A novel chromatographic system is introduced. The system internally generates a concentration gradient of ammonium sulfate (AS) along a long channel to fractionate proteins according to their solubility in AS solution. The separation column consists of a pair of disks with mutually mirror-imaged spiral channels that are separated by a semipermeable membrane. The disk assembly is mounted on a sealless continuous flow centrifuge. Concentrated As solution is introduced into the upper channel while a water solution is passed through the lower channel in the opposite direction in a rotating column. A mixture of proteins injected into the water channel moves along an AS gradient of increasing concentration that has been established in the water solution. Each protein species precipitates at a different AS concentration along the gradient. The eluate is continuously monitored and collected using a fraction collector. A series of basic experiments was performed to study the rates of AS transfer and osmosis through the membrane, and the operational parameters including elution time, revolution speed, inclination of the gradient and sample size were optimized using stable protein samples. Preliminary applications were successful in purification of monoclonal antibody from cell culture supernatnat and affinity separatoin of recombinant ketosteroid isomerase from a crude E. coli lysate. - purification, precipitation, ammonium sulfate, proteins, centrifugation, sealless continuous flow centrifuge
