CHEMISTRY · Multi-part · A Level · Chemistry · Acids and bases · Acids and bases
Problem
An enzyme assay must be carried out at pH 7.40. The experimental reaction mixture is expected to generate up to 0.0020 mol of strong acid per 100 mL of solution during the course of the assay. A researcher selects the HEPES buffer system (pKₐ = 7.55 at the assay temperature) to maintain pH control.
Assume that volume changes due to added reagents are negligible.
(1) Determine the required initial ratio of the base form to the acid form of HEPES that will produce pH 7.40.
(2) Using qualitative capacity reasoning, propose a total buffer concentration (in mol L⁻¹) that would restrict the pH drift to no more than ±0.05 units after the expected acid is generated. Clearly state any assumptions you make in your proposal.
(3) If, contrary to expectations, the reaction instead produced the same amount of strong base, predict the direction of pH drift and explain why the buffer would be less or more effective under this reversed condition.
(4) Evaluate, with reasoning, whether increasing the total buffer concentration by a factor of two would symmetrically improve resistance to both acid and base perturbations, or whether asymmetry would remain. Support your conclusion with reference to buffer-capacity principles.