CHROMATOGRAPHY

Principle

Chromatography is based on the principle of separation of solute between two phases. It generally
contains a mobile phase and a stationary phase.

Mobile phase- Mixture of substances to be separated dissolved in a liquid or gas.

Stationary phase-A porous solid/liquid matrix through which the sample contained in the mobile
phase percolates.

The interaction of the substances in the mixture between the mobile and stationary phase leads to
the separation of compounds

These interactions maybe adsorption, partition, ion exchange, molecular sieving or affinity.

Chromatogram

A graphical representation of detector response of

concentration of analyte in the effluent or other
quantity used as a measure of effluent
concentration. Data represented by the
chromatogram helps to identify and quantify the
solute. When the eluting solute is displayed
graphically as a series of peaks they are called as
chromatographic peaks.

Types of chromatography
➢ Paper chromatography
➢ Thin layer chromatography
➢ Column chromatography
➢ High performance liquid chromatography
➢ Ion exchange chromatography
➢ Gel permeation
 ➢ Gas chromatography (Gas-liquid/Gas solid)
➢ Affinity chromatography
Factors affecting selection of technique:
✓ Polarity of sample
✓ Solubility and volatility of sample
✓ Resolution required
✓ Concentration of analyte
✓ Detection limit
✓ Physical and chemical properties of sample
Chromatography is used to:
Analyze:
Identify:
Purify:
Quantify:
General applications of chromatography
✓ Separation of amino acids, proteins, carbohydrates
✓ Analysis of drugs, hormones and vitamins.
✓ Qualitative and quantitative analysis of complex mixtures
✓ Molecular weight of proteins
✓ Food and beverages
✓ Criminal forensics
✓ Environmental monitoring
Advantage of chromatography over other methods
➢ Separation of compounds with closer chemical and physical properties
➢ Simple method
➢ Fast and efficient technique
➢ Low concentrations can be separated.
➢ Economical
➢ Reproducible and accurate
➢ High sensitivity

Paper Chromatography
Principle:It is a type of planar chromatography in which the paper is the support for the stationary
phase.
The mechanism of separation is partition. It is dependent on the polarity of the solvent and the
solubility of the mixture in the liquid phase.
It is liquid-liquid chromatography. (LLC)
Stationary phase: Water held in the network of cellulose fibers
Mobile phase: Liquid- Solvent (single or mixture)
As the non-aqueous mobile phase moves along the paper, the components of the mixture distribute
themselves between the two phases in a ratio characteristic of their distribution coefficient.
The component which is more soluble in the stationary phase moves more slowly while the
component which is more soluble in the mobile phase moves faster.
As a result of the differential movement, the components get separated.
Distribution coefficient = solute in stationary phase
solute in mobile phase
Quantitative analysis:
Direct method- Comparison of visible spots, photo densitometry, Fluorimetry, radio tracer
Indirect method- spot cut and eluted with solvents and analyzed by spectrophotometry,
electrochemical method etc.
Applications:
❖ Separation of amino acids, peptides, alkaloids, sugars, lipids etc.
❖ Structure and amino acid composition of proteins
❖ Analysis of blood, hemoglobin, urine.
Advantages:
 ✓ Simple, inexpensive, easy to handle
✓ Separation can be carried out under lab conditions
Disadvantages:
✓ Time consuming
✓ Cannot be use as preparative method
THIN LAYER CHROMATOGRAPHY
✓ The separation of moderately volatile or nonvolatile substances based on differential
adsorption on an inert solid (stationary phase) immersed in an organic solvent or solvent
mixture (mobile phase).

✓ Mechanism of separation is adsorption or partition

✓ The components with more affinity to the stationary phase move slower. Components with
less affinity to stationary phase move faster.

