , d i r e c t v i s u a l i s a t i o n o f d i s t r i b u t i o n s i n h i g h e r dimensional space i s , o f course, i m p o s s i b l e .

However, as l o n g as t h e h i g h e r

d i m e n s i o n a l space i s m e t r i c , c a l c u l a t i o n o f d i s t a n c e s between p o i n t s obeys t h e same m e t r i c laws as c a l c u l a t i o n o f d i s t a n c e s i n two- o r t h r e e - d i m e n s i o n a l space.

There-

f o r e , c o n s i d e r t h e s i t u a t i o n shown i n F i g u r e 28, where each o f t h e two p o i n t s r e p r e s e n t s t h e r e s u l t o f a measurement i n v o l v i n g t h e same t h r e e parameters, x, y , and z.

I n terms o f Py-MS d a t a , t h i s would correspond t o two mass s p e c t r a c o n t a i n i n g

masses x, y, and z w i t h t h e i o n i n t e n s i t i e s f o r t h o s e p a r t i c u l a r masses p l o t t e d along t h e c o r r e s p o n d i n g axes.

Since t h e values found f o r each o f t h e s e parameters a r e

d i f f e r e n t i n t h e two measurements, t h e p o i n t s occupy d i f f e r e n t p o s i t i o n s i n t h r e e d i m e n s i o n a l space.

O b v i o u s l y , t h e more d i f f e r e n t t h e v a l u e s measured, t h e f a r t h e r

a p a r t a r e t h e p o i n t s , and v i c e versa.

Thus, t h e d i s t a n c e between t h e p o i n t s i s a

n u m e r i c a l e x p r e s s i o n o f t h e degree o f s i m i l a r i t y between t h e m u l t i p a r a m e t e r measurements, The most d i r e c t way o f measuring t h e d i s t a n c e between t h e p o i n t s i s t o c a l c u l a t e t h e distance along a s t r a i g h t l i n e through both p o i n t s , the so-called Euclidean distance.

A seemingly more c o m p l i c a t e d way o f measuring t h e d i s t a n c e i s t o sum t h e

d i s t a n c e when t a k i n g t h e s h o r t e s t r o u t e f r o m one p o i n t t o a n o t h e r o n l y f o l l o w i n g l i n e s p a r a l l e l t o one o f t h e t h r e e axes, t h e s o - c a l l e d c i t y b l o c k d i s t a n c e (see F i g u r e 28).

The a t t r a c t i v e p r o p e r t y o f t h i s d i s t a n c e measurement i s t h a t i s i n v o l v e s

o n l y s i m p l e a d d i t i o n s and s u b t r a c t i o n s .

F r e q u e n t l y , q u a d r a t i c and o t h e r n o n - l i n e a r

f u n c t i o n s (e.g. c o r r e l a t i o n c o e f f i c i e n t s , s t a n d a r d d e v i a t i o n s , c h i squared c o e f f i c i e n t s o r g e n e r a l i z e d Mahalanobis d i s t a n c e s ) a r e used t o d e r i v e d i s t a n c e values ( r e f . 132).

A l l o f t h e s e f u n c t i o n s a r e g e n e r a l l y r e f e r r e d t o as " s i m i l a r i t y c o e f f i c i e n t s " .

Y /

X

Z F i g u r e 28. Three-dimensional r e p r e s e n t a t i o n o f two h y p o t h e t i c a l mass s p e c t r a l p a t t e r n s w i t h f e a t u r e s X, Y and Z, showing E u c l i d e a n d i s t a n c e and c i t y b l o c k distance.

A l l m e t r i c s i m i l a r i t y c o e f f i c i e n t s i n two- o r t h r e e - d i m e n s i o n a l space can be v i s u a l i s e d p r o v i d e d t h a t s u i t a b l e l i n e a r o r n o n - l i n e a r axes a r e chosen.

For a mathemati-

c a l d e s c r i p t i o n o f t h e v a r i o u s s i m i l a r i t y c o e f f i c i e n t s used i n m u l t i v a r i a t e s t a t i s t i c a l a n a l y s i s , t h e r e a d e r i s r e f e r r e d t o s p e c i a l i s e d t e x t b o o k s ( r e f s . 132, 133) o r t o t h e SPSS User Manual ( r e f . 128). I n o u r e x p e r i e n c e , t h e m o d i f i e d E u c l i d e a n d i s t a n c e a l g o r i t h m d e s c r i b e d by Eshuis

e t al. ( r e f . 45) and t h e c l o s e l y r e l a t e d c h i squared a l g o r i t h m p r o v i d e t h e most s a t i s f a c t o r y r e s u l t s f o r p y r o l y s i s mass s p e c t r a , p r o v i d e d t h a t t h e p r o p e r f e a t u r e s c a l i n g t e c h n i q u e s a r e used f i r s t .

Again r e f e r r i n g t o t h e DMD d a t a , T a b l e 6 shows

t h e d i s t a n c e m a t r i x o b t a i n e d f o r t h e Py-MS s p e c t r a when . u s i n g t h e 40 mass peaks w i t h h i g h e s t " c h a r a c t e r i s t i c i t y " v a l u e s and employing t h e c h i squared f o r m u l a .

The

d i a g o n a l o f t h e m a t r i x shows d i s t a n c e v a l u e s between d u p l i c a t e s p e c t r a o f t h e same sample.

Examination o f t h e s e v a l u e s a l l o w s t h e d e t e c t i o n o f b a d l y r e p r o d u c i n g

s p e c t r a , which s u b s e q u e n t l y may be d e l e t e d and t h e m a t r i x r e - c a l c u l a t e d .

If there

a r e no more o b v i o u s o u t l i e r s among t h e d u p l i c a t e s p e c t r a , t h e m a t r i x can be subs t a n t i a l l y reduced by l i s t i n g o n l y t h e d i s t a n c e v a l u e s between t h e average spectrum o f each sample, as i l l u s t r a t e d i n T a b l e 7.

A close s c r u t i n y o f t h e values i n Table

7 enables a c r u d e mental p i c t u r e t o be formed o f t h e r e l a t i v e p o s i t i o n s o f t h e average s p e c t r a i n m u l t i - d i m e n s i o n a l space.

O b v i o u s l y , t h e s p e c t r a o f t h e non-

dystrophic c o n t r o l s form a small c l u s t e r w i t h t h e spectra o f t h e dystrophic p a t i e n t s moving p r o g r e s s i v e l y away f r o m t h i s c l u s t e r w i t h i n c r e a s i n g age o f t h e p a t i e n t . f o r m such m e n t a l p i c t u r e s becomes i n c r e a s i n g l y d i f f i c u l t , i f n o t i m p o s s i b l e , w i t h

To

m

P

TABLE 6 D i s t a n c e m a t r i x showing c h i squared c o e f f i c i e n t v a l u e s f o r p y r o l y s i s mass s p e c t r a o f muscle biopsy samples from t h r e e d y s t r o p h i c p a t i e n t s ( D , E , F ) and three c o n t r o l s ( A , B , C). A1

1.1 1.1

-

A3

.8

-

A4

1.1

.8

.8

B1

2.9

2.3

2.6

2.6

-

82 B3

3.5

2.8

3.0

3.1

2.4

2.6

2.8 2.6

1.8 1.0

c1

3.94.03.8

c2

5.65.05.25.3 5 . 2 4.7 4.9 5.1

3.7

4.2

3.7

1.4

-

c3

5.6

4.9

5.2

5.3

3.8

3.9

3.6

1.0

1.3

-

D1 D2

6.6

6.1

6.3

6.3

4.7

4.5

4.5

3.9

6.6 7.3 7.2

6.0 6.6

6.2

4.6 5.0

6.6

El

9.5

E2 E3

10.2 8.6

8.2

8.3

E4

8.7

8.3

8.4

A2

D3 D4

-

-

1.4

-

-

4.8

4.2

4.4

6.8 6.8

6.2 6.8

4.4 4.7

5.3

4.7

4.9

5.2

5.5

4.2 4.7

1.3 1.8 1 . 2

-

6.8

5.3

4.7

4.9

5.0

5.4

4.6

1.6

1.2

.7

-

9.1

9.1

9.1

8.1

7.3

7.7

8.4

8.7

8.0

9.8

9.8

9.9

8.8

8.1

8.5

9.2

9.4

8.8

4.7 5.4

4.1 4.8

4.2 4.8

8.3

7.2

6.6

6.9

7.6

7.8

7.2

3.9

4.3 5.0 3.6

3.4

8.4

7.3

6.7

7.0

7.8

8.0

7.4

4.0

3.7

3.4

9.7

9.4

9.0

1.1

-

3.4

1.4

2.0

-

3.5

1.4

1.9

.8

-

9.1

5.4

4.6

6.2

6.1

-

F1

14.5 14.1 14.1 14.1

13.2 1 2 . 4 12.9

13.4 13.7 13.1

F2

13.1 12.7 12.7 12.7 13.6 1 3 . 3 13.3 1 3 . 4

1 1 . 8 11.1 11.4

12.0 12.2 1 1 . 6

8.2

7.S

2.0

-

8.6

4.6

3.9

4.7 5.3

4.6

8.8

7.6 8.3

3.3

1 2 . 5 12.7 12.1

7.5 8.2

4.0

1 2 . 4 11.7 12.0

5.2

1.8

1.5

-

Dl

D2

D3

D4

El

E2

E3

E4

F1

F2

F3

F3

A1

A2

A3

A4

B1

82

83

C1

C2

C3

65 TABLE 7 Reduced d i s t a n c e m a t r i x showing t h e average d i s t a n c e v a l u e s f o r each s e t o f analyses g i v e n i n T a b l e 6 f o r c o n t r o l s (A,B,C) and p a t i e n t s (D,E,F).

)

A

1.0

B

2.8

1.4

C

5.2

3.8

1.2

D

6.6

4.7

4.8

1.3

E

9.0

7.5

8.2

4.2

1.4

F

13.5

12.1

2.6

8.5

4.8

1.8

A

B

C

D

E

F

'

l a r g e r m a t r i c e s and more complex r e l a t i o n s h i p s .

In o r d e r t o o b t a i n an a c c u r a t e

i m p r e s s i o n i n t h e s e cases, s p e c i a l t e c h n i q u e s have t o be used t o e x t r a c t t h e most s i g n i f i c a n t i n f o r m a t i o n f r o m t h e d i s t a n c e m a t r i x and t o d i s p l a y t h i s s e l e c t e d i n f o r m a t i o n i n an e a s i l y v i s u a l i s e d form.

The loss of i n f o r m a t i o n i n c u r r e d can be

m i n i m i z e d by u s i n g a number o f complementing v i s u a l i s a t i o n t e c h n i q u e s r a t h e r t h a n a s i n g l e technique.

V i s u a l i s a t i o n t e c h n i q u e s o f t e n used a r e :

nearest neighbour

t a b l e s , dendrograms, r e - o r d e r e d m a t r i c e s and n o n - l i n e a r maps ( s e e F i g u r e 2 9 ) .

Of

t h e s e we most o f t e n use t h e n o n - l i n e a r maps and n e a r e s t neighbour t a b l e s , and occasionally t h e re-ordered matrices.

CORREL COEFF.

q-1

ORDEREI MATRICES

MAPS *:

F i g u r e 29. Schematic o v e r v i e w o f r e l a t i o n s h i p s between m u l t i v a r i a t e s t a t i s t i c a l a n a l y s i s procedures a p p l i c a b l e t o p y r o l y s i s mass s p e c t r a .

66 6.5.

VISUALISATION TECHNIQUES

N e a r e s t n e i g h b o u r t a b l e s a r e s i m p l e l i s t s o f t h e K n e a r e s t neighbours of each sample i n m u l t i - d i m e n s i o n a l space.

Table 8 shows such a l i s t f o r t h e DMD d a t a .

I n s p e c t i o n o f t h i s t a b l e demonstrates t h a t each sample r e c o g n i z e s o t h e r s i m i l a r C o n t r o l s always f i n d o t h e r c o n t r o l s as f i r s t neighbours and t h e d y s t r o p h i c

samples.

samples always f i n d o t h e r d y s t r o p h i c samples. TABLE 8 N e a r e s t neighbour t a b l e , d e r i v e d f r o m d i s t a n c e m a t r i x i n T a b l e 7, f o r c o n t r o l (A,B,C) and Duchenne muscular d y s t r o p h y (D,E,F) muscle b i o p s y samples N e a r e s t Neighbour Sample

1st

2nd

3rd

B A B E

C C D B F D

D D A C B B

D E

An o r d e r e d m a t r i x i s a d i r e c t f o r m o f r e p r e s e n t a t i o n o f t h e d i s t a n c e m a t r i x i n w h i c h v a r i o u s c o l o u r s o r degrees o f shading a r e used t o r e p r e s e n t d i f f e r e n t d i s t a n c e ranges.

The samples i n t h e m a t r i x a r e arranged i n such a way as t o p u t n e a r e s t

n e i g h b o u r s as c l o s e t o g e t h e r as p o s s i b l e . o r d e r e d m a t r i x f o r t h e DMD data.

F i g u r e 30 g i v e s an example o f a s i m p l e

An e x c e l l e n t way t o a r r i v e a t t h e b e s t p o s s i b l e

sequence o f neighbours f o r t h e o r d e r e d m a t r i x i s t o use Kruskal I s m u l t i d i m e n s i o n a l s c a l i n g t e c h n i q u e ( r e f . 134), which i s s t r o n g l y based on n e i g h b o u r s h i p c r i t e r i a , t o s c a l e t h e n-dimensional d i s t a n c e m a t r i x t o a one-dimensional sequence. N o n - l i n e a r mapping t e c h n i q u e s have been used e x t e n s i v e l y i n t h i s work and a r e s u f f i c i e n t l y complex t o be c o n s i d e r e d as m u l t i v a r i a t e s t a t i s t i c a l a n a l y s i s procedures i n t h e i r own r i g h t .

I n f a c t , t h e n o n - l i n e a r mapping t e c h n i q u e most o f t e n used by us,

which i s based on K r u s k a l I s m u l t i d i m e n s i o n a l s c a l i n g method ( r e f . 134), has sometimes been compared w i t h p r i n c i p a l component f a c t o r a n a l y s i s t e c h n i q u e s ( r e f . 132) because o f i t s remarkable a b i l i t y t o d i s p l a y t h e most prominent numerical t r e n d s i n t h e data. The c o n c e p t o f n o n - l i n e a r mapping i s c o m p l i c a t e d and t h e r e f o r e o f t e n a s o u r c e o f confusion.

F o r i n s t a n c e , a K r u s k a l - t y p e two-dimensional n o n - l i n e a r map ( t h r e e -

d i m e n s i o n a l v e r s i o n s a l s o e x i s t ) i s n o t a p r o j e c t i o n o f m u l t i d i m e n s i o n a l space on t o two-dimensional space, b u t t h e r e s u l t o f an i t e r a t i v e computer procedure ( r e f . 45) aimed a t m i n i m i z i n g t h e " s t r e s s " , an e m p i r i c a l g o o d n e s s - o f - f i t v a l u e ( r e f . 135) which compares t h e c o n f i g u r a t i o n i n two-dimensional space w i t h t h e o r i g i n a l c o n f i g u r a t i o n i n m u l t i d i m e n s i o n a l space.

Low s t r e s s values (e.g.

t

rT)

Z

J

w I-

114

z

60

z4

0 -J a t

58

\

2 s

112

4

20

60

40

80

100

120

m/z

SAMPLE

: ARABINAN (from apple j u i c e )

CHEMICAL DETAILS SAMPLE ORIGIN

: poly[ (1+5)~Ara0];see i n s e r t in spectrum

SAMPLE PREP./SIZE

PYROL. CONDITIONS TOTAL ION COUNTS

140

160

: g i f t from Dr. A.G.J.

Voragen, Agricult. Univ. Wageningen, The Nether1 ands : suspension in methanol, 10 p g : standard : 2.2

x

lo5

m/z 112 and 114. Especially m/z 114 appears t o be c h a r a c t e r i s t i c o f pentose residues ( e . g . compare with Spectrum C . l ( R N A ) ) .

REMrlRKS - Note t h e high r e l a t i v e i n t e n s i t i e s of t h e peaks a t

127

SPECTRUM A , 12 30

32

'L

43

67

95

59

20

40

60

80

100

120

m/z

SAMPLE

: CHITOSAN

CHEMICAL STRUCTURE

: p o l y [ ( l + 4 ) ~ G l c N ~ ] ; see i n s e r t i n spectrum

SAMPLE O R I G I N

: Polysciences I n c . ,

SAMPLE PREP./SIZE

: suspension i n methanol; 10 pig

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

:

3

x

140

160

Warrington, PA, USA

lo4

mm8.a - C h i t o s a n i s a s e m i - s y n t h e t i c compound o b t a i n e d on d e - a c e t y l a t i o n o f c h i t i n . The s t r u c t u r e and c o m p o s i t i o n o f c h i t o s a n i s l e s s w e l l d e f i n e d t h a n t h a t o f t h e p a r e n t compound (compare S p e c t r a A.12 and A.lZa, r e p r e s e n t i n g c h i t o s a n p r e p a r a t i o n s f r o m d i f f e r e n t manufacturers). The spectrum shows a r a t h e r complex p a t t e r n o f m a i n l y odd-numbered peaks ( N - c o n t a i n i n g p y r o l y s i s p r o d u c t s ) which i s s i g n i f i c a n t l y d i f f e r e n t from t h a t of c h i t i n (Spectrum A.13).

SPECTRUM A, 1 2 ~ 630

J

~il, 60

>

; 4Z w

t

z

Z

0 1

20

60

40

80

100

120

m/z

140

SAMPLE

: CHITOSAN

CHEMICAL DETAILS

: p o l y [ (1+4)~GlcNp]; see i n s e r t i n spectrum

SAMPLE O R I G I N

: Koch-Light L t d . , Colnbrook, Bucks., UK

SAMPLE PREP./SIZE

: suspension i n methanol; 10 p g

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

:

160

z x lo5

- Compare w i t h Spectrum A.12. The i n t e n s e peak a t m/z 60 may p o i n t t o t h e presence o f a c e t a t e c o u n t e r i o n s a t t h e amine groups. Note t h e s u l p h u r c o n t a i n i n g i o n s i g n a l s a t m/z 34 (HzS+'), 48 (CH3SH+' and/or SO+') and 64 (SO + ' a n d / o r S z t ' and/or CH3SOHt') i n d i c a t i n g t h e presence o f noncarbohydrate confami n a n t s .

liEMARKS

129

SPECTRUM A, 13 3.9%)

73 43

109 I

30

I

20

60

40

80

100

120

m/z

140

SAMPLE

: CHITIN

CHEMICAL DETAILS

: p o l y [ ( 1+4)oGlcNAc~]; see i n s e r t i n spectrum

SAMPLE O R I G I N

:

SAMPLE PREP./SIZE

: suspension i n methanol; 10 ug

PYROL. CONDITIONS

: standard

TOTAL I O N COUNTS

4 : 7.3 X 10

160

BDH Chemicals Ltd., Poole, UK

- This spectrum should be compared w i t h t h a t o f t h e glucan analogue, c e l l u l o s e (Spectrum A . l ; see a l s o P a r t 1, Figure 2 ) which shows s i m i l a r c h a r a c t e r i s t i c s e r i e s o f i o n s a t mass values d i f f e r i n g by 1 o r 41 amu, appare n t l y r e p r e s e n t i n g t h e presence o f NH2 o r NHAc groups i n s t e a d o f an OH f u n c t i o n a t C 2 i n t h e r e s p e c t i v e p y r o l y s i s fragments. This would seem t o p o i n t t o a s t r o n g s i m i l a r i t y between t h e p y r o l y s i s pathways o f b o t h polymers. The abunda n t fragment a t m/z 59, as a l s o found f o r o t h e r N-acetylamino sugars (see Spectra A.14-A.20, A.23, A.25), was i d e n t i f i e d as acetamide ( r e f . 80); i t i s much l e s s pronounced i n t h e c h i t o s a n p a t t e r n (Spectrum A.12).

REMARE

130

SPECTRUM A, 1 3 ~ 73

6

1

>

9

59

c

m 2 4

A

W

I-

z

85

z

125

0

55

-1

U

s)

b 2

s

20

40

60

80

100

120

m/z

140

SAMPLE

: CHITIN

CHEMICAL DETAILS

: poly[(l-.Q)DGlcNAc~]; see i n s e r t i n spectrum

SAMPLE O R I G I N

: BDH Chemicals L t d . ,

SAMPLE PREP./SIZE

: suspension i n PBS b u f f e r ;

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 4.3

x

160

Poole, UK

10 ug

lo5

Note t h e q u a n t i t a t i v e d i f f e r e n c e s i n t h e t y p i c a l n i t r o g e n - c o n t a i n i n g fragment s e r i e s (odd mass v a l u e s ) and t h e i n c r e a s e i n i n t e n s i t y a t m/z 136 ( s t r u c t u r e unknown). Peaks a t m/z 36, 38 r e p r e s e n t HC1+' (PBS b u f f e r ) .

REMARKS - Compare w i t h Spectrum A.13.

131

SPECTRUM A, 14 8

c

C H20H I

>-

43

t

I

m Z w I-

z 4. z 0

5

J

s0

109

t-

s

40 SAMPLE CHEMICAL DETAILS

60

80

100

120

m/z

140

160

: CAPSULAR POLYSACCHARIDE ( f r o m h’eisseria rneningitidis, group X) : poly[4~GlcNAc~-l-phosphate]; see i n s e r t i n spectrum

SAMPLE O R I G I N

: g i f t f r o m D r . E.C. Beuvery, N a t l . I n s t . Publ. H e a l t h , B i l t h o v e n , The Netherlands

SAMPLE PREP./SIZE

: suspension i n PBS b u f f e r ;

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 1.5

20 ug

x lo5

- Sample prepared f o l l o w i n g r e f . 49. Note t h e q u a l i t a t i v e c o r r e s pondence w i t h t h e spectrum o f o t h e r phosphate-bridqed (Spectrum A.15) and non. phosphate-bridged (Spectrum A. 13a) N-acetylhexosamine polymers. Peaks a t m/z 36, 38 r e p r e s e n t H C l t ’ (PBS b u f f e r ) .

REMARKS

SPECTRUM

43

A,15

59 i0 ONa

I

1?5 136

lI l

109

8P 20

'

40

'

60

80

I

97

.

