Solution Preparation and Calculations Flashcards

A buffer protocol requires 'Tris-HCl pH 8.0'. What does this notation specifically indicate about how the buffer was prepared?

Answer: Tris base was partially protonated by adding HCl until pH 8.0 was reached, creating a mixture of Tris base and Tris-HCl (the conjugate acid)

Tris-HCl pH 8.0 means Tris base was dissolved in water and HCl was added to titrate some base to its conjugate acid form (Tris-H+), establishing the buffer at pH 8.0.

You need 250 mL of a 50 mM EDTA solution (MW = 292.24 g/mol for EDTA disodium salt) at pH 8.0. How much EDTA disodium salt do you need, and why must you adjust to pH 8.0?

Answer: 3.65 g; pH adjustment ensures EDTA is fully dissolved since EDTA disodium salt requires alkaline conditions to go fully into solution

Mass = 0.050 mol/L x 0.250 L x 292.24 g/mol = 3.65 g. EDTA disodium salt is poorly soluble at low pH — adjusting to pH 8.0 with NaOH ensures complete dissolution.

What is the Henderson-Hasselbalch equation used for in laboratory buffer preparation?

Answer: Calculating the ratio of conjugate acid to base needed to achieve a target pH, or predicting pH from the ratio of buffer components present

The Henderson-Hasselbalch equation (pH = pKa + log([A-]/[HA])) relates pH to the ratio of conjugate base to acid, allowing calculation of required ratios to achieve a target pH.

A 100x antibiotic stock solution must be prepared for cell culture use. If the working concentration of penicillin is 100 U/mL, how many units of penicillin are in 1 mL of the 100x stock?

Answer: 10,000 U

A 100x stock is 100 times more concentrated than the working solution. Working = 100 U/mL x 100-fold = 10,000 U/mL in the stock.

When preparing a 1 M phosphate buffer at pH 7.2 using KH2PO4 and K2HPO4, what ratio of K2HPO4 to KH2PO4 is required? (pKa2 of phosphate = 7.2)

Answer: Approximately 1:1 (equal amounts of both salts)

At pH = pKa2 = 7.2, the Henderson-Hasselbalch equation gives log([K2HPO4]/[KH2PO4]) = 0, so ratio = 1. Equal moles of K2HPO4 and KH2PO4 needed.

A lab technician has a lysis buffer containing protease inhibitors that have been stored at 4°C for 3 weeks (stability: 1-2 weeks at 4°C). The buffer looks clear. What should the technician do?

Answer: Discard the buffer and prepare fresh; protease inhibitors degrade over time and the buffer would fail to protect proteins during extraction

Protease inhibitors are unstable at 4°C beyond their rated shelf-life; expired inhibitors would not prevent protease-mediated protein degradation during cell lysis, compromising experimental results.