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

Abstract

This paper presents the first study of the UV imaging of spots on thin-layer chromatographic plates whilst still wet with solvent. Imaging of spots of benzophenone during and after development was carried out using a charge-coupled device camera. Limits of detection were found to be 5ng on a wetted plate and 3ng for a dry plate and the relationship between peak area and sample loading was found to be linear in the low nanogram range over an order of magnitude for both wet and dry modes with r(2) values>0.99. It was found that UV measurements on wet glass-backed plates suffer from low sensitivity; however, the use of aluminium-backed plates gave increased sensitivity. The apparent absorption coefficient epsilon(app) of 10AUm(2)g(-1) at 254nm is consistent with reflection of the light from the aluminium surface with a double pass through the sorbent layer, and suggests that use of aluminium-backed plates should enable monitoring of separations by UV absorbance during TLC development.

Instrument platforms for thin-layer chromatography

“…For the detection of UV-absorbing compounds thin-layer plates with a fluorescent indicator are generally used. Alternatively, cameras with a UV-sensitive phosphor coating for detection below 400 nm can be used, but are expensive and not in common use [79].…”

Planar Chromatography

“…Spraying, dipping, and use of the AR2i Derivapress device were compared for application of phosphomolybdic acid (PMA) reagent in the detection and quantification of neutral lipids by silica gel HPTLC (118). A study of the UV imaging of zones with a charge coupled device (CCD) camera on TLC aluminum backed plates while still wet indicated that monitoring of separations by UV absorbance in real time was feasible (119). Validation of results is usually carried out for quantitative densitometric determinations, but procedures were described for specificity, reproducibility, and robustness validation of the qualitative identification of Hoodfia gordonii according to cGMP (current GMP) requirements (120).…”