Preparation of Phosphate Buffer and Measurement of pH Using Electrometric and

Colorimetric Method
Mercado, B.L., Miranda, M.A., Napo, V.M., Nolido, S.N., and Pampilon, N.M.
2D-PH, Group 6., Department of Pharmacy, Faculty of Pharmacy, University of Santo Tomas,
Espaa Boulevard, 1015 Manila, Philippines
ABSTRACT
All biological process are affected by pH, pH is a measure of hydrogen ion concentration
and it is also the measure of the acidity or alkalinity of a solution. The occurrence of the
hydrogen ion points the function and conformation of most biomolecules. Buffers, a buffer is a
solution mixture of a weak acid and its conjugate base or a weak base and its conjugate acid
which resists sudden change in pH when small amounts of base or acid is added to the solution.
The Henderson-Hasselbach equation relates the pH of the solution to the pK of an acid and the
ratio of the concentration of acid and its conjugate base. This equation is also used to calculate
the amounts of acid and its conjugate base. The objectives of this experiment were to prepare
different buffer solutions, to determine the pH of the buffers and samples colorimetrically using
different liquid indicators and electrometrically using pH meter and last, to calculate the buffer
capacity of the prepare buffer solutions. The buffer solution assigned was phosphate with pK of
7.21 and a desired pH of 7.50. The following are the acid-base indicators present: Thymol blue,
Bromophenol blue, Bromocresol green, Phenol red, Methyl red, Methyl orange,Phenolpthalein.
The sample liquid solution used was Smart C orange juice. The pH of the buffer solution was
measured electrometrically by using a pH meter and was adjusted by HCL to make it more acidic
and NaOH to make it more basic. The pH meter indicates the hydrogen ion concentration in a
solution by responding to the potential developed by the electrical cell. The pH of the buffer was
also measured colorimetrically by using acid base indicators, when these are added varying
colors are produced it an help identify substances by narrowing the pH range. The sample given
had similar color range for pH 3 indicating that the orange juice sample has a pH of 3. The pH
range of the unknown is the color change interval of an indicator, where the difference in color
happens the end of the interval.
INTRODUCTION

n an aqueous solution, knowing the

concentration of the hydrogen ion is
important in order to be able to find
out if the solution is acidic or basic.
Knowing its acidity and alkalinity has a
great effect on learning the different
properties of a given solution; its solubility,
rate of reaction, and reaction to other
chemicals. [1] pH measurement measures

the relative amount of the hydrogen ion (H+)

contained in a solution. Solutions that are
acidic contain a high amount of hydrogen
ion have low pH value and solutions that are
basic contain low amount of hydrogen ion
have higher pH value. pH of a solution can
be measured electrometrically using a pH
meter, which is an electric device that acts as
a volt meter that measures the electrical

potential difference between a pHresponsive electrode and a reference

electrode and displays the calibrated pH
value of solution of which it is immersed
into [2]
A buffer solution consists of a weak
conjugate acid-base pair that can resist
drastic change of pH upon the addition of
small quantities of an acid or a base. [3] A
buffer is made in order that the components
of the solution must not consume each other
so a buffer solution may be acidic or basic.
An acidic buffer solution has a pH lower
than 7 and is composed of a weak acid and
one of its salts. A basic buffer solution has a
pH higher than 7 and is composed of a weak
base and one of its salts. [3] The pH
measurement of the buffer solution can be
measured using colorimetric analysis.
Colorimetric analysis use the aid of different
color reagents to determine its pH value.
The objectives of this experiment is
to; prepare different buffer solutions,
determine the pH of the buffers and samples
colorimetrically using different liquid
indicators and electrometrically using the
pH meter and calculate the buffer capacity
of the prepared buffer solutions.
METHODOLOGY
First, one of our classmates prepared
500 mL solution that contains 6.0 M HCL
and 500 mL solution of 6.0 M NaOH and
labeled it properly. He mixed 500 mL of
distilled water and 6.0 M HCL. These
aqueous solutions are the ones we used to
obtain the assigned pH of our buffer
solutions.

Our group was assigned to prepare a

phosphate buffer solution having 7.50 pH.
We calculated the components of the
solution by using the HendersonHasselbalch equation. The components we
used was 3.45 g of NaH PO H O and
2

2.75 mL of 6M NaOH.

