Chemical Derivatization in Gas Chromatography (Journal of Chromatography Library)

JOURNAL OF CHROMATOGRAPHY LIBRARY- volume 19

chemical derivatization in gas chromatography

JOURNAL OF CHROMATOGRAPHY LIBRARY

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Chromatography of Antibiotics by G.H. Wagman and M.J. Weinstein

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Chemical Derivatization in Liquid Chromatography by J.F. Lawrence and R.W. Frei

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Volume 19

Chemical Derivatization in Gas Chromatography by J. Drord

JOURNAL OF CHROMATOGRAPHY LIBRARY- volume 19

chemical derivatization in gas chromatography J. Drozd Institute of Analytical Chemistry, Czechoslovak Academy of Sciences, Brno, Czechoslovakia with a contribution by J. Novik

ELSEVIER SCIENTIFIC PUBLISHING COMPANY 1981 Amsterdam - Oxford New York

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ELSEVIER SCIENCE PUBLISHERS B.V. Sara Burgerhartstraat 25 P.O. Box 21 1,1000 AE Amsterdam, The Netherlands Distributots for the United States and Canada: ELSEVIER SCIENCE PUBLISHING COMPANY INC. 52, Vanderbilt Avenue NewYork,NY 10017

First edition 1981 Second impression 1985

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Contents

Abbreviations used Preface .

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

VII

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IX

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XI

Introduction

1. Reasons for using chemical derivatives in gas chromatography . . . . . . . . . . . . . . . . 1.1. Volatility of sample compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Spurious adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Separation of closely related compounds . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4. Selective and sensitive detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5. Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

..

1

2 2 4 4 6 6

2 . Sample preparation and derivatization techniques . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Sampling techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Sample treatment prior to analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Preparation of derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Gas chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

9 9 10 18 21 22

3 . Identification and quantitation (by J . Novik) . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

25 26 40 50

4. Most frequent derivatives and methods for their preparation . . . . . . . . . . . . . . . . . . . 4.1. Esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Ethers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Acyl derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Silyl derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Oximes and hydrazones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. Cyclicderivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,. . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

53 53 64 66 69 75 76 78

5 . Derivatization of individual species of compounds . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Alcohols and phenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Aldehydes and ketones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Amines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Sulphur compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Carboxylic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6. Aminoacids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7. Thyroid hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.8. Steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.9. Sugars and related compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.10. Bases of nucleic acids, nucleosides and nucleotides . . . . . . . . . . . . . . . . . . . . 5.1 1. Insecticides and other pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 2 . Pharmaceuticals and drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V

83 84 92 97 109 111 126 148 151 165 175 177 182

VI

CONTENTS

5.13. Anions of mineral acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.14. Cations of metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.15. Miscellaneous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

188

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213

Appendix 1 . Purification of chemicals and solvents

Appendix 2 . A list of some suppliers of reagents and accessoires for derivatization Subject index

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191 198

199

221 223

Abbreviations used

AFID BSA BSTFA DEGA DEGS DMS DNP DNPH ECD EGA EGS FID

alkali flame-ionization detector N,O-bis(trimethylsily1)acetamide

N,O-bis(trimethylsily1)trifluoroacetamide

poly(diethy1ene glycol adipate) poly(diethy1ene glycol succinate) dimethylsilyl (derivative) dinitrophenyl (derivative) dinitrophenyl hydrazone electron-capture detector poly(ethy1ene glycol adipate) poly(ethy1ene glycol succinate) flame-ionization detector gas chromatography Gc combined gas chromatography-mass spectrometry GC-MS gas-liquid chromatography GLC heptafluorobut yryl (derivative) HFB hexamethyldisilazane HMDS HMDSO hexarnethyldisiloxane high-performance liquid chromatography HPLC inner diameter I.D. a methylsiloxane polymer JXR methoxime MO N-meth yl-N-trimethylsilylacetamide MSA N-meth yl-N-trimethylsilyltrifluoroacetamide MSTFA poly(neopenty1 glycol adipate) NGA poly(neopenty1 glycol succinate) NGS outer diameter O.D. pentachlorophenol PCP pentafluoropropionyl (derivative) PFP poly tetrafluoroethylene PTFE programmed-temperature gas chromatography PTGC thermal conductivity detector TCD trifluoroacetyl (derivative) TFA thin-layet chromatography TLC trimethylchlorosilane TMCS trimethylsilyl (derivative) TMS TMSDEA trimethylsilyldiethylamine TMSDMA trimethylsilyldimethylamine TMSIM trirnethylsilylimidazole V/V volume ratio w/w weight ratio

VII

This Page Intentionally Left Blank

Preface Since the first work on the gas chromatographic analysis of compounds in the form of their chemical derivatives there have been published innumerable papers dealing with the derivatization of diverse substrates for the purposes of gas chromatographic analysis. The writing of this book was suggested by the fact that there is n o comprehensive work that covers all of the problems of the preparation and use of chemical derivatives in gas chromatography. Several publications have dealt with the problems of certain species of derivatives (e.g., Pierce, Silylation of Organic Compounds, Pierce Chemical Co., Rockford, IL, 1968) and/or the derivatization of certain types of compounds [e.g., HuSek and Macek, “Gas Chromatography of Amino Acids”, J. Chromatogr., 113 (1975) 1391, but only a few authors have attempted to survey the entire range and variety of the problem. Our review on chemical derivatization in gas chromatography [J. Chromatogr,, 113 (1975) 3031 included over 600 references, but far from covered all of the work on this topic. As with this book, the review did not include papers on reaction gas chromatography, pyrolytic reactions and post-column derivatization (identification) techniques. This book is intended to complement the above review, with the inclusion of papers published up to the end of 1978 plus some of the most important papers from 1979, together with our own experience and the results of discussions with investigators in the field. The author realizes that he has covered scarcely a third of all the papers on these problems by including about 800 references, but he believes that this book surveys the most important procedures as they have been applied to various species of compounds. Especially in recent years there has been a sharp increase in the number of publications dealing with the chemical derivatization and gas chromatographic analysis of new substances that are particularly interesting from the biochemical and biomedical points of view. The contents of most of these papers, which usually are based on rudimentary procedures, differ from each other mostly in detail, and the aims of the work described lie not in the derivatization steps but elsewhere, so that their inclusion here would necessarily have led to an unrewarding enlargement of the book. The book is designed to introduce even a beginner to the whole extent of the problems, to acquaint him with all types of derivatives and methods employed, and to enable him to utilize them in practice without it being necessary t o consult the original sources; to this end full descriptions of the main procedures are provided. However, it is intended that workers proficient in the field will also find the book to be a source of useful information, and perhaps, inspiration. This intention is supported by the inclusion of some of the latest publications and a chapter on identification and quantitation, which may give hints for further investigations. A comprehensive book by &ng and Blau (Handbook of Derivatives, Heyden and Son, London, 1978), of which a major part is on derivatives for gas chromatography, has been published but its concept is quite different to that of this book. As the individual procedures are described according to the kinds of derivatives in their book, a search for all of the procedures available for a given group of compounds may be laborious. In addition, IX

X

PREFACE

much space was devoted to topics that can be found in books on general organic chemistry (e.g., reaction mechanisms). However, King and Blau’s book can indisputably be recommended as an excellent source of further detailed information. It is a pleasant duty t o aknowledge the assistance of all those who took part in the genesis of this book. The first thanks are due t o Dr. Josef Novik (Institute of Analytical Chemistry, Brno), who wrote Chapter 3, followed with interest the development of the whole manuscript, and helped with advice, comments and moral support. Further thanks go to Dr. Jaroslav Janak (Institute of Analytical Chemistry, Brno) for his critical comments on the form and contents of the book and t o Dr. Jaroslav Jonas (PurkynZ University, Brno) for carefully reading the manuscript and correcting the text from the point of view of the nomenclature of organic chemistry. Equal thanks are due to Dr. Radka RunCtukovi (Institute of Analytical Chemistry, Brno) for her enormous efforts in translating the manuscript into English, to Mrs. Marcela Pierovski (Institute of Biophysics, Brno) for drawing the illustrations, and to the staff of the Documentation Department of this Institute for the literature searches and other help with collecting the documentation material. Last, but not least, I am indebted t o my wife and children for their patience and understanding during the whole period of my work on the book.

Brizo, February 1981

JOSEF DROZD

Introduction Gas chromatography is a method suitable for the separation and analysis of substances that have a sufficiently high volatility in the chromatographic system used. Criteria for making this definition a true statement are rather loose, as there are several ways of controlling the volatility of a solute. The problems associated with these aspects have been studied since the very beginning of gas chromatography, and in many respects it were these problems that gave rise to new concepts in gas chromatographic techniques and instrumentation and the development of new chromatographic materials. High-temperature gas chromatography, temperature programming, the use of highly selective sorbents, operation in systems with very small amounts of sorbents, high-pressure and supercritical fluid chromatography (up to the transition towards liquid chromatography) and chemical conversions of sample compounds into their derivatives can be cited as examples. The aspect mentioned last differs from the others considerably: whereas in other instances the volatility of the solute and therefore also its suitability for gas chromatography are controlled by changing the characteristics and operating conditions of the chromatographic system, chemical derivatization changes the character of the sample compound itself. When considering the problems associated with chemical derivatization in gas chromatography, it is expedient t o distinguish between the two factors that influence the volatility of substances. Low volatility can be caused either by a bulky molecule of the compound or by associations among the molecules through their polar groups. In the former instance the intermolecular interactions result from dispersion forces and the volatility of these compounds can hardly be increased by derivatization. In the latter instance, however, even compounds with relatively small molecules can possess very low volatilities, provided that functional groups are present in their molecules that allow polar interactions, particularly hydrogen bonds or ionic bonds. A number of compounds of this type show measurable vapour pressures only at temperatures at which they decompose. Some of them are highly reactive and often change even on contact with the activated surface of the chromatographic support or the metal of the apparatus. Almost always these compounds provide asymmetric elution curves or “ghost” minor peaks. In these instances, a considerable increase in the volatility and suppression of the above undesirable influences can usually be achieved by derivatization, which eliminates or restricts considerably the range of polar intermolecular interactions and reduces the reactivity of the compound. Moreover, if the sample compound is converted into a suitable derivative, its molecules can be given properties that make selective separation or selective detection possible. The combination of gas chromatography and chemical derivatization has found a particularly wide range of applications in investigations of biochemical and biomedical processes; a great number of substances, enormously interesting from this viewpoint, could not have been analysed by gas chromatography without derivatization, often not even qualitatively. Thanks to the extensive applicability of gas chromatography and to various possibilities of coupling with other analytical methods, primarily with mass spectrometry, chemical derivatization is still a very important discipline of chromatographic methods XI

XI1

INTRODUCTION

with topical practical applications in spite of the rapid development and vast applications of other methods, e.g ,modern liquid chromatography. This book reviews the methods for and the most important papers dealing with the preparation and applications of chemical derivatives to analytical gas chromatography. The five chapters are constructed so that even a newcomer to the field, acquainted with the principles of gas chromatography, might become familiar with the problems, first in general by learning the reasons that have given rise to the applications of this method, and by featuring future possible progress. The introductory chapter is devoted to this aim in particular. The second chapter discusses in general some practical aspects of the preparation and analysis of various derivatives and ancillary techniques associated with them. Having anticipated the reader’s basic knowledge of chromatography, we did not pay much attention to the problems of gas chromatographic analysis itself and have simply highlighted some particular aspects that follow from the use of derivatives. Problems associated with identification and quantitative analysis are discussed in Chapter 3. Its scope, as the preceding case, allows only a brief discussion of ancillary techniques, such as spectral methods and data processing by computer, to be included. In Chapter 4, the problems of derivatization are classified according to the functional groups of the sample compounds; the most frequently used derivatization procedures and problems associated with them are described here. However, it is useful to be aware of the fact that this somewhat theoretical approach is suited for general considerations and for acquiring a knowledge of the problems as a whole. On the other hand, with various types of compounds other specific problems occur, resulting from the different chemical characteristics of the moieties or the presence of other functional groups, their interactions, etc. This is the reason why in Chapter 5 , which is the main chapter of the book, attention is paid to the derivatkation of various types of compounds on the assumption that there is more interest in the problems connected with a particular group or a few groups of compounds than in the problems with an individual derivative in general and in the differences in its applications to various substrates. For example, for the worker involved in steroid analysis a knowledge of procedures for the preparation of silyl derivatives of these compounds is essential; he can, however, easily do without a knowledge of the details of the preparation of silylated amino acids. The scheme of Chapter 5 and the classification of an enormous amount of material has been based on this consideration. It probably does not represent the best possible solution for all readers and there are certainly other opinions on the emphasis in various sections and on the classifications of certain compounds. However, the classification adopted here allows a rapid orientation within the extensive range of material and makes in possible to find all of the derivatives used for one type or compound under a single heading. Each section in Chapter 5 starts with a short introduction which discusses the reasons for and main problems in the derivatization of the particular groups of compounds, followed by a survey of the derivatives used, usually starting with those which are used most frequently. In some sections (Insecticides and Other Pesticides, Pharmaceuticals and Drugs, Cations of Metals) this arrangement has been abandoned as it has appeared more logical to classify derivatization procedures according to the various substrates. Methods for the purification of chemicals and solvents used most frequently for the preparation of derivatives are described in Appendix 1 . Throughout the text abbreviations are used, and these are listed on p.VII.It isnecessary

INTRODUCTION

XI11

to mention at this point that the author is not much in favour of expressing himself in this way and therefore tried to reduce the number of abbreviations as much as possible to make the text easier to understand. Mainly abbreviations commonly used in the relevant literature for long and frequently used technical terms, such as trimethylsilyl and gas chromatography (TMS and GC, respectively) are used. In principle, new abbreviations have neither been created nor used; for the names of stationary phases, abbreviations have been adopted in the form introduced by various authors and mznufacturers.

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Chapter 1

Reasons for using chemical derivatives in gas chromatography CONTENTS 1.1. Volatility o f sample compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Spurious adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Separation of closely related compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4. Selective and sensitive detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5. Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 2 4 4 6 6

Efforts to extend the method of gas chromatography (GC) to the analysis of the largest possible number of compounds were the main reason for the introduction and development of the use of derivatives. Conversion of sample compounds into volatile derivatives made it possible to separate and analyse by GC groups of compounds for which GC analysis would otherwise be impossible, e.g., amino acids, sugars and related compounds. The presence of different polar groups in the molecules of such parent compounds is the most significant source of the difficulties associated with their GC analysis. Carboxyl, hydroxyl, carbonyl, thiol and amino groups (particularly if several groups, whether of one or more types, are present in the molecule), owing to their polarity and tendency to form hydrogen bonds, are responsible both for the low volatility of the compounds and for other phenomena that make direct GC either difficult or impossible, e.g., strong adsorption on the support of the stationary phase and asymmetry of peaks eluted from it, and thermal and chemical instability of the compounds, which cause losses of the samplc compounds in the chromatographic system, i.e., their non-quantitative elution or the elution of decomposition products. Having initially led to the development and expansion of derivatization procedures, the above reasons remain important even nowadays, in spite of the fact that their significance for some compounds has decreased owing to the development of modern procedures for the preparation of columns, deactivation and the preparation of modern chromatographic packing. The separation of closely related compounds is another very important example of the application of derivatives in GC. The resolution of two solutes that can be chromatographed alone without difficulty can often be improved considerably by their conversion into suitable derivatives, whereas further modifications of the column and the chromatographic conditions do not result in any substantial improvement. During the development of derivdtization techniques, it'appeared that on being converted into a suitable derivative, a sample compound could also be given some properties that would make it suitable, e.g., for selective detection. This aspect is still being developed and its significance is increasing with the progress with selective detectors. A topic of particular importance is the combination of GC with mass spectrometry (GC-MS), for which special derivatives

2

REASONS FOR USING CHEMICAL DERIVATIVES IN GC

have recently been developed that give characteristic fragments that make identification and quantitative evaluation easier.

1.l. VOLATILITY OF SAMPLE COMPOUNDS Compounds that possess a high relative molecular mass are usually not accessible to direct GC analysis owing to their low volatility. Polar groups in the molecules of such compounds decrease the volatility even further, so that these compounds then have impracticably long retention times or they are not eluted from the column. However, even compounds with a low relative molecular mass that bear polar groups which make the formation of ions potentially possible can show similar behaviour. By eliminating the possibility of strong intermolecular interactions of polar groups or even by compensating their electrical charges, the volatility of such compounds may often be increased significantly. By blocking the function with a non-polar substituent, a derivative may be obtained which, in contrast to the non-volatile parent compound, can be chromatographed in the gaseous phase. Chapter 5 describes several examples of this type. It is noteworthy that in practice even the reverse of the above may occur. Often it is necessary t o analyse compounds that are too volatile. Significant losses during the preliminary treatment of the sample (e.g., extraction, removal of the solvent), due to this volatility, may introduce errors into the quantitative evaluation. Analysis of volatile carboxylic acids in biological samples is an example. Conversion of these compounds into less volatile derivatives is therefore advantageous from the viewpoint of both GC proper and preliminary isolation of the compounds and sample treatment. Many compounds cannot be analysed by GC because of their thermal instability. Such compounds decompose in the injection port of the chromatograph and give several peaks on the chromatogram due to decomposition products. These difficulties also can often be overcome by the use of suitable thermally stable derivatives.

1.2. SPURIOUS ADSORPT'ION

Almost always, compounds of high polarity and low volatility tend to undergo adsorption on the chromatographic support or decomposition on contact with it. These phenomena usually result in peak tailing and the quantitative evaluation of the chromatograms is difficult or even impossible. A wellknown example is the GC analysis of cholesterol, which can be analysed as such or as the TMS derivative (Fig. 1.1). If the support is not modified, free cholesterol provides a wide, tailing peak which can be evaluated quantitatively only with difficulty, whereas the TMS ether provides a sharp, symmetric peak the retention time of which is, however, substantially shorter [ 11. Tailing peaks may also originate if the amount of the solute in the chromatographic system is too great. If the linear range of the adsorption isotherm of the solute is exceeded, the chromatographic sorbent is overloaded, which results in asymmetry of the elution peak. By conversion into a derivative with other sorption properties, conditions may be attained that are suitable for operation in the linear range.

c

SPURIOUS ADSORPTION

3

G HOL EST EROL

0

15

30

TIME ( M I N I Fig. 1.1. Comparison of the chromatograms of free and trimethylsilylated cholesterol on nondeactivated support coated with FdO.(Reproduced from ref. 1 by courtesy of W.J.A. VandenHeuvel and the publisher.)

Similarly, the calibration graph may be non-linear, particularly if peak heights are used as a quantitative parameter, and during manipulations with low concentrations of the solute when its adsorption on the surface of the support, column walls, etc., occurs to a significant extent. Fig. 1.2 illustrates the improvement that was obtained by the conversion of the sample compound. In the direct determination of morphine by GC, the dependence of the ratio of the peak height of morphine to that of squalene on the amount of compound injected is non-linear and therefore quantitative evaluation is difficult. An analogous calibration graph for the TMS derivative, in contrast, is linear. Hence, if a suitable derivative is used a drawback that could interfere with the GC analysis itself can be overcome [ 2 ] .

50 -

40 30

-

Fig. 1.2. Effect of sdylation on the linearity of the plot of peak height ratio versus amount of solute. (Reproduced from ref. 2 by courtesy of K. Hammarstrand and the publisher.)

4

REASONS FOR USING CHEMICAL DERIVATIVES IN GC

In conclusion, it is necessary to add that a number of the problems mentioned, caused by adsorption in the chromatographic system, may be solved by new technological procedures for the preparation of columns and whole systems. Modern sorbents and procedures for deactivation have partly solved the initial difficulties in some instances, but the field is still wide enough for useful applications of derivatives.

1.3. SEPARATION OF CLOSELY RELATED COMPOUNDS Efforts to improve the separation of closely related compounds are a frequent reason for using derivatives, and their application often makes it possible tq separate compounds that otherwise cannot be separated. Chapter 5 gives various examples of this type, of which the separation of enantiomers of alcohols (p.90), carboxylic acids (p.129, amino acids (p.146) etc., are the most illustrative. The separation of sterols that differ in the position of the hydroxyl group may serve as another example. Isomers with a hydroxyl group in the a-position are not separated from 0-isomers on non-polar columns. However, if the hydroxyl group is converted into a suitable derivative, the two isomers can be separated well even on non-polar columns. The anomers of sugars can also be separated after their conversion into derivatives. Another example is illustrated in Fig. 1.3. Testosterone and epitestosterone are eluted as one peak using SE-30 as the stationary phase. By conversion of the hydroxyl group into a more bulky substituent, the slight difference in the structure is enhanced and the two epimers can be resolved. The same approach can be used for the separation of 16and 15-keto isomers of androstan3&ol. The initial ketones are not separated on SE-30, but after conversion into the corresponding N,N-dimethylhydrazones their separation is possible as these derivatives are eluted much more slowly than the initial compounds [3]. Fig. 1.4 shows GC separation of the three estrogens estrone, estradiol and estriol, as such and in the form of their TMS derivatives, on a non-polar column. Whereas the free compounds are only incompletely separated, with unsatisfactory peak shapes, the TMS derivatives are well separated and their peaks are symmetric [ 11. An improvement in the separation of some other compounds can be achieved in a similar manner.

1.4. SELECTIVE AND SENSITIVE DETECTION Derivatives of sample compounds are commonly used also for their detection by other chromatographic techniques and by other analytical methods. The basic difference in their use in GC is that in most instances the sample compounds are derivatized prior to the analytical process itself and their properties are thus changed to improve their chromatographic and detection characteristics. The significance of this aspect of the use of derivative; keeps increasing with the development of research into selective detectors, which provide a response to certain groups of compounds. These detectors sometimes allow the selective analysis of various compounds in complex mixtures without a prior separation. If mass spectrometry is considered as a means of detection, then there is a large group of the derivatives that are used for this particular purpose.

SELECTIVE AND SENSITIVE DETECTION

5

2

1

.Eli \

0

rY 0

u

w

LL

0

10

TIME (MIN

0

15

3[

TIME(MIN)

Fig. 1.3. Gas chromatographic analysis of a mixture of epitestosterone (1) and testosterone (2) before (upper) and after (below) preparation of their TMS ethers. Conditions: glass column, 6 ft. X 4 mm I.D.; 2% SE-30 on GasChrom P (100-120 mesh, AW, silanized); carrier gas inlet pressure, 1.12 atm; temperature, 235°C. (Reproduced from Med. Res. Eng., 7 (1968) 10, by courtesy of W.J.A. VandenHeuvel.) Fig. 1.4. Effect of silylation on the resolution and peak shapes of estrogens. Peaks: 1 = estrone; 2 = estradiol; 3 = estriol. Stationary phase, JXR; temperature, 210°C. (Reproduced from ref. 1 by courtesy of W.J.A. VandenHeuvel and the publisher.)

The electron-capture detector (ECD) has considerable selectivity and is the most frequently used. Its response depends considerably on the type of functional groups or even on the kind of elements that are present in the molecule of the compound to be detected [4]. It is fairly sensitive to halogens, particularly chlorine, bromine and iodine, but fluorinated derivatives, e.g., trifluoroacetates, heptafluorobutyrates, pentafluorobenzoates and other perfluoroacyl derivatives, and pentafluorophenyl derivatives, have mostly been used for practical reasons. The nitro group and some other arrangement of functional groups in the molecule (see Section 3.1.3, p.36) also provide a high ECD response, which is why, e.g., 2,4-dinitrophenyl derivatives are often used also for this purpose. The alkali flame-ionization detector (AFID) is considered to be specific for phosphorus and nitrogen but it provides a response also to other elements, such as bromine, chlorine, sulphur [5], lead, silicon, tin and boron 161. For detection with the aid of this detector, however, sample compounds are mostly converted into derivatives containing phosphorus

REASONS FOR USING CHEMICAL DERIVATIVES IN GC

6

(e.g., see pp.91 and 118) or sulphur (p.95), and these are used for a limited number of compounds only. Boron may be introduced into the molecules of compounds in the form of cyclic boronates which are selective only for a certain bifunctional arrangement in the molecule and may also be detected with other detectors. The use of mass spectrometry combined with GC does not necessitate derivatives other than those used in GC itself. In spite of this, particularly recently, special derivatives for GC-MS have been developed, some of which are mentioned in this book. Derivatives are considered that provide characteristic mass spectra and facilitate identification and quantitative evaluation. Various trialkylsilyl derivatives [7] are an example. They d o not offer any particular improvement over conventional TMS derivatives from the viewpoint of the chromatographic analysis, but their highly characteristic fragments permit the sample compounds under analysis to be identified unambiguously in GC-MS.

1.5. IDENTIFICATION The significance of the confirmation of the identity of compounds with the aid of their derivatives has decreased with the development of modern methods. However, this method has always represented a rapid and technically simple alternative if costly instrumentation is not available. With some groups of compounds (e.g., pesticides) it is still used fairly frequently and plays a significant part in their determination. Essentially, there are three fundamental ways of using derivatives for identification purposes: (i) if the substance can be chromatographed it is analysed both as such and as a derivative, and the compound is identified from the differences in retention behaviour; (ii) if the compound cannot be analysed as such it is usually converted into two derivatives and following procedure is analogous; in both instances the detection can be combined with the use of a selective detector and therefore also the qualitative information can be increased; (iii) the compound is chemically cleaved prior t o the analysis and its characteristic products are analysed after derivatization. Procedures, usually multi-step, have been developed for several particular compounds that lead to highly specific derivatives that can be utilized for identification. The study of structural differences of alcohols and phenols [8], using as a characteristic the ratio of the adjusted retention time of the alcohol or the phenol t o that of its TMS derivative, can serve as an example of the first aspect. Alcohols, thiols and primary and secondary amines in food flavours were identified successfully after the preparation and GC analysis of different derivatives [9]. The third method is fairly frequent in the analysis of residues of pesticides (see p.177). Post-column identification reactions constitute a special kind of ancillary GC technique [ l o ] that will briefly be discussed in Section 3.1.2, p.34.

REFERENCES 1 W.J.A. VandenHeuvel, in M.B. Lipsett (Editor), Gas Chrornatograplz~~ of Steroids iri Biological Fluids, Plenum Press, New York, 1965, p. 277.

REFERENCES

7

2 K. Hammarstrand and E.J. Bonelli, Derivative Formation in Gas Chromatography, Varian Aerograph, Walnut Creek, CA, 1968. 3 W.J.A. VandenHeuvel and E.C. Horning,Med. Res. Eng., 7 (1968)10. 4 J.E. Lovelock, Nature (London), 189 (1961)729. 5 M. Dressler and J. Jan&, J. Chrornatogr. Sci., 7 (1969)451. 6 R. Greenhalgh and P.J. Wood, J. Chrornatogr., 82 (1973)410. 7 C.F. Poole and A. Zlatkis,J. Chrornatogr. Sci., 17 (1979)115. 8 L. Tullberg, I.-B. Peetre and B.E.F. Smith, J. Chromatogr., 120 (1976) 103. 9 L. Gasco and R. Barrera, Anal. Chim. Acta, 61 (1972)253. 10 C. Merritt, Jr., in L.S. Ettre and W.H. McFadden (Editors), Ancillary Techniques of Gas Chromatography, Wiley-Interscience, New York, 1969,p.325.

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Chapter 2

Sample preparation and deriva tization techniques CONTENTS 2.1. Sampling techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Sample treatment prior to analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Drying of the sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2.Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. Concentration, evaporation of the extraction agent . . . . . . . . . . . . . . . . . . . . 2.2.4. Thin-layer chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Preparation of derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Gas chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

9 10 16 16 18 18 18 21 22

The preparation of a derivative of a sample compound prior to GC is a significant potential source of both qualitative and, in particular, quantitative errors. Almost all reactions that are used for derivatization are organic syntheses adapted to the micro-scale. This approach makes full use of an advantageous property of GC, namely the need to take only very small amounts of the sample for the analysis, but on the other hand, it makes heavy demands on the quality of the materials used and the precision of the operating procedures. As GC has especially been used in analyses of complex mixtures with large contents of various components, such as biological samples, the operations necessary for the preliminary separation of the compounds of interest from the sample, e.g., extraction or TLC, are often involved in the entire procedure, and make it even more complicated. With some reactions, the necessity for an anhydrous medium requires the application of drying (lyophilization) in the treatment of the sample. During the derivatization reaction, proper attention should be paid to its yield, the stability of the derivatives produced and their volatility, which can be the reason for losses and errors in the analysis. The GC of derivatives can be performed on common instruments, major modifications of which are usually not required. Only a few derivatives are sensitive to the activity of the chromatographic support or the material of the column, and some unstable derivatives are affected by contact with metals. This chapter describes the rules which should be observed when preparing derivatives, the experimental facilities most frequently used for this purpose and peculiarities of the instrumentation and performance of GC analysis proper when derivatives are used.

2.1. SAMPLING TECHNIQUES The method of sampling may be a serious source of errors in any analytical method and is particularly critical in GC. With the manipulation of small amounts of samples, it can easily arise that a non-homogeneous aliquot is taken for the analysis, which does not represent the composition of the sample. 9

10

SAMPLE PREPARATION AND DERIVATIZATION TECHNIQUES

Problems associated with representative sampling must be viewed as statistical [ 11. Non-homogeneity of liquid samples containing suspended matter can be largely compensated for by taking a series of samples at various levels, whilst providing sufficient agitation to keep the solid matter suspended as uniformly as possible. Solids in particulate form (discrete lots) are subjected to random sampling procedures. Special studies of the sampling of many types of various materials have been carried out by testing organizations and government agencies. For instance, recommended procedures for metals, non-metallic materials of construction, paper, paints, fuels, petroleum products and soils can be found in the Book of ASTM Standards [ 2 ] and other publications of the American Society for Testing and Materials. The Journal of the Association of Official Analytical Chemists regularly publishes tentative procedures for sampling and analysis of soils, fertilizers, foods, water, drugs, etc., and at 5-year intervals releases new editions of the Official Methods of Analysis [3]. Similar methods for vegetable fats, oils, soaps and related materials are published and revised periodically by the American Oil Chemists’ Society [4]. Many other references to the original literature can be found in Standard Methods o f Chemical Analysis [5]. In addition to the necessity for ensuring acceptable homogeneity and taking representative samples, a problem arises associated with the transfer of the sample into the chromatograph. It is necessary to ensure that the sample does not undergo decomposition or some other reaction prior to the preparation of derivatives or in the course of injection of the sample into the instrument after the derivatization. For this reason the chemical properties of the initial material, the kinetics of the reaction used and the possibility of the occurrence of side-reactions should be known and, moreover, all possible impurities and admixtures in chemicals used must be eliminated and, if necessary, stabilizers should be used. The simplest example of a stabilizer is the use of an excess of reagent, which protects labile derivatives against spurious effects. Some derivatives decompose owing to the action of light, heat, moisture etc., and therefore cannot be stored for a long period prior to analysis. It is advisable to prepare unstable derivatives immediately before the analysis, particularly derivatives that are sensitive to moisture (e.g., TMS derivatives).

2.2. SAMPLE TREATMENT PRIOR TO ANALYSIS Although nowadays chromatographic columns with very high efficiencies are widely available and various selective detectors can be used, it is not possible to eliminate the procedures by means of which interfering compounds and other components, which would be present in excess after the reaction, are removed from the sample after or prior to the derivatization procedure. Other operations, such as removal of the excess of the reagent or solvent with inconvenient chromatographic properties and its replacement with an alternative, are also sometimes required. In general, the more complicated is the sample mixture and the more specific the analysis must be, the more steps the sample treatment involves. For instance, if a single compound or a small group of compounds is to be analysed in a complicated system such as biological material, several preliminary separation steps may be necessary, such as multiple extraction, thin-layer chromatography and isolation of the sample or interfering compounds by means of a specific reaction.

11

SAMPLE TREATMENT

Each of these operations can affect adversely the final result of the analysis. Some procedures are given for various types of compounds in Chapter 5. Several examples of a complete sample treatment prior to analysis will be presented here for illustration. Maruyama and Takemori 161 isolated norepinephrine and dopamine from brain tissue prior to GC analysis by the procedure outlined in Fig. 2.1. A I-ml volume of 0.05 N oxalic acid (saturated with NaCI) and 3.5 ml of 25% n-butanol in isopropanol are added to a small homogenization test-tube containing one (380-480 mg) or two entire mouse brains and the mixture is homogenized. The homogenate is centrifuged at 2500 g for 5 min in a clinical centrifuge adapted for this purpose. Then 3 ml of the pure phase of

WAIN TISSUE 005 N OXALK K I D BUTANOL-ISOPROPANOL (1 3)

Centrifuge

AQUECUS

I nEXANE+WFFER pH

1

65

DISCARD

Extract Centrifuge

ACUEWS WANOL-ISOPROPANOL(1 3 1

SOLVENT

PH 2 0

DISCARD

i AQUECXlS

SOLVENT

Evapcrate

DISCARD RESIDUE Silylote

Fig. 2.1. Flowdiagram of procedure for extraction of norepinephrine and dopamine from brain tissue of mice.

