Optical and Electronic Aspects of Automating a Spectrofluorometric/liquid-Chromatographic System
Lyons, John William
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https://hdl.handle.net/2142/67246
Description
Title
Optical and Electronic Aspects of Automating a Spectrofluorometric/liquid-Chromatographic System
Author(s)
Lyons, John William
Issue Date
1980
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Analytical
Language
eng
Abstract
The techniques of liquid chromatography and spectrofluorometry were combined to facilitate qualitative analysis of multicomponent fluorescent mixtures. Electronic hardware was developed so that instrument control and data collection could be automated.
The optical design of the fluorometric system and the high levels of scattered light inherent in the small volume flow cells used to preserve the resolution of chromatographic effluents were of particular interest. A computer program based on the matrix methods of Gaussian optics was developed to determine the positions of the elements of the detection system so as to maximize its throughput. Ray tracing algorithms were developed in an investigation of the production of scattered light caused by reflection and refraction. The investigation showed that the use of double monochromators in both the exitation and emission systems and proper cell selection are the most important methods of scattered light reduction when temporal rejection is not possible.
The optical characteristics of the spectrofluorometer were studied. The temporal stability and spectral profile of the excitation source, a xenon arc lamp, were examined. The polarization of excitation light which occurs in the monochromator was catalogued and used to help explain experimentally determined scattered light levels. A simple polarization measurement device based on Brewster's angle for dielectrics was developed for the above purpose. The spectral sensitivity of the detection system was determined and used to correct the emission spectrum of quinine bisulfate. The corrected spectrum is compared to correct spectra from the literature.
The performance capabilities of the completed system were investigated. The detection limit of the fluorescence spectrometer was found to be 1.2 x 10('-12) M fluorescein. A mixture of phenothiazines was qualitatively analyzed by chromatographically resolving the elements of the mixture, obtaining fluorescence spectra of the effluents, and automatically comparing the obtained fluorometric data with a library of fluorescence data.
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