Plant Pigments and Photosynthesis

Introduction:
In this laboratory you will separate plant pigments using chromatography. You will also measure the rate of photosynthesis in isolated chloroplasts. The measurement technique involves the reduction of the dye DPIP. The transfer of electrons during the light-dependent reactions of photosynthesis reduces DPIP, changing it from blue to colorless.

Exercise 4A: Plant Pigment Chromatography:
Paper chromatography is a useful technique for separating and identifying pigment and other molecules from cell extracts that contain a complex mixture of molecules. The solvent moves up the paper by capillary action, which occurs as a result of the attraction of solvent molecules to the paper and the attraction of the solvent molecules to one another. As the solvent moves up the paper, it carries along any substances dissolved in it. The pigments are carried along at different rates because they are not equally soluble in the solvent and because they are attracted, to different degrees, to the fibers of the paper through the formation of intermolecular bonds, such as hydrogen bonds.

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.

Procedure:
1. Obtain a 250 mL beaker which has about 2 cm of solvent at the bottom. Cover the beaker with aluminum foil to prevent the vapors from spreading. It is also suggested this work be done under a fume hood.

2. Cut a piece of filter paper which will be long enough to reach the solvent. Draw a line about 1.0 cm from the bottom of the paper. See Figure 4.1 below.

 Figure 4.1

 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 to crush the leaf cells. Be sure the pigment line is on top of the pencil line. Use a back and forth movement exerting firm pressure through out.

4. Place the chromatography paper in the cylinder. See Figure 4.2 below. Do not allow the pigment to touch the solvent.

 Figure 4.2

 

5. Cover the beaker. 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 distance that each front, including the solvent front, moved in Table 4.1 Depending on the species of plant used, you may be able to observe 4 or 5 pigment bands.

Table 4.1

Distance moved by Pigment Band (millimeters)

Band Number

Distance (mm)

Band Color

1

 

 

2

 

 

3

                                    

                               

4

 

 

5

 

 

Distance Solvent Front Moved _________________

 Analysis of Results:
The relationship of the distance moved by a pigment to the distance moved by the solvent is a constant called Rf . It can be calculated for each of the four pigments using the formula:

 

Rf

=

distance pigment migrated (mm)_____

 

 

distance solvent front migrated (mm)

Record your Rf values in Table 4.2

Table 4.2

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= Rf for carotene (yellow to yellow -orange)

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= Rf for xanthophyll (yellow)

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= Rf for Chlorophyll a (bright green to blue green)

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= Rf for Chlorophyll b (yellow green to olive green)

Topics for Discussion:
1. What factors are involved in the separation of the pigments?

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

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3. What type of chlorophyll does the reaction center contain? What are the roles of the other pigments?

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 Exercise 4B: Photosynthesis / The Light Reaction:
Light is a part of a continuum of radiation or energy waves. Shorter wavelengths of energy have a greater amounts of energy. For example, high-energy ultraviolet rays can harm living things. Wavelengths of light within the visible spectrum of light power photosynthesis. when light is absorbed by leaf pigments, electrons within each photosystem are boosted to a higher energy level and this energy level is used to produce ATP and to reduce NADP to NADPH. ATP and NADPH are then used to incorporate CO2 into organic molecules, a process called carbon fixation.

Design of the Exercise:
Photosynthesis may be studied in a number of ways. For this experiment, a dye-reduction technique will be used. The dye-reduction experiment tests the hypothesis that light and chloroplasts are required for the light reactions to occur. In place of the electron accepter, NADP, the compound DPIP ( 2.6-dichlorophenol-indophenol), will be substituted. When light strikes the chloroplasts, electrons boosted to high energy levels will reduce DPIP. It will change from blue to colorless.

In this experiment, chloroplasts are extracted from spinach leaves and incubated with DPIP in the presence of light. As the DPIP is reduced and becomes colorless, the resultant increase in light transmittance is measured over a period of time using a spectrophotometer. The experimental design matrix is presented in Table 4.3.

Table 4.3: Photosynthesis Setup

Cuvettes

 

1

Blank

 

2

Unboiled Chloroplasts Dark

3

Unboiled Chloroplasts Light

4

Boiled Chloroplasts Light

5

No
Chloroplasts

Phosphate Buffer

1 ml.

1 ml.

1 ml.

1 ml.

1 ml.

Distilled Water

4 ml.

3 ml.

3 ml.

3 ml.

3 ml + 3 drops

DPIP

----

1 ml.

1 ml.

1 ml.

1 ml.

Unboiled Chloroplasts

3 drops

3 drops

3 drops

----

----

Boiled Chloroplasts

----

----

----

3 drops

----

Procedure:
1. Turn on the spectrophotometer to warm up the instrument and set the wavelength to 605 nm by adjusting the wavelength control knob.

2. While the spectrophotometer is warming up, your teacher may demonstrate how to prepare a chloroplast suspension from spinach leaves.

3. Set up an incubation area that includes a light, water flask, and test tube rack. The water in the flask acts as a heat sink by absorbing most of the light's infrared radiation while having little effect on the light's visible radiation.

Figure 4.2: Incubation Setup

Flood Light -------Water Heat Sink-------Cuvettes

 

4. Your teacher will provide you with two beakers, one containing unboiled chloroplasts. Be sure to keep these on ice at all times.

5. At the top rim, label the cuvettes 1,2,3,4, and 5, respectively. Using lens tissue, wipe the outside walls of each cuvette ( Remember: handle cuvettes only near the top). 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.

6. Refer to Table 4.3 to prepare each cuvette. Do not add unboiled or boiled chloroplasts yet. To each cuvette, add 1 ml of phosphate buffer.

7. Bring the spectrophotometer to zero by adjusting the amplifier control knob until the meter reads 0% transmittance. 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 by adjusting the light -control knob. Cuvette 1 is the blank to be used to recalibrate the instrument between readings. 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.

8. Obtain the unboiled chloroplast suspension, stir to mix, and transfer three 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 % 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.

9. Obtain the unboiled chloroplast suspension, mix, and transfer three drops to cuvette 3. Immediately cover and mix cuvette 3. Insert it into the spectrophotometer's sample holder, read the % 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 cuvette 1 occasionally to check and adjust the spectrophotometer to 100% transmittance.

10. Obtain the boiled chloroplast suspension, mix, and transfer three drops to cuvette 4. Immediately cover and mix cuvette 4. Insert it into the spectrophotometer's sample holder, read the % 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 cuvette 1 occasionally to check and adjust the spectrophotometer to 100% transmittance.

11. Cover and mix the contents of cuvette 5. Insert it into the spectrophotometer's sample holder, read the % 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 cuvette 1 occasionally to check and adjust the spectrophotometer to 100% transmittance.

Table 4.4: Transmittance (%)

Time (minutes)

Cuvette

0

5

10

15

2 Unboiled /Dark

               

              

              

              

3 Unboiled/ Light

 

 

 

 

4 Boiled / Light

 

 

 

 

5 No Chloroplasts

 

 

 

 

Analysis of Results:
Plot the percent transmittance from the four cuvettes on the graph below
.

a. What is the dependent variable? ____________________________________________

b. What is the independent variable? __________________________________________

Graph Title: __________________________________________________________________

Graph 4.1

Topics for Discussion:
1. What is the purpose of DPIP in this experiment?

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

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

4. What was measured with the spectrophotometer in this experiment? ____________________________________________________________________________

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

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

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7. 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?

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