Gradient reversed-phase high-performance liquid chromatography of saturated, unsaturated and oxygenated free fatty acids and their methyl esters

Abstract

Abstract Reversed-phase C18 high-performance liquid chromatography (HPLC) separation of 49 free fatty acids and their methyl esters is reported here. Separation is achieved using methanol/water gradients and detection by either UV absorbance or evaporative light scattering. The factors decreasing retention times are: keto group, hydroxy group, epoxy group, triple bond, shortened chain length (C2H4 shorter), cyclopropenyl group, double bond, and cyclopropanyl group, in approximate correspondence to the order of their decreasing polarity. Fatty acids with cis-double bond, cis-epoxy and threo-dihydroxy substituents elute slightly earlier than the corresponding trans-double bond, trans-epoxy and erythro-dihydroxy fatty acids. These geometric isomers are better separated as free fatty acids than their methyl esters. For monounsaturates, cis-double bond at C-10 of the methyl ester of octadecenoic acid decreased the retention times most, with slightly increasing retention time as the double bond shifts toward either end of the chain. The trans-double bond of methyl esters of octadecenoic acids showed similar results. The effect of single double bond position in the HPLC of free octadecenoic acids is similar except the shortest retention time occurs when the double bond is at C-11. Two of the C18 columns in series achieve the separation of double bond positional isomers (free fatty acids), cis-7-octadecenoic acid and petroselinic acid (cis-6) at a resolution Rs = 1.05.