BUNSEKI KAGAKU Abstracts

A fluorometric determination of boron was investigated by flow injection analysis with 2,3-dihydroxynaphtalene (2,3-DHN). In the determination of boron, a method using a boron-2,3-DHN-insulin ternary complex was attempted in order to remove any influence of excess reagent on the precision of boron determination. Catechol and chromotropic acid except 2,3-DHN as Huorometric reagents were examined from standpoint decreasing the effect of a large reagent blank value. As a result, 2,3-DHN was better than them as a fluorometric reagent. The flow system was composed of three channels, including the carrier (1.9 mL/min), reagent (a mixture of 2,3-DHN, Mn(II), H₂O₂) (0.9 mL/min) and insulin (0.2 mL/min). In the presence of Mn(II) and H₂O₂, 2,3-DHN formed a radical, which accelerated the formation of a ternary complex in an alkali solution at pH 11. With the reagent solution containing the radical, the ternary complex formed effectively at pH 7.6. The ratio of the boron-DHN complex composition in the ternary complex was boron: DHN＝1 : 2. On the other hand, the ratio between insulin and boron was 1 to above 1. A number of B-DHN complexes were connected to the surface of one insulin molecule. The B-DHN-insulin complex was determined in an aqueous solution by detecting the fluorometric intensity (λ_ex＝400 nm, λ_em＝500 nm). The calibration curve for the constructed boron was linear over the range of 0 to 0.5 ppm. The limits of detection and determination for boron were 3.7 and 12 ppb, respectively. The relative standard deviation at B 20 ppb was 2.6％ (n＝6). The determination results for boron in a river reference material showed good agreement with the certified value.

Three-dimensional excitation emission matrix (3-D EEM) spectroscopy was applied to deep groundwaters, which have low dissolved organic carbon (DOC) concentration below 1 mg L⁻¹ and collect a small volume of samples (about 500 mL) by a multiple piezometer system, to study validity on the convenient measurement of aquatic humic substances without the isolation and purification procedures. From comparison with deep groundwaters and isolated humic substances, 3-D EEM spectra of the groundwaters with the DOC concentration of 0.08 to 0.1 mg L⁻¹ can detect fulvic-like materials. Fluorescence peak position and relative fluorescence intensity (RFI) of groundwater fulvic-like materials at four depths in a borehole were different from those of groundwaters in sedimentary rock and granite. The RFI and RFI/absorbance at 280 nm of fulvic-like materials in sedimentary groundwaters also have some variations. These results indicate that 3-D EEM spectroscopy is a convenient and useful method for the measurements of concentration and characteristics of humic substances in deep groundwaters.

The size of the blank value is enumerated in one of the factors that influence the detection limit in the fluorescence determination method, and it is brought about by excess reagent. In general, removing the influence of the excess reagent is needed because a high blank value may often cause a decrease in the reproducibility. To remove the influence of excess reagent, the solvent-extraction method etc. is used besides a method of changing the pH. 1,8-Dihydroxynaphthalene (1,8-DHN) with a similar structure to chromotropic acid is a reagent that is easily extracted into organic solvent because it doesn't have a sulfo group. 1,8-DHN generates a complex of boron to be the 6-membered ring. This study attempted a fluorescence determination of boron by keeping the complex in the aqueous phase, and extracted the excess reagent in the organic phase. The boron-1,8-DHN complex in aqueous phase was detected by measuring the fluorescence intensities (λ_ex＝305 nm, λ_em＝360 nm). Moreover, the calibration curve by using a synchronous - derivative fluorescence method was linear over the range of 0 to 120 ppb. The limit of detection was 1.7 ppb. The relative standard deviation at 30 ppb B was 2.8％ (n＝5). The detection limit was improved to about 1/10 by using the synchronous - derivative fluorescence method. The result of applying this proposed method to standard river water showed good agreements with the certified values.

On-column dissociation rate constant (k_d) of the Ni(II) complex with pyridine-2-aldehydebenzoylhydrazone (PAB) has been determined using the high performance liquid chromatographic kinetic analysis method to estimate the effect of the properties of the stationary phase on the on-column dissociation reaction characteristics of Ni(II)-PAB complex. In the case of using the column with endcapped packing material, smaller k_d was obtained as compared with that in the case of using the column with non-endcapped one. The dissociation rate constants of the Ni(II)-PAB complex in the stationary phase and those of in the mobile phase (k_d,s, k_d,m) was estimated from the values of k_d. The value of k_d,s reduced with the addition of tetrabutylammonium ion to the eluent as the blocking agent of the silanol groups. It was thus suggested that the on-column dissociation reaction of the Ni(II)-PAB complex was accelerated by the silanol groups on the surface of the silica support. It is cleared that the dissociation reaction of the Ni(II)-PAB complex in the stationary phase dominates the total on-column dissociation reaction of the complex and the properties of the stationary phase are the important factor to determine the on-column dissociation reaction characteristics of the metal complexes.

