Process Steps

    TLC/HPTLC is a highly flexible analytical technique. The plate is the central element which is moved through the individual process steps. For each process step the parameters can be adjusted to the specific analytical task. The higher the demand on the final analytical result, the higher is the required level of automation.

In contrast to other chromatographic techniques, TLC/HPTLC offers the unique opportunity to “visualize” the chromatographic result directly for the human eye. Colored substances can be seen directly on the plate; others are conveniently changed into colored derivatives. Compounds that absorb UV light of UV 254 nm are visualized with the help of a fluorescence indicator (F254) embedded in the stationary phase of the plate and excited by a UV lamp. Substances excited to fluoresce by UV 366 nm either with or without derivatization can also be visualized. For electronic image acquisition a digital camera captures visible polychromatic light. The obtained data can be edited, archived and evaluated. For quantitation, either hyperspectral data (CAMAG® HPTLC PRO Module DETECTION), densitograms (CAMAG® TLC Scanner 4) or image profiles (CAMAG® TLC Visualizer 2) can be used. Hyperspectral data are obtained by using polychromatic light for excitation in conjunction with an imaging spectrometer (CAMAG® HPTLC PRO Module DETECTION). Densitograms are obtained by scanning the plate with monochromatic light and detecting the reflected light with a photomultiplier (CAMAG® TLC Scanner 4). Images are captured with a tri-colored CCD camera (CAMAG® TLC Visualizer 2). The spectral selectivity offers a great advantage over visual inspection. Each individual analyte can be evaluated at its absorption/fluorescence maximum. Furthermore, UV spectra of separated analytes can be recorded and used for identification purposes. For structure confirmation, selected target analytes can be transferred to a Mass Spectrometer (see MS-Interface). A non-target analysis can be achieved by effect-directed HPTLC, e.g. basis general toxicity for Alliivibrio fischeri analyzed by bioluminescence detection (CAMAG® BioLuminizer 2).

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