AP Biology Lab Four: Plant Pigments and Photosynthesis

Purpose: The purpose of this lab is to separate and identify pigments and other molecules within plant cells by a process called chromatography. We will also be measuring the rate of photosynthesis in isolated chloroplasts. Beta carotene, the most abundant carotene in plants, is carried along near the solvent front because it is very soluble in the solvent being used and because it forms no hydrogen bonds with cellulose. Another pigment, Xanthophyll differs from carotene in that it contains oxygen. Xanthophyll is found further from the solvent font because it is less soluble in the solvent and has been slowed down by hydrogen bonding to the cellulose. Chlorophyll's contain oxygen and nitrogen and are bound more tightly to the paper than the other pigments. Chlorophyll a is the primary photosynthetic pigment in plants. A molecule of chlorophyll a is located at the reaction center of the photo systems. The pigments collect light energy and send it to the reaction center. Carotenoids also protect the photosynthetic systems from damaging effects of ultraviolet light.

Hypothesis: I believe the rate of photosynthesis in plant cells is controlled by the light it receives and the temperature around it.

Procedure: 1) Obtain a 50 ml graduated cylinder which has about 1 cm of solvent at the bottom. 2) Cut a piece of filter paper which will be long enough to reach the solvent. Draw a line about

1.5 cm from the bottom of the paper. 3) Use a quarter to extract the pigments from spinach leaf cells. Place a small section of leaf on the

top of the pencil line. Use the ribbed edge of the coin to crush the leaf cells. Be sure the pigment line is on top of the pencil line.

4) Place the chromatography paper in the cylinder. 5) Cover the cylinder. When the solvent is about 1 cm from the top of the paper, remove the paper

and immediately mark the location of the solvent front before it evaporates.6) Mark the bottom of each pigment band. Measure the distance each pigment migrated from the

bottom of the pigment origin to the bottom of the separated pigment band. Record the distances. 7) Turn on the spectrophotometer to warm up the instrument and set the wavelength to 605 nm.8) Set up an incubation area that includes a light, water flask, and test tube rack. 9) Your teacher will provide you with two beakers, one containing unboiled chloroplasts and the

other containing a solution of boiled chloroplasts. Be sure to keep these on ice at all times.10) At the top rim, label the cuvettes 1,2,3,4, and 5, respectively. Using lens tissue, wipe the outside

walls of each cuvette. Using foil paper, cover the walls and bottom of cuvette 2. Light should not be permitted inside cuvette 2 because it is a control for this experiment.

11) Refer to Table 4.3 to prepare each cuvette. Add 4 ml of distilled water to cuvette 1. To 2,3, and 4, add 3 ml of distilled water and 1 ml of DPIP. To 5, add 3 ml plus 3 drops of distilled water and 1 ml of DPIP.

12) Bring the spectrophotometer to zero by adjusting the amplifier control knob until the meter reads 0% transmittance. Add 3 drops of unboiled chloroplasts and cover the top of cuvette 1 with Parafilm and invert to mix. Insert cuvette 1 into the sample holder and adjust the instrument to 100% transmittance. For each reading, make sure that the cuvettes are inserted into the sample holder so that they face the same way as in the previous reading.

13) Obtain the unboiled chloroplast suspension, stir to mix, and transfer 3 drops to cuvette 2. Immediately cover and mix cuvette 2. Then remove it from the foil sleeve and insert it into the spectrophotometer's sample holder, read the percentage transmittance, and record it as the time 0 reading in Table 4.4. Replace cuvette 2 into the foil sleeve, and place it into the incubation test tube rack. Turn on the flood light. Take and record additional readings at 5,10,and 15 minutes.

Mix the cuvette's contents just prior to each readings. Remember to use cuvette 1 occasionally to check and adjust the spectrophotometer to 100% transmittance.

14) Obtain the unboiled chloroplast suspension, mix, and transfer 3 drops to cuvette 3. Immediately cover and mix cuvette 3. Insert it into the spectrophotometer's sample holder, read the percentage transmittance, and record it in Table 4.4. Replace cuvette 3 into the incubation test tube rack. Take and record additional readings at 5, 10, and 15 minutes. Mix the cuvette's contents just prior to each readings. Remember to use cuvtte 1 occasionally to check and adjust the spectrophotometer to 100% transmittance.

