Electrophoresis, which derives from the Ancient Greek words v (lektron, “amber”) and phórsis, “the act of bearing,” is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. Electrophoresis is a technique for separating dispersed particles from a fluid. When applied to positively charged particles (cations), electrophoresis is sometimes referred to as cataphoresis, and when applied to negatively charged particles (anions), electrophoresis is often referred to as anaphoresis.

It was Russian professors Peter Ivanovich Strakhov and Ferdinand Frederic Reuss at Moscow University who made the first observation of the electrokinetic phenomenon of electrophoresis in 1807, when they noticed that the application of a constant electric field caused clay particles dispersed in water to migrate. The presence of a charged contact between the particle surface and the surrounding fluid is ultimately responsible for this phenomenon. It serves as the foundation for analytical techniques in chemistry that separate molecules according to their size, charge, or binding affinity.

Electrophoresis is a technique that is used in laboratories to separate macromolecules depending on their relative sizes. The approach uses a negative charge to cause proteins to migrate in the direction of a positive charge. Electrophoresis is a technique that is widely used in the study of DNA, RNA, and proteins.

Theory

In suspension, particles have an electric surface charge that is greatly impacted by surface adsorbed species, and an external electric field exerts an electrostatic Coulomb force on this charge, causing the particle to move. According to the double layer theory, all surface charges in fluids are screened by a diffuse layer of ions, which has the same absolute charge as the surface charge but the opposite sign when compared to the surface charge of the fluid in question. The electric field also exerts a force on the ions in the diffuse layer that is in the opposite direction as the force acting on the surface charge. This force is known as the ionisation force. It is not the particle itself that is subjected to this latter force; rather, it is the ions in the diffuse layer that are placed at a distance from the particle surface, and a portion of this force is transported all the way to the particle surface by means of viscous stress. 

ERF is an abbreviation for electrophoretic retardation force, which is the name given to this portion of the force. Because there is no net force produced when an electric field is supplied and the charged particle under investigation is moving steadily through the diffuse layer, the total force produced is zero:

When the drag on moving particles caused by the viscosity of the dispersant is taken into account, in the case of low Reynolds number and moderate electric field strength E, the drift velocity of a dispersed particle v is simply proportional to the applied field, leaving the electrophoretic mobility e defined as:

Smoluchowski devised the following hypothesis of electrophoresis in 1903, which is the most well-known and frequently utilised today:

the dielectric constant of the dispersion medium is r, the permittivity of free space is 0 (C2 N1 m2), the dynamic viscosity of the dispersion medium is (Pa s), the zeta potential is r, and (i.e., the electrokinetic potential of the slipping plane in the double layer, units mV or V).

Due to the fact that it works for scattered particles of any shape at any concentration, the Smoluchowski hypothesis is extremely powerful. Its validity is subject to certain restrictions. For example, it does not include the Debye length of one atom (units m).” The Debye length must be significant for electrophoresis to take place. Increasing the thickness of the double layer (DL) results in the point of retardation force being removed from the particle surface to a greater distance. The greater the thickness of the DL, the smaller the retardation force required.

It was demonstrated through a thorough theoretical investigation that the Smoluchowski theory is valid only for sufficiently thin DL when the particle radius and is significantly greater than the Debye length:

This “thin double layer” model simplifies not only electrophoresis theory, but also a wide range of other electrokinetic theories, which is extremely beneficial. For the vast majority of aquatic systems, where the Debye length is often only a few nanometers, this model is valid. It only breaks for nano-colloids in solution with an ionic strength close to that of water, according to the literature.

The Smoluchowski hypothesis also ignores the contributions from surface conductivity, which is another source of error. Modern theory expresses this as the requirement of a small Dukhin number, which is as follows:

In an effort to broaden the scope of electrophoretic theories’ applicability, the asymptotic scenario in which Debye length is greater than particle radius was considered: when Debye length is more than particle radius

Hückel anticipated the following relationship for electrophoretic mobility in the presence of a “thick double layer” under these conditions:

If the Debye length of a given nanoparticle or a non-polar fluid is significantly longer than it is in the usual circumstances, this model may be applicable.

As Overbeek pioneered, there are various analytical theories that incorporate surface conductivity and eliminate the limitation of a small Dukhin number, all of which may be found here.

Modern, rigorous theories that are applicable to any Zeta potential and, in many cases, result mostly from Dukhin–Semenikhin theory.

Principle of Electrophoresis

1. Because of their varied electrophoretic mobility, molecules having distinct charges will begin to separate when a potential difference is applied. Even molecules with comparable charges will begin to separate if their molecular sizes differ, since frictional forces would be different. As a result, some types of electrophoresis rely almost entirely on the various charges on molecules for separation, while others take advantage of differences in molecule size (molecular size).
2. Because the electric field is eliminated before the molecules in samples approach the electrode, electrophoresis is considered an incomplete type of electrolysis. However, the molecules will have already been sorted as per their electrophoretic mobilities.
3. The separated samples are subsequently found by staining with a suitable dye or, if the sample is radiolabeled, by autoradiography.

Process of Electrophoresis

An electrical current is used to separate these molecules, which is commonly done through a gel. This gel, which is commonly made of silica, is utilised to hold the charge and suspend the particles. Two electrodes are linked to the gel, and the current generated by them attracts molecules to one side of the gel and repels them from the other. The gel creates a friction force which prevents all of the molecules from flowing through it at the same time, however the larger molecules are able to overcome the friction & separate nevertheless. As the molecules pass through the gel, they form a stratum of different types of molecules.

Application

The separation of biological molecules, which includes molecules with comparatively lower relative molecular weights, such as amino acids, as well as molecules with larger relative molecular masses, such as proteins and polynucleotides, has been the primary use of electrophoresis (including RNA and DNA molecules). .Almost all laboratories that study proteins and other macromolecular electrolytes employ paper electrophoresis.

Conclusion

Electrophoresis is a widely used technique that works by passing an electric current through biological molecules (typically DNA, but it can also be protein or RNA) and separating them into larger or smaller bits. It’s utilised in a multitude of applications, from forensics to detecting the identity of people who may have been involved in a crime by comparing their DNA pattern, or electrophoresis pattern, to one in a database.