Optimization of a sensitive and robust strategy for micellar electrokinetic chromatographic analysis of sofosbuvir in combination with its co-formulated hepatitis C antiviral drugs.

Based on our previous work with "pseudostationary-ion exchanger sweeping", we use this strategy to develop a sensitive, reliable and robust method for the analysis of the newly-FDA approved hepatitis C antiviral drugs namely; sofosbuvir (SOV), daclatasvir (DAC), ledipasvir (LED) and velpatasvir (VEP) in their pure forms and co-formulated pharmaceutical dosage forms using micellar electrokinetic chromatography (MEKC) as a separation method. For the first time, a successful separation of all the investigated compounds was achieved in less than 8 min using a basic background electrolyte (BGE) composed of 25 mmol L-1 SDS + 20% (v/v) ACN (acetonitrile) in 10 mmol L-1 disodium tetraborate buffer (final apparent pH is 9.90). A special focus was given to optimize the composition of the sample matrix to maintain the solubility of the analytes within the sample zone while gaining additional benefits regarding analyte zone focusing. It was found that replacing phosphoric acid (as a sample matrix) with a zwitterionic/isoelectric buffering compound (L-glutamic acid) has a substantial positive impact on the obtained enrichment efficiency. The interplay of other enrichment principles such as the retention factor gradient effect (RFGE) is also discussed. A full validation study is performed based on the pharmacopeial and ICH guidelines. The obtained limits of detection and quantitation are as low as 0.63 and 1.3 µg mL-1; respectively for SOV and DAC and 1.3 and 2.5 µg mL-1; respectively for LED and VEP using UV-DAD as a detection method. The selectivity of the developed method for determination of the studied compounds in their pharmaceutical dosage forms or in the presence of ribavirin (RIB) or elbasvir (ELB), which are other prescribed medications in the treatment regimen of patients with hepatitis C virus infection, is demonstrated. It is shown that with acidic sample matrix and basic BGE, an efficient and precise approach was designed in which analyte adsorption on the capillary wall was minimized while keeping repeatable peak height, peak area and migration time together with the highest possible enrichment efficiency.

Determination of the Exact Particle Radius Distribution for Silica Nanoparticles via Capillary Electrophoresis and Modeling the Electrophoretic Mobility with a Modified Analytic Approximation.

In this study, we use aqueous dispersions of amorphous silica nanoparticles of various sizes to investigate whether electropherograms recorded from capillary electrophoresis experiments can be converted directly into exact number-based particle radius distributions, provided that there is a relaxation effect-based size selectivity of the electrophoretic mobility and provided that the electrokinetic potential ζ of the particles can be regarded to be homogeneous over the surface of the particles, independent of the particle size. The results of this conversion procedure are compared with number-based particle radius distributions obtained from a large set of transmission electron microscopy (TEM) data. For this specific example, it is shown that the modified analytic approximation developed by Ohshima adequately describes the mobility-dependent relaxation effect and the electrophoretic mobility of the particle as a function of the reduced hydrodynamic radius and electrokinetic potential, which is a prerequisite for the presented procedure. Simultaneously, we confirmed that for the given Debye length/particle diameter ratio the electrokinetic surface charge density can be regarded to be size-invariant (including spherical geometry and planar limiting case). It is shown that the accuracy of the results of the developed method is comparable to that gained by a large set of TEM data, which is important when a precise description of the particle size distribution is needed to deduce conclusions regarding the underlying mechanism(s) of particle growth. The values obtained for the dispersion (width) of the distribution show only a small negative deviation, when compared with the TEM data (4-16%).

Mixed-mode acrylamide-based continuous beds bearing tert-butyl groups for capillary electrochromatography synthesized via complexation of N-tert-butylacrylamide with a water-soluble cyclodextrin. Part I: Retention properties.

