University of Santo Tomas

Department of Pharmacy
General Biochemistry (Laboratory)

EXPERIMENT 1
pH MEASUREMENT AND BUFFER PREPARATION

Duran, R., Ebuen, E., Epistola, M., Fabriga, Z., Figueroa, E., Flores, D
Group 4 2A-PH

ABSTRACT

A buffer solution is a homogenous mixture of weak acid and its conjugate base to achieve its
components of neutralized excess hydrogen or hydroxyl ions. This experiment covers the preparation of
buffer solution to determine the pH of the sample alkaline water. The purpose of the experiment is to
determine the pH of buffers and sample colorimetrically using liquid indicators. In the experiment, the first
step is to prepare the correct buffer solution by computing the exact amount of Sodium phosphate and
Sodium Dibasic Phosphate. The buffer solution is placed in microplate along with the distilled water as the
control and the sample alkaline water. Addition of organic dyes are used to determine the pH of the alkaline
water by comparing the colors of the sample to the buffer. According to the gathered results, the indicated
pH in the sample alkaline water which is 9.0 does not match with the results in the colorimetric test which
is 6.8-8.0 pH.

OBJECTIVES

- Prepare different buffer solutions complementary coexistence between the

- Determine the pH of buffers and
samples colorimetrically using liquid
indicators and electrometrically using
the pH meter
- Calculate the buffer capacity of
prepared buffer solutions
INTRODUCTION
Even with the slightest changes in
pH, the different biological processes and
systems of the Earth may be greatly affected
and influenced by this. The importance of pH
lies within the fact that it indicates whether
the given environmental condition is suitable
for an organism to survive or not. For
example, when a water source becomes too
acidic, a risk of implications (e.g. death or
illnesses) may occur on its consumers. This
now becomes an issue in terms of
Earth and its inhabitants. A similar concept
can be said inside the human body. At a
cellular level, cells must maintain a normal
pH balance in order to perform its optimal
functions. Once an imbalance occurs, the
individual may be susceptible to health
repercussions such as acidosis and alkalosis.
With that said, the devices that can measure
different pH balances and mechanisms that
can help maintain it at a normal level gain
their significance.
pH, as defined by a Danish biochemist, Søren
Sørensen (as cited in Chemistry LibreTexts,
2019 ), is the negative logarithm of hydrogen
ion concentration [H+]. There are two main
types of determining pH balance. The more
traditional method of determining pH
concentration of a buffer is through
Colorimetry. It uses color standards or
indicators, and then visually compares the
color combinations of the sample based on
varying pH. On the other hand, a modern
