Electrokinetic Chromatography System Research Articles

The role of deep eutectic solvents in chiral capillary electrokinetic chromatography: A comparative study based on α-cyclodextrin chiral selector

• The first investigation of the effect of HBAs of DESs on chiral EKC. • The comparison of the performance of chiral and achiral DESs in chiral EKC. • The comparison of the performance of DESs and ILs as additives in chiral EKC. • The objective evaluation of the feasibility of DESs as additives in chiral EKC. Deep eutectic solvents (DESs) are emerging as potential candidates as additives/modifiers in chiral electrokinetic chromatography (EKC). However, the true role of DESs in the chiral separation process is far from being fully elucidated. Since most previous studies have been restricted to using choline chloride as the hydrogen-bond acceptor (HBA) in DESs, here we first evaluated the effect of five types of DESs (containing different HBAs) on the enantioseparation performance of an α-CD-based EKC system. The results demonstrate that these DESs are capable of moderately improving the enantioresolutions of several model drugs. A series of comparative experiments indicate that the improvement is mainly due to the contribution of the HBAs, rather than the selected hydrogen-bond donors (HBDs). Considering that most of these DESs are achiral, we further prepared five chiral DESs, aiming to investigate whether there is a synergistic effect between α-cyclodextrin and chiral DESs. However, these chiral DESs did not show obvious superiority compared with achiral DESs. This conclusion is different from the use of achiral or chiral ionic liquids (ILs) as additives in chiral EKC, in which chiral ILs usually exhibit significantly better enantioseparation performance than achiral ILs. Another important conclusion of this work is that there seems to be no significant difference between using DESs and their individual components, which is also different from the performance of most previously reported ILs. This conclusion indicates that the DESs may be virtually completely dissociated into their initial components (HBAs + HBDs) in the separation medium due to the destruction of their hydrogen-bond network. Therefore, a question which deserves particular attention of researchers in this field is raised here: whether it is absolutely necessary to use DESs in chiral EKC since adding HBAs and HBDs separately seems to have the same function?

Estimation of the octanol-water distribution coefficient of basic compounds by a cationic microemulsion electrokinetic chromatography system.

The octanol-water partition coefficient (Po/w), or the octanol-water distribution coefficient (Do/w) for ionized compounds, is a key parameter in the drug development process. In a previous work, this parameter was estimated through the retention factor measurements in a sodium dodecyl sulfate (SDS) - microemulsion electrokinetic chromatography (MEEKC) system for acidic compounds. Nonetheless, when ionized basic compounds were analyzed, undesirable ion pairs were formed with the anionic surfactant and avoided a good estimation of log Do/w. For this reason, an alternative MEEKC system based on a cationic surfactant has been evaluated to estimate Po/w or Do/w of neutral compounds and ionized bases. To this end, it has been characterized through the solvation parameter model (SPM) and compared to the octanol-water partition system. Results pointed out that both systems show a similar partition behavior. Hence, the log Po/w of a set of neutral compounds has been successfully correlated against the logarithm of the retention factor (log k) determined in this MEEKC system. Then, the log Do/w of 6 model bases have been estimated at different pH values and they have been compared to data from the literature, determined by the reference shake-flask and potentiometric methods. Good agreement has been observed between the literature and the estimated values when the base is neutral or partially ionized (up to 99% of ionization).

Evaluation of the enantioselectivity of capillary electrokinetic chromatography using ethanediamine-bonded poly (glycidyl methacrylate) microspheres as the pseudostationary phases.

In this work, a new capillary electrokinetic chromatography (EKC) approach using ethanediamine-bonded poly (glycidyl methacrylate) (Ami-PGMA) microspheres as pseudostationary phases (PSPs) for enantioseparation with a polysaccharide, chondroitin sulfate E (CSE), as the chiral selector. The CSE@Ami-PGMA EKC system was applied to enantioseparate basic drugs, and distinct improved separations of tested enantiomers were obtained while comparing with the single CSE system (the resolution increased from 0.41 to 1.26 for nefopam, from 1.24 to 2.15 for laudanosine, and from 0.92 to 2.36 for amlodipine). The Ami-PGMA microspheres were fully characterized by scanning electron microscopy (SEM) and Fourier Transform Infrared (FT-IR) spectroscopy, and the results showed Ami-PGMA microspheres were uniform and spherical in size (1μm). Several principal parameters were systematically investigated, and the optimal chiral separations were obtained with Tris/H3 PO4 (20mM, pH2.4, and 3.4 for NEF) containing 2.5% (w/v) CSE and 20-μg Ami-PGMA microspheres in 20°C. Subsequently, the concentrations of Ami-PGMA microspheres and CSE were proved to be the dominant factors for the separation in the CSE@Ami-PGMA EKC system by Statistical Product and Service Solutions (SPSS).

