Separations — Open Access Separation Science Journal

Mycotoxins are a very diverse group of natural products produced as secondary metabolites by fungi. Patulin is produced by mold species normally related to vegetable-based products and fruit, mainly apple. Its ingestion may result in agitation, convulsions, edema, intestinal ulceration, inflammation, vomiting, and even immune, neurological or gastrointestinal disorders. For this reason, the European Commission Regulation (EC) 1881/2006 established a maximum content for patulin of 10 ppb in infant fruit juice, 50 ppb for fruit juice for adults and 25 ppb in fruit-derived products. In this work, a rapid and selective method based on magnetic molecularly imprinted stir-bar (MMISB) extraction has been developed for the isolation of patulin, using 2-oxindole as a dummy template. The final extraction protocol consisted of simply pouring in, stirring and pouring out samples and solvents from a beaker with the MMISB acting inside. The magnetic device provided satisfactory recoveries of patulin (60%–70%) in apple samples. The successful MMISB approach has been combined with high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) to determine patulin. Full article

Lactoperoxidase (LP), which is a high-value minor whey protein, has recently drawn extensive attention from research scientists and industry due to its multiplicity of function and potential therapeutic applications. In this study, the separation and optimization of two similar-sized proteins, LP and lactoferrin (LF) were investigated using strong cation exchange column chromatography. A two-step optimization strategy was developed for the separation of LP and LF. Optimization was started with central composite design-based experiments to characterize the influences of different decision variables, namely, flow rate, length of gradient, and final salt concentration in the linear elution gradient step on the yield of LP. This was followed by a more accurate optimization of ion-exchange chromatography (IEC) separation of LP and LF based on an experimentally verified chromatographic model. The optimal operating points were found and the results were compared with validation experiments. Predictions respecting yield confirmed a very good agreement with experimental results with improved product purity. Full article

The performance of a miniaturized channel for the separation of polymer and metal nanoparticles (NP) using Asymmetrical Flow Field-Flow Fractionation (AF4) was investigated and compared with a conventional AF4 system. To develop standard separation methods, experimental parameters like cross flow, gradient profile and injection time were varied and optimized. Corresponding chromatographic parameters were calculated and compared. Our results indicate that the chromatographic resolution in the miniaturized channel is lower, whereas significantly shorter analyses time and less solvent consumption were obtained. Moreover, the limit of detection (LOD) and limit of quantification (LOQ) obtained from hyphenation with a UV-detector are obviously lower than in a conventional channel, which makes the miniaturized channel interesting for trace analysis. Full article

Byproducts of winery industries are treated, in many cases, as useless wastes constituting not only a major disposal problem but also not providing any additional profit to the industries. However, these byproducts could be utilized as a source of various phenolic compounds, such as anthocyanins, that could be used as nutraceuticals or natural colorants. Nine materials were tested and evaluated for their ability to retain and elute anthocyanins, total phenolics, and sugars from a grape pomace extract. The materials tested were the ion exchange Amberlite IRA 400 Cl⁻, Lewatit TP 208 and Lewatit TP 260, and the sorbing Chromosorb G-HP, Amberite XAD 2, Zeocros CA 150, Chemviron Carbon, Oasis HLB (hydrophilic-lipophilic balance) and Isolute C8 end-capped (EC). The two materials with the higher anthocyanins recovery rate, Oasis HLB and Isolute C8 (EC), were further examined for their anthocyanin capacities which were calculated as 5.76 mg·cm⁻³ and 3.06 mg·cm⁻³ respectively. Furthermore, their behavior pattern towards anthocyanins of various molecular weights was investigated using a liquid chromatography coupled with mass spectrometry (LC-PDA-MS) system. Full article

Simultaneous quantitative analysis of 10 indole compounds, including indole-3-acetic acid (IAA, one of the most important naturally occurring auxins) and some of its metabolites, by high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) after solid-phase extraction (SPE) was reported for the first time. The analysis was carried out using a reverse phase HPLC gradient elution, with an aqueous mobile phase (containing 0.1% formic acid) modified by methanol. Furthermore, a novel SPE procedure was developed for the pre-concentration and purification of indole compounds using C₁₈ SPE cartridges. The combination of SPE, HPLC, and LC-MS was applied to screen for the indole compounds present in sugar cane (Saccharum officinarum L.) juice, a refreshing beverage with various health benefits. Finally, four indole compounds were successfully detected and quantified in sugar cane juice by HPLC, which were further unequivocally confirmed by LC-MS/MS experiments operating in the multiple reaction monitoring (MRM) mode. Full article

