Biochemistry Essay

Aim:
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.

Method:
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.

Results.
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 5.9 6.9 7.9 H20
Initial pH 5.76 6.8 7.7 6.7
3mL NaOH 6.18 7.01 9.72 12.61
Change in pH 0.42 0.21 2.02 5.91

The pka values for each of these amino acids are similar to the table below.
Amino acid Pka 1 Pka 2 Pka 3
Cysteine 1.71 8.33 10.78
Lysine 2.18 8.95 10.53
Tyrosine 2.02 9.11 10.07

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.

Discussion:
When measuring the buffer capacity of the phosphate solution using the pH meter, the addition of an alkali to the solution created a minor shift in pH (0.8 average) in the right direction as alkalis increase pH levels. “Buffer solutions….resists change in pH when small quantities of an acid or alkali are added.” (D.L. Nelson and M.M Cox, 2008). This shift is pH is significantly minor when compared to the shift in pH of the distilled water where the shift in pH was 5.91, this significant shift pH is also a result of an error, the initial amount of NaOH that was supposed to be added was 2mL but instead 3mL was added which may result in larger than expected shifts in all pH values.
By creating a titration curve (plot of pH against the amount of 0.1M NaOH added), it was revealed the unknown solution was a weak acid and that three possible amino acids were conceivable; cysteine, lysine or tyrosine. However lysine displayed the closest resemblance to the graph and hence the unknown solution is expected to be lysine due to the pKa values. A possible sources of error could have been the accuracy of the amount of 0.5mL aliquots added, adding more or less than 0.5 might have slightly altered the shape of the graph but even so, this wouldn’t be significant enough to alter or question the identity of the unknown amino acid.

Reference
Nelson D.L, Cox M.M. (2008), Principles of Biochemistry , Titration Curves Reveal the pKa of Weak Acids, 58.
Conclusion
The buffer capacity of a phosphate solution was verified as only an average minor shift of 0.8 in pH had occurred when compared to the change in pH of distilled water 5.91 and the through the construction of a titration curve, an unknown amino acid was identified as lysine.