We weighed 3.45 g of NaH 2PO4

H2O in a paper box by using the triple beam
balance. Next, we measured 2.75 mL of 6M
NaOH in a graduated cylinder. And 20 mL
of distilled water. We mixed all of the three
components in a beaker and stirred it. After
that, we checked the pH using an
electrometer/pH meter. It was not the
desired pH so we added drops 6M HCL to
make the pH acidic (lower) and 6M NaOH
to make it basic (higher). We continued
adding drops of the two aqueous solutions
until we reached the desired pH of 7.5.

Then, we put the buffer solution in a

volumetric flask and added distilled water
up to 250 mL. After that, we transferred the
solution in an amber bottle.

ELECTROMETRIC DETERMINATION OF
pH

We calibrated the pH meter at 4, 7

and 10. Then, we measured 20 mL of the
buffer solution, 20 mL of distilled water and
20 mL of SmartC Orange Juice. we put each
one of them in an individual beakers, we
used the electrometer to determine the pH of
the three samples and solved for the value of
H+ of each. We also collected data from
other groups the pH of their buffer solutions
because we are assigned with different
buffer solutions. After we collected, we
solved the value of H+.

COLORIMETRIC DETERMINATION OF
pH

A Preparations of standards using the

buffer solutions
We prepared 8 vials and pipetted 5mL of
the buffer solution and transferred it into
each vial by using a serological pipette. We
added in each vial two drops of the
following acid-base indicator: thymol blue,
bromophenol blue, bromocresol green,
bromocresol purple, phenol red, methyl red,
methyl orange and phenolphthalein. And
labeled it properly with gum labels. We
shook the vials with a vortex mixer. And
noted down the color in the corresponding
table. We noted it down under pH 7.5.

After that, we prepared another 8 vials

and pipetted 5 mL of distilled water and
transferred it to each vial. We added two
drops of the acid-base indicators: thymol

blue, bromophenol blue, bromocresol green,

bromocresol purple, phenol red, methyl red,
methyl orange and phenolphthalein. And
labeled it properly with gum labels. We
shook the vials with a vortex mixer. We
noted down its color under distilled water
column.

B Determination of the pH of the

samples
We repeated the procedure again but this
time we used the SmartC Orange Juice. We
prepared another 8 vials and pipetted 5 mL
of SmartC Orange Juice and transferred it to
each vial. We added two drops of the acidbase indicators: thymol blue, bromophenol
blue, bromocresol green, bromocresol
purple, phenol red, methyl red, methyl
orange and phenolphthalein. And labeled it
properly with gum labels. We shook the
vials with a vortex mixer. We noted down its
color under sample column. Then, we
compared it with the buffer solutions to
know its pH.

After that, all groups put their buffer

solutions with acid-base indicators in the
first table of the room. They labeled it with
the corresponding pH of the groups. Then,
we compared the vials with smartc orange
juice with the buffer solutions. After
comparing, we noticed that the buffer
solution with a pH of 3.00 have the same
colors with the colors of the vials containing
the SmartC sample. Therefore, we
concluded that our SmartC Orange Juice has

a pH of 3.00. Then, we compared it with the

buffer solutions to know its pH.

RESULTS AND DISCUSSION

Buffer has a weak acid (HA) and a
conjugate base(A-). A complete dissociation
in water will note happen between weak
acids and base rather an equilibrium is
produced, therefore the resultant solution
will resist the change in pH [4]. Buffers
have the pH range that enables them to
balance the change in hydrogen ion
concentration. The dissociation constant of
an acid buffer is called as the pka which can
be
determined by the
Henderson
Hasselbalch equation [4].

In
the
preparation of a buffer there are factors that
need to be considered: the change in
temperature is correlated with the change in
dissociation. [5] Prepare the buffer solution
at room temperature to avoid a change of
pH, but if your experiment involves a shift
in temperature choose a buffer that has the
range capacity if a shift in dissociation
happens.[4]Change in concentration can
affect the shift in pH, so to maintain the
buffer make sure to check the pH after
dilution. After achieving the desired pH
place the buffer in a amber bottle to prevent
the absorption of UV light absorption. [4]

Electrometric determination of PH
Table 1.0 Electrometric Determination of
pH
Samples
pH
Distilled
5.10
Water
Assigned
3.40
Sample
(Smart
C
Orange
Juice)
Buffers
Phosphate;
7.50
pK=7.21

