AP Biology
Name: __________________________
Photosynthesis (Lab 4B)
OVERVIEW
In the first part of this laboratory you separated plant pigments using chromatography. You will now measure the
rate of photosynthesis in isolated chloroplasts. The measurement technique involves the reduction of the dye, DPIP.
The transfer of electrons during the light-dependent reactions of photosynthesis reduces DPIP and changes its color
from blue to colorless.
AP Biology LAB 4 OBJECTIVES
Before doing this laboratory you should understand:

how chromatography separates two or more compounds that are initially present in a mixture

the process of photosynthesis

the function of plant pigments

the relationship between light wavelength or light intensity and photosynthetic rate
After doing this laboratory you should be able to:
 separate pigments and calculate their Rf values
 describe a technique to determine photosynthetic rates
 compare photosynthetic rates at different temperatures, different light intensities, and different wavelengths
of light in a controlled experiment
 explain why the rate of photosynthesis varies under different environmental conditions
The process of photosynthesis involves the use of light energy to convert carbon dioxide
and water into sugar, oxygen, and other organic compounds. This process is often summarized
by the following reaction:
6 H2O + 6 CO2 + light energy C6H12O6 + 6 O2
This process is an extremely complex one, occurring in two stages. The first stage, called
the light reactions of photosynthesis, requires light energy. The products of the light reactions
are then used to produce glucose from carbon dioxide and water. Because the reactions in the
second stage do not require the direct use of light energy, they are called (perhaps incorrectly) the
dark reactions of photosynthesis.
In the light reactions, electrons derived from water are “excited” (raised to higher energy
levels) in several steps, by pigment groups called photosystems I and II. In both steps,
chlorophyll absorbs light energy that is used to excite the electrons. Normally, these electrons
are passed to a cytochrome containing an electron transport chain. In the first photosystem, these
electrons are used to generate ATP. In the second photosystem, excited electrons are used to
produce the reduced coenzyme nicotinamide adenine dinucleotide phosphate (NADPH). Both
ATP and NADPH are then used in the “dark” reactions to produce glucose.
In this experiment, a blue dye (2,6-dichlorophenol-indophenol, or DPIP) will be used to
replace NADP in the light reactions. When the dye is in an oxidized state, it is blue. When
reduced, however, it turns colorless. Since DPIP replaces NADP in the light reactions, it will
turn from blue to colorless when reduced during photosynthesis. This will allow you to monitor
the rate of photosynthesis. In order to allow the DPIP to come into contact with chloroplasts, the
cells will need to be carefully disrupted. You will test for photosynthetic activity in isolated
spinach chloroplasts. The intensity of color and its change through time, measured as
absorbance, will be detected by a colorimeter.
1
Photosynthesis
Some useful definitions and background information:
• Transmittance--the amount of light that gets through the sample, as compared with the
amount that gets through the clear “blank.” It is usually expressed as a percentage (%T). If the
percentage is to be used in a calculation, it should be converted to a decimal fraction
(75% to 0.75, etc.).
• Absorbance--the logarithm of the reciprocal of the transmittance, where transmittance is
expressed as a decimal fraction. In the formulas below “A” indicates absorbance and “T” is the
transmittance.
A = log 1/T
T = 10(-A)
%T = 10(-A) x 100
Why are absorbance readings used? When you plot transmittance readings against
concentration for sample, a curve will be produced. When absorbance is plotted against
concentration, a straight line is produced. It is easier to work with straight lines in most cases.
The amount of light absorbed by the sample in the cuvet is directly proportional to the
concentration of the sample. That is really common sense: the more concentrated a solution, the
more light it absorbs. This fact is called the Beer-Lambert Law.
These properties can be useful in science. In chemistry, the measured absorbance of a
few solutions of known concentration of a substance X can be used to create a standard curve
(line). Solutions of X whose concentrations are unknown can be determined by simply
measuring their absorbance in a colorimeter or spectrophotometer and reading the standard curve
to reveal their concentrations. In biology, the same technique can be used to determine
population growth sizes of bacteria growing in solution, for instance. The more light absorbed
by the bacterial suspension, the more bacteria are in the solution.
In this lab, the LabPro will take the light intensity readings it senses coming through your
specimens and compare them with the light that was transmitted through the “blank.” It will then
produce absorbance readings. You can read the % Transmittance and Absorbance off the top of
the screen using Logger Pro3, or just enter the absorbance into the Excel worksheet called
“APBio Lab 4B Absorbance Data Table” which will calculate the %T automatically in the AP
Biology Labs and Tables on “Instruction on Milledge” in the RPatterson folder..
MATERIALS
Windows PC Laptop & Vernier Colorimeter
LabPro System & Power Cord
Logger Pro3 Software
USB Cable
cuvettes with lids
0.1 M phosphate buffer
5-mL syringe
floodlight
Beaker for distilled water
2
DPIP solution
two chloroplast suspensions (boiled & unboiled)
large glass vessel filled with water (heat sink)
Ice container
KimWipes
watch or clock with second hand
1-mL syringe for the buffer & DPIP
two Pasteur pipettes
Marker pen
Photosynthesis
PROCEDURE
1. Obtain and wear goggles and an apron.
2. Connect the LabPro to your computer and connect the colorimeter to the LabPro in the CH1
port. Then boot up Logger Pro3. Click on the LabPro menu button. Click on CH1 (channel
1) and go to “choose sensor” and left click on “colorimeter.”
3. Prepare the computer for data collection by clicking Open in the File Menu, and opening the
file “Exp 07 Colorimeter” in the Experiment 7 folder of Biology with Computers. You
should see absorbance and % transmittance values on the screen.
4. Calibrate your Colorimeter.
a. You are now ready to calibrate the colorimeter. First prepare a “blank” by filling a
cuvette with the mixture described in Table 1 at the top of page 4—see cuvette #1. Cover
it with one of the translucent caps. To correctly use a colorimeter cuvette, remember:

