JP7307812B2 - Method for quantifying urea and analyzer for urea - Google Patents

Description

TECHNICAL FIELD The present invention relates to the determination of urea in water, and more particularly to a method for determining urea using urea- free water and an analyzer for urea.

There is a demand for accurate analysis and quantification of trace amounts of urea in water. For example, when producing pure water from raw water using a pure water production system, it is difficult to remove urea from the raw water with an ion exchange device or an ultraviolet oxidation device that constitutes the pure water production system, so urea is removed in advance. Raw water must be supplied to the water purification system. As a method for removing urea, a method of selectively oxidizing urea by adding an agent that generates hypobromous acid to raw water and using hypobromous acid to selectively oxidize urea is known. Since it becomes a load on the pure water production system provided in the apparatus, the smaller the amount of the chemical to be added, the better. Therefore, it is desired to quantify the urea concentration in raw water to determine the necessity of urea removal treatment, and to add an appropriate chemical when urea removal treatment is required. In addition, there is a need to measure urea concentration in pure water obtained from water purification systems.

As a quantification method for urea, a quantification method based on a colorimetric method using diacetylmonoxime, which is a method described in, for example, "Sanitary Test Method" (Non-Patent Document 1) is known. In the colorimetric method using diacetyl monoxime, other reagents can be used together for the purpose of accelerating the reaction. Reagents used in combination here include, for example, a sulfuric acid solution of antipyrine, an aqueous solution of semicarbazide hydrochloride, an aqueous solution of manganese chloride and potassium nitrate, a sulfuric acid solution of sodium dihydrogen phosphate, and the like. When antipyrine is used in combination to quantify urea, diacetylmonoxime is dissolved in an acetic acid solution to prepare a diacetylmonoxime acetic acid solution, and antipyrine (that is, 1,5-dimethyl-2-phenyl-3-pyrazolone) is added. is dissolved in, for example, sulfuric acid to prepare an antipyrine-containing reagent solution. Then, the diacetylmonoxime acetic acid solution and the anripyrin-containing reagent solution are sequentially mixed with the sample water, the absorbance at a wavelength of around 460 nm is measured, and the urea in the sample water is quantified by comparison with the standard solution.

The colorimetric method for the determination of urea using diacetyl monoxime is intended for the determination of urea in e.g. swimming pool water and public bath water. Poor sensitivity for quantification of urea. Therefore, in Patent Document 1, by measuring the absorbance by applying flow injection analysis based on the colorimetric method using diacetyl monoxime, urea in sample water can be continuously measured online in the concentration range of ppb or less to several ppm. A method for quantification is disclosed. WO 2005/010103 describes a long-term, continuous, automated, on-line, quantification of urea by refrigerating the reagents used in the reaction when flow injection analysis is applied while being based on a colorimetric method using acetylmonoxime. It discloses that the measurement can be performed stably.

JP-A-2000-338099 JP 2018-179545 A

Edited by The Pharmaceutical Society of Japan, Sanitary Test Method and Commentary 1990.4.1.2.3 (13) 1 (1990 edition, 4th printing supplement (1995), p1028), 1995

The methods described in Patent Document 1 and Patent Document 2 are methods that can quantify urea with high sensitivity by using flow injection analysis. If urea is contained in the water used to prepare the standard solution or the carrier water in the flow injection analysis, the urea contained in such water will cause an analytical error. Even when urea is quantified by an analytical method other than flow injection analysis, urea contained in any of the water used in the quantification operation causes an analytical error. For example, even when performing analysis by liquid chromatography, if water is used as the mobile phase, urea is contained in the water, that is, the carrier water, an analytical error occurs. In particular, when analyzing minute amounts of urea at the μg/L level contained in sample water, if the water used to prepare the standard solution or the carrier water contains urea even at the μg/L level, the effect will be big.

