Upon the completion of this exercise the student should be able to:
1. write the summary equation for the process of photosynthesis;
2
. determine the effect of
light
intensity on the rate of photosynthesis of elodea leaves;
3. predict the effect of varying the light intensity on the rate of photosynthesis;
4. separate a mixture of
photosynthetic pigment
s by paper chromatography;
5. describe the mechanism of pigment separation and identify the factors that contribute to the separation of pigments in paper chromatography;
6. use a spectrophotometer to collect data and graph the absorption spectrum of spinach leaf chloroplast extract; and
7. describe and interpret the absorption spectrum of a sample of spinach leaf chloroplast extract.
Photosynthesis is the process by which light energy is converted into chemical energy. It occurs in all plants, algae, and some bacteria. This process is the source of nearly all of the organic molecules in living organisms, as well as the source of oxygen in the atmosphere. In this process, plants use light energy to convert carbon dioxide and water into organic molecules.
2C3H6O3 (a sugar) + 6O2 + 6H2O
light
chlorophyll
6CO2 + 12H2O
and oxygen. Except for cyanobacteria (blue-green algae) and other photosynthetic bacteria, the reactions of photosynthesis occur in specialized structures called chloroplasts. The overall process may be summarized as follows:
Contrast this equation with the summary equation for cellular respiration on page 177. The photosynthesis equation may be further elaborated by dividing photosynthesis into two general sub-processes—the light reactions and the Calvin cycle.
In the
light reactions
, light energy is absorbed and converted into chemical energy which is stored in molecules of ATP and NADPH. These reactions involve the splitting of water molecules, with the consequent liberation of molecular oxygen (O2). Light is a very small part of the electromagnetic spectrum, and the only part that is visible to the eye. This portion of the spectrum lies between the wavelengths of 380 nanometers (nm) and 760 nanometers, as seen in the diagram below (1 nanometer = 1/1,000 of a micrometer or 1×10-9 meters). Light is radiated in very small packets called
photons
, which travel at specific
wavelengths
. We can distinguish among the different wavelengths as differences in color. A prism is often used to separate white light into its component colors. The energy of light is inversely proportional to the wavelength, i.e., the shorter the wavelength, the greater the energy in each photon.
In order to capture the energy from light, it must first be absorbed by the plant. This is accomplished by the organelles called
chloroplasts
, which contain special
photosynthetic pigments
. These pigments are capable of absorbing photons having the wavelengths of light which are used in photosynthesis. The principal photosynthetic pigment is
chlorophyll a
, while the secondary (accessory) pigments are
chlorophyll b
(also c and d in certain organisms), the carotenoids, and the
xanthophylls
. Since the pigments do not absorb all wavelengths of light equally, they have characteristic colors. Chlorophyll is responsible for the green color of plants because chlorophyll reflects green wavelengths of light. The carotenoids reflect much of the orange and red wavelengths; thus, they provide coloration for plant organs such as tomatoes, carrots, pumpkins, and the striking colors of the autumn leaves.
In the
Calvin Cycle
, the energy which was stored in ATP and NADPH molecules is used to convert carbon dioxide into organic molecules. One of these organic molecules is
glucose
, a carbohydrate which can be condensed to form starch or other storage or structural compounds. The glucose can also be used as the starting point for the construction of every other organic molecule that the plant produces (e.g., amino acids, fatty acids, etc.). The dark reactions do not require light to proceed. They do, however, require the ATP and NADPH produced by the light reactions.
2
10-6 nm 10-3 nm
Nanometers (nm) are billionths of a meter (1 nm = 10-9 m)
Visible light
Wavelength
s are often expressed in meters or nanometers
Violet
Blue
Green
Yellow Orange
Red
Radio waves
Micro- waves
Infrared (IR)
Ultraviolet (UV)
X-
rays
Cosmic and Gamma rays
1 nm
103 nm
106 nm 1 m
103 m
400 nm
500 nm
600 nm
700 nm
THE VISIBLE LIGHT SPECTRUM AS PART OF THE OVERALL ELECTROMAGNETIC SPECTRUM
The purpose of this lab exercise is to examine several factors involved in photosynthesis. We will examine the effect of the intensity of light (number of photons per time) on the rate of photosynthesis. We will isolate and identify the photosynthetic pigments found in the chloroplasts of spinach leaves. Finally, we will construct an absorption spectrum of chloroplast extract from spinach leaves.
