表面粗糙开管毛细管电色谱柱的制备及在蛋白质分析中的应用
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摘要
采用原位聚合方法制备了一种表面粗糙型开管毛细管电色谱柱,具体方法为：以甲基丙烯酸酯为功能单体,二甲基丙烯酸乙二醇酯(EDMA)为交联剂,2-丙烯酰胺-2-甲基丙磺酸(AMPS)提供正向电渗流,偶氮二异丁腈(AIBN)为引发剂,通过热引发聚合反应,在毛细管内壁形成均匀的多孔涂层,并可以通过致孔剂的种类和加入量控制表面的粗糙程度。实验结果表明在合成原料中加入致孔剂所制备的开管柱表面更粗糙,更适合蛋白质的分离分析。由于一元致孔剂比多元致孔剂更易形成均一的孔径结构,因此一元致孔剂比多元致孔剂所制备的开管柱分离效率更高,更有利于分离。甲基丙烯酸丁酯为功能单体时,采用正丙醇作为一元致孔剂得到的开管柱分离效果(R=2.02)最好,而甲基丙烯酸十二酯为功能单体时,采用正十二醇作为一元致孔剂得到的开管柱分离效果(R=2.00)最好,这是由于正丙醇,正十二醇的溶解度与各自的功能单体相近,易于致小孔,比表面积较大。另外,聚合温度越高,制备的涂层越稳定,孔径越小,而从分离效果看,分离蛋白质的最佳聚合温度为65℃。制备的两种表面粗糙型开管电色谱柱连续进样5次,保留时间的RSD%均小于1%,峰面积的RSD小于9%,不同批次开管电色谱柱的保留时间的RSD%(n=3)均小于3%,峰面积的RSD小于11%,说明本研究所采取的制备方法具有良好的稳定性和重复性。与聚乙烯吡咯烷酮动态涂覆的毛细管柱进行比较,尽管两者的分离效率相当,但本方法所制备的开管电色谱柱重复性略好,灵敏度则得到大大提高；与整体柱相比,分离时间大大缩短,结果表明这种新型开管毛细管电色谱柱在蛋白质快速分离分析中具有明显的优势
Open tubular capillary electrochromatographic (OTCEC) column using porogen to form rough inner surface was developed. The rough layer was coated on the capillary inner wall by in situ polymerization of methacrylate and ethylene glycol dimethacrylate in the presence of porogen. The roughness of polymer was optimized by different types and proportions of porogenic solvents in polymerization mixture in this study. The results show that the column prepared with porogenic solvent is rougher and more appropriate for separation than the column without porogenic solvent. Furthermore, the columns with sole porogen have higher separation efficiency than the column with co-porogen due to more uniform surface. The best separation of BSA and Cty-c with higher resolution is obtained in the poly (butyl methacrylate) OTCEC column using 1-propanol as sole porogen and in the poly (lauryl, methacrylate) OTCEC column using 1-dodecanol as sole porogen. Hildebrand solubility parameter (8) of 1-propanol and 1-dodecanol close to their polymer would bring about a small pore size with a large specific surface area. In addition, the higher the temperature, the more stable coating and the smaller pore size. The results showed that the best separation performance was achieved when the polymerization temperature was at 65℃. The two types of prepared OTCEC columns show good column-to-column (less than 3.0%,11.0%) and run-to-run (less than 1.0%,9.0%) repeatability of retention times and peak areas. Additionally, the result of protein separation by methacrylate-based porous OT column and dynamically coated column with polyvinylpyrrolidone (PVP) is compared. The separation efficiency is similar for the two columns. While column-to-column repeatability of the OT column is slightly better than the dynamically coated column, the sensitivity of the OT column is obviously improved than the dynamically coated column. When compared to the methacrylate-based monolithic column, the OT column significantly shortened the separation time. The results show that the proposed OTCEC system is useful for the fast separation of simple protein samples.
Open tubular capillary electrochromatographic (OTCEC) column using porogen to form rough inner surface was developed. The rough layer was coated on the capillary inner wall by in situ polymerization of methacrylate and ethylene glycol dimethacrylate in the presence of porogen. The roughness of polymer was optimized by different types and proportions of porogenic solvents in polymerization mixture in this study. The results show that the column prepared with porogenic solvent is rougher and more appropriate for separation than the column without porogenic solvent. Furthermore, the columns with sole porogen have higher separation efficiency than the column with co-porogen due to more uniform surface. The best separation of BSA and Cty-c with higher resolution is obtained in the poly (butyl methacrylate) OTCEC column using 1-propanol as sole porogen and in the poly (lauryl, methacrylate) OTCEC column using 1-dodecanol as sole porogen. Hildebrand solubility parameter (8) of 1-propanol and 1-dodecanol close to their polymer would bring about a small pore size with a large specific surface area. In addition, the higher the temperature, the more stable coating and the smaller pore size. The results showed that the best separation performance was achieved when the polymerization temperature was at 65℃. The two types of prepared OTCEC columns show good column-to-column (less than 3.0%,11.0%) and run-to-run (less than 1.0%,9.0%) repeatability of retention times and peak areas. Additionally, the result of protein separation by methacrylate-based porous OT column and dynamically coated column with polyvinylpyrrolidone (PVP) is compared. The separation efficiency is similar for the two columns. While column-to-column repeatability of the OT column is slightly better than the dynamically coated column, the sensitivity of the OT column is obviously improved than the dynamically coated column. When compared to the methacrylate-based monolithic column, the OT column significantly shortened the separation time. The results show that the proposed OTCEC system is useful for the fast separation of simple protein samples.
引文
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