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.
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