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. : 2010131019
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)
Figure 1: Motion of a charged particle by electrophoresis
*
* A useful method to separate
substances based on their
charge – to – mass ratios

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

11. 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

12. Low voltage electrophoresis :
* Two compartments to hold
the buffer and electrodes
Figure 6: Apparatus for low voltage electrophoresis
*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

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) :
* 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

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
*
No Joule–Thompson effect
*
No band broadening
*
Most prominently used because of its faster results and
high resolution separation
*
Large range of detection methods available

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