Artifact Two: How Light/Dark and Boiled/Unboiled Environments Affect the Rate

of Photosynthesis

Lab partner(s): Elizabeth, Braden, Nathan

Date(s): February 23rd, 2021

Background:
The goal of this lab is to use the Colorimeter to measure color changes, study the effect of light on
photosynthesis, study the effect that the boiling of plant cells has on photosynthesis and compare the rates of
photosynthesis for plants in different light conditions. Photosynthesis is a complex process which involves the
use of light energy to convert carbon dioxide and water into sugar, oxygen, and other organic compounds.
This process occurs in two stages: light-dependent reactions and light-independent reactions. Light-dependent
reactions require light. Light-independent reactions do not require direct light. The light-dependent stage
produces glucose from carbon dioxide and water. During these types of reactions, water electrons are
“excited” as they pass through an electron transport chain in the membrane of the thylakoid. Chlorophyll
absorbs light energy that is used to excite the electrons in two different photosystems when they are in the
electron transport chain. The first photosystem (photosystem II) excited electrons generate ATP. In the second
photosystem (photosystem I) excited electrons produce reduced NADPH. ATP and NADPH are used in light-
independent reactions and the Calvin Cycle which produce glucose. During this specific experiment, DPIP (a
blue dye) is replacing NADPH in light reactions. When the dye is oxidized, it’s blue, but when reduced, it is
colorless. The DPIP should change from blue to colorless when reduced during photosynthesis. DPIP will come
into contact with chloroplasts if the cells are carefully and properly disrupted. The “chlorophyll” used in this
experiment is blended spinach leaves. Using the Colorimeter, color intensity and absorbance will be
measured. There are many factors that affect the rate of photosynthesis: light intensity, carbon dioxide
concentration, and temperature (Rate of Photosynthesis 1). Without proper light intensity, carbon dioxide
concentration, and temperature, photosynthesis cannot be performed. The light used in this experiment
should affect the photosynthetic rate because it affects both lighting and temperature, as the light produces
heat. Naturally, chlorophyll levels fluctuate over time (Chlorophyll Concentrations 1). Chlorophyll
concentrations are higher in the summer months, when the water temperatures rise.

Purpose:
The purpose of this lab is to determine how light levels and temperature, via boiling, affect the rate of
photosynthesis.

Hypothesis:
Part 1 (EFFECT OF DARKNESS): If the chloroplast is in the darkness, then photosynthesis cannot be performed,
because the chloroplast is what helps the plant convert light into energy.

Part 2 (EFFECT OF BOILING): If the chloroplast is boiled, then the rate of photosynthesis will slow or stop
because the nature of the chloroplast will be changed.

Materials:
● LabQuest 2
● Colorimeter
● Three cuvettes with lids
● Aluminum foil covered cuvette with lid
● Desk lamp
● 100 watt (or equivalent) bulb
 ● Stopwatch or clock with second hand
● 600 mL beaker
● 250 mL beaker
● Beral pipette
● 10 mL DPIP solution
● Tissues
● Distilled water
● Unboiled chloroplast suspension
● Boiled chloroplast suspension
● Ice
● Googles

Procedure:
1. Obtain and wear goggles.
2. Fill a 250 mL beaker with ice.
3. Obtain two plastic Beral pipets, three cuvettes with lids, and one aluminum foil covered cuvette with a
lid.
a. Mark one Beral pipet with a U (unboiled) and one with a B (boiled).
b. Mark the lid for the cuvette with aluminum foil with a D (dark).
c. For the remaining two cuvettes, mark one lid with a U (unboiled) and one with a B (boiled). Note:
One cuvette lid is left unmarked; use it for the blank.
4. Prepare a blank by filling the unmarked cuvette with 2.5 mL distilled water. Seal the cuvette with a lid. To
correctly use cuvettes, remember:
● Wipe the outside of each cuvette with a lint-free tissue.
● Handle cuvettes only by the top edge of the ribbed sides.
● Dislodge any bubbles by gently tapping the cuvette on a hard surface.
● Always position the cuvette so the light passes through the clear sides.
5. Connect the Colorimeter to your LabQuest2.
6. Calibrate the Colorimeter.
a. Place the blank in the cuvette slot of the Colorimeter and close the lid.
b. Press the < or > button on the Colorimeter to select a wavelength of 635 nm (Red).
c. Press the CAL button until the red LED begins to flash, then release. When the LED stops flashing, the
calibration is complete.
7. Obtain a lamp and a 600 mL beaker filled with water. Arrange the lamp and beaker as shown in Figure 1.
The beaker will act as a heat shield, protecting the chloroplasts from warming by the lamp. Do not turn the
lamp on until Step 10.
Figure 1

