EXERCISE 2

Buffer System

Maganaka, Klimpol C.
Group 4 Sec. DEFG-1L

July 10, 2013

____________________
1A scientific paper submitted in partial fulfillment of the requirements in Chemistry 160.1
laboratory under Prof. Noreen Fundador, 1st sem., 2013-2014.

INTRODUCTION
Almost every biological process is pH dependent; a small change in pH produces a large
change in the rate of the process. Cells and organisms maintain a specific and constant
cytosolic pH, keeping biomolecules in their optimal ionic state, usually near pH 7. In multicellular
organisms, the pH of extracellular fluids is also tightly regulated. Constancy of pH is achieved
primarily by biological buffers: mixtures of weak acids and their conjugate bases (Lehninger,
2004). According to Chang (2010), a buffer is a solution of (1) a weak acid or a weak base and
(2) its salt; both components must be present. The solution has the ability to resist changes in
pH upon the addition of small amounts of either acid or base. A buffer solution must contain a
relatively large concentration of acid to react with any OH- ions that are added to it, and it must
contain a similar concentration of base to react with any added H+ ions. Furthermore, the acid
and the base components of the buffer must not consume each other in a neutralization
reaction. In order for a buffer solution to be suitable in an experiment or application, it has to be
soluble in water, should not react with each other and has a characteristic pH zone in which it is
effective by the formula: pH=pKa 1
The buffering capacity, that is, the effectiveness of the buffer solution, depends on the amount of
acid and conjugate base from which the buffer is made. The larger the amount, the greater the
buffering capacity (Chang, 2010). On the other hand, the Henderson-Hasselbalch equation is
simply a useful way of restating the expression for the dissociation constant of an acid. For the
dissociation of a weak acid HA into H+ and A- which is:
This study aimed to reinforce the understanding of buffer systems. The specific objectives were:
1. to create buffer systems of different buffering capacities, 2. to add an acid or a base to the
generated buffer,
3. to measure the buffers pH using the pH meter,
4. to make a titration curve for glycine, identify the pKas and to compute its pI.
METHODOLOGY
The experiment has three sections, the first one is the factors affecting the buffering capacity. It
is divided into two parts: effect of concentration of buffer and effect of acid to conjugate base
pair ratio. In the former, a stock solution of 100mL of 0.5M phosphate buffer was prepared at pH
7. An aliquot was taken from it and then diluted with distilled water to achieve a concentration of
0.05M and 0.001M, this was done by using the formula: c1v1=c2v2. The actual pH of the three
buffer solutions was recorded using the pH meter. Furthermore, 2mL of 0.1M NaOH was added
to the buffer solutions and their pH was again recorded. These steps were repeated using an
acetate buffer with pH 4.7. For the latter, a phosphate buffer having pH 5.2, 6.2, 7.2, 8.2 and 9.2
with a concentration of 0.5M were created. 2mL of 0.1M NaOH was added to 25mL of each
buffer and the pH was recorded. The buffer was made using Henderson-Hasselbach equation.
The same is true for the addition of 0.1M HCl instead of NaOH. In making the 0.1M NaOH and
0.1M HCl, the molarity equation was applied (M=mole/liter). These steps were repeated for
acetate buffer having a pH of 2.7, 3.7, 4.7, 5.7 and 6.7.

