Arlette Baillet

Determination of alkylphosphonic acids by capillary zone electrophoresis using indirect UV detection

Abstract Capillary zone electrophoresis with indirect UV detection was used for the determination of a series of alkylphosphonic acids. For this purpose, a few UV-absorbing background electrolytes were tested and phenylphosphonic acid, which has a mobility close to that of the analysed compounds, was shown to be the most suitable. The influence of several parameters such as concentration of the UV-absorbing background electrolyte and concentration of borate on both sensitivity and efficiency was investigated. An increase in the borate concentration produced an improvement of the signal-to-noise ratio. Conversely, the sensitivity decreased with increasing concentration of the phenylphosphonic acid. The reproducibility of the method was very satisfactory and limits of detection were less than 0.21 pmol injected.

Mechanism of response enhancement in evaporative light scattering detection with the addition of triethylamine and formic acid

Triethylamine with an equimolar amount of formic acid added to the mobile phase provides an enhancement of the evaporative light scattering detector (ELSD) response. After characterization of the influence of various parameters on the ELSD response, a sequential strategy was defined to elucidate this response enhancement. The response enhancement was more marked at low mobile phase flow rate, and was highly dependent on solutes and solvents. The influence of drift tube temperature on response enhancement with various solutes demonstrated that triethylamine and formic acid mainly act as mass amplifiers by the inclusion of triethylamine-formic acid clusters inside the droplets.

Assessment of the retention properties of poly(vinyl alcohol) stationary phase for lipid class profiling in liquid chromatography.

Potentialities of polymerized vinyl alcohol on silica gel were assessed for class separation of simple lipids, sphingolipids, glyceroglycolipids and phospholipids by high-performance liquid chromatography. A screening of pure solvents in binary gradient elution and a chemometric approach was used to define a rugged two segment linear gradient formed from four solvents for total lipid class separation. Triethylamine and formic acid were added in all mobile phase components for acidic phospholipid separation and evaporative light scattering response enhancement. Simple analytical procedures are described for the analysis of complex lipid materials.

Structural analysis of commercial ceramides by gas chromatography-mass spectrometry.

A simple method using gas chromatography-mass spectrometry was applied to analyse structures of ceramides. Identification of trimethylsilylated ceramides were obtained in short analysis times (derivatization of ceramides in 30 min at room temperature and 20 min gas chromatography mass spectrometry run) even for complex mixtures. For example in ceramide Type III, 18 peaks were observed which represent 27 various structures. The coeluted compounds were ceramides containing the same functional groups and the same carbon number but with a different distribution on the two alkyl chains of the molecule. They were accurately differentiated by mass spectrometry. Therefore, 83 structures of trimethylsilylated ceramides were identified in 11 different commercial mixtures. For 52 structures of these, mass spectral data were not described in the literature, neither full mass spectra nor characteristic fragments.

Retention behaviour of ceramides in sub-critical fluid chromatography in comparison with non-aqueous reversed-phase liquid chromatography

This study was devoted to the development of an analytical method for ceramide analysis in packed subcritical fluid chromatography (pSubFC). Monofunctional grafted silica support was found to be more suitable for ceramide analysis. Five Kromasil columns were coupled and the parameters, temperature, pressure and percentage of organic modifier in CO2 were optimised, considering selectivity and analysis time. The final conditions were 31 degrees C, 6% of methanol (MeOH) and 13 MPa. In these conditions the selectivity for structural differences (methylene group, unsaturation or two different bases) were studied. As classically observed, the methylene selectivity decreased with the increase of the eluotropic strength. Moreover, unlike in non-aqueous reversed-phase liquid chromatography (NARP-LC), adding a further unsaturation and two further methylene groups on ceramide results to an increase of retention in pSubFC. Moreover, this last technique allowed to separate ceramides with the same total number of carbons containing unsaturated fatty acids, when the distribution of carbon number of the two chain is very different. These results had enabled to plot retention chart in order to predict ceramide structure in view to identify additional ceramide. This retention chart was finally compared with the one already obtained in NARP-LC.

Eluotropic strength in non-aqueous liquid chromatography with porous graphitic carbon

Porous graphitic carbon is an attractive packing for the chromatographic analysis of highly hydrocarbonaceous compounds with non-aqueous mobile phase. An eluotropic-strength scale of 10 pure organic solvents was established using the methylene selectivity from the fatty acid methyl ester homologous series (chain length between 18 and 31 carbon atoms). Eight binary mobile phases combining a weak solvent: methanol or acetonitrile with a strong solvent: toluene, chloroform, dichloromethane or tetrahydrofuran at different volume fractions phi of strong solvents (ranging from 0.3 to 1.0) were tested and their eluotropic strengths were then compared with those of pure solvents. The curves of the eluotropic strength versus the volume fraction of the strong solvent followed two different trends: linear or curved. The knowledge of the pure solvent strength is not sufficient to predict the eluotropic strength of solvent in the mixture. Then modelling of the eluotropic strength for binary mobile phases was envisaged in order to provide a prediction tool. This model was assessed for the establishment of the composition of eight iso-eluotropic mobile phases. Good assessment was found except in the case of toluene with acetonitrile where the difference between the predicted and the real value was the highest.

