Download MBBS (Bachelor of Medicine and Bachelor of Surgery) Latest Thin Layer Chromatography Lecture PPT
INTRODUCTION
TLC is one of the simplest, fastest, easiest, and least
expensive of several chromatographic techniques
used in qualitative analysis to separate organic
compounds and to test the purity of compounds.
TLC is a form of liquid chromatography consisting of :
A mobile phase (N-Butanol, Glacial acetic acid and
water)
A stationary phase (a plate or strip coated with silica
gel)
Principle of TLC
It is mainly based on the principle of
partition chromatography.
The components with more affinity towards
stationary phase travels at slower pace
compare to those with less affinity towards
stationary phase.
Reagents
1. A plate or a strip coated with silica gel
2. Solvent
N-Butanol , Glacial acetic acid and water mixed in the ratio
of 4:1:5
3. Visualizing agent
0.2% Ninhydrin
4. Standard amino acids and sample containing unknown
amino acid
Procedure
The mobile phase is added to TLC tank so that it is
about 5mm in height.
The lid of the chamber is closed for half an hour for
saturation
Use a soft pencil to mark the origin line 2cm from the
bottom of silica gel coated strip.
Spot is marked at least with 1 cm distance from each
other
Add 5l of standard amino acid solutions and sample
on the marked spot.
Dry the spot with hair drier
Place the silica gel coated strip in to TLC tank filled
with solvent
Close the TLC tank lid and the solvent is allowed to
ascend
The run is continued till the solvent reaches 2/3rd of
the strip which approximately takes 1 hour
The silica gel coated strip is removed and solvent front
is marked.
Dry the wet silica gel coated strip with hair dryer
Spray ninhydrin solution carefully to identify the spot
Calculate the Rf value
Clinical Application of TLC
Screening of inborn errors of metabolism like
Phenylketonuria, Maple syrup urine disease,
Cystinuria etc.
Identification, purity testing and determination of
active ingredients in drugs.
Determination of pesticides and fungicides in
drinking water
This post was last modified on 30 November 2021