With the aim to improve the understanding of morphology and efficiency properties, we investigate in this series the impact of the complex formation constant of the hydrophobic monomer with respect to statistically methylated-ß-cyclodextrin (Me-ß-CD) on the electrochromatographic properties of highly crosslinked amphiphilic mixed-mode acrylamide-based monolithic stationary phases. Based on our previous work on amphiphilic mixed-mode monolithic stationary phases for capillary electrochromatography (CEC) using N-(1-adamantyl)acrylamide (Ad-AAm) as hydrophobic monomer that forms an extremely strong water-soluble inclusion complex with Me-ß-CD, we now selected N-tert-butylacrylamide (NTBA) as hydrophobic monomer forming an inclusion complex with Me-ß-CD with a much lower value of the formation constant. Mixed-mode monolithic stationary phases are synthesized by in-situ free radical copolymerization of cyclodextrin-solubilized N-tert-butylacrylamide, a water soluble crosslinker (piperazinediacrylamide), a hydrophilic neutral monomer (methacrylamide), and a negatively charged monomer (vinylsulfonic acid) in aqueous medium in bind silane pre-treated fused silica capillaries. The synthesized monolithic stationary phases have both hydrophobic and hydrophilic moieties and can be employed in the reversed-phase mode, in the normal-phase mode, in a mixed-mode or in the hydrophilic interaction liquid chromatography (HILIC) mode (depending on the composition of the mobile phase and on the properties of the solute). Morphology and retention properties of this new type of stationary phase are compared to those reported in our previous series. With a homologues series of alkylphenones it is confirmed that the hydrophobicity (methylene selectivity αmeth) of the stationary phase is strongly dependent on the type of hydrophobic monomer employed. The studies reveal a significant influence of the formation constant of the involved host-guest inclusion complex on the morphology (i.e., the domain size) of the produced monoliths.

Elucidation of the Enantiodiscrimination Properties of a Nonracemic Chiral Alignment Medium through Gel-based Capillary Electrochromatography: Separation of the Mefloquine Stereoisomers.

Enantiodiscrimination and enantioseparation are two highly important processes in chemistry, often performed by using NMR spectroscopy and chromatography. For a better understanding of the mechanistic details, the same system should be studied by both methods. In addition, isotropic and anisotropic NMR parameters should be obtained, the latter using alignment media so that residual dipolar couplings and chemical-shift anisotropies can be measured. Consequently, a chiral alignment medium was used for the first time in chiral gel-based capillary electrochromatography with the four stereoisomers of the antimalaria drug mefloquine as test compounds. Chromatographic data verify that enantiodiscrimination obtained with this alignment gel is caused by differences in the equilibrium constants related to associate formation. Hence, the chromatographic separation provides physicochemical data that form a basis for the understanding and optimization of alignment processes, and vice versa.

In-capillary derivatization with o-phthalaldehyde in the presence of 3-mercaptopropionic acid for the simultaneous determination of monosodium glutamate, benzoic acid, and sorbic acid in food samples via capillary electrophoresis with ultraviolet detection.

For the rapid simultaneous determination of monosodium glutamate (MSG), benzoic acid (BA), and sorbic acid (SA) in canned food and other processed food samples, we developed a method that combines in-capillary derivatization with separation by capillary electrophoresis. This method employs the rapid derivatization of MSG with o-phthalaldehyde (OPA) in the presence of 3-mercaptopropionic acid (3-MPA) and enables the detection of the resulting OPA-MSG derivative and of non-derivatized BA and SA at 230nm. The composition of the background electrolyte and the parameters of derivatization and separation are as follows: 25mM borax containing 5mM OPA and 6mM 3-MPA, separation voltage 25mV, injection at 30mbar for 20s, and column temperature 25°C. Because of the high reaction rate and suitably adapted effective electrophoretic mobilities, band broadening due to the derivatization reaction at the start of the separation process is kept to a minimum. The optimized method is validated with respect to LOD, LOQ, linearity, recovery, and precision. This method can be applied to real samples such as soy, fish, oyster and sweet and sour chili sauces after application of appropriate clean-up steps. Mechanisms of zone broadening and zone focusing are discussed showing the validity of the employed theoretical approach regarding the dependence of the peak shape for OPA-MSG on the concentration of MSG in the sample.

Characterization of hydrophilic coated gold nanoparticles via capillary electrophoresis and Taylor dispersion analysis. Part II: Determination of the hydrodynamic radius distribution - Comparison with asymmetric flow field-flow fractionation.

