10 electrophoresis jntu pharmacy

electrophoresis

1. ELECTROPHORESIS
BY
Dr. Suman Pattanayak
Associate Professor
Department of Pharma Analysis & QA.
Vijaya Institute of Pharmaceutical Sciences for Women
IV B. Pharm/ I Sem
Pharmaceutical Analysis

2. CONTENTS
Introduction
Principle And Theory
Factors
Classification
Moving Boundary Electrophoresis
Zone Electrophoresis
Isoelectric Focussing

3. INTRODUCTION
The word electrophoresis is derived from a Greek word,
which means borne by electricity.
It is a separation technique in which the components are
separated due to their varying behaviour under the influence
of applied electric field.
It is defined as the migration of charged molecules under the
influence of external electric field.
The major requirement of the component to be subjected to
electrophoresis is that the component should be charged.

4. INTRODUCTION (cont..,)
It is mostly used for the separation of complex biological
substances such as:
Proteins
Polysaccharides
Nucleic acids
Peptides
Aminoacids
Oligosaccharides
Nucleosides
Organic acids
Small anions and cations in body fluids

5. PRINCIPLE
This technique is mainly used for separation of complex
mixtures of biological substances which possess ionisable
functional groups.
Therefore they can be made to exist as electrically charged
species, either as cation/anion.
Molecules with similar charges will have different m/e ratio.
This forms basis for differential migration when these ions in
solution are subjected to an electric field.

6. PRINCIPLE (cont..,)
Therefore electrophoresis can be applied to any mixture in which
the components carry a charge & have differential mobilities in an
electrical field.
The migration in an electrophoretic system depends on properties
of particle as well as instrumental system
Based on Stoke’s law the mobility of particle (µ) can be calculated
from
µ = Q/π r η
Where,
Q= charge of particle
µ= mobility of ion
r= radius of particle in cm
η = viscosity of medium

7. TECHNIQUES OF
ELECTROPHORESIS
It can be carried out by using either:
1.Low voltage
2.High voltage
Low voltage electrophoresis:
•It consists of two compartments to hold the buffer &
electrodes & a suitable carrier for support medium, such that
its ends are in contact with buffer compartments

8. TECHNIQUES OF ELECTROPHORESIS
(cont..,)
•The design of carrier depends on the medium
•The medium doesn’t dip into electrode compartments, but
into separate compartments connected by wicks with anode
& cathode cells
•The apparatus is enclosed to avoid evaporation
•LVE can be used in principle to separate any ionic substances
•Its main application is examination of biological and clinical
specimens for aminoacids and proteins

10. TECHNIQUES OF
ELECTROPHORESIS (cont..,)
High voltage electrophoresis:
•The construction of apparatus is similar to that of LVE except
that it contains additional cooling system.
•It was found that much reduced analysis time could be
achieved by using high voltage gradient
There are three approaches:
•Use of direct cooling systems in electrophoresis unit.
•Reduction in concentration of buffer solution.
•The sheet can be immersed in non conducting liquid and heat
exchangers.

11. CLASSIFICATION
A.Moving boundary electrophoresis.
B.Zone electrophoresis
C.Isotachophoresis
D.Isoelectric focussing
E.Capillary electrophoresis
F.Immuno electrophoresis

12. MOVING BOUNDARY
ELECTROPHORESIS
Principle:
It allows charged species to migrate in a free moving
solution, without the supporting medium
The main features of this method are:
•The formation of sharp boundaries
•Large electrode vessels containing reversible electrodes
•An optical system for following movement of boundaries
•Thermostatic control

13. Construction & working:
The apparatus consists of U-shaped glass cell of
rectangular cross-section consisting of 2 parts, one of which
can be made to slide over the other
The lower part of cell is filled with lypophilic solution
under examination sometimes the sample solution is
introduced into bottom of U-tube through a capillary side
arm, usually in buffer medium, while the upper part only
buffer solution
The 2 limbs are connected to 2 large electrode vessels

14. Care must be taken to minimize the disturbing effect of
convection currents caused by an increase in temperature during
the passage of current through the solution.
For this purpose, the apparatus is placed in a constant
temperature bath at 4˚c (at this temperature, the density of water
is maximum, hence density differences and convection currents
can be minimised)

15. By applying current of suitable potential difference, the
differential migration of charged particles, towards one or
another electrodes is observed
Separation occurs due to difference in mobility of
molecules. Mobility is proportional to m/e ratio.
The position of moving ions, which forms a boundary,
which is detected by measuring the changes in refractive
index throughout solution.
The concentration gradients which are formed during
electrophoresis are usually detected by Schleiren optical
method.

16. In Philpot-Stvenson or shadow method the boundary
between the solute and buffer appears as dark line on light back
ground.
These are photographed by cylindrical lens, where boundaries
are seen as peaks.
The height of area under peak is proportional to amount of
protein causing number of electrophoretically different
components.

