2.8.4 Skills: Separating Photosynthetic Pigments

Separation of photosynthetic pigments by chromatography

• Plants contain several different photosynthetic pigments, which absorb different wavelengths of light
• There are two groups of pigments: chlorophylls and carotenoids
• Carotenoids surround the chlorophyll and absorb both similar and different wavelengths of light to chlorophyll
  • This expands the range of wavelengths that can be absorbed from light for use in photosynthesis

Chloroplast Pigments Table

• 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 (R_f value) 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 (TLC)– 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
• Paper chromatography can be used to separate photosynthetic pigments although TLC gives better results

Apparatus

• Leaf sample
• Distilled water
• Pestle and mortar
• Filter paper
• Capillary tube
• Chromatography solvent
• Propanone
• 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 photosynthetic cells

• Add 20 drops of propanone and use the pestle to grind up the leaf sample and release the pigments
  • Propanone is an organic solvent and therefore fats, such as the lipid membrane, dissolve in it
  • The combination of propanone and mechanical pressure breaks down the cell and chloroplasts 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

• • 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 R_f value
• The R_f 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 R_f value
  • Molecules that are more soluble in the mobile phase will travel faster and therefore have a larger R_f value
• Although specific R_f values depend on the solvent that is being used, in general:
  • Carotenoids have the highest R_f values (usually close to 1)
  • Chlorophyll b has a much lower R_f value
  • Chlorophyll a has an R_f value somewhere between those of carotenoids and chlorophyll b
  • Small R_f 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 R_f values. In this example, a line of the 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 R_f 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