Investigations for photosynthesis:

Rules of Investigation.

Investigations need controls:

  • Control plant (or leaves) has all substances it needs.
  • Test plant lacks one substance (light/chlorophyll/Carbon dioxide).
  • Plants must be destarched.

   What is a destartched plant/leaf?

It is very important that the leaves you are testing should not have any starch in them at the beginning of the experiment. So, first of all, it is must to destarch the plants.

How can you destarch the plant/leaf?

  • Leave the plant in dark for 48 hours.
  • The plants use up all stored starch in its leaves.
  • Starch test with Iodine solution can be conducted then.

How can a starch test/iodine test will be conducted?

  • First of all boil the leaf in water for 30 second. This kill the cells in the leaf and break down the membrane so that iodine solution cross the cell membrane to reach starch inside the chloroplasts and react with it.
  • Then boil the leaf in alcohol (ethanol) in a water bath to remove chlorophyll. Leave the leaf in alcohol until all the chlorophyll has come out of the leaf. Eventually the colour of alcohol become green.
  • Rinse the leaf in water because boiling the leaf in ethanol makes it brittle so the water softens it.
  • Now to see the result spread the leaf on white tile.
  • When Iodine solution is used; a blue-black colour on the leaf shows it as positive test. It means it contain starch. While brown colour shows that it does not contain starch anymore.


Is chlorophyll and light necessary for photosynthesis?

Photosynthetic cells contain special pigments (chlorophyll) that absorbs light energy. Different pigments colours have different wavelengths of light. Chlorophyll, the primary green pigment used in photosynthesis, reflects green light and absorbs red and blue light most strongly. The process of photosynthesis occurs in chloroplasts, which contain a green pigment called chlorophyll.


What is a variegated leaf?

A variegated leaf is a leaf which has both green and non-green parts. As the green parts contain chlorophyll they photosynthesize, but the non-green parts don’t contain chlorophyll, so they cannot absorb sunlight. These leaves are partly green and partly yellow or white in color.

A variegated leaf is useful in conducting experiments to show that chlorophyll is needed for photosynthesis to happen.

Investigation to check the presence of starch in a variegated leaf:

  • Take a variegated leaf and dip in boiling water to kill it and for further use.
  • To remove the chlorophyll the leaf is left in hot ethanol for ten minutes.
  • Again the leaf is dipped in boiling water to soften it.
  • The leaf is spread out on a white tile and covered with iodine solution.
  • The areas turn blue-black shows the presence of chlorophyll.
  • While the areas of the variegated leaf that had no chlorophyll remain pale or brown.

What are the Control variables in the process of photosynthesis?

  • Presence or absence of chlorophyll is an independent variable.
  • Starch is present or not is a dependent variable.
  • Methods and equipment used in this investigation are the Control variables.

Precautions taken during the investigation:

  • Be careful when boil water.
  • Must be very careful when dealing with alcohol because ethanol (alcohol) is highly flammable.
  • Iodine solution is an irritant so keep it away from your eyes.
  • Goggles must be worn to protect the eyes.

Results of the investigation:

During the process of photosynthesis Starch is a stored food that is produced from glucose.

After adding the iodine solution if the areas of the leaf turn blue–black then it shows that starch is present. This proves that photosynthesis has taken place.

While white areas of the leaf without chlorophyll could not photosynthesise food. This shows the absence of starch which means that the process of photosynthesis did not occur. So, when these areas will be tested with iodine, will remain pale or brown.

Is carbon dioxide necessary for the process of photosynthesis?

Cells of the plants use carbon dioxide and energy from the light to make glucose and oxygen during the process of Photosynthesis. These sugar molecules (glucose) are the basis for more complex molecules made by the photosynthetic cell, such as glucose.


  • Arrange two destarched (without starch) potted plants as shown in the following picture.



  • In set up X, a container contains some saturated sodium hydrogen carbonate which helps the plant to provide carbon dioxide.
  • In set up Y, soda lime is placed to absorb the carbon dioxide inside the polythene bag as well as the carbon dioxide given off by microorganisms in the soil.
  • For two hours approximately keep both the set ups to bright sunlight.
  • After 2 hours, take out one leaf from both set up and test each leaf with iodine solution for the presence of starch.


  •  When checked with iodine solution the leaf taken from set up X turns blue black where as the leaf taken from set up Y remain unchanged.


  • The conclusion of the experiment shows that carbon dioxide must be present for plants to photosynthesize.

What is Hydrogen Carbonate indicator used for?

Hydrogen carbonate is an indicator is used to measure carbon dioxide levels in aquatic systems or we may say Hydrogencarbonate indicator is used to show the amount or concentration present in the different solutions. Different levels of carbon dioxide concentration show different colours of indicator.


Concentration of carbon dioxide Indicator turns
Highest Yellow
Higher Orange
Atmospheric level Red
Low Magenta
Lowest Purple


How can Hydrogencarbonate indicator be used to investigate the effect of light intensity?

A leaf is placed in a sealed boiling tube that contain small quantity of indicator solution (hydrogen carbonate). Now the effect of light intensity can be investigated.

The table shows some typical results.


Test tube A Test tube B Test tube C Test tube D
Light turned on
Paper on tube Black paper Tissue paper None None
Leaf Living Living Living Dead (boiled)
Indicator color at the end yellow magenta purple Red
Carbon dioxide concentration Highest Low lowest Atmospheric level
Photosynthesis ✓ ✓


  • Tube D was a control. The results in tubes C and D show that the leaf has to be alive for the carbon dioxide concentration to change.
  • Results of Tubes A, B and C are showing the effect of increasing the light intensity/concentration.
  • The black paper didn’t allow light reaching the leaf in tube A, so it shows that only respiration possibly will occur.
  • The tissue paper clogged some of the light reaching the leaf in tube B, but the leaf in tube C received the most of the sunlight.
  • Photosynthesis and respiration simultaneously in tubes B and C, so a net amount of carbon dioxide absorbed.
  • The rate of photosynthesis was the greatest in the leaf in tube C, and it had the greatest net absorption of carbon dioxide.

Investigating about the production of oxygen gas:

If the number of oxygen gas bubbles or volume of oxygen increased by increasing the light intensity then it means that the rate of photosynthesis has been increased.

It is suggested that measuring the volume of oxygen produced is more accurate than counting the bubbles of oxygen produced.

An oxygen electrode connected to a data logger can be used to measure the change in oxygen concentration.


  1. Take a stem of an aquatic plant and remove some leaves from the lower part.
  2. Cut a part of the stem at an angle and slightly crush the cut.
  3. In a test tube put a small amount of Sodium Bicarbonate.
  4. Place the plant into the test tube, from down to up direction.
  5. Now fill the test tube with distilled water so that the stem is completely submerged.
  6. Observe the bubbles evolved and count them in a fixed intervals of time as the set up  open to the source of sunlight.
  7. Alternatively, the gas can be collected in micro syringe and then its amount can be measured eventually.
  8. By keeping the light source at different distances the rate of photosynthesis can also be investigated.


  1. Small gas bubbles are released from the stem. The rate/speed of evolving bubble shows the rate of photosynthesis.
  2. When the setup is placed near to a light source, the rate of bubble production  will speed up, and as the plant is taken further away, the bubbles will slow down again.