PHOTOSYNTHESIS
Separation of the Plastid Pigments in a Plant Extract – Chromatography
INTRODUCTION
*Read the information below prior to performing the experiment. It provides
you with what you need to know to develop your hypotheses and form
conclusions.*
The Chemical equation for photosynthesis may be written:
6C02 + 6 H2O + Sunlight Energy
C6H1206 + 6O2
Photosynthetic Pigments
OR
6C02 + 12H20 + Sunlight Energy C6H1206 + 6O2 + 6H20
Photosynthetic Pigments
The above equations for photosynthesis are greatly simplified, merely indicating
the overall nature of the photosynthetic process. Actually, the process is far more
complex, involving many reactions and several intermediate products which you will
discuss during lecture. For this laboratory however, it will be sufficient to consider only
the simplified summary reaction.
In the absence of the photosynthetic process, all living things (with a few
exceptions) would not long exist and would soon disappear from the surface of the
earth. All of our food and many other plant products (i.e. sugars, starches, oils, some
drugs and fibers, etc.) are produced largely by the process of photosynthesis. Even the
flesh-eating animals depend upon plants via the plant-eating animals upon which they
prey. This dependence upon plants or more specifically photosynthesis is universal,
being equally or even more prevalent in the seas as on the land. It has even been
estimated that as much as 80% of all photosynthesis occurs within the oceans.
As is easily seen from the generalized equation, 6 molecules of carbon dioxide
and 6 molecules of water along with the addition of sunlight energy produces 6
molecules of oxygen and one molecule of glucose (a carbohydrate). The reaction,
however, occurs only in the presence of photosynthetic pigments. The photosynthetic
pigments absorb energy from sunlight to power the reaction. Thus, the whole process is
dependent upon the ability of the photosynthetic pigments to absorb energy from
sunlight. The green pigment, chlorophyll, seems to be the principle absorber of radiant
radiation in land plants. Although chlorophyll is generally considered to be a single
pigment, in higher plants, there are actually two major types: chlorophyll a - a bluegreen chlorophyll (C55H7205N4Mg); and chlorophyll b - a yellow-green chlorophyll
(C55H70O6N4Mg). These 2 chlorophylls differ only by a minor substitution at one edge of
the chlorophyll molecule. Additionally, other pigments known as the orange carotenes
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and the yellow xanthophylls are present in plants to “assist” the chlorophylls in
absorbing energy from sunlight. Chlorophyll a and chlorophyll b are commonly spoken
of as simply chlorophyll. Carotene and xanthophyll are often referred to as the
carotenoid pigments; as a matter of fact, there are at least two carotenes and several
xanthophylls. In summary, the photosynthetic pigments are:
a. Chlorophyll a (C55H72O5N4Mg)
b. Chlorophyll b (C55H70O6N4Mg)
c. Carotenes (C40H56)
d. Xanthophylls (C40H5602)
Using prisms or grids, electromagnetic radiant light energy of sunlight can be
separated into the various color bands of the visible spectrum (red, orange, yellow,
green, blue, indigo, and violet). Each color is a function of the wavelength of the
energy. The reds have the greatest wavelengths of about 660-680 nm (nanometers),
the oranges and yellows with wavelengths around 600 nm, greens about 550 nm and
the violets having the shortest wave lengths of about 400 nm. Recall that the colors
perceived by the human eye represent those wavelengths reflected off the object
viewed because the ones not visible have been absorbed by the pigment.
Since photosynthesis is so very important to life as we know it, the logical
question becomes exactly what parts of the solar electromagnetic light spectrum are
absorbed most by the photosynthetic pigments? It would seem that the light energy
which is most readily absorbed would then be the energy most available and perhaps
the most necessary for the photosynthetic process. Likewise, the light energy least
readily absorbed (that which is transmitted, or reflected back to one’s eyes) would be
the least necessary for photosynthesis.
