MODULE 5 - PRODUT IDENTIFICATION AND

RECOVERY
• Chromatographic separation processes: Principles
and protocols of Chromatographic techniques
– Partition chromatography
– Single dimensional (Both Ascending and Descending) and
two dimensional chromatography
– Thin layer chromatography,
– Gas liquid Chromatography,
– High Performance liquid chromatography (HPLC) –
analytical and preparative.
– Adsorption column chromatography.
– Ion Exchange Chromatography:
– Gel Filtration Chromatography,
– Affinity Chromatography
• Principle and Applications of Electrophoresis- their
types
 CHROMATOGRAPHIC SEPARATION PROCESS

• Chromatography is the process of separation of

components of the mixture dissolved in mobile phase
according to the rate of transport of these components in
the stationary phase.
• Individual components migrate through the stationary
phase and shows a peak profile that becomes broader
as a function of time and distance it travels.
• Chromatography is a widely used separation technique
in bioprocess industries for isolation and purification of
product of interest.

2
3
• During scaling up of analytical scale into preparative
purpose, the ratio of sample mass to column volume is
to be kept constant
Sample Mass
 Cons tan t
Column Volume

• Straightforward way to reach this is to keep the column

length constant and increasing the column cross-
sectional area
 Radius j 
2
Mass j
 
Massi  Radiusi 

where i, j = species i and species j

4
• Maintaining constant linear flow rate helps in achieving
reproducible separation conditions.
• This indicates that the volume flow rate should be
proportional to the column cross section and therefore to
the mass of the solute, as
Volume flowj Massj

Volume flowi Massi

5
 DEFINITIONS OF TERMS

1. Partition Coefficient (K)

• This is the basis of all chromatographic techniques
• It describes the way in which a solute entering the
chromatographic system, distributes itself between
stationary and mobile phases
• Partition coefficient is given as

Cs
K
Cm
where Cs = Molar concentration of solute in
stationary phase
Cm= Molar concentration of solute
in mobile phase
6
2. Retention time (tR)
• It is defined as the time taken by solute to reach detector
from moment of its injection into column
• Has two components:
 Time it takes the analyte molecule to pass through free
spaces between stationary phase matrix, known as dead
time tm
 The time the analyte is retained by stationary phase is
given as tR
• The adjusted retention time is given as

t R'  t R  t m

7
 tR2

tR1
tm
Detector signal

Sample injection

Time

On – line chromatogram of two component mixture

8
• The retention time of solute is affected by several
factors:
 Nature of stationary phase
 Composition of mobile phase
 Column dimensions like length and diameter
 Mobile phase flow rate
 The retention time of solute increases with increasing
length of column and with decreasing flow rate of the
mobile phase

9
3. Retention volume (VR)
• Defined as volume of mobile phase required to transport
a solute from the point of injection into the column and
its passage through the column to the detector
• It is given as

VR  t R F
where F = Flow rate of mobile phase
 The corrected or adjusted retention volume is given as

VR'  VR Vm
10
4. Elution time and volume
• It is used to plot a chromatograph which is a pictorial
record of detector response as a function of elution
time or volume
• This chromatograph consists of a series of peaks,
representing the elution of individual analytes
• Elution time and volume gives the time and volume the
eluent takes
a) To pass through free spaces between stationary
phases and
b) To be retained by the stationary phases

11
5. Capacity factor (k’)
• It is the most important factor in chromatography
• It is a measure of the time spent by a solute in the
stationary phase relative to the time spent in the mobile
phase
• It is given as

t R  tm or k' VR Vm or k' K VS

k'
tm Vm VM
where VS = Volume of stationary phase
VM = Volume of mobile phase

• k’ values normally range from 1 to 10

• Capacity factors are independent of physical dimensions
of column and rate of flow of mobile phase through it
12
6. Resolution (Rs)
• Chromatographic resolution of two peaks is a measure
of their separation
• It is defined as distance between peak maxima
compared with the average base widths of the two
peaks and is given as

 t R 2  t R1 
Rs  2  
 b1  wb2 
w

• Rs = 1.0 ------ separation of two peaks is 97.7 %

• Rs < 1 -------- separation is incomplete
• Rs >1.5 ------- complete separation of peaks
13
7. Selectivity or Separation Factor (α)
• Measure of inherent ability to discriminate between two
analytes.
• It is defined as relative retention ratio for the two
analytes
k ' t R' B
  B
'
k t' RA
A

• It is influenced by chemical nature of stationary and

mobile phases.
• Some chromatographic mechanisms are inherently
highly selective like affinity chromatography.

14
 A. PARTITION CHROMATOGRAPHY

• Based on differences in Capacity Factor and Partition

Coefficient of analytes using liquid stationary and mobile
phases
• Sub – divided into two types:
– Liquid - Liquid Chromatography where liquid stationary phase is
attached to supporting matrix by purely physical means. Eg:
Water supported by cellulose, starch or silica matrix
– Bonded – Phase Chromatography where liquid stationary phase
is covalently attached to supporting matrix. Eg: Silica is
derivatized to immobilize the stationary phase by reaction with
organochlorosilane

15
 Modes Of Partition Chromatography
PROPERTIES NORMAL REVERSE PHASE
PHASE
Polarity of Stationary Polar Non-polar and Inert
phase
Polarity of Mobile Non-polar Polar
phase
Eg. of Stationary Alkylamine Alkylsilane
phase bonded to silica chemically bonded
to silica
Eg. of Mobile phase Organic solvent Water or aqueous
like heptane, buffers, methanol,
ethylacetate acetonitrile
16
PROPERTIES NORMAL PHASE REVERSE PHASE

Elution of Analytes Least polar - First Polar – First

Most polar - Last Non-polar – Last

Requirement for Polar analytes Non – polar

gradient elution need gradient analytes need
elution with gradient elution
increasing polarity with increasing
with methanol or proportions of low
dioxine polarity solvent like
hexane

17
 B. SINGLE AND TWO – DIMENSIONAL (PAPER)
CHROMATOGRAPHY

• It was developed in 1941 by Archer Martin and Richard

Synge
• It uses simple equipment
• It is an analytical technique for separating and identifying
mixtures
• It plays an indispensable role in biochemical analysis
and can efficiently separate small molecules like amino
acids and oligopeptides
• A few drops of solution containing a mixture of
components to be separated are applied 2 cm above
one end of strip of filter paper

