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Spectral Curve Of Leaf Pigments
Materials:
Visible spectrophotometer Distilled water in squeeze bottle
Autumn Leaves Tissues or KimWipes
Cuvettes Excel graphing program
Cuvette rack Graduated cylinder
Rubbing alcohol (91% Isopropyl alcohol) Beakers
Plastic wrap and rubber bands (to reduce Spectro-Pro Software (optional)
Alcohol evaporation from beakers) Computer (optional)
Introduction:
Plants produce their food for survival from water, carbon dioxide, and nutrients in the soil and sunlight.
Sunlight provides the energy necessary for the plants to make their food out of the available ingredients.
To change this form of energy (light energy) into chemical energy (which is easier for the plant to use),
the leaves of the plant contain a chemical called chlorophyll. In addition to transforming the energy
from sunlight into chemical energy, chlorophyll is responsible for making the leaves green.
Leaves are green because the chlorophyll absorbs all colors of the visible sunlight except for the green
colored light. This light is reflected off of the leaves and into our eyes; therefore, we see leaves as green.
However, in the fall, some leaves that are green become different colors: red, yellow, orange, and brown.
These colors are due to a chemical called carotenoid.
The question investigated by this experiment is: What causes a green leaf to turn a different color? We
can predict what is happening, then perform our experiment and see what information the experiment
gives us. The experiment’s results may not give us enough information for us to be sure that our
explanations are correct, and thus we may need to think of other experiments to perform.
Let’s list our predictions as to what causes a green leaf to change color:
1. The chlorophyll is altered (somehow) so that it now reflects a new color.
2. The chlorophyll disappears (somehow) so that other chemicals in the leaf reflect new color.
Now it’s your turn to predict:
1.
2.
Lab #535, Spectral Curve of Leaf Pigments (3/20/03), By Advancing Science, Gettysburg College
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In this experiment, you will extract the leaf colors (if not already done) and determine the
spectral curve of these colors. The purpose of creating a spectral curve is to visually and numerically see
at which wavelengths colors reflect and absorb light. You will create spectral curves for one of four
colors: green, red-green, yellow, or orange leaf extracts. The procedure is simple. You will record the
absorbance values of the colored extract at a number of different wavelengths. Then, plot these values on
a graph, or graph your data using the software program such as Excel (see Excel instructions). It is also
possible to collect data using the Spectro Pro program, which will automatically graph for you.
Inside the spectrophotometer [See Figure 1] there is a light bulb, which produces white light.
The white light is separated into the different colors of light by either a prism or diffraction grating (An
example of a diffraction grating would be the surface of a CD ROM. The reflective surface has tiny
grooves etched into it, which separate white light in a manner similar to light passing through a prism).
After the light is separated, it passes through a filter (to block out unwanted light, such as light of a
different color) and a slit (to narrow the beam of light - making it in the shape of a rectangle). Next the
beam of light passes through the sample that is in the sample holder. The amount of light that passes
through the sample is measured and the spectrophotometer displays this quantity in one of two ways:
(1) Absorbance -- a number between 0 and 2. This is a measure of how much light is absorbed
by the solution, in other words, now much does not pass through.
(2) Transmittance -- a number between 0 % and 100 %. This is a measure of how much light
passes through the solution (this is transmitted light).
Figure 1
Lab #535, Spectral Curve of Leaf Pigments (3/20/03), By Advancing Science, Gettysburg College
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Procedure :
(Steps 1-4 can be performed from 1 to 30 days in advance. Some strategic planning can allow for many
species of leaves to be collected at peak color intensity. Leave can be frozen in a zip lock bag until
needed.)
1. Collect your leaves and separate them according to color. For best results, pick one tree that has a
variety of colored leaves. Sugar maple and sweet gum trees have green, red, yellow, and orange
leaves. (For best chlorophyll peaks, collect green leaves prior to mid-September.)
2. Get the same number and size of leaves for each color and tear them into small pieces, still keeping
the colors separate.
3. Place the separated leaf pieces in small plastic beakers (100 mL). Fill the beakers approximately
one-half full with rubbing alcohol. Cover the beakers with plastic wrap and secure with a rubber
band. Let stand for 24 – 48 hours for optimum color. Be sure to label the beaker with leaf type and
color. Pour the leaf extract into a labeled, sealed container. Discard the leaves.
4. Once the solutions become colored, you can perform the rest of the experiment. Solutions can be
kept in a sealed bottle in the refrigerator for many weeks to allow for analysis even after peak autumn
color is past.
