Chromatography of Photosynthetic Pigments (AQA A-Level Biology): Revision Notes
Chromatography of Photosynthetic Pigments
Purpose and principle
This practical uses chromatography to separate and identify photosynthetic pigments from plant leaf samples. The technique works by exploiting differences in how various pigments interact with the chromatography paper and solvent. Different pigments will migrate at different rates based on their solubility, molecular mass, and affinity to the paper. This allows comparison between pigments from shade-tolerant versus shade-intolerant plants, or leaves of different colours.
The underlying principle involves a mobile phase (the solvent) carrying dissolved pigments up through a stationary phase (the chromatography paper). Pigments with higher solubility in the solvent and lower affinity for the paper will travel further, creating distinct bands of colour at different heights.
Apparatus and materials
The following equipment is required for this investigation:
- Philtre paper (chromatography paper)
- Fresh leaf samples from different plant types
- Distilled water
- Pestle and mortar for grinding
- Pencil for marking
- Ruler for measurements
- Capillary tube for applying pigment
- Chromatography solvent (specific solvent mixture)
- Acetone for pigment extraction
Method
- Prepare the chromatography paper by drawing a straight pencil line approximately 1cm above the bottom edge. Avoid using pen as the ink will interfere with results and obscure the pigment separation.
- Extract the pigments by cutting a section of fresh leaf and placing it in the mortar. Add exactly 20 drops of acetone to the leaf sample. Use the pestle to thoroughly grind the leaf material, which releases the photosynthetic pigments into the acetone.
- Apply the pigment extract. Use a capillary tube to collect the coloured acetone solution. Touch the tube to the centre of your pencil line. Allow the spot to dry, then reapply 2-3 times to concentrate the pigments
- Set up the chromatography by suspending the paper vertically in the solvent. Ensure the liquid level sits below the pencil line where you applied the pigment. Allow the solvent to migrate up the paper until it reaches near the top.
- Stop the separation by removing the paper from the solvent once adequate separation has occurred. Immediately mark with a pencil line where the solvent front reached, as this boundary will fade as the solvent evaporates.
- Calculate Rf values for each separated pigment spot. Measure the distance from the original pencil line to the centre of each pigment band. The Rf value equals the distance travelled by the pigment divided by the distance travelled by the solvent front.
Rf Value Calculation:
Safety considerations
This practical involves several chemical hazards requiring appropriate precautions:
Biohazards from plant material may cause allergies, soil bacteria exposure, or contamination. Always wash hands thoroughly after handling leaf samples and seek assistance if allergic reactions occur.
Chromatography solvents are typically flammable and can irritate eyes and skin. They may also be harmful if inhaled. Work in well-ventilated areas, avoid naked flames, wear eye protection, and keep solvents in fume cupboards when possible.
Acetone presents similar risks as it is flammable and causes eye irritation. Inhalation may lead to dizziness and drowsiness. Follow the same precautions as for chromatography solvents.
Data collection and analysis
Record your results in a table showing each pigment colour, the distance it travelled, the solvent front distance, and the calculated Rf value. Always measure to the centre of each spot for consistency.
Compare your calculated Rf values to published databases to identify specific pigments. Ensure you reference databases that used the same solvent system and chromatography paper type, as these variables significantly affect results.
Factors affecting pigment mobility
Two main factors determine how far each pigment travels:
- Affinity describes how strongly pigments bind to the chromatography paper. Pigments with lower affinity to the paper experience less resistance and travel further distances, resulting in higher Rf values.
- Solubility in the mobile phase determines how readily pigments dissolve and move with the solvent. More soluble pigments travel faster and further up the paper, ending up closer to the solvent front.
Pigments that migrate the greatest distances will have the highest Rf values, while those that barely move from the origin will have Rf values close to zero.
Expected results and interpretation
Typical leaf extracts separate into several distinct bands representing different photosynthetic pigments. Chlorophyll a and chlorophyll b are usually prominent, along with various carotenoids such as beta-carotene and xanthophylls.
Expected Pigment Separation Pattern:
From top to bottom (highest to lowest Rf values):
- Carotenes (orange) - highest Rf value (~0.9)
- Xanthophylls (yellow) - medium-high Rf value (~0.7)
- Chlorophyll a (blue-green) - medium Rf value (~0.6)
- Chlorophyll b (green) - lowest Rf value (~0.4)
Shade-tolerant plants often show higher concentrations of chlorophyll b relative to chlorophyll a, as this adaptation helps capture light more efficiently in low-light conditions. Different coloured leaves may reveal varying pigment compositions that reflect their specific adaptations.
Links to photosynthesis theory
This practical directly connects to photosynthesis by demonstrating the diversity of light-harvesting pigments in plant leaves. Each pigment absorbs different wavelengths of light, allowing plants to maximise energy capture across the visible spectrum. The pigment composition revealed by chromatography helps explain how different plants adapt to varying light conditions in their natural environments.
Key Points to Remember:
- Chromatography separates pigments based on their solubility and affinity to the paper
- Rf values are calculated as:
- Higher Rf values indicate pigments that travelled further up the paper
- Safety precautions are essential when handling flammable solvents like acetone
- Compare results to databases using the same solvent system for accurate pigment identification