Download laboratory exercise: photosynthesis: isolation and characterization of

LABORATORY EXERCISE:
PHOTOSYNTHESIS: ISOLATION AND CHARACTERIZATION
OF CHLOROPLAST PIGMENTS
Introduction:
The capacity to carry out photosynthesis is found in a wide spectrum of organisms, including both
prokaryotes and eukaryotes. The photosynthetic eukaryotes include not only the higher green plants but
also “lower” forms such as the green, brown, and red algae, and organisms such as euglenoids,
dinoflagellates, and diatoms. The photosynthetic prokaryotes include the blue-green algae (cyanobacteria),
the green bacteria, and the purple bacteria.
During the process of photosynthesis, light energy is transformed into chemical (bond) energy.
Light energy is a type of electromagnetic radiation; visible light is radiation of wavelengths ~4000 to ~7000
nanometers (nm) (Fig. 6.1, below). Light energy is emitted from the sun in the form of photons, or
quanta, of light. Because only absorbed light can transfer its energy, photosynthetic cells have substances
that can absorb visible light. Substances that have the ability to absorb light selectively are called
pigments.
Wavelength (cm)
10 -12
10 -10
10 -8
10 -6
X rays
Cosmic rays
Gamma rays
10 -4
Ultra
violet
rays
Visible light
Violet
Blue
400
Orange
Green
Yellow
10 -2
10 0
Infrared
rays
1 meter (m)
1 centimeter (c)
1 millimeter (mm)
1 micrometer (µm)
1 nanometer (nm)
1 angstrom ( )
10 2
10 4
10 6
Radio waves
= 100 centimeters
= 0.01 (10-2 ) meter
= 0.001 (10-3 ) meter
= 0.000001 (10-6 ) meter
= 0.000000001 (10 -9 ) meter
= 0.0000000001 (10-10 ) meter
Red
600
500
Wavelength (nm)
700
Figure 6.1 Electromagnetic spectrum
All photosynthetic cells contain 1 or more types of the class of green pigments known as
chlorophylls. Higher plants contain 2 forms of chlorophyll, chlorophyll a and chlorophyll b. Brown
algae, diatoms, and dinoflagellates contain chlorophyll a and chlorophyll c; red algae contain chlorophyll a
and chlorophyll d.
The structure of chlorophyll a was established by the German scientists Wilstatter and Fischer.
Chlorophyll a (Fig. 6.2) contains a 4-ring structure in which the 4 central nitrogen atoms are bonded with a
Mg2+ ion. Both chlorophyll a and chlorophyll b have a long, hydrophobic side-chain, consisting of the
alcohol phytol.
The ability of a substance to absorb light depends on its atomic structure, particularly on the
arrangement of electrons surrounding the nucleus. It is, in fact, the electrons that absorb light energy.
When a photon strikes an atom capable of absorbing light, energy is absorbed by some of the electrons and
the atom goes into an energy-rich (“excited”) state. Only photons of certain wavelengths can excite a
given atom. Only absorbed light can excite atoms.
Chlorophyll a in pure form dissolved in acetone has an absorption maximum at 663 nm. In the
intact cell, however, chlorophyll a may show 2 or more different absorption maxima-- for example,
672 nm and 683 nm. The exact position of the absorption peak of chlorophyll a differs from one species
of plant to another. The differences apparently reflect the relative proximity of chlorophyll
a molecules to other pigment molecules in the thylakoid membranes of the chloroplasts.
Photosynthetic cells also contain a very small amount of a pigment with a light absorption
maximum at 700 nm. This pigment, designated P700, is a specialized chlorophyll molecule making up
only 1/400 of the total chlorophyll in the cell. It serves as the photoreactive center of an assembly of
chlorophyll molecules, absorbing energy from the sunlight and, in the process, losing an “excited”
electron. The photoreactive center of the light-absorbing chlorophyll assembly is connected with a
chain of electron carriers. This chain serves to lead electrons away from the excited photoreactive center in
an energetically “downhill” direction toward the coenzyme NADP + , causing its reduction to NADPH.
