IIT/FIELD MUSEUM—High School Transformation Project
Biology
Using Chromatography to Separate Pigments
Biology: Illinois edition (Glencoe)
Unit 2 The Cell
Chapter 8 Cellular Energy
Section 8.2 Photosynthesis
Guiding Question
What are the different pigments found in leaves and how do we separate them?
Context of Lesson
The object of this lesson is to examine the different types of pigments found in common plants by
separating the pigments from leaves. This lesson will build on the students’ study of Sec.8.1 How
Organisms Obtain Energy. This activity is used to introduce the unit on photosynthesis. In order for
plants to use the energy in sunlight, they must first trap light energy. Pigments are the molecules that
absorb light for the plant.
Plants contain a variety of pigments. Chlorophyll a, found in all plants, is the major photosynthetic
pigment. It captures the energy found in red and blue wavelengths of light and uses it to produce
glucose during photosynthesis. Additional pigments such as chlorophyll b and carotenoids, known as
"accessory pigments", capture different wavelengths of light and pass the energy to the chlorophyll a for
use during photosynthesis. These accessory pigments often have additional functions such as protecting
plant cells from sunlight damage.
The lab activity will allow students to observe the pigments present in a leaf. The term chromatography
comes from the Greek language: "chroma" (color) and "graphein" (writing). The solubility of each
pigment, plus its affinity for the chromatography paper will determine how quickly the different
pigments separate.
Main Goals / Objectives
Students will be able to:
 Explain the relationship between pigments and photosynthesis.
 Predict which pigment colors would be best for photosynthesis.
Nature of Science: Integrated Theme
 Explain that scientific knowledge is tentative due to new evidence or new interpretation.
 Distinguish observations from inferences, explain that inferences should be based on
observations and explain that the development of scientific knowledge involves both
observations and inferences so scientific knowledge is partially inferential.
 Explain that scientific knowledge should be based on empirical data.
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Scientific Inquiry: Integrated Theme
 Explain that scientific investigations all begin with a question, but do not necessarily test a
hypothesis.
 Explain that there is no single scientific method and provide at least two different methods.
 Explain that inquiry procedures are guided by the question asked.
 Explain that not all scientists performing the same procedures may get the same results.
 Explain that inquiry procedures can influence the results of an investigation.
 Explain that research conclusions must be consistent with the data collected.
General Alignment to Standards
ILS Goals
STATE GOAL 11: Understand the processes of scientific inquiry and technological design to investigate
questions, conduct experiments and solve problems.
A. Know and apply the concepts, principles and processes of scientific inquiry.
ILS 11.A.1c Collect data for investigations using measuring instruments and technologies
STATE GOAL 12: Understand the fundamental concepts, principles and interconnections of the life,
physical and earth/space sciences.
A. Know and apply concepts that explain how living things function, adapt, and change.
ILS 12.A.4b Describe the structures and organization of cells and tissues that underlie basic life
functions including nutrition, respiration, cellular transport, biosynthesis, and reproduction.
PSAE Goals
STANDARD 12 – LIVING THINGS
12.11.07 Understand the chloroplasts in plant cells capture useable energy from sunlight and store it for
future use by synthesizing sugar out of carbon dioxide and water.
12.11.09 Understand that the chief energy-storing compound used by organisms is ATP.
Materials (per group)
Fresh Spinach, 1-2 leaves (One bag is enough for 5 classes) and autumn leaves
Chromatography paper strip (Can be cut from filter paper: approx. 15 cm x 1.5 cm)
Penny
100 mL beaker
Solvent: 2% Acetone or 97 % Isopropyl Alcohol (700 mL is enough for 5 classes)
Pencil
Ruler
Scissors
Teacher Notes
It is a good idea to put 1cm (approximately 10 mL) of solvent into each beaker before class. Cover the
beakers with plastic wrap.
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Safety
 Before you select items for this activity, determine if the students have any known food
allergies or chemical sensitivities.
 Emphasize that under no circumstances should students taste any substances in the laboratory.
Remind students to use lab materials ONLY as directed. Clean your equipment and dispose of
your solvent in the designated container.
