Analysis of mixtures containing free fatty acids and mono-, di- and triglycerides by high-performance liquid chromatography coupled with evaporative light-scattering detection

Commercial monostearates of glycerol, generally used as antistatic agents for thermoplastic polymers, consist of a complex mixture of mono-, di- and trisubstituted glycerides and the corresponding fatty acids. Thus far, the glycerides and the fatty acids have been analyzed separately by high-performance liquid chromatography (HPLC). In fact, the simultaneous analysis of glycerides and fatty acids requires esterification of the acids in order to avoid overlapping of the chromatographic peaks. This paper describes an HPLC method which allows the direct separation and identification, simultaneously and in a single run, of the variously substituted glycerides, and also the corresponding saturated fatty acids that are found as by-products in commercial glycerol monostearates. The procedure is based on the use of a ternary gradient HPLC instrument equipped with an evaporative light-scattering detector; the stationary phase was a reversed-phase RP-8 end-capped (Merck) column; the mobile phase was two consecutive binary gradients consisting of acetonitile-water plus acetic acid (0.1%, v/v) and acetonitrile-methylene chloride. The method is fast and shows high sensitivity and selectivity, being able to separate also the positional isomers of mono- and diglycerides in addition to the mixed di- and triglycerides.