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Photosynthesis Experiments

These experiments feature at Leaving Certificate with great regularity at both higher and ordinary level. Ignore these experiments at your peril.

Testing A Leaf For Starch - The 'KISS' experiment: Kill, Intoxicate, Soften, Stain.


  1. Place the leaf in boiling water - kills the leaf stopping all biochemical activity and breaks open the cells making easier the removal of chlorophyll and the entry of iodine stain.
  2. Place the leaf in boiling alcohol - this removes the green chlorophyll from the leaf and so the colour of the iodine stain, when added to the leaf, can be clearly seen.
    The alcohol is not to be heated by a Bunsen flame. Heat the alcohol in a water bath of very hot water.
  3. Place the leaf into boiling water - alcohol made the leaf brittle and unreceptive to the iodine stain, the alcohol is replaced with water softening the leaf.
  4. Apply iodine solution to the leaf - to check for the presence or absence of starch.

    1. A blue-black colour indicates starch is present (iodine enters the spiral starch molecule changing its colour).
    2. A yellow-brown, the colour of the iodine solution, indicates that starch is absent.

The iodine stain is iodine dissolved in a solution of potassium iodide.


Evidence of Photosynthesis

The two major tests for photosynthesis in school laboratories are carried out to show the formation of starch and the production of oxygen gas. The formation of starch can only be truly demonstrated by using a plant, the leaves of which do not have starch. The de-starching of plant leaves is done to deprive the plant of light for at least 24 hours.

Controlled Experiments
A controlled experiment must be carried out to show that light, chlorophyll and carbon dioxide are needed for photosynthesis. The procedure known as the experiment includes the factor under test. The procedure, known as the control, differs from the experiment in that it does not include the factor being tested, e.g., no light. The results of the two procedures are then compared. If there is a difference then the effect of the factor under test can be determined.

To show that Light is necessary for Photosynthesis

  1. Place a plant in darkness for at least 24 hours - this de-starches the leaves.
    Test a leaf with iodine to verify that de-starching was successful.
  2. Cover part of a number of leaves with lightproof material.
    Covered part of the leaf is the control - no light.
    Using a number of leaves, repeat the experiment to verify the results.
  3. Place the plant into good light for six hours at room temperature (20°C).
    Good light and warm temperature: to give a high rate of photosynthesis.
    Six hours: There will be plenty of excess glucose for storage as starch.
  4. Test the leaves for starch using iodine.

Results:
Light: exposed parts »» blue-black with iodine »» starch present »» photosynthesis occurred.
No Light: covered parts »» yellow-brown with iodine »» starch absent »» photosynthesis did not take place.
Therefore, light is necessary as photosynthesis only occurs when it is present.


To show that Chlorophyll is necessary for Photosynthesis

  1. Place a plant with variegated leaves in darkness for at least 24 hours - this de-starches the leaves.
    Test a leaf with iodine to verify that de-starching was successful.
    Variegated: areas of different colour, green areas have chlorophyll, white areas without chlorophyll.
    The non-chlorophyll areas are the control.
    Suitable plants with variegated leaves: ivy, spider plant.
  2. Draw a 'map of a leaf' to record the green and non-green areas.
    Chlorophyll is removed when testing a leaf for starch turning the leaf completely white before staining.
  3. Place the plant into good light for six hours at room temperature (20°C).
    Good light and warm temperature: to give a high rate of photosynthesis.
    Six hours: There will be plenty of excess glucose for storage as starch.
  4. Test the leaves for starch using iodine.
  5. Compare the iodine stain pattern to the original 'green/non-green' pattern.

Results:
Chlorophyll: green parts »» blue-black with iodine »» starch present »» photosynthesis occurred.
No Chlorophyll: white parts »» yellow-brown with iodine »» starch absent »» photosynthesis did not take place.
Therefore, chlorophyll is necessary, as photosynthesis only occurs when it is present.


