PREPARATION OF BUFFER AND ELECTROMETRIC AND COLORIMETRIC

DETERMINATION OF PH
Eunice Aurelle T. Basco, Ian Lindley C. Cabral, Aira Mina A. Cayago,
Jardine Mariel L. Ching, Leomariss M. Chua and Filjosh R. Cucueco
Group 2
2A-Medical Technology
Biochemistry Laboratory
ABSTRACT
Preparation of buffer solution, determination of pH of the buffer and samples electrometrically and colorimetrically
were done in the experiment. The standard reagent was prepared by diluting 60g of NaOH pellets. The primary
phosphate buffer was prepared by using 1.69 mL of 14.8 M H3PO4 and 0.88g NaOH pellets. To obtain the desired pH of
3.0, the buffer was measured electrometrically using the pH meter and was manipulated by using either 6.0 M HCl or
6.0 M NaOH. The pH of the prepared buffer solution was measured colorimetrically using acid-base indicators such as
thymol blue, bromophenol blue, bromocresol green, bromocresol purple, phenol red, methyl red, methyl orange, and
phenolphthalein. The sample, distilled water with a pH of 5.7, was subjected to colorometric determination giving the
result colors of yellow for thymol blue, violet for bromophenol blue, blue for bromocresol green, yellow for
bromocresol purple, yellow for phenol red, yellow for methyl red, orange for methyl orange, and colorless for
phenolphthalein.

INTRODUCTION
pH is defined as a way of expressing low
concentration of hydrogen ions. [1] The term was
first described by Danish biochemist Sren Peter
Lauritz Srensen in 1909. [2] pH is an
abbreviation for "power of hydrogen" where "p" is
short for the German word for power (potenz)
and H is the element symbol for hydrogen. The H
is capitalized because it is standard to
capitalize element symbols. [2]
A buffer system/solution is a mixture of a weak
acid and its conjugate base, or a weak base and
its conjugate acid. [1] Since its components
neutralize the excess hydrogen (H+) and
hydroxyl ion (OH-), it allows solutions to resist
large changes in pH. Therefore, a buffer helps
maintain a near constant pH upon the addition of
small amounts of H+ or OH- to a solution. [3]
For buffers, The Henderson-Hasselbach equation
shows the relationship of the pH of a solution to
the pK of an acid and the ratio of the
concentrations of the acid and its conjugate base.
[1] The equation provides a convenient way to
think about buffers and pH:

pH= pKa+ log

The Henderson-Hasselbalch equation is used to

determine if an aqueous solution of a conjugate
acid/base pair is functioning as a buffer. [3]
With these in mind, we prepared different buffer
solutions, determined the pH of the buffers and
samples colorimetrically (by using liquid
indicators) and electrometrically (by using the pH
meter), and calculated the buffer capacity of the
prepared buffer solutions in this experiment.

EXPERIMENTAL
A. Compounds, solvents, and solutions
utilized and consumed
For the preparation of reagents, the samples
used, solvents or compounds tested in the
experiment were distilled water (H2O), Sodium
hydroxide pellets (NaOH) and 12.2 M
concentrated Hydrochloric Acid (HCl).
For the preparation of primary phosphate
buffer, the samples used, solvents or compounds
tested in the experiment were distilled water
(H2O), Sodium hydroxide pellets (NaOH) and
14.8 M concentrated Phosphoric Acid (H3PO4).
For the electrometric determination of pH, the
samples used, solvents or compounds tested in
the experiment were distilled water (H2O), Coca
Cola soft drink (assigned sample), prepared 250
mL Primary phosphate buffer, prepared 250 mL
6.0 M Hydrochloric acid reagent, and prepared
250 mL 6.0 M Sodium hydroxide reagent.
For the colorimetric determination of pH, the
samples used, solvents or compounds tested in
the experiment were the prepared 250 mL
Primary phosphate buffer and the different acidbase indicators: Thymol blue (C27H30O5S),
Bromophenol blue (C19H10Br4O5S), Bromocresol
green (C21H14Br4O5S), Bromocresol purple
(C21H16Br2O5S), Phenol red (C19H14O5S), Methyl
red (C15H15N3O2), Methyl orange (C14H14N3NaO3S),
and Phenolphthalein (C20H14O4).

