JOURNAL OF CHROMATOGRAPHY LIBRARY - VOLUME 24 chemical methods in gas chromatography

The application of GC in chemical analysis has substantially advanced many branches of analytical chemistry and brought forth radical changes in the organic analysis of volatile compound

JOURNAL OF CHROMATOGRAPHY LIBRARY - VOLUME 24

chemical methods in gas chromatography

JOURNAL OF CHROMATOGRAPHY LIBRARY - volume 24

Molenwerf 1

P.O Box 21 1, 1000 A E Amsterdam, The Netherlands

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ELSEVIER SCIENCE PUBLISHING COMPANY I N C

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Library of Congress Cataloging in Publication Data

Eerezkin, V G (Viktor Grigor'evich), 1931-

Chemical methods in gas chromatography

( J c u r n a l of chromatography library ; v 24)

Includes hiblicgraphical references and index

1 Gas chroiuatograpu I Title 11 Series

QD79.Ch5B45 1983 543l.0696 ,53-1667

ISBN 0-LU-41951-9 (U.S.)

ISBN 0-444-41951-9 (Val 24)

ISBN 044441616-1 (Series)

0 Elsevier Science Publishers B.V., 1983

A l l rights reserved NO part of this publication may be reproduced, stored in a retrieval system or transmitted in any f o r m or b y any means, electronic, mechanical, photocopying, recording or other- wise, without the prior written permission of the publisher, Elsevier Science Publishers B.V., P.O

Box 330,1000 AH Amsterdam, The Netherlands

Printed in The Netherlands

Contents

Journal of Chromatography Library , , VII

Introduction 1

References 14

Chapter 1 Application of chemical methods to the pre-treatment of samples before chromato- graphic separation

1.1, Introduction

1.2 Peculiarities of the elaboration of techniques

1.3 The main methods used to obtain derivatives for gas chromatography

1.3.1 Silyl derivatives

1.3.2 Esters

1.3.2.1 The diazomethane method

1.3.2.2 The methanol method ,

1.3.2.3 The pyrolysis method

1.3.3 Ethers

1.3.4 Acyl derivatives

1.3.5 Oximes and hydrazones

1.3.6 Derivatives of inorganic compounds

1.4 Application of the chemical formation of derivatives method

1.4.1 Alcohols and phenols

1.4.2 Aldehydes and ketones ,

1.4.3 Amines

1.4.4 Carboxylic acids

1.4.5 Analysis of inorganic acids and metals

1.5 Conclusion

References

15 15 30 45 47 41 41 47 48 49 49 50 50 51 51 51 53 53 54 55 56 Chapter 2 Kinetic methods in gas chromatography

References 67 82 Chapter 3 Pyrolysis-gas chromatography

3.1 Introduction ,

3.3 Identification of substances

3.4 Determination of the composition of polymer systems and the structure of polymers

3.5 Conclusion

References

85 85 a9 112 121 131 132 3.2 Equipment and experimental procedure

Chapter 4 Determination of the carbon skeleton in organic compounds

References , 139 154 Chapter 5 Subtraction method

5.1 Principle of the method

5.2 Application to the identification of the components of test mixtures

5.3 Application t o impurity analysis

5.4 Prospects for development of the method

References

157 157 164 178 180 181 Chapter 6 Chemically selective stationary phases References 204 185

Chapter 7 Reaction gas chromatographic methods of elemental analysis

7.1 Introduction

7.2 Determination of carbon and hydrogen

7.3 Determination of nitrogen

7.4 Determination of sulphur

7.5 Determination o f oxygen

7.6 Determination of other elements

7.7 Eleniental analysis of gas chromatographically separated fractions

7.8 Conclusion ~ References

Chapter 8 Reaction methods of trace analysis

8.1 Introduction

8.2 Increasing the retention time of the main and trace components or their derivatives

8.3 Decreasing the retention time o f t h e main and trace components or their derivatives

8.4 Improvement of detection characteristics

8.5 Conclusion

References

Chapter 9 Functional group analysis

9.1 Introduction

9.2 Specific features o f chemico-chromatographic methods o f functional group analysis

9.3 Preliminary reactions

9.4 R e a d i o n s after gas chromatographic separation

9.5 Methods of analysis of main functional groups

9.5.1 Determination of active hydrogen

9.5.2 Determination of amino groups

9.5.3 Determination of main oxygen-containing groups

9.5.4 Determination of some sulphur-containing groups

9.5.5 Determination of alkoxy groups

9.5.6 Determination of sulphur-, nitrogen- a n d silicon-alkyi groups

9.5.7 Determination of vinyl and alkyl groups

9.5.8 Determination of compounds with unsaturated bonds

9.6 Conclusion

References

209 209 213 218 225 229 2 3 2 233 243 243 249 249 250 261 267 279 280 287 287 288 288 292 296 296 297 298 299 300 301 302 302 304 305 Subject lndex 311

JOURNAL OF CHROMATOGRAPHY LIBRARY

A Series of Books Devoted to Chromatographic and Electrophoretic Techniques and their Applications

Although complementary to the Journal of Chromatography, each volume in the library

series is an important and independent contribution in the field of chromatography and electrophoresis The library contains no material reprinted from the journal itself

edited by T Braun and G Ghersini

Liquid Column Chromatography A Survey of Modern Techniques and

Applications

edited by Z Deyl, K Macek and J Janik

Detectors in Gas Chromatography

by J SevEik

Instrumental Liquid Chromatography A Practical Manual on High-

Performance Liquid Chromatographic Methods

by N.A Parris

Isotachophoresis Theory, Instrumentation and Applications

by F.M Everaerts, J.L Beckers and Th.P.E.M Verheggen

Chemical Derivatization in Liquid Chromatography

by J.F Lawrence and R.W Frei

Chromatography of Steroids

by E Heftmann

HPTLC - High Performance Thin-Layer Chromatography

edited by A Zlatkis and R.E Kaiser

Gas Chromatography of Polymers

by V.G Berezkin, V.R Alishoyev and 1.B; Nemirovskaya

Liquid Chromatography Detectors

Volume 1 5 Antibiotics Isolation, Separation and Purification

edited by M.J Weinstein and G.H Wagman

Volume 16 Porous Silica Its Properties and Use as Support in Column Liquid

Chromatography

by K.K Unger

75 Years of Chromatography - A Historical Dialogue

edited by L.S Ettre and A Zlatkis

Electron Capture Theory and Practice in Chromatography

edited by A Zlatkis and C.F Poole

Environmental Problem Solving using Gas and Liquid Chromatography

by R.L Grob and M.A Kaiser

Volume 22A Chromatography Fundamentals and Applications of Chromatographic and

Electrophoretic Methods Part A: Fundamentals

edited by E Heftmann

Volume 22B Chromatography Fundamentals and Applications of Chromatographic and

Electrophoretic Methods Part B: Applications

edited by E Heftmann

Volume 23A Chromatography of Alkaloids Part A: Thin-Layer Chromatography

by A Baerheim Svendsen and R Verpoorte

Chemical Methods in Gas Chromatography

by V.G Berezkin

Volume 17

Volume 18A Electrophoresis A Survey of Techniques and Applications

Volume 18B Electrophoresis A Survey of Techniques and Applications

Volume 19

Volume 20

Volume 2 1

Volume 24

The author dedicates this book

to the memoly of his parents

Introduction

The advances made in science and technology in the second half of the twentieth century are closely associated with the development of analytical methods ‘Science progresses in quantum leaps depending on the advances in methods Every forward step

in the development of methods brings us to a new height from which we observe a broader vista with previously unseen objects’ - such was the vivid and accurate assess- ment of the significance of new methods by Pavlov

