Countercurrent Chromatographic Separation of Proteins by Cross-Axis Coil Planet Centrifuge: Choice of Polymer Phase Systems and Revolution Speed

Abstract

Countercurrent chromatographic separation of proteins by the cross-axis coil planet centrifuge was maximized by selecting the suitable polymer phase system and revolution speed. Polymer phase systems composed of polyethylene glycol (PEG) 1000 and several inorganic salts were examined to determine partition coefficient values (K) for various proteins. The overall results indicated that the polymer phase system composed of 12.5% (w/w) PEG 1000 and 12.5% (w/w) dibasic potassium phosphate yielded suitable K values for most proteins except for cytochrome C and apo-transferrin which may be separable with a solvent system composed of 12.5% (w/w) PEG 1000 and 24% (w/w) potassium citrate. A series of experiments with the PEG 1000 - potassium phosphate system under various revolution speeds revealed that the best separation was achieved at 850 rpm. The above optimized conditions may be applied to separations of other protein samples.