Porous Column Research Articles

Wall modified photonic crystal fibre capillaries as porous layer open tubular columns for in-capillary micro-extraction and capillary chromatography

Wall modified photonic crystal fibre capillary columns for in-capillary micro-extraction and liquid chromatographic separations is presented. Columns contained 126 internal parallel 4 μm channels, each containing a wall bonded porous monolithic type polystyrene-divinylbenzene layer in open tubular column format (PLOT). Modification longitudinal homogeneity was monitored using scanning contactless conductivity detection and scanning electron microscopy. The multichannel open tubular capillary column showed channel diameter and polymer layer consistency of 4.2 ± 0.1 μm and 0.26 ± 0.02 μm respectively, and modification of 100% of the parallel channels with the monolithic polymer. The modified multi-channel capillaries were applied to the in-capillary micro-extraction of water samples. 500 μL of water samples containing single μg L−1 levels of polyaromatic hydrocarbons were extracted at a flow rate of 10 μL min−1, and eluted in 50 μL of acetonitrile for analysis using HPLC with fluorescence detection. HPLC LODs were 0.08, 0.02 and 0.05 μg L−1 for acenaphthene, anthracene and pyrene, respectively, with extraction recoveries of between 77 and 103%. The modified capillaries were also investigated briefly for direct application to liquid chromatographic separations, with the retention and elution of a standard protein (cytochrome c) under isocratic conditions demonstrated, proving chromatographic potential of the new column format, with run-to-run retention time reproducibility of below 1%.

Ultra-High Performance Method on Superficially Porous Stationary Phase for the Determination of Related Substances in Pitavastatin Calcium by HPLC

A simple reverse-phase method for the selective quantification of pitavastatin calcium (PIT) and its related substances was developed. The method demonstrated an excellent separation between PIT and each of 15 impurities (including its isomers and degradants) within a short run time of 12 min by HPLC. A rapid resolution similar to that of UHPLC was achieved using high flow rate on superficially porous C18 stationary phase. A synergistic combination of quality by design approach and use of a superficially porous column delivered a HPLC method with ultra-high performance. Forced degradation studies proved the method to be highly specific (mass balance > 98 %) and the structures of major degradation products were proposed based on LC–MS analysis. The results of validation proved the method to be highly precise (%RSD 0.999) and sensitive (LOQ ≤ 0.02 % and LOD ≤ 0.005 %) for all the impurities and drug. Use of multivariate analysis helped to incorporate high robustness in the method. The method is valuable for quantification of PIT and its related substances in both drug substance and oral solid dosage form.

Investigation of poly(styrene-divinylbenzene-vinylsulfonic acid) as retentive and electroosmotic flow generating phase in open-tubular electrochromatography

In this work, a new sulfonated polystyrene based porous layer was synthesized on the wall of a capillary by a single step in situ polymerization process. To obtain a capillary suited for electrochromatography, vinylsulfonic acid (VSA) was, next to divinylbenzene (DVB), copolymerized to induce charges for the electroosmotic flow (EOF) generation. The VSA ratio in the monomer mixture and the polymerization time were optimized while the chromatographic characteristics of the obtained open tubular columns were investigated in electrochromatography. To allow unambiguous study of only chromatographic processes, evaluations were performed with a mixture of sufficiently retained and electrophoretically neutral parabens. Comparison of SEM pictures and chromatograms revealed that the polymerization time had a great influence on the polymer layer morphology and on the chromatographic performance. An increase in the VSA ratio, led to an increase in the mobile phase velocity but simultaneously lowered paraben retention. The novel optimized stationary phase could generate a stable and significant electro-osmotic flow (EOF) of 1.1mm/s over a wide pH range which could be produced in a reproducible manner. Minimal plate heights of 10μm, equivalent to the capillary internal diameter, were obtained. The open-tubular character of this optimized porous layer column allowed successful analyses at elevated temperature, resulting in a maximum efficiency of 85,500 plates for a 75cm capillary and linear velocities up to 1.4mm/s. Finally, a thermal gradient was successfully applied, leading to artificial sharpened peaks with a peak capacity of 55 in a 20min time span.

Facilitated transport of anatase titanium dioxides nanoparticles in the presence of phosphate in saturated sands.

