Determining the absorption spectrum for leaf pigments

Biology Photosynthesis Lab

Exercise 10

PHOTOSYNTHESIS Student Learning Outcomes At the completion of this exercise you should: (1) Be able to define the following linear metric system units: meter, centimeter, millimeter,

micrometer, nanometer, and Angstrom. (2) Be able to describe, using both nanometers and color descriptions, the wavelengths of

sunlight that the human eye can see. (3) Be able to describe how the “paper chromatography” technique can be used to separate out

the different pigments present in plant leaves. (4) Be able to describe, using both nanometers and color descriptions, the most important

wavelength of sunlight used in photosynthesis. (5) Be able to describe an experimental design that could be used to measure the relationship

between light intensity and the rate of photosynthesis. (6) Be able to diagram the summary equation for photosynthesis. I. The Nature of Light Striking the Surface of a Leaf Sunlight is a relatively small but very important part of the vast electromagnetic spectrum of energy. Visible light, together with a small amount of invisible radiation at its boundaries, is the only part of this spectrum which is useful to life. Close examination of visible natural light reveals that it, too, contains a spectrum, which is revealed to our eyes in the colors of a rainbow. Two physical properties of light are of special interest to biologists: wavelength and energy. Procedure: 1. Point the slit end of the #125 Wabash Spectroscope at a white light source, such as a

small light bulb. The prism within the spectroscope will separate the various wavelengths (colors) of which the white light is composed.

2. The spectrum of visible light and the wavelengths of the colors can be seen when you

look through the eyepiece. Each white numbered marking denotes the wavelength of the color above it. The wavelength is the actual length of a wave of light of that color, measured in units called angstroms (Å).

Table 1. Metric System: Length Measurement Units

Quantity Numerical Value English Equivalent Converting to Metric kilometer (km) 1,000 m 1 km = 0.62 mile 1 mile = 1.609 km

meter (m) 100 cm 1 m = 3.28 feet

= 1.09 yard 1 yard = 0.914 m 1 foot = .305 m

centimeter (cm) 0.01 m 1cm = 0.394 inch 1 foot = 30.5 cm

millimeter (mm) 0.001 m 1 mm = 0.039 inch 1 inch = 2.54 cm

micrometer (1 µm), also called a "micron (µ)"

0.000001 m

nanometer (nm), also called a "millimicron(mµ)"

0.000000001 m

angstrom (Å) 0.0000000001 m

*Note: In other sources, you will sometimes see another unit, the nanometer (nm), used to measure wavelength. Each nanometer is equal to ten angstroms: for example, 7000 Å=700 nm. Although both the angstrom and the nanometer are used to measure wavelength, the nanometer is currently the more frequently used unit.

Question 1. Using Table 1, complete the following:

a. There are _________ centimeters (cm) in a meter (m).

b. There are _________ millimeters (mm) in a meter (m).

c. There are _________ micrometers (microns or µm) in a meter (m).

d. There are _________ nanometers (nm) in a meter (m).

e. There are _________ Angstroms (Å) in a meter (m). Question 2. Look at the chart on the wall. It gives nanometer (nm), rather than angstroms as the until of wavelength. Since one nanometer is equal to 10 angstroms, 3500 (Å) = 350 nm. Write down the wavelengths in nanometers, using this simple conversion. Nanometer is currently the more frequently used unit.

In the table below, arrange the six visible light colors in a sequence starting with the one with the shortest wavelength and ending with the color with the longest wavelength. Also, indicate the approximate wavelengths of the colors. You will fill in the last column using the information in the next question.

Wavelength Observed Color

Approximate Range of

Wavelengths (nm)

Relative Energy

Shortest 1. 2. 3. 4. 5.

Longest 6. Question 3. It is known that shorter wavelength light possesses more energy than longer wavelengths. Indicate in the table in, Question 2, which color has the highest energy and which color has the lowest energy. II. The Photosynthetic Pigments Pigments are light absorbing substances. They are useful to humans in decoration because they absorb certain wavelengths and reflect others, thus coloring our environment. In this case, we appreciate the pigments for the energy they reflect to our eyes. Plants contain several pigments, e. g., chlorophyll, each pigment having its own light absorbing qualities. The colors which we see on plants represent the wavelengths reflected by their pigments. Today the absorbed light is the more important to us, since this energy is used to produce glucose through photosynthesis. As time passes, energy absorbed by plant pigments flows through the entire food web, providing energy for nearly all forms of life.

