Aquinas University of Legaspi City

College of Health Sciences

S.Y 2015-2016
PH MEASUREMENT AND BUFFER PREPARATION
Rommel Joy Aspe, Alfie Benedict Espedido, Gregory
Vincent Mendoza, Jobelle Ramirez ,Tan, Gilberto Tan, Alyssa Royce Tero
Group 3

BS-Pharmacy II

---------------------------------------------------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 (H3PO4) as weak
acid and Primary sodium phosphate dihydrate (NaH2PO4.2H2O) as conjugate base.
Using 6M HCl (to make the buffer more acidic) and 6M NaOH (to make the buffer more
basic) the pH of the buffer solution was measured electrometrically using the pH meter
and manipulated. The pH meter indicates the concentration of the hydrogen ion in a test
solution by responding to the potential developed by an electrical cell. It showed
accurate pH readings of the prepared buffer solution. With slightest addition of HCl and
NaOH, It result to a variation in readings. Thus, the pH meter shows more accuracy in
reading pH levels rather in using a pH paper. Using acid-base indicators the pH of the
prepared buffer solution was measured colorimetrically (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 be used to
narrow down the pH range of a substance. The pH range is the color-change interval of
an indicator, where pronounced color 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 (811).
I. INTRODUCTION
In chemistry, pH is the measure of the concentration of hydrogen ions [H +] in a given
substance. To compute for the pH, we calculate the negative logarithm of the number of
moles of the hydrogen ion. The formula is given below:
pH= -log[H+]

or

pH= log 1/ [H+]

Generally, a low pH indicates acidity of a substance and a high pH indicates a basic

characteristic of the substance. The lower the pH, the more acidic a substance is.
Inversely, the higher the pH, the more basic a substance is. Thus, a pH of 3 for
example, is more acidic than a pH of 4. And a pH of 10 is more basic than a pH of 9.
The determination of pH is a fundamental analytical procedure in biochemistry and even
in the study (and practice) of the science of pharmacy. The conformation and important
functions of most biomolecules are affected by the pH of their environment. Even
intracellular and extracellular fluids of living organisms have different pH values. In
pharmacy, drug absorption and drug elimination are both affected by pH. The pH is very
important in most biological processes.
On the other hand, buffer solutions are aqueous solutions that consist of a weak acid
and its conjugate base or vice versa. It can resist or minimize changes in pH.
Physiologically, buffers play an important role in many bodily processes. The internal pH
of our cells is approximately equal to 7.0. The pH of human blood is 7.4. A blood pH of
below 7.0 or above 7.8 can cause death within minutes. To stabilize blood pH, the body
uses buffers.
The dominant buffers in our body include the following:
1) Bicarbonate buffers
2) Phosphate buffers
3) Protein buffers
.
II. MATERIALS AND METHODS
Materials and Reagents:
Volumetric flasks
Beakers
Serological pipettes
Aspirators
Vials/ test tubes
Conc. H3PO4 (85% w/w, sp.gr. 1.70, MM 98)
Conc. HCl (37.3% w/w, sp.gr. 1.18, MM 36)
Glacial acetic acid (99.7% w/w, sp.gr. 1.05, MM 60)
pH paper
NaOH pellets (MM 40)
NaH2PO4.H20 (MM 138)
Na2HPO4.7H2O (MM 268)
Acid-base indicators:
o Bromophenol blue
o Bromcresol green
o Bromcresol purple
o Bogen indicator

o Phenol red
o Methyl red
o Methyl orange
o Phenolphthalein
o Thymol blue

o Preparation of Reagents
1. Prepare 500 mL of the following aqueous solutions:
a. 6.0 M HCl
b. 6.0 M NaOH
2. Label the containers properly.
o
o Buffers
1. Prepare 250 mL 0.10 M buffer solutions:
o
o
o
o
o
o
o
o

2.

1.
2.

3.
4.

