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 <1 l), 30 ml of benzene are added and the mixture is stirred for 45 min. The benzene phase is transferred into a separating funnel and the extraction is repeated with 30 ml of benzene for 30 min and 10 ml of benzene for 10 min. Sodium sulphate, isopropanol and/or methanol is used to break any emulsion. The combined benzene extracts are further extracted once with 40 ml and twice with 30 ml of 0.1 MK2C03solution. The aqueous phase is used for derivatization. The magnitude of the distribution coefficient and therefore the recovery of the extraction can be controlled by changing the pH, adding salts, etc. Three examples are given below for illustration. Extraction of urinary phenols [ 131. An aliquot of a 24-h urine sample corresponding to 5-10 min excretion was adjusted to pH 5 with acetic acid, incubated at 37°C for 16 h with 100 p1 of Helix pomatia juice, then 100 p1 of aqueous 4-chlororesorcinol solution (1 mg/ml) and 2 ml of phosphate buffer (pH 7,O.S M ) were added. The mixture was saturated with sodium chloride and extracted with three 10-ml volumes of diethyl ether followed by three 10-ml volumes of ethyl acetate. In this instance, the addition of a salt did not significantly improve the recovery, but it considerably reduced the amount of water taken up by the solvents. Extraction of aromatic acids from urine [ 141. Portions of 10 ml of urine were added to 20 ml of saturated sodium chloride solution in a separating funnel, and the pH was adjusted to 1.O with 6 N HC1. The solution was extracted with three 25-ml portions of redistilled ethyl acetate followed by extraction with three 25-ml portions of diethyl ether. The combined organic extracts were dried with magnesium sulphate and filtered. The filtrate was evaporated to dryness under reduced pressure. Extraction of the amines from tissues [ 151. Tissues were homogenized in 4 volumes of cold 0.0 1 N HC1 containing 50 mg/l of pargyline (N-methyl-N-propenylbenzylamine) hydrochloride. The homogenates were centrifuged at 10000 g for 15 min in a refrigerated centrifuge. To a 1 .O-ml aliquot of the supernatant were added 0.6 mg of NaCl and 10 pl of thioglycolic acid, as an antioxidant, and the pH was adjusted to 8 with 2.5 N NaOH solution. The amines were extracted with two successive portions of n-butanol, using a vortex-type mixer, centrifuging to separate the layers after each extraction. The combined n-butanol layers were transferred into a 15-ml centrifuge tube containing 0.5 ml of 0.01 N HCl and 4 ml of n-heptane and the amines were extracted back into the aqueous phase by mixing and centrifuging. The aqueous layer was separated and evaporated to dryness in vacuo over P z 0 5 . For phenylethylamines the recoveries varied from 30 to 80%. For further consideration it is necessary to take into account that the distribution coefficient is never zero, so that theoretically the conditions for a single-step quantitative extraction cannot be set. It is well known from theory [16] that the extraction yield is much greater in repeated extractions with smaller volumes of extraction agent than in a single-step extraction with the whole volume. This consideration can easily be derived from the relationship for the concentration of the compound under extraction in the

18

SAMPLE PREPARATION AND DERIVATIZATION TECHNIQUES

sample, Clf, after the nth extraction step: CE =Ct(1 + VEJKLEVL)-"

where Ct is the initial concentration of the extracted compound in the liquid sample, V , and VL are the volumes of the extraction agent and the liquid sample, respectively, and KLE = CL/C, is the distribution coefficient. In practice, an extraction yield higher than 99% is usually considered to be quantitative. With the use of the same volumes of the extraction agent and the sample, this result can be obtained even in a single extraction step if KLE< 0.01. Sometimes the entire procedure can be complicated by a chemical reaction taking place, e.g., in the extractive alkylation (see p.59) or in the preparation of volatile metal chelates (see p.194), and the total yield of the extraction then involves, in addition to the interphase distribution of the initial compounds and products, also the chemical equilibrium which is attained by the reaction. If the quantitative yield of the extraction cannot be predicted on the basis of the character of the system, the extraction efficiency must be determined, otherwise the quantitative evaluation is questionable. 2.2.3. Concentration, evaporation of the extraction agent

The same rules apply to this operation as to the drying. Possible losses should be prevented, particularly if relatively volatile compounds are being treated. Extreme caution is necessary with the commonly used technique of removal of the solvent with a stream of a gas, which can again be a significant source of errors. Concentration of the extract also results in the enrichment of impurities accidentally present in the extraction agent, which is why extreme purity of the solvents used and the performance of blank experiments are prerequisites for success.

2.2.4. Thin-layer chromatography TLC can be applied to the preliminary isolation of the compound under analysis from complex mixtures or to the purification of the products after a derivatization reaction. Several examples of TLC conditions for various substances and/or derivatives are given in Table 2.1. In all instances TLC must be carried out in such a way that it will contribute to the solution of a given analytical problem and that it should not become a source of difficulties and errors. As in the preceding instance, contamination of the sample with incidental impurities from the solvents used should be prevented. Chromatographic materials should also be tested for the presence of substances that could interfere with the compounds under analysis in the chromatogram. The quantitative recovery of individual zones from the layer for further treatment is obviously a prerequisite for reliable results.

2.3. PREPARATION OF DERIVATIVES Derivatization procedures follow the rules for the performance of organic synthetic reactions on the micro-scale. Some special requirements associated with the type of application led to some peculiarities in the performance of the reactions and to the develop-

19

PREPARATION OF DERIVATIVES TABLE 2.1 EXAMPLES OF TLC CONDITIONS Substance

Support material

Solvent system

Reference

Thiohydantoins of amino acids

Silica gel, activated at 100°C for 1 h

(1) n-Heptane-1-butanolanhydrous formic acid (10 : 7 : 3) (2) Chloroform-95% ethanolacetic acid (100 : 50 : 15)

17

Alditols and alditol acetates

Silica gel G

Benzene-ethyl acetate (1.5 : 1)

18

Metabolites of 3deoxysteroids

Silica gel G

Benzene followed by ethyl acetate

19

Androstanediol

Silica gel Fz

Benzene-ethyl acetate (1 : 1 ) n-Hexane-ethyl acetate (1 : 1)

20

Testosterone

4

ment of some specialized devices. Depending on the type of reactions involved, they can be performed in various reaction test-tubes and vessels and/or sealed ampoules. As most of the reactions are not carried out under very extreme conditions but in the absence of moisture, different septum-closed reaction vials are used for this purpose, some of which are illustrated in Fig. 2.5. Most of the shapes shown are available commercially with volumes from 1 to 20 ml and with different stoppers and septa made of various materials, including with FTFE and metal linings. The sample and the reagents are placed in the vial and as the final products are taken from it through the septum, with the aid of an injection (micro)syringe. Heating is performed in a thermostated bath or with the aid of a heating block, stirring manually, in an ultrasonic bath or with a miniature magnetic stirrer. For the treatment of very small volumes of reaction mixtures (units to tens of microlitres) vials with conical-shaped bottoms are used, which make scattering of the sample over a large area of the vial bottom impossible and facilitate withdrawal of the sample from the reaction mixture for GC analysis. Dunges [21] developed the device illustrated in Fig. 2.6 and used it for the performance of micro-reactions that require refluxing. The reactions are carried out (even several at a time) in small test-tubes with various volumes and conical-shaped bottoms. The lower parts of the test-tubes are immersed in a bath and heated at an appropriate temperature; the upper parts are cooled and the contents of the vials are shaken with a vibrational device. The equipment is available commercially (e.g., Jenaer Glaswerk Schott & Gen., Mainz, G.F.R.). More complicated and multi-step reactions are carried out with the aid of other devices commonly used for micro-preparations, micro-distillations, micro-filtrations and others. Special procedures have been developed for a limited number of derivatives, such as esterification on thin layer (“sandwich layer reaction”, see p.65) and some others described in Chapter 5 under applications.

20

SAMPLE PREPARATION AND DERIVATIZATION TECHNIQUES

Fig. 2.5. Reaction vials for the preparation o f derivatives.

The “on-column” technique for the preparation of derivatives consists in simultaneous injection of the sample and the reagent (or injection in a rapid sequence), so that the derivative is formed immediately before the chromatographic process in the injection port of the chromatograph or in the front part of the column. This technique has been applied to simple reactions only, such as silylation (see p.72) and acylation (see p.66), for the rapid identification of compounds. A quantitative reaction is often difficult to define as it depends on a number of parameters. The “flash-heater reaction” technique for the preparation of derivatives is closely related to the preceding one as it is usually

5 cm

water cooling

Fig. 2.6. The micro-refluxer according to Dunges [21]. (Reproduced from Anal. Chern., 45 (1973) 963, by courtesy o f the American Chemical Society.)

GAS CHROMATOGRAPHY

21

impossible to distinguish whether the reaction proceeds in the heated injection port of the chromatograph or only on the column. A typical example of this type is exhaustive alkylation by thermal decomposition of quaternary ammonium salts. The results of the reaction depends on a number of parameters, such as the temperature and geometry of the injection port. Some workers prefer temperatures of about 260°C and glass-woo1 in the injection port, whereas others use an empty injection port at temperatures of 360°C and higher. Another technique is to perform the reaction in a sealed capillary in a modified injection port. Details are given under various applications in Chapter 5 (see pp.116, 177,183 and 188). It is apparent that all glass and other apparatus used for the preparation of derivatives must be perfectly clean in order to prevent contamination. If septa are used t o close reaction vessels, they should be used repeatedly only with caution in order that substances accidentally adsorbed on the septum do not cause the same difficulties. The surface of glass vessels is modified in special instances only when the reagents used for the derivatives are extremely sensitive to the activity of the glass. Silanization is a common modification of the surface of glass vessels. It is performed, e.g., by treatment with a 5% solution of dichlorodimethylsilane in toluene [ 2 2 ] .The silanized glass is then rinsed with methanol and dried. Other silanization agents can also be applied. If the quantitative evaluation of an analysis is performed, the degree of conversion attained in the reaction used must be known. Two ways of achieving this are applied in practice: (i) a large excess of the reagent is used for the reaction and the reaction equilibrium is then shifted virtually completely towards the products, and a quantitative yield is then assumed; (ii) the analysis is evaluated by means of reference samples. In the latter instance the yield is not known and identical reaction rates must be assumed for both the standard and the compound under analysis. All reaction conditions, such as temperature, reaction time, type of catalyst and concentrations of reactants, must, however, also be identical in both instances. In this event it must be appreciated whether a “matrix effect” can cause a difference in the reaction yield in the treatment of a real sample in comparison with the standard. Some hints for eliminating the influence of the matrix are proposed in Section 3.2.4. (p.47). Products from the decomposition of the reagents can also adversely affect the reaction yield and therefore maximal purity of all reagents must again be emphasized.

2.4. GAS CHROMATOGRAPHY

Gas chromatographic analysis proper does not differ from the conventional procedure in its arrangement for derivatives, as the derivatives used are often common compounds which can, in other circumstances, be the object of analysis themselves (e.g., carboxylic acids versus esters). It is beyond the scope of this book to deal with the detailed theory and instrumentation of GC and readers are referred elsewhere [23-251; only some peculiarities introduced into GC as a result of the application of derivatives will be discussed here. The injection of derivatives in a suitable solvent or directly together with the resulting reaction mixture is performed by conventional means. Microsyringes of various volumes

22

SAMPLE PREPARATION .AND DERIVATIZATION TECHNIQUES

are suitable and are often used. In general, any sampling system for liquid samples can be used. Among others, the “falling needle” injection system developed for the injection of high-boiling compounds dissolved in a volatile solvent into a capillary column has been used [ 2 6 ] .In principle, a liquid sample is applied on the tip of a glass needle and a volatile solvent is evaporated under a stream of the carrier gas, the pathway of which is split into the column and along the needle into the atmosphere so that the column is not exposed to large amounts of the solvent. When the solvent has evaporated, the needle is inserted into a heated zone of the system and the vaporized sample is swept into the column with the carrier gas. The whole system is made of glass and thus inert and can be combined with instruments of various types. Other special injection devices have been applied in combination with derivatization only rarely, if the reaction is carried out directly in the injection port. The method of thermal degradation of quaternary ammonium salts has already been mentioned. This can be performed in a sealed capillary directly in the injection port of the apparatus. The port should then be modified in order that when the reaction is finished, the capillary can be crushed and the products swept into the column. Another method of modification of the injection port in the thermal decomposition of hydrazones by a-ketoglutaric acid is shown in Fig. 4.3 (p.77). The activity of the metallic parts of the instrument can be suppressed by surface modification (e.g., by nickel plating) or by replacement with glass. In exceptional cases with very labile substances, borosilicate-glass columns are used. On injecting the reaction mixture, the possibility of corrosion by residues of aggressive agents should also be considered. Column packings do not require any modifications at present as a wide range of supports deactivated and modified in various ways are commercially available. If none of them is obtainable, supports can be modified by silanization performed in the same way as in the modification of glass surfaces. Deactivation of glass capillary columns is mostly performed by treatment with Carbowax 20M in solution or in the vapour phase. A comparison of various methods has recently been published by De Nijs et al. [27]. With detectors, it is also necessary to pay attention to their protection against corrosion and various deposites which can affect quantitative results. The katharometer is usually equipped with resistant filaments, usually nickel- or gold-plated. Silyl derivatives decompose in the flame of the FID into silicon oxide, which deposits on the electrodes and reduces the response of the detector significantly. Hence, when analysing silyl derivatives, the electrodes must be cleaned more often than usual.

REFERENCES 1 H.A. Laitinen and W.E.Harris, Chemical Analysis, McCraw-Hill, New York,2nd ed., 1975, pp. 563-582. 2 Book of ASTM Standards, ASTM, Philadelphia, PA, annual. 3 Official Methods ofAnalysis o f the Association o f Official Analytical Chemists, AOAC, Washington, DC, 10th ed., 1965. 4 Official arid Tentative Methods of the American Oil Chemists’Society, AOCS, Chicago, IL, 1951-, updated with supplements. 5 Standard Methods o f Chemical Analysis, 3 vols., Van Nostrand Reinhold, New York, 6th ed., 1962-1966.

REFERENCES

23

Y. Maruyama and A.E. Takemori, Anal. Biochem., 49 (1972) 240. F. Berthou, L. Bardou and H.H. Ploch,J. Chromatogr., 93 (1974) 149. R.F. Kaiko and C.E. Inturrisi, J. Chromatogr.. 100 (1974) 63. H.L. Davis, K.J. Palk and D.C. Bailey, J. Chrornarogr., 107 (1975) 61. B.L. Karger, L.R. Snyder and C. Horvath, A n Introduction to Separation Science, Wiley-lnterscience, New York, 1978, p. 557. 11 C.W. Gehrke and D.L. Stalling, Separ. Sci., 2 (1967) 101. 12 A.S.Y. Chau and J.A. Coburn, J. Ass. OjJic. Anal. Chem., 57 (1974) 389. 13 V. Fell and C.R. Lee,J. Chromatogr., 121 (1976)41. 14 M.C. Homing, K.L. Knox, C.E. Dalgliesh and E.C. Horning,AnaL Biocbem., 17 (1966) 244. 15 D.J. Edwards and K. Blau, Anal. Biochern., 45 (1972) 387. 16 L. Alders, Liquid-Liquid Extraction, Elsevier, Amsterdam, 1955, p. 65. 17 F.E. Dwulet and F.R.N. Gurd,Anal. Biochem., 82 (1977) 385. 18 L.J. Griggs, A. Post, E.R. White, J.A. Finkelstein, W.E. Moeckel, K.G. Holden, J.E. Zarembo and J.A. Weisbach, Anal. Biochem., 43 (1971) ,369. 19 T. Nambara and Y.Ho Rae, J. Chromatogr., 64 (1972) 239. 20 G. Charransol, F. Robas-Mason, S. Cuillemant and P. Mauvais-Jarvis,J. Chromatogr., 66 (1972) 55. 21 W. Diinges, Anal. Chem., 45 (1973) 963. 22 W.J.A. VandenHeuvel and E.C. Homing, Blochim. Biophys. Acta, 64 (1962) 416. 23 S. Dal Nogare and R.S. Juvet, Jr., Gas-Liquid Chromatography: Theory and Practice, Interscience, New York, 1962. 24 L.S. Ettre and A. Zlatkis (Editors), The Practice of Gas Chromatography, Interscience, New York, 1967. 25 A.B. Littlewood, Gas Chromatography, Academic Press, New York, 2nd ed., 1970. 26 P.M.J. van den Berg and Th.P.H. Cox, Chromatographia, 5 (1972) 301. 27 R.C.M. de Nijs, J.J. Franken, R.P.M. Dooper, J.A. Rijks, H.J.J.M. de Ruwe andF.L. Schulthg, J. Chromatogr., 167 (1978) 231. 6 7 8 9 10

This Page Intentionally Left Blank

Chapter 3

Identification and quantitation (by JOSEF NOVAK)

CONTENTS 3.1. Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 . l . Correlations of retention data with the structural and physico-chemical properties of solute compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1.I. Physicochemical basis of the retention behaviour of compounds . . . . . . . . . 3.1.1.2. Identification methods based on the retention behaviour of compounds . . . . . 3.1.2. Use of chemical reactions in connection with GC . . . . . . . . . . . . . . . . . . . . . . 3.1.3. Use of selective detectors . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.4. Combination of GC with other analytical methods . . . . . . . . . . . . . . . . . . . . . 3.1.5. Prospects of performing identification by virtue of retention behaviour only . . . . . . 3.2. Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . 3.2.1. General concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Specificity of detection, response factors . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3. Techniques of quantitative chromatographic analysis . . . . . . . . . . . .. . . . . . . . 3.2.3.1. Absolute calibration method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3.2. Internal standard method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3.3. Standard additions method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3.4. Internal normalization method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3.5. Manual processing of the chromatogram . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3.6. Automatic processing of the chromatogram . . . . . . . . . . . . . . . . . . . . . . 3.2.4. Special problems of quantitation in derivatization GC . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

26 26 26 27 34 36 38 39 40 40 41 43 44 44 44 45 46 46 47 50

The main reason for the chemical derivatization of solutes in gas chromatography is to make their chromatography easier or even feasible at all. However, the conversion of solutes into appropriate derivatives may also greatly facilitate their identification and determination. There are many papers in which GC combined with chemical derivatization has been utilized successfully to identify certain compounds and/or classes of compounds and to determine their contents in complex materials. For instance, Zarazir et al. [ I ] measured the retention indices of a series of aliphatic alcohols and their trimethylsilyl ethers, acetates, trifluoroacetates, propionates, pentafluoropropionates, butyrates and heptafluorobutyrates on three stationary phases and showed that these compounds can be identified by calculating their retention-index fractions and plotting them in a triangular diagramme according to the concept of Brown [ 2 ] .Simpson [3] determined synthetic glucocorticosteroids in rat muscles by converting the corticosteroids into their trimethylsilyl ethers and analysing them by GC using an ECD. It was possible t o determine triamcinolone acetonide, triamcinolone, betamethasone and prednisolone in muscle after administration of doses of 20 mg/kg body weight. Brooks et al. [4] compared the 2s

26

IDENTIFICATION AND QUANTITATION

MS data for certain steroids and their TMS ethers in order to study the neutral urinary metabolites of the anabolic steroid 17ol-ethyl-l7P-hydroxy-4-en-3-one by GC-MS. As far as quantitative chemical derivatization GC analysis is concerned, it is necessary to mention especially the work of Gehrke and his collaborators, who specified the fundamental concepts of quantitative GC analysis combined with the chemical derivatization of sample compounds and applied them to the accurate determination of the twenty natural protein amino acids and other non-protein amino acids as their N-TFA-n-butyl esters [S],the urinary excretion level of methylated nucleic acid bases as their TMS derivatives [6], TMS nucleosides [7] and other investigations. Further examples include a computer program for processing the quantitative GC data obtained for seventeen triglyceride fatty acids after their transesterification by 2 N K O H in n-butanol [8], a study of the kinetics of the transesterification reactions of dimethyl terephthalate with ethylene glycol [9] and the GC-MS determination of chlorophenols in spent bleach liquors after isolation of the chlorophenols by a multi-step extraction, purification of the final extract by HPLC and derivatization with diazoethane [lo]. This chapter gives a brief survey of the concepts and methods employed in the identification of compounds and their determination by GC. The scope is to provide the reader readily with basic information on the analytical aspects proper of GC. Those interested in a more detailed treatment of these problems are referred to the specialized literature on qualitative [ll-131 and quantitative [14-161 analysis by GC.

3. I . IDENTIFICATION Procedures for the identification of compounds by GC constitute a very broad and varied discipline. There are many possibilities in this respect, and the proper choice in a particular instance is dependent on the nature of the problem and the equipment available. Basically, GC is a separation method, but its very high separation efficiency confers upon retention characteristics a significant identification virtue and greatly facilitates the use of various ancillary identification techniques. It is hardly possible to present any procedure that would be generally applicable to any identification problem; the decisive factors in this respect are the complexity and amount of the sample available to be analysed, knowledge of the history of the sample and the standard of the available analytical inctrumentation. On the other hand, any analytical problem can usually be approached successfully in several ways, from which the analyst can make a deliberate choice. In this respect, the professional experience of the analyst is another important factor. 3.1 . I , Correlations of retention data with the structural and physico-chemical properties of solute compounds

3.1.1.1. Physico-chemical basis of the retention behaviour of compounds

Often one reads the statement that chromatographic retention data are characteristic of the compounds being chromatographed, but this statement is not completely correct

IDENTIFICATION

21

unless it is referred to a particular chromatographic sorbent and the physical conditions of the measurement. The best way of reporting this situation is to consider the chromatographic distribution constant, which is a reduced and physically well defined chromatographic quantity. However, it is necessary to point out that the correlations discussed below also apply to all kinds of adjusted retention data (retention time, retention volume, specific retention volume, capacity ratio, relative retention). The distribution constant of a solute i in a GC system, Ki, can be expressed as

where R and Tare the universal gas constant and the absolute column temperature, vi, yis and us are the fugacity coefficient of solute i in the mobile (gaseous) phase, nonideal compressibility coefficient of the carrier gas, Raoult's law activity coefficient of solute i in the liquid stationary phase and molar volume of the stationary liquid respectively, all at the temperature and mean pressure in the column, Pi" and up are the saturation vapour pressure of solute i at the column temperature and the fugacity coefficient of the single solute vapour at the column temperature and saturation vapour pressure P,", respectively, and 0 is given by ZG,

PP

(uklR7') dP

0 = exp

(2)

P

where uf is the molar volume of the single liquid solute i and P is the mean column pressure. If the second-order quantities vi, up, ZG and 0 are neglected in eqn. 1 (they all approach unity under the usual GC conditions), Ki consists of a property (P,") of the single solute i , a property (us) of a single liquid stationary phase and the interaction parameter yis, a property of the solute-liquid stationary phase mixture. Hence, the distribution constant can be considered as either a characteristic of solute i for a given liquid stationary phase or a characteristic of the liquid stationary phase for a given solute, and, owing to the parameter yis, neither of the two characteristics is unambiguous. Hence, the unequivocal identification of an unknown compound merely by virtue of a knowledge of its distribution constant (and/or any other retention quantity) on some stationary phase is problematical, even when a wide range of reference compounds and/or a library of reference data are available, and it is therefore necessary to utilize some complementary identification dimension. Such dimensions include various relationships between the retention data and the physico-chemical properties of solute compounds. The identification of a compound is confirmed if its retention data logically fit some of the relationships determined with the use of several stationary phases.

3.1.1.2. Identification methods hased on the retention behaviour of compounds The most important correlations are those based on regularities between retention data and the number of carbon atoms in the solute molecule, existing within homologous series of compounds [ 171. All of these correlations have a certain thermodynamic basis,

28

IDENTIFICATION AND QUANTITATION

which can be demonstrated by showing the relationship between the chromatographic distribution constant and the standard molar Gibbs function of sorption and employing Martin's theorem [ 181 of additivity of the retention increments corresponding to the groups that constitute the solute molecule. Employing standard states of a single solute in a physical state of infinite dilution in the liquid stationary phase at the temperature and pressure of the system and a single solute in the perfect gas state at unit pressure and the temperature of the system for the solute in the stationary and in the gaseous phase, respectively, we obtain for the standard molar Gibbs function of sorption of solute i, AG,o,(i) [ 1 9 ]: AG,0,(i) = -R T ln(KpslR1")

(3 )

With regard to further discussion, it is more convenient to consider the standard Gibbs function of desorption, AG&(i): AG&(i) = -AG&(i)

(4)

Considering a solute molecule of the type i = CH3(CH2),X, where X is a functional group, and applying Martin's additivity theorem we can write AG&(i) = AG&(CH3) + n AG&(CH2) t AG&(X)

(5)

and combining eqns. 3 , 4 and 5 we obtain

which implies that log Ki = log K(CH3) + n log K(CH2) t log K(X) t log C

(7)

where C i s a constant. Hence, plotting log K i against n should give a straight line with a slope and intercept of log K(CH2) and log [CK(X)],respectively. If log K(CH2) were a quantity independent of both the position of the given methylene group with respect to other groups and the nature of the latter in the molecule, the plots of log K i versus n for different homologous series of compounds theoretically should be parallel straight lines. Indeed, when plotting the logarithms of adjusted retention data against methylene number, one obtains a family of nearly parallel lines, each representing a particular homologous series of compounds (Fig. 3.1) [ 2 0 ] .The identification proper by using this plot is then carried out by fitting the retention data of the unknowns to the lines in such a manner that the respective coordinates correspond to integral carbon numbers. There can obviously be two or more such fits with a single plot, which makes the identificatioii ambiguous. Unequivocal results can be obtained by repeating the procedure using several kinds of sorbent. The identity is confirmed if the compound in question displays positive identifications with all of the sorbents employed. A more refined method is to plot against each other the logarithms of the retention data measured on two different sorbents [21]. This again results in almost parallel lines for the individual homologous series (Fig. 3.2) [ 2 2 ] .Such a plot provides fairly reliable identification, as the necessary condition for the indication of identity is agreement for both carbon number and retention data on both sorbents. The confirmation of identity

IDENTIFICATION

29

0 1 2 3 4 5 6 7 8 9101112131415 C H, NO

Fig. 3.1. Relationship between the specific retention volume (logarithmic scale) and the number of methylene groups for various types of monofunctional homologous compounds on Carbowax 1000 at 100°C.

can again be carried out with the aid of another pair of sorbents. This method affords correlations that may reveal very fine structural features, as illustrated in Fig. 3.3 1231. Such procedures obviously presuppose high-precision measurement. A very refined version of this kind of correlation was developed by Keulemans’ school. From thermodynamic concepts, Ladon and Walraven [24] suggested the equation log 4 = f logr:

+ bs + c t +dq + e

(8)

where the r values denote relative retention data, superscripts A and B denote the two sorbents employed, subscripts i, s, and u represent the compound under analysis (i) and reference substances, f is the slope of the lines in the plot, b, c, d and e are empirical constants, and s, t and q are the numbers of secondary, tertiary and quaternary carbon atoms, respectively, in the molecule. The equation represents a whole family of lines; the agreement between calculated and measured data is shown in Fig. 3.4 [24]. The significance of this procedure consists in the possibility of predicting from the number of carbon

30

IDENTIFICATION AND QUANTITATION

100

10’

102 103 Vg ,CARBOWAX 1000

14

Fig. 3.2. Relationship between the specific retention volumes (logarithmic scales) on Carbowax 1000 and isooctyl decyl adipate for the same compounds as in Fig. 3.1 at 100°C; the numbers denote the number of methylene groups (n).

J

>-

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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12 13 14 15 16 17 18 19 20

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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

*

;
butanone 80'

~

108

DERIVATIZATION OF COMPOUNDS

dure. A sample containing 0.5-25 pmol of acetylcholine and a known amount of hexyldimethylammonium chloride (ca. 10 nmol) is placed into a conical centrifuge tube and evaporated to dryness, then 0.5 ml of the reagent (6 mg/ml of sodium thiophenolate in n-butanol) is added and the atmosphere over the solution is replaced with dry nitrogen. The tube is stoppered and then heated on a water-bath at 80°C for 30 min, being shaken every 5 min. When the reaction is completed, the tube is cooled, 0.1 ml of aqueous citric acid (0.5 M) and 2 mi of pentane are added and the solution is shaken vigorously and centrifuged. The upper organic phase is separated and the aqueous phase is washed twice with 1 ml of pentane. Pentane residues are removed with a gentle stream of dry nitrogen, then 50 pl of chloroform, 0.1 ml of a mixture of ammonium citrate (2 M) and ammonia solution (7.5 M) are added to the aqueous residue and the solution is shaken vigorously and centrifuged; ca. 5 p1 of the organic phase is injected. The authors modified the method for sub-microgram amounts [ 1 1 1] and demonstrated its application to the determination of choline in brain tissue [ 1101. C H, /

C H2 - C - C H

CH3-C0

I,

0

3 +

NH2-NH2

-

CHj

+

2 H20

I

H

Scheme 5.11.

Hydrazine and methylhydrazine were determined in aqueous solution at 0.1 -50 ppm levels in the form of the corresponding pyrazoles [ 1121 produced by a condensation reaction with 2,4-pentanedione (Scheme 5.1 1). The pH.of the aqueous solution was adjusted to 6-9 with the aid of 1 N NaOH or 1 N &SO4 and 50 pl of 2,4-pentanedione were added to a 100-ml portion. The mixture was mixed thoroughly and allowed to stand at room temperature for at least 1 h. Samples of 5 pl were analysed on 30% mixture of Amine 220 and Apiezon L (1 : 5) at 130°C. Neurath and Liittich [ 1131 described a GC separation of asymmetric dialkyl-, aralkyl-, diaryl- and cyclic hydrazines up to C l z after their conversion into 5-nitro-2-hydroxylbenzal derivatives. A column with 2.5% of silicone grease was employed for the separation. R-NH2 + C2 H5 O-CO-O-CO-OC2H5-

R-NH-CO-OC2H5

+

C02 + CZH50H

Scheme 5.12.

Gejvall [ 1141 analysed low-molecular-weight amines in the form of urethanes produced by their reaction with diethyl dicarbonate (Scheme 5.1 2 ) . The reaction can be carried out in aqueous solution. A 10-nig aniount of diethyl dicarbonate was allowed to react with 0.05-0.7 mg of the amine at rooni temperature for 30-40 min, the pH being adjusted to 9.5 with NaOH. A good separation with symmetric peaks was obtained by using SE-30 as the stationary phase. Hoshika [115] separated 13 lower aliphatic amines on a column packed with Tenax GC. Primary amines (methyl-, ethyl-, n-propyl-, isopropyl-, ii-butyl-, isobutyl-, n-amyland isoamylamine) were converted into the corresponding Schiff's bases by reaction with inol) were benzaldehyde. The aniines ( I -6 . l o 4 mol) and benzaldehyde (5 .

SULPHUR COMPOUNDS

109

mixed with 2 ml of n-hexanol at room temperature. The formation of Schiffs bases was rapid and exothermic and the reaction time of several minutes was therefore sufficiently long for the reaction t o be completed. Secondary amines (dimethyl-, diethyl-, and di-n-propylamine) and tertiary amines (trimethyl- and triethylamine) do not react with benzaldehyde and are therefore analysed as such.

5.4.SULPHUR COMPOUNDS This section is devoted mainly to the derivatization and GC of thiols, sulphonic acids and related compounds. The GC of other sulphur compounds (sulphones, sulphonamides, etc.) is also mentioned in other sections, e.g., Pharmaceuticals, Insecticides. Thiols are converted into derivatives for GC analysis by procedures analogous t o those for alcohols, i.e., mainly by acylation and trimethylsilylation. However, the thiol functional group is generally less reactive than the hydroxyl group. Kawahara [ 1 161 applied the condensation reaction with cw-bromo-2,3,4,5,6-pentafluorotolueneto the preparation of tho1 derivatives and utilized them for their sensitive analysis (cf., p. 87). Korolczuk et al. [ 1171 described the conditions for the preparation and GC separation of dinitrophenyl and benzyl derivatives of a number of normal and branched thiols up t o CB. Alkylthiobenzoates were found to be more suitable. The following procedure was elaborated for their preparation in aqueous medium. A 4-pl volume of each thiol and 0.1 ml of benzoyl chloride were transferred into 1 ml of 0.1-2.5 N sodium hydroxide solution and 2 ml of acetone were added to dissolve the benzoyl chloride. The reaction tube was stoppered well and the reaction proceded at room temperature or at 100°C for 10-90 min. After cooling, 10 pl of water were added and thiobenzoates were separated by a three-fold extraction with diethyl ether. The combined extracts were evaporated to dryness and the residue was dissolved in 1 ml of ethyl acetate and subjected to GC analysis. A good separation was achieved on 4% of SE-30 at 150°C with temperature programming. Jellum et al. [ 1 181 analysed biologically significant thiols and disulphides after their reaction with pivalaldehyde (Scheme 5.13). About 100 pl of an extract of a tissue were

CH -CH2

I 2 1 S H NH2

+

(CH3)3C-CH=0

CH -CH2

I 2 lNH S

+

H20

\ /

CH

S - C H2 - C

H2 -NHZ

I S - C H2 - C H2 -NH2 Scheme 5.13.

+

2 (CH3)3C-CH=0

-I

s -C H2 - C H

~N-= C H -C(C H

~

S-CH - C H 2 - N = C H -C(CH313 2

) +

~

2 H20

DERIVATIZATION OF COMPOUNDS

110

evaporated to dryness and the residue was dissolved in methanol (0.1 ml) and pivalaldehyde (0.1 ml). Ionex beads (AG 1-X8, HCO;) were added until the pH of the solution was 7-8 and molecular sieve (3A) is added to remove trace amounts of water. After 10 min a l-pl sample was taken for GC analysis on 5% of SE-30. Sulphur-containing amino acids can be converted into derivatives after prior esterification with HC1-methanol by the same procedure. R-S03H

+

SOC12

-

R-S02CI

+

SO2

+

HCI

Scheme 5.14.

Sulphonic acids and their salts are analysed by CC after esterification with diazomethane or after chlorination with thionyl chloride or phosgene [ 1191. Reaction with thionyl chloride proceeds according to Scheme 5.14. A 0.5-g sample of sulphonic acid or its salt is placed into a round-bottomed flask fitted with a magnetic stirrer and a reflux condenser, 0.5 ml of dimethylformamide and 20 ml of thionyl chloride are added and themixtureis refluxed for several minutes up to 2 h (according to the character of the sample) until the evolution of gas from the reaction mixture ceases (detection with the aid of a bubbler filled with chlorobenzene). If a salt is chlorinated, solid chloride produced in the reaction mixture must be removed by dilution with dichloromethane and by careful filtration through a fine glass filter. Excess of thionyl chloride and solvent is evaporated carefully under decreased pressure. The residue is dissolved in a suitable solvent (CCb) and analysed by GC (silicone stationary phase, temperature 16OOC). Parsons [ 1201 converted sulphonyl chlorides into fluorides prior to GC analysis. A I-ml volume of a solution of sulphonyl chloride in benzene (ca. 50 mg) and 1 g of potassium fluoride dihydrate were placed in a reaction flask fitted with a reflux condenser. Benzene was refluxed on an oil bath at 105-1 10°C for about 1.5 h and, after cooling, the benzene solution was injected into the chromatograph or fluorides were isolated by removing benzene. Fluorides of sulphonic acids are more stable than chlorides and their volatility is higher. GC separation was executed with satisfactory results on 3.8% of SE-30. Sulphonic acids are usually esterified with diazomethane [ 121] by methods analogous to those used for the preparation of esters of carboxylic acids (see p. 54). If the reaction starts from the salts, it is necessary to convert them into acids either by direct acidification or with the aid of a cation-exchange resin in the H ' form. Baker and Boyce [ 1221 determined isomers of toluenesulphonic acid as ethyl esters prepared by the reaction with triethyl orthoformate. About 0.1 ml of sulphonic acid was added to a known amount of toluene (1 2 ml) and the water produced was removed by azeotropic distillation with about 5 ml of toluene. o-Terphenyl (0.05 g in 3 ml of toluene) was added as an internal standard, followed by a 20-fold excess of triethyl orthoformate, and the solution was refluxed for 15 min. After this period the reaction proceeds quantitatively and the reaction mixture can be injected directly into the chromatograph. The separation was carried out on 3% of OV-101. N,N-Dialkyl dithiocarbamates were subjected to S-alkylation prior to GC analysis and the derivatives have been used even for GC-MS. S-alkylation is performed by the reaction with diazomethane, iodoethane or iodopropane. GC data on Apiezon L and mass spectra of a number of these compounds have been published [ 1231.

CARBOXYLIC ACIDS

-

111

NaBHL

R-CY-C-SCoA

II

R-CH2-CH2-OH

+

HS-COA

0

Scheme 5.15.

Identification and quantitative analysis of long-chain acyl thioesters after their reduction with sodium borohydride were performed by Barron and Mooney [124]. With acetyl-CoA derivatives, the corresponding alcohol, which is analysed either directly or after the derivatization, and coenzyme A are produced (Scheme 5.15). In order to increase the solubility, 0.3 ml of tetrahydrofuran was added to 1 nil of an aqueous solution (or suspension) of acetyl-CoA. About 10 mg of NaBH4 were added and the sample was reduced at 38°C for 15 min. The reaction was terminated by adding slowly 0.7 ml of 1 N HCl, which decomposed the residue of NaBH4, and the reaction mixture was then re-extracted with three 1.5-ml portions of CHC13. (If it is necessary to remove free fatty acids, the sample is rendered alkaline prior to the extraction.) The extract was washed with water, CHC13 was evaporated at 40°C in a stream of nitrogen, and the residue was dissolved in suitable amount of benzene and chromatogaphed. The method is specific for thioesters, as normal esters are not reduced under these conditions.