100

120

m/z

140

160

SAMPLE

: CAPSULAR POLYSACCHARIDE (from fleisseria meningitidis , Group A)

CHEMICAL DETAILS SAMPLE O R I G I N

: poly[6~ManNAca-l-phosphate] ; see i n s e r t i n spectrum

SAMPLE PREP./SIZE

: suspension i n PBS b u f f e r ; 20 pg

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 1

REMKS

-

: g i f t f r o m D r . E.C. Beuvery, N a t l . I n s t . Publ. H e a l t h , B i l t h o v e n , The N e t h e r l a n d s

x lo5

Sample prepared f o l l o w i n g r e f . 49.

See Remarks on spectrum A.14.

133

SPECTRUM

A,16

43 N

125

32 N

83

I

20

40

60

80

100

SAMPLE

: HYALURONIC ACID,

CHEMICAL DETAILS

: poly[

SAMPLE O R I G I N

: Boehringer, Mannheim, GFR

SAMPLE P R E P . / S I Z E

: suspension i n methanol; 10 Fig

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 1.7 X 10

120

m/z

140

160

K-salt

(1-.4)DGlcAg(l-+3)nGlcNAc~]; see i n s e r t i n spectrum

5

- Note t h e presence o f t h e even-numbered mass peaks a t m/z 60, 68, 9 6 , 112, 114 and 126 a p p a r e n t l y d e r i v e d from t h e GlcA m o i e t i e s (see Spectrum A.8) and t h e odd-numbered mass peaks a t m/z 59, 73, 83, 97, 109, 125, 137, 139 and The fragment a t m/z 151 d e r i v e d from t h e GlcNAc m o i e t i e s (see Spectrum A.13). 59 can be i n t e r p r e t e d as acetamide d e r i v e d from t h e NAc groups.

REMARKS

SPECTRUM A, 17 I

19

COONa

43

N

20

40

60

80

100

120

140

160

m/z

SAMPLE

: CHONDROITIN SULPHATE C , Na-salt

CHEMICAL DETAILS

0-sulphated a t position 6 o f GalNAc; see i n s e r t i n spectrum : Seikagaku Kogyo Go. Ltd., Tokyo, Japan

SAMPLE ORIGIN

: poly[(l+4)~GlcAg(l-3)nGalNAc~];

SAMPLE PREP./SIZE

: suspension in methanol; 10 pg


: standard

5

: 1.2 X 10

- As i s t o be expected, this spectrum resembles t h a t o f hyaluronic acid (Spectrum A.16). Peaks a t m/z 96, 112 and 114 i n d i c a t e t h e GlcA residues; t h e peaks a t m/z 59 (acetamide) and 125 a r e i n d i c a t i v e o f t h e GalNAc residues. The signal a t m/z 64 (S02+') apparently represents t h e sulphate moieties, the r e l a t i v e l y low i n t e n s i t y might be connected with t h e f a c t t h a t t h e compound i s analysed i n t h e s a l t form. The presence of ion s i g n a l s a t m/z 34, 48, 7 6 , 92, 94, 108 and 1 1 7 probably indicates proteinaceous contaminants. Compare a l s o w i t h Spectrum A.18, showing t h e 4-sulphated analogue.

REMARKS

135

SPECTRUM A , 1 7 ~

'

OH

H N - CII- C H ~

0

N

59

*

85 A

A

96

72

N

125

I

N

167

40

60

80

100

120

m/z

140

...........1. 160

SAMPLE

: CHONDROITIN SULPHATE C, N a - s a l t

CHEMICAL DETAILS

: poly[(1-+4)~GlcA~(1-3)~GalNAc~]; 0 - s u l p h a t e d a t p o s i t i o n 6 o f GalNAc; see i n s e r t i n spectrum

SAMPLE O R I G I N

: Sei kagaku Kogyo Co. L t d . , Tokyo, Japan

SAMPLE PREP./SIZE

: suspension i n PBS b u f f e r ; 10 pg

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 4.5

x lo5

REMARG - Peaks a t m/z 36, 38 r e p r e s e n t HC1" Spectrum A. 17 (methanol suspension).

(PBS b u f f e r ) .

Compare w i t h

136

SPECTRUM A 18 I

8

B.5%)

0w... COONa

1 9 I

85

CH20H

--.o OH

HN-C-CH3 I1 0

> c ln

z

N

c

97

W

z

z s4 -I

a

I-

e

s

N

20

40

J

80

0

100

120

SAMPLE

: CHONDROITIN SULPHATE A, Na-salt

CHEMICAL DETAILS

:

SAMPLE O R I G I N

: Seikagaku Kogyo Co. Ltd., Tokyo, Japan

SAMPLE PREP./SIZE

: suspension i n methanol; 10 pg

PYROL. CONDITIONS

: standard

TOTAL I O N COUNTS

: 1.3 X 10

140

160

poly[(1+4)~GlcA~(1+3)~GalNAc~]; 0-sulphated a t p o s i t i o n 4 o f GalNAc; see i n s e r t i n spectrum

5

REMARKS - The r e l a t i v e l y l a r g e d i f f e r e n c e between t h i s spectrum and t h a t o f t h e 6-sulphate analogue (Spectrum A.17) i s remarkable. Probably t h i s d i f f e r e n c e i s a t t r i b u t a b l e t o d i f f e r e n c e s i n t h e i o n i c s t a t e o r p u r i t y o f t h e samples ( t h e above spectrum shows much fewer p r o t e i n fragment peaks than Spectrum A. 17) r a t h e r than t o t h e d i f f e r e n c e i n the p o s i t i o n o f t h e sulphate s u b s t i t u e n t s . These d i f f e r e n c e s are l e s s pronounced i n t h e spectra obtained from PBS suspensions o f t h e compounds (Spectra A.17a, A.18a).

137

SPECTRUM

A, 1 8 ~ CH20H

43

6-

--0

85 >-

6H

1

I

t ln z

HN-C-CH~

II

0

$4-

z 0

I

-1

32

s

s 2-

55

l!6

20

40

60

80

100

120

m/z

140

160

SAMPLE

: CHONDROITIN SULPHATE A, N a - s a l t

CHEMICAL DETAILS

: poly[(l+4)~GlcA~(1+3)nGalNAc~]; 0 - s u l p h a t e d a t p o s i t i o n 4 o f Ga1NAc;see i n s e r t i n spectrum

SAMPLE O R I G I N

: Seikagaku Kogyo Co. L t d . , Tokyo, Japan


: suspension i n PBS b u f f e r ; 10 ug

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 3.6

x lo5

REMARKS - Peaks a t m/z 36, 38 r e p r e s e n t H C l f ’

Spectrum A.18 (methanol suspension).

(PBS b u f f e r ) .

Compare w i t h

SPECTRUM A,19 '3

1

N

1 !5

59

43

A

96 30 N

55

lul 80

20

60

40

SAMPLE CHEMICAL DETAILS

100

120

140

160

: TEICHURONIC ACID (from BaciZZus subtiZis) : poly[(l+4)DGlcA~~,~(l-+3)DGalNac~~,B]; see i n s e r t in

SAMPLE ORIGIN

:

SAMPLE PREP./SIZE PYROL. CONDITIONS TOTAL ION COUNTS

: :

:

Spectrum A.19a g i f t from Dr. J.T.M. Wouters, Lab. of Microbiology, Univ. of Amsterdam, The Netherlands suspension in methanol; 10 p g standard 3 x lo5

Abundant fragment ions a t m/z 59, 73, 83, 109, 125, 139 and 151 i n d i c a t e the GalNAc moieties and t h e peaks a t m/z 96, 102, 112, 114 and 126 t h e GlcA moieties. Note the large difference between t h i s fragment pattern and t h a t obtained f o r the neutral PBS solution (Spectrum A . 19a).

REMARKS - Sample prepared following r e f . 172.

139

SPECTRUM A, 1 9 ~ 1

COONa

CHzOH

OH

'

HN-C-CH~

II 0

!

* A

P-J

20

60

40

80

100

120 m/z

140

160

SAMPLE

: TEICHURONIC ACID (from BacSZZus subtiZis)


see i n s e r t in spectrum : g i f t from Dr. J.T.M. Wouters, Lab. o f Microbiology, Univ. of Amsterdam, The Netherlands


: suspension in PBS buffer; 10 pg

:

poly[(1+4)nGlcAa,p(1+3)nGalNAca,B1;

: standard : 2

x

lo5

- Sample prepared following ref. 172. This spectrum shows a marked decrease in intensity a t m/z 83, 96, 109 and 125 and an increase a t m/z 102 with respect t o Spectrum A.19. The i d e n t i t i e s o f these fragments have n o t yet been established. Peaks a t m/z 36, 38 represent H C l t ' (PBS buffer).

REMARKS

140

SPECTRUM A 20 I

8

>

cm z

W

t

z

z 0 J 4 a !-

2

s

I

20

40

60

80

100

120

m/z

140

160

,

SAMPLE

: CAPSULAR POLYSACCHARIDE ( f r o m Neisseria meningitidis Group 29E)

CHEMICAL DETAILS

: p o l y [ (2-+3)nGal NAca( 1+7)KD08];

SAMPLE O R I G I N

: g i f t f r o m D r . E.C. Beuvery, N a t l . I n s t . Publ. H e a l t h , B i l t h o v e n , The N e t h e r l a n d s

SAMPLE PREP./SIZE


PYKOL. CONDITIONS

: standard

TOTAL ION COUNTS

: 2.2

see i n s e r t i n spectrum

x lo5

- Sample r e p a r e d f o ! l o w i n g r e f . 49. I o n s e r i e s a t m/z 59, 73, 95, 97, The 109 and 125 a r e c l a r a c t e r i s t i c o f hexNAc r e s i d u e s (see Spectrum A . 1 3 ) . peaks a t m/z 110, 112, 122 a r e i n d i c a t i v e o f t h e KDO ( k e t o d e o x y o c t o n a t e ) r e s i d u e s (see Spectrum A.21) b u t a r e much more pronounced i n a phosphate b u f f e r environment (compare w i t h Spectrum A.20a).

REME

141

SPECTRUM A, 2 0 ~

1

' '

/

122

43

I 1{a

N

t

*

94

0

40

L.h

60

15;

J

140

J 8

100

120

m/z

140

SAMPLE

: CAPSULAR POLYSACCHARIDE (from Neisseria meningitidis ,

CHEMICAL DETA I LS SAMPLE ORIGIN

: pol y [ (2+3)DGal NAca ( 1+7) KDO 61


Group 29E)

: g i f t from Dr. E.C.

Beuvery, Natl. I n s t . Publ. Health, Bi 1thoven, The Nether1 ands : suspension i n PBS buffer; 10 U g : standard : 1 x lo5

REMAI~KS - Compare with Spectra A.20 and A.21.

The c h a r a c t e r i s t i c KDO peak s e r i e s a t m/z 110, 112, 122 and 140 i s much more pronounced here. The peak a t m/z 94 may be a t t r i b u t a b l e t o residual phenol used i n t h e i s o l a t i o n procedure although i t i s a l s o present in the KDO spectrum (A.21).

142

SPECTRUM A , 21 8

J I12

>

43

c

m z

81

W

c

98

z -4 z 2

I 1'

-I

U c

J

P s

140

20

60

40

80

100

1 ; 120

140

160

m/z

SAMPLE

: 2-KETO-3-OEOXYOCTONATE

(KDO), NHq+-sal

t (3-deoxy-manno-

o c t u l osonic a c i d ) CHEMICAL DETAILS

: see i n s e r t i n spectrum; C8H,407,

SAMPLE O R I G I N

: Sigma Chemical Corp.,


: s o l u t i o n i n PBS b u f f e r ; 10 1~g

PYROL. CONDITIONS

: standard

TOTAL I O N COUNTS

: 6 X

MW=222

S t . L o u i s , USA

lo4

REMARKS - Note t h e c h a r a c t e r i s t i c peak s e r i e s a t m/z 110, 112, 122 and 140 (see a l s o S p e c t r a A.20 and A.20a). The h i g h m/z 17 o r i g i n a t e s f r o m t h e ammonium ions.

143

SPECTRUM A 22 T CH20H

3

126

20

60

40

80

100

140

120

160

ill/Z

SAMPLE

: TEICHOIC ACID ( f r o m BaciZZus s d t i Z i s )

CHEMICAL DETAILS

:

SAMPLE O R I G I N

: g i f t from D r . J.T.M.

SAMPLE PREP./SIZE PYROL. CONDITIONS

: suspension i n methanol; 10 p g : standard

TOTAL ION COUNTS

: 1. z

w i t h DGlcB-units l i n k e d t o p o s i t i o n 1 o f g l y c e r o l ; see i n s e r t i n spectrum

poly[2,3-glycerol-phosphate]

Wouters, Lab. of M i c r o b i o l o g y , U n i v . o f Amsterdam, The N e t h e r l a n d s

x

lo5

REMARKS - Sample prepared f o l l o w i n g r e f . 172. The t e r m i n a l s i d e c h a i n g l u c o s e u n i t s appear t o g i v e r i s e t o peaks a t m/z 96, 98, 110 and 126 (see S p e c t r a A. 1-A.4), whereas t h e most pronounced fragments f r o m t h e g l y c e r o l m o i e t i e s a r e t h o u g h t t o be m/z 61 ( p r o b a b l y an E I f r a g m e n t ) and m/z 44. Peaks a t m/z 36, 38 (HCl+') i n d i c a t e t h e presence o f c h l o r i d e i n t h e sample.

144

SPECTRUM 8

A,23

kl

1

H~LOH

).

k cn

z

W

I-

z

z4 0 -I a I-

J

67 32

109

E s

20

60

40

80

do0

120

m/t

140

160

CHEMICAL DETAILS

: CAPSULAR POLYSACCHARIDE (from Neisseria rneningitidis, group B ) : poly[ ( 2 & ) ~ ~ - N - a c e t y l n e u r a m i n i c acid]; see i n s e r t in

SAMPLE ORIGIN

: g i f t from Dr. E . C . Beuvery, Natl. I n s t . Publ. Health,


: solution in PBS buffer; 10 p g

SAMPLE

spectrum Bil thoven, The Netherlands : standard : 1

x

lo5

REMRKS - Sample prepared following r e f . 49. The c h a r a c t e r i s t i c , odd-numbered peak s e r i e s a t m/z 59, 67, 73, 97, 109, 123, 135 and 151 i s r e l a t e d t o t h a t

observed f o r hexNAc-polymers. Marked differences, e.g. with the spectrum of c h i t i n (Spectrum A.l3a), a r e observed a t m/z 67 and in the peak g r o u p around m/z 81, 123, 125 and 135. The spectrum of t h i s a c i d i c polysaccharide (and a l s o t h a t of the a(2+9)-linked analogue) i s considerably influenced by the ionic strength and pH of the s t a r t i n g solutions (see a l s o Part I , Figure 1 1 ) . A comparison o f Py-mass spectra of a ( 2 4 ) and a(2+9)-linked po1y-Nacetylneuraminic acids and 0-acetylated derivatives i s made i n r e f . 49.

145

SPECTRUM A, 24

I

8 /

9

---6 6 H H2CO-

P---

I

OH

J 68 I

43

J

82J I 84

J

114

20

40


60

80

100

120

m/z

140

160

: CAPSULAR POLYSACCHARIDE ( f r o m HaernophiZus infZuenzae, t y p e B) : poly[3DRibg(l+l)Ribitol-5-phosphate]; see i n s e r t i n spectrum

SAMPLE O R I G I N

: g i f t f r o m D r . E.C. Beuvery, N a t l . I n s t . P u b l . H e a l t h , B i l t h o v e n , The Netherlands

SAMPLE PREP./SIZE

: suspension i n methanol; 10 1-19

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 5

x

lo4

REMS- The s e r i e s o f i o n s a t m/z 68, 82, 84, 96, 98 and 114 i s i n d i c a t i v e o f

pentose polymers; compare w i t h Spectrum C . l

(RNA).

SPECTRUM A 25 CHIOH

CH20H

...o 0

,

0

H,C-C-C=O

H

95

I

43

w

I

NH

I

L-Ala

I

D-GIU

I

DAP---

109

I

D-A la - - -

125

20

40

100

120

m/z

140

160

SAMPLE

: PEPTIDOGLYCAN ( f r o m BaciZZus subtiZis)

CHEMICAL DETAILS

: p o l y [ ( l 4 ) ~ G l c N A c ~ ( 1 4 ) M u r N A cw ~ ]i t h A l a - G l u - d i a m i n o p i me1 i c a c i d - A l a c r o s s - l i n k s bound t o t h e p o l y s a c c h a r i d e c h a i n v i a t h e muramic a c i d c a r b o n y l groups; see i n s e r t i n spectrum

SAMPLE O R I G I N

: g i f t f r o m D r . J.T.M. Wouters, Lab. o f M i c r o b i o l o g y , U n i v . o f Amsterdam, The Netherlands

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 5.5

x lo4

REMARKS - Sample p r e p a r e d f o l l o w i n g r e f . 172. The p o l y s a c c h a r i d e p a r t o f t h i s g l y c o c o n j u g a t e can be regarded as a c h i t i n c h a i n w i t h 0 - l a c t i c a c i d s u b s t i t u e n t s on a l t e r n a t i n g monomeric r e s i d u e s . The spectrum i s dominated by N - a c e t y l sugar fragments f r o m t h e GlcNAc and MurNAc r e s i d u e s (compare w i t h c h i t i n Spectrum A.13). No s t r o n g s i g n a l s a r e found which can be unambiguously a s c r i b e d t o t h e amino a c i d r e s i d u e s . A p p a r e n t l y t h e s m a l l N - c o n t a i n i n g p y r o l y s i s fragments o f t h e s e a l i p h a t i c amino a c i d s c o n t r i b u t e t o t h e same mass values as t h e amino sugars.

147

SPECTRUM A , 2 5 ~

59

43 P

56

20

60

40


80

100

'*O m/z 140

160

: PEPTIDOGLYCAN (from BaciZZus subtiZis) : poly[(l4)~GlcNAc~(l+4)MurNAc6l w i t h Ala-Glu-

SAMPLE ORIGIN

:


: : :

diaminopimelic acid-Ala cross-links, bound t o the polysaccharide chains via the muramic acid carbonyl groups; see i n s e r t in spectrum A.25 g i f t from Dr. J.T.M. Wouters, Lab. of Microbiology, Univ. of Amsterdam, The Netherlands suspension in PBS buffer; 10 ug standard 1 X 105

- Sample prepared following r e f . 172. In comparison with the spectrum of the methanol suspension (Spectrum A.25) t h i s spectrum shows a marked peak a t m/z 56 possibly derived from the a l i p h a t i c amino acid and/or the Mur-lactic acid moieties. Also note the increased i n t e n s i t y a t m/z 125 as well as the decreased i n t e n s i t i e s a t m/z 95 and 109 a s compared t o Spectrum A.25. Peaks a t m/z 36, 38 represent HClt' (PBS b u f f e r ) . REMRKS

148

SPECTRUM A 26 I

1126

Gal/3[l-3)GalNAc/?(l~4~Gal/?~l-4)Glc~-cf?ramide

: >

t-4

ln

z

I

8

43

w I-

z Z

0 J

a

32

E 2

s

;i

20

40

SAMPLE CHEMICAL DETAILS SAMPLE ORIGIN SAMPLE PREP./SIZE PYROL. CONDITIONS TOTAL ION COUNTS

144

60

80

100

1zu

14u

IOU

m/r

: TETRAHEXOSYLCERAMIDE (gl ycosphi ngol i pi d ) : see i n s e r t i n spectrum : g i f t from Dr. R.W.

Veh, R u h r Universitkit, Bochum, GFR : suspension in PBS buffer; 10 ug : standard : 2 x lo5

- Sample prepared from bovine brain. This glycolipid spectrum i s dominated by l a r g e peaks a t m/z 97, 98, 110 and 126, a l l of which appear t o be derived from the hexose and N-acetylhexosamine moieties. The o r i g i n of t h e unusually l a r g e peak a t m/z 136 i s y e t unknown. Although contributions from t h e l i p i d moiety might be expected t o appear, e.g. a t m/z 28, 42, 56, 70 and 84 (alkene fragments), such mass peaks a r e not pronounced.

REmRm

149

SPECTRUM

A 27

Galp(l-3)GalNAcp(l-4)Gal~~l~4)Glcp-Ceramide NeuAcu(2-3)

T

t

NeuAca(2+8)NeuAca(2 - 3 )

43 I

97

73

55

20

40

60

120

100

80

SAMPLE

: GANGLIOSIDE GT1

CHEMICAL DETAILS

: see i n s e r t i n spectrum

SAMPLE O R I G I N

: g i f t f r o m D r . R.W.

SAMPLE PREP./SIZE

: suspension i n PBS buffer;

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 3

m/z

140

160

( T r i s i a l o s y l -tetraglycosylceramide) Veh, Ruhr U n i v e r s i t z t , Bochum, GFR

10 pg

x lo5

- Sample prepared f r o m b o v i n e b r a i n . Compared w i t h t h e p a t t e r n o f t h e a s i a l o - a n a l o g u e (Spectrum A.26) t h e spectrum o f t h i s g a n g l i o s i d e shows s t r o n g l y i n c r e a s e d s i g n a l i n t e n s i t i e s a t m/z 59, 67, 123, 135 and 151. T h i s appears t o r e f l e c t t h e presence o f t h e N - a c e t y l n e u r a m i n i c a c i d ( s i a l i c a c i d ) r e s i d u e s Furthermore, t h e l o w e r i n t e n s i t i e s a t m/z 97, 98, 110, (see Spectrum A.23). 125 and 126 r e f l e c t t h e decreased percentage o f hexose and N-acetylhexosamine r e s i d u e s . As i n t h e case o f Spectrum A.26 t h e l i p i d m o i e t y is h a r d l y expressed i n t h e spectrum.

REMARKS


GROUP B PEPTIDES AND PROTEINS


153

SPECTRUM B , 1

117

69

SAMPLE

: ADRENOCORTICOTROPHIC HORMONE, fragment ACTH 1-24

CHEMICAL DETAILS

: H-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-

SAMPLE O R I G I N

: g i f t f r o m D r . H.M.

