Determination of fatty acids in vegetable oil by reversed-phase liquid chromatography with fluorescence detection

The effect of temperature and organic solvent composition (acetonitrile and methanol) on the reversed-phase separation of coumarin-derivatized fatty acids according to their carbon number (C14 to C22), the degree of unsaturation, as well as cis/trans (C18:1 c/t, C18:2 cc/tt, C18:3 ccc/ttt) configuration was investigated to find out the effective separation condition. Based on the linear plots of the logarithm of the capacity factor of saturated fatty acids versus their carbon number, the equivalent chain length (ECL) of unsaturated fatty acids was calculated. The ECL values were found to be significantly altered and the differentiation between cis and trans fatty acids was increased when either the temperature or organic solvent composition was decreased. These results generally led to a better resolution at the expense of separation time. A ternary gradient composed of water, acetonitrile, and methanol was then developed to elute the solutes at 55 degrees C within a separation time of 40 min with a minimum resolution of 1.0 for the worst pair. This method was demonstrated to resolve the fatty acids in a vegetable shortening.