method of pH determination known as the
Electrometry involves the use of a pH meter.
It uses the glass electrode of the pH meter
to sense the acidity or basicity of the
solution.
A buffer is an aqueous solution that
has a highly stable pH. It is composed of a
weak acid and its conjugate base or a weak
base and its conjugate acid. There are two
kinds of buffer systems, the acidic buffers,
pH <7, and the alkaline buffers, pH>7. When
hydrogen ions are added to a buffer, they will
be neutralized by the base in the buffer.
While Hydroxide ions, on the other hand, will
be neutralized by the acid (Helmenstine,
2018). Buffer solutions are very important in
the environment and inside the human body.
It prevents the sudden change in pH and
thus sustain the normal functioning of life.
The significance of pH can also be
seen in the pharmaceutical field. In terms of
drug stability, pH plays an important role in
the prevention of drug degradation. Drug
degradation mostly occur through hydrolysis.
With this said, hydrolysis heavily depends on
the concept of pH and thus the presence of
buffer solutions are integral for a drug to
exhibit maximum stability (Allen, 2011).
METHODOLOGY
Materials needed
Volumetric flasks, vials/test tubes and
beakers are needed to contain the
solution.Serological pipettes and aspirators
are also essential to deliver the liquid sample
from one container to another. The reagents
needed are: conc. H3PO4 (85.0% w/w,
sp.gr. 1.70, MM 98), conc. HCl (37.3% w/w,
sp.gr. 1.18, MM 36), glacial acetic acid
(99.7% w/w, sp.gr. 1.05, MM 60), NaOH
pellets (MM 40), NaH2PO4 . H2O (MM 138),
NaH2PO4 . 7H2O (MM 268). pH paper and
pH meter is used to determine the pH of the
sample. A weighing balance is used to weigh
the solid reagents. Lastly, these acid-base
indicators are used to test the sample pH:
bromophenol blue, bromocresol green,
bromocresol purple, phenol red, methyl red,
methyl orange, phenolphthalein, and thymol
blue.
Preparation of Buffers
The buffer assigned to the group is a
pH7 phosphate buffer with a pka of 7.21. The
group identified dihydrogen phosphate as
the weak acid and hydrogen phosphatE as its
conjugate base. To compute for the amount
of the identified components, the
Henderson-Hasslebach formula is needed:
pH = pka + log
[B]
[A]
The sample given is a “pH9” alkaline
drinking water (Figure 1). Since the sample
is drinking water, the procedure of diluting it
in a small amount of water was skipped.
Less than 50 mL of the sample was then put
in a beaker.
Figure 1: “pH9” alkaline drinking water
Using a pH meter, the sample was checked
for its pH. The pH measured was 6.6. 15
drops of 6.0M NaOH and 8 drops of 6.0M HCl
were added to achieve the desired pH of the
sample. After the addition of reagents, the
pH meter measured a pH of 7.2 (Figure 2).
 Table 1: Downward Columns

