OCR Specification focus:
‘Explain light-harvesting systems and photosystems; separate photosynthetic pigments using thin layer chromatography (TLC).’
Photosynthetic pigments absorb light energy to drive photosynthesis. These pigments are organised into light-harvesting systems and photosystems, whose components can be separated experimentally by thin layer chromatography (TLC).
Photosynthetic Pigments
Overview of Pigments
Photosynthetic pigments are coloured molecules found in the thylakoid membranes of chloroplasts that absorb specific wavelengths of light. Each pigment has a distinct absorption spectrum, allowing plants to capture a broad range of the light available for photosynthesis.
The major pigments include:
Chlorophyll a – the primary pigment directly involved in the light-dependent reactions.
Chlorophyll b – an accessory pigment transferring absorbed energy to chlorophyll a.
Carotenoids (including carotenes and xanthophylls) – accessory pigments that protect chlorophyll from photooxidation and extend the range of absorbed light.
Absorption Spectrum: The range and efficiency of wavelengths of light absorbed by a pigment.
Function of Pigment Diversity
Different pigments absorb light most efficiently at different wavelengths, creating an action spectrum that reflects the overall rate of photosynthesis across the light spectrum. The combined effect of all pigments allows plants to utilise more of the visible light spectrum, particularly blue and red wavelengths, while reflecting green light.
Absorption spectra for chlorophyll a and chlorophyll b, showing strong peaks in the blue and red regions and a trough in green. This visual explains why leaves appear green and why pigment diversity broadens usable light. SVG format provides scalable, high-resolution detail suitable for study. (Carotenoids are not shown here; use with Image 2/3 to complete the pigment picture.) Source.
Action Spectrum: The rate of photosynthesis plotted against wavelength, showing the effectiveness of different wavelengths in driving photosynthesis.
Localisation in the Chloroplast
Pigments are embedded in the thylakoid membranes of chloroplasts, bound to specific protein complexes forming photosystems. This structural organisation maximises the capture and transfer of light energy to the reaction centre where photochemical reactions occur.
Light-Harvesting Systems
Structure and Function
A light-harvesting system (antenna complex) consists of clusters of accessory pigments (chlorophyll b and carotenoids) surrounding a primary pigment (chlorophyll a) in the reaction centre. Their arrangement ensures efficient absorption and funneling of light energy.
Light-Harvesting System: A group of pigment molecules and proteins that capture light energy and transfer it to the reaction centre of a photosystem.
Energy absorbed by accessory pigments is transferred through resonance energy transfer, a process where excitation energy, rather than electrons, moves between pigment molecules until it reaches the reaction centre. This ensures minimal loss of energy and efficient excitation of electrons in chlorophyll a.
Importance of Organisation
The spatial arrangement of pigments allows plants to absorb light efficiently under varying light conditions. Shaded leaves, for instance, often contain more chlorophyll b to absorb light of wavelengths not captured by chlorophyll a, enhancing overall photosynthetic performance.
Photosystems
Composition and Types
Photosystems are large complexes of proteins, pigments, and cofactors embedded in the thylakoid membrane. There are two main types involved in the light-dependent reactions: Photosystem II (PSII) and Photosystem I (PSI).
Diagram of Photosystem II and Photosystem I showing antenna pigments funnelling energy to a chlorophyll a reaction centre (P680 and P700 respectively). Labels highlight photon capture, energy transfer, and the reaction centre where electrons are excited. This figure also shows electron-transport context (b₆f/ETC), which is extra detail beyond the pigments/photosystems focus but supports accurate placement in the thylakoid membrane. Source.
Photosystem: A complex of pigments and proteins that absorb light to initiate the light-dependent reactions of photosynthesis.
Each photosystem contains:
A reaction centre with a specific chlorophyll a molecule:
PSII contains P680 (absorbs light at 680 nm).
PSI contains P700 (absorbs light at 700 nm).
Antenna pigments forming the light-harvesting system.
Electron carriers transferring excited electrons to subsequent stages of the light-dependent reactions.
Role in Photosynthesis
In Photosystem II, photons excite electrons in chlorophyll P680. The excited electrons are passed to an electron transport chain, while water molecules are split (photolysis) to replace the lost electrons and release oxygen.
In Photosystem I, photons re-excite electrons reaching P700, which are then transferred to form reduced NADP (NADPH) used in the Calvin cycle.
These processes together constitute the foundation for non-cyclic photophosphorylation, while cyclic photophosphorylation involves only PSI to generate additional ATP.
Thin Layer Chromatography (TLC)
Principle of TLC
Thin layer chromatography is an experimental method used to separate and identify photosynthetic pigments based on their differing affinities for the stationary and mobile phases. The stationary phase is a thin layer of silica or alumina coated on a plate, while the mobile phase is a solvent mixture carrying the dissolved pigments upward by capillary action.
Thin Layer Chromatography (TLC): A separation technique that distinguishes pigments based on their solubility and adsorption properties on a stationary phase.
Method for Separating Photosynthetic Pigments
Pigment extraction – Grind a leaf with a suitable solvent (e.g. propanone) to dissolve pigments.
