Cobb K A, Dolnik V, Novotny M
Department of Chemistry, Indiana University, Bloomington 47405.
Anal Chem. 1990 Nov 15;62(22):2478-83. doi: 10.1021/ac00221a013.
A procedure for obtaining highly stable coated capillaries for use in capillary electrophoresis (CE) is described. Reaction of surface-chlorinated fused silica capillaries with the Grignard reagent, vinyl magnesium bromide, followed by reaction of the vinyl group with acrylamide, results in an immobilized layer of polyacrylamide attached through hydrolytically stable Si-C bonds. This method is an extension of the capillary coating procedure described previously by Hjerten, differing in the means by which the polyacrylamide layer is bonded to the capillary walls. Capillaries treated in the manner described here can be used over a pH range of 2-10.5, without noticeable decomposition of the coating. In comparison to uncoated capillaries, separations of proteins using such coated capillaries are improved due to a reduction in protein adsorption to the capillary walls, although interaction is still present to some degree as evidenced by an inability to obtain plate counts as high as those predicted by theory. Electroosmotic flow is virtually eliminated in the coated capillaries, resulting in improved reproducibilities of protein migration times in comparison to uncoated capillaries. Additionally, peak skew is evaluated for model proteins and improvements are noted for the coated capillaries. Results are presented for separations of model protein mixtures, comparing the performance of the vinyl-bound polyacrylamide coated capillaries and uncoated capillaries at both high and low pH extremes.
本文描述了一种制备用于毛细管电泳（CE）的高度稳定涂层毛细管的方法。表面氯化的熔融石英毛细管与格氏试剂溴化乙烯基镁反应，然后乙烯基与丙烯酰胺反应，形成通过水解稳定的Si-C键连接的聚丙烯酰胺固定层。该方法是Hjerten先前描述的毛细管涂层方法的扩展，不同之处在于聚丙烯酰胺层与毛细管壁结合的方式。按此处所述方式处理的毛细管可在pH值为2至10.5的范围内使用，涂层不会明显分解。与未涂层的毛细管相比，使用此类涂层毛细管分离蛋白质的效果有所改善，这是因为蛋白质对毛细管壁的吸附减少，不过仍存在一定程度的相互作用，这可从无法获得理论预测的高塔板数得到证明。涂层毛细管中的电渗流几乎消除，与未涂层的毛细管相比，蛋白质迁移时间的重现性得到提高。此外，对模型蛋白的峰拖尾进行了评估，发现涂层毛细管有所改善。给出了模型蛋白混合物分离的结果，比较了在高pH值和低pH值极端情况下乙烯基结合的聚丙烯酰胺涂层毛细管和未涂层毛细管的性能。
