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Photosynthesis Chloroplasts DPIP

Laboratory Experiment 4:

Plant Pigments and Photosynthesis

Part A: Plant Pigment Chromatography

Table 4.1

Band Number

Distance (mm)

Band Color

1

17

Olive green

2

31

Blue green

3

46

Yellow

4

170

Yellow orange

Distance Solvent Front Moved: 176 millimeters

Table 4.2

Rf

Value

Pigment

0.96591

Carotene

0.26136

Xanthophyll

0.17614

Chlorophyll a

0.09659

Chlorophyll b

Analysis

1. The factors involved in the separation of the pigments from the spinach plants are the pigments' solubility in the solution, how much they bind to the paper based on their chemical structure, and the size of the pigment particles. The greater the solubility, the faster it ascends the paper. Smaller molecules can travel faster than larger ones. Other factors include the temperature of the room, time, and the amount of pigment.

2. No. I would not expect the Rf values to be the same because the pigments will dissolve differently in different types of solvents. If chloroplasts from a spinach cell were placed on the paper in a hydrophobic solution, chlorophyll b would travel the highest distance along the paper instead of the lowest distance, because chlorophyll b is very soluble in hydrophobic solutions.

3. The pigments absorb light, harnessing energy to raise an electron to a higher energy level. Chlorophyll a is in the reaction center, which absorbs the energy from light to excite electrons in the light reactions of photosynthesis. Chlorophyll b absorbs light energy from other wavelengths that chlorophyll a cannot absorb light from and then transfers the energy to chlorophyll a, providing more energy to be used in photosynthesis. Carotenoids, consisting of carotenes and xanthophylls, capture light energy and transfer it to the chlorophyll a at the reaction center. They protect the photosynthetic system from the damaging effects of ultraviolet light.

Part B: Photosynthesis / The Light Reaction

Purpose:

The purpose of this experiment is to determine the effect of light intensity and boiling the chloroplasts on the rate of photosynthesis.

Hypothesis:

The cuvette (3) containing unboiled chloroplasts and placed in light will show the greatest transmittance and rate of photosynthesis. The cuvette (2) containing unboiled chloroplasts and placed in the dark will show the lowest transmittance rate of photosynthesis and no change of transmittance, thus showing that no photosynthesis will occur. The cuvette (4) containing boiling chloroplasts will show no change in transmittance and the cuvette (5) containing no chloroplasts will also show no change in transmittance, thus showing no photosynthesis will occur. In the light reactions of photosynthesis, light is required to the reduction of NADP, or in this experiment, DPIP, because light provides the energy required to excite the electrons in the chlorophyll, which reduce DPIP and NADP.  Darkness does not provide the light energy required to be absorbed by the pigments on the chloroplasts and excite the electrons which would reduce DPIP. Thus, cuvette 2 will experience the lowest transmittance, cuvette 5 will show no change in transmittance, and cuvette 3 will experience the highest transmittance. Boiling chloroplasts prevents the DPIP from being reduced because the enzymes for photosynthesis are denatured by the high temperature and are no longer present in the chloroplasts. The heat denatures the organelle, destroying the photosynthetic membranes that contain the carrier molecules which transport electrons from chlorophyll to NADP or DPIP, and stripping it of its photosynthetic capacity. Since photosynthesis cannot be performed by the denatured chloroplasts, the DPIP cannot be reduced. Thus, cuvette 4 would experience no change in transmittance. Live chloroplasts incubated in the light would carry on photosynthesis and reduce the DPIP, resulting in a color change from blue to clear, which leads to a higher transmittance. In the dark, there is no photosynthesis, no reduction of DPIP, no changing of the DPIP from blue to clear, and thus, a lesser transmittance results. For photosynthesis to occur in plant cells, active chloroplasts must be present and light must be available.

Variables:

The independent variables of this experiment are the amount of light and the condition of the chloroplasts (boiled or unboiled).

