PH MEASUREMENT AND BUFFER PREPARATION

Kriziaoumo P. Orpia, Michael Gavin G. Quinto, Nico Joy C. Ridulme

Kathryn Chemaine L. Samorano and Ryza Ruba
Group 8 2E Medical Technology Biochemistry Laboratory

ABSTRACT
Phosphate buffer solutions with desired pH and concentrations were prepared by mixing a weak acid phosphate to its
conjugate phosphate base. The desired pH of the buffer was adjusted and determined by means of Electrometric and
Colorimetric methods.
INTRODUCTION
Phosphate buffer solutions with different
pH and concentration were prepared for the
experiment. pH was introduced in 1909 by
Sörensen who defined it as the negative
logarithm of the hydrogen ion concentration [1].
pH in the experiment is measured and
determined using different kinds of methods
available in the laboratory. These methods are
colorimetry which uses color standards to
determine the pH of the Buffer Solution, and
electrometry via pH potentiometer which uses a
glass electrode to sense Hydrogen potential of
the solution.
concentrated Hydrochloric Acid (12.0M) was
diluted first. For 6.0 M Sodium Hydroxide,
certain amount of NaOH pellets were dissolved
together with distilled water in a 500 mL
volumetric flask. Each standard was placed in
an amber bottle and was labeled properly.
2. Buffers
For the buffer solutions, six phosphate
buffers were prepared instead of the original
procedure where only five phosphate buffers
and one TRIS –HCl buffer were produced. The
buffers were assigned with desired pH and
concentration and are stated as followed.
Table 1. List of buffer solution

VOLUME CONCENTRATION BUFFER pH

EXPERIMENTAL 0.5L 0.5M Phosphate 2.0
I. Compounds Used 0.5L 0.5M Phosphate 3.0
1. For Buffer Preparation 0.5L 0.5M Phosphate 7.0
 Sodium Primary Phosphate 0.5L 0.05M Phosphate 7.5
Monohydrate (NaH2PO4.H2O) 0.5L 0.5M Phosphate 8.0
 Sodium Secondary Phosphate 0.5L 0.5M Phosphate 12.0
Heptaphydrate (Na2HPO4.7H2O)
 Concentrated Phosphoric Acid 3. Electrometric Determination of pH
In measuring pH via electrometry, the
(H3PO4)
apparatus used was a pH meter. The pH meter
2. Acid-Base Indicator
was calibrated was then calibrated at pH 4, 7
 Thymol blue
and 10. After calibrated, the pH of the six
 Bromphenol blue
buffer solutions were measured and adjusted to
 Bromcresol green
their desired pH using the prepared standards,
 Bromcresol purple
Hydrochloric Acid (HCl) and Sodium Hydroxide
 Phenol red
(NaOH).
 Methyl red
 Methyl orange
4. Colorimetric Determination of pH
 Phenolphthalein
Using a spot plate, small amounts of the
3. Standard Reagent Preparation
primary phosphate buffer where placed on
 Conc. HCl
eight spots of the spot plate. The first spot was
 Conc. NaOH
treated with the first acid-base indicator,
Thymol blue, the second spot was treated with
II. Procedure bromphenol blue. Each spot was treated with
1. Preparation of different acid-base indicator, one different from
Reagents the other. Each spot was noted for the color it
The reagents to be prepared were 6.0M produced after the acid-base indicator
Hydrochloric Acid (HCl) and 6.0M Sodium treatment. It was also done to the other five
Hydroxide (NaOH). In preparing 500mL 6.0M buffers left and was noted for their color
Hydrochloric Acid, certain amounts of reaction.
1
 Phosphate buffer is 2.12, Secondary phosphate is
7.21 and Tertiary Phosphate Buffer is 12.32. The
RESULTS AND DISCUSSION Henderson- Hasselbalch Equation will give the
1. Preparation of Reagents experimental amount of primary phosphate and
A. Prepare 6.0 M Hydrochloric Acid secondary phosphate buffer needed. The
Using the dilution formula, the phosphate buffer of interest in the preparation is
hydrochloric acid standard was prepared by a secondary phosphate buffer with a pka value of
adding 0.25L of distilled water to 0.25 L of 7.21 and a desired pH of 7.5.
concentrated (12 M) Hydrochloric Acid.
pH = pka + log [HPO42-]
Concentrated HCl = 12M [H2PO4-]

7.5 = 7.21 + log [HPO42-]

Dilution: C1V1 = C2V2
[H2PO4-]
(12M)(x) = (6.0M)(0.5L)
7.5 -7.21 = log [HPO4 ]
2-
x = (6.0M)(0.5L)
[H2PO4-]
(12M)

V1 = 0.25L of conc. HCl 0.29 = log [HPO42-]

