Physical and Chemical methods:

Following are some of the physical and chemical methods employed.

1. Visual comparison of spots: The standards containing known amounts and samples must be run on the same sheet or plate, and their relative intensity or area may be compared visually.

2. Physical measurement of colored spots: The color intensity may be measured by transmission or reflectance using spectrophotometer.  Scanning photo densitometers which measure spot intensity are also available. For organic fluorescent substances, the fluorescence intensity may be measured under illumination with ultraviolet radiation. Full spectrum recording and multiple wavelength scanning capabilities are available commercially.

3. Spot area measurement: The spot area determined by using transparent graph paper and counting the squares within a spot, is proportional to the logarithm of the amount of substance. The standard is run under identical and controlled conditions and a calibration plot is made among area vs log of concentration.

4. Radioactivity measurements: In this method, radiotracers or radiolabelled substances are used for spotting. The plate or paper strip after development could be scanned for activity. A comparison of the activity for the sample along with that of standard might provide quantitative results. Instead, before spotting pencil lines are drawn on the paper strip at an interval of one half cm and after development these strips are cut and then counted for radioactivity using GM counter or scintillation gamma ray spectrometer. Also, an X-ray film may be kept in contact with the developed paper or thin layer plate so that black spots will appear at the location of the separated constituents.

5. Removal of spots: The spot is cut from the paper or removed from the plate by scrapping of the adsorbent. A substance is eluted or extracted from the spot paper or adsorbent (using dil hydrochloric acid and slight warming for inorganic substance and suitable organic solvent for organic substance). The solution is made up to a standard volume and then measurements are made by spectrophotometry or any other technique.

CAMAG, Switzerland, pioneers in modern equipments of TLC, have come up with a Planar Chromatography Manager winCATS which offers a new approach to thin layer chromatography. That is a 32bit Windows software designed to control, monitor and document all steps of TLC analysis. CAMAG instruments such as the automatic TLC sampler, scanner etc. could be connected to winCATS through means of a software interface and offer unique features.