18
 Glass jar

Support rod Clip

Paper
Solvent front

Sample
Solvent for
development

Ascending Paper Chromatography

49
Descending Paper Chromatography

50
• After drying, that end of paper is dipped into a solvent
mixture consisting of aqueous and organic components like
water/butanol/acetic acid in 4:5:1 ratio
• The paper should be in contact with equilibrium vapours of
solvent
• Solvent soaks into paper by capillary action because of
fibrous nature of paper
• Aqueous component of solvent binds to cellulose of paper
and forms stationary gel like phase with it
• Organic component of solvent continues migrating, forming
the mobile phase

51
• Rates of migration of substances are governed by their
relative solubilities in the polar stationary and non polar
mobile phases
• The chromatogram is removed from solvent and dried
• Uncoloured separated components are detected by:
1. Radiation detection methods
2. Ultraviolet light
3. Reagent solution that forms coloured product upon reaction
with substance under investigation
Example: α- amino acids and primary amines react with
ninhydrin to form intense purple colour
Secondary amines like proline forms yellow compound

22
 Direction of flow of
1st solvent

Origin Separation
if only 2nd
solvent is
Direction used
of flow of
2nd solvent Separation
if both
solvents are
used
sequentially
Separation if only 1st
solvent is used
Two – dimensional paper chromatography
23
• Used when a complex mixture is incompletely separated
in a single paper chromatogram
• Sample is spotted on corner of a sheet of filter paper
• Chromatogram is run parallel to edge of paper
• The paper is then dried and chromatogram is rotated 900
• It is then chromatographed parallel to the second edge
using another solvent system
• This enhances the separation of the mixture into its
components
• Movement of analyte is expressed by retardation factor,
Rf
Dis tan ce moved by analyte from origin
Rf 
Dis tan ce moved by solvent front from origin

24
 C. THIN - LAYER CHROMATOGRAPHY

• Principle
• Sample application
• Plate development
• Analyte detection

25
 Principle
• A thin layer of stationary phase is formed on a suitable flat
surface
• Stationary phase slurry is applied to glass, plastic or foil
plate, 20 cm2, as uniform thin layer by means of spreader
• For preparative separation, the layer is up to 2mm
• For analytical separation, the layer is 0.25 mm thick
• Once slurry layer is prepared, plates are dried
• Since layer is thin, movement of mobile phase across the
layer is by simple capillary action
• Analytes are transferred at a rate determined by their
partition coefficient between stationary and mobile phases

26
• Movement of analyte is expressed by retardation factor,
RF
Dis tan ce moved by analyte from origin
Rf 
Dis tan ce moved by solvent front from origin

Sample Application
• Sample is applied to the plate 2.0 to 2.5 cm from edge
by means of micropipette or micro syringe
• Solvent is removed from the spot by heating or by use of
an air blower
• Care should be taken in case of volatile or thermo –
labile compounds
• Then it is possible to apply more samples to spot

27
Plate development

28
• Separation commonly takes place in glass tank that
contains mobile phase to a depth of about 1.5 cm
• This is allowed to stand for 1 hour with lid over the tank so
that tank is saturated with solvent vapour (equilibration)
• Then lid is removed and thin layer plate is placed vertically
in tank such that it touches the solvent
• Lid is replaced and separation of compounds occur as
solvent travels up the plate
• Separation occurs at a moderate speed of 30 to 90
minutes

29
 Analyte Detection
• Several detection methods available are:
1. Fluorescent compounds are analyzed under ultraviolet light
2. Unsaturated compounds are investigated by iodine vapours
3. Amino acids and peptides are analyzed with specific colour
reagents like ninhydrin
4. Radiolabelled compounds are analyzed by autoradiography,
which detect spots as dark areas on X-ray film or plate is
scanned by radiochromatograph
5. General non-specific technique is to spray the plate with 50 %
(v/v) sulphuric acid or 25 % (v/v) sulphuric acid in ethanol and
heating to 110 0C, where sample is charred and brown spots
are seen

30
 D. GAS LIQUID CHROMATOGRAPHY

• Principle
• Column selection
• Preparation and application of samples
• Detectors

31
 Principle
• This is based upon partitioning of compound between
liquid and gas phase
• It is widely used method for qualitative and quantitative
analysis of a large number of compounds because of its
 High sensitivity
 Speed of resolution
• It is valuable for the separation of compounds of relatively
low polarity
• A stationary phase of high boiling point liquid material like
silicone grease, polyethylene glycols, supported on inert
granular solid

32
• Most commonly used support is Celite (diatomaceous
silica), which is often treated with compounds like
hexamethyl disilazane to modify hydroxyl groups of celite
• Stationary phase must be involatile and thermally stable at
the temperature used for analysis
• These phases are of two types
 Selective -------- where separation occurs by utilization of different
chemical characteristics of components
 Non – selective -------- where separation occurs on the basis of
differences in boiling points of sample components

33
 Amplifier

Diagram of Gas Liquid Chromatographic system

34
A gas chromatography oven to show a capillary column

35
• GLC system consists of a narrow coiled glass or steel
column 1 to 3 m long and 2 to 4 mm internal diameter
• Column is maintained in an oven at an elevated
temperature so that compounds for separation are in
vapour state
• Inert carrier gas like nitrogen, helium or argon is passed
and it carries the compounds through the column
• Compounds leaving the column pass through a detector,
linked to amplifier to chart recorder which records a peak
when analyte passes through detector

36
 Column selection

• GLC is performed in capillary columns made of glass or

metal with diameters between 0.03 and 1.0 mm and up to
100 m in length

Capillary column
system

Wall coated open Support coated open

Tubular (WCOT) Tubular (SCOT)

37
i. Wall coated open tubular columns
• Here stationary phase is coated directly onto the walls of
capillary tubing
• Small amounts of sample is chromatographed
• A splitter system is used at the sample injection port so
that small fraction of injected sample reaches column
• Some instruments have on – column injectors that
require considerable skill in their operation

38
ii. Support coated open tubular column
• Also known as porous layer open tubular (PLOT)
columns
• Support material is bonded to walls of capillary
columns
• Stationary phase is coated on to the support
• Capacity is higher than WCOT
• Small samples can be injected directly onto columns
without splitter system
• Simpler to use than WCOT
• Efficiency is less

39
 Preparation and application of samples

Preparation of samples
• Majority of non and low-polar compounds are directly
amenable to GLC
• For compounds with polar groups, derivatization is carried
out
• Example: Methylation, silanization and perfluoracylation
are done for fatty acids, carbohydrates and amino acids

40
 Application of samples
• The sample is dissolved in a suitable solvent like acetone,
heptane or methanol
• It is injected onto the column using a microsyringe in the
injection port
• Normally 0.1 to 10 mm3 of solution is injected
• The injection region is at slightly higher temperature to
ensure complete volatilization of the sample
• Sample injection is automated in many commercial
instruments