As mentioned above, it is difficult to remove urea by ion exchange treatment or ultraviolet oxidation treatment. Ultrapure water produced may contain trace amounts of urea and its concentration may fluctuate due to such processing. Therefore, even when ultrapure water is used as the concentration standard solution or as the carrier water, the results of microanalysis of urea may be inaccurate.

An object of the present invention is to provide a quantification method and analyzer capable of accurately quantifying trace amounts of urea in sample water by using urea- free water in which the urea concentration has been reduced to the extent that it does not affect the analysis at least. is to provide

Urease (EC 3.5.1.5) is an enzyme that hydrolyzes urea. Urease can decompose urea into carbon dioxide and ammonia in the presence of water, even at very low concentrations, as shown in the following formula.

( _NH2 ) _2CO + _H2O- > _CO2 + _NH3

According to the studies of the present inventors, as is clear from the examples described later, the urea concentration in water can be reduced to 1 μg/L or less by treating water with urease.

The urea quantification method of the present invention is a quantification method for quantifying urea in sample water. Urea-free water having a urea concentration of 1 μg/L or less obtained by passing water through the manufacturing department is used. The urea-free water production unit has at least a carrier on which urease is immobilized, and urea-free water is obtained by passing water through the carrier on which urease is immobilized.

The urea analyzer of the present invention is an analyzer that introduces a certain amount of sample water into the flow of carrier water and quantifies the amount of urea in the sample water. The carrier water is treated water having a urea concentration of 1 μg/L or less, which is obtained by passing the water through the urea-free water production unit, and the urea-free water production unit contains at least a carrier on which urease is immobilized. and water is passed through the carrier on which urease is immobilized to obtain treated water .

According to the present invention, urea-free water in which the urea concentration is reduced to the extent that it does not affect the analysis can be easily obtained, so that a trace amount of urea in the sample water can be accurately quantified. .

It is a figure showing composition of an analysis device of one embodiment of the present invention. 4 is a graph showing the results of Example 3. FIG.

Next, an embodiment of the invention will be described. A method for producing urea-free water according to the present invention is to produce urea-free water with a urea concentration of 1 μg/L or less by passing water through a carrier on which at least urease is immobilized. The carrier on which urease is immobilized is hereinafter referred to as "immobilized enzyme". By passing water through the immobilized enzyme, urea in water is selectively hydrolyzed at high speed and removed. The water flow 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 1 μg/L or less.

As urease, for example, urease derived from jack bean (Canavalia gladiata) is commercially available, and in the present invention, commercially available urease can be used favorably. As the carrier for immobilizing urease, those commonly used for immobilizing enzymes can be used, for example, ion exchange resins, synthetic adsorbents, entrapping immobilization carriers, etc. can be used. . The immobilized amount of urease in the immobilized enzyme is expressed in enzyme units (units) per 1 g of carrier, that is, expressed in U/g-R, and should be 300 U/g-R or more and 7500 U/g-R or less. is preferred, and more preferably 750 U/g-R or more and 3000 U/g-R or less. When the immobilized amount of urease is less than 300 U/g-R, it becomes necessary to reduce the space velocity (SV) during passage of water in order to sufficiently reduce the urea concentration. On the other hand, when the immobilized amount of urease exceeds 7500 U/g-R, the production cost increases.

Water having a low urea concentration is preferred as the water that is passed through the immobilized enzyme for the production of urea-free water. In order to prevent problems such as an increase in the differential pressure of water flow due to turbidity contained in water, the water that is passed through the immobilized enzyme is preferably pure water, ultrapure water, distilled water, or the like. The lower the water flow rate to the immobilized enzyme, the higher the ureolytic performance of the immobilized enzyme. From the viewpoint of the quality of the urea-free water to be produced, the water flow rate to the immobilized enzyme is preferably 500 h ⁻¹ or less, more preferably 300 h ⁻¹ or less, and more preferably 100 h ⁻¹ or less, in terms of space velocity. preferable.