In the first paper of this series we have shown for hydrophilic coated Au nanoparticles that capillary electrophoresis in combination with Taylor dispersion analysis in fused silica capillaries with an inner diameter of 75 µm allows for the unbiased precise determination of the number-weighted mean hydrodynamic diameter, the zeta potential and the effective charge number, although mobility corrected double layer polarization has to be taken into account. In this second paper we investigate whether the modified approximate analytic expression developed by Ohshima (2001) permits the calculation of calibration lines and the concomitant conversion of electropherograms into number-weighted particle radius distributions. We show that with the method developed size distributions are obtained which are independent of the measurement conditions. These size distributions are much narrower than those obtained via dynamic light scattering and data evaluation by the CONTIN algorithm. Capillary electrophoresis together with the proposed data evaluation method reveals that the analyzed nanoparticle populations have very narrow size distributions with a width of 2-4 nm. The hydrodynamic radius distributions of the coated NPs are only slightly broader than the solid particle radius distribution of the Au-NP cores. The presence of monomodal/bimodal size distributions is confirmed by asymmetric flow field-flow fractionation.

Characterization of gold nanoparticles with different hydrophilic coatings via capillary electrophoresis and Taylor dispersion analysis. Part I: determination of the zeta potential employing a modified analytic approximation.

Taking gold nanoparticles with different hydrophilic coatings as an example, it is investigated whether capillary electrophoresis in combination with Taylor dispersion analysis allows for the precise determination of mean electrophoretic mobilities, electrophoretic mobility distributions, and zeta potentials in a matrix of exactly known composition and the calibration-free determination of number-weighted mean hydrodynamic radii. Our experimental data confirm that the calculation of the zeta potential for colloidal nanoparticles with ζ>25 mV requires to take the relaxation effect into account. Because of the requirement to avoid particle-wall interactions, a solution of disodiumtetraborate decahydrate (borax) in deionized water had been selected as suitable electrolyte. Measurements of the electrophoretic mobility at different ionic strength and application of the analytic approximation developed by Ohshima show that in the present case of a buffered solution with a weak electrolyte co-ion and a strong electrolyte counterion, the effective ionic drag coefficient should be approximated with the ionic drag coefficient of the counterion. The obtained results are in good agreement with theoretical expectations regarding the dependence of the zeta potential and the electrokinetic surface charge density on the ionic strength. We also show that Taylor dispersion analysis (besides estimation of the number-weighted mean hydrodynamic radius) provides additional information on the type and width of the number-weighted particle distribution.

Boronate affinity-assisted MEKC separation of highly hydrophilic urinary nucleosides using imidazolium-based ionic liquid type surfactant as pseudostationary phase.

In this work, we extend our investigations regarding the separation of urinary nucleosides by MEKC with the ionic liquid type surfactant 1-tetradecyl-3-methylimidazolium bromide (C14MImBr). We study the impact of adding alkyl- and arylboronic acids (in the presence of C14MImBr micelles) to the separation of these highly hydrophilic metabolites and investigate the mechanism of interaction between the negatively charged nucleosides (the negative charge is acquired either due to deprotonation of the amidic group and/or complexation with boronate) and the positively charged pseudostationary phase. This interaction is not only due to electrostatic (Coulombic) forces, but also due to hydrophobic interaction of the alkyl or aryl group of the boronate that forms a complex with the cis-diol group of the nucleoside. In this case, alkylboronates can act as a cosurfactant that increases the partitioning coefficient of the analytes into the micelles. In the presence of an alkylboronate in the BGE (employing only 20 mmol/L C14MImBr), the retention factors of the studied analytes are increased considerably when compared to a BGE without this additive. It is shown that the concept of one-site hydrophobically assisted ion exchange can be applied to describe the observed retention behavior. The high selectivity of boronates toward cis-diol-containing compounds can be used to adjust selectively the migration behavior of members of this compound class. By adding alkylboronic acid to the BGE, the separation selectivity is fine-tuned so that interferences from matrix components can be avoided in real sample analysis.

Determination of urinary nucleosides via borate complexation capillary electrophoresis combined with dynamic pH junction-sweeping-large volume sample stacking as three sequential steps for their on-line enrichment.

