Pseudostationary Phase Research Articles

Investigations of the Mechanism of Gold Nanoparticle Stability and Surface Functionalization in Capillary Electrophoresis

Covalently functionalized gold nanoparticles influence capillary electrophoresis separations of neurotransmitters in a concentration- and surface-chemistry-dependent manner. Gold nanoparticles with either primarily covalently functionalized carboxylic acid (Au@COOH) or amine (Au@NH(2)) surface groups are characterized using extinction spectroscopy, transmission electron microscopy, and zeta potential measurements. The impact of the presence of nanoparticles and their surface chemistry is investigated, and at least three nanoparticle-specific mechanisms are found to effect separations. First, the degree of nanoparticle-nanoparticle interactions is quantified using a new parameter termed the critical nanoparticle concentration (CNC). CNC is defined as the lowest concentration of nanoparticles that induces predominant nanoparticle aggregation under specific buffer conditions and is determined using dual-wavelength photodiode array detection. Once the CNC has been exceeded, reproducible separations are no longer observed. Second, nanoparticle-analyte interactions are dictated by electrostatic interactions which depend on the pK(a) of the analyte and surface charge of the nanoparticle. Finally, nanoparticle-capillary interactions occur in a surface-chemistry-dependent manner. Run buffer viscosity is influenced by the formation of a nanoparticle steady-state pseudostationary phase along the capillary wall. Despite differences in buffer viscosity leading to changes in neurotransmitter mobilities, no significant changes in electroosmotic flow were observed. As a result of these three nanoparticle-specific interactions, Au@NH(2) nanoparticles increase the mobility of the neurotransmitters while a smaller opposite effect is observed for Au@COOH nanoparticles. Understanding nanoparticle behavior in the presence of an electric field will have significant impacts in separation science where nanoparticles can serve to improve either the mobility or detection sensitivity of target molecules.

Aptamer-Modified Micellar Electrokinetic Chromatography for the Enantioseparation of Nucleotides

In this paper, a new aptamer-based capillary electrophoresis (CE) method, which was able to separate the enantiomers of an anionic target (adenosine monophosphate, AMP) displaying the same electrophoretic mobility as that of the oligonucleotidic chiral selector, is reported. The design of the aptamer-modified micellar electrokinetic chromatography (MEKC) mode consisted of nonionic micelles which acted as a pseudostationary phase and a hydrophobic cholesteryl group-tagged aptamer (Chol-Apt) which partitioned into the uncharged micellar phase. Under partial-filling format and suppressed electroosmotic flow conditions, the strong mobility alteration of Chol-Apt permitted AMP enantiomers to pass through the micelle-anchored aptamer zone and promoted the target enantioseparation. The influence of several electrophoretic parameters (such as concentration and nature of the nonionic surfactant, preincubation of the Chol-Apt and surfactant, capillary temperature, and applied voltage) on the AMP enantiomer migration was investigated in order to define the utilization conditions of the aptamer-modified MEKC mode. The chiral resolution, in a single run, of three adenine nucleotides, i.e., AMP, ADP (adenosine diphosphate), and ATP (adenosine triphosphate), was further accomplished using such methodology. This approach demonstrates the possibility to extend the CE applicability of aptamer chiral selectors to potentially any target, without restriction on its charge-to-mass ratio.

Recent developments in capillary EKC based on carbon nanoparticles

This paper provides an overview of the use of carbon nanoparticles (CNPs) as pseudo-stationary phases (PSPs) in EKC. Specifically, it describes the characteristics and properties of the major types of CNPs used as PSPs in EKC separations, namely C(60) fullerenes, carbon nanotubes and covalently modified carbon nanotubes. Based on such properties, a plausible mechanism for the interactions governing EKC separation with these materials is proposed. Also, the most salient uses of CNPs as PSPs are outlined. Finally, CNPs are compared in terms of performance with other well-established types of nanostructures used to enhance selectivity in EKC over the past decade.

Cover Picture: Electrophoresis 1'09

AbstractThis issue is a paper symposium on “CE and CEC Reviews” with 21 comprehensive reviews describing the latest developments in methodologies, applications, stationary phases in CEC, pseudo stationary phases in EKC, detection and sensitivity enhancement approaches, and proteomics and metabolomics.In addition, issue 1 has a “Fast Track” contribution on two dimensional CE using multiple detection points for chiral analysis of native amino acids.

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

This review is an account of the development, utilization, and characterization of polymeric pseudostationary phases (PSPs) for EKC since the previous review in this series in 2007. Improvements in performance by means of new polymeric phases and modifications to previously reported materials are described. The use of EKC and spectroscopic studies to characterize the interactions between polymeric PSPs and analytes is discussed. The application of polymeric PSPs for techniques or approaches to which they provide particular advantages is reviewed. Chief among these is the application of polymeric PSPs for the combination of EKC with MS detection.

