Plant Pigments and Photosynthesis
Adapted from J. Smith by A. Lyman-Buttler
Overview
In this lab you will separate plant pigments using chromatography, determine the absorption spectra of different plant
pigments, and measure the rate of photosynthesis in isolated chloroplasts. The measurement technique involves the
reduction of the dye 2,6-dichlorophenol-indophenol (DPIP). The transfer of electrons during the light reactions of
photosynthesis reduces DPIP and changes its color from blue to colorless. Any color changes will be monitored using a
colorimeter. (For oxidation and reduction reactions, remember OIL RIG!)
Plant Pigments
Paper chromatography is a useful technique for separating and identifying pigments 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 (adhesion) and the attraction of solvent molecules to
each other (cohesion). As the solvent moves up the paper, it carries along any substances dissolved in it, in this case
plant pigments. 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 cellulose in the paper (polar) through the formation of hydrogen
bonds. Examination of the molecular structure of the pigments may reveal chemical functional groups that help
determine the pigment's solubility in the solvent (nonpolar).
Four pigments are typically found in leaf extracts from plants. They are: Chlorophyll a (bluish green), Chlorophyll b
(yellow to olive green), carotenes (orange-yellow), and xanthophylls (lemon yellow). The molecular structures of each
pigment are shown on the next page. Note that the difference between chlorophylls is a slight difference in the
functional group on the ring. Beta-carotene and lutein (a xanthophyll) are also similar chemically, differing only slightly
in their functional groups.
Objectives





Explain the principles of chromatography.
Calculate Rf values.
Design an experiment in which chromatography is used as a separation technique.
Describe how light intensity, light wavelength, and temperature can affect photosynthesis.
Design an experiment to measure how light intensity, light wavelength, and temperature can affect
photosynthetic rates.
I. Chromatography of Plant Pigments
Procedure
Students will work in groups of two in this section. Each group should do the following:
1. Each student should obtain a 6-10 cm strip of chromatography paper. Using a pencil (not a pen—why not?), draw a
baseline approximately 1.5 cm from the bottom of the paper. Try to touch the paper as little as possible because skin
oils can interfere with the chromatogram development. (You may wish to have one group member wear gloves to
handle the paper.)
2. Using a capillary pipette, streak the plant extract (prepared by the instructor) along the baseline.
3. Repeat the application of the pigment several times until a dark streak is present. Allow the paper to dry between
applications and try to keep the streak as narrow as possible.
4. Each group will be given a chromatography development flask. Keep the flask sealed until ready to use. (Caution avoid inhaling the solvent fumes from the flask). The solvent used for this experiment is a mixture of petroleum ether,
a highly nonpolar solvent, and acetone. (9:1)
5. Place the completely dried chromatography paper into the flask and use the stopper to hold the paper in place
against the flask mouth. The chromatograph paper should hang so that the bottom touches the solvent in the bottom
of the flask, but the pigment strip must be above the solvent. Hold the strip next to the flask and determine were the
length is satisfactory before opening the flask. Each flask may be able to hold multiple papers.
6. When the solvent is about 1 cm from the top margin of the paper (several minutes), remove the paper and
immediately mark the location of the solvent front in pencil before it evaporates. Your instructor will help you identify
the spots. Label your pigment spots (1-4) from top to bottom. Reseal the flask after removing the paper.
Note – Some individuals may find more than 4 spots. If more are present, adjust your data recording as needed.
Analysis of Results
Calculate the Rf (Rate of flow) value for each of the pigments using the formula:
Rf = distance pigment migrated
distance solvent front migrated
Note - because Rf is a relative value, there are no units.
Report your data by completing Table 1. Use the colors, Rf values and molecular structure to match each spot with a
pigment name.
Spot Number
Color
Rf Value
Pigment Name
1
2
3
4
Answer the following questions:
1. What factors are involved in the separation of the pigments? (e.g. molecular weight, intermolecular forces, etc.)
Explain your reasoning.
2. What functional group difference separates the almost identical pigments of Chlorophyll a and Chlorophyll b?
Why and how does the functional group change affect the Rf values?