12

SAMPLE PREPARATION AND DERIVATIZATION TECHNIQUES

the solvent are transferred into a small test-tube and 0.5 ml of 0.5 M NaH2P04-Na2HP04 buffer (pH 6 . 5 ) and 3 ml of n-hexane are added. The test-tube is closed and agitated for 5 min. After centrifugation for 5 min at 2500 g , 0.5 ml of the aqueous phase is separated, its pH is adjusted to 2.0 with 6 N HC1, then it is saturated with NaCl and re-extracted with 0.5 ml of 25% n-butanol in isopropanol. The upper layer of the solvent is transferred quantitatively by means of a Pasteur pipette into a small test-tube and evaporated to dryness in a mild stream of air. The residue is used for silylation and GC analysis. The total corrected recovery was stated to be 92.2 f 9.3% for dopamine and 78.8 f 6.7% for norepinephrine (eight determinations). Even more complicated is the procedure for the determination of isomers of androstanediols and pregnanediols with particular emphasis on the specific determination of 5a-androstane-3P,17/3-diols[7]. As shown in Fig. 2.2, the procedure starts with incubation of the sample with Helix pomatia a-glucuronidase at 37OC and free steroids are extracted with diethyl ether. Phenolic steroids are removed from the sample with 1 N sodium hydroxide solution and keto steroids, which could interfere, are removed by means of Girard-T reagent (see Scheme 5.5, p.92). Hydroxy steroids themselves are then purified by adsorption chromatography on alumina. The sample is then divided into two halves, one of which is subjected to TLC. Substances from the two zones obtained are subjected to GC analysis after being silylated. Steroids in the other half are epoxidized with 3-chloroperbenzoic acid and the product is chromatographed on paper. The zone corresponding to Sa-androstane-3P,17/3-doil is eluted and purified on a thin layer and the final extract is silylated and analysed by GC. The average recovery for the entire procedure measured by means of substrates labelled-with 14C were 65 k 5% and 32 f 7% for 5a-androstane-3a,l7/3-diol and 5a-androstane-30,I7S-diol respectively. The significantly lower recovery of the latter compound is caused by the purification by paper chromatography. Kaiko and Inturrisi [8] followed the procedure outlined in Fig. 2.3 to determine cyclazocine and its metabolites in human urine. To urine (1-4 ml) in a siliconized 15-ml centrifuge tube with a PTFE-lined screw-cap are added 0.2 ml of an aqueous solution of internal standard (levallorphan, 20 pg/ml), 0.5 ml of carbonate-hydrogen carbonate buffer (1 M, pH 9.8) and 1 drop of 1-octanol. After thorough mixing the sample is extracted with 5.0 ml of n-butyl chloride-isobutanol (7 : 3) by shaking for 5 min followed by centrifugation at 350 g for 5 min. The upper, organic phase is removed and the extraction is then repeated. The compounds are extracted into acid by adding 5.0 ml of 0.2 N HCl to the combined organic phases and shaking for 7 min, followed by centrifugation at 350 g for 3 min. The organic phase is removed and the acid phase is washed by addition of 5.0 ml of n-hexane and shaking for 5 min followed by centrifugation. The n-hexane phase is removed and the washed aqueous phase is made alkaline by adding 0.4 ml of concentrated ammonia solution (pH adjusted to ca. 10). The compounds are extracted into 7.0 ml of the n-butyl chloride-isobutanol solvent mixture by shaking for 5 min followed by centrifugation. The organic phase is transferred into a 12-ml siliconized centrifuge tube; after evaporation to dryness with the use of a multiple flash evaporator with the bath at 65"C, the sample extract is concentrated in the lower tip of the tube by rinsing the lower sides of the tube with 50 pl of chloroform and allowing this to evaporate. The sample extract is dissolved in 10-20 pl of TFA-imidazole-chloroform (1 : 4)

SAMPLE TREATMENT

13

Gircrd

-

T reogent

I

Msaptm Chromatogmphy

1

Epoxidatm

1 TLC on silica gel F254

Pnper chromatography

TLC on silica gel F2%

Fig. 2.2. Flowdiagram of the method for the simultaneous determinationof urinary androstanediols and pregnanediols.

and between 1 and 4 pl are injected immediately into the gas chromatograph. The mean recovery, determined using tritiated compound, was 88.5 f 3.1%. The procedure illustrated in Fig. 2.4 was used to determine anticonvulsant drugs (phenobarbital, primidone and diphenylhydantoin) in serum [9]. To a serum sample (2.0 ml) in a 15-ml stoppered glass centrifuge tube is added 0.2 ml of a methanolic solution of internal standard [5-(p-methylphenyl)-5-phenylhydantoin,200 pg/ml] and 0.2 ml of 2 N HCl to make the solution acidic. After thorough mixing, the sample is extracted with two 5.0-ml portions of chloroform by shaking for 5 min followed by centrifugation at 160 g for 5 min. The lower organic layers are removed with a Pasteur pipette, combined in a 15-ml test-tube and concentrated to approximately 5 ml by a stream of dry

14

SAMPLE PREPARATION AND DERNATIZATION TECHNIQUES URNE

+

Irk. StaKXrd

Extract With n-butyl chloride-isobutanol (7 3 )

1

I

AQUEOUS PHASE

CRGANIC PHASE I

Discard Extract w t h

0.2 N HCl

dI

t

LXJECUS PHASE

ORGANIC PHASE Discard

Wash with n-hexane

I n-HEYANE

Extract with n-butyl chloride- lSObUtMol(7 3 )

I

ACUECUS PHASE Discard

i RESIDUE

Fig. 2.3. Flowdiagram of the procedure for extraction of cyclazocine and norcyclazocine from urine.

nitrogen at 70°C. The compounds are then extracted into an aqueous medium by vortexing for 1 min with 2.0 ml of 0.5 N NaOH solution followed by centrifugation. The upper, aqueous layer is removed and the extraction is repeated. The combined aqueous phase is acidified with 1.5 ml of 2 N HCl and extracted with 4.0 ml of diethyl ether by vortexing for 1 min followed by centrifugation. The upper, ethereal layer is transferred into a 15-ml centrifuge tube and the extraction is repeated. The combined extract is evaporated to dryness by a stream of nitrogen and concentrated in the lower tip of the tube by rinsing the sides of the tube with 0.2 ml of methanol followed by evaporation to dryness. The residue is dissolved in 20 p1 of 50% trimethylanilinium hydroxide and 1 pl of the solution is injected into the gas chromatograph.

SAMPLE TREATMENT

15

i lnternol standard

2 N HCI

Extract with chla’oform

t

1

AQUEOUS P H E

ORGANIC PHASE

Discard Extract with

0 5 N NaOH

I ORGANIC PHASE +

2 N HCI

Discard

*

Extract With diethyl ether

MUECUS PHASE Discard

ORGANIC PHASE

Evapcrate

Dissoke n reagent

Fig. 2.4. Flowdiagram of the procedure for extraction of phenobarbital, primidone and diphenylhydantoin from serum.

A number of other interesting examples of multi-step separations of complex mixtures can be found in the book by Karger et al. [lo]. From the complexity of the above procedures, it is obvious that the result of the analysis depends on a large number of factors. The most general approach to the solution of the problem consists in elaboration of a standard procedure, its strict observation and its testing by means of an independent method, eg., with the use of the sample compounds labelled with I4C. The determination of the total recovery by means of an internal standard, i.e., a chenlically very closely related compound, a defined amount of which is added to the sample before the start of the procedure, is not exact as it is difficult to ensure that all of the properties of the standard that contribute to the final result are completely identical with the properties of the compounds being determined. With simpler procedures, when only a few different steps are applied, it is usually sufficient if a

16

SAMPLE PREPARATION AND DERIVATIZATION TECHNIQUES

few principles are observed in order t o obtain satisfactory results. Some of these principles are considered below. 2.2.1. Drying of the sample

A number of reactions for the preparation of derivatives require anhydrous conditions and therefore drying is a frequent operation. When performing the drying procedures it must be known what type of material is being treated. If obviously non-volatile compounds are concerned, there is no risk of losses. However, not even in this instance can the possibility of stripping of compounds out of the sample, azeotropic distillation or similar phenomena be neglected. Therefore, evaporation and drying must be performed carefully and slowly. In the presence of relatively volatile compounds (eg., volatile fatty acids), considerable losses can occur during the drying of the sample. In this event it is necessary t o decrease the volatility of the compounds in question either b y using different conditions (in the present example by increasing the pH) or by conversion into less volatile derivatives (e.g., higher esters and/or salts), if the drying step cannot be omitted. The application of reduced pressure during sample drying can suppress the losses of volatile compounds if a lower temperature is used and the compounds d o not form an azeotropic mixture under these condifions. The most reliable procedure is vacuum drying a t very low temperatures (lyophilization or freeze-drying). Several devices are commercially available for this technique, which is particularly advantageous if thermally labile material is to be treated. Good results are obtained at the expense of a longer time necessary for drying, however. For example, the removal of water from protein hydrolysates was carried out [ 111 by two methods, vacuum evaporation and lyophilization. (i) In vacuum evaporation, an aqueous aliquot of the protein hydrolysate, containing 5-25 mg of total amino acids, was transferred into a 125-ml flat-bottomed boiling flask with a PTFE-coated magnetic stirring bar. The sample flask was placed on a rotary vacuum evaporator and immersed in a water-bath at 60-70°C. Then the water was removed by slowly lowering the pressure (to prevent bumping) until the minimum pressure was attained. (ii) In lyophilization of the sample, an aliquot was placed in a 125-ml flat-bottomed flask as above and shellfrozen prior t o being placed on an efficient lyophilizer to remove the water. Four procedures can then be applied t o the final drying of the sample: (i) desiccant drying over Pz05for 24 h at room temperature and under vacuum; (ii) desiccant drying over Pz05 for 6 h at 60 f 5°C and under vacuum; (iii) azeotropic distillation, in which I0 ml of CH2C12 are added and removed by vacuum evaporation at 6 5 f 5"C, then the procedure is repeated; and (iv) chemical drying, in which 2.0 ml of 2,2-dimethoxypropane are added t o 10 ml of methanol-HC1 reagent, the solvents are removed b y vacuum evaporation at 60"C, then the procedure is repeated. In any event, regardless of the drying procedure used, the possibilities of losses should always be tested. 2.2.2. Extraction

Solvent extraction is one of the most frequently used procedures for preliminary isolation of compounds of interest from the sample. An extraction agent is selected that does

SAMPLE TREATMENT

17

not mix with the sample phase and for which the distribution coefficient of the compound under analysis (the ratio of its concentration in the sample phase to its concentration in the extraction agent) is as low as possible. For example, pentachlorophenol can be extracted from water samples with benzene [ 121. To a preserved sample (volume -

N o of double bonds

LOg,oRETENTION VOLUME R E L A T I V E TO M E T H Y L M Y R I S T A T E I N REOPLEX 4 0 0 AT 197 ‘C

Fig. 3 . 3 . Relationship between log (relative retention volumes) on Apiezon M and Reoplex 400 for various fatty acid esters at 197°C. (Reproduced from J. Chromatogr., 2 (1959) 552, by courtesy of A.T. James.)

IDENTIFICATION

31

I

10 - 0 D -1

P

I 05

00

I

-

4020A

-4101’

-05

1

DMS

” I

-05

I

00

!

+

05

Fig. 3.4. Relationship between log (relative retention volumes) of a range of alkanes on octadecene-1 (OD-1) and dimethylsulpholane (DMS). (Reproduced from ref. 24 by courtesy of the Swiss Chemists Association)

atoms in given functionalities the intercept and in the fact that isomers with the same code number (a function of the number of primary, secondary, tertiary and quaternary carbon atoms) fall on a single line in the graph. As a rough guide, useful information may be gained from plots of logarithm of retention data versus boiling points. Such a plot is shown in Fig. 3.5 [25]. This method of correlating retention data is especially useful in the identification of hydrocarbons. The points for all hydrocarbons, i.e., branched, unbranched, saturated and unsaturated, lie close to a single line provided that a non-polar stationary phase is employed. Even aromatics fit the line, and only alicyclics display larger positive deviations. This plot provides a quick determination of the boiling point of the unknown, thus reducing the number of possible identities that might be assigned to a solute compound. A completely different picture is obtained if the same mixture of hydrocarbons is chromatographed on a polar stationary phase. Such a situation is shown in Fig. 3.6 [ 2 6 ] .Lines A, B, C and D represent saturated compounds, monoolefines and alicyclics, dienes and alicyclics with a double bond, and triply bonded unsaturated compounds and aromatics, respectively. This plot may give very valuable data complementary to those obtained with a non-polar stationary phase. In addition to the above correlations, plots of logarithm of retention data against the inverse of the absolute column temperature can be used t o obtain retention data at a desired temperature from those measured at different temperatures.

32

IDENTIFICATION AND QUANTITATlON

0

LO 80 BOILING POINT Tbl'c)

120

Fig. 3.5. Relationship between log (retention time) and boiling point for a range of hydrocarbons on qualane at 43°C. A, aliphatics (and benzene); B, alicyclics, 1-4 = C 5 - c ~n-alkanes; 5 = 2-methylbutane; 6 = 2-methylpentane; 7 = 2,3-dimethylbutane; 8-12 = c4-cS 1-olefms; 13, 15, 17 = trans@ut-2-ene, pent-2-ene and hept-2-ene); 14, 16, 18 = cis-(but-2-ene, pent-2-ene and hept-2-ene); 19 = 2-methylbut-l-ene; 20 = 2-methylpent-1-ene; 21 = 4-methylpent-l-ene; 22 = 2-methylbut-2-ene; 23 = cyclopentane; 24 = cyclohexane; 25 = methylcyclopentane; 26 = methylcyclohexane; 27 = cyclopentene; 28 = cyclohexene; 29 = 4-methylcyclohexane; 30 = 1,3-butadiene; 31, 32 = trans- and cis1,3-pentadiene; 33 = diallyl; 34, 35 = trans- and cis-2-methyl-l,3-pentadiene; 36 = cyclopentadiene; 37 = propyne; 38 = pent-1-yne; 39 = pent-2-yne; 40 = benzene. (Reproduced from ref. 25 by courtesy of J.H. Purnell and Wiley.)

The situation becomes involved if retention data measured with temperature programming are to be processed. There is a rule [27] according to which correlations analogous to the linear correlations of logarithm of isothermal retention data apply roughly to the differences between the retention and initial temperatures (not the logarithms) as measured by temperature-programmed GC. In view of correlations carried out for identification purposes, retention data measured by temperature-programmed GC are less reliable than isothermal data, which is unfortunate considering the great importance of temperature-programmed GC. A most important contribution to the above means of identification is the Kovats retention index system [28]. The Kovats retention index of a compound is 100 times the number of carbon atoms in a hypothetical n-alkane that would display in the given system the same retention as the compound in question. Hence, the retention index system essentially is also based on the regularities between the retention data and number of carbon atoms in homologous compounds. The concept of the Kovats retention index system is illustrated by the model in Fig. 3.7, which shows a plot of log K i values for homologous compounds of the type CH3(CH2),X and for n-alkanes against carbon number. It is apparent that the retention index of, e.g., CzHsX is 560, i.e., Z(C2H5X) = 200 +

IDENTIFICATION

33

I

I

I

1

I

I

I

I

I

I

I

1

I

I

1.0 -

0

40 80 BOILING POINT Tb('C 1

I

120

Fig. 3.6. Relationship between log (retention time) and boiling point for the same hydrocarbons as in Fig. 3.5 on p,P'-oxydipropionitrileat 43°C. A, saturated compounds; B, G and F, monoolefis and alicyclics; C, dienes; D, triple-bonded unsaturated compounds. The numbers denote the same hydrocarbons as in Fig. 3.5. (Reproduced from ref. 26 by courtesy of J.H. Purnell and Wiley.)

360 = looni + I(HX), where ni and I(HX) are the number of carbon atoms in the molecule of compound i and the retention index increment of the moiety HX, respectively. The analytical expression for calculating the retention index of compound i, I f ,by employing two reference n-alkanes with the carbon numbers n and n + 6 is [ 2 8 ]

where Kp,fl and K p , n + are ~ the distribution constants of the two reference n-alkanes. Of course, adjusted retention times, retention and/or specific retention volumes, capacity ratios and relative retention data can be employed instead of the K values. It has been shown from an analysis of the thermodynamic significance of retention index [29] that the retention index of a compound i = CH3(CH2),X (cf., eqn. 5) can be expressed as

Eqn. 10 indicates that the difference in the retention indices of compound i on two different stationary phases A and B, I: - I F , is

He&, the difference Zp - I? is a characteristic of the chemical functionality, independent of the carbon number of the given compound.

34

IDENTIFICATION AND QUANTITATION

I

0

100

200

I

300

400

500

600

700

RETENTION INDEX

Fig. 3.7. Schematic representation of the concept of Kovits retention indices.

All of the above correlations involving logarithmic retention data (isothermal) also apply to retention indices (not their logarithms).

3.1.2. Use of chemical reactions in connection with GC This discipline pertains to the field of classical organic analysis rather than to GC, and the gas chromatographer may find many useful hints in standard textbooks thereon. However, the combination of GC and chemical reactions is a very powerful and relatively inexpensive means of identification, particularly because each discipline complements the deficiencies of the other, i.e., the failure of GC to determine unambiguously the chemical functionality and the need for efficient separations of more complex mixtures in classical organic analysis. A simple approach involves the use of classification reagents. The first work dealing with the use of chemical reagents to classify GC eluates was by Dubois and Monkman [30]. The chromatographic fractions are led into vials containing test reagents, the vials being exchanged according to the indication provided by the detector. It is possible to use several reagents simultaneously, employing an appropriate splitter at the column outlet. Examples of the use of group classification reactions suitable for testing GC eluates are given in Table 3.1 [3 11 . A very useful source of information on chemical structure is the so-called carbonskeleton chromatography, introduced by Beroza and co-workers [32-371. With this method the substance being analysed is stripped of all its functional groups by hydrogenolysis (Pd catalyst, 300°C) carried out in an on-line arrangement with the GC analysis. It depends on the nature of the substance being hydrogenolysed whether the resulting skeleton will be the parent hydrocarbon or the next lower homologue. The possible reactions are summarized in Table 3.2. However, it is necessary to allow for possible rearrangements of the hydrocarbons.

IDENTIFICATION

35

TABLE 3.1 FUNCTIONAL GROUP CLASSIFICATION TESTS [31] Compound type

Reagent

Type of positive test

Alcohols

KzCrz07-HN03 Cerum (IV) nitrate 2,4-DNP Schiff s 2,4-DNP Iron (111) hydroxamate Sodium nitroprusside Isatin *(OAc)z Sodium nitroprusside Sodium nitroprusside Isatin Hinsberg Sodium nitroprusside

Blue colour

Aldehydes Ketones Esters Mercaptans

Sulphides Disulphides

Arnines

Nitriles

Aromatics Unsaturated aliphatics Alkyl halides

Amber colour Yellow precipitate Pink colour Yellow precipitate Red colour Red colour Green colour Yellow precipitate Red colour Red colour Green colour Orange colour

Minimum detectable amount bg)

Compounds tested

20 100 20

50 20 40

50 LOO 100 50 50 100 100

Red colour, primary Blue colour, secondary

50

Iron (111) hydroxamatepropylene glycol HCHOlHzS04

Red colour Red-wine colour

40 20

HCHO-H2S04 Ak. AgN03

Red-wine colour White precipitate

40 20 -~

Under somewhat milder conditions (2OO0C), the reaction does not proceed as far as removing the functional groups, and the result is merely the hydrogenation of multiple bonds [38,39]. This is an efficient means of structure elucidation, especially when combined with ozonolysis [40,41 J to establish the locations of multiple bonds in the molecule. In ozonolysis the substance supposed t o contain a double bond is dissolved in CS2, ozonized at about -70°C and the ozonide is reduced with triphenylphosphine t o produce aldehydes and/or ketones characteristic of the moieties linked by the double bond, Very useful adjuncts in the group classification and in verifying the identity of organic compounds are the so-called subtractive techniques [42]. With these techniques, a loop containing a chemical that either entraps or substantially retards a certain group of substances is included in the GC pathway. Such a procedure may be considered as an extreme

36

IDENTIFICATION AND QUANTITATION

Sulphides

R-CH~+S-

See.- or terf.-0 or -N

R-CH-R'

R-CH-R'

.-+--

_.I_ 0

N

/ \

I

Ketones

Parent hydrocarbon

R-C-R'

..[I__ 0

Primary 0 or N, such as in: Aldehydes

R+HO

Acids

R+COOH

Alcohols

R+C~,-OH

Ethers

R+cH~-)o~cH~+R'

I

I

I

Esters

I

,

Parent and/or next lower hydrocarbon

I

R~CH,+NH, I

Amides

I

R-~COO~CH~+R' '

Amines

I

I

,

RIC--NH2 o

I1 0

Unsaturated compounds are saturated

case of using selective sorbents in normal chromatography. The procedures in which a reactor constitutes a part of the GC pathway have been called reaction GC. An important aspect of this discipline is elemental analysis performed with the aid of GC [43-451.This kind of analysis may be conducted either quantitatively, in order to establish the formula of the substance, or merely to detect the presence of the elements. The reactions used in the elemental analysis of compounds are summarized in Table 3.3. When considering reaction GC, the techniques of pyrolysis GC [46] ought to be mentioned. Although these techniques are occasionally utilized to identify volatile compounds, their main application is to virtually non-volatile substances.

3.1.3. Use of selective detectors

All GC detectors are more or less selective, which is a complicating factor in quantitative analysis by GC. However, some of them display such a high selectivity towards certain elements or functionalities that they can be used advantageously for identification

IDENTIFICATION

37

TABLE 3.3 REACTION GC PROCESSES USED IN ELEMENTAL ANALYSIS [45] Products

Elements (compounds)

Reagents

C

CuO, Co oxides; AgMn04, 700- 1000°C Hz, Ni, 350-450°C [ 0 J as in C analysis Fe, 750°C; CaHz [OI CU, 500-800°C charcoal, 1 120°C Pr-carbon (1 : l), 920°C 0 2 , Pt, 850°C H z , Pt, 800-1000°C [O], Pt, 800°C H z , Pt, 750-1000°C Hz ,950"C

~~

S C1, Br

P

~~

~

COZ CH4

Hz 0 HZ Nitrogen oxides NZ

co co

Sulphur oxides Hz s Clz, BIZ HCl, HBr PH3

purposes. As pointed out above, the properties of selective detectors are often utilized in combination with the preparation of derivatives containing selectively detectable groups or elements. A selective and quantitative detector is the acid-base automatic titration detector used in the first work on the GC of volatile fatty acids and bases by James and Martin [ 171. The column effluent enters a cell containing a solution of an acid-base indicator. The change in the pH and thence the colour of the solution is titrated automatically by means of a photocell relay. The amount of titrant added to the cell is plotted against time, thus producing a selective integral chromatogram [47]. Highly selective and quantitative devices are those based on electrochemical principles, namely the microcoulometric [48] and electrolytic conductivity [49] detectors. With these detection methods, the column effluent is mixed with reactant gases (oxygen or hydrogen) and processed in a pyrolytic furnace. When employing a microcoulometric detector the products of combustion and/or reduction enter a four-electrode microcoulometric titration cell. The change in the concentration of the titrant in the cell, brought about by the ions produced from the sample, is sensed by the sensor/reference pair of electrodes, thus producing a signal to the coulometric amplifier. The amplifier proportionately supplies a voltage to the generator anode-cathode pair of electrodes, which generate ions to replace those lost by the reaction. By selecting properly the decomposition procedure and the electrodes-electrolyte system, it is possible to determine selectively the contents of halogens, sulphur, nitrogen and phosphorus in the molecule or t o detect selectively the compounds that contain these elements. The sensitivity of microcoulometric detectors is about 1 ng for compounds containing halogens, sulphur and nitrogen. With electrolytic conductivity detectors the stream of the reaction products is continuously scrubbed by a stream of deionized water, and the ionogenic species transferred into the water stream are detected by measuring the electrical conductivity of the

38

IDENTIFICATION AND QUANTITATION

water by using a d.c. bridge. The sensitivity of this device is about 1 ng for sulphur-containing compounds and about 0.1 ng for compounds containing chlorine and nitrogen. Another important means of selective detection is the flame photometric detector. This detector essentially has the properties of a flame photometer and has been constructed as a unit that can be attached directly to the burner jet of an FID gas chromatograph [50]. The component containing the elements to be detected is fed into the flame, the optical emission produced is transmitted via a glass window and an optical filter to a photomultiplier and the signal is sensed by a photocell. A high degree of selectivity can be attained if narrow-wavelength bands are selected by the filter. This detector is used to detect phosphorus-, sulphur- and halogen-containing compounds and metals in complexes [51]. The reported sensitivity is g/sec for phosphorus and lo-'' g/sec for sulphur. The spectral emission as a source of selective signal is also obtained by a microwave discharge, which constitutes the principle of the microwave emission detector [52]. The conventional flame-ionization detector (FID) can be made remarkably selective towards phosphorus- and/or halogen-containing compounds by inserting a tip of an alkali metal salt over the burner jet. This arrangement is called the thermionic or alkali flame-ionization detector (AFID). When using sodium sulphate [53] the response to phosphorus-containing substances is about 600 times and that to chlorine compounds about 20 times higher than with an ordinary FID. An enhanced selectivity towards sulphur- and nitrogen-containing compounds can be attained by employing potassium and rubidium salts, respectively [54,55]. The detection limit for phosphorus in pesticides is about g/sec with the AFID. Of the more conventional detectors, it is particularly the electron-capture detector (ECD) [56] that possesses an extremely high degree of selectivity towards functionalities exerting an affinity to electrons, such as alkyl halides, conjugated carbonyls, nitriles, nitrates, organometallic and sulphur compounds, particularly polysulphides. In this respect, very useful information can be obtained from ECD and FID chromatograms recorded simultaneously [57]. It is interesting that the FID fails t o produce a sufficient response to higher fused-ring polycyclic aromatics whereas the ECD is very sensitive to these compounds [58]. 3.1.4. Combination of GC with other analytical methods The combination of GC with other analytical methods may be considered as the most advanced approach to the identification of compounds in mixtures. This subject constitutes a self-contained discipline that far exceeds, in its extent rather than its nature, the scope of this chapter. Therefore, only a brief discussion of the possibilities will be given here. A detailed treatment of the ancillary techniques of GC can be found in the book by Ettre and McFadden [ 131. A very useful identification tool is the combination of GC and thin-layer chromatography (TLC). The first work on combined GC-TLC appears to have been by Janik [59]. The GC column effluent is split into two streams, one of which enters the detector and the other, led via a heated conduit, impinges on the chromatographic thin layer carried by a moving plate. The GC fractions sampled in this way are subsequently developed and the TLC spots detected in the usual manner. The result is a kind of two-dimensional thin-

IDENTIFICATION

39

layer chromatogram. Such a chromatogram provides two additional items of information: R F values and colour or other properties. Other important combinations of GC are those with mass spectrometry, infrared spectroscopy and proton magnetic resonance spectrometry, of which the first is most important. A typical problem incidental to all these combinations is the interfacing of the gas chromatograph with the spectral instrument. Of all the ancillary techniques mentioned, it is only the mass spectrometer that has a sensitivity compatible with that of high-sensitivity GC detectors, i.e., about pg of the substance being analysed. For the examination by IR spectroscopy, about 10-pg samples are necessary. The least sensitive is proton magnetic resonance spectrometry, in which the sample size required varies from tenths to units of milligrams, depending on the method of signal processing. Spectral methods can provide the deepest insight into the constitution of GC eluates. However, the spectral laboratory has to cooperate closely with a library providing stored reference spectra, and efficient communication between these two facilities is hardly practicable without the use of a computer. 3.1.5. Prospects of performing identification by virtue of retention behaviour only

Some chrornatographers have tried or are still trying to find methods that would provide the identification of substances by exclusively GC means. It is difficult t o say whether these efforts will be completely successful. On the other hand, the question may be raised as to whether it is expedient to try to use merely a single technique to perform identification; current practice shows that the combination of several techniques is usually more effective. However, it must be admitted that the potentialities offered by GC retention data are still not being utilized to full advantage. The main problem is the reliability of retention data measured by conventional procedures. Much attention has been paid to the precision of measurement of retention data in a certain laboratory and on a given instrument, but great discrepancies are encountered between retention data measured, although with very high precision, at two different places and under different conditions, considering of course only those conditions the variations of which are believed to have a negligible effect on the data measured. This is largely due to the fact that retention data depend on certain factors the effects of which are difficult to eliminate completely or control and which are normally neglected. These factorsare the imperfections in the gas phase and the compressibility of the stationary phase (cf., the quantities vi, $, ZG and B in eqn. I), the finite rate of equilibration of the solute, variations in the composition of the sorbent, spurious sorption of the solute, solubility of the carrier gas in the stationary phase, etc. Hence, even relative retention volumes and/or retention indices must depend to some extent on the kind, flow-rate and absolute pressure of the carrier gas, the load of the liquid stationary phase on the support, which production batch of the stationary phase has been used and the kind of support. The absolute column pressure will obviously vary with the column length and particle size of the support. Moreover, adjusted retention data are required in all instances, which renders it necessary to measure the dead retention time. This is a crucial step in obtaining accurate retention data and presents a problem per se.

40

IDENTIFICATION AND QUANTITATION

These second-order effects are only slight in comparison with those of the column temperature and sample size, but they become bery significant in high-precision measurement of retention data. It would be very beneficial if analytical chromatographers adopted the methods currently practised by physical chemists when dealing with GC, such as extrapolating retention data to zero sample size, zero or a standard mean column pressure and carrier gas velocity and correcting the rough data for spurious sorption effects. A detailed survey of the problems indicated here and a wealth of valuable hints on how to approach these problems can be found in recent books on physico-chemical applications of GC [60,61]. These rather complicated procedures would be adequate only when employing well defined stationary phases.

3.2. QUANTITATION

3.2.1. General concepts The basis of quantitative analysis by modern methods of column elution chromatography (gas and liquid chromatography) can be specified as the chromatographic separation of an n-component mixture into n binary (or pseudo-binary) component-mobile phase mixtures and the continuous measurement of the contents of the separated components in these mixtures with the aid of a special analyser. The function of this analyser is performed by the chromatographic detector, together with the system for recording and processing the chromatographic data. The range of applicability of analytical chromatography is qualified above all by the standard of chromatographic instrumentation; the role of instrumentation is particularly important from the point of view of the definition of chromatography as a quantitative analytical method. The chromatogram of a given compound is a record of the time course of the detector response to the presence of the compound in the column effluent. Depending on the character of the detector employed, this record may represent either the time course of the absolute or relative concentration of the compound in the column effluent as it passes through the sensor, the time course of the absolute amount of the compound within the space of the sensor or the time course of the rate at which the compound is introduced into the sensor. In any case (provided the response is linear), the shape of the time course of the response is determined by the concentration profile of the compound in the effluent and corresponds approximately to a Gaussian distribution [62]. The position of the peak in the chromatogram (retention time, retention volume) is associated with the quality of the substance being chromatographed, whereas the area of the peak is proportional to the total amount of the substance in the eluted chromatographic zone. If the response is linear, the peak area corresponding to a given amount of solute is independent of the shape of the peak. It is sometimes advantageous to characterize the size of the chromatographic peak by its height. With symmetrical peaks recorded for various amounts of a given solute compound under constant conditions the peak height is proportional to the peak area. However, whereas the peak area corresponding to a given amount of solute is independent of zone broadening, the peak height depends on the degree to which the chromatographic

QUANTITATION

41

zone has been broadened. As the broadening of zone is a function of a number of experimental parameters, the analytical significance of the peak height is rather limited. With asymmetric peaks the applicability of the peak height as a quantitative analytical quantity is doubtful, as there is no linear proportionality between the height and area of the peak in this instance. When considering the question of whether to carry out calculations by using peak heights or peak areas, it is expedient to take into account the properties of the detector employed [15,16,63-651. When employing a detector that responds to the rate at which the mass of solute is introduced into the detector (e.g., the FID and its modifications), the peak area corresponding to a given amount of solute is theoretically independent of the rate of introduction of solute into the detector, whereas the peak height is proportional to this rate. With a given charge of solute, the rate of introduction of solute into the detector can be varied by changing the column temperature and the carrier gas flowrate, provided that the other working conditions are kept constant. Hence, if the column temperature and/or the carrier gas flow-rate cannot be stabilized precisely enough, it is not suitable to carry out calculations by using peak heights when employing a mass-rate sensitive detector. On the other hand, with detectors that respond to the concentration of solute in the column effluent (e.g., the thermal conductivity detector), both the peak height and peak area depend, at a given carrier gas flow-rate, on the column temperature in the same manner as with mass-rate sensitive detectors but, at a given column temperature, the peak height is independent of the carrier gas flow-rate and the peak area is inversely proportional to the carrier gas flow-rate. Therefore, with a stable column temperature and a non-stable carrier gas flow-rate it is more suitable to work with peak heights when employing concentration-sensitive detectors.