A1-H1 pH 2.0
A2-H2 pH 3.0
A3-H3 pH 5.0
A4-H4 pH 7.0
A5-H5 pH 7.5
A6-H6 pH 8.0
Figure 2: Measurement of the pH A7-H7 pH 12.0
A8-H8 Water sample
After the desired pH was reached, the
sample is then transfered to a 50-mL A9-H9 Water sample
volumetric flask then filled to volume with
distilled water (Figure 3). 10µl of each indicator is then added in such
arrangement (Figure 4):

Table 2: Across rows

A1-A9 Thymol blue

B1-B9 Bromophenol blue
C1-C9 Bromocresol green
D1-D9 Bromocresol purple
E1-E9 Methyl red
F1-F9 Methyl orange
G1-G9 Phenolphthalein
Figure 3: Transfering of sample to volumetric H1-H9 Phenol red
flask

The addition of neutral distilled water should

not affect the pH of the sample. The solution
was mixed thoroughly. The flask was labeled
after.

Preparation of Reagents

The class was assigned to prepare 500 mL of

6.0M HCl and 6.0M NaOH. The bottles were Figure 4: Addition of indicators
labeled accordingly.

RESULTS and DISCUSSION

Colorimetric Determination of pH
Preparation of Buffers
200µl of each buffer solution, water and
solution is transferred to a microplate as Concentra Buffer pKa Desired
shown below: -tion Solution pH
0.50 M Phosphate 7.21 7.00
Table 3: Components of the Buffer Solution Phosphate buffer with a desired pH of 7.00
and 0.50 M concentration was assigned to
Given: the group. Buffers are solutions that can
resist pH change upon the addition of an
V = 50 ml acidic or basic components.
Na2HPO4.7H2O (MM 268)
NaH2PO4.H2O (MM 138) They can able to neutralize small
amounts of added acid or base, thus
1. Preparation of Reagents maintaining the pH of the solution relatively
stable. A buffer consists of a weak acid
(proton donor) and its own conjugate base
A. Prepare 6.0 M Hydrochloric
(proton acceptor). For the experiment, the
Acid
weak acid is the Primary Phosphate ion in
Sodium Primary Phosphate Monohydrate
The Sodium hydroxide standard was (NaH2PO4.H2O) and its conjugate base is
prepared using 500mL of 6.0 M Hydrochloric
the Secondary Phosphate in Sodium
Acid (37.3%w/w ; 118 sp gr; MM36)
Secondary Phosphate Heptahydrate
(Na2HPO4.7H2O).
37.7 𝑔 1.18 𝑔
𝑥 = 0.44014 𝑔/𝑚𝐿 Phosphate buffers have 3 pKa values.
100 𝑔 1 𝑚𝐿𝑚𝐿
The pKa value is one method used to indicate
𝑔 the strength of an acid. pKa is the negative
𝑀 = 𝑀𝑊 𝑥 𝐿
log of the acid dissociation constant or Ka
𝑀 = 12.23 value. A lower pKa value indicates a stronger
𝐶1 𝑉1 = 𝐶2 𝑉2 acid. That is, the lower value indicates the
(𝟔 𝑴)(𝟓𝟎𝟎) = (𝟏𝟐. 𝟐𝟑𝑴)(𝑽𝟐 ) acid more fully dissociates in water. Their 3
pKa values make them Triprotic acids.
𝑽𝟐 = 𝟐𝟒𝟓. 𝟑𝟎 𝒎𝑳
The pKa is needed in the Henderson-
B. Prepare 6.0 M Sodium Hasselbalch equation that is being shown
Hydroxide below:

[𝑐𝑜𝑛𝑗𝑢𝑔𝑎𝑡𝑒 𝑏𝑎𝑠𝑒]
The Sodium hydroxide standard was 𝑝𝐻 = 𝑝𝐾𝑎 + 𝑙𝑜𝑔 [𝑎𝑐𝑖𝑑]
prepared using 500mL of 6.0 M Sodium
Hydroxide: Triprotic acid is an acid that can donate
6.0 𝑚𝑜𝑙 𝑥
= 0.500 𝐿 3 hydrogen ions per molecule during
1𝐿
dissociation. Triprotic weak acid such as
Phosphate has 3 plateau regions in its
𝑥 = 3.0 𝑚𝑜𝑙 𝑁𝑎𝑂𝐻 titration curve. Every half-neutralization of
each plateau region indicates pKa value. The
40 𝑔 pKa values of Primary Phosphate buffer is
3 𝑚𝑜𝑙 𝑥 = 120 𝑔 𝑁𝑎𝑂𝐻
1 𝑚𝑜𝑙 2.12, Secondary Phosphate is 7.21 and
Tertiary Phosphate Buffer is 12.32.

2. Buffers The Henderson Hasselbalch Equation

will give the experimental amount of Primary
The two buffers namely HCl and Phosphate and Secondary Phosphate buffers
NaOH, were assigned to the 2 groups. needed. The Phosphate buffer of interest in
the preparation is the Secondary Phosphate based solutions, indicating its acidity or
buffer with a pKa value of 7.21 and a desired alkalinity expressed as pH.
pH of 7.00.