BUFFER SOLUTIONS
Phosphate; pK= 2.12
Phosphate; pK= 2.12
Acetate; pK= 4.70
Phosphate; pK= 7.21
Phosphate; pK= 7.21
Phosphate; pK= 7.21
Phosphate; pK=12.32

o
o
o
o
o
o
o
o

DESIRED pH
2.00
3.00
5.00
7.00
7.50
8.00
22.00

o
o Identify the weak acid and conjugate base components of each
buffer. Calculate the amounts of the buffer components using the
Henderson-Hasselbalch equation. Select the appropriate reagents
from the list of reagents and materials.
Label the containers properly.
o
o Electrometric Determination of pH
Calibrate the pH meter at pH 4, 7 and 10.
Measure pH of a 20-mL portion of the following samples:
a. Prepared buffer solutions
b. Distilled water
c. Assigned sample (pineapple juice, soda, and shampoo)
Calculate the [H+] of the samples used.
Adjust the pH of the prepared buffer solution to its desired value by adding
in portions of either 1.0 M HCl or 1.0 M NaOH as monitored by the pH
meter. Refer to table.

o
o Colorimetric Determination of pH
A. Preparation of colour standards using the buffer solutions
a. Prepare 6 vials or test tubes. Label the vial or test tube with the pH
of the buffer and acid-base indicator to be added.
b. Pipet 5 mL of a buffer to a certain pH into the vial and add 2 drops
of an acid base indicator. Shake the mixture and note down its
colour. Pipet 5 mL of a buffer of a different pH into another vial and
add 2 drops of the same acid-base indicator. Perform this
procedure on all prepared buffers.

c. Prepare another set of 6 vials or test tubes. Repeat step 2 but use
a different acid-base indicator. Perform this procedure using the
following acid-base indicators:
i. Thymol blue
ii. Bromophenol blue
iii. Bromcresol green
iv. Bromcresol purple
v. Phenol red
vi. Bogen indicator
vii. Methyl red
viii. Methyl orange
ix. Phenolphthalein
o
o
B. Determination of Buffer Capacity
a. Calibrate the pH meter at pH 4, 7 and 10.
b. Prepare 100 mL each of 0.10 M HCl and 0.10 M NaOH.
c. Determine the buffer capacity by adding 0.10 M HCl in 1 mL
portions from a buret to 20 mL of the buffer until its pH changes as
monitored with a pH meter. Record the volume added. Do the same
using 0.10 M NaOH. Perform this procedure in all buffers.
o
o III. RESULTS AND DISCUSSION

o
o ELECTROMETRIC DETERMINATION OF PH
o
o Results of Electrometric Determination of pH (Samples)
o
Samples
o
pH
o
[H+]

o
Distilled
Water
o
Assigned
Sample
o
Shampoo
o

Fruit Juice
o
o
o
o
o
o
o

7.0

1.0 x 10-7

10.8

1.58 x 10-11

1.0 x 10-8

5.6

2.51 x 10-6

o
o
o
o
o
o
o

o
o
o Results of Electrometric Determination of pH (Buffers)
o
o
Buffers
o
pH
o
[H+]
o
Phosphate
o
7.5
3.16 x 10-6
o
Acetate
o
5
1.1 x 10-5
o
Phosphate
o
8
1.0 x 10-8
o
Phosphate
o
11
1.0 x 10-11
o
Phosphate
o
7
1.0 x 10-7
o
Phosphate
o
3
1.0 x 10-3
Phosphate
o
2
o
0.01
o
o In Electrometric Determination of pH, the pH meter is used to
measure the electrochemical properties of liquids, pastes and semisolids. It indicates the concentration of hydrogen ion in a test
solution by responding to the potential developed by an electrical
cell. An accurate pH readings of the prepared buffer solution
are shown by the pH meter. With the slightest addition of HCl and
NaOH it indicates variations in readings. Addition of the former will
result to the decrease in pH and by 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.
Hence, the pH meter is more accurate in reading pH levels
compared to pH paper.
o
o In the electrometric method, the shampoo which has a pH of 8 and
the soda which has a pH of 10.8 are basic and the pineapple juice
with a pH of 5.6 is acidic. Meanwhile, the phosphate buffers with
pH's of 3.2 and an acetate buffer of 5 are good buffers for basic
substances because of its low pH and the phosphate buffer of 7.5
and 11 are good for acidic substances because of the high pH. The
phosphate buffer with the pH of 7 can be good for both acidic and
basic meaning it is amphoteric.
o
o
o
o
o
o
o
o
o
o

