Characterization of semi-packed columns with different cross section in high-pressure gas chromatography.

Hydrodynamics, efficiency, and loading capacity of two semi-packed columns with different cross sections (NANO 315 µm x 18 µm; CAP 1000 µm x 28 µm) and similar pillar diameter and pillar-pillar distance (respectively 5 µm and 2.5 µm) have been compared in high-pressure gas chromatography. A flow prediction tool has been first designed to determine pressure variations and hold-up time across the chromatographic system taking into account the rectangular geometry of the ducts into the semi-packed columns. Intrinsic values of Height Equivalent to Theoretical Plate were determined for NANO and CAP columns using helium as carrier gas and similar values have been obtained (30 µm) for the two columns. Loading capacity of semi-packed columns were determined for decane at 70 °C using helium, and the highest value was obtained from CAP column (larger cross section and stationary phase content). Finally, significant HETP improvement (down to 15 µm) and peak shape were observed when carbon dioxide was used as carrier gas, suggesting mobile phase adsorption on stationary phase in high pressure conditions.

Scout triggered multiple reaction monitoring mass spectrometry for the rapid transfer of large multiplexed targeted methods in metabolomics.

The field of metabolomics based on mass spectrometry has grown considerably in recent years due to the need to detect and, above all, quantify a very large number of metabolites, simultaneously. Up to now, targeted multiplexed analysis on complex samples by Liquid Chromatography coupled with tandem Mass Spectrometry (LC-MS/MS) has relied almost exclusively on compound detection based on absolute retention times, as in the Scheduled-MRM (sMRM) approach. Those methods turn out to be poorly transferable from one instrument to another and result in a time-consuming and tedious method development involving a significant number of critical parameters that need specific re-optimisation. To address this challenge, we introduce a novel acquisition mode called scout-triggered MRM (stMRM). In stMRM, a marker transition is used to trigger MS analysis for a group of dependent target analytes. These marker transitions are strategically distributed throughout the chromatographic run, and the dependent analytes are associated based on their retention times. The result is a targeted assay that remains robust even in the presence of retention time shifts. A 3 to 5-fold increase in the number of detected transitions associated to plasma metabolites was obtained when transferring from a direct application of a published sMRM to a stMRM method. This significant improvement highlights the universal applicability of the stMRM method, as it can be implemented on any LC system without the need for extensive method development. We subsequently illustrate the robustness of stMRM in modified chromatographic elution conditions. Despite a large change in metabolite's selectivity, the multiplexed assay successfully recovered 70% of the monitored transitions when consequently modifying the gradient method. These findings demonstrate the versatility and adaptability of stMRM, opening new avenues for the development of highly multiplexed LC-MS/MS methods in metabolomics. These methods are characterized by their analytical transparency and straightforward implementation using existing literature data.

Dual detection chromatographic method for fast characterization of nano-gravimetric detector.

Nano-gravimetric detector (NGD) has been recently introduced as miniaturized gas chromatography detector. The NGD response is based on an adsorption-desorption mechanism of compounds between the gaseous phase and the NGD porous oxide layer. The NGD response was characterized by hyphenating NGD in-line with FID detector and a chromatographic column. Such method led to the full adsorption-desorption isotherms of several compounds in a single run. Langmuir model was used to describe the experimental isotherms, and the initial slope of the isotherm (Mm.KT) obtained at low gas concentration was used to compare the NGD response for different compounds (good repeatability was demonstrated with a relative standard deviation lower than 3%). The column-NGD-FID hyphenated method was validated using alkane compounds according to the number of carbon atoms in the alkyl chain and to the NGD temperature (all results agreed with thermodynamic relations associated to partition coefficient). Furthermore, relative response factor to alkanes, for ketones, alkylbenzenes, and fatty acid methyl esters have been obtained. These relative response index values led to easier calibration of NGD. The established methodology can be used for any sensor characterization based on adsorption mechanism.

Identification of Molecular Fragments in Equilibrium with Polysiloxane Ultrasmall Nanoparticles.

During recent decades, ultrasmall inorganic nanoparticles have attracted considerable interest due to their favorable biodistribution, pharmacokinetics and theranostic properties. In particular, AGuIX nanoparticles made of polysiloxane and gadolinium chelates were successfully translated to the clinics. In an aqueous medium, these nanoparticles are in dynamic equilibrium with polysiloxane fragments due to the hydrolysis of Si-O-Si bonds. Thanks to high-performance liquid chromatography coupled with electrospray ionization mass spectrometry, all these fragments were separated and identified.

