TLC View software

Sorbfil TLC View runs the visualiser and both Sorbfil densitometers — the light-cabinet and the flatbed-scanner one. The instrument sends the plate image to the computer, where the software stores and processes it. An image can also be opened from a file or pasted from the clipboard.

How it calculates. For every track the software builds a densitogram — the deviation of spot brightness from the plate background — finds the peaks on it and computes their area. The model assumes that the size and brightness of a spot relative to the background are proportional to the amount of substance in it.

Two quantitative calculations:

• mixture composition — approximate content in per cent by simple normalisation: each peak's share of the total peak area in the track;
• analyte concentration in a sample — by absolute calibration (external standard): the standards' spots give a calibration curve of amount against peak area, linear or quadratic, and the sample is read off it, taking into account the sample mass or volume, the extract volume and the aliquot. The result is reported in mg/kg or mg/L.

Workflow:

• image capture — from the video camera as a series of frames (typically 10–16) averaged into one, from a scanner or from a file;
• marking up — start and front lines; tracks by hand, automatically within a selected area, as a regular grid or from a saved layout; a skewed image can be straightened so that Rf comes out right;
• track calculation — Rf, spot travel, peak area and height and their shares, number of theoretical plates, peak asymmetry; peak boundaries and apex can be adjusted by hand, the sensitivity threshold and smoothing tuned;
• colour-composition analysis and colour correction of the image — raise spot contrast and help separate merged spots;
• report — results are compiled into a printable report, and tables go through the clipboard to, for example, Excel.

The spot the software "sees" is about twice as wide as the one the eye sees, so a track is set about two diameters of its largest spot wide. Worth keeping in mind at the application stage: tracks applied too close together merge and the calculation loses accuracy.

Stored chromatograms stay available for re-calculation at any time. To rule out any change to an image, save it as a "closed BMP".