3.2.2. Specificity of detection, response factors

If the detector response is linearly proportional to the concentration of solute in the column effluent, there is also a linear proportionality between the peak area and the total mass of solute in the eluted chromatographic zone. Hence, for the peak areas of the compound under determination, i, calibration standard s and the reference compound r (cf., the definition of the relative specific response), A i , A , and A , , respectively, we have

A i= RgPmi A , =RiPm, A , = R;Pmr

(14)

where RfP,Rip and RSp are the specific responses (detector response to unit mass of compound) to compounds i, s and r, and mi,m, and m , are the masses of the compounds in the corresponding chromatographic zones. The specific response involves an apparatus constant and a substance-specific constant. It is apparent from eqns. 12-14 that it is necessary to know R S Pin order to determine the mass of the compound being chromatographed from the peak area. This quantity can either be determined by the calibration of the apparatus, i.e., from the peak area corresponding to a known amount of the compound under analysis, or predicted theoretically by virtue of the analysis of the processes

42

IDENTIFICATION AND QUANTITATION

taking place in the detector. With a given detector and under constant operating conditions (temperature, pressure and flow-rate of the mobile phase, electrical and/or other operating parameters of the detector, sensitivity-attenuation setting, etc.) the apparatus constant is the same for all of the compounds chromatographed, but the substancespecific constant is generally different with different compounds. This variability in the sensitivity of a given detector towards different species of compounds can be utilized for selective detection, but in quantitative analysis it gives rise to problems associated with the definition and application of response factors. The apparatus constant can be eliminated by using the so-called relative specific response; the relative specific response of a compound is the ratio of the specific responses of the compound and of a deliberately chosen reference compound (r). Hence, the relative specific responses of the compound under determination and of the calibration standard, R7f and R:; (cf., eqns. 12-14), respectively, are

The relative specific response characterizes unambigously the specificity of detection (with a given detector and under given conditions) and is relatively little dependent on the variations in instrumental parameters (cancellation of the apparatus constant). Therefore, the relative specific response is the most suitable basis for the definition of the mass-specific response factor. This factor can be defined as the inverse of the relative specific response; hence, for compounds i and s we have

and eqns. 12 and 13 can be rewritten as

where the superscript W indicates that the product of the peak area and response factor of a given component is proportional unambigously to the mass proportion of the component in the material being analysed. With some techniques (absolute calibration and internal standard method), the relationship mi/m, =Aifiy/As occurs. In such instances the calibration standard also fulfils the role of the reference compound, as

fz

The response factors can be determined experimentally by chromatographing known amounts and/or a defined mixture of the substance under determination and a reference substance and evaluating the chromatograms; it follows from eqns. 15-18 that it is suffiwe can write cient to know the ratios mi/mr and ms/mr only. For

fiy

43

QUANTITATION

It obviously applies that mi/ms = gi/g, = qi/q,, g and q denoting mass fractions and mass concentrations (mass/volume). If molar proportions of the components under analysis are to be determined, the peak areas have to be multiplied by the respective molar response factors; the following relationships between molar (superscript n) and mass specific response factors hold:

where M i , M, and Mr are the molar masses of components i, s and r respectively. In some instances (with some detectors and some compounds) it is possible to specify the analytical property [66] of the analyte with respect to the given detector and predict the response factor theoretically.

3.2.3. Techniques of quantitative chromatographic analysis

In this section, all of the data concerning concentration will refer to the material under analysis. Subscripts i and s will again designate the component under analysis and a calibration standard, respectively. Symbols with which the subcripts are without parentheses refer to single compounds i and s, and symbols with the subscripts in parentheses refer to materials containing the compounds designated by the subscripts. W and V represent the masses and volumes treated in the preparation of sample before its introduction into the chromatograph, and w and u designate the masses and volumes of the material injected into the chromatograph, respectively. With the individual techniques, relationships for calculating the mass concentration (the mass of compound in unit volume of the sample), qi, and the mass fraction, g j , of compound i in the sample will be considered. With the symbols for mass-specific response factors the subscript r will be omitted hereafter. According to the above notation, the quantities qi and g j are defined by qi = Wi/ V(i)= wi/u(i)

(26)

gi = WJC W i = Wi/W(i, = wi/Zwi = wi/w(i)

(27)

In order to express the results in molar concentrations (the number of moles of compound in unit volume of sample), p i , or molar fractions, xi, the following relationships can be used:

where M iis the molar mass of component i and M(i) is the average molar mass of the sample (containing component ?).The quantities qi and gi are related to each other by the equation

q . =g.d . 1

1

(1)

where d ( i )is the density of the sample.

(30)

44

IDENTIFICATION AND QUANTITATION

3.2.3.1. Absolute calibration method Calculation procedure. Defined amounts of the material under analysis and the calibration material with known contents of a standard compound are injected separately and chromatographed under identical conditions, thus giving chromatograms with peaks of compound i and s having the areas Ai and A,, respectively. The results are calculated by using the equation

Graphicalprocedure. Several different defined amounts of a standard compound are chromatographed under identical conditions; by plotting 4,u(,) and/or g,w(,) against A , f Y , a calibration graph is constructed. The amount of the compound to be determined in the given (injected) amount of the sample under analysis is then determined from the corrected peak area of compound i , A i f w , and the calibration graph. If use is made of a calibration standard identical with the compound under determination, the response factors can be neglected with both the above variants and, in addition, it is also possible to employ peak heights instead of peak areas under certain circumstances. 3.2.3.2. Internal standard method Calculation procedure. A defined amount of the material under analysis is mixed with a defined amount of the calibration material with known contents of a reference compound (internal standard), and some amount of this mixture is injected into the chromatograph, thus providing a single chromatogram with peaks of compound i and the standard having areas A and A,, respectively. The results are calculated by using the equations 4i = 4 4 i f i W v ( s j ~ s f Yv(i) gi g J i fiww ( s ) / ~s

fsW W ( i )

(33) (34)

Graphical procedure. Several model materials are prepared having various defined contents of the compound under determination and each of these materials is mixed in a defined ratio with the material containing a standard. Samples of these mixtures are then chromatographed and a calibration graph is constructed by plotting the concentrations of compound i in the initial model samples against the corresponding numerical values of expressions q J i V ( s ) / A s V ( i )and/or gJiW(s)/AsW(i).In the analysis proper, a defined amount of the material under analysis is mixed with a defined amount of the standard material, a sample of this mixture is chromatographed, the value of 4 J i V ( s ) / A s V ( j ) and/or gJiW(s)/AsW(i)is again calculated and the corresponding 4i or gi is read from the calibration graph. By using the graphical procedure the response factors are eliminated and, with certain limitations, it is also possible to employ peak heights instead of peak areas in the calculations. 3.2.3.3.Standard additions method Although this technique permits a graphical procedure to be used it will not be discussed here as it is relatively insignificant. There are two variants of the calculation proce-

QUANTITATION

45

dure, based either on direct measurement of sample charges or on the use of a reference compound. Direct measurement of sample charges. (a) A defined amount of the material under analysis is injected into the chromatograph and the chromatogram is recorded; the symbols for the amount of material injected and the corresponding peak area will be indicated by subscripts ( i ) and i, respectively. (b) A defined amount of the initial material under analysis is mixed with a defined amount of the standard material with a known content of compound i, the latter serving the function of calibration standard with this technique. A defined amount of the mixture is injected into the chromatograph under the same conditions as in (a) and the chromatogram is again recorded; the symbols denoting the amount of the initial material that has been mixed with the standard and the amount of the standard material will be indicated by subscripts ( i ) and (s), and those denoting the amount of the sample of the mixture injected into the chromatograph and the corresponding peak area by subscripts (is) and is, respectively. The results are calculated by using the equations

Use of a reference compound. This version makes it possible to obviate the necessity of measuring the amounts of the samples injected into the chromatograph. Instead, the peak areas are measured of an auxiliary reference compound in the chromatograms of the initial sample and of its mixture with the standard. Any component, either present already in the initial material under analysis or added to it, can serve as a reference compound. The amount of the reference compound need not be defined. The analytical procedure proper is the same as with the preceding alternative, except for the necessity of measuring the sample charges. The results are calculated by using the equations

where A , and A ; designate the peak areas of the auxiliary reference compound in the chromatograms of the initial sample and of the sample enriched with the standard, respectively. Provided that V(i) and Wu) are always the amounts of the initial material as such, eqns. 37 and 38 hold irrespective of whether the reference compound was an original component of the material being analysed or whether it was added to the material. With the various alternatives of the standard additions technique it is possible to carry out calculations with both peak areas and peak heights. 3.2.3.4. Internal normalization method This technique provides data on the relative contents of the components under determination, i.e., the mass or molar fractions. It is necessary with this technique that all of

46

IDENTIFICATION AND QUANTITATION

the components of the material being analysed are identified and provide a measurable peak in the chromatogram, otherwise the results are dubious. A sample of the material is chromatographed, the areas of all peaks in the chromatogram are multiplied by the pertinent response factors and the results are calculated by using the equations

where the summations involve all the components of the material under analysis. All of the above techniques can be employed to analyse materials in any state, i.e., gases, liquids and even solid compounds. Also procedure are conceivable in which the material under analysis and the calibration material have different states of aggregation.

3.2.3.5,Manual processing o,f the chromatogram The area of a peak of any shape can be determined by planimetering or by cutting the peak from the chromatogram and weighing the cutting. The areas of symmetrical chromatographic peaks can also be determined from their linear parameters. It follows from the analysis of the Gaussian curve and from the theory of chromatography that the area of a symmeirical chromatographic peak can be calculated according to the relationships A = (n/4 In 2)”2 hB1,, = 1.06hB1,2

(41)

A = (2ne)”’/4hfB‘ = I ,033A’

(42)

A = (2df)’” hbtR

(43)

where h is the peak height, B , , , is the peak width at the half height, e is the base of natural logarithms (2.718),h’ is the height of the triangle bounded by the tangents at the inflection points of the peak and the intersections of the tangents with the baseline, B‘ and A’ are the base width and the area of this triangle, N is the number of theoretical plates of the column, b is the chart speed and t R is the retention time of the component being chromatographed.

3.2.3.6.Automatic processing of the chromatogram The above manual procedures are laborious and are therefore being replaced by modern methods of automatic integration. The detectors used in modern GC provide an electric signal, the magnitude of which is proportional to the concentration in the column effluent of the compound being chromatographed. The automatic processing of the output information from the chromatograph can be performed on several levels, from the simplest mechanical analogue integrators up to the introduction of sophisticated and expensive dedicated computer systems. The practical selection of a suitable level is usually given by a compromise between the price of the apparatus and the performance required. The individual levels can be arranged into the following sequence: analogue integrators, digital integrators, small dedicated computers and large computer systems. A more detailed discussion of the problems of automatic processing of chromatograms exceeds the scope of this chapter. The interested reader is referred to the specialized literature on this topic [67].

41

QUANTITATION

3.2.4. Special problems of quantitation in derivatization GC Derivatization GC essentially is a kind of indirect analysis. Not only a chemical derivative rather than the original compound is the subject of GC determination proper, usually the derivative is isolated from the reaction mixture, purified by diverse techniques and concentrated before it is introduced into the gas chromatograph (cf., Chapter 2). Thus, the final analytical step is carried out with a material completely different from the original one, and the overall recovery of the compound in the form of its derivative may depend in a decisive manner on the composition of the matrix of the original material. If merely the identification of compounds is required, the above situation does not cause any serious problems. However, from the point of view of quantitation it is very important whether the composition of the matrix can or cannot be determined and simulated. Let us consider a situation in which N mol of compound i (Ni)react in an excess of the derivatizing agent to produce stoichiometrically N mol of derivative D (ND),the coefficient of conversion being a < 1 :

N i -+ reagent

-+ olND

-+ (1 - a) N i

Hence it follows that

where Wi is the overall mass of compound i in the processed amount of the material being analysed, W; is the corresponding mass of the derivative in the parent reaction mixture and Mi and M D are the molar masses of compounds i and D,respectively. Let the derivative be subjected to a series of consecutive operations (isolation, purification, concentration) I , 11, 111, ...,f,for which we can write

W h=klWB 1I-k wl wD-

I1

D

w21 = kII& w&

= kfWL-1 (45) where kI, k11,k l l I , ..., kr are the recovery constants of the individual operations, fdenoting the final operation. Eqns. 45 can be rewritten as

and on combining the latter with eqn. 44 we have

where K~ is the overall recovery constant. In quantitative analysis, it is necessary either to determine in some way the value of K,Q or to arrange that K,CY approaches unity, and/or to choose an analytical procedure in

48

IDENTIFICATION AND QUANTITATION

which the necessity to know K,OL is obviated. Procedures that involve chemical derivatization and subsequent multi-step preparative operations and in which K,OL = 1 are very rare. In more favourable circumstances the composition of the matrix of the material under analysis is known and can be simulated, which makes it possible to employ the reference model system method and/or to determine explicitly the value of K,OL. For a model mixture with some known contents of compound i, W;, we can write

Provided both the model mixture and the material under analysis have identical matrices and are processed in exactly the same way, it can be assumed that K&* = ~ , a and , it follows from eqns. 47 and 48 that

Under constant chromatographic conditions

where SfD and qfd are the concentrations of the derivative in the final materials obtained by processing the analysed and the model materials, and V{g) are the volumes of the materials, and are the volumes of the materials charged into the gas chromaand wfd are the masses of the derivative contained in the volumes &) and tograph, dD and A D and A ; are the peak areas corresponding to the masses wfD and df; , respectively. Hence, combining eqns. 49 and 50 we have

dD) 4:)

Vb)

4;)

D eqn. 48 and to employ it as an additional It is also possible to calculate M i / ~ ; a * M from note correction factor to the peak area, together with the detector response factor (j,”>; that the mass of compound i corresponding to the charge of final material introduced into the gas chromatograph is proportional to ( M i / ~ , d DA)D f t . The reference model system method can be combined with the internal standard method. In tlus instance, the model system contains known amounts of the compound under determination and a calibration standard, a known amount of the standard is added to the system to be analysed and both systems are processed in the same way and under the same conditions. With this combination there obviously apply exactly the same qualifications as specified with the plain reference model system method described above. The

QUANTITATION

49

results are calculated by using the equation (53)

It is also possible (cf. eqn. 33) to employ the relationship

In eqns. 53 and 54, 4 , is the concentration of standard s in the standard solution employed and V(s)is the volume of this solution, mixed with the volume V(i)of the original material t o be analysed. The subscripts i and s indicate that the respective derivatives refer to the compound under determination and the calibration standard, respectively. If the standard is a non-reactive compound under the given conditions, then obviously f,, [MD] Z M , and (Y = 1. [ A D ]= A s , Frequently the composition of the matrix of the material being analysed is unknown and impossible to simulate. In such instances it may be advantageous to use the standard additions method. It can be assumed that the addition of a relatively small amount of compound i, already present in the system, will not alter substantially the properties of the matrix of the system. The procedure is as follows. Step A: a defined volume [ V(i)]of the material to be analysed is subjected to the derivatization and subsequent preparation procedures, thus obtaining a volume V b )of the final material to be introduced into the gas chromatograph; a volume &) of this material is charged into the gas chromatograph, the corresponding peak area being A D . This stage is represented by eqn. 47. Step B: the same volume of the original material as in step A is mixed with a defined volume [ V(,)] of the standard (compound i) solution and the mixture is treated in strictly the same manner as in step A; for this stage there holds:

rg],

where WL' is the mass of the derivative in the total amount of the final material obtained in step B. Combining eqns. 47 and 55 and solving for Wi results in

The ratio W h ' / W L can again (cf., eqn. 50) be expressed as

However, owing to the above-specified requirements concerning the working procedure,

50

IDENTIFICATION AND QUANTITATION

V& = V&), and eqn. 56 can be expressed as

(58)

Hence, the standard additions method is unique in that it actually employs the very material under analysis as a reference matrix material, thus providing for efficient elimination of very complex matrix effects even when the final material is the result of a multi-step preparative procedure and the composition of the matrix of the original material is completely unknown. These advantageous features of the standard additions technique have been discussed and verified in context with quantitative headspace gas analysis [68].

REFERENCES D. Zarazir, P. Chovin and G. Guiochon, Chromatographia, 3 (1970) 180. I. Brown, J. Chromatogr.. 10 (1963) 284. P.M. Simpson, J. Chrornatogr., 77 (1973) 161. C.J.W. Brooks, A.R. Thawley, P. Rocher, B.S. Middleditch and W.G. Stillwell, J. Chromatogr. Sci., 9 (1971) 35. C.W. Gehrke and D.L. Stalling,Separ. Sci., 2 (1967) 101. D.B. Lakings, C.W. Gehrke and T.P. Waalkes, J. Chrornatogr., 116 (1976) 69. C.W. Gehrke and A.B. Patel, J. Chrornatogr., 123 (1976) 335. J.W. Atson, J. Chromatogr., 131 (1977) 121. J . Yamanis, R. Vilenchich and M. Adelman, J. Chromatrogr., 108 (1975) 79. 10 K. Lindstrom and J. Nordin, J. Chromatogr., 128 (1976) 13. 11 D.A. Leathard and B.C. Shurlock, in J.H. Purnell (Editor), Progress in Gas Chromatography, Wiley-Interscience, New York, 1968, p. 1. 12 D.A. Leathard and B.C. Shurlock, Identification Techniques in Gas Chromatography, WileyInterscience, New York, 1970. 13 L.S. Ettre and W.H. McFadden, Ancillary Techniques of Gas Chromatography, Wiley-Interscience, New York, 1969. 14 H.W. Johnson, Jr., Advan. Chromatogr., 5 (1968) 175. 15 J.C. Sternberg, in L. Fowler (Editor), Gas Chromatography, Proceedings of the 4th Intern. Symp. ISA, June 17-21, 1963, Academic Press, New York, 1963, p. 161. 16 J. No&, Quantitative Analysis b.v Gas Chromatography, Marcel Dekker, New York, 1975. 17 A.T. James and A.J.P. Martin, Biochem. J., 50 (1952) 679. 18 A.J.P. Martin, Biochem. Soc. Symp., 3 (1949) 4. 19 M.R. James, J.C. Giddings and R.A. Keller, J. Gas Cllrornatogr., 3 (1965) 57. 20 W.O. McReynolds, Gas Chromatographic Rtvention Data, Preston Technical Abstracts Co., Evanston, IL, 1966, p. 42. 2 1 A.T. James,Biochem. J . , 52 (1952) 242. 22 W.O. McReynolds, Gas Chromatographic Retention Data, Preston Technical Abstracts Co., Evanston, IL, 1966, pp. 4 2 and 98. 23 A.T. James, J. Chromatogr., 2 (1959) 552. 24 A.W. Ladon and J.J. Walraven, in E. Kovats (Editor), Column Chromatographj,, Sauerlander AG, Aarau, 1970, p. 167. 25 J.H. h r n e l l , Gas Chromatography, Wiley, New York, 1962, p. 334. 26 J.H. h r n e l l , Gas Chromatography, Wiley, New York, 1962, p. 390.

REFERENCES

51

27 W.E. Harris and H.W. Habgood, Programmed Temperature Gas Chromatography, Wiley, New York, 1966, p. 142. 28 E. Kovits, Helv. Chim. Acta, 41 (1958) 1915. 29 J. Nov& and J. R%Ekovh, J. Chromatogr., 91 (1974) 79. 30 L. Dubois and J.L. Monkman, in H.J. Noebels, R.F. Wall and N. Brenner (Editors), Gas Chromatography, Academic Press, New York, 1961, p. 237. 31 J.T. Walsch and C. Merritt, Jr., Anal. Chem., 32 (1960) 1387. 32 M. Beroza, Nature (London), 196 (1962) 768. 33 M. Beroza, Anal. Chem., 34 (1962) 1801. 34 M. Beroza and R. Sarmiento, Anal. Chem., 35 (1963) 1353. 35 M. Beroza and F. Acree, J. Ass. Offic. Agr. Chem., 47 (1964) 1. 36 M. Beroza and R. Sarmiento, Anal. Chem., 36 (1964) 1744. 37 M. Beroza and R. Sarmiento, Anal. Chem., 37 (1965) 1040. 38 T.L. Mounts and H.J. Dutton, Anal. Chem., 37 (1965) 641. 39 M. Beroza and R. Sarmiento, Anal. Chem., 38 (1966) 1042. 40 O.S. Privett and E.C. Nickell, J. Amer. Oil Chem. SOC.,43 (1966) 393. 41 V.L. Davison and B.J. Dutton, Anal. Chem., 38 (1966) 1302. 42 N. Brenner and V.J. Coates, Nature (London), 181 (1958) 1401. 43 V. Rezl and J. Jan&, J. Chromatogr., 81 (1973) 233. 44 V. Rezl and J. Uhdeovi, Int. Lab., Jan./Feb. (1976) 11. 45 L.S. Ettre and A. Zlatkis, The Practice of Gas Chromatogrphy, Wdey-Interscience, New York, 1967, p. 480. 46 C.E.R. Jones and C.A. Cramers, Analytical Pyrolysis, Elsevier, Amsterdam, Oxford, New York, 1977. 47 G.W. Langan and R.B. Jackson, J. Chromatogr., 17 (1965) 238. 48 D.M. Coulson, L.A. Cavanagh, E. de Vries and B. Walther, J. Agr. Food Chem., 8 (1960) 399. 49 D.M. Coulson, J. Gas Chromatogr., 3 (1965) 131. 50 S.S. Brody and J.E. Chaney, J. Gas Chromatogr., 4 (1966) 42. 51 R.S. Juvet, Jr. and R.P. Durbin, J. Gas Chromatogr., 1/12 (1963) 14. 52 A.J. McCormack, S.S.C. Tong and W.D. Cooke, Anal. Chem., 37 (1965) 1470. 53 L. Giuffrida, J. Ass. Offic. Anal. Chem., 47 (1964) 293. 54 L. Giuffrida and N. Ives, J. Ass. Offic. Anal. Chem., 47 (1964) 1112. 55 L. Giuffrida and J. Bostwick, J. Ass. Offic. Anal. Chem., 49 (1966) 8. 56 J.E. Lovelock and S.R. Lipsky, J. Amer. Chem. SOC.,82 (1960) 431. 57 D.E. Oaks, H. Hartmann and K.P. Dimick, Anal. Chem., 36 (1964) 1563. 58 Varian Aerograph Research Note Previews and Reviews, Varian Aerograph, Walnut Creek, CA, April 1965. 59 J. Jan&, J. Chromatogr., 15 (1964) 5. 60 R.J. Laub and R.L. Pecsok, Physicochemical Applications of Gas Chromatography, Wiley-Interscience, New York, 1978. 6 1 J.R. Conder and C.L. Young, Physicochernical Measurement by Gas Chromatography, WileyInterscience, New York, 1979. 62 A.J.P. Martin and R.L.M. Synge, Eiochem. J., 35 (1941) 1358. 63 S. Dal Nogare and R.S. Juvet, Jr., Gas-Liquid Chromatography, Wiley-Interscience, New York, 1962, p. 187. 64 I. Halasz, Anal. Chem., 36 (1964) 1428. 65 D.J; David, Gas Chromatographic Detectors, Wiley-Interscience, New York, 1974. 66 F. Cita, Lectures on Physical and Special Anal-vrical Methods, Technological University, Prague, 1963, p. 6. 67 F. Caesar, Topics in Current Chemistry, Springer Verlag, Berlin, 1973. 68 J. Drozd and J. Nov&, J. Chromatogr., 165 (1979) 141.

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Chapter 4

Most frequent derivatives and methods for their preparation CONTENTS 4.1.Esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Diazomethane method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Methanol method: catalysis with boron trifluoride . . . . . . . . . . . . . . . . . . . . . 4.1.3. Methanol method: catalysis with hydrochloric or sulphuric acid . . . . . . . . . . . 4.1.4. Decomposition of tetramethylammonium salts . . . . . . . . . . . . . . . . . . . . . . . 4.1.5. Extractive alkylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.6. Other methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.7. Higher esters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.Ethers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Acyl derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Silyl derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Oximes and hydrazones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. Cyclic derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

..

53 54 55 56 58 59 60 63 64 66 69 75 76 78

As already mentioned, it is carboxyl, hydroxyl, thiol, amino, imino and carbonyl groups that cause difficulties in GC anaiysis. The high polarity of these groups (exclusively in the case of C = 0) gives rise to major interfering interactions and also a strong tendency to form hydrogen bonds. Derivatives commonly used to protect these groups are usually less polar than the original groups. The presence of acidic hydrogen, the acidity of which decreases in the above sequence of groups, is mostly utilized for the preparation of such derivatives. Esterification is a reaction typical of carboxyl groups and various types of esters are often used in GC in order to eliminate the interfering effects of t h s group. Similarly, the effect of the hydroxyl group can be suppressed by conversion into an ether and that of the carbonyl group by condensation with various substrates; these are characteristic analytical reactions. Efforts aimed at developing a simple procedure, in which several or all functional groups in the molecule could be converted into a suitable derivative in one reaction step, led to the development of methods for the preparation of silyl, acyl, isopropyl and other derivatives. Their general utility is restricted to a certain extent, however, by the limitations placed on the successful application of particular derivatives, such as anhydrous conditions for their preparation and decomposition on contact with metals. Owing to similar limitations and the varying reactivities of individual functional groups, so far efforts aimed at finding derivatives that are universally applicable have not been successful, and therefore the problem of derivatization must always be approached from the viewpoint of the particular circumstances of individual cases. 4.1. ESTERS Esters are common derivatives of carboxyl groups. Methyl esters are the most often used as they have a sufficient volatility even for the chromatography of higher fatty acids 53

DERIVATIVES AND THEIR PREPARATION

54

contained in fats [ 1 1 . A number of methods have been developed for their preparation, often exclusively for the purpose of GC determination. Neglecting some detailed modifications, they can be classified into a few procedures: elegant methods for esterification with diazomethane [2,3] and methanolic solutions of BF3 [4-61 or BC13 [7] are fairly widespread. Reactions with methanol can be catalysed even with hydrochloric [8,9] or sulphuric acid [lo]. Methyl esters can also be prepared by pyrolysis of tetramethylammonium salts in the injection port [ l l ] ,by esterification on an ion exchanger [12] and by other methods [ 131. 4.1 .l.Diazomethane method

This is based on the reaction shown in Scheme 4.1. Diazomethane is prepared by decomposing N-nitroso-N-methylurea, N-nitroso-N-methyl-p-toluenesulphonamide or other substances with a similar configuration with a lye solution. An ethereal solution of diazomethane is added gradually to an ethereal solution of the sample until a permanent yellow colour is obtained [ 141. To prepare diazomethane solutions, Fales et al. [IS] used the apparatus shown in Fig. 4.1, available commercially in two sizes: 1 mmol +

R-COOH

CH

2

-

==id

R-COOCH3

+ N2

Scheme 4.1.

a

"O'Ring

L

Fig. 4.1. Arrangement for methylation with diazomethane. (Reproduced from Anal. Chem., 45 (1973) 2302, [ 151, by courtesy of H.M. Fales and the American Chemical Society.)

ESTERS

55

(1 33 mg) of the reagent (N-methyl-N-nitroso-N'dtroguanidine is used as it is reactive enough even at room temperature) and 0.5 ml of water for the removal of the heat evolved are placed in an inner tube with an open septum. Diethyl ether (3 ml) is placed in the outer tube, the apparatus is closed with a butyl rubber O-ring and mounted in a holder. Its lower part is placed in an ice-bath and about 0.6 ml of 5 N sodium hydroxide solution is injected through the septum with the aid o f syringe. The yield of diazomethane depends on the reaction time. It reaches 20% of the theoretical yield after 15 min, and in order to obtain the maximal yield of 60% 45 min are required. The apparatus has no ground-glass joints, which are said t o be one of the reasons for explosions. Another method [ 161 uses an apparatus consisting of three bubblers: dry nitrogen passing through the apparatus at a flow-rate of about 6 ml/min is saturated with diethyl ether in the first vessel and passes to the second vessel containing 0.7 ml o f 2-(2-ethoxyethoxy)ethanol, 0.7 ml of diethyl ether and I rnl of potassium hydroxide solution (6 g per 10 ml). In this vessel, after adding the reagent diazomethane is produced and swept with a stream of nitrogen into the third vessel, containing 5--30 mg of the sample dissolved in 2-3 ml of diethyl ether containing 10%of methanol. Using methanol labelled with 14C, methanol was proved not to participate in the esterification and to act only as a catalyst, accelerating the reaction t o such an extent that it is quantitative within a few minutes. The excess of diazomethane is again indicated by a yellow colour of the reaction mixture; the residue in the second vesse) can be neutralized by adding an acid. The whole procedure takes 10-20 min. The diazomethane method is simple and usually no by-products are produced. The formation of polymethylene polymers, which is sometimes observed, can be avoided b y using vessels with clean and smooth surfaces. Anhydrous conditions must be maintained during the reaction, as diazomethane decomposes on contact with water. The disadvantages of diazomethane are its toxicity and high reactivity, the latter often being the reason for explosions. Therefore, when working with diazomethane solution, it is better to prepare it freshly prior t o use. If storage is necessary, it should be kept for only a short period at -20°C. 4.1.2. Methanol method: catalysis with boron trifluoride

The principle of the method is based on the reaction shown in Scheme 4.2. MethanolBF3 reagent, prepared by bubbling boron trifluoride through methanol [4], is added t o the sample and the mixture is boiled for 2 min. In the presence of more volatile components, a reflux condenser must be used. After cooling, esters are extracted from the reaction mixture with diethyl ether and the extract is concentrated at room temperature and chromatographed. The reagents used are commonly available and the reagent is sufficiently reactive even towards strongly hindered groups. A high reactivity, on the other hand, brings about the possibility of undesirable side-reactions if the substrate contains, e.g., double bonds or other reacti¢res. Lough [17] reported that methanol-BF3 gives rise t o losses of unsaturated esters and that oleic acid provides a high yield of isomers of R-COOH

+

Scheme 4.2.

CH30H

BF3

R-COOCH3

+

H20

56

DERIVATIVES AND THEIR PREPARATION

methoxymethyl stearate. This inconsistency with commonly observed effects seems to be caused by the extremely high concentration of BF3 which was used (50%, w/v, compared with the usual 12.5-14%, w/v). Table 4.1, from a paper by Morrison and Smith [S], shows the effects of commonly used methylation reagents on unsaturated esters. A mixture of three different unsaturated esters (18 : 1 , 18 : 2, 18 : 3 ) with methyl palmitate as internal standard was heated for 90 min with 14%(w/v) methanol-BF3, anhydrous 3 N HC1-methanol and 5% (w/v) HzS04-methanol. Prior to extraction, methyl myristate was added as an external standard and the recoveries of individual esters were determined by GC and compared with that of a reference sample. The losses are comparable with all three methods, and increase as the degree of unsaturation of the esters increases. Further experiments showed that the losses increase with increasing reaction time and catalyst concentration. As the conditions given in Table 4.1 are very severe (they are used, e.g., for the methanolysis of particularly resistant lipids) and are usually unnecessary; the losses of unsaturated esters caused by methanol-BF3 can usually also be neglected. The formation of by-products is also brought about by the presence of a cyclopropane ring in the molecule of the substrate, e.g., some bacterial fatty acids [7]. The aggressive methanol-BF3 reagent must then be replaced with a milder reagent, e.g., 10%(w/v) BC13 in methanol. The method, for different modifications of the procedure, is comparable t o the diazomethane method (see Table 4.2). 4.1.3. Methanol method: catalysis with hydrochloric or sulphuric acid The reaction scheme is similar to that given above (Scheme 4.3). If hydrogen chloride is used as a catalyst, the reaction can be accomplished in two ways, the first of which offers better yields of esters [9] : (i) a mixture of acids containing 5-6 mg of each acid is heated with 2 ml of 5% methanol-HC1 for 4 h at 5SoC in a water-bath; (ii) the same reaction mixture is refluxed for 4 h. When the reaction is finished, 1 ml of deionized water is HCL/H~SO& R-COOH

+

CH30H

i

R-COOCH3

+

H20

Scheme 4.3. TABLE 4.1 RECOVERIES OF UNSATURATED ESTERS AFTER TREATMENT OF METHYL ESTERS FOR 90 MIN AT 100°C WITH METHANOLIC BF3, HC1 OR HzSO4 151 ~~

Treatment

Controls 14%(w/v) BF3-CH30H (ca. 2 M ) 3 N HCl-CH30H 5%(v/v) HzSOq-CH30H (ca. 1.8 N )

No. of samples

31 16 12 16

Recovery (%)

18:l

18:2

18:3

100 t 3.4 95.9 f 2.1 95.6 i 3.2 95.2 i 4.6

100 * 3.5 91.7 i 3.6 92.4 i 3.3 91.8 f 4.5

100 i 3.8 91.8 i 6.3 90.5 * 3.4 90.8 5.5

*

Controls consisted of untreated esters, and esters mixed with each of the reagents without heat treatment, which were then extracted as usual.

