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Name: Joanne T.
Status: Student
Age: 18
Location: N/A
Country: N/A
Date: 2001


Question:
As part of a lab, I separated spinach extract using chromatography, and then read the absorption and graphed it. I am then supposed to determine which band is chlorophyll-a , chlorophyll-b, and which is beta-carotene by comparing my graph with that of one in a textbook. However, my answers are inconclusive. Is there a web-site I could search for a better graph of the absorption spectrum for these three pigments. Any help would be greatly appreciated.



Replies:
Dear Joanne,

I found a pretty decent WWW resource for you from the PhotochemCAD database of absorption and fluorescence spectra, posted by the Oregon Health & Sciences Univ.:

http://omlc.ogi.edu/spectra/PhotochemCAD/html/alpha.html

It shows representative spectra for:

Beta-carotene -

http://omlc.ogi.edu/spectra/PhotochemCAD/html/beta-carotene.html

Chlorophyll b -

http://omlc.ogi.edu/spectra/PhotochemCAD/html/chlorophyll-b.html

Chlorophyll a -

http://omlc.ogi.edu/spectra/PhotochemCAD/html/chlorophyll-a(ether).html

These spectra are somewhat solvent-dependent & 2 such examples are shown for chlorophyll a, but there do not appear to be any big differences. It seems as though you can actually download the PhotochemCAD program for spectral comparison & calculations if you really want to get into it, but that shown on the Oregon Health & Sciences Univ. website appears to have already been organized into a more user-friendly format.

Best of luck with your project,

Jeff Buzby, Ph.D.


I might suggest almost any general Botany , biology or biochemistry text for the absorption spectra of the chlorophylls. Chlorophyll a absorbs most strongly in the 400-440 and 660-680 while b absorbs best around 450 (pretty steep narrow peak trailing off quickly to either side). Your results depend on a number of possible problems...poor separation because of inappropriate solvents, or matrix. I am assume you are using thin layer i.e., silica gel you can also get some breaking of the chlorophyll phytol tails which if I remember correctly results in trailing on the gel...but its been a long time. Carotenoids are bimodal with two main peaks just on either side of the chlorophyll b (but they don't absorb nearly as well...~ 50% of the b.

Peter Faletra Ph.D.
Assistant Director
Science Education
Office of Science
Department of Energy


You can find U.V./vis. absorption spectra, fluorescence spectra, and extinction coefficients of these compounds in a couple of solvents on the

Web Site: http://www.kean.edu/~biology/botlab7.html

There are also links that may provide you even more details.

Vince Calder



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