Retention mechanism of peptides on a stationary phase embedded with a quaternary ammonium group: a liquid chromatography study.

We investigated the mechanisms involved in the retention of various peptides on a stationary phase embedded with a quaternary ammonium group (BS C23), by high-performance liquid chromatography. This was compared with peptide retention on a conventional reversed-phase C18 (RP C18) column under isocratic conditions, to understand better the various mechanisms involved. Chromatographic characterization of the two stationary phases with "model" compounds showed that BS C23 is less hydrophobic than RP C18 and induces electrostatic interaction (attraction or repulsion) with ionized compounds. If reversed-phase partitioning was the predominant retention phenomenon, for both stationary phases, the retention mechanisms in BS C23 provided different selectivity to that of RP C18. Electrostatic attraction or repulsion was clearly observed between peptides and the permanent positively charged group embedded in BS C23 depending on the pH. For most of the peptides, a weak anion-exchange mechanism was observed on the quaternary ammonium-embedded stationary phase if mobile phases at neutral pH and low ionic strengths were employed.

Quantification of deformed peaks in capillary gas chromatography (CGC): application to simultaneous analysis of free fatty acids and less polar compounds in aqueous distillery effluent.

The purpose of this study was to find a simple and rapid method allowing the simultaneous quantification of some alcoholic fermentation inhibitors present in aqueous distillery effluent in order to evaluate its recycling properties. A capillary gas chromatography (CGC) method was tested for the quantification of both short chain fatty acids (acetic to hexanoic) and neutral compounds (butane 2,3-diol, 2-furaldehyde, phenyl-2-ethane1-ol). A polyvalent column coated with trifluoro-propyl-polysiloxane, allowing water injection, was tested and experiments were performed directly on untreated samples. During the development of the method, a deformation of acid peaks was observed; that could be explained by a secondary equilibrium, added to the chromatographic equilibrium. Although the acid peaks were deformed, calibration curves were produced and rigorously validated, proving that quantification is possible even when the best chromatographic conditions have not been achieved. Eventually, the method enabled the concentration of eight major fermentation inhibitors in distillery effluent to be measured.

Chromatographic behaviour of peptides on a mixed-mode stationary phase with an embedded charged group by capillary electrochromatography and high-performance liquid chromatography.

Retention behaviour of biological peptides was investigated on a stationary phase bearing an embedded quaternary ammonium group in a C21 alkyl chain by both high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). In HPLC experiments, variation of acetonitrile (ACN) content in the mobile phase showed that peptides are mainly separated by RP mechanism. The weak or negative retention factors observed as compared to C18 silica stationary phase suggested the involvement of an electrostatic repulsion phenomenon in acidic conditions. Comparison of HPLC and CEC studies indicated that (i) ion-exclusion phenomenon is more pronounced in HPLC and (ii) higher ACN percentage in mobile phase induce for some peptides an increase of retention in CEC, pointing out the existence of mechanisms of retention other than partitioning mainly involved in chromatographic process. This comparative study demonstrated the critical role of electric field on peptide retention in CEC and supports the solvatation model of hydrolytic pillow proposed by Szumski and Buszewski for CEC using mixed mode stationary phase in CEC.

Classification of special octadecyl-bonded phases by the carotenoid test.