3. Is it possible to have a Rf value greater than 1.00? Explain.
4. Would the Rf value of a pigment be the same if a different solvent were used? Explain.
5. A molecule has the same general formula as lutein, but has methyl groups in place of the hydroxyl groups.
Where would this molecule like be on the chromatography paper relative to lutein? Explain.
6. Where would a hydrophilic pigment such as anthocyanin (from red cabbage) wind up on the chromatography
paper in this experiment? Explain.
II. Photosynthesis
Light is a part of a continuum of radiation or energy waves. Shorter wavelengths have greater amounts of energy (e.g.
high-energy ultraviolet rays can harm living cells). Wavelengths of light within the visible part of the radiation spectrum
are used in photosynthesis. Visible light has enough energy to be useful but not enough energy to break chemical
bonds. (Of course, the evolutionary explanation shows that defining part of the spectrum by its visibility is backwards.
Plants evolved to use this part of the spectrum because of its physical properties—energetic enough to do work, not
energetic enough to do much damage—and animals like us can see it because our eyes evolved to help us find plants!)
The photosystems found in the chloroplasts of leaf cells contain pigments that absorb light. These chloroplasts have two
different kinds of pigment systems, Photosystem I (PS I) and Photosystem II (PS II). PS I contains a specialized type of
chlorophyll a molecule called P700 (its absorption spectrum peaks at 700 nm), and PS II contains a specialized
chlorophyll a molecule called P680 (its absorption spectrum peaks at 680 nm).
When light is absorbed by leaf pigments, electrons within each photosystem are boosted to a higher energy level and
the energy is captured in the chemical bonds of ATP and NADPH (the light reactions). These high-energy products are
then used to incorporate CO2 into organic molecules in a separate set of reactions (the Calvin cycle).
In 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-accepting compound NADP+, a dye
DPIP (2,6-dichlorophenol-indophenol), will be substituted. If the dye accepts electrons from the chloroplasts, it will
become colorless. In this experiment, chloroplasts are extracted from spinach leaves and incubated with DPIP in the
presence of light. As DPIP is reduced and becomes colorless, the resultant increase in light transmittance can be
measured over time using a colorimeter.
Read the directions carefully before starting the experiment in order to avoid mistakes.
The experimental treatments are:
1) a blank cuvette containing everything but the DPIP
2) a cuvette with chloroplasts, incubated in the dark
3) a cuvette with chloroplasts, incubated in the light
4) a cuvette using heated chloroplasts, incubated in the light
5) a cuvette using no chloroplasts, incubated in the light
Cuvette 1
Cuvette 2
Cuvette 3
Cuvette 4
Cuvette 5
Blank
Dark
Light
Heated
No Chloroplasts
Phosphate Buffer
1 ml
1 ml
1 ml
1 ml
Distilled Water
2.5 ml
2 ml
2 ml
2 ml
DPIP* (see note)
_____
0.5 ml
0.5 ml
0.5 ml
0.5 ml
Chloroplasts
5 drops
5 drops
5 drops
5 drops,
heated
_____
1 ml
2 ml + 5 drops
* The amounts of DPIP and water are suggestions. The instructor may give directions to alter the ratio of these two
items depending on the concentration of DPIP. The goal is to have a starting %T value around 30%.
Procedure
Students should work in groups; group size will be determined by the instructor. Your instructor will provide the class
with the regular and heated chloroplast suspensions made by grinding spinach leaves. The suspensions should stay on
ice until ready to use. An incubation area will be prepared using the light from an over-head projector or a light
fixture.
Comment - With adequate planning and team cooperation, it is possible to stagger the readings so that treatments 2,
3, 4 and 5 can be run simultaneously, saving lab time.
1. Connect the Colorimeter to Channel 1 of the LabPro. Connect the LabPro to the computer. Turn on the
computer and start Logger Pro. Set Logger Pro to work with the colorimeter probe if needed. Set up the
computer in Meter format for recording data. Press on the < or > button on the colorimeter to select the
wavelength to 635.
2. Prepare five cuvettes by adding the phosphate buffer, water and DPIP to each cuvette as indicated above, or as
altered by the instructor (DPIP isn’t always the same strength and the amount of DPIP and water may need
some adjustment). Cover the walls and bottom of cuvette 2 with foil* and make a foil cap* to cover for the top.
