Electrophoresis
Electrophoresis
Electrophoresis
2022
A Seminar on
Semester: 1
Vallabh Vidhyanagar
INDEX
1 Overview on Electrophoresis 3
2 Principle of Electrophoresis 4
3 Instrumentation 4
5 Detection 5
6 Types of Electrophoresis 6
7 Application 10
8 Reference 10
Overview on Electrophoresis
Introduction
Many substances exist in solution as electrically charged species. Once ionized, cations (+) or anions
(-) can be separated in an electrical field where they will migrate toward the cathode or anode
depending on their charge. ‘Electro’ means electric and ‘phoresis’ means to carry.
The rate at which ions move toward the attracting electrode is dependent on the balance between the
impelling force of the electric field on the charged ion and the frictional and electrostatic retarding
effects (i.e., the drag) between the sample and the surrounding medium. The degree of the drag
depends on the size and shape of the molecule and on the viscosity of the matrix. An estimate of the
velocity of a molecule in an electric field, in a solution, is given by the following equation:
v = Eq/d6πrn
where, v is the velocity at which the molecule is moving, E the electric field, q the net charge on the
molecule, d the distance between the electrodes, r the radius of the molecule, and Z the solution
viscosity.
The movement of an ion is proportional to its charge and the electric field, and inversely proportional
to its size and the viscosity of the surrounding medium.
Electrophoresis is a physical method of analysis which involves separation of the compounds that are
capable of acquiring electric change in conducting electrodes.
Electrophoresis may be defined as the migration of the charged particle through a solution under the
influence of an external electrical field. Ions that are suspended between two electrodes tend to travel
towards the electrodes that bear opposite charges.
“Electrophoresis” literally means running in the electric field. The charged molecule moves to their
counter charge electrodes but electric field is removed before it reaches the electrode. Movement of
charged species in an electric field gives differential mobility to the sample based on the charge and
consequently resolve them. Movement of the charged particle is retarded with the addition a
polymeric gel so that a sufficient time is available for resolving the sample. The polymeric gel is inert,
uncharged and does not cause retardation by binding the molecule. Instead it, forms pores of different
size and sample pass through these pores and as a result their electrophoretic mobility is reduced.
Principle of Electrophoresis
When a charged particle of an ith species with charge number z i is placed in the electric field with an
intensity E (in Vm-1), the Coulombic force Fc = z ieE acting on the particle causes its movement; e
=1.602 * 10-19 C is the elementary charge. As in electrophoresis the particles to be separated move
in liquid or gel form, they face obstacles during the movement – mostly molecules of the solvent or
polymer chains of the gel. The collisions and/or interactions with them causes the friction force F f,
which is proportional to the velocity of the moving particle, and which acts against the Coulombic
force. After a very short time (10-13–10-10 s) both forces are balanced, Fc = Ff, and a mean velocity of
the particle attains a constant value. The net movement of charged particles in the electric field is
called electromigration and the velocity of such movement is called electrophoretic velocity. Various
species have, under given conditions, a different electrophoretic velocity. When moving for a certain
time, they are spatially separated. This is a separation principle of the majority (but not all)
electrophoretic separation techniques.
Instrumentation
The supporting medium where electrophoresis takes place is called the electrophoretic system or the
separation system. The separation systems are solutions of electrolytes that are filled into a separation
column or a separation channel. Both ends of the column are connected with vessels, which are also
filled with the solution of electrolytes and serve as a stock of the solution. The electric voltage (500–
30,000 V), which is called driving voltage, is provided by two electrodes in the two vessels and rises
the driving electric field. The analyzed species (analytes) are introduced through a narrow sample
plug, which then migrate across the longitudinal axis of the column in the liquid, the negatively
charged analytes (anions) in the direction of the anode and the positively charged ones (cations) in the
direction of the cathode. As various species have different velocities in the electric field, they are
spatially separated from each other and are detected by a suitable detector, which is located at a
certain position of the column or, sometimes, at its end.
The separation column is often a very thin tube, with an inner diameter of 10–500 mm, which is
called the capillary; the electrophoretic methods that utilize such a capillary are called the capillary
electrophoretic methods. The separation column can also be formed by a channel in various materials
(glass, plastic, silica, etc.), which is fabricated by means of technologies normally used for the
production of electronic microchips. These approaches are called ‘lab-on-a-chip’ technologies and
electrophoresis that uses this approach is called chip or microchip electrophoresis.
A hydrophilic cross-linked gel (mostly polyacrylamide or polysaccharide gel), which is soaked with a
solution of electrolytes, or a viscous solution of a linear polymer with an electrolyte, can also serve as
the separation medium. Electrophoresis using such gels or polymeric media is called gel
electrophoresis. The polymeric chains of the gel serve in most cases not only as a supporting medium
but also influence to a great extent the movement of the separated species.
Samples are generally applied using a micropipette or syringe. The position of application will depend
on whether the sample contains components that will migrate to both electrodes. In this case the
sample is usually applied to the center of the support medium as a spot or narrow streak and
components will migrate in both directions. For samples where the components are all positively or
negatively charged, sample application should be at one end of the medium farthest away from the
attracting electrode. For a gel support medium the sample is usually applied to a well cast in the gel.
