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Peer Reviewed: Micellar Electrokinetic Chromatography
Abstract
MEKC belongs to a mode of CE but also to micro-LC
... The addition of ionic surfactants above the CMC has proven to be an effective technique for electrokinetic separation of neutral compounds in capillary electrophoresis. [15][16][17][18] In MEKC, micelles act as a pseudostationary phase, into which analytes partition based on their affinity for the functional groups comprising the core (e.g., through hydrophobic or pi-pi interactions). In this scenario, compounds that are not ionic under experimental conditions can be incorporated into the micelle and then electrophoretically separated by virtue of the charge on the micelles. ...
... Most relevant to the work reported here is that MEKC has been utilized for sample preconcentration by field-induced sample stacking and sweeping. [16] ...
... When BODIPY concentration surpasses the micelle concentration, the neutral species saturate the micelles, resulting in a sharp decrease in extraction efficiency(Figure 3b,c).Figure 3cshows extraction efficiency as a function of neutral species concentration. These results clearly demonstrate the limitation of CMEKF at high guest-host ratios and follow the previous finding in MEKC that the capacity of the extraction is directly proportional to the micelle concentration and the solubilization number.[16] ...
Ion concentration polarization (ICP) has been broadly applied to accomplish electrokinetic focusing of charged species. However, ICP-based separation and enrichment of uncharged (neutral) compounds, important for pharmaceutical, biological, and environmental applications, has not yet been reported. Here, we report the ICP-based continuous extraction of two neutral compounds from aqueous solution, by their partition into an ionic micellar phase. Our initial results show that the efficiency of the separation increases with the concentration of the surfactant comprising the micellar phase, reaching >99%, and drops precipitously when the concentration of the target compound exceeds the capacity of the micelles. As a key feature relevant to the practical application of this method, we show that focusing occurs even an order of magnitude below the critical micelle concentration through the local enrichment and assembly of surfactants into micelles, thus minimizing their consumption. To underscore the relevance of this approach to water purification, this method is applied to the extraction of pyrene, a model for polyaromatic hydrocarbons. This approach provides access to a broad range of strategies for selective separation that have been developed in micellar electrokinetic chromatography.
... According to Terabe et al. [63,64], it is required to exceed a certain concentration of surfactant molecules for self-aggregation. This minimum concentration of the surfactant is called critical micelle concentration (CMC). ...
... Aggregation Number and CMC of Selected Surfactants[64] ...
Hydrogen is considered as an alternative, clean and sustainable energy carrier for reducing the usage of global fossil fuel in large-scale process industries. However, the existing large-scale separation and purification of H2 for these industries requires large capital investment as well as large production cost. In this regard, Pd- and its alloy based membranes have the potential to play an important role in separation and purification of hydrogen. The dense Pd-Ag composite membranes are particularly attractive for the superior performance in terms of perm-selectivity and stability compared to pure Pd membranes of similar thickness. In this work, dense Pd-Ag composite membranes on microporous stainless steel (MPSS) substrates were fabricated by sequential deposition of Pd and Ag using the surfactant induced electroless plating (SIEP) process, a modified electroless plating process (EP). A cationic surfactant, dodecyl trimethyl ammonium bromide (DTAB), is used to provide a controlled grain-size distribution and agglomeration process. In this work, 4xCMC and 1xCMC of DTAB were used in Pd- and Ag- bath, respectively. It gave an average particle size in the range of 0.6 – 1.3 μm. Prior to the metal deposition, the MPSS substrates were oxidized at 600 °C for 12 hrs to create an oxide layer as an intermetallic diffusion barrier. A perm-selectivity study was performed at temperature and pressure ranges from 250 – 450 °C and 20 – 100 psig, respectively. The fabricated membrane by SIEP method was also tested for long term (42 days) hydrogen permeation measurements at temperatures cycled between 350 °C and 450 °C. These membranes were characterized by SEM, XRD and EDS to study physical and morphological features before and after stability tests.
... The wider the window, the larger is the peak capacity, which is the number of peaks that can be separated during a run. Migration time can be measured by using markers such as methanol for EOF and dodecanophenone for the micelle [5][6][7][8]. ...
... General chromatographic parameters can be employed to describe the migration parameters of the analytes in MEKC [5,18]. ...
owing to low reagent consumption, high resolution and efficiency as well as short analysis times. In CE, the chiral analytes (positively or negatively charged) can be separated based on their electrophoretic mobility differences. However, owing to the lack of electrical charge, neutral chiral molecules cannot be separated by CE. In order to solve this problem, charged chiral surfactants above their critical micelle concentration (CMC) are used in the CE running buffer, which allows the separation of uncharged molecules along with the charged ones. Chiral surfactants in general are comprised of a hydrophobic portion, usually a long alkyl chain, attached to hydrophilic or water-soluble enhancing functional groups. Chiral surfactants can be categorized according to the charge present in the hydrophilic portion of the molecule (after dissociation in aqueous solution): (1) anionic surfactants; (2) cationic surfactants; (3) nonionic surfactants; and (4) zwitterionic surfactants. Anionic surfactants have been more extensively used in chiral separations than any other surfactants, because of their very high success rate as chiral selectors and ease of synthesis. Unlike cationic surfactants (which interact with the negatively charged capillary wall), anionic surfactants do not interact with the capillary wall and hence do not lose the site of chiral interaction with the racemic analytes. Conventional surfactants form a pseudostationary phase in a solution-filled capillary column at a concentration above their CMC, with which molecules can partition. The micelles are made of aggregates of surfactant monomers. Owing to different solutes partitioning with the micelles, separation of solutes take place. This micelle-modified CE was termed micellar electrokinetic chromatography (MEKC) by Terabe et al.
... Atorvastatin and related substances 28 Introduction 29 Cholesterol-lowering statin drugs are 30 among most frequently prescribed agents 31 [1]. Statins can be grouped into naturally 32 derived and chemically synthesized. ...
... Even though CE is a rather good 106 alternative to the well-established and 107 widely used LC technique for evaluation 108 of impurity profile and enantiomeric 109 purity of a drug [28,29], it is seldom 110 applied. Micellar electrokinetic capillary 111 chromatographic (MEKC) is the most 112 appropriate electrophoretic technique 113 for impurity profiling because neutral 114 compounds and charged components 115 that have similar electrophoretic mobil-116 ities can be separated simultaneously 117 [30]. Separation of the lipid-lowering 118 drug pravastatin from its degradation 119 products was achieved by MEKC [31]. ...
A new micellar electrokinetic capillary chromatographic (MEKC) method has been developed for simultaneous quantitation of
atorvastatin (AT) and its related substances. The separation was carried out in an extended light path capillary at applied
voltage of 30kV using a background electrolyte consisting of 10mM sodium tetraborate buffer pH 9.5, 50mM sodium dodecyl
sulphate and 20% (v/v) methanol. The addition of methanol to the running buffer resulted in a very effective choice to achieve resolution between
the peaks of charged substances adjacent to AT as well as the peaks of neutral drug-related substances. Linear calibration
curves were established over the concentration range 100–1,200μgmL−1 for AT and 1.0–12.5μgmL−1 for related substances. The proposed MEKC procedure has been validated with respect to selectivity, precision, linearity,
limits of detection, and quantitation, accuracy and robustness. The method has been successfully applied to the determination
of AT and purity evaluation of bulk drug and formulated products.
... Several well-established chromatographic techniques relying on the use of electric fields have been developed, including electrochemically modulated liquid chromatography [24,25], capillary electrochromatography [24,26,27], and micellar electrokinetic chromatography [27,28]. However, dielectrophoretic chromatography has only been studied by a few groups: Holmes and Morgan [29], the Washizu group [30][31][32], and more recently, the Agah group [33] and Giesler et al. [34]. ...
The development of insulator-based dielectrophoresis chromatography is proposed here as a novel hybrid technique that capitalizes on the simplicity of insulator-based dielectrophoresis (iDEP) and the well-known chromatographic theory. Chromatographic parameters are employed to characterize dielectrophoretic separation of particles with particles being eluted from the system as enriched particle peaks. By varying the characteristics of the insulating posts, it was possible to manipulate the interactions of the particles with the insulating post array which acted as the stationary phase. The present work studied how the characteristics of the particles affected the particle retention. Different types of particles have distinct interactions with the post array; these interactions depend on particle properties (size, electrical charge, and polarizability). This work includes mathematical modeling with COMSOL and extensive experimentation. Particles ranging from 1 to 10 μm in diameter were tested for retention time and eluted as peaks in the iDEP chromatography devices. Separation results were reported in the form of dielectropherograms including the estimation of retention time (tR), separation efficiency (N/meter), and separation resolution (Rs). Two full separations were demonstrated: a separation by charge between two types of particles of similar size (~ 10 μm) with different electrical surface charges and a separation by size between 2- and 5-μm particles with similar surface charge (difference in ζP of 4 mV). The achieved separation resolutions were Rs = 1.8 and Rs = 3.5, respectively. This is the first study on DEP chromatography to assess performance in terms of resolution and separation efficiency, demonstrating the unique potential of iDEP chromatography.
