Name
Period
Date
AP Biology Lab 4: Plant Pigments and Photosynthesis, Converting
Solar Energy to Chemical Energy
Driving Questions
 How can the various photosynthetic pigments in plants be separated and visualized?
 Do rates of photosynthesis vary under different environmental conditions?

“Different Conditions” = dark, and extreme heat (= boiling!)

Why do rates of photosynthesis vary under different environmental conditions?
Background
Plants are photosynthetic organisms, able to harness light energy from the sun to convert carbon
dioxide gas from the atmosphere into sugar through photosynthesis.
6CO2 + 6H2O + light energy  C6H12O6 + 6O2
Chloroplasts, the organelles where photosynthesis occurs, contain pigments that absorb varying
wavelengths of light. In the leaves of the plant, the pigments are indistinguishable, but they
may be separated using chromatography.
Paper chromatography separates a mixture into its various components. The mixture is placed
onto a piece of chromatography paper and then a solvent is allowed to migrate (through
diffusion) up the paper. As the solvent migrates it carries the components of the mixture along
with it. Each of the components will migrate at varying rates based on their solubility, size and
hydrogen bonding with the paper.
The photosynthetic pigments absorb light energy from the sun. As light hits the chloroplasts
electrons are excited and then passed along an elaborate electron transport chain within the
thylakoid membrane of the chloroplasts. This electron flow does two things. It drives protons
against their concentration gradient, setting up an electro-chemical gradient used for ATP
synthesis; and it delivers electrons and protons to an electron acceptor, NADP +,, reducing the
molecule to NADPH. (NADPH is then used to reduce CO 2 to sugar in the light independent
reactions.)
The technique used in this lab to prepare the chlorophyll suspension physically breaks apart the
thylakoid membrane so that the natural electron transport chain is nonfunctional. DPIP is
added to the suspension to accept the excited electrons. When DPIP is reduced it changes color
from dark blue to colorless. The colorimeter will measure these color changes by determining the
percent transmittance of light in each sample prepared. This will indicate the rate of
photosynthesis in the sample.
Draw a picture of your experimental set up here:
1
AP Biology Lab 4: Plant Pigments and Photosynthesis
Materials and Equipment

 Distilled water, 13 ml
 Colorimeter and cuvettes
 Chloroplast suspension,2 ml
 Spinach
 0.1 M phosphate buffer,4 ml
 #1 Whatman chromatography paper
 DPIP in a small amber bottle,3 ml
 1 Small glass jar, 10-12 cm high
 100-watt floodlight
 1 quarter (or other coin)
 Heat Sink: large beaker or flask filled with water
 Chromatography solvent, 5 ml
 Aluminum foil
 Disposable 1-ml graduated pipettes, 2
 Ice
 Kim wipes or other lint-free tissue
 Cheesecloth
Lab Safety
 Due to the volatility of the chromatography solvent, all containers should remain
tightly capped.
Sequencing Challenge
The steps below are part of the Procedure for this lab activity. They are not in the right order.
Determine the proper order and write numbers in the circles that put the steps in the correct
sequence.
Calibrate the
colorimeter with a
mixture of the
chloroplast
suspension
without DPIP.
Separate the
various
photosynthetic
pigments in
spinach leaves
using paper
chromatography.
Create a graph of
percent
transmittance
versus time for
each of the four
samples.
Measure the initial
percent
transmittance for
each sample.
Allow the samples
to incubate for
five minute
intervals and
measure percent
transmittance
after each interval.
Procedure
After you complete a step (or answer a question), place a check mark in the box () next to that step.
Set Up Equipment - Plant Pigments
1.  Obtain a small glass jar and add 1 cm of solvent to the bottom.
2
AP Biology Lab 4: Plant Pigments and Photosynthesis
2.  Obtain a piece of chromatography paper about the length of the glass jar. Cut one end of
the paper into a point and draw a line in pencil 1.5 cm above point.
3.  Place a spinach leaf over the pencil line. Deposit the plant pigments onto the paper by
firmly rolling a quarter over the leaf about 15 times until a heavy green line appears on
the paper.
4.  Lower the paper into the jar ensuring that only the tip of the paper touches the solvent.
The green line must be above the solvent. Tightly close the lid of the jar.
