Reagent composition for analysis of phosphate ion and kit for analysis of phosphate ion

Abstract

To provide a reagent composition for phosphate ion analysis capable of simply and rapidly analyzing phosphate ions contained in a sample liquid and enhanced in use safety.SOLUTION: The reagent composition for analysis of phosphate ions contains the following components (A), (B) and (C): (A) an acidic compound represented by the formula (1): (B): molybdic acid (VI) or a salt thereof (C): tin (II) salt SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a reagent composition for analyzing phosphate ions, which can easily and quickly analyze phosphate ions contained in sample solutions such as environmental waters such as rivers and closed water bodies, industrial wastewater, domestic wastewater, and drinking water. [Background technology]

[0002] Conventionally, the so-called molybdenum blue method has been used to analyze phosphate ions (phosphate phosphorus; PO4-P) contained in a sample solution. For example, in the industrial wastewater testing method according to JIS K 0102, sulfuric acid is added to the sample solution to make the pH of the sample solution strongly acidic (2 or less), hexaammonium heptamolybdate (VI) to react phosphate ions and molybdate ions in the sample solution to form a molybdophosphate complex, and ascorbic acid, a reducing agent for forming molybdenum blue (reduced molybdophosphate), and potassium antimonyl tartrate, a reaction accelerator, to the sample solution, and the absorbance of the resulting molybdenum blue is measured to quantify the amount of phosphate ions in the sample solution (Non-Patent Document 1).

[0003] Phosphorus-induced eutrophication is a problem in rivers and closed water bodies, and effluent standards for total phosphorus (TP) have been established under the Water Pollution Control Act. Therefore, the ability to analyze the phosphate ion content of environmental water, industrial wastewater, and other wastewater samples on-site would be useful for water quality management. The present applicant has developed a simple analytical kit that allows for convenient and rapid on-site analysis of phosphate ions in sample solutions, and has commercialized it as "Packtest (registered trademark) Phosphoric Acid" (Non-Patent Document 2). This simple analytical kit uses the molybdenum blue method as its measurement principle. It consists of two reagents: a liquid reagent containing sulfuric acid and molybdate to form a molybdophosphate complex, and a powder reagent containing a reducing agent to reduce the molybdophosphate complex to molybdenum blue. The intensity of the resulting molybdenum blue can be visually measured by colorimetry or absorbance, allowing for simple quantification of phosphate ions in a sample solution. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Japanese Industrial Standards, JIS K 0102, Testing Methods for Industrial Wastewater [Non-patent document 2] "Pack Test Phosphoric Acid," [online], Kyoritsu Chemical Research Institute, Inc. website, [searched July 1, 2024], Internet<URL:https: / / packtest.jp / products / wak_po4> Summary of the Invention [Problem to be solved by the invention]

[0005] As described above, the simplified phosphate ion analysis kit described in Non-Patent Document 2 consists of two components: a liquid reagent containing sulfuric acid and molybdate, and a powder reagent containing a reducing agent. The reason for the two-component configuration is that the sulfuric acid used to adjust the pH of the sample solution is a strongly acidic liquid, and the reducing agent is easily degraded. Therefore, when performing an analysis using this simplified analysis kit, a first step is required: adding the strongly acidic liquid reagent to the sample solution to adjust the pH of the sample solution to a strongly acidic state. A second step is then required: drawing the strongly acidic sample solution into a transparent resin tube containing the powder reagent and mixing it with the powder reagent containing the reducing agent. Because the liquid reagent contains sulfuric acid, its pH is below 2.0, and therefore it is classified as a corrosive liquid under the GHS classification of Category 1, which is corrosive to the skin and irritating to the eyes. Therefore, careful attention was required during the analysis to avoid contact with the strongly acidic liquid reagent or the strongly acidic sample solution.

[0006] The present invention has been made in view of the above-mentioned points, and an object of the present invention is to provide a reagent composition for analyzing phosphate ions, which can analyze phosphate ions simply and quickly and has improved safety in use. [Means for solving the problem]

[0007] To improve the safety of the phosphate ion analysis kit, the inventors investigated the use of a liquid reagent containing a low concentration of sulfuric acid, adjusting the sample solution to an acidic condition with a pH exceeding 2. Furthermore, the JIS K 0102 industrial wastewater testing method uses ascorbic acid as a reducing agent in the molybdenum blue method, and the use of antimony potassium tartrate as a reaction accelerator is essential. However, antimony compounds are highly toxic and are designated as deleterious substances even in compositions containing trace amounts. Therefore, the inventors considered using tin(II) salts as reducing agents in the molybdenum blue method, which do not require accelerators, are low in toxicity, produce molybdenum blue with high color intensity, and exhibit a rapid color development rate at room temperature.