A. Pigment Extraction from Grass Leaves: Procedure: (Steps 1 - 4 to be done by the biology technician)

1. Place a handful of lawn grass in a fireproof blender with enough acetone to allow the blender to do its work.

2. Filter the extract through two layers of facial tissue which have been placed in a glass funnel over a flask. Fold the tissues over the top and squeeze the extracted pigment from the grass.

3. Label the flask "Pigment Extract from Grass Leaves." 4. Place a small quantity of the pigment extract (1"- 2" deep) into a test tube.

(Step 5 to be done by students or the instructor) 5. Allow a bright white light source to shine from the closed end of the tube to the open end.

View the surface of the extract.

Question 4. Describe the color change when you shine the light on the pigment extract: You are viewing a phenomenon known as fluorescence, a process during which energy absorbed at one wavelength is emitted at another wavelength. Various wavelengths of light are being absorbed by the mixture of pigments. These energies are passed to chlorophyll a, in which electrons are excited by the influx of energy. The high energy electrons are then forced* to release their energy as lower energy light in the red range. *Since the cells have been disrupted and the acetone has dispersed all membranes, the normal electron acceptors of photosynthesis are not available. B. Paper Chromatography Paper chromatography is a technique for separating components of complex mixtures quickly and cheaply. The process in this case, includes three separation phases: the paper, the petroleum ether and the acetone. Separation occurs because of the differential affinities of the components of the pigment mixture for the three different phase substances. Those pigments favoring the solvent system will race ahead with the solvent as it moves up the paper, while those favoring the paper will lag behind. In addition smaller (lighter) pigments will move up faster than larger ones.

https://www.youtube.com/watch?v=BFJAT7BHLMw&list=PLr27cjn y01Ut_4Wl9qMXQCLQc45CgJw8k&index=3&t=0s

URL: https://www.youtube.com/watch?v=u6jD0hJO-28 Procedure: 1. Obtain a piece of chromatography paper. Pick it up by the straight cut end, not the angled cut

end or the center of the paper.

2. Place the chromatography paper on a clean space of the lab bench. Obtain a bright green spinach leaf, and place it on top of the chromatography paper near the angled cut, as shown in Figure 1. Then, roll the coin firmly in a straight line over the spinach, so that the coin presses the spinach leaf’s pigments into the paper.

3. Attach the stopper and paper clip to the straight end of the chromatography paper, as shown in

Figure 1, and place on your group’s lab bench.

4. Obtain test tube rack with two large test tubes for your group. Carry the rack from the bottom. You only need one of the tubes but leave the other in the rack. At the side lab bench, pour the 90% petroleum ether: 10 % acetone solvent mixture to fill the tube to an approximate depth of 2 cm. Be sure not to breathe in this solvent mixture, as it is not good for you. Walk back to your group’s lab bench with your test tubes and rack. Place your test tube rack in the place you want to leave it in for the remainder of this exercise.

5. Place the chromatography paper, paper clip, and stopper set-up inside the test tube, so that the solvent just touches and soaks the end of the chromatography paper, as shown in Figure 1. Adjust the paper clip so that the solvent does not soak the pigment line but continues to wick up the paper.

6. Place the test tube in as vertical position as

possible. Do not move the test tube rack or test tube during the duration of the exercise, or the banding patterns will not be distinct. Start your timer.

Figure 1. Paper Chromatography

Pigment Extract

Solvent

(Direction of Solvent Migration)

Figure 2. Paper Chromatography

7. When the fastest moving pigment approaches the top at around 15 – 20 minutes, remove the strip from the tube. Otherwise the pigments may crowd together at the top. Allow the paper to dry. Immediately, pour the solvent back into the original container, being careful not to breathe it in.

8. When developed, the chromatogram should show 4-5 fairly distinct bands. The bands

from top to bottom are: the orange-yellow carotene; the greenish-yellow bands are xanthophylls; and the blue-green band is chlorophyll a and the yellow-green band is chlorophyll b. As the strip dries and is exposed to light and oxygen, some of the pigments will fade.

9.

Question 5. In the space below, sketch your paper chromatogram using colored pencils. Then label the pigment bands with their respective pigments. Question 6. Based on how far it migrated, which pigment must have the greatest affinity for the chromatography solvent? Give the name of the pigment. Which must have the least affinity? Give the name of the pigment. C. Determining the Absorption Spectrum of the Leaf Pigments As light strikes a pigment, certain wavelengths will be reflected or transmitted, and some will be absorbed. Figure 3 shows a theoretical absorption spectrum.

Figure 3. Theoretical Absorption Spectrums of Two Plant Pigments

Question 7. Generally speaking, which wavelength ranges and their corresponding colors are most strongly absorbed by the pigments in Figure 3? (See your answer from Question 2.)

