Gas chromatographic analysis of fatty acid methyl esters

Abstract

The full process of fatty acid methyl ester (FAME) analysis consists of esterification of lipids, and of injection, separation, identification and quantitation of the FAMEs. In order for the required accuracy and precision to be attained, each of these steps has to be optimized. Esterification of lipids can be carried out with several reagents based on acid-catalysed or base-catalysed reactions. The advantages and disadvantages of these reagents are discussed. The most critical step in the gas chromatographic analysis of FAMEs is sample introduction. The classical split injection technique, which is the most widely used technique in the analysis of FAMEs, has the potential disadvantage of boiling-point-dependent sample discrimination. Cold injection of the sample, either on-column or by programmed-temperature vaporization, does not present this problem and should therefore be preferred. Modern, commercially available fused-silica capillary columns offer excellent separation of FAMEs from biological samples. Very polar stationary phases give excellent separation of all FAMEs but have relatively low thermal stability, resulting in long retention times. Non-polar phases have a much greater thermal stability but inferior selectivity. For many analyses, phases of intermediate polarity, which combine the advantages of a relatively high resolution capability with relatively high thermal stability, are the most suitable. FAMEs can be identified by comparison of their retention times with those of individual purified standards or secondary standards based on lipids that have been well characterized in literature. Relative retention times and equivalent chain-length values also provide useful information. FAMEs can be quantitated by peak areas via calibration factors, and absolute concentrations can be determined by adding an internal standard. Among numerous applications in biomedical research, the analysis of fatty acids from body tissues may contribute to the understanding of the link between the dietary intake of fatty acids and the diseases with which these acids are associated.