Pseudostationary Phase Research Articles

Microchip electroseparation of proteins using lipid‐based nanoparticles

Porous liquid crystalline lipid-based nanoparticles are shown here to enable protein analysis in microchip electroseparation by reducing sample adsorption. Additionally, higher stability and reproducibility of the separations were observed. The method was tested by separating green fluorescent protein (GFP) in hot embossed cyclic olefin polymer microchips with integrated fiber grooves for LIF detection. The sample adsorption was indirectly quantified by measuring the height, width and asymmetry of the separation peaks for various concentrations of nanoparticles in the sample and background electrolyte. Without nanoparticles, electropherograms displayed typical signs of extensive adsorption to the channel walls, with low, broad tailing peaks. Higher, narrower more symmetric peaks were generated when 0.5-10% nanoparticles were added, showing a dramatic reduction of sample adsorption. The current through the separation channel decreased with nanoparticle concentration, reducing to half its value when the nanoparticle concentration was increased from 0.5 to 4%. Addition of nanoparticles enabled separations that were otherwise hindered by extensive adsorption, e.g. separation of GFP mutants differing by only one amino acid. It was also observed that increasing the nanoparticle concentration increased the number of impurities that could be resolved in a GFP sample. This indicates that the adsorption is further reduced, and/or that the nanoparticles provide an interacting pseudostationary phase for electrochromatography.

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.

The use of novel ionic liquid‐in‐water microemulsion without the addition of organic solvents in a capillary electrophoretic system

In this work, a new ionic liquid-in-water (IL/W) microemulsion without requiring toxic organic solvents was investigated as a pseudostationary phase (PSP) in CE. As observed during the IL/W microemulsion system, a fast and an efficient separation of eight phenolic acids was achieved using 1-butyl-3-methylimidazolivmhexa fluorophosphate (bmimPF(6)) as oil drops, Tween 20 as the surfactant, and borate as the BGE. The effects of oil phase, surfactant, buffer and pH on the separation were explored in detail to evaluate the novel PSP. In contrast, the detection efficiency of these same analytes was markedly decreased using oil-in-water (O/W) MEEKC. We have also validated the practicality of the IL/W microemulsion method by quantitative determination of acidic compounds in pharmaceutical injection. The results obtained indicated that an additional association between the IL cations and analytes tested seemed to play a prominent role in the separation mechanism exhibited by this novel PSP compared with the conventional O/W MEEKC.

NaDDBS as a dispersion agent for multiwalled carbon nanotubes in capillary EKC separation of nucleotides

A novel pseudostationary phase (PSP) of multiwalled carbon nanotubes (MWCNTs) dispersed with sodium dodecylbenzenesulfonate (NaDDBS) was used for the EKC separation of nucleotides. NaDDBS has a long hydrophobic chain and a benzylsulfonate group. It suspends more MWCNTs (about 100-fold) than SDS, and the π-π interaction between the benzene ring of NaDDBS and MWCNTs prolongs the slurry suspension time. Using NaDDBS as a surfactant can reduce the required amount of MWCNTs and decrease the baseline noise. To produce a stable suspension, the optimum ratio (w/w) of MWCNTs to NaDDBS was investigated with turbidimetry. In this context, several parameters affecting EKC separation were studied, including buffer pH, composition, concentration, and the organic modifier. Use of NaDDBS (8 mg/L)/MWCNTs (0.8 mg/L) as the PSP in a phosphate buffer (30 mM, pH 8) yielded complete resolution of seven geometric isomers of a nucleoside monophosphate. In stacking mode, with 10% MeOH in the sample plug, the mixture of nucleoside mono-, di-, and tri-phosphates was satisfactorily separated in phosphate buffer (50 mM, pH 9). The results indicate that nucleotides with bases containing more electron-withdrawing groups interact more strongly with MWCNTs. The system has been used to separate oligonucleotides, and to analyze nucleotides in a complex matrix sample.

Dual CD system‐modified MEEKC method for the determination of clemastine and its impurities

A dual system of CDs was used for the first time in MEEKC with the aim of determining clemastine and its three main related impurities in both drug substances and tablets. The addition of methyl-β-cyclodextrin and heptakis(2,6-di-O-methyl)-β-cyclodextrin to the microemulsion pseudo-stationary phase was essential to increase the resolving power of the system to obtain a baseline separation among the compounds. The best microemulsion composition was identified by mixture design and the effects of the factors concentrations of CDs and voltage were investigated by a response surface study applying a Central Composite Design. In both cases, Derringer's desirability function made it possible to find the global optimum, which corresponded to the following combination: microemulsion, 89.8% 10 mM borate buffer pH 9.2, 1.5% n-heptane and 8.7% of SDS/n-butanol in 1:2 ratio; 18 mM methyl-β-cyclodextrin, 38 mM heptakis(2,6-di-O-methyl)-β-cyclodextrin, 17 kV. By applying these conditions, the separation was completed in about 5.5 min. The method was validated following International Conference on Harmonisation guidelines and was applied to a real sample of clemastine tablets.

