Comparison of single and double detection points Taylor Dispersion Analysis for monodisperse and polydisperse samples

International audienceCompariThe purpose of this work was to compare single and double detection points Taylor Dispersion Analysis (TDA) of monodisperse and polydisperse samples for low injected volumes. For that, the hydrodynamic radii of human and bovine serum albumins, a poly(styrene sulfonate) standard and two dendrigraft polyl-lysines were determined by TDA on a capillary electrophoresis instrument using a modified detection interface allowing the analysis on two detection points. The results were compared to values obtained via a single detection point TDA with adequate corrections for the finite injected volume. The results showed no significant difference between the double and single detection points when Taylor conditions were respected and when the injected volume was less than 1% of the capillary volume till the detection window. The issue of the determination of the peak variance of the Taylorgram in the case of polydisperse sample with non-Gaussian peak is addressed. A criterion is proposed to define the interval of time on which thepeakvarianceiscalculatedbyintegrationasafunctionofthesignaltonoiseratio

Conception de supports monolithiques de silice pour la chromatographie

L objectif de ce travail de thèse a été de développer des colonnes monolithiques, d une part en étudiant les différents paramètres influençant la formation des phases associées et d autre part en développant une méthode de mise en colonne pour permettre leur utilisation en chromatographie. La nouvelle voie de synthèse proposée, utilisant le polymère F68 dans la génération de ces monolithes, s est révélée plus avantageuse que celle du PEG, permettant l obtention de phases de faible taille de domaine et présentant une structure mésoporeuse en l absence de traitement basique. D autre part, une méthode originale de mise en colonne a été développée et a ainsi permis l utilisation de ces monolithes en chromatographie. Les performances obtenues ont été améliorées par rapport à la bibliographie. Cependant, des élargissements de pics liés à l hétérogénéité de ces phases et à leur mise en colonne sont encore présents. La forte perméabilité des colonnes monolithiques a été mise à profit en couplant des colonnes afin de réaliser la séparation efficace de phospholipides de soja.This thesis aimed at developing monolithic phases, on the one hand by studying the different parameters influencing the formation of the monolithic structure and on the other hand by developing a cladding procedure in order to apply these monoliths in chromatography. A new procedure based on the use of the F68 triblock copolymer was compared to the classical PEG, in the formation of silica monoliths, and was demonstrated to be promising since it leads to co-continuous structures presenting both smaller throughpores, in a wider composition range, and mesporous structures without any alkaline treatment. Moreover, an original cladding method was developed and allowed use of such monoliths in chromatography. The obtained separation performances were improved seven times if compared to those reported in the literature. Nevertheless, peak broadening due to phase heterogeneity and cladding still occurs. The high permeability of the columns was used by coupling several columns to achieve the efficiency required for the separation of phospholipids.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Separation and Characterization of Highly Charged Polyelectrolytes Using Free-Solution Capillary Electrophoresis

The characterization of statistical copolymers of various charge densities remains an important and challenging analytical issue. Indeed, the polyelectrolyte (PE) effective electrophoretic mobility tends to level off above a certain charge density, due to the occurrence of Manning counterion condensation. Surprisingly, we demonstrate in this work that it is possible to get highly resolutive separations of charged PE using free-solution capillary electrophoresis, even above the critical value predicted by the Manning counterion condensation theory. Full separation of nine statistical poly(acrylamide-co-2-acrylamido-2-methylpropanesulfonate) polymers of different charge densities varying between 3% and 100% was obtained by adjusting the ionic strength of the background electrolyte (BGE) in counter electroosmotic mode. Distributions of the chemical charge density could be obtained for the nine PE samples, showing a strong asymmetry of the distribution for the highest-charged PE. This asymmetry can be explained by the different reactivity ratios during the copolymerization. To shed more light on the separation mechanism, effective and apparent selectivities were determined by a systematic study and modeling of the electrophoretic mobility dependence according to the ionic strength. It is demonstrated that the increase in resolution with increasing BGE ionic strength is not only due to a closer matching of the electroosmotic flow magnitude with the PE electrophoretic effective mobility, but also to an increase of the dependence of the PE effective mobility according to the charge density

Taylor dispersion analysis with two detection points on a commercial capillary electrophoresis apparatus

