Steroidal carbocations have been demonstrated to be chromophore and/or fluorophore in the reactions of steroids such as estrogens and testosterone with strong acids. In this report, the behavior of unsaturated ketosteroids in sulfuric acid was investigated in order to elucidate the mechanism of the reaction of a strong acid with testosterone and corticosteroids, which have α, β-unsaturated carbonyl function. Dissolution of androst-4-en-3-one(I), androsta-4, 6-dien-3-one (II) or 17, 17-dimethyl-18-norandrosta-4, 6, 8(14)-trien-3-one(IIIb)into 80 % H₂SO₄ gave rise to a maximum absorption at 294 nm, 360 nm and 484 nm respectively. Measurement of NMR spectra of the solutions confirmed that these absorptions were due to hydroxyalkenyl cation(IV), hydroxyalkadienyl cation(V) and hydroxyalkatrienyl cation(VIb) formed from the corresponding unsaturated ketosteroids in 80% H₂SO₄ by protonation of the oxygen atom of carbonyl function. The apparent pK_BH⁺ of these cations had a tendency to increase with extension of the conjugate system. Hydrogen-deuterium exchange of vinyl protons of VIb was observed in 80% D₂SO₄. On the other hand, the vinyl protons of IV and V did not exchange even in 97% D₂SO₄. Although hydroxyal-katrienyl cation(VIa) was also produced from 17β-methyl-18-norandrosta-4, 6, 8 (14)-trien-3-one (IIIa) in 80% H₂SO₄, the cation(VIa) was unstable in the acid and deconjugation of hydroxyalkatrienyl cation moiety of VIa was gradually induced. These differences in the reactivity of the cations were intimately correlated with the order of their apparent pK_BH⁺ values.

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A kinetic method for the determination of gallium(III) was studied on a basis of the substitution of the copper(II) complexes of ethylenediamine-N, N, N', N'-tetraacetic acid (edta) for gallium(III). Portions each 5 ml of 1 M sodium perchlorate and 5.0×10^-3M copper(II)-edta adjusted the pH to 3.0 were taken into a 50 ml measuring flask, added 25 ml of a sample solution containing (0.357.0) μg/ml of gallium(III) adjusted the pH to 2.3, and diluted to 50 ml with water. The reaction was followed by the measurement of the change in absorbance at 240 nm. The amount of gallium was found from the calibration curve of (A₀-A_∞) against gallium(III) concentration, where A₀ and A_∞, denote the absorbances at times 0 and infinity. The following amounts (μg/ml) of diverse ions did not cause interferences with the determination of 1.39 μg/ml of gallium(III): Bi(III) 42, In(III) 12, Fe(III) 1, Hg(II) 40, Cu(II) 1271, Ni(II) 12, Pb(II) 4, Cd(II) 450, Co(II) 236, Zn(II) 262, Al(III) 54, Mn(II) 2198, Be(II) 901, Mg(II) 2431, ZrO(II) 2, Sb(III) 4, Cl^- 177000, SO₄^2- 10800, and NO₃^- 1240. This method was successfully applied to the determination of gallium in bauxite after separating gallium(III) as its chlorocomplex by the extraction with methyl isobutyl ketone.

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A method for the determination of copper in rabbit plasma, red cells and ethanol-chloroform (EtOH-CHCl₃)-treated fraction of red cells, was studied by flameless atomic absorption spectrometry (A.A.S.). A large portion of copper in the last sample was shown to arise from superoxide dismutase (SOD). Plasma and red cells were diluted 20 times with 0.05 N NH₄OH-1 mM EDTA aq. solution, while EtOH-CHCl₃-treated fraction 6 times, and these solutions were submitted to flameless A.A.S. Analytical conditions by flameless A.A.S. were as follows: Dry, 15 A-30 s; Ash, 60A-40 s; Atomize, 250 A-3 s. Ten μl of each solution was injected into a graphite tube. Under these conditions, calibration curves for standard copper solutions were linear in the range of 0100μg/l. The recoveries obtained by addition of standard copper solutions were (90106)%, (96110)% and (93106)% for rabbit plasma, red cells and EtOH-CHCl₃-treated fraction, respectively. The coefficients of variation (n=6) for each sample were 2.3%, 2.9 % and 3.2%. The contents of copper in rabbit plasma, red cells and EtOH-CHCl₃-treated fraction were (502±94) μg/l, (572±109) μg/l and (374±36) μg/l, respectively. Copper concentrations in EtOH-CHCl₃-treated fractions obtained from red cells of some rabbits were relatively constant.