B.iProcedure
1. Preparation of Reagents
The group was assigned to prepare a 250 mL
6.0 M NaOH aqueous solution. First, the amounts
needed to prepare the reagents were computed

using the dilution factor as well as the formula for

getting the molar concentration.
For the preparation of 250 mL 6.0M NaOH
solution, 60 g of Sodium hydroxide (NaOH)
pellets were measured using a triple beam
balance and was then dissolved with distilled
water (H2O) in a 250 mL beaker. The aqueous
solution was transferred to a 250 mL volumetric
flask were additional distilled water (H2O) was
added to fill up to the 250 mL mark. Afterwards,
the 250 mL 6.0M NaOH reagent was transferred
to a reagent bottle and then labeled properly.
Given:
NaOH pellets (40 MM)
250 mL 6.0 M NaOH aqueous solution

moles Buffer Solution=( L solution)( M solution )

moles Buffer Solution=(0.250 L)(0.1 M )
moles Buffer Solution=0.0250 moles
[conjugate base]
pH= pKa+ log
[weak acid]

3.00=2.12+ log

[ primary phosphate]
[ phosphoric acid]

[ primary phosphate] 7.5858 M

=
[ phosporic a cid ]
1M

[Buffer solution ]=[ primary phosphate ] +[ phosphoric acid ]

[Buffer solution]=7.5858 M+ 1 M
moles NaOH =(Molarity NaOH )(Volume NaOH )moles NaOH
=( 6.0solution]=8.5858
M ) (0.250 L)
[Buffer
M
moles NaOH =1.5 moles NaOH

Computation:

Moles of Primary Phosphate (H2PO4-1)

mass NaOH=(moles NaOH )(molar mass NaOH )

mass NaOH =(1.5 moles)(40 g /moles)
mass NaOH=60 g NaOH
2. Preparation of Buffer Solution
The group was assigned to prepare a 250 mL
primary phosphate buffer solution (pK=2.12)
with a desired pH of 3.00. After identifying the
weak acid and conjugate base components of the
buffer solution, the amounts needed to prepare
the buffer were computed using the HendersonHasselbalch equation, dilution equation, and as
well as the formula for getting the molar
concentration.
For the preparation of 250 mL primary phosphate
buffer solution, 0.88 g of Sodium hydroxide
(NaOH) pellets was reacted with 1.69 mL of 14.8
M concentrated Phosphoric Acid (H3PO4). It was
then diluted with distilled water (H2O) in a 250
mL beaker. Then, the aqueous solution was
transferred to a 250 mL volumetric flask were
additional distilled water (H2O) was added to fill
up to the 250 mL mark. Afterwards, the 250 mL
primary phosphate buffer solution was
transferred to a reagent bottle and then labeled
properly.

[Primary phosphate] moles primary phosphate

=
[buffer solution]
moles buffer solution
7.5858 M moles primary phosphate
=
8.5858 M
0.0250 moles
Moles Primary phosphate=0.022 moles
Moles of Phosphoric Acid

[Phosphoric acid ] moles phosphoric acid

=
[ buffer solution ]
moles buffer solution
1M
moles primary phosphate
=
8.5858 M
0.0250 moles
Moles Phosphoric acid=0.0029moles
Volume of Acid Used

Volume Phosphoric acid=

0.0250 moles
14.8 M

Volume Phosphoric acid=0.00169 L1.69mL

Mass of Base Needed

Given:
14.8 M Concentrated H3PO4 (pK = 2.12)
NaOH pellets (40 MM)
250 mL 0.10 M buffer solution
Desired pH = 3.00
Weak acid = Phosphoric acid (H3PO4)
Conjugate base = Primary Phospate (H2PO4-1)

Mass Sodium hydroxide=(0.022moles)(40 g/moles)

Mass Sodium hydroxide=0.88 g
3. Electrometric Determination of pH
The pH meter was calibrated first at pH 4, 7,
and 10. First, the pH and temperature of the
distilled water (H2O) was measured and noted.

