Supercritical fluid chromatography hyphenated to bidimensional gas chromatography in comprehensive and heart-cutting mode: design of the instrumentation.

A new multidimensional chromatographic instrument has been developed to perform both SFC-GC×GC and SFC×GC×GC, in response to the challenge of complex matrices characterization. The design of this online system is fully described and enriched by theoretical and practical discussions. A new interface has been investigated: this interface allows the temporary storage of SFC fractions inside sampling loops before their quantitative transfer toward the GC×GC. This innovative design allows flexible and automatic hyphenated approaches such as single, multiple, total heart-cutting and comprehensive modes. An overview of the interfacing experimental conditions is also presented. Thanks to all the hyphenation possibilities of the three dimensions, this instrument opens up new prospects for the quantitative analysis of very complex matrices.

Impact of the probe solutes set on orthogonality evaluation in reverse phase chromatographic systems.

Two-dimensional liquid chromatography (2DLC) is a very attractive technique for the characterization of complex samples due to its separation power obtained via the coupling of two separation modes exhibiting different mechanisms, i.e. orthogonality. In reverse phase, orthogonality is mainly governed by three factors: the pH of the mobile phase, the structure of the stationary phase and the nature of the organic modifier. In the present paper, we studied the impact of the nature of the probe solutes on orthogonality evaluation. A set of 63 compounds with various physicochemical properties was used to evaluate 32 reverse phase chromatographic systems (2 pH × 8 stationary phases × 2 organic modifiers). Principle component analysis revealed that the solutes could be split into three subsets according to their charge in the experimental conditions. The factors affecting orthogonality and the magnitude of their effect were shown to depend on the charge of the compounds. For positively charged (basic) compounds, the pH was the most important factor, followed by the nature of the stationary phase. For negatively charged (acidic) compounds, the nature of the stationary phase had the highest impact. For neutral compounds, only the nature of the stationary phase and, to a smaller extent, the organic modifier had an influence. The present study also showed that a reduced set of only 9 test compounds instead of whole set of the 63 could enable an appropriate orthogonality evaluation.

Kinetic analyses and performance of a colloidal magnetic nanoparticle based immunoassay dedicated to allergy diagnosis.

In this paper, we demonstrate the possibility to use magnetic nanoparticles as immunosupports for allergy diagnosis. Most immunoassays used for immunosupports and clinical diagnosis are based on a heterogeneous solid-phase system and suffer from mass-transfer limitation. The nanoparticles' colloidal behavior and magnetic properties bring the advantages of homogeneous immunoassay, i.e., species diffusion, and of heterogeneous immunoassay, i.e., easy separation of the immunocomplex and free forms, as well as analyte preconcentration. We thus developed a colloidal, non-competitive, indirect immunoassay using magnetic core-shell nanoparticles (MCSNP) as immunosupports. The feasibility of such an immunoassay was first demonstrated with a model antibody and described by comparing the immunocapture kinetics using macro (standard microtiter plate), micro (microparticles) and nanosupports (MCSNP). The influence of the nanosupport properties (surface chemistry, antigen density) and of the medium (ionic strength, counter ion nature) on the immunocapture efficiency and specificity was then investigated. The performances of this original MCSNP-based immunoassay were compared with a gold standard enzyme-linked immunosorbent assay (ELISA) using a microtiter plate. The capture rate of target IgG was accelerated 200-fold and a tenfold lower limit of detection was achieved. Finally, the MCSNP-based immunoassay was successfully applied to the detection of specific IgE from milk-allergic patient's sera with a lower LOD and a good agreement (CV < 6%) with the microtiter plate, confirming the great potential of this analytical platform in the field of immunodiagnosis.

Comparison of different statistical approaches to evaluate the orthogonality of chromatographic separations: application to reverse phase systems.

