Centrifugal Precipitation Chromatography

离心沉淀色谱法的发展仍在继续。研制了一种新的离心沉淀色谱制备系统并进行了试验。将透析管（降解纤维素，4毫米内径，Spectrum， CA， USA）插入一个长弯曲的特氟龙管（平均内径0.6毫米，长220厘米）（Zeus Industrial Products, ariitan， NJ， USA），从而将管空间纵向划分为两个通道：透析管内部的一个通道（AS通道）用于洗脱浓硫酸铵溶液，透析管外部的另一个通道（样品通道）用于洗脱磷酸盐缓冲液和蛋白质样品。柱被安置在塑料离心机头上的螺旋槽中。透明的盖子应用于柱上，允许在分离期间对柱进行立体观察。该方法基本上采用与原系统相同的原理，即在离心力场作用下沿螺旋通道产生沉淀剂（硫酸铵）的浓度梯度。随着梯度中硫酸铵浓度的逐渐降低，注入样品通道的蛋白质经过反复沉淀和溶解，最终根据其在硫酸铵溶液中的溶解度从色谱柱中洗脱出来。利用该制备系统，在硫酸铵线性浓度梯度下（饱和度76% ~ 19%，持续6小时）分离人血清白蛋白和γ -球蛋白作为测试样品进行了基础研究。结果表明，采用硫酸铵线性浓度梯度分步法均可成功分离人血清白蛋白和γ -球蛋白。

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 accommodated into a spiral groove made in the plastic centrifuge head. The transparent lid is applied over the column which permit storoboscpic observation of the column during the separation. 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 human serum albumin and gamma-globulin using either stepwise of linear concentration gradient of ammonium sulfate.