Separations, Volume 5, Issue 1 (March 2018) – 20 articles

Understanding the aroma profile of wines made from cold climate grapes is needed to help winemakers produce quality aromatic wines. The current study aimed to add to the very limited knowledge of aroma-imparting compounds in wines made from the lesser-known Frontenac and Marquette cultivars. Headspace solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) with simultaneous olfactometry was used to identify and quantify selected, aroma-imparting volatile organic compounds (VOC) in wines made from grapes harvested at two sugar levels (22° Brix and 24° Brix). Aroma-imparting compounds were determined by aroma dilution analysis (ADA). Odor activity values (OAV) were also used to aid the selection of aroma-imparting compounds. Principal component analysis and hierarchical clustering analysis indicated that VOCs in wines produced from both sugar levels of Marquette grapes are similar to each other, and more similar to wines produced from Frontenac grapes harvested at 24° Brix. Selected key aroma compounds in Frontenac and Marquette wines were ethyl hexanoate, ethyl isobutyrate, ethyl octanoate, and ethyl butyrate. OAVs >1000 were reported for three aroma compounds that impart fruity aromas to the wines. This study provides evidence that aroma profiles in Frontenac wines can be influenced by timing of harvesting the berries at different Brix. Future research should focus on whether this is because of berry development or accumulation of aroma precursors and sugar due to late summer dehydration. Simultaneous chemical and sensory analyses can be useful for the understanding development of aroma profile perceptions for wines produced from cold-climate grapes. Full article

Τhe crucial role of mobile phase pH for optimizing the separation of a mixture of ionized and non-ionized compounds on a Phenomenex extended pH-range reversed-phase column (Kinetex 5 µm EVO C18) was examined. A previously developed Excel-spreadsheet-based software was used for the whole separation optimization procedure of the sample of interest under isocratic conditions as well as under single linear organic modifier-gradients in different eluent pHs. The importance and the advantages of performing a computer-aided separation optimization compared with a trial-and-error optimization method were realized. Additionally, this study showed that the optimized separation conditions for a given stationary phase may be used to achieve successful separations on new columns of the same type and size. In general, the results of this work could give chromatographers a feel of confidence to establish desired separations of a mixture of ionizable and neutral compounds in reversed-phase columns. Full article

Non-invasive diagnostics and finding biomarkers of disease in humans have been a very active research area. Some of the analytical technologies used for finding biomarkers of human disease are finding their use in livestock. Non-invasive sample collection from diseased cattle using breath and headspace of fecal samples have been reported. In this work, we explore the use of volatile organic compounds (VOCs) emitted from bovine nasal secretions and serum for finding biomarkers for bovine respiratory disease (BRD). One hundred nasal swabs and 100 serum samples (n = 50 for both ‘sick’ and ‘healthy’) were collected at the time of treatment for suspected BRD. Solid-phase microextraction (SPME) was used to collect headspace samples that were analyzed using gas chromatography-mass spectrometry (GC-MS). It was possible to separate sick cattle using non-invasive analyses of nasal swabs and also serum samples by analyzing and comparing volatiles emitted from each group of samples. Four volatile compounds were found to be statistically significantly different between ‘sick’ and ‘normal’ cattle nasal swabs samples. Five volatile compounds were found to be significantly different between ‘sick’ and ‘normal’ cattle serum samples, with phenol being the common marker. Future studies are warranted to improve the extraction efficiency targeting VOCs preliminarily identified in this study. These findings bring us closer to the long-term goal of real-time, animal-side detection and separation of sick cattle. Full article

