The leaves of most plants are green because of a special pigment called chlorophyll which is contained in the leaf cells. This pigment captures light and turns it into energy. It is essential for plant growth.
There are several different types of pigments found in a leaf. Each has a characteristic color:
Chlorophyll a = bright-green
Chlorophyll b = dull or olive-green
Xanthophyll = yellow
Carotene = orange
The leaves in trees are typically green because of the high amounts of chlorophyll a and b.
During the fall, the trees prepare for winter and stop making chlorophyll. When this happens, the other pigments in the leaves begin to show up. Depending on the tree, the leaves may turn yellow, red, or orange. These colors have always been present, but they have been overpowered by the green pigment in chlorophyll. In this lab, we will attempt to separate these pigments.
Paper chromatography works on the principle that different pigments, when dissolved in a solvent (such as water or ether), will move (travel) through a piece of paper at different rates of speed. The rate is determined by (1) how soluble the pigment is in the solvent and (2) the degree of adhesion of the pigment to the surface of the paper.
You will be preparing a chromatogram in this lab. This simple tool is used to separate different pigments. You will transfer a small amount of leaf pigment onto a piece of filter paper by rubbing leaf material with a coin. You will then dip the filter paper in alcohol which will separate out the different pigments.
The key to identifying the different pigments in the leaf is measuring how far the pigment has traveled from its starting point. Be very precise in your measurements. You will calculate the R f
(retardation factor) value for each pigment. This factor is calculated by comparing the distance the solvent has moved with the distance the pigment has moved.

1.
Gather one 200mL glass jar.
2.
Obtain a piece of filter paper 12cm long and 3cm wide.
3.
Make a pencil mark ( do not use a pen!
) on the coffee filter 2cm from the bottom.
4.
Obtain a leaf and using a coin, rub the leaf onto the line that you made in step #3. Keep rubbing until a significant amount of pigment has been transferred.
5.
Carefully place the bottom portion of the filter paper (closest to the line you drew) into the jar. The bottom of the paper should come into contact with the solvent. Use a piece of tape to secure the filter paper to the rim of the jar.
6.
Caution: the solvent in the jar alcohol which is highly flammable and gives off potentially harmful fumes. Do not directly smell the solution and notify your teacher if there are any spills.
Observe the solvent as it is drawn upward toward the spinach extract.
Wait 15 to 20 minutes for the chromatograms to develop.
7.
When instructed, remove your filter paper from the jar and place onto the table. Use a pencil ( do NOT use a pen!
) to mark where the solvent stopped as it moved up the filter paper (should be colorless, but wet). This is called the solvent front.
8.
Caution: Your filter paper will have a small amount of alcohol on it. After placing it on the table, let it dry for 1 minute before measuring.
Use a pencil to mark the highest point of each pigment (color). Some discussion will likely take place regarding the different shades of the pigment. Using a ruler, measure the distance (in cm) between the pigment origination (the line you drew on the bottom of the filter paper) and the highest point of each pigment. Record your data in the chart provided.
9.
Measure the distance between the pigment origination and the solvent front (the highest point the solvent traveled). This number should be the same for each pigment. Record data in the chart provided.
10.
Using the formula provided, calculate the R f
value for each pigment. Use the provided date to label your filter paper with the four pigments.

Pigment Origin
Chlorophyll a Chlorophyll b
2cm 2cm
Name: ________________________
Hour: ____________
Xanthophyll
2cm
Carotene
2cm
Pigment front
Solvent front
R f
Value
R f
= distance moved by pigment from original spot
distance moved by solvent from original spot
Chlorophyll a= blue-green
Chlorophyll b = Olive green
Xanthophyll = yellow
Carotene = orange yellow
Post-Lab Questions:
1.
Why is chromatography useful?
2.
Why is are chlorophyll a & b green?
3.
Why do leaves change colors in the fall?
Staple Filter
Paper Here

4.
How could you predict the color a tree’s leaves will turn in the fall?
5.
Use the chromatogram below to complete the data table.
Solvent Front (12cm)
Xanthophyll (9cm)
Carotene (7.7cm)
Chlorophyll a (5.2cm)
Chlorophyll b (4.7cm)
Pigment/Solvent Origin (2cm)
Pigment origin
Pigment front
Solvent front
R f
Value
Chlorophyll a
2cm
Chlorophyll b
2cm
Xanthophyll
2cm
Carotene
2cm