Curriculum topics:
 Chromatography
 Mixtures
 Photosynthesis
 Pigments
 Properties of
Materials
 Solutions
LEAF MIXTURE
CHROMATOGRAPHY
Cooking up a colorful pigment separation!
Subjects:
Life Science,
Physical Science
Grade range: 3 – 12
Photosynthesis is the process by which green plants combine carbon
dioxide, water, and solar energy to create sugars for support and for
food, releasing oxygen as a by-product. This activity reveals the
pigments in leaves that enable plants to harness the Sun’s energy, and
gives students valuable hands-on experience using a common
laboratory technique.
Who we are:
Resource Area for
Teaching (RAFT) helps
educators transform the
learning experience
through affordable
“hands-on” activities
that engage students
and inspire the joy and
discovery of learning.
For more ideas and to
see RAFT Locations
www.raft.net/visit-raft-locations
© 2014, RAFT
http://www.raft.net/raft-idea?isid=620
Materials required
For each activity station:
Cylindrical container, at least 14 cm
(5 ½”) diameter and 16.5 cm (6 ½”) tall
 Straw, stir stick, or stirring rod, ~4 cm (1 ½”)
longer than the diameter of the container
 Aluminum pan or equal that fits under the
container
 Hand lens, 1.5x – 3x or better
 Isopropyl alcohol, 70%
 Clear plastic wrap
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Paperclip
Scissors
Eye protection
Hot water
Fresh green leaves (any type)
Metric ruler
Filter paper or equal, cut into 2 cm (1”) wide
strips as long as the container height
Book or other flat weight, if needed
Safety Message: This activity uses isopropyl alcohol and hot water. Adult supervision is
required. Isopropyl alcohol is flammable. Keep away from heat and open flame. Avoid
prolonged exposure. Wear eye protection when handling isopropyl alcohol. Wash hands
thoroughly using soap and water after handling.
Dispose of isopropyl alcohol in accordance with federal, state and local regulations.
Teaching tip: This activity requires time for leaves to soak (3 or more hours) and time for the
pigments to travel upward in the filter paper. Start early in the day or hold over a 2 day period.
How to build it
1
Figure 1
Cut or tear leaves into small pieces
and place in container. Add enough
pieces to cover bottom of container.
See figure 1.
Leaf
pieces
Figure 2
2
Leaf/alcohol
mixture
Pour enough isopropyl alcohol into
container to cover leaf pieces and
stir. See figure 2.
Alcohol
3
Cover container with plastic wrap
and set inside of aluminum pan.
See figure 3.
Plastic
wrap
Pan
Figure 3
Figure 4
4
Fill pan half full of hot water. If
container floats in pan, set a book
or other weight on top of container
to weigh it down. Allow the leaf
mixture to soak for 3-4 hours to
release the pigments into the
alcohol. See figure 4.
Leaf Mixture Chromatography, page 2
Pour hot
water
into pan
Use book as weight
if necessary
© 2014, RAFT
5
Draw horizontal line 1 cm from end of paper strip. This serves as base line of
chromatogram. See figure 5
Figure 5
1 cm from edge
6
Remove water from pan and plastic wrap from container.
Lay straw across the opening of container. See figure 6.
Figure 6
7
Attach paper strip to center of straw with paperclip. Lower straw making sure baseline
is at bottom and exactly at the surface of the liquid. Allow alcohol and pigments to
travel upward through paper for 45 minutes. See figure 7.
Lower
onto
container
Baseline level
8
Figure 7
Remove paper strip from straw and draw horizontal line indicating maximum distance
alcohol traveled on chromatogram. Lay flat on a paper towel to dry. See figure 8.
Distance alcohol
traveled
Pigments
Dried
chromatogram
Figure 8
Base line
Leaf Mixture Chromatography, page 3
© 2014, RAFT
Curriculum
Standards:
To do and notice
Teaching Tip: For younger students, focus the discussion on the
importance of pigments in photosynthesis and reserve math and
chemistry concepts for older students.
Plant structures
(Next Generation Science
Standards: Grade 4,
Life Science 1-1)
Plants get material chiefly
from air and water
(Next Generation Science
Standards: Grade 5,
Life Science 1-1)
Cycles of matter / energy
(Next Generation Science
Standards: Life Science,
Grade 5, 2-1;
Middle School, 1-6)
Structure of matter
(Next Generation Science
Standards: Grade 5,
Physical Science 1-1)
Property of materials
(Next Generation Science
Standards: Grade 5,
Physical Science 1-3)
Energy in food comes
from the sun
(Next Generation Science
Standards: Grade 5,
Physical Science 3-1)
Science & Engineering
Practices
(Next Generation Science
Standards: Grades 3-12)

1
Count and list the different bands of color present on the
chromatogram. Which color is most common? Observe with hand lens
and discuss observations with a partner.