1. Effect of
Light Intensity
on the Rate of Photosynthesis
One of the products of photosynthesis is molecular oxygen (O2). Oxygen is only slightly soluble in water, so in a saturated solution, the excess oxygen will form gaseous bubbles. The rate of oxygen evolution by a plant immersed in water can be a reasonable measure of the rate of photosynthesis. We will set up a system that can measure the amount of oxygen gas released from plant leaves. We will use the system to determine the effect of varying light intensity (using light bulbs of differing wattage) on the rate of photosynthesis.
· PROCEDURE – This is the experimental protocol you would follow if we met in the lab. You will be given the data in the tables below. Use the data for your calculations and to graph your data.
1. Place two sprigs (about 3″ long) of E1odea into a test tube with the cut end up. Be certain the cuts are fresh and look healthy.
2. Completely fill the tube with 3% sodium bicarbonate solution. This solution provides the carbon dioxide source necessary for photosynthesis.
3. Insert a stopper (which contains a bent piece of glass tubing) into the test tube. The stopper should be tight. This will force some of the sodium bicarbonate solution out of the test tube and into the glass tubing. The stopper works best if you insert it when it is dry and when the lip of the glass tube is dry.
4. Use a long syringe to remove the liquid from the glass tubing so that the liquid front is between the bend in the tubing and the first mark on the tubing.
5. Position the test tube in the holder or in a test tube rack in front of the low wattage lamp. Place a large beaker of water between the lamp and the test tube to act as a heat sink. Place the lamp and beaker as close to the test tube as possible.
6. Turn on the lamp. As photosynthesis proceeds, oxygen (O2) will be produced. This increased volume of oxygen gas emitted by E1odea leaves into the tube will force the liquid up and out of the test tube into the glass tubing. The amount of movement of the liquid in the glass tubing should be proportional to the amount of oxygen gas released.
7. When the edge of the solution reaches the first major mark on the glass tubing nearest the bend, note the time and enter it as the initial time in Table 1 on the next page. Note the time again when the edge reaches the second major mark and enter it as the final time in Table 1 on the next page. Record the elapsed time in the fourth column of Table 1. The volume between the two major marks (or 10 minor marks) equals
0.1
mL. Note: this data is provided for you in Table 1.
8. Repeat procedures 4–8 two more times, first replacing the low wattage bulb with a medium wattage bulb, then, on the last trial, replacing the medium wattage bulb with a high wattage bulb. Note: this data is provided for you in Table 1 on the next page.
9. Use Table 1 data to plot a graph of the photosynthetic rate versus light intensity
on the next page. Use it to answer the questions.
Table 1: Effect of Light Intensity on the Rate of Photosynthesis
(Time should be in minutes, not minutes & seconds)
Light Intensity
Elapsed time (minutes + seconds)
Elapsed time (minutes)
mL O2
Rate of Photosynthesis (mL O2/hour)
45 watts
25 min 15 sec
0.1
75watts
14 min 45 sec
0.1
150 watts
16 min 30 sec
0.1
Calculating the Rate of Photosynthesis: The procedure is much the same as the one you used to calculate the metabolic rate. Divide the mL O2 released (0.1 mL) by the number of minutes (elapsed time) it took, then multiply that by 60 minutes/hour, as shown in this example
:
Plot the rate data on the graph below:
Questions:
1. Is the relationship between the intensity of light and the rate of photosynthesis a direct or an inverse relationship?
2. Would you expect the rate of photosynthesis to increase indefinitely with increasing light intensity? Explain why or why not.
2. Separation of
Chloroplast
Pigments by Paper Chromatography
As was noted earlier, the primary photosynthetic pigment is chlorophyll a; however, other accessory pigments (e.g., xanthophylls and
carotenes
) are also typically present in the chloroplast, albeit in smaller quantities. There are several techniques by which these primary and accessory pigments can be separated. In this exercise, the separation will be accomplished by
paper chromatography
. When a solution of these pigments is placed on a
strip
of filter paper, the pigment molecules adsorb onto the cellulose fibers of the paper. When the tip of the strip of paper is then immersed into a suitable solvent, the solvent is absorbed by the paper and moves up the paper by capillary action. As the solvent passes the spot on which the pigment solution was placed, the pigments are dissolved by the moving solvent. The pigment molecules are generally unable to move as fast as the solvent, but some of them move faster than others. The differential movement of the pigments is caused by three main factors that differ among the pigments:
Faster movement is associated with low molecular weight, high solubility in the solvent, and low affinity for the paper. The distance traveled by a particular pigment relative to the distance traveled by the solvent is specific for any given pigment and set of conditions. This relationship is called the
Rf
, and is calculated as follows:
distance from pigment origin to solvent front
Rf = distance moved by pigment
The Rf for any given substance is constant in a specific solvent separation system; thus, it can be used to characterize the pigment molecules.
