BUFFER PREPARATION AND ANALYSIS WITH THE USE OF ELECTROMETRIC AND COLORIMETRIC

DETERMINATION
Jealeka Abesamis​, Vanessa Agraviador, Fitz Banez,
Natalie Regina Cu, and Coleen De Guia
Group 1 - 2E Medical Technology Biochemistry Laboratory

ABSTRACT
This experiment showed how to prepare different buffer solutions, determine the buffer’s pH value
through the use of electrometric and colorimetric determination, and adjust the pH value of a solution. First,
the solution was identified as a primary phosphate buffer solution. The solution was prepared using
phosphoric acid (H​3​PO4) as the weak acid and primary sodium phosphate (NaH​2​PO4) as the conjugate base.
Then, the pH of the buffer solution was measured using the pH meter and was calibrated to the desired pH of
2.0 using 6M NaOH. The electrometric determination method shows accurate readings of the prepared buffer
mixture by determining the hydrogen ion activity. After which the buffer solution pH was also measured
colorimetrically using multiple acid-base indicators to narrow down the pH margin of a the buffer. Acid-base
indicators used and their respective ranges are as follows: thymol blue (1.2-2.8 and 8-11), bromophenol blue
(3.0-4.6), bromocresol green (3.8-5.4), bromocresol purple (5.2-6.8), phenol red (6.4-8.4), methyl orange
(3.1-4.4), methyl red-methylene blue (5.2-5.6), and phenolphthalein (8.2-10).

INTRODUCTION
PH is expressed as the measure of the
concentration of hydrogen ions present in a
solution. A solution with higher concentration of
hydrogen ions would have a lower PH value. At the
same time, a solution with lower concentration of
hydrogen ions but has higher concentration of
hydroxide ions will have a higher pH value. [1]
Mathematically, pH can be calculated and
expressed as the negative log in base of 10 of the
hydrogen ion concentration as shown below:
pH =− log [H+]
Buffer solutions are defined as one that
resist a change in its pH when small amounts of
acids or base is being added to the solution. Buffer
solutions commonly contain a weak acid and its
conjugate base or a weak base and its conjugate
acid. [2]
The Henderson-Hasselbalch equation
associates the pH of a solution to the pKa of an
acid and the ratio of the concentration of the acid
and its conjugate base. This can be expressed in
the equation shown below where the amounts of
the acid and its conjugate base is computed based
on the desired PH [3] :
[Conjugate base]
pH = pKa + log [Acid]
Buffer capacity refers to the measurement
of the strength of a buffer against an acid or a
base. It is expressed as the amount of acid or base
added to a buffer in order to change the PH of a
liter of the buffer by 1 unit. [3]
In this experiment, electrometric
determination and colorimetric determination was
used as a method to determine the PH.
Electrometric determination detects the hydrogen
ion activity of a solution. Here, the pH value is
presented through measuring the difference in
potential between two special electrodes in contact
with the solution by means of a null potentiometer.
One electrode develops a potential according to
the hydrogen ion activity of the solution, whereas
the other electrode has a constant, definite
potential which is independent of the hydrogen ion
activity of the solution. [4] Contrary to this,
colorimetric determination is the measurement of
the wavelength and intensity of electromagnetic
radiation in the visible region of the spectrum, and
is based on hydrogen ion concentration. It is
commonly used for identification and
determination of concentrations of substances that
absorb light. [5]
PH and buffers play an important role in the
biological system because all biological processes
depend on pH to maintain their optimum state.
Functionally, buffers are important as chemical
substances which help maintain the consistency of
pH in a solution. In relation to the biological
system, buffers aid in maintaining the consistency
of the internal environment, also known as
homeostasis. In addition to this, it also gives
qualitative measure for many problems in cell H​2​PO​4​-
biology and related fields. [6]
0.7586 x
The experiment aims to (1) show the 1.7586 = 0.125
preparation process of buffer solutions; (2) x= 0.0539 mol
determine the pH of buffers and samples through
colorimetric and electrometric determination; and
(3) show how to adjust the pH of a buffer solution. H​3​PO​4

