Make a Buffer
A recipe for the buffer you need. Choose the buffer, the pH, the concentration and the volume, and get the grams of the acid and base form to weigh out — or the grams of one form and the volume of NaOH or HCl to bring it to the right pH.
How Do You Make a Buffer?
There are two common ways. Weigh both forms: dissolve the right amounts of the acid form (for example NaH₂PO₄) and the base form (Na₂HPO₄) in a little less water than the final volume, check the pH, then fill up to the mark. Or titrate one form: dissolve the full amount of the acid form and add NaOH until the pH is right (or the base form with HCl, as is usual for Tris). Each mole of NaOH turns one mole of HA into A⁻.
First choose a buffer with a pKa within about one unit of the pH you need. The calculation gives a good starting point, but real solutions differ a little from the ideal equation (ionic strength, temperature), so always check the final pH with a pH meter and adjust.
The Recipe
recipeWorked Techniques
Find the ratio r first, then divide the total amount C·V in that ratio. Multiply each by its molar mass for the grams.
Weigh out the whole amount as the acid form, then add as much NaOH as the base form you need. For Tris it is the other way round: Tris base plus HCl, as much HCl as the acid form you need.
Sources & Verification
- D. L. Nelson & M. M. Cox, Lehninger Principles of Biochemistry, 8th ed., W. H. Freeman, 2021 — Ch. 2 (buffers; the Henderson–Hasselbalch equation).
- N. E. Good et al., “Hydrogen Ion Buffers for Biological Research”, Biochemistry 5(2), 1966, pp. 467–477 — MES, HEPES; pKa and temperature dependence.
- IUPAC, Atomic weights of the elements 2021 — the molar masses, calculated from the formulas.
Frequently Asked Questions
How do I make a phosphate buffer at pH 7.4?
Example: 500 mL of 100 mM sodium phosphate buffer, pH 7.4, using NaH₂PO₄ (acid form) and Na₂HPO₄ (base form), pKa 7.20.
1. Ratio Na₂HPO₄ : NaH₂PO₄ = 10^(7.4 − 7.20) = 1.585.
2. Total amount: 0.100 mol/L × 0.500 L = 50 mmol, split as 19.34 mmol NaH₂PO₄ and 30.66 mmol Na₂HPO₄.
3. Weigh 2.321 g NaH₂PO₄ (119.98 g/mol) and 4.352 g Na₂HPO₄ (141.96 g/mol), both anhydrous. Dissolve in about 400 mL water, check the pH, then make up to 500 mL.
How do I make a Tris buffer?
Tris is usually weighed out as Tris base and brought to the right pH with HCl. For 1 L of 50 mM Tris at pH 8.0: dissolve 6.057 g Tris base and add about 26.7 mL of 1 M HCl while you watch the pH meter, then make up to 1 L. Note that the pKa of Tris falls by about 0.028 per °C: a Tris buffer set to pH 8.0 at 25 °C is about pH 8.6 at 4 °C, so set the pH at the temperature you will use it.
Should I weigh both forms or add NaOH?
Both give the same buffer. Weighing both forms is quick and needs no titration, but you need both chemicals. Weighing one form and adding NaOH (or HCl) lets you fine-tune the pH with a meter, which corrects for small deviations from the ideal equation. Each mmol of NaOH turns one mmol of the acid form into the base form, so the calculator gives the volume to start from.
What if my chemical is a hydrate?
Then use the molar mass of the hydrate, or you weigh out too little. Na₂HPO₄·7H₂O, for example, has a molar mass of about 268.1 g/mol instead of 141.96 g/mol for the anhydrous salt. Choose Other in the calculator and enter the pKa and the molar masses of the forms you actually have.
Why is the measured pH a little different?
The pKa values hold at 25 °C and low salt concentrations. Temperature, ionic strength, the purity and water content of the chemicals and the calibration of the pH meter all shift the result a little. That is why the recipe is a starting point: dissolve in a bit less water than the final volume, adjust the pH with a few drops of NaOH or HCl, and then make up to the mark.