Name: ANDAL, Daniel Seth Date Performed: Jan.

27, 2020
Group number: 2 Date Finished: Jan. 27, 2020

Exercise 1
pH AND BUFFER SYSTEMS
I. RESULTS
Table Error! No text of specified style in document.1.1. Effect of concentration of buffer on buffering
capacity.

Actual pH
Concentration
Case Before addition After addition of ∆pH
(M)
of NaOH NaOH
0.005 6.3 12.3 +6.0
1 (phosphate
0.05 6.2 6.4 +0.2
buffer system)
0.10 6.1 6.3 +0.2
0.005 5.1 11.9 +6.8
2 (acetate buffer
0.05 4.7 5.0 +0.3
system)
0.10 4.7 4.8 +0.1

Table 1.2. Effect of adding NaOH to buffer with different pH.

Actual pH
Calculated Before After
Test tube no. [A-]/[HA] ∆pH
pH addition of addition of
NaOH NaOH
1 0.1 / 1 6.2 5.2 5.8 +0.6
Case 1 2 1/1 7.2 6.1 6.4 +0.3
3 10 / 1 8.2 6.9 11.6 +4.7
4 0.07943282347 / 1 3.7 4.0 4.4 +0.4
Case 2 5 0.7943282347 / 1 4.7 4.7 4.8 +0.1
6 7.943282347 / 1 5.7 5.8 11.6 +5.8

Table 1.3. Effect of adding HCl to buffer with different pH.

Actual pH
Calculated Before After
Test tube no. [A-]/[HA] ∆pH
pH addition of addition of
HCl HCl
1 0.1 / 1 6.2 5.1 3.0 -2.1
Case 1 2 1/1 7.2 6.1 5.7 -0.4
3 10 / 1 8.2 6.9 6.2 -0.7
4 0.07943282347 / 1 3.7 3.7 2.4 -1.3
Case 2 5 0.7943282347 / 1 4.7 4.7 4.4 -0.3
6 7.943282347 / 1 5.7 5.8 5.0 -0.8
Table 1.4. Titration 10.5 1.4 24.0 1.7 37.5 2.1
of an unknown 11.0 1.4 24.5 1.7 38.0 2.1
amino acid.
11.5 1.4 25.0 1.7 38.5 2.1
Volume of 12.0 1.5 25.5 1.7 39.0 2.1
0.1 M pH 12.5 1.5 26.0 1.7 39.5 2.2
KOH (mL)
13.0 1.5 26.5 1.7 40.0 2.2
0 1.5
13.5 1.5 27.0 1.8 40.5 2.2
0.5 1.7
14.0 1.5 27.5 1.8 41.0 2.3
1.0 1.5
14.5 1.5 28.0 1.8 41.5 2.3
1.5 1.5
15.0 1.5 28.5 1.8 42.0 2.3
2.0 1.6
15.5 1.5 29.0 1.8 42.5 2.4
2.5 1.6
16.0 1.5 29.5 1.8 43.0 2.4
3.0 1.4
16.5 1.5 30.0 1.8 43.5 2.5
3.5 1.4
17.0 1.5 30.5 1.9 44.0 2.5
4.0 1.4
17.5 1.6 31.0 1.9 44.5 2.6
4.5 1.4
18.0 1.6 31.5 1.9 45.0 2.7
5.0 1.4
18.5 1.6 32.0 1.9 45.5 2.8
5.5 1.4
19.0 1.6 32.5 1.9 46.0 2.9
6.0 1.4
19.5 1.6 33.0 1.9 46.5 3.1
6.5 1.4
20.0 1.6 33.5 1.9 47.0 4.7
7.0 1.4
20.5 1.6 34.0 1.9 47.5 6.5
7.5 1.4
21.0 1.6 34.5 2 48.0 10.4
8.0 1.4
21.5 1.6 35.0 2 48.5 11.2
8.5 1.4
22.0 1.6 35.5 2 49.0 11.7
9.0 1.4
22.5 1.7 36.0 2 49.5 11.9
9.5 1.4
23.0 1.7 36.5 2 50.0 12.1
10.0 1.4
23.5 1.7 37.0 2.1

14

12

10 pKa2 = 11.2

8
PH

6
ipH = 6.45
pKa1 = 1.7
4

0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50
VOLUME OF KOH (ML)

Figure 1.2. Titration curve of the unknown amino acid sample.

II. SAMPLE CALCULATIONS
Calculations for ∆pH
∆pH = pHf - pHi
12.3 - 6.3 = 6.0 5.8 - 5.2 = 0.6 3.0 - 5.1 = -2.1
6.4 - 6.2 = 0.2 6.4 - 6.1 = 0.3 5.7 - 6.1 = -0.4
6.3 - 6.1 = 0.2 11.6 - 6.9 = 4.7 6.2 - 6.9 = -0.7
11.9 - 5.1 = 6.8 4.4 - 4.0 = 0.4 2.4 - 3.7 = -1.3
5.0 - 4.7 = 0.3 4.8 - 4.7 = 0.1 4.4 - 4.7 = -0.3
4.8 - 4.7 = 0.1 11.6 - 5.8 = 5.8 5.0 - 5.8 = -0.8

Calculations for ipH

𝑝𝐾𝑎1+𝑝𝐾𝑎2
ipH = 2

pKa1 of unknown amino acid = 1.7 pKa2 of unknown amino acid = 11.2
1.7+11.2
ipH = = 6.45
2