Quantitative analysis:
Direct methods
➢ Visual assessment
➢ Measurement of spot area
➢ Densitometry
➢ Spectrophotometry
Indirect methods
➢ Gravimetry
➢ Fluorimetry
➢ Colorimetry
➢ Calorimetry
➢ Flame photometry
Stationary phase: Silica Gel, Alumina, cellulose powder
Selection of adsorbent:
✓ Solubility of compound- hydrophilic/ lipophilic.
✓ Nature of substance to be separated- acidic, basic or amphoteric
✓ Adsorbent particle size
✓ Reactivity of compound with solvent/adsorbent
✓ Reactivity with binders
Commonly used solvents: (increasing polarity)
n-hexane, cyclohexene, toluene, benzene, diethyl ether, chloroform, dichloromethane, acetone, ethyl
acetate, acetonitrile, propanol, methanol, acetic acid, water
Selection of solvent system:
✓ Adequate purity
✓ Stability
✓ Low viscosity
✓ Neither too high or low vapour pressure
✓ Low toxicity
Applications:
✓ Purity of samples
✓ Identification of organic compounds
✓ Separation of amino acids
✓ Separation of inorganic ions
✓ Kinetics of chemical reaction
✓ Pharmaceuticals and drugs
✓ Clinical chemistry, forensic chemistry and biochemistry
✓ Cosmetology
✓ Food analysis
✓ Environmental analysis
Advantages of TLC over paper chromatography
✓ Wider choice of stationary phase (adsorbents)
✓ Adsorbent layer of variable thickness and thus, variable sample load.
 ✓ Faster process (TLC-1 hr, PC-18-24 hrs)
✓ Separated spots are compact and detection at lower concentrations possible.
✓ Since it is simple, rapid and inexpensive, it can be used to check the purity of synthesized and
isolated chemicals.
Normal Phase TLC: Stationary phase is polar (silica gel) and mobile phase is an organic solvent or
mixture which is less polar.
Reverse Phase TLC: Stationary phase is silica bonded with organic substrate (long chain aliphatic acid
like C-18) and mobile phase is a mixture of water and organic solvent which is more polar than
stationary phase.
COLUMN CHROMATOGRAPHY
It is a separation technique in which components of a mixture are separated by
using a glass column packed with a stationary phase and the liquid mobile phase
flowing continuously through the column.
First chromatographic column developed by Mikhail Tsvet in 1901.
Types of column chromatography:
➢ Gravity column chromatography
➢ Flash column chromatography
Principle:
Mechanism of separation is Adsorption or partition
The sample mixture is mixed into the mobile phase and added into the column
Components travel due to their relative affinities
A component more soluble in the mobile phase will move rapidly through the
column while a component strongly attracted to the stationary phase will move more slowly.
Experimental:
Adsorbents:
Weak- Sucrose, starch, talc, sodium carbonate
Medium- Calcium carbonate, calcium phosphate, Magnesium carbonate, magnesium oxide, calcium
hydroxide.
Strong- Activated silica gel, alumina, charcoal, magnesia, Fuller’s earth.
Alumina is suitable for less polar compounds while silica gel is used for compounds with polar
functional groups.
Particle size is generally in the range of 50-220 um.
Weak adsorbents: Used for few components, different affinities, longer column
Strong adsorbents: More components, similar affinities, shorter columns.
Characteristics of the adsorbent:
✓ Particles should be spherical and uniform in size
✓ High mechanical stability
✓ Not chemically reactive
✓ Useful for a wide variety of compounds
✓ Economical
Solvent used:
✓ Choice of solvent depends on solubility of the components of the mixture. The must have
low boiling point for recovery of the eluted material.
✓ Polarity is taken into account for adsorption.
✓ Used in pure form or mixture of solvents.
✓
✓ Increasing polarity or elution strength:
✓ Cyclohexane <Carbondisulphide < ether < Benzene < toluene < Ester < Alcohol < Chloroform
< Acetone < Water < pyridine < organic acids.
Column:
Material- Generally glass
Dimensions: 10:1, 30:1, 100:1 (length:diameter)
Length of column depends on:
o Number of components to be separated
o Type of adsorbent used
 o Quantity of sample
o Affinity of compound towards adsorbent used.
Better separation with long narrow column than short thick column.
Column Preparation:
Long glass tube packed at the base with cotton wool/glass wool.

Dry packing- Adsorbent packed in the dry powder form after which the solvent is filled till
equilibrium is reached. Tapping done to remove void spaces.
Disadvantage- Air bubbles may get trapped and cracks may appear.
Wet packing- A slurry of the adsorbent is prepared with the solvent and added into the column. The
solid settles down and the excess solvent can be removed. No cracks appear.
Factors affecting column efficiency:
✓ Dimensions of column
✓ Particle size of column packing
✓ Activity of adsorbent
✓ Temperature
✓ Nature of solvent
Development (Elution):
✓ Isocratic elution- The same solvent composition/ solvent of same polarity is used throughout
the process
✓ Gradient elution- Solvents of increasing polarity or increasing elution strength used in the
process.
Detection of components:
➢ If the components are coloured, detected visually.
➢ If colourless, the eluted mobile phase is collected in small fractions sequentially and each
fraction is analyzed.
➢ Spot tests on paper or TLC
➢ By passing UV light
➢ Addition of reagents
Partition chromatography

Applications:
✓ Separation of mixtures
✓ Purification of compounds
✓ Isolation of active constituents
✓ Estimation of drugs in formulations
✓ Separation of diastereomers
✓ Isolation of metabolites from biological fluids
Advantages:
➢ Any type of mixture can be separated
➢ Any quantity of mixture
➢ Wider choice of mobile phase
➢ In preparative, sample can be separated and reused.
➢ Automation is possible
Disadvantages:
➢ Time consuming
➢ Quantity of solvents required is large
➢ Drying and presence of bubbles is possible in the column.
 ➢ Automation can make the technique complicated and expensive.

HPLC- High Performance liquid chromatography Also called as High Precision/Pressure liquid
chromatography
HPLC is a type of column chromatography which pumps a sample mixture or an analyte in a solvent
(mobile phase) at high pressure through a column with chromatographic packing material (stationary
phase)
Term coined by Prof. Csaba Horvath in 1970.
Normal phase HPLC: The separation is based on polarity.
Polar stationary phase and non-polar mobile phase.
Generally, stationary phase is silica and mobile phase is hexane, methylene chloride, chloroform,
diethyl ether or a mixture.
Polar substances are retained on the polar surface of column packing.
Reverse phase HPLC:
Stationary phase is non-polar (hydrophobic) mobile phase is polar such as water, methanol,
acetonitrile.
Due to hydrophobic interactions, the non polar substance will be retained longer.
PARTS of HPLC
Solvent reservoir:
In NP-HPLC non-polar solvents like hexane, heptane, iso-octane are used in combination with slightly
polar solvents like isopropanol, ethyl acetate or chloroform. Retention increases as the amount of
non-polar solvent increases in mobile phase.
In RP-HPLC- Water is the base solvent along with other polar solvents like methanol, acetonitrile,
tetrahydrofuran. pH is adjusted by buffers.
Degasser
The presence of air bubble may block the flow of the mobile phase or make its flow erratic which
would cause problem in the retention time.
Air is an optical detector (UV, fluorescence, RI) which will scatter the light causing noise spikes.
Solvent delivery system (PUMP)
The role of the pump is to force the mobile phase at a specific flow rate (ml/min). Normal flow rate is
1-3 ml/min, pressure about 2000 psi. They can reach a pressure upto 6000-9000 psi.
The pump can deliver a constant mobile phase composition (isocratic) or increasing mobile phase
composition (gradient).
Types of pumps:
Pneumatic- Mobile phase driven through the column using pressure produced form a gas cylinder.
Syringe type- syringe like plunger with activated by screw mechanism
Reciprocating- Transmits alternative pressure to the solvent via flexible diaphragm which is
hydraulically pumped.
Injector:
Manual- User loads the sample manually using a syringe and then turn the handle to inject the
sample into the mobile phase which transfer the sample to the head of the column at high pressure.
Auto sampler- Vials are loaded with sample solution in the auto sampler tray (100 samples) which
automatically measure the sample volume, injects, flushes until all the samples are processed.
Sample volume- 0.1 – 500 uL.
Column:
Made of polished steel, 50-300 mm length, internal diameter 2-5 mm. Stationary phase with particle
size of 3-10 um. Temperature of column and mobile phase must be maintained constant.
Normal phase columns- Porous silica gel, polymer gel
Reverse phase column- Silica gel coated with ODS, C18
Detectors:
➢ UV detector
➢ Fluorescence detector
➢ Refractive index detector
➢ Electrochemical detector
➢ Mass spectrometer
Recorder
A recorder draws the chromatogram and gives a visual representation. Time scale of chart movement
is from 1 cm/sec to 1cm/hr.
Retention time:
Time taken for the sample to travel through the column to the detector.
Time at which the sample is injected to the point on the display where it shows the maximum peak

