GRADIENT REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF SATURATED, UNSATURATED AND OXYGENATED FREE FATTY-ACIDS AND THEIR METHYL-ESTERS

Reversed-phase C-18 high-performance liquid chromatography (HPLC) sepa ration 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 d ecreasing retention times are: keto group, hydroxy group, epoxy group, triple bond, shortened chain length (C2H4 shorter), cyclopropenyl gro up, double bond, and cyclopropanyl group, in approximate correspondenc e to the order of their decreasing polarity. Fatty acids with cis-doub le bond, cis-epoxy and threo-dihydroxy substituents elute slightly ear lier than the corresponding trans-double bond, trans-epoxy and erythro -dihydroxy fatty acids. These geometric isomers are better separated a s 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-doubl e bond of methyl esters of octadecenoic acids showed similar results. The effect of single double bond position in the HPLC of free octadece noic acids is similar except the shortest retention time occurs when t he double bond is at C-11. Two of the C-18 columns in series achieve t he separation of double bond positional isomers (free fatty acids), ci s-7-octadecenoic acid and petroselinic acid (cis-6) at a resolution R( s) = 1.05.