Electrophoresis , principles and application

1. Principle:
Any charged ion or
molecule migrates when
placed in an electric field
the rate of migration
depend upon its net
charge size,shape and the
applied electric current.
 Mechanism:
• Definition=“migration of charged particles or
molecules under the influnce of electric current.”
• Literally= greek word means transport by
electricity.
Electrophoresis

2. Factors affecting the rate of ion
mobility
– Net charge of
molecule
– Size & shape of
molecule
– Electric field
strength
– Properties of
supporting medium
– Temperature
– pH of the buffer

3. Capillary electrophoresis:
Sample vial Destination vial
Definition;
It is a seperation
technique in which
electrophoresis is
performed in narrow bore
capillaries typically 25-
100um inner diameter.
• Difference from
electrophoresis;
In traditional
electrophoresis,electricall
y charged analytes move
in a conductive liquid
medium under the
influence of an electric
field,whereas cappilary
electrophoresis is
designed to separate
species based on their
charge to size ratio in the
interior of small capillary
filled with an electrolye.

4. Instrumentation
 A fused silica capillary
 Sample vial and destination vial
 Two buffer reserviors
 A high voltage power supply
 Detector
 Data output and handling device

5. Working of capillary electrophoresis

6. 1-Capillary zone electrophoresis
(CZE)
PRINCIPLE “In
CZE,analytes
are seperated
in a capillary
containing only
buffer,without
any anti
convective
medium”
Mechanism:
EXAMPLE:
Separation of small
ions, some proteins,
amino acids, and
carbohydrate.

7. 2-Capillary gel electrophoresis(CGE)
 Principle;
“seperation takes place
inside a capillary filled
with a gel which acts
as a molecular sieve”
Mechanism;
 Gels:
 linear noncrosslinked
polyacrylamide
(CH2=CH-CO-NH2) (C-
PAGE)
 dextran,
 agarose, and
 poly(ethyleneoxide)
• Gel matrix viscosity, density, and
pore size are all factors in
determining the ‘speed’ of
separation.
• Two types of separation can be done
using GE:
o Native: separation by size
and
o charge (charge/mass)
o Denaturing: separation by
size

8. Instrumentation
• Power supply
• Cooling Apparatus
• Electrophoresis gel
apparatus
• White Light/UV Light Box
• Digital Camera/Gel
Documentation System
• Working:
• Prepare samples
• Prepare gel and buffers
• Load samples onto gel
• Run gel
• Stain gel
• Interpret/analysis of gel
• Archive (photograph, dry
gel)

9. Application
• Determinatio
n of DNA
sequences….
• Formation of
gel
(agarose+buff
er)
• Heat it and
allowed to
cool down
• Place the gel
in buffer
• Application of
samples

11. Applications of capillary
electrophoresis
• Following are the fields in which electrophoresis play
important role:
• Pharmaceutics
• Forensic
• Foods
• Bioscience
• Agricultural field
• Pesticide analysis
• Surfactants analysis
• Transition metal analysis
• Organic compound analysis