Biochemical Techniques and Instrumentation

2.6.4. Technique

1. Sample application: Sample dissolved in a solvent is applied by means of micropipette or syringe as spots. Spot is placed 2-2.5 cm from the edge of plate. Solvent in the sample can be removed by gentle heating or by use of air blower. If a non-volatile solvent is used, the plate should be dried in a vacuum chamber.
2. Plate development: The separation takes place in a TLC glass chamber which contains the developing solvent or mobile phase to a depth of about 1.5 cm. The solvent is allowed to stand for at least 1 h with top cover plate to saturate the atmosphere within the chamber with solvent vapour. Failure to saturate the chamber will result in poor separation and non- reproducible results. Then, the plate is placed vertically in the tank and the cover plate is replaced.The separation occurs as the solvent moves up the plate by capillary action. When the solvent front reaches the top of the plate in the chamber, the plate is removed and air-dried. The speed of separation is 10-30 min commonly and > 90 min occasionally.
3. Detection: Compounds separated on TLC are mostly colourless and there are several detection methods to visualize the colourless spots.

• Spray of 50% H₂SO₄ or 25% H₂SO₄ in ethanol followed by application of heat to visualize as brown spots
• Addition of a fluorescent compound such as manganese - activated zinc silicate to the adsorbent to visualize the spots as fluorescent green under a UV₂₅₄ light.
• Subject the plates to iodine vapour to identify the unsaturated compounds.
• Spray of specific color reagents to identify certain compounds eg. ninhydrin for aminoacids
• Subject the plates to autoradiography to detect spots as dark areas on X ray film
• Scan the plates by radio chromatogram scanner, if the compounds are radioactive.