Name: _______________________________ Period: ________

Separation of Photosynthetic Pigments Using Paper Chromatography

Objective: To use a technique called chromatography to separate the chemical compounds of a mixture.

Background Information:

Why do leaves change color?

Chlorophyll is the main pigment found in leaves. Chlorophyll traps the sun’s light energy to enable the plant to make
its own food in the form of glucose. During winter, there is not enough light or water for photosynthesis. The trees
will rest, and live off the food they stored during the summer. They begin to shut down their food-making factories.
The green chlorophyll disappears from the leaves. As the bright green fades away, we begin to see yellow and
orange colors. Small amounts of these colors have been in the leaves all along. We just can't see them in the
summer, because they are covered up by the green chlorophyll.

Pigments are chemical compounds which reflect only certain wavelengths of visible light. Chromatography is a
technique used to separate the chemical compounds (pigments) of a mixture. One of the most common types is
called paper chromatography. It works as follows: the mixture is placed at the bottom of a strip of chromatography
filter paper, the same end is immersed in a liquid called a solvent, the other end is held up in the air. Now, as the
solvent moves up the paper (through capillary action), the chemicals are carried up according to mass (heavier stay
at the bottom). The process works to separate pigments in inks as well as those found in leaves.

Pre- Lab Questions (answer before you start the lab):

1. What are pigments? ______________________________________________________________________
2. What is the main pigment in photosynthesis called? _____________________________________________

Materials:
Chromatography paper Glass slide Black transparency marker
2 Flasks with rubber stoppers Thumb tacks Scissors
Solvents (water and alcohol) Spinach

Procedure:
Part 1: Black Marker
1. Cut the bottom of the chromatography paper into a “V” shape.
2. Measure approximately 2cm from the bottom of your chromatography paper and draw a line using the black
transparency marker.
3. Fold the top of your chromatography paper and use a thumbtack to attach the paper to the rubber stopper
(as demonstrated by the teacher).
4. Put the chromatography paper and stopper into the flask labeled WATER so that the bottom of the paper
touches the solution, but the black line DOES NOT get into the solution. IMPORTANT: DO NOT SHAKE
THE FLASK. THE DARK PIGMENT LINE CANNOT GET SOLVENT ON IT. IF IT DOES, START OVER! It
should look Figure 1 below.
5. Let it sit for about 10-15 minutes. The solvent will move up the paper, separating the pigments in the marker.
6. Remove the chromatography paper to dry and attach it to your lab handout.

Part 2: Spinach Leaf

1. Cut the bottom of the chromatography paper into a “V” shape.
2. Obtain a leaf of spinach and fold it over the glass slide. Gently rub the leaf about 2cm from the bottom of the
chromatography paper to make a dark green line (similar to what you did with the marker).
3. Fold the top of your chromatography paper and use a thumbtack to attach the paper to the rubber stopper
(as demonstrated by the teacher).
4. Put the chromatography paper and stopper into the flask labeled ALCOHOL so that the bottom of the paper
touches the solution, but the green pigment line DOES NOT get into the solution. IMPORTANT: DO NOT
SHAKE THE FLASK. THE DARK PIGMENT LINE CANNOT GET SOLVENT ON IT. IF IT DOES, START
OVER! It should look Figure 1 below.
5. Let it sit for 8 minutes. The solvent will move up the paper, separating the pigments in the leaf.
6. Remove the chromatography paper to dry and attach it to your lab handout.
 Figure 1

Conclusion Questions:
1. What color(s) were present in your marker line before the experiment? ______________________________

2. What color(s) were present in your spinach line before the experiment? ____________________________

3. What process did you use to separate the pigments? ____________________________________________

4. What colors appeared after you completed the procedure with the marker? ___________________________

5. What colors appeared after you completed the procedure with the spinach? __________________________

6. What color is present in the highest quantity in the marker? _______________________________________

7. What color is the present in the highest quantity in the spinach? ___________________________________

8. Which pigment in the marker in the greatest mass? _____________________________________________

How do you know? _______________________________________________________________________

9. Why are leaves green even though other pigments are present? ___________________________________
______________________________________________________________________________________

10. Why do plants change colors in the fall? Explain in detail (think about the Autumn leaves assignment).
_________________________________________________________________________________________
_________________________________________________________________________________________
_________________________________________________________________________________________

11. Below are the other pigments present in the leaves of plants other than chlorophyll. Complete the 3rd column
of the chart based on your spinach experiment.
color pigment Found on your spinach strip
(yes or no)
yellow-green chlorophyll b
blue-green (darker) chlorophyll a
light orange carotenoid
light yellow xanthophyll