1. Paper Chromatography 1Dr. Nikhat Siddiqi

2. Paper chromatography and TLC are examples of adsorption chromatography. 2Dr. Nikhat Siddiqi

3. Paper chromatography Cellulose support is in the form of sheet of paper which has large amount of water bound to it. Partitioning occurs between the bound water and developing solvent. The solvent used is water. There is one effluent and substances are distinguished by their positions in the paper after the solvent has moved a given distance. Uses: To identify unknown samples Isolation of components of mixtures. 3Dr. Nikhat Siddiqi

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5. Paper chromatograms can be developed by either ascending or descending solvent flow. 5Dr. Nikhat Siddiqi

6. Ascending Paper Chromatography Dr. Nikhat Siddiqi6

7. Descending Paper Chromatography Dr. Nikhat Siddiqi7

8. Descending chromatography Faster For quantitative separations of substances with very small Rf values which require long runs the solvent can run off paper. 8Dr. Nikhat Siddiqi

9. Two Dimensional Paper Chromatography Dr. Nikhat Siddiqi9

10. Detection of spots in the paper By colour Fluorescence Chemical reaction after the paper is sprayed with various reagents Radioactivity. Identification is based on comparision with standards of known Rf or by elution. Elution is accomplished by cutting out the spot and soaking the paper in appropriate solvent. 10Dr. Nikhat Siddiqi

11. Thin Layer Chromatography (TLC) Originally used to separate lipids. Substances used include inorganic substances like silica gel, aluminium oxide, diatomacious earth, magnesium silicate organic substances like cellulose, polyamide, polyethylene powder. Used for separation of small molecules. 11Dr. Nikhat Siddiqi

12. Stationary phase is a layer (0.25-0.5 mm) of sorbent spread uniformly over the surface of a glass or plastic plate. A slurry of sorbent is made in a solvent for that particular sorbent. The plate is coated with the slurry and allowed to dry at 100-120 oC in an oven. 12Dr. Nikhat Siddiqi

13. The TLC plate is placed in a chamber containing the solvent and developed by ascending chromatography. After the solvent has reached the top the plate is removed from the chamber and dried. Spots are located as on paper by colour, fluorescence or by spraying. 13Dr. Nikhat Siddiqi

14. Thin Layer Chromatography (TLC) Dr. Nikhat Siddiqi14

15. Detection Commonly used sprays are Ninhydrin for amino acids Rhodamine B for lipids Antimony chloride for steroids and terpenoids Sulfuric acid plus heating for organic substances Potassium permanganate in sulfuric acid for hydrocarbons Anisaldehyde in sulfuric acid for carbohydrate etc. 15Dr. Nikhat Siddiqi

16. Elution By scraping off the sorbent and eluting the powder with a suitable solvent. 16Dr. Nikhat Siddiqi

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