ELECTROPHORESIS

BYDr. Suman PattanayakAssociate ProfessorDepartment of Pharma Analysis & QA.

Vijaya Institute of Pharmaceutical Sciences for Women

IV B. Pharm/ I SemPharmaceutical Analysis

CONTENTS

Introduction Principle And Theory Factors Classification Moving Boundary Electrophoresis Zone Electrophoresis Isoelectric Focussing

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.

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

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.

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 ηWhere,

Q= charge of particleµ= mobility of ion r= radius of particle in cmη = viscosity of medium

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

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

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.

CLASSIFICATION

A. Moving boundary electrophoresis.B. Zone electrophoresisC. IsotachophoresisD. Isoelectric focussingE. Capillary electrophoresisF. Immuno electrophoresis

MOVING BOUNDARY ELECTROPHORESISPrinciple:

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

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

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)

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 by Schleiren optical method.

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 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 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.

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.

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 ELECTROPHORESIS1. 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

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.

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.

At this point ampholyte is in its zwitter ion form and is neutral.

Thus it losses electrophoreticmobility and becomes focused in narrow zone at this point.

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.

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