PH MEASUREMENT AND BUFFER PREPARATION

Paul Benjomin G. Agregado, Maria Kristene D. Alba, Ana Kristiana Louise A. Banzon, Rovileen M.
Barroquina, Margaret Eve L. Bartolome

Group 1 2F Pharmacy General Biochemistry Laboratory

ABSTRACT
The experiment was done to achieve the following: prepare different buffer solutions, determine the pH of the buffers
and samples colorimetrically using different liquid indicators and electrometrically using the pH meter. Primary
phosphate buffer solution was used in the experiment. It was prepared using Phosphoric acid (H 3PO4) as weak acid
and Primary sodium phosphate dihydrate (NaH 2PO4.2H2O) as conjugate base. The pH of the buffer solution was
measured electrometrically using the pH meter and manipulated using 6M HCl (to make the buffer more acidic) and
6M NaOH (to make the buffer more basic). This instrument indicates the hydrogen ion concentration in a test solution
by responding to the potential developed by an electrical cell. [2] The pH meter showed accurate pH readings of the
prepared buffer solution. It showed fluctuations in readings with the slightest addition of HCl and NaOH. Thus, the pH
meter is more accurate in reading pH levels compared to a pH paper. The pH of the prepared buffer solution was
measured colorimetrically using acid-base 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. This property of an
acid-base indicator can therefore be used to identify different substances by narrowing their pH range. This can help
in the identification of a substance since different substances exhibit different pH levels. Acid-base indicators can also
be used to narrow down the pH range of a substance. The colour-change interval of an indicator is the pH range,
where pronounced colour change takes place and it was determined in the experiment: Thymol blue (2-3/8-11),
Bromophenol blue (3-5), Bromocresol green (3-5), Bromocresol purple (5-7), Phenol red (5-8), Methyl orange (5-7),
Phenolphthalein (8-11).

INTRODUCTION Sorensen proposed in 1909 that [H+] be

We frequently encounter acids and bases in our
daily life. Fruits, such as oranges and apples,
contain acids. Household ammonia, a cleaning
agent, and Liquid Plumber are bases. [4]
According to the 1923 definition of Thomas M.
Lowry of England and Johannes N. Bronsted of
Denmark, acids release protons (hydrogen ions)
in their reactions whereas bases are substances
wich accepts protons (hydrogen ions). [1] 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. [1]
expressed logarithmically as follows: [1]
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.
These include the separation, purification, and
assay for biological activity of several
biomolecules. [1]

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.
There are certain solutions that resist change in
pH even when we add to them acids or bases.
Such systems are called buffers. [4] Buffers
resist changes in pH because of the Le Chatelier
Principle governing equilibrium conditions. [4]
Buffers contain relatively high concentrations of
weak acids or bases and their conjugate partners
which are generally present as salts of the weak
acid or base. [1]
 Because pH is dependent on ionic activity, a
property which cannot be measured easily or
fully predicted theoretically, it is difficult to
determine an accurate value for the pH of the
solution. [6] The pH reading of a solution is
usually obtained using a pH meter or pH indicator
paper/liquid.
The experiment was done due to the following
objectives: (1) to prepare different buffer
solutions (2) to determine the pH of the buffers
and samples colorimetrically using different liquid
indicators and electrometrically using the pH
meter and (3) to calculate the buffer capacity of
ther prepared buffer solution.
500mL beaker. A paperbox was made and
weighed to contain the Primary sodium
phosphate dihydrate (NaH2PO4.2H2O) powder.
reagent. A mass of 1.716g Primary sodium
phosphate dihydrate (NaH2PO4.2H2O) was
weighed using an analytical weighing scale then
was transferred to the 500mL beaker. A certain
volume of distilled water was added to the beaker
to make 250mL. The prepared solution was
transferred to a 500mL amber bottle that was
labeled properly.
Computations:
Molesbuffer = (0.250L)(0.10M) = 0.025 moles

Henderson Hasselbach equation:

EXPERIMENTAL pH = pKa + log¿ ¿

A. Compounds tested (or Samples used) H 2 PO 4−1

2.00 = 2.12 +log
H 3 PO 4
Distilled water, Phosphoric acid (H 3PO4),
Primary sodium phosphate dihydrate
H 2 PO 4−1
(NaH2PO4.2H2O), 6M HCl, 6M NaOH, Acid-base = antilog [2.00 – 2.12]
indicators (Thymol blue, Bromophenol blue, H 3 PO 4
Bromocresol green, Bromocresol purple, Phenol
red, Methyl orange, Phenolphthalein)
0.76 M [ H 2 PO 4¿¿−1]
= ¿
1M [ H 3 PO 4]
B. Procedure
Mbuffer = 0.76MH2PO4-1 + 1MH3PO4 = 1.76M
1. Preparation of reagents
0.76 M x
A volume of 120mL conc. HCl (12.2M) was MolesH2PO4-1 = =
1.76 M 0.025 M
measured using a graduated cylinder to prepare
a volume of 250mL of 6M HCl. The reagent was = 0.011 moles
transferred to a suitable container that was
labeled properly. 1M x
Moles H3PO4= =
1.76 M 0.025 M
Computation:
= 0.014 moles
(12.2M)(xL) = (6.0M)(0.250L)

x = 0.12L or 120mL conc. HCl

0.014 moles
mL H3PO4 = 14.7M H3PO4 =
L
2. Preparation of Buffer solution
= 0.000952L
The buffer solution was prepared using the
following guidelines: = 0.95mL

Table 1. Guidelines for buffer solution gNaH2PO4.2H2O = (0.011M)(156g/mol)

preparation
= 1.716g
Weak Conjugate Volum Conc. pH pKa
acid base e H3PO4
H3PO4 H2PO4-1 250mL 0.10M 2.0 2.12
0 3. Electrometric Determination of pH

The pH meter was calibrated at pH 7 by

A volume of 0.95mL (19 drops) Phosphoric acid soaking the electrode in neutral reagents like
(H3PO4) was measured using a serological pipette distilled water. The electrode was lifted out of the
and an aspirator. It was then dropped into a neutral solution and dried using a tissue paper.
The electrode was then immersed in the prepared
buffer solution (Primary phosphate buffer). The
standard buffer should have a pH within two (2)
pH units of the expected pH of the test solution.
[3] The bulb of the electrode must be completely
covered with solution. [3] The pH reading was
read. If the reading was within the proper pH
range of the desired, remove the electrode from
the buffer solution, rinse it with distilled water
using a water bottle and then dried using a tissue
paper. And if the pH reading is not within the
desired pH range, adjust the pH by removing the
electrode from the buffer solution, cleanse and
dry it then add in portions of either 6.0M HCl or
6.0M NaOH depending if you want to make the
buffer solution more acidic (use former) or more
basic (use latter). The process was continued
until the pH of the buffer solution is within the
proper range.
Single probe
combination
electrode
Electrode
Handle
Buffer
Electronic
meter
Figure 1. pH meter
4. Colorimetric Determination of pH
A certain number of test tubes (7) was
prepared and properly labeled with the acid-base
indicators. Each test tube was filled with 5mL of
the prepared buffer solution using a serological
pipette and an aspirator. A certain amount (2
drops) of acid-base indicator was dropped in the
corresponding labeled test tubes. The test tubes
were shaken and the color was taken down.
Table 2. Acid-base indicators used
Thymol blue (A)
Bromophenol blue (B)
Bromocresol green (C)
Bromocresol purple (D)
Phenol red (E)
Methyl orange (F)
Phenolphthalein (G)
A B C D E F G EAC
H
+
5mL of prepared buffer
Solution
+
2 drops of acid-base indicator corresponding to the
labelled test tubes
Shake
Note color
Figure 2. Colorimetric determination of pH
procedure
RESULTS AND DISCUSSION
1. Electrometric Determination of pH
The pH meter is used to measure the
electrochemical properties of liquids, pastes and
semi-solids. [2] This instrument indicates the
hydrogen ion concentration in a test solution by
responding to the potential developed by an
electrical cell. [2] The pH meter showed accurate
pH readings of the prepared buffer solution. It
showed fluctuations in readings with the slightest
addition of HCl and NaOH. Adding the former will
decrease the pH and adding the latter will
increase the pH of the buffer, this is because the
electrode is sensitive to change in the
concentrations of [H+] and [OH-] ions. Thus, the
pH meter is more accurate in reading pH levels
compared to a pH paper.
2. Colorimetric Determination of pH
Certain organic substances change color in
dilute solution when the hydrogen ion
concentration reaches a particular value. For
example, phenolphthalein is a colourless
substance in any aqueous solution with a
hydrogen ion concentration greater than
1.0x10-8 M (pH less than 8.0). In solutions with
a hydrogen ion concentration less than 1.0x10 -8
M (pH greater than 8.0), phenolphthalein is red
or pink. Substances like phenolphthalein, which
can be used to determine the pH of a solution,
are called acid-base indicators. Acid-base
indicators are either weak organic acids, HA, or
weak organic bases, BOH, where the letters A
or B stand for complex organic group. [6]
The equilibrium in a solution of the acid-
base indicator methyl orange, a weak acid, can
be represented by the equation
HA ↔ H+ + A -
red yellow
The anion of methyl orange is yellow, and
the non-ionized form is red. If acid is added to
the solution, the increase in the hydrogen ion
concentration shifts the equilibrium toward the
red form in accordance with the law of mass
action.
values. In general, the colour-change interval
of an indicator is the pH range, where
pronounced colour change takes place; the
borders of this interval can be estimated by
pKa-1 and pKa+1.[6]
Acid-base indicators also show molecular
characteristics of a substance. Color changes in
molecules can be caused by changes in electron
confinement. More confinement makes the light
absorbed bluer (darker), and less makes it
redder (lighter). [7]
Colorimetric Analysis uses the variation as a
means of determining the pH since the
intensity of the color of a solution changes with
its concentration or pH. The color may be 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
acid-base indicator. The pH of a solution can be
determined by comparing the color intensities
of the solution with unknown pH with the
intensities of the solutions with known pH. [6]
Table 3. Color-change intervals of acid-
base indicators used (from resources)