5.5. CARBOXYLIC ACIDS Carboxylic acids are analysed by GC almost exclusively in the form of derivatives. GC separation of free carboxylic acids cannot be achieved without special modification of the column in order to suppress the strong adsorption and tailing caused by the presence of strongly acidic and polar carboxyl groups. Mixed stationary phases, containing phosphoric or some other acid, are usually used for this purpose. Porous organic polymers, e.g., of the Porapak N type, have recently been used successfully. Despite these rare attempts, derivatization is usually used when analysing carboxylic acids by GC [ 125-1 271. On derivatizing monocarboxylic acids, protection of the - - O H group is effected and esters are the most frequently used. With polyhydric acids the situation is complicated by the different reactivities of various carboxyl groups. As substituted acids (e.g., hydroxy acids, phenolic acids and keto acids), which are important in the study of biological processes were the first of these compounds to be examined, various other derivatives were considered, including TMS derivatives, different common alkylation reactions, and also combined derivatives, depending on the type of other functional groups present. 5.5.1. Esters Esters and some procedures for their preparation have been discussed in the previous chapter (see p. 53). This section will therefore deal with some modifications of the procedures described there (e.g., for micro-scale analysis), higher esters, derivatives with a high ECD response and further special procedures and applications. Methyl esters are probably the most frequently used esters for the GC analysis of

112

DERIVATIZATION OF COMPOUNDS

acids. They have been used for a number of applications: GC of fatty acid isomers in capillary column [ 1281, analysis of &-branched carboxylic acids by capillary GC-MS [ 1291 and analysis of acidic fractions of tobacco and marihuana smoke by capillary GC-MS [ 1301. Schwartz and Bright [ 131] performed esterification with diazomethane at the picogram level using a reaction column. A capillary, ca. 50 mm X 1.5-2.00 mm O.D., was filled with Celite 545 to a length of about 2.5 cm and the filling was compressed to about 1.5 cm. Diethyl ether (2 pl) containing 0.5-5 pg of acids was applied on to the capillary filling and the walls of the capillary were washed with two 2-pl portions of pure diethyl ether. The capillary (about 2 cm) was then pierced through a septum. A 10-pl volume of a solution of the reagent (1 g of N-methyl-N-nitroso-p-toluenesulphonamide and 2 ml of ethanol dissolved in 3 ml of diethyl ether) and 10 pl of 50% aqueous potassium hydroxide were placed in a 1-ml reaction vial provided with a magnetic stirrer, the vial was stoppered with the septum carrying the capillary and the solution was stirred for 2 min. The capillary was then removed and methyl esters were eluted from it by injecting 10-15 p1 of dichloromethane, diethyl ether or carbon disulphide into the capillary. The first 5-6 p1 (5-6 mm) of the solvent leaving the capillary contained all of the esters and was withdrawn from the capillary with a syringe for GC analysis. Williams .[1321 tested several procedures for the preparation of various derivatives of dihydroxybenzoic acids. The procedures leading to diacetoxymethyl esters and bis-TMSmethyl esters did not always give a uniform product. The author recommended permethylation of carboxyl and hydroxyl groups by dissolving the acids in methanol and allowing the solution to react with an etherical solution of diazornethane for 16 h. GC analysis is performed on different stationary phases, such as SE-30, XE-60 and EGA. An analogous procedure was suggested for the rnethylation of the Krebs cycle acids [133]. The derivatives of some acids (pyruvate, fumarate) show thermal instability, however. Reaction with an alkyl halide, catalysed with silver oxide, can be used not only for the preparation of ethers (see Scheme 4.1 1, p. 64) but also for the preparation of esters prior to GC analysis. Johnson and Wong [ 1341 performed this reaction in a trapping column, specially adapted, packed with Ag20 reagent, where they trapped the acids being analysed. They applied the method to the analysis of volatile acids in mutton. The same esterification reaction was applied by Gloor and Leidner [135] to the preparation of ethyl esters and the determination of C1-CZ4 carboxylic acids in aqueous solution at parts per billion levels. KOH (1 IV) was added to an aqueous solution of the sample to give a pH of 10-1 1. Silver salts of the acids were precipitated in such a manner that the excess of free Ag' ions was 10-3-10-2 mol/l. The solution was evaporated to dryness, 2.5 ml of pentane containing 5 ppm of n-tridecane as the internal standard and 5-10 ml of ethyl iodide were added to the residue and the solution was allowed to stand in an amber-coloured stoppered bottle at about 10°C for 3 h. The pentane phase can be chromatographed directly. An example of the separation of phenylcarboxylic acids after their conversion into ethyl esters is demonstrated in Fig. 5.10. The utilization of methyl esters and higher alkyl esters for various applications of the determination of carboxylic acids has been described by a number of workers. Jones and

CARBOXYLIC ACIDS

113

r

1

3

A

L

4

30

20

10

TIME C M i n l

Fig. 5.10. Gas chromatogram of ethyl esters of phenylcarboxylic acids (2.5-7.5 pg). Peaks: 1 = benzoic; 2 = phenylacetic; 3 = 2-phenylpropionic; 4 = 2-phenylbutync; 5 = 3-phenylpropionic; 6 = 3-phenylbutyric; 7 = 4-phenylbutyric; 8 = 4-phenylvaleric; 9 = 5-phenylvaleric acid; S = n-tridecane (internal standard); x = blanks of reagent and products from side-reactions. Conditions: column, 20 m x 0.38 mm I.D., SE-54;hydrogen flow-rate, 4.5 ml/min; temperature programme, 4"C/min (30-150°C); injection, 1.5 pl, splitless, 30 sec. (Reproduced from Chromatographin, 9 (1979) 618, by courtesy of the authors and the publisher.)

Davison [ 1361 investigated the position of the double bond in unsaturated acids. After an oxidative cleavage they isolated the acids produced from the fragments as salts and converted them into esters prior to GC analysis. The total contents of saponifiable and free acids in small biological samples were determined by MacCee and Allen [137]. A sample of tissue (3-5 mg) or serum (5-100 pl) was hydrolysed with a lye, acidified and extracted with n-hexane. Free acids were extracted from the n-hexane into a small volume (5-10 1.11) of trimethyl-(a,a,a-trifluorom-toly1)ammonium hydroxide. The quaternary salt was injected together with methyl propionate, and methyl esters produced by the pyrolysis were subjected to GC analysis (injection port temperature 24OoC, column temperature 180°C, 10% EGSS-X). Comparison of this method with other esterification methods in Table 5.9 shows that icoffers at least equally good results. Determination of the composition of different ojls and fats is a very common application of the GC analysis of fatty acids. The samples under analysis are usually hydrolysed first and free fatty acids are esterified. Kleiman et al. [ 1381 used the methanol-BF3 method for determining acyl groups in oils. Barnes and Holaday [ 1391 started directly with ground peanuts when analysing the composition of their fats. After hydrolysis for 8 min by heating at 8OoC with a methanolic solution of NaOH they carried out the esterification with 10%of methanol-BF3 at 95OC for 5 min. n-Propyl esters were utilized for the analysis of fatty acids in soaps [140]. After evolving fatty acids with the aid of orthophosphoric acid, the esters were prepared by heating with n-propanol at 9OoC for 2 min. Aston [141] selected for the preparation of butyl esters transesterification with the aid of 2 N KOH in n-butanol (5 min at room temperature). He evaluated the analyses of fats and oils by computer using a Fortran program.

114

DERIVATIZATION OF COMPOUNDS

TABLE 5.9 RECOVERIES OF FATTY ACIDS AFTER SAPONIFICATION AND ESTERIFICATION OF A REFERENCE MIXTURE [ 137J Applied Science Fat and Oil Reference No. 6 Fatty acid

14 : 0 16 : 0 16 : 1 18 : 0 18 : 1 18 : 2 18 : 3

Label data (!%)

Found (% f standard deviation) after saponification and esterification with

2.0 30.0 3.0 14.0 41.0 7.0 3.O

BF3

HCI

H2S04

TMTFTH

1.97c 0.06 30.01 t 0.40 3.06 f 0.28 13.99 f 0.12 41.20 f 0.26 6.92 i 0.12 2.98 f 0.06

1.86 f 0.11 30.07 t 0.50 2.72 f 0.06 14.43 t 0.20 41.29 t 0.38 6.72 f 0.13 2.84 ?I 0.08

2.36 t 0.10 29.61 f 0.80 3.33 f 0.11 10.87 ?: 0.43 42.61 t 0.50 7.87 ?: 0.20 3.67 f 0.17

1.97 f 0.07 30.17 t 0.36 2.99 c 0.06 13.69 c 0.08 41.15 f 0.36 7.03 -t 0.10 3.05 f 0.06

* TMTFTH = trimethyl(a,a,a-trifluoro-ni-tolyl)ammoniumhydroxide. Higher esters of carboxylic acids are often prepared by reaction with higher diazoalkanes. Wil.cox [ 1421 analysed different biological interesting phenolic acids. Diazoalkanes with the addition of 0.007% of BF3 alkylate these substances almost quantitatively within 30 min, hydroxyl groups being converted into ethers. The use of higher diazoalkanes makes it possible to resolve the acids with free phenolic groups from methoxy derivatives. The separation of derivatives and their retention data are given for SE-30. Other workers [143] performed the separation of up to 18 acids on Chromosorb W, the surface of which was modified with Carbowax 20M (see Fig. 5.1 l), in the form of

2LO

210

180

150

120

90

60

45

TEMPERATUREK~

Fig. 5.1 1. CC separation of butyl esters of carboxylic acids. Peaks: 1 = glycolic; 2 = sorbic; 3 = leucic; 4 = mevalonic; 5 = salicylic; 6 = n-hexadecane; 7 = succinic; 8 = itaconic; 9 = adipic; 10 = pimelic; 11 = suberic; 12 = sebacic; 13 = hippuric; 14 = citric; 15 = isocitric; 16 = homogentisic; 17 = kynurenic acid. Conditions: Pyrex glass column, 175 cm X 2 mm I.D., Chromosorb W (60-80 mesh, AW) surface modified with Carbowax 20M; nitrogen flow-rate, 30 ml/min; temperature programme, 3"Clmin (45-240°C). (Reproduced from J. Chromatogr., 82 (1973) 382, by courtesy of K.O. Gerhardt.)

CARBOXY LIC ACIDS

115

acid butyl esters which were prepared by the following procedure. A solution of a mixture of acids was placed in a reaction vial with a PTFE cup and the solvent was evaporated under a stream of nitrogen. A 2-ml volume of 3 N HC1-n-butanol was added t o the dry sample and the stoppered vial was shaken in an ultrasonic bath at room temperature for 2 min. Esterification was performed by heating at 75°C in a water-bath for 30 min; after cooling, the excess of the reagent was removed with a stream of nitrogen. The residue was dissolved in 100 gl of dry acetone, and 1 pl was injected into the apparatus. Analysis of fatty acids with a short and medium chain lengths can also be performed after their conversion into benzyl esters [ 144-1461. The esterification is carried out with diazotoluene, which is prepared by decomposing N-nitroso-N-benzyl-p-toluenesulphonamide with sodium methoxide in an alkaline medium. The reagent is obtained by introducing a nitroso group into readily available N-benzyl-p-toluenesulphonamide with the aid of sodium nitrite and glacial acetic acid. Compared with diazomethane, diazotoluene is stable and much less explosive and can be stored in n-pentane at -20°C for several weeks. The esterification is performed by the following procedure. Diazotoluene in n-pentane is added to a 1% solution of carboxylic acids in n-pentane at room temperature. The esterification proceeds with the evolution of nitrogen and the red colour fades. After 10-15 min (or 30 min at 0°C) the excess of diazotoluene is decomposed by adding a 1% ethereal solution of phosphoric acid. A 0.5-1-pl volume can be injected without any further purification. Possible competitive reactions (e.g., cycloadditions on the double bond) proceed only very slowly with diazotoluene; dibenzyl ether is produced by the reaction with water so that strictly anhydrous conditions are not necessary. Similarly, the presence of traces of water does not interfere with the esterification with the aid of N,N’-dicyclohexyl-0benzylisourea, which reacts with water with the production of benzyl alcohol. The reagent is synthesized from dicyclohexylcarbodiimide and benzyl alcohol with copper(1) chloride as the catalyst. The esterification proceeds according to Scheme 5.16. C H - N = C - N H - C H 11 6 11

I 0 - C H2 - C6H5

+

R-COOH

-

R-COO-CH

-C H

t

C6Hl

-NH-C-NH-C6H1,

II

0

Scheme 5.16.

A solution of carboxylic acids in benzene (ca. 1 g per 100 ml) is refluxed with a slight excess of N,N’-dicyclohexyl-0-benzylisourea for 1 h. After cooling to room temperature and sedimentation of dicyclohexylurea, 0.5-1 .O pl of the supernatant is injected without any further purification. GC separation can be performed on an SE-30 or ECSS-X column. Corina [ 1471 reported retention data of benzyl esters of a number of acids from various biological materials on E-30 silicone stationary phase. Richards et al. [148] developed a rapid method for determining acetate in tissues and serum. A sample of a tissue (liver) is frozen with liquid nitrogen and powdered; 0.5-1.5 g is homogenized in 3 ml of distilled water containing 0.405 pmol of propionate as an internal standard. The homogenate is acidified with 0.3 ml of 5 M perchloric acid, stirred and allowed to stand for 10 min, then centrifuged at 1500 g for 10 min, decanted and the supernatant is centrifuged for 5 min. A 1-ml volume of the final solution is trans-

116

DERIVATIZATION OF COMPOUNDS

ferred into a 15-ml centrifuge tube containing 5 ml of toluene and 1 g of anhydrous sodium sulphate. The tube is shaken vigorously by hand and centrifuged at 600 g for 1 min, the toluene phase is transferred into a 5-ml conical centrifuge tube and 10 p1 of a 0.1 M aqueous solution of benzyldimethylphenylammonium hydroxide are added. The solution is stirred as previously and centrifuged. The lower phase is taken for analysis with a microsyringe. As the extraction into toluene is not quantitative, a calibration graph must be plotted for model samples analysed by the same procedure in order to effect a quantitative evaluation. Problems with avoiding too volatile methyl esters in the GC of carboxylic acids led Umeh [ 1491 to use p-bromophenacyl and p-phenylphenacyl esters (Scheme 5.17).

Scheme 5.17.

A 5-ml volume of an ethanolic solution of acids (0.1 mol of each) is placed in a 50-ml flask and 5 ml of distilled water and several drops of 0.5% phenolphthalein solution are added. The solution is neutralized with several drops of 5 N KOH solution and made just acidic with 3 drops of 1 N HC1. Then 0.56 g of p-bromophenacyl or p-phenylphenacyl bromide is added, the mixture is refluxed for 10 min and the solid residue is dissolved by adding several millilitres of ethanol. The solution is cooled, 20 ml of ethyl acetate are added, followed by cold distilled water, the organic phase is separated, a portion of it is transferred into a test-tube with anhydrous sodium sulphate and the pure solution is injected. The advantage of this procedure is that it can be carried out in an aqueous medium, but the residues of the reagent and the derivatives of formic, acetic and propionic acids are poorly separated on the stationary phases used (2.5% sodium dodecyl benzenesulphonate or 1% Apiezon L or their 1 : 1 mixture). Combined derivatives, TMS-methyl esters, were applied by Horning et al. [ 1501 to the determination of aromatic acids in urine. The acids, dissolved in 0.5 ml of methanol, were esterified with a solution of diazomethane in diethyl ether (the reaction time is not longer than 1 min) and excess of the reagent, diethyl ether and methanol were quickly removed with a stream of nitrogen. The residue was dissolved in 0.15 ml of pyridine and free hydroxyl groups were converted into the trimethylsilyl ether by adding 0.1 ml of HMDS and 0.05 ml of TMCS. The mixture was allowed to stand at room temperature overnight, the precipitate was removed by centrifugation and the pure solution was analysed directly on 10%F-60 with temperature programming from 100 to 240°C. Esters for the selective and sensitive detection of carboxylic acids contain atoms or functional groups for which the detector is specific. As with substances of other types, preferably derivatives containing halogens are used for electron-capture detection. Karmen [ 15 1] suggested 2-chloroethyl esters for specific detection with the aid of the alkali FID. They are much less volatile than methyl or ethyl esters, and are therefore interesting for the analysis of short-chain fatty acids. GC separation was carried out on EGA polyester or SE-30. Trichloroethyl esters were proposed for the sensitive analysis of aromatic acids [ 152J.

CARBOXY LIC ACIDS

117

The esterification is performed by reaction with 10%2,2,2-trichloroethanol in trifluoroacetic anhydride in a water-bath for 10 min. For electron-capture detection TFA anhydride and the excess of the reagent must be removed from the sample. OV-17 was used as the stationary phase. With the use of the ECD, the detection limit is reported to be in the range of units to tenths of nanograms for various benzoic acids. Alley et al. [153] determined aliphatic C2-Cs acids in various biological materials at the nanogram level in this way. They carried out the esterification in chloroform in the presence of HFB anhydride. They removed the excess of the reagent by adding palmitic acid, the ester of which does not interfere in the chromatogram with the acids under analysis. The analysis was performed on 3% OV-1 with temperature programming.

I

C H30

Scheme 5.18.

Acids with other functional groups can acquire the properties necessary for specific detection by derivatization. Sjoquist and Anggard [ 1541 first esterified homovanillic acid with diazomethane and then protected the hydroxyl group by reaction with HFB anhydride according to Scheme 5.18. The resulting derivative is highly specific for the given compound and permits a very sensitive analysis to be performed. Dziedzik et al. [ 1551 proceeded according to an inverse procedure and increased the electron affinity of the derivative by using a fluorinated alcohol for the esterification. In the first step the hydroxyl group is acylated by reaction with TFA anhydride, and then the esterification is performed with hexafluoroisopropanol. As the catalytic activity of TFA anhydride is not sufficient for quantitative esterification, this is carried out with the addition of BF3 at 100°C. Stationary phases of the OV-17, QF-I, XE-60 type, etc., are recommended for the GC analysis. Kawahara [ 1561 introduced pentafluorobenzyl esters, prepared by the following procedure. A mixture of four acids (0.8 mg of each) was dissolved in 100 ml of acetone, and 250 mg (25-fold excess) of a-bromo-2,3,4,5,6-pentafluorotoluene and 50 mg (10-fold excess of potassium carbonate were added (it can be replaced with an ethanolic solution of potassium hydroxide). After refluxing for 3 h, the mixture was diluted with 500 ml of diethyl ether and 20 ml of ethyl acetate, washed with I0 ml of water and dried with 8 g of anhydrous sodium sulphate. After filtration, the sulphate and the filter were washed with 50 ml of diethyl ether, the solvent was removed and the residue was dried at 40°C and 50 mmHg; it was further dissolved in 100 ml of n-hexane and, after an additional 100-fold dilution, 6 pl were injected. The ECD response to pentafluorobenzoate was almost the same as that to aldrin. A method for the preparation of p-substituted benzyl esters of lower monocarboxylic acids on the micro-scale [ 1571 is based on the same reaction scheme. A 10-pl volume of an ethanolic solution of carboxylic acids (ca. 1 p g / p l )

DERlVATIZATION OF COMPOUNDS

118

was transferred with the aid of a microsyringe into a capillary for measuring the melting point, and 3 pl of an ethanolic solution of potassium hydroxide (3 pg/pl) and 5 pl of an ethanolic solution of benzyl halide (3 pg/pl) were added. The capillary was sealed and heated at 110°C for 1 h and, after cooling, 2 pl of the reaction mixture were injected into the chromatograph. OV-17 stationary phase and temperature of lOO"C, 12OoC and 145°C were used for the analysis of tolyl esters, p-bromobenzyl esters and p-nitrobenzyl esters of Cl-C3 acids, respectively. 0 OH

II I I I

R, 0 - P -C - R ,, R 2 0 R,

Scheme 5.19.

Derivatives containing phosphorus for the GC analysis of carboxylic acids were described by Schulz and Vilceanu [ 158J .cu-Hydroxyphosphonic acid ester (Scheme 5.19), which reacts with the acids in the presence of dicyclohexylcarbodiimide under mild conditions, was used for the esterification. The following procedure is optimal. A 100-pl volume of a benzene solution of trace amounts of fatty acid is placed into a 1-ml vessel, 150 pl of a benzene solution containing 25 g per, 100 ml of dicyclohexylcarbodiimide and 12.5 g per 100 ml of pyridine are added, followed by 50 pl of a-hydroxyphosphonic acid ester, and the mixture is shaken vigorously. After heating for 1 h at 500C the reaction is completed and, after cooling, the solution can be analysed directly. Before entering the analytical column, excess of the reagent is vented into the atmosphere through a multiway valve. Retention data of these derivatives with various combinations of Rl-% have been published [ 1581 for C1-C7 monocarboxylic acids on Apiezon L, Carbowax 20M and neopentylglycol succinate. Using the alkali FID, these acids can be determined at the level of 10 pg. 5.5.2. Silyl derivatives

The carboxyl group reacts relatively easily even with mild silylating agents, so silylation is often used as a derivatization reaction for carboxylic acids. The advantage of these derivatives applies particularly to substituted acids, which are almost always involved when the analysis of biochemically important acids is concerned. The usually necessary two-step preparation of the derivatives may be obviated; the disadvantages are the sensitivity of the derivatives towards moisture and sometimes their low stability. This is the case, e.g., with TMS derivatives of Krebs cycle acids, containing keto groups. These derivatives do not provide a simple peak, probably owing to on-column decomposition and, therefore, if keto acids are present in the sample conversion into an oxime [159] is recommended. A 10-mg amount of acids and 10 mg of hydroxylammonium chloride are placed in a 5-ml test-tube fitted with a ground-glass stopper and are dissolved in 1 ml of dry pyridine. After standing for 10 min at room temperature 0.1 ml of TMCS and 0.1 ml of HMDS are added. The reaction is completed within a few minutes

119

CARBOXYLIC ACIDS

at room temperature. The resulting mixture can be analysed directly on SE-52 stationary phase. For the quantitative determination of Krebs cycle acids, Horning et al. [160] recommended methoxime-TMS esters. A 10-mg amount of methoxylammonium chloride is added to a solution of 1 mg of a keto acid (or a mixture of acids) in 0.5 ml of pyridine. After standing for 2-3 h, pyridine is removed with a stream of nitrogen and 0.2 ml of BSTFA is added t o the residue containing methoxime and unreacted methoxylammonium chloride. After standing for 1-2 h at room temperature, 0.5-1 .O pl of the solution is taken directly for GC analysis. If silylation alone (0.2 ml of BSTFA t 0.05 ml of TMCS) without the preceding methoximation is carried out, TMS enof ether-TMS esters are produced from keto acids. Using the procedure described, methoxime-TMS esters of keto acids and TMS ether-TMS esters of hydroxy acids are produced. Unsubstituted acids give TMS esters. The procedure eliminates possible losses of the derivatives, which can be caused by, e.g., evaporation of the solvent between the esterification and the silylation steps, and is quantitative. SE-30, OV-17 and OV-22 can be used and retention data on these stationary phases have been reported for 15 acids [ 1591. An example of the separation of the derivatives of some acids prepared by this procedure is illustrated in Fig. 5.12. Chalmers and Watts [ 1611 preferred ethoxime-TMS esters for the analysis of some physiologically important keto- and aldo-carboxylic acids. These derivatives were said t o be more stable than their methoxime analogues and had good chromatographic properties on ov-101. Different silylating agents have been utilized for the preparation of TMS derivatives of phenolic acids and related substances. Shyluk et al. [162] used the following procedure for the GC of shikimic acid and related compounds. A 2-mg amount of the acid was dissolved in 0.5 nil of dry acetone and 0.2 ml of HMDS and 0.1 ml of TMCS were added. After shaking for 30 min the mixture was allowed to stand for 10 min and 1 was injected into the chromatograph. SE-30, QF-1 and XE-60 were used as stationary phases. Stronger silylating agents (e.g., BSA), which react with hindered groups, are used more

T I M E I MIN 1

Fig. 5.12. Gas chromatogram of TMS derivatives of organic acids. Peaks: 1 = lactic; 2 = P-hydroxybutyric; 3 = fumaric; 4 = succinic; 5 = malic; 6 = oxaloacetic; 7 = a-ketoglutaric; 8 = citric; 9 = isocitric acid. Conditions: glass column, 12 ft. x 4 mm, 5% OV-22 on GasChrom P (80-100 mesh); nitrogen flow-rate, 40-50 ml/min; temperature programme, 2"C/min from 50°C. (Reproduced from Aria/. L e t f . , 1 (1968) 713, by courtesy of M.G. Horning and Marcel Dekker.)

DERIVATIZATION OF COMPOUNDS

120

I

w

co

z

0

a co W a (L

0 bu W

IW

0

5

10

15

20

25

30

TIME ( M I N )

Fig. 5.13. Gas chromatogram of silylated phenolic acids. Peaks: 1 = p-hydroxybenzoic;2 = vanillic; 3 = syringic;4 = p-coumaric;5 = ferulic; 6 = sinapic acid; 7 = ndocosane. Conditions: glass column, 9 ft. X 1/4 in. O.D., 3% UCW-98 on Chromosorb W HP(100-120 mesh);nitrogen flow-rate, 40 ml/ min; temperature programme, 6"C/min from 100°C. (Reproduced from J. Chromatogr., 71 (1972) 149, by courtesy of H. Morita.)

often [163-1653. An anhydrous sample of acid is mixed with an excess of BSA (ca. 100 pl per 1 mg of the acid) and is allowed to react at room temperature. The reactior, will proceed quantitatively within a few hours even in the presence of strongly hindered groups and can be accelerated significantly by adding 10-20% of TMCS as a catalyst. Fig. 5.13 illustrates the separation of TMS derivatives of some phenolic acids on UCW-98. Vande Casteele et al. [ 1661 separated 36 naturally occurring phenolic substances, including acids, on a mixed SE-30 and SE-52 stationary phase after their conversion into TMS derivatives. They carried out the silylation with the aid of BSTFA (minimally 10 pl per 4 mg of the compound or the mixture), by heating for 10 min at 125°C in a sealed vial. They reported mass, W, IR and NMR spectra. Laik Ali [167] applied BSTFA and MSTFA to the silylation of trace amounts of salicylic acid in acetylsalicylic acid. A 0.5-1-ml volume of the reagent and 50-100 mg of acetylsalicylic acid were heated at 70-80°C in a closed vessel for 30-60 min. As the TMS derivative of acetylsalicylic acid decomposes, an exact evaluation of the analysis is difficult and therefore the author recommended stable methyl derivatives prepared by reaction with methyl iodide and potassium carbonate. In order to resolve phenolic acids better, Scott [ 1681 developed sulphonyl-TMS derivatives. Several milligrams of a free acid were dissolved in 2 mi of ethyl acetate and 0.3 ml of chlorosulphonic acid was added. The mixture was heated at 60°C for 5 min, cooled and 2 ml of water were slowly added. The mixture was then extracted with three 3-ml portions of ethyl acetate and the extracts were combined and re-extracted with two

CARBOXYLIC ACIDS

121

2-ml portions of 1 M sodium carbonate solution. The aqueous phase was acidified with 5 M HCl and extracted with three 4-ml portions of diethyl ether. The extract was evaporated to dryness, 0.1 ml of BSTFA was added to the residue and, after standing for 10 min at 6OoC, the mixture can be analysed directly. Stationary phases of the OV type can be applied and according to their polarities the selectivity for the respective acid or a pair of closely related compounds can be controlled. Kahane et al. [169] first oxidized vanillylmandelic acid, after its extraction from urine by the action of sodium periodate, to vanillin. The vanillin was then converted into the methyloxime by reaction with methoxylammonium chloride and finally silylated. The recovery of the total procedure, including extraction from urine, is said to be minimally 95%, with the resulting derivative being highly specific for the compound under analysis. The GC analysis was performed on QF-1 as stationary phase. Of a number of other applications of TMS derivatives to the analysis of carboxylic acids, only some of the most interesting and illustrative will be mentioned. These derivatives were employed so that the acids in wines [170] and organic acids in vanilla extract [171] and in fruits [I721 might be determined. Prior to silylation, the acids under analysis were isolated from matrix material in the form of lead salts and thus separated from interfering sugars and other compounds. The procedure, which differs for various materials only in detail, is outlined here for the analysis of wine. A 15-ml volume of the sample is pipetted into a 250-ml centrifuge tube and diluted to 100 ml with water. Then 3 ml of 1 N H2S04 are added and the lead salts are precipitated by adding 20 ml of a solution of lead acetate (75 g of acetate and 1 ml of acetic acid in 250 ml of solution) and ca. 0.2 g of Celite 545; the mixture is shaken vigorously for about 2 min and centrifuged. The supernatant is decanted and the lead salts are washed with two 100-ml portions of 80%ethanol with thorough stirring of the precipitate, centrifugation and decantation. The solid residue is dried at 100°C for ca. 2 h and the dried residue is suspended in 5 ml of pyridine containing 20 mg of undecanoic acid as the internal standard. Several pieces of the desiccant, 1 ml of TMCS and 2 ml of HMDS are added. After stirring for 2 min the reaction is completed and the mixture is decanted into a 5-ml centrifuge tube, centrifuged and the supernatant is injected. Non-polar and non-selective stationary phases, e.g., SE-30 and Apiezon L, are used for the GC analysis. Further applications involves the analysis of both free fatty acids and fatty acids as constituents of fats, oils, etc. Tallent and Kleiman [I731 analysed products from the lipolysis of oils (in addition to acids also mono-, di- and triglycerides). BSA is more suitable than an HMDS-TMCS mixture for the silylation of these compounds as more uniform products are produced. The reaction is carried out in a reaction vial in which 2-3 mg of dry lipolysis products are placed and 0.2-0.3 ml of the silylation agent (BSA-pyridine, 1 : 5) is added. The mixture is shaken for 1 min and then analysed directly; OV-1 is a suitable stationary phase. Wood et al. [ 1741 analysed long-chain fatty hydroxy acids. After esterification with diazomethane, hydroxyl groups were silylated with HMDS. DEGS and Apiezon L were used as the stationary phases and even the partial resolution of some diastereoisomers was achieved. TMS esters have similarly been applied to the analysis of resin acids, and retention data have been reported for several of them on SE-30, Apiezon L, QF-I, etc. [175].

DERIVATIZATION OF COMPOUNDS

122

Silyl derivatives containing halogens have been used, as for other substances, also for carboxylic acids with sensitive electron-capture detection. Chloromethyldimethylsilyl derivatives have been applied to phenolic acids, as follows [ 1761. An amount of 1 mg of phenolic acids was dissolved in pyridine (600 pl), bis(chloromethy1)tetramethyldisilazane (200 pl) and chloromethyldimethylchlorosilane (1 00 pl). The mixture was diluted with n-hexane after 30 min and 0.1-0.2 pl was analysed (UCW-98,200"C). The limit of detection was reported to be 4 ng. fH3 R-C-OH

II

t

Br-CH2-Si-CI

0

I

H3

-.--

FH3

R-C-0-CH -Si-0-Si-CH2Br

II

0

2 1

CH3

I

Nal

-.

..

CH3

Scheme 5.20.

Brooks et al. [ 1771 suggested for the ECD of lower carboxylic acids iodomethyltetramethylmethyldisiloxane esters, produced by the above reaction (Scheme 5.20). The procedure starts with a dried ethereal extract, which is evaporated almost to dryness (1 drop or less) in a test-tube in a stream of dry nitrogen. One drop of diethylamine in chloroform (1 : 11) is added as the catalyst and 1 drop of bromomethyldimethylchlorosilane in chloroform (1 : 11) is added. The mixture is stirred and the test-tube is stoppered and heated at 80°C for 1 h. After cooling,'the chloroform is evaporated with a stream of air and 0.1 ml of a saturated solution of sodium iodide in acetone is added. The content is again stirred and the test-tube is stoppered and incubated at 35°C for 30 min. After cooling, 0.9 ml of chloroform is added, the mixture is centrifuged, decanted and evaporated almost to dryness with a stream of air. After dissolving the residue in 1 ml of xylene, 0.5 pl is taken for the analysis. Derivatives of CI-Cs carboxylic acids were separated successfully on OV-1 with temperature programming from 125 to 225°C at 3"C/min. The siloxane structure was confirmed from the mass spectra, but the detection limit for the ECD was not given. The presence of water in the reaction mixture reduces significantly the yield of the reaction.

5.5.3. Other derivatives Derivatives other than esters and silyl compounds were mostly developed for particular groups of acids, if not for individual substances, containing in addition to carboxylic also other functional groups, in an attempt to obtain derivatives possessing convenient properties, e.g., derivatives of lower acids less volatile than methyl esters, or more stable, or derivatives suitable for selective and sensitive detection. Dinitrophenylhydrazones (DNPHs) were applied to the GC analysis of keto acids. As with carbonyl compounds, they are prepared by reaction with 2,4-dinitrophenylhydrazine and are also used mainly for the preliminary isolation of keto acids. They can be isolated from a dilute aqueous sample by adsorption on activated carbon and selective desorption [ 1781 : hydrazones of aldehydes with a methyl formate-dichloromethane mixture and hydrazones of keto acids with a pyridine-water azeotropic mixture. Hydrazones of acids are released from their pyridine salts with methanol containing hydrogen chloride. After

CARBOXYLIC ACIDS

123

evaporation of the solvent at O"C, hydrazones are esterified with diazomethane in diethyl ether. The esters are isolated and dissolved in 1 ml of dichloromethane-methanol (1 : 4) and, at the temperature of a solid carbon dioxide-ethanol bath, oxygen containing 2% v/v of ozone is bubbled through at a rate of 10-20 ml/min for 2.5 h and pure oxygen for 30 min. Methyl esters evolved in this manner, after being heated t o room temperature, are chromatographed on DEGA with 3% of orthophosphoric acid. The liberation of keto acids from DNPHs by ozonolysis failed with dicarboxylic acids. With some monocarboxylic keto acids, by-products can also be produced during the analysis. Kallio and Linko [ 1791 proposed a direct analysis of 2,4-DNPH methyl esters of keto acids on SE-30 at 220°C using temperature programming. Under these conditions, pairs of 2-0x0butyric and 2-0x0-3-methylbutyric acids, 2-0x0-3-methylvaleric and 2-0x04-methylvaleric acids were separated poorly; the separation could be improved by decreasing the temperature at the expense of a longer analysis time. As with DNPHs of carbonyl compounds, secondary peaks of isomers appeared. With an FID, up t o 10-ng amounts of keto acids may be analysed; with the ECD the sensitivity may be increased 100-fold, but the response is linear in a only very narrow range. Anilides and toluides of low-molecular-weight carboxylic acids were analysed by GC by Umeh [ 180,18 11, as follows. A 0.5-ml volume of a solution of CI-C8 acids (each at a concentration of 0.5 M) in ethyl acetate was pipetted into a dry test-tube containing 0.1 5 ml of thionyl chloride, and 0.5 ml of aniline was added. The open test-tube was heated in a sand-bath at 60-80°C for 5 min, cooled in an ice-bath for 2 min and, after mixing with 10 ml of 1 M sodium hydrogen carbonate solution, 10 ml of a 0.1% solution of methyl myristate in ethyl acetate were added and the test-tube, still open, was stirred until effervescence ceased. Then the test-tube was stoppered, shaken vigorously for 30 min and allowed to stand until the phases separated. It was possible to inject 2 p1 of the acetone layer or t o isolate the layer completely and dry and store it. An example of the separation of the anilides of the eight lowest straight-chain carboxylic acids on sodium dodecylbenzene sulphonate is shown in Fig. 5.14. The problem of the procedure described above consists in that formanilide cannot be prepared as CO, SOz and HCl are produced by the action of thionyl chloride on formic acid. Formanilide must, therefore, be prepared in an aqueous medium, the other acids must be extracted into a non-aqueous medium. The yield of the extraction varies considerably depending on the type of the acid, and the presence of water in the reaction medium may affect the reaction yield significantly, which makes quantitative analysis difficult. Condensates with toluidine, which were used for the determination of formic acid by GC, are prepared by an analogous procedure. As other toluidine isomers may serve as the reagents, formyl derivatives have been suggested for the separation of these substances [ 1811. Dimethylformamide was suggested for the GC detection of microgram amounts of formic acid [ 1821. After esterification of acids with diazomethane the sample was treated with a dimethylamine-water mixture (1 : 2). Dimethylformamide was thus produced from formic acid ester, while other acids present were analysed as methyl esters. n-Butyl boronates prepared by reaction with n-butylboronic acid may be used for GC analysis of bifunctional carboxylic acids [ 1831. Reactions with a-hydroxy, aliphatic, alicyclic and aromatic 0-hydroxy acids proceed very quickly at room temperature. They are performed in dimethylformamide, except when the latter possesses the same reter:-

124

DERIVATIZATIONOF COMPOUNDS

I

30

25

20 15 TIME ( M I N )

10

5

Fig. 5.14. Separation of anilide derivatives of C1 -Cs straight-chain carboxylic acids. Peaks: 1 = propionic; 2 = n-butyric; 3 = acetic; 4 = n-valeric;5 = n-hexanoic; 6 = formic and n-heptanoic; 7 = n-octanoic acid; STD = methyl rnyristate. Conditions: glass column, 12 ft. X 3 mm I.D., 2.5%(w/w) sodium dodecylbenzene sulphonate on Chromosorb G (60-80 mesh, NAW); nitrogen inlet pressure, 15 p.s.i.; temperature, 200°C. (Reproduced from J. Chromatogr., 51 (1970) 147.)

tion time as that of one of the derivatives; then ethyl acetate, pyridine or acetone is used. The derivatives give well shaped peaks and may be separated on 1% of OV-17. However, some derivatives are not stable, particularly in the presence of moisture. n-Butyl boronates are also formed with phenylpyruvic acid, owing to the stabilizing effect of the phenyl groups. Other keto acids either form these derivatives with difficulty, or not at all. Quinoxalinones have been suggested for the chromatography of keto acids [ 184,1851. They are produced by reaction with aromatic o-diamines, as shown in Scheme 4.21 (p. 77). A solution of 60 mmol of sublimed o-phenylenediamine in 100 ml of 10% acetic acid was mixed with a solution of 30 mmol of the keto acid in 30 ml of water. The precipitated quinoxalinone was filtered after 15 min, washed with water, dried and crystallized from methanol. Prior to the GC analysis proper, it had to be converted into a silyl derivative: 5-50 pi of about a 1% solution of quinoxalinone in dry pyridine was mixed with 20 pl of a pyridine solution of the internal standard (6-methyl-2-naphtho1, p-nitrophenyl phenyl ether). A 200-pl volume of BSA and 50 pl of pyridine were added. As the silylation proceeded very quickly, the reaction mixture could be injected immediately. Another silylation procedure consists in a 30-min treatment with acetonitrile-BSTFA (1 : 1) at 20°C, when the enolized keto group is also silylated, as demonstrated by IR and mass spectra [ 1851. SE-30 or OV-17 were utilized and the temperature was programmed from 100 to 20OoC. By condensation with diamines with various substituents, derivatives possessing specific properties can be obtained. More complicated procedures, involving more extensive chemical changes of the acids under analysis, have been developed for special purposes. As an example, the determination of 3-methoxy-4-hydroxymandellic acid in urine, described by Dekirmenjian and

CARBOXYLIC ACIDS

125

Maas [186], can be considered. A 3-ml sample of urine was acidified with 0.3 ml of 3 N HCl and extracted with three 10-ml portions of ethyl acetate, and the organic phases were withdrawn carefully, combined and extracted with 1 ml of 1 M K2C03 solution. The layer of K2C03 separated quantitatively, 0.2 ml of 2%periodate solution was added and the substrate was oxidized at 50°C for 30 min. The reaction was stopped by the addition of 0.2 ml of 10% sodium metabisulphite, the reaction mixture was cooled with water and vanillin was reduced to vanillyl alcohol by reaction with 100 ml of potassium borohydride at room temperature for 10 min. The pH was then adjusted to 7.0 with 5 N acetic acid and 0.6 ml of phosphate buffer @H 7.2) was added. After mixing, the mixture was extracted with two 10-ml portions of ethyl acetate and the extracts were combined and evaporated to dryness at decreased pressure. In order to remove borate, methanol was added and the mixture again evaporated to dryness. The residue was transferred into a small vial with 2 ml of ethyl acetate and was treated with 0.5 ml of trifluoroaceticanhydride at room temperature for 1 h. The contents of the vial were evaporated to dryness in a n evacuated desiccator, the residue dissolved in 1 ml of ethyl acetate and 1 pl analysed on 3% OV-17 at 150°C. The method is said to be more reproducible than a direct vanillin acylation as uncontrolled losses of this volatile intermediate product are obviated. Esselman and Clagett [187] determined the position of the oxygen atom in the chain of polyfunctional fatty acids. The method is based on the reduction of keto, hydroperoxy, epoxy and carboxyl groups to the corresponding alcohols with LiAlH4,-subsequent silylation with BSA and the analysis by GC-MS using OV-1 at 225°C. 5.5.4. Separation of enantiomers

Enantiomers of carboxylic acids may sometimes be separated by GC as methyl esters, but special derivatives are mostly prepared for this purpose. Ackman et al. [ 1881 resolved enantiomers of isoprenoid fatty acids after their conversion into L-menthyl esters. The acids under analysis were chlorinated by refluxing with distilled freshly prepared thionyl chloride and the chlorides produced were treated with L-menthol in the presence of pyridine under strictly anhydrous conditions. GC separation was carried out in a capillary column coated with butanediol succinate polyester. Annett and Stumpf [I891 made use of L-menthyloxycarbonyl derivatives for the separation of enantiomers of methyl esters of hydroxy acids. The derivatization reagent, L-menthyl chloroformate, was prepared by the reaction of L-menthol with phosgene, with cooling with ice. Diastereoisomers of different hydroxy acids were thus separated on 1.5% OV-210. D-Phenyl propionates of methyl esters of racemic hydroxy acids were prepared by Hammarstrom and Hamberg [190] and used for the separation of enantiomers of these compounds on QF-1. The separation was successful with methyl 3-, 15-, 16- a n d 17hydroxyoctadecanoates, whereas diastereoisomers of methyl 4-, 7- and 13-hydroxyoctadecanoic acids were not separated. The derivatives were prepared at room temperature reaction of the ester of the hydroxy acid with D-2-phenylpropionylchloride in the presence of pyridine for 2 h.