Soil and water environments are inevitably contaminated by the excess of artificial nanoparticles (NPs) and phosphorus (P) fertilizers. There is a possibility of phosphate facilitating or inhibiting the transport of nanoparticles titanium dioxides (nTiO2). It is a great urgency and high priority to investigate the nTiO2 retention mechanisms and accurately describe the transport of nTiO2 in the presence of phosphate. Anatase nTiO2 with two sizes of 20 and 50nm through the saturated porous sand columns were observed under the conditions (0-50mM NaNO3 electrolyte, influent P concentrations of 0.10mM and 2.0mM, pH 6.5 and 7.5). The experimental results show the phosphate favor the dispersion of nTiO2, and consequently improve their transport patterns. The likely mechanism is that phosphate adsorption increasing the negative charge on the surface promotes the transportability of nTiO2 resulting from the low deposition rate and attachment efficiency of NPs. In particular, the facilitated transport of nTiO2 (50nm) is greater than those relative smaller as 20nm. In addition, this enhancement of nTiO2 transportability by phosphate at pH 6.5 is increased at higher pH of 7.5 due to the more negative zeta potential of surface, which indicates the potential risks to groundwater systems.

Systematic profiling of indole-3-acetic acid biosynthesis in bacteria using LC-MS/MS.

Indole-3-acetic acid (IAA) is produced from tryptophan through five synthesis pathways. A comprehensive method for the quantification of IAA and biosynthesis-related intermediates in a culture medium was developed. Sample preparation was simple with protein precipitation. The analytes were separated on a superficially porous C18 silica column and detected by electrospray ionization-tandem mass spectrometry in the positive ion multiple reaction monitoring mode. The limit of detection was 0.05 μM, and the lower limits of quantification ranged from 0.05 to 2 μM. The intra-day and inter-day precision and accuracy were less than 13.96%. Ion suppression was observed, and the deuterated internal standards were used to compensate for the matrix effect. The method was applied to analyze changes in tryptophan catabolism in a culture medium of Pseudomonas putida. The proposed method is robust and suitable for the systematic profiling of IAA biosynthesis in culture supernatant.

Permanent gas analysis using gas chromatography with vacuum ultraviolet detection

The analysis of complex mixtures of permanent gases consisting of low molecular weight hydrocarbons, inert gases, and toxic species plays an increasingly important role in today's economy. A new gas chromatography detector based on vacuum ultraviolet (VUV) spectroscopy (GC–VUV), which simultaneously collects full scan (115–240nm) VUV and UV absorption of eluting analytes, was applied to analyze mixtures of permanent gases. Sample mixtures ranged from off-gassing of decomposing Li-ion and Li-metal batteries to natural gas samples and water samples taken from private wells in close proximity to unconventional natural gas extraction. Gas chromatography separations were performed with a porous layer open tubular column. Components such as C1–C5 linear and branched hydrocarbons, water, oxygen, and nitrogen were separated and detected in natural gas and the headspace of natural gas-contaminated water samples. Of interest for the transport of lithium batteries were the detection of flammable and toxic gases, such as methane, ethylene, chloromethane, dimethyl ether, 1,3-butadiene, CS2, and methylproprionate, among others. Featured is the capability for deconvolution of co-eluting signals from different analytes.

The role of ultra high performance liquid chromatography with time of flight detection for the identification of synthetic cannabinoids in seized drugs

