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Saturday, December 31, 2011

Thin Layer Chromatography (TLC)


This is a technique more or less similar to paper chromatography. In thin layer chromatography (TLC) a thin layer of finely divided substance is deposited on to a flat plate. The sample to be separated is spotted at one end. The plate is then dipped in a glass jar having suitable solvent.

Preparation of Thin Layer:
The plate on to which thin layer is prepared should be uniform and should be thoroughly washed and dried before layer application. Plastic or foil plates can be used. The material of which thin layer is to be made is mixed with water to make a slurry. This slurry is applied on to plate uniformly using plate spreader.

Detection / Development:
There are several methods for detection of compounds in a TLC plate
Spraying the plate with 25 – 50% H2SO4 in ethanol and heating the plate. Spots will show up on the plate.
Iodine vapor is used for organic compounds. The iodine spots disappears rapidly but can be make it permanent by spraying with benzidine solution in absolute alcohol.
Even UV light can be used for detection of certain compounds.

Retention Factor (Rf)
The retention factor, or Rf, is defined as the distance traveled by the compound divided by the distance traveled by the solvent.

Rf = Distance Travelled by compound / Distance Travelled by the solvent

Thin Layer Chromatography Advantages
It is more versatile, Faster and more reproducible.
Often been used to detect drugs, contaminants and adulterants
It has been widely used to resolve plant extracts and many other biochemical preparations.