Microemulsion electrokinetic chromatography: An application for the simultaneous determination of suspected fragrance allergens in rinse-off products

A mixture of 18 neutral UV-active compounds with different characteristics of polarity was determined by capillary electrophoresis using a pseudostationary phase constituted by a microemulsion. The test analytes were volatile fragrance compounds, included in a list of 24 chemicals classified as suspected allergens according to Directive 2003/15/CE. The considered compounds were detected at 195 nm and p-anisaldehyde was chosen as internal standard. The background electrolyte consisted of a standard microemulsion made of 90.95% 10 mM borax buffer, pH 9.2, 1.05% n-heptane, 8.00% SDS/ n-butanol in 1:2 ratio, to which 40 mM methyl-β-cyclodextrin was added. Temperature and voltage were set at 20 °C and 25 kV, respectively. These experimental conditions allowed separation of the compounds to be obtained in about 20 min. The method was applied to real samples made up of rinse-off scented products. The results obtained using the standard microemulsion as pseudostationary phase showed its high resolution power, capable of effectively separating a complex mixture of analytes. Microemulsion electrokinetic chromatography was confirmed to have a great potential for different analytical challenges, holding up the possibility of using this technique as a good and complementary alternative to HPLC methods for routine analysis.

Stacking and separation of aspartic acid enantiomers under discontinuous system by capillary electrophoresis with light-emitting diode-induced fluorescence detection

We describe the stacking and separation of d- and l-aspartic acid (Asp) by capillary electrophoresis (CE) with light-emitting diode-induced fluorescence detection (LEDIF). In the presence of cyanide, d- and l-Asp were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) to form fluorescent derivatives prior to CE-LEDIF. The separation of NDA-derivatized d- and l-Asp was accomplished using a discontinuous system – buffer vials contained a solution of 0.6% poly(ethylene oxide) (PEO), 150 mM sodium dodecyl sulfate (SDS), and 60 mM hydroxypropyl-β-cyclodextrin (Hp-β-CD), while a capillary was filled with a solution of 150 mM SDS and 60 mM Hp-β-CD. The role of PEO, Hp-β-CD, and SDS is to act as a concentrating media, as a chiral selector, and as a pseudostationary phase, respectively. This discontinuous system could be employed for the stacking of 600 nL of NDA-derivatized d- and l-Asp without the loss of chiral resolution. The stacking mechanism is mainly based on the difference in viscosity between sample zone and PEO as well as SDS sweeping. The limits of detection at signal-to-noise of 3 for d- and l-Asp were down to 2.4 and 2.5 × 10 −10 M, respectively. Compared to normal sample injection volume (25 nL), this stacking approach provided a 100- and 110-fold improvement in the sensitivity of d- and l-Asp, respectively. This method was further applied for determining d- and l-Asp in cerebrospinal fluid, soymilk, and beer.

ChemInform Abstract: Carbon Nanotubes: Solid‐phase Extraction

AbstractReview: 181 refs.

Cationic gemini surfactants as pseudostationary phases in micellar electrokinetic chromatography. Part I: Effect of head group

Two cationic gemini surfactants with pyrrolidinium or alkyl ammonium head groups with but-2-yne spacers, but with the same length hydrocarbon chain have been characterized with respect to their aggregation behaviors and separation power as pseudostationary phases (PSPs) for micellar electrokinetic chromatography (MEKC). They were compared with a commonly used PSP, sodium dodecylsulfate (SDS). The results suggest that the head groups of the surfactants have some effect on physicochemical properties such as critical micelle concentration (CMC), C 20, γ CMC, partial specific volume, methylene selectivity and mobilities of the surfactants. CMC values of G1, G2 and SDS in pure water were found to be 0.82, 0.71, and 8.08 mM, respectively; they were reduced to 0.21, 0.11, and 3.0 mM when measured in 10 mM phosphate buffer at pH 7.0. G1 ( α C H 2 = 2.74 ) and G2 ( α C H 2 = 2.48 ) provided the most and the least hydrophobic environment, respectively. According to their partial specific volumes, geminis were found to have more flexible structures as compared with sodium dodecylsulfate. The effects of the head group structure were also characterized with the linear solvation energy relationship (LSER) model, which was able to evaluate the role of solute size, polarity/polarizability, and hydrogen bonding on retention and selectivity. The cohesiveness, hydrogen bond acidic and basic character of the surfactant systems were found to have the most significant influence on selectivity and MEKC retention of the gemini surfactants. It should be noted that with their large positive coefficient a values, G1 and G2 were found to be stronger HB acceptors than anionic and most of the cationic surfactants studied in the literature.

