pH Measurement and

Buffer Preparation
Pharmaceutical Biochemistry
PHA 6112

Prepared by:
Pharmaceutical Biochemistry Laboratory Instructors (A.Y. 2020-2021)
Faculty of Pharmacy
Department of Pharmacy
Learning Objectives
At the end of this chapter, the students should be able
to:
• Prepare different buffer solution
• Determine the pH of buffers and samples
colorimetrically using liquid indicators and
electrometrically using the pH meter; and
• Calculate the buffer capacity of prepared buffer
solutions.
Expt 1: pH Measurement and Buffer Preparation
Acid and Base Concept
1. Arrhenius Dissociation Theory
Acid: liberates H+ ion in aqueous soln
HCl + H2O → H3O+ + Cl-

Base: liberates OH ions in aqueous solution.

NaOH → Na+ + OH-
Expt 1: pH Measurement and Buffer Preparation
2. Bronsted- Lowry Theory
Acid: substance (charged or uncharged) that is capable of donating
proton.
Base: substance (charged or uncharged) that is capable of accepting a
proton from an acid.
HA + H2O → H3O+ + A-
acid base conj. Acid conj. Base
HNO2 + H2O → H3O+ + NO2-
• Water is amphoteric, which means it can act as both a Bronsted-Lowry
acid and a Brønsted-Lowry base.
• Strong acids and bases ionize completely in aqueous solution, while weak
acids and bases ionize only partially in aqueous solution.
Expt 1: pH Measurement and Buffer Preparation
3. Lewis Theory
Acid: a molecule or ion that accepts a pair of electrons
from another atom.

Base: a substance that donates a pair of electrons to be

shared with another atom.

NH3 + H + → NH4+
Expt 1: pH Measurement and Buffer Preparation
•
Expt 1: pH Measurement and Buffer Preparation
Sample problems:
1. What is the pH of a solution containing 0.00017 M of
hydronium ion? (Ans. pH is 3.77)
2. Calculate the pH of a solution if the hydrogen-ion
concentration is 1.92 x 10 – 5 mol/L. (Ans. 4.71)
3. If the pH of a solution is 7.36, what is the hydrogen-ion
concentration of the solution? (Ans. 4.3 x 10 -8)
Expt 1: pH Measurement and Buffer Preparation
Sample problems:
1. What is the pH of a solution containing 0.00017 M of
hydronium ion?
2. Calculate the pH of a solution if the hydrogen-ion
concentration is 1.92 x 10 – 5 mol/L.
3. If the pH of a solution is 7.36, what is the hydrogen-ion
concentration of the solution?
Expt 1: pH Measurement and Buffer Preparation
Buffers
- Compounds or mixture of compounds which by their
presence in solution resists change in pH upon addition of
small quantities of acid or alkali.
-Mixture of weak acid and its conjugate base or a weak base
and its conjugate acid.
-Acetic acid and sodium acetate (HAc & NaAc)
-Ammonium hydroxide and ammonium chloride (NH4OH & NH4Cl)
-Characteristics: Has definitive pH; Buffer action and Buffer
capacity
Expt 1: pH Measurement and Buffer Preparation

The pH of a buffer is determined by two factors;

1) The equilibrium constant Ka of the weak acid

2) the ratio of weak base [A-] to weak acid [HA] in

solution.

pH = pKa + log([A-)/(HA-)]
 Expt 1: pH Measurement and Buffer Preparation
I. Preparation of Buffers

A. 250mL 0.50M B. 250mL 0.50M C. 250mL 0.50M Acetate D. 250mL 0.50M

Phosphate Phosphate Buffer pKa: 4.75 | pH: Phosphate
Buffer pKa: 2.12 | pH: Buffer pKa: 2.12 | pH: 5.00 Buffer pKa: 7.21 | pH:
2.00 3.00 7.00

E. 250mL 0.050M F. 250mL 0.010M G. 250mL 0.50M Phosphate

Phosphate Phosphate Buffer pKa: 12.32 | pH:
Buffer pKa: 7.21 | pH: Buffer pKa: 7.21 | pH: 12.00
7.50 8.00
Expt 1: pH Measurement and Buffer Preparation
I. Preparation of Buffers Conc H3PO4 (85.00% w/w, sp.gr. 1.70 MW: 98g/mol)

Conc HCl (37.3% w/w, sp.gr. 1.18 MW: 36g/mol)

Glacial Acetic acid (99.7% w/w, sp.gr. 1.05 MW:

60g/mol)

NaOH (MW: 40g/mol)

CH3COONa (MW: 82 g/mol)