One of the most significant and spectacular achievements in analytical chemistry over the past quarter of a century has been the development and wide practical appli- cation of gas chromatography (GC) GC marked dramatic progress in the studying of complex mixtures of organic compounds and inorganic gases

The application of GC in chemical analysis has substantially advanced many branches

of analytical chemistry and brought forth radical changes in the organic analysis of volatile compounds Many determinations that had been practically impossible or demanded several days of arduous work have become accessible to any investigator

in an ordinary laboratory with the aid of GC

GC was a gigantic leap forward in studies of complex mixtures of organic compounds and inorganic gases A tremendous improvement has been achieved in such basic charac- teristics of analytical experiments as sensitivity (detection limit), selectivity (resolving power), accuracy, speed and reliability

The extensive use of GC in science and industry has given rise to a large volume of literature Fig 1 shows diagramatically the distribution of publications on individual analytical methods in the analysis of organic compounds and gases [ l ] It can be seen that in 1975 45% of all publications on the analytical chemistry of organic compounds

and gases dealt with chromatographic methods, GC accounting for 26% of the total

number of works on chromatography Table 1 lists analytical methods in decreasing

order of their frequency of use by subscribers to AnaZyticaE Chemistry [ 2 ] The table

demonstrates that GC is currently one of the most widely used techniques Another indication of the wide popularity of GC is the volume of production of gas chromato- graphs For example, in the U.S.A alone one (American) billion dollars worth of chro- matographs are produced each year and 47.9% of all research laboratories in that country use GC [3]

The development of GC methods and the extension of areas of their application are continuing even today, with emphasis on combined or, to use Zolotov’s definition, hybrid methods [4] incorporating two and more techniques The promising nature of the combined use of several chemical methods has been pointed out by Ostwald, who wrote that, if separation cannot be achieved directly by physical methods, one has the alterna- tive of providing the right conditions for the isolation of a new phase by appropriately transforming the separated substances and other compounds, the new phase containing a derivative of the sample substance and enabling its mechanical separation [5] The attractive idea of combining chemical and physical methods was later elaborated by

Liquid colunl,i chromatography

Ion-select ive electrodes

Nuclear magnetic resonance spectroscopy

Molecular sieve chromatography

INTRODUCTION 3 Siggia [6] and Zolotov [4] in analytical chemistry, and by Ettre and McFadden [7]

and Berezkin [8] in GC

In considering combined methods one should proceed from a classification of analytical methods The methods adopted in analytical chemistry lend themselves to the following classification into three groups: (1) methods for separating mixtures of elements or compounds, (2) methods for determining the amount or concentration of elements or compounds, and (3) methods for the chemical conversion of elements or

compounds The first two groups have been covered at length by Zolotov [4] We have extended his classification to include also methods for the chemical conversion of sub-

stances or chemical methods [9] These methods imply controlled chemical transforma- tions involving components of the sample mixture of elements or compounds with a view to obtaining chemical derivatives suitable for the solution of a given analytical task under given conditions

Analytical GC is essentially a combined, or hybrid, method based on the simultaneous application of two methods: (1) a method for the chromatographic separation of components of the sample mixture in a gaseous flow moving with respect t o a stationary

phase and (2) a method for the quantitative (and qualitative) determination of the

zones of the separated components The role of the second, or detection, method is no less important than that of the first As early as 1962 Zhukhovitsky and Turkeltaub

wrote that ‘the history of advances in gas chromatography is in fact the history of devel-

opment of the detector’ [lo] Initially, analytical GC was regarded as a physical separa- tion technique [lo, 1 1 1 However, such a restricted approach, although justified in the

early years of GC, inevitably imposed certain limitations on its development and appli-

cation, namely (1) the range of substances that can be analysed is confined to volatile

compounds and compounds thermally stable at the separation temperature, (2) the selectivity of separation, determined only by physical factors, is not always sufficient and

(3) the sensitivity (detection limit) of the widely employed thermal conductivity-type detectors and the gas density balance, based entirely on physical principles, is not high The implementation of chemical methods in GC will help to obviate these limitations The application of chemical methods has made it possible to broaden the scope of GC (e.g., determination of metals, polymers, anions, ultimate analysis), to develop new effective techniques of identification and group determination of components in complex mixtures of compounds, to improve separation and to enhance the sensitivity and, in some instances, the accuracy of determination of sample compounds Hence the intro- duction into GC of a new ‘chemical variable’ provided the analyst with ‘chemical vision’ and contributed to advances in analytical chromatographic methods and the solution of practically important problems

Chemical methods in GC, which can also be termed ‘analytical reaction GC’, represent

a GC technique combining chemical and chromatographic (physical) analytical methods

in which chemical transformations can be conducted within and without the chromato- graphic system This definition is broader than that proposed earlier [8] ; it takes into account the wider experience in and further development of analytical reaction GC, which also includes chemical transformations occurring outside the chromatographic scheme

Analytical reaction GC combines three basic methods of analytical chemistry:

separation, determination (detection j and chemical transformation methods Therefore,

it may also be called using Zolotov’s terminology [4] , a polyhybrid method

Analytical reaction GC is characterized by specific experimental techniques, a par-

ticular, area of application and distinctive design features of the instruments used It

should be emphasized that when chemical methods are used in GC, the efficiency of

chromatographic separation, sensitivity and other characteristics of the detector remain

virtually the same However, as a result of chemical reactions, or transformations of

the sample mixture, newly formed compounds are subjected to determination or separa-

tion, and the separation factors and detection sensitivity can be varied in a controlled

manner It should also be noted that the chemical transformation method is applicable

in other fields of analytical chemistry ( e g , spectroscopy, electrochemistry)

The tasks accomplished by analytical reaction GC are varied but basically they fall

into four distinct categories

{I) Broadening of the scope of application (analysis of polymers, metals, etc.)

(2) Detailed analytical study of a substance This task is accomplished by the following

GC analyses: ultimate, molecular (component), isotopic functional group, kinetic and

others

( 3 j Improvement of analytical determination characteristics: separation, sensitivity,

speed, etc

(4) Production of standard mixtures for qualitative and quantitative analysis

Table 2 compares classical ‘physical’ chromatography with analytical reaction GC

It suggests that the rational application of chemical methods in chromatography permits

all of its basic characteristics t o be substantially improved

Chemical reactions are conducted at all of the main stages of the analytical chromato-

graphic experiment, including (1) sample preparation, (2) separation and ( 3 ) detection

or measurement of the separation result The employment of chemical transformations

as early as the initial stage makes it possible to extend drastically the area of application

of the GC method as a whole As is well known, GC can be applied directly only to

resin 1 11 ] Pyrolysis temperature: 900°C 1-8: identified pyrolysis products

General Stable and volatile compounds Plus unstable and non-volatile

Detection limit is restricted

compounds (amino acids, metals, polymers, etc.) Possible

Possible Efficient enough as a result of additional use of (1) sufficiently strong interactions between the analyte and the stationary

phase (e.g., use of complexing

agents) and (2) chemical reactions whose products are separated more easily than the initial substances

(2) To ensure high sensitivity undetectable compounds are converted into those detectable

by high-sensitivity detectors samples that can be vaporized at the separation temperature without any chemical changes To apply GC to such objects one must, using appropriate chemical methods, convert them into stable volatile compounds that can be analysed successfully by GC

techniques For example, in polymer pyrolysis, GC may be used for identifying polymers

and determining the monomeric composition of copolymers Fig 2 illustrates as an example a chromatogram obtained in the pyrolysis of phenol-formaldehyde resin and showing macromolecular fragments corresponding to particular pyrolysis products [ 1 11 Fig 3 shows chromatograms obtained in the determination of beryllium in some rocks, including rock samples from the moon [ 121 The chromatogram in Fig 4 [ 131 represents

amino acid derivatives Free amino acids cannot be analysed directly by GC Fig 5 shows

a chromatogram of halogenated heptanes, obtained by thermal decomposition of tetra- heptylammonium salts of iodine, bromine and chlorine in the sample injector of a

chromatograph The halogen derivatives of tetraheptylammonium were produced by extraction of aqueous solutions with a solution of tetraheptylammonium carbonate

in toluene-undecanol solution [14]