Separation and mass spectrometric techniques are integral parts of forensic drug analysis for both screening and confirmation. The Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG), which is responsible for setting standards for drug analysis, requires for drug identification a Category A test such as mass spectrometry with an additional test from either Category B or C. If a Category A method is not used at least two uncorrelated tests from Category B must be included, for which separation techniques such as gas chromatography and liquid chromatography would qualify. The utility and validity of using ultra high performance liquid chromatography (UHPLC) and time-of-flight (TOF) mass spectrometry (MS) for the analysis of synthetic cannabinoids is presented. The separation of 32 solutes, including 23 controlled substances and nine non-controlled positional isomers of JWH-018, are compared using UHPLC with TOF detection and capillary GC with electron ionization (EI). For these solutes, the reversed phase UHPLC separation on three different 2.1mm×150mm×2.7μm superficially porous (SPP) columns (C18, Phenyl-Hexyl and Dimethylpentafluorophenylpropyl (PFP)) compared favorably with the capillary gas chromatography (GC) separation using an Elite-5MS column 0.25mm×30m×0.25μm. Principal component analysis revealed that all three UHPLC separations for the separation of the controlled substances are orthogonal to the capillary GC separation. It was also revealed by principal component analysis that the separation of JWH-018 and the nine non-controlled positional isomers for the various techniques were significantly more correlated than the separation of the controlled substances. Although most of the controlled synthetic cannabinoids gave unique TOF in-source collision-induced dissociation MS spectra and EI spectra, it was not possible to discriminate among the geometric isomers (CP47, 497, Epi CP47, 497; Cp47, 497 C8 homologue, Epi CP47, 497 C8 homologue). JWH-018 could be distinguished from the non-controlled isomers based on its EI spectra. In contrast, several of the non-controlled JWH-018 isomers give identical TOF in-source collision-induced dissociation MS spectra to JWH-018.

Recognition of pyrrolizidine alkaloid esters in the invasive aquatic plant Gymnocoronis spilanthoides (Asteraceae).

The freshwater aquatic plant Gymnocoronis spilanthoides (Senegal tea plant, jazmín del bañado, Falscher Wasserfreund) is an invasive plant in many countries. Behavioural observations of pyrrolizidine alkaloid-pharmacophagous butterflies suggested the presence of pyrrolizidine alkaloids in the plant. To determine whether the attraction of the butterflies to the plant is an accurate indicator of pyrrolizidine alkaloids in G. spilanthoides. The alkaloid fraction of a methanolic extract of G. spilanthoides was analysed using HPLC with electrospray ionisation MS and MS/MS. Two HPLC approaches were used, that is, a C18 reversed-phase column with an acidic mobile phase, and a porous graphitic carbon column with a basic mobile phase. Pyrrolizidine alkaloids were confirmed, with the free base forms more prevalent than the N-oxides. The major alkaloids detected were lycopsamine and intermedine. The porous graphitic carbon HPLC column, with basic mobile phase conditions, resulted in better resolution of more pyrrolizidine alkaloids including rinderine, the heliotridine-based epimer of intermedine. Based on the MS/MS and high-resolution MS data, gymnocoronine was tentatively identified as an unusual C9 retronecine ester with 2,3-dihydroxy-2-propenylbutanoic acid. Among several minor-abundance monoester pyrrolizidines recognised, spilanthine was tentatively identified as an ester of isoretronecanol with the unusual 2-acetoxymethylbutanoic acid. The butterflies proved to be reliable indicators for the presence of pro-toxic 1,2-dehydropyrrolizidine alkaloids in G. spilanthoides, the first aquatic plant shown to produce these alkaloids. The presence of the anti-herbivory alkaloids may contribute to the plant's invasive capabilities and would certainly be a consideration in any risk assessment of deliberate utilisation of the plant. The prolific growth of the plant and the structural diversity of its pyrrolizidine alkaloids may make it ideal for investigating biosynthetic pathways or for large-scale production of specific alkaloids.

Preparation of octadecyl methacrylate-based polymer stationary phase by in-situ polymerization for open tubular capillary electrochromatography

The preparation of porous polymethacrylate-based open tubular capilary columns by in-situ copolymerization of octadecyl methacrylate (OMA), and ethylene dimethacrylate (EDMA) in N,N-dimethylformamide (DMF) solvent are proposed. The parameters of the preparation procedure of the stationary phase are discussed in detail. The surface of the cross-section of an open tubular (OT) column by scanning electron microscope (SEM) showed wrinkle configuration, which is expedient to enhance proportion of stationary phase and column capacity. Efficient separation of six alkyl benzene homologous series (namely, toluene, ethylbenzene, propylbenzene, butylbenzene, amylbenzene and hexylbenzene) were performed by CEC using the prepared column, and their elution order was in agreement with their hydrophobicity. So, the OT columns bonded with octadecyl ligands yielded reversed-phase retention behavior toward nonpolar solutes. In addition, the separation of four antiepileptics was also investigated with satisfactory effectiveness. The column efficiency range was 35 300-49 800 plates/m. The results showed that the C18-OT column of organic matrix for reversed-phase chromatography was successfully prepared via in-situ polymerization in this work. Hereby, this new column thus offers a promising new alternative in OT-CEC and will be useful in separation science.