SAMPLE PREP./SIZE

: suspension i n PBS; 10 pg

Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-OH

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

:

Greven, Organon, Oss, The N e t h e r l a n d s

z x lo5

REMARKS - The almost complete absence o f a peak a t m/z 34 (H2S") i s due t o t h e l a c k o f Cys r e s i d u e s . The r e l a t i v e l y s m a l l amount o f H2S i s formed f r o m Met r e s i d u e s ( r e f . 58) which m a i n l y y i e l d s CH3SHf' (m/z 48). The p r o m i n e n t peak a t m/z 69 p o i n t s t o t h e h i g h Lys and Pro c o n t e n t s . The peak a t m/z 60 ( i n combinat i o n w i t h m/z 43) i s t h o u g h t t o r e p r e s e n t a c e t i c a c i d d e r i v e d f r o m t h e a c e t a t e c o u n t e r i o n s on t h e NH2-functions o f t h e Lys and Ar3 r e s i d u e s . The r e l a t i v e l y i n t e n s e peak a t m/z 117 i n combination w i t h t h e peak a t m/z 131 i s m a i n l y due t o ( m e t h y 1 ) i n d o l e d e r i v e d f r o m t h e Trp r e s i d u e .

154

SPECTRUM B,2

>8 c fn

17

z -z z 0 W

c

I

a

6 54 c

s

J 60

30

D

'

94

I

40

60

80

100

L

liO m;z

140

SAMPLE

: ADRENOCORTICOTROPHIC HORMONE, fragment ACTH 1-10

CHEMICAL DETAILS

: H-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-OH

SAMPLE O R I G I N

: g i f t f r o m Dr. H.M.

SAMPLE PREP./SIZE

: suspension i n PBS; 10 p 9

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 2

,

160

Greven, Organon, Oss, The N e t h e r l a n d s

x lo5

REMRKS - Compare w i t h Spectra 8.1 and B.3. T h i s spectrum i s now e n t i r e l y dominated by massive Met and T r p fragment peaks a t m/z 48 ( m e t h a n e t h i o l ) and m/z 117 ( i n d o l e ) , r e s p e c t i v e l y . The a c e t i c a c i d peaks (m/z 60, 43) a r e reduced w i t h r e s p e c t t o Spectrum B . l due t o t h e absence o f Lys and t h e l o w e r Arg c o n t e n t . The s m a l l peak a t m/z 64 may p o i n t t o s l i g h t o x i d a t i o n o f Met r e s i d u e s ( r e f . 96).

155

SPECTRUM B,3

J

u

69

1

Irl I


:

SAMPLE ORIGIN SAMPLE PREP./SIZE PYROL. CONDITIONS TOTAL ION COUNTS

:

83 94

I

80

"0 m/z 140

I

160

ADRENOCORTICOTROPHIC HORMONE, fragment ACTH 11-24

: H-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-

Pro-OH gift from Dr. H.M. Greven, Organon, Oss, The Netherlands : suspension i n PBS; 10 ug : standard : 2.1 x lo5

- Compare with Spectra 6.1 and 8.2. For dominant peaks at m/z 60, 43 see remarks on Spectrum B.l. Spectra B.2 and B.3 when compared with Spectrum B.l show a high degree o f additivity.

REMARKS

SPECTRUM B,4 3

4

s

1 8

43

60 83 71

I

17 120

40

60


:


:

:

: : :

80

100

120

m/z

140

160

INSULIN (bovine) for amino acid composition,see Table 13; proinsulin-like components 5 1 ppm; total salts < 2% Novo Res. Inst., Bagsvaerd, Denmark suspension in methanol; 10 ug standard 3 x lo5

- The pyrolysis pattern is dominated by fragments derived from the aromatic amino acids Phe and Tyr (m/z 92, toluene; m/z 94, phenol; m/z 108, cresol). The lack of Met residues explains the almost complete absence of a peak at m/z 48 (CH3SH"). The minor peak at m/z 117 should be interpreted as phenylacetonitrile from Phe, as Trp residues (yielding indole at this mass value) are absent (ref. 46). The marked peak at m/z 76, tentatively assigned to CS2" probably involves the Cys-Cys-Cys tripeptide linkage region between the A and B chains.

REMARKS

157

SPECTRUM

B,4~

1”4

1 I

43

56 I 71

I

J7

120

1 40

J, 60

80

100

120

m/z

140

160

SAMPLE

: INSULIN ( p o r c i n e )

CHEMICAL DETAILS

: f o r amino a c i d c o m p o s i t i o n see T a b l e 13; p r o i n s u l i n - l i k e components 5 1 ppm; t o t a l s a l t s < 2%

SAMPLE O R I G I N

: Novo Res. I n s t . ,

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL I O N COUNTS

: 3.5 X 105

Bagsvaerd, Denmark

- Note t h a t t h i s spectrum i s v i r t u a l l y i d e n t i c a l t o t h a t o f b o v i n e i n s u l i n (Spectrum 8.4). The o n l y s i g n i f i c a n t d i f f e r e n c e s i n r e l a t i v e peak i n t e n s i t i e s a r e f o u n d a t m/z 44, and 60 and a r e p r o b a b l y connected w i t h t h e s u b s t i t u t i o n o f Thr and I l e r e s i d u e s f o r A l a and Val r e s i d u e s r e s p e c t i v e l y i n t h e A chain.

REMARKS

158

SPECTRUM B,5

20

40

60

80

100

120

miz

SAMPLE

: RIBONUCLEASE-A (from bovine pancreas)


: f o r amino a c i d composition see Table 13

SAMPLE PREP./SIZE

: suspensionsin methanol; 10 ug

PYROL. CONDITIONS

: standard 5 : 1.1 X 10

TOTAL I O N COUNTS

140

'

160


REM~.RK,S- The absence o f Trp residues (producing i n d o l e and Me-indole) i s i n d i cated by t h e very low peaks a t m/z 117 and 131, whereas the high abundance o f Cys, and t o a l esser e x t e n t Met residues,is evidenced by the high i n t e n s i t i e s a t m/z 34 (H 9') and 48 (CH3SH+'). Although most RNase samples c o n t a i n a few p e rcent o$ hexose and N-acetyl hexosamine residues, t h i s carbohydrate content does n o t c o n t r i b u t e s i g n i f i c a n t l y t o t he spectrum as judged by t h e absence of pronounced peaks a t m/z 32 and 126.

159

SPECTRUM B,6

S

34

I

44 1 I

4 20

40

60

80

100

120

140

160

rn/t

SAMPLE

: DEOXYRIBONUCLEASE I (from bovine pancreas)


: f o r amino a c i d composition see Table 13 : 8DH Chemicals Ltd., Poole, U K

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 1.7 X 105

B E W E - I n comparison w i t h RNase (Spectrum B.5) t h e decrease i n Cys and Met content as w e l l as t h e appearance o f Trp residues produce t h e expected changes a t m/z 34, 48, 117 and 131. I t should be noted, however, t h a t t h e marked d i f f e r e n c e s i n t h e percentage c o n t r i b u t i o n o f some a l i p h a t i c amino a c i d r e s i dues, e.g. Leu, I l e a Val and A l a produce f a r l e s s c l e a r l y d e f i n e d d i f f e r e n c e s i n t h e spectrum. The peak a t m/z 64 (S02+* and/or CH3SOH”) may p o i n t t o s l i g h t o x i d a t i o n o f sulphur-containing amino a c i d residues.

SPECTRUM B,7 3.3%)

117

4 3

1

S

48 131

40

60

80

100

120

m/z

140

160

: LYSOZYME ( f r o m c h i c k e n egg y o l k )

CHEMICAL DETAILS

: f o r amino a c i d c o m p o s i t i o n see Table 13

SAMPLE O R I G I N


SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 1 .z

x lo5

- Most n o t a b l e a r e t h e v e r y l a r g e peaks a t m/z 34, 117 and 131 i n d i c a t i v e o f t h e h i g h Cys and T r p c o n t e n t .

REMARKS

161

SPECTRUM B,8

44

I

56 7

119

0

60

40

80

100

120

m/z

SAMPLE

: HEMOGLOBIN ( f r o m bovine e r y t h r o c y t e s )

CHEMICAL DETAILS


SAMPLE O R I G I N

: J.T.

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 1

140

160

Baker Chemicals, P h i l l i p s b u r g , NJ, USA

x lo5

- T h i s p r o t e i n shows a r a t h e r balanced p a t t e r n . S p e c i a l l y n o t e w o r t h y i s t h e obvious absence o f peaks which can be d i r e c t l y a s c r i b e d t o t h e heme m o i e t y . However, i t s h o u l d be r e a l i z e d t h a t t h e p o r p h y r i n u n i t r e p r e s e n t s o n l y about 1% by w e i g h t o f t h e o r g a n i c m a t e r i a l . Noreover, t h e heme m o i e t y tends n o t t o g i v e c h a r a c t e r i s t i c fragments, e.g. p y r r o l e and p y r r o l i n e d e r i v a t i v e s , such as formed f r o m b i l i r u b i n (Spectrum G.lO).

REMARKS

162

SPECTRUM B,9 6--

z

5

4-

94

w I-

1 3

z z

92

0

117

J

e2 2- ,

s

20

1

40

60

80

100

120

m/z

SAMPLE

: CYTOCHROME C (from equine h e a r t )

CHEMICAL DETAILS


SAMPLE O R I G I N

: Boehri nger, Mannheim, GFR

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 1

x

140

160

lo5

REMARE - High i n t e n s i t i e s a t m/z 69, 83 w i t h respect t o 6.8) may p o i n t t o t h e h i g h Lys content; t h e h i g h e r peaks h i g h e r T y r content and t h e lower peak a t m/z 119 t o l e s s hemoglobin no marked c o n t r i b u t i o n o f t h e p o r p h y r i n group

hemoglobin (Spectrum a t m/z 94, 108 t o t h e Phe. As i n t h e case of appears t o be v i s i b l e .

163

SPECTRUM B , l o

56

20

60

40

80

100

120

m/z

140

SAMPLE

: MYOGLOBIN, Type 1 ( f r o m e q u i n e s k e l e t a l muscle)

CHEMICAL DETAILS

: f o r amino a c i d c o m p o s i t i o n see T a b l e 13

SAMPLE O R I G I N


SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 4

x

160


lo4

- The v e r y low peak a t m/z 34 i n d i c a t e s t h e absence o f Cys r e s i d u e s and p r o b a b l y r e p r e s e n t s a m i n o r fragment o f Met ( m a i n l y p r o d u c i n g m e t h a n e t h i o l , m/z 48). As i n t h e case o f Cytochrome C (Spectrum B.9) t h e l a r g e peaks a t m/z 69 and 83 p r o b a b l y r e p r e s e n t t h e abundant Lys r e s i d u e s .

REMRES

SPECTRUM B , 11 (1L%I

3s4

J

43

60 56

4L

20

40

I

60

SAMPLE

: KERATIN

80

100

120

m/z

CHEMICAL DETAILS


SAMPLE O R I G I N

: Merck AG, Darmstadt, GFR

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 1

140

160

x lo5

REMARKS - T h i s spectrum shows t h e e x t r e m e l y h i g h H2St‘

peak a t m/z 34 so c h a r a c t e r i s t i c f o r t h e abundant Cys r e s i d u e s i n k e r a t i n . A l t h o u g h t h i s peak i s o f f - s c a l e i t s a c t u a l h e i g h t can be j u d g e d f r o m t h e i s o t o p e peak a t m/z 36 ( 5 % 3% abundance). The h i g h Cys c o n t e n t a l s o appears t o be r e s p o n s i b l e f o r t h e marked peak a t m/z 76 t h o u g h t t o r e p r e s e n t CS2”.

165

SPECTRUM B , 12

20

40

60

80

100

120

m/z

SAMPLE

: COLLAGEN (from bovine A c h i l l e s tendon)

CHEMICAL DETAILS


SAMPLE O R I G I N

: Koch-Light Ltd.,

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL I O N COUNTS

: 6 X lo4

-

140

160

Colnbrook, Bucks., UK

REUKS T h i s s p e c t r a l p a t t e r n i s very unusual compared t o those o f most o t h e r p r o t e i n s due t o t h e extremely h i g h content o f Gly, Ala, Pro and Hyp residues, t h e complete absence o f Trp and t h e v e r y low c o n t r i b u t i o n s o f aromatic and Sc o n t a i n i n g amino a c i d residues. The a l i p h a t i c c h a r a c t e r o f t h i s p r o t e i n causes t h e dominance of small p y r o l y s i s and E I fragments ( w i t h exception o f t h e marked p y r r o l e and d i h y d r o p y r r o l e peaks a t m/z 67, 69 and 83 from t h e Pro, Lys and Hyp m o i e t i e s ) . Small amounts o f carbohydrate a r e i n d i c a t e d by peaks a t m/z 31, 32, 96, 98, 110 and 124.

SPECTRUM B 13 I

6.6%)

1 1

i0 A

117

! i

I

a S

20

6769

40

60

80

100

120

m/z

140

160

SAMPLE

: CERULOPLASMIN ( p o r c i n e )

CHEMICAL DETAILS


SAMPLE O R I G I N

: g i f t from Dr. R. Wever, I n s t . o f Biochemistry, Univ. o f Amsterdam, The Netherlands

SAMPLE PREP./SIZE

: s o l u t i o n i n H20, 10 pg

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 3.4

x lo5

REMARKS - The aromatic character o f t h i s g l y c o p r o t e i n i s expressed by t h e h i g h peaks a t m/z 92, 94, 108, 117 and 131. Although carbohydrate c o n s t i t u t e s up t o 7% by weight no obvious sugar s i g n a l s a r e present. Peaks a t m/z 43 and 60 are thought t o stem m a i n l y from acetate.

GROUP C NUCLEOTIDES AND NUCLEIC A C I D S


169

SPECTRUM C,1 9f 6 > t m

z

84

W

I-

z Z L

s

J

58

a

I-

s2

8

* '"114

*

126

I

20

40

60

80

100

* 120

m/z

140

SAMPLE

: RIBONUCLEIC A C I D (RNA) ( f r o m b a k e r ' s y e a s t )

160

-

CHEMICAL DETAILS

:

SAMPLE O R I G I N

: A l d r i c h Chem. Corp. I n c . , Milwaukee, USA

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL I O N COUNTS

4 : 3.5 X 10

REMARKS - As discussed i n P a r t I,S e c t i o n 3.3, C u r i e - p o i n t p y r o l y s i s mass s p e c t r a of RNA a r e c o m p l e t e l y dominated by r i b o s e m o i e t y fragments (compare a l s o w i t h Spectrum C.2 (DNA)). S i g n a l s which can be a s c r i b e d t o complete base m o i e t i e s o r base m o i e t y fragments a r e absent due t o t h e loss o f more o r l e s s i n t a c t base m o i e t i e s by condensation on t h e w a l l o f t h e r e a c t i o n t u b e w i t h an e x c e p t i o n o f t h e peak a t m/z 17 which may r e s u l t f r o m deamination o f base m o i e t i e s . Such base fragments become more prominent when t h e e n t i r e p y r o l y s a t e t r a n s f e r l i n e i s heated ( r e f . 205) i n d i c a t i n g t h a t i n t h e p r e s e n t spectrum t h e absence o f such fragments i s due t o condensation l o s s e s . The marked peaks a t m/z 110, 126 and 128 p o i n t t o t h e presence o f r e s i d u a l hexose i m p u r i t i e s i n t h i s sample.

SPECTRUM C,2 '0

6 82

20

40


L.lh-60

I_ 80

100

120

m/z

140

160

: DEOXYRIBONUCLEIC ACID (DNA) , Na-salt (from calf thymus) :

-

Sigma Chemical Corp., St. Louis, USA : suspension in methanol; 10 ug : standard :

: 1

x

lo5

- The spectrum is dominated by deoxyribose moiety fragments, as discussed in Part I, Section 3.3 (compare also with Spectrum C.l (RNA)). The peak at m/z 116 is often more pronounced in other DNA preparations. Note the characteristic (methy1)furan condensation products at m/z 148, 160 and 162, also described by Schulten e t aZ. (ref. 62). At present it i s not certain whether these products originate from recombination of (methy1)furan radicals or through successive eliminations of base and phosphate residues from the polymer chain.

REMARKS

171

SPECTRUM C,3

20

40

SAMPLE

60

80

100

: ADENOSINE-5'-PHOSPHATE (5'-AMP)

120

rniz

, free

CHEMICAL DETAILS

: see i n s e r t i n spectrum; CIOHl4N5O7P;

SAMPLE O R I G I N

: Serva, H e i d e l b e r g , GFR



PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 4

140

160

acid

MW = 347

x lo4

REMARS - As i n t h e spectrum of RNA (Spectrum C . l ) , r i b o s e

m o i e t y fragments a r e a l m o s t e x c l u s i v e l y p r e s e n t i n t h i s spectrum. The peak a t m/z 114 can be r a t i o n a l i z e d as r e p r e s e n t i n g t h e l o s s of water f r o m t h e r i b o s e m o i e t y . Compare a l s o w i t h t h e spectrum o f t h e N a - s a l t o f 5'-AMP (Spectrum C.4).

SPECTRUM C,4 43

J

OH

OH

J

82

1 40

20

60

80

100

SAMPLE

: ADENOSINE-5’-PHOSPHATE (5’-AMP),

CHEMICAL DETAILS

: see i n s e r t i n spectrum;

SAMPLE O R I G I N

: Serva , Heidel berg, GFR

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 3.2

-

120

m/z

140

160

Na-sal t

C10H,2N5Na207P; MW = 391

x lo5

REMARKS I n comparison w i t h t h e spectrum o f t h e corresponding f r e e a c i d (Spectrum C.3) marked q u a n t i t a t i v e changes are observed a t m/z 43, 60, 72, 82, 85 i n d i c a t i n g t h e importance o f t h e i o n i c s t a t u s o f t h e compound.

173

SPECTRUM C , 5 9

116%)

98 / t

"

O

0C

d OH

I

68

HO-P=O

I

OH

58

114

20

60

40


80

100

120

m/z

140

160

ADENOSINE-3'-PHOSPHATE (3' -AMP), f r e e a c i d C10H14N507P; MW = 347 : Serva, H e i d e l b e r g , GFR

:

: see i n s e r t i n spectrum;

: suspension i n methanol, 10 ug : standard : 5

x lo4

- In comparison w i t h t h e spectrum o f 5'-AMP (Spectrum C.3) t h e i n t e n s i t y o f t h e peaks a t m/z 85 and 114 i s reduced whereas a m a r k e d l y h i g h e r peak i s observed a t m/z 84. T h i s i n d i c a t e s t h e s t r o n g i n f l u e n c e o f t h e p o s i t i o n o f t h e phosphate group on t h e p y r o l y t i c f r a g m e n t a t i o n pathways.

REMARKS

SPECTRUM C,6 I12 .o

%,I

96 98

O=P-O

OH

I

OH

I 114

20

40

60

80

100

SAMPLE

: ADENOSINE-3' ,5'-PHOSPHATE

120

( c y c l i c AMP)

CHEMICAL DETAILS

: see i n s e r t i n spectrum; C10H12N506P;

SAMPLE O R I G I N


SAMPLE PREP./SIZE

: suspension in methanol, 10 v g

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 4

-

x

m/z

140

, free

160

acid

MW = 329

lo4

T h i s spectrum c l o s e l y resembles t h a t of 3'-AMP (Spectrum C.5), a l - ' t h o u g h t h e i n t e n s i t y o f t h e peak a t m / t 114 appears t o be f u r t h e r reduced, p o s s i b l y as a r e s u l t o f b l o c k i n g o f t h e e l i m i n a t i o n o f t h e r i b o s e m o i e t y f r o m t h e m o l e c u l e d e s c r i b e d by Posthumus e t aZ. ( r e f . 7 2 ) .

REMARE

175

SPECTRUM C,7 3.2%)

'0

I OAN

0 OH

J 162

20

60

40


: : : : : :

L

80

116

100

120

m/z

140

THYMIDINE-5'-PHOSPHATE (5'-TMP), Na-sal t see insert in spectrum; C10H13N2Na208P; MW Serva, Heidelberg, GFR suspension in methanol, 10 p g standard 1.2 X 105

=

160

366

- As is to be expected this pattern closely resembles that of DNA (see Spectrum C. 2) ; both spectra are dominated by deoxyribose moiety fragments. Note the marked peak intensity at m/z 162 apparently caused by recombination of methylfuran radicals. Occurrence of such recombination reactions is rarely observed in filament pyrolysis and can be further reduced by using smaller sample amounts.

REMARKS

SPECTRUM C,8 0 II QC f .-

Qc)

100%)

b - -cd ONa I

ONa

-P-0 -P-0

II

II

0

0

ONa I

OH

6H

OH

22

106

I

0-P-ONa

0

96 I

!

J

4

20

40

60

80

100

120

..41 m/z

140

160

SAMPLE

: NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE, reduced ( NADPH)

CHEMICAL DETAILS

: see i n s e r t i n spectrum; C21H26N7Na4017P3; MW = 833

SAMPLE O R I G I N


SAMPLE PREP./SIZE

: suspension i n methanol, 10 pg

PYROL. C O N D I T I O N S

: standard

TOTAL ION COUNTS

: 3

x lo5

REMARKS - A p a r t f r o m t h e t y p i c a l r i b o s e fragment s e r i e s (compare S p e c t r a C . l , C.3) t h i s spectrum shows marked c o n t r i b u t i o n s f r o m t h e n i c o t i n a m i d e (m/z 79, 95, 104, 106, 1 2 2 ) and adenine (m/z 135) m o i e t i e s . The presence o f adenine i s e s p e c i a l l y n o t e w o r t h y s i n c e no adenine s i g n a l i s observed i n Py-mass s p e c t r a of A p p a r e n t l y , i n t h e l a t t e r cases no f r e e base DNA o r AMP (see S p e c t r a C.2-C.4). m o i e t i e s a r e produced as discussed i n P a r t I, S e c t i o n 3.3.

177

SPECTRUM C m 9 0

3 ONa

bo I

-P-0 II

0

0

OH

z

OH

OH

OH

9E 68

h,

20

40


60

J

80

100

120

rntz

140

160

NICOTINAMIDE ADENINE DINUCLEOTIDE, reduced (NADH) MW = 7 0 9 : Boehringer, Mannheim, GFR : suspension i n methanol; 10 119 :

: see i n s e r t i n spectrum; C2,H27N7Na20,4P2;

: standard : 2

x

lo5

REMARKS - I n comparison w i t h the-srpectrum o f NADPH (Spectrum C . 8 ) , t h e absence o f t h e phosphate group i n p o s i t i o n 2 o f t h e a d e n i n e - b e a r i n g r i b o s e m o i e t y causes marked q u a n t i t a t i v e d i f f e r e n c e s i n 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 peak s e r i e s o f t h e r i b o s e , adenine and n i c o t i n a m i d e m o i e t i e s .