Generalized model of the linear theory of electromigration and its application to electrokinetic chromatography: Theory and software PeakMaster 6-Next Generation.

The linear theory of electromigration, including the first-order nonlinear approximation, is generalized to systems with any equilibria fast enough to be considered instantaneous in comparison with the timescale of peak movement. For example, this theory is practically applied in the electrokinetic chromatography (EKC) mode of the CZE. The model enables the calculation of positions and shapes of analyte and system peaks without restricting the number of selectors, the complexation stoichiometry, or simultaneous acid-base equilibria. The latest version of our PeakMaster software, PeakMaster 6-Next Generation, implements the theory in a user-friendly way. It is a free and open-source software that performs all calculations and shows the properties of the background electrolyte and the expected electropherogram within a few seconds. In this paper, we mathematically derive the model, discuss its applicability to EKC systems, and introduce the PeakMaster 6 software.

Moment analysis for mass transfer kinetics at the interface of spherical molecular aggregates

New moment equations were developed on the basis of the principle of relativity for explaining some characteristics of elution peaks measured by electrokinetic chromatography (EKC) using spherical molecular aggregates. Basic equations representing mass balance and mass transfer kinetics in EKC system in a Galilean coordinate system S were transformed to those in another coordinate system S’, which imaginarily moved with respect to S. Moment equations for EKC peaks in S’ in the time domain were derived from the analytical solution of the modified basic equations in the Laplace domain. Moment equations for EKC peaks in S were derived from those in S’ by the inverse Galilean transformation. The moment equations were used to the re-analysis of EKC data previously measured. The values of permeation rate constants of thymol at the interface of sodium dodecylsulfate micelles were fairly in agreement with those determined in a previous study. The moment equations were also used to the numerical simulation of elution peaks in EKC systems. The influence of some experimental parameters on elution peak profiles was quantitatively analyzed. The moment equations are useful for determining the rate constants of interfacial solute permeation from elution peak profiles measured by EKC.

Moment Analysis of Mass Transfer Kinetics in Micellar Electrokinetic Chromatography 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.

Evaluation of the suitability of chromatographic systems to predict human skin permeation of neutral compounds

Several chromatographic systems (three systems of high-performance liquid chromatography and two micellar electrokinetic chromatography systems) besides the reference octanol–water partition system are evaluated by a systematic procedure previously proposed in order to know their ability to model human skin permeation. The precision achieved when skin-water permeability coefficients are correlated against chromatographic retention factors is predicted within the framework of the solvation parameter model. It consists in estimating the contribution of error due to the biological and chromatographic data, as well as the error coming from the dissimilarity between the human skin permeation and the chromatographic systems. Both predictions and experimental tests show that all correlations are greatly affected by the considerable uncertainty of the skin permeability data and the error associated to the dissimilarity between the systems. Correlations with much better predictive abilities are achieved when the volume of the solute is used as additional variable, which illustrates the main roles of both lipophilicity and size of the solute to penetrate through the skin. In this way, the considered systems are able to give precise estimations of human skin permeability coefficients. In particular, the HPLC systems with common C18 columns provide the best performances in emulating the permeation of neutral compounds from aqueous solution through the human skin. As a result, a methodology based on easy, fast, and economical HPLC measurements in a common C18 column has been developed. After a validation based on training and test sets, the method has been applied with good results to the estimation of skin permeation of several hormones and pesticides.

