Abstract: Supercritical fluid chromatography (SFC) was used to study the adsorption equilibrium and mass transfer properties for eicosapentaenoic acid(EPA) and docosahexaenoic acid(DHA) on C 18 by means of impulse response technique. In order to estimate the adsorption constant and rate constants, a kinetic model was established. Chromatographic curves were calculated by using the parameters obtained from experiments, and the agreement between the experimental and calculated curves was good. It was indicated that adsorption equilibrium, axial dispersion, mass transfer and effective diffusion are important factors that influence the separation of EPA and DHA by SFC.