What is Electrophoresis?
Electrophoresis in analytical chemistry is the technique of analysis or separation process of dispersed particles present on fluid under the influence of the electric field due to different rates of migration. Capillary and gel electrophoresis are the most common types of electrophoresis which we used for the separation of ions. In recent years exciting discoveries have been made in the field of gel and capillary electrophoresis. The choice of gel largely depends upon the molecular size and chemical properties of substances to be separated. Gel and capillary electrophoresis principle or apparatus are widely used for the analysis and separation of biomolecules such as DNA, RNA, colloids, proteins, and enzymes.
Gel Electrophoresis Process
Gel electrophoresis is a technique that works for fractionating substances such as proteins, enzymes, DNA, RNA according to their molecular sizes. The following process is used in the gel electrophoresis technique,
- It has a stationary phase consisting of a heterogeneous cross-linked polymer gel. Agar, agarose, polyacrylamide are the most common types of gel which we used in electrophoresis instruments. They are available in the market under the trade names such as BioGel, Sephadex, Styragel, Agarose, etc. The mechanism is based upon the different permission rates of each solute molecule into the interior gels.
- Using an electric field, proteins, enzymes, DNA, RNA can move through a gel made by agar, agarose, polyacrylamide, etc. The electric field contains a negative electrode at one end which pushes the molecules through the gel and a positive electrode at the other end which pulls the molecules through the gel.
- Therefore, when the electric field is applied, the larger molecules move more slowly through the gel while the smaller molecules move faster through the gel.
- By the flow of liquid, molecules diffused all the parts of the gel. Only those molecules having larger sizes remain behind the gel.
- Buffers in gel carry current and are used to maintain the pH value. It contains plenty of ions that carry a passage of electricity. For analysis of nucleic acids such as DNA or RNA, we used Tris/Acetate/EDTA (TAE) or Tris/Borate/EDTA (TBE) as a buffer.
Principle of Electrophoresis
If a charged foreign phase is subjected to a potential gradient, the foreign phase migrates through a continuous medium to the electrode according to the sign of the charge on the particle. Therefore, electrophoresis of positively charged particles or cations is called cataphoresis, while electrophoresis of negatively charged particles or anions is called anaphoresis. It establishes the basic concentration gradient across the system. Colloids, proteins, DNA, RNA, and enzymes show particular electrophoretic mobilities and isoelectric points. These properties can be used for the identification of substances.
Generally, the term electrophoresis is used for the transport of charged solutes through paper or gels under the influence of potential gradients. The migration of charged particles through the instrument depends on the surface, changes, applied voltage, electrolytic concentration, ionic strength, pH scale, viscosity, and other properties of migration medium. Ions migrating in one direction are affected by the ionic mobility of other ions moving in the opposite direction.
Applications of electrophoresis
Large biological molecules can be separated and purified by electrophoretic instruments. Gel or capillary electrophoresis is widely used in forensic, genetics, microbiology, and biological chemistry for the analysis and separation of samples. It is applied for the separation of proteins, enzymes, DNA, RNA, and biopolymers. Electrophoresis instruments are also used widely in the separation of inorganic ions and in clinical diagnosis. Inorganic separations are most readily achieved in the presence of complexing agents like EDTA. Some important applications of the techniques are given below,
Application on DNA fragments analysis
One of the most important applications of electrophoresis is the analysis of DNA and the study of DNA fragments. On the application of electric field, DNA or deoxyribonucleic acid breaks down into large and small fragments. It occurs because different parts of DNA fragments are affected by different scales of electric current. Now gel electrophoresis is used to separate and analysis of different fragments of DNA samples.
Application of electrophoresis on proteins
Immunoelectrophoresis is a common type that we used to analyze the unusual behavior of proteins. Irregular proteins are formed due to different types of medical conditions such as kidney failure, multiple sclerosis, and certain types of cancers. These irregular proteins can be detected with the help of immunoelectrophoresis of blood and urine samples.
Application on testing antibiotics
To detect the purity of antibiotics, we used the electrophoresis principle. Capillary and paper electrophoresis instruments are used to test the purity of antibiotics. It is also used for the determination of strength and exact dose of antibiotics.