Novel, selective sample stacking microemulsion electrokinetic capillary chromatography induced by reverse migrating pseudostationary phase for the determination of the new ultra-short acting hypnotic “HIE-124” in mice serum

Microemulsion electrokinetic capillary chromatography (MEEKC) with sample stacking induced by reverse migrating pseudostationary phase (SRMP) technique in a suppressed electro-osmotic flow (EOF) strategy was investigated for analysing the new ultra-short hypnotic HIE-124 in mice serum. The proposed method utilized fused-silica capillary with a total length of 50 cm (effective length 40 cm), applied voltages for stacking and separation were 5.0 kV for 4.30 min and subsequently 25 kV, respectively, with a sample injection of 0.5 psi for 90 s. All the runs were carried out at 25 °C and detected at 213 nm. The optimum microemulsion background electrolyte (BGE) solution consisted of 0.8% (v/v) ethyl acetate, 6.6% (v/v) butan-2-ol, 1.0% (v/v) acetonitrile, 2.0% (w/v) sodium dodecyl sulfate (SDS), and 89.6 mL with 25 mM phosphate buffer pH 8. When this preconcentration technique was used, the sample stacking and the separation processes took place successively with changing the voltage with an intermediate polarity switching step. The proposed method was validated carefully with respect to high specificity of the method, good linearity ( r = 0.9994), fair wide linear concentration range (66–1500 ng mL −1), limit of detection and quantitation were 21.6 and 65.5 ng mL −1, respectively. The mean relative standard deviation (RSD) of the results of intra- and inter-day precision and accuracy were less than 6.0%, and overall recovery higher than 95% of HIE-124 in mice serum. The developed method could be used for the trace analyses of HIE-124 in serum and was finally used for the pharmacokinetic study investigation of HIE-124 in mice serum.

Evaluation of the performance of single‐walled carbon nanohorns in capillary electrophoresis

This paper describes for the first time the use of single-walled carbon nanohorns (SWNHs) as pseudostationary and stationary phases for EKC and CEC, respectively, taking advantage of their characteristic features, such as conical-end termination, formation of spherical assemblies dahlia-flower like superstructure and easy functionalization. The use of SWNHs as pseudostationary phase for EKC required the study of their dispersion in different surfactants as well as their compatibility with the electrophoretic system. The carboxylation and subsequent immobilization of carboxylated SWNHs in fused-silica capillary to obtain useful, reproducible and stable stationary phases for CEC has also been investigated, with promising results. The electrophoretic separations obtained for water-soluble vitamins in both modalities (EKC and CEC) have been systematically compared with those obtained with single-walled carbon nanotubes.

Liposomes for entrapping local anesthetics: A liposome electrokinetic chromatographic study

Bupivacaine is a lipophilic, long-acting, amide class local anesthetic commonly used in clinical practice to provide local anesthesia during surgical procedures. Several cases of accidental overdose with cardiac arrest and death have been reported since bupivacaine was introduced to human use. Recent case reports have suggested that Intralipid (Fresenius Kabi) is an effective therapy for cardiac toxicity from high systemic concentrations of, e.g. bupivacaine, even though the mechanism behind the interaction is not fully clear yet. Our long-term aim is to develop a sensitive, efficient, and non-harmful lipid-based formulation to specifically trap harmful substances in vivo. In this study, the in vitro interaction of local anesthetics (bupivacaine, prilocaine, and lidocaine) with Intralipid or lipid vesicles containing phosphatidylglycerol, phosphatidylcholine, cardiolipin, cholesterol, and N-palmitoyl-D-erythro-sphingosine (ceramide) was determined by liposome electrokinetic chromatography. The interactions were evaluated by calculating the retention factors and distribution constants. Atomic force microscopy measurements were carried out to confirm that the interaction mechanism was solely due to interactions between the analytes and the moving pseudostationary phase and not by interactions with a stationary lipid phase adsorbed to the fused-silica wall. The heterogeneity of the liposomes was also studied by atomic force microscopy. The liposome electrokinetic chromatography results demonstrate that there is higher interaction between the drugs and negatively charged liposome dispersion than with the commercial Intralipid dispersion.

