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3.3.2Inquiry Process

Separating Photosynthetic Pigments- Skills — IB Biology HL Revision Notes & Topical Questions

5 min read 5 parts Lesson 117 of 160

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Part 1 of 5The Pigments

3.3.2 Separating Photosynthetic Pigments- Skills

A leaf contains two groups of pigments, chlorophylls and carotenoids, with different colours.

Chlorophyll a
  • A blue-green pigment, the main pigment of the reaction centres of the photosystems.
Chlorophyll b
  • A yellow-green pigment that passes absorbed light energy to chlorophyll a.
Carotene
  • An orange carotenoid that absorbs blue-violet light and is dissolved easily by organic solvents.
Xanthophyll
  • A yellow carotenoid that absorbs mostly in the blue-violet region and helps protect chlorophyll from damage.
Extending the range
  • Carotenoids absorb some different wavelengths, and so widen the range of light that can be used.

Part 2 of 5Method

1
Draw a pencil line about 1 cm above the base of the chromatography paper; pen ink would itself separate into coloured components.
2
Grind a healthy leaf with a little propanone in a pestle and mortar, which dissolves the membranes and releases the pigments.
3
Spot the extract repeatedly on the middle of the pencil line using a capillary tube, drying between each application.
4
Stand the paper in solvent so that the solvent level is below the pencil line and leave it until the solvent nears the top.
5
Mark the solvent front at once in pencil, dry the paper and mark the centre of each pigment spot.

Part 3 of 5Analysing Results

Analysing Results
Rf value
  • Rf = distance moved by the pigment divided by distance moved by the solvent front, measured from the pencil line.
Meaning
  • A pigment that dissolves well in the mobile phase and sticks little to the stationary phase travels far and has a high Rf.
Order
  • The usual order from the top is carotene, xanthophyll, chlorophyll a, then chlorophyll b.
Better results
  • Thin-layer chromatography with silica gel separates pigments more clearly than paper.
Safety
  • Propanone is flammable and volatile, so use it away from flames and in a ventilated place.
PigmentColourDistance moved (cm)Rf (solvent front 8.0 cm)
CaroteneOrange7.20.90
XanthophyllYellow5.60.70
Chlorophyll aBlue-green3.60.45
Chlorophyll bYellow-green2.80.35

Part 4 of 5Worked Example

Question

  • In a chromatogram the solvent front moved 8.0 cm above the pencil line.
  • The centre of the chlorophyll a spot was 3.6 cm above the line.
  • Calculate its Rf value.

  1. Rf = distance moved by pigment / distance moved by solvent front.
  2. Rf = 3.6 / 8.0.
  3. 3.6 / 8.0 = 0.45.

Answer The Rf value of chlorophyll a is 0.45 (no units).

Part 5 of 5Exam Checklist & Tips

3.3.2 – Key Points for the Exam

  • Chromatography A technique that separates the components of a mixture.
  • Rf value The ratio of the distance moved by a substance to the distance moved by the solvent front.
  • Mobile phase The solvent that carries the mixture.
  • Stationary phase The paper or silica layer on which the components are separated.

Common Mistakes

  • Using a pen to draw the starting line, so that the ink separates and confuses the results.
  • Letting the solvent level cover the pencil line, which washes the pigments into the solvent.
  • Measuring distances to the edge of the spot rather than to its centre.
  • Writing Rf as a value greater than 1; it is a ratio that is always less than 1.

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