Preparation of a porous polymer monolithic column with an ionic liquid as a porogen and its applications for the separation of small molecules in high performance liquid chromatography

A monolith based on an ionic liquid as a porogen was prepared to enhance the column efficiency of small molecule separation in HPLC.

Comparison of core–shell particles and sub-2 μm fully porous particles for use as ultrafast second dimension columns in two-dimensional liquid chtomatography

The peak capacity of small columns packed with 2.7μm core–shell particles and 1.8μm fully porous particles were compared at high temperatures using very steep (fast) gradient conditions and quite high linear velocities using the same instrument configuration as used to transfer first dimension effluent to the second dimension column as done in on-line comprehensive two-dimensional liquid chromatography. The experimental peak capacities of small columns (2.1mm×30mm) packed with both types of particles were measured with fast gradients (9s to 2min) at high temperature (95°C) using both the same flow rate (1.75mL/min) and then at different flow rates at the same pressure (400bar). Equal or slightly better peak capacities were achieved with the core–shell particle columns as compared to the fully porous particle columns at the same backpressure or the same flow rate. However, core–shell particles offer a real advantage over the smaller, fully porous particles because they can be operated at higher flow rates thus gradient mixer flush out and column reequilibration can be done in less time thereby allowing a greater fraction of the second dimension cycle time to be dedicated to the gradient time.

Nanostructured porous polymer monolithic columns for capillary liquid chromatography of peptides

The macroporous structure of poly(styrene-co-divinylbenzene) monolithic capillary columns has been optimized for the gradient separation of peptides. To exploit monolithic supports with porosity exceeding 70%, the thermodynamic properties of the polymerization mixture were carefully tailored to yield homogeneous monolithic materials featuring macropore and polymer microglobule sizes in the range of 50–200nm. The effects of (i) initiator content, (ii) composition of porogenic mixture, comprising tetrahydrofuran and 1-decanol, (iii) percentage of divinylbenzene crosslinker, and (iv) monomers to porogen ratio on the morphology was investigated. The resulting column structures were investigated using scanning electron microscopy and the prepared monolithic columns were tested for the separation of a tryptic digest of cytochrome c while applying a fixed flow rate and gradient time. To obtain a better understanding of the effects of macropore and microglobule size, and structure homogeneity on the separation performance in gradient elution, both in terms of peak capacity and gradient plate height, separations were also carried out at different flow rates while maintaining a constant gradient steepness. Furthermore, performance limits were determined applying ultra-high pressure conditions up to the maximum system pressure of 80MPa. The potential of monolithic nanostructured columns is demonstrated for the separation of tryptic digests of cytochrome c and bovine serum albumin.

Quantification of aflatoxin M1 in raw milk by a core-shell column on a conventional HPLC with large volume injection and step gradient elution

The immunoaffinity column cleanup method coupled with HPLC separation and fluorescence detection is still one of the most frequently used quantitative methods for routine analyses of AFM1. In this study, aflatoxin M1 (AFM1) was quantified with a chromatography column packed with 2.6 μm core-shell particles on a conventional HPLC. A large volume of solvent (100 μL) was injected into the highly efficient column without any noticeable reduction in separation performance with the help of stepwise gradient elution. The instrumental conditions were optimized by response surface analysis methodology (RSM) with a three-level three-factor Box–Behnken design.The use of core-shell columns on conventional HPLC for AFM1 analysis under the optimized instrumental conditions leads to increased analytical performance compared with traditional totally porous columns without the heavy costs associated ultra-high-pressure instruments. Moreover, the improved instrumental sensitivity enables simplified sample preparation by avoiding any solvent replacement. The method could be easily applied to enhance the sensitivity of HPLC-FLD for AFM1 analysis that is based on isocratic elution and is more widely used. The method was successfully applied to 40 raw milk samples collected in summer from 20 cattle ranches located in two different provinces in southwestern China.

