pH Measurement and Buffer Preparation

Dela Rosa, M.L., Diaz, K.I.M, Domingo, M.A., Dungca, M.K., Gabayan, M.
2H Pharmacy, Faculty of Pharmacy
University of Santo Tomas
Abstract
The experiment was done to determine the electrometric pH of the buffers
and samples with the use of the pH meter and to differentiate them by
colorimetric determination method through using different acid-base
indicators. The indicators used were thymol blue, which is a diprotic acid,
bromophenol blue, bromocresol green, bromocresol purple, phenol red,
methyl red, methyl orange, and phenolphthalein. The buffer that was
assigned to be made was an acetate buffer with a pH of 5 which was
checked by the pH meter. The samples used were the coconut juice and
distilled water. Distilled water had a pH of 5 given that one of the columns in
the pH meter matched into that of the distilled water. On the other hand, the
coconut juice which had a pH of 5.3 was determined by the pH meter by
using the electrometric determination method.
Introduction
All biological processes are
greatly affected by pH (Crisostomo
A.C.,et al.2010). The term pH is
used to identify the concentration
of [H+], thus the pH of a solution is
simply the negative logarithm of
[H+].
The pH of a solution is
greatly
dependent
on
the
concentration of [H+] ions, thus
buffers are required to maintain the
pH by binding the [H+] ions which
in turn stabilizes the changes in pH.
(Concepts in Biochemistry,n.d.)
A buffer solution is a mixture of a
weak acid and its conjugate base,
or a weak base and a conjugate

acid. (Crisostomo A.C.,et al.2010).

If the pH of a buffer solution is less
than 7 then the solution is acidic, if
the buffer solution exceeds the
range of 7 then the solution is
considered as basic but is the
buffer solution remains to be at pH
7, then the solution is considered
neutral. In this instance, the
[base]
pH = pKa + log
[acid]

Henderson-Hasselbach
equation
was used given that it shows the

relationship between pH and the

pKa of a weak acid:

There are two methods in

this experiment that were used in
order to determine the pH namely
the Electrometric determination
method and the Colorimetric
determination
method.
The
electrometric
determination
method is the most accurate of the
methods
employed
for
the
determination of hydrogen ion
concentration and the accepted
method for research and laboratory
work
necessitating
pH
measurements accurate to 0.1 to
0.001
pH (Braun Corporation
(1934) pp. 437 & 440) while
the colorimetric
determination
method measures color wherein
chemicals are added to the sample
and those chemicals react with
sample produces a color change
(Physical
Properties:
Water
Chemistry: pH, from Discovery of
Estuarine
Environments
(DOEE),n.d., para. 1).
In the
colorimetric determination method,
these chemicals were used to
determine the pH of a solution, and
are called acid-base indicators.
Acid-base indicators are either
weak organic acids, HA, or weak
organic bases, BOH, where the
letters A or B stand for complex
organic group (Determination of
the Average Molecular Weight of a
Liquid.n.d.). In this experiment,

acid-base indicators are as follows

together with their pH range:
Thymol blue with 1.2-2.8 and 8.09.6, Bromophenol blue with 3.04.6, Bromocresol green with 3.85.4, Bromocresol purple with 5.26.8, Phenol red with 6.8-8.4, Methyl
red with 4.2-6.3, Methyl orange
with 3.1-4.4, and Phenolphthalein
with 8.0-9.8.

Procedure
Preparation of reagents and buffer:
500 mL of 6.0 M of HCl and
6.0 M of NaOH was prepared and
properly
labeled.
Acetic
acid
(CH3COOH) and sodium acetate
(Na2CH3COO)
were
used
in
preparing the 250 mL buffer. 6
grams of acetic acid was mixed
with 0.01 L of NaOH and was added
with water to fill up the 250mL
pK = 4.80 pH = 5.00
pH = pKa + log [X]/[Y]
5.00 = 4.70 + log (x)
X = 0.50
1.50 = 0.10
1
Y
Y = 0.06L
Y = 10-0.06
Y = 0.04
pH = pKa + log [salt]/[acid]
5.00 = 4.70 + log X
0.3 = log X
X = 0.50

mark. Then the

labeled properly.

container

was

Acetate

Electrometric Determination of pH

1.0 M NaOH in portion while

being monitored by the pH
meter.

The pH meter was calibrated

to 5 and was measured using 20
mL portion of buffer solutions,
distilled water and coconut juice.
The [H+] of the sample used was
calculated. The pH of the buffer
solution was adjusted to the

Calorimetric Determination of pH
A. Preparation
of
color
standards using the buffer
solutions:
Six test tubes was prepared and
labeled with the pH of the buffer
and acid-base indicator to be
added. 5 mL of the buffer was
placed in each test tube and 2
drops of an acid-base indicator was
added. The mixture was shaken
and the resulting color was noted.
The procedure was repeated using

X = 0.50 moles salt

+
1 mole acid
1.50 moles buffer
X = (0.5 mol salt) (0.025 mol buffer)
1.5 mol buffer
X = 8.33 x 10-3 mole salt
0.025 mol buffer 8.33 x 10-3 =
0.167 mole
8.33 x 10-3mole x 59.05g/mole
= 0.492g
Acid-base
Acetate (salt)
indicator

pH

0.167mole x 59.04 2.0

= 9.86g Acetate
3.0
5.0
(acid)
Thymol blue
orange yellow
yellow

7.0
yellow

7.5
yellow

Distill
ed
Water

Sampl
e

Bromophenol
Blue
Bromocresol
Green
Bromocresol
Purple
Phenol Red
Methyl Red

yellow

yellow

blue

blue

blue

8.0
yello
w
blue

yellow

yellow

blue

blue

blue

blue

blue

blue

blue

yellow

yellow

yellow

purple

purple

yellow

yellow
pink

yellow
orange

orange
yellow

orange
yellow

yellow
orange

yellow
orange

Methyl
Orange
Phenolphthal
ein

yellow

yellow

orange

orange

orange

orange

orange

colorle
ss

colorle
ss

colorle
ss

colorle
ss

colorle
ss

purpl
e
red
yello
w
oran
ge
pink

yellow

yellow
pink

purpl
e
red
yello
w
oran
ge
pink

colorle
ss

colorle
ss

desired pH by adding 1.0 M HCl or

12.0
blue

yellow

yellow

blue

blue

blue

the following acid-base indicators:

