Laboratory Objectives
After completing this set of exercises, you should be able to

Describe the roles played by light and pigment in photosynthesis.

Name and describe pigments found in photosynthesizing tissues.

Explain the separation of pigments by paper chromatography, based on their molecular
structure.
Demonstrate an understanding of the process of spectrophotometry and the procedure for
using the spectrophotometer.

Introduction
Without photosynthesis, there could be no life on Earth as we know it. The Earth is an open
system constantly requiring an input of energy to drive the processes of life. All energy entering
the biosphere is channeled from the sun into organic molecules via the process of photosynthesis.
As the sun’s hydrogen is converted to helium, energy in the form of photons is produced. These
photons pass to Earth’s surface and are absorbed by pigments in the chloroplasts of plants,
initiating the process of photosynthesis.
Photosynthesis ultimately produces glucose and oxygen from carbon dioxide and water.
Glucose, the primary source of energy for all cells, may be converted to sucrose and transported
or stored in the polymer starch. These organic molecules are building blocks for plant growth
and development. Animals consume plants and convert the plant molecules into their own
organic molecules and energy sources – the ultimate in recycling. Oxygen, also produced by
photosynthesis, is necessary for aerobic respiration in the cells of plants, animals, and other
organisms.
In this set of exercises, you will investigate cellular and environmental components utilized in
the process of photosynthesis. This will be accomplished by monitoring different variables
within the photosynthetic system.
EXERCISE 8.1
Separation of Plant Pigments by Paper Chromatography
Introduction
Your instructor has prepared an extract of chloroplast pigments from fresh green grass. A
blender ruptured the cells, and the pigments were extracted with acetone, an organic solvent.
Begin this exercise by separating the pigments extracted using paper chromatography. To do
this, you will apply the pigment extract to a cylinder of chromatography paper. You will then
8-1
Morgan Community College
place the cylinder in a jar with the organic solvents petroleum ether and acetone. The solvents
will move up the paper and carry the pigments along; the pigments will move at different rates,
depending on their different solubilities in the solvents used and the degree of attraction to the
paper. The leading edge of the solvent is called the front. Discrete pigment bands will be
formed from the front back to the point where pigments were added to the paper.
The following information will be helpful to you as you make predictions and interpret results:
1.
Polar molecules or substances dissolve (or are attracted to) polar molecules.
2.
Nonpolar molecules are attracted to nonpolar molecules to varying degrees.
3.
Chromatography paper (cellulose) is a polar (charged) substance.
4.
The solvent, made of petroleum ether and acetone, is relatively nonpolar.
5.
The most nonpolar substance will dissolve in the nonpolar solvent first.
6.
The most polar substance will be attracted to the polar chromatography paper; therefore, it
will move last.
Figure 8.1. Molecular structure of major leaf pigments. The molecules, in order from most to least
polar, are chlorophyll b, chlorophyll a, xanthophylls, and carotene. Use this information to determine the
separation patterns for the pigments.
8-2
Morgan Community College
Materials
capillary tube
disposable gloves
beakers
safety goggles
extractions of leaf pigments in acetone
petroleum ether/acetone mixture
forceps
scissors
chromatography paper stapled into a cylinder marked with a pencil line about 1 cm from one end
quart jar with lid, containing solvent of petroleum ether and acetone
Hypothesis & Prediction
Develop a hypothesis relating polarities and solubilities of plant pigments. Predict the results of
the experiment based on your hypothesis (if/then).
Procedure
1. Using rubber gloves and forceps, obtain a pre-cut piece of chromatography paper. Place this
piece onto a paper towel and draw a pencil line across the length of the paper, 1 cm from the
bottom. NOTE: make sure to keep the paper clean, otherwise contaminants will spoil the
experiment.
2. Using a capillary tube, streak the leaf pigment extract on the pencil line of the paper cylinder.
Allow the chlorophyll to dry. Repeat this step six to ten times, allowing the extract to air-dry
each time. You should have a band of green pigments along the pencil line. The darker your
band of pigments, the better the results of your experiment will be.
 Perform the next step in a hood or a well-ventilated room. Do not inhale the fumes of the
solvent. NO SPARKS! Acetone and petroleum ether are extremely flammable. Avoid contact
with all solutions. Wash hands with soap and water. If a spill occurs, notify the instructor. If
an instructor is not available, do not attempt to clean up. Leave the room.
3. Obtain the jar containing the petroleum ether
and acetone solvent. Using forceps, carefully
lower the loaded paper cylinder into the
solvent, and quickly cover the jar tightly with
the lid (Figure 8.2). Avoid inhaling the solvent.
