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Photosynthesis Workbook – Background/Instructions

In this exercise you will investigate several aspects of light, pigments, and

photosynthesis. You will separate pigments from a plant leaf and observe the

absorption of CO2 during photosynthesis. Finally, you will measure the photosynthetic

output of a plant under different wavelengths of light.

Activity 1: Observation of the visible light spectrum

Light comes from the sun as white light which contains all the colors

of the visible spectrum

When white light strikes an object, such as a leaf, some of the light photons are

absorbed by the pigments in the leaf and some are reflected. The color that we

perceive an object to be is due to the light that is reflected back to our eyes.

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The banana absorbs all of the colors except yellow, which is reflected back

to our eyes.

Separation of plant pigments by paper chromatography

Paper chromatography is a technique used to separate and examine individual

pigments from the mixture of pigments found in plant leaves. There are several

differently colored pigments in plant leaves. Green is usually the overriding color

because of two pigments: chlorophyll a and chlorophyll b. In fact, the two chlorophylls

are slightly different shades of green, as you will see at the end of this activity. Other

pigments present in the leaf, such as xanthophylls and beta- carotene, are yellow and

orange. All of these pigments are hydrophobic (water-hating) and are only soluble in

nonpolar solvents such as ether and acetone. Each pigment has a different molecular

ANSWER QUESTIONS 1-3 BEFORE PROCEEDING

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structure resulting in a different degree of solubility for each pigment in any given

solvent.

The molecular structure of each pigments influences its solubility.

During paper chromatography, a mixture of pigments from a leaf is placed onto one

end of a piece of chromatography paper which is then placed into a small amount of

chromatography solvent as shown below. As the solvent is absorbed by the paper and

migrates up the paper, it carries the pigments up the paper as well. If a pigment is

more soluble in the solvent (more nonpolar pigment) it will move as quickly as the

solvent does. Less soluble pigments (less nonpolar pigments) move more slowly. Over

time, the different migration rates of the pigments cause them to separate into distinct

bands on the paper. Each of the bands represents one pigment that is normally found

in a plant leaf.

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Paper chromatography set-up. The pigment has been deposited on the

paper and placed in the solvent.

Paper chromatography virtual lab

To simulate this process, go to

http://www.phschool.com/science/biology_place/labbench/lab4/intro.html

Conduct the first exercise titled “4-I CHROMATOGRAPHY”, answer the quiz questions

and check your answers.

In this exercise you simulated depositing pigments from a spinach leaf onto

chromatography paper. We do it the same way in lab as they demonstrate, by rolling a

coin on top of a spinach leaf until the pigments are on the paper. We then set the

paper into a vial with the nonpolar solvent and allow it to travel up the paper. Below

is an example paper chromatography strip that resembles what you would have seen

in lab.

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Now, look at the example chromatography strip below. Record in Table 1 the

distance each pigment and the solvent front moved. Then calculate the Rf values

using the following formula.

%

When calculating the Rf value for each pigment remember that this is a characteristic

value for each pigment and is calculated by dividing the distance each pigment moved

from the pigment starting line by the distance the solvent moved from the starting

line. Your result should be recorded to two decimal places. Note that the distance

units cancel out and that the Rf value does not have a unit. It is a percentage.

ANSWER QUESTIONS 4-7 BEFORE PROCEEDING

COMPLETE TABLE 1 BEFORE PROCEEDING

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Carbon dioxide uptake and the Calvin Cycle

In this part of the lab you will be observing the activity of the light independent

reactions, also known as the Calvin cycle, using a piece of Elodea (an aquatic plant) and

several tubes of the pH indicator phenol red. Phenol Red is an acid/base indicator that

is red when neutral or basic and yellow when acidic (Figure 11).

During the Calvin cycle, a plant absorbs carbon dioxide from its environment and uses

the energy from ATP and NADPH produced during the light dependent reactions to

convert the inorganic carbon dioxide into glucose. If the Calvin cycle is functioning

properly, there should be a gradual decrease in the amount of carbon dioxide in the

environment around the Elodea.

When carbon dioxide is added to water, some of it forms carbonic acid and lowers the

pH of the water (makes it more acidic).

Addition of CO2 to water causes an acid to form.

