Separation and quantitation of mono-, di-, and triglycerides and free oleic acid using thin-layer chromatography with flame-ionization detection.

alpha-Monoolein was prepared from glycidol and oleic acid by the regioselective opening of glycidol in the presence of an anionic resin. During the reaction, the lipochemical synthesis medium becomes enriched in monoolein, the effective emulsifying agent. This mixture can be analyzed by thin-layer chromatography coupled with flame-ionization detection (FID). The products and reagents do not need to be derivatized. Diglyceride and triglyceride by-products affecting the selectivity of the reaction also could be detected using this technique. Cholesterol was used as an internal standard. The factors influencing the separation, including the hydrogen flow rate, scan speed, and the composition of the developing solvent, were investigated. The degree of separation is highly sensitive to the hexane/diethyl ether ratio of the developing solvent. Good separation of triglyceride, oleic acid, the two diglycerides, cholesterol, alpha-monoolein, and glycidol was obtained with the mixture hexane/diethyl ether/formic acid (65:35:0.04, by vol). Detector response, detection limits, and rod-to-rod variations also were examined. A range of rod loads giving a straightforward relationship between FID response and amount of compound loaded (relative to oleic acid and alpha-monoolein) was defined. The accuracy of the quantification was illustrated by analysis of a mixture of oleic acid and alpha-monoolein standards of known composition.