Pierre Gareil

Capillary electrochromatography : operating characteristics and enantiomeric separations

Abstract Some fundamental aspects of capillary electrochromatography (CEC) (electroosmotic flow, capacity factor, plate height) were studied by carrying out the separation of some neutral compounds with capillaries packed with octadecylsilica particles (ODS). No loss of efficiency up to a linear electroosmotic velocity of 1.3 mm/s was observed for retained analytes (capacity factors varying from 0.7 to 2.5). The extra-column dispersions caused by the frit and the unpacked section separating the frit from the detection window were estimated. Chiral separations of the neutral enantiomers of chlorthalidone by packed capillary electrochromatography was successfully achieved using two approaches: (a) use of the chiral agent, hydroxypropyl-β-cyclodextrin (HPβCD) directly in the mobile phase with an achiral stationary phase (3 μm ODS); (b) use of a chiral stationary phase (5 μm HPβCD-bonded silica particles) with an achiral mobile phase. As with liquid chromatography, the second method achieves higher selectivity and resolution in a shorter analysis time.

Selectivity in Capillary Electrophoresis: Application to Chiral Separations with Cyclodextrins

In order to accurately evaluate the performances of any electrolyte medium, a clear concept of selectivity in capillary electrophoresis and related electroseparation techniques is proposed. Selectivity is defined as the ratio of the affinity factors of both analytes for a separating agent (phase, pseudophase, or complexing agent present in the background electrolyte). When in the presence of a complexing agent and if only 1:1 complexation occurs, selectivity corresponds to the ratio of the apparent binding constants and is independent of the concentration of the complexing agent. This concept is illustrated through the separations of neutral and anionic enantiomers in the presence of a cationic cyclodextrin, the mono(6-amino-6-deoxy)-β-cyclodextrin, as a chiral complexing agent. The values obtained for different pairs of enantiomers are discussed with regard to the functional groups that distinguish them. When the analytes have the same mobilities in free solution and in their complexed form, then the resolution equation developed in micellar electrokinetic chromatography may be applied and optimum conditions (affinity factors, chiral agent concentration) can be predicted.

Intrinsic selectivity in capillary electrophoresis for chiral separations with dual cyclodextrin systems

Defined as the ratio of the affinity factors of the analytes for a complexing agent, the intrinsic selectivity is representative of the very nature of the complexing agent. When more than one complexing agent are present in the background electrolyte, it is possible to define several intrinsic selectivities according to whether complexing agents are considered separately or all together. A theoretical model with respect to selectivity is presented for separations that involve two complexing agents, using the concept of apparent constant for complex formation. When only independent complexation occurs (absence of mixed complexes), then the intrinsic selectivity of a complexing agent X in the presence of a complexing agent Y can be easily related to the intrinsic selectivity of each complexing agent and to complex formation constants. Dual systems of cyclodextrins (CDs), implementing the cationic mono(6-amino-6-deoxy)-β-cyclodextrin (β-CD-NH(2)) and a neutral CD (trimethyl-β-CD (TM-β-CD) or dimethyl-β-CD (DM-β-CD)), were studied to illustrate this model and to offer an alternative to the separation of neutral enantiomers when β-CD-NH(2) shows no or insufficient stereoselectivity. With a dual β-CD-NH(2)/TM-β-CD system at pH 2.3, arylpropionic acid enantiomers were baseline resolved and benzoin derivatives were partially resolved. For the arylpropionic acids, β-CD-NH(2), which is not stereoselective, confers on them a nonzero mobility, while TM-β-CD allows the chiral recognition. A study of the respective influence of ΤM-β-CD and β-CD-NH(2) concentrations was performed to determine the optimal conditions with respect to resolution. This theoretical approach allowed characterization of the intrinsic selectivity of neutral CDs for pairs of neutral enantiomers and therefore identification of the potential of neutral chiral agents for neutral enantiomers.

