AP Biology Lab

Plant Pigments and Photosynthesis Lab Activity

Background Information:
For many centuries, people believed that the increase in the size of plants was caused by the intake
of material from the soil. It was not until a Belgian physician, Jan Baptista van Helmont (circa 1577-1644),
performed an experiment that demonstrated conclusively what we accept today: the increase in the size of a
plant is not due simply to the plant obtaining a mystery substance from the soil; plants gain what they
require through the process of photosynthesis.
Photosynthesis uses energy from light captured by photosynthetic pigments. Water molecules are
split in the process; the plants fix carbon from carbon dioxide into glucose and fructose chains and oxygen,
a byproduct, is released. In many plants the sugars then combine to form long chains known as starches.
Many plants store their photosynthetic products this way, in the roots and stems.
Most plants produce their own organic molecules and do not need to obtain them from another
organism; through photosynthesis these plants are known as autotrophs. However, not all plants are able to
carry out photosynthesis. Many parasitic plants, without photosynthetic pigments, rely entirely on other,
host species for nourishment; these plants are heterotrophs.
Examples of photosynthetic pigments are chlorophyll a, chlorophyll b, and carotene. Green plants
usually have high chlorophyll content; in a typical plant, approximately three-quarters of the chlorophyll is
chlorophyll a and the rest is chlorophyll b. The presence of other pigments becomes apparent in some
plants in the fall when the chlorophyll begins to break down, and no longer masks their presence. Some
plants are high in pigments that mask the chlorophylls during the whole growing season (e.g., red cabbage
remains red because of the presence of anthocyanin). Each pigment absorbs light from a specific range of
wavelengths. Chlorophyll a, the primary pigments used in photosynthesis, absorbs blue and red light.
Chlorophyll b absorbs light in the blue-green and orange-red portions of the spectrum. Carotenoids absorb
light in the blue and blue-green regions. Remember, the color that is reflected is the color you see.
Chlorophyll, which absorbs blue and red light, would reflect from the green spectrum, and thereby appear
green in color. When color is absorbed, you do not see it.
Plants may adapt to this by capturing sunlight’s energy during the day, storing the energy
until night, and exchanging gases at that time when water loss is loser due to the lower temperatures. This
is known as the “light phase” of photosynthesis. The “dark phase” of photosynthesis can occur during the
light or the dark, while the light phase can occur only when wavelengths of light that will stimulate
photosynthetic pigments are present.
The first step in the conversion of light to chemical energy is the absorption of light by a pigment
system. In all photosynthetic cells, except photosynthetic bacteria, the pigment system includes
chlorophyll a. Chlorophyll a occurs in all photosynthetic eukaryotes and in prokaryotic blue-green algae.
In vascular plants, bryophytes, green algae, and euglenoid Protistans, chlorophyll b, and accessory
pigment, is also found. In the leaves of green plants, chlorophyll b generally constitutes about one-fourth
the total chlorophyll content. Chlorophyll b absorbs light wavelengths different from chlorophyll a,
extending the range of light that can be used for photosynthesis. It shares with chlorophyll a the ability to
absorb light energy and produce an excited state in the molecule. The excited molecule of chlorophyll b
transfers its energy to a molecule of chlorophyll a, which then transforms it into chemical energy.
Chlorophyll c or chlorophyll d takes the place of chlorophyll b in other groups of plants.
Carotenoids are also accessory pigments involved in the capture of light energy in
photosynthesis. Carotenoids are red, orange, or yellow fat-soluble pigments found in all chloroplasts and
also, in association with chlorophyll a, in the prokaryotic blue-green algae. There are two classes of
Carotenoids: those that do not contain oxygen are called carotenes, and those that do contain oxygen are
called the xanthophylls. In green leaves, the color of the carotenoids is masked by the much more
abundant chlorophylls; in some tissues, such as those of a ripe tomato or the petals of an orange flower, the
carotenoids predominate. During autumn, chlorophyll begins to break down as the leaf begins to senesce,
allowing the carotenes and xanthophylls to display the brilliant colors we associate with fall.
Carotenoids, which are not water soluble, are found free in the cytoplasm, but like the
chlorophylls are bound to proteins within the plastids. Only certain carotenoids serve as accessory
pigments, but these are important for the overall process of photosynthesis in the green plant.
A third class of pigments is anthocyanin. Unlike chlorophylls and carotenoids, anthocyanin do
not participate in photosynthesis and may appear red, purple or blue. AAnthocyanin occur widely among

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higher plants and are the pigments that generally give color to flowers, but also occur in leaves and fruit. In
leaves, these pigments often help protect against excessive sunlight that can damage some leaf tissues. This
is one reason why a young, newly developing leaf is often redder than when it reaches its mature size.
Chromatography is a technique for analyzing or separating mixtures of gases, liquids, or dissolved
substances such as chlorophyll pigments. There are many types of chromatography, including column,
paper, thin-layer, gas-liquid, ion exchange, and gel filtration. In general, all types of chromatography
involve two distinct phases: the stationary phase and the mobile phase. The separation depends on
competition for molecules of sample between the mobile phase and the stationary phase.
Column, paper, and thin-layer chromatography can be used to separate extracted plant and algal
pigments. In paper chromatography, the separation takes place through absorption and capillary action. A
drop of the mixture to be separated is placed at the bottom of a strip of chromatography paper, which holds
the substance by absorption. The chromatography paper and developer (chromatography solvent) are then
placed in a chamber. The paper acts as a wick, drawing the developer upward by capillary action and
dissolving the mixture as it passes over it. The components of the spotted mixture move upward at
differing rates, determined by both the solubility of the pigments in the solvent and their relative attractions
to the cellulose of the chromatography paper, resulting in the different pigments in the mixture showing up
as colored streaks or bands. The pattern formed on the paper is called a chromatogram.
To establish the relative rate of migration for each pigment, the R f value of each pigment is
calculated. The Rf value represents the ratio of the distance a pigment moved on the chromatogram relative
to the distance the solvent front moved. It is calculated using the following formula:

Rf = Distance Pigments Traveled

Distance Solvent Traveled
Solvent Front
Any molecule in a given solvent matrix has the uniquely consistent Rf. The Rf value is
used by scientists to identify molecules.

