A preconcentration method of arsenic(III) using ammonium pyrrolidinedithiocarbamate (APDC) and a finely divided anion-exchange resin is described for the determination by graphite-furnace atomic absorption spectrometry (GFAAS). The method is based on the extraction of the As(III)-APDC complex on the resin phase at pH 1.2, separation of the resin particles by filtration, dispersion of them in 1.0 ml of 0.1 mol l^-1 nitric acid containing 50 µg of nickel(II), and introduction of the suspension to a graphite furnace. The detection limit of arsenic in a 150-ml sample aliquot was 1.0 ng (6.7 ppt), based on three times the standard deviation of the blank. Total inorganic arsenic(III,V) was extracted similarly after the reduction of As(V) to As(III) with sodium thiosulfate. The interfering ions, such as Cu(II), Pb(II) and Sn(II), could be removed by extraction as the respective APDC complexes at pH 5, where neither As(III) nor As(V) were extracted. Arsenic(III) and total As(III,V) in the filtrate were then extracted at pH 1.2 in the absence or presence of sodium thiosulfate, respectively. The proposed method was applied to the determination of As(III) and total inorganic As(III,V) in hot spring water, seawater, river and tap water samples.

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A highly sensitive flow-injection spectrophotometric method has been developed for the determination of copper(II). It is based on the catalytic effect of copper(II) on the oxidative coupling reaction of 3-methyl-2-benzothiazolinone hydrazone with N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline to form a red compound (λ_max=525 nm) in the presence of hydrogen peroxide and pyridine as an activator. A higher sensitivity and sampling rate were achieved by adding ben-zyldimethyltetradecylammonium chloride (Zephiramine) as a surfactant to this reaction system. The calibration graph for copper(II) was linear over the range 0.005 - 0.75 ng cm^-3 at a rate of 40 samples h^-1. The relative standard deviations for ten determinations of 0.5 and 0.05 ng cm^-3 were 0.21 and 2.0%, respectively. The proposed method was successfully applied to the determination of copper in pepperbush.

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Peroxidase-like activity of anion-exchange resins modified with metal-tetrakis(sulfophenyl)porphine (M-TSPP_r, M=Fe³⁺, Co³⁺ and Mn³⁺) were evaluated in the flow injection analysis of hydrogen peroxide using a fluorescent dimmer-formation reaction from chromogens, such as tyramine. A column (2.1 mm×50 mm) packed with Fe³⁺-TSPP_r and a tube (0.8 mm×50 mm) packed with Mn³⁺-TSPP_r accelerated the dimmer-formation reaction, indicating that they exhibit peroxidase-like activity just like the peroxidase-immobilized-column in the flow injection system.

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A flow-injection system with spectrophotometric detection involving the in-line concentration was developed for lead determination in natural water samples. Lead concentration was carried out in a mini-column packed with novel stuff chitosan, and sorption occurred at pH=6 - 9. The amount of lead flowing through the chitosan was controlled by time, the CTAB surfactant was passed through a mini-column and a 0.2 M NaOH stream was used as the eluent. The eluted lead merged with 2,2′-dipyridyl-2-pyridylhydrazone (DPPH) reagent synthesised in the author’s lab and with a borate solution; complex formation took place at pH 11 - 12 inside a following reactor and monitoring was carried out at 468 nm. The detection limit was 0.13 µg l^-1 and, the sample throughput was 25 h^-1. The accuracy was assessed by running spiked river-water samples.

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Highly sensitive catalytic waves of OsO₄-bromate linear sweep voltammetric and square wave voltammetric techniques were proposed for the determination of ultratrace amounts of osmium. Under optimized conditions, the catalytic linear sweep voltammetric procedure gives excellent reproducibility and limits of detection and a linear dynamic range and gives a low signal for background, whereas the square wave voltammetric technique gives higher sensitivity, but the background signal was high and the linear dynamic range was narrower. The methods are suitable for the determination of osmium as osmium tetroxide as low as 1.5×10^-10 mol/l. The currents are linear for the concentration ranges of 0.10 - 210.0 ng/ml and 0.20 - 2.0 ng/ml for linear sweep voltammetry and square wave voltammetry, respectively.

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Self-assembled monolayers consisting of 3,3′-thiodipropionic acid and n-decyl mercaptan formed on gold electrodes have been studied for sensitive and selective determination of Cu²⁺ and Ag⁺ ions. The monolayers were found to provide both superior sensitivities and stabilities even in the presence of protein. Various factors, such as solution pH and concentration time, were optimized to obtain detection limits of 2×10^-9 M and 6×10^-8 M for Cu²⁺ and Ag⁺ ions at an S/N ratio of 2 by linear-sweep anodic stripping voltammetry. In the presence of albumin, the currents of the modified electrode, 1.75 times as great as those for the bare electrode, show a significant performance improvement over the previously reported cellulose coated electrode (1.13 times). One of the main advantages of using this electrode is an excellent antifouling activity against protein adsorption, coupled with highly selective metal-ion suppression/concentration capabilities, which can be attained without using a mercury film for preconcentration.

