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The importance of column design for protein separation using aqueous two-phase systems on J-type countercurrent chromatography

机译：在J型逆流色谱上使用水性两相系统分离蛋白质的色谱柱设计的重要性

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The separation of lysozyme and myoglobin has been used as a model system with which to study protein separation in aqueous two-phase systems (ATPS) containing 12.5% (w/w) PEG1000 and 12.5% (w/w) K2HPO4 in Brunei multilayer J-type countercurrent chromatography (CCC) centrifuges, where wave mixing pertains. Poor separations of the proteins with this type of CCC columns were observed in ATPS and the proteins did not elute as predicted from their equilibrium partition coefficients. As mass transfer of the proteins appeared to be a limiting factor, methods of improving mixing of the phases in the J-type CCC were explored. This has led to the design, construction and application of a toroidal column fitted to the Brunei Dynamic Extraction Centrifuge, providing pseudo-hydrostatic CCC in which much improved mixing for the stationary and mobile phases was obtained. In this toroidal column the protein mixture was separated with the locations of the protein peaks being consistent with those predicted by the partition coefficients. This toroidal coil system will form the basis for optimisation studies and scaling up separations.
机译：溶菌酶和肌红蛋白的分离已用作模型系统，用于研究文莱多层膜J中含有12.5％（w / w）PEG1000和12.5％（w / w）K2HPO4的水两相系统（ATPS）中的蛋白质分离型混流式（CCC）离心机，其中涉及混波。在ATPS中观察到用这种类型的CCC色谱柱分离的蛋白质差，并且蛋白质没有如其平衡分配系数所预测的那样洗脱。由于蛋白质的质量转移似乎是一个限制因素，因此探索了改善J型CCC中各相混合的方法。这导致了设计，建造和应用适合文莱动态萃取离心机的环形色谱柱，该色谱柱提供了拟静液压CCC，在其中可以大大改善固定相和流动相的混合。在该环形柱中，蛋白质混合物被分离，蛋白质峰的位置与分配系数预测的一致。这种环形线圈系统将构成优化研究和扩大分离的基础。

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