126

DERIVATIZATION OF COMPOUNDS

5.6. AMINO ACIDS

Amino acids cannot be analysed by GC unless suitable derivatives are prepared, which obviously is a disadvantage over other methods, such as paper, thin-layer and ion-exchange chromatography. On the other hand, the GC analysis of derivatives of amino acids is rapid, instrumentation can be used for a wide range of applications, the sensitivity of the analysis if high and there is the possibility of working with small amounts of samples. Also there is the possibility of combining GC with MS as a sensitive detector or a means of identification. The preparation of derivatives suitable for GC is complicated by the presence of different functional groups with different reactivities in the molecules of amino acids. Of amino acids that form proteins, simple amino acids with an aliphatic chain [glycine (Gly), alanine (Ala), valine (Val), leucine (Leu) and isoleucine (Ile)] contain carboxyl and amino groups only; proline (Pro), methionine (Met) and phenylalanine @he) are substituted with a non-reactive group. An additional hydroxyl group is present in serine (Ser), threonine (Thr) and tyrosine (Tyr), and another carboxyl group in aspartic (Asp) and glutamic (Glu) acids, with a carbamide group in their amides [asparagine (Asn) and glutamine (Gln)] . Basic amino acids [lysine (Lys), arginine (Arg), tryptophan (Trp) and histidine (His)] contain another amino (or imino) group. Cystine (Cys) contains a disulphidic bond, being composed of two molecules of its reduced form, cysteine (CysH), with a thiol group, and both compounds are easily converted into each other. The lability of the thiol group often results in cysteic acid (Cys03H) and S-methylcysteine (CysM) being determined instead of CysH. In addition to structural protein amino acids, other biologically important amino acids exist that can be determined simu!taneously with the acids mentioned above, e.g., hydroxyproline (Hypro), ornithine (Om), 0-alanine @-Ala), hydroxylysine (Hylys), y-aminobutyric acid (y-ABA), norvaline (Nval), norleucine (Nleu) and sarcosine (Sar). Iodoamino acids are dealt with separately in Section 5.7. One can conclude from this incomplete list that the quantitative preparation of a uniform derivative of all amino acids will be very difficult owing to the great variety of structures and chemical properties of amino acids. Since 1956, when the first papers devoted to the GC of amino acids appeared, a number of workers have tried to solve the problem. One of the most detailed reviews [I911 covers 415 publications up to 1974 and states that about 100 different chemical procedures for the preparation of derivatives suitable for GC analysis were tested. In spite of the fact that there has not been agreement about the most suitable derivatives, the most preferred derivatives are generally those which require two derivatization steps, i.e., esterification of the carboxyl and acylation of the amino and other functional groups. Difficulties mainly appear when preparing derivatives of Arg, His and Trp. Acylation of the guanidine group of Arg and the imidazole group of His is complicated owing to the formation of salts in a strongly acidic medium. The indole nitrogen atom in Trp is also acylated with difficulty and, although the other basic centre is acylated easily, the monoacyl derivative with a free indole nitrogen requires a high temperature in order to be eluted. Similarly, Lys necessitates the use of an acidic medium for the acylation of the other amino group. The oxidation of CysH during the derivatization can be prevented by an inert atmosphere. One-step procedures, advantageous as they may seem owing to their simplicity, have not been completely successful owing to the different reactivities of the various groups

AMINO ACIDS

127

present. TMS derivatives are often not stable and in the presence of different functional groups in the molecule the silylation can, depending on reaction conditions, proceed to different extents. Other procedures, e.g., preparation of diisopropyl derivatives, also suffer from a non-quantitative course of the reaction. The separation of enantiomers of amino acids is accomplished by using the same derivatives and optically active stationary phases or by using chiral reagents for the preparation of the derivatives. Selection of stationary phases suitable for the separation of derivatives of amino acids is another very difficult problem in their GC analysis, and is often so serious that it prevents the more general use of some derivatives and these are then used for only a limited number of amino acids. 5.6.1. N-Acyl alkyl esters

These derivatives for the analysis of amino acids have been widely investigated and various combinations of acyl and alkyl groups have been tested in order to find the properties most suitable from the viewpoint of chromatography, the reaction yields and the reaction time for the whole group of amino acids. Their preparation is based on the first, i.e., esterification step, when common esterification reactions are applied with minor modifications, and the second step, when amino and other functional groups are acylated with anhydrides, chlorides or other acylating agents. N-Acetyl alkyl esters with different C1-Csalkyl groups have been adopted for the GC of amino acids. Acetic anhydride offers the advantage of being commonly available, but in some instances it is not reactive enough, e.g., for acylation of the dihydrochloride of Arg alkyl ester; the guanidine group in the form of the hydrochloride is also difficult to acylate. In order to secure a quantitative course of the acylation, amino acids must sometimes be converted into free bases with the aid of an anion-exchange resin or by neutralization with an alkali metal carbonate. Methyl and ethyl esters [192-1971, (iso)propyl esters [193,195,198-2001, (iso)butyl esters [192,193,195,196,201] and (iso)amyl esters [202,203] of amino acids were modified by acylation with acetic anhydride. The esterification is mostly performed by the action of an anhydrous alcohol acidified with HCl with concentrations varying from 1.25 N up to saturation. Generally, the use of higher alcohols for the esterification necessitates a higher temperature and a longer reaction time. The reagent is prepared by bubbling dry hydrogen chloride through the alcohol until the required concentration is obtained. The use of high HCl concentrations combined with a long reaction time can, however, lead to decomposition of some amino acids, e.g., Trp. Lower solubilities of amino acids in higher alcohols cause problems when preparing higher esters; Stalling et al. I2041 avoided these difficulties by preparing methyl esters by reaction with methanol containing HCl at a concentration of 1.25 N at room temperature for 30 min. Then they transesterified the methyl esters by treating them with a 1.25 N HCl solution in n-butanol at 100°C for 150 min. Esterification with higher diazoalkanes (2051 has also been suggested. A 2-ml volume of 50% potassium hydroxide solution was added to 5 ml of diethyl ether in a small flask. N-n-Butyl(or propy1)-N-nitrosoguanidine (1 g) was suspended in diethyl ether and added to the flask through a separating funnel. The reaction was carried out in a water-bath at 45°C. The diazo compound have been passed through a cooler, was trapped in diethyl ether. Hydrochlorides of amino acids (about 5 mg) were dissolved in 4 ml of methanol

DERIVATIZATION OF COMPOUNDS

128

and the ethereal solution of the diazo compound was added until a permanent yellow colour persisted (5 ml). After 5 min, the excess of the reagent was removed under vacuum. Polyesters and polyglycols are mostly used as stationary phases for the separation of acyl alkyl esters of amino acids. Fourteen protein amino acids were separated on Carbowax 1540 in the form of their acetyl butyl esters [ 1931, and a comparative study of the preparation and analysis of acetyl butyl and amyl esters of 17 naturally occurring amino acids was published by Johnson et al. [202]. As the separation of all protein amino acids in the form of acetyl alkyl esters on a simple stationary phase was not successful, mixed stationary phases were tested. The most promising were, e.g., 0.7% Carbowax 6000 (half of the column) and 0.7% Carbowax plus 0.05% tris(cyanoethy1)pentaerythritol on Chromosorb G [198], and 0.31% Carbowax 20M, 0.28% Silar 5CP and 0.06% Lexan on Chromosorb W AW [ 199,2001. Coulter and Hahn [ 1981 chromatographed amino acids using acetyl n-propyl esters, which were prepared in an apparatus designed especially for the purpose, by the following procedure. A solution of amino acids or hydrolysate of proteins (ca. 0.1 ml) was placed in a ground-glass test-tube (volume about 2 ml) and quickly dried at 100°C under a stream of nitrogen. After drying, 0.4 ml of 8 N HCl in n-propanol was added and the mixture was incubated at 100°C for 10 min and then evaporated to dryness under nitrogen. This esterification step was repeated. After drying, the esters were acylated with 0.4 ml of a freshly prepared pyridine-acetic anhydride (4 : 1, v/v) mixture. The reaction was finished after 5 min at room temperature and the excess of the reagent was evaporated. The residue was dissolved in 1 ml of ethyl acetate and 1 pl was injected. Fig. 5.1 5 demonstrates the results of the analysis of 17 protein amino acids. As Arg and His are acylated only with difficulty by this procedure, the authors recommended either decreasing the acidity by adding sodium or lithium carbonate or converting Arg into Orn with the aid of the enzyme arginase and His into aspartic acid by ozonolysis.

leu

JJJ 240

ISOTHERMAL

200

PROGRAMMED TEMPERATURE 6'C/ MIN

Fig. 5.15. Gas chromatogram of N-acety1-n-propyl esters of 17 amino acids. Conditions: glass column, 106 cm X 3 mm I.D., packed with a mixture (1 : 1) of 0.7%Carbowax 6000 o n Chromosorb G (80100 mesh, HP) and 0.7%Carbowax 6000 plus 0.05% tetracyanoethyl pentaerythritol o n the same support; nitrogen flow-rate, 30 mI/min; temperature programme, 6"C/min, 100-240°C. (Reproduced from J. Chromatogr., 36 (1968) 42, by courtesy of J.R. Coulter.)

AMINO ACIDS

129

The above esterification reagent, however, decomposes Trp considerably (up to 75% after 1 h) and therefore it is essential not to exceed the optimal time in order t o obtain reproducible yields. The possibilities of acetylating all amino acids were evaluated by Adams [ 1991. A strongly basic acylating agent, consisting of acetone-ethylamine-acetic anhydride (5 : 2 : l), permitting all of the groups present to be derivatized at 60°C within 30 sec, converted the guanidine group only up to 78%. This degree of conversion was said, however, to be reproducible. Other workers [200] recommended for this procedure preliminary conversion of CysH into S-carboxymethylcysteine by the reaction with sodium iodoacetate. The derivatization was performed as follows. The solution of amino acids was evaporated to dryness at 110°C under a stream of nitrogen, 50 p1 of dimethoxypropane and 1 ml of an 8 N HC1 solution in n-propanol were added to the residue and the mixture was heated in a closed vial at 110°C for 20 min. After cooling, the excess of the reagent was evaporated at 60°C under a stream of nitrogen, 50 mg of anhydrous sodium carbonate and 1 ml of the acetylating reagent, freshly prepared, were added and after shaking for 15 sec the mixture was heated at 60°C for 3 min. Finally, the excess of the reagent was evaporated under a moderate stream of nitrogen at 60"C, the residue was dissolved in 0.5 ml of dry ethyl acetate and, after removing Na2C03 by filtration, the solution was analysed. N-Trifluoroacetyl derivatives were applied in the GC analysis of amino acids in combination with different alkyl esters. TFA anhydride serves as a strong acylating agent, which is very efficient in the derivatization of all protonic groups except carboxyl. An acylation medium, usually a mixture of TFA anhydride and methylene chloride, may be injected into the GC column without any preliminary evaporation. This is a very important fact as TFA derivatives are very sensitive towards moisture and mere evaporation can lead to decomposition, particularly of acylated hydroxy and thiol groups. Possible losses of more volatile derivatives are also eliminated. N-Trifluoroacetyl methyl esters were used by Weygand et al. [206] for the analysis of amino acids and dipeptides, their properties were described by Makisumi and Saroff [207] and the conditions for their quantitative preparation and analysis were investigated by various workers [197,208-2111. A common procedure for their preparation is the following. Esterification is accomplished by reaction with methanolic HCl at 70°C for 30 min, and acylation with TFA anhydride at room temperature for 30 min; Arg requires, however, a higher reaction temperature (140°C for 10 min) and His, even under these conditions, gives only low yields. A temperature of 120°C for 20 min was therefore recommended for acylation [212]. Teuwissen and Darbre [2 131 described an apparatus for the preparation of TFAmethyl esters of amino acids. An aqueous solution of amino acids is applied on a platinum wire, evaporated, and the derivatization is performed in a closed system. TFA-methyl esters are very volatile. Their retention times on polyester stationary phases are up to six times shorter and on silicone stationary phases up to three times shorter than those of the corresponding acetyl methyl esters. A high volatility can also result in losses of the derivatives during their preparation, and therefore esterification with diazomethane is not recommended as mere evaporation of ethereal solution can cause significant losses of the derivatives of Ala, Val, Gly and Leu. If evaporation of the

130

DERIVATIZATIONOF COMPOUNDS

acylating agent is to be carried out, then a temperature of 0°C and a reduced pressure are essential. TFA derivatives of hydroxy and thiol groups are labile and can decompose, e.g., under the action of alcohol [209], and also on some column packings, such as polyesters (except EGA), polyethylene glycols and the polar cyanoethylenesilicone XE-60 [210]. These restrictions have hindered to a certain extent the more general use of TFA methyl esters of amino acids for their GC analysis and have led to different results being obtained by various workers. Different chromatographic conditions, sometimes very unusual ones, have been tested for the analysis of TFA-methyl ester derivatives. Hagen and Black [208 J applied Carbowax 20M as the stationary phase, injected the sample at room temperature, and within 2-3 min increased the temperature to 27OoC; they detected in the chromatogram even Lys, Arg, Trp and His, thus succeeding where other workers failed, especially as far as His is concerned. The best separation of TFA-methyl esters of amino acids was obtained on a glass column (3.25 m X 2.5 mm I.D.) packed with Diatoport S coated with 2.5% of a mixed silicone phase (XEdO-QF-l-MS-200,46 : 27 : 27), both with a temperature programme and under isothermal conditions [2 111. Cliffe et al. [212] studied the use of TFA-methyl esters of amino acids for quantitative analysis. The yields of the derivatives depend on the reaction conditions: esterification was peEformed with 4 M methanolic HCl for 9 0 min at 65'C and acylation with 20% TFA anhydride in dichloromethane at 12OoC for 20 min. Injection into the chromatograph was carried out with the aid of a pre-column and the analysis was accomplished on the above-mentioned mixed stationary phase. Fig. 5.16 shows a typical analysis of a known mixture performed by tlus procedure. Replicate analysis showed a poor reproducibility for Met, Tyr, Arg and Cys (coefficient of variation +20%) and His was not acylated at all. This result is mainly caused by the strong dependence of the yield on the reaction conditions and the instability and high volatility of the derivatives (XE-60 is present in the stationary phase). Trifluoroacetyl butyl esters were studied as possible derivatives of amino acids by Zomzely et al. [214] and were described in detail in a number of papers by Gehrke et al. [2 15 and references cited therein]. These derivatives are obviously the most commonly

0

10

20

30

40

50

60

70

80 min

Fig. 5.16. Gas chromatogram of N-TFA methyl esters of amino acids. Peaks: 1 = .a-amino-n-butyric acid; 2 = biphenyl; 3 = phenanthrene;4 = methyl stearate. Conditions: glass column, 3.25 m X 2.5 mm I.D., 1.5% (w/w) of XE40,QF-1 and MS-200 mixed stationary phase (46 : 27 : 27) on Diatoport S (80-100 mesh); nitrogen flow-rate, 15 ml/min; temperature programme, (A) l"C/min from 90°C for 20 min, (B) 3"C/min for 7 min, (C) hold for 22 min, (D) 6"C/min for 12 min, (E) hold for 6 min, (F) 4"C/min for 7 min, (G) fmal hold at 231'C. (Reproduced from J. Chromatogr., 78 (1973) 333, by courtesy of A.J. Cliffe.)

AMINO ACIDS

131

used in the GC analysis of amino acids. Various acylation and esterification conditions were tested in efforts aimed at the quantitative preparation of all amino acid derivatives and the solution of the problems associated with their !ow solubility in n-butanol, and the following derivatization procedure was proposed [2 151. A sample of amino acids (e.g., alkaline eluate of protein hydrolysate from an ion-exchange resin) was evaporated to dryness under vacuum at 60°C. About 15 ml of n-butanol-3 N HC1 were added and the reaction flask was closed with a drying tube, sonicated for 1 min and then a magnetic stirrer was applied at 100°C for 15 min. After the esterification the flask was cooled and the excess of the reagent distilled under vacuum at 60°C. Residues of water were removed with the addition of 10 ml of dry dichloromethane and by azeotropic distillation. The samples was cooled, about 2 ml of dry dichloromethane and 1 ml of TFA anhydride were added and the sample was transferred into two 8-ml test-tubes which were closed with PTFE-lined screw-caps. Acylation was executed at 150°C for 5 min. Until take for analysis, the samples were stored in a freezer at 0°C. As an alternative procedure for the preparation of the derivatives already mentioned, esterification with diazobutane [205] and transesterification [204] have been suggested. Cancalon and Klingman [216] reported that having used the procedure described above for nanomole amounts of amino acids they did not obtain satisfactory results, and suggested the use of dichloromethane in n-butanol-3.5 N HCl(1 : 9) and 5 min in an ultrasonic bath. The influence of the presence of salts on the yield and the reliability of the results of GC analysis was also evaluated (2171 ;the presence of inorganic salts in an amount comparable to &hetotal weight of amino acids has no significant influence on qualitative analysis, but in some instances it can interfere in quantitative determinations. A number of packings with different stationary phases have been tested for the GC separation of TFA-butyl esters of amino acids. Polyester stationary phases, e.g., 1% NPGS on Gas-Chrom, have often been used and almost complete separations of all protein amino acids, except for one pair (Asp-Phe), have been achieved [214]. Using mixed stationary phases or less coating, an improvement can be obtained. Stefanovic and Walker [218] tested the suitability of EGA on Chromosorb W AW with 0.5-2.0% coating. When a temperature programme was used in the range from 80 to 230"C, the elution sequence changed, depending on the amount of stationary phase: a 0.65% coating appeared to be the best for the separation of 17 amino acids. McBride and Klingman [2 191 also used a temperature programme with a 1.1 m X 4 mm I.D. column packed with 1.2% phenyldiethanolamine succinate on Gas-Chrom A and obtained similar results. The use of Apiezon resulted in as good a separation as with other stationary phases. Gehrke and Takeda [220] separated all 20 protein amino acids on a column with 10% of Apiezon M; the chromatogram is shown in Fig. 5.17. Certain amino acids, however, give unexpected losses in the column (Met 14%,His 37%, Cys 54%). Silicone stationary phases are recommended for use in mixtures and in dual-column systems, including one column packed with 2% of OV-17 and 1% of OV-210 on Gas-Chrom Q and the other with 0.65% of EGA on Chromosorb W AW [2 151. The columns can either be switched independently to provide information for identification, or the signals from both columns are combined after a simultaneous injection in order to obtain the separation of all of the solutes and a reduction in the analysis time [221]. GC for the analysis of TFA-butyl esters of amino acids has also been applied to samples

132

DERIVATIZATION OF COMPOUNDS

I

Fig. 5.17. GC separation of N-TFA butyl esters of protein amino acids. Peaks: 1 = Ala; 2 = Thr; 3 = Gly; 4 = Ser; 5 = Val; 6 = Leu; 7 = Ile; 8 = CysH; 9 = HyPro; 10 = Pro; 11 = Met; 12 = Om (internal standard); 13 = His; 14 = Asp; 15 = Phe; 16 = Lys; 17 = Tyr; 18 = Glu; 19 = Arg; 20 = tranexamic acid (internal standard); 21 = Trp; 22 = Cys; 23 = n-butyl stearate (internal standard). Conditions: glass column, 2.5 m X 2 mm I.D., 10%Apiezon M on Chromosorb W (80-100 mesh, HP); nitrogen flow-rate, 20 ml/min; temperature programme, 90°C for 6 min, then 6"C/min to 260°C. (Reproduced from J. Chromatogr., 76 (1973) 6 3 , by courtesy of C.W. Gehrke.)

containing nanogram to picogram amounts of these substances [222,223]. At this level, contamination of amino acids is a serious problem. The most significant sources are laboratory facilities (water, butanol, methylene chloride, hydrochloric acid), human body (fingerprints, scraps of skin, hair, saliva, etc.), dust and cigarette smoke. The injection of a large volume of the sample (up to 100 PI) on to a conventional packed column may be achieved by the injection system, which separates the bulk of the solvent and the excess of the reagent from the derivatives proper on a short pre-column packed with a mixed silicone phase and is led outside the analytical column proper [215]. The derivatives are trapped in a cooled section of the column. When the solvent and other interfering compounds have been vented, the analysis is started by switching on a temperature programme. Hence, the concentration effect need not be effected by evaporation and therefore there is no risk of losses of the derivatives. Using the FID 5 ng and with the ECD 1-50 pg of a particular amino acid can be analysed. Certain principles must be adhered to for TFA-butyl esters to be used successfully in the analysis of amino acids. The most important are the use of chemicals free of water and other impurities, observance of temperature and reaction time during the esterification and acylation and elimination of contact with metals, e.g., in the injection port. Amico et al. [224] applied these derivatives to 30 non-protein in addition to protein amino acids. Trifluoroacetyl amyl esters were studied by Teuwissen [225] and were described in a number of papers by Darbre and Blau [ 197,226-229 1. The main reason for the development of these derivatives was their lower volatility and thus minimal losses during their preparation. Table 5.10 compares the volatility of N-TFA-alanine esterified with alcohols from methanol up to n-amyl alcohol [ 1971. A known amount of each derivative was

AMINO ACIDS

133

TABLE 5.10 LOSSES OF AMINO ACID DERIVATIVES OWING TO VOLATILITY [ 1971 Treatment: argon at 250 ml/min, diameter of the jet 1.1 mm, distance from ester 3 cm in a tube of 1.0 cm I.D. GC conditions: 1%PEGA on Celite 560 (100-120 mesh, AW, sihized); carrier gas, nitrogen at 38 ml/min; temperature, 108°C N-TFA-alanine ester

Methyl Ethyl nPropy1 nButyl n-Amy1

Recovery (%) after exposure to the stream of gas for

5 min

20 min

66 61 100 96 99

21

6 61 13 93

Retention time (rnin)

3.1 3.2 4.9 1.0 11.5

exposed to a stream of gas (250 ml/min) at room temperature, and at various time intervals the remaining percentage of the sample was determined by GC. Under these conditions, methyl and ethyl esters can be evaporated completely whereas n-amyl esters merely show small losses only even with longer action of the stream of gas. With shorter reaction times, not even losses of n-propyl and n-butyl esters are significant, and when using an appropriate treatment for their preparation their volatility does not interfere in the quantitative determination. The retention times given also demonstrate the differences in the volatilities of various esters. Based on experience, the following procedure was suggested for the preparation of TFA-amyl esters of amino acids. A sample of amino acids (0.5-2 nig of each acid) was placed into a test-tube and dissolved in 0.2 ml of trifluoroacetic acid. Amy1 alcohol (2 ml) was added and dry hydrogen chloride was bubbled through the reaction mixture continuously at 108°C for 25 min. Excess of the reagent was removed under vacuum, dissolved in a small amount of methanol, transferred into a small test-tube and the methanol was evaporated by standing freely at 7OoC. TFA anhydride (1 ml) was added and the stoppered test-tube was allowed to stand at room temperature for 1 h. As some of the amino acids (e.g., Arg) were not acylated quantitatively by this procedure, it was recommended that the sealed test-tube be heated at 140°C for 5 min [229]. Excess of anhydride was removed under vacuum and the residue was dissolved in a known volume of dry methyl ethyl ketone. The selection of a stationary phase suitable for the GC analysis of all amino acids seemed to be the main problem hindering the wider use of these derivatives, which were always applied to a limited number of amino acids only. For the separation of TFA-amyl esters about 100 stationary phases have been tested, most of which were rejected owing to the decomposition of the acyl derivatives of hydroxyl and thiol groups that proceeds on some stationary phases with Tyr, Ser, Hypro, Thr and CysH. The application of 25% DEGS led to the elution of only eight derivatives of amino acids out of thirteen that were analysed [225]. The polyester PEGA and the silicones QF-1 and MS-710 [197,227, 2281 were reported as the most suitable liquid stationary phases. For the analysis of

DERIVATIZATION OF COMPOUNDS

134

twenty protein amino acids in the form of TFA-amyl esters, two packings were used [229] : the nine most volatile derivatives were separated on a 3-5 m glass column packed with 5% of a mixture of XE-60 and MS-550 (3 : 2) on Anakrom ABS at 135OC; for the analysis of the remaining eleven less volatile derivatives, the best coating was 5% of a mixture of QF-1 and MS-710 (53 : 47). However, the separation of some derivatives was poor; Arg, Trp and Cys were eluted fmm the column only after about 6 h. Pentafluoropropionyl and heptafluorobutyryl derivatives have also been used for the GC analysis of amino acids in combination with esterification with various alcohols. In comparison with TFA derivatives, they are much more stable and resitant to hydrolysis, their retention times are shorter and all twenty protein amino acids can be separated on common phases (OV, SE, etc.). The price and restricted availability of the acylation reagents obviously hinder their wider application. Pollock [230] chromatographed PFP-butyl and HFB-butyl esters of fourteen protein amino acids in a capillary coated with Carbowax 20M and compared the results with those for TFA-butyl esters. Under isothermal conditions (100, 140 and 17OoC) HFB esters have retention times about 35% lower and PFP esters about 30% lower than those of the corresponding TFA esters. Using the ECD, a 3-10-fold increase in sensitivity can be achieved, e.g., the minimal detectable amount of HFB-butyl esters is 1 pg for Met and 2 pg for CysH. N-HFB-propyl esters of all protein amino acids were separated by Moss et al. [231] on 3% OV-1 (Fig. 5.18). They performed propylation in a similar manner to Coulter and Hann [198] (see p. 128), but in a test-tube that withstands high temperatures. After esterification, 0.2 ml of HFB anhydride and 0.1 ml of ethyl acetate were added to the dry sample. The test-tube was sealed and heated to 150°C for 10 min. After cooling, the contents were evaporated to dryness under a moderate stream of nitrogen and the residue was dissolved in 0.1 ml of ethyl acetate. A 3-1.11volume together with 2 pl of acetic anhydride was injected. In the absence of anhydride in the injection, the His derivative decom-

3 ,

I

1

1

1

1

1

1

1

1

1

1

1

1

I

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 TIME I MIN 1

Fig. 5.18. Gas chromatogram of N-HFB n-propyl esters of 20 protein amino acids. Peaks: 1 = Ma; 2 = Gly; 3 = Val; 4 = Thr; 5 = Ser; 6 = Leu; 7 = Ile; 8 = Pro; 9 = CysH; 10 = HyPro; 11 = Met; 12 = Asp; 13 = Phe; 14 = Glu; 15 = Lys; 16 = Tyr; 17 = Arg; 18 = His; 19 = Try; 20 = Cys. Conditions: glass column, 12 ft. X 1/4 in.O.D., 3% OV-1 on Chromosorb W (80-100 mesh, HP, AW, DMCStreated); helium flow-rate, 4 0 ml/min; temperature programme, 100°C for 5 min then 4"C/min. (Reproduced from J. Chromatogr., 60 (1971) 134, by courtesy of C.W. Moss.)

AMINO ACIDS

135

poses into the rnonoacyl derivative and tails significantly. Jonsson et al. [232] injected 2 p1 of a solution of the derivatives together with 2 pl of freshly prepared HFB anhydride and chromatographed it on a 6-m glass capillary column with chemically bonded dimethylsiloxane polymer as the stationary phase. The analysis of all the amino acids took about 35 min with temperature programming. March [233] modified the method as follows. Esterification was performed in 6 M HCl in n-propanol at 15OoC for 3.5 min with subsequent acylation at the same temperature for 12 min. Analysis was carried out in a column packed with 2.5% of OV-101 and 0.5% of OV-7 with temperature programming. An improved separation was obtained at the expense of a longer analysis time (up to 55 min), however. N-HFB-butyl (isobutyl) esters are said to be the least volatile and their treatment at room temperature does not lead to losses. MacKenzie and Tenaschuk [234] separated these derivatives in a simple column packed with 3% SE-30 and applied the method to the proteins in plant seeds. Poole and Verzele [235] applied SE-30,0V-17 and OV-210 in glass capillary columns and demonstrated their use in various applications. Felker [236] drew attention to an unfavourable influence of peroxides with which reagents and solvents can often be contaminated, which decreases the yield of the acylation reaction, mainly of Met and Trp. He used 3% of SP 2100 for the separation. Zanetta and Vincendon [237] preferred N-HFB-isoamyl esters of amino acids, which reputedly can be concentrated without any loss of material. They obtained a very good separation of these derivatives of all protein amino acids, including kynurenine and ornithine, on a 3.5-m column packed with 3% of SE-30 on Gas-Chrom Q. The analysis took about 40 min with temperature programming. Fu and Mak [238,239] dealt with acyl alkyl esters of amino acids with various combinations of acyl and alkyl moieties in comprehensive studies. They evaluated and determined optimal conditions for their preparation: they selected acylation with the aid of the anhydride or chloride of the appropriate acid as the first step. They performed the reaction at 20-30°C with vigorous stirring for 10 min, with subsequent esterification with the appropriate alcohol in dry benzene in the presence of Amberlite IR-120 (H'). They further evaluated their retention properties on both Carbowax 20M and GE-XE-60, and the relationships between these properties and their structure. Kolb and Hoser [240J described a microdevice in which the derivatives were prepared with the aid of opened aluminium or gold capsules (2O-pl volume) which acted as a microreactor in an automatic capsule-dosage ,system (MS 41). As an example they demonstrated the application of their device to the preparation of HFB-methyl esters. Other acyl alkyl esters have been utilized only sporadically. Makita et al. [241] analysed N-isobutyloxycarbonyl methyl esters of protein amino acids. During the first step of the preparation, the amino group reacts with isobutyl chloroformate according to Scheme 5.21. The reaction is accomplished in 10 rnin in an aqueous medium in the presence of sodium carbonate at room temperature. Excess of the reagent is extracted with diethyl ether and the reaction mixture is saturated with NaCl, acidified with orthophosphoric acid to pH 1-2 and extracted with diethyl ether. Methanol is added to the ethereal extract and the carboxyl group is esterified with diazomethane at room temperature for 5 min. The solvent is removed under a stream of nitrogen at 50°C and the residue is dissolved in ethyl acetate. Arg does not provide a volatile derivative when sub-

136

DERIVATIZATION OF COMPOUNDS

-

R-CH-COOH

I

,LH3

NH-COO-CH2-CH

+

NoCL

\

H3

Scheme 5.2 1.

jected to this method and prior to the derivatization it must therefore be converted into Orn by using arginase. The yields of the derivatives of all amino acids are higher than 94%. The separation of twenty protein amino acids was achieved in a dual column system using a 0.65% Poly-A-1O1A column and a 0.70% FFM-Poly-A-1OlA (1 : 1, w/w) column. The N-benzoyl methyl ester of Gly has been analysed on QF-1 [242] and N-benzyloxycarbonyl methyl and ethyl esters and N-palmitoyl ethyl esters of Leu on SE-30 [243]. N-Propionyl isoamyl derivatives of fourteen amino acids were prepared by allowing isoamyl esters to react with propionyl chloride and were also analysed on QF-I and applied to the analysis of amino acids in bacterial cultures [244].

5.6.2. Trimethylsilyl derivatives The advantage of a one-step preparation of the derivatives is reduced considerably by the varying reactivities of the different functional groups in the molecule of amino acids, which cause non-uniformity of the reaction products and lead to non-quantitative results. The presence of various functional groups in the molecule of amino acids and their varying affinities towards silylating agents result in the formation of different products depending on the silylating agent and the reaction conditions (Scheme 5.22). R-CH-COOH

I

TMS

donor

TMS

1

donor

HZ

H2

-

-

R-CH-COO-Si(CH3)3

R - C H - COO - S i ( C H3)3

I

TM S

donor

R - C H - C 00 - S i(C H3I3

'silt H3l3

I

N

NH

( C H3I3 S<

' S i ( C H3I3

Scheme 5.22.

It must further be taken into consideration that in the molecule of some amino acids another amino or imino or hydroxy group may be present. In order that a higher degree of conversion may be obtained, a longer reaction time (2.5 h) and a higher temperature

AMINO ACIDS

137

are usually necessary. Polar solvents, which are required for some amino acids (Arg), lead to the formation of two or three products. Also, TMS derivatives of amino acids are very sensitive to moisture, particularly the derivatives of amino groups; the materials of the instruments, columns and packings used can also cause their partial decomposition. Despite these discouraging properties, several workers have studied the applications of TMS derivatives of amino acids to their GC analysis, attempting to make use of the advantage of a one-step operation and to develop the knowledge obtained in this way into a general method. In the early works Rdhlmann and co-workers [245,246] and later also other workers [247,248] used TMCS, HMDS, TMSDEA and their combinations as silylating agents and drew some conclusions from the results. (i) Silylation of amino acids can be considered as a two-step process - carboxyl groups are silylated faster than amino groups. As carboxyl groups are silylated much more readily than amino groups, two products may be produced by the silylation. Mild silylating agents, e.g., HMDS or TMCS, are sufficient for silylating carboxyl groups. The silylation of other functional groups, such as hydroxy and thiol groups, necessitates more drastic reaction conditions; amino and guanidine groups are the most difficult to silylate. (ii) TMSDEA was evaluated as a preferred reagent; HMDS either alone or in a mixture with TMCS and pyridine as a solvent did not lead to sufficiently good results. (iii) The labilities of various silylated groups in the molecule of completely silylated amino acid differ considerably. The Si-N bond is less stable and hydrolyses very easily, whereas the 0-Si bond is relatively stable and resistant to hydrolysis, and the silylated carboxyl group shows medium stability. In order to separate TMS derivatives of amino acids, methylsilicone stationary phases have been used. The introduction of stronger silylating agents, BSA and BSTFA, resulted in their use for amino acids, and particularly the latter reagent is preferred although some workers do not consider it to be statisfactory. Klebe et al. [249] applied BSA and acetonitrile as a solvent and prepared the derivatives of 22 amino acids by heating the reaction mixture at temperatures close to the boiling point. They obtained simple chromatographic peaks on SE-30. However, Arg decomposed on the column and the peaks of the derivatives of Ala and Gly overlapped the peak of the by-product, N-TMS-acetamide. When BSTFA was used, the mono-TMS-amide produced was more volatile and was eluted from the column before Ala and Gly. Different procedures have been suggested for achieving the quantitative silylation of amino acids with these reagents. Shahrokhi and Gehrke [250] prepared quantitatively the derivatives of twelve sulphur-containing amino acids by heating them with BSA or BSTFA in acetonitrile (1 : 3) in a closed test-tube at 150°C for 5 min. BSA gave yields ranging from 95.5 to 99.2%; BSTFA was recommended for all sulphur amino acids except Met, Met sulphoxide and CysM. Smith and Shewbart [251] compared TMSDMA, TMSDEA, BSA, BSTFA, MSA and TMSI as silylating agents for the quantitative preparation of the derivatives of Phe, Tyr and Lys as representative amino acids under different reaction conditions. They concluded that TMS-amines are to be preferred to TMS-amides as silylating agents, mainly because of the stability of the resulting solution of the derivatives and the higher volatilities of the reagents. Acetonitrile was not used as a polar solvent, however, and optimal reaction conditions were thus not secured. Albro and Fishbein [252] reported a similar comparative study. They silylated Tyr, Try and their metabolites with different reagents

138

DERIVATIZATION OF COMPOUNDS

in various combinations with pyridine as a solvent or without it. They achieved the best results using TMSDEA-TMCS (8 : 1) and BSTFA-TMSDEA-TMCS-pyridine (99 : 30 : 1 : 100) mixtures at 20°C for 3 h or at 100°C for 10 min. Other workers [253] reported that for eleven amino acids BSA alone or in acetonitrile (1 : 3) is equally efficient if it reacts at 75°C for 15 min; for Phe and Glu, BSA alone is better, however. The silylation of amino acids with BSTFA was studied in detail by Gehrke and coworkers [254-2561. BSTFA-acetonitrile (1 : 1) was applied first and fourteen amino acids were silylated at 135°C for 15 min. Glu, Arg, Lys, Trp, His and Cys, however, require up to 4 h, in order for measurable peaks to be obtained in the chromatogram. Despite such a long reaction, Gly and Glu gave two peaks and also it was difficult to separate the tris-TMS derivative of Gly from the derivatives of Ile and Pro. The influence of polar and non-polar solvents was demonstrated later and was decisive mainly with respect to uniformity of the products. Only the bis-TMS derivative was produced in hexane, methylene chloride, chloroform and 1,2-dichloroethane; bis- and tris-derivatives were produced in six more polar solvents. On the other hand, Arg did not provide any peak in the less polar solvents that were used and only one peak in the six more polar solvents. The best and most reproducible results were obtained when silylating seventeen amino acids with BSTFA-acetonitrile (1 : 1) at 150°C for 15 min; 2.5 h at 150°C were necessary for the reproducible derivatization of Gly, Arg, and Glu. These reaction conditions were recommended for the analysis of all twenty amino acids. The TMS derivatives of amino acids were found to be stable on storing them in a sealed vial at room temperature for 8 days, with no decomposition. Particular attention should be devoted to the selection of the column packing for the separation of TMS derivatives of amino acids as they are, as already mentioned, very sensitive towards moisture and can decompose on supports that have not been deactivated sufficiently. Silicone stationary phases of the SE-30, OV-l,OV-17 and DC-550 type and supports such as Gas-Chrom Q, Chromosorb W HP and Diatoport S have mostly been applied. One of the best GC separations of TMS derivatives of amino acids was obtained by Gehrke and Leimer [256] on a 6 m X 2 mm I.D. column packed with 10% OV-I1 on Supelcoport (100-120 mesh), and is illustrated in Fig. 5.19. With this column, all twenty protein amino acids can be analysed quantitatively. Pocklington [257] applied the method to the determination of nanomole amounts of amino acids in sea water. Dry marine salt was extracted with acidified ethanol. Interfering components having been removed, the extract was purified on an ion-exchange resin. TMS derivatives were prepared by the action of pure BSTFA (10-fold excess) at 78°C for 1 h. The analysis was performed on 3% of OV-1 or OV-17 with temperature programming. Suitable conditions were found for the detection of down to lo-' mol of all protein amino acids (except Arg and His) plus several non-protein amino acids. Applications of the method to o-amino acids were discussed and existing knowledge was summarized by Maiik et al. [258], Silylation was executed at 80°C for 1-2 h or at 90°C for 0.5-1 h, with the use of BSA either alone or with the addition of triethylamine or acetonitrile. BSA alone was recommended as tris-TMS derivatives were produced in practically 100%yield. The presence of amine and solvent led to the formation of bisand tris-derivatives in various ratios, changing with time. However, these conclusions were valid only if the reaction was started with amino acid hydrochlorides. The presence of

AMINO ACIDS

0

139

154 22

194 30

244 LO

265 La

285-c 57 MIN

Fig. 5.19. Gas chromatogram of TMS derivatives of 20 protein amino acids. Peaks: 1 = Ala; 2 = Glyz; 3 = Val; 4 = Leu; 5 = Ile; 6 = Gly3; 7 = Pro; 8 = Ser; 9 = Thr; 10 = HyPro; 11 = Asp; 12 = Met; 13 = Glu; 14 = Phe; 15 = Arg; 16 = Lys; 17 = Tyr; 18 = His; 19 = phenanthrene (internal standard); 20 = Trp; 21 = Cys. Conditions: glass column, 6 m x 2 mm I.D., 10% OV-11 on Supelcoport (100-120 mesh); nitrogen flow-rate, 20 ml/min; temperature programme, 2"C/min from 110°C for 22 min, then 5"C/min to 285°C. (Reproduced from J. Chrornatogr., 5 7 (1971) 219, by courtesy of C.W. Gehrke.)

hydrogen chloride was indispensable for this reaction as it catalysed the formation of persilylated products. GC analysis was carried out on 1% of SE-30 on Chromosorb W (AW, DMCS treated). TMS derivatives of amino acids were also combined with other procedures and some difficulties were thus avoided. N-TMS-methyl and -ethyl esters of most protein amino acids were prepared b y the action of TMSDEA on alkyl esters o f amino acids and were chromatographed on methylsilicone stationary phases [246]. Their retention times were found t o be 15-20% lower than those of the corresponding TMS derivatives. Despite having an additional step in comparison with direct silylation, the procedure was applied by Hardy and Kerrin [259] t o the GC analysis of twenty protein amino acids, including Hypro and CysH. Amino acids were esterified with a 3 N HC1 solution in n-butanol at 150°C for 15 min with subsequent silylation with BSTFA for 90 min at the same temperature. Acetonitrile and methylene chloride were used as solvents for the silylation. In the former solvent double derivatives of Gly and Lys (bis- and tris-) were produced, whereas in the latter the less silylated form only was produced. As Arg, in contrast t o direct silylation, also leads to one peak in this instance, methylene chloride is recommended as the silylation solvent. The separation of all twenty amino acids was achieved on a simple column with 2% of OV-7 on GLC-110 textured glass beads (100-120 mesh).

