Buffer Preparation Calculator (Henderson-Hasselbalch)

The pH of a buffer is set by the ratio of the conjugate acid-base pair, not by the total concentration:

pH = pKa + log₁₀( [A⁻] / [HA] )

Working backwards, if the target pH is known: [A⁻]/[HA] = 10^(pH − pKa). Combined with total concentration and volume, you can calculate how many moles — and grams — of each component you need.

Choosing a buffer pair: the target pH should fall within pKa ± 1. Outside this range, buffering capacity drops sharply. Capacity is maximal at pH = pKa; at 1 unit away the ratio is already 10:1, and beyond that the buffer is essentially ineffective.

Key points:

• pKa shifts with temperature. Tris is especially sensitive, roughly −0.028 pH/°C: a Tris buffer equilibrated at 4 °C will be about 0.9 pH units lower at 37 °C. Always calibrate at the working temperature.
• Ionic strength shifts the effective pKa. At high concentrations, calculated and measured values can diverge. Always verify with a pH meter after preparation.
• This tool assumes ideal conditions; the result is a starting point, not a final answer.

Worked example

Preparing 1 L of 0.1 mol/L phosphate buffer at target pH 7.4 (pKa₂ = 7.21):

[A⁻]/[HA] = 10^(7.4 − 7.21) = 1.549
Base fraction = 1.549 / (1 + 1.549) = 60.77%

Na₂HPO₄  0.1 × 0.6077 = 0.0608 mol
NaH₂PO₄  0.1 × 0.3923 = 0.0392 mol

Temperature sensitivity: Tris vs. phosphate differ tenfold

pKa shifts with temperature, and the sensitivity varies enormously between buffer systems.

A full order of magnitude difference. The practical consequence: a Tris buffer adjusted to pH 8.0 at 25 °C will read approximately pH 8.6 in a 4 °C cold room — a shift of 0.6 units. The same temperature drop shifts a phosphate buffer by only 0.06 units, which is negligible.

When running cold-temperature experiments (chromatography, cold-room incubation) with Tris, adjust pH at the working temperature, not at room temperature. This is one of the most common hidden sources of error in low-temperature experiments.

Where buffering capacity is greatest

Capacity is maximal at pH = pKa, where acid and base are present in equal amounts. At 1 unit away the ratio is already 10:1 and capacity falls sharply; at 2 units away there is essentially no buffering. This is the origin of the rule of thumb “target pH should fall within pKa ± 1” — not an arbitrary convention, but a direct consequence of the equation above.