LEC PHARM ANALYSIS MODULE 4 & 5

2. Mobile phase
CHROMATOGRAPHY ➢ Pure liquid or gas or a mixture of solutions
that moves through or over the fixed phase
● Most widely used separation technique in Ex.
chemical laboratories, where it is used in ➔ Petroleum ether
analysis, isolation, and purification and it is ➔ Carbon tetrachloride
commonly used in the chemical process ➔ Benzene
industry as components of small and ➔ Chloroform
large-scale production ➔ Diethyl ether
➔ Ethyl acetate
● It is the versatility of chromatography in its
➔ Pyridine
many variants that is behind its ubiquitous ➔ Acetone
status in separation science, coupled with ➔ N- propanol
simplicity of approach and a reasonably ➔ Ethanol
well-developed framework in which the ➔ Methanol
different chromatographic techniques ➔ Water
operate.
● Use for separation, identification, and ● The word chromatography is derived from
determination of the chemical components in the Greek words “chroma” and “graphein”
complex mixture meaning “color” and “to write” or “to
● A separation of colored compounds on a represent”
suitable adsorbent ● While studying the coloring materials in plant
● Defined as a procedure by which solutes are life, a Russian botanist invented
separated by a differential migration process chromatography in 1903. His name was
in a system consisting of two phases, one of Mikhail S. Tswett
which moves continuously in a given ● It is a collective term for a family of
direction and in which the individual laboratory techniques for the separation of
substances exhibit different mobilities by mixtures
reason of differences in adsorption, partition, ● It involves passing a mixture dissolved in a
solubility, vapor pressure, molecular size or “mobile phase” through a “stationary phase”
ionic charge density(USP). which separates the analyte to be measured
from other molecules in the mixture and
TWO PHASES OF CHROMATOGRAPHY allows to be isolated
● It may be preparative or analytical
➢ In chromatography science, the solvent is
called “the mobile phase” or the “carrier
fluid” and the medium is called “the PREPARATIVE CHROMATOGRAPHY
stationary phase” ➢ The purpose of preparative
chromatography is to separate the
1. Stationary phase components of a mixture for further use
➢ Fixed phase may be porous or finely (and is thus a form of purification). It is
divided solid or a liquid that has been done normally with larger amounts of
coated in a thin layer on inert supporting material
material.
Ex. ANALYTICAL CHROMATOGRAPHY
➔ Silica gel ➢ The purpose of analytical chromatography
➔ Activated alumina is to measure the relative proportions of
➔ purified siliceous earth analytes in a mixture. It is done normally
➔ Calcium carbonate with smaller amounts of material.
 LEC PHARM ANALYSIS MODULE 4 & 5
PRINCIPAL OBJECTIVES OF
CHROMATOGRAPHY:
● Resolution of mixtures into constituent
parts,
● Determination of homogeneity
● Comparison of substances suspected of
being identical.
● Purification.
● The concentration of substances from dilute
solutions.
● Identification and control of technical
products.
● Quantitative separation from complex
mixtures.
● Indication of molecular structure.
RETENTION:
● Measure of speed at which a substance
moves in a chromatographic system.
● In continuous development systems (HPLC
& GC), where the compounds are eluted
with an eluent, it is expressed as retention
time (Rt).
● In interrupted development systems ( TLC &
PC ), It is expressed as retention factor (Rf)
PRINCIPLES OF SEPARATION:
ADSORPTION
● Retention and separation depends on the
ability of the atoms on the surface to
remove analytes from the mobile phase
and adsorb them temporarily by means of
electrostatic forces
PARTITION
● Retention and separation occur due to the
relative solubility of the analytes in the two
fluids as determined by their partition
coefficients
ION-EXCHANGE
● Retention and separation is mainly due to
the electrostatic bonds with the functional
groups
MOLECULAR EXCLUSION
● Also known as “size exclusion”, “gel
permeation” or “gel filtration”
● Retention and separation depends on the
