THIN LAYER CHROMATOGRAPHY.pptx
- 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
- 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
- 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