Method and apparatus for producing urea-free water, and method and apparatus for quantifying urea

Abstract

To provide urea-free water substantially rid of urea, the urea-free water capable of being used, for example, for preparing a concentration standard solution when quantifying urea in sample water and as carrier water of an analyzer and not affecting a measured value of urea concentration in the sample water.SOLUTION: Urea-free water is produced by performing a step of obtaining treatment water by passing water through carriers (immobilized enzymes 51) in which enzymes including at least urease are immobilized, and a post-treatment step of obtaining the urea-free water by removing or inactivating enzymes leaked into the treatment water. The post-treatment step is, for example, a step of passing the treatment water through anion exchange resin 53.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to the quantitative determination of urea in water, and in particular to a method and apparatus for producing urea-free water that can be used when quantifying urea, and a method and apparatus for quantifying urea using the urea-free water thus obtained. [Background technology]

[0002] There is a demand for accurate analysis and quantification of minute amounts of urea in water. For example, when pure water is produced from raw water by a pure water production system or an ultrapure water production system, it is difficult to remove urea in the raw water using an ion exchange device or an ultraviolet oxidation device that constitutes the pure water production system, so it is necessary to supply raw water from which urea has been removed in advance to the pure water production system. As a method for removing urea, a method is known in which a chemical that generates hypobromous acid is added to the raw water and urea is selectively oxidized by hypobromous acid, but since the chemical that generates hypobromous acid also becomes a load on the pure water production system, it is better to input the smaller the amount of chemical. Therefore, it is desired to determine the necessity of urea treatment by quantifying the urea concentration in the raw water, and to input an appropriate amount of chemical when treatment is necessary. In addition, there is a demand for measuring the urea concentration in the pure water obtained from the pure water production system.

[0003] As a method for quantifying urea, a quantitative determination method based on colorimetry using diacetyl monoxime (for example, the method described in Hygienic Test Methods (Non-Patent Document 1)) is known. In the colorimetry using diacetyl monoxime, other reagents (for example, antipyrine + sulfuric acid solution, aqueous solution of semicarbazide hydrochloride, aqueous solution of manganese chloride + potassium nitrate, sodium dihydrogen phosphate + sulfuric acid solution, etc.) can be used in combination for the purpose of promoting the reaction. When antipyrine (1,5-dimethyl-2-phenyl-3-pyrazolone) is used in combination, diacetyl monoxime is dissolved in an acetic acid solution to prepare a diacetyl monoxime acetate solution, antipyrine is dissolved in, for example, sulfuric acid to prepare an antipyrine-containing reagent solution, the diacetyl monoxime acetate solution and the antipyrine-containing reagent solution are mixed with the sample water in sequence, the absorbance at a wavelength of about 460 nm is measured, and the quantitative determination is performed by comparing with a standard solution.

[0004] The method for quantifying urea by colorimetry using diacetyl monoxime is intended to quantify urea in, for example, pool water or public bath water, and therefore has poor sensitivity as a method for quantifying urea in raw water supplied to a pure water production process. Patent Document 1 discloses a method for continuously quantifying urea in sample water in a concentration range from below ppb to several ppm by applying flow injection analysis based on the colorimetry using diacetyl monoxime to measure absorbance. Patent Document 2 discloses that when quantifying urea by applying flow injection analysis based on the colorimetry using diacetyl monoxime, continuous automatic measurement can be performed online for a long period of time with stability by refrigerating the reagent used in the reaction. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2000-338099 A [Patent Document 2] JP 2018-179545 A [Non-patent literature]

[0006] [Non-Patent Document 1] The Pharmaceutical Society of Japan, Hygienic Test Methods, Commentary 1990.4.1.2.3(13)1 (1990 edition, 4th printing, supplement (1995), p1028), 1995 Summary of the Invention [Problem to be solved by the invention]

[0007] The methods described in Patent Documents 1 and 2 are capable of quantifying urea with high sensitivity by using flow injection analysis. However, if urea is contained in the water used in carrying out this quantification method, for example, the water used in preparing a concentration standard solution or the carrier water in flow injection analysis, the urea contained in the water will be a cause of analytical errors. Even when urea is quantified by a method other than flow injection analysis, if urea is contained in any of the water used in the quantification operation, it will be a cause of analytical errors. For example, even when performing analysis by liquid chromatography, if urea is contained in the water, i.e., the carrier water, when water is used as the mobile phase, analytical errors will occur. In particular, when analyzing trace amounts of urea at the μg / L level contained in sample water, if urea is contained in the water used to prepare the standard solution or the carrier water, even at the μg / L level, the impact will be large.

[0008] As described above, it is difficult to remove urea by ion exchange treatment or ultraviolet oxidation treatment, and even when ultrapure water is produced by an ultrapure water production system, traces of urea may be contained in the ultrapure water produced due to incomplete treatment caused by fluctuations in the quality of the raw water or malfunctions of the equipment, and the concentration of urea may vary. Therefore, even if ultrapure water is used to prepare concentration standards or as carrier water, the results of the urea trace analysis may be inaccurate. In addition, even if water from which urea has been removed can be prepared for the quantitative determination of urea in sample water, if the components contained in the water affect the measured value of the urea concentration in the sample water, the quantitative determination result of urea will be inaccurate.

