Aminopropyl-controlled Pore Glass Research Articles

Flow injection on-line solid phase extraction for ultra-trace lead determination with hydride generation electrothermal atomic absorption spectrometry

The efficiencies of two types of chelating resins were evaluated and optimized for the extraction and determination of lead by flow injection graphite furnace and hydride generation atomic absorption spectrometry (FI-HG-ETAAS) in environmental water samples. The optimum analytical conditions were examined and the analytical features of each method were revealed and compared in terms of sensitivity, selectivity and accuracy. Lead determination by FI-HG-ETAAS using [1,5-bis(2-pyridyl)-3-sulphophenyl methylene] thiocarbonohydrazide immobilised on aminopropyl-controlled pore glass (PSTH-cpg, 55 nm) provides a detection limit of 3.0 ng l−1 lead with a relative standard deviation (R.S.D.) of 2.5% at the 50 ng l−1 level (n = 10) compared to silica gel funtionalized with methylthiosalicylate (TS-gel) which provides a detection limit of 82 ng l−1. Under the optimal experimental conditions, with the use of PSTH-cpg and a tungsten–rhodium coated graphite furnace, an enhancement factor of 20 was obtained with a sample consumption of 6.4 ml, while an enhancement factor of 3.5 was obtained with the use of TS-gel. Both methods have been applied to the determination of low levels of lead in environmental water samples. The results obtained show good agreement with certified or expected values.

Multi-element determination of Pt, Pd and Ir traces in environmental samples by ICP-MS after pre-concentration

An automated flow analysis method with on-line column pre-concentration/ inductively coupled plasma mass spectrometry (ICP-MS) was developed for the simultaneous determination of Pt, Pd and Ir in environmental samples. The system is based on retention of the analytes onto a column filled with 1,5-bis (2-pyridyl)-3-sulphophenyl methylene thiocarbonohydrazide immobilized on an aminopropyl-controlled pore glass (PSTH-cpg) placed in the injection valve of a simple flow manifold. The retained platinum group metals (PGMs) were subsequently eluted with a mixture of HNO3 and thiourea. The effects of chemicals and flow variables were investigated. The optimized operating conditions were: sample pH 3.2, sample flow rate: 1.1mLmin−1; eluent flow rate: 2.1mLmin−1 and eluent 0.03% m/v thiourea solution in 3.2% (v/v) HNO3. The detection limits obtained were 78.5ngL−1 for Pt, 55.5ngL−1 for Pd and 0.1ngL−1 for Ir. The enrichment factors were 18, 2.3 and 43 for Pt, Pd and Ir, respectively. The accuracy of the method was checked by the analysis of certified reference materials and by determining the analytes content in spiked environmental samples. Recovery was found to be in the range 93–107% in all cases.

Development of a solid phase extraction method for the multielement determination of trace metals in natural waters including sea-water by FI-ICP-MS

An automated solid phase extraction method by flow analysis on-line inductively coupled plasma mass spectrometry (FI-ICP-MS) for the determination of cobalt, chromium, nickel, cadmium, manganese, zinc, copper and lead in sea-water and other natural waters is described. The system is based on retention of the analytes onto a minicolumn packed with a chelating resin, 1,5-bis (2-pyridyl)-3-sulphophenyl methylene thiocarbonohydrazide immobilized on aminopropyl-controlled pore glass, placed in the injection valve of a simple flow manifold. The effects of chemical and flow variables were investigated and selected as a compromise between sensitivity and sampling frequency. So, the sample solutions (adjusted to pH 8.0±0.5) were passed through the column. After washing the column with water, the adsorbed metals were subsequently eluted into the plasma with 4% m/m nitric acid. Detection limits of the trace metals (180s sample loading time at a sample flow rate of 0.7mLmin−1; sampling frequency 8.6h−1) were 0.002μgL−1 for Co, 0.057μgL−1 for Cr, 0.117μgL−1 for Ni, 0.004μgL−1 for Cd, 0.210μgL−1 for Mn, 0.260μgL−1 for Zn, 0.030μgL−1 for Cu and 0.020μgL−1 for Pb, with enrichment factors between 2.2 and 6.8. The accuracy of the proposed method was checked with certified reference materials (CRMs) of sea-water SLEW 3, LGC6016 and CASS-5, river water SLRS-5 and fortified lake water TMDA-54.4. The results from the determination of these metals were in agreement with the certified values and recovery values ranged between 92.2 and 110.6%. The method was applied to the determination of these metal ions in sea-water samples collected in the Málaga Bay in order to realize a pilot study necessary to generate preliminary information on which to base a more detailed pollution study by heavy metals of the Bay.