To show that Carbon Dioxide is necessary for Photosynthesis

  1. Place a plant in darkness for at least 24 hours - this de-starches the leaves.
    Test a leaf with iodine to verify that de-starching was successful.
  2. Cover some leaves with a clear plastic bag containing some water.
    Clear plastic so light will be available to each leaf.
    Carbon dioxide is available to these leaves - there is carbon dioxide in the air inside the bag.
  3. Cover other leaves with a clear plastic bag containing some sodium hydroxide solution.
    Sodium hydroxide absorbs carbon dioxide from the air.
    These leaves will be the controls - no carbon dioxide.
  4. Place the plant into good light for six hours at room temperature (20°C).
    Good light and warm temperature: to give a high rate of photosynthesis.
    Six hours: so there will be plenty of excess glucose for storage as starch.
  5. Test the leaves for starch using iodine.

Results:
Carbon dioxide present »» blue-black with iodine »» starch present»» photosynthesis occurred.
No carbon dioxide »» yellow-brown with iodine »» starch absent »» photosynthesis did not take place.
Therefore, carbon dioxide is necessary, as photosynthesis only occurs when it is present.


To show that Oxygen is produced by Photosynthesis

  1. Photosynthesis occurs in light but not in darkness.
    The procedure in light is the experiment - photosynthesis.
    The same procedure in darkness is the control - no photosynthesis - cover the apparatus with a lightproof material.
  2. Place a funnel over Elodea, pondweed, in a beaker of freshwater.
    The funnel is raised off the bottom on pieces of blue-tack to allow unhampered diffusion of CO2 to Elodea.
  3. Invert a test tube full of water over the stem of the funnel to collect any gas from the Elodea.
  4. Leave for 6 hours at room temperature (20°C).
  5. A colourless gas is collected in each. Test to identify the gases.

Results:
Photosynthesis »» the gas ignites a glowing splint »» the gas is oxygen.
No photosynthesis »» the gas turns limewater milky »» the gas is carbon dioxide (produced by respiration).
Therefore, oxygen is produced by photosynthesis.


Photosynthesis Experiments (B)

Three of these experiments are variations of an experiment done at Junior Certificate - the one that shows oxygen produced by photosynthesis. This procedure is carried out to check the effect of environmental factors on the rate of photosynthesis such as (1) light intensity; (2) temperatures; (3) carbon dioxide concentrations. So really, these first three experiments are not that difficult.

When checking the effect of each of the above factors it is important that the other two factors are both at a suitable value and are kept constant at that value.

Temperature: 25°C and kept constant by using a heated water bath monitored with a thermometer.
Light Intensity: use a lamp close to the plant at a fixed distance and monitored with a light meter.
Carbon Dioxide Concentration: a saturated CO2 solution by using excess sodium bicarbonate in the water, it dissolves moderately in water releasing carbon dioxide gas, sodium bicarbonate is known now as sodium hydrogen carbonate.

Remember to tell how the variable factor is varied.
Temperature: water baths at different temperatures.
Light Intensity: change the distance of the lamp from the plant.
Carbon Dioxide Concentration: different dilutions of a saturated solution.


To show the effect of varying the Light Intensity on the Rate of Photosynthesis

  1. Place a funnel over Elodea, pondweed, in a beaker of freshwater at 25°C.
    The funnel is raised off the bottom on pieces of blue-tack to allow unhampered diffusion of CO2 to Elodea.
  2. Invert a test tube full of water over the stem of the funnel to collect any gas from the Elodea.
  3. Place the beaker on a hot plate at 25°C to maintain and monitor the temperature of the water with a thermometer.
  4. Excess sodium bicarbonate is placed in the water to give a constant saturated solution of CO2.
  5. Place the lamp (the only light source) at predetermined distance from the plant.
  6. Use a light meter to measure the light intensity at this distance. Record the light intensity.
  7. Allow the plant five minutes to adjust to the new conditions.
  8. Count the number of oxygen bubbles given off by the plant in a five-minute period.
    Repeat the count twice more and calculate the average of the three readings.
    This is the rate of photosynthesis at that particular light intensity.
    The gas should be checked to prove that it is indeed oxygen - relights a glowing splint.
  9. Repeat at different light intensities by moving the lamp to different distances.
  10. Graph the results placing light intensity on the x-axis.

