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. Plants may have a fifth pigment type, the anthocyanins. These pigments have a red-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 as bright green to blue-green in color to the human eye while chlorophyll β appears to be olive green in color. 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. These wavelengths that have been absorbed by the plant will energize the plant’s photosynthetic process, setting energy transfer events into action. Measuring the amount of actual photosynthesis occurring in various colors of light is called the plant’s action spectrum. It seems logical, then, that plants should do photosynthesis best when in red or blue light and very little or none in green light. When comparing the two spectra, one finds that photosynthesis does indeed work best in red and blue light, however, it is surprising to find that some photosynthesis does occur in green light. This is due to the aid of the yellow pigments, carotenes and xanthophylls. They have the ability to capture the energy of green photons and contribute to the light reaction.
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 carries 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 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 anthocyanin bands when other solvents do not.
Purpose
In this activity you will first prepare a chromatogram using pigments of Swiss chard leaves and three solvents. In the second portion of the activity, you will devise a plan to make a chromatogram containing as many bands as possible from a multicolored leaf of Coleus, spinach, or Swiss chard.
Materials
Safety
Methods
Results
Analysis
Chlorophyll α appears as bright green to blue-green in color to the human eye while chlorophyll β appears to be olive green in color. 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. These wavelengths that have been absorbed by the plant will energize the plant’s photosynthetic process, setting energy transfer events into action. Measuring the amount of actual photosynthesis occurring in various colors of light is called the plant’s action spectrum. It seems logical, then, that plants should do photosynthesis best when in red or blue light and very little or none in green light. When comparing the two spectra, one finds that photosynthesis does indeed work best in red and blue light, however, it is surprising to find that some photosynthesis does occur in green light. This is due to the aid of the yellow pigments, carotenes and xanthophylls. They have the ability to capture the energy of green photons and contribute to the light reaction.
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 carries 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 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 anthocyanin bands when other solvents do not.
Purpose
In this activity you will first prepare a chromatogram using pigments of Swiss chard leaves and three solvents. In the second portion of the activity, you will devise a plan to make a chromatogram containing as many bands as possible from a multicolored leaf of Coleus, spinach, or Swiss chard.
Materials
- Test tubes
- Test Tube Rack
- Filter Paper
- Coin
- Acetone
- 70% isopropyl alcohol
- Spinach, Coleus, and Swiss Chard leaves
- Pipettes
- Ruler
Safety
- Alcohol is flammable. Do not expose to an open flame.
- Avoid inhalation of fumes from solvents
- Aprons and goggles must be worn during this activity
Methods
- Obtain a piece of filter paper and cut it to fit into the test tubes. Draw a pencil line 1.5 cm from the bottom of the paper. This will be your starting line. See figure below for an example.
- Obtain a spinach leaf from your teacher. Place the spinach leaf on top of the line on the filter paper. Using a coin, press the pigment into the filter paper by rolling the coin over the surface of the leaf several times.
- Obtain a test tube hold the solvent and filter paper
- Add 10 drops of acetone in the bottom of the test tube. Fold the filter paper in half longwise (hot dog style) and insert into the test tube until the bottom of the filter paper is touching the bottom of the test tube. Avoid inhaling the solvent fumes.
- Repeat steps 1-5 for the isopropyl alcohol.
- Place the test tube in the test tube rack and leave it undisturbed until the solvent has wicked ¾ of the way up the filter paper. DO NOT LET THE SOLVENT RISE ABOVE THE TOP OF THE PAPER.
- Remove the filter paper and mark the level the solvent has traveled, using a pencil.
Results
- Use a pencil to mark the location of each pigment on your filter paper. Sketch the chromatogram on your own paper. Include the colors of the pigments in your diagram.
- Using a ruler, measure the distance the solvent moved from the starting line to the solvent pencil mark. Record the distance in a data table (see below for an example) in your notebook.
- Measure the distance traveled by each of the pigments from the starting line. Record the measurements in your data table.
- Calculate the Rf value for each pigment using the formula: Rf = Distance pigment migrated/distance solvent front migrated
- Using the information provided in the introduction, determine the type of pigment found in each band of the chromatogram and record this in your data table.
Analysis
- What types of pigments are typically found in leaves?
- What causes the pigments to move up the filter paper different distances?
- Why do some of the pigments appear yellow to our eyes while others appear green?
- How is an action spectrum different from an absorption spectrum?
- Why are all of the pigments not visible in a typical leaf?
- Compare the bands produced in all your different tests. How are the different? Similar?