22-03-2011, 04:37 PM
[attachment=10774]
ELECTROPHOROSIS
Migration of colloidal particles through a solution under the influence of electric field is called electrophoresis.
When a potential difference is applied between the two electrodes in a the colloidal solution it is been observed that the colloidal particles are earned to either the positive or negative electrode this phenomenon is called electrophoresis
Applied to separation of
Inorganic anions and cations
Amino acids
Catecholamines
Drugs vitamins carbohydrates peptides protein nuclic acids
Theory and principles
Principles
Electrophoretic separation is performed by injecting a small amount of sample in to an aqueous buffer solution contained in a narrow tube on a porous support medium (paper/semi solid /gel)
A high voltage applied across length of buffer by electrodes located at end of buffer field causes ions from the sample to migrate towards one another of electrode
Rate of migration depends
1. Characteristic of the particle
2. Properties of the electrical field
3. Temperature
4. Nature of the suspended medium
Separation is based on charge to ratio of various analytics in sample
Charge size ratio is proportional to ion migration in electric field
THEORY
All fluids contain ions-both cations and anions.
An object placed in the fluid, interacts with the ions à adsorption of ions to its surface .Gains positive or negative charge called Electrical Surface Charge.
When External electric field (E) applied to solution electrostatic Coulomb force (F) is exerted on the dispersed particles with charge q.
F=qe
Particles migrate towards the oppositely charged electrode.
Velocity of migration
V=E×q/F
v = velocity of migration of the molecule.
E = electric field in volts per cm
q = net electric charge on the molecule
f = frictional coefficient
This migration of the charged particle in the electric field is called Electrophoretic mobility, denoted as (μ)
µ=v/e or q/f
ELECTROPHORETIC RETARDATION FORCE
• Surface charges in fluids à surrounded by diffuse layer of ions à same magnitude but opposite sign
• Electric field exerts a force on the ions in diffuse layer à direction opposite to that acting on the surface charge à Electrophoretic Retardation Force
Classification
I . MOVING BOUNDARY ELECTROPHOROSIS/ FREE SOLUTION METHOD/ FREE BOUNDARY ELECTROPHOROSIS
In a free solution the motion of charged particles are under influence of an electric field
Separation is done in absence of a supporting medium .In this, the movement of the boundary is measured between a solution of the molecule being investigated and the solvent
Apparatus: A u-shaped cell filled in buffer solution electrodes immersed at its ends
Sample applied mixture of charged compounds (eg.protein mixture)
Apply voltage lumps migrate to anode/cathode depending on changes. Changes in refractive index at Boundaries of separated compounds are detected using Schleiren Optics –at both ends of the solution in cell ( Based on changes in refractive index at boundary)
ELECTROPHOROSIS
Migration of colloidal particles through a solution under the influence of electric field is called electrophoresis.
When a potential difference is applied between the two electrodes in a the colloidal solution it is been observed that the colloidal particles are earned to either the positive or negative electrode this phenomenon is called electrophoresis
Applied to separation of
Inorganic anions and cations
Amino acids
Catecholamines
Drugs vitamins carbohydrates peptides protein nuclic acids
Theory and principles
Principles
Electrophoretic separation is performed by injecting a small amount of sample in to an aqueous buffer solution contained in a narrow tube on a porous support medium (paper/semi solid /gel)
A high voltage applied across length of buffer by electrodes located at end of buffer field causes ions from the sample to migrate towards one another of electrode
Rate of migration depends
1. Characteristic of the particle
2. Properties of the electrical field
3. Temperature
4. Nature of the suspended medium
Separation is based on charge to ratio of various analytics in sample
Charge size ratio is proportional to ion migration in electric field
THEORY
All fluids contain ions-both cations and anions.
An object placed in the fluid, interacts with the ions à adsorption of ions to its surface .Gains positive or negative charge called Electrical Surface Charge.
When External electric field (E) applied to solution electrostatic Coulomb force (F) is exerted on the dispersed particles with charge q.
F=qe
Particles migrate towards the oppositely charged electrode.
Velocity of migration
V=E×q/F
v = velocity of migration of the molecule.
E = electric field in volts per cm
q = net electric charge on the molecule
f = frictional coefficient
This migration of the charged particle in the electric field is called Electrophoretic mobility, denoted as (μ)
µ=v/e or q/f
ELECTROPHORETIC RETARDATION FORCE
• Surface charges in fluids à surrounded by diffuse layer of ions à same magnitude but opposite sign
• Electric field exerts a force on the ions in diffuse layer à direction opposite to that acting on the surface charge à Electrophoretic Retardation Force
Classification
I . MOVING BOUNDARY ELECTROPHOROSIS/ FREE SOLUTION METHOD/ FREE BOUNDARY ELECTROPHOROSIS
In a free solution the motion of charged particles are under influence of an electric field
Separation is done in absence of a supporting medium .In this, the movement of the boundary is measured between a solution of the molecule being investigated and the solvent
Apparatus: A u-shaped cell filled in buffer solution electrodes immersed at its ends
Sample applied mixture of charged compounds (eg.protein mixture)
Apply voltage lumps migrate to anode/cathode depending on changes. Changes in refractive index at Boundaries of separated compounds are detected using Schleiren Optics –at both ends of the solution in cell ( Based on changes in refractive index at boundary)