Spectral Curve of Leaf Pigments

Spectral Curve Of Leaf Pigments

Materials:

Visible spectrophotometer

Autumn Leaves

Cuvettes

Distilled water in squeeze bottle

Tissues or KimWipes

Excel graphing program

Cuvette rack

Rubbing alcohol (91% Isopropyl alcohol)

Plastic wrap and rubber bands (to reduce

Alcohol evaporation from beakers)

Graduated cylinder

Beakers

Spectro-Pro Software (optional)

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.

1.

2.

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:

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


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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.)


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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.


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Table 1 – Absorbance Readings

Species of Leaves



Filter Setting Wavelength (nm) Green Leaf

Red-Green

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

Yellow

Leaf

Orange

Leaf


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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?


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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


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Spectral Curve of Leaf Pigments

Spectral Curve Of Leaf Pigments

QUESTIONS