differential migration of solute molecules
based on molecular siz
APPLICATION OF CHROMATOGRAPHY:
LIQUID CHROMATOGRAPHY
- TEST WATER SAMPLES FOR
POLLUTION.
GAS CHROMATOGRAPHY
- BOMB DETECTION IN AIRPORTS
- IDENTIFY / QUANTIFY DRUGS &
ALCOHOL.
- IN FORENSICS TO COMPARE FIBERS
FOUND ON A VICTIM.
THIN LAYER CHROMATOGRAPHY
- DETECTION OF PESTICIDES &
INSECTICIDES IN FOOD.
- IN FORENSICS TO ANALYZE DYE
COMPOSITION OF FIBERS.
PAPER CHROMATOGRAPHY
- SEPARATION OF AMINO ACIDS AND
ANIONS.
- RNA FINGERPRINTING
- SEPARATION AND TESTING OF
HISTAMINES & ANTIBIOTICS.
TYPE OF CHROMATOGRAPHY
➢ COLUMN CHROMATOGRAPHY
➔ Is a separation technique in which the
stationary bed is within a tube
➔ The particles of the solid stationary phase
or the support coated with a liquid
stationary phase may fill the whole inside
volume of the tube or be concentrated on
or along the inside tube wall leaving an
open, unrestricted path for the mobile
phase in the middle part of the tube
➔ It is a method used to purify individual
chemical compounds from mixtures of
compounds
COMPONENTS OF CC
Chromatographic Column
● The simplest type is consists of a suction
flask and a cylindrical glass or quartz
 LEC PHARM ANALYSIS MODULE 4 & 5
constricted at one end with a stopcock or
flow regulator.
● The size of the column is determined by
the quantity and adsorbability of the
substance being separated.
Stationary Phase
● Adsorbent or packing materials placed
inside the column to adsorb the sample
● Usually a solid material, finely ground
powders or gels which are microporous for
an increased surface
Evaluation of Chromatogram
➔ For colored samples that fluoresce under
UV light, the adsorbent column is forced
intact from the tube and the fractions are
easily divided by a knife.
➔ For colorless samples, visualization is
done by first spraying or painting the
extrude column with a color forming agent.
➔ For radioactive samples, their position in
the column is determined using a
Geiger-muller counter.
FLASH COLUMN CHROMATOGRAPHY
● A modified version of column
chromatography introduced by W.C. Still in
1978
● It is very similar to the traditional column
chromatography, except for that the
solvent is driven through the column by
applying positive pressure
PLANAR CHROMATOGRAPHY
➔ A separation technique in which the
stationary phase is present as or on a
plane.
➢ PAPER CHROMATOGRAPHY
➔ A method invented by the British
biochemists, Archer John Porter Martin
and Richard Laurence Millington Synge.
➔ An analytical technique for separating and
identifying mixtures that can be colored
especially pigments.
THREE METHODS IN PREPARING PAPER
PARTITION CHROMATOGRAM:
1. DESCENDING CHROMATOGRAPHY
➔ Which is accomplished by allowing the
mobile phase to flow downward on the
paper slip
2. ASCENDING CHROMATOGRAPHY
➔ In which the mobile phase is allowed to
rise upward on the paper by capillary
attraction
3. RADIAL CHROMATOGRAPHY
➔ In which the mobile phase moves out in
concentric circles from the center of a
circular piece of paper
★ PRINCIPLE: Differences of partition
coefficients of substances between two
immiscible liquids.
★ Solid adsorbent: Filter paper (Whatman,
Schleicher, schwell)
★ Chromatogram is determined by the Rf value,
used for identification.
Rf is a measure of the fraction of its total elution
time that any compound spends in the mobile
phase
Elution – cutting the spot and soaking the paper
in an appropriate solvent
Rf - means Retention factor, Retardation factor,
or Ratio front.
➢ THIN-LAYER CHROMATOGRAPHY
➔ A widely employed technique similar to
paper chromatography.
➔ It involves spotting of a sample or a
mixture of samples at one end of an
adsorbent-coated glass plate followed by
passage of a solvent through the
adsorbent for the purpose of separating
the components of a mixture.
➔ Involves the spotting of a sample of a
mixture of components at one end of aan
adsorbent-coated glass plate followed by
passage of a solvent through the
adsorbent separating the components of a
sample.
➔ Separation takes place on a planar
surface and the mobile phase moves
 LEC PHARM ANALYSIS MODULE 4 & 5