height.
Advantages:
➢ Simple, rapid, reproducible
➢ High sensitivity
➢ Less requirement of stationary phase
➢ Used for qualitative and quantitative analysis
➢ Variety of stationary phases
➢ High resolution and separation capacity
Disadvantages:
➢ Cost
➢ Complexity
➢ Low sensitivity for some compounds
➢ Irreversibly adsorbed compounds not detected
➢ Co-elution difficult to detect
Applications:
✓ Pharmaceutical applications- Drug stability, dissolution of pharmaceutical dosage forms,
quality control
✓ Environmental applications- phenolic compounds in water, bio-monitoring of pollutants
✓ Food industry- Quality of water and soft drinks, sugar analysis in fruit juices, polycyclic
compounds in vegetables, preservative analysis.
✓ Clinical tests- Urine analysis, antibiotic analysis, bilirubin in hepatic disorders,
✓ Forensic- drugs in biological samples, steroids in blood and urine, forensic analysis of textile
dyes, cocaine in drug abuse.
GC
Types:
Gas-Solid chromatography: The mobile phase is gas, and the stationary phase is a solid.
Used for separation of low molecular weight gases example, H2S, CO2, oxides of nitrogen, CO.
Gas-Liquid chromatography: The mobile phase is a gas while the stationary phase is a liquid retained
on a surface as inert solid by adsorption/ chemical bonding.
Principle:
The mixture to be separated is converted into vapour phase and mixed with gaseous mobile phase.
The components which are more soluble in the stationary phase travel slower and eluted later while
the components which are less soluble travel faster and eluted first.
The components are separated based on their partition coefficient.
Partition coefficient- The ratio of solubility of a substance distributed between two immiscible liquids
at constant temperature.
Carrier Gas:
The cylinder is fitted with a pressure controller to control the pressure of the gas, a pressure gauze to
indicate the pressure, a molecular sieve to transfer filtered dry gas and a flow regulator to ensure
constant rate of flow of mobile phase to the column.
Characteristics:
➢ Chemically inert
➢ Cheap and readily available
➢ Good quality and not cause fire hazards
➢ Suitable for sample to be analyzed and detector also
Gases used: Hydrogen, Nitrogen, Helium
Inlet pressure: 10-50 psi
Flow rate: 25-150 ml/min (packed column) 2-25 (open tubular)
Injection port:
Attached to the column head
Port is placed in an oven maintained at 20-50ᵒC above boiling point of sample, to maintain the
sample in a vapourized state.
For gaseous samples- tight hypodermic syringe of 0.5-10 ml capacity. For liquid samples, 0.1 -100 uL
capacity.
Types of injection port:
➢ Split injection
➢ Splitless injector
➢ On column injector
➢ Automatic injector
Column:
➢ Different shapes and sizes- U tube or coiled helix
➢ Made of copper, stainless steel, aluminum, glass, nylon and other synthetic plastics.
Support material
➢ Provides mechanical support to liquid phase. It must have large surface area, chemically
inert, uniformly wet with liquid phase, thermostable.
➢ Commonly used solid support: Diatomaceous earth or Kieselguhr, glass beads, porous
polymers, sand etc.
Characteristics of liquid phase:
✓ Non-volatile
✓ High decomposition temperature
✓ Chemically inert
✓ Low vapour pressure at column temperature
✓ Chemically and structurally similar to solute (polar for polar solute)
Non polar hydrocarbon phases- Paraffin oil (nujol), silicon oil, silicon rubber gum.
Polar compounds- Polyglycols
Liquids having hydrogen bonding- Glycol, glycerol, hydroxy acids.
Types of columns:
1) Packed columns- In GLC, densely packed with finely divided, inert, solid support material coated
with liquid stationary phase.
In GSC, packed with adsorbents or porous polymers.
Length: 1.5 -10 m Internal diameter: 2-4 mm
2) Capillary column:
Length: 10-100 m Internal diameter: 0.1-0.5 mm
Wall coated column (WCOT)
Support coated column (SCOT)
Equilibration of column:
Continuous flow of heated carrier gas for specific duration of time at given temperature.
Column temperature:
Maintained by jackets equipped with vapours of boiling liquid, electrically heated metal blocks, or
circulating air baths.
Compounds of low B Pt eluted at lower temperature
Compounds of higher B Pt boil at higher temperature giving broader and shallower peaks
(temperature programming required)
Detectors:
Characteristics of a detector:
➢ Sensitive
➢ Operate at high temperature (upto 400ºC)
➢ Stable and reproducible
➢ Linear response
➢ Simple
➢ Reliable
➢ Fast response
➢ Non-destructive
➢ Uniform response to all analytes
1) Thermal conductivity detector
➢ Advantages- Simple, inexpensive, durable, long life, accurate, non-selective
➢ Disadvantages- Low sensitivity, affect by temperature fluctuation
2) Electron capture detector

