Name ________________________
Other group members:
Bio1400
Biological Inquiry
Plants contain many different molecules directly or indirectly involved with photosynthesis, which may also impart color to the plant. The mixture of chlorophyll molecules found in spinach, for example, absorbs several wavelengths of visible light, with distinct absorbance peaks in the blue range (400 –500 nm) and in the yellow-red range (600–700 nm). The combination of visible light that is not absorbed appears green to the human eye. Chlorophyll contains a porphyrin ring in its structure with a magnesium ion in the center. The porphyrin ring accounts for much of the molecule’s light absorbance. Chlorophyll is found in the thylakoid plate of a plant chloroplast.
Carotenoids, accessory pigments produced in chromoplasts, are associated with many colors observed in vegetation. There are hundreds of different types of carotenoids. Carrots get their color, which is often orange but is not restricted to orange, from carotene. Carotene is a family name for several compounds that also go by the name terpene.
Another type of carotenoid phyto-pigment is called anthocyanin. The purplish color of a red cabbage and the rusty red of the flesh of a blood orange are a result of the presence of anthocyanins, which also have the property of changing color with changes in pH. Anthocyanins absorb UV light, which is used by plants to perform two important functions: to attract insects, which augment pollination, and as a “sunscreen” to protect the other parts of the plant cells such as DNA from harmful UV radiation.
Chlorophyll is a fluorescent substance. Fluorescent substances can absorb light of one wavelength and then reemit a new and longer wavelength of light. Chlorophyll absorbs light in the violet and blue regions of the visible spectra. If a violet or blue light is shined through a sample of spinach extract, the solution turns red in color. The intensity of the red color is an indication of how much chlorophyll is in the sample.
In this activity, first you will use Paper Chromatography to separate the various pigments in a leaf, then you will use a SpectroVis Plus spectrophotometer to determine and analyze the visible light absorbance spectrum of leaf extract. You will also determine and analyze the fluorescence spectrum of the same leaf extract sample. In fluorescence spectroscopy, a sample can be “excited” with a chosen wavelength of light and the resulting fluorescence from the sample can be measured and quantified.
EQUIPMENT mortar & pestles
50 mL graduated cylinders
50 mL beakers
Pasteur pipettes and bulbs test tube racks
Chromatography Paper (Whatman #1)
Residue container for pigment extract containing petroleum ether and acetone
300mL 10% NaCl
100 mL petroleum ether
200 mL 70% isopropyl alcohol
100 mL acetone
100 mL 9 parts petroleum ether:1 part acetone
10mL graduated cylinders glass funnels & cotton to make plugs
Falcon Tubes small test tubes goggles glass capillary tubes to transfer extract to paper
Parafilm
SpectroVis Plus spectrophotometer
USB cord
Computers
Vernier LabQuest interface spinach leaves

Plant Pigments
PIGMENT EXTRACTION FOR CHROMATOGRAPHY
1. Obtain and wear goggles at all times during this lab.
2. Remove the petiole from the leaf blade. Tear the remaining leaf material into small pieces.
A razor blade may be used to cut the pieces. Weigh about 2 g of leaf pieces.
3. Pour into the mortar 8 ml of acetone.
4. Add the leaf material to the mortar and grind with the pestle.
5. Filter the liquid extract through a glass funnel with a cotton plug.
6. Transfer the filtered extract to a Falcon tube.
7. Add 5 ml of petroleum ether. Mix thoroughly.
8. Add 10 ml of 10% NaCl. Cap the tube and mix. After mixing, allow the layers to separate.
9. After separating, remove the lower aqueous layer with a Pasteur pipette and discard it.
10. Repeat the 10% NaCl wash three more times (i.e., repeat steps 8 & 9).
11. Carefully pour the liquid portion of the pigment extract into the labeled container.
CHROMATOGRAPHY
1. Obtain a piece of Whatman #1 Chromatography Paper and cut to a size that will fit into a
Falcon tube. With a pencil, draw a line across the width of the paper, 2cm from the bottom.
2. Place the tip of a glass capillary tube into the pigment extract. The extract will move into the pipette to a height of about 2cm.
3. Beginning at the left edge of the paper, touch the tip of the pipette to the line on the chromatography paper and with a rapid movement, move the pipette along the line until all of the extract is absorbed onto the paper.
4. Allow the extract on the paper to dry, then repeat the extract application 3 more times.
5. Place a small amount of chromatography solvent (9 parts petroleum ether:1 part acetone) in a Falcon tube.
6. Place the chromatography paper in the Falcon tube with the extract end of the paper in the solution. The extract you placed on the paper should not be in the solvent.
