The Huntington Library, Art Collections, and Botanical Gardens
Fast Plant Chromatography
Overview
A solvent is used to separate pigments from a leaf extract, and Rf values are used to
identify those pigments.
Introduction
If asked to define a plant, many of us would start by keying in on their relatively
consistent color: “They’re green.” Indeed, the importance of the chlorophyll that gives plants
their green color can’t easily be overstated. As a result of the characteristics of these pigments,
plants have the ability, through a harvesting of the sun’s energy, to produce their own food. They
are autotrophs. Ultimately, this process, photosynthesis, is the source of food for the other life on
earth as well. On some level, we are all dependent on photosynthesis.
Good thing we know a lot about it. Photosynthesis is series of light-driven reactions that
converts carbon dioxide and water into energy-rich sugars. The process begins when pigments in
the plant absorb the light energy of the sun. Chlorophyll a is the most important pigment in
photosynthesis, and absorbs light in the blue and red zones of the visible light spectrum. This
pigment reflects green light and plants appear green because we see that reflected light. When
the chlorophyll a molecules absorb light photons, they channel that energy and excite electrons
that begin a series of reactions to create chemical energy. Accessory pigments, like chlorophyll
b, carotenoids, and xanthophylls help optimize the harvesting of light energy, as they absorb
wavelengths of the visible spectrum not absorbed by chlorophyll a.
Because chlorophyll a is much more abundant than any of the accessory pigments, it
tends to mask their presence in the plant. In this lab, students will separate the pigments present
in a leaf. When a solution of pigments is applied to chromatography paper and submerged in the
proper solvent, the solvent moves through the pigments and up the paper. The pigments differ in
molecular size, polarity, and solubility, and so move up the paper at different speeds.
Motivation
If you were painting a landscape and had a selection of white, navy, red, green, yellow,
white, brown, and black colors available, how would you go about painting a sky? What colors
would you use?
When artists create paintings, they mix the colors on the palates to get just the right
shades. To obtain the correct sky blue, they may add white and grey to a dark navy and mix
everything together. The individual colors that are part of the mix are no longer visible on their
own, but they are important for the end result. Similarly, the leaves of plants are all different
shades of colors, usually a form of green. The color we see is the result of a mix. The mix of
pigments is important to help the plant harvest a broad spectrum of light wavelengths. In this lab,
we will separate out the individual pigments in a leaf.
Objectives
Upon completion of the lab, students should be able to
1. Visually identify the pigments involved in photosynthesis in Fast Plant leaves.
2. Calculate the Rf value for each pigment.
3. Discuss the role pigments play in photosynthesis.
Materials
• Chromatography paper
• Fast Plant leaves and/or cotyledons
• Paper clip
• Parafilm or foil
• Petroleum ether/ethanol solution 9:1
(sold as “chromatography solvent”)
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•
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•
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50 ml graduated cylinders
Pencil
Ruler
Penny
Disposable pipettes
Associated California State Biology Standards
1f. Students know usable energy is captured from sunlight by chloroplasts and is stored through
the synthesis of sugar from carbon dioxide.
1i. Students know how chemiosmotic gradients in the mitochondria and chloroplasts store
energy for ATP production.
Procedure
1. Give each student a strip of chromatography paper. Make sure you and your students handle
the paper by its edges so that oil on your fingers does not contaminate the paper. Each
student or student group can complete the rest of the procedure but you may wish to be
particularly careful with the extremely flammable chromatography solvent.
2. Make a faint pencil mark across the paper approximately 2cm from the tip of the paper.
3. Place a leaf of cotyledon of a Fast Plant along the pencil line. Use a penny to roll along the
line, in the process transferring pigment extract to the chromatography paper. Let dry
completely and repeat 3-5 times.
4. Straighten a paper clip and bend one end through the top of the chromatography paper. Bend
the other end over the top of the graduated cylinder so that the bottom of the chromatography
paper is suspended just above the bottom of the tube (about at the 2-5 ml mark).
5. Remove the paper and pipette in chromatography solvent to just above where the end of the
paper was in the last step. Caution: the solvent is extremely flammable and should not be
inhaled.
6. Place the paper strip in the cylinder so that the end nearest the pigment is submerged in the
solvent, but the 2cm line is above the solvent level.
7. Immediately cover the cylinder, containing the chromatography paper, with foil or parafilm.
Wait and watch for 10 minutes.
8. Remove the paper and reuse or properly discard of the solvent or properly store for re-use.
9. Mark the position of the solvent front with a pencil and set the strip aside to dry. Observe the
bands of color and draw your results. Four bands should appear: a yellow band of carotenes,
a yellow band of xanthophylls, a blue-green band of chlorophyll a, and a yellow-green band
of chlorophyll b.