Electrokinetic Chromatography and Mass Spectrometric Detection Using Latex Nanoparticles as a Pseudostationary Phase

The utility of novel latex nanoparticles as pseudostationary phases for electrokinetic chromatography with UV and mass spectrometric detection is demonstrated. The nanoparticles are synthesized using ab initio RAFT (reversible addition-fragmentation chain transfer) in emulsion polymerization, which yields small (63 nm) particles with a narrow size distribution, a hydrophobic core, and an ionic shell. The nanoparticles are shown to provide efficient and selective separations, with retention and separation selectivity dominated by hydrophobic interactions. The nanoparticles are highly retentive, such that they are effective at relatively low concentrations. Addition of the nanoparticles to the background electrolyte at these concentrations has a minor effect on the noise with UV detection, no measurable effect on the separation current, and minor effects on analyte ionization efficiency during electrospray ionization. The nanoparticles do not cause fouling or degradation of the electrospray-mass spectrometer interface even after several weeks of use. The combination of online sample preconcentration via sweeping and selective mass spectrometric detection yields low detection limits (10-16 ppb), particularly for more hydrophobic compounds.

Experimental modelling of drug membrane permeability by capillary electrophoresis using liposomes, micelles and microemulsions

Capillary electrophoresis (CE) was evaluated as an in-vitro format for experimental modelling of membrane permeability using only nanogram quantities of drug compounds. The rationale for the CE technique emanates from emulation of a lipid-like pseudo-stationary phase that governs separations mainly as a result of differences in molecular size, lipophilicity, hydrogen bonding and charge, all of which also have a strong influence on in-vivo drug absorption. By means of micellar, microemulsion and liposome electrolytes, the migration behaviour was studied at 37 degrees C for 22 model drug compounds. The generated CE retention factor data were then compared with membrane permeability reference data. Both simple log D and more common Caco-2 cell parameters were evaluated. In addition, permeation through intestinal segments of rat ileum and rat colon was included. An improved correlation was obtained in the order: micellar < microemulsion < liposome systems. Although the correlation for the best liposome CE system was only R(2)=0.77, the evaluation results for all emphasized the strength and flexibility of CE for assessing specific drug-membrane interaction through tailor-made lipophilic media.

Marker compounds for the determination of retention factors in EKC

EKC and its sub-techniques, such as MEKC and microemulsion EKC, have attracted wide interest in recent years. Investigations on this topic have covered several analytical applications, but attention has also been paid more and more to basic studies. This review provides an overview of the different approaches to calculating retention factors, which express the ratio of the amount of sample component in the pseudostationary and mobile phases. Special attention is given to the selection of markers for the determination of the electrophoretic mobility or migration time of a marker describing the behavior of the pseudostationary phase in EKC. Introduction of a hydrophobic marker is by far the most common approach, but the use of a homologous series of compounds is also quite popular. In addition, other possible approaches found in the literature will be described.

Solute–solvent interactions in micellar electrokinetic chromatography: VII. Characterization of sodium cholate–sodium deoxycholate mixed-micellar systems

Micellar electrokinetic chromatography (MEKC) systems with mixed pseudostationary phases of the bile surfactants sodium cholate (SC) and sodium deoxycholate (SDC) have been characterized by means of the solvation parameter model. The importance of characterizing systems with an appropriate set of solutes that embrace a wide range of descriptor values has been proven as they can significantly influence the value of the system constants. The fit of the solvation parameter model to the experimental log k data has been compared for each SC–SDC system when the Abraham descriptors and the Poole optimized descriptors, recently proposed, are used. In both cases, the variation in MEKC surfactant composition results in similar changes in the coefficients of the correlation equations, which in turn leads to similar information on solute–solvent and solute–micelle interactions. It is demonstrated that SDC is more hydrogen-bond acidic and hydrophobic but slightly less polarizable than SC. Systems with intermediate selectivity are obtained through mixtures of both surfactants.

Nanoparticle-based capillary electroseparation of proteins in polymer capillaries under physiological conditions

Totally porous lipid-based liquid crystalline nanoparticles were used as pseudostationary phase for capillary electroseparation with LIF detection of proteins at physiological conditions using unmodified cyclic olefin copolymer capillaries (Topas, 6.7 cm effective length). In the absence of nanoparticles, i.e. in CE mode, the protein samples adsorbed completely to the capillary walls and could not be recovered. In contrast, nanoparticle-based capillary electroseparation resolved green fluorescent protein from several of its impurities within 1 min. Furthermore, a mixture of native green fluorescent protein and two of its single-amino-acid-substituted variants was separated within 2.5 min with efficiencies of 400 000 plates/m. The nanoparticles prevent adsorption by introducing a large interacting surface and by obstructing the attachment of the protein to the capillary wall. A one-step procedure based on self-assembly of lipids was used to prepare the nanoparticles, which benefit from their biocompatibility and suspension stability at high concentrations. An aqueous tricine buffer at pH 7.5 containing lipid-based nanoparticles (2% w/w) was used as electrolyte, enabling separation at protein friendly conditions. The developed capillary-based method facilitates future electrochromatography of proteins on polymer-based microchips under physiological conditions and enables the initial optimization of separation conditions in parallel to the chip development.