[0009] The object of the present invention is to provide a method and apparatus for producing urea-free water that can be used when analyzing urea in sample water, the urea concentration of which has been reduced to a level that does not affect at least the analysis and does not affect the measured value of the urea concentration in the sample water, and a quantitative determination method and analytical apparatus that can accurately quantify trace amounts of urea in sample water by using this urea-free water. [Means for solving the problem]

[0010] Urease (EC 3.5.1.5) is an enzyme that hydrolyzes urea. In the presence of water, urease can break down even very low concentrations of urea into carbon dioxide and ammonia. (NH2)2CO + H2O → CO2+NH3

[0011] According to the studies of the present inventors, as shown in the Examples described later, by treating water with urease, the urea concentration in the water can be reduced to 1 μg / L or less, and urea-free water can be obtained from which urea has been removed to a level that does not affect analysis. However, if urease is contaminated in urea-free water, when the urea-free water is used to quantify urea in a water sample, the urease will decompose the urea in the water sample, resulting in a quantitative result that is lower than the actual urea concentration.

[0012] Therefore, the method for producing urea-free water of the present invention includes a treatment step of contacting urea-containing water with an enzyme containing at least urease to obtain treated water in order to prevent urease from being mixed into the urea-free water, and a treatment step of removing or inactivating the enzyme leaked into the treated water. , urea concentration is 1μg / L or less and a post-treatment step of obtaining urea-free water. The treatment step is, for example, a step of passing the urea-containing water through a carrier on which an enzyme including at least urease is immobilized (i.e., an immobilized enzyme) to obtain treated water. The post-treatment step is, for example, a step of passing the treated water through an anion exchange resin, or a step of removing the enzyme from the treated water using a reverse osmosis membrane.

[0013] The urea-free water producing apparatus of the present invention includes a treatment means for contacting urea-containing water with an enzyme including at least urease, and an anion exchange resin through which treated water treated by the treatment means is passed, and the treated water discharged from the anion exchange resin is converted into urea-free water. This is a urea-free water production device that produces urea-free water with a urea concentration of 1 μg / L or less. The treatment means is, for example, an immobilized enzyme in which at least an enzyme including urease is immobilized on a carrier.

[0014] The urea quantification method of the present invention is a method for quantifying urea in sample water, in which urea-free water produced by the urea-free water production method of the present invention is used for at least one of the water used to prepare a urea standard solution and the carrier water.

[0015] The urea analyzing apparatus of the present invention is a urea analyzing apparatus that introduces a fixed amount of sample water into a flow of carrier water and quantifies urea in the sample water, and is equipped with the urea-free water producing apparatus of the present invention, and uses urea-free water having a urea concentration of 1 μg / L or less obtained from the urea-free water producing apparatus as the carrier water. Effect of the Invention

[0016] According to the present invention, it is possible to easily obtain urea-free water that can be used when analyzing urea in sample water, the urea concentration of which has been reduced to a level that does not affect the analysis, and which does not affect the urea concentration in the sample water, thereby making it possible to accurately quantify trace amounts of urea in the sample water. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 is a diagram illustrating an example of an apparatus for producing urea-free water based on the present invention. [Diagram 2] FIG. 2 is a diagram illustrating another example of an apparatus for producing urea-free water based on the present invention. [Diagram 3] 1 is a diagram showing a configuration of an analysis device according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Next, an embodiment of the present invention will be described. The method for producing urea-free water according to the present invention produces urea-free water in which the urea concentration is reduced to a level that does not affect the analysis of urea in sample water, for example, urea-free water with a urea concentration of 1 μg / L or less, by treating water that may contain urea with urease. In the present invention, urea-free water can be obtained using free urease, but it is preferable to use urease immobilized on a carrier in order to prevent the dissipation of urease, particularly to prevent the urease from being mixed into the urea-free water. Hereinafter, the carrier on which urease is immobilized is referred to as an "immobilized enzyme." By passing water through the immobilized enzyme, urea in the water is selectively hydrolyzed at a high speed and removed. The water passing conditions at this time may be determined in advance in a test so that the urea concentration in the water that has passed through the immobilized enzyme is, for example, 1 μg / L or less.

[0019] The urea-free water produced by such a production method and having a urea concentration of, for example, 1 μg / L or less can be used, for example, for preparing a urea standard solution used in quantifying urea, or as carrier water when quantifying urea using a flow injection method or liquid chromatography method. Methods for quantifying trace amounts of urea include the LC / MS method, which combines liquid chromatography (LC) and mass spectrometry (MS), and the LC / MS / MS method, which performs mass spectrometry in two stages, and the urea-free water produced in this manner can also be used as the mobile phase (i.e., carrier water) in the liquid chromatograph when carrying out these analytical methods. The flow injection method and the liquid chromatography method are the same in that they are quantified by introducing a fixed amount of sample water into the flow of carrier water.

