Separations, Volume 3, Issue 4 (December 2016) – 7 articles

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

(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

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