Thin Layer Chromatography and Column Chromatography

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Chromatography is a technique used to separate a mixture into its individual components. In this experiment, chromatography is done by using a thin layer chromatography (TLC) plate and filter paper. The word ‘chromatography’ itself means the separation of colours. However, nowadays, both coloured and colourless compound are used in chromatography by various methods. In chromatography, the separation process of mixture is mainly based upon the polarity and molecular weight of sample. Then, the separation involves mobile phase and stationary phase. The mixture to be separated is applied to an immovable solid surface, which is the stationary phase, such as TLC plate and the filter paper. Then, suitable solvent is then added to ‘wash’ the porous solid by the flow of solvent, which is the mobile phase. The mobile phase can be liquid (in liquid chromatography) which is in this experiment, or gas (as in gas chromatography). The porous solid, which is the stationary phase, adsorbs different substances depending on the polarity of the substance adsorbed to it. The term “adsorption” means the adhesion or stickyness of a substance to the surface of another substance. As mentioned, chromatography is based on polarity. So, a mixture of compound is separated or adsorbed differently on porous solid (stationary phase) based on the polarity of both the compound itself and also the stationary phase. By the concept of “like attracts like”, a polar stationary phase will adsorb a polar components more strongly than a non-polar component, which also indicates that a non-polar components will be removed along the solvent more easily and faster than the polar compounds. It is the same case as where a polar solvent is used. The polar component will be moved along the solvent more quickly and hence, leave behind the non-polar component which is move more slowly and removed later. The solvent (mobile phase) moves through the stationary phase (porous solid) by capillary action. The solvent ‘attracts’ or pulls the molecules of mixture, which is then separated at different rates. Molecular weight also affects the separation of mixture into its components. Components having higher molecular weight will be left behind. In contrast, component of lowest molecular weight will be the fastest to be removed.

Part 1: TLC analysis of analgesic drugs
1. Prepare a TLC plate with measurement shown in figure 1.

Diagram 1
2. A developing chamber was developed in a 250 mL beaker, with a half-piece of filter paper inside, and aluminium foil to cover. The eluent is a 15 mL mixture of 2:1 mixture of ethyl acetate: hexane. 3. The eluent must not exceed depth of 1 cm in the beaker. 5 drops of acetic acid was added into the beaker and mix. The TLC plate was then placed in the beaker and ensured the solvent level is below the pencil mark. 4. After the solvent was risen to near the top of the plate, the plate was removed and the solvent front was marked with a pencil. The solvent was allowed to evaporate in the fume hood. 5. By using 5 different capillary tubes for 5 different samples, the sample was spot on the origin line. The sequence from the left (first spot) is acetaminophen solution, caffeine, unknown A, aspirin and unknown B. 6. The TLC plate was then placed in the iodine chamber for few minutes. 7. The plate was then observed under UV lamp.

8. The spot was outlined with a 2B pencil.
9. The plate was then sketched and the retention factor, Rf value is calculated. 10. The unknown drug based on the Rf value is determined. Apparatus
UV lamp, capillary tube, 250 mL beaker
Aspirin, acetaminophen, caffeine, unknown A, unknown B, TLC plates, ethyl acetate, hexane, iodine Part 2: The separation of Pyrene and p-Nitroaniline by Column Chromatography
Part A:
1. A large amount of solvent was prepared, with volume of about 600 cm3. The ratio of solvent mixture is ethyl acetate:...
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