THIN LAYER CHROMATOGRAPHY

Thin layer chromatography technique - easier, cheaper.
Handling is easy. Used as an identification test also purity test. It comprises of stationary and mobile phase. There are various types of chromatography technique. TLC consists of three steps - spotting, development, and visualization. The Rf value is used to quantify the movement of the materials along the plate. Rf is equal to the
distance traveled by the substance divided by the distance traveled by the solvent. Its value is
always between zero and one. A TLC analysis might be summarized something like, "Using a silica
gel plate and ethyl acetate as the development solvent, unknown mixture X showed three spots
having Rf's of 0.12, 0.25, and 0.87". CThere are three components in TLC:
(1) the TLC plate (stationary phase), the development solvent (mobile phase), and the sample to be
analyzed (solute). In our experiment the TLC plate consists of a thin plastic sheet covered with a
thin layer of silica gel.

1. THIN LAYER CHROMATOGRAPHY PRESENTED BY - NIDHI BANSAL M.PHARMACY 2004610003

2. CHROMATOGRAPHY . Chromatography is a technique for the separation of a mixture of compounds by passing it through a medium between two phases, one of which is stationary phase while the other mobile phase moves in a definite direction. Types of Chromatographic techniques : Technique Stationary Phase Mobile Phase Thin Layer Chromatography (TLC) Liquid/ Solid Liquid Column/Adsoption Chromatography Solid Liquid Paper Chromatography Liquid Liquid Gas- Liquid Chromatography (GLC) Liquid Gas Gas- Solid Chromatography(GSC) Solid Gas Partition Chromatography Liquid Liquid Ion Exchange Chromatography Solid Liquid

3. INTRODUCTION Thin layer chromatography (TLC) is one of the simplest, fastest , easiest and least expensive of several chromatographic techniques used in qualitative and quantitative analysis to separate organic compound and to test the purity of compounds. TLC is a form of liquid chromatography consisting of: - A mobile phase (developing solvent) - A stationary phase ( a plate or strip coated with a form of silica gel) - Analysis is performed on a flat surface under atmospheric pressure and room temperature.

4. PRINCIPLE OF TLC It is based on the prinicple of adsorption or partition chromatography or combination of both , depending on adsorbent. One or more compounds are spotted on a thin layer of adsorbent coated on a chromatographic plate. The mobile phase solvent flows through because of capillary action. The compound with greater affinity towards stationary phase moves with slower rate and compounds with lesser affinity moves fast. Identification of compounds is done by calculating the Rf value for each compound. Retention factor (Rf) = Distance travelled by component/ Distance travelled by solvent

5. EXPERIMENTAL TECHNIQUES . COATING MATERIAL PREPARATION OF THIN LAYERS IN PLATE ACTIVATION OF ADSORBENTS SAMPLE APPLICATION MOBILE PHASE SPOTTING DEVELOPING CHAMBER DEVELOPEMENT OF TLC PLATE DETECTING AGENTS

6. STATIONARY PHASE/COATING MATERIALS A large number of coating materials are used which are commercially produced as a thin adsorbents. Their composition and ratio in which they are mixed with water or other solvents to form slurry for preparing TLC plates. Adsorbents Acidic or Basic Components to be separated Silica gel Acidic Acidic and neutral substances Alumina Basic Basic and neutral Cellulose Powder Neutral Soluble compounds

7. PREPARATION OF THIN LAYER IN PLATES Pouring Technique - The adsorbent of finely divided and homogenous 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 - Used for small plates by dipping the two plates at a time, back to back in a slurry of adsorbent in chloroform. Evenness of layer may not be good. Spraying - The suspension of adsorbent or slurry is sprayed on a glass plate using a sprayer.

8. . Spreading - It is the best technique where a TLC spreader is used. The glass plates of a specific dimensions (20cm *20 cm / 10 cm/5cm) are taken. The prepared slurry is poured inside the reservoir of TLC spreader. The thickness is adjusted by using a knob in the spreader. Normal thickness of 0.25 cm is used for analytical purpose and 2mm for preparative purpose. Then the spreader is rolled only once on the plate. The plates are allowed to air drying. This is done to avoid cracks. The plates are activated by keeping in an oven.

9. . ACTIVATION OF ADSORBENTS Activation of plates is removing the water/moisture from the surface of adsorbent by heating . This is done by drying the thin layer plate in oven at 110 ℃ for 30 minutes. The activated plates can be stored in thermostatiscally controlled or in desiccator and can be used whenever required. .

10. MOBILE PHASE It is developing liquid which travels up the stationary phase carrying the samples with it. It depends on : Nature of the substance to be separated that is 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.