o
o
o
o
o
o
o
o COLORIMETRIC DETERMINATION OF PH
o
o Color-Change Intervals of Acid-Base Indicators
o
o
Acido
Color
o
pH
o
Color
Base Indicator
in Acidic
Range
in Basic
Range
Range
o
Thymol
o
Pinkish o
1.2-2.8 o
Yellow
Blue
red
o
Thymol
o
Yellow
o
8.09o
Blue
Blue
9.6
o
Bromphe o
Yellow
o
3.0-4.6 o
Violet
nol Blue
o
Bromcres o
Yellow
o
4.5-5.5 o
Blue
ol Green
o
Bromcres o
Yellow
o
5.2-6.8 o
Purple
ol Purple
o
Phenol
o
Orange o
6.8-8.4 o
Red
red
o
Methyl
o
Red
o
3.1-4.4 o
Yellow
Orange
o
Phenolph o
Colorle o
8.0-9.8 o
Pink
thalein
ss
o
o When the hydrogen ion concentration reaches a particular value
there are certain organic substances change in color in dilute
solution. An example of this is phenolphthalein, it is a colorless
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 concentration of hydrogen ion less than 1.0x10 -8 M
(pH greater than 8.0), result to the red or pink color of
phenolphthalein. It is also can be used to determine the pH of a
solution, which 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.
o

o The equilibrium in a solution of the acid-base indicator methyl

orange, a weak acid, can be represented by the equation
o
o HA

H+ + Ao red
yellow
o
o The color of the anion of methyl orange is yellow, and the color of
the non-ionized form is red. An addition of acid to the solution,
result to the increase in the hydrogen ion concentration and shift
the equilibrium toward the red form in accordance with the law of
mass action.
o
+
H

o
A

Ka=
o
o
o
o The visible result of the ratio of the concentrations of the two
species A- and HA is called the indicator's color. For methyl orange:
o
o

o
o
o If the Hydrogen ion has the same numerical value as Ka, it will
result to the equal ratio of [A-] to [HA], meaning that 50% of the
indicator is present in the red acid form and 50% in the yellow ionic
form, and the color of the solution appears in color orange. An
increase of the hydrogen ion concentration to a pH of 3.1, about
90% of the indicator is present in the red form and 10% in the
yellow form, and which result to the red color of the solution. There
will be no change in color that will be visible for any further increase
in the concentration of hydrogen ion.
o
o Adding a base to the system will result to the reduction of 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 concentration of the
hydrogen ion will not yield visible color change. The color-change
interval of methyl orange is the pH range between 3.1 (red) and 4.4

(yellow); between these pH values a pronounced color change

takes place. In general, the pH range is the color-change interval of
an indicator, where pronounced color change takes place; the
borders of this interval can be estimated by pKa-1 and pKa+1.
o
o Acid-base indicators show the molecular characteristics of a
substance. By the changes in electron confinement can cause the
change in color in molecules. More confinement makes the light
absorbed bluer (darker), and less makes it redder (lighter).
o
o Colorimetric Analysis uses the variation as a means of determining
the pH since the color intensity of a solution changes with its pH.
The color result to the inherent property of a substance in the
solution, or the formation of a product as a result of adding acidbase indicator. By comparing the color intensities of the solution
with unknown pH with the intensities of the solutions with known
pH, the pH of a solution can be determined
o
o In the colorimetric method, a sample was given to us. It has a pH of
8.0 in thymol, a pH range of 7.5-12.0 in Bromophenol Blue, 7.512.0 in Bromophenol Green, 8.0 in Bromophenol purple, 5.0 in
methyl red, 2.0 in Methyl Orange and 8.0 in phenolphthalein.
Having seen these results, majority of it shows that it is basic.
o
o
o
o
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o Reference:
o
o 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.
o
o http://www.scribd.com/doc/25375272/Ph-Measurement-andBuffer-Preparation
o
o http://www.codental.uobaghdad.edu.iq/uploads/lectures/Pharm
a%20lectures/2%20Pharmacokinetics%20finishing%20with
%20bioavailibility.pdf
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o http://environmental-realm.blogspot.com/2012/04/importanceof-buffers-in-physiological.html
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o BIOCHEM LAB MANUAL

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