The complex formation reactions of Eriochrome Cyanine R (ECR, H₄L) with aluminum in the absence and presence of zephiramine (ZCl) were studied by spectrophotometry using the equilibrium concentration method. The properties of this complexes were compared with that of Chromazurol S (CAS) complexes with aluminum.
Aluminum and ECR formed a 1: 1 acidic complex, AlHL, (the absorption maximum at 550 nm) below pH 4 and a 1: 3 acidic complex, Al(HL)₃^6-, (the absorption maximum at 535 nm) above pH 5. In the presence of zephiramine, they formed a 1 : 2 acidic complex, Al(HL)₂Z₃, (the absorption maximum at 585 nm) below pH 5.8 and a 1: 3 acidic complex, Al-(HL)₃Z₆, above pH 6.
The calibration curves obey Beer's law for 0.010.3 ppm of aluminum in solutions containing 6 × 10^-4M ECR and at pH 6.0 or 3 × 10^-4M ECR, 2.7 × 10^-3 M zephiramine and pH 5.5 where the formation of the complexes were almost completed. Thus their molar absorptivity was 8 × 10⁴at 535 nm and 1.1 × 10⁵ at 585 nm, respectively.
As the acidic dissociation constants of ECR were the lower values than that of CAS in the absence and presence of zephiramine, the stability constants of aluminum-ECR complex were the higher values than that of the CAS complexes as shown in Table I and II.
In the absence of zephiramine, the 1 : 3 acidic complex of aluminum and ECR formed directly from the 1 : 1 acidic complex. It was considered that the 1 : 3 acidic complex was stabilized by a resonance effect for ECR molecule as shown in Fig. 12.
However, in the presence of zephiramine, the equilibrium constant of the 1 : 2 acidic complex formation reaction between aluminum and CAS was the higher values than that for the ECR complex. This result showed that an action of zephiramine for the CAS complex formation was more effective than that for the ECR complex. Then it was considered that the density of electron around carbon of methane in CAS was concentrated by a positive inductive effect of chlorine atom in CAS and consequently, the π-π transition of electron in the CAS molecular was accelerated by an action of the micelle having a positive charge.

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A highly sensitive method for the spectrophotometric determination of aluminum with Chromazurol S (CAS) and zephiramine have been developed as described in the previous paper {Bunseki Kagaku, 21, 997 (1972)}. Though interference with iron was masked by an addition of thioglycolic acid in that case, a negative effect by the absorbance of aluminum complex was caused. The writer found that the negative influence by thioglycolic acid could be removed by additions of some electrolytes, especially ammonium salts and its derivatives such as hexamine, ammonium chloride and trimethylbenzylammonium chloride. But ammonium acetate and urea which were a sort of acetate and of non-electrolyte respectively was ineffective. Optimum volumes of these reagents were above 2 ml for 1 M hexamine solution, above 3 ml for 1 M ammonium chloride solution and above 3 ml for 1 M trimethyl benzylammonium chloride solution. The absorbance had the highest and fixed value in the range of pH 4.755.2. However the color of the complex was faded catalytically by thioglycolic acid in the presence of iron at pH above 5.1, the pH was adjusted to 4.9. In the case of the alkaline solution of hexamine the absorbance was faded slightly with standing, so the pH of the hexamine solution had to be adjust to 5 with hydrochloric acid. Interference with up to 10 mg of iron was masked by adding 0.81.5 ml of 8% thioglycolic acid.
This method was applied to the determination of aluminum in malleable cast iron. The recommended procedure was as follows.
Take 0.2 g of sample into a 100 ml quartz beaker and dissolve in 1 ml of concentrated hydrochloric acid and 510ml of water with heating. Add 0.5 ml of concentrated nitric acid and boil up in order to remove nitrogen oxide. Filter the solution, wash the residue on the filter paper with hydrochloric acid (1+100), and dilute the filtrate to 100 ml with water. To an aliquot of the solution containing up to 5 μg of aluminum, add 1 ml of 8% thioglycolic acid, 1 ml of 0.2% CAS solution, 5 ml of 0.5% zephiramine solution, and 4 ml of 7% hexamine solution. Adjust the pH to 4.9 with 0.5 Msodium hydroxide solution and dilute to 25 ml with water. After the solution has stood for 30 minutes, measure the absorbance at 620 nm against the reagent blank which prepared from aluminumless iron by the same procedure as used for the sample.
The calibration curve was quite linear for 0.15μg of aluminum in 25 ml. The molar absorptivity was 1.15×10⁵, The relative standard deviation was 15% for the determination of 0.0020.06% of acid soluble aluminum in malleable cast iron.
In the case of determination of aluminum by Eriochrome Cyanine R(ECR) and zephiramine method interference with thioglycolic acid and the acetate could be also masked by an addition of ammonium chloride at pH 5.9.