41
Detectors

Types of detectors

Flame Ionization Nitrogen - Phosphorus

Detector Detector

Electron Capture
Detector

42
 1. Flame Ionization detector (FID)

Collector electrode
Igniter

Outlet

Air supply
Amplifier

Hydrogen supply
Flame

Carrier gas
43
• It is the most widely used detector
• It responds to almost all organic compounds
• It can detect as little as 1 ng
• Mixture of hydrogen and air gives a flame
• One electrode is jet of flame ad other is brass or
platinum wire mounted near tip of flame
• Sample components emerging from column are
ionized in the flame
• It has a minimum detection quantity of order of 5 X
10-12 gs-1 and upper temperature limit of 400 0C

44
2. Nitrogen – Phosphorus detector (NPD)
• Also called thermionic detector
• It is similar in design to FID
• Has a crystal of sodium salt fused onto electrode system
or burner tip embedded in ceramic tube containing
sodium salt or rubidium chloride
• It has excellent selectivity towards nitrogen and
phosphorus containing compounds
• It has a detection limit of 10-11 gs-1 and upper
temperature limit of 300 0C

45
3. Electron Capture Detector (ECD)
• It responds only to substances that capture electrons like
halogen containing compounds
• Widely used in the analysis of polychlorinated compounds
like pesticides, DDT, dieldrin and aldrin
• Has a sensitivity of 10-11 gs-1 and upper temperature limit
of 300 0C
• It works by means of radioactive source ionizing the
column gas
• The electrons produced give current across the electrodes
to which suitable voltage is applied
• When electron capturing compound emerges from
column, ionized electrons are captured and the drop in
current is recorded

46
 E. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY

• Principle
• Columns and packing
• Matrices and stationary phase
• Mobile phase and pumps
• Application of samples
• Detectors
• Applications

47
 Principle
• This is an important development in column
chromatography
• It has resulted in availability of new smaller particle size
stationary phases that can withstand pressures
• Smaller the particle size, better is the resolution
• This results in greater resistance to flow of mobile phase
• This creates back – pressures sufficient to damage matrix
structure of stationary phase
• HPLC has emerged as the most popular, powerful and
versatile form of chromatography

48
• It is used to separate components of a mixture by using a
variety of chemical interactions between the analyte and
the chromatography column and includes partition, ion –
exchange, adsorption, exclusion and affinity
chromatography, which gives better and faster resolution
• Commercially available HPLC systems are microprocessor
controlled to allow continuous chromatographic
separations

49
 Sample
injection loop
Guard
column
Detector

High Sample
Solvent
pressure collector
reservoir
pump
Recorder
Column and data
system
Diagram of isocratic HPLC system
50
 9060 Polychrom Computer
9050 Variable (Diode Array) Workstation
UV/Vis Detector Detector

Rheodyne
Injector

HPLC
Column
HPLC Solvent 9010 Solvent
Reservoirs Delivery System
51
Columns

• Columns used in HPLC are generally made of stainless

steel and can withstand pressures up to 5.5 X 107 Pa

Column system

Straight Microbore Preparative

52
1. Straight columns
• These are 15 to 50 cm in length and 1 to 4 mm in
diameter
• These are generally used

2. Microbore columns
• These are 25 cm in length and 1 to 2 mm in diameter
• Can sustain flow rates of 0.05 to 0.20 cm3min-1

3. Preparative columns
• These have internal diameters of 25 mm
• Can sustain flow rates up to 100 cm3min-1

53
• Best columns are precision bored with internal mirror
finish that allows efficient packing of columns
• Methods available for packing depend on nature of
packing material and dimensions of particles
• The packing should be of uniform bed of material with no
cracks or channels
• Hard gels swell in the solvent to be used in
chromatographic process and then are packed under
pressure
• Most widely used technique for column packing is high
pressure slurrying technique
• Slurry is then pumped rapidly at high pressure into
column

54
• Porous plugs of stainless steel or teflon are used in the
ends of column to retain packing material
• These plugs must be homogeneous to ensure uniform
liquid flow through column

Matrices and stationary phases

• The various matrices used are:
i. Microporous support in which micropores are generally 5 to
10 μm in diameter
ii. Pellicular (superficially porous) supports in which porous
particles are coated onto inert solid core like glass beads of
about 40 μm in diameter
iii. Bonded phases in which stationary phase is chemically
bonded onto inert support like silica

55
• For adsorption chromatography
 Adsorbents like silica and alumina are available as
microporous or pellicular forms
 Pellicular systems have high efficiency and low sample
capacity
 Microporous supports are preferred

• For partition chromatography

 Stationary phase is coated onto inert microporous or
pellicular support
 Supports coated with liquid phase may be washed off by
developing mobile phase
 Bonded phases are used to overcome this problem

56
• For ion exchange chromatography
 Cross linked microporous polystyrene resins are used
 Pellicular resins are also available
 Bonded phase exchangers contain covalently bonded
cross-linked silicone network

• For exclusion chromatography

 Stationary phases are porous silica, glass, polystyrene
beads
 In HPLC, semi-rigid gels like sepharose, sephadex are
used

• For affinity separations

 Spacer arm and ligands are attached to support by
chemical means
57
• For normal and reversed phase chromatography
 Normal phase stationary phase is alkylnitrile or alkylamine
 Reversed phase stationary phase is octasilane or
octadecysilane

Mobile phase and pumps

• Choice of mobile phase depends on type of separation to
be achieved
• Isocratic separations are done with single pump using
single solvent or two or more solvents premixed in fixed
proportions
• Gradient elution requires separate pumps to deliver two
solvents in proportions
58
• All solvents used must be purified since traces of
impurities affect column and interfere with detection
system
• Also purified solvents must be degassed to prevent
alteration in column resolution and interference with
continuous monitoring of effluent
• Various methods for degassing are:
 Warming the solvent
 Stirring vigorously with magnetic stirrer
 Subjecting to vacuum
 Ultrasonication
 Bubbling helium gas through solvent reservoir

59
• Pumps are the most important feature of HPLC systems
for delivery of mobile phase
• Should not have cyclic variations in pressure
• Must have flow capability of 10 cm3min-1 and upto 100
cm3min-1 for preparative separations
• Various pumping systems are available that operate on
the principle of constant pressure or constant
displacement
• Constant displacement pumps maintain constant flow
rate through the column
• These pumps have in-built safety mechanisms so that if
pressure in columns change, pump is inactivated
automatically

