DVANTAGES: TWO-DIMENSEONAL GEL ELECTROPHORESIS

(2-1) ELLCTROPHORESIS
As spreading of bands is minimized due to application of the applied field and In the first dimension, proteins are resoved in according to their Isoelectric
the PH gradient, high resolution can be achieved. points (PI) using immobilized pH gradient electrophoresis (IPGE), isoelectric
focusing (IEF), or non-equilibrium pH gradient electrophoresis. (Horizontal
Proteins that differ by as little as 0.001 PH units can be separated. seperation)
I n the second dimension, proteins are separated according to their approximate
SADVANTAGES: molecular weight using SDS-PAGE. (Vertical seperation).

Pratein H 40
Because carrier ampholytes are generally used in high concentration, a
high Two DimensionalElectrophoresis morture
voltage (upto 2000v ) is necessary. As a result the electrophoretic matrix must &pert Isoelocric
tosing (EF)
dfmension
by charg
be cooled which sometimes makes it difficult. 1gh 10.0

otop o ond
PPLICATIONS: MW.

For separating proteins and peptides.

crophoresis

For research in enzymology, immunology, cytology and taxonomy. (1)JEF or IPG (2) SDS-PAGE
7-72 hr,

PAPPLICATIONS OF ELECTROPHORESIS
DNA Sequencing
Medical Research

Protein research/purification
Agricultural testing
Separation of organic acid, alkaloids, carbohydrates, amino acids, alcohols, phenois, nucieic

acids, insulin.
In food industry
i.e. in the study of protein mixtures such as blood
It is employed in biochemical and clinical fields

haemoglobins and in the study of antigen- antibody interactions.

serum,
is used to study the binding of iron to serum
Electrophoresis in combination with autoradiography
proteins.
Used for analysis of terpenoids, steroids and antibiotics.

For testing purity of thyroid hormones by zone electrophoresis.

from plasma proteins
Paper chromato-electrophoresis is used to seperate free Insulin
diseases of kidney, liver and CYS.
t is used for diagnosis of various

buffer at PH 4.2.
is also used for separation of Scopolamine arid Ephedrine using
t
for separation of carhohydrates and vitamins.
Electrophoresis is also used
etc is
fractions of cellular entities, antibiotics, RBC, Er.zymes possible.
Quantitaive separation of all
 METHUD
1s usually carried out in 2% agar gel medium.
PRINCIPLE: 1SOELACTRIC FDCUSING4)
The antigen mixture is applied into a small circular wells cut out of
agar and the initlal All proteins have an isoelectric point pH.
electrophoretic seperation is carried out depending on their charge and molecular welght. When electrophoresis is run in a solution buffered at constant pH, proteins having a net charge will
PAfter the initial seperation; the antibody mixture is then introduced into a narrow slot in migrate towards the opposite electrode so long as the current flovs.
the The use of pH gradient across the supporting medium causes each protein to migrate to an area of
gel about 0.5 to 1.0 cm from the separated antigens.

During this period the antigen components diffuse radialy autwards, towards the sgecific pH.The pH of the protein equals the pH ofthe gradient,thus resuting in sharp well defined
,
diffusing protein bands.
antibody and precipitation takes place in elliptical arcs as related antigens and antibodies A procedure to determine the isoelectrie point (P) a
ofproteins thus, mixture of protelins
ean
be
diffuse into one another. electrophorised through a solution having a stable pH gradient in from the anode to the
cathodo and a each protein will mlgrate to the position in the pH gradlent according to its
ADVANTAGES: isoelectric polnt. This is called isoelectric focusing.
Spreading of bands is minimized due to the application of the applied field and the pH
gradient, high resolution can be achieved. Protein migrate into tine point where its net charge is zero- isoelectric pH.
Protein is positively charged in solutions at pH below its Pl and will migratetowards the cathode.
DISADVANTAGES: Protein is negatively charged in solution at pH abové its Pl will migrate towards the anode.
Carrier ampholytes generally are used in relatively high concentration, a high voltage power
source ( up to 2000V) is necessary and power is in the vicinity of 2 to 50 WAs a result the
They will be in the Zwitter jon form with no net charge so the further movement will cease.
electrophoretic matrix must be cooled. Ampholytes (amphoterlc electrolytes low molecular mass (600-900D) coligomers wth aliphatic
amino and carboxylic acid groups with a range of isoelectric points. Ampholytes help maintain the
APPLICATION pH gradiennt in the presence of high voltage
Mainly used for separating protein and peptides. Can also use gels with immobilized pH gradients made of acrylamide derivatives tha an
Used in clinical, forensic and human genetics laboratories for the separation and covalently linked to ampholytes.
identification of serum Protein in reseach in enzymology, membrane
biochemistry, microbiology and immunology.