NaH2PO4 ● H2O (MW: 138g/mol)

Na2HPO4 ● 7H2O (MW: 268g/mol)

Na3PO4 ● H2O (MW: 182 g/mol)

Expt 1: pH Measurement and Buffer Preparation
I. Preparation of Buffers

pKa WA CB
2.12 H3PO4 H2PO4-1
Na2HPO4 + NaOH =
7.21 H2PO4-1 HPO4-2 Na3PO4 + H2O
12.32 HPO4-2 PO4-3

pKa WA CB CH3COOH + NaOH =

4.75 CH3COOH CH3COONa CH3COONa + H2O
Expt 1: pH Measurement and Buffer Preparation
I. Preparation of Buffers
Conc H3PO4 (85.00% w/w,
pH = pKa + log([A-)/(HA-)]
sp.gr. 1.70 MW: 98g/mol

Conc HCl (37.3% w/w, sp.gr.

1.18 MW: 36g/mol

Glacial Acetic acid (99.7% w/w,

sp.gr. 1.05 MW: 60g/mol

A. 250mL CH3COONa (MW: 82 g/mol)

0.50M
NaH2PO4 ● H2O (MW:
Phosphate 138g/mol)
Buffer
pKa: 2.12 Na2HPO4 ● 7H2O (MW:
pH: 2.00 268g/mol)

Na3PO4 ● H2O (MW: 182 g/mol)

 Expt 1: pH Measurement and Buffer Preparation
I. Preparation of Buffers M=B+A
Conc H3PO4 (85.00% w/w, 2. Compute for the base and acid
sp.gr. 1.70 MW: 98g/mol
component
Conc HCl (37.3% w/w, sp.gr.
1.18 MW: 36g/mol I. Acid II. Base
Glacial Acetic acid (99.7% w/w, Component Component
sp.gr. 1.05 MW: 60g/mol
A. 250mL
0.50M CH3COONa (MW: 82 g/mol) M=B+A M=B+A
Phosphate 0.5 = 0.758A + A B=M–A
Buffer NaH2PO4 ● H2O (MW:
138g/mol) 0.5 = 1.758 A B = 0.5 –
pKa: 2.12
pH: 2.00 A = 0.286…M 0.284…
Na2HPO4 ● 7H2O (MW:
268g/mol) B = 0.215…M
Na3PO4 ● H2O (MW: 182 g/mol)
Expt 1: pH Measurement and Buffer Preparation
I. Preparation of Buffers
Conc H3PO4 (85.00% w/w,
sp.gr. 1.70 MW: 98g/mol

Conc HCl (37.3% w/w, sp.gr.

1.18 MW: 36g/mol

Glacial Acetic acid (99.7%

w/w, sp.gr. 1.05 MW: 60g/mol

A. 250mL CH3COONa (MW: 82 g/mol)

0.50M
Phosphate NaH2PO4 ● H2O (MW:
Buffer 138g/mol)
pKa: 2.12
Na2HPO4 ● 7H2O (MW:
pH: 2.00
268g/mol)

Na3PO4 ● H2O (MW: 182

g/mol)
 Expt 1: pH Measurement and Buffer Preparation
I. Preparation of Buffers
Conc H3PO4 (85.00% w/w,
sp.gr. 1.70 MW: 98g/mol

Conc HCl (37.3% w/w, 4. Compute for the actual grams of the base
sp.gr. 1.18 MW: 36g/mol

Glacial Acetic acid (99.7%

A. 250mL w/w, sp.gr. 1.05 MW:
0.50M 60g/mol
Phosphate
Buffer NaOH (MW: 40g/mol)
pKa: 2.12
pH: 2.00 NaH2PO4.H2O (MW:
138g/mol)

Na2HPO4.7H2O (MW:
268g/mol)
Expt 1: pH Measurement and Buffer Preparation
II. Preparation of Reagents

500mL 6.0M HCl 500mL 6.0M NaOH

Expt 1: pH Measurement and Buffer Preparation
III. Electrometric Determination of pH
Samples:
Group 1 -
Group 2 –
Group 3 –
Group 4 –
Group 5 –
Group 6 –
20mL buffer soln 20mL water 20mL sample + Group 7 –
+ 20mL water 20mL water

1. Calibrate pH meter at pH 4, 7 and 10.

2. Measure pH of the samples
3. Calculate the [H+] of samples used
4. Adjust pH of buffer soln to desired value by adding in portions of 6.0M
NaOH and 6.0M HCl.
Expt 1: pH Measurement and Buffer Preparation
IV. A Colorimetric Determination of pH IV. B Determination of the pH of the samples
BUFFER pH pH pH pH pH pH pH W SA
2 3 5 7 7.5 8 12 AT MP +10 µL indicator
ER LE