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Anodic stripping polarography of Ni(II), Co(II) and Mn(II) was studied by using their aliphatic polyamine complexes.
Ni(II) gave an anodic stripping polarogram in 1, 3-propanediamine, Co(II) gave that in ethylenediamine and also in 1, 3-propanediamine and Mn(II) gave that in triethylenetetramine medium.
Calibration curve was obtained with amounts 0.05 to 0.5 ppm for Ni(II) in 0.1 M 1, 3-propanediamine and 0.2 M KCl, and for Mn(II) in 0.03 M triethylene tetramine and 0.2 M KCl, and with amounts from 0.005 to 0.05 ppm for Co(II) in 0.1 M ethylenediamine and 0.2 M KCl.
The pre-electrolysis potential was -1.3 V vs. SCE for Ni(II), -1.50 V vs. SCE for Co(II) and -1.70 V vs. SCE for Mn(II), and the preelectrolysis time was 5 min for all the metals studied.
The peak potential was -0.16 V vs. SCE for Ni(II) in 0.1 M 1, 3-propanediamine and 0.2 M KCl medium, -0.31 V vs. SCE for Co(II) in 0.1 M ethylenediamine and 0.2 M KCl medium, and -1.01 V vs. SCE for Mn(II) in 0.03 M triethylenetetramine and 0.2 M KCl medium.
The peak current for 0.5 ppm of Ni(II) and of Mn(II), and for 0.05 ppm of Co(II) were linearly proportional to the square root of scanning rate in a range from 4 V/1.1 min to 0.5 V/1.1 min.
Anodic stripping polarogram of Ni(II), Co(II) and Mn(II) in polyamine solution were recorded in the presence of various electrolytes (KNO₃, KCl, KBr, KSCN and KNO₃+KI). Mn(II) was not affected by change in the supporting electrolytes, but Ni(II) and Co(II) was affected by their change. The peak current of Co(II) in 0.1 M ethylenediamine was increased by an addition of KI solution when the concentration is in a range from 0.002 to 0.006 M in a 0.2 M KNO₃ solution. The peak potential of Co(II) was shifted to the more negative potential side with in creasing peak current and the difference between the peak potential and the half-peak potential (E_p-E_p/2) became smaller with increasing peak current.
The peak current of Ni(II) in 0.1 M 1, 3-propanediamine was not so much affected as that of Co(II) in 0.2 M KNO₃, 0.2 M KCl and 0.2 M KBr. The peak potential and the peak current of Ni(II) in 0.1 M 1, 3-propanediamine were almost equal to those in 0.2 M KSCN solution. However, the peak current in 0.1 M 1, 3-propanediamine, 0.2 M KNO₃ and 0.002 M KI medium was larger than that in 0.1M 1, 3-propanediamine and 0.2M KNO₃ medium and also the peak potential in a medium containing iodide ion was shifted to the more negative potential side than that in the medium containing no iodide ion.

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In a previous paper we reported the preparation of the silica gel and alumina sintered plates. They consited of a layer of silica gel or alumina on various kinds of glass or metal base plate fixed with several kinds of sintered glass powder.
In the present paper we reported that the previously reported welding procedure was successfully applied to the preparation of silica gel or alumina sintered sticks. As reported previously, several kinds of glass or metal sticks were used as support and several kinds of glass powder were tested as binders. While these three materials are very much different from each other in their expansion coefficient (α) as shown in Table I, the welding among these materials does occur.
Thin-layer chromatographic separation of the following test mixtures was performed by using these silica gel and alumina sintered sticks: azodyes (indophenol, Sudan red G and p-dimethylaminoazobenzene, and p-aminoazobenzene, Sudan yellow and azobenzene), estrogens (estriol, estradiol and estrone), alkaloids (quinine, codeine, brucine and thebaine)and lipids (cholesterol, cholesteryl stearate, monostearin, distearin, tristearin, stearic acid and methyl oleate). All these sticks are thermo-stable, and those with glass stick supports are acid-resistant and, moreover, can be repeatedly used without reactivation after being in the hydrogen flame ionization detector. As shown in Table IV and IX, these sintered sticks have very superior reproducibility of separation.
By using the silica gel quartz sintered sticks, a quantitative determination of lipids was attempted. Among the five kinds tested, two kinds of stick with glass ceramic or borosilicate as binder were proved to be useful owing to their small baseline noise in response (Fig. 3 and 4). The response reproducibility of lipids was very good when scanning was performed with TLC-flame ionization detector (FID). The coefficient of variation was less than 5% in three weight ratios (Table VIII).
The silica gel sintered sticks and thd silica gel Laboratorymade sticks when used together with TLC-FID apparatus will facilitate the qualitative and quantitative analysis of lipids in biological fluids, heavy oil fractions in petroleum industry and plant components in phytochemistry.

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The amount of cholesterol in serum has a close relationship with the functions of liver, pancreas, thyroid gland and other organs. Thus its determination is very important in clinical examinations. The Zak, Pearson, and other methods have been used as routine analysis of total cholesterol in serum, but hitherto no method allows rapid analysis of total and free cholesterol in serum. Recently, cholesterol analysis in serum by gas chromatography has been done by hydrolysis of its ester, but since this method requires complicated procedure, it is not suitable for routine work.
We have studies on rapid analysis of cholesterol in serum by reaction gas chromatography. Serum sample was injected into a gas chromatograph and the hydrolysis of choelesterol ester was carried out in a KOH reaction column. The first 7 cm of the column was packed with KOH on quartz powder, and the rest packed with 2% OV-1. Thus the total cholesterol was detected as a single peak.
Apparatus used was Shimadzu Seisakusho model GC-5AP, dual column system, with flame ionization detector. A glass column, 0.5 cm × 3 mm i.d., packed with 2% OV-1 on Shimalite W was used for determination of free cholesterol and the other column (same size) packed with KOH on quartz powder and 2% OV-1 on Shimalite W was used for total cholesterol as shown in Fig. 2. The conditions for the analysis were: column temperature 230°C, detector and sample injection port temperature 300°C, nitrogen flow rate 60 ml/min, and electrometer sensitivity setting 0.32× 10^-10 A per millivolt.
From the tested concentration of KOH in the reaction column, 15% on quartz powder was found to yield best cholesterol ester hydrolysis. Optimum column temperature (injection port temperature) was 300 °C. Cholestane was used as a internal standard.
Five μl of serum and 10 μl of 0.02% cholestane in tetrahydrofuran were put into microtest tube, and after shaking up, it was left on for 2 min., and the upper phase was used as a sample for analysis. Value of total cholesterol in serum obtained by gas chromatographic analysis was compared with that obtained by colorimetric determination, and no significant difference was observed. This most rapid method for the determination of free and ester forms of cholesterol in serum was applied to the analysis of serum cholesterol of patients.