➢ Advantages-Selective, sensitive, non-

destructive,
➢ Disadvantages- Less sensitive to compounds
which have less affinity to electrons, carrier gas
must be pure.
3) Flame ionization detector- analytes combust in an air-hydrogen flame producing carbon ions which
induce current
4) Flame photometric detector (S/P)
Applications:
✓ Qualitative analysis
✓ Quantitative analysis
✓ Pharmaceutical applications
✓ Analysis of food
✓ Analysis of pollutants
✓ Analysis of dairy products
✓ Separation and identification of volatile materials, polymers, paints etc.

Ion exchange chromatography is a process by which a mixture of similarly charged ions can be
separated by using an ion exchange resin which exchanges ions based on their relative affinities.
Principle:
Reversible exchange of ions between ions present in the solution and those present on the ion
exchange resin.
Types of ion exchange:
Cation exchange:
X+ + R-Y → R-X + Y+
Anion exchange
A⁻ + R-B → R-A + B ⁻
 Classification of ion exchange reins:
1) Based on source of resin
Natural:
Cation- Zeolite, clay
Anion- Dolomite
Synthetic:
Inorganic and organic resins
(Polymer matrix- styrene divinyl
benzene)
2) Based on chemical nature/ Functional
group
Strong cation exchange resin- SO3H
Weak cation exchange resin- COOH, OH
Strong anion exchange resin- R3N+, R2HN+
Weak anion exchange resin- NHR, NH2
Ion exchange resin
Two main materials: Styrene divinylbenzene and cellulose

Cation exchange resin Anion exchange resin

Characteristics of ion exchange resin
✓ Chemically stable
✓ Insoluble in commonly used solvents
✓ High degree of cross linking
✓ Swollen resin must be denser than water
✓ Sufficient number of ion exchange groups.
Column material and dimensions
✓ Glass, stainless steel or polymer columns resistant to strong acids and alkalis
✓ Length: diameter 20:1 to 100:1
Type of exchange resin
✓ Type of ions- cation/anion
✓ Nature of ions- strong/weak
✓ Efficiency of resin- Ion exchange capacity
Ion exchange capacity
It is the capacity in terms of exchangeable functional groups expressed as milli equivalents per gram
of the ion exchange resin.
Ion exchange capacity = Normality x Volume/ wt of resin
For cation exchange resin = Nbase x Vbase/wt of resin
For anion exchange resin = Nacid x Vacid/wt of resin
Ion exchange capacity depends on
➢ Particle size of resin
➢ Extent of cross linking
Packing of column- Wet packing
Mobile phase: Generally acids, alkalis or buffers are used Organic solvents are usually not suitable.
Elution:
The process of extracting a substance by washing with a mobile phase with the exchange of ions.
Analysis of elute
▪ Spectrophotometric
▪ Polarographic
▪ Conductometric
▪ Amperometric
▪ Flame photometric
▪ Radiometric
Regeneration of the resin
Cation exchange- Acids (HCl, HNO3)
Anion exchange- Alkali (KOH, NaOH)
Factors affecting ion exchange
Nature and property of resin
Cross linking and swelling
Nature of exchanging ions
Valency
Na+ < Ca2+ < Al3+ < Th4+
Size
Li+ < H + < Na + < K + < Rb + < Cs + <Tl +
F⁻ < OH ⁻ < HCO3 ⁻ < Cl ⁻ < CN ⁻ < Br ⁻
Polarizability
Concentration
(If resin has higher positive charge and solution has lower positive charge exchange is favoured at
higher concentration. If resin has lower positive charge and solution has higher positive charge,
exchange is favoured at lower concentration.)
Advantages:
➢ Cost effective
➢ Reusable
➢ Low maintenance cost
➢ Easy to use
➢ Easy separation
Applications:
✓ Softening of hard water
✓ Conversion of salts
✓ Preparation of deionized water
✓ Separation and purification of metals
✓ Separation of inorganic ions
✓ Separation of additives in drug and food samples
Gel permeation
o Gel permeation chromatography
o Gel chromatography
o Size exclusion chromatography
o Gel filtration
o Molecular sieve chromatography
Principle:
Separation of components is based on the difference in molecular weight and size. Can be used to
analyze biomolecular substances.
Separates molecules in solution by their “effective size” in solution.
The mobile phase flow through a large number of highly porous, rigid particles (stationary phase)
tightly packed in a column
Theory of separation
Vt= Vo + Vi + Vm
Vt= Total volume of the column (can be measured)
Vo = Volume of liquid outside gel matrix
Vi = Volume of liquid inside matrix
Vm= Volume of gel matrix
Components
Stationary phase
Composed of semipermeable, porous polymer gel beads with well defined range of pore sizes.
Properties of gel beads
➢ Chemically inert
➢ Mechanically stable
➢ Ideal and homogenous porous structure
➢ Uniform particle and pore size
Mobile phase
Liquid used to dissolve the biomolecules allowing high detection response and wet the packing
surface.
Synthetic elastomers (polybutadiene, polyisoprene)- Toluene
PS, PVC, styrene-butadiene, Epoxy resin- Tetrahydrofuran
Polyolefins- Trichlorobenzene
Polyurethane- Di-methylformamide
Proteins, polysaccharides- Water/buffers
Columns
Analytical column: 7.5 – 8 mm diameter
Preparative column: 22-25 mm diameter
Usual column length: 25, 30, 50, 60 cm
Narrow bore column: 2-3 mm diameter
Pump:
Syringe pump or reciprocating pump with constant flow rate
Detector:
Concentration sensitive detector
Bulk property detector (RI)
Solute property detector (UV)
Evaporative detector (ELSD)
Molar mass sensitive detector
Light scattering detectors (LALS/MALS)
Viscosity detectors
Procedure:
• Preparation of column
• Loading the sample
• Eluting the sample
• Detection of components

