Pseudostationary Phase Research Articles

Determination of distribution constants of antioxidants by electrokinetic chromatography

AstractLiposome electrokinetic chromatography and micellar electrokinetic chromatography were used for studying the lipophilic properties of natural antioxidants, specifically phenolic acids and flavonoids. The employed negatively charged liposomes were composed of mixtures of 1-palmitoyl-2-oleyl-sn-glycerophosphatidylcholine and 1-palmitoyl-2-oleoyl-sn-phosphatidylserine or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol. In micellar electrokinetic chromatography, sodium dodecyl sulphate micelles were used as the pseudostationary phase. The retention factors of the studied compounds were determined at pH 7.4. The corresponding distribution constants were calculated from the experimentally determined retention factors and the phase ratios of the liposome dispersions and the micellar system. The distribution constants between the aqueous phase and the liposomes or micelles were compared with octanol/water partition or distribution constants of the studied compounds, which were predicted using the AC...

Performance and selectivity of cationic nanoparticle pseudo‐stationary phases in electrokinetic chromatography

Electrokinetic chromatography (EKC) is a powerful analytical technique that uses an ionic pseudo-stationary phase (PSP) to separate neutral compounds. Although anionic surfactants are the most common choice for PSP, cationic latex nanoparticles are an attractive alternative. Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize several types of diblock copolymers that self-assemble into latex nanoparticles, which were characterized by a variety of techniques including diffusion NMR. The performance of each nanoparticle as a PSP was studied by using a homologous series of ketones and linear solvation energy relationships (LSER) analysis. A cationic homopolymer coating was found to be necessary to prevent band broadening caused by analyte interactions with nanoparticles adsorbed to the capillary surface. No significant difference in methylene selectivity or LSER parameters was observed between nanoparticles with different cationic shells, but differences were observed between nanoparticles with different hydrophobic cores. Cationic latex nanoparticles behaved more like anionic latex nanoparticles than like cationic surfactants, suggesting that selectivity is primarily driven by the hydrophobic portion of a PSP. Cationic latex nanoparticles in combination with a homopolymer cationic capillary coating are an excellent choice for EKC analyses where an anodic electroosmotic flow is required.

Phospholipid bilayer affinities and solvation characteristics by electrokinetic chromatography with a nanodisc pseudostationary phase.

Phospholipid bilayer nanodiscs composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and synthetic maleic acid-styrene copolymer belts have been introduced as a pseudostationary phase (PSP) in electrokinetic chromatography and demonstrated good performance. The nanodiscs provide a suitable migration range and high theoretical plate counts. Using this nanodisc pseudostationary phase, the affinity of the bilayer structure for probe solutes was determined and characterized. Good correlation is observed between retention factors and octanol water partition coefficients for particular categories of solutes, but the general correlation is weak primarily because the nanodiscs show stronger affinity than octanol for hydrogen bond donors. This suggests that a more appropriate application of this technology is to measure and characterize interactions between solutes and lipid bilayers directly. Linear solvation energy relationship analysis of the nanodisc-solute interactions in this study demonstrates that the nanodiscs provide a solvation environment with low cohesivity and weak hydrogen bond donating ability, and provide relatively strong hydrogen bond acceptor strength.

Distribution of local anesthetics between aqueous and liposome phases

Liposomes were used as biomimetic models in capillary electrokinetic chromatography (EKC) for the determination of distribution constants (KD) of certain local anesthetics and a commonly used preservative. Synthetic liposomes comprised phosphatidylcholine and phosphatidylglycerol phospholipids with and without cholesterol. In addition, ghost liposomes made from red blood cell (RBC) lipid extracts were used as pseudostationary phase to acquire information on how the liposome composition affects the interactions between anesthetics and liposomes. These results were compared with theoretical distribution coefficients at pH 7.4. In addition to 25°C, the distribution constants were determined at 37 and 42°C to simulate physiological conditions. Moreover, the usability of five electroosmotic flow markers in liposome (LEKC) and micellar EKC (MEKC) was studied. LEKC was proven to be a convenient and fast technique for obtaining data about the distribution constants of local anesthetics between liposome and aqueous phase. RBC liposomes can be utilized for more representative model of cellular membranes, and the results indicate that the distribution constants of the anesthetics are greatly dependent on the used liposome composition and the amount of cholesterol, while the effect of temperature on the distribution constants is less significant.

