Michael Lancaster | PubMed Health

PubMed2006

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.

Azo Compounds↗

PubMed2005

Quantitative measurements on wetted thin layer chromatography plates using a charge coupled device camera.

This paper presents the first study of imaging of spots on thin-layer chromatographic plates whilst still wet with solvent. Imaging and quantification of Sudan II after development with dichloromethane was carried out in both reflectance and transmission modes, using a charge coupled device (CCD) camera. The relationship between peak area and sample loading was established at low sample loading, and found to be linear over an order of magnitude for both wet and dry modes with r2-values > 0.99. All data processing was carried out using the Beer-Lambert equation. Curvature at high loadings in the plots of integrated absorbance as a function of sample loading was accounted for using an empirical expression designed for use with the Kubelka-Munk treatment and apparent absorbance of the stationary phase due to scattering. Results are consistent with an effective pathlength significantly longer than the thickness of the sorbent layer. The limit of detection on a dry plate (0.5 ng) was found to be lower than on a wetted plate (2 ng). Precision was found to be 1-4% RSD intra-plate and 8-14% RSD inter-plate. Results are compared with quantification of the same analyte on dried plates.

Azo Compounds↗