Vesicles as pseudostationary phase for enantiomer separation by capillary electrophoresis

The suitability of noncovalently bilayer-coated capillaries for the analysis of proteins by capillary electrophoresis (CE) at medium pH was investigated. Fused-silica capillaries were coated simply by successively flushing with a polybrene (PB) and a poly(vinyl sulfonate) (PVS) solution. A protein test mixture was used to evaluate the performance of the coated capillaries. Comparisons with bare fused-silica capillaries were made. Several background electrolytes (BGEs) were tested in combination with the PB-PVS coating, showing that optimum performance was obtained for the proteins using high BGE concentrations. With a 300 mM Tris phosphate buffer (pH 7.0), good plate numbers (150,000-300,000), symmetrical peaks, and favorable migration-time repeatabilities (RSDs below 0.8%) were obtained for the proteins. Using bare fused-silica capillaries, the protein peaks were significantly broadened and the migration-time RSDs often exceeded 5%. It is concluded that the PB-PVS coating effectively minimizes adverse protein adsorption and provides a very stable electroosmotic flow (EOF). We also investigated the potential of a commercially available bilayer coating (CEofix) for protein analysis. It is demonstrated that with this coating, good plate numbers and peak symmetries for proteins can be achieved when the CEofix BGE ("accelerator") is replaced by a common BGE such as sodium or Tris phosphate. Apparently, the negatively charged polymer present in the "accelerator" interacts with the proteins causing band broadening. The utility of the bilayer coatings is further illustrated by the separation of proteins such as interferon-alpha 2b, myoglobin and carbonic anhydrase, by the analysis of a degraded insulin sample in time, and by the profiling of the glycoprotein ovalbumin. In addition, it is demonstrated that even in the presence of concentrations of human serum albumin in the sample of up to 60 mg/mL, the PB-PVS coating still provides reproducible protein separations of good performance.     

Organically modified sols as pseudostationary phases for microchip electrophoresis

We demonstrate that the selectivity of microchip electrophoresis separations is greatly improved by the presence of organically modified silica (Ormosil) sols in the run buffer. A negatively-charged N-(trimethoxysilylpropyl)ethylenediamine triacetic-acid (TETT)-based sol is used for improving the selectivity between nitroaromatic explosives and a methyltrimethoxysilane (MTMOS)-based sol is employed for enhancing the microchip separation of environmental pollutants, aminophenols. These sols are added to the run buffer and act as pseudostationary phases. Their presence in the run buffer changes the apparent mobility of studied solutes, and leads to a higher resolution. The observed mobilities changes reflect the interactions between the Ormosil sols and the solutes. Relevant experimental variables have been characterized and optimized. The diverse chemistry of Ormosil sols should be extremely useful for tailoring the selectivity of a wide range of electrophoresis microchip separations.     

Nanoparticle-based pseudostationary phases in capillary electrochromatography

During the past decades, research has been performed to enhance selectivity in CE by introducing different types of additives into the electrolyte. Research concerning this has taken many directions, especially during the last 5 years. A promising technique, which benefits from no packing or frits, is to use nanoparticles as the pseudostationary phase (PSP) in CEC. PSPs have the advantage of introducing a novel interaction phase for every analysis, which greatly simplify column exchange and circumvent contamination inherited from complex mixtures, e.g., biological samples. The field of nanoparticle-based PSPs used in CEC is covered in this review. The term CEC will be used consequently throughout this review, although some authors used the term EKC to categorize their work. Important requirements for the nanoparticles used and possible reasons for band broadening will be discussed. Applications with silica nanoparticles, polymer nanoparticles, molecularly imprinted polymer nanoparticles, gold nanoparticles, dendrimers, and polymeric surfactants as PSP will also be discussed.     

