流动相组成、浓度和pH对蛋白质在金属螯合柱上的保留特性的影响

Influences of the Mobile Phase Constitution,Salt Concentration and pH Value on Retention Characters of Proteins on the Metal Chelate Column

系统研究了流动相中盐的性质和浓度、溶液 pH以及竞争配体对蛋白质在金属螯合色谱中保留值的影响。导出了描述蛋白质在金属螯合色谱中保留特征的数学表达式 ,提出用洗脱强度指数ε表征盐溶液的洗脱能力。根据不同色谱条件下蛋白质的保留特性 ,发现蛋白质在金属螯合色谱中的保留是配位、静电和疏水的协同作用。对与蛋白质强结合的金属螯合柱 ,以配位作用为主 ,静电作用为辅 ;对弱结合的金属柱 ,以静电作用为主 ,配位作用为辅。在流动相中加入高浓度非成络盐 ,可增强蛋白质和固定相间的疏水作用。利用金属螯合配体和蛋白质间的作用力与色谱条件的相关性 ,对不同类型金属螯合柱可选择不同的洗脱方式。对弱结合的金属螯合柱可用低浓度盐或 pH梯度洗脱 ,对强结合的金属螯合柱通常用竞争洗脱剂洗脱。实验证明 ,NH3 是一种优秀的竞争洗脱剂 ,它不仅效能高 ,而且价廉 ,固定金属流失少 ,浓度易控制。改变色谱条件可改变金属螯合配体和蛋白质间的作用力 ,提高金属螯合色谱的选择性。

The effects of the nature and concentration of salts, pH value and competitive eluent in the mobile phase on the protein retention have been systematically investigated. A mathematical expression describing the protein retention in metal chelate chromatography has been derived. It is proposed that the eluting power of the salt solution can be expressed by the eluent strength exponent ε . According to the retention characters of protein under different chromatographic conditions, the interaction between the various metal chelate ligands and proteins is discussed. The protein retention on the metal chelate column is a cooperative interactions of coordination, electrostatic and hydrophobic interaction. For the strong combined metal column with proteins such as IDA Cu, the coordination is the most important, and the electrostatic interaction is secondary in chromatographic process. However, for the weak combined metal columns with proteins such as IDA Ni, IDA Co and IDA Zn, the electrostatic interaction between the metal chelate ligands and proteins is the chief one, while the coordination is the next in importance. When the mobile phase contains high concentration of salt which can't form complex with the immobilized metal, the hydrophobic interaction between the protein and stationary phase will be increased. As the interaction between the metal chelate ligand and proteins relates to chromatographic operating conditions closely, different elution processes may be selected for different metal chelate columns. The gradient elution is generally performed by the low concentration of salt or different pH for weakly combined columns with proteins, however the competitive elution procedure is commonly utilized for strongly combined column. The experiment showed that NH 3 is an excellent competitive eluent. It isn't only give the efficient separation of proteins, but also has the advantages of cheapness, less bleeding of the immobilized metals and ease of controlling NH 3 concentration. The interaction between the metal chelate ligand and proteins and the selectivity of metal chelate chromatography can be changed through changing chromatographic conditions.