The combination of dynamic pH junction, sweeping (using borate complexation), and large volume sample stacking (LVSS) is investigated as three consecutive steps for on-line focusing in the sensitive quantitation of urinary nucleosides by CE-UVD. A low conductivity aqueous sample matrix free from borate and a high conductivity BGE (containing borate, pH 9.25) are needed to fulfill the required conditions for dynamic pH junction, LVSS, and sweeping. Parameters affecting the separation and the enrichment efficiency are studied such as buffer concentration, separation voltage, capillary temperature, sample composition, and sample injection volume. Prerequisite for the developed strategy is the extraction of the nucleosides from urine using a phenylboronate affinity gel, which is described to be a unique means for the selective enrichment of cis-diol metabolites under alkaline conditions. The impact of ionic constituents remaining in the eluate after extraction on focusing efficiency and resolution is investigated. The developed method is applied to the analysis of blank and spiked urine samples. Fundamental aspects underlying the proposed enrichment procedure are discussed. A detection limit as low as 10 ng mL(-1) is achieved. To the best of our knowledge, this LOD represents the lowest LOD reported so far for the analysis of nucleosides using CE with UV detection and provides a comparable sensitivity to CE/MS. Because of the high sensitivity, the proposed method shows a great potential for the analysis of nucleosides in human urine and other types of biological fluids.

Adamantyl-group containing mixed-mode acrylamide-based continuous beds for capillary electrochromatography. Part IV: investigation of the chromatographic efficiency dependent on the retention mode.

In our previous work we have described the synthesis, characterization, and optimization of the chromatographic efficiency of a highly crosslinked macroporous mixed-mode acrylamide-based monolithic stationary phase synthesized by in situ free radical copolymerization of cyclodextrin-solubilized N-adamantyl acrylamide, piperazinediacrylamide, methacrylamide and vinylsulfonic acid in aqueous medium in pre-treated fused silica capillaries of 100µm I.D. In the present work, we study with different classes of neutral analytes (with varied hydrophobicity) the impact of the type of retention mode (influenced by the type of analyte and the mobile phase composition) and the impact of the solute functionality on the chromatographic efficiency and peak symmetry with a monolith synthesized under optimized synthesis parameters. With this monolithic capillary high separation efficiencies (up to ca. 220,000m(-1)) are obtained for the separation of different analyte classes (alkylphenones, nitrotoluenes, and phenolic compounds with k=0.2-0.55) in the reversed-phase mode, in the normal-phase mode, and in the mixed mode. For neutral alkylanilines (k<0.25) plate numbers of about 300,000m(-1) are routinely reached in the reversed-phase elution mode. For phenolic solutes separated in a mixed mode there is a solute-specific influence on peak symmetry and chromatographic efficiency. With increasing efficiency of the monolith, axial diffusion becomes an important mechanism of band broadening. For those peaks, which do not show a significant asymmetry (asymmetry factor ≤1.05), it is confirmed that plate heights gained via the tangent method are equivalent to those gained via moment analysis.

Adamantyl-group containing mixed-mode acrylamide-based continuous beds for capillary electrochromatography. Part II. Characterization of the synthesized monoliths by inverse size exclusion chromatography and scanning electron microscopy.

In our previous article we have described the synthesis of a new amphiphilic monolithic stationary phase by in situ free radical copolymerization of cyclodextrin-solubilized N-adamantyl acrylamide, piperazinediacrylamide, methacrylamide and vinylsulfonic acid in aqueous medium in pre-treated fused silica capillaries of 100µm I.D. In the present work, we study the morphology of different monolithic stationary phases synthesized under variation of the concentration of ammonium sulfate in the polymerization mixture. The pore size distribution is determined with inverse size exclusion chromatography (ISEC) using the retention data of a series of polystyrene standards with narrow molecular size distribution and known average molar mass ranging from 1560 to 2010000gmol(-1). The impact of the concentration of the lyotropic salt ammonium sulfate in the polymerization mixture on the formed morphology, the pore size distribution, and the fractional volume of mesopores and macropores is determined. The homogeneity and uniformity of the formed monolith over the length of the capillary and the covalent attachment to the confining walls are confirmed. Repetition of the synthesis procedure shows that these morphology parameters are well controlled as there is an excellent capillary-to-capillary, day-to-day, and run-to-run reproducibility reached for the electroosmotic mobility and the retention factor determined with alkylphenones in the reversed-phase mode.

