C. Metabolic Processes · Metabolic Processes

Investigation: Detecting the Products of Photosynthesis

C2.3 C2.1 C3.2 A1.1 A1.4 A1.5 A1.6 A1.8 A1.10 A1.11 A1.13About 75 minutes

Learning goals

I can plan and carry out investigations that detect O₂ production, CO₂ uptake and starch formation in photosynthesis.
I can design proper controls for photosynthesis experiments.
I can record and display qualitative and quantitative observations in suitable formats.
I can interpret results, identify sources of error, and relate the products to the light reactions and the Calvin cycle.

Words to know

floating leaf disk assay
A method in which leaf disks with their air removed sink in bicarbonate solution and float again as O₂ from photosynthesis builds up in their air spaces.

ET₅₀
The time it takes for 50% (half) of the leaf disks to float; a shorter ET₅₀ means a faster rate of photosynthesis.

sodium bicarbonate (baking soda)
A safe compound that releases CO₂ (as bicarbonate ions) in water; used as a CO₂ source for aquatic plants and leaf disks.

iodine test
A test for starch: iodine solution turns from brown-yellow to blue-black when starch is present.

variegated leaf
A leaf with green and white (or pale) areas; only the green areas contain chlorophyll.

glowing splint test
A school-lab test for O₂: a glowing wooden splint relights in a high concentration of oxygen.

Proving what a leaf makes

If the equation 6CO₂ + 6H₂O + light → C₆H₁₂O₆ + 6O₂ is right, then a photosynthesizing plant should release O₂, take up CO₂ and make sugar (which plants often store as starch), and it should do these only in light and only in green (chlorophyll-containing) parts. Expectation C2.3 asks you to investigate the process of photosynthesis to identify its products, interpret qualitative observations and display them appropriately. This lesson shows you three classic investigations and how to analyse them.

Remember from Lessons 9, 11, 12 and 13

From Lessons 11 to 13: O₂ is released by photolysis in the light reactions; CO₂ is used by rubisco in the Calvin cycle; G3P is made into glucose and then starch for storage. From Lesson 9: BTB is yellow in acid (high CO₂) and blue in base (low CO₂). From Lesson 12: the floating leaf disk method.

Investigation 1: CO₂ uptake (BTB and an aquatic plant)

Add a few drops of BTB to water; it is blue. Gently blow through a straw into it until it turns yellow-green or yellow (your exhaled CO₂ makes it acidic).
Put a sprig of an aquatic plant (such as Elodea) into tubes 1 and 2; leave tubes 3 and 4 without a plant. Stopper all four.
Put tubes 1 and 3 in bright light; wrap tubes 2 and 4 in foil (dark).
Observe after 30 to 60 minutes and after 24 hours.

Expected results: tube 1 turns blue: the plant removed CO₂ by photosynthesis. Tube 2 stays yellow (and may become more yellow over time, because the plant is still respiring but not photosynthesizing). Tubes 3 and 4 stay yellow: without a plant, nothing changes. Tubes 3 and 4 show that light alone doesn't change BTB; tube 2 shows that the plant needs light to remove CO₂.

The BTB tube experiment. Only the plant in light (tube 1) removes enough CO₂ to turn the BTB blue. Tubes 2 to 4 are controls. (The tubes are drawn in their final colours.) (*Tube 2 may turn even more yellow as the plant respires in the dark.)

Investigation 2: O₂ production

Floating leaf disks (Lesson 12): disks sink in bicarbonate solution once the air is removed; in light, O₂ from photolysis fills their air spaces and they float. The ET₅₀ (time for half to float) is a measure of rate: shorter ET₅₀ means faster photosynthesis.

Bubble counting: a sprig of Elodea upside down in bicarbonate solution under a lamp releases a stream of bubbles from its cut stem. Counting bubbles per minute at different lamp distances shows how light intensity affects the rate.

Glowing splint (school only): gas collected from an aquatic plant in an inverted test tube over several hours relights a glowing splint, showing it is rich in O₂. This test uses fire and is done only with a teacher.

Sample floating-leaf-disk data. With the lamp 10 cm away, half the disks floated at 8 min (ET₅₀ = 8 min); at 30 cm, ET₅₀ = 16 min; in the dark, none floated.

