Separations, Volume 3, Issue 2 (June 2016) – 9 articles

More and more studies are dedicated to termites and their symbionts, to better understand how they efficiently produce energy from lignocellulose. In that context, a powerful analytical method was developed to perform the detection, separation and identification of compounds in the 1 µL fluid volume of the gut of the termite Reticulitermes flavipes. Comprehensive two-dimensional gas chromatography (GC×GC) coupled to time-of-flight mass spectrometry (TOFMS) was tested with three different column combinations: (1) low-polar/mid-polar; (2) polar/low-polar and (3) mid-polar/low-polar. The column set (3) offered the best separation and was chosen for further analysis and comparison study. Metabolites were detected in the samples, including amino acids, sugars, amines and organic acids. Samples collected from termites fed for 30 days on Avicel cellulose or xylan powder diets were analyzed and compared with the wood diet. Principal component analysis (PCA) of metabolite profiles demonstrated a separation of different clusters corresponding to the three different diets, with a similar trend for diets containing cellulose. The Analysis of variance (ANOVA) (one way-ANOVA and Tukey’s test) was used to compare compound levels between these three different diets. Significant differences were observed, including higher levels of aromatic derivatives in the wood diet and higher levels of sugar alcohols in the xylan diet. A higher accumulation of uric acid was observed with the artificial diets (cellulose and xylan), likely to be related to the nitrogen deficiency. The present study highlighted the capability of adaptation of the termite system to non-optimal carbon sources and the subsequent modification of the metabolite profile. These results demonstrate the potential interest to investigate metabolite profiling with state-of-the-art separation science tools, in order to extract information that could be integrated with other omics data to provide more insight into the termite-symbiont digestion system. Full article

Anthocyanins are a group of phenolic compounds with great importance, not only because they play a crucial role in a wine’s quality, but also due to the fact that they can have beneficial effects on human health. In this work, a method was developed for the detection and identification of these compounds in solid wastes of the wine-making industry (red grape skins and pomace), using liquid-liquid extraction (LLE) prior to the liquid chromatography-mass spectrometry technique (LC-MS). The complete process was investigated and optimized, starting from the extraction conditions (extraction solution selection, dried matter-to-solvent volume ratio, water bath extraction duration, and necessary consecutive extraction rounds) and continuing to the mobile phase selection. The extraction solution chosen was a methanol/phosphoric acid solution (95/5, v/v), while three rounds of consecutive extraction were necessary in order to extract the maximum amount of anthocyanins from the byproducts. During the LC-MS analysis, acetonitrile was selected as the organic solvent since, compared with methanol, not only did it exhibit increased elution strength, but it also produced significantly narrower peaks. To enable accurate identification of the analytes and optimization of the developed method, kuromanin chloride and myrtillin chloride were used as standards. Furthermore, the wine variety (Syrah) from which the specific byproducts were produced was analyzed for its anthocyanin content, leading to interesting conclusions about which anthocyanins are transferred from grapes to wine during the vinification procedure, and to what extent. The results of this study showed that the total concentration of anthocyanins estimated in wine byproducts exceeded almost 12 times the equivalent concentration in Syrah wine, while the four categories of detected anthocyanins, simple glucosides, acetyl glucosides, cinnamoyl glucosides, and pyroanthocyanins, were present in different ratios among the two samples, ranging from 18.20 to 1, to 5.83 to 1. These results not only confirmed the potential value of these byproducts, but also indicated the complexity of the anthocyanins’ transfer mechanism between a wine and its byproducts. Full article

The wheat proteins soluble in chloroform-methanol mixtures are associated with several kinds of food allergies. A separation method based on capillary zone electrophoresis (CZE) with UV detection was developed for the analysis of these mixtures. An acidic phosphoric acid/β-alanine (pH 2.5) buffer containing HPMC, urea and acetonitrile was used for the separation. The capillary electrophoresis (CE) was able to complete the analysis in six minutes. The electrophoregrams of extracts of both durum and common wheat commercial cultivars were compared. The registered cultivar (cv.) Kamut^® was included as a representative of rustic cereal species. A different number of peaks were detected in the profile relative to the tetraploid and exaploid analyzed cultivars. Three main peaks were observed for all tetraploid cultivars, while four peaks were detected for the common wheat cultivars. The peak corresponding to the α-amylase inhibitor type III was identified in the common wheat electrophoregram. The possibility of quantitative determination of this inhibitor has been investigated. Full article

Headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) have traditionally been used, in combination with other analyses, for the chemical characterization of organic residues recovered from archaeological specimens. Recently in many life science fields, comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) has provided numerous benefits over GC-MS. This study represents the first use of HS-SPME-GC×GC-TOFMS to characterize specimens from an experimental modern reference collection. Solvent extractions and direct analyses were performed on materials such as ivory, bone, antlers, animal tissue, human tissue, sediment, and resin. Thicker film column sets were preferred due to reduced column overloading. The samples analyzed by HS-SPME directly on a specimen appeared to give unique signatures and generally produced a higher response than for the solvent-extracted residues. A non-destructive screening approach of specimens may, therefore, be possible. Resin and beeswax mixtures prepared by heating for different lengths of time appeared to provide distinctly different volatile signatures, suggesting that GC×GC-TOFMS may be capable of differentiating alterations to resin in future studies. Further development of GC×GC-TOFMS methods for archaeological applications will provide a valuable tool to uncover significant information on prehistoric technological changes and cultural behavior. Full article