Stable pH gradiient
METHOD 1 7 to 11 12 13 14
p H gradient is established in gel by addition of ampholytes which increases the pH from anode to cathode. anple ppiedt ptngon
A protein mixture ls placed in a well on
the gel.
Wth an applied electric field, proteins enter the gel migrates until each reaches lts pH equivalent to its (Pl).

Each specles of proteins Is therby focussed into a narow band about its Pl.
The Anode of the column is connected to a reservoir containing an acidic solution like phosphoric acid and
Cathode is connected to a reservolr containing alkalline solution lke sodlum hydroxlde.

allowed to diffuse Into the column from their

the two reservoir valves the two solutlons A lowpH, mos protebie arve a posidva charge while m high
are
On opening
respective ends, setingup a PH gradlent between the
acidic anode and the alkaline cathode. pH imod pAotein: have negtve cuange.
T h e vaves are then closed and the curent is switched on , causing the camer ampholytes to mlgrate until they reach

ine P H regions where they have no net charge. They will then remain statonany at these points.

SnN comporen

Ampholytes ncasngp
polyacrylamide
When an electric nedd is prese, he cahode indauode endnN
the poleu*
prota
(o
dho oelecaicPOM vge #cli imIidhal
pOWHeSNeS. A Nentml clharge
Cathode
()
electrode
solution

The rotelna otoiyedanlgrtiug becusa theyve unchedhar

4OelociiS pont a a AdquepHlevl.
Anode Cod
(+)
electrode
 The
IsOTACHOPHORESIS
technique of isotachophoresis depends on the development of potential gradient.
Electric Freld
We fil one well with slow trailing
electrolyte (T) mixed with
samples (S1,S2), and the other
well with fast leading electrolyte
(). When we apply an electric
PRINCIPLE:
field, ions electromigrate
Based on principle of moving boundary electrophoresis. through a microchannel
according to their electrophoretic
A leading electrolyte(e.9. chloride) with a higher mobility than the analytes, and a traling mobilities.
electrolyte(eg. glycinate) with a lower mobility are used.

Solution in which the separation takes place is normally an aqueous medium, which contains sucrose to
provide a higher density to the solution. concentatlon Sample ions overspeed the slow
trailing electrolyte, but cannot
Where the separation by Isoelectric focusing depends on the existence of a pH gradient in the Overspeed the fast leading
system. The technique of Isotachophoresis depends on the development of a potential gradient. electrolyte: consequently, they
focus at the interface.
Separation of the ionic components of the sample is achieved through stacking them into discrete zones

in order of their mobilities, producing very high resolution.

Gons (MI| In the example shown here three Conceniratjon

Sample continues to accumulate.
particle classes with different If sample concentration

charges are being separated and approaches the concentration of

via the leading electrolyte, sample
preconcentrated into discr
electrophoresis. After the self-segregate
separation is concluded a l Zones.
particles move at a constant
speed (lsotachophoresis)

