Experiment 1: pH Measurement and Buffer Preparation

Boligor, Lea Marie L., Bonguit, Sharah Kristine G., Boongaling Maryrose O., Capistrano, Nikki Cyril C.,
Cruz, Dana Jean B.

Department of Biochemistry
Faculty of Pharmacy, University of Santo Tomas
Espaa, Manila

Abstract
A buffer solution is one which resists changes in pH when small quantities of an acid or an alkali
are added to it. The objectives of this experiment are to prepare different buffer solutions as assigned to
the group by the instructor and to determine the pH of assigned buffers and samples colorimetrically using
liquid and electrometrically using the pH meter. The buffer solution assigned to the group was 250 mL of
0.50 M phosphate at pH 3 and it was prepared by mixing 1 mL concentrated H3PO4 and 15 g of NaH2PO4
H2O. A calibrated pH meter was used in order to electrometrically determine the pH of the buffer solution
made and the read-out was 2.9. For the colorimetric determination of pH, several indicators were used
namely: thymol blue, bromophenol blue, bromocresol green, bromocresol purple, methyl red, methyl
orange, phenolphthalein, and phenol red. The assigned sample of the group, water, was subjected to
electrometric determination and its pH was determined to be at 7.7. Through this experiment, it can be
inferred that the electrometric determination of pH is more accurate than the colorimetric determination.

Introduction The readout from the pH meter will give the pH

value of the assigned solution. In addition, the
In the field of Chemistry, the potential of acid-base indicators namely: thymol blue,
hydrogen or pH value determines the acidity or bromophenol blue, bromocresol green,
alkalinity of a specific solution. It denotes that bromocresol purple, phenol red, methyl red,
when the pH is 7, the solution is considered methyl orange, and phenolphthalein, were
neutral. On the other hand, pH values which are utilized to identify the acidity or basicity of the
lower or higher than 7 are considered more acidic solution through observing the occurrence of a
or more basic, respectively. The pH value could color change in the sample upon the addition of
be obtained through the use of a pH meter or the said indicators.
computed by getting the log[H+] or utilizing the
Henderson-Hasselbalch equation expressed as: Methodology
[]
pH = pKa + log
[]
A. Compounds Tested
Some experiments require aqueous
1. Preparation of buffers
solutions which are not greatly affected by the
Conc. H3PO4
addition of a strong acid or base for the said
NaH2PO4
experiment to run smoothly. This objective is
2. Electrometric Determination
fulfilled with the aid of buffer solutions. A buffer
0.50 M Phosphate buffer, pH 3
solution is an aqueous solution which exhibits
3. Acid-base indicators
minimal changes in pH even upon the addition of
Thymol blue
a strong acid or base.
In this experiment, the pH value is Bromophenol blue
determined by electrometric determination with Bromocresol green
the aid of a digital pH meter and colorimetric Bromocresol purple
determination with the aid of acid-base indicators. Methyl red
 Methyl orange Results and Discussion
Phenolphthalein
Phenol red A. Preparation of Buffer

4. Electrometric Determination A buffer solution is composed of a weak

Distilled water acid and its conjugate base or a strong base
its conjugate acid. This mixture would
B. Procedure maintain its acidic and basic components
since they will not undergo any reactions that
1. Preparation of buffers would alter their concentrations.
The Henderson-Hasselbalch equation
The buffer components were was used to know the components for the
computed using the Henderson- preparation of a 250 mL, 0.50 M phosphate
Hasselbalch equation. It was known that buffer solution with pH 3.00. The reagents
to prepare 0.50 M phosphate of ph 3.00, used were concentrated H3PO4 solution with
15.0 g of NaH2PO4 and 1.00 mL of Conc. a calculated 14.74 M and NaH2PO4 H2O.
H3PO4 will be needed. These
components were then mixed in a 250-
mL volumetric flask. Given the equation:
[]
pH = pKa + log
[]
2. Electrometric Determination
[]
3 = 2.12 + log
The laboratory apparatus used in []
[]
electrometric determination was pH 3 2.12 = log
[]
meter. The pH meter was calibrated at 4, []
7, and 10. After the calibration of the pH 0.88 = log[]
meter, a small amount of the assigned []
100.88 = []
buffer solution was diluted with 20-mL []
distilled water and pH was measured by 7.59 = []
dipping the pH meter in the solution.
The ratio is determined to be 7.59.
3. Acid-base indicators Since,

