Name _________________________________________________________ Per ________ Date ________________
Plant Chromatography
Background Information:
The plant life around us is full of wonderfully rich and vibrant colors. From red to violet, the entire color spectrum
can be found in plant materials. These colors come from a surprisingly small number of plant pigments. The three primary
groups of plant pigments are anthocyanins, carotenoids, and chlorophyll. Each of these absorbs light at different parts of
the visible light spectrum and has a different function in the plant.
Anthocyanins attract insects for pollination by absorbing light in the UV spectrum, serve as a sunscreen to protect
plants from sun damage, and provide protection against predators because of their foul taste.
Carotenoids and chlorophyll both serve as light-harvesting pigments in the photosynthetic processes of plants.
It is possible to separate these pigments from one another by the use of paper chromatography. In this process,
plant tissue is applied to filter paper and an organic solvent is allowed to move up the filter paper. Pigments move at
different rates up the filter paper. 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 is done by dividing the distance the pigment traveled by the distance the solvent
traveled.
Eqn. 1
Rf = distance pigment traveled
distance solvent traveled
Chlorophyll is the pigment in plants that captures sunlight energy and uses it to drive photosynthesis. While chlorophyll
does give plants their characteristic green color, chlorophyll actually comes in many colors and subtypes ranging from green
to yellow to orange to red. In this experiment, students use paper chromatography to separate the many pigments from
one another. First the pigments are extracted from the plants by simply crushing the plant cells open on the filter paper
with the edge of a penny. When the filter paper is then immersed in chromatography solvent, the pigments are carried
upwards through capillary action. The smallest pigments travel more quickly and thus separate from the larger pigments
that remain closer to the origin line.
Chlorophyll is an amazing chemical that is the essential ingredient in the process of photosynthesis, the process through
which plants capture light from the sun to make glucose. When photons of light hit chlorophyll, the electrons in the central
magnesium atom donate an electron to a series of chemical reactions – the electron transport chain – that produce ATP the
cells’ primary unit of energy currency. The chlorophyll gets this electron back by taking one from water, resulting in the
release of oxygen gas as a byproduct.
Chlorophyll comes in two main forms: chlorophyll a and chlorophyll b. Each has a slightly different chemical structure and
therefore absorbs light of different wavelengths. In this paper chromatography experiment, chlorophyll a will appear as a
bright yellow-green band while chlorophyll b will appear as a dull green band. In addition, there are many other accessory
pigments in plants that absorb light and help transfer photons to the chlorophyll. These include carotene (an orange band)
and xanthophyll (a yellow band). A final pigment that may be detected is anthocyanin (a red-brown band) that acts as a
type of sunscreen for plants, protecting the plant from UV damage.
Materials:
__chromatography paper
__ ruler
__ pencil
__chromatography solvent in glass vial with lid
__ penny
__ leaf (variety)
Procedure:
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One person from group should get materials from teacher.
Caution: DO NOT OPEN VIAL UNTIL TIME TO PLACE CHROMATOGRAPHY PAPER INSDE
Use the ruler to measure 1 cm from tip of chromatography paper. Make a pencil line across the end of the strip.
Use the penny to extract the pigments from the leaf. Place a small section of the leaf on top of the pencil line. Use
the edge of the penny to crush the cells by rolling the penny over the leaf. Repeat this about 8-10 times using a new
portion of the leaf until you get a dark line at the bottom.
Place the chromatography paper into the vial so that the pointed end barely touches the solvent liquid. Do not
allow the pigment to be in the solvent.
Put the lid on the vial. When the solvent is about 1cm from the top of the paper, remove the paper and
immediately mark where the solvent reached. DO this before the solvent evaporates and is not visible.
Once the paper is dry, draw a line at the bottom of each pigment band.
By measuring the point of origin from the bottom of each band, calculate the Rf value for each pigment.
9. Distance solvent moved in mm _________________________
Example of chromatography paper for a green leaf
Sample Rf Problem
Band# Distance moved (mm)
1
12 mm
2
21 mm
3
27 mm
4
53 mm
Band Color
olive green
bright green
bright yellow
yellow-orange
Solvent moved 53 mm
Pigment
Carotene
Xanothophyll
Chlorophyll a
Chlorophyll b
DRAW YOUR CHROMATOGRAPHY SAMPLE
Rf Value
53mm/ 53mm =1
27mm / 53mm = 0.51
21 mm / 53 mm = 0.40
12 mm/ 53 mm = 0.22
Student Data and Calculations:
After the filter paper has dried, you are ready to begin calculating Rf values using Equation 1.
Using your ruler, fill in the following tables and calculate Rf values for one of your pieces of filter paper.
Band Distances
Band
Distance (mm)
Rf values for pigments
Band Color
Pigment
1
Carotene (yellow orange)
2
Xanthophylls (yellow)
3
Chlorophyll A (bright green)
4
Chlorophyll B (olive or yellow green)
5
Anthocyanin (red)
Rf
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 suggesting a
reason why. Explain why the Rf values should be close amongst all your classmates.
3. Why do you think some pigments move farther than others?
4. What is the advantage to a plant of having several different pigments rather than just one?
5. Can different pigments in plants have any benefits for humans, what do you think?
6. Could you use paper chromatography to separate anything else? Think about shows like CSI.