On-line fluorescence detection for mechanistic study of capillary electrophoresis and capillary electrochromatography.

摘要

The interaction between the solute molecules and the C18 stationary phase under electrically and pressure driven conditions was probed using on-the-fly time-resolved fluorescence detection. Fluorescence lifetime recovery of pyrene molecules on packed C18 bonded phase was studied using nonlinear least-squares (NLLS) and maximum entropy method (MEM). The results indicated a distribution of heterogeneous microenvironments in the stationary phase. Effects of various separation conditions on the C18 stationary phase were investigated.;Dynamic two-dimensional fluorescence correlation spectroscopy (2D-FCS) was presented in the general form. 2D FCS evaluates the time correlation function between two wavelength axes when an external perturbation is applied to the sample. The correlation analysis combines both the spectral and fluorescence lifetime information, enabling resolution of strongly overlapping species in chemical systems. The power of 2D-FCS was illustrated from the aspects of excitation-emission correlation, excitation-excitation correlation, emission-emission correlation, and two dimensional phase map.;The technique of phase-resolved fluorescence lifetime detection (PRLD) was introduced for capillary electrophoresis. The detection scheme is based on the integrated nature of phase-resolved fluorescence intensity. The limit of detection of PRLD represents a two-orders-of-magnitude improvement over the detection limits previously reported in the UV-visible region. Whether the fluorescence lifetime of the background is longer or shorter than the solute molecules of interest, fluorescence background can be eliminated by setting the detector out of the phase from the background signal. Protein-ligand binding, protein-surface interactions in CE were studied using PRLD.;By exploring the small angle between the capillary and the incident laser beam, fluorescence signals from the center and the wall of the capillary were differentiated by scanning the laser beam across the column. Both computer simulation and experimental results proved the feasibility of this cross-capillary detection technique. The efficiency of pyrene excimer formation across the capillary was discussed.