Studies on the effect of column angle in centrifugal helix counter-current chromatography

The performance of the coiled column of centrifugal counter-current chromatography was investigated by changing the angle between column axis and centrifugal force in the separation of dipeptides or DNP-amino acids each with suitable two-phase solvent systems. In general, retention of the stationary phase (Sf) decreased, and peak resolution (Rs) increased as the column angle was increased. The first series of experiments was performed using a polar two-phase solvent system composed of 1-butanol–acetic acid–water (4:1:5, v/v/v) to separate two dipeptide samples, Trp-Tyr and Val-Tyr, at a flow rate of 1 ml/min at 1000 rpm. When the column angle was changed from 0° to 90°, Rs increased from 1.05 (Sf = 60.1%) to 1.17 (Sf = 38.7%) with the lower phase mobile and from 1.02 (Sf = 67.8%) to 1.14 (Sf = 47.4%) with the upper phase mobile, respectively. The second series of experiments was similarly performed with a more hydrophobic two-phase solvent system composed of hexane–ethyl acetate–methanol–0.1 M hydrochloric acid (1:1:1:1, v/v/v/v) to separate three DNP-amino acids, DNP-glu, DNP-β-ala and DNP-ala, at a flow rate of 1 ml/min at 1000 rpm. When the column angle was changed from 0° to 90°, Rs increased from 1.38 (1st peak/2nd peak) and 1.20 (2nd peak/3rd peak) (Sf = 61.1%) to 1.66 and 1.45 (Sf = 34.4%) with the lower phase mobile and from 1.14 and 0.63 (Sf = 72.2%) to 1.53 and 0.87 (Sf = 51.1%) with the upper phase mobile, respectively. The overall results of our studies indicate that increasing the column angle against the radially acting centrifugal force enhances the mixing of two phases in the column to improve the peak while decreasing the stationary phase retention by interrupting the laminar flow of the mobile phase.