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G. Umamaheswara rao 1 ELECTROPHORESIS


CONTENTS 2 Introduction History Definition Why we have to use Electrophoresis? Principle Theory Types of electrophoresis References


INTRODUCTION 3 Electrophoresis literally means ‘borne by electricity’ from Greek word. Electrophoresis is a separation technique, in which components are separated due to varying behavior under the influence of an applied electric field. The major requirement of the component to be subjected to electrophoresis is that- They must be charged since otherwise no movement can occur in the electric field and separation is impossible. Most of the substances in aqueous solvent acquires either a positive charge or negative charge. This results from partial or complete ionization of the substances itself, or from adsorption on dispersed substances of ions/charged species in the solution or from ion pair formation.


HISTORY 4 The name ‘ Electrophoresis ’ was coined by Mr. Michealis in 1909 to describe the migration of colloid ions in an electric field. In 1945 Martin and Synge defined in their review Ionophoresis as the electromigration of low molecular weight ions in stabilizing media. The first sophisticated electrophoretic apparatus was developed by Tiselius in 1937 . He was awarded the 1948 Nobel prize for his work in protein electrophoresis. He developed the "moving boundary," which later would become known as zone electrophoresis, and used it to separate serum proteins in solution.


DEFINITION 5 Electrophoresis is defined as the migration of charged molecules under the influence of an external electric field .

Why we have to use Electrophoresis?:

Why we have to use Electrophoresis? 6 Electrophoresis is a versatile separation method because, Electrical charge on polyelectrolyte can be modified by change of pH. Even if species have same charge, differences in molecular size and conformation affect mobility. Mild, non denaturing technique for proteins . High sensitivity with small sample size.


PRINCIPLE 7 Molecules of similar charge will have different e/m or charge mass ratio, because of the differences in the molecular weights. This differences form the sufficient bases for the differential migration when the ions in the solution are subjected to an electric field. So electrophoresis can be applied to any mixture in which the components carry a charge and have differential motilities in an electric field. The mobility of the particle can be evaluated from the Stokes law, µ = Q/6 rn where Q = charge on particle. r = radius of particle in ions. n =viscosity of the medium. µ = velocity of the particle.


THEORY 8 Electrophoresis is based on migration of charged particles. For a particle to migrate in electric field it is necessary that it posses a charge. Most of the polar substances acquire a surface electric charge when brought in contact with a polar medium by Ionization Ion adsorption Ion dissolution Ions of opposite charges known as counter ions are attracted towards the surface and ions of like charges (co-ions) are repelled away from the surface. This leads to formation of electric double layer.

Types of Electrophoresis:

Types of Electrophoresis 9 Frontal method or moving boundary electrophoresis. Zone electrophoresis (Ionophoresis). Free solution method. Powdered and porous solid support Paper electrophoresis Gel electrophoresis Density gradient electrophoresis Iso – Tachophoresis (Displacement method). Iso – Electric focusing. Two dimensional methods Special Techniques Immuno electrophoresis Capillary electrophoresis Thin layer electrophoresis High layer electrophoresis

Moving Boundary Electrophoresis:

Moving Boundary Electrophoresis 10 PRINCIPLE : The moving boundary electrophoresis method allows the charged species to migrate in a free moving solution in the absence of a supporting medium. The sample is fractionated in a ‘U’ tube filled with unstabilized buffers. The sample is carefully injected into the bottom of the ‘U’ tube through a capillary side arm. The position of the moving ions from a boundary, which is detected by measuring changes in refractive index throughout the solution.


Contd… 11 Method The apparatus consist of a ‘U’ tube with electrodes located at the two ends used to apply an electric field. By applying current of suitable potential difference the differential migration of charged particles, towards one or other electrodes is observed. Separation occurs due to the difference in migration of the molecule and the mobility is proportional to mass : charge ratio.