[0020] Urea-free water obtained by passing water through an immobilized enzyme having at least urease immobilized thereon can be used as, for example, a standard solution or carrier water for quantifying a trace amount of urea in test water. However, if urease is contaminated in urea-free water, the urease affects the quantitative value of urea. For example, when urea-free water is used to prepare a urea standard solution, the urease in the urea-free water decomposes and removes the urea in the urea standard solution, so that the linearity of the calibration curve cannot be obtained and quantification of urea becomes impossible. In addition, when urea-free water is used as carrier water into which test water is injected, the urease in the urea-free water decomposes and removes the urea in the test water, so that the quantitative result of urea using this urea standard solution gives a value smaller than the original amount of urea in the test water. Therefore, when urea-free water is produced by passing water through an immobilized enzyme having at least urease immobilized thereon, it is necessary to prevent the urease released from the immobilized enzyme from being mixed into the urea-free water while maintaining its enzymatic activity.

[0021] In the method for producing urea-free water according to the present invention, the treated water is first passed through an immobilized enzyme containing at least urease to obtain treated water, and then a post-treatment step is carried out to remove or inactivate the enzyme leaked into the treated water to finally obtain urea-free water. The post-treatment step is, for example, a step of passing the treated water through an anion exchange resin or a step of removing the enzyme from the treated water using a reverse osmosis membrane.

[0022] FIG. 1 shows an example of the configuration of a urea-free water production apparatus in the case where treated water is passed through an anion exchange resin in the post-treatment process. An immobilized enzyme column 52 containing an immobilized enzyme 51 and an anion exchange resin column 54 filled with an anion exchange resin 53 are provided, and water that may contain urea (hereinafter referred to as urea-containing water) is supplied to the immobilized enzyme column 52. As the urea-containing water passes through the immobilized enzyme column 52, the urea contained in the urea-containing water comes into contact with the urease immobilized as the immobilized enzyme 51 and is decomposed and removed, and treated water from which urea has been removed is discharged from the immobilized enzyme column 52. Enzymes such as urease released from the immobilized enzyme 51 may be contained in the treated water. This treated water is then supplied to the anion exchange resin column 54. As will be apparent from the examples described later, the urease in the treated water is inactivated or removed by contacting with the anion exchange resin 53 in the anion exchange resin column 54. As a result, urea-free water from which both urea and enzymes such as urease have been removed is obtained as the outlet water of the anion exchange resin column 54. At this time, when the spatial velocity of the treated water relative to the anion exchange resin 53 is 50 h -1 It is preferable to pass the treated water through the anion exchange resin 53 as described below. Either a strongly basic anion exchange resin or a weakly basic anion exchange resin can be used as the anion exchange resin 53. The ion form of the anion exchange resin 53 may be the OH form or other ion form such as Cl form, but the OH form is more preferable. When the ion form is the OH form, 10 to 99% of the ion exchange groups per total ion exchange capacity are OH. - It is preferable that ions are bonded, and 70 to 99% of the ion exchange groups are OH. -It is more preferable that ions are bonded to the anion exchange resin 53. When the treated water is passed through the anion exchange resin 53, air bubbles may be generated from the anion exchange resin 53. Therefore, it is preferable to cool the anion exchange resin column 54 or apply pressure to the treated water passed through the anion exchange resin column 53.

[0023] FIG. 2 shows an example of the configuration of a urea-free water production apparatus in which enzymes such as urease are removed from treated water by a reverse osmosis membrane in the post-treatment process. The apparatus shown in FIG. 2 is provided with a reverse osmosis membrane device 56 instead of the anion exchange resin column 54 in the apparatus shown in FIG. 1, and treated water discharged from the immobilized enzyme column 52 is supplied to the reverse osmosis membrane device 56. A reverse osmosis membrane 55 is provided inside the reverse osmosis membrane device 56. Since the enzymes have a large molecular weight and cannot permeate the reverse osmosis membrane 55, urea-free water from which urea and enzymes such as urease have been removed is obtained as the permeate of the reverse osmosis membrane device 56. The enzymes that could not permeate the reverse osmosis membrane 55 are discharged as wastewater from the reverse osmosis membrane device 56 in the form of concentrated water. As the reverse osmosis membrane 55, a reverse osmosis membrane made of cellulose acetate or a reverse osmosis membrane made of polyamide can be used. When using a new reverse osmosis membrane, it is used after removing eluates that affect the quantification of urea, more specifically, interfering substances that cause positive errors, by washing in advance.

