A buffer holds pH steady against added acid or base, and almost every biochemical assay depends on one. This generator turns a target pH, concentration, and volume into a concrete weigh-out recipe, so you can go straight from the bench plan to the balance.
How it works
The total amount of buffer is fixed by your concentration and volume:
moles = concentration (mol/L) x volume (L)
mass = moles x molecular weight (g/mol)For conjugate-pair buffers the recipe must split those moles between the acid and base forms to hit the target pH. That split comes from the Henderson-Hasselbalch equation:
pH = pKa + log10([base] / [acid])
ratio = [base] / [acid] = 10^(pH - pKa)The tool uses this ratio to portion the total molarity, then multiplies each fraction by its own molecular weight to give the grams of acid and base. Single-component buffers such as Tris-HCl are weighed as one compound and titrated to pH.
Tips and notes
Always prepare in roughly 80 percent of the final volume, adjust the pH precisely with acid or base, and only then top up to the mark, because adding titrant changes the volume. Stay within about one pH unit of the buffer’s pKa for meaningful buffering capacity; the tool flags targets that fall outside the effective range. Finally, remember temperature: Tris in particular shifts about 0.03 pH units per degree, so set the pH where the buffer will actually be used.
Common buffers, their pKa, and typical applications
| Buffer | pKa | Effective range | Common use |
|---|---|---|---|
| Acetate | 4.76 | pH 3.6–5.6 | Protein purification at low pH, cell-free assays |
| Citrate | 3.13 / 4.76 / 6.40 | pH 3–7 (broad) | Antibody production, ELISA, low-pH dialysis |
| MES | 6.15 | pH 5.5–6.7 | Cell culture, enzyme assays near neutral |
| Phosphate (PBS) | 7.20 | pH 6.2–8.2 | Cell biology, immunology, the most widely used buffer |
| HEPES | 7.55 | pH 6.8–8.2 | Cell culture, sensitive to metal chelation |
| Tris-HCl | 8.06 | pH 7.0–9.0 | Molecular biology, electrophoresis, protein assays |
| CHES | 9.30 | pH 8.6–10.0 | Alkaline enzyme assays |
| CAPS | 10.40 | pH 9.7–11.1 | Western blotting transfer at high pH |
Tris: the most common source of buffer preparation errors
Tris is the default buffer for molecular biology but also the most frequently misused. Its pKa changes approximately 0.03 pH units per degree Celsius — more than almost any other common buffer. A Tris buffer set to pH 7.5 at room temperature (20°C) will read around pH 7.8 in the cold room (4°C) and around pH 7.2 at 37°C. If your assay runs at physiological temperature, set the pH at 37°C or choose a buffer with lower temperature dependence such as HEPES.
Tris also absorbs CO₂ from air, which can drift the pH of improperly sealed solutions. Store Tris buffers capped, and if pH stability is critical, prepare fresh or use sealed containers.
PBS: most common formulation
The standard 1× PBS formulation used in most cell biology labs is approximately 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄. This produces a pH near 7.4. Because PBS is a phosphate buffer and not a conjugate acid/base pair in the single-compound sense, the generator handles it as a two-phosphate-salt recipe. The ratio of monobasic (KH₂PO₄) to dibasic (Na₂HPO₄) salt is what sets the pH; adjusting this ratio shifts pH across the 6.2–8.2 effective range.
Practical preparation checklist
- Calculate the recipe using the generator for your concentration and volume.
- Weigh out the components — use an analytical balance for small volumes.
- Dissolve in 80% of the final volume in a suitable vessel.
- Measure pH at the temperature where the buffer will be used.
- Adjust to target pH using the appropriate acid or base.
- Top up to the final volume mark and re-measure pH.
- Filter sterilise if required (0.22 μm), label with buffer name, concentration, pH, date, and initials.
- Store appropriately and discard if pH drifts more than 0.1 units from preparation.