... 1,2 MEKC improved the separation selectivity of charge and allowed the separation of neutral analytes by using the beneficial features of micelles, which are formed by the addition of a surfactant above the critical micelle concentration (cmc) into the background solution (BGS). 3 MEKC with UV detection generally needs sensitivity improvement for its application for analysis of real samples, due to its inherent sensitivity limitations. 4,5 Thus, different stacking techniques have been rigorously developed to improve MEKC sensitivity including field-enhancement/ amplification, 6−10 sweeping, 11−14 dynamic pH junction, 15−17 analyte focusing by micelle collapse, 18−21 and micelle-tosolvent stacking (MSS). ...
Analyte focusing by micelle to cyclodextrin stacking (MCDS) in micellar electrokinetic chromatography (MEKC) using sodium dodecyl sulfate (SDS) and fused silica capillaries is demonstrated for neutral, cationic, and chiral analytes. The stacking was at a dynamic boundary formed between the injected charged SDS micelles and neutral γ-cyclodextrin (γ-CD) zones, where the analytes bound inside micelles were released due to the formation of stable SDS-CD inclusion complexes. The complex formation reduced or eliminated the affinity of the analytes to the micellar phase. There was reversal (for charged) or nulling (for neutrals) of the analyte’s effective electrophoretic mobility that caused the analytes to accumulate at the boundary. Under the conditions where the SDS micelles velocity is faster than the electroosmotic flow (using acidic buffer), MCDS was conducted by injection of a long plug of sample in a micellar diluent after injection of a CD solution plug into a capillary that was filled with MEKC background solution. By simply extending the length of the CD plug, chiral separations of chlorpheniramine and phenoxyacid herbicides were achieved without optimizing the MEKC conditions. The analytical figures of merit including linearity and repeatability for the tested compounds were found acceptable and the sensitivity enhancement factors were up to 171. The stacking strategy in MEKC was applied to metabolic stability studies of small molecules with HepG2 cell line, where the samples were only treated with acetonitrile and then diluted with the micellar diluent (demonstrating the reduction of tedious sample preparation requirements for biological samples prior to chemical analysis).
... 1 Surfactant micelles have also been used as a pseudostationary phase to improve the separation characteristics of micellar electrokinetic chromatography (MEKC). [2][3][4][5][6] Even if objective solutes are electrically neutral, they can be separated by capillary electrophoresis (CE) on the basis of the difference in the degree of their distribution between a running buffer solvent and surfactant micelles. Although MEKC shows a high separation efficiency in many cases, the mass transfer resistance of solute permeation across the interface between a bulk running buffer and surfactant micelles causes peak broadening in MEKC systems. ...
Moment equations were developed for quantitatively studying the separation characteristics of micellar electrokinetic chromatography (MEKC). They explain how the first absolute and second central moments of elution peaks are correlated with some fundamental parameters of the partition equilibrium and mass transfer kinetics in MEKC systems. In order to discuss the influence of the mass transfer kinetics on peak broadening, the moment equations were used to analyze the separation behavior in MEKC systems. Separation conditions were chosen on the basis of practical MEKC experiments previously conducted. It was quantitatively clarified that both the solute permeation at the interfacial boundary of surfactant micelles and axial diffusion of solute molecules in a capillary had a predominant contribution to the spreading of the elution peaks in MEKC systems. This is a preliminary study for the analytical determination of rate constants concerning solute permeation at the interface of surfactant micelles from elution peak profiles measured by MEKC. In addition, it was also indicated that the experimental conditions of MEKC systems could be controlled so that the interfacial solute permeation would have a predominant role for the band broadening. For example, the contribution of the interfacial permeation was about 33 times larger than that of the axial diffusion of solute molecules under the MEKC conditions in a previous study. This means that the rate constants could appropriately be determined for the interfacial solute permeation.
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... [1][2][3][4][5][6] Capillary tubes also support interesting and useful physical and hydrodynamic phenomena, such as electro-osmotic flow and laminar flow. The electro-osmotic flow in a capillary tube is used in capillary electrophoresis [7][8][9] and capillary electrochromatography, [10][11][12] while laminar flow conditions enable hydrodynamic chromatography. [13][14][15] Recently, it was proposed that a ternary solvent mixture of water with hydrophilic and hydrophobic organic solvents would be radially distributed in a capillary tube under laminar flow conditions. ...
An open-tubular capillary chromatography system (tube radial distribution chromatography, TRDC) was developed using a ternary solvent (water-acetonitrile-ethyl acetate; volume ratio, 3:8:4) containing 10 mmol L(-1) 8-quinolinol for the separation of nitrate, chloride, and sulfate compounds of Ni(II), Al(III), and Fe(III). When a mixed solution of the Ni(II) compounds was injected into an untreated fused-silica capillary tube (90 cm × 75 μm i.d.) with a ternary solvent flow rate of 0.8 μL min(-1), the compounds were eluted in the following order: [Ni(II)-(8-quinolinol)3] complex, [Ni(II)-(8-quinolinol)]-nitrate ion interaction complex, [Ni(II)-(8-quinolinol)]-chloride ion interaction complex, and [Ni(II)-(8-quinolinol)]-sulfate ion interaction complex. The elution of mixtures of the Al(III) and Fe(III) compounds showed similar trends.
... Capillary tubes with inner diameters less than several hundred micrometers that provide micro-flow are known to exhibit interesting and useful physical or hydrodynamic phenomena, such as electro-osmotic flow and laminar flow. The electro-osmotic flow in a capillary tube promotes capillary electrophoresis [1,2] and capillary electrochromatography [3], while laminar flow conditions enable hydrodynamic chromatography [4,5]. Recently, our group reported the tube radial distribution phenomenon of carrier solvents in a micro-flow [6][7][8], which we call the tube radial distribution phenomenon (TRDP). ...
Capillary chromatography using an untreated open tubular capillary tube and a ternary solvent mixture consisting of water-hydrophilic/hydrophobic organic solvent as a carrier solution has been developed. The system is called tube radical distribution chromatography (TRDC). Separation performance of the TRDC system using a fused-silica capillary tube was examined through the phase diagram for the ternary water-acetonitrile-ethyl acetate solvent mixture. The TRDC system required homogeneous carrier solutions with solvent component ratios around the boundary curve between homogeneous and heterogeneous solution in the phase diagram. The data obtained using the fused-silica capillary tube were compared with those obtained using a polytetrafluoroethylene capillary tube in our previous study.
Soil is a complex mixture of minerals and organic matters in which microbes, plants, and animals interact. In the natural environment, soil constantly undergoes physical, chemical, and biological transformations under the influences of environmental factors such as humidity and temperature. Studies on soil chemical compositions, microbes, and abundances of plants and animals provide useful information on the soil property for proper land use planning. Since soil is a complex mixture, soil studies require the effective separation of its various components, which can be achieved with electrophoresis, a powerful method that exploits the inherent differences in the physical and chemical properties of these components. By combining electrophoresis with other technologies such as chromatography, mass spectrometry, polymerase chain reaction (PCR), and DNA sequencing, substances including humic acids, amino acids, environmental pollutants, nutrients, and microbial, plant, and animal DNA can be identified and quantified. In this chapter, the applications of different electrophoresis-based technologies will be discussed with respect to soil research, and their principles, advantages, and limitations will be addressed.
When ternary mixed solutions of water/acetonitrile/ethyl acetate are delivered into a microspace under laminar flow conditions, the solvent molecules show specific microfluidic flows, such as microfluidic inverted flow and tube radial distribution flow, which have been applied to novel analytical methods. In this paper, inverted flow was examined using various Y-type microchannels that had mixing angles of 0°, 90°, 180°, and 270°. Inverted flow was experimentally observed and the trigger phenomenon was also successfully expressed through computer simulations. Tube radial distribution flow, that is, annular flow, in a capillary tube is reported to cause exchange of the inner and outer phases based on the solvent composition of the ternary mixed solution. Tube radial distribution flow for an organic solvent-rich inner and a water-rich outer phases, as well as for a water-rich inner and an organic solvent-rich outer phases, could be well recreated by computer simulations for a ternary mixed solution. This highlights the effectiveness of computer simulations for such flow scenarios and will allow optimization of the operating conditions and design of microfluidic analytical devices.
