Buffer preparation
What to weigh out for a buffer, with the pKa corrected for temperature.
Better suited to pH 7.4:
The temperature of the meter when you titrate.
Where the experiment actually runs.
Weigh out
Tris·HCl
C4H12ClNO3 · 157.59 g/mol · 20.51 mmol
Tris base
C4H11NO3 · 121.14 g/mol · 4.488 mmol
pKa 8.06 at 25 °C · base fraction 0.18 · reads pH 7.4 at 25 °C
About TrisThe largest temperature coefficient in common use. Adjust the pH at the temperature you will work at, not on the bench. Tris also binds some metal ions and interferes with protein assays that use the Bradford reagent.
pH = pKa(T) + log₁₀([base]/[acid]); pKa(T) = pKa₂₅ + (dpKa/dT)(T − 25)- Hydrogen ion buffers for biological research — Biochemistry, 1966
- The Henderson–Hasselbalch relationship — Journal of Chemical Education, 2001
No correction for ionic strength is applied. Activity effects shift real pH by roughly 0.1 units at physiological salt, so use this to get close and then check with a meter at your working temperature.
When to use this
Use this to get a recipe for a buffer at a target pH, from either two salts or a single salt titrated with acid. It corrects the pKa for temperature, which is the reason it exists — a buffer adjusted warm and used cold is not at the pH you set.
Worked example
You need 500 mL of 50 mM Tris at pH 8.0, adjusted at 25 °C but used at 4 °C.
- Buffer
- Tris, pKa 8.06
- Target pH
- 8.0
- Concentration / volume
- 50 mM, 500 mL
- Adjust at / use at
- 25 °C / 4 °C
Result
The tool warns that this buffer will read about pH 8.59 in the cold room — adjust at 4 °C instead if that matters.
What people get wrong
- Adjusting the pH at bench temperature for an experiment that runs in the cold. Tris moves about 0.028 units per degree, so a 21 degree drop is more than half a pH unit.
- Choosing a buffer whose pKa is far from the target. Outside about one unit either side the buffering capacity collapses and the pH drifts on the first addition.
- Trusting the calculated recipe to the second decimal. No ionic strength correction is applied, which is worth roughly 0.1 units at physiological salt.
Questions
+Two salts or titration — which should I use?
Two salts is more reproducible and needs no meter. Titration is easier when you only stock one form, and it is what most published protocols describe.
+Why does the tool still tell me to use a pH meter?
Because activity effects, temperature and the exact salt you weighed all move the real pH. The calculation gets you close; the meter confirms it.
+Which buffer should I pick?
One whose pKa is within a unit of your target. The tool suggests better-suited buffers when your target sits outside the selected one’s useful range.
Related tools
- Molarity calculator — Convert between mass, molar concentration and volume for a solution.
- Molecular weight calculator — Compute molar mass from a chemical formula, including hydrates.
- Dilution calculator — Work out how much stock and diluent to combine for a target concentration.
Science last reviewed .