The dependent variable of this experiment is the rate of photosynthesis. A colorimeter is used to determine how much light can pass through the sample. The transmittance of light in percent determines how much of the DPIP has changed from blue to colorless, and thus, how much DPIP was reduced to measure photosynthetic activity.

The controlled variables of this experiment include adding the same amount of DPIP (1 milliliter) and phosphate buffer (1 milliliter) to each of the experimental cuvettes and adding the same number of drops (3) of either unboiled chloroplasts or boiled chloroplasts to the appropriate experimental cuvettes that require chloroplasts.

Procedure:

An incubation area that includes a light and a 1000 milliliter beaker filled with water was arranged. An ice bucket was filled three quarters full with ice. Five cuvettes were labeled as 1, 2, 3, 4, and 5, respectively. A foil container was constructed for cuvette 2 that could be easily removed and replaced so that the cuvette could be placed in the colorimeter to obtain readings. Since cuvette 2 was experimented in the dark, the foil container prevented light from entering the cuvette. Using pipettes, boiled and unboiled chloroplasts were obtained separately and placed in the ice bath. The chloroplasts from spinach leaves were prepared by shining a light on the leaves for several hours before the experiment and churned in a blender in a cold sucrose buffer, which keeps the enzymes in the chloroplasts from being denatured too quickly. A portion of the resulting chloroplast suspension was heated in boiling water for 5 minutes, which denatures the chloroplasts, stripping it of its photosynthetic capacity. The pipettes holding the boiling and unboiled chloroplasts were kept on ice at all times. Then, to each cuvette, phosphate buffer, distilled water, and DPIP were added as shown in the following table:

Cuvette 1

Cuvette 2

Cuvette 3

Cuvette 4

Cuvette 5

Phosphate Buffer

1 mL

1 mL

1 mL

1 mL

1 mL

Distilled Water

2.5 mL

1.5 mL

1.5 mL

1.5 mL

1.5 mL + 3 drops

DPIP

-

1 mL

1 mL

1 mL

1 mL

The colorimeter was calibrated by preparing cuvette 1 by adding the substances indicated in the data table and three drops of unboiled chloroplasts and placing it in the cuvette slot of the colorimeter. Cuvette 1 was the blank to be used to recalibrate the colorimeter between readings. Cuvettes 3 and 4 were placed behind the beaker that is in front of the light source. Three drops of unboiled chloroplasts were added to cuvette 2 and the transmittance of light through the solution was measured by the colorimeter. Cuvette 2 observes the rate of photosynthesis without the presence of light and observes the role of chloroplasts in photosynthesis. Three drops of unboiled chloroplasts were added to cuvette 3 and the transmittance of light through the solution was measured by the colorimeter. Cuvette 3 observes the normal rate and role of photosynthesis with light and the presence of chloroplasts. Three drops of boiled chloroplast were added to cuvette 4 and the transmittance of light through the solution was measured by the colorimeter. Cuvette 4 observes the rate of photosynthesis in chloroplasts that have been boiled and to observe the effect of stability of the chloroplasts on photosynthesis. The transmittance of light of cuvette 5, which is the control because it was not placed in either light or chloroplasts, was also measured using the colorimeter. Errors in the experiment's data may have been due to DPIP breaking own its own.

Data:

Time (min)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Average

Cuvette #2

0

13.9

20.3

14.8

15.3

16.2

0

16.5

18.4

25.5

18.4

21.3

21.6

21.2

21.7

17.499

Unboiled

5

15.6

17.2

17.2

22.3

16.5

0

16.9

19.1

35.9

19.3

22.3

23.5

22.7

22

19.318

Dark

10

16.2

22.9

17.1

21.7

16.5

0

16.6

18.9

36.8

20.8

22.7

24.3

22

22.2

19.901

15

16.2

22.2

16.6

21.4

15.7

0

16.2

18.1

34.4

20.4

21.7

18.8

21.3

21.8

18.916

Cuvette #3

0

13.8

17.4

14.5

20.6

16.9

17.8

14.1

18.9

24.9

17.3

20.1

14.8

21.2

21

18.088

Unboiled

5

99.9

97.4

50.6

106

101

91

43.9

98.2

102

57.2

92.2

59.5

92.5

93.3

84.631

Light

10

102

96.7

104

122

102

90.3

90.2

99.6

103

78.1

94.2

74.4

95.7

96.1

96.26

15

98.1

95.5

104

120

103

89.2

89.2

97

102

79.6

95.4

92.6

95.1

95.8

96.834

Cuvette #4

0

19.1

22

23.1

33.5

24.2

27

25.2

20.9

27.9

22.6

24.5

16.9

26.6

27.1

24.319

Boiled

5

23.3

18.3

28

28.5

24

32.2

31.1

26.4

32.5

25.3

25.4

17.1

36.6

36.8

27.547

Light

10

26.2

20.3

29

30.8

28

32

31.5

27.1

32

25

25.4

12.9

39.7

38.7

28.466

15

25.8

27.9

29

32.4

31.8

32.4

31.4

31.4

32.5

26.4

27.1

12.3

40.4

40.4

30.075

Cuvette #5

0

23.4

19

21.5

32.7

29.4

21.2

18.5

22.7

34

24.5

26.2

29.4

25.8

24.8

25.221

No CP

5

20.3

22.4

21.4

28.2

31

21.8

17.7

22.7

33.4

21.4

23.8

28.1

25.7

24.4

24.452

10

22.1

17.7

21.1

27

23

20.9

17.1

22.7

32.8

21.2

21.9

17.3

25.8

25.7

22.595

15

22.1

21.5

20.9

26.8

22.6

20.5

17

22.3

32.3

21.1

21.3

18.9

25.9

25.5

22.768

Conclusion:

Cuvette 3, which is in the presence of light and contains unboiled chloroplasts showed the greatest transmittance at 96.834 percent, and thus, the highest rate of photosynthesis. The great increase of transmittance from 18.088 percent to 84.631 after five minutes to 96.834 percent after 15 minutes in cuvette 3 demonstrates that photosynthesis did not slow down and DPIP was quickly used up. In the light reactions of photosynthesis, light is required to the reduction of NADP, or in this experiment, DPIP, because light provides the energy required to excite the electrons in the chlorophyll of the chloroplasts, causing the electrons to produce ATP and reduce DPIP and NADP. Live chloroplasts incubated in the light in cuvette 3 would carry on photosynthesis and reduce the DPIP, resulting in a color change from blue to clear, which leads to a higher transmittance. Light is necessary to excite electrons, which, in turn, reduces the DPIP. Cuvette 2 experienced the lowest transmittance at 18.916 percent. The small change in transmittance in cuvette 2 from 17.499 percent to 18.916 percent after 15 minutes indicates that there was little or no photosynthesis occurring. Darkness does not provide the light energy required to be absorbed by the pigments on the chloroplasts and excite the electrons which would reduce DPIP. In the dark, there is no photosynthesis, no reduction of DPIP, no changing of the DPIP from blue to clear, and thus, a lesser transmittance results. The slight change may have been caused by errors in the experiments, such as DPIP breaking on its own and the cuvette may have been exposed to light while transferring cuvettes. Cuvette 4 experienced a small change in transmittance from 24.319 percent to 30.075 percent after 15 minutes. This small change in transmittance indicates that the light reactions of photosynthesis did not occur in cuvette 4. Boiling chloroplasts prevents the DPIP from being reduced because the enzymes for photosynthesis are denatured by the high temperature and are no longer present in the chloroplasts. The heat denatures the organelle, destroying the photosynthetic membranes that contain the carrier molecules which transport electrons from chlorophyll to NADP or DPIP, and stripping it of its photosynthetic capacity. Since photosynthesis cannot be performed by the denatured chloroplasts, the DPIP cannot be reduced. Thus, cuvette 4 should have experienced no change in transmittance. However, because energy from the light source directly reduced DPIP, the transmittance of light increased slightly. Cuvette 5 experienced a small decrease in transmittance from 25.221 percent to 22.768 percent, which demonstrates that photosynthesis cannot occur without chloroplasts. Active chloroplasts incubated in the light would carry on photosynthesis and reduce the DPIP, resulting in a color change from blue to clear, which leads to a higher transmittance. In the dark, there is no photosynthesis, no reduction of DPIP, no changing of the DPIP from blue to clear, and thus, a lesser transmittance results. This explains why cuvette 3 experienced a higher transmittance of light after 15 minutes than cuvette 2 by 77.918 percent, cuvette 4 by 66.759 percent, and cuvette 5 by 74.066 percent. Thus, the hypothesis is supported: active chloroplasts must be present and light must be available for the light reactions of photosynthesis to occur.