A strategy to modulate the electrophoretic behavior in plastic microchips using sodium polystyrene sulfonate

Plastic microchips have been broadly used as disposable microfluidic devices, but the poorly defined surface properties limit their application. Herein, we proved that an anionic polymer could be used as the background electrolyte (BGE) to provide a strong and stable cathodic electroosmotic flow (EOF) and modulate the electrophoretic behavior for efficient separation in relative thicker microchannels (∼75μm id). A cathodic EOF of ∼3.3×10−4cm2V−1s−1 was maintained using sodium polystyrene sulfonate (PSSNa) with a molecular weight of 5×105 as the BGE, which ensured fluorescein isothiocyanate labeled biogenic amines (BAs) appeared ahead of other components in the electropherograms obtained with microchips of cyclic olefin copolymer. Four selected BAs appeared within 50s and theoretical plate numbers of 8.0×105/m were achieved. The role of PSSNa was evaluated with streaming potential, dynamic light scattering, contact angle and atomic force microscopy. Its functionalities as surface modifier, viscosity regulator and pseudostationary phase were also confirmed. The proposed electrophoretic method was applied in the fast determination of BAs in fish meat samples.

Advances in the Use of Cyclodextrins as Chiral Selectors in Capillary Electrokinetic Chromatography: Fundamentals and Applications

Capillary electrokinetic chromatography is generally recognized as a versatile and robust capillary electromigration technique for the separation of enantiomers. In this mode, one or more chiral selectors are added to the background electrolyte acting as pseudostationary phases. Within the various chiral selectors that have been applied to enantioseparations in capillary electrokinetic chromatography, cyclodextrins are by far the most often used selectors because of their versatility, structural variety and commercial availability. This is reflected in the large number of applications of cyclodextrins to analytical enantioseparations that have been reported between January 2012 and July 2016, the period of time covered by this review. Many of these applications cover aspects of life sciences such as drug analysis, bioanalysis or food analysis. Despite the large number of commercially available cyclodextrins, new derivatives have been developed in order to achieve altered enantioselectivities or to further broaden the application range. Cyclodextrins have also been used to demonstrate the validity of theoretical models of electromigration as well as complex formation equilibria in enantioseparations. Finally, recent studies for an understanding of the molecular basis of the chiral recognition between cyclodextrins and the analytes are discussed.

Enantioselective capillary electrophoresis-mass spectrometry of amino acids in cerebrospinal fluid using a chiral derivatizing agent and volatile surfactant

The sensitivity of coupled enantioselective capillary electrophoresis-mass spectrometry (CE-MS) of amino acids (AAs) is often hampered by the chiral selectors in the background electrolyte (BGE). A new method is presented in which the use of a chiral selector is circumvented by employing (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC) as chiral AA derivatizing agent and ammonium perfluorooctanoate (APFO) as a volatile pseudostationary phase for separation of the formed diastereomers. Efficient AA derivatization with FLEC was completed within 10 min. Infusion experiments showed that the APFO concentration hardly affects the MS response of FLEC-AAs and presents significantly less ion suppression than equal concentrations of ammonium acetate. The effect of the pH and APFO concentration of the BGE and the capillary temperature were studied in order to achieve optimized enantioseparation. Optimization of CE-MS parameters, such as sheath-liquid composition and flow rate, ESI and MS settings was performed in order to prevent analyte fragmentation and achieve sensitive detection. Selective detection and quantification of 14 chiral proteinogenic AAs was achieved with chiral resolution between 1.2 and 8.6, and limits of detection ranging from 130 to 630 nM injected concentration. Aspartic acid and glutamic acid were detected, but not enantioseparated. The optimized method was applied to the analysis of chiral AAs in cerebrospinal fluid (CSF). Good linearity (R2 > 0.99) and acceptable peak area and electrophoretic mobility repeatability (RSDs below 21% and 2.4%, respectively) were achieved for the chiral proteinogenic AAs, with sensitivity and chiral resolution mostly similar to obtained for standard solutions. Next to l-AAs, endogenous levels of d-serine and d-glutamine could be measured in CSF revealing enantiomeric ratios of 4.8%–8.0% and 0.34%–0.74%, respectively, and indicating the method's potential for the analysis of low concentrations of d-AAs in presence of abundant l-AAs.

Studying the interaction between peptides and polymeric nanoparticles used as pseudostationary phase in capillary electrochromatography

In this work, the interaction between synthetic bioactive peptides and polymeric nanoparticles (NPs) used as pseudostationary phase in capillary electrochromatography was studied. NPs were prepared from methacrylic acid and ethylenglycol dimethacrylate with benzoyl peroxide by utilizing a precipitation polymerization technique. The reaction was monitored by infrared spectroscopy. Polymer characterization was performed by dynamic light scattering and transmission electron microscopy. Experimental running conditions were tested, including organic solvent proportion in the background electrolyte, capillary conditioning, applied voltage, sample introduction amount, and how NPs were incorporated into the system. A continuous full filling technique in which the NPs were suspended in the entire electrolyte volume as well as a conventional partial filling technique were used. Results obtained at pH7.0 suggest that the NPs have a very strong interaction with more basic peptides. The interaction between analytes and NPs was found to be predominantly ionic.

