Title: Chromatography of Pigments from a Spinach Leaf

Background Information
The incredible variety of plant colors is produced by a surprisingly limited number of organic
pigments. They can be divided into three primary groups: chlorophylls, carotenes, and
anthocyanins. Each has different properties and performs different functions for the plant,
including absorbing light in different parts of the spectrum. More light absorbed = more energy for
a plant. Most plant parts, especially leaves, contain some combination of these three pigments,
even if only one is especially obvious. It is possible to separate these pigments from each other
using a technique called paper chromatography. In this process, plant tissue extract is applied to a
piece of chromatography (filter) paper. A solvent is allowed to travel up the paper, and if the
pigment is soluble in the solvent, it will be carried along with it. Different pigments have different
affinities for the solvents and will travel at different rates. Because of these differences, several
color bands would be expected if there is more than one pigment present. Based on the bands
formed on the filter paper, the retention factor, or Rf, value can be calculated for each pigment;
this measures the distance the pigment traveled versus the distance the solvent traveled, or the
solubility of the pigment in the solvent. Rf values are characteristic of specific pigments and are a
reliable identifier.

Chlorophylls: Chlorophylls are perhaps the most familiar plant pigment to most people. They are
polar (water-soluble) pigments that act as electron-transporters in photosynthesis and provide
green colors to plants. Two types of chlorophyll are usually visible with paper chromatography:
chlorophyll a and chlorophyll b. Chlorophyll a is more soluble than chlorophyll b and will travel
farther up the paper with the solvents we are using. Chlorophyll a produces a bright green to
blue-green band, and chlorophyll b produces a dull olive to yellow-green band.

Carotenes: Carotenes are non-polar (fat-soluble) hydrocarbons. This class of pigments ranges in
color from yellow to red. They are also essential as helpers in photosynthesis. Most likely, two
carotene bands will be visible with the chromatography you will be using. Carotene itself
produces a yellow-orange band. Xanthophyll produces a lighter yellow band.

Anthocyanins: Anthocyanins are polar (water-soluble) pigments that provide red, blue, and purple
colors to plants. They don’t have critical photosynthetic roles for the plant, but rather are
considered to be metabolites. Depending on what type of plant material you use, you may or may
not see anthocyanins. Anthocyanins tend not to travel very far up the paper and form a reddish
band along the bottom.

Materials
Spinach leaves (other leaves-optional)
250mL beaker
 Chromatography solvent
Chromatography paper
Pencil and ruler
Penny
Safety glasses

Procedure
1. Obtain a 10cmx10cm square of chromatography paper.
2. Make a pencil line all the way across one end of the paper,~1.0cm up from the bottom.
3. Use a coin to extract the pigments from the green leaf. Place a small section of leaf on top of the
pencil line. Use the edge of the coin to crush the cells. Be sure that the pigment line is on top of the
pencil line. Repeat this using a new portion of the leaf until it covers the line.
4. Create a cylinder with your paper. Staple both ends to ensure it maintains this shape.
5. Place the chromatography paper into the beaker with the pencil line closest to the bottom. Do not
allow the pigment to be in the solvent.
6. Put aluminum foil over the beaker. When the solvent is about 1 cm from the top of the paper,
remove the paper and immediately mark where the solvent reached before it evaporates.
7. Once the paper is dry, use the light box to draw a line across the bottom of each pigment band.
Depending on the species of plant used, you may be able to see 4 to 5 pigment bands.
8. Attach the chromatography paper to your lab notebook. To one side of the paper, label the solvent
front, the different pigments, and the point of origin.
9. If time and materials allow, repeat the above procedure with a different colored leaf.
10. By measuring from the point of origin to the bottom of each pigment band, the Rf value for each
pigment can be calculated using the following formula: Do the math for the spinach leaf only.

Rf = distance of pigment from origin

Distance in millimeters the solvent moved _____________

Pigments Extracted from a Spinach Leaf Using Chromotography

Band Distance (mm) Band Color Pigment Rf Value

Analysis Questions
1. Which pigments did you discover in your leaf sample?
2. Compare your Rf values to another group. Are they close? They should be. If they aren’t suggest a
reason why.
3. Why do you think some pigments move farther than others? (Hint: think polarity and size)
4. Based on what you have seen in this activity, come up with an explanation for how green leaves turn
such brilliant colors in the fall.
5. What is the advantage to a plant of having several different pigments rather than just one?
6. Could you use paper chromatography to separate pigments for anything else? (Hint: Think about
shows like CSI, NCIS and Cold Case)