Analysis of triglyceride isomers by silver-ion high-performance liquid chromatography

Silver ion chromatography (Ag-HPLC), utilizing columns packed with silver ions bonded to a silica or similar substrate, has proven to be a powerful technique for the analytical separation of cis and trans geometric and positional fatty acid methyl ester (FAME) and triacylglycerol (TAG) isomers. In this manuscript, we utilize an HPLC column chiller/heater to study the effects of Ag-HPLC column temperatures on elution rates of FAME and TAG isomers. Two Varian ChromSpher lipids columns connected in series and isocratic solvent systems of 1.0% or 1.5% acetonitrile (ACN) in hexane were used to analyze FAMEs (zero to six double bonds; cis/trans isomers), TAGs (homogeneous (triacetyl-, tristearoyl-, trioleoyl-, trilinoeyl- and trilinolenoyl-glycerols) and positional (1,3-distearoyl,2-monolinolenoyl- and 1,2-distearoyl, 3-monolinolenoylglycerol, etc.)) mixtures at four different temperatures (10 °C, 20 °C, 30 °C or 40 °C). Unexpectedly, the unsaturated FAME and TAG samples were found to elute more slowly at higher temperatures, a result just the opposite to the usual temperature effect (where samples elute more rapidly at higher temperatures) noted in gas and most liquid (reversed-phase or silica gel substrates) chromatography systems. This effect in Ag-HPLC may be limited to hexane-based solvent systems (it does not seem to occur with chlorinated hydrocarbon-based solvents); its magnitude is directly related to the total number of double bonds in the sample. It is also more evident with cis than with trans double bonds.