Isolation and Characterization of Plant Photosynthetic Pigments

BCHM 322 - fall 2008
Isolation and Characterization of Plant Photosynthetic Pigments
Experiment 5
From: R. F. Boyer, Modern Experimental Biochemistry, Second Edition, Benjamin/Cummings Publishing Co.
Background. The major photosynthetic pigments of higher plants can be divided into two groups: Chlorophylls and carotenoids. They are found in chloroplasts weakly associated with proteins where they function in light harvesting during photosynthesis. The pigments can be solublized with organic solvents such as acetone, and hence can be classified as lipids.
The most abundant plant pigments are chlorophyll a and chlorophyll b that are usually present in ratio of about 3:1. They absorb light in the blue region (450 nm) and the red region (650-700 nm) and initiate the photosynthetic process producing NADPH and ATP. The structure of chlorophylls is based on a porphyrin ring chelating a magnesium atom.
Figure 1
Structures of chlorophyll a and chlorophyll b.
Carotenoids can be divided into two major groups: carotenes, that contain only carbon and hydrogen, and xanthophylls, that also contain oxygen atoms. Carotenoid function is
1
not clear, but two probable functions have been described. Carotenes are believed to protect chlorophylls from photo-oxidation and destruction. Xanthophylls are believed to participate in light harvesting and may pass the energy on to chlorophylls. These compounds can have a variety of colors and are responsible for plant coloring such as that seen in flowers. Carotenoids have a single major peak of absorbance in the range of 350-500 nm.
Figure 2
Structures of major carotenoids: β-carotene, lutein, violaxanthin, and neoxanthin
2
Experiment. We will extract photosynthetic pigments from plant leaves (spinach) and characterize them by thin layer chromatography and spectral characteristics (absorption profiles). We will determine the total pigment content of the crude extract by its absorption spectrum. We will also fractionate the extract by thin layer chromatography and one pigment (of your choice) will be isolated and characterized by it absorption spectrum.
Procedure.
Pigments are sensitive to light and are subject to photo-bleaching, so minimize the exposure of these compounds to room lighting.
1. Extraction of pigment from leaves.
Mince one large spinach leaf, or several small ones into small bits and place in a mortar. Add about 0.5 g sand and 15 ml acetone and grind throughly until the acetone has a deep color. This may take some effort since plant cell walls are tough. Take care not to breathe acetone or get on skin. Add 1-2 g Na2SO4 to absorb any water released from the cell. Withdraw supernatant with a pasteur pipette and place in a glass tube and stopper to prevent evaporation.
2. Absorption spectroscopy of crude extract.
Zero the scanning spectrometer against acetone. Scan the sample from 400 to 700 nm according to instructions from the T.A.
3. Thin layer chromatography of pigments.
Application of pigment to thin layer plates.
Mark a cellulose thin layer plate 1 cm from the bottom with a pencil (gently, don’t scratch the cellulose). With a pasteur pipette, apply the pigment solution in a fine line along your pencil mark. Keep the line as fine as possible to maximize the resolution of the pigments. Allow the acetone to evaporate until dry. Repeat the application of pigments once or twice more until the line has a deep green color. Allow to dry.
Place the thin layer into a chromatography chamber (in the fume hood) that has been equilibrated with petroleum ether:acetone, 9:1. Make sure that the solvent is below the application line on the plate. Cover the chamber with its lid and allow chromatography to proceed until the solvent front is 1 cm from the top. Mark the level of the solvent front with a pencil and allow the plate to dry briefly in the hood. Pigments should be visible as colored bands on the plate. Select a pigment that appeals to you. Measure the distance from the application line to the solvent front and a selected pigment band on the plate and record the distance in cm. (You will need this information to calculate the Rf of your pigment).
3
Extraction of the purified pigment.
Cut the selected band from the thin layer plate and scrape into a glass tube using a funnel to collect the scrapings. Add 4 ml acetone and cover with a stopper. Mix for 5-10 min. to solublize the pigment. Centrifuge the tube briefly to separate the cellulose from the soluble pigment. Remove the supernatant to a new tube and scan in the scanning spectrophotometer in range 400 to 700 nm as described above.
Reports.
1. Calculate the concentrations of chlorophyll a, chlorophyll b, and total xanthophylls in the crude extract according to the equations:
Ca = 11.24 A661.6- 2.04 A644.8
Cb = 20.13 A644.8 – 4.19 A661.6
Ca+b = 7.05 A661.6 + 18.09 A644.8
Cx+c = 1000 A470 – 1.90Ca – 63.14 Cb
214
2. Report the Rf value for the pigment you selected to purify. Explain your Rf value based on the polarity of the pigment.
3. Try to assign identity to the pigment you purified based on its Rf and absorption spectrum.
where:
Ca = concentration of chlorophyll a
μg/mL of extract
Cb = concentration of chlorophyll b μg/mL
Ca+b = total chlorophyll μg/mL
Cx+c = concentration of total carotenoids
xanthophylls + carotenes μg/mL
Note: these equations are only valid in 100% acetone solvent
4
Absorption Spectra of Plant Pigments
Figure 3
Visible absorption spectra
of chlorophyll a (A) and
chlorophyll b (B).
Figure 4
Visible absorption spectra
for four carotenoids: β-
carotene (—) a, lutein (--) b,
violaxanthin (…) c, and
neoxanthin (. . .) d.
a
b
c
d
5