8. Locate the unboiled and boiled chloroplast suspension prepared by your instructor.
a. Before removing any of the chloroplast suspension, gently swirl to resuspend any chloroplasts which
may have settled out.
b. Using the pipet marked U, draw up ~1 mL of unboiled chloroplast suspension.
c. Using the pipet marked B, draw up ~1 mL of boiled chloroplast suspension.
d. Set both pipettes in the small beaker filled with ice at your lab station to keep the chloroplasts
cooled.
9. Add 2.5 mL of DPIP/phosphate buffer solution to each of the cuvettes. Important: Perform the following
steps as quickly as possible and proceed directly to Step 10.
a. Cuvette U: Add 3 drops of unboiled chloroplasts. Place the lid on the cuvette and gently mix; try not
to introduce bubbles in the solution. Place the cuvette in front of the lamp as shown in Figure 1.
Mark the cuvette’s position so that it can always be placed back in the same spot.
b. Cuvette D: Add 3 drops of unboiled chloroplasts. Place the lid on the cuvette and gently mix; try not
to introduce bubbles in the solution. Place the foil-covered cuvette in front of the lamp as shown in
Figure 1 and mark its position. Mark the cuvette’s position so it can always be returned to the same
spot. Note: Make sure that no light can penetrate the cuvette.
c. Cuvette B: Add 3 drops of boiled chloroplasts. Place the lid on the cuvette and gently mix; try not to
introduce bubbles in the solution. Place the cuvette in front of the lamp as shown in Figure 1. Mark
the cuvette’s position so it can always be returned to the same spot.
10. Take absorbance readings for each cuvette. Invert each cuvette two times to resuspend the chloroplast
before taking a reading. If any air bubbles form, gently tap on the cuvette lid to knock them loose.
 a. Cuvette U: Place the cuvette in the device and close the lid. Allow 10 seconds for the readings to
stabilize, then record the absorbance value in Table 1. Remove the cuvette and place it in its original
position in front of the lamp.
b. Cuvette D: Remove the cuvette from the foil sleeve and place it in the device and close the lid. Wait
10 seconds. Record the absorbance value in Table 1. Remove the cuvette and place it back into the
foil sleeve. Place the cuvette in its original position in front of the lamp.
c. Cuvette B: Place the cuvette in the device and close the lid. Allow 10 seconds for the readings to
stabilize, then record the absorbance value in Table 1. Remove the cuvette and place it in its original
position in front of the lamp.
11. Turn on the lamp.
12. Repeat Step 10 when 5 minutes have elapsed.
13. Repeat Step 10 when 10 minutes have elapsed.
14. Repeat Step 10 when 15 minutes have elapsed.

Data Methods:

The data that will be collected are the absorbances rates of chloroplasts that are boiled, unboiled, and in the
dark. The independent variables for this experiment are darkness and the boiled chloroplast. The dependent
variable is the rate of photosynthesis. The control is unboiled chloroplasts in the light. The unboiled
chloroplasts in the light will stay the same throughout the entire lab.

Data:
Table 1. The effect of darkness and boiling on the light absorbance value of spinach @ 635 nm.

Time (min.) Absorbance Unboiled Absorbance in Dark Absorbance Boiled

0 .86 .88 .78 .86 .71 .71

5 .46 .25 .76 .78 .64 .63

10 .25 .23 .75 .77 .64 .63

15 .19 .23 .76 .77 .64 .63

Table 2. The effect of darkness and boiling on the photosynthetic rate of chloroplasts in spinach.

Chloroplast Condition
Rate of Photosynthesis

Unboiled 0.044 0.0394

Dark 0.0014 0.0056

 Boiled 0.0042 0.0048

Graph 1.

Analysis
For our lab, we tested two different variables: chloroplasts in different lighting and chloroplasts that
were boiled/unboiled. The first part of our lab was focused on the lighting. When the chloroplasts were
exposed to no light, photosynthesis could not be performed. The rate of photosynthesis was extremely low. In
our first trial, we got 0.0014 as our rate of photosynthesis when the cuvette was covered in foil, therefore
exposed to little to no light. In our second trial, it was higher but still low. Overall, the rate of photosynthesis in
the dark was lower than 0.01 at the end. My hypothesis was correct for the first half of the experiment. I
predicted that, without light, photosynthesis would not occur.
When observing what boiled and unboiled chloroplasts do, we found that unboiled chloroplasts have
the highest rate of photosynthesis. The chloroplasts could actually perform photosynthesis here. The boiled
chloroplasts could not perform photosynthesis due to the temperature and manipulation they had been
through. The untouched chloroplasts that were exposed to the light did the best because the chloroplasts
were not manipulated and they were exposed to light, which is a necessity in the process of photosynthesis. In
the end, the unboiled chloroplasts had a rate of over 0.03 both trials. This shows us that photosynthesis did
occur. My hypothesis was correct for the second half of the experiment as well. I predicted that the boiled
chloroplasts would not perform photosynthesis because of their changed nature.

Evaluation
There are three possible mistakes or errors that could have occurred during this experiment: a cuvette could
have been murky/not properly cleaned, cuvette could be placed incorrectly in the Colorimeter, and the
unboiled/boiled chloroplasts were sitting out longer for our class so it could have affect numbers. With careful
procession, the first two of these errors could have been avoided. My group avoided both of those issues,
which is why I called them possible errors. However, the last one could not have been avoided. That happened
because we are the second class of the day.

