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Picking out the Pigments
Picking out the Pigments
Isolating and Comparing Plant Pigments
Plants contain a variety of pigments that vary in color and chemical characteristics. Chlorophyll α and
chlorophyll β, carotenoids, and xanthophylls are four pigments found in the chloroplasts of plants.
These pigments play important roles in the process of photosynthesis. Chlorophyll α has a blue-green
appearance. The yellow-green pigment is chlorophyll β. The orange-yellow pigment is carotene and the
light yellow pigments are called xanthophylls. These four pigments are non-polar and are not soluble in
water. A fifth pigment type, the anthocyanins, have a redish purple color, are polar and soluble in water.
The anthocyanins, found in the vacuoles of leaves, serve a protective role, help to attract pollinators, and
are thought to be distasteful to predators.
Chlorophyll α appears bright green to a bluish green color to the human eye. The color detected by
human eye results from the green wavelength of light being reflected by this pigment. As sunlight
strikes the leaf, the other wavelengths of light in the visible light spectrum (red, orange, yellow, blue,
indigo, and violet) are absorbed by the leaf while the chlorophyll pigments reflect the green
wavelengths. The wavelengths that are absorbed by the various pigments in the plant’s leaves are
collectively referred to as the absorption spectrum. Some of the wavelengths that have been absorbed
by the plant will energize the plant’s photosynthetic process, setting energy transfer events into action.
The wavelengths of light that stimulate the plant’s chemical processes are referred to as the plant’s
action spectrum. The action spectrum for many plants is limited to the red and blue wavelengths of
light. See Figure 1.
Fig. 1
The various pigments in a cell extract are separated and identified using a technique called paper
chromatography. In this technique, the solvent moves up the chromatography paper and carry the
dissolved pigments with it. The pigments move up the paper at unequal rates. One reason for this
unequal rate is that the pigments vary in their solubility. Another reason for this unequal rate is due to
the different degrees of attraction between the pigments and the paper. The fewer intermolecular
attractive forces formed with the paper, the faster the rate of movement of the pigment. For example,
when cell extracts containing beta carotene are placed in an acetone/ether solvent, few hydrogen bonds
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form between the paper and the pigment and beta carotene is very soluble in acetone/ether. As a result
beta carotene will move rapidly up the chromatogram.
Plant leaves contain varying amounts of chlorophyll, carotene, xanthophylls and anthocyanin. However,
most plants have a higher concentration of chlorophyll than the other pigments giving them an overall
green appearance. Some plants like Coleus have leaves that reveal a variety of colors including green,
yellow, and red due to the variation in pigments. During the fall, deciduous plants will decrease their
levels of photosynthesis in response to reduced hours of sunlight per day and the amount of chlorophyll
present will decline. This results in revealing the carotenes and xanthophylls which make the leaves
appear orange and yellow. The bright red and purple colors come from anthocyanin pigments. Brown
colors come from tannin, a bitter waste product. Different combinations of these pigments give us a
wide range of colors each fall.
The arrangement of these five types of pigments on the chromatography paper and the relative locations
of the pigments on the paper will change in different solvents. For example, solvents containing water
will produce produce anthocyanin bands when other solvents do not.
PURPOSE
In this activity you will first prepare a chromatogram using pigments of swiss chard leaves and the
solvent acetone. In the second portion of the activity, you will devise a plan to make a chromatogram
containing as many bands as possible.
MATERIALS
3 strips of chromatography paper
3 large test tubes (25 x 200mm)
3 rubber stopper with hook or pin
1 leaf from spinach
1 small glass beaker 14 rulers
100 mL isopropyl alcohol
100 mL each of various solutions to be chosen
from in Part II.
isopropyl alcohol, acetone, ethanol, water
1 leaf from Coleus or swiss chard
10 mL graduated cylinder
Safety Alert
1. Aprons and goggles must be worn during this activity.
2. CAUTION: Acetone, alcohols, and organic solvents are flammable.
3. Avoid inhalation of fumes from solvents.
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PROCEDURE
PART I
1. Three solvents are provided for you to use to make a chromatogram from spinach leaves. Solvent A
is made of petroleum ether and acetone, Solvent B is made of acetone, and Solvent C is acetone and
water. Which solvent will produce the most bands on a chromatogram of spinach leaf pigment?
