A protein is broken down into a mixture of amino acids, which is then separated by paper chromatography. Because the amino acids are colourless, a locating ...
Question 1 Report
A protein is broken down into a mixture of amino acids, which is then separated by paper chromatography. Because the amino acids are colourless, a locating agent is used. The Rf values of four known amino acids in the same solvent are shown in the table.
| amino acid | Rf value |
|---|---|
| glycine | 0.26 |
| alanine | 0.42 |
| valine | 0.65 |
| leucine | 0.73 |
On the student's chromatogram the solvent moved 10.0 cm from the baseline. Spot 1 moved 4.2 cm and spot 2 moved 6.5 cm.
(a) State why a locating agent, such as ninhydrin, is needed in this experiment. [1]
(b) Calculate the Rf value of spot 1 and of spot 2. Show your working. [4]
(c) Use the table to identify the two amino acids present in the mixture. [2]
(d) Explain why Rf values, rather than the distance a spot has moved, are used to identify a substance. [2]
(e) Suggest why two amino acids with very similar Rf values might not be distinguished by this method. [1]
(f) State one condition that must be kept the same if Rf values are to be compared. [1]
Answer Details
This question applies Rf values to identify colourless amino acids separated by chromatography.
(a) A locating agent such as ninhydrin is needed because the amino acids are colourless; the agent reacts with them to give coloured spots that can be seen and measured [1].
(b) Rf is the distance moved by the spot divided by the distance moved by the solvent (10.0 cm) [4]:
\[ R_f(\text{spot 1}) = \frac{4.2}{10.0} = 0.42 \] \[ R_f(\text{spot 2}) = \frac{6.5}{10.0} = 0.65 \]One mark for each correct working and one for each correct value.
(c) Comparing with the table: spot 1 (Rf 0.42) is alanine [1]; spot 2 (Rf 0.65) is valine [1].
| Amino acid | Rf | Match |
|---|---|---|
| glycine | 0.26 | |
| alanine | 0.42 | spot 1 |
| valine | 0.65 | spot 2 |
| leucine | 0.73 |
(d) Rf values are used rather than raw distances because the distance a spot moves depends on how far the solvent runs, which varies from one experiment to the next [1]; the Rf value is a ratio that stays constant for a given substance and solvent, so it can be compared with reference values [1].
(e) Two amino acids with very similar Rf values might not be distinguished because their spots would overlap / appear at almost the same height, so they cannot be told apart [1].
(f) One condition that must be kept the same: the same solvent (or the same temperature, or the same type of paper) [1].
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