Chemical speciation has become an important and challenging area in the geochemical, toxicological and environmental fields. Metal-humic complexes are typical and important species because humic and fulvic acids dominate naturally occurring organic matter in fresh water. This article describes the separation and determination of metal-humic complexes, with information about physicochemical forms of the complexes. Typical preconcentration techniques for characterizing aquatic humic substances are also reviewed briefly.

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Methodologies have been developed for the efficient extraction and fractionation of organic components present inambient air particulate matter. A protocol of ultrasonic solvent extraction, solvent exchange, and solid-phase extraction (SPE) results in five organic fractions which account for more than 95% of the mass of the dichloromethane (DCM) extractable organic components. These fractionated samples are amenable to subsequent bioassay studies and identification of potentially toxic compounds. A National Institute of Standards and Technology (NIST) ambient "Air Particles" sample (SRM 1649) was used to help validate this procedure. The unoxidized lubricating oil, polymeric components (Mw/Mn=4700/1300) and hydrated inorganic salts were effectively separated from the polar organic compound fractions. The recovery of mutagenic activity for the fractionation procedure was 72 and 78% (with and without S9 activation enzymes) using a TA98 Salmonella typhimurium microsuspension bioassay. Further analysis shows that the chemical integrity of the extract is maintained using this fractionation procedure.

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A metal-binding metallothionein-like protein in a cyanobacterium, Anacystis nidulans R-2, has been spectated by high- performance gel permeation liquid chromatography combined with inductively coupled plasma mass spectrometry (HPLC-ICP/MS). Cadmium-stressed culturing induced a characteristic water-soluble protein in the cyanobacterium. The protein had an apparent molecular weight of ca. 10000, and contained predominantly cadmium as well as smaller amounts of copper and zinc. The cysteine residues in the protein were quantitated on ²⁰²Hg chromatograms after modifying the sulfhydryl groups with p-chloromercuribenzoate. The present findings demonstrate that the HPLC- ICP/MS technique has high potential for the trace level speciation of heavy metal-binding proteins of environmental interest in living organisms.

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The applicability of isotope dilution-inductively coupled plasma mass spectrometry (ID-ICP-MS) was examined for Th in biological samples. A naturally occurring isotope of Th (Th-230) was used as the spiking isotope (called "tracer" in the text). The concentration of Th-230 in the final sample solution was about 50-60pg/ml; an isotope ratio of 232/230 could be measured with a relative standard deviation of less than 2%. The error magnification depended on the amount of Th-232 being concomitant with the Th-230. Though it was shown that one ng of Th-232 could be determined with reasonable precision with a tracer of the present purity, more care should be taken to reduce any source of systematic error.

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We developed a chemiluminescent (CL) assay for polyadenylic acid (poly A) based on the enzymatic cycling reaction following the conversion to ATP by pyruvate kinase/polynucleotide phosphoryrase enzyme reaction. Poly A was also measured with the bioluminescent (BL) assay using luciferase from fireflyPhotinus pyralis. The detection limits of poly A by CL and BL assay were 10amol and 2amol, respectively. We prepared the biotinylated poly A using biotin hydrazide and glutaraldehyde as a bifunctional coupling reagent to apply the poly A assay to a general avidin-biotin reaction system. When the CL assay and the BL assay of biotinylated poly A are applied to the DNA probe assay of λ- phage DNA and the immunoassay of thyroid-stimulating hormone (TSH) using avidin-biotin reaction as detection system, 500pg assay^-1 well of λ-phage DNA and 3μU ml^-1 of TSH were determined.

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Histidine oligopeptides which differ by one histidyl residue in length up to a total of 20 amino acid residues were separated by high-performance liquid chromatography on a commercially available strong cation-exchange column (TSK gel SP- 25W) using isocratic elution with a NaCl solution.

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The technique described here improves the purity determination of organic compounds through the use of the differential scanning calorimetry (DSC). The dispersion in the slope of a DSC curve has been observed to have great impact on the measurement results. The causes of this dispersion are the bulkiness and high thermal resistance of organic compounds. The sampling method is therefore directly related to the characteristics of the generated DSC curve. Pressing the sample onto the bottom of a sealed sample container improves the reproducibility of the slope. Sample was pressurized at 100kgf for one minute by using a correction jig. The slope of the DSC curve representing a pure sample was obtained from the thermal conductivity of the sample. This value was used for calibration during computation of the sample purity. When conducted in this manner, the determination of purity based on the DSC method yields values with a maximum error of 0.1mol% for samples with a minimum purity of 99mol%.

