Micellar electrokinetic chromatography,

The micellar pseudostationary phase is produced by adding a surfactant to the buffer at concentrations that exceed its critical micelle concentration 

Retention, expressed by the capacity factor k of nonionic analytes, is a function of the partition coefficient and the volume of pseudostationary phase (micelles), the volume of the mobile phase, the retention time of the analyte, the dead time (corresponding to the migration velocity of the EOF), and the retention time of the micelles. If the micelles were immobilized in the capillary (i.e., if the pseudostationary phase were stationary ), the capacity factor would be similar to the standard equation of retention in chromatography. 

Therefore, in MEKC, the only difference from chromatography, for nonionic solutes, is that the pseudostationary phase (the micelles) is not actually stationary, but slowly migrates toward the detector, eluting at a characteristic time. That time is determined experimentally by injecting a water-insoluble dye (e.g., Sudan III or Sudan IV), which is completely included in the micelles, and measuring its elution time. 

For ionizable solutes, separation mechanisms rely not only on the partitioning of the nonionized forms in the micelles, but also on the electrophoretic mobility of the ionized form of the compounds in the aqueous phase. Electrostatic interactions between the ionic analytes and the charged surface of the micelles are also to be taken into consideration. 

Common surfactants that have been used in MEKC, are listed in Table 3.1 with the respective critical micelle concentrations the most popular are SDS, bile salts, and hydrophobic chain quaternary ammonium salts. Selectivity can also be modulated by the addition to the aqueous buffer of organic solvents (methanol, isopropanol, acetonitrile, tetrahydrofuran, up to a concentration of 50%). These agents will reduce the hydrophobic interactions between analytes and micelles in a way similar to reversed-phase chromatography. Organic modifiers also reduce the cohesion of the hydrophobic core of the micelles, increasing the mass transfer kinetics and, consequently, efficiency. Nonionic 

The electrodriven separation of the isohavonoids is optimized using UV absorbance detection, and complete separation is achieved in 12 min using a micellar system of 50 mM aqueous borate buffers at pH 8.7 containing 50 mM SDS (Eigure 1.18). Severe conditions are necessary to hydrolyze and extract all phytoestrogens 

FIGURE 1.18 Electropherogram of an aqueous solution of 5 nm of daidzein (1), genistein (2), formononetin (3), biochanin A (4), and coumestrol (5) using micellar electrokinetic chromatography-ultraviolet (MEKC-UV). (Erom Beekman, M.C., Lingeman, H., Brinkman, U.A.Th., and Gooijer, C., J. Microcolumn Sep., 11, 347-350,1999.) 

MS conditions SIM positive ion mode capillary voltage, 4.5 kV fragmentor, 40 V d7ing gas N2 flow and temperature, 4 L min and 200 °C nebulizer pressure 4 psi sheath liquid, 0.1 % formic acid in water-isopropanol (50/50,v/v) sheath flow, 3 pL min .  

Despite the widespread use of CE-MS for qualitative analysis, few quantitative applications have been pubhshed for routine analysis, and the vahdation of CE-MS methods according to generally accepted criteria is very uncommon. To our knowledge, only a few validation procedures are reported in the hterature. Although CE methods can be validated like chromatographic techniques, there are some specific characteristics to be discussed when quantitative determinations are expected. 

Capillary cassettes designed for CE-MS allow capillaries to exit the instrument. They generally present a UV detection window at a short distance from the inlet position. The total length of the capillary varies from 120 to 60 cm if the UV detection is by-passed. In this case, maximum field strength is available but does not allow simultaneous UV and MS detection. 

In order to expand the CE potential for quantification, the selected ion monitoring (SIM) mode is to be preferred for its high selectivity and sensitivity. It can be noted that sensitivity improvement over UV-VIS spectrometry is closely related to the nature of the compound (molar absorptivity or protonation or deprotonation capacity) the awaited gain in sensitivity can diverge from about 10 to IO . Tandem mass spectrometry (MS ) appears to be relevantly advantageous for quantitative purposes [23, 37]. In fact, the selectivity issue is of crucial importance in chiral CE due to the complex composition ofthe BGE. 

Some electrospray parameters are known to be critical for achieving stable conditions and thereby good quantitative results. These parameters are the sheath liquid composition and flow rate, the nebulizing gas pressure, the applied electrospray voltage and the capillary outlet position. On the other hand, in previous studies, the impact of drying gas flow rate and temperature on stability and sensitivity were demonstrated to be moderate [3, 76]. Most of the quoted parameters are well described in the literature, apart from the capillary position which is disregarded for CE-ESI- 

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Micellar Electrokinetic Capillary Chromatography One limitation to CZE is its inability to separate neutral species. Micellar electrokinetic chromatography... 

The elution order for neutral species in MEKC depends on the extent to which they partition into the micelles. Hydrophilic neutrals are insoluble in the micelle s hydrophobic inner environment and elute as a single band as they would in CZE. Neutral solutes that are extremely hydrophobic are completely soluble in the micelle, eluting with the micelles as a single band. Those neutral species that exist in a partition equilibrium between the buffer solution and the micelles elute between the completely hydrophilic and completely hydrophobic neutrals. Those neutral species favoring the buffer solution elute before those favoring the micelles. Micellar electrokinetic chromatography has been used to separate a wide variety of samples, including mixtures of pharmaceutical compounds, vitamins, and explosives. 

The last set of experiments provides examples of the application of capillary electrophoresis. These experiments encompass a variety of different types of samples and include examples of capillary zone electrophoresis and micellar electrokinetic chromatography. 

