Improving the resolution of overlapping peaks by heartcut two-dimensional countercurrent chromatography with the same...

The countercurrent chromatographic (CCC) isolation of structurally related compounds with high chemical purity can be challenging, especially in the field of lipids, were the range of biphasic solvent systems is limited. In the past, special elution modes like recycling CCC or re-injection of pooled fractions into a subsequent CCC (off-line re-injection CCC) were applied successfully to improve the separation of interfering compounds. However, isolation of minor compounds is often hampered by overlapping major compounds in natural samples. In this study we used heartcut two-dimensional CCC (2D CCC) to transfer the analyte completely to the next dimension while the interfering compound was partly removed. This procedure not only reduced the amount of the interfering major compound but also its peak width by up to 20% in the second dimension despite the longer elution time. Since the same solvent system and coil dimensions were used in the second dimension, this positive effect was attributed to the "transfer volume" which can be compared to an injection volume into the second dimension. As a consequence, improved peak resolution was generally observed for peaks which were partly transferred to the next dimension. This unexpected beneficial effect of 2D CCC on peak resolution was demonstrated by means of two examples in comparison with CCC in recycling and off-line re-injection mode. Applying the same CCC conditions, heartcut 2D CCC yielded >14 mg of the pentacyclic triterpenol lupeol, isolated from the unsaponifiable matter of shea butter, while recycling CCC and one-dimensional CCC only yielded ∼5 and ∼1 mg, respectively. Moreover, the resolution of three different ergosterol derivates (ergosta-5,7-dienol, ergosta-7,22-dienol and ergost-7-enol) which were separated from the very abundant ergosterol in the unsaponifiable matter of freeze-dried button mushrooms could be improved with heartcut 2D CCC compared with off-line re-injection CCC.