The analytes are positioned between the electrolytes and, when the voltage is applied, they migrate in
order of decreasing mobility.
IMMUNO ELECTROPHORESIS
Antibodies are produced by immune system in response to foreign macromolecules. Each antibody
This establishes the potential gradient; from that point on, all the analytes move at the same speed.
binds specifically to one feature(epitope) on ane macromolecule(antigen).This alows the use of
border one another but represent completely separated components
with out overlap.
Individual zones antibodies for detection and quantitation of specific proteins in a complex mixture.
is mixed with the sample, so current flow is carried
In isotachophoresis no background electrolyte(buffer) aWhen Electrical potential is applied to study of antigen-antibody reactions, it is called
only by charged sample ions. Immunoelectrophoresis. The antibody are electrophoretically separated and antigens diffuse
Once a faster moving component separates completely from a
slower moving one, It createssa towards each other resulting in precipitin arcs where antigen antibody complexes fom. This
local
region of depleted charge between the two that increases the resistance and therefore technique has been referred to as Immunoelectrophoresis.

voltage in that region. Antibody is placed in trough cut parallel to the direction of the electrophoresis.
faster and close the gap, thereby R u n the electrophoresis as a resut preclpltn arcs wll be formed due to Ab-Ag complex
This increased voltage causes the slower component to migrate
of its z o n e until it matches that of the faster ion.
concentrating it and increasing the conductivity formation.
that differ in thickness, depending on
well in thick layer of bufered agar and an electric
Uitimately all ions migration at the rate of the faster ion in the zones
A fluid containing proteins antigens is placed in a a

their original concentrations. Current is applied, antigens will be distributed in separate spots along a line passing through the wel
and parallel to the direction of current flow.
APPLICATION: p a n d are
O Andaen

well as
sotachophoresis that been used for the separation of proteins
as 38
AnlOOCE

inorganic substances. 2 Antgon

 CAPILLARY ELECTROPHORESIS Capllary Electrophoresis
L1ght
30urce
The principle behind electrophoresls Is that charged molecules will migrate toward the opposlte
pole and separate from each other based on physical characterlstics.
Capllary electrophoresis has grown to become a collectilon of a range of
separation techniques Computor
which Involve the application of high
voltages across buffer flled caplllaries to achleve Dete In Photocathode
separatlons. DateChart
Out Recorder
Capillary electrophoresis, then, is the technique of performing electrophoresis Buffer
In buffer
filled, narrow-bore capillaries, normally from 25 to 100 mm in Internal dlameter (10).
Cathode Anode
A high voltage (typlcally 10-30 kV) is applied.
Capillaries are typically of 50 um inner diameter and 0.5 to 1 m in length.
Due to electroosmotic flow, all sample components migrate towards the negative electrode. me (min)_
Sample applicatlon is done by either
The capllay can also be flled with a gel, which eliminates the electroosmotilc flow. Separatlon Is a)High voltage injoction-potentlal is applied causing the sample to enter capillary by,
accomplished in conventlonal gel electrophoresls but the capllary allows combination of ionlc attraction and electroosmotic flow.
as
hlgher b)Prossure injoction-pressure dfference is used to drive the sample into capillary by
resolution, greater sensitivity, and on-line detection.
applying vaccum.
The capillary is filled with electrolyte solution which conducts current through the inside of the When PD is applied net migration occurs in the direction of cathode.
capillary. The ends of the capillary are dipped into reservoirs flled with the electrolyte Even substance with net negative migrate in the direction of cathode due
charge to the
phenomenon called as Electro Osmotic Flow.
Electrodes (platinum) are inserted into the electrolyte reservoirs to complete the electrical rt Neutral molecule moves at the same speed as the EOF. Positively charged species
faster, speed is sum of EOF and Electrophoretic mobility. Negatively charged molecules
lag behind.

A small volume of sample is moved into one end of the capil ary. The capil ary passes
ELECTROOSMOTICFLOW through a detector, usually a UV absorbance detector, at the opposite end ofthe capillary.