If carbon dioxide is removed from the water, the pH of the water will increase

(becomes more basic). By using the pH indicator dye phenol red, we can determine if

the plant is absorbing carbon dioxide from the water by watching the color of the

indicator dye. When a solution of phenol red is above pH 7.2, the indicator will be

pink. If the pH is below 7.2, the indicator will be yellow.

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The appearance of phenol red under basic and acidic conditions.

We will set up three tubes containing phenol red mixed with tap water as shown below.

The pH of tap water is usually around 7.8, so the solution in each tube will be pink. If

we exhaled through a straw (like blowing bubbles) into two of the tubes, the carbon

dioxide concentration of exhaled air is about 100X higher than that of room air, so the

phenol red in those two tubes will quickly turn yellow. A piece of Elodea will then be

placed into one of the tubes with the yellow phenol red. The other tubes (one yellow

and one still pink) will be set up as controls. This set of tubes will be set up under bright

lights for 45 minutes. We will also set up a second set of three identical tubes and place

them in the dark under a foil lined box.

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Tubes number 1 and 2 have had carbon dioxide added to them through a

straw. The carbon dioxide has reacted with the water present to form

carbonic acid. This has caused the phenol red to turn yellow because the

solution is now acidic. Tube number 3 has not had any carbon dioxide

added and remains pink.

Consider the equation for photosynthesis; 6 CO2 + 6 H2O + light ———> C6H12O6 + 6

O2.

The plant Elodea has been added to tube number 2. If it is placed beneath a bright light

it will conduct photosynthesis.

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As you can see, after 45 minutes the solution in tube number 2 has

started to turn pink.

ANSWER QUESTIONS 8 & 9 BEFORE PROCEEDING

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Now imagine a second set of tubes that started out the same way but instead were placed under a box so that no light could reach them.

These tubes are set up as before. However, instead of

being exposed to light, they will be placed in a foil lined

box. This is the dark treatment.

ANSWER QUESTIONS 10 & 11 AND COMPLETE TABLE 2 BEFORE PROCEEDING

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The effect of light color on photosynthesis

During the following experiment, you will investigate the role of light in

photosynthesis. Specifically, you will be testing the effect of the light wavelength or

color on the photosynthetic output of Elodea (an aquatic plant) by measuring the

amount of oxygen produced under white light and green light treatments. Our

hypothesis for this experiment is that green light will not be as effective as white light

for driving photosynthesis.

Oxygen is produced during the light dependent reactions of photosynthesis by the

photolysis (splitting) of water molecules. In nature, the majority of the oxygen

produced by a photosynthetic organism would be released into the environment.

Watch the video linked below to see an example of this process. In the video the

investigator is measuring the effect of the intensity of light. In the video she refers to

Elodea as pondweed and sodium bicarbonate as sodium hydrogen carbonate. We will

investigate the role of green vs white light but the set up she uses is similar to what we

would do.

https://www.youtube.com/watch?v=id0aO_OdFwA&feature=youtu.be

In the video we observed Elodea conducting photosynthesis and releasing oxygen

bubbles as a byproduct. As mentioned in the video, counting bubbles is not the most

precise way to measure the photosynthetic output. For one, we are assuming all of the

bubbles are the same size (same amount of oxygen) and for another we are discounting

all of the smaller bubbles released by the leaves. A more exact way is to trap all of the

FORMULATE A HYPOTHESIS (QUESTION 12) BEFORE PROCEEDING

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oxygen being released and measure the volume of gas released.

In our experiment, we place a piece of Elodea into a device called a volumeter (pictured

below) that allows us to capture and measure all of the oxygen produced by the plant.

The volumeter consists of a large glass tube (the volumeter tube) capped with a rubber

stopper. The rubber stopper is fitted with a syringe and a plastic 1ml pipet (the pipet

sidearm) that has been bent to a 90 degree angle. The volumeter tube will be filled with

a dilute solution of sodium bicarbonate (baking soda and deionized water) and a piece

of Elodea will be placed into the tube. When the tube is capped with the rubber

stopper, a small volume of air will be trapped in the top of the volumeter tube. When

the Elodea is exposed to light and begins to photosynthesize, it will release oxygen gas

into the liquid in the volumeter. Since oxygen is not very soluble in water, it will form

bubbles that will rise to the top and add to the air trapped in the volumeter.

