GAS CHROMATOGRAPHY AND LIPIDS
AN INTRODUCTION TO LIPIDS AND GAS CHROMATOGRAPHY
INTRODUCTION AND SUMMARY
A. A HISTORICAL PERSPECTIVE ON GAS CHROMATOGRAPHY AND LIPIDS
The initial stages in the development of gas chromatography were in the hands of lipid analysts, and this technique probably above all others so facilitated the separation of lipids, and especially their fatty acid components, that it must be considered one of the major factors in the explosive growth of knowledge about these natural products over the last thirty years. In the hands of a well-organised analyst, it is now possible to convert a lipid sample of a fraction of a milligram in size to the methyl ester derivatives, separate these by gas chromatography, and have a quantitative result in under one hour. This was not always so. In the 1940s and early 1950s, with even simple mixtures as in seed oils, it was necessary to start with a sample of 20 to 200g of the methyl esters. These were subjected to a preliminary fractionation by low-temperature crystallisation, steam distillation or as the urea adducts, then the individual fractions were subjected to fractional distillation under high vacuum. Further chemical reactions, many calculations and perhaps several weeks later, an approximate result was obtained  (or see this webpage). For a brief period, a form of column partition chromatography in the reversed phase mode came into use that would appear rather crude to the modern observer, but it reduced the time required to complete an analysis to one or two days.
This technique was based on the classic paper by Martin and Synge of 1941 , in which liquid-liquid column chromatography was used to separate amino acids. The paper contained the significant phrase - "the mobile phase need not be a liquid but may be a vapour". However, the idea was not pursued until A.T. James joined Martin in 1949. James has provided an entertaining account of their joint collaboration . In the initial work, they could only attempt to separate those compounds which could be detected and quantified by titration. The first essays at separations were made with a column packed with celite coated with liquid paraffin as the liquid phase and with short-chain fatty acids as the solute, apparently for the reason simply that G. Popjak in an adjacent laboratory was working on the biosynthesis of these compounds in mammary gland.
Success eluded them initially, but no complications were experienced with low molecular weight amines and the first successful application was to the resolution of mono- and dimethylamine. They soon realised that the difficulty with the fatty acids was because of the occurrence of dimerization, and when a long-chain fatty acid was incorporated into the liquid phase the problem was resolved. In fact, the application to the resolution of volatile fatty acids (formic to dodecanoic) was chosen for illustrative purposes, when the first paper on the new technique was published in 1952 . From the theoretical standpoint, they showed that a series of straight-chain homologues had a constant increment in retention volume for each additional methylene group.
The instrumentation became increasingly sophisticated with the addition of provision to heat the column, an automatic titrating device and then the first successful sensitive and universal detector, the gas-density balance, all of which required to be hand-crafted . In a companion paper to the description of the detector, the technique was demonstrated with the first separations of methyl ester derivatives of fatty acids . It appears that the initial contact with the problem of the resolution of fatty acids must have inspired James to continue with the study of lipids, and in 1957 he independently published a paper on the determination of the structures of longer-chain fatty acids using gas chromatography and micro-chemical methods . He subsequently went on to a distinguished career in lipid biochemistry.
The scientific instrument companies rapidly took up the technique of gas chromatography, bringing it to the high state of development described in this book. The technique began with a lipid problem, and lipid analysts have been at the forefront at every stage. Now, all but those lipids of the highest polarity or molecular weight can be separated by the technique.
Of course, many other instrumental procedures are utilised by lipid analysts, and the author has published a monograph on "High-Performance Liquid Chromatography and Lipids" . My latest book "Lipid Analysis - 4th Edition" (co-authored with Xianlin Han, 2010), also published by the Oily Press, is certainly more up-to-date as well as being much more comprehensive in terms of applications to lipids in general.
Perhaps the most important technique to be used with gas chromatography directly, however, is mass spectrometry. It may come as a surprise to many readers, but mass spectrometry is in fact a much older technique than gas chromatography, the basic principle and applications to the separation of atomic masses first having been demonstrated towards the end of the last century. Methods for separating the isotopes of the elements dominated the development of the technique after the First World War, but it was the demands of the petroleum industry during the Second World War together with improvements in electronics that led to the construction of instruments we would recognise today. When it became possible to couple the mass spectrometer to the outlet of the column of a gas chromatograph, lipid analysts were not slow to recognise the possibilities that lay before them. Because of further progress in computerisation and micro electronics, together with the introduction of wall-coated open-tubular columns of fused silica that greatly reduces the problem of interfacing, the price of gas chromatography-mass spectrometry systems has plummeted, and their reliability and ease of use have increased immeasurably. They have become the routine research and analytical tools for many, rather than the prized possessions of a few, and this trend can only accelerate.
