Chlorophyll Chromatography
Subject Area: Chemistry
Grade Level: Middle School Chemistry
Lesson Title: Chlorophyll Chromatography
National Science Education Standards:
 Science as Inquiry: 5–8
 Properties and Changes of Properties: 5–8
Suggested Prior Knowledge: concepts of solutions, mixtures, separation of mixtures, solubility
Purpose: To give students an understanding of paper chromatography and to allow students to
separate a mixture of pigments extracted from leaves.
Key Vocabulary:
absorbent—material used in chromatography that will attract and absorb the compounds
being separated
chlorophyll—one of many pigments used by plants to absorb energy from sunlight in the
process of photosynthesis
chromatography—method used to separate a mixture of compounds based on differing
solubilities of the compounds in the solvent being used
eluent (solvent)—material used in chromatography which carries the compounds to be
separated through the absorbent
photosynthesis—process by which plants convert energy from sunlight, water, and carbon
dioxide gas into sugar
solute—substance dissolved in a solution
solution—homogeneous mixture of two or more substances
solvent—substance dissolving the solute in a solution
Objectives:
1. Students will be able to design and carry out an investigation to separate the pigments
from a leaf by paper chromatography.
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2. Students will identify a mixture by separating it into different compounds.
Materials:
- safety goggles
- chromatography paper or filter paper cut into strips
- chromatography solvent (commercially available: 90% petroleum ether and 10% acetone or
50/50 mixture of acetone and water)
- isopropyl alcohol, acetone, water and/or ethanol (optional additional solvents)
- pencil
- ruler
- wooden splint
- 250 ml Erlenmeyer flasks
- 50 ml graduated cylinder
- aluminum foil (or parafilm)
- stapler or tape
- spinach leaf (fresh) or other leaves
- penny
- scissors- UV light source (optional)
Procedure:
1. Discuss with students photosynthesis and the pigments that plants use to carry out this process.
Plants use chlorophyll and other pigments to collect light energy. Review solutions and mixtures.
Discuss with students how we can design an experiment that allows us to visually sparate these
pigments from a leaf. Begin with a leading question, and follow up:
 What makes leaves green or other colors?(pigments such as chlorophyll)
 What kind of experiment can we do to find out how many pigments are in a leaf?
 What kind of experiment could we do to compare leaves of different colors or different
kinds of plants to see if they have different pigments?
 Do you think fall leaves that are colored yellow or orange still contain green
chlorophyll?
 What about multi-colored leaves? Do they contain the same pigments?
2. Lab safety equipment and protocols should be followed. The chromatography solvent is highly
flammable and mildly toxic by inhalation and ingestion. This lab investigation should only be
carried out in a well-ventilated room with no open flames. Wear goggles at all time. Wash
thoroughly if solvent gets on skin. Do not dispose of left over chromatography solvent, save it
for future use. This solvent should be stored clearly labeled in a specific flammables storage
cabinet.
3. Work with students to develop a hypothesis or question to test and design an investigation to
answer their questions or test their hypotheses. Some possible topics include:
 Do all leaves have the same number and kinds of pigments?
 Do different colored leaves contain different pigments?
 Do darker green leaves contain just green pigments?
 Do red or orange fall leaves contain green chlorophyll?
 Do other solvents give the same results?
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
How do the pigments compare in leaves from plants that prefer lots of sun to those that
like the shade?
4. Guide students to develop an investigation that is valid and that allows them to visually test
their hypothesis or question.
5. Be sure that proper safety procedures are followed and that students all wear goggles.
6. Students might benefit from doing an initial investigation with the chromatography solvent
and spinach leaves (control) then developing a plan to test various variables (different leaves,
different solvents). Special note: Some students might also benefit from an introductory
investigation of this chromatography procedure using water-soluble markers for the pigment
mixture and water as the solvent. Different brands of brown, green, and purple markers often
give interesting results.