Extra: at compensation point no bubbles will emerge from the Elodea (photosynthesis = respiration).
at less than compensation point carbon dioxide bubbles will be given off (respiration > photosynthesis) and at greater than compensation point oxygen bubbles are given off (photosynthesis > respiration).


To show the effect of varying the Temperature on the Rate of Photosynthesis

  1. Place a funnel over Elodea, pondweed, in a beaker of freshwater at 25°C.
    The funnel is raised off the bottom on pieces of blue-tack to allow unhampered diffusion of CO2 to Elodea.
  2. Invert a test tube full of water over the stem of the funnel to collect any gas from the Elodea.
  3. Place the lamp (the only light source) at a fixed distance from the plant - check its constancy with a light meter.
  4. Excess sodium bicarbonate is placed in the water to give a constant saturated solution of CO2.
  5. The temperature is 20°C - room temperature; thermostatically controlled room-heating system.
  6. Allow the plant five minutes to adjust to the new conditions.
  7. Count the number of oxygen bubbles given off by the plant in a five-minute period.
    Repeat the count twice more and calculate the average of the three readings.
    This is the rate of photosynthesis at that particular temperature.
    The gas should be checked to prove that it is indeed oxygen - relights a glowing splint.
  8. Repeat at different temperatures: 0°C - surround the beaker with an ice jacket; greater than room temperature (25°C, 30°C, 35°C, 40°C, 45°C, etc.,) by using a hot plate.
  9. Graph the results placing temperature on the x-axis.


To show the effect of varying the Carbon Dioxide on the Rate of Photosynthesis

  1. Place a funnel over Elodea, pondweed, in a beaker of freshwater at 25°C.
    The funnel is raised off the bottom on pieces of blue-tack to allow unhampered diffusion of CO2 to Elodea.
  2. Invert a test tube full of water over the stem of the funnel to collect any gas from the Elodea.
  3. Place the lamp (the only light source) at a fixed distance from the plant - check its constancy with a light meter.
  4. The temperature is 20°C - room temperature; thermostatically controlled room-heating system.
  5. Excess sodium bicarbonate is placed in the water to give a constant saturated solution of CO2.
  6. Allow the plant five minutes to adjust to the new conditions.
  7. Count the number of oxygen bubbles given off by the plant in a five-minute period.
    Repeat the count twice more and calculate the average of the three readings.
    This is the rate of photosynthesis at that particular temperature.
    The gas should be checked to prove that it is indeed oxygen - relights a glowing splint.
  8. Repeat at different lower carbon dioxide concentrations by using different dilutions of a saturated solution.
  9. Graph the results placing carbon dioxide concentration on the x-axis.

Compensation Point

The compensation point of a plant is the light intensity at which the rate of photosynthesis is equal to the rate of respiration;
(i) Food made = food used, (ii) no change in dry weight, (iii) CO2 used = CO2 made, (iv) O2 made = O2 used.
(v) No gas exchange with external environment.

Bicarbonate Indicator is used for the compensation point experiment and gas exchange experiments.
Passing fresh air through the indicator makes its carbon dioxide concentration equal to the atmosphere - it is said to be 'equilibrated' and has a red-orange colour. The colour will change if the pH changes.
Loss or gain of CO2 changes the pH of the indicator because CO2 is reasonably soluble in water forming carbonic acid.
Therefore bicarbonate indicator responds to changes in its carbon dioxide concentration.
If equilibrated indicator gains CO2 it becomes more acidic turning yellow.
If equilibrated indicator loses CO2 it becomes more basic turning purple.

