Photosynthesis

Photosynthesis in Elodea

CO₂ and pH

Before beginning this exercise, it is necessary to understand that photosynthesis uses light energy to synthesize carbohydrate from carbon dioxide. The equation is below.

6CO₂ + 6H₂O + Energy ® C₆H₁₂O₆ + 6O₂

This process requires light for some of the reactions.

It is also necessary to understand that the plant is constantly undergoing cellular respiration according to the equation below.

C₆H₁₂O₆ + 6O₂ ® 6CO₂ + 6H₂O + Energy

Notice that these two equations appear to be opposites. 

When plants are exposed to light, photosynthesis and cellular respiration both occur. In the dark, only cellular respiration occurs. 

We will study photosynthesis in an aquatic plant (Elodea) We can measure the rate of photosynthesis and cellular respiration by measuring the amount of CO₂ given off or taken up by the plant. 

Carbon dioxide combines with water to form carbonic acid (H₂CO₃) which dissociates into hydrogen ions (H⁺) and bicarbonate ions (HCO₃^-). The pH drops due to the presence of hydrogen ions.

          CO₂ + H₂O  H₂CO₃ H⁺ + HCO₃^- 

Respiring plants release CO₂ into the water, causing the pH to decline. During photosynthesis, plants take up CO₂ and the pH increases.

Using the pH Probe

Set up the pH probe by following the instructions given in the link below.

Instructions for setting up and using the pH probe.

Procedure for Measuring Photosynthesis

After the probe is set up (see the step above), obtain two large test tubes for this experiment. A beaker can be used to hold the tubes as shown in the photograph below.

Rinse both tubes and stoppers thoroughly with tap water to remove any traces of contaminants that might affect pH. A final thorough rinse can be obtained by putting some tap water in each tube, pushing the stoppers onto the top, and shaking the tubes vigorously. Discard the rinse water.

Wrap a piece of aluminum foil around one of the tubes so that no light is able to enter the tube.

Fill each tube with tap water to 3 cm from the top and then measure the pH of the water and record these values in Table 1 in the answer sheet.

Cut a two sprigs of Elodea that are long enough to fill the entire length of the tubes but not protrude from the water. The length of the stem can be adjusted, if necessary, by cutting a piece from the base of the stem. Put one of the plants in each of the tubes.

Cap both tubes with a piece of aluminum foil. The dark tube should be entirely covered so that no light can enter the tube.

Place the tubes in the test tube rack or beaker to hold them. 

Fill a two-liter beaker to near the top with water and place it between the lamp and the two test tubes. The water will serve as a heat sink, preventing the tubes with plants from getting too hot. Adjust the placement of the tubes so that the uncovered tube will receive as much light as possible.

Click on the photograph to view an enlargement.

Turn on the lamp and begin timing the experiment.

Before measuring the pH, mix the tube by inverting it several times, using a stopper to prevent water from spilling out. It is important to rinse the pH probe thoroughly before transferring it to the test tube containing the plant. Measure the pH of the water in each tube every 10 minutes for one hour. After mixing, insert the pH probe and wait until the reading stabilizes before recording your results. Record the pH to the nearest 0.01 units in the space provided on the answer sheet..

You should begin the chromatography procedure (below) while waiting to take pH readings.

When finished, record your data on the answer sheet and answer the questions on the answer sheet.

Chromatography

During photosynthesis, light energy is absorbed by photosynthetic pigments. Chlorophyll A is the main photosynthetic pigment but chlorophyll B, carotenes, and xanthophylls also absorb light. Each pigment absorbs a specific range of colors but all of them together enable the plant to use a larger amount of light. These pigments absorb red and blue light best and absorb green the least. Plants look green; because the green light is not absorbed by the plant; it is reflected. 

Chromatography is a technique used to separate the components of a mixture. In this investigation, you will use chromatography to separate and identify several photosynthetic pigments in a solution prepared from spinach leaves.

Paper chromatography can be used to separate the components of a mixture based on their polarity. Some of the mixture is placed on one end of a piece of paper and that end of the paper is immersed in a nonpolar liquid (see the diagram below). 

Click on the photograph to view an enlargement.

 

As the liquid moves up the paper, the molecules of the sample mixture will also move. Polar molecules within the sample will spend most of their time bound to the polar surface of the paper and will therefore not move very much. Nonpolar molecules, however, will spend most of their time dissolved in the liquid as it moves up the paper. When the liquid reaches the top of the paper, these molecules will also have traveled most of the way to the top. The two types of molecules (polar and nonpolar) are now separated.

R_f

It is useful to determine the relative distance moved by a particular kind of molecule using chromatography. This value is known as R_f. For example, if the molecules move half as far as the solvent traveled, the R_f = 0.5. If the molecules moved 1/4 the distance, the R_f = 0.25. The maximum value for R_f is therefore 1.0.

Procedure

Using a pencil, put a small dot in the center of a strip of chromatography paper 2 cm. from one end.

Put a hole in the other end of the paper so that it can be suspended on a wire clip inserted in a cork stopper as shown in the photograph above. The paper should be able to reach to within 1 cm of the bottom of the tube but not touch the bottom.

When the apparatus is adjusted properly, remove the paper so that you can add pigment extract.

Caution - The remainder of this procedure should be conducted under the hood because the vapors from the chemicals used are toxic and carcinogenic (cause cancer).

Use a capillary tube or small-diameter pipette to place a small amount of pigment extract on the dot that you marked on the paper. The extract should be placed directly on top of the dot. It will spread, producing a small circular spot. Add enough extract so that the spot becomes approximately 1 cm in diameter but not larger. Ideally, you want as much extract on the paper as you can get while keeping the spot as small as possible. In order to accomplish this, you should allow the spot to dry for 5 minutes and repeat this procedure 4 more times for a total of 5 applications of pigment extract. Allow the spot to dry between each application.

The paper in the top photograph has had one application of extract. After 5 applications, your paper should look like the one in the bottom photograph. Click on the photographs to view enlargements.

While waiting for the paper to dry after the fifth application, add chromatography solvent to the bottom of the chromatography tube. Add enough solvent so that the end of the chromatography paper will be immersed in the solvent but the spot with pigment extract will remain above the solvent. It is important not to immerse the pigment spot. This tube should be kept under the hood at all times.

When the spot has dried, suspend the paper vertically in a chromatography tube. If possible, keep the paper from touching the sides of the tube.

Check the movement of the solvent after 10 minutes and then every few minutes after that until the solvent reaches to within 2 cm of the top of the paper. Then, remove the paper and use a pencil to mark the location reached by the top of the solvent. Allow the paper to dry under the hood. After the paper is dried, you may bring it out from under the hood.

Do not discard the chromatography solution down the drain, discard it in the beaker provided under the hood.

The photosynthetic pigments will be separated in the following order beginning with the highest R_f: beta-carotenes, xanthophylls, chlorophyll a, chlorophyll b. Beta-carotenes are orange or orange-yellow, xanthophylls are yellow, chlorophyll A is blue-green, and chlorophyll B is yellow-green.

Calculate R_f values and record these values in table 2.

One member of your group should attach the chromatogram to the lab report before submitting it. The names of the members of your group should accompany the chromatogram.