Yves-Claude Guillaume

A novel stationary phase based on amino derivatized nanotubes for HPLC separations: theoretical and practical aspects.

A novel column based on silica containing immobilized carbon nanotubes (CNTs) was developed and evaluated in terms of its binding efficiency and resolution. First, CNT functionalized with amino groups (CNT-NH(2)) were prepared via chemical modification of carboxylic groups introduced on the CNT surface. Secondly the covalent immobilization of CNT-NH(2) was carried out by using glutardialdehyde activating agent on aminopropyl (AP)-silica surface. This CNT stationary phase was applied to the HPLC separation of two molecule series, i.e. polychlorinated biphenyl (PCB) isomers with different degrees of substitution in the ortho-position (non-ortho to tetra-ortho substituted) and terpenes (linalool, geraniol, thymol, alpha-terpineol). The retention behavior of these solute molecules was measured under isocratic conditions with different mobile phase compositions, ranging from 0.05-0.70 v/v of toluene in cyclohexane. The retention factors of the solute molecule do not depend linearly on the toluene fraction but follow a quadratic relationship. This CNT stationary phase was a very useful column for the separation of PCB congeners and terpenes. It was demonstrated that a planar conformation of the solute molecule enhanced the solute retention on this CNT stationary phase. As well, a quantitative structure relationship derived, demonstrated the significant input to retention was due to the structurally selective dipole-dipole and charge transfer interactions with the solutes. These results were compared with those obtained on the AP stationary phase. The proposed CNT stationary phase for the separation possess distinctive and interesting retentive properties, and chemometric analysis of retention data of appropriate designed series of test solutes appears to be a convenient, objective and quantitative method to prove a new phase specificity.

Comparison of quantitative high performance thin layer chromatography and the high performance liquid chromatography of parabens

A method is described for the densitometric determination of the p-hydroxybenzoic esters and p-hydroxybenzoic acid in mixtures or in drugs. This method is compared with the one used in high performance liquid chromatography (HPLC). The calibration curves were linear in interval 0.250-3.60 mumol ml-1 per 200 nl per spot. The limit of detection and the relative standard deviation (RSD) are higher than in HPLC (RSD is 6% in HPTLC. 3% in HPLC; limit of detection about 40 pmol in HPTLC and 25 pmol in HPLC) but HPTLC quantitative determination of parabens in drugs is faster.

Analysis of the progesterone displacement of its human serum albumin binding site by β-estradiol using biochromatographic approaches: effect of two salt modifiers

The mechanisms of (i) the binding of two sex-hormones (i.e. progesterone and beta-estradiol) to human serum albumin (HSA) and (ii) the progesterone displacement of its HSA binding cavity by beta-estradiol were studied by biochromatography using three different methods. In the first time, zonal elution method was used to prove the direct competition effect between the two sex-hormone. In the second time, the competition effect between beta-estradiol and progesterone to bound on the same HSA site was analysed by the competitive bi-Langmuir approach. Finally, the thermodynamic data of these two binding processes were studied. The Gibbs free energy value (Delta(approximately)G degrees) of the displacement equilibrium was negative demonstrating that beta-estradiol displaced progesterone of its HSA binding cavity. Moreover, the effect of two chloride modifiers (i.e. Na(+), Mg(2+)) on these two binding processes were analysed. Results showed that in the salt biological concentration ranges, the Mg(2+) cation enhanced strongly the bioavailable progesterone, whereas the Na(+) cation interacted slowly on the progesterone displacement of its HSA binding site by beta-estradiol. This study showed that it must be useful to carry out more in vivo test on the magnesium supplementation effect for women who suffer from estrogen dominance syndrome.

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.

A novel chiral column for the HPLC separation of a series of dansyl amino and arylalkanoic acids

In a previous paper [C. Andre, M. Thomassin, A. Umrayami, L. Ismaili, B. Refouvelet, Y.C. Guillaume, Talanta 71 (2007) 1817] a novel cyclic hexapeptide molecule dissolved in the mobile phase was evaluated as a chiral selector (CS) for the enantiomer separation of a series of dansyl amino and arylalkanoic acids using high performance liquid chromatography (HPLC). In this paper, this CS was immobilized to the surface of a monolithic support and the enantioselectivity and the performance of this novel column are discussed.

Role of the magnesium cation on antihypertensive molecule-human serum albumin binding: affinity chromatography approach.

The role of the Mg2+ cation on antihypertensive molecule binding on human serum albumin (HSA) was studied by affinity chromatography. The thermodynamic data corresponding to this binding were determined for a wide range of Mg2+ concentrations (c). For the nifedipine molecule, an increase in the Mg2+ concentration produced a decrease in binding due to a decrease in the electrostatic interactions. For verapamil and diltiazem, which have the highest solvent accessible surface area, the solute binding on HSA was divided into two Mg2+ concentration regions. For a low c value below c(c) (approximately 1.6 mmol/l), the binding dependence with c was similar to that of nifedipine. For c above c(c) the hydrophobic effect created in the bulk solvent associated with a decrease in the van der Waals interactions between the solute molecule and the HSA implied a decrease in its binding. These results showed that for patients with hypertension, an Mg2+ supplementation during treatment with these antihypertensive molecules can increase the active pharmacological molecule concentration.

