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1.3.2Biological Molecules

Chromatography- Amino Acids — AQA A Level Biology 7402 Revision Notes & Topical Questions

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📚 AQA A Level Biology 7402 Hub / Topical Revision
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1.3.2 Chromatography- Amino Acids — AQA A Level Biology 7402 notes: Explain how chromatography separates amino acids, locate them with ninhydrin, and use Rf values to identify the amino acids in a mixture.Proteins are hydrolysed into free amino acids by boiling with acid.To find …

  • Exam-style question 1
  • Exam-style question 2
  • Exam-style question 3
  • Hydrolysate
  • Solvent front
  • Two-dimensional chromatography

Section 1 of 9Why chromatography for amino acids

Proteins are hydrolysed into free amino acids by boiling with acid.

Chromatography
To find out which amino acids a protein contains, the mixture is separated by chromatography.
Amino acids are colourless, so they need to be made visible.
They differ in their R groups (polar, non-polar, charged),
  • so they dissolve to different extents in the solvent and are held to different extents by the paper, and travel different distances.

Section 2 of 9Method

Draw a pencil baseline 2 cm from the bottom edge of chromatography paper or a TLC plate. Handle by the edges, since fingerprints contain amino acids.
Use a capillary tube to apply small spots of the hydrolysed protein sample and known standard amino acids along the baseline, drying each spot before adding more.
Place in a tank with a solvent such as a butanol-ethanoic acid-water mixture, below the baseline, and put on a lid to saturate the air with vapour.
When the solvent front is near the top, remove the paper and mark the solvent front at once in pencil, then dry in a fume cupboard.
Ninhydrin
Spray with ninhydrin solution and warm gently in an oven.
  • Amino acids appear as purple or brown-pink spots.
  • Mark the centre of each spot in pencil as the colours fade.

Ninhydrin is harmful and is applied in a fume cupboard with gloves.

Section 3 of 9Calculating Rf values

Rf = distance travelled by the spot ÷ distance travelled by the solvent front, both measured from the baseline to the centre of the spot and to the solvent front.

Calculating Rf values
Rf is a ratio so it has no units, and is less than 1.
  • The same amino acid has the same Rf in identical conditions of solvent, paper and temperature,
  • so the unknown can be identified by comparing Rf values with standards.
Running standards on the same sheet is more reliable than relying on published values.
Amino acid (example)Distance (cm)Solvent front (cm)Rf
A3.010.00.30
B6.510.00.65

Section 4 of 9Interpreting results

1
Spots that match a standard in position (Rf) and colour indicate the same amino acid.
2
Polar amino acids with charged R groups are strongly attracted to the aqueous stationary phase and move less; non-polar amino acids dissolve better in the organic solvent and travel further.
3
If a spot stays on the baseline, the amino acid is very polar or the solvent is unsuitable.
4
If two amino acids have very similar Rf values, a second solvent in a second direction (two-dimensional chromatography) can separate them.

Chromatography can also be used to show that protein digestion has released amino acids, or that an organism makes a particular amino acid.

Section 5 of 9Limitations

Overloaded spots tail and overlap; amino acids with similar R groups may not separate; ninhydrin does not distinguish all of them by colour; the paper must not be touched; and only relatively small amounts can be detected and compared. Measurements to the centre of a spot are subjective, which is a source of uncertainty of perhaps ±1 mm.

Section 6 of 9Further context and exam approach

Chromatography of amino acids is often asked as a data question, so make sure you can handle the numbers.

Further context and exam approach
Measure from the baseline to the centre of the spot and from the baseline to the solvent front.
Show your working, including the formula, and give Rf to two decimal places.
Compare unknown spots with standards both by Rf and by colour,
  • since some amino acids,
  • such as proline, give a yellow rather than a purple colour with ninhydrin,
  • which helps to identify them.

Free amino acids come from the complete hydrolysis of a protein, for example by heating with 6 mol dm-3 hydrochloric acid for 24 hours.

Further context and exam approach
The number and variety of spots indicate how many different amino acids the protein contains, but not their order.
The primary structure, the sequence, requires other techniques such as mass spectrometry or DNA sequencing of the gene.

In evaluating the method, consider that the technique is qualitative and cannot easily show the proportion of each amino acid.

Further context and exam approach
Amino acids with similar R groups, such as leucine and isoleucine, have nearly identical Rf values and are hard to tell apart,
  • which is why two-dimensional chromatography with a second solvent gives better resolution.
Using the same paper type and the same volume of solvent for each run makes comparisons across experiments more reliable.

Section 7 of 9Exam-style questions

  1. Describe how you would identify the amino acids in a protein hydrolysate using chromatography. [5]5 marks

  2. A spot travelled 4.8 cm when the solvent front travelled 12.0 cm. Calculate its Rf value. A standard serine travelled 4.9 cm in the same run; could the spot be serine? [3]3 marks

  3. Explain why the baseline is drawn in pencil and the paper handled by the edges. [2]2 marks

Section 8 of 9Key terms

TermMeaning
Amino acidThe monomer of proteins, with an amino group, carboxyl group and R group.
NinhydrinA reagent that reacts with amino acids to give purple or brown colours.
Rf valueDistance moved by a spot divided by distance moved by solvent front.
HydrolysateThe mixture of smaller molecules produced when a polymer is hydrolysed.
Solvent frontThe furthest point reached by the solvent.
R groupThe variable side chain of an amino acid.
Two-dimensional chromatographyRunning the plate in a second solvent turned through 90 degrees.

Section 9 of 9Exam technique

1
Using ink for the baseline so the ink itself separates.
2
Leaving the paper in the tank until the solvent front runs off the top.
3
Measuring Rf from the edge of the paper instead of from the baseline.
4
Forgetting that ninhydrin is needed because amino acids are colourless.

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