Chapter 2

Tris-hydroxymethyl aminomethane (TRIS) is widely used for the preparation of buffers in biochemical research. Shown here is the structure of TRIS in its protonated form: Its acid dissociation constant, \(K_{\mathrm{a}},\) is \(8.32 \times 10^{-9} M .\) You have available at your lab bench a \(0.1 \mathrm{M}\) solution of TRIS in its protonated form, 0.1 \(M\) solutions of \(\mathrm{HCl}\) and \(\mathrm{NaOH}\), and ample distilled water. Describe the preparation of a 1 L solution of 0.02 M TRIS buffer, pH 7.8.

a. Draw the titration curve for Bicine, assuming the \(\mathrm{p} K_{\mathrm{a}}\) for its free COOH group is 2.3 and the \(\mathrm{p} K_{\mathrm{a}}\) for its tertiary amino group is 8.3 b. Draw the structure of the fully deprotonated form (completely dissociated form) of bicine. c. You have available a \(0.1 ~ M\) solution of Bicine at its isoelectric point \(\left(\mathrm{pH}_{\mathrm{I}}\right), 0.1 \mathrm{M}\) solutions of \(\mathrm{HCl}\) and \(\mathrm{NaOH}\), and ample distilled \(\mathrm{H}_{2} \mathrm{O}\) Describe the preparation of 1 L of 0.04 M Bicine buffer, pH 7.5 d. What is the concentration of fully protonated form of Bicine in your final buffer solution?

When a \(0.1 \mathrm{M}\) solution of a weak acid was titrated with base, the following results were obtained: $$\begin{array}{cc}\begin{array}{c}\text { Equivalents of } \\\\\text { base added }\end{array} & \text { pH observed } \\\\\hline 0.05 & 3.4 \\\0.15 & 3.9 \\\0.25 & 4.2 \\\0.40 & 4.5 \\\0.60 & 4.9 \\\0.75 & 5.2 \\\0.85 & 5.4 \\\0.95 & 6.0\end{array}$$ Plot the results of this titration and determine the \(\mathrm{p} K_{\mathrm{a}}\) of the weak acid from your graph.