The jar should now contain a saturated
atmosphere of the solvent.
Allow the
chromatography to proceed until the solvent
front has reached to within 3 cm of the top of
the cylinder.
Figure 8.2. Paper chromatography setup.
4. Remove the cylinder from the jar, allow it to dry, and remove the staples.
5. Save your paper with the separated pigments for the next exercise.
8-3
Morgan Community College
Results
Sketch the chromatography paper in the space provided on your worksheet. Label the color of
the various bands. The front, or leading edge of the paper, should be at the top. The pencil line
where pigment was added originally should be at the bottom.
Discussion
Based on your hypothesis and predictions, identify the various pigment bands. Label your
drawing in the Results section, indicating the correct identification of the pigment bands.
EXERCISE 8.2
Determining the Absorption Spectrum for Plant Pigments
Introduction
Using the results from exercise 8.1, you will now determine the absorption spectrum of plant
pigments separated by paper chromatography. The absorption spectrum is the absorption
pattern for a particular pigment, showing relative absorbency at different wavelengths of light.
For example, we know that chlorophyll a is a green pigment, and we know that it reflects or
transmits green wavelengths of light. We do not know, however, the relative proportions of
wavelengths of light absorbed by chlorophyll a. This information is of interest because it
suggests that those wavelengths showing greatest absorbency are important in photosynthesis.
The absorption spectrum can be determined with an instrument called a spectrophotometer.
This measures the proportions of light of different wavelengths absorbed and transmitted by a
pigment solution. It does this by passing a beam of light of a particular wavelength (designated
by the operator) through the pigment solution being tested. The spectrophotometer then
measures the proportion of light transmitted, or conversely, absorbed by that particular pigment
and shows the reading on the calibrated scale.
Materials
spectrophotometer
Kimwipes®
2 cuvettes
pigment solutions
150-ml beaker to hold cuvettes
acetone
squirt bottle with distilled water
Hypothesis & Prediction
Develop a hypothesis that describes the general relationship of each of the pigments to the color
of light that it absorbs. Predict the results of the experiment based on your hypothesis (if/then).
8-4
Morgan Community College
Procedure
1. Chromatography paper with plant pigments, similar to what you produced in exercise 8.1,
has already been eluted into separate band solutions. Each group will receive a band solution
with a number. Record that solution number in your worksheet. All of the groups will pool
their data, in order to determine the relative absorption spectra of the different pigments.
2. Fill one cuvette two-thirds full with pure acetone. This is your reference material, for
comparison with the unknown. This is cuvette A.
3. Fill the second cuvette two-thirds full with the band solution. This is cuvette B. Wipe both
cuvettes with a Kimwipe to remove fingerprints, and handle cuvettes only with Kimwipes as
you proceed.
Figure 8.3. The spectrophotometer has two scales. “Percent Transmittance” refers to how much light
is passing through the sample (how ‘clear’ the sample is). “Absorbance” refers to the inverse; how
much light is being absorbed by the sample (how ‘opaque’ the sample is).
4. Measure the absorption spectrum. Record your measurements in Table 8.1 of your
worksheet. The instructions that follow are for a Bausch & Lomb Spectronic 20
spectrophotometer (Figure 8.3). Turn on the machine (power switch C) for at least 5 minutes
before using.
a. Select the beginning wavelength using the wavelength control knob (A). Begin
measurements at 400 nm.
b. Zero the instrument by adjusting the 0 control knob (same as the power switch C) so that
the meter needle reads 0% transmittance. There should be no cuvette in the instrument,
and the sample holder cover must be closed.
c. Calibrate the instrument. Insert cuvette A into the sample holder and close the lid (be
sure to align the etched mark on the cuvette with the line on the sample holder). Adjust
the light control (F) until the meter reads 100% transmittance, or 0 absorption. You are
now ready to make your first reading.
d. Begin your readings. Remove cuvette A and insert cuvette B (align the etched mark).
Close the cover. Record the reading on the absorbency scale. Remove cuvette B.
8-5
Morgan Community College
e. Recalibrate the instrument. Insert cuvette A into the sample holder, and set the
wavelength to 420 nm. Again, calibrate the instrument to 100% transmittance (0
absorption) with cuvette A in place, using the light control (F).
f. Take the second reading. Remove cuvette A and insert cuvette B. Record absorbency at
420 nm. Remove cuvette B.
g. Continue your observations, increasing the wavelength by 20-nm increments until you
reach 720 nm. Be sure to recalibrate each time you change the wavelength.