Moment equations were developed on the basis of the Einstein equation for diffusion and the random walk model to analytically determine the rate constant for the interfacial solute permeation from a bulk solvent into molecular aggregates (kin) and the inverse rate constant from the molecular aggregates to the bulk solvent (kout). The moment equations were in good agreement with those derived in a different manner. To demonstrate their effectiveness in one concrete example, the moment equations were used to analytically determine the values of kin and kout of three electrically neutral solutes, i.e., resorcinol, phenol, and nitrobenzene, from the first absolute (μ1A) and second central (μ2C) moments of their elution peaks, as measured by electrokinetic chromatography (EKC), in which the sodium dodecyl sulfate (SDS) micelles were used as a pseudostationary phase. The values of kin and kout should be determined with no chemical modifications and no physical action with the molecular aggregates because they are dynamic systems formed through weak interactions between the components. The moment analysis of the elution peak profiles measured by EKC is effective to unambiguously determine kin, kout, and the partition equilibrium constant (kin/kout) under appropriate experimental conditions. This article is protected by copyright. All rights reserved
A simple, fast, sensitive and reproducible MEKC‐UV method for the determination of nikethamide (NKD) in human urine and pharmaceutical formulation has been developed and validated. The method exhibits high trueness, good precision, short analysis time and low reagent consumption. NKD is an organic compound belonging to the psychoactive stimulants used as an analeptic drugs. The proposed analytical procedure consists of few steps: dilution of urine or drug in distilled water, centrifugation for 2 min (12,000 × g), separation by MEKC and ultraviolet‐absorbance detection of NKD at 260 nm. The background electrolyte used was 0.035 mol/L pH 9 borate buffer with the addition of 0.05 mol/L SDS and 6.5% ACN. Effective separation was achieved within 5.5 min under the voltage of 21 kV (~90 μA) using a standard fused‐silica capillary (effective length 51 cm, 75 μm id). The determined LOD for NKD in urine was 1 μmol/L (0.18 μg/mL). The calibration curve obtained for NKD in urine showed linearity in the range 4‐280 μmol/L (0.71‐49.90 μg/mL), with R2 0.9998. The RSD of the points of the calibration curve varied from 5.4 to 9.5%. The analytical procedure was successfully applied to analysis of pharmaceutical formulation and spiked urine samples from healthy volunteers.
We applied the moment analysis (MA) with chromatographic capillary electrophoresis (CCE) method to the analytical determination of rate constants (kin and kout) of solute permeation at the interface between a bulk solvent and surfactant micelles. CE experiments were carried out in the MEKC system using thymol and sodium dodecylsulfate (SDS) as the solute and surfactant, respectively, under CCE conditions. In CCE mode, experimental conditions are controlled so that the migration of micelles, which dissolve single or plural solute molecules, is stopped in the axial direction of a capillary. Only solute molecules migrate in the capillary and are detected to record elution peak profiles. The information about mass transfer kinetics of solute molecules in the capillary is determined from the elution peak profiles by the MA theory. The values of kin and kout were respectively determined as 1.5 x 10⁻⁶ and 2.6 x 10⁻⁹ m s⁻¹ for the permeation of thymol molecules at the interfacial boundary of SDS micelle. It was demonstrated that MA-CCE was effective for the kinetic study of solute permeation across the interface between a bulk solvent and spherical molecular aggregates, e.g., surfactant micelles.
A new, robust, and reliable sweeping-micellar electrokinetic chromatography (sweeping-MEKC) method with dynamic pH junction and retention factor gradient effect (RFGE) is developed for the simultaneous determination of four different analytes in two ternary mixtures of pharmaceutical importance. The studied analytes are drotaverine hydrochloride and caffeine combined with paracetamol (Mixture-I) or dipyrone (Mixture-II). Different experimental parameters are carefully investigated in order to achieve the highest possible resolution and sensitivity in a short analysis time. A special interest is given to the effect of sample matrix composition on the attainable enrichment efficiency with a focus on dynamic pH junction and RFGE. Up to 8 times increase in sensitivity is achieved by the modification of the sample matrix. The theoretical considerations of the underlying effects regarding the separation and the enrichment processes are thoroughly discussed. A full validation study of the developed method based on the pharmacopeial guidelines is performed. The method is successfully applied to the analysis of the studied drugs in their co-formulated or single-ingredient tablet preparations with a total run time of less than 11 min.
Glycoprotein are one of the most complex biopolymer encountered in biological samples. This high complexity originates from the presence of possibly multiple glycosylation sites and their immense microheterogeneity, accounting for the difficulties associated with resolving fine structural differences in large biopolymers, and rendering their analysis very challenging. Capillary electrophoresis high resolving power has promted its numerous use for both at the intact glycoprotein and glycan levels. The utility of capillary electrophoresis, in its different modes, for the analysis of protein glycoforms and their glycan structures is highlighted here. Moreover, the ability to integrate multiple steps such as sample preparation, purification, separation and detection in to a small analytical unit, such as a microchip is invaluable to minimizing sample losses and, consequently enhancing sensitivity. This is very intriguing in the case of the analysis of glycoproteins since they commonly encountered at minute quantities. Therefore, chip-based approaches employed in glycoprotein analysis which involves devices allowing sample injection, preconcentration and separation are also discussed.
Since the introduction of micellar electrokinetic chromatography by Terabe, several authors have paid attention to the fundamental characteristics of this separation method. In this chapter the theoretical and practical aspects of resolution optimization, as well as the effect of different separation parameters on the migration behavior are discussed. These among others include fundamentals of separation, retention factor and resolution equation, efficiency, selectivity, and various surfactants and additives. Initial conditions for method development and instrumental approaches such as mass spectrometry detection are also mentioned covering the proposals for overcoming the difficulties arising from the coupling micellar electrokinetic chromatography with mass spectrometry detection.
This paper reports the use of methyl chloroacetate (MCA) as an extraction solvent for coupling liquid-liquid semimicroextraction (LLsME) with micellar electrokinetic chromatography (MEKC) through on-capillary decomposition for the separation of neutral compounds with concentration enhancement. Alkylphenones butyrophenone, valerophenone and acetophenone were chosen as modeling compounds. Aqueous samples of the alkylphenones were extracted by LLsME into MCA. The organic phase was directly subjected to separate by MEKC. The detection limits (S/N ≥ 3) were 50, 50 and 100μg/L for butyrophenone, valerophenone and acetophenone in water, respectively, and the concentration factors of the extraction were in a range of 63 to 151-fold in terms of peak area. The method developed has no requirement for any special experimental apparatus other than a basic CE setup and is time-saving and easy-ope-rated. Recoveries of 4-t-BP and BPA in drink water were examined. The recoveries were obtained as 99.87% and 102.8%, respectively. Therefore, it would be readily acceptable for routine analysis, especially for environmental samples.
Use of 1-adamantanecarboxylate as desorbing displacer for eluting solutes retained with the sorbent of β-cyclodextrin bonded silica in coupling solid phase extraction with micellar electrokinetic chromatography was studied. The sorbent showed strong capacity of retaining 4-tert-butylphenol and 2,2-di(4-hydroxyphenyl)propane spiked in double-distilled water. Using the desorbing displacer of 1-adamantanecarboxylate was more effective in eluting 4-tert-butylphenol and 2,2-di(4-hydroxyphenyl)propane retained from the b- cyclodextrin bonded silica sorbent in the solid phase extraction cartridges than common organic solvents. The eluate collected can be directly introduced to micellar electrokinetic chromatography for separation. The results of evaluation on analytical performance of coupling the solid phase extraction with micellar electrokinetic chromatography have been presented.
The critical micelle concentration (CMC) is the single most useful quantity for characterizing surfactants and the most significant parameter when working with ionic micellar phases, such as micellar electrokinetic chromatography (MEKC). Sodium dodecyl sulphate (SDS) was the first and is the most popular surfactant used in MEKC. Acetonitrile is often used as an organic additive for selectivity adjustment of solutes and solubility enhancement of hydrophobic compounds and phosphate as an inorganic additive for obtaining desired pH and buffer capacity in MEKC. The effect of acetonitrile on the CMC of SDS was studied by conductometry and the effect of phosphate buffer on the CMC of SDS in acetonitrile-water binary solvent was studied by fluorometry with the use of pyrene as a probe. It was found that the CMC of SDS first decreased up to 3 % (v/v) and then increased with increasing of the volume ratio of acetonitrile to water up to 5 % (v/v). The minimum of the CMC of SDS in acetonitrile-water binary solvent was found at the volume ratio of 3 % (v/v) acetonitrile to water and was determined to be 7.7 mM. The CMC value of SDS in acetonitrile-water (3 %, v/v) binary solvent decreased with increasing of phosphate in the concentration range of 10-50 mM. The information provided in this paper is expected to be useful for optimization of separation and interpretation of migration behaviour of solutes in MEKC.
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We demonstrate that the use of wormlike nonionic micelles as drag-tags in end-labeled free-solution electrophoresis ("micelle-ELFSE") provides single-base resolution of Sanger sequencing products up to 502 bases in length, a nearly two-fold improvement over reported ELFSE separations. "CiEj" running buffers containing 48 mM C12E5, 6 mM C10E5, and 3 M urea (32.5°C) form wormlike micelles that provide a drag equivalent to an uncharged DNA fragment with a length (α) of 509 bases (effective Rh = 27 nm). Runtime in a 40-cm capillary (30 kV) was 35 mins for elution of all products down to the 26-base primer. The CiEj buffer has a low viscosity (2.7 cP) and appears to be in the semi-dilute regime. We also show that smaller Triton X-100 micelles give a read length of 103 bases in a 4-min run, so that a combined analysis of the Sanger products using the two buffers in separate capillaries could be completed in 14 mins for the full range of lengths. A van Deemter analysis shows that resolution is limited by diffusion-based peak broadening and wall adsorption. Effects of drag-tag polydispersity are not observed despite the inherent polydispersity of the wormlike micelles. We ascribe this to a stochastic size-sampling process that occurs as micelle size fluctuates rapidly during the runtime. A theoretical model of the process suggests that fluctuations occur with a time scale less than 10 msec, consistent with the monomer exchange process in nonionic micelles. The large drag-tag size of the CiEj buffers leads to steric segregation of the DNA and tag for short fragments and attendant mobility shifts.
