ELECTROPHORESIS

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ELECTROPHORESIS:

ELECTROPHORESIS By Chikkulla. Priyanka, Nalanda Institute of Pharmacy, Kantepudi(village), Sattenapalli( Mandal ), Guntur(District). 1

CONTENTS:

2 CONTENTS Introduction Principle Theory Electrophoretic Techniques 1.Moving boundary electrophoresis 2.Zone electrophoresis Free solution method Gel electrophoresis 3.Paper electrophoresis 4.Immunoelectrophoresis 5.Isoelectric focusing 6.Isotachophoresis 7.Capillary Electrophoresis Applications

INTRODUCTION :

3 INTRODUCTION Introduced by TISELIUS in 1937, for the separation of proteins. Defined mainly as the migration of charged molecules under the influence of an external magnetic field. for the separation of Proteins Nucleic acid Polysaccharides

Principle :

4 Principle Rate of migration (separation) depends upon e/m (charge to mass) ratio The migration of particles or the rate of travel of particle, in electrophoretic system depends on properties of the particles as well as the instrumental system Characteristic of particles Property of electric field Temperature Nature of suspended medium

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5 Mobility of particle is calculated by Strokes law :- μ = Q/6 π r η where Q = charge on the particles μ = mobility of particle r = radius η = viscosity of the medium μ = Q/A π r2 η where A has a value ranges 4 to 6 and is related to particle shape

Solution condition:

6 Solution condition The solution conditions are important variables. For eg. An acidic pH would favors protonation of basic centers of a protein, producing a –ve charged molecule. It is not desirable to choose a pH such that the protein is at its isoelectric point and exists as the uncharged Zwitterions( a species not mobile in the imposed electrical field)

Ionic strength :

7 Ionic strength Electrophoretic mobility decreases with the supporting electrolyte ionic strength Generally, the ionic strengths employed in electrophoresis range from 0.01 to 0.10. The temperature of the solution is important because the solution viscosity varies with temperature and the mobility inc. with temperature Temperature

THEORY :

8 THEORY Migration of charged particles on potential gradient and space in between 2 electrodes. Hence field strength (x) = E/S Where, E = Potential gradient applied S = space between 2 electrodes

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9 Movement of charged particles under the influence of applied electrical field Forces acting on charged molecule moving in an electric field Factors affecting migration of ions in electrophoresis Resolution

ELECTROPHORETIC TECHNIQUES :

10 ELECTROPHORETIC TECHNIQUES Moving boundary electrophoresis Zone electrophoresis Immunoelectrophoresis Iso- tachophoresis Capillary electrophoresis

Moving boundary electrophoresis :

11 Moving boundary electrophoresis Principle: - method involves movement of charged particles in free moving solution in absence of supporting medium.

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12 Method The apparatus consists of a U shaped tube with provision for introducing the cathode and anode electrodes into each of the arms. The sample solution is introduced and each arm is filled carefully with a buffered solution. If the sample consists of compounds with different mobilities , their migration may be observed as several moving boundaries

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13 Initial position A +B +C Substance to be separated U tube Buffer sol . After migration A +B +C C B+C A A+B +ve -ve -ve +ve Fig 1 Fig 2

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14 Detection:- Position of ions detected by measuring changes in refractive index throughout the solution. Advantages:- Biologically active fractions can be recovered without using denaturing agent. As a reference method for measuring electro-mobility. Gives information on isoelectric point and mobility of compounds

Disadvantages:- :

15 Disadvantages :- Complete separation is rarely achieved. Maintaining sharply defined boundaries (stabilization of boundaries is needed) Only fastest and slowest components can be separated in pure form. Not used for preparative and quantitative analysis. Several problems are associated with the technique, including stabilization of ion boundaries, boundaries anomalies, and the need for specialized equipment

Zone electrophoresis :

16 Zone electrophoresis Zone electrophoresis makes use of a stabilizing medium to minimize the problems associated with free-boundary electrophoresis. It involves migration of charged particles which are supported on a relatively inert and homogenous solid or gel frame work. Separated components are distributed into different zone in a stabilizing media. Make use of stabilizing media like paper, agar, cellulose, starch, gels, polyacrylamide gels.