Characterization of surfactant and phospholipid vesicles for use as pseudostationary phases in electrokinetic chromatography

The physical, electrophoretic and chromatographic properties (mean diameter, electroosmotic flow, electrophoretic mobility, elution range, efficiency, retention, and hydrophobic, shape, and chemical selectivity) of three surfactant vesicles and one phospholipid vesicle were investigated and compared to a conventional micellar pseudostationary phase comprised of sodium dodecyl sulfate (SDS). Chemical selectivity (solute-pseudostationary phase interactions) was discussed from the perspective of linear solvation energy relationship (LSER) analysis. Two of the surfactant vesicles were formulated from nonstoichiometric aqueous mixtures of oppositely charged, single-tailed surfactants, either cetyltrimethylammonium bromide (CTAB) and sodium octyl sulfate (SOS) in a 3:7 mole ratio or octyltrimethylammonium bromide (OTAB) and SDS in a 7:3 mole ratio. The remaining surfactant vesicle was comprised solely of bis(2-ethylhexyl)sodium sulfosuccinate (AOT) in 10% v/v methanol, and the phospholipid vesicle consisted of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and phosphatidyl serine (PS) in 8:2 mole ratio. The mean diameters of the vesicles were 76.3 nm (AOT), 86.9 nm (CTAB/SOS), 90.1 nm (OTAB/SDS), and 108 nm (POPC/PS). Whereas the coefficient of electroosmotic flow (10(-4) cm2 V(-1) s(-1)) varied considerably (1.72 (OTAB/SDS), 3.77 (CTAB/SOS), 4.05 (AOT), 5.26 (POPC/PS), 5.31 (SDS)), the electrophoretic mobility was fairly consistent (-3.33 to -3.87 x 10(-4) cm2 V(-1) s(-1)), except for the OTAB/SDS vesicles (-1.68). This resulted in elution ranges that were slightly to significantly larger than that observed for SDS (3.12): 3.85 (POPC/PS), 8.6 (CTAB/SOS), 10.1 (AOT), 15.2 (OTAB/SDS). Significant differences were also noted in the efficiency (using propiophenone) and hydrophobic selectivity; the plate counts were lower with the OTAB/SDS and POPC/PS vesicles than the other pseudostationary phases (< or = 75,000/m vs. > 105,000/m), and the methylene selectivity was considerably higher with the CTAB/SOS and OTAB/SDS vesicles compared to the others (ca. 3.10 vs. < or = 2.6). In terms of shape selectivity, only the CTAB/SOS vesicles were able to separate all three positional isomers of nitrotoluene with near-baseline resolution. Finally, through LSER analysis, it was determined that the cohesiveness and hydrogen bond acidity of these pseudostationary phases have the greatest effect on solute retention and selectivity.     

Amphiphilic silica nanoparticles as pseudostationary phase for capillary electrophoresis separation

Amphiphilic silica nanoparticles surface-functionalized by 3-aminopropyltriethoxysilane (APTES) and octyltriethoxylsilane (OTES) were successfully prepared and characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectrometry (FT-IR) and thermogravimetry (TG) techniques. The potential use of these bifunctionalized nanoparticles as pseudostationary phases (PSPs) in capillary electrophoresis (CE) for the separation of charged and neutral compounds was evaluated in terms of their suitability. As expected, fast separation of representative aromatic acids was fulfilled with high separation efficiency, because they migrate in the same direction with the electroosmotic flow (EOF) under optimum experimental conditions. Using a buffer solution of 30mmol/L phosphate (pH 3.0) in the presence of 0.5mg/mL of the synthesized bifunctionalized nanoparticles, the investigated basic compounds were baseline-resolved with symmetrical peaks. Due to the existence of amino groups on the surface of nanoparticles, "silanol effect" that occurs between positively charged basic analytes and the silanols on the inner surface of capillary was greatly suppressed. Furthermore, the separation systems also exhibited reversed-phase (RP) behavior when neutral analytes were tested.     

