OCR · A-Level · Biology
Course version: H420 · Version from Sept 2025
(ii) practical investigations using thin layer chromatography (TLC) to…
Full specification wording
Photosynthesis: (ii) practical investigations using thin layer chromatography (TLC) to separate photosynthetic pigments
TLC separates photosynthetic pigments because each pigment partitions differently between a stationary phase (a thin silica layer on a plate) and a moving solvent.
Checked against the OCR H420 specification (opens in a new tab) on .
- Point 4 of 9
- 3 key terms
Study notes
From the checked teaching notes for this specification point.
The idea
TLC separates photosynthetic pigments because each pigment partitions differently between a stationary phase (a thin silica layer on a plate) and a moving solvent. A concentrated leaf extract is spotted as a small origin spot, the plate is developed in a shallow solvent so the solvent front rises by capillary action, and pigments travel at different rates according to their solubility in the solvent and their attraction to the stationary phase. More soluble pigments move further; Rf values (distance moved by pigment ÷ distance moved by solvent front) identify pigments such as chlorophyll a, chlorophyll b, carotene and xanthophyll. Good technique uses a pencil origin line, a small spot, a covered tank and a solvent front marked before it dries.
What earns the marks
- TLC uses a stationary phase (thin layer of silica on a plate) and a mobile phase (solvent) so pigments partition between them.
- Pigments separate because they differ in solubility in the solvent and in attraction to the stationary phase; more soluble pigments travel further.
- A concentrated extract is spotted on a pencil origin line and the plate is developed in solvent kept below the origin line, in a covered container.
- Rf value = distance moved by pigment ÷ distance moved by solvent front, and is used to identify pigments by comparison with known values.
- The solvent front must be marked immediately and the origin line drawn in pencil, not ink, to avoid contamination and to allow measurement.
Common mistakes and how to put them right
Thinking pigments separate because of their colour alone.
Put it right: Separation depends on differential solubility and adsorption, not colour.
Spotting the extract below the solvent level.
Put it right: The origin spot must sit above the solvent line so pigments travel up the plate.
Using ink for the origin line.
Put it right: Ink dissolves and contaminates the chromatogram, so a pencil line is used.
Measuring Rf from the top of the spot rather than its centre.
Put it right: Measure to the centre of each spot for consistency.
Exam tips
- State both phases explicitly: stationary silica layer and mobile solvent.
- Explain separation in terms of solubility and adsorption, not just 'different colours'.
- Give the Rf formula and note that it is a ratio with no units.
- Describe practical precautions: covered tank, solvent below origin, pencil line, mark solvent front.
Try the lesson
The start of the Photosynthesis: (ii) practical investigations using thin layer chromatography (TLC) to separate photosynthetic pigments lesson, made from checked teaching notes for this specification point.
Have a go first
In a TLC investigation of leaf pigments, a pigment spot travels 4.0 cm from the origin while the solvent front travels 8.0 cm. What is the Rf value of this pigment?
Listen
TLC separates photosynthetic pigments because each pigment partitions differently between a stationary phase (a thin silica layer on a plate) and a moving solvent. A concentrated leaf extract is spotted as a small origin spot, the plate is developed in a shallow solvent so the solvent front rises by capillary action, and pigments travel at different rates according to their solubility in the solvent and their attraction to the stationary phase. More soluble pigments move further; Rf values (distance moved by pigment ÷ distance moved by solvent front) identify pigments such as chlorophyll a, chlorophyll b, carotene and xanthophyll. Good technique uses a pencil origin line, a small spot, a covered tank and a solvent front marked before it dries. A strong answer earns marks for these points. TLC uses a stationary phase (thin layer of silica on a plate) and a mobile phase (solvent) so pigments partition between them. Pigments separate because they differ in solubility in the solvent and in attraction to the stationary phase; more soluble pigments travel further. A concentrated extract is spotted on a pencil origin line and the plate is developed in solvent kept below the origin line, in a covered container. Rf value = distance moved by pigment ÷ distance moved by solvent front, and is used to identify pigments by comparison with known values. A common slip: Thinking pigments separate because of their colour alone. Instead, separation depends on differential solubility and adsorption, not colour. Exam tip: State both phases explicitly: stationary silica layer and mobile solvent.
Key words
Say what each word means before you turn the card.
Partition between phases
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Rf values and pigment identification
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Practical technique and precautions
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See every definition
- Partition between phases
- Pigments partition differently between a stationary phase (a thin silica layer on a plate) and a moving solvent, which is the mobile phase.
- Rf values and pigment identification
- Rf value = distance moved by pigment ÷ distance moved by solvent front, and is used to identify pigments by comparison with known values.
- Practical technique and precautions
- A concentrated extract is spotted on a pencil origin line, the plate is developed in solvent kept below the origin line in a covered container, and the solvent front is marked before it dries.
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