Photosynthesis Lab - Northern Kentucky University
Photosynthesis Lab
INTRODUCTION
The importance of photosynthesis cannot be overstated. Indeed, all life depends
on this metabolic process. Plants themselves depend on photosynthesis as a novel method of producing carbohydrate. Animals at all other trophic levels also depend, either directly or indirectly, on the organic molecules that comprise plant tissue. They consume the plants and then convert this tissue into animal tissue. Photosynthesis is a complex anabolic process that occurs in the chloroplasts of plant leaves. In its simplest form, photosynthesis captures the energy of sunlight and uses this to drive the formation of carbohydrates.
Plant Pigments
Absorption of light energy in plants is made possible by the presence of pigments. Plants typically appear green to the human eye. This results from the reflection or transmission of green light from the pigments chlorophyll a and chlorophyll b. Other pigments, the carotenoids and xanthophylls, also participate in photosynthesis and these may appear yellow or orange to the human eye. Reflected/transmitted light energy is generally not available to photosynthesis. It is the absorbed light energy that can do useful work in photosynthesis.
In the first part of this laboratory you will prepare a pigment extract from the chloroplasts of spinach leaves. Using a technique called chromatography you will then separate the various pigments based on differences in their molecular structures. In the
second part, you will determine with a spectrophotometer what wavelengths of visible light are actually absorbed by plant pigments.
Analysis of Photosynthetic Pigments
The pigments identified above (chlorophylls a and b, carotenoids, and xanthophylls) can be extracted and separated for analysis by several methods. We will do a simple chemical extraction using an acetone-water solvent followed by filtration to remove tissue debris. The extract obtained by this method will contain all of the pigments; keep this in mind as you proceed with the analyses.
A. Extraction of pigments from spinach leaves.
Laboratory Procedure:
1. Each group of students should obtain about 15 g of spinach leaves (about 4
leaves). Tear the leaves into large pieces, excluding large veins and obviously damaged portions of leaves
2. Using a graduated cylinder, place 50 ml of an 80% (v/v) solution of acetone and water into a mortar. To this add a very small amount (1 scoop) of CaCO3 and a small (3 scoops amount of fine quartz sand (use the scoops provided for the CaCO3 and sand).
3. Use a pestle to grind the leaves in the mortar into a coarse slurry.
4. Set up a filtration apparatus consisting of a Buchner funnel (with two layers of wet filter paper) and vacuum flask; attach a vacuum hose to the aspirator on the water faucet in one of the sinks.
5. Pour the slurry into the Buchner and gently draw the liquid extract into the flask by turning on the cold water faucet Rinse with 25 ml of acetone water
solution.
6. After the liquid has been collected in the flask, turn off the vacuum and
remove the funnel. Pour the liquid into a small, dark screw top glass bottle and save for chromatographic and spectrophotometric analysis.
B. Chromatography of extracted pigments.
Chromatography allows the separation of similar compounds utilizing their slightly different solubility and adsorption characteristics. Extracts of these compounds are applied to various media (such as paper, ion-exchange resins, or silica gel) and allowed to migrate throughout the medium. The rates of migration for individual compounds differ according to the chemical structure of the compounds, the nature of the chromatographic medium, and physical factors such as pH and temperature. By varying composition of the solvent and medium used and controlling physical conditions, the compounds can be physically separated and identified by various means. Chromatographic techniques can also be used for large-scale preparation
of purified extracts.
You will use a technique known as “thin-layer chromatography”. The medium in this case is a fine slurry of silica gel spread on a glass, aluminum, cardboard, or plastic support. Silica gel is a colloidal suspension of Si02 that has many small pores making it very adsorbent when dried. Fortunately, you don’t have to prepare the thin-layer medium; plastic strips on which silica gel has been pre-spread are available for this exercise.
Laboratory Procedure.
1. Pour enough solvent into a chromatographic chamber to make a pool about 0.5
cm deep. Put the top on the chamber and set it aside to allow the vapor phase to equilibrate.
2. Obtain a hematocrit capillary tube and, holding the red-marked end in your
hand, draw some of the pigment extract, which you have just prepared, into the tube.
3. Apply several drops of extract in a spot about 2 cm from the end of a plastic
silica gel strip. Allow the spot to dry before applying a repeat spot. The spot should be as dense as possible, but no larger than 3-4 mm. Gently wave the strip about 4 or 5 times in the air to accelerate drying of the spot.
4. Make a faint scratch on the edge of the strip to indicate the location of the
spot so that you can calculate migration distances later.
5. Open the chamber and gently place the silica gel strip in the solvent; close
the chamber quickly to minimize vapor loss.
6. Allow the chromatogram to “run” (about 10 minutes) until the solvent moves to
within 2 cm of the top and then remove the strip, being sure to replace the top.
7. Make another faint scratch on the strip--this one corresponding to the point
reached by the solvent (i.e., the solvent front ).
Data Analysis:
1. With a pencil, circle the spots that represent each of the pigments. Measure the distances that each of the pigments has moved from the origin and calculate an Rf value for each using the formula:
Rf = distance substance moves/distance solvent front moves
2. Which spot corresponds with each of the pigments mentioned earlier in this
exercise and what criteria are you using for this determination?
