Huang X, Zhang J, Horváth C
Department of Chemical Engineering, Yale University, New Haven, CT 06520-8286, USA.
J Chromatogr A. 1999 Oct 8;858(1):91-101. doi: 10.1016/s0021-9673(99)00795-5.
Capillary electrochromatography (CEC) of basic proteins and peptides was carried out with porous-layer open-tubular (PLOT) columns which had a functionalized rugulose polymeric porous layer grafted to the innerwall of 20 microm I.D. fused-silica capillaries. The porous layer was highly crosslinked and prepared by in situ polymerization of vinylbenzyl chloride and divinylbenzene in the presence of 2-octanol as a porogen inside a pretreated fused-silica capillary. The chloromethyl functions at the surface of the porous polymeric support layer were reacted with N,N-dimethyldodecylamine to obtain a positively charged chromatographic surface with fixed C12 alkyl chains. A mixture of lysozyme, cytochrome c, ribonuclease A and alpha-chymotrypsinogen A was separated isocratically by counterdirectional CEC with hydro-organic mobile phases containing acetonitrile and phosphate buffer, pH 2.5. The overall migration behavior of the four proteins was the result of an interplay of chromatographic retention and electrophoretic migration, and was different from that observed in capillary zone electrophoresis or in reversed-phase chromatography under similar conditions. The separation of three basic peptides by CEC also exhibited the same behavior. The stability of the PLOT column was tested by measuring electroosmotic mobility during continual use.
使用多孔层开管（PLOT）柱对碱性蛋白质和肽进行毛细管电色谱（CEC）分析，该柱具有接枝到内径为20微米的熔融石英毛细管内壁的功能化皱纹聚合物多孔层。多孔层高度交联，通过在预处理的熔融石英毛细管内，以2-辛醇作为致孔剂，使乙烯基苄基氯和二乙烯基苯原位聚合制备而成。多孔聚合物支撑层表面的氯甲基官能团与N,N-二甲基十二烷基胺反应，以获得带有固定C12烷基链的带正电的色谱表面。通过反相CEC，使用含有乙腈和pH 2.5磷酸盐缓冲液的水-有机流动相，等度分离溶菌酶、细胞色素c、核糖核酸酶A和α-胰凝乳蛋白酶原A的混合物。这四种蛋白质的整体迁移行为是色谱保留和电泳迁移相互作用的结果，与在类似条件下毛细管区带电泳或反相色谱中观察到的行为不同。通过CEC对三种碱性肽的分离也表现出相同的行为。通过在连续使用过程中测量电渗迁移率来测试PLOT柱的稳定性。
