Sample preparation of free sterols from vegetable oils by countercurrent chromatography in co-current mode

Countercurrent chromatography (CCC) is a preparative instrumental method where both the mobile and stationary phases are liquids and which are predominantly used for the isolation of natural products. In this study, we widened the scope of CCC by using it as an instrumental method for the direct enrichment of the free sterol fraction from plant oils to which they contribute with ~ 1%. For the enrichment of sterols in a narrow band, we employed the so-called co-current CCC (ccCCC) mode in which both liquid phases of the solvent system (here: n-hexane/ethanol/methanol/water (34:11:12:2, v/v/v/v)) are moved at different flow rates in the same direction. Different from previous applications of ccCCC, the lower and predominant "stationary" phase (LPs) was pumped twice as fast as the mobile upper phase (UPm). This novel reversed ccCCC mode improved the performance but also required a higher demand of LPs compared to UPm. Therefore, the exact phase composition of UPm and LPs was determined by gas chromatography and Karl Fischer titration. This step enabled the direct preparation of LPs which considerably reduced the waste of solvents. Internal standards (phenyl-substituted fatty acid alkyl esters) were synthesised and utilised to frame the free sterol fraction. This approach allowed a fractionation of free sterols based on the UV signal and compensated run-to-run variations. The reversed ccCCC method was then applied to the sample preparation of five vegetable oils. In addition to free sterols, free tocochromanols (tocopherols, vitamin E) were also eluted in the same fraction as free sterols.