5.  What do you notice happening to the solvent in the jar?
________________________________________________________________________________
________________________________________________________________________________
Collect Data - Plant Pigments
6.  When the solvent has traveled to about 1 cm below the top of the paper, remove the
paper from the jar. Using a pencil, quickly mark the location of the solvent's furthest
point of travel before the solvent evaporates. Measure the distance the solvent traveled
(from the first line you drew to the second line you drew) and record this value in Table
4.1.
7.  On the paper, mark the location of the bottom of each of the pigments. Measure the
distance each pigment traveled from the origin to the bottom of each band. Record these
measurements in Table 4.1.
8.  How many different pigments bands do you see? Why do some of the bands migrate
further than others?
________________________________________________________________________________
________________________________________________________________________________
9.  How do your measurements compare with other groups?
________________________________________________________________________________
________________________________________________________________________________
Set Up Equipment - Photosynthesis
10.  Connect the colorimeter to the data collection and display device. Set the colorimeter to
measure 610nm percent transmittance only. Create a digits display of percent
transmittance.
11.  Prepare the incubation area. You will need a flood light and a heat sink (a 1000 ml
beaker or flask filled with water). Place the flood light directly in front of the heat sink.
The heat sink will absorb the heat from the flood light while still allowing light to pass
through to the cuvettes which will be placed a few inches behind the heat sink.
12.  Obtain five cuvettes from the colorimeter box and label the tops of each 1, 2, 3, 4 and 5
3
AP Biology Lab 4: Plant Pigments and Photosynthesis
with tape or a wax pencil.
13.  Cover the sides and top of cuvette number 2 with aluminum foil. Cover it loosely so you
can remove the cuvette and place it in the colorimeter every five minutes.
14.  Fill each of the cuvettes according to Table 4.2 on next page, but do not add either the
unboiled or boiled chloroplasts yet.
Table 4.2: Setup for photosynthesis experiment
Cuvette 1
Blank
Cuvette 3
Unboiled
chloroplasts
(Light)
Cuvette 4
Boiled
chloroplasts
(Light)
Cuvette 5
(no DPIP)
Cuvette 2
Unboiled
chloroplasts
(Dark)
Phosphate buffer
1 mL
1 mL
1 mL
1 mL
1 mL
Distilled water
4 mL
3 mL
3 mL
3 mL
3 mL + 4 drops
DPIP
None
1 mL
1 mL
1 mL
1 mL
Unboiled
chloroplasts
4 drops
4 drops
4 drops
None
None
Boiled
chloroplasts
None
None
None
4 drops
None
Contents
No
Chloroplasts
(Light)
Collect Data - Photosynthesis
15.  Add 4 drops of unboiled chloroplasts to cuvette 1. Screw the lid onto the cuvette and mix
by inverting the cuvette several times. Wipe the sides of the cuvette gently with lens
paper and insert into the cuvette holder on the colorimeter. Tightly close the lid.
16.  Press the green calibrate button on the top of the colorimeter. The light will turn green
while the calibration is in progress. When the light turns off you may remove the
cuvette. The sensor is calibrated.
17.  What is the purpose of cuvette 1?
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
18.  Add 4 drops of the unboiled chloroplast suspension to cuvette 2. Screw on the lid and
invert to mix. Remove the aluminum foil from the cuvette and insert into the cuvette
holder on the colorimeter. Tightly close the lid.
19.  Begin collecting data. Record the percent transmittance in Table 4.3. Remove the
4
AP Biology Lab 4: Plant Pigments and Photosynthesis
cuvette from the colorimeter. Re-cover the cuvette with the aluminum foil.
Note: Be sure to mix the contents of the cuvettes before each reading.
20.  Predict how the rate of photosynthesis will change for the sample in cuvette 2 (unboiled
chloroplasts - dark).
________________________________________________________________________________
________________________________________________________________________________
21.  Add 4 drops of the unboiled chloroplast suspension to cuvette 3. Screw on the lid and
invert to mix. Insert into the cuvette holder on the colorimeter and tightly close the lid.
Record the percent transmittance in Table 4.3. Remove the cuvette from the colorimeter.