Characterization of phosphatidylcholine/polyethylene glycol-lipid aggregates and their use as coatings and carriers in capillary electrophoresis

PEG-stabilized lipid aggregates are a promising new class of model membranes in biotechnical and pharmaceutical applications. CE techniques, field-flow fractionation, light scattering, quartz crystal microbalance (QCM), and microscopic techniques were used to study aggregates composed of 1-palmitoyl-2-oleyl-sn-glycero-phosphatidylcholine (POPC) and PEG-lipid conjugates. The PEG-lipids, with PEG molar masses of 1000, 2000, and 3000, were 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(PEG)] derivatives with either dimyristoyl (DM, 14:0) or distearoyl (DS, 18:0) acyl groups. The 80/20 mol% POPC/PEG-lipid dispersions in HEPES at pH 7.4 were extruded through 100 nm size membranes. Asymmetrical flow field-flow fractionation (AsFlFFF), photon correlation spectroscopy (PCS), and dynamic light scattering (DLS) were used to determine the sizes of POPC and the PEGylated aggregates. All methods demonstrated that the DSPEG-lipid sterically stabilized aggregates were smaller in size than pure POPC vesicles. The zeta potentials of the aggregates were measured and showed an increase from -19 mV for pure POPC to -4 mV for the POPC/DSPEG3000 aggregates. Atomic force microscopy (AFM), electron cryo-microscopy (EM), and multifrequency QCM studies were made to achieve information about the PEGylated coatings on silica. Lipid aggregates with different POPC/DSPEG3000-lipid ratios were applied as capillary coating material, and the 80/20 mol% composition was found to give the most suppressed and stable EOFs. Mixtures of low-molar-mass drugs and FITC-labeled amino acids were separated with the PEGylated aggregates as carriers (EKC) or as coating material (CEC). Detection was made by UV and LIF.     

Varying nanoparticle pseudostationary phase plug length during capillary electrophoresis

Capillary electrophoresis based separations of the hypothesized Parkinson's disease biomarkers dopamine, epinephrine, pyrocatechol, L-3,4-dihydroxyphenylalanine (L-DOPA), glutathione, and uric acid are performed in the presence of a 1 nM 11-mercaptoundecanoic acid functionalized gold (Au@MUA) nanoparticle pseudostationary phase plug. Au@MUA nanoparticles are monitored in the capillary and remain stable in the presence of electrically-driven flow. Migration times, peak areas, and relative velocity changes (vs. no pseudostationary) are monitored upon varying (1) the Au@MUA nanoparticle pseudostationary phase plug length at a fixed separation voltage and (2) the separation voltage for a fixed Au@MUA nanoparticle pseudostationary phase plug length. For instance, the migration times of positively charged dopamine and epinephrine increase slightly as the nanoparticle pseudostationary phase plug length increases with concomitant decreases in peak areas and relative velocities as a result of attractive forces between the positively charged analytes and the negatively charged nanoparticles. Migration times for neutral pyrocatechol and slightly negative L-DOPA did not exhibit significant changes with increasing nanoparticle pseudostationary plug length; however, reduction in peak areas for these two molecules were evident and attributed to non-specific interactions (i.e. hydrogen bonding and van der Waals interactions) between the biomarkers and nanoparticles. Moreover, negatively charged uric acid and glutathione displayed progressively decreasing migration times and peak areas and as a result, increased relative velocities with increasing nanoparticle pseudostationary phase plug length. These trends are attributed to partitioning and exchanging with 11-mercaptoundecanoic acid on nanoparticle surfaces for uric acid and glutathione, respectively. Similar trends are observed when the separation voltage decreased thereby suggesting that nanoparticle-biomarker interaction time dictates these trends. Understanding these analyte migration time, peak area, and velocity trends will expand our insight for incorporating nanoparticles in separations.     

