Exercise #6 PHOTOSYNTHESIS

In the process of photosynthesisphotosynthesis, several energy transformationstake place.

-Light energy is captured by plant cells and is converted intoelectrical 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 bondsof 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 PIGMENTSPHOTOSYNTHETIC PIGMENTS

The initial step of photosynthesis involves the capture of lightenergy by the photosynthetic pigment, chlorophyllchlorophyll.

However, the plant does not absorb all of the light energy inthe visible portion of the electromagnetelectromagnetic spectrumspectrum.

-Figure 6.1 of your lab manual represents the visibleportion of the electromagnetic spectrum.

In this exercise, you will use the spectrophotometer to determinewhich wavelengths of light are absorbed and which ones are reflected by the chlorophyll molecule.

Exercise #6 PHOTOSYNTHESIS

PROCEDUREPROCEDURE

In order to study the absorbance of various wavelengths of lightby 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 thechlorophyll extract.

Exercise #6 PHOTOSYNTHESIS

At this point, we wish to determine which wavelengths of lightare absorbed by the chlorophyll extract. However, we must firstensure 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 tocontinue with the experiment.

Exercise #6 PHOTOSYNTHESIS

Use your chlorophyll extract along with an ethanol blank anddetermine the absorbance of the chlorophyll solution at variouswavelengths.

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 characteristicsof your chlorophyll extract, you will plot your data using graphicanalysis.

Remember that wavelength is the independent variableindependent variable andthat absorbance is the dependent variabledependent variable. Indicate with arrows on the x-axis the wavelengths of light most stronglyabsorbed by the chlorophyll extract.

Exercise #6 PHOTOSYNTHESIS

LIGHT INTENSITY AND PHOTOSYNTHESISLIGHT INTENSITY AND PHOTOSYNTHESIS

In general, as the intensity of light increases, the rate ofphotosynthesis 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 effectof light intensity on oxygen production and then make a generalizationon plant survival based upon the data you have collected.

Exercise #6 PHOTOSYNTHESIS

PROCEDUREPROCEDURE

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 syringe4) 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)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. Record

your data in Table 6.4.

Exercise #6 PHOTOSYNTHESIS

The changes observed in the tube containing Elodea are theresult of two simultaneous happenings:

1) the production of oxygen by Elodea which will always result in an increase in volume, and2) 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 mustTo obtain a true reading of oxygen production, you mustsubtract the control reading from the subtract the control reading from the ElodeaElodea reading. reading.

Exercise #6 PHOTOSYNTHESIS

Graphic AnalysisGraphic Analysis

Plot all three cumulative movements of fluid as a function of timeon a sheet of graph paper.

It is suggested that different plotting symbols be used for eachdistance 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 greaterthe greaterthe light intensity, the greater the rate of oxygen production the light intensity, the greater the rate of oxygen production by the plantby the plant.

Exercise #6 PHOTOSYNTHESIS

CONCLUSIONSCONCLUSIONS

1) You have seen that plants contain photosynthetic pigments.1) You have seen that plants contain photosynthetic pigments.2) Such pigments (chlorophyll) absorb only specific 2) Such pigments (chlorophyll) absorb only specific wavelengths of light.wavelengths of light.3) The amount of light that the plant is exposed to has an3) The amount of light that the plant is exposed to has an influence on photosynthetic rate.influence on photosynthetic rate.

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