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PH MEASUREMENT AND BUFFER PREPARATION

Christine Dharryl F. Leal, Carl Nicanor V. Lontoc, Kristine Anne A. Mabansag, Janick Briand E. Mallare,
Justin Shaquille C. Manipon
Group 6 2F Medical Technology Biochemistry Laboratory

ABSTRACT
The objective of the experiment is to prepare different buffer solutions and to determine the pH of the
buffers and samples colorimetrically using different liquid indicators and electrometrically using the pH
meter. A buffer solution is a mixture of a weak acid and its conjugate base, or a weak base and its
conjugate acid. It resists a sudden change in pH whenever small amounts of acid or base are added. In
the experiment, a 250 mL phosphate buffer solution with pH of 7.5 with a molar concentration of 0.10M
was prepared. The pH of the buffer solution was measured electrochemically using the pH meter and
manipulated using 6M HCl (to make the buffer more acidic) and 6M NaOH (to make the buffer more
basic). The pH meter uses an electrode to measure the hydrogen ion concentration and the pH of an
unknown solution. The prepared buffer solution was subjected to colorimetric determination using acidbase indicators (Thymol blue, Bromophenol blue, Bromocresol green, Bromocresol purple, Phenol red,
Methyl orange, Phenolphthalein). Colorimetric determination of pH showed the varying color changes an
acid-base indicator undergoes when added to a solution of certain pH. The sample, iced tea and distilled
water were also subjected to Colorimetric determination. Acid - base indicators or simply indicators are
one of the easiest ways to determine the pH of a solution is by adding a substance whose color is
dependent on the pH.[1] Colorimetric determination of pH showed the varying color changes an acid-base
indicator undergoes when added to a solution of certain pH.
INTRODUCTION
According to Thomas Lowry and Johannes
Bronsted, acids release protons (hydrogen ions)
in their reactions whereas bases are substances
which accepts protons (hydrogen ions). [3]
Strong acids release protons readily and almost
completely in dilute aqueous solutions but weak
acids do not so that, at equilibrium, in most
cases, less than 1% of a weak acid is ionized to
yield protons. Strong bases have a great capacity
for accepting protons whereas weak bases are
poor acceptors of protons. [3]
Since weak acids dissociate only to a small
extent
in
dilute
aqueous
solution,
the
concentration of H+ in dilute solutions of these
acids is small. Frequently, the concentration of
hydrogen ions in solutions of weak acids is less
than 10-6 mol/L. It can be somewhat inconvenient
mathematically to work with values of this small
magnitude. To permit easier handling of such low
values of [H+], the Danish chemist S.P.L.
Sorensen proposed in 1909 that [H+] be
expressed logarithmically as follows: [3]
pH = -log10H+
The pH of solutions is important in the
biomedical sciences for two main reasons. First,
the proper functioning of biomolecules depends
to an important degree on the control of pH.
Second, changes as small as 0.1 or 0.2 pH unit
can cause significant metabolic disturbances in
certain cells, tissues, and organs. Because of the

pH sensitivity of many biomolecules, control of


pH also is important for the success of several
procedures used in the biomedical laboratory.

Experimental
A.) Compounds Tested
1. Preparation of Buffer Solution
Sodium Primary Phosphate Monohydrate
(NaH2PO4.H2O)
Sodium
Secondary
Phosphate
Heptahydrate (Na2HPO4.7H2O)
2. Acid-Base Indicators
Thymol blue
Bromophenol blue
Bromocresol green
Bromocresol purple
Phenol red
Methyl red
Methyl orange
Phenolphthalein
3. Samples Used
Distilled Water
Real Leaf Fruity Lemon Iced Tea

B.) Procedure
1. Preparation of Buffer Solution
A 250 mL 0.10 M Phosphate Buffer with a pH of
7.50 was assigned for our group to prepare.
Based on our computations for the buffer system,
a 1.32 grams of Sodium Primary Phosphate
Monohydrate (NaH2PO4.H2O) and 6.5 grams of

