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Henderson-Hasselbalch Buffer Preparation Calculator

Calculate the masses of acid and base forms needed to prepare a buffer at your desired pH.

Result:

💡 Quick Summary

Prepare any common biological buffer using the Henderson-Hasselbalch equation. Select a buffer system (Phosphate, Tris, HEPES, Acetate, and more), choose your acid and base salt forms, set the desired pH, concentration, and volume — the calculator tells you exactly how many grams of each component to weigh out.

📋 How to Use

Select a buffer from the dropdown, or choose Custom Buffer to enter your own pKa and molecular weights.
For a preset buffer, choose the specific acid (HA) and base (A⁻) salt forms from the sub-dropdowns.
Enter the Desired pH within the effective buffering range of your chosen buffer (pKa ± 1).
Enter Buffer concentration (in M or mM) and Buffer volume (in L or mL).
Click CALCULATE to get the mass of each component to weigh.
Click Reload calculator to reset the page, or Clear all changes to reset the form.

🧮 Formulas & Logic

Henderson-Hasselbalch

pH = pKa + log([A⁻] / [HA])

Ratio

ratio = 10^(pH − pKa)

Acid concentration

[HA] = C / (1 + ratio)

Base concentration

[A⁻] = C − [HA]

Mass of acid

m(HA) = [HA] × V × MW(acid)

Mass of base

m(A⁻) = [A⁻] × V × MW(base)

📊 Result Interpretation

Effective buffering range

A buffer works best within ±1 pH unit of its pKa. Outside this range the buffering capacity drops sharply.

Ratio meaning

ratio = [A⁻]/[HA]. At pH = pKa, ratio = 1 (equal acid and base). Above pKa the base form dominates; below it the acid form dominates.

Salt forms

Many buffers come as multiple hydrated salts. Always select the exact form you are weighing — anhydrous and hydrated forms have different molecular weights.

🔬 Applications

Preparing PBS, Tris, HEPES, acetate, and other common lab buffers
Enzyme assays requiring precise pH control
Cell culture media and biological experiments
Protein purification and chromatography buffers
Electrophoresis running buffers
Custom buffer design using any acid–conjugate base pair

⚠️ Common Mistakes & Warnings

Verify pH with a calibrated pH meter

Calculated masses give a good starting point, but always measure and fine-adjust the pH of your final solution with a calibrated pH meter before use.

pKa shifts with temperature and ionic strength

The pKa values used are at ~25 °C in dilute solution. Tris in particular shifts ~0.03 pH units per °C — prepare Tris buffers at the temperature they will be used.

Make up to final volume, not add solvent volume

Dissolve both components, then add water to reach the final target volume in a volumetric flask. Do not add a fixed volume of water to the solutes.

❓ Frequently Asked Questions

What is the Henderson-Hasselbalch equation?

pH = pKa + log([A⁻]/[HA]). It relates the pH of a buffer solution to the pKa of the weak acid and the ratio of the concentrations of its conjugate base (A⁻) and acid (HA) forms.

Why does the effective buffering range matter?

A buffer resists pH changes most effectively within ±1 unit of its pKa. At pH = pKa ± 1 the ratio [A⁻]/[HA] is 10:1 or 1:10 — the minority species is nearly depleted and can no longer neutralise added acid or base.

Can I use this for Tris buffers?

Yes. Select Tris from the dropdown (pKa 8.1). Remember that Tris pKa is temperature-sensitive — if you are working at 37 °C, the effective pKa is about 7.4, shifting the required ratio. Always measure the final pH at working temperature.

What if my buffer is not in the list?

Choose Custom Buffer. Enter the buffer name, its pKa, and the molecular weights of the acid and base forms. The calculator uses the same Henderson-Hasselbalch equation.