The main influencing factors of electrophoresis-Electrophoretic Materials

The charge, molecular size, and properties of the biomolecules to be separated will have a significant impact on electrophoresis. Generally speaking, the larger the charge of a molecule, the smaller its diameter, and the closer its shape is to a sphere, the faster its electrophoretic migration speed.

The pH value of the buffer solution can affect the degree of dissociation of the biomolecules to be separated, thereby affecting their charged properties. The further the pH value of the solution is from its isoelectric point, the greater the net charge it carries and the faster the electrophoresis speed. Especially for amphiphilic molecules such as proteins, the pH value of the buffer solution can also affect their electrophoresis direction. When the pH value of the buffer solution is greater than the isoelectric point of the protein molecule, the protein molecule carries a negative charge and its electrophoresis direction points towards the positive electrode. In order to maintain the charge of the biomolecules to be separated during electrophoresis and the stability of the pH value of the buffer solution, the buffer solution usually needs to maintain a certain ionic strength, usually between 0.02-0.2

If the ion strength is too low, the buffering capacity will be poor. However, if the ion strength is too high, a strong ion diffusion layer with opposite charges (i.e. ion atmosphere) will form around the molecules to be separated. Due to the opposite movement direction of the ion atmosphere and the molecules to be separated, electrostatic attraction will be generated between them, resulting in a decrease in electrophoresis speed. In addition, the viscosity of the buffer solution can also affect the electrophoresis speed.

Electric field strength (V/cm) is the potential drop per centimeter, also known as potential gradient. The higher the electric field strength, the faster the electrophoresis speed. However, increasing the electric field strength will cause an increase in the current intensity passing through the medium, resulting in an increase in the heat generated during the electrophoresis process. The work done by current in a medium (W) is: W=I2.R.t

In the above equation: I is the current intensity, R is a resistor, T is the electrophoresis time.

The majority of the work done by current is converted into heat, causing an increase in the temperature of the medium, which can have many effects:

① The increase in diffusion rate of samples and buffer ions leads to the widening of the sample separation band;

② Generate convection, causing mixing of the substances to be separated;

③ If the sample is sensitive to heat, it can cause protein denaturation;

④ Causing a decrease in medium viscosity, resistance, and so on. The heat generated in electrophoresis is usually emitted from the center to the periphery, so the temperature at the center of the medium is generally higher than that at the periphery, especially in tubular electrophoresis. This causes a decrease in the viscosity of the central part of the medium relative to the peripheral part, a decrease in the friction coefficient, and an increase in the migration speed of electrophoresis. Due to the faster electrophoresis speed at the center than at the edges, the electrophoresis separation band is usually arched. Reducing the current intensity can reduce heat generation, but it will prolong the electrophoresis time and increase the diffusion of biomolecules to be separated, which will affect the separation efficiency. So in electrophoresis experiments, it is necessary to choose an appropriate electric field strength, and at the same time, cooling and lowering the temperature appropriately can achieve better separation results.

The relative movement of a liquid in an electric field to a solid support medium is called electroosmotic phenomenon. Due to the presence of some charged groups on the surface of the supporting medium, such as carboxyl groups on the surface of filter paper, sulfate groups on agar, and Si OH groups on the surface of glass, etc. After ionization, these functional groups will make the surface of the supporting medium charged, adsorb ions with opposite charges, and move towards the electrode under the action of an electric field, forming a flow of solution on the surface of the medium. This phenomenon is called electroosmosis. When the pH value is higher than 3, the glass surface is negatively charged, adsorbing positive ions in the solution, causing the solution layer near the glass surface to be positively charged

Under the action, it migrates towards the negative electrode, driving the electrode liquid to generate electroosmotic flow towards the negative electrode. If the direction of electroosmosis is the same as the direction of electrophoresis of the molecules to be separated, the electrophoresis speed will be accelerated; If the opposite is true, reduce the electrophoresis speed.

The pore size of the supporting medium has a significant impact on the electrophoretic migration speed of biomolecules to be separated. Swimming speed is fast in media with large sieve holes, and vice versa, swimming speed is slow.