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This set up gives the Elodea everything it needs to conduct photosynthesis; light from

the lamp, water, and carbon dioxide from the sodium bicarbonate (NaHCO3). The

sodium bicarbonate ensures that CO2 is not a limiting resource. We have added a

beaker of water between the lamp and the plant as a heat sink.

As the plant conducts photosynthesis oxygen will be released and this will increase the

volume (and pressure) of the trapped air in the volumeter tube, which will push the

water level (sodium bicarbonate solution) in the volumeter tube down (away from the

plant as pictured below). The base of the bent pipet sticks down into the liquid of the

volumeter and is open at its tip, so the liquid will move up into the pipet and out

towards the tip of the pipet side arm. The pipet side arm is marked in 0.01mL

increments, so if we note the position of the water level in the pipet at the start and end

of each light treatment interval, we can determine the exact volume of oxygen produced

by the plant during each treatment.

Heat is one factor that must be controlled when using a volumeter. The 150 watt flood

lamps that we would use as our light source for this experiment emits a considerable

amount of heat. If the air space within the volumeter is heated, it will expand and cause

the water level in the pipet to move. This would give us a falsely high value for oxygen

production. To prevent this, we will use a 2000mL beaker of cold water set between the

lamp and the volumeter to absorb as much of the heat from the lamp as possible. The

water in the beaker will also give us a convenient way to change the color of the light by

adding green food coloring to the water. Remember, in our investigation we are

comparing photosynthetic rates of Elodea receiving white light vs green light.

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A second complicating factor in this experiment is oxygen consumption by cellular

respiration. Cellular respiration is the process by which plants and other organisms

consume glucose and oxygen to fuel their metabolism. It is carried out in the

mitochondria on a continuous basis as long as the plant is alive. This means that our

measurement of oxygen production by the plant is actually an underestimate, since

some of the oxygen that is produced by photosynthesis in the chloroplasts is almost

immediately consumed by respiration in the mitochondria. Since this oxygen is never

released from the plant, it can’t be measured.

The oxygen production that we do measure using the volumeter is only that amount of

oxygen that is in excess of what the plant used for its respiration. This is referred to as

the plant’s net oxygen production. If we can determine the amount of oxygen

consumed by the plant during respiration, we can add this to the net oxygen

production and determine the gross (total) oxygen production of the plant. A

good analogy for net and gross oxygen production is your paycheck. Your gross pay is

the total amount that you earned and your net pay is what you have left over after you

pay your taxes. The oxygen that the plant uses during respiration can be thought of as

the “taxes” that is has to pay for living. To continue the analogy, if someone knows

your net pay and how much you pay in taxes, they can calculate your gross pay by

adding the two together.

Measuring oxygen consumption by the plant will be relatively simple using the

volumeter. As outlined above, respiration is being carried out all the time, but

photosynthesis only happens in the light. If we block light from the volumeter by

wrapping it in aluminum foil, only oxygen consumption by respiration should be

happening. If no oxygen is available from photosynthesis, the Elodea will have to

consume oxygen from the water. Oxygen will then diffuse from the air space into the

water. This will reduce the volume (and pressure) of the air space in the volumeter

causing the water level in the volumeter tube to rise and the liquid in the pipet sidearm

to move back away from the tip.

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To summarize this experiment, each group would set up a volumeter to measure net

oxygen production by Elodea under white light, then measure oxygen consumption

under dark conditions, followed by net oxygen production under green light. The net

oxygen production and respiration data will be used to calculate gross oxygen

production by Elodea under both white and green light.

Activity 4: Setting up the volumeter

A piece of Elodea and would be placed into the volumeter tube and the tube filled with

sodium bicarbonate solution. The volumeter would be capped with the plug/

pipet/syringe assembly. The base of the pipet must extend down into the liquid in the

volumeter tube as shown in Figure 4.

The 2000ml beaker would be filled with cold tap water and set up using Fig. 4 as a

guide. The volumeter tube should go to one side of the beaker and the light should be

placed on the other side of the beaker.

