THIN LAYER CHROMATOGRAPHY.pptx

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1. THIN LAYER CHROMATOGRAPHY Presented By : Sabir Hussain M. Pharm 1 st Sem Roll No. 230624010 Dept. of Pharmacy Tripura University Guided By : Dr. Rajat Ghosh, PhD Assistant Professor Dept of Pharmacy, Tripura University 1

2. Contents : History Introduction Chromatography Principle of TLC Stationary phase Glass plates Preparation of glass plates Mobile Phase Spotting Developing chamber Application References 2

3. History : The first reported use of a thin layer was in 1938 by two Russian scientists, N.A. Izmailov and M.S. Schreiber. They separated plant extracts on a slurried adsorption medium spread to a 2-mm-thick layer by spotting an alcoholic plant extract in the center of the layer and observing rings as the solution spread. 3

4. Chromatography : Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. 4

5. TLC Definition : TLC is defined as the method of separation identification of a mixture of compound into individual components by using finely divided adsorbent solid/liquid spread over a glass plates as a stationary phase and Liquid as a mobile phase. On completion of the separation, each components appears a spots. Each spot has a retention factor(Rf ) 5

6. Introduction : TLC is a liquid chromatography consisting of : A mobile phase (developing solvent) A stationery phase (a plate or strip coated with a form of silica gel). Analysis is done under normal atmospheric pressure and room temperature. 6

7. Principle : The principle of TLC is where mixture components are separated between a fixed stationary phase and a liquid mobile phase by differential affinities between the two phases. The separation is through adsorption. Component with less affinity towards stationary phase travels fast. Component with more affinity towards stationary phase travels slow. 7

8. Stationary Phase : TLC plates also known as Chromatoplates can be prepared in the lab but mostly purchased. Silica gel and alumina are among the most common stationary phases, but others are available as well.  Many plates incorporate a compound which fluoresces under short-wave UV (254 nm). The backing of TLC plates is often composed of glass, aluminum, or plastic.  8

9. Glass backed TLC plates Aluminium backed TLC plates 9

10. Glass Plates : Glass plates which are specific dimensions like 20 cm X 20 cm (Full plate), 20 cm X 10 cm (Half plate ), 20 cm X 5cm (Quarter plate) can be used. These dimensions are used sincethe width of the commercially available TLC spreader is 20 cm. Microscopic slides can also be used for some applications like monitoring the progress of a chemical reaction. The development time is much shorter like 5 minutes. Glass plates of different dimensions can also be used when the TLC plates are prepared without the use of TLC spreader. In general, the glass plates should be of good quality and should withstand temperatures used for drying the plates. 10

11. Preparation and Identification of TLC plates : Pouring technique : The slurry is prepared and poured on to a glass plate which is maintained on a leveled surface. The slurry is spread uniformly on the surface of the glass plate. After setting, the plates are dried in an oven is used for spotting. 11

12. Dipping technique : In this two plates (either of standard dimensions or microscopic slides) are dipped in to slurry and are separated after removing from slurry and later dried. The disadvantage is that a larger quantity of slurry is required even for preparing fewer plates. 12

13. Spraying technique : Resembles that of using a perfume spray on a cloth. The suspension of adsorbent or slurry is sprayed on a glass plate using a sprayer. The disadvantage is that the layer thickness cannot be maintained uniformly all over the plate. 13

14. Mobile Phase : It is a developing liquid which travels up the stationary phase, carrying the samples with it. It depends on; Nature of the substance to be separated i.e polar or non polar. Nature of stationary phase used Mode of chromatography Solvent used should be of high purity. Solvents used:- petroleum ether benzene carbon tetrachloride chloroform 14

15. Spotting : 1% solution of sample or standard is spotted using a capillary tube or micropipette. The spots should be kept at least 2cm“4 above the base of plate and the spotting area should not be immersed in mobile phase in a developing chamber. The width of the band must be as narrow as possible. 15

16. Developing Chamber : It is used for the purpose of TLC plate run in mobile phase. After the mobile phase is poured into the chamber it is kept closed with lid. This is done to equilibrate the atmosphere of empty space in chamber with the mobile solvent. This is also known as saturation of TLC chamber, Edge effect occurs when the solvent front in the middle of TLC plate moves faster than that of edge edge of plate. 16

17. Development of TLC plates : Different development techniques are used for efficient separations. They are - 1. One dimensional development (ascending or descending technique). 2. Two dimensional development 3. Horizontal development 4. Multiple development 17

18. One dimensional development : Like conventional type, the solvent flows against gravity. The spots are kept at the bottom portion a kept in a chamber with mobile phase solvent at the bottom. 18

19. Two dimensional development : This technique is similar to 2-Dimensional TLC. The paper is developed in one direction and after development, the paper is developed in the second direction allowing more compounds or complex mixtures to be separated into individual spots. In the second direction, either the same solvent or different solvent system can be used for development. 19

20. Detecting Agent : After the development of chromatogram, the spots should be visualized. Detecting of coloured spots can be done visually. But for detecting of colourless spots, any one of the following techniques can be used. Non specific method : Where the number of spots can be detected but not exact nature of compound Example 1. lodine Chamber Method : Where brown or amber spots are observed when the paper is kept tank with few iodine crystals at the bottom. 2. UV Chamber for fluorescent compounds : When compounds are viewed under UV chamber at 245 nm or at 365 nm fluorescent compounds can be detected. 20

21. Specific Method : Specific spray reagents or detecting agents visualizing agents used to find out the nature of compounds for identification purposes Examples;. Ferric chloride for phenolic compounds Ninhydrin in acetone for amino group Dragen droff’s reagent for alkaloid 3,5-Dinitro benzoic acid for cardiac glycosides 2,4-Dinitrophenyl hydrazine for aldehyde and ketones 21

22. Advantages : Less cost required. Less time required. High sensitivity. High resolution. Simple equipment required. 22

23. Disadvantages : Since the separation takes place in open chamber so it can affect by humidity. Uneven migration of solvent. Over large spot formation. 23

24. Application : Separation of mixtures of drug of chemical or biological origin’ Separation of carbohydrates (sugars), vitamins, antibiotics, proteins alkaloids, glycosides, amino acids etc. Identification of related compounds in drugs. Purity of samples. 24

25. References : Instrumental Methods of Chemical Analysis, By Dr. H.KAUR 2 nd Edition Vogel's - Textbook of quantitative chemical analysis (5th Edition) https://chem.libretexts.org/Courses/SUNY_Oneonta/Chem_221%3A_Organic_Chemistry_I_(Bennett)/2%3ALab_Textbook_(Nichols)/02%3A_Chromatography/2.03%3A_Thin_Layer_Chromatography_(TLC)/2.3E%3A_Step-by-Step_Procedures_for_Thin_Layer_Chromatography https://www.researchgate.net/publication/277703425_Thin_Layer_Chromatography 25

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