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Spectrophotometric determination was studied for creatine (CT), 1-methyl-1-benzylguanidine (MBG), guanidine (GD), glycocyamine (GC) and arginine (AR) by the Voges-Proskauer (V-P) reaction using acetylbenzoyl (AB) and α-naphthol. According to the procedure shown in Table I, the maximum absorption was found at 565 nm for CT, at 570 nm for MBG, and at 560 nm for GC, GD and AR (Fig. 1, Fig. 2). All the guanidine derivatives examined showed the strongest coloration at 6% alkali concentration (Fig. 3). The most suitable reaction time was found to be 30 min. for CT and GD, 20 min for MBG, and 60 min for GC and AR, at this alkali concentration.At the respective reaction time, the greatest coloration was obtained at the following molar ratio of AB to guanidines: 10 for CT, 5 for MBG and 20 for GC, GD and AR (Fig. 4). The α-naphthol concentration for the best coloration was 1% for CT, 0.6% for MBG, and 0.8% for GC, GD and AR (Fig. 5).
iso-Propanol or tert-butanol at a final concentration of 40% was found to give the color intensity 1.1 to 2.1 times greater than that without the alcohol for the guanidines used except for MBG (Table II). The shift of the wavelength of the maximum absorption to the longer wavelength was observed with increasing the concentration of iso-propanol or tert-butanol (Table III). The absorbance at the respective wavelength of the maximum absorption reached maximum at 35 to 40 min for CT, at 50 to 60 min for GD and at 60 to 80 min for GC and AR in the presence of iso-propanol or tert-butanol (Fig. 6, Fig. 7). The greatest color intensity in the presence of an alcohol relative to that in its absence was obtained at a final concentration of 40% of tert-butanol (Table III). In the presence of tert-butanol the most suitable reaction temperature and time were 35 min at 25°C for CT and 20 min at 25 °C for MBG, 45 min at 30°C for GC, 35 min at 30 °C for GD and 50 min at 30°C for AR (Fig.8).
The most suitable procedures for the determination of guanidenes are summarized in Table IV. Beer's law held for a concentration range of 2.5 μM to 40 μM and the coefficient of variation (CV) ranged from 0.3% to 1.0% (Table V).
It was clarified by TLC that one of the reasons why the color intensity is decreased in the presence of primary alcohols, is the consumption of an intermediate, 4-methylene-4H-imidazol (I) by its conversion into 4-alkoxymethylimidazole (II) (Fig. 9).

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Potentiometric titration of triethylenetetraminehexaacetic acid (TTHA) with lead tetraacetate was investigated.
The titration procedure is as follows. An aqueous solution (100 ml) containing 40 ml of glacial acetic acid and 10.00 ml of 0.05 M lead tetraacetate in glacial acetic acid was titrated with a TTHA solution by using a platinized platinum wire electrode as indicator electrode. The potential of the electrode was measured against a saturated calomel electrode, which was connected to the solution through an agar bridge of 30% KNO₃. The measurement of the potential was made at 2 minutes after each addition of the titrant.
The titration curve indicated that Pb(IV) ion reacts with TTHA in molar ratio of 2:1. The potential change in the vicinity of the end point was about 100 mV per 0.10 ml of the titrant. TTHA could be determined over the concentration range of 0.01 M0.05 M within the error of ± 1.0% and with the relative standard deviation of ± 0.05%. The addition of Pb(II) ion into the titrated solution up to 20% of the concentration of Pb(IV) ion did not interfere with the determination. Interference of Pb(II) ion with the determination was observed, as shown in Fig. 7 and Table III, when its concentration was larger than 20% of that of Pb(IV) ion in the titrated solution. The conditional formation constant of the chelate of Pb(II) ion with TTHA in which the molar ratio was 2 : 1, was calculated to be about 1 M^-2 under the experimental condition.
Effect of the change in the concentration of acetic acid or sodium acetate and temperature of the titrated solution was examined.
When the volume fraction of acetic acid in the titrated solution increased to more than 50%, a positive error became remarkable, as shown in Fig. 3. When the concentration of sodium acetate in the titrated solution increased to 0.04 M, a negative error became remarkable as shown in Fig. 4.
When the temperature of the titrated solution was higher than 25°C, a negative error was noted as shown in Fig. 5, but when it was lower than 25°C, a positive error was noted as shown in Fig. 6.
It was considered that the potential of the indicator electrode beyond the end point of the titration curve would be determined predominantly by the platinum ion-platinum system, which was produced by the dissolution reaction of the platinum oxide layer in excess TTHA (Fig. 8).
The present potentiometric method is as satisfactory for the standardization of TTHA as the color indicator method.

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An indirect atomic absorption spectrometric method for germanium was studied. Molybdate of molybdogermanic acid, which was formed under a specified condition and extracted into methyl iso-butyl ketone (MIBK), was determined by atomic absorption spectrometry (using Hitachi 207 type atomic absorption spectrophotometer).
It was found that the optimum condition for the extraction of molybdogermanic acid was markedly influenced by the order of adding reagents. Molybdogermanic acid was extracted into MIBK from 0.60.7 M hydrochloric acid solutions containing molybdate. When germanium was added to molybdate solution and the acidity of the sample was then adjusted, molybdogermanic acid was extracted from hydrochloric acid solutions of relatively wide range of acidity, i.e., 0.61.0 M. The proposed procedure is as follows: 10 ml of ammonium molybdate solution (0.2 M as Mo) is transferred into a 100 ml separatory funnel, and 2 ml of 6 M hydrochloric acid is added. Then, a given volume of germanium solution is added and made up to 20 ml with water. After the solution is left to stand for 15 min, 20 ml of MIBK is added and shaken vigorously for 3 min. After the two layers separate, the organic layer. is washed with 20 ml of 0.6 M hydrochloric acid three times in order to eliminate the excess of molybdate, and dehydrated by using a centrifugal separator. The molybdate is determined by aspirating the dehydrated organic layer in airacetylene flame and measuring the atomic absorption at 3133Å against the reagent blank prepared according to the same procedure.
The back-extraction method, in which the molyb-ate extracted in the organic layer was extracted into an aqueous buffer solution (NH₃+NH₄Cl, pH 9.5) and was determined by atomic absorption spectrometry, was also examined.
The calibration curve was linear in the range of 2.5 ×10^-62.0×10^-5M of germanlum.The sensitivityby the extraction method was 1.3 times that of the back-extraction method. Silicate and tantalate interfered seriously and arsenate, niobate, phosphate and tungstate also did.