Approaches for enantioselective resolution of pharmaceuticals by miniaturised separation techniques with new chiral phases based on nanoparticles and monolithis.

This article discusses new developments in the preparation of nanoparticles and monoliths with emphasis upon their application as the stationary and pseudo-stationary phases for miniaturised liquid phase separation techniques, which have occurred in the last 10 years (from 2006 to the actuality). References included in this review represent current trends and state of the art in the application of these materials to the analysis, by EKC, CEC and miniaturised chromatography, of chiral compounds with environmental interest such as pharmaceuticals. Due to their extraordinary properties, columns prepared with these new chiral stationary or pseudo-stationary phases, based on materials such as gold nanoparticles, metal-organic frameworks, ordered mesoporous silicas, carbonaceous materials, polymeric-based and silica-based monoliths or molecularly imprinted materials, can usually show some improvements in the separation selectivity, column efficiency and chemical stability in comparison with conventional chiral columns available commercially.

The effects of organic modifier on physicochemical and chromatographic characteristics of self‐assembled micelle from poly (stearyl methacrylate‐co‐methacrylic acid) in electrokinetic chromatography

In our previous work, organic solvents, especially 1-butanol, play a key role to separate highly hydrophobic analytes in EKC using the polymeric micelle self-assembled from amphiphilic random copolymer poly (stearyl methacrylate-co-methyl acrylic acid) (P(SMA-co-MAA)) as a novel pseudostationary phase. Herein, the influences of organic solvents on polymeric micelle physicochemical properties including environmental micropolarity and dimension, as well as chromatographic characteristics containing elution window, hydrophobic selectivity and polar group selectivity were investigated in detail. P(SMA-co-MAA) has extremely low CMC of 1.26 × 10(-6) g/mL, and the self-assembled micelles with selective solvent method had regular spherical structure with diameter about 50 nm. The experimental results showed that methanol, isopropanol or acetonitrile molecules could not penetrate into the interior of the polymeric micelles, and mainly affected the properties of surrounding running buffer. Too much these organic solvents led to elution window narrowed down, methylene selectivity and group selectivity weaken, even the micelles collasped. Whereas, addition of 2% 1-butanol did not influence the elution window, instead, improved the hydrophobic selectivity. Furthermore, both better group selectivity and faster migration for relatively hydrophobic analytes could be achieved simultaneously. It indicated that 1-butanol could insert into the polymeric micelle and 2% 1-butanol was enough to modify the structure of the micelles.

Enantioseparation and impurity determination of ambrisentan using cyclodextrin-modified micellar electrokinetic chromatography: Visualizing the design space within quality by design framework

A capillary electrophoresis method for the simultaneous determination of the enantiomeric purity and of impurities of the chiral drug ambrisentan has been developed following the Quality by Design principles. The selected separation system consisted of a micellar pseudostationary phase made by sodium dodecyl sulphate with the addition of γ-cyclodextrin. The effects of critical process parameters (capillary length, temperature, voltage, borate concentration, pH, sodium dodecyl sulphate concentration, γ-cyclodextrin concentration) on enantioresolution of ambrisentan and analysis time were extensively investigated by multivariate strategies involving a screening phase and Response Surface Methodology. The Design Space was defined with a desired probability level π≥90%, and the working conditions, with the limits of the Design Space, corresponded to the following: capillary length, 64.5cm; temperature, 22°C; voltage, 30kV (26–30kV); background electrolyte, 100mM borate buffer pH 9.20 (8.80–9.60), 100mM sodium dodecyl sulphate, 50mM (43–50mM) γ-cyclodextrin. A Plackett-Burman design was applied for robustness testing, and a method control strategy was established. The method was fully validated according to the International Conference on Harmonisation guidelines and was applied to ambrisentan coated tablets.

