Mini-column System Research Articles

On-line determination of the trace amount of cobalt(II) in real samples by flame atomic absorption spectrometry method after pre-concentration by modified polyvinyl chloride

This work reports a simple and sensitive on-line solid-phase extraction system for the pre-concentration and determination of a trace amount of Co(II (ions by flame atomic absorption spectrometry (FAAS). This method is based on the on-line retention of cobalt ions with polyvinyl chloride modified by 3-(2-thiazolylazo)-2,6-diaminopyridine in a mini-column system. The retained Co(II) ions were eluted with 1.0 mol L−1 HCl, and direct aspiration to flame atomic absorption spectrometry (FAAS) for an accurate determination of it. The important experimental parameters such as the solution pH, length of column (adsorbent mass), sample flow rate, solvent eluent, type and concentration of the eluent, and co-existing ion effects were investigated in detail and optimized. Under the optimum conditions, the calibration graph was linear over the concentration range of 2–150 µg L−1 for pre-concentration of 10.0 mL of the sample solution. The interassay precession (n = 6) was in the range of 1.8–5.2% at the concentration levels of 10.0, 50.0, 100.0, and 140.0 µg L−1. The limit of detection and enhancement factor were 1.3 µg L−1 and 40, respectively. The method was applied for determination of trace levels of Co(II) ions in different real samples such as tap water, waste water, vegetable, fruit, and drug with satisfactory results.

Inhibition of Inflammatory Cytokines and Induction of Myeloid-Derived Suppressor Cells by the Effects of Granulocyte and Monocyte Adsorption Apheresis.

Pro-inflammatory cytokines are involved in the pathogenesis of inflammatory skin diseases attributable to activated neutrophils and macrophages. Myeloid-derived suppressor cells (MDSCs) play an important role in the regulation of the immune response and possess strong immunosuppressive and anti-inflammatory properties. Granulocyte and monocyte adsorption apheresis (GMA), an extracorporeal apheresis instrument featuring columns containing cellulose acetate (CA) beads, is designed to remove pathogenic myeloid lineage cells. The purpose of this study was to investigate the effects of GMA on cytokine production and MDSC induction. The serum level of various inflammatory cytokines and the incidence of MDSCs in peripheral blood before and after GMA treatment were recorded. Cytokines were assayed with the suspension-array method in 38 patients. The incidence of MDSCs was analyzed by FACS in eight patients and the effect of GMA on in vitro MDSC induction was examined using a mini-column system that mimics GMA. The serum level of IL-2Rα (P = 0.030), IL-8 (P = 0.018), and MIF (P = 0.0002) was significantly decreased by GMA and the incidence of MDSCs was increased (P = 0.030). With the mini-column system, MDSCs were induced in the peripheral blood of five healthy volunteers; the in vitro induction was significantly inhibited by inactivation of the complement component iC3b. The clinical effectiveness of GMA may be attributable to the inhibition of pro-inflammatory cytokines and the induction of anti-inflammatory MDSCs by iC3b activation via the CA beads in the GMA column.

379 Granulocyte and monocyte adsorption apheresis is effective for refractory netrophilic disorders and induces myeloid-derived suppressor cells

Granulocyte and monocyte adsorption apheresis (GMA) is an extracorporeal apheresis unit designed to remove pathogenic myeloid lineage cells. GMA involves a column containing cellulose acetate (CA) beads that serve as adsorptive leukocyte apheresis carriers. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population comprised of cells at several stages of myeloid lineage differentiation, that differentiate into macrophages, neutrophils, and dendritic cells. MDSCs play an important role in the regulation of the immune response and have immunosuppressive and anti-inflammatory properties. We previously demonstrated that CA beads activate complement component iC3b. Recently, iC3b was shown to induce differentiation of myeloid cells to MDSCs. Based on these facts, we hypothesized that GMA induces MDSC differentiation. The purpose of this study was to investigate the clinical effectiveness of GMA for neutrophilic skin diseases and related arthropathy including pustular psoriasis and psoriatic arthritis and whether GMA induces MDSC. For the assessment of clinical effectiveness, 68 patients with skin lesions and 41 with arthropathy were enrolled. Skin and joint lesions were assessed by overall lesional assessment (OLA) and visual analog scale (VAS), respectively. The incidence of MDSCs was analyzed by FACS in 8 patients and the effect of GMA on MDSC induction was examined using an in vitro mini-column system that mimics GMA. GMA was effective in 56 of 68 patients (82.4%) with skin lesions and 36 of 41 (87.8%) with arthropathy. The incidence of MDSCs in the patients was significantly increased by GMA. In the in vitro mini-column system, MDSCs were induced in the blood of 5 healthy volunteers and the induction was inhibited by inactivation of iC3b. GMAis effective for neutrophilic disorders and the effect might be attributed, at least in part, to the induction of MDSCs via iC3b activation by the CA beads in the GMA column.

