Department of Pure and Applied Chemistry

College of Arts and Sciences

Visayas State University

Date Performed: June 8, 2017 Score: ________

Date Submitted: June 16, 2017 Prepared by: BS Chem-2

Exercise No. 1
pH and BUFFER SYSTEM

Objectives:
 To calibrate the pH meter.
 To choose and prepare appropriate buffer systems.
 To titrate an amino acid.

Results:
Factors Affecting Buffer Capacity
1. Effect of Concentration of Buffer

Table 1.1. Phosphate Buffer (0.1M), pH of 7.2

Concentration Buffer + Change in
Buffer Type Original pH Actual pH
(M) NaOH (2mL) pH
Phosphate 0.005 7.2 5.58 6.07 0.49
Phosphate 0.05 7.2 5.77 7.74 1.97

Table 1.2. Acetate Buffer (0.1M), pH of 4.7

Concentration Buffer + Change in
Buffer Type Original pH Actual pH
(M) NaOH (2mL) pH
Acetate 0.005 4.7 3.09 3.57 0.48
Acetate 0.05 4.7 3.36 4.23 0.87

2. Effect of the Ratio of the Conjugate Base to the Weak Acid

Table 2.1. Ratio of Dihydrogen phosphate and Monohydrogen phosphate
Concentration Expected Ratio Buffer + NaOH Change
Buffer Type (Base:Weak Acid, in moles)
(M) pH (2mL) in pH
Phosphate 0.1 8.2 9.77:1 4.02 0.32
Phosphate 0.1 6.2 0.0977:1 11.37 5.67

Table 2.2. Ratio of Acetic acid and Acetate

Concentration Expected Ratio Buffer + NaOH Change
Buffer Type (Base:Weak Acid, in moles)
(M) pH (2mL) in pH
Acetate 0.1 3.7 0.0902:1 4.02 0.59
Acetate 0.1 5.7 9.016:1 11.37 4.53
Titration of an Amino acid (Alanine)
Table 3. Amount of NaOH added into the Alanine solution

Volume of NaOH (mL) pH of the Solution

0.00 1.49
0.50 1.58
1.00 1.61
1.50 1.65
2.00 1.69
2.50 1.74
3.50 1.80
4.50 1.88
5.50 1.97
6.50 2.05
8.50 2.16
10.50 2.33
12.50 2.62
14.50 2.82
16.50 3.15
18.50 4.91
20.50 8.71
22.50 9.14
24.50 9.46
26.50 9.69
28.50 9.91
30.50 10.03
32.50 10.23
34.50 10.46
36.50 10.69
38.50 11.01
40.50 11.41
42.50 11.78
44.50 12.00
Graph 1. Tritration of an Amino acid

Titration of Amino acid (Alanine)

14

12

10
pH of the Solution

0
0 5 10 15 20 25 30 35 40 45 50
Volume of NaOH

Discussion
First task that was performed was to identify the effect of concentration of buffer. Two different
buffers namely phosphate and acetate buffers with pHs of 7.2 and 4.7, and each buffer (both with
initial concentrations of 0.1M) were diluted to prepare two solutions of each buffer of different
concentrations, 0.005M and 0.05M. First case was the phosphate buffer. As it can be seen in the table,
at 0.005M the pH was 5.58. After adding NaOH, the pH was 6.07, therefore, the pH has increased by
0.49. At 0.05M, the pH recorded was 5.77, and after adding NaOH the pH was 7.74. So, the pH has
increased by 01.97. For acetate, at 0.005M, the pH was 3.09 and after adding NaOH the pH became
3.57. At 0.05M the pH was 3.36 and became 4.23 after adding NaOH. So, the pHs at 0.005M and 0.05M
had increased by 0.48 and 0.87.
Base on the results from tables 1.1 and 1.2, the pHs of the buffers increase with increasing
concentrations. Upon addition of a strong base namely sodium hydroxide, NaOH, big changes on the
pHs occurred indicating that the pHs of the buffer solutions are already outside the range of their
buffering capacities, due to the acids present in the buffer systems are almost used up to react with
the added base causing the pHs of the buffers to rise.
As shown in table 2.1, 9.77 moles dihydrogen phosphate is to 1 mole monohydrogen ratio, in
enough water, is required to make a 250 mL buffer solution with a pH of 8.2 and ratio of 0.0977 mole
dihydrogen phosphate is to 1 mole monohydrogen phosphate ratio, in enough water, is required to
make a buffer solution with a pH of 6.2, both were prepared from a stock phosphate buffer solution
with an initial concentration of 0.1M. After adding 2 mL of NaOH into the two different buffers, the pHs
were 4.2 (at pH 8.2) and 11.37 (at pH 6.2), therefore, the two pHs of the buffers changed by 0.32 and
5.67. In table 2.2, it is also shown that 1 mole acetic acid is to 0.0902 mole acetate and 1 mole acetic
acid is to 9.016 moles acetate ratios, in enough water, were required to make two 250 mL acetate
buffer solutions with pHs of 3.7 and 5.7 respectively. Each acetate buffer was prepared from a stock
acetate buffer solution with an initial concentration of 0.1M.
After adding 2 mL 0.1M NaOH, the pHs of the buffer solutions had increased, except for the one
phosphate buffer (pH of 8.2) where its pH decreased after adding 2 mL 0.1M NaOH. Their pHs changed
by 0.32 and 5.67 for phosphate buffers with pHs of 8.2 and 6.2, and 0.59 and 4.53 for acetate buffers
with pHs of 3.7 and 5.7. As it can be seen in the values of pHs, it can be said that there were some errors
committed upon preparing the buffer solutions with corresponding pHs. One error is maybe the
volumes of the stock buffer solutions used to prepare the said buffers were not precise in order to
achieve the required pHs and another one is improper use of the pH meter, or the glass electrode was
not washed properly after dipping it from one solution to the other.
Table 3 shows the volume of NaOH added into the amino acid buffer solution, specifically
alanine, resulting to a change in pH of the buffer little by little after increment addition of the strong
base. By plotting the values for volumes of NaOH and pHs, the student has obtained a curve shown in
graph 1. From the graph, the first pKa is 2.25 and the second pKa is 8.71 and these pka values are close
to the theoretical pKas of alanine which are 2.33 (pKa1) and 9.1 (pKa2). By looking at table 3, the pH is
increasing as the amino acid was being titrated with NaOH and it is evident in the titration curve in the
graph. The 2.33 is the pKa of carboxylic group, which is lower than the second pKa (9.1). This means
that carboxylic group is the first one to give up a proton the most readily. While the amine group is the
last one to give up a proton because amine group is relatively stable with a positive charge and
generally prefer to have more hydrogens attached.