7. The following basic procedure can be used:
a. Obtain the leaf or leaves to be tested.
b. Obtain chromatography paper strips, pencil, ruler, penny, large test tube, wooden splint, and
scissors.
c. Cut the chromatography paper into a long thin strip that is slightly narrower than the mouth of
the test tube and is long enough to extend from the bottom of the test tube out the mouth.
d. Make a pencil line 1 cm in from one narrow end, as in the diagram below. This end will be the
bottom of your strip.
e. Place a piece of the spinach or other leaf over this line and use the edge of the penny to rub
across the leaf along the pencil line drawn on the strip so that the pencil line is now covered with
pigments from the leaf. It is important that the chromatography strip contain a single, narrow,
horizontal green line.
f. Stand the strip of chromatography paper in the empty test tube and staple it to the wooden
splint so that it hangs freely from the stirrer into the test tube but does not touch the bottom. It
should hang very close (0.5 cm or so) to the bottom of the test tube when the splint rests across
the opening.
g. Remove the chromatography strip from the flask and put enough solvent in the test tube to
completely cover the bottom of the test tube to a depth of less than 1.0 cm. When you hang the
strip back into the test tube it should touch the solvent but the solvent should be below the pencil
line on the strip.
h. Carefully place the chromatography strip in the test tube so that the bottom of the strip is in the
solvent yet the solvent level is below the pencil line. See the diagram below for positioning of
the strip in the tube.
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http://www.funsci.com/fun3_en/exper1/exper1.htm
i. Allow the solvent to move up the chromatography strip. This movement is caused by capillary
action. As the solvent is drawn up the strip, it will carry the pigments in the sample at different
rates depending on the characteristics of the individual compounds. When the solvent level gets
close to the top of the strip (not to the staple and splint yet) remove the strip from the solvent to
stop the movement, and make a light pencil mark at the solvent top. It may also help to use a
pencil to mark the separated bands on the strip in case the colors fade as the paper dries.
Let the strip dry. You should be able to see the pigment spots for each pigment in the leaf
separately.
j. If you have access to an ultraviolet (UV or “black”) light, have students shine the light on their
strips and record their observations. Some pigments will fluoresce under UV light.
8. Have the students record the data and observations from this experiment. They should record
the color (be descriptive!) of each pigment and compare the distance each travelled. They should
also use colored pencils to color in the bands for their lab write up or presentations. If they test
different leaves, they can try to see if the leaves share some similar pigments and look for
different pigments.
9. Have students draw a conclusion (or conclusions) about their results.
10. Students may present their results to the class in a slideshow or on a poster.
11. After students have drawn their conclusions, the teacher may want to lead a discussion about
the similarities and differences found. Explain that different plants use different pigments
depending on their needs (varying amounts of sunlight, leaf type, etc).
Additional Resources:


http://orgchem.colorado.edu/hndbksupport/chrom.html
http://en.wikipedia.org/wiki/Photosynthetic_pigment
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


http://biology.wsc.ma.edu/biology/courses/concepts/labs/pigments/
http://en.wikipedia.org/wiki/Chlorophyll
http://www.rpi.edu/dept/chem-eng/Biotech-Environ/CHROMO/chromintro.html
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Student Worksheet for Chlorophyll Chromatography
Experiment Title: _____________________________Date: __________Name: _____________
Student Hypothesis or Question:
_____________________________________________________________________________
Materials:
_______________________________________________________________________
________________________________________________________________________
________________________________________________________________________
_______________________________________________________________________
Procedure: (Include all safety procedures)
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
Data: Colors/Pigments Observed
Leaf 1
Leaf 2
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Leaf 3
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Diagram of strips:
Analysis of Data:
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Conclusion: ___________________________________________________________________
_____________________________________________________________________________
_____________________________________________________________________________
_____________________________________________________________________________
_____________________________________________________________________________
_____________________________________________________________________________
_____________________________________________________________________________
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