Optimal elution modes were determined for four typical two-phase solvent systems each with different physical parameters to achieve the best peak resolution and retention of the stationary phase by spiral tube high-speed countercurrent chromatography using a suitable set of test samples. Both retention of the stationary phase and partition efficiency are governed by an interplay between two forces, i.e., Archimedean Screw force and radial centrifugal force gradient of the spiral channel. In the polar solvent system represented by 1-butanol./acetic acid/water (4:1:5, v/v/v) with settling time of over 30 s, the effect by the radial centrifugal gradient force dominates giving the best separation of dipeptides either by pumping the lower phase from the inner terminal or the upper phase from the outer terminal of the spiral channel. In the moderately hydrophobic two-phase solvent system represented by hexane/ethyl acetate/methanol/0.1 M HCl (1:1:1:1) with settling time of 19 s, and two hydrophobic solvent systems of hexane/ethanol/water (5:4:1, v/v/v) and non-aqueous binary system of hexane/acetonitrile both having settling time of 9, the effect of the Archimedean screw force play a major role in hydrodynamic equilibrium, giving the best separations by pumping the lower phase from the head or the upper phase from the tail of the spiral channel.
The original spiral tube support (STS) assembly is improved by changing the shape of the tubing, with 1-cm presses perpendicularly along the length. This modification interrupts the laminar flow of the mobile phase. The tubing in the 4 return grooves to the center of the rotor is flattened by a specially made pressing tool to decrease the dead volume and thus increase the column efficiency. The performance of this spiral tube assembly was tested in separations of dipeptides and proteins with suitable polar two-phase solvent systems. The results revealed that the present system yields high partition efficiency with a satisfactory level of stationary phase retention in a short elution time. The present high-speed counter-current chromatographic system will be efficiently applied to a broad spectrum of two-phase solvent systems including aqueous-aqueous polymer phase systems which are used for separation of biopolymers such as proteins and nucleic acids. . Keywords spiral tube support assembly; high-speed counter-current chromatography; Separation of proteins and peptides; polymer phase system (TPAS)
A novel counter-current chromatographic system is developed by mounting a vortex column on a type-I coil planet centrifuge. The column is fabricated from a high-density polyethylene disk (16 cm diameter and 5 cm thick) by making multiple holes of various diameters (3-12.5 mm) each arranged in a circle and connected with narrow transfer ducts. The performance of this vortex column is tested with three different two-phase solvent systems with a broad range in hydrophobicity. The results indicated that the smallest diameter column (3 mm diameter, 120 units with 42.8 ml capacity) yielded the best separation with the height equivalent to a theoretical plate of 2 cm compared with 20 cm required by the conventional multilayer coil column of high-speed CCC. By avoiding the use of an Archimedean Screw force, the system shows a low column pressure which would permit safe operation of a large preparative column without a risk of leakage of solvent and column damage.
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