Potentiometric Measurements

In pH-metric titration, precisely known volumes of a standardized strong acid (e.g., HCl) or base (e.g., KOH or NaOH) are added to a vigorously-stirred solution of a protogenic substance, during which pH is continuously measured with a precision combination glass electrode, in a procedure confined to the interval pH 1.5-12.5. The substance ( 50-500 p.M or higher) being assayed is dissolved in 2-20 mL of water or in a mixed solvent consisting of water plus an organic water-miscible cosolvent [e.g., methanol, dimethylsulfoxide (DMSO), acetonitrile, or 1,4-dioxane]. An inert water-soluble salt (0.15 M KCl or NaCl) is added to the solution to improve the measurement precision, and to mimic the physiological state. Usually, the reaction vessel is thermostated at 25°C and a blanket of a heavy inert gas (argon, but not helium) bathes the solution surface.

The plot of pH against titrant volume added is called a potentiometric titration curve. Figure 3.1a shows two examples. The shape of such a curve can suggest the amount of substance present and its characteristic acid-base ionization properties. The left curve in Fig. 3.1a represents a strong acid-base titration, containing no sample species. The curve on the right side of Fig. 3.1a is that of morphine-6-glucuronide (M6G), which has three pKa values (XH^ ! XH^ ^ XH— ^ X2—) [151]. The inflection points corresponding to where the slope in such plots is maximum in size are called endpoints (pH 7 in the left curve, pH 5.5 and 10 in the right curve). At the endpoint the sample is almost completely in one state of ionization (e.g., XH^ zwitterion at pH 5.5). The inflection points where the slope is at a minimum size designate regions of maximum buffering (pH 8.8 in the morphine metabolite curve). At such a point the molecule is present in two states of protonation of equal concentration (pH = pKa), unless two or more overlapping pKa values are in the buffer region. So by inspection of Fig. 3.1a, one can say that apKa of M6G may be ^8.8. (We will see in the next section that such a simple interpretation of the titration curve can lead to the wrong conclusion, because M6G has two overlapping pKa values centered about pH 8.8.) Where are the other pKa values of M6G? Unfortunately, a titration curve does not always reveal all the pKa values that a molecule may have. To reveal the other two pKa values of M6G and to test for overlapping pKa values, it is necessary to transform the titration curves into Bjerrum plots [112,116,118,153,163-165].

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