Processes involved in sweeping as sample enrichment method in cyclodextrin-modified micellar electrokinetic chromatography of hydrophobic basic analytes.

Sweeping is an enrichment method in MEKC, which includes following steps: stacking/destacking of the micelles, sweeping of analyte by the stacked/destacked micelles, destacking/stacking of the swept analyte zone and additional focusing/defocusing due to the retention factor gradient effect (RFGE). In this study, we investigate additional processes, regarding online focusing in cyclodextrin-modified MEKC (CD-MEKC) of hydrophobic basic analytes: dynamic pH junction (sample with pH different from that of BGE) and adsorption of analyte onto the capillary wall within the sample zone. It is demonstrated that the developed method for the assessment of the sweeping efficiency is also applicable to CD-MEKC taking ethylparaben as an example of acidic analytes and desloratadine as an example of basic analytes using different types of ß-cyclodextrin. Our previous results regarding RFGE as an additional focusing/defocusing effect in sweeping-MEKC are confirmed for the case that the apparent distribution coefficient differs for the sample and the BGE due to a different content of the complex-forming agent cyclodextrin and due to a pH difference between the sample and the BGE. Despite being significantly more hydrophobic than ethylparaben, desloratadine shows an unexpectedly low enrichment factor. This enrichment factor is nearly unaffected by the addition of CD to the BGE. This unexpected behavior is attributed to wall adsorption of the protonated hydrophobic basic analyte within the sample zone, which significantly counteracts the sweeping process. This assumption is corroborated by an improvement in the enrichment factor achieved via addition of a dynamic coating agent (triethylamine) to the sample solution.

Adamantyl-group containing mixed-mode acrylamide-based continuous beds for capillary electrochromatography. Part III. Optimization of the chromatographic efficiency.

In a previous article we described the synthesis of amphiphilic monolithic stationary phases by in situ free radical copolymerization of cyclodextrin-solubilized N-adamantyl acrylamide, piperazinediacrylamide, methacrylamide and vinylsulfonic acid in aqueous medium in pre-treated fused silica capillaries of 100µ.m I.D. In this work, a series of N-adamantyl-group containing acrylamide-based continuous beds is synthesized under variation of different synthesis parameters. The studied synthesis parameters are (i) concentration of the lyotropic salt ammonium sulfate, (ii) concentration of the initiator ammonium persulfate, and (iii) concentration of the negatively charged monomer vinylsulfonic acid in the polymerization mixture. The influence of the synthesis parameters on the chromatographic efficiency is studied under isocratic conditions for a homologues series of alkylphenones in the reversed-phase mode at constant composition of the mobile phase via capillary electrochromatography with varied electric field strength. With varied concentration of the lyotropic salt ammonium sulfate or varied concentration of the initiator ammonium persulfate in the polymerization mixture, a strong impact on the chromatographic efficiency is observed, while there is only a minor influence when varying the molar fraction of the charged monomer VSA. The absence of a significant influence of extra-column band broadening effects on the determined efficiency is confirmed. There is a good repeatability (with respect to capillary-to-capillary variation and run-to-run variation) reached for the theoretical plate heights obtained for DMF and selected alkylphenones in the reversed-phase mode.

Imidazolium-based ionic liquid-type surfactant as pseudostationary phase in micellar electrokinetic chromatography of highly hydrophilic urinary nucleosides.

