Pigment Extraction and Spectrophotometry

Photosynthetic pigments utilize various parts of the visible light spectrum by converting the energy from photons into chemical energy.  Plants and other photosynthetic organisms even utilize multiple pigments to capture as much of the visible light spectrum as possible for chemical energy production. In the Labster simulation, pigments were extracted from algae to determine what parts of the visible light spectrum the pigments absorb. Instruments like the spectrophotometer allow scientists to determine the amount of light that was absorbed at certain wavelengths.

Part 1:

1. Using online resources, in 4-5 sentences explain how spectrophotometry works as a technique. Use full sentences and make sure they are in your own words. (3 pts.)

2. What solution is used to blank a spectrophotometer? Why is it important to blank a spectrophotometer? (2pts.)

3. Spectrophotometers can be used to create absorption spectra for pigments, including those important for photosynthesis. After extraction with appropriate solvents and instrument preparation (i.e. blanking), the absorption at each wavelength can be determined for the sample of the pigment extracts.  When this is plotted with wavelength on the x-axis and absorbance on the y-axis, it is possible to see which wavelengths (colors) of light are absorbed, and which are reflected or transmitted.

Phycoerythrin, phycocyanin, and allophycocyanin (which all belong to a class of pigments called phycobilins), and chlorophyll a are all commonly found in cyanobacteria and red algae.  The absorption spectra for these pigments are shown in the figure below.

Use the figure below and the following facts to predict where you would expect to find red algae and cyanobacteria (deep vs. shallower/surface waters).  Include an explanation for your prediction.  (5 pts.)

• Green light (shorter wavelengths, higher energy) penetrates deeper into water than red light (longer wavelengths, lower energy).
• Cyanobacteria have a lower proportion of phycobilins among their photosynthetic pigments than red algae.

Part 2:

While a spectrophotometer is a helpful instrument used to study pigments, there are other methods, including chromatography.  The set-up of this technique involves extracting pigments using a solvent, and allowing the extract and solvent to upward through porous paper or other material.  As the solvent is absorbed, it carries soluble pigments with it.  The pigments are separated and can be visualized on the resulting chromatograph.

While some chromatography techniques involve toxic solvents and specialized materials, it is possible to do chromatography at home.  Follow the steps to select samples, extract the pigments, create a chromatograph, and analyze the results.

4. Select two (or more) samples to compare. You might be interested in comparing leaves of different plant species, plants from different areas, or leaves from the different part of the tree.  You can compare samples from the outdoors around your home, or maybe from your refrigerator if you have some produce in there.  Flower petals or vegetable shavings might be interesting too.  Indicate what samples you are comparing and what you expect to find.  (Will they be different?  If so, in what way?) (2 pts.)

Extract pigments using the following materials and steps.*

Materials:

• Paper from a coffee filter (or other porous paper)
• Acetone (a common nail polish remover). You could also use rubbing alcohol, but acetone tends to work better.
• Glass container, such as an empty pickle jar with a lid. (This can be substituted with a glass and aluminum foil.)
• Pencil or popsicle stick
• tape
• fresh spinach, flower petals, vegetable shavings, etc.

Suggested Method:

1. Cut a strip of paper that is a little bit longer than the distance needed reach the bottom of jar.
2. Make a mark with a pencil on the paper about 1 cm above the edge of the strip to indicate where your pigment will be placed.
3. Use a coin to extract the pigments from the leaves directly onto the paper where you drew the line. Place the plant material on top of the paper and roll the edge of a coin over it.  You might need to repeat this process a few times to make sure there is enough pigment deposited.
4. Cover the bottom of the jar with a thin layer of acetone. Fix the paper to a pencil or stick with tape, and suspend the bottom edge of the paper into the acetone.
5. After a few minutes, remove the paper and make a pencil mark where the acetone reached.
6. Take a look at the colors on your chromatographs!

*Note: There are many at-home chromatography set ups that can be found using an Internet search.  Feel free to try others you find.

5. When you are finished, photograph your chromatographs and insert the pictures. Calculate R_f values for the different pigments visible on your chromatograph by dividing the distance traveled by the pigment divided by the distance traveled by the solvent.  (5 pts.)

6. Compare your chromatographs to the one pictured below and/or use the information below to try and identify common pigments that may be in your samples. (3 pts.)

• Colors that are yellow-green to olive-green correspond to chlorophyll b.
• Colors that are bright green to blue green correspond to chlorophyll a.
• Colors that are yellow correspond to xanthophylls.
• Colors that are yellow to yellow-orange correspond to carotenes.