GROUP D LIPIDS


181

SPECTRUM D,1 37

,

20

60

40

80

100

120

SAMPLE

:

CHEMICAL DETAILS SAMPLE ORIGIN SAMPLE PREP./SIZE PYROL. CONDITIONS TOTAL ION COUNTS

: CH3(CH2)10CH20S03Na; MW = 288 : Hahmes, Maastricht, The Netherlands :

m/z

140

160

SODIUM LAURYL SULPHATE

suspension in methanol; 20 pg

: standard : 1 x lo5

- The spectrum is dominated by two homologous molecular ion series, namely at m/z 42, 56, etc. (alkenes) and at m/z 54, 68, etc, (alkadienes and/or alkynes) as well as two homologous fragment ion series at m/z 43, 57, etc. (alkyl fragments) and at m/z 55, 69, etc. (alkenyl fragments). Comparison with polyethylene (Spectrum H . l ) shows that the alkenyl and alkadiene (alkyne) ion series are much more pronounced, probably as-a result of unsaturation o f the aliphatic hydrocarbon chain upon pyrolytic elimination o f the sulphonate end group. Note the complete absence of obvious aromatic and sulphur-containing ion series.

REM4RKS

SPECTRUM D,2

20

40

60

80

100

140

120

160

m/z


: LINOLEIC ACID


: Lipid Supplies, St. Andrews Univ., UK

: cis-9, cis-12 octadecadienoic acid; C18H3202; MW = 280 : solution in methanol; 10 ug : ferromagnetic tube; T

5

: 2.6 X 10

eq

770°C

- Compared t o sodium lauryl sulphate (Spectrum D.l), a saturated a l i phatic acid, the l i n o l e i c acid pattern shows a higher abundance of relatively short chain polyunsaturated hydrocarbon ion s e r i e s . To some extent t h i s d i f f e r ence may be due t o the e f f e c t of oven pyrolysis. Also noteworthy i s the absence of prominent fragment ion s e r i e s , apparently as a r e s u l t of the low abundance of l a b i l e , saturated, long-chain hydrocarbons i n the pyrolysate. Whether the ion s e r i e s a t m/z 78, 92, e t c . represents aromatic or multiple unsaturated a l i p h a t i c ions cannot be decided from t h i s spectrum.

REMARKS

183

SPECTRUM D,3

_-.--

20

60

40

C

80

100

120

m/z

140


: a-LINOLENIC ACID METHYL ESTER


: Unilever Res. Lab., Vlaardingen, The Netherlands

-

160

: cis-9, cis-12, cis-15 octadecatrienoic acid methyl e s t e r ;

C19H3202; MW = 292

: solution in methanol; 10 pg

: ferromagnetic tube; T : 1.7

x

lo5

eq

770°C

The strong homologous ion s e r i e s a t m/z 66, 80, e t c . may well represent cyclic dienes and/or aliphatic trienes, especially i n view of the multiple unsaturation i n the parent molecule. A minor series of homologous ions a t m,’z 87, 101, e t c . , also observed in the spectrum o f y-linolenic acid methyl e s t e r (Spectrum D.4), could be connected with the presence of the methyl e s t e r function.

REMARG

SPECTRUM D,4

i

20

40

60

80

100

120

m/z

140

160


: y-LINOLENIC A C I D METHYL ESTER : cis-6, cis-9, cis-12 o c t a d e c a t r i e n o i c a c i d methylester; C19H3202; MW = 292

SAMPLE O R I G I N

: Unilever

SAMPLE PREP./SIZE

: s o l u t i o n i n methanol; 10 ug

PYROL. CONDITIONS

: ferromagnetic tube;

TOTAL I O N COUNTS

: 1.5 X 10

Res. Lab.,

5

Vlaardingen, The Netherlands

T

eq

770°C

- Noteworthy i s the strong d i f f e r e n c e o f t h i s spectrum and t h a t o f the a - l i n o l e n i c a c i d analogue (Spectrum D.3) p o i n t i n g t o t h e s t r o n g i n f l u e n c e of t h e p o s i t i o n o f the double bond on the p y r o l y t i c fragmentation pathway.

REMRILS

185

SPECTRUM D15 10.7%)

54.

1

I

,’

20

40

SAMPLE CHEMICAL DETAILS SAMPLE ORIGIN SAMPLE PREP./SIZE PYROL. CONDITIONS TOTAL ION COUNTS REMARKS -

80

60

100

120

140

160

miz

OCTADECADIYNOIC ACID CH ,(CH2)7-CaC-CH2-C3C-(CH2)4-COOH; Ci8H2802; MW3= 276 : Unilever Res. Lab. Vlaardingen, The Netherlands : solution in methanol; 10 vg : ferromagnetic tube; Teq 770°C

: :

:

5

1.5 X 10

Surprisingly, multiply unsaturated homologous ion series are not as prominent as might be expected on the basis o f the presence o f the two triple bonds in the parent molecule.

SPECTRUM D,6 18

I::

HCO -C-(CH&-CH3

I

I I :

H2CO -C-(CH2),-CH,

56 1

I

J

127

LL-4 20 4 SAMPLE CHEMICAL STRUCTURE

: see i n s e r t i n spectrum; (C7H15C00)3C3H5; MW = 470

SAMPLE O R I G I N


SAMPLE PREP./SIZE

: s o l u t i o n i n methanol; 10 pg

PYROL. CONDITIONS

: f e r r o m a g n e t i c tube; T 770°C 5 eq : 3.5 X 10

TOTAL I O N COUNTS

- A prominent s e r i e s o f m o l e c u l a r i o n peaks r e p r e s e n t s alkenes up t o C7H14 (m/z 98). I n a d d i t i o n , marked E I fragments a r e observed a t m/z 67 and 127. Note t h a t t h e peak a t m/z 67 i s a l s o p r e s e n t i n t h e s p e c t r a o f o t h e r g l y c e r i d e s (see S p e c t r a D.7 and D.8). The peak a t m/z 127 p r o b a b l y r e p r e s e n t s an E I fragment o f t h e i n t a c t f a t t y a c i d m o i e t y . REMARKS

187

SPECTRUM D,7 0

I1

1 8 \

H 2 C 0 -C-(CHZ),,,-CH3

I ?

\

HCO- C-(CHz),o-CH,

I 1

/

\ll*

100

120

m/z

140

SAMPLE

: TRILAURIN


: see i n s e r t i n spectrum; (Cl,H23C00)3C3H5; MW = 638


: solution in methanol; 10 pg : ferromagnetic tube; Teq 770°C : 4 x lo5

160

: Serva, Heidelberg, GFR

- Up to m/z 100 the spectrum i s very similar to t h a t o f tricaprylin (Spectrum 0 . 6 ) . The alkene s e r i e s of fragments, however, now appears t o extend i t s e l f to CllH22+' (m/z 154). Also the absence o f the prominent fragment i o n a t m/z 127 i s noteworthy.

REMRXT

SPECTRUM D,8

0

II H,CO-C-(CH,)

-CH3

14

I B

HCO -C-(CH,/4-CH3

I::

HaCO -C-(CH2),qCH3

\

\12

20

40


60

80

0

120

m/z

140

160

: TRIPALMITIN : see insert in spectrum;

(C15H31COO)3C3H5; MW

= 806


in methanol; 10 ug : ferromagnetic tube; T 77OOC eq : 2.7 x lo5 : solution

- This spectrum is virtually indistinguishable from that o f trilaurin (Spectrum D.7) within the mass range scanned.

REMARKS

189

SPECTRUM D 9 I

J

/

/ ! \

\

\ \

, \

\

', $09

'\

20

40

J

60

80

100

120

m/z

CHEMICAL DETAILS

: CHOLESTEROL : see i n s e r t i n spectrum; cholest-5-en-36-01; MW = 386

SAMPLE O R I G I N

: O.P.G.,

SAMPLE PREP./SIZE


SAMPLE

160

C27H460;

Utrecht, The Netherlands

PYROL. CONDITIONS

: ferromagnetic tube; T

TOTAL I O N COUNTS

: 5 X 10

-

140

4

eq

770'C

REMARKS The prominent i o n s e r i e s a t m/z 28, 42, e t c . and a t m/z 54, 68, e t c . ( a p p a r e n t l y r e p r e s e n t i n g a1 kene and a1 kadiene s e r i e s o f p y r o l y s i s fragments w i t h up t o 8 C atoms), are probably d e r i v e d from the hydrocarbon side-chain o f t h e molecule, whereas t h e peaks a t m/z 95, 109 and 123 may w e l l r e p r e s e n t E I fragments from t h e more o r l e s s i n t a c t parent molecule.

SPECTRUM D , l o 36

I:")r;.

I

\

I

> 6b

I

\\\\\

Ln

z

2 z

\

\

* 40

\

-I

a +

'Vll0

e

s 2-

I

H*

\ \ \ \

60

0

'

80

100

'

'

120

'

m/z

140

'

160

: STIGMASTEROL

CHEMICAL DETAILS

: see i n s e r t i n spectrum; stigmasta-5,22-dien-38-01;


: Sigma Chemical Corp., S t . Louis, USA

C29H480; MW = 412 : solution in methanol; 10 pg : ferromagnetic tube; T : 7.5

x

lo4

eq

770°C

- In comparison w i t h cholesterol (Spectrum D.9) the homologous ion s e r i e s a t m/z 54, 68, e t c . i s now more pronounced, apparently reflecting the unsaturated nature o f the aliphatic hydrocarbon side-chain. Also an EI fragment is now observed a t m/z 81, probably derived from o l e f i n i c pyrolysis fragments, such as the molecular i o n species a t m/z 96 (compare with ergosterol (Spectrum D.11)).

REWKS

191

SPECTRUM D,11 I2 I

‘ II I

I

I

I

I I

42

I

I \

I I I I

I I I

7b I

\ I

\

\ \

/

20

40

60

100

8

120

140

160

W Z

SAMPLE

: ERGOSTEROL

CHEMICAL DETAILS

: see i n s e r t i n spectrum; C28H440; MW = 396

SAMPLE O R I G I N


SAMPLE PREP./SIZE

: s o l u t i o n i n methanol; 10 ug

PYRUL. CONDITIONS

: f e r r o m a g n e t i c tube; T 770°C eq 5 : 1.2 X 10

TOTAL I O N COUNTS

ergosta-5,7,22-trien-38-01;


REMARXS - Note t h e correspondence w i t h s t i g m a s t e r o l (Spectrum D.10) w i t h r e g a r d t o t h e homologous i o n s e r i e s a t m/z 28, 42, e t c . However, t h e s e r i e s a t m/z 54, 68, e t c . now a t t a i n s i t s maximum i n t e n s i t y a t m/z 82 i n s t e a d o f m/z 96, a p p a r e n t l y r e f l e c t i n g t h e s u b s t i t u t i o n o f a methyl group f o r t h e e t h y l group i n t h e o l e f i n i c s i d e - c h a i n . A prominent fragment i o n i s observed a t m/z 67, p r o b a b l y d e r i v e d f r o m t h e m o l e c u l a r i o n species a t m/z 82 (compare w i t h s t i g m a s t e r o l (Spectrum D. 10) )

.

SPECTRUM D ,12

20

40

SAMPLE

: L-a-LECITHIi4 ( d i p a l m i t o y l )

CHEMICAL DETAILS

: C40H820gNP; MW = 792

SAMPLE O R I G I N


SAMPLE PREP./SIZE


PYROL. CONDITIONS

: ferromagnetic tube; T 77OOC eq : 7 x lo5

TOTAL I O N COUNTS

REMARKS - Compared o t r i p a l m i t i n (Spectrum D.8), a s i m i l a r s e r i e s of alkene molecular i o n s a t m z 28, 42, e t c . i s observed, as ,well as t h e c h a r a c t e r i s t i c f a t t y a c i d e s t e r f r gment i o n a t m/z 67. The prominent s i g n a l s a t m/z 58, 59 and 71 r e p r e s e n t we 1-known p y r o l y s i s fragments of t h e c h o l i n e moiety (see P a r t I, S e c t i o n 3.5, and Spectrum D.12a). Whether the odd-numbered i o n s i g n a l s a t m/z 87 and 143 represent N-containing i o n s o r hydrocarbon fragment i o n s i s as y e t unknown.

193

SPECTRUM D,1 2 ~ (=-2O*I

71

5

O-CH~-CH,-&~CH

I

)

33

O=P-OH

I

0

5

H H2C -C-CH2 I

0 I

c=o I

(CHZl

I

CH3

89

80

II

100

.I

I

0 I

CEO

1 (CH2),4

'4

I

CH3

.

120

m/z

140

SAMPLE

: L-a-LECITHIN ( d i p a l m i t o y l )

CHEMICAL DETAILS

: see i n s e r t i n spectrum; C40H820gNP; MW = 792

SAMPLE O R I G I N


SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL I O N COUNTS

: 1.6 X

-

160

lo5

Comparison w i t h the oven p y r o l y s i s p a t t e r n (Spectrum D.12) shows t h e complete absence of a l i p h a t i c hydrocarbon s e r i e s , apparently as a r e s u l t o f condensation losses. Instead, t h i s spectrum i s e n t i r e l y dominated by t h e c h o l i n e - d e r i v e d fragments a t m/z 58, 59 and 71 thought t o represent C3H8Nt, t r i m e t h y l a m i n e and dimethylvinylamine r e s p e c t i v e l y (see P a r t I , Section 3.5). The absence o f marked peaks a t m/z 50, 52 (CH3Clt') and a t m/z 89 (N,Ndimethylethanolamine) often observed i n Py-mass spectra of c h o l i n e - c o n t a i n i n g phospholipids (see P a r t I, Figure 9 ) i s probably due t o differences i n t h e i o n i c s t a t u s o f t h e sample.

REMARKS


GROUP E NATURAL PRODUCTS


197

SPECTRUM E,1 3

32

60

1,

20

40

60

80

SAMPLE

: COTTON

CHEMICAL DETAILS

: almost pure c e l l u l o s e

100

120

rn/z

140

SAMPLE O R I G I N

: F i b r e I n s t i t u t e , TNO, D e l f t , The Netherlands

SAMPLE PREP./SIZE

: ground i n glass mortar w i t h methanol; 10 ug

PYROL. CONDITIIONS

: standard 5 : 4 X 10

TOTAL I O N COUNTS

-

160

The spectral p a t t e r n i s e n t i r e l y o f a polysaccharidic nature w i t h o u t detectable c o n t r i b u t i o n s from other p l a n t c o n s t i t u e n t s such as p r o t e i n s o r l i g n i n s . The p a t t e r n s t r o n g l y resembles t h a t o f c e l l u l o s e (Spectra A.1, A.2). Compare a l s o w i t h f l a x (Spectrum E.2).

REMARKS

SPECTRUM E n 2

60

J.20

40

60

80

SAMPLE

: FLAX


: largely cellulose

100

120

miz

140

160

: Fibre I n s t i t u t e , TNO, Delft, The Netherlands

: ground in glass mortar with methanol; 10 pg : standard : 3

x

lo5

- This spectrum shows a polysaccharide pattern virtually identical t o t h a t of cotton (Spectrum E . l ) , indicating a high degree of similarity in composition and structure between these two n a t u r a l fibers.

REMARKS

199

SPECTRUM E,3 .6%1

12

3 I

r

t ln z w

85

fi

z

55

98

z 0 2 a fi

J

4

0

126

4 60

40

80

100

120

m/2

140

160

: BURLAP


: largely cellulose

: Fibre I n s t i t u t e , TNO, Delft, The Netherlands : ground in glass mortar with methanol; 10 pg : standard : 5

x

lo5

- This spectrum also shows a typical polysaccharide pattern with only minor indications f o r the presence o f other plant materials. However, t h i s polysaccharide pattern appears to be somewhat more complex t h a n t h a t of cotton (Spectrum E.l) and flax (Spectrum E.2). For instance, the relatively h i g h peak i n t e n s i t i e s a t m/z 114 and 128 may well be derived from hemicellulose constituents (compare with the relatively high i n t e n s i t i e s of m/r 114 and 128 in the spectra o f wood (Spectrum E . l l ) and cork (Spectrum E.12).

REMaRKs

SPECTRUM E,4

43

20

40

60

SAMPLE

: SILK

80

100

120

m/z

140

CHEMICAL DETAILS

: a l m o s t pure f i b r o i n

SAMPLE O R I G I N

: F i b r e I n s t i t u t e , TNO, D e l f t , The Netherlands

SAMPLE PREP./SIZE

: ground i n g l a s s m o r t a r w i t h methanol; 10 pg

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 4

160

x lo5

- I n c o n t r a s t w i t h t h e t h r e e p r e v i o u s s p e c t r a of n a t u r a l f i b e r s ( S p e c t r a E . l - E.3) t h i s spectrum shows a t y p i c a l p r o t e i n p a t t e r n w i t h l i t t l e o r no i n d i c a t i o n of n o n - p r o t e i n c o n s t i t u e n t s . The marked i n t e n s i t y o f peaks 94 and 108 r e p r e s e n t s t h e h i g h abundance o f t y r o s i n e m o i e t i e s i n f i b r o i n .

REMARKS

201

SPECTRUM E,5 14

2

94

1 J

I6

20

J 40

117

60

80

CHEMCAL DETAILS

: SHEEP WOOL : largely keratin

SAMPLE

100

120

m/z

140

SAMPLE ORIGIN

: F i b r e I n s t i t u t e , TNO, D e l f t , The N e t h e r l a n d s

SAMPLE PREP./SIZE

: ground i n g l a s s m o r t a r w i t h methanol; 10 ug

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 2.4

-

x

160

lo5

The spectrum of sheep wool as w e l l as of most o t h e r animal h a i r s , e.g. camel h a i r (Spectrum E.6) i s c o m p l e t e l y dominated by t h e p r o t e i n p a t t e r n o f k e r a t i n (see Spectrum B.11). T h i s i s evidenced by t h e e x t r e m e l y h i g h peak a t m/z 34 (H2Sf') d e r i v e d f r o m t h e c y s t e i n m o i e t i e s which a r e u n u s u a l l y abundant i n t h i s p r o t e i n . A l t h o u g h m/z 34 has gone o f f s c a l e , i t s t r u e i n t e n s i t y can be c o n v e n i e n t l y j u d g e d f r o m t h e i n t e n s i t y o f t h e 5% i s o t o p e peak a t m/z 36 (H234S+' ) .

REMARKS'

SPECTRUM E,6

3

I7

11

J

117

\

20

40

60

80

100

120

m/Z

140

SAMPLE

: CAMEL H A I R


: largely keratin : F i b r e I n s t i t u t e , TNO, D e l f t , The Netherlands

SAMPLE PREP./SIZE

: ground i n glass mortar w i t h methanol; 10 ug


: standard : 5 x lo5

- This However, small 92 and 117 may which i s t o be

REMARKS

160

spectrum i s h i g h l y s i m i l a r t o t h a t o f sheep wool (Spectrum E.5). b u t s i g n i f i c a n t increases i n peak i n t e n s i t i e s a t m/z 30, 48, 67, represent small amounts o f non-keratin proteins, t h e presence o f expected i n animal h a i r .

203

SPECTRUM E 1 7 20.4%)

i8 4ONOMER CH

93

, \

,

-

1

9 80

-

100

120

m/t

140

SAMPLE

: NATURAL RUBBER ( p a l e crepe, I)

CHEMICAL DETAILS

: see i n s e r t i n spectrum; cis-l,4-polyisoprene

SAMPLE O R I G I N

: KRI-TNO,

SAMPLE PREP./SIZE

: ground a t minus 170°C i n methanol; 10 ug

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 8.8 X

160

D e l f t , The Netherlands

lo4

REMARKS - See P a r t I,Section 3.5. carbohydrate s i g n a l s .

The spectrum shows no obvious p r o t e i n and

204

SPECTRUM E,8 LO*/.)

1C8%)

10

126

94

J

82

I

28

J 152

I

b 120


m/z

140

160

: TANNIN : e s t e r s o f hexoses and trihydroxybenzene c a r b o x y l i c a c i d s : Merck

AG, Darmstadt, GFR

: s o l u t i o n i n methanol; 10 ug : standard

: 2.5

x lo4

REMARE - Although t h e s t r u c t u r e o f t a n n i n s i s n o t w e l l defined, t h e presence o f hydroxybenzene moieties, e.g. g a l l i c acid, i s evidenced by t h e h i g h peak i n t e n s i t i e s a t m/z 94, 110 and 126 apparently representing phenol, dihydroxybenzenes and trihydroxybenzenes r e s p e c t i v e l y . The i o n s e r i e s a t m/z 55, 68, 82, 84, 96, 98, 102, 110 and 126 may represent ( e s t e r i f i e d ) hexose m o i e t i e s .

205

SPECTRUM E,9 32 30

43

#

124

I

20

111

111

60

40


I

94

EC

80

100

120

1?R

m/z

140

160

WHEAT STRAW LIGNIN prepared according to the Bjfirkman procedure gift from Dr. K. Haider, F.A.L., Braunschweig, GFR : suspension in methanol ; 10 pg : standard : 1.5 X 105

:

: :

- The pyrolysis mass spectra of lignins are characterized by the presence of pyrolysis products derived from each of the main phenolic building blocks. In the case o f grass lignins these are the parahydroxybenzoic, quaiacylic and syringylic acid moieties, giving rise to three discrete series of phenolic signals, namely m/r 94, 108, 120, 134, m/z 124, 138, 150, 164 and m/z 154, 168, 180, 194 respectively (signals beyond m/z 160 not shown). As confirmed by the Py-GC-MS studies of Bracewell e t aZ. (ref. 186), the syringylic acid series is particularly prominent in hardwood lignins (e.g. beech lignin) but absent in softwood lignins (e.g. Douglas fir lignin). See also Part I, Section 3.6.

