BIOL 1406
PreLab 7.2
How can I separate pigments using thin layer chromatography (TLC)?
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In the first part of this lab, you will be using thin layer chromatography to
separate a mixture of pigments extracted from spinach leaf cells. Before
applying the mixture to the silica gel, your chromatography plate should be thoroughly
dried in a desiccator. If this isn’t done, the silica gel may contain
a significant amount of water, which can interfere with the migration of the
pigments through the gel. Similarly, never touch the silica gel with your bare
hands because oils from your skin can affect the
migration of the pigments. The mixture of pigments should be applied to the silica gel in a very thin line, so that the pigments separate into distinct bands that do not overlap as they migrate up the plate. The thinner the line, the better the separation will be. At the same time, it is important to apply enough sample so that as the pigment bands separate from each other they don’t become too faint. Unfortunately, if a large amount of sample is applied all at once, it will spread over a large part of the plate, producing a thick line. Therefore, in order to get a thin, yet heavy, sample line at the origin, the sample should be applied in multiple, small applications, allowing the sample to dry thoroughly between each application. After the pigment sample has been applied to the silica gel, the chromatography plate will be placed in a jar with developing solution. The level of the developing solution should be a couple of mm below the lowest part of the pigment line. If the pigment becomes immersed in the developing solution, it will dissolve in the developing solution at the bottom of the jar, rather than be carried up through the silica gel. Once the chromatography plate is resting at the bottom of the jar, the cover should be tightly closed. It is important to keep your chromatography jar tightly sealed, for two reasons. First, the organic solvents used in the mobile phase are highly volatile and will rapidly evaporate when the chamber is open. The exact ratio of solvents in this mixture is very important to the successful separation of pigments, and if one solvent evaporates faster than the others, the ratio will change. Second, volatile organic solvents tend to be unhealthy, and breathing these vapors should be avoided. For this reason, containers with developing solution should ALWAYS remain inside the fume hood.
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Separation of spinach pigments using TLC |
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As the pigments are carried up through the silica gel by the developing solution, they will also be diffusing into a wider and wider band over time. To minimize this spreading of the pigment bands, the chromatography should be terminated as soon as the solvent front approaches the end of the chromatography plate. When removing the plate from the chamber, however, be aware that the solvents will dry rapidly, so it is important to mark the solvent front as soon as you open the jar. Once the solvents have dried, the solvent front will be invisible. |
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Your Turn | |
Based on your knowledge of potential problems during TLC, trouble shoot the following results and explain what might have gone wrong in each case: | |
Final chromatogram has fewer bands than
expected and the bands appear very faint. Why did this happen?
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Check your answer. |
Final chromatogram has bands that are dark
enough to see, but the bands are very broad and overlap. What are some
reasons that this could have happened?
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Check your answer. |
There are no bands in the chromatogram,
but the solvent at the bottom of the chromatography chamber has taken on a
very bright color. What went wrong here?
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Check your answer. |
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