Video-densitometric quantitation of fluorescence quenching on totally irradiated thin-layer chromatographic plates

The influence of the instrumental settings of a video-imaging system on the quality of captured images was studied. The performance of the Camag video documentation system was evaluated by measurements of fluorescence quenchi ng of a model compound (the pesticide atrazine) in the UV spectral region. The effects of different camera settings (aperture and number of accumulate d frames) on background response, baseline noise, and sensitivity and repro ducibility of detection were studied for different commercial thin-layer ch romatographic (TLC) and high-performance TLC (HPTLC) plates. The results sh owed that as the number of frames, in other words, the image brightness and background response, increased, the sensitivity also increased, reaching a maximum at settings that gave an image of moderate luminosity. Additional frame accumulation caused a decrease in sensitivity. A similar pattern was observed by measuring the limit of detection (LOD), If the f-stop number (a perture) was too large, the image was too bright, and if the aperture was t oo small, the image was too dark. Both of these extremes resulted in a high er LOD, However, good sensitivity does not necessarily mean a low detection limit because of the high baseline noise at the same capturing conditions. Evaluation of dark or moderately luminous video images gave move repeatabl e results than did evaluation of very bright images, With HPTLC silica gel plates, the relative standard deviation ranged from 1.8 to 2.5% for setting s that resulted in moderately bright images, and from 5 to 8% for settings that resulted in bright images.