Method 2 : Preparing buffer with NaH₂PO₄ and Na₂HPO₄

NaH₂PO₄ and Na₂HPO_4^-7H₂O completely dissociate in water to provide conjugate acid and conjugate base . As is evident from the Henderson-Hasselbalch equation (equation 3.8), the pH of the solution is determined by the ratio of conjugate base to the conjugate acid. Let us calculate this ratio:

(3.8)

For the given pair of conjugate acid and base, pK_a2 of phosphoric acid is to be used:

(3.20) (3.21) (3.22)

Therefore, Na₂HPO_4^-7H₂O: NaH₂PO₄ ratio for preparing the required buffer is 1.5488:1. For preparing one liter of buffer, the required number of moles of phosphate species = 0.2 i.e. 200 mmoles .

Let us now see how to prepare the buffer:

1. Weigh accurately 4.68 g of NaH₂PO₄ and 16.35 g of Na₂HPO_4^-7H₂O.
   
   
2. Transfer the salts into a 1 liter conical flask and add 400 ml distilled water.
   
   
3. Shake the flask well to achieve complete dissolution of the salts
   
   
4. Wash the electrode of the pH meter thoroughly with distilled water.
   
   
5. Place the pH electrode into the phosphate solution and measure its pH.
   
   1. Although the amounts of the conjugate acid and conjugate base are weighed so as to achieve a pH of 7.4, the pH of the solution is seldom 7.4.
      
6. If the pH of the solution is <7.4, add 1 N NaOH dropwise, shake the flask well and measure the pH. Keep doing this until a pH of 7.4 is achieved.
   
   
7.   If the pH of the solution is >7.4, add 1 N H₃PO₄ dropwise, shake the flask well and measure the pH. Keep doing this until a pH of 7.4 is achieved.
   
   
   1.   Addition of H₃PO₄ increases the concentration of a phosphate species in the solution; you will therefore end up with a buffer that will have a pH of 7.4 but the concentration of the buffer will be slightly more than 200 mM .