Conclusion
To calibrate a pH meter, it is appropriate to use buffer solutions with pHs of 4, 7, and 10, which
are the ideal pHs of buffer solutions. Rinse first the electrode with distilled water and dry it with a piece
of tissue paper before placing it in a buffer solution with pH 7, 4, or 10, and rinse again with distilled and
dry the electrodes with tissue paper before and after placing it different acids/bases to avoid
contaminating the buffer solution and errors when obtaining the pHs of the solutions.
To have an effective buffer solution, one must make it out of a weak acid/base and its
conjugate acid/base or its salt with a pH close/equal to the pKa in order to achieve a maximum
buffering capacity.
Upon titrating an amino acid, there will be two or more pKas that can be obtain. Depending on
what amino acid is used in titration because amino acid has many functional groups that can be
deprotonated.

Reference(s) and Link(s):

https://prezi.com/qlglmiohar-I/determinie-pka-values-for-amino-acid-functional-groups/
Chang, Raymond. Chemistry. Ninth Edition.
Ferrier, Denise. Lippincott’s Illustrated Reviews Biochemistry. Sixth edition.
Answers to Questions.
1. In protein precipitation, 2 L of 5 mM buffer solution with pH 5.2 is needed in the isolation of
albumin. Which among the following buffer solutions is best fitted for the said purpose? Justify
your answer.
Buffer solutions pKa
Acetate buffer 4.73
Tri-(hydroxymethyl)-aminomethane 8.08
Phosphate buffer 7.20
Acetate buffer is the one best fitted buffer because its pKa is the one closest to the needed pH
of 5 mM buffer solution. If pH is close or equal to pKa, a buffer solution can achieve its maximum
buffering capacity. Thus, it can absorb hydronium or hydroxide ions without a significant change in pH.
2.
Appendix

Calibration of pH Meter Prepared 0.05M and 0.005M Titration of Amino Acid

Phopsphate Buffers (Alanine)
 NaOH
pH Change Vol. Change pH dVol/dpH eq vol. pKa Vol pKa
Vol.
0.00 1.49 0.50 0.090 5.56 6.50 9.50 2.25
0.50 1.58 0.50 0.030 16.67 12.50 20.50 8.71
1.00 1.61 0.50 0.040 12.50 28.50 0 0
1.50 1.65 0.50 0.040 12.50 0 0 0
2.00 1.69 0.50 0.050 10.00 0 0 0
2.50 1.74 1.00 0.060 16.67 0 0 0
3.50 1.80 1.00 0.080 12.50 0 0 0
4.50 1.88 1.00 0.090 11.11 0 0 0
5.50 1.97 1.00 0.080 12.50 0 0 0
6.50 2.05 2.00 0.110 18.18 0 0 0
8.50 2.16 2.00 0.170 11.76 0 0 0
10.50 2.33 2.00 0.290 6.90 0 0 0
12.50 2.62 2.00 0.200 10.00 0 0 0
14.50 2.82 2.00 0.330 6.06 0 0 0
16.50 3.15 2.00 1.760 1.14 0 0 0
18.50 4.91 2.00 3.800 0.53 0 0 0
20.50 8.71 2.00 0.430 4.65 0 0 0
22.50 9.14 2.00 0.320 6.25 0 0 0
24.50 9.46 2.00 0.230 8.70 0 0 0
26.50 9.69 2.00 0.220 9.09 0 0 0
28.50 9.91 2.00 0.120 16.67 0 0 0
30.50 10.03 2.00 0.200 10.00 0 0 0
32.50 10.23 2.00 0.230 8.70 0 0 0
34.50 10.46 2.00 0.230 8.70 0 0 0
36.50 10.69 2.00 0.320 6.25 0 0 0
38.50 11.01 2.00 0.400 5.00 0 0 0
40.50 11.41 2.00 0.370 5.41 0 0 0
42.50 11.78 2.00 0.220 9.09 0 0 0
44.50 12.00 0 0 0 0 0 0

20.00
Titration of Amino acid (Alanine)
18.00 6.5,18.18
28.50,16.67
16.00
pH of the Solution

14.00
12.00
10.00 12.50,10.00
Vol.-pH
8.00
6.00 dvol/dpH
4.00
2.00
0.00
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00
Volume of NaOH