Claire Vidal-Madjar

Separation of basic proteins by capillary zone electrophoresis with coatings of a copolymer of vinylpyrrolidone and vinylimidazole

Capillary zone electrophoretic (CZE) separation of basic proteins has been achieved with capillary columns modified with copolymers of vinylpyrrolidone (VP) and vinylimidazole (VI). The copolymerization reaction is performed inside the capillary column and involves chemical bonding of the polymer to silica. The electroosmotic flow (EOF) is greatly decreased by this surface modification. The presence of positive charges on the coating surface, due to the cationic property of vinylimidazole at pH below 7, reduces the adsorption of basic proteins onto the silanol groups of the capillary surface. Acidic proteins are irreversibly adsorbed, but rapid separation and good performance reproducibility are obtained with basic proteins. In the case of capillaries modified with VP, the acidic and basic proteins are eluted within 10 min. In this work, we studied the effects of pH and buffer concentration on the magnitude of the EOF, as well as the effect of copolymer composition on the separation efficiency.

Anion-exchange chromatographic properties of α-lactalbumin eluted from quaternized polyvinylimidazole : study of the role of the polymer coating

The anion-exchange elution behaviour of alpha-lactalbumin was studied on cross-linked and quaternized polyvinylimidazole, deposited on various high-performance liquid chromatographic supports (porous silica and diol silica). The influence of the nature and thickness of the coating layer on the retention and band-width properties of the protein elution peak was examined by isocratic elution. The retention properties of alpha-lactalbumin were studied from the plot of log k vs. log([NaCl]), where k is the capacity factor and [NaCl] the displacer salt concentration in the aqueous phase. The retention depends on the amount of stationary phase deposited on the support, but an increased hydrophobic effect is found when the polymer films do not coat the chromatographic support uniformly. Band broadening of the elution peaks was studied in terms of plots of plate height vs. mobile phase velocity. An important mass-transfer contribution is found, which decreases with increasing k and increases with the thickness of the coating layer. These effects reveal that the diffusion into the polymer layer is the controlling step of the ion-exchange process with non-uniform polymer layers of large mean thickness.

Optimisation of fast protein separations on non-porous silica-based strong anion exchangers

SummaryThe adsorbed coating technology using various vinylpyrrolidone-vinylimidazole copolymer compositions was carried out on 1.7 μm non-porous monodisperse silica. It was shown that the retention properties and the loading capacity for bovine serum albumin (BSA) increases with the amount of vinylimidazole in the copolymer composition. The retention behavior of various proteins as a function of the salt composition in the eluent has been applied to find the optimal conditions for the synthesis of the anion-exchange stationary phase. The suitability of these supports for the fast separation of biological molecules is demonstrated. The best resolution and the highest speed in protein analysis was obtained by using an anion exchanger of low capacity.

Retention and enantioselectivity properties of β-cyclodextrin polymers and derivatives on porous silica for reverse-phase liquid chromatographic separation of enantiomers

SummaryChiral stationary phases (CSP) based on porous silica coated with β-cyclodextrin polymer and methylated or acetylated derivatives have been prepared. Their enantiomer separation capability was tested and compared under reversed-phase mode elution conditions. Noticeable changes in retention properties werenoted and in many cases a reverse enantioselectivity was evidenced, as a consequence of a strong modification of the energy, geometry and number of points of contact between solutes and stationary phase sites of molecular recognition.

Gas chromatography of Titan's atmosphere VIII. Analysis of permanent gases with carbon molecular sieve packed capillary columns

Abstract An alternative for an in situ GC analysis of permanent gases in Titans atmosphere, given the severe constraints on space instrumentation, is used of microcolumns packed with carbon molecular sieve. The differential adsorption enthalpies are determined from the variation of retention volume with temperature. A linear variation with the polarisability of solutes is obtained and the relative retentions are similar to the adsorptions observed on graphitized carbon black. Two columns having different porosities are compared with respect to efficiency, resolution and detection limit. They all provide high efficiency, fast analysis and a detection limit of CO in N 2 suitable for the quantification of Titans atmosphere composition. This detection limit is also studied with a vacuum column outlet using a mass spectrometer under conditions close to those of the flight instrument of the Huygens probe.

