High-performance liquid chromatography with post-column 2,2′-diphenyl-1-picrylhydrazyl radical scavenging assay : methodological considerations and application to complex samples

An improved post-column 2,2´-diphenyl-1-picrylhydrazyl (DPPH•) radical scavenging assay for the screening of antioxidants in complex matrices was developed. Experimental parameters believed to be influential to DPPH• response were studied in a univariate approach. Optimum conditions were found to be: 5×10⁻⁵M DPPH• reagent prepared in a 75% methanol: 25% 40mM citric acid–sodium citrate buffer (pH 6) solution, degassed with nitrogen; reaction coil of 2m×0.25mm i.d. PEEK tubing; detection at 521 nm; analysis at room temperature. The analytical utility of this protocol was evaluated by screening for antioxidants in thyme and green tea, in comparison with two commonly employed methodologies.

High performance liquid chromatography with two simultaneous on-line antioxidant assays : evaluation and comparison of espresso coffees

The antioxidant profiles of various espresso coffees were established using HPLC with UV-absorbance detection and two rapid, simultaneous, on-line chemical assays that enabled the relative reactivity of sample components to be screened. The assays were based on (i) the colour change associated with reduction of the 2,2´-diphenyl-1-picrylhydrazyl radical (DPPH•); and (ii) the emission of light (chemiluminescence) upon reaction with acidic potassium permanganate. Results from the two approaches were similar and reflected the complex array of antioxidant species present in the samples. However, some differences in selectivity were observed. Chromatograms generated with the chemiluminescence assay contained more peaks, which was ascribed to the greater sensitivity of the reagent towards minor, readily oxidisable sample components. The three coffee samples produced closely related profiles, signifying their fundamentally similar chemical compositions and origin. Nevertheless, the overall intensity and complexity of the samples in both UV absorption and antioxidant assay chromatograms were aligned with the manufacturers description of flavour intensity and character.

In vitro osteoblast-like cell proliferation on nano-hydroxyapatite coatings with different morphologies on a titanium-niobium shape memory alloy

The morphology of nanomaterials significantly affects their physical, chemical, and biological properties. In the present study, nano-hydroxyapatite coatings with different morphologies were produced on the surface of a titanium-niobium shape memory alloy via a hydrothermal process. The effect of the nano-hydroxyapatite coatings on the in vitro proliferation of SaOS-2 osteoblast-like cells was investigated. Factors including crystallinity, surface micro-roughness, and surface energy of the nano-hydroxyapatite coatings were discussed. Results show that in vitro proliferation of the osteoblast-like cells was significantly enhanced on the nano-hydroxyapatite-coated titanium-niobium alloy compared to the titanium-niobium alloy without coating. The cell numbers on the nano-hydroxyapatite-coated titanium-niobium alloy changed consistently with the surface energy of the hydroxyapatite coatings. This study suggests that surface energy as a characteristic parameter influencing the in vitro proliferation of osteoblast-like cells was predominant over the crystallinity and surface micro-roughness of the nano-hydroxyapatite coatings.

An investigation into the ion-exchange properties of chemically modified wool for use in water treatment and chromatography

This study was undertaken to investigate the suitability of natural and chemically treated wool fibres for use in water treatment and in the separation of constituents for monitoring contaminants in water.

Experimental work was carried out to determine the ability of natural and treated wool fibres to remove these constituents from water,

This study provided information on the characteristics of the wool fibre as a medium in water treatment.

Immobilized artificial membrane chromatography : quantitative structure-retention relationships of structurally diverse drugs

The chromatographic capacity factors (log k‘) for 32 structurally diverse drugs were determined by high performance liquid chromatography (HPLC) on a stationary phase composed of phospholipids, the so-called immobilized artificial membrane (IAM). In addition, quantitative structure-retention relationships (QSRR) were developed in order to explain the dependence of retention on the chemical structure of the neutral, acidic, and basic drugs considered in this study. The obtained retention data were modeled by means of multiple regression analysis (MLR) and partial least squares (PLS) techniques. The structures of the compounds under study were characterized by means of calculated physicochemical properties and several nonempirical descriptors. For the carboxylic compounds included in the analysis, the obtained results suggest that the IAM-retention is governed by hydrophobicity factors followed by electronic effects due to polarizability in second place. Further, from the analysis of the results obtained of two developed quantitative structure-permeability studies for 20 miscellaneous carboxylic compounds, it may be concluded that the balance between polarizability and hydrophobic effects is not the same toward IAM phases and biological membranes. These results suggest that the IAM phases could not be a suitable model in assessing the acid-membrane interactions. However, it is not possible to generalize this observation, and further work in this area needs to be done to obtain a full understanding of the partitioning of carboxylic compounds in biological membranes. For the non-carboxylic compounds included in the analysis, this work shows that the hydrophobic factors are of prime importance for the IAM-retention of these compounds, while the specific polar interactions, such as electron pair donor−acceptor interactions and electrostatic interactions, are also involved, but they are not dominant.

