INSTRUMENTAL TECHNIQUES IN FOOD ANALYASIS : Study of gel electrophoresis as analytical technique

 

Title: Study of gel electrophoresis as analytical technique

Introduction 

Many biologically important molecules like amino acids, peptides, proteins and nucleic acids possess ionizable groups and may therefore be made to exist in solutions as electrically charged species, either as cation (+) or anions (-). The magnetitude of opposite polarity i.e. cations move to the cathode (-) and anions move to the anode (+) when an electrical field is applied. This principle is employed in electrophoresis to separate molecules of differing charges.

Electrophoresis may be a technique that permits separation and analysis of charged molecules in an electrical field. Gel electrophoresis is most ordinarily used for separation and purification of proteins and nucleic acids that differ in size, charge, or conformation. The gel consists of polyacrylamide or agarose.

Gel electrophoresis may be a method for separation and analysis of macromolecules and their fragments, supported their size and charge.

Gel electrophoresis:

Gel electrophoresis is a widely used type of electrophoresis in which molecules are separated.

     

by movement through a porous gel under theinfluence of an electrical field. Separation is

brought about through molecular sieving technique, based on the molecular size of the

substances. Gel material acts as a "molecular sieve”. Different types of gels which can be used are; Agar and Agarose gel, Starch, Sephadex, Polyacrylamide gels.

• A porous gel acts as a sieve by retarding or, in some cases, by completely obstructing the movement ofmacromolecules while allowing smaller molecules to migrate freely. It is used to separate nucleic acids

 (DNA and RNA), nucleic acid fragments, proteins and amino acids.

• Gel is a colloid in a solid form (99% is water). It is important that the support media is electrically neutral.

Principle: 

The electromotive force (EMF) generated across the electrodes pushes or pulls the molecules (nucleic acids or proteins) through the gel matrix. The molecules move towards the anode if it is negatively charged or towards the cathode if positively charged.

Factor affecting migration rate: 

The electrophoretic mobility of a molecule depends upon many factors as described below. Separation of molecules can therefore be affected by selecting the appropriate conditions. The nature of charged compounds affects their migration rates in several ways: 

1) Charge: The rate of migration increases with an increase in net charge. The magnitude of charge is generally pH dependent.

 2) Size: The rate of migration deceases for larger molecules, due to the increased frictional and electrostatic forces which are exerted by the surrounding medium. 

3) Shape: Molecules of similar size but different shapes such as fibrous and globular proteins exhibit different migration characteristics because of the differential effect of frictional and electrostatic forces.

Electrophoresis apparatus: 

The equipment required for electrophoresis consists basically of two items, a power pack and an electrophoresis unit. 

1) power pack The power pack provides a stabilized direct current and has controls for both voltage and current output. For low voltage use, power packs are available with an output of 0-500V and 0-150mA or can give either constant voltage or constant current 

2) Electrophoresis unit The electrophoresis unit contains the electrodes, buffer reservoirs, a support for the electrophoresis medium and a transparent insulating cover. Stainless steel electrode can be used, but some buffers cause corrosion and platinum electrodes are more satisfactory. 

3) Buffer reservoirs The two buffer reservoirs are normally partitioned into two sections, the electrode and wick compartments. Electrical contact between the buffer in the two compartments is maintained by small holes or slots in the partition between the compartments or by means of porous wicks. Contact between the supporting medium and the buffer in the reservoir is normally maintained by wicks of several layers of filter papers or gauze. Wicks can be dispensed with low voltage paper electrophoresis and contact maintained by having the paper dipping into the buffer directly. 

4) Supporting medium The supporting medium, saturated with buffer and loaded with sample, is normally arranged horizontally on a flat surface of insulating material such as Perspex. Horizontal electrophoresis units are available for use with paper, cellulose acetate, metal cooling plates are required as for high voltage electrophoresis. Gels and thin layers are sometimes covered with a plate of insulating material to prevent evaporation. Simple vertical paper electrophoresis units are also available for low voltage work and slabs of starch gel can be used vertically in special apparatus.

Samples are separated on horizontal slabs, for preparative polyacrylamide gel electrophoresis, whereas for analytical investigations, vertical cylindrical columns of gel are supported in precision bores glass tubes in specially designed apparatus

Applications: 

1) Separation of DNA fragments for DNA fingerprinting 

2) To analyze results of polymerase chain reaction 

3) Determination of size, molecular weight of protein and amino acid 

4) Separation of organic acid, alkaloids, carbohydrates, amino acids, phenols nucleic acid and insulin 

5) Used for analysis of terpenoids and antibiotics 

6) Also used in separation of carbohydrates, vitamins. 

7) Determination of specific enzymatic activity