11. SPOTTING Spotting is done using a capillary tube or micropipette. The spots should be kept at least 2 cm above the base of plate and the spotting area should not be immersed in mobile phase in developing chamber. The sample is applied on the narrow band. The width of the band must be as narrow as possible.

12. DEVELOPING CHAMBER It is used for ther 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 aslo known as saturation of TLC chamber. Edge effect occurs when the solvent is in the middle of TLC plate moves faster than that of edge of plate.

13. DEVELOPEMENT OF TLC PLATE The plate is placed in the closed container saturated with developing solvent. Different developement techniques are used for efficient sprartions. They are: 1. One dimensional developement 2. Two dimensional developement 3. Horizontal developement 4. Multiple developement

14. DETECTING AGENTS After the developement of chromatograph, the spots should be visualised. Detecting coloured spots can be done visually,but for detecting 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. Iodine Chamber Method - where brown or amber spots are observed when the paper is kept with few iodine crystal at the bottom. 2. UV Chamber for fluorescent compound - when compounds are viewed under UV chamber at 245nm or at 365nm fluorescent compounds can be detected.

15. . Specific methods : Specific spray reagents or detecting agents visualising agents are used to find out the nature of compounds for identification purposes. Examples- 1. Ferric Chloride - For phenolic compounds and tannins 2. Ninhydrin in acetone - For amino acids 3. Dragondroff's Reagent - For alkaloids 4. 3,5- Dinitrobenzoic acid - For cardiac glycosides 5. 2,4- Dinitrophenyl hydrazine - For aldehdes and ketones

16. HOWTO RUN THIN LAYER CHROMATOGRAPHY Step 1: Prepare the developing chamber Step 2: Prepare the TLC plate Step 3: Spot the TLC plate Step 4: Develop the plate Step 5: Visualize the spots

17. STEP 1 : PREPARE THE DEVELOPING CONTAINER Take a beaker with watch glass on top. Required quantity of solvents are taken into the beaker. Coverthe beaker with watch glass and mix the solvents. Keep them aside until the plate is prepared.

18. STEP 2: PREPARE THE TLC PLATE Take the TLC plate and cut it into required length and width. Now mark a line about 1 cm from the bottom. On the line place two dots at equal space.

19. STEP 3 : SPOT THE TLC PLATE Take the capillary tube and by the help of heat make it into two, so that the end of the capillary tube will be thin. It helps to place a small amountof sample. Take the rquired solutions and spot them at the marked points.

20. STEP 4 : DEVELOP THE PLATE Put the TLC plate carefully into the beaker. The solution should not touch the marked line. Close the beaker with watch glass. Do not allow the solvent to run off the top of the plate.

21. STEP 5 : VISUALIZE THE SPOTS Take off the TLC plate from the beaker carefully. Mark the solvent front level. Let it dry. Spray solution. Observe the spot and round it with pencil carefully.

22. QUANTITATIVE ANALYSIS Direct Technique: Densitometer is an instrument which measues quantitavely the density of the spots. When the optical density of the spots for the standard and test solution are determined, the quantity of substance can be calculated. Indirect Technique: In this technique, the spots are cut into portions and eluted with solvents. This solution can be analyzed by any conventional techniques of analysis like spectrophotometry electrochemical methods etc.

23. QUALITATIVE ANALYSIS Rf Value The Rf value is calculated for identifying the spots i.e. in qualitative analysis. Rf value is theratio of distance travelled by the solute to the distance travelled by the solvent front. The Rf values ranges from 0 to 1. But ideal values are from 0.3 to 0.8 . Rf value is constant for every compound in a particular combination of stationary and mobile phase. When the Rf value of a sample and reference compound is same the compound is identified.

24. QUALITATIVE ANALYSIS Rx Value Rx value is nothing but the distance traveeled by the sample and the distance travelled by the standard. Rx value is always closer to 1.

25. ADVANTAGES OF TLC It is the simple process with short developement time. It helps in visualization ofseparated compound spots easily. This method helps to identify the individual compounds. It helps in isolation of most of the compounds. The separation process if faster and the selectively for compounds is higher even small differences in chemistry is enough for clear separation. It is a cheaper chromatographic technique.

26. APPLICATIONS OF TLC It is used for separation of all classes of natural prodcuts and is established as an analytical tool in modern pharmacopoeias. Eg-Acids, alcohols, glycols, alkaloids, amines, macromolecules like amino acids, proteins and peptides, and antibiotics. - for checking the purity of samples - as a purification process - for identifying organic compounds Extensively used as an identificaion test and test for purity. To detect the presence of foriegn substances in drugs.

27. CONCLUSION TLC is rapid, low cost. It is easy method for checkin sample purity. To detect the presence of foreign substances, decomposition matter in drugs. So it is widely used technique in laboratory for separation of compounds.

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