International audienceThis work describes a simple technical modification for doing Taylor dispersion analysis with two UV detection points on a commercial capillary electrophoresis apparatus. So far, double UV detection was only possible using specific detectors that are external to the capillary electrophoresis apparatus. In this work, the detection interface of the capillary electrophoresis apparatus was easily modified to allow the introduction and the superposition of two capillary windows in the same interface (at the same detection point). This modification made possible the double detection of the sample zone in Taylor dispersion analysis using a loop. The peak dispersion using the modified interface was similar to that obtained on a non-modified UV interface. Diffusion coefficients (and the corresponding hydrodynamic radii) of small molecule andproteinsweredeterminedingoodagreementwithvaluesoftheliteratureandwithRSD lower than 5

Hydrodynamic size characterization of a self-emulsifying lipid pharmaceutical excipient by Taylor dispersion analysis with fluorescent detection

International audienceIn this work, the sizing of microemulsion droplets of a lipid-based pharmaceutical excipient (Labrasol® ALF) is performed by Taylor dispersion analysis (TDA) using fluorescent detection. An hydrophobic fluorescent marker is used to tag the microemulsion droplet and to increase the sensitivity of detection (compared to UV detection). Combined with the frontal TDA mode, fluorescent detection was mandatory for an accurate sizing of microemulsions containing large coacervates. Microemulsion sizing of Labrasol was performed at various concentrations from 1 to 70g.L-1 and at two different temperature (25°C and 37°C). Results obtained by TDA are compared to those derived from DLS measurements. The combination of both techniques allows estimating the size and proportion of coacervates in the microemulsion, as well as the polydispersity in size of the sample

A simple cladding process to apply monolithic silica rods in high performance liquid chromatography

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Preparation and characterisation of silica monoliths using a triblock copolymer (F68) as porogen

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Quantitative Analysis in Capillary Electrophoresis: Transformation of Raw Electropherograms into Continuous Distributions

Quantitative analysis in capillary electrophoresis based on time-scale electropherograms generally uses time-corrected peak areas to account for the differences in apparent velocities between solutes. However, it could be convenient and much more relevant to change the time-scale electropherograms into mass relative distribution of the effective mobility or any other characteristic parameter (molar mass, chemical composition, charge density, ...). In this study, the theoretical background required to perform the variable change on the electropherogram was developed with an emphasis on the fact that both <i>x</i> and <i>y</i> axes should be changed when the time scale electropherograms are modified to get the distributions. Applications to the characterization of polymers and copolymers by different modes of capillary electrophoresis (CE) are presented, including the molar mass distribution of poly-l-lysine oligomers by capillary gel electrophoresis (CGE), molar mass distribution of end-charged poly-l-alanine by free solution CE, molar mass distribution of evenly charged polyelectrolytes by CGE, and charge density distribution of variously charged polyelectrolytes by free solution CE

Size characterization of commercial micelles and microemulsions by Taylor dispersion analysis

International audienceIn this work, Taylor dispersion analysis was applied to the measurement of micelles (or microdroplets) molecular diffusion coefficient in micellar (or microemulsion) systems based on neutral/anionic/cationic or zwitterionic surfactants. The choice of the micellar marker and the influence the surfactant/marker concentrations on this determination are studied. Experimental results are compared to those derived from the literature using other experimental techniques. Taylor dispersion analysis, experienced in narrow capillaries, was found to be an efficient and suitable method for micelle (or microdroplet) size measurement due to: the low sample consumption, the absence of filtration requirement of the sample, the broad range of size determination (with no lower limit down to angstroms), the simplicity of the protocol, the possibility to measure the viscosity of surfactant solutions in given conditions and the determination of the weight-average micelle hydrodynamic radius. Application to the size-characterization of commercial microemulsions (Gelucire(®) 44/14), used as an excipient in the pharmaceutical formulation, is provided with a comparison to DLS measurements. It was found that the polydispersity in size of the micelle did not influence the Gaussian peak shape of the taylorgram due to rapid surfactant exchange compared to the time-scale of the experiments (a few minutes)

Generalized polymer effective charge measurement by capillary isotachophoresis.

International audienceIn this work, we have generalized the use of capillary isotachophoresis as a universal method for determination of effective charge of anionic and cationic (co)polymers on ordinary capillary electrophoresis instruments. This method is applicable to a broad range of strong or weak polyelectrolytes with good repeatability. Experimental parameters (components and concentrations of leading and terminating electrolytes, capillary diameters, constant electric current intensity) were optimized for implementation in 100 μm i.d. capillaries for both polyanions and polycations. Determined values of polymer effective charge were in a very good agreement with those obtained by capillary electrophoresis with indirect UV detection. Uncertainty of the effective charge measurement using isotachophoresis was addressed and estimated to be ∼5–10% for solutes with mobilities in the 20–50 × 10−9 m2 V−1 s−1 range