Physicochemical characterization of phosphinic pseudopeptides by capillary zone electrophoresis in highly acidic background electrolytes

Phosphinic pseudopeptides (i.e., peptide isosteres with one peptide bond replaced by a phosphinic acid moiety) were analyzed and physicochemically characterized by capillary zone electrophoresis in the pH range of 1.1-3.2, employing phosphoric, phosphinic, oxalic and dichloroacetic acids as background electrolyte (BGE) constituents. The acid dissociation constant (pK(a)) of phosphinate group in phosphinic pseudopeptides and ionic mobilities of these analytes were determined from the pH dependence of their effective electrophoretic mobilities corrected to standard temperature and constant ionic strength of the BGEs. It was shown that these corrections are necessary whenever precise mobility data at very low pH are to be determined. Additionally, it was found that the ionic mobilities of the phosphinic pseudopeptides and pK(a) of their phosphinate group are affected by the BGE constituent used. The variability of migration behavior of the pseudopeptides can be attributed to their ion-pairing formation with the BGE components.     

Investigation of the effect of ionic strength of Tris-acetate background electrolyte on electrophoretic mobilities of mono-, di-, and trivalent organic anions by capillary electrophoresis

The effect of ionic strength of the background electrolyte (BGE) composed of tris(hydroxymethyl)aminomethane (Tris) and acetic acid on the electrophoretic mobility of mono-, di- and trivalent anions of aliphatic and aromatic carboxylic and sulfonic acids was investigated by capillary zone electrophoresis (CZE). Actual ionic mobilities of the above anions were determined from their CZE separations in Tris-acetate BGEs of pH 8.1 to 8.2 in the 3 to 100 mM ionic strength interval at constant temperature (25 degrees C). It was found that the ionic strength dependence of experimentally determined actual ionic mobilities does not follow the course supposed by the classical Onsager theory. A steeper decrease of actual ionic mobilities with the increasing ionic strength of BGE and a higher estimated limiting mobility of the anions than that found in the literature could be attributed to the specific behavior of the Tris-acetate BGEs. Presumably, not only a single type of interaction of anionic analytes with BGE constituents but rather the combination of effects, such as ion association or complexation equilibria, seems to be responsible for the observed deviation of the concentration dependence of the actual ionic mobilities from the Onsager theory. Additionally, several methods for the determination of limiting ionic mobilities from CZE measured actual ionic mobilities were evaluated. It turned out that the determined limiting ionic mobilities significantly depend on the calculation procedure used.     

Nonaqueous CE using contactless conductivity detection and ionic liquids as BGEs in ACN

N,N'-Alkylmethylimidazolium cations have been separated in NACE when one of the N,N'-dialkylimidazolium salts (ionic liquids (ILs)) was used as an electrolyte additive to the organic solvent separation medium. The separated species were 1-methyl-, 1-ethyl-, 1-butyl-, 1-octyl-, 1-decyl-3-methylimidazolium and N-butyl-3-methylpyridinium cations and BGE composed of 1-ethyl-3-methylimidazolium ethylsulfate or 1-butyl-3-methylimidazolium trifluoroacetate [BMIm][FAcO] (A6; B2) diluted in ACN. It was demonstrated that contactless conductivity detection (CCD) may be applied to monitoring the separation process in nonaqueous separation media, allowing to use the UV light-absorbing imidazolium-based electrolyte additives. There could be marked three concentration regions of added ILs; at first ionic strength of BGE below 1-2 mM, and then the actual electrophoretic mobility of analytes rises from 0. At concentrations above 1-2 mM, the added IL facilitated separation. In concentration region of 1-20 mM, the actual electrophoretic mobility of analyzed imidazolium cations was increasing with decrease in separation medium ionic strength. At higher concentrations of BGE (above 30 mM), the conductivity of the separation media became too high for this detector. Some organic dyes were also successfully separated and detected by contactless conductivity detector in a 20 mM A6 separation electrolyte in ACN.     