60
 Types of constant
Displacement pumps

Constant volume Reciprocating

61
 Inlet valve Outlet valve
Pump chamber

From To column
reservoir
Seal
Piston
Drive belt
Drive motor

Screw - jack

Constant volume displacement pump

62
• It is motor driven syringe – type pump that delivers a
fixed volume of solvent on to the column by piston
• It contains screw – jack driven by stepper motor
• Down stroke of piston closes outlet valve and opens
inlet valve to release eluent in pump chamber
• Up stroke of piston closes inlet valve and opens outlet
valve to release eluent on to the column

63
 Inlet valve Outlet check valve

Piston

Cam follower
Cam
Drive motor

Reciprocating displacement pump

64
• It is the most commonly used pump
• Piston is moved by a motorised crank and entry of solvent
into column is regulated by check valves
• On compression stroke ------- solvent is forced from pump
chamber onto the column
• During return stroke ------- exit check valve closes and
solvent enters via entry valve to the pump chamber
• These pumps produce small pulses of flow and pulse
dampeners are incorporated to minimize pulsing effect

65
 Application of samples
• Correct application of sample onto HPLC column is an
important factor in successful separations

Methods of sample
application

Microsyringe Loop injector

66
 1. Microsyringe
• Samples are injected directly onto column packing or onto
small plug of inert material above column packing
• Injection can be done
 While the system is under pressure
 By stop flow injection method -------- switching off the pump,
dropping the pressure to near atmospheric and injecting the
sample after which pump is again switched on

67
2. Loop Injector

Eluent from pump

6 1

5 2 To column
Loop of 4
fixed 3
Inlet port for
volume loading loop via
Waste microsyringe

Loading position
68
69
 Eluent

6 1
To column
5 2
4 3

Waste

Injecting position

70
71
• This consists of a metal loop of fixed small volume that is
filled with sample
• It has appropriate valve switching system
• The eluent from pump is channeled through the loop,
outlet of which leads directly onto column
• Thus sample is flushed onto the column by eluent
without interruption of flow to column

72
Detectors
Types of
Detectors

Variable wavelength Fluorescence

Scanning wavelength Electrochemical

Coupling to mass spectrometer

73
 1. Variable wavelength detector
• It is most commonly used detector
• Based on ultraviolet – visible spectrophotometry
• Capable of measuring absorbances from 190 nm
• Has sensitivities as low as 0.001 absorbance units for
full scale deflection (AUFS)

2. Scanning wavelength detector

• Can record complete absorption spectrun of each
analyte and helps in identification
• It is possible by temporarily stopping the effluent flow or
by using diode array techniques which measure
absorbance at all wavelengths within 0.01 s

74
 3. Fluorescence detectors
• Extremely valuable for HPLC
• Has high sensitivity
• Limited acess because only few substances fluoresce

4. Electrochemical detectors
• Selective for electroactive species
• Potentially highly sensitive
• Successful in the assay of catecholamines, vitamins
and antioxidants
• Consists of flow cell fitted with two electrodes:
a. A stable counter current Ag/AgCl or calomel electrode
b. Working electrode

75
• A constant potential is applied to working electrode
• When analyte flows through flow cell, molecules at
electrode surface undergo oxidation or reduction,
resulting in current flow between two electrodes
• Potential applied to counter electrode is sufficient to
ensure that the current will give full scale deflection on
recorder
• Compounds capable of undergoing oxidation are
hydrocarbons, amines, catecholamines, phenols
• Compounds capable of undergoing reduction are
esters, ketones, aldehydes

76
 5. Coupling of HPLC to mass spectrometer
• This is the greatest technological advance in detection
using HPLC
• Problems of removing bulk of mobile phase before
sample is introduced into mass spectrometer is
resolved in number of ways:

a. Direct liquid insertion interface (DLI)

 Consists of flow of effluent via capillary into direct insertion
probe in mass spectrometer

b. Thermospray ionization (TSI)

 Requires use of aqueous mobile phase containing ammonium
acetate
 End of heated capillary (400 0C)from effluent flow enters a
chamber exposed to electron bombardment and to vacuum
pumping system

77
  High temperature creates supersonic expanding jet of droplets
which are ionized and gradually reduced in size due to
pumping system
 Ions of analyte are then ejected from droplet into mass
analyser

c. Electrospray ionization (ESI)

 Capillary containing effluent is held at high voltage and effluent
is mixed with nitrogen gas
 Voltage creates charge on liquid surface that causes it to
disperse into very fine spray
 Solvent in fine droplets evaporates and is flushed away by
nitrogen gas
 Ions of analyte are swept as supersonic stream into vacuum
chamber and then into mass analyzer

78
Applications

• Used for assay or purification of all types of biological

molecules
• Also used in separation of oligopeptides and proteins
• Enables complex mixtures as tryptic digests of proteins
and culture supernatant of microbes to be applied directly
to column
• Protein mixtures from cell extracts need some form of
preliminary fractionation prior to study

79
 F. ADSORPTION CHROMATOGRAPHY

• Principle
• Simplified theory of adsorption
• Operational procedure for batch wise adsorption
• Hydroxylapatite chromatography
• Hydrophobic interaction chromatography

80
Principle

• It is based on the principle that solid materials

(adsorbents) have ability to hold molecules at their
surfaces
• It involves weak, non ionic attractive forces of van der
waals and hydrogen bonding type, which occur at
specific adsorption site
• These sites are occupied by molecules of eluent or
analytes present in mixture in proportions depending
upon relative strengths of their interaction
• Strength of binding of particular analyte depends upon
functional groups present in its structure

81
 Solute particles

Solute adsorbed
on surface of
stationary matrix

Stationary matrix

82
• This chromatography is influenced by presence of
specific groups which interact with adsorption site
• When eluent is passed through column, differences in
binding strengths eventually leads to separation of
analytes
• Adsorbents are silica, alumina and carbon
• Silica is acidic and good for separation of basic materials
• Alumina is more basic and better suited for resolution of
acidic materials
• Selection of correct eluent (mobile phase) is essential for
good resolution since it influences the capacity factor of
analytes

83
• Examples: Alcohols are selected when analytes contain
hydroxyl groups
 Acetone or esters are selected for analytes containing carbonyl
groups
 Hydrocarbons like hexane, heptane and toluene are selected for
predominantly non – polar analytes
• This chromatography is most commonly used to separate
non ionic, water insoluble compounds like triglycerides,
vitamins and many drugs

84
Simplified theory of adsorption
• The dissociation constant for protein – matrix interaction is
given as
m.p
Kp 
q
where p = concentration of protein in free solution
solution at equilibrium with adsorbent
m = concentration of free effective binding sites

q = concentration of matrix – bound protein

85
 mt (1  )
Kp 

where mt = total effective concentrations of adsorption
sites
α = partition coefficient of solute adsorbed as
fraction of total
• For useful adsorption on column chromatography, α needs
to be at least 0.8
• There are three possible fates for a particular protein:
 α = 1 ------- it adsorbs totally and immovably
 α = 0 ------- it does not adsorb at all
 0 < α < 1------ it adsorbs partially and moves down the column

