Question

You are planning to carry out an enzyme assay. The stoichiometry of the reaction indicates that the reaction will produce 0.25 mmol of hydrogen ions (H+), in an assay medium which will contain 10 ml of a 0.02 M phosphate buffer, pH 7.4. With this information, please answer the following questions:

a) What will be the amounts of A- and HA in mmol before and after H+ is produced?

b) What will be the ratio of [A-]/[HA] in mmol before and after H* is produced? c) What will be the pH at the end of the assay?​

Answer 1

Explanation:

Firstly you need to do the calculations via Handerson Hasselbach equation:

pKa's of phosphoric acid are 2.3, 7.21 and 12.35. If required pH is 6, then, 7.21 will be used. This means, monopotassium dihydrogen phosphate and dipotassium monohydrogen phosphate (diprotic (H2PO4-) and monoprotic (HPO4--) potassium salts) will be used.

pH = pKa + log ([A-]/[HA])

Now, for A- you put K2HPO4 concentration, and for HA you put KH2PO4 concentration:

6 = 7.21 + log(A/HA)

log(A/HA)=-1.2

A/HA = 0.063