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Biochemistry Laboratory Manual  
Titration of Amino Acids - Relevance

Playing an integral role in the structure and function of proteins, amino acids are perhaps one of the most important biomolecules as proteins themselves participate in nearly every physiological event within a cell. In order to understand the acid-base properties of proteins and their resulting behavior as polyionic macromolecules, we will begin by investigating the properties of their constituent amino acids. With each containing at least one amino and carboxylic acid group, amino acids are classified as amphoteric substances and react with both acids and bases as follows:

Titration 1

Titration 2

The ionic form of the amino acid present in an aqueous solution is dependent upon the solution’s pH. In this experiment you will identify an unknown amino acid via an acid-base titration. Titration curves of amino acids are very useful for identification as you can see in the example for glycine given below.

Titration 3

Simple amino acids, like glycine, have two dissociation steps: (1) the loss of H+ from the acidic carboxyl group at low pH; and (2) the loss of H+ from the more basic amino group at high pH. The pKa value for each dissociable group of an amino acid can be determined from such a titration curve by extrapolating the midpoint of each buffering region (the plateau) within the curve. Also revealed from the diagram is a point on the curve where the amino acid behaves as a neutral salt. Specifically, this point is known as the isoelectric point (pI), and is loosely defined as the pH where the amino acid is predominantly a zwitterion. Furthermore, the pI can be approximated as halfway between the two points of strongest buffering capacity and can be estimated by:

Titration 4

where K1 and K2 are the dissociation constants for the deprotonation of glycine’s carboxylic acid and amino groups.

Charged amino acids possess R-groups having acidic or basic side chains giving them more than two dissociable H+ ions. For example, glutamic acid has a carboxylic acid side chain in addition to its α-carboxyl and α-amino groups resulting in a titration curve more complex than that observed for glycine. Where glycine’s titration curve possessed only two plateau regions and thus two pKa values, glutamic acid possesses three—two in the acidic pH region, pKa1 (α-carboxyl group) = 2.2 and pKa2 (γ-carboxyl group) = 4.3; and one in the basic pH region, pKa3 (α-amino group) = 9.7. Members of the basic family of amino acids, like lysine, will also exhibit three pKa values. However, due to the extra amino group, they will have only one pKa in the acidic region and two pKa values in the basic region.

Tables of pKa and pI values of each amino acid are readily available and can be used as standards to identify an unknown amino acid. Furthermore, identification of the regions of the titration curve require a thorough knowledge of the protonation and deprotonation process of an amino acid and an understanding of the definition of an isoelectric point. In summary, titration curves are helpful in the identification of amino acids as follows:

  1. The number of pKa values differentiates polar and nonpolar amino acids from charged amino acids.
  2. The position of the pKa values for charged amino acids allows one to identify positively charged from negatively charged amino acids.
  3. Comparisons between experimental and literature pKa values can allow the identification of a specific amino acid.

Nonetheless, a meticulous titration of an amino acid unknown will produce a titration curve that is distinctive to the amino acid as shown in the figures below:

Titration 5

Titration 6


As these concepts will be used repeatedly during the course of this lab, now is a good time to review if you are not confident in your understanding. A titration is a fast, inexpensive way to determine the identity of an unknown sample; it would not however be a good way to determine the contents of a mixture of amino acids. To that end, you will each be assigned a single unknown amino acid to identify by titration versus a standardized solution of NaOH. Please review the information in the background and watch the video on titration prior to coming to lab so that you are well prepared to perform the experiment.