7. Cover the Falcon tube and place it in an area that will not be disturbed. The solvent will dissolve the pigments in the chlorophyll extract and the pigments will move up the paper at different rates depending upon the molecular size and their interaction with the paper. The chromatography "front" (the leading edge of the solvent) will remain visible.
8. Remove the paper from the jar when the front is 0.5cm from the top of the paper.
9. Allow the paper to dry then photograph the resulting chromatograph illustrating the position of each pigment. The various pigments will separate and can be identified by colors and by the order of separation (from top to bottom) as follows: carotenes (orange-yellow), xanthophylls (yellow), chlorophyll a (blue-green), chlorophyll b (yellow-green).
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Plant Pigments
PIGMENT EXTRACTION FOR SPECTROPHOTOMETRY
1. Obtain and wear goggles.
2. Prepare the spinach extract. a. Measure out 0.5 g of fresh spinach and cut or tear the spinach into tiny pieces. b. Grind the spinach pieces with a mortar and pestle. c. Add 20 mL of 70% isopropyl alcohol (IPA) and transfer the mixture to a small beaker. d. Allow the mixture to sit for about ten minutes. e. After ten minutes have elapsed, use a funnel and filter paper to filter the spinach extract into a clean beaker.
3. Connect the SpectroVis Plus spectrophotometer to the USB port of LabQuest or a computer. Start the data-collection program, and then choose New from the File menu.
Part I Collect a Visible Light Spectrum of Spinach Extract
4. Calibrate the spectrophotometer. a. Prepare a blank by filling an empty cuvette 3/4 full with 70% isopropyl alcohol and place it in the spectrophotometer. Make sure to align the cuvette so that the clear sides are facing the light source of the spectrophotometer. b. Choose Calibrate from the Sensors menu of LabQuest or the Experiment menu of
Logger Pro . c. Select Finish Calibration, and then select OK.
5. Conduct a full spectrum analysis of the spinach extract. a. Empty the blank cuvette and rinse it twice with small amounts of the spinach extract. Fill the cuvette 3/4 full with spinach extract. b. Place the cuvette containing the spinach extract into the spectrophotometer. c. Start data collection. A full spectrum graph of the spinach extract will be displayed. d. Stop data collection. e. Examine the graph, noting the absorbance peak ranges for chlorophyll described in the introductory remarks. If any of the peak absorbance values are greater than 1.5, dilute your sample to an extent that will bring the peaks below 1.5 and repeat the data collection.
6. Before continuing with data collection, answer Question 1. Save your experiment file. You can copy and paste the graph into a Word document or take a screenshot image of the graph using the Shift-Command-4 keystroke combination. An image file should appear on the desktop.
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Plant Pigments
Part II Collect a Fluorescence Spectrum of Spinach Extract
7. Ensure that the cuvette containing the spinach extract is in the cuvette slot of the spectrophotometer.
8. Choose New from the File menu.
9. Set up the SpectroVis Plus to measure fluorescence.
* In Logger Pro , choose Change Units ►Spectrometer from the Experiment menu of and select Fluorescence 405 nm.
* In LabQuest App, choose Change Units ► Fluorescence 405 nm from the Sensors menu.
10. Adjust the Sample Time.
* In Logger Pro , ch oose Set Up Sensors ►Spectrometer from the Experiment menu.
Change the Sample Time to 150 ms.
* In LabQuest App, choose Data Collection from the Sensors menu. Change the Sample
Time to 150 ms and choose OK.
11. Start data collection. A full spectrum graph of the fluorescence of the sample will be displayed. Note that one area of the graph contains a peak at approximately 675 nm. This peak is from chlorophyll.
12. Stop data collection. The height of the peak should be between 0.6 and 1.0. If necessary, adjust the sample time to increase or decrease the size of the fluorescent peak and repeat the data collection.
13. Answer Question 2 below.
14. Save your experiment file. You can copy and paste the graph into a Word document or take a screenshot image of the graph using the Shift-Command-4 keystroke combination. An image file should appear on the desktop.
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Plant Pigments
QUESTIONS
1. Describe the visible light absorbance spectrum of the spinach extract, identifying the absorbance peaks and other distinguishing features.
2. Describe the fluorescence spectrum of the spinach extract, identifying the fluorescence peaks and other distinguishing features.
3. List five colorful plant flowers, fruits, leaves, or other parts whose extracts might contain plant pigments of interest in visible light spectra and/or fluorescence spectra investigations.
4. Develop at least one researchable question concerning the spectroscopic investigation of plant pigment.
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