Formamide as an organic modifier in MEKC with SDS

The effect of formamide (FA) as a modifier on the retention in MEKC with SDS as the detergent was investigated. The mobility of a series of alkylphenones and of a zwitterionic fluorescent compound as a function of the FA and the SDS concentration was determined for this purpose. Buffering electrolyte was borate, pH 9.23, with total ionic strength of 50 mM. The dependence of the mobility on the FA content – up to 63% w/w – of the BGE (at 10 mM SDS) allows the conclusion that the micelles are destabilized, and the CMC is shifted to higher values. In the system containing 33% FA or more no micelles are present anymore, and the retention factors of all compounds tend to zero. In an MEKC system with 27% v/v FA the CMC of SDS is increased from 2.4 mM in the aqueous BGE with the same buffer composition to 9.7 mM, a behavior that is in contrast to electrolyte‐free FA–water systems. The partition constants of free analytes and the formation constants of the adduct between analyte and detergent monomer (assuming 1:1 stoichiometry) were derived from the dependence of the mobility on the SDS concentration. In addition, the involved equilibria were extended by that from the distribution of the analyte–monomer adduct between aqueous and micellar phase, and the according partition constants were derived as well. A selective change in the extent of partitioning was observed for the zwitterionic compound. In general, all binding constants were decreased upon addition of FA, though to a different extent. Although the binding constants of the analyte–monomer associate were only slightly influenced, the most pronounced decrease is found for their partitioning into the micelles.     

Cation effects in the separation of calix[4]pyrroles by nonaqueous capillary electrophoresis with tetraalkylammonium chloride salts as background electrolytes

The migration behavior of three calix[4]pyrroles (C4Ps) in acetonitrile (ACN) and in the mixtures of ACN/ethanol (EtOH) (95:5; v/v)with tetraalkylammonium chloride as background electrolyte (BGE) was studied. Electroosmotic flow (EOF) mobilities and absolute mobilities in ACN and mixture of ACN/EtOH (95:5; v/v) were investigated. A decrease of EOF mobilities in EtOH/ACN was observed compared with that of in pure ACN. Variation of the C4Ps effective mobilities as a function of the square root of concentration of tetraakylammonium chloride salts such as tetramethylammonium chloride (Me4NCl), tetraethylammonium chloride (Et4NCl) and tetrabutylammonium chloride (Bu4NCl) in both solvents was plotted, and straight lines were exhibited when Bu(4)NCl was used as BGE. Absolute mobilities of C4Ps were obtained by extrapolation from the mentioned plots. It was found that the absolute mobilities of C4Ps is independent of the nature of BGE and the values are: meso-octamethylcalix[4]pyrrole (OMCP)>meso-tetracyclopentylcalix[4]pyrrole (TPCP)>meso-tetracyclohexylcalix[4]pyrrole (THCP). The ion-pairing constants follow the order under given solvent: Me4N+>Et4N+>Bu4N+, and ion-pairing constant of C4P follows the order: THCP>TPCP>OMCP. As though, ion-pairing association is intensified by steric hindrance.     

Capillary electrophoresis of boron cluster compounds in aqueous and nonaqueous solvents

The electrophoretic properties of boron cluster compounds were determined in water, methanol and ACN as solvents of the BGE and discussed based on the principles of ion migration. Two types of boron cluster compounds were investigated. One type consisted of derivatives of the nido-7,8-dicarbaundecaborate cluster, the other types are derivatized cobalt bis(dicarbollide) ions (COSANs) whose central cobalt atom is sandwiched by two 7,8-dicarbaundecaborate clusters. The BGE in all solvents was acetate/acetic acid buffer with pH 4.75 in water, 9.7 in methanol and 22.3 in ACN, respectively, at different ionic strength between 5 and 30 mM. The dependence of the mobility on ionic strength could not be explained by the theory of Debye, Hückel and Onsager, but good agreement was found upon considering an ion size parameter. Limiting mobilities were derived by curve fitting, and by the aid of the solvent viscosities the hydrodynamic radii of the analyte anions were calculated. They are between 0.25 and 0.48 nm, and were nearly independent of the solvent. Electrophoresis of the analytes in a BGE consisting of 6 mM perchloric acid in ACN allows the conclusion that the present boron cluster compounds behave as stronger acids than perchloric acid.     

