 The term Electrophoresis comes from greek

and means ""transport by electricity.

 This electrokinetic phenomenon was
observed for the first time in 1807 by
Russian physicist Alexander Reuss .
 When electricity was passed through a glass
tube containing water and clay , the colloidal
particles move towards the positive
electrode. Thus electrophoresis describe the
migration of charged particle under the
infuence of electric feild.
 In 1955, Oliver smithies found that
separation of human tissue extract with high
resolution by starch gel electrophoresis.
 Electrophoresis is defined as the
migration of the colloidal particles
through a solution under the
influence of an electrical feild.
 The collodial particle transfer either
to positive electrode or negative
electrode . They behave like as if
they are electrically charged
 Migration towards positive or
negative electrode depends on the
charge on the sample.
 Substances that are soluble and have
charges or can be made soluble and
given charges by ionization or by
attaching charged substance to them
they become suitable subject for
electrophoresis .
 Electrophoresis methods are powerful
analytical tools for fractionating the
components of a mixture ,irrespective
of weather they are aggregates or
mono -dispersed
 Two main types of electrophoresis methods
1. Free solution electrophoresis
2. Zone electrophoresis

Depending upon whether the

separation is carried out in a absence or
presence of a supporting or stabilizing
medium
When the separation is carried
out in the absence of a
supporting medium, the
method is called free solution
method.
This method was first proposed
by Picton and Linder (1892),
but was fully developed until
 In free solution electrophoresis, the sample
solution is introduced as a bond at the
bottom of the U tube that has been filled with
unstabilized buffer solution through a
capillary. An electric field is applied by means
of electrodes located at the ends of the tube.
The differential movements of the charged
particles towards one or the other electrode
is observed. Separation takes place as a result
of differences in mobilities. The mobility of
the particle is proportional to its charge. The
free solution method was perfected by
Tiselius. He applied method for the
separation of protiens
 The electrophoresis of lyophilic solution
has been studied by both microscopic and
as well as microscopic method Tiselius
1937 permit the separation and possibly
the identification of proteins and other
colloids in biological fields. A number of
improvements were later made by Philpot
1948, Svenson 1939, Longswerth 1939 and
others.
The apparatus essentially consist of a U
shaped glass cell of a rectangular cross-
section consisting of two parts, one of
which can be made to slide over the other.
The lower part of the cell is filled with the
lyophilic solution under examination
 The two limbs of the U cell are
connected to two large electrode
vessels. Electrophoresis takes place and
sensitive photographic devices are used
for directing the movement of the
colloid. Care must be taken to
minimise as far as possible the
distribution effect of convection caused
by an increase in temperature during
the passage of electric current through
the solution. For this purpose,
apparatus is usually placed in a
constant temperature bath at 4 degree
celcius
 Many experimental difficulties associated
with free solution electrophoresis are avoided
, if the separation are carried out in a
stabilizing medium . Ex:-paper
 such preparations are made possible by
using a supporting medium to keep
convection current from disorting the
electrophoric pattern
 The supporting media in
electrochromatography are numerous and
varied as found in the other chromatography
methods.
 Eg:- agarose gel , synthethic gel , agar gel ,
starch gel , cellulose powder , glass fiber
paper , acetate strips , starch powder etc.
 The kind of supporting media that has to be
used depends upon the sepration to be carried
out.
 The electrophoresis with papers as the
stabilizing material is called as paper
electrochromatography.
 Most analytical separations can be made on
strips of filter paper or cellulose acetate.
Paper are widely used for routine clinical
fractionation of proteins in serum. With
paper , the proteins in human blood serum
separate easily into five fractions where as in
cellose acetate medium is capable of
resolving proteins into nine or more fraction.
 Occasionally , when the solvent or sample
attacks organic material, glass fibers paper
has been used.
 Paper is unsuitable sometimes for dilute
olutions or for substances that are difficult to
detect, this is probably due to the fact that
papers may carry only small volume of the
sample. So paper is replaced by starch , agar and
synthetic gel.
 Gels are recommended for high degree
resolution , because they offer maximum
stabilization against convection currents.
Moreover gels can be cast into thick beds with
sample capacities upto 0.1 mL which is several
times larger than the capacitiy of paper strips.
 INTRODUTION :~
 Paper Electrophoresis is one of the zone
electrophoresis.
 The technique is simple and inexpensive and
requires only micro quantities of plasma for
separation.
 The electrophoresis apparatus in its simplest form
consist of two troughs to contain buffer solution,
through which electric current is passed.
 Historical significance :first media used for
electrophoresis by pioneering investigation like
Tiselius.
 Used in clinical investigation of serum and other
body fluids
 Adsorbs protein
 Poor conductivity
 Background staining
 PRINCIPLE:
“The charge carried by a molecule depends on the
pH of the medium. Electrophoresis at low voltage
is not usually to separate low molecular weight
compounds because of diffusion, but it is easier
to illustrate the relationship between charge and
pH with amino acids than with proteins (or) other
macromolecules”.
 FILTER PAPER :
 It is the stabilizing medium. We can use Whatman
filter paper, cellulose acetate filter paper or
chromatography paper.
 Filter paper such as whatmann no1 and no
3 mm in strip of 3 or 5 cm wide have been
used to good effect.
 Separation takes place in 12 to 14 hrs.