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Six of bisazochromotropic acid analogues (arsenazo III, carboxyarsenazo, sulfonazo III, dimethylsulfonazo III, dinitrosulfonazo III and chlorophosphonazo III) were compared as indicators for the precipitation titration of sulfate with barium perchlorate solution.
The effect of various sulfates on those indicators was investigated in aqueous solution (Table I). Sulfonazo III, dimethylsulfonazo III and dinitrosulfonazo III indicated a good agreement with the gravimetric values. Arsenazo III and carboxyarsenazo could not be used for the determination of sulfuric acid solution, because the color change did not occure at the end point, and chlorophosphonazo III showed lower values comparing with the gravimetric value.
Direct volumetric determination of sulfate by barium ion encountered a difficulty by somewhat slow rate of formation of barium sulfate precipitation. To enhance the formation of barium sulfate, the author carried out the titration with an addition of organic solvent (i.e. acetone). As shown Fig. 1, the volume of the titrant decreased gradually with increasing content of acetone, but when the ratio acetone/water was one or more, the value was constant. Although the constant value was somewhat lower than the gravimetric value, difference between the end point and the gravimetric value could be corrected by the experimental factors.
From the effect of pH on the color change at the end point, sulfonazo III, dimethylsulfonazo III and chlorophosphonazo III were chosen as most suitable indicators.
The recommended procedure for the determination of sulfate was as follows : Ten ml of sample containing ca. 10 mg of sulfate ion was taken in a 100 ml beaker, and added 5 ml of buffer solution (pH 3.0), 23 drops of indicator (sulfonazo III, dimethylsulfonazoIII or chlorophosphonazo III) and 1525 ml of acetone. stirring continuously, it was titrated with 0.01 M barium perchlorate solution. At the end point, the color changed from pink to blue.
The effect of some interfering ions was examined (Table II) and the results of the analysis of commercial sodium sulfates were shown in Table III.
An attempt to clear the purity of the bisazochromotropic acid analogues was carried out by paper chromatography (Fig. 2). They contained always some impurities such as monoazo compound and so on.

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The sensitivity enhancement of atomic absorption analysis was examined. Nickel and cadmium as test of elements, C₂H₂-air and C₂H₂-N₂O as the flames, and methanol, ethanol, propanol, butanol, aceton, acetic acid, and ethylacetate as solvents were used. The absorption, the aspirated volume (ml/min), the atomized percent, the amount of spray volume reached burner(ml/min) were measured in detail using various degrees of solvent mixtures. Here the ratio between absorbance and the amount of spray volume reached burner was defined as the relative sensitivity, and were plotted the relative sensitivity for the ordinates and the percent of the carbon (or the volume of organic solvent) for the abscissas. As a result, direct ratio was found between them. By the leastsquares method, the coefficient of a straight line was got. The coefficient was defined as the enhancement's factor. The value was -1424 and the straight line could be showed as form (1).
T=A+BX……………(1)
T: Relative sensitivity, A : Relative sensitivity at aqueous solution, B: Enhancement's factor, X: Percent of carbon (or volume of organic solvent).
In the smoll value of factor, as Allan pointed, it is probable that the enhancement was effected by the amount of spray volume reached burner, and it's phenomenon was observed in aceton, acetic acid and ethylacetate mixture at using C₂H₂-air flame. In the large value of factor, as Robinson pointed, it is probable that the enhancement was effected by the mechanisms of the formation of atoms in the flame, and it's phenomenon was observed in alcohol mixture at using C₂H₂-air flame, and in aceton, acetic acid and ethylacetate at using C₂H₂-N₂O flame. To study the mechanism of enhancement in the flame, adding the colloidal carbon (0.51.0 μ) 0.0003%, 30 ml to make 100 ml aqueous solution, the absorption increased 5% in cadmium but adding the carbon over 1.0 μ, the enhancement did not occured. From this fact, as one factor of enhancement in organic solvent, it is probable that the enhancement was occured on account of a chain reactional re-dividing spray by the heated carbon and high heat before organic solvent evaporates and burns and that the mechanism was different on each solvent and each flame. As an insoluble solvent, the relative sensitivity of methylisobutylketon (MIBK) was measured with cadmium and the enhancement's factor was calculated by form (1). It's value was 1.9 in C₂H₂-air flame, from that, and it is probable that the enhancement in MIBK was effected by the amount of spray volume reached burner more than by the mechanisms of the formation of atom in the flame.

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For the purpose of investigating lead distribution in rat tissues after administration of lead compounds, lead in biological materials is determined by atomic absorption spectrophotometry. As pretreatment of the determination ammonium pyrrolidine dithiocarbamate (APDC)-methyl isobutyl ketone (MIBK) extraction is applied for concentrating lead in samples. The lead is extracted quantitatively at a pH range 1.05.0. There is no interference with coexisting ions except a large quantity of iron, which should be removed by cupferron-MIBK extraction prior to APDC-MIBK extraction. It is considered that the method is applicable to the determination of lead in biological materials, since recovery of lead from rat tissues is 98.0102.3%, the coefficient of variation is 1.15.6%, and the bias is not significant.
The procedure is as follows: (1) Destruction of sample. Samples are destructed with a mixture of nitric acid and perchloric acid, and the residue obtained is dissolved in 5 ml of hydrochloric acid (1 : 1). (2) Removal of iron. For samples containing more than 160 μg of iron the sample solution is transferred into a 25 ml volumetric flask, and to the solution 1 ml of 10% cupferron solution and 1 ml of MIBK are added. The flask is shaked for 30 seconds and the organic layer is discarded. The cupferron-MIBK extraction is repeated until the organic layer becomes colorless. (3) Extraction of lead. The aqueous solution obtained by the procedure (1) or (2) is transferred into extraction-centrifuge bottles, and to the solution 3.5 ml of 10% ammonium citrate solution and 3 drops of thymol blue are added. After the pH of the solution was adjusted to 3 with ammonia water, 0.5 ml of 4% APDC solution and 2 ml of MIBK are added. The bottle is shaked mechanically for 3 minutes and centrifuged. The organic layer separated is applied to atomic absorption spectrophotometry for the determination of lead.