Diagnosis of inherited diseases or cancer predisposition involves analysis of specific mutations or polymorphisms. The number of characterized monogenetic and polygenetic diseases is rising significantly every year. In order to apply genetic analyses for large groups of patients or population screening, automation of a sensitive and precise method was necessary. This chapter provides an overview of capillary electrophoresis (CE), which developed analytical techniques that can rapidly process large numbers of patient samples in an automated fashion. In the area of proteomics separation, analysis and characterization of proteins by CE is a challenging task to understand the molecular basis of biological processes and to discover new biomarkers and drug targets. In principle, all CE techniques are based on the movement of molecules in an electric field. The acceleration and rapid success of the Human Genome Project (HGP), leading to the generation of the draft human genomic sequence, became possible after the introduction of CE-based sequencers. For clinical diagnostics, CE is widely used for analyzing proteins in physiological matrices, such as serum, urine, and cerebrospinal fluid. It also finds wide use in the analysis of pesticides, food content and composition, and pollutants in water and soil samples.
Lipophilicity is a key physicochemical property which affects pharmacokinetic and pharmacodynamics processes of a drug. This parameter is particularly important for dermatological drugs which should penetrate through the skin barrier. Micellar electrokinetic chromatography (MEKC) method was applied for determination of lipophilicity of salicylic acid and antibiotics commonly used in dermatology. The results of MEKC analysis were compared to lipophilicity coefficients determined by RP-TLC (RM0) and computational (AlogPs,AClogP, logPPallas) methods using principal component analysis (PCA). Thanks to PCA method, a high correlation between the results obtained by both experimental: logk(MEKC) and RM0 (RP TLC) parameters was observed, but the relationship between them and results of the theoretical values were poor.
As described throughout this book, capillary electrophoresis (CE) has many advantages, but some shortcomings as well. The objective of this chapter is to describe the versatile roles of surfactants in solving problems encountered in CE. First, background information will be given for the structure and properties of surfactant monomers and their aggregates. Then, the use of surfactants as capillary wall coatings to increase separation efficiency, as a separation buffer modifier to tune selectivity, and as a pseudostationary phase to preconcentrate analytes to improve sensitivity and limits of detection will be described. Finally, the impact of surfactants on detection techniques coupled to CE will be discussed.
The derivatization of bovine serum albumin with fluorescamine was examined utilizing the tube radial distribution phenomenon of the ternary mixed solution of wateracetonitrileethyl acetate (3:15:8 volume ratio) in a fused-silica capillary tube. Double capillary tubes having different inner diameters (100 and 250 m i.d.) were designed to promote the tube radial distribution phenomenon. The smaller tube was inserted into the larger one through a T-type joint. A wateracetonitrile mixture (3:1 volume ratio) including bovine serum albumin was delivered into the large tube from the inside through the small tube, and an acetonitrileethyl acetate mixture (7:4 volume ratio) including fluorescamine was delivered from the outside through the joint. The solutions were mixed through the large tube to perform the derivatization reaction. The fluorescence intensity of the fluorescamine-derivatized bovine serum albumin obtained under the tube radial distribution phenomenon was greater than that obtained through a batch-reaction using a homogeneous solution of wateracetonitrile (1:5 volume ratio).
The aim of this study was to develop a micellar electrokinetic chromatography method (MEKC) in order to quantify voriconazole in tablets. The optimized conditions of the method were: background electrolyte composed of sodium tetraborate 10 mM and SDS 40 mM, pH 9.0; voltage of +27 kV; hydrodynamic injection of 5 s (50 mBar), and detection wavelength 207 nm. The separation was carried out in a fused-silica capillary (48.5 cm × 50 μm i.d., effective length 40 cm), maintained at 32 °C, using potassium diclofenac as an internal standard. The method was validated in accordance with the ICH requirements showing specificity, linearity (r = 0.9998, range of 30-100 μg mL−1), precision (relative standard deviation lower than 2%), accuracy (mean recovery 100.44%), and robustness, which was evaluated by 2-Level 25-2 fractional factorial design. The proposed MEKC method was successfully applied for the quantitative analysis of voriconazole in tablets. Results were compared to those obtained by high performance liquid chromatography and ultraviolet spectrophotometric methods previously developed showing non-significant difference (p > 0.05).
Open-tubular capillary chromatography was developed based on tube radial distribution of the water–acetonitrile mixture solvents containing sodium chloride under laminar flow conditions. The specific homogeneous carrier solvents were radially distributed in the capillary tubes, generating inner and outer phases. This distribution behavior was observed with fluorescence microscope-CCD camera system and considered with the phase diagram constructed with the solvents composition and sodium chloride concentration. Hydrophilic 1-naphthol and hydrophobic 2,6-naphthalenedisulfonic acid as model analytes were separated in this order with the fused-silica capillary tube (100 µm inner diameter), while, in the reverse order with the PTFE capillary tube (100 µm inner diameter). The outer phase created in the capillary tube functioned as a pseudo-stationary phase in chromatography. The mixture solution including five analytes was examined with the present capillary chromatography. The opposite elution order was observed between the fused-silica and PTFE tubes.
Tube radical distribution chromatography (TRDC) uses an untreated open tubular capillary tube and a ternary mixture of solvents (water and hydrophilic/hydrophobic organic solvents) as a carrier solution. A model analyte mixture comprising 1-naphthol, 1-naphthoic acid, 1-naphthalenesulfonic acid, 2,6-naphthalenedisulfonic acid, and 1,3,6-naphthalenetrisulfonic acid was examined by the TRDC and capillary zone electrophoresis (CZE) systems that comprised mainly a capillary tube and a detector. In the TRDC system the elution order of analytes could be changed by altering the component ratios of the solvents, whereas in the CZE system the elution order was changed by altering the electroosmotic flow direction. The experimental data obtained provide clues about the features and utility of TRDC as a new separation method.
A new method for the determination of trans-resveratrol in grape seed was developed by coupling liquid extraction (LE) and micellar electrokinetic chromatography (MEKC). LE was employed for sample preparation and MEKC for analytical separation. The solvent used for LE was ethyl acetate and the buffer for MEKC consisted of 25 mM borate containing 25 mM sodium dodecyl sulfate (SDS) and 2.5 mM sodium salt sulfated-β-cyclodextrin at pH 9.0. The limit of detection (S/N = 3) was found to be 4.62 × 10−7 g ml−1. Linear regression of peak area versus concentration of resveratrol was checked with the r squared being greater than 0.99 for resveratrol at a concentration range of 9.25 × 10−6 g ml−1 to 4.63 × 10−2 g ml−1. The recoveries were over 90% for the determination of resveratrol in grape seed samples. The method developed can be an attractive alternative to the HPLC methods reported in the literature for the determination of resveratrol in grape seed with the advantages of a reduction of analysis time and operation cost. To the best of our knowledge, this is the first report on the determination of resveratrol in grape seed based on coupling LE and MEKC.
In this study, a micellar electrokinetic chromatography method was developed and validated for the analysis of dronedarone in film-coated tablets. Electrophoretic conditions were investigated by changing factors such as pH, buffer concentration, SDS concentration, capillary temperature, injection time and applied voltage. Separation was performed using a bare fused-silica capillary of 40.0 cm effective length (48.5 cm total length; 50 μm internal diameter) maintained at 30 °C and detection was set at 216 nm. Optimal conditions were obtained using 40 mM borate buffer and 50 mM SDS at pH 9.2 as running buffer with an applied voltage of 28 kV (positive polarity) and using hydrodynamic injection at 50 mbar for 7 s. The method was validated by evaluating typical validation characteristics such as specificity, linearity, accuracy, precision, the limit of detection, the limit of quantitation and robustness. The analytical curve was linear in the concentration range of 25 to 150 μg mL−1 (r = 0.9995). The accuracy was 99.9% and the relative standard deviations of repeatability and intermediate precision were lower than 2%. The limit of detection and limit of quantitation were 0.88 μg mL−1 and 2.66 μg mL−1, respectively. The method proved to be robust by a fractional factorial design evaluation. Forced degradation studies were performed by exposing dronedarone sample solution to stress conditions (acidic, basic, oxidative, thermal and photolytic) in order to verify the stability-indicating capability of the method. The MEKC method was successfully applied for the quality control of dronedarone hydrochloride in commercial film-coated tablets.
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.
The addition of surfactants to the separation electrolyte is one of the most convenient ways to modify the electro‐osmotic flow (μEOF) in capillary electrophoresis. However, surfactants spontaneously adsorb to most surfaces; therefore, their presence in the running electrolyte may also affect the electrochemical detection. Changes in selectivity and sensitivity due to dynamic coating of electrode surfaces have been systematically reported during the last 30 years. In this review, some pertinent papers related to the use of surfactants to perform dynamic coatings of the capillary surface are discussed. The proposed mechanisms to explain the enhancements produced by surfactants at the detection step are also discussed along with some specific applications. A particular emphasis was placed on recent reports from our group dealing with the effects of anionic surfactants on the separation and detection of phenolic compounds.
