Pseudostationary Phase Research Articles

Michrochip chromatography using an open‐tubular microchannel and a ternary water–ACN–ethyl acetate mixture carrier solution

A capillary chromatography system has been developed using a ternary mixed-solvents solution, i.e. water-hydrophilic/hydrophobic organic solvent mixture as a carrier solution. Here, we tried to carry out the chromatographic system on a microchip incorporating the open-tubular microchannels. A model analyte solution of isoluminol isothiocyanate (ILITC) and ILITC-labeled biomolecule was injected to the double T-junction part on the microchip. The analyte solution was delivered in the separation microchannel (40 μm deep, 100 μm wide, and 22 cm long) with the ternary water-ACN-ethyl acetate mixture carrier solution (3:8:4 volume ratio, the organic solvent rich or 15:3:2 volume ratio, the water-rich). The analyte, free-ILITC and labeled BSA mixture, was separated through the microchannel, where the carrier solvents were radially distributed in the separation channel generating inner and outer phases. The outer phase acts as a pseudo-stationary phase under laminar flow conditions in the system. The ILITC and the labeled BSA were eluted and detected with chemiluminescence reaction.

Application of molecularly imprinted polymer particles in capillary electrochromatography

Molecularly imprinted polymer (MIP) particles prepared by molecular imprinting technique (MIT) possess the ability of specific identification and selective affinity towards template molecules and their structural analogues. They also have a large specific surface area and rapid mass transfer kinetics, thus they have been widely used as stationary phases in liquid chromatography and matrixes in solid phase extraction. Using MIP particles as capillary electrochromatography (CEC) stationary phase, which combines the high speed and efficiency of CEC with the high affinity and selectivity of MIP, has become one of the most promising separation technique in analytical science. There are several different strategies of MIP particles applied in CEC: as packing materials packed into a capillary column; as entrapping materials entrapped into different matrix frameworks inside a capillary column; as pseudostationary phases (PSPs) added into the running buffer of CEC. This review focuses on the recent developments of MIP particles in CEC and prospects the future developments in this field.

Separation of DNA Fragments by Capillary Electrophoresis Using a Mixture of Poly-β-CD and Poly(ethylene oxide) as a New Sieving Matrix

In this study, we present the first example of the separation of DNA by capillary electrophoresis (CE) using a new sieving matrix, a mixture of the poly-β-cyclodextrin (poly-β-CD) and poly(ethylene oxide) (PEO). The poly-β-CD and PEO were used as a pseudostationary phase to improve the resolution of DNA fragments. CE separation was performed using a DB-17 coating capillary at an applied voltage of −10 kV (detector at anode side). Ammonium acetate buffer (25 mmol L−1, pH 6.0) was used as a background electrolyte and all sample solutions were prepared by the buffer. The best CE separation was obtained at a concentration of 5 mg mL−1 poly-β-CD and 1 mg mL−1 PEO. Under these conditions, 13 DNA fragments with sizes ranging from 200 bp to 4000 bp were completely resolved within 12 min, and the peak efficiencies ranged from 91000 to 234000 plates m−1. The results presented in this study show the advantage of PEO containing poly-β-CD for DNA separation, including rapidity, high resolving power, and ease of analysis.

Capillary electromigration techniques for studying interactions between analytes and lipid dispersions

CE has matured into a well-known and much used separation technique, with applications covering more or less all types of samples. EKC, which originally was developed for the separation of uncharged compounds, is still today under much development, with main focus on finding the perfect or ideal carriers (pseudo-stationary phase) for a broad range of analytes. In this review, the use of lipid dispersions as pseudostationary phases in EKC performed in capillaries and microchips, in addition to CE frontal analysis and partial filling EKC using lipid dispersions is discussed. Various types of lipid dispersions including liposomes, PEG-stabilized aggregates, proteoliposomes, lipid-based nanoparticles, and commercial lipid emulsions are described. The purpose of the review is to give the reader an overview of how EKC, CE frontal analysis, and partial filling EKC, have been applied to the study of interactions between analytes and lipid membranes.

