Capillary Electrophoresis

Capillary electrophoresis is an experimental technique that uses the instrumentations of electrophoresis. It separates ions based on their electrophoretic mobility using an applied voltage. 

The charge of the molecule, viscosity, and radius of the atom all affect electrophoretic mobility. 

The particle’s velocity is proportional to the strength of the applied electric field; the greater the field, the faster the particle’s mobility. The electric field affects just ions; neutral species are unaffected. 

If two identically sized ions have the same charge, the one with the larger charge will move quicker. When comparing ions of the same charge, the smaller particle has less friction and a quicker total migration rate. 

Capillary electrophoresis is the most often utilised method because it produces quicker results and delivers high-resolution separation. It is a handy strategy because so many different detecting methods are accessible.

Methods for Capillary Electrophoresis

1. Capillary zone electrophoresis (CZE)

2. Capillary isotachophoresis (CITP)

3. Micellar electrokinetic capillary chromatography (MECC)

4. Capillary gel electrophoresis (CGE)

5. Capillary isoelectric focusing (CIEF) (CIEF)

High Performance Capillary Electrophoresis’s (HPCE) default operating mode is CZE. One often uses borate buffers and phosphate for charged compound separation and analysis. We adjust the experimental variables such as pH, voltage, modifiers, temperature,  buffer concentration, and (acetonitrile, methanol, etc.) accordingly (proteins, drugs, peptides, etc.). CZE may not be useful to distinguish between aggressive and non-aggressive chemicals.

MECC is a new liquid chromatography technology depending on micellar electrokinetic migration and solubilisation. As micellar phases, MECC utilises CZE-based surfactants. Solute has unique coefficients of partition in the aqueous and micellar phases.

When subjected to an electric field, the micelles and the phase of water migrate at distinct rates. It is because of the flow of electroosmotic solution inside the capillary and the electrophoresis of the micelles.

The compounds are supposed to be divided and disseminated several times in the water phase and the micellar phase simultaneously. The influence of the flow of electroosmotic and the distribution process separates them.

MECC expands the use area of CZE, is excellent for neutral material separation, and can identify chiral chemicals. It can analyse peptides, amino acids, chiral chemicals, medication samples, small molecule compounds, and bodily samples of fluids.

CITP forms a discontinuous buffer system from a leading electrolyte and the following electrolyte, divided depending upon the different areas of the electrophoretic mobility of the solute. 

CITP is often used (such as organic acids) for ionic compounds. We may use it for micro-preparation because it is suited for capillaries with a wider inner diameter.  This approach, however, has a low spatial resolution.

For samples containing zwitterions, we employ CIEF (proteins, peptides). It helps determine the isoelectric point of amphoteric compounds to accomplish isoelectric point separation. It also helps detect distinct components in the sample.

CGE separates macromolecular compounds based on their molecular weight and is mostly used to separate proteins and nucleotide fragments.

Separation Methods and Principles of  Electrophoresis

There are six different types of capillary electrophoretic separation techniques.

• Capillary zone electrophoresis (CZE) – The conductive fluid is a free solution.

• Capillary gel electrophoresis (CGE) – The conducting fluid is a gel.

• Micellar electrokinetic capillary chromatography (MEKC) – Partitioning between micelles and the solvent/conductive fluid separates the components of a mixture.

• Capillary electrochromatography (CEC) — We utilise a packed column except for the conducting fluid.

• A mobile liquid is transported through the column with the mixture to be separated.

• We commonly use capillary isoelectric focusing (CIEF) to separate zwitterionic components containing positive and negative charges, such as peptides and proteins.

• We utilise a conductive fluid with a pH gradient to separate the protein solution. Each protein migrates to the portion of the pH gradient that contains its isoelectric point. Proteins’ net charge becomes zero at the isoelectric point.

• Capillary isotachophoresis (CITP) is a discontinuous system. Each component migrates in successive zones. We determine the amount of the component by measuring the migration duration.

Conclusion

Capillary electrophoresis separates ions of substance based on their mobility when subjected to voltage. The energy of the molecule, the viscosity, and the atom’s radius all influence electrophoretic mobility. 

Capillary electrophoresis is the most often utilised method because it produces quicker results and delivers high-resolution separation.