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Photosynthesis
Photosynthesis is the process of converting light energy into chemical energy that can be used by plants, animals and other organisms (yes there are other organisms!). Photosynthesis is important not just because of its role in energy transfer, but also because of the chemical products, oxygen for respiration, and glucose, the ultimate source of carbon for all the organic molecules that make up all organisms. Photosynthesis can be summarized in the following reaction:
In this laboratory, we will be examining the pigments involved in photosynthesis as well as the actual substrates and products of the reactions involved.
Exercise 1: Carbon dioxide consumption during photosynthesis
When carbon dioxide (CO2) is dissolved in water it forms carbonic acid in the following reaction:
You may recall from the pH laboratory that carbonated drinks are quite acidic, dissolved CO2 in the form of carbonic acid is the primary reason for this. In this exercise, you will put CO2 into a solution containing an indicator that changes color when the pH goes down. If CO2 is removed from the solution by the action of plants doing photosynthesis, the pH will go back up and this should be observable in the form of a color change in the indicator solution.
1.Obtain three 10 ml test tubes and number them 1 - 3 then add 9 ml of distilled water to tube 1 and 8 ml of distilled water to tubes 2 and 3.
2.Add 1 ml of Brom Thymol Blue indicator solution to tubes 2 and 3.
3.Blow though a straw into all three tubes so that carbon dioxide in your breath is absorbed in the water. If the solutions in tubes 2 and 3 are not already yellow, they should turn yellow at this point.
4.Place a sprig of Elodea into each tube endeavoring to place approximately the same amount of plant tissue into each tube.
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5.Cork each tube and wrap tube 3 in aluminum foil.
6.Place all three tubes under a bright light and record the time. When the indicator changes color in tube 2, record the time again so that you can note the total time in your lab report. Note: This may take some time and will vary depending on the amount of light and the quantity of plant material in your tubes.
7.Record the color of all three tubes then try bubbling your breath through tube 2 again just to ensure that the indicator is working properly.
Be sure to explain in your lab report which tubes were controls and what type of control each control tube was.
Exercise 2: Oxygen evolution during photosynthesis
1.Obtain 2 10 ml graduated cylinders and 2 sprigs of approximately the same amount of elodea. The elodea should be approximately 7 cm long.
2.Mark each graduated cylinder so that you can easily identify it, then wrap one tube in aluminum foil. Put one sprig of elodea into each cylinder with the top of the plant facing the bottom of the cylinder. The elodea should reach approximately to the 2 ml mark on the graduated cylinder.
3.Take both cylinders to the aquarium and fill them with water, then invert them so that the bottom of each cylinder is resting on the bottom of the tank and each tube is filled with water. Measure the distance from the light source to the cylinder, not the tank. Any oxygen released by the elodea should be trapped in the top of the tube.
4.Record the time and check to see how much oxygen has been released by the elodea in 1 hours time. If the amount is so small that it can not be easily read, you may need to use a longer period of time.
Exercise 3: Sugar production
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This exercise takes advantage of the fact that different molecules have solubilities that vary depending on the solvent being used. As a rule, polar molecules or ions, like sugar or salt (NaCl), tend to dissolve in polar solvents, like water. Nonpolar molecules, like safranin, will dissolve in nonpolar solvents, like cooking oil. Some red and blue pigments produced by plants, particularly anthocyanins, dissolve readily in water, while others like chlorophylls (green), carotenes (yellow and orange), and xanthophylls (yellow), dissolve in ethanol, a less polar solvent than water.
CAUTION: Please be careful not to burn yourself or your colleagues. If you are burned, run the burn under cold water IMMEDIATELY and inform you laboratory instructor.
1.Start a boiling water bath by placing 50 ml of distilled water in a 100 ml beaker and placing the beaker on a hot plate turned to high.
2.Obtain a variegated coleus leaf taking care to pick a leaf showing all four colors: White, green, red, and purple and carefully record the location of each color on the leaf by doing a detailed drawing (remember the rules for doing illustrations).
3.Place the leaf into the boiling water bath for 5 min. and record the pattern of colors left on the leaf by removing it and spreading it out in a petri dish containing a small amount of distilled water. Save the water, you will need it for the next exercise.
CAUTION: Ethanol is highly flammable and has a low flashpoint extreme care should be exercised when handling it. If it does catch on fire do not try to put it out with water, cover the container with something that is not flammable and call your laboratory instructor.
