Buffer pH Calculator

Calculate the pH of a buffer solution using the Henderson-Hasselbalch equation.

pKa of the weak acid

Acetic acid: 4.76, Phosphate: 7.21, HEPES: 7.55

Moles or concentration of conjugate base [A⁻] (mol/L)

Moles or concentration of weak acid [HA] (mol/L)

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How Buffers Resist pH Change

A buffer contains appreciable amounts of both a weak acid and its conjugate base. Added acid is consumed by the base form; added base is consumed by the acid form. Because only the ratio of the two appears in the equation, moderate additions barely move the pH.

pH = pK_a + log([A⁻]/[HA])

Two consequences follow directly from that logarithm. First, when the two concentrations are equal the log is zero and pH = pK_a. Second, because it is a ratio, dilution does not change buffer pH — both concentrations fall together and the ratio is preserved. Dilution does reduce capacity, however.

Ratio [A⁻]/[HA]	log term	pH relative to pK_a
1:10	−1	pK_a − 1
1:1	0	= pK_a
4:1	+0.60	pK_a + 0.60
10:1	+1	pK_a + 1

Choosing a Buffer

Select the buffer whose pK_a is closest to your target pH, and stay within roughly one unit either side. Beyond that, the ratio becomes so lopsided that one component is nearly exhausted and capacity collapses.

Buffer	pK_a (25°C)	Useful range	Notes
Citrate	3.13 / 4.76 / 6.40	2.1–7.4	Polyprotic; chelates metal ions
Acetate	4.76	3.8–5.8	Simple, inexpensive
MES	6.15	5.5–6.7	Biological, minimal metal binding
Phosphate	2.15 / 7.20 / 12.35	6.2–8.2	Physiological; precipitates with Ca²⁺
HEPES	7.55	6.8–8.2	Cell culture standard
Tris	8.06	7.1–9.1	Large temperature coefficient

Two practical warnings. Phosphate is the obvious choice near pH 7.2 but precipitates with calcium and magnesium, making it unsuitable for many cell culture media. Tris has a pK_a that shifts by roughly −0.03 per °C, so a Tris buffer adjusted to pH 8.0 at 25°C sits near 7.7 at 37°C — adjust it at the working temperature.

Worked Examples

Example 1: Acetate buffer: pKa=4.76, [NaOAc]=0.1, [HOAc]=0.1

pH = 4.76 + log(1)

Result: pH = 4.76 (equal concentrations = pKa)

Maximum buffer capacity at pH = pKa

Example 2: Phosphate: pKa=7.21, [HPO4²⁻]=0.08, [H2PO4⁻]=0.02

pH = 7.21 + log(4)

Result: pH = 7.82

Shift toward base raises pH

Example 3: Preparing a buffer at a target pH

Need pH 5.2 from acetate, pK_a = 4.76

Result: Ratio [A⁻]/[HA] = 10^0.44 = 2.75

Rearranging gives the required ratio directly. Using 0.1 M total buffer means about 0.073 M acetate and 0.027 M acetic acid.

Example 4: Effect of adding strong acid

100 mL of buffer with 0.05 mol each form, plus 0.005 mol HCl

Result: pH falls from 4.76 to 4.67

The acid converts 0.005 mol of acetate to acetic acid, changing the ratio to 0.045/0.055. The shift is under 0.1 pH units — unbuffered water would drop to about pH 1.3.

Example 5: Why Tris shifts with temperature

Tris adjusted to pH 8.0 at 25°C, used at 37°C

Result: Actual pH about 7.7

The pK_a falls roughly 0.03 per degree. Over 12 degrees that is about 0.36 units — enough to affect enzyme assays significantly.

Common Mistakes

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Adjusting Tris pH at the wrong temperature

Its pK_a changes about −0.03 per °C. A buffer set to pH 8.0 on the bench is near 7.7 in a 37°C incubator. Always adjust at the temperature of use.

⚠️

Choosing a buffer more than one pH unit from its pK_a

The ratio becomes extreme and one component is nearly exhausted, so capacity collapses. Pick a buffer whose pK_a is near your target instead.

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Using phosphate with divalent cations

Phosphate precipitates calcium and magnesium salts. In media containing them, HEPES or MES is the better choice despite the higher cost.

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Assuming dilution changes buffer pH

It barely does, since only the ratio matters. What dilution reduces is capacity — the diluted buffer is far more easily overwhelmed by added acid or base.

Frequently Asked Questions

What is buffer capacity?

Buffer capacity is maximum at pH = pKa (ratio 1:1). A buffer works best within ±1 pH unit of its pKa. Outside this range, pH shifts dramatically with small acid/base additions.

Which buffer for biological pH 7.4?

HEPES (pKa 7.55), MOPS (pKa 7.2), or phosphate buffer (pKa 7.21) are ideal. Phosphate is most common but can interfere with some enzymatic reactions.

Why does buffer pH equal pK_a when concentrations are equal?

Because the ratio is 1 and log(1) = 0, leaving pH = pK_a. This is also the point of maximum buffer capacity.

Does diluting a buffer change its pH?

Barely. Only the ratio of conjugate base to acid appears in the equation, and dilution preserves it. Capacity falls in proportion to concentration, however.

Which buffer should I use at pH 7.4?

Phosphate (pK_a 7.20) or HEPES (7.55) are standard. HEPES is preferred when calcium or magnesium is present, since phosphate precipitates them.

How far from pK_a can a buffer work?

About one pH unit either side. Beyond that the ratio exceeds 10:1, one component approaches exhaustion, and capacity falls off steeply.

Why is Tris temperature sensitive?

Its protonation enthalpy is unusually large, giving a pK_a coefficient near −0.03 per °C. Buffers must be adjusted at their working temperature.

Formula Explorer connections

Interpretation: This relationship connects hydrogen-ion activity, dissociation, conjugate ratios or titration stoichiometry to acid–base behavior. Assumption: Concentration approximates activity mainly in dilute solutions. Temperature, ionic strength, acid strength and equilibrium approximations affect accuracy.

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