Spiral Disk Assembly For Countercurrent Chromatography

Heart, Lung, And Blood Institute

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Abstract

A set of four separation disks equipped with spiral channel(s) was designed for our type-J HSCCC centrifuge to improve retention of the stationary phase of polar solvent systems. Four different spiral disks were tested: two had a single spiral channel with different depths and the two had four spiral channels connected in series to provide a greater spiral pitch. Performance of each disk was tested in terms of partition efficiency and/or stationary phase retention using three different two-phase solvent systems including 1-butanol/acetic acid/water (4:1:5, v/v/v)for dipeptide separation; 12.5%(w/w)polyethylene glycol(PEG)1000-12.5%(w/w)dibasic potassium phosphate for protein separation; and 4%(w/w)PEG8000-5%(w/w)dextranT500 in 10mM dibasic sodium phosphate for determination of stationary phase retention. The results show that the spiral column retains a satisfactory amount of stationary phase for all solvent systems even at a relatively high mobile phase flow rate where the spiral pitch plays a significant role. This is particularly important in retention of the PEG-dextran polymer phase system. Separation of dipeptides with the butanol solvent system was possible using a high flow rate of the mobile phase and 4-spiral-disks with their greater spiral pitch. In protein separations with the PEG-phosphate system, the single-spiral disks yielded the best separation using the upper phase in head to tail elution mode, while the 4-spiral disks show higher retention of the stationary phase. The retention of the PEG-dextran system is much improved in the 4-spiral disks which exceed 60% at a flow rate of 0.5 ml/min. Preliminary separations of a set of dipeptide samples and proteins were successfully performed using a spiral disk assembly consisting of 8 units of a single spiral disk.