Peak modeling approach to accurate assignment of first-dimension retention times in comprehensive two-dimensional chromatography

Modeling of first-dimension retention of peaks based on modulation phase and period allows reliable prediction of the modulated peak distributions generated in the comprehensive two-dimensional chromatography experiment. By application of the inverse process, it is also possible to use the profile of the modulated peaks (their heights or areas) to predict the shape and parameters of the original input chromatographic band (retention time, standard deviation, area) for the primary column dimension. This allows an accurate derivation of the firstdimension retention time (RSD 0.02%) which is equal to that for the non-modulated experiment, rather than relying upon the retention time of the major modulated peak generated by the modulation process (RSD 0.16%). The latter metric can produce a retention time that differs by at least the modulation period employed in the experiment, which displays a discontinuity in the retention time vs modulation phase plot at the point of the 180° out-ofphase modulation. In contrast, the new procedure proposed here gives a result that is essentially independent of modulation phase and period. This permits an accurate value to be assigned to the first-dimension retention. The proposed metric accounts for the time on the seconddimension, the phase of the distribution, and the holdup time that the sampled solute is retained in the modulating interface. The approach may also be based on the largest three modulated peaks, rather than all modulated peaks. This simplifies the task of assigning the retention time with little loss of precision in band standard deviation or retention time, provided that these peaks are not all overloaded in the first or second dimension.

Effects of variation in modulator temperature during cryogenic modulation in comprehensive two-dimensional gas chromatography

Many modulation systems in comprehensive 2D GC (GC×GC) are based on cryogenic methods. High trapping temperatures in these systems can result in ineffective trapping of the more volatile compounds, whilst temperatures that are too low can prevent efficient remobilisation of some compounds. To better understand the trapping and release of compounds over a wide range of volatilities, we have investigated a number of different constant temperature modulator settings, and have also examined a constant temperature differential between the cryo-trap and the chromatographic oven. These investigations have led us to modify the temperature regulation capabilities of the longitudinally modulated cryogenic system (LMCS). In contrast to the current system, where the user sets a constant temperature for the cooling chamber, the user now sets the temperature difference between the cryo-trap and the chromatographic oven. In this configuration, the cooling chamber temperature increases during the chromatographic run, tracking the oven temperature ramp. This produces more efficient, volatility-dependent modulation, and increases the range of volatile compounds that can be analysed under optimal trap-and-release conditions within a single analytical run. This system also reduces cryogenic fluid consumption.

Determination of intracellular glutathione and glutathione disulfide using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection

Measurement of glutathione (GSH) and glutathione disulfide (GSSG) is a crucial tool to assess cellular redox state. Herein we report a direct approach to determine intracellular GSH based on a rapid chromatographic separation coupled with acidic potassium permanganate chemiluminescence detection, which was extended to GSSG by incorporating thiol blocking and disulfide bond reduction. Importantly, this simple procedure avoids derivatisation of GSH (thus minimising auto-oxidation) and overcomes problems encountered when deriving the concentration of GSSG from ‘total GSH’. The linear range and limit of detection for both analytes were 7.5 × 10−7 to 1 × 10−5 M, and 5 × 10−7 M, respectively. GSH and GSSG were determined in cultured muscle cells treated for 24 h with glucose oxidase (0, 15, 30, 100, 250 and 500 mU mL−1), which exposed them to a continuous source of reactive oxygen species (ROS). Both analyte concentrations were greater in myotubes treated with 100 or 250 mU mL−1 glucose oxidase (compared to untreated controls), but were significantly lower in myotubes treated with 500 mU mL−1 (p < 0.05), which was rationalised by considering measurements of H2O2 and cell viability. However, the GSH/GSSG ratio in myotubes treated with 100, 250 and 500 mU mL−1 glucose oxidase exhibited a dose-dependent decrease that reflected the increase in intracellular ROS.

Chemiluminescence detection flow cells for flow injection analysis and high-performance liquid chromatography

We have examined a range of new and previously described flow cells for chemiluminescence detection. The reactions of acidic potassium permanganate with morphine and amoxicillin were used as model systems representing the many fast chemiluminescence reactions between oxidising agents and organic analytes, and the preliminary partial reduction of the reagent was exploited to further increase the rates of reaction. The comparison was then extended to high-performance liquid chromatography separations of α- and β-adrenergic agonists, with permanganate chemiluminescence detection. Flow cells constructed by machining novel channel designs into white polymer materials (sealed with transparent films or plates) have enabled improvements in mixing efficiency and overall transmission of light to the photodetector.