This article details the elements used in the verification method for the simultaneous high performance liquid chromatography (HPLC) assay of Pentoxifylline, Bupivacaine HCl, Levocetirizine HCl, Tranilast, and Fluticasone Propionate in Humco™ Sanare Advanced scar base. The method was proven to be linear over 50%–150% of the nominal concentration of the standard. The method was proven to be accurate over 50%–150%, with 98%–102% recovery of the actives from spiked placeboes over that range. The method exhibited specificity to the analytes listed, and it was shown to be precise, yielding acceptable results for system reproducibility and method repeatability. The method, as written, is considered to have been verified. Full article

The benzene metabolite trans,trans-muconic acid (tt-MA) is widely used as a biological indicator of exposure to this xenobiotic. An analytical method was developed and validated for the determination of urinary tt-MA using solid phase extraction onto a Strata SAX cartridge. The analysis was performed by HPLC with an Aminex HPX-87H ion exclusion column and a UV diode array detector. The sample preparation conditions were optimized using a 2⁴⁻¹ factorial design. Comparison of the slopes of standard analytical curves prepared in aqueous solution and urine showed that the biological matrix suppressed the tt-MA signal by around 50%, so the analytical curves were prepared with tt-MA standard at low concentrations in pooled urine. The analytical curves in the range of 5–500 μg·L⁻¹ showed determination coefficients values (R²) > 0.99 for tt-MA standard in water and R² > 0.98 for tt-MA standards in pooled urine. The coefficients of variation obtained using seven replicates were lower than 3.6%, and recoveries of tt-MA from solutions containing 5, 25, 50 and 100 μg·L⁻¹ of the analyte were in the range 85%–90%, demonstrating the satisfactory precision and accuracy of the method. The limits of detection and quantification were 0.11 and 0.36 µg ·L⁻¹, respectively. The benefits of this new method developed is the possibility of complete chromatographic peak separation for the determination of tt-MA at baseline, without matrix components’ interference as normally found in the C18 column. This is the first time that this chromatographic column has been used for the analysis of tt-MA in urine. Full article

Aspergillus flavus produces dangerous secondary metabolites known as aflatoxins, which are toxic and carcinogenic, and their contamination of agricultural products results in health issues and economic hardships in the U.S. and around the world. Early identification of aflatoxigenic isolates of A. flavus is the key in the management of these fungi. An emerging detection method for specific fungi identification involves the analysis of microbial volatile organic compounds (MVOCs) released by the fungi. Complicating this approach is the understanding that many factors influence metabolic production, including growth parameters, such as growth media, temperature, spore counts and oxidation stress. In addition, analytical and data analysis methods can also influence the results. Several growth and analysis methods were evaluated and optimized in order to better understand the effect of the methods on fungi MVOC signatures. The results indicate that carboxen/polydimethylsiloxane (CAR/PDMS) has the best extraction efficiency for the MVOCs emitted by A. flavus. Both chemical defined agar (CDA) and chemical defined liquid (CDL) are suitable growth media for MVOC emission studies. The highest MVOC production was found at 30 °C. Log transformation was considered one of the best data pretreatment methods when analyzing MVOC data and resulted in the best principal component analysis (PCA) clustering in the experiments with different growth media. This study aims to elucidate fungal volatile organic compounds (VOCs) differences due to variations in growth parameters as a first step in the development of an analytical method for the monitoring of aflatoxigenic A. flavus contamination in crop storage facilities. Full article

Over the last two decades, miniaturization, integration, and automation have made microfluidic systems popular. Core to advances in microfluidics are numerous electrophoretic separation and preconcentration strategies, some finding their origins on bench-top systems. Among them, gradient-based strategies are especially effective in addressing sensitivity challenges. This review introduces several gradient-based techniques according to a broad definition, including conductivity, field, and concentration, organized by the method of gradient generation. Each technique is introduced and described, and recent seminal advances explored. Full article

The plate model proposed by Martin and Synge has been used for the characterization of columns up-to-date. In this approach, the column is divided into a large number (N) of identical theoretical plates. Mobile phase transference between plates takes place in infinitesimal steps with mixing of the solutions in the adjacent plates during the flow. The plate height is related to the band broadening that occurs in the mixing process due to the microscopic heterogeneities in the mobile phase flow. According to the original Martin and Synge model, solutes reach the equilibrium instantaneously in each theoretical plate, where dispersion is produced by: (i) convection or mixing of the mobile phase reaching a theoretical plate with that existing in that plate; and (ii) the equilibrium of the solute that is partitioned between mobile phase and stationary phase. In this work, a general method is proposed to solve the problem of chromatographic elution by means of an extended plate model assuming slow mass transfer, longitudinal diffusion in both mobile phase and stationary phase, and the extra column dispersion. The final equation was validated by comparing the results with those obtained through the numerical simulation of the solute migration using the finite differential approach. Experimental data were also used to check the validity of the derived equations. Full article