Chromatography of Plant Pigments
Background:
All life on earth depends on the process of photosynthesis. Photosynthesis is a process that plants use to
convert carbon dioxide (CO2) and water (H20) in the presence of light energy into carbohydrates (sugar).
Photosynthesis requires the presence of special pigments that can absorb the energy of light. A pigment is a substance
that absorbs light of a particular wavelength. Its color depends upon the color of light that it reflects. For example, a
green substance appears green because it reflects green light and absorbs all other colors, especially red and blue.
The most important plant pigments in photosynthesis are chlorophylls. There are two major types:
chlorophyll a, which are a light green color, and chlorophyll b, which are a green to dark green color. Green plants
contain both types. In addition to chlorophyll, the leaves of many green plants also contain one or more other
pigments, including carotenes which are orange, and xanthophylls, which are yellow. The presence of these other
pigments is masked by the abundance of chlorophyll during most of the year. In the fall, however, when chlorophyll
production decreases, the other pigments show up, giving leaves their bright red, orange, and yellow autumn colors.
As temperatures during autumn drop and the days shorten, cells at the base of the leaves break up which block the
passages from leaves to branches and cause chlorophyll to decompose. As the chlorophyll decreases, colors in other
pigments (carotenoids and anthocyanins) come to the surface. Trees with leaves that have an abundance of carotenoids
– like beech, birch, and willow – turn varying shades of yellow. When anthocyanins in leaves predominate – as in the
case of many maples, dogwoods, and sumacs – reds and purples appear.
The pigments in plant cells can be separated from one another by a technique known as “chromatography”.
Chromatography is a technique for separating and identifying substances in a mixture, based upon their solubility in a
solvent. It is one of the most valuable techniques chemists and biochemists use to determine the ingredients that give
flavor or scent, analyze environmental pollutants, identify drugs in urine, and even separate proteins that can identify
evolutionary relationships.
The name “chromatography” is derived from the Greek words “chroma” and “graph” which means “color
writing”. Chromatography was invented in 1910 by a Russian botanist Mikhail Tswett who used it to separate plant
pigments. When a dye mixture is placed on a strip of chromatography paper and then placed in a solvent solution, the
individual substances in the mixture will migrate up the chromatography paper at different rates. The rate of migration
is based upon the absorption capacity of the chromatography paper and the solubility of the sample (solute) in the
solvent. In this investigation, you will separate individual pigments involved in photosynthesis using chromatography.
Purpose/Objectives:
- To separate and observe the pigments that gives a leaf its color using chromatography
- To describe the function of plant pigments during photosynthesis
Materials:
Chromatography Paper
10 ml Graduated Cylinder
Capillary tube or pipette
Chromatography Developing Solution
Reaction Chamber with cap
Plant Pigment Extract
Pencil
Ruler
Scissors
Procedure:
1) Use scissors to cut the bottom end of the chromatography paper strip to a tapered end, as
shown in Figure 1.
2) At the end of the chromatography paper, place a faint line 2 cm from the bottom with a
pencil (NO PEN!!!!!!)
3) Use the capillary pipette to gather a small amount of plant pigment extract. Place a small
drop of plant pigment in the middle of the line drawn on the chromatography paper.
4) Let the pigment drop dry completely for 1-2 minutes. Then reapply two (2) more drops,
allowing the drop to dry thoroughly between applications.
5) Place about 3 ml of Chromatography Developing Solution into a reaction chamber and place
the cap on top. This will saturate the atmosphere of the chamber insuring better separation.
Keep the cap on the reaction chamber till the drops of plant pigment extract are completely dry.
Figure 1
6) Place the chromatography paper strip into the reaction chamber and adjust the length of the strip so that a small
portion of the end tip is immersed. MAKE SURE THE DROP OF PLANT PIGMENT REMAINS ABOVE THE
SOLVENT IN THE CYLINDER OR ELSE IT WILL DISSOLVE OFF THE PAPER. The top of the reaction
chamber is tapered so that it will hold the chromatography paper
strip at the desired distance. Ensure that the paper strip is level
and does not touch the walls of the reaction chamber. Place the
Figure 2
screw cap back on and do not agitate the reaction chamber. See
Figure 2 for details.
7) Once the solvent travels approx. ¾ of the way up the paper,
remove the chromatography paper from the chamber. Place the
paper on the table to dry.
8) Draw a line where the solvent stopped moving up the paper.
This is the distance the solvent traveled.
9) Draw a line at each place you see a different colored pigment.
You should see about 4 different colors (orange, yellow, and two
shades of green)
10) Clean up lab area. Rinse and place materials in appropriate areas. DO NOT throw away your
chromatography paper.
Calculate the Rf (Relative Mobility Factor) Value of each pigment using the following equation and fill out
the table.
Rf = Distance pigment traveled / Distance solvent traveled
*Round Rf values to 2 decimal places
Data/Results
Pigment Color
Pigment Type
Distance Traveled by
Pigment (cm)
Distance Traveled by
Solvent (cm)
Rf Value *
**** Make a full size drawing of your chromatography paper. Use a ruler to be accurate
and color to represent each pigment. ****
Discussion
1) What is a pigment and how do plants use them?
2) Describe what happened to the original spot of plant pigment extract.
3) Why must the line be drawn in pencil and not pen?
4) Why do plants appear to be green?
5) Why are the other colored pigments in leaves not seen during the summer months? What allows these
other colored pigments to be seen during the fall months?
6) Why do plants have pigments other than chlorophyll a?
7) Green pigments are essential for photosynthesis to take place. How do you think that a Japanese maple
tree, which has only red leaves, can carry on photosynthesis?