[0024] As the urease that can be used in the present invention, a commercially available product can be preferably used. For example, urease derived from sword bean (Canavalia gladiata) is commercially available, and urease derived from sword bean can be preferably used in the present invention. As the carrier for immobilizing urease, a carrier generally used for immobilizing enzymes can be used, for example, ion exchange resin, synthetic adsorbent, entrapping immobilization carrier, etc. can be used. The immobilized amount of urease in the immobilized enzyme is expressed in enzyme units (units) per 1 g of carrier, i.e., U / gR, and is preferably 300 U / gR to 7500 U / gR, more preferably 750 U / gR to 3000 U / gR. In the case of urease, 1 U is the amount of enzyme that hydrolyzes 1 μmol of urea per minute at 37° C. If the immobilized amount of urease is less than 300 U / gR, it becomes necessary to reduce the space velocity (SV) during water flow in order to sufficiently reduce the urea concentration. On the other hand, if the amount of urease immobilized exceeds 7500 U / gR, the production cost becomes high.

[0025] The water passed through the immobilized enzyme to produce urea-free water is preferably water with a low urea concentration. In order to prevent problems such as an increase in the differential pressure due to turbidity contained in the water, the water passed through the immobilized enzyme is preferably pure water, ultrapure water, distilled water, or the like. The lower the water flow rate through the immobilized enzyme, the higher the urea decomposition performance of the immobilized enzyme. From the viewpoint of the quality of the produced urea-free water, the water flow rate through the immobilized enzyme is preferably 500 h 2 in terms of spatial velocity. -1 Less than 300h is preferable. -1 Less than 100h is more preferable. -1 The following is more preferred:

[0026] The temperature of water passed through the immobilized enzyme, i.e., the temperature of the water passed through the immobilized enzyme, is, for example, 0°C or higher and 60°C or lower. The higher the water passing temperature, the faster the reaction rate of urea hydrolysis in the immobilized enzyme, but the faster the deactivation rate of urease. On the other hand, if the water passing temperature is low, the reaction rate of urea hydrolysis is slow, but the deactivation rate of urease is small, making it possible to extend the life of the immobilized enzyme. From the viewpoint of the life of the immobilized enzyme, the water passing temperature is preferably 2°C or higher and 30°C or lower (room temperature), more preferably 4°C or higher and 20°C or lower. In order to lower the water passing temperature, a method such as installing a heat exchanger for cooling at the inlet side of the immobilized enzyme or placing a column filled with the immobilized enzyme in a refrigerator can be used.

[0027] According to the production method of the present invention, urea-free water can be continuously produced, so that the present invention is more useful for continuous quantitative analysis of urea or monitoring of urea than for quantitative determination of urea by batch analysis. By using the urea-free water produced according to the present invention, it is possible to accurately quantify urea in sample water containing a trace amount of urea at the μg / L level. In the production method described above, treated water is obtained by passing urea-containing water through an immobilized enzyme having at least an enzyme including urease immobilized therein, and the enzyme leaked into the treated water is removed or inactivated to obtain urea-free water, but the production method of urea-free water according to the present invention is not limited thereto. As long as the method includes a treatment step of contacting urea-containing water with an enzyme including at least urease to obtain treated water, and a post-treatment step of removing or inactivating the enzyme leaked into the treated water, the method falls within the scope of the production method of the present invention, regardless of whether the enzyme is immobilized on a carrier in the treatment step.

[0028] Fig. 3 shows a urea analyzer to which the method for producing urea-free water based on the present invention is applied. Here, an example is described in which raw water used for producing pure water or pure water itself is used as sample water, and trace amounts of urea contained in the sample water are continuously quantified online. Of course, the sample water to be analyzed for urea is not limited to raw water used for producing pure water or pure water.

[0029] As shown in Fig. 3, a raw water line 20 used for producing pure water is provided, and the raw water is pumped through this line 20 by a pump P0. A sample water pipe 21 is provided branching off from the raw water line 20. The sample water pipe 21 is a pipe for sample water branching off from the raw water, and is equipped with an on-off valve 22 and a flow meter FI. A sampling valve 10 (also called an injector or injection valve) is provided at the end of the sample water pipe 21. The portion downstream of the sampling valve 10, including the sampling valve 10, is configured as a flow injection analysis (FIA) device and is the portion actually involved in the quantitative determination of urea.

[0030] The sampling valve 10 has a configuration generally used in the FIA ​​method, and includes a six-way valve 11 and a sample loop 12. The six-way valve 11 includes six ports indicated by circled numbers in the figure. A sample pipe 21 is connected to port 2. A pipe 23 to which carrier water is supplied via pump P1 is connected to port 6, and a pipe 25 for discharging sample water via pump P4 is connected to port 3. A sample loop 12 for collecting a predetermined volume of sample water is connected between ports 1 and 4. One end of a pipe 24, which serves as an outlet of the sampling valve 10, is connected to port 5. Carrier water is supplied to pump P1 via pipe 19, and is sent from pump P1 to port 6 via pipe 23.