Biodegradation of metal-based ultra-small nanoparticles: A combined approach using TDA-ICP-MS and CE-ICP-MS.

The knowledge of the fate of metal-containing nanoparticles in biological media in aqueous media is of utmost importance for the future use of these promising theranostic agents for clinical applications. A methodology based on the combination of TDA-ICP-MS and CE-ICP-MS was applied to study the degradation pathway of AGuIX, a phase 2 clinical ultrasmall gadolinium-containing nanoparticle. Nanoparticle size measurements and gadolinium speciation performed in different media (phosphate buffer, urine and serum) demonstrated an accelerated dissolution of AGuIX in serum, without any release of free gadolinium for each medium.

Behavior of micro pillar array column in high pressure gas chromatography.

Micro pillar array column with interpillar distance of 2.5 µm for pillars diameter of 5 µm has been introduced in high pressure gas chromatographic systems for online industrial analysis. Separation of gas mixtures have been performed under carrier gas pressure as high as 60 bar using rotating valve for gas injection without sample decompression stage prior to injection. A very low intrinsic height equivalent to a theoretical plate value of 14 µm has been obtained in few seconds. Instead of conventional gas chromatography, carrier gas nature such as helium, argon and carbon dioxide and pressure can be used to tune the selectivity. Liquid hydrocarbon samples have been successfully introduced in the column using a septum based split/splitless injector modified to work up to 40 bar. Separations of VOCs and gasoline samples have been successfully performed.

Taylor Dispersion Analysis Coupled to Inductively Coupled Plasma-Mass Spectrometry for Ultrasmall Nanoparticle Size Measurement: From Drug Product to Biological Media Studies.

During past decade, special focus has been laid on ultrasmall nanoparticles for nanomedicine and eventual clinical translation. To achieve such translation, a lot of challenges have to be solved. Among them, size determination is a particularly tricky one. In this aim, we have developed a simple hyphenation between Taylor dispersion analysis and inductively coupled plasma-mass spectrometry (ICP-MS). This method was proven to allow the determination of the hydrodynamic radius of metal-containing nanoparticles, even for sizes under 5 nm, with a relative standard deviation below 10% (with a 95% confidence interval) and at low concentrations. Moreover, its specificity provides the opportunity to perform measurements in complex biological media. This was applied to the characterization of an ultrasmall gadolinium-containing nanoparticle used as a theranostic agent in cancer diseases. Hydrodynamic radii measured in urine, cerebrospinal fluid, and undiluted serum demonstrated the absence of interaction between the particle and biological compounds such as proteins.

Characterization of Nano-Gravimetric-Detector Response and Application to Petroleum Fluids up to C34.

A nano-gravimetric detector (NGD) for gas chromatography is based on a nanoelectromechanical array of adsorbent-coated resonating double clamped beams. NGD is a concentration-sensitive detector and its sensitivity is analyte-dependent based on the affinity of the analyte with the porous layer coated on the NEMS surface. This affinity is also strongly related to the NGD temperature (NGD working temperature can be dynamically set up from 40 to 220 °C), so the sensitivity can be tuned through temperature detector control. An adsorption-desorption model was set up to characterize the NGD response on a large set of n-alkanes from C10 to C22 at different NGD temperatures. For fast identification of petroleum mixture based on chromatogram fingerprint, a general strategy for NGD temperature program design was developed leading to a constant relative response factor between 0.96 and 1.03 for all the alkanes, and then chromatograms are very similar to those obtained with a flame ionization detector (FID). The analysis of a real petroleum fluid was also performed and compared to FID results: quantitative results obtained for all the analytes were satisfactory according to precision (<5%) and accuracy (average relative error = 4.3%). Based on such temperature control strategy, NGD sensitivity and the dynamic linear range can be adjusted and detection limits at a picogram level can be easily achieved for all n-alkanes.

Off-line coupling of capillary isotachophoresis separation to IRMPD spectroscopy for glycosaminoglycans analysis: Application to the chondroitin sulfate disaccharides model solutes.

