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Chromatography
Light
Chloroplast
Structure
CHROMATOG
RAPHY
Photosynthesis
Objective:
-
Tasks:
1. Compare and contrast the different pigments in leaves.
2. Analyze a chromatogram
Name:
(or down) the paper by capillary action. Compounds which are very soluble in the solvent
move along with the advancing solvent front, while less soluble compounds travel slowly
through the paper, well behind the solvent front. As a result, the different compounds are
separated on the basis of their solubilities in the chosen solvent. Chlorophyll a is slightly
soluble in a 3:1:1 mixture of petroleum ether, acetone, and water.
Carotenoids are very soluble in this solvent system. These solubility differences will
allow the separation of chlorophyll a from the carotenoids and chlorophyll b on a paper
chromatogram.
Light visible to the human eye occupies only a small portion of the electromagnetic
spectrum, namely from about 350 to 750 nanometers, or from violet to red. The color of light
is also related to the energy of the light as shown below.
1.
Which wavelength of visible light possesses the greatest energy value, and what is its
color?
2.
Which wavelength possesses the least energy value, and what is its color?
3.
4.
Where are Pigments located? Label the following diagram: Inner-membrane, outermembrane, stroma, thylakoid, thylakoid membrane (provide label description).
Procedure:
Time required: 50 minutes
Materials:
Chromatography Paper (4 strips)
Test Tube Rack
Graduated Cylinder
2 green leaves
Quarter
One-holed Rubber Stoppers
Internet Access
Pipette
Obtain four large test tubes, four paper clips, and four one-holed rubber stoppers
Unbend the paper clip and insert through the hole in the stopper.
Create a hook at the end of the paper clip located on the inside of the stopper
Place the paper clip/stopper apparatus in an empty test tube to test its stability
Testing for Pigments
1. Hook the chromatography strip (the far end away from the pigments) to the hook of the paper
clip. The chromatography strip should now be hanging securely.
2. Place the strip and paper clip/stopper apparatus into the empty test tube. Make sure the strip
is not touching the walls.
3. There should be two centimeters of unmarked paper below the pigment line. Using the vis a vis
marker, create a line one centimeter below the pigment.
4. Remove the paper and apparatus from the test tube.
5. Using a pipette, fill the large, empty test tube with ethanol up the line marked in step three.
6. After ethanol is added, gently add your strip and apparatus into the test tube. This must be
done slowly so that the pigments are not in direct contact with the ethanol.
7. Gently place the test tube in the rack and let sit for 20 minutes.
REPEAT PROCEDURE FOR EACH LEAF. BE SURE TO DO THE PROCEDURES FIRST SO YOU
HAVE TIME TO WAIT FOR EACH LEAF.
1. After 20 minutes, remove the chromatography strip gently from the test tube and place on a
sheet of plane white paper.
2. Fill in the chart provided
3. Based on your results, use print/online resources to determine what kind of tree the leaf came
from.
4. Create your own wavelength chart using the information that your have found. Refer to the
wavelength chart presented earlier in the lab.
Physical
Characteristics
Color
Size
Shape
Chromatogram
Description
Surprising Finds
Green Leaf A
Green Leaf B
Non-Green A
Non-Green B
WAVELENGTH CHART:
Assessment Questions:
What is the purpose of this lab?
Will red leaves contain the same amount of chlorophyll as green leaves? Why or why not?
Based on your results, do non-green leaves undergo photosynthesis? Explain your answer.
During the fall, the chlorophyll in the leaves of many plants starts to break down. The colors of other
pigments present in the leave are revealed. How do you think a chromatogram of a leaf that just turns
red in the fall would compare with your chromatogram of a leaf that is red all year?
What advantage could there be for a leaf to have pigments other than chlorophyll?