Porous layer open tubular columns in capillary liquid chromatography.

This review covers the latest developments and applications of porous layer open tubular columns in capillary liquid chromatography. Here, the authors provide a concise background on the fundamentals of porous layer open tubular columns, their fabrication and application. Over the past two decades, growing interest in the areas of proteomics and hyphenated LC-MS techniques have played a large part in the development of porous layer structures within capillary formats due to their high permeability, excellent efficiency and exceptional peak capacity. This review gives a brief overview of open tubular columns in general, however, it focuses predominantly on the applications of covalently bonded porous layer open tubular columns in liquid chromatography. Open tubular columns containing non-bonded or electrostatically attached layers will not be discussed in detail.

Focussed ion beam serial sectioning and imaging of monolithic materials for 3D reconstruction and morphological parameter evaluation.

A new characterisation method, based on the utilisation of focussed ion beam-scanning electron microscopy (FIB-SEM), has been employed for the evaluation of morphological parameters in porous monolithic materials. Sample FIB serial sectioning, SEM imaging and image processing techniques were used to extract the pore boundaries and reconstruct the 3D porous structure of carbon and silica-based monoliths. Since silica is a non-conducting material, a commercial silica monolith modified with activated carbon was employed instead to minimise the charge build-up during FIB sectioning. This work therefore presents a novel methodology that can be successfully employed for 3D reconstruction of porous monolithic materials which are or can be made conductive through surface or bulk modification. Furthermore, the 3D reconstructions were used for calculation of the monolith macroporosity, which was in good agreement with the porosity values obtained by mercury intrusion porosimetry (MIP).

In-process phase growth measurement technique in the fabrication of monolithic porous layer open tubular (monoPLOT) columns using capacitively coupled contactless conductivity.

A technique for the in-process measurement of polymer stationary phase growth inside fused silica capillaries during the fabrication of monolithic porous layer open tubular (monoPLOT) columns is presented. In this work, capacitively coupled contactless conductivity detection (C4D) was applied as an online measurement tool for porous polymer layer growth within fused silica capillaries. The relationship between effective capillary diameter and C4D response was investigated for two polymers, butyl methacrylate-ethylene dimethacrylate (BuMA-EDMA) and polystyrene-divinylbenzene (PS-DVB) over a range of capillary diameters and layer thicknesses. The described technique can be used with both thermal and photo-initiated approaches for monoPLOT fabrication and provides an accurate, real-time measurement of the porous layer growth within the capillary, which should vastly improve column-to-column reproducibility. The technique was shown to be very precise, with a measured %RSD < 10%.

Nano-particle modified stationary phases for high-performance liquid chromatography.

This review covers the latest developments and applications of nano-materials in stationary phase development for various modes of high-performance liquid chromatography. Specific attention is placed upon the development of new composite phases, including the synthetic and immobilisation strategies used, to produce either encapsulated nano-particles, or surface attached nano-particles, layers, coatings and other structures. The resultant chromatographic applications, where applicable, are discussed with comment upon enhanced selectivity and/or efficiency of the nano-particle modified phases, where such effects have been identified. In the main this review covers developments over the past five years and is structured according to the nature of the nano-particles themselves, including carbonaceous, metallic, inorganic, and organopolymer based materials.

Iminodiacetic acid functionalised organopolymer monoliths: application to the separation of metal cations by capillary high-performance chelation ion chromatography.

Lauryl methacrylate-co-ethylene dimethacrylate monoliths were polymerised within fused silica capillaries and subsequently photo-grafted with varying amounts of glycidyl methacrylate (GMA). The grafted monoliths were then further modified with iminodiacetic acid (IDA), resulting in a range of chelating ion-exchange monoliths of increasing capacity. The IDA functional groups were attached via ring opening of the epoxy group on the poly(GMA) structure. Increasing the amount of attached poly(GMA), via photo-grafting with increasing concentrations of GMA, from 15 to 35%, resulted in a proportional and controlled increase in the complexation capacity of the chelating monoliths. Scanning capacitively coupled contactless conductivity detection (sC(4)D) was used to characterise and verify homogenous distribution of the chelating ligand along the length of the capillaries non-invasively. Chelation ion chromatographic separations of selected transition and heavy metals were carried out, with retention factor data proportional to the concentration of grafted poly(GMA). Average peak efficiencies of close to 5,000 N/m were achieved, with the isocratic separation of Na, Mg(II), Mn(II), Co(II), Cd(II) and Zn(II) possible on a 250-mm-long monolith. Multiple monolithic columns produced to the same recipes gave RSD data for retention factors of <15% (averaged for several metal ions). The monolithic chelating ion-exchanger was applied to the separation of alkaline earth and transition metal ions spiked in natural and potable waters.

