BIOCHEM 1 L Experiment # 01 2A BC Group 2

PH MEASUREMENT AND BUFFER PREPARATION

Aquino, M. G. , Badua, A. A., Belardo, A. J. S., Cabasag, A. S. D.
Cantal, I. N.,
Department of Biochemistry, Faculty of Pharmacy
University of Santo Tomas

ABSTRACT

pH stands for potential of Hydrogen. It measures the acidity and alkalinity of

water soluble substances and ranges from 1-14. 7 indicates neutrality, below 7
means the substance is acidic while above 7 implies a basic substance. In this
experiment, two methods were used to determine the pH of a phosphate buffer. First
method used is the Electrometric Method which is done by using a pH meter
containing an electrode while Colorimetric Method works with the use of acid-base
indicators. The objective of this experiment is to compare and contrast the two
methods and identify which one is best to use. The pH of the mixture of 0.50M buffer
and sample H2O2 was determined using the electrometric method and the goal was
to make it pH 3.00 by adding HCl since the buffer is already basic. Other buffer
solutions with different pH were then determined by observing the color change after
adding acid-base indicators. At the end of the experiment, it was confirmed that
Electrometric method works more efficiently than Colorimetric Method because it is
more sensitive, not tedious, and easier to use.

INTRODUCTION METHODOLOGY

The pH of a solution is the measure I. Preparation of Buffer

of the molar concentration of hydrogen
ions present in the solution. This
measures the acidity or basicity of a
solution. [2] In measuring the pH, a
device called pH meter is used to
accurately determine the pH of a
solution.
A pH buffer or a buffer solution is
an aqueous solution which consists of a
weak base and its conjugate acid or a
weak acid and its conjugate base. A
buffer is capable of resisting sudden
changes in pH of a substance so it is
very vital to any biological processes.
One way to calculate the pH or
concentration of the buffer is through
Henderson-Hasselbalch equation.
The objectives of this experiment is
to prepare different buffer solutions and
determine its pH together with the
samples colorimetrically using different
indicatory and electrometrically using
the pH meter.
For the preparation of buffer, the
amounts of each buffer components
were determined using the Henderson-
Hasselbalch equation. An amount of 4.4
g NaOH pellets were weighed using an
analytical balance and 8.4 mL of 85%
w/w concentrated H3PO4 solution were
measured using a pipette. The two
components were mixed in a small
beaker. The pH meter was used to take
note of the initial pH of the buffer.
II. Electrometric Determination of pH
For the electrometric determination
of buffer, the pH meter to be used was
first calibrated to pH 4.00, 7.00, and
10.00. After calibration, small amount of
0.50 M Phosphate buffer, distilled water,
and hydrogen peroxide sample was
diluted with 20 mL distilled water to
calculate for the concentration of H +
ions. The pH of 0.50 M phosphate buffer
 was calibrated using A set of test tubes were prepared
the pH meter by using 2 mL of distilled water and another
using 6.0 M HCl or set for 2 mL of hydrogen peroxide
6.0 M NaOH until sample. Two drops of acid-base
the pH reading was indicators shown above were also
3.00. added in each test tubes for both
distilled water and hydrogen peroxide
Figure 1. sample. Vortex mixer was used to mix
Calibration of Phosphate buffer to pH the solution evenly. The color was
3.00 recorded as data.

III. Colorimetric Determination of pH RESULTS AND DISCUSSION

A. Preparation of color standards using In using the electrometric method

the buffer solutions
Seven sets of 2mL of each solution
was prepared in test tubes:
0.50 M Phosphate buffer pH
2.00
0.50 M Phosphate buffer pH
3.00
0.50 M Phosphate buffer pH
5.00
0.50 M Acetate buffer pH 7.00
0.050 M Phosphate buffer pH
7.50
0.010 M Phosphate buffer pH
8.00
0.50 M Phosphate buffer pH
12.00
for determining the pH, a pH of 3.00
was recorded from reading the buffer
solution prepared, 5.54 for distilled
water and a pH of 3.25 was recorded
from reading the pH of the hydrogen
peroxide sample.
Table 1. shows the data in using the
colorimetric determination of pH. It
shows the different colors for the
different pH of buffers with different
indicators.
Table 1. Colorimetric Determination of
pH Data

In each set of the prepared

buffer solutions, 2 drops of the following
acid-base indicators were added:

Thymol blue
Bromophenol blue
Bromocresol green
Bromocresol purple
Methyl red
In using this method, the students
Methyl orange observed that the changes in color of
Phenolphthalein the hydrogen peroxide sample were
Phenol red similar to those of the buffer solution
that had a pH of 3 and the distilled water
The color of each test tubes had similar colors with the buffer
observed was recorded as data. solution with a pH of 5. From this
observation, the students concluded
B. Determination of the pH of the that these samples have a similar pH
samples. value.
 Based on the colorimetric
determination of pH, the pH of distilled
water is 5.00 while the pH of the
hydrogen peroxide sample is 3.00 which
were quite close to the values obtained
from the electrometric method which
was 5.54 and 3.25, respectively.

CONCLUSION

In conclusion, the electrometric method in determining the pH of our sample,

hydrogen peroxide, was more efficient and accurate than the colorimetric
method in determining the pH of a substance. In using the colorimetric method,
the pH of the sample was still identified but it took more time and it was less
accurate. The colors observed from the buffer solution at pH 3.0 were very close
to the colors observed when the indicators were added to the hydrogen peroxide
sample. Therefore, the pH of the prepared buffer solution is pH 3.0.

REFERENCES

[1] Experiment on Determination of pH (n.d.). Retrieved from

http://nitttrc.ac.in/Four%20quadrant/eel/Quadrant%20-
%201/Exp1_pdf.pdf on February 24 2017.
[2] pH as a Measure of Acid and Base Properties
hyperphysics.phy-astr.gsu.edu/hbase/Chemical/ph.html