Investigation 3: starch production (school lab)

Sugar made in the Calvin cycle is stored in leaves as starch, so the iodine test can show where photosynthesis happened.

De-starch a potted plant by keeping it in the dark for 48 hours.
Cover part of a leaf with a foil strip, or use a variegated leaf (green and white). Expose the plant to bright light for a day or two.
Remove the leaf. Dip it in boiling water for about 30 seconds (this kills the cells and softens the leaf).
Put it in a tube of ethanol standing in a hot water bath (never over a flame; ethanol is flammable) until the chlorophyll dissolves and the leaf is pale.
Rinse in warm water, spread on a dish, and add iodine solution.

Expected results: only the parts that were green and in light turn blue-black. The foil-covered part and the white parts of a variegated leaf stay brown-yellow. This shows that starch formation needs both light and chlorophyll.

Safety (A1.4): wear goggles; ethanol is flammable (hot water bath only, no open flames); iodine stains and is harmful if swallowed; handle boiling water with tongs.

The starch test in four steps. After de-starching, only the parts of a leaf that got light make starch, which turns blue-black with iodine; the foil-covered strip stays brown.

Displaying your results

Qualitative data (colour changes, whether disks floated, iodine colour) go in a table with a clear title, one row per set-up, columns for each time or observation, and precise colour words. A labelled diagram of the leaf after the iodine test is also an appropriate format.
Quantitative data (disks floating vs time; bubbles per minute vs distance) go on a line graph: independent variable on the x-axis, dependent on the y-axis, units, and a title. Use the graph to find ET₅₀.
Always include controls in the display, so the reader can compare.

Worked example

Let's analyse a leaf disk investigation

Use the graph data: lamp at 10 cm, ET₅₀ = 8 min; lamp at 30 cm, ET₅₀ = 16 min; dark, no disks floated after 20 min. (a) What are the independent and dependent variables? (b) Calculate a relative rate (1/ET₅₀) for each lamp distance. (c) Write a conclusion. (d) Suggest one source of error.

(a) Independent: light intensity (lamp distance). Dependent: time for disks to float (ET₅₀), a measure of O₂ production.
(b) Rate ∝ 1/ET₅₀: 10 cm: 1/8 = 0.125 min⁻¹; 30 cm: 1/16 = 0.0625 min⁻¹. The closer lamp gives twice the rate.
(c) Higher light intensity increased the rate of photosynthesis (O₂ released by the light reactions). With no light, no O₂ was produced, so the disks didn't float.
(d) A lamp close to the cup can warm the solution, which also affects the rate; place a container of water between the lamp and the cup as a heat shield, or use an LED lamp. Other errors: disks of different sizes or from different parts of the leaf; incomplete infiltration.

Rate at 10 cm (0.125 min⁻¹) was twice the rate at 30 cm (0.0625 min⁻¹): brighter light → faster O₂ production. Controlling temperature would make the test fairer.

Worked example

Let's interpret a variegated leaf result

A variegated leaf from a plant kept in light is tested with iodine. The green parts turn blue-black; the white edges stay brown-yellow. A second variegated leaf from a plant kept in the dark for 48 hours stays brown-yellow everywhere. What do the results show?

First leaf: starch only where there is chlorophyll, so chlorophyll is needed for photosynthesis.
Second leaf: no starch anywhere, even in green parts, so light is needed (and the de-starching worked: any starch at the start was used up).
The second leaf is an important control: without it, you couldn't be sure the starch in the first leaf was made during the experiment rather than before it.

Starch (a product of photosynthesis) forms only in parts with chlorophyll that receive light.

Just for fun

Joseph Priestley's 1771 experiment showed that a sprig of mint could "restore" air that a candle had burned out. Two and a half centuries later, students are still proving that plants are excellent roommates: quiet, tidy, and constantly freshening the air.

Hands-on activity

Home investigation: light intensity and the floating leaf disk assay

You need: fresh spinach leaves, a drinking straw, baking soda, dish soap, a 10 mL needle-free syringe, 3 clear cups, a desk lamp (LED if possible), a ruler, a timer, foil, a bowl of water.

Safety note: use a needle-free syringe; keep the lamp's cord and bulb away from water and don't let a hot bulb touch anything.