Linear Free Energy Relationship Correlation of The Distribution of Solutes Between Water And Cetytrimethylammonium Bromide (CTAB) Micelles

AbstractData have been assembled from the published literature on the water‐to‐micellar Cetyltrimethylammonium Bromide (CTAB) partition coefficient data for more than 60 compounds, and on the Micellar Electrokinetic Chromatographic (MEKC) retention factors for more than 50 compounds measured on a CTAB pseudostationary phase. In total more than 200 experimental data have been compiled. The water‐to‐micellar CTAB partition coefficients and MEKC retention factor data from three separate studies have been combined into a single database and correlated with the Abraham model. The derived correlation described the 200 experimental values to within a standard deviation of 0.175 log units. Also, the previously derived Abraham model correlation for water‐to‐micellar Sodium Dodecylsulfate (SDS) was updated to include MEKC retention factor data from the five recent experimental studies. The new repression correlation for SDS is based on 706 experimental values. Principal component analysis showed that water‐to‐micellar surfactant partition systems are good models for non‐polar and polar narcotic toxicities of organic compounds toward fish, water fleas, and other aquatic organisms.

Genotyping of α-thalassemia deletions using multiplex polymerase chain reactions and gold nanoparticle-filled capillary electrophoresis

A gold nanoparticle-filled capillary electrophoresis method combined with three multiplex polymerase chain reactions (PCRs) was established for simultaneous diagnosis of five common α-thalassemia deletions, including the -α 3.7 deletion, -α 4.2 deletion, Southeast Asian (- - SEA), Filipino (- - FIL) and Thai (- - THAI) deletions. Gold nanoparticles (GNPs) were used as a pseudostationary phase to improve the resolution between DNA fragments in a low-viscosity polymer. To achieve the best CE separation, several parameters were evaluated for optimizing the separation conditions, including the capillary coating, the concentrations of polymer sieving matrix, the sizes and concentrations of GNPs, the buffer concentrations, and the pH. The final CE method for separating a 200-base pair (bp) DNA ladder and α-thalassemia deletions used a DB-17 capillary, 0.6% poly(ethylene oxide) (PEO) prepared in a mixture of GNP 32nm solution and glycine buffer (25 mM, pH 9.0) (80:20, v/v) as the sieving matrix with 1 μM YO-PRO-1 for fluorescence detection; the applied voltage was −10 kV (detector at anode side) and the separation temperature was 25 °C. Under these optimal conditions, 15 DNA fragments with sizes ranging from 0.2 kb to 3.0 kb were resolved within 11.5 min. The RSDs of migration times were less than 2.81%. A total of 21 patients with α-thalassemia deletions were analyzed using this method, and all results showed good agreement with those obtained by gel electrophoresis.

Hydrophobic Interaction Capillary Electrochromatography of Protein Mutants. Use of Lipid-Based Liquid Crystalline Nanoparticles as Pseudostationary Phase

Nanoparticle-based hydrophobic interaction-capillary electrochromatography was utilized for separation of proteins with similar mass-to-charge ratio at neutral pH without organic modifier. Lipid-based liquid crystalline nanoparticles were prepared and used as pseudostationary phase,benefiting from their high biocompatibility, ease of preparation,and suspension stability at high concentrations.Use of laser-induced fluorescence enabled detection at high nanoparticle concentrations. Green fluorescent protein(GFP) and mutants of GFP harboring single or double amino acid substitutions with the same charge were separated in the described system but not in conventional capillary electrophoresis. Separation was achieved by increasing the salt concentration to promote hydrophobic interactions by shielding of the repulsive electrostatic interactions. In addition, the method was adapted to a capillary with an effective length of 6.7 cm, enabling fast separations and future applications on chip.

Sodium cholate aggregation and chiral recognition of the probe molecule (R,S)-1,1'-binaphthyl-2,2'-diylhydrogenphosphate (BNDHP) observed by 1H and 31P NMR spectroscopy.

