Multicomponent analysis of biologically important compounds by laser-induced molecular fluorescence spectroscopy

Laser-Induced Fluorescence Spectroscopy (LIFS) with applications to multicomponent analysis techniques and fluorescence probe developments is described.;LIFS is recognized as a real-time, non-hazardous fluorescence-based DNA sequencing technique. Due to the broad band nature of fluorescence, peak overlap is commonly encountered for multicomponent mixtures. Time-resolved LIFS is one of the techniques proposed in resolving the multicomponent peak overlap based on the fluorescence lifetime difference. LIFS incorporated with statistical methods for resolving two- and three-component systems is another technique. Principle Component Regression (PCR) was employed to identify and predict concentration of components having severe peak overlap. PCR analysis was also applied to Fourier transformed spectra for improving the concentration prediction.;Laser-induced fluorescence probe applications for measuring solution pH and correlating surface tension on polymer surfaces were investigated. A dual-wavelength fluorescent dye, derivatized from sulforhodamine 101, showed full pH range indication capability through the ratio of 585/525-nm fluorescence. Ratioing makes the technique more immune to fluorescence photobleaching and excitation source power fluctuations. PE and PP films subjected to corona-discharge treatment to modify film surface tension can be characterized by time-resolved pyrene fluorescence. The pyrene fluorescence at 373- and 384-nm, designated as bands I and III was correlated with the surface tension of the treated films. The surface tension of the films were also validated with the ASTM D2578 standard method. Pyrene I/III ratio can serve as a sensitive and on-the-fly routine in the film/coating industry.;A quantitative study of the extremely fast laser-induced fluorescence photofading of fluorescein and the FITC-butylamine conjugate, in various sample matrices was investigated. Three other DNA sequencing dye-conjugates were also examined in less detail. With 5.0 mW focused excitation, fluorescein and FITC were found to have severe fast photofading (85%) while NBD-amine and Texas Red showed slight (10%) signal loss and only TRITC showed resistance to photofading. Results indicated the importance of addressing the fast photofading in quantitative fluorescence applications.