化学学报 ›› 2012, Vol. 70 ›› Issue (15): 1631-1636.DOI: 10.6023/A12050217 上一篇    下一篇

研究论文

关于混合模式色谱固定相对蛋白保留特征的研究

加晓丹a, 柯从玉b, 孙萱a, 耿信笃a   

  1. a 西北大学现代分离科学研究所 现代分离科学省级重点实验室 教育部合成与天然功能分子化学重点实验室 西安 710069;
    b 西安石油大学化学化工学院 西安 710065
  • 收稿日期:2012-05-18 出版日期:2012-08-14 发布日期:2012-06-21
  • 通讯作者: 耿信笃
  • 基金资助:

    项目受陕西省重点实验室基金(No. 11JS099)资助.

Studies on the Characterization of Stationary Phase for Protein Retention on Mixed-mode Chromatography

Jia Xiaodana, Ke Congyub, Sun Xuana, Geng Xindua   

  1. a Institute of Modern Separation Science, Northwest University, Provincial Key Laboratory of Modern Separation Science of Shaanxi, Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education of China, Xi'an 710069, China;
    b College of Chemistry & Chemical Engineering, Xi'an Shiyou University, Xi'an 710065, China
  • Received:2012-05-18 Online:2012-08-14 Published:2012-06-21
  • Supported by:

    Project supported by the Foundation of Shaanxi Province Key Labaratory (No. 11JS099).

为使混合机理色谱(MMC)得到广泛地应用, 合成、表征和评价MMC固定相就成了首先要解决的问题. 依据离子交换色谱柱也具有疏水色谱(HIC)保留机理的特征, 选了4种弱阳离子交换(WCX)柱和一根二维[2D(WCX,HIC)]色谱柱, 研究了标准蛋白在这两类色谱柱上的保留行为. 这四种WCX色谱柱中的两种能在WCX和HIC两种分离模式下分离蛋白, 虽不如2D色谱柱效果好, 但有可能当成“准2D柱”来使用. 发现蛋白在这四种WCX柱上所显示的HIC分离特征各不相同, 且保留值随盐浓度变化呈现出的“U型”曲线也有大的差异. 实验结果显示, “U型”曲线的宽度和临界点分别与色谱动力学和热力学因素相关. 还对这两类色谱固定相的峰容量表征方法和命名提出了建议和说明.

关键词: 二维色谱固定相, 混合模式色谱, 蛋白分离, 离子交换色谱, 疏水色谱, 计量置换理论

Mixed-mode chromatography (MMC) is a type of chromatographic method in which multiple-mode take place between the stationary phase and solute in the feed. MMC has been found to have many advantages and thus became a research hot point. However, before going wide applications, the synthesis of new MMC column and/or the selection of some commercial columns having the character of MMC need to be established. Based on the fact that some commercial ion-exchange columns perform the separation behavior of hydrophobic interaction chromatography (HIC), one of four commercial weak-cation exchange (WCX) column, a two-dimensional mode (WCX, HIC) column called as 2D column, and standard proteins were selected to investigate and compare their retention behaviors. The two type columns have some common characters: proteins can be separated by either WCX, or HIC mode, but have some quite different column efficiencies, and the profiles of “U-shape” elution curve. The width of the “U-shape” curve was experimentally found to relate to the chromatographically dynamic factor, i.e., the broader the “U-shape” curve, the narrower the chromatographic peak obtains, while the magnitude of the critical point of the “U-shape” curve relates to the protein retention, a larger value of a critical point corresponds to a stronger retention. An intensive comparison between the two types of column efficiency was carried out by the peak capacity of proteins. The experimental results showed that the 2D column has much more number of peak capacity than the commercial WCX column, either for one-dimensional, or two-dimensional separation. However, for the separation of a group proteins, the elution order is dominated by the comprehensive consideration of the peak capacity, the dynamic, and thermodynamic factors. Finally the nomenclature of the various stationary phases of MMC and/or 2D column employed in two-dimensional liquid chromatography were suggested. Although, in terms of column efficiency, none of the four commercial WCX column could be compared to the 2D column, at least, two of them as “quasi-2D column” may be employed for protein separation with both of WCX and HIC modes by means of either off-line in all cases, or on-line in some cases. However, none of them has been found to approach to the criteria of the 2D column. The nomenclature and expressions of MMC and 2D columns were suggested and the difference between the MMC and 2D columns were also explained.

Key words: two-dimensional chromatographic stationary phase, mixed-mode chromatography, protein separation, ion-exchange chromatography, hydrophobic interaction chromatography, stoichiometric displacement theory