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 quenching of a model compound (the pesticide atrazine) in the UV spectral region. The effects of different camera settings (aperture and number of accumulated frames) on background response, baseline noise, and sensitivity and reproducibility of detection were studied for different commercial thin-layer chromatographic (TLC) and high-performance TLC (HPTLC) plates. The results showed 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 (aperture) was too large, the image was too bright, and if the aperture was too small, the image was too dark. Both of these extremes resulted in a higher 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 more repeatable 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 settings that resulted in moderately bright images, and from 5 to 8% for settings that resulted in bright images.