 APPARATUS:
 The equipment required for
electrophoresis consists basically of two
items, a POWER PACK and an
ELECTROPHORETIC CELL.
 Power pack : direct current of 0 to 500V
and 0 to 150mA .
 Electrophoretic cell : electrodes, buffer
reservoirs, a support for paper and a
supporting transparent insulating cover. The
electrodes are usually made of platinum.
 the types of buffer used depends upon the
type of separation. Once removed, the paper
is dried in vaccum oven@110 °C
 Detection : by comparing with standards
 Individual compounds are usually identified
by physical properties by the following
methods.
 1. Fluorescence 2. UV-absorption(260-
280nm) 3. Staining
 i) Fluorescence:
 a) Staining with “Ethidium bromide” and
subsequent visualization of the
electrophoreticgram under UV light makes
DNA & RNA fluoresce and thus facilitates their
detection.
 b) Flourescamine staining is utilized for
detecting amino acids, amino acid derivatives,
 ii) UV absorption:
Proteins, Peptides & nucleic acids absorb in
the range of 260 to 280nm, this property
these can be detect.
iii)) Staining:
 iv) Detection of Enzymes in situ:
If the component to be separated is an enzyme.
Special techniques may be used to detect it.
 The paper strip, which have separated enzyme,
is impregnated with the substrate for the
enzyme desired to be separated.
 The paper strip is now placed in a suitable
buffer along with electrophoretogram. The
color bands will appear which indicates the
position of enzyme.
 v) Quantitative estimation:
The color density of the zone may be multiplied
with the area of the zone and the resulting value
would be a rough estimate of the concentration
of the component.
 ADVANTAGE :
 It is economic
 Easy to use

 DISADVANTAGE :
 Ceratain compounds such as protein,
hydrophilic molecule cannot be resolved due to
the absorptive and inorganic properties of paper
which results in tailing and distortion of
component bands.
 Electro osmosis
 APPLICATION:
 Serum analysis for diagnostic purpose is
routinely carried about by paper
electrophoresis.
 Muscle proteins, egg white proteins, milk
proteins & snake, insect venom analysis
done by this technique.
 Separation of peptides, plasma proteins,
nucleic acids, charged carbohydrates.
 It is employed in biochemical and clinical
field.
 Antigen antibody reaction
 In fractioning protein.
 In analysis of lipoprotein.
 Hemoglobin
 In combination with autoradiography.
 In food industry
 Separation of organic acid, alkaloids,
alcohol, phenol, insulin.
• The technique has widely been used in
clinical, Diagnosis ,for the analysis of
serum,urine,
Spinal fluid,gasrtic juices and other body
fluids.
• Electrochromatography has also been applied
by biochemists for fractionation of smaller
molecules,such as alkaloids antibiotics,nucleic
acids,vitamins,natural pigments,amino
acids,organic acid and carbohydrates.
 The techniques also provides a convenient
method of separating the inorganic ions.
 Paper electrochromatography is special type of
paper chromatography where separations are
enhanced by the application of electrical field.
 Generally paper chromatographic techniques
can not be applied to substance of high
molecular weight such a proteins because the
separations are very slow.In such cases
electrochromatographic techniques is the
alternative.

 Certain diagnostically important electrophoretic

applications ,eg.the detection of abnormal
heamoglobin as an aid to diagnosis the
hereditary anemias ,can not easily be done with
filter paper.
 Electrochromatography is applicable to
substances such as acids, bases ,amino acids
,proteins etc.which carry charge in a buffer of
 The method of paper electrophoresis has
been used extensively in almost every
laboratory where proteins other similar
macromolecular electrolytes are investigated.
 The appratus used for separation of serum
proteins consists of two electrode chambers
placed 15 cm.
• It is also equipped with a device to support
up to fix 1 inch strips of filter paper between
them
• The appratus is also equipped with a dc
supply source which provides current variable
 The two electrodes chamber are filled with
the buffer solution of equal heights with one
liter each in both the containers.the common
buffer used for this purpose is diethyl
barbiturates buffer.
 The buffer was proposed by Aronsson &
Gronwell ,(1958) .and borate buffer was
proposed by consden & powell,(1955).
 It is a technique used for the separation of
DNA, RNA or protein molecules according to
their size and electrical charge using an
electric current applied to a gel matrix
Types of gel
1. Agarose gel
2. Polyacrylamide gel
 The gels are used as beds and formed by
casting the gel in a trough or tube that forms a
bridge between the electrode chambers. When
electrophoresis is carried out in tubes, the
phenomenon is called as disc electrophoresis.
The gel bed may be horizontal or vertical. The
voltage gradient are measured within the gel.
The trough is usually constructed from acrylic
plastic, while for tube casting of gel, glass
tubing is employed.
Techniques for preparing gels may with the
matrix, but in all cases the liquid used is a
buffer to be used during electrophoresis.
 The main difference between the synthetic and
natural gels
 Synthetic gels are formed by polymerisation
 Natural gels are already polymers that are
poured while hot and allowed to set by cooling

Gel is allowed to stand in the open

air for a few min drying out of its
surface causes formation of a thin
skin that is impermeable to large
molecules. Injecting a sample into
a gel is not desirable because
instead of blending into the bed,
the sample liquid splits the gel.
When the bed is formed in a tube,
sample loading is difficult.
 Estimation of size of DNA molecules
following restriction enzyme digestion. Eg: in
restriction mapping of cloned DNA
 Analysis of PCR product eg: in molecular
genetic diagnosis or genetic fingerprinting
 Used in forensics, molecular biology,
genetics, microbiology and biochemistry
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