HPTLC chromatograms showings peaks of the separated compounds can be recorded with scanning densitometry. In the densitometer, a vertical beam of monochromatic light between 190-900 nm moves along the individual tracks of the plate. Part of that light is reflected from the plate and measured by a detector. The obtained signal is processed and plotted as a function of position (RF), generating the densitogram or Peak Profile from Scanning Densitometry (PPSD) used for qualitative and quantitative evaluation.
Modern scanning densitometers allow measurements in two modes:
- Absorption mode: Measures the amount of light absorbed by the zones. The particles of the stationary phase reflect light (baseline). Zones present in the track can absorb part of that light lowering the signal received in the detector. For plotting the PPSD, the signal is typically inverted.
- Fluorescence mode: Measures the fluorescence of the zones excited at a specific wavelength. From a molecular point of view, fluorescence happens when a photon is absorbed, causing an electronic transition from the ground state to an excited state. When the molecule returns to its ground state, the energy is dissipated in a higher wavelength. For selectively detecting that response, densitometers are usually equipped with a cutoff filter placed between the plate and the detector (yellow rectangle in the image below). That filter will block the reflected short wave light used for excitation so that only longer wavelengths can reach the detector.
Fluorescence measurements are up to 100 times more sensitive than absorption measurements and generally feature a straight baseline because the cutoff filter blocks signals from the plate. Only a few substances are naturally fluorescent, but fluorescence can be induced by chemical derivatization, and the fluorescence of zones decreases with time (not stable).
Commercial densitometers feature three light sources:
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