Reverse Phase High-Performance Liquid Chromatographic Analyses of Fatty Acids by a Low Wavelength Detector

Abstract

Analyses of free fatty acids (saturated, branched and unsaturated) by reverse phase high-performance liquid chromatography have been studied at low wavelengths (196220nm) by using a variabe wavelength UV detector, and aqueous methanol containing 0.1% phosphoric acid as eluent. Plots of logarithm of the corrected retention volume against the carbon number of saturated straight chain fatty acid gave good linear relationship, from which C values of branched and unsaturated fatty acids were estimated. C values of branched and some unsaturated fatty acids were constant independent of water content of eluent, however, those of polyunsaturated fatty acids increased with increasing the water content. C values of branched and unsaturated fatty acids were smaller than their total carbon numbers, respectively. This shows that these acids are more polar than the corresponding straight chain fatty acids, respectively. Resolution of geometrical and positional isomers of unsaturated fatty acids was performed. Movement of double bonds toward the polar end and cis form increased the polarity of the acids to a slightly greater than the corresponding isomeric forms, respectively. These observations were discussed by the interaction between the acids and the reverse phase. Relative response of unsaturated fatty acids against dodecanoic acid (12 : 0) has been estimated. The plots of S (unsaturated fatty acid) /S (12 : 0) against Wt (unsaturated fatty acid) /Wt (12 : 0) gave linear relationship, demonstrating a possibility of stable and easily available saturated fatty acids as internal standards for the quantitative HPLC analyses of unsaturated fatty acids and the related compounds that have the absorption only at low wavelengths. The relative response of unsaturated fatty acids against 12 : 0 increased with decreasing the detection wavelength. Unsaturated fatty acid compositions of some vegetable oils were determined using the relative response, in agreement with the results in the GLC analyses.