ASI, Ranelle Janine L.

Chemistry 160.1 Section 3L

2015

Date performed: June 22, 2015

Date submitted: June 24,

EXERCISE 2. pH and Buffer Systems

Post-Laboratory Report
Buffer systems keep the pH of a solution within a narrow range to maintain a
stable environment for many biological processes, the reactions of which occur
optimally at around pH 7. Buffers resist drastic change in optimal pH levels even
upon addition of strongly acidic or basic species involved in living systems. Buffers
are also used in biochemical experiments to produce in vitro conditions of living
systems under study or to simply set-up a nearly pH-constant medium for chemical
applications.
In this laboratory exercise, two variables were studied to determine their
effects on acetate (pH 4-6) and phosphate (pH 6-8) buffers. The first variable was
the concentration of the components of the two buffer systems at constant
acid/base-to-conjugate base/acid ratio (hence same pH). Table 2.1 shows the
changes in pH of acetate and phosphate buffers at varying concentrations after
addition of NaOH, a strong alkali species.
Table 2.1. Effect of concentration of buffer on buffering capacity.
Actual pH
Concentration
Before
addition
After addition
Buffer used
(M)
of NaOH
of NaOH
0.005
4.65
11.33/11.85
0.05
4.65
5.42
Acetate
0.1
4.57
4.83
buffer
0.005
7.00
12.00
Phosphate
0.05
6.98
10.71
buffer
0.1
6.98
7.47

pH
7.08/7.20
0.77
0.26
5.00
3.73
0.49

Results showed a noticeable trend of increasing pH change as concentrations

decrease for both buffers. Since buffer efficiency is measured by its resistance to
drastic pH changes, the lower the difference, the more efficient the buffer is at the
set concentration. Both acetate and phosphate buffers are most efficient at 0.1M
and least at 0.005M.
The addition of NaOH to the solutions produced hydroxide ions (OH -) that
reacted with the weak acid components of the systems in the reactions:
CH3COOH + OH- <==> CH3COO- + H2O
H2PO4- + OH- <==> HPO42- + H2O
Reactions 2.1.
At 0.1M buffer concentration, enough weak acid molecules could react with
the hydroxide ions to prevent them from drastically changing the pH. However, at

0.005M buffer concentration, the small amount of weak acid molecules was
consumed and the rest of the hydroxide ions from NaOH brought the hydronium
(H3O+) concentration down, therefore increasing the pH significantly.
The second variable studied was the pH levels or the ratio of the conjugate
base to the weak acid determined by addition of a strong base, NaOH, (Table 2.2)
and a strong acid, HCl (Table 2.3). In this set-up, the varying concentrations of weak
acid per 1 part conjugate base influenced the pH changes of buffers with the same
concentrations due to equilibrium dynamics.
Upon addition of NaOH (Reactions 2.1), both buffers showed highest
efficiency when the amount of the conjugate base is equal to the amount of the
weak acid as almost all OH- ions are neutralized by the weak acid present in
sufficient amounts unaffected by equilibrium shifting.
In the solutions where the weak acid has higher concentration, buffer
efficiency is also maintained as the OH- ions from NaOH combine with the H+ ions
dissociating from the weak acid to form water molecules. As a result, the
equilibrium shifts to favour the production of more H+ ions until all OH- ions are
neutralized and pH change is minimized. However, the equilibrium shift resulted to
a lower concentration of the weak acid than in the original buffer, leading to a
decrease in H+ ions, hence the final solutions have slightly higher pH compared to
those with equimolar components.
In the solutions where the conjugate base has higher concentration, buffer
efficiency is weakest as the presence of excess acetate or monohydrogen
phosphate suppresses the ionization of acetic acid and dihydrogen phosphate,
decreasing the amount of H+ ions and raising the pH of the solutions. Upon addition
of NaOH, pH changes significantly as the weak acids, already in low concentration,
are mostly unable to neutralize the OH- ions.
Table 2.2. Effect of adding NaOH to buffer with different pH.
Actual pH
Before addiAfter
Test tube no.
[A ]/[HA]
Calculated
tion
of
addition
of
pH
NaOH
NaOH
1
0.1/1
6.2
5.86
6.77
Phospha
2
1/1
7.2
6.85
7.46
te buffer
3
10/1
8.2
7.56
8.85
4
0.1/1
3.7
3.73
4.36
Acetate
5
1/1
4.7
4.67
5.11
buffer
6
10/1
5.7
5.57
11.67
On the other hand, upon addition of HCl, protons are donated to the
conjugate base of each buffer through the reactions:
CH3COO- + HCl <==> CH3COOH + ClHPO42- + HCl <==> H2PO4- + ClReactions 2.2.

pH

0.91
0.61
1.29
0.63
0.44
6.1

In the solutions where the concentration of the conjugate base is higher, the
increase of H+ ions from addition of HCl is counteracted by association with the
excess of conjugate base ions to form unionized weak acids. Hence, the added H+
ions are neutralized and the pH changes are minimized, resulting to high buffer
efficiency.
However, in the solutions with higher weak acid concentrations, HCl
consumes the scarce conjugate base and the rest of the protons are donated to
water molecules, forming hydronium ions that dramatically decrease pH.
The set-up for acetate buffer shows inconclusive data as all solutions
changed pH drastically. This may be due to contamination or improper pH readings.
Table 2.3. Effect of adding HCl to buffer with different pH.
Actual pH
Before
addiAfter
Test tube no.
[A ]/[HA]
Calculated
tion of
addition of
pH
NaOH
NaOH
1
0.1/1
6.2
5.96
3.09
Phospha
2
1/1
7.2
6.92
6.28
te
3
10/1
8.2
7.51
7.17
buffer
4
0.1/1
3.7
3.73
2.50
Acetate
5
1/1
4.7
4.67
2.28
buffer
6
10/1
5.7
5.57
3.50

pH

2.87
0.64
0.34
1.23
2.39
2.07

In addition to the study of pH and factors that affect buffer action, a sample
of an unknown amino acid (either Alanine or Leucine) was characterized using
titration with 0.1M KOH. The resulting titration curve shown in Figure 2.1 implies
that the amino acid has two pKa values.

14
12

pKa2
8.9

10
8
pH

IpH
5.6

pKa1
2.2

4
2
0
0

10

12

14

Volume 0.1M KOH (mL)

Figure 2.1. Titration curve of an unknown amino acid sample.

The resulting pKa1 and pKa2 values based on the curve are 2.2 and 8.9,
respectively. These pKa values are the optimal pH where they can act as buffers.
Theoretically, buffer action by this amino acid will be observed at the ranges of 1.2 3.2 pH and 7.9 - 9.9 pH.
IpH was computed using the formula (pKa 1 + pKa2)/2, yielding to 5.6 which
falls correspondingly near the middle of the region with drastic pH change. At this
range, this amino acid cannot act as buffer as it exists in a zwitterionic form, a state
in which an amino acid acts as both an acid and a base due to the presence of both
positive and negative charges, rendering it reactive to addition of any strong acid or
base.
Although the value for pKa2 is inconclusive, since the unknown is only
between Alanine and Leucine, Leucine is the more probable candidate as its
theoretical pKa1 and IpH are 2.33 and 5.97, respectively.