GENERAL BIOLOGY LAB II (BSC1011L)
Lab #3: Photosynthesis
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OBJECTIVES:
 Compare and contrast photosynthesis with cellular respiration.
 Separate photosynthetic pigments by paper chromatography and calculate Rf
values.
 Review light-dependent and light-independent reactions.
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INTRODUCTON:
Photosynthesis is a series of chemical reactions that use the energy from sunlight
to convert water (H2O) and carbon dioxide (CO2) into glucose (C6H12O6) and oxygen
(O2). Through this process autotrophs generate Adenosine Triphosphate (ATP), an
essential component for synthesis of organic molecules and growth. While only
organisms in the Kingdoms Bacteria, Protista and Plantae (Fig. 1) obtain energy through
photosynthesis, the oxygen that they produce sustains life on Earth, making organismal
diversity possible.
Figure 1. Organisms that photosynthesize
In addition to removing approximately 200 billion tons of carbon from the
atmosphere, the oxygen autotrophs produce as a byproduct of photosynthesis is utilized
by aerobic organisms for cellular respiration. In fact, cellular respiration and
photosynthesis are the exact opposite of each other (Fig. 2); during photosynthesis the
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electrons generated from the oxidation of water are used to reduce carbon dioxide to
glucose while in cellular respiration, glucose is oxidized to carbon dioxide and oxygen is
reduced to water.
Photosynthesis: 6H2O + 6CO2 + light  C6H12O6 + 6O2
Cellular respiration: C6H12O6+ 6O2  6CO2 + 6H2O
Figure 2. Comparison of photosynthesis and cellular respiration
In plants, photosynthesis occurs in the leaves in specific structures called
chloroplasts (Fig. 3). Chloroplasts, like mitochondria, are believed to have arisen via the
endosymbiotic theory from cyanobacteria (See Lab 2 Task sheet). As a result of this
symbiotic partnership, chloroplasts resemble bacteria structurally, i.e., they are
encapsulated by a double membrane system and they contain their own circular DNA. In
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chloroplasts, the outer layer encloses the entire organelle, which is filled with a semifluid matrix called the stroma while the inner membrane is composed of thylakoid disks
stacked into columns referred to as grana (singular = granum).
Figure 3. General structure of a chloroplast
Photosynthesis is subdivided into three stages: (1) light-capturing events, (2)
light-dependent (photochemical/light) reactions and (3) light-independent
(biochemical/dark) reactions (Fig. 4). During the light-capturing portion of
photosynthesis, photons of light are absorbed by the chlorophyll present in the thylakoids,
ultimately resulting in excitation of the electrons present. Once excited, the electrons are
taken up by electron acceptors to generate ATP and NADPH during the light reactions.
These products are then utilized in the dark reactions for the synthesis of glucose from
the “fixation” of carbon dioxide. The light and dark reactions are compared in Figure 5.
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Figure 4. General Overview of Photosynthesis
Figure 5. Comparison of light and dark reactions of photosynthesis
Light Reactions:
The light-dependent reactions are further divided into cyclic and noncyclic
photophosphorylation. For noncyclic photosynthesis to occur, the light capturing events
must take place in Photosystem II (P680), which absorbs wavelengths of light around 680
nanometers (nm). The excited electrons are then passed through a series of electron
acceptors [i.e., the electron transport chain (ETC)] until they reach Photosystem I (P700),
whose peak absorption of light is approximately 700nm. At this point, water molecules
are split to release 2 Hydrogen molecules, 1 Oxygen molecule and 2 electrons (used to
replace those being lost during the light capturing events in Photosystem II). Meanwhile,
as the excited electrons move through the ETC from Photosystem II to Photosystem I,
they cause Hydrogen molecules to move across the thylakoid membrane into the lumen
via an electrochemical gradient, ultimately resulting in the production of ATP via
chemiosmosis. Subsequent excitation of the electrons in Photosystem I results in the
reduction of NADP+ to NADPH (Fig. 6).
