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ABSORBANCE OF CHLOROPHYLL
PRE-LAB
Answer the pre-lab questions on the Student Data Sheet.
INTRODUCTION
Light from the sun is used as fuel for photosynthesis. The light is absorbed by special molecules
in plants called pigments. Using a spectrophotometer, specific wavelengths (colors) of light will
pass through a solution containing pigments extracted from a plant.
SAFETY
1. Keep the pigment solution away from your mouth and eyes.
2. Do not consume any of the solution.
3. The solution will stain your clothing if spilled.
4. Keep the electrical cords away from water sources.
5. Do not spill any of the solution in the spectrophotometer.
MATERIALS
spectrophotometer
cuvettes
distilled water
pigment solution
spinach
funnels
test tubes w/stoppers
colored pencils
white paper strips
test tube holders
mortar and pestle
acetone
filter paper
black light
PROCEDURE
Extracting Chlorophyll
1. Using the mortar and pestle crush your plant material.
2. Add 5-10 ml of acetone to the crushed material and crush for an additional minute.
3. Filter and cap the solution.
4. Set aside for later use.
Using the Spectrophotometer to record color
1. Turn the power on.
2. Place a small piece of white paper in the cuvette holder.
3. Using the side arrows set the bottom indicator to %T (Transmittance).
4. Press the Calibration button on the bottom (CAL).
5. Adjust the wavelength to the values indicated on your data sheet using the top and bottom
arrows.
6. Record on the data sheet each observation of color reflected onto the paper for each
wavelength indicated.
Using the Spectrophotometer to record absorbance
1. Pipette approximately 2 ml of chlorophyll solution into the cuvette.
2. Fill the rest of the cuvette with distilled water.
3. Using your pipette, gently mix the water and chlorophyll solution.
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ASIM Biology: Botany
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4. Place a cuvette of distilled water in the cuvette holder (clear sides towards the light
source). Close the lid.
5. With the setting still on %T, calibrate the distilled water to the wavelength indicated on
the data table.
6. Remove the distilled water and place the cuvette with the chlorophyll solution in the
cuvette holder (clear sides towards the light source). Close the lid.
7. Using the side arrows change the bottom setting to absorbance.
8. Record the absorbance on the data table.
7. Repeat steps 7-11 for each wavelength given on the data table. You must calibrate
using distilled water every time the wavelength is changed.
8. Graph your results.
After you have completed the student data sheet, hold your original sample of chlorophyll up to
the UV light and observe.
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STUDENT DATA SHEET
Absorbance of Chlorophyll
NAME________________________
DATE________________________
PRE-LAB QUESTIONS
1. What color is the chlorophyll solution?
2. Why is it this color?
Observe the chlorophyll solution under the UV light.
3.
Is it still chlorophyll?
4. What is different? Why?
DATA
WAVELENGTH
COLOR
ABSORBANCE
420
_______________
___________
440
_______________
___________
460
_______________
___________
480
_______________
___________
500
_______________
___________
520
_______________
___________
540
_______________
___________
560
_______________
___________
580
_______________
___________
600
_______________
___________
620
_______________
___________
640
_______________
___________
660
_______________
___________
680
_______________
___________
700
_______________
___________
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400
440
480
520
560
(nm)
600
640
Absorbance vs. Wavelength
680
720
740
.
Absorbance
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ANALYSIS QUESTIONS
1. For which wavelengths is absorbance the greatest?
2. What colors are associated with these wavelengths?
3. What wavelength is not absorbed by the pigment solution?
4. What color is associated with this wavelength?
5. Predict which wavelengths would have a higher rate of photosynthesis. Explain your
prediction.
6. The chlorophyll pigment converts light energy to food energy which is used by the plant for
growth and reproduction. Based on your data which wavelengths of light will be the most
useful to the plant for this purpose?
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Figure 5.5
Visible spectrum
Figure 5.6
Action spectra and absorption spectra of photosynthesis
Absorption spectra of photosynthetic pigments
Rate of Photosynthesis
Action spectrum of
photosynthesis
400
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500
600
Wavelength (nm)
700
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