The second section of the experiment was the titration with amino acid. 40mL of 0.1M glycine
was obtained. This is possible by also using the molarity equation. The pH of the solution was
recorded and 1mL of 2.5M NaOH was added to it and the pH was then measured again. There
was a continuous addition of 1mL 2.5M NaOH to the solution and the pH was recorded after
each addition until it reaches a pH of 12.
The last section was the selection of an appropriate buffer. A sample was obtained from the
instructor and its pH was measured. 10mL of 0.5M phosphate buffer with a pH of 7.2 was mixed
with 5mL of the sample. Additional 1mL of 0.1M NaOH was placed in the solution and the pH
was recorded. Lastly, these steps were repeated using 0.5M acetate buffer having a pH of 4.7.
RESULTS AND DISCUSSION
As seen in Table 1, there is no distinct pattern to see the effect of the concentration of the buffer
except for 0.001M phosphate buffer which exhibits an extreme change in pH which is an ideal
one because upon addition of a strong base having a much lower concentration than the buffer
itself, there should be no great change in the pH of the buffer. Since 0.001M is much lower than
the 0.1M NaOH, then there is a change in pH because the strong base would use up the acid
thereby increasing the buffers pH. But these results are not reliable including to those that are
shown in Table 2 because the conductors of the experiment failed to measure the pH of 0.05M
and 0.001M phosphate and acetate buffers after an aliquot was taken from the stock solution
which is the 0.5M buffers. All solutions must have a constant or same pH which is 7.2 and 4.7
for the phosphate and acetate buffers, respectively to be able to see the differences. So there
was a human error upon conducting the experiment which in effect failed to distinguish the
effect of the buffers concentration.
A buffer works in a pH range equal to 1 unit from its pKa. In Table 3, the pH 7.2 should work
effectively as a buffer ranging from pH 6.2 to pH 8.2 but results showed that it is not because
there is a drastic change in the pH 8.2 which became pH 9.040 after addition of NaOH and
7.970 after the addition of HCl. And the pH 5.2 as well as pH 9.2 should show drastic changes in
pH after addition of the strong base and acid since it doesnt belong in the range anymore. In
Table 4, the pH 4.7 of acetate buffer having a range of 3.7 to 5.7 exhibits a good working buffer
since the values are closer to each other and therefore, it didnt undergo drastic change. On the
other hand, the pH 6.7 underwent drastic change upon addition of 0.1M NaOH and this is an
ideal one but upon addition of 0.1M HCl, it underwent little change. For pH 2.7, it also went
through a little change in pH. The ideal one is that there should be a drastic change. There
should be no extreme change in pH when the buffer solution is at its 1 range in pKa.
Table 5 shows the pH of glycine for every addition of 1mL 2.5M NaOH until reaches a pH of 12.
As one adds NaOH, the pH of the solution increases. This is also shown graphically in Figure 1.
Glycine is a kind of a buffer because it contains a carboxyl and amino groups. As measured, the
actual pH of glycine is 5.480 and as 1mL of 2.5M NaOH was added, it raised to pH 8.215. From
this data, there is a great discrepancy between the pH values. Because ideally, in Figure 2, the

starting pH must be lower than 3 which is very unlikely in the obtained pH which is 5.480. In the
ideal titration curve of glycine, it has two pKas. The 1st pKa value is 2.34 and the 2nd pKa value
is 9.60 according to literature. Since the data obtained is not coherent with the theoretical one,
the 1st pKa value cant be plotted since the starting pH jump up to 5 but the 2nd pKa value can
be plotted on the curve. The effective buffer region of glycine is from pH 1.34 to pH 3.34 for the
1st pKa while pH 8.60 to pH 10.60 is the effective buffer region of glycine. Structurally, there is a
loss of H+ from the acidic carboxyl group at low pH which is shown by diagram of anionic form
of glycine and a loss of H+ from the more basic amino group at high pH which is the cationic
form of glycine. The calculated pI of glycine is 5.97 where in this point, it behaves as a neutral
salt and the pH where the amino acid is predominantly a zwitterion.
On the other hand, two different unknown samples having 0.5M phosphate and acetate buffers
are shown respectively in Table 6. The most appropriate buffer solution is the one that contains
the 0.5M phosphate buffer because 7.296 is much more closer to 6.048 than the 4.707 which is
way too far from the given sample. The buffer range of the sample must be from 5.048 to 7.048
since the acetate buffer is away from the range, it is not suitable. On the other hand, the
phosphate buffer didnt fall in the range, but still, it is relatively closer to the given sample pH.
I. Factors affecting the buffering capacity
A. Effect of concentration of a buffer
Table 1.Actual pH of the 3 phosphate buffer solutions and after addition of 0.1M
NaOH. Phosphate buffer
Actual pH
pH after addition of 0.1M NaOH
0.5M
7.158
7.383
0.05M
7.002
11.873
0.001M
6.652
11.495
Table 2.Actual pH of the 3 acetate buffer solutions and after addition of 0.1M NaOH.
Acetate buffer
Actual pH
pH after addition of 0.1M NaOH
0.5M
4.675
4.653
0.05M