REMARKS

206

SPECTRUM

20

40

SAMPLE CHEMICAL DETAILS SAMPLE ORIGIN SAMPLE PREP./SIZE PYROL. CONDITIONS TOTAL ION COUNTS REMARXS

-

60

80

E,10

100

120

m/z

140

160

WHEAT STRAW LIGNIN : prepared according to the Brauns procedure : gift of Dr. K. Haider, F.A.L., Braunschweig, GFR : suspension in methanol; 10 pg : standard :

:

I.z x

lo5

Comparison of this spectrum with that of BjPrkman lignin (Spectrum

E.9) shows the strong influence of the preparation procedure on composition and

structure of the lignin obtained. This reflects a basic problem in obtaining pure, undegraded lignin samples as the lignin moieties in plant matter are intricately connected to the hemicellulose fraction (ref. 85). As shown in the spectrum of Douglas fir (Spectrum E.11) , however, characteristic lignin patterns are easily obtained from whole wood samples by Py-MS. This should enable fast characterization of lignins without lengthy and risky separation procedures as well as provide a practical guide to comparing the effectiveness and reliability of various extraction and separation techniques for lignins.

207

SPECTRUM E 11 I

4%)

85 55

I

I

I:j

98

4 40

60

80

100

120

m/z

140

160

SAMPLE

: DOUGLAS FIR WOOD

CHEMICAL DETAILS

:

SAMPLE O R I G I N

: g i f t f r o m F l a m m a b i l i t y Research Center, S a l t Lake City, USA

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 2.5

-

-

x 1o5

REMRKS The o v e r a l l p y r o l y s i s mass s p e c t r o m e t r y p a t t e r n s o f whole wood samples can be e x p l a i n e d t o a l a r g e e x t e n t by adding t h e p a t t e r n o f c e l l u l o s e (Spectrum A . l ) t o a t y p i c a l l i g n i n p a t t e r n ( S p e c t r a E.9, € . l o ) , t a k i n g i n t o account t h e softwood o r hardwood n a t u r e o f t h e sample (see Remarks on Spectrum E.9). The m a j o r peak i n t e n s i t i e s n o t e x p l a i n e d by t h i s procedure, u s u a l l y f o u n d a t m/z 114 and 128, a p p a r e n t l y r e p r e s e n t h e m i c e l l u l o s e fragments. Comparison w i t h s p e c t r a o f whole wood samples r e p r e s e n t i n g d i f f e r e n t t y p e s o f wood shows c h a r a c t e r i s t i c d i f f e r e n c e s w i t h r e g a r d t o l i g n i n and h e m i c e l l u l o s e p a t t e r n s which s h o u l d be o f c o n s i d e r a b l e p r a c t i c a l v a l u e i n i n d u s t r i a l a p p l i c a t i o n s and t e c h n i c a l uses of wood and wood products.

208

SPECTRUM E, 12 8

>.

c

Y,

z W

c

I

2 4

P

30 I

4

a t-

e

r, 96

5;

s

20

60

40

80

100

CHEMICAL DETAILS

: CORK : -

SAMPLE O R I G I N

: b o t t l e stopper

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 1

SAMPLE

120

m/z

140

160

x lo5

REMARKS - The o v e r a l l c h a r a c t e r o f t h i s spectrum appears t o be s i m i l a r t o t h a t o f whole wood (see Spectrum E . l l ) . A more c l o s e i n s p e c t i o n , however, shows t h e presence of s u l p h u r c o n t a i n i n g s i g n a l s a t m/z 34, 48, and 64 which c o u l d be d e r i v e d from s u l p h u r c o n t a i n i n g p l a n t c o n s t i t u e n t s , e.g. p r o t e i n s . A l t e r n a t i v e l y , these s i g n a l s m i g h t p o i n t t o some form o f chemical pretreatment o f t h e sample. The low i n t e n s i t i e s of t h e peaks a t m/z 102 and 126 i s a l s o noteworthy s i n c e t h i s i n d i c a t e s a much lower c e l l u l o s e c o n t e n t than i n whole wood samples such as Douglas fir (Spectrum E . l l ) . F i n a l l y the characteristic ion series a t m/z 124, 138, and 150 appears t o p o i n t t o t h e presence o f q u a i a c y l i c a c i d t y p e l i g n i n components. According t o some views, however, " t r u e l i g n i n s " a r e g e n e r a l l y n o t found i n bark ( r e f . 85).

209

SPECTRUM E n13

32

70 58

J

I

120

I:

!O

60

40

80

100

SAMPLE

: POLLEN (from Pinus s y h e s t r i s )

CHEMICAL DETAILS

120

m/z

140

160

-

SAMPLE O R I G I N

: : c o l l e c t e d i n The Netherlands

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 7.2

x lo4

REMRW- Although dominated by s i g n a l s t y p i c a l of carbohydrate products, e.g. a t m/z 32, 43, 72, 74, 84, 96, 98, 102, 110, 112, 114, 126, t h e r e a r e small b u t d e f i n i t e i o n s e r i e s which can be a t t r i b u t e d t o p r o t e i n s e.g. a t m/z 34, 43, 92, 94 and 117. Strong aromatic s i g n a l s a r e present a t m/z 94, 106, 108 and 120. E s p e c i a l l y t h e l a r g e peak a t m/z 120 i s noteworthy since i t i s a l s o found i n some s p o r o p o l l e n i n preparations ( n o t shown i n t h i s A t l a s ) and t h e r e f o r e might be r e p r e s e n t a t i v e f o r t h i s r a t h e r i l l z d e f i n e d t y p e of biopolymer ( r e f . 7 4 ) .

21 0

SPECTRUM E, 14

32

60

P

34 I

20

60

40

80

100

120

m/z

SAMPLE

: BREWER'S YEAST (Succharomycea cerevisiue)


: see NBS p u b l i c a t i o n s

SAMPLE PREP./SIZE

: suspension i n methanol; 10 vg

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 8.3

140

160

: N a t l . Bureau of Standards (SRM 1569), Washington, USA

x lo4

REMARKS - As i n t h e case o f p o l l e n (Spectrum E.13) carbohydrate s i g n a l s

dominate, e.g. a t m/z 32, 43, 55, 60, 72, 74, 84, 96, 98, 110, 112, 126, b u t p r o t e i n s i g n a l s a r e a l s o w e l l represented e.g. a t m/z 34, 48, 69, 92, 94,108, 117. It should be r e a l i z e d t h a t some o f t h e carbohydrate s i g n a l s may be d e r i v e d from t h e r i b o s e and deoxyribose m o i e t i e s o f RNA and DNA r e s p e c t i v e l y (see Spectra C . l and C.2). Under t h e Py-MS c o n d i t i o n s employed these classes o f compounds f a i l t o produce d e t e c t a b l e s i g n a l s o f t h e more c h a r a c t e r i s t i c base m o i e t i e s Also s i g n a l s r e p r e s e n t i n g l i p i d m o i e t i e s w i l l t e n d (see P a r t I, S e c t i o n 3.3). t o be underrepresented i n these s p e c t r a (see P a r t I , S e c t i o n 3.5).

211

SPECTRUM E ,15 I 'I.)

3

60

20

40

60

SAMPLE

: WHEAT FLOUR

CHEMICAL D E T A I L S

:

80

100

120

m/z

140

160

PYROL. CONDITIONS

see NBS publications : Natl. Bureau of Standards (SRM 1567), Washington, USA : suspension in methanol; 10 pg : standard

TOTAL ION COUNTS

:9

SAMPLE O R I G I N SAMPLE PREP./SIZE

x lo4

- Somewhat surprisingly, this spectrum is more similar to that of cellulose (Spectrum A.l) than that o f amylose (Spectrum A . 2 ) . No explanation for this finding can be given yet. In addition, relatively weak protein signals appear at m/z 34, 48, 92.

REMARKS

SPECTRUM E 16 I

i3

32

IL

126

20

60

40

80

100

SAMPLE

: SPHAGNUM MOSS

CHEMICAL DETAILS

:

SAMPLE O R I G I N

: Marsh by L e i f e r d e , GFR

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 7.3

-

120

m/z

140

160

-

x lo4

REMARKS This p l a n t i s o f considerable i n t e r e s t as a major source and cons t i t u e n t o f t h e extensive sphagnum peat d e p o s i t s i n some p a r t s o f t h e world. As i n most p l a n t samples, we f i n d a dominant complex carbohydrate p a t t e r n p o i n t i n g t o t h e presence o f c e l l u l o s e as w e l l as hemicellulose c o n s t i t u e n t s . Most remarkable however a r e t h e l a r g e s i g n a l s a t m/z 120 and 134. These appear t o represent fragments o f sphagnol, a p r i m i t i v e type o f l i g n i n c h a r a c t e r i z e d by t h e absence o f methoxylated phenol m o i e t i e s ( r e f . 85). Accordingly, parahydroxybenzoic a c i d m o i e t i e s a r e t h e o n l y l i g n i n b u i l d i n g blocks present, y i e l d i n g a fragment s e r i e s a t m/z 94, 108, 120, 134 (see a l s o Remarks on l i g n i n Spectra E.9 atia E.10 and on sphagnum peat (Spectrum F.15).

GROUP F

HUMIC MATERIALS AND GEOPOLYMERS


21 5

SPECTRUM F , 1 3

32

96

1l4 84

20

40

60

80

98

100

I

120

m/z

140

160

SAMPLE

: GRANITE BROWN SOIL, A-1 h o r i z o n ( t y p i c Xerochrept)

CHEMICAL DETAILS

: see r e f . 188; carbon c o n t e n t 3.5%; n i t r o g e n c o n t e n t 0.4%; pH 5.6 i n H20

SAMPLE O R I G I N

: Huelva, SW Spain

SAMPLE PREP./SIZE

: suspension i n methanol; approx. 50 ug

PYROL. CONDITIONS

: standard

TOTAL I O N COUNTS

: n o t determined

REMARKS - T y p i c a l o f a s o i l o f t h i s t y p e i s t h e extremely r i c h m i x t u r e o f

o r g a n i c compounds comprising carbohydrate (e.g. m/z 43, 72, 74, 84, 86, 96, 98, 102, 112, 114, 126, 128, 144) as w e l l as proteinaceous (e.g. m/z 34, 48, 117) and l i g n i n - t y p e (e.g. m/z 120, 124, 134, 138, 150) compounds. Compare a l s o t o bog peat (Spectrum F.14) and t o r i v e r c l a y (Spectrum F.2).

21 6

51

SPECTRUM F,2

I

70

30

20

'

40

60

80

SAMPLE

: R I V E R CLAY (Holocene)

100

140

CHEMICAL DETAILS

: see r e f . 51; 30.6% c l a y m i n e r a l s ; 0.45% o r g a n i c

SAMPLE ORIGIN

: Lienden, Marspolder, The N e t h e r l a n d s

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL I O N COUNTS

: n o t determined

160

c

R E M K S - I n c o n t r a s t t o t h e humus-rich brown s o i l (carbon c o n t e n t 3.5%) (Spectrum F . l ) , t h e r i v e r c l a y shown h e r e i s v e r y l o w i n o r g a n i c m a t e r i a l s Moreover, t h e o r g a n i c m a t t e r appears t o be composed p r i m a r i l y of (0.45% C ) . a l i p h a t i c and a r o m a t i c hydrocarbons. The main heteroatom c o n s t i t u e n t s appear t o be f o u n d i n t h e r e l a t i v e l y l o w peaks a t m/z 17 (NH3+'), m/z 64 (S02" and/or S2") and, p o s s i b l y , i n t h e i o n s e r i e s a t m/z 68, 82, 96, 110, 124 which may r e p r e s e n t compounds w i t h oxygen f u n c t i o n a l groups.

21 7

SPECTRUM F,3

*.

c m

z

2 z z 0 -I

s e

s

20

60

40

80

100

140

160

SAMPLE

: R I V E R SEDIMENT ( r e c e n t )

CHEMICAL DETAILS

: S i 0 2 51%; MgO 4%; A1 03 4%; CaO 4%; K j e l d a h l n i t r o g e n 0.08%; phosphorus 0.65%; f r e o n s o l u b l e s 1.7%

SAMPLE O R I G I N

: N a t l . Bureau o f Standards (SRM 1645), Washington, USA

SAMPLE PREP./SIZE

: suspension i n methanol; 25

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 4

REMARKS

-

x

LIB

lo4

The r e l a t i v e l y s t r o n g i n t e n s i t i e s o f s u l p h u r - c o n t a i n i n g i o n s i g n a l s a t

m/z 34, 48, 64 and 76 a r e t y p i c a l o f o r g a n i c m a t e r i a l s exposed t o aqueous environments. Note t h e r e l a t i v e monotony of t h e marked i o n s e r i e s , t h u s p r e s e n t i n g a p a t t e r n n o t u n l i k e some kerogen p a t t e r n s (e.g. Spectrum F.12).

SPECTRUM F , 4

z

J

1

114

43

1

84 821

:i '

20

40

60

80

102

100'

6

J

128

1;

J 120

m/z

140

160

SAMPLE

: SOIL POLYSACCHARIDE (water e x t r a c t a b l e f r a c t i o n from Granite Brown Soi 1 ; see Spectrum F . 1 ) .


: see r e f . 188; C 37.8%; H 6.8%; N 2.1%; 0 53.3%; ash 22%


: Huelva, SW Spain : suspension i n methanol; approx. 20 p g : standard : not determined

- The spectrum shows a pattern c o n s i s t e n t with a r e l a t i v e l y pure, b u t highly complex carbohydrate f r a c t i o n containing neutral sugar residues as well as N-acetyl aminosugars. The presence o f t h e l a t t e r i s evidenced by ion s e r i e s a t m/z 1 7 , 67, 81, 109, 125, 137 and 151. H i g h s i g n a l s a t m/z 114 and 128 appear t o i n d i c a t e the presence, of pentose and deoxyhexose residues respecti v e l y . Note t h e r e l a t i v e l y low i n t e n s i t y of sulphur-containing ion s i g n a l s a t m/z 34, 48 and 64.

REMARKS

21 9

SPECTRUM F,5

-

\

2f

I

I2

96

13

I

3

4> ! I Y,

I

w

4

z

t--

110

z z

2

4

I

2 2-,f

e

s -

J

4 20

41

J c

80

100

120

m/z

SAMPLE

: FULVIC A C I D (from a Podzol)

CHEMICAL DETAILS

: see r e f . 185

SAMPLE ORIGIN

: NW Spain

SAMPLE PREP./SIZE

: suspension i n methanol; approx. 25 vg

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: n o t determined

-

140

160

REUk5' The o v e r a l l p a t t e r n i s t h a t o f a carbohydrate m a t e r i a l . Compared w i t h t h e s o i l polysaccharide (Spectrum F.4), however, t h i s spectrum i s f a r more simple s i n c e i t l a c k s i o n s e r i e s i n d i c a t i v e o f N-acetyl aminosugars and pentose o r deoxyhexose residues. Moreover, t h e absence o f a w e l l marked peak a t m/z 102 warns t h a t we may n o t be d e a l i n g w i t h a simple hexose polymer. Also, major peaks a t m/z 16, 28, 41, 42, a p p a r e n t l y i n d i c a t i n g t h e presence o f a l i p h a t i c hydrocarbon m o i e t i e s , a r e unusual f o r polysaccharide p a t t e r n s . Although v a r y i n g amounts o f carbohydrate m a t e r i a l are o f t e n found i n f u l v i c acids, depending on t h e p r e p a r a t i o n techniques used, t h e "core" o f f u l v i c a c i d s may be made up of p o l y c a r b o x y l i c , e.9. polymaleic, acids r a t h e r than carbohydrates as shown by r e c e n t Py-GC/MS s t u d i e s ( r e f . 181 )

.

220

SPECTRUM F,6

28

96 13 68

112

20

40


60

80

100

120

m/z

140

160

: HUMIC ACID (from a Podzol)

see ref. 185 NW Spain : suspension in methanol; approx. 25 pg : standard : not determined : :

- Although superficially rather similar to fulvic acid (Spectrum F.5), this humic acid spectrum shows much higher peak series indicating the presence of nitrogen-containing (m/z 17, 67, 79, 117), sulphur-containing (m/z 34, 48) and aromatic (m/z 78, 92, 94, 104, 106, 124) fragments. As most of these signals are also encountered in pyrolysis mass spectra of proteins this suggests a significant contribution of protei naceous compounds.

REMRE

221

SPECTRUM F,7 24

10

L

L

L

150

120

-Y

20

40

60

80

100

120

m/z

SAMPLE

: HUMIC A C I D ( f r o m a f o r e s t s o i l )

CHEMICAL DETAILS

: see r e f . 185

SAMPLE O R I G I N

: Canada

SAMPLE PREP./SIZE

: suspension i n methanol; approx. 25 ug

PYROL. CONDITIONS

: standard

TOTAL I O N COUNTS

: n o t determined

140

160

REMARKS- I n s p i t e o f t h e s t r o n g q u a l i t a t i v e resemblance t o hurnic a c i d f r o m

Podzol (Spectrum F.6) a marked s e r i e s o f i n t e n s e peaks a t m/z 124, 138, 150, 164 e a s i l y d i s t i n g u i s h e s t h e two s p e c t r a . These s i g n a l s appear t o be d e r i v e d f r o m g u a i a c y l i c l i g n i n m o i e t i e s , o b v i o u s l y c o r r e s p o n d i n g t o t h e presence o f s i z e a b l e amounts o f i n c o m p l e t e l y degraded p l a n t m a t e r i a l i n t h e upper h o r i z o n s o f the forest soil.

222

SPECTRUM F,8 i%)

3

3

96 >

c

m

z W +z 2

z

s

4:

-I

a +

28

e

$ 1

20

40

60

80

100

120 rnfz 140

160

SAMPLE

: COORONGITE

CHEMICAL DETAILS

: p o s s i b l y formed t h r o u g h o x i d a t i v e p o l y m e r i s a t i o n o f t h r e e a1 kadienes w i t h t h e s t r u c t u r e CH2=CH(CH2)nCH=CH(CH2)7-CH3 (n=15, 17 and 19) produced by Botryococcus braunii ( r e f . 206)

SAMPLE O R I G I N

: Coolaway, A u s t r a l i a



PYROL. CONDITIONS

: standard

TOTAL I O N COUNTS

: 2.6 X 10

5

REMARE - C o o r o n g i t e , r e l a t e d t o b u t p r o b a b l y n o t a d i r e t p r e c u r s o r o f Torbani t e , shows a p a t t e r n r a t h e r s i m i l a r t o t h a t o f T o r b a n i t e (Spectrum F.9). However, compared t o T o r b a n i t e t h e maxima o f each homologous i o n s e r i e s have s h i f t e d t o a h i g h e r mass range, i n d i c a t i n g l o n g e r average a l i p h a t i c c h a i n l e n g t h s . A l s o t h e i o n s e r i e s a t m/z 44, 58, 72, 86 and a t m/z 46, 60, 74 a r e much more pronounced t h a n i n T o r b a n i t e , a p p a r e n t l y i n d i c a t i n g a h i g h e r abundance o f o x y g e n - c o n t a i n i n g a l i p h a t i c compounds.

223

SPECTRUM F , 9 5E

\70

42

20

40

60

80

100

120

rn/z

SAMPLE

: TORBANITE ( ~ o t r ~ o c o ~braunii cus alginite)

CHEMICAL DETAILS

: see r e f . 206

SAMPLE O R I G I N

: Coolaway, A u s t r a l i a

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 1

140

160

x lo5

- I n comparison w i t h i t s " p e a t s t a g e " , C o o r o n g i t e (Spectrum F.8) t h i s T o r b a n i t e spectrum shows s h o r t e r average a l i p h a t i c c h a i n l e n g t h s and l e s s pronounced o x y g e n - c o n t a i n i n g compound s e r i e s . Furthermore t h e a l k e n e s e r i e s a t m / z 42, 56, e t c . i s r e l a t i v e l y more pronounced i n T o r b a n i t e , p o s s i b l y i n d i c a t i n g a more s a t u r a t e d n a t u r e o f t h e carbon skelet-on.

REM~W

SPECTRUM F , l o

*

28

60

40


:

: : : :

80

too

120

m/z

140

160

TORBANITE see ref. 206 Ermelo, S. Africa suspension in methanol; 20 1-19 standard

: 2.5

x lo4

- This spectrum is highly similar to that of the Coolaway Torbanite (Spectrum F.9) in spite of the enormous geographic distance. The major difference appears t o be a stronger preponderance of sul phur-containing products at m/z 34, 48 and 64 as well as of short chain aliphatic fragments.

REMARXS

225

SPECTRUM F ,11 9.5%)

34 S

56

z 0

20

40

60

a0

100

120

m/z

140

SAMPLE

: GREEN R I V E R SHALE

CHEMICAL DETAILS

: 40% o r g a n i c m a t t e r ; C 64.5%; H 8.5%; 0 6.8%; N 2.3%

SAMPLE O R I G I N

: Wyoming, USA



PYROL. CONDITIONS

: standard 4 : 6.3 X 10

TOTAL I O N COUNTS

160

R E ~ R K S- The p y r o l y s i s p a t t e r n o f t h i s s h a l e i s a l m o s t i d e n t i c a l t o t h a t o f t h e Coolaway T o r b a n i t e (Spectrum F.9). Apparently t h e l a c u s t r i n e o r i g i n o f

b o t h samples p r e v a i l s o v e r d i f f e r e n c e s i n age, e t c . A l s o , t h i s c o n f i r m s t h e d o m i n a n t l y a l g a l o r i g i n of t h e o r g a n i c m a t t e r i n Green R i v e r s h a l e . Note, however, t h e much h i g h e r i n t e n s i t y of t h e s u l p h u r - c o n t a i n i n g p r o d u c t s a t m/z 34, 48 and 64 compared t o T o r b a n i t e (Spectrum F . 9 ) .

226

SPECTRUM F ,12 * 16.3%I 34 S

20

40

SAMPLE


60

80

100

120

m/z

140

160

: MESSEL SHALE : 50% organic matter; C 68.5%; H 7.9%; 0 17.5%; N 2.4% : Darmstadt, GFR : suspension in methanol; 20 pg : standard : 3 x lo5

- In comparison t o Torbanite (Spectra F.9, F.10) and Green River Shale (Spectrum F . l l ) , Messel shale shows r e l a t i v e l y high peak i n t e n s i t i e s a t m/z 94, 110, 124, 138 and 150. These s i g n a l s can be explained by phenols and methoxyphenols ( r e f . 52). Most l i k e l y , these compounds a r e derived from l i g n i n moieties present i n t e r r e s t r i a l plant materials which a r e known t o contribute t o t h e organic matter i n Messel shale. In f a c t , s i m i l a r ion s e r i e s a r e observed i n l i g n i t e (Spectrum F.16). Another i n t e r e s t i n g phenomenon i s the marked abundance o f CH3C1+’ ions a t m/z 50 and 52, i n d i c a t i v e of t h e presence o f methylating functional groups.