86
• Adsorption occurs on outer portions of bead particles
• As larger proteins bind, these block further access to sites
• When saturated, beads do not have homogeneous
penetration of proteins
Outer 20 % of bead
contains 49 % of total
volume

Less protein penetrates

centre

Cross section of spherical bead after protein has adsorbed

87
• Smaller proteins are more capable of penetrating the
outer layer and are selectively adsorbed
• Outer 20 % of bead, measured by radius, constitutes 49
% of total bead volume
• Less proteins penetrate to centre and this explains how
actual adsorption kinetics are faster

Eg. 1.Hydroxylapatite Chromatography

• Crystalline hydroxylapatite [Ca5(PO4)3(OH)] is an
insoluble form of calcium phosphate
• It is an adsorbent used to separate mixtures of proteins
or nucleic acids
• Mechanism of adsorption involves dipole – dipole
interactions and electrostatic attractions

88
• It is available commercially in a range of forms like
crystalline or spheroidal
• Adsorption capacities of these forms is maximum around
neutral pH and includes 20 mM phosphate buffer for
adsorption process
• Elution is achieved by increasing phosphate buffer
concentration to 500 mM
• Most important application is the separation of single –
stranded from double stranded DNA
• Both forms of DNA bind at low phosphate buffer
concentrations
• As buffer concentration is increased, single stranded DNA
is selectively desorbed

89
• When concentration of buffer is further increased, double
stranded DNA is released
• Affinity of hydroxylapatite for double stranded DNA is so
high that it can be selectively removed from RNA and
proteins in cell extracts

90
 Hydrophobic Interaction
Chromatography

• This purifies proteins by exploiting their surface

hydrophobicity, which is related to the presence of non –
polar amino acid residues
• Here the presence of hydrophobic groups attached to
suitable matrix facilitates protein – matrix interaction
• Commonly used stationary phases are alkyl (hexyl, octyl)
or phenyl groups attached to agarose matrix
• This method is commonly used after fractionation of
proteins with ammonium sulphate

91
• Once proteins are adsorbed to stationary phase, selective
elution is achieved by:
a. Gradually decreasing ionic strength
b. Increasing pH
c. Selective displacement by displacer which has stronger affinity
for stationary phase than has the protein
• Examples:
– Non – ionic detergents like triton X-100
– Aliphatic alcohols like butanol, ethylene glycol
– Aliphatic amines like butylamine
• Proteins purified include aldolase, cytochrome c,
thyroglobulin
• Potential problems of HIC:
a. Some elution conditions may cause protein denaturation
b. Its non predictability in that it works well for some proteins and
not for others

92
 G. ION EXCHANGE CHROMATOGRAPHY

• This relies on the attraction between oppositely charged

particles
• Used for separation of mixtures of biological molecules
like amino acids and proteins
• It utilizes the fact that biological molecules have ionisable
groups and carry net positive or negative charge
• Ion – exchange separations are carried out mainly in
columns packed with an ion exchanger
• Various proteins bind to ion exchanger with different
affinities
• Affinity with which protein binds to given ion exchanger
depends on the identities and concentrations of the other
ions in solution

93
• As the column is washed, a process known as elution,
proteins with relatively low affinities for ion exchanger
move through column faster than proteins that bind with
higher affinities
• Proteins that bind tightly to ion exchanger can be eluted
by changing elution buffer
1. With higher salt concentration
2. Different pH

94
 Negatively charged
proteins
Layer sample Elute negatively charged
Positively charged
onto column protein with salt solution
proteins

Cl-
Na+

Collect positively
Positively charged charged proteins
gel bed
95
Types of ion exchangers

Types

Cation Anion

Weakly acidic Weakly basic

Strongly acidic Strongly basic

96
1. Cation exchangers
• Possess negatively charged groups which attract
positively charged cations
• These are also called acidic ion exchangers since their
negative charge results from ionization of acidic groups
• Example: Weakly acidic exchanger like carboxymethyl-
cellulose
Strongly acidic exchanger like sulphomethyl
cellulose

2. Anion exchangers
• Possess positively charged groups which attract
negatively charged anions

97
• These are also called basic ion exchangers since their
positive charge results from association of protons with
basic groups
• Example: Weakly basic exchanger like
diethylaminoethyl-cellulose
Strongly basic exchanger like
triethylaminoethyl cellulose

• Choice of ion exchanger depends on:

1. Stability of sample components
2. Relative molecular mass
3. Specific requirements of separation like effect of pH on sample
charge
• Weak electrolytes requiring very high or low pH for
ionisation is separated on strong exchangers as these
operate under wide pH range

98
• Strong electrolytes are separated using weak
exchangers for number of reasons:
a. Reduced tendency to cause sample denaturation
b. Inability to bind weakly charged impurities
c. Enhanced elution characteristics
• pH of buffers used should be one pH unit above or
below its isoionic points of compounds to be separated
• Examples of cationic buffers include pyridine and
alkylamines used in anion exchangers
• Examples of anionic buffers are acetate, phosphate with
cation exchangers

99
Applications
• Separation of amino acids in a protein hydrolysate
achieved in strong acid cation exchanger
• Sample introduced onto column at a pH of 1 to 2 results
in complete binding of various types of amino acids.
Gradient elution results in sequential elution of amino
acids
• Acidic amino acids like aspartic and glutamic acids are
eluted first
• Neutral ones like glycine and valine are eluted next
• Basic amino acids such as lysine and arginine retain net
positive charge up to pH values of 9 to11and are eluted
last

100
• Buffers of lowest ionic strength that effects elution is used
initially for subsequent elution of components
• Generally with anion exchanger, pH gradient decreases
and ionic strength increases
• For cation exchanger, both pH and ionic gradients
increase

101
 H. GEL FILTRATION CHROMATOGRAPHY

• Also known as exclusion chromatography or permeation

chromatography
• Separation of molecules take place on the basis of their
molecular size and shape
• It utilizes the molecular sieve properties of a variety of
porous materials
• Most commonly used material is a group of polymeric
organic compound that possesses three-dimensional
network of pores that confers gel properties upon them
like Sephadex
• Gel consists of polysaccharide dextran cross-linked to
form a three-dimensional network in shape of beads