A. Acid Component [𝑯𝟐 𝑷𝑶−𝟏 ]: The pH meter measures the difference in

electrical potential between a pH electrode
0.50 𝑀 = 0.6165950019𝐴 + 𝐴 and a reference electrode, and so the pH
𝟎. 𝟓𝟎 𝑴 = 1.6165950019𝐴 meter is sometimes referred to as a
"potentiometric pH meter".
𝑨
= 𝟎. 𝟑𝟎𝟗𝟐𝟗𝟐𝟎𝟔𝟏 𝑴
A. Basic Component [𝑯𝑷𝑶𝟒 −𝟐 ]: Samples pH [H+]
Distilled 2.5 3.16 x 10-3
0.50 𝑀 = 𝐴 + 𝐵 Water
𝟎. 𝟓𝟎 𝑴 = 𝟎. 𝟑𝟎𝟗𝟐𝟗𝟐𝟎𝟔𝟏 + 𝑩 Assigned 8.0 1 x 10-8
𝐵 = 0.190707939 𝑀 Sample
Buffer 7.0 1 x 10-7
A. Acid Component [𝑯𝟐 𝑷𝑶 ]: −𝟏
Prepared
Table 4: Electrometric test for samples and
𝟎. 𝟑𝟎𝟗𝟐𝟗𝟐𝟎𝟔𝟏𝑴(𝟎. 𝟎𝟓𝒎𝒍) = buffer
𝟎. 𝟎𝟏𝟓𝟒𝟔𝟒𝟔𝟎𝟑𝟎𝟓 𝒎𝒐𝒍𝒆𝒔
𝟎. 𝟎𝟏𝟓𝟒𝟔𝟒𝟔𝟎𝟑𝟎𝟓𝒎𝒐𝒍(𝑴𝑴𝟏𝟑𝟖) = 4. Colorimetric determination of pH
𝟐. 𝟏𝟑𝟒𝟏𝒈
[𝐻2 𝑃𝑂−1 ] The very basis for what the Chemist calls
Colorimetric analysis is the variation in the
intensity of the solution’s color with changes
B. Basic Component [𝑯𝑷𝑶𝟒 −𝟐 ]:
in concentration (or pH). Calorimetric
0.190707939 𝑀 (0.05 𝑚𝑙) determination is done by using a spot plate
= 0.00953539695 𝑚𝑜𝑙 and an acid base indicator. The results for
0.00953539695𝑚𝑜𝑙(𝑀𝑀 268) = 2.5555𝑔 the Colorimetric determination of pH are
[𝐻𝑃𝑂4 −2 ] shown on Table 5.

Table 5: Colorimetric Test of

The molarity of the solution is 1.6166 M. The Buffers
number of acid component’s moles is 0.0154 The table below shows the colorimetric test of
moles, and the base component is 2.5555 buffers. See full picture on the last page.
moles. By ratio and proportion, 2.1341 grams
of the Primary Phosphate and 2.5555 grams
of Secondary Phosphate must be used to
form the buffer solution.

3. Electrometric determination of
pH

A pH meter is a scientific instrument that

measures the hydrogen-ion activity in water- Figure 5: pH9 drinking water
 For Phenol red, the pH range is 6.8 - 8.2. The
Table 6: Colorimetric Test Colour buffer had shown a Tan coloration.
Chart

The table below shows the colorimetric test of
buffers’ legend (colour chart).
This report concludes that the pH of the
group’s secondary buffer solution with a
desired pH of 7.00 is approximately within
the pH range generalized by those Acid-Base
Indicators having a pH that ranges from 6.8
- 8.0.

Based on the results, the sample product,

ACID-BASE INDICATORS
The group experiment worked on the
Secondary Phosphate buffer with a pH of
7.00.
which is 7 select drinking water has a pH of
8.0, indicating that it is an alkaline drinking
water. However, it claims to have a pH of 9.
The colorimetric test clearly shows the
original pH of 7 select drinking water -
negating the pH claim of the drinking water’s
brand.

At Thymol blue, the buffer indicated a yellow

orange coloration. Thymol blue has a pH
range of 1.2 - 2.8 acid range.

At Bromophenol blue, the buffer indicates a

dark violet coloration. Bromophenol blue has
a pH range of 3.0 - 4.6.

At Bromocresol green, the buffer also

indicates a light blue coloration. Bromocresol
green has a pH range of 3.8 - 5.4.

For Bromocresol purple, the buffer gave a

violet coloration. The pH range of
bromocresol purple ranges from 5.2 - 6.8.

For Methyl red, having a pH range of 4.4 -

6.2, the buffer indicated a yellow coloration.

Methyl orange with a pH range of 3.1 -4.4,

had shown orange coloration.

Phenolphthalein gave a colorless solution,

indicating a lower pH range of 8.2. At pH 12,
Phenolphthalein gave a colorless coloration.
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