5.6.3.Condensation products This section describes some derivatives of amino acids prepared by the condensation of a reagent with the amino group only or with both functional groups with the formation of a cyclic derivative. A number of reactions of this type have been tested for the derivatization of amino acids [191], mostly for a limited number. Some of the main reactions only will be mentioned here.

DERIVATIZATION OF COMPOUNDS

140

Azomethines (Schiff bases) have been prepared in order to block the amino group, using different carbonyi compounds as reagents. Aqueous formaldehyde condenses with amino acids in the presence of palladised charcoal in a hydrogen atmosphere with the production of a dimethylamino group (the reduction time is 3-12 h at 2OoC). The free carboxyl group is esterified with diazomethane [260]. The general application of this procedure is limited, however, and the N,N-dimethylaminomethyl esters produced are very volatile so that mere evaporation of the ethereal solution of diazomethane at room temperature results in losses and low yields of the derivatives of Ala, Gly, Val and Leu. R - C H - C O O C Hg

+

(C H312C H - C H = O

R-C H-COOCH3

I

I

N=CH-CHlCH3)2

NH2

Scheme 5.23.

Isobutyraldehyde is another reagent that has been applied to condensation with amino acids [261]. The reaction of methyl ester hydrochlorides of amino acids with isobutyraldehyde and sodium hydrogen sulphite proceeds in a solution of sodium carbonate according to Scheme 5.23. The azomethines produced were chromatographed either as such or as N-alkyl esters after reduction with zinc powder in methanolic hydrogen chloride. N-Isobutylidene methyl esters of most of amino acids, except His, Arg, Trp, Tyr and Hypro, were separated on a capillary column coated with Carbowax 1540 at 102-165OC. On a preparative scale (gram amounts) thz yields ranged from 50 to 98%. The application of pivalaldehyde to the preparation of the derivatives is illustrated by Scheme 5.13 (p. 109). Benzaldehyde reputedly reacts with amino acids under moderate conditions [ 1621. Mere mixing of the methyl esters of amino acids with a pyridinebenzaldehyde-methanol mixture (1 : 1 : 10) is reported to be sufficient for the formation of the derivatives. N-Benzylidene methyl esters of amino acids were analysed on SP-400 at 100-280°C. Pro and Hypro did not produce condensation products, however. The condensation of amino acids esters with diketones leads to cylic derivatives. Limited success was achieved in this respect with acetyl acetone [262] ;amino acids were condensed prior to GC analysis with 2,5-hexanedione by Walle [263] (Scheme 5.24). The free carboxyl group of the reaction product was esterified with methanolic HC1. However, the procedure was applied to only a few amino acids.

I CH3

I

H3

Scheme 5.24.

Two functional groups in the molecule of amino acids offer the possibility of the formation of cyclic derivatives. On combining two molecules of the same amino acid, diketopiperazines are produced [264] (Scheme 5.25). With amido groups present in the

AMINO ACIDS COOH / R-CH \

NH2

+

141

-

H2N\ JH-R HOOC

CO - NH / \ R-CH CH-R \

+

/

2H20

NH- CO

Scheme 5.25.

molecule of the products, however, these compounds are not sufficiently volatile and prior to GC analysis they must be further modified (e.g., by silylation). Moreover, the possibility of producing mixed products in the presence of more amino acids must be taken into account. That is obviously why these derivatives have been used only sporadically. Similar difficulties appeared when attempts were made to apply morpholinones as volatile derivatives of amino acids. Coussement and Renard [265] prepared them by reaction of two molecules of propylene oxide with an amino acid (Scheme 5.26). Simple peaks were obtained from simple amino .acids only; multifunctional molecules led to multiple peaks. OH

I

c H~ - i H - C 30- 35' R-CH-COOH + 2 CH3-CH-CH2

I

NH2

\/

I

H ~

4 M NaOH

0

Scheme 5.26.

Substituted oxazolinones (see Scheme 4.23, p. 78) were synthesized directly by heating amino acids with TFA anhydride at 150°C for 10 min or by the action of dicyclohexylcarbodiimide on N-acylated amino acids [266]. Oxazolinones of eleven amino acids were chromatographed on 0.325% EGA on Chromosorb G at 40-140°C. 2-H-, 2-methyland 2-phenyloxazolinones of Leu were analysed on OV-17 together with 2-trifluoromethyloxazolinone, which has the shortest retention time and is very volatile. Substituted oxazolidin-5-ones are produced by the reaction of amino acids with substituted acetone. Simmons and Wiley [267] applied 1,3-dichlorotetrafluoroacetone,and it is this reagent that has been most often used for the preparation of oxazolidinones [268] (Scheme 5.27). The use of hexafluoroacetone as a reagent is limited as it is gaseous, expensive and the derivatives of the simplest amino acids are too volatile. The former reagent is therefore preferred for cyclizations.

Scheme 5.27.

Engelhardt [269] prepared and chromatographed oxazolidinones of simple amino acids. However, difficulties were caused by the solubility of the acids in the reaction medium as amino acids as such are not soluble in dichlorotetrafluoroacetone. In a solvent, e.g., acetonitrile, sufficient conversion was achieved only at increased temperature after several hours. In addition, hydrochlorides of amino acids do not react under the same conditions.

142

DERIVATIZATION OF COMPOUNDS

Hugek [268] searched for optimal conditions for the condensation reaction with respect to solvent, catalyst, reaction time and temperature. He selected Tyr and its monoand diiodinated analogues as model substrates. He succeeded in the GC analysis of the oxazolidinones of these compounds after subsequent acylation or silylation in the same reaction medium and evaluated the molar responses of both an FID and ECD. The application of this unique and selective chemical reaction was extended to the quantitative GC determination of other protein amino acids [270]. A reaction mixture consisting of 1-4% (v/v) of pyridine in acetonitrile and dichlorotetrafluoroacetone (the reagent to solvent ratio being 1 : 5 to 1 : 15) was adequate for the derivatization of all protein amino acids at 50°C within a few minutes. The reaction yields were high (>95%) and the same results were obtained for both free amino acids and their hydrochlorides. Half of the protein amino acids can be analysed after condensation for 10 min at 40°C. The other reactive groups in the side-chains can be acylated by adding TFA or HFB anhydride to the reaction mixture, the reaction being completed within 5 min. The other carboxyl group in the molecule of Asp and Glu is esterified with a small amount of methanol in the course of the second acylation step. Extraction of the derivatives in a light potroleum (boiling range 30-7O0C)-methylene chloride mixture enables undesired reagents to be removed and His to be converted into a suitable form for GC, e.g., by an instantaneous reaction of the imidazolyl group with isobutyl chloroformate. The subsequent short acylation with a drop of the corresponding anhydride at 70°C also permits Arg, Asp, and Glu to be analysed successfully. The whole procedure, including GC analysis of all protein amino acids, takes 1 h. OV-17 can be used as the stationary phase to separate the derivatives in a well deactivated capillary column. Tliiohydantoins (see Scheme 4.25, p. 78) play an important role as derivatives of amino acids, particularly in the sequential analysis of peptides. When the sequence is being determined from the carboxyl end, ammonium thiocyanate is dissolved in acetic acid and acetic anhydride and this mixture is allowed to react with the carboxyl end of the peptide with the formation of 1-acyl-2-thiohydantoin.2-Thiohydantoin is released from the peptide by the action of a base, and a new carboxyl end of the aniino acid is exposed. Prior to GC analysis, thiohydantoins must be further modified, e.g., by silylation [271], as follows. A 25-pl volume of ethyl acetate and 25 p1 of BSA are added to 500 nmol of 2-thiohydantoin and the mixture is shaken until it is homogeneous. The mixture is then heated in a stoppered test-tube at 80°C for 5 min, cooled and centrifuged before injection. This procedure is reputedly more suitable than silylation with BSTFA under the same conditions or with BSA-pyridine at room temperature. Other workers [272] accomplished the silylation of thiohydantoins with the aid of BSTFA-pyridine (1 : 1) at 50°C for 10 min. Gly and Thr, however, provide two products which are not stable. A good separation of silylated 2-thiohydantoins was obtained on a 4 ft. X 2 mm I.D. column packed with 10%of SP-400 on Chromosorb W HP with temperature programming (145260°C). The analysis was also performed on 1% of Dexsil300 GC and 5% of OV-I7 with identification by means of ,,lass spectrometry. A number of mass spectra of thiodantoins and their TMS derivatives have been published [271]. In the sequential degradation according to the Edman procedure methyl or phenylisothiocyanate is used as the reagent and the reaction starts from the amine end of the

AMINO ACIDS

143

10

08

VI

3

06

0

2 _1

=’ I:

04

02

00 ,

Fig. 5.20. GC separation of TMS-methylthiohydantoins of amino acids. Conditions: borosilicate-glass column, 165 cm x 4 mm I.D., 2% (w/w) OV-17 on GasChrom Q (80-100 mesh);nitrogen flow-rate, 50 ml/min; temperature programme as indicated. (Reproduced from Anal. Biochrm., 58 (1974) 549, by courtesy of Academic Press.)

peptides or proteins. Methylthiohydantoins possess properties more favourable for GC analysis, mainly owing to their higher volatility. Attrill et al. [273] demonstrated the GC separation of these derivatives of amino acids without any further modification in two columns coated with SE-30 and OV-17. Methylthiohydantoins of the remaining amino acids had to be silylated before the analysis (Asp, Ser, Arg, CysM, Cys0,H); the derivative of CysH was not analysed successfully. These derivatives are mostly chromatographed after being silylated with BSA or acylated with TFA anhydride in the injection port [274] or with BSA-acetonitrile (5 min at 20°C) [275]. A suitable column packing is 5% of QF-1, OV-1 or OV-17. The derivative of His is unstable and injection together with BSA is recommended. Lamkin et al. [276] studied in detail the GC analysis of silylated methylthiohydantoins of all protein amino acids. They effected the silylation with BSA-acetonitrile (1 : 3) at 100°C for 10 min. They separated the products in a simple column packed with 2% of OV-17 on Gas-Chrom Q at 145-230°C, and Fig. 5.20 illustrates the results. The authors used a flame photometric detector, sensitive to sulphur-containing compounds, in order to ensure sensitive and selective detection. Minor incidental peaks that were often noticed during the analysis of the samples obtained by the Edman degradation of proteins with the use of an FID did not appear and the peak of the solvent was not detected. The baseline stability was good and the response was linear over a range of two orders of magnitude of concentration. Asn and Phe were the only unresolved pair; Arg, as in previous instances, did not form a volatile derivative. Free phenylthiohydantoins are chromatographed only with difficulty and none of the various methylation procedures for simple amino acids are satisfactory. Strong silylating agents (BSA and BSTFA) were applied successfully, e.g., by Harman et al. [277]. All amino acids, including Ser and Thr, gave products that could be chromatographed, with

144

DERIVATIZATION OF COMPOUNDS

the hydantoin ring in the molecule being silylated in addition to the remaining protonic groups. Similarly, the phenylthiohydantoin ring was acylated with acetic or TFA anhydride [278] (Scheme 5.28).

Scheme 5.28.

The properties and GC behaviour of TMS phenylthiohydantoins were also studied by Guerin and Shults [279]. They investigated 26 derivatized amino acids by using a detector sensitive to sulphur capable of detecting nanogram amounts of thiohydantoins. Pisano et al. [280] studied the analysis and conditions for the preparation of these derivatives in detail. The technique of silylation was tested so that it might be extended to all amino acids; BSA and BSTFA were used with solvents of different polarity. Satisfactory conditions were 10-15 min reaction at 50°C of a mixture containing acetonitrile or pyridine (1 : 1). Depending on the volatility and the number of the functional groups that are to be silylated, the compounds were classified into three groups: (i) the derivatives of Ala, Val, Ro, Gly, Leu, Ile, Met and Phe are the most volatile and generally provide symmetric peaks; (ii) Asp, Glu, Tyr, His and Trp are the least volatile; phenylthiohydantoins of His, Asp and Glu show the strongest tendency towards sorption on the column, and towards the origin of asymmetric peaks and provide low responses; (iii) Asp, Glu and Cys03H, for which the silylation was necessary, whereas for Ser, Thr, Lys and CysM it was useful. For the GC separation of TMS-phenylthiohydantoins different, mainly silicone, stationary phases have been utilized. A mixture of 7.33% of SP400,5.33% of OV-210 and 0.66% of OV-225 at 190-270°C was recommended as the most suitable. SP-400 was also fairly suitable for the separation of phenylthiohydantoins of simple amino acids, including Tyr and Trp in a GC 55/65 system of a Beckman 890c sequence analyser. Although the phenylthiohydantoin of Arg does not provide any peak after being silylated, it can be analysed after subsequent acylation. Inglis and Nicholls [281] studied the conditions required for successful acylation. The phenylthiohydantoin of Val, as the model compound, was acetylated with acetic anhydride-pyridine (4 : 1): complete conversion was achieved after 6-14 h at 20°C, 1 h at 50°C or 5 min at 100°C. For His and Arg, which otherwise require 3 h-6 days for satisfactory derivatization, only an oncolumn technique was suitable. In this technique the acetylating agent was injected together with the sample and good yields of the acetylated product were obtained; the hydantoin ring could not be acetylated, and with Val 30% of the derivative did not undergo acylation. Compared with free phenylthiohydantoins, the retention times of acetylated derivatives are by about 6% shorter. Lequin and Niall [282] described GC analysis of more volatile analogues, pentafluorophenylthiohydantoins, which were prepared by modifying the Edman degradation using pentafluorophenylisothiocyanate as a reagent. Except for Arg and His, the derivatives of all amino acids could be chromatographed and separated satisfactorily in a simple column (1.22 m X 2 mm I.D.) packed with either 10% of DC-560 or 2% of OV-25 on Chromo-

AMINO ACIDS

145

sorb W (100-1 20 mesh) with temperature programming (160-240°C). Silylation with BSA was applied with the aim of determining Asp and Glu (and also Asn and Gln) and also to resolve the pair Leu-Ile. Although these derivatives possess some suitable properties from the viewpoint of GC analysis, the perhalogenated reagent did not find more general application. 5.6.4. Other derivatives

Dinitrophenyl (DNP) derivatives of amino acids are used to identify terminal amino acids in proteins. They are determined by GC after the conversion of free carboxyl groups into methyl esters [283-2851 with the aid of either diazomethane or BF3-methanol. As the molecular weight of the resulting derivatives is relatively high, temperatures above 200°C are usually necessary for the analysis, and therefore mostly silicones (SE-30, XE-60, XE-61, QF-1)and polyesters are used as stationary phases. Satisfactory results were obtained with simple and acidic amino acids, whereas His, Ser, Thr, Tyr and basic amino acids were not analysed successfully, obviously as a consequence of their low volatility and decomposition in the column. However, Landowne and Lipsky [284] described a successful analysis of bis-DNP-Lys, -0rn and -Cys on a column coated with 3% NPGA or NPGS or XE-60. DNP derivatives of these amino acids were chromatographed together with fifteen other simple amino acids (including Asp and Glu) at 220-240°C with the use of an ECD. For all common amino acids identical sensitivities of 3 . mol/sec were obtained, which shows that the ECD response is a function of the DNP group only and does not depend on the remaining part of the molecule, so that calibration for individual amino acids is not required. Bis- and mono-DhT derivatives of Lys, Orn and Cys give the same response, however. Ikekawa et al. [285] successfully separated thirteen amino acids on a 4-m column coated with 1.5% of SE-30 with temperature programming (170-230°C). Only Thr and Ser were not separated on this column; they can, however, be separated on 1.5% of XE-61. Prior to the analysis, DNP derivatives of these amino acids were silylated by using HMDS-TMCS in pyridine (2 : 1 : 5) at room temperature for 20 min. The method was applied to the analysis of amino acids in serum, as follows. To 0.5 ml of serum, 1.5 ml of ethanol was added in order to deproteinate it. The mixture was centrifuged, the liquid layer drained off and the residue washed with 0.5 ml of water and 1.5 ml of ethanol. Combined extracts were adjusted t o pH 3 and washed twice with 5 ml of chloroform. To the aqueous layer, 2 ml of phosphate buffer (pH 8.8), 1 ml of 5%dinitrofluorobenzene in ethanol and 3 ml of ethanol were added and the mixture was shaken at 40°C for 2 h, then extracted three times with 30 ml of diethyl ether. The ethereal layer was washed with the buffer solution and the washings were added to the aqueous layer, which was then acidified with 15 ml of 10%HCl and extracted with three 20-ml portions of ethyl acetate. After washing with water, the organic layer was dried over anhydrous sodium sulphate and evaporated to dryness. The DNP derivatives thus obtained were dissolved in a small amount of methanol and esterified with an ethereal solution of diazomethane. N-Thiocarbonyl alkyl esters of amino acids were chromatographed by Halpern et al. [286]. Their preparation proceeds according to Scheme 5.29. The reaction sequence includes esterification of amino acids followed by reaction with carbon disulphide and triethylamine, giving rise to dithiocarbamate. Reaction of the latter with chloroformate

DERIVATIZATION OF COMPOUNDS

146

R-CH-COOR,

I

CS2

NIC2H513

H2

R-CH-COOR1

I

CICOOR2

NH-CSS F S H ( C ~ H ~ ) ~

R-CH-COOR1

I

acid

NH-CSS-COOR2

R-CH-COOR,

I

N=C=S

Scheme 5.29.

ester gives carbalkyloxydithiocarbamate, from the decomposition of which the N-thocarbonyl derivative is produced. Dry protein hydrolysate (0.2-0.5 mg) is esterified twice with 1 ml of HCl-n-propanol(25-30%, w/w) at 80°C. After removing the solvent under vacuum, 100 pl of methylene chloride, 10 p1 of carbon disulphide and 8 pl of triethylamine are added at -5°C. After 1 h at 20"C, the reaction mixture is cooled to -5°C and 2 p1 of methyl chloroformate are added. After a further 1 h at 2OoC, the mixture is diluted with 5 ml of rnethylene chloride and washed with 1 ml of citric acid (20%, w/w). After additional washing with 1 ml of water, the solution is dried and concentrated to 0.2 ml and 3-pl samples are injected. In the course of the reactions, the other functional groups are also derivatized. Hydroxyamino acids and Tyr react with alkyl chloroformate with the formation of carbonate esters, Cys and CysH provide thiocarbonates, and the imidazole nitrogen of His is also protected. The indole functional group of Try does not change, Pro forms a stable carbalkoxydithiocarbarnate and Arg does not provide any volatile product. The derivatives were separated successfully (except the pair Leu-Ile) on 5% of QF-1 with temperature programming (94-235°C). The reproducibility of the method was stated to be 5%. Using an FID and ECD, lo-'' and mol of amino acids, respectively, can be determined. Isopropyl derivatives were introduced by Pettitt and Stouffer [287] and later studied by other workers [288]. They are prepared by reaction with 2-bromopropane in the presence of sodium hydride in dimethyl sulphoxide. The reaction scheme and the preparation procedure were given in Chapter 4 (see p. 64). Except for Arg, all amino acids under study provided the expected derivatives. The hydroxyl group of Hypro was, however, not protected. The derivatives were found to be stable for a reasonable period of time and were analysed on 3% of OV-17. The extension of this promising one-step method to all protein amino acids did not fulfill expectations, however [288]. Some amino acids (Gly, Gln, Asp and Asn) did not provide detectable derivatives and the others led to multiple peaks. Moreover, significant amounts of by-products were produced, which may interfere. Arg provided a single peak, the mass spectrum of which was identical with that of Orn; both derivatives resulted from lactam formation. Isoproy; derivatives of 23 common amino acids were separated on 5% of.Carbowax 20M on silanized Chromosorb G with temperature programming (50-240°C).

5.6.5.Separation of enantiomers Essentially two basic approaches to the separation of enantiomers of amino acids have been applied: (i) the derivatives described in the preceding sections are chromatographed on optically active stationary phases, e.g., N-acyl alkyl esters or alkylamides of amino acids, ureides or N-acyl alkyl esters of dipeptides; (ii) GC separation is performed on conventional stationary phases and the derivatives of amino acids are prepared by reaction

AMINO ACIDS

147

with optically active reagents. It is obvious that the bifunctionality of amino acids offers the use of either optically active esterification or an acylating agent. Both derivatization procedures have been applied to the separation of amino acid enantiomers. Secondary alcohols, particularly 2-butanol and 2-octanol, have mainly been used for this purpose [289,290]. Although the derivatives of higher secondary alcohols were more suitable for the separation, sec.-butanol represents the best compromise between solubility in lower alcohols and higher volatility. The preparation of the derivatives .involves the esterification with a strongly acidic (4-8 M HCl) optically active alcohol at 100°C for at least 1 h, followed by solvent removal and acylation with TFA anhydride. Diastereoisomers of N-TFA-2-butyl and -2-octyl esters of simple amino acids, including Hypro, Asp, Glu and Lys, were separated on a capillary column (50 m X 0.25 mm I.D.) coated with polypropylene glycol or FS-I 265 fluorosilicone. The latter stationary phase was more suited as it provided longer retention times and higher separation factors (ratio of the retention time of LD- and LL-diastereoisomers). Pollock and co-workers [291,292] applied a 50-m capillary column coated with Ucon LB-550-X and/or Carbowax and resolved optical antipodes of 21 amino acids as their N-TFA-2-butyl esters. Under strictly anhydrous conditions the derivatives were stable; the 0-TFA bond was cleaved owing to the action of water or methanol. N-TFA-3,3-dimethyl-2-butyl esters and N-pentafluorobenzoyl-2-butyl esters were used for Asp. It can be generally said that (i) the greater is the difference in the size of the groups bound to an asymmetric centre of an alcohol, the greater is the separation factor, and (ii) the closer to the asymmetric centre is the branching of the chain, the greater is the contribution to the separation of diastereoisomers. These conclusions were proved by Ayers et al. [293], who achieved the best possible separation using 3,3-dimethyl-2-butanol for esterification. On a 3-m column packed with 10%of OV-17 a resolution of diastereoisomers better than 93% was achieved with fourteen amino acids; 70% and 82% resolution were achieved for Asp and Pro, respectively. However, a packing that would resolve all diastereoisomers completely has not been found. N-TFA-L-menthyl esters were described for the resolution of optical antipodes of the most volatile amino acids (Ala, Val, Leu, Nleu) on a polyester or PEG stationary phase [294]. Modified chlorides of proline are mainly applied as optically active acylation reagents, with dipeptides as the resulting derivatives. The reagent is prepared by the action of TFA anhydride on L-Pro with subsequent chlorination with thionyl chloride (Scheme 5.30).

A

COCF3

COCF3

Scheme 5.30.

N-TFA-L-prolyl methyl esters have been most frequently used for the separation of diastereoisomers of amino acids [295,296], although other procedures have been suggested for blocking the imino group of Pro using functional groups such as a-chloropropionyl and a-bromopropionyl. The preparation of the derivatives consists in conversion of amino acids into methyl ester hydrochlorides by the action of methanol and thionyl chloride and subsequent reaction with TFA-L-prolyl chloride in dichloromethane

148

DERIVATIZATION OF COMPOUNDS

in the presence of triethylamine. Bonner [296] drew attention, however, to possible racemization during this procedure and recommends that the second step be performed at a lower temperature (solid carbon dioxide) and triethylamine added to the reaction mixture in a theoretical amount and in a very dilute solution. Packed columns of the usual length (1.5-2 m) are sufficient for the GC separation of the resulting diastereoisomeric dipeptides. Silicones (SE-30,OV-1) or polyesters (DEGS, EGA) have mostly been used as stationary phases. Bonner [296] applied a 15-m SCOT column coated with NPGA. The chiral reagent used for the preparation of the derivatives should not undergo racemization easily; e.g., proline reagent, owing to its cyclic structure, has such a tendency. Other reagents proposed for this purpose by Nambara et al. [297] possess very rigid molecular skeletons. They are substances related to camphor d-isoketopinyl chloride (I), I-dihydroteresantalinyl chloride (11) and I-teresantalinyl chloride (111) (Scheme 5.3 1).

Scheme 5.31.

Acid chloride (about 4 mg) is added to a solution of the amino acid ester (about 1 mg) dissolved in tetrahydrofuran (0.8 ml) and containing pyridine (0.2 ml). The reaction products are injected directly together with an excess of the reagent; 1.5% of SE-30, 1.5% of OV-1, 1.5% of OV-I 7 and 0.5% of PEGA were tested as stationary phases for methyl and butyl esters of these derivatives of amino acids. On the last-mentioned stationary phase, the best resolution of diastereoisomeric pairs of l-teresantalinyl derivatives of twelve amino acids was obtained. However, the restricted availability of the reagent will prevent the wider application of this procedure.

5.7. THYROID HORMONES

This section should have a more precise title, “iodoamino acids”. Six amino acids are concerned, for which derivatization procedures prior to their GC analysis have been studied: mono- and diiodotyrosine (MIT and DIT), diiodo-, 3,5,3’- and 3,3’,5‘-triiodothyronine and thyroxine (Tz, T3, T$ and T4, respectively) have been considered. Of these, T3 and T4 are clinically important and their accurate determination in biological materials, particularly blood and pharmaceuticals, is of great importance. The concentration of T3 in blood is very low (a few nanograms per millilitre); the concentration of T4 is 50-fold higher and its GC analysis necessitates the prior isolation of the substances under analysis from the sample material. Moreover, the derivatization step which is necessary can cause, however negligible it may be, deiodination of T4 to T3 and, consequently, a large error in the determination of T3. These effects have meant that GC has not been generally adopted for the determination of thyroid hormones and that radio-

THYROID HORMONES

149

immunoassay procedures have often been preferred. However, it cannot be disregarded that for the GC analysis of these substances only a limited number of derivatives have been tested, if compared with amino acids, so that further efforts in this respect need not be in vain. 5.7.1. Acyl methyl esters

Esterification of the carboxyl group is usually performed by heating with a 25% solution of HCl in methanol at 70°C for 30 min and the residue is acylated with TFA anhydride [298,299]. Although the acylation proceeds under very mild conditions (20°C) and satisfactory results have been obtained for micromole amounts with the use of the FID, decomposition of the derivatives during the process has been observed when working a t the picomole level with the ECD. The derivatives prepared by reaction with pivalic anhydride (trimethylacetyl) were much more suitable and have been studied more thoroughly. Stouffer et al. [300] analysed these derivatives first by using the FID with temperature programming. Later, they applied the ECD and isothermal conditions and modified the procedure for the analysis of a sample of serum by using 3-5 ml of the serum [301] and finally only 1 ml [ 3 0 2 ] . Solvent extraction and chromatography on a layer of Sephadex LH-20 was suggested for the isolation of the substances from serum. Acylation is carried out with a mixture of 0.2 ml of pivalic anhydride, 10 p1 of methanoi and 10 pl of triethylamine and is completed within 30 min at 110°C. For the analysis a 90 cm X 2 mm I.D. column packed with 2% OV-17 on Gas-Chrom Q was used at 272 or 285°C. T3 was detected at concentrations down to 20 pg with a pulsed ECD. The derivatives were stable against the action of moisture, heat and light and could be stored without any significant changes for several months in dilute methanolic solution. Hollander and co-workers [303-3051 dealt with the problem in detail and developed a method for the isolation of hormones from blood, using Bio-Rad AG 50W-X2 (100-1 20 mesh) ion-exchange resin. Acylation with pivalic anhydride-methanol-triethylamine (20 : 1 : 1) was performed at 70°C for 10 min. The derivatives were purified with the aid of Amberlite IR-45 resin and benzene as a solvent. The dry residue was dissolved in 100 pl of benzene and 5 pl were injected directly on to a 60 cm X 4 mm I.D. column packed with 5% OV-1 on Chromosorb W HP;after an isothermal period at 220°C for 12 min, the temperature was increased at 3"C/min up to 300°C. Calibration standards were injected immediately after the sample. Almost identical results were obtained for T3 by GC and radioimmunoassay [304]. Other workers [306] applied the same procedure to the seeds and analysed pivalyl methyl esters of T3 and T4 on an 81 cm column packed with 3% of Dexsil on Chromosorb W HP at 305°C. HFB anhydride has been used for the acylation of methyl esters of thyroid hormones to a lesser extent, in spite of the fact that the resulting derivatives are sufficiently stable and the most volatile and provide the highest ECD response, to which the acyl moiety also contributes. Acylation is said t o be completed within 5 min at 50°C [307] ;however, other workers [308] performed acylations at 60°C for 1 h with a mixture of HFB anhydride (400 pl) and acetonitrile (500 pl) per 1 mg of the substrate. The derivatives of all of the thyroid hormones were separated on a capillary column (20 m X 0.15 mm I.D.) coated

150

DERIVATIZATION OF COMPOUNDS

with 1% OV-101 at 290 or 275°C. The method was applied to physiological samples, and mass spectra of all of the derivatives were obtained [309]. 5.7.2. Silyl derivatives Trimethylsilyl derivatives are prepared by treatment with BSA alone [310,311] or with the addition of TMCS [312,314] in a suitable solvent (acetonitrile, pyridine, tetrahydrofuran) or even without a solvent. For completion of the reaction, 10-20 min at 50°C are necessary [312], but as little as 30 min at 150°C has been reported for a stoppered vial with the use of a solvent [31 I ] . BSA alone can be used to advantage if picomole amounts are to be derivatized. The reaction products are said to decompose in dilute solutions even though pure BSA is used for dilution. At concentrations around 1 ng/pl, up to 40%decomposition of the products is observed; if diluted with BSAacetonitrile (1 : 4), 100%decbmposition occurs in 20 min. Of other silylating agents, e.g., HMDS and TMCS have been tested, but conversion into derivatives was not complete [311]. Silicone stationary phases of the SE-30,OV-1 and similar types have been used in the analysis. In most instances, temperature programming is required. Using the FID (in almost all instances), the detection limit is about 20 ng for T4 and 5-20 ng for TB, whereas with the aid of an ECD amounts about two orders of magnitude smaller can be detected [310,314]. Fig. 5.21 demonstrates a typical separation of five iodoamino acids and Tyr on 0.5% of SE-30. The silylation procedure has been accepted as a routine method for the trace analysis of preparations of thyroid hormones and drugs containing them. Quantitative evaluation was achieved by using Tz as an internal standard [3 141. The method has not been applied to the analysis of hormones in serum. Silylation does not seem suitable for this purpose as the derivatives partially decompose if sub-nanogram amounts are injected.

5

10

15

20

25

30

35

TIME 1 MIN 1

Fig. 5.21. Gas chromatogram of TMS derivatives of five iodinated amino acids and tyrosine. Peaks: 1 = Tyr; 2 = MIT; 3 = DIT; 4 = Tz ; 5 = T3 ;6 = T4. Conditions: borosilicate-glass column, 1 m x 3.5 mm I.D., 0.5% SE-30 on Chromosorb G (60-80 mesh, AW, DMCS-treated);nitrogen flow-rate, 40 ml/min; temperature programme, 4.6"C/min from 70°C. (Reproduced from Anal. Biochern., 24 (1968) 281, by courtesy of the authors and Academic Press.)

STEROIDS

15 1

5.8. STEROIDS

The high molecular weight of steroid compounds and the presence of polar groups in their molecules, which, moreover, are often not stable under chromatographic conditions, are the properties that make the direct GC analysis of free steroids difficult. The reasons for using derivatives are almost the same as those mentioned elsewhere: the symmetry of GC peaks is improved substantially by conversion into suitable derivatives which, amongst other factors, facilitates quantitative evaluation, and the volatility and stability of the compounds under analysis are also increased. The improvements in these properties are often so significant that derivatization prior to GC analysis is essential for various steroid compounds. It also makes it possible to distinguish steric differences among various steroids. The separation of epimers of testosterone and epitestosterone, which can be separated only after their conversion into TMS or acetyl derivatives, is a well known example. The retention characteristics of different derivatives may serve for the identification of steroids that differ only slightly in their structures. Fine structural differences, e.g., the extent of the hindrance of the functional group, may be estimated on the basis of the different rates of formation of the derivatives and investigated with the aid of gas chromatography. The preparation of selective derivatives, permitting the substances under analysis to be separated from interfering components, is of great significance for the analysis of steroid compounds in complex biological materials. The necessity for determining trace concentrations often involves the preparation of derivatives producing high responses with certain detectors; of importance also are the derivatives developed for combined GC-MS. In the steroid molecule four main polar groups occur: hydroxyl, phenolic hydroxyl, carbonyl and carboxyl. Because sometimes all of them are present in one molecule, procedures are necessary for their chemical blocking that will convert all of them into suitable derivatives. This is particularly important if steroids are profiled in various biological samples. Silylation procedures, enabling the derivative to be obtained in one reaction step, have been exploited widely. In addition to TMS, silyl derivatives with other substituents, e.g., halomethyl and pentafluorophenyl, have been prepared and chromatographed in order that the sensitivity of detection might be improved and the derivatives utilized in combination with mass spectrometry. Other derivatives of the above-mentioned functional groups, such as acyl, ether and cyclic derivatives, have also been exploited but mostly as specific derivatives for smaller groups of compounds. The necessity for blocking labile carbonyl groups led to the use of their derivatives also for the GC analysis of steroids: alkyl or aryloximes and hydrazones are mainly applied in combination with TMS, acyl and other derivatives. Several books, e.g., that by Eik-Nes and Homing [3 151, dealt in detail, and with particular respect to biological materials, with the GC analysis of free steroids and the preparation and analysis of their derivatives. 5.8.1. Silyl derivatives

Trimethylsilyl derivatives for the analysis of steroids were first described by Luukkainen et al. [316]. HMDS in tetrahydrofuran with the addition of TMCS as a catalyst

152

DERIVATIZATIONOF COMPOUNDS

TIME I MIN

Fig. 5.22. Gas chromatogram of TMS derivatives of androsterone (A), etiocholanolone (B) and dehydroepiandrosterone (C) standards (left), and of the same compounds obtained from a urine extract (right). Conditions: glass column, 6 ft. x 6 mm O.D., 2%GE XEdO on GasChrom P (100-140 mesh, silanized); argon inlet pressure, 20 p.s.i.; 195°C. (Reproduced from Clin. Chem., 12 (1966) 399, by courtesy of C.R. Berrett.)

was selected for the silylation of a number of steroids. The reaction mixture was allowed to stand at room temperature overnight. GC analysis was performed on silicone stationary phases (SE-30, QF-1) and very closely related compounds, such as cholestanol, epicholestanol, estrone and estradiol, could be resolved even on these non-selective stationary phases. The same reaction conditions were applied by Berrett and McNeil [3 171 to 17ketosteroids. A method was devised for determining androsterone, etiocholanolone and dehydroepiandrosterone in urine (see Fig. 5.22). After extraction of conjugates, hydrolysis and extraction of free steroids, the latter were purified on a thin layer and after silylation chromatographed on a column packed with 2% of GE XE-60 at 195°C. Chambaz and Homing [318] studied in detail the silylation conditions for steroids by applying different reagents. They tested TMSDMA, HMDS, BSA and TMSIM separately and in mixtures and with TMCS as a catalyst. They concluded that for the silylation of the hydroxyl groups of steroids any silylating agent could be utilized; however, depending on its reactivity and on the reactivity of the group (extent of its hindrance) considerable differences in the conversion rate would occur, i.e., different yields would be obtained in the same reaction time. Fig. 5.23 illustrates the course of the reaction of the 3a-hydroxyl group of androsterone with TMSDMA and the influence of the addition of the catalyst. The same effect was found if the reaction temperature was increased. Under the initial conditions (TMSDMA, room temperature) a 100% yield was achieved after an impractically long time even if a non-hindered hydroxyl group was involved. The following silylation conditions were therefore recommended by the authors, depending on the degree of hindrance of the hydroxyl group: (i) BSA at room temperature for non-hindered hydroxyl groups; (ii) BSA-TMCS or BSTFA-TMCS (4-50% of the latter reagent) at room temperature or at 60°C for groups with medium hindrance; and (iii) TMSIM-BSATMCS (3 : 3 : 2) at 60-80°C for the derivatization of strongly hindered groups. The reaction times necessary for quantitative conversion were then half an hour to several hours.

