PURPOSE: To enable the economical separation and purification of the titled compound by liquid chromatography, by using a column packing agent consisting of an aromatic vinyl compound copolymer containing monovinyl aromatic monomer unit and polyvinyl aromatic monomer unit.

CONSTITUTION: The titled compound such as eicosapentanoic acid useful as a preventive and remedy for thrombosis is separated and purified from a mixture of long-chain fatty acids or their lower alkyl esters and containing the titled compound, using a liquid chromatography. The chromatography is carried out by using a column packing material consisting of an aromatic vinyl compound copolymer containing monovinyl aromatic monomer unit and polyvinyl aromatic monomer unit. Preferably, a liquid chromatography treatment using a solvent having lower polarity than the packing material and the one using higher polarity solvent are combined together in arbitrary order. The objective compound can be separated and purified in high purity, recovery, and efficiency, at a relatively low cost.