An-Lac Nguyen

Separation and determination of polycyclic aromatic hydrocarbons by solid phase microextraction/cyclodextrin-modified capillary electrophoresis

A sensitive method for the determination of polycyclic aromatic hydrocarbons (PAHs) by solid phase microextraction coupled with cyclodextrin (CD)-modified capillary electrophoresis (CE) using UV detection has been developed. A glass fiber was prepared and used for absorbing 16 EPA priority PAHs from diluted samples until equilibrium was reached. After the glass fiber was connected to a separation capillary via an adapter, the absorbed analytes were directly released into the CE buffer stream, and electrophoretic separation was effected using a 50 mM borate, pH 9.2, buffer containing 35 mM sulfobutyloxy-β-CD, 10 mM methyl-β-CD, and 4 mM α-CD. Separation was effected since neutral PAHs differentially partitioned between the neutral and charged CD phases. Under 30 kV applied potential, separation was achieved in less than 15 min with high resolution and number of theoretical plates. Pyrene as low as 8 ppb was detected, while the highest limit of detection was 75 ppb for acenaphthene. Very satisfactory reproducibility with respect to migration time and peak area was obtained for repetitions using the same separation capillary and adapter, where only the extraction fiber was discarded after each analysis.

Development of Electrokinetic Capillary Electrophoresis Equipped with Amperometric Detection for Analysis of Explosive Compounds

Cyclic voltammograms of trinitrotoluene (TNT) and other related explosive compounds obtained by using glassy carbon, platinum, nickel, gold, and silver electrodes revealed the applicability of gold and silver in capillary electrophoresis (CE) amperometric detection. The selected electrode, gold or silver, was inserted into a specially designed detection cell that was easily adapted to a commercial CE apparatus. The electrochemical reduction of TNT, octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and 10 other explosives could be reliably monitored at −700 mV vs Ag/AgCl. CE analyses were performed with a borate buffer (15 mM, pH 8.7) containing 25 mM sodium dodecyl sulfate (SDS) to attain baseline resolution of the selected compounds. A bimetal electrode, prepared by depositing silver on gold, offered a superior performance by exploiting the sensitivity of gold while suppressing its response toward acetonitrile to achieve a 10-fold lower detection limit...

Determination of explosives in soil and ground water by liquid chromatography–amperometric detection

Electrochemical reduction of trinitrotoluene (TNT) and several nitroaromatics has been exploited toward the development of an amperometric detector for liquid chromatography (LC). Up to a ten-fold increase in sensitivity was accomplished for the explosives using amperometric detection instead of conventional UV measurement. A working glassy carbon electrode (poised at -0.80 V vs. Ag/AgCl) offered a detection limit of 9, 44 and 550 nM for trinitrobenzene, TNT and 1,4-dinitrobenzene, respectively. Separation of eleven TNT-related compounds in a mixture was achieved within 15 min using a C18 column and a mobile phase consisting of acetonitrile-50 mM phosphate buffer pH 5 (1:2, v/v) and 18 mM sodium dodecylsulfate. The LC-amperometric detection system was applicable for analyzing soil extracts and ground water and the results obtained agreed well with that of the US Environmental Protection Agency recommended procedure. Extension to analysis of HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) and RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) was accomplished with a silver working electrode instead of a glassy carbon electrode installed in a thin channel cell.

Achiral selectivity in cyclodextrin-modified capillary electrophoresis

Torus-shaped, circular, and hydrophilic cyclodextrins (CD) have been frequently used in capillary electrophoresis (CE) as buffer modifiers to effect chiral separation of enantiomers of drugs and specialty chemicals. Although less common, both neutral and charged cyclodextrins have also been exploited in CE to optimize the achiral separations of peptides, proteins, small molecules and a variety of positional isomers. Nonionic CDs are only useful for separations of net charged analytes through judicious partitioning of such guest molecules into their hydrophobic cavity of the former. However, they can be used with a surfactant for an effective resolution of neutral solutes as a result of a differential partitioning of such solutes in the micellar and the cyclodextrin-modified buffer phase. Ionic cyclodextrins, particularly, negatively charged derivatives with their own electrophoretic mobilities, increase the separation window and enable better resolution of analytes which weakly complex with or are poorly differentiated by neutral cyclodextrins.

Utilization of TiO2 deposited on glass plates for removal of metals from aqueous wastes

Glass plates coated with TiO2 were used in a photocatalytic process to collect mercury, lead, copper and cadmium from aqueous solutions containing individual metals and mixtures. Stripping voltammetry, verified to achieve 1-10 ppb detection limits, was used to show that individual metals at concentrations of 1000 to 5200 ppb were reduced to undetectable levels in 3 to 55 min. Capillary electrophoresis (CE) with 8-hydroxyquinoline-5-sulfonic acid as complexing agent was used when appropriate, since it could quantitate all four metals under study in one run although it was less sensitive. It was demonstrated that 100 mL solutions containing 10 ppm of each of the four metals could be treated with a 10 cm2 TiO2-coated plate to leave undetectable metal concentrations in one hour. Stripping voltammetry using carbon electrodes coated with mercury films was estimated to generate daily about 1.1 L of aqueous waste containing 0.1 ppm of each metal. The results indicate the feasibility of assembling an apparatus capable of treating the waste generated by stripping voltammetry to render the latter suitable for routine on-site analyses without environmental concern. Data were also obtained to show the effectiveness in treating silver containing solutions, indicating suitability of the photocatalytic process in treating photographic processing wastes.

