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5.1.7Unit 5: Respiration, Internal Environment, Coordination and Gene Technology

Separation of Photosynthetic Pigments with Chromatography — IAL Edexcel Linear Revision Notes & Topical Questions

5 min read 6 parts Lesson 14 of 36 Unit quiz

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Part 1 of 6Principle

5.1.7 Separation of Photosynthetic Pigments with Chromatography

Pigments are carried by a solvent through a stationary phase at different speeds.

Stationary phase
  • Chromatography paper or a thin layer of silica gel holds the pigments back by different amounts.
Mobile phase
  • A solvent mixture moves up the plate by capillary action, carrying the pigments with it.
Solubility
  • Pigments that dissolve well in the solvent and stick less to the plate travel further.
Result
  • A series of coloured spots separate in a vertical line above the starting point.

Part 2 of 6Method

Care in setting up gives clear separation.

1
Grind leaves with a little sand and propanone, then filter or decant to get a concentrated pigment extract.
2
Draw a pencil line near the bottom of the paper or plate, and place a small spot of extract on it.
3
Dry the spot and add more extract on top several times to build up a concentrated spot.
4
Stand the plate in a shallow solvent with the solvent below the pencil line, in a covered container.
5
Remove the plate before the solvent reaches the top and mark the solvent front at once.

Part 3 of 6Interpreting the Chromatogram

Interpreting the Chromatogram
Carotene
  • An orange-yellow pigment that travels furthest, with the highest Rf.
Xanthophyll
  • A yellow pigment that travels a middle distance.
Chlorophyll a
  • A blue-green pigment lower down than the xanthophylls.
Chlorophyll b
  • A yellow-green pigment that travels least of the four.

Part 4 of 6Safety and Accuracy

Pencil line
  • Pencil does not dissolve in the solvent, whereas ink would separate into its own colours.
Solvent
  • Propanone is flammable and harmful, so no flames and good ventilation are essential.
Spot size
  • A small, concentrated spot gives sharper, more clearly separated pigments.
Rf comparison
  • Compare Rf values to published values to identify each pigment.

Part 5 of 6Worked Example

Question

  • A pigment spot is 4.2 cm above the start line and the solvent front is 6.0 cm above the start line.
  • Calculate its Rf value.

  1. Rf = distance moved by the pigment / distance moved by the solvent.
  2. Rf = 4.2 / 6.0.
  3. Rf = 0.70.

Answer Rf = 0.70, with no units.

Part 6 of 6Exam Checklist & Tips

5.1.7 – Key Points for the Exam

  • Chromatography A technique that separates a mixture based on movement through a stationary phase.
  • Rf value Distance moved by a spot divided by distance moved by the solvent front.
  • Solvent front The furthest point reached by the solvent.
  • Stationary phase The paper or silica layer on which the separation takes place.

Common Mistakes

  • Drawing the start line in ink, which will itself separate.
  • Allowing the solvent level to cover the pencil line so the pigments wash into the solvent.
  • Measuring the Rf value from the bottom of the plate rather than from the start line.
  • Writing an Rf value larger than 1; it is always between 0 and 1.

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