Combination of conventional and co-current countercurrent chromatography for the separation of compounds with very...

Countercurrent chromatography (CCC) is an instrumental liquid-liquid chromatographic method that separates analytes according to their partitioning between the stationary and mobile phases (K_sm value). CCC is valuable for the isolation of compounds in a biphasic solvent system, which provides K_sm values between 0.4 and 2.5. However, limitations exist for very high K_sm values, as this leads to long elution times and a high solvent consumption. Here, we show that these disadvantages can be overcome by a combination of conventional (CCC) and co-current (ccCCC) countercurrent chromatography. Specifically, separations were started in normal CCC mode followed by the fast elution in ccCCC mode (CCC+ccCCC). CCC+ccCCC experiments were performed with oleic acid methyl ester (18:1n-9-ME) and palmitic acid methyl ester (16:0-ME), which are difficult to separate by CCC and have K_sm values of >10 in n-hexane/acetonitrile (1:1, v/v). Tests with different switching times between CCC to ccCCC were used to determine the linear range of CCC+ccCCC runs, which was crucial for the separation efficiency and the stability of the solvent system. By combining simulated data with the obtained experimental data, it was possible to reliably predict chromatographic parameters in CCC+ccCCC mode. As a result, 18:1n-9-ME and 16:0-ME were sufficiently separated with CCC+ccCCC. The yield of pure 18:1n-9-ME and 16:0-ME was further improved by performing CCC+ccCCC in heart-cut mode. Given their reproducibility and predictability, the combination of normal CCC mode, followed by the ccCCC mode (CCC+ccCCC), represents a promising complement to existing approaches to make compounds with unfavorable K_sm values amenable to CCC.