Fig 3 Chromatogram of beryllium trifluoroacetylacetonates, obtained in analysis of moon rocks

A, Crystalline rock (Apollo 11); B, breccia rock (Apollo 11); C, standard containing 100 g of beryllium; peak 1, beryllium trifluoroacetylacetonate From ref 12

Fig 4 Chromatogram of separation on a silicone stationary phase of methyl esters of trifluoro- acetylated amino acids of hydrolysate of human fingernail Sorbent: silicone stationary phase Tem-

perature programme: A, 100°C, isothermal; B, heating from 100°C at l.S"C/min; C, heating from 116.5"C at 4°C/min; D, 140"C, isothermal; E, heating from 140°C at 6"C/min to 210°C Peaks: 1 = afanine; 2 = valine; 3 = glycine; 4 = isoleucine; 5 = threonine; 6 = leucine; 7 = norleucine; 8 = internal standard; 9 = p r o h e ; 10 = asparagine; 11 = ghtamine; 12 = phenylakdnine; 13 = tyrosine;

14 = lysine From ref 1 3

INTRODUCTION

Time (rnin)

Fig 5 Chromatogram of separation of halogenated heptanes Peaks: 1 = heptene-1; 2 = toluene;

3 = 1-chloroheptane; 4 = 1-bromoheptane; 5 = liodoheptane; 6 = dodecanol; 7 = triheptylamine (1 and 7 are by-products; 2 and 6 are solvents) From ref 14

The numerous physico-chemical applications of reaction GC, which constitute a separate field of experimental physical chemistry, are not considered in this paper The application of GC in catalysis has been treated at length by Roginsky et al [15] andh other reviews [16-181; GC methods as applied to studies of the kinetics of homogeneous

reactions are discussed in reviews [19,20]

The main purpose of a chromatographic study is the separation of compounds with closely similar properties The factor separation Rji of the chromatographic zones of

two compounds j and i is described by the equation

where aji is the relative retention volume for compounds j and i (sorbent selectivity),

Ni is the number of theoretical plates for component i and K i is the distribution coef-

ficient of substance i between the stationary and mobile phases The equation suggests that the separation is determined primarily by the value of the selectivity factor, aji A

slight increase in aji, particularly in the region where it is close to unity, leads to a sharp decrease in the number of theoretical plates required for separation As the required number of theoretical plates is almost proportional to the separation time, by enhancing the selectivity one can substantially reduce the latter, which is why the search for new selective stationary phases has always received a great deal of attention

A good example of highly selective stationary phases is those based on the complexing

reaction between the separated volatile compounds and the non-volatile active com- ponent of the stationary phase Complexing in the stationary phase is a particular case

of reversible chemical interaction The selectivity of such phases is extremely high; it is sufficient for the separation of the isomers of some compounds, particularly nuclear isomers of unsaturated organic compounds whose molecules differ only in the content and position of deuterium atoms

Solutions of silver salts in polar solvents were the first t o be used as complexing

Fig 6 Chromatogram of separation of unsaturated C , -C, compounds Column, 2 m X 1.75 mm I.D.;

stationary phase, 15% 0.01 M dicarbonylrhodium 3-(trifluoroacetyl) camphorate on Chromosorb P

'l'emperature, 30°C Peaks: 1 = acetylene; 2 = isobutene; 3 = trans-butene-2; 4 = cis-butene-2; 5 = butene-1 From ref 26

agents in liquid stationary phases for the selective separation of isomers of unsaturated hydrocarbons in GC [21-251 Later, the range of complexing agents used for separation

in GC was extended considerably More stable agents have been developed Of particular

interest, for example, is the use of 0.01M solutions of dicarbonylrhodium 3-(trifluoro- acety1)camphorate (DRTC) in squalane as a selective liquid stationary phase The results

of employing this phase in the separation of butenes are presented in Fig 6 [26] It can

be seen that it takes 4 min to separate all butenes Note that when only squalane is used

as the stationary phase, the separation of some compounds (e.g., trans- and cis-butenes)

is much more difficult, and some compounds (e.g., isobutene and butene-1) are not separated at all Fig 7 [27] is a chromatogram of deuterated ethylenes on DRTC,

separated using a capillary column The separation of isomers is good Hence the method

of analytical reaction GC can also be used in isotopic analysis

The application of chemical methods makes it possible in some instances to eliminate one of the typical difficulties encountered in trace analysis, namely masking of the peaks

of trace components by the broad zone of the main component To separate the zone of trace components from that of the main components, which may contain various types

of compounds, the subtraction method is often recommended This consists in incorpora- tion into the chromatographic system of a reactor containing a reagent that forms non- volatile compounds with the main component; the zones of trace components pass

INTRODUCTION 9

Time(rnin)

Fig 7 Chromatogram of separation of deuterated ethylenes Column, 200 m X 0.5 mm I.D Stationary

phase, 0.02M solution of dicarbonylrhodium 3-(trifluoroacetyl) camphorate in squalane Temperature,

22°C Peaks: 1 = C z 2 H 2 ; 2 = C 2 H , ; 3 = C 2 H , ' H Z ; 4 = C 2 H 2 2 H 2 ; 5 = C 2 H 2 H , ; 6 = C Z 2 H ,

From ref 27

through the reactor without any changes To illustrate this method Fig 8 [28] shows

a chromatogram of aromatic hydrocarbons in pyridine The use of a reactor containing copper chloride permits pyridine to be removed (subtracted), with the result that the chromatogram shows trace peaks that were previously masked by the broad zone of pyridine

The subtraction method is also used in functional analysis, which is of particular value

as far as the individual identification of chromatographic zones is concerned Fig 9 [29]

shows two chromatograms derived in different ways The top chromatogram (A) was

obtained during the analysis of an initial mixture on a column filed with 20% of

Carbowax 20M on Chromosorb P, whereas the bottom one (B) was obtained with a separation scheme including the same column connected in series with a reactor filled with 3% of boric acid on Chromosorb P Alcohols react with boric acid to yield non- volatile compounds Therefore, chromatogram B shows only non-alcohol components

of the sample mixture

Rational application of the chemical transformation method also improves the speed

of determination Fig 10 [30] shows chromatograms of alcohols derivatized to fluoro- propionates (A) and propionates (B); free alcohols cannot be eluted under these con- ditions These chromatograms demonstrate that, the selection of a volatile derivative (pentafluoropropionate) substantially reduces the separation time

The application of chemical transformations permits both the accuracy and the sensitivity of the experimental data to be controlled It is generally considered that the introduction of an additional stage or step into the chromatogram procedure reduces the accuracy of determination This, however, applies only in instances where the intro- duction of an additional stage does not affect the other steps of the analysis In analytical

reaction GC the introduction of an additional stage often produces a positive effect on the other stages, and therefore the accuracy and reliability of determination are in most instances improved For example, Fig 11 [31] shows two calibration graphs of peak height versus sample size for (1) morphine and (2) its trimethylsilyl derivative The

13 A

I I 1 I I I

€ig 8 Chromatograms of traces of aromatic hydrocarbons in pyridine: (A) direct determination;

(B) determination using a reactor containing copper chloride Peaks: 1 =benzene; 2 = toluene;