Capillary zone electrophoretic determination of C2C18 linear saturated free fatty acids with indirect absorbance detection

Abstract The potential of capillary zone electrophoresis for the separation of linear saturated fatty acids in their free form was evaluated. With increasing chain length, difficulties arise from decreasing analyte solubility in aqueous media, increasing occurrence of analyte aggregation and decreasing separation selectivity between successive homologues. A solution was to use electrolytes containing cyclodextrins (CDs) and methanol. The separation of C 2 C 14 fatty acid homologues differing in only one carbon atom was achieved in less than 10 min using a purely aqueous electrolyte with trimethyl-β-CD as an additive. The separation of C 7 C 18 homologues was completed in under 20 min with electrolytes containing up to 60% methanol, in addition to the aforementioned CD. In the presence of the CD, analyte solubility is enhanced through the inclusion of the alkyl chain of the acid in the CD cavity, while separation selectivity is improved because the stability constants of the inclusion complexes increase with increasing chain length of the acid. The lack of a suitable chromophore moiety was circumvented through the optimization of indirect absorbance detection conditions. p -Anisate was selected as the chromogenic species. The minimum detectable concentrations are of the order of (1–2)·10 −6 mol l −1 (0.2–0.5 ppm) and detection linearity was established over at least three orders of magnitude of concentration. The quantitative analysis of a coco oil extract sample is presented, showing results almost identical with those obtained by gas chromatography. Owing to the close values for response factors resulting from the indirect detection principles, a rapid percentage composition can be obtained by corrected peak area normalization.

Chiral separations of underivatized arylpropionic acids by capillary zone electrophoresis with various cyclodextrins Acidity and inclusion constant determinations

Abstract Chiral separations of non-steroidal anti-inflammatory drugs of the family of arylpropionics acids (carprofen, flurbiprofen, indoprofen, ketoprofen, naproxen, pranoprofen and suprofen) were studied by capillary electrophoresis in different pH buffers (pH 4, 6, 8 and 10) containing various neutral cyclodextrins (CDs) β-CD, hydroxypropyl-β-CD, dimethyl-β-CD, trimethyl-β-CD and hydroxypropyl-γ-CD). Baseline resolution of all the racemates was only achieved with trimethyl-β-CD at pH 4.0. The solute acidity constants and apparent formation constants of their inclusion complexes were also derived from these experimenst as an aid for understanding chiral recognition and selectivity optimization. It is clearly shown that no conclusion with regard to the stereoselectivity of a CD can be drawn from the strength of the fit between the CD and the enantiomers.

Determination of nanoparticle diffusion coefficients by Taylor dispersion analysis using a capillary electrophoresis instrument

The collective diffusion coefficient D(C) of diluted suspensions of positively charged iron oxide maghemite particles was experimentally investigated using a capillary electrophoresis instrument on the grounds of Taylor dispersion theory. Conditions for this approach to be applicable to nanoparticles of mean solid diameter below 10nm were set in this work, enabling precisions on D(C) determination of less than 2% relative standard deviation (RSD). Significantly different D(C) values were thus measured for particle populations differing in solid number mean diameter by only 2 nm. The obtained values were compared to the z-average diffusion coefficient derived from dynamic light scattering (DLS) experiments and used for the calculation of the Stokes radius. The measured diffusion coefficients appeared to be dependent on particle volume fraction and electrolyte ionic strength. These observations were eventually discussed in terms of particle interactions.

Separation and determination of warfarin enantiomers in human plasma samples by capillary zone electrophoresis using a methylated β-cyclodextrin-containing electrolyte

A methyl beta-cyclodextrin with a degree of substitution of 1.8 proved to be an effective chiral selector, among other cyclodextrins tested, for the separation of warfarin enantiomers by capillary electrophoresis. The operating conditions were optimized with respect to electrolyte composition (buffer pH, ionic strength, cyclodextrin concentration, methanol content) and applied voltage. The influence of a high ionic strength on the resolution was clearly shown. A baseline separation can be obtained in less than 15 min with an efficiency of ca. 250,000 theoretical plates. These conditions were applied to the determination of warfarin enantiomers in the plasma of patients under warfarin therapy. The limit of detection for the whole procedure (dichloromethane extraction followed by evaporation to dryness and capillary electrophoresis) was of the order of 0.2 mg/l (6.5.10(-7) M) of each enantiomer.

Interaction of fucoidan with the proteins of the complement classical pathway.

