Postlab Report on

Exercise no. 2:
pH AND BUFFER SYSTEMS

Jonathan C. Lim
CHEM 161.1-1L
Midyear AY 2016-2017

Groupmates:

Joshua Gemperoso
Angelica Marie Ramos
Aaron James Rolando Santiago
Andrew Exequiel Tabilog

Performed on June 14, 2017

Submitted on June 16, 2017

Ms. Rochelle Ibabao

I. Results and Discussion

Buffers are solutions that can resist drastic changes in pH when a small amount of an acid or

a base is added to it. It has two main components- the acid/base and its salt (Kuchel, 2009). The

mechanism by which a buffer system resists drastic changes upon addition of small amounts of acids

or bases are shown below:

Dissolution reaction:

𝐻𝐴 + 𝐻2 𝑂 ⇌ 𝐴− + 𝐻3 𝑂+ (2.1)

Reaction with acid:

𝐴− + 𝐻3 𝑂+ → 𝐻𝐴 + 𝐻2 𝑂 (2.2)

Reaction with base:

𝐻𝐴 + 𝑂𝐻 − → 𝐴− + 𝐻2 𝑂 (2.3)

The pH of a buffer solution may be calculated using the Henderson Hasselbach equation:

[𝐵𝐴𝑆𝐸]
𝑝𝐻 = 𝑝𝐾𝑎 + log [𝐴𝐶𝐼𝐷] (2.4)

The pH of a solution may be obtained through the use of indicator dyes, pH papers or pH meters.

For a buffer to be effective and efficient in carrying out chemical reactions, a buffer system

must possess these characteristics: a) freely soluble in water for easy preparation, b) relatively inert

so that it may not interfere to the chemical reactions occurring in the solution, c) does not form metal

complexes to avoid drop in the pH of the system, d) should be non-absorbing in the UV-Vis region

for spectrophotometric analyses, e) should be available in pure form, also for easier preparation, f)

should be nontoxic and g) should not be costly. In addition, buffers in biological systems must: a)

have a pH within 6 to 8 since many biological processes are carried out in neutral pH; b) impermeable
to prevent accumulation in cell or its organelles; and c) have an ionic strength that cannot alter

biological processes (AppliChem, 2008).

Buffer systems have intensive and extensive properties. Buffering range, which refers to the

pH range where it can work to resist changes brought by the addition of small amounts of acids or

bases, is an intensive property of a buffer system. Most of the simple buffer systems known is

effective at pKa±1.0. This is due to the fact that the buffering capacity at pH equals pKa is most

effective. Going one unit beyond the ranges means a severe decrease in the buffering capacity of the

buffer system. Buffering capacity, on the other hand, is an extensive property of buffer systems which

is a measure of the buffer efficiency in resisting changes. It is defined as the amount of acid or base

that is required to raise or decrease the pH of 1 liter of solution by 1 unit (Mohan, 2003). The buffer

capacity may be computed by the formula below

∆𝐵
𝛽 = ∆𝑝𝐻 (2.5)

Where β is the buffering capacity, ΔB is the gram equivalent of strong acid/base to change pH of 1

liter of buffer solution, and ΔpH is the pH change. Buffering capacity can be affected by two factors.

These are the buffer concentration and the conjugate base to acid ratio.

In reality, buffers are found in all biological organisms such as plants, animals and

microorganisms. Most of the biological processes in our body involve acid-base reactions, which

may easily disrupt equilibrium in the body since the molecules in our body are pH-sensitive. For

example, deoxyribonucleic acids (DNA) in our body are stable at neutral pH. Once the pH of its

environment comes close to 9, the DNA dissociates until it fully dissociates at pH 10. If this happens,

severe damages to the health of a person may occur. Buffers work to minimize possible impairments

caused by slight changes in pH in the concentration of acids or bases in the body, such as this, and

establish homeostasis in the body (Berg, 2015).

