Centrifugal Precipitation Chromatography

离心沉淀色谱的发展和应用一直在继续。 该系统内部产生一个浓度梯度的硫酸铵（AS）沿着一个长通道，以破碎蛋白质根据其在AS溶液中的溶解度。 分离柱由一对圆盘组成，圆盘上有相互镜像的螺旋通道，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。 浓缩的As溶液被引入到上部通道中，而水溶液在旋转柱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。每种蛋白质物质在不同的AS浓度下沿梯度沿着沉淀。使用馏分收集器连续监测和收集馏分。通过一系列的基础实验研究了AS在膜中的迁移和渗透速率，并以稳定的蛋白质样品为研究对象，对洗脱时间、转速、梯度倾角和样品量等操作参数进行了优化。为提高分离效率，设计了一套不同通道结构的新型柱，并初步应用于从细胞培养上清和腹水中纯化单克隆抗体，以及从大肠杆菌粗品中亲和分离重组甾酮异构酶。大肠杆菌裂解物。该方法也被成功地应用到其他生物聚合物，如透明质酸，硫酸软骨素，和聚合的儿茶素使用合适的有机溶剂和水之间的梯度的分馏。

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, and polymerized catechins using a gradient between suitable organic solvents and water.