Chromatogram evaluation with classical densitometry or electronic image acquisition?

Simplified presentation of measurement middle), and 16 bit (right).

A laboratory fully equipped for instrumental Thin-Layer Chromatography should be able to resort to both classical densitometry and electronic image acquisition. If for cost reasons a choice between the two must be made, their advantages and limitations must be considered in the light of the intended tasks.

Quantitative evaluation of a TLC/HPTLC plate is always performed densitometrically, either in absorption or fluorescence mode. The signal of each substance zone is compared to the substance free plate background. For calibration and result calculation the obtained peak data of the unknowns are compared against data obtained for standards on the same plate. Quantitative evaluation can be performed with data from classical densitometry or with those from electronic image acquisition.

Classical densitometry uses monochromatic light and a slit of selectable length and width to scan the tracks of a chromatogram, measuring the diffusely reflected light. The CAMAG TLC Scanner 3 uses the entire spectral range from 190 to 800 nm with high spectral selectivity for data acquisition. Absorption spectra for substance identification and for selection of the most suitable measurement wavelength can be recorded within this range.

Further information about the CAMAG TLC Scanner 3 and about classical densitometry are found in the corresponding brochure.

The strengths of classical densitometry are the spectral resolution of the light source and the higher reproducibility of quantitative determinations.

Currently electronic image acquisition works only in the visible range. The UV region – exceptionally useful for Thin-Layer Chromatography – is only indirectly accessible to image acquisition. In this respect this technology exactly parallels the human eye.

Spectral selectivity is only accessible through the classical densitometer. UV-absorbing substances can be detected by image acquisition technology vi the quenching of a fluorescence indicator embedded in the layer, i.e. detection is shifted to the visible region. The more a substance to be quantified absorbs at or near the excitation maximum of the fluorescence indicator (254 nm), the higher is sensitivity and accuracy of image processing but the lower the absorbance at 254 nm, the less sensitive and less accurate it becomes.
Recording of UV spectra with their information about absorption maxima, identity and purity, is only possible with classical densitometry.
Sensitivity and reproducibility are strongly dependent on the resolution of the measurement. The Scanner 3 with an AD converter of 16 bit (65535 values) exceeds by far the resolution of a simple digital camera with 8 bit (255 values) or that of a high-resolution camera with 12 bit (4095 values).

Chromatogram under white light (top), UV 254 nm (middle), and UV 366 nm (bottom)

Electronic image acquisition uses polychromatic light (white light, UV 254 or UV 366) to illuminate the entire object and to capture an electronic image with a digital camera. For the documentation of Thin-Layer Chromatograms, electronic image acquisition has essentially replaced photography. Electronic images can easily be archived and can be retrieved at any time, for review or to perform a quantitative evaluation.

Further information about the system Reprostar 3/Digistore 2 and electronic image acquisition can be found in the corresponding brochure.

The strength of electronic image acquisition is the view of the complete image of the chromatogram.

This possibility to get a »visual impression« of the chromatogram is one of the principal advantages of Thin-Layer Chromatography over all other chromatographic techniques.

Further strengths are:

Quick and intuitive operation
Access to archived chromatograms for later verification or quantitation
Low cost: If a system Reprostar 3 / DigiStore 2 is available for documentation and archiving, for quantitative evaluation only the evaluation software is required.

Requirements for high precision of evaluation:

Use of HPTLC plates. Small layer thickness, narrow particle size distribution and the homogenous packing of the HPTLC layer result in less fraction broadening and low background noise
Automatic spray-on sample application technique. Only by spraying does the size of the starting zone remain independent of the application volume and the sample is homogenously distributed across the application position. Data acquisition can be based on larger substance amounts.
Use of a chamber providing good reproducibility of chamber conditions
Choosing a working range for calibration according to the absorption / fluorescence behavior of the substances. The evaluation software offers suitable calibration functions
Optimization of light and measurement parameters, such as slit dimensions, measuring wavelength, scanning speed for the substances to be analyzed
Suitable baseline correction to maximize the signal to noise ratio
Derivatization can contribute to the overall error of the determination The more homogenous the reagent is applied the smaller the error.