Leaf Chromatography
Leaves are nature's food factories. Plants take water from the ground through their roots. They take carbon dioxide from the air. Plants use sunlight energy to convert water and carbon dioxide into glucose.
· Glucose is a type of sugar.
· Plants use glucose as food for energy and as a building block for growing.
· The process of converting CO2 and water to glucose is called photosynthesis.
· A pigment called chlorophyll helps make photosynthesis happen.
· Chlorophyll is a pigment that gives plants their green color. Pigments are compounds that absorb and reflect light.
Plants have several kinds of pigments. They play an important role in capturing light energy for the photosynthesis. Pigments are responsible for the colors we see in leaves because they reflect light at that wavelength.
· Chlorophyll reflects green light
o Chlorophyll a reflects bright green and blue-green light.
o Chlorophyll b reflects yellow-green and olive green light.
· Anthocyanins reflect red light
· Carotenoids reflect orange light
· Xanthophylls reflect yellow light
Beta carotene, the most abundant carotenoid in plants, is carried along near the solvent front because it is very soluble in the solvent being used and because it forms no hydrogen bonds with cellulose. Carotenoids also protect the photosynthetic systems from damaging effects of ultraviolet light. Another pigment, Xanthophyll differs from carotene in that it contains oxygen. Xanthophyll is found further from the solvent front because it is less soluble in the solvent and has been slowed down by hydrogen bonding to the cellulose.
Chlorophyll's contain oxygen and nitrogen and are bound more tightly to the paper than the other pigments. Chlorophyll a is the primary photosynthetic pigment in plants. A molecule of chlorophyll a is located at the reaction center of the photosystems. The pigments collect light energy and send it to the reaction center.
As summer ends and autumn begins in the northern hemisphere, the Earth revolves around the sun so that the tilt of the Earth’s axis is pointed further away from the sun. This means that days get shorter and shorter. The decrease in sunlight is how trees "know" to begin getting ready for winter.
During winter, there is not enough light or water for photosynthesis. The trees will stop producing as much glucose; they live off the food stored during the summer. The green chlorophyll breaks down in the leaves. As the bright green pigment fades away, yellow, red, and orange pigments become visible. Small amounts of these colors have been in the leaves all along. We just can't see them in the summer, because the green chlorophyll hides them.
Paper Chromatography is a method to separate mixtures into their individual parts. Chromatography means "color writing." The mixture of pigments in a leaf may be separated into bands of color by paper chromatography. The parts of a mixture can be separated with this process. The pigments are mixed with a solvent and separated on a piece of paper.
Paper chromatography is a useful technique for separating and identifying pigment and other molecules from cell extracts that contain a complex mixture of molecules. The solvent moves up the paper by capillary action, which occurs as a result of the attraction of solvent molecules to the paper and the attraction of the solvent molecules to one another.
As the solvent moves up the paper, it carries along any substances dissolved in it. The pigments are carried along at different rates because they are not equally soluble in the solvent and because they are attracted, to different degrees, to the fibers of the paper through the formation of intermolecular bonds, such as hydrogen bonds.
In this experiment, we will use various solvents including rubbing alcohol, acetone, chromatography solvent and water. Pigments are separated on the paper and show up as colored streaks. The pattern of separated components on the paper is called a chromatogram.
We can tell the size of pigment molecules by the distance they travel. Lighter pigment molecules travel farther relative to the solvent, while heavier pigment molecules travel less distance relative to the solvent.
We measure this distance using the following ratio, called an rf (for relative front):
(Distance traveled by pigment in cm)
(Distance traveled by solvent in cm)
The closer this value is to 1, the more affinity the pigment molecules have for the solvent.
The closer this value is to 0, the less affinity the pigment molecules have for the solvent.
Exercise 4A: Plant Pigment Chromatography
Procedure
1. Obtain a large beaker.
2. Cut a piece of filter paper that will be long enough to reach the solvent. Draw a horizontal line about 1.0 cm from the bottom of the paper.
3. Use a quarter to extract the pigments from spinach leaf cells or another plant’s leaf cells Place a small section of leaf on the top of the pencil line. Use the ribbed edge of the coin to crush the leaf cells. Be sure the pigment line is on top of the pencil line. Use a back and forth movement exerting firm pressure through out.
OR Paint a line of extracted pigment on the line using a glass rod onto the line. Allow it to dry. (Gently shaking the paper will accelerate this.) Repeat until the line is dark!
4. Tape the strip of filter paper to a pencil and lay the pencil across the top of the beaker. Pour a small amount of solvent in the bottom of the beaker. Wind the paper around the pencil so that the tip of the paper just barely touches the solvent. Do not allow the pigment to touch the solvent.
5. Cover the beaker. When the solvent is about 1 cm from the top of the paper, remove the paper and immediately mark the location of the solvent front before it evaporates.
6. Mark the bottom of each pigment band. Measure the distance each pigment migrated from the bottom of the pigment origin to the bottom of the separated pigment band. Record the distance that each front, including the solvent front, moved. Depending on the species of plant used, you may be able to observe several pigment bands.
7. Compare your results to your tablemates’ results and to other groups.
Data:
Draw & color a diagram of your filter paper.
Set up a table such as the one below on a separate sheet of paper. Use the following formulas to fill out the data table below:
1. Enter each pigment band’s color in the first column. (You will have 3-4 bands.)
2. Measure the distance traveled by each pigment. Enter this in the second column.
3. Measure the distance traveled by the alcohol. (This will be the same for all the bands.) Enter this in the third column.
4. Divide the distance traveled by the pigment by the distance traveled by the alcohol. Enter this in the fourth column.
Pigment Band (by color) / Distance Traveled by Pigment (cm) / Distance Traveled by Alcohol (cm) / rf= Distance traveled by pigment/distance traveled by alcohol. (Column 2/Column 3)Post-Lab Analysis Questions (Answer in advance of a quiz on the material!)
1. 1. What materials do plants use during photosynthesis? What is produced by photosynthesis?
2. How many pigment bands did you observe? What colors were they? Which pigments did these represent?
3. Rank your pigment molecules in order from greatest affinity for the solvent to least affinity for the solvent. Why did you place them in this order?
4. What factors are involved in the separation of the pigments?
5. Would you expect the Rf value of the pigment to be the same if a different solvent were used? Explain.
6. What type of chlorophyll does the reaction center contain? What are the roles of the other pigments?
7. Why do leaves change color in the fall? Connect this to your results! Your answer should be AT LEAST paragraph.