Understanding operational system differences for transfer of miniaturized chromatography column data using simulations

In this paper, a simulation tool was employed to identify and appropriately incorporate differences between MiniColumns and benchtop column systems. It was first demonstrated that including multi-step gradients and fraction collection into the simulations resulted in improved agreement between simulated and experimental linear gradient profiles as well as calculated first moments in the MiniColumn experiments. Step elution experiments of binary mixtures (a monoclonal antibody and one of three model proteins) were then carried out to examine comparability of the MiniColumns to the benchtop system. Although the peak shapes were qualitatively similar, peak elution began earlier in the MiniColumn system while improved separation was observed between overlapping peaks using the benchtop format. Simulations were then carried out to demonstrate that increased dispersion of the eluent breakthrough in the benchtop system could readily explain these observed differences. Importantly, by incorporating these system differences into the simulations, we were able to predict benchtop step elution performance using the parameters solely obtained from the MiniColumns. The findings presented in this paper illustrate that the appropriate consideration of system differences can facilitate the implementation of miniature chromatography columns as scale-down models for bioprocess development.

Using isothermal microcalorimetry to measure the metabolic activity of the mineral-associated microbial community in bioleaching

Microbial colonisation of mineral ore is critical in efficient mineral solubilisation for metal recovery in heap bioleaching processes. A better understanding of the processes and behaviour by which microorganisms attach to, and colonise, mineral surfaces may help to enhance bioleaching processes. The direct quantification of metabolic activity of mineral associated cells is difficult and has not been demonstrated adequately in ore beds typical of heap leaching. In this investigation, isothermal microcalorimetry (IMC) was used to measure metabolic activity of a mixed mesophilic culture colonising the mineral surface. IMC is non-destructive and measures continuous heat flow from chemical or biological processes. Our results showed an increase of heat output from the colonised surfaces of mineral concentrate in the biotic system with respect to the abiotic system, caused by oxidative reactions facilitated by the mineral-microbial biofilm. This confirmed that the attached microorganisms were metabolically active and facilitated ongoing mineral leaching through regeneration of lixiviants. The progression of mineral colonisation in a mini-column system was monitored using IMC, scanning electron microscopy and conventional wet chemistry measurements.

Use of MiniColumns for linear isotherm parameter estimation and prediction of benchtop column performance

In this paper, a comparison between experimental chromatography data and column simulations is carried out to determine the efficacy of using miniaturized chromatography columns (MiniColumns) for both column modeling parameter estimation and process development. Normalization of the data with respect to column volumes along with appropriate translations to account for system differences is shown to result in comparability of the experimental data for the MiniColumn and benchtop systems. A parameter estimation protocol is then employed to determine the linear steric mass-action (SMA) isotherm and lumped mass transport parameters for two cation exchange resins. The models are then validated and simulations using different parameter sets from the MiniColumn and benchtop systems are shown to result in similar predicted chromatography profiles and calculated retention volumes. The parameters generated from the MiniColumn system are demonstrated to be well suited for predicting experimental data from the benchtop system. These simulation results, the ability to operate MiniColumns in parallel, and the significantly lower material requirements per experiment support an industry trend toward increased usage of miniaturized chromatography columns as a scale-down model for process development.

Multi-walled carbon nanotubes as solid-phase extraction adsorbents for the speciation of cobalamins in seafoods by liquid chromatography

Multi-walled carbon nanotubes (MWCNTs) were evaluated as potential adsorbents for miniaturized solid-phase extraction coupled to liquid chromatography. The adsorption capacity of this sorbent was applied to assess the speciation of four cobalamins representing the various forms of vitamin B(12). The preconcentration on the MWCNTs was based on the retention of analytes by introducing the sample online into the mini-column system. Dimethyl sulfoxide was used to elute the retained vitamins for liquid chromatographic analysis. The experimental conditions of the continuous flow device, which affect the enrichment procedure, such as the type and amount of nanotubes, the volume, pH and flow rate of the sample solution, and the eluent and its volume, were optimized. For detection purposes, a diode array device was used and good resolution was obtained with a mobile-phase acetonitrile-phosphate buffer and gradient elution. Specificity was demonstrated by the retention characteristics and UV spectra and by comparing the peak purity index with commercial standards. Linearity, precision, recovery, and sensitivity were satisfactory. Detection limits ranged from 0.35 to 30 ng mL(-1). The method was successfully applied to the determination of cobalamins in seafoods, which were extracted from the sample with a buffer solution using an ultrasonic probe. The reliability of the procedure was checked by analyzing a certified reference material.