Ionic liquid (IL)-type surfactants have been shown to interact more strongly with polar compounds than traditionally used quaternary ammonium cationic surfactants. The aim of this study is to provide an alternative micellar electrokinetic chromatographic method (MEKC) for the analysis of urinary nucleosides in their ionic form at low surfactant concentration. This approach could overcome the use of high surfactant concentrations typically associated with the analysis of these highly hydrophilic metabolites as neutral species, which is frequently accompanied by high electric current, Joule heating and long analysis time. The investigated IL-type surfactant; 1-tetradecyl-3-methylimidazolium bromide (C14MImBr) is similar to the commonly employed cationic surfactant; tetradecyltrimethylammonium bromide (TTAB) but it provides a different separation selectivity. We employed C14MImBr micelles for the MEKC analysis of seven urinary nucleosides. The studied analytes possess a negative charge at pH 9.38 (exceptions are adenosine and cytidine which are neutral at this pH value). Borate imparts an additional negative charge to these compounds after complexation with the cis-diol functionality of the ribose unit, which in turn enables them to interact with the oppositely charged C14MImBr micelles via electrostatic (Coulomb) forces. The effect of the concentration of borate (the complexing, competing and buffering ion) on the effective electrophoretic mobilities and on the retention factors was investigated. The effective electrophoretic mobility data show that complexation between these nucleosides and borate occurs with high degree of complexation even at very low borate concentration (2.5 mmol L(-1) disodium tetraborate). In addition, we found that the retention factors are strongly dependent on the borate concentration being the highest when using the lowest borate concentration and they can be regulated by variation of either tetraborate concentration or the pH of the background electrolyte using only 20 mmol L(-1) C14MImBr. We confirmed also that the main mode of interaction between these analytes and the C14MImBr micelles is electrostatic interaction. Our experimental results reveal that the cationic surfactant C14MImBr exhibits superior selectivity and higher reproducibility relative to that of TTAB, which makes this surfactant a promising cationic surfactant for the MEKC separation of other hydrophilic polar analytes.

Robust analysis of the hydrophobic basic analytes loratadine and desloratadine in pharmaceutical preparations and biological fluids by sweeping-cyclodextrin-modified micellar electrokinetic chromatography.

The analysis of hydrophobic basic analytes by micellar electrokinetic chromatography (MEKC) is usually challenging because of the tendency of these analytes to be adsorbed onto the inner capillary wall in addition to the difficulty to separate these compounds as they exhibit extremely high retention factors. A robust and reliable method for the simultaneous determination of loratadine (LOR) and its major metabolite desloratadine (DSL) is developed based on cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) with acidic sample matrix and basic background electrolyte (BGE). The influence of the sample matrix on the reachable focusing efficiency is studied. It is shown that the application of a low pH sample solution mitigates problems associated with the low solubility of the hydrophobic basic analytes in aqueous solution while having advantages with regard to on-line focusing. Moreover, the use of a basic BGE reduces the adsorption of these analytes in the separation compartment. The separation of the studied analytes is achieved in less than 7min using a BGE consisting of 10mmolL(-1) disodium tetraborate buffer, pH 9.30 containing 40mmolL(-1) SDS and 20mmolL(-1) hydroxypropyl-ß-CD while the sample solution is composed of 10mmolL(-1) phosphoric acid, pH 2.15. 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 tablets without interference of tablet additives as well as the analysis of spiked human urine without any sample pretreatment. Furthermore, DSL can be detected as an impurity in LOR bulk powder at the stated pharmacopeial limit (0.1%, w/w). The selectivity of the developed method allows the analysis of LOR and DSL in combination with the co-formulated drug pseudoephedrine. It is shown that in CD-MEKC with basic BGE, solute-wall interactions are effectively suppressed allowing the development of efficient and precise methods for the determination of hydrophobic basic analytes, whereas the use of a low pH sample solution has a positive impact on the attainable sweeping efficiency without compromising peak shape and resolution.

Sweeping as a multistep enrichment process in micellar electrokinetic chromatography: the retention factor gradient effect.

The application of a new method developed for the assessment of sweeping efficiency in MEKC under homogeneous and inhomogeneous electric field conditions is extended to the general case, in which the distribution coefficient and the electric conductivity of the analyte in the sample zone and in the separation compartment are varied. As test analytes p-hydroxybenzoates (parabens), benzamide and some aromatic amines are studied under MEKC conditions with SDS as anionic surfactant. We show that in the general case - in contrast to the classical description - the obtainable enrichment factor is not only dependent on the retention factor of the analyte in the sample zone but also dependent on the retention factor in the background electrolyte (BGE). It is shown that in the general case sweeping is inherently a multistep focusing process. We describe an additional focusing/defocusing step (the retention factor gradient effect, RFGE) quantitatively by extending the classical equation employed for the description of the sweeping process with an additional focusing/defocusing factor. The validity of this equation is demonstrated experimentally (and theoretically) under variation of the organic solvent content (in the sample and/or the BGE), the type of organic solvent (in the sample and/or the BGE), the electric conductivity (in the sample), the pH (in the sample), and the concentration of surfactant (in the BGE). It is shown that very high enrichment factors can be obtained, if the pH in the sample zone makes possible to convert the analyte into a charged species that has a high distribution coefficient with respect to an oppositely charged micellar phase, while the pH in the BGE enables separation of the neutral species under moderate retention factor conditions.

