# How to make a pH 6.8 Tris buffer?

To preface this, I am a biologist.

I need to make a $\mathrm{pH}=6.8$, $\pu{1 M}$ Tris buffer. We have access to Tris base and Tris Hydrochloride. (Unfortunately, none of the fancy Tris compounds will work in my situation.)

I tried making a straight Tris base buffer, but before I could lower the $\mathrm{pH}$ to 6.8 (using concentrated $\ce{HCl}$), it crashed drastically. I assume that I got past the buffering capabilities.

Are there any recommendations for this? Thank you.

• You need $\mathrm{pH}$ of your Tris buffer @ 37 degrees, I believe. – Mathew Mahindaratne Mar 31 '18 at 3:10

From Wikipedia...

The conjugate acid of tris has a pKa of 8.07 at 25 °C, which implies that the buffer has an effective pH range between 7.5 and 9.0.

So you're outside the range where TRIS is an effective buffer.

I assume by "crashing" you mean that a precipitate formed. A buffer is also rated based on its "buffer capacity." Effectively this depends on the the concentration of the buffer. Thus solubility of the buffers comes into consideration.

Tris is short for tris(hydroxymethyl)aminomethane. Tris has a $\mathrm {pK_a}$ of $\mathrm {8.1}$ and a $\mathrm {pH}$ level between $\mathrm {7}$ and $\mathrm {9}$. Although that range may change at physological temperature, I doubt it go as low as $\mathrm {pH} = 6.8$ without sacrificing $\mathrm {[B]/[A]}$, which may go as low as $\mathrm {\approx0.05}$.

Thus, I think you should use $\mathrm {BIS-TRIS-propane}$ instead. $\mathrm {BIS-TRIS-propane}$ (1,3-Bis[tris(hydroxymethyl)methylamino]propane) is two Tris molecules connected by 1,3-diaminopropane. It has $\mathrm {pK_a}$s of $\mathrm {6.8}$ and $\mathrm {9.0}$, and effective $\mathrm {pH}$ range of $\mathrm {6.3-9.5}$.

I'm not sure how soluble it in deionized water, but worth trying ($\mathrm {$1/g}$). Probably a good idea to keep your stock solution in$\mathrm {0.5 M}$range. Another choice: Ypu may use N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) (CAS Number: 10191-18-1), instead. It has p$$K_\mathrm{a}$$ of $$7.193$$ at $$\pu{25 ^{\circ}C}$$ (Ref.1) and useful pH range of $$6.4-7.8$$. BES is therefore better suited for pH control in the buffer region of biochemical interest (Ref.1,2). Sigma-Aldrich sells it in 99% pure form (~$1.0/g).

BES is soluble to the extent of $$\pu{3.2 M}$$ at $$\pu{0 ^{\circ}C}$$ (Ref.2) and can be easily purified from aqueous alcohol. Two equimolal buffer solutions consist of BES and sodium besate (sodium salt of BES), each at a molality of $$\pu{0.02 mol kg^{-1}}$$ and $$\pu{0.10 mol kg^{-1}}$$, respectively have shown p$$K_\mathrm{a}$$ value of each solution as $$6.97$$ at $$\pu{37 ^{\circ}C}$$ (Ref.1). On the other hand, a solution prepared with $$\pu{0.03 mol kg^{-1}}$$ of BES and $$\pu{0.02 mol kg^{-1}}$$ of sodium besate has shown p$$K_\mathrm{a}$$ value of $$6.76$$ at $$\pu{37 ^{\circ}C}$$ (Ref.1).

References:

1. R. N. Roy, E. E. Swensson, G. LaCross, Jr., C. W. Krueger, “Standard buffer of N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (Bes) for use in the physiological pH range 6.6 to 7.4,” Anal. Chem. 1975, 47(8), 1407–1410 (DOI: 10.1021/ac60358a076).
2. N. E. Good, G. D. Winget, W. Winter, T. N. Connolly, S. Izawa, R. M. M. Singh, “Hydrogen Ion Buffers for Biological Research,” Biochemistry 1966, 5(2), 467–477 (DOI: 10.1021/bi00866a011).