Adsorption of water from aqueous acetonitrile on silica-based stationary phases in aqueous normal-phase liquid chromatography

Excess adsorption of water from aqueous acetonitrile mobile phases was investigated on 16 stationary phases using the frontal analysis method and coulometric Karl–Fischer titration. The stationary phases include silica gel and silica-bonded phases with different polarities, octadecyl and cholesterol, phenyl, nitrile, pentafluorophenylpropyl, diol and zwitterionic sulfobetaine and phosphorylcholine ligands bonded on silica, hybrid organic-silica and hydrosilated matrices. Both fully porous and core-shell column types were included. Preferential uptake of water by the columns can be described by Langmuir isotherms. Even though a diffuse rather than a compact adsorbed discrete layer of water on the adsorbent surface can be formed because of the unlimited miscibility of water with acetonitrile, for convenience, the preferentially adsorbed water was expressed in terms of a hypothetical monomolecular water layer equivalent in the inner pores. The uptake of water strongly depends on the polarity and type of the column. Less than one monomolecular water layer equivalent was adsorbed on moderate polar silica hydride-based stationary phases, Ascentis Express F5 and Ascentis Express CN column at the saturation capacity, while on more polar stationary phases, several water layer equivalents were up-taken from the mobile phase. The strongest affinity to water was observed on the ZIC cHILIC stationary phases, where more than nine water layer equivalents were adsorbed onto its surface at its saturation capacity. Columns with bonded hydroxyl and diol ligands show stronger water adsorption in comparison to bare silica. Columns based on hydrosilated silica generally show significantly decreased water uptake in comparison to stationary phases bonded on ordinary silica. Significant correlations were found between the water uptake and the separation selectivity for compounds with strong polarity differences.

Porous polymer monolithic columns with gold nanoparticles as an intermediate ligand for the separation of proteins in reverse phase-ion exchange mixed mode

A new approach has been developed for the preparation of mixed-mode stationary phases to separate proteins. The pore surface of monolithic poly(glycidyl methacrylate-co-ethylene dimethacrylate) capillary columns was functionalized with thiols and coated with gold nanoparticles. The final mixed mode surface chemistry was formed by attaching, in a single step, alkanethiols, mercaptoalkanoic acids, and their mixtures on the free surface of attached gold nanoparticles. Use of these mixtures allowed fine tuning of the hydrophobic/hydrophilic balance. The amount of attached gold nanoparticles according to thermal gravimetric analysis was 44.8wt.%. This value together with results of frontal elution enabled calculation of surface coverage with the alkanethiol and mercaptoalkanoic acid ligands. Interestingly, alkanethiols coverage in a range of 4.46–4.51molecules/nm2 significantly exceeded that of mercaptoalkanoic acids with 2.39–2.45molecules/nm2. The mixed mode character of these monolithic stationary phases was for the first time demonstrated in the separations of proteins that could be achieved in the same column using gradient elution conditions typical of reverse phase (using gradient of acetonitrile in water) and ion exchange chromatographic modes (applying gradient of salt in water), respectively.

Determination of quinolones in fish by ultra-high performance liquid chromatography with fluorescence detection using QuEChERS as sample treatment

A simple and sensitive method is proposed for the simultaneous determination of quinolones (marbofloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid and flumequine) in different fish samples. Ultra-high performance liquid chromatography using a partially porous C18 column coupled to fluorescence detection with a wavelength excitation/emission program was used. The sample treatment consisted of extraction and clean-up using the QuEChERS methodology, showing a high efficiency without interferences in the chromatographic determination. The method was characterized in fish tissue in terms of linearity, precision, trueness and limits of detection and quantification. Limits of detection between 0.1 and 4.7 μg/kg were obtained, with recoveries between 72 and 108%. The proposed method has been tested in bass, trout, panga and sturgeon, showing its simplicity, sensitivity and suitability for routine analysis in that complex matrix.

Comparison of superficially porous and fully porous silica supports used for a cyclofructan 6 hydrophilic interaction liquid chromatographic stationary phase

A new HILIC stationary phase comprised of native cyclofructan-6 (CF6) bonded to superficially porous silica particles (2.7μm) was developed. Its performance was evaluated and compared to fully porous silica particles with 5μm (commercially available as FRULIC-N) and 3μm diameters. Faster and more efficient chromatography was achieved with the superficially porous particles (SPPs). The columns were also evaluated in the normal phase mode. The peak efficiency, analysis time, resolution, and overall separation capabilities in both HILIC and normal phase modes were compared. The analysis times using the superficially porous based column in HILIC mode were shorter and the theoretical plates/min were higher over the entire range of flow rates studied. The column containing the superficially porous particles demonstrated higher optimum flow rates than the fully porous particle packed columns. At higher flow rates, the advantages of the superficially porous particles was more pronounced in normal phase separations than in HILIC, clearly demonstrating the influence that the mode of chromatography has on band broadening. However, the minimum reduced plate heights (hmin) were typically lower in HILIC than in the normal phase mode. Overall, the superficially porous particle based CF6 column showed clear advantages over the fully porous particle columns, in terms of high throughput and efficient separations of polar compounds in the HILIC mode.