[H+]
7.94x10-6
3.98x10-4

3.16x10-8

Table 1.0 shows the pH and the

concentration of Hydrogen ions in distilled
water, assigned sample and assigned buffer.
Electrometric
method
of
determination of pH made use of an
electrometer or pH meter [2]. A PH
meter, electric device used to measure
hydrogen-ion activity (acidity or alkalinity)
in solution.[2] Fundamentally, a pH meter
consists of a voltmeter attached to a pHresponsive electrode and a reference
(unvarying) electrode. [2]
The pHresponsive electrode is usually glass, and the
reference is usually a mercurymercurous
chloride (calomel) electrode, although a
silversilver chloride electrode is sometimes
used. [2] When the two electrodes are
immersed in a solution, they act as a battery.
.[2] The glass electrode develops an electric
potential (charge) that is directly related to

the hydrogen-ion activity in the solution

(59.2 millivolts per pH unit at 25 C [77
F]), and the voltmeter measures the
potential difference between the glass and
reference electrodes. [2]

The pH meter is calibrated at pH 4, 7 and 10

these pH numbers help achieve the best
accuracy of the device.[7] The numbers
serve as a range for the desired pH for the
experiment, it will eliminate the other values
that are beyond what you need.[7]

Table 2.0 Colorimetric determination of

PH.
Acid-base
indicator
Thymol

2.0
peach

Bromopheno
l blue

yellow

Bromocresol
green
Bromocresol
purple
Phenol red
Methyl red
Methyl
orange
Phenolphthal
ein
pH

colorle
ss
yellow
yellow
violet
pink
colorle
ss

3.0
light
yellow
light
yelllow
orange
pale
yellow
light
yellow
yellow
pink
orange
colorless

7.0
yellow

pH
7.5
light yellow

blue

light indigo

blue

light blue

violet

light purple

red
yellow
yellow
orange
colorless

light scarlet
pale yellow
light orange
colorless

Table 2.0 Shows change in color of the

sample after two drops of the acid-base
indicator was added. The assigned buffer,
Phosphate buffer with pH 7.5 formed a light
yellow solution with thymol, light purple
solution with bromocresol purple, light
scarlet solution with phenol red,pale yellow
solution with methyl red, light orange
solution with methyl orange and colorless
solution with phenolphthalein.

Colorimetric analysis is a process for

determining the pH with the use of color
variations in relation with the change in
concentration.[8] The color is formed due to
the addition of acid base indicators and
reacting with the property itself or a
formation of a colored compound as a result
of the reaction.[8] Acid base indicators are
either weak organic acids or weak organic
bases. [8] Some organic substances change
color in dilute solutions when the
concentration of the hydrogen ion reaches a
specific value. [8]Comparison of the
unknown solutions variation in color with
the color variation of the given pH will give
a basis on what is the correct concentration

of the unknown. [8]

Table 3.0 Acid base indicators

The sample given had similar color range

for pH 3 indicating that the orange juice
sample has a pH of 3. The pH range of the
unknown is the color change interval of an
indicator, where the difference in color
happens the end of the interval can be
estimated by:

In preparation of a 7.5 pH buffer we

concluded the different factors that may
affect buffer system. In finding the pH of the
unknown sample two methods were used,
first is the electrometric which gave a more
accurate reading of pH compared to the
calorimetric method.[8]

REFERENCES:
[1] Buffer solution (n.d.) retrieved at
http://webhost.bridgew.edu/c2king/CHEM1
42/Lab/7_Buffers%20and%20Properties.pdf
[2] pH meter (n.d.) retrieved at
http://www.britannica.com/technology/pHmeter
[3] Acid and bases and buffer solutions

(n.d.)
retrieved
at
http://chemwiki.ucdavis.edu/Core/Physical_
Chemistry/Equilibria/AcidBase_Equilibria/7._Buffer_Solutions
[4] Buffers for niochemical reactions(n.d)
retrieved on February 18 2016 at

https://www.promega.com/~/media/files/res
ources/paguide/letter/chap15.pdf?la=en
[5] Effect of temperature on Buffers (n.d)
retrieved on February 18 2016 at
http://reagecon.com/pdf/technicalpapers/Eff
ects_of_Temperature_on_pH_v4-_TSP-012.pdfBuffers
[6]PH meter.(2016).Retrieved on February
18,2015
at
http://www.britannica.com/technology/pHmeter.
[7] Guide care and calibration
(n.d)
retrieved on February 18 2016 at
http://www.mbhes.com/ph_meter_guide_car
e_and_calibration.htm
[8] Determination of buffer solution and
calorimeteric determination of pH (n.d)
retrieved on February 18 2016 at
http://iris.inc.bme.hu/en/subjects/genchem/p
hdet2.pdf