pH MEASUREMENT AND BUFFER PREPARATION

Airika P. Muhi, Charlot P. Navarro, Kristin D. Oanes,

Jhunabelle D. Pablo, Irish Jane M. Patron, Margaret Corinne U. Ramos
Group 6
2G Pharmacy
PH Biochemistry Laboratory

ABSTRACT
This experiment focused on achieving the following objectives: To prepare different buffer solutions, to determine the
pH of the buffers and samples colorimetrically using different indicators and electrometrically using the pH meter, and
to calculate the buffer capacity of the prepared buffer solutions. Buffer solution of phosphate was prepared by adding
NaH2PO4 . H2O and of Na2HPO4 . 7 H2O to sufficient amount of distilled water. With the use of the pH meter, the pH of
the buffer solution was electrometrically determined. To successfully control the basicity and acidity of the buffer
solution and keep it neutral, either 6M HCl or 6M NaOH was added to make the solution more acidic or basic. The
buffer solutions pH was also determined colorimetrically using eight different acid-base indicators; Thymol blue,
Bromothymol blue, Bromocresol green, Bromocresol purple, Methyl red, Methyl orange, Phenolphthalein, and Phenol
red. These acid-base indicators are organic molecules that are mostly weak acids. They make use of color dyes, and
the pH range is usually determined by identifying the different changes in color of the indicators. In this experiment
the following results were obtained: Thymol blue orange yellow, Bromothymol blue purple, Bromocresol green
blue, Bromocresol purple violet, Phenol red orange, Methyl red yellow, Methyl orange orange, Phenolphthalein
colorless. When comparing the two different methods of determining the pH, the pH meter is more effective.

INTRODUCTION
Acid and bases are of great importance in our
daily life. They are present everywhere, in the
food we eat, in the commercially available
products, and even in our body. [4] According to
Bronsted-Lowry definition of acids and bases, an
acid releases proton (hydrogen ion) whereas a
base accepts proton (hydrogen ion). In an
aqueous
solution,
these
hydrogen
ion
concentrations are measured using pH.
Mathematically, pH is expressed as the negative
log in base of 10 of the hydrogen ion
concentration.
pH = - log [H+]
Due to large changes in pH, our biological system
cannot withstand this therefore a buffer solution
is needed. Generally, buffer solutions are
concentrations of weak acids and its conjugate
base, or a weak bae and its conjugate acid. It
resists a sudden change in pH whenever small
amounts of acid or base are added. [2]
Because pH is dependent on ionic activity, a
property which cannot be measured easily or
fully predicted theoretically, it is difficult to
determine an accurate value for the pH of the
solution. [2] The pH reading of a solution is
usually obtained using a pH meter or pH indicator
paper/liquid. [3]

The objectives of this experiment were as

follows:
(1) to prepare secondary phosphate
buffer with a pH of 7.5 (2) to determine the pH of
the buffers and samples colorimetrically using
different liquid indicators and electrometrically
using the pH meter.

EXPERIMENTAL
PROCEDURE
1. Preparation of Reagents
1. 500 mL of the 6.0 M HCl and 6.0 M NaOH was
prepared.
Buffers
1. 250 mL 0.10 M buffer solutions of the following
were prepared.
BUFFER SOLUTIONS
Phosphate; pK=2.12
Phosphate; pK=2.12
Acetate; pk=4.70
Phosphate; pK=7.21
Phosphate; pK=7.21
Phosphate; pK=7.21
Phosphate; pK=12.32

Weak acid and conjugate base components of

each buffer were identified. The amounts of the
buffer
components using the HendersonHasselbalch equation were calculated. The

appropriate reagents from the list of reagents

and materials were selected.
2. Containers were labelled properly by indicating
the name of the solution, date prepared, pH and
group number.
2. Electrometric Determination of pH
1. The pH meter was calibrated to pH 4, 7 and
10.
2. The pH of a 20-mL portion of the prepared
buffer solutions and distilled water were
measured.
3. The [H+] of the samples used were calculated.
4. The pH of the prepared buffer solution to its
desired value was adjusted by adding in portions
of either 6.0 M HCl or 6.0 M NaOH as monitored
by the pH meter.

RESULTS AND DISCUSSION

1. Computations
The computation below shows how much of
the available reagents used were determined in
order to prepare 0.10M secondary phosphate
buffer with the pH of 7.5. By using the
Henderson-Hasselbach equation, the ratio of
HPO42- and H2PO4- were computed. From the
ratio, the moles of HPO42- and H2PO4- were
calculated. From moles, the amounts in grams of
the reagents were determined. 1.242g NaH 2PO4
H2O and 4.88g Na2 HPO4 7H2O were dissolved in
sufficient distilled water to make 250mL
secondary phosphate buffer.

pH=pKa+ log

3. Colorimetric Determination of pH
A. Preparation of color standards using the buffer
solutions
1. 8 test tubes were prepared and labelled with
the pH of the buffer and acid-base indicator to be
added.
2. 3 mL of the buffer of a the assigned pH was
pipetted into the vial. 2 drops of an acid-base
indicator were added. Mixture was shaken and
its color was noted. 3mL of a buffer of a different
pH was pipetted into another vial and 2 drops of
the same acid-base indicator were added. This
procedure was performed on all the prepared
buffers.
3. Another set of 8 test tubes were prepared.
Step 2 was repeated but a different acid-base
indicator was used. This procedure was
performed with the use of the following acid-base
indicators:
Thymol blue
Methyl red
Bromophenol blue
Methyl orange
Bromocresol green
Phenolphthalein
Bromocresol purple
Phenol red

log

CB
acid

7.5=7.21+ log

HPO2-4
=0.29
H 2 PO-4

HPO24
= 100.29
H2 PO 4

2-

2-

HPO4
=1.9
H2 PO 4
0.25L x

HPO2-4
H2 PO -4

Ratio:

1.9 HPO4
1 H2 PO 4

10 mol
=0.025 mol total buffer
L

1 H2 PO 4 x H2 PO 4
=
2.9
0.025

x=0.009 H2 PO -4

1.9 HPO2-4 x HPO 2-4

=
2.9
0.025

x=0.016 HPO2-4

0.009 mol x

138g
=1.242g Na H 2 PO 4 H2 O
mol

0.016 mol x

268g
=4.88g Na 2 HPO4 7 H2 O
mol

2. Preparation of reagents
250mL 6M HCl

37.3g x

1 mol
=1.04 mol
36g

100g x

0.001L
=0.08L
1.18g

1.04 mol
=12.2M conc HCl
0.08L

reading of the pH through the activity of the

hydrogen ions.

C1 V1 = C2 V 2

( 12.2M ) x= (6M ) ( 250mL )

Figure 1. pH meter

x=122.95mL 12.2M HCl

4. Colorimetric Determination of pH
The available reagent in the laboratory was
concentrated HCl (37.3%). This was diluted to 6M
by diluting 122.95mL of concentrated HCl with
enough water to make 250mL reagent.
250mL 6M NaOH

6mol x

40g
=240g
mol

240g
xg
=
1000mL 250mL

x=60g
The available reagent in the lab was NaOH
pellets. 60g of NaOH pellets were dissolved in
enough water to make 250mL of NaOH solution.

3. Electrometric Determination of pH
Electrometric method is a way of determining the
pH of the buffer with the use of the pH meter. In
this experiment, a secondary phosphate buffer
solution was utilized. The first reading of the pH
of the solution was 6.5. NaOH was added to the
solution to increase of the pH to the desired level
which is 7.5. The discrepancy between the
calculated desired pH and the pH of the prepared
buffer was affected by the container used,
apparatus and the presence of the carbon dioxide
in the environment that increases the hydrogen
ion concentration of the solution thus decreasing
pH. Addition of a base was necessary in order to
achieve the desired pH. The pH of distilled water
was also measured using a pH meter which
resulted to 6.8. The pH meter gives an accurate

Colorimetric determination of pH is a method

used in determining the pH of the solutions with
the use of pH indicator also called acid-base
indicators. These pH indicators change its color
and measures pH in terms of concentration of the
hydronium ions that determines whether the
solution is acidic or basic. [1]
The results of the experiment showed varying
changes in the colors of the pH indicators
depending on the pH of the sample. These color
changes occur in pH levels which are higher or
lower than their pH range.
Table 1. Tabulated results of the colorimetric
determination of pH uising acid-base indicators
AcidBase
Indica
tor

2.0
2.2

3.0
3.0

5.0
4.6

7.0
7.1

Tb

Salmon

YO

OY

Bb

Color
less

Purple

Purple

Bg

GY

Bp
Pr
Mr
Mo

Y
OY
P
R
Color
less

Y
Y
P
RO
Color
less

Y
Y
Pink
O
Color
less

V
RO
Y
O
Color
less

Pp

pH color Standards

Table 2. Continuation of the tabulated results of

the colorimetric determination of pH using acidbase indicators
AcidBase
Indica
tor

Tb
Bb
Bg
Bp
Pr
Mr
Mo
Pp

pH color Standards

REFERENCES

7.5

7.5

8.2

12.2

OY
Purpl
e
R
V
OR
Y
O
Color
less

OY

Dis
tilled
water
pH =
6.8
Y

Purple

Purple

R
V
OR
Y
O
Color
less

B
V
R
Y
O

B
V
Pink
Y
O

Pink

Pink

G
GY
Y
Pink
O
Color
less

7.5

8.0

12.0

Based on the results, the

changes that occurred on the
closely matched to the solution
value of 4.6. Therefore, the pH
water is at approximately 4.6.

indicators. Electrometric method is said to be

more accurate since it measures the activity of
hydrogen ions in the sample compare to the use
of acid-base indicators which measures the pH in
terms of the concentration of hydronium ions.

resulted color
distilled water
which had the
of the distilled

The pH of the distilled water is 6.8 when

measured using the electrometric method while a
pH of 4.6 was obtained with the use of acid-base

[1] Biology Dictionary

http://www.biology-online.org/dictionary/
3/1/2016
[2] Crisostomo, A.C., et. al. (2010). Laboratory
Manual in General Biochemistry. Quezon City: C
& E Publishing, Inc. Pages 1-4.
[3]Cecil,
J.R.
(1995).
Basic
Biochemical
Laboratory Procedures and Computing with
Principles, Review Questions, Worked Examples,
and Spreadsheet Solutions. (1st ed.). New York:
Oxford University Press. Pages 40-65.
[4] The editors of Encyclopedia Britannica,
Bronsted-Lowry theory
http://www.britannica.com/science/BronstedLowry-theory 3/1/16