Buffer Preparation Calculator
Preparing a buffer at a precise pH requires knowing the correct ratio of weak acid to its conjugate base. This calculator uses the Henderson-Hasselbalch equation (pH = pKa + log10([A-] / [HA])) to determine the mole fraction of each component. Enter the target pH, the pKa of your buffer system, and the total molarity required. The calculator outputs the ratio, the molar concentrations of each component, and the mass of each to dissolve in the final solution volume.
Henderson-Hasselbalch equation
pH = pKa + log10([A-] / [HA])
[A-] / [HA] = 10^(pH - pKa)
Rearranging: fraction of base = 10^(pH - pKa) / (1 + 10^(pH - pKa)). The Henderson-Hasselbalch equation was derived from the dissociation equilibrium of a weak acid and is the standard method for buffer preparation in biochemistry and clinical science.
Common buffer pKa values (at 25 degrees Celsius)
- Citrate: pKa1 = 3.13, pKa2 = 4.76, pKa3 = 6.40 (useful pH range 3.0 to 6.2)
- Acetate: pKa = 4.76 (useful pH range 3.8 to 5.8)
- MES: pKa = 6.15 (useful pH range 5.5 to 6.7)
- Phosphate: pKa2 = 7.20 (useful pH range 5.8 to 8.0)
- HEPES: pKa = 7.55 (useful pH range 6.8 to 8.2)
- Tris: pKa = 8.06 (useful pH range 7.0 to 9.0)
- Glycine: pKa = 9.78 (useful pH range 8.6 to 10.6)
Buffer preparation: frequently asked questions
What is the Henderson-Hasselbalch equation?
The Henderson-Hasselbalch equation is pH = pKa + log10([A-] / [HA]), where pKa is the acid dissociation constant of the weak acid, [A-] is the molar concentration of the conjugate base, and [HA] is the molar concentration of the weak acid. It allows calculation of the pH of a buffer solution.
How do I choose the right buffer for a target pH?
Select a buffer whose pKa is within 1 pH unit of your target pH. Within this range (pKa +/- 1) the buffer has significant buffering capacity. Common choices: phosphate (pKa1 = 2.15, pKa2 = 7.20), acetate (pKa = 4.76), Tris (pKa = 8.06), HEPES (pKa = 7.55), MOPS (pKa = 7.20).
What is buffering capacity?
Buffering capacity is the ability of a solution to resist pH changes upon addition of acid or base. Maximum buffering capacity occurs at pH = pKa, where concentrations of the weak acid and conjugate base are equal (ratio = 1). Buffering capacity decreases as pH moves more than 1 unit from pKa.
How do I account for temperature changes affecting buffer pH?
The pKa of many biological buffers changes with temperature. Tris-HCl is particularly sensitive: its pKa decreases by approximately 0.028 per degree Celsius rise in temperature. Phosphate buffers are more stable with temperature. Always re-measure pH at the working temperature.
Can I use this calculator for Tris-HCl buffer preparation?
Yes. Tris has a pKa of approximately 8.06 at 25 degrees Celsius. Enter 8.06 as the pKa, your target pH (e.g., 7.4 or 8.0), and your total buffer concentration to get the ratio of Tris base to Tris-HCl to combine.
Official sources
- NIST Standard Reference Database, Henderson-Hasselbalch: NIST WebBook: Acid Dissociation Constants.
- NIH/NCBI Biochemistry textbook buffer chapter: NCBI Bookshelf: Biochemistry, 5th ed., Chapter 2.
Reviewed by the CalculatorHub team, edited by James Graham, 14 June 2026. See our methodology.