B. A SUMMARY
In Chapter 2, the structures, chemistry and compositions of lipids in animals, plants and microorganisms are summarised, since a knowledge of the lipid types, which may be encountered in an analysis, might assist in determining the best analytical procedure to adopt. The first step in the analysis of lipids generally involves the preparation of a lipid fraction, relatively free of non-lipid contaminants, by means of solvent extraction of the tissue. Methods of achieving this are described here. Unwanted degradation of lipids can occur during the storage and handling of tissues and lipid samples, and autoxidation of unsaturated fatty acids can be especially troublesome. Methods of avoiding these difficulties during extraction are described that are applicable to all stages of analytical procedures, and they are relevant to each of the subsequent chapters. Some preliminary fractionation of lipid samples into simple lipid, glycolipid and phospholipid groups may then be desirable to facilitate their analysis or the isolation of single lipid classes on a small scale. The last objective can be accomplished by high-performance liquid, thin-layer or ion-exchange chromatography.
In order to make the best use of the technique, it helps to have some knowledge of the theory of gas chromatography, and this is outlined in Chapter 3. Many important aspects of chromatography are governed by a few simple equations, so a highly detailed account of the mathematics and physics of the subject is not necessary here. Novices on the subject or newcomers to capillary gas chromatography are confronted by a bewildering array of different columns, injection systems and detectors, and these are discussed systematically in relation to specific analytical problems. Various precautions are described to extend column life, and to ensure reproducibility in quantification, and details of these obviously cannot be repeated in each of the subsequent Chapters.
The important complementary technique of mass spectrometry is discussed briefly here, but applications are dealt with at length in later Chapters. Some modern mass spectrometry techniques are not directly compatible with gas chromatographic separations, requiring direct-probe inlet systems, and these are not treated in depth in this book.
Part 2 of the book deals with the analysis of fatty acids. This has grown to be a rather large subject, and for convenience has been divided here into four chapters. However, aspects of the subject matter in each may be helpful for particular problems, and it is never safe to rely on evidence from a single analytical technique.
The first of these chapters (Chapter 4) is concerned with the preparation of volatile non-polar derivatives of fatty acids. Methyl ester derivatives are used almost universally for fatty acid analysis, although many analysts continue to employ older cumbersome procedures for their synthesis rather than the simple safe ones now available. It should be noted that no single method can be used in all circumstances. In addition, fatty acids with a wide range of different substituent groups in the aliphatic chain occur in nature, and their analysis by means of gas chromatography can also be facilitated by the preparation of specific derivatives.
Gas chromatography on stationary phases consisting of polar polyesters of various kinds has become the standard technique for the separation of the common range of fatty acids encountered in most animal and plant tissues (Chapter 5). Packed columns afford adequate resolution for most purposes, but the high resolving power, stability and inertness of wall-coated open-tubular (capillary) columns made from fused silica have made these the standard in increasing numbers of laboratories. Fatty acid derivatives differing according to degree of unsaturation, that have been separated by the technique, can frequently be identified with a reasonable degree of certainty by their relative retention times alone, provided that these are measured with care. When other functional groups, such as hydroxyl, branched or cyclic moieties, are present in the alkyl chain, such methods can also be used provided that a sufficient range of model compounds are available for comparison of their chromatographic properties. Modern flame ionisation detectors have sufficient range and sensitivity to cope with most problems of quantification, provided that they are set up correctly and calibrated with care.
There are many circumstances when it is not possible to rely on gas chromatographic retention data for the identification of a fatty acid, for example in the analysis of material from a new natural source or when the interpretation of a metabolic experiment hinges on the identity of some specific component. It is then necessary to isolate the fatty acids by an appropriate method, nowadays high-performance liquid chromatography in the reversed-phase or silver ion modes is chosen most often, and then determine the structures by chemical and/or spectroscopic procedures (Chapter 6). Chemical degradative methods are available for locating double bonds and many other functional groups in fatty acids on a micro-scale. If the material can be isolated in sufficient amount, then spectroscopic techniques, especially proton and carbon-13 nuclear magnetic resonance spectroscopy, can be substantial non-destructive aids to structure determination.