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Figure 6. Noncyclic photophosphorylation
For some species however, noncyclic photophosphorylation does not generate
sufficient ATP. Therefore, to increase ATP yield, these organisms can bypass
Photosystem II entirely, i.e., they carry out cyclic photophosphorylation instead. During
this process, the excited electrons from Photosystem I are used to synthesize ATP only.
Consequently, NADP+ is not reduced and, in contrast, to noncyclic
photophosphorylation, there is no need to split water molecules (Fig. 7).
Figure 7. Cyclic photophosphorylation
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Dark Reactions:
Plants use the products (ATP and NADPH) of the light-dependent reactions to
build carbohydrates during the light-independent reactions. The dark reactions, also
known as the Calvin Benson cycle, take place in the stroma of the chloroplasts and, as
their name implies, do not require light directly. During this stage of photosynthesis, 6
molecules of CO2 are captured by ribulose bisphospate carboxylase (RuBP, the most
abundant enzyme on Earth) to produce 1 molecule of glucose through a series of carbon
fixation reactions. This process is shown in detail in Figure 8.
Figure 8. The Calvin-Benson cycle
TASK 1: Paper Chromatography of photosynthetic pigments
Pigments are substances that absorb light in the visible spectrum (400 -700 nm) and
transfer the light energy to electrons. There are two types of pigments: primary and
accessory. Primary pigments, i.e., chlorophylls a & b, are an integral part of
Photosystems I & II and are used during the light-dependent reactions to absorb photons
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of light. Accessory pigments (e.g. carotenes and xanthophylls), on the other hand, act
as magnifiers that capture and transfer light energy to chlorophyll a.
Different pigments absorb different wavelengths of light as shown in Figure 9;
chlorophylls absorb light at wavelengths in the ranges of 425-450nm (blue) and 625-675
nm (orange-red) whereas carotenoids have a much broader absorption peak, ranging from
400-525 nm (blue-green). Also note that the color of a pigment originates from the
wavelengths of light that are reflected, i.e. those that are not absorbed. As illustrated in
Figure 9, chlorophylls a and b do not absorb light between 550 and 600 nm, the yellowgreen portion of the spectrum, thus, organisms in which these pigments predominate
appear green in color.
Figure 9. Absorption spectrum of primary and accessory pigments
Because most photosynthetic organisms generally have more than one pigment,
and many pigments reflect similar wavelengths, it is not possible to judge which
pigments a plant of interest contains simply by noting its color. However, we can
examine which pigments the plant possesses by chemically extracting the pigments and
then using paper chromatography to separate them. Paper chromatography is based on
the principle that different compounds adhere to (adsorb) and move across the
chromatography strip based on their molecular size, polarity and solubility in a particular
solvent. Applying a plant’s pigment extract to chromatography paper causes the pigments
to adsorb to the cellulose fibers on the strip. Subsequent immersion of the strip in a
solvent will cause the solvent to move up the strip as it becomes adsorbed by the fibers in
the paper. As the solvent moves up the strip, the pigments dissolve and move along with
it. The relative distance traveled by the solvent from the pigment origin compared to the
distance traveled by the different pigments in the extract (also measured from the same
point) is used to calculate the Rf (Retention factor) value of each pigment. Retention
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factor, calculated using the equation below, is dependent upon the compound’s chemical
properties; a small Rf value indicates a strong affinity of the compound for the
chromatography paper, i.e., the compound adsorbs strongly to the paper and will move
very slowly, while a large Rf value is indicative of a fast moving compound that
demonstrates a decreased affinity for the fibers in the strip. In addition, larger molecules
tend to move slower and adsorb more strongly to the chromatography paper than smaller
ones. Under identical conditions Rf values for each pigment should remain constant.