Glycans analysis is challenging due to their immense structural diversity. Isotachophoresis was investigated as separation method for the purification of isobaric sulfated disaccharides prior to their characterization by Mass Spectrometry (MS) and tunable IR multiple photon dissociation (IRMPD). This proof of feasibility study was applied to the separation and characterization of chondroitin sulfate (CS) disaccharides. ITP separation conditions were optimized. Separation starts using a 2.5 mM chloride ions and 10 mM glycine at pH 3.2 solution as leading electrolyte and a terminating electrolyte composed of formic acid 2.5 mM and glycine 10 mM at pH 3.5. The CS disaccharides sample were prepared in the terminating electrolyte. The length of injection was also investigated in order to create longer plateau-like bands of pure solutes. This strategy was helpful for collecting fraction at such microseparation scale. Indeed, capillary ITP affords the injection of few tens of nanoliter of sample. Fractionation of the CS disaccharides mixture in isolated ITP bands and collection of solutes were successfully done using a HPC coated fused silica capillary of 1m-length and 75 µm of internal diameter. Collected fractions in a final of volume 10 µL were analyzed by CZE, tandem MS and IRMPD spectroscopy. The purity of each fraction is higher than 90% and is well-adapted to IRMPD characterization.

Development of a new in-line coupling of a miniaturized boronate affinity monolithic column with reversed-phase silica monolithic capillary column for analysis of cis-diol-containing nucleoside compounds.

In-line coupling of capillary columns is an effective means for achieving miniaturized and automated separation methods. The use of multimodal column designed to allow the direct integration of a sample preparation step to the separation column is one example. Herein we propose a novel in-line coupling at the capillary scale between a boronate affinity capillary column (µBAMC unit) and a reversed-phase separation column. This has been made possible due to the elaboration of a new and efficient µBAMC unit. A thiol-activated silica monolithic capillary column was functionalized through thiol-ene photoclick reaction. This simple and fast reaction allows to prepare stable µBAMC units having grafting densities of 1.93 ± 0.17 nmol cm-1. This grafting strategy increases the surface density by a factor 4 compared to our previous strategies and opens the frame to in-line coupling with reversed-phase capillary column. Proof of concept of the in-line coupling was done by coupling a 1-cm length µBAMC unit to a 7-cm length reversed phase capillary column. The conditions of loading, elution and separation were optimized for cis-diol nucleosides analysis (uridine, cytidine, adenosine, guanosine). A loading volume (at pH 8.5) of up to 21 hold volume (i.e 1 µl) of the µBAMC unit can be loaded without sample breakthrough. For the least retained nucleoside (uridine) a limit of detection of 50 ng mL-1 was estimated. Elution and full separation of the four nucleosides was triggered by flushing the multimodal column with an acetic acid (50 mM) / methanol (98/2, v/v) mobile phase.

Hyphenation of short monolithic silica capillary column with vacuum ultraviolet spectroscopy detector for light hydrocarbons separation.

Compared to conventionnal bench top instruments, on-line GC analyzers require specific characteristics. On one hand, for some applications operating with a reactor pressure as high as several tens of bars, sample pressure has to be reduced before GC separation, or specific valves and columns have to be designed to perform separation with high carrier gas inlet pressure. On the other hand, informative detectors such as mass spectrometer are valuable but low maintenance detectors are prefered. To fit these two requirements (sampling at high pressure without decompression stage, and informative detector with low maintenance), short monolithic silica capillary column operated with inlet pressure as high as 60 bar has been hyphenated to VUV detector. Injection and column performance have been first investigated. The system has been optimized by adjusting split ratio at high pressure and by tuning two main VUV detector parameters ("average number" linked to data point averaging and make-up gas pressure) to decrease the limit of quantification. The optimization stage led to a set of experimental parameters which is a good compromise between signal-to-noise ratio and chromatographic efficiency. Finally, the hyphenated monolithic column has be used to partially separate a mixture of methane, ethane, carbon monoxide and carbon dioxide within 15 s, and the VUV deconvolution capabilities have been exploited to overcome coelution and finally separate individual signals.

Monolith weak affinity chromatography for µg-protein-ligand interaction study.

Affinity monolith columns of 375 nL (effective length 8.5 cm, internal diameter 75 µm) were developed for protein-ligand affinity investigations needing only 3 µg of human serum albumin (HSA). To promote specific interactions and avoid non-specific ones, different combinations of monolithic supports and bio-functionalization pathways were evaluated. Silica and glycidylmethacrylate based monoliths were in-situ synthesized and grafted with HSA. Two direct grafting methods epoxy-amine and Schiff Base plus the streptavidin-biotin method were compared. The columns were evaluated by frontal analysis with ligands of known affinity for HSA. It is shown that a classical capillary electrophoresis instrument equipped with an external pressure device can be used to do weak affinity chromatography at low pressure (less than 1.2 MPa) in a fully automated way and with very low reagent consumption. The grafting pathways were compared in terms of (i) total and active amounts of immobilized protein, (ii) non-specific interactions, (iii) protein denaturation. According to these criteria, the organic monoliths combined with the streptavidin-biotin approach provided the best results. This immobilization pathway led to the highest active protein content (40 pmol of HSA per 8.5-cm column) with less than 10% non-specific interactions and 84% protein activity. The target grafting step lasts only 10 min and is UV-monitored, the UV breakthrough curve giving the exact amount of bound protein. This novel approach was validated by Kd measurements of 3 known ligands of HSA. Streptavidin generic monolith columns could be stored at 4 °C for 3 months maintaining activity. µg of a biotin modified sensitive protein could be attached to a stable streptavidin monolith for immediate interaction studies avoiding stability problems. This development was subsequently extended to another protein of higher pharmaceutical interest: the N-terminal domain of HSP90. Affinity was measured for two known ligands and determined Kd values were in accordance with the literature, proving that our technique is applicable to other proteins.

