2. BY
DR. KOMAL M. JADHAV
1st YEAR PG SCHOLAR
DEPT. OF AGADATANTRA
01-10-2021
THIN LAYER CHROMATOGRAPHY 2
3. CONTENTS
• Objectives
• Introduction
• History
• Principle of TLC
• Types of Chromatography
• Description of technique
• Procedure
• Interpretation of the test
• Applied aspects
• When and where TLC is used
• Precautionary measures
• Advantages
• References
• Summary
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4. OBJECTIVES
• To study in detail about Thin Layer Chromatography, its
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THIN LAYER CHROMATOGRAPHY 4
Principle Procedure
Types Application
5. TYPES OF CHROMATOGRAPHY TECHNIQUE
TECHNIQUE STATIONAY PHASE MOBILE PHASE
Column/Adsorption Chromatography solid Liquid
Partition Chromatography Liquid Liquid
Paper Chromatography Liquid Liquid
Thin layer Chromatography Liquid/solid liquid
Gas- liquid Chromatography Liquid Gas
Gas- solid Chromatography Solid Gas
Ion exchange Chromatography solid liquid
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THIN LAYER CHROMATOGRAPHY
5
Type of chromatography differ in the mobile and stationary phase used
7. INTRODUCTION
• THIN LAYER of Adsorbent ( silica, alumin)
• CHROMATOGRAPHY – study of light / colour.
TLC used for - a physical method of separation in which the contents to be
separated are distributed between two phases, one of which is stationary phase
while the other ( mobile phase) moves in a definite direction.
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8. HISTORY
Chromatography method is first discovered by Tswett, a Russian botanist, in 1906
for the separation of coloured substances into individual components.
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9. PRINCIPLE OF TLC
TLC is an adsorption chromatography which involves separation of the
substances of a mixture over a thin layer of adsorbent which is supported on a
glass plate or other supporting medium under the influence of mobile phase.
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11. • Adsorbent is thinly coated onto a suitable support – Glass plate/ polyster/
aluminu sheet
• Glass plate – rigid, transparent and chemically resistant to mobile phase.
• Glass capillaries – to mark the sample on TLC plate
• Oven – used to activate the layers after coating (Silica gel plates become
activated when all the water has been removed by drying in an oven)
• Spraying agents – aluminum chloride for flavonoids. Carbon tetrachloride –
organophosphorus pesticides.
• Fume hood – while preparing samples (toxins, pesticides, poison etc) – being
adsorbed through skin or inhaled, which may cause allergies.
• UV combinet – UV light wave length 254 / 366nm.
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12. PROCEDURE
o Select suitable TLC plate.( if we prepare manually – it should be air dried for 20 mints. Then
placed in oven at 100degree Celsius for 30 mints in order to activate it.)
o The solvent should be added to at least 30 mints before the chromatogram is developed to
saturate the atmosphere with solvent vapors.
o The sample should be prepared.
o With the help of capillary pippete apply the sample over the TLC plate as a spotting.
o The sample and standard should be applied at the line with sufficient distance.
o If solution is less concentrated then apply for several times.
o Allow them to dry out for a short time between each application.
o Number the spotting from left to right and record the substance that have been applied.
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13. CONTI…..
• TLC chamber to be filled with mobile phase and cover with glass plate.
• The coated plate is placed layer side down on the TLC chamber
• Ensure that the level of solvent should be below the level of spots applied.
• The set up must not be disturbed in order to obtain effective result.
• When chromatogram has been developed then the plate is air dried.
• Spraying reagent – for the compounds not characterized by the possession of colour,
strong absorption to UV have to be rendered visible by special detection reagents applied
to the layer by means of a spray.
• The chromatogram should be examined under UV light. The substances present appear
as dark areas.
• Calculate Rf value of sample and standard.
• If values are same then we qualitatively confirm the presence of compound.
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14. DESCRIPTION OF TECHNIQUE
Sample preparation
Selection of chromatographic phase
and mobile phase
Application of sample on plate and
development
Drying of chromatographic plate
and detection
Visual examination and
documentation
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17. INTERPRETATION OF TESTS
• Rf - RETENTION FACTORS
• Rf = distance analyte traveled
distance solvent traveled
This is used to compare with standard value.
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18. EXAMPLE
• SRUNGHATAKA – DRIED SEED
• T L C of alcoholic extract of the drug on silica gel ‘G’ plate using n- Butanol: Acetic acid: Water
(4:1:5)v/v shows under U V (366mm) one fluorescent zone at Rf.0.60(blue). On spraying with
5% methanolic – sulphuric acid reagent and heating the plate for about ten minutes at Rf. 105
degree Celsius three spots appear at Rf 0.30 (grey), 0.43(grey) and 0.93(violet).
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19. WHEN TLC IS USED
• Non volatile / low volatile substance.
• The substance are strongly/medium/non polar .
• ( adsorbent is the polar compound and solvent is non polar)
• If solvent is non polar, it moves rapidly on polar media( stationary phase) and
vice versa.
• The substance cannot be detected by the method of LC or GC
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20. WHERE TLC IS USED
• PHARMACEUTICALS AND DRUG - determination of the conc of active ingredients,
preservatives in drugs.
• CLINICAL CHEMISTRY,FORENSIC CHEMISTRY AND BIOCHEMISTRY –
determination of active substances and their metabolites in biological matrices,
diagnosis of metabolic disorders such as PKU( phenylketonuria), cystinuria.
• COSMETOLOGY – dye raw materials and end products, preservatives, fatty acids,
constituents of perfumes.
• FOOD ANALYSIS – Determination of pesticide and fungicides in drinking water,
residues in vegetables, salad, meat etc… banned additives etc
• Analysis of inorganic substances – ions(metals)
• Environmental Analysis – Ground water analysis, determination of pollutants from
abandoned armaments in soil and surface waters, decomposition products from azo dyes
used in textiles.
• Pesticides, barbiturates, narcotics, tranquilizers.
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21. FORENSIC SCIENCE
• Analysis of barbiturates – barbital, phenobarbital and secobarbital
• Analysis of tranquilizers – diazepam, chloropromazine
• Analysis of narcotics – morphine, cocaine, heroin etc
• Analysis of pesticides – organophosphorus, carbamates, pyrethroids etc..
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