To verify the buffering capacity of a phosphate solution using a pH meter and construct a titration curve of an amino acid to determine the pka values of its ionisable groups to identitfy an unknown amino acid.
The ratio of [HPO42-] to [H2PO4-] required to produce buffer solutions at pH values 5.9, 6.9 and 7.9 were calculated. 0.1M of H2PO4- and 0.1M HPO42- were used to mix appropriate volumes to 25mL of each of the buffer solutions. The calibrated pH meter was used to measure and record the pH of each buffer solution and then were compared to the pH of 25mL of distilled water. 3.00 mL of 0.1M NaOH was added to each of the 25mL buffer samples and to the distilled water and were mixed well in each tube. The pH of each solution was then recorded after the addition of the alkali. The results were recorded in a table.
20mL of an unknown sample was pipetted into a 100mL beaker. A burette was used to add 0.5mL aliquots of 0.1M NaOH and the pH was recorded from the pH meter. This procedure was repeated until pH of around 11 or 60mL of NaOH had been added with each 0.5mL aliquots being recorded.
Testing of the Henderson-Hasselbach equation
The results in Table 1 reveal the effects on a buffer pH when an alkali solution is added. Table 1.
Buffer pH 22.214.171.124H20
3mL NaOH 6.187.019.7212.61
Change in pH0.42 0.212.025.91
The pka values for each of these amino acids are similar to the table below. Amino acidPka 1Pka 2Pka 3
Information gathered from the results indicates that three different amino acid possibilities; cysteine, lysine or tyrosine. Keeping in mind the possibility of errors having occurred which may have effect the credibility of the graph, it can be assumed unknown amino acid is lysine.
When measuring the buffer capacity of the...