Vogt, C. Conradi, S. Rhode, E. Determination of Caffeine and Other Purine Compounds in Pood and Pharmaceuticals by Micellar Electrokinetic Chromatography, /. Chem. Educ. 1997, 74, 1126-1130. 

The effects of pH on electrokinetic velocities in micellar electrokinetic chromatography was studied by using sodium dodecyl sulfate solutions [179]. Micellar electrokinetic capillary chromatography with a sodium dodecyl sulfate pseudostationary phase has been used to determine the partition constants for nitrophenols, thiazolylazo dyes, and metal chelate compounds [180]. A similar technique was used to separate hydroquinone and some of its ether derivatives. This analysis is suitable for the determination of hydroquinone in skin-toning creams [181]. The ingredients of antipyretic analgesic preparations have also been determined by this technique [182], The addition of sodium dodecyl sulfate improves the peak shapes and resolution in chiral separations by micellar electrokinetic chromatography [183]. 

WORTH c c, wiESSLER M and SCHMITZ o J (2000) Analysis of catechins and caffeine in tea extracts by micellar electrokinetic chromatography . Electrophoresis, 21 (17), 3634-... 

CE was recently used for anthocyanin analysis because of its excellent resolution. This technique has different modes capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), micellar electrokinetic chromatography (MEKC), capillary electrochromatography (CEC), capillary isoelectric focusing (CIEE), and capillary isotachophoresis (CITP)."° CZE is the most popular method for anthocyanin... 

Capillary electrophoresis is increasingly used in food analysis due to its separation performance combined with the short time of analysis. - CapiUary electrophoresis recently applied to colorant measurements includes technical variants such as capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography. ... 

Watanabe, T. et al.. Analysis of elderberry pigments in commercial food samples by micellar electrokinetic chromatography. Anal. Sci., 14, 839, 1998. 

Trone, M. D., Leonard, M. S., Khaledi, M. G. Congeneric behavior in estimations of octanol-water partition coefficients by micellar electrokinetic chromatography. Anal. Chem. 2000, 72, 1228-1235. 

R., Khaledi, M. G. Quantitative structure-activity relationships studies with micellar electrokinetic chromatography. Influence of surfactant type and mixed micelles on estimation of hydrophobicity and bioavailability. J. Chromatogr. A 1996, 727, 323-335. 

S. L, Carlucci, A., Bregni, C., Kenndler, E. Comparison of the retention charaderi sties of different pseudostationary phases for microemulsion and micellar electrokinetic chromatography of betamethasone and derivatives. Electrophoresis 2003, 24, 984-991. 

A variety of formats and options for different types of applications are possible in CE, such as micellar electrokinetic chromatography (MEKC), isotachophoresis (ITP), and capillary gel electrophoresis (CGE). The main applications for CE concern biochemical applications, but CE can also be useful in pesticide methods. The main problem with CE for residue analysis of small molecules has been the low sensitivity of detection in the narrow capillary used in the separation. With the development of extended detection pathlengths and special optics, absorbance detection can give reasonably low detection limits in clean samples. However, complex samples can be very difficult to analyze using capillary electrophoresis/ultraviolet detection (CE/UV). CE with laser-induced fluorescence detection can provide an extraordinarily low LOQ, but the analytes must be fluorescent with excitation peaks at common laser wavelengths for this approach to work. Derivatization of the analytes with appropriate fluorescent labels may be possible, as is done in biochemical applications, but pesticide analysis has not been such an important application to utilize such an approach. 

Saitoh, K., Micellar electrokinetic chromatography, Kaguka (Kyoto), 45, 884, 1990. 

Saitoh, K., Kiyohara, C., and Suzuki, N., Mobilities of metal [i-diketonato complexes in micellar electrokinetic chromatography, ]. High Resolut. Chromatogr., 14, 245, 1991. 

Nishi, H. and Terabe, S., Application of micellar electrokinetic chromatography to pharmaceutical analysis, Electrophoresis, 11, 691, 1990. 

Nishi, H. and Matsuo, M., Separation of corticosteroids and aromatic hydrocarbons by cyclodextrin-modified micellar electrokinetic chromatography, J. Liq. Chromatogr., 14, 973, 1991. 

For many applications, diode array detection has become routine. A photodiode array was used for simultaneous detection of 100 capillaries in zone electrophoresis and micellar electrokinetic chromatography (MEKC).1516 Deflection of a laser beam by acoustic waves was reported as a means to scan six capillary channels on a microchip.17 The design of a low-noise amperometric detector for capillary electrophoresis has been reported.18... 

Xue, G., Pang H.-M., and Yeung E.S., Multiplexed capillary zone electrophoresis and micellar electrokinetic chromatography with internal standardization, Anal. Chem. 71, 2642, 1999. 

Palmer, C.P. and Tanaka, N., Selectivity of polymeric and polymer-supported pseudo-stationary phases in micellar electrokinetic chromatography, /. Chromatogr. A, 792, 105, 1997. 

FIGURE 15.1 One-dimensional capillary electrophoresis separation of a protein homogenate prepared from the hTERT cell line. Both separations were preformed in 30 pm ID, 145 pm OD, 20 cm long capillaries at 20,000 V. (a) Micellar electrokinetic chromatography performed with a 100 mM CHES, 100 mM Tris, and 15 mM SDS buffer at pH 8.7. Sample is electro-kinetically injected with 0.25 kV for 1 s (b) Capillary sieving electrophoresis performed in 5% Dextran (513 kDa), 100 mM CHES, 100 mM Tris, 3.5 mM SDS, pH 8.7. 

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