During the next three parts of the exercise, you would have subjected the plant to white

light, darkness, and green light. During each of these parts, you would allow a 5

minute stabilization period for the plant to adjust to the new conditions (no data is

collected). This would be followed by a 10 minute experimental period (you will

collect data here). The stabilization period allows for the plant to adjust to each set of

conditions so that when we measure during the experimental portion we are getting

accurate results.

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Activity 5: Net oxygen production under white light

Once your volumeter was set up properly, we would look closely at the pipet that

makes the sidearm of the volumeter (Fig. 5). The pipet is a 1ml pipet and is labeled in

0.1mL increments. The small increments are 0.01mL.

The starting liquid level was 0.5 mL. You would not use this level in your calculations.

It is only for reference so that you know when the liquid level starts to move.

A 5 minute stabilization period would be timed. This is when we would monitor the

liquid level in the pipet. Oxygen production will cause the liquid level to move

outward toward the tip of the sidearm (shown in Figure 4, above). This is an

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indication that photosynthesis had started.

After 5 minutes, compare the liquid level to that recorded in step 2. You note that

the water has moved to 0.13mL, record this new liquid level in Table 3 as “starting

liquid level”, reset the stopwatch, and begin the 10 minute experimental period.

After the 10 minute experimental period you note that the water has moved to 0.29

mL. Record this liquid level in Table 3 as the “ending liquid level”. The net oxygen

production is the absolute value of the difference between the starting and ending

levels. This should not be a negative number. If you get a negative number, ignore the

sign as it will cause an error in your later calculations if included.

Activity 6: O2 consumption under dark conditions

COMPLETE TABLE 3 BEFORE PROCEEDING

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Next we would turn the light off and wrap the tube of the volumeter in foil. The water

level is now at 0.4mL. You would not use this level in your calculations. It is only for

reference so that you know when the liquid level starts to move.

Start the stopwatch and begin the 5 minute stabilization period for the respiration

measurement. You should monitor the liquid level in the sidearm during this time.

After 5 minutes, you will compare the liquid level to that recorded in step 2 and note

the water has moved down to 0.39mL. Record the new water level in Table 4 as

“starting liquid level”, reset the stopwatch, and begin the 10 minute experimental

period.

After the 10 minute experimental period, you note the new level is 0.35mL. Record

the liquid level in Table 4 as the “ending liquid level”. The oxygen consumption by

respiration is the absolute value of the difference between the starting and ending

levels.

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Activity 7: Net oxygen production under green light

For the next step we empty the 2000ml beaker and replace the water with cool tap

water. We would then add several drops of green food coloring.

ANSWER QUESTION 13 AND COMPLETE TABLE 4 BEFORE PROCEEDING

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The foil would be removed so the plant was once again exposed to the light. The water

level in the pipet is now 0.1 mL. You would not use this level in your calculations. It is

only for reference so that you know when the liquid level starts to move.

Turn on the light, start the stopwatch, and begin the 5 minute stabilization period. You

should monitor the liquid level in the sidearm during this time. As in the white light

treatment, oxygen production should cause the liquid level to move outward toward

the tip of the sidearm.

After 5 minutes, you note the liquid level has moved forward to 0.18mL. Record the new

water level in Table 5 as

“starting liquid level”, reset the stopwatch, and begin the 10 minute experimental

period.

After the 10 minute experimental period you note the water has moved to 0.2mL.

Record the liquid level in Table 5 as the “ending liquid level”. The net oxygen

production is the absolute value of the difference between the starting and ending

levels.

Activity 8: Calculate gross (total) oxygen production

COMPLETE TABLE 5 BEFORE PROCEEDING

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You have calculated the rate of photosynthesis (O2 production) under white and green

light and the rate of respiration (net O2 consumption) in Tables 3-5. Now, transfer

these values to Table 6. Assume respiration is the same regardless of light color.

Calculate the gross oxygen production rate by adding the respiration rate to the net

oxygen production rate under white and green light. The gross oxygen production rates

should be greater than the net oxygen production rates.

Activity 9: Comparison of treatments and conclusions

COMPLETE TABLE 6 BEFORE PROCEEDING

COMPLETE THE REST OF THE QUESTIONS