Electrophoresis MODULE
Biochemistry
21
Notes
ELECTROPHORESIS
21.1 INTRODUCTION
The movement of particles under spatially uniform electric field in a fluid is
called electrophoresis. In 1807, Ferdinand Frederic Reuss observed clay
particles dispersed in water to migrate on applying constant electric field for the
first time. It is caused by a charged interface present between the particle surface
and the surrounding fluid. The rate of migration of particle depends on the
strength of the field, on the net charge size and shape of the molecules and also
on the ionic strength, viscosity and temperature of medium in which the
molecules are moving. As an analytical tool, electrophoresis is simple, rapid and
highly sensitive. It is used analytically to study the properties of a single charged
species and as a separation technique. It provides the basis for a number of
analytical techniques used for separating molecules by size, charge, or binding
affinity, example- for the separation of deoxyribonucleic acid (DNA), ribonucleic
acid (RNA), or protein molecules using an electric field applied to a gel matrix.
Gel matrix used mainly is polyacrylamide and agarose. DNA Gel electrophoresis
is usually performed for analytical purposes, often after amplification of DNA
via PCR, but may be used as a preparative technique prior to use of other
methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or
Southern blotting for further characterization.
OBJECTIVES
After reading this lesson, you will be able to:
z define the electrophoresis
z describe the principle and important types of electrophoretic methods
z explain the principle and components of a electrophoresis
z explain various uses of electrophoresis
BIOCHEMISTRY 269
, MODULE Electrophoresis
Biochemistry
21.2 PRINCIPLE
The surface adsorbed sample strongly affects suspended particles by applying
electric surface charge, on which an external electric field exerts an electrostatic
coulomb force. According to the double layer theory, all surface charges in fluids
are screened by a diffuse layer of ions, which has the same absolute charge but
opposite sign with respect to that of the surface charge. The electric field also
exerts a force on the ions in the diffuse layer which has direction opposite to that
Notes
acting on the surface charge This force is not actually applied to the particle, but
to the ions in the diffuse layer located at some distance from the particle surface,
and part of it is transferred all the way to the particle surface through viscous
stress. This part of the force is also called electrophoretic retardation force. When
the electric field is applied and the charged particle to be analyzed is at steady
movement through the diffuse layer, the total resulting force is zero:
Ftoto = 0 = Fel + Ff + Fret
Considering the drag force on the moving particles due to the viscosity of the
dispersant, in the case of low turbulence and moderate electric charge strength E,
the velocity of a dispersed particle ν is simply proportional to the applied field,
which leaves the electrophoretic mobility μe defined as:
ν
μe =
E
The most known and widely used theory of electrophoresis was developed in
1903 by Smoluchowsky
εr ε0 ζ
μe = ,
η
where εr is the dielectric constant of the dispersion, ε0 is the permittivity of free
space (C² N–11 m–2), η is dynamic viscosity of the dispersion medium (Pa s),
and ζ is zeta potential (i.e., the electrokinetic potential of the slipping plane in
the double layer).
The Smoluchowski theory is very powerful because it works for dispersed
particles of any shape at any concentration. Unfortunately, it has limitations on
its validity. It follows, for instance, from the fact that it does not include Debye
length κ–1. However, Debye length must be important for electrophoresis, as
follows immediately from the Figure on the right. Increasing thickness of the
double layer (DL) leads to removing point of retardation force further from the
particle surface. The thicker DL, the smaller retardation force must be.
Detailed theoretical analysis proved that the Smoluchowski theory is valid only
for sufficiently thin DL, when particle radius a is much greater than the Debye
length :
270 BIOCHEMISTRY
Biochemistry
21
Notes
ELECTROPHORESIS
21.1 INTRODUCTION
The movement of particles under spatially uniform electric field in a fluid is
called electrophoresis. In 1807, Ferdinand Frederic Reuss observed clay
particles dispersed in water to migrate on applying constant electric field for the
first time. It is caused by a charged interface present between the particle surface
and the surrounding fluid. The rate of migration of particle depends on the
strength of the field, on the net charge size and shape of the molecules and also
on the ionic strength, viscosity and temperature of medium in which the
molecules are moving. As an analytical tool, electrophoresis is simple, rapid and
highly sensitive. It is used analytically to study the properties of a single charged
species and as a separation technique. It provides the basis for a number of
analytical techniques used for separating molecules by size, charge, or binding
affinity, example- for the separation of deoxyribonucleic acid (DNA), ribonucleic
acid (RNA), or protein molecules using an electric field applied to a gel matrix.
Gel matrix used mainly is polyacrylamide and agarose. DNA Gel electrophoresis
is usually performed for analytical purposes, often after amplification of DNA
via PCR, but may be used as a preparative technique prior to use of other
methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or
Southern blotting for further characterization.
OBJECTIVES
After reading this lesson, you will be able to:
z define the electrophoresis
z describe the principle and important types of electrophoretic methods
z explain the principle and components of a electrophoresis
z explain various uses of electrophoresis
BIOCHEMISTRY 269
, MODULE Electrophoresis
Biochemistry
21.2 PRINCIPLE
The surface adsorbed sample strongly affects suspended particles by applying
electric surface charge, on which an external electric field exerts an electrostatic
coulomb force. According to the double layer theory, all surface charges in fluids
are screened by a diffuse layer of ions, which has the same absolute charge but
opposite sign with respect to that of the surface charge. The electric field also
exerts a force on the ions in the diffuse layer which has direction opposite to that
Notes
acting on the surface charge This force is not actually applied to the particle, but
to the ions in the diffuse layer located at some distance from the particle surface,
and part of it is transferred all the way to the particle surface through viscous
stress. This part of the force is also called electrophoretic retardation force. When
the electric field is applied and the charged particle to be analyzed is at steady
movement through the diffuse layer, the total resulting force is zero:
Ftoto = 0 = Fel + Ff + Fret
Considering the drag force on the moving particles due to the viscosity of the
dispersant, in the case of low turbulence and moderate electric charge strength E,
the velocity of a dispersed particle ν is simply proportional to the applied field,
which leaves the electrophoretic mobility μe defined as:
ν
μe =
E
The most known and widely used theory of electrophoresis was developed in
1903 by Smoluchowsky
εr ε0 ζ
μe = ,
η
where εr is the dielectric constant of the dispersion, ε0 is the permittivity of free
space (C² N–11 m–2), η is dynamic viscosity of the dispersion medium (Pa s),
and ζ is zeta potential (i.e., the electrokinetic potential of the slipping plane in
the double layer).
The Smoluchowski theory is very powerful because it works for dispersed
particles of any shape at any concentration. Unfortunately, it has limitations on
its validity. It follows, for instance, from the fact that it does not include Debye
length κ–1. However, Debye length must be important for electrophoresis, as
follows immediately from the Figure on the right. Increasing thickness of the
double layer (DL) leads to removing point of retardation force further from the
particle surface. The thicker DL, the smaller retardation force must be.
Detailed theoretical analysis proved that the Smoluchowski theory is valid only
for sufficiently thin DL, when particle radius a is much greater than the Debye
length :
270 BIOCHEMISTRY