Speciation of antimony(iii) and antimony(v) in seawater by flow injection solid phase extraction coupled with online hydride generation inductively coupled plasma mass spectrometry

An automated low pressure flow analysis method with online columns pre-concentration/hydride generation (HG) inductively coupled plasma mass spectrometry (ICP-MS) was developed for the simultaneous determination of Sb(III) and Sb(V) in aqueous environmental samples. For this purpose a chelating resin [1,5-bis(2-pyridyl)-3-sulfophenyl methylene] thiocarbonohydrazide immobilised on aminopropyl-controlled pore glass (550 A, PSTH-cpg) and an anion exchanger (Amberlite IRA-910) were used. A three column system was accordingly designed, using the two adsorbents in tandem, for the separation and preconcentration of the antimony species. The method was developed on the basis of selective sorption of Sb(III) on PSTH-cpg resin. After removal of Sb(III) by PSTH-cpg, Sb(V) was collected on Amberlite. The effects of chemicals and flow variables were investigated. The optimized operating conditions were: sample pH 8.4, a sample flow rate of 1.1 mL min−1, an eluent flow rate of 1.3 mL min−1, and an eluent 0.04% thiourea in 5% HNO3. The relative standard deviations (n = 10) were 4.6% for Sb(III) and 3.0% for Sb(V), demonstrating a good precision for the analysis. The enrichment factors for Sb(III) and Sb(V) were 5.5 and 3.9, respectively. Detection limits (2 min sample loading time) were 0.013 µg L−1 for Sb(III) and 0.021 µg L−1 for Sb(V).The accuracy of the proposed method was checked with two certified reference materials (CRMs): SLRS-5 River Water and TMDA-54.4 Fortified Lake Water. Values obtained were in accordance with those reported for the certified materials. Recovery was found to be in the range of 97–105% for a suite of seawater samples collected in Malaga, Spain.

Optimization of DNA Hybridization on Aminopropyl-Controlled Pore-Glass Particles: Detection of Non-Labeled Targets by PicoGreen Staining

A rapid and cost effective strategy was developed to detect the hybridization of DNA sequences, covalently attached to aminopropyl-controlled pore glass particles (ACPG) with non-labeled targets. The optimization of parameters, such as immobilization chemistry, hybridization time, and washing conditions, was first accomplished using a direct method based on the detection of fluorescein-labeled complementary targets. Under the optimized conditions, a methodology based on the post PicoGreen staining of the duplex molecules formed upon hybridization was demonstrated. This approach eliminates the requirements for pre-target labeling, while providing a rapid detection (less than 20 minutes) of specific complementary targets present in the solution.

Study of Two Different Immobilized Acid Proteases for Wine Application

This study compares the hydrolytic efficiency in model wine (pH 3.2 tartaric acid buffer with 12% v/v ethanol) of two immobilized enzymes. Food-grade acid proteases (from Aspergillus saitoi and stem bromelain) were covalently immobilized on two solid supports, aminopropyl controlled pore glass beads and Eupergit®C. The effects of pore diameter, immobilization procedure, and protein properties on the activity of biocatalysts were evaluated to determine the feasibility of immobilized proteases in wine. The results expressed in terms of yield of immobilized enzyme, enzyme activity, and kinetic parameters (Vmax and Km) demonstrated that the best performance of both proteases was when immobilized in Eupergit®C.

