Experiment 11 used a titration curve to determine the identity of an unknown amino acid. The initial pH of the solution was 1.96, and the pKa's found experimentally were 2.0, 4.0, and 9.85. The accepted pKa values were found to be 2.10, 4.07, and 9.47. The molecular weight was calculated to be 176.3 while the accepted value was found to be 183.5. The identity of the unknown amino acid was established to be glutamic acid, hydrochloride.
Amino acids are simple monomers which are strung together to form polymers (also called proteins). These monomers are characterized by the general structure shown in figure 1.
Although the general structure of all amino acids follows figure 1, the presence of a zwitterion is made possible due to the basic properties of the NH2 group and the acidic properties of the COOH group. The amine group (NH2) is Lewis base because it has a lone electron pair which makes it susceptible to a coordinate covalent bond with a hydrogen ion. Also, the carboxylic group is a Lewis acidic because it is able to donate a hydrogen ion (Kotz et al., 1996). Other forms of amino acids also exist. Amino acids may exists as acidic or basic salts. For example, if the glycine reacted with HCl, the resulting amino acid would be glycine hydrochloride (see fig. 2). Glycine hydrochloride is an example of an acidic salt form of the amino acid. Likewise, if NaOH were added, the resulting amino acid would be sodium glycinate (see fig. 3), an example of a basic salt form.
Due to the nature of amino acids, a titration curve can be employed to identify an unknown amino acid. A titration curve is the plot of the pH versus the volume of titrant used. In the case of amino acids, the titrant will be both an acid and a base. The acid is a useful tool because it is able to add a proton to the amine group (see fig. 1). Likewise the base allows for removal of the proton from the carboxyl group by the addition of hydroxide. The addition of the strong acid or base does not necessarily yield a drastic jump in pH. The acid or base added is unable to contribute to the pH of the solution because the protons and hydroxide ions donated in solution are busy adding protons to the amine group and removing protons from the carboxyl group, respectively. However, near the equivalence point the pH of the solution may increase or decrease drastically with the addition of only a fraction of a mL of titrant. This is due to the fact that at the equivalence point the number of moles of titrant equals the number of moles of acid or base originally present (dependent on if the amino acid is in an acidic or basic salt form). Another point of interest on a titration curve is the half-equivalence point. The half-equivalence point corresponds to the point in which the concentration of weak acid is equal to the concentration of its conjugate base. The region near the half-equivalence point also establishes a buffer region (Jicha, et al., 1991). (see figure 4).
The half-equivalence point easily allows for the finding of the pKa values of an amino acid. A set pKa values can be extremely helpful in identifying an amino acid. Through a manipulation of the Henderson-Hasselbalch equation, the pH at the half-equivalence point equals the pKa. This is reasoned because at the half-equivalence point the concentration of the conjugate base and the acid are equal. Therefore the pH equals the pKa at the half-equivalence point (see figure 5.)
Fig. 5 [base]
pKa= pH - log -------
log -------- = log 1 = 0
therefore, pH = pKa
However, many substances characteristically have more than one pKa value. For each value, the molecule is able to give up a...