|Abstract or Summary
- 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
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.