For an aquatic plant like Elodea (pondweed) in an equilibrated bicarbonate solution the following colours indicate
Red-orange: P = R - the light intensity is at compensation point, no loss or gain of CO2 by the plant.
Yellow: P < R - the light intensity is less than compensation point, release of CO2 from the plant into the indicator.
Purple: P > R - the light intensity is greater than compensation point, plant absorbs CO2 from the indicator.

To determine the Compensation Point of a plant

  1. Set up at different distances (light intensities) from a single constant light source a pair of clear glass airtight bottles as follows:
    Bottle 1: control - red-orange equilibrated bicarbonate indicator.
    Bottle 2: experiment - same volume of red-orange indicator with Elodea.
  2. All bottles have the same volume of equilibrated indicator.
  3. The samples of Elodea are from the same plant and same mass.
  4. Temperature to remain constant at room temperature, 20°C.
  5. Leave the set up for an hour.
  6. Control Results: the indictor colour has not changed, all red-orange.
  7. The plant at compensation point is still red-orange - unchanged indicator.
  8. Use a light meter to measure the light intensity at this distance and record it.
  9. This reading of light intensity is the compensation point.


To show Gas Exchange during Photosynthesis

  1. Set up at different distances from a single constant light source, a pair of clear glass airtight bottles as follows:
    Bottle 1: control - red-orange equilibrated bicarbonate indicator.
    Bottle 2: experiment - same volume of red-orange indicator with Elodea.
  2. All bottles have the same volume of equilibrated indicator.
  3. he samples of Elodea are from the same plant and same mass.
  4. Temperature to remain constant at room temperature, 20°C.
  5. Leave the set up for an hour.
  6. Control Results: the indictor colour has not changed, all red-orange.
  7. The plant at compensation point is still red-orange - unchanged indicator.
    P = R: no loss or gain of CO2 by the indicator.
  8. The plants closer to the light will have turned the indicator purple. The light intensity is greater than compensation point. P > R. Indicator lost carbon dioxide to the plant so the indicator went more basic.
  9. The plants further from the light will have turned the indicator yellow. Light intensity is less than compensation point. P < R. Indicator gained carbon dioxide from the plant so the indicator went more acidic.


The effect of the following on Equilibrated Bicarbonate Indicator.

  1. Fresh green leaf in bright light: goes purple because P > R. Light intensity above compensation point.
  2. Fresh green leaf in dim light: goes yellow because P < R. Light intensity below compensation point.
  3. Fresh green leaf in green light: goes yellow because P < R. Absorption of green light is very low.
  4. Fresh green leaf in darkness: goes yellow - no photosynthesis, respiration produces CO2.
  5. Small animals, e.g., woodlice: goes yellow - respiration produces CO2.
  6. Soda lime: goes purple as soda lime (sodium hydroxide) absorbs CO2 from the air.


To make a Chloroplast Extract

  1. Tear up fresh green leaves and place the small pieces into a mortar.
  2. Add a little sand - helps to break open the plant cells walls during the grinding.
  3. Cover the leaf pieces with acetone - the clear acetone dissolves the pigments.
  4. Grind the leaf pieces with a pestle.
  5. The acetone goes 'green' with the dissolved pigments.
  6. Decant the chloroplast extract from the fibrous residue into a beaker.

To separate the pigments of a Chloroplast Extract by simple Chromatography
  1. Place a drop of chloroplast extract about 2 cm from the bottom of a strip of chromatography paper.
    Dry it fast - stop it spreading.
  2. Repeat about 20 times at the same point to build up a concentrated spot of chloroplast extract.
  3. Suspend the strip in an airtight jar containing pigment solvent (petroleum ether + acetone) with the concentrated spot about 1 cm above the solvent surface.
  4. Observe the solvent rise up the paper by capillary action.
  5. The solvent dissolves the pigments and carries the up the paper.
  6. The different pigments are carried at different speeds and separate out.
  7. Remove the paper when the solvent front is close to the top.
  8. Dry the paper so the pigments stop moving.
  9. Observe the separated coloured bands.
    From the bottom to the top: green chlorophyll b, blue-green chlorophyll a, yellow xanthophyll, yellow carotene.