CAUTION: Ethanol is a highly toxic chemical, but it is the alcohol found in may beverages. The DENATURED ethanol you are using is not the same thing, it has aviation fuel added to it. Drinking it will make you violently ill and the laughing stock of your friends.
4.Place 50 ml of denatured 95 % ethanol in a 100 ml beaker and very carefully bring it to a boil. As the boiling point of water (100 oC) is higher than the boiling point of ethanol (65 oC), the ethanol should begin to boil soon after being placed on the hot plate. As soon as it begins to boil, turn down the heat.
5.Place the leaf into the boiling ethanol for 5 min. At this point, all the pigments should be removed from the leaf.
6.Spread the now white leaf out in a petri dish again, but this time use Lugol's solution to aid your spreading. The leaf should be covered with Lugol's solution.
7.Record the pattern that develops on the leaf. Remembering what Lugol's solution is used to test for, what causes this? What does this indicate about the function of the pigments that you extracted?
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Exercise 4: Absorption spectrum of plant pigments
Pigments are chemicals that absorb some wavelengths of light while reflecting others. The reflected wavelengths are the color that we see. For example, a red pigment absorbs all wavelengths except those in the 700 nm range so they appear red when we look at them. If absorbance is measured for a pigment, the lowest absorbance should be at the wavelengths of the color the pigment appears. In this exercise you will be using a spectrophotometer to measure absorbance of the extracts you made in Exercise 3.
1.Turn on the Spec 20 by turning the knob on the left clockwise. It should go click and a red light on the top of the machine should come on. The machine takes about 20 min. to warm up.
2.You will need 4 cuvettes for this exercise. While cuvettes resemble test tubes, they are significantly different, they are made of special glass that transmits light over a wide range of wavelengths and are very uniform in thickness and diameter. They must be very clean before use so make sure they are clean.
3.Number the cuvettes 1 - 4. Into 1 put 5 ml of distilled water. Into 2 put 5 ml of the water extract from Exercise 3. Into 3 put 5 ml of 95 % denatured ethanol. Into 4 put 5 ml of the ethanol extract from Exercise 3.
4.Calibration of the Spec 20 is essential if you are to get meaningful results. Cuvettes 1 and 3 are your blanks for calibration. To calibrate do the following:
a.Turn the knob on top of the Spec 20 to the wavelength you wish to measure absorbance at (You will be starting with 400 nm so turn to 400).
b.If the number on the wavelength dial is black, check to ensure that the red filter is not in place (ask you lab instructor to help you with this). If the number is red, make sure that the red filter is in place.
c.Make sure that the top to the cuvette holder is closed and adjust the knob on the lower left of the machine until the needle on the absorbance meter reads 0 % transmittance/ absorbance.
d.Insert your blank into the cuvette holder, close the top and set the absorbance meter to 100 % transmittance/0 absorbance using the knob on the lower right of the Spec 20.
e.Measure the absorbance at that wavelength of the solution you wish to measure by removing the blank, inserting the cuvette with the pigment solution into the cuvette holder and closing the top.
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5.Measure the absorbance of both extracts obtained in Exercise 3 starting at 400 nm and going up to 750 nm in 25 nm increments. Make sure that you calibrate with the blank containing the same solvent as the extract for each extract or your results will be meaningless.
6.Graph your results for your lab report, and compare them with the absorption spectrum for chlorophyll given in your text book.
Exercise 5: For A students only
Design an experiment of your own to investigate photosynthesis. Here are a few suggestions:
1.Repeat Exercise 3 on leaves that are all red, or are showing fall colors.
2.Try doing Exercise 2, but wrap the experimental tube with colored cellophane so that only one wavelength of light falls on the elodea. You may want to get together with several other people so that you can compare the results using different colors.
3.Try adding sodium bicarbonate (NaHCO3) to the water in Exercise 2. NaHCO3 acts as a carbon dioxide source for the elodea. Should this affect the rate of O2 release?
4.Separate the pigments obtained in Exercise 3 using paper chromatography. This may help to explain why the results obtained in Exercise 4 were not identical to those given in your text book.
Materials
Equipment
Colored cellophane sheets
Cuvettes
Fish tank
Graduated cylinders, 10 ml 2/student
Light source
Petri dishes
Spec 20
Chemicals
Brom thymol blue indicator solution, To make 100 ml, 0.04 g brom
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thymol blue in 50 ml 95 % ethanol and 50 ml distilled water.
Distilled water
Ethanol, denatured 95 %
Lugol's solution (Gramm's iodine, IKI)
Sodium bicarbonate NaHCO3
Supplies
Elodea
Straws
Variegated coleus plant