1. Thin Layer Chromatography

2. Thin layer chromatography The common adsorbent materials are silica, alumina, kieselguhr, celite, cellulose powder, ion exchange cellulose, starch and Sephadex. The thin layer of adsorbent is known as the stationery phase. A chromatography technique used to separate different analytes that ascend through a layer of adsorbent coated on a glass or plastic plates at different rates, as the mobile phase is drawn up the plate through capillary action.

3. Principle Different compounds travel at different rates due to the differences in their attraction to the stationary phase, and due to the differences in solubility in the solvent. Separation of compounds is based on the competition of the solute and the mobile phase for binding places on the stationary phase. Normal phase silica gel is polar. More polar compound has a stronger interaction with the silica and more capable to dispel the mobile phase from the binding places. Consequently, less polar compound moves higher up the plate with higher Rf value. If the mobile phase is changed to a more polar, it is more capable to dispel the solutes from the binding places and all compounds will move higher up the plate. It is common that "strong" solvents push the analyzed compounds up the plate, while "weak" solvents barely move them. Order of strength/weakness depends on the stationary phase.

4. Strength of solvents For silica gel plates: The strength of solvent increases in the following order: Perfluoroalkane, hexane, pentane, carbon tetrachloride, benzene/ toluene, dichloromethane, diethyl ether, ethylacetate, acetonitrile, acetone, 2-propanol/n-butanol, water, methanol, triethylamine, acetic acid, formic acid. For C-18 coated plates the order is reverse. If a mixture of ethyl acetate and heptane is used as the mobile phase, addition of ethyl acetate results in higher Rf values for all compounds. Changing the polarity of the mobile phase will normally not result in reversed order of running of the compounds on the TLC plate. An eluotropic series can be used as a guide in selecting a mobile phase.

5. Plate preparation A slurry of stationery phase in water is applied to a glass, plastic or foil plate as a uniform thin layer by means of plate ‘spreader’. For analytical separation, layer thickness is 0.25 mm and for preparatory separation it is up to 5 mm. A binding agent calcium sulphate is added to slurry to facilitate adhesion of absorbent to plate. Plates are dried in an oven at 100-120 o C to activate the adsorbent.

6. Sample application Sample is applied by means of micropipette or syringe. Spot is placed 2-2.5 cm from the edge of plate. Solvent can be removed by gentle heating or by use of a air blower. Sample is applied as band across the plate rather than as a single spot for preparatory TLC.

7. Plate development Separation takes place in a glass tank which contains the developing solvent to a depth of about 1.5 cm. Solvent is allowed to stand for at least 1 h with top cover plate to saturate the atmosphere within the tank with solvent vapour. Plate is then placed vertically in the tank. Separation occurs as the solvent travels on the plate. Speed of separation is 10-30 min commonly ; sometimes >90 min

8. Component detection Several methods are available for detection of components 1.Spraying with 50% H 2 SO 4 and 25% H 2 SO 4 in ethanol and heating to get brown spots 2.Examination of plate under UV light to show position of fluorescent compounds i.e blue, green or black areas. 3.Subjecting the plate to iodine vapour is useful to identify unsaturated compounds 4.Spraying with specific color reagents to identify certain compounds eg. ninhydrin for aminoacids 5.Subjecting to autoradiography to detect spots as dark areas on X ray film or plates can be scanned by radio chromatogram scanner, if compounds are radioactive.

9. Relative factor (Rf) value Movement of compounds on TLC is characterized by the relative factor (Rf value). Compounds are identified based on reference compounds separated along side the sample. Rf value is defined as the ratio of distance moved by solute to the distance moved by solvent front. Distance moved by solute Rf value = ------------------------------------------------- Distance moved by solvent front Rf value is always constant for a particular compound under standard conditions.

10. Quantification Off-plate quantification Scrap off the spot and elute the compound with suitable solvent. The amount of compound is determined by colorimetry. On-plate quantification 1.Radio chromatogram scanner for radio-labeled compounds 2.Densitometry for UV or visible absorption of compounds

11. TLC is also used on a small semi-preparative scale to separate mixtures of up to a few hundred milligrams. Sample mixture is not "spotted“ as spots, but applied as a thin horizontal band. When developed, the compounds separate in horizontal bands. Each band is scraped off with the backing material, extracted with a suitable solvent (e.g. dichloromethane) and filtered to get the compound upon removal of the solvent. Preparative TLC is far more efficient in terms of time and cost than column chromatography. It is best for compounds that are visible under UV light. Alternatively, a small section of the plate can be cut and chemically developed by exposing to a chemical developer like iodine. Preparative TLC

12. Two-dimensional TLC This is performed to improve resolution of a particular separation. Sample is placed towards one corner of plate as single spot and developed in one direction and allowed to dry. Then, it is again developed by another solvent system in the direction at right angles to first development.

13. High Performance TLC Whatman HPTLC plates is used for most sensitive separations. Plates consist of a 4.5 µm particle size silica gel plus an inert binder in a uniform 200 µm layer on glass. They exhibit product characteristics like narrow particle size distribution, homogeneity, and overall uniformity. The results are performance and reproducibility, giving the ultimate in TLC resolution and sensitivity. Short development distance and times Low band diffusion provides very compact sample bands and increased detection sensitivity Micro samples (nanograms and picograms) can be analyzed

14. Applications NoCompoundsAdsorbentSolvent system (v/v) 1.AminoacidsSilica gel G 90% ethanol/water (70:30) Butanol/acetic acid/water (80:20:20) 2. Mono and di- saccharides Kieselguhr G Ethylacetate/ propanol (65:35) Butanol/ acetone/ Phosphate buffer pH 5 (40:50:10) 3.Neutral lipidsSilica gel G Petroleum ether/ diethyl ether/ acetone (90:10:1) 4. Cholesterol esters Silica gel GCarbon tetra chloride/ chloroform (95:5) 5.CarotenoidsKieselghur GPetroleum ether / propanol (99:1) 6.PhospholipidsSilica gel GChloroform/ methanol/ water (65:25:4)