[HPO42-]
B. Preparation of 6.0 M Sodium Hydroxide
For the Sodium hydroxide standard, it was 100.29= [HPO42-]
[HPO42-]
prepared by dissolving 120g of Sodium
Hydroxide pellets to 0.5L of distilled water. 1.9498446 = [HPO42-]
1 [HPO42-]
6.0M = (x)mol 3.0 mol = 40g
0.5L 1 mol
The theoretical mole of the buffer is
X = 3.0 mol m=120g NaOH pellets 2.9498446 moles. Actual Moles of the buffer was
calculated from 0.05 M and 0.5 L in volume to be
2. Buffers 0.0025 moles. By ratio and proportion, 0.228 g
Each of the buffers was assigned to two of Primary Phosphate and 0.2271g of secondary
groups. Secondary Phosphate buffer with a phosphate must be used to from the buffer.
desired pH of 7.5 and 0.05M concentration was
3. Electrometric Determination of pH
assigned to the group. Buffers are aqueous
The buffer was prepared by manipulating the
systems that tend to resist changes in their pH
amount of weak acid and its conjugate base in
when small amounts of acid (H +) or base (OH-)
the solution. In electrometric Determination of
are added [3]. A buffer consists of a weak acid
pH, the pH of the buffer was read by means of a
(proton donor) and its corresponding conjugate
pH meter. A pH meter is a potentiometer. It
base (proton acceptor). For the experiment, the
consist of a Glass electrode which acts as the
weak acid is the Primary Phosphate ion in Sodium
cathode and a Reference electrode in this case
Primary Phosphate Monohydrate (NaH2PO4.H2O)
Standard Calomel Electrode which acts as the
and its conjugate base is the Secondary
anode. Calomel electrode is a platinum electrode
Phosphate in Sodium Secondary Phosphate
in contact with a paste of mercury, mercurous
Heptaphydrate (Na2HPO4.7H2O).
chloride and potassium chloride [4]. The pH of
each buffer prepared in the laboratory was
H2PO4- HPO 4
2-
+ H+
(1° Phosphate) (2° Phosphate) (Hydrogen Ion) measured and adjusted to their desired pH using
the pH meter.
Phosphate Buffers have three pka values
(acid dissociation constant) making it a triprotic
If the actual pH was less than the desired pH,
acid [4]. pka is needed in the Henderson
the buffer would be treated with a small amount
Hasselbalch equation which is.
of the Standard Sodium Hydroxide (NaOH). And
if the actual pH was greater than the desired pH,
pH = pka + log [HPO42-]
the buffer would be treated the same but with a
[H2PO4-]
small amount of the Standard Hydrochloric Acid.
The group adjusted the pH of the buffer to 7.51
A triprotic weak acid such as phosphate has
which is approximately 7.5
three plateau regions in its titration curve. Every
half-neutralization of each plateau region
indicates a pka value. The pka values of Primary
2
 For bromcresol purple, the buffer gave a
4. Colorimetric Determination of pH violet coloration. The pH range of bromcresol
Colorimetric determination is done by using a purple ranges from 5.2-6.8. Yellow color indicates
spot plate and an acid base indicator. The results that it is above the pH range and pink indicates a
for the colorimetric determination of pH are high pH value. The color of the buffer might have
shown on Table 2. been affected by other environmental factors
hence turning pink to violet. Nevertheless, it
Table 2. Colorimetric Test of the Buffers narrows the pH range of the buffer to 6.8-8.0.
Acid-base pH pH pH pH pH pH For Phenol Red, the pH range is 6.8-8.2. The
Dis
indicator 2 3 7 7.5 8 12 H2O buffer showed a red coloration. Red for a lower
pH than the pH range and yellow for a higher pH
Thymol blue R YO Y Y B B YO than the pH range.
Bromphenol For Methyl red which has a pH range 4.4-6.2,
Y YG B B BG BV GL
blue the buffer indicated a yellow coloration.
Bromcresol Methyl orange with a pH range 3.1-4.4,
Y Y BL B BV BD B
green showed orange red colorization.
Bromcresol As so as phenolphthalein which gave a
Y Y V V V V Y
purple colorless solution that indicates lower the pH
range of 8.2.
Phenol red Y Y R R RV RV Y
This further concludes that the pH of the
Methyl red P P Y Y Y Y P group’s secondary buffer solution with a desired
pH of 7.5 is approximately within the pH range
Methyl orange R RO YO O O O O generalized by those Acid-Base Indicators which
is within 6.8-8.0.
Phenolphthalein X X X X P RV X
Y=yellow, R=red,V=violet, O=orange, P=pink, B=blue, G=green, REFERENCES
X=colorless; L=light, D=dark [1] Murray, R.K., Granner D.K., and Rodwell,
ACID-BASE INDICATORS V.W. (2006). Harper’s Illustrated
Biochemistry. 27th ed. Singapore:
The group experiment was focused on the McGraw-Hill Companies Inc. (Asia)
Secondary Phosphate buffer with a pH of 7.5. [2] Ninfa A.J., Ballou, D.P., and Benore, M.
(2010). Fundamental Laboratory
At Thymol blue, the buffer indicated a yellow Approaches for Biochemistry and
coloration. Thymol blue has a pH range of 1.2- Biotechnology 2nd ed. United States of
2.8 at acid range. In this range red is the color America: John Wiley & Sons Inc.
below pH-range and yellow is color above. [3] Lehninger A. L., Nelson D.L., and Cox
Thymol blue has a second pH range wherein at M.M. (1993). Principles of Biochemistry 2nd
pH range 8.0-9.6 it is at basic range and yellow ed. New York: Worth Publishers.
indicated below the pH range and blue is above [4] Wilson K., and Walker J. (2005).
the pH range. This means the pH of the buffer is Principles and Techniques of Biochemistry
below the pH 8.0 because exceeding 8.0 would and Molecular Biology 6th ed. New York:
indicate a blue color. Cambridge University Press.

At Bromphenol blue, the buffer indicates a

blue coloration. Bromphenol blue has pH range of
3.0-4.6 and yellow indicates it is below the pH
range and blue violet indicates it is above the
range. This further narrows the pH range of the
buffer from 4.6- 8.0 as the pH range of the
Phosphate buffer.

At Bromcresol green, the buffer also indicates

a blue coloration. Bromcresol green has a pH
range of 3.8-5.4 wherein the yellow is also the
below pH range and blue as above the pH range.
The pH range is now narrowed down to 5.4-8.0.