across the plate by capillary action. ●

Readily bonds phenol, carboxylic acids,
ADVANTAGE OF TLC OVER COLUMN AND quinones and nitro compounds of which
PAPER CHROMOTOGRAPHY requires polar solvents like methanol and
dimethylformamide to displace them.
A. Achieving separation in a relatively short time, CELLULOSE
30 mins or less ● A polysaccharide has numerous neutral
B. Accomplishing a complete analysis with as hydroxyl groups on its surface and can
little as 20 ng of material absorb water or solvents (polar) by
C. Providing a complete separation of hydrogen bonding.
components in complex mixtures.
D. Rapid NOTE:
E. Sensitive In order to ensure that the stationary phase
F. Excellent resolution adheres firmly to the backing plate and does not
G. Simplicity flake off during the development, binders such as
H. Cheap calcium sulfate (gypsum), starch or carboxyl
methylcellulose are added to the adsorbent.

DIFFERENT KINDS OF STATIONARY PHASE

SILICA GEL ABSORBENTS APPLICATION

● Surface is acidic due to the presence of
many silanol hydroxyl groups and best Silica gel Steroids, amino acids, alcohols,
suited to the analysis of acidic compounds (adsorption) hydrocarbons, lipids, aflatoxins,
such as amino and sugars. vitamins alkaloids. etc
● The surface of silica gel is acidic due to
Silica gel Fatty acids, vitamins, steroids,
the presence of silanol hydroxyl group
(partition) hormones, carotenoids
● Use in the analysis of acidic compounds
and polar compounds such as amino acids Cellulose, Carbohydrates, sugar alcohols, amino
and sugars. kieselguhr acids, carboxyl acids, fatty acids
● The most frequently used stationary phase
Aluminum Amines, alcohols, steroids, lipids,
in adsorption TLC
oxide aflatoxins, bile acids, vitamins,
● Common adsorbents used: Silica gel G alkaloids
and Alumina
● Usually dimension of the plate: 20 X 20 cm Polyamide Phenol, carboxylic acids, steroids,
● Layer thickness: 250 um quinones
● Selica gel coated with silicone oil and ODS
PEI cellulose Nucleic acids, nucleotides,
(octadecylsilyl) are reversed-phase nucleosides, purines, pyrimidines
separation.
● The solvents used as mobile phase in TLC
must be of high purity and use of a single
ALUMINA (ALUMINUM OXIDE)
solvent to develop chromatogram is
● Has a basic surface
preferred rather than a multi-component
● Use for the separation of basic and weakly
mixture.
polar compounds

POLYAMIDE (NYLON) MOBILE PHASE IN TLC

● A long chain polymer
● Has many free amide and carboxyl groups ● Heptane
● Ethyl ether
on its surface
● Acetonitrile
● An adsorbent with strong hydrogen ● Hexane
bonding capabilities ● Chloroform
 LEC PHARM ANALYSIS MODULE 4 & 5

● Pyridine
● Isoctane
● Acetone
● Ethylene chloride
● Cyclohexane
● Ethanol
● Methanol
● CCl4
● 1-Propanol Acetic acid
● Toluene
● Dioxane
● Water SPECIAL DETECTION METHOD
● Benzene
● Ethyl acetate CHARRING
● Involves the spraying of concentrated
sulfuric acid and heating the plate.
STATIONARY AND MOBILE PHASES USED IN TLC:
● The result is seen by the charring of the
Technique STATIONARY MOBILE spots.
PHASE PHASE
USE OF IODINE VAPOR
I. Adsorption Silica gel G Non-polar or ● In this method, the chromatogram is
Alumina polar organic
placed in a closed container holding a few
Charcoal solvents
Polyamide iodine crystals.
● The sample spots react with the iodine
II. Partition Cellulose Mix aqueous vapor and form brown spots.
reversed P. Silica gel Organic ● The reaction is reversible.
ODS silica gel solvent
Coated silica Mixed
EXAMINATION UNDER UV RADIATION
Acetylated aqueous polar
Cellulose solvent ● Useful for compounds that fluoresce.
● Two UV light sources are useful and
III. Ion exchange Ion Buffered commercially available, these are the UV
resins exchange-solution aqueous short-wave [254 nm] and long-wave
DEAE and [360nm].
CM-Cellulose

