Popular LabKit · Recipe
Tris-HCl Buffer pH 8.0: Preparation and Recipe
How to prepare Tris-HCl buffer pH 8.0 at 20 mM, 50 mM or 0.1 M: grams of Tris base and millilitres of 1 M HCl, and how much the pH of Tris changes with temperature.
Recipe: 50 mM Tris-HCl, pH 8.0 at 25 °C, 1000 mL
| Component | Amount |
|---|---|
| Tris base, C₄H₁₁NO₃ (MW 121.14) | 6.057 g |
| 1 M hydrochloric acid | about 29.0 mL (adjust to pH 8.0) |
| Purified water | to 1000 mL |
Other concentrations (pH 8.0 at 25 °C)
| Concentration | Tris base | 1 M HCl (approx.) |
|---|---|---|
| 20 mM | 2.423 g | 11.3 mL |
| 50 mM | 6.057 g | 29.0 mL |
| 0.1 M | 12.11 g | 59.1 mL |
Method
- Dissolve the Tris base in about 800 mL of purified water.
- Bring the solution to the temperature you will use it at.
- Add 1 M HCl while stirring and watching the pH meter until it reads 8.0. The volumes above are a guide.
- Make up to 1000 mL in a volumetric flask and mix.
Tris pH changes with temperature
The pKa of Tris falls by about 0.028 per °C. A buffer set to pH 8.0 at 25 °C reads about pH 8.6 at 4 °C and pH 7.7 at 37 °C. Always set the pH at the temperature of use. To get pH 8.0 at each temperature (50 mM):
| Temperature of use | 1 M HCl for pH 8.0 |
|---|---|
| 4 °C | about 42.3 mL |
| 25 °C | about 29.0 mL |
| 37 °C | about 19.1 mL |
Use a pH electrode that is compatible with Tris (a double-junction or Tris-compatible electrode); ordinary Ag/AgCl electrodes can give drifting readings in Tris buffers.
Recipes are calculated with the Popular LabKit buffer model (ionic strength and temperature corrected) and checked against published buffer tables. For a pharmacopoeial test, follow the reagent or buffer recipe in your monograph. Always confirm pH with a calibrated pH meter.
Frequently asked questions
How do I make 1 M Tris-HCl pH 8.0 stock?
Dissolve 121.1 g Tris base in about 800 mL water, cool to room temperature and adjust to pH 8.0 by adding concentrated (37%) HCl slowly while watching the pH meter – typically 42–50 mL – then make up to 1 L. At this strength the calculator warns that the ionic-strength correction is less exact, so rely on the pH meter.
Can I use Tris hydrochloride instead of Tris base?
Yes. Mix Tris base and Tris-HCl (MW 157.60) so that the HCl-salt fraction equals the mmol of HCl shown in the calculator, then check the pH.
Why does my Tris buffer pH keep drifting?
Usually temperature (0.03 pH per °C) or an incompatible electrode. Let the solution reach room temperature before adjusting.