Separation Modes of High Performance Capillary Electrophoresis

1 Capillary Zone Electrophoresis (CZE)

Separation principle: capillary zone electrophoresis is called free solution capillary electrophoresis, which is the simplest form of capillary electrophoresis. The separation mechanism is based on the difference in the ratio between the net charge and the mass of each substrate. Different ions move in the mesosolute at different speeds according to the difference in their surface charge density, resulting in separation. It requires that the buffer solution has uniformity, and that there is a constant electric field strength everywhere in the capillary, which is the most widely used separation mode at present. It is applicable to the analysis of proteins, amino acids, peptides and ions. Except electrolyte, the capillary does not need to be filled with any substance, which is easy to operate and highly automated.

2 Capillary gel electrophoresis (CGE)

Separation principle: gel electrophoresis is a zone electrophoresis in which gel is moved into the capillary to be used as a support for separation. Gel is a solid dispersed system. It is porous, similar to the role of molecular sieve. The separated substances are separated one by one according to the size of their respective molecules through the gel force loaded into the capillary tube. The large molecules are separated first. It is suitable for the analysis of biological macromolecules. It can be separated purely according to the size of molecular species (SDS-PAGE) to determine the molecular weight of proteins, identify an oligonucleotide with different bases, and separate DNA fragments, For example, microsatellite (STR) analysis can change the concentration of gel without controlling the separation range, such as the analysis of PCR products.

3 Micellar Electrokinetic Capillary Chromatography (MECC)

Separation principle: It is the intersection of electrophoresis technology and chromatography technology. When ionic surfactant is added to the buffer solution and its concentration is large enough, the monomers of this surfactant will be combined to form a sphere (micelle). At present, sodium dodecyl sulfonate (SDS) micelles are most commonly used. MECC has two phases distributed between them. Because of their retention ability in the micelles, they have different retention times. As soon as CZE increases, the buffer solution forms a positive charge at the tube wall, producing a strong percolation that moves toward the negative pole. The surface SDS micelles have a tendency to migrate toward the positive pole due to the negative shell, In general, the velocity of EOF is higher than the migration velocity of micelles to the positive electrode, which forces the micelles to move to the negative electrode at a lower velocity. The separation between neutral particles is achieved according to their own hydrophobicity, and the interaction between particles with different hydrophobicity and micelles is different. The hydrophobic force is strong and the retention time is long.

MECC is the only HPCE mode that can separate neutral ions and charged components.

4 Isoelectric Focusing (IEF)

Separation principle: the migration of amphoteric electrolytes in the separation medium forms a pH gradient in the capillary tube. Various peptides and proteins with different isoelectric points migrate to their different isoelectric positions according to this gradient and stop, thus creating a very narrow focus zone. The separation is carried out by taking advantage of the subtle differences of isoelectric points. After focusing different proteins at different positions, the buffer solution of the cathode is replaced with salts, and the high pressure is added, The gradient at the end is reduced so that the components can pass through the detector one by one to obtain accurate isoelectric points.

5 Isotachophoresis (ITP)

Separation principle: an electrophoretic method for focusing separation when the separated component and electrolyte move forward together. Like IEF, the electroosmotic flow of ITP in the capillary is zero, and the buffer system is composed of two electrolytes with different mobility. During separation, the trailing electrolyte is first introduced into the capillary (mobility is lower than that of the separated component). Under the action of strong electric field, the separated components will focus and separate in the gap between the two electrolytes.

Both the treated and untreated silica capillary tubes were selected, and the electroosmotic flow could be inhibited by 0.25% hydroxyproline methyl cellulose The leading electrolyte is 5nM phosphoric acid, and the following electrolyte is valine. At the beginning of separation, the current will increase rapidly as the electrolyte with high mobility completely fills the capillary. When entering the separation process, the current will decrease as the electrolyte with low mobility enters the capillary

6 Capillary electrochromatography (CEC)

Separation principle: It is a chromatographic process in which a large number of stationary phases of high performance liquid chromatography are filled and flushed into the capillary, with the interaction between the sample and the stationary phase as the separation mechanism and the current flow phase as the driving force

7 Affinity capillary electrophoresis (ACE)

Separation principle: It has biological specific affinity in the electrophoresis process, that is, the specific affinity between receptor and ligand, forming a complex of receptor ligands The electrophoretogram changes of receptors and ligands before and after the affinity action can obtain information about the products of the changes in the size and structure of the affinity of receptors and ligands

8 Electrokinetic chromatography (EKC)

Separation principle: an electrophoretic mode named according to electrokinetic phenomena, involving the principles of electroosmosis, electrophoresis and chromatography, mainly used for the separation of chiral compounds

9 non aqueous capillary electrphoresis (NACE)

Separation principle: It is a mode of electrophoretic separation of analytes in organic solvents. The use of nonaqueous phase media can increase the selectivity of the method and facilitate the separation of water-insoluble substances