 There are several buffers that are found in biological systems. Such buffers are the following:

carbonate-carbonic acid buffer which aims to maintain blood pH at 7.4 (easily regulated by the release

of CO2 in breathing and the release of HCO3- in urination). When blood pH decreases, the organism

has a condition called acidosis. On the other hand, an organism with high blood pH has a condition

called alkalosis; protein buffer systems which regulate pH in extracellular and intracellular fluids and

interact with other buffer systems; and phosphate buffer which buffers the intracellular fluids and is

used in the laboratory to mimic biological chemical processes (Hrycyna, 2013). In the second and the

third parts of this exercise, the phosphate buffer was used for the observation and determination of

the effects of the aforementioned factors on the buffer capacity of a buffer system. The pertinent

reactions involving the phosphate buffer are as follows:

Dissociation:

𝐻2 𝑃𝑂4 − + 𝐻2 𝑂 ⇌ 𝐻𝑃𝑂4 2− + 𝐻3 𝑂+ (2.6)

Reaction with acid:

𝐻𝑃𝑂4 2− + 𝐻3 𝑂+ → 𝐻2 𝑃𝑂4 − + 𝐻2 𝑂 (2.7)

Reaction with base:

𝐻2 𝑃𝑂4 − + 𝑂𝐻 − → 𝐻𝑃𝑂4 2− + 𝐻2 𝑂 (2.8)

Calibration of the pH pen

In the first part of the exercise, a pH pen was calibrated against two solutions with

standardized pH. The pH of the standardized solutions were 4.00 and 7.00. Calibration was done to

minimize possible sources of error that may arise from the experimentation
Effect of buffer concentration on the buffer capacity

In the exercise, 50 mL of the 0.1 M phosphate buffer was obtained by the students and was

adjusted to pH 7.2. After that, 2.00 mL of 0.1 M NaOH was added to the solution. The pH of the

solution was recorded. This was also done to buffers that are diluted to 1:10, 1:50 and 1:100. The

results are shown in Table 2.1.

Table 2.1. Effect of buffer concentration on buffer capacity

No dilution 1:10 dilution 1:50 dilution 1:100 dilution

Initial pH 7.2 7.2 7.2 7.2
pH after
addition of 0.1
7.5 8.3 10.3 12.1
M NaOH
solution
Change in pH 0.3 0.9 3.1 4.9

It can be observed from Table 2.1 that as the dilution of the buffer solution increases the

change in pH also increases. This phenomenon is attributed by the fact that the concentration of the

buffer solution decreases as it is being diluted thus the number of reacting buffer species is less

therefore there is a greater change in pH. This can also be proven by equation 2.5, in which the

buffer capacity is inversely proportional to the change in pH.

Effect of conjugate base to weak acid ratio on the buffer capacity

In the third part of the exercise, five 100 mL beakers containing 20 mL of 0.1 M phosphate

buffers with different conjugate base to weak acid ratios were prepared in the amounts shown on

Table 2.2. Also, the pH values per beaker were calculated using the Henderson Hasselbach equation

(equation 2.4). The actual pH of the buffer solutions were measured. 2 ml of 0.1 M NaOH was

added to each beaker. The ph were measured afterwards. The same procedure was done with 2 ml

of 0.1 M HCl instead of NaOH. The results are tabulated at Table 2.3.
 Table 2.2. Preparation of phosphate buffer.
Amount of 0.1 M Amount of 0.1 M
Beaker Na2HPO4 NaH2PO4 [𝑯𝑷𝑶𝟒 𝟐− ]
pH
no. Vol., Conc., Vol., Conc., [𝑯𝟐 𝑷𝑶𝟒 − ]
mL M mL M
1 0.60 0.003 19.40 0.097 5.70034952 0.03092783505
2 1.80 0.009 18.20 0.091 6.205201117 0.0989010989
3 10.00 0.05 10.00 0.05 7.21 1
4 18.20 0.091 1.80 0.009 8.214798883 10.1111111111
5 19.40 0.097 0.60 0.003 8.71965048 32.3333333333
6 20 mL distilled water 7.0 -

It can be seen in Table 2.2 that the pH of the solution is influenced by the base to acid ratio

of the phosphate buffer. This can also be proven by equation 2.4, wherein the logarithm of the ratio

directly affects the pH of the buffer. Moreover, when the ratio of the base to the acid is 1:1, the pH

is equivalent to the pKa which means that at this ratio, the buffering capacity is optimal.

Furthermore, the buffering capacity is still generally dependent on the concentration of its

component.

Table 2.3. Effect of conjugate base to weak acid ratio.