For capillaries the sample is applied at one end furthest from the detector. This is usually achieved by
dipping the capillary into the sample vial, the vial is then pressurized causing a volume to be forced
into the capillary. Sample volumes used in electrophoresis can vary from 1*10 -5 cm3 (in CE) to 5 cm3
for some preparative separations.
Detection
Detection of unknown compounds or molecules after electrophoresis is usually in situ. Most
biological molecules are colorless and need to be treated to produce stable colored compounds.
Examples are the visualization of proteins in polyacrylamide gel with Coomassie Blue or silver stain.
The medium can also be treated with compounds that bind to the sample causing them to fluoresce
under ultraviolet (UV) light. Enzymes can be detected by histochemical methods where substrates are
converted into insoluble colored products. It is possible to detect many biological compounds by
immunological methods; for example, in western blotting the separated components are transferred
from a polyacrylamide gel onto another suitable medium (e.g., nitrocellulose) that can then be treated
with antisera and the conjugated product detected by various methods. If a sample contains
radioactive components then the radioactive element can be detected by several different methods,
e.g., phosphor imaging, fluorography. In CE, there are detectors built into the instrument to detect the
peaks as they are resolved. The most frequently used is a UV absorbance detector. There are also
Types of Electrophoresis
Paper Electrophoresis :
Paper electrophoresis is the simplest and cheapest form of electrophoresis. A strip of commercially
available chromatography paper is soaked in buffer and placed with one end in each buffer reservoir
(connecting wicks may be used). It is important that the paper is saturated with the buffer since it is
the buffer that conducts the majority of the current. A spot of sample is placed in the center of the
strip. When the current is applied ions separate out and migrate toward the attractive electrodes
Paper electrophoresis has several limitations. Only small charged molecules can be reliably separated
since many macromolecules adsorb on to the paper, although it is possible to reduce the adsorption by
using a buffer that is more alkaline than the isoelectric point of the sample. Paper systems are
associated with high electrical resistance causing heating that dries out the paper. Although this
method has been largely superseded by gel methods for many samples it is still useful for separation
of small molecules such as amino acids, small peptides, nucleotides, and inorganic ions. An important
limitation to this technique is that considerable diffusion of small molecules occurs at low voltage.
Zone Electrophoresis:
This mode is characterized by two features: (1) the whole separation system together with electrode
vessels is filled with a homogeneous solution of electrolytes, which is called the background
electrolyte (BGE), (2) the mixture of analyzed species to be separated is introduced or injected in a
rather low amount as a short plug at one end of the separation column. The mode of zone
electrophoresis is often used in capillary columns, when it is called capillary zone electrophoresis
(CZE). The BGE is mostly formed by a suitable buffer to maintain a certain pH, which is optimal for
the separation of a given mixture. This mode is also almost exclusively used for gel electrophoresis:
here the whole separation system is formed by a buffer-soaked hydrophilic slab gel and the mixture to
Isotachophoresis:
Figure 5: Isotachophoresis
Isoelectric Focusing
Capillary Electrophoresis:
CE separates samples by applying voltage across buffer-filled capillaries. The capillaries are usually
made of fused silica with an external polyimide coating for increased mechanical strength. Capillaries
are between 25 and 100 cm long with an internal diameter of 25–100 mm. After passing through a
detector the separated ions are seen as peaks. The area of each peak is proportional to the
concentration of the molecule, which enables quantitative studies to be carried out. Advantages of CE
are that analysis time is rapid: 1–30min and operating costs are low. Detection can be by a variety of
methods depending on the sample. CE has become a range of techniques that include: capillary zone
electrophoresis where separation is based on size and charge differences; capillary gel electrophoresis
analogous to SDS-PAGE where separation is based solely on size; capillary isoelectric focusing,
separation of neutral compounds using surfactant micelles and capillary isotachophoresis. Using these
methods it is possible to use CE for separation of many different sample types, e.g., inorganic ions
such as metal ions and anions (chloride, sulfate, and nitrate), DNA, serum proteins. CE is routinely
used in a clinical setting, in the pharmaceutical industry, and also in forensic laboratories.
Application
For estimation of molecular weight of proteins and nucleic acids
Separation of organic acids, alcohols, insulin, alkaloid, carbohydrates, amino acids, phenol,
nucleic acids
Determination of structure of proteins
Purification of isolated proteins
Identifying disulfide bond between proteins
Agriculture testing
In food industry
In forensic science
Isolation of targeted DNA as well as to estimate size of DNA
Reference
1. R.J. Fritsch, I. Krause, in Encyclopedia of Food Sciences and Nutrition (Second Edition), 2003
3. R. Stringer, ELECTROPHORESIS | Overview, Editor(s): Paul Worsfold, Alan Townshend, Colin Poole,
Encyclopedia of Analytical Science (Second Edition), Elsevier, 2005, Pages 356-363