强阳离子交换整体柱的研制及其在电色谱多肽分离中的应用

以丙烯酸、2-丙烯酰胺-2-甲基丙磺酸为功能单体,N,N′-亚甲基双丙烯酰胺为交联剂,正十二醇、1,4-丁二醇及二甲基亚砜为致孔剂,偶氮二异丁腈为引发剂,原位聚合制备了丙烯酰胺类强阳离子交换整体柱。考察了驱动电压、有机调节剂、盐浓度、pH值等对电渗流的影响。结果表明,电渗流与驱动电压的线性关系良好,相关系数为0.9981;有机调节剂乙腈对电渗流的影响除与流动相的黏度有关外,还与固定相的溶胀有关,当浓度低时,电渗流随乙腈浓度的增加有反常的下降趋势;随着磷酸盐浓度逐渐增加,电渗流降低,与理论相符;在pH值为3～9范围内,电渗流基本上保持恒定,体现了整体柱使用酸碱范围宽的优越性。在优化的实验条件下,采用毛细管电色谱法在此整体柱上成功分离了5种多肽,体现了该类整体柱在多肽分离研究中的优势,为进一步将其应用于蛋白质分离研究打下了基础。

Preparation of strong cation-exchange monolithic column and its application in polypeptide separation by capillary electrochromatography

A strong cation-exchange monolithic column was prepared by polymerization inside the fused-silica capillary. The solution consisted of acrylic acid and 2-acrylamido-2-methyl-1-propanesulfonic acid as functional monomers, N,N'-methylenebisacrylamide as a cross-linking agent, dimethyl suiphoxide and dodecanol, 1 , 4-butanediol as organic porogenic solvents and azobisisobutyronitrile as a suitable initiator. The effects of the applied voltage, concentrations of organic modifier and salt solution, pH value on the electroosmotic flow were investigated. The experimental results showed that there existed a good linear relationship between the applied voltage and electroosmotic flow with a correlation coefficient of 0.9981; When the concentration of organic modifier (acetonitrile, ACN) was less than 70%, the swelling degree of stationary phase played a main role and the electroosmotic flow was decreased abnormally with the increase of ACN concentration; The electroosmotic flow was decreased with the increase of the concentration of phosphate. When the pH value was in the range of 3-9, it did not exert a significant change in electroosmotic flow. These results were consistent with the theoretical role. At the same time, five peptides were separated successfully under the optimal experimental conditions on the monolithic column for capillary electrochromatography. The column has obvious advantages in polypeptide separation and will be favorable for the protein investigation.