Enzymatic flow injection method for rapid determination of choline in urine with electrochemiluminescence detection

In order to determine trace choline in human urine, a flow injection analysis (FIA) system has been developed by coupling an enzyme reactor with an electrochemiluminescence (ECL) detector. The enzyme reactor is prepared by covalently immobilizing choline oxidase (ChOx) onto the aminopropyl-controlled pore glass beads, which are then carefully packed into a micro column. The enzyme reactor catalyzes the production of hydrogen peroxide that is in direct proportion to the concentration of choline. In this study, the enzymatically produced hydrogen peroxide was detected by an ECL detector positioned at the down stream of an enzyme reactor based on the luminol/H 2O 2 ECL system. Under the optimized condition, the enzymatic FIA/ECL provided high sensitivity for the determination of choline with the detection limit as low as 0.05 µM (absolute detection limit was at sub pmol level). The method was successfully applied in the determination of choline in the samples of human urine, and the analytical results were in good agreement with those obtained by using the microbore HPLC with an immobilized enzyme reactor-electrochemical detection system.

Quantitation of non-amplified genomic DNA by bead-based hybridization and template mediated extension coupled to alkaline phosphatase signal amplification

Klenow I polymerase activity was combined with solid phase DNA hybridization to detect non-amplified genomic DNA (gDNA) sequences from Escherichia coli. Aminopropyl-controlled pore glass surface-bound oligonucleotides were hybridized to fragmented gDNA. The template-mediated extension at the 3'-terminus of the immobilized probe was then promoted in the presence of Klenow I polymerase and digoxigenin-labeled nucleotides. Detection of the extended probes was accomplished with an anti-digoxigenin alkaline phosphatase conjugate protocol coupled to colorimetric or fluorescent detection. Using the colorimetric protocol, the proof-of-concept was established. The fluorescence-based methodology, on the other hand, provided the basis for a quantitative interpretation of the data, affording a detection limit of 5 pM gDNA.

Tyrosinase immobilized reactor as a fast tool for polyphenolic index of tea

A new approach for the determination of polyphenolic index of black and green teas has been developed employing a bioreactor with mushroom tyrosinase immobilized on aminopropyl-controlled pore glass (AMP-CPG). Initially a linear correlation ( r 2 = 0.997) of catechin content between the Folin-Ciocalteu (FC) and the enzymatic assays is obtained. Our method appears to be more specific and rapid. Successively the inhibition of the enzymatic oxidation of catechin by some tea components like gallic acid (GA), epigallocatechin (EGC), epigallocatechingallate (EGCG) and epicatechin (EC) is investigated. Finally when tea samples are analyzed by using the new method it appears that green tea is the strongest inhibitor followed by black tea and flavored tea. The level of polyphenols, which is correlated to the extent of inhibition, is reported as epigallocatechin equivalents and the results are compared with those obtained using the FC assay and the vanillin index. The differences in phenol content found by applying the three methods are discussed in terms of the different specificities of the analytical basis.

Determination of an Antioxidant Capacity Index by Immobilized Tyrosinase Bioreactor

Immobilized tyrosinase on aminopropyl-controlled pore glass support (AMP-CPG) was used to determine the antioxidant capacity index and the total phenol content in juices, integrators, infusions, jams, and drugs containing bilberry fruits. The method is based on the chromatographic determination of the decrease of total ingredients content, absorbing at 280 nm, by mushroom tyrosinase oxidation. In comparison with the widely used Folin-Ciocalteu method (FC), this enzymatic method appears to be more specific and rapid and as whole is not affected by interfering compounds such as citric acid and sugar. The results were also compared with those obtained by 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)/persulfate antioxidant assay, expressed as Trolox equivalents (TEAC) . Using all 17 samples, a good linear correlation (r(2) > 0.94) was observed between the enzyme assay and both the FC and TEAC assays. Poor correlation (r(2) < 0.4) with the other tested methods (pH-differential, vanillin index, and butanol-HCl assays) was evidenced. This shows that the enzymatic assay cannot be applied for a specific phenol class determination, but it is sensitive to the total phenolic content.

