Direct Quantitative Photometry on Thin Layer Chromatograms

Abstract

The principal methods for the direct photometric evaluation of thin layer chromatograms are discussed with the aid of examples. Since light is strongly scattered by TLC adsorbents, absorption measurements in reflection are preferable to transmission measurements. In the reflection measurements, an approximation method for the Kubelka-Munk function leads to satisfactory straight calibration lines. The working range for quantitative reflection measurements is in the pg range, and the detection limit is of the order of lo-". The standard deviation for spots of equal concentration is less than f 1%. The signal-to-background ratio is improved by simultaneous measurement of reflection and transmission. The measurement of diminution in fluorescence ("fluorescence quenching") is found to be less sensitive than the reflection at the absorption maximum. However, combined measurement of the reflection and the diminution of fluorescence gives an increase in sensitivity as compared to pure reflection measurements. As in solution photometry, fluorescence measurements are generally much more sensitive than absorption measurements. The working range is normally in the ng region, and the detection limit is around lo-' ' g. All the investigations described in this paper were carried out on the Zeiss chromatogram spectrophotometer.