Kleindienst G, Huber C G, Gjerde D T, Yengoyan L, Bonn G K
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens-University, Innsbruck, Austria.
Electrophoresis. 1998 Feb;19(2):262-9. doi: 10.1002/elps.1150190220.
High-resolution capillary electrophoretic separation of proteins and peptides was achieved by coating the inner wall of 75 microm ID fused-silica capillaries with 40-140 nm polystyrene particles which have been derivatized with alpha-omega-diamines such as ethylenediamine or 1,10-diaminodecane. A stable and irreversibly adsorbed coating was obtained upon deprotonation of the capillary surface with aqueous sodium hydroxide and subsequent flushing with a suspension of the positively charged particles. At pH 3.1, the detrimental adsorption of proteins to the capillary inner wall was suppressed efficiently because of electrostatic repulsion of the positively charged proteins from the positively charged coating which enabled protein separations with maximum efficiencies of 400000 plates per meter. A substantial improvement of separation efficiency in particle-coated capillaries was observed after in-column derivatization of amino functionalities with 2,3-epoxy-l-propanol, resulting in a more hydrophilic coating. Five basic and four acidic proteins could be separated in less than 7 min with efficiencies up to 1900000 theoretical plates per meter. Finally, coated capillaries were applied to the high-resolution analysis of protein glycoforms and bioactive peptides.
通过用40 - 140纳米的聚苯乙烯颗粒涂覆内径为75微米的熔融石英毛细管内壁实现了蛋白质和肽的高分辨率毛细管电泳分离，这些聚苯乙烯颗粒已用α,ω - 二胺（如乙二胺或1,10 - 二氨基癸烷）进行了衍生化。在用氢氧化钠水溶液使毛细管表面去质子化并随后用带正电荷的颗粒悬浮液冲洗后，获得了稳定且不可逆吸附的涂层。在pH 3.1时，由于带正电荷的蛋白质与带正电荷的涂层之间的静电排斥，蛋白质对毛细管内壁的有害吸附得到有效抑制，这使得蛋白质分离的最大效率达到每米400000理论塔板数。在用2,3 - 环氧 - 1 - 丙醇对氨基官能团进行柱内衍生化后，观察到颗粒涂覆毛细管的分离效率有了显著提高，从而得到更亲水的涂层。五种碱性蛋白质和四种酸性蛋白质能够在不到7分钟的时间内分离，效率高达每米1900000理论塔板数。最后，涂覆毛细管被应用于蛋白质糖型和生物活性肽的高分辨率分析。