[0008] However, when tin(II) salts are used as reducing agents, the dissolved oxygen typically present in sample solutions such as environmental water and wastewater oxidizes tin(II) to tin(IV), forming suspended solids derived from tin(IV), causing the color-developing solution to become turbid. This turbidity is particularly pronounced when the sulfuric acid concentration in the liquid reagent is adjusted to a low level so that the sample solution has an acidic pH of over 2. Therefore, when tin(II) salts are used as reducing agents, the visual colorimetry and absorbance analysis of molybdenum blue are hindered.

[0009] As a result of extensive research, the inventors have found that by using a specific acidic compound, phosphate ions can be analyzed accurately without causing turbidity in the color-developing solution, even when the pH of the sample solution is not set to a strongly acidic condition (pH 2 or less) and a tin(II) salt is used as a reducing agent. Based on this finding, the present invention has been completed.

[0010] In order to solve the above problems, the reagent composition for phosphate ion analysis of the present invention contains the following components (A), (B), and (C): component (A): an acidic compound represented by formula (1), component (B): molybdic acid (VI) or a salt thereof, and component (C): a tin (II) salt, wherein in formula (1), R represents a hydrogen atom or a methyl group.

[0011] [ka]

[0012] By preparing a reagent composition for analyzing phosphate ions containing an acidic compound represented by formula (1), molybdic acid (VI) or a salt thereof, and a tin (II) salt, a reagent composition for analyzing phosphate ions with improved safety can be obtained. The acidic compound represented by formula (1) is malonic acid (where R in formula (1) is a hydrogen atom) or methylmalonic acid (where R in formula (1) is a methyl group), both of which are dicarboxylic acids and highly safe compounds that do not fall under the category of deleterious or hazardous substances. The use of malonic acid or methylmalonic acid as the acidic compound makes it possible to easily adjust the pH of a sample solution to an acidic condition of above pH 2, which allows the formation of a molybdophosphate complex. Furthermore, even when a tin (II) salt is used as a reducing agent, the formation of suspended solids derived from tin (IV) can be suppressed, preventing the color-developing solution from becoming cloudy, enabling accurate analysis.

[0013] In the present invention, the term "phosphate ion" refers to "PO4 3- " refers to the inorganic phosphate ion (orthophosphate ion) expressed as HPO4 2- , H2PO4 - and H3PO4, etc., all of which are included in the phosphate ion.

[0014] In addition, it is also preferable that component (A) in the reagent composition for phosphate ion analysis of the present invention is malonic acid. This allows the selection of an acidic compound that is preferably used in the reagent composition of the present invention. That is, by using malonic acid, color development in the blank control is further reduced, allowing phosphate ion to be analyzed with high accuracy.

[0015] In addition, the tin(II) salt of component (C) in the reagent composition for phosphate ion analysis of the present invention is preferably tin(II) sulfate or tin(II) chloride. This allows the selection of a tin(II) salt suitable for use in the reagent composition of the present invention. These salts are used as reducing agents to reduce molybdophosphate complexes to molybdenum blue, and the color development intensity and speed of the molybdenum blue are excellent.

[0016] In addition, in the reagent composition for phosphate ion analysis of the present invention, the molybdic acid (VI) or its salt as component (B) is preferably ammonium molybdic acid (VI) or sodium molybdic acid (VI), thereby allowing selection of a molybdic acid salt suitable for use in the reagent composition of the present invention.

[0017] Furthermore, the reagent composition for analyzing phosphate ions of the present invention is preferably in the form of powder, granules or tablets.

[0018] A conventional phosphate ion analysis kit (Non-Patent Document 2) consists of two components: a liquid reagent containing sulfuric acid and a molybdate salt, and a powdered reagent containing a reducing agent. Therefore, analysis using this analysis kit requires a first step of adding the liquid reagent to a sample solution, followed by a second step of drawing the sample solution containing the liquid reagent into a transparent resin tube containing the powdered reagent and mixing it with the powdered reagent. Therefore, the two steps, the first and second steps, are time-consuming and laborious. However, according to the present invention, the reagent composition for phosphate ion analysis is a powdered, granular, or tablet-like reagent composition containing an acidic compound represented by formula (1), molybdic acid (VI) or a salt thereof, and a tin (II) salt. This allows molybdenum blue to be formed in a single step of adding the reagent composition of the present invention to the sample solution, thereby enabling the amount of phosphate ion in the sample solution to be analyzed more simply and quickly. Malonic acid or methylmalonic acid represented by formula (1) is an acidic compound that is solid at room temperature and has high water solubility, so when added to a sample solution, it dissolves rapidly and adjusts the pH of the sample solution to an acidic condition of above pH 2, at which a molybdophosphate complex can be formed. Furthermore, since the reagent composition according to the present invention is in the form of a powder, granule, or tablet, it is lightweight, highly portable, and easy to carry, and has excellent formulation stability and is easier to handle than a liquid reagent, allowing for safe analysis.