[0031] Generally, pure water or the like is used as the carrier water, but since it has a large effect on the accuracy of urea quantification, it is required that the urea concentration is extremely low. As will be clear from the examples described later, when quantification of urea at the μg / L level in sample water is performed, the urea concentration in the carrier water must be 1 μg / L or less. In this embodiment, urea-free water produced by the production method of the present invention is used as the carrier water. For this purpose, the analyzer is provided with an immobilized enzyme column 52 containing an immobilized enzyme 51 in which an enzyme including at least urease is immobilized on a carrier, and an anion exchange resin column 54 provided after the immobilized enzyme column 52 and filled with anion exchange resin 53. A pipe 23 for carrier water is connected to the outlet of the anion exchange resin column 54. Pure water is supplied to the immobilized enzyme column 52 through a pipe 62. As described above, it is preferable to lower the temperature of water when it is passed through the immobilized enzyme 51, so in the urea-free water producing section 50, a heat exchanger 63 to which cooling water is supplied is provided in the pipe 62, and the pure water flowing through the pipe 62 is cooled to a predetermined temperature, for example, 20° C., by the heat exchanger 63. Instead of providing the heat exchanger 63, the urea-free water producing section 50, and in particular the immobilized enzyme column 52, may be provided inside a cooler.

[0032] In the urea-free water producing section 50, pure water supplied through a pipe 62 passes through the immobilized enzyme 51 in the immobilized enzyme column 52, during which urea in the pure water is hydrolyzed. The water that has passed through the immobilized enzyme 51 then passes through an anion exchange resin 53 in an anion exchange resin column 54, where enzymes such as urease in the water are inactivated or removed. As a result, urea-free water that is substantially free of enzymes such as urease in a state in which the urea concentration is 1 μg / L or less and is active is supplied from the urea-free water producing section 50 to the pipe 23 as carrier water, and is used in the quantification of urea by the FIA ​​method. Note that in the urea-free water producing section 50, a reverse osmosis membrane device equipped with a reverse osmosis membrane may be provided instead of the anion exchange resin column 54 filled with the anion exchange resin 53.

[0033] In the six-way valve 11, the communication between port X and port Y is represented as (XY). The six-way valve 11 can be switched between a first state of (1-2), (3-4), and (5-6) and a second state of (2-3), (4-5), and (6-1). In FIG. 1, the connection between the ports in the first state is shown by solid lines, and the connection between the ports in the second state is shown by dotted lines. In the first state, the carrier water flows from the pipe 23 to port 6 to port 5 to the pipe 24 and flows out of the sampling valve 10 to the downstream side. The sample water flows from the sample water pipe 21 to port 2 to port 1 to the sample loop 12 to port 4 to port 3 and is discharged from the pipe 25. When the first state is switched to the second state, the sample water flows from the sample water pipe 21 to port 2 to port 3 and is discharged from the pipe 25, and the carrier water flows from the pipe 23 to port 6 to port 1 to the sample loop 12 to port 4 to port 5 to the pipe 24 and flows out to the downstream side. At this time, the sample water that has already flowed in and filled the sample loop 12 when the first state was in effect flows from port 5 into the pipe 24 before the carrier water and flows to the downstream side of the sampling valve 10. The volume of the sample water flowing into the pipe 24 is determined by the sample loop 12. Therefore, by repeatedly switching between the first state and the second state, for example by rotating the six-way valve 11 in the direction of the illustrated arrow, a predetermined volume of sample water can be repeatedly sent into the pipe 24. The first state and the second state can be switched at predetermined intervals, taking into consideration the residence time required for the reaction and the time until urea is detected by the detector 32. The switching can also be performed by detecting that the sample water introduced into the detector 32 has been discharged from the detector 32. In this way, by automatically switching between the first state and the second state, urea can be continuously quantified.

[0034] In this analytical device, the FIA ​​method is applied to the quantitative determination of urea by the colorimetric method using diacetyl monoxime. Therefore, diacetyl monoxime acetic acid solution (hereinafter also referred to as reagent A) and antipyrine-containing reagent solution (hereinafter also referred to as reagent B) are used as reaction reagents used for the quantitative determination of urea. Here, the case where antipyrine-containing reagent solution is used as a reagent used in combination with diacetyl monoxime will be described, but the reagent used in combination with diacetyl monoxime is not limited to the antipyrine-containing reagent solution. Reagent A and reagent B are stored in storage tanks 41 and 42, respectively.