102
• Cross linked dextran is highly polar because of its large
content of hydroxyl groups and swells considerably when
placed in water
• Extent of cross-linking during manufacture of gel controls
pore size within gel beads
• Column of gel particles or porous granules will be in
equilibrium with suitable mobile phase for molecules to
be separated
• Distribution of analyte in the column is determined solely
by total volume of mobile phase both inside and outside
the gel particles

103
 Matrix

Large molecules
are excluded

Small molecules
penetrate pores of
matrix

104
 Layer sample Large proteins Small proteins
onto column
Add
buffer
to wash
proteins

Collect
Polymer gel fractions
bead

3 2 1

Gel filtration chromatography 105

106
• Distribution coefficient Kd of particular analyte between
inner and outer mobile phase is a function of its
molecular size
– Kd = 0 ------------- Analyte is large and completely excluded from
mobile phase within gel
– Kd = 1 ------------- Analyte is sufficiently small to gain complete
access to inner mobile phase
– Kd > 1 ------------- Analyte has adsorbed on gel and this should be
avoided
• On eluting column, large molecules completely excluded
from pores will pass through interstitial spaces and will
appear in effluent first
• Small molecules entering pores of gel will be eluted last
• Separation is due to different amount of time the different
solutes take to stay within the liquid phase that is
entrapped in matrix

107
• This time is related to fraction of pores that are
accessible to the solute
• Total volume of column of gel is given as
Vt  V0 Vi V g
where V0 = Void volume
Vi = Pore volume
Vg = volume of gel matrix

• Inner volume of gel is given as

Vi  WS
where W = Dry weight of gel
S = Solvent regain
108
• Elution volume of compound is given as

Ve  V0  K dVi
• Rearranging the above equation,

Ve V0
Kd 
Vi
– For large molecules which are completely excluded from the gel,
Kd = 0 and Ve = V0
– For molecules of very small size which have complete
accessibility to the gel pores, Kd = 1 and Ve = V0 + Vi

109
• The relationship between elution volume and molecular
weight is given as

Ve  a  b log M
Intercept = a
Ve / tr

Slope = -b

Log Molecular Weight, kDa 110

• For multiple components with different molecular
weights,
– At different concentrations, the weight average molecular weight
of the entire mixture is given as

Mw   cM i i

c i

– With varying number of each component, the number average

molecular weight of the entire mixture is given as

Mn   nM
i i

n i

111
Materials
• Gels commonly used are
a. Cross-linked dextrans (Sephadex)
b. Agarose (Sepharose)
c. Polyacrylamide (Bio-gel P)
d. Polystyrene (Bio-Beads S)
• Columns used are long in order to increase the
amount of stationary phase and hence pore volume

112
Applications

Types

Fractionation

Estimation of
Desalting molecular weight

Determination of
equilibrium constant

113
1. Fractionation
• It is possible to use a gel which will exclude protein of
interest and include contaminants or vice-versa
• This requires gel of optimal properties of high pore
volume and suitable pore size to elute protein of
interest
• Example: Purification of anti IgE-β-galactosidase
conjugate on a sepharose-6 gel

2. Desalting
• Highly useful for desalting as biomacromolecules differ
greatly in size from salts and other small molecules

114
• Porosity of gel is selected to exclude solute to be
desalted and particle size is chosen to give low surface
area
• Desalting is carried out with distilled water or volatile
buffer salts
• Used for termination of reactions between
macromolecules with low molecular weight substances
• Also used for the removal of
a. Phenol from preparations of nucleic acids
b. Unincorporated nucleotides during DNA sequencing
c. Free low molecular weight labels from solutions of labelled
proteins
d. Products, cofactors and inhibitors from enzymes

115
 3. Determination of equilibrium constants
• This can be used to study chemical equilibria
• In slow reactions, reactants and products separated in
gel filtration column are quantitatively determined for
finding equilibrium constant
• In fast reactions, one reactant is chromatographed in an
eluent containing the other reactant and reaction is
studied from elution curves
• This is useful in study of
a. Protein binding of low molecular weight substances like drugs
b. Competition of two molecules for the same site
c. Estimation of reaction rates

116
4. Estimation of molecular weight
• Used for determining molecular weight or size of native
or denatured globular proteins under different conditions
like pH, ionic strength and temperature
• Can be carried out in the presence of urea or guanidine
hydrochloride which transforms polypeptides and
proteins to a random coil configuration and reduces
structural differences
• Mixture of molecular weight markers or standards with
known molecular weights is applied to column and
chromatogram is obtained
• Test sample containing protein whose molecular weight
is to be determined is then eluted under identical
conditions of eluent composition, flow rate and column
dimensions
• Based on elution volume of test sample, a rough
estimate of molecular weight is obtained
117
 I. AFFINITY CHROMATOGRAPHY

• It exploits unique property of extremely specific

biological interactions to achieve separation and
purification
• Does not rely on differences in physical properties of
molecules to be separated
• Material to be isolated should be capable of binding
reversibly to a specific ligand that is attached to an
insoluble matrix

M + L ML
(Macromolecule) (ligand) (Complex)

118
 Only one kind of
molecule in complex
mixtures of
molecules get
attached to the
stationary matrix

All other molecules

wash through
• Column procedures are carried out where substances
bind to ligands
• In case of enzymes, ligands can be:
a. Substrate
b. Reversible inhibitor
c. Allosteric activator
149
 Wash Elute with
Load in pH 7 buffer pH 3 buffer
Protein
recognized
by antibody

Protein not
recognized
by antibody

Antibody
3 2 1

Antibody affinity chromatography

150
Matrix
• An ideal matrix must possess the following
characteristics:
a. Must contain suitable and sufficient chemical groups to which
ligand is covalently coupled
b. Must be stable during binding of macomolecule and its
subsequent elution
c. Should exhibit good flow properties
d. Must interact only weakly with other macromolecules to minimize
non specific adsorption
e. Uniform, spherical and rigid particles like cross – linked dextrans
(Sephacryl S), agarose (Sepharose), polyacrylamide gels (Bio-
Gel P), cellulose, silica are used

151
Selection and attachment of ligands
• It is possible to select a ligand that displays
a. Absolute specificity where it will bind exclusively to one
particular compound
b. Group selectivity where it will bind to a closely related group of
compounds with similar chemical specificity
 Example: ligand 5’ – AMP can bind reversibly to many NAD+ dependent
dehydrogenases since it is structurally similar to part of NAD+ molecule

• Ligand should have suitable chemical groups like –

COOH, –OH, which are used to attach ligand to matrix
• Most common method of attachment of ligand to matrix
involves preliminary treatment of matrix with cyanogen
bromide, sulphonyl chloride, sodium periodate