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The electrochemical behavior of biotin labeled with ferrocene was investigated at a perfluorosulfonated ionomer (Nafion^®) film-coated glassy carbon electrode (GCE). The electrode response of the labeled biotin (LB) was improved by about fifty-fold in sensitivity by using a Nafion^®-coated GCE, compared with a plain GCE. It was found by a solid-phase avidin-biotin binding assay that the labeling with ferrocene hardly affected the affinity of a biotin moiety in the labeled biotin to avidin. The avidin-biotin interaction was estimated from the change of the electrode response caused by the formation of a complex with avidin and the labeled biotin. Furthermore, the detection of biotin was performed by a competitive reaction between the labeled biotin and unlabeled biotin for the limited binding sites of avidin.

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A chemiluminescence biosensor combined with flow injection analysis has been developed for determining cholesterol. Cholesterol oxidase was immobilized onto amine-modified silica gel via glutaraldehyde activation and packed in a column. The analytical reagents, including luminol and ferricyanide, were electrostatically coimmobilized on an anion-exchange column. Cholesterol was sensed by the CL reaction between hydrogen peroxide released from the enzymatic reaction and luminol and ferricyanide, which were released from a column with immobilized reagents by elution. The calibration graph was linear over the range 5×10^-6 to 1×10^-4 g ml^-1 and the detection limit was 5×10^-6 g ml^-1. The pro-posed method has been successfully applied to the determination of cholesterol in human serum.

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Heme-undecapeptide (HUP, microperoxidase-11) labeled insulins, in which the Lys(3) amino group of HUP was cross-linked to one amino group of insulin (Gly(A1), Phe(B1) or Lys(B29)) via a disulfide linkage, were prepared. Lys(3)-[3-(2-pyridyldithio) propionoyl]-HUP (Lys(3)-PDP-HUP) was synthesized by the reaction of HUP with N-succinimidyl 3-(2-pyridyldithio)propionate and then conjugated with Gly(A1)-thioglycoloyl- (Gly(A1)-TG-), Phe(B1)-TG- or Lys(B29)-TG-insulin. Each TG-insulin was generated by deacetylation of Gly(A1)-acetylthioglycoloyl-(Gly(A1)-ATG-), Phe(B1)-ATG- or Lys(B29)-ATG-insulin, that was prepared by reacting native insulin, Phe(B1)-3,4,5,6-tetrahydrophthalyl-insulin (Phe(B1)-THP-insulin) or Gly(A1),Phe(B1)-(THP)₂-insulin with N-succinimidyl S-acetylthioacetate (SATA), respective-ly, followed by deprotection of THP, a reversible amino-protecting group. Both preparative reversed-phase and anion-exchange HPLC were used in the syntheses of PDP-HUP and the insulin derivatives (THP-, ATG- and HUP-insulin), respectively. The optimal reaction conditions for these syntheses were extensively studied using HPLC separation. In addition, immunoreactivity of Phe(B1)-HUP-insulin was manifested by using a solid-phase antibody method with chemiluminescence detection.

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The use of an aggregate of amphiphilic block copolymer 1, which consists of poly[(N-acetylimino)ethylene] and poly[(N-pentanoylimino) ethylene], for a pseudo-stationary phase in capillary electrophoresis has been examined. From gel-filtration chromatography, the aggregate from 1 (1-AG) was found to incorporate phenol. When the running solution contains 1-AG and sodium dodecyl sulfate (SDS), the electrophoretic mobility of phenols increases as the concentration of SDS is raised. In the absence of either 1-AG or SDS, the electrophoretic mobility becomes nearly zero. Thus, it is found that when 1-AG and SDS are added to the running solution, SDS is incorporated in 1-AG, giving negative charges on the 1-AG, which works as a pseudo-stationary phase. By the addition of 1-AG and a low concentration of SDS in the running solution, six phenols are separated and the migration order of phenols is basically dependent on their lipophilicities. However, the migration order of some phenols is different from that in the micellar electrokinetic chromatography (MEKC) using a high concentration of SDS.

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A direct chemiluminescent method for the determination of nucleic acids has been developed. The method is based on the enhancement of nucleic acids on the chemiluminescence light emission of the reaction between Ru(bipy)₃²⁺ and Ce(IV). Under the optimum conditions, the calibration graphs are linear over the ranges 7.0×10^-7 - 5.0×10^-5 g/ml for calf thymus DNA, 4.0×10^-7 - 5.0×10^-5 g/ml for fish sperm DNA and 5.0×10^-7 - 5.0×10^-5 g/ml for yeast RNA, respectively. The limits of detection are 1.8×10^-7 g/ml for calf thymus DNA, 1.1×10^-7 g/ml for fish sperm DNA and 1.2×10^-7 g/ml for yeast RNA, respectively. The final procedue allows the sucessful determination of calf thymus DNA, fish sperm DNA and yeast RNA in six synthetic samples. This method is simple, rapid and specific. A possible reaction mechanism is discussed.

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Various mimetic enzymes have been systematically studied concerning the color reaction for hydrogen peroxide oxidizing 4-aminoantipyrine (4-AAP) and N,N-diethylaniline (DEA) to yield a dye. The results have shown that β-CD-hemin is the best mimetic enzyme for peroxidase among those tested, and that DEA is a quite good substrate for β-CD-hemin. Meanwhile, the chromogenic reaction rate and sensitivity of 4-AAP-DEA-β-CD-hemin system for determination of hydrogen peroxide were investigated. Zero to 8.4×10^-5 M hydrogen peroxide was determined with good accuracy and reproducibility. The molar-absorption coefficient of the chromogenic compound for hydrogen peroxide determined was found to be 1.65×10⁴ mol l^-1 cm^-1.

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