Amongst the numerous base-deactivated ODS phases obtained by increasing the bonding density or/and by efficient endcapping treatments, some particular stationary phases have been developed, to limit the additional interactions of basic compounds with residual silanols, to work at extreme pH or with rich water mobile phases. Horizontal polymeric phases, sterically protected ones, hybrid silicas, propylene bridge, are particularly used for this purpose. Octadecyl chains with embedded polar groups and hydrophilic endcapping are also used in this goal. The properties of these phases were studied with a simple test consisting in the injection of carotenoid pigments in Subcritical Fluid Chromatography. The molecules used and the nature of the mobile phase allow the determination of hydrophobicity, polar site accessibility and type or/and bonding density of the stationary phases. Whatever the type of the phases, the particular stationary phases do not show any remarkable property, in comparison to other base-deactivated C18-bonded phases. On the other hand, embedded and polar-endcapped phases display a specific behaviour in regard of hydrophilic interactions, which are highlighted by the absence of water in the subcritical fluid. Additional properties of these phases are described, such as steric recognition and retention performances. As expected, polar-embedded phases are less retentive than classical ODS ones, but are sometimes able to provide greater steric recognition. On the other hand, the polar-endcapped phases display greater hydrophobicity than polar-embedded ones. From a simple classification diagram based on chromatographic properties, differences can be noticed between the polar-embedded groups (amide, carbamate, ether, sulfonamide) and between embedded and endcapped phases. Surprising behaviours are also noticed for some on the tested phases.

Atheroprotective effect of adjuvants in apolipoprotein E knockout mice.

Strategies aimed at treating atherosclerosis by immunization protocols are emerging. Such protocols commonly use adjuvants as non-specific stimulators of immune responses. However, adjuvants are known to modify various disease processes. The aim of this study was to determine whether adjuvants alter the development of atherosclerosis. We performed immunization protocols in apolipoprotein E knockout mice (E degrees ) following chronic administration schedules commonly employed in experimental atherosclerosis. Our results point out a dramatic effect of several adjuvants on the development of atherosclerosis; three of the four adjuvants tested reduced lesion size. The Alum adjuvant, which is the adjuvant currently used in most vaccination protocols in humans, displayed a strong atheroprotective effect. Mechanisms accounting for atheroprotective effect of Freund's adjuvants included their capacity to increase both Th2 responses and anti-MDA-LDL IgM titers, and/or to impose atheroprotective lipoprotein profiles. The present study indicates that adjuvants have potent atheromodulating capabilities, and thus, implies that the choice of adjuvant is crucial in long-term immunization protocols in experimental atherosclerosis.

Retention characteristics of porous graphitic carbon in subcritical fluid chromatography with carbon dioxide-methanol mobile phases.

Numerous relationships usually used in high-performance liquid chromatography (HPLC) for describing the retention on porous graphitic carbon (PGC) have been applied in subcritical fluid chromatography, with CO2-methanol mobile phases. As reported in HPLC, octanol-water partition coefficient failed to fit the retention, whereas satisfactory results were obtained with the sum of partial negative charges. A better fit was reached by using the solvation parameter model, allowing a better understanding of the interactions developed between the solute, the stationary and the mobile phases. Results show that the dominant contribution to retention was given by the polarizability (E) and the volume (V), while the hydrogen-bond basicity (B) was not selected in the retention model, whatever the methanol content. The increase in methanol percentage favours the retention decrease, mainly through the volume for hydrophobic compounds, and through the hydrogen-bond acidity for polar compounds.

Advantages of the use of monolithic stationary phases for modelling the retention in sub/supercritical chromatography. Application to cis/trans-beta-carotene separation.

The low viscosity of supercritical fluids enables the coupling of columns, which favours both the high efficiency of separation and the ability of tuning the selectivity. However, it increases the inlet pressure then modifies the fluid density, i.e. the eluotropic strength of the mobile phase. In this case, the latter is rather different depending on the number of coupled columns. This fact prevents the calculation of the chromatographic parameters for coupled columns from the results obtained from one. In subcritical conditions, by using silica rod columns, which have bimodal porous structure, the flow resistance parameter is dramatically reduced. Consequently, the addition of monolithic columns induces only slight internal pressure changes and the fluid density does not vary with the column length. In this case, the calculation of retention factor and selectivity based on retention values obtained on each separate column provides accurate results allowing to determine the optimum column length in regard to the studied separation. After a better characterisation of the stationary phase included in the Chromolith column, this paper describes the beta-carotene isomers separation obtained by coupling up to six Chromolith columns to an octadecyl bonded particulate one. These compounds were studied because of the difficulty to separate these cis/trans isomers. No abnormal apparent dead volume change due to fluid density variation was reported, and good correlations between experimental and calculated retention factors and selectivities were observed. The optimum separation requires five highly porous columns coupled to a YMC Pack Pro. Moreover, the use of monolith packing allows to decrease both the retention factor and the analytical time by comparison to previous studies.

