Effects of the Planetary Motion of a Coiled Column on Protein Separation by the Nonsynchronous Coil Planet Centrifuge

Abstract

The effects of rotational speed and direction of revolution of the coiled separation column on protein separation were examined using a rotary‐seal‐free nonsynchronous coil planet centrifuge (CPC) fabricated in our laboratory. This apparatus has a unique feature that allows a freely adjustable rotational speed of the coiled separation column at a given revolution speed. The separation was performed using a set of stable proteins including cytochrome C, myoglobin, and lysozyme with an aqueous–aqueous polymer phase system composed of 12.5% (w/w) polyethylene glycol (PEG) 1000 and 12.5% (w/w) dibasic potassium phosphate. A series of experiments revealed that the head to tail elution mode produced better stationary phase retention and higher peak resolution regardless of the choice of the mobile phase. The best result was obtained in the head to tail elution mode by the clockwise (CW) coil rotation for the lower mobile phase or by the counterclockwise (CCW) coil rotation for the upper mobile phase, both under CCW revolution of the rotor.

Keywords:

• Nonsynchronous coil planet centrifuge
• Planetary motion
• Protein separation
• Countercurrent chromatography
• Polymer phase system
• Retention of stationary phase
• Cytochrome C
• Myoglobin
• Lysozyme

Acknowlegments

The authors would like to thank Misters Tomoaki Tanaka and Hayato Watanabe for their technical assistance, and Mr. Haruo Sasaki (Machining Technology Center, College of Science and Technology, Nihon University, Chiba, Japan) for the electric adjustment of the nonsynchronous CPC apparatus. This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.