Isolation of lanosterol and dihydrolanosterol from the unsaponifiable matter of lanolin by urea complexation and countercurrent chromatography in heart-cut recycling mode

•Detailed GC/MS analysis of the unsaponifiable matter of lanolin.•Preparative enrichment of desmethyl- and 4,4-dimethylsterols by urea complexation.•Partition coefficients (KL/U values) of sterols in 14 biphasic solvent systems.•CCC runs of urea-complexed lanolin with the two best suited solvent systems.•Isolation of lanosterol and dihydrolanosterol by CCC in heart-cut recycling mode. 4,4-Dimethyl-substituted sterols are bioactive minor sterols of most animal fats and plant oils, but higher shares are present in lanolin (wool grease). Here, the isolation of the 4,4-dimethyl-substituted sterols dihydrolanosterol and lanosterol from lanolin by countercurrent chromatography (CCC) is described. An initial examination of the hexane extract of saponified lanolin showed the presence of relatively high portions of fatty alcohols which were known to co-elute with the target analytes in CCC. Hence, fatty alcohols were precipitated by urea complexation. Unexpectedly, 4,4-dimethyl-substituted sterols were also found in the crystalline fraction, while cholesterol and other desmethylsterols were detected in the liquid phase. Urea complexation represented a useful preparative method for the separation of desmethylsterols and 4,4-dimethyl-substituted sterols from lanolin. Shake flask experiments of 4,4-dimethyl-substituted sterols and fatty alcohols with 14 biphasic solvent systems indicated suitable partition coefficients (K values) with n–hexane/ethanol/water (12:8:1, v/v/v) and n–hexane/benzotrifluoride/acetonitrile (20:7:13, v/v/v). After initial tests with conventional CCC, the application of CCC in heart–cut recycling mode provided 4,4-dimethyl-substituted sterols with purities of 99 % (dihydrolanosterol) and 95 % (lanosterol).