EXPERIMENTAL
1 x
A. Test Compound/s (or Sample/s) used 1.7586 = 0.125
For this experiment, the following x= 0.0711 mol
compounds used were:
1. Concentrated H​3​PO​4
2. NaH​2​PO​4​- d. H​3​PO​4​ ⇔ H​2​PO​4​-
3. 6M NaOH
4. Distilled water 0.125 mol - 0.0539 mol= ​0.0711 mol H​3​PO​4​-
H​2​PO​4​- ​= 0.0539 mol
B. Procedure
The experiment was divided into four parts
namely preparation of the buffer, adjustment of pH e. Reagent
of the buffer, electrometric determination, and
lastly, colorimetric determination. gNa​2​HPO​4​ x 2H​2​O​ = (0.0539) (156)
= 8.4084 ≈ 8.41 g
1. Preparation of Buffer
Concentration Volume Buffer pKa Desired 0.0711 mol
L​H​3​PO​4​ =
14.7 M
Solution pH
= 0.00484 L ≈ 4.84 mL
0.50 M 0.250 L Phosphate 2.12 2.00
Table 1. Assigned Values for Buffer In a beaker containing 5-10 mL distilled
Preparation water, an amount of 8.41 g NaH​2​PO​4 • H​2​O was
added and mixed. Then, it was placed in a
The following calculations were made to volumetric flask through the use of a funnel. The
start the experiment. Given the molarity and the beaker used earlier was rinsed with distilled water
volume of the assigned buffer, it was possible to thrice, added in the volumetric flask, and was set
solve for the amount of weak acid and conjugate aside.
base needed to produce the buffer.
An amount of 4.8 mL concentrated H​3​PO​4
a. Moles Buffer was pipetted and added to a beaker which
contained 5-10 mL distilled water, then it was
(0.50 M) (0.250 L) mixed and was placed inside a volumetric flask.
= 0.125 mol The beaker which contained the previous solution
was rinsed thrice and was added to the volumetric
b. WA: H​3​PO​4​ ; CB: H​2​PO​4​- flask until 250 mL mark was reached. Lastly, it was
sealed and was mixed well.
[H2P O4−]
pH=pKa​ + ​log [H3P O4]
2. Electrometric Determination of pH
2.12 + log [H2P O4−]
= 2.00 The buffer was transferred to a 500mL
[H3P O4]
beaker and was tested for its pH value. Using the
[H2P O4−] pH meter, the group was able to record the actual
= antilog (2-2.12)
[H3P O4]
pH of the buffer solution.

= 0.7586
c. 3. Adjustment of Buffer pH Value
 Since the buffer solution prepared did not
Blue
match the assigned pH value, 6M NaOH was added
to increase the pH value until it reached pH 2.00. Bromoph
Y Y V V V V V V Y
enol Blue
4. Colorimetric Determination of pH
With the knowledge of the pH values of Bromocre
Y Y G B B B B B Y
distilled water and the buffer solution from the sol Green
electrometric determination, it was then tested for
its pH value using the colorimetric method. Using a
chart on acid-base indicator ranges, the group Bromocre
Y Y Y V V V V V Y
determined the pH value of the distilled water and sol Purple
the buffer.

RESULTS AND DISCUSSION Phenol

Once the buffer was prepared and mixed Y Y Y O R R R R Y
Red
well, it was transferred to a beaker and was
measured for its pH value. Using the electrometric Methyl
determination method, the glass electrode tip of Red-Meth
R R V G G G G G R
ylene
the pH meter was dipped in the buffer solution.
green
From there, the pH of the buffer was recorded as
1.7 in 22.4°C. However, since the assigned buffer Methyl
was supposed to be 2.0, the group titrated the R R Y Y Y Y Y Y R
Orange
solution with sufficient amount of 6M NaOH. Once
the reading gave a value of 2.0 at 23.4°C, the Phenolpht
C C C C C C F C C
addition of NaOH was halted. halein

7.5-
Samples pH [H+] pH - 8.1 2.0
0
Distilled Water 7.9 (21.5°C) 1.19 x 10−8 M Legend:
R: red Y: yellow B: blue
Buffer Prepared
1.7 (22.4°C) 0.020M V: violet C: colorless O: orange
(actual)
G: green
Buffer Prepared Table 2. Colorimetric Determination of pH
2.0 (23.4°C) 0.01M
(post-adjustment)
Based on the two sets of pH values
Table 1. Electrometric Determination of pH
obtained, the electrometric determination gives a
more specific value compared to the colorimetric
Using the colorimetric determination
determination. Furthermore, the colorimetric
method, the buffer solution and distilled water
method requires multiple acid-base indicators to
were tested for the pH values given multiple
improve accuracy, and to be able to narrow down
acid-base indicators. The indicators used were
the pH value of the sample. However, as seen in
thymol blue, bromophenol blue, bromocresol
the results of the experiment, even with eight
green, bromocresol purple, phenol red, methyl red,
acid-base indicators, the specific pH of the distilled
methyl orange, and phenolphthalein. The following
water remains a range of values instead of a single
observations were made.
value only. This difference is due to the fact that
these two determination processes rely on
Colors with Standard Buffers different factors. Electrometric determination relies
on the activity of the hydrogen ions while
Acid-Base pH Sample
colorimetric determination relies on the
Indicator
D. concentration of hydrogen ions present in the
2 3 5 7 7.5 8 12 Buffer solution.
H​2​O

Thymol R Y Y Y Y Y B Y R
REFERENCES
From books
[1] Tan, Y., Chen, L., Sadler, J., & Sadler, E.
(2013). ​Discover Chemistry GCE’O’ level science
2nd edition​. Singapore: Marshall Cavendish
Education.
[2] Zumdahl, S.S., & Zumdahl, S.S. (2015).
Chemistry: An atoms first approach. B ​ oston,
Massachusetts, United States: Cengage learning.
[3] Crisostomo, Angelica C., et al. (2010).
Laboratory Manual in General Biochemistry.
Quezon City: C&E Publishing, Inc.

From the internet (on-line)

[4] Hydrogen Ion Determination. (1934). Retrieved
from
http://users.humboldt.edu/rpaselk/MuseumProject
/Instruments/Poten-Braun/pHPot-Braun.htm
[5] Colorimetry Chemistry. (2012). Retrieved from
https://www.britannica.com/science/colorimetry
[6] Biological Application of PH. (2014). Retrieved
from
https://groups.chem.ubc.ca/courseware/pH/sectio
n19/index.html