Normal-phase separation effects with lipids on a silver ion high-performance liquid chromatography column

The separation of saturated fatty acids (as the methyl esters and as triacylglycerols) of differing chain lengths by silver ion chromatography (ChromSpher Lipids; containing silver ions; Chrompack, Middelburg, Netherlands) was studied. The retention characteristics (isocratic solvent system of acetonitrile in hexane) of saturated fatty acid methyl esters and homogeneous, saturated triacylglycerols were found to be inversely related to the number of carbon atoms in the fatty acid(s). Thus methyl stearate eluted first and methyl acetate eluted last; for triacylglycerols, tripalmitoylglycerol eluted first while triacetin was strongly retained (11 min vs. 70 min). While mixtures of fatty acid methyl esters and of triacylglycerols could be separated, resolution was found to improve at higher concentrations of acetonitrile, a characteristic of normal-phase separations. In the absence of carbon-carbon double or triple bonds, the effects of the more polar silanol groups of the column packing tended to override the silver ion contributions (interaction of silver ions with the unpaired electrons of the carbonyl oxygens), resulting in separations similar to those observed in silica thin layer and normal-phase high-performance liquid chromatography.