Chromatography Using Ternary Water–Acetonitrile–Ethyl Acetate Mixture as a Carrier Solution on a Microchip Incorporating Microchannels

Abstract An open-tubular capillary chromatography system has been developed using a ternary mixed solvent solution, i.e., water–hydrophilic/hydrophobic organic solvent mixture as a carrier solution. The system is called “tube radial distribution chromatography (TRDC).” In this study, we tried to carry out the TRDC on a microchip incorporating microchannels. A model analyte solution of isoluminol isothiocyanate (ILITC) and ILITC-labeled bovine serum albumin (BSA) was injected to the double T-junction part (0.5 mm length, ca. 2 nL) on the microchip. The analyte solution was then delivered in the separation microchannel (40 µm in depth, 100 µm in width, and 22 cm in length) with a ternary water–acetonitrile–ethyl acetate mixture carrier solution (3:8:4, volume ratio). The model analytes, the ILITC and the labeled BSA were separated through the microchannel, where the carrier solvents were radially distributed in the separation channel generating inner and outer phases. This phenomenon of the solvents is called “tube radial distribution phenomenon (TRDP).” The outer phase functions as a pseudo-stationary phase under laminar flow conditions in the chromatography, i.e., TRDC. Finally, the ILITC and the labeled BSA were eluted in this order and detected with chemiluminescence reaction.

Comments on: A Comparative Study of Diamine-Modified and Amine-Modified Silica Nanoparticles as Pseudo-Stationary Phases for CE Separation by Li-Juan Hui and Yong Ma

Comments on: A Comparative Study of Diamine-Modified and Amine-Modified Silica Nanoparticles as Pseudo-Stationary Phases for CE Separation by Li-Juan Hui and Yong Ma

Processes involved in sweeping under inhomogeneous electric field conditions as sample enrichment procedure in micellar electrokinetic chromatography

Sweeping under inhomogeneous electric field conditions has been described as a process that includes stacking or destacking of the micelles when entering the sample zone, sweeping of analytes by the stacked or destacked micelles, and destacking or stacking of the swept analyte zone. However, there is ongoing debate that not only the retention factor of the analyte but also the electric conductivity of the sample solution or the concentration of an added salt can have an impact on the enrichment efficiency. Revisiting the equations describing sweeping, a factor θ (phase ratio shift factor) is defined to quantitatively describe the change of the retention factor between the sample and separation zones. The influence of the sample matrix composition on the experimentally obtained sweeping efficiency is studied with SDS as pseudostationary phase taking parabens, benzamide and anilines as model analytes. To this end, a robust and reliable method for the assessment of the sweeping efficiency is developed. The values obtained via this method are very precise and agree well with theoretically predicted ones. The results obtained for varied buffer concentration and varied concentration of NaCl in the sample solution show that under the conditions of our experimental study, the approximation of assuming θ to be equal to the reciprocal value of the field strength enhancement factor γ is valid. Accordingly, the sweeping efficiency for neutral analytes is independent of the electric conductivity of the sample matrix. It is also shown that under specific conditions unexpectedly high enrichment factors are obtained which are ascribed to the focusing of neutral analytes by micellar transient isotachophoresis (mtITP). The results obtained in this study can be used as a guide for better understanding of the sweeping process and the factors affecting the sweeping efficiency in micellar electrokinetic chromatography (MEKC).

Metal‐organic framework ZIF‐8 nanocrystals as pseudostationary phase for capillary electrokinetic chromatography

The outstanding properties such as large surface area, diverse structure, and accessible tunnels and cages make metal organic frameworks (MOFs) attractive as novel separation media in separation sciences. However, the utilization of MOFs in EKC has not been reported before. Here we show the exploration of zeolitic imidazolate framework-8 (ZIF-8), one of famous MOFs, as the pseudostationary phase (PSP) in EKC. ZIF-8 nanocrystals were used as the PSP through dispersing in the running buffer (20 mM phosphate solution containing a 1% v/v methanol (pH 9.2)) to enhance the separation of the phenolic isomers (p-benzenediol, m-benzenediol, o-benzenediol, m-nitrophenol, p-nitrophenol, and o-nitrophenol). ZIF-8 nanocrystals in the running buffer were negatively charged, and interacted with the phenolic hydroxyl groups of the analytes, and thus greatly improved the separation of the phenolic isomers. Inclusion of 200 mg L-(1) ZIF-8 in the running buffer as the background electrolyte gave a baseline separation of the phenolic isomers within 4 min. The relative standard deviations for five replicate separations of the phenolic isomers were 0.2-1.1% for migration time and 4.5-9.7% for peak area. The limits of detection varied from 0.44 to 2.0 mg L-(1) . The results show that nanosized MOFs are promising for application in EKC.