Oxysterols are metabolites produced in the first step of cholesterol metabolism, which is related to neurodegenerative disorder. They can be detected by testing blood, plasma, serum, or cerebrospinal fluid. In this study, some cholesterol precursors and oxysterols were determined by gas chromatography coupled to mass spectrometry. The selected cholesterol-related compounds were desmosterol, lathosterol, lanosterol, 7α-hydroxycholesterol, 7β-hydroxycholesterol, 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 7-ketocholesterol, and 27-hydroxycholesterol. A powerful method was developed and validated considering various analytical parameters, such as linearity index, detection and quantification limits, selectivity and matrix effect, precision (repeatability), and trueness (recovery factor) for each cholesterol-related compound. 7α-hydroxycholesterol, 7β-hydroxycholesterol, and desmosterol exhibited the lowest detection and quantification limits, with 0.01 and 0.03 μg/mL, respectively, in the three cases. 7-ketocholesterol and lathosterol showed matrix effect percentages between 95.5% and 104.8%, respectively (demonstrating a negligible matrix effect), and very satisfactory repeatability values (i.e., overall performance of the method). Next, the method was applied to the analysis of a very interesting selection of mouse plasma samples (9 plasma extracts of non-transgenic and transgenic mice that had been fed different diets). Although the number of samples was limited, the current study led to some biologically relevant conclusions regarding brain cholesterol metabolism. Full article

Wastewater analysis is an important area in analytical and environmental chemistry. It can be performed with both the classic wet methods and instrumental techniques. The development of new methods, and modification of the existing ones, constitute a major task for researchers. Ion chromatography plays a predominant role in ion determinations with the instrumental methods. It offers several advantages over the conventional methods, such as simultaneous determinations of alkali and alkaline earth cations and ammonia. Ammonium ions cannot be determined by spectroscopic methods. Ion chromatography has been accepted world-wide as a reference method for analyzing anions and cations in water and wastewater due to the fact that it enables the replacement of several individual wet chemistry methods for common ions with one instrumental technique. The following article describes the principles of ion chromatography, such as stationary phases, eluents, detectors, and sample preparation methods. Moreover, the applications of ion chromatography in wastewater analyses and international standards are presented. Full article

This paper introduces a poly(octadecyl acrylate) (pODA)-based organic phase on silica, which is assisted by 2-vinyl-4,6-diamino-1,3,5-triazine (AT), for a chromatography stationary phase. The ODA-AT copolymer grafting onto silica surface was characterized by elemental analysis, FT-IR spectroscopy, scanning electron microscopy, thermo gravimetric analysis and differential scanning calorimeter (DSC). An endothermic peak top of the copolymer-grafted silica was increased to 46 °C from 38 °C, which was a peak top of pODA homopolymer. For high performance liquid chromatography (HPLC) application, the molecular selectivity increased with an increase in the AT contents of the ODA-AT copolymer as organic phase. The co-existence of an aminotriazine moiety in the copolymer promoted side-chain ordering of the poly(octadecyl) moiety, thus enhancing molecular planarity selectivity for PAHs in reversed-phase liquid chromatography. Full article

Casein is the major protein fraction in milk, and its cross-linking has been a topic of scientific interest for many years. Enzymatic cross-linking has huge potential to modify relevant techno-functional properties of casein, whereas non-enzymatic cross-linking occurs naturally during the storage and processing of milk and dairy products. Two size separation techniques were applied for characterisation of these reactions: gel electrophoresis and size exclusion chromatography. This review summarises their separation principles and discusses the outcome of studies on cross-linked casein from the last ~20 years. Both methods, however, show limitations concerning separation range and are applied mainly under denaturing and reducing conditions. In contrast, field flow fractionation has a broad separation range and can be easily applied under native conditions. Although this method has become a powerful tool in polymer and nanoparticle analysis and was used in few studies on casein micelles, it has not yet been applied to investigate cross-linked casein. Finally, the principles and requirements for absolute molar mass determination are reviewed, which will be of increased interest in the future since suitable calibration substances for casein polymers are scarce. Full article

