Chromatography: Leaf Pigments
As you may have noticed, plants display a variety of colors depending on what season of the year it is. These color displays are created by a variety of pigments. A pigment is a substance the will absorb light. For example, if you have a leaf that is red in color, it will reflect light wavelengths in the red portion of the visible light spectrum and abosorb the other wavelengths.
Some leaf colors are produced by only one pigment, while a greater diversity of colors are produced by the accumulation of different pigments. Chlorophyll-a will produce a bright green to blue green color . Cholorphyll-b will produce a color which is yellow green to olive green. Yellow to yellow orange or possibly red will be produced by carotenoids. Pale yellow is produced by xanthophylls. All of these pigments are located in the chloroplasts. These pigments are not water soluble. Other colors such as the blues, purples, violet, pinks and dark reds are usually produced by a water soluble pigment called the anthocyanins. These are located in the vacuoles and are not part of the photosynthetic processes.
You will be using the process of chromatography to separate and identify the type of pigments found in leaf samples. The solvent will travel up the paper by capillary action. This is due to the attraction of solvent molecules to the paper and the attraction of solvent molecules to one another. As the solvent moves up the paper, the dissolved pigments will be carried along. These pigments will be carried along at different rates because they are not equally soluble and will have different degrees of attractions due to hydrogen bonding. The less the degree of hydrogen bonding, the farther the pigment will travel. If molecules contain oxygen or nitrogen, they tend to bond more tightly to the paper and not move as far up the solvent line.
Some leaf colors are produced by only one pigment, while a greater diversity of colors are produced by the accumulation of different pigments. Chlorophyll-a will produce a bright green to blue green color . Cholorphyll-b will produce a color which is yellow green to olive green. Yellow to yellow orange or possibly red will be produced by carotenoids. Pale yellow is produced by xanthophylls. All of these pigments are located in the chloroplasts. These pigments are not water soluble. Other colors such as the blues, purples, violet, pinks and dark reds are usually produced by a water soluble pigment called the anthocyanins. These are located in the vacuoles and are not part of the photosynthetic processes.
You will be using the process of chromatography to separate and identify the type of pigments found in leaf samples. The solvent will travel up the paper by capillary action. This is due to the attraction of solvent molecules to the paper and the attraction of solvent molecules to one another. As the solvent moves up the paper, the dissolved pigments will be carried along. These pigments will be carried along at different rates because they are not equally soluble and will have different degrees of attractions due to hydrogen bonding. The less the degree of hydrogen bonding, the farther the pigment will travel. If molecules contain oxygen or nitrogen, they tend to bond more tightly to the paper and not move as far up the solvent line.
Engaging Questions
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Based on results, which band would be the least polar?
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Based on results, which band would be the most polar?
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Would you expect the Rf value of a pigment to be the same if a different solvent were used? Explain.
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What factors are involved in the separtion of the pigments?
Teacher Goals
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By using the process of chromatography, you will be able to identify the pigments involved in photosynthesis.
Required Resources
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Jar with a lid or graduated cylinder with a stopper
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Leaf samples
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Quarter or dime
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Pre-cut chromatography paper
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5 ml of solvent (9 parts petroleum ether/ 1 part acetone)
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Use this chromatography data sheet to record results that later may be entered online for sharing.
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Rf and pigment chart may be used as needed.
Optional Resources
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For background on paper chromatography and Rf values read more at Wikipedia.
Steps
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Leaf Location
Consider creating a Location resource to document the place the leaf was collected for this activity. Be prepared to attach it to the form provided for this activity. Use the Help option if needed. -
Jar with Solvent
Obtain the chromatography jar which has 5 ml of solvent in the bottom. Make sure the lid is on tight because the solvent is volatile. -
Chromatography Paper
Obtain the pre-cut chromatography paper and lay it on a clean surface. Touch the paper as little as possible. -
Pencil Mark
Use a pencil to mark a straight line 1.5 cm from and parallel to the bottom of the paper. -
Pigment Mark
Place a small section of leaf on top of the pencil line. Use the rough edge of a quarter to crush the leaf cells by pressing down hard and rubbing back and forth. Be sure the pigment is on top of the pencil line. Repeat this process 8 to 10 times. -
Solvent
Use forceps to hold the paper and lower it into the solvent. Do NOT allow the pigment to be in the solvent. The paper should stand up straight and NOT touch the jar. Cover the jar and leave it undisturbed. Depending on resources available to you, the setup should look similar to graphic.DO NOT SMELL THE SOLVENT!!
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Remove Chromatography Paper
Check the jar every few minutes. When the solvent is about 1 cm from the top of the paper (about 20 minutes), remove the paper. -
Solvent Front
Using your pencil, IMMEDIATELY mark the solvent front (edge of the part wet by the solvent) before it evaporates. -
Pigment Band
Mark the bottom of each pigment band. Note the color of each band. Record this information on the chart located in the Rubrics section. -
UV Light
Take your chromatogram into a dark room, or place it in a dark box, and view it under UV light. NEVER LOOK DIRECTLY AT THE UV LIGHT. Note the bright pink fluorescence of chlorophyll. Outline the spots in pencil. Note whether there are any dark areas that absorb UV. Outline these in pencil. Record this information on the chart. -
Measure
Measure in cm the distance travelled from the pigment origin to the bottom of the pigment band. Record this information on the chart. -
Rf Value Calculations
Calculate the Rf value. Rf is equal to distance pigment migrated (cm) divided by distance solvent front migrated (cm). Record the Rf value in chart. Tape your chromatogram to the chart. -
Share Results
Use the attached form to share results with others. OPTIONAL: If resources and time is provided, consider scanning the chromatography paper after it dries and upload to site in order to attach to form.
Teacher Notes
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The following information may be helpful:
- Chromatography paper is polar.
- The solvent, made of petroleum ether and acetone, is nonpolar. It can be purchased pre-mixed.
- The most nonpolar substance will dissolve in the nonpolar solvent first.
- The most polar substance will be attracted to the polar chromatography paper so it will move last.
You could also look at the molecular structure of each pigment. To determine polarity, count the number of polar oxygens present in each molecule.
- Chlorophyll-b: six polar groups (most polar)
- Chlorophyll-a: five polar groups
- Xanthophyll: two polar groups
- Carotene: no polar groups (least polar) so most soluble in the solvent.
Learner Notes
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Polar molecules or substances dissolve (or, are attracted to) polar molecules.
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Nonpolar molecules are attracted to nonpolar molecules to varying degrees.
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Chromatography paper is polar.
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The solvent, made of petroleum ether and acetone, is nonpolar. It can be purchased pre-mixed.
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The most nonpolar substance will dissolve in the nonpolar solvent first.
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The most polar substance will be attracted to the polar chromatography paper so it will move last.
Standards
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PASS: Science > Biology I > Biology I > 5 > 5.1
The complexity and organization of organisms accommodates the need for obtaining, transforming, transporting, releasing, and eliminating the matter and energy used to sustain the organism (i.e., photosynthesis and cellular respiration).
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