Sodium Secondary Phosphate Heptahydrate


(Na2HPO4.7H2O) were carefully weighed using an
analytical balance. The weighed reagents were
dissolved in a beaker using distilled water. After
completely dissolving, the solution was then
transferred to a volumetric flask together with
the rinsings and was filled with distilled water
until it reaches the mark or its desired volume.
2. Calorimetric Determination of ph
Six test tubes were prepared and labeled with
the pH of the buffer and acid-base indicator that
will be added. A 1 mL of a buffer of a certain pH
was pipetted into the test tube and was diluted
with 4 mL of distilled water. Two drops of an
acid-base indicator was added. The mixture was
shaken and the change in color was noted down.
The procedure was repeated on all the prepared
buffers using the acid-base indicators that were
specified above.
In determining the pH of the iced tea sample
and distilled water, 5 mL of the sample was used
and was added with two drops of an acid-base
indicator. The mixture was shaken and the
change in color was noted down. The procedure
was repeated using the acid-base indicators that
were specified above.

RESULTS AND DISCUSSIONS


Phosphate Buffer with a pH of 7.50 was
assigned for our group to prepare. Below was the
computed mass of Sodium Primary Phosphate
Monohydrate and Sodium Secondary Phosphate
Heptahydrate that was used for us to obtain the
desired pH of our buffer system.

[]
= 10
[]

[]
= 107.57.21
[]
[]
1.95
=
[]
1
Total mol=
+ 1.95 = 0.025
2.95 = 0.025
= 8.47103
1.95 = 0.0165

2 4 = 8.47103 156.01

2 4 = 1.32
2 4 = 0.0165 394 /
2 4 = 6.5
Table 1: Results of Colorimetric
Determination of PH
Acid Base
Indicator

pH
2.0

3.0

7.0

7.5

8.
0
Y

12
.0
B

Thymol
Y
Y
Y
Y
Bromophenol
Y
Y
P
P
P
P
Blue
Bromocresol
Y
Y
B
B
B
B
Green
Bromocresol
Y
Y
P
P
P
P
Purple
Phenol
Y
Y
Y
R
R
R
Red
Methyl
R
R
Y
Y
Y
Y
Red
Methyl
R
R
Y
Y
Y
Y
Orange
Phenolpht Color Color Color Color
P
P
less
halein
less
less
less
Colorimetric determination of pH showed the
different color changes acid-base indicator
undergoes when added to a solution of a certain
pH. Acid base indicators are either weak organic
acids, HA, or weak organic bases. The molecular
characteristics of a substance can be shown
through the use of acid-base indicator. The
changes of color in a molecule may be caused by
changes
in
electron
confinement.
Less
confinement makes the light absorb redder
(lighter), and more confinement makes it bluer
(darker). A property of the acid-base indicator
helps us to identify different substances by
narrowing their pH range. For example, using
Phenolphthalein as the acid-base indicator, the
solution turned to the color of pink. By such
observation we can say that the pH is 8.0. (Table
1)
Table 2: Results of colorimetric
Determination of distilled water and Sample
Acid Base
Indicator
Thymol

pH
Distilled
Water
Yellow

Sample
Peach

Bromophenol
Blue
Bromocresol
Green
Bromocresol
Purple
Phenol Red
Methyl Red

Blue-Violet

Yellow
Yellow
Green

Light Blue

Pale Yellow

Yellow

Yellow

Yellow
Light
Orange

Yellow

Methyl Orange

Orange

Phenolphthalein

Colorless

Pale Pink
Salmon
Pink
Flesh

REFERENCES
From Books:
[1]Bernas, G.C., Ysrael, M.C., & Bernaldez, A.T.
(1994). Basic Laboratory Studies in Biochemistry
(3rd ed.). Manila: UST Publishing House. Pages 59 and 17.
[2]Boyer, R.F. (2006). Biochemistry Laboratory:
Modern Theory and Techniques. (1st ed.). San
Francisco: Pearson Education Inc. Pages 55-69.
[3]Cecil,
J.R.
(1995).
Basic
Biochemical
Laboratory Procedures and Computing with
Principles, Review Questions, Worked Examples,
and Spreadsheet Solutions. (1st ed.). New York:
Oxford University Press. Pages 40-65.
From the internet(on-line)
[1] Estimation of pH - Acid-Base Indicators.
(n.d.). Retrieved February 15, 2015, from
http://www.harpercollege.edu/tmps/chm/100/dgodambe/thedisk/ph/abind.htm

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