electrophoresis a chormatographic techniques

1. BY PRATIKSHA SRIVASTAVA
M.PHARMA
GALGOTIAS UNIVERSITY

2.  The word electrophoresis is derived from a Greek word,
which means borne by electricity.
 Electrophoresis may be defined as separation of charged
compounds using electric field.
 Rate of movement of compound depends upon charge and
size of the compound
 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.

3.  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.  Any charged ion and molecule migrates when
palced in an electric field
 The rate of migration dependsupon its net
charge,size, shape and the appilied electric
current
 can be represented by
V=Eqf
 where, v= velocity of migration of molecule
E = electric field in volts per cm
q = net electric charge on the molecule
f = frictional coefficient

6. The movement of charged particle in an electric
field is expressed in terms of electrophoretic
mobility ,denoted by µ.
where,
µ = v/E OR
µ = q/f
For molecules with similar conformation f varies
with size but not with shape. Thus
electrophoretic mobility (µ) of a molecule is
directly proportional to charge
density(chargemass ratio).

7.  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. • 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. 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. 1. Charge – higher the charge greater the
electrophoretic mobility.
2. Size – bigger the molecule greater are the
frictional and electrostatic forces exerted on
it by the medium. Consequently, larger
particles have smaller electrophoretic
mobility compared to smaller particles.
3. Shape – rounded contours elicit lesser
frictional and electrostatic retardation
compared to sharp contours. Therefore
globular protein move faster than fibrous
protein.

12.  Adsorption :- if sample is adsorbed on carrier only non-
adsorbed part of substance migrate and a reduction of
migration speed occurs.
 Diffusion :- Dissolved substance in a concentration gradient
tend to a diffuse. The path is always fortuous. So, carrier
material should have uniform particle size.
 Zone flows :- due to inhomogenecity of support, non uniform
moisture content, electrolytic concntration, temperature
differences.
 Electrolysis :- Electrolysis which cause microscopic bubbles to
accumulate on the electrodes. When bubble formation
occurs- electrical contact with buffer is lost.
 Ionic Strenght :- The electrophoretic mobility is approximately
proportional to the reciprocal of the square root of ionic
strenght.

13.  Heat :- Mobility increases with heat,the central portion
of each zone migrates in advance of its edges.
 Interaction With Supporting Medium :- Electrophoretic
migration has ionic side chains there can inherent with
the particles being separated by electrophoresis. Ionic
side chains are usually undesirable because they
produce Trailing.
 pH and other chemical characteristics:- The
electrophoretic mobility is greatly affected by the pHof a
buffer, particularly when the sample is either a weak acid
or a weak base because the ph establishes its degree of
ionization.

15. A. Moving boundary electrophoresis.
B. Zone electrophoresis
c. Isoelectric focusing

16. 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

17. 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

18. 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 minimized)

19. 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.

20. 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.

21.  Any electrophoretic technique in which component are separated
into zones and bands in buffer.
 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

22. 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.

23.  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.

24.  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.

25. 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.

26.  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.

27.  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

30. 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.

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

32. 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.

33.  Isoelectric focusing (IEF), also known as
electrofocusing, is a technique for separating
different molecules by differences in their
isoelectric point (pI). It is a type of zone
electrophoresis, usually performed on proteins in
a gel, that takes advantage of the fact that
overall charge on the molecule of interest is a
function of the pH of its surroundings.

34.  IEF involves adding an ampholyte solution into immobilized
pH gradient (IPG) gels. IPGs are the acrylamide gel matrix
co-polymerized with the pH gradient.
 A protein that is in a pH region below its isoelectric point
(pI) will be positively charged and so will migrate towards
the anode (negatively charged electrode). As it migrates
through a gradient of increasing pH, however, the protein's
overall charge will decrease until the protein reaches the
pH region that corresponds to its isoelectric point.

35.  At this point it has no net charge and so migration ceases
(as there is no electrical attraction towards either
electrode). As a result, the proteins become focused into
sharp stationary bands with each protein positioned at a
point in the pH gradient corresponding to its pI. The
technique is capable of extremely high resolution with
proteins differing by a single charge being fractionated
into separate bands
 Molecules to be focused are distributed over a medium
that has a pH gradient (usually created by aliphatic
ampholytes). An electric current is passed through the
medium, creating a "positive" anode and "negative"
cathode end. Negatively charged molecules migrate
through the pH gradient in the medium toward the
"positive" end while positively charged molecules move
toward the "negative" end

36.  As a particle moves towards the pole opposite of its
charge it moves through the changing pH gradient
until it reaches a point in which the pH of that
molecules isoelectric point is reached. At this point
the molecule no longer has a net electric charge.
.

38. 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.