Speciation of inorganic arsenic in a sequential injection dual mini-column system coupled with hydride generation atomic fluorescence spectrometry

The separation and speciation of inorganic arsenic(III) and arsenic(V) are facilitated by employing a novel sequential injection system incorporating two mini-columns followed by detection with hydride generation atomic fluorescence spectrometry. An octadecyl immobilized silica mini-column is used for selective retention of the complex between As(III) and APDC, while the sorption of As(V) is readily accomplished by a 717 anion exchange resin mini-column. The retained As(III)–PDC complex and As(V) are effectively eluted with a 3.0 mol L −1 hydrochloric acid solution as stripping reagent, which well facilitates the ensuing hydride generation process via reaction with tetrahydroborate. With a sampling volume of 1.0 mL and an eluent volume of 100 μL for both species, linear ranges of 0.05–1.5 μg L −1 for As(III) and 0.1–1.5 μg L −1 for As(V) are obtained, along with enrichment factors of 7.0 and 8.2, respectively. Precisions of 2.8% for As(III) and 2.9% for As(V) are derived at the concentration level of 1.0 μg L −1. The practical applicability of the procedure has been demonstrated by analyzing a certified reference material of riverine water (SLRS-4), in addition to spiking recovery in a lake water sample matrix.

Granulocyte and Monocyte Adsorption Apheresis (GCAP) for Refractory Skin Diseases Caused by Activated Neutrophils and Psoriatic Arthritis: Evidence that GCAP Removes Mac‐1‐Expressing Neutrophils

In the present study, we have shown that granulocyte and monocyte adsorption apheresis (GCAP), an extracorporeal apheresis instrument whose column contains cellulose acetate (CA) beads, is useful for skin diseases attributable to activated granulocytes and psoriatic arthritis (PsA). We assessed the clinical effectiveness of GCAP and investigated the mechanisms underlying the adsorption of pathogenic granulocytes. The effect of GCAP was assessed in 14 patients with neutrophilic dermatoses and 16 with PsA. The mechanisms by which the instrument adsorbs activated granulocytes were investigated using an in vitro mini-column system that mimics the GCAP. Skin lesions and arthropathy improved in 22 of 29 patients (75.9%) and 14 of 18 (77.8%), respectively. Mac-1 (CD11b/CD18) expression on the peripheral neutrophils, increased compared with normal subjects, was reduced by GCAP. In the mini-column system, CA beads adsorbed 50% neutrophils; and adsorption was inhibited significantly by treating plasma with EDTA and blood cells with antihuman CD11b monoclonal antibody. GCAP was useful for treating neutrophilic dermatoses and PsA. GCAP adsorbs Mac-1-expressing neutrophils to the CA beads by the binding of complement component (iC3b) on CA beads and CD11b expressed on activated neutrophils.

Flow-injection microcolumn on-line preconcentration for the determination of chromium (VI)

A rapid and sensitive on-line preconcentration method for spectrophotometric determination of chromium (VI) in nature water is described. Preconcentration and determination are based on (i) the quantitative and fast adsorption of chromium (VI) on the high surface area nanometer-size TiO2 (anatase) powders, which prepared by a sol-gel method from hydrolysis of TiCl4 and (ii) the quantitative and reproducible elution of Cr (VI) by 2.0 mol·L−1 HCl. A mini-column system for preconcentration is developed, Cr(VI) on the mini-column is eluted and merged with a stream water and DPCB (1,5-diphenylcarbazide) as the chromogenic reagent. The proposed system permits throughputs of 6 sample h−1 (0.001 μg·mL−1 Cr(VI)) or 20 sample h−1 (0.1 μg·mL−1 Cr(VI)). The preconcentration factor is 55. The detection limit is 0.8 ng · mL−1 Cr(VI). The reproducibility is satisfactory with a relative standard deviation of less than 3.35% (0.01 μg·mL−1 Cr(VI),n=5).

