CHROMATOGRAPHIC-PHOTODENSITOMETRIC QUANTITATION OF THE SOLANACEOUS ALKALOIDS
Date
1970
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
Type
Degree Level
Masters
Abstract
The thesis work has involved an attempt to improve the accuracy and precision of direct photodensitometric quantitation of thin-media chromatograms.
Direct photodensitometric evaluation of paper and thin-layer chromatograms has been used increasingly in the past decade and is now accepted as a standard method of estimation. The bulk of the methods published have been highly empirical in nature. Recently, there has been considerable disenchantment with all types of in situ analysis and there is now considerable interest in developing a theoretical basis for optical assays performed in situ.
Little attention has been paid to the fact that the absorbance varies considerably throughout a spot and that the geometry of "'inhume spots may vary considerably from that of standard spots be-
cause of the greater complexity of unknown samples. It was felt that the only way to reduce errors of non-linearity due to non-uniform distribution of substance over the illuminating slit and compensate
for distortions in spot geometry would be to replace fixed-slit scanning by two-dimensional scanning of spots with a small point of illumination.
Because a two-dimensional or "flying-spot" scanner was not available, two-dimensional scanning was simulated by manual collection of density data using a 1 x 1 mm. optical slit over the entire area of the spot, integration being accomplished by summing all data points.
Experimental work was done on the Solanaceous alkaloids which were separated electrophoretically on cellulose thin-layers and stained with Dragendorff's reagent. The chromatograms were made
translucent with mineral oil before scanning in order to reduce the high light-scattering properties of the support media.
In order to expedite analysis and make the scanning process more continuous, an analog computer was later built to convert the output potential of the existing photometer (output varying with
transmittance) to absorbance (negative log of the transmittance). Two-dimensional scanning was semi-automatic in nature--scanning across the spot was motorized but the chromatogram had to be
advanced manually and the scanning stage had to be returned manually for the next pass over the chromatogram. Output of the density signals Was on a strip-chart recorder with continuous, automatic, digital integration of density peaks.
Calibration curves (integrated absorbance vs. concentration) produced were quite linear and assay results on "simulated" unknowns and comparative assays on natural products containing the
alkaloids under study were encouraging.
Further work in this area necessitates better stabilized equipment as well as rapid flying-spot scanning with continuous electronic integration. This would allow improvements in accuracy, not only by allowing more precision in scanning; but would allow the collection and averaging of more data. Well stabilized equipment is necessary for meaningful study of the proportionality of radiative transfer to concentration.
Description
Keywords
Direct photodensitometric evaluation
Citation
Degree
Master of Science (M.Sc.)
Department
Pharmacy and Nutrition