Investigations into Tie Lines and Solubility Curves on Phase Diagrams in Open-Tubular Capillary Chromatography Using Ternary Mixed-Carrier Solvents

Open-tubular capillary chromatography using a ternary solvent mixture consisting of a water-hydrophilic-hydrophobic organic solvent as a carrier solution has been developed. When the ternary carrier solution is fed into the capillary tube, the carrier solvents are radially distributed and generate inner and outer phases in the tube. The outer phase functions as a pseudo-stationary phase in chromatography. In this study, investigations proceeded with reference to the tie lines and solubility curves on the phase diagram of the ternary mixed solvents. Model analytes, 1-naphthol and 2,6-naphthalenedisulfonic acid, were separated in this order with ternary water-acetonitrile-ethyl acetate solvent mixtures (organic solvent-rich solutions) that possessed various solvent compositions on the tie lines. In addition, fluorescence photographs of the dyes dissolved in the ternary solvents in the capillary tubes were observed with a fluorescence microscope-CCD camera system. It was found that the separation performance on the chromatograms and the phase formation observed in the fluorescence photographs were related to data provided through the tie lines and solubility curves on the phase diagram. The solvent compositions on the same tie line that gave different volume ratios of upper and lower phases in a vessel influenced the chromatographic separation, or the resolutions of the analytes, and also the inner and outer phase formation in the chromatography.

Phosphonium-based ionic liquids in electrokinetic capillary chromatography for the separation of neutral analytes

In this work we elucidated the applicability of phosphonium-based ionic liquids (ILs) as pseudostationary phase in electrokinetic capillary chromatography (EKC) with UV-detection. The phosphonium ILs studied contain bromide, chlorine, or tosylate ions, as counter ions, and alkyl side chains of variable length on the phosphorous atom. The effects of the type and concentration of the IL, pH, ionic strength, and type of background electrolyte solution on the electroosmotic flow (EOF) and on the effective electrophoretic mobilities of some neutral model analytes were investigated and large variations in the migration times were observed. Especially the IL employed remarkably affected the strength and direction of the EOF Successful separations were obtained for neutral aromatic singly substituted analytes, namely benzene, toluene, phenol, and nitrobenzene. The results demonstrated the potential of capillary electromigration methods for rapid interaction studies between ILs and analytes, which is useful for the development of novel materials for sample preparation and separation purposes or for novel catalyst and chemical processing studies.

Diamino moiety functionalized silica nanoparticles as pseudostationary phase in capillary electrochromatography separation of plant auxins

A novel and simple method for the preparation of silica nanoparticles having surface-functionalized diamino moiety (dASNPs) was reported in our paper and characterized using scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectrometry, and thermogravimetry techniques. To test this method practically, in this contribution we describe the enhanced separation of four plant auxins - indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (dCPAA), and 2-(1-naphthyl) acetic acid (NAA) - by capillary electrochromatography using diamino moiety functionalized silica nanoparticles as pseudostationary phase (PSP) in the running buffer. The effect of pH, buffer concentration, and diamino moiety functionalized silica nanoparticles concentration on the selectivity of separation was investigated. A combination of the nanoparticles and running buffer reversed the electroosmotic direction making possible the rapid and efficient separation of the auxins from the auxins migrated in the same direction with the EOF under optimum experimental conditions. A good resolution of four auxins was obtained within 5.5 min under optimum experimental conditions. The precision (RSD, n = 5) was in the range of 0.72-0.91% and 1.89-2.23% for migration time and peak area response, respectively. The detection limits were 0.48, 0.44, 0.46, and 0.42 μM for NAA, IBA, IAA, and dCPAA, respectively. Furthermore, the method was successfully tested for the determination of IAA in the grapes.