Write your answer in the form of a hypothesis on the student answer page.
2. Obtain a piece of chromatography paper that has been precut to fit the large test tube.
3. Use a pencil to draw a line across the width of the chromatography paper 1.5 cm from the bottom of
the paper. This will be your starting line. See Figure 2.
Fig. 2
4. Obtain a spinach leaf from your teacher. Place the spinach leaf on top of the line on the
chromatography paper. Using a coin, press the pigment into the chromatography paper by rolling the
coin over the surface of the leaf several times.
5. Obtain a large test tube and stopper to serve as your chromatography chamber.
6. Attach your pigment stained chromatography paper to the stopper and insert the paper into the test
tube. Observe the position of the paper. It should be suspended in such a way that the tip is almost
touching the bottom of the test tube. Do not allow your chromatography paper to touch the sides of
the test tube. Adjust the position of your chromatogram as needed.
7. Remove the stopper and paper from the tube, and place approximately 5 mL of Solvent A in the
bottom of the test tube. You will need enough solvent to wet the bottom of the paper without
covering the pigment line. See Figure 2 above. Avoid inhaling the solvent fumes.
8. Repeat steps 4 through 7 for Solvent B and Solvent C.
9. Place the test tubes in an upright position and leave them undisturbed until the solvent has wicked at
least 3/4 of the way up the chromatography paper. DO NOT LET THE SOLVENT RISE TO THE
TOP OF THE PAPER.
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10. Remove the stopper and chromatography paper from the tube. Quickly mark the level that the
solvent has traveled, using a pencil.
11. Use a pencil to mark the location of each pigment on your chromatogram. Sketch your
chromatogram in the data section of the student answer page. Include the colors of the pigments in
your diagram.
12. Using a ruler, measure the distance the solvent moved from the starting line to the solvent pencil
mark. Record this distance in the data table on the student answer page.
13. Measure the distance traveled by each of the pigments from the starting line. Record the
measurements in the data table.
14. Using the information provided in the introduction, determine the type of pigment found in each
band on the chromatogram.
15. Answer the Part I conclusion questions on the student answer page.
PART II
1. Using the chromatography technique learned in Part I, determine which solvent combination will
allow you to extract the largest number of pigments from Coleus.
2. Write your hypothesis in the space provided on the student answer sheet.
3. Design an experiment to test your hypothesis. Describe your experiment in the space provided on
the student answer page. Limit your experimentation for this portion of the lab to two
chromatograms.
4. Record the distanced traveled by the pigments in your chromatograms in the data table provided on
the student answer page.
5. Label the probable pigment types for each band in your chromatograms. Place the pigment names in
the data table.
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Picking out the Pigments
Name______________________________________
Period _____________________________________
Picking out the Pigments
Isolating and Comparing Plant Pigments
HYPOTHESIS
Part I Hypothesis
Part II Hypothesis
DATA AND OBSERVATIONS
Part I Data: Sketch your chromatogram(s) in the space below:
Part II Data: Sketch your chromatogram(s) in the space below:
Part II: Experimental Design
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ANALYSIS
Data Table 1: Acetone Chromatogram Measurements
Band #
Color
Distance Traveled
Pigment Type
Rf*
1 (top)
2
3
4
5
Data Table 2: Part II Chromatogram #1 Measurements
Band #
Color
Distance Traveled
Pigment Type
Rf*
Data Table 3: Part II Chromatogram #2 Measurements
Band #
*R f =
Color
Distance Traveled
Pigment Type
Rf*
Distance pigment migrated
Distance solvent front migrated
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CONCLUSION QUESTIONS
1. What types of pigments are typically found in leaves?
2. What causes the pigments to move up the chromatogram different distances?
3. List the pigments in order of solubility from most soluble to least soluble in solution A.
4. Why do some of the pigments appear yellow to our eyes while others appear green?
5. How is an action spectrum different from an absorption spectrum?
6. Why are all of the pigments not visible in a typical leaf?
7. Compare the bands produced in solutions A, B, and C. How are they alike? How are they different?
8. How do the chromatograms made from Coleus (or swiss chard) compare to those made from spinach
leaves?
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