ESTERS

51

TABLE 4.2

GC RESULTS FOR FATTY ACIDS METHYL ESTERS FOLLOWING VARIOUS ESTERIFICATION METHODS [ 7 ] Fatty acids: cis-9,10-methyleneoctadecanoicacid (Cyc C19 ) and heptadecanoic acid (C1 T ) , internal standard. Esterifcation: 1 mg of each acid. Results (range of 3 determinations): peak areas for fatty acid methyl esters relative to C I 7 taken as 1.00 Esterification method

Relative peak area CYCC19

Diazomethane, 30 min, 0°C Open tube, 0.5 min, 100°C: 14%(w/v) BF3 in CHjOH Open tube, 2 min, 100°C: 10%(w/v) BC13 in CH30H 14%(w/v) BF3 in CH3OH Closed tube, 5 min, 100°C: 10%(w/v) BCl3 in CH30H 14%(w/v) BF3 in CH30H

Other esters

0.99-1.01 0.45-0.59

0.14-0.15

0.93 - 1.OO 0.12-0.13

0.31-0.32

0.96-0.98 0.10-0.11

0.46-0.50

added to the mixture and the solution is extracted four times for 3 min each time with 2 ml of chloroform irl a 10-ml separating funnel. The extracts are dried with anhydrous sodium sulphate, filtered and diluted to 10 ml with chloroform. Concentrated sulphuric acid can be used for the same purpose as a mixture with absolute methanol (1 : 10). This procedure also requires a subsequent extraction. Esterification with HC1-methanol can also be carried out on an ion-exchange resin [12]. A 10-g amount of Amberlite IRA400 is stirred with 25 ml of 1 N sodium hydroxide solution for 5 min, the resin is allowed to sediment and the supernatant is decanted. After several-fold agitation of the resin with distilled water, three-fold agitation with 25 ml of anhydrous ethanol and three-fold agitation with 25 ml of light petroleum, a sample is dissolved in light petroleum and applied on an ion exchanger and, after stirring for 5 min, the resin is again washed with three 25-ml portions of light petroleum (boiling range, 30-70°C). After decanting, 25 ml of anhydrous HC1-methanol are added and the mixture is stirred for 25 min and filtered. The resin is then washed by stirring for 5 min with two portions of 15 ml of the reagent and the methanolic extracts are combined, diluted with 10 ml of distilled water and extracted with 50 ml of light petroleum. Extraction with 20 ml of light petroleum is subsequently twice and the extracts are washed with 50 ml of water until neutral, dried with anhydrous sodium sulphate and concentrated in a water-bath under a stream of dry nitrogen. The concentrate is then transferred into a 1-ml volumetric flask and aliquots are chromatographed. Vorbeck et al. [ I ] compared the above methylation methods, and some of their results are shown in Table 4.3. Methods requiring more complicated procedures give lower values of concentrations found. The losses caused by the volatility of methyl esters are most significant with lower acids, with unacceptable standard deviations. The diazomethane method gives good agreement of results for both lower and higher acids. Vorbeck et al.

58

DERIVATIVES AND THEIR PREPARATION

TABLE 4.3 COMPARISON OF YIELDS OBTAINED IN DIFFERENT METHODS OF METHY LATION [ 1 ] For butyric to caproic and for myristic to linoeic acids the standard deviations were 0.32, 26.5 and 13.8, and 0.25,0.77 and 0.52%with diazomethane, CH30H-HCl and CH30H-BF3, respectively Acid

Concentration eiven (wt.%)

Concentration found (wt.%) Diazomethane

CH30H-HCl

CHBOH-BF~

38.7 30.0 31.1 14.2 16.1 17.8 17.8 17.8

38.6 29.8 31.6 14.7 15.9 17.6 17.6 17.8

4 .O 14.8 29.6 13.8 15.7 18.7 18.7 17.8

30.1 24.4 30.4 13.9 15.6 18.0 18.0 17.8

-

Butyric Valeric Caproic Myristic Palmitic Stearic Oleic Linoleic

[ I ] further reported a small significance of side-reactions during the esterification of unsaturated acids with diazomethane. Double bonds are obviously only slightly polarized in a long chain, so that the possibility of the reaction occurring at the double bond will be negligible. 4.1.4. Decomposition of tetramethylammonium salts

One of the methods is based on the reaction shown in Scheme 4.4. A sample containing the acid is usually titrated with a methanolic solution of tetramethylammonium hydroxide using phenolphthalein as indicator. After adjustment of the volume, the solution is either injected directly into a GC injection port heated to 360400°C [ 181 or filled into a 3-1.11capillary, dried at 100°C and pyrolysed at a higher temperature. With direct injection of the solution of the salt the conversion depends strongly on the conditions used and, in order to secure acceptable reproducibility three main conditions should be observed: (i) the injection port temperature should be in the range 360-400°C; (ii) the injection port should be filled loosely with glass-wool; and (iii) piercing of the septum should be reproducible. The use of other solvents, e.g., chlorinated hydrocarbons, leads to inhibition of the methylation. The use of chloroform as a solvent for the methylation of bifunctional acids gives rise to the formation of a mixture of dimethyl and monomethyl esters (1 : 1). On the other hand, even with a 50%water content in the sample, the methylation is claimed to be complete (99.9%) [l 11. Some substrates can pyrolyse irreproducibly into a number of products at a high temperature of the injection port. The use of trimethylanilinium hydroxide with the injection port temperature being only 265°C [ 191 was therefore suggested. Dimethylaniline is R-COOH

+ + ( C 5 J 4 N OH

Scheme 4.4.

- t

-C

R-COO N(C%Ljh

A A

R-COOCH3

+

(CH313N

59

ESTERS

released more easily than trimethylamine from the quaternary salt. Decomposition of a quaternary trimethyl (a,a,a-trifluoro-m-tolyl)ammonium salt 1201 requires an even lower temperature (240°C). Differences in the basicities of individual reagents are also important and probably, as a result, the use of tetramethylammonium hydroxide causes almost complete decomposition of polyunsaturated fatty acids, whereas trimethyl(trifluorotoly1)ammonium hydroxide leads to only very mild decomposition. The addition of methyl propionate “neutralizes” the basicity of the reagent and losses of polyunsaturated fatty acids do not occur [21]. The method of decomposition of quaternary ammonium salts in the injection port (so-called flash-heater alkylation) is used even for the chromatographic analysis of substances that also contain other functional groups, such as -NH and -OH. The active hydrogen in these groups reacts with the reagent in a similar manner as in the case of the carboxyl group and the quaternary salt is pyrolysed into a corresponding alkyl derivative and an amine. For instance, barbiturates, alkaloids and xanthines [22], substituted ureas [23] and other substances [24] were chromatographed in this way. Neutral tetramethylammonium and trirnethylanilinium acetates were recommended as reagents for the derivatization of some compounds as they suppress the origin of by-products [25]. The decomposition of quaternary ammonium salts can also be achieved by the action of alkyl halides (Scheme 4.5). R-COOH

t

+ RiN OH

--

R-COO

-

+

NRi

K‘I A

+ R-COOR”+

-

RiN I

Scheme 4.5.

The reaction is carried out in a strongly polar solvent, e.g., in the following way. A 25-mg amount of stearic acid (0.088 mmol) is dissolved in 4 ml of N,N-dimethylacetamide and 0.95 ml of methanol, 0.05 ml of tetramethylammonium hydroxide (0.104 mmol) and 0.1 ml of iodobutahe (0.88 mmol) are added to the solution and the mixture is agitated vigorously. The yield exceeds 98%in 3-10 min, depending on the alkyl iodide used. Anhydrous conditions must obviously be ensured. The presence of 5% of water in the solvent results in prolongation of the reaction time to 1 h with the same yield. The sequence of reagents also must be maintained in the above order in order to suppress the decomposition of the alkyl iodide with the quaternary base to the smallest possible extent 1261. 4.1.5. Extractive alkylation

Extractive alkylation has a reaction scheme identical with that for the previous procedure. The substrate with a carboxyl group reacts in an aqueous solution with a quaternary base and is extracted in the form of an ion pair into a polar solvent of low solvation . capacity (dichloromethane) that contains alkyl halide. Low solvation of the anion of the acid and high solvation of the reaction product lead to increased reactivity of the anion and to a rapid reaction with the alkylation agent in the organic phase. Methyl iodide 1271 is used to prepare methyl esters and pentafluorobenzyl bromide 1281 is used for the preparation of esters providing a high ECD response.

60

DERIVATIVES AND THEIR PREPARATION

The course of the reaction is characterized by the following equilibrium:

and by an extraction constant, EQA, of the ionic pair given by

where [QA], is the concentration of the ionic pair in the organic phase and [Q'], and [A-], are the concentrations of the quaternary ammonium salt cation and acid anion, respectively, in the aqueous phase. The partition ratio of the substrate in the form of the ionic pair, DQA,can be expressed as

and the degree of extraction, P(%), is then given by

where Va and Vo are the volumes of the aqueous and organic phase, respectively. Equilibrium 1 is shifted to the right by the reaction of the ionic pair with alkyl halide. The degree of conversion (reaction time) depends on the degree of extraction and it is greater, the smaller is the ratio of the phase volumes (V,/Vo) and the larger is the partition ratio (DQA).It increases with increasing concentration of quateinary ammonium ion in the aqueous phase and with increasing extraction constant, which increases as the hydrophobicity of the quaternary anion increases. Consequently, for instance, tetrahexylammonium hydroxide is used for extractive akylation. Other possible equilibria in which the components present can participate, e.g., substrate hydrolysis, HA + A- t H', and possible dependence of the yield on the pH of the aqueous phase must obviously be taken into account. In addition to carboxyl groups, other groups containing active hydrogen can also be alkylated in this way [29]. 4.1.6. Other methods

Other methods are based on the reactions used in organic synthesis for the preparation of esters, which are modified for the purpose of GC. For example, esterification with an alkyl halide in the presence of potassium carbonate has been used (Scheme 4.6). 2 R-COOH

+ 2 CHjl + K 2 C 0 3

-

2 R-COOCH3t

2 K I + CO2

+

H20

Scheme 4.6.

For the analysis by GC, the reaction can be carried out in the following way: 1 ml of an acetone solution of the acid, containing ca. 1 mg of the acid, is pipetted into a roundbottomed flask and 2 ml of freshly distilled methyl iodide and about 50 mg of K2C03, dried in advance over P2OS at 170°C,are added. The mixture is refluxed for 30 min under a calcium trap and, after cooling, 1 pl of the solution is injected directly into the chromatograph [30,31].

ESTERS

61

Methyl iodide can be replaced with other alkylation agents, such as alkyl sulphates, chloromethyl ethers [30] and a-bromopentafluorotoluene [32]. By this reaction other groups containing active hydrogen are also alkylated, e.g., barbiturates [33]. With the aid of a micro-refluxer (see Fig. 2.6) the above reaction can be carried out on the micro-scale with volumes of units to tens of microlitres [34]. Taking into account the necessity for refluxing, the method is relatively time consuming, particularly if subsequent extraction of the reaction products is required. R-0 R-COOH

+

\

CH-NICH3J2

/

-

--JR'%HI

R-COOR'+

(CH3)2N-CH=0

RAJ

Scheme 4.7.

Esterification with the aid of N,N-dimethylformamide dialkylacetals was described by Thenot et al. [35] (Scheme 4.7). The reaction is carried out by heating the reaction mixture in pyridine at 6OoC for 10-15 min, or the derivatives can also be prepared on the column by injecting the components of the reaction mixture simultaneously by means of the same syringe. R' can be selected according to the particular requirements, and the quantitative yield of the reaction is said to be adequate. +R-OH

R,-COOH

+ RN=C=NR

A

RN=C-NHR

R,-C

Scheme 4.8.

0-0

2R,-COOR2

+ RNH-C-NHR

I1 0

l

Esterification with alcohols in the presence of pyridine was used by Felder et al. [36]. Water produced in the reaction mixture is bound by the addition of N,"-dicyclohexylcarbodiimide (Scheme 4.8). The acid (ca. 10 mequiv.) is dissolved in 25 ml of alcohol and 4 ml of pyridine and an excess of dicyclohexylcarbodiimide (12 mequiv.) are added (even more if the sample is not dry). The amount of pyridine can also be larger and it then acts as a solvent, e.g., if the alcohol used is solid (menthol). The mixture is then stirred at room temperature; only with some higher alcohols or acids must the mixture be heated at 4O-8O0C for 30-120 min. If a precipitate of N,N'-dicyclohexylurea is produced in the reaction, it is allowed to sediment and, after adding an internal standard, 0.5 1.11 of the pure solution is injected into the chromatograph. A rapid method for esterification using a mild agent was described by KO and Royer [37]. A substrate containing a carboxyl group reacts with N,N'-carbonyldiimidazole, and the acyl imidazolide produced in this way is decomposed with alcohol and the appropriate ester is produced (Scheme 4.9). The reaction proceeds very rapidly even at room temperature and is completed within several minutes. In the course of this procedure no transesterification of the esters occurs, e.g., triglycerides or cholesteryl esters. The method R-COOH

t

N=\ N -;-",J,I

,c=N 0

-

FN R C O - Nd

I R-COOR'

Scheme 4.9.

t

&OH

+

+

NL-

H

+

Co2

62

DERIVATIVES AND THEIR PREPARATION

also makes possible the preparation of any ester, depending on the alcohol used. If the carboxyl group has already been blocked and the derivative is not volatile, conversion into a volatile ester must be carried out prior to the chromatographic determination. This can be accomplished by saponification with an alkali, with subsequent esterification by one of the above methods or by transesterification. Some stronger esterification agents can be used at a higher catalyst concentration even for the transesterification (methanol-HC1, methanol-BF3). However, other direct procedures have aiso been elaborated. Transesterification with a “2 N methanolic base” is common. The base is prepared by dissolving an appropriate amount of sodium or potassium hydroxide in dry methanol. About a 10%ethereal extract of fats is mixed with this reagent in a 1 : 19 ratio. Withn 5 min the reaction mixture can reputedly be injected into a chromatograph without any further treatment [38]. Mason and co-workers [39,40] developed a method for the transesterification of fatty acids in triglycerides in the presence of 2,2-dimethoxypropane. To 35 pmol of the triglyceride in a 25 ml erlenmeyer flask 10 ml of benzene, 4 ml of dimethoxypropane, 5 ml of methanol and 1 ml of 2 N sodium methanolate are added. After mixing, the mixture is allowed to stand for 5 min and an amount of methanolic HCl is then added such that about a 0.3 mmol excess remains. The mixture is again stirred, allowed to stand for 50 min, about 1.5 g of a solid neutralizing agent is added and it is stirred for 30 min. The precipitate is allowed to sediment, the supernatant is decanted into a 25-ml volumetric flask and the volume is made up with methanol, with which the precipitate was washed. Aliquots are injected into the chromatograph. The total amounts of fatty acids in different fats and oils were determined by Mason and co-workers with an error of *3%. Glycerol is determined simultaneously as isopropylideneglycerol. The tendency of 2,2-dimethoxypropane to polymerize in the acidic medium of the reaction mixture is suppressed by adding 1% of dimethyl sulphoxide [41]. A comparison of the above esterification methods from the viewpoint of their reliability in the GC analysis of substances containing carboxyl groups is made difficult as a consequence of the variety of the substrates under analysis and different approaches to the problem taken by individual workers. In addition to the paper already cited [ I ] , other comparative studies [42-44] can be found that are in agreement with the opinion that all of the methods are approximately equally precise and function comparably well in solving the problems for which they were proposed. The differences in the reactivities of individual agents require differences in reaction times in order that quantitative yields may be obtained. Table 4.4, from a paper by Churric‘ek et al. [42], shows the minimal reaction times required for the quantitative esterification of various acids with diazomethane and BF3-methanol. In spite of the great differences in the properties ot’individual substrates, the reaction times are reasonable for practical purposes. A remarkable effect is the possibility of speeding up esterification with diazomethane by adding 10%of methanol to the reaction mixture. Some of the limitations of individual methods have already been mentioned. Further possible reaction centres in the molecule of the substrate (double bonds, carbonyl and other functional groups) must always be taken into consideration and the esterification procedure must be selected such that side-reactions take place to the smallest possible extent.

63

ESTERS TABLE 4.4 MINIMAL REACTION TIMES NECESSARY FOR QUANTITATIVE ESTERIFICATION [42 1 Acid

Propionic n-Caprylic Lauric Palmitic Benzoic p-Toluic Phthalic Isophthalic Terephthalic

Reaction time Esterification with methanol + 11% BF3

Esterification with 100%excess of diazomethane in diethyl ether

Esterification with 100%excess of diazomethane with 10%methanol

3min* 5min* 5min* 34 min * 4 min ** 75 min ** 5 min ** 12 min ** 5 0 min **

12 sec

10 sec 15 sec 30 sec 2 rnin 12 see 8 sec 9 sec 5 min 5 min ***

30 min 30 min 1 min 5 rnin 5 min 20 min 30 min

* Reaction temperature 60°C. ** *** Reaction temperature 100°C.

300% excess of diazomethane.

4.1.7. Higher esters

These are prepared by methods similar to those for methyl esters. Their use in GC is necessitated by the fact that methyl esters of compounds containing carboxyl groups are sometimes too volatile. This can lead to losses during treatment and to erroneous results. There have been described, for example, propyl [45], isopropyl and butyl esters [46] prepared by esterification with alcohols with the addition of BF3 or by catalysis with hydrogen chloride [47] or sulphuric acid [48]. The preparation of these esters has also been achieved by reaction with an appropriate diazoalkane and the addition of 0.007% of BF3 [49]. Benzyl esters, often used for short-chain acids, are usually prepared by reaction with phenyldiazomethane [50-521. Substituted benzyl esters are mostly prepared by reactions of alkali metal salts with halogen derivatives [53,54]. When using selective detectors, carboxyl groups are converted into halogen-containing esters. 2-Chloroethyl esters [55], trichloroethyl esters [56] and hexafluoroisopropyl esters [57] are prepared by an acid-catalysed reaction with an appropriate alcohol. Pentafluorobenzyl esters have already been mentioned (see p. 59). Enantiomers can be separated by GC after converting a carboxyl group into an L-menthyl ester [58]. The acid is converted into chloride by refluxing with freshly distilled thionyl chloride (S0Cl2). Chloride is esterified with menthol in the presence of pyridine. Anhydrous conditions must be maintained. Pettitt and Stouffer [59] described the use of isopropyl esters, prepared by reaction with 2-bromopropane and sodium hydride, for the GC of amino acids. The above reagent also reacts with other functional groups, which can be of practical significance for the

64

DERIVATIVES AND THEIR PREPARATION

derivatization of compounds containing various functional groups in their molecules (Scheme 4.10).

7

/--

( C H3 12C H B r

-c00cH[cH3’2 -NHC H(C H312

-S C H(C H3I2 -OCHICH3)2

Scheme 4.10.

In a reaction flask closed with a septum, 1-10 mg of the substrate are dissolved in 2 ml of dry dimethyl sulphoxide. An excess of sodium hydride is washed four times with 5 ml of dry n-hexane in another flask so that oil is removed and is added to the substrate. An excess of 2-bromopropane is added to the mixture and the flask is closed and allowed to stand overnight. After adding 3 ml of saturated NaCl solution the mixture is extracted with either benzene or chloroform, depending on the detector used. Prior to injection the extract is washed with saturated NaCl solution and dried with anhydrous sodium sulphate. In spite of the possibility of converting different functional groups in one step, this reaction has not found widespread use in GC analysis, obviously owing to the complexity of the procedure and the necessity of maintaining strictly anhydrous conditions. The GC separation of esters and the selection of the stationary phase represent problems of varying degrees of complexity in individual cases. An acceptable separation can usually be obtained on different polyester stationary phases (EGA, butanediol succinate polyester, EGS, etc.), Carbowax-type phases, OV-17, OV-225 and SE-30. Non-selective and non-specific stationary phases are preferred, The supports should not be acidic, and they are sometimes modified by silanization. In particular instances, when other groups are also derivatized prior to the analysis the selection of the stationary phase may be very difficult. These problems are discussed for individual types of compounds in Chapter 5.

4.2. ETHERS

Ethers are used for blocking hydroxyl groups. However, apart from TMS ethers, which will be discussed separately, they have not been of great significance and they are mostly used in special cases. Hydroxyl groups of polyhydroxy compounds with higher molecular weights (e.g., sugars, sterols) are converted into ethers by reaction with methyl iodide in the presence of silver oxide in dimethylformamide [60] (Scheme 4.1 1). 2 R 4 H + 2C%I

+ Ag20

2R-OCH3

t

2 Agl

+

H20

Scheme 4.11.

To etherify sterols, it is recommended that the reaction is carried out in diethyl ether in the presence of potassium twt.-butanolate. On the micro-scale the yields range from 78 to 90% [61]. Derivatives for the trace analysis of hydroxy compounds are prepared in a similar manner to esters (see p. 61) by reaction with a-bromopentafluorotoluene catalysed with potassium carbonate [62] (Scheme 4.12). The phenolic substrate is dissolved

ETHERS

65 F

F

F

F

Scheme 4.12.

in a 20-fold excess of acetone and heated with halide in the presence of K,C03. The yields range from 84 to 100%. A high ECD response is also provided by 2,4-dinitrophenyl ethers, for which various methods of preparation were reported by Cohen et al. [63]. A 4-ml volume of acetone containing phenols (ca. 10 pg), 0.1 ml of a saturated methanolic solution of sodium methanolate and 1 ml of I-fluoro-2,4-dinitrobenzenein acetone (l%, w/v) were refluxed in a 10-ml flask for 30 min. The reaction mixture was then added to 25 ml of sodium hydroxide solution ( 2 3 6 , w/v), diluted with a small volume of water and extracted with 25 ml of chloroform. After being dried with anhydrous sodium sulphate, the extract was carefully evaporated and the residue was dissolved in acetone and injected into the chromatograph. Another method starts from an aqueous solution, the mixture being agitated in a separating funnel and finally extracted with n-hexane. The reaction can also be carried out in such a way that an acetone solution of the substrate is applied on a thin layer or TABLE 4.5 COMPARISON OF THE YIELDS OF THE METHODS FOR THE PREPARATION OF 2,4-DINITROPHENYL ETHERS, THEIR RETENTION TIMES AND ECD SENSITIVITIES [63 j Procedures: (1) refluxing in acetone solvent; (2) cold aqueous reaction; (3) sandwiched layer reaction, chromatographic plate coated with Kieselguhr G; (4) sandwiched layer reaction, silica gel-loaded paper SG 81. Conditions: Glass column (140 cm X 1.5 mm I.D.) packed with 1%GE XE-60 and 0.1% Epikote 1001 on Chromosorb G, acid washed, dimethylchlorosilane coated, 60-80 mesh. Temperature: 215°C Parent phenol

pK,

Yield (%) 1

Phenol 4-Fluorophenol 4CNorophenol 4-Bromophenol 4-Iodophenol 2,4-Dichlorophenol 4-Nitr ophenol 1-Naphthol 2-Naphthol 4-Benzylphenol

10.0 9.88 9.42 9.34 9.10 7.82 7.20

6 5

23 38 61 34 54

2

3

4

15 25 20 19 20 0 0

4 10 41 62 91 32 100

51 46 69 63 76 51 100

Relative retention time

Sensitivity (g * 1091

16 19 38 56 92 58 238 100 (8.2 min) ** 141 285

0.10 0.10 0.10 0.10 0.10 0.10 0.50 0.20

*

0.20 0.40

Sensitivity expressed as the weight of derivative producing a peak with height equivalent to 10% full-scale deflection at an amplification producing a noise level of 5% f.s.d. ** Absolute retention time.

66

DERIVATIVES AND THEIR PREPARATION

on paper and the reagents are applied gradually in the form of sprays. The layer (the paper), fixed between two glass plates, is then heated for 40 min at 190°C and, after cooling, a portion of the layer is extracted. It is obvious from Table 4.5 that the yields fluctuate widely, depending on both the method and type of phenol. The derivatives are very stable, but their elution requires a high temperature, particularly for polyfunctional compounds, which can adversely affect the functioning of the detector. The sensitivity of the analysis with ECD detection is high, however, and permits the very sensitive analysis of traces of phenols. The possibilities of using the method even in combination with TLC for the identification of phenolic compounds are considerable.

4.3. ACYL DERIVATIVES Acyl derivatives are common derivatives of hydroxy, amino and thiol groups (Scheme 4.13). R-c-co R' ( R'-CO)20

R-SH

2

\

-

L

R-NH-COR' R-S-COR'

Scheme 4.13.

As they eliminate unfavourable properties of the above groups these derivatives are used in the GC of amines, phenols and substances containing several of these functional groups. The derivatives are usually less polar than the original substances and, with the replacement of active hydrogen in the groups, their tendency to form hydrogen bonds decreases substantially and their volatility increases so that even non-volatile or thermally unstable compounds can be chromatographed. Specific characteristics necessary for detection with an ECD are introduced into the molecule with the aid of halogenated reagents. The preparation of acyl derivatives is easy in most instances and consists in the reaction of an excess of an acylating reagent (usually anhydride of the corresponding acid) in pyridine, tetrahydrofuran or another solvent capable of binding the acid produced [64,65]. The reaction time and temperature depend on the properties of the substrate and the reactivity of reagent used, and can vary in the range from 15 min to 1 h and from room temperature to boiling point [66]. The amount and type of the solvent used often have a substantial influence on the reaction yield and, with polyfunctional compounds, even on the degree of acylation and the proportions of individual products [67]. The derivatives are also sensitive to moisture and therefore it is desirable to maintain anhydrous conditions even if traces of water can be removed with the reagent. The reaction mixture is then injected into the chromatograph directly or is concentrated in various ways and the concentrate so obtained is injected. This common procedure can vary substantially in individual cases and the methods used for particular purposes are therefore described in more detail in Chapter 5 . A direct method is known for the preparation of acyl derivatives on the column [ 6 8 ] . The injection of the anhydride follows the injection of the substrate and the peak of the derivative will then appear in the chromatogram. The time period between the injections

61

ACY L DERIVATIVES

affects the retention times of the derivatives. The method is useful for the characterization and identification of substances by the shift of the peak after conversion into a derivative, the so-called “peak shift technique”. In principle, all of the reagents known from organic synthesis as acylating reagents can be used; however, acid anhydrides are used in most instances. The use of other reagents is motivated by efforts to eliminate acidic media, which can decompose the derivatives. Acyl imidazoles react with basic groups according to Scheme 4.9 on p. 61. The imidazole by-product is relatively inert and does not cause decomposition of the derivatives [69]. As with the use of ethyl trifluoroacetate as an acylating agent, the unfavourable properties of trifluoroacetic acid produced are eliminated [70]. With the addition of hexamethylenediamine the reaction proceeds rapidly at 60-70°C and the yield is about 70%. If dry ammonia is added to the reaction mixture, the reaction proceeds quantitatively. Ammonia probably neutralizes the residues of trifluoroacetic acid produced by the hydrolysis of the ester and in this way eliminates a competitive acylation reaction. The acylation of -NH2, -OH and -SH groups with bis(acetylamines), e.g., N-methylbis(trifluoroacetamide), proceeds under mild conditions and does not require an acidic medium [71] (Scheme 4.14).

R--OH

+ CF3-CO-N

----c

R--O-CO-CF3

+

CF3CO-NH-CH3

\

Scheme 4.14.

CO-C

F3

Chromatographic conditions can be selected so that the by-product is eluted at the beginning of the chromatogram as a symmetrical peak which does not overlap those of lower derivatives. The excess of the reagent need not then be removed. As it is liquid at room temperature, another solvent need not be utilized and the excess of the reagent protects the derivatives against hydrolysis. Selective acylation of amino groups in the presence of -OH and -COOH groups is achieved after prior trimethylsilylation of the latter groups with a mild silylating agent. Although the common availability of acetic anhydride as an acylating agent has resulted in the widespread use of acetyl derivatives for blocking hydroxyl and amino groups of compounds and for increasing their volatility in GC analysis, it is halogenated acetyl derivatives that are the most significant. They are frequently applied in trace analysis because of their generally high ECD responses. Of haloacetates the most frequently used is the trifluoro derivative, despite its having the lowest response of such derivatives. Landowne and Lipsky [72] arranged haloacetates according to increasing ECD response in the order trifluoroacetyl < trichloroacetyl < bromoacetyl < chloroacetyl. This sequence was also confirmed by McCallum and Armstrong [73]. Monochloroacetates are, however, not commonly used, amongst other reasons, because of their disadvantageous chromatographic properties, such as asymmetric peaks [74]. Perfluorinated acyl derivatives derived from higher acids, such as propionic, butyric and benzoic acids, usually have even higher ECD responses and make possible more sensitive analyses. The last in the series, pentafluorobenzoate, has the highest response, as is obvious from Tablc 4.6, taken from the paper by McCallum and Armstrong [73], who compared the responses of seven thymol derivatives: 2,4-dinitrophenyl and pentafluorobenzyl ethers, heptafluorobutyryl (HFB),

68

DERIVATIVES AND THEIR PREPARATION

TABLE 4.6 RELATIVE SENSITIVITY OF THE ELECTRONCAPTURE DETECTOR TOWARDS DIFFERENT DERIVATIVES OF THYMOL [73] Conditions: 1 m x 2 mm I.D. glass column; 1% SE-52 on Diatoport S ; nitrogen caner gas, flow-rate 15-20 ml/min. Derivative

Column temperature C)

Retention time (min)

Relative sensitivity

70 70 100

1 .o 1.3

100 150 150

2.7 1.9 3.1 1.2 5.8 1.7 9.8

7. 5.9 6.9 0.3

120

3.0

7 . 1 0 4 **

e

Heptafluorobutyrate Pentafluoropropionate Monochloroacetate Monofluoroacetate Pentafluorobenzyl ether Pentafluorobenzoate 2,4-Dinitrophenyl ether Free thymol detected With FID

100

0.3

Relative to heptafluorobutyrate. ** Relative to the ECD response of thymol heptafluorobutyrate.

pentafluoropropionyl, chloroacetyl, fluoroacetyl and pentafluorobenzoyl derivatives. The most sensitive analysis was achieved with pentafluorobenzoate, which permits up to lo-'' g of thymol to be determined. Clarke et al. [74] compared the characteristics and ECDresponses of acyl derivatives of amines. Table 4.7 shows some of their results. It follows that a different type of acyl derivative is suitable for each type of amine and at the same time other characteristics, which are not reported in the table, must also be taken into consideration. TFA derivatives have better chromatographic characteristics than chloroacetates and usually are preferred, despite their lower responses. The highest sensitivity was obtained by Clarke et al. for HFB derivatives. Other workers [75] drew attention to the dependence of the responses of haloacyl derivatives on the detector ternperature. Its significance can be particularly important in trace analysis, when it is necessary to work at the maximal sensitivity of the detector. In each instance when an acyl derivative is used a compromise must be found among the sensitivity required, volatility of the derivatives, reagent availability and other factors. To block amino and hydroxyl groups of thyroid hormones, N,O-dipivalyl derivatives were used [76]. After esterifying the carboxyl group, 0.5-1 mg of ester is heated for 30 min with 0.2 ml of pivalyl anhydride and 0.05 ml of triethylamine. Both amino and hydroxyl groups are converted into the pivalyl derivative (Scheme 4.1 5). These derivatives are superior to others with respect to their ease of preparation, stability even against the action of moisture and particularly their thermal stability. This makes further prelim-0 H -N

H2

Scheme 4.15.

[(CH313C-C0]20

-0-C

0-C( C H3J3

-NH-

CO-C(C H313

69

SILYL DERIVATIVES TABLE 4.7 COMPARISON OF THE CHROMATOGRAPHIC PROPERTIES OF SOME ACYL DERIVATIVES OF AMINES 1741 Conditions: (A) 6 ft. x 4 mm I.D. glass column; 6%QF-1 on Anakrom ABS, 60-70 mesh; column temperature, 152°C; carrier gas (nitrogen) flow-rate, 30 ml/min; (B) column as in A; temperature 155°C; carrier gas (nitrogen) flow-rate, 80 ml/min. Amine

Derivative

Conditions

Retention time (min)

Peak shape

Benzylamine

Acetyl Monochloroacetyl

B

2.5

Asymm.

B

3.0

Slightly asymm.

Trifluoroacetyl Pentafluoropropionyl Heptafluorobutyryl a-Methylbenzylamine

Acetyl Monochloroacetyl Trifluoroacetyl Pentafluoropropionyl Heptafluorbbutyryl

Sensitivity of determination

*

0.04 30 0.8

A

1.6

Symm.

A

2.2

Symm.

229

A

2.3

Symm.

715

B

2.5

Asymm.

B

2.8

slightly asymm.

0.16 32

A

2.2

Symm.

0.5

A

1.9

Symm.

19.1

A

2.2

Symm.

563

~

Expressed as peak area (mm2) per

mol of the compound.

inary operations possible, e.g., purification on a thin layer. Problems with the selection of the stationary phase for the GC of acyl derivatives are similar to those associated with the GC of esters. In simple separations silicone and polyester phases are satisfactory but more complicated separations require special phases, low coatings, mixed phases, etc. Individual cases are discussed for specific types of compounds in Chapter 5.