The extraction yield of a microextraction technique depends on thermodynamic and kinetics factors. Both of these factors have been the focus of intensive research in the last few years. The extraction yield can be increased by synthesizing and using novel materials with favorable distribution constants (one of the thermodynamic factors) for target analytes. The extraction yield can also be increased by improving kinetic factors, for example, by developing new extraction modes. Microextraction techniques are usually non-exhaustive processes that work under the kinetic range. In such conditions, the improvement of the extraction kinetics necessarily improves the performance. Since the extraction yield and efficiency is related to how fast the analytes diffuse in samples, it is crucial to stir the sample during extraction. The stirring can be done with an external element or can be integrated with the extraction element in the same device. This article reviews the main recent advances in the so-called extraction/stirring integrated techniques with emphasis on their potential and promising approaches rather than in their applications. Full article

This work presents the first instance of reversed-phase liquid chromatographic separation of small molecules using graphene oxide nanoparticle-modified polystyrene-divinylbenzene polymeric high internal phase emulsion (GONP PS-co-DVB polyHIPE) materials housed within a 200-µm internal diameter (i.d.) fused silica capillary. The graphene oxide nanoparticle (GONP)-modified materials were produced as a potential strategy to increase both the surface area limitations and the reproducibility issues observed in monolithic stationary phase materials. GONP PS-co-DVB polyHIPEs were found to have a surface area up to 40% lower than unmodified polymeric high internal phase emulsion (polyHIPE) stationary phases. However, despite having a surface area significantly lower than that of the unmodified material, the GONP-modified polyHIPEs demonstrated superior analyte adsorption properties. Reducing the GONP material did not have any significant impact on elution order or retention factor of the analytes, which was most likely due to low GONP loading attributed to the 250-nm GONPs utilised. The lower surface area of GONP-modified polyHIPEs provided similar separation efficiency and increased repeatability from injection to injection resulting in % relative standard deviations (%RSDs) of less than 0.6%, indicating the potential offered by graphene oxide (GO)-modified polyHIPES in flow through applications such as adsorption or separation processes. Full article

As for the measurement of polycyclic aromatic hydrocarbons (PAHs), ultra-performance liquid chromatography (UPLC) is used for PAH identification and densitometry. However, when a solvent containing a substance to be identified passes through a column of UPLC, a dedicated high-pressure-proof device is required. Recently, a liquid chromatography instrument using a monolithic column technology has been proposed to reduce the pressure of UPLC. The present study tested five types of monolithic columns produced in experiments. To simulate the flow field, the lattice Boltzmann method (LBM) was used. The velocity profile was discussed to decrease the pressure drop in the ultra-performance liquid chromatography (UPLC) system. Full article

Coffee is a favorite and beverage in Western countries that is consumed daily. In the present study, capillary zone electrophoresis (CE) was applied for the separation and quantification of three isoflavones including daidzein, genistein and formononetin in coffee. Extraction of isoflavones from the coffee sample was carried out by extraction and purification process using ether after the acid hydrolysis with the antioxidant butylated hydroxy-toluene (BHT). The experimental conditions of the CE separation method were: 20 mmol/L Na₂HPO₄ buffer solution, 25 kV applied voltage, 3 s hydrodynamic injection at 30 mbar, and UV detection at 254 nm. The results show that the three compounds can be tested within 10 min with a linearity of 0.5–50 µg/mL for all three compounds. The limits of detection were 0.0642, 0.134, and 0.0825 µg/mL for daidzein, formononetin and genistein, respectively. The corresponding average recovery was 99.39% (Relative Standard Detection (RSD) = 1.76%), 98.71% (RSD = 2.11%) and 97.37% (RSD = 3.74%). Full article

Monoliths are continuous adsorbents that can easily be synthesised to possess tuneable meso-/macropores, convective fluid transport, and a plethora of chemistries for ligand immobilisation. They are grouped into three main classes: organic, inorganic, and hybrid, based on their chemical composition. These classes may also be differentiated by their unique morphological and physicochemical properties which are significantly relevant to their specific separation applications. The potential applications of monoliths for molecular separation have created the need to enhance their characteristic properties including mechanical strength, electrical conductivity, and chemical and thermal stability. An effective approach towards monolith enhancement has been the doping and/or hybridization with miniaturized molecular species of desirable functionalities and characteristics. Nanoparticles are usually preferred as dopants due to their high solid phase dispersion features which are associated with improved intermolecular adsorptive interactions. Examples of such nanomaterials include, but are not limited to, carbon-based, silica-based, gold-based, and alumina nanoparticles. The incorporation of these nanoparticles into monoliths via in situ polymerisation and/or post-modification enhances surface adsorption for activation and ligand immobilisation. Herein, insights into the performance enhancement of monoliths as chromatographic supports by nanoparticles doping are presented. In addition, the potential and characteristics of less common nanoparticle materials such as hydroxyapatite, ceria, hafnia, and germania are discussed. The advantages and challenges of nanoparticle doping of monoliths are also discussed. Full article

