Centrifugal Precipitation Chromatography

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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.