4.4. SILYL DERIVATlVES

These derivatives are probably the most commonly used in the GC of non-volatile substances and for blocking their functional groups. Trimethylsilyl derivatives can be prepared by the reaction of trimethylsilylating agent with groups containing active hydrogen (Scheme 4.1 6). If the enolized form of the carbonyl group is added to this range of func-

DERIVATIVES AND THEIR PREPARATION

70 -OH -COOH

7

-\

7-O-Si(CH313

/-

-COO-SilCH3)3

S i I C H313 / -NH-Si1CH3j3

=NH

-N Sl(Cty3

Scheme 4.16.

tional groups, almost all of the groups that could interfere in GC analysis because of their polarity are included. The advantage of these derivatives is evident with compounds that have different functional groups in the molecule: all groups are converted into the derivative in a one-step reaction. However, it should be noted that silyl derivatives have not always been successful and are not such ideal deiivatives as was originally expected. Many methods for the preparation of TMS derivatives have been developed. Pierce [77] reported a number of methods and their modifications depending on the type of substrate to be silylated. Commercial reagents prepared for immediate use are available. Mixtures of reagents with solvents are supplied for methods elaborated for individual substrates. Individual types of reagents can be classified into four groups (see Table 4.8): (i) trimethylchlorodisilane, pure or with an acceptor of the acid or with a catalyst; (ii) hexamethylsilazane, mostly with addition of TCMS as a catalyst; (iii) silylamines, such as trimethylsilyldiethylamine and trimethylsilylimidazole; (iv) silylamides and others; N,O-bis(trimethylsilyl)acetamide,N,O-bis(trimethylsily1)trifluoroacetamide and hexamethyldisiloxane are often used. A relatively mild reagent is HMDS with the addition of TMCS, used for the silylation of hydroxyl groups [78] ;stronger reagents such as BSA [79] and BSTFA [80] are used for the silylation of less reactive groups (-NH,,-NH-) and of sterically hindered groups. Piekos et al. [81] described the use of N-trimethylsilylacetanilide and its p-ethoxy derivative, which are inexpensive and have favourable GC properties. Birkofer and Donike [82-841 tested a number of other similar reagents, such as N-methyl-N-trirnethylsilylacetamide, N-methyl-N-trimethylsilyltrifluoroacetamide and N,N,N’,N’-tetrakis-TMS-1 , n-diaminoalkanes. Methylamides have symmetrical peaks and shorter retention times than their bis-TMS analogues. The same applies to N-methyltrifluoroacetamide, the by-product from silylation with MSTFA, so that derivatives with shorter retention times are not overlapped in the chromatogram (see Table 4.9). Pyridine or another solvent with a large solvation capacity (acetonitrile, dimethylformamide) are mostly used as solvents in the silylation reactions. Pyridine provides on some phases a broad tailing peak and can overlap lower components. Lehrfeld [85] therefore developed a procedure for the removal of pyridine from the sample before the analysis. During the derivatization anhydrous conditions are essential because the derivatives are decomposed by traces of water. However, a method has been described for the preparation of silyl derivatives even in the presence of water; its principle consists in the addition of such a large excess of the silylating agent that all of the water present is removed [86]. This can be of importance in the treatment of samples that cannot be previously dried as losses of more volatile components could occur. The extent to which the presence of water affects the reaction yield and whether or not a large excess of by-products has an adverse effect must be tested, however.

SILYL DERIVATIVES

71

TABLE 4.8 SURVEY OF SOME SILYLATING REAGENTS Name Trimethyichlorosilane Hexamethyldisilazane

Formula

Abbreviation

(CH,),St-CI

TMCS

(CH3),St -NH-St

HDMS

(CH,),

N-A1kylhexameth yldisilazane Hexamethyldisiloxane

HMDSO

Trimeth ylsilyldieth ylamine

TMSDEA

Trimethylsilylimidazole

TMSIM

(CH3),Si,

Tetrakis-TMSdiaminoalkanes

,N-(CH

“

[CH3),Si

N-Methyl-N-TMS-acetamide

CH,-CO-N’

,s(

(CHA

‘S

(CHJ,

1-N

CH3

MSA

\SI(CH,), ,N-Si(CH-,I,

N,O-Bis-TMS-acetamide

cy-c

N-Methyl-N-TMS-tritluoroacetamide

CF3-CO-N/CH3 \SI cH$3

N,O,Bis-TMS-trifluoroacetamide

CF3-C

BSA

b S t (CH3),

MSTFA

/ N-S(Cy3

\O-Sh

BSTFA

(CH,),

TABLE 4.9 COMPARISON OF RELATIVE RETENTION TIMES OF SILYLAMIDES I83 J Compound

3.8% SE-30

10% SE-52

5% Apiezon L

MSTFA BSTFA MSA BSA N-Methyltrifluoroacetamide TMS ester of butyric acid

0.45 1.oo 1.45 1.89 0.23 1.oo

0.53 0.83 1.57 1.64 0.47 1.oo

0.45 0.67 0.89 1.21 0.33

1.00

DERIVATIVES AND THEIR PREPARATION

72

Silylation is usually carried out in reaction vessels closed with stoppers made of silicone rubber. The reagent is added and the sample is taken for the injection via a septum by means of a syringe. Because of the possible sensitivity of the derivatives towards moisture, they should be prepared immediately prior to the analysis, even though they have been reported to be stable under anhydrous conditions for a few days [87]. A method has been described for the direct preparation of silyl derivatives on the column [88]. After injecting the sample, the silylating agent is injected; the conditions are adjusted so that the sample loses any water and alcohol present before coming into contact with the reagent. The derivatives so formed than proceed along the column and are separated. McCugan and Howsam [89] elaborated this principle and described an apparatus for the derivatization of substances on a trapping column packed with a chromatographic material (see Fig. 4.2). The substance enters the trapping loop, cooled to a low temperature, either directly from injection port B or from the effluent from the first column. The silylating reagent is charged through injection port B and the loop is closed. After heating the loop, the derivatives are chromatographed on the second column. Rasmussen [90] introduced solid plant material directly into the injection port heated at 300°C. Cannabinoids distilled at this temperature and were trapped on a column kept at 40°C. After injection of the silylating agent, the derivatives were chromatographed with temperature programming. Rasmussen achieved good reproducibility of peak heights and retention times with quantitative yields. Selection of the stationary phase for the separation of silyl derivatives is mostly not critical. Various stationary phases have been used, but preferred ones are non-selective and non-polar phases of the silicone oil type. The support is usually deactivated by washing with an acid and by silanization, which results in an increase in the separation effi-

Insulated section

-

I / 16-in.union

Column no 1

..... ..... ..... ..... .... ..... Trap .... .... ..... .. ...... ... ..... ... ..... ...

To GLC detector

INJECTION PORTS

f

To GLC detector or moss Spectrometer

Fig. 4.2. Schematic diagram of gas-liquid chromatograph and trap for derivatization. V 1 and Vz are Carle microvolume valves. (Reproduced from J. Chrornatogr., 82 (1973) 370 (891, by courtesy of W.A.McGugan.)

SILYL DERIVATIVES

73

ciency of the column [91]. The packing must not be acidic, otherwise decomposition of derivatives will occur. Decomposition can even occur on contact with metallic parts of the apparatus and therefore an all-glass apparatus is sometimes recommended; the column may be made of glass or stainless steel. Jansen and Baglan [92] investigated the decomposition and deposition of some TMS derivatives on a chromatographic column. The amount of the derivative that was injected into the chromatograph and the proportion of it that was eluted were determined by measuring the radioactivity of substrates labelled with 14C. The results reported (Table 4.10) are not very encouraging for quantitative determinations. Most of the decomposition products were trapped in the front part of the column (in about the first tenth of it). Using an FID, one must take account of the fact that an aerosol of silicone dioxide, produced by the decomposition of silyl derivatives in the flame, is deposited on the electrodes. The deposit can decrease the sensitivity of the detector or change the response factors. This effect is suppressed if BSTFA is used for the silylation; a more volatile silicone tetrafluoride is then produced. Of other silyl derivatives, trialkylsilyl derivatives [93], particularly for GC-MS, and dimethylsilyl derivatives [94] have been described. They are prepared by similar methods to TMS derivatives. DMS derivatives are more volatile and have shorter retention times than TMS derivatives (see Table 4.1 l), but they are less stable; they are suitable for com-

TABLE 4.10 RECOVERY OF TMS DERIVATIVES OF VARIOUS 14C-LABELLEDCOMPOUNDS [92] TMS derivative

Stationary phase

Column temperature ("0

Counts/min

Recovery (%)

Injected

Recovered

Cholesterol

SF 96-50

250

12,000 12,300 15,200 15,200 15,200

5 300 5500 7200 8200 8500

43 45 47 54 56

Glycerol

Carbowax

135

6800 6800 6800

4900 5900 5700

72 87 84

Stearyl alcohol

Carbowax

200

8900 8900 8900 8900 8900

2500 2700 2100 2000 2700

28 30 24 23 30

Glucose

SF 96-50

225

187,000 232,000 278,000

44,500 55,200 74,500

24 24 27

Fructose

SF 96-50

225

7800 7800 7800

2000 2800 2100

26 36 27

74

DERIVATIVES AND THEIR PREPARATION

TABLE 4.11 COMPARISON OF RETENTION TIMES OF SOME HIGHER ALCOHOLS, PHENOLS AND THEIR DMS AND TMS DERIVATIVES [ 94 ] Conditions: 6 ft. x 4 mm I.D. glass column; 15% Apiezon L on CasChrom P, 100-120 mesh; 120°C; carrier gas (nitrogen) flow-rate, 60 ml/min. Retention times are given relative to hexadecanol and its derivatives and phenol derivatives, respectively. Values in parentheses are absolute retention times Compound studied

Relative retention time Parent compound

DMS derivative

TMS derivative ~

Dodecanol Tetradecanol Hexadecanol

0.17 0.42 1.oo (14.0 min)

0.19 0.43 1.oo (8.9 min) 2.29 1.00 (8.5 min) 1.73 1.98 1.98

Octadecanol Phenol oCresol mCresol pCresol

0.19 0.44 1.oo (10.4 min) 2.28 1.oo (11.2 min) 1.74 1.98 1.98

pounds of high molecular weight. Halomethyldimethylsilyl derivatives, on the other hand, have higher retentions than TMS derivatives (Table 4.12) [95,96]. They often permit the separation of compounds which, after conversion into their TMS derivatives, overlap in

TABLE 4.12 COMPARISON OF RETENTION TIMES OF TRIMETHYLSILYL (TMS) ETHERS AND CHLOROMETHYLDIMETHYLSILYL (CDMS) ETHERS OF C19 AND Cz 1 STEROIDS [95 ] Steroid

Retention time (min) TMS ethers

Androsterone Etiocholanolone Dehydroepiandrosterone Pregnanediol 5-Pregnene-3,2O-diol 5-Pregnene-3,20-diol Cholestane

CDMS ethers

(1)

(2)

(3) *

8.7 10.3 11.8 8.3 9.3 8.7 8.9

8.7 9.2 10.5 9.2 9.4 8.4 9.3

6.7 7.6 8.9 18.7 22.6 20.1 2 -0

* Conditions: (1) 5-ft. column; 1%XE-60 on Gas-Chrom Q, 100-120 mesh; 198°C; carrier gas (nitrogen) flowrate, 35 ml/min. (2) 2% QF-1; other conditions as in (1). (3) 1%XE-60; 215°C; carrier gas (nitrogen) flow-rate, 60 ml/min.

OXIMES AND HYDRAZONES

75

the chromatogram. The increase in ECD response is significant only with brominated and iodinated derivatives. Flophemesyl (pentafluorophenyldimethylsilyl) derivatives were developed for sensitive detection with the ECD [97].

4.5. OXIMES AND HYDRAZONES Usually the presence of a carbonyl group in a sample compound does not give rise t o serious difficulties in GC analysis. However, sometimes it can be the reason for instability of the compounds. Owing to its polarity and strong interactions with the support, it can cause peak asymmetry and, in more complex samples, the peaks of carbonyl compounds in the chromatogram can be overlapped by those of interfering components. In these instances the carbonyl group must be converted into an inert derivative. Suitable properties of the derivatives are often utilized also for the preliminary isolation of carbonyl compounds from other components of the sample [98]. R-O-NY

+

IR1 0% \ R2

-

R-0-N=C

/R' \

+

H20

R2

Scheme 4.17.

Oximes have been used for this purpose (Scheme 4.17). They are usually prepared by the reaction of the reagent (hydroxylammonium, methoxylammonium or benzylammonium chloride) with the compound containing a carbonyl group in pyridine. The reaction is allowed to proceed either at room temperature for 20 h [99] or the reaction mixture is heated at 60-100°C and the reaction is then completed within 15 min [loo]. Pyridine is removed from the sample by heating or stripping in a stream of nitrogen and the residue is dissolved in some other solvent (ethyl acetate). The sample is injected into the chromatograph or other groups present are blocked, e.g., by silylation. Oximes as such (R = H> are rarely used. At high temperatures of the injection port aldoximes are not stable and decompose into the corresponding nitriles. If such a decomposition is to be avoided, glass apparatus is used [98]. Lohr and Warren [ l o l l drew attention to the fact that at 250°C the decomposition is complete and oximes can be chromatographed reproducibly in the form of nitriles. Methoximes have gained the widest application, particularly for blocking labile keto groups in the molecules of compounds of high molecular weight [99,100]. On comparison with the original compounds, methoximes are more stable, d o not decompose during the analysis and can be modified chemically, depending on other interfering groups present, e.g., by silylation. Higher oximes, such as butyloximes, pentyloximes and benzyloximes, of e.g., steroids, have sufficiently short retention times to be eluted within reasonable time intervals at about 200°C. The molecular weights of the derivatives lie in a range very suitable for mass spectrometry and characteristic mass spectra can easily be interpreted [ 1021. Pentafluorobenzyloxime hydrochloride condenses with carbonyl groups and the resulting derivative has a high ECD response and also enables the carbonyl compound to be separated preliminarily from the sample [103]. The other advantage of this reagent is the higher

DERWATIVES AND THEIR PREPARATION

16

thermal stability of the resulting oxime. The excess of the reagent can easily be removed by washing the sample with an acid prior to injection into the chromatograph, which is of considerable importance if an ECD is used. The bulky molecule of the reagent is indifferent towards strongly hindered carbonyl groups. R

J

\

N-NH2 /

+

O=d

P3

\

- \' R

P3

Scheme 4.18.

Hydrazones of compounds containing a carbonyl groups are also used for the GC of the latter (Scheme 4.18). They are prepared by the condensation of a substituted hydrazine with the carbonyl group, usually in the presence of an acid. The reaction conditions can vary; at room temperature the reaction may take several hours. The hydrazones are then mostly isolated from the reaction misture by extraction with a suitable solvent. Using Girard's T reagent for the preparation of these derivatives [ 1041, the interfering compounds that do not contain carbonyl groups can be removed from more complex samples by extraction into a non-polar solvent. Isolation of carbonyl compounds from the sample prior to the CC proper is usually the main reason for the preparation of hydrazones. Mostly they are injected into the instrument simultaneously with a-ketoglutaric acid or another keto compound that releases from the hydrazones at an increased temperature of the injection port the original carbonyl compounds, which are then chromatographed as such. Ralls [I051 filled a capillary with a mixture of hydrazones and a-ketoglutaric acid and placed it in the injection port of a chromatograph and, after heating at 250°C, chromatographed free carbonyl compounds. Halvarson [ 1061 modified the carrier gas inlet into the chromatograph in such a way that the gas passed through an exchangeable side-tube into which a mixture of hydrazones and a-ketoglutaric acid was placed (Fig. 4.3). After heating the loop at 250°C in a polyglycol bath, the carbonyl compounds released were swept with the carrier gas into the column. Direct CC analysis of hydrazones is carried out only with special derivatives, e.g., if the sensitivity of the determination is to be increased. GC retention data have been reported for phenylhydrazones [ 1071, dinitrophenylhydrazones [ 1081, and trichlorophenylhydrazones [ 1091 of different aldehydes and ketones. Particularly the last two types can be used to advantage in trace analysis using the ECD. 4.6. CYCLIC DERIVATIVES

If two or more functional groups which should be blocked occur in the molecule of the substrate, blocking can be accomplished with a bifunctional reagent producing a cyclic product. Cyclic boronates have been used for the GC of compounds containing cis-diol groups in the 1,2- and 1,3-positions (Scheme 4.19). Other substrates, e.g., 0-ketoamines and I I -C-OH HO,' -c* + 8-R I B-R + 2 H 2 0 I '

-C-OH

I Scheme 4.19.

HO'

-

-c-0'

I

CYCLIC DERIVATIVES

77

gloss wool

~

I

D NPH -derivatives + + a-ketoglutaric acid

Fig. 4.3. Schematic diagram of the reactor for reproducible regeneration of carbonyls from microamounts of DNPHs prior to GC analysis. A = injection port (gas chromatograph); B = injector; C = exchangeable PTFE tube; D = back-pressurevalve; E = connection to carrier gas source. (Reproduced from J. Chromatogr., 5 7 (1971) 406 (1061, by courtesy of H. Halvarson.)

hydroxy acids, react similarly. R can denote methyl, butyl or another alkyl group. The reaction is usually carried out with an excess of the reagent in ethyl acetate, pyridine or another solvent and a quantitative yield is usually obtained within 15 min. Boronates are stable enough for further chemical modifications of the substrate to be performed (methoxylation, silylation). As they have characteristic mass spectra, boronates are mainly used in combination with mass spectrometry [110,111]. Two hydroxyl groups also can be blocked if siliconides are prepared (Scheme 4.20).

I Scheme 4.20.

Dimethyldiacetoxysilane reacts in a similar manner to dimethyldichlorosilane. Pyridine, trimethylamine, etc., can serve as solvents [112,113]. In a similar way acetonides [114] are prepared from diols prior to GC. Both carbonyl and carboxyl groups in the a-position can be condensed with a suitable diamine with the formation of a heterocyclic derivative (Scheme 4.21). The properties of such a heterocycle are mostly unsuitable for GC and they are further modified by silylation. Properties suitable for ECD detection can be introduced into the molecule by using a diamine substituted with halogens on the benzene ring [ 1IS]. GC of dialdehydes of the malonaldehyde type was performed after condensation with urea [ 1 161 (Scheme 4.22). The 2-hydroxypyrimidine produced is chemically stable, but R'[40 C C 04'OH

+

HzND x G n '

H2N

I

H

Scheme 4.21.

+ 2 H20

DERIVATIVES AND THEIR PREPARATION

78

H c’

*

C H 4

+

\CH=O

H N 2 ‘c=o H~N/

-

Scheme 4.22.

. . R1

R2

R1

Scheme 4.23.

R’ “ = C S

OH

I o= c

‘C

+

H/ I

HZ

R2

Scheme 4.24.

-

R’

\

//

N-C

S

I

t

C

NH

+

H20

o4 I R

R

Scheme 4.25 ..

has a low volatility. It must therefore be converted into the TMS derivative prior to analysis. A wider range of possibilities of preparing cyclic derivatives is offered by the presence of carboxyl and a-amino groups in the molecule. Substituted 5-oxazolinone (Scheme 4.23) is prepared by refluxing with TFA anhydride, and substituted 5-oxazolidinone (Scheme 4.24) by reaction with (halogenated) acetone [117,118]. Thiohydantoins are formed by reaction with isothiocyanate (Scheme 4.25). If R’ is methyl or phenyl, then the corresponding methyl- or phenylthiohydantoin is produced. Thiohydantoins are usually not volatile enough and for the purposes of GC analyses must be further modified, e.g., by trimethylsilylation [ 1 19,1201.

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M.L. Vorbeck, L.R. Mattick, F.A. Lee andC.S. Pederson,Anal. Chem., 33 (1961) 1512. L.D. Quin and M.E. Hobbs,Anal. Chem., 30 (1958) 1400. M. Rogozinski, J. Gas Chromatogr., 2 (1964) 136. L.D. Metcalfe and A.A. Schmitz,AnaL Chem., 33 (1961) 363. W.R. Morrison and L.M. Smith, J. Lipid Res., 5 (1964) 600. L.D. Metcalfe, A.A. Schmitz and J.R. Peka, Anal. Chem., 38 (1966) 514. B.L. Brian, L.W. Gracy and V.E. Scholes, J. Chromatogr., 66 (1972) 138. W. Stoffel, F. Chu and E.H. Ahrens, Anal. Chem., 31 (1959) 307. H. Iwase, T. Kimura and T. Sugiyama, Bunseki Kagaku (Jap. Anal.), 24 (1975) 272. C. Litchfield, M. Farquhar and R. Reiser, J. Amer. Oil Chem. Soc., 4 1 (1964) 588. J.J. Bailey, Anal. Chem., 39 (1967) 1485.

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12 13 14 15 16 17 18 19 20

80 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80

DERNATIVES AND THEIR PREPARATION

I.C. Cohen, J. Norcup, J.H.A. Ruzicka and B.B. Wheals, J. Chromatogr., 44 (1969) 251. T. Imanari, Y.Arakawa and Z. Tamura, Chem. Pharm. Bull., 17 (1969) 1967. W.F. Lehnhardt and R.J. Winder, J. Chromatogr., 34 (1968) 471. R. Varma, R.S.Varma and A.H. Wardi, J. Chrornatogr., 77 (1973) 222. K.M. Rajkowski and G.C. Broadhead, J. Chromatogr., 69 (1972) 373. M.W. Anders and G.J. Mannering, Anal. Chem., 34 (1962) 730. M.G. Homing, A.M. Moss, E.A. Boucher and E.C. Homing,Anal. Lett., 1 (1968) 311. J.A. Lubkowitz, J. Chromatogr., 63 (1971) 370. M. Donike, J. Chromatogr., 78 (1973) 273. R.A. Landowne and S.R. Lipsky, Anal. Chem., 35 (1963) 532. N.K. McCallum and R.J. Armstrong, J. Chromatogr., 78 (1973) 303. D.D. Clarke, S. Wilk and S.E. Gitlow, J. Gas Chrornatogr., 4 (1966) 310. B.C. Pettitt, P.G. Simmonds and A. Zlatkis, J. Chromatogr. Sci., 7 (1969) 645. P.I. Jaakonmiiki and J.E. Stouffer, J. Gas Chromatogr., 5 (1967) 303. A.E. Pierce, Silylation of Organic Compounds, Pierce Chemical Co., Rockford, IL, 1968. C.C. Sweeley, R. Bentley, M. Makita and W.W. Wells,J. Amer. Chem. Soc., 85 (1963) 2497. J.F. Klebe, H. Finkbeiner and D.M. White, J. Amer. Chem. Soc., 88 (1966) 3390. D.L. Stalling, C.W. Gehrke and R.W. Zumwalt, Biochem. Biophys. Res. Commun., 31 (1968) 616. 81 R. Piekos, J. Teodorczyk, J. Grzybowski, K. Kobylczyk and K. Osmialowski, J. Chromatogr., 117 (1976)431. 82 L. Birkofer and M. Donike, J. Chrornatogr., 26 (1967) 270. 83 M. Donike, J. Chromatogr.,42 (1969) 103. 84 M. Donike, J. Chromatogr., 74 (1972) 121. 85 J. Lehrfeld,J. Chromatogr. Sci., 9 (1971) 757. 86 A.H. Weiss and H. Tambawala, J. Chromatogr. Sci., 10 (1972) 120. 87 M.G. Horning, E.A. Boucher, A.M. Moss and E.C. Horning, Anal. Lett., 1 (1968) 713. 88 G.G. Esposito, Anal. Chem., 40 (1968) 1902. 89 W.A. McGugan and S.G. Howsam, J. Chromatogr., 82 (1973) 370. 90 K.E. Rasmussen, J. Chromatogr., 114 (1975) 250. 91 V. Miller and V. Pacikovi, Chem. Listy, 67 (1973) 1121. 92 E.F. Jansen and N.C. Baglan, J. Chrornatogr., 38 (1968) 18. 93 C.F. Poole and A. Zlatkis, J. Chromatogr. Sci., 17 (1979) 115. 94 W.R. Supina, R.F. Kruppa and R.S. Henly, J. Amer, Oil Chem. Soc., 44 (1967) 74. 95 B.S. Thomas and D.R.M. Walton, J. Endocrinol., 41 (1968) 203. 96 C. Eaborn, C.A. Holder, D.R.M. Walton and B.S. Thomas, J. Chem. Soc. Part C , (1969) 2502. 97 A.J. Francis, E.D. Morgan and C.F. Poole, J. Chromatogr., 161 (1978) 111. 98 J.W. Vogh, Anal. Chem., 43 (1971) 1618. 99 N. Sakauchi and E.C. Homing,Anal. Lett., 4 (1971) 41. 100 J.P. Thenot and E.C. Homing, Anal. Lett., 5 (1972) 21. 101 L.J. Lohr and R.W. Warren, J. Chromatogr., 8 (1962) 127. 102 T.A. Baillie, C.J.W. Brooks and E.C. Homing, Anal. Lett., 5 (1972) 351. 103 T. Nambara, K. Kigasawa, T. Iwata and M. Ibuki, J. Chromatogr., 114 (1975) 81. 104 D.F. Gadbois, J.M. Mendelsohn and L.J. Ronsivalli,Anal. Chem., 37 (1965) 1776. 105 J.W. Ralls, Anal. Chem., 32 (1960) 332. 106 H. Halvarson, J. Chromatogr., 57 (1971) 406. 107 J. Korolczuk, M. Daniewski and Z. Mielniczuk, J. Chrornatogr., 88 (1974) 177. 108 J.B. Pias and L. Gascb, Chromatographia, 8 (1975) 270. 109 D.C. Johnson and E.G. Hammond, J. Amer. Oil Chem. Soc., 48 (1971) 653. 110 G.M. Anthony, C.J.W. Brooks, I. MacLean and I. Sangster, J. Chromatogr. Sci., 7 (1969) 623. 111 S.J. Gaskell, C.G. Edmonds and C.J.W. Brooks,AnaL Lett., 9 (1976) 325. 112 R.W. Kelly, Tetrahedron Lett., (1969) 967. 113 R.W. Kelly, J. Chromatogr.,43 (1969) 229.

REFERENCES 114 115 116 117 118 119 120

E. Bailey, Steroids, 10 (1967) 527. A. Frigerio, P. Martelli, K.M. Baker and P.A. Biondi, J. Chromatogr., 81 (1973) 139. M. Hamberg, W.G. Niehaus, Jr., and B. Samuelsson,Anal. Biochem., 22 (1968) 145. F. Weygand, 2. Anal. Chem., 205 (1964) 406. 0. Grahl-Nielsen and E. Solheim,J. Chromatogr., 69 (1972) 366. J.E. Attrill, W.C. Butts and W.T. Rainey, Jr., Anal. Lett., 3 (1970) 59. M. Rangarajan, R.E. Ardrey and A. Darbre,J. Chrornatogr., 87 (1973) 499.

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Chapter 5

Derivatization of individual species of compounds CONTENTS 5.1. Alcohols and phenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.1. Esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.2.Ethers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.3. Silyl ethers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.4. Derivatives of enantiomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.5. Other derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Aldehydes and ketones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1. Hydrazones and oximes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2. Other derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 . 3 . A m ~ e s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.1. Acyl derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.2. Silyl derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.3. Dinitrophenyl derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.4. Isothiocyanates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.5. Other procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Sulphur compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Carboxylic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.1. Esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.2. Silyl derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.3. Other derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.4. Separation of enantiomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6. Amino acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6.1. N-Acyl alkyl esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6.2. Trimethylsilyl derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6.3. Condensation products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6.4. Other derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6.5. Separation of enantiomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7. Thyroid hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7.1. Acyl methyl esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7.2. Silyl derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.8.Steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.8.1. Silyl derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.8.2. Acyl derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.8.3. Oximes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.8.4.Hydrazones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.8.5. Cyclic derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.8.6. Other derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.9. Sugars and related compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.9.1. Methyl ethers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.9.2. Trimethylsilyl derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.9.3. Acyl derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.9.4. Acetals, ketals and other derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.10. Bases of nucleic acids, nucleosides and nucleotides . . . . . . . . . . . . . . . . . . . . . . . 5.10.1 Silyl derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.10.2 Other derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

84 84 87 88

90 91 92 92 96 97 97 101 104 106 107 109 111 111 118 122 125 126 127 136 139 145 146 148 149 150 151 151 156 160 162 163 164 165 166 168 171 174 175 175 177

84

DERIVATIZATION OF COMPOUNDS

5.1 1. Insecticides and other pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 1.1. Carbamates and related compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 1.2. Organophosphorus insecticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 1.3. Organochlorine pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 1.4. Other substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.12. Pharmaceuticals and drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.12.1. Barbiturates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.12.2. Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.12.3. Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.12.4. Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.12.5. Other pharmaceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.13. Anions of mineral acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.13.1. Non-oxygen-containing anions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.13.2. Oxygencontaining anions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.14. Cations of metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.14.1. Metal halides and other derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.14.2. Metal chelates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.15.Miscellaneous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

177 178 179 180 180 182 182 184 185 186 186 188 188 189 191 191 194 198 199

5.1. ALCOHOLS AND PHENOLS The direct GC analysis of free alcohols, particularly low-molecular-weight compounds, is no longer a serious problem. Conversion into a'derivative prior to GC improves the peak symmetry, particularly for higher alcohols and substances that contain several hydroxyl groups. Phenols and their derivatives (e.g., chlorophenols) and natural phenolic substances also are analysed only after derivatization. Further reasons justifying the usage of this procedure and the development of special and sometimes unusual derivatives are the need for the specific detection of hydroxy compounds in complex mixtures and an increase in the sensitivity of the analysis and the separation of enantiomers. The derivatives commonly used for hydroxy compounds are esters and ethers, including TMS ethers. The methods and problems associated with the derivatization and GC analysis of polyalcohols (reduced sugars) are discussed in Section 5.9.

5.1.1. Esters Acetates of fatty [ l ] and polyhydric [ 2 ] alcohols, phenols [3] and chlorophenols [4] have been studied. Fell and Lee [3] described a GC method for the determination of polyhydric phenols in urine, which, having been extracted, were acetylated with acetic anhydride in the presence of 4-dimethylaminopyridine. According to these authors this substance shows much stronger catalytic effects than does the usually used pyridine. The derivatives are formed rapidly and quantitatively even in very dilute solutions. In the absence of the catalyst, bifunctional phenols provide more than one GC peak. Slightly polar OV-210 is recommended for the separation of phenol acetates, but analysis on nonpolar OV-101leads to tailing, probably as a consequence of insufficient deactivation of the column. Chau and Coburn [4] described the determination of pentachlorophenols (PCPs) in natural and industrial waters at 0.01 ppb levels. The PCPs are extracted into benzene and

85

ALCOHOLS AND PHENOLS

lmin

-t--

1

10

Fig. 5.1. Separation of chlorophenol acetates (2-0.02 ng). Peaks: 1 = 2chloro; 2 = 3-chloro; 3 = 4-ChlOrO;4 = 2,6-dichloro;5 = 2,5dichloro; 6 = 2,4dichloro; 7 = 3,rldichloro; 8 = 2,3-dichloro; 9 = 3,5-dichloro; 10 = 2,4,6-trichloro; 11 = 2,4,5-trichloro; 12 = 2,3,4,6-tetrachloro; 13 = pentachlorophenol acetates. Conditions: Pyrex glass column (25 m X 0.35 mm I.D.), dynamically coated with SE-30; temperature programme, 3"C/min (95-180°C); helium flow-rate, 2-3 ml/min; splitting flowrate, 0-60 ml/min. (Reproduced from J. Chromatogr.; 131 (1977) 412.)

from benzene into a solution of potassium carbonate. Addition of acetic anhydride to the aqueous solution produces acetyl derivatives, which are extracted into n-hexane and analysed by GC using an ECD; stationary phases of the OV type are used. The purity of the acetic anhydride is important (repeated distillation in an all-glass apparatus is recommended) as it may contain impurities that interfere with the peaks of the PCP derivatives in the chromatogram. An identical procedure was also used by Krijgsman and Van de Kamp [5] ,but the GC analysis was carried out in a glass capillary column coated with SE-30. Using the ECD, the detection limit of PCP acetate was 1 pg. The recovery of the extraction-acetylation step was 80-100%. An example of the analysis is shown in Fig. 5.1. A tribenzoyl derivative was used by Decroix et al. [6] for the determination of glycerol. The preparation of the derivative was carried out directly in the sample as it does not require strictly anhydrous conditions. After performing the extraction with diisopropyl ether and after evaporating the solvent, the derivative dissolved in chloroform was injected. A detection limit of 1 pg of glycerol was reported. The low thermal stability of the derivative is a drawback. Makita et al. [7] chromatographed simple phenols as their 0-isobutyloxycarbonyl derivatives. They were prepared by reaction with isobutyl chloroformate in aqueous

DERNATIZATION OF COMPOUNDS

86

alkaline medium, the yields being above 93%.They had good GC properties on OV-17 stationary phase, and their application to the determination of phenols in urine was demonstrated. Halogenated derivatives are frequently used in order to obtain sensitive and selective detection (cf. Table 4.6, p. 68). Argauer [8] proposed chloroacetates and tabulated retention data for 32 phenol derivatives on XE-60, with a detection limit of less than 0.01 ppm of phenols in water. However, the recovery of some phenols depends strongly on the reaction time, which makes quantitative evaluation of the analysis difficult. TFA esters have been utilized for the GC separation of phenols [9] and long-chain fatty alcohols [ l o ] . They are, however, rather labile and their decomposition is catalysed by traces of moisture and acids. HFB esters are more stable and were used by Ehrsson et al. [ 111 for the determination of picogram amounts of phenols. A 0.5-ml volume of a benzene solution was mixed with 100 pl of 0.1 M trimethylamine in benzene and 10 pl of HFB anhydride. After reaction for 10 min at room temperature, the organic phase was agitated with 0.5 rnl of phosphate buffer, pH 6.0 (p = l), for 30 sec. The mixture was centrifuged and 2 pl of the benzene phase were taken for analysis on OV-l,OV-17 and on a mixed stationary phase containing both of them. HFB derivatives were also applied to the determination of phenols in water [12] at the 10 ng/ml level. A 25 ml sample of water was acidified with concentrated hydrochloric acid to pH 1 and 25 rnl of benzene were added. 'Jke mixture was agitated for 15 min and allowed to stand until the layers separated. Portions of 2 ml of the benzene extract were dried by passage through a 5 cm X 5 rnm glass column packed with anhydrous sodium sulphate. A 1-ml volume of the eluent was taken into a 4-ml glass vial and 5 pl of HFBimidazole reagent were added. The vial was closed and the reaction solution was heated

2

7

0

2

4

6

a

10

12

1L

TIME(MIN1

Fig. 5.2. ECD chromatogram of heptafluorobutyryl derivatives of phenols. Peaks: 1 = phenol; 2 = 4-chlorophenol; 3 = 2-chlorophenol; 4 = 2-bromophenol; 5 = 2,4dichlorophenol; 6 = 2,6-dichlorophenol; 7 = p-fur.-butylphenol;8 = 2,4,6-trichlorophenoI; 9 = 2,4,5-trichlorophenol; 10 = 2,4-dibromophenol; 11 = o-phenylphenol. Conditions: glass column, 270 cm X 2 mm I.D.; nitro-DEGS bonded GC packing; nitrogen flow-rate, 33 ml/min; temperature, 80°C for 2 min, then programmed at 8"C/min to 170°C. Concentrations of phenols in the initial water sample ranged from 188 ppb (peak 7) to 44 ppb (peak 6). (Reproduced from J. Cbromatogr., 156 (1978) 143, by courtesy of L.L. Lamparski.)