Pigment: Ranges: Colors:

Chlorophyll a

Chlorophyll b

Question 8. Which wavelength ranges and corresponding colors are least absorbed? (See your answer from Question 2.)

Pigment Ranges: Colors:

Chlorophyll a

Chlorophyll b

Introduction to Spectrophotometers In today’s lab you will be using the Spectronic 200, a type of spectrophotometer, to perform an analysis. Spectrophotometers measure light transmission and absorption. For your lab activity, you will concentrate on the light transmission measurements. The spectrophotometer works by using a light source that emits light of various wavelengths (see Figure 4). An adjustable filter removes all but a single wavelength of light (chosen by the experimenter). This wavelength of light passes through the sample tube, and an analog scale in the spectrophotometer measures the percent transmittance.

Figure 4. Basic function of the spectrophotometer measuring transmittance of light

Emits light of various wavelengths

Adjustable filter removes all but a single wavelength of light

Sample tube

Analog Scale measures

% Transmittance

The amount of light a substance transmits is called the % Transmittance or %T for short. A substance that transmits no visible light is opaque and has a 0% T. A substance that is completely transparent, transmits all visible light, has a 100% T. We will use acetone as a calibration or blanking solution; it is completely transparent and therefore will have a 100% T. You will use range of light wavelengths. Then we will view a sample of plant pigments that have been prepared as solution. Since they are pigments, they will absorb some of the light and transmit other wavelengths of light. The Parts of the Spectronic 200 Spectrophotometer

Figure 5. Outside View of Spectrophotometer

Figure 6. Sample Chamber

Wavelength adjustment knob. The Greek letter lambda (λ) is the symbol for wavelength

Fine wavelength adjustment and “enter” (¿) key

Data screen: shows status, wavelength and %T

Blanking button

Sample Chamber in closed position

Figure 7. Control Panel

Data Screen λ = current wavelength In this image, the wavelength is 674nm which is in the red region of the visible spectrum. %T value = 100% Visible Spectrum: Violet, Blue, Green, Yellow, Orange, Red

Sample holder for cylindrical cuvettes

Sample holder for square cuvettes

Figure 8. Key Pad

Watch time lapse video to obtain your data: https://www.youtube.com/watch?v=zInwzmnPApE&list=PLr27cjny01U t_4Wl9qMXQCLQc45CgJw8k&index=2&t=0s

Procedure: Your instructor has done the following steps prior to lab.

1. Basic calibration and Dark Zeroing of Spectrophotometer 2. Set the Spectrophotometer to read %Transmittance (%T) 3. Set the initial wavelength for today’s experiment 4. Prepared samples for today’s experiment

Use a Spectronic 200 spectrophotometer to determine the absorption spectrum of your grass leaf pigment extract.

Collecting %T data for today’s experiment

Coarse Wavelength Adjustment Knob. Use this knob to set wavelength close to required wavelength.

Fine Wavelength Adjustment buttons Use these knobs to set wavelength to the exact value required. Left Arrow button reduces wavelength in 1 nm increments. Right Arrow button increases wavelength in 1 nm increments.

Blanking button. Press to set Blank Zero to 100%T.

ENTER button for freezing and unfreezing display.

1. Have one person in your group handle the spectrophotometer, while the other one writes

down the data in Table 1.

2. Check the wavelength and adjust as needed (according to Table 1). (See Figures 5, 7 and 8.)

3. Open the Sample Chamber Lid. (See Figure 5.)

4. Remove a lens paper from its booklet. Do not touch the lense paper anywhere you will be using it to clean the cuvette.

5. Pick up the Blanking Cuvette by the top rim to avoid contaminating the tube with oils from your fingers. Clean the cuvette throughly with lens paper. Be careful not to touch anywhere on the cuvette. a. The Blanking Cuvette contains 100% acetone b. The tube is sealed with a cork and waxy parafin film. Do not remove either.

6. Insert Blanking Cuvette in the cylindrical sample holder. (See Figure 6.)

7. Close Sample Chamber Lid.

8. Press the Blanking Button. When %T reads 100%, immediately press

ENTER (¿) to freeze the data screen. Check the display to see if it actually froze the data, as the Enter button is finicky. (See Figure 8.)

9. Open Sample Chamber Lid (See Figure 5), and remove Blanking Cuvette (remember to handle by the top rim to avoid contaminating the tube with oils from your fingers).

10. Pick up the Sample Cuvette by the top rim (not the stopper) and clean with lens paper, especially from below the white line to the bottom of the cuvette, as this is where the light will be passing through.

11. Insert Sample Cuvette in the cylindrical sample holder. Do not remove the rubber stopper (this will prevent spilling caustic fluids in the spectrophotometer chamber).

