The use of countercurrent chromatography in the separation of nonpolar lipid compounds

Isolation of lipophilic compounds by countercurrent chromatography (CCC) is a challenge because biphasic solvent systems in which these compounds distribute evenly are difficult to obtain. In this article we present novel applications of lipid compound isolation from natural sources. Conjugated linolenic acids (CLnAs, log K_OW ∼7) were isolated from pomegranate oil using a solvent system consisting of n-heptane/methanol/water 100:91:9 (v/v/v). The CLnA fraction was free of other fatty acids but consisted of different isomers which were not resolved from each other. In the less polar range (log K_OW ∼12), three tocotrienols (α-, γ- and δ-tocotrienol) were isolated from a vitamin E capsule produced from palm oil by using the solvent system n-hexane/acetonitrile/benzotrifluoride (BTF) at a ratio of 10:6.5:3.5 (v/v/v). Between 36 and 65 mg of each of the three tocotrienols were obtained in one injection with purities >97%. Advantages and disadvantages of the “BTF system” are discussed by comparing the phase composition with the simple n-hexane/acetonitrile system and by the fractionation of phytosterols (log K_OW ∼9.5) from rapeseed oil.