ELECTROPHORESIS

BY PRATIKSHA SRIVASTAVAM.PHARMA

GALGOTIAS UNIVERSITY

INTRODUCTION 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.

It is mostly used for the separation of complex biological substances such as:ProteinsPolysaccharidesNucleic acidsPeptidesAminoacidsOligosaccharidesNucleosidesOrganic acidsSmall anions and cations in body fluids

PRINCIPLE 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=Eq\f where, v= velocity of migration of molecule E = electric field in volts per cm q = net electric charge on the molecule f = frictional coefficient

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(charge\mass ratio).

TECHNIQUES OF ELECTROPHORESIS It can be carried out by using either:1. Low voltage2. 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

• 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

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.

FACTORS AFFECTING ELECTROPHORETIC MOBILITY1. 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.

FACTOR AFFECTING ELETROPHORHETIC MOBILITY 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.

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.

TYPES OF ELECTROPHORESIS:

TYPES OF ELECTROPHORESISA. Moving boundary electrophoresis.B. Zone electrophoresisc. Isoelectric focusing

MOVING BOUNDARY ELECTROPHORESISPrinciple: It allows charged species to migrate in a free moving

solution, without the supporting mediumThe 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

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 solutionThe 2 limbs are connected to 2 large electrode vessels

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)

By applying current of suitable potential difference, the differential migration of charged particles, towards one or another electrodes is observedSeparation 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.

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.

ZONE ELECTROPHORESIS 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 electrophoresis2. Cellulose acetate electrophoresis3. Thin layer electrophoresis4. Gel electrophoresis

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.

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.

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.

INSTRUMENTATION1. 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.

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.

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 and3. Continuous

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.

GEL ELECTROPHORESISThe separation here is brought about through molecular

sieving technique, based on molecular size of substances

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.

ISO ELECTRIC FOCUSSING 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.

ISOELECTRIC FOCUSING IN LABORATORY

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.

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

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.

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Advantages: Spreading of bands is minimized. Proteins that differ by little as 0.01pH can be

adequately resolvedDisadvantages:

As carrier ampholytes are used in high concentration, a high voltage power supply is necessary. As a result the electrophoretic matrix must be cooled.