A Neglected Issue: Stationary Phase Retention Determination of Classic High-Speed Counter-Current Chromatography Solvent Systems

Obtaining an ideal solvent system for target compounds is still an obstacle to the wide application of high-speed counter-current chromatography (HSCCC). The partition coefficient and retention of the stationary phase are two key parameters for solvent system selection. The retention of the stationary phase of the solvent system is roughly judged by settling time using a test tube, which is subjective and inaccurate. In this study, we demonstrated that high-resolution separation of HSCCC is tightly connected with the retention of the stationary phase. Notably, unlike the in vitro test of settling time, we investigated the retention of the stationary phase of classical biphasic solvent systems by a TBE300C HSCCC apparatus. Our results revealed that settling time is not always inversely proportional to the retention of the stationary phase. The n-hexane–ethylacetate–methanol–water solvent systems showed the highest correlation coefficient of settling time and retention of the stationary phase (r = −0.91, n = 16). N-heptane–n-butanol–acetonitrile–water solvent system showed the lowest correlation coefficient (r = −0.26, n = 7). These results may be helpful for HSCCC solvent system selection and accelerate the application of this technique