Beaker 1 Beaker 2 Beaker 3 Beaker 4 Beaker 5 Beaker 6
pH of solution
5.3 6.2 7.2 8.2 8.1 7.0
using pH meter
Calculated pH 5.70034952 6.205201117 7.21 8.214798883 8.71965048 7.0
pH difference
- 0.0 0.0 0.0 - -
after adjustment
pH after adding
6.5 6.9 8.4 11.0 11.1 12.0
0.1 M NaOH
Change in pH
after addition of 1.2 0.70 1.2 2.8 3.0 5.0
NaOH
pH after adding
3.1 5.9 7.0 7.6 7.6 2.4
0.1 M HCl
Change in pH
after addition of -2.2 -0.3 -0.2 -0.6 -0.6 -4.1
HCl
Titration of unknown amino acid
Amino acids are biomolecules with the an α-carbon that is linked to an amino group, a

carboxylic acid, a hydrogen and a side chain denoted by R. Amino acids are building blocks for

proteins which primarily functions in many biological process: they may act as catalyst,

transporters or structural frameworks (Berg, 2015).

Amino acids, as it names implies, are weak acids. Therefore, it may also serve as a

component for buffer solution. Also, since it is a weak acid, its identity may be determined through

plotting a titration curve. In this exercise, the instructor assigned an unknown amino acid to each

group. The groups must identify the unknown through this procedure. The titration curve for the

unknown is shown below. The experimental data for the unknown amino acid is summarized in

table 2.4.

ph<pKa1=2.36
mostly fully
protonated
ph<IpH=5.98 ph>pKa2=9.60
moistly mostly fully
zwitterions deprotonated

Figure 2.1. Titration curve for the unknown amino acid.

Table 2.4. Data on the comparison on the values obtained from references and from
experimentation.
LITERATURE EXPERIMENTAL PERCENT
VALUES VALUES ERROR (IpH)
pKa (α-NH3+) 2.36 2.25
pKa (α-COO-) 9.60 9.5
~ -1.7559%
IpH 5.98 5.875
IDENTITY LEUCINE
 In identifying the unknown, the IpH of the alkyl amino acids were computed. Amino acids

that deviated too much from the experimental data were eliminated. Then the pKa1 and pKa2 from

the literature values were obtained and compared to the experimental data. Among the 20 standard

amino acid, the most probable identity of the unknown is leucine.

The percent error from the IpH may have arisen from the fact that the materials used during

the titration was contaminated. Furthermore, the sample was also contaminated.

In titrating leucine, the following reaction occurred:

At very low pH, the prominent form of the amino acid is its fully protonated form. At very low pH,

the prominent form is the fully deprotonated form of the amino acid. At the pKa, the reactants and

the products are in equilibrium. At the IpH, the zwitterion, or the amino acid with zero net charge is

prominent. Figure 2.2 summarizes the concentration of the forms of leucine in increasing pH.

Figure2.1. Concentration vs pH plot for leucine (source: Google).

 As mentioned earlier, proteins may serve as buffers in biological systems. The same is true

for amino acids. However, there is only a certain pKa at which an amino acid can act as a buffer. For

example, leucine can act as a buffer in pKa1 and pKa2. This is because the weak acid/base and its salt

are present. Buffers are not possible in IpH since there are no components for the buffer system.

II. Sample Calculations

 Change in pH, ΔpH = pHfinal - pHinitial

= 6.5 – 5.3

=1.2

𝑝𝐾𝑎1 + 𝑝𝑘𝑎2
 IpH = 2

2.36 + 9.60
= 2

=5.98
III. References

AppliChem. (2008). Biological Buffers. Retrieved June 16, 2017, from

https://www.applichem.com/fileadmin/Broschueren/BioBuffer.pdf
Berg, J. M. (2015). Biochemistry (Eight ed.). New York: W.H. Freeman and Company.
Hrycyna, C. (2013, January 21). Retrieved June 16, 2017, from www.chem.purdue.edu
Kuchel, P. W.-S. (2009). Schaum's outlines: Biochemistry (Third ed.). New York City: McGraw-Hill
Companies, Inc.
Mohan, C. (2003). Buffers: a guide for the preparation and use of buffers in biological systems.
Darmstadt: Calbiochem.