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Abstract
A set of four separation disks equipped with spiral channel(s) was designed for our type‐J high speed countercurrent chromatography (J‐HSCCC) centrifuge to improve retention of the stationary phase of polar solvent systems. Four different spiral disks were tested: two had a single spiral channel with different depths and the other two had four spiral channels connected in series to provide a greater spiral pitch. Performance of each disk was tested in terms of chromatographic resolution and/or stationary phase retention using three different two‐phase solvent systems, including 1‐butanol/acetic acid/water (4:1:5, v/v/v) for dipeptide separation; 12.5% (w/w) polyethylene glycol (PEG) 1000–12.5% (w/w) dibasic potassium phosphate for protein separation; and 4% (w/w) PEG8000–5% (w/w) dextran T500 in 10 mM dibasic sodium phosphate for determination of stationary phase retention. The results show that the spiral column retains a satisfactory amount of stationary phase for all solvent systems, even at a relatively high mobile phase flow rate where the spiral pitch plays a significant role. Separation of dipeptides with the butanol solvent system was possible using 10 mL/min flow rate of the mobile phase and 4‐spiral‐disks with their greater spiral pitch. In protein separations with the PEG–phosphate system, the single‐spiral disks yielded the best separation using the upper phase in tail‐to‐head elution mode at 1 mL/min, while the 4‐spiral disks show higher retention of the stationary phase. The retention of the PEG–dextran system is improved in the 4‐spiral disks, which exceed 60% at a flow rate of 0.5 mL/min. Various parameters, which affect the performance of the spiral disk separation column are discussed.
Acknowledgment
The authors are indebted to Dr. Henry M. Fales of National Institutes of Health for editing the manuscript.