EXPERIMENT 1

PREPARATION OF BUFFER SOLUTIONS AND THE DETERMINATION OF THE pH USING DIFFERENT

LIQUID INDICATORS (COLORIMETRICALLY) AND USING THE pH METER
(ELECTROMETRICALLY)
Caa, Emmeline D., Dalmacio, Juan Severino B., Guzman, Gerome B., Doria,
Reina Mari Q., Elazegui, Monina J., Empaynado, Aira Louise E.
GROUP #3 of 2C-Medical Technology General Biochemistry Laboratory
Faculty of Pharmacy
Abstract
This experiment requires the researchers to prepare different buffer solution and to determine the pH of the
buffers and samples using different liquid indicators and using the pH meter. Primary phosphate buffer
solution of ph 8 was assigned to the group to be prepared, which is also utilized in the experiment. It was
prepared using Phosphoric acid (H 3PO4) as the weak acid and Primary sodium phosphate dihydrate
(NaH2PO4.2H2O) as conjugate base. 1.85 g NaOH pellets were dissolved in 1.6953 ml of phosphoric acid.
The pH of the buffer solution was determined using the pH meter and manipulated dropwise using 6M
NaOH. The pH meter showed accurate pH readings of the prepared buffer solution. On the other hand, the
pH of the prepared buffer solution was determined based on the colors produced upon applying the acidbase indicators (Thymol blue, Bromophenol blue, Bromocresol green, Bromocresol purple, Phenol red,
Methyl orange, Phenolphthalein). Colorimetric determination of pH showed the different color changes an
acid-base indicator undergoes when added to a solution of certain pH. This property of an acid-base
indicator enables one to identify different substances by narrowing their pH range. This enables one to
identify a specific substance as different substances exhibit different pH levels.
Introduction
Acids and bases compose the essential components in
disturbances in certain cells, tissues, and organs.
our daily life. From cleaning agents (basic) to fruits
Because of the pH sensitivity of many biomolecules,
(acidic), these contain acids and bases. Household
control of pH also is important for the success of
ammonia, a cleaning agent, and Liquid Plumber are
several procedures used in the biomedical laboratory.
examples of bases. According to Thomas M. Lowry
These include the separation, purification, and assay
and Johannes N. Bronsted, acids release protons
for biological activity of several biomolecules. The pH
(hydrogen ions) in their reactions whereas bases are
can
be
measured
electrometrically
and
substances which accepts protons. Strong acids
colorimetrically. One way of measuring pH is by using
release protons readily and almost completely in dilute
a device called pH meter, which consists of a pair of
aqueous solutions but weak acids do not so that, at
electrodes connected to a meter capable of measuring
equilibrium, in most cases, less than 1% of a weak
small voltages, on the order of millivolts. A voltage,
acid is ionized to yield protons. Strong bases have which
a
varies with the pH, is generated when the
great capacity for accepting protons whereas weak
electrodes are placed in a solution. This voltage is
bases are poor acceptors of protons.
read by the meter, which is calibrated to give the pH.
On the other hand, Acid-base indicators can also be
used to narrow down the pH range of a substance.
H refers to a measure of the hydrogen ion
The colour-change interval of an indicator is the pH
concentration of a solution. Solutions with a high
range, where emphasized colour change takes place
concentration of hydrogen ions have a low pH and
it was determined in the experiment.
solutions with low concentrations of H+ ions have and
a
high pH. Therefore, pH is also used as a measure of
There are certain solutions that resist change in pH
the acidity or basicity of a solution. Mathematically, pH
even when we add to them acids or bases. These are
is expressed as the negative log in base 10 of the
called buffers. A buffer is a solution which contains a
aquated hydrogen ion concentration.
weak conjugate acid-base pair that can resist drastic
pH = -log [H+]
changes in pH upon the addition of small amounts of a
The pH of solutions are important in the biomedical
strong acid or base. A buffer resists changes in pH
sciences for these reasons. First, the proper
because it contains both an acidic species to neutralize
functioning of biomolecules depend to an important
+
degree on the control of pH. Second, changes as small ions and a basic one to neutralize H ions. It is a
requirement that the components of a buffer must not
as 0.1 or 0.2 pH unit can cause significant metabolic

consume each other. For this reason, buffers are

prepared by mixing a weak acid or a weak base with a
salt of that acid or base.
The objectives of the following experiment are as to
prepare different buffer solutions and determine the
pH of the buffers and samples colorimetrically using
different liquid indicators and electrometrically using
the pH meter.
EXPERIMENTAL
A.

