Christiane Guinchard

Retention mechanism of weak polar solutes in reversed phase liquid chromatography.

The retention mechanism of a weak polar solute, a series of 10 benzodiazepines in reversed phase liquid chromatography, was investigated over a wide range of mobile phase compositions. The values of enthalpy (ΔH°) and entropy (ΔS°) of transfer from the mobile to the stationary phases were determined. The method studied each factor (water fraction Φ in the acetonitrile (ACN)/water mixture and column temperature) controlling the retention mechanism. The changes in ΔH° and ΔS° as a function of the water fraction Φ in the ACN/water mixture were examined. These variations are explained using the organization of organic modifier (ACN) in clusters in the ACN/water mixture. A change in the retention mechanism thus indicated when the ACN/water mixture was used instead of the hydrogen-bonded mobile phase such as CH(3)OH/water. Enthalpy-entropy compensation revealed that the retention mechanism was independent of the water fraction Φ but showed that differences between the molecular structures of the benzodiazepines contributed more significantly to changes in the retention process in the CH(3)OH/water mixture than in the ACN/water mixture.

Affinity chromatography study of magnesium and calcium binding to human serum albumin: pH and temperature variations.

The magnesium and calcium binding on human serum albumin (HSA) was studied using an affinity chromatography approach. The effects of the mobile phase pH, its ionic strength and column temperature on the transfer equilibrium constants were studied. The thermodynamic data corresponding to the electrostatic interactions occurring during the HSA-ion binding were determined. Enthalpy-entropy compensation revealed that the ion binding mechanism at HSA was independent of the ionic strength, the same at four pH values (6.5, 8, 8.5 and 9), but presented a weak change at physiological pH around 7-7.5 due to a HSA phase transition. A theoretical model based on the Gouy-Chapman theory allows to determine the relative charge density of the HSA surface implied in the binding process and the variation of the number of ions bound to one albumin molecule with the pH.

Rapid determination of sulbactam and tazobactam in human serum by high-performance liquid chromatography.

A simple and rapid HPLC method for the determination of tazobactam and sulbactam, two beta-lactamase inhibitors, in serum for the therapeutic follow-up of patients is described. The effect of the pH of the aqueous mobile phase and column temperature on column efficiency and retention were examined and equations for their dependences were derived. The use of a chromatographic response function showed that methanol-buffer (5:95, v/v) (pH 6.3) as the mobile phase and a 45 degrees C column temperature were optimum values for chromatographic separation. The analytical method was linear from 10 to 200 micrograms/ml. This assay limit range is sufficient for the analysis of human serum. The limit of detection was 10 micrograms/ml for sulbactam and 5 micrograms/ml for tazobactam. The coefficient of variation was less than 5%. The speed at which this assay can be performed makes it especially useful for estimating the levels of these drugs in human serum.

A New Approach to Study Benzodiazepine Separation and the Differences Between a Methanol/Water and Acetonitrile/Water Mixture on Column Efficiency in Liquid Chromatography

Abstract A chemometric methodology was used to study column efficiency and the separation of 10 benzodiazepines in reversed phase liquid chromatography. New simple mathematical models and the organic modifier (OM) organization of ACN in the water, explained differences on column efficiency observed when ACN is chosen instead of CH3OH. A new response function, which takes into account the separation quality and the analysis time, was proposed for the separation optimization. The result, a mobile phase ACN/water (60/40)(V/V), with a flow rate = 1.00 mL/min and a column temperature = 47°C were optimum values for a rapid chromatographic separation.

PECULIARITIES OF THE RETENTION MECHANISM OF CIRCULAR AND LINEAR DNA FRAGMENTS USING NON-EQUILIBRIUM CHROMATOGRAPHY

Two different methods have been used to investigate the retention mechanism of a series of DNA fragments in non equilibrium chromatography (NEC) over a range of column temperatures (T) and with different mobile phase flow-rates (F). The first approach was the separate study of each factor affecting the retention mechanism; the second method was the simultaneous variation of all these factors. The apparent relative retention time of each DNA fragment was used as a retention marker. The data obtained showed that the retention mechanism of the DNA fragment was dependent on the F and T values, and also on the DNA form, i.e., linear or circular form. For low F values, the retention mechanism was based on the classical hydrodynamic regime (HDC) specific to the circular form. On the contrary, for the linear forms, their retention mechanism was based on a slalom chromatographic process (SC) specifically for high F values at low column temperature. This confirms that the SC and HDC modes are interconnected and that the HDC⇄SC transition exists and was clearly visualized. In addition, using a simplex process connected to the chemometric methodology, the optimal conditions for F and T were calculated to obtain the more efficient separation of the linear DNA forms in a minimum analysis time.