Evaluation of boronate affinity solid-phase extraction coupled in-line to capillary isoelectric focusing for the analysis of catecholamines in urine.

In this study, a new miniaturized and integrated analytical system was developed based on the in-line coupling of boronate affinity solid phase extraction with capillary isoelectric focusing separation and UV detection. This original coupling takes advantage of the selective enrichment of cis-diol-containing compounds using a boronate affinity sorbent and the exceptional focusing features of isoelectric focusing process. Such coupling has been used for preconcentration/purification and separation of urinary catecholamines (dopamine, adrenaline and noradrenaline) as proof of concept. Poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic capillary column (8 cm) was chosen as solid phase extraction support due to its good chemical stability and its easy and versatile surface functionalization. Characterization of the miniaturized boronate affinity monolith column (µBAMC) was done by frontal affinity chromatography. An active-site amount of about 0.16 nmol cm-1 (75 µm i. d.) of phenyl boronic acid groups and Kd values ranging from 224 to 106 µM were obtained for catechol and catecholamines. A high loading volume (up to 15 times the affinity column volume) can be introduced with quantitative recovery yields. Optimization of the in-line coupling concerned the adaptation of (i) the µBAMC volume, (i.e. length and inner diameter of the monolithic column) for loading of large sample volumes and (ii) the CIEF experimental conditions. The ampholyte mixture was adapted (i.e. nature and concentration of carrier ampholytes, volume of sacrificial electrolytes) in order to ensure elution and separation of catecholamines and to decrease limit of detection down to 10-20 ng ml-1. The optimized method was applied to analyze urine samples.

Kinetic accumulation processes and models for 43 micropollutants in "pharmaceutical" POCIS.

The "pharmaceutical" polar organic integrative sampler (POCIS) is a passive sampler composed of an outer polyethersulfone (PES) membrane and an inner receiving Hydrophilic-Lipophilic Balance (HLB) phase. Target micropollutants can accumulate in the POCIS HLB phase following different uptake patterns. Two of the most common ones are a first-order kinetic uptake (Chemical Reaction Kinetic 1, CRK1 model), and a first-order kinetic uptake with an inflexion point (CRK2 model). From a previous study, we identified 30 and 13 micropollutants following CRK1 and CRK2 accumulation model in the POCIS HLB phase, respectively. We hypothesized that uptake in the outer PES membrane of POCIS may influence the uptake pathway. Thus, novel measurements of uptake in PES membrane were performed for these micropollutants to characterise kinetic accumulation in the membrane with and without the HLB phase. We determined, for the first time, the membrane-water distribution coefficient for 31 micropolluants. Moreover, the lag times for molecules to breakthrough the POCIS membrane increased with increasing hydrophobicity, defined by the octanol-water dissociation constant Dow. However, Dow alone was insufficient to predict whether uptake followed a CRK1 or CRK2 model in the POCIS HLB phase. Thus, we performed a factorial discriminant analysis considering several molecular physico-chemical properties, and the model of accumulation for the studied micropollutants can be predicted with >90% confidence. The most influent properties to predict the accumulation model were the log Dow and the polar surface area of the molecule (>70% confidence with just these two properties). Molecules exhibiting a CRK1 uptake model for the POCIS HLB phase tended to have log Dow>2.5 and polar surface area <50Ǻ2.

Calibration of silicone rubber rods as passive samplers for pesticides at two different flow velocities: Modeling of sampling rates under water boundary layer and polymer control.

