Process Steps

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).