The present invention provides a chromatographic separation process forrecovering apolyunsaturated fatty acid (PUFA) product from a feed mixture, whichcomprises: (a) purifyingthe feed mixture in a first chromatographic separation step using as eluent amixture of waterand a first organic solvent, to obtain an intermediate product； and (b)purifying the intermediateproduct in a second chromatographic separation step using as eluent a mixtureof water and asecond organic solvent, to obtain the PUFA product, wherein the second organicsolvent isdifferent from the first organic solvent and has a polarity index whichdiffers from the polarityindex of the first organic solvent by between 0.1 and 2.0, wherein the PUFAproduct is otherthan alpha-linolenic acid (ALA), gamma-linolenic acid (GLA), linoleic acid, anALA mono- di- ortriglyceride, a GLA mono- di- or triglyceride, a linoleic acid mono, di- ortriglyceride, an ALA C1-C4alkyl ester, a GLA C1-C4 alkyl ester or a linoleic acid C1-C4 or a mixturethereof.
