2 Thin Layer Chromatography Thin Layer Chromatography is a technique used for the separation of mixtures of chemical compounds on a thin layer of adsorbent material. It is one of the most widely used technique for the separation and final purification of organic compounds.
3 a) greater sharpness of separation b) short development time The TLC has distinct advantages over other chromatography because of itsa) greater sharpness of separationb) short development timec) high sensitivity in detection andd) convenience in use.In TLC, one side of a glass plate is uniformly coated with an adsorbent powder and the mixture applied to it is separated into its components by means of suitable solvents.
4 Thin Layer Chromatography Procedure Preparation of TLC plate1.Wash five TLC plates (20cm x 20cm) with soap and water.2. Place the plates in a TLC rack and dry them in an oven.3. Arrange the plates side by side on a TLC tray and clean the plates further with a peace of cotton soaked with acetone.
5 Make a slurry of TLC grade silica with water in a stoppered conical flask by vigorous shaking for three minutes. (Usually 40 g of silica is mixed with 80 ml of water to make 5 plates of thickness 0.5mm).Place the TLC spreader on the TLC plate and pour the slurry into the chamber.Draw the spreader over the plates. As a results the plates will be covered with a layer of slurry.After 5-10 minutes when the plates are dry,place the plates in the TLC rack.
6 Activation of TLC plate Activate the TLC plates by heating at 105o for 1th or at 60o for overnight.Sample applicationDissolve the sample to be analyzed or separated with a suitable solvent and then apply as a spot near the bottom of the TLC plate.10. For preparative TLC, where the purpose is to separate sufficient amount of components, apply the sample as a band.
7 Development of the chromatogram 11. Place the TLC plate in a shallow pool of a mobile phase in a developing chamber/TLC tank (previously saturated with the mobile phase) so that only the spotted end of the plate is in the liquid. The sample spot or band should be above the level of the mobile phase.12. Cover the tank with a lid.
8 13. The mobile phase slowly rises up the TLC plate by capillary action. 14. When the solvent has reached the top of the plate, remove the plate from the developing chamber and dry.
10 Visualization of Compounds 14 Visualization of Compounds 14. Obverve the plate if any colored spot or band present and mark with a needle. 15. Usually the compounds are not colored. So visualize the plates under UV lamp if any fluorescent or quenching compound present and mark. 16. Spray the plates with colour producing reagents to detect the compounds which are not visualized by the above procedure. (like some terpenoids which contain no chromophore in their structure). 17. In case of preparative TLC spray one side of the plate. When colour develops after heating with a hair drier, mark the whole band with a needle.
11 Recovery of Sample (for preparative TLC) 17. Scrap the bands separately from the preparative TLC plate.18. Elute each band with a suitable solvent.19. Check the purity on a precoated TLC plate.20. Evaporate the eluting solvent to obtain the pure compounds.
12 Detection of Compounds If the compounds are colored, visualization is straightforward. Usually the compounds are not colored. These compounds can be detected by various methods.
13 Ultraviolet light:The UV light at 254nm and 366 nm is usually used to detect the spot or band of fluorescent compounds.The plate itself contains a fluor which fluoresces everywhere except where a quenching compound is present on the plate. These compounds appear as black spot on the TLC plate. Compounds which do not contain any chromophore are not visualized under the UV light.
14 Spray Reagents:Most reagents which are used to produce colors with specific chemicals can be used in thin layer chromatography. The atomizers are used for spraying the reagents.Vanillin in sulfuric acid (1%) : It is most commonly used in the Laboratory to detect organic compounds.It is prepared by dissolving 1 g of vanillin in 100 ml of concentrated sulfuric acid. The TLC plate or the chromatogram is sprayed with 1% vanillin in sulfuric acid reagent and then heated for 2 minutes to develop colour.
15 2% Ninhydrin in ethanol: IodineThe plates are placed in an enclosed tank containing a few crystals of iodine. The compounds particularly those containing nitrogen absorbs the iodine and the spots appear as brown areas on a faint yellow background.2% Ninhydrin in ethanol:Amino acids produce mainly brown or purple color when the plate is sprayed with this reagent and heated to 120oC for 10 minutes.
17 Dragendorff’s Reagent: It is used to detect alkaloids. Modified Dragendorff’s reagent is prepared by mixing equal parts (v/v) of 1.7% bismuth subnitrate dissolved in 20% acetic acid in water and 40% aqueous solution of potassium iodide.Iron II Thiocyanate Reagent:It is used to detect peroxy compounds. The reagent is prepared by mixing 10 ml of 4% aqueous solution of ferrous sulphate with 15 ml of 3% ammonium thiocyanate in acetone. After spraying with this reagent the peroxides immediately develop a brown red colour.
18 Aniline phthalate reagent: It is prepared by dissolving 0.03 g of aniline and 1.6 g of phthalic acid in 100 ml of butanol (saturated with water). The reagent is used to detect sugars.2,4-dinitrophenyl hydrazine in ethanol:After spraying the plate with this reagent and heating to 120oC for 10 minutes aldehydes and ketones produce a variety of colors.
19 Precoated TLC Plate:TLC plates are usually commercially available, with standard particle size ranges to improve reproducibility.They are prepared by mixing the adsorbent, such as silica gel, with a small amount of inert binder like calcium sulfate (gypsum) and water.This mixture is spread as a thick slurry on an unreactive carrier sheet, usually glass, thick aluminum foil, or plastic.
20 Precoated TLC Plate:The resultant plate is dried and activated by heating in an oven for thirty minutes at 110 °C.The thickness of the adsorbent layer is typically around 0.1 – 0.25 mm for analytical purposes and around 0.5 – 2.0 mm for preparative TLC.In many cases, a fluorescent powder is mixed into the stationary phase to simplify the visualization later on (e.g. bright green when it is exposed to 254 nm UV light).
21 Identification of compounds by TLC The components, visible as separated spots, are identified by comparing the distances they have traveled with those of the known reference materials.Measure the distance of the start line to the solvent front (a). Then measure the distance of center of the spot to the start line (b).Divide the distance the solvent moved by the distance the individual spot moved. The resulting ratio is called Rf-value. The value should be between 0.0 (spot did not moved from starting line) and 1.0 (spot moved with solvent front) and is unitless.
22 The Rf (retardation factor) depends on the following parameters: solvent systemabsorbent (grain size, water content, thickness)amount of material spottedtemperatureDue to the fact that all those variables are difficult to keep constant, a reference compound is usually applied to the plate as well.
23 The Rf value for each dye is then worked out using the formula:
25 Application of Thin Layer Chromatography. Thin-layer chromatography (TLC) is a very commonly used technique in natural chemistry for -separating the components of an extract,identifying the isolated compounds anddetermining their purity.In synthetic chemistry, it is also used for following the progress of a chemical reaction.TLC can also be used in quantitative analysis since the diameter of a spot is depended on the concentration of solution.