Selectivity of phase system is of primary concern when designing a bidimensional chromatographic system and looking for the highest degree of orthogonality between the two separations. Several statistical or geometrical criteria can potentially be used to measure the degree of orthogonality. A comparison of eight candidate criteria has been carried out in this study. Analysis of variance (ANOVA) was used to evaluate the relevance of each criterion and its ability to reveal the significance of the influence of factors like pH, stationary phase, and organic modifier. Experimentally, a set of 32 chromatographic systems was evaluated by the same generic gradient with 63 probe solutes, likely to be present in biological and/or environmental samples and covering a wide range of physico-chemical properties: acidic, basic and neutral compounds with different pKa, molecular mass and hydrophobicity (logP). Each chromatographic system was defined by the nature of the stationary phase (8 different silica or grafting chemistries), the pH of the aqueous fraction of the mobile phase (2.5 or 7.0) and the nature of the organic modifier (acetonitrile or methanol). The orthogonality of the 496 couples of chromatographic systems was evaluated and ranked using the eight different approaches: the three correlation coefficients (Pearson, Spearman and Kendall), two geometric criteria characterizing the coverage of the 2D separation space, Slonecker's information similarity and two chi-square statistics of independence between normalized retention times. In fact, there were only seven distinct criteria, since we established the analytical equivalence between the rankings with the likelihood ratio statistics and Slonecker's information similarity. Kendall's correlation coefficient appeared to be the best measure of orthogonality since, according to ANOVA, it exhibited the highest sensitivity to all experimental factors. The chi-square measures, and hence Slonecker's information similarity, performed equally well provided the discretization of the separation space was carried out appropriately. Finally, from the compared study of the factors acting upon orthogonality carried out by ANOVA, it is possible to draw the conclusion that the pH of the mobile phases has the highest impact on the selectivity followed by the type of stationary phase and finally by the organic modifier.

Investigating comprehensive two-dimensional gas chromatography conditions to optimize the separation of oxygenated compounds in a direct coal liquefaction middle distillate.

Considering the global energetic context, diversifying fuels is of growing importance and many new alternatives are promising. Coal liquefaction products definitely appear among the new generation substitutes. These product's characteristics are very far from fuel specifications as they are mainly composed of naphthenes, aromatics, polycondensed naphthenic and aromatic structures and heteroatomic compounds (nitrogen and oxygen), with a very low paraffin content. Identification and quantification of oxygen-containing species in coal-derived liquids are of considerable importance to understand their behaviors in further processing. However, these species have not been characterized as fully as the predominant hydrocarbon components. Literature shows that these compounds consist mainly in alkylated phenolic and furanic structures. Therefore, comprehensive two-dimensional gas chromatography has been investigated to provide enhanced molecular characterization of these complex samples. Several different configurations involving innovative column configurations were tested. Each of them was optimized by testing different column lengths, modulation periods, and oven conditions. A comparison of the contribution of each column configuration was carried out regarding four main criteria: individual separation of oxygenates, group type separation, resolution, and space occupation. One of them enabled an outstanding separation of paraffins, naphthenes, monoaromatics, diaromatics and targeted O-compounds in a direct coal liquefaction product. It was therefore subjected to further experimentations using a time-of-flight mass spectrometer to validate the identification and unravel more than fifty oxygenated molecular structures. A group-type quantification was also established for four column arrangements and gives the distribution of paraffins, naphthenes and aromatics. It can be concluded from this study that a non-orthogonal arrangement involving a highly polar column in the first dimension was the most adapted one.

Challenge in the speciation of nitrogen-containing compounds in heavy petroleum fractions by high temperature comprehensive two-dimensional gas chromatography.

Extending the knowledge related to nitrogen-containing compounds presents an important interest for the petroleum industry due to their implication in atmosphere pollution as well as their inhibitive or refractive behaviour towards hydroprocessing. Most of the nitrogenated species are concentrated in heavy petroleum cuts. As no analytical method is resolutive enough for these heavy cuts, particularly regarding nitrogen-containing compounds, a new approach is needed. For this reason, this study focuses on the development of a GC×GC technique, through the hyphenation of a specific NCD detector with a GC×GC system at high temperature. The performances of highly polar thermally stable stationary phases, in particular those composed of promising ionic liquids, were monitored in normal and reversed configurations. Subsequently, after the extraction of neutral or basic compounds by adsorption on an ion-exchange resin, a first quantitative determination was attempted for a straight-run and a direct coal liquefaction vacuum gas oils. This study led to a better understanding of the behaviour of highly aromatic N-compounds by 2D-GC including an ionic liquid phase as well as a deeper N-characterization of heavy petroleum fractions.