STEROIDS

153

I

loo 100

1 -

80-

W

p"

TMSDMA

60 -

>

z

0 LJ

S

40

-

q-/4-I

0 '

1

2 TIME

,

/

4

/r r4 4 20

(HOURS)

Fig. 5.23. Time course of conversion of androsterone (SLY-androstan-3a-01-1 7-one) into the TMS ether with TMSDMA alone and with TMCS added at point A. Reaction conditions: 2 mg of androsterone, 1.0 ml of TMSDMA, 0.2 ml of TMCS added at point A to half (0.5 ml) of the solution; room temperature. (Reproduced from Anal. Biochem., 30 (1969) 7 , by courtesy of E.M. Chambaz and Academic Press.)

Slightly hindered hydroxyl groups of bile acids can be silylated according to the procedure of Makita and Wells [319]: methyl esters of bile acids, prepared by treatment with diazomethane, are dissolved in anhydrous pyridine (ca. 1 ml per 10 mg) and 0.1 ml of HMDS and 0.03 ml of TMCS are added to this solution. After standing for 1 0 min at room temperature an aliquot of the reaction mixture is injected directly. The method was applied to the determination of faecal bile acids in the rat. Other workers [320] investigated the retention behaviour of 52 persilylated methyl esters of urine acids on QF-1,OV-1 and OV-17 stationary phases. They correlated the data expressed as relative retention times with the structures of the compounds. Brooks et al. [321] characterized the urine metabolites of anabolic steroids on the basis of the different retention properties and mass spectra of free steroids and their TMS derivatives prepared by the same procedure. In addition to MS data they reported the retention indices of 30 170-hydroxysteroids of estrane and androstane groups obtained on OV-21O,OV-17 and OV-1 stationary phases. Steroids of the cardenolide group can be silylated by using the same procedure [322,323 J . Ethers of relatively non-hindered hydroxyl groups (e.g., 30, 120, 160) are produced by treatment with HMDS-TMCS. An unsaturated lactone ring is cleaved with the formation of TMS enol ethers by silylation with BSA-TMCS. All of the hydroxyl groups, including the terf.-140 group, can be silylated with TMSIM-BSA-TMCS at 60°C. The analysis is performed in a column packed with 1% SE-30 or 1%OV-17 on Gas-Chrom P with temperature programming (200-260°C and 23O-29O0C, respectively). Of other applications t o biological samples we can cite the determination of urine metabolites of androstd-en-17-ones [324]. Some saturated substrates did not separate from their unsaturated analogues on OV-17 and therefore preliminary epoxidation with m-chloroperbenzoic acid was used in order to resolve them. Simpson [325] analysed corticosteroids in rat muscles and also reported mass spectra of their derivatives. Berthou

154

I

DERNATIZATION OF COMPOUNDS

II

104

c

96 88

80 72

6L

56 48

40

32

24

16

8

0

TIME(MIN)

Fig. 5.24. Gas chromatogram of an authentic steroid mixture as TMS ethers. Peaks: 1 = epietiocholanolone; 2 = androsterone; 3 = etiocholanolone; 4 = dehydroepiandrosterone;5 = 1l-oxoandrosterone; 6 = 1 1-oxoetiocholanolone; 7 = 5a-androstan-3&17P-diol; 8 = pregnanolone;9 = 1 10-hydroxyandrosterone;10 = 11-hydroxyetiocholanolone;11 = do-pregnanediol; 12 = pregnanediol; 13 = A5-pregnenediol;14 = pregnanetriol; 15 = epicoprostanol; 16 = cholesterol. Conditions: glass capillary column, 50 m X 0.25 mm I.D., dynamically coated with OV-101; argon flow-rate, 2 ml/min; temperature programme, 2 min at 150°C, then 32"C/min to 190°C and then l"C/min to 280°C. (Reproduced from J. Chromatogr., 96 (1974) 33, by courtesy of E. Bailey.)

et al. [326] published an extensive comparative study of the derivatives of urine androstanediols and pregnanediols. They presented retention data of free isomers, acetates, TMS,TFA, HFB and chloromethyldimethylsilyl derivatives on seven stationary phases (SE-30. OV-l,OV-l7, QF-1, XE-60, NGS and Hi-Eff 8 BP). The overall procedure, specific for above-mentioned steroids, includes enzymatic hydrolysis of the sample, extraction into diethyl ether, removal of ketosteroids with the aid of Girard T reagent, preliminary purification by adsorption and thin-layer chromatography and derivatization. Using QF-1 as the stationary phase, resolution of saturated and unsaturated steroids can be achieved and thus the prior epoxidation step is obviated. Similarly, Gupta et al. [327] compared the retentions of TMS, TFA and HFB derivatives on XE-60,0V-lOl,OV-l and OV-17 stationary phases. TMS derivatives also appeared very suitable for the combined GC-MS analysis of plant and other biologically interesting sterols [328,329]. Novotnjr and Zlatkis [330] demonstrated the separation of steroid TMS derivatives on a capillary column. Bailey et al. [331] analysed neutral urine steroids on a 50 m X0.25 mm I.D. capillary column coated with OV-101. The resulting chromatogram of a standard mixture is shown in Fig. 5.24. The derivatives were prepared by heating for 1 h with BSA (0.1 m1)-TMCS (0.02 ml) in chloroform (0.1 ml) at 60°C. The injection was carried out by a special injection system for solid samples, similar t o the "falling needle" system (see p. 22). Halomethyldimethylsilyl ethers of steroids possess longer retention times than TMS derivatives, which makes it possible to separate steroids with different numbers of hydroxyl groups. Thomas and co-workers [332,333] exploited chloromethyldimethylsilyl derivatives of steroids, the retention times of which are up to three times longer

STEROIDS

155

TABLE 5.11 RETENTION TIMES AND ECD RESPONSES OF TESTOSTERONE DERIVATIVES [ 3381 Conditions: 5-ft. glass column packed with 3% DC-410 on Supasorb (100-120 mesh, AW); column temperature, 220°C; carrier gas, Nz at 75 mllmin; 63Ni pulse ECD. Derivative

Relative retention time

Chloromethyldimethysilyl ether Chloroacetate Bromomethyldimethylsilylether Iodomethyldimethylsilyl ether

1.0 (19min) ca. 1.7 1.45 2.18

Relative molar response 1.o ca. 5 .O 18 49

than those of the TMS derivatives. They applied the following procedure for their preparation. Solutions of diethylamine (0.1 ml) in n-hexane (1 ml) and chloromethyldimethylchlorosilane (0.2 ml) in n-hexane (1 ml) were mixed and centrifuged in order t o remove the heavy white precipitate. The supernatant (0.2 ml) was added to an extract of steroids (ca. 20 pg) and the mixture was allowed to stand at room temperature for 1 h. Volatile reagents were removed in a vacuum desiccator over calcium chloride and the residue was dissolved for analysis in 0.2 ml of n-hexane. The analysis could be performed, e.g., on XE-60 or SE-30. The derivatives were said to be more stable than TMS ethers so that losses in the column were avoided. The ECD response did not differ much from that of the FID and hence the significance of these derivatives is mainly that they can be better separated from interfering components and used for identification. The derivatives have been used for the analysis of methyl esters of bile acids [334] and androstane in urine [335]. They also proved useful in combination with mass spectrometry [336,337] as they provided characteristic mass spectra. As follows from Table 5.1 1 for testosterone derivatives, bromo- and iodomethyldimethylsilyl ethers have even longer retention times, but they have also higher ECD responses, which makes it possible to use them in trace analysis [338,339]. Brominated derivatives are prepared in the same way as the preceding derivatives, using bromomethyldimethylchlorosilane as the silylating agent; iodinated analogues are prepared from brominated derivatives by refluxing for 15 min with sodium iodide in acetone or by incubation for 30 min at 37°C. Iodinated TMS derivatives permit up to 0.1 ng to be determined quantitatively, and for the GC determination of testosterone in plasma by this procedure a sensitivity of 6 ng per 100 ml was reported [340]. Fluorocarbonsilyl ethers were studied by Morgan and Poole [341] with steroids of the ecdysone group. Perfluorinated alkyldimethylsilyl ethers are thermally unstable and decompose at the temperatures required for the GC of sterols (Scheme 5.32). Derivatives F

F

I 1 I

R-C-C-Si-

1 1 ’

F

F

Scheme 5.32.

-

R-CF:CF2

I

+ F-SI-

I

DERWATIZATION OF COMPOUNDS

156 TABLE 5.12

RELATIVE RETENTION TIMES AND ECD SENSITIVITIES OF CHOLESTEROL SILYL ETHERS W11 Conditions: (A) 3-ft. column, 1%OV-101 on GasChrom Q; carrier gas, Nz at 75 ml/min; column temperature, 250°C; (B) 3-ft. column, 1%Dexsil300 GC on GasChrom Q; carrier gas, Nz at 75 ml/min; column temperature, 290°C; (C) 1.5-ft. column, 1.5%OV-101 plus 1.0% tetramethylcyclobutanediol adipate; carrier gas, N2 at 90 ml/min; column temperature, 250°C Derivative of cholesterol

A Free (CH3)3SiCF3(CH2)zSitCH3h CF3 (CF2)z (CHz )zSi(CH& C1CH2Si(CH3)2C6FSSi(CH3)2-

Detection limit 0%)

Relative retention time

1.00 (1.58 min) 1.17 1.47 1.60 2.54 3.68

B 1.oo

(1.03 min) 0.92 1.24 0.87 1.87 2.84

C 1.oo (2.45 min) 0.69 1.06 0.68 1.69 2.78

1500 115 75 4

Detection h i t is defined as nanograms of cholesterol giving a signal twice as great as the noise.

with an alkyl group which is not fluorinated in the a-and 0-positions were therefore proposed, such as 3,3,3-trifluoropropyldimethy~silyI, 3,3,4,4,5,5,5-heptafluoropentyldimethylsilyl and pentafluorophenyldimethylsilyl derivatives. Table 5.1 2 presents a comparison of the retention characteristics and detection limits for the electron-capture detection of these derivatives with those of chloromethyldimethylsilyl and TMS derivatives of cholesterol. Fluoroalkylsilyl ethers are stable and volatile derivatives but their ECD responses are poor. Pentafluorophenyldimethylsilylethers have advantages over chloromethyldimethylsilyl ethers of having the same stability but a higher ECD response and also a higher resistance to nucleophillic attack on silicon, which is possible in the chloromethyl group. The derivatives are prepared by treatment with a reagent synthesized by reaction of dimethylchlorosilane with the respective fluoroalkene in the presence of chloroplatinic acid. The reaction time necessary for the preparation of the reagent is 6-7 h at 210-240°C in a sealed ampoule [341]. Trialkylsilyl derivatives with other alkyl substituents were developed mainly for their higher stability, capability of being purified in advance on a thin layer, better GC separation and particularly for combination with mass spectrometry, where they provide a greater amount of structural information and facilitate quantification. rert. -Butyldimethylsilyl [342-3441 and allyldimethylsilyl ethers [345] and others, e.g., cyclotetramethyleneisopropylsilyl and cyclotetramethylene-terr.-butylsilyl ethers [344], have been described for steroids. The separation of epimers improves as the volume of the alkyl group grows, but retention times also increase. 5.8.2. Acyl derivatives

As hydroxyl groups are usually present in the molecules of steroids, it is the preparation of esters of these groups and of the enolized carbonyl group with different acylating

STEROIDS

157

agents that is involved. The ready availability of the reagent led to the wide use of acetyl derivatives, but of greater importance are halogenated acyl esters and some others developed for the specific and sensitive analysis of steroids. Acetyl derivatives prepared on the column by subsequent injection of anhydride were exploited by Anders and Mannering [346] for the characterization of some steroids by the “peak shift” technique. The simultaneous injection of an acetic anhydride-propionic anhydride mixture leads to the simultaneous formation of acetyl and propionyl derivatives. Chamberlain et al. [347] applied acetyl derivatives to the analysis of progesterone metabolites in urine. They performed the acetylation of substrates with acetic anhydride in pyridine at room temperature overnight and GC on a column packed with 6%QF-1 (250”), 5% SE-30 (250°C) or 3% NPGA (225°C). Brooks [348] studied acetylated corticosteroids and related 2O-oxopregnanes. Acetylation of 17a- and 1 10-hydroxyl groups was carried out with acetic anhydride with the addition of p-toluenesulphonic acid (10 mg/ml), as follows. The steroid (0.1-1 mg) was dissolved in the reagent (10-50 pl) and the solution was allowed to evaporate to dryness at room temperature in a desiccator. The residue was extracted with chloroform or methylene chloride and a suitable aliquot used for GC (1% SE-30,22S°C). The yields were reduced in some instances owing to the formation of by-products, which was more significant if larger initial amounts of the substrate were used; the 2 1-hydroxyl group in the configuration of a 17a,2 1-dihydroxy-20-ketone was acylated with acetic anhydride in pyridine in advance. Brooks concluded that completely acetylated corticosteroids are thermally stable and may be applied as suitable derivatives for GC even on the microscale. Mougey et al. [349] described a method for determining epitestosterone and testosterone in human and monkey urines. The method involves enzymatic hydrolysis, extraction with diethyl ether, TLC of neutral steroids, conversion into acetates, TLC of steroid acetates and separation of testosterone and epitestosterone by GC. Acetylation is performed by treatment with acetic anhydride in pyridine and GC analysis on a 6-ft. column packed with 0.6% of JRX plus 0.2% of Hi-Eff-8BP on Gas-Chrom Q. As the total recovery of the method was only 54-6776, the results of the determination had to be corrected with the aid of values obtained with ‘‘C-labelled substrates. The sensitivity of the method was stated to be 1 pg/l, the coefficient of variation being 7%. Nordby and Nagy .[3501 chromatographed acetates of twelve plant steroids on fourteen stationary phases. They evaluated the efficiency of the separation by studying five “critical pairs”: sitosterolstigmastanol, campesterol-stigmasterol, sitosterol-fucosterol, sitosterol-isofucosterol and fucosterol-isofucosterol. Carbowax SP-100 was the most suitable stationary phase. On the basis of this incomplete list of applications, it can be assumed that acetates are of great significance for the GC analysis of steroids and can be used to advantage in many instances, particularly if no other reagent is available. Halogenated acyl derivatives of steroids have been applied in order to increase sensitivity of the analysis. It follows from a comparison of the ECD responses of haloacetates of steroids that the highest sensitivity can be obtained with the aid of monochloroacetates [351]. Brownie et al. [352] applied them in the analysis of testosterone in blood. The method involves the extraction of blood plasma with diethyl ether, purification by TLC and derivatization. GC analysis is performed only after a preliminary separation on a thin layer. Preparation of the derivatives is carried out by treating a dried extract with

158

DERIVATIZATION OF COMPOUNDS

0.5 ml of a solution of monochloroacetic anhydride in tetrahydrofuran (100 mg per 10 ml) with the addition of 0.1 ml of pyridine. GC is carried out on 1% of XE-60 and testosterone concentration of 5 ng per 10 ml of plasma can be determined with an accuracy of 8%with the use of an ECD. Trifluoroacetates of 18 steroids were analysed by Voelter et al. [353] on OV-17,OV-1 and XEdO stationary phases and compared with the results for TMS derivatives. On the first two stationary phases TFA derivatives have shorter retention times, whereas on XE-60 the reverse applies. With the use of the FID, TFA derivatives gave 30-50% higher responses. These derivatives were also applied to the analysis of the bile acids [354]. In order to eliminate the treatment with diazomethane, the carboxyl group of bile acids was blocked by the reaction with hexafluoroisopropanol, as follows. A 100-p1volume of hexafluoroisopropanol and 200 p1 of trifluoroacetic anhydride were added to the dried extract of bile acids and the mixture was heated at 37°C for 30 min. The mixture was evaporated under reduced pressure at room temperature and the residue dissolved in 100 pl of acetonitrile; 5 pl were analysed on a 2 m X 3 mm I.D. column packed with 1% QF-1 on Chromorosb W (80-100 mesh). As the FID was applied, a high ECD response was not used to advantage. Comparison of various halogenated acyl derivatives verifies that the typical properties of the ECD.apply also for steroids [355].Table 5.13 compares the relative retention times and responses of different derivatives of testosterone and cholesterol obtained on TABLE 5.13 COMPARISON OF RELATIVE RETENTIONS AND FID AND ECD RESPONSES OF TESTOSTERONE AND CHOLESTEROL HALOGENATED ACYL DERIVATIVES [ 3551 Conditions: glass column, 6 ft. X 1/8 in. I.D., 2%XEdO on GasChrom Q (100-120 mesh); carrier gas, Nz at 90 ml/min, column temperature, 200°C Parent compound

Testosterone

Cholesterol

Derivative

Relative response FID

ECD

0.20 1.41 0.01 0.28 1.00 *** 2.10 2.30

Monochloroacetate Monochlorodifluoroacetate Trifluoroacetate Pentafluoropropionate Heptafluorobutyrate Perfluorooctanoate Diheptafluorobutyrate

6.90

0.99

2.25 1.08 1.01 1.00 1.22 0.19

1.02 1.00 1.04 1.00 ** 1.08 1.11

Trifluoroacetate Monochlorodifluoroacetate Heptafluorobutyrate Perfluorooctanoate

1.04 2.22 1.00 5 1.44

1.13 0.84 1.00

* 31 min. ** 2.7 coulomb/mol. *** 1.18. lo3 coulomb/mol. 5 22.3 min.

Relative retention time

0.90

0.09

2.08 1.00 1.85

STEROIDS

159

an XE-60 column and with an ECD and an FID. Perfluorinated acyl derivatives permit a more sensitive analysis to be performed and their volatilities increase up to the HFB derivative. Di-HFB esters (in testosterone the carbonyl group is enolized) have the highest ECD response and are also the most volatile. As further extension of the perfluorinated acyl chain does not increase the sensitivity of the analysis markedly and the volatility is lower, HFB derivatives are often used for the GC analysis of steroids, particularly for trace analysis. In spite of the fact that HFB derivatives have a number of properties suitable for GC analysis, their use for steroids suffers from various problems, as can be judged from the different results obtained by different workers. Wotiz and co-workers [356,357] reported a detection limit of 0.1 ng of steroids and stated that if estrogens are determined in plasma, 2 ng per 10 ml of plasma can be analysed. Derivatization was performed by the following procedure. A 0.1-ml volume of n-hexane, 1 pl of tetrahydrofuran and 2 pl of HFB anhydride were added to the estrogen fraction and the mixture was heated in a closed test-tube at 60°C for 30 min. The solvent was evaporated under a moderate stream of nitrogen at 40-50°C and the residue was dissolved in 50 pl of n-hexane. GC was performed on 3% QF-1 or 2% XE-60 at 240°C. Other workers [358] carried out the acylation of estrogens in acetone and reported the following conditions as optimal for the preparation of tris-HFB-estriol: 0.1-0.3 p1 of acetone per 1 pg of the'substrate and 0.05 ml of HFB anhydride at room temperature for 10 min. The use of a larger amount of another solvent (benzene, methylene chloride, dimethyl sulphoxide, diethyl ether, dioxane) was said to result in the formation of a number of by-products. Poole and Morgan [359], however, stated that the HFB derivatives of some steroids are not thermally stable and that only the decomposition product is detected, e.g., cholesta-3,5-diene is produced from cholesterol. This leads to a considerably lower ECD response, so that the detection limit, which under favourable circumstances can be as low as 0.005 ng, is usually not achieved. As steroids that form unstable HFB derivatives they reported cholesterol, lumisterol, ergosterol, estradiol, pregnanetriol and others. The enol form of the carbonyl group can also be acylated in the course of acylation of 3-ketosteroids with a double bond in position 4. Depending on the reaction conditions, particularly on the solvent used, derivatives of 2,4- and 3,5-dienols can, however, be formed. It is therefore necessary to find the conditions when only one of the above derivatives is produced exclusively, or at least predominantly. In order to prepare the di-HFB derivative of testosterone, reaction in benzene with the addition of pyridine or collidine [360] is recommended, as follows. A 2 0 4 volume of pyridine or collidine and 10 gl of HFB anhydride are added to 100 yl of a solution of testosterone in benzene (ca. 5 pg/pl). After 2 h at 60°C the mixture is dried under a stream of nitrogen and dissolved in benzene. Under these conditions di-HFB derivative of 2,4-diene-3,17/3-diol is produced as the main product and its amount does not change with time, so that the procedure can be applied for quantitative determination. Less than 30 pg of testosterone can be detected in the form of the di-HFB derivative. Challis and Heap [361] performed the acylation with an HFB anhydride-benzene mixture and controlled the reaction yield by altering the ratio of the two components. They did not mention the above problems and reported the detection limit for the deter-

160

DERIVATIZATION OF COMPOUNDS

mination of estradiol in plasma in the form of the di-HFB derivative to be 30 pg/ml. Watson and Kalman [362] described the determination of digoxin and related compounds in plasma at concentrations of 5-100 ng per 10 ml. The method involves extraction with methylene chloride, purification on a thin layer, derivatization, purification on silica gel and GC. They carried out the acylation with the aid of HFB-imidazole in benzene. A dried extract was dissolved in 100 p1 of benzene, 6 pl of HFB-imidazole were added and the mixture was heated at 90°C for 20 min. Hexadecafluorononanoates and eicosafluoroundecanoates of testosterone and estradiol were chromatographed by Kirschner and Taylor [363]. In comparison with HFB derivatives, they have longer retention times and the area responses are 2-2.5 time higher; for the di-derivatives the difference in responses is very small. Special derivatives were reported by Dehennin and Scholler [364]. By reaction with acetone in the presence of TMCS, a hemiketal is produced, which dehydrates to the corresponding vinyl ether and the latter forms with HFB anhydride 1-heptafluorobutanoyl-2propenyl ether (Scheme 5.33). The reaction is carried out as follows. The steroid mol) is dissolved in 0.1 ml of dry acetone and 0.1 ml of TMCS. Then HFB (2 anhydride (0.05 ml) is added and the mixture is allowed to stand at room temperature for 1 h. The derivative is selective for sec.-hydroxyl groups and provides a higher ECD response that the HFB ester (up to 40 times higher for 50-androstane-l7P-01).

U/J

- HCI

Scheme 5.33.

Methanesulphonyl derivatives of steroids are prepared by treatment with methanesulphonyl chloride in pyridine. They might be applied in selective analysis with a detector sensitive to sulphur, but they decompose during analysis into the corresponding olefin, the double bond of which indicates the position of the original hydroxyl group. They have been applied to bile acids [334]. Jacob and Vogt [365] prepared dimethylthiophosphonic esters of steroids for a very sensitive analysis of steroids with the aid of an AFID. Dimethylthiophosphonic chloride reacts with the hydroxyl groups of steroids in the presence of triethylamine (Scheme 5.34). The detection of 10-15 pg of steroids can be achieved at a signal-to-noise ratio of 4-5.

Scheme 5.34.

5.8.3. Oximes

The keto group of steroids usually does not cause serious problems in GC analysis. However, in some instances it can interfere, e.g., if its enol form contributes to the deri-

STEROIDS

161

20

30 TlMElMlN 1

Fig. 5.25. Separation of MO-TMS derivatives of epitestosterone and testosterone on a 1%OV-17 column and a 1%OV-1 column, with temperature programming at l"C/min from 2QD"C. (Reproduced from Anal. Biochem., 22 (1968) 284, by courtesy of M.G. Homing and Academic Press.)

vatization reaction. A characteristic retention shift due to the conversion of the carbonyl group into the derivative as well as characteristic mass spectra obtained by combined GC-MS may be exploited for identification. For this purpose, oximes substituted with methyl and benzyl group and in combination with derivatives of the hydroxyl group, especially TMS and acyl derivatives, are commonly used. Methoximes (MO) of 55 steroids were prepared by Fales and Luukkainen [366] by treatment with methoxylammonium chloride in pyridine and standing overnight at room temperature. They were analysed on SE-30 and NPGS stationary phases. Hydroxyl groups were blocked by silylation. This procedure was also used in further studies devoted to the analysis of MO-TMS derivatives of urine steroids [367,368] and dexamethasone [369]. For silylation different reagents were used, depending on the reactivity of the hydroxyl groups, mostly BSA, TMSIM alone and TMSIM in combination with TMCS catalysis, as already mentioned in Section 5.8.1. Non-selective stationary.phases, such as SE-30 and QF-1, were used. A common property of oximes, the formation of synlanti isomers, is also found with steroids; however, for their separation stationary phases possessing certain selectivity are necessary. Fig. 5.25 shows a chromatogram of MO-TMS derivatives of testosterone and epitestosterone. Each steroid provides a simple peak on OV-1 (upper chromatogram), even though the shape of the peak suggests the presence of more substances. On OV-17 both isomers of each steroid can be resolved [370]. MO-TMS derivatives were also used for the quantitative analysis of steroid profiles in urine [371,372]. The procedure involves extraction of steroids and conjugates by means of XAD-2 resin, enzymatic hydrolysis, solvolysis with acidified ethyl acetate and washing with alkali. The derivatives are prepared as follows. The organic solvent is evaporated under a stream of nitrogen at 60°C and 100 pl of a solution of methoxylammonium chloride in dry pyridine (100 mg/ml) are added to the dry residue. The mixture is heated in a closed vial at 60°C for about 1 h, then the excess of pyridine is removed under

162

DERIVATIZATION OF COMPOUNDS

nitrogen at 60°C. After adding 100 p1 of BSA-TMSIM-TMCS (3 : 3 : 2, v/v), persilylation of all of the steroids is achieved at 80°C. GC separation of 27 steroids was performed on a 3.5 m X0.25 mm I.D. WCOT column with SE-30 at 250"C, with identification by retention indices or MS. Methoximes combined with halogenated acyl derivatives have been used for a very sensitive analysis of steroids with an ECD. Aldosterone was analysed by Horning and Maunie [373] at nanogram and sub-nanogram levels in biological samples. The di-MO derivative is formed by treatment with methoxylammonium chloride in pyridine at room temperature overnight; prolongation of the reaction time t o 3 days results in the tri-MO derivative. The HFB-di-MO derivative possesses good chromatographic characteristics and provides a single peak on a non-selective stationary phase. However, up to four isomers may be distinguished on a selective stationary phase (OV-17,OV-22). Higher alkoximes are suitable for combined GC-MS. Their molecular weights vary within a range that is very suitable for mass spectrometry and characteristic fragments are highly informative. For a representative series of ketosteroids, pentoximes and butoximes were studied [374]. The corresponding alkoxyamines react with steroids quantitatively, the resulting derivatives are stable and their retention times lie between those of methoxy- and benzyloximes. Benzyloximes posses considerably longer retention times (MU values are 6-7 times larger than those of methoximes) and similar properties. Some groups of ketosteroids can be separated much better by using benzyloximes than by using MO derivatives, and in combination with TMS derivatives benzyloximes have been applied to the measurement of steroid profiles in the urine of newborns [375,376]. Peritafluorobenzyloximes also provide high ECD responses and have been applied to the determination of dehydroepiandrosterone in human plasma [377]. As in previous instances, the reaction is carried out in pyridine, as follows. Pentafluorobenzyloxyammonium chloride (0.2 mg) is added to a solution of 17-ketosteroids (ca. 1 pg) in pyridine ( 2 drops) and is heated for 1 h at 60°C. The reaction mixture is diluted with n-hexane (3 ml), washed gradually with water (1 ml), 0.1 N H C l (1 ml), 0.1 N N a O H solution (1 ml) and water (1 ml) and centrifuged. After evaporation of the solvent, the residue is silylated with HMDS (0.1 ml) + TMCS (0.1 ml) in pyridine (5 drops). After 5 min at room temperature, the mixture is analysed directly or the solvent is evaporated, the residue is dissolved in n-hexane (1 ml) and 2 p1 are analysed. Satisfactory linearity of the calibration graph was observed in the range from 0.6 t o 3 ng of the ketosteroid; the detection limit will undoubtedly be much lower, however. For the analysis a column packed with 1% of OV-1 on Gas-Chrom Q (80-100 mesh) at 220°C was used. 5.8.4. Hydrazones

The possibility of preliminary isolation of substances under analysis in the form of hydrazones is frequently utilized in the determination of ketosteroids in complex mixtures such as biological materials. In this form they are even sometimes subjected directly t o GC analysis. Charransol et al. [378] determined androstanediol and testosterone in urine. After hydrolysis the sample was extracted with methylene chloride, evaporated and treated with Girard T reagent. Free hydroxysteroids were extracted with diethyl ether and

STEROIDS

163

Girard T complexes of ketosteroids were hydrolysed and also extracted. Steroids were further purified on a thin layer, silylated and subjected to analysis. VandenHeuvel and Homing [379] dealt with the preparation of dimethylhydrazones of ketosteroids: an 0x0 group in position 3, non-conjugated with a double bond, reacts with dimethylhydrazine alone. The preparation of hydrazones in positions 16, 17 and 20 and 3 conjugated with a double bond requires acid catalysis; the 1 1-keto group does not react. SE-30 or QF-1 (1%) were satisfactory stationary phases. Condensation with other reagents and preparation of substituted hydrazones can serve for the characterization and identification of ketosteroids [380]. Pentafluorophenylhydrazones permit up to 0.1 ng of estrone to be determined with the ECD [381]. A hydroxyl group in position 3 is blocked by methylation with dimethyl sulphate and the reaction with pentafluorophenylhydrazine is performed in methanol in the presence of acetic acid. A column packed with 1% of XEdO on Gas-Chrom Q at 205°C is suitable for the analysis. The determination of free estrone in blood plasma by this procedure provides good results provided that the measurement is corrected for the total recovery.

5.8.5.Cyclic derivatives Application of these derivatives is considered for steroids with two functional groups, firstly with vicinal hydroxyl groups and functional groups on the 17,20,21-dihydroxyacetone side-chain. CH2-OH

I

w

c=o ---

0H

CH2=0 H+

Scheme 5.35.

Bismethylenedioxy derivatives of hydroxycorticosteroids are produced by the reaction of the dihydroxyacetone function with formaldehyde (Scheme 5.35). Cortisone (10 mg) is suspended in 5.0 ml of chloroform and 1.3 ml of 12 N hydrochloric acid and 1.3 ml of a 37% solution of formaldehyde are added. The reaction mixture is stirred at 5°C for 48 h and during this time the steroid dissolves. After washing with 1 N sodium hydroxide solution and drying over sodium sulphate, the mixture is filtered through glass-wool and analysed directly. The derivatives are stable and provide symmetric peaks on SE-30, and make it possible to analyse selectively complex mixtures of steroids with a dihydroxyacetone moiety in the molecule [382]. Acetonides 1383,3841 and siliconides [385,386] are prepared by the reactions of neighbouring hydroxyl groups, e.g., in positions 16,17 and 17,21, with acetone or dimethylchlorosilane. The reaction with acetone proceeds under acid catalysis with hydrogen chloride or TMCS, as follows. Steroids are dissolved in 10 ml of freshly distilled acetone and 100 pl of TMCS are added. The mixture is agitated at room temperature for 2 h, 1 ml of 1 N sodium hydroxide solution is added and the solvent is evaporated at

164

DERIVATIZATION OF COMPOUNDS

40°C under reduced pressure. The residue is dissolved in 25 ml of 1 N sodium hydroxide solution and extracted twice with half its volume of chloroform. The combined extracts are evaporated down to the required volume and, after conversion of the remaining hydroxyl groups into TMS ethers, the solution is subjected to analysis. Dimethyldiacetoxysilane can also be used as a reagent for the preparation of siliconides, as it reacts simultaneously with other unprotected hydroxyl groups and blocks them. A sample of steroids is evaporated in a 2 X 75 mm test-tube, 20 pl of a solution of dimethyldiacetoxysilane (2%) and triethylamine (2%) in dry n-hexane are added and the test-tube is sealed in a flame. After incubation for 2 h at 40°C the test-tube is opened and the mixture is injected. The derivatives may be analysed on 1% of OV-1 at 245°C. Their significance again consists in the possibility of selective characterization of steroids with the above-mentioned arrangement of functional groups. The situation is similar with cyclic boronates, which are prepared by the following procedure. Steroid (10 pmol) and the respective substituted boric acid (10 pmol) are dissolved in ethyl acetate (1 ml) and the mixture is allowed to stand for 5 min at room temperature. Under these conditions, 17,20-diols, 20,21-diols and 17,20,21-triols are convertedcompletely into boronates. Cyclic boronate was mainly produced from 17,21dihydroxy-20-ketone, but side-products also appeared, the formation of which could be suppressed by adding a 10%excess of the reagent [387-3891. Different substituents on the boron atom, such as methyl, n-butyl, terr. -butyl, cyclohexyl and phenyl, are interesting from the viewpoint of GC-MS application. They are further suitable for converting isolated hydroxyl groups into TMS or acetyl derivatives.

5.8.6. Other derivatives Methyl ethers were applied firstly to the GC of cholesterols [390,391]. They are prepared by the method common for the preparation of ethers, e.g., by treatment with dimethyl sulphate or methyl iodide in the presence of potassium rut.-butoxide (cf., p. 64). Their good chromatographic properties, e.g., on SE-30 and PEGS stationary phases, are usually obtained only after further treatment with a silylating or acylating agent. Ketals were applied by Sarfaty and Fales [392] to the GC analysis of steroid alcohols and to their combined GC-MS. The resulting derivative is prepared by the reaction of a hydroxyl group of the substrate with halogenated acetone and by subsequent methylation of the free hydroxyl group of hemiketals with diazomethane (Scheme 5.36). The steroid is dissolved in a solution of haloacetone in benzene (20-60%, v/v) and allowed to stand overnight. The reagent is removed with a stream of nitrogen at 60°C and the residue is methylated by treatment with ethereal diazomethane at 10°C for 5-8 h. GC was performed with good results on XE-60 or OV-17.

Scheme 5.36.

SUGARS AND RELATED COMPOUNDS

165

TABLE 5.14 ECD RESPONSES (PEAK AREAlMASS OF DERIVATIVE) OF HALOGENATED STEROID DERIVATIVES RELATIVE TO TESTOSTERONE 17p-HFB [ 3921 Parent compound

Derivative

Testosterone

17P-Heptafluorobutyrate 17p-(O-Methyldichlorotetrafluoro)ketal 17pChloroacetate 17~-(O-Methylhexafluoro)ketal

Androsterone

3ol-(O-Methyldichlorotetrafluoro)keta! 3aCNoroacetate 3a-Heptafluorobutyrate

Relative ECD response ~

100 33 22 2 16 16 22

Under these conditions, the sterically hindered (2-20 hydroxyl group does not react, but the less hindered C-17 group of testosterone is derivatized successfully. sym-Dichlorotetrafluoroacetone, I ,1,3-trichlorotrifluoroacetoneand hexafluoroacetone were tested as initial reagents. As illustrated by Table 5.14, the ECD responses of ketals prepared with these reagents are comparable to or lower than those of HFB derivatives. Oxidation of corticosteroids prior to GC analysis makes it possible to analyse steroids of various nature simultaneously [393,394]. The oxidation is performed with periodic acid in dioxane at room temperature. Progesterone and androstenedione and their derivatives do not change owing to the action of this reagent. Deoxycorticosterone, dehydrocorticosterone and corticosterone are converted into the corresponding etiocholenic acids. Aldosterone and 18-hydroxydeoxycorticosteroneform lactones of the etiocholenic acids produced. Action of the oxidation reagent for longer than 3 h can, however, lead to slow oxidation of the 1 lp-hydroxyl group into a keto group. GC separation of the products after oxidation and subsequent esterification with diazomethane provides information on all of the corticosteroids that are present. SE-30 stationary phase can be applied at 245°C.

