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Concepts:

Chromatography

Light

Chloroplast
Structure

CHROMATOG
RAPHY

Photosynthesis

Objective:
-

Students will produce a chromatogram of four different leaves


(2 green/2 orange, red, or yellow) and evaluate the leaves
pigment composition by the end of the lesson.

Tasks:
1. Compare and contrast the different pigments in leaves.
2. Analyze a chromatogram

Name:

3. Evaluate your findings


Hour:

Paper chromatography separates compounds on paper as solvent carries the mixture up

(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.

Which pigment(s), chlorophyll a, chlorophyll b, and/or carotenoids, will travel the


farthest on the chromatography paper?

4.

Which pigment(s) is least soluble in the solvent?

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

Four large test tubes


Ethanol (10 mL each)
Vis a Vis marker
2 leaves of another color (yellow, red, brown, orange)
Paper Clips
Lab Handout
Colored Pencils
Sheet of blank white paper

Generating the Pigments:


1. Obtain the lab handout and complete pre-lab exercises.
2. Obtain four strips of chromatography paper that are approximately one-two centimeters in
width.
3. Separately place one of the four leaves by each strip. Label the top of each strip in the top
corner to remember which belongs to which leaf.
4. Place the leaf bottom side down two centimeters above the bottom of the chromatography strip.
5. Gently roll the quarter back and forth along the bottom of the leave. This is the extraction of
the pigments, so be careful not to ruin the leaf.
Constructing the Apparatus
1.
2.
3.
4.

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.

Analyzing Chromatography Strip:

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

COMPARE THE LEAVES:

CONTRAST THE LEAVES:

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?

Identify the pigments that are visible on your chromatograms.

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?