Novel analytical spiral tube assembly for protein separation by high-speed countercurrent chromatography

National Heart, Lung, And Blood Institute

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Abstract

1. A new spiral column assembly for analytical separation by high-speed countercurrent chromatography is described. The column is made from a plastic spiral tube support which has 12 interwoven spiral grooves. The PTFE tubing of 1.6 mm ID was first flattened by extruding through a narrow slit and inserted into the grooves to make 5 spiral layers with about 60 ml capacity. The performance of the spiral column assembly was tested with the separation of three stable protein samples including cytochrome C, myoglobin and lysozyme in a polymer phase system composed of polyethylene glycol 1000 and dibasic potassium phosphate each at 12.5 % (w/w) in water. At 2 ml/min, three protein samples were well resolved in one hour. The separation tme may be further shortened by application of higher revolution speed and flow rate after improving the strength of the spiral tube support in the future. 2. Novel spiral tube assembly for semipreparative sparation of proteins by high-speed countercurrent chromatography A new spiral column assembly for preparative separation of proteins by high-speed countercurrent chromatography is described. The column is made from a plastic spiral tube support which has 4 interwoven spiral grooves with 12 radial grooved to accommodate interconnecting tubing. Two separation columns was fabicated. One was made from PTFE tubing of 1.6 mm ID by inserting it into the spiral grooves to form 13 spiral layers with 135 ml capacity. The other column was made from the same tubing by first flattening by extruding it through a narrow slit followed by twisting along its axis to form flat-twisting tubing by inserting it into spiral grooves to form 10 sprial layer with a total capacity of 90 ml. The performance of these spiral column assemblies were first tested on the separation of three stable protein samples including cytochrome C, myoglobin and lysozyme in a polymer phase system composed of polyethylene glycol 1000 and dibasic potassium phosphate each at 12.5 % (w/w) in water at flow rates of 1, 2, 3 and 5 ml/min and the revolution speeds of 800 and 1000 rpm. Three protein samples were well resolved in both columns at 1000 rpm, while the modified flat-twisted column yielded slightly higher separaton efficiency. The separation may be further improved by application of higher revolution speed and flow rate after improving the strength of the spiral tube support in the future.