Question and hypothesis. Write a question about light intensity and the rate of photosynthesis, and a hypothesis ("If..., then..., because...").
Prepare bicarbonate solution (1/8 teaspoon baking soda in 300 mL water + 1 drop of dish soap) and infiltrate 30 leaf disks as in Lesson 12, so they all sink.
Put 10 disks in each cup with equal volumes of solution. Cup A: 10 cm from the lamp. Cup B: 30 cm from the lamp. Cup C: wrapped in foil (dark), same distance as A. If you use an incandescent bulb, put a bowl of water between the lamp and cup A as a heat shield.
Count floating disks every minute for 20 minutes.
Display: make a data table, then a line graph of disks floating vs time for all three cups. Find ET₅₀ for each.
Analyse: calculate 1/ET₅₀ for each cup, write a conclusion, and list two sources of error and improvements.

What to look for: cup A floats fastest, cup B more slowly, and cup C (dark) not at all, showing that O₂ production needs light and is faster in brighter light. Typical ET₅₀ values are a few minutes to about 20 minutes. If the dark cup's disks float, check that the foil fully blocks light. Errors: uneven disk size, temperature change, solution concentration, counting mistakes.

Your turn

In the BTB experiment, why does the tube with Elodea in the light turn from yellow to blue?

Why is a tube with no plant kept in the light?

What gas makes leaf disks float, and which stage of photosynthesis produces it?

Why is baking soda added to the leaf disk solution?

List the steps of the starch test on a leaf, with one safety precaution.

Why must a plant be de-starched before the starch test?

What is the best way to display data on the number of disks floating over time?

More practice: warm-up

What colour does iodine turn in the presence of starch?

What does ET₅₀ stand for?

Which test shows that a gas is O₂?

Why is the leaf dipped in hot ethanol in the starch test?

In the starch test, which parts of a variegated leaf turn blue-black?

More practice: on your own

In the BTB experiment, tube 2 (plant in the dark) turns a deeper yellow after 24 hours. Explain.

A student finds ET₅₀ values of 6, 7 and 20 minutes in three trials at the same lamp distance. How should she handle the data?

Suggest why leaf disks eventually float in a cup with bicarbonate in bright light, but not in a cup with plain water in bright light.

Explain why an incandescent lamp placed very close to the cup could make light intensity seem more important than it is.

Write a hypothesis and identify variables for an investigation of the effect of bicarbonate concentration on ET₅₀.

Challenge

Bubble counts from an Elodea sprig: lamp at 10 cm: 40 bubbles/min; 20 cm: 12/min; 40 cm: 3/min. Light intensity is roughly proportional to 1/d². Do the data fit a direct relationship between rate and light intensity? Explain.

Design an investigation using leaf disks and coloured filters to test whether green light is less effective than red or blue light. Identify a key variable that is hard to control and how you would deal with it.

A classmate concludes from the BTB experiment that "plants only photosynthesize in the light and only respire in the dark." Evaluate this conclusion.

Check your understanding

Quiz

In a BTB experiment, an aquatic plant in light turns yellow BTB blue. This shows that the plant:

In the floating leaf disk assay, disks float because of:

Which result would show that chlorophyll is needed to make starch?

Why should leaves be placed in hot ethanol in a water bath, not heated over a flame?

At a lamp distance of 10 cm, ET₅₀ = 5 min; at 20 cm, ET₅₀ = 15 min. The rate at 10 cm is about:

A cup of leaf disks is wrapped in foil as a control. What does it test?

For parents and teachers

Big idea: C2.3 requires a laboratory investigation of the products of photosynthesis (O₂, CO₂ uptake, starch), with careful controls and clear display of qualitative and quantitative observations (A1.1, A1.4 to A1.6, A1.8, A1.10, A1.11, A1.13).

How to help: The floating leaf disk investigation is safe and works well at home; you only need a needle-free syringe, baking soda and spinach. The starch test with boiling ethanol and iodine is for the school lab only. Ask your teen to explain what each control rules out.

Common difficulties:

Forgetting controls, or not explaining what each one shows.
Thinking plants don't respire in the light.
Confusing "faster ET₅₀" with a larger number; a smaller ET₅₀ means a faster rate.
Ignoring heat from the lamp as a confounding variable.