TLC Thin Layer Chromatography

....

1. THIN LAYER CHROMATOGRAPHY Submitted by : HRISHAV VARDWAJ 1 ST SEMESTER M.PHARM Dept. of Pharmaceutics

2. Introduction to Chromatography DEFINITION Chromatography is a separation technique based on the different interactions of compounds with two phases, a mobile phase and a stationary phase, as the compounds travel through a supporting medium . Components : mobile phase : a solvent that flows through the supporting medium stationary phase : a layer or coating on the supporting medium that interacts with the analytes supporting medium : a solid surface on which the stationary phase is bound or coated

3. Basic terms Chromatogram: It is the visual output of the chromatograph. Chromatograph: It is equipment that enables a sophisticated separation . Stationary phase: It is a phase that is covalently bonded to the support particles or to the inside wall of the column tubing.

4. Basic terms Mobile phase: It is the phase which moves in a definite direction. Analyte (Sample): It is the substance to be separated during chromatography. Eluate: It is the mobile phase leaving the column.

5. Basic terms Retention time: It is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions. Eluent: It is the solvent that will carry the analyte.

6. Basic terms Retardation Factor( R f value): It is defined as the distance traveled by the compound divided by the distance traveled by the solvent.

7. Types of Chromatography

8. TLC Thin Layer Chromatography can be defined as a method of separation or identification of a mixture of components into individual components by using finely divided adsorbent solid / (liquid) spread over a glass plate and liquid as a mobile phase . Principle: - Adsorption or retention or partition or both or any other principle of a substance (s ) on the stationary phase - Separation of the adsorbed substances by the mobile phase - Recovery of the separated substances by a continuous flow of the mobile phase (elution) - Qualitative and quantitative analysis of the eluted substances

9. Steps in TLC analysis The following are the important components of a typical TLC system: – Preparation of developing chamber – Stationary phase and mobile phase – Methods of plate preparation – Development of the plate – Detection of analyte

10. Preparation of the developing chamber It can be a specially designed chamber, a jar with a lid, or a beaker with a watch glass on the top Pour solvent into the chamber to a depth of just less than 0.5 cm. To aid in the saturation of the TLC chamber with solvent vapours , you can line part of the inside of the beaker with filter paper.  Cover the beaker with a watch glass Allow it to stand while you prepare your TLC plate.

11. Why silica is used? Silica ( SiO 2 ) is a solid with an extended structure of tetrahedral silica atoms bridged together by bent oxygen atoms . On the surface of the silica particles , the solid terminates in very polar silanol groups ( Si-O-H) . The silica is the stationary phase because it remains adhered to the glass plate and does not move during the chromatography process .

12. Adsorbent : Water ratio

13. Mobile Phase The eluting solvent should also show a maximum of selectivity in its ability to dissolve the substances being separated. A more important property of the solvent is its ability to be itself adsorbed on the adsorbent. Mixtures of solvents can be used and, since increasing eluting power results (0.5 to 2% by volume)

14. Methods of plate preparation • Pouring : The adsorbent of finely divided and homogeneous particle size is made into slurry and is poured on a plate and allowed to flow over it so that it is evenly covered . Dipping : This technique is used for small plates by dipping the two plates at a time, back to back in a slurry of adsorbent in chloroform or other volatile solvents. Exact thickness of layer is not known and evenness of layer may not be good .

15. • Spraying : Slurry is diluted further for the operation of sprayer. But this technique is not used now a days as it is difficult to get uniform layer. • Spreading : All the above methods fail to give thin and uniform layers. Modern methods utilize the spreading devices for preparation of uniform thin layers on glass plates. Commercial spreaders are of two types (a) Moving spreader, (b) Moving plate type. It gives layer thickness from 0.2 to 2.0 mm.

16. ACTIVATION OF PLATES • After spreading plates are allowed to dry in air and further dried and activated by heating at about 110 o C for 30 min in hot air oven. • By removing the liquids associated with layer completely, the adsorbent layer is activated.

17. Development of TLC plate TLC plates used are purchased as 5 cm x 20 cm; 10cm x 20cm; 20cm x 20cm sheets. Measure 0.5 cm from the bottom of the plate. Using a pencil, draw a line across the plate at the 0.5 cm mark. This is the origin : the line on which you will spot the plate. Under the line, mark lightly the samples you will spot on the plate . Leave enough space between the samples so that they do not run together; about 4 samples on a 5 cm wide plate is advised.

18. Thin Layer Chromatography Development Place the prepared TLC plate in the developing beaker, cover the beaker with the watch glass, and leave it undisturbed on your bench top. The solvent will rise up the TLC plate by capillary action. Make sure the solvent does not cover the spot. Allow the plate to develop until the solvent is about half a centimeter below the top of the plate. Remove the plate from the beaker and immediately mark the solvent front with a pencil . Allow the plate to dry

19. Factors affecting R f value It depends on following factors : Nature adsorbent Mobile phase Thickness of layer Temperature Saturation Dipping zone Chromatographic techniques

20. TLC development

21. DETECTING OR VISUALISING AGENTS Non specific methods Iodine chamber method Sulphuric acid spray reagent UV chamber for fluorescent compounds Using fluorescent stationary phase Specific methods Spray reagents or Detecting agents or Visualizing agents Same as Paper chromatography

22. Advantages of TLC TLC is very simple to use and inexpensive. There are little materials needed for TLC Therefore, once the best solvent is found, it can be applied to other techniques such as HPLC More than one compound can be separated on a TLC plate as long as the mobile phase is preferred for each compound. The solvents for the TLC plate can be changed easily and it is possible to use several different solvents depending on your desired results.

23. Disadvantages of TLC TLC plates do not have long stationary phases. Therefore, the length of separation is limited compared to other chromatographic techniques. If you would need a lower detection limit, one would have to use other chromatographic techniques. TLC operates as an open system, so factors such as humidity and temperature can be consequences to the results of your chromatogram. Edge Effect : occurs due to improper saturation of TLC chamber

24. Applications of TLC It is used for separation of all classes of natural products and is established as an analytical tool. - E.g. Acids, alcohols, glycols, alkaloids, amines, macromolecules like amino acids, proteins and peptides, and antibiotics . Extensively used as an identification test and test for purity.

25. Applications Applications of TLC for separation of Inorganic Ions – Used for separating cationic, anionic, purely covalent species and also some organic derivatives of the metals. Separation of Amino Acids- two dimensional thin – layer chromatography Separation of vitamins – vitamin E, Vitamin D3, vitamin A

26. Thank You