A new chromatographic response function for use in size-exclusion chromatography optimization strategies: Application to complex organic mixtures

A new chromatographic response function (CRF) is presented aiming at designing an optimal chromatographic separation protocol for assessing the molecular size distribution of complex organic mixtures, such as those of natural organic matter from different sources (atmospheric, aqueous, and terrestrial). This CRF can be applied to mixtures of unknown solutes, being well suited for describing separation processes of pair of peaks of highly unequal area, and also for overlapping and asymmetric peaks. The performance of the developed CRF was compared to that of an existing response function, using simulated chromatograms. The capability of the new function to qualify the resolution degree that it is attained under different chromatographic conditions was further assessed through a size-exclusion chromatography study of a variety of different organic compounds, via a two-level full factorial design. It was proved that this function is a reliable alternative to optimize simultaneously the composition of the mobile phase (pH, ionic strength, and organic modifier concentration) and the instrumental variables (flow rate).     

Advancement in optimization tactic achieved by newly developed chromatographic response function: application to LC separation of raloxifene and its impurities

In this paper a new chromatographic response function (CRF) is designed and proposed for utilization in the optimization strategies. The function capability to represent the overall quality of a experimentally obtained chromatograms was compared to the other two objective functions and proved to give more accurate and reliable results. The new CRF has improved concept of separation and time term estimation. It reflects all important defects of the chromatogram such as the appearance of asymmetrical or overlapping peaks and prolonged elution time and allows the appropriate weighting of each of them. The LC separation of raloxifene and its four impurities was evaluated through the central composite design experimental plan choosing the new CRF to be the only output of the system. The function demonstrated the ability to judge the impact of the complex interactions of the selected chromatographic parameters (acetonitrile content in the mobile phase, sodium dodecyl sulfate concentration in the water phase, pH of the mobile phase and column temperature) on the mixture behavior and led to the determination of the optimal separation conditions. The newly developed CRF proved to have the advanced performances and it presents the important step forward in the optimization of the chromatographic separation.     

A novel ion-pairing chromatographic method for the simultaneous determination of both nicarbazin components in feed additives: chemometric tools for improving the optimization and validation

The development, optimization and validation of an ion-pairing high performance liquid chromatography method for the simultaneous determination of both nicarbazin (NIC) components: 4,4′-dinitrocarbanilide (DNC) and 2-hydroxy-4,6-dimethylpyrimidine (HDP) in bulk materials and feed additives are described. An experimental design was used for the optimization of the chromatographic system. Four variables, including mobile phase composition and oven temperature, were analyzed through a central composite design exploring their contribution to analyte separation. Five responses: peak resolutions, HDP capacity factor, HDP tailing and analysis time, were modelled by using the response surface methodology and were optimized simultaneously by implementing the desirability function. The optimum conditions resulted in a mobile phase consisting of 10.0 mmol L⁻¹ of 1-heptanesulfonate, 20.0 mmol L⁻¹ of sodium acetate, pH = 3.30 buffer and acetonitrile in a gradient system at a flow rate of 1.00 mL min⁻¹. Column was an INERSTIL ODS-3 (4.6 mm × 150 mm, 5 μm particle size) at 40.0 °C. Detection was performed at 300 nm by a diode array detector. The validation results of the method indicated a high selectivity and good precision characteristics, with RSD less than 1.0% for both components, both in intra and inter-assay precision studies. Linearity was proved for a range of 32.0-50.0 μg mL⁻¹ of NIC in sample solution. The recovery, studied at three different fortification levels, varied from 98.0 to 101.4 for HDP and from 99.1 to 100.2 for DNC. The applicability of the method was demonstrated by determining DNC and HDP content in raw materials and commercial formulations used for coccidiosis prevention. Assays results on real samples showed that considerable differences in molecular ratio DNC:HDP exist among them.     

Measurement and computation of zinc binding to natural dissolved organic matter in European surface waters

The zinc binding characteristics of natural dissolved organic matter (DOM) from five representative European surface freshwater sources were studied by square wave anodic stripping voltammetry (SWASV) and model simulation. Water samples were titrated with zinc and free zinc ion activity {Zn²⁺}, was calculated from the measurement of labile zinc by SWASV and other system conditions. Measured values of {Zn²⁺}, which were in the range 10⁻⁷ to 10⁻⁵ M, were compared with those simulated using Humic Ion-Binding Models V and VI. It was assumed that zinc speciation was controlled by the organic matter, represented by fulvic acid (FA), together with inorganic solution complexation. The models were calibrated by adjusting the parameter DOMFA, the proportion of DOM considered to behave as FA. Two modeling scenarios were used to obtain DOMFA values, both considering and not considering the competitive effects of Al, Fe(II) and Fe(III). The default Zn-DOM binding strength in Model VI (log K_MA = 1.6) was not able to provide realistic values of DOMFA and a log K_MA of 1.8 was tentatively proposed as a more plausible value in these waters. Models V and VI gave very similar fits to the data after optimization of DOMFA, in contrast to recent findings for copper. This may be due to the fact that the additional strong binding sites provided by Model VI are not important in complexing Zn in the Zn concentration range investigated in this study. Computed free Zn activities from both modeling scenarios were very similar; however, the consideration of Al and Fe competition is more realistic for natural waters.     