Capillary electrophoresis is a powerful analytical technique which is increasing in utility in the pharmaceutical industry. It is used as an alternative or complementary technique to HPLC due to its high efficiency, speed of analysis, reduction in solvent and sample consumption, and low operating cost compared to HPLC methodology. Various modes have been developed and used for pharmaceutical analysis including capillary zone electrophoresis, non‐aqueous capillary electrophoresis, micellar electrokinetic capillary electrophoresis, capillary gel electrophoresis, capillary isotachophoresis, chiral capillary electrophoresis, and capillary electrochromatography. This review summarizes the principles and gives recent pharmaceutical analysis applications used for each mode of capillary electrophoresis. The review will also describe recent developments for enhancing concentration limits of detection.
The present work describes the retention behavior of structurally related corticosteroids separated with SDS based microemulsion. The analytes include cortisone acetate (CA), hydrocortisone (H), hydrocortisone acetate (HA), prednisolone (P), prednisolone acetate (PA), and prednisone (Ps), which are not easily separated by the conventional MEKC. Surfactants with linear and planar structures have been compared for the separation of these analytes. By fine tuning the parameters, the results revealed that complete baseline separation could be achieved with a microemulsion consisted of 0.8% (w/w) n-octane, 3.6% (w/w) SDS, 6.6% (w/w) 1-butanol, and 89% (w/w) phosphate buffer (40 mM, pH 8.0), under an applied voltage of +7 kV and a column dimension of 53 cm (effective length, 30 cm) × 75 μm id. Although the composition of the microemulsion has a high recognition property for these structurally related compounds, the high affinity of these analytes with the pseudostationary phase made the analysis time very long. The effects of the addition of acetonitrile, methanol, and isopropanol have also been studied. Acetonitrile was found to have the greatest impact on this separation.
A sensitive and simple micellar electrokinetic chromatography (MEKC) method was developed for the determination of the antiepileptic drug carbamazepine (CBZ) using a sweeping on‐line concentration method with photodiode array detection. The effect of pH, concentration of the running buffer solution, organic modifier, applied voltage and injection time on the concentration efficiency and separation was investigated. An untreated fused‐silica capillary was used (50 cm; effective length, 40 cm, 75 µm i.d.) for the analysis. The background solution (BGS) was 50 mmol · L NaH2PO4 (pH 3.0) containing 100 mmol · L SDS and 20% acetonitrile (5.82 ms · cm) with an applied voltage of −20 kV at 25°C. Sample introduction was performed at 0.5 psi for 90 s with diode array detection at 214 nm. For the method, the calibration curve was linear over a range of 0.5–40 µg · mL for CBZ with a correlation coefficient of 0.998. The detection limit (S/N=3∶1) of CBZ was 0.10 µg · mL. About 100‐fold improvement in concentration sensitivity was achieved in terms of peak height by the sweeping method compared to conventional injection method. The sweeping‐MEKC method has been successfully applied to the analysis of CBZ in tablet and human serum.
a b s t r a c t The commercial dye C.I. Acid Black 194 was analyzed by reversed-phase HPLC, Capillary Zone Electro-phoresis and Micellar ElectroKinetic Chromatography under different operative conditions, with the scope to detect the impurity distribution typical of any production processes and synthetic batch. The three chrome(III) complexes deriving from the industrial synthesis of C.I. Acid Black 194, and one of the main impurities were isolated by silica flash chromatography and identified by mass spectrometry and NMR. More than twelve compounds present in the commercial mixture, but undetectable by the analytical protocols known in literature, were fully separated by the MECK mode capillary electropho-resis with low % Relative Standard Deviation of the main electrophoretic parameters. Two of them were identified by isolation from the commercial mixture. As additional examples two other commercial metal-based dyes, C.I. Acid Brown 432 and C.I. Acid Brown 434, were analyzed by the same protocol with very good results.
Metabolomics offers a revolutionary framework for phenotyping individuals at a molecular level that is needed for new breakthroughs in cell biology and personalized medicine. Separation science plays several critical roles in metabolomics for reliable quantification and improved identification of metabolites in complex sample mixtures. Separation efficiency and peak capacity are two important parameters for determining the performance of a separation in terms of the total number of unique compounds that are baseline resolved prior to detection. Higher peak capacity within a wider separation window reduces solute coelution that can complicate relative quantification or spectral matching for unknown identification. The use of a wider bore/thicker nonpolar stationary phase film as the second dimension column enhances separation performance by increasing sample loadability that is important when quantifying low abundance metabolites in complex biological samples.
Instrumentation has been developed for the implementation of ultra-high voltage capillary electrophoresis (UHVCE) with potentials up to and exceeding 300kV. Several separations have been used to demonstrate the utility of higher applied voltages for improving the resolution of peptide, protein, and nucleic acid separations. Previously reported instrumentation was limited to 120kV and required submersion in a bath of transformer oil to prevent corona and high voltage arcing between the components of the instrument [Hutterer, 1999, 2000, 2005] [1-3]. A modular design that uses plastic dielectric materials to overcome these obstacles enabling simplified operation of the instrument in air is described here in detail. A forced air system developed to control the temperature of the instrument to within a few degrees over a range of 25-60°C for use with ultra-high voltage capillary gel electrophoresis is also described. UHVCE instrumentation and its applications with UV absorption and laser induced fluorescence detection are further developed, and the first demonstration of UHVCE coupled to electrospray ionization-mass spectrometry is shown.
With capillary electrophoresis, ionic analytes can be separated in free solution under the influence of an electric field. The capillary or microchannel format offers several advantages over classical electrophoresis, mainly the much lower time of analysis, low sample, chemical and solvent consumption, on-column detection and quantification, and the possibility for on-line combination with a mass spectrometer. Supporting media like porous material or gels are not needed. Thus the potential analytes cover the size range from 0.1 nm to several μm and reach from small molecules like drugs to viruses, bacteria, and eukaryotic cells. The high voltage that can be applied due to the capillary dimensions and the composition of the buffer leads, together with the high charge numbers of biomolecules like proteins or polynucleotides, to very high separation efficiencies. The large variability of the working conditions allows for separation of charged and uncharged analytes, for the latter by using hybrid methods combining electrokinetic mass transport and chromatographic separation principles.
Micellar electrokinetic chromatography (MEKC) Applying chemometrics to MEKC Concluding remarks References
Capillary electrophoresis (CE) is being extensively applied for characterization of biotechnology derived products like peptides and proteins. Recently, the role of CE in the strategy of protein analysis increases, with an emphasis on multidimensional separation and the combination of mass spectrometric techniques. This article gives emphasis on the fundamental principles of CE and its application to the separation and characterization of peptides and proteins.
Keywords:
capillary electrophoresis (CE);
polyacrylamide gel electrophoresis;
high performance liquid chromatography;
protein pharmaceuticals
A method based on cyclodextrin-mediated micellar electrokinetic chromatography (CD-MEKC) was developed and validated for quantification
of the minor, undesired enantiomer (distomer) in the drug candidate PHA-549184, an oxazolidinone that was under development
as an antibiotic. The low intrinsic solubility of the compound (0.24 mg mL−1), combined with the poor solution concentration sensitivity of capillary electrophoresis, required extensive method development
to enhance the solubility of PHA-549184 while minimally degrading electrophoretic performance. A number of approaches were
investigated, including inclusion of neutral and charged cyclodextrins in the background electrolyte (BGE) and addition of
both charged and neutral surfactants. The final BGE contains the nonionic surfactant Brij 35: pH 2.5, 18.8 mM lithium phosphate
buffer/5% highly sulfated-γ-CD/7.5 mM Brij 35. Quantitation is versus an external standard in the presence of an internal standard. The assay is selective
for the distomer and proved linear with a mean recovery of 104.0% over the range 0.25–2.0%. The LOD was 0.1, and the LOQ 0.2%,
both slightly higher than customary in chiral analysis, a consequence of an upper bound of 1.5 mg mL−1 placed on the sample concentration in order to maintain high efficiency for the system. Precision examined over the range
0.2–1.0% varied between 3.8% and 8.0%. No batch of drug substance registered above the detection limit for the distomer.
The micellization of the ionic surfactant sodium dodecyl sulfate (SDS) has been investigated in the presence of neutral cyclodextrins
by means of capillary electrophoresis (CE). The measurements of electric current allowed the determination of the critical
micelle concentration of SDS in the presence of α-, β- and γ-cyclodextrin, and of (2-hydroxypropyl)-β-cyclodextrin and (2,6-di-O-methyl)-β-cyclodextrin. Measurements of the CE current also yields information on the binding of SDS by cyclodextrins. The
results are supported by electronic paramagnetic resonance spectroscopy and suggest that the methylated cyclodextrin affects
the micellization of SDS in an unconventional way compared to other considered cyclodextrins. The combination of SDS with
methylated cyclodextrin can have a profound effect on the reliable application of cyclodextrin-modified micellar electrokinetic
chromatography.