5.9. SUGARS AND RELATED COMPOUNDS

This section discusses derivatization procedures and the GC analysis of saccharides and related compounds, such as amino sugars, polyalcohols, aldonic and uronic acids and glycosides. Their low volatility, caused by the presence of several functional groups in one molecule and by their high molecular weight, and their thermal lability are the factors responsible for the fact that they cannot be analysed by GC as such. In addition t o some hydroxyl groups, it is necessary to take into consideration the presence of other polar groups, such as carbonyl and amino groups. The difficulty in derivatizing and preparing a uniform product lies firstly in polyfunctionality of the substrate and in the different reactivities of various groups, and gives rise to several products from one compound, which is, e.g., for siiylation, fairly common. Another complication is brought

166

DERIVATIZATION OF COMPOUNDS

about by the formation of a-and P-anomers and pyranose and furanose rings, occurring either during the preparation of the derivative or in the analysis proper. In the analysis of mixtures this may lead to very complicated chromatograms and make their evaluation difficult; however, in spite of this derivatization procedures are commonly used in the GC analysis of sugars. If a uniform product cannot be obtained, efforts must be aimed to control the derivatization reaction, maintaining the ratio of different reaction products constant. In addition to silylation procedures, the preparation of ethers, especially methyl ethers, and acyl derivatives offer has been widely applied. Wells et al. [395] and Sloneker [396] described the derivatization problems and GC analysis of sugars in detail. 5.9.1. Methyl ethers

Methyl ethers have been used extensively for the GC analysis of sugars as natural derivatives of hydroxyl groups. McInnes et al. [397] and Bishop and Cooper [398] seem to be the first who tested them. They prepared these derivatives by treatment with dimethyl sulphate in the presence of sodium hydroxide and chromatographed them on Apiezon M. Completely methylated derivatives of thermally labile monosaccharides appeared to be sufficiently stable and volatile for GC. Pentoses were well separated from hexoses, but the latter were only partly separated from one another. Methyl glycosides methylated to different extents could also be separated from one another. Their volatilities decrease and the retention times increase with increasing number of methyl groups so that, e.g., even the monomethyl ether of methylglucopyranose did not have a sufficient volatility for the analysis; however, a heavy coating (20%) and a relatively low temperature (1 50°C) without temperature programming were applied. Kircher [399] performed methylation by treatment with methyl iodide in dimethylformamide in the presence of silver oxide. He tried some methylated polysaccharides as stationary phases for the separation of the methyl derivatives, the best of which was methylated hydroxyethylcellulose, which was capable of separating even some a-and 0-anomers. A number of stationary phases were later applied successfully. Fig. 5.26 shows the separation of glucopyranosides methylated to various extents on 5% Carbowax 20M esterified with 2-nitroterephthalic acid [400]. For the GC of methylxylofuranosides, Anderle and co-workers [401,402] applied several stationary phases (10%Apiezon L, 5% XE-60,3% ECN SS-M, 10% diethylene glycol succinate, 10% Carbowax 20M terephthalate), each with temperature programming in the range 100-240°C. On all of these stationary phases the retention sequence was determined by the number of methyl groups in the molecule of the sugar, with the most methylated derivative eluting first. Permethylated alditols and aldonic acids were analysed on 10% SE-30 (140°C), 1% OV-17 (13OoC), 3% QF-1 (1 10°C) and 1.5% XE-60 t 1.5% EGS ( I 30"C), with success varying for individual groups of compounds [403]. The significance of methyl ethers for the GC analysis of sugars is emphasized by the fact that they are direct products of the methanolysis of oligosaccharides and glycosides. Methanolysis is often applied as a suitable alternative method for the cleavage of these substances prior to analysis, particularly in those instances when more stringent condi-

SUGARS AND RELATED COMPOUNDS

167

W

Ln

z

0

a w Ln

(L (L

0

+

V LLI

tW 0

0

4

8 12 16 2 0 24 28 32 TIME ( M I N )

Fig. 5.26. Gas chromatogram of a mixture of methyl glucopyranosides. Methoxyl groups in the following positions: 1 = p-2,3,4,6-;2 = 01-2,3,4,6-;3 = p-2,3,4-;4 = p-2,3,6-;5 = a-2,3,4-; 6 = a-2,3,6-;7 = p-2,4-; 8 = 01-2,4-;9 = p-2,3-; 10 = a-2,3. Conditions: stainless-steel column, 8.5 ft. X 0.125 in. O.D., 5%Carbowax 20M esterified with 2-nitroterephthalic acid on Chromosorb G (80-100 mesh, DMCStreated); helium flow-rate, 22 ml/min, temperature programme, 4"C/min, 150-270°C. (Reproduced from Anal. Letr., 3 (1970) 151, by courtesy of Marcel Dekker.)

tions of hydrolysis could result in chemical changes of the monosccharides [404]. A solution of the glycoside or the carbohydrate (1 -500 pg) is placed in a 2-nil conical reaction test-tube with a PTFE-lined screw-cap. After adding an internal standard (e.g., 40 pg of mesoinositol), the sample is lyophilized and placed in a desiccator over P 2 0 5 overnight. The residue is dissolved in 250-500 p1 of 0.5 M methanolic HC1, prepared by the dissolution of dry gaseous hydrogen chloride in dry methanol. The test-tube is stoppered, shaken vigorously and heated at 80°C for 20 h. The excess of the reagent is removed under a stream of nitrogen at 50°C. 0-Methylglycosides obtained in this way are further treated, prior t o GC analysis, either by the rnethylation procedures described above or, more often, by acylation and silylation. Alditols [405] and amino sugars [406] have been analysed by GC-MS as combined methyl ether-acetyl derivatives. Acetylation was performed with acetic anhydride in pyridine, as follows. A 0.1 5-ml volume of re-distilled dry pyridine was added t o 0.5 ml of 1.5 N acidic methanolysate containing 0.025-2.5 pmol of amino sugars. The apparent pH of the methanolic pyridinium hydrochloride varied in the range 4-5. Acetic anhydride (0.1 ml) was added and the mixture was stirred thoroughly and allowed t o stand at room temperature for 30 min. Evaporation t o dryness was performed in vacuum over solid KOH with subsequent vacuum drying for 4 h over P2OS. SE-30 was used as the stationary phase. Trifluoroacetates of 0-methylglycosides possess shorter retention times than acetates, which makes possible a shorter analysis time and properties that even allow the use of more polar stationary phases (5% OV-210). Acylation with T F A anhydride is executed at 150°C for 5 min or at 100°C for 3 11. Under these conditions amino groups, if present, are also acylated t o advantage [404]. For improvement of the separation of mono-0methylglucoses, Anderle and Kovac' '(4071 recommended prior reduction with sodium borohydride into the corresponding glucitols. Trifluoroacetylation of these substances with T F A anhydride in the presence of pyridine is sufficiently rapid (1 h at room temper-

168

DERIVATIZATION OF COMPOUNDS

ature) and results in acylation of all of the hydroxyl groups in the substrates. XE-60 (1%) was used as the stationary phase. Partly methylated alditols can be acylated after their conversion into TMS ethers [408]. Non-polar stationary phases of the OV-101 and SE-30 types are the most suitable for the separation of these derivatives. 5.9.2. Trimethylsilyl derivatives

As the functional groups of saccharides and related compounds are silylated relatively easily, TMS derivatives have been widely applied to this group of substances. Any silylating agent may be used for the preparation of the derivatives. As even mild silylating agents are sufficient, Sweeley et al.'s original method [409] has been used in most instances, sometimes with minor modifications only. Carbohydrate (10 mg) is treated with 1 ml of anhydrous pyridine (dried over KOH), 0.2 ml of hexamethyldisilazane and 0.1 ml of trimethylchlorosilane and the mixture is shaken vigorously and allowed to stand for 5 min or longer at room temperature. The mixture usually becomes turbid, probably owing to the formation of ammonium chloride, the precipitate of which, however, does not interfere in the analysis. If the substrate is not soluble in the reaction medium, the mixture must be heated at 75-85OC for 2-3 min. The resulting product is analysed either as such or after the removal of pyridine, which gives a broad tailing peak on some stationary phases that may overlap the peaks of substances with short elution times and make quantitative evaluation difficult. Lehrfeld [410] suggested a simple apparatus for this purpose by means of which the solvent may be removed under a stream of dry nitrogen. Under the above conditions, with an excess of the silylating agent, blocking of all of the hydroxyl groups is secured and one sugar then usually gives two peaks (a- and 0-anomers), provided that they can be resolved in the column used. A chromatogram of TMS derivatives of monosaccharides, amino sugars, disaccharides and other substances in a mixture that was prepared in this way is demonstrated in Fig. 5.27. Incomplete silylation gives rise to several products, e.g., the tetra-TMS ether of glucose can theoretically have up to 10 isomeric forms, Kim et al. [411] proposed the use of the mixtures of the derivatives thus obtained for identification purposes. Strictly anhydrous conditions are not critical, in the opinion of some workers, as an excess of the reagent reacts with water and removes it. Weiss and Tambawala [412] reported that with a sufficient excess of the reagent even quantitative results could be obtained with samples containing up to 90% of water. Further elaboration of this idea led to the development of a two-phase method for the concentration of the sample [413 J applied to the analysis of monosaccharides. The principle of this method consists in the use of a solvent for the silylation reaction in which the derivatives produced are hardly soluble (e.g., dimethylformamide, dimethyl sulphoxide). A separate phase is then isolated from the reaction mixture, into which TMS derivatives are transferred. It is supposed that this phase is hexamethyldisiloxane, produced by the decomposition of HMDS and TMCS with traces of water, as in the absence of these two silylating reagents the phase is not formed. Lablanc and Ball [414] modified this method and applied it to the silylation of sugars

SUGARS AND RELATED COMPOUNDS

169

c

TIME I MIN 1

Fig. 5.27. GC separation of TMS derivatives of sugars. Peaks: 1 = erythrose; 2 = p-arabinose; 3 = ribose; 4 = a-xylose; 5 = methyl a-rnannoside;6 = agulose; 7 = agalactose; 8 = aglucose; 9 = L-ascorbic acid; 10 = p-glucose; 11 = N-acetylglucosamine; 12 = N-acetylgalactosarnine;13 = D-glyceroD-guloheptonolactone; 14 = sucrose; 15 = a-maltose; 16 = p-maltose; 17 = cellobiose; 18 = gentiobiose; 19 = raffmose; 20 = melezitose. Conditions: stainless-steel column, 6 ft. X 0.25 in.O.D., 3% SE-52 on Chromosorb W (80-100 mesh, AW, silanized); argon flow-rate, 75 ml/min; temperature programme, 2.3"C/rnin, 125-250°C. (Reproduced from J. Amer. Chern. Soc., 85 (1963) 2497, by courtesy of the American Chemicai Society.)

and their phosphates, as follows. A 10-mg amount of a dry sample was placed into a vial and closed with a FTFE-lined septum. The reagents were added in the following sequence: 1.O ml of dimethyl sulphoxide, 1 ml of cyclohexane, 0.2 ml of HMDS and 0.1 ml of TMCS. The mixture was then shaken in an ultrasonic bath until the sample had dissolved (heating of the sample should be avoided). The reaction took about half an hour for sugar phosphates, and the silyl derivatives were found in the upper layer. Simple sugars 2nd their phosphates are said to give uniform derivatives with the use of this method. Other workers [415,416] developed an analogous method for the analysis of isomers of trisaccharide kestose in aqueous solutions, as follows. A 0.5-ml volume of reagent containing TMSIM in dry pyridine (4 : 1, v/v) was added to 8 mg of the sample in a reaction vial. A violent reaction started if the sample contained a significant amount of water. The mixture was shaken and allowed to stand for 10 min, then it was saturated with imidazole and the binary phase was produced by adding 40-1 00 ftl of hexamethyldisiloxane or n-hexane. The mixture was finally shaken and centrifuged. The upper layer was reported to contain almost all of the silyl derivatives. However, for accurate quantitative determination the distribution of the product into the two phases will undoubtedly have to be taken into consideration. The advantage of this procedure is that it does not involve evaporation of the solvent, with the risk of losses of the derivatives. Other and possibly stronger silylating agents, such as BSA, are suitable particularly in those instances when it is necessary also to modify other functional groups. They have also been used for these applications (e.g., analysis of amino sugars), but they give rise to substantial anomerization. Galactosamine and glucosamine can be converted into completely silylated derivatives by treatment with BSA-TMCS-HMDS-pyridine (1 : 0.5 : 1 : 10) at room temperature withm 30 min. On SE-30 at 187OC, the anomers of both

170

DERIVATIZATION OF COMPOUNDS

substances are separated. However, the anomers that have longer retention times for both of the compounds partly overlap [417]. Both non-polar (SE-30, SE-52, OV-101, etc.) and polar stationary phases (EGS, Carbowax) have been used for the separation of TMS derivatives, depending on the character of the problem. TMS derivatives of saccharides provide very long retention times on polar stationary phases, but on non-polar stationary phases some pairs are not separated at all (eg., a-lactose and sacharose). Therefore, Larson et al. [418] suggested a composite column consisting of a 20-ft. section packed with 1.5% of XE-60 on Chromosorb G (100-120 mesh) and a 7-ft. section packed with 3% of SE-52 on Anakrom ABS (70-80 mesh). This column showed long-termed good efficiency. Tesaiik et al. [419] separated TMS ethers of hexoses on capillary columns coated with SE-30,0V-101, XE-60 and Apiezon K. Decomposition of TMS derivatives of sugars on columns packed with polar stationary phases was observed with the use of I4C-labelled sugars [420] (see Table 4.10, p. 73). This decomposition is, however, said to be reproducible and much greater than with TFA derivatives. Of innumerable applications of TMS derivatives to the GC analysis of sugars, only a few examples will be selected. Copenhaver [421 J analysed galactose and glucose in blood plasma. A dried sample was silylated according to Sweeley et al. [409] and the derivatives were analysed on Carbowax 20M. The same procedure was applied by Dutton et al. [422] to the simultaneous analysis of polyhydric alcohols and sugars which occur in natural polysaccharides, using an 8 ft. X 0.25 in. I.D. column packed with 20% of SF-96 on Diatoport S (60-80 mesh) with a temperature programme of 3'Clmin from 130 to 220°C. Other workers [423] oxidized aldoses first into aldonic acids, as follows. A mixture of aldoses (10 mg) in water (1 ml) was oxidized at room temperature for 24 h by the addition of 4 ml of a 0.1 N solution of iodine and 6 ml of 0.1 N sodium hydroxide solution. The resulting I ,4-lactones gave stable and volatile derivatives after silylation. GC was performed on a polyester stationary phase (10% of neopentyl glycol sebacate or Carbowax 20M) at 170°C. The method was applied to a mixture of aldopentoses, aldohexoses and aldoheptoses in biological material. The determination of sugars in fruits and other materials involves extraction of the sample with 80%ethanol, transfer of sugars into an aqueous phase with the aid of a chloroform-methanol-water system (64 : 32 : 24) and drying. The analysis is carried out on SE-54 or SE-52 stationary phase after silylation [424,425]. The concentrations of monosaccharides can similarly be determined in the course of a fermentation process [426] and, after hydrolysis, also in polysaccharides from cellular walls and apple tissues [427,428]. SE-30 served as a stationary phase for these analyses. The silylation can be effected with an HMDS-TMCS mixture also when analysing glucosamine, galactosamine and other amino sugars in biological materials. The same stationary phases as in the preceding instances can be utilized for GC [429,430]. Hexuronic acids were reduced prior to analysis in the form of their barium salts with sodium borohydride into the corresponding aldonic acids, and their 1,4-1actones were silylated and chromatographed [43 11. Kagan and Mabry [432] and Furuya [433] analysed the composition of flavonoid glycosides by chromatographing TMS derivatives of plant glycosides based on phenols, coumarin, isoflavone, anthraquinone and others. Silylation of sugar phosphates requires more drastic conditions [434,435], as follows.

SUGARS AND RELATED COMPOUNDS

171

Several micromoles of sugar phosphate (in the form of sodium or cyclohexylammonium salt) are treated with 50 p1 of BSA, 20 pl of TMCS and 30 p1 of dry pyridine. After being shaken thoroughly, the mixture is allowed to stand at room temperature for 30 min to 3 h. If BSTFA is used, the procedure is as follows: 1 mg of phosphate (free or a salt) reacts with 0.1 ml of BSTFA and 0.05 ml of TMCS in 0.1 ml of acetonitrile. The mixture is heated at 80°C for about 10 min in order that the salts might also react. Another silylation procedure is performed with 0.1 ml of TMSIM in 0.1 ml of acetonitrile by standing at room temperature for 5 min. The analysis is carried out on a column packed with 1-576 of SE-30 containing 1%of OV-17, with temperature programming. 5.9.3. Acyl derivatives

The high molecular weight and polyfunctionality of sugars are the factors that limit the selection of the acylating agent. For GC analysis of sugars acetates and trifluoroacetates are mostly used. Alditols are acylated as such; for the other compounds, the other functional groups are usually converted into derivatives: the carbonyl group is derivatized by reduction into hydroxyl group or by reaction with hydroxylamine into oxime and further into nitrile, and the amino group is acylated simultaneously with hydroxyl groups. Reduction of sugars into alditols is effected by treatment with sodium borohydride, and acetylation by treatment with acetic anhydride containing sulphuric acid (2%) at 80°C for 15 h [436] or for 4 h by refluxing with a mixture of acetic anhydride and pyridine (1 : 1) [437]. The excess of reducing agent is usually decomposed prior to the acylation by treatment with an acid. The boric acid so produced should be removed, as it forms a complex with alditols and retards the acylation. Polar stationary phases (e.g., Carbowax 20M), on which strong sorption and decomposition of the derivatives occur, are not very suitable for the GC separation of acetates. Carbowax 20M modified with terephthalic acid and XE-60 provides good results, but some derivatives do not separate. A stationary phase possessing medium polarity between the above two, e.g., organic silicone polyester composed of ethylene glycol succinate combined with cyanoethyl silicone, is recommended as the most suitable [437]. Albersheim et al. [438] introduced a method for the analysis of sugars in polysaccharides from plant cellular walls, as follows. A 20-mg amount of sample material and 1 mg of myoinositol (internal standard) were added to 2 ml of 2 N trifluoroacetic acid. After hydrolysis for 1 h in a sealed test-tube at 121"C, the sample was evaporated to dryness at 50°C under a stream of nitrogen. Reduction was carried out with 10 mg of sodium borohydride in 0.5 ml of 1 N ammonia solution at room temperature for 1 h, and excess of the reagent was decomposed by the dropwise addition of glacial acetic acid. Five 1-ml volumes of methanol were added, each time evaporating to dryness at 50°C. The acetylation was carried out by heating at 121°C with 1 ml of acetic anhydride in a sealed test-tube. The best separation was obtained on a mixture of 0.2% of polyester glycol succinate, 0.2% of polyethylene glycol adipate and 0.4% of silicone XF-1150. Fig. 5.28 shows a chromatogram of a standard mixture of eight sugars. Lehnhardt and Winzler [439] proceeded in a similar manner in determining neutral sugars in glycoproteins. Hydrolysis of 0.1 -3 mg of the sample was performed on a

DERIVATIZATION OF COMPOUNDS

172 I

TEMPERATURE ('Cl

Fig. 5.28. Gas chromatogram of a mixture of sugars after reduction and acetylation. Peaks: 1 = L-rhamnose; 2 = D-fucose; 3 = L-arabinose;4 = D-xylose; 5 = D-mannose, 6 = D-galactose; 7 = Dglucose; 8 = myo-inositol. Conditions: copper column, 4 ft. x 1/8 in.O.D., 0.2%EGS, 0.2%EGA and 0.4% XF 1150 on GasChrom P (100-120 mesh); temperature programme 120°C for 10 min, then l"C/min. (Reproduced from Curbohyd. Res., 5 (1967) 340, by courtesy of P. Albersheim.)

column packed with ion-exchange resin in the H' form. Derivatization of the dried hydrolysate was carried out as follows. The sample was dissolved in 50 pl of water and treated with 50 p1 of 0.22 M NaBH4 at room temperature for 1 h. The excess of the reagent was decomposed with 50 pl of glacial acetic acid and the mixture was evaporated to dryness. Borate was removed by three-fold addition of 100 pl of HC1-methanol (1 : 1000, v/v) and evaporation to dryness at reduced pressure. The samples were acetylated at 100°C for 15 min with a mixture of 50 pl of pyridine and 50 pl of acetic anhydride. After further mixing, the mixture was heated for 15 min, than 1-10 pl of this solution were chromatographed on a column packed with 0.75% of HiEff-1 BP, 0.25% of EGSS-X and 0.1% 144-B (phenyldiethanolamine) with temperature programming at I.3"C/min from 160 to 210°C. The chromatogram in Fig. 5.29 illustrates the analysis of twelve sugars in a standard mixture by this procedure. Modification of this procedure was applied to the analysis of neutral and amino sugars in mucins [440]. A mixture of 0.2% of ethylene glycol succinate, 0.2% of ethylene glycol adipate and 1.4% of silicone XE-60 was used as the stationary phase and the analysis was effected with temperature programming at l"C/min from 150 to 205°C. Similar results could be obtained, however, on a column packed with 3% of OV-225. Partially ethylated alditol acetates were used to determine the components of polysaccharides [441]. Peracetylated derivatives of alditols and reduced sugars are sufficiently stable for GC analysis and possessing good properties, but a relatively complicated procedure is required for their preparation. They also cannot be applied to the simultaneous determination of the sugar and the corresponding alditol; with xylose, 2- and 4-substituted isomers cannot be resolved owing to the symmetry of xylitol. This gave rise to the use of combination with other derivatives, firstly with nitriles [442]. The procedure for a standard mixture

SUGARS AND RELATED COMPOUNDS

173

W

8

L?

Z 0

a

ul w LL CT

0 Iw u IW 0

0

20

10

30

TIME I MIN 1

Fig. 5.29. Gas chromatogram of alditol acetates from a standard mixture of the parent carbohydrates. Peaks: 1 = erythrose; 2 = 2deoxyribose; 3 = rhamnose; 4 = fucose; 5 = ribose; 6 = arabinose; 7 = xylose; 8 = 2deoxyglucose + 2deoxygalactose; 9 = mannose; 10 = galactose; 1 1 = glucose. Conditions: glass column, 1.83 m X 4 mm I.D., 0.75%HiEFF-IBP, 0.25%EGSS-X and 0.1% 144-B on GasChromosorb Q (60-80 mesh); nitrogen flow-rate, 40 ml/min; temperature programme, 1.3"C/min, 160-210°C. (Reproduced from J. Chrornotogr.,34 (1968) 471, by courtesy of W.F. Lehnhardt.)

is as follows. A dry mixture of twelve monosaccharides containing 1 mg of each sugar is dissolved in 0.6 ml of pyridine and is heated at 90°C with 12 mg of dry hydroxylammonium chloride in a sealed ampoule for 30 min. After cooling, I .8 ml of acetic anhydride are added and the re-sealed ampoule is heated for 30 min. The cooled solution is evaporated to dryness under reduced pressure at 40°C. The residue is dissolved in 0.1 ml of chloroform and 1 1.11 is taken for analysis. Aldonitrile acetates of sugars have been applied t o their GC analysis in different polysaccharides 14421 on LAC-4R-886 polyester stationary phase (190°C) and to the analysis of polyols and aldoses in urine and crystalline lenses [444] on a capillary column of borosilicate glass (60 m X 0.3 mm I.D.)coated with SE-30 containing a dispersion of Silanox 101 (temperature programming at 1"C/min from 150°C). These derivatives were very stable and a uniform product was formed from every individual substrate. Trifluoroacetates have been applied to the analysis of sugars in the same way as acetates. Imanari et al. [445] analysed aldoses after a prior reduction, as follows. A 0.5-ml volume of 1% NaBH4 in water was added to 0.5 ml of an aqueous solution containing 100-500 pg of a mixture of aldoses. The solution was allowed to stand at room temperature for 30 min and the excess of borohydride was decomposed by adding 0.5 ml of Amberlite CG-120 (H'). The resin was removed by filtration and the filtrate was evaporated to dryness. Borate was removed by the three-fold addition of 1 ml of methanol and subsequent evaporation. The residue was vacuum dried and dissolved in 0.1 ml of ethyl acetate and 0.1 ml of TFA anhydride. After 30 min at room temperature, 1-2 pl were taken for analysis. The resulting derivatives were more volatile than acetates and provided symmetric peaks on a column packed with 2% of XE-1105 (see Fig. 5.30). The application of a capillary column (OV-l01,85"C), combined with the use of an

174

DERIVATIZATION OF COMPOUNDS

I

5

6

20 0

a

TIME ( M I N I

Fig. 5.30. Gas chromatogram of alditol trifluoroacetates produced from a standard mixture of the parent aldoses. Peaks: 1 = fucose; 2 = ribose; 3 = arabinose;4 = xylose; 5 = mannose; 6 = glucose; 7 =galactose. Conditions: glass column, 1.8 m x 4 mm I.D., 2% XF-1105 on GasChrom P (80-100 mesh); nitrogen flow-rate, 70 ml/min; temperature, 140°C. (Reproduced from Chem. Pharm. Bull., 17 (1969) 1967.)

ECD, permits the analysis to be carried out at the picogram level, as demonstrated for the determination of sugars in sea water [446]. Prior to the analysis it was necessary to remove excess of the reagent in order to avoid overloading the detector. Direct acylation, without the prior conversion of carbonyl groups, leads, of course, to multiple derivatives. Mee [447] applied TFA derivatives to the determination of hexosamines in body fluids. The amino group was also acylated in the course of the procedure and the derivatives were analysed on 0.3% of EGA with temperature programming at S"C/min from 110 to 190°C. Wrann and Todd [448] carried out the derivatization reaction in a capillary (50 X 1.6 mm I.D.) and GC separation on a micropacked column coated with OV-210 with temperature programming at 1-2"C/min from 120 to 21OoC. 5.9.4. Acetals, ketals and other derivatives

Kircher [399] also described the GC analysis of acetals, in addition to the abovementioned derivatives (see p. 166). He demonstrated the analysis of 4,6-O-ethylidene-Dglucose in the form of the 1,2,3-triacetate on a column packed with completely methylated hydroxyethylcellulose. The diisopropylidene derivative of glucose (retention time ca. 50 min at 200°C) was sufficiently volatile for the analysis and its methyl, ethyl and vinyl ether in the 3-position was even more volatile (retention time 16 min). Mutual separations of these derivatives have not been achieved, however, on this column. Jones et al. [449] described a detailed study of the GC separation of acetal and ketal derivatives of sugars possessing either free hydroxyl groups or further substituted with acetyl, benzoyl, methanesulphonyl, methyl and p-toluenesulphonyl groups. A column packed with 1% of SE-30 on glass beads was applied with a short column, with 20% of Apiezon M and 20% of butanediol succinate on Chromosorb W (60-80 mesh) arranged before it. Some compounds with bulky substituents, e.g., benzyl or two or more p-toluenesulphonyl groups, have impractically long retention times, however. Acetylated diethyldithioacetals of xylose were analysed by Lance and Jones [442]. On a column coated with 5% of LAC4R-886 polyester stationary phase, a single large

BASES OF NUCLEIC ACIDS, NUCLEOSIDES AND NUCLEOTIDES

175

peak accompanied by one or more minor peaks was always obtained for one substance. These derivatives have not gained more extensive use in the GC analysis of sugars, however, as they do not provide any particular advantages over other derivatives that have been utilized with respect to ease of their preparation, uniformity of the products or the separation of various substances.

5.10. BASES OF NUCLEIC ACIDS, NUCLEOSIDES AND NUCLEOTIDES

Functional groups present in the molecules of the components of nucleic acids make their direct GC analysis impossible. As a rule, both hydroxyl and amino groups (or imino groups) or several of these groups together are usually present, which for the preparation of derivatives leads logically to the utilization of reactions that modify functional groups of all types, particularly alkylation and silylation. The latter procedure is used much more frequently, and several studies have been devoted to searches for the most suitable silylation conditions. Problems associated with non-uniformity of the products caused by the lower reactivity of amino group led to the use of combined derivatives. 5.10.1. Silyl derivatives

Hancock and Coleman [450,451] and Hashizume and Sasaki [452454] were the first to apply these derivatives to the analysis of the components of nucleic acids. They prepared the derivatives by treatment for 1 h with pyridine-HMDS-TMCS (7 : 2 : 1) at reflux temperature or at 145-155°C (temperature of an oil-bath). SE-30 and XE-60 stationary phases at temperatures from 200 to 250°C or silicone DC-430 with temperature programming were applied. Nucleosides [450], adenosine derivatives [451], ribonucleotides [452], bases and nucleosides simultaneously [453] and the composition of the hydrolysate of nucleosides and nucleic acids [454] were analysed in this way by GC. Hydrolysis was effected by heating for 1 h at 100°C with 70% perchloric acid. The problems associated with the uniformity of the resulting product, particularly in the presence of amino groups in the molecule, and the quantitativeness of the yields led to the testing of other silylating reagents. Trialkyl or triphenylsilyl derivatives, prepared with the aid of the corresponding chlorosilanes and tested on adenosine, did not result in any considerable improvement in this respect [455]. Only stronger silylating agents resulted in progress and have been applied to the preparation of TMS derivatives of different components of nucleic acids. BSA was effective for the derivatization of purine and pyrimidine bases [456] and nucleosides [457]. Bases were silylated by heating at 150°C with BSA-acetonitrile (1 : 3) for 45 min. It was stated that under these conditions the TMS derivative of guanine can be prepared reproducibly, but both cytosine and 5-methylcytosine provided two peaks. Silylation of nucleosides, including pseudouridine, was carried out by heating at 1 2OoC with a 100-fold excess of BSA for 2 h. With the use of OV-17 as the stationary phase, this procedure was adopted for the determination of the composition of ribonucleic acids. BSTFA has frequently been used for the silylation of the components of ribonucleic acids. After treatment for 15 min with a mixture containing pyridine (1 : 4) at room

176

DERIVATIZATION OF COMPOUNDS

temperature, it was possible to resolve two peaks for pseudouridine on SE-30 stationary phase, which were ascribed to a-and 0-anomers [458]. Butts [459] came to the same conclusion and modified the derivatization procedure by reaction with methoxylamine, by which he obtained MO-TMS derivatives of cytidine and deoxycytidine that possess more suitable chromatographic properties. Pyridine (I ml) containing 15-20 mg of methoxylammonium chloride was added to 1-2 mg of nucleoside in a 3-ml reaction vial. After heating for 3 h at 75°C the reaction was completed and the sample was evaporated to dryness under a stream of dry nitrogen. A 0.2-ml volume of pyridine and 0.3 ml of BSTFA containing 1% of TMCS were added to the residue and the reaction mixture was heated for 3 h in the case of ribonucleosides, and overnight in the case of desoxyribonucleosides. Volumes of 2 pl were analysed on a column packed with 5% of SE-30 with temperature programming. Gehrke and Patel [460] looked for optimal reaction and chromatographic conditions in the analysis of silyl derivatives of nucleosides prepared by reaction with BSTFA. They recommended performing the derivatization with a 225-fold molar excess of BSTFA at 150°C for 15 min in a closed vial and analysis on a 1 m X 4 mm I.D. column packed with 4% of OV-1 1 on Supelcoport (100-120 mesh) with temperature programming at 5'C/ min from 140°C. The conditions were similarly optimized for rnethylated bases and the whole method was applied to the analysis of these bases in urine [461]. The silylating reaction was pre-

TABLE 5.15 RETENTION INDICES OF TMS-PYRIMIDINE AND TMS-PURINE BASES [462 ] Conditions: Glass column, 2 m x 0.4 cm I.D., 3% of stationary phase on Chromosorb W HP (100-120 mesh, AW, DMCS-treated); carrier gas, argon at 60 ml/min; column temperature, 160 and 190°C for pyrimidine and purine bases, respectively. Compound

Uracil 5,6-Dihydrouracil 6-Azauracil Thymine 5,6-Dihydrothymine 6-Azathymine Cytosine 5-Methylcytosine

Hypoxanthine Adenine Xanthine Guanine Allopurinol

ov-101

OV-17

r160°C

r160°C

1325.5 1328.2 1449.9 1396.7 1395.9 1459.8 1509.6 1536.3

1448.4 1448.0 1573.9 1507.9 1508.0 16 15.4 1696.4 1706.9

122.9 119.8 124.0 111.2 112.1 155.6 186.8 170.6

I19OoC

1190°C

Al

1798 1849 2010 2106 1611.7

2037 2073 226 1 2298 1722.7

239 224 25 1 192 110

AI

INSECTICIDES AND OTHER PESTICIDES

111

ceded by a purification procedure aimed at the removal of salts and interfering components, involving hydrolysis of the urine sample with the aid of 1 NHCl (4 h at 1lO"C), fdtration and purification by adsorption on activated carbon and an ion exchanger. The dried sample was then silylated for 15 min by heating at 150°C with BSTFA-acetonitrile (1 : 1) in a closed test-tube. For separation a column packed with 5% of OV-3 with temperature programming at 7.5"Clmin from 90°C was more suitable. Pyrimidine and purine bases were also silylated with the aid of BSTFA because lower yields were obtained with HMDS-TMCS-pyridine (3 : 1 : 3) [462]. The following conditions were reported as optimal: heating for 15 min at 150°C in a sealed ampoule with a 100-fold molar excess of BSTFA. The derivatives were analysed on OV-101 and OV-17 stationary phases and retention data of some of them, expressed in the form of retention indices, are given in Table 5.15. 5.10.2. Other derivatives

Only a limited number of other derivatives have been tested for the GC analysis of the components of nucleic acids. Miles and Fales [463] blocked polar groups by combining acetyl, methyl and isopropylidene derivatives and used SE-30 and QF-1 as stationary phases. They obtained a good separation of various components and reproducible chromatograms; the peaks were asymmetric, however, and tailed badly. MacGee [464] determined the ratios of the bases in nucleic acids after methylation by decomposition of their tetramethylammonium salts. Nucleic acids were decomposed by hydrolysis by heating for 40 min at 100°C in 70% perchloric acid. After neutralization and purification on anion-exchange resin, the sample was dried and dissolved in 100 p1 of 1 M tetramethylammonium hydroxide in ethanol and 5 pl were injected (the temperature of the injection port was 360°C). The analysis, however, suffered from a non-quantitative reaction course and the formation of numerous products. Moreover, the same derivative may be produced by the methylation of different substances: xanthine, theobromine and theophylline give rise to caffeine after complete methylation.

5.1 1 . INSECTICIDES AND OTHER PESTICIDES This group of substances includes chemically very different compounds, such as ureas and carbamates, organic phosphorus and sulphur compounds, chlorinated hydrocarbons and chlorophenoxy acids and heterocyclic compounds. The wide range of applications of pesticides gave rise to a requirement for the analysis of their residues in various materials, particularly biological. As mainly trace analysis is concerned, GC has proved useful for this purpose and a number of pesticides have been analysed by GC even in the free state. The lability of some pesticides, caused by either poor column deactivation or their thermal instability, led to the preparation of derivatives prior to the analysis proper. The requirement for determining ultra-trace amounts of substances necessitated the preparation of specific derivatives. The identification of these substances by means of their derivatization also plays a significant role [465]. Depending on the type of functional groups in the molecule, common derivatives of these groups are applied. Another approach

178

DERNATIZATION OF COMPOUNDS

utilizes decomposition of the substances and, after derivatization, the products formed by using the procedures described in previous sections (e.g., amines and phenols from carbamates) are taken for analysis. 5.1 1.1. Carbamates and related compounds As carbamates often tend to decompose during GC analysis, prior to the analysis they have been modified by acetylation [466,467], silylation [468], alkylation [467] and acylation with fluorinated acylating agents [467,469-4711. Other procedures involve hydrolytic decomposition of carbamates and GC analysis of the phenols or amines so produced by procedures common for these substances.

Scheme 5.37.

Bowman and Beroza [472] hydrolysed carbamates by heating with 10% NaOH sohtion and condensed the phenol produced with chlorothiophosphate in the presence of pyridine (Scheme 5.37). The resulting derivative is highly specific for the compound under analysis and, with the use of a flame photometric detector, it permits its sensitive analysis (at the level of hundredths of 1 ppm in foodstuffs). A column packed with 10% of DC-200 was used for the analysis at 190°C. Holden et al. [473] also obtained a high sensitivity in the analysis of N-methyl- and dimethylcarbamates if they condensed amines released by alkaline hydrolysis with 1-fluoro-2,4-dinitrobenzene and applied an ECD for detection (2% of XE-60, 190°C). Different substituted anilines were similarly analysed as products from the decomposition of different pesticides [474] in the form of different perfluorinated acyl derivatives. Lawrence [475] and Ryan and Lawrence [476] chromatographed insecticidal carbamates and other agricultural chemicals in the form of perfluorinated acyl derivatives. Of these they recommended HFB derivatives as most suitable. They prepared them by treatment with HFB anhydride in benzene in the presence of trimethylamine, as follows. A 15-pl volume of HFB anhydride and 0.4 ml of 0.1 M trimethylamine in benzene were added to 1-10 pg of insecticide (dried extract of a food sample) in a 20-ml test-tube. The test-tube was closed, stirred and allowed to stand at room temperature for 30 min. The contents of the test-tube were mixed with 4.5 ml of benzene, 10 ml of distilled water were added and the test-tube was shaken vigorously. Aliquots of the benzene phase were analysed on 3% of OV-1 at 170°C. These derivatives were stable even towards the action of water and with the use of an ECD picogram amounts of carbamates could be analysed. An electrolytic conductivity detector, which was also applied to these analyses, permitted the analysis to be performed with a 10-100-fold lower sensitivity. In addition to hydrolysis into anilines, substituted phenylureas can also be converted,

INSECTICIDES AND OTHER PESTICIDES

179

prior to GC analysis, into methyl derivatives. Tanaka and Wien [477] applied a flashheater method: they injected the sample together with trimethylanilinium hydroxide (1 : 2.5) into the injection port heated at 320°C. A good separation was achieved on mixed silicone stationary phases. Another methylation method makes use of a reaction with iodomethane and a catalyst [478], as follows. About 30 mg of an oil dispersion of sodium hydride was washed three times with anhydrous diethyl ether, dried under a mild stream of nitrogen and suspended in 1 ml of freshly distilled dimethyl sulphoxide. A 200-p1 portion of this suspension was added to ca. 1 pmol of phenylurea dissolved in 100 pl of pure dimethyl sulphoxide followed immediately by 50 pl of iodomethane. Reaction was allowed to proceed for 5-10 min with continuous stirring. Then 1 ml of water was carefully added and the mixture was extracted with three 1-ml portions of diethyl ether or light petroleum. The extracts were dried with anhydrous sodium sulphate, filtered and evaporated t o dryness under a stream of nitrogen. Silicone stationary phases [OV-225 at 108°C or a mixture of DC-200 and QF-1 (1 : 3) a t 170°C] were used. The disadvantage of the methylation procedures is that some metabolites give, after methylation, the same derivative as the initial substituted urea and they therefore cannot be determined simultaneously. 5.1 1.2. Organophosphorusinsecticides

There are two main procedures for the derivatization of these,substances: (i) alkaline hydrolysis and derivatization of the products and (ii) derivatization of the intact insecticide. As the products from the hydrolysis are mostly derivatives of phosphorus acid and phenols, the former procedure involves the preparation of esters and ethers of these compounds. In the latter instance derivatives are applied, depending on interfering functional groups present, and further oxidation, reduction and other reactions. Esterification of the phosphorus product from the hydrolysis is performed with the aid of diazomethane and has been applied to 32 organophosphorus insecticides [4794811. The analysis was performed, e.g., on 20% Versamid 900 on Gas-Chrom Q . Substances that provide the same hydrolysis products with the use of this procedure cannot be resolved and confusion with organic phosphates can also occur. In order to avoid these difficulties, simultaneous conversion into methyl and ethyl esters is performed. If the other product from the hydrolysis is phenol, the procedures described in Section 5.1 are applied to the preparation of the derivatives, particularly those which permit a very sensitive analysis. Perfluorinated acyl derivatives and 2,4-dinitrophenyl-, 2,6-dinitro4-trifluoromethylphenyl and pentafluorophenyl ethers are frequently used [482], when concentrations of 0.01-0.1 ppb of the insecticide in water can be determined. Vilceanu et al. [483] performed acetylation of the free hydroxyl group of ( 1 -hydroxy2,2,2-trichloroethyl)dialkylesters of phosphoric acid prior to analysis, as unchanged original substances often decompose in the column. They prepared the derivatives by reaction with acetic anhydride in acetonitrile with pyridine as catalyst and performed the analysis on a column packed with Apiezon L (15% on Chronlosorb W) on which acetates showed good chromatographic properties. Reduction of substances containing nitroacyl or cyanoacyl groups into the correspond-

180

DERIVATIZATION OF COMPOUNDS

ing amines was carried out with the aid of CrCl?, PdClz and Zn-HCl as reducing agents, of which the last has been used the most frequently [484]. The amine produced was analysed as such or after additional derivatization. Singh and Lapointe [485] analysed six organophosphates with a P=S group after oxidation with a neutralized solution of sodium hypochlorite. The substrates were thus converted into 0x0 compounds and, with the aid of a flame photometric detector sensitive t o phosphorus, were determined in vegatables at levels of tenths of 1 pprn. Other derivatization procedures involve alkylation and silylation [486]. The latter is carried out with the aid of BSTFA and provides simple products, whereas alkylation with a methyl iodide-sodium hydride-dimethyl sulphoxide mixture (50"C, 10 min) leads to two peaks due to various products. 5.1 1.3. Organochlorine pesticides

These substances can usually be chromatographed directly even though some of them are sensitive to the activity of the column and derivatization is also applied, particularly for identification purposes. Pionke et al. [487] analysed organochlorine insecticides either free or after treatment with a 2% ethanolic solution of KOH or concentrated HC1 in ethanol. Different retention times of the original substances and the products on DC-200 and DEGS stationary phases were utilized for identification. Chau and Cochrane [488] refluxed organochlorine pesticides with potassium terr.-butoxide in [err.-butanol, where dehydrochlorination of cis- and trans-chlordane, isomerization of heptachlorepoxide and hydroxylation of heptachlor occurred. In combination with subsequent silylation or acetylation, all of the above-mentioned pesticides can be identified simultaneously at a level of 0.01 ppm. A mixture of 4% DC-11 and 6% QF-1 (1 : 1) on Chromosorb W was applied as the column packing. Phenolic groups of hexachlorophene were blocked by Ferry and McQueen [489] by reaction with diazomethane. The derivatives were analysed on 5% of QF-1 on Varaport.30 at 185"C, and with an ECD a sensitivity of down to units of picogram was achieved. 5.1 1.4. Other substances

Triazines can be chromatographed as such and their GC behaviour has been studied on several stationary phases [490]. Hydroxytriazines, which are the main metabolites of triazine herbicides, cannot be analysed directly and must be converted into more volatile substances by chlorination with the aid of PCls after prior separation of the residues of these substances in the sample [491]. The conversion is reported to vary in the range 75-85% at the nanogram level and the sensitivity is down to 0.03 ppm in foodstuffs with the use of the AFID. Silylation and alkylation are other methods of modifying triazines. An HMDS-TMCS mixture was used for the preparation of TMS derivatives [492]. Flint and Aue [493], who evaluated several reagents, recommended performing silylation with the aid of BSTFA by heating at 150°C for 15 min. Under these conditions, hydroxy derivatives of simazine, atrazine and propazine provided one large peak and few by-products, identified as mono-, di- and trisilyl derivatives; 10% of OV-17 was used as the stationary phase.

