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.

Figure 1

Schematic illustration of the newly designed locular tubing.

Figure 2

CCC separations of proteins obtained by 1.0 mm I.D., 2.0 mm O.D. PTFE locular multilayer coiled columns with a lower mobile phase. Experimental conditions: apparatus: small-scale X-axis CPC with three multilayer coil assemblies; total column capacity: (A) 54.0 mL, (B) 53.0 mL and (C) 52.0 mL; sample: cytochrome C (2 mg), myoglobin (8 mg) and lysozyme (10 mg); solvent system: 12.5% (w/w) PEG 1000 – 12.5% (w/w) dibasic potassium phosphate; mobile phase: lower phase (outward elution); flow rate: 0.4 mL/min; revolution: 1000 rpm (counterclockwise). SF = solvent front.

Figure 3

CCC separations of proteins obtained by locular multilayer coiled columns of 1.5 mm I.D., 2.5 mm O.D. PTFE tubing with a lower mobile phase. Experimental conditions: total column capacity: (A) 102.0 mL, (B) 98.0 mL and (C) 97.0 mL; flow rate: 0.8 mL/min. Other experimental conditions are same as those described in Figure 1 caption. SF = solvent front.

Figure 4

CCC separations of proteins obtained from the locular multilayer coiled columns of 1.0 mm I.D., 2.0 mm O.D. PTFE tubing with an upper mobile phase. Experimental conditions: sample: myoglobin (8 mg) and lysozyme (10 mg); mobile phase: upper phase (inward elution); revolution: 1000 rpm (clockwise). Other experimental conditions are same as those described in Figure 1 caption. SF = solvent front.

Figure 5

CCC separations of proteins obtained from the locular multilayer coiled columns with 1.5 mm I.D., 2.5 mm O.D. PTFE tubing with upper phase mobile. Experimental conditions: total column capacity: (A) 102.0 mL, (B) 98.0 mL and (C) 97.0 mL; sample: myoglobin (8 mg) and lysozyme (10 mg); mobile phase: upper phase (inward elution); revolution: 1000 rpm (clockwise). Other experimental conditions are same as those described in Figure 1 caption. SF = solvent front.