Liquid Chromatography of Biological Substances

这项建议旨在进一步扩大现代液相色谱的范围，以满足医学和生命科学对高速、高分辨率分离方法的需求。这一需求是由蛋白质组学框架内复杂蛋白质混合物研究的最新进展产生的。毛细管电色谱(CEC)是一种很有潜力的新型分离技术，我们的研究重点是建立对毛细管电色谱分离过程中迁移过程的物理化学基础的坚实的理论理解。这种新的高效分离方法可以认为是层析和电泳法的混合体。换句话说，我们可以将CEC看作是在一个电泳平台上的层析，体现了两种领先的生物分析技术的优势。毛细管色谱柱采用20-125微米的小内径毛细管柱和高电场，通过电雾化来驱动流动相流。它有助于实现极高的柱效。我们在毛细管电泳色谱理论和应用方面的基础研究有望为我们计划在实验室定制的新型固定相的设计、制备和使用提供支持。在许多方面，与CEC开发相关的技术问题与开发基于芯片的分析系统所遇到的技术问题非常相似。毛细管和芯片都代表了分析仪器小型化的趋势。因此，我们在毛细管柱研究方面的进展也有望有助于芯片集成分析系统的发展。我们研究的这一部分将极大地受益于我们在过去几十年中在实验室为高效液相色谱制备和表征固定相方面所获得的经验。我们的理论研究结果以及我们在柱工程和CEC方面的经验将用于计划中的研究的第三部分：蛋白质组学。它代表了蛋白质化学的一个新领域，一旦成功，预计将对各种疾病的诊断方式产生重大影响。我们的努力将得到耶鲁大学人类遗传学教职员工和耶鲁大学凯克核心设施的支持。我们将从改进一种被称为ICAT技术的有前途的分析方法的分离步骤开始。此外，我们计划通过使用高温来提高这种方法的分析速度。最后，我们计划通过使用置换层析步骤来提高该技术的灵敏度。在这个程序的更高级的开发步骤中，我们将设计一个半自动，甚至是全自动的蛋白质组分析仪。它应具有较高的分离效率，有利于加快蛋白质组学和蛋白质层析的分析分离。

This proposal is aimed at further expanding the scope of modem liquid chromatography in a major way to meet the needs in medicine and life sciences for separation methods of high speed and resolution. The need is engendered by recent advances in the study of complex protein mixtures within the framework of proteomics. The main focus of our research is the establishment of a solid theoretical understanding of the physico-chemical underpinning of the migration processes that are involved in the separation by capillary electrochromatography (CEC), a novel separation technique of great potential. This new high performance separation method can be considered to be a hybrid of chromatography and electrophoresis. In other words, we can look at CEC as chromatography on a electrophoretic platform, that embodies the advantages of the two leading bioanalytical techniques. CEC employs capillary columns of small inner- diameter, in the range of 20-125 mum, and a high electric field to drive the mobile phase flow by electrosmosis. It can facilitate the realization of extraordinary high column efficiencies. Our fundamental studies on the theory and application of CEC are expected to support the design, preparation and use of the new stationary phases we plan to tailor make in our laboratory. In many respects the technical problems associated with the development of CEC are very similar to those encountered in the development of chip based analytical systems. Both capillaries and chips represent the trend of miniaturization of analytical instrumentation. Therefore advancements in our research with capillary columns are expected to benefit the development of chip based integrated analytical systems as well. This part of our study would greatly benefit from the experience we have gained with preparing and characterizing stationary phases made in our laboratory for HPLC over the last decades. The results of our theoretical studies together with our experience in column engineering and CEC will be utilized in embarking on the third part of the planned investigation: proteomics. It represents a new area of protein chemistry and upon success it is expected to have a great impact on the way in which various illnesses will be diagnosed. Our endeavor will be supported by Yale faculty members in human genetics and in the Keck core facility at Yale. We shall start with improving the separation steps of a promising analytical method referred to as ICAT technology. Further we plan to enhance the speed of analysis of this approach by using elevated temperature. Lastly, we plan to increase the sensitivity of the technique by employing a displacer chromatographic step. In the more advanced development steps of this procedure we shall design a semi, or even, a fully automated proteomic analyzer. It should have high separating efficiency and facilitate speeding up the analytical separation in proteomics and in protein chromatography at large.