The availability of various high-purity unsaturated fatty acids has a wide range of needs due to their different activities. The nonlinear preparative chromatography behavior and principle for purification of palmitoleic acid with octadecyl bonded stationary phases were studied. The peak broadening and the concentration distribution of the target compounds were used to compare different C18 stationary phases. In preparative liquid chromatography, the C18 stationary phases with low, medium, and high bonding density showed different peak broadening and concentration distribution results. Medium bonding density C18 was suitable for the purification of ethyl palmitoleic acid. The forward broadening was much greater than the backward broadening on medium bonding density C18 column. And the highest concentration distribution of impurities and the main peak was not crossed in this column. Due to the low content of crude ethyl palmitoleic acid sample, a two-step purified method yields an oily product with purity of 96.57% in the GC method. This method would be universal and extensible for constructing purification method for other unsaturated fatty acids.
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