A student carries out paper chromatography to identify the amino acids present in a protein hydrolysate. After running the chromatogram, the student calculates the Rf value for each spot. Explain how the student would carry out the chromatography experiment and how Rf values are used to identify the amino acids present.
Written & reviewed by James Millett — Biology (Imperial College London), PGCE Science (University of Cambridge).
Paper chromatography can be used to separate mixtures of compounds. Each compound travels a different distance up the paper depending on how strongly it is attracted to the stationary phase compared to the mobile phase. An Rf value can be calculated for each compound using: Rf = distance travelled by spot ÷ distance travelled by solvent.
Model answer (5 marks)
1. Draw a pencil baseline near the bottom of a sheet of chromatography paper.
2. Spot a small drop of the protein hydrolysate onto the baseline.
3. Place the paper in a solvent chamber so that the solvent level is below the baseline.
4. Allow the solvent (mobile phase) to rise up the paper; the amino acids will migrate at different rates.
5. Remove the paper before the solvent reaches the top, mark the solvent front, and note the positions of the separated spots.
6. Measure the distance from the baseline to each spot and to the solvent front.
7. Calculate each Rf value as (distance spot ÷ distance solvent front).
8. Compare the calculated Rf values with reference Rf values for standard amino acids to identify which amino acids are present.
2. Spot a small drop of the protein hydrolysate onto the baseline.
3. Place the paper in a solvent chamber so that the solvent level is below the baseline.
4. Allow the solvent (mobile phase) to rise up the paper; the amino acids will migrate at different rates.
5. Remove the paper before the solvent reaches the top, mark the solvent front, and note the positions of the separated spots.
6. Measure the distance from the baseline to each spot and to the solvent front.
7. Calculate each Rf value as (distance spot ÷ distance solvent front).
8. Compare the calculated Rf values with reference Rf values for standard amino acids to identify which amino acids are present.
Examiner tips
- Use a pencil for the baseline to avoid smudging; mark the solvent front clearly. Show the Rf calculation for at least one spot. Mention that reference Rf values are obtained from a standard run under identical conditions. Explain that identical Rf values indicate the same amino acid.
Common mistakes
- Drawing the baseline too high or too low, affecting Rf. Not keeping the solvent level below the baseline. Using a coloured marker for the baseline, which can bleed into the spots.
Mark scheme (5 marks)
- A baseline (start line) is drawn in pencil near the bottom of the chromatography paper and the protein hydrolysate sample is spotted onto it.
- The paper is placed in a suitable solvent so that the solvent level is below the baseline, and the solvent (mobile phase) travels up the paper.
- The chromatogram is removed before the solvent reaches the top, the solvent front is marked, and the positions of the separated amino acid spots are marked.
- The Rf value is calculated by dividing the distance travelled by the spot by the distance travelled by the solvent front.
- The Rf values calculated are compared to known / reference Rf values for amino acids to identify which amino acids are present.
Key terms in this question
Related
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