How to make 1 M Tris-HCl buffer
Step-by-step recipe for 1 M Tris-HCl at any pH from 7.0 to 9.0, with the weigh-out calculation, the temperature correction everyone forgets, and why you titrate before making up to volume.
1 M Tris-HCl 1 L of 1 M stock
Preparation time about 20 minutes.
| Component | Formula weight | Amount |
|---|---|---|
| Tris base (Trizma base) | 121.14 g/mol | 121.14 g |
| Deionised water | — | to 1 L |
| Concentrated HCl (~12.1 M) | 36.46 g/mol | to pH |
Procedure
- Weigh 121.14 g of Tris base into a beaker with a stir bar.
- Add about 800 mL of deionised water and stir until fully dissolved. Dissolution is endothermic, so the solution will feel cold and will warm back to room temperature as it equilibrates — wait for that before you touch the pH.
- Calibrate your pH meter at the temperature you will actually use the buffer at. Tris changes pH more with temperature than almost any other common buffer.
- Titrate to the target pH with concentrated HCl, adding slowly near the endpoint. Reaching pH 8.0 from Tris base takes roughly 42 mL of concentrated HCl per litre; pH 7.5 takes about 60 mL.
- Make up to exactly 1 L with water. Do this after titrating — the acid you added contributes real volume.
- Filter through 0.22 µm if the buffer is for cell culture, chromatography or long storage. Autoclaving works but darkens the solution slightly.
What matters here
Temperature is the big one
Tris has a dpKa/dT of about −0.028 per °C. A buffer titrated to pH 8.0 at 25 °C sits near pH 8.6 in a 4 °C cold room and near pH 7.7 at 37 °C. If your protein cares about pH, titrate at the working temperature.
Tris base vs Tris-HCl
Tris base (121.14 g/mol) is the free amine and needs acid to reach neutral pH. Tris-HCl (157.60 g/mol) is the pre-protonated salt and needs base. Mixing the two in the right ratio avoids titration entirely — the buffer calculator will give you the split.
Do not use Tris where the amine matters
The primary amine reacts with aldehydes (so no glutaraldehyde fixation), with NHS esters (so no amine-coupling chemistry), and it interferes with the Bradford and BCA assays at high concentration. Use HEPES or phosphate instead.
Frequently asked questions
pH drifts upward overnight
Usually CO₂ absorption is not the culprit with Tris — check that the meter was calibrated at the same temperature, and that the buffer had fully equilibrated thermally before you read it.
Solution turns yellow after autoclaving
Normal for Tris and harmless for most uses, but it indicates some degradation. Filter-sterilise instead for anything sensitive.
White precipitate on dilution
Almost always a divalent cation in the water reacting with something else in the buffer. Use fresh deionised water and add MgCl₂ or CaCl₂ last.