Extra:
Chlorophyll a is the primary pigment supplying the electrons to drive the light phase of photosynthesis.
Chlorophyll b, xanthophyll and carotene enhance photosynthesis by passing the light energy they absorb to chlorophyll a.
Xanthophyll and carotene also protect the chlorophyll from destruction by excessive light.


Photosynthesis Graphs and Limiting Factors

You are expected to know the shapes of the following graphs.

  1. Absorption Spectrum of a Chloroplast Extract and Chlorophyll. (Higher Only)
  2. The Effect of Varying the Light Intensity on the Rate of Photosynthesis.
  3. The Effect of Varying the Temperature on the Rate of Photosynthesis.
  4. The Effect of Varying the Carbon Dioxide Concentration on the Rate of Photosynthesis.
  5. The Difference Between Shade Adapted and Bright Light Adapted Plants.

Be Careful: explaining graphs is usually not done well by students in exams.

Absorption Spectrum of Chlorophyll
This is a graph that shows how much light is absorbed by chlorophyll at each wavelength of light.

graph1


  1. Chlorophyll a absorbs light very strongly in the violet, blue and red wavelengths. Chlorophyll a absorbs very little of the green, yellow and orange wavelengths are absorbed.
  2. Chlorophyll b absorbs light very strongly in the blue; reasonably well in the violet and orange wavelengths. Chlorophyll b absorbs very little of the green, yellow and red wavelengths.
  3. The chloroplast extract shows very strong absorption in the violet, blue and red; reasonably good in orange; very little absorption in the green and yellow.

The action spectrum is not mentioned in the syllabus but is in all the textbooks. This graph shows the amount of photosynthesis carried out at each wavelength of light. The absorption and action spectra of a chloroplast extract are closely parallel indicating that the wavelengths absorbed are the ones responsible for photosynthesis.

Chlorophyll appears green because it only weakly absorbs green and strongly reflects it; chlorophyll absorbs red and blue.
The three primary colours of light are red, green and blue. The colour of an object is the colour of the reflected light.
Green objects reflect only green; they absorb red and blue.
Red objects reflect red only; they absorb green and blue.
Yellow objects reflect red and green; they absorb blue.

The graphs on temperature, light intensity and carbon dioxide are really 'enzyme graphs'; light intensity and carbon dioxide are substrate concentration graphs - revise enzymes.


The Effect of Varying the Light Intensity on the Rate of Photosynthesis

graph2

  1. No light - no photosynthesis. The light phase does not take place.
  2. Increasing the light intensity to value A causes photosynthesis to increase. The more light the greater the light phase and the greater the production and supply of Hs and ATP to the Dark Phase.
    to light intensity A light is a limiting factor.
  3. At light intensity A the rate of photosynthesis reaches its maximum and levels off. Some factor other than light intensity is limiting the rate of photosynthesis: it may be low temperature, low carbon dioxide, low chlorophyll content or the enzyme system is deficient (enzymes at maximum turnover number).
    Light intensity A is known as the 'saturation point' - the value beyond which light intensity is not a limiting factor.
  4. The rate of photosynthesis remains constant at maximum beyond light intensity A. The Increase in light intensity has no effect on the new limiting factor so photosynthesis stay the same.

Limiting Factor
This is the independent agent that is restricting an increase in the process because it is the factor in shortest supply.
An increase in this agent will result in an increase in the rate of the process.

During daylight CO2 concentration is usually the limiting factor for photosynthesis.

