Investigating Photosynthetic Pigments with a Pasco Spectrometer

Photosynthesis depends on a range of pigments that capture light energy from different parts of the spectrum. While chlorophyll dominates, other pigments, such as carotenoids and xanthophylls, also contribute, extending the range of light that plants can use. Using a PASCO spectrometer and coloured filters, students can investigate how different wavelengths affect light absorption — and discover why plants aren’t simply “green.”

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The Experiment

Students set up a PASCO light sensor with a white light source and a series of coloured filters (red, blue, and green).
They:

1. Measure the light intensity passing through a pigment extract or leaf sample at each wavelength.

2. Record how much light is absorbed (low transmission) or reflected (high transmission).

3. Plot a spectral absorption graph, showing how pigment extracts respond to different colours of light.

Alternatively, a PASCO spectrometer can be used to collect continuous absorption data across the visible spectrum.

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The Science

Each pigment absorbs specific wavelengths of light due to the arrangement of its electrons.

• Chlorophyll a absorbs mainly red and blue light, reflecting green.

• Chlorophyll b, carotenoids, and xanthophylls absorb in slightly different regions, broadening the plant’s overall light-harvesting ability.

By comparing absorption and photosynthesis rates, students can link pigment properties to plant adaptation and efficiency in different environments.

Plants contain a variety of photosynthetic pigments, primarily

chlorophylls and carotenoids (which include carotenes and xanthophylls), that allow them to absorb a broader range of light wavelengths for photosynthesis. These different pigments can be separated and identified using chromatography.

Plants with Different Photosynthetic Pigments

While most green plants contain the same primary pigments, the relative abundance and specific types can vary, particularly across different plant and algal groups: 

Different plants to test could include:

• Green leaves (e.g., spinach, grass) for typical chlorophylls and carotenoids.
• Red or purple leaves (e.g., red cabbage, some Ficus benjamina cultivars) to observe anthocyanins (though these are not photosynthetic pigments, they co-exist).
• Brown algae (seaweed) contain chlorophyll c and fucoxanthin.
• Carrots or corn for high amounts of carotenes and xanthophylls, respectively.

Testing Photosynthetic Pigments 

The standard method for separating and identifying these pigments is chromatography (paper or thin-layer chromatography, TLC), often followed by spectrophotometry. 

Materials 

• Leaf samples (e.g., spinach, a red leaf variety)
• Pestle and mortar
• Acetone (organic solvent)
• Chromatography paper or TLC plate
• Chromatography solvent (e.g., a mixture of petroleum ether, acetone, and trichloromethane)
• Capillary tube
• Pencil and ruler
• Beaker or test tube with a cover
• 
  

Procedure (Thin-Layer Chromatography) 

Extract the pigments: Grind a piece of leaf tissue in a mortar and pestle with a small amount of acetone to break open the cells and dissolve the pigments.

1. Spot the plate: Draw a pencil line near the bottom of a TLC plate. Use a capillary tube to repeatedly spot the pigment extract onto the line, allowing each spot to dry before applying the next, to create a concentrated spot.
2. Develop the chromatogram: Place the plate in a beaker containing a shallow layer of chromatography solvent, ensuring the solvent level is below the pencil line. Seal the container to saturate the atmosphere with solvent vapour.
3. Separate the pigments: Allow the solvent (mobile phase) to move up the plate by capillary action. Different pigments travel at different speeds because they vary in size and solubility in the mobile phase compared to their affinity for the stationary phase (the plate material).
4. Analyse the results: Once the solvent has nearly reached the top, remove the plate and immediately mark the solvent front with a pencil. You will see colored spots (bands) at different heights.
5. Identify pigments:
   • Colour: Carotenes (orange) travel furthest, followed by xanthophylls (yellow), chlorophyll a (blue-green), and chlorophyll b (yellow-green).
   • Rf value: Calculate the retention factor (Rf) for each pigment using the formula:
     
     

     $R_f=\frac{\text{distance travelled by pigment}}{\text{distance travelled by solvent}}$
   • Compare the calculated Rf values to known standards for identification.
6. Further testing: The individual pigment bands can be scraped off the TLC plate, dissolved in a suitable solvent (e.g., alcohol), and analysed using a spectrophotometer to determine their specific light absorption spectrum. This confirms which wavelengths each pigment absorbs most effectively. 

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Skills Highlight

• Using spectrometers to measure light absorption.

• Plotting and interpreting graphs of intensity vs wavelength.

• Relating pigment chemistry to photosynthetic efficiency.

• Understanding experimental design and controlled variables.

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Why It Works in Teaching

This investigation turns colour into data. Students can see the relationship between wavelength, absorption, and plant adaptation — a clear, visual link between physics and biology that strengthens understanding of photosynthesis.