Real-time image acquisition for absorbance detection and quantification in thin-layer chromatography

This paper presents the first quantitative study of real-time acquisition of images of spots on thin-layer chromatographic plates during development. Procedures are described for imaging using a CCD camera and for image processing, incorporating corrections for fixed pattern effects and compensation for the moving solvent front, to measure the absorbance of the analyte. Imaging of Sudan II was carried out in transmission mode, and peak areas were found to be time-independent. Quantification of the relationship between peak area and sample loading was established over the range 1−50 ng. After averaging 55 images obtained during a single chromatographic run, which attenuates noise contributions from local nonuniformities in the sorbent layer, precision and detection limits were found to be comparable with values obtained in previous work using offline measurements.