[0035] As already disclosed in Patent Document 2, the present inventors have found that when these reagents are prepared and then kept at room temperature for a long period (for example, several days or more) for continuous quantification of urea, the peak intensity in the absorbance measurement decreases, and that this decrease in peak intensity can be prevented by refrigerating the reagents (particularly reagent B). Since it is preferable that the peak intensity in the absorbance measurement does not decrease in order to perform stable quantification, in the analyzer of this embodiment, the storage tanks 41 and 42 are provided in the refrigerating section 40. Reagent A is prepared by dissolving diacetyl monoxime in an acetic acid solution, but when the refrigerating section 40 is provided, the preparation itself is performed in the storage tank 41, or reagent A is prepared and then stored in the storage tank 41. Similarly, reagent B is prepared by dissolving antipyrine in, for example, sulfuric acid, but the preparation itself is performed in the storage tank 42, or reagent B is prepared and then stored in the storage tank 42. The refrigeration section 40 shades the storage tanks 41 and 42 from light and cools the storage tanks 41 and 42, thereby maintaining the temperature of the reagents A and B in the storage tanks 41 and 42 at 20°C or lower, preferably 3°C to 20°C, more preferably 5°C to 15°C. The storage tank 41 for storing the reagent A does not necessarily need to be placed in the refrigeration section 40 as long as it can be stored in a shaded environment. The refrigeration temperature of the reagent may be lower than 5°C as long as no crystals are precipitated in the reagent. The Hygienic Test Method (Non-Patent Document 1) describes that an antipyrine sulfuric acid solution in which antipyrine is dissolved in sulfuric acid can be used for 2 to 3 months if stored in a brown bottle, and that refrigerated storage is not suitable because crystals are precipitated and do not re-dissolve even when the temperature is returned to room temperature. However, the present inventors have confirmed through experiments that an antipyrine sulfuric acid solution prepared according to the Hygienic Test Method does not crystallize even at 3°C. If a dilution operation is carried out when preparing reagent A and reagent B, it is preferable to use urea-free water produced according to the present invention as the water used for dilution.

[0036] One end of the pipe 26 is connected to the storage tank 41, and the other end of the pipe 26 is connected to the pipe 24 by a mixing unit 43. The pipe 26 is provided with a pump P2 for feeding the reagent A to the pipe 24 at a predetermined flow rate. Similarly, one end of the pipe 27 is connected to the storage tank 42, and the other end of the pipe 27 is connected to the pipe 24 by a mixing unit 44. The pipe 27 is provided with a pump P3 for feeding the reagent B to the pipe 24 at a predetermined flow rate. The mixing units 43 and 44 have the function of uniformly mixing the reagent A and the reagent B with the liquid flow in the pipe 24, respectively. The other end of the pipe 24 is connected to the inlet of a reaction coil 31 provided in the reaction thermostatic bath 30. The reaction coil 31 causes a color-developing reaction between urea and diacetyl monoxime in the presence of antipyrine, and its length and the flow rate inside the reaction coil 31 are appropriately selected according to the residence time required for the reaction. The reaction thermostatic chamber 30 heats the reaction coil 31 to a temperature suitable for the reaction, for example, to a temperature of 50° C. or higher and 150° C. or lower, preferably 90° C. or higher and 130° C. or lower.

[0037] At the end, i.e., at the outlet, of the reaction coil 31, a detector 32 is provided for measuring the absorbance of the color generated in the liquid by the color reaction, for example, at a wavelength of 460 nm. The detector 32 determines the peak intensity or peak area of ​​the absorbance at a wavelength of 460 nm. The absorbance when the carrier water is flowing is used as a baseline, and a calibration curve is determined from the absorbance of a standard solution with a known urea concentration, so that the concentration of urea in the sample water can be determined from the absorbance of the sample water. At the outlet of the detector 32, a back pressure coil 33 is provided for applying back pressure to the pipe line from the pump P1 through the sampling valve 10, the pipe 24, and the reaction coil 31 to the detector 32. A pressure gauge PI is connected to a position between the outlet of the detector 32 and the inlet of the back pressure coil 33. The wastewater from this analyzer flows out from the outlet of the back pressure coil 33.

[0038] When quantifying urea using the analyzer of this embodiment, it is necessary to prepare a calibration curve using a urea standard solution in advance. When preparing the urea standard solution used to prepare the calibration curve, urea-free water that has been treated with an immobilized enzyme and further post-treated with an anion exchange resin or reverse osmosis membrane to have a urea concentration of 1 μg / L or less is used.

[0039] According to the analytical device of this embodiment, flow injection analysis is performed using carrier water with a urea concentration of 1 μg / L or less, so that urea contained in sample water can be accurately quantified at the μg / L level. EXAMPLES

[0040] Next, the present invention will be described in more detail with reference to examples.

[0041] [Example 1] 660 mg of urease (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) with an enzyme activity of 150 U / mg was dissolved in 80 mL of pure water to obtain an aqueous urease solution. AMBERLITE (registered trademark) XAD-7HP, an acrylic synthetic adsorbent manufactured by Organo Corporation, was used as a carrier for preparing the immobilized enzyme. This aqueous urease solution was contacted with 20 g of the carrier at room temperature for 5 minutes to immobilize urease on the carrier, obtaining an immobilized enzyme with an immobilization amount of 830 U / gR.