Use a marker
to make a reference mark on the lids of the cuvettes in line with the clear
sides of the cuvette, and on the ribbed side of each cuvette write the appropriate number
1– 5. The “blank” will be #1.
 All cuvettes should be wiped clean and dry on the outside with a tissue. Use a KimWipe.
 Handle cuvettes only by the top edge of the ribbed sides.
 All solutions should be free of bubbles, so to suspend the chloroplasts and mix the
contents of the cuvettes, gently invert them two or three times--don’t shake them.
Calibration Procedure
a. Insert the “Blank” (Cuvette #1) into the colorimeter with its index mark in line with the
white arrow in the colorimeter. The index mark on the lid of the cuvette should be in line
with the clear sides of the cuvette.
b. Close the lid and use the arrows to set the colorimeter on the 635 nm wavelength.
c. Push the CAL button. When the red light stops flashing, the device is ready to go.
5. Obtain a large beaker, fish bowl or flask and fill it with water. This will act as a heat shield
(heat sink) for the cuvettes with the chloroplasts, protecting them from warming due to the
flood lamp.
6. Place a flood lamp on one side of the heat shield (heat sink) as shown in the diagram below.
7. Obtain a sample of boiled and another of unboiled chloroplasts from Mr. Patterson. Keep
both samples on ice at all times.
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Photosynthesis
Table 1: Cuvette Ingredients
1
Blank
2
Unboiled
Chloroplasts
Light
3
Unboiled
Chloroplasts
Dark
4
Boiled
Chloroplasts
Light
5
No
Chloroplasts
Phosphate Buffer
.5 mL
.5 mL
.5 mL
.5 mL
.5 mL
Distilled Water
2 mL
1.5 mL
1.5 mL
1.5 mL
8 drops*
8 drops
8 drops
–
1.5 mL
+ 8 drops
–
Boiled Chloroplasts
–
–
–
8 drops
–
DPIP (Add this stuff
LAST to all cuvettes
2-5 sequentially)
–
.5 mL
.5 mL
.5 mL
.5 mL
Unboiled Chloroplasts
*If 8 drops is too many, we should know within the first 5 minutes or so if we see the blue
DPIP decolorizing too fast. We will then stop, refill the cuvettes and start again.
8.
Get the other four numbered cuvettes with lids. Put the ingredients listed in Table 1 into
each--EXCEPT FOR THE DPIP. Follow directions carefully. Swirl the chloroplast
samples before adding the drops to the cuvettes. Cover each cuvette with a translucent
plastic lid except for #3 that should have a blue opaque one
9.
Use the 1mL syringe to put in the phosphate buffer, then use the 5 mL syringe to add the
distilled water. Use one Pasteur pipette for the unboiled chloroplasts and the other for the
boiled chloroplasts. Remember, don’t shake the cuvettes—just cap them and invert gently a
couple of times.
10. Place the cuvettes on marked masking tape in front of the light in spots they will occupy
throughout the experiment. TURN ON THE SPOTLIGHT. Then, in order (cuvettes #2–
#5), use the 1 mL syringe to add 0.5 mL of DPIP solution, gently invert each cuvette to
resuspend the chloroplasts, and take an transmittance reading for each as the time = 0 data
point. Timing is important, keep track of it! Measure the transmittance in this same order
each time so that each cuvette is measured at precisely the same intervals. (Once you add the
DPIP to cuvette #3, after taking you initial reading, you will place it into the 135 mm film
canister and close the canister’s lid to put it into total darkness. Care should be exercised
when removing the canister’s lid to take later readings so that the cuvette is not
unnecessarily jostled. Always use two hands to open and close the canister.)
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Photosynthesis
11. Take absorbance & transmittance readings for each cuvette as follows:
aFor cuvettes 2, 4 & 5: Invert the cuvette a couple of times then place the cuvette in the
cuvette slot of the colorimeter and close the lid. Allow about 3-4 seconds for the readings
