used in qualitative analysis to separate organic
compounds and to test the purity of compounds.
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TLC is a form of liquid chromatography consisting of :
A mobile phase (N-Butanol, Glacial acetic acid and
water)
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A stationary phase (a plate or strip coated with silica
gel)
Principle of TLC
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It is mainly based on the principle of
partition chromatography.
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The components with more affinity towardsstationary phase travels at slower pace
compare to those with less affinity towards
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stationary phase.
Reagents
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1. A plate or a strip coated with silica gel2. Solvent
N-Butanol , Glacial acetic acid and water mixed in the ratio
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of 4:1:53. Visualizing agent
0.2% Ninhydrin
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4. Standard amino acids and sample containing unknownamino acid
Procedure
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The mobile phase is added to TLC tank so that it isabout 5mm in height.
The lid of the chamber is closed for half an hour for
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saturation
Use a soft pencil to mark the origin line 2cm from the
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bottom of silica gel coated strip.Spot is marked at least with 1 cm distance from each
other
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Add 5l of standard amino acid solutions and sample
on the marked spot.
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Dry the spot with hair drierPlace the silica gel coated strip in to TLC tank filled
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with solvent
Close the TLC tank lid and the solvent is allowed to
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ascendThe run is continued till the solvent reaches 2/3rd of
the strip which approximately takes 1 hour
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The silica gel coated strip is removed and solvent front
is marked.
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Dry the wet silica gel coated strip with hair dryerSpray ninhydrin solution carefully to identify the spot
Calculate the Rf value
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Clinical Application of TLCScreening of inborn errors of metabolism like
Phenylketonuria, Maple syrup urine disease,
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Cystinuria etc.
Identification, purity testing and determination of
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active ingredients in drugs.Determination of pesticides and fungicides in
drinking water
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