D
C
Materials and Procedures B
A
Materials needed per group Safety Precautions

1 vial Protective gloves, goggles, and aprons

1 chromatography paper strip may be worn throughout this activity.
Chromatography solvent, 1 mL When working with chromatography
Quarter solvent, a fume hood should be used to
Spinach and alternate leaves ensure proper ventilation.
forceps

Procedure
1. Obtain a chromatography vial
2. Perform step two under a fume hood, or well ventilated area. Remove the cap from
the chromatography vial. Using a disposable pipet, add 1 mL of chromatography
solvent to your vial. The pipets are marked with mL markings to make the
measurement easier. Replace the cap and allow the chamber to sit undisturbed until
needed in step 7. This will ensure that the atmosphere within the vial is saturated with
solvent vapors (equilibration).
3. Obtain 3 chromatography strips. Be sure to handle the chromatography strip by the
edges. Do not touch the surface of the strip. The oils from your fingers can interfere
with the chromatogram.
4. Cut each strip to 10 cm in length. Label one strip spinach another kale and the last one
mustard green.

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5. Measure 1.5 cm from one end of the chromatography strip and draw a pencil line across the
width of the strip.
6. Use a pair of scissors to cut two small pieces below the pencil line to form a pointed end. The
pointed end will be referred to as the bottom end of the chromatogram.
7. Obtain a fresh piece of spinach, kale and green. Place it over the line on the chromatography
strip. Rub the ribbed edge of a coin (dime or quarter) over the spinach leaf (or alternate item) to
extract the pigments. Repeat 5 to 10 times with different portions of the spinach leaf, making
sure you are rubbing the coin over the pencil line. Do the same for the other leaves on their own
strip.
8. Remove the cap from your vial and carefully place the chromatography paper strip into the vial
so that the pointed end is BARELY immersed in the solvent. Make sure not to immerse the
pigments in the solvent.
9. Cap the vial and leave it undisturbed. Observe as the solvent is drawn up the chromatography
strip by capillary action. You will be able to see the plant pigments separating along the strip.
Notice the different colors that you see during this process.
10. When the solvent reaches approximately 1 cm from the top of the strip, remove the cap
from the vial. Using forceps, remove the strip from the vial. This is a chromatogram.
11. Immediately mark the location of the solvent front (place where the solvent reached on the
paper) the solvent will evaporate quickly.
12. Fill in the data tables for the corresponding plants. Be sure to identify the type of leaf
you used. Once the strip has dried, mark the middle of each pigment band on your
chromatography strip with a pencil.
13. Using a metric ruler, measure the distance from the original pencil line with the spinach
(or alternate) extract to the solvent front and each mark you have made for each pigment
band. Record these distances in millimeters in your data table.
14. Calculate the Rf value for each pigment on your chromatogram using the following
formula and record your answers in your data table.
15. Use the expected results and your data to calculate the % error for each pigment. Be sure
to include the expected and % error results in your data table.

Rf = Distance pigment traveled

Distance solvent traveled

16. Staple the chromatography strip to the lab report.

17. If there is any chromatography solution left in the vial, leave it there for the next class.

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Name _____________________________________________________________ period _________

Leaf Pigment Lab

Chromatography strips must be stapled to this paper.

Data Tables

Spinach

Pigments Color Expected Rf Observed Rf % error

Chlorophyll a Bright green .59
Chlorophyll b Olive, Yellow green .42
Carotene Yellow orange .98
Xanthophyll yellow .35

Kale

Pigments Color Expected Rf Observed Rf % error

Chlorophyll a Bright green .59
Chlorophyll b Olive, Yellow green .42
Carotene Yellow orange .98
Xanthophyll yellow .35

Greens

Pigments Color Expected Rf Observed Rf % error

Chlorophyll a Bright green .59

Chlorophyll b Olive, Yellow green .42

Carotene Yellow orange .98

Xanthophyll yellow .35

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 Analysis Questions
Read the background information and answer the following:

1. List color of each pigment:

a. Chlorophyll a
b. Chlorophyll b
c. Carotenoids
d. Anthocyanin

2. In part A, which pigment migrated the farthest? Why?

3. During the summer months, leaves are generally bright green. What would you hypothesize that
this indicates about the role of green light wavelengths, chlorophyll, and the photosynthetic
process?

4. Why do leaves change colors in the autumn?

5. What is the function of chlorophylls in photosynthesis?

6. What are the accessory pigments and what are their functions?

7. How does the absorption rate of different wavelengths relate to a plant’s appearance?

8. What is the benefit for a young plant to have red leaves?

9. What does the Rf value represent? If you were to perform your experiment on a chromatography
strip that was twice the length of the one you used, would your Rf values still be the same?
Explain.