Instrumental Parameters
Voltage
Separation time is inversely proportional to applied voltage. An increase in voltage can cause excessive heat production that gives rise to temperature gradients and viscosity gradients of the buffer in the cross section of the capillary. This effect can be significant with high conductivity buffers, such as those containing micelles. Poor heat dissipation causes band broadening and decreases resolution.
Temperature
Variations in capillary temperature affect the partition coefficient of the solute between the buffer and the micelle, the critical micelle concentration, and the viscosity of the buffer. These parameters contribute to the migration time of the solutes.
Capillary
Length and internal diameter contribute to analysis time and efficiency of separations. Increasing both effective length and total length can decrease the electrical fields, working at constant voltage, and will increase migration time and improve the separation efficiency. The internal diameter controls heat dissipation, at a given buffer and electrical field, and provides a broadening of the sample band.
Electrolytic Solution Parameters
Surfactant Type and Concentration
The type of surfactant, as the stationary phase in chromatography, affects the resolution because it modifies separation selectively. The log
K¢ of a neutral compound increases linearly with the concentration of detergent in the mobile phase. When
K¢ approaches the value of
resolution in MEKC reaches a maximum. Modifying the concentration of surfactant in the mobile phase changes the resolution.
Buffer pH
pH does not modify the partition coefficient of non-ionized solutes, but it can modify the electroosmotic flow in uncoated capillaries. A decrease in the buffer pH decreases the electroosmotic flow and, therefore, increases the resolution of the neutral solutes, giving rise to longer analysis time.
Organic Solvents
To improve separation of hydrophobic compounds, organic modifiers (methanol, propanol, acetonitrile, etc.) can be added to the separation electrolytic solution. The addition of these modifiers generally decreases migration time and selectivity of the separation. The addition of organic modifiers affects micelle formation, thus, a given surfactant concentration can be used only with a certain percentage of organic modifier before the micellezation equilibrium is eliminated or adversely affected, resulting in the absence of micelles and, therefore, the absence of the partition mechanism of MEKC. The elimination of micelles in the presence of a high content of organic solvent does not always mean that the separation will no longer be possible, because, in some cases, the hydrophobic interaction between the ionic surfactant monomer and the neutral solutes form solvophobic complexes that can be separated electrophoretically.
Additives for Chiral Separations
A chiral selector is included in the micellar system, either covalently bound to the surfactant or added to the micellar separation electrolyte. Micelles that have a moiety with chiral discrimination properties include salts, N-dodecanoyl-l-amino acids, bile salts, etc. Chiral resolution can also be achieved using chiral discriminators, such as cyclodextrins, added to the electrolytic solutions that contain micelliced achiral surfactants.
Other Additives
Selectivity can be modified by adding chemicals to the buffer. Addition of several types of cyclodextrins to the buffer is also used to reduce the interaction of hydrophobic solutes with the micelle, increasing the selectivity for this type of compound. The addition of substances able to modify solute-micelle interactions by adsorption on the latter has been used to improve the selectivity of the separations in MEKC. These additives may consist of a second surfactant (ionic or non-ionic), which gives rise to mixed micelles, metallic cations that dissolve in the micelle and give co-ordination complexes with the solutes.