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# How to prepare the balance solution

### #1 YUANZHILIU297

YUANZHILIU297

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Posted 04 March 2003 - 04:40 PM

When I prepared the balance solution,I found
Tris.Cl (PH 8.0)
EDTA (pH8.0)
Why does it have two pH?Is it the final pH?

and Tris.Cl (PH 7.6)
EDTA (pH8.0)
Please tell me how to prepare it?
thank you.

### #2 tfitzwater

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Posted 05 March 2003 - 05:41 PM

EDTA does not dissolve until is is approximately pH 8. Typically, 500 mM EDTA is about 680 mM NaOH by the time you get it dissolved.

I assume you are preparing 10 mM Tris-HCl, pH 8.0, 1 mM EDTA or 10 mM Tris-HCl, pH 7.6, 1 mM EDTA.
Since Tris is a buffer, the pH of the Tris is the final pH.
Prepare 1 M Tris-HCl at the desired pH.
You must take into account the temperature sensitivity of Tris when preparing it. For every increase in degrees Celcius, the pH drops 0.031. So if the Tris you are preparing is pH 7.60 at 20.1 degrees C, but you want to use it at 37 degrees C, multiply the difference in temperature (37 - 20.1 = 16.9 degrees) x 0.031 pH units/degree = a reduction of 0.524 pH units. The actual pH of the buffer at 37 degrees will be 7.60 - 0.524 = pH 7.076 rather than the pH 7.6 stock you wanted to prepare.