Extended characterization of a vacuum gas oil by offline LC-high-temperature comprehensive two-dimensional gas chromatography.

In a context of environmental preservation, purification and conversion of heavy petroleum cuts into high-quality fuel becomes essential. The interest for the characterization of those very complex matrices becomes a trendy analytical challenge, when it comes to get molecular information for the optimization of industrial processes. Among new analytical techniques, high-temperature 2-D GC has recently proved its applicability to heavy petroleum matrices, but lacks in selectivity to separate all chemical groups. To gain resolution, heart cutting is demonstrated for LC separation of saturated, aromatic and polar compounds prior to high-temperature 2-D GC. Therefore, an extended global resolution was obtained, especially by a better distinction of saturated compounds. This includes iso-paraffins and biomarker polynaphthenic structures, which are impossible to quantify with MS methods. This new way to analyze heavy petroleum fractions gives innovative opportunities for the construction of global weight distributions by carbon atoms number and by chemical families. This can right now be employed for quantitative analysis of heavy petroleum fractions and for studying conversion processes.

A chemometric approach for optimizing protein covalent immobilization on magnetic core-shell nanoparticles in view of an alternative immunoassay.

A chemometric approach was developed to optimize the grafting of a bovine milk allergen: alpha-Lactalbumin (alpha-Lac) on colloidal functionalized magnetic core-shell nanoparticles (MCSNP). Such nanoparticles, functionalized with polyethyleneglycol and amino groups, exhibit a 30nm physical diameter and behave as a quasi-homogeneous system. The alpha-Lac immobilization was achieved through the covalent binding between MCSNP amino groups and alpha-Lac carboxylic moieties using the well-known tandem carbodiimide (EDC) and hydroxysulfosuccinimide (NHS). In this study, a chemometric approach was employed to highlight the parameters influencing the number of grafted proteins on the MCSNP. Three factors were evaluated: the ratio in concentration between EDC and alpha-Lac, between NHS and EDC and the concentration of alpha-Lac. After a first full factorial design to delimit the region of the space where the optimum could be located, a central composite design was then carried out to predict the best grafting conditions. It was established and experimentally confirmed that the optimum parameters are [EDC]/[alpha-Lac]=25; [NHS]/[EDC]=1.55 and alpha-Lac=24.85nmolmL(-1). In these optimal conditions, MCSNP surface was successfully saturated with alpha-Lac (34 alpha-Lac/MCSNP) with a high reproducibility (RSD=2%). The colloidal stability of MCSNP grafted with alpha-Lac as well as the immunological interactions using anti alpha-Lac antibody were then investigated in different buffers. The results emphasized that a 50mM MES buffer (pH 6) allows an efficient immune capture and a satisfying colloidal stability which provide an immunological interaction in homogeneous liquid phase.

Supercritical fluid chromatography hyphenated with twin comprehensive two-dimensional gas chromatography for ultimate analysis of middle distillates.

This paper reports the conditions of online hyphenation of supercritical fluid chromatography (SFC) with twin comprehensive two-dimensional gas chromatography (twin-GCxGC) for detailed characterization of middle distillates; this is essential for a better understanding of reactions involved in refining processes. In this configuration, saturated and unsaturated compounds that have been fractionated by SFC are transferred on two different GC x GC columns sets (twin-GCxGC) placed in the same GC oven. Cryogenic focusing is used for transfer of fractions into the first dimension columns before simultaneous GCxGC analysis of both saturated and unsaturated fractions. The benefits of SFC-twin-GC x GC are demonstrated for the extended alkane, iso-alkane, alkene, naphthenes and aromatics analysis (so-called PIONA analysis) of diesel samples which can be achieved in one single injection. For that purpose, saturated and unsaturated compounds have been separated by SFC using a silver loaded silica column prior to GC x GC analysis. Alkenes and naphthenes are quantitatively recovered in the unsaturated and saturated fractions, respectively, allowing their identification in various diesel samples. Thus, resolution between each class of compounds is significantly improved compared to a single GCxGC run, and for the first time, an extended PIONA analysis of diesel samples is presented.