The same procedure was done for the Coca Cola

soft drink (assigned sample) and prepared
Primary phosphate buffer. For each of the pH
measured, the [H+] were calculated using the
formula 10-pH.
Using a pH meter, the pH of the prepared
buffer solution was measured. The buffer solution
was then manipulated to pH 3.0 (which is the
desired pH) using the 6.0M NaOH solution (to
make it more basic, increase pH) and 6.0M HCl
solution (to make it more acidic, decrease pH).
4. Colorimetric Determination of pH
Table 1. Organic Dyes Used for Colorimetric
Determination of pH
Indicator

pH Range
Acidic: 1.2-2.8

Thymol Blue
Alkaline: 8.0-9.6
Bromophenol
Blue
Bromocresol
Green
Bromocresol
Purple

3.0-4.6
3.8-5.4
5.2-6.8

Color Change
Acidic: Red to
yellow
Alkaline: Yellow to
blue
Yellow to Blueviolet

The preparation of the buffer was done by

altering the amount of weak acid and its
conjugate base in the solution. The pH of the
buffer was obtained by the use of a pH meter.
This is a device utilized to measure the
expression of the degree of activity of an acid or
base according to hydrogen ion [H+] activity or
the acidity or alkalinity of a solution - also known
as pH [5]. pH is the unit of measure that
describes the degree of acidity or alkalinity of the
given solution and is measured on a scale of 0 to
14 [5].
In the experiment, if the actual pH was less than
the desired pH, the buffer would be treated with
an appropriate amount of the standard Sodium
hydroxide (NaOH). However, if the actual pH was
greater than the desired pH, the buffer would be
treated with a small amount of the standard
Hydrochloric acid (HCl).

Yellow to Blue
Yellow to Purple

Phenol Red

6.4-8.2

Methyl Red
Methyl Orange
Phenolphthalei
n

4.8-6.0
3.2-4.4

Yellow to Redviolet
Red to yellow
Red to yellow

8.2-10.0

Colorless to pink

a. Preparation of Color Standards Using the

Buffer Solutions
A certain number of vials (8) was prepared and
properly labeled with their respective acid-base
indicators. Using a serological pipette and an
aspirator, each vial was filled with 5mL of the
prepared primary phosphate buffer solution. A
certain amount (2 drops) of acid-base indicator
was dropped in the corresponding labeled vials.
The vials were shaken and the color was noted
down.
b. Determination of the pH of samples
The sample used in this experiment was
distilled water (H2O). A certain number of vials
(8) was prepared and properly labeled with their
respective acid-base indicators. Using a
serological pipette and an aspirator, each vial was
filled with 5mL of distilled water (H2O). A certain
amount (2 drops) of acid-base indicator was
dropped in the corresponding labeledvials. The
vials were shaken and the color was noted down.

RESULTS AND DISCUSSION

1. Electrometric Determination of pH

Figure 1. A pH meter connected

combination pH electrode.

to

2. Colorimetric Determination of pH
Colorimetry is a technique that is commonly used
in biochemistry. This process makes use of a
device called colorimeter that measures the
absorbance of specific wavelengths of light by a
specific
solution
and
thus, involves
the
quantitative estimation of colors [4] [6]. The
produced difference in color results in the
variation in the absorption of light, which is made
use of here in this technique. Moreover, this
method is usually applied to determine the
concentration of a known solute in a given
solution by the adaptation of the Beer-Lambert
law
[4].
The colorimeter is based on Beer-Lambert's law,
according to which the absorption of light
transmitted through the medium is directly
proportional to the medium concentration. The
results depicted in Table 1 shows that at pH=8,
the acid-base indicators, when mixed with the
solution, yielded their corresponding colors and
those colors are due to the concentration of the