ACE applied to the quantitative characterization of benzo‐18‐crown‐6‐ether binding with alkali metal ions in a methanol–water solvent system

ACE was applied to the quantitative evaluation of noncovalent binding interactions between benzo‐18‐crown‐6‐ether (B18C6) and several alkali metal ions, Li⁺, Na⁺, K⁺, Rb⁺ and Cs⁺, in a mixed binary solvent system, methanol–water (50/50 v/v). The apparent binding (stability) constants (K_b) of B18C6–alkali metal ion complexes in the hydro‐organic medium above were determined from the dependence of the effective electrophoretic mobility of B18C6 on the concentration of alkali metal ions in the BGE using a nonlinear regression analysis. Before regression analysis, the mobilities measured by ACE at ambient temperature and variable ionic strength of the BGE were corrected by a new procedure to the reference temperature, 25°C, and the constant ionic strength, 10 mM . In the 50% v/v methanol–water solvent system, like in pure methanol, B18C6 formed the strongest complex with potassium ion (log K_b=2.89±0.17), the weakest complex with cesium ion (log K_b=2.04±0.20), and no complexation was observed between B18C6 and the lithium ion. In the mixed methanol–water solvent system, the binding constants of the complexes above were found to be about two orders lower than in methanol and about one order higher than in water.     

Hydrogen-bond effect and ion-pair association in the separation of neutral calix[4]pyrroles by nonaqueous capillary electrophoresis

A study on the migration behavior of four neutral macrocyclic compounds calix[4]pyrroles (C4Ps) by nonaqueous capillary electrophoresis (NACE) with tetrabutylammonium halide salts as background electrolyte (BGE) is described. Systematic studies were carried out with different BGE in acetonitrile (ACN) to elucidate the involved phenomena. The effect of BGE concentration on analytes effective mobilities mu eff,i was studied. Rough values of analytes absolute mobilities mu infinity,i were obtained by extrapolation of effective mobilities to zero ionic strength of BGE. Stokes radius of C4Ps in the form of C4PF(-) and C4PCl(-) was calculated. The complex constants of C4Ps with F(-) anion and Cl(-) anion and ion-pair association constants of each C4PF(-) and C4PCl(-) with tetrabutylammonium cations was evaluated and confronted. Hydrogen bonding effect is prerequisite in the separation; meanwhile ion-pair association which is intensified by steric hindrance effect plays an important role.     

Nonaqueous capillary electrophoretic behavior of 2-aryl propionic acids in the presence of an achiral ionic liquid. A chemometric approach

Ionic liquids (ILs) appear really attractive as electrolyte additives in nonaqueous capillary electrophoresis (NACE). These salts may offer new possibilities of interactions to modulate analyte effective mobilities. The presence of 1-n-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide (BMIM NTf2) in acetonitrile/alcohol background electrolytes (BGEs) was investigated in this work. The aim of this study was to elucidate the influence of the IL concentration on the electrophoretic behavior of four arylpropionic acids and to identify the interactions between the analytes and the IL cation. The influence on mobility of the IL concentration, the nature and the proportion of the organic solvents, and the concentration of the ionic components of the BGE was first studied by a univariate approach. A four-factor D-optimal experimental design was then applied to provide a deeper insight into analyte interaction with IL cation present both free in BGE and adsorbed onto the capillary wall.     

Determination of synthetic polypeptide conformations and molecular geometrical parameters by nonaqueous CE

