Tris Buffer pH Calculation: Henderson–Hasselbalch for Tris and Tris-HCl

This calculator estimates the pH of a Tris buffer with the Henderson–Hasselbalch equation. Enter the pKa (8.07 for Tris at 25 °C) and the concentrations of Tris base and of the protonated acid form (Tris-HCl) in mM; you get the pH, the base-to-acid ratio and the total Tris concentration.

Formula

pH = pK_a + log₁₀([base] ÷ [acid]).

• pK_a: about 8.07 for Tris at 25 °C; it falls by about 0.028 per °C rise, so a buffer made at room temperature has a lower pH when warmed
• [base]: concentration of Tris base (the free amine), in mM
• [acid]: concentration of the protonated form, TrisH⁺, supplied as Tris-HCl or made by adding HCl to Tris base, in mM

Worked Example

A buffer with 30 mM Tris base and 70 mM Tris-HCl at 25 °C.

1. Ratio: 30 ÷ 70 = 0.429
2. log₁₀(0.42857) = −0.36798
3. pH = 8.07 − 0.36798 = 7.70
4. Total Tris: 30 + 70 = 100.0 mM

Assumptions

• The pKa of 8.07 applies at 25 °C and low ionic strength; at 4 °C it is roughly 0.6 units higher, at 37 °C roughly 0.3 units lower. Enter the pKa for your temperature if you know it.
• Concentrations stand in for activities, which is an approximation; ionic strength from added salt shifts the effective pKa.
• The useful buffer range is about pKa ± 1, that is pH 7.1 to 9.1 at 25 °C.

How to read the result

When the base and acid concentrations are equal the pH equals the pKa; more base raises the pH and more acid lowers it. The calculated pH is a starting point: the reading of a calibrated pH meter is the real check, and pH depends on temperature and ionic strength, so adjust with small additions of acid or base while you measure. Tris buffers are usually made by titrating Tris base with HCl at the temperature of use.

Limits and common mistakes

• Setting the pH at room temperature and using the buffer cold or warm: the pH moves by about 0.028 per °C.
• Calibrating the pH meter at a different temperature from the buffer.
• Outside about pKa ± 1 the buffer capacity is small and the equation becomes unreliable.

Frequently Asked Questions

What is the pKa of Tris?

About 8.07 at 25 °C, and it drops by roughly 0.028 units for every degree Celsius of warming.

What is the pH of 30 mM Tris base with 70 mM Tris-HCl?

About 7.70 at 25 °C by the Henderson–Hasselbalch equation; check it with a pH meter.

Why does my Tris buffer pH change with temperature?

Because the pKa of Tris itself depends on temperature more than most buffers do.

How much base to acid gives pH 8.07?

Equal concentrations, a ratio of 1, give pH = pKa.

in pka | pKa of Tris | - | 8.07 | 0 | 14
in b | Tris base concentration | mM | 30 | 0.001
in a | Tris-HCl (acid form) concentration | mM | 70 | 0.001
out ph | Buffer pH | - | pka + log10(b / a) | 2
out ratio | Base to acid ratio | - | b / a | 3
out tot | Total Tris concentration | mM | a + b | 1