Demonstration or Class practical

This procedure kills a leaf, disrupts the cell membranes and softens the cuticle and cell walls. This makes it possible to extract the chlorophyll with hot ethanol and also allows the iodine solution to penetrate the cells and react with any starch present.

Lesson organisation

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You can run this as a teacher demonstration, or with students carrying out the procedure in pairs.

Apparatus and Chemicals

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Health & Safety and Technical notes

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Ethanol (IDA), iodine solution and hot liquids require safety precautions to be taken. Wear eye protection.

1 Ethanol (IDA) – refer to CLEAPSS Hazcard 40A and student safety sheet 60 – is highly flammable (flash point 13 °C) and harmful (because of the presence of methanol). The risks in this procedure are reduced by using hot water from kettles or in water baths rather than heating with a Bunsen burner flame. Some protocols recommend propanol (Hazcard 84A) in place of ethanol, as it removes chlorophyll more effectively. However, it has the additional risk of eye damage, its flashpoint is very similar to that of ethanol (IDA), and it may be more expensive.

2 Kettles are a safer source of hot water than heating with a Bunsen burner because of the presence of flammable ethanol (IDA) in this procedure. Students are familiar with the hazards of using kettles. Consider how to limit the movement of students around the laboratory with kettles or beakers of near-boiling water. Electrically-heated and thermostatically-controlled hot water baths may be a safer alternative.

3 Iodine solution – refer to CLEAPSS Hazcard 54B and Recipe card 39. A 0.01M solution is suitable for starch testing. Make this by 10-fold dilution of 0.1M solution. Once made, the solution is a low hazard but may stain skin or clothing if spilled, and may irritate the eyes.

4 Save the waste ethanol as a source of chlorophyll for future work. Make sure it cannot be tipped over and is in a safe place so it is not a fire hazard.

5 If the teacher or technician snips the leaves from the plants to give to the students, the plants are more likely to survive to be used again. Variegated Pelargonium (pot geranium) are good subjects for this experiment as are Tradescantia and Impatiens (busy lizzie).

6 Ensure that the plants have been well-illuminated for 24-48 hours. In winter, it might be worth using a halogen lamp to ensure the illumination is adequate.

Ethical issues

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There are no ethical issues associated with this procedure.

Procedure

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SAFETY: Ensure the ethanol is kept away from naked flames. Students should wear eye protection when working with ethanol or iodine solution. Take care with hot liquids. Be aware that plant sap may irritate the skin.

Investigation
a Collect leaves from the plants to be tested.

b At your desk, pour some boiling water from a kettle into a large beaker.

c Using forceps, pick up one of your leaves and hold it in the hot water for about one minute.

d Using forceps, remove the leaf from the boiling water and note how it has changed.

e Drop the leaf into a boiling tube and push it to the bottom with a glass rod. Add some anti-bumping granules (optional). Label this tube with your initials if you will be placing it in a hot water bath.

f Put on your eye protection.

g Add enough ethanol to cover the leaf, and stand the boiling tube in your beaker of hot water, or in the hot water bath.

h Watch as the ethanol boils and the green colouring (chlorophyll) is removed from the leaf. This will take a few minutes.

i Replace the hot water with freshly-boiled water from the kettle after 5 minutes if there is still some green colour in the leaf.

j Using forceps, remove the leaf from the boiling tube and rinse the leaf in cold water.

k Put the leaf in a Petri dish on a white tile.

l Add iodine solution to the leaf from the dropper bottle. Make sure the leaf is completely covered with iodine.

m Watch for a few minutes to see if a blue-black colour develops in any part of the leaf. A blue-black colour with iodine solution indicates that starch is present.

n Wash your hands to remove any traces of plant sap, or the chemicals that you have used.

Teaching notes

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The familiar word equation for photosynthesis is:

carbon dioxide + water → oxygen + glucose

In the leaf, excess glucose is rapidly converted to starch, so we test leaves for starch to show that photosynthesis has happened, rather than testing for glucose.

We often indicate that light and chlorophyll are required for the process by adding them to the equation near the arrow.

You could demonstrate the effect of iodine solution on starch by adding a few drops of solution to a starch powder or starch suspension in a boiling tube. You can then discuss the fact that this colour change might be hard to see in a dark green leaf until the chlorophyll is removed.

Depending on student’s prior knowledge, you could discuss the structure of plant cells and explain that the starch is within the cells, the cells are surrounded by cell membranes and tough cellulose cell walls and that some leaves also have a protective waxy cuticle. The hot water treatment softens up the protective structures, and disrupts the cell membranes to let the chlorophyll out and the iodine solution in.

Having established the technique, students can apply it in a further practical – Identifying the conditions needed for photosynthesis.

Health & Safety checked, December 2008

Related experiments

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Identifying the conditions needed for photosynthesis
This protocol applies the technique of testing leaves for starch to plants that have been kept in controlled conditions in order to establish the conditions needed for photosynthesis to occur.

Investigating factors affecting the rate of photosynthesis
This procedure allows you to quantify the effect of some of the factors that affect the rate of photosynthesis by following the changing rate of photosynthesis in pond weed as conditions such as temperature, carbon dioxide concentration and light intensity change.

Investigating photosynthesis using immobilised algae
This procedure uses a more sophisticated method to follow the process of photosynthesis by directly measuring the changes in carbon dioxide levels caused by photosynthetic activity.