152
• Reaction conditions and relative proportions of reagents
will determine number of ligand molecules that can be
attached to each matrix particle
• Spacer arms of six to ten carbon atoms or equivalent are
interposed between ligand and matrix to prevent
interfering with ability of ligand to bind to the
macromolecule
• Spacers are mostly purely hydrophobic consisting of
methylene groups
• Some are hydrophilic possessing carbonyl groups
• Spacers are most important for small immobilized ligands
– Examples of spacer arms include 1,6-diaminohexane; 6-
aminohexanoic acid

123
 Spacer arm

Ligand
Matrix Enzyme Bound enzyme-wash free
of contaminating proteins
OR
Non – specific
Specific elution with
elution

Dialysis Restore optimum

conditions

Purified enzyme
Purification of enzyme by affinity chromatography
124
a. Specific elution
• This is affinity elution and involves addition of high
concentration of substrate or reversible inhibitor if it is
enzyme
• It involves addition of ligands for which macromolecules
have higher affinity than it has for immobilized ligand

b. Non-specific elution
• It is achieved by changes in pH or ionic strength
• pH shift elution using dilute acetic acid or ammonium
hydroxide results from change in state of ionization of
groups in ligand or macromolecule
• pH of collected fractions are readjusted to optimum
values to minimize protein denaturation
• Change in ionic strength also causes elution due to
disruption of ligand macromolecule interaction
125
 Applications
• Purification of wide range of enzymes and other proteins
like receptor proteins and immunoglobulins
• Immobilized nucleotides are useful for isolation of
proteins involved in nucleic acid metabolism
• Messenger RNA is routinely isolated by selective
• hybridization on Sepharose 4B
• Immobilized single stranded DNA is used to isolate
• complementary RNA and DNA

156
Examples
i. Lectin affinity chromatography
• Lectins are groups of proteins produced by animals,
plants and slime moulds
• These are tetrameric structured with molecular weights
between 40000 to 400000
• Their subunits may be identical or of two types
• Have the ability to recognize and bind specific
saccharides
• These are valuable in the purification of glycoproteins
especially membrane receptor proteins
• Most widely used lectins are from leguminous plants due
to their abundance
• These can be immobilized to agarose matrix
157
• Once glycoproteins are bound to immobilized lectins,
elution is done in number of ways:
a. Affinity elution using simple monosaccharide for which lectin has
an affinity
b. Use of borate buffer which forms complex with glycoprotein
c. Changes in pH (not below pH 3 and not above pH 10)
d. Addition of reagent like ethylene glycol to reduce ligand
hydrophobic interaction
• This can be carried out in presence of high salt concentrations since it does
not rely on ionic interactions
• It can be applied directly after salt fractionation

ii. Immunoaffinity chromatography

• Uses antibodies as immobilized ligand
• These are linked to agarose matrix by cyanogen bromide
technique
158
• Protein binding to immobilized antibody is achieved in
neutral buffer solution containing moderate salt
concentrations
• Elution is done by forceful conditions
• Elution procedures include use of high salt
concentrations with or without use of detergents and use
of urea, sodium dodecyl sulphate ----- lead to protein
denaturation
• Use of chaotropic agents like thiocyanate, perchlorate
and trifluoroacetate or lowering pH to 3 avoids
denaturation
• Used in isolation and purification of range of proteins

159
 ELECTROPHORESIS
Principle
• Electrophoresis is the migration of charged particles or
molecules in a medium under the influence of an applied
electric field
• Upon suspension in aqueous solvent, particles like red
blood cells, bacteria and important biomolecules like
nucloetides, amino acids, peptides, proteins aquire
positive or negative charges depending upon the nature
of particles and solvent
• Net charge density of molecules makes them to move in
electric field in a direction and at a velocity depending
upon its sign and quantity
• Smaller particles move faster than larger ones.

130
• The velocity of charged particle in an electric field is
– Directly proportional to the
• Voltage (ΔV)
• Charge on the protein (X)
– Inversely proportional to the
• Distance between the electrodes (ẟ)
• Size of the protein

Where D = Diffusion coefficient of the protein and it is inversely proportional to

the size of protein and viscosity of solution.
Π= Constant
= 96500 current / mol

131
• First measurements of electrophoretic phenomenon
were recorded in 1861 by Quincke
• Usual purpose of electrophoresis are:
– To determine the number, amount and mobility of the
components in a given sample or to separate them
– To obtain information about electrical double layer surrounding
the particles
• Electrophoretic mobility is given as

where v = velocity of ion

ε=electric field strength

132
• Electrophoresis is carried out in appropriate buffer which is
essential to maintain constant state of ionization of molecules
being separated
• Any variation in pH alters the overall charge and hence
mobilities of molecules being separated

Applications
• Most widely used in biochemistry and molecular biology lab
• Health and medical field including antibiotic and vaccine
analysis
• Protein and DNA analysis
• Purification, processing, analysis of influenza, hepatitis and
polio vaccine
• Gel electrophoresis is used in forensics

133
• Types of Electrophoresis
– Paper
– Cellulose acetate
– Gel
– SDS-page
– Capillary
– Isoelectric focussing

134
 1. Paper Electrophoresis
• Equipment consists of power pack and electrophoretic cell
• Power pack provides a stabilized direct current with
controls for both voltage and current output
• Electrophoretic cell contains electrodes of platinum, buffer
reservoirs, support for paper and transparent insulating
cover
• Two buffer reservoirs are each partitioned into two
interconnected sections
 One containing electrodes
 Other in contact with supporting medium
• Separate compartments are necessary so that any
change in pH occurring at electrodes does not affect
buffer in contact with supporting medium

132
• Wicks made of several layers of filter paper are made to
dip in buffer
• Paper is placed on insulating material like perspex sheet
• Whole unit is covered with insulating material to minimize
evaporation during
• run and ensure electrical insulation
• Two arrangements of filter strips commonly used
 Horizontal
 Vertical

136
 Power pack

Perspex cover Filter paper

Wick

- +

Buffer reservoir Insulating plate

Glass plate
Reservoir
Horizontal paper electrophoresis
137
 - +

Buffer reservoir
Electrodes

Filter paper

Buffer reservoir

Vertical paper electrophoresis

138
Buffers used
• Depends on the sample to be separated
• Examples include
 Borate at pH 9.0 and ionic strength 7.63 for proteins
 Acetate at pH 4.5 and ionic strength 0.1 for mucoproteins
 Phosphate at pH 4.6 and ionic strength 0.15 for amino acids
 Tris acetate at pH 7.8 for nucleic acids

139
 2. Cellulose acetate electrophoresis
• This was introduced by Kohn in 1958
• Developed from bacteriological cellulose acetate
membrane filters
• Commercially available as high purity cellulose acetate
strips, which are thin and have uniform micropore
structure
• Used for clinical investigations such as the
separation of glycoproteins, lipoproteins and
haemoglobin from blood

