5-1-3-required-practical-investigating-photosynthetic-pigments-with-chromatography

Required practical: investigating photosynthetic pigments with chromatography

• Chloroplasts contain several different photosynthetic pigments within the thylakoids,

• Different pigments absorb different wavelengths of light, maximising the light energy that can be absorbed by a plant

  • 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

Investigating photosynthetic pigments with chromatography

• Chromatography is an experimental technique used to separate mixtures

• Two of the most common techniques for separating photosynthetic pigments are:

  • paper chromatography: the mixture of pigments is passed through paper

  • thin-layer chromatography (TLC): the mixture of pigments is passed through a thin layer of adsorbent, e.g. silica gel, through which the mixture travels faster and separates more distinctly

Apparatus

• Leaf sample

• Distilled water

• Pestle and mortar

• Chromatography paper

• Capillary tube

• Liquid chromatography solvent

• Acetone

• Pencil

• Ruler

Method

1. Draw a straight line in pencil approximately 1 cm 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

2. 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

3. 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 those present in cell membranes, dissolve in it

   • Acetone and mechanical pressure are used to break down the cell, chloroplast and thylakoid membranes to release the pigments

4. Extract some of the pigment using a capillary tube and spot it onto the centre of the pencil line you have drawn

5. 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 pigments have separated

   • Remove the paper before the solvent has run all the way to the top

   • There should be separate spots on the paper at different heights above the initial pencil line; these are the separate pigments

6. Remove the paper from the solvent and draw a pencil line marking the point reached by the solvent

   • This is sometimes described as the solvent front

7. Calculate the R_f value for each spot; always measure to the centre of each spot

R_f value = distance travelled by pigment ÷ distance travelled by the solvent

Results

• The calculated R_f value is a measure of the distance moved by each pigment through the stationary phase, in relation to the distance moved by the solvent

  • A higher Rf value indicates that molecules have a higher affinity with the liquid mobile phase, e.g. due to being:

    • non-polar

    • highly soluble in the solvent

    • small

  • A lower Rf value suggests that molecules have a higher affinity with the solid stationary phase, e.g. due to being

    • polar

    • less soluble

    • large

• 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