HPLC Buffer Preparation Calculator (Phosphate, Acetate, Formate)

Get the weights (or volumes) of each buffer component for a target pH.

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Buffer system

Target pH

Total concentration mM

Final volume mL

Example: 20 mM ammonium formate, pH 3.50, 1 L (LC-MS friendly).

Acid form–

Base form–

Base : acid–

pH = pKa + log([base]/[acid]) · n(acid) = C·V/(1 + 10^(pH − pKa)) · n(base) = C·V − n(acid)

How it works

A buffer works best within about ±1 pH unit of its pKa. Outside that range nearly all of it is in one form and it can no longer resist pH changes from the sample or the column.

This calculator splits the total buffer concentration between the acid and base forms with the Henderson–Hasselbalch equation. Liquids (phosphoric, acetic and formic acid) are given as volumes using their density and assay. Ionic strength makes the measured pH differ slightly from the calculated one, so always finish by adjusting with a calibrated pH meter, in the aqueous portion only.

For LC-MS, use volatile systems (ammonium formate, ammonium acetate, formic or acetic acid). Phosphate and Tris are not volatile and will contaminate the source.