Application of a voltage causes movement of sample ions towards their appropriate

The surface of the silicate glass capillary contains negatively-charged functional electrode usually passing through the detector.

groups that attract positively-charged counterions. The positively-charged ions A plot of detector response with time is generated which is termed an electropherogram.
migrate towards the negative electrode and carry solvent molecules in the same
direction. This overall solvent movement is called electroosmotic flow. During a

separation, uncharged molecules move at the same velocity as the electroosmotic Capilary tupe aa aquistion
flow (with very little separation). Positively-charged ions move faster and
negatively-charged ions move slower. Sample inlet

Glass capillary

Bauifer electrole

&88888. Anode Cathode 34

Electroosmotic flow
Eledtoosnoti low
 THIN LAYER ELECTROPHORESIS XCELLULOSE ACETATE ELECTROPHORES
It contains 2-3 acetyl groups per glucose unit and its adsorption capacity is less than
that of paper
o Studies can be carried out in thin layer of silica, keisulguhr, alumina.
It gives sharper bands.

Provides a good background for staining glycoproteins.

ADVANTAGE:
ADVANTAGES: No tailing of proteins or hydrophilic materials.
o Less time consuming and good resolution. o Available in wide range of particle size and layer thickness.
o Give sharp bands and offer good resolution.

APPLICATION: oHigh voltage can be applied which will enhance the resolution.
DISADVANTAGE:
o
Widely used in combined
electrophoretic-chromatography studies in two aExpensive.
dimensional study of proteins and nucleic acid hydrolysates. a Presence of sulphonic and carboxylic residue causes induced electroosmosis during
electrophoresis.
APPLICATION:
o Widely used in analysis of clinical and biological protein samples
(albumin and globulins).
Alternative to paper electrophoresis.

MoVING BOUNDARY ELECTROPHORESIS

PRINCIPLE: ADVANTAGES:
The moving boundary method allows the charged species to migrate in a free moving Biologically active fractions be recovered without the
solution without the supporting medium.
o can use
of denaturing
agents.
INSTRUMENTATION:
Consists of a U shaped glass cell of rectangular cross section, with electrodes placed on o Areference method for measuring electrophoretic mobilities.
the one each of the limbs of the celil.
Sample solution is introduced at the bottom or through the side arm, and the apparatus is
o Minute concentrations of the sample can be detected.(0.05mg/ml by
placed in a constant temp. bath at 40° C. Interferometric optical system).
Detection is done by measuring refractive index throughout the solution.(Schlieren optical
system).
Anode Cathoae
DISADVANTAGES:
o Costlier.
Ascending
Dounday Initlal boundarkes Burer Bulor

Boundary
a Elaborate optical system are required.
Descending
boundary

Prote in molecules
APPLICATION:
-Saumple inlet
o To study homogenecity of a macromolecular system. 30
-observ
boundarles by
optical system o Analysis of complox biological mixtures.
Moving Boundary Eechrophore is
 STARCH VERTICAL AND HORIZONTAL
A suspension of granular starch should be boiled
clear colloidal suspension.
in a buffer to give a
Hhor pape oUp .
GEL ELECTROPHORESIS
The suspension on cooling sets as a semisolid Methods for application of
the branched chains of
gel due to intertwining of Trough
e a r a go
amylopectin. sample:
o In order to avoid swelling and
shrinking petroleum jelly is used. mpl
a) Sample is mixed ith the

gel.
ADVANTAGES: b) By soaking a bit of filter
High resolving power and sharp zones are obtained.
paper in sample and
The components resolved can be recovered in reasonable yield pehroleuan ely pressing it into the gel.
especially proteins. Fiter paper
Can be used for analytical as well as preparative electrophoresis. connedng st
e Detection by staining or
by Direct gravimetry or by
DISADVANTAGES: UV absorption, etc.
Electro osmotic effect.
Variation in pore size from batch to batch.

PULSED-FIELD GEL ELECTROPHORESIS

Pulsed field gel electrophoresis is a technique used for the separation of large

DNA molecules by applying to a gel matrix an electric field that periodically changes

direction.
While in general smal fragments can find theirway through the gel matrix more easily than
large DNA fragments, a threshold length exists above 30-50 kb where all large fragments will
run at the same rate, and appear in a gel as a single large diffuse band. However, with

periodic changing of field direction, the various lengths of DNA react to the change at
differing rates. Over the course of time with the consistent changing of directions, each band
wilbegin to separate more and more even at very large lengths. Thus separalion of very large
DNA pieces using PFGE is made posslble.
The voltage ls perlodically switched among three directlons; one that runs through the
central axis of the gel and two that run at an angle of 60 degrees eithor side. The pulse
times are equal for each direction resulting In a net forward migratlon of the DNA.