The interplay of metal oxide nanoparticles, environmental pollution, and health risks is key to all industrial and drinking water treatment processes. In this work we present a study using gel filtration chromatography for the analytical investigation of metal oxide nanoparticles in water, their coating with polydopamine, and their encapsulation within lecithin liposomes. Polydopamine prevents TiO₂ and ZnO nanoparticles from aggregation during chromatographic separation. Lecithin forms liposomes that encapsulate the nanoparticles and carry them through the gel filtration column, producing an increase of peak area for quantitative analysis without any change in retention time to affect qualitative identification. To the best of our knowledge, this is the first report that demonstrates the potential application of lecithin liposomes for cleaning up metal oxide nanoparticles in water treatment. Encapsulation of graphene quantum dots by liposomes would allow for monitoring of nanoparticle-loaded liposomes to ensure their complete removal by membrane ultrafiltration from treated water. Full article

Bisphenol-A, a synthetic organic compound with estrogen mimicking properties, may enter bloodstream through either dermal contact or ingestion. Probiotic bacterial uptake of bisphenol can play a major protective role against its adverse health effects. In this paper, a method for the quantification of BPA in bacterial cells of L. lactis and of BPA and its potential metabolites 4-hydroxybenzoic Acid, 4-hydroxyacetophenone and hydroquinone in the culture medium is described. Extraction of BPA from the cells was performed using methanol–H₂O/TFA (0.08%) (5:1 v/v) followed by SPE. Culture medium was centrifuged and filtered through a 0.45 μm syringe filter. Analysis was conducted in a Nucleosil column, using a gradient of A (95:5 v/v H₂O: ACN) and B (5:95 v/v H₂O: ACN, containing TFA, pH 2), with a flow rate of 0.5 mL/min. Calibration curves (0.5–600 μg/mL) were constructed using 4-n-Octylphenol as internal standard (1 > R² > 0.994). Limit of Detection (LOD) and Limit of Quantification (LOQ) values ranged between 0.23 to 4.99 μg/mL and 0.69 to 15.1 μg/mL respectively. A 24 h administration experiment revealed a decline in BPA concentration in the culture media up to 90.27% while the BPA photodegradation levels were low. Our results demonstrate that uptake and possible metabolism of BPA in L. lactis cells facilitates its removal. Full article

State-of-the-art purification of biomolecules, as well as separation of complex omic mixtures, is crucial for modern biomedical research. Mass spectroscopy (MS) represents a technique that both requires very clean biomedical samples and can substantially assist liquid chromatography (LC) separations, using either LC-MS or LC-MS/MS methods available. Here, a brief overview of the applicability of LC-MS/MS methodology for structural analyses of complex omic mixtures without prior purification of each sample component will be given. When necessary bioinformatic tools are available, these can be carried out quite quickly. However, manual data analysis of such complex mixtures is typically very slow. On the other hand, the need for high-level purity of protein samples for modern biomedical research will be discussed. Often, modification of protein purification protocols is needed, or additional purification steps may be either required or preferred. In the context of mass spectroscopy-related biomedical research, purification of pmol and subpmol amounts of biomedical samples, as well as commercial availability of pmol amounts of purified standards will be discussed. Full article

In the present work the extraction efficiency of in-tube solid-phase microextraction (IT-SPME) for polar herbicides has been evaluated using extractive capillaries coated with different polymeric sorbents. For this purpose, aqueous solutions of herbicides with a wide range of polarities, including some highly polar compounds (log K_ow < 1), have been directly processed by IT-SPME coupled on-line to capillary liquid chromatography with UV-diode array detection. For extraction, commercially available capillary columns coated with polydimethylsiloxane (PDMS) and polyetilenglicol (PEG)-based phases have been used, and the results have been compared with those obtained with a synthesized tetraethyl orthosilicate (TEOS)-trimethoxyethylsilane (MTEOS) polymer, as well as the same polymer reinforced with silica nanoparticles (SiO₂ NPs). The SiO₂ NPs functionalized TEOS-MTEOS coating provided the best results for most herbicides, especially for the most polar compounds. On the basis of the results obtained, conditions for the quantification of the herbicides tested are described using a SiO₂ NPs reinforced TEOS-MTEOS coated capillary. The proposed method provided satisfactory linearity up to concentrations of 200 μg/L. The precision was also suitable, with relative standard deviations (RSDs) values ≤9% (n = 3), and the limits of detection (LODs) were within the 0.5–7.5 µg/L range. The method has been applied to different water samples and the extract obtained from an agricultural soil. Full article