Discussion
Going into this lab, I had some knowledge about the nature of photosynthesis. I knew the basics:
photosynthesis uses light energy to convert carbon dioxide into sugar, oxygen and other organic compounds.
When first introduced to photosynthesis, I understood that light was an essential part of the process.
Therefore, my hypothesis about the cuvette in the dark was correct. I knew photosynthesis could not be
performed without light. When it came to the boiling and unboiling of chloroplasts, my hypothesis was mostly
a guess, not based on prior knowledge. I assumed chloroplast would not perform well under changed nature.
However, I knew that chloroplast would perform when it was in its natural state, considering it does in nature.
A further area of study we could explore is what chloroplasts do when they are frozen.

References
RSC | Advancing the Chemical Science. (n.d.). Rate of photosynthesis: limiting factors. Reckitt

Benckiser.https://edu.rsc.org/download?ac=12620#:~:text=The%20main%20factors%20affecting

%20rate,carbon%20dioxide%20concentration%20and%20temperature.

OzCoasts. Chlorophyll A Concentrations.

ozcoasts.org.au/indicators/biophysical-indicators/chlorophyll_a/.
 RUBRIC FOR SELF-ASSESSMENT AND GRADING:
THIS RUBRIC WILL BE USED TO GRADE YOUR REPORT. PLEASE REVIEW PRIOR TO SUBMITTING YOUR ASSESSMENT.
PHOTOSYNTHESIS LAB
Key: Highlighted Yellow are the ‘correct’ items, red font color are the ‘missing’ items.

Score Description
RS7: SE2 Scientific Investigations & Data ● Planning and Carrying Out Investigations
● Analyzing and Interpreting Data

In addition to a 3.0 score, student demonstrates

sophisticated applications such as:
☐ In depth analysis and explanations of the effect of darkness
4 and boiling on the rate of photosynthesis in spinach cells.
☐ Connections made beyond the context of the topic such as:
● Effect of temperature on photosynthetic enzyme structure
and function
● Effect of darkness on specific step(s) of the light dependent
reactions
☐ Multiple references used to provide additional
background information, explanation of results, or
procedural improvements.
☐ Formal, elaborate, and colorful language and sentence
structure demonstrates mastery.

While engaged in grade appropriate tasks, the student

demonstrates the following:
☐ All required lab report sections are present and completed
3 accurately.
☐ Sentence structure, grammar, and scientific vocabulary
used appropriately.
☐ Qualitative data is correctly presented in both table and
graph format.
 ☐ Analysis of data provides claims, supported with evidence
and backed up with reasoning.
☐ Explanation of hypotheses are supported or rejected.
☐ Evaluation of procedural errors and improvements is
thorough.
☐ Connections made within the context of the topic.
☐ At least one APA format reference used to support and
enhance background information or analysis
☐ Little to no errors present in APA format for in-text citations
and references.

No major errors or omissions regarding the simpler details

such as:
☐ One or more lab report section is missing or incomplete.
2 ☐ Basic sentence structure and/or vocabulary used.
☐ Data is presented in table and graph format with minor
errors.
☐ Basic analysis of data; Analysis lacks data, evidence, and/or
reasoning.
☐ Evaluation of procedural errors and/or improvements lacks
detail.
☐References missing, incomplete, or improperly formatted

1 Major errors or omissions present.

RS2: ● Construct an explanation based on evidence for how

Matter & Energy energy and matter change and/or flow into, out of, and
within the system during the processes involved in
photosynthesis.

4 In addition to a 3.0 score, student demonstrates

sophisticated applications such as:
☐ In depth analysis and explanations of changes in energy
and matter such as:
● Use of explicit details, names of structures, and steps
involved in the photosynthetic process throughout
the report
☐ Connections made beyond the context of the topic such as:
● Relating and explaining the processes of
photosynthesis based on prior knowledge such as
enzyme structure and function, pigment and
wavelength absorption and reflection, cellular
transport mechanisms, and membrane structure
● Connection to the carbon cycle and how human-
caused changes to that cycle may affect
photosynthesis and/or cellular respiration
● How plants that live in extreme environments are
specially adapted to be able to perform
photosynthesis under stress

3 While engaged in grade appropriate tasks, the student

demonstrates the following:
☐ Thorough explanation of changes in energy and matter
 during photosynthesis
☐ Discuss how the molecules involved in the process of
photosynthesis relate to those of cellular respiration
☐ Explain the production and use of ATP during
photosynthesis
☐ Explain the role of DPIP as a replacement for NADPH in the
light reactions

2 No major errors or omissions regarding the simpler details

such as:
☐ Basic explanation of photosynthesis and its relationship to
cellular respiration
☐ Basic explanation of the roles of ATP and DPIP

1 Major errors or omissions present.