R~MclRxs

227

SPECTRUM F, 13 '0

\

\84

4;

*

32

20

60

40

80

100

120

140

160

rn/Z

SAMPLE

: GILSONITE

CHEMICAL DETAILS

: l a r g e l y long chain p a r a f f i n s

SAMPLE O R I G I N

: Utah, USA

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 6

X lo4

- The saturated a l i p h a t i c hydrocarbon nature of G i l s o n i t e i s r e f l e c t e d i n the dominant i o n s e r i e s a t m/z 42, 56, etc., apparently representing alkenes, and a t m/z 43, 57, etc., thought t o represent E I fragments o f alkanes. The c o n t r i b u t i o n o f heteroatom compounds appears t o be n e g l i g i b l e as i n d i c a t e d by t h e very low peaks a t m/z 17 (NH3+') and m/z 34 (H2S") and t h e absence o f prominent oxygen-containing i o n series.

RE MAR^

228

SPECTRUM F ,14

61 >

!z

9:

m

110

s 4

z z

Q -I a I-

s %

20

60

40


:

80

100

120

m/z

140

160

BOG PEAT

: see ref. 50

Bad-Zwischenahn GFR; depth >250 cm suspension in methanol; approx. 25 : standard : not determined : :

pg

- Bog peat i s composed to a large extent of wool grass and other plant residues. Therefore it contains grass lignins characterised by the presence of methoxylated phenolic series derived from guaiacylic (m/z 124, 138, 150) as well as syringylic (weak signal at m/z 154) building blocks, in addition to the nonmethoxylated phenols (m/z 94, 108, 120, 134) also found in sphagnum peat (Spectrum F.15). Note also the characteristic methane peak (m/z 16) encountered in pyrolysis mass spectra o f methoxylated lignins (Spectra E.9, E . l O ) .

REMARKS

229

SPECTRUM F , 15 '2

t3

96

J 114 110 84

I

/

128

4 S

48

4-

d

20

m/z

SAMPLE

: SPHAGNUM PEAT

CHEMICAL DETAILS

: see r e f . 50


160

: Bad-Zwischenahn, GFR; t o p 60 cm o f p r o f i l e

. : suspension i n methanol; approx. 25 ug

PYROL. CONDITIONS

: standard

TOTAL I O N COUNTS

: n o t determined

- I n c o n t r a s t t o bog p e a t (Spectrum F,14),sphagnum p e a t e x h i b i t s an almost s t r i c t l y p o l y s a c c h a r i d e p a t t e r n w i t h s t r o n g peaks a t m/z 114 and 128 i n d i c a t i n g t h e presence o f sugar r e s i d u e s o t h e r t h a n hexose, e.g. pentose and deoxyhexose. The absence o f a t y p i c a l l i g n i n p a t t e r n such as i n bog p e a t i s The e x p l a i n e d by t h e l a c k of t r u e l i g n i n s i n Sphagnum moss (Spectrum E.16). o n l y l i g n i n - l i k e c o n s t i t u e n t o f Sphagnum moss, sphagnol ( r e f . 85), a p p a r e n t l y g i v e s r i s e t o t h e nonmethoxylated ( a l k y 1 ) p h e n o l s e r i e s a t m/z 94, 108, 120 ana 134. A l s o n o t e t h e absence o f p y r o l y t i c methane (m/z 1 6 ) .

REMKS

230

SPECTRUM F ,16

42 s

I

I

L

0

40

60

80

SAMPLE

: LIGNITE (Miocene)

CHEMICAL DETAILS

: see r e f . 52; 90% o r g a n i c m a t e r i a l ; C 61.6%; H 4.6%; 0 32.4%; N 0.6%

SAMPLE O R I G I N

: Herzogenrath, GFR

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL I O N COUNTS

: n o t determined

REM~RE - T h i s l i g n i t e spectrum i s dominated by p h e n o l i c peak s e r i e s w i t h s i g n a l s a t m/z 124, 138, 150, 164 a p p a r e n t l y r e p r e s e n t i n g more o r l e s s unchanged g u a i a c y l i c b u i l d i n g b l o c k s (compare w i t h l i g n i n Spectra E.9 and E.lO). However, t h e s t r o n g dihydroxybenzene peak a t m/z 110 p o i n t s t o t h e l o s s of methoxy groups by d e m e t h y l a t i o n . A l s o t h e m u l t i p l e peaks a t even mass numbers between t h e m a j o r " l i g n i n " peaks i n t h e h i g h mass range p r o v i d e evidence f o r a p r o g r e s s i v e chemical m o d i f i c a t i o n o f t h e o r i g i n a l biopolymer m a t r i x . Note t h e complete absence o f t y p i c a l c e l l u l o s i c fragments (compare w i t h wood spectrum E . l l ) as a r e s u l t o f t h e more r a p i d d e g r a d a t i o n o f t h e p o l y s a c c h a r i d e m o i e t i e s o f t h e o r i g i n a l wood. F u r t h e r n o t e t h e low s u l p h u r s i g n a l s which c o n f i r m s t h e nonm a r i n e o r i g i n o f t h i s l i g n i t e l a r g e l y composed o f redwood remains.

231

SPECTRUM F , 17

108 !

., ”

’G.,

SAMPLE

: HIGH VOLATILE C BITUMINOUS COAL (from Hanna Bed #2)

CHEMICAL DETAILS

: C 78.8%; S 0.73%; m i n e r a l m a t t e r 20.4%; v i t r i n i t e s 94.9%; e x i n i t e s 2.1%; i n e r t i n i t e s 3.0%

SAMPLE O R I G I N

: Wyoming, USA (Penn S t a t e Coal Sample Bank; PSOC 514)

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: Teq 610°C; t 5s; t, 10s; Ee, PY-Ms sys tern]

TOTAL ION COUNTS

: 1

15eV (Extranuclear 5000-1

x lo5

REMARKS - Compared t o Spectrum F.16 ( l i g n i t e ) t h e methoxyphenol s e r i e s ( a t m/z 124, 138, 150, 164) appears t o have been r e p l a c e d by a dihydroxybenzene s e r i e s w i t h prominent peaks a t m/z 110 and 124, a p p a r e n t l y as a r e s u l t o f f u r t h e r demethylation. Moreover, a l i p h a t i c hydrocarbon s e r i e s s t a r t appearing a t m/z 42, 56, 70 e t c . (alkenes) and 54, 68, 82 (dienes; perhaps mixed w i t h f u r a n s ) . Note t h e r e l a t i v e l y low sulphur-containing i o n peaks a t m/z 34, 48 and 64.

232

SPECTRUM F a18

40

60

80

100

=,,

120

140

160

180

200 m k

HIGH VOLATILE C BITUMINOUS COAL (from New Mexico Seam #7) C 77.9%; S 0.92%; mineral matter 9.1%; vitrinites 83.5%; exinites 2.0%; inertinites 14.5% : New Mexico, USA (Penn State Coal Sample Bank; PSOC 310) : suspension in methanol; 40 pg : Teq 610°C; t 5s; t, 10s; Eel 15eV (Extranuclear 5000-1 Py-MS system]

:


:

: 1

x lo5

- Compared to the Hanna Field coal o f the same ASTM rank (Spectrum F.17) the phenolic series are less prominent whereas the aliphatic hydrocarbons series appear to be more pronounced.

REMARKS

233

SPECTRUM F ,19

r\

108

' I I\

'.'.

'.

40

60

80

100

120 m,z 140

170

160

180

200

220

SAMPLE

: H I G H VOLATILE B BITUMINOUS COAL ( f r o m B l i n d Canyon Seam)

CHEMICAL DETAILS

: C 79.4%; S 0.76%; m i n e r a l m a t t e r 9.2%; v i t r i n i t e 82.3%; e x i n i t e s 2.1%; i n e r t i n i t e s 15.6%

SAMPLE O R I G I N

: Utah, USA (Penn S t a t e Coal Sample Bank; PSOC 238)

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: Teq 610OC; t 6s; tz 10s; Eel 15 eV ( E x t r a n u c l e a r 5000-1 Py-MS system]

TOTAL ION COUNTS

: 1

x lo5

- Compared t o t h e High V o l a t i l e C Bituminous c o a l from t h e Rocky Mount a i n Coal Province (Spectrum F.18), t h e dihydroxybenzene peak s e r i e s (m/z 110, 124, 138) i s c l e a r l y reduced whereas t h e naphthalene s e r i e s (m/z 142, 156, 170, 184, 198) s t a r t s t o dominate t h e h i g h mass region.

REME

234

SPECTRUM F ,20

J

u S

64

108 /’

A

: HIGH VOLATILE

CHEMICAL DETAILS

: C 80.5%; S 1.78%; mineral m a t t e r 10.8%; v i t r i n i t e s 85.2%; e x i n i t e s 2.9%; i n e r t i n i t e s 11.9%

SAMPLE O R I G I N


SAMPLE PREP./SIZE


PYROL. CONDITIONS

: Teq 610°C; t 6s; t z 10s; Eel 15eV ( E x t r a n u c l e a r 5000-1 Py-MS system] 5 : 1.3 X 10

TOTAL ION COUNTS

BITUMINOUS COAL ( f r o m Utah

A

SAMPLE

Seam)

BEMIIKS - T h i s spectrum i s v e r y s i m i l a r t o t h a t o f t h e h i g h v o l a t i l e B b i t u m i n ous c o a l o f t h e same r e g i o n ( U i n t a h Region o f Rocky Mountain Coal Province) w i t h t h e e x c e p t i o n o f t h e markedly h i g h e r s u l p h u r - c o n t a i n i n g i o n s i g n a l s a t m/z 34 and 64. T h i s c o r r e l a t e s w i t h t h e much h i g h e r S c o n t e n t (1.78% vs. 0.76%). Note, however, t h a t t h e peak a t m/z 48 (CH3SHt’) remains low. The absence of obvious r a n k - r e l a t e d d i f f e r e n c e s between t h e p y r o l y s i s mass spectra o f h i g h v o l a t i l e B and A bituminous ranks i s t y p i c a l . C h a r a c t e r i s t i c rank r e l a t e d changes occur most s t r o n g l y &tween t h e HVCB and HVBB ranks and again between t h e HVAB and MVB ranks.

235

SPECTRUM F, 21

I

40

80

0

'

100

I

I20 m,z 140

I

160 '

60

'

260

220

SAMPLE

: MEDIUM VOLATILE BITUMINOUS COAL ( f r o m Colorado B Seam)

CHEMICAL DETAILS

: C 89.9%; S 0.56%; 0 2.07%; mineral m a t t e r 7.6%; v i t r i n i t e s 92.0%; e x i n i t e s 0.1%; i n e r t i n i t e s 7.9%

SAMPLE ORIGIN

: Colorado, USA (Penn S t a t e Coal Sample Bank; PSOC 157)

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: Teq 610°C; t T 6s; t c 10s; Eel 15eV ( E x t r a n u c l e a r 5000-1 Py-MS system)

TOTAL ION COUNTS

: 1

-

x lo5

REMARKS Compared t o t h e h i g h v o l a t i l e .bituminous c o a l s i n Spectra F.17-F.20, t h e p h e n o l i c s e r i e s a t m/z 94, 108, e t c . and a t m/z 110, 124, e t c . a r e d r a s t i c a l l y decreased and t h e spectrum i s now e n t i r e l y dominated by a l i p h a t i c and aromatic hydrocarbons. Note e s p e c i a l l y t h e s t r o n g alkene (m/z 42, 56, etc.), benzene (m/z 92, 106, e t c . ) and naphthalene (m/z 142, 156, e t c . ) s e r i e s . F u r t h e r n o t e t h e decrease i n s u l p h u r - c o n t a i n i n g i o n peaks. Some o f t h e d i f f e r ences w i t h t h e h i g h v o l a t i l e bituminous coal s may be exaggerated s i n c e t h e Colorado B Seam c o a l s underwent a c c e l e r a t e d m a t u r a t i o n because o f exposure t o h i g h geothermal g r a d i e n t s .

236

SPECTRUM F ,22

82

SAMPLE

: BOGHEAD COAL ( f r o m Cannel King Seam)

CHEMICAL DETAILS

: C 80.2%; S 1.82%; 0 11.1%; m i n e r a l m a t t e r 29.1%; v i t r i n i t e s 26.7%; e x i n i t e s 69.1% ( a l g i n i t e ! ) ; i n e r t i n i t e s

SAMPLE O R I G I N


SAMPLE PREP./SIZE


PYROL. CONDITIONS

: Teq 610°C;

TOTAL ION COUNTS

: 1.2

3.9%

t T 6s; t, 10s; Eel 15eV ( E x t r a n u c l e a r 5000-1 Py-MS system)

x

- The spectrum o f t h i s a l g i n i t e - r i c h c o a l i s r a d i c a l l y d i f f e r e n t f r o m t h a t o f t h e v i t r i n i t e - r i c h c o a l s shown i n s p e c t r a F.16-F.21. A l i p h a t i c hydroc a r b o n s e r i e s dominate, e.g. alkenes (m/z 42, 56, e t c . ) and dienes (m/z 54, 68, e t c . ) , whereas a r o m a t i c s i g n a l s a r e inconspicuous. Note t h e h i g h s u l p h u r c o n t a i n i n g i o n peaks a t m/z 34 and 64 which c o n f i r m t h e a q u a t i c c h a r a c t e r o f t h e d e p o s i t i o n a l environment. I n many r e s p e c t s t h i s Boghead c o a l spectrum resembles t h a t o f , t h e a l g i n i t e - r i c h T o r b a n i t e s (Spectra F.9, F.10) and shales ( S p e c t r a F . l l , F.12).

REMRKS

237

SPECTRUM F ,23

SAMPLE

: BOGHEAD COAL, s e v e r e l y weathered ( f r o m Cannel King Seam)

CHEMICAL DETAILS

: C 67.6%; S 2.39%; 0 26.2%; m i n e r a l m a t t e r 32.7%

SAMPLE O R I G I N


SAMPLE PREP./SIZE


PYROL. CONDITIONS

: Teq 610°C; t 6s; t z 10s; Eel 15eV ( E x t r a n u c l e a r 5000-1 PY-Ms system)

TOTAL ION COUNTS

: 7

x lo4

REMARS - T h i s spectrum i s i n c l u d e d t o show t h e e f f e c t s o f severe w e a t h e r i n g ( o x i d a t i o n ) . Comparison w i t h t h e r e l a t i v e l y non-weathered sample f r o m t h e same seam (Spectrum F.22) shows a marked i n c r e a s e i n t h e i n t e n s i t i e s o f t h e peaks a t m/z 44 (COz+') and m/z 78 (C6H6+'). T h i s i s t h o u g h t t o i n d i c a t e t h e presence o f benzene c a r b o x y l i c a c i d s as a r e s u l t of o x y d a t i o n o f a r o m a t i c r i n g s . The r e l a t i v e i n c r e a s e i n phenols (m/z 94, 108) may a l s o r e f l e c t r i n g o x y d a t i o n . F u r t h e r n o t e t h e h i g h e r peak a t m/z 60, p r o b a b l y a c e t i c a c i d , which may w e l l r e p r e s e n t o x i d a t i o n o f a1 i p h a t i c m o i e t i e s .

SPECTRUM F , 24 0%)

I2 k

\

A

16

\ i4

-4

100

40

60

: FUSINITE


:

ORIGIN

SAMPLE PREP./SIZE PYROL. CONDITIONS

TOTAL

ION COUNTS

80

120

m/z

140

160

(from Westfield Fusain) (see ref. 206) : Scotland : suspension in methanol; 20 1-19 : standard

SAMPLE SAMPLE

6c

142

: 3

x

lo4

- This spectrum is dominated by (alkyl)benzenes, m/z 78, 92, etc. and (alkyl) naphthalenes, m/z 128, 142, etc.. This agrees well with a possible charcoal nature and origin o f fusinite (ref. 194). Note further the relatively low sulphur-containing ion signals at m/z 34 and 48, although m/z 64 and 76 show we1 1 pronounced peaks.

REMARKS

239

SPECTRUM F ,25

1

*

\6

32

I

20

60

40

80

100

120

m/z

140

160

SAMPLE

: SPORINITE ( f r o m Deep Hard Seam)

CHEMICAL DETAILS

: H/C r a t i o 0.98; s p o r i n i t e 93.0%; m i c r i n i t e and f u s i n i t e 7.0% (see r e f . 206)

SAMPLE O R I G I N

: England

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 5

x lo4

- T h i s p a t t e r n appears t o be more o r l e s s i n t e r m e d i a t e between t h a t o f To some e x t e n t , t h i s v i t r i n i t e (Spectrum F.26) and f u s i n i t e (Spectrum F.24). may r e f l e c t t e c h n i c a l d i f f i c u l t i e s i n o b t a i n i n g s p o r i n i t e f r a c t i o n s f r e e o f v i t r i n i t e and f u s i n i t e . O l e f i n i c s e r i e s a t m/z 42, 56, e t c . and m/z 54, 68, e t c . appear t o dominate t h e o v e r a l l p a t t e r n as m i g h t be expected f r o m a f r a c t i o n r i c h i n e x i n i t i c p l a n t residues.

REMARKS

240

SPECTRUM F ,26

42

1 I'

;

/

j

4

LI 20

0

1

4

'

60

'

80

'

100

120 m,z 140

160

SAMPLE

: VITRINITE (from High Hazles Seam)

CHEMICAL DETAILS

: H/C r a t i o 0.80; v i t r i n i t e 97.0%; e x i n i t e 0.7%; i n e r t i n i t e 2.3% (see r e f . 206)

SAMPLE O R I G I N

: England

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL I O N COUNTS

: n o t determined

- Not s u r p r i s i n g l y , t h i s spectrum resembles t h a t o f a h i g h v o l a t i l e bituminous coal (e.g. Spectra F.19, F.20). Not o n l y was t h i s v i t r i n i t e p r e pared from a h i g h v o l a t i l e bituminous coal, b u t a l s o v i t r i n i t e s account f o r t h e b u l k (e.g. up t o 95%) o f most coals.

REMARKS

241

SPECTRUM F ,27

i

32

20

40

SAMPLE

60

80

100

120

m/z

140

160

180

: AMBER ( r e s i n i t e )

CHEMICAL DETAILS

: see r e f . 207, 208

SAMPLE O R I G I N

: B a l t i c coast

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL I O N COUNTS

: 1.7

X 105

REWS - The o r i g i n a l b u i l d i n g blocks o f t h i s f o s s i l n a t u r a l r e s i n a r e m a i n l y terpenoids c o n t a i n i n g c a r b o x y l i c acid, e s t e r , aldehyde, keto and/or hydroxyl f u n c t i o n a l groups, which on f o s s i l i s a t i o n have been condensed and v i n y l polymerised. F u r t h e r m o d i f i c a t i o n s may have been introduced by dehydration, d e c a r b o x y l a t i o n and dehydrogenation. The complex Py-MS p a t t e r n s o f ambers ( r e f . 207) c o n t a i n s e r i e s o f unsaturated hydrocarbon and aldehyde fragments. Promine n t i o n s e r i e s a t odd mass numbers ( E I fragments) p o i n t t o a mainly brancheda l i p h a t i c c h a r a c t e r o f t h e p y r o l y s i s fragments.

SPECTRUM F, 28 i i /

I

! i

L

152

-I

60562

J 160

20

40

60

80

100

SAMPLE

: FOSSIL WOOD (Eocene)

CHEMICAL DETAILS

: carbonified


: Hengelo, The Netherlands

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 1.4

120

m/z

140


x lo5

REMARXS - This p a t t e r n o f c a r b o n i f i e d wood i s s i m i l a r t o t h a t o f many l i g n i t e s (see Spectrum F.16) and i s dominated by aromatic fragments obviously derived 'from l i g n i n - l i k e moieties. It d i f f e r s from t h a t o f recent wood (see Spectrum E . l l ) by t h e almost complete absence o f c e l l u l o s e (Spectrum A . l ) and hemicellul o s e fragments, as w e l l as by the high sulphur-containing peaks a t m/z 34, 48, 64, 76 and ( p o s s i b l y ) 62.

243

SPECTRUM F ,29 91

08 I

i

i \

8 >.

c

m

i

z

\ \i

2 z

\

i

z 0 $4

I-

2 s

D

40


60

80

120

m/z

140

160

FOSSIL CONIFER WOOD (Upper Cretaceous) silicification phase: Opal CT : Aix-en-Province, France; obtained from the Inst. Botanique et Palaeobotanique, Fac. des Sciences, Univ. Paris : suspension in methanol; 50 ug : standard

:

:

: 2.2

x lo4

- This much older specimen of silicified wood shows a dominant (alky1)phenol pattern. The absence of more complex lignin-derived fragments, e.g. methoxyphenols, appears to indicate much higher degrees of chemical transformation. Also note relatively low signals for sulphur-containing ions.

REMARKS


GROUP

G

OTHER BIOCHEMICALLY IMPORTANT COMPOUNDS (drugs, vitamins, metabolites, e t c . )


247

58

4’

i J3

dH

84

12

60

JIlL

20

60

40

100

80


:


: Serva, H e i d e l b e r g ,

L 120

m/z

140

160

DIGITOXIN

: see i n s e r t i n spectrum; digitoxigenin-(digitoxose)3; C41H64013; MW = 764

GFR

: suspension i n methanol; 20 p g : standard :

I

x

lo4

REME- The spectrum i s dominated by p y r o l y s i s fragments o f t h e sugar m o i e t i e s (compare w i t h p o l y s a c c h a r i d e p a t t e r n s , Group A). The peak a t m/z 130 may w e l l r e p r e s e n t a dideoxyhexose-minus-H20 u n i t . NO obvious p y r o l y s i s p r o d u c t s o f t h e s t e r o i d s k e l e t o n a r e observed b u t n o t e t h e h i g h peak a t m/z 44 (C02+’?).