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The dual-wavelength spectrophotometry was applied to a separation and determination of a three components system containing 3-, 5-nitrosalicylic acids (3- and 5-NSA) and salicylic acid (SA), and operational conditions were investigated in detail.
In the case of the dual-wavelength spectrophotometry, the difference ΔA_λ2-λ1 between the absorbances at λ₁ and λ₂ can be directly determined, and at least one component can be masked instrumentally by selecting an appropriate λ₁ and λ₂ as shown in Figs.1, 6, 7, 8, 9, 10 and 11, because the absorbance atλ₁ is equal to that at λ₂.
As shown in Figs. 2, 3 and 4, the absorption bands of 3- and 5-nitrosalicylic acids were shifted to longer wavelengths thanthat of salicylic acid in sodium hydroxide solution, therefore, the former absorption bands were notquite interfered by that of salicylic acid.
Beer's law was obeyed at least up to 30 μg of 3-NSA in sodium hydroxide solution containing less than 12 μg of 5-NSA, and was up to 4 μg/ml of 5-NSA in sodium hydroxide solution containing less than 30 μg/ml of 3-NSA, respectively.
The equations used are as follows,
ΔA_430.0-391.5=k_3-NSA×C_3-NSA=0.00927×C_3-NSA
ΔA_410.0-454.0=k_5-NSA×C_5-NSA=0.0676×C_5-NSA
where k and C are an absorption coefficient in ΔA/μg/ml and a concentration in μg/ml, respectively.
The difference ΔA_295.5-333.2 between the absorbances at λ₁ ( =333.2 nm) and λ₂( =295.5 nm) is equal to the sum of the absorbances of SA, 3- and 5-NSA.
On the other hand, 5-NSA can be masked instrumentally by selecting λ₁ and λ₂ described above, therefore, the difference ΔA_{295.5 - 333.2} is ultimatly the sum of the absorbance for 3-NSA and SA.
The absorbances ΔA_295.5-333.2 for 3-NSA containing 5-NSA and SA are proportional to the concentration of 3-NSA as shown in Fig. 13, and the following equation holds up to 50 μg/ml of 3-NSA.
ΔA_{3-NSA(295.5-333.2)}=k_{3-NSA(295.5-333.2)}×C_3-NSA=0.0118×C_3-NSA
The following equation holds up to 5 μg/ml of SA in sodium hydroxide solution containing less than 25 μg/ml of 3-NSA and 50 μg/ml of 5-NSA(Fig.12).
ΔA_{SA(295.5-333.2)}=k_{SA(295.5-333.2)}×C_SA=0.0240×C_SA
Therefore, the concentration of SA containing 3- and 5-NSA can be calculated from the following equation.
ΔA_295.5-333.2=ΔA_{SA(295.5-333.2)}+ΔA_{3-NSA(295.5-333.2)}
=k_SA×C_SA+k_{3-NSA(295.5-333.2)}×C_3-NSA
=0.024×C_SA+0.0118×C_3-NSA
This method can be satisfactorily applied to the determination of a three components system where a molar ratio of 3-NSA to SA is up to 50 as shown inTable II.

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The method for routine analysis of benzotriazol (BT) and methylbenzotriazol (MBT) were established by using potentiometry and gas chromatography.
When the sample was dissolved in 1.5% aqueous solution of sodium bicarbonate, the most definitive end point in the potentiometric titration with silver nitrate solution was obtained (Fig. 1 and 2).
If the sample contains chloride, the interference with chloride can be avoided by addition of an excess of EDTA (Fig. 3).
As Table I shows, 2 to 30 mg of BT or MBT was determined successfully by this method.
Each content in mixtures of BT and MBT is determined by the potentiometric titration combined with extraction procedure. For example, 100 mg of the mixtures was dissolved in 10 ml of a mixed solvent (chlorobenzene : carbon tetrachloride =1 : 1, v/v), and 10 ml of water was added to it. After the mixture had been shaked for 30 min at a room temperature (20°C), an aliquot of aqueous phase was transferred to a beaker, and 100 ml of 1.5% aqueous solution of sodium bicarbonate was added to it. The solution was titrated by the potentiometric method, in which a silver and glass electrode were employed as an indicator and reference electrode, respectively.
As Fig. 5 and 6 show, the extractability reveals a good linear relation with the composition of sample, therefore, the calibration line can be applied to determine the mixtures of BT, 4-MBT and 5-MBT.
In case of the mixture, gas chromatography was also useful in the microanalysis. The optimum conditions were as follows-glass column; 1 m×3 mφ, supporter; Chromosorb W(60 to 80 mesh) coated with 1.5% of neopentyl glycol sepagate, column temperature; 180°C, injection temperature; 200°C, carrier gas; nitrogen (30 ml/min), FID gas presser; hydrogen (0.5 kg/cm²) and air(1.0 kg/cm²), sensitivity and range; 10³×128, sample; 1 to 5 μl of acetone solution.
A chromatogram and a calibration line obtained with the above conditions were shown in Fig. 7 and 8.
These methods were simple and rapid, and successfully applied to determine BT, MBT and the mixture in various commercial products(Table II, III and IV).

