| The analytical challenge brought by the complexity and dynamic range of component concentrations in biological systems has created an imperative need for new separation techniques with high speed, high resolution and high sensitivity. Traditional separation methods, mostly comprised of one- or two-dimensional chromatography or various electrophoretic methods, offer limited separation efficiency, peak capacity and detection range. Low abundance species with biological importance in complex mixtures are often undetected. There is an urgent need for novel approaches combining features of selective enrichment with high efficiency and high resolution separation. Such capability is particularly important in view of the wide dynamic concentration range of the proteome, which is estimated to be as high as 1012 in human plasma, for example. Emerging high sensitivity mass spectrometry and various ion sources serve as well-matched detectors for sample identification and structure elucidation after separation. The work presented here includes developing a novel non-linear chromatography technique coupled with mass spectrometry, termed elution modified displacement chromatography (EMDC), which combines characteristics of elution and displacement chromatography in order to selectively enrich and detect minor trace peptides from complex proteomes, and in another work, establishing a theoretical understanding of the fundamentals of capillary electrochromatography (CEC), a hybrid of chromatography and electrophoresis which is expected to be very useful for protein and peptide separation. The EMDC technique allows fast, high-resolution separation of peptides and enables detection of low levels (femtomol) of components in mixtures over the concentration range of 1:106. Our work on the fundamental studies of CEC is focused on the clarification of the source of band spreading in CEC and investigation of the migration mechanism of charged sample species. The results are expected to facilitate the interpretation of CEC data, the design of the chromatographic system and the optimization of the operating separation conditions. |
