The aim of this experiment was to determine the titration curves exhibited by two amino acids (Glutamic acid and Alanine) and use them to determine their Pka values and their isolectric point. Results from the experiment reveal several interesting facts as will be seen throughout this paper. As ampoteric compounds, this paper seeks to investigate these base-acid properties found in amino acids. Therefore, this paper will have the following basic parts; introduction, procedure for titration, discussion of titration results with reference to the titration curves as well as a conclusion. The introduction part will give an in-depth look at the sources, functions and properties of amino acids and more specifically Glutamic acid and Alanine. The discussion part will give a detailed analytical report of the experiment whereas the conclusion part will concentrate on the findings or deductions made from the experiment. For this experiment, Glutamic acid and Alanine will be titrated against NaOH. Subsequently, the changes in Ph will be observed and a curve plotted against the concentration of the base. Experimental results will also be used to explain PKa, and PI values which are actually very important in the identification of the various amino acids. The pKa values which are determined experimentally through titration are very important in differentiating nonpolar and polar amino acids. The identification of these values is also used in identifying negative and positive amino acids.
Amino acids form the building blocks of proteins. Amino acids are naturally occurring are acquired in the body through ingestion of food. They are thus vital bio-molecules since proteins take part in almost all cellular physiological processes. Amino acids are polyionic molecules, which are amphoteric. An amino acid is essentially an organic acid that contains an amino group (NH2) as well as a carboxyl group (-COOH). Due to the fact an amino acid contains both a basic and an acid group; it usually undergoes an intra molecular acid base reaction and exists mostly as a dipolar ion also referred to as zwitterions.
Because of its amphoteric properties, an amino acid can react with both a base and an acid..Amino acids react with bases and acids as follows.
Majority of amino acids have two phases of disassociation. These are;
- Loss of hydrogen ions to the carboxyl group (acidic)
- Loss of hydrogen from the amino group at extreme pH.
Then this implies that during titration of amino acids, there is a neutral point that is characterized by PI. When a titration curve is drawn for the amino acids, there is a point of inflection between the anionic wave and the cationic wave that represents this neutral point.
In an aqueous solution, the form the present amino acid depends on the solution’s pH. Titration becomes a very valuable tool in determining the reactivity of the side chains of any amino acid. A titration curve of an amino acid is the plot of the amino acids against the neutralization degree of the acid by a strong base such as NaOH. The titration with NaOH occurs in two stages as shown in the equations below.
+NH3CH(R)COOH + OH- +NH3CH(R)COO- + H2O
+NH3CH(R)COO- + OH- NH2CH(R)COO- + H2O
The titration of amino acids using NaOH shows the effect that pH has on the structure of an amino acid. Additionally, the titration curve of an amino acid is often used to approximate the pKa values of the amino acids ionizable groups as well as its pI.
The titration curves are usually gotten when in when the pH of an amino acid solution fluctuates after the successive addition of an alkali or an acid. In many cases, the curves are plots of pH against the volume of the titrant being added or in other cases like in this experiment against the number of equivalents that are added per mole of the given sample.
As stated earlier, the main aim of this experiment was to determine the titration curves of two amino acids and use these curves to approximate the pKa values of the amino acids’ ionizable groups. The experiment also sought to comprehend the acid base tendencies of an amino acid.
In this experiment, two amino acids were (Glutamic acid and Alanine) were titrated using a standard base (NaOH).
- 100 ml of 0.1M Glutanic acid was prepared in H20.
- 25 ml of this solution was further diluted with water to form a new solution
- The solution was then titrated with standard hydrochloric acid up to a pH of 1.5
- Another portion of this solution was then titrated with standard NaOH up to a ph of 11.5
- The experiment was repeated again using Alanine as the amino acid
- The results were recorded and the titration curves of the two amino acids were then constructed using Microsoft Excel and the pKa and pI values derived from the curves.
Results and Discussions
Data acquired from the titration of glutamine was used to plot titration curve for glutamine. Being amphoteric, Glutamic has two titrable parts. This is because glutamine is partly basic and partly acidic. Between the basic phase and acidic phase in glutamine stands the isoelectric point. As seen in the titration curve for glutamine, the inflection point of the curve that lies between the PKa for both the anionic and the cationic phases. The curve has three waves and the point of inflection (PI) is 3. The two PKa values are 2 and 4 respectively. The graphical results agree with mathematical results since PI is calculated as the mean of the two pKa values. Consequently, the first PKa represents the Ph of glutamine in the anionic phase whereby the second PKa represents the Ph of the amino acid in the cationic phase. As earlier mentioned this is because glutamine is amphoteric exhibiting both acidic and basic phase. In the anionic part, the amino group is protonated by the addition of OH- whereby in the cationic phase deprotonation of the carboxylic group takes place. PI marks the beginning of the cationic (zwitterions phase) phase and the end of the anionic phase. In other words during titration, protonation represents the dissociation of the amino part and deprotonation represents the disassociation of the carboxylic group.
After recording the observations from the experiment, a titration curve of Alanine was drawn. The titration curve of Alanine has two waves. This is because it is a diprotic acid. In Alanine, the two titrable groups are the amino group and the carboxyl group. At a very low pH, Alanine contains an amino group that is positively charged and a protonated carboxyl group (uncharged). Alanine therefore has a positive charge of one at the beginning of the experiment. As the sodium hydroxide (base) is added, the carboxyl group of the Alanine loses the proton (that is, it becomes deprotonated) and hence turns into a negatively charged carboxylate group. In fact, this is the first equivalence point of the entire titration. The pH of the equivalence point can be calculated as an average of the pka’s of the amino and the carboxyl groups. At this particular pH, Alanine does not carry any charge as seen from the titration curve and is the isoelectric point of the amino acid. The ph of the solution thus increases. In addition, the alpha-carboxyl of the solution disassociates at the first pKa. The alpha-amino also disassociates at the second pKa. At each pKa, Alanine solution is buffered. This means that it resists changes in its pH base is added. It is also important to note that the pi happens where the Alanine has no net charge. From the graphs, the pka values of the COOH and the NH3 groups 2.34 and 9.91 respectively. The isoelectric point form the graph is around 5.46.
The results from the above experiment were clearly accurate because they were quite consistent to what would be expected for the two amino acid groups. In fact, the pKa and the pI from the two sets of acids were very close to the scientific tabulated values. The behaviour of the two main subgroups of amino acids, that is the carboxyl and the amino groups were clearly shown in the experiment and the nature of their titration curves were also shown.
Campbell, M. K. (1999). Biochemistry. Philadelphia: Saunders College Pub.