Capillary Electrophoresis

Capillary Electrophoresis

When the ions are separated which are based on their electrophoretic mobility, then we can use the capillary electrophoresis technique. It is an analytical technique that we used to separate the molecules. The electrophoretic mobility depends on the charge of the molecule, the viscosity, and the radius of the atom. The particle’s rate moves are directly proportional to the applied electric field.

The greater the field strength, the faster the mobility of the ions. If the size of the ion is the same, the one with the greater charge will move the fastest. If the ions have the same charge, the smaller particle has less friction and a faster migration rate.

Capillary electrophoresis is used most commonly because it gives faster results. This technique provides high-resolution separation. It is a very useful procedure because there is a large range of detection methods available.

Capillary electrophoresis (CE) began in the late 1800s. The experiments start with the use of glass tubes and trials of both gel and free solutions.
Arnes Tiselius first showed the capability of electrophoresis in his experiment in the 1930s. He showed the separation of proteins in the solutions.

Then, Hjerten introduced the use of capillaries in the 1960s. But his work is not recognized until Jorgenson and Lukacs published papers that show the ability of capillary electrophoresis to perform separations that seemed unachievable. Its increased productivity and the separating capabilities of capillary electrophoresis encouraged a growing interest in the scientific society to perform further developments in the technique.

Principle of Capillary Electrophoresis

Capillary electrophoresis (CE), is also known as high-efficiency capillary electrophoresis. It is a type of liquid phase micro-separation analysis technology. It is used as the separation channel, high-voltage direct-current electric field as the driving force, and various characteristics of the sample as the basis. It can be used to separate components ranging from organic ions to biological macromolecules such as proteins and nucleic acids. It is another important development in analytical science after high-performance liquid chromatography. It can be used to examine body fluid samples such as serum or plasma, urine, cerebrospinal fluid, and saliva, and even single-molecule analysis.

Theory

Electrophoretic Mobility

In the Electrophoresis technique, the ions migrate under the influence of an applied electric field. The ion undergoes a force, and this is equal to the product of the net charge and the electric field strength. It is also affected by a drag force that is equal to the product and the velocity.
The electrophoretic mobility equation is as follow;

μEP=qf=q6πηr(R)(R)μEP=qf=q6πηr

where;
• f is a spherical particle is given by Stokes’ law
• η is the viscosity of the solvent
• RR is the radius of the molecule.

Electroosmotic Flow

When we apply the high voltage to the electrolyte-filled capillary, the electroosmotic process can occur. This electroosmotic flow starts when the buffer began running through the silica capillary. The SiOH groups lose a proton to become SiO- ions and the pH value is greater than three. Now, the wall of the capillary contains a negative charge and it produces a double layer of cations that are attracted towards it. The inner layer of the cation is stationary, while the outer layer is free to move with the capillary.

When the electric field is applied, it causes the free cations to migrate towards the cathode and it creates a powerful bulk flow.

The rate of the electroosmotic flow is shown by the following equation:

μEOF=ϵ4πηEμEOF=ϵ4πηEζ

Where,
• ε is the dielectric constant
• η is the viscosity of the solution
• E is the field strength
• ζ is the zeta potential.

Instrumental Setup

Capillary electrophoresis system consists of a;
• High-voltage power supply
• A sample introduction system
• A capillary tube
• A detector
• An output device

Some devices include a temperature control tool to assure reproducible results. In this technique, the sample is dependent on the electrophoretic mobility and the viscosity of the solutions. When it decreases as the column temperature rises.

Each side of the high voltage power supply is connected to an electrode and these electrodes help to induce an electric field to initiate the migration of the sample from the anode to the cathode in the capillary tube. The capillary is made of fused silica and it is dipped in a vial containing the electrode and an electrolytic solution, or aqueous buffer.

The capillary must be clean with the desired buffer solution before introducing the sample. The cathodic end of the capillary has a small window that allows the UV visible light to pass into the sample and measure the absorbance.

Generally, A photomultiplier tube, which is a type of detector, is also connected at the cathodic end of the capillary. This detector enables the construction of a mass spectrum and provides reliable information about the mass to charge ratio of the ionic species.

Capillary Electro-separation Methods

There are six types of capillary electro separation methods:
• Capillary zone electrophoresis (CZE)
• Capillary gel electrophoresis (CGE)
• Capillary electrochromatography (CEC)
• Capillary isoelectric focusing (CIEF)
• Capillary isotachophoresis (CITP).

Another Classification is:

Continuous System- It has a background electrolyte that acts throughout the capillary as a buffer.

Discontinuous systems- It has a system that keeps the sample in different zones.

Capillary Zone Electrophoresis (CZE)

It is also known as free solution capillary electrophoresis. It is the most commonly used technique in which a mixture can be separated into its components quickly and easily.

The separation depends on the differences in electrophoretic mobility, which is directed proportional to the charge on the molecule, and inversely proportional to the viscosity of the solvent and radius of the atom. The velocity of the moving ion is directly proportional to the electrophoretic mobility and the magnitude of the electric field.

These silica capillaries have SiO- groups that become ionized in the buffer. The SiO- ions have a negative charge and it attracts towards the positively charged cations and forms the two layers—the first is a stationary layer and the other is a diffuse cation layer. When the electric field is applied then, the diffuse layer migrates towards the negatively charged cathode and produces an electrophoretic flow that drags bulk solvent along with it. In this solution, anions are attracted to the positively charged anode. Cations with the largest charge-to-mass ratios separate first, followed by cations with reduced ratios, neutral species, anions with smaller charge-to-mass ratios, and finally anions with greater ratios.

Capillary Gel Electrophoresis (CGE)

It is the separation technique that is based on the difference in solute size as the particles migrate through the gel.

In this type of method, gels are used because they reduce solute diffusion that creates zone broadening.

The heat transfer is limited because they slow down the molecules. SDS-PAGE is a gel apparatus that is used for the separation of proteins. It is a highly sensitive system. This technique requires only a minimum amount of samples.

Capillary Electrochromatography (CEC)

This method of capillary electrochromatography is similar to the chromatography technique. The liquid is passed over the silica surface which acts as a mobile phase. CEC and CZE, both techniques have a plug-type flow that is compared to the pumped parabolic flow that increases the band broadening.

Capillary Isoelectric Focusing (CIEF)

Generally, this technique is commonly used to separate the peptides and proteins. Those molecules which contain both positive and negative charges are called zwitterionic compounds. The charge depends on the functional groups and these functional groups are attached to the main chain and the surrounding pH of the environment. Each molecule has a particular isoelectric point (pI). The molecule carries no net charge when the surrounding pH is equal to this pI. When the pH is below the pI, the molecule is positive, and when it is negative, then the pH is above the pI. Because the charge changes with pH. It can also be used to separate the molecules in a mixture. The capillary is filled with the sample in solution during a CIEF separation and typically no EOF is used because it is removed by using a coated capillary.

Advantages

The advantages of capillary electrophoresis are as follows;
• Capillary electrophoresis is efficient and automation technique.
• The diameter of the capillary is very small. It ranges from 20–100 µm.
• This means 30000V high voltages, can be applied.

Application

• Capillary electrophoresis is used to examine the various sample matrices in the industrial sector.
• Capillary electrophoresis is used to examine the products such as; food additives, herbicides, animal nutrition, and detergents.
• CZE can be used to separate and recognize the small molecules that are present in the samples.