The aim of this work was to study changes in homopolypeptide chain conformation as a function of the number of residues by the modeling of the electrophoretic mobility. For this purpose, the frictional coefficients of poly(N(epsilon)-trifluoroacetyl-L-lysine) with different number of residues (up to 11) were determined from the absolute ionic mobilities and modeled by the hydrodynamic frictional coefficient of an equivalent cylinder. This approach allowed determination of geometrical parameters of the polypeptide chain in a liquid phase (nonaqueous solution of the BGE). The fact that the BGE and analyte are dissolved in mixed (methanol-ACN) organic solvent implied to take into account different effects and corrections that are generally not considered in aqueous solvent: namely, the effect of ion-pairs between constituents of the BGE for the calculation of the ionic strength, the effect of ion-pairs between the solutes and the electrolyte counterions and the correction due to the dielectric friction (Hubbard-Onsager equations). In addition, the influence of the ionic strength on the electrophoretic mobility was corrected using the Pitts equation, and the effect of lateral charges due to a slight deprotonation of the -NH- group in the lateral chain was also considered. From this modeling, molecular geometrical parameters relative to the linear and helico?dal conformations were obtained with very good correlation coefficients. Interestingly, this work also points out that the use of ionic mobility modeling for extracting molecular geometrical parameters can also be applied to end-charged polypeptides with slightly charged lateral chains (3% of elementary charge per residue).     

Synergistic effects of ion-pairing in the enantiomeric separation of basic compounds with cyclodextrin derivatives in nonaqueous capillary electrophoresis

The enantiomeric separation of various kinds of basic pharmaceuticals has been investigated in nonaqueous capillary electrophoresis (NACE) systems using an ion-pairing reagent in combination with cyclodextrins (CDs). The simultaneous addition to the methanolic background electrolyte (BGE) of (+)-S-camphorsulfonate or alkanesulfonates and an anionic beta-cyclodextrin derivative, heptakis(2,3-dimethyl-6-sulfato)-beta-cyclodextrin (HDMS-beta-CD), led to partial or complete enantioresolution in most cases. In the absence of ion-pairing reagent, the enantiomeric resolution obtained with this CD derivative was most often completely lost or strongly reduced, indicating the important role of ion-pairing in the chiral recognition mechanism in these NACE systems. The influence of the nature and concentration of the counterion and the anionic CD derivative on the enantioseparation of basic compounds was studied. Synergistic effects between these two kinds of charged additives were clearly observed.     

High-performance size-exclusion chromatography of polypeptides on a TSK G2000SW column in acidic mobile phases

Polypeptides subjected to high-performance liquid chromatography on a TSK G2000SW column in acidic mobile phases of low ionic strength underwent a combination of size exclusion and ion exclusion from the matrix. The distribution coefficient (Kd) of each polypeptide was dependent upon the concentration of ion-pairing agent present and increased as the concentration of ion-pairing agent increased. The fractionation range of the column could thus be manipulated by altering the concentration of acid in the mobile phase. The nature of the ion-pairing acid, in terms of hydrophilic or hydrophobic ion pairing, also had an effect on the chromatography, such that hydrophobic ion-pairing agents lead to increased Kd values, especially in the case of the most basic polypeptides. To account for these results it is proposed that the TSK GSW matrix is cationic at low pH, and that ion pairing suppresses the ion exclusion experienced by cationic polypeptides under these conditions.     

Optimization of the chiral resolution of baclofen by capillary electrophoresis using beta-cyclodextrin as the chiral selector

The chiral resolution of baclofen was achieved by capillary electrophoresis using a fused-silica capillary (60 cm x 75 microm ID). The background electrolyte (BGE) was phosphate buffer (pH 7.0, 50 mM)-acetonitrile (95:5 v/v) containing 10 mM beta-cyclodextrin. The applied voltage was 15 kV. The values of alpha and R(s) were 1.06 and 1.00, respectively. The electrophoretic conditions were optimized varying the pH and the ionic strength of the BGE, concentrations of beta-cyclodextrin and acetonitrile and the applied voltage.     