10. Measure the distance from the pigment origin to the solvent front and from the pigment
origin to each pigment band. Calculate the Rf for each pigment: Rf is the distance traveled by
the pigment divided by the distance traveled by the solvent front (from the pigment’s origin).
Record the Rf values for each pigment. Rf values differ depending on the solvent used, but
the order should remain the same: yellow-colored carotenes travel the farthest, followed by
yellow xanthopylls, blue-green chlorophyll a, then the yellow-green chlorophyll b. The Rf
values for petroleum ether by pigment are listed below:
Pigment
Carotenes
Xanthophylls
Chlorophyll a
Chlorophyll b
Approx. Rf Value
0.95
0.71
0.65
0.45
Evaluation
The following questions are listed under the Analysis section of the student handout and may be
used as part of a report, class discussion or assessment.
1. Which pigment travels the farthest? What does that tell you about the pigment?
2. Why aren’t the pigments made apparent by chromatography normally visible?
3. Why do plants have more than one pigment?
4. How are pigments involved in photosynthesis?
Extension Activities
1. Using the Huntington Botanical Gardens’ sunflower photosynthesis poster and
accompanying booklet, conduct the “Charge of the Light Brigade” activity in which the class
acts out the pathways involved in photosynthesis.
2. Have students research recent scientific articles about fall color change in plants and present
the varying views on why and how this happens. Example news articles (not journal articles)
on the subject are available here: http://www.ljmu.ac.uk/NewsCentre/63012.htm and
http://www.sciam.com/article.cfm?articleID=0005DF26-7ABD-1D949275809EC5880105&pageNumber=2&catID=4 . One example journal article on the topic is
located here:
http://harvardforest.fas.harvard.edu/research/leaves/pdf/Schaefer_and_Wilkinson_TREE_20
04.pdf . Please note that thought the article was published in December 2004, this version is
an unpublished one, and so may not reflect peer reviewing.
Test Preparation
1. When electrons of a chlorophyll molecule are raised to a higher energy level
(A)
they become a photon of light.
(B)
they form a glucose bond.
(C)
they enter an electron transport chain.
(D)
carotenoids are converted to chlorophyll.
2. Chlorophyll is green because
(A)
it absorbs green wavelengths of light.
(B)
it absorbs blue and yellow wavelengths, which make green.
(C)
it reflects green wavelengths of light.
(D)
of an optical illusion cause by transmitted light.
Student Sheet: Fast Plant Chromatography
Name:________________________
Procedure
1. Make a faint pencil mark across a strip of chromatography paper approximately 2cm from
the tip of the paper. Make sure you handle the paper by its edges so that oil on your fingers
does not contaminate the paper.
2. Place a leaf of cotyledon of a Fast Plant along the pencil line. Use a penny to roll along the
line, in the process transferring pigment extract to the chromatography paper. Let dry
completely and repeat 3-5 times.
3. Straighten a paper clip and bend one end through the top of the chromatography paper. Bend
the other end over the top of the graduated cylinder so that the bottom of the chromatography
paper is suspended just above the bottom of the tube (about at the 2-5 ml mark).
4. Remove the paper and pipette in chromatography solvent to just above where the end of the
paper was in the last step. Caution: the solvent is extremely flammable and should not be
inhaled.
5. Place the paper strip in the cylinder so that the end nearest the pigment is submerged in the
solvent, but the 2cm line is above the solvent level.
6. Immediately cover the cylinder, containing the chromatography paper, with foil or parafilm.
Wait and watch for 10 minutes.
7. Remove the paper and reuse or properly discard of the solvent.
8. Mark the position of the solvent front with a pencil and set the strip aside to dry. Observe the
bands of color and draw your results on the back of this paper. Four bands should appear: a
yellow band of carotenes, a yellow band of xanthophylls, a blue-green band of chlorophyll a,
and a yellow-green band of chlorophyll b.
9. Measure the distance from the pigment origin to the solvent front and from the pigment
origin to each pigment band. Calculate the Rf for each pigment. Rf is the distance traveled by
the pigment divided by the distance traveled by the solvent front (from the pigment’s origin).
Record the Rf values for each pigment. Rf values differ depending on the solvent used, but
the order should remain the same: yellow-colored carotenes travel the farthest, followed by
yellow xanthopylls, blue-green chlorophyll a, then the yellow-green chlorophyll b. The Rf
values for petroleum ether by pigment are listed below. Label the pigments in your drawing
and include the calculated Rf values.
Pigment
Carotenes
Chlorophyll a
Chlorophyll b
Xanthophylls
Approx. Rf Value
0.95
0.65
0.45
0.71
Analysis
On a separate sheet of paper, please complete the following:
1. Which pigment travels the farthest? What does that tell you about the pigment?
2. Why aren’t the pigments made apparent by chromatography normally visible?
3. Why do plants have more than one pigment?
4. How are pigments involved in photosynthesis?