Download Biology Chapter 2: Chemistry of Life

Biology
Mrs. Douma
Plant Pigments and Chromatography Lab
2017
Plant Pigments and Chromatography
Background Information
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
Objectives
1. Separate plant pigments using chromatography
2. Observe and identify the different pigments found in a leaf.
3. Understand how chromatography separates two or more compounds that are
initially present in a mixture
4. Calculate the Rf value of a pigment
Biology
Mrs. Douma
Plant Pigments and Chromatography Lab
2017
Procedure
1. 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.
2. 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.
3. 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.
4. 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 the solvent.
5. 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.
6. Mark the bottom of each pigment band with a light pencil.
7. 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.
Biology
Mrs. Douma
Plant Pigments and Chromatography Lab
2017
Biology
Mrs. Douma
Plant Pigments and Chromatography Lab
2017
Analysis
1. What does a small Rf value indicate about
the characteristics of moving molecules?
Hint – Read the background information
2. Which are more soluble in the
chromatography solvent, xanthophylls or
chlorophyll a?
3. Would you expect the Rf value of a pigment to change if we altered the
composition of the solvent? Why or why not?
4. If yellow xanthophylls were present in the extract (crushed leaf) why did the
extract appear green?
5. Is it possible to have an Rf value greater than 1? Why or why not?