[0019] The phosphate ion analysis kit of the present invention preferably includes the above-described reagent composition for phosphate ion analysis and has a simple analytical device in which the above-described reagent composition for phosphate ion analysis is sealed in a resin tube. This provides a phosphate ion analysis kit that can safely analyze phosphate ions in a sample solution. Furthermore, since the phosphate ion analysis kit of the present invention includes a simple analytical device in which the above-described reagent composition for phosphate ion analysis is sealed in a resin tube, analysis can be performed simply, quickly, and safely by simply drawing the sample solution into the resin tube and mixing the reagent composition in the resin tube with the sample solution. [Effects of the Invention]

[0020] According to the present invention, it is possible to provide a reagent composition for analyzing phosphate ions and a kit for analyzing phosphate ions that have the following excellent effects. (1) It is highly safe to use because it does not use any reagent composition designated as a deleterious or hazardous substance and analysis can be performed without making the pH of the sample solution a strongly acidic condition (pH 2 or less). (2) The reagent composition for phosphate ion analysis can be formulated into a powder, granule, or tablet form, which allows for simple and rapid analysis of phosphate ions contained in a sample solution in a single operation. [Brief explanation of the drawings]

[0021] [Figure 1] 1 is a graph showing the results of examining acidic compounds used in the measurement of phosphate ions by the molybdenum blue method in Example 1, showing the absorbance (measurement wavelength: 580 nm, optical path length: 2 cm) of the color-developing solution when various types of acidic compounds and amounts of molybdate added were varied. The data are from the examination of the acidic compounds: (a) malonic acid, (b) methylmalonic acid, (c) ethylmalonic acid, (d) phenylmalonic acid, (e) citric acid, and (f) tartaric acid. [Figure 2] 1 is a graph showing the results of examining acidic compounds used in the measurement of phosphate ions by the molybdenum blue method in Example 1, showing the absorbance (measurement wavelength: 580 nm, optical path length: 2 cm) of the color-developing solution when the type of acidic compound and the amount of molybdate added were varied. The data are obtained by examining the following acidic compounds: (g) malic acid, (h) succinic acid, (i) maleic acid, (j) sulfamic acid, and (k) sodium hydrogen sulfate monohydrate, as well as (l) sulfuric acid as a comparative example. [Figure 3] 1 is a graph showing a calibration curve obtained by measuring the absorbance of a molybdenum blue color-developing solution produced using the reagent composition of the present invention and standard solutions with phosphate ion concentrations of 0 to 5 mg / L in Example 2. [Figure 4] 1 is a photograph showing the degree of blue color of a molybdenum blue color-developing solution produced using the reagent composition of the present invention and standard solutions with phosphate ion concentrations of 0 to 20 mg / L in Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0022] The reagent composition for phosphate ion analysis and the kit for phosphate ion analysis according to the present invention will be described below. The reagent composition for phosphate ion analysis according to the present invention is mainly composed of component (A): an acidic compound represented by the following formula (1) [in formula (1), R represents a hydrogen atom or a methyl group], component (B): molybdic acid (VI) or a salt thereof, and component (C): a tin (II) salt.

[0023] [ka]

