ISOLATION AND HYDROLYSIS

Hazel Joy Krissen F. Capulong, Allyson Denise N. Carlos, Shoula Marie G. Constantino,
Patricia Danielle N. Chong, Nia Bernadette U. Del Rosario
Group 3 2J-Pharmacy Biochemistry Laboratory

ABSTRACT
A buffer solution is a mixture of a weak acid and its conjugate base, or a weak base and its conjugate acid. This
experiment requires to prepare different buffer solution and to determine the pH of the buffers and samples using
different liquid indicators and using the pH meter. Phosphate buffer solution of pH 3 was assigned to the group to be
prepared, which is also utilized in the experiment. Buffer solution of phosphate was prepared by adding NaH 2PO4H2O
and of Na2HPO4 7H2O to sufficient amount of distilled water. With the use of the pH meter, the pH of the buffer
solution was electrometrically determined. To successfully control the basicity and acidity of the buffer solution and
keep it neutral, either 6M HCl or 6M NaOH was added to make the solution more acidic or basic. 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 acid-base indicators (Thymol blue, Bromophenol blue,
Bromocresol green, Bromocresol purple, Phenol red, Methyl 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 of the fluids and tissues of living organisms to

In biochemistry, buffer preparation and pH
measurement are essential for certain chemicals
to react with one another or to produce new
products. Due to the fact that biochemical
reactions are often defined in terms of hydrogen
ion (H+) concentrations, a Danish biochemist
named Soren Sorensen utilized a logarithmic
scale for expressing the H+ concenctration which
he called the pH, where p stands for power and H
for hydrogen ion concentration [1]. He then
defined the pH of a solution as the negative
logarithm of concentration (in moles/litre) of
hydrogen ions as shown in the equation below:
sustain the normal functioning of life which is
why buffer systems in the body are exceedingly
important.
The strength of a buffer against an acid or a
base is measured by buffer capacity. It is
described as the number of an acid or a base
added to a buffer to change the pH of a liter of
the buffer unit by 1.
In this experiment, the group aimed to prepare
a buffer solution; and determine the pH of buffers
and samples colorimetrically using liquid
indicators and electrometrically using the pH
meter.

pH= -log[H+]
EXPERIMENTAL
Equation 1. Sorensens definition of pH A. Samples used
The samples subjected to electrometric
The symbol p denotes negative logarithm of. determination were distilled water, lemon tea
All biological processes are greatly affected by pH (Snapple), sports drink (Gatorade), commercial
and the term mentioned is a convenient way of soda (Sprite), and the prepared phosphate buffer
expressing the concentration of hydrogen ions. with pH of 3. While in the colorimetric
The function and conformation of most determination, distilled water, commercial soda
biomolecules are determined by the presence of (Sprite), and the prepared phosphate buffer were
used.
hydrogen ions as well.

On the other hand, a buffer solution is a B. Procedure

mixture of a weak acid and its conjugate base or
a weak base and its conjugate acid. Its function
is to resist changes in hydrogen ion concentration
as a result of internal and environmental factors,
and when there is an addition of small amounts
of acid or base. Excess hydrogen or hydroxyl ions
neutralize its components. It also controls the pH
1. Preparation of Buffer
In a volumetric flask, 13.2 g of NaHPO4 7H2O
was weighed accurately using an analytical
balance by subtracting the weight of the empty
volumetric flask from the total weight of the flask
and the chemical. The salt component (NaHPO4
7H2O) in the volumetric mixed thoroughly with
0.97 ml of the acid component ( NaH2PO4 H2O).
Distilled water was added to until it reached the Samples pH [H+]
250-mL mark. In order to check if the buffer had Distilled Water 7 1 x 10-7
a correct pH, it was measured using the pH Assigned Samples
meter. The prepared buffer, with a pH of 3, was 1. Lemon Tea 2.9 1.26 x 10-3
placed in a 250-mL amber bottle and was 2. Gatorade 2.9 1.26 x 10-3
properly labelled. 3. Commercial 3.1 8.0 x x10-4