Types of supporting or stabilizing medium:

17 Types of supporting or stabilizing medium Free solution method porous solid support gel electrophoresis paper electrophoresis

Free solution method :

18 Free solution method

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19 Rotating tube apparatus Migration occurs in horizontal tube. 10 revolution / min Micro syringe (small sample can be applied) Profiles of zones are determined by scanning devices. Advantages:- Complete separation of electrophoretically different components. Small sample

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20 Hydrogen lamp filter motor Electrophoretic tube photomultiplier Recorder Seal Sample Cathode Free solution method

Gel electrophoresis :

21 Gel electrophoresis Electrophoresis in compact gels, which depends at least in part on size-exclusion effects to achieve separation, is used frequently for the separations of proteins and nucleic acids The overall migration in these gels is a combination of movement under the influence of the electric field and size separation by the pores of the gel It is carried out by using Agar Starch Polyacrylamide

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22 The most frequently used techniques of polyacrylamide gel electrophoresis (PAGE) Is the discontinuous buffer system developed by Laemmli . In this procedure sample is placed on a stacking gel with a low level of cross linking and therefore a large pore size. During movement through this gel, the sample is concentrated into a narrow band and then deposited onto a separating gel that has a higher cross linking and smaller pore size. The separation of the solutes occurs in this phase.

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23 In a special modification of this technique used for separation of proteins, a detergent, such as sodium dodecylsulfate (SDS), is introduced in the buffer. This interacts with the proteins to produce particles of consistent shape and uniform negative charge so that separation occurs according to size alone. This enables the simple determination of molecular weight because the migration distance is proportional to the logarithm of molecular weight, as in size exclusion chromatography.

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Agarose gel electrophoresis:

25 Agarose gel electrophoresis Agarose gel electrophoresis is a method used in biochemistry and molecular biology to separate DNA strands by size, and to estimate the size of the separated strands by comparison to known fragments.

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Paper electrophoresis :

27 Paper electrophoresis One of the simplest procedures in electrophoresis involves spotting a mixture of solutes in the middle of a paper strip, moistening the paper with some electrolyte and placing it between two sheets of glass. The ends of the paper strips are immersed in beakers of electrolyte Electrophoresis is allowed to continue for a period of several hours.

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Immunoelectrophoresis :

29 Immunoelectrophoresis Enzymatic and immunological methods also have been used to detect proteins following electrophoresis in gels. Immunochemical methods add an additional dimension to protein identification. Following electrophoresis in an agar gel backed with a microscope slide, an antibody is placed into a trough cut parallel to the direction of electrophoresis.

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30 The antibody and electrophoretically separated antigens diffuse towards each other resulting in precipitin arcs where antigen antibody complexes form. This technique has been referred to as immunoelectrophoresis.

Isoelectric focusing :

31 Isoelectric focusing Technique based on moving boundary electrophoresis Amphoteric substances such as amino acids and peptides are separated in a specially designed vertical column; down to which there is both pH and voltage gradient. Each compound migrates towards the region in the column, where the pH corresponds to that of its Isoelectric point and is immobilized here.

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32 Acidic pH Isoelectric pH Basic pH pH gradient reservoir Strongly acidic solution e.g phosphoric acid Strongly alkaline solution e.g tetranol amine

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33 Advantage:- In separation and characterization of proteins in one step. High resolution – (identifying iso enzymes) Application:- Useful for microanalysis of proteins Identifying isoenzymes

Isotachophoresis :

34 Isotachophoresis Based on principle of moving boundary electrophoresis. Separation is achieved either horizontally or vertically. Solution in which the separation takes place is normally an aqueous medium, which contains sucrose to provide a higher density to the solution. Where the separation by Isoelectric focusing depends on the existence of a pH gradient in the system, the technique of Isotachophoresis depends on the development of a potential gradient. A leading electrolyte (e.g. Chloride) with a higher mobility than the analytes , and a trailing electrolyte (e.g. Glycinate ) with a lower mobility are used

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35 In the example shown here three particle classes with different charges are being separated and preconcentrated via electrophoresis. After the separation is concluded all particles move at a constant speed (Isotachophoresis).

Capillary Electrophoresis :

36 Capillary Electrophoresis The principle behind electrophoresis is that charged molecules will migrate toward the opposite pole and separate from each other based on physical characteristics. The two limiting factors of traditional electrophoresis were: 1) detection of molecules upon completion of electrophoretic separation and 2) only low voltages could be used to prevent heat damage of the samples.

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37 Capillary electrophoresis solved both of these problems. Because the capillary tube has a high surface to volume ratio (25-100 µm diameter), it radiates heat readily and thus samples do not over heat. Detection of the migrating molecules is accomplished by shining a light source through a portion of the tubing and detecting the light emitted from the other side.

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Applications :

39 Applications Electrophoresis is employed in biochemical and clinical field. In the study of protein mixtures Antigen antibody reactions In fractioning protein. In analysis of lipoprotein Hemoglobin In combination with autoradiography Separation of organic acid, alkaloids, carbohydrates, amino acids, alcohols, phenols, nucleic acids, insulin. In food industry

References:

40 References Notes of Dr. M.N.Noolvi sir Pharmaceutical analysis, by “P.Parimoo” Introduction to chemical analysis, by “H.K.Kaur” Pharmaceutical analysis, by “Robert.D.Braun” Instrumental methods of chemical analysis, by “B.K.Sharma” From Internet source

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41 Thank you

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