Electrophoresis is a separation technique where different molecules of a substance are separated depending on their capacity to travel through a stationary phase with respect to the mobile phase.
The term electrophoresis is derived from two words i.e., ‘electro’ meaning electricity, and ‘phoresis’ meaning separation.
Thus, it can be defined as the separation of serum proteins by the effect of electric current.
Arne Tiselius won Nobel prize in Chemistry in 1948 and is considered as the Father of electrophoresis for his research and adsorption analysis for the discovery of the complex nature of serum proteins.
Types of Electrophoresis
Contingent on the nearness or non-appearance of supporting media the technique can be classified as Moving Boundary and Zone electrophoresis.
Moving Boundary Electrophoresis
- Capillary Electrophoresis
- Immuno Electrophoresis
- Paper Electrophoresis
- Gel Electrophoresis
- Thin Layer Electrophoresis
- Cellulose Acetate Electrophoresis
Moving Boundary Electrophoresis
This helps in the separation of molecules in a free solution without the support of any medium. It is of 4 types which include:-
Capillary Electrophoresis (CE)
CE is a method in which charged particles move towards their opposite poles and separates on the basis of a physical characteristic such as size of particles.
The instrument of this method consists of a capillary tube, high voltage power supply, sample, buffer solutions, detector, and output device. The capillary tube is filled with an electrolyte solution and the ends are dipped in the reservoirs filled with an electrolyte solution.
Both ends of the reservoirs are connected with electrodes to complete an electric circuit so that the charged particles move towards the opposite charge and get separated based on the size and charge of particles/molecules.
CE depends on Electro-osmotic flow and electrophoretic mobility.
Unlike in chromatographic techniques where the mobile phase pushes the sample through a stationary phase or mobile phase is pumped. In CE, the electric circuit maintained pushes the buffer solution to move from one reservoir to another. The flow is called as electro-osmotic flow.
In this method, the sample ions move under the influence of applied voltage. The sample ions undergo a force equal to the product of net charge and strength of the electric field which is also affected by drag force equal to the product of friction coefficient and velocity. The resultant expression is known as electrophoretic mobility.
CE is used to detect bacterial/microbial contamination, determination of ions in saliva, amplification and detection of DNA fragments.
This technique is based on the principle of moving-boundary electrophoresis and the term “Isotachophoresis” is derived from three words i.e., ‘Iso’ meaning equal, ‘tacho’ meaning speed, and ‘phoresis’ meaning migration.
ITP is a technique in which separation and concentration of ionic samples are done based on the ionic mobility factor which tells how fast the ionic analytes migrated in the presence of the electric field.
The separation due to this technique is achieved by stacking them in discrete zones based on their mobilities producing very high resolution.
Separation of ionic analytes in this technique involves a leading anion with high mobility than sample ion, trailing anion with lower mobility and a common cation for anions. When the electric field is started, leading anions move towards appropriate electrode followed by sample ion and trailing anion in order of their mobilities.
All these anions will move at the same speed once equilibrium is established between them and during that time the separated is done on the basis of their mobility.
Isotachophoresis is used in determining lactate ions taken from the person usually encountered by the Forensic Pathologist, detecting detergents and inorganic ions in the effluent water, food quality control, separating charged substances such as proteins, organic acids, etc.
The term ‘Immuno-electrophoresis’ was first coined in 1953 by Grabar and Williams. The process is a combination of Immuno-diffusion and electrophoresis which refers to precipitation in agar under electric field.
This technique is based on the principle that when an electric field is applied to the slide which is layered by gel, the antigen mixture placed in the gel is separated based on the charge and size of particles.
After the separation of the particles, the antigen-mixture is separated with anti-sera placed in the troughs allowing diffusion. In the presence of electric field, anti-sera moves towards antigen mixture marking a precipitin line within a day to indicate the reaction between antibody and its protein.
It is a powerful technique which has a high resolving power as it is a combination of separation of antigens and diffusion with anti-sera and a large number of antigens can be identified in a particular serum.
This technique helps in the forensic identification of blood stains for diagnosing age of blood stain and determining the species to which the blood stain belongs. Apart from this, it is also used in identification of proteins and determining quantity.
It also aids in the diagnosis and evaluation of therapeutic response in many diseases affecting immune system.