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Fluorescent reaction of tin(IV) with 3-hydroxyflavone and its twelve hydroxyl and methoxyl derivatives in acid solution was studied. These reagents react with tin(IV) to form the soluble complexes, several of which show intense fluorescence in phosphoric acid and medium fluorescence in sulfuric or perchloric acid. All complex does not fluoresce in hydrochloric acid. The insertion of hydroxyl or methoxyl group into 2'- or 3'-position and hydroxyl group into 4'- or 5-position of 3-hydroxyflavone decreases the fluorescence intensity, whereas the insertion of methoxyl group into 4'-position and hydroxyl or methoxyl group into 7-position increases the intensity. The recommended acid (concentration) and reagent for the fluorometric determination of tin(IV) are phosphoric acid(3 M) and 3, 7-dihydroxyflavone, respectively, and the complex has a maximum excitation at 398 nm and emission at 446 nm. Analytical procedure is as follows: To a solution containing (0.020.5) μg of tin(IV), 3 ml of methanolic 1×10^-3 M solution of reagent, 9.5 ml of methanol (the final content is 50% v/v) and 5 ml of 85% phosphoric acid are added, and the mixture is diluted to 25 ml with water. The fluorescence intensity is measured with an excitation wavelength of 405 nm (mercury line), using a secondary filter passing above ca. 430 nm. The calibration line is linear in the range mentioned above when an aqueous solution of 2 μg/ml sodium fluorescein is used as a reference standard. The complex is stable for at least 2 h. The coefficient of variation obtained in five measurements was 1.7 % for 0.36 μg of tin(IV). The molar ratio of tin(IV) to reagent in the complex is 2:3. In the determination of 0.3 μg of tin(IV), sulfate, nitrate and perchlorate (up to 100 mg), chloride (10 mg), acetate, tartrate and citrate (1 mg), oxalate (10 μg), EDTA (1 μg), Ag, Mg, Cd, Pb, As(III, V), Se(IV) and Mn(II) (10 mg), Cu, Be, Ca, Sr, Ba, Zn, Hg(II), Sc, La, Te(IV), Co and Ni (1 mg), Au, Al, In, Hf, Bi, Fe(III) and Pd (100 μg), Ti, V, Mo, W and Pt (10 μg), Ga, Zr and Cr(III) (1 μg) do not interfere. However, Ge and Sb(III) give positive errors to a large extent.

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Four different methods of digestion were examined for biological, environmental and geological materials in order to determine arsenic by the method of arsine evolution-flameless atomic absorption spectrophotometry described in a previous report. The methods studied were: A) digestion with acids in a Teflon container kept in a stainless bomb at 150°C for 3 h, B) digestion with acids under reflux in a quartz flask for 3 h, C) Kjeldahl digestion with a mixture of HNO₃/(1+1)H₂SO₄ in a quartz flask up to SO₃ fuming, and D) digestion with acids in a Teflon (or conical glass) beaker with cover on a hot-plate (150°C) for 3 h. Examination of arsenic retention during digestions by using a standard solution of As(III) proved that arsenic was not lost in the methods A), B), C) and D) with a mixture of HNO₃-H₂SO₄-HF-KMnO₄, while in the method D) significant loss of arsenic was found when no KMnO₄ was added. Digestion of biological samples by the method C) gave reproducible results and good recoveries in standard addition experiments. It is, however, noteworthy that the methods A) and B) gave lower values suggesting incomplete decomposition of biological materials. Methods B), C) and also D) combined with HF-treatment were examined for geological materials, and it was found that the method C) gave reasonable arsenic data although some siliceous residues remained, but in the method B) complete extraction of arsenic was difficult when diluted HCl or concentrated HNO₃ was used as the extractant. The method D) employing a mixture of HNO₃-H₂SO₄-HF-KMnO₄ as the digesting solution also gave the arsenic values similar to those obtained by the method C). However, it is unfavorable in this method that a large blank value is caused by contaminations by arsenic in the reagents added and that glass and quartz wares can not be used in the subsequent procedures because of corrosions by the hydrofluoric acid. Arsenic in eleven standard reference samples were analyzed by the Kjeldahl digestion-flameless atomic absorption spectrophotometry procedures, and it was concluded that arsenic contents observed in present study were in good agreement with those certified by NBS, and those reported by IAEA and other authors.