(NADH and NADPH are very similar in that they both carry high energy electrons. While NADH usually
carries electrons from degradative processes, NADPH is often used to carry electrons that will be used for
syntheses. In the process of photosynthesis, NADPH carries electrons to the Calvin Cycle, where they will
be used to produce glucose.)
Despite having chlorophyll, not all photosynthetic cells are green; photosynthetic algae and bacteria
may be brown, red, or purple. This variety of colors exists because, besides chlorophylls, most
photosynthetic cells contain members of 2 other classes of light-trapping pigments, often called accessory
pigments: the yellow carotenoids and the blue or red phycobilins. These accessory pigments, which
have absorption maxima at wavelengths other than those of the chlorophylls, serve as supplementary light
receptors for portions of the visible spectrum not completely covered by chlorophyll.
The phycobilin pigments, such as phycoerythrobilin (Fig. 6.3), occur only in red algae and
blue-green algae. Phycobilins lack Mg2+ , and unlike chlorophylls and carotenoids, they are conjugated to
specific proteins.
Among the carotenoids found in photosynthetic cells are beta-carotene, spirilloxanthin, and
lutein (Fig. 6.4). In addition to functioning as supplementary light receptors, carotenoids function to
protect chlorophyll from degradative attack by molecular oxygen.
Basic structure of chlorophyll
figure 6.2
CH
In chlorophyll a, X = -CH 3
In chlorophyll b , X = -CHO
X
CH2
H3 C
CH2 CH3
N
N
Mg
H3 C
N
N
CH3
H
H H
CH3 OOC
O
O
O
phytol
side
chain
Basic structure of phycobilin
figure 6.3
H
O
In phycoerythrobilin (red pigment in algae)
X = CH2 CH
CH3
NH
CH2 CH3
In phycocyanobilin (blue pigment in algae)
X = CH3 CH2
CH3
NH
HOOCCH2 CH2
HOOCCH2 CH2
N
CH3
H
CH3
NH
X
O
Basic structures of carotenoids
figure 6.4
Spirilloxanthin
CH3
CH3
OH
Carotene
O
CH3
Lutein
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
OH
O
CH3
CH3
In this exercise, you will examine light-absorbing photosynthetic pigments, and determine the absorption
spectrum of a chloroplast pigment solution.
A. ISOLATION OF CHLOROPLAST PIGMENTS: PAPER CHROMATOGRAPHY
Complex mixtures of chemical substances can be separated by chromatography. The separation of
the constituents of the mixture is based on differences in their solubilities in various solvents. In this study,
you will use the technique of paper chromatography to analyze the pigment composition of fresh spinach
leaves.
In paper chromatography, special filter paper is used to separate components of mixtures. A streak
of the substance to be chromatographed is placed at 1 end of the paper. This end is then immersed in a
solvent, which separates the components of the mixture as it migrates upward, through the spot, to the top
of the paper. After you dry the paper, materials that have separated may be observed directly if they are
colored, or you can make them visible by using various spray reagents.
CAUTION: Both petroleum ether and acetone are very flammable. There should be NO open
flames anywhere nearby!
1) Using a lead pencil (not a pen), draw a horizontal line near the bottom of the filter paper . Using a
small camel's hair brush, apply a narrow strip of chlorophyll extract to the pencil line. Dry the
paper thoroughly by blowing on it or waving it in the air.
2) Apply the extract 5 or 6 more times, but be sure the paper is completely dry before adding each
new application.
3) Place the strip in a test tube containing 2 to 3 ml of the petroleum ether/acetone mixture, as shown
in figure below. Immediately record the time.