17. ZONE ELECTROPHORESIS
It involves migration of charged particles, which are supported on
relatively inert and homogeneous solid or gel framework.
In this method the separated components are distributed into
discrete zones on stabilizing media.
The zones are heterogeneous and physically separated from one
another.
It is classified based on supporting material used.
They are:
1.Paper electrophoresis
2.Cellulose acetate electrophoresis
3.Thin layer electrophoresis
4.Gel electrophoresis

18. Principle :
Basically a supporting media is saturated with buffer
solution and a small amount of sample solution is applied as
narrow band.
On application of potential difference between the ends
of strip, each component migrates at a rate determined by its
electrophoretic mobility.

19. ADVANTAGES AND
DISADVANTAGES
Useful in biochemical investigation.
Very small quantity of samples can be analysed.
Useful to study both simple and complex mixtures equally.
Equipment cost is low and maintenance is easy.
Unsuitable for accurate mobility and isoelectric point
determination.
Complications such as capillary flow, electro osmosis,
adsorption and molecular sieving are introduced.

20. GENERAL METHOD OF
OPERATION
Saturation of medium: the supporting medium other than gel
must be saturated with a buffer so that it can conduct
current.
Sample application: sample is applied as spot or streak.
Electrophoretic separation: the power is switched on at
required voltage.
After completion of separation the power is switched off
before supporting media is removed.
Removal of supporting medium: paper, cellulose acetate
strips and thin layer plate are removed and air dried or in
oven. The gels are removed by forcing water from
hypodermic syringe.

21. INSTRUMENTATION
1.Electrophoretic chamber: It contains buffer solution.
2.Electrodes : Ag/AgCl reversible electrodes can be used.
3.Diffusion barriers: The electrode should be separated from
the electrophoretic bed by a barrier such as gel, filter paper,
sponge.
4.Supporting media: It should have low resistance to electric
current, inert to sample, electrolyte and developing
reagents.

22. PAPER ELECTROPHORESIS
One of the simplest process in electrophoresis involves
spotting a mixture of solute in middle of paper , moistening
the paper with some electrolyte and placing it between two
sheets of glass.
The ends of paper strip extending beyond glass plate are
immersed in beakers of electrolyte.
A potential of 5V/cm of paper length is placed from a DC
source.
It is allowed to continue for a period of several hours.

23. ADVANTAGES AND
DISADVANTAGES
It is economical and also easy to use.
Some compounds such as proteins can not be adequately
resolved.
There are three types of paper electrophoresis:
1.Horizontal
2.Vertical and
3.Continuous

26. CELLULOSE ACETATE
ELECTROPHORESIS
1.Cellulose acetate strips, which are used widely in clinical
laboratories produce excellent separations of 7 to 9 protein
fractions in a few hours.
2.this material is exceedingly fine and homogeneous, and
little tailing is encountered due to adsorption.
3.It is especially useful for separating alpha immunoglobulins
from albumin.
4.It contains 2 to 3 acetyl groups per glucose unit and its
absorption capacity is less than that of paper.

27. GEL ELECTROPHORESIS
The separation here is brought about through molecular sieving
technique, based on molecular size of substances

28. THIN LAYER
ELECTROPHORESIS
1.Electrophoretic studies can be also carried out on thin
layers of silica, keisulghur,alumina.
2.The studies with these materials offer advantages of
speed and resolution when compared with paper.
3.They have greatest application in combined
electrophoretic-chromatography studies in two-
dimensional study of proteins and nucleic acid
hydrolysates.

29. ISO ELECTRIC FOCUSSING
It is mainly used for a separation of electrolytes such as
proteins.
When electrophoresis is run in solution buffered at constant
pH, proteins having net charge will migrate towards opposite
electrode.
The use of pH gradient across supporting medium causes
each protein to migrate to an area of specific pH.
A sharp well defined protein bands occur at the point where
iso electric point equals to pH of gradient.

30. Separation is carried out on gels on which a stable a pH
gradient has been established.
An ampholytic compound has a pH at which it is neutral.
The pH gradient is achieved by impregnating the gel with
polyamino-polycarboxylic acids.
When subjected to electric field these migrate and come to
rest in order of their pH.
Thus each ampholyte migrates in applied field until it reaches
a position on the plate where pH of medium is equal to iso
electric point.

31. At this point ampholyte is in its zwitter ion form and is
neutral.
Thus it losses electrophoretic
mobility and becomes focused
in narrow zone at this point.

32. Advantages:
Spreading of bands is minimized.
Proteins that differ by little as 0.01pH can be adequately
resolved
Disadvantages:
As carrier ampholytes are used in high concentration, a
high voltage power supply is necessary. As a result the
electrophoretic matrix must be cooled.

33. THANK
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