IV. Size Dextral gels Aqueous

exclusion buffer

FACTORS AFFECTING Rf

➔ Moisture content of adsorbent

➔ Chamber saturation
➔ Type and purity of adsorbent
➔ Thickness of adsorbent layer
➔ Temperature
➔ Sample size
➔ Depth of developing phase
➔ Solvent parameters
 LEC PHARM ANALYSIS MODULE 4 & 5
GAS CHROMATOGRAPHY
● Also known as “gas-liquid chromatography” or
“gas-liquid partition chromatography”.
● A separation technique in which the mobile
phase is a gas.
● In GC the separation of mixtures into individual
components depends on the differential
partition of the sample components between
the involatile liquid stationary phase and the
carrier gas or mobile phase
● Thus the technique is called gas liquid partition
chromatography or gas liquid chromatography
or gas chromatography
PRINCIPLE OF SEPARATION
A. The sample is introduced into the carrier
gas stream at the head of the column
through the injector part.
B. This injector part is enclosed by a
self-sealing rubber septum so that once
the sample is introduced, backflow is
prevented.
C. The injector part is heated to a
temperature above that of the column
oven temperature to ensure vaporization
but so high as to decompose the sample.
D. The vaporized sample is moved by the
carrier gas through the oven-enclosed
column onto the stationary phase. (The
oven temperature must be high enough to
maintain all the components in the vapor
state)
E. When the solute reaches the stationary
phase, distribution occurs between the 2
phases. The molecules not absorbed or
dissolved in the stationary phase are
swept down the column by the flow or the
carriers gas.
F. At the column exit, there is a continuous
flow of the carrier gas. When the solute is
eluted, a binary mixture of solute and gas
emerges and passes into the detection
unit, And this produces an electrical signal
which in turn is recorded by an integrator
or a recorder.
Although Gas chromatography is limited to volatile
materials, the availability of column temperature
up to 450℃ pyrolytic techniques and the
possibility of converting non-volatile material into a
volatile derivative, extended the application of the
method.
PATS OF GAS CHROM
1. CARRIER GAS
➔ Containing Helium, nitrogen, or hydrogen
➔ Separate solute one by one
MOBILE PHASE IN GAS CHROMATOGRAPHY
CARRIER GAS
 LEC PHARM ANALYSIS MODULE 4 & 5
PURITY
● One of the most important considerations
is the purity of the gas
● An impure gas will result in not only in an TUBING MATERIAL
unsteady or elevated baseline but may
cause negative or inverted peaks
● The negative can introduce both
qualitative and quantitative errors.
INERTNESS
● Carrier gas must also be inert. This is
especially important in GSC where the ADVANTAGES
reactive gas would compete with the
solute for binding sites on the column.
Theoretically, any gas can be used as a carrier
but the carrier gases are practically limited to the
following choice: H2, He, N2, Ar because of the
following consideration or requirements.
Nitrogen (N2) - the most common and best
recommended
Hydrogen (H2_ is dangerous while Helium and
argon are expensive.
2. INJECTION POINT
➔ Where the sample is introduced into the
system
➔ The primary requirement of the injection
system is that the sample be vaporized as
nearly instantaneously as possible so that a
narrow band of vapor is introduced into the
beginning of the column.
➔ The injector port is equipped with a
self-sealing rubber septum to prevent the
sample from escaping.
3. OVEN and COLUMN
➔ Like HPLC, the heart of the gas
chromatographic system is also the
column
➔ GC columns are long tubes packed with a
sorbent material
COMMON DETECTORS
PARTS OF COLUMN
● Tubing material
↪ Glass
↪Steel
● Solid supports
● Coating or liquid stationary phase
● Tubing material of column are generally 1 -
15 m long and about 0.5cm in internal
diameter
● Glass and stainless steel tubings are the
most commonly used material for the
construction of high temperature columns
● GLASS COLUMN
➔ It is possible to see how well the column is
packed
➔ The extent to which the packing has
deteriorated with extended use
➔ The most inert
● METAL COLUMN
➔ The advantage of durability
➔ It is unbreakable
4. SOLID SUPPORT
● Qualities of good support
- Large surface area
- Uniform pore size
- Inert
● The solid used to support the stationary
phase is usually porous in order to absorb
large quantities of solvent and still remain
dry to the touch and free-flowing
● Non porous supports, such as glass
microbeads are occasionally used
especially for analysis of high boiling
liquids.
● The normal mesh size of solid is around
60-100 mesh
5. DETECTOR
● Measure the concentration of the sample
injected in the column
● It generates a signal proportional to the
sample concentration.
★ FID - Flame ionization detector
★ ECD - Electron capture detector
★ TCD - Thermal conductivity detector
 LEC PHARM ANALYSIS MODULE 4 & 5