Rapid isolation of double-stranded RNAs from entomopathogenic species of the fungus Paecilomyces using a commercial minicolumn system

A method is described for rapid extraction of double-stranded RNAs from entomopathogenic fungi. Lyophilised and ground mycelium is incubated with 6 M guanidine thiocyanate, centrifuged, and the cleared lysate applied to a QIAGEN silica-based mini-spin column. Following washing with 70% isopropanol, bound nucleic acids are eluted under low salt conditions and treated with DNAse I prior to analysis by non-denaturing agarose gel electrophoresis.

Flow injection-mini-column technique with ICP-AES detection for the isolation and preconcentration of the fast reactive aluminium fraction in waters

A flow injection mini-column system based on short reaction times with 8-hydroxyquinoline (oxine) with ICP-AES detection is described for the isolation and preconcentration of the “fast reactive” or “toxic” aluminium fraction in water samples. Using a 3 s reaction time with oxine (5 × 10–4 mol/l) at pH 5.0, the “fast reactive” aluminium fraction is shown not to include the non-toxic AlF2+ species at low F–: Al3+ molar ratios (0.3 : 1). The complexed aluminium is isolated in a stable and recoverable form on mini-columns of Amberlite XAD-2 (0.3 cm × 5.0 cm, resin particle size range 0.08 mm–0.16 mm)). The retained aluminium is recovered by “back-flushing” the analytical column with 1 mol/l HCl for final element specific detection by ICP-AES. Detection limits (after preconcentration) of 2 μg/l, a linear range of 0–500 μg/l, and possible preconcentration factors of up to 18 times are demonstrated with the present system. Implications for the possible solution of sample stability problems encountered with labile aluminium species analysis and the development of a “field sampling” technique are discussed, where the desired Al fraction is quantitatively retained in a stable form on mini-columns.

Field sampling technique for the ‘fast reactive’ aluminium fraction in waters using a flow injection mini-column system with inductively coupled plasma atomic emission spectrometric and inductively coupled plasma mass spectrometric detection

A novel field sampling strategy for the ‘fast reactive’ Al fraction in natural waters is described. The method is based on the isolation of the Al fraction which reacts with 8-hydroxyquinoline in under 3 s at pH 5.0. The resulting Al–8-hydroxyquinoline complex is sorbent-extracted from the sample stream onto mini-columns containing Amberlite XAD-2 non-ionic resin. The retained ‘fast reactive’ Al is then eluted with 1.0 mol l–1 HCl for on-line element-specific detection by inductively coupled plasma atomic emission spectrometry or inductively coupled plasma mass spectrometry (ICP-MS). Data from field sampling trials show good agreement with those for the toxic Al fraction as measured by an established high-performance liquid chromatography technique. Between-column relative standard deviations of 8% at the 100 µg l–1 level, a limit of detection of 1.8 µg l–1(ICP-MS) and ease of use are the main advantages of the proposed method. The mini-column technique has the potential for solving many of the sample storage problems associated with Al-speciation studies, particularly for the determination of the more toxic (or ‘fast reactive’) fraction.

An economic water-jacketed vacuum-enforced mini-column system for quick separation of double and single stranded DNA

A jacketed column system that can be applied in separation of double and single stranded DNA by hydroxylapatite chromatography is described. The construction of the column allows high flow rates and a short manipulation time. The system is simple, inexpensive, and easy to construct.

Effect of temperature on quantifying glycated (glycosylated) hemoglobin by cation-exchange chromatography.

As a consequence of nonideal chromatographic conditions, values for stable glycated hemoglobin (HbA1c) determined by cation-exchange chromatography in a commercial minicolumn system (y) or by "high-performance" liquid chromatography (x) differ markedly, yielding the regression line y = 0.82x + 0.6. With use of the protocol specified by the manufacturer, 20% of the HbA1c peak is not collected in the HbA1c fraction. Increasing the ionic strength of the eluting buffer by increasing the operating temperature to 28 degrees C increases the rate of elution from the minicolumn, making results of the two methods more closely comparable (y = 0.98x - 0.22). Because at a given pH the elution volume is determined primarily by the ionic strength, close limits on the composition of the eluting buffer are set by the temperature-dependence of its ionic strength. At a specified temperature and pH the position of a peak can be judged to within a volume of 1 mL if the conductivity of the eluent does not vary by more than +/- 0.05 mS.