12. Close Sample Chamber Lid.

13. Press ENTER (¿) to unfreeze. Check the display to see if it actually froze the data, as the Enter button is finicky.

14. As soon as the %T value changes from 100% press ENTER (¿) to freeze the data screen. This should take no more than 1-3 seconds.

15. Record %T in your Table 1.

16. Repeat steps 1-15 for each wavelength in your lab’s procedures.

Table 1. Percent TRANSMITTANCE at Wavelengths 400 nm - 700 nm

400 425 450 475 500 525 550 575 600 625 650 675 700

17. Subtract the above values from 100%, since % T + % A = 100%. Write the results in the

Table 2 blanks below:

Table 2. Percent ABSORPTION at Wavelengths 400 nm - 700 nm

400 425 450 475 500 525 550 575 600 625 650 675 700

18. Using the data from Table 2, plot your team’s leaf pigment absorption spectrum curve on

Figure 10.

80%

60%

40%

20%

0%

400 500 600 700

Wavelength (nm)

Figure 10. Observed Absorption Spectrum for Grass Leaf Pigments Question 9. On the graph above, at what two wavelength ranges do you see the "peaks" of light absorption? Label colors of light that are found in these two ranges, based on your answer from Question 2.

Question 10. a. In what general range of wavelengths is absorption low?

b. What colors of light are found in this range? Question 11. a. What happens to the light that is not absorbed in a solution? How does this relate to the fact

that leaves appear green? b. What happens to the light that is not absorbed in the solutions we used in the

spectrophotometers? III. Leaf Anatomy: Model of a Leaf Cross-Section Procedure: 1. Locate a laboratory model of a leaf cross-section. You may also wish to consult a diagram in

your textbook. 2. Study the leaf diagram on the next page (Figure 5) and be able to identify the following:

stomata (plural), guard cells, mesophyll, palisade mesophyll (cells), epidermis, and vascular tissues.

Question 12. Although a plant’s leaf is its primary organ for photosynthesis, not all the cells in a leaf are photosynthetic.

a. Observe the epidermal peel diagram on the bottom left side of Figure 5, as well as the model. Do all of the cells in the epidermal peel have chloroplasts? ____________ (Hint: Look for the cells that have chloroplasts in them! They appear as small, dark dots or ovals inside the cells drawn in Figure 5.)

b. What is the name of the cells in the surface of the leaf that have chloroplasts? ___________________________________

c. Now observe the diagram of the leaf cross section on the bottom right of Figure 5, as well as the model. Which cells have chloroplasts in this part of the leaf? ____________________________________

Question 13. Observe the openings in the leaf’s epidermis in Figure 5 and on the model. What are they called? __________________________ If plants take in gaseous carbon dioxide (CO2) from the air and release oxygen (O2), how do the gases enter and leave the leaf? Question 14. Observe Figure 5 on the bottom right. Is the cellular material inside the leaf arranged so that the leaf is a solid mass, or are there spaces left inside? Question 15. If we could somehow remove the gases from a leaf without killing all the cells and then expose that leaf to sunlight, water and CO2 for a given amount of time, what gas would be produced that would refill the spaces within the leaf? ________________ Question 16. Write the summary equation for photosynthesis: Question 17. How does your answer to Question 15 relate to your answer in Question 16?

Figure 5. Diagram showing epidermal peel (left) and cross section (right) through a leaf.

IV. Photosynthesis and Light Intensity Many questions may come to your mind when you first study photosynthesis. Perhaps you have never really thought about plants respiring and using oxygen. Just how much do they use in a given period of time? Is it about the same amount as they produce during photosynthesis? Is the photosynthetic rate the same in a plant on a cloudy day as it is on a sunny day? The questions are endless. Doubtless there are many that you may be pondering right now. Some of these questions may require considerable time and sophisticated pieces of equipment to answer, but many may be studied in our lab, with minimum equipment and a little thought. The question that you are trying to answer today is: “Does the intensity of the light striking a plant affect the rate at which plants produce oxygen?? Let’s identify the components of the hypothesis. Question 18. What is the independent variable? (The one you will let vary in the different “treatment levels”)? Question 19. Name a dependent variable (or variables) (the variable(s) that you will measure to see if the Independent variable has an effect). Question 20. Now that you have the independent and dependent variables, create a hypothesis. Question 21. List the most important “controlled variables” (the ones that might wrongly affect your results if they are not “controlled.” These are also called confounding variables in the Understanding Experimental Design simulation).