Mercury speciation in sea food by flow injection cold vapor atomic absorption spectrometry using selective solid phase extraction

An on-line inorganic and organomercury species separation, preconcentration and determination system consisting of cold vapor atomic absorption spectrometry (CV-AAS or CV-ETAAS) coupled to a flow injection (FI) method was studied. The inorganic mercury species was retained on a column (i.d., 3 mm; length 3 cm) packed to a height of 0.7 cm with a chelating resin aminopropyl-controlled pore glass (550 Å) functionalized with [1,5-bis (2 pyridyl)-3-sulphophenyl methylene thiocarbonohydrazyde] placed in the injection valve of a simple flow manifold. Methylmercury is not directly determined. Previous oxidation of the organomercurial species permitted the determination of total mercury. The separation of mercury species was obtained by the selective retention of inorganic mercury on the chelating resin. The difference between total and inorganic mercury determined the organomercury content in the sample. The inorganic mercury was removed on-line from the microcolumn with 6% (m/v) thiourea. The mercury cold vapor generation was performed on-line with 0.2% (m/v) sodium tethrahydroborate and 0.05% (m/v) sodium hydroxide as reducing solution. The determination was performed using CV-AAS and CV-ETAAS, both approaches have been used and compared for the speciation of mercury in sea food. A detection limit of 10 and 6 ng l −1 was achieved for CV-AAS and CV-ETAAS, respectively. The precision for 10 replicate determinations at the 1 μg l −1 Hg level was 3.5% relative standard deviation (R.S.D.), calculated from the peak heights obtained. Both approaches were validated with the use of two certified reference materials and by spiking experiments. By analyzing the two biological certified materials, it was evident that the difference between the total mercury and inorganic mercury corresponds to methylmercury. The concentrations obtained by both techniques were in agreement with the certified values or with differences of the certified values for total Hg 2+ and CH 3Hg +, according to the t-test for a 95% confidence level. It is amazing how this very simple method is able to provide very important information on mercury speciation.

Chemiluminescent bead-based hybridization assay for the detection of genomic DNA from E. coli in purified plasmid samples

A bead-based hybridization assay was developed for detection of traces of E. coli genomic DNA (gDNA) present in purified plasmid DNA (pDNA) samples. Standards of gDNA and pDNA samples were sheared by sonication and adsorbed onto aminopropyl controlled pore glass (CPG) particles (130 microm). A preliminary study was conducted to optimize the amount of DNA adsorbed on the particles. Results indicated that maximum attachment efficiency was obtained by adsorbing DNA for 2 h in 0.2 x SSC, pH 5.7. The DNA-bound particles were hybridized overnight with a 181-bp digoxigenin-labeled probe, specific for gDNA. Following a chemiluminescent detection protocol, signal intensities of the standards were plotted as a function of initial gDNA concentration. The calculated detection limit (LOD) was 1.4 pM of gDNA. The assay was able to detect gDNA in pure plasmid preparations at the 1% level even in the presence of 1,000-fold excess of noncomplementary target. Hybridization results were compared with a quantitative real-time PCR assay. Both methods afforded similar accurate results at the 95% confidence level.

Amperometric Enzyme Biosensor Based on the Controlled Pore Glass

A new amperometric biosensor based of glucose oxidase immobilized in aminopropyl-controlled pore glass (CPG) is reported. The glucose oxidase was linked to the CPG by covalent bonds with glutaraldehyde. The effect of analytical variables on the biosensor response was studied using experimental design methodology. Analytical properties such as linearity, detection limit, quantitation limit, range, and precision are reported. Interferences caused by compounds usually present in biological samples were eliminated.