5. To turn on the Spectronic 20D spectrophotometer, simply plug the instrument in and turn the left
knob on the front (0% T) of the instrument clockwise (it will click). Allow the spectrophotometer to
warm up for 20 minutes. This step may have been done ahead of time by your teacher. The Spec 20
automatically comes on in the Transmittance mode, but if it is not in this mode, simply push the
Mode button one time to change it. The mode is indicated with a red light on readout panel. Rotate
the on/off knob to adjust the transmittance to 0 % transmittance. This MUST be done with the
sample compartment empty!! {NOTE: After this is done, do NOT turn this knob for the rest of
the lab activity!!!!}
6. In the meantime, you can prepare your sample. In a cylindrical cuvette, put about 5 mL of rubbing
alcohol. This will serve as the solvent blank for this experiment. Note: the amount is not really
important; just be sure the cylindrical cuvettes are about half full.
7. Fill a different cuvette half full with the provided extract from an autumn leaf. Add 20 drops of leaf
extract, then dilute to half-full with the isopropyl alcohol. Be sure to mix thoroughly. Absorbance
values at 350 nm should be between 1.000 and 1.500, if not, the concentration of the extract should
be adjusted. If you do not get an absorbance reading greater than 0.5, you should make a more
concentrated solution. Do this by adding additional drops of leaf extract. If the solution is too
concentrated (> 1.5) it must be diluted.
8. Turn the Wavelength Control on the right-hand (top) side of the machine to set the proper
wavelength. We will start at 350nm, so be sure the Wavelength on the digital readout, reads 350
nm. Flip the Filter Lever to the left position (located at the bottom, left, front of the instrument.)
Lab #535, Spectral Curve of Leaf Pigments (3/20/03), By Advancing Science, Gettysburg College
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9. Zeroing the Spectrophotometer: Pick up the cuvette with the alcohol blank and wipe the bottom
half of the cuvette with a tissue or KimWipe. Place the cuvette into the sample holder of the
spectrophotometer. Align the white line on the cuvette (if there is one) with the line on the sample
holder lip. Close the cuvette door. Using the right knob (100%T/0A) on the front of the instrument,
rotate until the instrument reads 100.0 % transmittance. This means that you are telling the
spectrophotometer that ALL of the light is passing through the blank. Press the MODE button one
time; this will change the mode to absorbance. As one might expect, since all of the light is
transmitted, the absorbance reads 0.000 (The instrument must be re-zeroed every time the
wavelength is changed.) Open the cuvette door and remove the cuvette. [The purpose of zeroing
the spectrophotometer is to tell the instrument what "colorless" is. The only time we want to have an
absorbance reading (i.e. a number greater than 0.000) is when there is a solution of leaf pigment in
the instrument, not when there is colorless rubbing alcohol].
10. Taking an Absorbance Reading: Pick up the sample cuvette, wipe it off, place it into the sample
holder, close the cuvette door and read the digital display.
11. When you have a sample that is giving good readings, wait until the absorbance value stabilizes, then
record the absorbance using the table below. Repeat this procedure for each color of leaf extract that
you are using.
12. When you are finished recording data at 350 nm, remove the leaf extract cuvette from the sample
compartment and close the cuvette door. You will be recording absorbance values every 25 nm, so
the next setting is going to be 375 nm. Turn the wavelength knob on the top of the instrument until
the wavelength window reads 375. Now open the cuvette door and put the “blank” cuvette back in
the cuvette hole; turn the 100%T/0A knob again to zero the instrument at this wavelength. [Note: It
is not necessary to change the mode back to Transmittance.] Record the leaf extract absorbance value
for each sample and move on to the next setting, 400 nm. Repeat the steps every 25 nm up to 700
nm. When 600 nm is reached, remember to flip the Filter Lever from left to right position.
Between 650 and 700 nm, increase the wavelength in increments of 5 nm. Remember to insert the
Blank and zero the spectrophotometer every time the wavelength is changed!
13. Plot your data on a graph. Complete a scatter graph if you are using a graphing program to plot your
data. Choose the option that connects the dots to draw a curve. Wavelength is plotted on the x-axis
and Absorbance is plotted on the y-axis. Remember to put units on your axes and to give your graph
a title that tells the purpose of the graph.
Lab #535, Spectral Curve of Leaf Pigments (3/20/03), By Advancing Science, Gettysburg College
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Table 1 – Absorbance Readings
Species of Leaves
Red-Green Yellow Orange
Filter Setting Wavelength (nm) Green Leaf Leaf Leaf Leaf
Left 350
Left 375
Left 400
Left 425
Left 450
Left 475
Left 500
Left 525
Left 550
Left 575
Right 600
Right 625
Right 650
Right 655
Right 660
Right 665
Right 670
Right 675
Right 700
Lab #535, Spectral Curve of Leaf Pigments (3/20/03), By Advancing Science, Gettysburg College
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QUESTIONS
Name _____________________________
1. Compare the spectral curves of each leaf color to one another. What do you observe? Please
identify similarities and differences.
