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Fundamental Study on Micellar Electrokinetic Chromatography
Capillary electrophoresis (CE) is a relatively new analytical separation technique. Terabe et al. published the first paper on micellar electrokinetic chromatography (MEKC) in 1984 and they have exerted much effort to develop MEKC as a new microseparation technique from the viewpoint of fundamental studies. MEKC is performed by adding micelles in the running solution of CE without any modification of the instrument and enables the separation of neutral analytes, which was thought to be impossible in electrophoresis theretofore, having dramatically extended the application fields of CE. His academic achievement is briefly introduced below.
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Prof. Sugeru Terabe
University of Hyogo
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1.Fundamental theory and extension of the separation principle
Terabe et al. have developed MEKC based on the proposal by Dr. Toshio Nakagawa (Div. Colloid Surf. Chem., Chem. Soc. Jpn. 1981, 6, No. 3, 1) on the utilization of the micellar solubilization phenomenon in chromatography, by employing CE which was just developed on these days. They demonstrated successful separation of neutral analytes by electrophoresis with high efficiencies and named the technique MEKC. The separation principle is explained below when using an anionic surfactant such as sodium dodecyl sulfate (SDS). The surfactant is added to the running solution at a higher concentration than critical micelle concentration and the micelle formed migrates toward the anode due to electrophoresis but the micelle is simultaneously subject to the electroosmosis which transfers the bulk solution inside the capillary toward the cathode. The electroosmotic flow (EOF) is stronger than the electrophoretic migration of the micelles under neutral or alkaline conditions and hence, the micelle also migrates toward the cathode with a retarded velocity. When the neutral analyte is introduced into the running solution, a portion of the analyte is incorporated into the micelle and migrates at the velocity of the micelle, which is slower than the EOF velocity. While the analyte is free from the micelle, it migrates at the EOF velocity. Since the partition equilibrium of the analyte between the micelle and the surrounding aqueous phase can be established quickly compared with the electrophoretic separation process, e.g., 10 min, the migration velocity depends on the ratio of the fraction of the analyte incorporated into the micelle over that in the aqueous phase. Since the zone broadening is mainly caused by the longitudinal diffusion of the analyte inside the capillary, the separation efficiency is high, easily reaching several hundred-thousands theoretical plates.
The separation principle of MEKC is similar to that of chromatography and Terabe et al. have developed fundamental equations to describe the migration behavior of the analyte by employing the chromatographic theory. Since the micelle corresponds to the stationary phase in chromatography, it is called the pseudostationary phase in MEKC. They have shown that charged polymers, large molecules, or molecular aggregates can be utilized as pseudostationary phases and the technique has been generally named electrokinetic chromatography (EKC).
2.Solutions of important issues in MEKC
Several issues were found to be solved during the fundamental study on MEKC: (1) the analytes which interact strongly with the micelle cannot be separated, (2) the use of mass spectrometry (MS) for detection is difficult, (3) although several sample preconcentration techniques were developed for CE to compensate the poor detection sensitivity in terms of concentration, these techniques were not applicable to the neutral analytes.
The issue (1) was relatively easily solved by reducing the retention factor or distribution coefficient in micellar solubilization through several techniques: addition of a water-miscible organic solvent such as methanol to the running solution, addition of a cyclodextrin (CD) to the running solution, the use of a surfactant having a different structure from that of SDS as the pseudostationary phase. CD, if it is neutral, does not migrate by electrophoresis and migrates in the bulk solution at the EOF velocity, but it can include the analyte into its cavity, reducing the apparent partition of the analyte to the micelle. CD can also enhance the selectivity in MEKC particularly for chiral or isomeric analytes. For example, MEKC can separate even polycyclic aromatic hydrocarbons by employing SDS and a CD derivative.
The issue (2) was difficult to solve. Electrospray ionization (ESI) is usually employed as an interface between liquid phase separation method and MS, as liquid chromatography-MS is routinely used as a indispensable analytical separation technique today. To use ESI-MS for the MEKC detection, the presence of a high concentration of the surfactant in the running solution causes two major problems: reducing the ionization efficiency of ESI and the contamination of the instrument by the non-volatile surfactant. These problems were not completely solved yet but they proposed and showed several possible solutions. The partial filling technique was the most promising technique, which is useful not only for MEKC-MS but also for other purpose to avoid the problem in detection caused by the additives in the running solution.
To solve the issue (3), they were successful to develop a new and powerful on-line sample preconcentration technique named sweeping. The poor concentration sensitivity in CE detectors is mainly due to a short light-pathlength of the absorbance detector, which is nearly equal to the capillary diameter, e.g., 50 μm, and a minute amount of the sample solution injected. A larger volume of the sample solution than that of the conventional procedure is introduced in on-line sample preconcentration techniques and the analytes are concentrated inside the capillary and then, the focused zone is separated. Sweeping is based on the principle that the micelle can efficiently incorporate the analyte in a dilute solution, which is different from principles of other sample preconcentration techniques based on the modification of electrophoretic velocities. Sweeping is useful for sample preconcentration of neutral analytes as well as charged ones and up to several thousand-fold increases in detection sensitivity have been shown.
As mentioned above, Terabe has greatly contributed to the development of MEKC and the technique has been widely accepted as a useful analytical separation technique of CE.
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