colored complex that was formed. The color or

the amount of light absorbed is also related to
the concentration of the absorbing compound,
therefore it is one of the most useful techniques
in obtaining the pH.
According to Beers law when monochromatic
light passes through the colored solution, the
amount
of
light
transmitted
decreases
exponentially with increase in concentration of
the colored substance [6]. In addition, the
Lamberts law states that the amount of light
transmitted decreases exponentially with increase
in thickness of the colored solution [6].
Colorimetric determination of pH exhibits the
varying color changes an acid-base indicator
undergoes when added to a solution of a certain
pH. This property is used to distinguish different
substances by adjusting their pH ranges.

Figure 1.
Indicators

Distilled water with Acid-Base

Figure 2. Buffer solution with AcidBase Indicators

Table 2. Results of Colorometric

Colorimetric Analysis use the acquired various

Determination of pH (R = Red, O=
colors as a means of determining the pH since
Orange, G= Green, Y = Yellow, b = Blue,
the intensity of the color of a solution changes
P=Pink, V= Violet, C= Colorless, YO=
with its concentration or pH. The color may be
Yellow Orange, OY= Orange Yellow,
due to inherent property of a substance in the
solution or due to the formation of a product as a
GB= Green Blue, BV= Blue Violet, VB=
result of the addition of a suitable reagent or
Violet Blue) (Tb = Thymol Blue, Bb=
acid-base indicator. The
Acid
pH Color Standard
Bromophenol
pH of a solution can be
Base
2
3
5
7
7.5
8
12
D.
blue, Bg=
determined
by Indic. 2.2
3
4.6 6.9 7.4 7.5 7.9 12.1 H2O Bromocresol
comparing
the
color
green, Bp =
Tb
O
YO
Y
Y
Y
Y
Y
B
Y
intensities
of
the
Bromocresol
Bb
G
Y
B
VB
V
V
V
V
V
solution with unknown
purple, Pr =
Bg
Y
Y
GB
B
B
B
B
B
B
pH with the intensities
Phenol Red, Mr
Bp
Y
Y
Y
BV
V
V
V
V
Y
of the solutions with
Pr
Y
Y
Y
YO
R
R
P
P
Y
known pH.
= Methyl Red,
Mr
P
P
R
Y
Y
Y
Y
Y
Y
Mo= Methyl
Table 1. List of AcidMo
R
R
OY YO
O
O
O
O
O
Orange, Pp =
Base Indicators with
Pp
C
C
C
C
C
C
C
P
C
their
corresponding
Phenolphthalein)
colors
Acid - Base Indicator

pH 8.0

Thymol blue

Yellow

Bromophenol blue

Purple

Bromocresol green

Blue

Bromocresol purple

Purple

Phenol red

Red

Methyl red

Yellow

Methyl orange

Orange

Phenolphthalein

Colorless

REFERENCES
[1] Crisostomo, A. D., Daya, M. L., de Guia, R.
M., Farrow, F. L., Gabona, M. G., Ysrael, M.C.
(2009). Laboratory Manual in General
Biochemistry. Ph Measurement and buffer
preparation, 1, 1.
[2] Anne Marie Helmenstine, Ph. D. (2015,
November 13). What Does pH stand for?
Retrieved from
http://chemistry.about.com/od/ph/f/What-DoesPh-Stand-For.htm
[3] Chemistry Department-Xavier University of
St. Louisiana (2014, December 3). Preparation
of buffers at a desired pH. Retrieved from

www.xula.edu_Chemistry_documents_biolab_Car
oll...(14-12-03pdf).pdf
[4] Chhabra, N. (September, 2015). ColorimetrySimplified. Retrieved from
http://www.namrata.co/colorimetry-simplified/
[5] No author (2003). pH Meter. Retrieved from
http://www.omega.co.uk/prodinfo/ph-meter.html
[6] No author (June, 2011). Principles of
Colorimetry. Retrieved from

https://ecoplants.wordpress.com/2011/06/23/pri
nciples-of-colorimetry/