Bile salt micelles can be employed as a pseudostationary phase in micellar electrokinetic capillary chromatography (MEKC) separations of chiral analytes. To improve MEKC separations of chiral analytes, a molecular level understanding of micelle aggregation in the presence of analyte is needed. Here, aggregation of sodium cholate has been observed by exploiting the presence of a model analyte molecule. The 31P and 1H nuclear magnetic resonance spectroscopy (NMR) chemical shifts of (R,S)-1,1'-binaphthyl-2,2'-diylhydrogenphosphate ((R,S)-BNDHP), a model analyte in chiral MEKC separations, are demonstrated to be very sensitive to the aggregation state of the bile salt sodium cholate. In addition to probing micellar aggregation, the NMR spectral resolution of enantiomeric species is also stronglycorrelated with chiral separations in MEKC. In this work, the aggregation of sodium cholate in basic solutions (pH 12) has been observed over the concentration range 0-100 mM. The primary critical micelle concentration (cmc) was found to be 14 +/- 1 mM for basic solutions of sodium cholate. In addition, a primitive aggregate is clearly observed to form at 7 +/- 1 mM sodium cholate. The data also show pseudo-cmc behavior for secondary aggregation observed in the regime of 50-60 mM cholate. Finally, the H5-H7 edge of BNDHP is shown to be sensitive to chirally selective interactions with primary cholate micelles.

Neutral analyte focusing by micelle collapse in micellar electrokinetic chromatography

The operating parameters that affect the performance of analyte focusing by micelle collapse (AFMC) to neutral analytes (i.e., dialkyl phthalates) in normal migration micellar electrokinetic chromatography (NM-MEKC) are examined. NM-MEKC is characterized by an electroosmotic flow greater than the electrophoretic velocity of the micellar pseudostationary phase, and was performed using sodium dodecyl sulfate (SDS) with neutral to high pH electrolytes in fused silica capillaries. AFMC is a recently introduced on-line sample preconcentration technique in capillary electrophoresis that can provide hundreds fold improvement in detection sensitivity. The mechanism of AFMC is based on the analyte transport, release, and accumulation by the moving surfactant micelles [e.g., SDS at concentrations closer to the critical micelle concentration (CMC)] in the sample that dilutes below its CMC into a liquid phase zone. The sample is prepared in a matrix that contains SDS micelles and high mobility anions, where the conductivity of the sample matrix is higher compared to that of the separation solution. The sample injection length, sample and separation solution conductivity ratio, and surfactant micelle concentration in the sample were found to affect the AFMC performance, as well as the effective separation length in NM-MEKC. The use of a different electrolyte salt in the sample and separation solution also affected AFMC NM-MEKC results. In particular, sodium chloride in the sample matrix can induce a micelle-mediated neutral analyte isotachophoretic concentration that is detrimental to the technique.

Different sample stacking strategies for the determination of ertapenem and its impurities by micellar electrokinetic chromatography in pharmaceutical formulation

Ertapenem, a Group 1 carbapenem, is most recently introduced into the market. It is a β-lactam antibiotic that possesses a broad antibacterial spectrum including common community-acquired Gram-positive and Gram-negative aerobic and anaerobic pathogens, but low activity against some nosocomial pathogens such as Pseudomonas aeruginosa, Acinetobacter spp., enterococci and methicillin-resistant staphylococci. The elaborated method of micellar electrokinetic chromatography (MEKC) of ertapenem separation from its impurities was successfully performed using normal stacking mode (NSM) and stacking with reverse migrating micelles (SRMM), followed by UV absorption detection at 214 nm. The best results were obtained with 60 mM sodium dihydrogen phosphate and 20 mM boric acid buffer pH 6.0, as background electrolyte. Uncoated fused-silica capillary and neutral-coated capillary with normal and reverse polarity, and voltage values of +18 and −12 kV, respectively, were used throughout the investigation. Sodium dodecyl sulfate was employed as the pseudostationary phase. A comparison of applied techniques, including sensitivity enhancement factors and limits of detection (LOD), is presented. The optimized method was validated in terms of linearity, accuracy and precision. Comparable LOD was obtained using both stacking methods (0.3 μg/mL) but better efficiency of ertapenem peak was obtained using NSM. Under the optimum stacking conditions, about 183–4.75-fold and 1289–4.07-fold improvements in peak areas were obtained for NSM and SRMM, respectively, compared to the usual hydrodynamic sample injection (10 s). The reproducibility, expressed by relative standard deviations (RSD) of the migration times, for NSM was about 0.96–1.25 and for SRMM was 0.32–0.45. The RSD of corrected peak areas, for NSM was about 1.07–8.14 and for SRMM was 0.74–8.12. The difference in separation time between the two techniques was not obvious. Satisfactory separation was possible after less than 11 min of electrophoresis. The evaluated MEKC method was applied in the analysis of medicinal product containing ertapenem: Invanz—ertapenem for injection.

