Development of centrifugal precipitation chromatography has been continued. A new preparative system of centrifugal precipitation chromatography is developed and tested. The column is made by inserting a dialysis tubing (degenerated cellulose, 4 mm ID, Spectrum, CA, USA) into a long convoluted Teflon tube (0.6 mm average ID and 220 cm long) (Zeus Industrial Products, Raritan, NJ, USA), thus dividing the tube space longitudinally into two channels: one channel (AS channel) inside the dialysis tubing is used to elute a concentrated ammonium sulfate solution and the other channel (sample channel) outside of the dialysis tubing to elute phosphate buffer and protein samples. The column is mounted on an aluminum plate in a spiral configuration so that the ammonium sulfate gradient is preserved by the radial centrifugal force gradient. The column is rotated rotated at 1000 rpm. The method uses basically the same principle of the original system, in that a concentration gradient of precipitant (ammonium sulfate) is generated along the spiral channel under a centrifugal force field. Proteins injected into the sample channel are subjected to repetitive precipitation and dissolution as the ammonium sulfate concentration in the gradient is gradually reduced, and finally elute out from the column according to their solubility in the ammonium sulfate solution. Using this preparative system a basic study was performed to separate human serum albumin and gamma-globulin as test samples under a linear concentration gradient of ammonium sulfate (76% to 19% saturation for 6 hours). The results show that the present method can successfully separate 500 mg of proteins. The sample loading capacity may be further increased using a longer and/or larger ID column.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Intramural Research (Z01)
Project #
Application #
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
U.S. National Heart Lung and Blood Inst
United States
Zip Code
Baldermann, Susanne; Fleischmann, Peter; Bolten, Mareike et al. (2009) Centrifugal precipitation chromatography, a powerful technique for the isolation of active enzymes from tea leaves (Camellia sinensis). J Chromatogr A 1216:4263-7
Inui, Masafumi; Fukui, Akimasa; Ito, Yuzuru et al. (2006) Xapelin and Xmsr are required for cardiovascular development in Xenopus laevis. Dev Biol 298:188-200
Cao, Xue-Li; Xu, Ya-Tao; Zhang, Guang-Ming et al. (2006) Purification of coenzyme Q10 from fermentation extract: high-speed counter-current chromatography versus silica gel column chromatography. J Chromatogr A 1127:92-6
Shibusawa, Yoichi; Takeuchi, Naoko; Sugawara, Kazusa et al. (2006) Aqueous-aqueous two-phase systems composed of low molecular weight of polyethylene glycols and dextrans for counter-current chromatographic purification of proteins. J Chromatogr B Analyt Technol Biomed Life Sci 844:217-22
Shibusawa, Yoichi; Yamakawa, Yutaka; Noji, Ryoko et al. (2006) Three-phase solvent systems for comprehensive separation of a wide variety of compounds by high-speed counter-current chromatography. J Chromatogr A 1133:119-25
Takeda, Naoya; Kondo, Masashi; Ito, Satoru et al. (2006) Role of RhoA inactivation in reduced cell proliferation of human airway smooth muscle by simvastatin. Am J Respir Cell Mol Biol 35:722-9
Ito, Yoichiro (2005) Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography. J Chromatogr A 1065:145-68
Yu, Henry; Ito, Yoichiro (2004) Preparative separation of proteins using centrifugal precipitation chromatography based on solubility in ammonium sulfate solution. Prep Biochem Biotechnol 34:1-12
Ito, Y (2000) Centrifugal precipitation chromatography: principle, apparatus, and optimization of key parameters for protein fractionation by ammonium sulfate precipitation. Anal Biochem 277:143-53