Clarke N J, Tomlinson A J, Schomburg G, Naylor S
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA.
Anal Chem. 1997 Jul 15;69(14):2786-92. doi: 10.1021/ac961283+.
Capillary isoelectric focusing within capillaries (cIEF) is a powerful and practical method for high-resolution separation of components within complex biological mixtures. However, a major problem has always existed; separation performance is usually degraded by the presence of salts within the sample. Normally this requires the removal of these components by some off-line sample cleanup method, prior to analyte separation by cIEF. In this study, we have shown it is possible to efficiently remove high salt levels from samples by on-line voltage ramping of the applied CE voltage. To allow this technique to be used effectively, a customized version of an existing method to internally coat a fused-silica capillary has been developed and examined for interexperimental reproducibility. We describe the systematic examination of the desalting process and its optimization through the use of model protein systems. Furthermore, we demonstrate the automated application of this on-line desalting cIEF scheme to studies of whole human blood and human cerebrospinal fluid which have undergone no manipulation or work up prior to cIEF analysis.
毛细管内毛细管等电聚焦（cIEF）是一种用于高分辨率分离复杂生物混合物中各组分的强大而实用的方法。然而，一个主要问题一直存在；样品中盐的存在通常会降低分离性能。通常，在通过cIEF进行分析物分离之前，需要通过一些离线样品净化方法去除这些成分。在本研究中，我们已经表明，通过对施加的CE电压进行在线电压斜坡调节，可以有效地从样品中去除高盐含量。为了使该技术能够有效使用，已经开发了一种定制版本的现有方法来对熔融石英毛细管进行内部涂层，并对实验间的重现性进行了研究。我们描述了对脱盐过程的系统研究及其通过使用模型蛋白质系统的优化。此外，我们展示了这种在线脱盐cIEF方案在未经cIEF分析前任何处理或预处理的全血和人脑脊液研究中的自动化应用。