 Everyone should wear chemical splash goggles and gloves to protect eyes and skin.
 Wash hands thoroughly after handling material and before eating or drinking.
 Use ventilation to keep airborne concentrations below exposure limits. Have approved eyewash
facility and fire extinguishers readily available.
 Acetone/Isopropyl alcohol Health Effects: Overexposure may cause stinging pain, watering of
eyes, inflammation of eyelids. Repeated/prolonged skin contact may cause dryness or rashes.
Facilitation
Conduct this activity 1-2 days prior to introducing the Photosynthesis model lesson. It may take 1-2 days
to complete the activity and delivery of content.
The Lesson
Guiding Question
What are the different pigments found in leaves and how do we separate them?
Day 1
Bell Ringer
Ask students to brainstorm the following questions:
1. How do plants get energy?
2. Why do leaves change color in the fall?
Discuss with students how plants get energy. They should explain that plants do not eat food. Instead,
plants use the energy from the sun to produce their own food. Plants need to absorb light energy in
order to do this. Explain that plants have pigments that absorb light energy from the sun.
Activity
At this point students will have just finished their study of why organisms need energy. They will
understand what ATP is and how cells use energy for life processes. Now it is time to learn about how
plants trap light energy in order to begin the process of converting that light energy into ATP and then
into energy-rich food.
Distribute the Chromatography lab worksheet. Explain what is chromatography paper and how they
will use it to separate the pigments found in plant leaves. Inform students that you will continue this
discussion once they have gotten their lab set up and working.
Distribute lab equipment to pair of students. . Have the students sketch a chromatography strip in their
lab notebook (As shown on the handout).
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Assignment
Distribute the “Connection to Chemistry” (p. 240) handout for homework. Students should read the
article and answer the “Think Critically” question in their Science notebooks.
Assessments
Complete the lab worksheet. See answer key for more information. Throughout the class discussion, as
the teacher is eliciting responses from students, the teacher can monitor student understanding and
adjust his/her strategies to ensure student understanding. Students will answer, and turn in for grading,
the lab analysis questions for both lab activities.
Debriefing
Discuss the results from the activity. Again, the goal is to have the students express the “big idea” of the
lesson without simply repeating words. De-brief the students on their knowledge of inquiry and the
aspects of Nature of Science and Scientific Inquiry. Refer to your charts posted in the classroom. Probe
the students on which aspects are applicable in this lesson. Refer to the lists at on the first page of the
model lesson. The students should be able to come up with most, if not all, of the items on the list.
Modifications/Accommodations
• Provide assistance with the set up and making predictions if the student has an IEP.
• Conduct class discussion of the results and conclusions.
Attachments
 Worksheet – Using Paper Chromatography to Separate Pigments
 Answer key for worksheet
 “Connection to Chemistry – Plant Pigments”, Biology: The Dynamics of Life, p. 240
Extensions
Conduct the activity using leaves from different trees to see how their leaf pigments compare to those
of spinach. Compare and contrast the chromatograms for different types of leaves.
Procedure: Select different types of leaves and shred them. Put the pieces in the mortar bowl. Using a
pestle, students grind and squish the leaves into smaller pieces. Before adding the acetone, have
students observe the clear color in the bottle. Walk around to each group and add a spoonful of
acetone to extract the pigment from the leaves. Students keep grinding the leaves with the pestle until
they see that the liquid has gained some color from the leaf. Let the liquid sit for 1-2 minutes.
Preparing the Chromatogram
1. Draw a line in pencil across the chromatography paper 1.5 cm from the bottom.
2. Starting at the line, taper the bottom of the paper with scissors.
3. The chromatogram should resemble Figure A.
Students use a Q-tip to pick up a drop of the colored liquid from the leaf grinding. Students place the
drop one inch from the end of the chromatogram. Let it dry. Use the same Q-tip again to add a few
additional drops to the same spot. Students should let each drop dry before adding the next drop. Fill
the beaker with approximately 10mL of alcohol. Students carefully put the end of the chromatogram—
the end with the drops of dried leaf extract on it—into the alcohol, but do not let the colored drop touch
the alcohol. Students can discuss what they observe between the different chromatograms.