Integrated microdevice for preconcentration and separation of a wide variety of compounds by electrochromatography.

An integrated microdevice was developed to couple on-chip SPE to separation by channel electrochromatography. An acrylate-based monolith was synthesized within a glass microdevice by photoinitiated polymerization. It was used for both separation and preconcentration by direct injection on the head of the stationary phase or by confining the preconcentration step in a given zone of the stationary phase. The composition of the polymerization mixture was chosen to achieve a monolithic material containing both hydrophobic and charged moieties to ensure an electroosmotic flow for separation. As a consequence the extraction procedure occurs via hydrophobic and ionic interactions. Neutral, ionizable and charged compounds were successfully preconcentrated and separated within the microdevice through electrochromatographic mechanisms, highlighting the versatility of this device. The performance of the integrated microdevice was demonstrated with the preconcentration and separation of a mixture of PAHs for which a signal enhancement factor (SEF) of 270 was achieved within 120 s of preconcentration. In the case of charged and ionizable compounds, according to the electrolyte composition, contributions of both reverse-phase and ion-exchange mechanisms were used to perform effective electrochromatographic preconcentration. A SEF of 250 was obtained for the model-charged compound within 20 s of preconcentration. Finally, the potentials of on-chip preconcentrate and separate both neutral and ionized compounds have been demonstrated using a mixture of model compounds.

High-temperature two-dimensional gas chromatography of hydrocarbons up to nC60 for analysis of vacuum gas oils.

In a tense energetic context, the characterization of heavy petroleum fractions becomes essential. Conventional comprehensive two-dimensional gas chromatography (2D-GC or GCxGC) is widely used for middle distillates analysis, but only a few applications are devoted to these heavier fractions. In this paper, it is shown how the optimization of GCxGC separation allowed the determination of suitable high-temperature (HT) conditions, adjusting column properties and operating conditions. 2D separations were evaluated using 2D separation criteria and a new concept of 2D asymmetry (As(2D)). New HT conditions allowed the extension of GCxGC range of applications to heavier hydrocarbons, up to nC(60). A first application of high-temperature two-dimensional gas chromatography (HT-2D-GC) to a full vacuum gas oil (VGO) feed stock is described. Comparisons with other standardized methods illustrate the high potential of HT-2D-GC for heavy fractions analysis.

Using comprehensive two-dimensional gas chromatography for the analysis of oxygenates in middle distillates I. Determination of the nature of biodiesels blend in diesel fuel.

In the current energetic context (increasing consumption of vehicle fuels, greenhouse gas emission etc.) government policies lead to mandatory introduction in fossil fuels of fuels resulting from renewable sources of energy such as biomass. Blending of fatty acid alkyl esters from vegetable oils (also known as biodiesel) with conventional diesel fuel is one of the solutions technologically available; B5 blends (up to 5%w/w esters in fossil fuel) are marketed over Europe. Therefore, for quality control as well as for forensic reasons, it is of major importance to monitor the biodiesel origin (i.e. the fatty acid ester distribution) and its content when it is blend with petroleum diesel. This paper reports a comprehensive two-dimensional gas chromatography (GC x GC) method that was developed for the individual quantitation of fatty acid esters in middle distillates matrices. Several first and the second dimension columns have been investigated and their performances to achieve (i) a group type separation of hydrocarbons and (ii) individual identification and quantitation of fatty acid ester blend with diesel are reported and discussed. Finally, comparison of quantitative GC x GC results with reference methods demonstrates the benefits of GC x GC approach which enables fast and reliable individual quantitation of fatty acid esters in one single run. Results show that under developed chromatographic conditions, quantitative group type analysis of hydrocarbons is also possible, meaning that simultaneous quantification of hydrocarbons and fatty acid esters can be achieved in one single run.

Integration of various stacking processes in carrier ampholyte-based capillary electrophoresis.