Adamantyl-group containing mixed-mode acrylamide-based continuous beds for capillary electrochromatography. Part I: study of a synthesis procedure including solubilization of N-adamantyl-acrylamide via complex formation with a water-soluble cyclodextrin.

A new synthesis procedure for highly crosslinked macroporous amphiphilic N-adamantyl-functionalized mixed-mode acrylamide-based monolithic stationary phases for capillary electrochromatography (CEC) is investigated employing solubilization of the hydrophobic monomer by complexation with a cyclodextrin. N-(1-adamantyl)acrylamide is synthesized and characterized as a hydrophobic monomer forming a water soluble-inclusion complex with statistically methylated-ß-cyclodextrin. The stoichiometry, the complex formation constant and the spatial arrangement of the formed complex are determined. Mixed-mode monolithic stationary phases are synthesized by in situ free radical copolymerization of cyclodextrin-solubilized N-adamantyl acrylamide, a water soluble crosslinker (piperazinediacrylamide), a hydrophilic monomer (methacrylamide), and a negatively charged monomer (vinylsulfonic acid) in aqueous medium in bind silane-pretreated fused silica capillaries. The synthesized monolithic stationary phases are amphiphilic and can be employed in the reversed- and in the normal-phase mode (depending on the composition of the mobile phase), which is demonstrated with polar and non-polar analytes. Observations made with polar analytes and polar mobile phase can only be explained by a mixed-mode retention mechanism. The influence of the total monomer concentration (%T) on the chromatographic properties, the electroosmotic mobility, and on the specific permeability is investigated. With a homologues series of alkylphenones it is confirmed that the hydrophobicity (methylene selectivity) of the stationary phase increases with increasing mass fraction of N-(1-adamantyl)acrylamide in the synthesis mixture.

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).

Sensitivity enhancement in near-field photothermal-lens detection in capillary electrophoresis using laser-induced online precipitation.

This paper reports simultaneous photoinduced precipitation-based online preconcentration of target analytes at the inner walls in capillary zone electrophoresis (CZE) and surface-enhanced near-field crossed-beam photothermal-lens detection of the preconcentrated analytes. A simple technique using online readjustment of the optical scheme of the thermal-lens detector in the course of the separation for gaining optimum sensitivity for both water-soluble and precipitated analytes is proposed. It provides a considerable decrease in the limits of detection (LOD) with good concordance with the previously developed theoretical approach to this combination (D. A. Nedosekin, W. Faubel, M. A. Proskurnin, and U. Pyell, Talanta, 78, 682-690 (2009)). As a result, an enhancement of more than an order of magnitude in the limit of detection of the photoactive 4-aminoazobenzene compared to conventional thermal-lens detection in CZE is achieved while retaining very good sensitivity for unabsorbed analyte (Mordant Yellow 7). The application of the thermal-lens detector to the investigation of laser-induced reactions in flow in capillaries is discussed.

Regulation of the retention factor for weak acids in micellar electrokinetic chromatography with cationic surfactant via variation of the chloride concentration.

For tetradecyltrimethylammonium bromide in boric acid/borate or acetic acid/acetate buffer and NaCl or CaCl2 as the added salt, it is investigated whether the retention behaviour of weak acids in MEKC with cationic surfactant can be modelled by assuming for the deprotonated species predominantly electrostatic interaction with the micelles acting as a pseudostationary ion-exchanger. The retention of (partially) charged solutes by oppositely charged micelles is analyzed by applying the classical theory of IEC (plotting lg k against lg(c(Cl⁻)) assuming a fixed concentration of ion-exchange sites. When plotting the absolute slopes of the regression lines against the absolute effective charge numbers of the analytes, correlation coefficients of 0.968-0.998 were obtained. It is shown that the dependence of the retention factor on the concentration of chloride (the competing ion) in the separation electrolyte and on the degree of dissociation of the analyte corresponds to what would be expected for mixed-mode retention (hydrophobic and ion-exchange interaction) on a pseudostationary ion-exchanger.