Bromophenol blue
Bromophenol green
Bromophenol purple
Phenol red
Methyl red
Methyl orange
Phenolphthalein
Thymol blue
pH Measurement and
Buffer Preparation
Post Lab Discussion
Pharmaceutical Biochemistry
PHA 6112
Expt 1: pH Measurement and Buffer Preparation
Buffer solution
• resists changes in the activity of an ion on the addition of substances
that are expected to change the activity of that ion
• contains an acid and its conjugate base, or a base and its conjugate
acid

Characteristics:
1. Definitive pH value
2. Buffer capacity
Expt 1: pH Measurement and Buffer Preparation
Importance of Buffers
Biological: To maintain normal functioning of the cells.
• Blood – pH 7.4 and maintained by the so-called primary
buffer in the plasma and secondary buffers in the
erythrocytes. NaHCO3 & H2CO3; NaH2PO4 & Na2HPO4
• Plasma proteins – behave as acids in blood combines
with bases to act as buffer.
• Erythrocytes – hemoglobin/oxyhemoglobin, acid/alkali
potassium salts of phosphoric acid
Expt 1: pH Measurement and Buffer Preparation

Significance of Buffers
To protect pharmaceutical preparations from any sudden
change in pH even when limited concentrations of acid or
base are added
Minimize drug degradation
Improve patient comfort and compliance
Improve the efficacy of delivery
Expt 1: pH Measurement and Buffer Preparation

Significance of Buffers
Expt 1: pH Measurement and Buffer Preparation
Application of Buffers
Buffered isotonic solution to be
prepared and administered should
have the same solute concentration
as that of the biological fluid
Eyes
Soft tissues
Nasal cavities
Anal applications
Vaginal cavity applications
Expt 1: pH Measurement and Buffer Preparation

Uses of Buffers
1. To establish hydrogen-ion activity for the
calibration of pH meters
2. To prepare isotonic dosage forms
3. To maintain stability of dosage forms
4. For analytical procedures
Expt 1: pH Measurement and Buffer Preparation
Buffers in Pharmaceutical Systems
Solid Dosage Semisolid Parenteral Ophthalmic
Forms Dosage Forms Products Preparations
Gastric irritation Stability issues Tissue necrosis (pH Solubility and
>10) stability issues
Pain on site of action
(pH <3)
Sodium Citric acid Citrate Citrate
bicarbonate
Magnesium Sodium citrate Glutamate Phosphate
carbonate
Sodium citrate Phosphoric acid / Phthalate Borate
sodium phosphate Acetate
Expt 1: pH Measurement and Buffer Preparation
pH Indicator Dyes
Organic dyes pH range Color change Color change
Acid Base
Thymol blue (acid range) 1.2 – 2.8 Red Yellow
Bromophenol blue 3.0 – 4.6 Yellow Blue
Bromophenol green 3.8 – 5.4 Yellow Blue
Bromophenol purple 5.2 – 6.8 Yellow Violet
Phenol red 6.8 – 8.4 Yellow Red
Thymol blue (alkaline range) 8.0 – 9.6 Yellow Blue
Methyl red 4.8 – 6.0 Yellow Red
Methyl orange 3.2 - 4.4 Red Yellow
Phenolphthalein 8.2 – 10.0 Colorless Pink
Expt 1: pH Measurement and Buffer Preparation
How the Color Change of the Indicators Happens
• Caused by the dissociation of the H+ ion from the
indicator itself since they are weak acids

HIn+H2O ⇌ H3O++In−
• HIn = the acidic pH indicator
• In- =the conjugate base of the pH indicator
 Expt 1: pH Measurement and Buffer Preparation
pH Determination
Specifications Electrometric Colorimetric
Principle Automated sensing hydrogen ion Based on property of acid-
electrode that calculates the difference base indicator dyes to
between the reference electrode and produce a color depending on
sensing electrode potentials to pH of a sample
measure pH
Limitations Need for equipment calibration, Rough pH estimation
maintenance and protection from dirt
and extreme heat or freezing
conditions
during use and storage
Solution interference Not subjected to solution interferences Strong color and additives of
from color, turbidity, colloidal matter, sample may interfere with
oxidants, reductants or high salinity measurement
Expt 1: pH Measurement and Buffer Preparation

Buffer Capacity
• The measure of the efficiency of a buffer in
resisting pH changes
• The quantity of a strong acid or strong base
that must be added to one liter of a solution to
change it by one pH unit