Micelles in MEKC

To obtain a true k in MEEKC, it is important to trace the migration of the pseudostationary phase accurately. Sudan III, timepidium bromide, and quine, which have generally been used as tracers for micelles in MEKC, could not be employed as tracers for microemulsions consisting of sodium dodecylsulfate salt (SDS) or cetyltrimethylammonium bromide (CTAB), n-butanol and heptane (12). An iteration method based on a linear relationship between log k and the carbon number for alkylbenzenes (13) seems to provide a reasonable value of the migration time of the microemulsions. Dodecylbenzene shows a migration time larger than the value calculated by the iteration method and those of other hydrophobic compounds, such as phenanthrene, fluoranthrene, and Sudan III (Table 1). Methanol and ethanol were used as tracers for the aqueous phase. 

Figure shows electropherograms of a mixture of five barbiturate standards it can be observed that the addition of micelles in MEKC clearly resulted in a different separation mechanism, reflected in various changes in elution order, compared to CZE. The migration behavior in MEKC depends largely on the hydro-phobic interaction of the analytes with the micelles. Hydrophobic components are more solubilized in the micelles, resulting in a slower migration compared to less hydrophobic compounds. 

As in chromatography, selectivity factor is the most important parameter in optimizing the separation. Since the micelle corresponds to the stationary phase in chromatography, the selection of the surfactant is of primary importance. SDS is a good initial choice and, if not successful, bile salts or CTAB/CTAC should be the second choice. If other surfactants are available, they may be tested. However, the number of different surfactants available for MEKC is rather limited and, hence, the selection of additives to modify selectivity should be considered when expected separation is not obtained by changing the surfactant. Some of the additives suggested above should be considered, as well as the use of mixed micelles. In MEKC, the efficiency is much higher than that of HPLC, the minimum a that provides successful separation may be as low as 1.02. 

Various V-alkanoyl-L-amino acids, such as sodium N-dodecanoyl-L-valinate (SDVal), sodium V-dodecanoyl-L-alaninate (SDAla), sodium V-dodecanoyl-L-glutamate (SDGlu), V-dodecanoyl-L-serine (DSer), V-dodecanoyl-L-aspartic acid (DAsp), sodium V-tetradecanoyl-L-glutamate (STGlu), and sodium V-dodecanoyl-L-threoninate (SDThr) have been employed as synthetic chiral micelles in MEKC several enantiomers have been successfully separated (Fig. 1). In each case, the addition of SDS, urea, and organic modifiers such as methanol or 2-propanol were essential to obtain improved peak shapes and enhanced enantioselectivity. 

Because micelles are negatively charged, they migrate toward the cathode with a velocity less than the electroosmotic flow velocity. Neutral species partition themselves between the micelles and the buffer solution in much the same manner as they do in HPLC. Because there is a partitioning between two phases, the term chromatography is used. Note that in MEKC both phases are mobile. ... 

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. 

In order to separate neutral compounds, Terabe et al. [13] added surfactants to the buffer electrolyte. Above their critical micellar concentration (cmc), these surfactants form micelles in the aqueous solution of the buffer electrolyte. The technique is then called Micellar electrokinetic capillary chromatography, abbreviated as MECC or MEKC. Micelles are dynamic structures consisting of aggregates of surfactant molecules. They are highly hydrophobic in their inner structure and hydrophilic at the outer part. The micelles are usually... 

FIGURE 12 Schematic overview of the separation principle in MEKC. Compound N is partitioned between aqueous phase represented by the EOF that moves toward the cathode in a fused silica capillary and the typical SDS micelles M. Reconstructed typical electropherogram with three peaks, t = I A neutral compound with no affinity for the micelles migrates with the velocity of the EOF. t = 2 A neutral compound with an affinity for both the micellar and the aqueous phase migrates with an intermediate velocity, t = 3 A fully solubilized neutral compound migrates with the velocity of the micelles. 

Like in MEKC, CEC combines the principles of CE and chromatography, with the major difference that the micelles are replaced by very small, i.e., less than 3 pm, solid or semi-solid... 

