Partition Efficiency of Newly Designed Locular Multilayer Coil for Countercurrent Chromatographic Separation of Proteins Using Small-Scale Cross-Axis Coil Planet Centrifuge with Aqueous-Aqueous Polymer Phase Systems

Abstract

Countercurrent chromatographic performance of the locular multilayer coil separation column newly designed in our laboratory, was evaluated in terms of theoretical plate number, peak resolution, and retention of the stationary phase in protein separation with an aqueous polymer phase system, using the small-scale cross-axis coil planet centrifuge (X-axis CPC) fabricated in our laboratory. The locular column was made from 1.0 mm I.D., 2.0 mm O.D., or 1.5 mm I.D., 2.5 mm O.D. PTFE tubing, compressed with a pair of hemostat at 2 or 4 cm intervals. The protein separation was performed using a set of stable proteins including cytochrome C, myoglobin, and lysozyme with the 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate system under 1000 rpm of column revolution. The 1.5 mm I.D., 2.5 mm O.D. locular tubing compressed at 2 cm intervals yielded better partition efficiencies than the non-clamped tubing, using both lower and upper mobile phases with satisfactory retention of the stationary phase. The overall results suggest that the newly designed locular multilayer coil is useful to the preparative separation of proteins with aqueous-aqueous polymer phase system using our small scale X-axis CPC.

Keywords:

• Aqueous-aqueous polymer phase system
• Countercurrent chromatography
• Cross-axis coil planet centrifuge
• Locular multilayer coil
• Protein
• Separation

Notes

Abbreviations: Cyt C = cytochrome C; Myo = myoglobin; Lys = lysozyme. The average K values (C_U/C_L) for each protein were 0.09 for Cyt C, 0.56 for Myo and 1.73 for Lys, respectively.

Abbreviations: Lys = lysozyme; Myo = myoglobin. The average K values (C_L/C_U) for each protein were 0.92 for Lys and 2.41 for Myo, respectively.