pH MEASUREMENT AND BUFFER PREPARATION

Jon Ellis G. Datu, Athina Darla B. Deala, Alan Nathan D. Derige, Mary Kimberly L. Espaldon,
Ma. Theresa Angeli M. Estabillo, and Jemielle Patricia A. Estrada
Group 3 2F Medical Technology General Biochemistry Laboratory
ABSTRACT
In the experiment the group have prepared different buffer solutions, determined the pH the buffers and samples
calorimetrically using different liquid indicators and electrometrically using the pH meter. Acetate buffer solution was
used in the experiment. The buffer solution was prepared by using acetic acid (CH 3COOH) and sodium acetate
(CH3COO-). The desired pH for the acetate buffer solution is 5. The buffer solution was measured using a pH meter
and adjusted using 6.0 M HCl to make the buffer more acidic or 6.0 M NaOH to make it more basic. Once the desired
pH of the buffer is reached the pH of the buffered solution is measured calorimetrically using acid-base indicators such
as thymol, bromophenol, bromocresol green, bromocresol purple, phenol red, methyl red, methyl orange. This shows
the changes what an acid-base indicator undergoes when added to a solution of a certain pH. This can also be used to
narrow down the pH range of a substance.

INTRODUCTION
Power of hydrogen or better known as pH is
defined as the logarithmic measure of the
hydrogen ion concentration. It is most commonly
used to compare the respective acidities of
solutions. Because of this pH and its changes, it
can greatly affect the function and processes of
most bimolecular activity [5].
There is a variety of ways that can determine
the pH of a substance one of it being the
electrometric
determination
method.
The
electrometric determination of the pH is one of
the most accurate methods used in measuring
the pH of a substance. The method consists of
measuring the difference between the potential
of two special electrodes in contact with the
substance through the use of a potentiometer.
The pH value is obtained by measuring the
potential of the combination and deducting the
potential of the known electrode [1].
Another method used in measuring the pH of a
substance
is known as the colorimetric
determination method. Through this method, the
pH range of a substance is given with the aid of a
color reagent. The acid-base indicators will
transition in color at a certain range of pH [4].

EXPERIMENTAL
A. Compounds tested (or Samples used)
Distilled water, Glacial acetic acid (99.7% w/w,
sp.gr. 1.18, MM 60), Acid- base indicators
(Thymol blue, Bromphenol blue, Bromcresol
green, Bromcresol purple, Phenol red, Methyl
red, Methyl orange, Phenolphthalein)

B. Procedure
1. Preparation of Reagents

The group prepared 500 mL of 6.0 M HCl and

6.0 M NaOH to be used as reagents in the
experiment and labeled the separate containers
properly.

2. Buffers
In order to prepare a buffer solution, the group
prepared 250 mL 0.10 M acetate buffer; pk =
4.70 with a desired pH of 5.00 and identified the
weak acid and its conjugate base and calculated
the amounts of the buffer components using the
Henderson-Hasselbalch equation.

3. Electrometric Determination of pH
In the electromagnetic determination of pH,
first, the pH meter was calibrated at pH 4,7,10
then measure the pH of a 20 mL portion of the
following samples: Distilled water, the prepared
buffer and the assigned sample. The [H+] of the
sample was calculated and the pH of the
prepared buffer solution was adjusted to its
desired value by adding in portions of either 1.0
M HCl or 1.0 M NaOH while being monitored by
the pH meter.

4. Calorimetric Determination of pH
a. Preparation of color standards using
the buffer solutions
In the calorimetric determination of pH, which
involves the preparation of color standards using
the buffer solutions, the group prepared 6 test
tubes then labeled it with the pH of the buffer
and acid-base indicator to be added. After that, 5
mL of a buffer of a certain pH was pipetted into
the test tubes and two drops of an acid-base
indicator was added. Then, the mixture was
subjected to a vortex mixer and the color was
noted. Next, 5 mL of a buffer of a different pH
was put into another test tube and another 2
drops of the same acid-base indicator was added.
The group then prepared another set of 6 test
tubes then used a different acid-base indicator:
thymol blue, bromophenol blue, bromocresol
green, bromocresol purple, phenol red, bogen
indicator, methyl red, methyl orange, and
phenolphthalein.

b. Determination of the pH samples

The group determined the pH of samples by
adding distilled water or the assigned sample to 5
mL of the sample then two drops of an acid-base
indicator was added. The mixture was subjected
to the vortex mixture. After that, the color was
observed and noted down.

5. Determination of Buffer Capacity

In the determination of buffer capacity, first,
the pH meter was calibrated at pH 4, 7, and 10.
Then 100 mL each of 0.10 M HCl and 0.10 M
NaOH was prepared. After that, the buffer
capacity was determined by adding 0.10 M HCl in
1 mL portions from a burette to 20 mL of the
buffer until its pH changes as it is being
monitored with a pH meter. The group recorded
the volume which was added and continues to do
the same using 0.10 M NaOH.