Fucoidan inhibits complement by mechanisms that so far remain to be unraveled, and the objective of this work was to delineate the mode of inhibition by this sulfated polysaccharide. For that purpose, low molecular weight fractions of algal (Ascophyllum nodosum) fucoidan containing the disaccharide unit [-->3)-alpha-L-Fuc(2SO3(-))-(1-->4)-alpha-L-Fuc(2,3diSO3(-))-(1-->](n) have been studied. Gel co-affinity electrophoresis and a new affinity capillary electrophoresis (ACE) method have been implemented to characterize fucoidan-complement protein complexes. Fucoidan binds C1q, likely to its collagen-like region through interactions involving lysine residues, and then prevents the association of the C1r(2)-C1s(2) subunit, required to form the fully active C1. In addition to C1q, fucoidan forms a complex with the protein C4 as observed by ACE. The fucoidan inhibits the first steps of the classical pathway activation that is of relevance in view of the proinflammatory effects of the subsequent products of the cascade. This study shows that a high level of inhibitory activity can be achieved with low molecular weight carbohydrate molecules and that the potential applicability of fucoidan oligosaccharides for therapeutic complement inhibition is worthy of consideration.

A semi-empirical approach to the modeling of the electrophoretic mobility in free solution: application to polystyrenesulfonates of various sulfonation rates.

This work focuses on the understanding of the electrophoretic behavior of flexible chains of polystyrenesulfonates (PSSs) in free solution. It deals mainly with the variation of the electrophoretic mobility with (i) the polymerization degree (N) of fully sulfonated PSSs and (ii) the sulfonation rate of randomly sulfonated PSSs. In both cases, the electrophoretic mobility was modeled following a semi‐empirical approach which involves parameters retaining a physical meaning. Fully sulfonated PSS oligomers, having a length smaller than or similar to the Debye length, exhibit a particular electrophoretic behavior, in‐between that observed for multicharged small molecules and that for polyelectrolytes. The electrophoretic mobility of these oligomers increases strongly with N, which is attributed to a hydrodynamic coupling between monomers. Then the mobility is maximum for an N of about 10, for which the PSS oligomers are still in a rod‐like conformation. Afterwards, as N increases and the PSSs are larger than the Debye length, the electrophoretic mobility decreases slowly until it reaches a constant value corresponding to the free‐draining behavior. Next, the electrophoretic behavior of long PSS (N about 1200) differing in their sulfonation rates was investigated. The effective charge rates were determined independently by conductimetric measurements and the mobilities were modeled as a function of the sulfonation rate. The PSS behavior observed was compared to the one previously reported for classical polyelectrolytes having hydrophilic backbones, such as copolymers of poly(acryamide‐co‐acrylic acid). A specific behavior has been pointed out for these partially sulfonated PSSs, which is attributed to the hydrophobicity of their backbone. Finally, it is shown that separations of PSSs of different sulfonation rates can be obtained with electrolytes containing an anionic surfactant or methanol.

A new insight into suction and dilution effects in capillary electrophoresis coupled to mass spectrometry via an electrospray ionization interface. Part I‐Suction effect

The hyphenation of CE with MS is nowadays accepted as a powerful analytical approach. Employing ESI, the most common interface, one challenge is to provide quantitative information, which is quite a difficult task, as it is linked, among other factors, to suction and dilution effects. In the coaxial ESI configuration, the suction effect has been presented in literature as stemming from nebulizing gas (NG) flow rate and drying gas temperature. But as this interface consists in three concentric capillaries, allowing for BGE, sheath liquid (SL) and NG mixing, it is demonstrated herein that other parameters are also involved in this suction effect: the CE capillary protrusion from the interface needle, SL flow rate, and overall BGE flow rate and velocity profile. Whereas NG flow rate is the parameter affecting suction to a greater extent, separation capillary protruding length, SL, and overall BGE flow rate have a significant additional impact on this phenomenon. It is shown that SL flow rate can affect suction differently according to the NG velocity, which may be explained by modification of the Taylor cone geometry. Furthermore, it appears that suction effect is noticeably favored by a parabolic velocity profile of the BGE, again probably due to the Taylor cone shape modification. Finally, the temperature gradient created by the contact between the heated NG and the separation capillary enhances this effect.