Capillary electrochromatography of proteins and peptides with porous layer open-tubular columns
Capillary electrochromatography of proteins and peptides with porous layer open-tubular columns
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DOI：
10.1016/s0021-9673(99)00795-5
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发表时间：
1999-10-08
影响因子：
4.1
通讯作者：
Horváth, C
中科院分区：
文献类型：
--
作者：
Huang, X;Zhang, J;Horváth, C
采用多孔层开管（PLOT）柱对碱性蛋白和多肽进行了毛细管电色谱（CEC）分析，该柱在20 μ m的熔融二氧化硅毛细管内壁上接枝功能化聚糖聚合物多孔层。在2-辛醇作为致孔剂存在的情况下，将乙烯基氯化苄和二乙烯基苯原位聚合制备了多孔层。将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，得到带正电的层析表面，并得到固定的C-12烷基链。用含乙腈和磷酸盐缓冲液的有机流动相（pH为2.5），用双向CEC对溶菌酶、细胞色素c、核糖核酸酶A和α -胰糜蛋白酶原A的混合物进行等压分离。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，与毛细管区带电泳或反相色谱在相似条件下观察到的结果不同。CEC对三种碱性肽的分离也表现出相同的行为。在连续使用过程中，通过测量电渗透迁移率来检测PLOT色谱柱的稳定性。(C) 1999 Elsevier Science B.V.版权所有
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 mu m 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 C-12 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. (C) 1999 Elsevier Science B.V. All rights reserved.