PROCEDURE:
1. Obtain the chloroplast extract. Either it will be available at your desk or you will have to prepare it yourself. To prepare the extract,
a. cut one spinach leaf into small pieces and place into a mortar,
b. using the pestle, thoroughly grind the cut spinach leaf into a paste,
c. add a small amount of acetone to the mortar and mix with the ground spinach,
d. fill the mortar about ¾ full of acetone and blend thoroughly, and
e. strain the mixture by pouring it through cheese cloth into a small beaker
Alternative Procedure for Transferring Pigments onto Paper:
1. Draw faint pencil line approximately 2 cm from tip of paper.
2. Be sure strip is long enough so that tip of paper is just above bottom of tube, but pencil line will not be submerged in solvent.
3. Cut or tear strip of spinach leaf 1-2 cm wide. Place spinach leaf over pencil line. Use edge of coin to roll over spinach leaf while pushing down so that line of spinach extract is transferred to paper. Roll over leaf several times until you have a dark green line about 2 mm wide.
2. Refer to the illustration above to visualize the setup of the paper chromatographic strip. Since oil from the skin may interfere with the procedure, handle the chromatographic strip by one end at all times. Cut a strip of paper about 6″–7″ long.
3. Cut notches and a point on one end of the paper strip with scissors, as seen in the illustration.
4. Using the drawn-out end of a Pasteur pipette, put a small dab (much less than a drop) of the chloroplast extract on the paper between the notches, and allow the solvent to evaporate. Gentle blowing on the paper or fanning it in the air will speed this up. Build up the spot (called the origin) by repeated applications of the extract at the exact same point. This will require a minimum of 20 applications but may require up to 30.
5. Obtain a paper chromatography apparatus, a corked test tube with the petroleum ether-acetone (92 % and 8%, respectively) solvent at the bottom. If necessary, pour the solvent in the test tube; this should be done in a fume hood. Care should be taken not to breathe the vapor of the solvent. To reduce evaporation of the solvent, the test tube should be kept closed with the cork.
6. Hang the strip from the paper clip in the cork and place the strip into the test tube allowing just the tip of the strip to touch the solvent. The origin spot must not touch the solvent
. From this point on, it is imperative that you do not jiggle or bump the test tube. The solvent should not splash onto the origin spot. It needs to migrate upwards on its own. Make necessary adjustments so the cork will fit tightly and the spot will not contact the solvent. Stand the tube upright in the test tube rack.
7. Over the next 25 minutes, periodically examine the strip. Remove the strip before the solvent reaches the top of the strip. Average migration time is about 20 minutes. If the leading edge of the solvent, which is called the solvent front, reaches within one centimeter of the top of the paper, you should remove the paper. Do not allow the solvent to migrate up over the top of the paper.
8. Immediately after removing the paper strip, mark the solvent front with pencil. Do not use ink. Do this right away before the solvent evaporates. After the solvent evaporates, you will not be able to locate the solvent front.
9. Four pigment bands corresponding to the following four colors should be observed on the strip. Outline the areas occupied by these pigments with a pencil. Place a dash mark where the color seems the brightest; this will be in the approximate center of the pigment band. Do not use ink.
These pigments, from top to bottom, will be:
Color
Pigment(s)
yellowish orange
carotenes
pale yellow
xanthophylls
bluish green
chlorophyll a
yellowish green
chlorophyll b
10. To calculate the Rf value for each pigment, measure the distance (in millimeters) from the origin spot to the solvent front and from the origin to the pencil mark you placed near the center of each of the pigment band . Note this data has been provided for you in Table 2 below. Use it and the equation on page 5 to calculate the Rf value for each pigment. Enter the values on Table 2 and use them to answer the questions.
11. Find out from your instructor what you should do with the remaining solvent. You may be told to re-stopper the tube, leaving the solvent for future use. Or you may be asked to pour the remaining solvent into the waste jar. Do not pour the solvent down the sink
. Do not wash the test tube with water.