This procedure takes longer than normal gel olectrophorosis due to the slze of the fragments APPLICATIONS:
being resolved and the fact that the DNA does not move in a stralght line through the gel.
26
PFGE may be used
forgenotyping or genetic fingerprinting. It is commonly
considerod a gold standard in epidemiological studies af pathogenic organisms.
 SLAB PAGE SDS-POLYACRYLAM:DE GEL
The Polyacrylamide gel is cast as
thin rectangular slab inslde a plastic frame and this slab is ELECTROPHORESIS (SDS-PACGE)
olaced vertlcally on a buffer solution 'aken in a reservoir.
Several samples dissolved in dense sucrose solution SDS-PAGE, sodlum dodecyl sulfate polyacrylamide gol oloctrophoresls,
or glycerol are placed in separate is
walls cut In to the upper edge of the slab and are technique widely used in blochemistry, forensics, genetics and molecular biology to
oovered by the same buffer
Cathode. and anode are abovo and below to solutlon, 8eparate protelns according to thelr electrophoretlc mobillty.
produce electric fleld effect. Different
components migrate simultaneously down parallel lanes in the slab and When a detergent SDS added to PAGE the combined procedure is termed as SDS PAGE
get separated into
bands. SDS coats protein molecules glving all proteins a constant charge-mass ratlo.

VISUALIZATION Due to masking of charges of proteins by the large negative charge on SDS binding
with them, the protelns migrate along the gel in order of
increasing_sizes or
After the electrophoresis is complete, the molecules In the molecular welghts.
gel can be : lained to make
them
visible. SDS is an anlonlc detergent which denatures secondary and non-disuflde-linked
tertiarý
Ethidium bromide, silvor, or coomassio bluo dye may be used for this process.
structures by wrapping around the polypeptide backbone. n
negative charge to the polypeptlde in proportion to lts length.
so doing, SDS confers a
ne
If the analyte molecules fluoresce under ultraviolet light,
photograph can be taken of the
a
Moiecules in solution wlth SDS have a net negative charge within a wide pH range.
gel under ultraviolet lightling conditions. If the molecules to be separated contain
added for visiblity, an autoradlogram can be recorded of the
radiaacyy A polypeptide chain binds amounts of SDS in proportion to its relative molecular mas20
gel. The negative charges SDS destroy most of the complex structure of proteins,
on
andare
strongly attracted toward an anode in an electric field.

SODIUM DODECYL SULFATE Denature anple wlth

o d u m ciodeayisurato
3 nto luailze
sopurated bende

(SDS-PAGE) SDS-coated
proreins
SDS Is an lonic detergent that binds Dacreasino
Native protein is unfolded by heating in the stlochiometrlcallyto númber of peptlde bonds,
Native protein thus separates on basis of sze (MW
presence of B-mercaptoethanol and SDS.
2Placemixture of proteins
on gel, applyelectrio field

SDS binds to the protein so that it stays in

Heat
solution and denatures.
Cross-linked
ylamide
Portiully

Large polypeptides bind more SDS than Reductant Separated-

proteins Direction of migration

small polypeptides, so proteins end up with

SDS
negative charge in relation to their_size.

When treated with SDS and a reducing

agent, the polypeptides become rods of
negative charges with equal "charge densities"
or charge per unit length.
Denatured protein
o

