Pseudo-stationary phase

MEKC is a CE mode based on the partitioning of compounds between an aqueous and a micellar phase. This analytical technique combines CE as well as LC features and enables the separation of neutral compounds. The buffer solution consists of an aqueous solution containing micelles as a pseudo-stationary phase. The composition and nature of the pseudo-stationary phase can be adjusted but sodium dodecyl sulfate (SDS) remains the most widely used surfactant. 

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 pseudo stationary phase, Electrophoresis, 20, 1987, 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. 

Besides CZE and NACE, micellar electrokinetic chromatography (MEKC) is also widely used, and ionic micelles are used as a pseudo-stationary phase. MEKC can therefore separate both ionic and neutral species (see Chapter 2). Hyphenating MEKC with ESI/MS is problematic due to the non-volatility of micelles, which contaminate the ionization source and the MS detector, resulting in increased baseline noise and reduced sensitivity. However, MEKC—ESI/MS was applied by Mol et al. for identifying drug impurities in galantamine samples. Despite the presence of non-volatile SDS, all impurities were detected with submicrogram per milliliter sensitivity and could be further characterized by MS/MS. 

Tanaka, Y, and Terabe, S. (1995). Partial separation zone technique for the separation of enantiomers by affinity electrokinetic chromatography with proteins as chiral pseudo-stationary phases. J. Chromatogr. A 694, 277—284. 

Y Tanaka, M Yanagawa, S Terabe. Separation of neutral and basic enantiomers by cyclodextrin electrokinetic chromatography using anionic cyclodextrin derivatives as chiral pseudo-stationary phases. J High Res Chromatogr 19 421-433, 1996. 

Y Ishihama, Y Oda, N Asakawa, Y Yoshida, T Sato. Optical resolution by electrokinetic chromatography using ovomucoid as a pseudo-stationary phase. J Chromatogr A 666 193-201, 1994. 

Separation by MEKC is based on the differential partitioning of solutes into a pseudo -stationary phase. In general, the pseudo-stationary phase is... 

The alkylphosphonic acids, MPA, EPA, and PPA, have been determined by direct fluorescence after derivatization with 4-(9-anthroxyloxy)phenacyl bromide (panacyl bromide) (18). The 325-nm line of the HeCd laser was used for excitation. The neutral derivatives required MEKC for the separation and 50 mM sodium cholate was used as the pseudo-stationary phase. Separation was achieved in 33 min and limits of detection ranged from 0.13-0.14 xM (12-17ppb). 

Chankvetadze et al. have demonstrated the potential of flow-counterbalanced capillary electrophoresis (FCCE) in chiral and achiral micropreparative separations [27], Unlimited increase of separation selectivity can be achieved for binary mixtures, such as (+ )-chlorpheniramine with carboxymethyl-(3-cyclodextrin chiral selector, or a- and (3-isomers of a asparatame dipeptide. The carrier of the chiral selector or pseudo-stationary phase, electroosmotic flow (EOF), pressure-driven flow, or hydrodynamic flow can be used as a counterbalancing flow to the electrophoretic mobility of the analyte or vice versa, resulting in dramatic changes of the effective mobilities of the sample mixture components [28], This approach can be used for micropreparative CE, stepwise separations, and fraction collection of multicomponent mixtures [27],... 

The first electrically driven enantioseparations involved the addition of a chiral selector to the mobile phase in CE. This selector is usually a complexing agent and acts as a pseudo-stationary phase. The separation is accomplished by the difference in the distibution equilibria between the pseudo-stationary phase and the enantiomers [134], The most common additives incorporated into these CE experiments were cyclodextrins and cyclodextrin derivatives [135-138], However, these experiments required the replacement of the chiral selector after each electrophoretic run. 

The thick acetonitrile layer adsorbed on the bonded phase surface acts as a pseudo-stationary phase, thus making retention in acetonitrUe/water systems a superposition of two processes partitioning into the acetonitrile layer and adsorption on the surface of the bonded phase. Based on the model described in reference 166, analyte retention could be represented in the following form ... 

The multilayered character of acetonitrile adsorption creates a pseudo-stationary phase of significant volume on the surface, which acts as a suitable phase for the ion accumulation. In the low organic concentration region (from 0 to 20 v/v% of acetonitrile), studied ions show significant deviation from the ideal retention behavior (decrease in ion retention with increase in acetonitrile composition) due to the formation of the acetonitrile layer, and significant adsorption of the chaotropic anions was observed. This creates an electrostatic potential on the surface in which there is an adsorbed acetonitrile layer, which provides an additional retentive force for the enhancement of the retention of protonated basic analytes. When the dielectric constant is lower than 42 [167], this favors the probability of ion pair formation in this organic enriched layer on top of the bonded phase. 

The concept of direct emmtioseparation is based on the formation of reversible transient diastereomeric SOSA associates between a chiral SO and the SA-enantiomers with different equilibrium constants. As a consequence of the enantioselective SOSA complexation, differences in retention of the corresponding SA-enantiomers are observed, assuming that the SO successfully acts as a chiral (pseudo) stationary phase. 

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). 

The stationary phase can he filled into the capillary, bonded as a film to the capillary walls (open tubular CEC) or introduced into the capillary as a suspension or solution of the mobile phase (pseudo-stationary phase). The separation is, in the case of neutrally charged analytes, based on partitioning between the stationary phase and the mobile phase or, in the case of charged analytes, based on both partitioning and electrophoretic mobility (charge to friction coefficient ratio). 

We take, here, only protolysis into consideration and do not discuss such important other equilibria such as complexation or interactions with pseudo-stationary phases. It follows from Eq. (3) that the effective mobility depends on the actual mobility (that of the fully charged particle at the ionic strength of the experiment), on the pK value of the analyte, and on the pH of the BGE. It follows that all these properties determine the selectivity term in the resolution. 

An ionic chiral micelle is used as a pseudo-stationary phase it works as a chiral selector. When a pair of enantiomers is injected to the MEKC system, each enantiomer is incorporated into the chiral micelle at a certain extent determined by the micellar solubilization equilibrium. The equilibrium constant for each enantiomer is expected to be different more or less among the enantiomeric pair that is, the degree of solubilization of each enantiomer into the chiral micelle would be different for each. Thus, the difference in the retention factor would be obtained and different migration times would occur. 

An ionic achiral micelle [e.g., sodium dodecyl sulfate (SDS)] and a neutral CD are typically used as a pseudo-stationary phase and a chiral selector, respectively. When a pair of enantiomers is injected into this system, two major distribution equilibria can be considered for the solutes or enantiomers (a) the equilibrium between the aqueous phase and the micelle (i.e.. 

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