Study of the mechanism of acetonitrile stacking and its application for directly combining liquid-phase microextraction with micellar electrokinetic chromatography

Acetonitrile stacking is an online concentration method that is distinctive due to its inclusion of a high proportion of organic solvent in sample matrices. We previously designed a universal methodology for the combination of liquid-phase microextraction (LPME) and capillary electrophoresis (CE) using acetonitrile stacking and micellar electrokinetic chromatography (MEKC) mode, thereby achieving large-volume injection of the diluted LPME extractant and the online concentration. In this report, the methodology was extended to the analysis of highly substituted hydrophobic chlorophenols in wines using diethyl carbonate as the extractant. Additionally, the mechanism of acetonitrile stacking was studied. The results indicated that the combination of LPME and MEKC exhibited good analytical performance: with ∼40-fold concentration by LPME, a 20-cm (33% of the total length) sample plug injection of an eight-fold dilution of diethyl carbonate with the organic solvent-saline solution produced enrichments higher by a factor of 260–791. Limits of qualification ranged from 5.5 to 16.0ng/mL. Acceptable reproducibilities of lower than 1.8% for migration time and 8.6% for peak areas were obtained. A dual stacking mechanism of acetonitrile stacking was revealed, involving transient isotachophoresis plus pH-junction stacking. The latter was associated with a pH shift induced by the presence of acetonitrile. The pseudo-stationary phase (Brij-35) played an important role in reducing the CE running time by weakening the isotachophoretic migration of the analyte ions following Cl− ions. The combination of acetonitrile stacking and nonionic micelle-based MEKC appears to be a perfect match for introducing water-immiscible LPME extractants into an aqueous CE system and can thus significantly expand the application of LPME-CE in green analytical chemistry.

Separation and stacking of iodine species from seafood using surfactant-coated multiwalled carbon nanotubes as a pseudo-stationary phase in capillary electrophoresis

This article describes an on-line technique for hydrodynamic injections of long sample plugs with simultaneous stacking of iodine species in capillary electrophoresis (CE) with diode array detection. Surfactant-coated multi-walled carbon nanotubes (SC-MWNTs) were used as a pseudostationary phase for the separation of iodate, tetraiodothyronine,triiodothyronine, diiodothyronine, and diiodotyrosine. The effects of MWNTs, concentration of SC-MWNTs and salt, of buffer pH value, injection times were examined. Under the optimized conditions, i.e. detection at 230 nm, a separation voltage 25 kV, a borate running buffer of pH 7.5 and a SC-MWNT concentration of 9 μg · mL−1, the method gave relative standard deviations of the retention times and peak areas in intra-day assays (for n = 6) and for inter-day assays (for n = 3) of less than 4.49 and 5.80 %, respectively. The CE method was then applied to the analysis of the above iodine species in (spiked) kelp (kunbu) and porphyra, and recoveries ranged from 81.6 to 98.4 % with RSDs (n = 3) for extraction repeatability of <3.39 % in all cases.

Performance of a moving bed-membrane bioreactor treating saline wastewater contaminated by hydrocarbons from washing of oil tankers

This work analyses the performance of a moving bed-membrane bioreactor (MB-MBR) in the treatment of saline wastewater contaminated by hydrocarbons from washing of oil tankers with seawater (slops). In order to allow a biomass acclimation, a gradual increase in salinity and total petroleum hydrocarbons (TPHs) were adopted during six experimental phases. The results showed that acclimation of heterotrophic strains to TPHs occurred during Phase IV (30% by volume of slop). This is confirmed by an evident increase in the biological removal efficiencies of chemical oxygen demand, total organic carbon and TPHs. In particular, the TPHs removal efficiency increased from about 8% up to the range of 35–70%. No inhibition of the ammonia oxidizing bacteria (AOB) was noted except for Phase VI (100% by volume of slop) characterized by a collapse of ammonium removal efficiency from about 95% to about 21–24%. An accumulation of nitrite at the end of Phase IV, suggested an inhibition of nitrite-oxidizing bacteria (NOB), while a simultaneous “salt-in” of bromide indicated the acclimation of the same strains to salinity. Moreover, the hydraulic performance showed a high increase in total resistance to filtration (RT) during the pseudo-stationary phases (from Phase I to Phase III) and during Phase VI, due to the high salinity and hydrocarbons concentration. The results showed that the MB-MBR system has high potentiality for the treatment of saline oily wastewater.

Ionic Liquids as Modifiers of Chromatographic and Electrophoretic Systems

The application of ionic liquids to chromatography and capillary electrophoresis as modifiers of background electrolytes, chiral selectors, and components of mobile, stationary, and pseudostationary phases in the separation of different classes of organic compounds is discussed; the effect of ionic liquids on the efficiency of separation and selectivity is considered.

Microemulsion Electrokinetic Chromatography.