Table 2: Photosynthetic Pigments Separated by Paper Chromatography
Substance that migrated
Distance from the origin
Rf
petroleum ether-acetone solvent
10.0 cm
photosynthetic pigment
carotenes
9.3 cm
xanthophylls
4.3 cm
chlorophyll a
5.3 cm
chlorophyll b
1.3 cm
Questions:
1. What can be deduced about a molecule that has a very high Rf?
2. Which pigment in spinach leaves do you think is most soluble
in the petroleum ether-acetone solvent we used in today’s lab?
3. What is the maximum value that the Rf can be for any molecule?
4. Would you expect the Rf of chlorophyll a to be the same or different if the solvent were something other than that used in this exercise? Explain why or why not.
III. Absorption Spectrum of Chloroplast Extract
An instrument called
spectrophotometer can be used to pass light of specific wavelengths through a sample of chloroplast pigments dissolved in a solvent (please read the basic principles of spectrophotometry in page 21 and 22). The degree of absorption of each of the wavelengths of light by the pigments can then be measured. The resultant data can be plotted on a graph, thus producing an
absorption spectrum of the pigment mixture. Since all green plants don’t have exactly the same type and amount of photosynthetic pigments, the spectra may vary from one species of plant to another.
The pigment solution is prepared by mixing several plant leaves with acetone and then homogenizing them in a blender. Acetone is a nonpolar organic solvent in which the pigment molecules are soluble. This mixture is then filtered to remove all debris. The biology lab technician has already prepared the mixture for you. The solvent is extremely flammable, so use caution in handling both the pigment solution and the solvent.
The particular model of the spectrophotometer may vary from campus to campus and from lab to lab. Your instructor will familiarize you with the instrument and teach you how to use it. In the appendix section of this manual you may find a “how to use” guide for the particular model of the spectrophotometer you are given to work with in the lab.
Here is a video on how to use the spectrophotometer we have in the lab:
Ask your instructor how to calibrate (set the absorbance to zero) the spectrophotometer using the control cuvette containing the colorless solvent and how to obtain absorbance data by using the cuvette containing pigment solution. You should be recording the absorbance at different wavelengths from
380 nm
through
720 nm
. Record the absorbance for the specific wavelengths given in the Table 3.
You should calibrate the spectrophotometer every time you change the wavelengths. Note this data is provided for you in Table 3. Use the data to construct an absorption spectrum on the next page. Remember to label your axis correctly.
Table 3: Absorption Spectrum Data for Spinach Chloroplast Extract
Wavelength
Absorbance
Wavelength
Absorbance
Wavelength
Absorbance
380 nm
0.725
500 nm
0.139
620 nm
0.100
400 nm
0.834
520 nm
0.074
640 nm
0.135
420 nm
0.971
540 nm
0.047
660 nm
0.406
440 nm
0.844
560 nm
0.041
680 nm
0.049
460 nm
0.678
580 nm
0.059
700 nm
0.029
480 nm
0.338
600 nm
0.079
720 nm
0.000
Absorption Spectrum Graph of Spinach Chloroplast Extract
Questions:
1. Your graph should have two “peaks.” At the highest of the two, what wavelength was most strongly absorbed by the chloroplast pigments? _________
What wavelength corresponds the top of the second peak? _________
What wavelength was least absorbed by the chloroplast pigments? _________
2. Based on your observations, which wavelength (color) of light might be expected to generate the lowest
rate of photosynthesis? _________
3. Based on your observations, which wavelength (color) of light might be expected to generate the
highest
rate of photosynthesis? _________
4. Why are plants typically green?
5. Photosynthesis produces the oxygen (O2) on which all aerobic organisms depend for survival. What other factors make photosynthesis essential to the maintenance of virtually all life on earth?
6. In the experiment that tested the effect of light intensity on the rate of photosynthesis, we did
not run a control tube during the experiment. Why is a control important in an experiment? How would you design a control for this experiment? Be specific
7. If during the experiment, you found that the control tube generated pressure and had a measurable rate of “photosynthesis” just like the experimental tube did, what would this fact tell you about the experiment?
Chromatographic
strip
Chloroplast
extract spot
Solvent
Solvent Front
Carotene (yellow)
Xanthophylls (yellow)
Chlorophyll a (blue green)
Chlorophyll b (yellowish green)
Chloroplast extract origin
Chromatographic
strip
Chloroplast
extract spot
Solvent
Solvent Front
Carotene (yellow)
Xanthophylls (yellow)
Chlorophyll a (blue green)
Chlorophyll b(yellowish green)
Chloroplast extract origin
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