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Evaluation: Detection

 

The chromatogram is evaluated under white or ultraviolet light. Options range from visual inspection of electronic images to quantitative determinations using video or scanning densitometry.

Chromatogram evaluation with classical densitometry

A laboratory fully equipped for instrumental Thin-Layer Chromatography should be able to resort to both classical densitometry and 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 uses the entire spectral range from 190 to 900 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.

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

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 the size of the starting zone does 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.

Products

TLC Scanner 4
The CAMAG TLC Scanner 4 is the most advanced workstation for densitometric evaluation of TLC/HPTLC chromatograms and other planar objects.
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