0.50 x 0.250L = 0.125 mol buffer

0.50 M phosphate buffer with pH
3.00 was assigned to the researchers to
0.125 = [HA] + [A]
be used with the different indicators. The
0.125 = 1[HA] + 7.59[HA]
8 test tubes were labeled with thymol
0.125 = 8.59 [HA]
blue, bromophenol blue, bromocresol
[HA] = 0.01455
green, bromocresol purple, methyl red,
[A] = 0.125 0.01455
methyl orange, phenolphthalein and
[A] = 0.1104
phenol red. Each test tubes were added
with 5.0 mL of buffer solution and 2-3
Therefore,
drops of the acid-base indicators. Each 1000
test tubes were mixed and results were 0.01455 HA x x
14.74
observed. = 0.987 mL 1mL H3PO4

138
0.1104 A x
1
= 15.24 g 15 g NaH2PO4 H2O
 Phenolphthalein X X X X X Pink Pink
An amount of 1 mL concentrated H3PO4
and 15 g of NaH2PO4 H2O are needed for R= red, O= orange, Y= yellow, B=blue, P=purple,
X=colorless
the preparation of the buffer solution.

B. Electrometric and Colorimetric

C. Electrometric Determination of Samples
Determination of Buffer
The pH of the assigned sample, distilled
In the electrometric determination of
water, was determined through
buffer, a pH meter was simply immersed in
electrometric determination. The calibrated
the buffer solution. Since the desired pH of
pH meter was immersed in the sample and
the buffer was 3.00, the read-out of the pH
the read-out was 7.7.
meter should be 3.00 or near 3.00. The pH of
The pH of all the assigned samples are
the buffer prepared by the group was 2.59
summarized on Table 2.
which is near the desired buffer. The
concentration of the buffer was also
Table 2. pH of Assigned Samples
determined to be 1.26 x 10-3 M by using the
Sample pH
equation:
Tap Water 7.6
[H] = antilog(-pH)
Distilled Water 7.7
Colorimetric determination of pH showed Coconut Juice 5.2
the change in color when exposed to a Soda (Sprite) 3.3
certain pH. These changes occur in narrow Shampoo (Pantene) 7.0
ranges of pH due to the equilibrium shift that Dishwashing Soap 9.2
took place in the reaction. The color may be Facial Wash (Nivea) 7.5
due to the inherent property of a substance in
the solution or the formation of a product as a
result of the addition of a suitable reagent or Conclusion
acid-base indicator.
The results of the colorimetric The objectives of the experiment were
determination of the buffer are shown on met. Through the use of a calibrated pH meter,
Table 1. electrometric determination of the buffer solution
and sample was accomplished. Colorimetric
Table 1. Colorimetric determination of buffer determination was attained by using different
liquid indicators. Based on the data obtained, it
Acid-base
2.0 3.0 5.0 7.0 7.5 8.0 12.0 was inferred that the electrometric means of
indicator
determining the pH is more accurated compared
Light Light Light
Thymol blue Y Y Y B to the colorimetric method.
O Y Y
Bromophenol Light Clear
P P P P P References:
blue Y Y
Bromocresol Light
Y B B B B B
green Y [1]
Bromocresol Light
Y P P P P P Crisostomo, A. C., et al. (2010). Laboratory Manual
purple Y
in General Biochemistry. Quezon City: Philippines.
Phenol red Y Y RO RO RO Pink R
Pale Pale Pale [2]
Methyl red Pink Pink Y Y
Y Y Y Clark, J. (2002). Buffer Solutions. Retrieved
Methyl orange
Pink
O O O O O O February 27, 2017 from:
O
http://www.chemguide.co.uk/physical/acidbasee
qia/buffers.html
[3]
Lancashire, R.J. (2002). EXPERIMENT 36-
COULOMRIMETRIC DETERMINATION OF
PHOSPHATE. Retrieved February 27, 2017
from:
http://wwwchem.uwimona.edu.jm/lab_manuals/c
10expt36.html
[4]
Chemteam. Introduction to the Henderson-
Hasselbalch Equation. Retrieved February 27,
2017 from:
http://www.chemteam.info/AcidBase/HH-
Equation.html