III. DISCUSSION

Buffer systems are solutions composed of a weak base and its salt/conjugate acid or a
weak acid and its salt/conjugate base. These solutions are capable of resisting drastic changes in
pH level by adding limited amounts of acid or base. In this exercise, two buffer systems were
used namely, phosphate and acetate buffer. The components of these systems:
Upon addition of limited amount of H3O+:
HPO42- + H3O+ ⇄ H2PO4- + H2O (PB)
CH3COO- + H3O+ ⇄ CH3COOH +H2O (AB)
Upon addition of limited amount of OH-:
H2PO4- + OH- ⇄ HPO42- + H2O (PB)
CH3COOH + OH- ⇄ CH3COO- + H2O (AB)
Whereas H2PO4- serves as the weak acid with HPO42- as the conjugate base in phosphate
buffer and CH3COOH as the weak acid with CH3COO- as the conjugate base in acetate buffer.
These buffer components can affect the buffering capacity depending on their total
concentration. Based on the trend that can be observed in Table 1.1, the buffering capacity and
the total concentration of its components are directly proportional. Higher concentration of buffer
enables it to resist drastic pH change better than having low concentration. This is applicable for
both the phosphate and acetate buffer where 0.10M concentration records the lowest change in
pH after the addition of NaOH.
 Buffering capacity can be influenced by the relative amount of base and acid to each
other. As recorded on Tables 1.2 and 1.3, the addition of NaOH and HCl, respectively, show which
ratio of acid and conjugate base or base and conjugate acid is more effective against drastic pH
change. Theoretically, pH change is at its most minimal if the concentration of the acid and the
base is equal. An effective buffer has a pH equal to its pKa but it does not mean that if these are
equal, the pH would not change upon any addition of acid or base (Bettelheim, Brown, Campell,
Farrell, 2007, p. 157). Both cases of phosphate and acetate buffer conforms to this concept as
Table 1.2 suggest that phosphate buffer has 1/1 concentration of its buffer components and Table
1.3 shows that acetate buffer has 0.7943282347/1 concentration of its buffer components where
it is relatively closer to equal concentration compared to the other pH set-ups in this case. If the
concentration of the conjugate base in the buffer is higher than the weak acid, then the H + ions
from HCl will be neutralized effectively, hence, minimizing changes in pH and have higher
buffering capacity. However, when the conjugate base concentration is higher than the weak
acid, buffer capacity is lowered since there is less H + to neutralize the additional OH- ions from
NaOH (Bettelheim et.al, 2007, p. 157).
Based on the graph on Figure 1.2, the pKa1 is 1.7 and the pKa2 is 11.2 which gives an ipH
of 6.45. The identity of the unknown amino acid is determined to be proline since it is within the
range of the pKa ±1 values and the ipH being relatively close to the actual ipH of proline which
is 6.475. The structures illustrated on the graph were the structure of the amino acid throughout
the experiment whereas the structure at pH 6.5 is where the amino acid exists as a zwitterion
where addition of strong acid or base can react with it by either receiving a proton or donating a
proton. As observed on the graph, the buffer had relatively greater record of resisting drastic pH
change on the range of pH 1.5 to 2.3, where the line graph show a constant result for 42 mL
addition of KOH the, which also supports the pKa range of proline. The ipH was observed to be
at 6.45 since it is the middle point of the slope on the phase where the pH starts to rise drastically.
It cannot act as a buffer at the ipH since a zwitterion has both positive and negative charges and
any addition of an acid or base will favor the reaction to whichever is added in the solution.
Buffers in living systems is of great essence as it comes in the form of blood. Buffer action
is utilized in the capillaries during the exchange of oxygen and carbon dioxide whereas pH in the
blood must be maintained to prevent the occurrence of acidosis or alkalosis. Another example of
buffer action is the relationship of bicarbonate buffer system in the lungs and the kidney
(Castellion and McMurry, 1999, p. 270) as organisms rely on these to maintain homeostasis by
providing optimum environments and pH levels for various biochemical processes inside the cell
(Berg, Tymoczko, Stryer, and Gumport, 2006).
With the note of the importance of buffer systems in living systems, it is also important in
the concept of biochemistry and experiments with regards to it. Mistakes in buffer computations
led unusual discoveries such as the correct number of human chromosomes (Arduengo, 2010)
but the importance of grasping the concept and calculations regarding buffers provides a better
understanding of the trends and properties of different biochemical processes that are conducted
and worked on as an experiment in laboratories and lectures. As it is essential to living beings, it
is also essential to know the way substances work within them in a biochemical perspective.
IV. REFERENCES/LITERATURE CITED

ARDUENGO, P.M. 2010. Sloppy technicians and the progress of science. Promega Connections
http://promega.wordpress.com/2010/03/15/sloppy-technicians.

BERG, J., TYMOCZKO, J., STRYER, L., and GUMPORT, R. 2006. Biochemistry, 5th edition.
Basingstoke: W.H.Freeman & Co Ltd.

BETTELHEIM, F.A., BROWN, W.H., CAMPBELL, M.K., FARRELL, S.O. 2007. Introduction to Organic
and Biochemistry, 6th edition. Belmont, CA: Thomson Brooks/Cole.

CASTELLION, M.E., MCMURRY, J. 1999. Fundamentals of General, Organic, and Biological

Chemistry, 3rd edition. Upper Saddle River, NJ: Prentice Hall.