Chromatography (paper chromatography and tlc)

test analytical

1. 1 Paper Chromatography AND Thin Layer Chromatography

2. 1. Paper Chromatography Paper chromatography is a technique that involves placing a small dot of sample solution onto a strip of chromatography paper. 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. The paper is placed in a jar containing a solvent such as ethanol or water then sealed. 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. 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. Different compounds in the sample mixture travel at different rates due to differences in solubility in the solvent differences in their attraction to the fibers in the paper. 6 1. Paper Chromatography

7. 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

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9. 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. 9 Paper Chromatography 1.2 Descending

10. 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. 10 Paper Chromatography 1.2 Descending

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12. After development, the spots corresponding to different compounds may be located by: their color , ultraviolet light , ninhydrin or by treatment with iodine vapors. The paper remaining after the experiment is known as the Chromatogram . 12 Paper Chromatography Analysis

13. R ƒ value ( Retention Factor) 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. 13

14. The final chromatogram can be compared with other known mixture chromatograms to identify sample mixes, using the Rf value in an experiment . 14 R ƒ value ( Retention Factor)

15. 2. Thin layer chromatography (TLC) TLC is a widely used. Stationary phase solid+ adsorbent Adsorbent like silica gel (polar), alumina  ( aluminium oxide ), or  cellulose . Traveling of solvent and elute via capillary action 15

16. Silica is a typical adsorbent. It has silanol (Si-OH) groups on its surface, which are slightly acidic, and can interact with polar functional groups of the analyte or eluent . In general, an eluent with a polarity comparable to that of the most polar analyte in the mixture is chosen. Thus, alcohols would be selected if the analytes contained hydroxyl groups, acetone or esters would be selected for analytes containing carbonyl groups, and hydrocarbons such as hexane, heptane and toluene for analytes that are predominantly non-polar. Mixtures of solvents are commonly used in the context of gradient elution. 16

17. Plate preparation TLC plates are made by mixing the adsorbent + small amount of inert binder calcium sulfate (gypsum) + water . This mixture is spread as a thick slurry on an unreactive carrier sheet, usually glass , thick aluminum foil, or plastic (support) 17 2. Thin layer chromatography (TLC)-Procedure

18. 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. 18 2. Thin layer chromatography (TLC)-Procedure

19. 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. 19 2. Thin layer chromatography (TLC)-Procedure

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21. 21 2. Thin layer chromatography (TLC)- Analysis The scan of TLC plate (silica gel G) with 10 essential oils developed with mobile phase toluene - ethyl acetate (93:7 v/v), next sprayed with vanillin in H 2 SO 4 and heated. From left to right oils from: bergamot, cedar, eucalyptus, syzygium , lavandula , mint, orange, pine, spruce. Identified components: B1 and L1 - linalol , B2 and L2 - linalyl acetate, E1 - cinneol , G1 - eugenol , G2 - carryophyllene . Doubtfully identified components - C1 - cedrol , M3 - menthol, P1 - limonene..

22. Some samples are colorless, it can be visualize by: Manganese - activated zinc silicate (fluorescent compound ( is added to the adsorbent Iodine vapors 22 2. Thin layer chromatography (TLC)- Analysis

23. 23 IODINE has been used as a colour reagent for paper chromatography of amino-acids and their derivatives 1,2 , imidazoles 3 , and other nitrogen-containing compounds 1,4 . Steroids are also revealed by this technique, which is sufficiently sensitive 5 . The method has not gained wide acceptance possibly only because it cannot be relied on to yield a colour diagnostic of a class of compounds—some nitrogen-containing compounds are not revealed—but its application is simple, and if unsuccessful, other colour reagents can be applied to the same chromatogram. In the most convenient procedure, the dried paper is exposed to iodine vapour (supplied by a few iodine crystals in a closed battery jar) for 10–30 sec.; or the paper may be sprayed with a solution of iodine in carbon tetrachloride 3 , or may be dipped into a saturated solution of iodine in petroleum ether ( b.p . 60°–80°) 5 . Compounds are revealed usually as brown spots , which fade rapidly.

24. 3. Specific color reagents are sprayed onto the plate ( ninhydrin ) 4. In the case of lipids, the chromatogram may be transferred to a PVDF ( Polyvinylidene fluoride) membrane and then subjected to further analysis like mass spectrometry 24 2. Thin layer chromatography (TLC)- Analysis

25. B. Calculate the Rf value These values depend on the solvent the type of TLC plate. 25 2. Thin layer chromatography (TLC)- Analysis

26. It is faster runs than paper, It has better separations, Wide choice between different adsorbents. It has better resolution and to allow for quantitation 26 2. Thin layer chromatography (TLC)- Advantages

27. identifying compounds present in a given substance determination of the pigments a plant contains detection of pesticides or insecticides in food analyzing the dye composition of fibers in forensics monitoring organic reactions Assaying the radiochemical purity of  radiopharmaceuticals identification of medicinal plants and their constituents  [ 27 2. Thin layer chromatography (TLC)- Applications

28. Hydroxylapatite chromatography Crystalline hydroxylapatite (Ca10(PO4)6(OH)2) is an adsorbent used to separate mixtures of proteins or nucleic acids. One of the most important applications of hydroxylapatite chromatography is the separation of single-stranded DNA from double-stranded DNA. Both forms of DNA bind at low phosphate buffer concentrations but as the buffer concentration is increased single-stranded DNA is selectively desorbed. As the buffer concentration is increased further, double-stranded DNA is released. This behaviour is exploited in the technique of Cot analysis (Section 5.3.4). The affinity of doublestranded DNA for hydroxylapatite is so high that it can be selectively removed from RNA and proteins in cell extracts by use of this type of chromatography. 28