6. RECORDER/INTEGRATOR
● The main function of the recorder is to INJECTOR PORT
graphically reproduce the output of the ● The choice of the inlet type and injection
detector and record the result called a technique depends on the type of sample
chromatogram and solvent matrix.
● The signal comes directly from the ● Dissolved samples are introduced directly
detector and usually goes through the onto the column via COC injector
attenuator which changes the ● If a solvent matrix has to be vaporized and
amplification. partially removed, a S/SL injector is used
● Gaseous samples are usually injected
using a gas switching valve system.
GC SYSTEM: COMPONENTS AND FUNCTIONS
COLUMN OR COLUMN OVEN
CARRIER GAS TANK
● The “heart” of the GC system, it is
● A chamber made of stainless steel that
contained in an oven, the temperature of
supplies the carrier gas needed in the
which is precisely controlled.
analysis
● Its sole function is to maintain the
constancy and uniformity of the column
PRESSURE REGULATOR
temperature at the desired value.
● A suitable two-stage diaphragm controlled
● An airbath is used to maintain this
pressure regulator that reduces the
requirement.
pressure level compatible with the
requirement of the instrument
COLUMN OR COLUMN OVEN
● Column temperature is selected to
FLOW CONTROLLER
compromise between the length of the
● Contained within a thermostated chamber
analysis and the level of separation
capable of maintaining a constant
● The rate at which a sample passes
temperature as high as 400C
through the column is directly proportional
● Controls gas flow rate, the gas flow rate is
to the temperature of the column.
selected to compromise between the
● The higher the column temperature, the
length of the analysis and the level of
faster the sample moves through the
separation
column.
● The rate at which a sample passes
● However, the faster a sample moves
through the column is directly proportional
through the column, the less it interacts
to the gas flow rate in the column
with the stationary phase and the less
● The higher the flow rates the faster the
analytes are separated.
analysis, but the lower the separation
between analytes.
DETECTOR
● A number of detectors are used in GC
INJECTOR PORT
The most common detectors are the following:
● A small chamber where the sample is
● Thermal Conductivity Detector [TCD]
introduce into the system
- A universal detector
● The primary requirement of the injection
- Simple, inexpensive and
system is that the sample be vaporized
nondestructible to the sample
instantaneously so that a narrow band of
vapor is introduced into the beginning of
● Flame Ionization Detector [FID]
the column
- Highly sensitive detector
● It is equipped with a self-sealing septum
- Almost a universal detector
made of rubber or silicone to prevent the
sample from escaping
 LEC PHARM ANALYSIS MODULE 4 & 5
RETENTION TIME [Rt]
● The time required by an average molecule
of component to pass from the injector port
through the column to the detector
RETENTION VOLUME [Rv]
● The volume of the carrier gas necessary to
carry an average molecule of the
component from the point of injection to
the detector
ADVANTAGES OF GAS CHROMATOGRAPHY
➔ Fast analysis
➔ Efficient, proving high resolution
➔ Sensitive
➔ Non-destructive
➔ Highly accurate quantitative analysis
➔ Requires small samples
➔ Reliable,, relatively simple and
inexpensive
➔ GC can be run 1,000 times faster than lc.
➔ Larger preparative GLC's can be used for
purification of samples.
DISADVANTAGES OF GAS
CHROMATOGRAPHY
➔ Limited to volatile samples
➔ Not suitable for thermally labile samples
➔ Fairly difficult for large, preparative
samples.
➔ Requires elaborate instrument such as
mass spectroscopy, for confirmation of
peak identity.
APPLICATION OF GAS CHROMATOGRAPHY
➔ Limit test for solvent residues and other
volatile impurities in drug substances
➔ Characterization of volatile oils.
➔ In analytical chemistry and biochemistry
➔ In petrochemical environmental monitoring
➔ In chemistry research
➔ Measurement of toxic substances in air,
water and soil
➔ In drug quality control, to assure the
quantitative/qualitative features of
pharmaceuticals
➔ Assay of volatile oil content
HIGH-PERFORMANCE CHROMATOGRAPHY
High Performance Liquid Chromatography /High
pressure liquid chromatography
● It is a form of column chromatography used
frequently in biochemistry and analytical
chemistry to separate, identify and quantify
compounds.
● Advantages - greater speed, precision,
accuracy, ease of separation
● Prerequisite Prior to HPLC Analysis -
Sample must be soluble with solvent
● Most advance chromatographic method
● Most commonly used analytical method in drug
testing
● Results are obtained in the recorder chart
showing the peak in the chromatogram
Applications:
1)Nonvolatile substances,
2)Substances with high polarity or highly tonic,
3)Substances with high molecular weight and
4)Thermally unstable and decomposable
substances
APPLICATION FIELDS OF HPLC
1. Organic chemistry - Analytical &
preparative
2. Pharmaceutical chemistry - Analytic &
preparative
3. Clinical chemistry - Analytic
4. Forensic chemistry - Analytic
5. Food chemistry - Analytic & preparative
6. Cosmetics - Analytic & preparative
7. Biochemistry - Analytic & preparative
8. Polymer chemistry - Analytic &
preparative
9. Environmental chemistry - Analytic &
preparative
TYPICAL ANALYSES
1. Aflatoxin
2. Aldehydes
3. Amines/amino acids
4. Antibiotics
5. Antioxidants/preservatives
6. Carboxylic acid
7. Dyestuff
8. Fatty acids/ F.A. esters/lipids
 LEC PHARM ANALYSIS MODULE 4 & 5

9. Food additives / Artificial fatterners 2. PNEUMATIC - which produces a constant

10. Flavourings / Aroma compound /
Constituents
11. Herbicides/pesticides
12. Hormones/steroids
13. Mycotoxins
14. Nitrates/Nitroamines
15. Plasticizers/Phthalate
16. Polyaromatics/polychlorinatead biphenyl
17. Proteins
18. Sugars/Carbohydrates/sugar substitute
19. Vitamins
20. Water
21. Xanthines/purines
MOBILE PHASE
● Aqueous and non-aqueous solvents may be
used.
● Solvents must be degassed before they can
be used or the detector will absorb bubbles
and erroneous readings will be obtained
● Degassing is the best accomplished by
subjecting the solution to a vacuum system
containing suitable traps.
pressure
INJECTOR
● The sample solution is injected through a
self-sealing rubber or Teflon disc using a
microliter syringe.
HPLC COLUMN
● Guard column - this contain solid support
coated with higher percent of liquid phase
than the analytical column in order to
saturate the mobile phase and retard
dissolution
ANALYTICAL COLUMN
● In which the actual separation takes place,
is a stainless steel tube, usually, 25 cm in
length with an internal diameter of 2 to 4.6
mm
DETECTOR
● Measures the concentration of the sample
injected on the column
● It generates a signal proportional to the
sample concentration

COMPONENTS OF HPLC

HPLC SOLVENT
● Glass or stainless steel containers capable
of holding up to a liter of mobile phase
which may consist of pure organic solvents
or aqueous solutions of salts or buffers

HPLC PUMPS DATA ACQUISITION

● Needed to force the mobile phase through INTEGRATOR
the column ● The main function is to graphically
reproduce the output of the detector and
2 TYPES OF PUMPS record the result called a chromatogram.

