Chlorophyll Chromatography
Subject Area: Chemistry
Grade Level: High School Chemistry
Lesson Title: Chlorophyll Chromatography
National Science Education Standards
 Science as Inquiry: 9–12
Physical Science Standards:
 Properties and Changes of Properties: 5–8
 Structure of Atoms: 9–12
 Structure and Properties of Matter: 9–12
 Chemical Reactions: 9–12
Suggested Prior Knowledge: concepts of solutions, mixtures, separation of mixtures, solubility,
photosynthesis
Purpose: To give students an understanding of the process of chromatography and to allow
students to separate a mixture of photosynthetic pigments extracted from leaves.
Key Vocabulary:
absorbent—solid 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
solubility 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 glucose and carbon dioxide
solute—substance dissolved in a solution; the substance there is less of in solution
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solution—homogeneous mixture of two or more substances
solvent—substance dissolving the solute in a solution; the substance there is more of in
solution
Objectives:
1. Students will be able to design and carry out an investigation to separate the pigments
from a leaf by paper chromatography.
2. Students will identify a mixture by separating it into the different compounds.
3. Students will compare pigments found in different leaves to see similarities and
differences.
Materials:
- safety goggles
- column chromatography kit (commercially available from Flinn Scientific, AP7392)
- spinach leaf (fresh) and other leaves
- UV light source (optional)
- other optional materials if column chromatography kit is not used:
 chromatography solvent (commercially available: 90% petroleum ether and 10% acetone)
*note: this solvent can be reused; do not throw it away, save it!
 chromatography paper
 pencil
 ruler
 wooden splint
 large test tube
 50 ml graduated cylinder
 parafilm or aluminum foil
 stapler or tape
 penny
 scissors
Procedure:
1. Review with students the process of photosynthesis and the pigments that plants use to carry
out this process. Two of these pigments are chlorophyll a and chlorophyll b (see illustration
below). Review solutions and mixtures.
Separation of Photosynthetic Pigments by Chromatography (High School Level)
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http://www.bio.miami.edu/dana/226/226F08_10.html
2. Discuss with students how we can design an experiment that allows us to visually separate
these pigments from a leaf and try to identify each pigment. Begin with a leading question, and
follow up:
 What pigments do leaves contain to carry out photosynthesis?
 What kind of experiment can we do to find out how many pigments are in a leaf?
3. Lab protocols should be followed, incorporating safety equipment. Goggles must be worn at
all times.
4. The basic lab procedure from the column chromatography kit from Flinn Scientific can be
completed as a teacher demonstration or as a class experiment to introduce the column
chromatography technique. This procedure uses column chromatography to separate pigments in
spinach powder. The kit also includes information on how to analyze the relative polarity of the
compounds separated.
5. Once students have familiarized themselves with this technique, guide them to design an
investigation using this technique to explore photosynthetic pigments in different leaves. Some
leading questions include the following:
 Do leaves from various plants contain different pigments or do they all contain the same
pigments?
 Do leaves of plants that thrive in different environments (sun vs. shade etc.) contain
different pigments?
6. If column chromatography is not used, the following basic procedure for paper
chromatography can also 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. A
rectangular strip about 15 cm long and 2 cm wide is generally good.
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.
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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 when the splint rests across the opening of
the test tube (see the diagram below for positioning of the strip in the tube).
http://www.funsci.com/fun3_en/exper1/exper1.htm
g. Remove the chromatography strip from the flask and put enough solvent in the flask to
completely cover the bottom of the flask to a depth of less than 1.0 cm. When you hang the strip
back into the flask 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 flask so that the bottom of the strip is in the
solvent yet the solvent level is below the pencil line.
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.
j. Let the strip dry. You should be able to see the pigment spots for each pigment in the leaf
separately (see diagram of spinach paper chromatography results below).
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http://www.ableweb.org/volumes/vol-16/6-motten/6-motten.htm
k. 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. The
fluorescent properties of a particular chlorophyll pigment are functions of the structure of the
molecule and its immediate environment. This is a characteristic of chlorophyll that can help
students to compare and identify the pigments separated. Different pigments may not fluoresce
or may produce specific colors as characteristic properties. Chlorophyll pigments emit red
fluorescence.
7. Students can then work to design an investigation in which they can use this technique to
explore the photosynthetic pigments in various leaves. Guide them to develop a sound question
and an investigation that is valid and that will help them learn about these compounds and their
properties. Green plants have six closely related photosynthetic pigments (listed from least to
most polar): carotene (orange), xanthophyll (yellow), phaeophytin a (gray-brown), phaeophytin b
(yellow-brown), chlorophyll a (blue-green), and chlorophyll b (yellow-green). Chlorophyll a is
the most common pigment, present in all plants. Some possible questions to investigate include
the following:
 Do “shade loving” plants have different pigments than “sun loving” plants?
 Do plants with different colored leaves contain different pigments?
 Do all plants contain chlorophyll?
 Do autumn leaves that are red or orange still contain chlorophyll?
8. Guide students as they develop their questions and procedures to be certain that they include
all safety concerns and that they focus on a limited number of variables so their results are valid.
They can use the spinach investigation from the Flinn Kit or from the basic procedure of paper
chromatography as a control.
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9. Have the students record the data and any calculations from this experiment. If they do paper
chromatography they can calculate Rf values for each separated pigment:
Rf = the distance traveled by the pigment
the distance traveled by the solvent.
10. Students should include analysis of the relative polarity of the compounds found in each leaf;
this will allow them to be able to draw some valid comparisons between pigments from different
sources.
11. Have students draw conclusions about their results. They can then present their results to the
class using slideshows, movies, or poster boards.
Additional Resources:
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http://www.flinnsci.com
http://www.flinnsci.com/store/Scripts/prodView.asp?idproduct=22281&noList=
http://orgchem.colorado.edu/hndbksupport/chrom.html
http://en.wikipedia.org/wiki/Photosynthetic_pigment
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 Chromatography Investigation
Experiment Title: _____________________________Date: __________Name: _____________
Student Question or Hypothesis:
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Materials:
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Safety Concerns:
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Procedure: ____________________________________________________________________
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_______________________________________________________________________
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Data:
Observations: __________________________________________________________________
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Analysis of Data:
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Conclusion: ___________________________________________________________________
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