Performance of chromatographic systems to model soil–water sorption

A systematic approach for evaluating the goodness of chromatographic systems to model the sorption of neutral organic compounds by soil from water is presented in this work. It is based on the examination of the three sources of error that determine the overall variance obtained when soil–water partition coefficients are correlated against chromatographic retention factors: the variance of the soil–water sorption data, the variance of the chromatographic data, and the variance attributed to the dissimilarity between the two systems. These contributions of variance are easily predicted through the characterization of the systems by the solvation parameter model. According to this method, several chromatographic systems besides the reference octanol–water partition system have been selected to test their performance in the emulation of soil–water sorption. The results from the experimental correlations agree with the predicted variances. The high-performance liquid chromatography system based on an immobilized artificial membrane and the micellar electrokinetic chromatography systems of sodium dodecylsulfate and sodium taurocholate provide the most precise correlation models. They have shown to predict well soil–water sorption coefficients of several tested herbicides. Octanol–water partitions and high-performance liquid chromatography measurements using C18 columns are less suited for the estimation of soil–water partition coefficients.

Micellar electrokinetic chromatography systems for the separation of mixtures of charged and uncharged compounds

In this study, the migration behavior of charged and uncharged analytes was investigated under different conditions. Effective mobilities - electrophoretic mobilities under the influence of micelles - of cations, anions, and neutrals were measured at neutral, basic, and acidic pH (7.5, 11, and 2.2) using background electrolytes containing different sodium dodecyl sulfate (SDS) concentrations (0-90 mM) and acetonitrile (ACN) proportions (0-75%). SDS concentration and ACN proportion were found to have a tremendous effect on the effective mobilities and migration order of the model compounds. Although the SDS micelles preferably interact with neutrals and cations, hydrophobic bonds can also occur with anions. Cations, anions, and neutrals having rather different migration behaviors, it is possible to considerably enhance the selectivity of the method by adjusting properly the SDS concentration and the ACN proportion. These observations confirm the interest of using micellar electrokinetic chromatography not only for the separation of neutral substances but also to analyze charged compounds.

Modeling Nonspecific Toxicity of Organic Compounds to the Fathead Minnow Fish by Means of Chromatographic Systems

The performance of chromatographic systems to mimic aquatic toxicity to the fathead minnow fish is evaluated taking into account the factors that contribute to the variance of biological-chromatographic correlations. These factors are the precision to measure the fathead minnow toxicity, the precision of the surrogate chromatographic system, and the error from the dissimilarity between the fathead minnow and chromatographic systems. The precisions are estimated through the characterization of the systems by the solvation parameter model. Several chromatographic systems as well as the common reference octanol-water partition system have been selected to test their ability to model the nonspecific toxicity to the fathead minnow by means of the proposed approach. Predictions and experimental tests show that the micellar electrokinetic chromatography system of sodium taurocholate and chromatographic measurements using an immobilized artificial membrane column provide the most precise estimations of this biopartitioning property. The octanol-water partition system, the conventional C18 high-performance liquid chromatography systems, and the micellar electrokinetic chromatography system of sodium dodecylsulfate show worse performances.

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

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

Linear Free Energy Relationship Analysis of Retention Factors in Cerasome Electrokinetic Chromatography Intended for Predicting Drug Skin Permeation

The retention factors of neutral, positively charged, and negatively charged solutes were determined in a liposome electrokinetic chromatography (EKC) system, where cerasome was used as the investigated liposome. The Abraham linear free energy relationship (LFER) for neutral and ionized solutes gave a good account of the retention factors (N = 71, R(2) = 0.814, and SD = 0.29 log units). It was shown that the calculated retention factors for 16 neutral acids were about four times higher than those of the corresponding anions, whereas the calculated retention factors for neutral bases were less than those for the corresponding cations by a factor of 0.36. The LFER equation for neutral species, anions, and cations was compared with those for partition from water into a number of solvents and for n-octanol-water distribution coefficients. It was shown that the cerasome EKC system is substantially different to the other systems and consequently it could be a very useful additional model system, possibly for predicting skin permeation. It was further shown that there are considerable advantages in the use of Abraham LFERs that can encompass not only neutral molecules but also ionic species.

