1. Welcome
To my
presentation

2. Shahjalal University of Science & Technology, Sylhet
Department of Chemistry
Course No. : CHE 300
Course title : Seminar and Oral
A presentation on Electrophoresis
Presented by,
Tanjila Islam
Reg. No. :
Semester : 3/2

3. electrophoresis

4. 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) *
A useful method to separate substances based on their charge – to – mass ratios *
Figure 1: Motion of a charged particle by electrophoresis

5. 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 = q 6πηr (when electric force = frictional drag)
showing that small highly charged species have high mobility and vice versa. * *

6. 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

7. 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

8. Separation of different ions migrating at different speeds
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

9. Factors affecting electrophoretic mobility :*
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

10. Types of electrophoresis :
Frontal
Micro electrophoresis
Moving boundary
Zone electrophoresis
Paper electrophoresis
Cellulose acetate electrophoresis
Gel electrophoresis
Figure 4: Types of electrophoresis

11. Techniques of electrophoresis:
Low voltage (LVE)
High voltage (HVE)
SDS polyacrylamaide gel (SDS-PAGE)
Isoelectric focusing
Immunoelectrophoresis
Discontinuous electrophoresis
Figure 5: Different techniques of electrophoresis

12. Low voltage electrophoresis :
Two compartments to hold the buffer and electrodes *
A suitable carrier for support medium
ending in contact with the buffer
medium *
To provide voltage gradient ̴ 5 Vcm-1, a power pack supplying up to 500 V or even 1000 V and 0 – 150 mA *
Figure 6: Apparatus for low voltage electrophoresis

13. Application of LVE : 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

14. High voltage electrophoresis :*
To obtain voltage gradients up to 100 Vcm-1, high voltage and current supplying *
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

15. Capillary electrophoresis (CE) :
zone electrophoresis
(CZE)
Capillary gel electrophoresis (CGE)
Isoelectric focusing
Isotachophoresis
(ITP)
Miscellar electrokinetic capillary chromatography
(MECC)
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). *
Figure 9: Separation modes of capillary electrophoresis

16. 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

17. Advantages of CE : *
Power dissipation minimized by high electrical resistance
Having voltage gradients up to 100 – 500 Vcm-1 necessary for rapid separations * *
Most prominently used because of its faster results and high resolution separation *
Large range of detection methods available *
No Joule–Thompson effect *
No band broadening

18. Applications of electrophoresis :*
DNA analysis
Protein analysis *
Antibiotic analysis * *
Vaccine analysis
Detection of damaged genes by gel electrophoresis * *
To use in forensic research
Figure 11: A simple view of protein separation

19. 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. *

20. Thank you