Improvements in high-performance liquid chromatography of biological compounds are sought through further development of microcolumn chromatographic techniques and the use of highly sensitive detectors. As compared to earlier investigation from this laboratory, when developments in column technology were the primary objective, the proposed research will emphasize new developments in the areas of detection and ancillary techniques for microcolumn liquid chromatography. Their biochemically important applications will also be stressed. A laser-based fluorescence detector will be further developed for the femtogram (10-15g) level detection of steroids, prostaglandins, primary amines, carbonyls, peptides and polar lipids. Fluorescence-tapping approaches will be developed for substances lacking suitable chromophores. Chromatographic separation efficiences on the order of 100 thousand theoretical plates will be used to resolve extremely complex biological mixtures, while the individually resolved components will be characterized by a newly built miniaturized optical activity detector and a fluorimetric intensified photodiode array device. Further characterization of the separated components will be accomplished through a combination of highly sensitive ancillary techniques. The merits of precolumn concentration and miniaturized multidimensional chromatography will be studied for the complex mixtures extracted from physiological fluids, and various column systems will be explored. The proposed research will result in effective multicomponent analyses of biologically important mixtures. The ultrahigh sensitivities obtained with the laser-based detector will provide new possibilities for monitoring biomedically important substances (e.g., hormones and their mediators) at extremely low levels, as is necessary in modern biology and medicine for problems in which sample amounts are limited and micromanipulations are desirable. Significant improvements in sensitivity are also expected in the area of protein determination and sequencing. Structural characterization of the constituents in physiological fluids should lead to an improved understanding of various metabolic processes.
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