Biology Plant Pigments and Chromatography Lab
Mrs. Douma 2017
Plant Pigments and Chromatography
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 the
paper by capillary action (adhesion) carrying with it dissolved
solutes – in this case plant pigments. The pigments are carried along
at different rates because they are different sizes, not equally
soluble in the solvent and are attracted in different degrees to the
paper (adhesion and hydrogen bonding).
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.
Beta carotene, the most abundant carotene in plants, is carried along
near the solvent front because it is very soluble in the solvent being
used and because it forms no hydrogen bonds with cellulose
(non-polar). Xanthophyll is found further from the solvent front
because it is less soluble in the solvent and has been slowed down by
hydrogen bonding to the cellulose. Chlorophylls contain oxygen and
nitrogen and are bound more tightly to the paper than are the other
pigments (so they will move more slowly.)
Chlorophyll a is the primary photosynthetic pigment in plants.
Chlorophyll b and the carotenoids (xanthophylls and carotene) are also
present. Carotenoids also protect the photosynthetic system from the
damaging effects of ultraviolet light
Separate plant pigments using chromatography
Observe and identify the different pigments found in a leaf.
Understand how chromatography separates two or more compounds that
are initially present in a mixture
Calculate the Rf value of a pigment
Obtain a graduated cylinder that has 1 cm of solvent in the
bottom. The cylinder should stay stoppered because the solvent is
very volatile. Avoid breathing into the cylinder.
Cut a piece of filter paper that is just long enough to reach the
solvent. Cut one end of the paper into a point and then draw a
light pencil line 1.5 cm above the point.
Use a coin to extract the pigments from the spinach leaf cells.
Place a small section of leaf on the 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. Make two or three passes, using
a different part of the leaf each time.
Place the chromatography paper in the cylinder so that the pointed
end is immersed in the solvent. Do NOT allow the pigment to be in
Stopper the cylinder. When the solvent is about 1 cm from the top
of the paper, remove the paper and immediately mark the location
of the solvent front before it evaporates.
Mark the bottom of each pigment band with a light pencil.
Measure the distance each pigment migrated from the bottom of the
pigment origin (pencil line) to the bottom of the separated
pigment band. Record the distance in mm that each pigment moved.
What does a small Rf value indicate about the characteristics of
moving molecules? Hint – Read the background information
Which are more soluble in the chromatography solvent, xanthophylls
or chlorophyll a?
Would you expect the Rf value of a pigment to change if we altered
the composition of the solvent? Why or why not?
If yellow xanthophylls were present in the extract (crushed leaf)
why did the extract appear green?
Is it possible to have an Rf value greater than 1? Why or why not?