Preparation of a novel magnetic resin for effective removal of both natural organic matter and organic micropollutants

A novel,bifunctional,hypercrosslinked.magnetic resin W2 was prepared using divinylbenzene(DVB) and glycidyl methacrylate(GMA) as comonomers in three steps(i.e.,suspension polymerization, amination and post-crosslinking reactions).To evaluate the adsorption of natural organic matter(NOM) and organic micropollutants(OMPs) on the obtained resin W2,two magnetic resins Wl(the precursor of W2 before post-crosslinking) and WO(the precursor of Wl before amination) were chosen for comparison.The results indicated that W2 would be a promising material for the removal of both NOM and OMPs from aquatic environments.     

Influence of salinity and natural organic matter on the solid phase extraction of sterols and stanols: application to the determination of the human sterol fingerprint in aqueous matrices

Faecal sterols have been proposed as direct chemical markers for the determination of faecal contamination in inland and coastal waters. In this study, we assess the impact of (a) the concentration of dissolved organic carbon (DOC), (b) the nature of DOC, (c) the salinity and (d) the concentration of sterols and stanols on their solid phase extraction. When natural organic matter (NOM) is modelled by humic acid, increasing DOC concentration from 2.7 to 15.4 mg/L has no significant impact on the recovery of sterols and stanols. The modelling of NOM by a mixture of humic acid and succinoglycan induces a significant (24%) decrease in the recovery of sterols and stanols. For all concentrations of target compounds, no significant increase in recovery is associated with increasing the salinity. Moreover, an increase in the recovery of target compounds is induced by an increase in their concentration. The nine target compounds and the recovery standard (RS) exhibit the same behaviour during the extraction step. Thus, we propose that (a) the concentration of target compounds can be corrected by the RS to calculate more realistic concentrations without modifying their profile and (b) the sterol fingerprint can be investigated in the colloidal fraction of aqueous samples without altering the information it could provide about the source. The application of this analytical method to waste water treatment plant influent and effluents yields results in agreement with previous studies concerning the use of those compounds to differentiate between sources of faecal contamination. We conclude that this analytical method is fully applicable to the determination of sterol fingerprints in the dissolved phase (<0.7 μm) of natural aqueous samples.     

Use of a photocatalytic membrane reactor for the removal of natural organic matter in water: Effect of photoinduced desorption and ferrihydrite adsorption

The photocatalytic degradation of natural organic matter (NOM) would be an attractive option in the treatment of drinking water. The performance of a submerged photocatalytic membrane reactor (PMR) was investigated with regard to the removal of NOM and the control of membrane fouling. In particular, this work focused on the adsorption and desorption of humic acids (HA) and lake water NOM at the surface of TiO₂ photocatalyts and ferrihydrite (FH) adsorbents in the PMR for water treatment. The addition of FH particles with a large sorption capacity helped remove the NOM released from TiO₂ particles, but FH suspended in water affected the photocatalysis of lake water NOM with a low specific UV absorbance (SUVA) value. To prevent the UV light being scattered by FH without any photocatalytic activity, FH particles were attached to a submerged microfiltration (MF) membrane, which contributed to a greater removal of NOM during long-term PMR operation. The further removal of NOM from aqueous solution was achieved due to the synergistic effect of TiO₂ photocatalysis and FH adsorption in PMR while minimizing the influence of photoinduced desorption of NOM. No significant membrane fouling occurred when the submerged PMR was operated even at high flux levels (>25 L/m² h), as long as photocatalytic decomposition took place.     

Determination of molecular formulas of natural organic matter molecules by (ultra-) high-resolution mass spectrometry: Status and needs

Electrospray ionization (ESI) combined with ultra-high-resolution mass spectrometry on a Fourier transform ion cyclotron resonance mass spectrometer has been shown to be a very powerful tool for the analysis of fulvic and humic acids and of natural organic matter (NOM) at the molecular level. With this technique thousands of ions can be separated from each other and their m/z ratio determined with sufficient accuracy to allow molecular formula calculation. Organic biogeochemistry, water chemistry, and atmospheric chemistry greatly benefit from this technique. Methodical aspects concerning the application of Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) to NOM isolated from surface water, groundwater, marine waters, and soils as well as from secondary organic aerosol in the atmospheric are reviewed. Enrichment of NOM and its chromatographic separation as well as possible influences of the ionization process on the appearance of the mass spectra are discussed. These steps of the analytical process require more systematic investigations. A basic drawback, however, is the lack of well defined single reference compounds of NOM or fulvic acids. Approaches of molecular formula calculation from the mass spectrometric data are reviewed and available graphical presentation methods are summarized. Finally, unsolved issues that limit the quality of data generated by FTICR-MS analysis of NOM are elaborated. It is concluded that further development in NOM enrichment and chromatographic separation is required and that tools for data analysis, data comparison and data visualization ought to be improved to make full use of FTICR-MS in NOM analysis.     

