反相毛细管整体柱的制备及其在多肽混合物分离中的应用

Preparation of a reversed-phase capillary monolithic column and its application in the separation of polypeptide mixtures

采用甲基丙烯酸月桂酯为基础功能单体,乙二醇二甲基丙烯酸酯为交联剂,正十二醇、1,4-丁二醇及二甲基亚砜为致孔剂,在内径为75μm的石英毛细管内制备了具有良好机械性能及化学稳定性的反相毛细管整体柱。考察了致孔剂的种类、比例以及交联剂在单体混合物中的比例对柱压和分离效果的影响;以单体15%、交联剂15%、致孔剂70%(均为质量分数)作为优化配方,在70℃条件下反应24h;并对所合成的毛细管整体柱进行了电镜表征,测试了流速、柱长与柱压的关系。结果表明,毛细管整体柱的通透性良好,可通过延长柱长的方法提高分离效果。将所制备的毛细管整体柱装于纳升级高效液相色谱仪上进行牛血清白蛋白及血浆样本的胰蛋白酶酶切液的分离,获得了比较理想的分离效果。

Capillary monolithic columns（75 μm i.d.） were prepared by the copolymerization of lauryl methacrylate as the basic monomer,ethylene dimethacrylate as the cross-linking agent and 1-dodecyl alcohol,1,4-butanediol and dimethyl sulfoxide as the porogenic mixture.The synthetic stationary phases had better mechanical properties and chemical stabilities.A series of characterization and evaluations were performed on the capillary monolithic columns including the scanning electron microscope（SEM） images,the influences of pressure and the effects on the separation of peptide mixtures by changing the proportions of the porogen solution and cross-linking agent.The final prescription contained 15%（w/w） monomer,15%（w/w） cross-linking agent,and 70%（w/w） porogenic agent.Then the solution was heated at 70 ℃ for 24 h.The test of relationship between column length and back pressure showed that the capillary monolithic columns prepared have superior permeability,so a longer column can be used to improve the effects of separation.The prepared capillary monolithic columns are fitted on the nano-scale high performance liquid chromatography for the separation of tryptic digests of bovine serum albumin（BSA） and human plasma samples,and better results have been obtained.