Postcolumn fluorescence as an alternative to evaporative light scattering detection for ceramide analysis with gradient elution in non-aqueous reversed-phase liquid chromatography.

Ceramide analysis was developed with gradient elution in non-aqueous reversed-phase liquid chromatography with evaporative light scattering detection (ELSD) or postcolumn fluorescence detection. Fluorescence detection (excitation, 360 nm; emission, 425 nm) after postcolumn formation of mixed assemblies between eluted ceramides and 1,6-diphenyl-1,3,5-hexatriene was developed. In comparison with ELSD, fluorescence detection allows a better detection of the minor species ceramide from ceramide type III (commercial mixture of non-hydroxy fatty acid-sphingosine) and appears to be more sensitive for quantitation of ceramides at low concentrations. The fluorescence response is linear over a wide range of injected amount of ceramide III (expressed as stearoyl-phytosphingosine): 10 ng to 1000 ng. The response of ELSD is non linear but can be linearized in double logarithmic coordinates for calculations over a narrow range, e.g. between 10 to 350 ng ceramide III injected. The lower quantitation limits of these two detectors are similar: 5 ng ceramide III was injected.

Isolation of ceramide fractions from skin sample by subcritical chromatography with packed silica and evaporative light scattering detection.

Separative method of lipid classes from the stratum corneum was developed with packed silica and supercritical CO2 containing 10% of methanol at 15 degrees C, 15 MPa and 3 ml min(-1). The elution order of lipid classes was first esterified cholesterol, triglycerides, squalene co-eluted in a single peak, then free fatty acids, free cholesterol, ceramides and finally glycosylceramides. The ceramides were eluted in several fractions which depended on the number of hydroxyl groups in the molecule, i.e. more hydroxyl groups were contained in ceramides, more important was the retention. Moreover, the retention was not altered by the presence of carbon double bond and variation of the alkyl chain length. The ceramide response with the evaporative light scattering detector was improved by turning the influence of the solvent nature on the response to advantage. Therefore, addition of various solvents with or without triethylamine and formic acid were tested in post-column due to the incompatibility of such modifiers with silica stationary phase. Thereby the solvent conditions for the separation and the detection can be adjusted almost independently. The response was greatly increased by post-column addition of 1% (v/v) triethylamine and its equivalent amount of formic acid in dichloromethane introduced at 0.1 ml min(-1) into the mobile phase. This device had allowed the detection of 400 ng of ceramide with a S/N = 21, whereas no peak was observed in absence of the post-column addition. Finally, the method was applied to the treatment of skin sample which led to highly enriched ceramide fraction.

Structure–retention diagrams of ceramides established for their identification

Molecular species analysis of ceramides was carried out using porous graphitic carbon with gradient elution: chloroform-methanol from 45:55 to 85:15 with a slope at 2.7%/min. These conditions gave a linear relationship between retention data and structure of ceramides. It was demonstrated that linearity occurred when a high slope value of linear gradient elution was used. Thereby the linear diagram was evolved by plotting the adjusted retention time against the total number of carbon atoms of ceramide molecules. Each line represents one ceramide class. Such a Structure-Retention Diagram describes ceramide retention and thus constitutes an identification method using only retention data. This Structure-Retention Diagram was assessed and compared to another obtained from octadesyl-grafted silica in terms of their reproducibility, precision and ability to provide ceramide identification. Better identification was obtained using the results from both Structure-Retention Diagrams. This approach with a two-dimensional separation system allowed to take advantage of the specificity of both identification models.

Étude par chromatographie liquide haute performance des antioxydants phénoliques présents dans les matériaux plastiques: Comparaison de trois méthodes de détection: spectrophotométrique dans l'ultra-violet, electrochimique, et évaporative à diffusion de la lumière

Abstract Comparison of three detection systems for reversed-phase high-performance liquid chromatography of traces of antioxidants in plastics: UV spectroscopy, electrochemical detection and light-scattering diffusion. National and international regulations require the identification of penolic antioxydants in plastic materials used for the packaging of pharmaceuticals, food and cosmetics, and the detection of their possible migration into the contents. The objective of this paper is to propose a method for the identification of these compounds by high-performance liquid chromatography and to determine their limit of sensitivity by UV spectrophotometry, electrochemistry and light scattering diffusion. The results are discussed for four phenolic antioxidants generally used in plastic materials.