During this experiment, you will extract photosynthetic pigments from spinach
leaves and separate them using chromatography. Since these pigments are not watersoluble, polar solvents will not remove them and non-polar solvents must be used to
extract them from the leaves. There are various methods employed to separate the four
plastid pigments in a plant extract. A fairly quick method founded upon the differential
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adsorption (“clinginess” of molecules to a substrate, based on their physical
characteristics) of molecules of various substances to the surface of a solid has found
considerable use in biology and biochemistry. This method is referred to as
“CHROMATOGRAPHY.” When an extract of leaf pigments dissolved in a solvent is
passed up a strip of filter paper, the pigment most readily adsorbed will be separated
out first, followed by those less readily adsorbed. Molecular weight is one significant
characteristic determining how far up the chromatography paper a pigment molecule is
carried. The idea is very similar to what happens during gel electrophoresis. Smaller
molecular weight molecules move farther up the chromatography paper.
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PROTOCOL
*Work in your group.*
*Record everything in your lab notebook as you typically would.*
*The solvents used in this experiment are highly flammable and potentially
toxic. Wear labcoat and gloves and follow all hazardous waste disposal
precautions described in the handout provided.*
MATERIALS
Labcoat
Gloves
Goggles
Loose-leaf spinach
Mortar and pestle
Sand
Forceps
Chromatography paper
Capillary dropper
Paper towels
Test tube
Cork
Fume hood
CHEMICALS
Acetone
Solvent mixture (9 parts petroleum
ether: 1 part acetone)
PROCEDURE
1. Put on a labcoat and gloves and fillout your lab notebook. State two hypotheses:
1)What order do you predict the photosynthetic pigments will appear along the strip
of chromatography paper? 2)Which regions of the light spectrum are the most
useful to the process of photosynthesis?
2. Place 2 – 3 spinach leaves in a mortar.
3. Add a pinch of sand and about 1 ml of acetone to the mortar, and create a slurry of
spinach leaf pigments by grinding and liquefying the spinach leaves using the pestle.
Make sure to grind until the leaves are no more than a fine powder.
4. Use a forceps to handle the chromatography paper for all further steps.
5. Place a strip of chromatography paper flat on several layers of paper towels.
6. Use capillary action to fill the capillary dropper with your spinach slurry.
7. With the capillary dropper, paint a very narrow band of spinach slurry about one
inch from the pointed end of the strip of chromatography paper.
8. After the liquid has evaporated, repeat the application at least 50 times so that the
band is a very dark green. Make sure to allow the solvent to evaporate before each
application.
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9. In the fume hood and with proper ventilation, pour the solvent mixture (9
parts petroleum ether: 1 part acetone) into a test tube to a depth of about one-half
inch.
10. After filling, stand the test tube in a vertical position and allow all of the solvent to
disappear from the sides of the tube.
11. Bring the strip of chromatography paper to the fume hood and carefully insert the
strip of paper into the tube, painted region down. Avoid creasing or wrinkling the
paper. Make sure to keep the tube upright.
12. Seal the tube with a cork.
13. You may bring the tube back to your lab bench for observation.
14. Allow the tube to stand upright undisturbed until the solvent front is about one inch
from the top of the paper.
15. Return to the fume hood, remove the strip of chromatography paper from the test
tube and allow all of the solvent to evaporate from it.
16. Once dry, return to the lab bench and place your chromatography paper you’re your
lab notebook. Record the position, color, and thickness of each pigment band.
Identify each specific band by color --- carotenes appear pale orange, xanthophylls
are bright yellow, chlorophyll a is dark bluish-green, and chlorophyll b is yellowishgreen/army green in appearance.
17. Properly dispose of the spinach slurry and waste acetone and solvent mixture as
specified in the Chemical Disposal Procedure handout.
18. In your lab notebook, for your discussion, answer the following questions:
a. Were your hypotheses supported by the results? Why or why not?
b. Fluorescent lights lack most of the red wavelengths but have higher
intensities of wavelengths from the rest of the spectrum, especially the
blues and violets; incandescent lights (light bulbs) are rich in red
wavelengths and heat (infra red) but have far less total light. What
effect would these facts have on photosynthesis of house plants?
Which lamp type or types would be preferable, and why?
c. Why is grass green? (or, why is your sweater green? why is the sky
blue?)
d. Why do some greenhouses sometimes add CO2 into their
greenhouses?
e. How would light affect the buildup of starchy (polymer of glucose)
reserves in plants? What would you expect would happen to a plant’s
stored food in the dark? Why?
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