Documentation and evaluation are the last steps in TLC/HPTLC-workflow. They give you the results or inspirations for new experiments.

You can record images reflecting the real optical impression by your eyes or by a camera or you generate chromatograms by a densitometer showing absorption or fluorescence curves of the separations.

Sometimes, it is sufficient to check the results by eyes only, independent on the light source. In most cases, UV light is required -> the UV detection methods belong to the most sensitive methods for the detection in thin-layer chromatography. At long-wavelength UV light 366 nm, substances fluoresce brightly on a dark background. This method becomes even more sensitive if the light intensity increases. In order to boost the contrast, it is important that the visible light is filtered out with a special filter. At 254 nm, when using TLC/HPTLC plates containing a fluorescent indicator, the substances reveal themselves as dark spots on a bright fluorescent background. The light intensity and the filtering of visible light are less critical for this form of detection. Our UV-Boxes, HP-UVIS and CabUVIS are very suitable devices for this purpose.

With camera-based systems, you have not only the opportunity of recording images under white light but also under UV light. Therefore, the fluorescent substances can be checked and help to identify the substances. It is advantageous to record the images of a TLC/HPTLC plate under white light, UV 254 nm and 366 nm without changing the position of the plate so that the spectral properties of a given spot can be monitored precisely. For this reason, it is important that the darkhood is equipped with light sources for white light, UV 254 and 366 nm which can be switched on/and off optionally. Another point is the need for a high-resolution camera with high color reproduction. Images recorded in this way can be quantified by an appropriate software similar to the process with electrophoretic gels.

For an accurate quantitative analysis of TLC/HPTLC separations, densitometers are used which produce chromatograms of the lanes measured at suitable wavelengths. This can be done in absorption or fluorescence mode. Calibration is feasible by defined standard substances and therefore, quantification of unknown samples is possible. Such instruments offer the opportunity of recording adsorption spectra of desired spots to assist substance identification. Measurements with reference wavelength (two-wavelengths measurements) serve for background reduction. With multi-wavelength scans, it is possible to detect the best measurement wavelengths for chromatograms of unknown samples. Our HPTLC-densitometer CD60 is the perfect instrument for the generation of reliable quantitative data.