22.  Predict how the rate of photosynthesis will change for the sample in cuvette 3 (unboiled
chloroplasts - light). How will the change in photosynthetic rate change the percent
transmittance?
________________________________________________________________________________
________________________________________________________________________________
23.  Add 4 drops of the boiled chloroplast suspension to cuvette 4. Screw on the lid and invert
to mix. Insert into the cuvette holder on the colorimeter and tightly close the lid. Record
the percent transmittance in Table 4.3. Remove the cuvette from the colorimeter.
24.  Will this cuvette show signs of photosynthesis? Why or why not?
________________________________________________________________________________
________________________________________________________________________________
25.  Screw on the lid to cuvette 5 and invert to mix. This cuvette does not receive any
chloroplasts. Insert into the cuvette holder on the colorimeter and tightly close the lid.
Record the percent transmittance in Table 4.3. Remove the cuvette from the colorimeter.
26.  Place all cuvettes in the incubation area. Begin keeping track of the time that the
cuvettes are in the incubation area. Measure the percent transmittance of all five
cuvettes again at 5, 10, 15, and 20 minutes. Record all data in Table 4.3.
27.  What is the purpose of cuvette 5?
________________________________________________________________________________
________________________________________________________________________________
5
AP Biology Lab 4: Plant Pigments and Photosynthesis
Data Analysis
28.  Within your class results you may notice some variation in the distance traveled by the
same pigments. While the migration distance of each pigment may vary, the distance
relative to the migration of the solvent does not. The migration of the pigment relative to
the solvent is expressed at the constant Rf. In the space below, calculate the Rf value for
each of the pigments you observed and record these values in Table 4.1.
Rf = distance traveled by the pigment (mm)
distance traveled by the solvent
Table 4.1: Distance moved by pigment band (millimeters)
Band
Number
Distance (mm)
Band color
Pigment Name*
1.
2.
3.
4.
5.
SOLVENT
* Potential Pigments are as follows: Carotene, Chlorophyll a, Chlorophyll b, Xanthophyll
6
Rf
AP Biology Lab 4: Plant Pigments and Photosynthesis
Table 4.3: Percent Transmittance
Cuvette
Conditions
%T
610nm
% T 610nm
% T 610nm
% T 610nm
% T 610nm
(5 min)
(10 min)
(15 min)
(20 min)
(Initial)
#2
Unboiled Dark
#3
Unboiled Light
#4
Boiled – Light
#5
No
Chloroplasts Light
29.  Use a graphing template to draw a graph of the percent transmittance over time for each
of the five experimental cuvettes. Be sure to title the graph, label both the X and Y axis,
and include units on each axis.
30.  Gather data from the other four groups and determine the effects of various wavelengths
of light on the rate of DPIP reduction (the rate of light reactions). You will need to graph
these data on a separate graphing template, include all the appropriate information and
graph each line in the appropriate color. Please include a legend on your graph.
Analysis Questions (AQ) and Synthesis Questions (SQ) on next page.
7
AP Biology Lab 4: Plant Pigments and Photosynthesis
Analysis Questions
1. Which spinach leaf pigments did you observe in your chromatography
experiments? Hopefully you observed some accessory pigments. If so, what are
the roles of these pigments? Be as specific as possible.
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
2. What type of chlorophyll is at the reaction center of Photosystems I and II?
What is the role of this pigment?
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
3. How do all the pigments described above work together to insure survival
of plants? How do they work together to increase the efficiency of the
conversion from solar energy to chemical energy?
_________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
8
AP Biology Lab 4: Plant Pigments and Photosynthesis
4. What were the effects of the following on the light dependent reactions?
For each variable, cite evidence to support your claim and explain why you
obtained your results.
light ___________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
darkness _______________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
boiling _________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
Synthesis Questions
1. What is the function of DPIP in this experiment? Explain how it is an
adequate “surrogate” for measuring the light reactions of photosynthesis.
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
9
AP Biology Lab 4: Plant Pigments and Photosynthesis
2. What is the source of electrons that will directly reduce DPIP? What is the
ultimate source of electrons in this experiment?
______ __________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
3. How do the results from the choromotography experiment and the light
reaction experiment correlate? How do they help explain the action spectrum
of photosynthesis? Cite specific data from your experiments to support your
explanations.
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
10