Graduate Thesis Or Dissertation
 

Design and development of an intensified diode array system and its application to new fluorometric reaction rate methods of analysis

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  • Improvements in instrumentation for fluorometric kinetic measurements and the application of a new intensified diode array (IDA) rapid scanning system to low level transient fluorescence measurements are reported. This instrumentation is applied to a new fluorometric kinetic method for thiamine (TM). Modifications to the existing fluorometric reation rate instrument include a mechanical arm to reproducibly position a reagent delivery tube over the sample cell. Also a new circuit provides a trigger signal which synchronizes the reagent delivery from an automatic dispenser with the rate measurement. A new KIM ratemeter was designed and constructed which uses only a single board microcomputer and a minimum of external hardware to acquire and store up to 100 data points and to perform rate or signal magnitude calculations. A significant advantage of the KIM ratemeter is that, since the data are stored in memory, the data may be reexamined and further calculations performed after the experiment is over. The software programmability of the KIM ratemeter provides greater flexibility of measurement times and functions than a hardwired ratemeter. Data points can represent time periods from 0.25 s to 64 s. The time between data points is between 64 μs and 0.44 s depending on the variables selected. Subroutines to perform a hexidecimal to BCD conversion and hardware to output the numbers to a digital printer are described. The IDA system is composed of the commercial IDA detector, an in house control module, a PDP 11/20 computer, and software. The IDA system can obtain spectra from 200-800 nm in as little as 5 ms, or 15 ms when the data are stored in the computer. The IDA detector was characterized and S/N expressions are presented. The design of the control module, computer interface, and interactive BASIC and assembly language programs are described. Using the IDA system under software control one can automatically store data from any portion of a spectrum, control integration times, acquire multiple spectra which may be added together or stored separately, and time periods between integrations. Spectra may be displayed and manipulated on the graphics terminal and added or subtracted. Also summations, means, standard deviations, and rates of change of signals in specific wavelength regions of the spectra may be calculated. Results of the application of this system to situations involving only test equipment or to real analytical situations are presented. A new fluorometric kinetic method for thiamine in which thiamine reacts with Hg(II) to form fluorescent thiochrome (TC) is presented. An extensive absorption study of the reactions of thiamine in basic aqueous solutions is described which provides evidence of the formation of Hg(II) complexes with thiamine. The results of an extensive interference study of species in multivitamin tablets and of the successful analysis of multivitamin and cereal samples are presented. The kinetic method for thiamine has a dynamic range of over four orders of magnitude, a detection limit of 2 x 10⁻⁹ M, and is faster and simpler than the standard method. The IDA system was used to study cereal and urine matrices and provided information impossible or very difficult to obtain with a typical scanning monochromator. The IDA system was also applied to a novel simultaneous analysis of thiamine and riboflavin involving both steady state and kinetic fluorescence measurements. The multichannel, computer control, and data storage capabilities of the IDA system were used to automatically initiate the reaction, and time and acquire successive spectra from which both the steady state fluorescence of riboflavin and the thiamine reaction rate are obtained.
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Déclaration de droits
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