Micellar electrokinetic chromatography (MEKC) is a mode of capillary electrophoresis (CE) introduced by Shigeru Terabe in 1984 that extends the separation capabilities of CE to neutral analytes. In MEKC, surfactant molecules added to the running buffer at concentrations above the critical micelle concentration (CMC) self-assemble into micelles that function as a pseudostationary phase. Analyte separation results from the differential partitioning of solutes between the aqueous buffer (mobile phase) and the hydrophobic interior of the micelles (pseudostationary phase), combined with the differential migration of the charged micelles under the applied electric field. MEKC bridges the gap between classical electrophoresis, which requires charged analytes, and chromatography, which relies on differential partitioning between phases.
The Principle of Micellar Solubilization and Partitioning
Surfactants are amphiphilic molecules containing both a hydrophilic head group and a hydrophobic tail. In aqueous solution at concentrations above the CMC, surfactant monomers aggregate to form micelles with the hydrophobic tails oriented toward the interior and the hydrophilic heads facing the aqueous buffer. For sodium dodecyl sulfate (SDS), the most common surfactant in MEKC, the CMC is approximately 8 mM in water, and each micelle contains about 60 to 70 monomers. Neutral analytes partition between the aqueous phase and the micellar pseudophase based on their hydrophobicity: hydrophobic solutes are retained more strongly by the micelles and migrate at the same velocity as the micelles, while hydrophilic solutes spend more time in the aqueous phase and migrate with the bulk electroosmotic flow. Charged analytes experience an additional electrophoretic mobility component that influences their overall migration behavior.
The Pseudostationary Phase
The pseudostationary phase in MEKC is not physically fixed but moves with a velocity determined by the electrophoretic mobility of the micelles and the electroosmotic flow (EOF). Anionic surfactants such as SDS migrate toward the anode, but under typical CE conditions the EOF toward the cathode is stronger, causing the micelles to migrate in the same direction as the bulk flow but at a reduced velocity. This creates a migration window between the fastest analyte (one that does not interact with micelles and migrates with the EOF) and the slowest analyte (one that is fully retained by the micelles). All neutral analytes elute within this window, known as the elution range. The width of the elution range is determined by the migration time ratio of the micelles to the EOF and is a critical parameter affecting resolution. Surfactants other than SDS, including cationic surfactants such as cetyltrimethylammonium bromide (CTAB), nonionic surfactants such as Brij-35, and bile salts such as sodium cholate, can be used to alter selectivity for specific applications.
Resolution and Efficiency in MEKC
Resolution in MEKC is governed by three factors: separation efficiency (plate count), selectivity (the relative difference in retention factors between two analytes), and the width of the elution range. The plate count in MEKC is similar to that of CZE, typically ranging from 100,000 to 300,000 plates per meter, because the plug-like flow profile of the EOF minimizes band broadening. Selectivity is manipulated by changing the type and concentration of surfactant, adding organic modifiers such as methanol or acetonitrile to the buffer, or incorporating cyclodextrins, urea, or ion-pairing reagents into the separation medium. The elution range is expanded when the micellar migration velocity differs significantly from the EOF velocity, which can be achieved by adjusting the buffer pH, ionic strength, or capillary wall chemistry. Optimization of resolution in MEKC involves balancing these parameters to achieve adequate separation of all target analytes within a reasonable analysis time.
Operating Parameters and Method Development
The choice of surfactant type and concentration is the primary variable in MEKC method development. SDS at concentrations of 20 to 100 mM is the default starting point because of its low cost, low UV absorbance, and well-characterized behavior. Buffer pH controls both the EOF and the ionization state of acidic or basic analytes, with pH 7 to 9 being the most common range for SDS-based separations. Organic solvents such as methanol, acetonitrile, and 2-propanol are added at 5 to 30 % to reduce the retention of highly hydrophobic analytes and alter selectivity. Temperature control is essential because the CMC, micellar size, and buffer viscosity all vary with temperature. Voltage is typically set between 15 and 30 kV, with higher voltages providing faster separations but increased Joule heating. Sample injection is performed hydrodynamically to avoid the bias introduced by electrokinetic injection in the presence of micelles.
Applications of MEKC
MEKC is widely applied in pharmaceutical analysis for the determination of drug purity, content uniformity, and stability, particularly for neutral or weakly ionized drugs that are difficult to separate by CZE. In the food and beverage industry, MEKC is used to analyze preservatives, antioxidants, sweeteners, and caffeine in complex matrices. Environmental applications include the determination of polycyclic aromatic hydrocarbons (PAHs), pesticides, and phenols in water and soil samples. In clinical and biomedical analysis, MEKC has been employed for the quantification of steroids, bile acids, porphyrins, and vitamins in biological fluids. The technique is also used in the analysis of natural products, including flavonoids, alkaloids, and coumarins in plant extracts. The combination of MEKC with mass spectrometry detection (MEKC-MS) via electrospray ionization extends its applicability to the identification and structural characterization of unknown compounds, requiring the use of volatile surfactants and buffers compatible with MS detection.
