Centrifugal Precipitation Chromatography

离心沉淀层析法的发展一直在继续。开发了一种新的离心沉淀色谱制备系统，并对其进行了测试。通过将透析管(退化纤维素，4 mm ID，Spectrum，CA，USA)插入长的卷曲的特氟龙管(平均内径0.6 mm，长220 cm)(Zeus Industrial Products，Raritan，NJ，USA)，从而将管空间纵向分成两个通道，从而将管空间纵向分成两个通道：透析管内部的一个通道(AS通道)用于洗脱浓缩的硫酸铵溶液，而透析管外部的另一通道(样品通道)用于洗脱磷酸盐缓冲液和蛋白质样品。该柱以螺旋形安装在铝板上，从而通过径向离心力梯度保持硫酸铵梯度。柱子以每分钟1000转的速度旋转。该方法采用了与原系统基本相同的原理，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。进入样品通道的蛋白质随着梯度中硫酸铵浓度的逐渐降低而经历重复沉淀和溶解，最后根据它们在硫酸铵溶液中的溶解度从柱上洗脱出来。利用该制备体系，在硫酸铵的线性浓度梯度下(76%~19%饱和6h)，对人血清白蛋白和丙种球蛋白进行了基础研究。结果表明，该方法可以成功地分离出500 mg蛋白质。使用更长和/或更大的ID柱可以进一步增加样品装载能力。

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.