Plant Pigments and Photosynthesis0809 - Pingry School

Plant Pigments and Photosynthesis

Overview:
In this lab you will:

1. Separate plant pigments using paper chromatography
2. Measure the rate of photosynthesis in isolated chloroplasts by the reduction of the blue

dye 2,6-dichlorophenol-indophenol (DPIP).

Before doing this lab you should understand:
… How paper chromatography works to separate compounds in a mixture.
… The function of plant pigments.
… The relationships between light intensity, wavelength and photosynthetic rate.
… The light reactions of photosynthesis.
… How a spectrophotometer works to measure absorption and transmittance of light.
… How reduction changes the color of DPIP.

After completing this lab, you should be able to:
… Separate pigments using paper chromatography and compare their Rf values.
… Compare the value of 1- and 2- dimensional paper chromatography in separating
plant pigments.
… Measure the rate of photosynthesis.
… Compare the rate of photosynthesis at different light intensities or different
wavelengths of light using controlled experiments.
… Explain why the rate of photosynthesis varies under different environmental
conditions.

Exercise A: Plant Pigment Chromatography
Paper chromatography is a useful technique for separating and identifying pigment and

other molecules from cell extracts that contain a complex mixture of molecules. The solvent
moves up the paper by capillary action, which occurs as a result of the attraction of solvent
molecules to the paper and the attraction of the solvent molecules to one another. As the solvent
moves up the paper, it carries along any substances dissolved in it. The pigments are carried
along at different rates because they are not equally soluble in the solvent and because they are
attracted, to different degrees, to the fibers of the paper through the formation of intermolecular
bonds, such as hydrogen bonds.

Beta carotene, the most abundant carotene in plants, is carried along near the solvent
front because it is very soluble in the solvent being used and because it forms no hydrogen bonds
with cellulose. Another pigment, Xanthophyll differs from carotene in that it contains oxygen.
Xanthophyll is found further from the solvent font because it is less soluble in the solvent and
has been slowed down by hydrogen bonding to the cellulose. Chlorophylls contain oxygen and
nitrogen and are bound more tightly to the paper than the other pigments.

Chlorophyll a is the primary photosynthetic pigment in plants. A molecule of chlorophyll
a is located at the reaction center of the photosystems. The pigments collect light energy and
send it to the reaction center. Carotenoids also protect the photosynthetic systems from damaging
effects of ultraviolet light.

Procedure:

1. Obtain a mortar and pestle, a sample fresh spinach or red cabbage, a capillary tube, 2

square pieces of filter paper, a paper clip, and 2 chromatography jars. One of the

chromatography jars should contain diethyl ether, and the other a petroleum ether:acetone

mixture.

2. Grind the sample with the mortar and pestle. You may add a small amount of water if

necessary. The goal is to obtain a concentrated sample of pigment.

3. Use a capillary tube to spot a sample of pigment onto a

square of chromatography paper about 1cm above and to

the left of the bottom right hand corner of the paper (see

fig. a).

4. Roll the chromatography paper into a loose coil with the

spotted side toward the center. Secure the roll at the top

with the paper clip. The roll should be just tight enough so

that the paper fits into the chromatography jar and does not

touch the sides.

5. Uncap the diethyl ether chromatography jar, place the

coiled paper inside and replace the cap. Do this quickly

and avoid inhaling the fumes.

6. When the solvent is about 1cm from the top of the paper,

remove the paper from the chromatography jar, avoiding

releasing and avoiding fumes as before.

7. Uncoil the paper and immediately mark the top of the

solvent front with a pencil.

8. Allow the paper to dry. Diethyl ether is very volatile. This

will happen quickly.
9. Turn the chromatography paper 90o clockwise (see fig. b),

coil and paper clip.

10. Insert the coil into the petroleum ether:acetone

chromatography jar.

11. When the solvent is about 1cm from the top of the paper, remove the paper from the

chromatography jar, avoiding releasing and avoiding fumes as before.

12. Uncoil the paper and immediately mark the top of the solvent front with a pencil

13. Create a table such as the one shown below in your lab notebook

Band (name Pigment Distance in Rf1 Distance in Rf2
of pigment) Color dimension 1 dimension 2

(mm) (mm)

Solvent front Clear

14. Using a ruler, measure the Dimension 2 Dimension 1 Solvent
front for
distance from the pigment diethyl
origin to each solvent front ether

in millimeters (see diagram Pigment origin
at right). Solvent front from petroleum ether:acetone

15. Measure the distance each
pigment spot migrated in

each dimension. Your
measurements should be

parallel to the edge of the
page and start

approximately 1cm in from
the edge.