Gas chromatography and mass spectrometry in combination make a particularly powerful technique for the identification of fatty acids, and this is the subject of Chapter 7. In some instances, the methyl ester derivatives alone give adequate mass spectra for identification purposes. More often, it is preferable to prepare pyrrolidides or picolinyl esters, since these give characteristic fragmentations that permit the location of many functional groups, including double bonds and methyl branches, in aliphatic chains. Sometimes it is necessary to prepare derivatives of other functional groups in order to facilitate chromatography and to ensure that interpretable mass spectra are obtained.
Lipid classes do not occur in nature as single pure entities, but rather they are a complex mixture of molecular species in which the fatty acids and other aliphatic moieties are present in different combinations. One excellent method of resolving this complexity is to use high-temperature gas chromatography, and this is the subject of Chapter 8. With lipids of high molecular weight, such as triacylglycerols, it is possible to obtain fractions differing in size by one or two carbon atoms on short packed columns containing non-polar methylsilicone phases. By using wall-coated open-tubular columns of glass or fused silica coated with similar phases or even, as in recent work, with more polar phases, greatly improved resolution is obtained, including components differing in degree of unsaturation.
This methodology is perhaps stretching current technology to its limits, and it is a much simpler task to resolve molecular species of phosphoglycerides, after they have been hydrolysed to diacylglycerols with the enzyme phospholipase C and then chemically derivatized (Chapter 8, Part 2). Similarly, gas chromatography is an excellent procedure for the separation of molecular species of monoacylglycerols, cholesterol esters and wax esters. Glycosyldiacylglycerols can be analysed by this methodology, but it has not been used a great deal for the purpose. Ceramides derived from sphingomyelin are easily resolved by high-temperature gas chromatography, but glycosphingolipids are not readily converted to ceramides and the technique has been little used for these compounds for which high-performance liquid chromatography is now generally favoured (Chapter 8, Part 3). In all such work, gas chromatography coupled with mass spectrometry has proved invaluable for identifying fractions, and also for the quantification of unresolved components in a single chromatographic peak. The last section of this Chapter deals with methods for obtaining a profile of all the main lipid classes in body fluids, such as plasma, by gas chromatography.
Gas chromatographic methods are only capable of separating a limited number of molecular species in complicated lipid samples, but greatly improved resolution can be obtained if they are used in conjunction with a complementary technique, such as adsorption or silver ion chromatography, as described in Chapter 9. Adsorption chromatography is of most value for the separations of species containing fatty acids with polar substituents. Silver ion chromatography, which has historically been associated with thin-layer chromatography although high-performance liquid chromatographic methods are now being actively developed, gives separations of triacylglycerols, diacylglycerol acetates and even of intact phospholipids by degree of unsaturation. On the other hand, high-performance liquid chromatography in the reversed-phase mode is best regarded as an alternative separatory procedure to high-temperature gas chromatography and recent developments are summarised to indicate the wider choice available to the analyst.
In the final Chapter (Ten), some miscellaneous but none the less important topics, which do not conveniently fit into other Chapters, are gathered together. Fatty alcohols, for example, are major components of waxes in addition to having other biological functions, and they are best analysed by gas chromatography in the form of various non-polar derivatives. Most glycerolipids exist in alkyl and alkenyl forms in addition to having esterified fatty acids; each of these ether-linked residues is conveniently analysed by gas chromatography in the form of alkylglycerol and aldehyde derivatives respectively. Platelet-activating factor is a special case of an ether lipid with vital metabolic properties, and the analysis of this compound is described here also. In addition to the fatty acid constituents, which are considered earlier, sphingolipids contain a range of different sphingoid bases. Procedures for the isolation of these, and for their analysis by gas chromatography are considered here. With all of these compounds, gas chromatography-mass spectrometry is of immense value for identification purposes. Cholesterol and glycerol are important lipids or components of lipids, and gas chromatographic methods for their analysis are likewise described in this Chapter.
The following abbreviations are employed at various points in the text of these chapters:
Amu, atomic mass units; BDMS, tert-butyldimethylsilyl; BHT, 2,6-di-tert-butyl-p-cresol; CI, chemical ionisation; DNP, dinitrophenyl; ECL, equivalent chain-length; ECN, equivalent carbon number; EI, electron-impact ionisation; FCL, fractional chain-length; GC, gas chromatography; GLC, gas-liquid chromatography; HPLC, high-performance liquid chromatography; IR, infrared; MS, mass spectrometry; NMR, nuclear magnetic resonance; PAF, platelet-activating factor; ODS, octadecylsilyl; TLC, thin-layer chromatography; TMS, trimethylsilyl; UV, ultraviolet.
William W. Christie, James Hutton Institute (and Mylnefield Lipid Analysis), Invergowrie, Dundee (DD2 5DA), Scotland.
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