Rf = Distance moved by pigment / Distance moved by solvent
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Procedure A: Pigment extraction (performed by TA)
1. Homogenize 2 handfuls of spinach leaves (Spinacia oleracea) in 100mL of
80% acetone: 20% water in a blender.
2. Filter the homogenate through 4 layers of cheesecloth into a beaker.
 Suggestion: The smell of the extraction solution is very strong so this
step should be performed under the fume hood.
3. Transfer the extract into a large capped glass test tube.
4. Repeat steps 1-3 using 2 handfuls (3oz) of carrot sticks (Daucus carota subsp.
sativus).
-----------------------------------------------------------------------------------------------------------Procedure B: Separation of Pigments by Paper Chromatography:
NOTES:
a. The chromatography solvent is EXTREMELY dangerous and volatile.
b. All students MUST wear protective equipment including lab coats, safety
goggles and gloves.
c. If any solvent is spilled, inform your TA IMMEDIATELY.
d. Dispense the chromatography solvent in the fume hood using a glass
pipette.
e. At the end of the experiment, dispose of the used solvent in the bottle
labeled "chromatography solvent waste," also located in the fume hood.
Procedure:
1. Wearing gloves, obtain 2 strips of chromatography paper from the front of
the room. Make sure not to touch the strips with your bare hands or else
you will contaminate the paper.
2. Measure the chromatography strip to make sure that it fits properly in the
tube when placed on the hook. Keep in mind that you will have 2mL of
solvent in the tube which should not touch the pigment origin as shown in
figure 3.9. You will need to make a hole at the top of your strip for the
hook to be placed. You can use a hole puncher.
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3. Add 2mL of chromatography solvent to each of the two large glass test
tubes at your station and close each tube with the rubber stopper.
4. Using a pencil and a ruler, mark a line approximately 2cm from the tip of
the paper as well as a dot in the middle of that line, as shown in the figure
below (Figure 3.9).
5. With the capillary tube at your station, apply the spinach extract onto your
first chromatography strip directly on top of the penciled dot and then
blow on the strip to dry.
a. Repeat application of the extract at least 15 times to ensure that the
extract is sufficiently concentrated for separation to occur properly.
6. You should repeat step 5 but with the carrot extract using your second
chromatography strip and a different capillary tube.
Figure 3.9. Labeling the chromatography and measuring Rf
7. Remove the rubber stoppers from the test tubes containing the
chromatography solvent. Hook each chromatography strip on the pin in
each stopper so that the tip (but not the dot of plant extract) of the paper is
submerged in the solvent.
8. Place both test tubes in a test-tube rack and leave them undisturbed as
you observe the movement of the solvent up the paper.
9. Remove the strips before the solvent reaches the top of the paper (about 35 minutes). Immediately, mark the position of the solvent with a pencil
and set the strips aside to dry.
10. Dispose of the used chromatography solvent in the "chromatography
solvent waste” bottle which is found under the fume hood.
11. Once the strips are dry, calculate the Rf (see formula in the tables below)
for each pigment observed on each strip. Note: All distances are measured
from the point of pigment origin. Record your results in Tables 3.2 and 3.3
for the spinach and carrot extracts, respectively.
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12. Staple your chromatography strips or draw it in the spaces provided in
Tables 1 and 2.
Results:
Table 3.2: Spinach Extract
Distance solvent traveled in mm ____________
Pigment
Distance Pigment
Moved (mm)
Carotene (yellow-orange)
Xanthophylls (yellow)
Chlorophyll a (blue-green)
Chlorophyll b (yellow-green)
Chromatography Strip:
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Rf = distance pigment traveled
distance solvent traveled
Table 3.3: Carrot Extract
Distance solvent traveled in mm ____________
Pigment
Distance Pigment
Moved (mm)
Rf = distance pigment traveled
distance solvent traveled
Carotene (yellow-orange)
Xanthophylls (yellow)
Chlorophyll a (blue-green)
Chlorophyll b (yellow-green)
Chromatography Strip:
Questions:
1. What does a small Rf tell you about the characteristics of the moving
molecules?