ALCOHOLS AND PHENOLS

ai

at 65’C. After cooling, the excess of the reagent was hydrolysed by adding 20 p1 of 0.01 N HCl and agitating vigorously for 1 min. The excess of water was removed prior to GC by the addition of ca. 250 mg of anhydrous sodium sulphate. As non-polar phases of the OV type are not suitable for separating derivatives of closely related phenols, a polar stationary phase, nitro-DEGS, and a very low coating (0.5%, w/w) were applied. Fig. 5.2 illustrates the results obtained for a standard mixture of phenols in water (the values in parentheses are individual concentrations). Nose et al. [ 131 determined o-phenylphenols in citrus fruit as pentafluorobenzoates. The reaction proceeds optimally in an aqueous medium at pH 10 (1% NaHC03), and at room temperature quantitatively within 5-1 0 min. The recovery varies from 90 to 98%. Esters of pyruvic acid 2,6-dinitrophenylhydrazonewere described by Bassette et al. [ 141 for the sensitive electron-capture detection of primary, secondary and tertiary alcohols. CI-CI5 primary alcohols were separated within 15 min by using a temperature programme from 150 to 240°C on a 2-ft. column (5% SE-30). Primary amines and thiols can be analysed in this way as the respective amides:and thioesters. Neurath and Luttich [ 151 studied the GC separation of esters of 4‘-nitroazobenzene4-carboxylic acid with saturated and non-saturated aliphatic and aromatic alcohols and phenols. These derivatives may be useful for identification purposes. 5.1.2. Ethers

Although the CC separation of methyl ethers [ 16)of phenols has been described, these derivatives are nowadays mainly employed only in special instances, e.g., for increasing the sensitivity of the analysis. The determination of chlorinated phenols in spent bleach liquors from paper mills was reported by Lindstrom and Nordin [ 171.A water sample was extracted stepwise with diethyl ether at various pH values, after preliminary purification by means of HPLC, phenols were converted into ethyl ethers by reaction with diazoethane in isooctane-ethanol (9 : 1) and good reproducibility was achieved. GC analysis was performed in a glass capillary coated with SE-30 using temperature programming. Kawahara [I81 chromatographed a number of phenols after their reaction with a-bromo-2,3,4,5,6-pentafluorotoluene. In the presence of potassium carbonate in acetone pentafluorobenzyl ethers are produced in high yield (84-10096). They are stable in water and provide a high response of the ECD, permitting the analysis of nanogram amounts. Other phenol derivatives with a high ECD response are 2,4-dinitrophenyl ethers. Cohen et al. [I91 prepared these derivatives by the reaction of phenols with I-fluoro-2,4-dinitrobenzene in the presence of sodium methoxide. They compared the yields obtained by methods based on aqueous solution, acetone solution and a so-called “sandwich layer” reaction proceeding on a thin layer or a paper on which the reagents are applied (cf., Chapter 4, p. 6 5 ) . The last method provides the best results for various types of phenols, despite depending on the character of the material of the thin layer or the paper and not exceeding 40%.The minimum detectable amount with the use of the ECD varies in the range 0.5-0.05 ng, depending on the type of phenol. Seiber et al. [20] compared the ethers of phenols (Table 5.1 ) above derivatives with 2,6-dinitro4-trifluoromethylphenyl and tried to optimize the reaction conditions. They suggested the following procedure

88

DERIVATIZATION OF COMPOUNDS

TABLE 5.1 GAS CHROMATOGRAPHIC RETENTION AND ELECTRONCAPTURE RESPONSE FOR PENTALPHENYL (DNT) AND 2,4FLUOROBENZYL (PFB), 2,6-DINITR04-TRIFLUOROMETHY DINITROPHENYL (DNP) ETHERS OF PHENOLS [20] Conditions: glass column, 6 ft. x 1/8 in. O.D., 5% SE-30 on Chromosorb W (60-80 mesh, AW, DMCS treated); column temperature, 195,230 and 250"C, for the PFB, DNT and DNP derivatives, respectively; carrier gas, N? at 40-60 ml/min. All values are relative to aldrin (1.00) Phenol

PFB

DNT

Retention

DNP

Response

Retention

Response

Retention

Response

0.14 0.30

0.18 0.29

0.48 0.89

0.14 0.19

1.5 2.5

0.23 0.31

0.49 0.5 1 0.92 0.94

0.36 0.30 0.29 0.39

1.19 1.17 2.14 2.25

0.16 0.13 0.10 0.20

3.8 3.7 5.5 6.3

0.20 0.25

~

Phenol pChloropheno1 3,4,5-Trimethylphenol Carbofuranphenol p-Nitrophenol 1-Naphthol

0.06 0.19

for derivatization on the micro-scale. A 20-p1 volume of a 5% solution of potassium hydroxide or carbonate is added to a solution of '10-25 pg of phenol in 0.2 ml of acetone in a 10-ml volumetric flask. After agitating, 9 ml of acetone and 0.25 ml of a stock solua-bromo-2,3,4,5$-pen tation (5 mg/'ml) of a,a,a-trifluoro4-chloro-3,5-dinitrotoluene, fluorotoluene or 1-fluoro-2,4-dinitrobenzene in acetone are added. The volume is made up to 10 ml with acetone, the stoppered flask is shaken vigorously for 30 sec, allowed to stand in darkness for 2 h and 1-3-pl samples are injected directly into the gas chromatograph with 5% SE-30 as the stationary phase. 5.1.3. Silyl ethers

The reactivity of hydroxyl groups of alcohols and phenols towards silylating agents is sufficiently high in most instances, and for the preparation of the silyl ethers prior to GC analysis even milder silylating agents, such as HMDS with the addition of TMCS, usually in a 2 : 1 ratio, are utilized. Strong silylating agents inust be used very carefully as they may result in attack upon further reaction centres in the molecule and in the formation of non-uniform products. On silylating phenolic ketones with BSA, the enol-form of the carbonyl group also takes part in the reaction, with the ratio of mono- to disilyl ether depending on the reaction conditions [21]. BSTFA leads to a uniform product, and was applied by vande Casteele et al. [22] to the silylation of some naturally occurring nonvolatile phenolic compounds and related substances. The reaction mixture (0.2-0.4 mg of the compound together with 400 pl of BSTFA) was heated in a sealed vial at 125°C for 10 min. Hoffman and Peteranetz [23] recommend the catalysis of silylation by the addition of trifluoroacetic acid, which, in contrast to catalysis with TMCS, makes it possible t o silylate even sterically hindered hydroxyl groups with mild silylating agents. Some of their results are shown in Table 5.2. Although the reaction was performed at room

ALCOHOLS AND PHENOLS

89

TABLE 5.2 COMPARISON OF THE REACTION RATES OF THE SILYLATION OF STERICALLY HINDERED PHENOLS WITH DIFFERENT SILYLATING AGENTS [23] ~

Silylating agent

HMDS TMCS BSA BSTFA

~~

Ratio of peak height of TMSether to peak height of free phenol 2,6-Dimethylphenol

o-tert.-Butylphenol

o-tert.-Butylphenol *

5min

10min

5min

10min

5min

10min

1.1

0.4 0.8 2.8 0.6

0.5 1.o 4.2 0.8

21 10

m

1.0 60 110 23

m m

m

m

5

m

m Do

Addition of trifluoroacetic acid to the reaction mixture.

TABLE 5.3 METHYLENE UNIT (MU) VALUES OF MENTHOL STEREOISOMERS MENTHOGLYCOL AND NEOMENTHOGLYCOL [ 35 ] Conditions: glass column, 9 ft. X 2 mm I.D., 5 % SE-30 or OV-17 on GasChrom P (120-140 mesh, AW, silanized); carrier gas, Nz at 25 ml/min; temperature programme, l"C/min from 70°C Compound

Menthol p- Menthan-3-01 3-Trimethylsilylox y Neomenthol p-Menthan-3-01 3-Trimethylsilyloxy Isomenthol p-Menthan-3-01 3-Trimethylsilylox y Neoisomenthol p-Menthan-3-01 3-Trimethylsilylox y Menthoglycol 3-Trhethylsilyloxy 3,8-Di(trirnethylsilyloxy) 3-Heptafluorobu tyrate 3,8-Di(heptafluorobutyrate) Neomenthogly col 3-Trheth ylsilyloxy 3,8-Di(trimethylsilyloxy) 3-Heptafluorobutyrate 3,8-Di(heptafluorobutyrate)

MU value SE-30

OV-17

13.41 12.5 1

12.61 12.60

13.32 12.25

12.48 12.19

13.40 12.41

12.60 12.46

13.51 12.65

12.72 12.67

14.10 15.35 11.96 13.88

14.88 15.09 12.02 13.29

13.79 15.12 11.86 13.45

14.49 14.81 11.92 12.79

90

DERIVATIZATION OF COMPOUNDS

temperature, a distinct accelerating effect of the acid might be observed with HMDS. 2,6-Dinietliylphenol is not included in the table as it is converted quantitatively into the TMSether by all silylating agents on the addition of trifluoroacetic acid in less than 1 min. Langer and co-workers [24,25] converted alcohols and phenols prior to GC analysis into TMS ethers by the action of HMDS. The method using silylation with HMDS-TMCS (2 : 1) was used for the GC of ethylene and diethylene glycols on Apiezon L [26], glycerol on SE-30 [27], polyethers of glycols on XE-61 [28], hydroxystilbenes on SE-52 [29], some flavonoides and related substances on SE-30 [30], plant phenols and related substances on OV-l,OV-l7 and OV-25 [3 1 1, anthocyanines on OV-225 [32] and aminochromes on SE-30 [33]. Different terpene alcohols were silylated by Seidenstucker [34] within 10 min by heating at 40-60°C with a 6-&fold excess of TMS-acetamide. The separation was performed on a stainless-steel column (50 m X 0.25 mm I.D.) coated with Apiezon L. Other workers [35] have described several methods for the silylation of different stereoisomers of menthol and separated them on a column coated with SE-30. They recommend n-butyl boronates for the GC analysis of menthoglycol and neomenthoglycol and HFB esters for sensitive analysis at the nanogram level. Retention data for some derivatives of these substances are listed in Table 5.3. In addition to TMS ethers, dimethylsilyl ethers [36] were studied for the analysis of alcohols and phenols. They were prepared by reaction with reagents analogous to those used for TMS ethers [dimethylmonochlorosilane and tetramethyldisilazane in pyridine (1 : 3 : 9)] and they provide shorter retention times in comparison with TMS ethers on Apiezon L. Grant [37] converted phenols into bromomethyldimethylsilyl ethers. In addition to a higher sensitivity, selective detection, permitting, e.g., phenolic substances to be determined in tars without any preliminary separation, was even achieved by using an ECD. 5.1.4. Derivatives of enantiomers

Pereira [38] separated enantiomers of set-alcohols on Carbowax 20M after their conversion into carbamates by treatment with optically pure R-(+)-N-1-phenylethylisocyanate. This reagent is prepared from commercially available I?-(+)-]-phenylethylamine and phosgene. 3fl-Acetoxy-As-etienic acid esters [39] were used for the same purpose. The alcohol (1 8 pmole), 3(3-acetoxy-As-etienicacid chloride (50 pmole) and pyridine (10 11) were dissolved in 1 ml of benzene and injected into the chromatograph. The derivatives were well separated on OV-17 and DC-200. Enantiomers can be separated in an analogous manner after reaction with 2-phenylpropionyl chloride [40]. Brooks et al. [41] used drimanoyl and chrysanthemoyl chlorides as chiral reagents for series of enantiomeric alcohols. The alcohol (1 mg) in dry toluene (20 p i ) was treated with 10 pl of a solution of freshly prepared drimanoyl chloride (3 molar excess) in dry toluene and the mixture was heated at 60°C for 1-2 h. The injection was performed without using any other purification. Good separation of enantiomers of chrysanthemoyl esters, which are prepared in an analogous manner, was achieved on a 5-m column packed with 1% of SE-30 on Gas-Chrom Q (100-120 mesh) at 143°C. Enantiomers of 3,3-dimethyl-2-butanol and other alcohols were separated by preparative GC on OV-type phases after their conversion into esters of N-TFA-L-alanyl [42].

ALCOHOLS AND PHENOLS

91

5.1.5. Other derivatives

For the sensitive and selective detection of trace amounts of low-molecular-weight alcohols and other hydroxyl-containing compounds by means of the alkali FID, Vilceanu and Schulz [43] prepared phosphorus-containing derivatives. Derivatives 5 .I and 5.2 were prepared by the reaction with 2-chloro-l,3,2-dioxaphosphorinane (5.3) and 2-chloro1,3,2-dioxaphospholane (5.4), respectively, in the presence of triethylamine in benzene, which proceeds very quickly, and were particularly suitable for the determination of trace amounts of alcohols in non-alcoholic and anhydrous media. Retention indices of these derivatives of alcohols up to Cs are listed in Table 5.4.

Scheme 5.1.

Scheme 5.2.

Scheme 5.3.

Scheme 5.4.

The GC separation of dihydric alcohols of natural origin may be carried out after their conversion into alkyl boronates. Methaneboronates of ceramides [44] were prepared by treatment with methaneboronic acid in pyridine solution. They offer excellent GC properties on stationary phases of the OV-1 and 5e-30 types and are particularly suitable for GC-MS. TABLE 5.4 RETENTION INDICES OF HETEROCYCLIC PHOSPHORUSCONTAINING DERIVATIVES OF ALCOHOLS (431 Conditions: glass column, 1.85 m column temperature, 120°C Compound

X

5 mm I.D., 10%Apiezon L on Chromosorb G (60-80 mesh);

R

1120OC

)-0-R 0

Methyl Ethyl n-Propyl n-Butyl n-Pentyl

792 856 967 1022 1096

L>P-O-R

Methyl Ethyl n-Propyl n-Butyl n-Pentyl

821 884 1002 1065 1124

Methyl Ethyl n-Propyl n-Butyl n-Pentyl

895 96 1 1078 1131 1180

~-

~

0

(

H3

C I P - 0 - R

92

DERIVATIZATION OF COMPOUNDS

5.2. ALDEHYDES AND KETONES The direct GC analysis of these compounds is often complicated by the presence of a strongly polar carbonyl group in their molecules and its strong interactions with column packing. The conversions into derivatives were originally applied in most instances only to a prelimhary separation of carbonyl compounds from complex sample mixtures and GC analysis proper was performed after releasing the original compounds from their derivatives. The direct analysis of the derivatives of aldehydes and ketones is aimed at increasing the sensitivity and selectivity of their detection. In order to prepare derivatives of monofunctional aldehydes and ketones, common condensation reactions of carbonyl group, used also for the preparation of Schiff s bases, are applied. For bifunctional compounds cyclization reactions are applied, etc. (cf., Chapter 4 , pp. 77 and 78).

5.2.1. Hydrazones and oximes Preliminary isolation and concentration of volatile carbonyl compounds from foodstuffs are performed via 2,4-dinitrophenylhydrazones [45,46]. The sample material analysed is steam distilled and volatiles and the condensate are introduced into a reaction mixture coasisting of 2,4-dinitrophenylhydrazine (2 g/l) and 2 N HCl. When the distillation is finished, the reaction proceeds at room or higher temperature for several hours and 2,4-DNPHs are separated by filtration or extraction (with benzene). The GC analysis proper is executed after releasing the original carbonyl compounds from their derivatives, e.g., according to Ralls [47] by heating with wketoglutaric acid (1 : 3, w/w) in a sealed capillary or in a modified injection port of the chromatograph (see Fig. 4.3, p. 77). A temperature of 25OoC is applied for 5-10 sec, although at this temperature some 2,4-DNPHs may undergo thermal decomposition. a-Ketoglutaric acid alone is reported by Jones and Monroe [48] to lead, under conditions of regeneration, to products with retention times identical with those of some lower aldehydes (C1--C3). A mixture of oxalic acid dihydrate and p-dimethylaminobenzaldehyde (5.3 : 6.0) served as a regenerator and was added to 8 mg of a mixture of 2,4-DNPHs and Celite; 50 pl of the DNPH of butyraldehyde were added as an internal standard. An error of *lo% was obtained in a quantitative evaluation. For separating released carbonyl compounds by GC, Carbowax or LAC-446 (glycoladipate polymer) may be used as a stationary phase. Halvarson [46] recommends, for combination with MS, a column packed with Porapak S which provides very small changes in the MS background even under a temperature programme up to 18OoC.Gadbois et al. [49] modified the method for the preparation of Girard-T derivatives to the isolhtion of carbonyl compounds before GC analysis. Scheme 5.5 illustrates the preparation of Girard-T reagent and Scheme 5.6 the derivatization reaction, for which the following procedure is recommended by the authors. A standard mixture of aldehydes (1.7 * mol) was subjected to reaction with [CH31 N + CI-CH2-COO-C2H5

3

Scheme 5.5.

+ N H -NH

2

2

-

[[CH31 i!4-CH2-CO-NH-NH2]

3

ik

ALDEHYDES AND KETONES

KH

I ~ - C H ~ - C O - N H - N 61 H ~+

33

93

d, R'

-

+

c=o

-

(CH313N-CH2-CO-NH-N=C

,d

-

CI

i

H20

'R

Scheme 5.6.

300 mg of Girard-T reagent (1.8 mol) in the presence of 250 mg of Rexyn 102 (&) and 50 ml of tert.-butanol azeotropic solution. The mixture was refluxed at 80°C for 1 h, filtered through glass-wool and concentrated in Buchler rotational evaporator to ca. 5 ml at room temperature. The concentrated solution was transferred into a 25-ml volumetric flask and made up to the mark with tert.-butanol. Aliquots of 10 mi were pipetted into a 25-ml erlenmeyer flask and evaporated almost to dryness at 40°C in a stream of pure nitrogen. The residue was dissolved in 0.5 ml of tert.-butanol, transferred into a 1-ml volumetric tube and made up to the mark with tert.-butanol. Aliquots of 5 pl were transferred into a capillary (90 X0.8 mm) with 5 pl of a solution of paraformaldehyde or methylolphthalimide. The capillary was sealed and heated at 200°C for 2 min, cooled and 2 pl were injected for GC analysis. The whole procedure is complicated and unless the yields of both derivatization and regeneration reaction are loo%, the total recovery must be known in order to achieve accurate quantitative analysis. It varies within the range 80-100% with aliphatic alcohols (acrolein 33%). The application of Girard-T reagent, however, offers the possibility of isolating non-carbonyl interfering components from the sample, e.g., by extracting with n-hexane after preparing the derivatives. More attention has been devoted, particularly in recent years, to the direct GC separation of hydrazones of carbonyl compounds. Phenylhydrazones of aldehydes can be separated successfully in a column packed with SE-30 on Chromosorb W at temperatures ranging from 120 to 190°C [50]. Korolczuk et al. [51] considered this problem in more detail. They described the separation of phenylhydrazones of 27 aldehydes and-ketones using different temperature programmes and studied the influence of the initial temperature on the retention of the derivatives. The analysis time is less than 15 min for carbonyl compounds with up to 11 carbon atoms with programming at lO"C/min in the range 150-280°C. Some derivatives provide two peaks which can be ascribed to their syn-anti isomerism, although even the decomposition of the derivatives cannot be eliminated as a cause, particularly with the use of a metallic column. The GC separation of 2,4-dinitrophenylhydrazoneshas been studied by a number of workers [52-55]. Non-polar stationary phases of the SE-30 and SF-96 type were utilized for this purpose at temperatures of 2O0-25O0C, mostly with temperature programming. Retention indices of the 2,4-DNPHs of some carbonyl compounds on OV-type stationary phases are presented in Table 5.5. Using columns with a higher separation efficiency, some 2,4-DNPHs provide two peaks. The discussion of whether these artifacts are caused by thermal decomposition or syn-anti isomerization of the derivatives seems to favour the latter. The ratio of the areas of the peaks of the two derivatives depends on the polar-

94

DERIVATIZATION OF COMPOUNDS

TABLE 5.5 OF ALDEHYDES AND RETENTION INDICES OF 2,4-DINITROPHENYLHYDRAZONES KETONES (541

Conditions: stainless-steel column, 1 m x 2 mm I.D., 1%OV-3 or OV-7 on Chromosorb G (80-100 mesh, AW, DMCS treated); carrier gas, argon at 10-45 ml/min; column temperature, 205 or 245°C 2.4-DNPH

Methanal Ethanal 2-Propanone Propanal Propenal 2-Butanone Butanal 3-Pentanone 2-Pentanone 3-Methyl-3-buten-2-one Pentanal 2-Hexanone . Hexanal 2-Methyld-hexanone Heptanal Furan-2-aldehyde Phenylacetaldehyde Octanal Nonanal

Benzaldehyde Acetophenone Decanal Undecanal p-Anisaldehyde

OV-3

OV-7

I2QS0C

I245'C

I2Q5"C

I245OC

2070 2198 2272 2290 2279 2356 2378 2399 2423 2450 2472 2527 2568 2584 2666 266 1

2137 225 1 2333 2336 2344 2417 2428 2455 2470 2490 2513 2578 2611 2628 2706 2723 2781 2802 2897 2927 2994 2994 3099 3243

2174 2302 2363 2385 2385 2437 2472 2490 25 10 2531 2569 2607 2666 2659 2763 2780

225 1 2370 2438

2763 2857 295 1 305 2 -

-

2859 2955 2982

3050 3154 -

-

2522 -

2624 -

2719 2814 2910 3004 3098 3096 3202 -

ity of the solvent used and on the period of time for which the derivative was subjected to its effects [56]. A typical chromatogram of 2,4-DNPHs of ten aliphatic aldehydes is illustrated in Fig. 5.3, taken from the paper by Hoshika and Takata [55].It was obtained with a glass capillary (30 m X 0.25 mm I.D.) coated with SF-96 at a column temperature of 200-240°C. The authors utilized 2,4-DNPHs for the analysis of carbonyl compounds in exhaust gases and in cigarette smoke. The components contained in 30 ml of sample gas were condensed in a trap cooled with liquid nitrogen and dissolved in 5 ml of ethanol. The solution obtained was poured into a 0.1% solution of 2,4-dinitrophenylhydrazine in 2 N HCI and allowed t o crystallize overnight at room temperature. The resulting precipitate was extracted with carbon tetrachloride and then dried in vacuum. The residue was dissolved in 0.5 ml of acetone and anthracene was added as an internal standard. VandenHeuvel et al. [57] suggested substituted hydrazones for the GC characterization and identification of carbonyl compounds, e.g., condensation products with N-amino-

ALDEHYDES AND KETONES

I

0

20

10

95

30

I

TIME IMIN)

Fig. 5.3. Gas chromatogram of 2,4-dinitrophenylhydrazones of ten aliphatic aldehydes. Peaks: 1 = formaldehyde; 2 = acetaldehyde; 3 = propionaldehyde; 4 = acrolein; 5 = isobutyraldehyde; 6 = n-butyraldehyde; 7 = isovaleraldehyde; 8 = n-valeraldehyde;9 = crotonaldehyde;10 = n-capronaldehyde. For conditions see text. (Reproduced from J. Chromatogr., 120 (1976) 379, by courtesy of Y. Hoshika.)

piperidine, N-aminohomopiperidine, pentafluorophenylhydrazine and phenylhydrazine, the retention times of which are different on F-60 stationary phase. An increase in the selectivity and sensitivity of the detection of carbonyl compounds may be achieved by their conversion into special derivatives. Johnson and Hammond [58] condensed carbonyl compounds with 2,4,6-trichlorophenylhydrazineand, prior to the analysis, separated the products by means of thin-layer chromatography. Using an ECD, they were able to determine by GC 10-7-10-10 g of the substance. They prepared the derivatives in a reaction column as follows. A 0.40-g amount of 2,4,6-trichlorophenylhydrazine was dissolved in 40 ml of 1 N HC1 with heating and mixed with 40 g of Celite 545. n-Hexane was added to the wet mixture until a paste consistency was obtained, and the column (30 X 2 cm I.D.) was filled with the paste. In order to prepare the derivatives, the carbonyl compound was applied to the column in an amount corresponding to half of the theoretical column capacity and the column was eluted with 75-100 ml of n-hexane. The n-hexane was distilled off at decreased pressure and the viscous derivatives were stored in 10 ml of n-hexane at -27°C. However, these derivatives are sometimes not separated satisfactorily on silicone phases. Pentafluorophenylhydrazine, in a condensation reaction with 27 carbonyl compounds, gave quantitative results for most of them [59]. The minimum detectable amount was 0.01 ng, the derivatives being separated in a column coated with SE-30 or PEG 20M. Hoshika [60] used derivatives containing sulphur for the GC analysis of benzaldehyde, which were prepared by condensation with 2- and 3-methylthioaniline (Scheme 5.7).

O C H = O + H::b

Scheme 5.7.

*

D

C

H

=

:

b

96

DERIVATIZATION OF COMPOUNDS

0

5

10

15

20 25 30 35 LO L5 50 RETENTION TIME, MINUTES

55

60

65 70

Fig. 5.4. Gas chromatogram of gasoline engine exhaust sample. Peaks: 1 = formaldehyde; 2,2' = acetaldehyde; 3 = acetone; 4,4' = propanal; 5 = butanone; 6 = 2-methylpropenal; 7 = benzyl alcohol; 8 = 0-cresol; 9 = phenol; 10 = m-cresol; 11 = p-cresol; 12 = benzaldehyde; 13 = tolualdehyde; 14 = I-naphthol (internal standard). Conditions: borosilicate glass column, 42 in. X 4 mm I.D., 0.1% Carbowax 20M on glass beads; nitrogen flow-rate, 20 ml/rnin; temperature programme, 2"C/min from 75°C. (Reproduced from A n d . Chem., 43 (1971) 1618, by courtesy of J.W.Vogh and the American Chemical Society.)

Using a flame-photometric detector sensitive to sulphur, selective detection can be obtained at the nanogram level, with the possibility of carrying out the reaction on the micro-scale with only 50 pl of the sample. These derivatives give symmetric peaks on SE-30. Oximes decompose during GC analysis at higher temperatures into the corresponding nitriles [61], and this decomposition is catalysed by metal parts of the injection port and the apparatus. The decomposition can be eliminated by using an all-glass apparatus (borosilicate glass) and oximes can then be chromatographed directly. Their slightly acidic character (pK 10-12) can, however, be utilized for a preliminary separation of carbonyl compounds from complex mixtures by extraction with a dilute base or for the elimination of interfering hydrocarbons and other neutral components by extracting a basic sample with a paraffinic solvent. Using this principle, Vogh [62] developed a procedure for the determination of carbony1 compounds in exhaust gases. The sample components were trapped in a methanolic solution of hydroxylamine, the pH of the mixture was adjusted to alkaline and unwanted components were extracted with n-pentane. Some higher aliphatic ketoximes in which both alkyl groups are bulky can also be extracted to a significant extent. By direct GC analysis of oximes Vogh eliminated the regeneration of carbonyl compounds, which can introduce further errors into the procedure, and which is essential when using, e.g., Girard-T derivatives. He used an all-glass apparatus for GC and performed the separation on Carbowax 20M or UCON-50-HB-660 with a 0.1% coating on glass beads. Some oximes provided double peaks corresponding to syn- and anti-isomers. Fig. 5.4 illustrates a profile of carbonyl compounds found in exhaust gases of a petrol engine.

5.2.2. Other derivatives Malonaldehyde in biological samples, e.g., produced in the microsomal peroxidation of fats, was converted for GC analysis into 2-hydroxypyrimidine by condensation with urea

AMINES

91

(see Scheme 4.22, p. 78). A non-volatile, although chemically stable, product may be isolated from the samples in microgram amounts and must be converted into the volatile TMS ether prior to GC analysis [63]. Fatty aldehydes with long chains were separated by Gray [64,65] as dimethylacetals on Apiezon L and Reoplex 400 stationary phases. Aldehydes were converted into their dimethylacetals by refluxing with 2% anhydrous methanolic HCl(20 : 1, v/v) for 2 h. The conversion was quantitative ( B 5 % ) . The methanolic solution was cooled and neutralized with a small excess of sodium carbonate. Acetals were extracted from methanol with light petroleum. For structural studies, acetals were oxidized to the corresponding acids by the action of chromic oxide in glacial acetic acid. The esters produced by the esterification with methanolic HCl were chromatographed and compared with standards. Schogt et al. [66] used silver oxide for the oxidation and diazomethane for the esterification.

