Thin Layer Chromatography and High Performance Thin Layer Chromatography

Thin-layer chromatography is a method of separation, or identification of a mixture of components by using finely divided adsorbent solid/ liquid over a glass plate, and liquid as a mobile phase. The distinction depends on the relative affinity between the stationary and mobile phases of the compounds.

1. A Presentation on the Topic T HIN LAYER CHROMATOGRAPHY AND HIGH PERFORMANCE T HIN LAYER CHROMATOGRAPHY MPL101T Presented by AVINASH KUMAR 1 st semester . DEPARTMENT OF PHARMACEUTICAL CHEMISTRY

2. Content - Introduction of TLC. Principle of TLC. Steps involved in TLC Application of TLC. Introduction of HPTLC . Principle of HPTLC. Instrumentation Application of HPTLC. References 2

3. Chromatography Chromatography is an important technique that enables the separation , Identification and purification of the components of a mixture for qualitative and quantitative analysis . The Russian botanist Mikhail tswett coined the term chromatography in 1906 . The first analytical use of chromatography for the analysis of fatty acids mixtures 3

4. Types of chromatography 4 4

5. Introduction of TLC One of the most popular and widely used separation technique for all classes of natural products .important analytical test for qualitative and quantitative analysis . TLC is a planner chromatography in which them layer of adsorbent (stationary phase)and coated to a solid support (glass sheet / aluminium sheet) by pouring ,dipping, spreading or spraying technique. Mobile phase-solvent system , moves through the stationary phase by capillary action ,some times by gravity and electrical potential. 5

6. Principle of TLC Based on adsorption chromatography. The components with more affinity towards stationary phase travels slower. The components with more affinity towards mobile phase travels faster. 6

7. Adsorbents for TLC Silica gel –G , Alumina Kieselguhr Silicates- calcium ,magnesium ,phosphate Active carbon Glass powder Cellulose Ferric oxides. 7

8. Solvent system with increasing polarity n-hexane Cyclohexane Carbon tetrachloride Benzene Chloroform Diethyl ether Ethyl acetate Acetone Ethanol Methanol and water 8

9. Steps involved in TLC Selection of stationary phase Coating of TLC plates( chromaplate ). Selection of mobile phase Sample preparation Application of sample Development of chromatogram Detection of spot Rf value calculation 9

10. 10

11. 11

12. 12

13. Calculation for Rf value Rf= Distance travelled by the solute Distance travelled by solvent Rf values- less than or equal to1 Rf depends upon a number of factors: The solvent employed. The medium used for separation. The nature of the mixture. The temperature 13

14. Sintering -when silica gel is heated to higher temperature 100-1000 degree Celsius the cavity system under goes change . The porous material thickens. Silica gel sinters and the causes this sintering are-elimination of chemically linked (structural) water from SiOH groups. The silica gel framework is reptured when water is split out. Chamber Saturation -In a ordinary chamber the observation is made that substances near the edge of a thin layer migrate further than the same substances in the center. This “edge effect” occurs especially when mixtures of solvents are used which differ considerably in polarity, vapor density and density. The effect is due to inadequate chamber saturation . 14

15. Terms that are used in Chromatography- Chromatography - is a technique. Chromatograph -is the instrument that is used to generate chromatogram. Chromatogram -is the output(product of that technique. Chromatoron - is a preparative , centrifugal accelerated radial thin layer chromatography desinged by the authors of the compendium of organic synthetic methods.it replaces prep TLC plates , small columnsand HPLC over all dimensions are 30 x 35 x 30 cm. Craig tube - for the recovery of component from TLC plates. 15

16. Application of TLC As a check on process Checking the purity of sample Examination a reaction Identifying organic compounds like acids ,alcohols alkaloids, amino acids, Separation of vitamins. 16

17. High Performance Thin Layer Chromatography HPTLC(High Performance Thin Layer Chromatography)is the automated , sophisticated form and improved method of TLC . It is a powerful analytical method equally suitable for qualitative and quantitative analytical tasks . It is also known as planner or flat bed chromatography. HPTLC is very popular for many reasons such as - Visual chromatogram Multiple sample handling Enables the most complicated separation . 17

18. Principle- Same theoretical principle of TLC ( Adsorption chromatography ) i.e. the principle of separation is adsorption . the component are separated on a chromatographic plate according to their affinity and separation also based on their solubility in mobile phase . 18

19. Steps involved in HPTLC Stationary phase : Silica gel GF- 254 ( SO 2 + CaSO 4 + zinc silicate ) Particle size in 7 μ m . Thickness of plate 100 μ . Decreased particle size , increased surface area , so increasing efficiency increasing resolution & decreasing analysis time. Sample preparation : It is important to prepare proper sample for successful separation. Sample and reference substances should be dissolved in the same solvent to ensure comparable distribution at starting zones . 19

20. Sample Application Usual concentration range is 0.1-1 μ g/l . Above this causes poor separation . Volume recommended for HPTLC is 0.5 – 5 μ l. The size of sample spot must be not large than 1mm in diameter . The problem of overloading can be overcome by applying the sample as band . The major criteria is that it should not damage the surface while applying sample . 20

21. Selection of mobile phase- Chemical properties of analytes & adsorbent layer should be considered while selection of mobile phase . The less amount of mobile phase is required then TLC Due to small amount of mobile phase , it prevents errors in result which is occur due to mobile phase Pre-conditioning (chamber saturation) 21

22. Detection and visualization- Detection under UV light is first choice . Fluorescent compounds can be seen at 254 nm (short wavelength ) or at 366 nm(long wavelength ). Non UV absorbing compounds are visualized by dipping the plates in 0.1% iodine solution . 22

23. 23 Instrumentation-

24. Factors affecting HPTLC : Types of stationary phase Types of mobile phase Layer thickness Temperature Amount of sample 24

25. Applications of HPTLC Pharmaceutical industry : Quality control , purity test . Food analysis : QC , stability testing Clinical applications : Metabolism studies , drug screening etc. Biomedical analysis : Separation of different proteins. Forensic : Poisoning investigation Environment analysis : pesticides in drinking water etc. Analysis of drug in blood etc . 25

26. References- Stahl Egon , Thin Layer Chromatography-A Laboratory Handbook, Springer International Edition , Second edition,56-102. Chatwal G.R.and Anand S.K. , Instrumental Methods of Chemical Analysis,Himalaya publishing house,2007,2.599-2.609 . Kasture Dr. A.K.and Mahadik Dr.K.R . Pharmaceutical Analysis Nirali Publishers ,Volume -II , 28-30 . Sethi.P.D ,HPTLC – Quantitaive analysis of pharmaceutical formulation. Beckett A.H. and Stenlake J.B.,Practical Pharmaceutical Chemistry,Collier Books,Fourth Edition,Volume-II,56-59. 26

27. THANK YOU 27