Acid-base Color in pH range Color in

indicator the (color- the
more change more
The indicator's colour is the visible result of acidic interval) basic
the ratio of the concentrations of the two range range
species A- and HA. For methyl orange: Thymol Blue Pinkish 1.2-2.8 Yellow
red
Thymol blue Yellow 8.0-9.6 Blue
Bromphenol Yellow 3.0-4.6 Violet
blue
When [H+] has the same numerical value as Bromocresol Yellow 4.5-5.5 Blue
Ka, the ratio of [A-] to [HA] is equal to 1, green
meaning that 50% of the indicator is present in Bromocresol Yellow 5.2-6.8 Purple
the red acid form and 50% in the yellow ionic purple
form, and the solution appears orange in Phenol red Orange 6.8-8.4 Red
colour. When the hydrogen ion concentration Methyl orange Red 3.1-4.4 Yellow
increases to a pH of 3.1, about 90% of the Pheolphthalei colorless 8.0-9.8 Pink
indicator is present in the red form and 10% in n
the yellow form, and the solution turns red. No
change in colour is visible for any further
increase in the hydrogen ion concentration. [6]

Addition of a base to the system reduces the

hydrogen ion concentration and shifts the
equilibrium toward the yellow form. At a pH of
4.4 about 90% of the indicator is in the yellow
ionic form, and a further decrease in the
hydrogen ion concentration does not produce a
visible colour change. The pH range between
3.1 (red) and 4.4 (yellow) is the colour-change
interval of methyl orange; the pronounced
colour change takes place between these pH
 Table 4. Results of the colorimetric determination of pH

pH
Acid-base indicator 2.0 3.0 5.0 7.0 7.5 8.0 11.0
Thymol blue Dull pink Dull Light Dull Light Light Dark
yellow yellow yellow yellow yellow blue
Bromphenol blue Dull Yellow Lavender Blue Blue Lavender Blue
yellow violet violet
Bromocresol green Light Dull Light blue Blue Blue Faded Light
yellow yellow blue blue
Bromocresol purple Yellow Bright Yellow Purple Purple Purple Purple
yellow
Phenol red Yellow Fuchsia Yellow Dull Light Pinkish Dark
orange orange orange red pink
Methyl orange Neon Orange Red Orange Orange Yellow Orange
pink red orange
Phenolphthalein Colorless Colorless Colorless Colorless Colorless Colorless Red
violet

REFERENCES

From books:

[1]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.

[2]Bernas, G.C., Ysrael, M.C., & Bernaldez, A.T.

(1994). Basic Laboratory Studies in Biochemistry
(3rd ed.). Manila: UST Publishing House. Pages 5-
9 and 17.

[3]Boyer, R.F. (2006). Biochemistry Laboratory:

Modern Theory and Techniques. (1st ed.). San
Francisco: Pearson Education Inc. Pages 55-69.

[4]Bettelheim, F.A., & Landesberg, S.M. (2010).

Laboratory Experiments for Introduction to
General, Organic, and Biochemistry. (7th ed.).
USA: Brooks/Cole. Pages 207-210.

[5]Crisostomo, A.C., et. al. (2010). Laboratory

Manual in General Biochemistry. Quezon City: C
& E Publishing, Inc. Pages 1-4.

From the internet:

[6]images.kimme08.multiply.multiplycontent.co
m/attachment/0/ (12/31/11)

[7]http://www.scribd.com/doc/25375272/Ph-
Measurement-and-Buffer-Preparation (12/31/11)