Highly reproducible fabrication of porous layer open tubular (PLOT) structures in fused silica capillaries is often challenging; thus, methods to measure layer thickness growth in real time represent a powerful tool for the production of such columns. The work presented herein demonstrates the application of optical absorbance in the near-infrared (near IR) range for the in-process measurement of polymer layer growth inside fused silica capillaries during the fabrication of PLOT columns. The proposed technique can be used for both on- and off-line measurements of layer thickness for thermal- and photo- initiated polymerisation methods, performed in either polytetrafluoroethylene (PTFE)- or polyimide-coated capillaries. Measurements of layer thickness were carried out at λ 700 nm, using 100 μm and 8 μm optical fibres, yielding relative standard deviation (%RSD) values of 27% and 22%, respectively. Full article

High performance liquid chromatography-ultraviolet (HPLC-UV) was applied to the analysis and characterization of olive oils and other vegetable oils. A chromatographic separation on a Zorbax Eclipse XDB-C8 reversed-phase column was proposed under gradient elution, employing 0.1% formic acid aqueous solution and methanol as mobile phase, for the determination of 14 polyphenols and phenolic acids, allowing us to obtain compositional profiles in less than 20 min. Acceptable sensitivity (limit of detection (LOD) values down to 80 µg/L in the best of cases), linearity (r² higher than 0.986), good run-to-run and day-to-day precisions (relative standard deviation (RSD) values lower than 11.5%), and method trueness (relative errors lower than 6.8%) were obtained. The proposed HPLC-UV method was then applied to the analysis of 72 oil samples (47 olive oils and 27 vegetable oils including sunflower, soy, corn, and mixtures of them). Analytes were recovered using a liquid–liquid extraction method employing ethanol:water 70:30 (v/v) solution and hexane as extracting and defatting solvents, respectively. HPLC-UV polyphenolic profiles using peak areas were then analysed by principal component analysis (PCA) to extract information from the most significant data contributing to the characterization and classification of olive oils against other vegetable oils, as well as among Arbequina and Picual olive oil varieties. PCA results showed a noticeable difference between olive oils and the other classes. In addition, a reasonable discrimination of olive oils as a function of fruit varieties was also encountered. Full article

Countercurrent distribution based on liquid–liquid partition is a powerful separation method with minimal incurrence of loss of solutes, but its industrial application has been limited by cumbersome shifting of immiscible solvents. Although centrifugation has been employed to facilitate equilibration between phases, process scaling-up remains difficult. In this study, a dispersed mobile-phase countercurrent chromatography (DMCC) method has been developed to adapt the countercurrent distribution principle to a continuous column chromatography format. Continuous solute exchange between two immiscible phases within a series of separation columns is achieved by mechanical dispersion of an influx of mobile phase into an upward stream of small droplets travelling through the columns filled with stationary phase. The diameter, length, and number of columns, and the number of stationary phases employed in the different columns can be varied to match the requisite scale and resolution of operation. Illustrations of DMCC were provided by examples of solute separations where the fractionated solutes could be collected either from the eluate of the series of columns, or from drainage of the stationary phases in the individual columns at the end of a chromatographic run. Full article

High-speed countercurrent chromatography with a spiral tube assembly can retain a satisfactory amount of stationary phase of polymer phase systems used for protein separation. In order to improve the partition efficiency, a simple tool was fabricated to modify the tubing shapes, and the following four different tubing modifications were made: intermittently pressed at 10 mm width, flat, flat-wave, and flat-twist. Partition efficiencies of the separation column made from these modified tubings were examined in protein separation with an aqueous–aqueous polymer phase system at flow rates of 1–2 mL/min under 800 rpm. The results indicated that the column with all modified tubing improved the partition efficiency at a flow rate of 1 mL/min, but at a higher flow rate of 2 mL/min, the columns made of flattened tubing showed lowered partition efficiency, apparently due to the loss of the retained stationary phase. Among all the modified columns, the column with intermittently pressed tubing gave the best peak resolution. It may be concluded that the intermittently pressed and flat-twist improve the partition efficiency in a semi-preparative separation, while other modified tubing of flat and flat-wave configurations may be used for analytical separations with a low flow rate. Full article