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Polarographic behavior of both tris(acetylacetonato)-iron (III), [Fe(acac)₃], and tris (thenoyl trifluoroacetonato) iron ( III), [Fe(TTA)₃], were investigated in methyl isobutyl ketone (MIBK) containing 0.1 M tetrabutylammonium perchlorate (TBAP) as a supporting electrolyte.
Both chelates can be reduced at the dropping mercury electrode or the platinum disc electrode. The half wave potential of Fe(acac)₃ is -0.74 V (vs. SCE) and that of Fe(TTA)₃ is -0.05V (vs. SCE) in anhydrous MIBK. From the dependence of the limiting current on the height of the mercury reservoir, the log plot analysis, the a. c. polarographic wave height and the cyclic voltammogram, the electrode reaction of Fe(acac)₃ is reversible and is diffusion-controlled involving one electron reduction process in the same manner as in other solvents. The diffusion current constant is 1.71 μA·mg^-2/3·sec^1/2·mM^-1. In MIBK containing 0.1 M acetylacetone solution saturated with water, the half wave potential shifts to anodic side by 120 mV and the limiting current is slightly increased. The d.c. wave height is proportional to the concentration of the Fe(acac)₃ in the range 8×10^-5 M to 1×10^-3 M.
It was found that the electrode reaction of Fe(TTA)₃ was also reversible and is diffusion-controlled involving one electron process in both anhydrous MIBK and MIBK containing 0.1 M TTA saturated with water. The diffusion current constant obtained is 1.46μA·mg^-2/3·sec^1/2·mM^-1. The d.c. wave height is proportional to the concentration of the TTA chelate in the range 8×10^-5 M to 5×10^-4 M.
Iron(III) is quantitatively extracted from 0.1 M citrate buffer solution into MIBK solution of 0.1 M acetylacetone in the pH range 8.8 to 9.1. After TBAP as the supporting electrolyte was added to the MIBK extract, the extracted iron(III) acac chelate in the organic phase is directly analyzed polarographically. From the analysis of the polarograms, the extracted chelate is found to be Fe(acac)₃. Iron(III) in the range 8×10^-5 M to 1×10^-3 Min aqueous phase can be determined by this method.
In case of TTA, iron(III) could not be extracted quantitatively. A remarkable result was obtained that the polarograms of chelate extracted from the acetate buffer solution at pH 5.8 showed not only the cathodic wave but anodic wave. The plot of log(i-i_d.a)/(i_d.c-i) against potential E for this oxidation-reduction wave is linear and has a slope of 65mV. The half wave potential is identical with that of Fe(TTA)₃.Therefore, the extracted species may be a mixture of iron(III) and iron(II)TTA chelates.

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Chlorine occurs as an impurity in sodium as a result of the commercial production of sodium metal by the Downs process, which involves electrolysis of fused sodium-calcium chloride mixtures. In order to develop a simple analytical method for trace amounts of chlorine in sodium, an application of extraction spectrophotometric method with mercury(II)-diphenylcarbazone complex was studied. The method is based on the fact that the extent of mercury(II)-diphenylcarbazone complex is decreased by a trace amount of chlorine.
The recommended procedure is as follows: Take 0.31 g of sodium sample in a 50-ml beaker and transfer the beaker to a sodium decomposition apparatus (Fig. 1) containing dilute sodium hydroxide solution warmed at 40°C. After the decomposition of sodium with water vapour under a reduced pressure, neutralize the solution with nitric acid (1+1). Add 2.1×10^-7 mol of mercury(II) and 1.3×10^-7 mol of potassium bromide to the solution, and adjust pH of the solution to 3.0±0.2 by using 1 ml of 0.1 M phosphoric acid and a suitable amount of 0.1 M sodium hydroxide. Transfer the solution to a separatory funnel shielded from light with green plastic tape, add 10.0 mlof benzene and 0.8 mlof 0.02% diphenylcarbazone-ethanol solution, and extract the mercury(II)-diphenylcarbazone complex with benzene by shaking for a minute. In parallel with the procedure described above, take sodium nitrate solution, corresponding to 1 g of sodium to another beaker and evaporate the solution with 3 ml of concentrated nitric acid to dryness for removing residual chloride in sodium nitrate. Run the extraction procedure as described for the sample solution. Measure the absorbances of the extracts in 1 cm cells at 560 nm using benzene as the reference and determine the amount of chlorine from the difference in the absorbance. Because the slope of the calibration curve is affected by the presence of sodium, construct the calibration curve by taking, for example, 0, 2.0, 4.0, 6.0, 8.0 and 10.0 μg of chlorine and sodium nitrate corresponding to 1 g of sodium and carrying out the entire procedure.
The effects of the foreign ions on the analysis for chlorine are shown in Table II. As shown in Table V, analytical results obtained by the proposed method are in good agreement with those obtained by the coulometric method.