The Effect of Varying the Temperature on the Rate of Photosynthesis


graph3

  1. At 0°C the rate of photosynthesis is low. Enzyme activity is low. Photosynthesis is an enzyme-controlled process.
  2. Increasing the temperature to 30°C increases the rate of photosynthesis. Enzyme activity increases.
  3. Maximum photosynthesis at 30°C. Enzyme activity at it maximum - maximum collision frequency between native enzymes and substrates.
  4. Photosynthesis declines beyond 30°C. Enzyme activity slowing due to denaturing of enzymes.
  5. No photosynthesis at 50°C. No enzyme activity - enzymes are denatured.

The Effect of Varying the Carbon Dioxide Concentration on the Rate of Photosynthesis.

graph4

  1. No carbon dioxide - no photosynthesis. Dark phase does not take place.
  2. Increasing the carbon dioxide concentration to value A causes photosynthesis to increase. The greater the supply of CO2, the faster the rate of enzyme activity - increasing collision frequency between enzymes and substrates.
    Up to CO2 concentration A carbon dioxide is a limiting factor.
  3. At CO2concentration A the rate of photosynthesis reaches its maximum and levels off. Some factor other than CO2 is limiting the rate of photosynthesis: it may be low temperature, low light intensity, low chlorophyll content or the enzyme system is deficient (enzymes at maximum turnover number).
    CO2 concentration A is known as the 'saturation point' - the value beyond which CO2 is not a limiting factor.
  4. The rate of photosynthesis remains constant at maximum beyond CO2A. Increase in CO2 has no effect on the new limiting factor so photosynthesis stays the same.


The Difference Between Shade Adapted and Bright Light Adapted Plants

graph5

Plant A is the shade adapted plant.


  1. It has a lower compensation point - light intensity X.
  2. It reaches it maximum rate of photosynthesis at a lower light intensity, i.e., its light saturation point is lower.
  3. Its rate of photosynthesis declines at high light - intensities not causing a decline in the photosynthesis of B.

Remember: compensation point is the light intensity at which the rate of photosynthesis is equal to the rate of respiration.
Photosynthesis and respiration are equal where their graph lines intersect. Drop a perpendicular line from the intersection point to the light intensity x-axis to find the compensation point.


Extra Graphs on Limiting Factors

graph6

  1. Up to A1, A2 and A3 the concentrations of CO2 is the limiting factor for the respective light intensities.
  2. Low Light Intensity: beyond A1 light intensity is the limiting factor because increasing the light intensity (medium value) increases the rate of photosynthesis.
  3. Medium Light Intensity: beyond A2 light intensity is the limiting factor because increasing the light intensity (high value) increases the rate of photosynthesis.
  4. High Light Intensity: beyond A3 the limiting factor could be light intensity, chlorophyll content, temperature or the enzyme system. It cannot be CO2 concentration because increase in CO2 concentration does not lead to an increase in photosynthesis.

graph7


  1. Up to A1, A2 and A3 the light intensity is the limiting factor for the respective CO2 concentrations.
  2. Low CO2 concentration: beyond A1 CO2 concentration is the limiting factor because increasing the CO2 concentration (medium value) increases the rate of photosynthesis.
  3. Medium CO2 concentration: beyond A2 CO2 concentration is the limiting factor because increasing the CO2 concentration (high value) increases the rate of photosynthesis.
  4. High CO2 concentration: beyond A3 the limiting factor could be CO2 concentration, chlorophyll content, temperature or the enzyme system. It cannot be light intensity because increase in light intensity does not increase photosynthesis.


Links

Experiments to show light and carbon dioxide in photosynthesis - it also shows some diagrams and pictures of the effects of these experiments on leaves.
http://web.ukonline.co.uk/webwise/spinneret/plants/psfac2.htm

This is the same idea as the last and from the same site - this time showing chlorophyll.
http://web.ukonline.co.uk/webwise/spinneret/plants/psfac1.htm

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