2. How many peaks are there with each leaf color? At what wavelength are those peaks at maximum?
What do these peaks indicate or identify?
3. What do the spectral curves suggest as to what causes the leaves to change color? Please explain.
4. Based on this experiment and the information in the beginning, can you predict why leaves fall off of
trees once they are no longer green?
Lab #535, Spectral Curve of Leaf Pigments (3/20/03), By Advancing Science, Gettysburg College
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Spectral Curve of Leaf Pigments - Teacher's Notes
Due to time constraints it may not be possible for each group to do all four colors. In this case, groups should share
data. Data collection proceeds more quickly if all solutions are analyzed before proceeding to the next wavelength.
Students may get an occasional absorbance reading that is “slightly” negative. They should record the actual value,
but replace the negative value with a “0” when graphing. Theoretically, a negative reading means that the sample
absorbed less light than the blank. This is usually due to using two different cuvettes; they are not shaped exactly
the same where the light travels through the cuvette. When you blank the spectrophotometer, you not only tell the
instrument that the alcohol is not to absorb any light, but the cuvette is not to either. Imperfections in the cuvette as
well as electronic noise can cause slightly negative readings. Larger negative readings (> -0.010) are usually
due to student error, such as not zeroing or changing the 0%T knob.
Additional Background Information (See also The Chemistry of Changing Colors, Changing Color, and Why
Leaves Change Color, attached.)
Leaves change color in the fall as the amount of daylight decreases. When the leaf receives less light, it produces
less chlorophyll. As the amount of chlorophyll produced decreases, the color of the carotenoids can show through.
The carotenoids are always present in the leaf but are not seen until the amount of chlorophyll decreases. (This can
be verified by separation of the extract by thin layer chromatography.) Thus a green leaf spectrum will look like a
colored leaf spectrum with additional absorbance for chlorophyll.
Once the leaf stops producing chlorophyll, the leaf no longer has a way to change solar energy into chemical
energy and therefore cannot make food. The leaf subsequently dies and falls off the tree. The best time to collect
leaves is mid-August to mid-September. At this time green leaves still contain chlorophyll while the colored leaves
do not.
Extension
You can perform the following experiment anytime of the year and with less planning than the autumn leaves
experiment requires. However, you need to refer to that experimental procedure before reading further.
Two vegetables are required: spinach and carrots. You can extract the pigments from these vegetables with
rubbing alcohol. From the carrots, you will get an orange solution, which will contain beta-carotene. From the
spinach, you will get a dark green solution, which will contain a mixture of beta-carotene as well as that of
chlorophyll. (If you are interested, the spinach solution can be physically separated into orange beta-carotene and
green chlorophylls using silica in thin-layer chromatography separation.)
For additional fun, have the students bring in other vegetables from which they would extract pigments, such as
beets or purple cabbage.
As for the spinach versus carrots experiment, you can ask the students why the carrots only contain beta-carotene
and not chlorophyll. Guide them into discovering that the leaves of spinach are exposed to the sun and therefore
they contain chlorophyll so that the plant can make food/energy for survival. The carrot is a root of the plant and
therefore it is not exposed to the sun and thus does not need chlorophyll to make food. Carrots do have leaves,
which contain chlorophyll, and thus the food production occurs here. Pigment can be removed from carrot leaves
for analysis, which also produces nice results. (What about beets and purple cabbage)?
Spectral Curve of Leaf Pigments, written by Jack W. Sipe, Jr. Tracy Schoolcraft, Paula Rupert and Gregory J. Anderson for the Advancing Science Program at
Gettysburg College, Gettysburg PA 17325 http://www.advancingscience.org
Lab #535, Spectral Curve of Leaf Pigments (3/20/03), By Advancing Science, Gettysburg College
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Equipment Suppliers:
Spectronic – Spec 20D+ Flinn Scientific $995.00
Test Tube racks Fisher Scientific $13.00
Spectro – Pro Software Vernier $50.00
Spectro – Pro Cable Vernier $25.00
Dell Notebook Computer Dell $1800.00
Isopropyl Rubbing Alcohol 91% Giant $2.00/liter
Lab #535, Spectral Curve of Leaf Pigments (3/20/03), By Advancing Science, Gettysburg College
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