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The condensation product of ο-phenylenediamine(OPD) with dehydroascorbic acid (DAA), which was formed from ascorbic acid (AA) by oxidation with bromine. gave two reduction waves at -0.35 and-0.72V vs. SCE in 0.2 M acetate buffer of pH 3.8.Both of the waves could be employed for the determi-nation of AA and/or DAA in foods, but the first one would be preferred in order to avoid interference with coexisting substances in foods.
Polarographic behavior of various organic substances that were supposed to have the possibility of interfering with the determination was studied in the presence of OPD in the acetate buffer, after oxidation with bromine. The substances were reductones {triosereductone (TR), dehydrotriosereductone (DTR), reductic acid (RA) and dehydroreductic acid (DRA)}, carbonyl compounds (methylglyoxal, diacetyl, pyruvic acid), amino acids (tyrosine, tryptophane and histidine), and others. Sugars and some organic acids, such as glucose, glucuronic acid, glucuronolactone and nicotinic acid, gave no reduction wave in the above media. Behavior of some inorganic ions was also studied under the same condition.
Among the substances studied the condensation products DTR-OPD and DRA-OPD interfered with the measurement of the first wave of DAA-OPD, but these condensation products were easily eliminated by extracting them with chloroform. Accordingly a new polarographic OPD-method for the determination of AA and/or DAA in foods was proposed. The method was based on oxidation of AA to DAA with bromine, formation of addition of compound DAA-OPD, extraction of interfering substances with chloroform, and then the measurement of the first wave of the polarogram of DAA-OPD in the acetate buffer.
Analytical results of AA in artificial samples by dinitrophenylhydrazine method by Roe or its modified method by Matsushita were discussed in view of the results by the present polarographic OPD-method, and a possible source of the errors in the dinitrophenylhydrazine methods was estimated.
The proposed method could be applied to the determination of AA with a standard deviation of 0.50 mg% for an artificial sample containing 44.0 mg% AA, together with 110 mg% TR, 48.1 mg% histidine, 21.9 mg% pyruvic acid and 68.8 mg% hydroquinone. The lowest limit of AA content which could be determined by the present method was around 2 mg% in the sample. In conclusion the proposed method had the advantage of rapidity and disadvantage of lower sensitivity in comparison with the mordified dinitrohydrazine and chromatography. The present method was successfully applied to the determination of AA and DAA in foods, such as fruits, teas, canned foods, and AA-enriched foods.

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In order to examine the amount of scopolamine contained as inpurity in scopolamine-n-butyl bromide (BSB), determination method employing ultraviolet absorption was studied.
About 2 g of BSB was accurately measured, and dissolved in 4 ml of water. The solution, to which 1 ml of ammonia test solution was added, was saturated with salt, and extracted five times with 10 ml of mixture of carbon tetrachloride and chloroform(1 : 2). After dehydration with anhydrous sodium sulphate, the extract was evaporated to remove the solvent on a water bath under reduced pressure. The residue was extracted five times with successive 10 ml of carbon tetrachloride. The extract was filtered and the filtrate was evaporated on a water bath under reduced pressure to remove the solvent as complete as possible. The residue, dissolved in about 10 ml of ethanol, was evaporated again to remove the solvent under reduced pressure. Another 10.0 ml of ethanol accurately measured was added to the residue, 1.5 ml of the resultant solution was accurately measured and water was added to it to make 100 ml. This was served as test solution. In another place, about 10.00 mg of reference standard of scopolamine was measured and dissolved in 4 ml of water. After addition of 1 ml of ammonia test solution, the mixture was treated just as in the case of test solution to make standard solution. The absorptions at 205 nm of the test solution and standard solution were measured using 1.5% of ethanol as a blank, it made A_t and A_s, respectively. Weight of scopolamine (mg) was calculated from following formula.
weight of scopolamine (mg)=10.00 (mg)×f×A_t/A_s
A_s: absorbance at 205 nm of standard solution,
A_t: absorbance at 205 nm of sample solution, f: purity of standard scopolamine
The experimental results revealed that recovery from the prepared test material (2.0 g of BSB with 9.75 mg of scopolamine added) was 96.5± 1.63 (average± standard deviation) by our method, and these corresponds with the result of gas chromatography.

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In the presence of excess cetylpyridinium chloride (CPC) in an acidic solution molybdenic acid(MoO₄^2-) forms a water soluble complex with phenylfluorone, whose color was very stable. The color development of a water soluble reddish brown complex between phenylfluorone and Zr(IV) enhanced by the catalytic action of sodium fluoride, particluary in the coexistence of excess CPC which increased the absorbance of the complex. Their absorption maximum wavelength were at about 540 nm (MoO₄^2-), 560 nm {Zr(IV)} against the reagent blank, and the coloration was stable over the pH range from 1.2 to 1.9 (pH was adjusted with NaCl glycocohol-HCl buffer solution) and pH range from 4.2 to 5.2 (pH was adjusted CH₃COONa-CH₃COOH buffer solution). A linear calibration curve was obtained for 460 μg MoO₄^2-/10 ml and 07 μg Zr(IV)/10 ml. According to Sandell's expression the sensitivities were 0.01 μg/cm² of MoO₄^2- and 0.002 μg/cm² of Zr(IV) for absorbance of 0.001. From the data, the recommended analytical procedures of MoO₄^2-, and Zr(IV) are as follows.
A sample containing 460 μg MoO₄^2- is taken in a 10.0 ml measuring flask, and 1.0 ml of 1.0 × 10^-2 M CPC solution, 2.0 ml of buffer solution(pH 1.5), and 2.0 ml of 1.0 × 10^-3 M phenylfluorone methanolic solution are added. The solution is diluted to 10.0 ml with water, and allowed to stand at 45°C for 30 minutes, the absorbance of phenylfluorone-MoO₄^2--CPC complex is measured at 540 nm against the reagent blank. To the solution of Zr(IV) is added each 1.5 ml of 1.0 × 10^-2 M CPC solution, 0.8 ml of 1.0 × 10^-2 M sodium fluoride solution, 1.0 ml of 2.0 × 10^-3 M methanolic phenylfluorone solution, and 1.5 ml of buffer solution (pH 4.6). After making up to the volume to 10.0 ml and allowing it to stand for 10 minutes, the absorbance is measured at 560 nm against the reagent. The effects of diverse ions on the absorbance of the complex were examined and shown.