4.513
4.854
0.001M
4.603
11.455
B. Effect of acid to conjugate base pair ratio
Table 3. The pH of different phosphate buffers after the separate addition of 2mL
0.1M
NaOH and 0.1M HCl.
pH of 0.5M Phosphate buffer
Actual pH
pH after addition of 2mL 0.1M NaOH
pH after addition of 2mL 0.1M HCl
5.2
5.244
5.401
5.063
6.2
6.207
6.245
6.155
7.2
7.235
7.305
7.168
8.2
8.231
9.040
7.970
9.2
9.186
9.933
8.430
Table 4. The pH of different acetate buffers after the separate addition of 2mL 0.1M
NaOH and 0.1M HCl.
pH of 0.5M Acetatate buffer
Actual pH
pH after addition of 2mL 0.1M NaOH
pH after addition of 2mL 0.1M HCl
2.7
2.744

3.705
2.286
3.7
3.704
3.78
3.533
4.7
4.695
4.704
4.530
5.7
5.695
5.740
5.467
6.7
6.652
11.508
5.889
II. Titration with amino acid
Table 5. The pH of 1.0M glycine every additional 1mL of 2.5M NaOH. Additional 1mL
of 2.5M NaOH
pH
0mL
5.480
1mL
8.215
2mL
8.625
3mL
8.918
4mL
9.101
5mL
9.298
6mL
9.437
7mL
9.582
8mL
9.709
9mL
9.824

10mL
9.914
11mL
10.091
12mL
10.225
13mL
10.371
14mL
10.751
15mL
11.173
16mL
12.125
Volume of NaOH
Figure 1. The titration curve of glycine.
Figure 2. Ideal titration curve of glycine.
Calculation:
= (2.34 + 9. 60)
= 5.97
III. Selection of an appropriate buffer
Table 6. The pH of the sample with phosphate and acetate buffers, separately and
0.1M NaOH. pH of the sample
6.048
pH of the sample with 10mL 0.5M phosphate buffer and 1mL 0.1M NaOH 7.296
pH of the sample with 10mL 0.5M acetatate buffer and 1mL 0.1M NaOH 4.707
CONCLUSION
Buffer solutions are the ones that resist drastic changes in pH when a strong acid or
a strong base is introduced in the solution. The buffering capacity which is the
ability of the solution to keep the pH at a narrow range, increases as the molar
concentration (molarity) of the buffer salt/acid solution increases. Therefore, the
ratio of the acid and its conjugate base influences the pH of a solution while its
actual concentrations influence the effectiveness of a buffer. On the other hand, a
buffer is effective when its pH range from 1 pKa. The more the acid and its
conjugate base molecules available, the less of an effect addition of a strong acid or
base will have on the pH of a system.

LITERATURE CITED
Chang, R. 2010. Chemistry.10th ed. New York: McGraw-Hill.p.717-718.
Cox, M.M and Nelson,D.L. Lehninger Principles of Biochemistry.4th ed. p.72-73.
http://chemed.chem.wisc.edu/chempaths/GenChem-Textbook/pH-and-ConjugateAcid-Base-Pairs-619.html accessed on June 09, accessed on June 09, 2013
https://www.boundless.com/chemistry/acid-base-equilibria/buffereffectiveness/relative-amounts-of-acid-and-base/, accessed on June 09, 2013