A spectrophotometric method to determine nitrate-nitrogen was developed based on the reaction of anisole with nitrate in the presence of sulfuric acid and sodium chloride. Anisole was added as its methanol solution to give homogeneous color-developed solutions. Color development is probably ascribed to the formation of a nitroso-dervative of anisole caused by nitrosyl chloride, which resulted from the reaction of nitrate and chloride under strongly acidic conditions. The nitrate-nitrogen contents in well-water samples determined by the present method were in good agreement those determined by ion chromatography. The sensitivity of the present method is higher than those of similar methods based on water-soluble aromatic compounds.

The carbazole-sulfuric acid method (determination of D-glucuronic acid) has been used as a general determination method for sodium chondroitin sulfate (ChS-Na) in dosage forms. However, this method requires a complicated column pretreatment to isolate ChS-Na in the dosage form. Among the various determination methods for ChS-Na, we focused on a determination method using Alcian Blue (AB method), and examined the analytical conditions necessary to apply to tablets. As a result, we not only found that this was a simple determination method, but also established it as an excellent method, which shows good specificity, linearity, accuracy and precision. The ChS-Na content in bulk powder products, measured by the AB method, had a comparatively high correlation with the sulfur content (r＝0.81) measured by oxygen flask combustion and the D-glucuronic acid content (r＝0.87), measured by the carbazole-sulfuric acid method, which are the conventional determination methods.

The determination of the total nitrogen in soy sauce by the Dumas method was studied. Eleven samples of soy sauce were used and a half gram of each sample was combusted at 870°C in an oxygen atmosphere. The resulting nitrogen oxides (NO_x) were reduced to nitrogen by a copper column at 600°C, and nitrogen was detected with a thermal conductivity detector. Analyses of each sample were repeated 3 times on 2 different days. The samples were also analyzed by the Kjeldahl method under the optimum digestive condition in order to know the differences between the methods. The standard lysine solution was analyzed by the Dumas method, and accurate results were obtained. Two sets of data by the Dumas and the Kjeldahl methods were tested with a two-sided Welch t-test. There were significant differences in six samples between the methods (p＜0.05). However, the differences between the methods were within the repeatability limit, calculated by the Horwitz equation. Also, the correlation between the methods was 0.9999. The Dumas method had excellent intermediate precision, and was equivalent to, or more accurate than, the Kjeldahl method under the optimum digestive condition.

In order to confirm the formation of ternary aluminum-citrate-phosphate complexes, which have been predicted by a potentiometric study on the reaction between aluminum ion, citrate and phosphate in aqueous solution, ²⁷Al and ³¹P NMR spectra for solutions containing aluminum ion (Al) citrate (L) and phosphate (P) {Al : L : P mole ratio＝1 : 1 : n (n＝0〜5)} at pH 3.0 and 4.0 were measured. In a solution of pH 3.0 with Al : L＝1 : 1 in the absence of P, a mononuclear Al-L complex in addition to [Al(H₂O)₆]^3＋ existed, while in a solution with Al : L : P＝1 : 1 : 1 of the same pH, the formation of a ternary complex (P-Al-L) was confirmed by ²⁷Al and ³¹P NMR measurements. The broad ²⁷Al and ³¹P NMR peaks assigned to the P-Al-L were observed around 0 ppm and −4.8 ppm, respectively. The intensity of both peaks increased with increasing the proportion of P. In a solution with the same composition of pH 4.0, the formation of a trinuclear complex, (Al₃-L₃), the major species in the solution with Al : L＝1 : 1 in the absence of P, was prevented due to the predominant formation of the P-Al-L.

Eight pyridylamino (PA) derivatives of fucose-containing oligosaccharides and ten high mannose-type PA-oligosaccharides were separated by high performance liquid chromatography (HPLC) on a triacontyl (C30) silica-gel column. The eight fucose-containing PA-oligosaccharides and eight out of ten high mannose-type PA-oligosaccharides were clearly separated by C30 reversed-phase HPLC at a column temperature of 40°C with a fluorescence detector; two high mannose-type PA-oligosaccharides, Man7-GlcNAc2 and Man8-GlcNAc2, were not separated. The two unresolved PA-oligosaccharides were finally separated by C30 reversed-phase HPLC at a column temperature of 10°C. The C30 reversed-phase HPLC is quite useful for the analysis of various types of PA-oligosaccharides, including positional isomers, because of excellent separation compared with that of C18 reversed-phase HPLC. The C30 reversed-phase HPLC system can be applied to HPLC-mass spectrometry for re-confirmation of the structures of the PA-oligosaccharides.