Evaluation of the performance of protein separation in figure-8 centrifugal counter-current chromatography

The performance of protein separation using the figure-8 column configuration in centrifugal counter-current chromatography was investigated under various flow rates and revolution speeds. The separation was performed with a two-phase solvent system composed of polyethylene glycol 1000/potassium phosphate each at 12.5% (w/w) in water and with lysozyme and myoglobin as test samples. In order to improve tracing of the elution curve, a hollow fiber membrane dialyzer was inserted at the inlet of the UV detector. The results showed that the retention of stationary phase (Sf) and resolution (Rs) increased with decreased flow rate and increased revolution speed. The highest Rs of approximately 1 was obtained at a flow rate of 0.01 mL/min under a revolution speed of 1200 rpm with a 3.4 mL capacity column.

► Protein separation was performed with figure-8 columns in centrifugal CCC.
► Tracing of the elution curve was improved with an on-line dialyzer.
► The highest peak resolution was achieved at 0.01 mL/min flow at 1200 rpm.