论文部分内容阅读
用二维(弱阳,疏水)色谱柱首次完成了在线单柱二维液相色谱法快速纯化牛胰腺中的细胞色素C.在将牛胰腺粗提液进样到该二维色谱柱后,在弱阳离子交换模式下,以梯度洗脱方式进行一维色谱分离,并将分离得到的细胞色素C样品液收集到色谱仪的附加样品储液管内.然后将储液管中样品液全部排出,并二次进样到同一根二维色谱柱中,与此同时也完成了对该样品液的缓冲溶液交换,按疏水色谱(HIC)分离模式进行分离.最终对细胞色素C完成了第二维的HIC纯化.上述全部操作均为在线,在一具有正压的封闭体系中进行并可在52分钟内完成.细胞色素C的最终产品纯度高达94.7%(RSD=1.91%),质量回收率为80.5%(RSD=2.20%).预计此在线单柱二维液相色谱法也可能用于牛胰腺中其他功能蛋白的快速纯化,并可能将其放大到制备和生产规模.
The online single-column two-dimensional liquid chromatography was used for the first time to purify the cytochrome C in bovine pancreas with a two-dimensional (weak-cation, hydrophobic) column C. After the bovine pancreatic crude extract was injected into the two-dimensional column, In the weak cation exchange mode, a one-dimensional chromatographic separation was performed by gradient elution, and the isolated cytochrome C sample liquid was collected into an additional sample reservoir of the chromatograph, and all the sample liquid in the reservoir was discharged, And the second injection into the same two-dimensional column, at the same time also completed the sample solution buffer exchange, according to the hydrophobic chromatographic (HIC) separation mode for the final completion of the cytochrome C second dimension HIC. All of the above operations were performed online and in a closed system with positive pressure and could be completed in 52 minutes.The final purity of cytochrome C was as high as 94.7% (RSD = 1.91%), and the recovery rate was 80.5% (RSD = 2.20%). This online single-column two-dimensional liquid chromatography is also expected to be used for the rapid purification of other functional proteins in bovine pancreas and may be scaled up to production and production scales.