5.3. AMINES The character of the amino groups makes the GC analysis of amines difficult owing to an interfering sorption that gives rise to asymmetric peaks. Using derivatization, the symmetry of the zones is improved and the compounds often acquire properties that result in selective and sensitive detection. Acylation, silylation and the formation of different condensation products (Schiff s bases) are common procedures. This section also describes methods for the derivatization of biogenic amines, such as catecholamines, phenylethylamines and indolylalkylamines. These compounds are analysed by GC only after their conversion into suitable derivatives because other polar groups, especially the hydroxyl group, are usually also present. The direct GC separation of biogenic amines is therefore very difficult and peaks tail even with the use of a non-polar stationary phase (SE-30) (e.g., ref. [67]). Derivatization procedures are preferred that protect the functional groups of both types, e.g., acylation and silylation. Halogenated acyl derivatives play a particularly important role in combination with the ECD as biogenic amines are present in the samples nearly always in trace concentrations. However, the different reactivities of various functional groups often affect the quantitativeness of the preparation of the derivatives and therefore combined derivatives, e.g., N-acyl-0-TMS, are often utilized. Isothiocyanates, dinitrophenyl derivatives and others have also been described. 5.3.1. Acyl derivatives

Acetyl derivatives, which may even be prepared directly in the CC column by using a subsequent injection of acetic anhydride [68], are the most readily available and can be used for the rapid characterization or identification of amines. Marmion et al. [69]used acetyl derivatives for the determination of small amounts of 2-naphthylamine in 1-naphthylamine. Propionyl derivatives have been studied for the CC of biogenic amines [70]. They are very stable and can even be prepared in aqueous medium. Their lipophilicity makes possible their quantitative extraction into ethyl acetate or other organic solvents. The follow

98

DERIVATIZATION OF COMPOUNDS

ing procedure was suggested for their preparation. A 5-pmol amount of amines is dissolved in 1 ml of 0.1 N hydrochloric acid and the solution is saturated with solid sodium carbonate. In the course of 5 rnin, with continuous shaking, three 0.05-ml portions of propionyl anhydride are added. The propionyl derivatives of the amines are then extracted three times with 1 ml of ethyl acetate. The combined organic extracts are evaporated to dryness at 25-3OoC in a stream of dry air, the residue is dissolved in 0.2 ml of pyridine-propionic anhydride (3 : 1, v/v) and the solution is heated at 100°C for 15 min. After cooling, the excess of pyridine and propionic anhydride is evaporated. For GC analysis the derivatives are dissolved in acetonitrile. Mixtures of up to 21 different biogenic amines can be separated on 3% OV-17 or OV-101 in the temperature range 100-280°C with temperature programming at 6"C/min. Of halogenated acetyl derivatives, trifluoroacetyl derivatives are mainly used for the sensitive analysis of arnines, particularly owing to their better chromatographic properties despite their ECD response being lower than that of chloroacetyl derivatives (cf., Table 4.7, p. 69). Trifluoroacetyl derivatives were exploited for the GC separation of a mixture of saturated and unsaturated homologues of amines up to CZ2on conventional packed columns [71]. Mori et al. [72] developed a method for the quantitative and qualitative GC analysis of m- and p-xylenediamines in polyamides in the form of their N,N'-trifluoroacetyl derivatives. An analogous method was elaborated for the analysis of ethanolamine for the presence of mono-, di- and triethanolamine [73]. A 1-ml volume of TFA anhydride is stoppered in a 2-ml vial, which is evacuated with the aid of a 10-ml injection syringe. The derivatives are prepared by adding slowly 0.05 ml of the sample by means of a 0.25-ml injection syringe with continuous stirring. The contents of the vial are then shaken at intervals for 5-10 rnin and 5-pl samples are injected into the chromatograph. On neopentyl glycol succinate, TFA derivatives of ethanolamines give symmetric peaks, making possible quantitative evaluation. Dove [74] analysed a complicated mixtuie of aromatic amines after their conversion into TFA derivatives on a mixed stationary phase containing 9.5% of Apiezon L and 3.6% of Carbowax 20M (Fig. 5.5). The derivatization was performed in a 25-ml vial in which the sample of a mixture of amines (0.2 g) and a standard (0.05 g of n-dodecane) were dissolved in 2 rnl of tetrahydrofuran and 5 drops of pyridine. The mixture was cooled in an ice-bath and 2 ml of TFA anhydride were added carefully. The contents of the vial were then heated at 50°C for 10 rnin, cooled and 5 ml of water were added. The mixture was extracted with 8 and 4 ml of dichloromethane. The extract was washed with 5 ml of saturated aqueous NaHC03 and 5 ml of water, dried over anhydrous Na2S04 and a 5-pl portion was chromatographed. Cyclohexylamine in blood was determined at levels of 0.1 pg/ml after trifluoroacetylation [75]. Lubkowitz [76] used ethyl trifluoroacetate for the preparation of TFA derivatives of 1,2-diaminocyclohexane in order to avoid the unpleasant properties of anhydride, such as its high reactivity, and the formation of a corrosive by-product. A three-fold excess of the reagent in anhydrous medium in the presence of ammonia leads to a yield of up to 99%. He resolved cis- and trans-isomers of 1,2-diaminocyclohexaneon Versamide 900 stationary phase. Conversion into a derivative with a high ECD response makes possible the sensitive

AMINES

I

99

1

3

Lo W

z

0

n W vl

11 11 W

n 11 0

u

W

n ~~

0

8

16

2L

MI N

40

48

56

64

Fig. 5.5. GC separation of TFA derivatives of arylamines. Peak: 1 = solvent (CH2CI2); 2 = N-methylaniline; 3 = N,N-dimethylaniline; 4 = Nethyfaniline; 5 = N-methyls-toluidine; 6 = N-methyl-mtoluidine; 7 = N-methyl-p-toluidine; 8 = o-toluidine; 9 = oethylaniline; 10 = aniline; 11 = 2,Sdirnethylaniline; 12 = 2,6-dimethylaniline; 13 = 2,4-dimethylaniline; 14 = m-toluidine; 15 = p-toluidine; 16 = 2,3dimethylaniline; 17 = 3,5-dimethylaniline; 18 = m-ethylaniline; 19 = pethylaniline; 20 = 3,4dimethylaniline. Conditions: stainless-steel column, 18 ft. X 0.125 in. O.D., 9.5% Apiezon L + 3.6% Carbowax 20M on Aeropack 30 (80-100 mesh); helium flow-rate, 100 ml/min; column temperature, 152°C. (Reproduced from Anal. Chem., 39 (1967) 1188, by courtesy of the American Chemical Society.) TABLE 5.6 RETENTION DATA AND SENSITIVITIES OF THE ANALYSIS FOR SOME DERIVATIVES OF PHENYLETHYLAMINE [ 8 21 Conditions: glass column, 12 ft. X 4 mm I.D. with 5% of stationary phase on GasChrom P (80-100 mesh); carrier gas, nitrogen and/or argon-methane (95 : 5 ) a t 60 ml/min; temperature programme, 2"C/min from 150°C (SE-30) or 100°C (OV-17) to 320°C Derivative *

Free base Acetyl Trifluoroacetyl Pentafluoropropionyl Heptafluorobutyryl N-(2,4-dinitrophenyl) Acetone SB Benzaldehyde SB Trifluoroacetone SB Heptafluorobutyraldehyde SB Perfluorooctanaldehyde S B Pentafluorobenzaldehyde SB p-Chlorobenzaldehyde SB p-Nitrobenzaldehyde SB

MU value

Sensitivity (mol/sec)

**

SE-30

OV-17

FID

ECD

10.87 14.74 12.65 12.69 12.99 26.89 12.73 17.73 12.02 11.35 13.27 16.81 19.71 22.20

12.82 18.03 14.81 14.45 14.54 32.00 14.57 20.48 13.43 12.17 13.25 18.81 22.66 26.15

4.2 . 10-13 4 . 2 . 10-l3 4.7 . 1 0 - l ~ 2.6. 10-13 3.4. 10-13 7.1.10-13 4.0 . 2.4 . 10-13 3.5 . 10-13 6.4 . 5.2. 10-13 2.4 . 10-13 4.5 . 10-13 4.1 . 10-13

2.1 ' 10-12 1.1 ' 10-11 1.1 .10-14 1.2 . 10-15 2.2 ' 10-16 4.3 . 10-16 7.1 ' 3.1 . 10-12 1.6 . 1 0 - l ~ 3.1 . 6.0 . lo-' 9.1 . 10-I 6.3 . lo-'' 5.3 . 10-1

* SB = Schiff base. ** Sensitivity is given as that amount of the derivative (niol) which gives a peak twice as high as the noise/peak width (sec).

100

DERIVATIZATION OF COMPOUNDS

analysis of trace concentrations of biogenic amines. This property is found with pentafluoropropionyl and heptafluorobutyryl derivatives, which are more stable than TFA derivatives [77] and their ECD response is higher. PFP and HFB derivatives of the compounds of the metanephrine and normetanephrine type are prepared by reaction with the corresponding anhydrides in ethyl acetate. The reagent and the solvent are removed by passage of a stream of nitrogen at room temperature for 15 min and the derivative is dissolved in ethyl acetate. A good separation of the derivatives may also be obtained on OV-17 and XE-60 and these stationary phases are also suitable for combining with mass spectrometry. These derivatives have also been applied to other biogenic amines [78,79] and for the determination of pseudoephedrines and related substances in blood [80]. Brooks et al. [81] studied the use of these derivatives in the sensitive analysis of N-nitrosodimethylamine. Moffat and Horning [82] compared the properties of different perfluoroacyl derivatives and Schiff s bases of phenylethylamine with respect to the sensitivity obtainable with the use of the ECD (Table 5.6). The FID response does not change much if the derivative and free bases are compared, while the sensitivity of the analysis may be increased by up to 200,000 times by using the ECD. The greatest response is provided by the condensation products of phenylethylamine with perfluorooctaaldehyde and pentafluorobenzaldehyde. The latter derivative leads to the highest ECD response in comparison with other pentafluorobenzene derivatives qnd is particularly recommended for primary amines [83]. Pentafluorobenzoyl derivatives, comparable to it, are preferred for sec.-amines and catecholamines, as a uniform product is formed by their reaction with pentafluorobenzoyl chloride. All of the derivatives compared were prepared by a common procedure. A 1-mg amount of amine was added to 0.2 ml of acetonitrile and 0.1 ml of the appropriate reagent (20 mg of solid reagent in 0.1 ml of acetonitrile) in a small screw-capped vial. After heating at 60°C for 1 h, the mixture was diluted with n-hexane and subjected to GC analysis on SE-30. Diperfluoroacylamides of primary amines offer higher ECD responses than the mono derivatives (Table 5.7). The catalytic action of trimethylamine 1841 is necessary in order to perform the acylation reaction successfully. A 200-p1 volume of a M benzene solution of the amine containing an internal standard (p-dibromobenzene or 1-bromonaphthalene) is mixed with 200 pl of 0.3 M trimethylamine in benzene and 25 p1 of anhydride. After 15 min at TABLE 5.7 COMPARISON OF THE ECD RESPONSES TO MONO- AND DIACYL DERIVATIVES OF DODECYLAMINE [ 8 4 ] Sensitivity is expressed as in Table 5.6 Derivative

Sensitivity (mol/sec)

Mono-TFA Di-TFA Mono-HFB Di-HFB

6.7.10-14 3.8 . 1.0. 10-l4 6.0 ’

AMINES

101

room temperature 2 ml of phosphate buffer, pH 6.0 (p = l), are added and the mixture is shaken for 15 sec and centrifuged. A 1-111volume of the organic phase is injected directly or after dilution with benzene. OV-l,OV-l7 and QF-1 stationary phases have been applied.

5.3.2. Silyl derivatives The amino group is not very reactive during silylation reactions and its conversion into a silyl derivative is difficult. In a mixture of hexuronic acid, 1-octanol and I-octaylamine [85], it is the amine that gives the lowest yield under the same silylation conditions. By modifying the reaction conditions and using stronger silylating agents and catalysts, however, silyl derivatives of amines can be prepared and in this form analysed by means of

GC . HMDS alone is seldom used for the silylation of amines and addition of a catalyst is usually necessary [86]. Particularly with polyfunctional amines, silylation does not proceed quantitatively and non-uniform products are obtained. Mori et al. [87] analysed the components of polyamide resins by GC after silylation with the aid of BSA. About 10-mg portions of the samples are dissolved in 0.15 ml of acetonitrile in a 10-ml flask and 0.03 ml of triethylamine and 0.15 ml of BSA are added gradually. The flask is connected to a reflux condenser and heated at 90°C for 30 min with the elimination of the excess of air humidity. Another 0.15 ml each of BSA and acetonitrile are added gradually and the flask is heated for 30 min. The solution is then made up to a known volume with acetonitrile and 10 pl are injected into the gas chromatograph. Binding of the hydrochloric acid produced makes necessary the addition of triethylamine to the reaction mixture. In order to separate TMS derivatives, Mori et al. used a 2-m column packed with 5% of neopentyl glycol succinate on Celite 545 at 160"C, and a relative error of 4.4% was obtained for the quantitative analysis. The same column coated with SE-30 or Apiezon L failed. Metcalfe and Martin [88] performed the silylation of primary amines in n-hexane. About 100 mg of the sample in a vial were dissolved in 2 ml of n-hexane and 0.2 ml of BSA was added. After shaking for 1 min the mixture was allowed to stand for 5 min, then 2 p1 of the solution were injected. Positional isomers of Cll-CI5 amines were resolved on a capillary coated with SF-96 silicone modified with trioctadecylammonium bromide. Butts [89] presented retention data of TMS derivatives of a number of biochemically important substances, such as amines, pyrimidines, purines, imidazoles, indoles, various acids and other substances on two stationary phases, OV-1 and OV-17. He prepared the derivatives using the following procedure. A I-mg amount of the substance was placed in a 3.5-ml septum-stoppered vial, then 100-p1portions of dry pyridine and BSTFA containing 1% of TMCS were added, the contents were stirred thoroughly and heated for 16 h at 60°C. Portions of 4 p1 were injected directly into the gas chromatograph. The problem with the silylation of different biogenic amines is that it is difficult to prepare uniform derivatives as these compounds usually contain amino groups of different reactivity, hydroxyl and other functional groups. Several procedures have been proposed for solving this problem. The determination of norepinephrine and dopamine in brain tissue was described by Maruyama and Takemori [ 9 0 ] . Dried residue from the extraction

102

DERIVATIZATION OF COMPOUNDS

was dissolved in 20 pl of a mixture of TMS-imidazole and acetonitrile (1 : 1) and heated at 50°C for several minutes on a water-bath. The degree of silylation when BSA in pyridine is used depends on the reaction conditions [91] and may be controlled by varying them. 3,4-Dimethoxyphenylethylamineis half silylated after reaction for 10 min at room temperature (or for 5 min at 6O0C), and is fully silylated after 20 min at 60°C with the addition of TMCS. Tryptamine under the former conditions gives almost entirely the mono-derivative and under the latter conditions the bis-derivative with traces of the trisderivative; by prolonging the reaction time to 45 min, the yield of the tris-derivative is increased so that it is comparable to that of the bis-derivative. A two-step silylation has been described for catecholamines [92]. All of the hydroxyl groups are converted into TMS ethers within 2-3 h by reaction with TMS-imidazole in acetonitrile at 60"C, BSA-TMCS (2 : 1) reagent is added and within the next 2 h all of the primary amino groups are converted into the N,N-di-TMS derivative. Secondary (N-methyl) amino groups do not react under these conditions. A distinct speed-up of the silylation reaction on the addition of a small amount (l%,v/v) of water to the reaction mixture (see Fig. 5.6) and different effects of the solvents are interesting. These derivatives possess excellent chromatographic properties and are well separated on OV-1. Albro and Fishbein [93] compared 11 different silylating mixtures for the derivatization of tyrosine and tryptophan metabolites. BSTFA with the addition of TMCS is the most suitable reagent as far as quantitative reaction is concerned; a mixture of BSTFA with TMSDEAand TMCS in pyridine (99 : 1 : 30 : 100,v/v) is recommended as a universal silylating agent. The presence of different functional groups in the molecules of biogenic amines led logically to the development of multi-step procedures for the preparation of combined, but uniform, derivatives for GC analysis. Holmstedt et al. [94] separated and identified

T I M E I HOURS)

Fig. 5.6. Conversion of norepinephrine (1 mg) into the fully silylated N,Ndi-TMS derivative using BSA (0.2 m1)-TMCS (0.1 ml) at 60°C in 0.1 ml of either acetonitrile (A,C) or pyridine (B,D). For curves A and B 2 pl of water were added to the reaction mixture. (Reproduced from Biochim. Biophys. A c f a , 148 (1967) 597, by courtesy of M.G. Homing.)

AMINES

103

I TEMPERATURE I'CI

Fig. 5.7. GC separation of acetone Schiff base-TMS derivatives of amines. Peaks: 1 = P-phenylethylamine; 2 = norephedrine; 3 = p-hydroxy-p-phenylethylamine;4 = tyramine; 5 = p-(3,4-dimethoxypheny1)ethylamine;6 = metanephrine; 7 = dopamine; 8 = epinephrine; 9 = normetanephrine; 10 = norepinephrine. Conditions: glass column, 6 ft. x 4 mm I.D., 10% F-60 on GasChrom P (80-100 mesh, AW, silanized); nitrogen inlet pressure, 12 p.s.i.; temperature programme, 11.S0C/min.(Reproduced from Anal. Chem., 38 (1966) 316, by courtesy of E.C. Horning and the American Chemical Society.)

tryptamine and related indole bases after their reaction with HMDS. Free primary amino groups were protected by acetone condensation. A mixed stationary phase (7% of F-60 with 1% of EGSS-Z) was used for the separation. The method was generalized for a number of biological amines [95]. A 0.5-1-mg amount of free amine in 0.05 ml of dimethylformamide is added to 0.15 ml of HMDS and the mixture is allowed to stand at room temperature for 30 min. By adding 1 ml of HMDS to 10 ml of acetone (or another ketone) and boiling, the ketone-HMDS reagent is prepared. A 0.4-ml volume of this reagent is added to the reaction mixture, which is then allowed to stand for 12 h. The precipitate is separated by centrifuging and 1-2 pl of the reaction mixture is injected directly into the chromatograph. The resulting chromatogram, obtained on a column with 10%of F-60, is presented in Fig. 5.7. Dimethyl sulphoxide with dioxane, used as a solvent for silylation, enables the reaction time to be reduced (10 min at 80°C) and condensation of dimethylformamide with some amines to be eliminated (it is this reaction in the above procedure that makes speed-up of the reaction by means of heating impossible) [96]. TMS derivatives of biogenic amines are used in combination with acyl derivatives for electron-capture detection. Horning et al. [97] presented retention data of TMS-N-acetyl and TMS-N-HFB derivatives of a number of these substances on SE-30,OV-1 and OV-17. The derivatives were prepared by the following procedure. A 1-mg amount of the aniine or amino hydrochloride was dissolved in 0.1 ml of acetonitrile and 0.2 ml of TMS-imidazole was added. After heating for 3 h at 60"C, 5 nig of N-acetylimidazole (or 0.1 ml of HFB-imidazole) were added and the solution was heated at 80°C for 3 h (30 min at 6OoC). The solution was used directly for the GC analysis. Schwedt and Bussemas [98] described a rapid method for the preparation of TMSTFA derivatives of 3-methoxytyramine, normetanephrine and metanephrine. A 20-p1 volume of a methanolic solution (concentration ca. 1 mg/ml) of amine or amine hydrochloride was evaporated to dryness at 60"C, then 50 pl of BSTFA were added and the solution was heated at 80°C for 5 min. After adding 5 p1 of N-methylbis(trifluoroaceta-

104

DERIVATIZATION OF COMPOUNDS

mide), the solution can be injected immediately. OV-17 (3%) is recommended as a suitable stationary phase for the separation of the derivatives.

5.3.3.Dinitrophenyl derivatives These are prepared by the reaction of primary or secondary amino groups with 2,4-dinitrofluorobenzene,and the main reason for their preparation is an increased sensitivity t o the ECD (Scheme 5.8). The reaction can be performed in aqueous solution and GC analysis can be carried out after removing the excess of the reagent and extracting the derivatives [99]. A 10-ml volume of the sample containing ca. 1 ,ug/ml of amine is pipetted into a 50-ml flask, 5 ml of a borate buffer (2.5% aqueous solution of Na2Bz0, . 10 HzO) and 2 ml of the reagent (2 ml of 2,4-dinitrofluorobenzene in 25 ml of p-dioxane) are added and the flask is heated on an air-bath at 60°C for 20 min. Then 2 ml of 2 N NaOH are added and the solution is heated for 30 min, cooled and transferred quantitatively into a 125-ml separating funnel with deionized water. Extraction is performed with 10 ml of cyclohexane and the extract is washed three times with 15 ml of 0.1 NNa2CO3and dried with anhydrous Na2S04.

NozQF

N 0, L

+

R / 1 H - N \ R2

-

No2-QNlR1

+ HF

\

NO, L

R2

Scheme 5.8.

This procedure leads, however, to different yields, particularly with low-molecular weight amines, which can make quantitative evaluation of the analysis difficult. Walle [ l o o ] carried out the derivatization after prior extraction of an aqueous sample (urine) with benzene. A 3-ml volume of the sample and 1 ml of an aqueous solution of an internal standard were mixed in a 50-ml centrifuge tube, 1 ml of 5 M K2C03and 30 ml of benzene were added and the mixture was shaken for 10 min. After the centrifugation, the benzene phase was separated and 25 ml of the benzene solution of the amine were mixed with a five-fold excess of 2,4-dinitrofluorobenzene dissolved in 1 ml of benzene. After standing for 5 min at room temperature, the solution was heated at 60°C for 15 min, cooled and 2-5 pl were chromatographed. The sensitivity of the analysis for different amines obtained by Walle for DNP derivatives using the ECD is demonstrated in Table 5.8. It varies in the range 2-20 pg. The reaction yields studied for diethyl-, mi.-butyl-, n-amyl- and di-n-butylamines are almost quantitative; the recovery of the extraction from an aqueous solution of diethyl-, isopropyl- and n-amylamines is also reported as being practically 100%. If this is the case with other amines also, the problem of quantitative analysis is thus made considerably easier. The extraction of amines with benzene prior to the derivatization makes possible their isolation from other substances that could react with the reagent (e.g., phenols, see p. 88) and make the analysis complicated. Weston and Wheals [ l o l l applied DNP derivatives to the determination of cyclohexylamine in cyclamates and in soft drinks at levels of 1 and 0.1 ppm, respectively. Isolation

105

AMINES TABLE 5.8 ECD SENSITIVITY TO 2,4-DINITROPHENYL DERIVATIVES OF AMINES [ 1001

Sensitivity is expressed as in Table 5.6. Conditions: 0.8% SE-30 + 0.1% NPGSe; carrier gas, nitrogen at 40 ml/min. Amine

Sensitivity (mol/sec)

Amine

Sensitivity (mol/sec)

Diethylamine tert. -Butylamine Isopropylamine 1-Methylpropylamine ti-Amylamine Di-n-butylamine Aniline

5.7.10-15 6.3 . lo-' 6.4 . lo-' 6.8 . lo-' 7.1 . lo-' 5.9.10-15 1.7 * lo-'

N-Methylaniline rn-Toluidine Benzylamine Amphetamine n-Nonylamine n-Decylamine

2.7 . 2.3 . lo-' 2.1. 1 0 - l ~ 2.0.10-14 2.3.10-14 1.8.10-14

of the amine by distillation from alkalinized sample preceded the derivatization reaction proper. DNP derivatives are substances sufficiently polar that their retention increases strongly during GC analysis with increasing polarity of the stationary phase. Non-polar stationary phases are therefore preferred. A typical chromatogram of a mixture of DNP derivatives of 12 aniines, obtained on 10%SE-30 with temperature programming, is shown in Fig. 5.8 [ 1021.

10

20

30

TIMEIMIN)

Fig. 5.8. Gas chromatogram of a mixture of 2,4dinitrophenyl derivatives of amines. Peaks: 1 = ammonia; 2 = diethylamine; 3 = isopropylamine; 4 = fur.-butylamine; 5 = sec-butylamine; 6 = isomylamine; 7 = ti-amylamine; 8 = TMSethanolamhe; 9 = aniline; 10 = cyclohexylamine; 11 = benzylm i n e ; 12 = P-phenylethylamine. Conditions: glass column, 3 m x 3 mm I.D., 10% SE-30 on Chromosorb W (60-80 mesh, AW, silanized); nitrogen flow-rate, 35 ml/min; temperature programme, 2"C/min (19O-22O0C), 3"C/min (220-250°C). (Reproduced from J. Chrottzafogr.,88 (1974) 373, by courtesy of S . Baba.)

106

DERIVATIZATION OF COMPOUNDS

GC analysis of different biogenic amines in biological tissues was carried out b y Edwards and Blau [ 1031 after converting them into N-2,4-dinitrophenyl-O-TMS derivatives. 2,4-Dinitrobenzenesulphonicacid (0.05 ml of 0.25 M solution in saturated sodium borate) was added t o either 0.05 nil of a standard solution of the amines containing 100-200 ng of each amine or t o a dried extract dissolved in 0.05 ml of water. Centrifuge tubes were stoppered and heated on a boiling water bath for 15 min and, after cooling, the derivatives were extracted with 0.4 and 0.2 ml of benzene. After centrifugation, the benzene phase was transferred with the aid of a Pasteur pipette into a 0.3-ml Microflex tube with a conical bottom and evaporated to dryness in a stream of dry nitrogen. The test-tubes were stoppered with Teflon-lined septa, 5-1.11 portions of BSA were added to each with the aid of a microsyringe and the tubes were heated a t 60°C for 30 min. After cooling, the excess of BSA and other volatile substances was removed under vauum for 1 h. In order t o protect the derivatives against hydrolysis, 1 1.11 o f 3SA was added and the solution was made up t o I0 pl with benzene. A 1-pl was injected for the GC analysis, which may be performed on a column packed with 1% OV-17 on Gas-Chrom Q at 230°C. Using this method, less than 0.1 ng of phenylethylamine can be determined. A microscale version of the procedure, however, allows the peak of the solvent and interferences caused by it to be minimized and only 30 mg of tissue are required for the analysis.

5.3.4. Isothiocyanates Isothiocyanates are prepared by the reaction of primary amines with carbon disulphide according t o Scheme 5.9. Brandenberger and Hellbach [lo41 made use of this derivative

Scheme 5.9.

Fig. 5.9. GC separation of isothiocyanate derivatives of amphetamines. Peaks: 1 = D-amphetamine; 2 = phenylethylamine; 3 = p-methoxyamphetami~e;4= 2J-dimethoxyamphetamine; 5 = 3,4-dimethoxyamphetamine; 6 = 3,4-dimethoxyphenylethyIamine;7 = 3,4,6-trimethoxyamphetamine; 8 = 2,3,4-trimethoxyamphetamine;9 = 3,4,5-trimethoxyphenylethylamine(mescaline). Conditions: column, 4 ft. X 4 mm I.D., 2.5%OV-225; carrier gas flow-rate, 40 ml/min; temperature programme, 2"C/min from 120°C. (Reproduced from Anal. Biochern., 45 (1972) 154, by courtesy of N. Narasimhachari and Academic Press.)

AMINES

107

to resolve amphetamine from methylamphetamine on SE-30, XE-60 and Carbowax 20M and described a method for the determination of these substances in urine. Other workers [105,106] made use of these derivatives even for the GC analysis of other biogenic amines and published many retention data (SE-30,OV-lOl,OV-225) and mass spectrometric data. Free hydroxyl groups present in some amines were protected by silylation. A solution containing 1 mg of a free base in 1 ml of ethyl acetate was shaken with 0.5 ml of carbon disulphide for 30 min. Under reduced pressure the solution was evaporated to dryness and the residue dissolved in 1 ml of ethyl acetate. Aliquots of 1 ~1 were taken for analysis. For phenolic and indolic amines 100-pg aliquots of isothiocyanate derivatives are subjected to reaction with a mixture of BSTFA and TMCS (99 : 1) at 90°C for 15 min. A typical chromatogram of isothiocyanates of different amphetamines is shown in Fig. 5.9.

5.3.5.Other procedures Oxidation can be applied in order to convert functional groups in the molecules of amines and thus give the substrate suitable chromatographic properties. Dimethylnitrosoamines were determined in smoked foodstuffs after oxidation to nitroamines [107]. A 1-5-pg amount of dimethylnitrosoamine in 2-10 pl of methylene chloride was added to 9 ml of a mixture of trifluoroacetic acid and 50% hydrogen peroxide (5 : 4). The solution was allowed to stand for 12-24 h, then poured over 10-15 g of ice, rendered alkaline by adding carefully 30-40 ml of 20% potassium carbonate solution and was extracted with two 50-ml portions of methylene chloride. The extract was dried with anhydrous sodium sulphate and concentrated to ca. 5 ml by evaporation on a water-bath. The concentrate was transferred quantitatively into a calibrated test-tube and 1 ml of n-hexane was added. Then it was further concentrated to 0.2 ml, made up to volume with n-pentane and 1-4 pl were injected. A high ECD response to nitroamines permits the analysis to be performed at the picogram level. Frere and Verly [lo81 oxidized normetanephrine and related amines with periodate in aqueous medium. The aldehydes produced were extracted with benzene and analysed by GC. Jenden and co-workers [109-1111 determined acetylcholine and other choline derivatives by GC after demethylating them with sodium thiophenolate. The reaction is described schematically by Scheme 5.10 and is carried out by using the following proceC H+I

0

3

"

C H - N - C H -CH - 0 - C - C H 3 ,

2

Scheme 5.10.

2

*

;" 1.5308 Iodomethane can be purified by shaking with dilute sodium carbonate solution and washing repeatedly with water. After preliminary drying with calcium chloride, the material is allowed to stand for I day over phosphorus pentoxide and fractionally distilled twice.

L-Menthol, m.p. 44-465°C; a;'--48.8 This can be purified by crystallization from chloroform, light petroleum or ethanolwater.

Methanol, b.p. 64.7"C; n&' 1.3284 Acetone (0.2%) is removed from methanol by treating it with sodium hypoiodite. Iodine (25 g) is dissolved in 1 1 of methanol and the solution slowly poured, with constant stirring, into 500 ml of I N sodium hydroxide solution. The iodoform is precipitated upon the addition of 150 ml of water. After standing overnight, the solution is filtered and the filtrate boiled under refliix until the smell of iodoform disappears. A single fractional distillation produces 800 ml of acetone-free methanol. Methanol containing no acetone is fractionally distilled through an efficient column, dehydrated with calcium hydride, and the distillation and drying are repeated three times. This process will yield about 50% of the starting material. One distillation of methanol over sodium reduces the water content to 0.003%; after the second distillation it is 0.00005%.

Pyridine, b.p. 115.3"C; n:; 1.5102 Pyridine can be dried by refluxing with solid potassium hydroxide, sodium hydroxide, calcium oxide, barium oxide or sodium, followed by fractional distillation. Other methods

PURIFICATION OF CHEMICALS AND SOLVENTS

219

of drying include standing with Linde type 4A molecular sieve, calcium hydride or lithium aluminium hydride, azeotropic distillation of the water with toluene or benzene, and treatment with phenylmagnesium bromide in diethyl ether, folllowed by evaporation of the ether and distillation of the pyridine. It can be stored in contact with barium oxide, calcium hydride or molecular sieve. Non-basic materials can be removed by steam distilling a solution containing l .2 equiv. of 20% sulphuric acid or 17%hydrogen chloride until about 10%of the base has been carried over together with the non-basic impurities. The residue is then made alkaline, and the base is separated, dried with sodium hydroxide and fractionally distilled. Silver oxide Silver oxide is prepared by pouring slowly and with stirring a hot, filtered solution of barium hydroxide octahydrate in water (1 : 10, w/v) into a hot solution of silver nitrate (1 : 5, w/v) and filtering the precipitated silver oxide. After thorough washing with hot water the product is dried in a vacuum oven at 60°C and stored in a tightly closed dark container. Freshly prepared, material is recommended.

Tetrahydrofuran,b.p. 66.0"C; nko 1.4072 Commercial material is allowed to stand for 48 h over freshly fused sodium hydroxide and 24 h over sodium wire, over which it is refluxed. It is fractionally distilled in an atmosphere of dry nitrogen, and finally vacuum distilled from lithium aluminium hydride, the last 25% being rejected.

Tetramethylammonium hydroxide, pentahydrate, m.p. 63°C (decomposition) This can be freed from chloride ions by passage through an ion-exchange column (Amberlite IRA-400, prepared in its hydroxide form by passing 2 M sodium hydroxide solution until the effluent is free from chloride ions, then washed with distilled water until neutral).

Triethylamine,b.p. 89.5"C; nko 1.4010 Commercial anhydrous triethylamine is distilled from acetic anhydride to remove trace amounts of primary and secondary amines, dried with activated alumina and distilled three times under reduced pressure. Preliminary drying may be carried out by storing the solvent with solid potassium hydroxide.

REFERENCES 1 D.D. Perrin, W.L.F. Armarego and D.R. Perrin, Purification of Laboratory Chemicals, Pergamon Press, Oxford, 1966. 2 J.A. Riddick and W.B. Bunger, in A. Weissberger (Editor), Techniques of Chemistry, Vol, II, Organic Solvents, Physical Properties and Methods of Purification, Wiley-Interscience, New York, 1970.

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Appendix 2

A list of some suppliers of reagents and accessories for derivatization Supplier

Reagents and accessories

Aldrich-Europe, Turnhoutsebaan 30, B-2340 Beerse, Belgium

Silylation reagents Acylation reagents Esterification and alkylation reagents Accessories

Alltech Associates Inc., 202 Campus Drive, Arlington Heights, IL 60004, U.S.A.

Silylation reagents Esterification reagents Other derivatizing reagents Various reaction (micro)vials and other accessories

Analabs Inc., A Unit of Foxboro Analytical, 80 Republic Dr., North Haven, CT 06473, U.S.A.

Silylation reagents Esterification reagents Reaction vials and other accessories

Applied Science Laboratories Inc., P.O. Box 440, State College, PA 16801, U.S.A.

Silylation reagents Esterification and alkylation reagents Acetylation and other reagents Vials and accessories

J.T. Baker Chemical Co., 222 Red School Lane, Phillipsburg, NJ 08865, U.S.A.

Acylation reagents Esterification and alkylation reagents Chelating reagents

BDH Chemicals Ltd., Poole, Dorset BH12 4 NN, Great Britain

Silylation reagents Acylation reagents Esterification and alkylation reagents Chelating agents

Carlo Erba, Chemicals Division, Via Carlo hnbonati 24, 20159 Milan, Italy

Silylation reagents Esterification and alkylation reagents

Chrompack Nederland B.V., P.O. Box 3, Middelburg, The Netherlands

Silylation reagents Acylation reagents BF3/Methanol reagents Reaction vials and accessories

Eastman Kodak Co., Eastman Organic Chemicals, 343 State St., Rochester, NY 14650, U.S.A.

Silylation reagents Acylation reagents Esterification and alkylation reagents Chelating agents

The names of manufacturers and products mentioned throughout this book reflect solely the personal experience of the authors and do not constitute any preferential endorsement or recommen221 dation.

222

APPENDIX 2

Supplier

Reagents and accessories

Fisher Scientific Co., 71 1 Forbes Ave., Pittsburgh, PA 15219, U.S.A.

Common silylation reagents Esterification and alkylation reagents

Fluka AG, CH-9470 Buchs, Switzerland

Silylation reagents Acylation reagents Esterification and alkylation reagents Specialized reagents

ICN Pharmaceuticals Ihc., K&K Labs Division, Life Science Group, 121 Express Street, Plainview, NY 11803, U.S.A.