15) Obtain the boiled chloroplast suspension, mix, and transfer 3 drops to cuvette 4. Immediately cover and mix cuvette 4. Insert it into the spectrophotometer's sample holder, read the percentage transmittance, and record it in Table 4.4. Replace cuvette 4 into the incubation test tube rack. Take and record additional readings at 5, 10, and 15 minutes. Mix the cuvette's contents just prior to each readings. Remember to use cuvtte 1 occasionally to check and adjust the spectrophotometer to 100% transmittance.

16) Cover and mix the contents of cuvette 5. Insert it into the spectrophotometer's sample holder, read the percentage transmittance, and record it in Table 4.4. Replace cuvette 5 into the incubation test tube rack. Take and record additional readings at 5, 10, and 15 minutes. Mix the cuvette's contents just prior to each readings. Remember to use cuvtte 1 occasionally to check and adjust the spectrophotometer to 100% transmittance.

Results:

Table 4.1 Distance Moved by Pigment Band

Band Number Distance (mm) Band Color

1. 0 mm Yellow-brown

2. 5 mm Light green

3. 30 mm Green

4. 48 mm Yellow

Distance Solvent Front Moved 60 mm.

Table 4.2 Rf Values

0.8 = Rf for xanthophyll (yellow)

0.5 = Rf for chlorophyll a (bright green to blue green)

0 = Rf for chlorophyll b (yellow green to olive green)

Table 4.4 Transmittance (%)

Cuvette 0 5 10 15

2 Unboiled/Dark 41% 43% 44% 43%

3 Unboiled/Light 35% 38% 39% 37%

4 Boiled/Light 51% 52% 53% 54%

5 No Chloroplasts 57% 57% 56% 55%

Lab 4B Color Chart

Cuvette Initial Color Final Color

1 Clear Clear

2 Light clear blue Blue/green

3 Light clear blue Dark clear blue

4 Light clear blue Light clear blue

5 Light clear blue Dark clear blue

Analysis:

1. What factors are involved in the separation of the pigments?

The solubility, size of particles, and their attractiveness to the paper are all involved in the separation.

2. Would you expect the Rf value of the pigment to be the same if a different solvent were used? Explain.

No, the different solubilities of the pigments would change the Rf values. For example chlorophyll b is only soluble to fat solutions.

3. What type of chlorophyll does the reaction center contain? What are the roles of the other pig-ments?

The reaction center contains chlorophyll a. The other pigments collect different light waves and transfer the energy to chlorophyll a.

4. What is the purpose of DPIP in this experiment?

DPIP is the electron acceptor in this experiment.

5. What molecule found in chloroplasts does DPIP "replace" in this experiment?

DPIP substitutes for the NADP molecules.

6. What is the source of the electrons that will reduce DPIP?

The electrons come from the photolysis of water.

7. What was measured with the spectrophotometer in this experiment?

The spectrophotometer measures the percentage of light transmittance through the cuvette due to DPIP reduction.

8. What is the effect of darkness on the reduction of DPIP? Explain.

The effect of darkness is that no reaction will occur.

9. What is the effect of boiling the chloroplasts on the subsequent reduction of DPIP? Explain.

Boiling denatures the protein molecules and stops the reduction.

10. What reasons can you give for the difference in the percent transmittance between the live chloroplasts that were incubated in the light and those that were kept in the dark?

In the dark cuvette, there was no light energy available, so there was no flow of electrons and no photol-ysis of water, while in the lighted cuvette these processes were allowed to continue.

Conclusion:

From lab 4a we discovered that the many pigments found in chloroplasts are all involved in gath-ering energy from sunlight. The spectrum of color displayed on the filter paper showed the pigments and the solubility of each. In lab 4b the spectrophotometer measured the light transmittance through the vari-ous cuvettes and the chloroplast solutions in each. The actual purpose of this was to observe the DPIP go from a blue color to a clear color. This indicated that photosynthesis was occurring and at what rate it was occurring. The cuvette with the unboiled chloroplasts that had been exposed to light showed the biggest change in % transmittance, which indicates that the amount of light available has a very big ef-fect on the rate at which the light reactions of photosynthesis occur .