Distilled water, Phosphoric acid (H 3PO4), Primary

sodium phosphate dihydrate (NaH2PO4.2H2O), 6M HCl,
6M NaOH, Acid-base indicators (Thymol blue,
Bromophenol blue, Bromocresol green, Bromocresol
purple, Phenol red, Methyl orange, Phenolphthalein)
B. Procedure/Methodology
1. Preparation of reagents
A volume of 500mL conc. HCl (6 M) was measured
using a graduated cylinder to prepare a volume of
500mL of 6M HCl. The reagent was transferred to a
reagent bottle that was labeled accordingly.
Computations:
(12.2M)(xL) = (6.0M)(0.250L)
x = 0.12L or 120mL conc. HCl
2. Preparation of Buffer solution
The buffer solution (Phosphate; pKa =7.21;
pH 8) was prepared using the following
guidelines:
Conjugate
base

Volume

<---> H2PO4 - + H +

Henderson Hasselbach equation:

pH = pKa + logconj. baseacid
8.00 = 7.21 +log(H2PO4 -H3PO4 )
(H2PO4 -H3PO4 ) = antilog [8.00 7.21]
(H2PO4 -H3PO4 ) = 6.16595
Let

x= H3PO4
6.16595x = H2PO4 -

Total Moles (nT) or Molesbuffer = (0.250L)

(0.10M) = 0.025 moles

Compounds Tested

Weak
acid

H3PO4

Conc.

pH

pKa
H3PO4

Molesbuffer = (Molesacid)+(Molesconj. base)

0.025 moles = x + 6.16595x
X= 3.4868 x 10-3
6.16595X= 0.021373

=MolesH3PO4
=MolesH2PO4

Molarity of H3PO4 :
85 g H3PO4 100 g H3PO4 X 1 mole 98 g X
1.70 g 1 cm3 X1 cm3 1 mole =14. 745
moleL
Volume H3PO4 :
0.025 mole(14.745 mole/L) X (1L/1000ml)=
1.6953 ml H3PO4
Mass of NaOH:
Mole NaOH= Mole total + Mole conj. base
0.025 + 0.023173 = 0.046373 mole
0.046373 mole NaOH x 40 g 1 mole = 1.85 g
NaOH

3. Electrometric Determination of pH
This
instrument
indicates the hydrogen ion
H3PO4
H2PO4-1
250mL 0.10M 8.0
7.21
concentration in a test solution by responding to the
0
potential developed by an electrical cell. The pH meter
was calibrated at pH 7 by soaking the electrode in
Table
1.
Guidelines/Data
for
buffer
solution
neutral reagents like distilled water. The electrode was
preparation
lifted out of the neutral solution and dried using a
tissue paper. The electrode was then immersed in the
A mass of 1.85 g NaOH pellets was weighed using an
prepared buffer solution. The standard buffer should
analytical weighing scale. It was then transferred into
have a pH within two (2) pH units of the expected pH
a beaker with a certain volume of distilled water to
of the test solution. The bulb of the electrode was
dissolve the pellets. In another beaker, a volume of
completely covered with solution. Upon immersing the
1.6953 ml (19 drops) Phosphoric acid (H 3PO4) was
electrode into the beaker containing the prepared
measured using a serological pipette and an aspirator.
buffer solution, the reading stopped fluctuating at the
After preparing the solutions, the acid was mixed with
reading 7.4. Amounts of NaOh were added dropwise
the NaOH solution. The mixture was then transferred
to bring the pH level up to the desired level, 8. After
to the 250mL volumetric flask. Distilled water was
8-10 drops of the strong base, the reading was
added into the volumetric flask until it reached the
8 and the desired pH level was attained. The
250mL mark. The prepared solution was stored into already
a
electrode was carefully removed from the buffer
500mL amber bottle that was labeled properly.
solution, rinse it with distilled water using a water
bottle and then dried using a tissue paper.
Computations of the Buffer Solution:

because the electrode is sensitive to change in the

concentrations of [H+] and [OH-] ions. In that
manner, the pH meter is more accurate in reading
pH levels compared to a pH paper.

2.

4. Colorimetric Determination of pH
Eight test tubes for each buffer solutions were
prepared and properly labeled with the acid-base
indicators. The following acid-base indicators were
utilized:
Thymol
blue,
Bromophenol
blue,
Bromocresol green, Bromocresol purple, Phenol
red,
Methyl
red,
Methyl
orange,
and
Phenolphthalein. Each test tube was then filled with
1mL (28 drops) of the prepared buffer solution
using a serological pipette and an aspirator. A
certain amount (2 drops) of acid-base indicator was
dropped in the corresponding labeled test tubes.
The test tubes were shaken and the color was
recorded.