1. MECHANICAL - which delivers at a

constant flow rate
 LEC PHARM ANALYSIS MODULE 4 & 5

FORMULA APPLICATION

● pH Measurement
● Measures the activity of H+ rather than the
concentration

POTENTIOMETRIC TITRATION: TYPES

● ACID-BASE TITRATIONS
● REDOX TITRATIONS
● PRECIPITATION TITRATIONS

POTENTIOMETRY FACTORS THAT INFLUENCE THE ACCURACY

● A branch of electrochemistry which deals
with the study and measurement of electrode
potential
● The measurement of electrode potential
finds wide application in pharmaceutical
sciences especially in potentiometric titration
and related measurements
POTENTIOMETRIC TITRATIONS
○ A technique similar to direct titration of a redox
reaction.
○ No indicator is use; instead the voltage across
the analyte, typically an electrolyte solution is
measured.
○ The monograph in the USP and NF usually
designate the electrode pair which is to be
used for a particular potentiometric titration.
○ For the purpose of measuring pH of
pharmaceutical solutions and products, a
properly standardized pH meter equipped with
glass and calomel electrodes may be used.
○ Potentiometry is a branch of electro-chemistry
which deals with the study and measurement
of electrode potential (E)
○ Potentiometric method gives the pH values
using a commercially available pH meter
○ The measured potential may be used to
determine the analytical quantity of interest,
generally the concentration of some
component of the analyte solution.
OF INDICATOR TITRATION
● Lighting conditions
● Color or turbidity of the solution
● Color blindness
● Fatigue of the operator
● Indicator or end point techniques may not
be available.
● Potentiometric is a technique for detecting
tthe endpoint of titration where there is a
change in the concentration of the reactants
and thus bug shift in the electrode potential.
● It involves the measurement of the changes
in the EMF of the cell brought about by
adding a titrant, i.e the monitoring of the
potential serves only to locate the
equivalence point for a titration.
ELECTRODES USED
For the purpose of measuring pH of pharmaceutical
solutions and products, a properly standardized pH
meter equipped with glass and calomel electrodes
may be used.
REFERENCE ELECTRODES USED IN
POTENTIOMETRY
ELECTRODE USE/DESCRIPTION
Hydrogen electrode Ultimate reference
(SHE) electrode

Calomel electrode Commonly used

(SCE) reference electrode

Ag/AgCl electrode Reference electrode

 LEC PHARM ANALYSIS MODULE 4 & 5

DC POLAROGRAPHY
INDICATOR ELECTRODES USED IN
POTENTIOMETRY
● Also known as “conventional polarography”
Electrode Use/Description
Simplest method consisting of:
An electrode that relies
on a glass membrane, Reference Electrode:Saturated calomel electrode
Membrane electrode
measures a potential
(Ex: pH electrode) ● Acts to maintain a constant potential
difference across a
membrane throughout the measurement

Similar to a membrane Indicator Electrode: Dropping mercury electrode

electrode except that
the membrane is an ● Assumes the potential impressed upon it
organic polymer from external source
Liquid membrane saturated with a liquid
electrode (Ex: Ca2+ ion exchanger, FOLLOWING PARTS:
electrode) measures potential
difference upon 1. ELECTROLYTIC CELL
interaction of the - An electronic chemical vessels where
exchanger with target experiments are performed and it contains
ions
electrolyte
A very popular type of
Solid state electrode 2. INDICATOR ELECTRODE
ion specific electrode
- It is a mean of converting the current to a
Measures a change in measurable signal
pH, it is a normal pH
electrode coated with a
urease impregnated 3. REFERENCE ELECTRODE
Enzyme electrode gel; urea will permeate - Electrode measuring electric potential of the
the gel where the working electrode, usually a saturated
enzyme will attack calomel electrode.
resulting to the
formation of ammonium 4. SUPPORTING ELECTRODE
Measures an - Added to the solution to suppress the
equilibrium change migration of electroactive species towards
Gas sensing electrode upon permeation of the the electrodes by electrostatic attraction
target analyte through a Ex. KCl
permeable membrane
POLAGRAM

