extracting chlorophyll from soil?

larson eric elarson at ux1.cso.uiuc.edu
Wed Feb 1 23:21:10 EST 1995

cmduran at whale.st.usm.edu (Charles Michael Duran) writes:

>I am working on a master's thesis project dealing with soil algae and
>chlorophyll in soil.  There is extensive literature on extracting
>chlorophyll from water, but I have been able to find very few references on
>extracting chlorophyl from soil.  I would appreciate any input.

>I am using a colleague's network connection to pose this question?
I'm guessing here.
Chlorophyll should readily extract into ethanol or acetone.  Either solvent
should cause a disruption of cellular membranes, allowing the release of
the chlorophyll.  I would suggest a rather long extraction period since 
algae can infiltrate into the pores of soil particles.  
The extract will probably contain algal pigments (chlorophylls,
xanthophylls, phycolbilins) and probably lots of other colored material
extracted from decayed organic material, soil bacteria, and even from the
rock that is soil (unlikely, but keep an open view.)
I do suggest bufferring the solvent extraction media to a neutral pH.
Chlorophyll will lose the Mg if exposed to acidic pH's.  Instead of using
100% acetone (or ethanol), try 90% or so with a little buffer (phosphate or
one of the "good" buffers.)  Do note that the pH of a high organic solvent
solution will be different than in water (pKa's usually increase as solvent
concentration is raised.).
How to detect the chlorophyll?  Good question.  If you're lucky, straight
absorption spectrophotometry might work, but I'll guess this will fail when
the algae concentration is low.  Still, it's worth a try -- especially if
you consider that you can use an acid treatment to force the loss of Mg
from Chlorophyll (which makes a compound called pheophytin.)  Pheophytin
has a different absorption spectra and if this is the only compound that
changes with acid treatment, you could end up with a very sensitive
difference method.
Fluorescence is an option, albeit a potentially difficult one.  The
instruments are expensive, difficult to corral into a linear assay and
likely to pick up fluorescence from a whole variety of compounds.

Pursuing proper analytical separtion techniques for surety of analysis
would seem to require the use of an HPLC or some other chromatographic
technique -- it will be easy to find methods, difficult to convince your
boss to buy the equipment.  If you're on a shoe-string budget, then
thin-layer (or paper) chromatography is probably the way to go.  Finding 
a method to separate the pigments should be easy, as will extracting the
band with solvent for spectrophotometry.
Extract a good chunk of soil early on.  If the pigment concentration is too
low, you can easily concentrate the extract -- life will be easiest working
from a position of plenty.

Good luck.

Eric Larson                  | University of Illinois at Urbana-Champaign
USDA/Agronomy                | 190 PABL; 1201 W. Gregory; Urbana, IL 61801
elarson at ux1.cso.uiuc.edu     | Voice 217.244.3079  Fax 217.244.4419
Fidonet: 1:233/4.1           | My opinions are my own, but correct :-) 

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