to stabilize, and then record the absorbance and the % Transmittance values displayed on
the computer screen into Table 2 below. Then remove the cuvette, invert it gently a
couple of times, and place it in its original position in front of the light.
bFor cuvette #3: Remove the cuvette from the film canister, invert it gently a couple of
times, and place it in the cuvette slot of the colorimeter. Close the colorimeter lid and
wait about 3-4 seconds. Record the absorbance value displayed on the laptop screen into
Table 2 below. Remove the cuvette, invert the cuvette a couple of times gently, and place
it back into the film canister. Place the canister in its original position in front of the
light.
12. Repeat Step 11 when 5 minutes have elapsed.
13. Repeat Step 11 when 10 minutes have elapsed.
14. Repeat Step 11 when 15 minutes have elapsed.
15. Repeat Step 11 when 20 minutes have elapsed. (Optional depending on time available)
16. Enter the Group absorbance and % Transmittance data into Data Table 2 below.
17. Enter your %T values onto the class computer at the front of the room. The class means will
be emailed to you later. This data table will probably have to be printed sideways (landscape
format).
18. Graph the data by entering your %T (not absorbance) group data (four curves in the same
graph) into Graphical Analysis. Then graph the four class means curves. Graphical Analysis
to help you calculate the rates for all four treatments for both your group data and the class
means. Report those rates on Data Table 3 on the next page. Data tables 2 and 3 are
available in the RPatterson folder on “Instruction on Milledge”
Data Table 2
Group Data: Absorbance & % Transmittance for DPIP
Time
(min)
Unboiled/Light
A
%T
Unboiled/Dark
A
%T
Boiled
A
%T
No
Chloroplasts
A
%T
0
5
10
15
20
5
Photosynthesis
Data Table 3
Data Table 5: Reaction Rate (%T) for Group and Class
Chloroplast
Suspension
Class Mean
Group Rate of
Rate of
Photosynthesis
Photosynthesis
(∆%T/minute)
(∆%T/minute)
Unboiled in
Light
Unboiled in
Dark
Boiled
Chloroplasts
No
Chloroplasts
QUESTIONS
1. What role does DPIP play in this experiment?
2. What molecule found in chloroplasts does DPIP “replace” in this experiment? Explain.
3. What is the source of the electrons that will reduce DPIP?
4. What was actually being directly measured with the colorimeter in this experiment?
5. What is the effect of darkness on the chloroplasts’ reduction of DPIP? Explain.
6. What is the effect of boiling the chloroplasts on the subsequent reduction of DPIP? Explain.
7. What reasons can you give for the differences seen in the shape of the time course curves
between unboiled chloroplasts incubated in the light and those kept in the dark?
8. Why was it necessary to use Cuvettes #1 and #5 in the experiment? What were their roles?
9. Why did we use 635 nM as the wavelength setting in this experiment rather than the other
two settings on the colorimeter?
10. Design an experiment using this technique to test for the effect of light of different
wavelengths on the rate of photosynthesis. Be thorough--this is a long answer.
11. How would you modify the experimental design you produced for question #10 to test for
the effect of light of different intensities on the rate of photosynthesis?
12. How would you modify the experimental design you produced for question #10 to test for
the effect of different temperatures (stay within the range of normal biological activity,
0-100oC) on the rate of photosynthesis?
*
6
Please write out the questions along with your answers in your write-up. You can obtain a
copy of this lab protocol in the RPatterson folder on the “Instruction on Milledge” server so
that you can simply cut and paste the questions into the write-up like you do the data tables.