3 = ethylbenzene: 4 = isopropylbenzene; 5 = n-butylbenzene; 6 = naphthalene Reprinted with per- mission from ref 28

Fig 9 Functional group analysis of alcohols Chromatograms of separation of (A) the initial mixture

a n d (B) the mixture after separation and ‘subtraction’ of alcohols in a reactor containing boric acid connected in series with the column Peaks: 1 = tert.-butanol; 2 = n-butanol; a = cumene; 3 =

linalool; 4 = menthol; b = methyl phenylacetate; 5 = benzyl alcohol; c = 2-acetylpyrrole Reprinted with permission from ref 29,

determination of morphine in the form of its trimethylsilyl derivative permits the detec- tion limit to be lowered from 7 to 2 p g , and a graph with a steep slope to be obtained The improvement of the quantitative determination characteristics in this instance results

from the conversion of the initial polar compound into non-polar compounds, which minimizes the irreversible adsorption of the analyte during chromatographic determi- nation

Chemical transformations also permit one to enhance substantially the sensitivity

of determination, for example, as a result of the transformation of a non-detectable compound into a detectable one As an example, Fig 12 [ 3 2 ] illustrates a chromatogram

of benzyl esters of organic acids The conversion of organic acids into non-polar esters not only dramatically improves the quantitative characteristics of analysis (lack of

memory effects, symmetrical peaks, etc.) but also substantially lowers the detection limit For example formic acid is not detectable by a flame-ionization detector, whereas

its benzyl ester is detected with a high degree of sensitivity It should also be pointed

out that most detectors used in GC are chemical ones For example, flame-ionization

detectors are used to measure the ionic current resulting from ion formation during combustion of organic substances in the hydrogen flame; electron-capture detectors are intended to measure the yield of the negative ion formed in the reaction of the electron with the electronegative molecule of an organic substance; electrochemical

detectors make use of various electrochemical reactions to measure the content of

substances in the gas flow emerging from the chromatographic column; mass spec- trometers are designed to determine the products of a reaction between the electron

(or ion) and the molecule of a sample component [33-351 Thus, even conventional

chromatography based on the above-mentioned widely used detectors can justifiably

be regarded as a variant of analytical reaction chromatography, as the detector also serves

as a chemical reactor and the detection basically involves measurement of the reaction products

To calibrate detectors and chromatographic columns one must have pure substances

or standard mixtures of pure compounds Chemical methods allow standard compounds

Fig 12 Chromatogram of benzyl esters of C , -C, organic acids obtained using a flame-ionization

detector Column, 3 m X 4 mm 1.D.; sorbent, 10% SE-130 on Chromosorb W AW DMCS; temperature: initially 130°C (4.7 rnin), increased to 150°C at 5"C/min Peaks: 1 = diethyl ether; 2 = n-hexanoic acid; 3-12 = benzyl esters of the following acids; 3 = formic; 4 = acetic; 5 = propionic; 6 = iso- butyric; 7 = n-butyric: 8 = lactic; 9 = isovaleric; 10 = n-valeric; 1 1 = by-product; 12 = n-hexanoic

To obtain acrylonitrile it has been recommended to use a solution of cyanethyltrimethyl- ammonium iodide in dimethylformamide The introduction of this solution, heated to 225°C into the sample injector causes thermolysis of the quaternary ammonium salt, yielding 97% of acrylonitrile These methods are described in greater detail elsewhere

[37] together with an outline of the main trends in the application of chemical methods

to obtain standard compounds

The areas of application of chemical methods in GC are most diverse They are put to

to be extremely important in analytical chemistry

In developing new reaction methods and testing known ones in order to choose optimal conditions, the following analytical characteristics are important: (1) selectivity,

(2) sensitivity (limit of detection), ( 3 ) level of chemical noise, (4) influence of the local environment on chromatographic characteristics, ( 5 ) speed, ( 6 ) possibility of automation,

(7) accuracy and precision and (8) field of application

Chemical methods should be used in analytical reaction GC only after all of the above criteria have been considered

The main task, as the author sees it, is to draw the attention of specialists (analytical chemists, physical chemists, biochemists, medical scientists, etc.) who use GC to the wide possibilities of reaction GC, and to characterize the main methods involved in important trends and hence to promote the development of the practical use of chemical methods

The main results in the theory, methods and applications of analytical reaction GC are presented in this book As the development of science and industry continuously leads to new demands, and current methods often quickly become obsolete, the author has tried to draw most attention to the strategy of the method and t o general solutions

of model analytical problems Particular methods are usually considered not in isolation but in connection with others that are used for the solution of the same or similar types

of problems

The tasks formulated above have determined the structure of the book, the first six

chapters of which are devoted to accounts of the main chemical methods (preliminary processing of samples, kinetic methods, pyrolysis GC, determination of carbon skeleton, subtraction method, chemically selective stationary phases, elemental analysis) The last two chapters are devoted to the solution of two tasks that are most important in analytical chemistry nowadays: the determination of impurities (Chapter 8) and the identification of components of complex mixtures by functional group analysis (Chapter The author hopes that the book will be useful for those who use GC as an analytical method and will perhaps promote the further development of analytical reaction GC The author considers the expression of his gratitude to colleagues who took part

in the discussion of the book and its separate chapters, S.V Vitt and Yu.A Zolotov,

and also to V.M Fateeva and L.A Novitskaya, who helped considerably in the prep- aration of the manuscript, to be a pleasant duty

The author also expresses his cordial gratitude to Elsevier’s desk-editor, R.J Marx, for his active help in improving this book and in preparing it for publication

In conclusion, the author hopes that the publication of this considerably enlarged new version in English will further widen the field of application of chemical methods

in GC

9)

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Gostoptekhizdat , Moscow, 1962

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1971, University o f Houston, Houston,TX, 1971, p 84

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S.Z Roginsky, M.I Yanovsky and A.D Berman, Osnovy Premeneniya Khrornatograjli v Katalize (Fundamentals of Application of chromatography in Catalysis), Nauka, Moscow, 1972

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graphy, Wiiey, New York, 1973, p 294

N.C Saha and D.R Matthews,J Chromatogr., 81 (1973) 207

V.G Berezkin, Usp Khim., 37 (1968) 1348

H Psheidl, K Bothke and D Haberland, 2 Chem., 18 (1978) 393

B.W Bradford, D Harvey and B.E ChakIey,J fnst Petrol., 41 (1955) 80

B Smith and R Ohlson, Acta Chem Scand., 13 (1959) 1253

B Smith and R Ohlson, Acta Chern Scand., 16 (1962) 351

A.D Zorin, A.Ye Yezheleva and G.G Devyatykh, Trudy Khim Khim Tekhnol., 1 (1958) 605 O.K Guha and J Janak, Usp Khim., 42 (1973) 1708

V Schurig and E Gil-Av, Chem Commun., (1971) 650

V Schurig, Angew Chem., 88 (1976) 336

C.D Chriswell, L.D Kissinger and J.S Fritz, Anal Chem., 48 (19763 1123

B.M Ykeda, D.E Simmons and J.D Grossman, Anal Chem., 36 (1964) 2188

W.J.A VandenHeuvel W.L Cardiner and E.C Horning, J Chromatogr., 19 (1965) 263

K Hammarstrend and E Bonelli, Derivative Formation in Gas Chromatography, Varian

Aerograph, Walnut Creek, CA, 1968

E.K Doms, J Chromatogr., 105 (1975) 79

V.A Rotin, Radiatsionnoye Detektirovanie v Gazovoy Khromatografii (Radiation Detection

in ?as Chromatography), Atomizdat, Moscow 1974

J SevEik, Detectors in Gas Chromatography, Elsevier, Amsterdam, 1976

D.J David, Gas Chromatographic Detectors, Wiley-Interscience, New York, 1974

D.1 Freed and A.M Mujsce, Anal Chem., 49 (1977) 139

V.G Berezkin, L Sojak and J Undeova,J Chromatogr., 98 (1974) 157

R.P Taylor (Editor), Gas and Liquid Chromatography Abstracts, Applied Science Publishers,