Supercoiled circular DNA and protein retention in non-equilibrium chromatography: Temperature and velocity dependence: testimony of a transition

Non-equilibrium chromatography (NEC) is a chromatographic mode for the rapid separation of polymers. The retention behavior of various proteins (human, chicken, bovine serum albumin) and supercoiled circular double-stranded DNA (plasmids) was investigated using a phosphate buffer as a mobile phase at different velocities and column temperatures with a C1 column with very low-packing particle diameter as a stationary phase. It was shown that the two factors (temperature and velocity) constituted important parameters in the retention mechanism of plasmids and proteins in NEC. The protein was retained more than the plasmid. At all the temperatures (5, 10, 15, 20, 25 degrees C) the plasmid retention increased over the entire flow-rate range (0.02-1.8 ml/min). For the protein, the retention curve presented a decrease in the relative retention time until a critical value of the mobile phase flow-rate, followed by an increase. The transition between the two well known NEC methods, slalom chromatography and hydrodynamic chromatography was clearly visualized for proteins at the lowest temperature, but did not appear for plasmids due to their strong compact structure.

HPLC RETENTION AND INCLUSION OF IMIDAZOLE DERIVATIVES USING HYDROXYPROPYL-β-CYCLODEXTRIN AS A MOBILE PHASE ADDITIVE

Using the results of high performance liquid chromatography (HPLC), this paper investigates the separation and inclusion of a series of weakly polar imidazole derivatives, with hydroxypropylβ -cyclodextrin (HP-β-CD) in the mobile phase over a wide range of column temperatures.These compounds are used for the treatment of onychomycosis. Gibbs Helmholtz parameters (Δ(ΔH) and Δ(ΔS)) of two adjacent imidazole peak on a chromatogram were determined from the graph of the logarithm of the separation factor,α, against the reciprocal of the temperature. A temperature dependent reversal of the elution order between a pair of imidazole derivatives was studied. The results revealed that the main parameter determining interactions between imidazole derivatives and HP-β-CD increased as follows:HP-β-CD ⇔solute hydrogen bonding interaction >HP-β-CD ⇔solute complexation.

High-performance liquid chromatographic determination of tazobactam by precolumn derivatization.

A new reversed-phase high-performance liquid chromatography (RP-HPLC) method for the detection and quantification of tazobactam in serum and haemofiltration fluid is described. The assay for these biological fluids involves an extraction with diethyl ether followed by derivatization using 1,2,4-triazole. The mobile phase consisted of phosphate buffer-methanol and the detection wavelength was 325 nm. The limit of detection was 0.05 microgram/ml in the two fluids and the calibration curves were linear over the range 0.1-50 micrograms/ml. For a tazobactam concentration equal to 1, 5 or 20 micrograms ml-1, the coefficients of variation were less than 5%. The assay was successfully applied to the analysis of samples from drug monitoring in a patient with renal insufficiency undergoing continuous venovenous haemofiltration (CVVH).

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.

Prediction of gas chromatographic retention times, column efficiency and resolution as a function of temperature and flow-rate Application for gas chromatographic separation of eight p-hydroxybenzoic esters

Using eight p-hydroxybenzoic esters, simple equations were developed to provide accurate gas chromatographic retention data and column efficiency represented by the height equivalent to a theoretical plate. With an experimental design, these equations have practical applications in the calculation of the resolution for a given pair of these compounds and for the determination of the isothermal conditions for their separation in minimum time. The method reduced the number of experiments to be carried out and provided good agreement between experimental results and predicted data.