University of Santo Tomas

COLLEGE OF PHARMACY

pH Measurement and Buffer Preparation

Alcarioto, Y., Alimane, K., Ambida, P., Arcangel, P., Cachero, C.
Group 1, 2C Pharmacy, Faculty of Pharmacy, University of Santo Tomas

ABSTRACT
The experiment aims to prepare different kinds of buffer solutions and to determine
their pH by two ways; colorimetrically and electrometrically. “Colorimetrically” basically
means using liquid indicators and “electrometrically” because the use of a pH meter was
needed. According to Bathan, et.al, 2017, biological processes are affected by the
body’s acidity or basicity, and measurement of hydrogen concentration is termed pH.
Determination of pH levels whenever necessary using a buffer is needed so the
experiment was done by placing various samples in a microplate.

INTRODUCTION upon the addition of acidic or basic

components (“Buffers,” 2019). These we
According to Dr. Anne-Marie
call buffers are useful for processes or
Helmenstine (2019), pH is a measure of
reactions that need to reach a desired
hydrogen ion concentration, a measure
pH or stabilize the pH of the sample. To
of the acidity or alkalinity of a solution.
be able to relate both the pH value and
Therefore, for the researchers to be able
the pKa constant of the solution given,
to identify if the aqueous solution that
the researchers used the Henderson-
they have is acidic or alkaline, we use
Hasselbalch equation.
the pH scale. Most of the students are
familiar with the pH scale, it usually [𝑐𝑜𝑛𝑗𝑢𝑔𝑎𝑡𝑒 𝑏𝑎𝑠𝑒]
ranges from 0 to 14; solutions with pH 𝑝𝐻 = 𝑝𝐾𝑎 + log
[𝑎𝑐𝑖𝑑]
less than 7.0 are determined as acidic,
while solution with a pH greater than
The researchers were also able to
7.0 are determined as alkaline and
compute for the ratio of the
lastly, neutral solutions are those who
concentrations of the acid and its
have pH of exactly 7.0. There would be
corresponding conjugate base with the
instances that we had to alter or
use of this equation. For this
neutralize the pH values of some
experiment, the mentioned terms above
aqueous solutions, and to be able to do
are what the researchers will use to be
that, we have to use buffers. A buffer is
able to meet the following objectives: a)
a solution that can resist pH change
to prepare different buffer solutions; b)
determine the pH of buffers and
samples colorimetric ally using liquid
indicators and electrometric-
ally using the pH meter; and c) calculate
the buffer capacity of prepared of buffer
solutions. Aside from these objectives,
the researchers also aim to determine
the most effective buffer out of all the
buffer solutions prepared.
METHODOLOGY
In this experiment, the
determination of the pH of the sample
assigned (i.e., alkaline water) can be
done by the process of reacting the
prepared buffer solutions and the
sample with the acid-base indicators
(i.e., bromophenol blue, bromocresol
green, bromocresol purple, phenol re,
methyl red, methyl orange,
phenolphthalein, thymol blue, pH paper,
pH meter) called Colorimetric
Determination of pH.
A. Preparation of Reagent
The reagents prepared were
500mL of 6.0M HCl and 500mL of 6.0M
NaOH. The 6.0M HCl was prepared by
adding 245.38mL of concentrated HCl in
200mL of distilled water. The 6.0M
NaOH was prepared by dissolving 120g
of NaOH in 300mL of distilled water.
Both solutions were transferred to a
500mL volumetric flask and were filled
up to volume with distilled water. The
computation for concentrated HCl and
NaOH are the following:
(𝑀𝑜𝑙𝑎𝑟𝑖𝑡𝑦)(𝑀𝑊 )(𝑉𝐿 )(𝑆𝑡𝑟𝑒𝑛𝑔𝑡ℎ)(𝜌)
100𝑔 1𝑚𝐿
= 6𝑀 ∙ 36 ∙ 0.5𝐿 ∙ ∙
37.3𝑔 1.18𝑔
= 245. 38 𝑚𝐿 𝐻𝐶𝑙
𝑔 = 𝑀 ∙ 𝑀𝑊 ∙ 𝐿
= 6 ∙ 40 ∙ 0.5
= 120𝑔 𝑁𝑎𝑂𝐻
B. Preparation of Buffers
In preparing 50 mL of the buffer
solution assigned, the researchers
identified the weak acid and conjugate
base components of the buffer to be
prepared. The researchers were
assigned to prepare buffer #1 using the
weak acid H3PO4 and the conjugate
base H2PO4-1. Computation of the
amounts of the buffer components was
done using the Henderson-Hasselbalch
equation.
o Formula:
pH = pK a + log
[B]
[A]
The computed acid component
was 0.9630mL of H3PO4 and the
computed base component weighed
1.4904g of NaH2PO4. Both components
were dissolved separately in an
Erlenmeyer flask and a beaker,
respectively in 10mL of distilled water.
Stirring was continuously done until
both components were completely
dissolved.
Concentration Buffer pKa Desired On an Erlenmeyer flask, the base
Solution pH
component, 0.9630mL of H3PO4 was
0.50M Phosphate 2.12 2.00 dissolved in 10mL of distilled water.
0.50M Phosphate 2.12 3.00 Continuous agitation was done until
0.50M Acetate 4.70 5.00
base component was completely
0.50M Phosphate 7.21 7.00
0.050M Phosphate 7.21 7.50 dissolved.
0.010M Phosphate 7.21 8.00
0.50M Phosphate 12.32 12.00
Once completely dissolved, both
Table 1. Buffer Solution the diluted acid component solution and
the base component solution was mixed
together in a separate beaker. The pH
of the buffer was determined via digital
pH meter to check if desired pH was
met. The 6.0M HCl and 6.0M NaOH
reagents were prepared to adjust the
pH of the buffer in case the desired pH
was not met. In the case of the
researchers’ prepared buffer, the
Figure 1. The base component to be used for the desired pH was met without adjusting
preparation of buffer the pH with the said reagents.

