A buffer only works close to its pKa. Most buffer problems start with ignoring that, or with a pH meter that nobody calibrated this week.
Choose by pKa
A buffer resists pH change best within about one unit of its pKa. Outside that range it is just salt water with a pH number on the label.
| Buffer | pKa (25 °C) | Useful range |
|---|---|---|
| Acetate | 4.76 | 3.8-5.8 |
| MES | 6.1 | 5.5-6.7 |
| Phosphate (H₂PO₄⁻) | 7.2 | 6.2-8.2 |
| HEPES | 7.5 | 6.8-8.2 |
| Tris | 8.1 | 7.0-9.0 |
| Glycine (amine) | 9.6 | 8.8-10.6 |
Then check compatibility. Phosphate precipitates with calcium and inhibits some kinases. Tris has a primary amine, so it reacts with NHS esters and aldehydes. HEPES can make peroxide under light in some media. The right buffer is the one your downstream step tolerates.
1X PBS, pH 7.4 (1 L)
| Component | Amount | Final |
|---|---|---|
| NaCl | 8.0 g | 137 mM |
| KCl | 0.2 g | 2.7 mM |
| Na₂HPO₄ (anhydrous) | 1.44 g | 10 mM |
| KH₂PO₄ | 0.24 g | 1.8 mM |
- Dissolve in about 800 mL of purified water.
- Check pH. It usually lands near 7.4. Adjust with HCl or NaOH only if needed.
- Make up to 1 L. Autoclave or filter (0.22 µm) for cell work.
If your Na₂HPO₄ is the heptahydrate or dodecahydrate, the mass changes. Heptahydrate: 2.72 g. Dodecahydrate: 3.63 g. Check the bottle.
Want more options, saved recipes and warnings? Open the full Buffer Recipe Generator in BenchMate.
1 M Tris-HCl (100 mL)
- Dissolve 12.11 g Tris base in about 70 mL water.
- Bring to the temperature you will use the buffer at.
- Add concentrated HCl slowly with stirring. Roughly 4.2 mL takes it to pH 8.0, about 6 mL to pH 7.6 and about 7 mL to pH 7.4 at room temperature. Treat these as starting points and trust the meter.
- Make up to 100 mL.
Temperature changes Tris pH
Tris pH drops about 0.028 units for every 1 °C rise. A buffer set to pH 8.0 at 25 °C reads about 8.6 at 4 °C and about 7.7 at 37 °C. That is not a small error for an enzyme with a narrow pH optimum. Set Tris at the temperature of use, or correct for it. Phosphate and HEPES shift much less.
Do not use a standard pH electrode in Tris without checking. Some silver/silver chloride reference junctions react with Tris and drift. Use a Tris-compatible electrode or a double-junction probe if you make Tris buffers daily.
pH meter habits
- Calibrate with two buffers that bracket your target, every day you use the meter.
- Stir gently and wait for a stable reading. Rushing is the common error.
- Approach the target slowly. Overshoot and back-titration add salt you did not plan for.
- Rinse the probe between solutions and store it wet in storage solution, never in water.
BenchMate's buffer generator scales 40 common recipes to any volume and pH, and flags the temperature effect for Tris.
Frequently asked questions
How do I make 1X PBS?
For 1 L: dissolve 8.0 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄ and 0.24 g KH₂PO₄ in about 800 mL water, check the pH is 7.4, then make up to 1 L and sterilise.
Why does my Tris buffer pH change in the cold room?
Tris pH falls about 0.028 units per °C rise, so it reads higher when cold. Set the pH at the temperature you will use the buffer.
How do I choose a buffer?
Pick a buffer with a pKa within about one unit of your target pH, then check it does not interfere with your enzymes, metals or chemistry.
Should I adjust pH before or after making up to volume?
Adjust pH before the final volume adjustment, because the acid or base you add takes up volume.
22 core bench techniques, from molarity to sterilisation. Volume 1 of The Biotechnology Techniques Bible.