describe and use chromatography to separate and identify chloroplast pigments (reference should be made to Rf values in identification of chloroplast pigments)
Cambridge A‑Level Biology 9700 – Photosynthesis as an Energy Transfer Process
Topic: Photosynthesis as an Energy Transfer Process
Learning Objective
Students will be able to describe how chromatography can be used to separate and identify the pigments present in chloroplasts, making reference to the use of \$R_f\$ values in the identification process.
1. Why Study Chloroplast Pigments?
Chloroplast pigments absorb light of specific wavelengths and convert that energy into chemical energy during photosynthesis. Understanding the pigment composition helps explain:
The range of light wavelengths that can drive photosynthesis.
How plants adapt to different light environments.
The role of accessory pigments in transferring energy to chlorophyll a.
2. Principle of Paper Chromatography
Paper chromatography separates pigments based on their differing solubilities in a mobile phase (solvent) and their affinities for the stationary phase (paper). As the solvent front moves up the paper, each pigment travels a characteristic distance.
The degree of migration is expressed as the retardation factor (\$R_f\$):
\$R_f = \frac{\text{distance travelled by pigment}}{\text{distance travelled by solvent front}}\$
\$R_f\$ values are reproducible under standard conditions and allow pigments to be identified by comparison with known standards.
3. Materials Required
Whatman No. 1 chromatography paper (or equivalent)
Solvent system (e.g., petroleum ether : acetone = 90:10 v/v)
Fresh spinach leaves (or other green plant material)
Mortar and pestle, acetone (80 % v/v) for pigment extraction
Pencil, ruler, and ruler‑marked chromatography chamber
Capillary tubes or micropipettes for spotting
4. Procedure (Step‑by‑Step)
Cut the chromatography paper into strips 2 cm wide and 15 cm long.
Draw a faint pencil line 2 cm from the bottom edge; this is the origin line.
Grind a small amount of fresh spinach in 5 ml of 80 % acetone to obtain a pigment extract.
Using a capillary tube, place a small drop of the extract on the origin line. Allow it to dry; repeat 2–3 times to concentrate the spot.
Place the strip in a sealed chamber containing a shallow layer of solvent (the solvent level must be below the origin line).
Seal the chamber and allow the solvent to rise until it is about 1 cm from the top of the strip.
Remove the strip, mark the solvent front immediately, and allow the paper to dry in a fume cupboard.
Measure distances travelled by each pigment band and calculate \$R_f\$ values.
5. Identification of Chloroplast Pigments Using \$R_f\$ Values
Typical \$Rf\$ values for common chloroplast pigments in the petroleum ether : acetone (90 : 10) system are shown below. Values may vary slightly with temperature, paper grade, and solvent purity, so students should compare their experimental \$Rf\$ values with these reference ranges.
Pigment
Colour (visible)
Typical \$R_f\$ range
Primary role in photosynthesis
Carotene (β‑carotene)
Orange
0.85 – 0.90
Absorbs blue‑green light; transfers energy to chlorophyll a
Lutein
Yellow‑orange
0.75 – 0.80
Photoprotection; accessory pigment
Violaxanthin
Yellow‑orange
0.70 – 0.75
Photoprotection; part of the xanthophyll cycle
Chlorophyll b
Blue‑green
0.55 – 0.60
Extends absorption spectrum; transfers energy to chlorophyll a
Chlorophyll a
Blue‑green (dark)
0.45 – 0.50
Primary pigment for photochemical reactions
6. Interpreting Results
Compare each measured \$R_f\$ with the reference ranges. A match within ±0.02 is generally acceptable.
If a band’s \$R_f\$ does not correspond to any listed pigment, consider experimental error or the presence of less common pigments (e.g., neoxanthin).
Record the order of pigment migration; the most non‑polar pigments (carotenes) travel furthest, while the most polar (chlorophyll a) travel least.
7. Linking Pigment Separation to Energy Transfer in Photosynthesis
The separation pattern reflects the polarity and, indirectly, the light‑absorption properties of each pigment. By visualising the pigment suite, students can:
Explain how accessory pigments capture photons that chlorophyll a cannot, broadening the usable spectrum.
Discuss the energy‑transfer cascade: high‑energy photons are absorbed by carotenoids → energy is passed to chlorophyll b → finally to chlorophyll a, where charge separation occurs.
Relate the efficiency of photosynthesis to the proportion of each pigment, which can be estimated from band intensity (qualitative).
8. Common Sources of Error
Uneven solvent front – leads to inaccurate \$R_f\$ values.
Over‑loading the sample spot – causes streaking and overlapping bands.
Using a solvent mixture of incorrect proportion – changes polarity and shifts \$R_f\$ values.
Temperature fluctuations – affect solvent evaporation rate.
9. Suggested Extension Activities
Repeat the experiment with different solvent systems (e.g., pure acetone, petroleum ether alone) and compare how \$R_f\$ values shift.
Quantify pigment concentration by densitometry (if equipment is available) and relate pigment ratios to leaf colour.
Investigate how light intensity during plant growth influences the relative amounts of chlorophyll a, chlorophyll b, and carotenoids.
Suggested diagram: A schematic of the chromatography setup showing the solvent chamber, paper strip, origin line, solvent front, and separated pigment bands with labelled \$R_f\$ values.
10. Summary
Chromatography provides a simple yet powerful method for separating chloroplast pigments. By measuring \$R_f\$ values and comparing them with known standards, students can accurately identify each pigment and understand its role in the overall energy‑transfer process of photosynthesis. Mastery of this technique underpins deeper investigations into plant adaptation, photoprotection, and the optimisation of photosynthetic efficiency.