Figure 2. Measure 1.4904g of Sodium dihydrogen

Figure 4. The 0.9630mL of conc. H3PO4 added in
phosphate to be placed in a beaker
an Erlenmeyer flask containing 10mL of distilled
water

Figure 3. The 1.4904g of NaH2PO4 added to the

beaker to be dissolved in distilled water
Figure 5. Mixing of the acid and base component
 Figure 6. Determination of the buffer’s pH
with the digital pH meter
The buffer solution that was mixed
in the beaker was transferred in a 50 mL
volumetric flask. Fill the flask until the
calibration mark with distilled water.
Figure 7. Buffer solution transferred in a 50 mL
volumetric flask
Figure 8. Buffer solution was transferred to the
volumetric flask and filled up to volume with
distilled flask
Figure 8. Volumetric flask filled to volume with
distilled water
C. Colorimetric Determination of pH
The researchers diluted 10mL of
the sample, i.e., alkaline water in 20mL
of distilled water to after which was
placed in the 9th column of a microplate.
The 1st up to the 8th column of the
microplate was filled with the buffers;
each buffer had a designated pH level,
using a 200µm micropipette. The acid-
base indicators were placed in each well
dropwise. The following acid-base
indicators were used to determine the
pH range:
• Thymol blue
• Bromophenol blue
• Bromocresol green
• Bromocresol purple
• Methyl red
• Methyl orange
• Phenolphthalein
• Phenol red

