Exercise #6
PHOTOSYNTHESIS
In the process of photosynthesis, several energy transformations
take place.
-Light energy is captured by plant cells and is converted into
electrical energy in the form of high energy electrons.
-The kinetic energy of the electron is transformed into
chemical energy in the bonds of ATP.
-The ATP, in turn, transfers the energy to the chemical bonds
of a glucose molecule.
Through this process, light energy from the sun is captured and
stored in the chemical bonds of sugar molecules.
Exercise #6
PHOTOSYNTHESIS
PHOTOSYNTHETIC PIGMENTS
The initial step of photosynthesis involves the capture of light
energy by the photosynthetic pigment, chlorophyll.
However, the plant does not absorb all of the light energy in
the visible portion of the electromagnetic spectrum.
-Figure 6.1 of your lab manual represents the visible
portion of the electromagnetic spectrum.
In this exercise, you will use the spectrophotometer to determine
which wavelengths of light are absorbed and which ones are
reflected by the chlorophyll molecule.
Exercise #6
PHOTOSYNTHESIS
PROCEDURE
In order to study the absorbance of various wavelengths of light
by chlorophyll, you must first separate the pigment from the leaf tissue.
This is accomplished by following the procedure below:
1) Place a large spinach leaf into a 50 ml beaker and add 30 ml of ethanol.
2) Place the beaker with ethanol on a hot plate and slowly heat the solution taking care not to crush the
leaf or boil the ethanol solution.
3) As the chlorophyll exits the leaf tissue it will enter the ethanol solution and turn the color green.
4) Fill the cuvette approximately 2/3 full will the chlorophyll extract.
Refer to the diagrams on the
right for a representation of the
chlorophyll extract.
Exercise #6
PHOTOSYNTHESIS
At this point, we wish to determine which wavelengths of light
are absorbed by the chlorophyll extract. However, we must first
ensure that the concentration of chlorophyll in the solution is
appropriate for the experiment.
To do this, follow the procedure below:
1) Set the spectrophotometer at 550 nm and depress the %T button.
2) Place a cuvette filled 2/3 full of ethanol into the sample compartment and calibrate to 100 %T.
3) In order to continue on with the absorption experiment you must obtain a reading between 65% - 85%.
-If you have a reading below 65%, your experimental solution is too concentrated--what
should you do to change its concentration.
Once you have reached an appropriate %T reading you are ready to
continue with the experiment.
Exercise #6
PHOTOSYNTHESIS
Use your chlorophyll extract along with an ethanol blank and
determine the absorbance of the chlorophyll solution at various
wavelengths.
NOTE: the procedure employed here is identical to that used in the determination of the
absorption spectrum for methylene blue (Exercise #3)
-remember to zero the machine after you change each wavelength.
In order to clearly visualize the actual absorbance characteristics
of your chlorophyll extract, you will plot your data using graphic
analysis.
Remember that wavelength is the independent variable and
that absorbance is the dependent variable. Indicate with
arrows on the x-axis the wavelengths of light most strongly
absorbed by the chlorophyll extract.
Exercise #6
PHOTOSYNTHESIS
LIGHT INTENSITY AND PHOTOSYNTHESIS
In general, as the intensity of light increases, the rate of
photosynthesis increases.
How does one measure the rate of photosynthesis?
As you should recall, oxygen is a by-product of photosynthesis.
Therefore, the photosynthetic rate can be determined by
measuring the rate of oxygen production by the plant.
During this portion of the laboratory you will observe the effect
of light intensity on oxygen production and then make a generalization
on plant survival based upon the data you have collected.
PROCEDURE
Exercise #6
PHOTOSYNTHESIS
To determine the effect of light intensity upon photosynthetic rate,
you will conduct the following experiment:
1) Obtain a piece of Elodea 5 to 6 inches in length and place it in a test tube with the cut end up.
2) Fill the test tube with NaHCO3 (serves as a source of CO2) and plug with the rubber stopper containing
the bent glass pipette an a syringe.---AVOID AIR BUBBLES!
3) The position of the fluid in the pipette can be adjusted by raising or lowering the plunger in the syringe
4) Prepare a second tube filled with NaHCO3 without Elodea to act as a control for temperature and
pressure fluctuations.
5) Place both tubes in a beaker filled with room temperature water.
6) Place a lamp 75 cm from the experimental beaker and set up a heat filter (additional beaker) filled
with tap water placed half way between the light source and the experimental beaker.
Refer to the picture on the following slide for reference.
Exercise #6
PHOTOSYNTHESIS
The following movie represents the experimental setup for the
light intensity experiment.
Exercise #6
PHOTOSYNTHESIS
PROCEDURE (continued)
75 cm - Move the lamp so it is 75 cm away from the experimental tubes and place the heat
filter half way between the lamp and tubes. Wait 5 minutes for equilibration. Adjust
the fluids in the pipettes so that they are at the 0.2 ml mark and recalibrate only when
changing the distance to the light source. Record the position of the fluid in the
pipettes at 2 minute intervals for 10 minutes.
50 cm - Move the lamp forward so it is 50 cm away from the experimental tubes and again place
the heat filter half way between the lamp and experimental tubes. Wait 5 minutes for
equilibration and adjust the fluid in the pipettes to 0.2 ml. Repeat the experiment and
record your data in table 6.4.
25 cm - Repeat the equilibration, adjustments and measurements at the 25 cm mark.
your data in Table 6.4.
Record
Exercise #6
PHOTOSYNTHESIS
The changes observed in the tube containing Elodea are the
result of two simultaneous happenings:
1) the production of oxygen by Elodea which will always
result in an increase in volume, and
2) fluctuations in temperature and pressure, which is why
the pipette is set at the 0.2 ml mark.
To obtain a true reading of oxygen production, you must
subtract the control reading from the Elodea reading.
Exercise #6
PHOTOSYNTHESIS
Graphic Analysis
Plot all three cumulative movements of fluid as a function of time
on a sheet of graph paper.
It is suggested that different plotting symbols be used for each
distance from the light source (e.g. * for 75 cm; + for 50 cm; and
- for 25 cm).
Using a clear plastic ruler, draw a straight line that best fits all
points for each distance. Your lines should indicate that the greater
the light intensity, the greater the rate of oxygen production
by the plant.
Exercise #6
PHOTOSYNTHESIS
CONCLUSIONS
1) You have seen that plants contain photosynthetic pigments.
2) Such pigments (chlorophyll) absorb only specific
wavelengths of light.
3) The amount of light that the plant is exposed to has an
influence on photosynthetic rate.
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