Advantages:
➢ Short analysis time
➢ Well defined separation
➢ Narrow bands
➢ Good sensitivity
➢ No sample loss
➢ Small amount of mobile phase required
➢ Flow rate can be controlled
Disadvantages:
➢ Limited number of peaks resolved in the short time of run
➢ Filtration required for removing interfering particulates
➢ Molecular mass of species is too close for effective separation
Applications:
✓ Protein fractionation
 ✓ Purification
✓ Molecular weight determination
✓ Separation of sugar, protein, peptides, rubbers etc
✓ Quaternary structure of purified proteins.
Electrophoresis
Definition:
Migration of charged particles through a solution under the influence of external electric field. Ions
that are suspended between two electrodes tend to travel towards the electrodes that bear opposite
charges.
Types:
➢ Zone electrophoresis
➢ Frontal/Moving boundary electrophoresis
Zone electrophoresis:
It involves the migration of charged particles on a supporting media. Components are separated into
discrete zones on the support media. Supporting media is saturated with buffer solution, small
volume of sample is applied as narrow band. It includes paper, Cellulose acetate and gel
electrophoresis.
Moving boundary electrophoresis
It allows the charged species to migrate in a free moving solution without a supporting medium.
Instrumentation:
Electrophoretic chamber/Buffer tank
Electrodes (Pt or C)
Diffusion barrier/Buffer- Phosphate, tris-EDTA, Tri-glycine buffer.
Supporting/Stabilizing media- Paper/Cellulose acetate/Gels (starch/agar/polyacrylamide)
Methodology:
➢ Saturation of medium with buffer
➢ Sample application
➢ Electrophoretic separation
➢ Removal of supporting media
Paper electrophoresis
Filter paper- Whatmann No 1/3, 3mm to 5 cm wide, separation in 12-14 hrs
✓ Economical and easy to use
✓ Proteins and hydrophilic molecules cannot be resolved due to adsorption and inorganic
properties leading to tailing and distortion.
Moving boundary electrophoresis

Used for biologically active

fractions
A reference method for
electrophoretic motilities
Expensive and elaborate
optical system required.

Factors affecting migration

of ions:
✓ Strength of electric field
✓ Size and shape of particles
✓ Relative hydrophobicity of sample
✓ Ionic strength and temperature of buffer
✓ Net charge on molecule
✓ Properties of supporting medium
✓ Temperature
✓ pH of solvent
Electrophoretic mobility
Rate of migration in cm/sec per unit field strength (volts/cm)
µ = Q/6πrη
µ- electrophoretic mobility
Q- net charge on ion
r- ionic radius of solute
η - viscosity of the medium
Advantages:
➢ Biochemical investigations
➢ Small quantity of sample can be analyzed
➢ Low cost and easy maintenance
Disadvantages:
➢ Unsuitable for accurate mobility and isoelectric point determination.
➢ Due to the presence of supporting medium capillary flow, electroosmosis, adsorption and
molecular sieving are observed.
Applications:
✓ DNA sequencing
✓ Protein research and purification
✓ Separation of organic acids, alkaloids, amino acids, alcohol, phenol, nucleic acids
✓ Purity of thyroid hormones
✓ Food industry
✓ Diagnosis of diseases
MODULE 2
Electrolytic conductance, terminology & relationships in conductance, variation of
conductance with concentration, Measurement of conductance.
It is an electrochemical method of analysis used for the determination or measurement of electrical
conductance of an electrolyte solution using a conductometer.
Electrical conductivity depends on:
➢ Type of ions
➢ Concentration of ions
➢ Mobility of ions
➢ Temperature
The movement of ions in a solution creates electrical conductivity which is dependent on the
concentration of the ions.
Electrical conductance works according to Ohm’s law which states that, strength of current passing
through a conductor is directly proportional to potential difference and inversely proportional to
resistance. I = V/R
Conductance: Ease with which the current flows per unit area of conductor per unit potential
applied. It is the reciprocal of resistance.
Unit- Siemens or (ohm-1)
Resistance (R): It is the measure of a conductor to oppose the flow of electric current.
Unit- Ohm
Specific resistance (ρ): Resistance offered by a substance of 1 cm length and 1 sq cm surface area.
Unit- Ohm cm ρ = R a/l
Specific conductivity (κv): Conductivity offered by a substance of 1 cm length and 1 sq cm surface
area.
Units- ohm-1 cm-1 κ = 1/ ρ
Equivalent conductivity (λv):
Conductivity of a solution containing equivalent weight of the solute between the electrodes 1 cm
apart and 1 sq cm surface area.
Units- ohm-1 cm-1 or S/m
Equivalent conductivity = specific conductivity (κv )x volume of solution containing 1 g equivalent
weight of electrolyte.
Molar conductivity (µv):
Conductivity of a solution containing molecular weight of the solute between electrodes 1 cm apart
and 1 sq cm surface area.
Units- ohm-1 cm2 mol-1 or S cm2 mol-1
Molar conductivity = specific conductivity (κv ) x volume of solution containing 1 molecular weight of
electrolyte.
Factors affecting conductance
✓ Temperature:
✓ Nature of ions: (size, molecular weight, number of charge carriers)
✓ Concentration of ions:
✓ Size of electrode: (Cell constant)
Effect of dilution:
Specific conductance- Decreases with increase in dilution
Molar conductance- Increases with increase in dilution.

