Sodium phosphate buffer - (Dec/01/2007 )

Hi,
Can someone tell me the exact procedure for preparation of sodium phosphate buffer, 1M, pH 8. I have the table and know that i have to add 93.2ml of Na2HPO4 and 6.8 ml of NaH2PO4. But i need to know the exact order of addition, is it the acidic solution first or the basic solution first. For some reason, whichever salt i add the pH seems to be reducing and never reachs 8
My3

---

I don't know what table you are using, but (assumng the solutions you are mixing are 1 M solutions) you can just adjust the pH by altering the amounts. If the pH is too low, add more Na2HPO4, if too high, add more NaH2PO4. The final volume you end up with is immaterial with this method, since it will always be 1 M phosphate. The order of addition makes no difference. If you have trouble reaching a pH of 8, then add less NaH2PO4 initially.

---

check the pH of your stock solutions; if the Na2HPO4 solution is not >pH 8 you cannot titrate a pH of 8 with the other solution;

in general, start with the solution that is nearer your pH of interest, and titrate with the other solution

---

One chart I have says 94 ml of Na2HPO4 and 6.0 ml of NaH2PO4 to generate pH 8.0, but a similar chart recommends 94.7 ml of Na2HPO4 and 5.3 ml of NaH2PO4.

Caution: phosphate buffers are not always effective above pH 7.5, as they may precipitate or bind polyvalent cations, and may behave as a participating or inhibiting metabolite.

dpKa/dT = 0.0028, so a pH 8.0 buffer at 21c is 7.96 @ 37C.

---

My requirement is to measure GSH (glutathione) in brain tissue and i use the fluorimetric assay. So my GSH can be measured at pH 8, for which i need sodium phosphate buffer.
Also can you explain the equation that u have put down in the end

---

My3 wanted to say that the pH of the buffer depend on the temperature.
you should check the pH at the same temperature as when you will use the buffer, and adjust the pH as said The Bearer

---