Comparison in partition efficiency of protein separation between four different tubing modifications in spiral high-speed countercurrent chromatography.

Robert Clary,Yoichiro Ito

doi:10.3390/separations3040031

Abstract

High-speed countercurrent chromatography with a spiral tube assembly can retain a satisfactory amount of stationary phase of polymer phase systems used for protein separation. In order to improve the partition efficiency a simple tool to modify the tubing shapes was fabricated, and the following four different tubing modifications were made: intermittently pressed at 10 mm width, flat, flat-wave, and flat-twist. Partition efficiencies of the separation column made from these modified tubing were examined in protein separation with an aqueous-aqueous polymer phase system at flow rates of 1-2 ml/min under 800 rpm. The results indicated that the column with all modified tubing improved the partition efficiency at a flow rate of 1 ml/min, but at a higher flow rate of 2 ml/min the columns made of flattened tubing showed lowered partition efficiency apparently due to the loss of the retained stationary phase. Among all the modified columns, the column with intermittently pressed tubing gave the best peak resolution. It may be concluded that the intermittently pressed and flat-twist improve the partition efficiency in a semi-preparative separation while other modified tubing of flat and flat-wave configurations may be used for analytical separations with a low flow rate.

Highlights

Countercurrent chromatography (CCC) uses two-phase solvent systems without solid support [1,2,3,4,5], and it has an advantage over the conventional column chromatography for eliminating the risk of sample loss on the solid support matrix used in liquid chromatography
Partition efficiency and retention of the stationary phase of these separations are all normalized to a 60 mL column capacity for fair comparison (Table 1)
The flattened portion of the tubing will interfere with the laminar flow of the mobile phase, which would cause the sample band broadening to decrease the peak resolution

Summary

Introduction

Countercurrent chromatography (CCC) uses two-phase solvent systems without solid support [1,2,3,4,5], and it has an advantage over the conventional column chromatography for eliminating the risk of sample loss on the solid support matrix used in liquid chromatography. CCC systems have been developed and their partition efficiency has been examined in terms of theoretical plate number, peak resolution, and retention of the stationary phase. Among those CCC systems, high-speed countercurrent chromatography (HSCCC) has been most widely used for the separation of natural and synthetic products [6,7,8]. This system uses a type-J planetary centrifuge, which rotates a coiled separation column in such a way that the column synchronously rotates about its own axis while revolving around the central axis of the centrifuge. The separation column is fabricated by winding a long piece of polytetrafluoroethylene (PTFE) tubing coaxially around the column holder, making multiple

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Results

Conclusion