How Buffers Work
A buffer is a solution that resists pH changes when small amounts of acid or base are added. It consists of a weak acid and its conjugate base (or a weak base and its conjugate acid) in equilibrium.
The Henderson-Hasselbalch equation describes this equilibrium: pH=pKa+log[HA][A−]. At the pKa, equal concentrations of acid and base form are present, and the buffer has maximum capacity.
Buffers are most effective within ±1 pH unit of their pKa. Outside this range, the ratio of acid to base becomes so skewed that the buffer loses its ability to neutralize added acid or base.
Temperature Effects on Buffer pH
All buffer pKa values change with temperature, but the magnitude varies dramatically. Tris buffers are notorious — a Tris-HCl buffer prepared at pH 7.5 at room temperature (25°C) will be pH 8.0 at 4°C and pH 7.2 at 37°C.
Good's buffers (HEPES, MOPS, MES) were specifically designed for minimal temperature sensitivity. HEPES, for example, shifts only −0.014 pH units per °C — five times less than Tris.
This calculator applies temperature corrections automatically. Enter your working temperature, and the recipe will produce the correct pH at that temperature — not just at 25°C.
Choosing the Right Buffer System
Select a buffer whose pKa is within ±1 pH unit of your target pH. Beyond that, consider:
• Cell culture: HEPES or MOPS (low temperature sensitivity, non-toxic to cells)
• Protein work: Tris (but prepare at working temperature) or phosphate (stable, but precipitates some metals)
• Electrophoresis: TAE (better for cloning — DNA recovery) or TBE (sharper bands, higher resolution)
• Low pH: Acetate (pH 3.7–5.6) or citrate (pH 3.0–6.2)
• High pH: Carbonate-bicarbonate (pH 9.2–10.8)
Avoid phosphate buffers when working with divalent cations (Ca²⁺, Mg²⁺) — they form insoluble precipitates. Use Good's buffers instead.