The Effect of Light Intensity on Photosynthesis: Experiment It is quite likely that the experiment you suggested would be suitable to test your hypothesis, but in order to give some uniformity to today's laboratory experiment, it will be set up for you. Leaf material, placed in a buffered water solution, will be subjected to a vacuum, removing gases from within the spongy mesophyll layer of the leaf. As the gas is drawn out of the spongy mesophyll layer, the leaf will become denser than the water, and it will sink. Light of a given intensity will then be shown upon the leaf and, due to the gas generated by photosynthesis, the leaf will regain its buoyancy and rise to the surface. The time taken by the leaf to come to the surface will be used as a rough measure of the photosynthetic rate. The rate will be compared for different light intensities. Procedure:

https://www.youtube.com/watch?v=- VimyQbMaEo&list=PLr27cjny01Ut_4Wl9qMXQCLQc4 5CgJw8k&index=4&t=0s 1. Preparation of Leaf Discs:

a. Your experimental material will be the leaves from freshly a cut plant which only moments before had been outside, photosynthesizing.

b. Obtain a 50 ml beaker and pour in a small amount of buffer solution. c. Using a #5 cork borer (punch) and cutting board, cut six or seven leaf discs of the same

size. In cutting your discs avoid the major veins and make them as evenly-sized as possible. As you cut the discs, place them into the buffer solution so that they do not dry out. The buffer solution resists changes in the acidity or the alkalinity of the water, thereby ensuring that the pH variable will not interfere with the experiment.

d. Your instructor will prepare a large vacuum flask containing buffer solution. Drop your

discs into the flask, along with those of the rest of the class. Be sure the discs are down in the buffer solution.

Question 22. Observe the leaf discs before vacuuming them: Are they now floating, or are they on the bottom of the flask?

2. Equipment Set-up

a. Obtain a ring stand with a flood lamp with a ring attached and a timer. b. Place a stacking dish half full of cold water on the ring. This will serve as a heat trap

(see Figure 7). The position of the heat trap should be one inch above the beaker.

c. Measure the distance from the top surface of the ring stand to the painted edge of the light bulb where the light shines from. One group at your table should use the 15 cm distance, and the other should use the 60 cm distance. Ask your instructor if he/she has assigned you one of these distances.

Figure 7. Photosynthesis Apparatus

15 or 60 cm

stacking dish + water

beaker + discs

white card

ring stand

White surface of base

3. Data Collection Use this video to collect you data.

https://www.youtube.com/watch?v=YQJuT9- bOmM&list=PLr27cjny01Ut_4Wl9qMXQCLQc45CgJw8 k&index=6&t=0s

a. Using forceps or a dissecting needle (not your finger), move the leaf discs around on the bottom of the beaker so that they are not resting on one another.

b. Add three drops of saturated sodium bicarbonate (NaHCO3 = baking soda) solution to

the beaker (not the stacking dish). This dissociates in water to form CO2.

a. Immediately place the beaker on ring stand base, move it into position and begin timing.

b. As gas is produced during photosynthesis, it will fill the spaces in the mesophyll and buoy up the discs in the solution. You will measure the apparent rate of photosynthesis by determining the number of seconds that it takes each leaf disc to rise to the surface. (Note: A disc does not need to be flat on the surface, as long as its edge reaches the surface.)

e. Obtain times (in seconds) for the first 3 leaf discs to rise. Enter the data in Table 3

below. Other teams will enter their data to be shared by the class.

Table 3. Leaf Disc Data

Distance Team Time (sec)

Team All Teams

Number Disc 1 Disc 2 Disc 3 Averages Average 15 cm 1 X

2 X X X X X 3 X X X X X 4 X X X X X

60 cm 1 X 2 X X X X X 3 X X X X X 4 X X X X X

C. Photosynthesis Data Analysis Question 23. According to your team's data, would you accept or reject your hypothesis regarding the effect of light intensity on photosynthetic rate? (Question 20) Describe exactly how your data lead to the above conclusion. Question 24. You also have data from all teams in your class. According to the class average from Table 3, would you accept or reject your hypothesis? Describe exactly how the data lead to the above conclusion. Question 25. If the class average data causes you to reject your hypothesis, present a new one in the space below.

Question 26. Perhaps your data were inconclusive or even contrary to the class average. List some sources of experimental error which could cause a team's data to not fit the class average pattern: a. b. c. d. Question 27. Those who designed this laboratory exercise have tried to use certain procedures in the performance of the experiment to minimize experimental error or "control the variables." Identify some of those procedures: a. b. c. d. Question 28. Why have we used uniform discs and not whole leaves? Question 29. Examine the formula for sodium bicarbonate. What ingredient, essential for photosynthesis, does it provide to the leaf discs?