Analysis of fatty acid mono- and diacylglycerol positional isomers by silver ion high-performance liquid chromatography

Mono- and diacylglycerol positional isomer pairs were separated (as acetates) by silver ion high-performance liquid chromatography on a commercially available column using an isocratic solvent system of 1.2% (v/v) acetonitrile in hexane and flame ionization detection. The acetate derivative(s) from the 1- and 2-monoacylglycerols and 1,2- and 1,3-diacylglycerols were prepared by acetic anhydride-pyridine. Conversion of the thermodynamically less stable 2-mono- and 1,2- diacylglycerols to triacylglycerols containing acetate(s) in the 1-, 3- and 2-positions, respectively, resulted in <3% isomerization. Conversion of the thermodynamically more stable 1-mono- and 1,3-diacylglycerol analogues yielded <1% isomers. Less than 0.5% inter-esterification by-products were noted. The triacylglycerol, the diacylglycerol-monoacetate isomer pair, the monoacylglycerol-diacetate isomer pair and triacetin were completely separated for the 16:0 and 18:1 fatty acid series. The triacylglycerols eluted first and the triacetin eluted last. The 16:0 elution pattern is unusual, since silver ion chromatographic separations are generally ascribed to the interaction of silver ions with carbon-carbon double bond pi-electrons, a condition absent in the 16:0 series.