Empirical models for change in pH and temperature within gravity-based reactor columns

Column reactor models of volume size 3,000 and 1,500 cm3 are made using organic materials such as sawdust and immature (drumstick) Moringa oleifera and other natural materials such as gravels (6 mm size) and ball clay available locally at Jodhpur, India. Water is passed through these porous reactors under gravity at once. The experiments were aimed at finding low-cost solutions for wastewater or sewage disposal at point of use. The change in pH during water filtration experiments is measured and modelled as functions of X1 (column height), X2 (flow rate), X3 (cumulative percolation time) and X4 (change in electrical conductivity). The parameters X1, X2, X3 and X4 are found to be highly correlated to each other irrespective of materials used for making the bioreactors. There is a hyperbolic relationship between temperature gradient within the porous material column bed through which water is percolating and time taken during that process. The temperature distribution in the gravel or sawdust media reactors is not influenced by the inflow rate or height of the reactor column used for experimentation. Distinct temperature distribution exists at each depth of the heterogeneous reactors. The multi-parameter model developed and the hyperbolic relationships help to characterize the efficacy of bioreactors. The effect of the materials on the wastewater treatment can now be individually evaluated using the multi-parameter approach presented in this paper.

Incorporation of metal-organic framework HKUST-1 into porous polymer monolithic capillary columns to enhance the chromatographic separation of small molecules

Metal-organic framework (MOF) HKUST-1 nanoparticles have been incorporated into poly(glycidyl methacrylate-co-ethylene dimethacrylate) (HKUST-1-poly(GMA-co-EDMA)) monoliths to afford stationary phases with enhanced chromatographic performance of small molecules in the reversed phase capillary liquid chromatography. The effect of HKUST-1 nanoparticles in the polymerization mixture on the performance of the monolithic column was explored in detail. While the bare poly(GMA-co-EDMA) monolith exhibited poor resolution (Rs<1.0) and low efficiency (800–16,300plates/m), addition of a small amount of HKUST-1 nanoparticles to the polymerization mixture provide high increased resolution (Rs≥1.3) and high efficiency ranged from 16,300 to 44,300plates/m. Chromatographic performance of HKUST-1-poly(GMA-co-EDMA) monolith was demonstrated by separation of various analytes including polycyclic aromatic hydrocarbons, ethylbenzene and styrene, phenols and aromatic acids using a binary polar mobile phase (CH3CN/H2O). The HKUST-1-poly(GMA-co-EDMA) monolith displayed enhanced hydrophobic and π–π interaction characteristics in the reversed phase separation of test analytes compared to the bare poly(GMA-co-EDMA) monolith. The experiment results showed that HKUST-1-poly(GMA-co-EDMA) monoliths are an alternative to enhance the chromatographic separation of small molecules.

Asymmetric transport of passive tracers across heterogeneous porous media

Experimental results on tracer transport in a porous column consisting of two adjacent segments of different (fine and coarse) glass bead packs having the same porosity value, have shown that the breakthrough curve behavior depends on the transport direction (Berkowitz et al. (2009)). It was found that tracers migrating in the fine-to-coarse (FC) direction arrive significantly faster than in the coarse-to-fine (CF) direction. In this work, we simulate Brownian tracers moving in a 2D random porous medium in a configuration similar to that of the referred experimental set-up. By considering only diffusive effects, the results confirmed the transport asymmetry across the heterogeneous interface. Arguments based on effective medium equations for the porous medium transport indicate that the asymmetry can be explained by subtle differences of effective diffusivity between the coarse and fine porous medium. In turn, such diffusivity differences are induced by differences in the tortuosity and porosity in the vicinity of the porous media boundary.