SPECTRUM G,2 44

58

0

I

7

3

BG

20

SAMPLE CHEMICAL DETAILS SAMPLE ORIGIN SAMPLE PREP./SIZE TOTAL ION COUNTS

60

40

80

100

120

m/z

140

DIGOXIN see insert in spectrum; digoxigenin-(digitoxose)3; C4,H640,4; MW = 780 : O.P.G., Utrecht, The Netherlands : suspension in methanol; 20 ug : :

: 1

x lo4

- Only minor quantitative differences are observed with digitoxin (Spectrum G.l), e.g. at m/z 112 and 130.

REMARKS

160

249

SPECTRUM G,3

3

32

HO

0

G I al-Glc-Xyl Gal-Glc I

20

40

60

80

100

120

m/z

140

160

CHEMICAL DETAILS

: DIGITONIN : see i n s e r t i n spectrum; G l c a ( l + 3 ) G a l a ( l + 2 ) [ XylB(1+3)]Glc~(l+4)GalB-digitogenin; C56Hg2029; MW = 1229

SAMPLE

SAMPLE O R I G I N


SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

4 : 2.5 X 10

- T h i s spectrum p r e s e n t s a t y p i c a l hexose p a t t e r n (compare w i t h p o l y s a c c h a r i d e p a t t e r n s , Group A). No obvious p y r o l y s i s fragments o f t h e s t e r o i d s k e l e t o n a r e observed.

REMARKS

SPECTRUM G,4 [ 6.3%1

8.1 %I

$3

126

NH

II

NH

HN-C-NH2

II

60 34

20

40

d 60

OH

95

80

109

100

139

120

m/z

140

160

SAMPLE

: STREPTOMYCIN SULPHATE

CHEMICAL DETAILS

: see i n s e r t i n spectrum; C21H39N7012(1 .5 H2S04); MW=728

SAMPLE O R I G I N


SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL I O N COUNTS

: 1 X 10

5

REMARKS - The spectrum shows t y p i c a l n e u t r a l sugar as w e l l as aminosugar f r a g The h i g h peak a t ment s e r i e s (compare w i t h p o l y s a c c h a r i d e p a t t e r n s , Group A ) . m/z 64 must r e p r e s e n t SO2+’ d e r i v e d f r o m t h e s u l p h a t e group.

251

SPECTRUM G , S

20

40

60

80

100

120

m/z

SAMPLE

: KANAMYCIN SULPHATE

CHEMICAL DETAILS

: see i n s e r t i n spectrum; C,8H36N4011(H2S04);

SAMPLE O R I G I N


SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 7

-

140

160

MW = 582

x lo4

REMARKS A h i g h l y c h a r a c t e r i s t i c spectrum w i t h unusual peak s e r i e s , o b v i o u s l y due t o t h e e x o t i c c h a r a c t e r o f t h e sugar m o i e t i e s . The peak a t m/z 64 must r e p r e s e n t SO2+' d e r i v e d from t h e s u l p h a t e group. The i d e n t i t y o f t h e "sugar f r a g m e n t s " i s as y e t unknown.

SPECTRUM G,6 9 L%)

126%

34

91

S

COOK

10.2%)

100

J

1 1

1 7

L 20

4 - L

40

60

80

100

120

m/z

140

SAMPLE

: PENICILLIN G, K - s a l t ( b e n z y l p e n i c i l l i n )

CHEMICAL DETAILS

: see i n s e r t i n spectrum; C16H17N204S(K); MW = 372

SAMPLE O R I G I N




PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 5

160

x lo5

R E M I L S - C o n s i d e r i n g t h e r e l a t i v e l y small s i z e of t h i s molecule, t h e spectrum i s u n u s u a l l y complex i n d i c a t i n g t h e occurrence o f c o m p l i c a t e d f r a g m e n t a t i o n pathways. W i t h t h e e x c e p t i o n o f t h e H$+' i o n a t m/z 34 t h e . i d e n t i t y and o r i g i n o f most of t h e peaks i s as y e t unknown. Nevertheless t h e marked peak s e r i e s a t m/z 34, 71, 74, 88, 100 and 115 i s h i g h l y c h a r a c t e r i s t i c f o r a l l p e n i c i l l i n s

t h u s f a r analysed.

253

SPECTRUM G,7 COOH

2.5%)

14

J

S

1

J

\

88

71

c-4 20

40


60 :

/

I

80

100

120

m/z

140

160

AMPICILLIN

: see i n s e r t i n spectrum; 6-[D-~~-aminophenylacetamido]MW = 403 p e n i c i l l a n i c a c i d ; Cl6HlgN3O4S(3H20); : Serva, H e i d e l b e r g , GFR : suspension i n methanol; 20 pg : standard

: 1.6 X

lo5

- Compared t o t h e spectrum o f b e n z y l p e n i c i l l i n t h e c h a r a c t e r i s t i c p e n i c i l l i n peaks a t m/z 100 and 115 a r e much more pronounced whereas no s i g n i f i c a n t peaks a t 147, 136, 113, 97, 91 and 8 1 a r e observed i n d i c a t i n g a r a t h e r d i f f e r e n t p y r o l y s i s mechanism. The s t r o n g peak a t m/z 125 may w e l l be characteristic f o r ampicillin.

REMARKS

SPECTRUM G ,8 134

J

119

60

1

/

20

40

60

80

100

SAMPLE CHEMICAL DETAILS SAMPLE ORIGIN

: CANNABIDIOL



120

m/z

140

160

: see i n s e r t in spectrum; C21H3002; MW = 314

: UNO, Narcotics, Vienna : standard

: 2

x

R E ~ K -S The strong peak a t m/z 134 i s thought t o represent the eliminated

Note t h e presence of the alkene s e r i e s (m/z cyclohexadienyl moiety (C10H14''). 28, 42, e t c . ) . NO obvious fragments of the pentyldihydroxybenzene moiety a r e observed in t h i s mass range. For pyrolysis products of cannabidiol see r e f . 209.

255

SPECTRUM GI 8~ 11.5%1

11.0%I

58

7

J

106

1

20

28

lylyw 20

40

60

100

80

SAMPLE

: CANNABIDIOL


: C21H3002; MW = 314

SAMPLE P R E P . / S I Z E PYROL. CONDITIONS


TOTAL IONCOUNTS

: 2

120

m/z

140

: UNO, Narcotics, Vienna : ferromagnetic t u b e ; T

x

lo5

eq

=770"C

- In comparison with t h e filament pyrolysis p a t t e r n (Spectrum 6.8) t h i s spectrum i s dominated by a peak a t m/z 68, possibly representing C5H8+ and a stronger aromatic hydrocarbon s e r i e s a t m/z 92, 106 and 120. In a d d i t i o n , phenolic peaks a t m/z 94 and 108 may represent pyrolysis fragments of t h e C5-alkyldihydroxybenzene moiety. The peak a t m/z 134 i s again thought t o be t h e C10H14+' (cyclohexadienyl) moiety.

REMARKS

160

SPECTRUM G.9

17

! 111

40

20

80

60

100

SAMPLE

: CYANOCOEALAMIN ( v i t a m i n 812)

CHEMICAL DETAILS

: C63H88CoN,40,4P;

SAMPLE O R I G I N

: Merck AG, Darmstadt, GFR

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 5

120

m/z

140

160

MW = 1355

x lo4

REMRKS - The amide s u b s t i t u e n t s o f the h e t e r o c y c l i c r i n g system may g i v e r i s e t o t h e f o r m a t i o n o f acetamide (m/z 5 9 ) . The peak a t m/z 67 probably represents p y r r o l e whereas t h e peak a t m/z 98 may be d e r i v e d from t h e r i b o f u r a n o s i d e I-.. k In 2

w !-

z z

I

0

CH3

28

. ,

20

'

40


.

60

80

C2H5

112 100

120

m/z

140

160

: ETIOPORPHYRIN I

see insert in spectrum; Phillips Petroleum Co., : suspension in methanol; : ferromagnetic tube; T eq :

:

: 1.3 x

C32H38N4; MW = 478 Bartlesville, OK, USA. 10 yg 770°C

lo5

- The intense peak group at m/z 67, 68 is thought to represent C5alkynes derived from the alkyl-substituted rings. This unusual pyrolysis mechanism may well be unique for this type o f compound.

REMARKS

GROUP H POLYMERS OF NON-BIOLOGICAL ORIGIN

( p l a s t i c s , resins, e t c . )


261

SPECTRUM H,1

20

60

40

80

100

120

140

160

m/z SAMPLE CHEMICAL DETAILS

: POLYETHYLENE (longitudinal f i bri 1 l a r c r y s t a l 1 i n e )

SAMPLE ORIGIN

: g i f t from Dr. A.J. Pennings, Lab. f o r Polymer Chemistry, Univ. o f Groningen, The Netherlands



REMARKS

-

: see i n s e r t in spectrum

: standard : 4

x

lo4

The spectrum shows the absence o f marked monomer a n d oligmer peaks due

t o a dominance o f thermal degradation r a t h e r t h a n depolymerisation reactions. Note the presence of homologous ion s e r i e s representing increasingly unsaturated

a l i p h a t i c hydrocarbons a n d even some degree o f aromatisation (m/z 7 8 ! ) . Dotted l i n e s connect alkane fragment ion s e r i e s a t m/z 43, 57, 7 1 , 85, e t c . . Heteroatom-containing ions a t m/z 18 (H20t'), 32 (CH30Hf'), 60 (C2H402") and 64 ( S O z t ' ) must represent sample contaminants.

262

SPECTRUM H12

&

MONOMER

82

70

I

42

28

DlMER

112

TRIMER

166 168

40

60

80

100

120 m,z 140

160

180

SAMPLE

: POLYBUTENE ( i s o t a c t i c )

CHEMICAL DETAILS

: see i n s e r t i n spectrum; l o w m o l e c u l a r w e i g h t ; d e n s i t y 0.91

SAMPLE O R I G I N

: S c i e n t i f i c Polymer Products; USA

SAMPLE PREP./SIZE

: s o l u t i o n i n cyclohexane;

PYROL. CONDITIONS

10 ug : Teq 610°C; t T 5s; t x 10s; Eel 12eV; ( E x t r a n u c l e a r 5000-1 Py-MS system)

TOTAL ION COUNTS

: 1

x lo5

REMARKS - I n comparison w i t h p o l y e t h y l e n e (Spectrum H.1), p o l y b u t e n e shows a c o m b i n a t i o n o f thermal d e g r a d a t i o n and d e p o l y m e r i s a t i o n r e a c t i o n s . The thermal d e g r a d a t i o n r e a c t i o n s r e s u l t i n t h e f o r m a t i o n of prominent a l k e n e and a l k a d i e n e s e r i e s . The d e p o l y m e r i s a t i o n r e a c t i o n s appear t o proceed v i a u n z i p p i n g , l e a d i n g t o t h e monomer peak a t m/z 56, as w e l l as v i a a b a c k b i t i n g mechanism, r e s u l t i n g i n t h e marked t r i m e r peak complex a t m/z 166/168 ( i t s h o u l d be i n t e r e s t i n g t o s t u d y t o what e x t e n t b a c k b i t i n g occurs i n a t a c t i c and/or s y n d i o t a c t i c p o l y b u t e n e ) . Whether t h e peak a t m/z 168 r e p r e s e n t s a l i n e a r o r c y c l i c t r i m e r i s unknown, a l t h o u g h t h e l a t t e r c o n f i g u r a t i o n would seem t o be t h e most p r o b a b l e . The peak a t m/z 166 must r e p r e s e n t a dehydrogenated t r i m e r , whereas t h e peak a t m/z 139 i s p r o b a b l y an e l e c t r o n i m p a c t fragment (M-29) of t h e t r i m e r .

263

SPECTRUM H m 3

104 MONOMER

+ 20

60

40

SAMPLE

: POLYSTYRENE

80

:

100

- --!-

140


: see i n s e r t in spectrum; MI4 = 270,000

SAMPLE O R I G I N

: Natl. Bureau o f Standards (SRM 706), Washington, USA


: solution in acetone; 2 1-19


: standard

TOTAL ION COUNTS

: 3

x

160

lo4

REMRKS - This simple spectrum shows a dominant styrene monomer peak a t m/z 104

a s well as r e l a t i v e l y minor benzene (m/z 78) and phenylpropene (m/z 118) peaks. A dimer peak appearing a t m/z 208 i s outside the mass range shown here.

SPECTRUM H,4 1%I

lO%l

I0

\

---CH2-CH2-

0---

\

55

L4 20

40

60

SAMPLE

: POLYETHYLENEGLYCOL (Carbowax 20M)

CHEMICAL DETAILS


SAMPLE O R I G I N

: Becker, D e l f t , The Netherlands

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 2.3

x lo5

- The p y r o l y s i s mass spectrum o f Carbowax 20M shows a r e l a t i v e l y complex p a t t e r n due t o thermal d e g r a d a t i o n mechanisms and E I f r a g m e n t a t i o n processes. The s t r o n g c o n t r i b u t i o n o f t h e l a t t e r r e s u l t s f r o m t h e f a c t t h a t t h e a l i p h a t i c CHO-fragments produced i n t h e thermal d e g r a d a t i o n process a p p a r e n t l y f a i l t o produce s t a b l e m o l e c u l a r i o n s a t 14eV e l e c t r o n energy, t h u s l e a d i n g t o i n t e n s e fragment i o n s e r i e s (e.g. a t m/z 45, 59, 73, 87, 101). The peak a t m/z 44 may be t h o u g h t t o r e p r e s e n t t h e b a s i c r e p e a t i n g s u b u n i t C2H40 ( e t h y l e n e g l y c o l minus H20).

REMARKS

265

SPECTRUM H,5

-

4-

30 32

> m

MONOMER

c

113

J 85

84 56

'

3

60

40

71

80

100

140

160

: POLYCAPROLACTAM ( N y l o n 6 )

CHEMICAL DETAILS


SAMPLE O R I G I N

: S c i e n t i f i c Polymer Products, USA

SAMPLE PREP./SIZE

: suspension i n methanol;

PYROL. CONDITIONS

: Teq 610°C, t 5s, t x lOs, Eel 12eV, ( E x t r a n u c l e a r 5000-1 Py-MS systernJ

TOTAL ION COUNTS

: 5

10 ug

x lo4

REht&'?E - The marked monomer peak a t m/z 113 p o i n t s t o a dominant u n z i p p i n g mechanism a l t h o u g h thermal d e g r a d a t i o n r e a c t i o n s a r e a l s o e v i d e n t , e.g. f r o m peaks a t m/z 17 (NH3+'), 30 (CH20+') and t h e s m a l l a l k e n e s e r i e s a t m/z 42, 56, e t c . The l a r g e peak a t m/z 85 p r o b a b l y r e p r e s e n t s CO e l i m i n a t i o n f r o m t h e monomer by e l e c t r o n impact o r p y r o l y s i s mechanisms. A l t h o u g h t h e mass spectrum was scanned up t o m/z 240 no obvious dimer peak o r o t h e r l a r g e fragments were observed.

266

SPECTRUM H ,6 8

(11.4 *I,

l11%1

78

3f

) .

c

m

z W

c

z

38

2 4

2

-I

s

92

n

e

s

J

I

J J W 20

40

60

80

100

SAMPLE

: POLYVINYLCHLORIDE


: see i n s e r t i n spectrum : S o l v i c 239, Solvay, France

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

5 : 1.7 X 10

120

m/z

140

160

REMARKS - T h i s spectrum shows dominant molecular i o n s f o r HC1 (m/z 36 and 38) and benzene (m/z 78). Other aromatic and/or unsaturated hydrocarbon products a r e observed as r e l a t i v e l y low s i g n a l s a t m/z 54, 68, etc., m/z 66, 80, e t c . and m/z 92, 106, 120. The peaks a t m/z 104, 116 and 128 probably r e p r e s e n t CgHg ( s t y r e n e ) , CgH (indene) and C10Hg (naphthalene). Note t h e absence of an obvious v i n y l c f l o r i d e monomer s i g n a l a t m/z 62.

267

SPECTRUM H,7

L 207

220

SAMPLE

: POLYDIMETHYLSILICONE (OV-1)

CHEMICAL DETAILS


SAMPLE O R I G I N

: Supelco, USA


: s o l u t i o n i n t o l u e n e ; 10 pg


: Teq 510°C; t T 5s; t x 10s; Eel 12eV; expansion chamber removed ( E x t r a n u c l e a r 5000-1 Py-I.4S system)

TOTAL ION COUNTS

: 2

x lo3

- The low i o n i n t e n s i t y may be caused by condensation l o s s e s o f l a r g e fragments on t h e r e a c t i o n chamber w a l l , by i n c o m p l e t e p y r o l y s i s , by l o w e r i o n t r a n s m i s s i o n i n t h e h i g h mass range o r by a c o m b i n a t i o n o f these f a c t o r s . The l a r g e peak a t m/z 207 must r e p r e s e n t t h e (M-15) e l e c t r o n i m p a c t fragment o f t h e t r i m e r a l t h o u g h no t r i m e r p a r e n t i o n i s observed a t m/z 222. T h i s i s due t o t h e presence o f s i x methyl groups i n t h e t r i m e r , each o f which has a s i g n i f i c a n t chance o f b e i n g l o s t i n t h e e l e c t r o n impact i o n i s a t i o n process i n s p i t e o f t h e l o w e l e c t r o n energy. Note t h e unusual i s o t o p e peak d i s t r i b u t i o n around m/z 207, due t o t h e presence o f t h r e e S i atoms. The absence o f obvious monomer o r dimer peaks as w e l l as o f thermal d e g r a d a t i o n p r o d u c t s i n d i c a t e s a s t r o n g dominance o f b a c k b i t i n g mec_hanisms. A peak o c c u r r i n g a t m/z 281 ( n o t shown) p o i n t s a t t h e occurrence o f t e t r a m e r f o r m a t i o n ,

REMRKS

268

SPECTRUM H,8

i8 -MONOMER

204 *

B-MONOMER

11,.

I.

I-DIMER

1

93 8-DIMER

8-TRIMER

162

I

20

40


60

80

100

120 m/z 140

160

180

: POLYISOPRENE/POLY (cis)BUTADIENE (50/50 copolymer) : see i n s e r t in spectrum ( X and Y represent i n t e g e r s , the

values of which may change throughout the polymer matrix) SAMPLE ORIGIN SAMPLE PREP./SIZE PYROL. CONDITIONS

TOTAL ION COUNTS

: B.F.

Goodrich, USA

: solution in tetrahydrofuran; 10 p g

t c 10s; Eel 12eV; expansion chamber removed (Extranuclear 5000-1 Py-MS system) : 5 x lo4 : Teq 510°C; t T 5 s ;

- The dominant pyrolysis mechanism appears t o be depolymerisation res u l t i n g in the formation of large monomer, medium-sized dimer and small trimer peaks. Many of the o t h e r signals including a l l odd-numbered mass peaks, appear t o be due t o residual electron impact fragmentation (mainly loss of 15 daltons from the monomer and oligomer i o n s ) . I n addition, some peaks may be due t o thermal degradation reactions. The conspicuous peaks a t m/z 220 and m/z 205 represent the molecular ion and (M-15) fragment ion of dibutylhydroxytoluene, a common s t a b i l i z e r in tetrahydrofuran solvents. Note the r e l a t i v e l y low abundance of mixed oligomer s i g n a l s (e.g. the lack of a marked butadieneisoprene dimer a t m/z 1 2 2 j indicating a block co-polymer s t r u c t u r e .

REMARKS

269

SPECTRUM H,9 2 @I*)

04

’8

f

36

S

92

34

20

40

60

80

100

120

m/z

140

160

SAMPLE

: DOWEX 50 WX8, Ht (cation-exchange r e s i n )

CHEMICAL DETAILS

: see i n s e r t i n spectrum; s t y r e n e - d i v i n y l b e n z e n e copolymer

SAMPLE O R I G I N

: F l u k a AG, Buchs, S w i t z e r l a n d

SAMPLE PREP./SIZE

: suspension i n water; 10 ug

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 2

w i t h s u l p h o n i c a c i d f u n c t i o n a l groups

x lo4

REMARKS - Compare w i t h t h e s p e c t r a o f o t h e r s t y r e n e - t y p e polymers (Spectrum H.3, A s t y r e n e monomer peak shows up a t m/z 104 b u t no c l e a r d i v i n y l H.lO, H . l l ) . benzene monomer (MW 130) i s seen. The peaks a t m/z 78, 92 and 118 must r e p r e s e n t benzene, t o l u e n e and phenylpropene. The h i g h peak a t m/z 64 (S02”) i s d i r e c t l y d e r i v e d f r o m t h e s u l p h o n i c a c i d s u b s t i t u e n t s . The presence o f a H2St’ peak a t m/z 34 and a p r o b a b l e thiobenzene peak a t m/z 110 appear t o i n d i c a t e t h e i n f l u e n c e o f r e d u c t i v e processes on t h e s u l p h o n i c a c i d groups, e i t h e r i n t h e o r i g i n a l polymer o r d u r i n g t h e p y r o l y s i s process.

SPECTRUM HI 10 8 %)

28%)

1

59

I8

I

145 I

I

132

L.t..-.

20

60

40

80

100

120

rn/z

140

160

SAMPLE

: DOWEX 1 x 2 , C 1 - (anion-exchange r e s i n )

CHEMICAL DETAILS

: cross1 inked styrene-type polymer w i t h Phe-CH2-N(Me)3 C1-

SAMPLE ORIGIN SAMPLE PREP./SIZE

functional groups : Fluka AG, Buchs, Switzerland : suspension in water; 10 pg


+

: standard : 1.7

x

lo5

REMARKS - Compare with s p e c t r a of other styrene-type polymers (Spectra H.3,

H . l l ) . The polymer backbone i s represented by the peaks a t m/z 9 2 , 104, 106, 118 (130) and 132 (alkylbenzenes). The peaks a t m/z 50, 52 (CH$l+’), 58 (MeZN=CHZ+) and 59 (Me3N+’) a r e derived from the alkylamine functional groups.

H.9,

271

SPECTRUM HI 11 9 *I.)

18 'I.)