[0024] (acidic compound) First, we will explain the acidic compound represented by formula (1) of component (A). This acidic compound adjusts the pH of the sample solution to an acidic condition above pH 2, thereby forming a molybdophosphate complex from phosphate ions and molybdate(VI) ions in the sample solution. The acidic compounds represented by formula (1) are malonic acid, where R in formula (1) is a hydrogen atom, and methylmalonic acid, where R is a methyl group. Both of these are solid acidic compounds at room temperature, but are readily soluble in water (malonic acid solubility: 140 g / 100 mL of water), so when added to a sample solution, they dissolve rapidly and adjust the sample solution to an acidic pH above pH 2. Furthermore, both malonic acid and methylmalonic acid are highly safe dicarboxylic acid compounds that are not classified as deleterious or hazardous substances. As shown in the examples below, when malonic acid or methylmalonic acid is used as the acidic compound, the formation of tin(IV)-derived suspended solids is suppressed, and the color-developing solution does not become cloudy, even when a tin(II) salt is used as a reducing agent in the molybdenum blue method. Therefore, a reagent composition for analyzing phosphate ions containing an acidic compound, molybdic acid or a salt thereof, and a tin(II) salt can be obtained, which has improved safety in use. Furthermore, since the acidic compound of component (A) is a solid at room temperature, the reagent composition for analyzing phosphate ions can be formulated in the form of a powder, granules, or tablet. In this case, molybdenum blue can be formed by a single operation of adding the powdered, granular, or tablet-form reagent composition to a sample solution, and the amount of phosphate ions contained in the sample solution can be analyzed simply, quickly, and safely.

[0025] In the reagent composition of the present invention, either malonic acid or methylmalonic acid alone or a combination of both can be used as the acidic compound of component (A). Furthermore, since malonic acid suppresses color development in a blank control containing no phosphate ions compared to methylmalonic acid, it is preferable to use malonic acid as the acidic compound from the viewpoint of enabling accurate analysis.

[0026] The amount of the acidic compound (component (A)) in the reagent composition of the present invention is preferably an amount that adjusts the pH of the sample solution to an acidic condition of greater than pH 2, sufficient to form a molybdophosphate complex, and allows smooth formation of the molybdophosphate complex from phosphate ions and molybdate (VI) ions in the sample solution. Specifically, the amount is preferably an amount that adjusts the pH of the sample solution to greater than 2 but not greater than 3, more preferably greater than 2 but not greater than 2.5, and particularly preferably greater than 2 but not greater than 2.2. The specific amount of component (A) is not particularly limited, as it depends on the pH and buffer capacity of the sample solution itself. For example, the amount of component (A) in the reagent composition of the present invention added to 10 mL of sample solution is preferably 50 mg to 1000 mg or 0.4 mmol to 10 mmol, more preferably 100 mg to 500 mg or 0.8 mmol to 5 mmol, and particularly preferably 150 mg to 300 mg or 1.2 mmol to 3 mmol.

[0027] (Molybdic acid (VI) or its salts) Next, we will explain molybdic acid (VI) or its salt, which is component (B). Molybdic acid or its salt generates molybdic acid ions, which then form a molybdophosphate complex with phosphate ions in the sample solution, and then form molybdenum blue (reduced molybdophosphate) with a reducing agent. The molybdic acid or its salt is not particularly limited as long as it is a compound applicable to the molybdenum blue method. Specific examples include molybdic acid (VI), lithium molybdate (VI), sodium molybdate (VI), potassium molybdate (VI), ammonium molybdate (VI), magnesium molybdate (VI), and calcium molybdate (VI). Of these, ammonium molybdate (VI) and sodium molybdate (VI) are preferred from the viewpoint of excellent analytical accuracy.

[0028] The amount of component (B) in the reagent composition of the present invention is preferably an amount that smoothly forms a molybdophosphate complex with phosphate ions in the sample solution and produces a molybdenum blue color with the tin(II) salt of the reducing agent. More specifically, when added to a phosphate ion standard solution having a median concentration within a predetermined range set as the analyzable phosphate ion concentration, the amount is preferably such that the absorbance at a measurement wavelength of 580 nm and a 2 cm path length is 1.0 or greater, more preferably 1.1 or greater, and particularly preferably 1.2 or greater, when added to a phosphate ion standard solution having a median concentration within the range set as the analyzable phosphate ion concentration. For example, when the analyzable phosphate ion concentration is set to 0 to 10 mg / L, the amount is preferably such that the absorbance at a measurement wavelength of 580 nm and a 2 cm path length is 1.0 or greater, more preferably 1.1 or greater, and particularly preferably 1.2 or greater, when added to a 5 mg / L phosphate ion standard solution. It is also preferable to select an amount that has little or no effect on the color development of a phosphate-free control (blank control), taking into consideration the color development level of the blank control.

[0029] For example, when the analyzable phosphate ion concentration is 0 to 10 mg / L and malonic acid is selected as the acidic compound, the amount of molybdic acid or a salt thereof (component (B)) in the reagent composition of the present invention added to 10 mL of sample solution is preferably 16.5 μmol to 27.5 μmol, more preferably 17.5 μmol to 25.0 μmol, and particularly preferably 19.0 μmol to 22.5 μmol. When methylmalonic acid is selected as the acidic compound, the amount of molybdic acid or a salt thereof (component (B)) in the reagent composition of the present invention added to 10 mL of sample solution is preferably 13.5 μmol to 22.5 μmol, more preferably 15.0 μmol to 19.5 μmol.