Spotting – Apply a small drop of the pigment extract near the base of the TLC plate and allow it to dry.
Development – Place the plate in a sealed chamber containing the solvent. The solvent rises through the stationary phase, carrying pigments at different rates.
Separation – Pigments move according to their solubility in the solvent and affinity to the stationary phase. Carotenes, being highly soluble, travel furthest; xanthophylls and chlorophylls move less.
Visualisation – When dry, pigment bands are visible as distinct colours or can be viewed under UV light.
Interpretation of TLC Results
Each pigment produces a characteristic Rf value, representing its relative movement compared with the solvent front.
EQUATION
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Rf Value = Distance moved by pigment ÷ Distance moved by solvent front
Distance moved by pigment = distance from origin to pigment centre (mm)
Distance moved by solvent front = distance from origin to solvent front (mm)
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Typical Rf values under common solvent systems:
Carotene: 0.95 (travels furthest)
Xanthophyll: 0.70
Chlorophyll a: 0.65
Chlorophyll b: 0.45
Significance of TLC in Photosynthesis Studies
TLC allows students and scientists to:
Identify the variety of pigments present in plant tissue.
Compare pigment composition between different species or leaf types.
Demonstrate the principle that multiple pigments contribute to the efficiency of light absorption.
By revealing distinct pigment profiles, TLC provides direct evidence for the complexity and cooperation of photosynthetic pigments in capturing light energy, as required for understanding light-harvesting systems and photosystems within the OCR A-Level Biology framework.
FAQ
The orientation of chlorophyll molecules within the protein matrix ensures efficient energy transfer toward the reaction centre.
Each pigment molecule is positioned so its absorption dipole aligns optimally with adjacent molecules. This allows resonance energy transfer to occur rapidly and directionally, minimising energy loss as heat.
This spatial organisation also supports the separation of Photosystem I and II within distinct thylakoid regions—PSII mainly in stacked grana and PSI in unstacked stroma lamellae—optimising light absorption and electron transport.
Several factors influence a pigment’s Rf value:
Solubility in the solvent: More soluble pigments travel further up the plate.
Affinity to the stationary phase: Pigments that adhere more strongly move less.
Solvent composition and polarity: Changing the solvent mixture alters pigment movement.
Temperature and plate preparation: Small variations can affect migration rates.
As a result, Rf values are reproducible only under identical experimental conditions, making standardisation essential for reliable pigment identification.
Carotenoids act as photoprotective pigments by safely dissipating excess light energy absorbed by chlorophyll when illumination is too intense.
They quench triplet-state chlorophyll and reactive oxygen species (ROS), preventing oxidative damage to chloroplast membranes and proteins.
Additionally, carotenoids stabilise thylakoid membranes by embedding within the lipid bilayer, reducing photobleaching and ensuring photosynthetic efficiency under variable light conditions.
Pigment–protein binding maintains the correct orientation and spacing required for efficient energy transfer.
The proteins also stabilise pigment molecules, prevent them from aggregating, and control their microenvironment to fine-tune absorption properties.
This arrangement ensures that absorbed light energy is funnelled predictably to the reaction centre rather than dissipated randomly. Without protein association, pigments would degrade more quickly and energy transfer would be inefficient.
Accuracy can be improved by:
Using fresh, finely ground leaf material to ensure full pigment extraction.
Applying narrow, concentrated spots to avoid overlap between pigment bands.
Maintaining consistent solvent depth so the origin remains above the solvent.
Using pre-marked plates and precise rulers to measure distances for Rf calculations.
Running replicate plates and comparing with known pigment standards.
These refinements increase reproducibility, allowing better distinction between pigments such as chlorophyll a, chlorophyll b, and carotenoids.
Practice Questions
Question 1 (2 marks)
State two functions of accessory pigments in the light-harvesting system of chloroplasts.
Mark Scheme:
1 mark for each correct function.
Accept any two of the following:
Absorb wavelengths of light not absorbed by chlorophyll a.
Transfer absorbed energy to chlorophyll a in the reaction centre.
Extend the range of light wavelengths used for photosynthesis.
Protect chlorophyll a from damage by excess light or photooxidation
Question 2 (5 marks)
Describe how thin layer chromatography (TLC) can be used to separate and identify photosynthetic pigments from a leaf extract. Include how the results can be interpreted.
Mark Scheme:
1 mark: Leaf pigment extract prepared by grinding leaves with an organic solvent (e.g. propanone) to dissolve pigments.
1 mark: A small sample of pigment extract is spotted onto a TLC plate near the base line/origin.
1 mark: Plate placed upright in a suitable solvent; pigments move up the plate as the solvent travels by capillary action.
1 mark: Pigments separate according to their solubility in the solvent and their affinity for the stationary phase.
1 mark: Distances travelled by pigments and the solvent front are measured to calculate Rf values for identification (Rf = distance moved by pigment ÷ distance moved by solvent front).
Award an additional mark (maximum 5 total) if the student correctly states that each pigment produces a distinct coloured band (e.g. carotene, xantho