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Name ___________________________________________ Date _______________
Using Paper Chromatography to Separate Pigments
BACKGROUND:
The leaves of most plants contain many different pigments. Chlorophyll is the most abundant and
important plant pigment for absorbing sunlight. It exists in two forms: chlorophyll a (bright green) and
chlorophyll b (dull green). Chlorophyll often hides the other pigments present in leaves. In autumn,
chlorophyll levels drop, allowing other pigments to show their colors. Yellow xanthophyll and orange
carotene are commonly visible at this time. Other pigments may also be present in leaves.
Question: What is the purpose of pigment?
___________________________________________________________________
___________________________________________________________________
The individual pigments in a leaf may be separated by the technique of paper chromatography.
Chromatography means, “color writing”. The separation takes place by absorption and capillarity. The
paper absorbs the mixture of pigments in a solvent. Capillarity pulls the substances up the paper at
different rates depending upon their specific properties. As a result, the pigments will separate on the
paper. They show up as colored streaks. The pattern of separated pigment colors on the paper is called a
chromatogram.
PURPOSE:
You are a plant specialist working for a natural clothing company. All the garments manufactured by your
company are made from natural plant fibers and are dyed with natural dyes. One of your company’s
designers wants you to develop a new yellow dye. You think one of the plant species growing in the
company greenhouse contains yellow pigment. You decide to separate the leaf pigments using paper
chromatography.
MATERIALS:
1-2 leaves per group
Chromatography paper strip
Penny
100 mL beaker
Solvent- Acetone or isopropyl alcohol, approximately 10 mL
Ruler
Pencil
Scissors
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PROCEDURE:
Part 1 – Preparing the Chromatogram.
A) Draw a line in pencil across the paper 1.5 cm from the bottom.
B) Starting at the line, taper the bottom of the paper with scissors.
C) Your chromatogram should resemble Figure A.
Part 2 – Adding the pigment.
A) Place leaf over the line on the filter strip.
B) Using a penny, rub the leaf to transfer pigment onto the line.
C) Move the leaf and repeat several times until a dark green
line of leaf pigment is visible on the pencil line.
Figure A
Chromatogram
Pencil
line
Place leaf over pencil
line and rub with penny
Part 3 – Running the experiment.
A) Uncover the beaker. DO NOT INHALE THE SOLVENT!
B) Place the filter strip into the beaker so that only the tip soaks in the solvent.
C) Fold the top of the paper over the edge of the beaker. Be sure the paper
doesn't sink further into the solvent. The green line CANNOT be in the solvent.
D) Wait fifteen minutes, or until the pigments fully separate. There should be different colors
of pigment visible on the strip.
E) Remove the filter strip and cover the beaker with plastic wrap.
F) Sketch your pigment bands in your lab notebook on the outline you made earlier.
G) Record the colors in TABLE 1.
Part 4 – Identifying the pigments.
A) Use the information below to identify which pigments are in your leaf:
Chlorophyll a: bright green-yellow
Chlorophyll b: dull blue-green or khaki
Carotene: orange or gold
Xanthophyll: pale yellow
B) Record your pigment names in TABLE 1.
C) Label the pigment names on your sketch in your lab notebook.
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TABLE 1
CONCLUSION / ANALYSIS:
Type of leaf
Color of pigment
Name of pigment
1. Would your company be able to use this plant as a source for yellow dye?
Explain your answer.
2. Does your data support this statement: “Chlorophyll is the most abundant and important
photosynthetic plant pigment”? Explain.
3. Why do most leaves contain more than one type of pigment?
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ANSWER KEY
Using Paper Chromatography to Separate Pigments
BACKGROUND:
The leaves of most plants contain many different pigments. Chlorophyll is the most abundant and
important plant pigment for absorbing sunlight. It exists in two forms: chlorophyll a (bright green) and
chlorophyll b (dull green). Chlorophyll often hides the other pigments present in leaves. In autumn,
chlorophyll levels drop, allowing other pigments to show their colors. Yellow xanthophyll and orange
carotene are commonly visible at this time. Other pigments may also be present in leaves.
Question: What is the purpose of pigment?