[0030] (Tin(II) salts) Next, the tin(II) salt of component (C) will be described. The tin(II) salt is a component for reducing the molybdophosphate complex to molybdenum blue (reduced molybdophosphate). In the reagent composition of the present invention, a tin(II) salt is selected as the reducing agent for the molybdenum blue method, because it has low toxicity, produces a high molybdenum blue color, and exhibits a fast color development rate. In particular, when the pH of the sample solution is not kept at a strongly acidic condition (pH 2 or less), the use of a tin(II) salt as a reducing agent for the molybdenum blue method can result in the oxidation of tin(II) to tin(IV) by the dissolved oxygen typically present in sample solutions such as environmental water and wastewater, resulting in the formation of tin(IV)-derived suspended solids, making analysis difficult. However, in the present invention, the action of malonic acid or methylmalonic acid, component (A) added as an acidic compound, suppresses the formation of tin(IV)-derived suspended solids, preventing the color-developing solution from becoming cloudy.

[0031] The tin(II) salt is not particularly limited as long as it is a compound capable of reducing the molybdophosphate complex to molybdenum blue. Specific examples include tin(II) sulfate, tin(II) fluoride, tin(II) chloride, tin(II) bromide, tin(II) iodide, and tin(II) acetate. Among these, tin(II) sulfate and tin(II) chloride are preferred for their superior analytical accuracy. The amount of component (C) in the reagent composition of the present invention is preferably an amount sufficient to sufficiently reduce the molybdophosphate complex to molybdenum blue and obtain a color-developing solution. More specifically, the amount of the tin(II) salt of component (C) in the reagent composition of the present invention added to 10 mL of sample solution is preferably 1 μmol to 40 μmol, more preferably 2 μmol to 30 μmol, and particularly preferably 5 μmol to 20 μmol, depending on the amount of phosphate ions and molybdic acid or a salt thereof contained in the sample solution.

[0032] (Other ingredients) Furthermore, the reagent composition for phosphate ion analysis according to the present invention may contain other components in addition to those described above, as long as the effects of the present invention are not impaired. Examples of other components include antioxidants such as ascorbic acid, bulking agents such as inorganic salts, and binders such as coating agents.

[0033] (Reagent composition for analyzing phosphate ions) The reagent composition for phosphate ion analysis according to the present invention is a reagent composition containing the above-described components (A) to (C). Therefore, the reagent composition according to the present invention is not designated as a deleterious or hazardous substance, and analysis can be performed without requiring the sample solution to be kept at a strongly acidic pH (pH 2 or less). This reagent composition is therefore highly safe to use, easy to handle, and allows for safe analysis. The reagent composition for phosphate ion analysis according to the present invention can be in any form, including liquid and non-liquid. Liquid refers to a form in which the reagent composition is dissolved or dispersed in water or other solvent. When the reagent composition for phosphate ion analysis according to the present invention is a liquid composition, the above-described components can be mixed and dissolved or dispersed in a solvent to form a single liquid composition, or the individual components can be separated and housed in multiple containers to prevent contact between easily reactive components. The reagent composition for phosphate ion analysis according to the present invention can also be a combination of a liquid reagent and a non-liquid reagent.

[0034] The reagent composition for phosphate ion analysis according to the present invention is preferably in a non-liquid form. Non-liquid forms include solid and gel forms, but a solid form is preferred. Solid forms include powder, granules, and tablets. By making the reagent composition for phosphate ion analysis a solid, more specifically, a powder, granule, or tablet form, it becomes easier to carry and handle, and analysis can be performed safely. This allows molybdenum blue to be formed by a single operation of adding the powder, granule, or tablet reagent composition to a sample solution, thereby enabling the amount of phosphate ions contained in the sample solution to be analyzed more simply and quickly. Furthermore, by making the reagent composition for phosphate ion analysis a non-liquid, preferably a solid, reagent composition, the reactivity between the components is weakened compared to when it is in a liquid form, making it possible to prepare a single-component reagent composition in which the components are mixed. The reagent composition for phosphate ion analysis according to the present invention is in a non-liquid form, preferably a solid form, more preferably a powder, granule or tablet form. Since all of the constituent components (A) to (C) are readily soluble in water, they dissolve quickly and exert their functions in the sample solution.

