Evaluation of the effect of column orientation in type-I high-speed counter-current chromatography

The performance of type-I high-speed counter-current chromatography was evaluated by changing the column inclination against the rotating centrifugal force field. The separations were performed with two different solvent systems composed of 1-butanol-acetic acid-water (4.75:0.25:5, v/v) (BAW) and hexane-ethyl acetate-methanol-0.1 M HCl (1:1:1:1, v/v) (HEMW) using dipeptides and DNP-amino acid as test samples, respectively. A set of short coiled columns connected in series is mounted around the holder hub in two different ways: in the parallel orientation, all column units are arranged in parallel to each other and mounted on the holder at various angles against the horizontal plane. In the zigzag configuration, the neighboring units of the same column are mounted symmetrically forming various angles apart. In the parallel configuration, for both the BAW and HEMW systems, Sf (the retention of stationary phase) first increased as the column angle decreased from 90 degrees to 60 degrees and then decreased, as the column angle further decreased from 60 degrees to 0 degrees, while Rs (peak resolution) continually declined over the entire column angle range from 90 degrees to 0 degrees. But, for both solvent systems, with the zigzag configuration, retention of stationary phase and resolution both decreased as the column angle decreased from 90 degrees to 0 degrees. In general, Sf and Rs for separation of dipeptides in the BAW system, from 90 degrees to 15 degrees, is better for the parallel orientation than for the zigzag configuration. However, at 0 degrees, Sf and Rs are better for the zigzag orientation. In the DNP-amino acid separation with the HEMW system, retention of the stationary phase and Rs for the parallel orientation is better than that for the zigzag orientation from 90 degrees to 30 degrees, whereas from 30 degrees to 0 degrees the results are opposite. Over all results of our studies revealed that the formally used column orientation at 90 degrees inclination yields the highest peak resolution in both solvent systems.