Understanding EDTA and how it works, decalcification considerations

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From:Gayle Callis <uvsgc@msu.oscs.montana.edu>
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I strongly suggest reading of the following chapter in order to understand
how EDTA works.  A bit chemical, but wow!  sure is worth the read.  Had to
thank my P Chem hubby for this info, plus some impromptu lectures on
chelators, particularly how they work as a function of pH.  

Harris DE  EDTA Titrations:  In Quantitative Chemical Analysis.  WH Freeman
and Co, 1982 pp 271-289.

I agree with Abizar about adding Mg and Ca to EDTA solutions, the EDTA will
chelate them, and that is why one has to add MgCl back into staining
reagents after EDTA decalcification in order to reactivate alkaline
phosphatase staining.

I often see (AND this is a huge pet peeve of mine) EDTA mentioned in
protocols. Well, Folks, what EDTA is being talked about??   People need to
be specific, either by giving the molecular weight of the EDTA they use or
say whether it is EDTA, EDTA disodium or EDTA tetrasodium.  The latter has
a different solubility AND pH when dissolved in water that EDTA/EDTA
disodium. The clue that gives away what EDTA is being used is often the
EDTA concentration, if they say 10% EDTA, I suspect EDTA or EDTA disodium
salt, if they say 14% EDTA, I suspect tetrasodium salt.  Making an educated
guess is the pits!!  

Adjusting the pH of EDTA tetrasodium must be done in a way to not lessen
the concentration of the EDTA itself, i.e. 80 mls water, 14 gms EDTA -
tetrasodium salt,dissolve, then adjust pH with concentrated Acetic acid,
and  bring to 100 ml volume with water.  

Adjusting pH of EDTA or disodium EDTA is usually done with sodium
hydroxide, have done it with NaOH pellets in large volume, stirring and
electrode down in the solution during stirring for continuous pH readout.  

Sorry to be gritchy about this, but paying attention to details lessens
confusion when one has to repeat a procedure.  

Another thought is why put EDTA in an HCl solution when it cannot chelate
below pH 3???  It is known that the pH of an acid solution goes up or
becomes more alkaline as bone decalcifies, and if the pH at which EDTA
begins to work (somewhere, but slowly around 4) then the EDTA will begin to
chelate the calcium ions released by the HCl when that spent acid solution
reaches ~ pH4 so EDTA can chelate the released Ca. 

Not only that, but the EDTA concentration in HCl or other acid solutions is
usually extremely low.   Consequently, is the EDTA really there as a
scavenger for Ca ions released by acid - the agent doing actual
decalcification?? I have never been convinced that EDTA is necessary in HCl
solutions, although decalcifying solution mfrs maintain otherwise plus say
the decalcification is better than with just acid alone.  When using these
HCl with EDTA reagents, it is the acid that concerns me as the active

I often wanted to do an experiment with a large piece of bone, acid
solution, and then test the pH to see what pH is at decalcifiation endpoint
with confirmation by xray endpoint test.  Could this be the ultimate
endpoint test, or close to it, using a simple pH meter??  

How's that for confusion, enough on EDTA   



Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303

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