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Download MBBS Thin Layer Chromatography Lecture PPT

Download MBBS (Bachelor of Medicine and Bachelor of Surgery) Latest Thin Layer Chromatography Lecture PPT

This post was last modified on 30 November 2021


INTRODUCTION

TLC is one of the simplest, fastest, easiest, and least

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expensive of several chromatographic techniques

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 towards

stationary 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 gel

2. Solvent
N-Butanol , Glacial acetic acid and water mixed in the ratio

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of 4:1:5

3. Visualizing agent
0.2% Ninhydrin

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4. Standard amino acids and sample containing unknown

amino acid
Procedure

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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

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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 drier



Place 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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ascend

The 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 dryer
Spray ninhydrin solution carefully to identify the spot
Calculate the Rf value


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Clinical Application of TLC

Screening 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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