6. Computation
Given:
pH= 7
0.5 M
250 ml

Weak acid= HPO4-2

Conjugate base= H2PO4pKa= 7.21

Handerson-Haselbach:
pH= pKa + log [WA]/ [CB]
pH = 7.21 + log [HPO4-2]/ [H2PO4-]
log-1 (7-7.21)= [HPO4-2]/ [H2PO4-]
0.616595001/ 1= [HPO4-2]/ [H2PO4-]
Total theoretical moles of buffer= 1.616595001
Total actual moles of buffer= (0.5M)(0.25L)=
0.125 moles
Actual
moles
[HPO4-2]:
(0.616595001/1.616595001) = ( x/ 0.125)
= 0.047676984 moles HPO4-2
Actual moles [H2PO4-]: (1/ 1.616595001)= (x/
0.125)
= 0.077323015 moles H2PO4Grams of HPO4-2:
Actual moles x MW = 0.047676984 moles x 268
g/mol
= 12.77743171 g HPO4-2
Grams H2PO4-:
Actual moles x MW= 0.077323015 moles x 138
g/mol
= 10.67057611 g H2PO4Figure 2.Computation for buffer preparation

RESULTS AND DISCUSSION

A. Electrometric Determination of pH
pH meter measures the potential difference
between the reference and the measuring pH
electrode. The potential of the pH electrode relies
on the activity of the hydronium ion.
The pH meter gives accurate measurement
although it needs to be calibrated beforehand.
Calibration can be done by dipping the meter into
a buffer and adjusting the meter accordingly.
To get the [H+] we use the formula:
10-pH = [H+]
Table 1. The pH, concentration and
temperature of the samples and the buffer
SAMPLES

pH

[H+]

Distilled H2O
Coca Cola
BUFFERS
Acetate

6.2
2.4

6.3x10-7
4.0x10-3

TEMP
(0C)
23
28

4.8

1.6x10-5

31

B. Colorimetric Determination of pH
Colorimetric determination which is the basis
for colorimetric analysis is the variation of color in
respect to the changes in pH of a solution. By
comparing the colors of unknown solutions with
the colors of known solution one may be able to
determine the pH of the unknown solution [3].
Acid base indicator which are usually weak acid
or bases are substances which changes color with
pH [2].
Table
2.
Results
Determination of pH

of

Colorimetric

ACID-BASE
INDICATOR

2.1

4.8

7.5

12.1

SX

Bb

Bg

Bp

Pu

Pr

Pi

Mr
Mo

Pi
R

Pi
O

Pi
O

Y
O

Y
O

V
Y

Y
O

Pi
O

Pp

pH

For pH 2.1, 3, 4.8, 7, 7.5, 8, 12.2 and SX

(distilled water), the following are the color
results for the acid base indicator Thymol blue:
orange, green, yellow, yellow, yellow, yellow,
blue and yellow.
For pH 2.1, 3, 4.8, 7, 7.5, 8, 12.2 and SX
(distilled water), the following are the color
results for the acid base indicator Bromophenol
blue: yellow, yellow, blue, violet, violet, blue,
blue and violet.
For pH 2.1, 3, 4.8, 7, 7.5, 8, 12.2 and SX
(distilled water), the following are the color

5.9

results for the acid base indicator Bromocresol

green: yellow, yellow, blue, blue, blue, blue, blue
and blue.
For pH 2.1, 3, 4.8, 7, 7.5, 8, 12.2 and SX
(distilled water), the following are the color
results for the acid base indicator Bromocresol
purple: yellow, yellow, yellow, violet, purple,
violet, violet and yellow.
For pH 2.1, 3, 4.8, 7, 7.5, 8, 12.2 and SX
(distilled water), the following are the color
results for the acid base indicator Phenol red:
yellow, yellow, yellow, red, red, red, pink, and
yellow.
For pH 2.1, 3, 4.8, 7, 7.5, 8, 12.2 and SX
(distilled water), the following are the color
results for the acid base indicator Methyl red:
pink, pink, pink, yellow, yellow, violet, yellow and
pink.
For pH 2.1, 3, 4.8, 7, 7.5, 8, 12.2 and SX
(distilled water), the following are the color
results for the acid base indicator Methyl
orange: red, orange, orange, orange, orange,
yellow, orange and orange.
For pH 2.1, 3, 4.8, 7, 7.5, 8, 12.2 and SX
(distilled water), the following are the color
results for the acid base Phenolphthalein:
colorless, colorless, colorless, colorless, colorless,
orange, magenta and colorless.

Figure 1. Distilled water after

variety of acid base indicator

using

REFERENCES
[1] Ackland, T., Kimbrough, D., Samal, P., Yoder,
C. Laboratory Instruments, Apparatus and
Supplies. Braun Corporation 34 (2007) pp. 437
& 440.
[2] Campbell, F. Acid - Base Indicators and
Titrations.
http://www.ch.ic.ac.uk/vchemlib/course/indi/indi
cator.html 02/28/16
[3] Cui, V. Preparation of Buffer Solutions and
Colorimetric Determination of the pH.
http://iris.inc.bme.hu/en/subjects/genchem/phde
t2.pdf 02/28/16
[4] Pahlavan, D. Preparation of Buffer Solutions
and Colorimetric Determination of the pH.
http://iris.inc.bme.hu/en/subjects/genchem/phde
t2.pdf 02/27/16
[5] Senese, A. What is pH? General Chemistry
Online.
http://antoine.frostburg.edu/chem/senese/101/a
cidbase/faq/what-is-pH.shtml 02/27/16