These factors specifically affect the migration rates of molecules in the sample during electrophoresis.
These are described in detail below (Figure 4).
1. Electric field
An electric field is a property that describes the space that surrounds electrically charged particles.
It is defined as the electric force per unit charge. This electric field exerts a force on other charged objects and is radially outward from a positive charge and radially in toward a negative point charge.
A movement of ions depends upon voltage, current, and resistance of the electric field (Figure 5).
However, voltages that are too high can possibly melt the gel or cause smearing or distortion of DNA bands. If the separation of the electrodes is d (meters) and the potential difference between them is V (volts), the potential gradient is V/d volts m-1.
The equation is Vq/d newtons, if force on the ion with a charge is q (coulombs).
The rate of migration is proportional to Vq/d, so it increases with increase in potential difference.
Voltage is the potential energy of electrical supply stored in the form of electrical charge.
Current is generated due to potential difference applied between the electrodes. It is a continuous and uniform flow of electrons around a circuit that are being pushed by the voltage source.
Current is measured in coulombs sec-1. The current is mainly conducted between the electrodes by buffer ions. Thus, increase in voltage will increase total number of charge towards the electrode.
The distance traveled by the ions is directly proportional to the current and the time.
Electrical resistance is a property of measuring the resistance to the flow of an electrical current.
An object of uniform cross-section has resistance proportional to its length, and resistivity of a material and is inversely proportional to its cross-sectional area.
Resistance of an electrophoresis unit depends on its size, gel thickness, amount of buffer, buffer conductivity, and temperature. This resistance normally decreases in time with increasing temperature.
The amount of resistance determines whether the circuit is a good conductor (low resistance), or a bad conductor (high resistance).
The resistance, R (measured in ohms, Ω) of an object can be defined as the ratio of voltage, V (measured in volts) to the current, I (measured in amperes), in accordance with Ohm’s law,
R = V/I
The rate of migration of ions is inversely proportional to resistance. Resistance increases with the length of supporting medium but decreases with its cross-sectional area and with increase in the buffer ion concentration.
The power dissipated in the supporting medium, W (measured in watts) during electrophoresis is as shown below.
An increase in temperature leads to decrease in resistance. This is due to increase mobility of ions and evaporation of the solvents from the supporting medium.
2. The Sample
Charge, size and shape of the sample being separated affect its own migration rate. A net increase in the charge increases the rate of migration.
In accordance with Henderson-Hasselbalch equation, magnitude of charge is pH-dependent.
Rate of migration is affected by increase in size of molecule (inversely proportional) and difference in shape of the sample.
3. The Buffer
Buffer affects migration rate of a compound and stabilizes the pH of the supporting medium.
It has been observed that zwitterionic buffers are able to withstand prolonged electrolysis much better in comparison to the traditional buffers especially in capillary zone electrophoresis.
Most commonly used buffers for the electrophoresis are formate, EDTA, pyridine, Tris, barbitone acetate, and citrate.
The buffer should never bind to the molecules being separated as it effects the migration of the sample. In its simplest form, a buffered solution contains a mixture of a weak acid and its conjugate base.
The position of acid/base equilibrium is represented by the acid dissociation constant, Ka. This number is large if the acid is stronger and the equilibrium tends toward dissociation.
While the value is small for an equilibrium that tends toward proton capture. Buffers used in life science range from 10-4 to 10-10 in their Ka values.
Ka = [ H+][A–]/HA
Where, Ka is usually expressed as its negative logarithm. So, pKa is
Proportion of current carried by buffer increases and the one carried by sample decreases with the ionic strength of the buffer.
Thus, at a low ionic strength the proportion of current carried by the buffer decreases and those carried by the sample increases.
It leads to overall reduction of current and results in heat production causing diffusion and loss of resolution.
The extent of ionization depends on pH, especially in organic compounds. The ionization increases with increase in pH of an organic compound and its just reverse for the organic bases therefore affecting its rate of migration.
These affects apply to the ampholytes.
Heat generation in electric fields
One of the practical problems encountered in electrophoresis is generation of heat from resistive dissipation of energy in the electrophoretic medium.
Heating not only changes viscosity and density of the electrophoretic media, it also damages equipment by warping, cooling blocks, melting plastics, or cracking glass plates.
It may also cause poor resolution and distortion in resolution. The generation of heat is given by:
W = E I
Where, W = power in watts
I = current in amperes
Current and electric field strength are related by the conductivity of the electrophoretic medium by Ohm’s law, where,
Where, C = medium conductivity (Ω cm-1)
If the conductivity of an electrophorectic medium is high, electrophoresis becomes difficult.
This is because high conductive solutions result in lower field strength per current as well as high heat load on the system. This load increases proportional to the current squared.
Electrophoresis is preferred in resistive media and adding polymer particles (such as, gels) increase the resistivity of media.
4. The Supporting Medium
Migration rate of compounds depends upon type of supporting medium. Inert medium is always preferred.
The medium might cause adsorption, molecular sieving, and electro-osmosis processes that affect the electrophoretic rate.
Adsorption causes tailing of the sample, leading to movement of sample in the form of comet rather than a band. This reduces rate as well as resolution of the separation.
Molecular sieving is affected by type of gel used.
Electro-osmosis depends upon the relative charge produced between water molecules in buffer and surface of supporting material.