Microemulsion electrokinetic chromatography (MEEKC) is a special mode of capillary electrophoresis employing a microemulsion as carrier electrolyte. Analytes may partition between the aqueous phase of the microemulsion and its oil droplets which act as a pseudostationary phase. The technique is well suited for the separation of neutral species, in which case charged oil droplets (obtained by addition of an anionic or cationic surfactant) are present. A single set of separation parameters may be sufficient for separation of a wide range of analytes belonging to quite different chemical classes. Fine-tuning of resolution and analysis time may be achieved by addition of organic solvents, by changes in the nature of the surfactants (and cosurfactants) used to stabilize the microemulsion, or by various additives that may undergo some additional interactions with the analytes. Besides the separation of neutral analytes (which may be the most important application area of MEEKC), it can also be employed for cationic and/or anionic species. In this chapter, MEEKC conditions are summarized that have proven their reliability for routine analysis. Furthermore, the mechanisms encountered in MEEKC allow an efficient on-capillary preconcentration of analytes, so that the problem of poor concentration sensitivity of ultraviolet absorbance detection is circumvented.

Enantiomer Separations by Capillary Electrophoresis.

Capillary electrophoresis (CE) is a versatile and flexible technique for analytical enantioseparations. This is due to the large variety of chiral selectors as well as the different operation modes including electrokinetic chromatography, micellar electrokinetic chromatography, and microemulsion electrokinetic chromatography. The chiral selector, which is added to the background electrolyte, represents a pseudostationary phase with its own electrophoretic mobility allowing a variety of different separation protocols. The present chapter briefly addresses the basic fundamentals of CE enantioseparations as well as the most frequently applied chiral selectors and separation modes. The practical example illustrates the separation of the enantiomers of a positively charged analyte using native and charged cyclodextrin derivatives as chiral selectors.

Determination of Total Apigenin in Herbs by Micellar Electrokinetic Chromatography with UV Detection.

Apigenin is a naturally occurring plant flavone that exhibits strong antioxidant, anti-inflammatory, and antitumor properties. A MEKC-UV based method was developed for the determination of total apigenin in selected herbs. Application of pseudostationary phase in the form of SDS micelles resulted in great repeatability of retention times and peak areas. A buffer solution consisting of 30 mmol/L sodium borate (pH 10.2), 10% acetonitrile, and 10 mmol/L sodium dodecyl sulfate was found to be the most suitable BGE for the separation. The method was validated and calibrated for total apigenin in the range of 1.0–100 μmol/L (R 2 = 0.9994). The limits of detection and quantification were 0.48 μmol/L and 0.92 μmol/L, respectively. This precise and robust method was successfully applied to the analysis of plant samples for total apigenin content.

Capillary electrochromatography using monoamine- and triamine-bonded silica nanoparticles as pseudostationary phases

Monoamine- and triamine-bonded silica nanoparticles were prepared using 3-aminopropyltrimethoxysilane and N1-(3-trimethoxysilylpropyl)diethylenetriamine, respectively, and used as pseudostationary phases for capillary electrochromatography. The amine-bonded silica nanoparticles were tightly adsorbed on the inner wall of a capillary and generated fast electro-osmotic flow (2.59×10−4cm2V−1s−1) toward the anode in an electric field. The electro-osmotic velocities obtained with 20nm triamine-bonded silica were three to five times larger than those generated by a fused silica capillary and two times faster than those for the commercial cationic polymer-modified capillary. Fast electro-osmotic flow enables rapid analysis. This method was applied to hydrophilic interaction chromatography (HILIC) mode separation of various samples including the size separation of glucose oligomer derivatives and the resolution of four nucleic acid bases.

New highly fluorinated polymers: Modifiers of chromatographic and electrophoretic systems

A set of four highly fluorinated copolymers that differ from each other in the length of perfluorinated pendants to the fluorine-containing polymer backbone chain, charge, and acid–base functions of terminal groups (sulfonate, carboxylate, sulfamide, and N,N-diethylcarbamide) is obtained for the first time. Packed columns for gas–liquid chromatography (GLC) are prepared on the basis of synthesized fluoropolymers as stationary phases. Evaluation of main types of interactions between fluoropolymers and analytes of different nature and relative polarity of phases obtained is performed using McReynolds constants and Kovats retention indices. It is found that, under the conditions of capillary electrophoresis (CE) and micellar electrokinetic chromatography (MEKC), the influence of fluoropolymer as a component of supporting electrolyte on the increase in the efficiency (by two to three times) and separation selectivity of neutral analytes (steroid hormones) is due to two reasons: modification of walls of quartz capillary by the polymer and formation of a pseudostationary phase. Under the conditions of high-performance thin-layer chromatography (HPTLC) the introduction of highly fluorinated polymers into eluent composition results in the increase in the efficiency by two to four times in the determination of amino acids and water-soluble vitamins.