Pigments absorb light energy.
The individual pigments in a leaf may be separated by the technique of paper chromatography.
Chromatography means, “color writing”. The separation takes place by absorption and capillarity. The
paper absorbs the mixture of pigments in a solvent. Capillarity pulls the substances up the paper at
different rates depending upon their specific properties. As a result, the pigments will separate on the
paper. They show up as colored streaks. The pattern of separated pigment colors on the paper is called a
chromatogram.
PURPOSE:
You are a plant specialist working for a natural clothing company. All the garments
manufactured by your company are made from natural plant fibers and are dyed with natural dyes. One
of your company’s designers wants you to develop a new yellow dye. You think one of the plant species
growing in the company greenhouse contains yellow pigment. You decide to separate the leaf pigments
using paper chromatography.
MATERIALS:
1-2 leaves
Chromatography paper strip
Penny
150 mL beaker
Solvent- Acetone or isopropyl alcohol, approximately 10 mL
Pencil
Ruler
Scissors
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PROCEDURE:
Figure A
Part 1 – Preparing the Chromatogram.
A) Draw a line in pencil across the paper 1.5 cm from the bottom.
B) Starting at the line, taper the bottom of the paper with scissors.
C) Your chromatogram should resemble Figure A.
Part 2 – Adding the pigment.
A) Place leaf over the line on the filter strip.
B) Using a penny, rub the leaf to transfer pigment onto the line.
C) Move the leaf and repeat several times until a dark green
line of leaf pigment is visible on the pencil line.
Chromatogram
Pencil
line
Place leaf over pencil
line and rub with penny
Part 3- Running the experiment.
A) Uncover the beaker. DO NOT INHALE THE SOLVENT!
B) Place the filter strip into the beaker so that only the tip soaks in the solvent.
C) Fold the top of the paper over the edge of the beaker. Be sure the paper
doesn't sink further into the solvent. The green line CANNOT be in the solvent.
D) Wait fifteen minutes, or until the pigments fully separate. There should be different colors
of pigment visible on the strip.
*See Teacher Notes below
E) Remove the filter strip and cover the beaker with plastic wrap.
F) Sketch your pigment bands in your lab notebook on the outline you made earlier.
G) Record the colors in TABLE 1.
*Teacher Notes:
While the pigments are separating, lead the students in a discussion about how pigments absorb
certain wavelengths of light and reflect others. They need to understand that white light is actually a
combination of all colors of light. When pigments absorb a color of light, it is no longer visible. The
color we see when we look at an object is the color that is reflected. Green plants look green because
green light is not very useful for photosynthesis so plants reflect it back out. Once plants absorb the
light energy, they can begin to convert it to usable food energy.
Ask students why plants might have more than one color of pigment – why not just have chlorophyll?
They should answer that the more light a plant can absorb the better! Every color of light has at least
some energy the plant can use. Point out that these ideas are explored further in the Photosynthesis
lesson.
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Part 4 – Identifying the pigments.
A) Use the information below to identify which pigments are in your leaf:
Chlorophyll a: bright green-yellow
Chlorophyll b: dull blue-green or khaki
Carotene: orange or gold
Xanthophyll: pale yellow
B) Record your pigment names in TABLE 1.
C) Label the pigment names on your sketch in your lab notebook.
Type of leaf
Spinach
Color of pigment
Orange (gold)
Name of pigment
Carotene
Pale yellow
Xanthophyll
Chlorophyll a
Chlorophyll b
CONCLUSION / ANALYSIS:
1. Would your company be able to use this plant as a source for yellow dye? Explain your answer.
Yes, there is at least one type of yellow pigment visible. (Students may note that there is not a large
amount of yellow.)
2. Does your data support this statement: “Chlorophyll is the most abundant and important
photosynthetic plant pigment”? Explain.
This may vary depending on the health of the spinach and other variables, but the answer should be
YES. There should be more color green than other colors (especially when both types of chlorophyll
are included).
3. Why do most leaves contain more than one type of pigment?
The more light a plant can absorb the better. Every color of light has at least some energy the plant
can use, so having more pigments is a way to absorb morelight energy.
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