Prediction of Elution Profiles in Countercurrent Chromatography With a Linear Loss of Stationary Phase

Countercurrent chromatography (CCC) is a liquid-liquid separation method that is predominantly used for the preparative isolation of natural products. Key indicators for the development of CCC methods are the partition coefficient (K value) between the two liquid phases and the properties of the chosen biphasic solvent system. Based on this information, it is possible to predict elution volumes and peak widths with the probabilistic model for immiscible separations and extractions (ProMISE). However, a common challenge in reliably predicting CCC elution profiles is a persistent loss of stationary phase (i.e., bleeding), which leads to a decrease in the percentile share of stationary phase (S_f value) in the column as the separation progresses. In this study, two carotenoids were isolated and used as test compounds to investigate the effect of bleeding with the n-hexane/benzotrifluoride/acetonitrile (20:7:13, v/v/v) solvent system. After showing that the stationary phase bleeding rate was linear (and not exponential), experiments were carried out both in head-to-tail and in tail-to-head mode. Next, a simple calibration method was developed to predict elution volumes and peak widths with the ProMISE2 software in CCC separations with linear bleeding. Finally, based on the collected data, a method was developed to accurately predict the elution volume and peak width during a (long) CCC run, particularly for analytes with comparatively high elution volumes.