Light should not be permitted inside cuvette 2. (Hint - make a loose foil jacket and a foil cap so that the cuvette
can be slipped in and out of the foil easily.)
3. Gently mix the regular chloroplast suspension and transfer five drops to cuvette 1.
4. Cover the top of cuvette 1 and invert to mix. Insert cuvette 1 into the colorimeter's sample holder (the clear
sides of the cuvette should line up with the arrow at the top of the cuvette slot) and close the lid. Press and
hold the calibration button until the indicator light blinks. Retain cuvette 1 as a blank to be used to check
calibrations between readings if needed.
Note – The Logger Pro program will probably show the reading in absorbance. Double left click on the word
“absorbance” in your graph to open a window. Change “absorbance” to “transmittance”. You may also wish to
double left click on the column heading and use “options” to set the number of decimal places to zero.
5. Transfer five drops of the regular chloroplast suspension to cuvette 2 and add the lid. Immediately invert
cuvette 2 to mix, remove the foil sleeve, and insert cuvette 2 into the sample holder. Read and record the %
transmittance as the time 0 value. Rewrap cuvette 2 in foil (including the foil cap), and place it in front of the
light in the incubation area. Take and record additional readings at 5, 10, and 15 minutes. Invert and mix the
cuvette prior to taking each reading.
6. Transfer five drops of the regular chloroplast suspension to cuvette 3 and add the lid. Invert to mix then
immediately insert the cuvette into the sample holder. Read the % transmittance and record as the time 0
value. Place the cuvette in the incubation area. Take and record additional readings at 5, 10, and 15 minutes.
Invert and mix the cuvette before each reading.
7. Add five drops of the heated chloroplast suspension to cuvette 4 and add the lid. Invert to mix and immediately
insert the cuvette into the sample holder. Read and record the % transmittance as the time 0 value. Place the
cuvette in the incubation area. Take and record additional readings at 5, 10, and 15 minutes. Invert and mix the
cuvette prior to taking each reading.
8. Place a lid on cuvette 5 and invert to mix. Insert the cuvette into the sample holder. Read and record the %
Transmittance as the time 0 value. Place the cuvette in the incubation area. Take and record additional readings
at 5, 10, and 15 minutes. Invert and mix the cuvette prior to taking each reading.
Time in minutes
Cuvette number
0
5
10
15
2 – dark
3 – light
4 – heated
5- no chloroplasts
Analysis - Plot the data from the four cuvettes with the independent variable (time) as the X axis and the dependent
variable (% Transmittance) as the Y axis. Be sure to label the axes and give units. Construct a key to identify each
treatment. Consider using Excel. Attach your graph. Answer the following questions, using separate paper if needed:
1. Why were chloroplasts added to cuvette 1 (the blank)?
2. Is there evidence of photosynthesis in cuvette 2? 3? 4? 5? For each cuvette, explain your evidence and discuss
what each result implies about photosynthesis (e.g. “photosynthesis requires ___”).
3. What would you expect to happen if green light had been used to incubate the chloroplasts in cuvette 3? Why?
4. What is the purpose of the phosphate buffer in the experiment?
5. What molecule in chloroplasts does DPIP "replace" in this experiment?
6. Why are the isolated chloroplasts in a solution of 0.5 M sucrose instead of distilled water?
7. Suppose the leaf material is ground in a blender for a long period of time. Speculate on the probable effect on
this experiment.
8. Why is the ground leaf material filtered with cheese cloth instead of filter paper?
III. Absorption Spectra of Pigments
Use the following procedure to determine the absorption spectra for chlorophyll (extracted from spinach) and carotene
(extracted from carrots).
1.
2.
3.
4.
Refer to Figure 10.8 (8th ed.), “Determining an Absorption Spectrum”, for background information.
Make sure the spectrophotometer is on, calibrated, and has been warmed up. (Mr. LB will do this.)
Place the pigment sample in a clean, clear cuvette. Load the cuvette into the spectrophotometer.
Press “Collect”.