Study of the size-based environmental availability of metals associated to natural organic matter by stable isotope exchange and quadrupole inductively coupled plasma mass spectrometry coupled to asymmetrical flow field flow fractionation

The determination of the isotopically exchangeable fraction of metals in environmental solid samples (soils, composts, sediments, sludges, etc.) is used to know the amount of metal potentially available (E-value). Stable isotopes can be used for determination of E-values through the analysis of the aqueous phases from spiked suspensions. However, the presence of isotopically non-exchangeable metal forms in the aqueous phase led to overestimation of the E-values. In this paper, a method for monitoring the degree of isotopic exchange in function of the molecular mass and/or size of the metal form has been developed based on the direct coupling of asymmetrical flow field flow fractionation (AsFlFFF) with inductively coupled plasma mass spectrometry (ICP-MS) for on-line isotope ratio measurements. ICP-MS data acquisition parameters were stressed to avoid degradation of isotope ratio precision. Two sets of fractionation conditions were selected: a colloids separation, which allowed the separation of substances up to 1 μm, and a macromolecules separation, designed to resolve small size substances up to 50 kDa. The methodology was applied to study the environmental availability of copper and lead in compost samples, where metals are mainly associated to different forms of organic matter. No significant differences on isotopic exchange were observed over the size range studied, validating the E-values determined by direct analysis of the aqueous phases.     

Interactions and stability of silver nanoparticles in the aqueous phase: Influence of natural organic matter (NOM) and ionic strength

The rapid development of nanotechnology and the related production and application of nanosized materials such as engineered nanoparticles (ENP) inevitably lead to the emission of these products into environmental systems. So far, little is known about the occurrence and the behaviour of ENP in environmental aquatic systems. In this contribution, the influence of natural organic matter (NOM) and ionic strength on the stability and the interactions of silver nanoparticles (n-Ag) in aqueous suspensions was investigated using UV–vis spectroscopy and asymmetrical flow field-flow fractionation (AF⁴) coupled with UV–vis detection and mass spectrometry (ICP-MS). n-Ag particles were synthesized by chemical reduction of AgNO₃ with NaBH₄ in the liquid phase at different NOM concentrations. It could be observed that the destabilization effect of increasing ionic strength on n-Ag suspensions was significantly decreased in the presence of NOM, leading to a more stable n-Ag particle suspension. The results indicate that this behaviour is due to the adsorption of NOM molecules onto the surface of n-Ag particles (“coating”) and the resulting steric stabilization of the particle suspension. The application of AF⁴ coupled with highly sensitive detectors turned out to be a powerful method to follow the aggregation of n-Ag particle suspensions at different physical–chemical conditions and to get meaningful information on their chemical composition and particle size distributions. The method described will also open the door to obtain reliable data on the occurrence and the behaviour of other ENP in environmental aquatic systems.     

Differentiation of organic and non-organic ewe's cheeses using main mineral composition or near infrared spectroscopy coupled to chemometric tools: a comparative study

Two independent methodologies were investigated to achieve the differentiation of ewes’ cheeses from different systems of production (organic and non-organic). Eighty cheeses (40 organic and 40 non-organic) from two systems of production, two different breeds of ewe, different sizes, seasons (summer and winter) and ripening times up to 9 months were elaborated. Their mineral composition or the information provided by their spectra in the near infrared zone (NIR) coupled to chemometric tools were used in order to differentiate between organic and non-organic cheeses. Main mineral composition (Ca, K, Mg, Na and P) of cheeses and stepwise lineal discriminant analysis were used to develop a discriminant model. The results from canonical standardised coefficients indicated that the most important mineral was Mg (1.725) followed by P (0.764) and K (0.742). The percentage of correctly classified samples was 88% in internal validation and 90% in external validation, selecting Mg, K and P as variables.Spectral information in the NIR zone was used coupled to a discriminant analysis based on a regression by partial least squares in order to obtain a model which allowed a rate of samples correctly classified of 97% in internal validation and 85% in external validation.     