16. Use the figure at right to
identify your pigment.

Analysis:

Retention factor (or retardation factor),
designated Rf, is a ratio between the distance the pigment
migrated to the distance the solvent moves. The Rf for a
given pigment-solvent pair is a constant and can be used

to identify the pigment. It can be calculated as shown
below:

Rf = Distance pigment migrated (mm)
Distance solvent front migrated (mm)

Calculate Rf for each pigment and record in your table.

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

2. Are the Rf values calculated for dimension 1 the same as those calculated for dimension 2?
Why or why not?

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

Exercise B: The Light Reactions of Photosynthesis:

Light is a part of the electromagnetic
spectrum. Electromagnetic radiation
with shorter wavelengths has higher
frequency and higher energy. Radiation
with longer wavelengths has lower
frequency and lower energy. Radiation
with wavelengths within the visible
spectrum of light power photosynthesis.

When light is absorbed by a molecule of chlorophyll
a in a photosystem, electrons are excited to a higher
energy level. In photosystem II, this energy is
harvested by passing the electrons through an electron
transport chain which pumps hydrogen ions into the
thylakoid interior of the chloroplast. The
electrochemical gradient produced is used to drive
ATPase to produce ATP. The electrons from
chlorophyhll a are replaced by oxidizing water to
molecular oxygen.

In photosystem I, the electrons excited by light are used to reduce NADP+ to NADPH. Electrons
excited when light is absorbed by accessory pigments
are funneled into one of the two photosystems. The

ATP and NADPH generated in this process are
used in the carbon fixation reactions of the
Calvin cycle (formerly called the “dark
reactions”—though they do not take place in
the dark).

Design of the Exercise:
Photosynthesis may be studied in a number of ways.
For this experiment, a dye-reduction technique will
be used. The dye-reduction experiment tests the
hypothesis that light and chloroplasts are required
for the light reactions to occur. In place of the
electron accepter, NADP, the compound DPIP (
2.6-dichlorophenol-indophenol), will be substituted.
When light strikes the chloroplasts, electrons
boosted to high energy levels will reduce DPIP. It
will change from blue to colorless.

In this experiment, chloroplasts are extracted from spinach leaves and incubated with DPIP in
the presence of light. As the DPIP is reduced and becomes colorless, the resultant increase in

light transmittance is measured over a period of time using a spectrophotometer. The
experimental design matrix is presented in the table below.

Procedure:

1. Obtain a spectrophotometer, plug it in and turn it on to warm up. Set the wavelength to
605 nm.

2. Obtain a test tube rack with 5 cuvettes. Label the cuvettes 1-5 at the top near the rim.
Wipe each cuvette with lint-free paper and hold at the top to avoid fingerprints on the

glass. Set up each cuvette as shown below except for the chloroplast suspensions.
Adding chloroplasts too early will ensure poor results.

Cuvettes

12 3 45

Blank Unboiled Unboiled Boiled No
Chloroplasts Chloroplasts
Chloroplasts Dark Chloroplasts Light
Light 1 ml.
Phosphate 1 ml. 1 ml. 1 ml. 1 ml.
Buffer 3 ml + 3
3 ml. drops
Distilled Water 4 ml. 3 ml. 3 ml. 1 ml. 1 ml.
----
DPIP ---- 1 ml. 1 ml. ----
3 3 drops 3 drops 3 drops
Unboiled drops ----
Chloroplasts ---- ----
----
Boiled
Chloroplasts

3. Using aluminum foil, cover the walls and bottom of cuvette 2. Make a cap for the top.

Light should not be permitted inside cuvette 2 because it is a control for this experiment.
Make this reusable since it will be necessary to remove this cuvette from the foil for each

reading in the Spec. 20.
4. Set up an incubation area where you will be able to place your rack of cuvettes in the

light.
5. Make a table in your notebook such as the one shown below.

