Buffer calculator: Henderson–Hasselbalch, pKa and recipes
Pick a buffer system, choose a pH, and get the acid/base split, the masses to weigh and the temperature correction.
Henderson–Hasselbalch pH ↔ ratio
Leave either the pH or the ratio blank and the other is calculated.
Two-component buffer recipe
Weigh out an acidic and a basic salt separately — the classic way to make a phosphate or acetate buffer without titrating.
Temperature correction
A buffer titrated on the bench is not the same pH in a cold room or a 37 °C incubator. Tris is the worst offender.
pH, pOH and [H⁺]
Straight conversions between pH, hydrogen-ion concentration and hydroxide-ion concentration at 25 °C.
Buffer pKa reference table
Useful pH range is roughly pKa ± 1. Temperature coefficients are dpKa/dT in units per °C.
| Buffer | pKa (25 °C) | Useful range | dpKa/dT | Acid form / base form |
|---|
Standard recipes worth memorising
| Buffer | Composition (1 L, 1×) | Final pH |
|---|---|---|
| PBS | 8.0 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄, 0.24 g KH₂PO₄ | 7.4 |
| TAE (50× stock) | 242 g Tris base, 57.1 mL glacial acetic acid, 100 mL 0.5 M EDTA pH 8 | ≈ 8.3 |
| TBE (10× stock) | 108 g Tris base, 55 g boric acid, 40 mL 0.5 M EDTA pH 8 | ≈ 8.3 |
| TE | 10 mM Tris·HCl, 1 mM EDTA | 8.0 |
| SSC (20× stock) | 175.3 g NaCl, 88.2 g sodium citrate | 7.0 |
| Tris-glycine running buffer | 3.0 g Tris base, 14.4 g glycine, 1.0 g SDS | ≈ 8.3 |
| Laemmli 4× sample buffer | 250 mM Tris·HCl, 8 % SDS, 40 % glycerol, 0.02 % bromophenol blue, 20 % β-ME | 6.8 |
| Sodium phosphate 0.1 M | see the recipe calculator above | 5.8–8.0 |
Practical buffer rules
- Titrate at the working temperature and at the working concentration. Diluting a 10× stock shifts the pH, sometimes by 0.2 units.
- Adjust pH before making up to volume. Acid or base adds volume, so a buffer brought to the mark first will end up dilute.
- Match the counter-ion to the experiment. Phosphate precipitates divalent cations and inhibits many enzymes; Tris has a primary amine that reacts with aldehydes and NHS esters; borate complexes cis-diols including ribose.
- Check optical interference. Good's buffers are transparent above 240 nm, but the ones with piperazine rings absorb in the far UV.
- Buffer capacity scales with concentration and peaks at pH = pKa. If your reaction generates protons, either buffer harder or use a pH-stat.
Why the calculated pH is never exactly right
Henderson–Hasselbalch uses concentrations where it should use activities. At the ionic strengths typical of a lab buffer (50–200 mM) the activity coefficient of a monovalent ion is around 0.75, and lower still for divalent species, so the observed pH can sit 0.1–0.3 units away from the calculation. Use the calculator to get close, then titrate with a calibrated meter.
Frequently asked questions
What is the Henderson–Hasselbalch equation?
pH = pKa + log₁₀([A⁻]/[HA]). It relates the pH of a buffer to the ratio of conjugate base to weak acid, and is accurate within roughly one pH unit of the pKa.
How do I choose a buffer for my experiment?
Pick one whose pKa is within about 1 unit of your target pH, then check that it does not chelate metals you need, does not absorb at your assay wavelength, and has a temperature coefficient you can live with.
Why does Tris buffer pH drift?
Tris has a large temperature coefficient (ΔpKa/°C ≈ −0.028), so a buffer set to pH 8.0 at 25 °C is nearer pH 8.6 at 4 °C. Always titrate at the temperature of use.
What is buffer capacity?
The amount of strong acid or base a buffer absorbs per unit pH change. It peaks when pH = pKa (equal acid and base) and scales with total buffer concentration.