Silylation reagents Acylation reagents Esterification and alkylation reagents Chelating agents Others

Koch-Light Laboratories Ltd., 2 Willow Rd., Colnbrook, SL3 OBZ Buckingharnshire, Great Britain

Silylation reagents Acylation reagents Esterification and alkylation agents Chelating agents

E. Merck, Frankfurter Str. 250, Postfach 41 19, D-61 Darmstadt, G.F.R.

Silylation reagents Esterification and acylation reagents Chelating agents

Packard Instrument Co., Inc., 2200 Warrenville Rd., Downers Grove, IL 605 15, U.S.A.

Silylation reagents Esterification and alkylation reagents Anhydrides and other reagents Reaction vials and other accessories

PCR Research Chemicals Inc., P.O. Box 1778, Gainesville, FL 32602. U.S.A.

Silylation reagents Acylation reagents Esterification and other reagents Chelating agents

Pierce Chemical Co., P.O. Box 117, Rockford, IL 61 105, U.S.A.

Silylation reagents Acylation reagents Esterification and alkylation reagents Specialized reagents Various reaction vials and accessories

Serva Heidelberg, Karl-Benz-Str. 7, D-6900 Heidelberg 1, G.F.R.

(Perfluoro)acyl anhydrides Common silylation reagents Other derivatizing agents Reaction tubes for GC-derivatization

Supelco Inc., Supelco Park, Bellefonte, PA 16823, U.S.A.

Silylation reagents Acylation reagents Esterification and alkylation reagents Special reagents Reaction vials and other accessories

Subject index

A

-, -,in fruits 121 -, -,in vanilla extract 121 -, -,in wines 121 -, -, tuluides 123

Acetals, dimethyl, of aldehydes 97 -, of sugars 174 Acetates, aldonitrile, of sugars 173 -, in tissues 115 -, of alcohols 84 -, of alditols 167, 171 -, of amines 97 -,of amino sugars 167 -, of carbamates 178 -, of chlorophenols 84 -, of phenols 84 -,of steroids 157 -, of vitamin A 185 Acetic anhydride, purification 21 3 Acetone, halogenated, reaction with amino acids 141 -, purification 213 Acetonides 77, 163 Acetonitrile, purification 21 3 Acetylacetone, chelates 194 -, condensation with amino acids 140 -,purification 213 N-Acetyl alkyl esters of amino acids 127, 128 Acetylation, of amino acids, evaluation 129 Acetylcholine 107 Acetylsalicylic acid, TMS 120 Acid, ascorbic, TMS 185 -, cacodylic 191 -, formic 123 -, homovanillic 117 -, 3-methoxy-4-hydroxymandellic, in urine 124 -, salicylic 120 -, shikimic 119 -, vanillylmandellic 121 Acids, ddonic 166 -, amino see Amino acids -, aromatic, in urine, extraction 17, 116 -, -, trichloroethyl esters 116 -, bifunctional, n-butyl boronates 123 -, bile, TFA-methyl esters 158 -, -, TMS methyl esters 153 -, carboxylic, anilides 123 -, -, disiloxane esters 122 -, -,esterification 111 -, -, in biological samples 113

-, chlorophenoxy, methyl esters

181

-, -, propyl esters 181 -, -,pentafluorobenzyl esters 182 -, -, trichloroethyl esters 181 -, dihydroxybenzoic, bis-TMS methyl esters 112

_ , _ ,diacetoxymethyl esters

1 12

-, fatty, in fats and oils 121 -, -, in triglycerides 62, 113 -, hexuronic 170

-, hydroxy, TMS methyl esters 121 -, keto, DNPHs 122

-, -, quinoxalinones 124 -, Krebs cycle, TMS 118 -, -, methoxime TMS esters 119 -, phenolic, alkyl esters 114 -, -, DMS derivatives 122 -, -, sulphonyl-TMS derivatives 120 -, -, TMS derivatives 120

-, -,TMS methyl esters 116 -, phenylcarboxylic, ethyl esters 112 -,resin 121

-, sulphonic, esterification 110 Acetonides of steroids 163 Acylation, catalysts 84 -,methods 66 -, on-column 66 -,reagents 67 -, selective, of amino groups 67 Adenosine, silyl derivatives 175 Alcohols 84 -, acetates 84 -, derivatives for identification 87 -, enantiomers, GC separation 90 -, phosphoruscontaining derivatives 91 -, silylation 88 Aldehydes 92 -, dimethylacetals 97 -, DNPHs 92,95 Alditols, methyl ether acetates 167 Aldonitrile acetates, of sugars 173 Aldoses, in urine 173 Aldosterone, methoxime HFB 162 Alkylation, agents 61 223

2 24

-,extractive, theory 59 -, flash heater 59 -, of barbiturates 183 -, of carbamates 178 -, of halides 188 -, of organophosphates 180 -, of triazines 180 Alphenal 184 Aluminium, chelates 194 -,in alloys 195 -,in mouse liver 195 -,in sea water 195 Amines 97 -, acetates 97 -,aromatic, TFA 98,99 -, biogenic, acetone SB TMS 103 -, -, dinitrophenyl TMS derivatives 106 -, -, propionates 97 -, -, silylation 101 -, -, TMS acyl derivatives 103 -, condensation with benzaldehydes 108 -, dinitrophenyl derivatives 104 -, extraction from tissues 17 -, GC as urethanes 108 -, in aqueous samples 104 -, silyl derivatives 101 Amino acids 126 -, N-acetyl alkyl esters 127, 128 -, N-benzylidene methyl esters 140 -, condensation with diketones 140 -, dinitrophenyl methyl esters 145 -, GC as, diketopiperazines 140 -, -, methylthiohydantoins 142 -, -, morpholinones 141 -, -, oxazolidinones 141 -, -, oxazolinones 141 -, -, thiohydantoins 142 -, N-HFB isoamyl esters 135 -, N-HFB n-propyl esters 134 -, in sea water 138 -,inserum 145 -, N-isobutylidene methyl esters 140 -, N-isobutyloxycarbonyl methyl esters 135 -, isopropyl derivatives 146 -, N-pentafluorobenzyl2-butyl esters 147 -, NPFP n-butyl esters 134 -, N-propionyl isoamyl esters 136 -, Schiff bases 140 -, silylation 136 -, N-TFA alkyl esters 129 -, -,volatility 133 -, N-TFA n-amyl esters 132 -, N-TFA 2-butyl esters 147 -, N-TFA Lmenthyl esters 147

SUBJECT INDEX

-, N-TFA-L-prolyl methyl esters 147 -, N-TMS alkyl esters 139

-, N-thiocarbonyl alkyl esters 145 Aminochromes, TMS 90 Amino sugars, in mucins 172 -, methyl ether acetates 167 Amitriptyline 187 Amphetamine, in urine 107 -, isothiocyanate 106 Amy1 esters of amino acids 127 Anabolic steroids, in urine 153 Analytical methods, combination with GC 38 Androstane, in urine 155 Androstanediols, comparison of derivatives 154 -,in urine 12, 162 Androstanol-3p, 16- and 15-keto isomers, separation 4 Androsterone, comparison of derivatives 165 -,TMS 152 Anilides, of carboxylic acids 123 Anions, non-oxygencontaining 188 -, oxygencontaining 189 Anthocyanines, TMS ethers 90 Antibiotics 184 Anticholinergics 186 Anticonvulsant drugs 187 Antihistaminics 187 Aromatic amines, TFA 98 Arsenate, TMS 190 Arsenic, triphenylarsine 194 Atrazine, TMS 180 Atropine 186 B Barbiturates 182 -, alkyl derivatives 183 -, aryl derivatives 183 -, in blood 183 Benzaldehyde, condensation with amino acids 140 -, purification 213 -, sulphur-containing derivatives 95 Benzene, purification 214 Benzoates, of thiols 109 N-Benzoyl methyl ester, of glycine 136 Benzyl alcohol, purification 214 Benzylamine, acyl derivatives, comparison 69 Benzyl esters 63, 115, 117 N-Benzylidene methyl esters, of amino acids 140 Benzyloximes, of steroids 162

SUBJECT INDEX Beryllium, chelates 195 -,inair 195 -, in blood 195 -,in plant extracts 195 -,in urine 196 Bile acids, TFA methyl esters 158 -, TMS methyl esters 153 Borate, TMS 190 Boronates 76 -, alkyl, of ceramides 91 -, n-butyl, of bifunctional acids 123 -, of steroids 164 Boron fluoride, GC 192 Boron trichloride, purification 214 Boron trifluoride, purification 214 Bromides 188 -, GC as dibromocyclohexane 189 2-Bromopropane, purification 215 Butabarbital 184 Butanol, purification 215 Butoximes, of steroids 162 Butyl derivatives, of barbiturates 183 n-Butyl esters, of amino acids 127 see also Esters 2-Butyl esters, of amino acids 147 see also Esters

C Calibration methods 43 Cambendazole 187 Carbamates, insecticidal 178 Carbohydrates, see Sugars Carbonate, TMS 189 Carbon disulphide, purification 215 Carbon tetrachloride, purification 216 Carbonyl compounds, 2,4-DNPHs 92 -, - , retention indices 94 -, Girard T derivatives 92 -, in cigarette smoke 94 -, in exhaust gase 96 -,isolation from foodstuffs 9 2 -, liberation from DNPHs 92 -, pentafluorophenylhydrazones 95 -, phenylhydrazones 92 -, 2,4,6-trichlorophenylhydrazones95 Cardenolide steroids, silylation 153 Catecholamines, pentafluorobenzoates 100 -, silylation 102 Ceramides, alkyl boronates 91 Chelate-forming agents 194, 198 Chelates of metals 194 Chemical reaction of eluates 34

225 Chloramphenicol, TMS 184 Chlorides 188 -,of metals 191 Chlorination of triazines 180 Chloroacetates of phenols 86 Chloroethyl esters 63, 116 see also Esters Chloroform, purification 216 Chlorophenols, acetates 84 -,ethyl ethers 87 -, extraction from water 17 -, GC separation 85 -, in water 84,87 Chlorthalidone 187 Cholesterol, fluoroalkylsilyl ethers 156 -, haloacyl derivatives, comparison 158 -, methyl ethers 164 -, silyl ethers, comparison 156 -,TMSethers 3 Choline 107 -,in brain tissue 108 Chromium, chelates 196 -, in blood 196 -, in serum 196 -,in steel 196 -, in tissues 196 -,inurine 197 Chrysanthemoyl derivatives, of alcohol enantiomers 90 Cobalt, chelates 197 Codeine, TMS 186 Combination of GC, with analytical methods 38 Copper, chelates 194 -, in alloys 197 -, in tissues 197 -, in water 197 Correlation of retention data 26 Corticosteroids, acetates 157 -, oxidation 165 -, TMS derivatives 153 Cyanides 189 Cyanozine 181 Cyclazocine 186 -,inurine 1 2 Cyclohexylamine, in blood 98 -, in cyclamates 104 -,in soft drinks 104 Cytidine, methoxime TMS derivatives 176 Cytosine, TMS 175

226

D Deactivation, of glass capillary columns 22 -, of surface of glass vessels 21 Dehydroepiandrosterone, in human plasma 162 Derivatives, for selective detection 5 -, preparation 19 - ,_ ,reaction vials 20 -, -, micro-refluxer 20 -, reasons for use in GC 1 Detectors, selective 5 , 36 Dexamethasone, methoxime TMS derivatives 161 Diaminocyclohexane, TMS 98 4,4’-Diaminodiphenyl sulphone 187 Diazoalkanes 114 -, esterification of amino acids 127 Diazomethane, preparation 54 Diazotoluene, preparation 115 Dibutylsulphoxide 198 Dichloromethane, purification 216 Diethyl ether, purification 216 Digoxin, in plasma 160 Dimethylformamide, purification 216 Dimethyl sulphoxide, purification 217 Dinitrophenyl derivatives, of anunes 104 -, -, GC, ECD sensitivities 105 -, of thiols 109 Dinitrophenyl ethers, comparison of methods 65 -, of phenols 65,87 _ , _ , preparation on the micro-scale 88 Dinitrophenylhydrazones 76 -, of aldehydes 95 -, of carbonyl compounds 92 -, - ,GC separation 93 Dinitrophenylhydrazine, purification 21 7 Dinitrophenyl methyl esters, of amino acids 145 Dinitrophenyl TMS derivatives, of biogenic amines 106 Diphenylthiohydantoin, in serum 13 N,O-Dipivalyl derivatives, of thyroid hormones 68 Disiloxane esters, of carboxylic acids 122 Distribution constant 27 -, characteristic of solute 27 -, characteristic of sorbent 27 -, Gibbs function of sorption 27 _ , _ ,Martin’s additivity theorem 28 Dithizonates 182 Dithizone, purification 217 DMS ethers, of phenols 90

SUBJECT INDEX Dodecylamine, fluoroacyl derivatives 100 Dopamine, in brain tissue 11, 101 Drimanoyl derivatives, of alcohol enantiomers 90 drugs, anticonvulsant, in serum 13 -, -, GC 182 Drying of the sample, procedures 16

E Enantiomers of, GC separation -, alcohols 90 -, amino acids 146 -, -, chiral reagents 148 -, carboxylic acids 125 Epitestosterone, acetate 157 -, methoxime TMS derivative 161 l,2-Epoxyalkanes 198 Epoxyglycerides 198 Esterification 54 -,comparison of methods 56,57,58,63 -, decomposition of quaternary ammonium salts 58 -, diazomethane method 54 -, ,on micro-vxtie 112 -, methanol-BF3 method 5 5 -, methanol-HCI/HzS04 method 56 -, on an ion exchanger 57 Esterification, of phosphates 190 -, of thyroid hormones 149 see also Esters - , with alkyl halide -silver oxide 112 -, with N,N’-carbonyldiimidazole 6 1 -, with diazoalkanes 63, 114 -, with diazotoluene 115 with N,N‘dicyclohexyl-0-benzylisourea 115 -, with N,N’dimethylformamide dialkylacetals 61 -, with higher alcohols 61 -,with inethyl iodide 60 -, with trichloroethanol 117 -, with triethyl orthoformate 110 Esters, amyl, of amino acids 127 -, benzyl 6 3 -, -, of fatty acids 115 -, -, preparation on the micro-scale 1 17 -, p-bromophenacyl 116 -, butyl 63 ~, ,of amino acids 127 -, -, of carboxylic acids 114 -, chloroethyl 63, 116 -, disiloxane, of carboxylic acids 122 -, ethyl, of amino acids 127

-.

SUBJECT INDEX

-, --, of phenylcarboxylic acids 112 -, -, of sulphonic acids 110 -, GC, selection of stationary phase 64

-, hexafluoroisopropyl 63 -, L-menthyl 6 3

-, -, of amino acids 147 -, -, of carboxylic acids 125 -,methyl 54

-, -,of amino acids 127,129,130 -, -,of bile acids 153 -, -, of carboxylic acids 111 -, -, of chlorophenoxy acids 181 -, -,of sulphonic acids 110 -, -, of thyroid hormones 149 -, methoxime TMS, of Krebs cycle acids 119 -, pentafluorobenzyl 1 1 7

-, -, of chlorophenoxy acids 182 -, p-phenylphenacyl 116 -, propyl 63, 113

-, -, of amino acids 127 -, TMS, of inorganic anions 190 -, -, of carboxylic acids 118 -, trichloroethyl, of aromatic acids 116 -, -, of chlorophenoxy acids 181 Estrogens, GC separation 4 HFB derivatives 159 Ethanol, purification 217 -, 2-(2ethoxyethoxy), purification 21 7 Ethanolamines, TFA derivat'ves 98 Ethers, dinitrophenyl 65, F I -,methyl 61, 164, 166,87 -, pentafluorobenzyl 64 Ethyl acetate, purification 21 7 Ethyl esters, see Esters Ethyl ethers, of chlorophenols 87 Ethyl trifluoroacetate, acylation with 98 Etiocholanolone, TMS derivative 152 Extracticn 16, 17 Extraction, of amines from tissues 17 -, of aromatic acids from urine 17 -, of cyclazocine from urine 14 -, of diphenylhydantoin from urine 15 -, of dopamine from brain tissue 11 -, of norepinephrine from brain tissue 11 -, of pentachlorophenols from water 17 -, of phenobarbital from serum 15 -, of phenols from urine 17 -, of primidone from serum 15 Extractive alkylation, theory 59

-.

F Fatty acids, see Acids Flavonoides, TMS 90

227 Flavonoid glycosides, TMS 170 Fluorides, GC as trialkylfluorosilanes 188 -, of metals 191 Fluorocarbonsilyl ethers, of sterols 155 Formic acid 123 Functional group classification tests 35 Fungicides, organomercury 182 Furosemide 187

G Galactosamine 169 Galactose, in blood 170 Germanium, chloride 192 Girard T reagent, preparation 92 -, purification 2 17 Glucosamine, TMS 169 Glucose, in blood 170 Glycerol, tribenzoyl derivative 85 Glycine, N-benzoyl methyl esters 136 Glycols, TMS ethers 90 Glycosides, methanolysis 166 Guanine, TMS 175

H Halides, GC as, haloethanols 188 -, -, alkyl halides 188 -,of metals 191 Heterocyclic derivatives 77 Hexafluoroisopropyl esters 63 2,5-Hexanedione, condensation with amino acids 140 Hexobarbital 184 Hexosamines, in body fluids 174 Hexuronic acids 170 N-HFB n-butyl esters, of amino acids 134 HFB derivatives, of amines 100 -,of carbarnates 178 -, of phenols 86 -, of steroids 159 -, of triazines 181 -, of vitamin D 185 N-HFB isoamyl esters, of amino acids 135 N-HFB methyl esters, of thyroid hormones 149 N-HFB n-propyl esters, of amino acids 134 Hydrazine, GC as pyrazoles 108 Hydrazones 76 -, of keto acids 122 -, of steroids 162, 163 Hydrox ycorticosteroids, bismethylenedioxy derivatives 163

228

I Identification 26 -, carbon skeleton GC 34,36 -, chemical reactions of GC eluates 34 -, combination of GC with, analytical methods 38 -, -, infrared spectroscopy 39 -, -, mass spectrometry 39 -, -,PMR spectrometry 39 -, -, thin layer chromatography 38 -, correlation of retention data 26 -, -,boiling point of solute 31, 32, 33 -, -, carbon number of solute 27,29 -, -,two different sorbents 28, 30, 31 -, distribution constant, major parameters 27 -, -,physicochemical meaning 27 -, -, second order parameters 27, 39 -, group classification reaction with eluates 34,35 -, hydrogenolysis of eluates 34 -, ozonolysis of eluates 35 -, reaction GC, elemental analysis 36,37 -, retention behaviour 27, 39 -,retention index 32, 33, 34 -, selective detectors, acid-base titrator 37 -, -, alkali flame-ionization detector 38 -, -, electrolytic conductivity detector 37 -, -, electron capture detector 38 -, -, flame photometric detector 38 -, -, microcoulometric detector 37 _ , - ,microwave emission detector 38 -, subtractive techniques 35 Imidazoles, silyl derivatives 101 Indoles, silyl derivatives 101 Injection system, falling needle 22 -, for DNPHs 77 -, for silyl derivatives 72 Insecticides 177 Iodides 188, 199 -,in milk 199,189 Iodine, GC as iodoacetone 199 -,inmilk 199 Iridium, fluoride 192 Iron, chelates 194 -, in ores 197 N-Isobutylidene methyl esters, of amino acids 140 0-Isobutyloxycarbonyl derivatives, of phenols 85 N-Isobutyloxycarbonyl methyl esters, of amino acids 135 Isobutyraldehyde, condensation with amino acids 140

SUBJECT INDEX Isopropyl derivatives, of amino acids 146 Isopropyl esters 6 3 Isothiocyanate, reaction with amino acids 78

K Kanamycin, TMS 184 Kestose, silylation 169 Ketals, of sterols 164 -, of sugars 174 Keto acids, DNPHs 122 -, quinoxalinones 124 Krebs cycle acids, methoxime TMS esters 119 -, TMS derivatives 118

L Lanthanides, chelates 198 Lead, chelates 197 Leucine, N-palmitoyl ethyl ester 136

M Malonaldehyde, condensation with urea 78, 96 -, in biological samples 96 Mandellic acid, 3-methoxy-rl-hydroxy, in urine 124 -, vaniuyl 121 Matrix composition, simulation 48 Matrix effects, elimination 48, 49, 50 L-Menthol, derivatives, retention data 89 -, esterification with 61 -, purification 218 L-Menthyl chloroformate, preparation 125 L-Menthyl esters 63 -, of amino acids 147 -, if isoprenoid acids 125 L-Menthyloxycarbonyl derivatives, of hydroxy acids 125 Mephobarbital, butyl derivatives 183, 184 Mercury, organo, GC 193 Metal chelates 194 Metal halides 191 Metals, in alloys 192 Methanephrine, HFB derivatives 100 -, TMS-TFA derivatives 103 Methanol, purification 218 Methanol-BF3 reagent, preparation 55 2 N Methanolic base, transesterification with 62

SUBJECT INDEX Methanolysis, of glycosides 166 -, of oligosaccharides 166 Methoxime TMS derivatives, of antibiotics 185 -, of Krebs cycle acids 119 -,of steroids 161 Methoxylation, of triazines 181 Methylation, of barbiturates 183 -,of carbamates 179 -, of cholesterol 164 -, of nucleic acids components 177 -, of phenols 87 -, of sugars 166 Methylbenzyl amine, acyl derivatives, comparison 69 Methyl derivatives, see Methylation Methyl esters, of thyroid hormones 149 see also Esters Methyl ethers, preparation 64 see also Methylation Methylglycosides, TFA 167 Methyl iodide, esterification with 60 -, methylation with 166 -,purification 218 Methylthioaniline, condensation with benzaldehyde 95 Methylthiohy dan toins 143 Molybdenum, fluoride 192 Morphine, in biological samples 186 -, TMS derivative 3,186 Morpholinones 141

N 2-Naphthylamine, in 1-naphthylamine 97 Neomycin, TMS 184 Neutral sugars, in glycoproteins 171 -, in mucins 172 Nickel, chelates 197 Nitrates, GC as nitrobenzene 190 Nitrosoamines, in smoked foodstuffs 107 Norepinephrine, in brain tissue 11, 101 Normetanephrine, HFB derivatives 100 -, TMS-TFA derivatives 103 Nucleosides, TMS 175 Nucleotides, TMS 175

0 Oligosaccharides, methanolysis 166 Organomercury 193 Osmium, fluoride 192 Oxalate, TMS 190

229 Oxazepam 187 Oxazolidinones, formation 78, 14 1 -, silylation 142 Oxazolinones, formation 78, 141 Oxidation, of corticosteroids 165 -, of organophosphates 180 -, of sugars 170 Oximes 75 -, GC, decomposition 96 20-Oxopregnanes, acetates 157

P Paromycin, TMS 184 Peak area, analytical significance 4 1 Peak height, analytical significance 4 1 Penicillins, TMS 184 Pentachlorophenol, in water 17, 84, 87 Pentafluorobenzoates, of phenylphenols 87 N-Pentafluorobenzoyl 2-butyl esters, of amino acids 147 Pentafluorobenzyl derivatives, of barbiturates 183 Pentafluorobenzyl ethers, preparation 64 Pentafluorophenylhydrazones of carbonyl compounds 95 Pentafluorophenylthiohydantoins 144 Pentafluorotolyl derivatives, of thiols 109 Pentafluorotolyl ethers, of phenols 87 Pentazocine 186 Pentyloximes of steroids 162 Peptides, Edman degradation 142 -, sequential analysis 142 Pesticides 177 -, organochlorine 180 -, phenolic, silylation 182 Pethidine 187 N-PFP n-butyl esters of amino acids 134 Phenobarbital, butyi derivatives 183, 184 -, in serum 13 Phenolic acids, DMS derivatives 122 -, sulphonyl-TMS derivatives 120 Phenols, acetates 84 -, chloroacetates 86 -, dinitrophenyl ethers 65, 87 -, dinitrotrifluormethylphenyl ethers 87 -, -, preparation on the micro-scale 88 -, DMS, TMS ethers 90 _ , - ,comparison 74 -, extraction from urine 17 -, H F B derivatives 86 -,in urine 86 -.in water 86

230

-,0-isobutyloxycarbonyl derivatives 85 -, methyl ethers 87 -, pentafluorotolyl ethers 87 -, silylation 88 -, -,comparison 89 -, trifluoroacetates 86 Phenyldiazomethane, esterification with 63 Phenylethylamine, derivatives, comparison 99 Phenylhydrazones 76,93 o-Phenylphenols, pentafluorobenzoates 87 Phenylthiohydantoins 144 Phenytoin 187 Phosphates, TMS 190 Phosphorus-containing derivatives, of alcohols 91

-,of carboxylic acids 118 -,of steroids 160 Piperazines, diketo 140 Pivalaldehyde, reaction with thioamines 109 N-Pivalyl methyl esters of thyroid hormones 149 Plant steroids, acetates 157 Platinum, fluoride 192 Pregnanediols, comparison of derivatives 154 -, in urine 12 Primidone, in serum 13 Processing of chromatograms, automatic 46 -,manual 46 Progesterone, acetate 157 Propazine, TMS 180 Propionates, of biogenic amines 97 N-Propionyl isoamyl esters, of amino aicds 136 Propranolol 187 Propylene oxide, reaction with amino acids 141 Propyl esters 63, 113 -,of amino acids 127 see also Esters Prostaglandins 186 Pseudoephedrines, in blood 100 Pseudouridine, separation of anomers 176 -, TMS 175 Pteridines, TMS 198 Purine bases, TMS 175,177 -, -, retention indices 176 Purines, silyl derivatives 101 Pyridine, purification 218 Pyridine bases, acylation 198 Pyrimidine bases, TMS 175, 177 -, - ,retention indices 176 Pyrimidines, silyl derivatives 101

SUBJECT INDEX

Q Quantitation 40 -, calibration methods 43 -, -,absolute calibration 44 -, -, internal normalization 45 -, -,internal standard 44,48 - ,_ ,notation 43 -, -, standard additions 44 -, expressing concentration 43 -, general concepts 40 -,nature of detector 4 1 -,peakuea 4 0 -, peak height 40 -, processing of chromatograms 46 -,relative specifrc response 42 -, response factors 4 1 -, special problems 47 -, -,matrix effects 47,48,49,50 -, -, preparative operations 47 -, -, recovery of analytes 47 -, -,reference model systems 48,49,50 -, -, standard additions method 49,50 -, specific response 42 Quinoline bases, acylation 198 Quinoxalinones, formation 124

R Reference model systems 48 -, internal standard method 48 -, standard additions method 49,SO Relative specific response 42 Resin acids 121 Resins, polyamide, components 101 Response factors, determination 41,42,43 -,molar 43 Retention behaviour 26,39 -, correlation with properties of solutes 26 -, distribution constant 27 -, physicochemical bases 26 Retention index 32,33, 34 -, thermodynamic significance 33 -, two different sorbents 33 Rhenium, fluoride 192 Ruthenium, thiosemicarbazide 193

S Saccharides, see Sugars Salicylic acid, silylation 120 Sampling, representative 10

SUBJECT INDEX Secobarbital 184 Selective detection, derivatives for 4 Selective detectors 36 Selenium, fluoride 192 -, GC as piazselenol 193 Silanization of, surface of glass vessels 21 Silicates, in siliceous rocks 189 Silicon, chloride 192 Siliconides, formation 77 -, of steroids 163 Silver oxide, catalyst 64,112 -, preparation 219 Silylation, conditions 72 -, of alcohols and phenols 88 -, of alkaloids 186 -,ofamines 101 -, of amino acids 136 -, -, methods comparison 137 -,of antibiotics 184 -, of carbamates 178 -, of carboxylic acids 118 -, of inorganic anions 189 -, of nucleic acids components 175 -, of organophosphates 180 -, of pharmaceuticals 186 -, of phenolic pesticides 182 -, of pteridines 198 -,of steroids 152 -, of sugars 168 -, of sugar phosphates 170 -, of thyroid hormones 150 -, of Mazines 180 -, of vitamins 185 -, on a trapping column 72 -, oncolumn 72, 186 Silyl derivatives, decomposition on GC column 73 -, of steroids, comparison 74, 155, 156 -, reagents 70,71 -,stability 72 see also TMS derivatives Simazine, TMS 180 Specificity of detection, response f x t o r 4 1 Specific response 42 Steroids 151 -,acetates 157 -, acetonides 77, 163 -, bis-methylenedioxy derivatives 163 -, boronates 164 -, chloromethyldimethylsilyl derivatives 154 -, comparison of silyl derivatives 74, 155,156 -,dimethylthiophosphonic derivatives 160 -, HFB derivatives 159 -, hydrazones 162, 163

231

-, methanesulphonyl derivatives 160 -,oximes 160 -,profdes in urine 161 -, siliconides 163 -, TMS derivatives 151 -, trialkylsilyl derivatives 156 -, trifluoroacetates 158 Sterols, acetates 157 -, fluorocarbonsilyl ethers 155 -, HFB derivatives 159 -,ketals 164 -, methyl ethers 164 -, TMS ethers 154,156 Stilbenes, hydroxy, TMS ethers 90 Sugar phosphates, TMS 169,170 Sugars 165 -, acetals 174 -,acetates 171 -, aldonitrile acetates 173 -, in fruits 170 -, in polysaccharides 171 -, in sea water 174 -, ketats 174 -, methyl ethers 166 -, oxidation 170 -, reduction 171 -, trifluoroacetates 173 -, TMS derivatives 168 Sulphate, TMS 190 Sulphonic acids, esterification 110 Sulphonyl TMS derivatives, of phenolic acids 120 Sulphur, fluoride 192 Sulphurcontaining derivatives, of benzaldehyde 95 -, of steroids 160

T Technetium, fluoride 192 Testosterone, acetate 157 -, derivatives, comparison 165 -, epimers, separation 4 -, haloacyl derivatives, comparison 158 -, HFB derivatives 159 -,inblood 157 -,in urine 157,162 -, methoxime TMS derivatives 161 -, silyl derivatives, comparison 155 Tetracycline, TMS 184 Tetrahydrofuran, purification 219 Tetramethylammonium hydroxide, purification 219

232 N-TFA namyl esters, of amino acids 132 N-TFA 2-butyl esters, of amino acids 147 N-TFA n-butyl esters, of amino acids 130, 131 -, -, GC separation 131 N-TFA Lmenthyl esters, of amino acids 147 N-TFA methyl esters, of amino acids 129 -, of thyroid hormones 149 N-TFA-L-prolyl methyl esters, of amino acids 147 Thiamphenicol, TMS 184 Thiazide, hydrochloro 187 N-Thiocarbonyl alkyl esters, of amino acids 145 Thiohydantoins 78 -, silylation 142 Thiols, derivatives 109 Thiosemicarbazide, of ruthenium 193 Thorium, chelates 198 Thymol derivatives, comparison 6 8 Thyroid hormones 148 -, acyl methyl esters 149 -, N,Odipivalyl derivatives 68, 149 -, in serum 149, 150 -, silyl derivatives 150 Tin, chloride 192 Titanium tetrachloride 192 TMS acyl derivatives, of biogenic amines 103 TMS derivatives, of amino acids 136, 138 -, - ,GC separation 138 -, of aminochromes 90 -, of antibiotics 184 -, of phenolic acids 120 -, of thyroid hormones 150 TMS ethers, of alcohols and phenols 88 -, of anthocyanines 90 -, of glycols 90 -, of flavonoids 90 -, of hydroxystilbenes 90 -,of sterols 154, 156 -, of sugars 168 N-TMS ethyl esters, of amino acids 139 TMS methyl esters, of amino acids 139 -, of aromatic acids I16 -, of bile acids 153 -, of hydroxyacids 121 see also Silylation Tolmetin 187 Toluides, of carboxylk acids 12 4 -, -, GC separation 124 Transesterificntion, methods 62 Trialkylsilyl derivatives, of steroids, GC-MS 156

SUBJECT INDEX Triazines 180 -, HFB derivatives 181 -,in foodstuffs 181 -, methoxylation 181 Tribenzoyl derivative, of glycerol 85 Trichloroethyl esters, see Esters Trichlorophenylhydrazones 76 Triethylamine, purification 219 Trifluoroacetates, of aromatic amines 98 -, of cyclohexylamine 98 -, of diaminocyclohexane 98 -, of ethanolamines 98 -, of pharmaceuticals 187 -, of phenols 86 -, of steroids 158 -, of sugars 173 -, of xylenediamines 98 Trifluoroacetylacetone, chelates 194- 198 Triglycerides, fatty acids in 6 2 Triphenylarsine 194 Tryptamine, silylation 102 Tryptophan, metabolites, silyl derivatives 102 Tungsten, fluoride 192 Tyramine, 3-methoxy, TMS TFA derivatives 103 Tyrosine, metabolites, silyl derivatives 102

U Uranium, chelates 198 -,fluoride 192 -, in water 198 Urea, condensation with dialdehyde 78, 96 Urine steroids, methoxime TMS derivatives 161 -,TMS derivatives 154

V Vanadium, fluoride 198 Vanillylmandelic acid 121 Vitamins 185

X Xylenediamines, TFA derivatives 98