A critical study of some parameters in quantitative in situ investigations of thin-layer chromatography by light absorption methods

Abstract

Reflectance spectroscopy has been found to be superior to transmission measurements for the in situ evaluation of thin-layer chromatograms, contrary to several other published results. Explanations for these experimental facts are given. In the visible region of the spectrum, simulation of infinite layer thickness by inserting empty chromatosheets as a background, shows advantages in the reflectance mode. A double beam instrument is recommended when chromogenic sprays have to be used for visualization of the spots. With regard to time, cost and simplicity of a method, scanning of chromatographic zones with a slit is still considered the optimum approach to deal with inhomogeneity problems. The potential of adjustable lightbeams for single measurement techniques has been critically evaluated and found promising in the absence of tailing and if used with a modified Kubelka-Munk function. In conclusion in situ reflectance spectroscopy is considered a truly quantitative method, with errors between 2–5% to be expected. Chromatographic rather than instrumental parameters are likely to be responsible for errors of this magnitude.