2. Electrometric Determination of pH
Soda
The pH meter was first calibrated at pH 4, 7
Buffer Prepared 3 1 x 10-3
and 10. In 20mL portions, the pH of the following
samples were measured: the prepared buffer
The pH of the samples were then obtained
solution, distilled water and the clear, colorless
colorimetrically using different acid-base
commercial soda as an assigned sample. The
indicators. The acid-base indicators were added
concentration of H+ of the samples were
to portions of each of the samples. The resulting
calculated. The pH of the prepared buffer solution
color of the solutions is shown in Table 2.
was adjusted accordingly to pH 3 by adding in
portions of either 6.0M HCl or 6.0M NaOH while Table 2. Colorimetric Determination of pH Using
being monitored by a pH meter. Different Samples

3. Colorimetric Determination of pH Samples

Six test tubes were prepared and labeled each Acid-base Clear
with the pH of the prepared buffer and an acid- Distilled Buffer
indicator Sample
base indicator to be added. The acid-base Water Prepared
(Sprite)
indicators used were the following: thymol blue, Light Light
bromophenol blue, bromocresol green, Thymol blue Yellow
orange yellow
bromocresol purple, methyl red, methyl orange, Bromophenol Light Light
phenolphthalein and phenol red. Then, 5 mL of Indigo
blue green green
the buffer was pipetted in each test tube and 2 Bromocresol Light
drops of specific acid-base indicator were added. Green Yellow
green yellow
The mixture was shaken thoroughly using a Bromocresol
vortex machine. The change in color was Yellow Yellow Yellow
purple
recorded. Phenol red Yellow Yellow Yellow
Light
The pH of both distilled water and the assigned Methyl red Pink Pink
orange
sample, which is a clear, colorless commercial
Red Red
soda were determined. Two sets of six test tubes Methyl orange Orange
orange orange
were prepared and labeled with distilled water
colorles colorles
and the acid-base indicator to be added. Then, Phenolphthalein colorless
s s
5mL of the distilled water was pipetted in each
Between
test tube and 2 drops of specific acid-base pH 3.0 3.0
3.0 -5.0
indicator were added. The mixture was shaken
thoroughly using a vector machine. The change
The principle of electrometric pH is the
in color was recorded. The same procedure was
determination of the activity of the hydrogen ions
done with the clear, colorless commercial soda.
by potentiometric measurement using a glass pH
indicating electrode coaxially joined to a
RESULTS AND DISCUSSION reference electrode [3]. While the colorimetric
determination of pH is based on Beer-Lambert's
The pH of the samples were first obtained law, according to which the absorption of light
electrometrically using a pH meter, which was transmitted through the medium is directly
first calibrated using standard liquids at pH 4, pH proportional to the medium concentration [4].
7 and pH 10. The results of the pH and [H+]
concentration of the samples are shown in Table
1.

Table 1. Electrometric Determination of pH

 A B C D E F
A B C D E F
G H
G H

Figure 1. Colorimetric Determination of Figure 3. Colorimetric Determination of the

Commercial Soda (Sprite). (A) Methyl Red (B) Prepared Phosphate Buffer. (A) Bromophenol blue
Phenol Red (C) Bromophenol Blue (D) Thymol (B) Phenolphthalein (C) Bromocresol purple (D)
blue (E) Bromocresol green (F) Phenolphthalein Phenol red (E) Thymol blue (F) Methyl orange (G)
(G) Methyl orange (H) Bromocresol purple. Methyl red (H) Bromocresol green.

REFERENCES
[1] Buffers for Biochemical Reactions. (n.d.).
Retrieved February 26, 2017, from
https://www.promega.com/resources/product-
A B C D E F guides-and-selectors/protocols-and-
G H applications-guide/buffers-for-biochemical-
reactions/
[2] Bathan, et al. (2017). Laboratory Manual
in General Biochemistry. Manila, Philippines.
C&E Publishing Inc.
[3] Britton, et al. (n.d.). Hydrogen Ions, Their
Determination and Importance in Pure and
Industrial Chemistry. New York, United States
of America.
[4] Principles of Colorimetric Measurement.
(2005)
Figure 2. Colorimetric Determination of Distilled Retrieved February 27, 2017, from
Water. (A) Phenolphthalein (B) Methyl orange (C) http://pubs.acs.org/doi/pdf/10.1021/j150334a
Methyl Red (D) Phenol red (E) Bromocresol green 005
(F) Bromocresol purple (G) Bromophenol blue (H)
Thymol blue.