Spiral disk assembly for HSCCC: Column design and basic studies on chromatographic resolution and stationary phase retention

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 I 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 How rate of 0.5 mL/min. Various parameters, which affect the performance of the spiral disk separation column are discussed.