Controlled ultraviolet (UV) photoinitiated fabrication of monolithic porous layer open tubular (monoPLOT) capillary columns for chromatographic applications.

An automated column fabrication technique that is based on a ultraviolet (UV) light-emitting diode (LED) array oven, and provides precisely controlled "in-capillary" ultraviolet (UV) initiated polymerization at 365 nm, is presented for the production of open tubular monolithic porous polymer layer capillary (monoPLOT) columns of varying length, inner diameter (ID), and porous layer thickness. The developed approach allows the preparation of columns of varying length, because of an automated capillary delivery approach, with precisely controlled and uniform layer thickness and monolith morphology, from controlled UV power and exposure time. The relationships between direct exposure times, intensity, and layer thickness were determined, as were the effects of capillary delivery rate (indirect exposure rate), and multiple exposures on the layer thickness and axial distribution. Layer thickness measurements were taken by scanning electron microscopy (SEM), with the longitudinal homogeneity of the stationary phase confirmed using scanning capacitively coupled contactless conductivity detection (sC(4)D). The new automated UV polymerization technique presented in this work allows the fabrication of monoPLOT columns with a very high column-to-column production reproducibility, displaying a longitudinal phase thickness variation within ±0.8% RSD (relative standard deviation).

Porous graphitized carbon monolith as an electrode material for probing direct bioelectrochemistry and selective detection of hydrogen peroxide.

For the first time, graphitized carbon particles with a high surface area have been prepared and evaluated as a new material for probing direct electrochemistry of hemoglobin (Hb). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging revealed that the carbon monolithic skeleton was constructed by a series of mesopores with irregular shapes and an average pore diameter of ~5.6 nm. With a surface area of 239.6 m(2)/g, carbon particles exhibited three major Raman peaks as commonly observed for carbon nanotubes and other carbon materials, i.e., the sp(3) and sp(2) carbon phases coexisted in the sample. A glassy carbon electrode modified with carbon monoliths and didodecyldimethylammonium bromide exhibited direct electron transfer between Hb molecules and the underlying electrode with a transfer rate constant of 6.87 s(-1). The enzyme electrode displayed a pair of quasi-reversible reduction-oxidation peaks at -0.128 and -0.180 V, reflecting the well-known feature of the heme [Fe(3+)/Fe(2+)] redox couple: a surface-controlled electrochemical process with one electron transfer. This reagentless biosensing approach was capable of detecting H(2)O(2), a simple molecule but plays an important role in analytical and biological chemistry, as low as 0.1 µM with linearity of 0.1-60 µM and a response time of <0.8 s, comparing favorably with other carbon based electrodes (5 s).

Versatile capillary column temperature control using a thermoelectric array based platform.

A new direct contact platform for capillary column precise temperature control based upon the use of individually controlled sequentially aligned Peltier thermoelectric units is presented. The platform provides rapid temperature control for capillary and microbore liquid chromatography columns and allows simultaneous temporal and spatial temperature programming. The operating temperature range of the platform was between 15 and 200 °C for each of 10 aligned Peltier units, with a ramp rate of approximately 400 °C/min. The system was evaluated for a number of nonstandard capillary based applications, such as the direct application of temperature gradients with both linear and nonlinear profiles, including both static column temperature gradients and temporal temperature gradients, and the formation of in-capillary monolithic stationary phases with gradient polymerization through precise temperature control.

Hydrophobicity of polymer based anion-exchange columns for ion chromatography.

The regularities of the retention of alkanoic and alkanesulfonic acids homologues were investigated for the set of 36 anion-exchange columns produced by various manufacturers. The role of hydrophobic and electrostatic interactions in the retention and separation of organic anions was studied. The methylene selectivity increments α(CH2) were measured for the studied columns with 10 mM sodium hydroxide eluent. The influence of matrix, surface area, polar group structure, ion-exchange capacity, the density of charged functional groups on the surface and other characteristics of anion-exchangers on resin hydrophobicity was considered. A unified approach for the measurements of hydrophobic properties of anion-exchange resins is proposed and the ratio of chloride retention factor (k Cl) to α(CH2) was introduced as mixed-mode factor. The synergetic effect of electrostatic and hydrophobic interactions was observed.

Holistic visualisation of the multimodal transport and fate of twelve pharmaceuticals in biosolid enriched topsoils.

