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Tris Buffer Calculator (Tris-HCl)

Calculate the Tris base and HCl (or Tris-HCl and Tris base) for a Tris buffer at a target pH, correcting the pKa for temperature and showing the pH at the temperature of use.

Formula

pKa(T)=8.07−0.028 (T−25 ∘C)\mathrm{p}K_{\mathrm{a}}(T) = 8.07 - 0.028\,(T - 25\,^\circ\mathrm{C})
nHCl=C1+10 pH−pKa+[H+]−[OH−]n_{\mathrm{HCl}} = \dfrac{C}{1 + 10^{\,\mathrm{pH}-\mathrm{p}K_{\mathrm{a}}}} + [\mathrm{H}^+] - [\mathrm{OH}^-]
m=C×V×MWm = C \times V \times \mathrm{MW}
pKa(T)\mathrm{p}K_{\mathrm{a}}(T)
pKa of protonated Tris at temperature T
CC
total Tris concentration

How it works

Tris buffers well between about pH 7 and 9. It is most often made by dissolving Tris base and titrating with hydrochloric acid to the target pH. The amount of HCl follows from the fraction of Tris that has to be protonated at that pH, which is set by the pKa, plus a small correction for the free hydrogen and hydroxide ions.

Tris has an unusually large temperature dependence: its pKa falls by about 0.03 per °C, so a buffer set to pH 7.4 at 25 °C reads about 7.06 at 37 °C, and higher in the cold. The page corrects the pKa for the temperature at which you set the pH, and reports the pH at the temperature of use. Set the pH at the temperature you will use it.

Worked example

1 L of 50 mM Tris at pH 7.4 (25 °C), from Tris base and 1 M HCl.

  1. pKa = 8.07; the fraction protonated at pH 7.4 is 1 / (1 + 10^(7.4 − 8.07)) = 0.8239, so 41.19 mmol of HCl.
  2. Tris base: 0.05 mol × 121.14 g/mol = 6.057 g.

6.057 g of Tris base and 41.19 mL of 1 M HCl in a litre. At 37 °C the pH of the same solution is about 7.06.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • An ideal solution; the pKa of 8.07 at 25 °C, falling linearly with temperature.
  • The HCl concentration is that stated, and its volume is small compared with the batch.
  • The pH is set at the temperature given.

Common mistakes

  • Setting the pH at room temperature for a buffer used at 4 °C or 37 °C, then finding it far from target.
  • Adjusting Tris with a large excess of HCl and overshooting, which adds chloride and raises the ionic strength.
  • Using Tris with aldehyde fixatives or in assays sensitive to primary amines.

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Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.