Figure 32.4: Events in spotting during thin layer chromatography.

Running of the TLC: Once the spot is dried, it is placed in the TLC chamber in such a way that spot should not be below the solvent level. Solvent front is allowed to move until the end of the plate.

Analysis of the chromatography plate- The plate is taken out from the chamber and air dried. If the compound is colored, it forms spot and for these substances there is no additional staining required. There are two methods of developing a chromatogram-

Staining procedure- In the staining procedure, TLC plate is sprayed with the staining reagent to stain the functional group present in the compound. Forx. Ninhydrin is used to stain amino acids.

Non-staining procedure- In non-staining procedure spot can be identify by following methods-

1. Autoradiography- A TLC plate can be placed along with the X-ray film for 48-72 hrs (exposure time depends on type and concentration of radioactivity) and then X-ray film is processed.

2. Fluorescence- Several heterocyclic compounds give fluorescence in UV due to presence of conjugate double bond system. TLC plate can be visualized in an UV-chamber (Figure 37.4) to identify the spots on TLC plate.