The use of municipal biosolids as agricultural fertilisers has raised significant concerns in recent years. As part of this, the presence of complex mixtures of pharmaceutical residues and their effects on soil ecosystems remains particularly under-researched. This study focuses on the transfer of a selection of pharmaceutical residues from municipal sewage sludge to agricultural topsoils and their fate therein after an accelerated 6-month rainfall event. Twelve pharmaceuticals encompassing antibiotics, analgesics, anti-inflammatories, beta-blockers, hyperlipidaemics and stimulants were invesigated by employing a combination of extraction techniques and liquid chromatography-tandem mass spectrometry. Both liquid- and solid-phase pharmaceutical contents were analysed and pharmaceutical and personal care products quantified at defined timepoints to elucidate transport behaviour and transformation potential. Results show the distribution and separation of pharmaceuticals over a 100-mm soil depth following typical biosolid enrichment. Using experimentally determined solid-water partition coefficients (K (d)) and hydrophobicity distribution ratios (D (ow)), mobility and modes of interaction under dynamic conditions are discussed. Finally, a brief study into the susceptibility of soil microbes is also presented. To our knowledge, this is the first investigation of pharmaceutical and personal care products release from amended biosolids to soils to include the factors and mechanisms governing their distribution and transformation even over relatively shallow depths. It applies multicompartmental and mass-balanced chemical analyses as well as microbiological approaches for a holistic view of these complex processes.

Preparation, characterisation and modification of carbon-based monolithic rods for chromatographic applications.

A range of porous carbon-based monolithic (PCM) rods with flow-through pore sizes of 1, 2, 5 and 10 mum, were produced using a silica particle template method. The rods were characterised using SEM and energy-dispersive X-ray spectroscopy, BET surface area and porous structure analysis, dilatometry and thermal gravimetry. SEM evaluation of the carbon monolithic structures revealed an interconnected rigid bimodal porous structure and energy-dispersive X-ray spectroscopy analysis verified the quantitative removal of the embedded silica beads. The specific surface areas of the 1, 2, 5 and 10 mum rods were 178, 154, 84 and 125 m(2)/g after pyrolysis and silica removal, respectively. Shrinkage of the monolithic rods during pyrolysis is proportional to the particle size of the silica used and ranged from 9 to 12%. Mercury porosimetry showed a narrow distribution of pore sizes, with an average of approximately 700 nm for the 1 mum carbon monolith. The suitability of bare and surface oxidised PCM rods for the use as a stationary phase for reversed and normal phase LC was explored. The additional modification of PCM rods with gold micro-particles followed by 6-mercaptohexanoic acid was performed and ion-exchange properties were evaluated.

Simultaneous separation of inorganic anions and metal-citrate complexes on a zwitterionic stationary phase with on-column complexation.

The retention and separation selectivity of inorganic anions and on-column derivatised negatively charged citrate or oxalate metal complexes on reversed-phase stationary phases dynamically coated with N-(dodecyl-N,N-dimethylammonio)undecanoate (DDMAU) has been investigated. The retention mechanism for the metal-citrate complexes was predominantly anion exchange, although the amphoteric/zwitterionic nature of the stationary phase coating undoubtedly also contributed to the unusual separation selectivity shown. A mixture of 10 inorganic anions and metal cations was achieved using a 20 cm monolithic DDMAU modified column and a 1 mM citrate eluent, pH 4.0, flow rate equal to 0.8 mL/min. Selectivity was found to be strongly pH dependent, allowing additional scope for manipulation of solute retention, and thus application to complex samples. This is illustrated with the analysis of an acidic mine drainage sample with a range of inorganic anions and transition metal cations, varying significantly in their concentrations levels.

Anion-exchange chromatography on short reversed-phase columns modified with amphoteric (N-dodecyl-N,N-dimethylammonio)alcanoates.

Short reversed-phase columns (50 mm x 4.6 mm Gemini C(18)) were dynamically coated with carboxybetaines of the general structure, C(12)H(25)N(+)(CH(3))(2)(CH(2))(n)COOH, namely (N-dodecyl-N,N-dimethylammonio)undecanoate, DDMAU (n=10) and (N-dodecyl-N,N-dimethylammonio)butyrate, DDMAB (n=3), and investigated for the separation of inorganic anions in ion chromatography. The role of the ionic strength of coating surfactant solutions on their adsorption and resultant column capacity was studied. The retention of inorganic anions was investigated with different eluents at various concentrations and pH. Interestingly, no retention for anions was found with pure water as the eluent, but the addition of small amounts of electrolytes, up to 0.1 mM, caused a sharp increase in the retention of analytes. The effect of increasing anion retention with an increase in eluent cation charge was also observed. Based on this effect a new cation charge gradient concept was proposed and applied to the separation of a standard mixture of anions.