Two Glutaraldehyde‐Immobilized Trypsin Preparations for Peptide Mapping by Capillary Zone Electrophoresis, Liquid Chromatography, and Mass Spectrometry

Trypsin was immobilized using glutaraldehyde either by covalent attachment to aminopropyl controlled pore glass (CPG) or by direct crosslinking without a carrier. As peptide mapping is a comparative method, reproducibility of the analytical separation techniques (liquid chromatography, HPLC, and capillary zone electrophoresis, CZE) and the proteolyses resulting from both enzyme preparations were evaluated. Elution time reproducibilities of 0.3 and 0.6% were found for HPLC and CZE maps, respectively. Proteolysis reproducibility was tested for each trypsin preparation and compared with solution phase proteolysis. Sequence coverages of ca 65% were obtained from matrix‐assisted laser desorption/ionization−time‐of‐flight (MALDI‐TOF) mass spectral mapping for the two solid phase preparations.

Comparison of Dansylated Aminopropyl Controlled Pore Glass Solvated by Molecular and Ionic Liquids

We compare how (i) four ionic liquids (ILs) (1-butyl-3-methylimidazolium tetrafluoroborate ([C4mim][BF4]), 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([C4mim][Tf2N]), 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide ([C4mpy][Tf2N]), and trihexyltetradecylphosphonium bis(trifluoromethylsulfonyl)imide ([P(C6)3C14][Tf2N])) and (ii) two conventional molecular liquids (methanol and 1-octanol) solvate/wet luminescent organic moieties that are covalently attached to the surface of silica controlled pore glass (CPG). A series of aminopropyl CPG particles that have been covalently tagged with the solvatochromic fluorescent probe group dansyl were used in this study. The results demonstrate that ILs solvate/wet the silica surface differently in comparison to molecular liquids (MLs). Specifically, when comparing ILs and MLs that appear to solvate the free probe, dansylpropylsulfonamide (DPSA), equally in solution, we find that ILs do not solvate/wet the silica surfaces as well as the corresponding MLs. The cation component in these ILs is the significant factor in how the ILs solvate/wet silica surfaces. Solvation/wetting of surface-bound species at a silica surface depends on the cation size. Chlorosilane end-capping of the surface silanol and amine residues attenuates the cation's affects.

Phenols removal by immobilized tyrosinase reactor in on-line high performance liquid chromatography

The development of an immobilized enzyme reactor (IMER) based on tyrosinase immobilized on aminopropyl-controlled pore glass (AP-CPG) for the removal of phenols from model aqueous solutions was reported. To elucidate the influence of the substrate nature, the apparent ( V ′ max , K ′ m ) and the inherent ( V max, K m) Michaelis–Menten constants were determined by Lineweaver–Burk method and the external diffusional contributions on measured enzyme activities were removed by a graphical method. The dephenolization process was realized by recycling the phenol solutions through the bioreactor connected to a chitosan trap in order to remove the colored quinone-type products of the tyrosinase reactions. The results indicated that a complete removal of phenol derivatives in the range of 150–300 min, with the exception of 60% removal for phenol reached in 400 min, was obtained. The observed sequence: cresol > 4-methylcathecol > catechol > 4-Cl-phenol ≫ phenol was in accordance to the V ′ max / K ′ m values.

