Anne Ravel

Dansyl amino acid enantiomer separation on a teicoplanin chiral stationary phase : effect of eluent pH

The retention and separation of a series of D,L dansyl amino acids (used as test solutes) on a teicoplanin stationary phase were investigated over a wide range of mobile phase (citrate buffer-methanol, 90:10, v/v) pH. An approach based on the development of various equilibria was carried out in order to describe the retention behavior of the solute in the chromatographic system. The equilibrium constants corresponding to the transfer of the anionic and zwitterionic forms of the dansyl amino acids from the mobile to the stationary phase were determined. These values allowed one to explain the decrease in the retention factor and the associated increase in the separation factor as the eluent pH was increased. Thermodynamic parameter variations were calculated so that the driving forces of the solute association with the teicoplanin phase were derived. This approach indicated that the chiral discrimination was principally controlled by the interaction between the anionic form of the solute and the stationary phase.

Reversal of the enantiomeric elution order of some aromatic amino acids using reversed-phase chromatographic supports coated with the teicoplanin chiral selector

In this paper, two chiral stationary phases were prepared by coating the surface of both C8 and C18 high-performance liquid chromatography (HPLC) supports with the teicoplanin chiral selector. The hydrophobic C11 acyl side chain, attached to the D-glucosamine group of teicoplanin, served as anchor moiety for the immobilization of the chiral selector on the apolar support material. The retention and enantioselectivity of these coated stationary phases were studied using some aromatic amino acids as probe solutes and an aqueous solution as mobile phase. It was found that the enantiomer elution order on the modified C8 and C18 stationary phases was reversed (L>D) relatively to that classically observed with a teicoplanin covalently immobilized on a silica support (D>L). Such a dynamic coating on the reversed-phase supports was found to be of interest since the apparent enantioselectivity was not significantly changed by the use during an extended period of time or following a long-term storage of the columns.

Effect of temperature on DNA fractionation in slalom chromatography.

Slalom chromatography is an alternative chromatographic procedure for the analysis of relatively large double-stranded DNA molecules and is based on a hydrodynamic principle. The retardation of the DNA fragments from the cleavage of the Lambda DNA by the KpnI restriction enzyme was studied using an acetonitrile-phosphate buffer as a mobile phase (flow rate equal to 0.3 ml/min) and a C1 column as a stationary phase at various temperatures. It was shown that the temperature constituted an important parameter for the separation of the DNA fragments in slalom chromatography. The DNA hydrodynamic behavior with the temperature was related to the variation in the fluid viscosity and the modification of the elastic properties of the biopolyrner.

FLOW RATE DEPENDENCE ON THE BIOPOLYMER RETENTION IN HYDRODYNAMIC CHROMATOGRAPHY. COMPARISON BETWEEN THE BEHAVIORS OF PROTEINS AND PLASMIDS

Hydrodynamic chromatography (HDC) in a packed column is a useful chromatographic mode for the rapid separation of polymers. This paper compared the retention of various circular double-stranded DNA molecules (3, 5 and 10 kbp) and proteins (keyhole lumpet hemocyanin, ferritin, thyroglobulin, and catalase) in a chromatographic system using a C1 stationary phase and a acetonitrile-phosphate buffer mixture as a mobile phase. For a similar molecular weight, the protein was retained more than the corresponding plasmid. This was attributed to the difference in the compactness between the two species. As well, the retention dependence on the flow rate (0.03 to 1.5 mL/min) exhibited a different behavior in relation to the type of biopolymer. The protein retention curve showed a decrease in the relative retention time until 0.2 mL/min, followed by an increase for the high size polymers, only, while the plasmid retention increased over the entire flow rate range. This observation confirmed that the migration in HDC was dependent on two antagonist phenomena, i. e. stress induced diffusion and polymer deformation. This work demonstrated that the HDC separation of protein was optimal for a flow rate equal to 0.2 mL/min.

A novel approach to study the inclusion mechanism of imidazole derivatives in micellar chromatography

A chromatographic approach was proposed to describe the existence of surfactant micelles in a surfactant/hydroorganic phosphate buffer mobile phase. Using this mixture as a mobile phase, a novel mathematical theory is presented to describe the inclusion mechanism of imidazole derivatives in surfactant micelles. Using this model, enthalpy, entropy and the Gibbs free energy were determined for two chromatographic chemical processes: (i) the transfer of the imidazole derivative from the mobile phase to the stationary phase; and (ii) the imidazole derivative inclusion in surfactant micelles. These thermodynamic data indicate that the main parameter determining chromatographic retention is distribution of the imidazole derivatives to micelles of surfactant while the interaction with the stationary phase play a minor role.

MIGRATION BEHAVIOR MODELING OF ANIONIC SPECIES IN A HYDROORGANIC BACKGROUND ELECTROLYTE

The migration of benzoate derivatives in a hydroorganic background electrolyte (BGE) was predicted using a novel mathematical model. In capillary electrophoresis (CE), with an acetonitrile (ACN)/buffer mixture as BGE, the influence of pH and the ACN cluster fraction can be quantitatively described by a general equation which was used to study the separation optimization. The cluster solute solvation energies in an ACN/buffer mixture over a 0.50–0.80 buffer fraction were calculated. The energetics of the ACN cluster exchange process in the BGE was investigated in relation to the difference in pKa (ΔpKa) between a solute used as reference and the other solutes. A linear correlation was found between the Gibbs free energy change of the solvent exchange process and ΔpKa confirming that the solute solvation by ACN clusters was enhanced for the lesser polar solutes. Enthalpy-entropy compensation revealed that the solute solvation mechanism was independent of both the benzoate derivative structure and the BGE ionic strength.

Sucrose effect on reversed-phase liquid chromatography solute retention

The reversed-phase liquid chromatography retention of a series of benzoate ester molecules was investigated over a wide range of sucrose concentrations (c) 0.01‐0.8 M using a C18 as the stationary phase and a methanol‐water mixture with high organic fraction (60/40, v/v) as the mobile phase. A theoretical treatment was developed to investigate the effect of sucrose molecules on the equilibrium between the solutes and the C18 phase and the methanol‐water medium, respectively. This was found to be adequate to accurately describe the benzoate ester retention behavior when c varied. It was expected that the addition of sucrose was responsible for two main opposite contributions to solute retention, (i) a net interaction between the solute and the modifier in the mobile phase determining a decrease in k 0 values with c up to 0.2 M; and (ii) an increase in the solute affinity for the C18 stationary phase due to the salting-out effect governing a retention increase above 0.2 M. Thermodynamic parameter variations were calculated using van’t Hoff plots and discussed in relation to this retention model to confirm the respective effects of the modifier on the solute affinity towards the two phases.