POLAROGRAPHY ● A plot of current flowing in the cell as a

function of the applied potential.
○ A method of analysis based on the
measurement of current resulting from the
electrolysis of an electroactive species at a
given electrode potential under controlled
conditions
 LEC PHARM ANALYSIS MODULE 4 & 5
● Half-wave potential is the potential at which
half of the maximum current is reached and
is characteristic of a given analyte in a given
medium and thus used for qualitative
analysis.
● For quantitative analysis, the diffusion
current is proportional to concentration of
analyte. Id is measured from the base line
recorded without analyte
● Residual current is due t o reduction
reactions of impurities in solution and
charging of Hg drop
THE SHAPE OF POLAROGRAM DEPENS ON
THE:
A. Method of analysis selected
B. Type of the indicator electrode
➢ Dropping mercury electrode (DME)
- which is use for reduction reactions
➢ Platinum electrode (stationary or
rotating) - is used for oxidation
reactions
C. Potential ramp that is applied.
METHODS OF POLAROGRAPHY
● Classical or linear sweep voltammetry
● Cyclic voltammetry or triangular wave
● Differential pulse polarography
● DC polarography or pulse
APPLICATIONS
● Water analysis of natural, waste and fresh
water
● Determination of nitratem nitrite, chloride,
iodide, cyanide, oxygen.
● Determination of organic and toxic
materials such as herbicides,pesticides
WATER CONTENT DETERMINATION
● Used to ensure uniformity of water contentt
limts in the drug. Drugs official in the USP
and N.F contain varying quantities of water
either as water of crystalization or as water in
the adsorbed form.
METHODS OF WATER CONTENT
DETERMINATION
GRAVIMETRIC
- For drugs containing no constituents other
than water, volatile at 105 C
AZEOTRPIC OR TOLUENE DISTILLATION IN
THE NF
XYLENE DISTILLATION IN THE USP
KARL FISCHER ELECTROMETRIC TITRATION
METHOD.
Reagent: Iodine, sulfure dioxide, pyridine and
methanol
● Uses coulometric or volumetric titration to
determine trace amounts of water in the
sample
● Invented in 1935 by german chemist Karl
fischer
● Widely applicable in the determination of
water content
ADVANTAGES
➔ Most rapid of the official methods
➔ Requires a small sample
➔ Specific for water
➔ High accuracy & precision
➔ Easy sample preparation
➔ Short time o f analysis
➔ Nearly unlimited measuring range
➔ Suitability for analysis of all forms of
samples
➔ Independence of presence of other
volatiles
➔ Suitability for automation
 LEC PHARM ANALYSIS MODULE 4 & 5
● Titration of the sample in methanol with
Karl Fischer reagent
COMPONENTS
➢ Pyridine, Iodine, Methanol, Sulfur dioxide
● Karl Fischer Reagent should be freshly
prepared and standardized with sodium
tartrate
KARL FISCHER PRINCIPLE
● Karl fischer discovered that this reaction
can b e modified to be used in water
content determination (in an aqueous
system) containing a n excess of sulfur
dioxide.
● It is based on the bunsen reaction
between iodine and sulfur dioxide in an
aqueous medium.
● He used Primary 1 Alcohol (methanol) as
a sovent and a base (pyridine) as buffering
agent.
FORMULA FOR KARL FISCHER
ADVANTAGE OF KF METHOD TO LOD
METHOD
● Loss on drying (LOD) will only detect any
volatile substance.
● KF method is very specific for water
detection
LOSS ON DRYING (LOD)
● A classic laboratory method of measurement
of high level moisture in solid or semisolid
materials.
● A relatively slow method of analysis
● A sample of the material is weighed, heated
in an oven, cooled in a dry atmosphere of a
dessicator, then re-weighed.
● If the volatile content of the sample is
primarily water, the LOD gives a good
measure of water conten
POLARIMETRY
● Deals with the study of the rotatory power of
substances
● Technique of measuring the polarization of light
● Measurement of optical rotation or optical
activity of a substance
● Measures the extent to which a substance
interacts with plane polarize light
● Rotatory Power - also known as “optical
activity”
○ Dextrorotatory – when the direction of the
rotation is toward the right [+]
○ Levorotatory – when the direction of the
rotation is toward the left [-]
Importance; Establish identity of substances,
Establish purity of samples, Indication of its
therapeutic value
Instruments:
○ Polariscope or Polarimeter – used for
liquid substances
○ Polarizing Microscope - used for solid
substances
 LEC PHARM ANALYSIS MODULE 4 & 5

OPTICAL ROTATION
APPLICATION OF POLARIMETRY

● Rotation of linearly polarized light as it ● Establish both its identity and purity
travels through certain materials. ● Indication of its therapeutic value

IT OCCURS IN SOLUTIONS OF CHIRAL ANGLE OF ROTATION

MOLECULES;

1. SUCROSE (SUGAR), PEPTIDES, VOLATILE ● The concentration of the solution

OILS ● The pathlength of the solution through which
2. SOLIDSW/ ROTATED CRYSTAL PLANES the light travels
(QUARTZ) ● The wavelength of light
3. SPIN-POLARIZED GASES OF ATOMS AND ● The chemical configuration of the substance.
MOLECULES
FACTORS THAT AFFECT THE ANGLE OF
EXAMPLES OF ORGANIC SUBSTANCES THAT ROTATION
ARE OPTICALLY ACTIVE
● The concentration of the optically active
● Volatile oils substance
● Alkaloids ● Solvent
● Sugars ● Temperature (25℃)
Specific rotation of official sucrose NLT +65.9 ● Wavelength of the polarized light
● Nature of the substance
● Length of sample tube
OPTICALLY ROTATION APPLICATION