[0042] In Example 1-1, a strongly basic anion exchange resin, AMBERJET (registered trademark) ESG4002(OH)-HG manufactured by Organo Corporation, which has an OH ion form, was placed downstream of the immobilized enzyme prepared by the above method, and an aqueous urea solution with a urea concentration of 5 μg / L was passed through the immobilized enzyme. The strongly basic anion exchange resin used in Example 1-1 has OH ion exchange groups for 90% of its total ion exchange capacity. -Similarly, in Example 1-2, a strongly basic anion exchange resin, AMBERJET (registered trademark) 4002 (OH) manufactured by Organo Corporation, which has an OH ion form, was placed downstream of the immobilized enzyme, and an aqueous urea solution with a urea concentration of 5 μg / L was passed through the immobilized enzyme. The strongly basic anion exchange resin used in Example 1-1 has OH ion groups for 75% of its total ion exchange capacity. - In both Examples 1-1 and 1-2, the SV (space velocity) of water passing through the immobilized enzyme was 2 h -1 In addition, the SV in the strongly basic anion exchange resin was 5 to 50 h. -1 The experiment was repeated while changing the concentration within the range. When the water that had been passed through the immobilized enzyme and the strongly basic anion exchange resin in sequence was analyzed by the LC / MS / MS method, it was confirmed that the urea concentration was 1 μg / L or less, which is the lower limit of quantification, in both Examples 1-1 and 1-2, regardless of the SV in the strongly basic anion exchange resin. In other words, urea-free water with a urea concentration of 1 μg / L or less was obtained.

[0043] [Example 2] Urea was added to the urea-free water obtained in Examples 1-1 and 1-2 so that the urea concentration was 10 μg / L, and the water was allowed to stand at 20° C. for 1 hour. The urea concentration of this water was then analyzed using an online urea meter (ORUREA, manufactured by Organo Corporation). The results are shown in Table 1. In Table 1, the SV column indicates the SV of the strongly basic anion exchange resin when the urea-free water was obtained in Examples 1-1 and 1-2. Since the urea concentration hardly changed even after 1 hour had passed, it was found that the urea-free water obtained in Examples 1-1 and 1-2 contained almost no urease in a form having decomposition activity against urea.

[0044] [Table 1]

[0045] [Comparative Example 1] SV of water passing through a strong basic anion exchange resin for 100 hours -1 and 200h -1 The same operation as in Example 1-1 was carried out, except that water was obtained by passing through the immobilized enzyme and the strongly basic anion exchange resin in sequence. The urea concentration of this water was measured in the same manner as in Example 1-1, and was found to be below the lower limit of quantification of 1 μg / L. Next, urea was added to the water thus obtained in the same manner as in Example 2, and the urea concentration after standing at 20° C. for 1 hour was measured in the same manner as in Example 2. The results are shown in Table 1. From the results shown in Table 1, it can be seen that the SV of the water passing through the strongly basic anion exchange resin was 100 h. -1 and 200h -1 It can be seen that in the case of Example 2, the urea concentration decreased from the initial amount of 10.0 μg / L. This is thought to be because urease leaked into the water that passed through the immobilized enzyme and the strongly basic anion exchange resin in sequence, and urea was decomposed by the leaked urease. From Example 2 and Comparative Example 1, when the space velocity (SV) of the water passing through the strongly basic anion exchange resin was set to 50 h -1 It is found to be preferable to do the following:

[0046] [Example 3] The difference in urease removal performance depending on the type of anion exchange resin was investigated. As the anion exchange resin, AMBERJET (registered trademark) 4400 Cl manufactured by Organo Corporation, which is a strongly basic anion exchange resin whose ion form is Cl (Example 3-1), AMBERLITE (registered trademark) IRA900J Cl manufactured by Organo Corporation, which is a strongly basic anion exchange resin whose ion form is Cl (Example 3-2), and AMBERLITE (registered trademark) IRA96SB manufactured by Organo Corporation, which is a weakly basic anion exchange resin (Example 3-3), were prepared. The strongly basic anion exchange resins whose ion form is Cl used in Examples 3-1 and 3-2 all had Cl-type ion exchange groups for almost 100% of their total ion exchange capacity. - The weakly basic anion exchange resin used in Example 3-3 has OH groups for 15% of the total ion exchange capacity. -Then, for each type of anion exchange resin, 12 mL of the anion exchange resin was packed into a column made of tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (PFA) with a diameter of 10 mm and a length of 200 mm to obtain an anion exchange resin column.

[0047] Urease (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) with an enzyme activity of 150 U / mg was dissolved in pure water to a concentration of 1 mg / L to obtain an aqueous urease solution. -1 and 20h -1 The aqueous urease solution was passed through the anion exchange resin column under both of the above water passing conditions, and a treated solution discharged from the anion exchange resin column was obtained. Then, urea was added to each treated solution so that the urea concentration was 10 μg / L, and the treated solution was left to stand at 20° C. for 1 hour. The urea concentration of this water was then analyzed using an online urea meter (ORUREA, manufactured by Organo Corporation). The results are shown in Table 2. In Table 2, the column for SV indicates the SV when the aqueous urease solution was passed through the anion exchange resin column. From the results shown in Table 2, whether a strong basic anion exchange resin in which the ion form is the Cl form or a weak basic anion exchange resin was used, the decrease in urea concentration after standing at 20° C. for 1 hour was slight, and it was found that urease could be inactivated or removed by passing the solution through the anion exchange resin.