Bilayer disk capillary electrophoresis: A novel method to study drug partitioning into membranes

CE in the presence of lipid bilayer disks was introduced as a new approach in membrane partitioning studies. The disks were used as a pseudostationary phase in the partial-filling mode of CE and the partitioning of cationic drugs was determined. The migration times of the analytes increased linearly with the lipid amount in the system. An appropriate algorithm for the calculation of a partition coefficient is presented. In the disk-shaped bilayers, which have excellent stability and shelf life, all of the lipids are readily available for interaction and the disks can be used as realistic cell membrane models.

Antibiotic fusidic acid has strong interactions with negatively charged lipid membranes: An electrokinetic capillary chromatographic study

Fusidic acid (FA), a narrow spectrum steroidal antibiotic, is useful for treatment of most skin, conjunctival, and corneal infections and also in infections caused by atypical microbes in the surface of the eye. Liposome electrokinetic capillary chromatography (LEKC) was used to study the interactions between FA and lipid membranes. Liposomes prepared by extrusion were composed of 1-palmitoyl-2-oleyl- sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleyl- sn-glysero-3-phosphor- l-serine (POPS), cholesterol, FA, and sphingomyelin (SM) in various molar ratios. 26 different liposome dispersions were studied as dispersed (pseudostationary) phase in LEKC. The hydrophobicities of the liposomes were evaluated by calculating the retention factors of model neutral steroids. The retention factors were calculated using the EOF and the effective electrophoretic mobilities of the analytes and the liposomes. The latter were separately determined by capillary electrophoresis with a polyacrylamide (PAA)-coated capillary. FA–lipid membrane interactions were studied by determining the retention factor of FA. In addition, liposomes prepared from lipids extracted from Escherichia coli bacterium were studied and used as dispersed phase in LEKC for interaction studies between FA and lipid membranes.

Linear solvation energy relationships of anionic dimeric surfactants in micellar electrokinetic chromatography: III. Effect of fluorination

The range of selectivities of pseudostationary phases (PSPs) available for micellar electrokinetic chromatography is limited, and there continues to be interest in the synthesis and evaluation of novel PSPs. With the unique selectivity of lithium perfluorooctanesulfonate (LPFOS) in mind, we synthesized anionic dimeric surfactants with fluorinated spacers of various lengths. Their selectivities, analyzed via linear solvation energy relationships, are similar to those of their non-fluorinated analogues, although the results indicated that they are somewhat less polarizable. While it can be concluded that fluorination has some influence on the behaviour of these dimeric PSPs, the influence on the selectivity is not as dramatic as in LPFOS. This can be explained by the inferred poor hydration of the fluorinated spacer and the positioning of the fluorine substituents, too distant from the surfactant head groups to fully exert the strong inductive effect required to significantly influence the head group chemistry.

Separation of very hydrophobic analytes by micellar electrokinetic chromatography: IV. Modeling of the effective electrophoretic mobility from carbon number equivalents and octanol–water partition coefficients

It is investigated whether those relationships derived within an optimization scheme developed previously to optimize separations in micellar electrokinetic chromatography can be used to model effective electrophoretic mobilities of analytes strongly differing in their properties (polarity and type of interaction with the pseudostationary phase). The modeling is based on two parameter sets: (i) carbon number equivalents or octanol–water partition coefficients as analyte descriptors and (ii) four coefficients describing properties of the separation electrolyte (based on retention data for a homologous series of alkyl phenyl ketones used as reference analytes). The applicability of the proposed model is validated comparing experimental and calculated effective electrophoretic mobilities. The results demonstrate that the model can effectively be used to predict effective electrophoretic mobilities of neutral analytes from the determined carbon number equivalents or from octanol–water partition coefficients provided that the solvation parameters of the analytes of interest are similar to those of the reference analytes.

Comparative study of carbon nanotubes and C 60 fullerenes as pseudostationary phases in electrokinetic chromatography

The use of carbon nanostructures in different modalities is a topic of growing interest. This article provides a systematic comparison of surfactant-coated single-wall carbon nanotubes, multi-wall carbon nanotubes and C 60 fullerenes as pseudostationary phases (PSPs) in electrokinetic chromatography. The differences on the electrophoretic behaviour as a function of the pseudostationary phase for phenolic compounds, triazines and nitroimidazole derivatives have been explored. Phenolic compounds, triazines and nitroimidazole derivatives has been selected as model compounds of aromatic compounds, π-exceeding heteroaromatic compounds and π-deficient heteroaromatic compounds, respectively. Resolution was also evaluated on the basis of the differences in the electrophoretic behaviour and in this sense the best PSP for each group of compounds can be proposed.

