5.6.4 Practical: Investigating photosynthetic pigments with chromatography

Question 1

What can chromatography be used for in investigating photosynthesis

Answer 1

Used to seperate and identify chloroplast pigments that have been extracted from a leaf

Question 2

What is chromatography?

Answer 2

An experimental technique that is used to seperate mixtures.
Different components within the mixture travel through the material at different speeds due to their size and charge - causing them to seperate

Question 3

What can be calculated for each component of the mixture?

Answer 3

An Rf value (retardation factor)

Question 4

What are the two most common techniques for seperating the photosynthetic pigments?

Answer 4

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 (e.g. silica gel), through which the mixture travels faster and seperates more distinctly

Question 5

Describe the method of this experiment (photosynthesis)

Answer 5

1. Draw straight line in pencil approx 1cm above bottom
2. Cut section of leaf, place in pestle and mortar. Important to select a healthy leaf that is been in direct sunlight so you can be sure it contains many active photosystems
3. Add 20 drops of acetone, grind with pestle.
   (Acetone is organic = fats, such as the lipid membrane, dissolve in it)
4. Extract some of the pigment using a capillary tube and spot it onto the centre of the pencil line
5. Suspend the paper in the chromatogarphy 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 - mobile. The dissolved mixture then passes through a static material - stationary)
6. Remove the paper from the solvent and draw a pencil line marking where the solvent moved up to
7. Calculate Rf value for each spot

Question 6

Describe the results of this experiment (photosynthesis)

Answer 6

Carotenoids - highest Rf values (close to 1)
Chlorophyll b - much lower Rf value
Chlorophyll a - Rf value somewhere between those of carotenids and chlorophyll b
Small Rf values indicate the pigment is less soluble and/or larger in size

Question 7

What are the limitations to this experiment (photosynthesis)

Answer 7

• Paper chromatography is not as specific as other chromatography techniques. It is sufficient to seperate and distinguish different pigments
• 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