FIGURE 6.5
Chromatography chamber setup
Cork
Paper clip
Chromatography
paper
Glass jar
Concentrated
extract
Running
solvent
4) Examine the chromatogram for the next several minutes. Record how long it takes for the solvent
to reach the top of the filter paper.
5) Remove the filter paper from the test tube and lay it on a paper towel to dry. Using a lead pencil
(not a pen), circle any pigment spots you can see. Use a ruler to measure the distance that each
pigment has traveled from the initial spot of extract.
B. ABSORPTION SPECTRA OF CHLOROPLAST PIGMENTS
The wavelengths of the visible spectrum that are absorbed by the chloroplast pigments can be
determined by using a Milton Roy Spectronic-20 colorimeter (spectrophotometer). In this instrument, a
highly polished plate with closely-spaced, etched lines disperses visible light into its component spectral
bands, and then projects them onto a scale. The disappearance from the spectrum of various colors
(wavelengths) as the light passes through a pigment solution indicates that those wavelengths were
absorbed by the pigments. A graph of the amount of light a substance absorbs versus the wavelength of
the light is called an absorption spectrum. The absorption spectrum for a hypothetical pigment is shown
in the figure 6.6 below.
FIGURE 6.6
Absorption Spectrum
In this study, you will determine the absorption spectrum of the spinach extract using the
Spec-20. Refer to How To Use A Spectrophotometer on the Biology lab web page to review the
instructions.
1) Beginning at a wavelength of 400 nm, blank the instrument to 0% absorbance and 100%
transmittance with the petroleum ether used to extract the chloroplast pigments.
2) Place the tube containing the extract into the sample holder and determine the percent
transmittance (%T).
3) Remove the sample and reset the wavelength control to 425 nm. Re-“blank” the instrument, and
then determine the percent transmittance of the sample.
4) Repeat Step 3 at 25-nm intervals. It will be necessary to insert an accessory red filter and
red-sensitive phototube for determinations above 625 nm.
5) Using the table in the “How To Use A Spectrophotometer” handout, convert the percent
transmittance (%T) into absorbance (A).
6) You should plot out your data to create an absorption spectrum for the spinach extract. For some
hints about how to set up your axes, look at Fig. 6.6. You can use Fig. 6.7 to make a rough draft
of your graph, but you should turn in your final draft made on a separate sheet of graph paper.
Figure 6.7
Student
Date
PHOTOSYNTHESIS LAB
STUDENT WORKSHEET
You should answer the questions below on a separate sheet of paper.
1. Look at the chemical structures of the pigments. Why do you suppose solvents such as petroleum
ether and acetone (rather than water) are used in this lab exercise?
2. In your paper chromatography experiment, how long did it take for the solvent to reach the top of
the paper?
3. Describe any separation that occured during yoru chromatography experiment. Give the colors of the
pigments that you observed and the distance that they traveled.
4. Are you able to identify any of the pigments that separated out during the chromatography? Look at
the introductory text in this handout for some hints on how to identify your pigments based on
their colors.
5. Why is it important that you use a pencil (rather than a ballpoint pen or a felt-tip pen) when marking
your chromatography paper?
6. What wavelengths are strongly absorbed by the hypothetical pigment in Fig. 6.6?
7. What wavelengths are weakly absorbed by the hypothetical pigment in Fig. 6.6?
8. At what wavelength(s) does the chlorophyll extract absorb maximally?
9. In your spectrophotometric determination of chloroplast pigments, why is the same solvent mixture
that was used to extract the pigments used to blank the colorimeter? Why couldn’t you use
another solvent, such as H2O?
10. Why do leaves of those plants in which chlorophyll is the predominant pigment appear green even
though there are many other pigments present?
11. Because the chloroplast pigments consist of both chlorophylls and carotenoids, you cannot tell from
the absorption spectrum which pigments are absorbing which wavelengths. Design and experiment
or a procedure that would enable you to determine which pigments in the spinach leaf extract are
contributing to the absorption spectrum of the extract.