KeywordsCritical micelle concentration-Capillary electrophoresis-Cyclodextrins-Electronic paramagnetic resonance
A universal micellar electrokinetic capillary chromatographic (MEKC) method with diode-array detection for the simultaneous
and short-time analysis of lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin and rosuvastatin was introduced.
Base hydrolysis was used to open lactone ring of lovastatin and simvastatin, administered as lactone prodrugs, in order to
transform these compounds to the corresponding β-hydroxyl acid forms before MEKC analysis. This approach offered shorter analysis
time due to a decrease of the migration times of negatively charged statin drugs in comparison to neutral lactone forms. Optimized
conditions were found to be a 25mM borate buffer pH 9.5 with 25mM sodium dodecyl sulphate and 10% methanol added as an organic
modifier, an applied voltage of 23kV and a separation temperature of 30°C. Ketoprofen was used as an internal standard.
The linearity of the detector response for each statin was within the concentration range from 10 to 100μgmL−1 with a correlation coefficient greater than 0.9994. Analyses of six statin drugs in pharmaceutical samples were carried out
in only 5min. The interference of the tablet sample matrix was not observed. The recovery values were in the range of 98.04–100.80%.
KeywordsMicellar electrokinetic capillary chromatography-Capillary electrophoresis-Statin drugs
Proteinuria, i.e. increased excretion of proteins in urine, is a common sign indicating renal or urinary tract diseases. In
this study, a fast and simple procedure for urine sample preparation and capillary micellar electrokinetic chromatographic
analysis is presented, without any sample pretreatment prior to the analysis. The developed MEKC method was employed for simultaneous
determination of albumin (ALB), haemoglobin (HGB), and myoglobin (MYO) in human urine samples obtained from patients with
diagnosed proteinuria. Optimum conditions for detection and separation of ALB, HGB, and MYO are 50mmolL−1 borate buffer containing 20mmolL−1 SDS (pH 9.3), injection 40mbar×20s, voltage 25kV, temperature 30°C, and detection wavelength 200nm. The method was
shown to be specific, accurate, linear (correlation coefficients r
2>0.99), and precise (RSD below 3.75 and 7.23% for migration time and peak area, respectively). Multi-variable-at-a-time
(MVAT) approach for robustness testing shows no significant variations in accuracy, specificity, and precision as RSD values
were lower than 5 and 10% for migration time and peak area, respectively. The presented method is applicable for routine analyses
of urine samples as a screening method for patients with excess ALB, HGB, and MYO.
When the ternary mixed solvents of a water-hydrophilic/hydrophobic organic solvent mixture are delivered under microspace under laminar flow conditions, the solvent molecules are radially distributed in the microspace, and generate a major inner phase and a minor outer phase. We call this fluidic phenomenon as the tube radial distribution phenomenon (TRDP). In this paper, phase formation in the TRDP was collectively considered based on experimental data, such as the inner and outer phase formation in a microchannel under laminar flow conditions, the phase diagram for the ternary mixed solvents, the solvent-component ratios required for the TRDP, and the phase transformation in a batch vessel above atmospheric pressure, which were mainly reported in our previous papers. Furthermore, the formation of inner and outer phases in a capillary tube was simulated with the two-component solvents mixture model of water and ethyl acetate. Phase formations in capillary tubes were expressed through computer simulations.
A micro-flow reaction system was developed in which liquid-liquid interface was created based on the tube radial distribution of ternary mixed carrier solvents. The system was constructed from double capillary tubes having different inner diameters (100 and 250 µm i.d.). The smaller tube was inserted into the larger one through a T-type joint. The reaction of a protein with a fluorescence derivatizing reagent was adopted as a model. A water-acetonitrile mixture (3:1 volume ratio) including bovine serum albumin (hydrophilic) was delivered into the large tube from the inside through the small tube and an acetonitrile-ethyl acetate mixture (7:4 volume ratio) containing fluorescamine (hydrophobic) as a derivatizing reagent was delivered from the outside through the joint. Solutions were mixed through the double capillary tubes to promote ternary mixed carrier solvents (water-acetonitrile-ethyl acetate; 1:2:1 volume ratio). The liquid-liquid interface was created based on the tube radial distribution of ternary solvents in the larger tube. The derivatization reaction was performed in the larger, or reaction, tube in the micro-flow system. The fluorescence intensity of the fluorescamine-derivatized bovine serum albumin obtained by the system, which specifically included the kinetic liquid-liquid interface in the tube, was greater than that obtained through a batch reaction using a homogeneous solution of water-acetonitrile (1:2 volume ratio).
When a neutral analyte zone is injected into a charged pseudostationary phase, the length of the zone is predicted to be narrowed
by 1/(1 + k), where k is the retention factor. The conditions for zone narrowing to occur assume negligible electroosmotic flow, a relatively constant
electric field along the capillary column, and no pseudostationary phase in the injected analyte mixture. The theoretically
expected concentration enhancement was demonstrated experimentally. Consequently, the detection sensitivity of analytes in
micellar electrokinetic chromatography (MEKC) can be improved significantly. For example, 9 to 18 parts per billion of an
environmentally important racemic herbicide spiked in lake water was separated by MEKC and detected by ultraviolet absorption.
Two strategies for the isotachophoretic preconcentration of samples were evaluated using a standard protein mixture as an example. In the first, on-column transient isotachophoretic migration permits the injection of relatively large volumes of sample into a commercial instrument. Depending on the composition, 30–50% of the column length can be filled with the sample while maintaining good resolution of the sample components. When proper electrolyte compositions are selected, the conventional single-column instrument can be used for an isotachophoretic sample preconcentration of 50-fold or more without any modification. In the second strategy, a coupled-column system was examined which, in principle, provides a higher degree of freedom in the selection of capillary zone electrophoretic running conditions and the possibility of injection of higher sample volumes. The gain in detection level is at least a factor of 1000 in the coupled-column arrangement; however, the instrumentation is more complicated.
Fundamental characteristics of a new type of chromatography with micellar solutions of ionic surfactants were examined. Electrokinetic phenomena in open-tubular capillaries move two phases, aqueous and micellar, with different velocities and micellar solubilization operates as the distribution process of solutes. Sodium dodecyl sulfate (SDS) solutions and a 0.05 mm i.d. x 650 mm fused silica tube were employed and high dc voltages up to 25 kV were applied. Linear relationships were observed between current and migration velocities of water, micelle, and any solute, but not between applied voltages and the velocities. This discrepancy can be reasonably interpreted in terms of the temperature rise of the solution in the tube resulting from Joule heating. Optimum resolutions can be obtained when the capacity factor is about 2, because retention times are limited between those of an insolubilized solute or water and a totally solubilized solute or micelle. Observed dependence of capacity factors on current can also be explained by the temperature rise. Thermodynamic parameters in micellar solubilization were presented. 22 references, 10 figures, 4 tables.
The separating power of micellar electrokinetic chromatography (MEC) for neutral solutes is critically examined. A theory for the optimization of resolution (Rs) and resolution per unit time (Rs/tR) is presented and evaluated. Equations are derived that predict the optimum retention factor (k′) and the corresponding surfactant concentration to use. Another equation is derived that relates the analysis time to five parameters: Rs, α (selectivity), k′, to/tmc, and H/veo (plate height/electroosmotic velocity). The capacity factor for the best resolution is given by k′ = (tmc/to)1/2, where to and tmc are the retention times of the aqueous and micellar phases, respectively. The capacity factor for the best resolution per unit time ranges from 1.2 to 2. The separations obtained by using the optimum capacity factor for resolution per unit time instead of resolution may be slightly poorer in terms of resolution but are much faster. Provided the optimum conditions are employed, the quality of MEC separations is independent of the solute hydrophobicity. The advantages of a surfactant gradient for the separation of solutes with a wide range of hydrophobicities are discussed. A brief comparison of MEC, micellar liquid chromatography, and conventional high-performance liquid chromatography is also made.
On-line combination of partial filling micellar electrokinetic chromatography (PF-MEKC) and electrospray ionization mass spectrometry (ESI-MS) is demonstrated for the analysis of triazine herbicides including atrazine, propazine, ametryne and prometryne. In comparison with conventional micellar electrokinetic chromatography (MEKC), PF-MEKC involves filling a small portion of the capillary with a sodium dodecyl sulfate (SDS) micellar solution for achieving the separation. In PF-MEKC, the triazine analytes first migrate into the micellar plug where the separation occurs and then into the electrophoresis buffer which is free of surfactant. Consequently, the electroosmotic transfer of neutral triazine herbicides to ESI-MS at the end of PF-MEKC capillary is comparable to conventional capillary zone electrophoresis ESI-MS. Therefore, PF-MEKC-ESI-MS provides a mechanism for the separation and mass detection of neutral molecules without the interference of surfactant.
