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Pigment Photosynthesis Chloroplasts

Lab 4: Plant Pigments and Photosynthesis

Purpose

Part B

The purpose of this experiment is to determine if light and functional chloroplasts are individually necessary for the process of photosynthesis.

Variables

Part B

Independent - chloroplasts boiled or unboiled, exposure to light or darkness, (presence of chloroplasts)

Dependent - percent light transmittance (measured by colorimeter)

Control- same volume of liquid in each cuvette, same type of container (important for transmittance measurements), calibration of colorimeter (using calibration cuvette 1 with unboiled chloroplasts)

Hypothesis

Part B

Cuvette 2 - No photosynthesis will occur without light present.

Cuvette 3 - Photosynthesis will occur in the presence of live chloroplasts and light.

Cuvette 4 - No photosynthesis will occur with boiled chloroplasts.

Cuvette 5 - (Control) No photosynthesis will occur without light or chloroplasts.

All four hypotheses are based upon the knowledge that photosynthesis is light-dependent, and that chloroplasts are vital organelles in the photosynthetic process. Photosynthesis cannot occur in the dark because the light is the energy source for reactions to occur. It also cannot occur without chloroplasts because they absorb and channel the energy so that it is useful to the plant. When plants absorb light (photons), the energy is transmitted to chlorophyll a in the reaction center of a photosystem. The photosystem is composed of chlorophyll, proteins, and small organic molecules contained in the chloroplast. The chlorophyll loses an energy-excited electron to an electron acceptor (normally NADP, in this case DPIP), which ends up producing ATP and reducing the electron acceptor. Therefore, without light the chloroplasts are incapable of carrying out a reaction, and without chloroplasts the light cannot be used.

Procedure

Part B

We began with the lab setup already in place, including our light source and heat sink, an ice bucket, clean pipettes, bottles of phosphate buffer and DPIP, the colorimeter (substituted for spectrophotometer), and clean cuvettes. The boiled and unboiled chloroplast solutions, derived from spinach leaves, had already been prepared. The first step was to label the cuvettes 1 through 5, handling them carefully so as not to smudge the sides, which could interfere with the transmittance data. We then made a foil wrapper for cuvette 2, because its contents needed to be kept in darkness as a control. Next we filled two separate pipettes with the boiled and unboiled chloroplast solutions, placing them in the ice while we prepared the rest of the materials. We added 1 mL of phosphate buffer to each cuvette, and 1.5 mL of distilled water to each except #1, which required 2.5 mL, and #5, which required 1.5 mL and three drops. Each cuvette except #1 also received 1 mL of DPIP. Before adding the chloroplasts, which would start the reactions, we opened up the data-logging software and hooked up the colorimeter to the computer to record out percent transmittance. Three drops of shaken unboiled chloroplasts were added to cuvette 1 and it was used to calibrate the colorimeter, to account for any change in light transmittance due to the water, phosphate buffer, or chloroplasts.

Three drops of unboiled chloroplasts were added to cuvette #2, and the stopwatch was started to keep track of measurement times. The transmittance of #2 was checked and recorded, and then the foil casing was wrapped around it and placed in the light behind the heat sink. Three drops of unboiled chloroplasts were added to #3, and three drops of boiled chloroplasts to #4, and they were both checked for transmittance and time of measurement, and then placed in the light. Cuvette #5 was also measured for transmittance and time and placed in the light, but as the control, no chloroplasts were added. Each cuvette was subsequently measured, recorded, and replaced at intervals of 5 minutes from the starting time, until 15 minutes had passed since chloroplasts were added to each cuvette. All equipment was cleaned and disassembled, and the data collected and analyzed.

Data

Part A

Data Table 4.1

Band Number

Distance (mm)

Band Color

1

5

Lime green

2

27

Grass green

3

56

Lemon

4

82

Lemon

5

163

Orange

Distance Solvent Front Moved 170 mm

Data Table 4.2

0.9588 = Rf value for Carotene (yellow to yellow-orange)

0.4059 = Rf value for Xanthophyll (yellow)

0.1588 = Rf value for Chlorophyll a (bright green to blue-green)

0.0294 = Rf value for Chlorophyll b (yellow-green to olive green)

Part B

Graph 4.1

Transmittance Data Table

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

Conclusion

Part B

The hypotheses were supported by the experimental data. No significant change in transmittance indicative of photosynthesis occurred in cuvette 2, 4, or 5, while cuvette 3 experienced a very rapid initial change in transmittance, which leveled off as the DPIP ran out. As expected, cuvette 2 could not photosynthesize and break down the DPIP because it had no energy source. Cuvette 4 could not photosynthesize because boiling denatures the proteins of the photosystem, rendering the chloroplasts unable to function. Cuvette 5 did not show photosynthesis because there were no chloroplasts at all. Measuring percent transmittance is an effective way of determining if photosynthetic processes are taking place because photosynthesis, which uses light energy to excite electrons, then reduces DPIP with those electrons. When DPIP is reduced, it changes color from blue to clear. A clear substance allows more light to pass through than a tinted substance, so the colorimeter reads a higher percent transmittance after photosynthesis has taken place. In cuvette 3, photosynthesis was able to occur in the presence of light and working chloroplasts, and it quickly reduced all the DPIP, with the transmittance change leveling off because the DPIP had been used up in the reaction.