Barking, 1979 - 80

Ozapter 1

Application of chemical methods to the pre-treatment

of samples before chromatographic separation

1.1 INTRODUCTION

Gas chromatography (GC) is one of the most widespread methods for analysing organic and inorganic compounds, owing to its high selectivity and efficiency, rapidity, simplicity and ready automation, availability of standard equipment and high sensitivity

of recording the separated compounds The method has certain limitations, however: (1) it can be applied only t o volatile compounds stable at the separation temperature,

(2) the identification of analysed compounds and their functional group analysis by

chromatographic methods only are complicated by the lack of reproducibility of the properties of chromatographic sorbents, (3) the separation of compounds with similar properties is a complicated and as yet unsolved problem and (4) the selective determi- nation of impurities when only the chromatographic methods are used and in the absence

of selective detectors is very complicated

An important step in solving these problems was made by utilising chemical methods aimed at achieving preliminary (prior to chromatographic separation) chemical conver- sions of (all or part of) the components of the sample Let us now consider the roIe and significance of these conversions in greater detail

GC is an efficient method for analysing volatile and stable (at the temperature of chromatographic separation) compounds As the technique and the apparatus are simple

and efficient, expansion of the range of applications of GC is highly desirable At present two main directions have been followed in order to expand the range of compounds that can be analysed by GC: (1) using vapours at temperatures and pressures above the critical values as the mobile phase in GC (supercritical, ‘fluid’ chromatography) [l-31, and GC with organic and inorganic vapour-phase eluents [4], and (2) converting non- volatile into volatile compounds and unstable into stable compounds Although the first solution is more universal its utilization requires more expensive and complicated equipment, and although the method of fluid chromatography has been known for more than 15 years, no company has so far produced any standard equipment for this method On the other hand, preliminary (pie-chromatographic) conversions of sample components into volatde, stable derivatives can be effected rapidly by using standard chemical reagents and simple glassware Many companies manufacture standard reagents

to perform these conversions (see, e.g., refs 5-7) Hence the second method of expand- ing the sphere of GC applications has gained wider acceptance, and the methods for the chemical formation of derivatives (CFD), primarily of organic compounds, are widely used in chromatographic practice

The simple and very fruitful idea of using chemical conversions of sample compounds

to obtain volatile and stable derivatives has been widely and successfully practiced from

the earliest period in the development of GC As early as 1953 Cropper and Heywood [8] were the first to use the CFD method to obtain methyl esters of CI2-C2* fatty acids for their subsequent GC separation The methyl esters obtained were separated at 230°C on a

90 cm x 25 mm I.D column packed with a silicone lubricant on Celite The methyl esters

of lauric, myristic, palmitic, stearic, arachidinic and behenic acids were separated One of the best examples of the development of this method is probably the analysis of amino acids [9, l o ] The elaboration of methods for their analysis is of great interest in indus-

try, medicine and scientific research The known methods for the analysis of amino acids and other compounds can be subdivided into two main groups: (1) methods of protecting the active functional groups (e.g., in the case of amino acids the amino and the carboxy groups are protected) and (2) methods for the chemical conversion (including the removal) of the active groups of the sample compounds (e.g., in the case of amino acids amino and carboxy groups are either converted into other functional groups or removed) When analysing CFD methods, the author considered it to be one of the main tasks to demonstrate their real advantages to chromatographers Unfortunately, in many reviews and books a comprehensive analysis of the advantages and characteristic features of this method is not given Many analysts, therefore, have a one-sided view of CFD methods, namely those which make it possible to analyse only polar and unstable organic com- pounds However, although the analysis of unstable and polar compounds is the first it

is not the only positive feature of the CFD method

The quantitative preparation of amino acid derivatives for subsequent GC analysis and separation of the derivatives obtained is very complicated The principal stages of the initial approaches to solving this problem are shown in Table 1.1 [ 1 11 , which demon- strates both the complexity of the problem and the variety of methods that were used

t o solve it

When analysing Table 1.1 we should draw the attention of the reader to the appli- cation of enzymatic methods in CFD [ 141 Enzymatic methods are now being used more and more widely in analytical chemistry in various determinations, from simple ions of the nitrate or phosphate type t o macromolecules; they are distinguished by high selec- tivity, moderate cost and simplicity of separation and insertion into the reaction mixture

[30-321 The use of immobilised enzymes in pre-chromatographic conversions is a

promising trend The main advantage of enzymatic methods lies in the high specificity

of the enzymatically catalysed reactions and the relatively high productivity These methods are used, e.g., in hydrolysis, in analysing various biologically active compounds

It is not difficult to see why so many different methods can be and have been applied

to the analysis of amino acids (see Table 1.1) Amino acids vary widely in reactivity, and when they are being derivatized undesirable side-reactions are often observed and high reac- tion yields are reproduced only with difficulty, which decreases the accuracy of analysis Thus, in the mid-1960s GC was regarded mainly as a method for the qualitative analysis

INTRODUCTION 17 TABLE 1.1

METHODS FOR DERIVATIZATION OF AMINO ACIDS FOR GAS CHROMATOGRAPHIC ANALYSIS PROPOSED IN THE INITIAL PERIOD OF DEVELOPMENT OF ANALYTICAL REACTION GAS CHROMATOGRAPHY [ 111

Type of reaction Derivatives References

Esterification with methanol- hydrochloric Methyl esters of amino acids 1 2 , 1 3

acid

Enzymatic decarboxylation Amines 14

Treatment with nitrous acid

Esterification with diazomethane

Methyl esters of 15,16

a-hydroxycarboxylic acids Treatment with a mixture of hydrochloric Methyl esters of 17

and nitric acids a-chlorocarboxylic acids

Esterification with diazomethane

Esterification with diazomethane

Treatment with acetic anhydride

Methyl esters of N-acetylamino acids

18,19

Esterification with diazomethane Methyl esters of 20-22

Treatment with trifluoroacetic anhydride N-tritluoroacetylamino acids

and N-trifluoroacetyldipeptides

Oxidation with ninhydrin Aldehydes 23-25

Reaction with formic acid in acetic

anhydride

Esterification with diazomethane

Methyl esters of N-formylamino acids

Reduction with lithium aluminium

Quantitative CFD methods have also been developed for analysing most amino acids

As an example, Fig 1.1 [33] shows chromatograms of 50 amino acids The GC analysis

of amino acids became possible mainly after chemical ‘protection’ of their functional groups, which allows the stability and volatility to be increased The ‘protection’ (con- version) of functional groups so as to make them more stable is widely used in CFD techniques Naturally, the functional groups protected are those whose presence in the molecules of sample compounds is responsible for their increased reactivity or adsorption capacity, thermal instability and the formation of associates Similar problems in pre- parative organic chemistry have for many years been solved by temporarily blocking or protecting those functional groups whose participation in the reactions with the other functional groups of a molecule is possible but undesirable Methods of protecting func-

tional groups have been surveyed by McOmie [34]

Using the CFD methods one can analyse polymeric and other involatile compounds by converting them, prior to chromatographic separation, into characteristically volatile products These volatile products can be obtained either by pyrolysis or by using more selective chemical conversions It is customary to regard the application of pyrolysis reaction to identify and analyse quantitatively involatile samples as an independent part

