ANALYSIS OF FATTY ACIDS
Before looking at the various strategies to study fatty acids you would like to learn details about the history of their discovery, so read the next chapter.
We owe a considerable debt to ancient investigators who, prior to about 1935, made enormous contributions to our knowledge of the fatty acid composition of natural lipids despite primitive equipments and analytical techniques. Since the first works of Chevreul and for about a century, chemists isolated lipids using only solubility properties of solvents, the formation of salts of fatty acids which were further characterized by their raw formula, and ebullition or fusion temperatures.
The period following 1935 has been marked by new and more efficient procedures for separating and studying fatty acid mixtures. These procedures include ester distillation, crystallization of urea complexes or of various metallic salts, various forms of chromatography and countercurrent distribution.
An overview of these ancient techniques applied to fatty acids can be found
The discovery in the mid-1950’s of gas-liquid chromatography (GLC) has revolutionized the analysis of fatty acids and, undoubtedly, this technique is the most frequently used. Indeed, for the quantification of individual fatty acids in any acylated lipids, GLC must be adopted.
In some other studies, complementary techniques should be considered. Metabolic studies involve the knowledge of the intensity of labelling of molecular species with radioactive atoms while identification studies require the separation and quantification of hydroxylated, branched-chain, trans or conjugated fatty acids. All these investigations are more easily run with HPLC than with GLC procedures since positional and conformational isomers are more easily separated by HPLC than by GLC. Furthermore, HPLC is the method of choice for preparative scale separations of particular fatty acids for further structural or metabolic studies. In contrast to GLC which preferred flame ionization detection (FID), the choice of the detector for HPLC analysis is important and determines the adopted procedure. Several detections are possible, the most used are light scattering, UV, fluorescence and radioactivity.
In general, fatty acids are separated by HPLC as derivatized molecules but unesterified forms can also be chromatographed if acidic solvent systems are used.
For some precise purposes only the amount of fatty acids is to be known. Global methods are useful when the fatty acid profile is not in the scope of the investigation.
– study of normal fatty acids
– study of trans fatty acids
– study of conjugated fatty acids
– study of dicarboxylic acids
– study of cyclic fatty acids