Required Practical: Investigating Photosynthetic Pigments with Chromatography
- Chloroplasts contain several different photosynthetic pigments within the thylakoids, which absorb different wavelengths of light
- The light-dependent stage of photosynthesis occurs in the thylakoid membranes and the thylakoid spaces (the spaces inside the thylakoids)
- The thylakoid membrane system provides a large number of pigment molecules in an arrangement that ensures as much light as necessary is absorbed
- The pigment molecules are arranged in light-harvesting clusters known as photosystems
- In a photosystem, the different pigment molecules are arranged in funnel-like structures within the thylakoid membrane (each pigment molecule passes energy down to the next pigment molecule in the cluster until it reaches the primary pigment reaction centre)
- There are two groups of pigments: primary pigments known as chlorophylls and accessory pigments known as carotenoids
- The primary pigment in photosystem II is chlorophyll b and the primary pigment in photosystem I is chlorophyll a
- Accessory pigments that surround the primary pigment absorb both similar and different wavelengths of light to chlorophyll, this expands the wavelength range that can be absorbed from light for use in photosynthesis
Chloroplast pigments table
| Pigment group | Name of pigment | Colour of pigment |
| Chlorophylls | Chlorophyll a | Blue-green |
| Chlorophyll b | Yellow-green | |
| Carotenoids | β Carotene | Orange |
| Xanthophyll | Yellow |
- Chlorophylls absorb wavelengths in the blue-violet and red regions of the light spectrum
- They reflect green light, causing plants to appear green
- Carotenoids absorb wavelengths of light mainly in the blue-violet region of the spectrum
Chlorophyll and carotenoids absorb light across the visible light spectrum to use in the light-dependent reaction of photosynthesis
Chromatography
- Chromatography is an experimental technique that is used to separate mixtures:
- Different components within the mixture travel through the material at different speeds
- This causes the different components to separate
- A retardation factor (Rf) can be calculated for each component of the mixture
- Two of the most common techniques for separating these photosynthetic pigments are:
- Paper chromatography – the mixture of pigments is passed through paper (cellulose)
- Thin-layer chromatography – the mixture of pigments is passed through a thin layer of adsorbent (eg. silica gel), through which the mixture travels faster and separates more distinctly
Apparatus
- Leaf sample
- Distilled water
- Pestle and mortar
- Filter paper
- Capillary tube
- Chromatography solvent
- Acetone
- Pencil
- Ruler
Method
- Draw a straight line in pencil approximately 1cm above the bottom of the filter paper being used
- Do not use a pen as the ink will separate into pigments within the experiment and obscure the results
- Cut a section of leaf and place it in a mortar
- It is important to choose a healthy leaf that has been in direct sunlight so you can be sure it contains many active photosystems
- Add 20 drops of acetone and use the pestle to grind up the leaf sample and release the pigments
- Acetone is an organic solvent and therefore fats, such as the lipid membrane, dissolve in it
- Acetone and mechanical pressure are used to break down the cell, chloroplast and thylakoid membranes to release the pigments
- Extract some of the pigment using a capillary tube and spot it onto the centre of the pencil line you have drawn
- Suspend the paper in the chromatography solvent so that the level of the solvent is below the pencil line and leave the paper until the solvent has reached the top of the paper
- The mixture is dissolved in the solvent (called the mobile phase) and the dissolved mixture then passes through a static material (called the stationary phase)
- Remove the paper from the solvent and draw a pencil line marking where the solvent moved up to
- The pigment should have separated out and there should be different spots on the paper at different heights above the pencil line, these are the separate pigments
- Calculate the Rf value for each spot
Rf value = distance travelled by component (pigment) ÷ distance travelled by the solvent
- Always measure to the centre of each spot
Results
- Chromatography can be used to separate and identify chloroplast pigments that have been extracted from a leaf as each pigment will have a unique Rf value
- The Rf value demonstrates how far a dissolved pigment travels through the stationary phase
- Molecules with a higher affinity to the stationary phase, such as large molecules, will travel slower and therefore have a smaller Rf value
- Molecules that are more soluble in the mobile phase will travel faster and therefore have a larger Rf value
- Although specific Rf values depend on the solvent that is being used, in general:
- Carotenoids have the highest Rf values (usually close to 1)
- Chlorophyll b has a much lower Rf value
- Chlorophyll a has an Rf value somewhere between those of carotenoids and chlorophyll b
- Small Rf values indicate the pigment is less soluble and/or larger in size
Paper chromatography is used to separate photosynthetic pigments. These pigments can be identified by their Rf values. In this example, a line of mixture (rather than a spot) is added to the paper.
Limitations
- Paper chromatography is not as specific as other chromatography techniques
- It is sufficient to separate and distinguish different pigments and to calculate their Rf value
- Chromatography does not give data on the amount of each pigment present or the wavelengths that they absorb
- Colorimetry can be used to calculate these values
Examiner Tip
Remember – the pigments themselves have colour (as described in the table). This is different from the colours of light that they absorb.
Make sure you learn the approximate Rf values for the different pigments within chloroplasts (or at least their values relative to each other). This means you should be able to identify different chloroplast pigments based on their Rf values alone.
