thin layer chromatography presentation.pptx

This presentation is about thin layer chromatography for M. Pharm students

1. TLC: Thin Layer Chromatography Presented By: Komal R. Chimkar M.Pharm First Year Dr. Rajendra Gode College of Pharmacy, Malkapur

2. Content Introduction Principle Methodology Advantages Disadvantages Applications References 2 TLC- by Komal Chimkar

3. Introduction The technique of TLC was first discovered by Izmailov & Schraiber (1938). Consden , Gorden and Martin (1944) started using filter paper. Williams carried out chromatography on adsorbent layer sandwiched between 2 glass plates. Kirchner in 1955 separate and identify terpenes . TLC for analytical adsorption chromatography was first introduced by Stahl in 1958. TLC is also called as Strip, Spread layer, Surface chromatography and Open column chromatography. 3 TLC- by Komal Chimkar

4. Principle The principle of separation is adsorption. On a chromatographic plate, one or more compounds are spotted on a thin layer of adsorbent. Through the capillary action, the mobile phase solvent passes through the TLC plate. The components are separated on a thin layer chromatographic plate based on the affinity of the components towards the stationary phase. Separation may result due to adsorption or partition or depending upon nature of adsorbent used on plates and solvent used for TLC. 4 TLC- by Komal Chimkar

5. Methodology Selection of plates Adsorbent Preparation and activation of plates Preparation and application of sample Selection of solvent Development chamber Detecting reagent Evaluation of chromatogram 5 TLC- by Komal Chimkar

6. 1. TLC plates Glass or flexible plates ( aluminium foil, polyethylene, or plastic strips) are commonly used for spreading adsorbent. Glass plates are widely used. Size depends on the type of separation carried out, the type of chromatographic tank and spreading apparatus available. Standard sizes are 20x5 cm, 20x10 cm, 20x20 cm. The glass plates should be of good quality and a surface of plates should be flat. Plates should be withstand to temperature. 6 TLC- by Komal Chimkar

7. 2. Adsorbent as a stationary phase a) Silica gel: Available commercially with or without binder. Binder (10% w/w) is added to provide good mechanical strength. Particle size should be 15 μ m with particle size range of 5-40 μ m. b) Alumina: Used without binders. Alumina produced with its surface either acidic, basic or neutral. Acidic alumina- used for separation of neutral or acidic materials. Basic alumina- used for separation of steroids, alkaloids and aromatic and unsaturated hydrocarbons. Various other materials also used for TLC like calcium phosphate, magnesium trisilicate , polyamide, silica gel-alumina, acetylated cellulose, ferric oxide hydrate, etc. 7 TLC- by Komal Chimkar

8. 3. Preparation and activation of plates For preparation of TLC plates thin layer of adsorbent slurry or suspension applied on plates. The various methods of preparing layers are as follows: Pouring Dipping Spraying Spreading An improved TLC spreader was developed by DESAGA ( W est Germany). By this spreader the layer thickness can be adjusted to 0.0 to 2.0 mm. The spreading method is the most important now-a-days to obtain thin and uniform layers. Fig. Operation of thin layer spreader 8 TLC- by Komal Chimkar

9. Activation of adsorbent e) Precoated plates These are quite expensive. Thickness varies from 0.1 to 0.2 mm . After making thin layer on plates, the activation of adsorbent is done. The plates are kept for 30 min in air and then in an oven at 110°C for another 30 min. In order to obtain very active layers, silica gel and alumina plates can be heated to 150°C for about 4 hours. Fig. P recoated plates used in TLC 9 TLC- by Komal Chimkar

10. 4. Preparation and application of sample Generally mobile phase used for preparation of sample solution or volatile and non-polar solvents are used. 1% solution of the sample is applied on the plates with the help of capillaries, micropipettes and microsyringes . Before applying the sample, the origin should be marked on the sheet by making a scratch or pencil mark off to the slide. The origin should be far enough from edge of the sheet. And then solution is applied as single spot in a row along one side of the plate. 10 TLC- by Komal Chimkar

