Alain Jaulmes

Determination of binding equilibrium constants by numerical simulation in zonal high-performance affinity chromatography

Abstract The equation of propagation of a signal of finite concentration through an affinity column is obtained by solving the mass conservation equations of liquid chromatography. Its numerical integration allows peak simulation, curve fitting and thence the determination of the equilibrium isotherm function. This method was applied to the measurement of ligand-protein interactions in zonal elution chromatography. In the range of concentrations studied, a three-parameter isotherm equation is convenient for characterizing the binding of phenylbutazone with human serum albumin on diol-silica. The data were analyzed with two different isotherm models: the two-independent-site model with specific and non-specific interaction and the stepwise-multiple-interaction model. The effects of stationary-phase coverage and temperature were studied. From these results were determined the amount of active immobilized protein and the equilibrium constant characterizing the affinity interaction (2.6 l/μmol), which is about twice as large as the value measured in solution.

Multivalent ion-exchange model of biopolymer chromatography for mass overload conditions

Abstract The simple model of multivalent ion-exchange biopolymer chromatography is analyzed on the basis of classical quasi-chemical temperature. The rigorous isotherm equation deduced from the stoichiometric displacement model (SDM) was used to stimulate the migration of a solute through the chromatographic column in the isocratic and gradient elution modes. The peak profiles generated for various sample sizes were compared with those obtained on the basis of a Langmuir isotherm. Peak tailing increases with the value of the exponent Z, defined as the ratio of the protein valency to the displacing counter-ion valency. For large Z the asymmetries due to the non-linearity of the isotherm are still present for small sample sizes, the ion-exchange model was applied to analyse the zonal elution behaviour of bovine serum albumin on a polymeric anion-exchange stationary phase deposited on silica. The effective charge of the protein (m = 8) was determined at infinite dilution with mono- and a divalent counter-ions. This value was introduced into the SDM isotherm to predict the elution behaviour in mass-overload conditions and the maximum loading capacity of the protein was determined. Good agreement between theory and experiment was obtained: for about the same capacity factor at infinite dilution, a larger peak asymmetry due to non-linear effects is found with a monovalent counter ion.

Adsorption kinetics of β-lactoglobulin on a polyclonal immunochromatographic support

Beta-Lactoglobulin is one of the main components of whey proteins. Among other reasons, its allergenicity makes its determination in hypoallergenic foods and bio-pharmaceutical products necessary. Immunoaffinity chromatography is a widely accepted technique for purification and analysis of proteins. Knowledge of the apparent kinetics of the adsorption of beta-lactoglobulin onto the anti-beta-lactoglobulin immunochromatographic column is important to optimize the analytical process. High-performance frontal affinity chromatography was used to study the apparent kinetics of the adsorption process. Langmuir and bi-Langmuir kinetic models, assuming one and two kinds of binding sites, respectively, were used to characterize the adsorption kinetics of beta-lactoglobulin B on a polyclonal immunoadsorbent. Very good fits were obtained with the bi-Langmuir model for two different concentrations of beta-lactoglobulin and this allowed us to calculate the apparent adsorption rate constants and the column capacities for both kinds of sites. Experimental results indicate the possibility that the adsorption process is not irreversible. The values of the apparent dissociation rate constants leading to the best fit were estimated and the affinity constants were calculated.

Adsorption kinetics of human serum albumin on various types of chromatographic support

SummaryThe adsorption of human, serum albumin (HSA) on various types of chromatographic support (C6 reversed-phase, anion-exchanger, immunoadsorbent) was studied by analysis of frontal breakthrough curves. These curves are first characterized by a sharp increase; a more diffuse profile is then observed at longer elution times. Data were analyzed using a bi-Langmuir kinetic model assuming irreversible adsorption on two types of binding site. Fitting the model to the experimental data enables determination of apparent adsorption rates and the saturation capacities of both types of binding site.

Determination of adsorption isotherms on graphitized carbon black by zonal gas chromatography

A method for extracting the physico-chemical parameters from the analysis of the chromatographic elution peak profile is developed. From the set of differential equations of gas-chromatography, an analy- tical expression of the elution peak is given in the case of low concen- trations, when the isotherm can be replaced by a two term expansion. The model is a function of 4 parameters: the sample size (peak area), the apparent axial dispersion (band broadening), the slope and the curvature of the isotherm at origin. The variation of peak maximum with increasing amounts injected provides a convenient way of determining the first and second surface virial coeffi- cients of the adsorption isotherm. We have examined the validity of the model by studying the elution behavior of benzene and hexane on graphi- tized carbon black at different temperatures. The parameters of the adsorption isotherm determined by chromatography are compared with those obtained from static methods (ref. 1). In the case of a Langmuir type isotherm, the monolayer capacity of the adsorbate can be measured.