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A polymeric cationic surfactant, poly(4-vinyl-N-dodecyl pyridinium bromide), prepared by quaternizing poly(4-vinyl pyridine) with dodecyl bromide, forms micelles in aqueous solution which associate with acidic dyes. This surfactant was found to promote the phenolic-proton dissociation of sulfophthalein dyes such as Xylenol Orange (XO) and Pyrocatechol Violet. Addition of the surfactant to the lanthanum-XO system results in a bathochromic shift as well as a remarkable increase in absorbance. The optimum conditions for the spectrophotometric determination of lanthanum were established as: pH 5.0, a 5-fold molar excess of XO over lanthanum, and a 2-fold molar excess of the surfactant over XO. Beer's law is obeyed over the range up to 1.04×10^-5M lanthanum at 613 nm, the molar absorptivity being 1.57×10⁵ cm^-1 mol^-1l. The composition of the lanthanum-XO complex in the presence of the surfactant was determined to be 1:3 by the continuous variation method. Aluminum and transition metals interfered with the determination of lanthanum.

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¹³C-NMR measurements of t-butylamine and benzylamine adsorbed on silica gel and octadecylsilyl (ODS)-silica added in various solutions were carried out. In the presence of silica gel, two well-resolved peaks were observed ascribable to species dissolved in solution and adsorbed on silica gel, respectively. An increase in salt concentration in aqueous solutions, however, provided no chemical shift difference between two states but only a narrowing of the ¹³C-NMR signals. Solvent shifts of the methyl and quaternary carbons were observed in the adsorption state of amines. These solvent shifts correlate closely with the values of the solvent strength for silica gel in the adsorption chromatography. The ¹³C-NMR shifts of amines in the adsorption state can be explained in terms of the protonation or quasi-protonation of amines with the silanol groups on the solid surface. Pretreatments of silica gel caused some changes of relative intensities between two signals but no changes in chemical shifts of these carbon resonances. On the other hand, in the solution containing ODS-silica the chemical shift difference between free and adsorbed amines was not observed. The above observations directly show that the adsorption of amine molecules on solid surface highly depends on the solubility of amines into solutions containing solid silicas.

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For the determination of ppb-level hexavalent chromium ion in water, the foam separation technique with cetylethyldimethylammonium bromide (CEDA-Br) as a foaming agent has been investigated. By batch operations on 1000 ml sample solutions, effects of pH, amount of CEDA-Br, flow rate of nitrogen gas bubbled for foaming, and flotation time on both recovery and concentration ratio of Cr(VI), were studied and the following procedure was recommended. Take a 1000 ml of sample adjusted at pH 5.5 into the flotation cell. Under bubbling of nitrogen gas at a flow rate of 100 ml/min, add 1% CEDA-Br solution continuously at a rate of 0.465 ml/min for 20 min, collecting successively the foam coming out of the cell. The whole Cr(VI) ion thus collected into the foam was analysed spectrophotometrically by the diphenylcarbazide method. The concentration ratio and recovery of Cr(VI) from 5×10^-8 g/ml solution were 120% and 94%, respectively, through above procedure. A large amount of sulfate ion and ferric ion in sample solutions interfered with the recovery of Cr(VI). The foam separation technique was also developed to be applicable to chromium (sample) solutions which flew continuously at rate of 2000 ml/h, 3000 ml/h, 4000 ml/h and 5000 ml/h. Satisfactory recovery was obtained at every flow rate in these continuous separations.

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A method of X-ray fluorescence spectrometric determination with sample preparation and enrichment procedure is described for some heavy metals such as iron, copper, nickel, zinc, lead, bismuth, cobalt and cadmium. This method is based on the preconcentration of metals by solvent extraction of the APDC chelates from hot sample solution into molten stearyl alcohol, the pressing of stearyl alcohol beads to form a suitable X-ray sample and the analysis of the sample by X-ray fluorescence. Stearyl alcohol was found to be an excellent media for collecting microgram quantities of APDC chelates and for subjecting the collected metals to an energy dispersive X-ray fluorescence spectrometer. The samples are treated as disks having intermediate masses per unit area and numerical matrix correction technique is used to linearized fluorescence X-ray intensities for stearyl alcohol disk. APDC extracts heavy metals cited above quantitatively from a 0.5 mol/l sodium acetate or 0.1 mol/l ammonium citrate solution. Iron was, however, not quantitatively extracted from citrate solution, and its chelate was not stable in hot solution. Cobalt was quantitatively extracted only in a narrow pH range (<pH 5). Recovery of the procedure was checked with standard solutions, and it was confirmed that more than 99 % of heavy metals were found in the stearyl alcohol beads. Standard reference materials, Japan standards of iron and steel, No. 506-2 chromium steel and NBS SRM 1570 Spinach, were analysed to test the accuracy of the method. In all case, the close agreement between the cirtificate values and X-ray results is considered to indicate no significant bias in the proposed method.