Conductivity cell
✓ Made of pyrex or quartz containing two platinum electrodes.
✓ Should be placed in a vessel containing water to maintain constant temperature.
Electrodes:
✓ Platinum sheets each of 1 cm2 fixed at a distance of 1 cm.
✓ Surface coated with platinum black to decrease polarization effect and increase surface area.
✓ Platinization is done by using solution of 3 chloroplatinic acid and lead acetate to get uniform
coating.
✓ If concentration is low, the electrodes should be large and closely packed.

Cell constant:
Ratio of the distance between the two electrodes (l) to the area of the electrodes(a).
Cell constant = l/a = specific conductance/ observed conductance
Units- cm-1
Primary standard solution: KCl
7.419 g in 100 g of water at 25ᵒC
Specific conductivity = 0.01286 ohm-1 cm-1
Applications of conductance measurements
✓ Water treatment
✓ Leakage detection in heat exchangers
✓ Clean-in-place
✓ Interface detection
✓ Desalination
✓ Solubility of sparingly soluble salts
✓ Kinetics of a reaction
✓ Agriculture and hydroponics
Conductometric titrations. :Applications of titrations for detection of end points.
Principle: Conductometric titration is the determination of end point of a titration by a
conductometric measurement.

As the number and mobility of ions changes, there is a change in the conductivity of the solution.

At the end point there is a sharp change in the conductivity of a solution shown by the intersection
of lines in a graph of conductivity Vs volume of titrant added.
\
Complexometric titrations:
Generally used for non-aqueous titrations
Example:
Titration of weak base vs perchloric acid in dioxan-formic acid
Titration of weak organic acid in methanol vs tetra methyl ammonium hydroxide in methanol-
benzene.
Advantages of conductometric titrations
➢ Determination of specific conductivity not required
➢ Suitable for dilute or coloured solutions
➢ No indicators required
➢ Instrumental methods helps to get accurate results with minimum error
➢ Can be used for weak acids/ bases, turbid solutions, mixture of acids etc.
➢ Temperature need not be known as long as it is maintained constant throughout the
experiment.
Disadvantages
➢ Increased salt level may mask the conductivity changes
➢ Only specific redox titrations can be carried out
➢ Accuracy is low when the concentration of the electrolyte is high.
Applications of conductometric titrations
 ➢ Solubility of sparingly soluble salts can be detected
➢ Alkalinity of fresh water
➢ Salinity of sea water
➢ Check water pollution
➢ Detection and quantitation of antibiotics
➢ Microbiology- tracing micro-organisms
➢ Purity of distilled and de-ionized water.
➢ Estimation of vanillin in vanilla flavor
Principle of potentiometry, Reference electrodes, metallic indicator electrodes.
When a metal strip is placed in a solution of its own ions there are two possibilities:
• Metal atom may dissolve in the solution as positive ions leaving behind the electrons on the
electrode
• Metal ions may take up electrons from the electrode and get deposited as neutral atoms.
Thus, a potential difference is setup between electrode and solution
It is the field of electroanalytical chemistry in which potential is measured under the condition of no
current flow.
The analysis is based on measurement of electrochemical cells without appreciable current.
Electrodes are used to measure voltage from chemical reactions.
Also, can be stated as the method of measuring the potential or emf of a solution by using a set of
indicator or reference electrodes.
Standard electrode potential:
Potential of a pure metal when dipped in 1 M solution of its own ions at 25ᵒC (298K).
Oxidation potential
The potential of a substance to get oxidized.
Reduction potential
The potential of a substance to get reduced.
Nernst Equation
The potential (E) of an electrode measured at 25ᵒC immersed in a solution of its own ions is given as
Where, Eᵒ is the standard electrode potential of the cell
R is the gas constant
T is temperature
F is faradays constant
N is the valency of the ions.
Components of Potentiometer
Potentiometer is an instrument used to measure the potential difference between reference and
indicator electrode. The measured potential can be used to determine the quantity of the electrolyte
in terms of concentration.
1) Reference electrode
2) Salt bridge
3) Analyte
4) Indicator electrode
5) Galvanometer
Reference Electrode Salt Bridge Analyte Indicator Electrode
E = Eref + Ej + Eind
 Liquid Junction potential
It is the interface between two
solutions containing different
electrolytes or different
concentrations of the same
electrolyte.
A junction potential is observed
at every liquid junction due to
the difference in mobilities of
the positive and negative ions

Types of reference Electrode

✓ Standard hydrogen electrode
✓ Saturated calomel electrode
✓ Silver/silver chloride electrode
The standard hydrogen electrode potential is the potential which is developed between H2 gas
adsorbed on the Pt metal and the H+ of the solution when the H2 gas at a pressure of 760 mm
pressure of Hg is in equilibrium with H+ of unit concentration.
✓ It can be used as indicator as well as reference electrode
✓ It has a platinum foil coated with platinum black and has a wire contact with mercury. Pt acts
as conductor, inert and facilitate in attaining equilibrium.
✓ The assembly is enclosed in a glass covering.
✓ Hydrogen gas is passed continuously at 1 atm pressure.
✓ At all times, the surface of the electrode and solution must be saturated with gas.
✓ The electrode potential (Eᵒ) is taken as 0 (zero).
Cell representation:
Pt, H2 (1 atm), H+ (1M)
2H+(aq) + 2 e⁻ ↔ H2(g)

