pH MEASUREMENT AND BUFFER PREPARATION

Sofia Alessandra N. Javier, Katherine F. Jayme, Patricia Eunice T. Juachon, ​Jocelle Hui Xing T.
Khoo​, Corin A. Lim, and Castor Carl Jacob V. Llamas
Group 5 2D Medical Technology Biochemistry Laboratory

ABSTRACT
A buffer is a combination of a weak acid and its conjugate base, or a weak base with its conjugate acid. It is a solution
that resists the pH change due to the addition of an acidic or basic compounds [1]. Buffers neutralize the small
amounts added to maintain the pH range of the solution, making it relatively stable. pH is the measure of the
concentration of hydrogen ions in a solution [5]. In this experiment, the researchers prepared a buffer solution with a
pH of 7 using Phosphate (pka = 7.21). The buffer solution was prepared using 6.44 mL of H​3​PO​4 and 7.98 mL of 6M
NaOH. The different pH indicators were used specifically the electrometric method and colorimetric method. The
electrometric method was more accurate since it was able to give the specific pH, while the colorimetric method gave
a range of the pH through the color change [8].

INTRODUCTION meter, and colorimetrically by usage of color

Acid and bases are relevant substances in one’s
everyday life. It is observable in the food and
drinks being consumed, in the medicines used to
treat the ill, and in one’s body. It is more
popularly defined by the Brønsted-Lowry acid
base theory that states, acids are proton donors
whereas bases are proton acceptors.
Perhaps the most important role of acids and
bases in one’s life is the maintenance of pH,
which is the measure of alkalinity or acidity of a
substance. It ranges from a value of 0 to 14, 7
being the neutral point, with lower values than 7
indicating acidity while greater values than 7
indicate alkalinity.
Having a neutral pH is essential for
homeostasis. The human body has a pH range of
7.35 to 7.45. Veering away from this optimal
range has implications on our bodily functions
and thus may be a cause for diseases to develop
[7].
This is where Buffer solutions come in. Buffers
are made from a mixture of a weak acid and its
corresponding conjugate base. Due to its
components it is able to resist a sudden change
in pH by neutralizing the sudden addition of
hydrogen or hydroxyl ions made possible by the
presence of an acid, and a conjugate base [1].
The human body has a buffer system that works
to maintain the pH of our overall bodily fluids and
biomolecules essential for the continuous
functioning of the body [7].
In this experiment, the Henderson-Hasselbalch
equation is utilized to determine the pH of the
buffer sample and assigned sample (Soda) to the
group as well as distilled water. This was done
with the help of pH meters and liquid indicators
such as Methyl-red, phenolphthalein, and
etcetera.
The objective of the experiment is the
preparation of a buffer solution with a specific pH
measured electrometrically with the help of a pH
indicators​.
EXPERIMENTAL
A. Test Compounds/Samples Used
The test compounds or samples used in the
experiment of pH determination and measure of
Buffer capacity are Distilled water (250 mL),
Soda (5 mL), 12 M Phosphoric Acid (H​3​PO​4​), 6 M
Sodium Hydroxide (NaOH), 6 M Hydrochloric
Acid (HCl), and Acid-Base Indicators (Thymol
Blue, Bromophenol Blue, Bromocresol Green,
Bromocresol Purple, Methyl Red, Methyl Orange,
Phenolphthalein, and Phenol Red)
B. Procedure
Preparation of Reagents
The 6M HCl was prepared by missing 54 mL of
aqueous HCl in 250 mL water. Whereas the 6M
NaOH was prepared by dissolving 60 g of NaOH
pellets in 250 mL.
Preparation of Buffer
H​3​PO​4​ ↔ H​2​PO​4 ↔
​ HPO​4 ↔
​ PO​4
Figure 1​. Reaction Equation of H​3​PO​4 to PO​4
​
​ The assigned buffer solution to the group is 250
mL with 0.50 M, a pKa value of 7.21 and a pH
value of 7.0 (neutral). The weak acid and
conjugate base component were first identified,
Secondary Phosphate Acid (H​2​PO​4​) being the
weak acid component and Primary Phosphate
Acid (HPO​4​) being the conjugate base
component. Using the Henderson-Hasselbalch
equation the amount of buffer components were
computed as well as the amount of NaOH needed
to be added:
Henderson-Hasselbalch Equation Figure 2​. Buffer Solution prepared during
pH = pKa + log [A] experiment
[HA]

7.0 = 7.21 + log [HP O4] 1. Electrometric Determination of pH

[H2P O4)]
[HP O4]
[H2P O4] = antilog (7.21 − 7) ​Using a calibrated pH meter that would
measure the hydrogen-ion activity in the
[HP O4] following samples: (1) prepared buffer solution,
= 0.6166
[H2P O4] 1 (2) distilled water, and (3) soda.
While the buffer solution’s pH was being
Theoretical Amount of Moles Present measured, small amounts of either 6.0M HCL or
0.6166 + 1 = 1.6166 6.0M NaOH were added to monitor and adjust the
pH of the buffer to its desired value (pH=7.0).
Amount of Moles of Buffer desired
M = molL
mol
0.50 M = 0.250 L
0.50 M (0.250 L) = 0.125 mol