Colorimetric Determination of pH

Colorimetric Analysis uses the variation as a

means of determining the pH since the
intensity of the color of a solution changes
with its concentration or pH. Research says
that the color may be due to the inherent
property of a substance in the solution, or the
formation of a product as a result of the
addition of a suitable reagent or acid-base
indicator. The pH of a solution can be
determined by comparing the color intensities
of the solution with unknown pH with the
intensities of the solutions with known pH.
Certain organic substances change color in
dilute solution when the hydrogen ion
concentration reaches a particular value. For
example, phenolphthalein is a colourless
substance in any aqueous solution with a
hydrogen ion concentration greater than
1.0x10-8 M (pH less than 8.0). In solutions
with a hydrogen ion concentration less than
1.0x10-8
M
(pH
greater
than
8.0),
phenolphthalein is red or pink. Substances
like phenolphthalein, which can be used to
determine the pH of a solution, 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.
The equilibrium in a solution of the acid-base
indicator methyl orange, a weak acid, can be
represented by the equation
HA
red

RESULTS AND DISCUSSION

1. Electrometric Determination of pH
In the process of adding 8-10 drops of the strong
base, the reading was already 8 and the desired pH
level was attained. It showed fluctuations in
readings with the addition of NaOH. This happens

H+ + Ayellow

For instance, the anion of methyl orange is

yellow, and the non-ionized form is red. If
acid is added to the solution, the increase in
the hydrogen ion concentration shifts the
equilibrium toward the red form in accordance
with the law of mass action.
The indicator's colour is the visible result of
the ratio of the concentrations of the two
species A- and HA. In the case of methyl
orange, when [H+] has the same numerical

value as Ka, the ratio

Simply put, the colourof [A-] to [HA] is
change interval of an
Acid Base
pH 8.0 Buffer
equal to 1, meaning
indicator is the pH
Indicator
that 50% of the
range, where
indicator is present
pronounced colour
Thymol
Light yellow
in the red acid form
change takes place; the
and 50% in the
borders of this interval
Bromophenol blue
Lavender
yellow ionic form,
can be estimated by
and the solution
pKa-1 and pKa+1. In
Bromocresol green
Faded blue
appears orange in
addition, acid-base
colour. When the
indicators also show
Bromocresol purple
Purple
hydrogen ion
molecular characteristics
concentration
of a substance. Color
Phenol red
Pinkish red
increases to a pH of
changes in molecules
3.1, about 90% of
can be caused by
Methyl orange
Yellow orange
the indicator is
changes in electron
present in the red
confinement. More
Phenolphthalein
Colorless
form and 10% in the
confinement makes the
yellow form, and the
light absorbed bluer
solution turns red. No change in color is
(darker), and less makes it redder (lighter). The
visible for any further increase in the
following table presents the expected color changes
hydrogen ion concentration.
that would occur depending on the given pKA values.
The addition of a base to the system will increase the
hydroxide ion concentration and decrease the
hydrogen ion concentration and shifts the equilibrium
The table above became the basis in order to
toward the yellow form. At a pH of 4.4 about 90% of
predict more or less the outcome of the
the indicator is in the yellow ionic form, and a further
experimental procedure. The researchers
decrease in the hydrogen ion concentration does not
were tasked to prepare a phosphate buffer
produce a visible colour change. The pH range
with a pH level of 8. Upon adding the acidbetween 3.1 (red) and 4.4 (yellow) is the colourbase indicators to this particular buffer, the
change interval of methyl orange; the pronounced
following were the resulting colors.
colour change takes place between these pH values.
TABLEINFOHERE

Acid-Base
Indicator

pH
Range

pka
Value
s

1.2 - 2.8
; 8.0 9.6

1.3 ;
8.9

Bromophenol
Blue

3.0 - 4.6

7.0

Yellow
Blue/Indigo

Bromocresol
Green

3.8 - 5.4

4.7

Yellow
Blue

Bromocresol
Purple

5.2 - 6.8

6.3

Yellow
Pink/Purple

Phenol Red

6.4 - 8.0

7.9

Yellow
Pink/Purple

Methyl Red

4.4 - 6.2

5.1

Red Yellow

Methyl
Orange

3.1 - 4.4

3.7

Red Yellow

8.0 10.0

9.4

Colorless
Pink

Thymol

Phenolphthal
ein

Expected
Color
change

Red
Yellow
Blue

The following table presents the

resulting colors upon the addition of
the acid-base indicators to the
different buffer solutions and to
distilled water. The legend below must
be referred to in order to interpret the
table of results.
Legend:
R-Red
Y-Yellow
PY-Pale Yellow
GY- Golden Yellow
YG- Yellow Green
O-Orange
RO- Red Orange
LB- Light Blue
B-Blue

BG-Blue Green
I- Indigo
V- Violet
P-Purple
LPI- Light Pink
PI- Pink
DPI- Dark Pink
DP-Dark Purple
C- Colorless

AcidBase
Indicato
r

pH 2.5

pH 3.0

pH
5.0

pH 7.0

pH
7.5

pH 8.0

pH
12.0

Thymol

Disti
lled
Wat
er
GY

PY

GY

Bromop
henol
Blue

PY

Bromocr
esol
Green

LY

GY

BG

DB

DB

DB

Bromocr
esol
Purple

PI

PY

GY

DP

Phenol
Red

GY

DPI

PI

Methyl
Red

LPI

LPI

DPI

LPI

LY

Methyl
Orange

RO

Phenolp
htalein

table of expected color outcomes:

C-PI

LPI

DP