In-Depth Distribution in Quantitative TLC

Among many techniques used for quantiRcation of thin-layer chromatograms, slit-scanning densitometry is the most common. It gives the opportunity to choose between either reSectance or transmission mode according to the nature of the supporting material. For instance, glass-backed plates can only be used for transmission measurements above 320 nm due to absorption of the UV radiation, while aluminium-backed plates are opaque at all wavelengths. The matrix of sample and stationary phase, which consists of small particles, is optically opaque and strongly light-scattering. Therefore, densitometric measurements of separated substances are much more difRcult than equivalent photometric measurements in solution. A further problem with densitometry is the fact that it is unable to detect the vertical and radial concentration proRles of the separated substance within the spot as a result of diffusion and the chromatographic process. ReSectance and transmission are particularly sensitive to changes in sorbent quality, thickness, spot shape and size, eluent and development conditions. To reduce the errors due to plate-to-plate variation, standards should always be applied to the same plate as the unknown samples. The errors due to migration differences as a result of edge effects, deviations in layer thickness and nonlinear solvent fronts can be further minimized using the data-pair technique introduced by Bethke and co-workers in 1974. This technique is based on an internal compensation, by pairing up the measurements of two spots on the same plate. Most thin-layer chromatography (TLC) analysis is performed in the UV and visible spectral region by applying reSectance scanning densitometry. However, the results of reSectance measurements are restricted to the surface of the sorbent on the TLC plate and are therefore strongly dependent upon the indepth distribution of the analysed compound inside the sorbent. Nonuniformity of the in-depth distribution of a compound inside the sorbent as a result of secondary chromatography can occur during the evaporation}drying stage. Differences in the indepth concentration proRles of the samples and standards can cause signiRcant errors and cannot be completely eliminated by the data-pair technique.