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The sample flow at (1525) ml/min is mixed with argon (40 ml/min) and oxygen (160 ml/min) and burned out in a 800°C quartz combustion tube. The combustion products are swept into a 400°C quartz absorption tube packed with silver wool. The sulfur compound reacts with the silver to form a compound (Ag_xS_yO_z). Then the absorption tube is pulled out from the absorption furnace and introduced into a 800°C decomposition furnace. A hydrogen flow of 110 ml/min is immediately passed through the absorption tube, and here Ag_xS_yO_z is hydrogenated into the metallic silver and gaseous compound (H_xS_yP_z). The H_xS_yO_z is swept together with the hydrogen into a 800°C reduction tube packed with platinum plate, and converted into hydrogen sulfide. The hydrogen sulfide is absorbed in an aqueous solution of zinc sulfate, sodium hydroxide and glycerin, and then determined spectrophotometrically at 665 nm by the Methylene Blue method. The percent recovery of sulfur from standard gaseous samples of sulfur dioxide was 99.6 % at 7.70 μg of sulfur and the coefficient of variation was 1.4% (n=11). This method is free from the interference of chlorine, nitrous oxide and carbon dioxide. The sulfur in propane for domestic fuel was 3.12 ppm, whereas in commercial ethylene less than 0.14 μg (absorbance 0.005), that is, 0.081 ppm.

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The separation of homologous compounds of aliphatic monoalcohols and glycols was investigated by high performance liquid chromatography as their 3, 5-dinitrobenzoates. The aliphatic alcohols were quantitatively converted to corresponding 3, 5-dinitrobenzoates in a pyridine solution. The molar absorbance of the derivatives at 254 nm, 1.0×10⁴ for monoalcohols and 2.08×10⁴ for glycols, facilitated the use of UV detection. The mixtures of alcohols of carbon numbers of C₁ to C₆ were chromatographed by an ODS-column. (RP-18, 25 cm) with methanol-water (55/45) as a mobile phase. The mixtures of alcohols of C₁ to C₁₂ were completely separated by a gradient elution technique. Glycols such as poly-methylene, poly-ethylene glycol were separated according to the corresponding methylene unit by a polymer column with methanol-2, 2, 4-trimethylpentane(95/5) as a mobile phase. This method was applied to clarify the molecular distribution in the commercially available PEG-200, PEG-400 and PEG-600, and could be applicable for the determination of trace amount of alcohols.

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A highly sensitive spectrophotometric method was developed for the determination of iron(II), by using a chelating agent containing sulfonic acid group and a cationic dye(C⁺) and measuring the absorbance of the dye in the ion associate formed. The chelate anion of iron(II) with 2-nitroso-l-naphthol-4-sulfonic acid (nitroso-NW acid, H₂R) could be extracted into chloroform in the presence of 1-alky1-4-(4-diethylaminophenylazo)pyridinium ion as a counter ion. The alkyl groups examined were methyl, ethyl, propyl, butyl and benzyl. The extraction constants, log K_E, for the chelate anions with the ethyl and propyl derivatives of the azo-dye cation were 17.83 and 19.44, respectively. The extractability of the iron(II) chelate, FeR₃^4-, and the deprotonated forms of nitroso-NW acid, HR^- and R^2-, into an organic solvent decreased in this order. For instance, R^2- was hardly extracted into chloroform but FeR₃^4- was very easily extracted. When propyl derivative was used at a concentration of 10^-4 M, iron(II) ion was selectively and quantitatively extracted into chloroform from a solution adjusted to pH>8.2. The molar absorptivity of the extracted species(C₄FeR₃) was 2.1×10⁵l mol^-1 cm^-1 at 555 nm.