In cuvette #2, there is little variation, with the class average transmittance ranging from 19.9% to 17.4%, hardly significant. Similar results are seen in cuvette #4, ranging from 24.3% to 30.1%. Any change in transmittance is likely due to the fact that DPIP tends to break down over time when exposed to light (which is why it must be stored in amber bottles). Even the dark cuvette was exposed to small amounts of light when it was being transferred into the colorimeter. The DPIP in cuvette 4 was exposed to more light, so it tends to break down more. In cuvette #5, the results are slightly unusual, as one would expect the light transmittance to increase because of DPIP breakdown. However, the difference isn't enough to be significant, and natural error associated with measuring and averaging the data probably accounts for it.

This experiment was a good review of the photosynthetic process from last semester, and served as an opportunity to become familiar with different types of lab equipment. I had never used a colorimeter before, and I was also unfamiliar with cuvettes. The lab also stressed the importance of taking things step-by-step and paying close attention to directions, so as to add chemical ingredients to the cuvettes at the proper time and not skew the results by starting the reaction too early.

Analysis

Part A

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

The solvent moves up the paper by capillary action, carrying with it the dissolved pigments. However, the pigments are not equally soluble in the solvent that is transporting them, so they move at different rates. They are also different in their degrees of molecular attraction and bonding to the paper fibers. Solutes that bind easily to cellulose are found farther away from the solvent front. The size of the solute molecules may also play a role in how easily they move up the paper.

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

No, the Rf value should be different when a new solvent is used. The placement of pigment lines is dependent on the attraction between the pigment molecules and the paper, which shouldn't change, but the placement is also dependent on how soluble the pigment is in the solvent. Pigments will respond differently depending on the solvent used. Some may travel farther while others stay closer to the bottom of the paper. Therefore, the Rf value, given as the distance of pigment migration divided by distance of solvent front migration, should change because pigment migration changes.

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

Chlorophyll a is the only chlorophyll molecule located in the reaction center of the photosystem, which is where the first light-driven photosynthetic reaction occurs. The other pigments serve to “gather” light over a larger surface and spectrum, which is then transferred to the chlorophyll a. Carotenoids also serve in photoprotection, taking up extra light that could damage chlorophyll, like UV rays.

Part B

1. What is the function of DPIP in this experiment?

DPIP (2,6-dichlorophenol-indophenol) functions as an electron receptor. Electrons boosted to high energy levels by exposure to light reduce the DPIP, which changes its color and visibly indicates the reaction taking place.

2. What molecule found in chloroplasts does DPIP “replace” in this experiment?

DPIP “replaces” the natural electron acceptor NADP (which then reduces to NADPH).

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

The electrons are part of the photosystem in plant leaf cells. The pigment cells, such as chlorophyll, absorb light, which excites the electrons to higher energy levels, and the energy is used to produce ATP and reduce NADP/DPIP.

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

The spectrophotometer (actually a colorimeter) measures light transmittance as a percentage through a medium.

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

Darkness caused no significant change in the amount of reduced DPIP. Without light as an energy source to excite the electrons, they are not rising to higher energy levels and subsequently reducing DPIP. They simply remain at their lower energy levels, and the DPIP also does not exhibit significant change.

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

Boiling denatures the enzymes of the chloroplasts, which inhibits their normal function, meaning they are unable to absorb light, boost electrons to a higher energy level, and subsequently reduce the DPIP. Any change in the DPIP is due to a small amount of natural breaking down that occurs when it is exposed to light, and not to action of the chloroplasts, which have been rendered useless.

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

The light is the energy source used to excite the electrons, which reduce the DPIP from blue to clear. The clearer the substance in the cuvette is, the more light is transmitted through it (a higher percent transmittance). If there is no energy source, like the dark setup, the DPIP cannot be reduced and will not change color, meaning that approximately the same amount of light penetrates the substance in each percent transmittance measurement.

8. Identify the function of each of the cuvettes.

Cuvette 1 - “Calibration Cuvette”, used to calibrate the colorimeter because it contained both independent variables in their unaltered forms (unboiled chloroplasts and light), but accounted for any effect of the water, phosphate buffer, or chloroplasts on the transmittance.

Cuvette 2 - Cuvette 2, which contained DPIP and unboiled chloroplasts, was used to determine the importance of light to the processes of photosynthesis. The cuvette showed that even healthy chloroplasts cannot function properly without light, their source of energy.

Cuvette 3 - Cuvette 3 contained DPIP, unboiled chloroplasts and was exposed to light. It showed that electron reduction occurs rapidly in a situation with both factors - light and functional chloroplasts. However, the other cuvettes were used to prove that both factors contributed and were necessary to the process.

Cuvette 4 - The fourth cuvette, containing DPIP and boiled chloroplasts exposed to light, showed that reduction cannot occur without functional chloroplasts. The boiling damaged them so that they were not able to absorb the light, although it was present.

Cuvette 5 - “Control Cuvette”, containing DPIP but no chloroplasts, was important to make sure that DPIP did not change significantly on its own, which could skew the results of the other experimental cuvettes.

Bibliography

(Unknown author and editor) Biology Lab Manual for Students, pp. 1-18. College Entrance Examination Board, 2001.

Campbell, Neil A.; Reece, Jane B. Biology: Sixth Edition, pp. 182-185. Benjamin Cummings, 2002. San Francisco, California.

Section

Points Earned

Out of

Part A

Data

3

3

Analysis Questions

9

9

Part B

Purpose

4

4

Variables

10

10

Hypothesis

9

10

Data

15

15

Conclusion

25

25

Analysis Questions

23

24

Total

98

100


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