An improved design of spiral tube assembly for separation of proteins by high-speed counter-current chromatography

A new spiral tube assembly was designed to improve the column capacity and partition efficiency for protein separation. This spiral tube assembly has greater column capacity than the original tubing because of an increase in radial grooves from 4 to 12 to accommodate more spiral layers and 12 narrow spots instead of 4 in each circular loop to interrupt the laminar flow that causes sample band broadening. Standard PTFE tubing (1.6 mm ID) and the modified flat-twisted tubing were used as the separation column. The performances of both assemblies were compared for separating three stable test proteins including cytochrome c, myoglobin, and lysozyme using a two phase aqueous-aqueous solvent system composed of polyethylene glycol 1000 (12.5% w/w) and dibasic potassium phosphate (12.5% w/w). All samples were run at 1, 2, 3, and 5 mL/min at both 800 rpm and 1000 rpm. The separation of these three protein samples produced high stationary phase retentions at 1, 2, and 3 mL/min, yet separated efficiently at 5 mL/min in 40 min. After comparing the separation efficiency in terms of the peak resolutions, theoretical plate numbers, and separation times, it was determined that the flat-twisted tubing was more effective in separating these protein samples. In order to validate the efficacy of this novel assembly, a mixture of five protein samples (cytochrome c, myoglobin, ovalbumin, lysozyme, and hemoglobin) were separated, under the optimal conditions established with these three protein samples, at 1 mL/min with a revolution speed of 1000 rpm. There were high stationary phase retentions of around 60%, with effective separations, demonstrating the efficiency of the flat-twisted spiral tube assembly. The separation time of 6 h was a limitation but can potentially be shortened by improving the strength of the column that will permit an increase in revolution speed and flow rate. This novel spiral separation column will allow rapid and efficient separation of mixtures with high yield of the constituent components.