Thus, we can separate the proteins based on

with bound SDS
heir mass.
 roLYACRYLAMIDE GEL ELECTROPHORESIS TYPES OF PAGE
(PAGE) PAGE can be classifled according the separation Pogerariaye ecirgkeesa
conditlons Into:
t ls propared by polymorlzing aaryl amidetomonomora In tho
cross link tho CH--CH
presenca of methylono-bls-acrylanmido
monomers. NATIVE-PAGE:
Structure of acrylamide (CH®CH-CO-NH,) are run in non-denaturing conditions, so that
Natlvo golo
Polyacrylamldo gol structuro hold togothor by covalont the analyte's natural structure is maintalned.
cross-lInks.
Polyacrylamide gels are tougher than agarose gols. of
Seporation ls based upon charge, slze, and shape
is thermostable, transparent, strong and relatively chemically
It macromolecules
inert.
Gels are uncharged and are prepared in a variety of pore Useful for separation or purification of mixture of
sizes.
Proteins are soparated on the basis of charaa to nass proteins. Annau
and molecular size, a phenomenon called Molecular This was the orlglinal mode of electrophoresls.
ratlo
sleving.
o DENATURED-PAGE OR SDs-PAGE: tar
of
ADVANTAGES: Separation is based upon the molecular welght aeomve
Gels are stable over wide range of pH and temperature.
Gels of different pore size can be formed. proteins
The conimon method for determining MW of proteins.
Simple and separation speed is good comparatively. Lnnal
Very useful for checking purity of protein samples.
+
tha
SLAB PAGE
PAGE-PROCEDURE PAGE PROCEDURE

is cast into cólumn inside a vertical tube, often with large

T h e gel of different pore sizes a

the bottom. Samplo wel

pore gel at the top and small pore gel at
column and covered
of the gel
oMicrogram quantity
by a buffer solution
of the sample
having such a
is placed

pH so as to
over the top

change sample components into anions. bufe

buffer In the bottom reservolr.
of the gel column is made to dip in the
same
oThe foot

Cathode and anode are kept above and below the column to impose an electric field
BuMera
through the column.

towards the anode down the gel column.

high voltage
So Macromolecular anions move

Co There is no external solvent space, all the migratory particles have

to pass through the Power supply Polyacrylamide Gel Electrophoresis
gel pores. (PAGE)
a) The gel is poured vertically
oRale of migration depends on the charge to mass ratio.
berween two glas plates.
Different sample components get separated into discrete migratory bands along theg b.) Protein bands are separated on88
column on the basis of electrophoretic mobility and gel filtration effect.
buiter 195 CHP
basis of relative molecular weight and
visualized with stains.
 GEL ELECTROPHORESIS
GEL ELECTROPHORESIS
thhe molecular
Separation is brought about through molecular sleving technique,
size of the substances. Gel material acts as a "molecular sleve".
based on
Gcl elcctrophoresis Gel Electrophoresis is
Gel is a colloid in a solid form (99% is water). caried out in two
It is important that the support media is electrically neutral.
Different types of gels which can be used are; Agar and Agarose methods
gel, Starch, Sephadex, Polyacrylamide gels.
A porous gel acts as a sieve by retarding or, In some cases, by completely
movement of macromolecules while allowing smaller molecules to
obstructing the 1. Vertical starch gel
migrate freely.
electrophoresis

*eiolalors 2. Horizontal starch

ww

gel electrophoresis

AGAR AND AGAROSE GEL DISADVANTAGES:

Electro osmosis is high.
o Agar is a mixture of poly saccharides extracted from sea weeds.
>Resolution is less compared to polyacrylamide gels.
Agarose is a highly purified uncharged polysaccharide derlved from agar.
Different sources and batches of agar tend to give different results and purification is
Agarose is chemically basic disaccharide repeating units of 3,6-anhydro-L-galactose.
oAgarose dissolves when added to bolling liquid. It remains in a liquld state unti the temperatureIs often necessary.
lowered to about 40°Cat which point it gels.
oThe pore size may be predetermined by adjusting the concentration of agarose in the gel.
APPLICATION:
Agarose gels are fragile. They are actually hydrocolloids, and they are held together bythe
formation of weak hydrogen and hydrephobic bonds.
Widely used in Immuno electrophoresis.
oThe pores of an agarose gel are large, agarose is used to separate macromolecules such as
nucleic acids, large proteins and protein complexes.
ADVANTAGES: GESTRuCTURE OF AGAROSE
Easy to prepare and small concentration of agar is required.
Resolution is superior to that of filter paper
Large quantities of proteins can be separated and recovered.
Adsorption of negatively charged protein molecule is negliglble.
It adsorbs proteins relatively less when compared to other medium. 5R
Sharp zones are obtained due to less adsorption.
Recovery of protein is good, good method for preparative purpose.
100°
initiel ge tinat.get strueture
$ostat
 HORIZONTAL PAPER ELECTROPHORESIS
PAPER ELECTROPHORESIS *****

