Plant Pigments & Paper Chromatography - fmfranco.com

Plant Pigments
& Paper Chromatography

Pre-Lab Discussion:

Photosynthesis begins when light is absorbed by pigments in the plant cell. One
technique for separating and identifying these pigments is paper chromatography. In
paper chromatography, solvent moves up paper carrying with it dissolved substances- in
this case plant pigments. The pigments are carried along different rates because they are
not equally soluble in the solvent and are attracted in different degrees to the paper. The
heavier the molecule the slower it moves up the paper.

Many green leaves contain pigment colors that are not seen until autumn because
they are hidden by the chlorophyll. A few plants have leaves that are red, orange, or
yellow all year long.

In this investigation, you will use paper chromatography to determine what
differences exist in the plant pigments of various colors of leaves. You will also
determine which leaves or which parts of leaves contain the chlorophyll necessary to
carry out photosynthesis.

Paper chromatography separates pigments but can also allow us to identify
compounds based on how far they move up the paper. The Rate of Flow or Rf value is
used to measure the distance traveled by various components in a substance. To calculate
the Rf value we use the formula:

Rf = Solute f - Solute i

Solvent Front

Beaker Solvent Front

Solute f
(Solute Front = point
where solute stops)

Solute i
(point of initial application,

origin of solute)

Solvent

I. Purpose:

1. To determine the number of components present in each sample.
2. To calculate the Rf value for the components in each sample.

1

II. Materials: Scissors
Pipet
Chromatography Paper Penny
Spinach Ruler
Chard Clock
Alcohol Food Coloring
Foil Covers
Pencil

III. Procedure:

1. Cut 4 pieces of chromatography paper 8cm in length.
2. Use a pencil to draw a baseline 1.5 cm from the bottom off the paper.
3. Label the top (about 1 cm down) of each o the three pieces of chromatography

paper with the names of the substances to be separated.
4. Cut 2 small pieces of each leaf. Place the lea directly over the pencil line.

Leave a small portion of the pencil line as a guide to the line’s position.
5. Roll a penny over the leaf so that a horizontal line is transferred to the

chromatography paper. Reposition the other leaf piece over the line again to
enhance the amount of leaf transferred to the chromatography paper.
6. Repeat Step 5 with the other leaf pieces.
7. Add one drop of food coloring to the center of the pencil line (or lightly go
over the line with a pipet in food coloring) on the fourth piece of
chromatography paper.
8. Add two squirts of alcohol to each beaker to cover the bottom. Be sure the
pigment line is not submerged when the paper is lowered into the beaker.
9. Crease the papers. Gently lower each piece into a beaker containing alcohol.
10. Cover each beaker with a piece of foil.
11. Do not disturb the beakers until the solvent is about 1 cm from the top of the
paper.
12. At that time remove the paper from the beaker, place it on a paper towel, and
mark the farthest point o the solvent’s (alcohol) progress. This is the solvent
front.
13. When samples dry, use a pencil to make a mark through the middle of each
separated component. These are the solute fronts.
14. Calculate the Rf value for each component and identify the possible pigments
present (leaves only).

Faint Yellow = Carotene
Xanthophyll
Yellow = Chlorophyll a
Chlorophyll b
Bright Green = Anthocyanin

Yellow-Green =

Pink-Red =

2

IV. Data/Observations:

Sample Line Color Probable Rf value Diagram of
Observed Pigment Chromatography Paper

Spinach 1(Base)
2
3
4
5(Front)

Chard 1(Base)
2
3
4
5(Front)

Food Color 1(Base)
2
3
4
5(Front)

1(Base)
2
3
4
5(Front)

3

V. Conclusions:

1. How many pigments were separated in each substance?

2. How did the pigments in the spinach compare with the pigments in the other
leaves.

3. Which of these leaves can carry on photosynthesis? Explain your answer.

4. Photosynthesis requires the green pigment chlorophyll. Explain how a
Japanese maple tree, having only red leaves, can carry on photosynthesis.

5. Many trees have leaves that are green in the summer and red, yellow, or
orange in autumn. Where were these colors during the summer? How can
they suddenly appear in autumn? (Hint: Chlorphyll breaks down without light)

6. In addition to separating plant pigments, what are some other possible
applications for paper chromatography?

4