Interaction mechanisms on octadecyl packed columns in subcritical fluid chromatography with CO2-modifier mobile phases.

An experimental design was carried out for describing the interaction mechanisms between solutes and octadecyl bonded silicas in subcritical fluid chromatography (SubFC), with CO2-methanol and CO2-acetonitrile mobile phases. The effects of modifier amount, temperature and outlet pressure were studied. The homologous series of alkylbenzenes was mainly used as probe, and results were in part assessed with other series. Curves between the methylene selectivity (alphaCH2) and the alkyl chain carbon number (Cn) were plotted, because changes of slope or discontinuity in these curves are yielded by interaction mechanism modifications. Moreover, the linearity of the Van 't Hoff curves with CO2-acetonitrile mobile phases has enabled one to calculate the transfer enthalpy (deltaH) for each homologue. The curves log k = f(-deltaH) allow a discrimination of the retention behaviors between the short and the long homologues for CO2-acetonitrile mobile phases. Depending on the analytical conditions, different oriented partition mechanisms occur for the long homologues, when the short ones seem to be fully embedded into the grafted chains near the silica surface. With methanol-CO2 mobile phases the discrimination between the homologues disappears and the methylene selectivity curves correspond to a bulk partition mechanism. The differences in the interaction mechanisms following the modifier nature are related to the adsorption the mobile phase onto the stationary phase, because the amount of adsorbed mobile phase modifies the bonded chain mobility. With methanol, an important adsorption of the mobile phase occurs, when this adsorption is reduced with acetonitrile. In this latter case, an anisotropy in the stationary phase mobility can explain the observed difference in the interaction mechanisms of homologues. Finally, effects of stationary phase chain length (from C18 to C22) and bonding density (from 2.5 to 3.4 micromol m(-2)) were also reported.

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.

Use of relationships between retention behaviors and chemical structures in subcritical fluid chromatography with CO2/modifier mixtures for the identification of triglycerides.

Satisfactory separations of vegetable oil triglycerides (TG) differing in fatty acid composition are obtained by subcritical fluid chromatography (SubFC) with octadecyl packed columns and CO2/modifier mobile phases. However, the identification of TG can be sometimes difficult due to the small retention differences between the compounds. A method of TG identification in SubFC was achieved, which does not require calculation of retention pattern but uses the differences in retention behavior related to TG structure and to the nature of the subcritical mobile phases. These retention differences were produced by the variation of either outlet pressure or modifier percentage or of temperature. Whatever the column aging, this method allows the determination of the triglyceride total chain length and double bond number. Among numerous structures, these two criteria restrict the structural hypothesis at worst to three or four possibilities and sometimes only to one. The validity of this relative identification method was confirmed by electronic impact mass spectrometry of triglyceride fractions collected from the analysis of a peanut oil. The analysis of concentrated fractions is favored by the spontaneous elimination after the pressure regulator of carbon dioxide, the main fluid of the subcritical mobile phase.

Study of dead volume measurement in packed subcritical fluid chromatography with ODS columns and carbon dioxide-modifier mobile phases.

Studies were done for providing a simple, rapid and reliable procedure of void volume measurement in packed subcritical fluid chromatography (pSubFC), with CO2-modifier mobile phases containing high modifier amounts. Methods used in RPLC with ODS columns were applied in pSubFC: gravimetric, homologous series linearisation and unretained marker injection. Results lead us to propose the method of marker injection to determine the void volume in pSubFC. Acetonitrile was chosen as the void volume marker among six tested markers. Furthermore, void volume variations vs. the modifier volume (from 5 to 45%) were studied for nine organic modifiers. The void volume variations were related both to adsorption-desorption phenomena between the mobile phase and the stationary phase and to mobile phase density changes. These variations allowed the classification of the modifiers into four groups on the basis of the molecular interactions.

