pH MEASUREMENT AND BUFFER PREPARATION

Ma. Rio Lauren M. Imbao, Jasmine Mary A. Jamorabon,

Kurt Raven T. Lai, Beatrice Andrea G. Lim, Danvel C. Liwag
Group 5 2C-BC Biochemistry Laboratory

ABSTRACT
A buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or
vice versa. Buffers are an important part of the biochemical processes of living things because they help
keep the pH within organisms' body stable. The objective of this experiment is to prepare a buffer
solution, and to determine its pH using both electrometric and colorimetric methods. The buffer will also
be used as a basis in determining the pH of the distilled water and Bllue water based sample using
colorimetric method. This experiment is consisted of four parts: (1) preparation of a buffer solution, (2)
preparation of standard reagents, (3) electrometric and, (4) colorimetric determination of pH. The group
of analysts was assigned to prepare a 0.050M Phosphate buffer with a pH of 7.50. Using the Henderson-
Hasselbach equation, it was calculated that 0.6611 g of NaH 2PO4H2O and 1.173 g of Na2HPO4 were needed
to prepare the buffer. The exact pH of the prepared buffer solution was determined to be 7.53 using a pH
meter. Likewise, the Blue water based sample and distilled water sample were subjected to colorimetric
and electrometric determination. The pH of the Blue water based sample was determined to be 3.68,
while the distilled water sample has a pH of 5.83, using the electrometric method. On the hand, the pH of
the distilled water sample and Blue water based sample was estimated to be 6.96 and 2.99, respectively,
using colorimetric method of analysis. And from the data gathered, it can be inferred that the
electrometric analysis gave a more accurate determination of pH compared to the colorimetric
determination.

INTRODUCTION
Potential of Hydrogen or pH is a numeric
scale in chemistry that determines if an aqueous
solution is acidic or basic. All biological systems
are dependent on pH levels. In terms of
Figure 2. Henderson-Hasselbach Equation
chemistry, it is the negative logarithm of the
concentration of the hydronium ion in moles per The Henderson-Hasselbalch equation is
liter (molarity). It is calculated using this valid when it contains equilibrium concentrations
expression: of an acid and a conjugate base [7].

