Development

A solvent or a mixture of solvents, called the eluant is taken in a wide-mouth container called the ‘development chamber'. A filter paper wetted with eluant is placed inside the development chamber to create a solvent saturated atmosphere. The dried TLC plate is placed in the development chamber such that the solvent front is slightly below the sample spot (ensure that the plate does not touch the filter paper placed in the development chamber). The solvent is taken up by the TLC adsorbent due to capillary action. The stationary phase is polar (silica or alumina gel) while the eluant (mobile phase) is relatively non-polar. The molecules are therefore partitioned between the stationary and the mobile phase based on their polarities. As the solvent front reaches within 1 cm of the top edge of the plate, the plate is taken out using tweezers and the solvent front is marked with a pencil. The plate is then air dried and spots are visualized.

Visualization

If the compounds under analysis are colored, they can easily be visualized without any assistance. Most organic compounds, however, are not colored. In most cases, the spots can be detected using ultraviolet light. TLC plates wherein silica gel is impregnated with fluorescent compounds are commercially available. The plates have compounds that fluoresce when excited with the UV light of wavelength 254 nm. The plate, after the developing step, is illuminated with the UV light. The analytes that absorb UV quench the fluorescence and appear as dark spots in the glowing green background. The spots are outlined using a pencil and the plate is taken out of the UV light. In case this method does not work, the bands may be observed by placing the plates with iodine vapors; a large number of organic compounds form a dark-colored complex with iodine. A large number of TLC stains are available for detecting specific classes of molecules some of which are listed in Table 21.1

Table 21.1 List of some of the TLC stains used for biomolecular analysis