8

118

8

104

>

c m

I

z

9

W t-

z

z 0

132

106

71

20

40

60

80

100

120

m/z


: DOWEX 2x8, C1- (anion-exchange resin)

SAMPLE ORIGIN SAMPLE PREP./SIZE PYROL. CONDITIONS

Fluka AG, Buchs, Switzerland : suspension in water; 10 ug : standard : 1.7 x lo5

TOTAL ION COUNTS

:

140

160

crosslinked styrene-t pe polymer with Phe-CH2-N(Me2,C2H40H) Y C1- functional groups

:

- Compare with spectra o f other styrene-type polymers (Spectra H.3, H.9, H.10). The polymer backbone is represented by the peaks at m/z 92, 104, 106, 118 (130) and 132 (alkylbenzenes). The peaks at m/z 50, 52 (CHsCl+'), 58 (MeZN=CHZ+), 59 (Me3N+'), 71 (N,N-dimethylvinylamine) and 89 (N,N-dimethylaminoethanol) are derived from the amino functional groups.

REMARKS

SPECTRUM HI 12

J

99

5558

135

97

?2

120

20

40

60

80

100

120

m/z

140

SAMPLE

: D I ETHYLAMINOETHYL-DEXTRAN

CHEMICAL DETAILS

: glucan w i t h e t h e r - l i n k e d -O-CH2-CH2-NH(C2H5)2

160

i

C1- groups;

MW = 2 X 106

SAMPLE O R I G I N

: Pharmacia, Sweden

SAMPLE PREP./SIZE

: s o l u t i o n i n H20; 10 i ~ q

PYROL. CONDITIONS

: standard

TOTAL ION COUNTS

: 1.5

-

x lo5

The spectrum shows a carbohydrate fragment p a t t e r n of t h e d e x t r a n backbone (e.g. m/z 32, 43, 55, 58, 60, 72, 102, 110, 126) somewhat overshadowed by i o n s i g n a l s d e r i v e d from the d i e t h y l a m i n o e t h y l u n i t s . The main molecular i o n s r e p r e s e n t i n g t h e l a t t e r group appear a t m/z 135 and 137 and presumably r e p r e s e n t a (C2H5)2 NC2H4C1 organochlorine compound formed d u r i n g t h e p y r o l y s i s process. For a d i s c u s s i o n o f o t h e r organochlorine products formed by p y r o l y s i s of quaternary ammonium compounds i n t h e present o f C1 i o n s see Spectra D . l O , D . l l and P a r t I , S e c t i o n 3.5. The i n t e n s e i o n s i g n a l s a t m/z 86, 99, 120 appear t o r e p r e s e n t e l e c t r o n impact fragments o f t h e molecular i o n a t m/z 135, formed by t h e loss of 15 (CH3), 36 (HC1) and 49 (CH2C1) daltons, r e s p e c t i v e l y . The h i g h i n t e n s i t y fragment a t m/z 86 has proven t o be a u s e f u l marker f o r d e t e c t i n g small q u a n t i t i e s o f DEAE-dextrans i n b i o l o g i c a l samples, e.g. v i r u s p r e p a r a t i o n s ( r e f . 63).

REMARKS

273

SPECTRUM HI 13

SAMPLE

: ETHYLCELLULOSE

CHEMICAL DETAILS

: e t h o x y l c o n t e n t 49%

SAMPLE O R I G I N

: S c i e n t i f i c Polymer Products, USA

SAMPLE PREP./SIZE


PYROL. CONDITIONS

: Teq 510°C; t T 5s; t z 10s; Eel 12eV; expansion chamber removed ( E x t r a n u c l e a r 5000-1 Py-MS system)

TOTAL ION COUNTS

: 1

x lo5

RE~R -E A t f i r s t s i g h t t h i s spectrum appears t o be q u i t e d i f f e r e n t f r o m t h a t o f c e l l u l o s e (Spectrum A . l ) because o f t h e dominance o f odd numbered mass peaks and t h e prominent s i g n a l s i n t h e h i g h mass range. O b v i o u s l y many p y r o l y s i s p r o d u c t s now possess an e t h o x y l group i n s t e a d o f a h y d r o x y l group. A l s o , t h e presence o f t h e e t h o x y l g r o u p s s i g n i f i c a n t l y reduces t h e o t h e r w i s e dominant d e h y d r a t i o n r e a c t i o n s . T h e r e f o r e , t h e peaks a t m/z 172 and 200 can be r a t i o n a l i s e d as t h e mono-ethylated and d i e t h y l a t e d analogues o f a n h y d r o l e v o g l u c o s a n ( n o r m a l l y a t m/z 144) r e s p e c t i v e l y , whereas t h e peaks a t m/z 143 and 171 may t h e n r e p r e s e n t t h e (M-29) fragments. I n t e r e s t i n g l y , e t h y l a t i o n ( g a i n o f 28 d a l t o n s ) f o l l o w e d by t h e l o s s o f an e t h y l r a d i c a l (29 d a l t o n s ) r e s u l t s i n t h e n e t l o s s o f 1 d a l t o n . Thus, some o f t h e m a j o r odd mass peaks may w e l l c o r r e s pond t o t h e n e x t h i g h e r mass peak i n t h e normal c e l l u l o s e spectrum, e.g. m/z 31 vs. 32, m/z 59 vs. 60, m/z 97 vs. 98, m/z 125 vs. 126. Furthermore, p y r o l y t i c d e g r a d a t i o n o f t h e e t h o x y l groups may r e s u l t i n t h e e l i m i n a t i o n o f ethene (m/z 28) and e t h a n a l (m/z 44) r e s p e c t i v e l y . Such p y r o l y t i c e l i m i n a t i o n s may e x p l a i n t h e occurrence o f t h e marked peaks a t m/z 156 (m/z 200 - 44) and 112 (m/z 200 2 x 44).


275

REFERENCES 1

P.D. Zemany, I d e n t i f i c a t i o n o f complex organic m a t e r i a l s by mass s p e c t r o m e t r i c a n a l y s i s of t h e i r p y r o l y s i s products. Anal. Chem., 24 (1952) 1709-1713.

2

W.H.T. Davison, S. Slaney and A.L. Wragg, A novel method o f i d e n t i f i c a t i o n o f polymers. &em. and Ind., (1954) 1356.

3

E.M. Wilson, V. Oyama and S.P. Vango, Design f e a t u r e s o f a l u n a r gas chromatograph, i n N.B. Brenner (Ed.), Proc. 3rd I n t . Symp. on Gas Chromatography, 1962, Acad. Press, New York, pp. 329-338.

4

V.J. Oyama, Use o f gas chromatography f o r t h e d e t e c t i o n o f l i f e on Mars. Nature, 200 (1963) 1058-1059.

5

E. Reiner, I d e n t i f i c a t i o n o f b a c t e r i a l s t r a i n s by p y r o l y s i s - g a s - l i q u i d chromatography. Nature, 206 (1965) 1272-1274.

6

E. Reiner, R.E. Beam and G.P. Kubica, Pyrolysis-gas-chromatography s t u d i e s f o r t h e c l a s s i f i c a t i o n o f mycobacteria. Am. Rev. Resp. Dis., 99 (1969) 750-759.

7

E. Reiner, J.J. Hicks, M.M. B a l l and W.J. Martin, Rapid c h a r a c t e r i z a t i o n o f Salmonella organisms by means o f pyrolysis-gas-1 i q u i d chromatography. A m Z . Chem., 44 (1972) 1058-1061.

8

D.O. Hummel and H.-D.R. Schlddemage,Thermischer Abbau von Polysulfonen I. Gaschromatographische Untersuchung der Pyrolyseprodukte von P o l y s t y r o l s u l f o n e n . Kolloid-2. u. Z. Polymere, 210 (1966) 97-102.

9

H.-D. R. Schuddemage, Karakterisierung von Hochpolymeren durch FyroZyse irn Feldionen-Massenspektrameter. Thesis, U n i v e r s i t y o f Cologne (1967).

10

R.L. Levy, P y r o l y s i s gas chromatography, review o f the technique. Chromatogr. Rev., 8 (1966) 48-89.

11

W.J. I r w i n and J.A. Slack, A n a l y t i c a l p y r o l y s i s i n biomedical s t u d i e s , a review. The Analyst, 103 (1978) 673-704.

12

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191 J.W. de Leeuw, p e r s o n a l communication, 1977. 192

K.J. Voorhees, F.O. Hileman, and S.M. Kunen, Pyrolysis-mass s p e c t r o m e t r y s t u d i e s of atmospheric p a r t i c u l a t e s , i n Proc. 26th Annual ASMS Conf. on Mass Spectrome t r y and A l l i e d Topics, S t . L o u i s , U.S.A., 1978, p. 437.

193 A.C. S i g l i o , Degraded l i g n i n compounds i d e n t i f i e d i n s i l i c i f i e d wood 200 m i l l i o n y e a r s o l d . Science, 200 (1978) 1054-1056. 194

H.L.C. Meuzelaar, G.R. H i l l , J.H. F u t r e l l , A.M. Harper, D.J. Iwamoto, D.L. Pope, G.S. M e t c a l f and J.H. Tomlinson, C h a r a c t e r i z a t i o n o f Rocky Mountain Coals and c o a l 1 i q u i d s by computerized a n a l y t i c a l t e c h n i q u e s , F i r s t Annual Progress Report (DOE c o n t r a c t number DE-FG22-8OPC30242), 1981.

195

M. T e i c h m u l l e r , R. T e i c h m u l l e r , i n E. Stach, G.H. Taylow, M-Th Mackowsky, D. Chandra, M. T e i c h m u l l e r , R. T e i c h m u l l e r ( E d s . ) , s t a c h ' ~ Testbook of CoaZ Petrology, Gebruder B o r n t r a e g e r , B e r l i n , 1975, pp. 5-53.

196

G. van Graas, J.W. de Leeuw and P.A. Schenck, C h a r a c t e r i z a t i o n o f c o a l s and sedimentary o r g a n i c m a t t e r by C u r i e - p o i n t p y r o l y s i s mass s p e c t r o m e t r y , P a r t I . J . Anal. AppZ. Pyrol., 2 (1980) 265-276.

197

L.H. Wojcik, D.L. Pope, L.R. Frank, C . Greenwalt, H . L . C . Meuzelaar and J.H. F u t r e l l , An i n v e s t i g a t i o n o f t h e p y r o l y s i s o f a composite Green R i v e r o i l s h a l e by TGA-MS and f a s t - s c a n n i n g Py-MS, i n Proc. 28th Annual ASMS Conf. on Mass Spectrometry and A l l i e d Topics, New York, 1980 pp. 458-459.

198

H. S o l l i , S.R. L a r t e r and A.G. Douglas, The a n a l y s i s of kerogens by p y r o l y s i s gas chromatography-mass s p e c t r o m e t r y u s i n g s e l e c t i v e i o n d e t e c t i o n . 2 . Use o f a l k y l b e n z e n e s f o r geochemical c o r r e l a t i o n s s t u d i e s , i n A.G. Douglas and J.R. Maxwell (Eds. ) Adv. i n Organic Geochemistry, 1979, Pergamon Press, Oxford, 1980, pp. 591-597.

199

H. S o l l i , S.R. L a r t e r and A.G. Douglas, The a n a l y s i s of kerogens by p y r o l y s i s gas chromatography-mass s p e c t r o m e t r y u s i n g s e l e c t i v e i o n m o n i t o r i n g 2. A l k y l napthalenes, J . Anal. AppZ. P y r o l . , 1 (1980) 237-241.

H. S o l l i and A.G. Douglas, A n a l y s i s o f kerogens by p y r o l y s i s - g a s chromatography-mass s p e c t r o m e t r y u s i n g s e l e c t i v e i o n d e t e c t i o n . J . Chromatog. 167 (1978) 421-431.

200 S.R.Larter,

2 01 D. van de Meent, S.C.

Brown, R.P. P h i l p and B.R.T. S i m o n e i t , P y r o l y s i s - h i g h r e s o l u t i o n gas chromatography and p y r o l y s i s gas chromatography-mass s p e c t r o m e t r y o f kerogens and kerogen p r e c u r s o r s . Geochim. Cosmochim. Acta, 44 ( 1 980) 999-1 01 3.

202 W.E. Robinson, Kerogen o f Green R i v e r f o r m a t i o n i n o r g a n i c geochemistry

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Methods and r e s u l t s . i n G. E g l i n t o n and M.T.J. Murphey ( E d s . ) , Organic Geochemistry, S p r i n g e r Verlag, B e r l i n , 1969, pp. 619-637. 203

P.H. van d e r Meide, P. Westbroek, E.W. de Jong, J.W. de Leeuw and H.L.C. Meuzelaar, C h a r a c t e r i z a t i o n o f macromolecules f r o m f o s s i l s h e l l s by immunolo y and C u r i e - p o i n t p y r o l y s i s mass s p e c t r o m e t r y , i n M. Omori and N. Watabe, (Eds32'he Mechanisms of Biomineralization i n Animals and PZants,(Proc. 3 r d I n t . Symp. on t h e Mechanisms o f B i o m i n e r a l i z a t i o n i n t h e I n v e r t e b r a t e s and P l a n t s , K a s h i k o j i m a , Japan, 1977),Tokai U n i v . Press, Tokyo, 1980, pp. 251-256.

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H.L.C. Meuzelaar and S.M. H u f f , C h a r a c t e r i z a t i o n o f leukemic and normal w h i t e b l o o d c e l l s by C u r i e - p o i n t pyrolysis-mass spectrometry. J . A n a l . Pyroz., 3 (1981) 111-129.

205

J . Haverkamp, W. Windig, D. V a l e r i o and P.G. Kistemaker, Advances i n p y r o l y s i s mass spectrometry, i n h o e . 28th Annual ASMS Conf. on Mass Spectrometry and AZZied Topics, New York, USA, 1980, pp. 49-50.

206

S.R. L a r t e r , A geochemical study o f kerogen and r e l a t e d m a t e r i a l s , Thesis, U n i v e r s i t y o f Newcastle upon Tyne, UK, 1978.

207

B.R. Thomas, Modern and f o s s i l p l a n t r e s i n s , i n J.B. Harborne (Ed.) PhytochemicaZ Phy Zogeny , 1970, Acad. Press, London , pp. 59-79.

208

G.O. P o i n a r and J. Haverkamp, Use o f p y r o l y s i s mass spectrometry i n t h e i d e n t i f i c a t i o n o f amber samples, Gems and GemoZogy, submitted f o r p u b l i c a t i o n .

209

F.J.E.M. KGppers, Pyrolyse van c a n n a b i d i o l , Ph.D. Thesis, U n i v e r s i t y o f U t r e c h t , The Netherlands (1973).

287

SUBJECT

INDEX

A A c e t i c acid, 19 A c e t y l c h o l i n e , 10 A c r o l e i n , 35 Adenosine phosphate, C. 3,C. 4,C.5,C.6 A d r e n o c o r t i c o t r o p h i c hormone (ACTH), B.l ,B.2,B.3 A d r e n o c o r t i c o t r o p i n , 22 Agarose, A.6 A i r p a r t i c u l a t e s , 77 - n o n - l i n e a r map, 92 A1 bumin , 3,12,32,34,35,41 A l g i n i c acid, A.9 A1 kenes , 27,40,94,95 Alkaptonuria, 84 A l k y l p y r r o l e s , 87 Amber, F.27 Amino acids, 10,15 - a l i p h a t i c , 21 aromatic, 21 sulphur-containing, 21 Amino sugars, 17 Ammonia, 13 A m p i c i l l i n , 6.7 Amylose, 12, A.2 Analogue i o n d e t e c t i o n , 45,46 A n c i l l a r y a n a l y t i c a l methods, 13,42 Anhydrosugars, 17,A.5,A.6 A n t i b i o t i c s , 7, 24 Antigens ( b a c t e r i a l ) , 85 Arabinan ( f r o m apple j u i c e ) , A . l l ArenicoZa marina, 86 Aromatic components, 87,89 Arthrobacter, 78 Automation, 6,7 Azo compounds, 31

-

B

Bacizzus, 78 - subtiZis, 86,A.lg,A.lga,A.22,A.25,A.25a Background ( i n s t r u m e n t ) , 8,31,47,48 Bacteria - c l a s s i f i c a t i o n and i d e n t i f i c a t i o n , 77 - colonies, 29,31 - s t r a i n s , 7,10,11 Base phosphate condensates, 19 Batch c o a t i n g o f sample wires, 32 Benzene, 27,93,95

Benzofurans, 96 Benzoic a c i d s ( s u b s t i t u t e d ) , 6 Beverages, 77 B i l e , 83 B i l i r u b i n , G.10 Bimolecular r e a c t i o n s , 10 Biochemical f r a c t i o n s , 77 Biogeochemical a p p l i c a t i o n s o f Py-MS, 92 ,105, F. 1-F.29 B i o l o g i c a l a p p l i c a t i o n s o f Py-MS, 77, 86,87.88 Bituminous c o a l , 12,F.17-F.21 Body f l u i d , 49,77,83 Bovine m i 1k (homogenized), 12 Bovine serum albumin, 12,51 B r a i n t i s s u e , 10 Burlap, E.3

C Cannabidiol, G.8,G.8a Capsular polysaccharide, 85 Neisseria meningitides, A. 14 ,A. 15, A.20,A.20a,A.23 HaemophiZus influenzae, A.24 C w b o h y d r a t e - p r o t e i n mixtures, 35,36 Carbohydrates, 35,36,71,11O,A. 1- A 2 7 - anhydrohexose, A.6 deoxyhexose, A.10 - hexosamine, A. 12 hexose, A.l-A.6,A.Z,A.26,A.27 - hexuronic a c i d , A.8,A. 9 ,A. 16-A. 19a keto-deoxyoctonate (KDO), A.20, A. 20a ,A. 21 - ketose, A.7 - rnuramic a c i d , A.25,A.Z5a - N-acetyl -hexosamine, A. 13-A.20a, A.25-A.27 - N-acetylneuraminic a c i d , A.23,A.27 pentose, A.ll,A.24 Carbon d i s u l p h i d e , 29 Carbowax 20M, H.4 Carragheenan, A.5 C a t a l y s t s , 15 C a t a l y t i c r e a c t i o n s , 31 Cataractous eye-lens t i s s u e , 84 C a t t l e food, 90 Cells, 9,29 C e l l i d e n t i f i c a t i o n , 5,84 C e l l w a l l composition, 86

-

-

-

-

288

A. 1 , C e l l u l ose , 12,15,16,17,18,93,94, A.la Ceruloplasmin, 8.13 Channel e l e c t r o n m u l t i p l i e r arrays, 6 Char, 22,37,41,49,96 C h a r a c t e r i s t i c i t y , 59,60 Chemical i o n i s a t i o n mass spectrometry, 4,7,14,24,41,42,43 Chemical i n t e r p r e t a t i o n , 11,68 Chi-squared c o e f f i c i e n t s , 62 - a l g o r i t h m , 63 Chi t i n , 17,18,86,A.13, A. 13a Chitosan, A.12,A.l2a C h o l e s t e r o l , 49,D.g Choline, 10,25,26,71, D12, D12a C h o n d r o i t i n sulphate, A.17-A.18a Chromic acid, 31 C l i n i c a l d e t e c t i o n and diagnosis, 85 Coal, 5,27,29,49,70,95,96 - bituminous, 12,96 - boghead, F.22,F.23 brown, 93,94 - conversion behavior, 27 - h i g h v o l a t i l e bituminous, F.17-F.20 - medium v o l a t i l e bituminous, F.21 subbituminous, 12,93 Coating techniques, 29-33 Coatings, 77 Coffee r u s t , 86,87 Collagen, 68,B.12 C o l l i s i o n a l induced d i s s o c i a t i o n , 11, 14,19 Computer-assi s t e d chemi ca 1 i n t e r p r e t a t i o n , 68 Computer-assisted q u a n t i t a t i v e a n a l y s i s , 70 Computerised data processing, 7,46,55 Computerised mass a n a l y s i s , 43,46 Condensation, 24,37,39,44,52 Connective t i s s u e , 4 Contaminants ( d e t e c t i o n o f ) , 86 Contamination o f ion-source, 37,39,40 Coorongite, F.8 Cork, E.12 Cotton, E.1 Cresol, 21,35 C u r i e - p o i n t p y r o l y s i s mass spectrometry, - automated, 44 diagram, 39 - technique, 29 Cyanocobalamin ( v i t a m i n B12), G.9 Cysteine, 21,69 Cytochrome C, 6.9

-

-

-

D DNA, 4,8,14,19,20,21 ,C.2 Data a n a l y s i s procedures, 55-76 DEAE-dextran, 10,70,72,H.12 Decarboxyl a t i o n , 16

Degradation r e a c t i o n s , 16,34,35 Dehydrated nucleosides, 19 Dehydration, 16 Dehydrogenated p y r o l y s i s products, 47 Dendrograms, 65 Deoxysugars, 17,A.lO,A.21 Deoxyribonuclease I , B.6 Deoxyribonucleic a c i d (DNA), 4,8,14,19 20,21 ,c.2 Deoxyribose , 19,20 Depolymerisation, 15,24 D i benzvl 7.10 D i e t h y i ami noe t h y l ( DEAE ) -de x t ran, 10,70 72, H.12 D i f f e r e n c e spectrum, 68 D i f f u s i o n l i m i t e d pumping, 43 D i g i t o n i n , 6.3 D i g i t o x i n , G.l Digoxin, 6.2 D i hydroxybenzene , 93,95 Diketopiperazine, 10 Dimethoxyphenyl , 25 Dimethylaminoethanol , 25,26 Dimethylvinylamine, 26, D12, D12a Dimet h y l v iny 1ammoni umchl o r i de , 26 D i n u c l e o t i de , 8,20 D i p a l m i t o y l , D.12,D.l2a Dipeptides, 23,24 D i r e c t probe p y r o l y s i s mass spectrome t r y , 3,4,7,19,23,24,25,41 D i s c r i m i n a n t a n a l y s i s , 55,75 Diseases ( p l a n t ) , 86 Distance - Euclidean, 63 c i t y block, 62,63 Douglas fir, 12,52,53 ,E. 11 Dowex, H.9-H.11 Drugs, 11,77,111 ,G.l-G.9 Duchenne muscular dystrophy (DMD), 55, 69,71

.

-

E Eddy c u r r e n t s , 33 E l e c t r o n energies, 41,52 E l e c t r o n impact io n i s a t i o n , 3,4,13,19, 20 ,24 E l e c t r o - o p t i c a l i o n d e t e c t o r s , 6,14 E l e c t r o p h o r e t i c bands, 24 Endogonaceae, 86 E q u i l i b r i u m temperature, 33,34,35,36 Ergosterol, D . l l Escher

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