Field-enhanced sample stacking, field-enhanced sample injection as well as electrokinetic supercharging have been successfully integrated in carrier ampholyte-based capillary electrophoresis. Through the analysis of different test sample mixtures, it has been shown that the carrier ampholyte-based background electrolytes, in spite of their very low conductivity, allow efficient online preconcentration of analytes by field-amplified techniques. Sensitivity enhancement factors of the same magnitude as those usually encountered with classical conductive background electrolytes have been obtained in such carrier ampholyte-based buffers. Depending on the online preconcentration method that has been integrated, sensitivity enhancement factors between 50 and several thousands have been reached.

Comprehensive two-dimensional gas chromatography for enhanced analysis of naphthas: new column combination involving permethylated cyclodextrin in the second dimension.

A new column association using comprehensive two-dimensional gas chromatography for the detailed molecular analysis of hydrocarbon mixtures is reported in this paper. In order to compare the impact of two different secondary columns, a novel column combination relying on a GC x 2GC system was used. This system is based on a non-polar first column (PONA) combined with both a permethylated beta-cyclodextrin (beta-Dex 120) stationary phase and a polysilphenylensiloxane (BPX 50) in the second dimension. Compared to BPX 50 stationary phase, the implementation of beta-cyclodextrin columns as the second dimension was found to improve the resolution between paraffins and naphthenes in the naphtha range but not in the middle distillate range. Attempts to improve the results and to understand the interaction mechanism remained unsuccessful. Therefore, the benefits of the beta-Dex 120-column are only demonstrated on heavy naphtha cut for the quantitation of hydrocarbons.

Online preconcentration using monoliths in electrochromatography capillary format and microchips.

Online preconcentration and separation of analytes using an in situ photopolymerized hexyl acrylate-based monolith stationary phase was evaluated using electrochromatography in capillary format and microchip. The band broadening occurring during the preconcentration process by frontal electrochromatography and during the desorption process by elution electrochromatography was studied. The hexyl acrylate-based monolith provides high retention for neutral analytes allowing the handling of large sample volumes and its structure allows rapid mass transfer, thus reducing the band broadening. For moderately polar analytes such as mono-chlorophenols that are slightly retained in water, it was shown that enrichment factors up to 3500 can be obtained by a hydrodynamic injection of several bed volumes for 120 min under 0.8 MPa with a decrease in efficiency of 50% and a decrease of 30% for the resolution between 2- and 3-chlorophenol. An 8 min preconcentration time allows enrichment factors above 100 for polyaromatic hydrocarbons. The interest of these monoliths when synthesized in microchip is also demonstrated. A 200-fold enrichment was easily obtained for PAHs with only 1 min as preconcentration time, without decrease in efficiency.

Comparison of different capillary isoelectric focusing methods--use of "narrow pH cuts" of carrier ampholytes as original tools to improve resolution.

Two capillary isoelectric focusing (CIEF) systems have first been optimized: one uses a bare silica capillary and 30% (v/v) of glycerol in the separation medium while the other uses a coated capillary and an aqueous background electrolyte. To perform permanent capillary coating, two neutral polymers have been compared: hydroxypropylcellulose (HPC) and polyvinylalcohol (PVA). HPC coating gave best results for electroosmotic flow (EOF) limitation on a wide pH range: as compared to a bare silica capillary, it allowed to decrease EOF by 96% at pH 7.2 after acidic and basic treatments, whereas PVA coating lead only to a 76% decrease. The glycerol CIEF system was more satisfying for the separation of model proteins classically used as pI markers. Finally, the use of "narrow pH cuts" of carrier ampholytes added to commercial ampholyte mixtures allowed increasing resolution up to a factor 2.4 at a chosen pH for the separation of pI markers and milk proteins.

New benchmark for basic and neutral nitrogen compounds speciation in middle distillates using comprehensive two-dimensional gas chromatography.