Cuvette % Transmittance % Transmittance % Transmittance % Transmittance
Time 0 min. Time 5 min. Time 10 min. Time 15 min.
2 unboiled/dark
3 unboiled/light
4 boiled/light
5 no chloroplasts/light

6. Obtain boiled and unboiled chloroplasts. Be sure to keep these on ice at all times.
7. Bring the spectrophotometer to zero by adjusting the amplifier control knob until the

meter reads 0% transmittance. Cover the top of cuvette 1 with Parafilm and invert to mix.
Insert cuvette 1 into the sample holder and adjust the instrument to 100% transmittance
by adjusting the light -control knob. Cuvette 1 is the blank to be used to recalibrate
the instrument between readings. For each reading, make sure that the cuvettes are
inserted into the sample holder so that they face the same way as in the previous reading.
8. To obtain good results, cuvettes 2-5 must be read every 5 minutes. A good way to ensure
enough time to read each sample, and to allow 5 minutes between readings is to stagger
the start times of each cuvette as shown below:

a. At time zero, do step 6, and immediately do step 8.
b. At time = 1 minute, do step 6 again and immediately do step 9.
c. At time = 2 minutes, do step 6 again and immediately do step 10
d. At time = 3 minutes, do step 6 and immediately do step 11.
e. Wait until time = 5 minutes, do step 6, and read cuvette 2.
f. Read cuvette 3 at t = 6 min, cuvette 4 at t = 7 min, and cuvette 5 at t = 8 min.
9. Stir the unboiled chloroplast suspension to mix it and transfer three drops to cuvette 2.
Immediately cover and mix cuvette 2. Then remove it from the foil sleeve and insert it
into the spectrophotometer's sample holder, read the % transmittance, and record it as the
time 0 reading. Replace cuvette 2 into the foil sleeve, and place it into the incubation test
tube rack. Turn on the flood light. Take and record additional readings at 5,10,and 15
minutes. Mix the cuvette's contents just prior to each reading. Remember to use cuvette 1
to check and adjust the spectrophotometer to 100% transmittance.
10. Stir the unboiled chloroplast suspension to mix, and transfer three drops to cuvette 3.
Immediately cover and mix cuvette 3. Insert it into the spectrophotometer's sample
holder, read the % transmittance, and record it. Replace cuvette 3 into the incubation test
tube rack. Take and record additional readings at 5,10,and 15 minutes. Mix the cuvette's
contents just prior to each reading. Remember to use cuvette 1 to check and adjust the
spectrophotometer to 100% transmittance.
11. Stir the boiled chloroplast suspension to mix, and transfer three drops to cuvette 4.
Immediately cover and mix cuvette 4. Insert it into the spectrophotometer's sample
holder, read the % transmittance, and record it. Replace cuvette 4 into the incubation test
tube rack. Take and record additional readings at 5,10,and 15 minutes. Mix the cuvette's
contents just prior to each reading. Remember to use cuvette 1 to check and adjust the
spectrophotometer to 100% transmittance.
12. Cover and mix the contents of cuvette 5. Insert it into the spectrophotometer's sample
holder, read the % transmittance, and record it in Table 4.4 . Replace cuvette 5 into the
incubation test tube rack. Take and record additional readings at 5,10,and 15 minutes.
Mix the cuvette's contents just prior to each reading. Remember to use cuvette 1 to check
and adjust the spectrophotometer to 100% transmittance.

Analysis
1. Obtain average transmittance values for the class.
2. Use Excel to prepare two graphs: one of your group’s transmittance data and one of the
class data. Be sure your graph includes all of the important features (make sure it
TALKS). Tape a copy of the graph into your lab notebook.

Answer the following questions:
1. What is the purpose of DPIP in this experiment?
2. What molecule found in chloroplasts does DPIP "replace" in this experiment?
3. What is the source of the electrons that will reduce DPIP?
4. What was measured with the spectrophotometer in this experiment?
5. What is the effect of darkness on the 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 difference in the percent transmittance between the
live chloroplasts that were incubated in the light and those that were kept in the dark?
8. Explain the function of each cuvette. What question does each provide the answer to?

Edited by D. Maxwell in 2005, 2006
This document was prepared for classroom use by D. Maxwell in 2004 from material found
at the following web sites:
http://central.saisd.org/dpts/science/biologyap/student/unit2/Unit%202%20Labs/photosynthesis
%20lab.htm
http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookPS.html
http://www.ias.ac.in/resonance/May2001/pdf/May2001p83-91.pdf