Electrophoresis

INTRODUCTION

This method is mainly based on the movement of the charged particles towards the opposite charge
by the influence of the direct current which is obtained by the electric field. Electrophoresis is first
proposed by Arne Tiselius for the separation of proteins. In this method, the charged particles
move towards the opposite charge. The mobility of the ions in the electric field is first observed
by Reuss in 1807. After that many scientists proposed different methods based on the principle of
electrophoresis.

In 1939, zone electrophoresis is proposed. In 1950, gel electrophoresis with agar is proposed.
In 1955, gel electrophoresis with starch is proposed. In 1983, capillary electrophoresis is proposed.

PRINCIPLE

The main principle involved in the separation of the compounds by electrophoresis is the
following:

 According to the size.

 According to the charge: When charged molecules are placed in the electric field, they
move towards to the anode or cathode based on their charge.

When the sample is placed in the constant electric field, based on the size, the particles are
separated that is high molecular weight compounds are eluted first and low molecular weight
compounds are eluted later.

Based on the charge, the positively charged molecules move towards the cathode which is
negatively charged and the negatively charged particles move to anode which is positively
charged.

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Separation of the particles

THEORY

The electrophoretic separations are based on the theory that the electric force (F) on a charged
particle in an electric field (E) is directly proportional to the charge of the particle (q)

F = qE

The mobility of the electrons is expressed by the following equation:

Mobility = (applied voltage) (net charge)/(frictional coefficient)

where E is the electric field strength; q is the net charge; 611 is the shape; r is the size; η is the
viscosity.

The value of the electric field depends on the following:

 Charge of the analyte ion.

 Size and shape of the ion.

 Viscosity of the medium

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TYPES OF ELECTROPHORESIS

There are two main classifications:

1. Based on the state:

o Solution electrophoresis.

o Example: Capillary electrophoresis.

o Supporting electrophoresis.

o Example: Paper, film and gel electrophoresis.

2. Based on the material used for the separation:

o Paper electrophoresis: Porous layer of 2-10 cm long paper is soaked in the
electrolyte buffer. In which, the electrodes placed produce a electric field. This
method is slow and poor quantification. It requires large quantity of sample to
analysis.

Paper electrophoresis

o Paper electrophoresis

o Capillary electrophoresis: Narrow silica capillary tube is filled with the electrolyte
buffer solution. When compared other techniques this technique is fast and requires
small sample solution.

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Capillary electrophoresis

o Capillary electrophoresis

o Gel electrophoresis: This method is mainly based on the application of the gel as a
support to the buffer solution. The commonly employed gels are agarose, cellulose
acetate, etc.

Gel electrophoresis

o Gel electrophoresis

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o Isoelectric focusing: The isoelectric point is maintained before the analysis.
Isoelectric point is defined as the point, where the net PH change is zero. This can
be attained by using the neutral solutions.

o SDS-PAGE: This is chemically known as sodium dodecyl sulphate-poly
acrylamide which is used as a supporting material. This is a common detergent
which denatures the protein. The negatively charged particles are attached to the
hydrophobic region.

INSTRUMENTATION

The following are main components and materials used in the electrophoretic instruments:

 Mobile phase: Commonly buffer solutions are employed as the mobile phase. The main
purpose of the mobile phase is it carries the current at constant pH and determines the
electric charge on the solute.

 Example: Barbital buffer, tris-EDTA buffer, tris-acetate-EDTA buffer, tris-borate-EDTA
buffer.

 Power supply: This is to supply the current at constant voltage.

 Support material or stationary phase: This is mainly used to support the mobile phase that
is a buffer and also used for carrying the sample.

Examples:  Paper.

 Starch.

 Agar or agarose is mainly used in the separation of
the large proteins.

 Cellulose acetate.

 Poly acrylamide gel is used in the blotting
technique.

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The stationary phase should be neutral. The presence of charged groups produces migration
retardation.

Detectors: This is mainly used for the detection of the charged particles concentration or
charges or masses. Detection is done by the following two main methods:

1. By staining: by using staining dyes.

2. Example: amido black, Coomassie blue, India ink, Silver stain, Colloidal gold. etc.

3. By using any spectroscopic detector.

4. Example: UV/VIS detector, mass detector, coulometric detector, etc.

ELUTION METHOD

It involves the mixing of the sample with the stationary phase and it is placed in the buffer
solution. Then the current is flowed by placing the electrodes. The charged particles are migrated
towards the opposite electrode and detected with the help of the detector. Finally, it is amplified
and recorded with the recorder.

FACTORS AFFECTING THE ELECTROPHORESIS

 Magnitude of its charge

 Charge density

 Molecular weight

 Tertiary and quaternary structure

 Solution pH

 Electric field

 Solution viscosity

 Temperature

ADVANTAGES

 Excellent degree of purity.

 Application to the wide variety of high molecular weight substances.

 Excellent resolution.

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 Rapid separation.

 Simple to operate.

 Easy to construct.

DISADVANTAGES

 Needs large quantities for analysis.

 Quantification is difficult.

APPLICATIONS

 Mainly used for the determination of high molecular weight compounds.

 Example: Amino acids, proteins and nucleic acids.

 Used for the DNA sequencing.

 Example: Recombinent DNA sequencing.

 Used for the DNA hybridisation.

 Used in the determination of biological fluid constituents.

 Example: Blood constituents determination.

 Used in antibiotics analysis.

 Example: Penicillin determination.

 Used in the purification of vaccines.

 Example: Influenza vaccine is purified.

 Used in the chiral analysis.

 Example: Technique is used in chiral chromatography.

 Used in the determination of impurities.

 Used in the atropine phosphate assay.

 Used in the codeine assay.

 Used in the diagnosis of genetic toxicology.

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 Used in the diagnosis of hereditary diseases.

 Used in the carbohydrates analysis.

 Example: Used in invert sugar.

 Used in the forensic science.

 Example: Used in composition finger print analysis determination.

 Used in the molecular biology

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