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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

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


I. Compounds Used 0.5L 0.5M Phosphate 2.0
1. For Buffer Preparation 0.5L 0.5M Phosphate 3.0
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
(H3PO4) In measuring pH via electrometry, the
2. Acid-Base Indicator apparatus used was a pH meter. The pH meter
• Thymol blue was calibrated was then calibrated at pH 4, 7
• Bromphenol blue and 10. After calibrated, the pH of the six
• Bromcresol green buffer solutions were measured and adjusted to
• Bromcresol purple their desired pH using the prepared standards,
• Phenol red Hydrochloric Acid (HCl) and Sodium Hydroxide
• 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 A triprotic weak acid such as phosphate has
reaction. three plateau regions in its titration curve. Every
half-neutralization of each plateau region
indicates a pka value. The pka values of Primary
RESULTS AND DISCUSSION Phosphate buffer is 2.12, Secondary phosphate is
1. Preparation of Reagents 7.21 and Tertiary Phosphate Buffer is 12.32. The
A. Prepare 6.0 M Hydrochloric Acid Henderson- Hasselbalch Equation will give the
Using the dilution formula, the experimental amount of primary phosphate and
hydrochloric acid standard was prepared by secondary phosphate buffer needed. The
adding 0.25L of distilled water to 0.25 L of phosphate buffer of interest in the preparation is
concentrated (12 M) Hydrochloric Acid. a secondary phosphate buffer with a pka value of
7.21 and a desired pH of 7.5.
Concentrated HCl = 12M
pH = pka + log [HPO42-]
Dilution: C1V1 = C2V2 [H2PO4-]
(12M)(x) = (6.0M)(0.5L)
x = (6.0M)(0.5L) 7.5 = 7.21 + log [HPO42-]
(12M) [H2PO4-]
7.5 -7.21 = log [HPO4 ]

V1 = 0.25L of conc. HCl [H2PO4-]

B. Preparation of 6.0 M Sodium Hydroxide 0.29 = log [HPO42-]

For the Sodium hydroxide standard, it was [HPO42-]
prepared by dissolving 120g of Sodium
Hydroxide pellets to 0.5L of distilled water. 100.29= [HPO42-]
6.0M = (x)mol 3.0 mol = 40g
0.5L 1 mol 1.9498446 = [HPO42-]
1 [HPO42-]
X = 3.0 mol m=120g NaOH pellets
The theoretical mole of the buffer is
2. Buffers 2.9498446 moles. Actual Moles of the buffer was
Each of the buffers was assigned to two calculated from 0.05 M and 0.5 L in volume to be
groups. Secondary Phosphate buffer with a 0.0025 moles. By ratio and proportion, 0.228 g
desired pH of 7.5 and 0.05M concentration was of Primary Phosphate and 0.2271g of secondary
assigned to the group. Buffers are aqueous phosphate must be used to from the buffer.
systems that tend to resist changes in their pH
3. Electrometric Determination of pH
when small amounts of acid (H+) or base (OH-)
The buffer was prepared by manipulating the
are added [3]. A buffer consists of a weak acid
amount of weak acid and its conjugate base in
(proton donor) and its corresponding conjugate
the solution. In electrometric Determination of
base (proton acceptor). For the experiment, the
pH, the pH of the buffer was read by means of a
weak acid is the Primary Phosphate ion in Sodium
pH meter. A pH meter is a potentiometer. It
Primary Phosphate Monohydrate (NaH2PO4.H2O)
consist of a Glass electrode which acts as the
and its conjugate base is the Secondary
cathode and a Reference electrode in this case
Phosphate in Sodium Secondary Phosphate
Standard Calomel Electrode which acts as the
Heptaphydrate (Na2HPO4.7H2O).
anode. Calomel electrode is a platinum electrode
in contact with a paste of mercury, mercurous
H2PO4- HPO 4
+ H+
(1° Phosphate) (2° Phosphate) (Hydrogen Ion) chloride and potassium chloride [4]. The pH of
each buffer prepared in the laboratory was
measured and adjusted to their desired pH using
Phosphate Buffers have three pka values
the pH meter.
(acid dissociation constant) making it a triprotic
acid [4]. pka is needed in the Henderson
If the actual pH was less than the desired pH,
Hasselbalch equation which is.
the buffer would be treated with a small amount
of the Standard Sodium Hydroxide (NaOH). And
pH = pka + log [HPO42-]
if the actual pH was greater than the desired pH,
the buffer would be treated the same but with a
small amount of the Standard Hydrochloric Acid.

The group adjusted the pH of the buffer to 7.51 At Bromcresol green, the buffer also indicates
which is approximately 7.5 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.
4. Colorimetric Determination of
pH For bromcresol purple, the buffer gave a
Colorimetric determination is done by using a violet coloration. The pH range of bromcresol
spot plate and an acid base indicator. The results purple ranges from 5.2-6.8. Yellow color indicates
for the colorimetric determination of pH are that it is above the pH range and pink indicates a
shown on Table 2. high pH value. The color of the buffer might have
been affected by other environmental factors
Table 2. Colorimetric Test of the Buffers hence turning pink to violet. Nevertheless, it
Acid-base pH pH pH pH pH pH Dis narrows the pH range of the buffer to 6.8-8.0.
indicator 2 3 7 7.5 8 12 H2O For Phenol Red, the pH range is 6.8-8.2. The
buffer showed a red coloration. Red for a lower
Thymol blue R YO Y Y B B YO
pH than the pH range and yellow for a higher pH
Bromphenol than the pH range.
blue For Methyl red which has a pH range 4.4-6.2,
Bromcresol the buffer indicated a yellow coloration.
green Methyl orange with a pH range 3.1-4.4,
Bromcresol showed orange red colorization.
As so as phenolphthalein which gave a
Phenol red Y Y R R RV RV Y colorless solution that indicates lower the pH
range of 8.2.
Methyl red P P Y Y Y Y P This further concludes that the pH of the
group’s secondary buffer solution with a desired
Methyl orange R RO YO O O O O
pH of 7.5 is approximately within the pH range
Phenolphthalein X X X X P RV X generalized by those Acid-Base Indicators which
is within 6.8-8.0.
Y=yellow, R=red,V=violet, O=orange, P=pink, B=blue, G=green,
X=colorless; L=light, D=dark
ACID-BASE INDICATORS [1] Murray, R.K., Granner D.K., and Rodwell,
V.W. (2006). Harper’s Illustrated
The group experiment was focused on the Biochemistry. 27th ed. Singapore:
Secondary Phosphate buffer with a pH of 7.5. McGraw-Hill Companies Inc. (Asia)
[2] Ninfa A.J., Ballou, D.P., and Benore, M.
At Thymol blue, the buffer indicated a yellow (2010). Fundamental Laboratory
coloration. Thymol blue has a pH range of 1.2- Approaches for Biochemistry and
2.8 at acid range. In this range red is the color Biotechnology 2nd ed. United States of
below pH-range and yellow is color above. America: John Wiley & Sons Inc.
Thymol blue has a second pH range wherein at [3] Lehninger A. L., Nelson D.L., and Cox
pH range 8.0-9.6 it is at basic range and yellow M.M. (1993). Principles of Biochemistry 2nd
indicated below the pH range and blue is above ed. New York: Worth Publishers.
the pH range. This means the pH of the buffer is [4] Wilson K., and Walker J. (2005).
below the pH 8.0 because exceeding 8.0 would Principles and Techniques of Biochemistry
indicate a blue color. and Molecular Biology 6th ed. New York:
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.