Stationary phases for capillary electrophoresis and capillary electrochromatography

An overview of the most recent developments in column technology employed in capillary electrophoresis (CE) and capillary electrochromatography (CEC), mainly for the separation of small molecules and ions, is presented. Particular emphasis is laid on permanent coating. The wall modification methods in CE include covalent modification, adsorbed coatings and polymeric coatings, while those in CEC include packed columns, open-tubular columns and fritless columns. A short discussion on the characterization and selectivity of the bonded phases is also given.     

Polyamidoamine‐grafted silica nanoparticles as pseudostationary phases for capillary electrochromatographic separation of proteins

Polyamidoamine‐grafted silica nanoparticles were synthesized, characterized and investigated for the feasibility as pseudostationary phases in alkaline buffer for separation of cationic and anionic proteins, viz., lysozyme, cytochrome C, gamma globulin, and myoglobin. Neither bare silica nanoparticles nor polyamidoamines nor their mixtures as pseudostationary phases could lead to simultaneous separation of the four proteins. However, polyamidoamine‐grafted silica nanoparticles not only suppressed the irreversible wall adsorption of the cationic lysozyme and cytochrome C, but also provided selectivity toward all the proteins. We found that polyamidoamine generation two‐modified silica nanoparticles were the optimum pseudostationary phases with respect to detection sensitivity and separation efficiency; presence of the nanoparticles at 0.01% in the running buffer of 12.5 mM tetraborate/phosphate at pH 9.1 resulted in baseline resolution of the four proteins.     

Characterization and separation of semiconductor quantum dots and their conjugates by capillary electrophoresis

Semiconductor quantum dots (QDs) are very important luminescent nanomaterials with a wide range of potential applications. Currently, QDs as labeling probes are broadly used in bioassays, including immunoassay, DNA hybridization, and bioimaging, due to their excellent physical and chemical properties, such as broad excitation spectra, narrow and size‐dependent emission profiles, long fluorescence life time, and good photostability. The characterization of QDs and their conjugates is crucial for their wide bioapplications. CE has become a powerful tool for the separation and characterization of QDs and their conjugates. In this review, some CE separation models of QDs are first introduced, mainly including CZE, CGE, MEKC, and ITP. And then, some key applications, such as the measurements of size, surface charge, and concentration of QDs and the characterization of QDs conjugates (e.g. QD–protein, QD–DNA, QD–small molecule), are also described. Finally, future perspectives are discussed.     

Recent progress in the use of ionic polymers as pseudostationary phases for EKC

This review concerns the introduction, characterization, and application of polymeric pseudostationary phases (PSPs) for EKC since 2004. Achiral and chiral polymers and separations are reviewed, as is the application of polymeric PSPs for the combination of EKC with mass spectrometric detection.     

Alkyl-modified siloxanes as pseudostationary phases for electrokinetic chromatography

Anionic siloxane polymers with novel linker arm structures have been synthesized and characterized with respect to their performance and selectivity as pseudophases for electrokinetic chromatography. The linker arm between the siloxane backbone and the sulfonate head group is shorter and does not have the tertiary amine structure found in the siloxane pseudophases studied previously. This change in the linker arm structure and chemistry has dramatic effects on the chemical selectivity of the pseudophases. Linear solvation energy relationship (LSER) studies show that the greatest contributor to the difference in selectivity is that the new polymers are not as nonpolar as those previously studied. This result indicates that siloxane polymers are not by their nature more nonpolar or hydrophobic than other pseudophases. The LSER studies also demonstrate that siloxane pseudophases have a strong tendency to accept hydrogen bonds that cannot be attributed to the presence of the tertiary amine in the linker arm.     

