Titration of Amino Acids and Peptides

Titration is the process used to analyze the acid-base behaviour of amino acids and peptides. A 0.25 g sample of unknown amino acid and aspartame were transferred quantitatively to separate 250-ml Erlenmeyer flask. 20.0 ml of distilled water was added to each flask using a volumetric pipette then the flask was swirled to dissolve the samples. Small increments of 0.200 M HCl were added to both samples to produce an acidic solution and then each sample was titrated by adding 0.200 ml of 0.200 M NaOH. The pH of the acidic solutions was measured from 1.5 until it reaches pH 11 and it was recorded in every addition of the base. On the Aspartame, the total volume of the base added was 8.0 ml and 29.6 ml on the unknown amino acid. Titration curves were constructed based on the pH values and volume added. The identity of the unknown amino acid was revealed to be Aspartic acid. A titrimetric profile of the amino acid and aspartame were drawn.

INTRODUCTION

Proteins are the most important class of biochemical molecules, next are carbohydrates and lipids. Proteins are the basis for the major structural components of animal and human tissue. Proteins are also the indispensable agents of biological function and amino acids are the building block of proteins. Amino acid is an organic compound containing an amino group (-NH2) and a carboxyl group (-COOH). Since amino acids contain both an acidic and a basic group, they undergo an intra molecular acid-base reaction and exist primarily in the form of a dipolar ion, or zwitterions. Amino acids are also amphoteric, meaning, they can react either as acid or base depending on the circumstances. There are basically 20 primary amino acids that serve as the backbone of most proteins.

Peptide is a molecule that consists of two or more amino acid linked together by bonds between the amino group and the carboxyl group. Although their structures are less complex than the larger protein molecules, peptides have significant biological activities.

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Titration is a useful tool in determining the reactivity of amino acid side chains. Since amino acids contain an ionisable group, the predominant ionic form of these molecules in solution depends on pH. Titration of amino acid shows the effect of pH on amino acid structure. Titration is also useful in determining the isoelectric pH of the sample.

MATERIAL AND METHODS

A 0.250 g sample of unknown amino acid powder was placed on a 250 ml beaker using an analytical balance. 20.0 ml of distilled water was added to each flask using volumetric pipette and then the flask was swirled to dissolve the sample. Small increments of 0.200 M HCl were added to the dissolved sample using a syringe until the pH value is 1.50. Both acidified solutions were titrated by adding 0.20 ml of 0.200 M NaOH. The pH was measured and recorded at each amount of base that was added. The procedure was repeated until the pH of the solution reached 11.0. Same process was done using commercial aspartame. Titration curves of the unknown sample and that of aspartame were constructed using MS Excel. The unknown sample of amino acid was identified based on the pKa values and pI that were seen in the constructed titration curves. Titrimetric profiles of aspartame and the identified amino acid were drawn. Then the total percentage errors of the pKa and pI were calculated.

RESULTS AND DISCUSSION

The unknown amino acid was recognized by comparing its experimental value to 20 amino acids. In able to do that, a titration curve must be drawn.

The unknown amino acid was recognized as glutamic acid and it was determined based on its pKa and pI value from the graph. In the titration curve of the unknown amino acid, it can be observed that at the pH reading of 2.16, 10.3 and 4.4 the graph shows an inflection curve making it the pKa values of the unknown sample. These reading were compared with the pKa values the 20 amino acids and it was observed that the experimental pKa values were close to the pKa values of glutamic acid.

Identity of Unknown Amino Acid: Glutamic Acid

Percentage errors between the experimental and theoretical pKa values of the Unknown amino acid were computed and the results were 4.11 %, 6.51 % and 3.53 % and the percentage error between the pI values of experimental and theoretical is 6.32%. The possible sources of errors were probably the inaccurate weighing of the sample and the measurement in adding HCl/NaOH.

During titration, the prototrophic groups lose H+ successively as the pH reach their respective pKa values. The net charge of glutamic acid at the start of the titration is +1 and as each group loses H+ the net charge will decrease by one unit.

The acidified Aspartame solution was titrated using 0.200 M of NaOH. The titration curve above shows the inflection points. These points are 2.5, 10.5 and 3.4.

The possible sources of errors were the inaccurate weighing of the sample and the measurement in adding HCl/NaOH is not precise.

During titration, the prototrophic groups lose H+ successively as the pH reach their respective pKa values. The net charge of aspartame at the start of the titration is +1 and as each group loses H+ the net charge will decrease by one unit.

Other possible source of errors on the experiment was the inaccurate plotting of pKa values on the titration curve. Wrong pKa will cause the identity of the unknown sample to be incorrect.

The buffering zones of glutamic acid are at 2.16 and at 10.3. In aspartame, the buffering zones are at 10.5 and at 3.4. Both are suitable as buffers on acidic pH.

Aspartame has a taste that is close to sugar. It enhances the flavour and does not cause the teeth to decay. There is a scientific study that shows that aspartame is beneficial in weight control and it is helpful for individuals with diabetes because it allows them to please their taste for sweets without affecting their blood sugar level. Aspartame can also result in fewer calories, which helps people with diabetes, manage their weight. Aspartame hydrolyzes into its respective amino acid when heated.

There are also unpleasant side effects of aspartame utilization which includes loss of memory, seizures, headache, blindness, protruding eyes, palpitation, depression, insomnia, diarrhea and skin rashes. Aspartame blocks and lowers the levels of serotonin, tyrosine, dopamine, nor epinephrine and adrenaline. As a result, it is typical that aspartame symptoms cannot be detected in laboratory tests and on x-rays.