Zhu et al. coupled OT-CEC to ESI/MS for the analysis of /i-blockers and benzodiazepines. The authors described the use of a polymeric surfactant as a stationary-phase coating that enabled minimal surfactant introduction in the MS compared to MEKC—ESI/MS, thus avoiding interferences from non-voIatile micelles in ESI/MS. ... 

In MEKC, mainly anionic surface-active compounds, in particular SDS, are used. SDS and all other anionic surfactants have a net negative charge over a wide range of pH values, and therefore the micelles have a corresponding electrophoretic mobility toward the anode (opposite the direction of electro-osmotic flow). Anionic species do not interact with the negatively charged surface of the capillary, which is favorable in common CZE but especially in ACE. Therefore, SDS is the best-studied tenside in MEKC. Long-chain cationic ammonium species have also been employed for mainly anionic and neutral solutes (16). Bile salts as representatives of anionic surfactants have been used for the analysis of ionic and nonionic compounds and also for the separation of optical isomers (17-19). 

Fig. 1 Schematic representation of the separation principle of MEKC. An EOF/ micelle marker and three solutes differing in lipophilicity in the presence of anionic micelles in the background buffer are present. The lipophilicity increases in the sequence Sj < S2 < S3 t—migration time of EOF (nonionic solutes) S (solute) me —micelle.

In MEKC, the supporting electrolyte medium contains a surfactant at a concentration above its critical micelle concentration (CMC). The surfactant self-aggregates in the aqueous medium and forms micelles whose hydrophilic head groups and hydrophobic tail groups form a nonpolar core into which the solutes can partition. The micelles are anionic on their surface, and they migrate in the opposite direction to the electroosmotic flow under the applied current. The differential partitioning of neutral molecules between the buffered aqueous mobile phase and the micellar pseudostationary phase is the sole basis for separation as the buffer and micelles form a two-phase system, and the analyte partitions between them (Smyth and McClean 1998). 

The separation of phospholipids by micellar electrokinetic capillary electrophoresis (MEKC) has been described (17-19). In this technique, solutes are separated based on their distribution between a mobile (usually aqueous) and a pseudostationary (micellar) phase. Szucs et al. found that the major soybean phospholipids were fully resolved in only 7 minutes using deox ycholic acid for micelle formation in combination with 30% n-propanol at 50°C (18). However, quantification of the separated compounds remains troublesome. This is due first of all to the fact that only UV detection can be used, thus making the response highly dependent on the degree of unsaturation of the phospholipids. Besides, the comparison of peak areas in MEKC is more complicated than in HPLC, because all compounds are moving with different velocities. 

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. 

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... 

Conceptually. CE enantioseparations are mainly applied to charged SAs. Micellar electrokinetic chromatography (MEKC) (introduced by Terabe et al. in 1984 488 ), in contrast, permits the separation of electrically neutral compounds. In enantiomer separation by MEKC. ionic pseudo-stationary phases, such as chiral micelles composed of chiral SO moieties, which migrate according to their electrophoretic mobility, may interact stereoselectively with the solutes to be separated. MEKC with synthetic (e.g. A-dodecoxycarbonylvalines, commercialized as SDVal by Waters) 1489.490) or naturally occurring chiral surfactants (e.g. bile salts) 1491-494). and cyclodextrin-moditied MEKC (most often SDS/CD combinations) 1495-498) are the mo.st widely used selector systems in MEKC. The topic of MEKC enantioseparation has been reviewed by Nishi )499). 

In this section, chiral separation by MEKC with chiral micelles is mainly treated. The development of novel chiral surfactants adaptable to pseudo-stationary phases in MEKC for enantiomer separation is continuously progressing. It seems somewhat difficult for a researcher to find an appropriate mode of CE when one wants to achieve a specific enantioseparation. However, nowadays, various method development kits for chiral separation have been commercially available and some literature on the topic is also available, so that helpful information may be obtained without difficulty. 

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