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Second-derivative spectrophotometry was applied to determine the binding constant (K) between chlorpromazine (CPZ) and β-cyclodextrin (CD) in aqueous solutions. An iterative calculation method which included a calculation of the free- ligand concentration was examined; two kinds of data were used in the calculation. One was the derivative intensity difference of CPZ at λmax of CPZ between, before and after the addition of CD; the other was an increase in the derivative intensity induced by the CD addition at the zero-crossing point of the second-derivative spectrum of CPZ. Although both data gave similar K values, the latter data were highly scattered. The reason was considered to be that the small uncertainty in the wavelength reproducibility of the spectrophotometer would severely affect the derivative intensities at the zero-crossing point. The derivative intensity changes of CPZ induced by binding to CD were well reproduced by a calculation using the obtained K value. The results are useful for applications of derivative spectrophotometry to determine the binding constants between drugs and large molecules.

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A simple spectrophotometric method is introduced for the determination of carbimazole in drug formulation. The method requires the use of 16mg ml^-1 potassium dichromate, 0.5M sulfuric acid and heating at 90°C for 25min. The method is easily applied to the determination of carbimazole in tablet formulations. The accuracy of the method was tested by a statistical comparison with the British Pharmacopeia official method.

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A micellar bile salt-coated stationary phase was used for separating alkali metal ions; copper(II) was employed as a lightabsorbing mobile phase for indirect UV detection at 200nm. Lithium, sodium, potassium, rubidium and cesium ions could be separated by the present system, though ammonium ions could not be separated from potassium ions. The detection limits of the analyte ions at a signal-to-noise ratio of 2 were 0.88pmol, or 44μM per injection (0.02μl). The present system was applied to the determination of potassium and sodium ions in beverages.

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The ion chromatographic separation of W(VI) and Mo(VI) oxoanions was investigated by changing the eluents. Although W(VI) and Mo(VI) oxoanions were eluted with alkaline eluents over a pH range from 9 to 11, the separation factors between these oxoanions were not remarkably different, since W(VI) and Mo(VI) oxoanions were eluted as monomeric anions. On the other hand, the elutions of W(VI) and Mo(VI) oxoanions and the separation factors between these oxoanions were remarkably influenced within the pH range from 3 to 8. In particular, citric acid among α-hydroxy carboxylic acids was the most effective eluent for separating W(VI) and Mo(VI) oxoanions. In the use of eluents without α-hydroxyl groups, W(VI) and Mo(VI) oxoanions were strongly retained, due to the formation of W(VI) and Mo(VI) isopolyanions. The composition of W(VI)-citrate complex was 1:1.

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2-Hydroxy-alkylsubstituted azorenes were synthesized to examine the extraction behavior of silver(I), mercury(II), copper(II), nickel(II), cobalt(II) and iron(III) from 0.1mol/dm³ of aqueous ammonium nitrate solution. The distribution ratio of these metals was determined as a function of pH. The order of selectivity is the following:Fe(III)>Ag(I)>Cu(II)>Ni(II)>Co(II). Mercury(II) was not extracted with the present extractants. A quantitative study on the extraction of copper(II) was carried out to elucidate the extraction stoichiometry and extraction equilibrium constant by examining the concentration effects of hydrogen ion, extractant and ammonium nitrate on the distribution ratio of copper ion. Copper(II) was found to be extracted according to the following chelating formation reaction: Cu²⁺+2HR_??_CuR₂+2H⁺; K_cu. The equilibrium constant of this reaction was evaluated as K_cu=1.6×10^-8.

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Spectrophotometric and calorimetric studies of the complexation of iron(III) with thiocyanate ions have been performed in an aqueous solution containing 1mol dm^-3 NaClO₄ or NH₄ClO₄ as a constant ionic medium at 25°C. Spectrophotometric titration data were well explained in terms of the formation of [Fe(SCN)]²⁺, [Fe(SCN)₂]⁺ and [Fe(SCN)₃], and their formation constants and individual electronic spectra were determined. The [Fe(SCN)]²⁺, [Fe(SCN)₂]⁺ and [Fe(SCN)₃] complexes exhibit absorption maxima at 450-500nm. Enthalpies and entropies for the formation of the [Fe(SCN)]²⁺, [Fe(SCN)₂]⁺ and [Fe(SCN)₃] complexes were also determined from calorimetric titration data. On the basis of these thermodynamic quantities, it is postulated that the [Fe(SCN)]²⁺, [Fe(SCN)₂]⁺ and [Fe(SCN)₃] complexes have a six-coordinate octahedral structure.

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