Figure 9. Microplate with the resulting colors of
the buffer-acid-base indicator reaction
The researchers then recorded the
all the colors produced by the buffer and
the corresponding acid-base indicator.
The colors were used as a basis in
determining the pH level of the alkaline
water.
RESULTS AND DISCUSSION
1. Electrometric Determination of
pH
The buffer solution was prepared by
mixing the phosphoric acid and its
conjugate base which is sodium
phosphate. The calculated amount of
these buffer agents is 0.96mL
phosphoric acid and 1.49g sodium
phosphate in order to obtain a pH of 2.
The electrometric determination of pH
using a pH meter showed an accurate
measurement on the buffer prepared.
Because the reading shows the exact
value of the desired pH, which is 2,
adjustment of the pH of the buffer
solution was not necessary in the case.
2. Colorimetric Determination of pH
Table 2.1 Listed below are the results
of the colorimetric determination of pH.
 Acid- Base pH pH pH pH pH pH pH Distilled Alkaline
Indicators 2.00 3.00 5.00 7.00 7.50 8.00 12.00 Water Water
Phenolphthalein Clear Clear Clear White White White Red- White White
violet
Methyl red Dark Red Red Dark Dark Dark Light Yellow- Dark
red yellow yellow yellow orange orange yellow
Thymol Blue Light Light Light Light Light Light Light Light Light
red yellow yellow yellow yellow yellow blue yellow yellow
Methyl Orange Dark Red Orange Light Orange Orange Light Orange Orange
red Orange orange
Bromocresol Yellow Yellow Blue- Blue Blue Blue Blue Blue Blue
Green green
Phenol Red Yellow- Yellow Yellow Yellow- Yellow- Light Light Yellow Yellow-
orange orange orange orange red orange
Bromophenol Yellow Green Dark Violet Violet Violet Violet Violet Violet
Blue violet
Bromocresol Yellow- Yellow- Purple Purple Purple Purple Purple Light Purple
Purple green green violet
than 7, it is acidic while greater than
7, it is basic.
On the colorimetric determination
of pH, acid-base indicators were The assigned sample, which is
used as color standards to determine alkaline water by the brand Nature’s
the pH value of the solutions. Acid- Spring, was observed and compared on
base indicators, also called pH the collection of different buffer
indicators, are usually weak acids or solutions ranging from pH 2, 3, 5, 7,
bases and changes color with pH 7.50, 8, and 12. As shown on Table 2.1,
(Clark, 2019). Phenolphthalein the students were able to compare the
changes from colorless to pink changes of colors between the assigned
indicating a pH range of 8.3 – 10, sample and the buffer solutions
methyl red changes from red to prepared by the other groups. It is seen
yellow indicating pH range 4.4 – 6.2, that the color changes of the sample
methyl orange changes from red to were the same with the buffer solution
yellow indicating pH 3.2 – 4.4, with the pH of 7.50. Therefore, based
bromocresol green changes from on the observations, it shows that the
yellow to blue indicating pH range pH value of alkaline water matched the
3.8 – 5.4, phenol red changes from buffer solution with 7.50 and with this
yellow to red indicating pH 6.6 – 8, result, the colorimetric method is less
bromophenol blue changes from accurate because the claim of the
yellow to purple indicating pH range sample, Nature’s Spring alkaline water,
3 – 4.6, and bromocresol purple which is supposed to be pH 9, does not
changes from yellow to purple correspond to the data.
indicating pH 5.2 – 6.8. The pH 7 is
neutral and as the pH becomes less
REFERENCES

Buffers. (2019). Retrieved

September 3, 2019, from
https://chem.libretexts.org/Bookshel
ves/Physical_and_Theoretical_Chemi
stry_Textbook_Maps/Supplemental_
Modules_(Physical_and_Theoretical_
Chemistry)/Acids_and_Bases/Buffers.

Clark, J. (2019, June 06). Truro

School in Cornwall. Retrieved from:
https://chem.libretexts.org/Bookshel
ves/Physical_and_Theoretical_Chemi
stry_Textbook_Maps/Supplemental_
Modules_(Physical_and_Theoretical_
Chemistry)/Equilibria/Acid-
Base_Equilibria/6._Acid-
Base_Indicators

Helmenstine, A. (2019). pH
Definition and Equation in Chemistry.
Retrieved September 3, 2019, from
https://www.thoughtco.com/definitio
n-of-ph-in-chemistry-604605

Bathan, G. et. al., (2017). Laboratory

Manual in General Biochemistry
Second Edition. Quezon City, NCR: C
& E Publishing, Inc.