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Formation of nitrosyl-ethylenediaminetetraacetatoiron (II) complex, Fe (I I)NO- EDTA, by absorption of nitrogen dioxide to the iron(II)-EDTA aqueous solution was studied by controlled potential electrolysis and spectrophotometry. The NO₂ introduced into the iron(II)-EDTA aqueous solution was converted to HNO₂ and HNO₃ as eqns. (1), (2), and Fe(II)NO-EDTA was formed from the reaction of HNO₂ and iron(II)EDTA but HNO₃ did not react with iron(II)EDTA.
2NO₂+H₂O→HNO₂+HNO₃……(1)
NO₂+Fe(II)EDTA+H⁺
→Fe(III)EDTA+HNO₂……(2)
In the reaction (1) one mole of HNO₂ was formed from two moles of NO₂, in the reaction (2) on the other hand, from one mole of NO₂. The experimental result of the conversion rate of NO₂ to Fe(II)NO-EDTA was increased with increasing iron(II)EDTA concentration and at high iron(II)EDTA concentration, 0.2 mol dm^-3, the conversion rate was increased up to about 1.0. The total determinations of NO and NO₂ should be carried out in the absorbent with high iron(II)EDTA concentration.

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The gas chromatographic separation of enantiomers of α-halocarboxylic acid in the form of amide on a 40 m glass capillary column coated with N, N' -[2, 4-(6- ethoxy- 1, 3, 5-triazine)-diyl]bis (L-valyl-L-valyl-L-valine isopropyl ester) (OA-300) has been studied. The separation factor for various amides of α-bromo-β, β-dimethylbutyric acid enantiomers were 1.0221.034 at 180°C. Therefore, the structure of amine was not so effective for the separation of enantiomers. Eleven α-halocarboxylic acid hexyl amides were resolved into their antipodes with separation factors ranging from 1.0201.032 in the same chromatographic conditions. As the differences of separation factors were quite small in a-chloro-, bromo- and iodo-alkyl carboxylic acids, it was concluded that halogen atoms did not produce a notable effect on the enantiomeric separation. The peak of R-isomer of α-chloro and α-bromo-β, β-dimethylbutyric acid appeared prior to that of S-isomer in this stationary phase. Although this order is reverse to that of α-alkyl phenylacetic acid enantiomers on the same optically active stationary phase, the results are seemed to be reasonable considering the absolute configuration of these compounds. Because, the absolute configurations on the α-carbon atoms to the both carbonyl groups in the R-isomer of α-haloalkyl carboxylic acid and the S-isomer of α-alkyl phenylacetic acid are superimposable.

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Spectrophotometric determination of iron(II) by utilizing solvent extraction of its complex with 2, 2'-dipyridylketoxime (DPK) was studied. DPK reacts with iron(II) to form red complex easily, which is extracted quantitatively into chloroform at a pH of about 5. Chloroform extract of the complex had two absorption maxima at 335 nm and 540 nm, and showed constant absorbance over the range of pH 4.55.8. Beer's law held over the range (2.040) μg Fe(II)/10 ml at 335 nm and (4.090) μg Fe(II)/10 ml at 540 nm, respectively. The apparent molar absorption coefficient of the complex at each absorption maximum is 1.94×10⁴ and 1×10⁴ dm³ mol^-1 cm^-1. Analytical procedure is as follows; To a sample solution containing up to 90 μg of iron(II) in a 100 ml separatory funnel, 2 ml of 10 % hydroxylamine hydrochloride solution is added, and a pH is adjusted to about 5 with an acetate buffer solution. The resulted solution is shaken for 7 min with 10 ml of 5×10^-3 M DPK solution in chloroform. The absorbance of the extract is measured at 335 nm or 540 nm against the reagent blank. The combining ratio of iron and DPK in this complex was estimated to be 1:3 by analysis of the isolated complex. Therefore, DPK acts as a bidentate ligand for iron(II).

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Titanium in commercially available ammonium thiocyanate was determined by spectrophotometric method with diantipyryl methane after ion-exchange chromatographic separation. Samples, weighing 83 g each, were dissolved in 11 of water, and mixed with 91 ml of concentrated hydrochloric acid. The resultant solutions were passed through a column of Amberlite CG400 (5g of SCN- form) 5 times repeatedly. The total amount of sample was 415 g. Trace amount of titanium was adsorbed on the column, and then eluted quantitatively with 80 ml of 2 M hydrochloric acid-1.5 % hydrogen peroxide solution. The concentration of titanium in the eluent was determined spectrophotometrically with diantipyryl methane. Titanium in reagent grade of ammonium thiocyanate contained not less than 21.8 ppb.

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