Photosynthesis Lab (online lab) (2024)

Photosynthesis (online lab activity)

Copyright © 2021 by Jeff Carmichael, Ph.D.

University of North Dakota

Name:

Learning Objectives:

After completing this lab you should be able to:

1. Calculate Rf values in order to identify unknown photosynthetic pigments
2. Explain the role of various light wavelengths in driving photosynthesis
3. Test whether or not plants absorb carbon dioxide during photosynthesis
4. Test the role of light during photosynthesis
5. Test the role of chlorophyll in producing starch in plants

Photosynthesis involves a series of chemical reactions that produces sugars from carbon dioxide, water, and light energy. Among other things, this process requires photosynthetic pigments (e.g., chlorophyll, carotenes, xanthophylls) capable of absorbing light energy and using that energy to produce chemical energy (sugars). Why is this important? Most plants are autotrophic- they synthesize their own food. Heterotrophs, organisms that cannot produce their own food, rely on plants (either directly or indirectly) to provide them with the food they need for energy, growth, and development. Plants also provide organisms with the oxygen needed for aerobic respiration. Although the complete series of reactions involved in photosynthesis is quite complex, the overall process can be summarized in the following chemical equation:

6 CO₂ + 12 H₂O + Light C₆H₁₂O₆ (sugar) + 6 H₂O + 6 O₂

With light and chlorophyll present, plants are able to take in carbon dioxide and water and convert them to sugar, which is used immediately for growth or metabolism, converted to other organic compounds, or stored as starch. The water and oxygen are then released as byproducts.

In today’s lab you will identify the pigments involved in photosynthesis using paper chromatography. In addition, you will observe the roles of light and carbon dioxide during the formation of sugars and starch.

Part 1- Photosynthetic Pigments

Plants have a variety of pigments that are involved in photosynthesis. These pigments, including chlorophyll a and accessory pigments such as chlorophyll b, carotene and xanthophyll, absorb light and use that energy for carbon fixation (converting carbon dioxide to an organic compound). The accessory pigments ultimately transmit the light energy they absorb to chlorophyll a (hence their designation as accessory pigments).

We can easily separate these pigments by using a technique called paper chromatography. Pigment extract is applied to one end of a piece of paper; that end is put into a solvent (just below the spot). The solvent moves up the paper, taking the pigment particles with it. The more soluble the pigment particles are, the further they travel, along with the solvent, up the paper.

We can identify each pigment by determining its R_f (ratio of fronts) value. The R_f value describes the relationship between the distance moved by the pigment and the distance moved by

the solvent and is calculated as follows:

R_f = Distance moved by pigment

Distance moved by solvent

Procedures for chromatography were carried out as follows:

1. Cut a piece of chromatography paper about eight inches long.
2. Make a mark on the paper about one inch from the end using a pencil (not a pen or marker!).
3. Lay a spinach leaf over the pencil mark and using the end of a blunt probe, gently press down and twist a few times. Repeat this 3 or 4 times in the same spot to get a nice concentrated pigment spot.

Working under the hood, carefully pour about 2 ml of chromatography solvent into a large jar (these will likely already be filled and ready for use). This is a powerful solvent, so use in a well ventilated area!!! The solvent is EXTREMELY flammable and can dissolve many plastics, including some CONTACT LENSES! Be sure to wear safety glasses. Place your chromatography paper into the jar with the pigment located near the bottom. Be sure to allow the tip of your paper to touch the solvent, but don’t cover the pigment with the solvent. Complete this under the hood and place the lid back on as soon as possible.

You may take the jar to your bench, but be sure to keep the lid on. Watch the edge of the solvent move up the paper; this is the solvent front. Remove the paper when the solvent front is about 2 cm from the top of the paper. Immediately mark where the solvent front is with a pencil and then allow the paper to dry.

A sample chromatogram is shown below. Note the following: The spinach extract was applied at region “A”. Pigments were carried up the paper along with the solvent. Pigment “F” and the solvent traveled essentially the same distance up the paper (10 cm). Although the pigments appear dim, the maximum distance traveled by each is indicated by the letters “B” through “F”.

1. ? Explain, in your own words, why the spinach pigments separated at different levels.

Calculate the approximate R_f values for each of the pigments and record them in the table below. Recall that the distance between “A” and “F” is about 10 cm. Use these values to identify each of the pigments based on the known Rf values provided below.

Known pigment Rf values:

• Chlorophyll a- 0.30
• Chlorophyll b- 0.40
• Xanthophyll 1- 0.50
• Xanthophyll 2- 0.70
• Carotene- 0.99

1. ? Which is more soluble in the chromatography solvent, xanthophylls or chlorophyll a?

1. ? Why do spinach leaves appear green if yellow and orange pigments were present?

1. ? Based on your observations during this experiment, what is the relationship between accessory pigments and the characteristic colors of autumn leaves?

Part 2- Colors of Light Absorbed by Chlorophyll

Light can be viewed through a spectroscope. A spectroscope can separate white light into its individual colors (much like a prism). When viewed through a spectroscope, white light is separated into the full spectrum of colors (red, orange, yellow, green, blue, violet). However, if a sample of chlorophyll (which is green) is placed in front of the light, certain colors are absorbed (and disappear from view).

