在线单柱二维液相色谱法快速纯化牛胰腺中细胞色素C

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.

On-line fast purification of cytochrome C from bovine pancreas by two-dimensional liquid chromatography using a single column

Cytochrome C （Cyt C） from bovine pancreas was fast purified by on-line two-dimensional liquid chromatography with a single column （on-line 2D-LC-1C）. The single column was called as two-dimensional column of weak-cation exchange chromatography （WCX） and hydrophobic interaction chromatography （HIC） denoted with 2D （WCX, HIC） column. After the coarse extract from bovine pancreas was directly injected into the 2D column, the WCX as the first separation mode under gradient elution was employed to purify Cyt C and on-line collecting with simultaneous storing the whole fraction containing Cyt C in the attached collection loops. Subsequently the whole stored fraction was also discharged and then re-injected into the same 2D column with simultaneously carrying out the sample buffer exchange for the second purification with HIC separation mode. During the foregoing process, the whole purification of Cyt C with on-line 2D-LC-1C was carried out in a closed system having positive pressure in 52 min. The purity and mass recovery of the finally obtained Cyt C were determined to be 94.7% and 80.5%, respectively. It would be expected that the presented method should lay on a methodological basis for the purification of other active proteins existed in pancreas, and also enlarged to preparative and productive scales.