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Photosynthesis Chloroplast Experiment

Purpose: The purpose of Lab 4 is to test whether chloroplast and/or light are required for photosynthesis to take place in plants.

Variables:

Independent-Chloroplast (boiled of unboiled), Light (absence or presence)

Dependent-Amount of photosynthesis (percent transmittance)

Controls-Same method of measuring transmittance, same plant chloroplast, same environmental temperature, same environmental pressure, same spectrophotometer settings, same size cuvettes

The dependent variable is ultimately the amount of photosynthesis occurring in the cuvettes. This was measured using the dye-reduction test using a spectrophotometer and the compound DPIP. The more photosynthesis occurs, the more DPIP is reduced. As the DPIP is reduced, the transmittance becomes higher. The higher the percent transmittance, the greater the amount of photosynthesis. Thus, the amount of photosynthesis occurring was measured by the percent transmittance using a spectrophotometer.

Hypothesis:

The cuvette with the unboiled chloroplasts that are exposed to the light will have the highest percent transmittance and thus have a higher amount of photosynthesis occurring. This is because chloroplasts and light are both necessary for a plant to go through photosynthesis. The cuvette with the boiled chloroplasts should experience little to no change in percent transmittance because the enzymes necessary for the function of the chloroplasts were boiled and denatured. The cuvette with unboiled chloroplast not exposed to light also should have little to no change in percent transmittance because light is necessary for photosynthesis to occur in plants.

Procedure:

Cuvettes, chloroplast, DPIP, aluminum foil and a spectrophotometer were obtained. An incubation area was set up with a flood light and heat sink. Using the aluminum foil, a container was made for Cuvette 2. The container blocked out all the light to the cuvette but was also made to be easily removed. The spectrophotometer was connected to a laptop computer with the proper reading program and set to 605 nm wave length. A cuvette without chloroplast was used to then calibrate the spectrophotometer. Cuvettes 2, 3, and 4 were filled with chloroplast, distilled water, and DPIP. No chloroplast was placed in Cuvette 5. The cuvettes were then placed into the spectrophotometer one by one and the percent transmittances were recorded. Each cuvette was then placed into the incubation area for 5 minute intervals of 5, 10, and 15 minutes. For Cuvette 2, each time it was taken out of the spectrophotometer it was made sure that it was fully enclosed in the aluminum foil container to block out light.

Data:

Group #/%Transmittance

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

 

Table 4.1:

Band #

Distance (nm)

Band Color

1

5

Yellow Orange

2

54

Yellow

3

18

Green

4

10

Yellow Green

Total: 146 nm

Table 4.2:

.034 = Rffor Carotene (yellow to yellow orange)

.37= Rf for Xanthophyll (yellow)

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

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

 

Conclusion:

Cuvette 3 went through the most photosynthesis. This can be seen in the data, as percent transmittance went from the initial 18.088 to 96.834 at 15 minutes. This went along with my hypothesis, as Cuvette 3 contained the unboiled chloroplast that was exposed to light. Cuvette 2 contained unboiled chloroplasts but was not exposed to the light and did not see significant increase in percent transmittance. Cuvette 4 had boiled chloroplasts and was exposed to light. Cuvette 4 also did not experience much change in percent transmittance. This is because Cuvette 4 had boiled chloroplast, and the majority of the enzymes in the chloroplast are denatured, thus not allowing the chloroplast to properly function. The negligible change in transmittance may have been due to some of the enzymes not being denatured upon boiling. Cuvette 5 contained no chloroplasts and experienced negligible amount of change in percent transmittance. The reason why percent transmittance would go up is due to the compound DPIP being reduced during photosynthesis. The DPIP is used in place for the naturally occurring NADP electron acceptors that are normally reduced during photosynthesis. As more photosynthesis occurs, the more DPIP compound is reduced, which results it in the contents in the cuvettes becoming clearer and allowing more light to transmit through. With little change in transmittance, as in Cuvette 2, 4, and 5, it can be inferred that the DPIP was not reduced, thus meaning that the amount of light transmitted through the cuvette did not increase, leading to the conclusion that photosynthesis did not occur or only occurred on a negligible scale. From interpreting the data, it can be inferred that both chloroplasts and light are necessary for photosynthesis to take place. Chloroplasts are the parts of the plant where the majority of photosynthetic actions take place. Chloroplasts are necessary for photosynthetic reactions, and because the chloroplasts are unboiled in Cuvette 3 and the enzymes are not denatured, the chloroplasts were functional and photosynthetic reactions occurred.

Analysis:

P.47 1-3

  • Solvent mixture, absorption mixture, and time are factors involved in the separation of the pigments.
  • No, if a different solvent were used, it would have created a different mixture with different solubility. This would affect the capillary action and rate of which capillary action proceeded and also how far the pigment traveled.
  • Chlorophyll A is contained in the reaction center. Another type of chlorophyll is chlorophyll B, which absorbs light energy. Carotene and xanthophylls aid in protection and photosynthetic reactions.

P.52 1-8

  • DPIP was used in place of NADP to accept electrons and be reduced. As DPIP was reduced, a change in transmittance occurred and allowed for observation on the how much photosynthesis took place.
  • DPIP replaced NADP in the experiment.
  • The electrons are from the water in the pigments.
  • The spectrophotometer measured the percent transmittance of light through the cuvettes.
  • Without light, the electrons were not excited and did not reduce the DPIP as much as when exposed to light. The light is needed to energize the electrons to reduce the DPIP.
  • Boiling of the chloroplasts denatures enzymes that are necessary for photosynthesis. The DPIP was then unable to be reduced as the enzymes that aid in the electron reactions reducing DPIP were denatured.
  • In the light, the electrons were excited and readily reacted with the DPIP This reduces the DPIP. In the dark, the electrons were not excited and thus did not react with the DPIP receptors and did not reduce the DPIP.
  • Cuvette 1: This cuvette was used to calibrate the spectrophotometer.

Cuvette 2: This cuvette was used to show the effect darkness has on photosynthesis.

Cuvette 3: This cuvette was used to show the effect light had on photosynthesis

Cuvette 4: This cuvette was used to show the effect nonfunctional boiled chloroplast had on photosynthesis

Cuvette 5: This cuvette was used as the control.

Bibliography:

Campbell and Reece. Biology. Boston: Pearson, Benjamin Cummings. 2002

Biology Lab Manual. The College Board. 2001

Section

Points Earned

Out of

Part A

Data

3

3

Analysis Questions

6

9

Part B

Purpose

4

4

Variables

10

10

Hypothesis

8

10

Data

13

15

Conclusion

20

25

Analysis Questions

24

24

Total

88

100

Additional Comments:


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