Re: End Point Determination when EDTA decalcified

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From:"J. A. Kiernan" <>
Date:Tue, 28 Sep 1999 12:40:00 -0400 (EDT)
Content-Type:TEXT/PLAIN; charset=US-ASCII

On Tue, 28 Sep 1999 wrote:

> Sorry I did not mention the pH of the buffer.  My procedure states that it
> should be at 3.2 - 3.6.  The problem is that I don't have the specific
> amounts needed to make up the citrate-phospahte buffer.  Do you happen to
> have a recipe that you could share.

     Sodium phosphate, dibasic, 0.2M:  57 ml
     Citric acid, 0.1M:               143 ml

  That's the recipe for pH 3.4.  As I said in my previous email,
  don't expect to get pH 3.4 by simply mixing the Na2EDTA solution
  with this buffer. However, it might not matter what the
  exact pH is, as long as it's below 5.5.

  The source of this method is   A.D. Rosen, 1981.
  End-point determination in EDTA decalcification using ammonium
  oxalate. Stain Technol. 56, 48-49. The rationale of the testing
  method is that the [Ca-EDTA] complex is unstable below pH 5.5,
  so that the solution contains free calcium ions, which can
  then form an insoluble oxalate. The sensitivity is said to
  be 10 micrograms Ca per ml.

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1

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