EDTA decalcification endpoint

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From:Gayle Callis <uvsgc@msu.oscs.montana.edu>
Date:Wed, 29 Sep 1999 09:14:51 -0600

John Kiernan gave the method found in literature, and the pH is critical,
as mentioned in one of todays messages, the pH must be acidic in order
that the calcium is released by the chelator, EDTA.

0.5 ml used 10% EDTA solution, add 1 ml citrate-phosphate buffer
(0.20 M (the reference did not say exactly if this was M or %, a mistake
in publication) citric acid 0.16M dibasic potassium phosphate, pH 3.2-3.6) and
2.5 ml saturated aqueous ammonium oxalate.

If solution is still clear after 20 min, it is considered negative,
test every 24 hours, remembering that EDTA will probably not ruin
your specimen like acids.  

Reference:  Rosen, AD Stain Technology, 1981, 56(1):48-49.  End Point
Determination in EDTA decalcification using ammonium oxalate.

This article has excellent discussion on how EDTA and why the
endpoint determination works.  EDTA chelates effectively as a function
of pH, at low pH calcium does not bind, at higher pH 7 and above, binds
more effectively, and that is why acid  pH of endpoint buffer works, 
rendering the EDTA-Calcium complex unstable, and binding capacity of
EDTA to calcium is pretty much zero. 

I think I repeated much of what John stated, good luck.

Gayle Callis

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