[0035] When the reagent composition for phosphate ion analysis according to the present invention is prepared in the form of a powder, granule, or tablet, the above-described components may be mixed together or other components, such as bulking agents or excipients, may be added and uniformly dispersed to form a reagent composition. Alternatively, the components may be separated into multiple tablets to prevent contact between the easily reactive components, or the components may be coated to reduce contact. For example, a reagent composition containing two small tablets containing the acidic compound (component (A)) and the molybdate (VI) salt (component (B)) and the tin (II) salt (component (C)) together with a bulking agent may be prepared. Alternatively, a granular reagent composition in which each component is coated in a multilayer structure may be prepared by mixing the tin (II) salt (component (C)) and the bulking agent to form granules, applying a coating of cyclic oligosaccharides or the like to the surface of the granules, followed by the molybdate (VI) salt (component (B)), and then applying a coating of cyclic oligosaccharides or the like to the surface of the granules, followed by the acidic compound (component (A)).

[0036] In the molybdenum blue method, to perform highly accurate analysis, it is important that molybdenum blue is formed by a reducing agent after sufficient formation of a molybdophosphate complex from phosphate ions in the sample solution. Therefore, it is preferable to prepare the reagent composition for phosphate ion analysis according to the present invention so that the tin(II) salt of component (C) dissolves after the acidic compound of component (A) and the molybdate(VI) salt of component (B) dissolve in the sample solution to form the molybdophosphate complex. For example, immediate-release granules are prepared by granulating the acidic compound of component (A) and attaching the molybdate(VI) salt of component (B) to the surface of the granules. Meanwhile, delayed-release granules are prepared by coating the surface of granules prepared by mixing the tin(II) salt of component (C) with a cyclic oligosaccharide or other coating agent. The thus-obtained immediate-release granules and delayed-release granules are preferably combined to form the reagent composition according to the present invention. When the reagent composition prepared in this manner is added to a sample solution, the acidic compound of component (A) and the molybdic acid of component (B) that constitute the rapid-release granules dissolve in the sample solution first, and then the tin (II) salt of component (C) that constitutes the slow-release granules dissolves in the sample solution, thereby improving analytical accuracy.

[0037] (Method for analyzing phosphate ions using a reagent composition for analyzing phosphate ions) A method for using the reagent composition for phosphate ion analysis according to the present invention, i.e., a method for analyzing phosphate ions using this reagent composition for phosphate ion analysis, will be described below. The analytical method generally comprises the steps of adding the reagent composition for phosphate ion analysis to a sample solution and dissolving the reagent composition in the sample solution, and measuring the color of the sample solution (color-developing solution) in which molybdenum blue has been produced.

[0038] When the reagent composition for phosphate ion analysis according to the present invention is added to a sample solution, the components (A) to (C) of the reagent composition react with the phosphate ions in the sample solution, producing molybdenum blue, causing the sample solution to develop a color. The color of the sample solution colored with molybdenum blue (color-developed solution) is measured preferably 30 seconds to 5 minutes, more preferably 1 to 5 minutes, and even more preferably 2 to 4 minutes after adding the reagent composition to the sample solution. The color measurement can be performed by visual colorimetry or by measuring absorbance with an absorptiometer. Specifically, the concentration of phosphate ions in the sample solution can be determined by comparing the color with a standard color or a calibration curve obtained by previously analyzing a standard solution of phosphate ions. The reagent composition according to the present invention uses a tin(II) salt as a reducing agent for component (C), but the action of component (A) used as an acidic compound suppresses the formation of suspended solids derived from tin(IV), enabling accurate analysis.

[0039] (Phosphate ion analysis kit) The phosphate ion analysis kit according to this embodiment includes the above-described reagent composition for phosphate ion analysis. Other components of the phosphate ion analysis kit may include a container or dispensing tool for measuring the amount of sample liquid, a kit instruction manual, and a colorimetric (colorimetric) sample. The reagent composition for phosphate ion analysis constituting the phosphate ion analysis kit may be provided as a simple analytical device enclosed in a transparent resin tube. More specifically, for example, a transparent cylindrical resin tube in which the reagent composition is enclosed, the upper and lower ends of the resin tube are fused, with the upper end sealed with a thin rod-shaped plug inserted therein, is preferably configured similarly to the Packtest (registered trademark; Kyoritsu Chemical Research Institute, Inc.) series. Therefore, by removing the thin rod-shaped plug inserted in the upper seal, an introduction channel for introducing sample liquid into the resin tube is formed, allowing the sample liquid to be drawn into the resin tube and mixed with and dissolved in the reagent composition. Therefore, phosphate ion analysis can be performed more simply, quickly, and safely with a single operation. [Example]

[0040] The present invention will be described in detail below using examples.