Use of basic amphiprotic organic solvents containing neutral-surfactant aggregates as pseudostationary phase in non-aqueous capillary electrophoresis

A new approach to non-aqueous capillary electrophoresis based on the addition of anionic carboxylic surfactants to the basic amphiprotic organic solvent in which form neutral-surfactant aggregates was developed with a view to improving the electrophoretic resolution of charged substances. These aggregates acts as a new pseudostationary phase. The presence of these aggregates allows the effective separation of four tetracyclines with increased selectivity. The efficiency of sodium caprylate, sodium laurate and sodium palmitate as surfactants was examined. The latter two proved more effective than the former as they provided migration times reproducible to within 7% or better in all cases. The additional use of an alcohol allows peak shape to be controlled, which expands the potential of this electrophoretic technique even further. The proposed method was used to determine tetracyclines in water samples. The sensitivity of the determination was improved by using a flow manifold coupled at-line to the capillary electrophoresis system in order to preconcentrate the analytes. The limits of detection thus achieved ranged from 50 to 90 μg/l. Under optimal operating conditions, recoveries ranged from 97 to 104%, and precision from 5.4 to 7.0%.     

Characterization of synthetic polyelectrolytes by capillary electrophoresis

Capillary electrophoresis in entangled polymer solutions was applied to determine the molecular mass and polydispersity of polyelectrolytes. The separation selectivities of different polyethylene glycols as buffer additive can be correlated to their average molecular mass. A universal curve correlating the selectivity and the molecular mass could be obtained by using the instrinsic viscosity of the polyethylene glycol. The separation of poly(2-vinylpyridine) standards was compared to the separation of poly(4-vinylpyridine) standards. An indirect detection system was developed to characterize the cationic polyelectrolyte polydiallyldimethyl ammonium chloride. Various polymers with oppositely charged groups (polycarboxybetaines) were investigated with respect to structure dependence, pH dependence and molecular mass dependence of interand intramolecular association.     

Characterization of glycerin-based polyols by capillary electrophoresis

Methods based on capillary electrophoresis (CE) have been developed to obtain the molar mass distribution (MMD) of glycerin-based polyols and details on the presence of mono- and difunctional byproducts in technical samples. Prior to the analyses the hydroxy end-groups of the trifunctional polyols were converted to chargeable and UV-active moieties with phthalic anhydride (PhAH) as the derivatization reagent. With a method of capillary zone electrophoresis (CZE) samples of glycerin-based polyols with average molar masses up to 6000 were separated according to their charge-to-size ratio. The separations were carried out with a buffer solution containing 50% (v/v) acetonitrile and 10 mM sodium tetraborate, and for detection UV absorption at 220 nm was measured. An approximately linear relation between the reciprocal of the effective mobilities and the degree of polymerisation of the glycerin-based polyols was found. Therefore, the proposed CZE system could be used to determine the degree of polymerisation and polydispersity of technical glycerin-based polyol samples. The effect of the presence of sodium dodecyl sulfate (SDS) in the buffer solution on the CE separation of linear polyethylene glycols (PEGs), polypropylene glycols (PPGs) and ethylene oxide-propylene oxide (EO-PO) copolymers with different molar masses was investigated. The interaction between the charged polymer derivatives and SDS ions in solution increased strongly with the degree of polymerisation and the amount PO in the chain of the polymeric compounds. This behaviour made it possible to invert the migration order of EO-PO containing polymers of different size. With a background electrolyte (BGE) composition of 10mM SDS and 25% (v/v) acetonitrile in borate buffer mono- and difunctional byproducts were separated from the main glycerin-based polyols based on their number of end-groups. Accurate quantities for the mono- and difunctional impurities in technical glycerin-based polyol products were determined.     

Characterization of humic substances by capillary electrophoresis

Capillary electrophoresis was used for the separation of humic acid (HA) from peat, soil, and vermicompost. The electropherograms show the presence of at least three peaks eluted between 6 and 11 min for all HA. The best analysis resolution was obtained with the use of borate buffers at pH 8.9. The HA analyzed have structural and charge similarity, which increases the difficulty of separation. Therefore, the shape of the peaks is broad and the CE profiles of all HA are similar. It is reasonable to assume that the broad band in the three regions is due to the acidic groups that have a similar structure. By comparing the results obtained for HA extracted from soil, peat, vermicompost, and the commercial sample, HA from peat had the major carbon content.     