Assessment of drug transport across the placenta is important in understanding the effect of drugs on placental and fetal health. These phenomena can be studied in both in vitro cell lines and ex vivo placental perfusions. We have successfully developed a sensitive yet simple high performance liquid chromatography (HPLC) method coupled with fluorescence detection to determine the concentration of doxorubicin (DXR) in cell culture media for transport studies in human trophoblast cells (BeWo, b30 clone) and in fetal media for placental perfusion experiments. The method was developed based on a protein precipitation technique and was validated in both media types for linearity, intra-day, and inter-day precision and accuracy. The relationship of peak area to concentration was linear with R² values of 0.99 or greater obtained over the concentration range of 1.5 to 15,000 ng/mL. Despite the high concentrations of albumin in fetal perfusion media (30 mg/mL), the lower limits of detection and quantification for DXR were found to be 1.5 and 5 ng/mL, respectively. This analytical method may be used to study the transport of DXR across BeWo cells and human placenta during placental perfusion studies. Full article

In this work, a combination of molecularly imprinted polymer (MIP) technology with micro solid-phase extraction in a hollow fibre device is described. MIP microspheres were synthesized and packed into polypropylene hollow fibre (HF) segments. The proposed device was used directly for trace enrichment and clean-up of triazines in soil sample extracts by hollow-fibre membrane-protected molecularly imprinted micro solid-phase extraction (HFM-protected-MI-MSPE). Analytes were extracted from soil by ultrasonic assisted extraction, evaporated to dryness and reconstituted in toluene. The proposed device was immersed in the toluene extracts, and migrations of analytes through the walls of the hollow fibre was achieved with the help of external agitation. Then, selective recognition of the target analytes by MIP-microspheres took place in the inner part of the HF. All parameters affecting the extraction were optimized. Under optimum conditions, quantitative recoveries were obtained for simazine, cyanazine, atrazine, propazine and terbutylazine in soil samples, with relative standard deviations lower than 11%. The detection limits (LODs) were lower than 5 ng g⁻¹ in all cases. Full article

The baker’s yeast Saccharomyces cerevisiae harbors multiple prions that allow for the creation of heterogeneity within otherwise clonal cell populations. However, in many cases, the consequences of prion infection are entirely unclear. Predictions of prion-induced changes in cell physiology are complicated by pleotropic effects, and detection is often limited to relatively insensitive cell growth assays that may obscure many physiological changes. We previously showed that silica gel high performance thin-layer chromatography-densitometry (HPTLC) can be used to empirically determine prion-induced changes in lipid content in yeast. Here, we conduct pair-wise quantifications of the relative levels of free sterols, free fatty acids, and triacylglycerols [petroleum ether-diethyl ether-glacial acetic acid (80:20:1, v/v/v) mobile phase and phosphomolybdic acid (PMA) detection reagent]; steryl esters, methyl esters, and squalene [hexane-petroleum ether-diethyl ether-glacial acetic acid (50:20:5:1, v/v/v/v) and PMA]; and phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol (chloroform-diethyl ether-acetic acid (65:25:4.5, v/v/v) and cupric sulfate-phosphoric acid) in otherwise clonal prion-infected ([RNQ⁺]) and prion-free ([rnq⁻]) cells in both stationary- and logarithmic-growth phases. We detected multiple statistically significant differences between prion-infected and prion-free cells that varied by growth phase, confirming our pr evious observations that prions exert distinct influences on cell physiology between stationary- and log-phase growth. We also found significant differences between cells expressing or lacking the Rnq1 protein which forms the [RNQ⁺] prion, providing new clues to the as yet unresolved normal biological function of this prion-forming protein. This investigation further emphasizes the utility of HPTLC-densitometry to empirically determine the effects of prions and other presumed innocuous gene deletions on lipid content in yeast, and we expect that additional analyses will continue to resolve the physiological effects of prion infection. Full article