[0041] [Example 1] 1. Examination of acidic compounds In this example, we investigated acidic compounds used to make the pH of a sample solution acidic when measuring phosphate ions using the molybdenum blue method. The acidic compounds investigated in this example are shown in Table 1 below. All of these are solid acidic compounds at room temperature. As a comparative example, we also conducted a similar test using sulfuric acid, a liquid acidic compound used in the molybdenum blue method of JIS K 0102.

[0042] [Table 1]

[0043] The test was performed as follows. As shown in Table 2, 30 mg of solid acidic compounds from each test group, a predetermined amount of sodium molybdate (Na2MoO4·2H2O, molecular weight: 241.97) was added in stages ranging from 0 mg to 2 mg, and 7.5 μL of a 1000 mg / L phosphate standard solution was added as the sample solution. The solution was then filled up to 1500 μL with purified water. Each acidic compound and sodium molybdate were dissolved in purified water as appropriate. The final phosphate concentration was 5 mg / L. This solution was measured using a spectrophotometer [Digital Pack Test Multi SP; Model No. DPM-MTSP] and a dedicated cell (2 cm path length, 1.5 mL capacity, both manufactured by Kyoritsu Chemical Research Institute, Inc.) and zero-corrected.

[0044] To the zero-corrected solution, 0.4 mg of tin sulfate (SnSO4, molecular weight: 214.77) was added and the solution was shaken approximately 30 times to thoroughly dissolve and mix the tin sulfate, forming molybdenum blue. Three minutes after adding the tin sulfate, the absorbance of this color-developing solution was measured at a wavelength of 580 nm using a spectrophotometer. After measuring the absorbance, the pH of the color-developing solution was also measured.

[0045] [Table 2]

[0046] As a blank control, the same test as above was carried out, except that the 1000 mg / L phosphate standard solution was replaced with pure water.

[0047] For the comparative sulfuric acid solution, 30 μL (30 μmol: equivalent to 2.9 mg) of 1 mol / L sulfuric acid (Fujifilm Wako Pure Chemical Corporation, volumetric grade) was added, and sodium molybdate (Na2MoO4·2H2O, molecular weight: 241.97) was added in predetermined amounts ranging from 0 mg to 2 mg. 7.5 μL of a 1000 mg / L phosphate standard solution was added as the sample solution, and the solution was then filled up to 1500 μL with purified water. The solution was zero-corrected under the same conditions and method as in the above-mentioned examples. Then, 0.4 mg of tin sulfate (SnSO4, molecular weight: 214.77) was added and the solution was shaken approximately 30 times to thoroughly dissolve and mix the tin sulfate and form molybdenum blue. Three minutes after the addition of the tin sulfate, the absorbance of this color-developing solution was measured at a wavelength of 580 nm using a spectrophotometer. After measuring the absorbance, the pH of the color-developing solution was also measured.

[0048] The results of each test are shown in Figures 1 and 2 and Tables 3 to 14. Because the upper limit of absorbance measurement for the spectrophotometer used for absorbance measurement is 2.0, the absorbance value "2.000" shown in the tables indicates that the value is above the detection limit. Table 15 also shows the results of observing the color development of molybdenum blue, the color development of the blank, and the turbidity of the color-developed solution for each test group and comparative example. In the graphs of Figures 1 and 2, square markers indicate the test results of sample solutions containing phosphate ions, while open square markers indicate the test results of blanks (containing no phosphate ions). The dotted line near an absorbance of 1.2 in each graph indicates the absorbance of the color-developed solution when a sample solution with a phosphate ion concentration of 5 mg / L was measured using a simple phosphate ion analysis kit developed by the applicant and shown in Non-Patent Document 2.