Double gradient ion chromatography using short monolithic columns modified with a long chained zwitterionic carboxybetaine surfactant.

The rapid separation of inorganic anions on short monolithic columns permanently coated with a long chained zwitterionic carboxybetaine-type surfactant is shown. The surfactant, N-dodecyl-N,N-(dimethylammonio)undecanoate (DDMAU), was used to coat 2.5, 5.0 and 10 cm long reversed-phase silica monoliths, resulting in a permanent zwitterionic exchange surface when used with aqueous based eluents. The unique structure of the surfactant results in a charge double layer structure on the surface of the stationary phase, with strong internal anionic and weak external cationic exchange groups. The dissociation of the weak external carboxylic acid group acts to shield the inner anionic exchange site, resulting in substantial effective capacity changes with eluent pH. Utilising this effect with the application of an eluent pH gradient, simultaneously combined with eluent flow-rate gradients, very rapid simultaneous separations of both weakly retained anions and strongly retained polarisable anions was possible, with up to 10-fold decreases in overall run times. Coating stability and retention times under isocratic and isofluentic eluent conditions were shown to be reproducible over >450 repeat injections, with peak efficiency values averaging 29,000 N/m for the 2.5 cm column and 42,000 N/m for the 10 cm monolithic column, again under isocratic elution conditions.

Chelation ion chromatography of alkaline earth and transition metals a using monolithic silica column with bonded N-hydroxyethyliminodiacetic acid functional groups.

A commercially available porous silica monolithic column (Onyx Monolithic Si, 100 mm×4.6 mm I.D.) was 'in-column' covalently functionalised with 2-hydroxyethyliminodiacetic acid (HEIDA) groups, and applied to the simultaneous and rapid separation of alkaline earth and transition metal ions, using high-performance chelation ion chromatography (HPCIC). With a 0.3mM dipicolinic acid (DPA) containing eluent, the baseline separation of various common transition and heavy metal ions and the four alkaline earth metal ions could be achieved in under 14 min with a flow rate of just 0.8 mL/min. Detection was achieved using spectrophotometric detection at 540 nm after post-column reaction (PCR) with 4-(2-pyridylazo)-resorcinol (PAR). Significant effects from variation of eluent nature, concentration and temperature upon selectivity and retention were demonstrated with the new monolithic silica chelating phase. Under optimised conditions (0.165 M LiNO(3) eluent, pH 2.5), peak efficiencies of 54,000, 60,000 and 64,000 N/m, for Zn(2+), Mn(2+) and Cd(2+), respectively, were recorded, far exceeding that previously reported for IDA based chelation ion exchange columns.

Fabrication and characterization of nanotemplated carbon monolithic material.

A novel hierarchical nanotemplated carbon monolithic rod (NTCM) was prepared using a novel facile nanotemplating approach. The NTCM was obtained using C60-fullerene modified silica gels as hard templates, which were embedded in a phenolic resin containing a metal catalyst for localized graphitization, followed by bulk carbonization, and template and catalyst removal. TEM, SEM, and BET measurements revealed that NTCM possessed an integrated open hierarchical porous structure, with a trimodal pore distribution. This porous material also possessed a high mesopore volume and narrow mesopore size distribution. During the course of carbonization, the C60 conjugated to aminated silica was partly decomposed, leading to the formation of micropores. The Raman signature of NTCM was very similar to that of multiwalled carbon nanotubes as exemplified by three major peaks as commonly observed for other carbon materials, i.e., the sp3 and sp2 carbon phases coexisted in the sample. Surface area measurements were obtained using both nitrogen adsorption/desorption isotherms (BET) and with a methylene blue binding assay, with BET results showing the NTCM material possessed an average specific surface area of 435 m2 g(-1), compared to an area of 372 m2 g(-1) obtained using the methylene blue assay. Electrochemical studies using NTCM modified glassy carbon or boron doped diamond (BDD) electrodes displayed quasi-reversible oxidation/reduction with ferricyanide. In addition, the BDD electrode modified with NTCM was able to detect hydrogen peroxide with a detection limit of below 300 nM, whereas the pristine BDD electrode was not responsive to this target compound.

A new N-hydroxyethyliminodiacetic acid modified core-shell silica phase for chelation ion chromatography of alkaline earth, transition and rare earth elements.