Characterization and application of molecular binary mixed molecular micelles of sodium 10-undecenyl sulfate and sodium N-undecenyl leucinate as pseudostationary phases in micellar electrokinetic chromatography

Poly (sodium 10-undecenyl sulfate) (poly SUS), poly (sodium 10-undecenyl leucinate) (poly SUL), and their five molecular binary mixed micelles with varied SUS:SUL composition were prepared. The purity of these molecular micelles was confirmed by elemental analysis. Their partial specific volume, aggregation number, methylene selectivity, polarity, phase ratio, mobility, and elution window values were determined using a variety of analytical techniques. These molecular micelles were then evaluated as pseudostationary phases in micellar electrokinetic chromatography (MEKC) for separation of benzene derivatives with a wide range of chemical properties. Elemental analysis results reveal that the ratio of the two surfactants in the binary mixture does not change significantly during the polymerization process. Poly SUS was found to have the lowest partial specific volume and it increases gradually with an increase of SUL mole fraction. Poly SUL was found to provide the most hydrophobic environment for test solutes. Based on the retention results, the strength of interaction between the molecular micelles and the analytes was found to follow the following order: NHB>HBA>HBD. This order indicates that the hydrophobic interaction plays a major role in retention of benzene derivatives.

Polymeric micelle as the pseudostationary phase in electrokinetic chromatography

A simple, green, and novel approach to prepare polymeric micelle with amphiphilic random copolymer P (MMA-co-MAA) via neutralization in aqueous medium has been developed, and the polymeric micelle was firstly applied as a pseudostationary phase (PSP) in electrokinetic chromatography (EKC) in the present work. Three structurally similar corticosteroids namely hydrocortisone, prednisolone, and prednisone were separated with EKC using polymeric micelle as PSP to assess the separation performance. The effects of polymeric concentration and pH on micellar microstructure including size, morphology, surface charge density and EKC performances have been investigated. TEM showed that amphiphilic random copolymers were self-assembled via neutralization to form micelles with well-defined size and shape. The size and shape of the micelle depended on the P (MMA-co-MAA) concentration and pH. At the concentration of 0.048mM and pH 9.2, the polymeric micelles were of monodispersity and perfect spheres. DLS showed the size of micelle was almost equal as polymer concentration in the range of 0.0096–0.048mM, and then enlarged sharply at the concentration larger than 0.048mM. However, the zeta potentials of micelle were nearly unchanged. The polymer concentration is also the key parameter for EKC separation. Under the optimum conditions, three analytes could be baseline separated within 7.4min. Compared with typical MEKC, MEEKC, and MEKC modified with IL ([Bmim]BF4), the developed method was more rapid, efficient, and higher selective. The separation mechanism using polymeric micelle as PSP was reverse-phase interaction. The actual cosmetic samples were analyzed with recoveries between 97.3% and 113%.

Proteoliposome-Based Capillary Electrophoresis for Screening Membrane Protein Inhibitors

A method for screening of monoamine oxidase (MAO) inhibitor was carried out using capillary electrophoresis (CE) based on the interaction of MAO and its substrate kynuramine (Kyn). Bioactive proteoliposome was reconstituted by liposome and MAO and then was applied as the pseudostationary phase (PSP) of CE to mimic the interaction between the enzyme and its substrate. N-prolmrgyl-R-2-heptylamine (R-2-HPA) and rasagiline [N-propargyl-1-(R)-aminoindan], which are two kinds of MAO inhibitors, were added into the running buffers containing proteoliposome. The results showed that the relative migration time ratio (RMTR × 10(-1)) values of Kyn were enhanced from 8.88 to 9.31 with an increase of the concentrations of rasagiline from 10(-6) to 1 mM. However, the RMTR values of Kyn were enhanced from 8.83 to 9.14 with an increase of the concentrations of R-2-HPA from 10(-6) to 1 mM. The RMTR value of Kyn in the presence of rasagiline was larger than that in the presence of R-2-HPA when rasagiline and R-2-HPA were at the same concentration. The results indicated that the interaction between Kyn and MAO was weakened with the increase of the inhibitors. In addition, the results of offline incubation showed that the inhibitions of rasagiline were 100.0, 72.1, 51.8 and 5.4% at the concentration of 1, 10(-2), 10(-4) and 10(-6) mM; moreover, the inhibitions of R-2-HPA were 70.0, 44.9, 4.1 and 0.9% at the concentrations of 1, 10(-2), 10(-4) and 10(-6) mM. The inhibition efficiency of rasagiline was stronger than that of R-2-HPA at the same concentration. Additionally, the interaction between Kyn and liposome was also investigated. This newly developed method might provide a potential tool for screening MAO inhibitor.

