Authors:Bernd Spangenberg, Klemens Lorenz, and Steffen Nasterlack
In-situ densitometry for qualitative or quantitative purposes is a key step in thin-layer chromatography. It offers a simple way of quantifying by measuring the optical density of the separated spots directly on the plate. A new TLC scanner has been developed which is able to measure TLC plates or HPTLC plates, at different wavelengths simultaneously, without destroying the plate surface. The system enables absorbance and fluorescence measurements in one run. Fluorescence measurements are possible without filters or other adjustments.The measurement of fluorescence from a TLC plate is a versatile means of making TLC analysis more sensitive. Fluorescence measurements with the new scanner are possible without filters or special lamps. Improvement of the signal-to-noise ratio is achieved by wavelength bundling. During plate scanning the scattered light and the fluorescence are both emitted from the surface of the TLC plate and this emitted light provides the desired spectral information from substances on the TLC plate. The measurement of fluorescence spectra and absorbance spectra directly from a TLC plate is based on differential measurement of light emerging from sample-free and sample-containing zones.The literature recommends dipping TLC plates in viscous liquids to enhance fluorescence. Measurement of the fluorescence and absorbance spectra of pyrene spots reveals the mechanism of enhancement of plate dipping in viscous liquids — blocked contact of the fluorescent molecules with the stationary phase or other sample molecules is responsible for the enhanced fluorescence at lower concentrations.In conclusion, dipping in TLC analysis is no miracle. It is based on similar mechanisms observable in liquids. The measured TLC spectra are also very similar to liquid spectra and this makes TLC spectroscopy an important tool in separation analysis.
In thin-layer chromatography the development step distributes the sample throughout the layer, a process which strongly affects the reflection signals. The essential requirement for quantitative thinlayer chromatography is not a constant sample concentration but constant sample distribution in each sample spot. This makes evaporation of the mobile phase extremely important, because all tracks of a TLC plate must be dried uniformly. This paper shows that quantitative TLC is possible even if the concentration of the sample is not constant throughout the layer or if the distribution of the sample is not known. With uniform sample distribution, classical
theory is valid for isotropic scattering only. In the absence of this constraint classical
theory must be extended to situations where scattering is asymmetric. This can be achieved by modification of the original
equation. Extended theory is presented which is not only capable of describing asymmetrical scattering in TLC layers but also includes a formula for absorption and fluorescence in diode-array TLC. With this new theory all different formulas for diode-array thin-layer chromatographic evaluation are combined in one expression.