Buffer cheat sheet

Everything you need to prepare a buffer at a target pH, in one page.

Henderson-Hasselbalch

For a weak acid HA with dissociation constant $K_a$:

$\mathrm{pH} = \mathrm{p}K_a + \log_{10}\!\left(\dfrac{[\mathrm{A^-}]}{[\mathrm{HA}]}\right)$

At [A⁻] = [HA] (equimolar), $\mathrm{pH} = \mathrm{p}K_a$ exactly — the point of maximum buffer capacity. Rearranged for the ratio:

$\dfrac{[\mathrm{A^-}]}{[\mathrm{HA}]} = 10^{\mathrm{pH} - \mathrm{p}K_a}$

pKa table — 12 common lab buffers (25 °C)

pKa values are 25 °C conventional values from the primary sources listed at the top of the page. Buffers marked Good’s are from the 1966/1972 Good buffer series, chosen for low metal binding, low UV absorbance at 260 nm, and biological inertness.

Temperature-sensitive buffers

Tris is famously temperature-sensitive: ΔpKa/°C ≈ -0.028. A Tris buffer calibrated to pH 7.4 at 25 °C reads about pH 8.1 at 4 °C. If the reaction runs on ice, calibrate the pH meter at the reaction temperature, not room temperature.

HEPES is milder (ΔpKa/°C ≈ -0.014) but still not negligible over a 20 °C swing.

For phosphate and the acetate/carboxylate-family buffers, the temperature dependence is small enough to ignore for routine work.

Preparing a buffer at a target pH

Three routes to the same buffer:

1. Titration — dissolve the acid (or the conjugate base), then add strong base (or strong acid) until the pH meter reads the target. Most forgiving; requires a calibrated meter.
2. Ratio calculation — Henderson-Hasselbalch tells you the [A⁻]/[HA] ratio for the target pH. Weigh out both the acid and its conjugate base directly at that ratio. Works only if both forms are available as separate reagents (e.g., NaH₂PO₄ + Na₂HPO₄ for phosphate).
3. Preset recipes — many textbook tables give exact gram amounts for a fixed volume of buffer at a fixed pH. Fast but inflexible; use the calculator for anything outside the standard entries.

Rules of thumb

• Stay within pKa ± 1. Farther than 1 pH unit from pKa, the ratio exceeds 10:1 and small additions of acid or base swing the pH by ≥ 0.1.
• Buffer at the working concentration you’ll use — a 1 M Tris stock diluted to 100 mM shifts pH slightly because the effective ionic strength changes.
• Ionic strength changes pKa. Textbook pKa values are for infinite dilution or specified ionic strength; a real buffer at 100 mM ionic strength is typically 0.1 – 0.2 pH units off the table value. Fine for cell culture, matters for enzyme kinetics.
• Autoclaving Tris is fine; autoclaving phosphate is fine; autoclaving anything with glucose is not (Maillard browning).

Common practical mistakes

1. Ignoring temperature on Tris. The pH drifts by ~0.03 per °C. A Tris buffer prepared at 25 °C and used at 4 °C is nearly 0.6 pH units higher than the label says.
2. Using a buffer 2+ pH units from its pKa. The buffering capacity is essentially zero; the solution behaves like water plus a spectator salt.
3. Not accounting for the counter-ion. “0.1 M sodium phosphate” is the sum of Na⁺ contributions from both NaH₂PO₄ and Na₂HPO₄, and the ionic strength scales with the ratio.