1. Chromatography Chapter 4-2 1 Dr Gihan Gawish

2. 1. Paper Chromatography Dr Gihan Gawish  Paper chromatography is a technique that involves placing a small dot of sample solution onto a strip of chromatography paper. The compounds within the mixture travel are non- polar.  Adsorbent: More polar substances bond with the cellulose paper more quickly. matrix = cellulose paper (polar substance) 2

3. Dr Gihan Gawish 3 1. Paper Chromatography

4. Dr Gihan Gawish 1. The paper is placed in a jar containing a solvent such as ethanol or water then sealed. 2. A small concentrated spot of solution that contains the sample of the solute is applied to a strip of chromatography paper about 2 cm away from the base of the plate 4 1. Paper Chromatography-Procedure

5. Dr Gihan Gawish 4. As the solvent rises through the paper, it meets the sample mixture which starts to travel up the paper with the solvent. 5. Paper chromatography takes from several minutes to several hours. 5 1. Paper Chromatography-Procedure

6. Dr Gihan Gawish  Different compounds in the sample mixture travel at different rates due to 1. differences in solubility in the solvent 2. differences in their attraction to the fibers in the paper. 6 1. Paper Chromatography

7. Dr Gihan Gawish  In this method, the solvent moves upward against gravitational force.  The only force that cause the motion is capillary force. So the speed of the process is slow. 7 Paper Chromatography 1.1 Ascending

8. Dr Gihan Gawish  In this method, the solvent is kept in a trough at the top of the chamber and is allowed to flow down the paper.  The liquid moves down by capillary action as well as by the gravitational force.  In this case, the flow is more rapid as compared to the ascending method. 8 Paper Chromatography 1.2 Descending

9. Dr Gihan Gawish  Because of this rapid speed, the chromatography is completed in a comparatively shorter time.  The developing solvent is placed in a trough at the top which is usually made up of an inert material.  The paper is then suspended in the solvent. Substances that cannot be separated by ascending method, can be separated by the above descending method. 9 Paper Chromatography 1.2 Descending

10. Dr Gihan Gawish  After development, the spots corresponding to different compounds may be located by: 1. their color, 2. ultraviolet light, 3. ninhydrin 4. or by treatment with iodine vapors.  The paper remaining after the experiment is known as the Chromatogram. 10 Paper Chromatography Analysis

11. R ƒ value ( Retention Factor) Dr Gihan Gawish  R ƒ =  If R ƒ value of a solution is zero, the solute remains in the stationary phase and thus it is immobile.  If R ƒ value = 1 then the solute has no affinity for the stationary phase and travels with the solvent front. the ratio of the distance traveled by the substance the distance traveled by the solvent. 11

12. Dr Gihan Gawish  The final chromatogram can be compared with other known mixture chromatograms to identify sample mixes, using the Rf value in an experiment.Rf 12 R ƒ value ( Retention Factor)

13. 2. Thin layer chromatography (TLC) Dr Gihan Gawish  TLC is a widely used.  Stationary phase solid+ adsorbent  Adsorbent like silica gel (polar), alumina.  Traveling of solvent and elute via capillary action 13

14. Dr Gihan Gawish Plate preparation 1. TLC plates are made by mixing the adsorbent + small amount of inert binder calcium sulfate (gypsum) + water. 2. This mixture is spread as a thick slurry on an unreactive carrier sheet, usually glass, thick aluminum foil, or plastic (support) 14 2. Thin layer chromatography (TLC)-Procedure

15. Dr Gihan Gawish 3. The resultant plate is dried and activated by heating in an oven for thirty minutes at 110 °C. 4. A small spot of sample is applied to a plate, about 1 cm from the base. 5. The plate is then dipped in to a suitable solvent, such as hexane or ethyl acetate, and placed in a sealed container.hexaneethyl acetate 15 2. Thin layer chromatography (TLC)-Procedure

16. Dr Gihan Gawish 6. The solvent (mobile phase) moves up the plate by capillary action and meets the sample mixture, which is dissolved and is carried up the plate by the solvent.solvent capillary action 16 2. Thin layer chromatography (TLC)-Procedure

17. Dr Gihan Gawish 17 2. Thin layer chromatography (TLC)- Analysis

18. Dr Gihan Gawish A. Some samples are colorless, it can be visualize by: 1. Manganese-activated zinc silicate (fluorescent compound (is added to the adsorbent Manganesezinc silicate 2. Iodine vapors Iodine 18 2. Thin layer chromatography (TLC)- Analysis

19. Dr Gihan Gawish 3. Specific color reagents are sprayed onto the plate (ninhydrin) 4. In the case of lipids, the chromatogram may be transferred to a PVDF membrane and then subjected to further analysis like mass spectrometryPVDFmass spectrometry 19 2. Thin layer chromatography (TLC)- Analysis

20. Dr Gihan Gawish B. Calculate the Rf value  These values depend on 1. the solvent 2. the type of TLC plate. 20 2. Thin layer chromatography (TLC)- Analysis

21. Dr Gihan Gawish  It is faster runs than paper,  It has better separations,  Wide choice between different adsorbents.  It has better resolution and to allow for quantitation 21 2. Thin layer chromatography (TLC)- Advantages

22. Dr Gihan Gawish 1. identifying compounds present in a given substance 2. determination of the pigments a plant contains 3. detection of pesticides or insecticides in food 4. analyzing the dye composition of fibers in forensics forensics 5. monitoring organic reactions 22 2. Thin layer chromatography (TLC)- Applications