Separate and identify chloroplast pigments by chromatography
Describe paper chromatography of chloroplast pigments and calculate Rf values to support identification.
Section 13.1 · Photosynthesis as an energy transfer process
A Level; Cambridge International Biology 9700, 2028–2030. Written practice is self-checked; practical investigations need supervised work and human subject-teacher review remains pending.
Before you begin
- Chloroplasts contain photosynthetic pigments, and enzymes catalyse metabolic reactions.
Work on paper. Try each question before revealing help, and explain why your method works.
Quick readiness check
What is the Rf formula?
What you will learn
- Describe a controlled chromatography method for leaf pigments
- Calculate and compare Rf values with reference data
Build the explanation
A concentrated leaf-pigment extract is spotted on a pencil baseline near one end of chromatography paper. The baseline must stay above the solvent. As solvent rises, pigments partition between the mobile solvent and the paper and separate into bands. Measure from the baseline to each band's centre and to the solvent front. Rf = distance travelled by pigment ÷ distance travelled by solvent front. Compare Rf values with reference values collected in the same solvent and conditions.
Apply it carefully
Rf is a ratio without units and is normally between zero and one for a band that stays on the paper. Do not use pen for the baseline, place the spot under the solvent, or compare reference Rf values from a different solvent system as if they were universal. Band colour can support, but does not alone prove, pigment identity.
Pause and explain
Why must the pigment spot start above the solvent level?
Worked example
A pigment band travels 3.6 cm while the solvent front travels 6.0 cm. Calculate Rf.
Show the worked solution
- Divide pigment distance by solvent-front distance.
- 3.6 ÷ 6.0 = 0.60.
Answer Rf = 0.60.
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Write your answer and reasoning first. Use a hint only if you are stuck. The model solution is for self-checking; your written work is not automatically marked.
Outline a chromatography method for separating leaf pigments and calculating one Rf value.
Give me a hint
Include baseline, solvent and distance measurements.
Compare my reasoning
- Spot concentrated extract on a pencil baseline above the solvent and allow the solvent to rise.
- Measure pigment and solvent-front distances from the baseline, then divide pigment distance by solvent distance.
Draw a pencil baseline above the solvent level, apply a concentrated chloroplast-pigment extract, and let solvent rise through the paper. Mark the solvent front, measure each band from the baseline, and calculate Rf = pigment distance ÷ solvent-front distance. Compare with reference values from the same solvent system.
Look for these in your work
- Baseline/solvent positions correct
- Separation described
- Rf method correct
- Reference conditions considered
A yellow band travels 4.2 cm and the solvent front travels 7.0 cm. Calculate Rf and state one reason the colour alone is not conclusive.
Give me a hint
Use the distance ratio; identity needs comparison evidence.
Compare my reasoning
- Rf = 4.2 ÷ 7.0.
- Rf = 0.60; a band colour may be shared or distorted, so compare Rf with reference values in the same solvent.
Rf = 4.2/7.0 = 0.60. Yellow colour supports a possible identity but is not conclusive; compare the Rf value with reference pigment data using the same solvent conditions.
Look for these in your work
- Calculation correct
- Rf unitless
- Identification qualified
A student starts the baseline below the solvent surface and uses a permanent marker. Explain two problems and correct the setup.
Give me a hint
Think about the sample dissolving and the marking dye moving.
Compare my reasoning
- The pigment spot can dissolve into the solvent reservoir instead of travelling up the paper.
- Marker dye may separate and contaminate the chromatogram; draw a pencil baseline above the solvent.
The spot may dissolve directly into the solvent pool, and marker ink may move as extra bands. Use a pencil baseline above the solvent and apply the pigment extract there before placing the paper in the chamber.
Look for these in your work
- Solvent-position problem identified
- Marker contamination identified
- Corrected procedure stated
Common mistakes
- Dividing solvent distance by pigment distance
- Using a baseline below the solvent
- Treating an Rf value as universal across solvents
What can you explain now?
What is the Rf formula?
Compare with the explanation
Distance travelled by pigment ÷ distance travelled by solvent front
Use the same baseline for both distances.
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Make it stick
How do you calculate an Rf value and use it to support pigment identification?
If you needed the solution, close it and solve the problem again from a blank page. A correct answer is stronger when you can explain the reason for each step.