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.
Authors:Bernd Spangenberg and Marion Weyandt-Spangenberg
A new formula is presented for transforming fluorescence measurements in accordance with
theory. The fluorescence signals, the absorption signals, and data from a selected reference are combined in one expression. Only diode-array techniques can measure all the required data simultaneously to linearize fluorescence data correctly. To prove the new theory HPTLC quantification of the analgesic flupirtine was performed over the mass range 300 to 5000 ng per spot. The fluorescence calibration curve was linear over the whole range. The transformation of fluorescence measurements into linear mass-dependent data extends the technique of in-situ fluorescence analysis to the high concentration range. It also extends
theory from absorption to fluorescence analysis. The results presented also emphasize the importance of
theory for in-situ measurements in scattering media, especially in planar chromatography.
Authors:Bernd Spangenberg, Peter Post, and Siegfried Ebel
High performance thin layer chromatography (HPTLC) is a frequently used separation technique which works well for quantification of caffeine and quinine in beverages. Competing separation techniques, e.g. high-performance liquid chromatography (HPLC) or gas chromatography (GC), are not suitable for sugar-containing samples, because these methods need special pretreatment by the analyst. In HPTLC, however, it is possible to separate ‘dirty’ samples without time-consuming pretreatment, because disposable HPTLC plates are used. A convenient method for quantification of caffeine and quinine in beverages, without sample pretreatment, is presented below. The basic theory of in-situ quantification in HPTLC by use of remitted light is introduced and discussed. Several linearization models are discussed.A home-made diode-array scanner has been used for quantification; this, for the first time, enables simultaneous measurements at different wavelengths. The new scanner also enables fluorescence evaluation without further equipment. Simultaneous recording at different wavelengths improves the accuracy and reliability of HPTLC analysis. These aspects result in substantial improvement of in-situ quantitative densitometric analysis and enable quantification of compounds in beverages.