The on-line coupling of micellar electrokinetic chromatography (MEKC) with mass spectrometry (MS) is hampered by the presence of high concentrations of surfactant in the electrolyte system. Partial-filling MEKC (PF-MEKC) provides a possible way to overcome this problem. In PF-MEKC only a part of the capillary is filled with an electrolyte solution containing micelles, thus allowing an MEKC separation without allowing surfactant to enter the mass spectrometer. The migration behaviour of the micellar zone was investigated applying indirect UV detection. It was demonstrated that micelles gradually break down under PF-MEKC conditions and migrate as surfactant monomers at a concentration at or below the critical micelle concentration. The influence of several experimental parameters on the separation performance is studied and the possibilities as well as the limitations of PF-MEKC are discussed. On-line PF-MEKC–MS with sodium dodecyl sulphate micelles is demonstrated for some pharmaceuticals applying an atmospheric pressure chemical ionisation interface.
Unlike recent studies that have depended on manipulation of separation buffer parameters to facilitate stacking of neutral analytes in micellar capillary electrophoresis (MCE) mode, we have developed a method of stacking based simply on manipulation of the sample matrix. Many solutions for sample stacking in MCE are based on strict control of pH, micelle type, electroosmotic flow (EOF) rate, and separation-mode polarity. However, a universal solution to sample stacking in MCE should allow for free manipulation of separation buffer parameters without substantially affecting separation of analytes. Analogous to sample stacking in capillary zone electrophoresis by invoking field amplification of charged analytes in a low-conductivity sample matrix, the proposed method utilizes a high-conductivity sample matrix to transfer field amplification from the sample zone to the separation buffer. This causes the micellar carrier in the separation buffer to stack before it enters the sample zone. Neutral analytes moving out of the sample zone with EOF are efficiently concentrated at the micelle front. Micelle stacking is induced by simply adding salt to the sample matrix to increase the conductivity 2-3-fold higher than the separation buffer. This solution allows free optimization of separation buffer parameters such as micelle concentration, organic modifiers, and pH, providing a method that may complement virtually any existing MCE protocol without restricting the separation method.
Elementary conditions for the on-line concentration of neutral analytes by field-enhanced sample injection with reverse migrating micelles for micellar electrokinetic chromatography is presented. Acidic phosphate buffers containing micelles of sodium dodecyl sulfate are utilized as both sample solvent and separation solution. After the capillary is conditioned with a separation solution, a water plug is hydrodynamically injected to achieve field enhancement at the injection end of the capillary during injection by application of voltage. A model is provided to give insight into the stacking scheme. Significant detector response improvements are confirmed experimentally. Moreover, utility of the technique for the analysis of a real sample is tested using urine spiked with testosterone and progesterone.
On-line concentration of neutral analytes by sample stacking in reversed migration micellar electrokinetic chromatography is presented. Micellar separation solutions of sodium dodecyl sulfate are prepared with acidic buffers to reverse the direction of the migration velocity of neutral analytes owing to a reduced electroosmotic flow. Samples are prepared in nonmicellar matrixes of low conductivity (i.e., water, diluted buffer, or dilute organic/aqueous solvent) to achieve field enhancement in the sample zone. Without polarity switching inherent in large-volume sample stacking, narrowing of analyte bands, removal of sample matrix, and separation of focused analyte bands are achieved. A model is proposed to describe the stacking technique and is supported by experimental results. In addition, equations are derived to describe band broadening associated with the technique. Detector response improvements reaching a 100-fold are confirmed experimentally. Concentration detection limits on the order of low-ppb levels (S/N = 3) are realized with model steroidal compounds.
Micellar electrokinetic chromatography (MEKC) permits the separation of electrically neutral analytes by chromatographic principles in a capillary electrophoresis system. The most effective way to obtain high resolution in MEKC is to increase the separation factor, as in conventional chromatography. The separation factor in MEKC depends on the molecular structure of the micelle and hence on the surfactant used, the pH of solution, and the nature of any additives to the micellar solution. The hydrophilic moieties of surfactant molecules generally affect selectivity more than do the hydrophobic moieties. Chiral surfactants enable the enantiomeric separation of mixtures of chiral solutes to be achieved. Mixed micelles consisting of ionic and nonionic surfactants display different selectivity from that of single ionic micelles. Additives such as cyclodextrins, ion-pair reagents, urea, organic solvents and metals can also serve as useful modifiers of the micellar solution for improving separation. In particular, cyclodextrins are useful for the separation of aromatic isomers and enantiomers. A general introductory guide to the design of successful separations by MEKC is proposed, based primarily on the author's work.
The influence of surfactant type on migration behavior and chemical selectivity in micellar electrokinetic chromatography (MEKC) is investigated through linear solvation energy relationships (LSER) and functional group selectivities. In LSER modeling, solutes' capacity factors are correlated with their structural descriptors such as size, dipolarity, and hydrogen-bonding abilities. Using the LSER methodology, useful information about the nature of solute interactions with different types of surfactant aggregates can be obtained since capacity factor in MEKC is directly related to solute distribution between the bulk aqueous solvent and micelles. High correlations were observed for different LSER models of migration behavior in MEKC for a group of 60 uncharged aromatic compounds of non-hydrogen bonding (NHB), hydrogen-bonding acceptor (HBA) bases, and hydrogen-bonding donor (HBD) acids. In two anionic, hydrocarbon micellar systems of sodium dodecyl sulfate (SDS) and sodium cholate (SC), retention is primarily influenced by the size of molecules and their hydrogen bond accepting basicity. Their dipolarity/polarizability and hydrogen bond donating acidity play minor roles. Capacity factors of solutes in SDS and SC systems increase with their size and decrease for stronger hydrogen bond acceptor bases. These results are similar to those observed for other systems where hydrophobic interactions play a major role, e.g., solute distribution in the 1-octanol-water solvent system or retention in reversed phase LC. In MEKC with an anionic fluorocarbon surfactant, lithium perfluorooctanesulfonate (LiPFOS), however, size and solute HBD acidity are the two predominant factors. The LSER results indicate that compounds find the SDS micellar environments slightly less cohesive (i.e., more apolar) than the SC micelles, while the LiPFOS micelles are the most cohesive among the three surfactant aggregates and 1-octanol provides the least cohesive environment. The fluorocarbon micelles of LiPFOS, on the other hand, are the strongest hydrogen bond donor acids, followed by SDS, SC, and 1-octanol, respectively. The SC micelles have the most hydrogen bond acceptor basic characteristics, followed by 1-octanol, SDS, and LiPFOS micelles. It can be concluded that selectivity differences between these surfactant types in MEKC is primarily due to hydrogen-bonding interactions rather than the dipolar interactions. Comparing the perfluorinated and the hydrocarbon surfactants, even solute size can play a role in selective migration patterns. In addition, information from polar and hydrophobic group selectivities confirm the LSER conclusions about the underlying interactions that control migration behavior and chemical selectivity in MEKC.(ABSTRACT TRUNCATED AT 400 WORDS)
From the definitions of retention time (tR) and resolution (Rs) in conventional chromatography, two fundamental equations for the retention behaviour and resolution of neutral solutes are derived and proved to be valid in all cases of micellar electrokinetic capillary chromatography (MECC). Two parameters, phase velocity ratio (Pr) and column availability (Aco), are introduced to reveal clearly the relationships and differences between MECC and conventional chromatography. The tR and Rs values may be either positive or negative in MECC. A negative tR indicates that the solute migrates toward the positive electrode and a positive tR toward the negative electrode. Rs > 0 means that the solute with a smaller value of the capacity factor (k') in the pair of solutes reaches the detector first, while Rs < 0 means that the elution order is the opposite. MECC can be classified into eight cases depending on the values of Pr for convenience of discussion. So far, MECC was usually performed in case IV and the resolution was poorer than that in conventional chromatography for given values of theoretical plate number, selectivity and k'. However, a better resolution can be obtained in cases II, VI and VIII when Pr < (1-k')/2. Cases VI, VIII and II are preferable to case IV for high resolution and should be more frequently employed in the future.
Closely related peptides such as neurotensin and angiotensin analogues were separated by capillary zone electrophoresis using a nonionic surfactant, sucrose monododecanoate, as a micelle forming reagent. These peptides were detected by an on-line coupled mass spectrometer using an electrospray ionization interface. However, the presence of the micelles in the separation solution drastically reduced the sensitivity of the mass spectrometer. Therefore, a partial filling technique was employed to prevent the micelles from entering the mass spectrometric interface. A part of the capillary from the injection end was filled with the micellar solution in this technique. Analytes passed through the micellar zone during the electrophoresis and when the separated analytes reached the detection end of the capillary, the micellar zone was still behind the analyte zones, because the nonionic surfactant moved very slowly in acidic conditions. Thus the technique was very useful for mass spectrometric detection for CE when the micellar solution was employed for separation. The optimization of separation and detection conditions was investigated.
A novel method that combines two on-line concentration techniques in capillary electrophoresis (CE), namely, sample stacking with electrokinetic injection (field-enhanced sample injection, FESI) and sweeping, afforded the detection of positively chargeable analytes in parts per trillion (ppt) levels. The main idea is to selectively introduce by FESI as many molecules of cationic analytes as possible from a very dilute sample solution and focus the resulting zone by sweeping. Limit of detection values (signal-to-noise ratio 3) of 4.1 and 8.0 ppt-the lowest concentration reported by direct UV detection in CE-with average plate numbers of 3.6 x 10(5) and 4.4 x 10(5) are obtained for laudanosine and naphthylamine (standard solutions), respectively. This translates to improvements in peak heights compared with usual injection approaching a million-fold. Optimization schemes and application to quantitative and qualitative analyses are also investigated.
