Chromatography
Chromatography is used to separate substances and show the various different substances that have been separated. Chromatography techniques are used in industry because they can be easily controlled very precisely and they only use very small amounts of a substance. Thin-layer chromatography is used for determining if two compounds are identical i.e. a sample of pure aspirin compared to the aspirin I made. A spot of the compound being investigated (my aspirin) is placed on a chromatography plate, and a spot of a pure manufactured sample (pure aspirin) of the same substance is placed next to it. The plate is then allowed to stand in the developing solvent (Ethyl Ethanoate), which travels up the plate. If the compound to be identified leaves exactly the same pattern on the chromatography plate as the known pure compound it is reasonable to conclude that they are the same. However, if extra spots are observed as well as the characteristic pattern of the known compound, then impurities are likely to be present in the sample.
Method
Do not touch the surface of the TLC plate with your fingers, Handle the plate only by the edges and use tweezers.
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Take a TLC plate and using a pencil (not a biro or felt tip pen) lightly draw a line across the plate about 1 cm from the bottom. Mark three equally spaced points on this line.
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Place about 1/3 of a spatula of your aspirin samples, and your pure sample of aspirin in separate test tubes. Label the test-tubes.
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Make up 5 cm3 of solvent by mixing equal volumes of ethanol and dichloromethane in a test-tube. Add 1 cm3 of the solvent to each of the test-tubes to dissolve the samples
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Use capillary tubes to spot each of your three samples onto the TLC plate. Allow the spots to dry and then repeat this three more times. The spots should be about 1–2 mm in diameter.
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After all the spots are dry, place the TLC plate in the developing tank making sure that the original pencil line is above the level of the developing solvent – ethyl Ethanoate. Put a lid on the tank and allow it to stand in a fume cupboard until the solvent front has risen to within a few millimetres of the top of the plate.
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Remove the plate from the tank and quickly mark the position of the solvent front.
Allow the plate to dry
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Observe the plate under a short wavelength UV lamp and lightly mark with a pencil any spots observed.
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Carefully place the plate in a jar or beaker containing a few iodine crystals. Put a cover on the jar and warm gently on a steam bath until spots begin to appear. Do this in a fume cupboard if possible.
Rf values from thin layer chromatography for the aspirin I made against an aspirin tablet. Rf A is the aspirin tablet Rf 1-5 is the samples of aspirin I made.
Rf values = distance travelled by substance/ distance travelled by solvent
Rf A = 5.8/7.3 cm’s = 0.794
Rf 1 = 6/7.3 cm’s = 0.822
Rf A = 6.4/7.3 cm’s = 0.877
Rf 2 = 6.5/7.3 cm’s = 0.890
Rf 3 = 6.6/7.3 cm’s = 0.904
Rf A = 7/8.1 cm’s = 0.864
Rf 4 =7.1/8.1 cm’s = 0.877
Rf 5 = 7.1/8.1 cm’s = 0.877
