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
Section 1 of 6Principle
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.
Section 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.
Section 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.
Section 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.
Section 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.
- Rf = distance moved by the pigment / distance moved by the solvent.
- Rf = 4.2 / 6.0.
- Rf = 0.70.
Answer Rf = 0.70, with no units.
Section 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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