A solution that resists changes in pH when an acid or base is added is known as a buffer. To create a buffer, a weak acid (HA) is mixed with its conjugate base (A-). When an acid, like HCl, is added to a buffer, the A- can neutralize it and become HA. Similarly, if a base is added, the HA can neutralize it and become A-. The maximum buffering capacity occurs when the pH is equal to the pKa, but a conjugate acid-base pair can still act as an effective buffer when the pH is within approximately ±1 unit of the pKa. If the amounts of HA and A- are equal, the pH is equal to the pKa. In Figure 1.9, a solution containing acetic acid (HA = CH3 - COOH) and acetate (A- = CH3 - COO-) with a pKa of 4.8 resists a pH change from 3.8 to 5.8, with maximum buffering at pH 4.8. When the pH is less than the pKa, the predominant species in solution is the protonated acid form (CH3 - COOH). On the other hand, when the pH is greater than the pKa, the predominant species is the deprotonated base form (CH3 - COO-).

The titration curve of an amino acid can be analyzed similarly to acetic acid. Let's consider alanine as an example, which contains an ionizable α-carboxyl and α-amino group. At a low pH, both of these groups are protonated. As the pH increases, the -COOH group can donate a H+ to the medium and dissociate, forming the carboxylate group (-COO-), which is shown as the dipolar form II or the isoelectric form of alanine. The dissociation constant of the carboxyl group of an amino acid is called K1, and it can be analyzed using the Henderson-Hasselbalch equation, similar to acetic acid. The amino group of alanine is the second titratable group, and it is a weaker acid than the -COOH group, with a smaller dissociation constant, K2. When a H+ is released from the protonated amino group, alanine transforms into the fully deprotonated form, form III. The pKa for the most acidic group (-COOH) is pK1, while the pKa for the next acidic group (-NH3+) is pK2. The pKa of the α-carboxyl group of amino acids is approximately 2, whereas the pKa of the α-amino group is around 9.
A buffer is a solution that resists change in pH following the addition of an acid or base. A buffer can be created by mixing a weak acid (HA) with its conjugate base (A−). If an acid such as HCl is added to a buffer, A− can neutralize it, being converted to HA in the process. If a base is added, HA can likewise neutralize it, being converted to A− in the process. Maximum buffering capacity occurs at a pH equal to the pKa, but a conjugate acid-base pair can still serve as an effective buffer when the pH of a solution is within approximately ±1 pH unit of the pKa. If the amounts of HA and A− are equal, the pH is equal to the pKa. As shown in Figure 1.9, a solution containing acetic acid (HA = CH3 – COOH) and acetate (A− = CH3 – COO−) with a pKa of 4.8 resists a change in pH from pH 3.8 to 5.8, with maximum buffering at pH 4.8. At pH values less than the pKa, the protonated acid form (CH3 – COOH) is the predominant species in solution.
At pH values greater than the pKa, the deprotonated base form (CH3 – COO−) is the predominant species.
C. Amino acid titration
The titration curve of an amino acid can be analyzed in the same way as described for acetic acid.
1.
Carboxyl group dissociation: Consider alanine, for example, which contains an ionizable α-carboxyl and α-amino group. [Note: Its –CH3 R group is nonionizable.] At a low (acidic) pH, both of these groups are protonated (Fig. 1.10). As the pH of the solution is raised, the −COOH group of form I can dissociate by donating a H+ to the medium. The release of a H+ results in the formation of the carboxylate group, −COO−. This structure is shown as form II, which is the dipolar form of the molecule (see Fig. 1.10). This form, also called a zwitterion (from the German word for “hybrid”), is the isoelectric form of alanine, that is, it has an overall (net) charge of zero.
2.
Application of the Henderson-Hasselbalch equation: The dissociation constant of the carboxyl group of an amino acid is called K1, rather than
Ka, because the molecule contains a second titratable group. The Henderson-Hasselbalch equation can be used to analyze the dissociation of the carboxyl group of alanine in the same way as described for acetic acid: where I is the fully protonated form of alanine and II is the isoelectric form of alanine (see Fig. 1.10). This equation can be rearranged and converted to its logarithmic form to yield: 3.
Amino group dissociation: The second titratable group of alanine is the amino (−NH3+) group shown in Figure 1.10. Because this is a much weaker acid than the –COOH group, it has a much smaller dissociation constant, K2. [Note: Its pKa is, therefore, larger.] Release of a H+ from the protonated amino group of form II results in the fully deprotonated form of alanine, form III (see Fig. 1.10).
4.
Alanine pKs: The sequential dissociation of H+ from the carboxyl and amino groups of alanine is summarized in Figure 1.10. Each titratable group has a pKa that is numerically equal to the pH at which exactly one half of the H+ have been removed from that group. The pKa for the most acidic group (−COOH) is pK1, whereas the pKa for the next most acidic group (−NH3+) is pK2. [Note: The pKa of the α-carboxyl group of amino acids is ~2, whereas that of the α-amino group is ~9.] 5.