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A solvent extraction-atomic absorption spectrophotometry for the determination of trace amount of iron in mercury for electrolysis has been investigated.
Isoamyl acetate was more suitable solvent than methyl isobutyl ketone or isopropyl ether, as for phase separation, flame stability and recovery of iron.
30 g of sample was dissolved with nitric acid and evaporated to dryness. It was dissolved with hydrochloric acid containing 12 drops of bromine, and evaporated to dryness again, and then dissolved with 8 N hydrochloric acid. Iron(III) in the solution was extracted by isoamyl acetate. The isoamyl acetate phase was washed by 8 N hydrochloric acid, and iron(III) in the solvent phase was determined by atomic absorption spectrophotometry at 2483.3 A. Iron(III) in the washed solvent phase, was also successfully determined by spectrophotometry after back-extraction with diluted hydrochloric acid. The absorbance of the back-extracted phase colored by ammonium thiocyanate was measured at 460 nm. By this method, many samples could be treated at the same time without any chelate reagent. The relative error of the results was less than 3%, and recovery was more than 95%.

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Recently the authors found that vanadium exhibits a marked depressive interference on the chromium absorption when the solution containing vanadium, chromium, and hydroxylamine is nebulized in an air-acetylene flame. This phenomenon was applied to the indirect determination of vanadium.
A sample containing 25300 μg of vanadium was taken in a 50 ml volumetric flask, and 5 ml of 250 μg/ ml of chromium, 5 ml of hydroxylamine hydrochloride solution (1.4M), and 2.5 ml of hydrochloric acid (1 M) were added to it, then the mixture was diluted to 50 ml with water. The solution was nebulized in the flame, and the chromium absorption was measured. The following conditions were used : lamp current, 10 mA; air flow, 6.6 l/min; acetylene flow, 1.6 l/min. The height in the flame at which the measurements were made was 8 mm above the burner top. The chromium line used was 3579 Å.
A small amount (0.56 μg/ml) of vanadium has been successfully determined by this method. The sensitivity of the method was 0.2 μg/ml.

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Determination of nitric acid in nitration solutions by means of the nitrate electrode was examined. For this purpose, the sample of nitration solution must be diluted to 100 times with water before the standard addition method is applied, because the assymmetric potential may change in the presence of nitrated organic compounds while the electrode slope (the increase of e.m.f. per ten-fold change in the nitrate content) remains almost unchanged if the concentration of sulfuric acid is kept constant.
The electrode slopes are much affected by the presence of sulfuric acid when the concentration of nitric acid is relatively high. It is also affected by the presence of nitric acid when the concentration of sulfuric acid is high. But, when the pH of solution is adjusted to about 10 by the addition of sodium hydroxide, the electrode slope is almost constant and approximately equal to the theoretical value.

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Commercially available iodide sensitive electrodes were found to be successfully used for the determination of mercury(II) ion, when the measurements were carried out in a 12 N nitric or sulfuric acid solution. A linear calibration curve was obtained within the concentration range of mercury 10^-7 to 10^-3 M, and the slope of the curve was about 49 mV per decade of the concentration.
The electrode potential reaches to a point of equilibrium within ten minutes after the electrode was immersed in the test solution having the concentration range of mercury presented above. A large amount of copper (II), lead, zinc, manganese (II), calcium, magnesium, aluminum, chromium (III, VI) and fluoride do not interfere the determination of mercury. Cyanide, bromide and chloride interfere to some extent.
The electrode was applied to detect the end point of the titration of thiocyanate with standard mercuric nitrate solution.

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A pink spot is revealed selectively by spraying the reagent of diacetylmonoxime-thiosemicarbazide-phosphoric acid to the urea which is isolated from several urine components by either paper or thin-layer plate chromatography. After development of the chromatograms, the reagent is sprayed on the chromatograms, and then they are heated at 80°C for 30 minutes in the case of paper, or heated at 105°C for 30 minutes in the case of thin-layer plate. The identification limit of urea is 3 μg. N-Monoalkyl and N-monoaryl derivatives of urea are also detected with the reagent as pink spots at a level of 310 μg. The spray reagent is prepared by the following procedure. A thiosemicarbazide solution is prepared by dissolving 0.1 g of thiosemicarbazide in 3 ml of water by heating in a water bath and by mixing the solution with 7 ml of ethanol. A diacetylmonoxime solution is prepared by dissolving 0.3 g of diacetylmonoxime in 1 ml of ethanol and by mixing the solution with 9 ml of phosphoric acid solution which consists of 85% phosphoric acid with water in a 1 : 6 volume ratio. The thiosemicarbazide solution is mixed with the diacetylmonoxime solution, allowed to stand for 20 minutes with occasional swirling, and then filtered. The filtrate is used as the spray reagent.

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Heterocyclic azo dyestuffs, 5-(2-pyridylazo)-2, 4-diaminotoluene(PADAT) and its analogs have been prepared, and their possible use as spectrophotometric reagents has been investigated. Cobalt(II) and PADAT in a slightly acid, neutral or alkaline medium form a yellowish brown complex. This complex, on addition of mineral acid, changes into a species of a deep violet color. The reagent itself and the violet complex are very stable even in strongly acid solutions, and the system follows Beer's law; the optimal range for measurements in a 1-cm cell is 0.0010.4 ppm cobalt in 27 N hydrochloric acid. The yellowish complex which is formed at pH 411 can be extracted with 3-methyl-butanol or n-butyl phosphate. The reagent blank is also negligiable at an absorption peak of the violet complex. Common anions and cations do not interfere. The molar absorption coefficient of 5-[ (3-benzyloxy-2-pyridyl) azo]-2, 4-diaminotoluene (3-Benzyloxy-PADAT), PADAT, 5-[(5-chloro-2-pyridyl) azo]-2, 4-diaminotoluene (5-Cl-PADAT), 5-[ (5-bromo-2-pyridyl) azo]-2, 4-diaminotoluene (5-Br-PADAT) and 5-[(3, 5-dichloro-2-pyridyl)azo]-2, 4-diaminotoluene (3, 5-diCl-PADAT) are 1.10×10⁵, 1.16×10⁵, 1.26×10⁵, 1.30×10⁵ and 1.38×10⁵ cm²mol^-1 at 591, 561, 573, 574 and 590 nm, respectively.

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[in Japanese]

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