[0048] [Table 2]

[0049] [Comparative Example 2] It was investigated whether urease could be removed when the urease aqueous solution was passed through a resin other than an anion exchange resin. Instead of the anion exchange resin packed in the PFA column in Example 3, the same column was packed with a synthetic adsorbent AMBERLITE (registered trademark) XAD-7HP (Comparative Example 2-1) manufactured by Organo Corporation and an H-type strong acid cation exchange resin AMBERJET (registered trademark) ESG1024 (H) (Comparative Example 2-2) manufactured by Organo Corporation, and the urease aqueous solution was passed through in the same manner as in Example 3. Urea was added to the treated liquid obtained by passing the water through the column, and the urea concentration was measured after leaving the liquid at 20°C for 1 hour. The results are shown in Table 2. As shown in Table 2, the urea concentration was significantly reduced in both the case of using the synthetic adsorbent and the case of using the cation exchange resin, which indicates that the treated liquid treated with the synthetic adsorbent or the cation exchange resin contains urease in an active state. Even if urea-free water is obtained by treating treated water from which urea has been decomposed and removed using urease with a synthetic adsorbent or cation exchange resin, urease will leak into the urea-free water, and it is understood that there is a risk of errors in the quantitative determination results when urea is quantified using the urea-free water.

[0050] [Example 4] We investigated whether urease can be removed by reverse osmosis membrane. Urease with an enzyme activity of 150 U / mg (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in pure water to a concentration of 1 mg / L to obtain an aqueous urease solution. This aqueous urease solution was passed through a reverse osmosis membrane FilmTec BW60-1812-75 manufactured by DuPont under the conditions of an inlet flow rate of 1.2 L / min, a concentration flow rate of 840 mL / min, and a permeation flow rate of 360 mL / min. Urea was then added to the permeated water so that the urea concentration was 10 μg / L, and the solution was allowed to stand at 20°C for 1 hour. The urea concentration of this water was then analyzed with an online urea meter (ORUREA manufactured by Organo Corporation). The urea concentration remained unchanged at 10 μg / L even after standing at 20°C for 1 hour, indicating that urease can be removed by reverse osmosis membrane. [Explanation of symbols]

[0051] 10 Sampling valve 11 Sample Loop 31 Reaction coil 32 Detector 33 Back pressure coil 50 Urea-Free Water Production Department 51 Immobilized Enzymes 52 Immobilized Enzyme Column 53 Anion exchange resin 54 Anion exchange resin column 55 Reverse osmosis membrane 56 Reverse osmosis membrane equipment

Claims

1. a treatment step of contacting the urea-containing water with an enzyme containing at least urease to obtain treated water; a post-treatment step of removing or inactivating the enzyme leaked into the treated water to obtain urea-free water having a urea concentration of 1 μg / L or less; The method for producing urea-free water comprises the steps of:

2. 2. The method for producing urea-free water according to claim 1, wherein the treatment step is a step of passing the urea-containing water through a carrier on which an enzyme including at least urease is immobilized to obtain the treated water.

3. The method for producing urea-free water according to claim 1 or 2, wherein the post-treatment step is a step of passing the treated water through an anion exchange resin.

4. The anion exchange resin was subjected to 50 h -1 The method for producing urea-free water according to claim 3, wherein the treated water is passed through at a space velocity of:

5. The method for producing urea-free water according to claim 3 or 4, wherein the anion exchange resin is an OH-type strongly basic anion exchange resin.

6. 3. The method for producing urea-free water according to claim 1, wherein the post-treatment step is a step of removing the enzyme from the treated water using a reverse osmosis membrane.

7. A treatment means for contacting the urea-containing water with an enzyme including at least urease; an anion exchange resin through which the treated water treated by the treatment means is passed; Equipped with The treated water discharged from the anion exchange resin is treated as urea-free water, and the urea-free water having a urea concentration of 1 μg / L or less is obtained by the urea-free water producing apparatus.

8. The urea-free water producing apparatus according to claim 7, further comprising a cooling means for cooling at least one of the urea-containing water supplied to the treatment means and the treatment means so that the temperature of the water passing through the treatment means is 30°C or lower.

9. A method for quantifying urea in a water sample, comprising the steps of: A quantitative determination method, comprising using urea-free water produced by the method for producing urea-free water according to any one of claims 1 to 6 as at least one of water used in preparation of a urea standard solution and carrier water.

10. 1. An apparatus for analyzing urea, comprising: introducing a constant amount of sample water into a flow of carrier water to quantify urea in the sample water, The apparatus for producing urea-free water according to claim 7 or 8 is provided, The analytical device uses urea-free water having a urea concentration of 1 μg / L or less obtained from the urea-free water production device as the carrier water.

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