Studies on the application of a newly synthesized polymer for trypsin purification

A water soluble ligand bound polymer has been synthesized for the purpose of purification and stabilization of trypsin, an easily autodigestible enzyme. The affinity polymer was formed by copolymerizing N-acryloyl-m-aminobenzamidine and acrylamide in the absence of oxygen. The binding efficiency of trypsin to this polymer was dependent upon the ratio of acrylamide to the monomer N-acryloyl-m-aminobenzamidine used in the polymer synthesis. The amount of acrylamide present in the polymer solution also greatly affected the trypsin binding efficiency. The total binding capacity of trypsin molecules to ligand molecules approached the theoretical value of 120 : 1 which was considerably higher than that of insoluble gel matrices. Bound trypsin could be easily eluted by the trypsin inhibitor arginine in comparison to acid, salt and benzamidine. At low temperatures, the polymer solution was very stable and retained its high capacity for trypsin binding over a long period of time. The polymer could also be reused with trypsin binding close to 90% for three consecutive runs. This study provides a sufficient background for the development of a continuous and one-step process for trypsin purification.

Application of polarography for monitoring the fish postmortem metabolite transformation

Abstract Based on the principle of polarography, a new technique has been developed for monitoring the degradation of adenosine triphosphate (ATP) in fish muscles. The technique consisted of a polarographic electrode attached to a temperature-controlled reaction chamber where the metabolites in fish muscles were enzymatically degraded to uric acid and hydrogen peroxide. If the electrode was polarized at +0.7 volts with respect to a silver cathode and a platinum anode, the electrode response to uric acid and hydrogen peroxide was found to be of an additive manner. Consequently, the newly proposed technique offered a simple and rapid determination of inosine, hypoxanthine, and inosine monophosphate in fish homogenates. Excellent agreement was observed between the results obtained by the new technique and those of a new commercial freshness meter, where oxygen consumption was followed during the enzymic reactions.

The development and application of a new affinity partitioning system for enzyme isolation and purification.

A reactive water-soluble polymer was synthesized by copolymerizing N-isopropylacrylamide and glycidyl acrylate. The reactive polymer could react with the amino groups of enzymes/proteins or other ligands to form an affinity polymer. As a model, the reactive polymer was allowed to react with paraaminobenzamidine, a strong trypsin inhibitor. The affinity polymer could easily form an aqueous two-phase system with either dextran or pullulan, and the phase diagram was compared favorably to that of the well-known polyethylene glycol-dextran system. Once trypsin was attracted to the affinity polymer dominant phase, the enzyme could be dissociated from the polymer at low pH. Owing to the N-isopropylacrylamide units, the affinity polymer could be isolated from the solution by precipitation at a low level of ammonium sulfate. The enzyme recovery was always greater than 50%, and the affinity polymer could be reused in several cycles of affinity partitioning and recovery.

Applications of pullulan in aqueous two-phase systems for enzyme production, purification and utilization

SummaryPullulan, a microbial polysaccharide, was employed with polyethylene glycol (PEG) to form an aqueous two-phase system. The phase diagram of the PEG-Pullulan systems as well as the partition of enzymes depended strongly on the molecular weight of PEG. This behavior provided a basis for the separation of cellulase from β-galactosidase. The extractive hydrolysis of lactose, using a commercial preparation of β-galactosidase, could also be performed in a PEG-Pullulan system (16% PEG 1400, 14% Pullulan). The enzyme was effectively contained in the bottom phase while a high percentage of the glucose produced could be removed in the top phase. The enzyme activity was preserved very well after several repeated hydrolyses.Furthermore, it was demonstrated that the PEG-Pullulan system was capable of retaining Trichoderma reesei in the bottom phase, allowing the production of cellulase which could be intermittently removed in the top phase. Such a system permitted a semicontinuous production scheme although the enzyme production was observed to decrease after five cycles.In view of the low cost and low viscosity of pullulan, this polysaccharide could be considered a suitable replacement for dextran, a component widely used in aqueous two-phase formulation.

Nonaqueous capillary electrophoresis equipped with amperometric detection for analysis of chlorinated phenolic compounds.

Nonaqueous capillary electrophoresis (NACE) equipped with amperometric detection has been developed for separation and detection of an 11-member model mixture of chlorinated phenolic compounds. With triacetyl-beta-cyclodextrin (TACD) as a novel selectivity selector, acetonitrile proved to be an excellent solvent for this water-insoluble cyclodextrin derivative. Resolution of the analytes was achieved by using an optimized acetonitrile medium consisting of 500 mM acetic acid, 10 mM sodium acetate, 12 mM TACD and 50 mM tetrabutylammonium perchlorate. Separation of analytes was attributed to differential electrostatic and/or inductive interactions of the analytes with the TACD/TBA+ complex and charged tetrabutylammonium phases. A simple end-column amperometric detector (Pt vs. Ag/AgCl, poised at +1.6 V) in conjunction with NACE was used to analyze chlorophenols. Amperometric detection of such target compounds in acetonitrile-based media offers high sensitivity and alleviates electrode fouling compared to aqueous buffers. The detection limits obtained, ranging from 30 nM to 500 nM, are 3-8-fold lower than those obtained with aqueous buffers.