An electrochemical characterization of a polymer inclusion membrane (PIM) fabricated with the ionic liquid (IL) Aliquat 336 (26%) and the polymer cellulose triacetate (CTA) (76%) is presented. Considering the use of PIMs in separation systems to remove pollutants from water, the characterization was performed with NaCl solutions by measuring membrane potential, electrochemical impedance spectroscopy, and salt diffusion and results were compared with those obtained from dry membranes. Results showed a significant reduction in the membrane diffusive permeability and electrical conductivity as well as the transport number of cation Na⁺ across the PIM when compared with solution values, which could be mainly related to the dense character of the membrane. Membrane application in the separation of different sulfonamides (sulfathiazole, sulfapyridine, sulfamethazine, and sulfamethoxazole) from water, with 1 M NaCl solution as striping phase, was also considered. These results indicated that the different chemical characteristics of the compounds, as well as the compact structure of the PIM, limited the transport of the organic molecules though it. Full article

Hot, subcritical and supercritical water, methanol, ethanol and their binary mixtures have been employed to treat fuels (desulfurize coal and recover liquid fuels from coal and oil shales) and to extract valuable solutes from biomass. The properties of these solvents that are relevant to their extraction abilities are presented. Various extraction methods: accelerated solvent extraction (ASE), pressurized liquid extraction (PLE), supercritical fluid extraction (SFE, but excluding supercritical carbon dioxide) with these solvents, including microwave- and ultrasound-assisted extraction, are dealt with. The extraction systems are extensively illustrated and discussed. Full article

DNA-immobilized silica gel TLC plate (DNA-TLC plate) by coating a mixture of double-stranded DNA and silica gel on a glass plate was stable in common organic solvents, such as alcohol, acetone, chloroform, and ethyl acetate. DNA-TLC plate showed different R_f values for the L- and D-form acidic amino acids, such as aspartic acid and glutamic acid. The chiral recognition of an acidic amino acid by the DNA-TLC was related to the interaction between the nucleic acid base, particularly guanine (G) and cytosine (C), and the amino acid. Furthermore, the DNA-TLC plate indicated the recognition of a chiral metal complex, such as the tris(1,10-phenanthroline)ruthenium(II) ([Ru(phen)₃]²⁺) salt, which has Λ- and Δ-forms. Therefore, the double-stranded DNA-TLC plate may have the potential to be utilized as a chiral separation material for amino acids, peptides, and a metal complex with a chiral property. Full article

Alzheimer’s disease is characterized by the accumulation of brain amyloid plaques composed of aggregates of amyloid β (Aβ) peptides. The present paper describes a novel and easy-to-run capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) method for the specific analysis of fibrillar forms of Aβ aggregates obtained after in vitro incubation of Aβ 1-40 monomer. For that purpose, an affinity CE-LIF approach in which the ligand thioflavine T was added to the running buffer has been used, leading to the separation and detection of various fibrillar aggregates which migrated as spikes. The procedure has been optimized to get spikes only corresponding to Aβ aggregates, through the careful elimination of interfering factors and the electrophoretic validation of the link between spikes and particulate material. This method exhibited semi-quantification capabilities, led to the separation of Aβ fibrillar aggregates of different sizes and showed that highly concentrated solutions of Aβ peptides led to the formation of aggregates of larger size than lower-concentrated solution did. Advances brought by this method as well as future development needed to overcome its present limitations are discussed. Full article

Although analytical scientists equivocally agree that “no sample preparation” would be the best approach, the fact is that all samples that are handled in any analytical laboratory need to undergo treatment to some extent prior to their introduction to the analytical instrument [...] Full article