Lead ultra-trace on-line preconcentration and determination using selective solid phase extraction and electrothermal atomic absorption spectrometry: applications in seawaters and biological samples

In this work, a new chelating resin [1,5-bis (2-pyridyl)-3-sulphophenyl methylene] thiocarbonohydrazide immobilised on aminopropyl-controlled pore glass (550 A; PSTH-cpg) was synthesised and packed in a microcolumn which replaced the sample tip of the autosampler arm. The system was applied to the preconcentration of lead. When microliters of 10% HNO3, which acts as elution agent, pass through the microcolumn, the preconcentrated Pb(II) is eluted and directly deposited in a tungsten-rhodium coated graphite tube. With the use of the separation and preconcentration step and the permanent modifiers, the analytical characteristics of the technique were improved. The proposed method has a linear calibration range from 0.012 to 10 ng ml(-1) of lead. At a sample frequency of 36 h(-1) with a 90 s preconcentration time, the enrichment factor was 20.5, the detection and determination limits were 0.012 and 0.14 ng ml(-1), respectively and the precision, expressed as relative standard deviation, was 3.2% (at 1 ng ml(-1)). Results from the determination of Pb in biological certified reference materials were in agreement with the certified values. Seawaters and other biological samples were analysed too.

Immobilized tyrosinase reactor for on-line HPLC application: Development and characterization

Mushroom tyrosinase was covalently bonded with glutaraldehyde, as activating agent, to aminopropyl-controlled pore glass support by “in situ” immobilization technique. The Schiff's base double bond reduction with sodium cyanoborohydride was made as innovation. Catalytic activity and stability of the chromatographic reactor were evaluated using d,l-3,4-dihydroxyphenylalanine as substrate. The tyrosinase-IMER was characterized by investigation of various parameters influencing the enzymatic activity (pH, temperature, ionic strength and organic solvents). In addition kinetic measurements showed that, by removal of the external diffusional limitation, the enzyme selectivity towards substrate was improved whereas the activity was comparable with respect to that of free enzyme.

Development of an on-line immobilized-enzyme reversed-phase HPLC method for protein digestion and peptide separation

This paper describes use of a novel glass bead-based immobilized-enzyme micro column for simple and swift on-line protein digestion then peptide separation by reversed-phase HPLC. The inexpensive and easily made immobilized-enzyme micro column was prepared from aminopropyl controlled-pore glass that was reacted first with glutaraldehyde then with trypsin in the presence of phosphate buffer. Tryptic digestion of bovine serum albumin (BSA) was achieved simply by passing pretreated protein solution through the laboratory made immobilized-trypsin column; the tryptic fragments were then separated by reversed-phase HPLC. The peptide separation was found to be identical to separation of a sample which had undergone conventional enzymatic protein digestion in solution. Digestion of BSA by the immobilized-trypsin column decreased with increasing flow rate of the solution through the column, and 1.0 muL min(-1) was found to be the optimum flow rate for on-line protein digestion with our system. It was also found that the sample required pretreatment with urea before injection, because of a change in the properties of the protein in the presence of urea, and the immobilized-trypsin column lost its function in the presence of acetonitrile. This on-line proteomics system enables simple and rapid protein digestion and was successfully applied to partially micro two-dimensional (2D) chromatographic separation of proteins.

DEVELOPMENT OF A BIOSENSOR FOR MEASUREMENT OF DIACETYL IN BEER

A selective and sensitive on-line monitoring system for diacetyl, based on a biosensor, was developed forapplication in the brewing industry. The biosensor consists of an enzyme reactor coupled with either amperometric orphotometric detection in a flow injection analysis (FIA) design. The reactor was packed with the enzyme diacetyl reductase(EC 1.1.1.5) immobilized onto aminopropyl controlled-pore glass (CPG) beads. An amperometric glassy carbon electrode,chemically modified using the organic dye toluidine blue (TB), was able to monitor the amount of coenzyme NADPH oxidizedin the reactor. The detection limit of the biosensor was 5.8 10-6 M of diacetyl with an error of 14%. An alternative opticaldetection scheme, based on absorbance at 340 nm, was also evaluated. This resulted in lowering the biosensors detectionlimit of diacetyl to 2.3 10-7 M with a 4% error at 5.8 10-6 M. The immobilized enzyme reactor columns were stable for45 days and gave consistent data for at least 30 diacetyl measurements on laboratory beer before there occurred a significantdecrease in enzyme activity.