2. Is it possible to have an Rf greater than 1? Why or why not?
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3. The accepted theoretical Rf values for each pigment in a green plant extract
are as follows: carotenes = 0.98; chlorophyll a = 0.59; chlorophyll b = 0.42;
xanthophylls = 0.67. Use these values to calculate the percent error of your
experiment separating the spinach extract. Show your work in the table below.
Percent Error:
|
Pigment
Carotenes
|* 100
Percent Error Calculations
Chlorophyll a
Chlorophyll b
Xanthophylls
4. Were the pigments present in the spinach extract also present in the carrot
extract? Is this what you would expect to see?
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TASK 2: Absorption spectrum
A spectroscope is an instrument that separates white light into its component
colors, ranging from violet (400 nm) to red (740 nm). Your TA will demonstrate how to
use the spectroscope at your station to observe this phenomenon. Wavelengths of light
that are no longer visible have been absorbed. Examine which colors are absorbed by the
spinach extract by placing it between the light and the spectroscope.
Questions:
1. Which color(s) are diminished or absent and therefore absorbed in the spinach
extract?
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2. Which color(s) are reflected in the spinach extract?
2. Complete the absorption spectrum below for both the spinach extract. For
each color, estimate the relative absorbance of that color by placing an X
above the color name at the appropriate position along the y-axis.
Absorbance
High
Low
Violet
Blue
Green
Yellow
Red
Color of Light
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TASK 3: Fluorescence (TA Demo)
As light strikes a plant, its chlorophyll molecules become excited causing
electrons to move into higher energy orbitals. This process initiates the light-dependent
portion of photosynthesis where the energy of the excited electrons is passed through a
series of pigment molecules until it reaches chlorophyll a. However, if these energized
electrons are unable to be passed onto the next pigment molecule in either Photosystem,
they fall back into their original lower energy orbital and release the remaining energy as
fluorescence. This excess energy is dissipated in the form of light with a larger, less
energetic wavelength.
Procedure:
1. Place the tube of spinach extract under a UV light.
2. View the tube from the side.
3. Note your observations in the space provided.
Observations:
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Questions:
1.
What color light does the extract fluoresce?
a. What wavelength does this color correspond to?
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TASK 4: Electron transport in chloroplasts
The purpose of this experiment is two-fold: (1) to indirectly measure
photosynthesis by observing the rate of electron transfer, and (2) to demonstrate that the
dark reaction is a separate and independent event from the light reaction. In order for the
light-dependent and light-independent reactions to be disparate events, the electron
transport chain should not require carbon dioxide to fix carbon. You will illustrate this
concept empirically by providing chloroplasts with an alternate electron-acceptor,
DCPIP, to transfer electrons.
DCPIP (2, 6-dichlorophenol-indolephenol) is a dye, which is dark blue in color
when oxidized and colorless when reduced, i.e., after accepting electrons. The rate of
DCPIP decoloration depends on its concentration and the rate of electron flow. Thus, by
using DCPIP as an artificial electron-acceptor, you will be able to monitor electron
transport, in the absence of CO2 fixation.
Note: DCPIP is very dangerous. Use extreme care and wear gloves.
-----------------------------------------------------------------------------------------------------------TA Preparation of Chloroplasts:
1. Please follow protocol given to you.
-----------------------------------------------------------------------------------------------------------Procedure:
1. Label the test tubes with tape with the test tube number and your group
number.
2. Add the buffer and water to each of the 4 test tubes with the amount
designated on Table 3.4.
3. Then add 0.5mL of the chloroplast mixture that your TA will give you to test
tubes 1, 2, and 4.
4. Add 0.5mL of DCPIP as your last step into test tubes 2, 3, and 4.
5. Cap all 4 of the test tubes.
6. Wrap tube 4 completely with aluminum foil.
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7. Mix the contents of each tube well.
