THIN LAYER CHROMATOGRAPHY ppt.pptx

Briefly discussed about the introduction, preparation, application of thin layer chromatography.

1. THIN LAYER CHROMATOGRAPHY SUBMITTED BY :- TARUN KUMAR DEPARTMENT OF PHARMACY M.PHARMACY CENTRAL UNIVERSITY OF SOUTH BIHAR

2. CONTENT INTRODUCTION PRINCIPLE METHOD EXPERIMENTAL TECHNIQUE RF VALUE DETERMINATION IMPORTANCE OF RF VALUE APPLICATION

3. CHROMATOGRAPHY It is defined as the method of separation of a mixture into an individual components through equilibrium distribution between two phases . Michael tswett is being the father of liquid chromatography. Tswett developed his idea in early 1900s. \\\\\\

4. The chromatographic method of separation in general involve in the following steps:- Adsorption or retention of substance, or substance on stationary phase. Separation of absorbed substance by mobile phase. Recovery of separated substance by continues flow of the mobile phase the method being called elution . Qualitative and quantitative analysis of eluated substance.

5. THIN LAYER CHROMATOGRAPHY It is a form of liquid chromatography consists of :- A mobile phase (developing solvent). A stationary phase ( a plate or strip coated with a form with a silica gel ) . Analysis is performed on a flat surface under atmospheric pressure or room temperature.

6. The two most commonly types of thin layer chromatography Normal phase Reverse phase Normal phase :- It is terminology used when the stationary phase is polar for e.g. silica gel and the mobile phase is organic solvent of mixture of organic solvents which is less polar than stationary phase.

7. In Reverse phase :- In the reverse phase when the stationary phase is a silica bonded with an organic substrate such as a long chain aliphatic acid . Mobile phase is mixture of water and organic solvent which is more polar than stationary phase.

8. PRINCIPLE • TLC technique involves the distribution of components of a mixture to be separated between two phases. • The components of the mixture are partitioned between an adsorbent (stationary phase), and a solvent (mobile phase). • Different compounds will have different solubility and adsorption to the two phases between which they are to be partitioned. • In TLC separation of the individual substances is based on their relative affinities towards stationary and mobile phases.

9. The stationary phase: is a thin layer of adsorbent (usually silica gel or alumina) coated on a plate. • The mobile phase: is a developing liquid which flows through the stationary phase, carrying the samples with it. Components with more affinity towards stationary phase travels slower. Components with less affinity towards stationary phase travels faster.

10. METHOD • Adsorbents used as Stationary Phase: - Inorganic: Silica Gel, Aluminium Silicate, Bentonite. -Organic: Cellulose & its acetylates, Charcoal & activated Charcoal, Dextran Gel, Polyamides. • Solvents used as Mobile Phase: - Petroleum ether, Benzene, Carbon tetrachloride. • Selection of Adsorbents and Solvents: -Adsorbent should not adhere to glass plate. -Solvents should be of high purity. -Selected on the nature of compound to be separated(polar or non polar).

11. TLC TECHNIQUE Step1. Preparation of Slurry • A plastic, glass or aluminum sheet is coated with a thin layer of silica gel (adsorbent). • Plates must be dried, activated and stored in desicator until used Step 2. Preparation of tank • Solvent mixtures should be freshly prepared for analysis. • Solvent is poured down side of the tank (1.5cm depth). • Tank is covered with the glass lid and kept for saturation.

12. Step 3. Application of sample (spot). A very small amount of sample (solution) to be analyzed is applied in a small spot with a capillary tube, ~1cm from the bottom of the TLC plate.

13. EXPERIMENTAL TECHNIQUE OF TLC The TLC is developed in a chamber which contains the mobile phase (solvent). When the mobile phase rises up the plate up by capillary action, the components dissolve in the solvent and move.

14. TLC TECHNIQUE Individual components in the sample move up at different rates. • More polar analytes interact more strongly with the stationary phase move very slowly up. More nonpolar analytes interact less strongly with the polar silica gel and more strongly with the less polar mobile phase move higher up. Once the solvent reaches the top (below ~1-2 cm) of the TLC sheet the plate is removed from the developing chamber and position of solvent front is marked.

15. TLC TECHNIQUE • The solvent is allowed to evaporate from the TLC sheet. • As the compound is colorless, it can be visualized by suitable methods. Lipids - Iodine vapors Amino acids - Ninhydrin reagent. • Also, manganese-activated zinc silicate (fluorescent compound), is added to the adsorbent that allows the visualization of spots under a black light (UV254 lamp). Once visible, the Rf value of each spot can be determined.

16. RF VALUE • Rf value indicates the position of migrated spots on chromatogram. • In TLC the results are represented by Rf value which represents the migration of solute relative to the solvent front. • The Rf value is calculated as:- Rf = distance travelled by the solute distance travelled by the solvent

17. RF VALUE DETERMINATION

18. IMPORTANCE OF RF VALUE Qualitative results of TLC – Rf values of any component lies between 0 to 1. – no more than two decimal places . Rf values can be used to aid in the identification of a substance by comparison to standards. Comparison should be made only between spots on the same sheet, run at the same time.

19. APPLICATIONS Test the purity of the sample: Thin layer chromatography helps to detect the purity of the sample by direct comparison with the standard or authentic sample. Any impurity in the sample shows up as extra spots in chromatography. Identify the components:   Thin layer chromatography can purify, isolate and identify the natural products like volatile oil or essential oil, fixed oil, waxes, terpenes, alkaloids, glycosides, steroids etc. in the test samples.

20. Biochemical analysis:   Biochemical metabolites from the body fluids, blood plasma, serum, urine etc. can be isolated using thin layer chromatography. In chemistry: TLC is used to separate and identify closely related compounds or cations and anions in inorganic chemistry. Pharmaceutical industries utilize TLC technique for qualitative analysis or detect impurities in various medicines like hypnotics, sedatives, anticonvulsants, tranquillisers, anti-histaminics, analgesics, local anaesthetics, steroids, etc. Yet another important application of TLC is to separate multi-component  pharmaceutical formulations  into its individual components. .

21. In food and cosmetic industry: Any artificial colour, preservatives, sweetening agent, and other impurities in food and cosmetic products can be detected and isolated by TLC technique.

22. REFERENCE 1. A . Archana, Bele and Anubha Khale, An overview on thin layer chromatography, IJPSR, 2(2), 2011, 256-267. 2. A. Mohammad, S.A. Bhawani and S. Sharma, Analysis of herbal products by thin-layer chromatography: a review, International Journal of Pharma and Biosciences, 1(2), 2010, 1-50. 3. A.H. Beckett, J.B.Stenlake, Practical pharmaceutical chemistry, thin layer chromatography, CBS publishers, 4th edition, 2005, 115-128. 4. A.V. Kasture, K.R. Mahadik, S.G.Wadodkar, H.N. More, A textbook of pharmaceutical analysis, instrumental methods, Nirali Prakashan, 9th edition, 2, 2005, 18-30. 5. B. Fried, J. Sharma, Thin-layer chromatography, fourth edition, revised and expanded, Marcel Dekker inc., New York - Basel, 1999, 499