Amino Acid Titrations Explained + Sample Graph Walkthrough

Amino acids are the functional monomers of proteins. The mechanism by which they connect to form proteins is rooted in their functional groups: the amino (NH2) and carboxyl (COOH) groups. These groups are ionizable, which means that they can become protonated or deprotonated, and therefore gain or lose charge.

The conditions for protonation and deprotonation for each functional group depend on the characteristics of the functional group. This characteristic is quantified by the pKa, or acid dissociation constant, of the functional group. The pKa can be thought of as a threshold of pH for which protonation and deprotonation status change. If the pH of a solution of amino acids is below the pKa of the functional group being inspected, the functional group will be protonated. If the pH of a solution of amino acids is above the pKa of the functional group being inspected, the functional group will be deprotonated. 

Inspecting protonation status of the various functional groups on a molecule allows us to determine the charge of that molecule at a given pH. For amino acids with neutral side chains (or R groups), the ionization status is relatively consistent at various pH values. In an amino acid with a neutral side chain, only the carboxyl and amino groups are ionizable. The pKa of the carboxyl group is around 2, and the pKa of the amino group is around 9. Therefore, when the pH of the solution is 6, the carboxyl group is mostly deprotonated and the amino group is mostly protonated. The deprotonated carboxyl group carries a -1 charge, and the protonated amino group carries a +1 charge. The presence of separate charges on the molecule at pH 6 demonstrates the zwitterionic status of amino acids. Because, at pH=6, these charges happen to be equal and opposite, the net charge of the molecule is 0.

However, zwitterions may not necessarily be neutral at pH=6. If the amino acid has a charged R group (aka the pKa of the side chain is not around 6), the amino acid will not be nearly neutral at pH 6. In other words, the isoelectric point (pI) of the amino acid is not 6. How, then, can we determine the isoelectric point of an amino acid with a charged R group? Let’s walk through a titration of a hypothetical charged amino acid to see what’s going on at the atomic level.

Suppose the pKa of the R group is around 3 (the amino acid is acidic). Let’s examine protonation status as we add strong base to the solution. Below pH=2 (which is below the pKas of the carboxyl, R, and amino groups), all three groups are protonated. Therefore, the charge is around +1. Between pH=2 and pH=3, the carboxyl group is deprotonated, but the other groups remain protonated. The overall charge in this pH range is around 0. However, this is an approximation, since the charge is not constant across the range specified. The charge is truly 0 at pH=2.5, because the amino acid has achieved a balance of protonated amino and deprotonated carboxyl groups, resulting in a neutral molecule. From pH=3 to pH=9, all groups other than the amino group are deprotonated, so the overall charge is -1. Above the pKa of the amino group, the charge is -2 because the amino group has been deprotonated. 

This concludes our discussion of amino acid titrations! Here are some cool sources if you’re interested in learning more about this subject:

https://www.youtube.com/watch?v=UT_YFQItvhM - I like this video’s animations and conciseness.

https://fac.ksu.edu.sa/sites/default/files/amino_acid_titration.pdf - This slideshow walks you step-by-step through the logic of the graphs. Additionally, it provides a procedure for this kind of titration experiment and some fun calculations at the end.

https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Organic_Chemistry_(McMurry)/26%3A_Biomolecules-_Amino_Acids_Peptides_and_Proteins/26.03%3A_Amino_Acids_the_Henderson-Hasselbalch_Equation_and_Isoelectric_Points - This brief article explains pI and some other titration stuff succinctly.