Advantages:
✓ It can be used over the entire pH range
✓ Can be used both as reference as well as indicator electrode
✓ It is used as the primary reference standard against which the potential of other electrodes is
measured.
Disadvantages:
✓ Affected by the presence of strong oxidizing and reducing agent
✓ Difficult to maintain the pressure of H2 gas at 1 atm and conc of HCl at 1M
✓ Platinum foil can get poisoned by the impurities present
✓ H2 gas is highly inflammable.
Saturated Calomel Electrode
➢ Consists of an inner jacket and outer sleeve
 ➢ Inner tube has a wire which maintains contact with Hg and contains a slurry of calomel
(Hg2Cl2) and KCl
➢ The space between inner jacket and outer sleeve is filled with satu KCl/ 1N KCl/ 0.1 N KCl
➢ The tip is filled with crystals of KCl and porous plug of asbestos
➢ The potential of the electrode depends on the concentration of KCl and temperature.
➢ Tube 15 cm long, 0.5-1 cm diameter.
➢ Ceramic fiber acts as salt bridge
➢ Cell reaction:
Hg2Cl2(s) + 2e⁻ ↔ 2 Hg(l) + 2Cl ⁻(aq)
Nernst equation:
E = EᵒHg2Cl2/Hg -0.0591/2 [log (Cl ⁻)2]
Cell representation:
Hg(l) Hg2Cl2(satu), KCl(aq,satu)
Potential at 25ᵒC is +0.2444 V (Satu)
Advantages:
➢ Easy to construct
➢ Easy to use
➢ Stability of potential
➢ Concentration of chloride ions do not change even when the solvent gets evaporated.
Disadvantages:
➢ Temperature dependent
➢ Toxic
Silver-Silver chloride reference electrode
✓ It has a silver wire coated with a paste of silver chloride dipped in an aqueous solution of KCl
and AgCl.
✓ Coating maybe done by electroplating or physical coating.
✓ Generally used with saturated KCl but can also be used in low concentration like 1M KCl or
directly in sea water.
Advantage:
Easy handling, cost effective
Disadvantage:
Maybe be reactive sometimes.
Cell reaction:
AgCl(s) + e⁻ ↔ Ag(s) + Cl⁻
Nernst equation:
E = Eᵒ -0.0591/1 [log (Cl ⁻)]
Cell representation:
Ag(s) AgCl(Satu), KCl(xM)
Potential (Saturated KCl) at 25ᵒC is +0.199 V
Indicator electrodes (IE)
Metallic IE
Electrodes of first kind
Electrodes of second kind
Inert metallic electrodes (Redox systems)
Membrane/Ion selective electrodes
Glass electrode
Liquid membrane
Crystalline membrane electrode
Gas sensing electrode
Enzyme electrode
Metallic indicator electrodes
Metal electrode develops electrical potential as a result of redox reaction at the surface.
Electrodes of the First kind
The pure metal is in direct equilibrium with its cation. (Ag, Ag+ or Zn, Zn2+)
Mn+(aq) + n e ⁻ ↔ M(s)
Eind = Eᵒ – 0.0591/n (log 1/ [Mn+])
Example: Ag+ + e⁻ ↔ Ag(s) E= 0.800 V
Cu2+ + 2e ⁻ ↔ Cu
Zn2+ + 2e ⁻ ↔ Zn
Electrodes of the First kind
Limitations:
➢ Not very selective (Ag+ interferes with Cu2+). May respond to other ions which are more
easily reducible.
➢ Maybe pH dependent (Zn and Cd dissolve in acidic solutions)
➢ Easily oxidized
➢ Non-reproducible response.
➢ Used only in neutral or basic solutions. They dissolve in the presence of acids.
Electrodes of the Second kind
Respond to anions by forming precipitates or stable complexes
Example:
Silver electrode for Chloride ion determination
Ag + Cl⁻ ↔ AgCl + e ⁻ E= 0.199 V
Hg electrode for EDTA determination
Inert Metallic redox electrodes
✓ They are inert conductors which respond to redox systems
✓ The electrodes are not involved in the half-cell reactions
✓ The inert metal is in contact with a solution containing soluble oxidized and reduced forms of
the redox half-reaction.
✓ May not be reversible
Example: Pt, Au, Pd, C
Glass membrane electrode
The Glass electrode is the most commonly used indicator electrode involving ion exchange reaction.
Membrane made of chemically bonded Na2O, SiO2, Al2O3
The glass bulb is filled with a solution of HCl and KCl, silver acetate coated with silver chloride is
inserted as electrode.
• The membrane is not limited to any particular shape.
• It must maintain contact with the solution
• Different styles have been developed to maximize pH
sensing ability and longevity
Properties of glass electrode
✓ Potential is not affected due to the presence of
oxidizing or reducing agent
✓ Operates over a wide pH range
✓ Instantaneous response
✓ Selective
✓ Long life span
✓ Can be used in physiological systems