Bare core-shell silica (1.7µm) has been modified with iminodiacetic acid functional groups via standard silane chemistry, forming a new N-hydroxyethyliminodiacetic acid (HEIDA) functionalised core-shell stationary phase. The column was applied in high-performance chelation ion chromatography and evaluated for the retention of alkaline earth, transition and heavy metal cations. The influence of nitric acid eluent concentration, addition of complexing agent dipicolinic acid, eluent pH and column temperature on the column performance was investigated. The efficiencies obtained for transition and heavy metal cations (and resultant separations) were comparable or better than those previously obtained for alternative fully porous silica based chelation stationary phases, and a similarly modified monolithic silica column, ranging from ∼15 to 56µm HETP. Increasing the ionic strength of the eluent with the addition of KNO3 (0.75M) and increasing the column temperature (70°C) facilitated the isocratic separation of a mixture of 14 lanthanides and yttrium in under 12min, with HETP averaging 18µm (7µm for Ce(III)).

Separation of selected transition metals by capillary chelation ion chromatography using acetyl-iminodiacetic acid modified capillary polymer monoliths.

Capillary housed laurylmethacrylate-co-ethylene dimethacrylate (LMA-co-EDMA) polymer monoliths were fabricated, functionalised with varying amounts of vinyl azlactone, followed by immobilisation of iminodiacetic acid (IDA), forming a range of acetyl-iminodiacetic acid (AIDA) functionalised monoliths, applied to the chelation ion chromatographic separation of selected transition and heavy metals. A number of monoliths of varying length and ligand density were prepared, resulting in increased cation retention and chromatographic resolution on those displaying the highest capacity. Ligand density and related column capacity were confirmed visually using scanning capacitively coupled contactless conductivity detection (sC(4)D) techniques. Column temperature studies to determine retention mechanism and the effect of temperature on the retention of Mn(II), Cd(II) and Cu(II) was investigated, showing an increase in retention with increased temperature for Cd(II) and Cu(II), whilst a decrease in retention was obtained for Mn(II). Isocratic capillary chelation ion chromatographic separations of Mn(II), Cd(II) and Cu(II) were obtained, with dual peak detection demonstrated using combined on-column C(4)D detection and UV-Visible detection following the post-capillary column reaction of the eluted metals with 4-(2-pyridylazo) resorcinol (PAR).

Comparative metal distribution in scalp hair of Pakistani and Irish referents and hypertensive patients.

The abnormal metabolism of metal ions plays an important role in health and disease conditions, and studies about them have been attracting significant interest. The aim of our study was to assess the heavy metals (cadmium (Cd), nickel (Ni), lead (Pb), and zinc (Zn)) in scalp hair samples of 50 Irish and 78 Pakistani hypertensive patients of an urban population together with 50 Irish and 96 Pakistani non-hypertensive male subjects in the age group of 30-50 years. The concentrations of trace and toxic elements were measured by inductively coupled plasma-atomic emission spectrophotometer and atomic absorption spectrophotometer before microwave-assisted acid digestion. The validity and accuracy of the methodology were checked using certified reference materials, and by the conventional wet acid digestion method on the same certified reference materials and on real samples. The recovery of all the studied elements was found to be in the range of 97.5-99.7% in certified reference material. The results of this study showed that the mean values of cadmium, nickel, and lead were significantly higher in scalp hair samples of both Pakistani and Irish hypertensive patients than in referents (p < 0.001); whereas, the concentration of zinc was lower in the scalp hair samples of hypertensive patients of both genders. The deficiency of zinc and the high exposure of trace and toxic metals may be the risk factors associated with hypertension.

Monolithic porous layer open tubular (monoPLOT) columns for low pressure liquid chromatography of proteins.

Glycidyl methacrylate-ethylene dimethacrylate (GMA-co-EDMA) based monolithic porous layer open tubular (monoPLOT) columns (0.05 mm I.D., monolithic layer thickness ≈ 5 µm) have been fabricated using an automated column scanning technique, providing UV polymerisation at 365 nm. Columns were chemically modified to obtain desired diol groups on the surface, and the longitudinal homogeneity of the stationary phase was profiled using scanning capacitively coupled contactless conductivity detector (sC4D), before and after such modification. Using the automated scanning polymerisation technique, column-to-column production reproducibility, including longitudinal phase thickness, was within ±5% RSD. The prepared columns were tested to evaluate their liquid chromatographic stationary phase selectivity, efficiency and reproducibility, with a series of test protein mixtures. Under optimised gradient conditions, the separation of up to 8 proteins was demonstrated on the open tubular column (510 × 0.05 mm I.D.), with a column pressure drop of <1.5 MPa.