[0049] These results demonstrate that the use of malonic acid or methylmalonic acid as an acidic compound enables accurate analysis of phosphate ions without turbidity in the color-developing solution, even when a tin(II) salt is used as a reducing agent in the molybdenum blue method. Furthermore, the pH of the sample solution was greater than 2.0 and less than or equal to 2.2. On the other hand, as shown in Figure 1 and Table 15, when using ethylmalonic acid and phenylmalonic acid, among the four malonic acid analogs, the absorbance of the blank control increased and the color-developing solution became turbid, indicating that these were unsuitable. Furthermore, no molybdenum blue color development was observed with citric acid, tartaric acid, or malic acid. Succinic acid, maleic acid, sulfamic acid, sodium hydrogen sulfate, and sulfuric acid (comparison example) all exhibited high absorbance in the blank control and generated turbidity in the color-developing solution due to suspended solids derived from tin(IV), indicating that these were unsuitable. These findings indicate that malonic acid and methylmalonic acid are suitable acidic compounds, and that malonic acid is a particularly suitable acidic compound, as the absorbance of the blank control was also kept low.

[0050] Furthermore, under the conditions set in this example, where the sample solution was 1.5 mL and the phosphate ion concentration was 5 mg / L, when malonic acid was selected as the acidic compound, it was found that the amount of sodium molybdate (Na2MoO4·2H2O, molecular weight: 241.97) added was preferably 0.6 mg to 1.0 mg (2.48 μmol to 4.13 μmol), more preferably 0.65 mg to 0.9 mg (2.69 μmol to 3.72 μmol), and particularly preferably 0.7 mg to 0.8 mg (2.89 μmol to 3.31 μmol). Furthermore, when methylmalonic acid was selected as the acidic compound, it was found that the amount of sodium molybdate (Na2MoO4·2H2O, molecular weight: 241.97) added was preferably 0.5 mg to 0.8 mg (2.07 μmol to 3.31 μmol), and more preferably 0.55 mg to 0.7 mg (2.27 μmol to 2.89 μmol).

[0051] [Table 3]

[0052] Table 4

[0053] Table 5

[0054] Table 6

[0055] Table 7

[0056] Table 8

[0057] Table 9

[0058] Table 10

[0059] Table 11

[0060] Table 12

[0061] Table 13

[0062] [Table 14]

[0063] [Table 15]

[0064] [Example 2] 2. Preparation of a calibration curve using phosphate standard solutions 150 g of malonic acid, 3.5 g of sodium molybdate(VI) dihydrate, and 2 g of tin(II) sulfate were placed in a mortar and thoroughly mixed to obtain a powdered reagent composition. Separately, standard solutions of various concentrations were prepared using the phosphate ion standard solution used in Example 1, with phosphate ion concentrations of 0 mg / L (no additives), 0.1 mg / L, 0.2 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 3 mg / L, and 5 mg / L. 1.5 mL of each standard solution was added to 75 mg of the powdered reagent composition, and the mixture was shaken approximately 30 times to thoroughly dissolve and mix the reagent composition. The absorbance of the solution was measured at a wavelength of 600 nm 3 minutes after the addition of the standard solution.

[0065] The calibration curves prepared based on the absorbance measurements are shown in Figure 3. These results demonstrate that the reagent composition of the present invention provides a linear proportional relationship between phosphate ion concentration and absorbance, making it possible to quantify phosphate ion. Furthermore, 3 minutes after the addition of the standard solution, the solution was clear and no turbidity due to tin(IV) was observed. Therefore, by visually comparing the blue color intensity of the molybdenum blue in the colored solution, the phosphate ion concentration could be quantified, as shown in Figure 4.

[0066] The present invention is not limited to the above-described embodiments or examples, and its technical scope also includes various modified designs within the scope that does not deviate from the gist of the invention described in the claims.

Claims

1. A reagent composition for analyzing phosphate ions, comprising the following components (A), (B) and (C): (A): an acidic compound represented by formula (1) 【Chemistry 1】 [In formula (1), R represents a hydrogen atom or a methyl group.] (B): Molybdic acid (VI) or its salt (C): Tin(II) salt

2. 2. The reagent composition for analyzing phosphate ions according to claim 1, wherein the component (A) is malonic acid.

3. 2. The reagent composition for analyzing phosphate ions according to claim 1, wherein the tin (II) salt of component (C) is tin (II) sulfate or tin (II) chloride.

4. 2. The reagent composition for analyzing phosphate ions according to claim 1, wherein the molybdic acid or a salt thereof of component (B) is ammonium molybdic acid (VI) or sodium molybdic acid (VI).

5. 2. The reagent composition for analyzing phosphate ions according to claim 1, which is in the form of a powder, granules or tablet.

6. A phosphate ion analysis kit comprising the reagent composition for phosphate ion analysis according to any one of claims 1 to 5.

7. A phosphate ion analysis kit comprising a simple analytical device in which the reagent composition for phosphate ion analysis according to any one of claims 1 to 5 is sealed in a resin tube.

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