AlR TIGHT HOUSING

45 V i Dc (5 batteries of9 v)n
ELECTROOE ELECTRODE-

two glass platea

FILTER PAPER STRIP
electrolyte
BUFFER SOLUTION

VERTICAL PAPER ELECTROPHORESIS

Pencl ine
Paper fiiter SAMPLE r'TIGHT
APPLICATION HausiNG
Figure 21 - Apparatus for paper electrophoresis. PINT

FILTER PAPER
STRIP ELECTRODE
ELECTRODE
1. Horizontal paper Electrophoresis

2. Vertical paper Electrophoresis

BUFFER SOLUTION-

o Filter paper such as Whatmann no1 and no 3mm in strip of 3 or 5cm CONTINUOUS ELECTROPHORESIS
wide have been used to good effect. sample reserviaur

o Separation takes place in 12 to 14hrs.

burier soludon

electrodes
ADVANTAGES:
sample camponents iter paper
oltis economical.

Easy to use.
ample collacton

DISADVANTAGESs: Used for preparative scale purpose.

Freáetermined sanpie voiune of 0.2 miiun üuvugia iue vaive device is apica
oCertain compounds such as proteins, hydrophilic molecules cannot
be continuously on the centre of the paper.
Silica gel, powdered glass, sand & agar gel can be used.
resolved due to the adsorptive and ionogenic properties of paper which APD of 500V is applied.
results in tailing and distortion of component bands. Purefractcompounds
ions
are collected in separate containers, the
are reused. solvent is evaporated and
After complete electrophoresis the flter is removed from apparatus and stained to locate |
oElectro osmosis. the separated components.
THEORY OF ELECTROPHORESIS TYPES OF ELECTROPHORESIS
INTRODUCTION: fenoonmka
Electrophoresis is a physical method of analysis which ) Zone Electrophoresis
a) Paper Electrophoresis
involves separation of the conmpounds that are capable of b) Gel Electrophoresis
acquiring electric charge in conducting electrodes. c) Thin Layer Electrophoresis
d) Cellulose acetate Electrophoresis

DEFINITION: 2) Moving Boundary Electrophoresis

Electrophoresis may be defined as the migration of the a) Capillary Electrophoresis
charged particle through a solution under the influence of b) Isotachophoresis
an external electrical field. c) Isoelectric Focussing
lons that suspended between two electrodes tends to d) Immuno Electrophoresis
are
travel
towards the electrodes that bears opposite charges.

ZONE ELECTROPHORESIS
t Involves the migration of the charged particle on the supporting media.
GENERAL METHOD OF OPERATION
Paper, Cellulose acetaie membrane, starch Gel, Poly acrylamide. Saturation of the medium with the buffer.
Components separated are distributed into discrete zone on the support media.
Supporting media is saturated with buffer solution, small volume ofthe sample is applied Samplè application.
as narrow band.

On
its
application
electrophoretic
of PD at the ends of
mobility.
a strip components migrates at a rate determined by Electrophortic separation.
Removal of the supporting media.
ADVANTAGES:
Useful in biochemical investigations.
o
INSTRUMENTATION
a Small quanity of sample can be analysed.
o Cost is low and easy maintenance. o Electrophoretic chamber.
oElectrodes.
DISADVANTAGES: o Diffusion barriers.
oUnsultablefor accurate mobility and isoelectric point determination. oSupporting/Stabilizing media.
Due to the presence of (inert to sample and to any developing reagents).
o
supportlng medlum, technical complicetions such as
capl
fow, electro osmosis, adsorptlon and molecular sieving are Introduced.