TLC requirements, procedure, indication etc

1. 01-10-2021
THIN LAYER CHROMATOGRAPHY 1

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
01-10-2021
THIN LAYER CHROMATOGRAPHY 3

4. OBJECTIVES
• To study in detail about Thin Layer Chromatography, its
01-10-2021
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
01-10-2021
THIN LAYER CHROMATOGRAPHY
5
Type of chromatography differ in the mobile and stationary phase used

6. THIN LAYER CHROMATOGRAPHY
01-10-2021
THIN LAYER CHROMATOGRAPHY 6

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.
01-10-2021
THIN LAYER CHROMATOGRAPHY 7

8. HISTORY
Chromatography method is first discovered by Tswett, a Russian botanist, in 1906
for the separation of coloured substances into individual components.
01-10-2021
THIN LAYER CHROMATOGRAPHY 8

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.
01-10-2021
THIN LAYER CHROMATOGRAPHY 9

10. REQUIREMENTS
01-10-2021
THIN LAYER CHROMATOGRAPHY 10

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.
01-10-2021
THIN LAYER CHROMATOGRAPHY 11

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.
01-10-2021
THIN LAYER CHROMATOGRAPHY 12

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.
01-10-2021
THIN LAYER CHROMATOGRAPHY 13

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
01-10-2021
THIN LAYER CHROMATOGRAPHY 14

15. 01-10-2021
THIN LAYER CHROMATOGRAPHY 15

16. 01-10-2021
THIN LAYER CHROMATOGRAPHY 16

17. INTERPRETATION OF TESTS
• Rf - RETENTION FACTORS
• Rf = distance analyte traveled
distance solvent traveled
This is used to compare with standard value.
01-10-2021
THIN LAYER CHROMATOGRAPHY 17

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).
01-10-2021
THIN LAYER CHROMATOGRAPHY 18

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
01-10-2021
THIN LAYER CHROMATOGRAPHY 19

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.
01-10-2021
THIN LAYER CHROMATOGRAPHY 20

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..
01-10-2021
THIN LAYER CHROMATOGRAPHY 21

22. 01-10-2021
THIN LAYER CHROMATOGRAPHY 22