19 INTRODUCTION

of analytical reaction GC, known as pyrolysis-GC (see Chapter 3) This is absolutely

justified if one takes into account the specificity of the pyrolysis reaction, the special equipment needed to conduct it and the characteristic areas of its application (see, e.g., refs 35 and 36) One of the main disadvantages of pyrolysis-gas chromatography (Py-

GC) is the complexity of the pyrolytic reactions, which proceed in various directions; this hinders the establishment of the structure of the sample compound in the presence

of the products of its pyrolysis The application of more selective chemical reactions to obtain volatile products characterizing, e.g., the polymer sample, is therefore a more informative and simpler method For the directionally controlled destruction of involatile organic compounds reagents are used that ensure the selective separation of the whole compound into its component parts This special area of the analysis of involatile com- pounds should, in our opinion, be called reagent-destructive chromatography as distinct from temperature-destructive or pyrolysis chromatography

Let us now consider the application of reagent-destructive or reagent-functional chromatography (the reactions proceed with certain functional groups of polymers) for the analysis of polyesters Methods for the analysis of linear polyesters and branched polyester and alkyd resins usually involve complete decomposition of the polymer into its initial components (e.g., by saponification, aminolysis or alcoholysis) and their GC

analysis As an example let us consider the work of Allen et al [ 3 7 ] , devoted to elabo-

rating a quantitative method for determining the monomers (repetitive structural units)

in polyesters, the first stage of which was alkaline hydrolysis by a solution of potassium hydroxide in 2-ethoxyethanol After hydrolysis the mixture was acidified by adding

~~

Fig 1.1 Chromatograms of 50 amino acids in the form of their isobutylestersof N-heptafluorobutyric derivatives (331 Conditions: glass column (6 m X 2 mm I.D.) packed with 3% OV-101 on GasChrom Q; temperature programming, 5min isothermal at 80"C, heated to 250°C at the rate of 6"C/min, then isothermal 250°C Each peak corresponds to approximately 2pmol of amino acid derivative Samples (1 p l of amino acid solution in ethyl acetate) were inserted in the column after the prelimi- nary insertion in the chromatographic system of 0.5 w l of acetic anhydride in A and C A, Analysis

of protein amino acids; B, some biologically interesting amino acids; C, some amino acids in the presence of protein amino acids A: ALA = alanine; GLY = glycine; VAL = valaine; THR = threonine; SER = serine; LEU = leucine; ILE = isoleucine; IS (internal standard) = NLE = norleucine; PRO = proline; CYSH = cysteine; HYP = 4-hydroxyproline; MET = methionine; ASP = aspargine; PHE =

phenylalanine; GLU = glutamine; LYS = lysine; TYR = tyrosine; ARG = arginine; HIS = histidine;

TRF' = tryptophan; C Y S = cystine B: 2-AIBA = 2-aminoisobutyric acid; 2-ABA = 2-aminobutyric acid; p-ALA = p-alanine; 3-ABA = DL-3-aminobutyric acid; IS = NLE = norleucine; HSER =

homoserine; AHBA = DL-4-amino-3-hydroxybutyric acid; PIP = DL-pipecolic acid; 2,4-ABA =

2,4-diaminobutyric acid; MSO = methionine sulphoxide; ORN = ornithine; HYL = 5-hydroxylysine;

TYR = tyrosine; 2-AAA = DL-2-aminoadipic acid; DOPA = 3,4-dihydroxyphenylalanine; DAP = DL-2,6-diaminopimelic acid; MIT = 34odotyrosine; HCYS = DL-homocystine; CTT = cystathionine; DIT = 3,s-diiodotyrosine C: ALA = alanine; GLY = glycine; 2-ABA = 2aminobutyric acid; 8-ALA = p-alanine; VAL = valine; THR = threonine; SER = serine; LEU = leucine; ILE = isoleucine;

IS = NLE = norleucine; PRO = proline; CYSH = cysteine.; PIP = DL-pipecolic acid; HYP =

4-hydroxyproline; MET = methionine; ASP = asparagine;~ PHE = phenylalanine; ORN = ornithine; GLU = glutamine; HYL = 5-hydroxylysine; LYS = lysine; TYR = tyrosine; 2-AAA = DL-&amino- adipic acid; DOPA = 3,4dihydroxyphenylalanine; ARG = arginine; HIS = histidine; TRP = trypto- phan; MIT = 3-iodotyrosine; CTT = cystathionine; CYS = cystine; DIT = 3,5-diiodotyrosine; HCYS = DL-homocystine

Fig 1.3 Chromatograms of trimethylsilyl ethers of high-boiling acids Reprinted with permission from ref 37

concentrated hydrochloric acid, and pyridine was added to dissolve the acids A 50-pl volume of the reaction mixture was then added to 5OOpl of N,O-bis(trimethylsily1)tri- fluoroacetamide (BSTFA) to obtain ethers and esters of the alcohols and acids formed The chromatographic analysis was performed on a 0.1 -pl sample

Fig 1.2 shows a chromatogram of the trimethylsilyl ethers of ethylene glycol ( l ) , nonyl

alcohol used as the internal standard (2), 1,4-butanediol (3) and cis (4) and trans (5)

isomers of 1,4-cyclohexanedimethano1 These products were obtained by analysing an experimental sample of polyether The GC analysis was conducted on a glass column

(1.8 m x 6.25 mm 1.D.) packed with 10% Versilube F-50 on Chromosorb W HP with the following temperature programme: 120°C for 8 min, then increased to 210°C at 4"C/min

Fig 1.3 shows a chromatogram of the separation of trimethylsilyl derivatives of

isophthalic acid (1) and terephthalic acid (2) obtained by alkaline hydrolysis of an

experimental polyether sample The separation was conducted at 183°C on a stainless- steel column (1.8 m x 3 mm I.D.) packed with 10% Versilube F-50 on Chromosorb

W H The relative standard deviations in quantitative analysis were 0.8% for 1,4-butane- diol, 2% for 1,4-cyclohexanedimethanol and 0.7-2.4% for isophthalic acid The method ensures a rapid and sufficiently accurate determination of acids and polyglycols that form the polyethers This method is described in greater detad elsewhere 136,381

CFD methods have acquired particular importance in the last 10-15 years in connec- tion with the development of methods for the GC analysis of labile biologically active compounds and pharmaceutical preparations Let us consider some examples of the application of CFD in this field

The analysis of barbiturates is an important practical task A technique was developed

[39] for the determination of a number of widely used barbiturates (see Table 1.2), based on extraction of the drug from the blood by adsorption on carbon, extractive ethylation of the m i n e group (the method of extractive alkylation was described by

INTRODUCTION 21 TABLE 1.2

STRUCTURAL FORMULAE OF BARBITURATES [39]

Ethyl Phenyl Hydrogen Allyl Phenyl Hydrogen Ethyl l-C y clopentenyl Hydrogen

Ehrsson [40]) and subsequent GC analysis on a glass surface-layer capillary column The temperature was initially 170°C (4min), then increased to 260°C at 4"C/min The separation was conducted on a 43 rn x 0.5 mm I.D column treated with SE-30

liquid stationary phase A flame-ionization detector (FID) was used

A chromatogram of the derivatives of barbiturates is shown in Fig 1.4 [39] The notation of the peaks corresponds to the numbers of the barbiturates in Table 1.2 The asterisk denotes the peak appearing in the analysis of blood plasma The total concen- tration of barbiturates was 20

A method was developed [41] for the analysis of natural penicillins after methylation