This paper reports an analytical method for the comprehensive two-dimensional gas chromatography (GC x GC) separation and identification of nitrogen compounds (N-compounds) in middle distillates according to their types (basicity). For the evaluation of the best chromatographic conditions, a non-polar x polar approach was chosen. The impact of the second dimension (stationary phase and column length) on the separation of basic and neutral N-compounds was evaluated by mean of two-dimensional resolution. This study revealed that the implementation of polar secondary column having free electron pairs improves drastically the separation of N-compounds. Indeed, the presence of permanent dipole-permanent dipole interactions between neutral N-compounds and the stationary phase was enlightened. The comparison of two different nitrogen chemiluminescence detectors (NCD) was also evaluated for GC x GC selective monitoring of N-compounds. Owing to higher resolution power and enhanced sensitivity achieved using developed chromatographic and detection conditions, it was possible to identify univocally and to quantitate N-compounds (i) by class of compounds and (ii), within a class, by carbon number. Finally, quantitative comparison of GC x GC-NCD with conventional gas chromatography illustrates the benefits of GC x GC leading to an excellent correlation with results obtained by American Society for Testing Materials (ASTM) methods for the determination of basic/neutral nitrogen ratio in diesel samples.

Molecularly imprinted polymers for the clean-up of a basic drug from environmental and biological samples.

A molecularly imprinted polymer (MIP) was synthesized and evaluated to selectively extract an alpha-blocker, i.e. alfuzosin, from human plasma. The synthesis of the MIP was performed in dichloromethane with methacrylic acid as monomer and the target drug as template. A first series of experiments was carried out in dichloromethane to estimate the potential of the MIP in its specific recognition medium, i.e. dichloromethane, by developing a selective procedure and by measuring the capacity of the sorbent. An optimized procedure was developed for the selective extraction of alfuzosin with a recovery close to 100% in this medium and a specific capacity of 1.3 micromol g(-1) of MIP was measured. A study in aqueous media was also carried out by a comprehensive approach of the retention mechanism in order to build a selective procedure of extraction. The effects of the amount and of the charge of cations were studied and an optimal pH value was defined to limit matrix effects. Then, the alfuzosin MIP was then directly used to selectively extract the target drug from human plasma with an extraction recovery of 60%. Lastly, a soil was extracted by a pressurized solvent and the resulting extract was cleaned up on the MIP, showing the possibility to use this selective sorbent for the sample treatment of various complex matrices.

Transient isotachophoresis in carrier ampholyte-based capillary electrophoresis for protein analysis.

Transient ITP (t-ITP) has been used in carrier ampholyte-based CE (CABCE) to enhance the sensitivity of protein analysis. The characteristics of carrier ampholytes (CAs) narrow pH cuts-based buffers, when used as BGEs in CE are compatible with t-ITP requirements. Indeed, being the sole buffering species of such solutions, CAs impose a pH close to their pI. Thus, in these solutions, the CAs possess low electrophoretic mobility. As a consequence, by adding an ionic component with high electrophoretic mobility either in the studied sample or in the BGE, a t-ITP step can be generated. This has first been demonstrated for protein test mixtures. Then, the combination of t-ITP with CABCE has been applied to study a real sample, the bovine milk.

Feasibility study of a reference material for water chemistry: long term stability of triazine and phenylurea residues stored in vials or on polymeric sorbents.

Matrix Reference Materials (MRM) are essential tools for the validation of analytical protocols. Nowadays, there are no such materials for the determination of herbicides in water. So, a feasibility study of a MRM for the analysis of triazines and phenylureas in water was carried out. Different kinds of candidates MRM were prepared: solutions of pesticides diluted in acetonitrile and stored in sealed vials or stored at the dry state after the evaporation of the solvent to dryness, pesticides stored on two different types of polymeric solid-phase extraction (SPE) sorbents after the percolation of drinking or river waters spiked with pesticides. The stability of these candidates MRM stored at various temperatures (room temperature, 0.5 degrees C or -18 degrees C) was studied over a period of approximately 1 year. Two different levels of concentration were studied for each kind of material. During the storage, some samples of each different MRM candidate were monthly analyzed by liquid chromatography. Results showed that, among the candidate materials, some of them presented satisfactory enough stability to consider a further certification. They were either pesticides in solution in sealed vials or pesticides stored on cartridges after the percolation of spiked water samples. However, it was shown that these different MRM candidates had to be stored at a temperature lower than 0.5 degrees C.