Actual Amount of Moles of Base Present

0.6166
1.6166
= 0.125
x

x = 0.04768

Actual Amount of Moles of Acid Present

1
1.6166
= 0.125
x

x = 0.07732

Volume of NaOH
6M = 0.04786
x
−3
x = 7.98 x10 L = 7.98 mL

Volume of H​3​PO​4 Figure 3​. Setup of pH meter

0.07732
12M = x
x = 6.44 x10
−3
L = 6.44 mL
2. Colorimetric Determination of pH
For the colorimetric method of determining pH
level, color standards are first prepared using a
The assigned buffer was prepared by adding
buffer solution. To do this, 6 vials or test tubes
7.98 mL of NaOH and 6.44 mL of H​3​PO​4​.
are prepared and labeled with the name of the
Afterwards, 250 mL of distilled water was added.
buffer, its pH, and the acid-base indicator to be
used. 5 mL of a buffer with a certain pH is
pipetted into a vial/test tube and is mixed with 2
drops of acid-base indicator, then the color
produced is recorded. The same procedure is
performed using a buffer with a different pH.
Then, the same steps are done but using
different acid base indicators.

To determine the pH of samples using the

colorimetric method, 5mL of samples are used
and is mixed with 2 drops of an acid-base
indicator. The mixture was shaken and the color
is recorded. The same procedure is done using
different acid-base indicators in the preparation
of color standards.

RESULTS AND DISCUSSION

Buffer solutions are able to resist changes in
pH as any acids added are neutralized by the
base present in the solution, while any bases
added are neutralized by the present weak acid
[1].
A. Electrometric Determination of pH
The pH of distilled water, buffer sample, and
the assigned sample (soda) was measured by the
use of electrometric method. This method is
dependent upon the concentration of Hydrogen
[H+] ions present in the sample, making it a
more accurate method of pH determination as
opposed to the colorimetric method of pH
determination [8].
acids or bases that would cause a shift in
equilibrium (either left or right) due to the
hydrogen ion concentration [2]. For example,
with a general formula of HIn, the chemical
equation below shows both the weak acid and its
conjugate base.
HIn​(aq)​ + H​2​O​(l)​ ↔ In​-​(aq)​ + H​3​O+​​ (aq)
​ (Color A) (Color B)
Figure 4​. Chemical Equilibrium Reaction

Table 1​. Electrometric Determination of pH

​HIn represents the weak acid which has a
corresponding color A, while its conjugate base
Samples pH [H+] In​-​(aq) would have a corresponding color B as well.
Given a low pH, hydronium ion (​H​3​O​+​)
Distilled Water 6 1.0x10​-6 concentrations are greater which pushes the
(22.3°C)
equilibrium to the left, leading to a solution with
Assigned 2.8 1.58x10​-8 color A [4]. The opposite happens when given a
Sample (soda) (22.4°C) high pH. Chemicals such as thymol blue,
phenolphthalein, and phenol red are all acid-base
Buffer Sample indicators with different corresponding colors for
a. Initial a. 0.681 a. 0.21 its acidic state and basic state. The table below
b. Desired b. 7 (35°C) b. 1x10​-7 shows the color standards for the common
acid-base indicators as well as the color results
from the experiment.
​At a temperature of 22.3°C, the pH of distilled
water was 6, ideally distilled water should have a
pH value of 7. However exposure to the Table 2.​ Colorimetric Determination of pH
atmosphere, specifically to carbon dioxide (CO​2​)
may make distilled water more acidic as it may
absorb carbon dioxide present in the air. Carbon
dioxide produces Carbonic acid (​H​2​CO​3​) when
exposed to water. making it more acidic over
time as opposed to when it was just distilled.
The assigned sample’s recorded pH is 2.8, at a
temperature of 22.4°C. Sodas are known for their
rather high acidity level [6]. Having a pH of 2.8 is
proof of soda’s high acid content.
The initial pH of the buffer, 0.681 was below
the desired pH value of 7, at 35°C. This
Based on the results and colors seen with using
discrepancy may be due to mistakes in the
distilled water, the results are a range between
calculated amounts of each reagent, improper
pH 5.0 and 7.0, which estimates the sample at
calibration of the pH meter, contaminants in the
around 6. Ideally, distilled water should have a
atmosphere and/or possibly temperature
pH of 7. However, the slight variation in color
fluctuation. To remedy this, small amounts of 6M
reveals it to be slightly more acidic with a value
NaOH and 6M HCl were added by usage of a
of around 6 due to the exposure to the
dropper until the buffer reaches the desired pH
atmosphere and carbon dioxide, as previously
value of 7.
explained in the discussion for electrometric
determination of pH. On the other hand, the color
B. Colorimetric Determination of pH
results of the buffer solution show it has a pH of
Colorimetric determination of pH involves using
7, which means it is a neutral solution.
acid-base indicators that change color under
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