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Welcome To my
presentation
Shahjalal University of Science &
Technology, Sylhet
Department of ChemistryCourse No. : CHE 300Course title : Seminar and OralA presentation on Electrophoresis
Presented by,Tanjila IslamReg. No. : 2010131019Semester : 3/2
electrophoresis
Basic of electrophoresis :
Differential rate of migration of ion molecule in an electrolyte solution under the influence of an applied electric field in a support medium (e.g. paper, gel or capillary tube)
Figure 1: Motion of a charged particle by electrophoresis
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* A useful method to separate substances based on their charge – to – mass ratios
Principle :
* Charged ion or molecule migrates when placed in an electric field
Rate of migration depends on its net charge, size, shape and the applied electric current v = μeE where, v = velocity of an ion E = electric field strength (Vcm-1)
μe = electrophoretic mobility = distance migrated in a certain time period
The electrophoretic mobility is given by μe = (when electric force = frictional drag)showing that small highly charged species have high mobility and vice versa.
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Driving force of migration :
* Resultant of the electrostatic force of attraction between the electric field and the charged molecule, and the retarding forces due to friction and electrostatic repulsion from molecules of the transport medium.
Figure 2: Illustration of electrophoresis retardation
Supporting media for electrophoresis :
* Paper- filter paper such as Whatman no.1 and no.3MM- Used to good effect
* Cellulose acetate- containing 2 to 3 acetyl groups- to give sharper bands- more easily rendered transparent- low solvent capacity- enhancing the resolution
Gels- 3 dimensional semisolid colloids- resolving power enhanced due to sieve effect operating- prepared from starch, agar, or polyacrylamide
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General procedure for electrophoresis :
Immersion of two electrodes in two separate buffer chambers but not fully
isolated from each other
Migration of charged particles from one chamber to the other by using an electric
field
Separation of different ions migrating at different speeds Figure 3: Fundamental of electrophoresis
Factors affecting electrophoretic mobility :
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Charge – higher the charge greater the mobility
Size – bigger the molecule greater the frictional and electrostatic forces exerted on it by the medium i.e. larger particles have smaller electrophoretic mobility compared to smaller particles
Electric field – increase of migration with the increase of voltage gradient
* Buffer – dependence of migration on pH of the buffer
* Ionic strength – greater the ionic strength of the buffer solution higher proportion of the current hence electrophoretic mobility
Types of electrophoresis :
Electrophoresis
Frontal Electrophoresis
Micro electrophoresis
Moving boundary
Zone electrophoresis
Paper electrophoresis
Cellulose acetate electrophoresis
Gel electrophoresis
Figure 4: Types of electrophoresis
Techniques of electrophoresis:
Techniques
Low voltage (LVE)
High voltage (HVE)
SDS polyacrylamaide gel (SDS-PAGE)
Isoelectric focusing
Immunoelectrophoresis
Discontinuous electrophoresis
Figure 5: Different techniques of electrophoresis
Low voltage electrophoresis :
* Two compartments to hold the buffer and electrodes
Figure 6: Apparatus for low voltage electrophoresis
*A suitable carrier for support mediumending in contact with the buffermedium
*To provide voltage gradient 8 5 Vcm-1, a power pack supplying up to 500 V or even 1000 V and 0 – 150 mA
Application of LVE :
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*
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To separate any ionic substances
The examination of biological and clinical specimens for amino acids and proteins
Separation of sugars
Figure 7: Electrophoretogram of plasma proteins on cellulose acetate at pH 8.6
High voltage electrophoresis :
To obtain voltage gradients up to 100 Vcm-1, high voltage and current supplying
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*
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Using cooling plates for heat dissipation generated by high voltage
Less than of 1h analysis time
* Working best with small ions deriving from small peptides and amino acids
Figure 8: HVE apparatus
Capillary electrophoresis (CE) :
* Separation of analyte species achieved on the basis of differential migration in an electric field through narrow bore fused silica capillary columns (25 – 100 μm).
Capillary electrophoresis
Capillary
zone electrophoresis
(CZE)
Capillary gel electrophoresis
(CGE)
Isoelectric focusing
Isotachophoresis
(ITP)
Miscellar electrokinetic
capillary chromatography
(MECC)
Figure 9: Separation modes of capillary electrophoresis
Overview of instrumentation of CE :
* A fused capillary column dipping into two electrolyte buffers containing Pt foil cathode or anode across 15 – 60 kV voltage applied
* Introducing a small volume of sample at one end of capillary
* Migration of sample through the capillary under the force of applied electric field
Figure 10: Schematic of a capillary zone electrophoresis
Advantages of CE :
Power dissipation minimized by high electrical resistance*
* Having voltage gradients up to 100 – 500 Vcm-1 necessary for rapid separations
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No Joule–Thompson effect
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No band broadening
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Most prominently used because of its faster results and high resolution separation
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Large range of detection methods available
Applications of electrophoresis :
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* DNA analysis
Protein analysis
* Antibiotic analysis
* Vaccine analysis
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Detection of damaged genes by gel electrophoresis
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To use in forensic research Figure 11: A simple view of protein separation
Conclusion :
* Although not in principle a chromatographic method, electrophoresis used in conjunction with paper chromatography and gel materials, proves an extremely useful method for separation of charged substances, ranging from small ions to large charged macromolecules, of biological and biochemical interest.
* It is widely used yet it has some limitations.
Thank you