Optimization of sample stacking for the simultaneous determination of nonsteroidal anti-inflammatory drugs with a wall-coated histidine capillary column

A wall-coated histidine capillary column was developed for the on-line preconcentration of nonsteroidal anti-inflammatory drugs (NSAIDs) in capillary electrochromatography (CEC). A wide variety of experimental parameters, such as the sample buffer, background electrolyte (BGE) composition, concentration, sample plug lengths, water plug, and the effect of organic modifiers were studied. The relationship between peak height and injection times for the NSAIDs by variation of sample and BGE buffer concentration was investigated. On addition of sodium chloride (0.3-0.6%) to the sample zone, the stacking efficiency was increased. With acetate buffer (100 mM, pH 5.0)/ethanol (20% v/v) as BGE and sample solution in acetate buffer (0.2 mM, pH 5.0)/ethanol (20% v/v)/NaCl (0.3% w/v), NSAIDs could be determined at low microM levels without sample matrix removal. The detection limit was 0.096 microM for indoprofen, 0.110 microM for ketoprofen, 0.012 microM for naproxen, 0.023 microM for ibuprofen, 0.110 microM for fenoprofen, 0.140 microM for flurbiprofen, and 0.120 microM for suprofen. The method could be successfully applied to the simultaneous determination of NSAIDs in urine. The recoveries were better than 82% for all the analytes. The present method enables simple manipulation with UV detection for the determination of NSAIDs at low concentration levels in complex matrix samples.     

Aptamer–Target Interaction: A Comprehensive Study by Microchip Electrophoresis in Frontal Mode

An original methodology based on microchip electrophoresis in a continuous frontal analysis mode (FACMCE) was employed to provide new insights into the interaction between an aptamer and its target (lysozyme), i.e., the influence of various experimental conditions on possible conformation change of the aptamer. The parameters evaluated were: background electrolyte (BGE) nature and ionic strength, nature and concentration of an added divalent cation, use of an aptamer thermal treatment, conditions classically used when employing aptamers. Increasing the BGE ionic strength led to a decrease in the dissociation constant highlighting the role played by the non-ionic interactions, and a decrease in the number of binding sites due to a change of binding mode and/or an amplification of selectivity. Divalent cation addition in the BGE improved the binding affinity. We demonstrated that this is not exclusively related to an increase in ionic strength but also certainly to an aptamer conformational change. Furthermore, changing the BGE nature permitted to modulate the binding parameters. Finally, we showed that an initial heating of the aptamer solution has been proved critical to stabilize the optimal conformation and thus to get a high binding affinity as well as a smaller standard deviation on the binding parameters values. This study has evidenced the influence of a wide range of parameters so as to better grasp the target/aptamer interaction, and thus highlight some phenomena involved in such an interacting system. This work will therefore help for further applications of this binding system for selectivity improvement in bioanalytical development.     

Electrophoretic mobilities of cationic analytes in non-aqueous methanol, acetonitrile and their mixtures. Influence of ionic strength and ion-pair formation

The mobilities of cationic analytes in organic solvents and water are compared, and the reasons for differences in the mobilities are discussed in detail. Actual mobilities (at background electrolyte concentration 10 mmol/l) of anilinium ions were determined by capillary zone electrophoresis in water, methanol, acetonitrile and mixtures of methanol and acetonitrile (in volume ratios 1:1, 1:3 and 3:1). The actual mobilities correlated with the viscosity of the organic solvent: the products of actual mobility and viscosity were constant within 7%. However, these products were significantly larger in water. Larger products of mobility and viscosity in water were also found for unsubstituted anilinium when the absolute mobility (at zero ionic strength) was taken into consideration. Thus, ion-solvent interactions must be responsible for the seemingly high mobility in water compared with that in organic solvents. This finding can be explained by the effect of the ion on the water structure. Based on equilibrium constant for ion-pair formation given in the literature, about 20% of the main background electrolyte constituent (tetrapropylammonium perchlorate) is associated at 10 mmol/l concentration in acetonitrile. Comparison of the plot of the measured mobilities of the analytes vs. the square root of the corrected ionic strength of the background electrolyte in acetonitrile with the prediction based on the Debye-Hückel-Onsager theory showed the measured mobilities deviate negatively from the theoretical line. This is apparently due to ion pairing, which takes place for the analytes as well.     