● In the sugar industry – to measure syrup PARTS OF POLARIMETER

concentration
● In optics – to manipulate polarization
● In chemistry – to characterize substances
in solution
● In optical mineralogy – identify certain
minerals,
● In thin sections in medicine – to
measure blood-sugar levels in diabetic
patients
OPTICAL ROTATION OF SUGARS
INVERT SUGAR SYRUP
- Gets its name from the fact that the
conversion causes the direction of rotation
to “invert” from right to left.
GLUCOSE / DEXTROSE
- It causes linearly polarized light to rotate to
the right or to the dexter side
FRUCTOSE
- It is more levorotatory than glucose is
dextrorotatory.
● LIGHT SOURCE
- Sodium lamp: 589 or 590 nm
● POLARIZING PRISM
- Polarizes the original light source
or plane-polarized
● OBSERVATION TUBE OR
POLARIMETER TUBE
- Contains the substance being
examined, usually consist of atube
of thick glass with accurately
ground ends, closed by circular
glass plates with parallel sides,
which are pressed against the ends
of the tube by means of scew caps.
➢ Since the unit of length, in polarimetric
measurements is 1 dm, the observation
tubes are generally 1 dm length
➢ The length of the tube used in any definite
determination usually depends upon the
depth of color in the liquid and the
magnitude of its power
➢ It is usually 10 or 20 cm lolng
➢ It may be cleaned with water, alcohol nad
ether
● ANALYZER PRIMS
- Used to examine the polarized light
after it passes through a solution or
 LEC PHARM ANALYSIS MODULE 4 & 5
the optically active species.
- Analyzer prism is rotated to permit
maximum passage of light and is
then and is then said to be lined up
- Thee degree (angle) of rotation (∝)
is measured
PRISM
● POLARIZER AND ANALYZER
➢ Calcite prism polarizers (Nicol prsm,
iceland spar) are widely used as polarizer
and analyzer in the visible range.
➢ Reflection polarizers (Stacks of 3-6
selenium films or silver chloride plates
place at the polarizing angle are commonly
used in the IR)
● VIEWER
● TELESCOPE
- Where the field of view is observed
● DETECTOR
- Photoelectric and photoconductive
detectors are widely used in the
visible and UV
SPECIFIC ROTATION OF A LIQUID
● It is defined as the angular rotation in
degrees through which the plane
polarization of polarized monochromatic
light is rotated by passage through 1 dm of
the liquid, calculated on the basis of
specific gravity of 1
● Temperature specified in official standards
for the measurement of optically activity is
25 C
REFRACTOMETRY
○ It deals with the study and measurement of
index of refraction of substances in order to
assess their composition of purity
○ A technique that measures how light is bent /
refracted, when it passes through a liquid
substance
○ The amount by which light is refracted
determines its “REFRACTIVE INDEX.”
Importance
○ To determine the identity of an unknown
substance based on its refractive index
○ To assess the purity of a particular substance
○ To determine the concentration of one
substance dissolved in another
Index of Refraction/“Refractive index”
○ It is defined as the ratio of velocity of light in air
to the velocity of light in the medium or the
ration of the sine of angle of incidence to the
sine of angle of refraction
○ The values vary with temperature and
wavelength, so they should be held constant.
● It is valuable in the identification of a
substance and the detection of impurities
● Refractive index is temperature dependent
FACTORS THAT AFFECT THE REFRACTIVE
INDEX
TEMPERATURE
- It is inversely proportional to the refractive
index
WAVELENGTH
- The sodium D-line at 589 nm (yellow light)
is the appropriate wavelength for the
determination of the refractive index
VISCOSITY
- It is inversely proportional to the refractive
index
★ Calibrated using distilled water (1.3325 at
250℃)
★ Temperature selected for measurement is at
25 C
★ D line of sodium is referred to as the
wavelength
REFRACTOMETER
 LEC PHARM ANALYSIS MODULE 4 & 5

○ An optical instrument used to measure the

Lower scale: FOR SOLID
refractive index - 0 to 85%
○ They are best known for measuring refractive ★ The scales are visible through the
indices of liquids but they also measure RI of instrument eyepiece when the power/lamp
gases and solids [such as glass and selector switch is depressed to its inner
gemstones] position
★ The eyepiece should be focused for the
best image of the reticle and scales

★ Turning the handwheel on the right hand

side of the instrument moves the scales
PARTS OF ABBE REFRACTOMETER and (total reflection) across the reticle
1. Refracting prism assembly with
illuminator
★ Upper prism case - Contains illuminating
prism
★ Lower prism case - Contains the
measuring prism and acts as sample
compartment
★ Prism field lamp - Provides the required
illumination (white light) located at the end of
the adjustable arm
★ Hinged lightshield - prevents stray light
from entering the front of the measuring
prism
2. An internal measurement scale
★ Eyepiece - Used to observe both the total 3. A compensating prism system
reflection borderline and the instrument’s ➢ If light source is not monochromatic
internal measurement light gets dispersed (blurred)
➢ To prevent dispersion, 2 amici
prisms were added to the design.
FINE ADJUSTMENT KNOB
- Removes the rainbow color seen in the
viewing film
COARSE ADJUSTMENT KNOB/HANDWHEEL
- Sets the total reflection borderline exactly
at cross hair intersection.

★ Measurement scale - The instrument

contains 2 coincident scales which read
from 1.30 to 1.71 𝑛𝐷 and from 0-85% total
dissolved solids.
Upper scale: FOR LIQUID
- 1.30 to 1.71 𝑛𝐷
LEC PHARM ANALYSIS MODULE 4 & 5