Study of conformational effects of recombinant interferon γ adsorbed on a non-porous reversed-phase silica support

Reversed-phase chromatography is a powerful method for separating recombinant interferon gamma and one of its analogues differing only by a single amino acid residue. Structural differences of the proteins explain this separation ability as demonstrated from adsorption studies on a non-porous reversed-phase support. To reveal the structural differences occurring in the adsorbed state, two different and independent methods were employed. The variation of the retention with the slope of the linear gradient gave information about the molecular contact area of the protein with the support. For different experimental conditions, these data were correlated with the adsorbent capacities measured on an n-octadecyl-modified non-porous silica support. These supports are useful for these types of experiments because the protein is adsorbed exclusively at the external surface of the beads. Moreover, a small amount of protein is necessary to saturate the column, owing to its low capacity.

Kinetic study of the adsorption of human serum albumin on immobilized antibody using the split-peak effect in immunochromatography.

The split-peak effect was used to determine the association rate constant of the antigen-immobilized antibody reaction. The amount of immobilized human serum albumin antibody on the chromatographic support was varied in order to find the optimal conditions to reduce the mass transfer contribution in the stagnant mobile phase fluid and measure the effective association rate constant of human serum albumin with the immobilized antibody. Kinetic studies as a function of flow-rate demonstrate the validity of the method consisting in determining the association rate constant from measurements performed on columns of various capacities. These experiments show that limitations due to mass transfer to the surface of the adsorbent are minimized at high flow-rates and for a low density of immobilized ligand.

Study of the retention properties of warfarin enantiomers on a β-cyclodextrin polymeric support

High-performance liquid chromatography was used to study the retention properties of (R)- and (S)-warfarins on a silica support coated with a beta-cyclodextrin polymer. The influence of the methanol content of the acetate buffer eluent was investigated at pH 4. The measure of the variations of retention time with temperature enables one to determine the enthalpy and the entropy of adsorption. The plot of the two thermodynamic functions shows a minimum around 30% (v/v) methanol. At low methanol contents, the decrease of the hydrophobic interactions with increasing methanol content explains the decrease of the enthalpic and entropic terms. Above 40% (v/v) methanol, the decrease of the adsorption enthalpy absolute value is due to the solvation by the organic component. From the analysis of peak shape in mass-overload conditions, the column capacity toward each enantiomer was determined. A lower capacity was found toward (S)-warfarin, the more retained enantiomer. Peak shape analysis in mass-overload conditions was used to determine the adsorption isotherm. A Langmuir-type adsorption isotherm accounts well for the experimental data.

Study of the adsorption of self-associating proteins on an anion exchanger: Application to the chromatography of β-lactoglobulin B

Abstract The stoichiometric displacement model (SDM) for the adsorption of proteins on an ion exchanger was extended to describe the adsorption of self-associated molecules. The model based on the mass-balance equations assumes a monomer-dimer equilibrium with a rapid interconversion rate. The role of protein self-association in solution and in the adsorbed state is discussed in terms of adsorption isotherm shapes and of zonal elution chromatographic behaviours. The influence of the parameter Z , defined as the ratio of the protein valency to the displacing counter-ion valency, is demonstrated. The model was applied to analyse the zonal elution behaviour of β-lactoglobulin B (β-lac B) on a polymeric anion-exchange stationary phase. The influence of the counter-ion-valency was studied by adding NaCl or Na 2 SO 4 to the eluent. The adsorption isotherm of β-lact B on the anion exchanger was determined at pH 7.5 from the non-linear zonal elution profiles observed with increasing sample size. Various aspects of the adsorption behaviour of β-lack B, such as positive cooperativity, were deduced from the isotherm shape and the corresponding Scatchard plot. The column capacity, the association constant in solution and the parameter defining the association in the adsorbed phase were determined from the best fit of the theoretical model to the experimental profiles. In agreement with the model, these parameters can be used to describe the experiments performed with a monovalent or a divalent counter ion. It is shown that protein-protein interactions exist in the adsorbed state and lead to an increase in the protein self-association.

Capillary electrophoretic behavior of milk proteins in the presence of non-ionic surfactants.

The electrophoretic behavior of alpha-lactalbumin and beta-lactoglobulins (A and B) in the presence of non-ionic surfactants was studied by capillary electrophoresis (CE), using a poly(ethylene glycol) coated capillary column. The surfactants (Tween 20, Brij 35 and 78) were used as buffer additives. The separation is based on the difference in the strength of protein-surfactant association complexes, which results in a change of the effective electrophoretic mobility. The modification of the electrophoretic mobilities of proteins was observed and this variation permitted the estimation of the interaction between protein and surfactant. The effect of surfactant type and concentration on the migration behavior of protein in CE is discussed. It is found that the retention behavior of the milk proteins (the alpha-lactalbumin and the beta-lactoglobulins) in CE is very different. The pH of the buffer and the surfactant type influence significantly the protein-surfactant interactions.