Contd… 12


Contd… 13 Advantages Biologically active fractions can be recovered without the use of denaturing agents. Reference methods for measuring electrophoretic mobilities. It is direct and convenient method to separate solute from a supernatant buffer solution. Conc. as low as 0.05mg/ml can be detected with an infterferometric optical systems. Give boundaries but not zones (zones would be gravitationally unstable leading to convection). Greater sensitivity and better separation.


Contd… 14 Disadvantages High equipment cost. Separated components tends to mix due to thermal and density gradients, so extra apparatus to counter these effects are required. This makes it costly. Difficult to isolate separated zones so elaborate optical systems are required Applications Analysis of complex mixtures – protein mixtures. To study the homogeneity of macromolecular system. To calculate the net charge of a dissolved protein molecule from its electrophoretic mobility, by comparing with acid-base titration data. To study the colloidal dispersion.

Zone Electrophoresis:

Zone Electrophoresis 15 Zone electrophoresis involves the migration of charged particles, which are supported on a relatively inert and homogenous solid or gel framework. Principle : Basically a strip of paper or some other supporting material is saturated with buffer solution and a small amount of a solution of the material is applied as a narrow band. On application of a potential difference between the ends of the strip, each component migrates at a rate determined by its electrophoretic mobility.


Contd… 16 Advantages Simple, inexpensive apparatus allows simultaneous analysis of several specimens. Simple procedure for visualizing of zones and for isolation of fractions. Higher resolution power. Adaptability to large scale or micro-separation. Very small quantity of sample can be analyzed.


Contd… 17 Disadvantages Use of supporting material will however introduces complicating variables like- Capillary flow Electro-osmosis Adsorption Molecular sieving It is unsuited for accurate mobility and Iso electric point determination.

Gel Electrophoresis:

Gel Electrophoresis 18 Definition: Gel electrophoresis is the movement of molecule through gel under the influence of an electric field. It is carried out using starch, agar, polyacrylamide.

Electrophoretic chamber:

Electrophoretic chamber 19

Paper Electrophoresis:

Paper Electrophoresis 20 Definition Paper electrophoresis refers to the migration of charged species on a paper impregnated with buffer solution in an electric field.


Contd… 21 The standard chromatographic filter papers with volume up to 20 µl and for larger volumes up to 60 µl extra thick papers are used. ex. SCHILECHER and SCHULL 2043A and B WHATMAN 1,2,3 EATON-DIKEMAN 301-85, 320 and 652. For extra thick paper WHATMAN 3mm or 31ET


Contd… 22 Various buffers for electrophoresis Constituents pH range Acetic acid – Formic acid mixture up to 2 Tri-ethanolamine – Formic/Acetic acid mixture 3.5-6 Ammonium carbonate – Amm. Hydroxide 8.0-9.5 Mono/Tri ethanolamine – HCl 6.8-11 Ammonium hydroxide – acetic acid mixture 6.0-10


Contd… 23 Basic electrophoretic techniques are as followed. Closed strip a. Solid support ex. Plastic or glass b. Non polar liquid ex. Chlorobenzene, CCl 4 c. Covering type Semi closed Open strip a. Horizontal type b. Hanging strip type / vertical type Also classified as Low voltage electrophoresis- 20 volt/cm High voltage electrophoresis- 20-200 volt/cm

Horizontal type:

Horizontal type 24 Electrode Electrode Sample application point Filter paper strip Buffer solution Buffer solution

Hanging strip / vertical type:

Hanging strip / vertical type 25 Electrode Electrode Sample application point Filter paper strip Buffer solution Buffer solution

Non polar liquid :

Non polar liquid 26 Buffer solution Buffer solution Electrode Electrode Filter paper strip Immiscible solvent such as Chlorobenzene

Solid support:

Solid support 27 Buffer solution Buffer solution Electrode Electrode Glass plate Filter paper strip

References :

References 28 Remington’s Pharmaceutical sciences. 21 st Edition. Principles of Instrumental Analysis by Skoog, Holler and Nieman. Paper Chromatography and electrophoresis by Zweig Whitakes. Instrumental methods of Chemical analysis by Gurdeep R, Chatwal and S.K. Anand www.google.com

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