Basic principles of electrophoresis technology

Electrophoresis technology is based on the basic principles of electrophoresis and is affected by internal factors of electrophoretic particles and external factors of the electric field.
Basic principles

Under normal circumstances, material molecules generally have no charge, that is, the amount of positive and negative charges they carry is equal, showing electrical neutrality. However, due to its own dissociation or the adsorption of other charged particles on its surface, it carries a negative or positive charge, and the substance will migrate toward the positive or negative electrode in the electric field. There are many types of charged material particles, which can be ions or biological macromolecules, such as proteins, nucleic acids, virus particles, and even organelles.
Protein molecules are composed of amino acids, which are typical amphoteric electrolytes and can be dissociated into positively charged amino groups (-NH3+) and negatively charged carboxyl groups (-COO-) in solution. The nature of the charge of protein molecules and the amount of charge they carry mainly depend on their properties and the ionic strength (ionic strength, I) and pH of the solution.
Under a certain pH condition, the number of positive and negative charges of protein molecules is exactly equal, and the net charge of protein molecules is zero. At this time, the pH of the solution is the isoelectric point (pI) of the protein.
When the pH of the solution = pI, the protein molecules are uncharged and do not move in the electric field;
When the pH of the solution> pI, the protein molecules are negatively charged and move toward the positive electrode;
When the pH of the solution < pI, the protein molecules are positively charged and move toward the negative electrode.

Nucleic acids are similar to proteins and are also amphoteric electrolytes. The polynucleotide chains of DNA and RNA molecules have both acidic phosphate groups and alkaline bases, but because the acidity of the phosphate group is stronger than the alkalinity of the base, in neutral or alkaline solutions, nucleic acid molecules usually appear acidic, carry negative charges, and migrate toward the positive electrode in a DC electric field.

Influencing factors
Internal factors
The electrophoretic velocity is related to the characteristics of the particles themselves, such as the positive and negative charge, the size and shape of the particles, the dissociation trend, the amphoteric properties, the degree of hydration, etc. Generally speaking, the more net charge a particle carries, the smaller the particle is, and the closer the shape is to a sphere, the faster the particle moves in the electric field; otherwise, the slower it moves.
Generally speaking, linear double-stranded DNA molecules do not have complex conformations that affect the electrophoretic speed. In gel electrophoresis, the commonly used logarithm of its relative molecular weight is inversely proportional to the electrophoretic speed; but the electrophoretic speed of plasmid DNA is greatly affected by the spatial conformation of the molecule. The relative speed of electrophoretic speed of plasmid DNA with the same molecular weight is: closed loop type > linear type > semi-open loop type. RNA molecules are single strands with local double helical structures, so the electrophoretic speed of RNA molecules depends not only on the size of the molecule, but also mainly on its spatial conformation.

External factors
1. Electric field strength The greater the electric field strength, the greater the electric field force on the charged particle, and the faster the swimming speed, and vice versa. According to the difference in electric field strength, electrophoresis can be divided into two categories:
① Normal pressure electrophoresis (2~10V/cm): The voltage is generally 100~500V. Suitable for separating macromolecules such as proteins and nucleic acids, the separation time is relatively long, ranging from several hours to several days.

② High-voltage electrophoresis (50~200V/cm): The voltage is generally 2000~10000V. It is mostly used to separate small molecules, such as amino acids, peptides, nucleotides, sugars, etc. The required electrophoresis time is very short, even just a few minutes.
2. Solution properties Mainly include the pH of the electrode buffer and sample solution, ionic strength and medium viscosity.
(1) Solution pH: During electrophoresis, a buffer must be used as the electrode solution to maintain a stable solution pH. The pH of the solution determines the degree of dissociation of the charged particles; it also determines the number of charges carried by the substance. For amphoteric electrolytes such as proteins, amino acids, and nucleic acids, the farther the pH of the buffer is from the pI of the substance to be separated, the more charge the particles carry and the faster the electrophoresis speed; otherwise, the slower it is. Therefore, choosing a suitable pH so that the number of charges carried by the substances to be separated is relatively different is conducive to their separation during electrophoresis. Most protein electrophoresis uses barbiturate or boric acid buffers with a pH of 8.2 to 8.8. At this time, serum proteins are generally negatively charged.

Nucleic acid electrophoresis often uses one of the following three buffer systems: TAE buffer [Tris-acetic acid-ethylenediaminetetraacetic acid (EDTA)], TBE buffer (Tris-boric acid) or TPE buffer (Tris-phosphoric acid). DNA molecules are negatively charged in these buffers.

(2) Ionic strength of the solution: The net electric force generated by all types of ions is called ionic strength. The level of ionic strength depends on the total number of ionic charges and has nothing to do with their properties. The higher the ionic strength of the solution, the slower the swimming speed of the charged particles; conversely, the faster the swimming speed of the charged particles. The reason is that the charged particles attract ions with opposite charges to gather around them, forming an ionic atmosphere. The ionic atmosphere reduces the charge of the particles and increases the resistance to particle movement, thereby reducing the electrophoretic speed. Moreover, if the ionic strength is too high, the heat generated when a large amount of current passes through can cause a large amount of water to evaporate. However, if the ionic strength is too low, the total concentration and buffer capacity of the buffer solution will be reduced, making it difficult to maintain the pH of the solution, thereby affecting the charge of the particles, resulting in a decrease in current, severe diffusion, and reduced electrophoretic resolution. During electrophoresis, the ionic strength of the solution is generally 0.02~0.20mol/L.
(3) Solution viscosity: As mentioned earlier, the electrophoretic mobility of the particles is inversely proportional to the viscosity of the solution medium. Therefore, if the viscosity of the solution is too low or too high, it will inevitably affect the speed of electrophoresis.
3. Electroosmosis In an electric field, the relative movement of a solution to a solid support medium is called electroosmosis. When the support medium is not an absolutely inert substance, the solution close to the support medium is often relatively charged, and the solution moves while carrying the particles. Therefore, the apparent swimming velocity of a charged particle is the vector sum of the swimming velocity of the particle itself and the swimming velocity of the particle carried by the solution. When the two directions are consistent, the swimming velocity of the particle is accelerated, and when the directions are opposite, the swimming velocity of the particle is reduced.
4. Adsorption The surface of the support medium has a certain adsorption effect on the sample, which can retain the substance to be separated and reduce the electrophoresis speed, thereby causing the sample to tail and reducing the resolution.

5. Joule heat The heat generated by the passage of current during electrophoresis is called Joule heat, and its value is proportional to the square of the current intensity. Joule heat can increase the temperature of the buffer solution, reduce the viscosity of the medium, intensify the molecular motion, and reduce the resolution. When the Joule heat is too high, it will also burn the filter paper, melt the agarose gel or burn the polyacrylamide gel support medium. The influence of thermal effect on electrophoresis can be reduced by controlling the voltage or current, or by installing a cooling and heat dissipation device.