Hydrostatic countercurrent chromatography (CCC) reported in the past used a coiled column mounted around the periphery of the centrifuge bowl to carry out analytical separation. In this separation the centrifugal force retains the stationary phase in each turn of the coil, while the mobile phase entirely occupies the other half. Since the mobile phase steadily percolates through the stationary phase by partially displacing it, the volume of the stationary phase retained in the separation column becomes substantially less than 50% or typically about 30 % of the total column space. This low retention of the stationary phase limits the partition efficiency of the system. the amount of the stationary phase retained in the column is one of the important parameters to determine the peak resolution in countercurrent chromatography. Ther higher the retention of the stationary phase, the better peak resolution is obtained. This project introduces new configurations of the toroidal column such as an equilateral triangular coil and various other shapes of tubing distributed on the rotary plate to improve both retention of the stationary phase and peak resolution. The present study uses a rotary-seal-free centrifuge fabricated by Pharma-Tech Research Corporation, Baltimore, Maryland, USA. It holds an aluminum rotary plate about 34 cm in diameter to hold a toroidal separation column. Three column configurations all made of 0.46 mm ID fluorinated plastic tubing (Zeus Industrial Products, Orangeburg, SC, USA) have been examined as follows: 1. triangluar helical column: The column was made by winding the tubing onto a 2 m length of equilateral triangular PTFE (polytetrafluoroethylene) tubing (6 mm each side) making about 2000 turns with a 6.7 ml capacity. The coiled column was arranged at the periphery of the rotary plate by making two and a half spiral turns in such a way that one side of the triangular column is facing to the plate. 2: Zigzag toroidal column: This column design was made by hooking the tubing onto upstanding screws forming sixteen units of zigzag patterns over the rotary plate. Each column unit consisted of 5 zigzag loops each about 3 cm long which was serially connected with a radial transfer tubing, and 16 zigzag units were made in each layer, and the whole column consisted of several layers of zigzag tubing to make a desired column capacity of several milliliteres. 3. Saw-tooth toroidal column: The column is made by hooking the tubing onto upstanding screws around the periphery of the rotary plate to form tubing layout pattern similar to the circular saw-tooth consisting of short radial segments (6 mm) and long spiral loops (ca 3 cm). The column consisted of multiple layers of tubing to make several milliliters of column capacity. Each terminal of all these columns is connected to 0.47 mm ID PTFE flow tube using a set of tubing connectors (Upchurch Scientific, Palm Spring, CA, USA). These flow tubes are put together and passed through the center of the central shaft downward, the hollow horizontal shaft of a miter gear, then led upward into the vertical hollow tube support, and finally exit the centrifuge from the center of the upper plate where they are tightly with a pair of clamps. Each column configuration is tested in separation of dipeptides, tryptophyl-tyrosine and valyl-tyrosone with a two-phase solvent system composed of 1-butanol-acetic acid-water (4:1: 5, v/v). Zigzag and saw-tooth columns were also testfed with an additional two-phase solvent system composed of hexane-ethyl acetate-methanol-0.1M HCl (1:1:1;1, v/v) for separation of DNP-amino acid samples. The overall results indicated that effective column space and stationary phase retention are improved by changing the configuration of the helical column originally used for toroidal coil countercurrent chromatography. The use of an equilateral triangular core for the helix column doubles effective column space and retains the stationary phase over 40% of the total column capacity without increasing the column pressure. Both zigzag and saw-tooth tubing configurations further improved the retention of the staitionary phase at a low flow rate of the mobile phase.
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