Quantitative Retention‐Activity Relationship Studies by Liposome Electrokinetic Chromatography to Predict Skin Permeability

AbstractLiposome electrokinetic chromatography (LEKC) provides a simple and facile approach for drug membrane interactions using liposome as a pseudostationary phase. This study evaluated the potential of LEKC for high‐throughput skin permeability profiled as an in vitro technique. A quantitative retention‐activity relationship (QRAR) model for the estimation of skin permeability was proposed. For the 16 structurally diverse chemicals, lg k correlated well with permeability values (R2=0.886). The predictive ability of the model was evaluated by cross‐validation. The result was compared to traditional quantitative structure‐activity relationship, QSAR, models using some molecular descriptors and physicochemical parameters. Interestingly, a single LEKC retention parameter was capable of describing the skin permeability, while three variables in QSAR were needed to achieve a similar correlation (R2=0.704). The QRAR models developed in this paper may be a useful method to screening new chemicals and in the early stage of development and selection of chemicals.

Separation of nonionic compounds by electrokinetic chromatography using an inorganic layered compound as a pseudostationary phase

The use of an inorganic layered compound as a pseudostationary phase (PSP) in EKC was investigated. A synthetic smectite, which is the most typical swellable clay mineral, with an average diameter of 130 nm was selected as the PSP. The retention characteristics of the smectite and on-line sample concentration by sweeping were examined for the analysis of polyoxyethylene mono phenyl ethers (PPEs) with different degrees of ethoxylation. The retention factor was increased with increase in the number of ethylene oxide groups and a good separation of the PPE homologs was achieved by smectite-EKC. The RSD of the migration time, plate number, and peak area were 0.60, 8.3, and 2.7% (n = 5), respectively. The developed method can be applied to the analysis of PPEs in commercially available consumer products without any sample pretreatments. In addition, ca. 100-fold sensitivity enhancements for the PPEs with high retention factors were obtained by sweeping.

Electrokinetic chromatography with polymeric pseudostationary phases

Recent research and development efforts concerning polymeric pseudostationary phases (PSPs) for electrokinetic chromatography are reviewed. The introduction of new materials, characterization of structural effects on performance and selectivity, applications, and the use of polymeric PSPs with mass spectrometric detection are considered. Very interesting results concerning the effects of polymer structure have been reported. Significant developments have also been reported in the development of novel applications of polymeric PSPs, particularly for sample preconcentration using micellar affinity gradient focusing. The use of mass spectrometric detection with electrokinetic chromatography has seen significant development, and recent reports indicate that this is a robust and sensitive approach.

Compositional effects on electrophoretic and chromatographic figures of merit in electrokinetic chromatography with cetyltrimethylammonium bromide/sodium octyl sulfate vesicles as the pseudostationary phase. Part 1: Effect of the phase ratio

The effect of the phase ratio on the electrophoretic and chromatographic properties of unilamellar vesicles comprised of cetyltrimethylammonium bromide (CTAB) and sodium octyl sulfate (SOS) was investigated in EKC. The surfactant concentration of the vesicles was 0.9, 1.2, 1.5, and 1.8% w/v, with a mole ratio of 1:3.66 (CTAB/SOS). Results were compared to those obtained using SDS micelles at concentrations of 1.0% (w/v, 35 mM) and 1.5% (52 mM). The CTAB/SOS vesicles (0.9-1.8% w/v) provided a significantly larger elution range (5.7 < or = t(ves)/t(0) < or = 8.7) and greater hydrophobic (methylene) selectivity (2.8 < or = alpha(CH2) < or = 3.1) than SDS micelles (3.1 < or = t(mc)/t(0) < or = 3.3; alpha(CH2) = 2.2). Whereas the larger elution range can be attributed to the 25% reduction in EOF due to the interaction of unaggregated CTAB cations and the negatively charged capillary wall, the higher methylene selectivity is likely due to the lower concentration of water expected in the CTAB/SOS vesicle bilayer compared to the Palisades layer of SDS micelles. For a given phase ratio, CTAB/SOS vesicles are somewhat less retentive than SDS micelles, although retention factors comparable to those observed in 1.0-1.5% SDS can be obtained with 1.5-1.8% CTAB/SOS. A linear relationship was observed between phase ratio and retention factor, confirming the validity of the phase ratio model for these vesicles. Unique polar group selectivities and positional isomer shape selectivities were obtained with CTAB/SOS vesicles, with both types of selectivities being nearly independent of the phase ratio. For four sets of positional isomers, the elution order was always para < ortho < meta. Finally, the thermodynamics of solute retention was qualitatively similar to that reported for other surfactant aggregates (micelles and microemulsions); the enthalpic contribution to retention was consistently favorable for all compounds, whereas the entropic contribution was favorable only to hydrophobic solutes.