140
• Advantages of cellulose acetate:
 No adsorption problems like paper
 Chemically pure and does not contain lignins, hemicellulose or
nitrogen
 Translucent and are suitable for direct photoelectric scanning for
separated bands of components
 Not hydrophilic and holds very little buffer and is very
conducive for better resolution in shorter time

141
 3. Gel Electrophoresis
• Gels are porous materials
• Size of pores relative to that of molecule determines
whether molecule will enter the pore and be retarded or
will bypass it
• Separation of molecules depend on size of molecules and
also charges
• Resolution of sample is sharper and better than any other
type of medium

a. Starch gel
• First introduced by Smithies in 1955
• Potato starch is hydrolyzed in acidified acetone at 37 0C

142
• Suspension is neutralized with sodium
acetate and washed with large amounts of distilled
water and dried with acetone
• Hydrolyzed starch when heated and cooled in
appropriate buffer sets as gel
• High porosity starch gels are obtained with 2 % w/v
starch

b. Agar
• First described by Gordon in 1949
• Consists of two galactose based polymers agarose and
agaropectin

143
• Agarose is sulphated and is charged and this gives rise
to severe electroosmosis detrimental for electrophoretic
separation
• Solution of agar at 80 0C is mixed with equal volume of
40 % polyethylene glycol, which precipitates agarose
• Agarose is collected, washed with large amounts of
distilled water and dried with acetone and is free of
sulphate
• Agar solubilizes in aqueous buffers above 40 0C and sets
to form gel at about 38 0C
• Useful in immunoelectrophoresis for detection of
antigenic properties
• Used to separate high molecular weight
macromolecules like proteins and nucleic acids

144
c. Agarose-acrylamide
• This is complex gel used to separate very high molecular
weight nucleic acids (200 Kd or more)
• So complex gel was first used by Peacock and Dingman
in 1968
• Prepared by dissolving 0.5 % agarose in boiling water
and allowing it to cool to 40 0C
• Components needed for polyacrylamide gel
formation mixture is transferred to either column or slab
gel rig
• Acrylamide used is as low as 0.5 %
• Gel sets on cooling yielding highly porous and rigid matrix
• Successfully used for isolation of 3.5 X 106 daltons DNA

145
4. SDS- page Electrophoresis
• Technique commonly used to separate proteins
according to electrophoretic mobility and is a function of
molecular weight and post-translational modifications.
• Molar mass of proteins can be estimated by
determination of electrophoretic mobility in
polyacrylamide gel, after unfolding protein using
denaturation agents like sodium dodecyl sulphate and β-
mercaptoethanol or dithiothreitol.
• Surface charge on long rod of constant width SDS-
protein complex is almost entirely due to exposed
sulphate ions.

146
• In SDS-PAGE, overall negative charge on protein is
obtained after denaturation and bonding with sodium
dodecyl sulphate molecules.
• Nearly one molecule of SDS binds to every two amino
acids via its hydrophobic alkyl chains, resulting in
production of denatured protein with large negative
charge proportional to its mass.
• SDS protein mixture is applied to sample wells on top of
polyacrylamide.
• Once desired separation has occurred, gel is removed
from apparatus and protein is visualized.

147
• Separation occurs as small molecules move faster
and larger molecules move slower through the gel.
• The mobility of most of the polypeptide chains is linearly
proportional to the logarithm of their mass
• Molecular mass of unknown protein can be determined
by comparing it with protein of known molar mass
• SDS-PAGE is rapid, sensitive and commonly used
technique to determine the degree of purity of protein
sample, molecular mass of unknown protein and
number of subunits within protein

148
149
150
5. Capillary Electrophoresis

151
• Consists of inlet and exit electrolyte reservoir, sample
container, capillary, detector and high voltage power
supply.
• Reservoir and capillary are filled with buffer solution,
electrolyte.
• Inlet of capillary is placed in sample container which
introduces microlitres of sample into capillary.
• Capillary inlet is placed back into inlet reservoir and
electric field is applied.
• Charged solutes migrate through capillary due to electric
field, detected by detector

152
• High voltage of 30 kV is used for fast and efficient
separation.
• Used to determine samples like vitamins, inorganic
acids, carbohydrates, environmental pollutants,
nucleotides, pharmaceuticals, amino acids, peptides
and proteins

153
 6. Isoelectric Focussing
• Discovered by H. Svensson in Sweden
• Also called electro focusing
• Has high resolution power and can resolve plasma
proteins into 40 bands
• Proteins are separated by an electric current that passes
through a gel containing a pH gradient
• Protein’s charge depends on the charge of its amino acid
side chains and these charges change with pH
• Stable pH gradient is arranged with pH increasing
gradually from anode to cathode

154
• Protein introduced into this system at point where pH is
lower than its isoionic point will possess net positive
charge and migrate in direction of cathode
• Presence of higher pH gradient influences ionization and
net charge of molecules
• Finally protein encounters a pH where its net charge is
zero (isoelectric point) and it stops migrating
• Every protein will migrate to and focus at its respective
isoelectric point in stable pH gradient, irrespective of
origin in apparatus at the time current was applied
• Point of application and volume of protein solution are
not critical
• Diffusion is not a problem with electro-focusing

155
Cathode
- Direction of
X-, Y-, Z- migration of
anions
X-

Y-

Z-

X
Y Zwitterions
Z

X+
Y+

Z+
Direction of
migration of
X+, Y+, Z+
+ cations Increasing
pH
Anode 156
• pH gradients are obtained by electro focusing special
buffer substances known as carrier ampholytes
• Carrier ampholytes are available commercially in
mixtures covering
– wide pH band (pH 3 – 10) or
– narrow bands (pH 5 – 6)
• Commercial ampholytes include
 Ampholine
 Pharmalyte
 Bio-lyte
• Carrier ampholytes should have the following properties:
 Certain buffering capacity at their isoelectric point
 Conductance at their isoelectric point
 Low molecular weight so that macromolecules can be separated
from them easily after electro focusing
157
  Soluble in water so that these do not bind to hydrophobic regions
of proteins
 Low light absorption at 280 nm which permits detection of
proteins after electro focusing by measurements at 280 nm
• Immobilized pH gradients (IPGs) can be used because
the pH gradients remain stable, at very high voltages for
a long time
• Buffering compounds are used to create pH gradients
• IPGs are cast strips of gel with plastic backing sheets
and are available in different pH ranges and lengths
• These offer
– High resolution
– Reproducibility
– High protein loads

158