Chiral discrimination of dansyl-amino-acid enantiomers on teicoplanin phase: sucrose-perchlorate anion dependence

Abstract The chiral recognition mechanism for a series of d , l -dansyl-amino-acids (test solutes) on a teicoplanin stationary phase was investigated in reversed phase liquid chromatography (RPLC). The effect of both a surface tension modifier (sucrose) and a chaotropic agent (perchlorate anion) on the enantiomeric separation was studied by varying their concentration, c , in the mobile phase. The thermodynamic data supported the fact that the sucrose molecule acted only on the hydrophobic part of the interaction teicoplanin/dansyl-amino-acid and not on the specific chiral part. It was demonstrated that the enhancement of the separation factor observed as the perchlorate salt concentration increased in the mobile phase was enthalpically controlled owing to stereoselective bonding interactions. Such behavior was used to optimize the chromatographic conditions for separation of dansyl-amino-acids on teicoplanin.

A biochromatographic framework to evaluate the calcium effect on the antihypertensive molecule-human serum albumin binding.

The Ca(2+) cation effect on the antihypertensive molecule binding on human serum albumin (HSA) was studied by biochromatography. The thermodynamic parameters corresponding to this binding were determined for a wide range of Ca(2+) concentration (x). For the two antihypertensive molecules under study, their binding to HSA can be divided into two Ca(2+) cation concentration regions due to a HSA phase transition. This result was confirmed by an enthalpy-entropy investigation. For a low x value (below x(c)=1.6 mmol l(-1)), the HSA cavity was in an ordered solid-like state leading to an increase in the interactions between the antihypertensive drugs and the HSA cavity and consequently, a solute-HSA affinity increase. For x above x(c), the HSA cavity was in a disordered solid-like state, implying a decrease in the antihypertensive drug-HSA binding.

Magnesium effect on the acetylcholinesterase inhibition mechanism: A molecular chromatographic approach

The acetylcholinesterase enzyme (AChE) was immobilized on a chromatographic support to study the effect of magnesium on the binding mechanism of five AChE inhibitors (donepezil, tacrine, galanthamine, physostigmine and huperzine). The determination of the enthalpy and entropy changes of this binding at different magnesium concentration values suggested that van der Waals interactions and hydrogen bonds predominated the donepezil and tacrine association to AChE. As well, hydrophobic and electrostatic forces seemed to be the major interactions controlling the huperzine, galanthamine and physostigmine association with AChE. In addition, it appeared that magnesium cation increased the binding affinity of galanthamine and physostigmine to the active site gorge of AChE. A comparison of the inhibitors hydrophobicity to their relative bound percentage with AChE showed an affinity enhanced with the increase in the molecule hydrophobicity and confirmed that the hydrophobic forces played an important role in the AChEI-AChE binding process. This novel biochromatographic column could be useful to find a specific inhibitor for this enzyme and so open new perspectives to be investigated.

Magnesium effect on testosterone–SHBG association studied by a novel molecular chromatography approach

A biochromatographic approach is developed to measure for the first time thermodynamic data and magnesium (Mg(2+)) effect for the binding of testosterone (TT) to sex hormone-binding globulin (SHBG) in a wide temperature range. For this, the SHBG was immobilized on a chromatographic support. It was established that this novel SHBG column was stable during an extended period of time. The affinity of TT to SHBG is high and changes slightly with the Mg(2+) concentration because the number of Mg(2+) linked to binding is low. The determination of the testosterone retention with the steroid hormone at different Mg(2+) concentrations and temperatures demonstrated that the Mg(2+) binding heat effect associated with this Mg(2+) release or uptake during this binding was in magnitude around 17kJ/mol corresponding to the model describing the electrostatic attraction that occurs between the negatively charged non specific areas of SHBG and the positively charged of magnesium. At all the magnesium concentrations studied, the DeltaH values were negative due to van der Waals interactions and hydrogen bonding which are engaged at the complex interface confirming strong TT-SHBG hydrogen bond networks. As well, the DeltaS values were all positive due to hydrophobic forces in the testosterone-SHBG complex formation. In addition our results suggest that adaptive conformational transitions contribute to the specific testosterone-SHBG complex formation. As well, in the biological Mg(2+) concentration domain, it was clearly demonstrated that there was an uncompetitive inhibition of Mg(2+) on TT-SHBG binding which led an enhancement of bioavailable TT. Our work indicated that our biochromatographic approach could soon become very attractive for study other SHBG-steroid (or phytoestrogen) binding.