11. 5. Selection of solvent: The choice of solvent depends on the following two factors: Nature of the substance to be separated. Material on which the separation is to be carried out. More polar solvents produce the greater migration, a better separation. It has also been observed that a combination of two solvents give better result than one solvent. 6. Development chamber: To hold TLC plates development chambers of different sizes and standards dimensions used. In TLC, the plate is placed in a development chamber at an angle of 45°. The bottom of chamber is covered upto nearly 1mm by solvent. Top is covered tightly with the lid. 11 TLC- by Komal Chimkar

12. Fig. Apparatus of thin layer chromatography 12 TLC- by Komal Chimkar

13. 7. Detecting reagent: Many compounds separated by thin layer chromatography are colourless ; their positions are thus located or detected with the help of some reagent known as locating or detecting reagent. One can detect colourless components either under UV light or by treating the plate with a visualizing reagent. C hromic acid/ sulphuric acid can be used in TLC. 8. Evaluation of the chromatogram: After locating the spots on plates and marking their position and size, they are evaluated either qualitatively or quantitatively. Qualitative: The Rf value of the sample is calculated and on comparison of Rf values for known and unknown the qualitative identification of sample is made. 13 TLC- by Komal Chimkar

14. Quantitative: Visual comparison: simple procedure in which the only detector is the human eye. Spot Areas and Weight Relationship: from the area of spot, amount of substance present is calculated. High random errors, results in variation of the spot shapes. Spot densitometry: intensity of the colour of a substance is measured directly on the chromatogram. Direct spectrophotometry: quantitative measurements are obtained by evaluation with wavelength of maximum adsorption of the substances. 14 TLC- by Komal Chimkar

15. Advantages of thin layer chromatography It requires simple equipment for separation. Rate of separation is faster as compared to column and paper chromatography. Small quantities of sample sufficient for analysis i.e. few µg. Detection for this technique is easy. It is used as adsorption and partition chromatography due to availability of wide range of adsorbents. Cost effective technique. Disadvantages of TLC TLC plates do not have long stationary phases and hence separation takes place only at certain length. Sample may be affected by the humidity and temperature. TLC- by Komal Chimkar 15

16. Application of thin layer chromatography The various application of TLC are as follow: 1. Identification of compound: TLC is increasingly employed in isolation, purification and identification of various class of organic compounds. In photochemistry it is used for identification of natural products like volatile oil (Essential oil), fixed oils, waxes, terpenes , alkaloids, glycosides, steroids, etc. 2. Purity of Sample: Shows extra spots and this can be detected very easily if impurities are present. For this, direct comparison of the sample and authentic sample is done. 16 TLC- by Komal Chimkar

17. 3. In Chemistry: For the separation and identification of compounds which are closely related to each other. It is also used for identification of cations and anions in inorganic chemistry. 4. In Pharmaceutical industry: For detection of impurity in Pharmacopoeial drug or chemical. Various drugs like hypnotics, sedatives, anticonvulsant, antihistamines, analgesics, local anesthetics, steroidal drugs have been tested qualitatively by TLC method. 5. Biochemical analysis: Useful in separation of biochemical metabolite or constituent from its body fluids, blood plasma, serum, urine, etc. 17 TLC- by Komal Chimkar

18. 6. Food and cosmetic: For separation and identification of colours , preservatives, sweetening agents and various cosmetic products. 7. As a check on processes: TLC has been used for checking of the other separation procedures and purification processes. It has been used for checking of distillation fraction and for checking the progress of purification by molecular distillation. 18 TLC- by Komal Chimkar

19. References Beckett and Stenlake - Practical Pharmaceutical Chemistry, Vol II, 4 th edition, CBS Publishers, New Delhi, 1997. Gurudeep R. Chatwal and Sham K. Anand - Instrumental Method Of Chemical Analysis , Himalaya Publishing House, 2011. P. Salunke , Md. Rageeb Md Usman and Dr.S.D.Barhate - Instrumental Method Of Analysis, Pee Vee . TLC- by Komal Chimkar 19

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