Characterization of carboxylated nanolatexes by capillary electrophoresis

Poly(styrene-co-acrylic acid) (St/AA) and poly(styrene-co-methacrylic acid) (St/MA) nanolatexes with different acid contents were prepared by emulsion copolymerization and were analyzed by capillary electrophoresis (CE) and by laser doppler velocimetry (LDV). Due to the intrinsic differences in the methodologies, CE (separative technique) and LDV (zetametry, nonseparative technique) lead to very different electrophoretic mobility distributions. Beyond these differences, the variation of the electrophoretic mobility is a complex and nonlinear function of the hydrodynamic radius, the ionic strength, and the zeta potential. To gain better insight on the influence of the ionic strength and the acid content on the electrophoretic behavior of the nanolatexes, the electrophoretic mobility data were changed into surface charge densities using the O'Brien, White, and Ohshima modeling. This approach leads to the conclusion that the surface charge density is mainly controlled at high ionic strength (～50 mM) by the adsorption of anionic surfactants coming from the sample. On the contrary, at low ionic strength, and/or in the presence of neutral surfactant in the electrolyte, the acid content was the main parameter controlling the surface charge density of the nanolatexes.     

Analysis of some anhydrides as their corresponding acids by capillary electrophoresis

Summary The potential of capillary electrophoresis for the indirect determination of anhydrides was demonstrated by the analysis of five closely-related anhydrides as their corresponding acids. Direct analysis of such substances is generally difficult because of their susceptibility to hydrolysis. In this work anhydrides were first hydrolyzed in water, hydrolysis being monitored using micellar electrokinetic chromatography (MEKC) which separated neutral anhydrides and anions of corresponding acids. After hydrolysis, separation of the acids was quickly achieved within two minutes by reversed electroosmotic-flow, capillary electrophoresis (REF-CE), with UV detection at 210 nm.     

Gold nanomaterials based pseudostationary phases in capillary electrophoresis: A brand‐new attempt at chondroitin sulfate isomers separation

In this work, a CE method with bare gold nanorods (GNRs) based pseudostationary phase was developed and applied for the separation of chondroitin sulfate (CS) isomers, CS, and dermatan sulfate (DS). The separation efficiency was investigated by varying the experimental parameters such as concentration and pH of the BGE, separation voltage, internal diameter of capillary, different size, and morphology of gold nanomaterials. Results showed that different size and morphology of gold nanomaterials had different effects on the separation of CS and DS. The best separation of CS and DS was achieved in the BGE composed of aqueous 150 mmol/L (mM) ethylenediamine + 20 mM sodium dihydrogen phosphate + 30% v/v GNRs, pH 4.5, at the separation voltage of ?10 kV. Capillary was 59.2 cm in length (effective length 49 cm), 50 μm id capillary thermostated at 25°C. CE with bare GNRs used as pseudostationary phase was shown to be a suitable technique for the separation of CS and DS mixtures with wider peaks. RSD of migration time and peak area of CS and DS were 0.13, 0.14 and 0.86, 1.07%, respectively.     

Use of ionic polymers as stationary and pseudo-stationary phases in the separation of ions by capillary electrophoresis and capillary electrochromatography

One of the problems with capillary electrophoresis is a lack of versatility regarding manipulation of the separation selectivity. A new and potentially universal concept is to introduce an ion-exchange component into a separation so that the migration of analyte ions is influenced by both their electrophoretic mobilities and their chromatographic properties. This may be accomplished by use of capillaries filled with or coated with solid ion-exchange polymers, or by addition of a soluble ionic polymer to the background electrolyte to create a pseudo-stationary phase. While each of these methods achieves the same result, they are not competitive, but rather complementary as the problems associated by one approach are overcome by the others. Recent highlights in the field are used to illustrate the flexibility that this approach provides to electrophoretic separation of ions.