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Slit-scanning densitometers

المؤلف:  Sue Jickells , Adam Negrusz (Editors)

المصدر:  Clarkes Analytical Forensic Toxicology

الجزء والصفحة:  359

2026-11-02

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Slit-scanning densitometers

Commercial instruments for scanning densitometry usually allow measurements in the reflectance mode by absorbance or fluorescence. Most instruments employ grating monochromators for wavelength selection and spectrum recording in the absorbance mode. For fluorescence measurements, a filter which transmits the emission wavelength envelope but attenuates the excitation wavelength is placed between the detector and the plate. The separations are scanned at selectable speeds up to about 10 cm/s by mounting the plate on a movable stage controlled by stepping motors. A fixed sample beam is shaped into a rectangular area on the plate surface, through which the plate is transported in the direction of develop ment. Each scan, therefore, represents a lane of length defined by the solvent-front migration distance and width by the slit dimensions of the source.

The data from scanning densitometry can be plotted as a densitogram, i.e. a plot of absorbance (y-axis) versus distance (x-axis). It resembles the chromatograms obtained by GC and HPLC separations, but in these separations the x-axis is time. Standards are typically run alongside samples. The area under the curve is computed for the samples, plotted to give a calibration line and then sample values interpolated from the calibration line. TLC with scanning densitometry permits the use of internal standards, provided an internal standard is selected that can be visualised in the same way as the samples (e.g. if it is derivatised in the same way or if it fluoresces). If an internal standard is used, the absorbance ratio of the peak area of the standard substance/peak area of the internal standard is plotted on the y-axis of the calibration. Scanning densitometers can also record a spectrum of the substance corresponding to each spot. This can help determine spot purity by comparing the sample spectrum with that of a standard analysed alongside and/or help in identifying the substance. Distorted chromatograms can be corrected by track optimisation, in which the sample zones are integrated as if the slit had moved along an optimum track from peak maximum to peak maximum. In modern TLC the relative standard deviation from all errors, instrumental and chromatographic, can be maintained below 2–3%, which makes it a very reliable quantitative tool.