Tricky buffer preparation - (Oct/22/2009 )

Hi guys!

I should prepare the following buffer:

5mM KH2PO4 pH 2.8; 30% Acetonitrile

Now I have a couple of questions:

1) Should the pH be measured before or after adding acetonitrile?... because the pH will be for sure affected by this apolar solvent.

2) How should I adjust the pH? With H3PO4? Wouldn't that change the final concentration as well? If yes, should I use the Anderson-Hasselbach equation?

3) Would that be a difference if I take H3PO4 and correct it with KH2PO4 instead of doing the opposite?

Thx a lot for your answer(s)!!!

Philip

adjust the pH before adding the acetonitrile.

with phosphate buffers, you can mix volumes of mono and dibasic (and acid and tribasic) that are the same concentration so that the final concentration will be the same but the volume will increase. order of addition of phosphate solutions does not matter.

that being said, what you should probably do is prepare a concentrated stock of pH adjusted phosphate, dilute to 5mM and then add acetonitrile.

(by the way, that's henderson-hasselbach)