Isoelectro Focusing (IEF)
Isoelectro Focusing is a commonly used technique for separation of proteins (amphoteric) molecules mainly by the difference in their isoelectric point.
The Isoelectric point is the point in the pH gradient at which the charge on protein is zero. A protein migrates into the point where its net-charge is zero isoelectric pH.
In this process, solid surface such as polyacrylamide is required and the protein (amphoteric) molecules to be separated are subjected to an electric field in a pH gradient. Angelika Gorg, Walter Weiss, and Michael J. Dunn introduced an immobilized pH gradient.
This technique is better for conventional electrophoresis for genetic marker typing, research in taxonomy, immunology, separation of peptides and proteins.
It is a powerful analytical technique for separation of proteins as it can separate the proteins with as little as 0.001 pH units, however, has limited stability of the solutions and inconsistent in providing better results.
A type of electrophoresis in which the separation of protein molecules takes place with the help of a supporting media such as Paper, Cellulose, Polyacrylamide, Starch, etc. It is of 4 types which include:-
It is one of the important and simple separation techniques requiring a filter paper as a medium of separation and can be done either horizontally or vertically.
The process includes the apparatus of two troughs containing buffer solution in it to pass the electric current for separation of proteins, oligopeptides, amino acids, etc.
The technique is based on the principle that “The charge carried by a molecule depends on the pH of the medium. Electrophoresis at low voltage is not usually to separate low molecular weight compounds because of diffusion, but it is easier to illustrate the relationship between charge and pH with amino acids than with proteins (or) other macromolecules”.
This technique is used for Serum analysis, clinical applications such as Sickle cell disease, hemoglobin C abnormalities, blood clotting factor separation, drug tests for detecting adulteration, etc.
However, lack of sensitivity and reproducibility are its common limitations.
Gel electrophoresis is a separation technique that is used to separate DNA/RNA fragments based on their molecular size is known as Gel electrophoresis which can be carried out vertically or horizontally.
In this technique, DNA samples are loaded into wells and an electric current is passed due to which negatively charged DNA particles move towards the positive charge at different speeds and in different locations thus, separating the DNA fragments.
Different types of gels are used in this technique as a supporting medium such as Starch, Agar and Agarose gel, Sephadex, Polyacrylamide gels, etc.
Agar is the commonly used supporting medium due to its good separation and less cost.
It is widely used in Immuno-electrophoresis to separate different kinds of proteins mixtures or nucleic acids.
Thin Layer Electrophoresis
Thin layer chromatography is a common term used for the separation of particles. However, Thin layer electrophoresis is a different term based on almost same principle.
In this technique, the a plate same as TLC is used with evenly spread gel as a medium for the separation of molecules. The plate is then attached with the electric field.
It is based on the principle that “When the electric field is applied on the plate, the protein sample molecules present on the plate starts moving in a direction at different speeds depending on their electric charge.”
This technique has an advantage over Paper electrophoresis that interfering adsorption effects can often be avoided through appropriate choice of adsorbent; the scope of the method can thus be markedly broadened.
In addition, the more uniform and finer structure of the adsorbent used
for this technique, compared with filter paper, leads to a better resolution.
This technique is widely used in electrophoretic-Chromatography two dimensional study of proteins and nucleic acid hydrolysates. It has an advantage of less-time consumption and providing good resolution.
Cellulose acetate electrophoresis
This technique is a modification in the Paper electrophoresis technique developed by Kohn in 1958.
Kohn used bacteriological acetate membrane filters in this technique rather than using chromatographic filter paper.
Cellulose acetate is an acetate salt of cellulose produced by treating cotton with acetic acid using Sulphuric acid as a catalyst. In this technique, migration takes place on the buffer film on the surface of the cellulose plate and the separation is mainly done by charge.
The advantage of using this technique is that it is available in a wide range of particle size and layer thickness. Being pure chemically, lignin, and hemicelluloses free, this kind of membrane filter acts as a barrier in the movement of large molecules.
Cellulose acetate electrophoresis is used for clinical investigation of hemoglobin, glycoprotein and lipoprotein from blood.
Electrophoresis is a common used technique for the separation of proteins and other molecules on the basis of electric charge. Different types of electrophoretic techniques are used in forensic investigation based on the type of evidence present in a crime scene.
These techniques are used in the research in proteins, immunology, taxonomy, cytology, medical research, agricultural testing, analysis of steroids, antibiotics, etc.
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