5. Pool data from all teams to complete Table 8.1.
Results
Using the readings recorded in Table 8.1, plot the absorption spectrum for each pigment as
Figure 8.4 in your worksheet. Choose appropriate scales for the axes, determine dependent and
independent variables, and plot data points. Draw smooth curves to fit the values plotted.
Prepare a legend for easy identification of pigments plotted.
Discussion
Based on your hypothesis and prediction, determine which pigment is most important in the
process of photosynthesis. Support this with evidence from your results.
EXERCISE 8.3
Visualization of Leaf Pigments
Introduction
You will be provided with plant leaves that contain a variety of pigments. Green is caused
primarily by chlorophylls (secondarily by carotenes and xanthophylls). Pinks and reds are due to
water-soluble pigments in the central vacuole of the plant cells. White or off-white is tissue that
contains no pigment. Brown is dead tissue. Dark pink is due to chlorophyll overlapping with
anthocyanin.
Materials
Coleus or Geranium plants
Glass beakers
Bunsen burners and strikers
Protective eyewear and gloves
Forceps
Ethyl alcohol
Petri dishes
Procedure
1. Obtain a fresh leaf and make a detailed drawing of the pattern of colors on the leaf.
8-6
Morgan Community College
 Wear protective eyewear and gloves during this experiment. Be careful of fumes and
open flames.
2. Boil the leaf in water until it is limp. Spread the leaf out in a Petri dish. Make sure it doesn’t
tear. Draw the details of colors again, noting any changes (the pink will have been removed
because the boiling has broken down cell membranes and the anthocyanin leaked out).
3. Prepare a boiling ethyl alcohol bath: place a small beaker of ethyl alcohol into a larger
beaker of water (the boiling point of alcohol is much lower than that of water). Only let the
water simmer; the alcohol will boil.
4. Boil the leaf in the alcohol until it loses its green color.
5. Place the leaf in a Petri dish and wash off the alcohol with water. Drain the water. Draw the
color pattern again.
6. Cover the leaf with iodine for a starch test (a positive test will occur only where the
chlorophyll had been). Rinse the leaf with water and observe the final color pattern.
Results
Record your observations as you perform the experiment.
Discussion
What results would you expect from different leaf types? From pine needles?
References
Dickey, J. 1995. Laboratory Investigations for Biology, Benjamin/Cummings Publishing Co., Inc., Menlo
Park, CA.
Lang, F. and S. Waldegger. “Regulating Cell Volume.” American Scientist 85: 456-463, 1997.
Morgan, J.G. & M.E.B. Carter, 1999. Investigating Biology, 3rd ed., Benjamin/Cummings Publishing Co.,
Inc., Menlo Park, CA.
Trammell, Vickie. Personal communication, 2004.
8-7
Morgan Community College
Name: _________________________
Due date: __________________
8. Photosynthesis
BIO 111 Laboratory Worksheet
EXERCISE 8.1
Paper Chromatography
1. State the hypothesis developed for this exercise:
2. Sketch the chromatography paper, as per the instructions in the exercise. Label the
appropriate bands and identify the pigments.
EXERCISE 8.2:
Absorption Spectrum
1. State the hypothesis & prediction developed for this exercise (if/then):
2. Table 8.1 will be given to you as a separate sheet, after everyone has pooled their data.
Staple that sheet to the end of this worksheet.
3. Plot the absorption spectrum for each pigment as Figure 8.4 (on a separate sheet of graph
paper), as per instructions in the exercise.
4. Discussion. In a brief paragraph, determine which pigment is most important in the process
of photosynthesis. Support this with evidence from your results.
8-8
Morgan Community College
EXERCISE 8.3:
Visualization of Leaf Pigments
1. Draw the leaf at the beginning of the experiment (label the colors).
2. What changes did you observe while the leaf boiled in the water bath?
3. Draw the leaf after the water bath (label the colors).
4. What changes did you observe while the leaf boiled in the alcohol bath?
5. Draw the leaf after the alcohol bath (label the colors).
6. Describe the results of the starch test. What does this mean?
8-9
Morgan Community College
Table 8.1. Absorbency of Photosynthetic Pigments.
Wavelength
400
Chlorophyll
a
Chlorophyll
b
Xanthophyll
Carotene
Total
Pigment
420
440
460
480
500
520
540
560
580
600
620
640
660
680
700
720
8 - 10
Morgan Community College