Zhi Wen-Bo, Deng Qiu-Yun, Song Jiang-Nan, Ouyang Fan
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, CAS, Beijing 100080, China.
Sheng Wu Gong Cheng Xue Bao. 2005 Jan;21(1):129-34.
High-speed counte-recurrent chromatography (HSCCC) is a continuous liquid-liquid partition chromatography without solid matrix, which has the significant features of high resolution and high recovery. The separation of bio-macromolecule in aqueous two-phase systems (ATPs) with HSCCC is still under research, and the establishment of high-speed counter-current aqueous two-phase chromatography (HSCCC-ATP) relies on the improvement of equipment structure and optimization of operation parameters. By using a multi-column high-speed counter-current chromatograph, the separation of protein mixture and the purification of ovalbumin from hen egg white were studied. The effects of pH and PEG concentration on the partition coefficients of proteins were tested in PEG1000-phosphate ATPs, and distinct differences among partition coefficients of proteins were found at pH 9.2 and 15.0% (W/W) PEG concentration in said system. The separation of protein mixture, consisting of cytochrome C, lysozyme and myoglobin was successfully performed in 15.0% (W/W) PEG1000-17.0% (W/W) potassium phosphate ATPs at pH 9.2 with high-speed counter-current chromatograph at rotation speed of 850r/min and flow rate of 0.8mL/min, using upper phase as stationary phase. pH and PEG concentration also had distinct effects on the partition coefficients of the major protein components in hen egg white, including ovaltransferrin, ovalbumin and lysozyme. The optimal pH value and PEG concentration for the purification of ovalbumin by HSCCC-ATP were found to be 9.2 and 16.0% (W/W) respectively. Ovalbumin was successfully purified to homogeneity from the hen egg white sample in 16.0% (W/W) PEG1000-17.0% (W/W) potassium phosphate ATPs at pH 9.2 with high-speed counter-current chromatograph at rotation speed of 850r/min and flow rate of 1.8mL/min, using upper phase as stationary phase. The purification recovery of ovalbumin was around 95%.
高速逆流色谱法（HSCCC）是一种无固体基质的连续液-液分配色谱法，具有高分辨率和高回收率的显著特点。利用HSCCC在双水相体系（ATPs）中分离生物大分子仍在研究中，而高速逆流双水相色谱法（HSCCC-ATP）的建立依赖于设备结构的改进和操作参数的优化。使用多柱高速逆流色谱仪，研究了蛋白质混合物的分离以及从鸡蛋清中纯化卵清蛋白。在聚乙二醇1000-磷酸盐双水相体系中测试了pH值和聚乙二醇浓度对蛋白质分配系数的影响，发现在该体系中pH值为9.2且聚乙二醇浓度为15.0%（W/W）时，蛋白质的分配系数存在明显差异。在pH值为9.2、聚乙二醇1000为15.0%（W/W）-磷酸钾为17.0%（W/W）的双水相体系中，以高速逆流色谱仪在850r/min的转速和0.8mL/min的流速下，以上相为固定相，成功分离了由细胞色素C、溶菌酶和肌红蛋白组成的蛋白质混合物。pH值和聚乙二醇浓度对鸡蛋清中主要蛋白质成分（包括运铁蛋白、卵清蛋白和溶菌酶）的分配系数也有明显影响。发现通过HSCCC-ATP纯化卵清蛋白的最佳pH值和聚乙二醇浓度分别为9.2和16.0%（W/W）。在pH值为9.2、聚乙二醇1000为16.0%（W/W）-磷酸钾为17.0%（W/W）的双水相体系中，以高速逆流色谱仪在850r/min的转速和1.