Look through the spectroscope (while pointing it toward white light) and observe the light spectrum from violet to red. A view through a spectroscope pointed at white fluorescent light is shown below (note the diffracted color patterns toward each side). Now imagine placing a sample of plant extract in front of the small slit at the end of the spectroscope.

1. ? Given that chlorophyll is green, which colors of light are absorbed by chlorophyll (and therefore would disappear when chloroplast extract was placed in front of the spectroscope)? Based on your observations, which color of light probably contributes the least to plant growth?

Part 3- Uptake of Carbon Dioxide During Photosynthesis

Recall that plants require CO₂ as part of the light-independent reactions (i.e., the Calvin Cycle). To detect the uptake of CO₂ by a plant, we can use a pH indicator. An indicator is a solution that changes color depending on pH. For this activity we used phenol red (phenol sulfonphthalein). Phenol red turns yellow in solutions below a pH of 8.2 and pink in solutions above 8.2.

By breathing into a tube of phenol red, CO₂ can be used to reduce the pH and demonstrate the color change from pink to yellow. This results in the following chemical reaction:

By adding a plant to the solution, it can “fix” the carbon dioxide, which will cause the pH to rise (become more alkaline). The solution should then turn back to pink.

Procedures for demonstrating carbon fixation were carried out as follow:

Two large test tubes were filled halfway with a solution of phenol red. Using a straw, we gently exhaled CO₂ into both of the test tubes, being careful to avoid splashing. We stopped blowing as soon as the solution turned yellow; otherwise, the experiment would have taken longer because of the additional carbonic acid formed.

A piece of the aquatic plant Elodea was added to one of the test tubes.

Both test tubes were placed in front of a bright light bulb for 30 minutes. Results are shown below.

1. ? Did the color in either test tube change? If so, explain.

1. ? What is meant by carbon fixation? What important role does Ribulose Bisphosphate Carboxylase (“RuBisCO”) play during this process?

Part 4- Oxygen Production

During this part of today’s lab we’ll examine the production of oxygen during the light reactions of photosynthesis.

The procedure for examining oxygen production was carried out as follows:

1. Obtain two each of the following: biochamber, oxygen probe, labquest module, and Elodea.
2. Fill the bottom of both biochambers with 3 or 4 sprigs of Elodea.
3. Then, fill the biochambers half-full with water.
4. Gently swirl.
5. Use the oxygen probe to and labquest module to measure oxygen levels at time zero.
6. Light Treatment- Place one of the biochambers with Elodea in front of a bright light.
7. Dark Treatment- Place the other biochamber with Elodea in the dark.
8. Continue to take oxygen readings in both biochambers every five minutes over a 30-minute period and record your results below.

Results:

1. ? Was there any evidence of oxygen production? Describe the results above and explain the changes in oxygen levels in the light and dark treatments.

1. ? List three factors that might influence the rate of oxygen production in plants out in nature and explain how you think each factor would affect the rate of oxygen production.

Part 5- Starch Production

Photosynthetic membranes called thylakoids found in plants and algae are the sight of photosynthesis during the light-dependent reactions. The thylakoid membranes are located within chloroplasts. Thylakoids are stacked in columns called grana. The grana, in turn, are connected by lamellae. A fluid called the stroma surrounds the thylakoid membrane and provides the enzymes that catalyze the light-independent reactions (this is where RuBisCO is found).

When sugars are produced during photosynthesis, they are often stored as starch. Light energy is needed to split water molecules, providing the hydrogen ions and electrons needed for the rest of the light reactions to take place. Chlorophyll is also required for photosynthesis to occur since this is where light energy is captured during the light- dependent reactions. However, it is during the light-independent reactions that the hydrogen ions and electrons produced during the light reactions are used to convert 5-carbon compounds into glucose and ultimately starch.

The following exercise will demonstrate the need for light and chlorophyll by detecting starch using iodine. Recall that a dark purple color is produced when iodine reacts with starch.

Leaves were obtained from the following plants:

1. Geranium kept under bright light for three days.
2. Geranium kept in the dark for three days.
3. Variegated Coleus (with green and yellow patches on the leaves).
4. Red leaf Coleus.

See image below for initial appearance of leaves.

The leaves were then processed for starch visualization as follows:

Leaves were immersed in boiling water for about one minute. They were then placed in boiling ethanol for 3 minutes.

Leaves were then removed from the ethanol and placed in a petri dish. Iodine was added to just cover the leaves.

1. ? Make predictions for the appearance of each of the four leaves after iodine treatment.

1. Geranium light-
2. Geranium dark-
3. Variegated Coleus-
4. Red leaf Coleus-

Results of iodine treatment are shown below.

1. ? What was the purpose of boiling leaves in water and ethanol?

1. ? Was there starch present in the leaf kept in the light? How about in the dark? Explain these results.

1. ? What was the pattern of starch deposition in the variegated leaves? Explain these results.

1. What was the pattern of starch deposition in the red leaves? Explain these results.

End of Lab