C. The Absorption of Light by Plant Pigments
As described earlier, we perceive pigments to have a particular color because of the portion(s) of the visible spectrum that are reflected. In this part of the exercise, we will measure the wavelengths of the spectrum that are absorbed and that, by implication, are involved in the photosynthetic process. The colors of the pigments that you should have extracted from spinach leaves earlier could be generally described as follows:
chlorophyll a- blue-green
chlorophyll b - yellow-green
carotenoids- yellow
xanthophylls- greenish-yellow
Re-examine your thin-layer chromatogram. Does this information change your
decision about the identification of the spots and how you might determine more precisely which pigment is present in each spot?
We will now examine one technique that can be used to characterize pigments by
the specific wavelengths of light that they absorb. The technique utilizes electronics to determine which wavelengths are absorbed and how much of those wavelengths are absorbed, therefore, giving both qualitative and quantitative information about the pigments. The instrument employed is known as a spectrophotometer, which is a relatively simple single-beam spectrophotometer that is sensitive over most of the visible spectrum. With modification, it will measure the amount of light absorbed (or transmitted) by a pigment sample over the entire 400-700 nm range of visible light.
Laboratory Procedure:
1. Pipette 1.0 ml of the extract you prepared earlier into a small test tube.
2. This extract is probably too concentrated to use directly in the spectrophotometer, so you will have to dilute it. Pipette 1 ml of the extract from the test tube you have just prepared into a second tube. Add 9 ml of the original 80% acetone solution to this second tube.
3. Proceed with 1:9 serial dilutions in this fashion until you have a solution that is still green, but is becoming transparent. Retain all the solutions you prepare and record the number of dilutions you made for later calculations.
4. Following the instructions on the spectrophotometer, set the wavelength at
400 nm and calibrate the instrument using a solvent blank (i.e., a spectrophotometric tube containing 80% acetone--the solvent used for your pigment extraction.
5. Remove the solvent blank and insert a spectrophotometric tube containing one
of your extract dilutions.
6. Read the absorbance scale on the face of the instrument and record the value.
7. Remove the sample tube, set the wavelength to 425 nm and proceed, as before,
to measure and record absorbance. Continue this process, in 25 nm increments, until you reach 700 nm.
8. Your maximum absorbance values should be in the range of 0.4 to 1 .4. If they
are outside this range, use a different dilution to obtain readings.
9. Record all your data and plot absorbance vs. wavelength on graph paper or use
the StatView program on the computers to generate a plot.
Data Analysis:
1. What are the absorption maxima for the spinach extract?
2. Can you determine from your data which pigments are responsible for this
absorption spectrum? Describe, in general, a procedure that would allow you to identify the absorption spectrum of each of the pigments in your extract.
D. Gas Exchange in Plants
Plant leaves constantly exchange gases with the surrounding environment. This
exchange of gases between the atmosphere and the leaves of terrestrial plants occurs through microscopic openings on the leaf surface called stomata. Gas exchange in aquatic plants may occur directly across cell membranes. Regardless of how gas exchange occurs, the two gases of prime importance to photosynthesis are CO2 (carbon dioxide) and O2 (oxygen). CO2 is scavenged from the atmosphere
or water and is used to build organic molecules (i.e., it is a reactant in the
process). O2 is released during the light-dependent reactions and must diffuse
from the leaf into the atmosphere or into the water (i.e., it is a product of
the process). Scientists interested in measuring the rate of photosynthesis have
two potential avenues: they can measure how much CO2 is taken up by the plant or
they can measure how much O2 is released from the plant. In the third and final
part of this laboratory we will determine the effect of light wavelength on the
photosynthetic rate. Release of O2 will be measured indirectly.
Laboratory Procedure:
1. Each group of students should obtain two manometer tubes. Lightly pack each
tube with equal amounts of fresh Elodea; try to pick single, healthy stems and insert them with the shoot tip toward the bottom of the tube. Then fill the tubes with 1 .5% sodium bicarbonate solution. Sodium bicarbonate will, through chemical reaction, supply the necessary CO2 for photosynthesis.
2. Obtain two stoppers for the tubes. These stoppers will be fit with a bent section of a graduated pipette. There will also be a syringe for manipulating the pressure. Insert the stoppers in the tubes and make sure they fit tightly with no leaks. There should be little or no air space remaining in the tubes.
3. Wrap one tube with aluminum foil. This prevents light from hitting the
Elodea. Use this tube as a control to ensure that your measurements of photosynthesis are reliable. Place both tubes in a test-tube rack.
4. Use the syringe to force some bicarbonate solution into the pipettes and note
the starting positions of the solution in the pipettes.
5. You want to shine the light on both tubes, but there must be a heat sink
between the tubes and the lamp to absorb infrared energy that would raise the
temperature of the tube contents. A beaker of water serves as an efficient heat
sink.
6. One group in class should use red light as the energy source,
another group should use green light, and a third group should use white light. In all cases, put the light source right up against the heat sinks. Record the time and the position of the fluid in the manometer. After 5 minutes, record the position of the fluid in the
manometer. Reset the manometer to zero using the syringes. Continue to take readings every 5 minutes for one hour. For each reading, both time and the volume of O2 released must be recorded.
Data Analysis:
1. Each group should calculate the rate of photosynthesis as measured in ml O2
generated per hour per clump of Elodea.
2. Did the rate of photosynthesis appear to vary with color of light? Did the
results coincide with your expectations?
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