Thermodynamics of Antimicrobial Peptide JCpep8 Binding to Living Staphylococcus aureus as a Pseudo-stationary Phase in Capillary Electrochromatography and Consequences for Antimicrobial Activity

To understand the details of the permeation pathways of antimicrobial peptide JCpep8, the antimicrobial processes were investigated step by step in this paper. First, the characterization of the initial binding process was explored by introducing the living Staphylococcus aureus cells (LSACs) into electrophoretic buffer used as pseudo-stationary phase in capillary electrochromatography (CEC), and the thermodynamic parameters were determined. The binding constants at 298, 303, and 309 K were 7.40 × 10¹¹, 1.43 × 10¹², and 2.6 × 10¹² M⁻¹, respectively, which indicated the evident interaction between JCpep8 and LSACs. This binding process was spontaneous. Both the electrostatic force and hydrophobic effect play major roles in this binding process. Second, antibacterial activity kinetics and outer membrane and inner membrane disruption assays were investigated. Data indicated that JCpep8 killed microbes principally by breaking their cell wall and membrane, followed by cell lysis. The results were confirmed by Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM). In summary, JCpep8 kills microbes mainly by wall-/membrane-targeting pore-forming mechanisms.

Study of chemical selectivity of molecular binary mixed micelles of sodium 10-undecenyl sulfate and sodium N-undecenyl leucinate using linear solvation energy relationships model

Poly(sodium 10-undecenyl sulfate) (poly-SUS), poly(sodium N-undecenyl leucinate) (poly-SUL) and their five molecular binary mixed micelles with varied SUS:SUL composition were prepared and used as pseudostationary phases in micellar electrokinetic chromatography (MEKC). Linear solvation energy relationships (LSERs) model and free energy of transfer studies were used to characterize the retention behavior and the selectivity differences among the seven surfactant systems. System constant differences and regression models for varied benzene derivative compounds are used to establish the selectivity differences of the seven pseudostationary phases. The cavity formation and dispersion interaction (the v system constant) and the hydrogen-bonding acidity (the b system constant) of the surfactant systems were found to have the most significant influence on selectivity and MEKC retention. The molecular micelle with sulfate head group, poly-SUS, was found to be more hydrogen-bond acidic than the molecular micelle with leucinate head group, poly-SUL. The other system constants (a, s and e) have modest effect on the retention and selectivity of the benzene derivatives. The model intercept coefficients (c system constants), which are negative for all surfactant systems have unusually large values. The free energy changes of transfer for the functional groups studied have all negative values except phenol and benzyl alcohol. Selectivity differences between pseudostationary phases were also compared by plotting the log k values against each other and were found to agree well with LSER results.

Separation of dansyl-dl-amino acids by open tubular capillary chromatography based on tube radial distribution phenomenon of the ternary mixed carrier solvents

Separation of enantiomers, dansyl-DL-amino acids, was carried out by open-tubular capillary chromatography based on the tube radial distribution of the carrier solvents. An untreated poly(tetrafluoroethylene) capillary tube (100 μm inner diameter and 90 cm effective length) as a separation column and a water–acetonitrile–ethyl acetate mixture containing cyclodextrin as a carrier solution were used in the chromatography. An analyte solution of dansyl-DL-amino acids, such as dansyl-DL-methionine, was injected into the capillary tube by a gravity method. The analyte solution was subsequently delivered through the capillary tube with the carrier solution by a microsyringe pump. The ternary mixed carrier solution (water-rich carrier solution) was radially distributed in the capillary tube based on the tube radial distribution phenomenon, causing the formation of inner (water-rich) and outer (organic solvent-rich) phases. The outer or capillary wall phase acted as a pseudo-stationary phase in the chromatography. The analytes were separated through the capillary tube with on-capillary detection by an absorption or a fluorescence detector. The D-enantiomer and the L-enantiomer were eluted in this order with a baseline separation. The separation mechanism of the enantiomers in the open-tubular capillary using cyclodextrin was discussed.