Occurrence of d-amino acids in living organisms is a useful indicator of various changes, diseases, or disorders. Determination of amino acid enantiomers, namely the enantiomeric ratio of amino acids or excess of certain d-amino acids, represents a useful tool in the studies of aging processes or biomarkers in disease/disorder diagnosis in humans. The amount of d-amino acids is usually very low. Therefore, suitable sample pretreatment, often derivatization, and highly selective and sensitive separation methods are essential for d-amino acid analysis in this field. Chromatographic techniques offer appropriate choices for solving these tasks. This review covers the advances in methodology and development of improved instrumental chromatographic methods focused on d,l-amino acid separation and determination. New findings in the area of possible d-amino acid biomarkers are also included. Full article

This review (4 tables, 88 references) describes current developments in the design and application of liquid chromatography electrochemical detection (LC ED) based approaches for the determination of drugs of abuse. Specific emphasis is placed on operating details and performance characteristics for selected applications. LC ED has been shown to be highly sensitive and specific as well being a more economic option. A wide range of abused substances have been determined using this approach, including: cannabinoids, ethanol, opiates, morphine, mushroom toxins, benzodiazepines and several legal highs. Reverse-phase liquid chromatography with either amperometric or coulometric determination has been the most commonly reported applications. However, coulometric arrays have been also reported. Detection limits in the ng/mL region have been reported for most target analytes. Full article

(1) Background: Countercurrent chromatography (CCC) is liquid-liquid partition chromatography without using a solid support matrix. This technique requires further improvement of peak resolution and shortening of separation time. (2) Methods: The long-pressed locular multilayer coils with and without mixer glass beads were developed for the separation of proteins and 4-methylumbelliferyl sugar derivatives using a small-scale cross-axis coil planet centrifuge. (3) Results: Proteins were separated from each other and the separation was improved when the flow rate of the mobile phase was decreased from 0.8 to 0.4 mL/min. On the other hand, 4-methylumbelliferyl sugar derivatives were separated at the resolution of almost over 1.5 in short separation time under satisfactory stationary phase retention when the flow rate of the mobile phase was increased from 1.0 to 1.4 mL/min. (4) Conclusion: Better peak resolutions over the previous results were achieved using the long-pressed locular multilayer coil for proteins with aqueous two-phase systems (ATPS) and for 4-methylumbelliferyl sugar derivatives with organic-aqueous two-phase solvent systems by inserting a glass bead into each locule. Full article

The development of separation methods for the analysis and resolution of chiral drugs and solutes has been an area of ongoing interest in pharmaceutical research. The use of proteins as chiral binding agents in high-performance liquid chromatography (HPLC) has been an approach that has received particular attention in such work. This report provides an overview of proteins that have been used as binding agents to create chiral stationary phases (CSPs) and in the use of chromatographic methods to study these materials and protein-based chiral separations. The supports and methods that have been employed to prepare protein-based CSPs will also be discussed and compared. Specific types of CSPs that are considered include those that employ serum transport proteins (e.g., human serum albumin, bovine serum albumin, and alpha₁-acid glycoprotein), enzymes (e.g., penicillin G acylase, cellobiohydrolases, and α-chymotrypsin) or other types of proteins (e.g., ovomucoid, antibodies, and avidin or streptavidin). The properties and applications for each type of protein and CSP will also be discussed in terms of their use in chromatography and chiral separations. Full article

At present, gas chromatography–quadrupole mass spectrometry (GC-qMS) is considered the gold standard amongst analytical techniques for fire debris analysis in forensic laboratories worldwide, specifically for the detection and classification of ignitable liquids. Due to the highly complex and unpredictable nature of fire debris, traditional one-dimensional GC-qMS often produces chromatograms that display an unresolved complex mixture containing only trace levels of the ignitable liquid among numerous background pyrolysis products that interfere with pattern recognition necessary to verify the presence and identification of the ignitable liquid. To combat these challenges, this study presents a method optimized to achieve a near-theoretical maximum in peak capacity gain using comprehensive two-dimensional gas chromatography (GC×GC) coupled to time-of-flight mass spectrometry (TOFMS) for the forensic analysis of petroleum-based ignitable liquids. An overall peak capacity gain of ~9.3 was achieved, which is only ~17% below the system’s theoretical maximum of ~11.2. In addition, through the preservation of efficient separation in the first dimension and optimal stationary phase selection in the second dimension, the presented method demonstrated improved resolution, enhanced sensitivity, increased peak detectability and structured chromatograms well-suited for the rapid classification of ignitable liquids. As a result, the method generated extremely detailed fingerprints of petroleum-based ignitable liquids including gasoline, kerosene, mineral spirits and diesel fuel. The resultant data was also shown to be amenable to chromatographic alignment and multivariate statistical analysis for future evaluation of chemometric models for the rapid, objective and automated classification of ignitable liquids in fire debris extracts. Full article