2
Identify the type of pigment in each band (anthocyanins are red/brown,
carotenoids are yellow/orange, and chlorophyll is green).
3
Calculate Rf values for each band using formula in “The science behind
the activity”.
4
Use Rf values to identify leaf pigments with highest and lowest rates of
upward movement and discuss with a partner how the rates might
relate to the size of the pigment molecules.
Learn more
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Investigate the colors and amounts of pigment in other types of leaves.
Use this activity to determine the season in which the leaves were picked.
Hint: Think about how leaf colors change during the year.
Use an acid/base indicator to compare pH of different leaf mixtures.
Measure Rf values for leaves gathered in different seasons.
Create a large-volume leaf mixture and store, then run activity once a
week to determine if chlorophyll degrades during storage (compare
chromatograms in terms of colors and Rf values).
Create mixture with food dyes instead of leaves, run chromatogram, and
discuss similarities/differences between food dye and real leaf pigments.
Related activities: See RAFT Idea Sheets:
Chromatography –
http://www.raftbayarea.org/readpdf?isid=38
Photosynthesis Review Books http://www.raftbayarea.org/readpdf?isid=628
Plant Cells http://www.raftbayarea.org/readpdf?isid=557
A Reason for the Seasons http://www.raftbayarea.org/readpdf?isid=523
Solar Path Across the Sky –
http://www.raftbayarea.org/readpdf?isid=518
Resources
Visit http://www.raft.net/raft-idea?isid=620 for “how-to” video demos & more
ideas! See these websites for more information on the following topics:

Additional standards at:
http://www.raft.net/raftidea?isid=620
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Leaf Mixture Chromatography, page 4
Chromatography details – www.rpi.edu/dept/chem-eng/BiotechEnviron/CHROMO/chromintro.html
Photosynthesis – biology.clc.uc.edu/courses/bio104/photosyn.htm
Video on Photosynthesis from the Khan Academy –
https://www.khanacademy.org/science/biology/photosynthesis
© 2014, RAFT
The science behind the activity
Plants absorb water through their roots and carbon dioxide into their leaves to make glucose (sugar).
Glucose is used as a food source and for growth and structure. Photosynthesis is the process plants
use to convert water and carbon dioxide into glucose. Plants absorb light energy using pigments to
catalyze (provide the necessary conditions for) the chemical reactions involved in photosynthesis.
Pigments like chlorophyll are large molecules responsible for different colors seen in leaves and stems.
Each pigment reflects a specific wavelength of light which the eye perceives as color. Chlorophyll
appears green because it reflects green light. Anthocyanins are pigments that reflect red light.
Carotenoids reflect orange light.
During the autumn and winter months deciduous trees shed their leaves. Chlorophyll begins to
chemically break down in fallen leaves and the green color fades, revealing the orange, yellow, and red
pigments that were always present. This is why autumn leaves can be various hues of yellow, orange,
and red.
Chromatography is a technique used to separate the parts of a mixture. The pigment solution is
separated into color bands due to absorption and capillary action. The rate of movement is dependent
on the molecular weights of the pigments. The rate of movement (Rf value) helps identify substances in
a solution. Chromatography is applied in the fields of chemistry, biology, marine ecology, and forensics.
Chromatography is used to separate mixtures into their components. Each type of chromatography
works on a two-phase principle. The stationary phase is typically a solid. The mobile phase is a liquid
that flows through the stationary phase carrying components of the mixture with it. This activity uses
paper (stationary) and alcohol with pigments (mobile).
Some compounds in a mixture move almost as far through the stationary phase as does the solvent
while other move less. The distance a compound travels through the stationary phase relative to that of
the solvent (same units) is called the Rf value for the compound, which is constant for each compound if
obtained under constant conditions. The closer the Rf value is to 1, the higher the rate of movement of
the compound. Rf values are calculated using the following formula:
Rf = distance traveled by compound
distance traveled by solvent
= A
B
B
A
Baseline Level
Leaf Mixture Chromatography, page 5
© 2014, RAFT