Name: Joanne T.
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.
I found a pretty decent WWW resource for you from the PhotochemCAD database
of absorption and fluorescence spectra, posted by the Oregon Health &
It shows representative spectra for:
Chlorophyll b -
Chlorophyll a -
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.
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.
Click here to return to the Molecular Biology Archives
Update: June 2012