Partial filling affinity capillary electrophoresis with cationic poly(vinylpyrrolidone)-based copolymer coatings for studies on human lipoprotein–steroid interactions

Human plasma lipoproteins have strong hydrophobic interactions with steroids and their fatty acyl derivatives such as estradiol fatty acyl esters. In this work, affinity capillary electrophoresis with the partial filling technique was applied to study the hydrophobic interactions between lipoproteins, which are nanometer-sized particles, and nonconjugated steroids. The capillaries were first rinsed with one of two novel poly(vinylpyrrolidone) (PVP)-based cationic copolymers that were strongly adsorbed onto the fused-silica surface via electrostatic interactions. This surface treatment greatly suppressed the adsorption of lipoproteins. Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) particles were then employed in the coated capillaries as pseudostationary phase in the partial filling mode. The changes in corrected migration times of steroids increased linearly with the filling time of the lipoproteins. The affinity constants between the steroids and lipoproteins were calculated, and the most hydrophobic steroid studied, progesterone, had stronger affinity than testosterone or androstenedione toward both LDL and HDL. Affinity between steroids and LDL was stronger than that between steroids and HDL. Interactions between the steroids and lipoproteins were mainly nonspecific with particle lipid components, whereas some were site specific with the apolipoproteins. The developed technique has great potential for determination of the affinity of various compounds toward lipoproteins.

Binary mixed micelles of chiral sodium undecenyl leucinate and achiral sodium undecenyl sulfate: I. Characterization and application as pseudostationary phases in micellar electrokinetic chromatography

Sodium 10-undecenyl sulfate (SUS), sodium 10-undecenyl leucinate (SUL) and their five different mixed micelles at varied percent mole ratios were prepared. The critical micelle concentration (CMC), C 20, γ CMC, partial specific volume, methylene group selectivity, mobilities and elution window were determined using a variety of analytical techniques. These surfactant systems were then evaluated as novel pseudostationary phases in micellar electrokinetic chromatography (MEKC). As a commonly used pseudostationary phase in MEKC, sodium dodecyl sulfate (SDS) was also evaluated. The CMC values of SUS and SUL were found to be 26 and 16 mM, respectively, whereas the CMC of mixed surfactants was found to be very similar to that of SUL. The C 20 values decreased dramatically as the concentration of SUL is increased in the mixed micelle. An increase in SUL content gradually increased the methylene group selectivity making the binary mixed surfactants more hydrophobic. Linear solvation energy relationships (LSERs) and free energy of transfer studies were also applied to predict the selectivity differences between the surfactant systems. The cohesiveness and the hydrogen bond acidic character of the surfactant systems were found to have the most significant influence on selectivity and MEKC retention. The SUS and SDS showed the strongest while SUL showed the weakest hydrogen bond donating capacity. The basicity, interaction with n and π-electrons of the solute and dipolarity/polarizability were the least significant factors in LSER model for the surfactant systems studied. Free energies of transfer of selected functional groups in each surfactant systems were also calculated and found to be in good agreement with the LSER data.

Detection of Agmatine and Octopamine in Rat Brain and Human Plasma by Microchip Electrophoresis

A microchip electrophoresis method with laser induced fluorescence detection was developed for the detection of agmatine (Agm) and octopamine (Oct). The fluorescent derivatization reagent, fluorescein isothiocyanate was used for precolumn derivatization of Agm and Oct. The sodium dodecyl sulfate (SDS) micelles was employed as pseudostationary phase for the separation of Agm and Oct with other endogenous compounds exist in biological samples. Some parameters including buffer concentration, buffer pH, SDS concentration and separation voltage were investigated in detail. Under the optimum conditions, the separation and determination of Agm and Oct was performed within 40 s. The calibration curves were linear for both Agm and Oct over the concentration range of 1.0 × 10−7 to 4.0 × 10−5 M and 1.5 × 10−7 to 4.5 × 10−5 M, respectively. The detection limits of Agm and Oct (S/N = 3) are 5.0 × 10−8 and 8.0 × 10−8 M, respectively. These values make the method very suitable for the determination of Agm and Oct in rat brain tissue and human plasma as demonstrated in this paper.