Mr Cole Chemistry

Thin Layer Chromatography Questions

Thin Layer Chromatography

Organic Analysis Worksheet

Amino Acid Analysis

1. A sample containing serine, alanine and one other amino acid underwent thin layer chromatography. The following chromatogram was obtained.

TLC of amino acids
a) The stationary phase was made of beads of aluminium oxide. Suggest a suitable mobile phase for the experiment and explain your choice.
Answer

Any solvent that is sufficiently low polarity will work. Examples include cyclohexane, ether, or ethyl ethanoate.

b) Explain why alanine has a higher Rf value than serine.
Answer

Alanine is non-polar whereas serine is polar. Serine has a greater relative affinity for the stationary phase (which is also polar) than alanine does; therefore, serine moves slower than alanine.

c) Suggest a possible identity for the unknown amino acid and explain your choice.
Answer

Serine is polar, alanine is non-polar. An amino acid that is somewhere between the two in terms of polarity will be between them in Rf value.

Glycine and threonine are both reasonable choices:

  • Glycine is non-polar but smaller than alanine, so it is more soluble.
  • Threonine is polar but has an extra methyl group, decreasing its solubility compared to serine.
d) At the end of the process, the spots need to be treated before being observed. Explain why this is the case, and state how they are treated.
Answer

The amino acids are colourless. They need to be treated with a developing agent to be visible. Chemicals such as ninhydrin or iodine, or UV light can be used.

2D Chromatography

2. Two dimensional thin layer chromatography was run on a sample, resulting in the following chromatogram.

2D TLC result
a) Describe how two dimensional TLC is conducted.
Answer
  1. Run TLC in one direction by placing the sample on a starting line (drawn in pencil) and placing the TLC plate in the solvent (solvent level below the starting line).
  2. Wait for the solvent to reach about 1cm before the top of the page and remove the plate from the solvent.
  3. Dry the TLC plate in the fume cupboard.
  4. Then, turn the TLC plate 90 degrees, draw a new starting line and repeat the process using a different solvent for the second run.
b) Explain why two dimensional chromatography was necessary to separate the compounds in the sample.
Answer

Some compounds have the same affinity for the mobile phase as each other. This results in the compounds moving the same distance and not being separated out. A different solvent can be used to separate out any compounds that did not separate in the first solvent.

Comparative Analysis

3. Ethyl ethanoate and butanoic acid are analysed using mass spectrometry and thin layer chromatography.

a) Explain whether the two compounds will be separated by mass spectrometry.
Answer

They will not be separated/distinguished easily by low-resolution mass spectrometry. This is because they have the same molecular formula (isomers) and therefore the same mass/charge ratio for the molecular ion peak.

b) Explain whether the two compounds will be separated by thin layer chromatography.
Answer

They will be separated by TLC because they have different relative affinities for the mobile and stationary phase due to having different structures. Butanoic acid is more polar so will have a greater affinity to the more polar phase.

c) While the chromatogram runs, there must be a lid on it. Explain why this is the case.
Answer

TLC solvents tend to be non-polar molecules with very low boiling points, making them very volatile. A lid prevents the solvent vapours from escaping, which saturates the atmosphere inside the container. This prevents excessive evaporation of the mobile phase from the plate surface.

d) When handling the TLC plate, you must wear gloves. Explain why this is the case.
Answer

There are molecules (amino acids/oils) on your hands which can contaminate the plate and affect the results.

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