Novel and highly sensitive mixed-polymeric electrokinetic chromatography system for determination of contaminants and impurities of heparin samples

A mixed-polymeric electrokinetic chromatography system has been developed for the simultaneous determination of a contaminant like oversulfated condroitin sulfate (OSCS) and impurities expressed as dermatan (Der) in heparin (Hep) samples. The EKC system consisted of 0.5% w/v polymeric β-CD, 0.4% w/v tetronic(®) 1107 and 400 mM tris-phosphate buffer at pH 3.5. The optimized electrophoretic conditions included the use of an uncoated-silica capillary of 50 cm of total length and 75 μm id, an applied voltage of -7 kV, a temperature of 30°C and 200 nm UV-detection. The highly sensitive method developed showed low values of LOD, 0.07% w/w (0.07 μg/mL) (OSCS) and 0.1% w/w (0.1 μg/mL) (Der), and values of LOQ 0.2% w/w (0.2 μg/mL) (OSCS) and 0.3% w/w (0.3 μg/mL) (Der) with a concentration level of Hep sample as low as 0.1 mg/mL. Additional parameters of validation such as specificity, linearity, accuracy, and robustness were evaluated according to international guidelines. Owing to its simplicity, high sensitivity, and reliability, the proposed method can be an advantageous alternative to the traditional methodologies for the analysis of Hep in raw material and specially in finished products because of the low amounts of Hep sample required.

Comparison of the retention characteristics of different pseudostationary phases for microemulsion and micellar electrokinetic chromatography of betamethasone and derivatives.

Five electrokinetic chromatography systems were compared concerning retention behavior and lipophilicity. Comparison was based on capacity (retention) factors of some steroidal drugs, and on log P(OW) values derived by the aid of reference substances. In all systems the aqueous buffer consisted of phosphate (20 mM, pH 7.5). Two systems had micelles, three systems microdroplets as negatively charged pseudostationary phases. The micelles were formed by sodium dodecyl sulfate (SDS) and sodium cholate, respectively. One microemulsion consisted (as usual) from octane as oil, butanol as cosurfactant and SDS as charged tenside. Two microemulsions were made from biosurfactants (phosphatidylcholine, isopropylmyristate) to better simulate biopartitioning of the drugs. Even for noncharged analytes a change in migration sequence and thus in log P(OW) was observed for the systems consisting of the biosurfactants, compared to the others. For the former systems, log P(OW) derived from the capacity factors agree for all analytes with those obtained from calculation by computer software based on the structure of the drugs, and with experimental data directly obtained from octanol/water partitioning.

On-line preconcentration and enantioselective separation of triadimenol by electrokinetic chromatography using cyclodextrins as chiral selectors

Enantioselective separation of triadimenol, a component of systemic agricultural fungicide, by electrokinetic chromatography (EKC) using cyclodextrins (CDs) as chiral selectors was investigated. Both a neutral CD derivative, hydroxypropyl-γ-CD (HP-γ-CD), and an ionic one, heptakis-6-sulfato-β-CD (HS-β-CD), were employed as an additive in cyclodextrin–modified micellar electrokinetic chromatography (CD–MEKC) and as a chiral pseudostationary phase in CDEKC, respectively. In each system, four stereoisomeric peaks were completely or partially separated from each other. To enhance the detectability or the concentration sensitivity, on-line preconcentration techniques were applied to both EKC systems. Sweeping was used in the CD–MEKC system under an acidic condition, whereas stacking with a reverse migrating pseudostationary phase (SRMP) in the CDEKC system. Around 10-fold increase in the detection sensitivity for each peak was attained with both sweeping and SRMP systems. Good repeatabilities in the migration time, corrected peak area, and peak height were recognized in terms of the relative standard deviation. The limit of detection for each peak in the SRMP-CDEKC system, calculated from the calibration curve, was found to be 0.8–3.8 ppm.

Electrokinetic stacking injection of neutral analytes under continuous conductivity conditions.