Previously only paper chromatographic methods for the analysis of penicillins were

known [42, 431 Penicillins were extracted with diethyl ether from aqueous solutions

at pH2 and esterified with a small excess of diazomethane at 0-2°C The solution of methyl esters was evaporated to dryness and the residue was dissolved in acetone Samples of 1-3 p1 were analysed on a glass column (150 cm x 4 mm I.D.) packed with 3%

OV-17 on Gas-Chrom (2 (100-120 mesh) with a carrier gas (nitrogen) flow-rate of 60ml/min After 10 min isothermal at 180°C the temperature was increased at 4"C/min for 10min and then kept constant until the end of the analysis All of the penicillins analysed under these conditions were separated within 45 min: methylpenicillin,

g/rnl

Fig 1.4 Chromatogram o f ethyl derivatives of barbiturates From ref 39

n-propylpenicillin , n-amylpenicillin, n-heptylpenicillin , benzyipenicillin, n-nonylpenicillin, phenoxymethylpenicillin (in order of elution from the column)

The application of CFD methods also makes it possible t o carry out the analysis of

such inorganic substances as anions and metals As an example let us consider the analysis

of trace amounts of nitrates in water Tan [44] proposed a simple and sensitive method for determining nitrates in aqueous solutions using GC-mass spectrometry (MS) with

an ionic detector-multiplier The method is based on the nitration of 1,3,5-trimethoxy- benzene (TMB) in sulphuric acid In this medium the nitration follows the hydrolysis

of the ether groups of TMB, and nitrobenzene is formed as the final product [44] The

reaction mixture is analysed by GC and detected with a mass spectrometer Hexamethyl-

benzene (HMB) is used as an internal standard To avoid interference from nitrates and chlorides, sulphamic acid and mercury(I1) sulphate are used

The procedure is relatively simple A 10-ml volume of an aqueous solution of nitrate

is transferred into a 50-ml erlenmeyer flask fitted with a stopper, and then 0.01 ml of 5%

sulphamic acid solution and 0.01 ml of mercury(I1) sulphate in 10% sulphuric acid are added A 20-ml volume of concentrated sulphuric acid is slowly added from a burette

INTRODUCTION 23

The mixture is then poured into another flask which is cooled in cold water to room temperature, and 2ml of TMB reagent (0.1 g of TMB in lOOml of benzene containing TMB at a concentration of 3 * g/l) is added with a pipette The mixture is shaken for 5min, and after separation of the phases the upper (benzene) layer is taken for GC-MS analysis Chromatographic analysis is performed on a glass column (0.9m x 0.2cm I.D.) packed with 2% SP-2300 on Chromosorb W HF' (100-120 mesh) with the temperature programmed from 110 t o 180°C at 32"Clmin Methane (12ml/min)

or helium (40 ml/min) is used as the carrier gas The sample volume is 1 PI Nitrobenzene was identified as the reaction product In the opinion of Tan [44] ,this can be explained

by the nitration of benzene or the formation of 1 -nitro-2,4,6-trimethoxybenzene

followed by its hydrolysis Fig 1.5 shows a chromatogram of a mixture of nitrobenzene

and TMB (internal standard) The analysis is sufficiently rapid, and the separation is

satisfactory This technique permits the determination of nitrates in water at concen- trations of ca 1 g/l It seems possible that the same technique may be applicable

to the analysis of nitrates with flame-ionization and electron-capture detectors

These and other methods of forming derivatives are summarized in Blau and King's

Handbook of Derivatives for Chromatography [45] As the GC analysis of anions never-

theless remains an insufficiently well known method, Table 1.3 lists the chemical reac- tions used to form the derivatives that are subsequently determined by GC The reactions listed indicate that known reaction chromatographic methods can be used to analyse most of the commonly encountered anions

In recent years increasing interest has been shown in the development of GC methods for the analysis of metals in the form of volatile complexes (see, e.g., refs 45 and 46)

An example is the separation of a mixture of cations of zinc, cadmium, cobalt, nickel and lead in the form of their complexes with bisdiethyldithiophosphinate [47] The complexes are formed readily in aqueous solution by reaction with sodium diethyldithio- phosphinate The separation was conducted on a column (7.8 m x 2.9 mm I.D.) packed with 3% OV-17 on Chromosorb W AW.DMCS at 220°C Under these conditions the bisdiethylphosphinate complexes of metals were characterized by the following retention

times: (1) zinc 9.8min; (2) cobalt 11.0min; (3) cadmium 16.0min; (4) nickel 23.2 min;

and (5) lead 60.8min; the chromatogram is shown in Fig 1.6 The content of each chelate in the sample (1 111) varied in the range 2 10-'-2 -

A good example of the application of this method is its use for the micro-determi- nation of beryllium, iron, cobalt, nickel and chromium in moon dust and moon rock Trifluoroacetylacetone and heptafluorodimethyloctanediol were used to obtain the chelates of metals in the moon samples [48] The examples cited are indicative of the wide possibilities of this method in inorganic microanalysis

The second positive feature of the CFD method is the improved separation as a result

of the individual differences in the derivatives formed being greater than those in the initial compounds Thus, e.g., racemates of amino acids can be separated into enantiomers

by GC if they have been converted using optically active reagents into diastereomers

that can be separated on optically inactive stationary phases [49-521 It should be noted that this method of analysing the enantiomers of amino acids is used much less often than the use of optically active stationary phases [9] The reason is the necessity to use very pure compounds as reagents, as optically active impurities cause the formation of many side products

g

PRELIMINARY CHEMICAL REACTIONS IN THE GAS CHROMATOGRAPHIC' DETERMINATION OF ANIONS

From W.C Butts, in ref 45, p 411

where R = CH,CH,, CH,CH,CH, or (CH,),CH, 6Br- + 2Mn0; + 8H+- 3Br, + 2Mn0, + 4H,O

3 0 r 2 + 3 - 3 01;

R,NX& R X + R,N

51- + 10; + 6H+ -31, + 3H,O

31, + 3CH,COCH,- 3ICH,COCH, + 3H' + 31- (CH,),SiCl + H,O-(CH,),SiOH + H' + C1-

F - + (CH,),SiOH + H+- (CH,),SiF + H,O

F - + (CH,CH2)3SiCl-(CH,CH,),SiF + CI-

y n - + H' + n BSTFA TMCS

where BSTFA = bis(trimethylsily1)trifluoroacetylamide

(TMS),,Y + nMSTFA TMCS = trimethylchlorosilane

TMS = trimethylsilyl MSTFA = mono(trimethylsily1) trifluoroacetylarnide

CH ) X

RX

ICH ,COCH ,

(CH,),SiI:

Y = SiO:-, Si,O6, -,

Si,O: -, Si,O,";,

Si,O,";

M,Y + nHC1- H,Y + pMZ + + nC1- H,Y + n(CH3,SiCl- [(CH,),Si],Y + nH' + nC1- where M = Mg, Fe, Zn, Ca, Na

(b) (CH,),AsO,H + 4H,-(CH,),AsH + 2H,O (CH,),AsH + CH, = CHCN- (CH,j,AsCH,CH,CN

S z - + 2H+- H,S (a) CN- + Br,- BrCN + Br-

(b) SCN- + 4Br + 4H2O-BrCN + SO:- + 7Br- + 8 H + CN- CN- + CH,C,H,SO,NClNa + 2H+-

ClCN ClCN + CH,C,H,SOzNHz + Na'

Fig 1.5 Chromatogram of the upper benzene laye1 after nitration 1 = Nitrobenzene; 2 = hexa-

methylbenzene (HMB) (internal standard) From ref 44

Fig 1.6 Chromatogram of fivecomponent mixture of metal complexes From ref 47

As a simpler example let us cite the separation of methyprylon ( 1 ) and pyrityldione

12) W1:

