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NotesBiologyTopic 3.3Separation and identification of photosynthetic pigments by chromatography
Back to Biology Topics
3.3.43 min read

Separation and identification of photosynthetic pigments by chromatography

IB Biology · Unit 3

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Contents

  1. 1Chromatography and Rf values
  2. 2Exam-style question (step by step)
The big idea: Leaves contain several pigments. Chromatography separates them: each pigment travels up the paper or plate at its own speed. Pigments are identified by colour and by their Rf value.
A chromatography strip drawn step by step: a spot of leaf extract on a pencil origin; solvent rising to the solvent front; coloured bands of carotene, chlorophyll a and chlorophyll b; then the distances a and b and Rf equals a divided by b
Chromatography, step by step.

The points to remember

  • Spot the extract on a pencil origin line, above the solvent level.
  • The solvent front is the furthest point the solvent reaches.
  • Rf = distance moved by pigment ÷ distance moved by solvent (0 to 1, no units).
  • Typical run: carotene (yellow-orange) highest; chlorophyll a (blue-green); chlorophyll b (yellow-green) lower.
  • Paper or thin-layer chromatography can be used.
Remember it as: Rf: how far the pigment ran, out of how far the solvent ran.

Real example: autumn leaves. Chromatography of a green leaf shows yellow-orange carotenoids hidden under the chlorophyll. In autumn the chlorophyll breaks down first, so those yellow and orange colours show.

Trap: Measure to the centre of each spot, and from the origin, not from the bottom of the paper.

Calculating an Rf value:

Worked example: an Rf value: The solvent moved 8.0 cm and a blue-green pigment moved 5.0 cm.

Rf = 5.0 ÷ 8.0 = 0.63 (no units).

A blue-green band with an Rf of about 0.6 matches chlorophyll a in a typical run.

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How it is asked: Expect an Rf calculation, identifying a pigment from a chromatogram and a table of Rf values, or naming the method used.
IB-style questionCalculate[2 marks]

The solvent front is 9.0 cm from the origin. A yellow-orange spot is 8.4 cm from the origin. Calculate its Rf value and identify the pigment.

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The solvent front of a chromatogram is 7.5 cm from the origin. A yellow-green spot is 3.4 cm from the origin.

the Rf value of the spot and which pigment it is.
[2 marks]

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