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1.1.6Biological Molecules

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

5 min read 9 parts Lesson 4 of 49 1 quiz

📚 AQA A Level Biology 7402 Hub / Topical Revision
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1.1.6 Chromatography- Monosaccharides — AQA A Level Biology 7402 notes: Explain how paper or thin-layer chromatography separates monosaccharides and calculate and use Rf values to identify them.Chromatography separates the components of a mixture according to how strongly each is…

  • stationary phase
  • mobile phase
  • Exam-style question 1
  • Exam-style question 2
  • Exam-style question 3
  • Solvent front

Part 1 of 7Principle of chromatography

Chromatography separates the components of a mixture according to how strongly each is attracted to a stationary phase compared with a mobile phase.

1
In paper chromatography the stationary phase is water held in the cellulose fibres of the paper.
2
In thin-layer chromatography (TLC) it is a thin layer of silica or alumina on a plate.
3
The mobile phase is a solvent that moves up the paper or plate by capillary action.
4
Components that are more soluble in the solvent, or less strongly held by the stationary phase, travel further.

Monosaccharides such as glucose, fructose and galactose are colourless and similar in size, so they need a suitable solvent and a visualising agent.

Part 2 of 7Method

1
Draw a pencil baseline about 2 cm from the bottom of the paper (pencil does not dissolve in the solvent).
2
Apply small spots of each unknown sugar solution and known standard solutions of glucose, fructose and so on the line, drying between applications to keep the spot small and concentrated.
3
Stand the paper in a tank with solvent that is below the baseline, and cover with a lid so the atmosphere is saturated with solvent vapour.
4
Remove the paper when the solvent front is near the top and immediately mark the solvent front in pencil.
5
Dry the paper,
  • then spray with a locating agent and heat to reveal coloured spots.
    • (for example aniline hydrogen phthalate, or a reagent that reacts with reducing sugars)

The method is a safe, small-scale technique, but locating agents can be harmful, so gloves and a fume cupboard may be required.

Part 3 of 7Calculating Rf

The Rf value is a ratio:

Rf = distance moved by the spot from the baseline ÷ distance moved by the solvent front from the baseline

Measure to the centre of each spot. Rf has no units and is always between 0 and 1. A given substance has a characteristic Rf in a given solvent at a given temperature, so unknown spots are identified by matching Rf values with the standards or by running them alongside the standards on the same plate.

Part 4 of 7Interpreting results

1
Spots at the same height as a standard indicate the same substance.
2
A spot with a different Rf from every standard suggests a sugar not included in the standards.
3
If one spot is present in the unknown at the same height as glucose and another at the height of fructose, the unknown contains both; sucrose, if hydrolysed first, would give both.
4
Streaks or overlapping spots suggest overloading; use less solution or a different solvent.

For a mixture that does not separate, a two-dimensional chromatogram is possible: the plate is turned through 90° and run in a second solvent.

Part 5 of 7Sources of error

Spot applied below the solvent level, so it dissolves into the tank; solvent front allowed to run off the top, so Rf cannot be calculated; fingerprints on the paper; baseline drawn in ink; and spots too large.

Sources of error
Rf values vary with temperature and solvent composition, so comparisons must be made under identical conditions.
Chromatography is qualitative or semi-quantitative: spot size can suggest relative amount, but not accurate concentration.

Part 6 of 7Chromatography in a wider setting

Chromatography is one of the most widely used separation techniques in biology.

1
In plant biology it separates photosynthetic pigments such as chlorophyll a, chlorophyll b, xanthophyll and carotene,
  • and the same Rf calculation is used.
2
In biochemistry it separates amino acids and sugars, and in forensic work it detects drugs in a sample.
3
In biotechnology, column chromatography purifies proteins.
4
The reasoning is always that components with different solubilities in the mobile phase and different attractions to the stationary phase travel at different speeds.

If a question asks you to explain why two sugars have different Rf values, give a full argument: the sugar with the higher Rf is more soluble in the solvent and/or forms fewer or weaker bonds with the stationary phase, so it spends more of its time in the mobile phase and moves further. A less soluble or more strongly attracted sugar moves less.

To get the best separation, use a small, concentrated spot, avoid disturbing the tank while the solvent runs, and use a suitable solvent. If spots move too quickly, a less polar solvent can slow them; if they do not move, a more polar solvent may help. A quick trial before running a precious sample is good practice.

Part 7 of 7Exam-style questions

  1. A glucose spot travelled 4.2 cm and the solvent front 12.0 cm from the baseline. Calculate the Rf value. [2]2 marks

  2. An unknown sample produced spots with Rf values of 0.35 and 0.42. Standards under identical conditions gave glucose 0.35, fructose 0.42, galactose 0.31. Identify the sugars and explain why the same conditions are needed. [3]3 marks

  3. Explain why the baseline is drawn in pencil and the spots are kept small. [2]2 marks

Key terms

TermMeaning
ChromatographyTechnique for separating a mixture by differences in attraction to a stationary and mobile phase.
Stationary phaseThe paper or plate coating that does not move.
Mobile phaseThe solvent that moves through the stationary phase.
Solvent frontThe furthest point reached by the solvent.
Rf valueDistance moved by a substance divided by distance moved by the solvent front.
Locating agentA reagent used to make colourless spots visible.
BaselinePencil line on which samples are applied.

Avoid these mistakes

1
Measuring to the bottom or top edge of a spot rather than its centre, giving inconsistent Rf values.
2
Putting the baseline below the solvent level so the spots wash into the tank.
3
Quoting Rf greater than 1 or with units. It is a ratio.
4
Saying the sugar 'dissolves in the paper'. Separation depends on relative attraction to the two phases.

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