Retention Behavior of Triglycerides in Octadecyl Packed Subcritical Fluid Chromatography with CO(2)/Modifier Mobile Phases.

A study of the retention behavior of vegetable oil triglycerides was carried out in subcritical fluid chromatography (SubFC) with CO(2)/modifier mobile phases. Analyses of 15 oils have enabled us to set up a retention diagram which includes ∼30 triglycerides. This diagram establishes a relation between triglyceride series retention and their unsaturation number. Furthermore, another relationship between retention and the triglyceride carbon number was studied. The retention properties were identical with those reported in nonaqueous reversed-phase liquid chromatography or in SubFC with neat CO(2): the linearity of these relationships and retention order that follows the partition number. Moreover, specific effects of modifier on these relationships or on retention variations were observed. Using octadecyl packed columns, these particular behaviors underline that the compound solubility is different between modified subcritical phases and nonaqueous liquid phases. Finally, these behaviors can be useful to increase the triglyceride separation and the previous relationships can be applied to improve the identification of unknown triglycerides.

Methylene Selectivity and Eluotropic Strength Variations in Subcritical Fluid Chromatography with Packed Columns and CO(2)-Modifier Mobile Phases.

This work has consisted of better understanding the chro-matographic behavior in subcritical fluid chromatography with CO(2)-acetonitrile and CO(2)-methanol mobile phases. To that end, the changes of solute solubility in the mobile phase have been studied by determination of methylene selectivity of a homologous series. Moreover, eluotropic strengths have been calculated for mobile phases containing from 5 to 45% modifier. To carry out the studies in all ranges of the subcritical area, experimental designs have been used. The influences of analytical temperature (from 10 to 30 °C) and outlet pressure (from 10 to 20 MPa) have also been investigated both on solubility and on eluotropic strength. A more general comparison of elu-otropic strengths of subFC and HPLC has been achieved.

Comparison of retention mechanisms of homologous series and triglycerides in non-aqueous reversed-phase liquid chromatography.

The retention behaviour of saturated homogeneous triglycerides was compared to that of single chain homologous compounds in reversed-phase liquid chromatography on silica-based octadecyl bonded phases with non-aqueous binary eluents containing chloroform. For both series, in all eluents and at all investigated temperatures, a discontinuity (1-6% slope change) in log k' vs. the carbon atom number, nc, of each solute hydrocarbonaceous chain was observed for a given critical number of carbon atoms, nc,crit approximately 12-13. Similar discontinuities were observed in delta H degrees and delta S degrees vs. nc for the same nc,crit value. These and other phenomena (existence of two convergence points for log k' vs. nc plotted at different eluent compositions; existence of two convergence temperatures in log k' vs. 1/T plotted for several members of a given solute series) reflect the mechanism of penetration of the solute aliphatic chains into the bonded layer. The eluotropic strength of acetonitrile-chloroform mobile phases was determined and seen to have a larger rate of variation with the solvent composition than that of some other non-aqueous binary eluents. The ratio of the slope of plots of log k' vs. nc curves for triglycerides and single chain homologous series is not a whole number but has a value lying between 2 and 3. This explains why triglyceride retentions cannot be predicted from retention data for the fatty acid methyl esters. More importantly, this indicates that the triglycerides may interact with the stationary phase using various conformations, one, two or three chains penetrating into the bonded layer. These conformations, in dynamic equilibrium with each other, contribute differently to the retention. This offers, in principle, the possibility to separate unsaturated triglycerides having the same number of carbon atoms and of double bonds but differing in the distribution of the unsaturations along the chains, if the double bonds located in a non-penetrating chain can selectively interact with a mobile phase component.