Question

Discuss capillary (zone) electrophoresis, micellar electrokinetic chromatography (MEKC) and capillary electrochromatography. Describe similarities and differences in working principle, injection, and separation. Discuss advantages and disadvantages of each of these techniques. Which compounds would you separate with each of these techniques?

Answer 1

Capillary electrophoresis is an analytical technique that separates ions based on their electrophoretic mobility with the use of an applied voltage. The electrophoretic mobility is dependent upon the charge of the molecule, the viscosity, and the atom's radius. The rate at which the particle moves is directly proportional to the applied electric field--the greater the field strength, the faster the mobility. Neutral species are not affected, only ions move with the electric field. If two ions are the same size, the one with greater charge will move the fastest. For ions of the same charge, the smaller particle has less friction and overall faster migration rate. Capillary electrophoresis is used most predominately because it gives faster results and provides high resolution separation. It is a useful technique because there is a large range of detection methods available.

There are six types of capillary electroseparation available: capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), micellar electrokinetic capillary chromatography (MEKC), capillary electrochromatography (CEC), capillary isoelectric focusing (CIEF), and capillary isotachophoresis (CITP).

Capillary Zone Electrophoresis (CZE), also known as free solution capillary electrophoresis, it is the most commonly used technique of the six methods.A mixture in a solution can be separated into its individual components quickly and easily.The separation is based 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 at which the ion moves is directly proportional to the electrophoretic mobility and the magnitude of the electric field.1

The fused silica capillaries have silanol groups that become ionized in the buffer. The negatively charged SiO- ions attract positively charged cations, which form two layers—a stationary and diffuse cation layer. In the presence of an applied electric field, the diffuse layer migrates towards the negatively charged cathode creating an electrophoretic flow (μep) that drags bulk solvent along with it. Anions in solution are attracted to the positively charged anode, but get swept to the cathode as well. Cations with the largest charge-to-mass ratios separate out first, followed by cations with reduced ratios, neutral species, anions with smaller charge-to-mass ratios, and finally anions with greater ratios. The electroosmotic velocity can be adjusted by altering pH, the viscosity of the solvent, ionic strength, voltage, and the dielectric constant of the buffer.

Micellar Electrokinetic Capillary Chromatography (MEKC):MEKC is a separation technique that is based on solutes partitioning between micelles and the solvent. Micelles are aggregates of surfactant molecules that form when a surfactant is added to a solution above the critical micelle concentration. The aggregates have polar negatively charged surfaces and are naturally attracted to the positively charged anode. Because of the electroosmotic flow toward the cathode, the micelles are pulled to the cathode as well, but at a slower rate. Hydrophobic molecules will spend the majority of their time in the micelle, while hydrophilic molecules will migrate quicker through the solvent. When micelles are not present, neutral molecules will migrate with the electroosmotic flow and no separation will occur. The presence of micelles results in a retention time to where the solute has little micelle interaction and retention time tmc where the solute strongly interacts. Neutral molecules will be separated at a time between to and tmc. Factors that affect the electroosmotic flow in MEKC are: pH, surfactant concentration, additives, and polymer coatings of the capillary wall.

Capillary Electrochromatography (CEC) : The separation mechanism is a packed column similar to chromatography. The mobile liquid passes over the silica wall and the particles. An electroosmosis flow occurs because of the charges on the stationary surface. CEC is similar to CZE in that they both have a plug-type flow compared to the pumped parabolic flow that increases band broadening.