Fig 1.7 shows two chromatograms of ( A ) compounds 1 and 2 and (B) their trirnethylsilyl

(TMS) derivatives obtained by treatment with N,O-bis(trimethylsily1)acetamide (BSA)

Separation was performed on a glass capillary column (3.5m x 0.25 mm I.D.) with SE-30 as the stationary phase at 200°C The initial compounds are not separated on a

non-polar stationary phase under these conditions, but their TMS derivatives are sepa- rated satisfactorily, with the time necessary for separation decreasing approximately

As an example of a clear improvement in separation we can cite the data on the

retention of steroids on a non-polar silicone stationary phase, SE-30, as presented by

Heftmann [ 541 Two monohydroxy-steroids, 5 (Y -cholest an-38 -01 and 5-cholesten-3P -01, have the same relative retention (2.85) on a column containing a non-polar stationary phase (internal standard cholestane), but the relative retentions of their trimethylsilyl derivatives are 2.60 and 2.55 and those of their chlorodichloroacetates are 3.79 and 3.62,

respectively The relative retention times of 3a-hydroxy-5a-androstan-17-one and

3/3-hydroxy-5a-androstan-l7-one are similar at 0.96 and 1 .OO respectively, whereas those of their TMS ethers are 0.46 and 0.61 respectively

two-fold

Fig 1.7 Chromatogram of (A) initial amines 1 and 2 (see text) and (B) thejr TMS derivatives From ref 53

Hence the application of the CFD method makes it possible in a number of instances

to improve separations as a result of the formation of derivatives with greater differences

in chromatographic properties Unfortunately, this area of application of the CFD method has not yet been sufficiently developed There is no doubt that its further development will help in the solution of many practical problems

The third positive feature of CFD methods is a substantial improvement in the quantitative characteristics of analytical determinations It is known (see, e.g., ref 55) that many mistakes in GC analysis are caused by irreversible or semi-reversible adsorption

of the sample compounds on the stationary phase-solid support interface and on the surface of the chromatographic apparatus The use of CFD methods makes it possible to convert the adsorptively active (e.g., carboxylic or hydroxylic) groups of the sample compounds that are mainly responsible for their irreversible or semi-reversible adsorption into inactive or only slightly active groups This results in a decrease in

irreversible adsorption and improves the symmetry of chromatographic zones, the independence of the quantitative characteristics of the zones (area, height) from the size of the sample and the previous history of the column, and improves the regularity

of the calibration graph The improved symmetry of chromatographic zones, resulting from the formation of weakly polar and thermally stable derivatives of the sample compounds, usually results simultaneously in an improvement in the quantitative

h

m

F i g 1.8 Calibration graphs for ( 1 ) morphine and (2) its trimethylsilyl derivative h is the relative

height of the peak of the sample compound (squalane = internal standard) and m is the amount

of substance in the sample (g) From ref 56

characteristics of analysis: systematic errors of analysis are reduced, the calibration graph becomes a straight line and its slope (sensitivity) increases As already mentioned,

all this is a result of smaller (or completely absent) irreversible or quasi-reversible adsorption of the derivatives on the surface of the solid support and the apparatus

As an example, Fig 1.8 [56, 571 shows the calibration graphs for morphine alkaloid and its TMS derivative The calibration graph for morphine is a curve, protrudes towards the abscissa and is characterized by a small slope that increases slightly with increasing size of the sample Moreover, as the graph intersects the abscissa when the size of the sample reaches ca 7 0 p g , this value represents the limiting amount of morphine that

can be determined in the sample The calibration graph for the TMS derivative is linear, with a greater slope and the smallest amount that can be determined is ca 2/45 i.e., the sensitivity is approximately three times greater than that for the direct determi- nation of morphine The advantages of derivatization are greater when the molecules

of the initial compounds contain active groups such as hydroxyl, carboxyl, amine or imine

It should be noted that the undesirable adsorptive effects and other complications in

GC separations are also observed in a number of instances when derivatives are analysed

In the analysis of some metals in the form of volatile compounds, for example, these phenomena can be the major complicating factor in the analysis [45]

The accuracy of analysis increases substantially if the compound used as the internal standard is chemicaliy similar t o the compound being determined In a limiting case it

is expedient t o use as the internal standard the same compound as that being determined, but with a different isotopic composition The combined use of GC and MS makes it possible to utilize this optimal variant In recent years, especially in the combined GC MS analysis of biologically active compounds, wide use has been made of com- pounds labelled with stable isotopes as internal standards [58], one of the optimal methods being the use of compounds having three or four I3C atoms A positive feature

of this method is the absence of isotopic effects in the course of the GC separation or

in detection during chemical ionization used in MS [59] Isotope effects also do not manifest themselves in the processes that result in the loss of the sample substance by

INTRODUCTION 29

adsorption, extraction, etc [59] In addition, no marked differences in the rates of chemical reactions conducted to form the derivatives have been observed The insertion

of three or four 13C atoms is desirable in order to avoid superposition of the mass spectra

of those compounds which also contain 13C in accordance with its natural distribution The use of the sample compound labelled with 13C as the internal standard thus simplifies substantially the quantitative estimation of the results of selected ion detection, and makes it possible to determine easily the quantitative content of the parent and the

labelled compound For instance, with chemical ionization (methane) in the mass spec- trum of the N,N-dimethyl derivative of phenylethylbarbituric acid (luminal) the main peak corresponds for the conventional (determined) compound to the ion with mass

261, and for the labelled derivative of [2,4,5-13C] phenylbarbituric acid (internal standard) to the ion with mass 264 The relative standard deviation in determining luminal in blood was 3.3% [59] In practice, other compounds used for the same purposes are those labelled with deuterium, and methylating agents labelled with deuterium are often used It should be noted, however, that in this instance the chro- matographic behaviour of labelled compounds can differ considerably from that of non- labelled compounds

The fourth positive feature of the application of CFD methods is an increase in the sensitivity of detection of the derivatives compared with that for the parent compounds

An example of a large increase in the sensitivity is the determination of formic acid in the form of its benzyl ester [60] with an FID Formic acid cannot be detected with an

FID, and the use of its derivatives enables one to determine it with very high sensitivity

Naturally, in the analysis of C1-C5 fatty acids the other advantages of the CFD

method also manifest themselves This method is of special importance in analysing the impurities and when using selective detectors In the latter instance it becomes possible

to achieve the high-sensitivity detection of impurities in the zone of a main component that does not form a derivative Similar problems have been discussed elsewhere [61] Often the CFD method is used to form derivatives that can be selectively detected with a high-sensitivity electron-capture detector (ECD) McCallum and Armstrong [62] gave examples of the dependence of the sensitivity of thymol(5-methyl-2-isopropylphenol)

derivatives on the content and nature of the heteroatom in the derivative formed The relative values of the sensitivity (with that of the heptafluorobutyrate taken as unity) for thymol derivatives are pentafluoropropionate 1.3, monochloroacetate 0.3, mono- fluoroacetate 7 ' pentafluorobenzyl ether 5.9, pentafluorobenzoate 6.9 and 2,4- dinitrophenyl ether 0.3

The successful application of the CFD method in combination with subsequent gas chromatographic separation and the use of an ECD has resulted in the extensive develop- ment of this technique However, other CFD methods aimed at obtaining derivatives that can be selectively detected by other selective detectors (e.g., sodium thermionic, flame photometric) have not been developed adequately, despite their obvious promise

It seems that the high selectivity of the method should be used for the elaboration of selective methods of functional group analysis in order to identify compounds at the picogram level This is especially pertinent to the analysis of microsamples on capillary columns

The fifth positive feature of the application of the CFD method is the possibility of obtaining compounds that are of interest for the qualitative and quantitative GC analysis