Capillary electrophoresis of inorganic anions in hydro-organic media. Influence of ion-pairing and solvation phenomena

The capillary electrophoresis separation of four inorganic anions (NO3-, I-, Br- and SCN-) was investigated over the whole range of methanol-water mixture composition. As the separation selectivity was strongly dependent on the solvent composition, the influence of ion-pairing and solvation phenomena was examined in depth in an attempt to explain this modification. First, a series of experiments was performed in methanolic background electrolytes, with counter-ions of different size. Ion-pair formation involving electrolyte ions was assessed to allow for a correction for free electrolyte ion concentration. Ion-pair formation constants between each inorganic anion and electrolyte counter-ion were next determined from the variations of the anion mobility as a function of the free counter-ion concentration. In view of the low values obtained, ion-pair formation alone failed to explain the selectivity variations. Solvation phenomena were then investigated with the help of a theoretical quantum model, the density functional theory (DFT), coupled with a polarizable continuum model to mime non-specific solvent effects. Whereas this model proved successful at predicting the mobility order at infinite dilution in water, it failed to predict the correct order in methanol.     

Low EOF rate measurement based on constant effective mobility in microchip CE

A new method for quickly determining low EOF rates (micro(EOF)) in microchip CE is described. The measurement is based on the notion that the effective mobility (micro(eff)) of an analyte is a constant in a certain BGE. The micro(eff) of an analyte is determined in a reference fast-electroosmosis microchip, and the apparent mobility (micro(app)) of the analyte can be determined in the microchip with unknown low electroosmosis, and then micro(EOF) in the low-electroosmosis microchip can be calculated according to the equation mu(EOF) = micro(app) - micro(eff). By an indirect method or other conventional methods, micro(eff) can be easily measured in the reference microchip. The proposed method is particularly useful for low-electroosmosis measurements in wall-modified microchannels.     

Capillary electrophoresis of anionic analytes in methanol: effect of counter-ions on electrophoretic mobility

Mobilities of 11 substituted benzoates and 3 nitrophenolates were determined in non-aqueous methanol with Li+, Na+, K+, Rb+, and tetrabutylammonium (Bu4N+) as counter-ions of the background electrolyte. The influence of the ionic concentration of the background electrolyte on the mobility of the analyte anions is more pronounced compared to aqueous solutions. The deviation from the dependence of the mobilities on the ionic strength from the Debye-Hückel-Onsager theory indicates the occurrence of ion-pair formation. For a given ion concentration (10 mmol/L), the decrease of the analyte mobility follows the counter-ion sequence Li+ < Na+ < K+ < Rb+, which is the inverse order of their Stokes radii. Bu4N+ as counter-ion has a similar effect on the analyte mobility than Li+ (which has the same Stokes radius, but a six times smaller crystal radius). Exceptions are some di- and trihydroxybenzoates. The mobilities in methanol and in water with the same counter-ion (Na+) at a given ionic concentration show very low correlation.     

Determination of nitrate in seawater by capillary zone electrophoresis with chloride-induced sample self-stacking

A capillary zone electrophoresis (CZE) method was established to determine low concentration nitrate which was online preconcentrated with chloride-induced leading-type sample self-stacking for seawater samples. The sample self-stacking was based on transient isotachophoresis in which chloride served as leading ion, and dihydrogenphosphate in the background electrolyte (0.1 M phosphate) as the terminating one. Due to the small mobility difference between nitrate and chloride, the isotachophoresis time was so long that nitrate could not separate from the rear sharp boundary between chloride and the background electrolyte (BGE) when it migrated to the detection window. A zwitterionic surfactant, 3-(N,N-dimethyldodecylammonio)propane sulfonate was added to the BGE to enlarge the mobility difference for its selective interaction with anions. Thus, a highly conductive sample could be injected in a large volume with about fourfold sensitivity enhancement compared to that of field amplification sample stacking in which nitrate was dissolved in pure water. The relative standard deviations (n=5) of migration time, peak area, peak height were 0.1, 3.0, 1.5%, respectively. The limit of detection (S/N=3) for nitrate was 35 microg/l in seawater samples with relatively low concentration BGE (0.1 M sodium phosphate, pH 6.2). The overall procedure consisting of online preconcentration and separation was as simple as routine CZE except for a slightly longer sample injection time (3-4 min).