Centrifugal Precipitation Chromatography

离心沉淀色谱法的发展和应用仍在继续。该系统内部沿长通道产生硫酸铵（AS）的浓度梯度，根据其在硫酸铵溶液中的溶解度对蛋白质进行分级。分离柱由一对圆盘组成，它们具有相互镜像的螺旋通道，由半透膜隔开。圆盘组件安装在无密封连续流离心机上。当溶液被引入上部通道时，水溶液在一个旋转的塔中以相反的方向通过下部通道。注入水通道的蛋白质混合物沿着在水溶液中建立的浓度增加的AS梯度移动。每种蛋白质沿着梯度以不同的AS浓度沉淀。洗脱液使用分馏收集器进行连续监测和收集。通过一系列的基础实验研究了AS在膜上的转移和渗透速率，并以稳定的蛋白质样品为研究对象，对洗脱时间、转速、梯度倾角和样品大小等操作参数进行了优化。为了提高分离效率，设计了一组具有不同通道结构的新型色谱柱。初步应用于从细胞培养上清和腹水中纯化单克隆抗体，以及从大肠杆菌裂解液中亲和分离重组酮类固醇异构酶。该方法还成功地应用于其他生物聚合物的分离，如透明质酸、硫酸软骨素、聚合儿茶素和葡聚糖，使用合适的有机溶剂和水之间的梯度。

Development and application of centrifugal precipitation chromatography has been continued. 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. A new set of columns with different channel configuration was made to improve the efficiency of separation. Preliminary applications were successful in purification of monoclonal antibodies from cell culture supernatant and ascitic fluid, and affinity separation of recombinant ketosteroid isomerase from a crude E. coli lysate. The method was also successfully applied to fractionation of other biopolymers such as hyaluronic acid, chondroitin sulfate, polymerized catechins, and dextran using a gradient between suitable organic solvents and water.