Pseudostationary phase

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]. 

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. 

Breadmore, M. C., Macka, M., and Haddad, P. R., Manipulation of separation selectivity for alkali metals and ammonium in ion-exchange capillary electrochromatography using a suspension of cation exchange particles in the electrolyte as a pseudostationary phase, Electrophoresis, 20, 1987, 1999. 

Several research groups used another interesting column technology as an alternative to the modification of the capillary surface. This method is inherited from the field of electrophoresis of nucleic acids and involves capillaries filled with solutions of linear polymers. In contrast to the monolithic columns that will be discussed later in this review, the preparation of these pseudostationary phases need not be performed within the confines of the capillary. These materials, typically specifically designed copolymers [85-88] and modified den-drimers [89], exist as physically entangled polymer chains that effectively resemble highly swollen, chemically crosslinked gels. 

Columns filled with polymer solutions are extremely simple to prepare, and the packing can easily be replaced as often as desired. These characteristics make the pseudostationary phases excellent candidates for use in routine CEC separations such as quality control applications where analysis and sample profiles do not change much. However, several limitations constrain their widespread use. For example, the sample capacity is typically very low, pushing typical detection methods close to their sensitivity limits. Additionally, the migration of the pseudostationary phase itself may represent a serious problem, e. g., for separations utilizing mass spectrometric detection. The resolution improves with the concentration of the pseudostationary phase. However, the relatively low solubility of current amphiphilic polymers does not enable finding the ultimate resolution limits of these separation media [88]. 

Three different interacting phases can be distinguished in ACE the stationary, pseudostationary, and mobile phases. First, the interaction can take place at the surface of a coated capillary wall or at a stationary phase present in the capillary. This approach is analogous to CEC, as discussed previously. Second, the interaction can take place in pseudostationary phases, such as micelles, microemulsions, and liposomes. Third, the interaction can take place when both the solute and the affinity molecule are in free solution. For studying these interactions, two analysis methods have been developed. 

Regarding other pseudostationary phases for measurement of lipophilicity or lipophilicity-related properties (e.g., intestinal absorption, brain penetration), there are several reports on the use of vesicles such as phospholipid bilayer liposome (56-58), lysophospholipid micelle (59), DTAB/SDS vesicle (60), and double-chain synthetic surfactant vesicle (61), which are described in other chapters. 

Electrokinetic chromatography (EKC) using microemulsion is one of the most powerful tools for the rapid measurement of log P w with high reproducibility. Because it is relatively easy to manipulate the pseudostationary phases of EKC, a lot of phases have been reported for the measurement not only of physicochemical properties but also of the separation selectivity, such as polymer micelles (64) and double-chain surfactant vesicles (56-58,60,61). These phases are also interesting in terms of the correlation to bioactivity. 

C Fujimoto. Application of linear solvation energy relationships to polymeric pseudostationary phases in micellar electrokinetic chromatography. Electrophoresis 22 1322-1329 (2001). 

K Bachmann, A Bazzanella, I Haag, K-Y Han. Charged native -cyclodextrin as a pseudostationary phase in electrokinetic chromatography. Fresenius J Anal Chem 357 32-36, 1997. 

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. 

The ACE analysis of interactions between drugs and phospholipid bilayers of liposomes present as a pseudostationary phase was performed by Zhang et al. (32). The capillaries were treated to eliminate electroendosmosis. Freshly prepared and essentially neutral small unilamellar liposomes composed of egg phosphatidylcholine were sucked into the capillary. These liposomes increased both the retention of four negatively charged drugs and the separation between the substances (Fig. 6). The chromatographic retentions of these drugs on immobilized phosphatidylcholine liposomes, ex-... 

S Terabe, H Ozaki, Y Tanaka. New pseudostationary phases for electrokinetic chromatography A high-molecular surfactant and proteins. J Chin Chem Soc 41 251-257, 1994. 

Electrokinetic chromatography (EKC) comprises a variety of electromigration techniques that use electrolyte solutions incorporating a separation carrier, which is called the pseudostationary phase, and are based on the distribution of the analytes between this phase and the surrounding solution. 

The migration behavior of neutral analytes in MEKC can be described by parameters similar to those employed in liquid chromatography [209], Hence, the ratio of the number of moles of the analyte in the micellar phase, the pseudostationary phase, (ttp p) to those in the surrounding solution (nip) defines the retention factor t... 

The equations reported above are related to MEKC and all other EKC separation modes performed with pseudostationary phase, analytes, and EOE moving in the same direction at different velocities. Such condition applies when the electroosmotic mobility is higher than the electrophoretic mobility of the pseudostationary phase migrating to the direction opposite to that of EOF. 

EKC in the reversed direction mode is performed when analytes and pseudostationary phase move at different velocities in the same direction, which is opposite to that of EOF. In this case, retention factor and resolution are expressed by the following equations [211] ... 

EKC is not restricted to the separation of neutral analytes, as it is widely employed for the simultaneous separations of charged and neutral analytes as well as of ionizable compounds having similar electrophoretic mobility. The separation of ionizable analytes by EKC is governed by differences in the partitioning between the pseudostationary phase and the surrounding electrolyte solution as well as electrophoretic mobility. For these analytes, the retention factor can be described by the following mathematical model ... 

FIGURE 6.11 Schematic representation of (A) partial-filling approach and (B) full-filling approach of EKC with pseudostationary phases consisting of nanoparticles dispersed into the BGE. 

The separation can be based on one or more of three possible mechanisms as follows (1) The two enantiomers of a solute have a tendency to form complexes with the selector in the mobile phase to different extents. The diastereomeric complexes formed and the free enantiomers have a different distribution to the achiral stationary phase. (2) The diastereomeric complexes formed have a different distribution to the achiral stationary phase. (3) The chiral selector adsorbs to the achiral stationary phase to form a chiral pseudostationary phase [49]. 

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