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Short answer. Tris-HCl buffer is a solution of tris(hydroxymethyl)aminomethane (Tris) partly neutralised with hydrochloric acid, so that Tris base and its protonated form coexist. Its pKa is about 8.07 at 25 °C, giving a useful range of roughly pH 7.1 to 9.1, and its pH shifts by about 0.03 units per °C, so it has to be adjusted at the temperature of use.
This page is for bench researchers and lab staff who need to name, prepare and record a Tris-HCl buffer correctly. It is general laboratory information, not safety or clinical advice.
Definition and scope
Tris (C4H11NO3, molar mass 121.136 g/mol) is an organic amine widely used as a biological buffer. The name “Tris-HCl” is used in overlapping ways, and mixing them up is the commonest source of confusion:
- Tris base: the free amine (121.14 g/mol). Dissolved alone it is basic.
- Tris hydrochloride (Tris-HCl): the solid salt, CAS 1185-53-1, molar mass 157.60 g/mol for the Sigma-Aldrich BioUltra product. A 4 M solution of that salt is acidic (pH 2.5 to 4.0), so the salt alone is not a buffer at working pH.
- “Tris-HCl buffer, pH 7.5”: a working solution in which Tris is brought to a stated pH with HCl. The label should state concentration, pH and the temperature at which the pH was set.
Manufacturer data give a pKa of 8.1 at 25 °C and a useful range of pH 7.0 to 9.0 (Sigma-Aldrich Trizma hydrochloride, T5941); Wikipedia gives 8.07 at 25 °C and about pH 7.1 to 9.1. A different manufacturer data sheet quotes 8.31 at 20 °C, a reminder that pKa values are only comparable at the same temperature.
How Tris-HCl buffer is prepared
How to prepare Tris-HCl buffer?
The method in the Fisher Scientific buffer preparation sheet is: dissolve Tris base, adjust to the target pH with HCl, then make up to final volume. Adjust the pH at the temperature at which the buffer will be used.
How do I make 50 mM Tris-HCl buffer?
The figures below are our own calculation from the molar masses above and the Henderson-Hasselbalch equation with pKa 8.07. They are not a manufacturer table, so confirm the final pH with a calibrated meter.
- 50 mM Tris base: 0.05 mol/L × 121.136 g/mol = about 6.06 g per litre.
- Target pH 7.5: the fraction present as protonated Tris is 1 / (1 + 10(7.5 − 8.07)), about 0.79, so roughly 40 mmol of HCl is needed per litre (about 79% of the Tris). In practice add HCl gradually while watching the meter rather than trusting the arithmetic.
- Starting from the 157.60 g/mol salt alone, 50 mM is 7.88 g per litre, but that solution is acidic, so base must be added to reach a buffering pH.
Record the pH-adjustment temperature on the bottle. For the generic steps (water quality, volumetric flasks, filtering, storage) see the buffer and solution preparation guide.
What does Tris-HCl do in a buffer?
It resists pH change in the mildly alkaline range around its pKa. Between about pH 7.1 and 9.1 the base and its conjugate acid are both present in useful proportions, so added acid or base shifts the pH only modestly. Tris is also a component of electrophoresis buffers such as TAE and TBE.
What is the buffering range of Tris-HCl?
Roughly pH 7.1 to 9.1, centred on the pKa of about 8.07 (Wikipedia, read 2026-11-07), or pH 7.0 to 9.0 per Sigma-Aldrich’s product page. Near pH 7.0 the buffer is weak; for targets near neutrality a buffer with a pKa closer to that value, such as HEPES or phosphate, is the more natural fit.
Temperature shift: the practical catch
Tris has a large temperature coefficient. Sigma-Aldrich’s data sheet reports about 0.03 pH units per °C between 5 and 25 °C and about 0.025 per °C between 25 and 37 °C; Fisher Scientific gives about 0.028. Cooling raises the pH and warming lowers it. By the 0.03 figure, a buffer set to pH 7.5 at 25 °C reads about 0.15 units higher at 20 °C, so a Tris buffer used in a cold room should be adjusted at that temperature. Wikipedia also notes that a ten-fold increase in concentration raises the pH by about 0.05.
Checklist: what to record for a Tris buffer
| Item | Why (source) |
|---|---|
| Concentration of Tris | Concentration changes pH slightly, about 0.05 per ten-fold (Wikipedia). |
| pH and the temperature at which it was set | About 0.025 to 0.03 pH units per °C (Sigma-Aldrich; Fisher Scientific). |
| Starting material: Tris base or Tris-HCl | Different molar masses (121.14 versus 157.60 g/mol) and different starting pH. |
| Electrode type | Silver-containing single-junction electrodes can be clogged by a silver-Tris precipitate; use a double-junction or non-silver electrode (Wikipedia). |
Related buffers
Bis-Tris is a related compound used in gel systems, HEPES is a zwitterionic buffer, and sodium phosphate buffer is the common alternative for near-neutral work. A broader introduction is in what a biological buffer is.
Common misunderstandings
- “Tris-HCl” powder is not the buffer. Dissolved alone it is acidic; it becomes a working buffer only once adjusted to the target pH.
- Fixing the pH at room temperature is enough. For use at 4 °C or 37 °C the pH will differ by several tenths.
- Tris is chemically inert. Its primary amine reacts with aldehydes, it can complex metals and it inhibits some enzymes (Wikipedia), so it does not suit every assay.
FAQ
What is the Tris pKa?
About 8.07 at 25 °C (Wikipedia); 8.1 per Sigma-Aldrich. It changes with temperature.
Is Tris-HCl the same as Tris buffer?
Not exactly. “Tris-HCl” usually means the hydrochloride salt or a Tris buffer titrated with HCl; state the pH, concentration and temperature.
Sources
- Wikipedia, “Tris”, en.wikipedia.org/wiki/Tris, read 2026-11-07.
- Sigma-Aldrich, Trizma hydrochloride T5941 product page, sigmaaldrich.com/product/sigma/T5941, read 2026-11-07.
- Sigma-Aldrich Tris data sheet 93362, PDF, read 2026-11-07.
- Fisher Scientific, buffer preparation sheet, PAGE744.PDF, read 2026-11-07.