Theory of glass membrane potential

The electrode is kept soaked in water during which the outer
membrane gets hydrated.
Sodium ions are exchanged for the protons in the solution,
which are then free to move and exchange with other ions
Working:
The glass absorbs water. The H+ ions move in the direction of lesser concentration and replace Na+
ions and others in the glass membrane.
SiO2-Na+ + H+ → SiO2-H+ + Na+
Due to diffusion and exchange process a potential develops on each side of the glass membrane
Advantages:
✓ Potential not affected by presence of strong oxidizing or reducing agents.
✓ Operates over a wide pH range
✓ Fast response and functions on physiological systems as well.
Liquid membrane electrode
A potential is developed across the interface between analyte solution and the liquid ion exchanger
Membrane is an organic polymer saturated with liquid ion exchanger
Used for polyvalent ions as well as some anions
Example: Calcium dialkyl phosphate insoluble in water but binds to Ca2+ ions strongly.
Characteristics of Ca2+ ISE
✓ High sensitivity
✓ Low LOD
✓ pH range 5.5-11
✓ Used for physiological processes
Solid state electrodes
✓ Contain ionic compounds
✓ It is crushed, powdered, melted and fabricated
✓ Sometimes doped to increase conductivity
✓ Working is similar to glass membrane
AgX membrane: For determination of X⁻
Ag2S membrane: For determination of S2⁻
LaF3 membrane: For determination of F⁻
Crystalline membrane electrode
F⁻ ion selective electrode
LaF3 is doped with EuF2
Eu2+ has less charge than La3+ and thus an anion vacancy is observed for every Eu2+
Neighbouring F⁻ occupied the vacancy and thus another vacancy is created, thus, F⁻ moves through
the lattice.
LaF3(s) → LaF2+(s) + F⁻(aq)
E cell = K- 0.0591 log aF⁻
Gas sensing electrode: It is a galvanic cell whose potential is related to the concentration of a gas
present in a solution
➢ Consists of a reference electrode, ion selective electrode and electrolyte solution
➢ A thin permeable gas membrane acts as barrier between the internal solution and the
analyte
➢ It allows only small gas molecules to permeate and pass into the internal solution
Example: O2, NH3/NH4+, CO2, HCO3-,CO32-
Enzyme Electrode: It is a miniature chemical transducer which works by combining an
electrochemical procedure with immobilized enzyme activity.
Example: Glucose oxidase immobilized on gel to measure concentration of glucose
Urea substrate coupled with the active surface of cationic electrode responsive to ammonium ion.
Urea diffuses from the membrane, reacts with the urease enzyme to produce ammonium ion at the
membrane surface which causes a gradient and diffusion of the ion back to the electrode where it is
sensed.
Quinhydrone electrode
❖ Solution contains quinones of hydroquinones and prepared from quinhydrone
 ❖ The electrode consists of inert metal electrode (Pt) in contact with quinhydrone crystals in a
water based solution.
❖ The potential depends on the ratio of activity of the two substances (quinone-hydroquinone)
and hydrogen ion concentration.
❖ Hydroquinone → Quinone + 2H+ + 2e-
❖ Simple and easily attains equilibrium
❖ Used in the presence of mild oxidizing and reducing agents at pH8.
❖ Sensitive to high concentration of salts and oxidising and reducing agents.
POTENTIOMETRIC TITRATIONS
Measurement of change in potential with a suitable indicator electrode as a function of the volume
of titrant added.
Two electrode system is used: a reference and an indicator electrode
The indicator electrode forms the half of the electrochemical cell where unknown ions is present in
the test solution
The reference electrode forms the other half of the cell, in which the potential is maintained
constant.
Types of potentiometric titrations
✓ Acid-base titrations (Aqueous/non-aqueous)
✓ Redox titrations
✓ Precipitation titrations
✓ Complexometric titrations
✓ Diazotization titrations
Acid-base titrations (Aqueous/non-aqueous)
✓ Titrations can be done in aqueous and non-aqueous medium
✓ Aqueous medium could be of any strength
✓ Acid base combinations: Strong acid Vs strong base, strong acid vs weak base, weak acid vs
strong base, weak acid vs weak base.
✓ Poly basic acids vs bases: Citric acid, tartaric acid vs strong base
✓ Non- aqueous medium: Weak acid vs potassium/Lithium methoxide
✓ Weak base vs perchloric acid

Acid-Base titration curves

Redox titrations:

Ce4+ + Fe2+ → Ce3+ + Fe3+

E= Eᵒ + 0.0591/n [log
(ox)/(red)]
Reference electrode: Ag/AgCl
or SCE
Indicator electrode: Pt wire or
foil

Ferrous Ammonium
sulphate in dil H2SO4 vs
KMnO4/K2Cr2O7
Diazotization titrations
Compounds containing
aromatic amino group
(Alkaloids, amines, sulpha
drugs) vs sodium nitrate in acidic medium . This leads to the formation of diazonium salt.
Reference electrode: SCE
Indicator electrode: Glass electrode
Precipitation titrations:
Determination of ions/compounds (Hg, Ag, Pb, Cu) which form precipitates with the titrant.
E= Eᵒ + 0.0591/n [log (Mn+)]
Reference electrode: Ag/AgCl, SCE, SHE
Indicators electrode: Silver wire
Complexometric titrations
Determination of metallic di/trivalent ions by titrating with EDTA.
Reference electrode: Ag/AgCl, SCE
Indicator electrode: Ag/Hg electrode
Precautions while using the potentiometer/pH meter
✓ The electrodes must be placed only on the electrode holder and not left lying around
✓ They must be rinsed at the end of each experiment
✓ The glass bulb must not be touched and only cleaned with a soft tissue paper.
✓ The bulb must be kept immersed in water.
✓ For storage the combined electrode can be stored in 2M HCl
✓ Buffer solutions (pH 4, 7, 9) used for calibration
✓ Temperature must be maintained constant for accurate results
Advantages of Potentiometric titrations:
✓ Provides more reliable and accurate results compared to the normal titrimetric analysis
✓ Can be used for coloured and turbid solutions
 ✓ No indicators required
✓ Used for dilute solutions
✓ Can be automated
✓ Less time consuming
Applications:
➢ Environmental pollution monitoring: S, F, CN etc in effluents.
➢ Agriculture: Determination of ions like Na, K, Ca, Cl in soil, fertilizers etc
➢ Sweat on skin, pH inside a living cell, Acidity of stomach
➢ Salt content in meat, fish, dairy, fruit juice etc