Analysis

1. DPIP is the electron acceptor in this experiment instead of NADP. The electrons boosted to high energy levels are used to produce ATP and reduce the DPIP, which will change its color from blue to colorless, showing that light reactions have occurred and increasing the light transmittance, which may determine how much photosynthesis has occurred. 2. DPIP replaces NADP molecules that are found in chloroplasts. 3. The electrons that reduce DPIP come from splitting water molecules that travel to the chlorophyll in the chloroplasts, and eventually transfer to the DPIP and reduce it. 4. A spectrophotometer usually measures light or specific wavelengths of light. The light transmittance was measured in this experiment, which really was the measure of how much the electrons in the chlorophyll reduced the DPIP.

5. In the light reactions of photosynthesis, light is required to the reduction of NADP, or in this experiment, DPIP, because light provides the energy required to excite the electrons in the chlorophyll, which reduce DPIP and NADP.  Therefore, darkness does not provide the light energy required to be absorbed by the pigments on the chloroplasts and excite the electrons which would reduce DPIP.

6. Boiling chloroplasts prevents the DPIP from being reduced because the enzymes for photosynthesis are denatured by the high temperature and are no longer present in the chloroplasts. The heat denatures the organelle, destroying the photosynthetic membranes that contain the carrier molecules which transport electrons from chlorophyll to NADP or DPIP, and stripping it of its photosynthetic capacity. DPIP is reduced from blue to colorless when light strikes the chloroplasts and the electrons are boosted to a higher energy level. Since photosynthesis cannot be performed by the denatured chloroplasts, the DPIP cannot be reduced.  

7. Live chloroplasts incubated in the light would carry on photosynthesis and reduce the DPIP, resulting in a color change from blue to clear, which leads to a higher transmittance. In the dark, there is no photosynthesis, no reduction of DPIP, no changing of the DPIP from blue to clear, and thus, a lesser transmittance results. In the light reactions of photosynthesis, light is required to the reduction of NADP, or in this experiment, DPIP, because light provides the energy required to excite the electrons in the chlorophyll, which reduce DPIP and NADP.  Light is necessary to excite electrons, which, in turn, reduces the DPIP.

8. Cuvette 2 observes the rate of photosynthesis without the presence of light and observes the role of chloroplasts in photosynthesis. Cuvette 3 observes the normal rate and role of photosynthesis with light and the presence of chloroplasts. Cuvette 4 observes the rate of photosynthesis in chloroplasts that have been boiled and to observe the effect of stability of the chloroplasts on photosynthesis. Cuvette 5 is the control because it was not placed in light and contained no chloroplasts, and it observes the reaction of the synthesis of water.

Section

Points Earned

Out of

Part A

Data

3

3

Analysis Questions

9

9

Part B

Purpose

4

4

Variables

10

10

Hypothesis

10

10

Data

15

15

Conclusion

24

25

Analysis Questions

24

24

Total

99

100

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