INSECTICIDES AND OTHER PESTICIDES

181

Alkylation gives fewer minor peaks and is performed, e.g., by treatment with methyl iodide in the presence of sodium hydride [494]. A good separation of the derivatives was obtained on 4% of SE-30 at 175°C and the method was applied to the analysis of triazine residues in foodstuffs at the level of 0.05 ppm. Acyl derivatives of triazines were used by Bailey et al. I4951 for their determination in potatoes, peas and tomatoes. Their preparation requires catalysis by trimethylamine or pyridine. A 15-pl volume of HFB anhydride was added to a dry residue of triazine (1-25 pg) in a 15-ml test-tube fitted with a screw-cap, 1.Oml of 0.5 M trimethylamine in benzene (or 1 ml of benzene plus 6 droplets of pyridine) was then added and the closed test-tube was stirred gently for 30 sec and then allowed to stand at room temperature for 30 min. Subsequently 4.0 ml of benzene were added and the contents were stirred for 1 min, then 10 ml of water were added and the contents were shaken vigorously for 1 min. The phases were separated and a suitable portion of the benzene phase was injected. Several stationary phases (OV-l,OV-101, OV-210 and a mixture of OV-101 and QF-I) were applied at temperatures of 180-200°C or with temperature programming. Exclusively mono-derivatives are said to be prepared by this procedure, with bis-derivatives in small amounts and for only some substrates. The sensitivity of the analysis using an ECD is from 300- to several thousand-fold higher that with free substances. Other procedures make use of the methoxylation [496], by which chlorine on a triazine ring is replaced with a methoxy group, basic hydrolysis of the side-chain and analysis of the released amine in the form of the dinitrophenyl derivative. Lau et al. [497] determined water-soluble cyanoazine metabolites quantitatively after simultaneous cleavage of the triazine ring and cyclization of the characteristic side-chain into 5 3 dimethylhydantoin. The latter was further modified by treatment with sulphenyl chloride to give trichloromethane. The whole procedure is highly specific for the group of substances concerned. Chlorophenoxy acids are chromatographed after esterification. Methyl esters [498] and propyl esters [499] prepared by transesterification from methyl esters by heating for 5 min with n-propanol and sulphuric acid are often used. For a very sensitive analysis, Mierzwa and Witek [500] proposed the following procedure. They esterified acids with 20%of 2,2,2-trichloroethanol in TFA anhydride in the presence of sulphuric acid by heating at 100°C for 15 min (or several hours at room temperature) and analysed the derivatives on a column packed with 15% of QF-1 plus 10% of DC-200 (1 : 1) at 195°C. Trichloroethyl esters permit down to hundredths of 1 ppb of chlorophenoxy acids to be TABLE 5.16 RELATIVE DETECTION LIMITS OF HALOETHYL ESTERS OF 4CHLORO-ZMETHYLPHENOXYACETIC ACID (MCPA) AND 2,4-DICHLOROPHENOXYACETICACID (2,4-D) WITH THE USE OF ECD [SO01 Ester

MCPA

2,4-D

2Chloroethyl 2,2,2-Trifluoroethyl 2,2,2-Trichloroethyl

100 41 1.2

100 66 12.5

182

DERIVATIZATION OF COMPOUNDS

determined in water; Table 5.16 compares the detection limits obtained for different haloethyl esters. De Beer et al. [Sol] reported an extensive comparative study of the chromatographic behaviour of methyl esters and pentafluorobenzyl esters of these substances. They correlated retention data (Kovats retention indices) on nine stationary phases with the structure of the derivatives and with the polarity of the stationary phases with the aim of utilizing these dependences for identification purposes. However, much more significant is the better separation obtained with pentafluorobenzyl esters and the possibility of increasing the sensitivity of the analysis. Phenolic and acidic pesticides mnd herbicides were chromatographed as chloromethyl and bromomethyldimethylsilyl derivatives [SO21 and the entire procedure was applied to the analysis of these substances in soil, as follows. A 1-ml volume of n-hexane, 0.075 ml of dimethylamine and 0.09 ml of halomethyldimethylchlorosilane were mixed in a 5-ml vial, which was then closed and shaken vigorously. The mixture was centrifuged and 0.4 ml of the supernatant was transferred into an 8-ml test tube and 0.1 ml of ethyl acetate containing 100 pg of the substrate was added. The contents were refluxed for 30 min at 6 5 ° C cooled and the reflux condenser was rinsed with 0.5 ml of n-hexane. The solution was diluted with n-hexane if necessary and chromatographed on the column with 10%of DC-200 under isothermal conditions at 110-200°C. With the aid of an ECD the method is said to be suitable for the determination of the compounds mentioned in the range of units to hundreds of nanograms. Residues of organomercury fungicides were determined by Tatton and Wagstaffe [503] after conversion into dithizonates. Having been extracted from the material under analysis, the substances are extracted from the aqueous phase into a 0.005%solution of dithizone in diethyl ether. After purification by column and thin-layer chromatography, the extract is analysed by GC. PEGS is a suitable stationary phase although tailing cannot be suppressed entirely. 5.12. PHARMACEUTICALS AND DRUGS

Gas chromatography has become widely used for the determination of these substances. Their conversion into derivatives prior to the analysis proper is often unavoidable as only a few of them can be analysed by GC as such. Selection of the derivatization procedure follows general rules depending on the type of functional groups present, and it includes the application of almost all of the reactions that are commonly used. Various substances active as medicaments or drugs differ much from the viewpoint of chemistry and several functional groups often occur in one molecule. In many instances individual procedures for particular substances differ only in the technique of preliminary isolation. Therefore, of numerous publications, the number of which has been rapidly increasing recently, this section will report only representative examples. Further details and information can be found in several reviews [504-5061 and a book [507]. 5.12.1. Barbiturates

Although some barbiturates can be subjected to GC analysis directly [508],strong adsorption often occurs in the column and peak tailing occurs, favouring the use of

PHARMACEUTICALS AND DRUGS

183

derivatives. Alkylation procedures have been applied most frequently. Methyl derivatives were used by Martin and Driscoll [509]; they were prepared by treatment with dimethyl sulphate and applied to the analysis of barbiturates in serum, as follows. A 2-ml volume of a saturated solution of sodium dihydrophosphate and 10 ml of diethyl ether were introduced into a test-tube containing 2 mi of serum. The test-tube was closed and shaken vigorously for 30 sec, centrifuged for 1 min, then 7 ml of the extract were transferred into another test-tube and the ether was removed in a stream of air in a water-bath. The residue was dissolved in 2 ml of methanol with 10% of water (v/v), saturated with potassium carbonate, and 0.1 ml of dimethyl sulphate were added and the mixture was heated in a water-bath at 60°C for 4 min. At this temperature methanol was removed in a stream of air (3 min) and the residue was extracted into 1.5 ml of n-heptane with the addition of 1 ml of 1 M acetate buffer (pH 6 ) . A 1-ml volume of the n-heptane extract was transferred into a small test-tube, evaporated carefully in order that losses of the derivatives might be avoided, and dissolved in 100 p1 of acetone (containing an internal standard if necessary); 5 pl were analysed on a column packed with 5% of SE-30 at 175°C. For the preparation of derivatives on a micro scale (at the microgram and picogram levels) reaction with methyl iodide was recommended [ 5 101: 100-200 pl of an acetone solution of barbituric acids was mixed with a 3- to 1000-fold molar excess of methyl iodide and refluxed with 3-5 mg of potassium carbonate. After about 30 min, 0.2 pl of the supernatant was injected directly into a column with 3% of OV-225, with temperature programming at 8"C/min from 100 to 240°C. The advantage of the method is that no toxic and hazardous chemicals are used. The method was further modified for the micro-determination of barbiturates in blood [51 11. Only 20-25 pl of blood were necessary for the determination, and with the use of a detector sensitive to nitrogen down to 1 ng of barbiturate could be determined in this sample. The derivatization reaction was performed in a micro-refluxer (see Fig. 2.6). The thermal decomposition of tetramethylammonium salts of barbiturates is a procedure used extensively for their methylation [ 5 12-5 151. Decomposition is performed at 240°C (temperature of the injection port) with analysis on SE-30, QF-1 and similar stationary phases. The use of trimethylanilinium hydroxide leads to better reproducibility of the preparation of the phenobarbital derivatives and to an improvement in the quantitative results [5 16,5171. The disadvantage of this procedure is the dependence of the course of the reaction on several parameters, e.g., geometry of the injection port, temperature. Butyl derivatives make possible the resolution of compounds the methylation of which lead to the same derivatives [518]. They were prepared in an analogous manner, by injection of substrates with a 25% methanolic solution of tetra-n-butylammonium hydroxide into the injection port heated at 270°C. The analysis can be performed on 3% OV-17 at 220°C; Fig. 5.31 illustrates an example of the separation of several barbiturates in the form of methyl and butyl derivatives. Mephobarbital and phenobarbital, which being methylated give rise to the same compound, could be resolved after their conversion into butyl derivatives; in addition, the different retention times of the derivatives could be utilized for the identification of barbiturates. Pentafluorobenzyl derivatives of barbiturates were chromatographed by Walle [5191.

184

DERIVATIZATION OF COMPOUNDS

TIME( MINI

Fig. 5.3 1. Gas chromatogram of barbiturates after flash-heater N-methylation (left) and N-butylation (right). Peaks: 1 = butabarbital; 2 = secobarbital; 3 = hexobarbital; 4 = mephobarbital;5 = phenobarbital; 6 = alphenal. Conditions: glass column, 6 ft. X 1/4 in. O.D., 3% OV-17 on Gas-Chrom Q (100-120 mesh); helium flow-rate, 60 ml/min, temperature, 22OoC. (Reproduced from J. Chromatogr., 76 (1973) 467.)

The reaction with pentafluorobenzyl bromide in the presence of an excess of triethylamine was quantitative. The derivatives possess good properties on OV-l,OV-l7 and WGSe stationary phases and provide high ECD responses. The minimal detectable amount of barbital diderivative is reported to be 0.05 pg. 5.12.2. Antibiotics

For the GC analysis of these substances trimethylsilyl derivatives are mostly prepared. Chloramphenicol is silylated with the aid of BSA [520,521]. Some workers [522], however, prefer an HMDS-TMCS mixture in pyridine as it is said to result in a more uniform product. The analysis is performed on silicone stationary phases of the SE-30 and OV-17 types. The related thiamphenicol is converted into the TMS derivative by treatment with BSA [523,524] and the same stationary phases are used for GC. If this method is applied to the determination of these substances in plasma and body fluids, and a selective detector is used (63Ni ECD), a sensitivity of determination of 0.1 pg/ml can be reached. Tsuji and Robertson analysed aminoglycoside antibiotics by GC of kanamycin, paromomycin and neomycin [525] and tetracyclines [526] after their silylation with a BSA-TMCS mixture in pyridine (1 : 1 : 2, v/v) or with commercially available Tri Sil Z reagent. The reaction was performed at room temperature for 24 h or at 75°C for 45 min. Good results with respect to peak symmetry and the separation of various compounds were obtained on silicone stationary phases at 260°C. Penicillins and related antibiotics were also modified by silylation prior to GC analysis [527]. A 50% (v/v) solution of HMDS in pyridine containing 0.375 mg/ml of 5-a-cholestane as an internal standard was used for the reaction. A 2-ml volume of a standard solution of penicillins (ca. 20 mglml), 8 ml of chloroform and 2 ml of a buffer (pH 2.2) were mixed, shaken vigorously for 1 min and centrifuged. A 2-ml aliquot of the organic phase was

PHARMACEUTICALS AND DRUGS

185

transferred into an 8-ml serum vial, 2 ml of organic reagent were added and the mixture was allowed to stand with intermittent stirring at room temperature. A 2-pl volume was analysed on 2% OV-17 at 245°C. The silylation was completed for penicillin G and V and D- and L-phenethicillin and -methicillin within 10 min, whereas oxacillin, cloxacillin and dicloxacillin required up to 60 min. Of the above series of penicillins two pairs had, however, similar retention times under the chromatographic conditions given. Naturally occurring antibiotics of the aglycone type were subjected to analysis by combined GC-MS as their pertrimethylsilyl methoxime derivatives [528]. Persilylation is performed by treatment for several minutes with BSTFA-TMCS-TMSIM (3 : 3 : 2) at room temperature. Preliminary methoximation is carried out by reaction with methoxylammonium chloride in pyridine at 75°C for 45 min. Some of the aglycones are methoximated incompletely, however, and provide two peaks for both derivatives. A column packed with 3% of OV-101 at 260°C was selected as the most suitable for GC. 5.12.3. Vitamins

Vitamins include compounds that differ considerably in their chemistry and therefore the range of possible derivatives is fairly wide. Vitamin A and related compounds were chromatographed in the form of acetates [529] on a column packed with 1 % of SE-30 at 150°C. Both retinol and retinyl acetate decomposed, however, on this column. This phenomenon can be suppressed substantially by treatment of the column with p-carotene. Vitamins of group B were analysed in different forms [530]. Isopropylidene derivatives showed selectivity of the chromatographic separation which was caused by even minor structural differences. Several compounds from the pyridoxine group can be analysed after their conversion into acetates; acetylation followed by GC also appeared suitable for three vitamins and 4-pyridoxic lactone. TMS derivatives were recommended for GC separation of the phosphate form of vitamins. When treated with BSTFA-pyridine (1 : 1) at 60°C for 15 min, biotin provides a completely silylated derivative, which was analysed on a column packed with 3% of OV-17 [531]. Ascorbic acid was silylated by means of HMDS and N-trimethylsilylacetamide [532]. Whereas the former reagent provided two products the concentrations of which decreased within a rather short period of time, the latter provided satisfactory results. With a 15-fold excess of the reagent a uniform derivative was produced at room temperature after reaction for 4 h and the results were reproducible. XE-60 and SE-30 silicone stationary phases were used at 135 and 170°C, respectively. Vitamin D and related compounds are derivatized as for compounds of steroidal character and by analogous procedures. HFB derivatives provide the possibility of analysis at the nanogram level [533]. Thermal instability of vitamin D and its derivatives is eliminated by prior conversion into isotachysterol. TMS derivatives were not stable, and reproducible results could be obtained only if the period of time between the preparation and the analysis of the derivatives was maintained constant [534] ;the most suitable column packing was OV-225 on Chromosorb W Hp. Walle et al. [535] determined calciferol in preparations containing an excess of vitamin A by means of their TMS ethers. De Leenheer and Cruyl [536] also carried out the derivatization in two steps: by reaction with 20% of

186

DERIVATIZATION OF COMPOUNDS

SbC13 in chloroform they converted the substrate into isotachysterol, and this was converted further into the methyl ether by treatment with methyl iodide in the presence of potassium rut-butoxide. The derivatives possessed good GC properties on SE-30,OV-1 and QF-1 stationary phases at temperatures from 220 to 265°C. 5.12.4. Alkaloids

In spite of the fact that a number of alkaloids can be analysed by GC in the free state, in some instances strong adsorption on the column and tailing peaks occur and derivatization is required [537]. Martin and Swinehardt [538] converted morphine and codeine into TMS derivatives by treatment with a TMCS-HMDS mixture in pyridine (1 : 1 : 3). GC of silyl derivatives instead of free bases permitted a lower temperature of the column to be used and more reproducible results to be obtained, particularly with morphine (one phenol and one hydroxyl group). Other workers [539] recommended 1% of TMCS in BSTFA as the silylating agent for the determination of morphine in biological fluids at the nanogram level. After extraction of the substrate from the sample and evaporation of the extractant, the silylation was performed by treatment for 20 min with 25 pl of the reagent at room temperature. Without the addition of TMCS, 30 min at 55°C were required. The problems associated with losses due to adsorption on the surfaces of glass vessels were solved by using polycarbonate test-tubes after evaporation of the extractant. Rasmussen [540] applied “on-column’’ silylation to the quantitative determination of morphine. A 1-p1 volume of a morphine solution in ethyl acetate (containing ca. 1 pg of the drug) and 2 pl of TMSIM were injected with temperatures of the injection port and of the column of 275 and 25OoC, respectively, using 3%of Dexsil300 as the stationary phase. It was stated that in this arrangement no free bases could be detected, i.e., the conversion was complete and the method provided reproducible results (the coefficient of variation was 1.3%). It was recommended preferrably for the rapid checking of pharmaceutical preparations. 5.12.5. Other pharmaceuticals

Anticholinergics, such as atropine and oxyphenonium bromide, which are esters of carboxylic acids, were analysed in plasma and urine as pentafluorobenzyl esters [541,542]. The method involves ion-pair extraction of the material under analysis, hydrolysis of esters and derivatization of the acid moiety. The minimal detectable amount was found to be 0.15 pg with the use of an ECD. Pentazocine, cyclazocine and related drugs, prior to analysis, were converted into pentafluorobenzyl ethers [543,544] or TMS derivatives [545]. The former make the determination of pentazocine possible at concentrations down to 1 ng/ml in human plasma. The method involves a several step extraction. Prostaglandins are preferably chromatographed in the form of their silyl derivatives, which are particularly suitable when profiling these substances in biological samples [506]. Sometimes they are combined with methyl esters [547] and n-butyl boronates [548 J with the aim of improving the separation and using a combination with MS. Albro and Fishbein [549] separated substrates into three fractions by column chromatography.

PHARMACEUTICALS AND DRUGS

187

They analysed B prostaglandins as acetylated methyl esters, and the others as TMS derivatives. Oxazepam and related compounds are converted by extractive alkylation into N,Odimethyl derivatives and are analysed on 3%of OV-225 at 250°C ISSO]. Another procedure involves the extraction of the sample with benzene, hydrolysis with a strong acid and acylation of the benzophenone amino derivative produced with TFA anhydride [55 11. Extractive alkylation is used to advantage also for other pharmaceuticals, especially for their determination in plasma, urine and other biological materials. By this procedure hydrochlorothiazide was analysed in plasma, blood corpuscules and urine [552], furosemide in plasma [553] and chlorthalidone and related drugs in plasma and other biological samples [554,555], Thermal decomposition of tetramethylammonium or trimethylanilinium salts was described for phenytoin methylation prior to its GC analysis in plasma [556] and diethylstilbestrol in biological fluids [557]. Usually a liquid or homogenized sample is extracted into toluene, the respective tetrasubstituted ammonium hydroxide is added and the liquid phase is injected at 260-290°C. Propranolol and related substances can be converted into TFA derivatives [558,559], which permit concentrations down to 10 ng/ml in plasma and similar samples to be analysed with the use of an ECD. The application of pentafluoropropionic anhydride [560] leads to a reduction in the time necessary for the analysis (acylation for 1 min at 7OoC) and enables higher sensitivity of the determination to be obtained. Silylation is often used for the preparation of the derivatives of pharmaceuticals. In addition to the above-mentioned examples, it was applied to the GC-MS of cambendazole [561], the TMS derivatives of which were prepared by heating for 30 min at 60°C with BSA in pyridine. Lauwereys and Vercruysse [562] analysed different hypnotica and carried out the silylation directly on the point of the needle in the “falling needle” injection system (see p. 22). In order to obtain good reproducibility of the method, they recommended conventional silylations as alternative procedures. Anticonvulsant drugs were also silylated with the aid of BSA and analysed on 5% of OV-101 [563]. Tolmetin, an anti-inflammatory drug, and its metabolites were chromatographed after esterification of the carboxyl group with diazomethane [564]. The extraction was performed with diethyl ether and was followed by purification and analysis on 3% of OV-l7.’The determination of pethidine in plasma [565] involves extraction, purification of the extract by partition chromatography and derivatization, which is performed by treatment with trichloroethyl formate in the presence of anhydrous sodium carbonate. The detection limit with an ECD was reported to be 5 pg. Antihistaminics, being tertiary amines, are analysed after reaction with an analogous reagent [566] ;with pentafluorobenzyl chloroformate a derivative is formed with a high ECD response and good GC properties, e.g., on OV-17. Hucker and Miller [567] subjected amitriptyline and other tertiary amines to exhaustive methylation and analysed the products from the reaction (Hoffman). Whereas the initial substances gave considerable tailing on 3% of QF-1, the derivatives provided sharp and symmetric peaks. 4,4‘-Diaminodiphenylsulphone and related drugs are chromatographed as 4,4‘-diiodo derivatives. The amino groups are diazotized by treatment with nitrous acid and substituted with iodine, and the resulting derivative provides a high ECD response [568].

188

DERIVATIZATIONOF COMPOUNDS

5.13. ANIONS OF MINERAL ACIDS The non-volatility of inorganic anions and efforts, initially unsuccessful, aimed at finding a universal derivative, have meant that the application of GC to the analysis of these substances is still a relatively new and far from thoroughly investigated discipline. In spite of this, a number of procedures have been reported for the GC of both groups of anions and individual anions; some of them have been summarized in reviews [569, 5701. With the development of existing procedures and new derivatives, the technique for the analysis of anions has been gaining in importance. The great advantages of its excellent sensitivity and specificity have been increasing with the development of detectors selective to particular elements present in inorganic anions, and therefore further developments in this direction may be expected. This section describes some procedures for the GC analysis of non-oxygenated and oxygen-containing anions; the GC of some substituted phosphates and sulphonic acids was discussed in preceding sections. 5.1 3.1. Non-oxygen-containinganions

The GC of halides is considered first. Fluorides react with trialkylchlorosilanes to form volatile trialkylfluorosilanes, which can be analysed by GC. Triethylchlorosilane [571,572] was used as a reagent for the analysis of fluorides in different materials; in teeth fluorides were determined after the treatment with trimethylchlorosilane [573], as follows. The sample was dissolved in 0.5 M perchloric acid and 5 p1 of the solution were transferred into a polyethylene test-tube coataining 50 fl of benzene in which 50 pg of trimethylchlorosilane and 65 ng of 2-methylbutane (internal standard) had been dissolved. The sample was stirred vigorously at 4°C for 20 min and 6-8 pl of the benzene layer were analysed (20% of DC-200/50,8OoC). With the use of an FID, less than 1 ng of fluoride could be determined in the sample. The simultaneous analysis of all halides is usually performed in the form of alkyl halides. MacGee and Allen [574] prepared tetramethyl-, tetraethyl- and tetrapropylammonium salts of halides on an ion-exchange resin. The corresponding alkyl derivatives and trialkylamine are produced by their thermal decomposition in the injection port (360°C). Methyl halides were analysed on Chromosorb 101 (Fig. 5.32). The yields of methyl iodide were, however, greater (86%)at higher reaction temperatures (up to 450°C). Tetraethyl- and tetrapropylammonium fluoride decompose with the formation of hydrogen fluoride. Matthews et al. [575] extracted an aqueous sample of halides with a solution of tetraheptylammonium carbonate in toluene with 10%(v/v) of undecanol. The quaternary ammonium salts of halides so produced were converted into 1-haloheptanes in the injection port at 225°C. Other procedures for the simultaneous determination of chlorides, bromides and iodides were suggested by Russel [576], He analysed 2-haloethanols produced by the reaction of ethylene oxide with HCl, HBr and HI. A sample of halides was converted into halic acids with the aid of a strongly acidic ion exchanger. Ethy1er.e oxide was allowed to pass through 10 ml of the acids cooled to 4°C for 15 min. In another procedure, 1 ml of liquid ethylene oxide was added to the acids. After 1 h, the sample was diluted to 15 ml and 1 p1 was analysed using 12% of EGS at 100°C. The method was illustrated on the

ANIONS OF MINERAL ACIDS

189

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c1

4

8

12 16 2 0 0 L TIME~MIN)

8

12 16 21

Fig. 5.32. Alkylation and GC of aqueous inorganic halides. A, authentic salts; B, solution prepared on a cation-exchange column. Peaks: 1 = methylfluoride; 2 = water; 3 = methyl chloride; 4 = methyl bromide; 5 = trimethylamine;6 = methyl iodide. Conditions: stainless-steel column, 10 ft. x 1/4 in. O.D., Chromosorb 101 (80-100 mesh); helium flow-rate, 75 ml/min; column temperature, 125°C; injection port temperature, 360°C; 6 pl of an aqueous solution of tetramethylammoniumhalides (0.25 M each) injected. (Reproduced from Anal. Chem., 42 (1970) 1672, by courtesy of J. MacCee and the American Chemical Society.)

example of the analysis of a 0.1 M solution of HC1, HBr and HI. Other methods were developed for various anions. Bromides were oxidized with permanganate and the bromine so produced reacted with cyclohexene to form 1,2dibromocyclohexane [577]. Similarly, iodides were analysed in milk as monoiodoacetone after oxidation with iodate and after reaction of the released iodine with acetone [578]. Pennington [579] utilized the same oxidation reaction for the analysis of iodates; the iodine released was analysed as such. Cyanides were chlorinated prior to analysis with chloramine-T and the cyanogen chloride so produced was subjected to GC [580]. Analogously, cyanides and isocyanates form cyanogen bromide with bromine water, which can be analysed by GC [581]. 5.13.2. Oxygen-containing anions

TMS esters are the derivatives of oxygen-containing anions that have been applied most frequently to GC analysis. They were first applied to the analysis of silicates [582]. Further, the method was developed for the analysis of five silicates in siliceous rocks [583], as follows. A 20-g amount of powdered sample was added to a mixture of 125 g of ice, 150 ml of concentrated HCl, 300 ml of 2-propanol and 200 ml of hexamethyldisilazane, which had been stirred for 1 h. The complete reaction mixture was then stirred for 16 h at room temperature. After filtration the silazane (upper) layer was separated, washed with water, mixed with 2.5 g of Amberlyst 15 for 1 h, filtered and stripped at 115°C. The liquid residue in the distillation flask was analysed on a column packed with SE-30 with temperature programming. The extension of the silylation reaction t o further anions, especially carbonate,

190

DERWATIZATION OF COMPOUNDS

2

4

6

8 10 TIME 1 MIN 1

12

14

Fig. 5.33. GC separation of TMS derivatives of seven anions. Peaks: 1 = phosphite; 2 = carbonate; 3 = oxalate; 4 = sulphate; 5 = vanadate; 6 = phosphate; 7 = arsenate. Conditions: glass column: 12 ft. x 0.25 in.O.D., 3% OV-17 on Chromosorb W (80-100 mesh, HP); helium flow-rate, 80 ml/min; temperature programme, 5"C/min, 70-150°C. (Reproduced from A n d . Chern., 43 (1971) 538, by courtesy of W.C. Butts and the American Chemical Society.)

oxalate, borate, phosphite, phosphate, arsenite, arsenate, sulphate and vanadate, was reported by Butts [584] and Butts and Rainey [585]. In order that better solubility might be obtained, ammonium anions were used for the reaction, which was carried out in dimethylformamide as follows. A sample (5-10 mg) of ammonium salts of anions was added to 200 p1 of dimethylformamide and the same amount of BSTFA and was allowed to react at room temperature overnight. Not having been present in the form of ammonium salts, anions were converted into that form with the aid of Dowex 50W-X8 (NH;) cation exchanger. An aliquot of the reaction mixture was injected directly into the chromatograph and analysed on 3% of OV-17 or 5% of SE-30. An example of the separation of seven anions is illustrated in Fig. 5.33. In addition to a better solubility, ammonium salts provide substantially better yields for some anions. Phosphates were analysed by Hashizume and Sasaki [586,587] in the form of their TMS esters, which were prepared by reaction with HMDS-TMCS in pyridine. Mono-, diand tributyl phosphates were silylated by the same reagent, but diethyl ether was used instead of pyridine [588]. The most powerful silylating agent for alkyl phosphonates is BSTFA with the addition of TMCS [589]. Phosphonate (1 mg) was dissolved in 0.1 ml of acetonitrile, 0.1 ml of BSTFA and 0.05 ml of TMCS. The derivatization was completed at room temperature after 15 min and an aliquot of the reaction mixture was injected directly into the chromatograph, 1% of SE-30 or OV-17 at 90-170°C was used as the stationary phase. Esterification with diazomethane [590-5931 is another possible derivatization procedure for substituted phosphates. However, it has been used for only a limited number of substrates. Some workers [592] obtained very good quantitative yields with it in comparison with other methods. Of other procedures, the GC determination of nitrates after their conversion into nitrobenzene will be mentioned [594]. A 5-ml volume of an aqueous solution of the sample was stirred vigorously with 5 ml of benzene, 15 ml of H2S04-H20 (3 : 1) were

191

CATIONS OF METALS

added and the mixture was stirred. After equalizing the pressure, the reaction flask was heated at 75°C for 5 min, then the mixture was again stirred and cooled to room temperature. The benzene solution was diluted to a suitable concentration, 2-nitrotoluene was added as an internal standard and the mixture was analysed on Apiezon M at 120°C. Soderquist et al. [595] analysed cacodylic acid [(CH3)2As02H] and applied several derivatization procedures. They concluded that the preparation of iododimethylarsine (see Scheme 5.38) was more suitable than silylation, which was not reproducible, or cyanoethylation, which was too slow. Iodomethylarsine provides a high ECD response, which makes it possible to detect nanogram amounts and it is directly extractable into n-hexane and sufficiently volatile. (CH3I2AsO2H

+

3 HI

-

(CH312Asl

t

2 H20

+

12

Scheme 5.38.

Srinivasan et al. [596] determined sulphates indirectly. They converted them on Dowex 50 (H') into the acid, which formed a stable salt with n-butylamine. After the removal of excess of amine, n-butylammonium sulphate was decomposed by treatment with a lye, and the butylamine released was subjected to analysis on Chromosorb 103 at 145°C.

5.14. CATIONS OF METALS

After conversion into a suitable volatile compound, inorganic cations can be analysed by GC. Except for several specific derivatives, volatile halides of metals are often analysed. but volatile complexes of metals with chelate-forming reagents are the most significant. All of these techniques have been applied to the analysis of a limited number of cations. Although their significance keeps increasing as a result of further developments, they can compete with other analytical techniques in only a few instances, e.g., sensitivity of the analysis and the amount of the sample required for the analysis. With beryllium, GC is for these reasons a preferred technique. A significant part is also played by the selectivity of GC, whose importance has also increased with the development of detectors and combined techniques. Of a number of reviews dealing with the theme of this section in detail, we can cite the book by Moshier and Sievers [597] and the review by Uden and Henderson [S98]. 5.14.1. Metal halides and other derivatives

The GC determination of metal halides is complicated by the instability in chromatographic systems and different analytical solutions. Fluorides and chlorides are the halides that are used most often for different groups of metals; the former are more volatile, but they also tend to be more reactive. The GC analysis of metal chlorides and fluorides necessitates highly inert column packings and inert chromatographic accessories, particularly the injection port, material of the column and the detector. Conversion of metal ions into halides involves different halogenation techniques. Direct reactions of metals

192

DERIVATIZATION OF COMPOUNDS '

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,

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8

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8

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S$i

ww LLLZ

6 - , , I ,

0

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8 10 12 14 16 18 20 22 24 26 28 30 32 34 TIMEIMIN)

Fig. 5.34.GC separation of metal fluorides. Peaks: 1 = fluorine plus reaction products; 2 = impurity in rhenium; 3 = WF6; 4 = ReF6; 5 = OsFg; 6 = ReOF5. Conditions: PTFE column, 22 ft. x 1/4in. O.D., 15% Kel-I: No. 10 oil on Chromosorb T (40-60 mesh); helium flow-rate, 28 ml/min; temperature, 75°C. (Reproduced from Anal. Chern., 38 (1966) 1860,by courtesy of R.S. Juvet and the American Chemical Society.)

with gaseous chlorine or hydrogen chloride and reactions of metal oxides with chlorinated hydrocarbons at increased temperatures have been used for chlorination. Phillips and Timms (5991 described a less general method. They converted germanium and silicon in alloys into hydrides and further into chlorides by contact with gold trichloride. They performed GC on a column packed with 13% of silicone 702 on Celite with the use of a gas-density balance for detection. Juvet and Fischer (6001 developed a special reactor coupled directly to the chromatographic column, in which they fluorinated metals in alloys, carbides, oxides, sulphides and salts. In these samples, they determined quantitatively uranium, sulphur, selenium, technetium, tungsten, molybdenum, rhenium, silicon, boron, osmium, vanadium, iridium and platinum as fluorides. They performed the analysis on a PTFE column packed with 15% of Kel-F oil No. 10 on Chromosorb T. Prior to analysis the column was conditioned with fluorine and chlorine trifluoride in order to remove moisture and reactive organic compounds. The thermal conductivity detector was equipped with nickel-coated filaments resistant to corrosion with metal fluorides. Fig. 5.34 illustrates the analysis of tungsten, rhenium and osmium fluorides by this method. Sie et al. [601-6031 analysed silicon and tin in different alloys and steel samples. A sample of the material (1-50 mg) was heated at 600-900°C in a quartz tube, w h c h was then washed with chlorine. Chlorides were trapped in a colum packed with 15% of Kel-F 40 on Haloport F and analysed on the same column at 75°C. With the use of a gas-density balance and PTFE-coated filaments a sensitivity of 50 ppm was obtained for silicon. The analysis time of 15-20 min can be reduced to 10 min. Germanium was determined in coal dust after conversion into the chloride [604] by treatment with hydrogen chloride at a high temperature on a mixture of coal powder and Chromosorb (1 : l), wetted with sulphuric acid. The analysis of a sample of 1.5 g was reported having a sensitivity of 3.3 ppm of germanium with an error of ?6%. Titanium was analysed in a mixture of oxides by Sievers et al. [605]. The tetrachloride was produced hy reaction with tetrachloromethane at increased temperature. The reagents were sealed in a small capillary, heated, and the capillary was crushed in a modifed injection port of the chromatograph. A stainless-steel column packed with 15% of Histowax on Gas Pak F at 77°C was used. The analysis of standard samples led to a relative error of 1.1%.

CATIONS OF METALS

193

Uranium can be analysed as the hexafluoride, but the procedure requires modification of the chromatographic apparatus, nickel coating of metallic parts and nickel filaments in the katharometer [606]. Tin in zirconium-tin alloys can be analysed as the chloride, prepared by treatment with chlorine [607]. Selenium and tellurium are converted into fluorides by treatment of their oxides with xenon difluoride [608J . Mercury, prior to the GC analysis, is converted by treatment with different organic reagents into organomercury compounds, particularly phenyl- and methylmercury. Jones and Nickless [609] determined mercury in different media after reaction with benzene sulphinate. The phenylmercury produced was extracted into toluene as the chloride and aliquots of the toluene layer were subjected to analysis at 185°C on a column packed with 5% of EGA polyester on Supasorb. A detection limit of 2 . lo-'' g of chloride was achieved with an ECD, and mercury was determined in the concentration range 0.05-50 ppm. The same authors [610] also utilized an organosilicon salt, sodium 2,2'-dimethyl2-silapentan-5-sulphonate,in order to convert organic mercury into a derivative that could be subjected to chromatography. The reagent was stable in strong acids and at high temperatures, and it was possible to determine quantitatively 0.003-10 ppm of mercury. The method was demonstrated satisfactorily in the determination of mercury in wetashed fish and sediment samples. Zarnegar and Mushak [611] used different organometallic reagents for the determination of mercury in various samples. They obtained the best results with methyl(pentacyano)cobalt(III) and tetraphenylboron(III), which gave rise to methyl- and phenylmercury, respectively. The detection limit for mercury as the corresponding organomercury was 10-50 ppb, e.g., in blood, urine and tissue homogenates.

Scheme 5.39.

Selenium forms a volatile derivative, piazselenol, which can be subjected to GC analysis (Scheme 5.39). Young and Christian [6 121 treated selenium with 2,3-diaminonaphthalene at pH 2.0 and extracted the resulting piazselenol into n-hexane. With the use of an ECD, down t o 5 * lo-'' g of selenium could be detected. The procedure, applied t o the analysis of selenium in human blood, urine and river water, led to results equivalent to those obtained by neutron activation analysis. Similarly, Nakashima and Tbei [613] performed the reaction of selenium (as selenious acid) with 4-chlorou-phenylenediamine at pH 1 and extracted the derivative into toluene. They reported a detection limit of 0.04 pg. Shimoishi [614] analysed the content of selenium in metallic tellurium by this method. The sample was dissolved in aqua regia, followed by reaction with 4-nitro-ophenylenediamine and extraction into toluene. Down to 10 ng of selenium could be determined using only a few milligrams of sample. Common ions did not interfere even when present in a large excess. Selenium in marine water was determined after the same derivatization step [615]. Ruthenium was determined as thiosemicarbazide [6 161. After a preliminary separation on a thin layer and extraction from the layer with ethanol-pyridine, the complex. decomposed reproducibly in the injection port of the chromatograph. Ruthenium was deter-

194

DERIVATIZATION OF COMPOUNDS

mined quantitatively by this procedure in the range 0.2-2 ng. Arsenic was analysed chromatographically by Schwedt and Russel [6171.It was first extracted from biological material into chloroform as the dithiocarbamate, which was converted into triphenylarsine(II1) by the treatment with phenyl-Grignard reagent. In this form it was subjected to GC analysis with the use of an FID. Concentrations of arsenic down to 2 pg per gram of biological sample was measured. 5.14.2. Metal chelates

Compounds of the 0-diketone type react with complex-forming cations to form volatile chelates that can be analysed by GC. Acetylacetone is commonly used as a chelateforming agent, and for sensitive analysis using the ECD its fluorinated derivatives. All of these substances have an active hydrogen atom on the methylene group, the acidities of which are expressed as pK, values: 8.9,6.7 and 4.6 for acetylacetone and the 1,I ,1 -trifluoro and hexafluoro derivatives, respectively. The acidic hydrogen is substituted with the corresponding metal cation and another coordination bond is formed with oxygen from the other 0x0 group, and a stable six-membered ring is thus formed. With four coordination bonds, two molecules of the diketone contribute to the chelate formation (Scheme 5.40).Other reagents used include 2,2-dimethyl-6,6,6-trifluoroheptane-3,5-dione, 2,2,6,6-tetramethylheptane-3,5-dione and other highly fluorinated ligands, e.g., heptafluorodimethyloctanedione, decafluoroheptanedione and dodecafluorooctanedione. Amino and thio analogues of these compounds, i.e., 0-ketoamines and thio-0-diketones, have also been used as chelate-forming agents.