8. Record the current colors of each test tube in Table 3.5 under “Before
Observed Results”.
9. Place the 4 test tubes in front of the light. Have someone keep an eye on them
as the reaction will occur within minutes (approximately 3-5 minutes).
10. Record expectations for each tube in Table 3.5 while you wait.
11. After 5 minutes, record the “After Observed Results” in Table 3.5.
Table 3.4: Test tube Preparation
Tube #
Chloroplasts
0.1M PO4 Buffer (pH 6.5)
H2 0
0.2mM DCPIP
1
0.5mL
3mL
2mL
0
2
0.5mL
3mL
1mL
0.5mL
3
0
3mL
1.5mL
0.5mL
4
Foil
0.5mL
3mL
1mL
0.5mL
Table 3.5: Results
Tube #
Expected Results
Before
Observed Results
(color)
After
Observed Results
(color)
1
2
3
4
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Explanation
Questions:
a. Which of the 4 tubes served as a negative control for chloroplast? Why?
b. Which of the 4 test tubes served as a negative control for DCPIP? Why
c. Which of the 4 test tubes served as a positive control? Why?
d. What was the purpose of covering tube 4 with foil?
e. Why was there some color change in the wrapped tube? (Hint: Does electron
transport occur without light?)
f. Are there electrons being accepted in tube #1 even though there’s no DCPIP
present? Explain your answer.
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TASK 5: Uptake of carbon dioxide during photosynthesis
In this experiment you will use Elodea, an aquatic plant, and a pH indicator, phenol
red, to demonstrate that plants use carbon dioxide (CO2). Phenol red is red in alkaline (basic)
solutions (pH > 7) and turns yellow in acidic solutions (pH < 7). To prepare for this
experiment, you will blow air into 3 tubes containing phenol red, releasing carbon dioxide
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into the liquid in each tube. The carbon dioxide will bind with the water in the phenol red
indicator to form carbonic acid (H2CO3).
Water
H2O
Carbon
dioxide
+ CO2
Carbonic
H2CO3
Hydrogen
acid
ion
H+ + HCO3-
Bicarbonate
ion
Questions:
1. Before blowing into the test tubes, what color would you expect the solution to be?
Explain.
You will then add Elodea to two of the tubes to begin carbon dioxide fixation process (i.e.
photosynthesis). As the amount of carbon dioxide decreases, the pH is going to change,
causing the phenol red indicator to change color.
2. In what direction (higher or lower) would you expect the pH to change as Elodea
fixes the CO2?
3. After blowing into the test tubes, what color should the phenol red indicator become?
Explain.
Procedure:
1. Fill three small screw cap test tubes half way with the phenol red indicator at your
station.
2. Label the tubes with tape with the test tube number and your group number.
3. Gently blow into each of the 3 tubes with a straw until the indicator turns yellow in
color. You should make sure that all 3 tubes are the same shade of yellow.
4. Add a piece of Elodea (approximately 7cm in length) to tubes 1 and 2 only.
5. Immediately screw the cap on each of the 3 tubes to prevent the escape of carbon
dioxide.
6. Completely cover tube 2 with aluminum foil.
7. Record your expectations for each tube in Table 3.6.
8. Place all 3 tubes in front of the light source.
9. Observe the tubes every 15 minutes for a total of 45 minutes. Record your
observations in Table 3.6.
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Table 3.6
Tube #
1
Contents
Elodea
Phenol Red
2
Elodea
Phenol Red
Foil
3
Phenol Red
Expected Results
Observed Results/Explanation
Questions:
1. What was the purpose of covering tube 2 with foil?
2. What was the purpose of tube 3?
3. Did tube 1 change color after the 45 minutes? Why?
4. Which one of the test tubes is considered the positive control? Why?
TASK 6: Check your fast plants and record any changes in your Fast Plant
appendix
TASK 7: Check and water your basil plants
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