A systematic study of selective analyte focusing in a multisection electrolyte system by capillary electrophoresis (CE) is presented. It was found that a dynamic pH junction between sample and background electrolyte zones can be used to focus zwitterionic catecholamines and weakly acidic compounds without the use of special ampholytes. Differences in pH and concentration of complexing agents, such as borate, in the sample and background electrolyte zones were determined to cause focusing through changes in the local velocity of the analyte in two different segments of the capillary. Velocity-difference induced focusing (V-DIF) of analytes using a dynamic pH junction allowed the injection of large sample volumes and significantly improved the concentration sensitivity of CE. Under optimized conditions, the limit of detection for epinephrine was determined to be about 4 x 10(-8) M (the original sample) with conventional UV absorbance detection. Moreover, separation efficiencies greater than a million theoretical plates can be achieved by focusing such large sample volumes into narrow zones. Multisection electrolyte systems, which lead to the formation of a dynamic pH junction, can be tuned toward improving the concentration sensitivity of specific analytes if their chemical properties are known.
The principles of stacking procedures are described and their properties are discussed, including the fundamentals of the behavior of zone boundaries and the consequences of the self-correcting properties of boundaries in moving boundary electrophoresis, isotachophoresis, and zone electrophoresis. Further, the diverse possibilities of stacking procedures and the unavoidable destacking are described, and several examples of practically applied stacking procedures are given, besides many references to applications. Some limitations in the use of stacking procedures are discussed. The paper is arranged in such a way that it can serve both as an introduction into the field and as a reference overview.
Two on-line sample concentration techniques, sample stacking and sweeping, were evaluated using cationic surfactants as pseudostationary phases in micellar electrokinetic chromatography. As cationic surfactant micelles, tetradecyltrimethylammonium bromide and cetyltrimethylammonium chloride were employed. About 10-fold and 1000-fold increases in detection sensitivity in terms of peak heights were observed by sample stacking and sweeping, respectively, without suppression of the electroosmotic flow. In particular, the concentration limits of detection (S/N=3) for test naphthalenesulfonic acids obtained with sweeping were from 0.96 to 0.47 ppb with UV detection without any preconcentration procedure.
Simple yet effective methods to enhance concentration sensitivity is needed for capillary electrophoresis (CE) to become a practical method to analyze trace levels of analytes in real samples. In this report, the development of a novel on-line preconcentration technique combining dynamic pH junction and sweeping modes of focusing is applied to the sensitive and selective analysis of three flavin derivatives: riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Picomolar (pM) detectability of flavins by CE with laser-induced fluorescence (LIF) detection is demonstrated through effective focusing of large sample volumes (up to 22% capillary length) using a dual pH junction-sweeping focusing mode. This results in greater than a 1,200-fold improvement in sensitivity relative to conventional injection methods, giving a limit of detection (S/N = 3) of approximately 4.0 pM for FAD and FMN. Flavin focusing is examined in terms of analyte mobility dependence on buffer pH, borate complexation and SDS interaction. Dynamic pH junction-sweeping extends on-line focusing to both neutral (hydrophobic) and weakly acidic (hydrophilic) species and is considered useful in cases when either conventional sweeping or dynamic pH junction techniques used alone are less effective for certain classes of analytes. Enhanced focusing performance by this hyphenated method was demonstrated by greater than a 4-fold reduction in flavin bandwidth, as compared to either sweeping or dynamic pH junction, reflected by analyte detector bandwidths <0.20 cm. Novel on-line focusing strategies are required to improve sensitivity in CE, which may be applied toward more effective biochemical analysis methods for diverse types of analytes.
Optimum conditions for the determination of the herbicides paraquat, diquat and difenzoquat by micellar electrokinetic chromatography (MEKC) using sweeping and cation-selective exhaustive injection (CSEI) as on-line concentration methods were developed. Sodium dodecyl sulfate (80 mM) in 50 mM phosphate buffer (pH 2.5) with 20% acetonitrile was used as a background electrolyte for the methods studied. The limits of detection, based on a signal-to-noise ratio of 3:1, were about 2.6-5.1 mg 1(-1) in purified water when MEKC was applied for the standards. By using an on-line preconcentration method known as sweeping-MEKC, up to a 500-fold increase in detection sensitivity was obtained whereas up to a 50 000-fold increase for CSEI-sweeping-MEKC was achieved. The limits of detection using optimum CSEI-sweeping-MEKC were lower than 1 microg 1(-1) and the method was validated obtaining good reproducibility (relative standard deviation lower than 22%) and linearity. CSEI-sweeping-MEKC was successfully applied to the determination of the three herbicides in spiked tap water below the levels established by the US Environmental Protection Agency.
An on-line method for the coupling of micellar electrokinetic chromatography (MEKC) and mass spectrometry (MS) is presented which allows conventional MEKC conditions to be employed without further modification. The MEKC system is coupled directly to electrospray ionization (ESI) MS using a triaxial interface. A systematic study of the influence of the surfactant concentration, the nature and concentration of buffer salts and presence of organic modifier on the interface performance indicated the feasibility of the MEKC-MS approach. Effective interfacing of MEKC was achieved with both single quadrupole and ion-trap MS instruments. Using a background electrolyte containing 20 mM sodium dodecyl sulfate (SDS) and 10 mM sodium phosphate buffer, it is demonstrated that full MEKC runs of test mixtures of mebeverine and related compounds can be monitored by ESI-MS with satisfactory sensitivity. Sub-microg/ml levels of the analytes can still be detected in full scan mode, while detection limits are in the 10-50 ng/ml range when selected ion monitoring is applied. It is shown that such sensitivity would allow full-scan MS detection of 0.1% (w/w) levels of potential impurities in mebeverine. With the ion-trap instrument successful MEKC-MS/MS experiments were carried out providing information-rich MS spectra of the related compounds. Repeated MEKC-MS analyses proved that in the course of 1 day the migration time of mebeverine remained fairly constant while the MS-signal intensity only gradually decreased to approximately 65% of its original value. Once-a-day cleaning of the first part of the ion source, which takes only 5 min, suffices to preserve an optimal interface performance for a prolonged period of time.
To improve the detection sensitivity of metal ions in capillary zone electrophoresis (CZE), a novel method that combines complex formation and on-line sample preconcentration by sweeping was developed. Sweeping is defined as the picking and accumulating of analytes by a carrier in the background solution, with which they have considerable affinity. In this sweeping method, using ethylenediaminetetraacetic acid as carrier, dynamic complexation to form a UV-absorbing chelate and on-line preconcentration occur simultaneously during a run. The technique was validated in terms of the limit of detection, reproducibility, and sensitivity enhancement. Detection responses of some divalent metal ions, in terms of peak heights, were improved from 60- to 180-fold, relative to conventional CZE which employed precapillary complexation. The limits of detection were in the range of (1.8-23.4) x 10(-8) M. This method was applied to the analysis of trace metal ions in factory wastewater. Furthermore, sweeping in conjunction with sample stacking accompanying electrokinetic injection, cation-selective exhaustive injection (CSEI-sweeping), was also examined. Up to 140 000-fold improvement in detector responses for some divalent and trivalent metal ions was realized by CSEI-sweeping. The limits of detection were in the range (2.4-25.2) x 10(-11) M.
A review of the research on micellar electrokinetic chromatography (MEKC), carried out mainly in our laboratory, is described from the viewpoints of (1) fundamental characteristics of MEKC, such as the separation principle, chromatographic parameters, selectivity and resolution, thermodynamic parameters, retention index, and band broadening; (2) selectivity manipulation, including effects of surfactants, temperature, pH, and various additives; (3) on-line coupling of MEKC with mass spectrometry; (4) applications, such as the separation of closely related compounds and enantiomer separations; and (5) on-line neutral sample concentration techniques.
Dynamic pH junction is an on-line preconcentration method in capillary electrophoresis (CE) based on electrokinetic focusing of weakly ionic analytes with in large sample volumes in a multisection electrolyte system. In this report, experiments and computer simulations were performed to gain a better insight of the analyte focusing mechanism when a dynamic pH junction was used. A computer program, SIMUL, was used to simulate the band-narrowing process of a group for phenol derivatives under optimized buffer conditions, which were compared with experimental results. Computer simulations revealed the formation of a sharp moving pH boundary within the sample zone causing efficient focusing of long plugs of weakly acidic analytes based on their pKa. These studies offered useful information for understanding the band-narrowing process by control of the depth and lifetime of the moving pH boundary as a function of analyte pKa, sample pH, and injection length. The change in pH of the sample within the capillary was also estimated by measuring the absorbances of an analyte at two different wave-lengths. Optimization of analyte focusing resulted in enhanced detection responses of about 60-450-fold in terms of peak heights for some phenol derivatives' relation to conventional injections. Dynamic pH junction represents a novel approach to control band dispersion (peak width) and selectivity (mobility) of specific analytes for high-resolution CE separations.
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