Scheme 5.40.

Aluminium was analysed in a mixture with gallium and indium [618].An aqueous solution of these ions was buffered with acetate at pH 4-7 and extracted into benzene in the presence of trifluoroacetylacetone as a chelate-forming reagent for 4 h. The analysis was performed on 5% of DC-550on silanized glass beads. At the milligram level of aluminium, the extraction yields obtained were 71-100%. From an aqueous medium of the acetate buffer, aluminium, copper and iron can be extracted at pH 4.5-5.5 with the aid of 1 M trifluoroacetylacetone in chloroform [619].At the 0.5 d l e v e l , a yield of 99.7% of aluminium was obtained. Scribner et al. [620]determined aluminium in nickel-copper alloys by using this method. Other workers [621]analysed alloys for their contents of aluminium, copper and iron. Aluminium and iron could be detected simultaneously on a column packed with 12% of Tissuemat E on Gas Pack F at 105"C,whereas copper necessitated a mod-

CATIONS OF METALS

195

ified procedure. Miyazaki and Kaneko [622] analysed aluminium in mouse liver at the nanogram level by this means. Wet ashing of the liver sample with a mixture of sulphuric and nitric acids was followed by neutralization of the sample, adjustment of the pH to 4.5 with buffer and extraction into a benzene solution (0.5%) of trifluoroacetylacetone. A glass column packed with 3% of OV-17 on Chromosorb G and an ECD were utilized. Of other applications, Genty et al. [623] analysed aluminium and chromium in uranium. Ions were extracted from a solution of uranyl nitrate with a 0.1 M benzene solution of trifluoroacetylacetone. The extraction efficiency was 100%at pH 5 after 1 h. A glass column packed with glass beads coated with 0.2% of DC-710 was recommended. A detection limit of 0.1 ppm was obtained with the use of an ECD. Lee and Burrell [624] applied the method to the determination of aluminium in sea water. Water samples (30 ml) were extracted with a 0.1 M solution of trifluoroacetylacetone in toluene and a detection limit at the picogram level for aluminium was obtained with the use of an ECD. A mixture of DC-710 and Carbowax 20M was used as the stationary phase. Minear and Palesh [625] published an extensive study on the GC analysis of aluminium and other metals (beryllium, chromium, copper) in water of various origins. With the same chelate-forming reagent and an ECD ('H), they achieved a sensitivity of 10-6-10-7 g/ml. A column packed with 5% of QF-I on Varaport 30 or 15% of Carbowax 20M on Chromosorb W made it possible to analyse aluminium, copper and chromium, but not iron. Sakamoto et al. [626] demonstrated the application of the microwave plasma detector to the analysis of trace amounts of aluminium and copper in zinc by GC in the form of their volatile chelates. A column packed with 0.5% of SE-30 on glass beads was used at 140°C. With the same chelates as above, the detection limit was 1 ng for copper and 0.5 ng for aluminium. Beryllium was analysed by GC as a volatile chelate the most frequently of all elements. A rapid micro-analytical procedure for the determination of beryllium in biological fluids was developed and published by Black and Sievers [627], who devoted their attention to urine, blood, liver homogenates and plant extracts. The sample in a,sealed glass ampoule was treated directly with trifluoroacetylacetone, by which means the losses of the sample or possible contamination that occur in conventional ashing procedures were eliminated. A sensitivity of lo-'' g of beryllium in a 50-pl sample was reported for a PTFE column packed with 5% of SE-52 on Gas-Chrom Z and an ECD. The average recovery from the samples of all types was 93.5% and no interference of common cations and anions was observed. The determination of beryllium contents in blood, urine, body organs, foodstuffs and sewage was dealt with by Kaiser et al. [628]. For the preparation of chelates, they applied both a direct reaction method and a preliminary ashing technique. The latter was performed by treatment with nitric acid or a mixture of nitric and hydrofluoric acids, and also by thermal decomposition using oxygen at low temperatures. The minimal detectable amount of beryllium was lo-" g in samples smaller than 1 g with the use of a 63Ni ECD. The reproducibility of the results was significantly better for the direct reaction method than for the conventional ashing method. A glass column packed with 5% of SE-52 on Gas-Chrom Z was used for the analysis. In environmental analyses, Ross and Sievers [629] analysed water and solid particles trapped on air sampling filters and for the detection of beryllium chelates used on ECD

196

DERIVATIZATION OF COMPOUNDS

and mass spectrometry. The strips of air filter were mineralized by using a lowtemperature asher, the residue was digested with a strong acid and the resulting solution was adjusted to pH 5.5-6.0. Further treatment of the sample involved extraction into a dilute solution (0.164 M) of trifluoroacetylacetone in benzene. Interfering ions were complexed with EDTA. By using a glass column packed with 2.8%of W-98 silicone on Diatoport S at 1 1O"C, a relative standard deviation of 3.0% was obtained. Wolf et al. [630] reported that with the use of MS for the detection, the analysis of picogram amounts of chromium and beryllium could be performed. Foreman et al. [631] compared the direct method of the chelate formation with the preliminary ashing method for the analysis of beryllium in rat urine. A detailed study showed that both of the methods are satisfactory, whereas testing of column material and packings showed the best results for a PTFE column packed with SE-52. Down to 1 ng/ml of the element could be detected in urine with the use of an ECD and EDTA as a masking reagent and a 0.05 M benzene solution of trifluoroacetylacetone. Beryllium in lunar, meteorite and terrestrial samples was determined successfully by Eisentraut et al. [632]. The sample was pulverized and fused with sodium carbonate, dissolved in dilute hydrochloric acid and transferred into a polyethylene bottle. After adjusting the pH to about 4, it was further adjusted to 5.0 with acetate buffer, both EDTA and trifluoroacetylacetone in benzene were added and the mixture was heated briefly at 95°C. A PTFE column packed with 10% of SE-30 on Gas-Chrom Z was used and a sensitivity of 4 . g of beryllium was reported for the measurement of peak heights with the use of a tritium-foil ECD. Chromium was measured in steels with high and low contents of carbon by Ross and Sievers [633]. A small amount of the sample ( 2 - 4 q) was allowed to react directly with trifluoroacetylacetone in the presence of nitric acid. If an undissolved residue occurred, it was dissolved in 70% nitric acid, evaporated to dryness and the procedure was repeated. The resulting red solution was extracted with benzene and excess of the chelating agent was removed by extraction with dilute sodium hydroxide solution. A PTFE column packed with 15% of SE-52 on Anakrom ABS and an ECD were used. The results showed a relative error of 1.4-1.7%. The determination of chromium in biological samples is made difficult by its very low concentrations. Hansen et al. [634] applied the method of direct reaction of series of fluorinated diketones to blood and plasma: the sample was heated in a sealed ampoule with an n-hexane solution of a chelate-forming reagent. A recovery of up to 98% was obtained with trifluoroacetylacetone, whereas hexefluoroacetylacetone and heptafluorodimethyloctanedione gave recoveries of less than 50%. The possibility was reported of determining chromium concentrations down to 5 ng/ml with the use of an ECD. A glass column packed with 5% of Dow Corning LS on Gas-Chrom P served well for the analyses. Savory et al. [635] determined chromium in serum. The sample was wet ashed by treatment with sulphuric, nitric and perchloric acids. Finally, the pH was adjusted to 6.0 with a buffer and chelation was effected with a dilute solution of trifluoroacetylacetone at 70°C. The analysis was carried out on 5% of QF-1 on Chromosorb W with the use of a 63Ni ECD. Booth and Darby [636] applied a very similar procedure to soft tissues and serum. They performed the chelation reaction also at 70°C for 1 h. Recoveries of chromium in serum and liver homogenates were 94 and 88-104%, respectively. Ross and Shafik

CATIONS OF METALS

197

[637] proceeded in the same way when analysing chromium in urine. They used a flame photometric detector with a 425.4-nm filter, which is highly specific for chromium. The sensitivity of detection was also sufficient for the analysis of biological samples and permitted amounts of chromium of units of nanograms to be determined. Wolf [ 6 3 8 ] reported on the combination of gas chromatography with atomic-absorption spectrometry. He led the column effluent directly into the burner of a commercial apparatus and achieved a detection limit of 1 ng. In addition to the above procedures by which copper was analysed simultaneously with other metals, GC has been applied to the determination of copper in drinking water and nickel-copper alloys [639]. In the latter instance copper is chromatographed as a bis(acetylpivaly1)ethylenediimine complex. For the determination of copper in alloys, the samples are dissolved in usual way, the pH is adjusted to 6-7 and chelating extraction g of is performed with an n-hexane solution of 0-ketoamine. Using an FID, down to copper could be determined. Uden et al. [640] investigated the contents of copper in the livers, kidneys and lungs of the animals fed with this element, by using butylenediamine derivative of trifluoroacetylacetone. The best chromatographic properties were obtained with 2% of Dexsil300 GC on Chromosorb 750. They concluded that GC provided poorer results for copper than atomic-absorption spectrometry. Barret et al. [641] studied the utilization of monothiotrifluoroacetylacetone for the analysis of nickel in alloys, tea and fats. The sample of the alloy was dissolved in 6 M HCl and HN03, the pH was adjusted with a buffer, then ammonia was added and the resulting solution was extracted with a dilute solution of the chelating reagent in n-hexane. The procedure for tea samples involved both wet and dry ashing. In the former instance the final residue was dissolved in dilute HN03 and in the latter in deionized water. Fats were first dried on a hot-plate and then ashed to 540°C. The following procedure was the same as that for alloys. A PTFE column packed with silicone E-350 on Universal B with temperature programming from 140 to 170°C and a tritium ECD were used for the analysis. As copper interfered significantly in the determination, it must be removed by preliminary treatment with hydrogen disulphide. Lead has been determined chromatographically in the form of its chelates with different 0-diketones [642]. A column packed with 15% of Apiezon L at temperatures around 200°C appeared to be the most suitable, but even with its use adsorption and peak tailing of chelates occurred. In combination with MS and using integrated ion-current curves g of lead. the detection limit was about Cobalt was determined in vitamin B I Z ,liver, blood and urine after chelation with heptafluorodimethyloctanedione [643]. The sample was rendered alkaline and, after the addition of hydrogen peroxide, was heated with a benzene solution of the chelating agent in a sealed test-tube at 75°C. After the removal of excess of the chelating agent by washing with alkali, aliquots were injected into a chromatograph equipped with an ECD. The detection limit was reported to be 4.4 . lo-" g of cobalt. Iron was determined in ores by Sievers et al. [644] with the use of the same chelating agent. Less than 1 mg of the sample was treated with the reagent in a sealed capillary, which was then crushed in a modified injection port of a gas chromatograph. A PTFE column packed with 10%of SE-30 on Gas-Chrom Z and a TCD were used. Quantitative data obtained by GC accorded well with those obtained by other methods.

198

DERNATIZATION OF COMPOUNDS

Uranium and thorium can be determined by GC in aqueous solutions as ternary complexes with hexafluoroacetylacetone and dibutyl sulphoxide [645]. The extraction was performed with a solution of both reagents at concentrations 2-5-fold higher than the concentration of the ions. The best separation was obtained on a column packed with 17.8% of QF-1 on Chromosorb W.With a TCD, the detection limits were 0.4 mg/ml of thorium and 0.6 mg/ml of uranium. Lanthanides of the yttrium group and tin were investigated by Burgett and Fritz [646] with the use of decafluoroheptane-3,5-dioneand dibutyl sulphoxide. An aqueous solution of lanthanides of pH 5.5 was extracted with a cyclohexane solution of both reagents in an amount three times higher than the total contents of the metals. Under these conditions, almost quantitative extraction of these elements was achieved.

5.1 5. MISCELLANEOUS

This section describes the derivatization procedures for those compounds which do not fit well into the preceding schemes, particularly heterocyclics, epoxides and iodine. Substituted pteridines were silylated by Lloyd et al. [647] with the aid of BSA and identified in biological material of different type? by combined GC-MS. The silylation was completed after 2 h at room temperature, and using this method down to 1 pg of pteridines could be identified. The analysis was performed on 1% OV-17 with temperature programming at 1S"C/min from 180 to 250°C. Pyridine and quinoline bases were analysed by Durbin and Zlatkis [648] after hydrogenation and acylation of the saturated products, as follows. Dry hydrogen chloride was bubbled through an anhydrous ethereal solution containing 0.5 g of nitrogen bases. The precipitate was dissolved in diethyl ether and the ether was then removed in a stream of nitrogen. The salt was dissolved in 15 ml of glacial acetic acid and hydrogenated with the use of Adams catalyst for 4 h under 3.5 atm of hydrogen. The pH was adjusted to 12 with the aid of sodium hydroxide and the solution was filtered and extracted with diethyl ether. The ethereal layer was washed and dried over MgS04 and the bases were acylated by reaction with an excess of pentafluoropropionic anhydride. The solution was neutralized with NaHC03, extracted with diethyl ether and the extract was dried and chromatographed. Capillary columns coated with GE SE-96 or Igepal CO-880 silicone stationary phases were applied at temperatures of 100-1 50°C and symmetric peaks were obtained. Epoxyglycerides react with ketones in the presence of boron trifluoride to give 1,3-dioxoIane derivatives. In this form they were analysed by GC on a column packed with 3% of OV-1 with temperature programming at 4"C/min from 260°C 16491. Cyclopentanone was recommended as the most suitable reagent, and acetone, methyl ethyl ketone, methyl isobutyl ketone and others can also be used. 1,2-Epoxyalkanes can be analysed directly but the quantitative results suffer from the thermal instability of these compounds and possible changes during the process [650]. Derivatization was performed by treatment with an alcohol in the presence of boron trifluoride and the analysis was carried out on the hydroxymethyl ethers or by reduction

MISCELLANEOUS

199

with lithium aluminium hydride with subsequent analysis of the alcohol produced. The latter procedure is more complicated and time consuming, but it is preferred for some systems, e.g., epoxides containing tertiary carbon. Free epoxides were analysed on a column packed with 5% of OV-22 and the derivatives on 5% of FFAP. McDonough and George [65 1] also chromatographed epoxides directly on four stationary phases: Apiezon L, cyclohexanedimethanol succinate, Carbowax 20M and DEGS. They' studied structural differences of olefins, particularly the contents of cis- and trans-isomers. Oxiranes produced from cis-isomers after stereospecific epoxidation of olefins usually possessed longer retention times than the corresponding trans-isomers. Iodine was determined by GC by Hasty [652]. He carried out the conversion into iodoacetone by reaction with an aqueous solution of acetone (0.5 M) and sulphuric acid (0.5 M) for 30 min. The reaction mixture was then extracted with n-hexane and aliquots were analysed on a column packed with 5% of SE-30 on Varaport. By using an ECD, iodine concentrations down to 1 pg/ml in an aqueous sample could be determined. The sensitivity could be increased by usingother ketones, e.g.,2-butanone or 2-pentanone [653]. The products from the reaction with these ketones provide higher ECD response and are more extractable with n-hexane. The utilization of:these properties makes it possible t o extend the determination of iodine down to concentrations of nanograms per millilitre. Grys [654] studied this method in more detail and applied it to the determination of iodine, present as a free element, iodide and firmly bound iodine in milk. A 0.4-ml volume of fresh milk was placed in a 35-ml Kjehldahl swan-necked flask, mixed with 1 .O ml of 2 M potassium carbonate solution, dried at 105°C overnight and then ashed at 600-610°C for 2 h. The next day the white residue was dissolved in 2 ml of 2 M HCl, diluted with 4 ml of distilled water and mixed with 0.5 ml of 5 M sulphuric acid, 0.5 ml of 5 M acetone and 0.05 ml of potassium iodate solution (concentration 100 ppm). After 30 min, the iodoacetone produced was extracted with 5 ml of n-hexane, the organic layer was decanted and 0.2-4 aliquots were subjected to analysis. It was verified that the column packed with 5% of 1,s-pentanediol succinate at 125°C provided better results than a column packed with SE-30. The total amount of iodine present in the sample in all forms was thus determined. If bromide-broniate reagent with 2 N acetic acid and 0.05 N sulphuric acid was applied to the liberation of bound iodine, the result did not include tightly bound iodine, which can be released only by wet or dry ashing. With the omission of this step from the procedure, a result was obtained which represented iodide and free iodine only; without the addition of iodate, free iodine only was determined.

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DERIVATIZATION OF COMPOUNDS

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58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78

202 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161

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204 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264

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561 W.J.A. VandenHeuvel, R.P. Buhs, J.R.Car1iqT.A. Jacob, F.R.Koniuszy, J.L. Smith, N.R. Trenner, R.W. Walker, D.E. Wolf and F.J. Wolf, Anal. Chem., 44 (1972) 14. 562 M. Lauwereys and A. Vercruysse, Chromatographia, 9 (1976) 520. 563 J.R. Watson, R.C. Lawrence and E.G. Lovering,.!. Pharm. Sci., 67 (1978) 950. 564 M.L. Selley, J. Thomas and E.J. Triggs, J. Chromatogr., 94 (1974) 143. 565 P. Hartvig, K.E. Karlsson, L. Johansson and C. Lindberg, J. Chromatogr., 121 (1976) 235. 566 L.A. Sternson and A.D. Cooper, J. Chromatogr., 150 (1978) 257. 567 H.B. Hucker and J.K. Miller, J. Chromatogr., 32 (1968) 408. 568 H.P. Burchfield, E.E. Storrs, R.J. Wheeler, V.K. Bhat and L.L. Green, Anal. Chem., 45 (1973) 916. 569 J.A. Rodrigues-Vazques, Anal. Chim. Aeta, 73 (1974) 1. 570 B.I. Anvaer and Y.S. Drugov, Zh. Anal. Khim., 26 (1971) 1180. 571 R. Bock and S. Strecker, Z . Anal. Chem., 266 (1973) 110. 572 K. Ranfft, Z. Anal. Chem., 269 (1974) 18. 573 E.C. Munksgaard and C. Bruun, Arch. Oral Biol., 18 (1973) 735. 574 J. MacGee and K.G. Allen, Anal. Chem., 42 (1970) 1672. 575 D.R. Matthews, W.D. Shultsand J.A. Dean,Anal. Lett., 6 (1973)513. 576 H.A. Russel, Angew. Chem., 9 (1970) 374. 577 A.W. Archer, Analyst (London), 97 (1972) 428. 578 S. Grys, J. Chromatogr., 100 (1974) 43. 579 S.N. Pennington, J. Chromatogr., 36 (1968) 400. 580 J.C. Valentour, V. Aggarwal and 1. Sunshine, Anal. Chem., 46 (1974) 924. 581 G. Nota and R. Palombari, J. Chromatogr., 84 (1973) 37. 582 C.W. Lentz, Inorg. Chem., 3 (1964) 574. 583 E.F.H. Wu, J. Gotz, W.D. Jamieson and C.R. Masson, J. Chromatogr., 48 (1970) 515. 584 W.C. Butts, J. Chromatogr. Sci., 8 (1970) 474. 585 W.C. Butts and W.T. Rainey, Anal. Chem., 43 (1971) 538. 586 T. Hashizume and Y. Sasaki, Anal. Biochem., 23 (1967) 316. 587 T. Hashizume and Y. Sasaki, Anal. Biochem., 24 (1968) 232. 588 J.W. Boyden and M. Clift, Z. Anal. Chem., 256 (1971) 351. 589 D.J. Harvey and M.G. Homing, J. Chromatogr., 79 (1973) 65. 590 C.J. Hardy, J. Chromatogr., 13 (1964) 372. 591 A.D. Horton, J. Chromatogr. Sci., 10 (1972) 125. 592 J.M.H. Daemen and W. Dankelman, J. Chromatogr., 78 (1973) 281. 593 A. Brignocchi, Anal. Lett., 6 (1973) 523. 594 D.J. Glover and J.C. Hoffsommer, J. Chromatogr., 94 (1974) 334. 595 C.J. Soderquist, D.G. Crosby and J.B. Bowers, Anal. Chem., 46 (1974) 155. 596 S.R. Srinivasan, B. Radhakrishnamurthy and E.R. Dalferes, Jr., Anal. Biochem., 35 (1970) 398. 597 R.W. Moshier and R.E. Sievers, Gas Chromatography of Metal Chelates, Pergamon Press, Oxford, 1965. 598 P.C. Uden and D.E. Henderson, Ana1,vst (London), 102 (1977) 889. 599 C.S.G. Phillips and P.L. Timms, Anal. Chem., 35 (1963) 505. 600 R.S. Juvet and R.L. Fischer, Anal. Chem., 38 (1966) 1860. 601 S.T. Sie, J.P.A. Bleumer and G.W.A. Rijnders, Separ. Sci., 1 (1966) 41. 602 S.T. Sie, J.P.A. Bleumer and G.W.A. Rijnders, Separ. Sci., 2 (1967) 645. 603 S.T. Sie, J.P.A. Bleumer and G.W.A. Rijnders, Separ. Sci., 3 (1968) 165. 604 M.L. Sazonov, J.E. A h o v a , M.S. Selinkina and A.A. Zhukhovitskii, Khim. Tverd. Topl., 3 (1968) 64. 605 R.E. Sievers, G . Wheeler, Jr., and W.D. Ross, Anal. Chem., 38 (1966) 306. 606 A.G. Hamlin, G. Iverson and T.R. Phillips, Anal. Chem., 35 (1963) 2037. 607 J.H. Becker, J. Chevalier and J. Spitz, Z. Anal. Chem., 247 (1967) 301. 608 N.N. Aleinikov, D.N. Sokalov, L.K. Golubena, B.L. Korsunskii and F.1. Dukovitskii, Izv. Akad. Nauk SSSR,Ser. Khim., (1973) 2614.

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DERNATIZATION OF COMPOUNDS P. Jones and G. Nickless, J. Chromatogr., 76 (1973) 285. P. Jones and G. Nickless,Proc. SOC.Anal. Chem., 10 (1973) 270. P. Zarnegar and P. Mushak, Anal. Chim. Acta, 69 (1974) 389. J.W. Young and G.D. Christian, Anal. Chim. Acta, 65 (1973) 127. S. Nakashima and K. Tbei, Talanta, 15 (1968) 1475. Y. Shimoishi,Bull. Chem. SOC.Jap., 44 (1971) 3370. Y. Shimoishi, Anal. Chim. Acta, 64 (1973) 465. K. Ballschmiter, J. Chromatogr. Sci., 8 (1970) 491. G. Schwedt and H.A. Russel, Chromatographia, 5 (1972) 242. G.P. Morie and T.R. Sweet, Anal. Chem., 37 (1965) 1552. G.P. Morie and T.R. Sweet, Anal. Chim. Acta, 34 (1966) 314. W.G. Scribner, W.J. Treat, J.D. Weis and R.W. Moshier, Anal. Chem., 37 (1965) 1136. R.W. Moshier and J.E. Schwarberg, Talanta, 13 (1966) 445. M. Miyazaki and H. Kaneko, Chem Pharm. Bull., 18 (1970) 1933. C. Genty, H. Horin, P. Malherbe and R. Schott, Anal. Chem., 43 (1971) 235. M.L. Lee and D.C. Burrell, Anal. Chim. Acta, 66 (1973) 245. R.A. Minear and C.M. Palesh, 165th ACSMeeting. Dallas, Texas, April 1973. T. Sakamoto, H. Kawaguchi and A. Mizuike,L Chromatogr., 121 (1976) 383. M.S. Black and R E . Sievers, Anal. Chem., 45 (1973) 1773. G. Kaiser, E. Grallath, P. Tschope and G. Tolg, 2.Anal. Chem., 259 (1972) 257. W.D. Ross and R.E. Sievers, Environ. Sci. Technol., 6 (1972) 155. W.R. Wolf, M.L. Taylor, B.M. Hughes, T.O. Tiernan and R.E. Sievers, Anal. Chem., 44 (1972) 616. J.K. Foreman, T.A. Gough and E.A. Walker, Analyp (London), 95 (1970) 797. K.J. Eisentraut, J.D. Griest and R.E. Sievers, Anal. Chem., 43 (1971) 2003. W.D. Ross and R.E. Sievers, Anal. Chem., 41 (1969) 1109. L.C. Hansen, W.G. Scribnsr, T.W. Gilbert and R.E.'Sievers, Anal. Chem., 43 (1971) 349. J. Savory, P. Mushak, F.W. Sunderman, Jr., R.H. Estes and N.O. Roszel, Anal. Chem., 42 (1970) 294. G.H.Booth, Jr., and W.J. Darby, Anal. Chem., 43 (1971) 831. R. Ross and T. Shafik, J. Chromatogr. Sci., 11 (1973) 46. W.R. Wolf, Anal. Chem., 48 (1976) 1717. R. Belcher, A. Khaiique and W.I. Stephen, in E. Wanninen (Editor), Essays in Analytical Chemistry, Pergamon Press, Oxford, 1977, p. 343. P.C. Uden, D.E. Henderson and C.A. Burgett,Anal. Lett., 7 (1974) 807. R.S. Barrett, R. Belcher, W.I. Stephen and P.C. Uden, Proc. SOC. Anal. Chem., 10 (1973) 167. R. Belcher, J.R. Majer, W.I. Stephen, I.J. Thomson and P.C. Uden, Anal. Chim. Acta, 50 (1970) 423. W.D. Ross, W.G. Scribner and R.E. Sievers, in R. Stock (Editor), Gas Chromatography, Institute of Petroleum, London, 1970, p. 369. R.E. Sievers, J.W. Connolly and W.D. Ross, J. Gas Chromatogr., 5 (1967) 241. R.F. Sieck, J.J. Richard, K. Iverson and C.V. Banks, Anal. Chem., 43 (1971) 913. C.A. Burgett and J.S. Fritz, Talanta, 20 (1973) 363. T. Lloyd, S. Markey and N. Weiner, Anal. Biochem., 42 (1971) 108. D.E. Durbin and A. Zlatkis, J. Chromatogr. Sci., 8 (1970) 608. J.A. Fioriti, M.J. Kanuk and R.J. Sims, J. Chrornatogr. Sci., 7 (1969) 448. C.R. Glowacki, P.J. Menardi and W.E. Link, J. Amer. Oil Chem. Soc., 47 (1970) 225. L.M. McDonough and D.A. George, J. Chromatogr. Sci., 8 (1970) 158. R.A. Hasty,Microchim. Acta, (1971) 348. R.A. Hasty,Microchim. Acta, (1973) 621. St. G u s , J. Chromatogr., 100 (1974) 43.

Appendix I

Purification of chemicals and solvents [ 1,2] Acetic anhydride, h.p. 138.0"C;nho 1.3904 Acetic anhydride can be purified by fractional distillation through an efficient column. To remove acid, 97% anhydride is allowed to stand for several days over thin slices of sodium, boiled in vacuum under reflux for several hours, and finally distilled over a mixture of sodium and sodium acetate. It is further purified by fractional distillation.

Acetone, b.p. 56.3"C;nho 1.3587 To 700 ml of acetone in a 1-1 bottle are added 3 g of silver nitrate dissolved in 20 ml of water, then 20 ml of 1 N sodium hydroxide solution are introduced and the mixture is shaken for about 10 min. The acetone is dried with calcium chloride, distilled, treated with phosphorus pentoxide and fractionally distilled. The distillate contains 0.01 -0.02% of water. A second distillation from phosphorous pentoxide reduces the water content to 0.001%or lower.

Acetonitrile, b.p. 81.6"C;nfp 1.3441 If a considerable amount of water is present (more than ca. 0.1 M), preliminary drying by shaking with silica gel or molecular sieve (e.g., Linde Type 3A) is advisable. The solvent is then shaken or stirred with sufficient calcium hydride to remove most of the remaining water, until hydrogen evolution ceases, and decanted. The distillation is carried out in an all-glass apparatus with a high reflux ratio from phosphorus pentoxide (not more than 5 g/l, otherwise gel formation may be excessive), while protecting the distillate from atmospheric moisture by means of a drying tube packed with anhydrous magnesium perchlorate. The first 5% of the distillate is discarded and the next 8 0 4 5 % is collected. The distillate is refluxed over calcium hydride (5 g/l) for at least 1 h, and then distilled slowly under the maximum reflux ratio (b.p. 81.6"C). Again the 5-90% fraction is collected. Acetylacetone, b.p. 45"C/30 mm, 138.9"C/750 mm; nb8.5 1.4518 Small amounts of acetic acid are removed by shaking with small portions of 2 M NaOH until the aqueous phase remains slightly alkaline. The sample, after washing with water, is dried with anhydrous sodium sulphate, and distilled through a modified Vigreux column. An additional purification step is fractional crystallization from the liquid.

Benzaldehyde, b.p. 178.9"C; nho 1.5455 To diminish its rate of oxidation, benzaldehyde usually contains additives such as hydroquinone or catechol. It can be purified via its bisulphite addition compound but 213

214

APPENDIX I

usually distillation under nitrogen at reduced pressure is sufficient. Prior to distillation it is washed with sodium hydroxide or 10%sodium carbonate solution (until no more carbon dioxide is evolved), then with saturated sodium sulphite solution and water, followed by drying with calcium sulphate, magnesium sulphate or calcium chloride.

Benzene, b.p. 80.l"C; nbo 1.5011 Benzene can be purified by storing the solvent over molecular sieve 4A, filtering and finally fractionally distilling. The water content determined by the Karl Fischer method is 5 . M. Highly pure benzene is most conveniently prepared by shaking a good commercial grade successively with concentrated sulphuric acid until free from thiophene, then with water, dilute sodium hydroxide solution, and finally two portions of water. The benzene can be dried by first shaking with anhydrous calcium chloride and removing the last traces of water by distilling over phosphorus pentoxide, shaking with a molecular sieve or passing through a column of silica gel.

Benzyl alcohol, b.p. 205.5"C; nbo 1.5404 The alcohol is purified by shaking with aqueous potassium hydroxide and extracting with diethyl ether that has been freed from peroxides with silver nitrate and sodium hydroxide. After being washed, the extract is treated with saturated sodium hydrogen sulphide solution, filtered, washed and dried over potassium carbonate. After removal of the ether, the alcohol is distilled under reduced pressure and the middle fraction dried over lime that has been burned in an atmosphere of nitrogen. The general method for purification is careful fractional distillation of the commercial alcohol, which is best carried out at reduced pressure with the exclusion of air.

Benzylrrimethylammonium hydroxide

A 38% aqueous solution (as supplied) is decolorized with charcoal, then evaporated under reduced pressure to a syrup, with final drying at 75°C and 1 mm pressure. Boron trichloride, b.p. O"C/476 mm This can be purified from chlorine by passage through two mercury-filled bubblers, then fractionally distilled under vacuum.

Boron trifluoride, b.p. - I I I .8"C/3OO mm The usual impurities (bromine, boron pentafluoride, hydrogen fluoride and nonvolatile fluorides) are readily separated by distillation.

Boron trifluoride diethyl etherate, b.p. 6 7"C/43 mm This is treated with a small amount of diethyl ether (to ensure an excess of this component), and then distilled under reduced pressure, from calcium hydride. It fumes in moist air.

PURIFICATION OF CHEMICALS AND SOLVENTS

215

2-Brornopropane,b.p. 59.4"C; n f p 1.4251 2-Bromopropane is washed with 95% sulphuric acid (the concentrated acid partially oxidizes it) until a fresh portion of acid becomes coloured after several hours. then with water, 10%sodium hydrogen sulphate solution, 10%sodium carbonate solution and water. (The H2S04 can be replaced by concentrated HC1.) Prior to this treatment, 2-bromopropane was purified by bubbling a stream of oxygen containing 5% of ozone through it for 1 h, followed by shaking with 3% hydrogen peroxide solution, neutralizing with aqueous sodium carbonate, washing with distilled water and drying. Alternatively, it was treated with elemental bromine and stored for 4 weeks, then extracted with aqueous sodium hydrogen sulphite and dried with magnesium sulphate. After the acid treatment, 2-bromopropane can be dried with sodium sulphate, magnesium sulphate or calcium hydride and fractionally distilled.

l-Butanol, b.p. I 1 7.7"C; n"$ 1.3992 The alcohol is washed with dilute sulphuric acid and with sodium bisulphite solution to remove bases, aldehydes and ketones. Esters are removed by boiling for 1.5 h with 20% sodium hydroxide solution. The alcohol is dried with potassium carbonate, followed by barium oxide, and finally distilled through an efficient column. Alternatively, 1-butanol is treated with sodium bisulphite solution, then boiled for 4 h with 10%sodium hydroxide solution and the separated alcohol is washed with water and neutralized with hydrochloric acid. The alcohol is dried overnight with lime and boiled three times with fresh lime for 3 h and. finally, fractionally distilled.

2-Butano1,b.p. 99.4"C; nD 1.3972 Optically active 2-butanol is prepared by converting the alcohol into the acid phthalate ester, 2.01 mol of which are thoroughly mixed with 2.0 mol of brucine. The mixture is charged into a 6-1 erlenmeyer flask with 2 1 of acetone and refluxed for 24 h. The solid changes in appearance during the reflux period and is kept broken up so that the acetone can come in contact with all of the solid. The mixture is filtered hot and recrystallized two or three times from methanol. The brucine salt is hydrolysed with sodium hydroxide to give a high purity (+)2-butanol.

Carbon disulphide, b.p. 46.2"C; nbo 1.6280 T h ~ can s be purified by first distilling over lime and then treating with potassium permanganate (5 g/l). The carbon disulphide is allowed to stand until the hydrogen sulphide is completely removed. After separating, the liquid is shaken with mercury to remove sulphur, poured off and shaken with 25 g/l of mercury(I1) sulphate until the unpleasant smell of carbon disulphide has disappeared. The liquid is poured off and distilled from calcium chloride, bright daylight being excluded. The purified material is stored in the dark.

216

APPENDIX 1

Carbon tetrachloride. b.p. 768°C;ni," 1.4631 One litre of solvent is shaken for 30 min at 60°C with a solution of 20 g of potassium hydroxide in 20 ml of water and 100 ml of ethanol. After cooling it is washed several times with water, dried with calcium chloride and distilled.

Chloroform, b.p. 61.2"C; nb5 1.4985 Chloroform can be purified by washing several times with concentrated sulphuric acid, tnen with dilute sodium hydroxide, and ice-water; it is then dried over potassiuni carbonate, stored in a completely filled brown flask and distilled shortly before use. Fused calcium chloride and phosphorus pentoxide can also be used as a drying agents.

Dichloromethane, b.p. 39.8"C; ng0 1.4242 The commercial product is washed with concentrated sulphuric acid, then with dilute sodium hydroxide and finally with water. The washed material is allowed to stand overnight over sodium hydroxide or calcium chloride and fractionally distilled in a 60-cm Widmer columr!.

Diethyl ether, b.p. 34.6"C; nbo 1.3524 Diethyl ether is shaken with one tenth of its volume of 10%sodium bisulphite solution intermittently for I h. The aqueous phase is removed and the ether is washed with saturated sodium chloride solution containing 0.5% of sodium hydroxide, then it is washed with saturated sodium chloride solution containing a small amount of sulphuric acid, followed by two portions of saturated sodium chloride solution, and finally distilled in an atmosphere of nitrogen. Substantially dry and alcohol-free diethyl ether can be prepared by fractional distillation. The distillate generally contains some aldehydes. Two methods of purification have been recommended for the preparation of high purity ethyl ether: (i) the ether is shaken with 50% sulphuric acid, distilled, dried for 8-15 days over calcium chloride and for 1 month over sodium, and then distilled; (ii) as above except that the ether is washed with saturated potassium permanganate solution containing 5% of sodium hydroxide; the final drying period over sodium is 2 months in the absence of light and air.

N,N-Dimethylformamide, b.p. 153.0"C; nb' 1.4305 The major part of the water from reagent-grade N,N-dimethylformamide may be removed as the benzene azeotrope. The product is stored in glass bottles with glass stoppers. About 800-ml portions are shaken with phosphorus pentoxide for 3 days; about 10 g of fresh oxide are added each morning. The amide is decanted and shaken with potassium hydroxide pellets to neutralize the formic acid. It is fractionally distilled at 50-64'C at 15-30 mmHg with a slow stream of dry nitrogen passing into the liquid.

PURIFICATION OF CHEMICALS AND SOLVENTS

217

Dimethyl sulphoxide, b.p. 189.0"C;nko 1.4783 This can be dried with Linde-type sieves 4 A or 13X, by prolonged contact and by passage through a column of the material, then distilled under reduced pressure. Other drying agents include calcium hydride, calcium oxide, barium oxide and calcium sulphate. It can also be fractionally crystallized by partial freezing.

2,4-Dinitrophenylhydrazine, m.p. 200°C This can be purified by crystallization from n-butanol, dioxan, ethanol or ethyl acetate. Dithizone Dithizone can be purified by shaking a carbon tetrachloride solution of the reagent with 1 : 100 ammonia (which extracts it), separating the layers, acidifying the aqueous layer with hydrogen chloride over a fresh portion of carbon tetrachloride and extracting the dithizone into the organic phase. Instead of carbon tetrachloride, chloroform can be used, and the final extract, after washing with water, can be evaporated in air at 40-50°C and dried in a desiccator.

Ethanol, b.p. 78.3"C;nbQ0.7894 The classical method for the preparation of dry ethanol is to reflux it over calcium oxide, then distil or fractionally distil it. Ethanol can be dried to a water content appraoching 0.005%if adequate precautions are taken to exclude water during the distillation. It has been found more convenient to take only common precautions and obtain ethanol containing not more than 0.05%water. The final drying can be accomplished with Type 3A or 4 A molecular sieve or Drierite.

2-(2-Ethoxyethoxy)ethanol,b.p. 202.0"C;nbQ1.4273 1,2-Ethanediol could best be removed by extracting 250 g of the alcohol in 750 ml of benzene with 5-ml portions of water, allowing 10 min for separation of the phases. The volumes of the aqueous extracts are accurately measured, the increase in volume calculated and the extraction continued until the increase in volume becomes constant.

Ethyl acetate, b.p. 77.1"C;nbQ 1.3724 One litre of the ester is refluxed for 6 h with 85 ml of acetic anhydride and then distilled through a Vigreux column. The distillate is shaken with 20 g of anhydrous potassium carbonate and redistilled.

Girard T reagent, m.p. 192°C This can be purified by crystallization from absolute ethanol.

218

APPENDIX 1

n-Hexane, b.p. 68.7"C; n&' 1.3749 n-Hexane is purified by fractional distillation in a 6-ft. column. Benzene is removed by treating it with an equal volume of nitrating mixture (58% concentrated sulphuric acid, 25% concentrated nitric acid and 17%,w/w of water) and shaking for 8 h. The hydrocarbon layer is washed with concentrated sulphuric acid, then with water, and dried. It is distilled over sodium and the distillate dried over sodium.

Hydroxylammonium hydrochloride, m.p. 151"C This is crystallized from aqueous 75% ethanol or boiling methanol, and dried under vacuum over calcium sulphate or phosphorus pentoxide. It can also be dissolved in a minimum volume of water and saturated with hydrogen chloride; after three such crystallizations it is dried under vacuum over calcium chloride and sodium hydroxide.

Iodomethane, b.p. 424°C; n:>" 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