Metabolic profiling of infant urine using comprehensive two-dimensional gas chromatography: Application to the diagnosis of organic acidurias and biomarker discovery

Comprehensive two-dimensional gas chromatography (GC × GC) time-of-flight mass spectrometry (ToFMS) was applied to the analysis of urinary organic acids from patients with inborn errors of metabolism. Abnormal profiles were obtained from all five patients studied. Methylmalonic academia and deficiencies of 3-methylcrotonyl-CoA carboxylase and medium chain acyl-CoA dehydrogenase gave diagnostic profiles while deficiencies of very long chain acyl-CoA dehydrogenase and mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase gave profiles with significant increases in dicarboxylic acids suggestive of these disorders. The superior resolving power of GC × GC with ToFMS detection was useful in separating isomeric organic acids that were not resolved using one-dimensional GC. A novel urinary metabolite, crotonyl glycine, was also discovered in the mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase sample which may be a useful specific diagnostic marker for this disorder. The quantitative aspects of GC × GC were investigated using stable isotope dilution analyses of glutaric, glyceric, orotic, 4-hydroxybutyric acids and 3-methylcrotonylglycine. Correlation coefficients for linear calibrations of the analytes ranged from 0.9805 to 0.9993 (R²) and analytical recoveries from 77% to 99%. This study illustrates the potential of GC × GC–ToFMS for the diagnosis of organic acidurias and detailed analysis of the complex profiles that are often associated with these disorders.     

Pyrolysis–GC–MS to trace terrigenous organic matter in marine sediments: a comparison between pyrolytic and lipid markers in the Adriatic Sea

The effectiveness of semiquantitative pyrolysis–gas chromatography–mass spectrometry (Py–GC–MS) as a rapid analytical technique for sourcing continental organic matter (OM) in marine sediments was examined by comparison with classical GC–MS analyses of solvent extractable lipid markers. Py–GC–MS was directly applied to HCl/HF de-ashed surface sediment samples collected in five stations located in north western Adriatic Sea. The resulting pyrolysates were characterised by compounds indicative of different biological precursors (e.g. proteins, carbohydrates, chlorophylls), including lignin methoxyphenols diagnostic for continental inputs. The relative abundance of pyrolytic markers was compared to the distribution of n-alkanes, n-alkanols and sterols extracted from the same sediments and determined by GC–MS analyses. For each class of molecular indicators, the terrigenous to aquatic ratio (TAR) was determined as follows: relative abundance of methoxyphenol/protein markers (TAR_PY), concentration ratios of (C27 + C29 + C31)/(C15 + C17 + C19) n-alkanes (TAR_HC), (C26 + C28+ C30)/(C14 + C16) n-alkanols (TAR_AL) and sitosterol/cholesterol (TAR_ST). A positive correlation was found between TAR_PY and both TAR_HC and TAR_AL indicating a decreasing contribution of land-plant-derived materials seaward in two investigated transects. TAR_ST values displayed a different trend suggesting a mixed origin for sitosterol. The distribution of TAR_PY values was also in good agreement with that of atomic C/N ratios. Considering the complexity of environmental systems (diagenetic alteration, different fractions of OM analysed) the obtained results indicate that the pyrolytic marker approach by Py–GC–MS is valuable for sourcing marine OM on a semiquantitative base, providing data consistent with GC–MS determinations of lipid markers and elemental bulk analyses.     

Spectrophotometric determination of zinc and copper in a multi-syringe flow injection analysis system using a liquid waveguide capillary cell: application to natural waters

This work exploits a multi-syringe injection analysis (MSFIA) system coupled with a long liquid waveguide capillary cell for the spectrophotometric determination of zinc and copper in waters. A liquid waveguide capillary cell (1.0 m pathlength, 550 μm i.d. and 250 μL internal volume) was used to enhance the sensitivity of the detection. The determination for both ions is based on a colorimetric reaction with zincon at different pH values. The developed methodology compares favourably with other previously described procedures, as it allows to reach low detection limits for both cations (LODs of 0.1 and 2 μg L⁻¹, for copper and zinc, respectively), without the need for any pre-concentration step. The system also provided a linear response up to 100 μg L⁻¹ with a high throughput (43 h⁻¹) and low reagent consumption and effluent production. The developed work was applied to natural waters and three certified reference water samples.     

Effect of organic modifier and temperature on the resolution of betamethasone and dexamethasone using a porous graphitic carbon column: application to their identification and confirmation in human urine by LC-ESI-MS/MS

The effects of temperature, organic modifier and the type of acid on the retention factor, the resolution and peak shape of betamethasone and dexamethasone are described. The study is performed using narrow bore porous graphitic carbon (PGC) columns online with diode-array detector (DAD) and ESI MS/MS. The results show that temperature affects the retention behaviour of the two compounds and ACN yields the best separation while no effect is obtained by changing the type of organic acid. The developed method is applied for the confirmation of dexamethasone and betamethasone in human urine.