Choline plasmalogen, non-choline plasmalogen and free plasmal can be separated by paper chromatography using development with acetone saturated with glycerol. The separation can be applied to phosphatide mixtures dissolved in organic solvents, aqueous phosphatide emulsions, blood serum and aqueous homogenates. Interference caused by aldehydic impurities in the solvents and by autooxidation of free plasmal and unsaturated compounds can be readily eliminated. Free plasmal can be determined by means of spot tests.