Electrophoresis

Capillary zone electrophoresis Gel electrophoresis Application

Presented by:

Hina Aslam

Electrophoresis:
Electrophoresis is a separation method based on the differential rate of migration of charged species in a buffer solution across which has been applied an electric field. Electrophoresis is a Greek term which literally means migration with electricity

Why electrophoresis?
The process has exhibited unparalleled resolution in the separation of charges species.
Electrophoresis may separate between the poly nucleotides which may differ by may be a single or just a few nucleotides. The Human Genome Project was possible only as a result of electrophoresis.

Principle:
Under the influence of an electrical field charged molecules and particles migrate in the direction of the electrode bearing the opposite charge. Because of varying charges and masses, different molecules and particles of a mixture will migrate at different velocities and will thus be separated into single fractions.

The rate of migration depend upon its net charge, size, shape and applied electric field. It can be represented by following equation =

v = velocity of migration of molecule E = electric current in volt per cm q = net electric charge on molecule f = frictional coefficient

Factors affecting electrophoretic mobility:

Sample:
i. Charge: Higher the charge, greater the electrophoretic mobility.

ii. Size: Bigger the molecule, greater are the frictional and electrostatic forces exerted on it by the medium.

iii. Shape: Rounder contours experience lesser frictional and electrostatic retardation compared to sharp contours. Therefore, globular protein move faster than fibrous protein.

Electric Field:
An increase in potential gradient increases the rate of migration.

The Buffer:
i. Ionic Strength: When increase ionic strength of the buffer means a larger share of the current being carried by the buffer ions & small proportion carried by the sample ions. When decrease ionic strength, a larger share of the current being carried by the sample ions leading to a faster separation. ii. The buffer pH: When increase pH, increases ionization of organic acids. Decrease in pH, increases ionization of organic bases.

Types of electrophoretic techniques:

Free solution electrophoresis
Capillary zone electrophoresis Capillary Isoelectric Focusing Capillary Isotachophoresis Capillary Gel Electrophoresis

Supporting medium electrophoresis

Paper electrophoresis

Gel electrophoresis
Thin layer electrophoresis

Capillary Zone Electrophoresis

Zone electrophoresis:
Any electrophoretic technique in which components are separated into zones or bands in a buffer.

Zone electrophoresis

Capillary zone electrophoresis

Capillary gel electrophoresis

Capillary Zone electrophoresis:

Capillary zone electrophoresis (CZE) was designed to separate species based on their size to charge ratio in the interior of a small capillary filled with an electrolyte.
Capillary electrophoresis yields high-speed high-resolution separation on exceptionally small sample volumes It separate ionic species by their charge and frictional forces.

Principle of separation:
Two processes operate in capillary: i. ii. Electrophoresis Electro osmosis

Electrophoretic separation:
The migration velocity v (cm/s) of a molecule in an electric field is equal to the product of the field strength E and the electrophoretic mobility e.

 = migration velocity e = electrophoretic mobility (cm/V/s) E = Electric field strength (V/s) The value of E depend upon Charge of analyte ion Frictional retarding forces which includes Size and shape of the ion Viscosity of medium in which migration occurs

Electro osmotic flow (EOF):

An important feature is the bulk flow of liquid and cause electroosmotic flow as follow Usually, the capillaries used are of fused silica. They contain charged sites created by ionization of silanol. Si-OH Si-O The inside wall of the capillary is covered by silanol groups (SiOH) that are deprotonated (SiO-) at pH > 2. SiO- attracts cations to the inside wall of the capillary.

Formation of layers for EOF:

Negatively charged silica surface Stern layer-The immobile layer Diffuse layer of cations (moves)

Net result of electrophoretic and electroosmotic flow:

A pictorial representation of the combined effect in a capillary, when EO is faster than EP (the common case):

Flow profile in electroosmotic flow:

EOF has flat profile because its flow velocity is uniform along the capillary.
In HPLC, frictional force at the column wall cause a pressure drop across the column yielding a parabolic or laminar flow profile. The flat profile of EOF minimizes zone broadening, yielding to high separation efficiencies that allow separation on the basis of mobility difference as small as 0.05 %.

Factors affecting efficiency of (zone broadening)

Longitudinal diffusion Injection length Joule heating Adsorption on capillary wall Detector cell size

Instrumentation:
A sample vial Catholyte Anolyte Cathode Anode High voltage power supply Capillary (25mm to 100mm ID) A Detection Method Data acquisition method

Main components

Working:
Capillary inlet Capillary out let Detector out put

Data out
Handling device Electropherogram

Schematic diagram of capillary electrophoresis system

Electropherogram:
Detectors are placed at the cathode since under common conditions, all species are driven in this direction by EOF. Detectors similar to those used in LC, typically UV absorption, fluorescence, and MS. Sensitive detectors are needed for small concentrations in CE. The general layout of an electropherogram:

Gel Electrophoresis

Gel electrophoresis:
Gel electrophoresis refers to the separation of charged particles on the basis of size and charge by movement through a gelatinous material when an electric current is applied. Charged particles polypeptides, etc. can include DNA, amino acids,

The electric current causes the protein or nucleic acid molecules to move through the gel, allowing for the separation of molecules of different sizes.

Gel electrophoresis

Types of gel:
Agarose
Most agarose gels are made between 0.8% and 2%. A 0.8% gel will show good resolution (separation) of large DNA fragments A 2% gel will show good resolution for small fragments

Polyacrylamide
It is used if sharper bands are required (protein separation)

Starch

Factors affecting separation:

Factors effecting separation are

Pore size of gel (resistance)

Buffer strength

Gel temperature

Nature of sample

Steps involved in separation:

Prepare samples

Interpret/analysis of gel

Electropherogram

Prepare gel

Stain gel

Load samples onto gel

Run gel

Steps involved:

Electropherogram:
A series of bands represent separated analytes.
Distance traveled is called migration time (dm), which can be compared to standards.

Applications

Gel electrophoresis of DNA:

Gel preparation

Sample loading into gel

Running gel

electropher ogram

Interpret/an alysis of gel

Staining the gel

Gel electrophoresis of DNA:

Applications:
Electrophoresis has been applied to a variety of difficult analytical separation problems:
Inorganic anions and cations Amino acids Vitamins Drugs Carbohydrates Peptides Nucleotides Proteins Polynucleotides

Source:
Modern analytical chemistry by David Harvey, pp 597-605 Analytical chemistry by Garry D Christain Principle of instrumental anlysis by Skoog edition V, pp779790 www.biotech.iastate.edu/publication/ppt-presentations Kreuzer, H., Massey, A., 2001, Recombinant DNA and Biotechnology, 2nd ed. ASM Press, Washington Turner, P.C., et al, 1997, Instant Notes in Molecular Biology, Bios, Oxford Photos - L. D. Macdonald, 2003