Thymol blue, Bromophenol blue,

Bromocresol green, Bromocresol
purple, Phenol red, Methyl red,
Methyl
orange
and
Phenolphthalein.
B. Determination of the pH of
the samples
Two drops of an acid-base
indicator was added to 5 mL of
distilled water and was shaken. The
resulting color was noted. The
same procedure was repeated but
the sample used was 5 mL of
coconut juice.

Results and Discussion

Colorimetric Determination of pH
Each
acid-base
indicator
showed different results in the
calorimetric determination of pH
test. These indicators were used as
the determinants of the pH of the
samples. Eight indicators were
used in the determination tests.
The samples that were also used
were coconut juice and distilled
water. Table 1 shows the results
that were obtained from the
experiment.

Table 1. Colorimetric Determination of pH results

Thymol blue changed the

color of the solution twice. First,
when the solution was at the pH
between 2.0 and 3.0 and second,
when the solution was at the pH
between 8.0 and 12.0. Thymol blue
underwent two different color
changes and this made it a diprotic
acid which means that at the lower
pH, around pH 2.0, the thymol blue
existed in its red color. As the pH
increases, its color was slowly
converting itself into yellow which
dominated the color of the solution
until around pH 8.0. As the pH
increases beyond pH 8.0 the
solution turned into blue. This
happened because thymol blue has
two protons and has two pKa
values, one that exists at around

pH 2.0 and the other at around pH

8.0.
Phenolophthalein at lower pH
was colorless and its ions were
pink. The indicator at lower pH
showed that it lacks hydroxide ions
which made it colorless because
adding hydrogen ions causes a
leftward shift and will turn the
indicator colorless. And as the pH
increases, hydrogen ions were
replaced by hydroxide ions from
the equilibrium and turned the
indicator to pink solution. The pH
range of the phenolophthalein was
between pH 8.0 and pH 12.0.
Bromophenol
blue,
bromocresol green, bromocresol
purple, phenol red, and methyl
orange were yellow at pH 2.0. Both

bromophenol blue and bromocresol

green changed color at pH 5.0
while bromocresol purple changed
its color at pH 7.0. Methyl orange
and phenol red both changed their
yellow initial color into orange but
at different
pH, phenol
red
dominated orange color from pH
7.0 while methyl orange changed
its color at pH 5.0.
These color
changes tell two things. One, if the
acid was added to the solution,
more H+ was produced which
resulted to change color of the
solution that matched that of an
HIn. On the other hand, if more
base was added, more OH- would
be the result causing the solution
to change color that would match
that of In-.
Distilled water which had a
yellow color results after the
additions of the thymol blue and

phenol red separately, blue color

was the result in additions of both
bromophenol blue and bromocresol
green, green color resulted when
distilled water was added with
bromocresol purple, orange was
the visible result after the additions
of methyl red and of methyl
orange, and then it was colorless
after adding with phenolophthalein.
These colors of each indicator
made the distilled water fall in
around pH 5.0 since its results were
visibly seen at the pH 5.0 column.
The sample, which was the
coconut juice, fell in the pH of
around 5.0. Similar case with
distilled
water,
the
samples
indicators matched at a given pH in
the table that had yellow color was
the result after additions of thymol
blue and methyl

red, purple color was seen after it

was being added with bromophenol
blue
and
bromocresol
purple
separately, orange color resulted
after phenol red and methyl orange

were separately added to it, a blue

color was witnessed after adding
with bromocresol green, and a
colorless was the result when it
was added with phenolophthalein.

Conclusion

that alter the physical properties of

color indicators are observed when
these are mixed with a particularly
acidic/basic
solution.
Color
indicators are sensitive enough to
change its physical color because
of
the
electron
confinement
happening in the process of

Determination of the pH of a
certain substance or solution
through acid-base color indicators
shows the different range of
varying colors when a color
indicator is added to a solution with
a certain pH. Chemical reactions

agitating the two liquids. More

confinement
makes
the
light
absorb darker shades and hues,
and less confinement makes it
absorb lighter shades.

Concepts in Biochemistry,n.d,
Retrieved
from:
http://www.wiley.com/college/boyer
/0470003790/reviews/pH/ph_ioniza
tion_ph.htm

References:

Braun Corporation, Catalog No. 34,

Laboratory Instruments, Apparatus
and Supplies, Braun Corporation
(1934) pp. 437 & 440

Crisostomo
A.C.,et
al.(2010).
Laboratory Manual in General
Biochemistry,
Quezon
City,
Philippines.
Physical
Properties:
Water
Chemistry: pH, from Discovery of
Estuarine
Environments
(DOEE),n.d., para. 1 Retrieved
from:
http://omp.gso.uri.edu/ompweb/do
ee/science/physical/chph2.htm

Determination of the Average

Molecular Weight of a Liquid.n.d.,
Retrieved
from:
http://iris.inc.bme.hu/en/subjects/g
enchem/phdet2.pdf