There is a need to determine time-weighted average concentrations of polar contaminants such as pesticides by passive sampling in environmental waters. Calibration data for silicone rubber-based passive samplers are lacking for this class of compounds. The calibration data, sampling rate (Rs ), and partition coefficient between silicone rubber and water (Ksw ) were precisely determined for 23 pesticides and 13 candidate performance reference compounds (PRCs) in a laboratory calibration system over 14 d for 2 water flow velocities, 5 and 20 cm s-1 . The results showed that an in situ exposure duration of 7 d left a silicone rubber rod passive sampler configuration in the linear or curvilinear uptake period for 19 of the pesticides studied. A change in the transport mechanism from polymer control to water boundary layer control was observed for pesticides with a log Ksw of approximately 3.3. The PRC candidates were not fully relevant to correct the impact of water flow velocity on Rs . We therefore propose an alternative method based on an overall resistance to mass transfer model to adjust Rs from laboratory experiments to in situ hydrodynamic conditions. We estimated diffusion coefficients (Ds ) and thickness of water boundary layer (δw ) as adjustable model parameters. Log Ds values ranged from -12.13 to -10.07 m2 s-1 . The estimated δw value showed a power function correlation with water flow velocity. Environ Toxicol Chem 2018;37:1208-1218. © 2017 SETAC.

Is vacuum ultraviolet detector a concentration or a mass dependent detector?

The vacuum ultraviolet detector (VUV) is a very effective tool for chromatogram deconvolution and peak identification, and can also be used for quantification. To avoid quantitative issues in relation to time drift, such as variation of peak area or peak height, the detector response type has to be well defined. Due to the make-up flow and pressure regulation of make-up, the detector response (height of the peak) and peak area appeared to be dependent on experimental conditions such as inlet pressure and make-up pressure. Even if for some experimental conditions, VUV looks like mass-flow sensitive detector, it has been demonstrated that VUV is a concentration sensitive detector.

Behavior of macroporous vinyl silica and silica monolithic columns in high pressure gas chromatography.

80% vinyltrimethoxysilane-based hybrid silica monoliths (80-VTMS), which have been initially developed for separation in reversed-phase liquid chromatography, have been investigated in high pressure gas chromatography separations (carrier gas pressure up to 60bar) and compared to silica monolithic columns. The behavior of both silica and 80-VTMS monolithic columns was investigated using helium, nitrogen and carbon dioxide as carrier gas. The efficiency of 80-VTMS monolithic columns was shown to vary differently than silica monolithic columns according to the temperature and the carrier gas used. Carrier gas nature was a significant parameter on the retention for both silica and vinyl columns in relation to its adsorption onto the stationary phase in such high pressure conditions. The comparison of retention and selectivity between 80-VTMS monoliths and silica was performed under helium using the logarithm of the retention factor according to the number of carbon atoms combined to Kovats indexes. The very good performances of these columns were demonstrated, allowing the separation of 8 compounds in less than 1min.

Development and application of a new in-line coupling of a miniaturized boronate affinity monolithic column with capillary zone electrophoresis for the selective enrichment and analysis of cis-diol-containing compounds.

An integrated, miniaturized and fully automated system was developed for the analysis (preconcentration/purification, separation and detection) of cis-diol containing molecules in complex matrices. This innovative in-line coupling system was achieved via the in-situ and localized synthesis of a short segment of silica-based monolith at the inlet of a 75-µm inner diameter fused silica capillary. The monolithic segment was locally functionalized with an acrylamide derivative of phenylboronic acid by free radical photopolymerization within 10min of irradiation time. Efficiency of the photopolymerization reaction was followed by frontal affinity chromatography of 1,2-dihydroxybenzene (catechol) as cis-diol model solute. An active-site amount of 0.43nmolcm-1 (9.8nmolµL-1) of phenylboronic acid moieties was obtained, with a Kd value of about 290µM close to reported value for the phenyl boronate-catechol complex. The optimal conditions of use of the miniaturized boronate affinity monolithic column (µBAMC) were determined and adapted to the in-line coupling with capillary electrophoresis. Catechol was specifically preconcentrated in a pH 8.5 phosphate buffer/MeOH (80/20, v/v) mixture. A volume up to 20 times the monolith volume can be percolated with a quantitative recovery yield. Three catecholamines were purified, preconcentrated and in-line separated. Elution from the µBAMC was performed with a small plug of acidic solution, allowing field amplified sample stacking of solutes within the plug before their in-line electrophoretic separation at pH 8.75. This unique in-line coupling was successfully used for the fully automated analysis of catecholamines neurotransmitters in urine samples, highlighting the purification efficiency of the µBAMC and the potential of such a fully integrated approach. In addition to the low sample volume required (less than 2µL), the limits of detection (LOD) accomplished with this coupling were estimated at 9.0, 9.5 and 4.8ngmL-1 for dopamine, adrenaline and noradrenaline respectively, which improves the LOD of theses solutes compared to other CE methods.