The objective of this experiment is to

prepare a buffer solution, and to determine its pH
using both electrometric and colorimetric
methods. The buffer will also be used as a basis
Figure 1. Negative Logarithm of H+ in determining the pH of the water and colorless
sample (Blue water based drink) using
All biological systems are dependent on colorimetric method.
pH. It is important that these systems maintain a
specific and constant pH in order to keep METHODOLOGY
enzymes in their optimum state of protonation [1]. A Compounds Tested
In order to maintain a constant pH, systems use
a certain buffer. A buffer is used to minimize the 1 Standard Reagents
change in pH when an acid or base is introduced NaOH pellets
into the system [9]. The preparation of a buffer Concentrated HCl
solution usually involves a weak acid and its 2 Electrometric Determination of pH and
conjugate base or a weak base and its conjugate Colorimetric Determination of pH
acid. In the preparation of a buffer solution, the 0.050M Phosphate buffer, pH = 7.50
Henderson-Hasselbach equation is used to Distilled Water
determine the amount of acid and conjugate base Colorless Sample (fBlue water based
needed to make the solution of a certain pH. drink)
3 Acid-base indicators
Thymol Blue
 Bromophenol blue
Bromocresol green
Bromocresol purple
Phenol red
Methyl red
Methyl orange
Phenolphthalein
B. Procedure
1. Preparation of Buffer
A 250 mL of 0.050M Phospate buffer solution
was prepared with a pKa value of 7.21 and
desired pH of 7.50. The weak acid and conjugate
base of the buffer solution were identified and
using the Henderson-Hasselbalch equation, the
amount of buffer component was determined.
NaH2PO4H2O and Na2HPO4 were used as the
starting material. Through series of calculations,
it was known that to prepare the desired buffer
solution, 0.6611 g of NaH 2PO4H2O and 1.173 g of
Na2HPO4 will be needed. (Refer to appendix B for
calculations.)
2. Preparation of Reagents
A 250mL of 6M NaOH and 6M HCl standards
were needed for the preparation and
neutralization of the buffer solution. For the 6M
NaOH standard, 59.9970 g of NaOH pellets were
dissolved in distilled water and was transferred to
a 250 ml volumetric flask, where it was diluted
and mixed for homogeneity. In the preparation of
6M HCl, a 122.65 ml of concentrated HCl were
transferred into a 250 ml volumetric flask and
was diluted until the graduation marking. (Refer
to appendix A for calculations.)
3. Electrometric Determination of pH
The apparatus used in electrometric
determination was a pH meter. It was calibrated
at pH 4, 7, and 10. After the calibration of the pH
meter, the pH of the buffer solution was
measured and adjusted to the desired pH using
the prepared standards, 6M HCl and 6M NaOH.
The pH of the distilled water sample and Blue
water based drink sample were also determined
using a pH meter.
4. Colorimetric Determination of pH
Three set of six test tubes were prepared and
labeled. The first set is composed of 5 mL of
0.050M Phosphate buffer (pH=7.50) with 2 drops
of an acid-base indicator. The mixture was mixed
and resulting color was noted.
The second set of test tubes was a mixture of
5 mL of distilled and 2 drops of the same acid-
base indicator.
The last set was for the 5ml colorless sample
and the same indicators. The procedure was also
performed on all the prepared buffers by the
other groups. All the colors were noted after
mixing. Afterwards, the color produced by the
samples in different acid-base indicators were
compared to the colors produced by each buffer
solution mixed with the same acid-base indicators
for pH determination.
RESULTS AND DISCUSSION
A. Electrometric determination of pH
A calibrated pH meter was used to
measure the pH of the prepared buffer solution,
the distilled water sample, and the Blue water
based sample. Table 1 shows the data gathered
from the pH meter.
Table 1. Electrometric determination of pH
Samples pH [H+]
Distilled
5.83 1.5x10-6
water
Assigned
sample (Blue
3.68 2.1x10-4
water based
drink)
Buffer
7.53 3.0x10-8
prepared
A pH meter provides a value as to how
acidic or alkaline a sample is. The basic principle
of the pH meter is to measure the concentration
of hydrogen ions. Note that the greater the
concentration of hydrogen ions, the stronger the
acid is. pH is equivalent to the log[H+]. When
the concentration of hydrogen ion increases, pH
decreases. That means that an acidic solutions
has a pH values less than 7 while a pH value
greater than 7 will indicate an alkaline solution. A
pH value of 7 indicates a neutral solution and this
could be observe in pure water. Distilled waters
are not pure. The moment it comes in contact
with air, carbon dioxide gas begins dissolving into
it, forming carbonic acid. The actual pH,
therefore, will often be slightly less than 7 as
seen from the table above.
On the other hand the results show that
the most acidic sample is the Blue water based
drink which has a higher hydrogen ion
concentration than the distilled water and the
prepared buffer solution. Flavored drinks
frequently includes citric acid and other fruit-
derived acids. This might cause the acidity of the
sample. In a recent study, the pH values
recorded for the flavored water drinks ranged
from 2.74 to 3.34, according to Dr. Jay Oltjen
from Oltjen orthodontics, USA. Thus, it is
advisable to limit the intake of flavored waters
due to its acidity.
B. Colorimetric determination of pH
Colorimetry refers to any technique by
which an unknown color is evaluated in terms of
standard colors. This utilizes complex organic
dyes which changes to distinctive colors through
a specific pH range of the aqueous solutions.
These dyes are called indicators.
The acid-base indicators are weak acid or
bases which can be used as a tool in colorimetry.
The indicators are halochromic chemical
compound. This means that it changes color
when a change in pH occurs. Hence, it can detect
changes in pH and dictates if the solution is acidic
or basic. Table 2 below presents the common
acid-base indicators and their respective pH
range.
Table 2. Acid-Base Indicators
of unknown concentration (or pH) with the
intensities of solutions of known concentrations
(or pH), the concentration (or pH) of an unknown
solution may be determined. Table 3 Below shows
the results of the colorimetric determination of
pH for the distilled water sample and Blue water
based drink sample. From the results of the
analysis, the pH of the distilled water sample was
determined to be 6.96. This means that the color
produced by the distilled water with different
indicators falls nearest to the colors produced by
the buffer solution of pH 6.96. The same goes
with the Blue water-based drink sample, which
according to the standards, has a pH of 2.99.
However, colorimetric determination of pH
is less accurate compared to a pH meter device.
A pH meter could exactly tell the pH while
colorimetric determination could only give the
possible range of the pH of a sample.
Colorimetric determination is also subjective and
its area of application is very limited. If the
solution to be measured is cloudy, the
measurements will be unreliable. So in order to
avoid having vague results, clear samples were
used in the analysis instead of colored ones.

CONCLUSION
The group of analysts was able to prepare
a buffer solution based on the reagents and
concentrations assigned to them. The analysts
were also able to determine the pH of buffers and
Table 3. Results of Colorimetric determination of pH samples by electrometric method using a pH
meter. The Blue water based sample was
pH identified to be acidic which has a pH of 3.68.
Acid-base
indicator 2.02 2.99 5.02 6.96 While the pH of the distilled water sample was
Pale Pale Pale Pale determined to be 5.83, a litter lower than 7
Thymol blue
pink yellow yellow yellow because of the presence of dissolved carbon
Bromophen Pale Pale Pale
Indigo
dioxide gas in the sample, forming carbonic acid.
ol blue yellow yellow indigo Colorimetric method was also done using
Bromocreso Pale Light Pale various acid-base indicators and buffers as the
Blue
l green yellow yellow blue standards. The color produced by the samples in
Bromocreso Light Pale Pale
Yellow different indicators were compared to the colors
l purple yellow yellow indigo
produced by each buffer solution mixed with the
Pale Orange
Phenol red Yellow Yellow same indicators for pH determination. The pH of
orange yellow
Light Pale Pale the distilled water sample and Blue water based
Methyl red Pink sample was estimated to be 6.96 and 2.99,
pink pink yellow
Methyl Light Yellow respectively. From the results aforementioned, It
Scarlet Orange
orange red orange can be inferred that the electrometric analysis
Phenolphth Colorles Colorles Colorles Colorles gave a more accurate determination of pH
alein s s s s compared to the colorimetric determination.
pH
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