Investigating the Need for Chlorophyll, Light & Carbon Dioxide (CIE IGCSE Biology)

• Although plants make glucose in photosynthesis, leaves cannot be tested for its presence as the glucose is quickly used, converted into other substances and transported or stored as starch.
• Starch is stored in chloroplasts where photosynthesis occurs so testing a leaf for starch is a reliable indicator of which parts of the leaf are photosynthesising.
• Leaves can be tested for starch using the following procedure:
  • A leaf is dropped in boiling water to kill the cells and break down the cell membranes
  • The leaf is left for 5-10 minutes in hot ethanol in a boiling tube. This removes the chlorophyll so colour changes from iodine can be seen more clearly
  • The leaf is dipped in boiling water to soften it
  • The leaf is spread out on a white tile and covered with iodine solution
  • In a green leaf, the entire leaf will turn blue-black as photosynthesis is occurring in all areas of the leaf
  • This method can also be used to test whether chlorophyll is needed for photosynthesis by using a variegated leaf (one that is partially green and partially white)
  • The white areas of the leaf contain no chlorophyll and when the leaf is tested only the areas that contain chlorophyll stain blue-black
  • The areas that had no chlorophyll remain orange-brown as no photosynthesis is occurring here and so no starch is stored

Testing a variegated leaf for starch

• Care must be taken when carrying out this practical as ethanol is extremely flammable, so at that stage of the experiment the Bunsen burner should be turned off.
• The safest way to heat the ethanol is in an electric water bath rather than using a beaker over a Bunsen burner with an open flame

• The same procedure as above can be used to investigate if light is needed for photosynthesis
• Before starting the experiment the plant needs to be destarched by placing in a dark cupboard for 24 hours
• This ensures that any starch already present in the leaves will be used up and will not affect the results of the experiment
• Following destarching, a leaf of the plant can be partially covered with aluminium foil and the plant placed in sunlight for a day
• The leaf can then be removed and tested for starch using iodine
• The area of the leaf that was covered with aluminium foil will remain orange-brown as it did not receive any sunlight and could not photosynthesise, while the area exposed to sunlight will turn blue-black
• This proves that light is necessary for photosynthesis and the production of starch

• Destarch two plants by placing in the dark for a prolonged period of time
• Place one plant in a bell jar which contains a beaker of sodium hydroxide (which will absorb carbon dioxide from the surrounding air)
• Place the other plant in a bell jar which contains a beaker of water (control experiment), which will not absorb carbon dioxide from the surrounding air
• Place both plants in bright light for several hours
• Test both plants for starch using iodine
• The leaf from the plant placed near sodium hydroxide will remain orange-brown as it could not photosynthesise due to lack of carbon dioxide
• The leaf from the plant placed near water should turn blue-black as it had all necessary requirements for photosynthesis

An example setup for an experiment to test whether carbon dioxide is necessary for photosynthesis in plants.