In capillary electrokinetic chromatography, neutral analytes can be injected by electroosmotic flow directly from a sample matrix into a separation buffer containing an electrokinetic vector with an opposite mobility. Analytes are injected at the velocity of electroosmotic flow but are retained at the interface of the sample matrix co-ion and separation buffer micelle zones as analyte/micelle complexes. A simple electrokinetic chromatography system containing sodium dodecyl sulfate as the micellar agent with borate as the buffering electrolyte included in the separation buffer and in the sample matrix to provide continuous conductivity was investigated. Concentrations of the micelle, methanol, and borate in the separation buffer were explored to increase maximum injection length of neutral analytes. Reducing the analyte velocity in the separation buffer without substantially decreasing the velocity of the analyte during injection from the sample vial allowed greatly extended sample plug injection lengths. It is presently possible to inject sample solvent volumes equivalent to approximately 7 effective capillary lengths (180 cm) with a 50-microm-i.d. capillary (24.5 cm effective capillary length), total volume of sample injection approximately 3.5 microL Equations describing the injection process and maximum injection lengths for this mode of stacking in electrokinetic capillary chromatography are introduced. The result of this work leads to a postulated generalization of electrokinetic stacking injection maximums for electrophoretic processes, and the concept of orthogonal analyte stacking/injection systems is discussed.

Solute–solvent interactions in micellar electrokinetic chromatography: III. Characterization of the selectivity of micellar electrokinetic chromatography systems

Several micellar electrokinetic chromatography (MEKC) systems (sodium dodecyl sulfate, lithium dodecyl sulfate, lithium perfluorooctanesulfonate, sodium cholate, sodium deoxycholate, tetradecyltrimethylammonium bromide and hexadecyltrimethylammonium bromide) have been characterized by means of the solvation parameter model. It has been observed that the coefficients of the correlation equations depend strongly on the particular set of compounds analyzed. Principal component analysis has been used to characterize the 2975 compounds with available solute descriptors and to select an appropriate subset of compounds to be analyzed by MEKC. With this set of compounds, the MEKC systems have been characterized. Principal component analysis has also been used to show the similarities and differences between the properties of the surfactants characterized by MEKC.

Enantioseparation of ofloxacin in urine by capillary electrokinetic chromatography using charged cyclodextrins as chiral selectors and assessment of enantioconversion.

A method was developed for the enantioseparation of ofloxacin, a member of the fluoroquinolones, using an anionic cyclodextrin-derivative with or without combination with a neutral cyclodextrin-derivative, as the chiral selector (s) in an electrokinetic chromatography system. The best results were obtained with 0.35 mM sulfated beta-cyclodextrin dissolved in a 50 mM phosphate buffer, pH 2.5, and at 15 degrees C. Under these conditions, a resolution of 2 was readily achieved. Furthermore, under adequate separation conditions, studies were performed in order to assess possible in vitro and in vivo enantioconversion of levofloxacin. The current method allows detection of 2 microg R-(+)-ofloxacine/mL diluted urine without the necessity of sample cleanup.

Open access capillary electrophoresis: A walk up capillary electrophoresis service for the synthetic chemist

A capillary electrophoresis (CE) system has been developed to provide routine purity information to support synthetic organic chemists. A single micellar electrokinetic chromatography system that produces good selectivity with short run times was developed. The instrument operating software has been modified to run separations in a custom open access mode. No expert knowledge of CE is required to run separations.

Microemulsion and micellar electrokinetic chromatography of steroids

A mixture of ten steroids was separated by microemulsion and micellar (SDS and glycodeoxyholate) electrokinetic chromatography systems. Separations were done on a 50 cm (to the detector) × 50 μm I.D. fused-silica capillary. Complete separation of all the test compounds in the micellar mode was obtained with glycodeoxycholate (50 m M) in 25 m M borate buffer, pH 6.5, as the micelle-forming agent. The best results, however, were obtained using microemulsion electrokinetic chromatography in which higher aliphatic alcohols were used as the microemulsion-forming modifiers. The system consisted of n-hexanol (0.81%), SDS (3.31%) and n-butanol (6.61%) in 20 m M phosphate buffer, pH 10.0 (89.28%, w/w). In the microemulsion mode, linear calibration for steroid standards was obtained in the concentration range 3 × 10 −4 − 3 × 10 −5 mol l −1 with a detection limit of 1 pmol. The method was validated and applied to an 11β-hydroxysteroid dehydrogenase assay in tissues.