How do I make a buffer at the pH I need?
Prepare a buffer
Choosing a buffer for a target pH, and the temperature trap that catches everyone.
Pick a buffer whose pKa is within about one unit of your target pH. Outside that range the buffering capacity falls away quickly and the solution stops doing the one job it has. Henderson–Hasselbalch gives the ratio of acid to base; from there it is two weighings, or one weighing and a titration.
The mistake that costs the most time is temperature. Many buffers shift pKa substantially with temperature, and Tris is the worst offender in common use at about −0.028 units per degree. A Tris buffer adjusted to pH 8.0 on the bench at 25 °C reads about 8.6 in a cold room at 4 °C. Adjust the pH at the temperature the experiment will run at, and if you cannot, at least know which direction it will move.
Phosphate barely moves with temperature, which is one reason it stays popular, but it precipitates with divalent cations and inhibits a number of enzymes. There is no buffer that is right for everything.
Calculated recipes get you close, not exact. Ionic strength shifts real pH by roughly 0.1 units at physiological salt, and no calculator here corrects for it. Check with a meter at the working temperature before you trust the number.
What to use
- Buffer preparation
What to weigh out for a buffer, with the pKa corrected for temperature.
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Compute molar mass from a chemical formula, including hydrates.
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