Dual polarization interferometric and capillary electrophoretic analysis of supported lipid bilayer constructed on silica-based surface: Evaluation of its anti-protein adsorption effect

Supported lipid bilayer (SLB) has been demonstrated as a model of cell membranes with prospective bioanalytical or biotechnological applications. In this study, the formation of SLB and their potential biofunctionality against protein adsorption were investigated by Dual Polarization Interferometry (DPI) and Capillary Electrophoresis (CE). DPI studies on different formulations of double-chained, zwitterionic phospholipidlipids, allow the process of bilayer formation to be followed in situ and in real time. Furthermore the anti-protein adsorption effect provided by the various formulated SLBs was examined by DPI. In addition, the SLB coatings of the same lipid formulations were subsequently employed in CE experiments as a pseudo-stationary phase for demonstrating more efficient separation of alkaline protein standard mixtures. SLB-assisted CE was found to be capable of separating 4 alkaline proteins (protonated at neutral pH). This study demonstrates the applicability of DPI to monitor the process of SLB formation; and our findings, obtained by both DPI and CE, confirm that the presence of the SLB reduced drastically the problematic interactions between cationic, alkaline proteins and the negatively charged silica capillary wall, leading to better recovery and efficient separation of the proteins under investigation.

Ionic liquids coated multi-walled carbon nanotubes as a novel pseudostationary phase in electrokinetic chromatography

A new CE system using ionic liquids coated multi-walled carbon nanotubes (ILs-MWNTs) as pseudostationary phase was developed for the simultaneous determination of four flavonoids, four phenolic acids and two saponins. Several parameters affecting the separation were studied, including the choice of ILs, ILs-MWNTs concentration, the respective use of ILs and MWNTs, buffer pH, SDS concentration and borate content. Results revealed that the addition of ILs-MWNTs in running electrolytes enhanced the separation of target compounds compared to conventional micelle because the surface of carbon nanotubes interacted favorably with the analytes. Under the optimum conditions, a baseline separation was achieved for these analytes within 11 min in a 41.5 cm of effective length fused-silica capillary. At a voltage of 28.0 kV, the separation was carried out in a 10mM borate buffer (pH 9.0) containing 100mM SDS, 6% propanol and 4 μg mL(-1) ILs-MWNTs. All calibration curves showed good linearity (r(2)>0.9990) within the test ranges. The intra- and inter-day precisions as determined from standard solutions were below 3.30% and 6.23%, respectively. The recoveries for ten compounds were found to range from 85.5 to 101.8%. The method was successfully applied for the determination of three types of compounds in Qishenyiqi dropping pills. Our experimental results indicated that the proposed method offered new opportunities for the analysis of complex samples.

Evaluation of the separation mechanism of electrokinetic chromatography with a microemulsion and cyclodextrins using NMR and molecular modeling

Electrokinetic chromatography (EKC) allows the separation of closely related substances by the detection of fine effects in analyte-separation system interactions. With the goal of understanding the fine effects involved in separation using a dual cyclodextrin-microemulsion EKC system, an integrated study of NMR and molecular modeling was carried out. The above dual cyclodextrin-microemulsion system was previously used in the separation of clemastine and its related substances and was prepared by the addition of methyl-β-cyclodextrin (MβCD) and heptakis(2,6-di-O-methyl)-β-cyclodextrin (DMβCD) to an oil-in-water microemulsion. The use of DMβCD was shown to be essential in the separation of clemastine from one of its related substance (I(B) ). A molecular modeling study allowed the different affinities of clemastine and I(B) for the two cyclodextrins to be explained. Furthermore, rotating-frame Overhauser effect spectroscopy NMR experiments clearly indicated that besides the primary pseudostationary phase, namely the ionic microemulsion, cyclodextrins acted as a secondary pseudostationary phase. In addition, it was shown that inclusion complexation of sodium dodecyl sulfate (SDS) monomers into the cyclodextrins cavity occurs; differently, the oil (n-heptane) used in the preparation of microemulsion system resulted to be not included into the macrocycle cavity. These experimental results were supported by molecular modeling, which highlighted the preferential inclusion of SDS into DMβCD. On the basis of these results, it was confirmed that, besides its primary role as the ionic carrier in EKC, SDS is involved in inclusion equilibria toward CDs, which can be effective in increasing the system selectivity.