The temperature at which water is passed through the immobilized enzyme, ie, the temperature of the water that is passed through the immobilized enzyme, is, for example, 0° C. or higher and 60° C. or lower. The higher the water flow temperature, the faster the reaction rate of hydrolysis of urea in the immobilized enzyme, but the higher the deactivation rate of urease. On the other hand, if the water flow temperature is low, the reaction rate of hydrolysis of urea is slowed down, but the deactivation rate of urease is slowed down, making it possible to extend the life of the immobilized enzyme. From the viewpoint of the life of the immobilized enzyme, the water flow rate is preferably 2° C. or higher and normal temperature or lower, more preferably 4° C. or higher and 20° C. or lower. Here, normal temperature means, for example, 30°C. In order to lower the water flow temperature, it is possible to install a cooling heat exchanger on the inlet side of the immobilized enzyme, or place a column filled with immobilized enzyme in a cold storage. can.

The method for producing urea-free water according to the present invention has been described above. Urea-free water produced by such a production method and having a urea concentration of 1 μg/L or less is prepared, for example, by preparing a urea standard solution used for quantification of urea, using a flow injection method or a liquid chromatography method. It can be used as carrier water when quantifying urea. Techniques for quantifying a trace amount of urea include an LC/MS method combining liquid chromatography (LC) and mass spectrometry (MS), and an LC/MS/MS method performing mass spectrometry in two steps. The urea-free water produced according to the present invention can also be used as the mobile phase (that is, carrier water) in the liquid chromatograph section when performing the analysis method of . The flow injection method and the liquid chromatography method agree in that quantification is performed by introducing a fixed amount of sample water into the carrier water flow. By using the urea-free water produced according to the present invention, it is possible to accurately quantify urea in sample water containing a minute amount of urea at the μg/L level. According to the production method according to the present invention, urea-free water can be produced continuously. Therefore, the present invention is more suitable for continuous quantitative analysis of urea or monitoring of urea than when quantitative analysis of urea is performed by batch analysis. useful when doing

FIG. 1 shows a urea analyzer according to one embodiment of the present invention. Here, an example will be described in which raw water or pure water used for producing pure water is used as sample water, and a minute amount of urea contained in the sample water is continuously quantified on-line. Of course, the sample water to be analyzed for urea is not limited to raw water or pure water used for producing pure water.

As shown in FIG. 1, a line 20 of raw water used for producing pure water is provided, and the raw water is fed through this line 20 by a pump P0. A sample water pipe 21 branched from the raw water line 20 is provided. The sample water pipe 21 is a sample water pipe branched from raw water, and includes an on-off valve 22 and a flow meter FI. A sampling valve 10 also called an injector or an injection valve is provided at the tip of the sample water pipe 21 . The portion downstream from the sampling valve 10, including the sampling valve 10, is configured as a flow injection analysis (FIA) device and is actually involved in the quantification of urea.

The sampling valve 10 has a structure commonly used in FIA equipment, and includes a hexagonal valve 11 and a sample loop 12 . The hexagonal valve 11 has six ports indicated by circled numbers in the figure. A sample pipe 21 is connected to port 2 . A pipe 23 for supplying carrier water via a pump P1 is connected to the port 6, and a pipe 25 for draining the sample water is connected to the port 3 via a pump P4. A sample loop 12 is connected between port 1 and port 4 for collecting a predetermined volume of sample water. One end of a pipe 24 serving as an outlet of the sampling valve 11 is connected to the port 5 . Carrier water is supplied to the pump P1 through the pipe 19 and sent from the pump P1 to the port 6 through the pipe 23 .

Pure water or the like is generally used as the carrier water, which greatly affects the accuracy of urea quantification, and is required to have an extremely low urea concentration. As is clear from the examples described later, the urea concentration in the carrier water must be 1 μg/L or less when quantifying urea at the μg/L level in the sample water. Therefore, in this embodiment, urea-free water, which is water from which urea is substantially removed, more specifically, urea-free water, which is water with a urea concentration of 1 μg/L or less, is used as carrier water. Urea-free water is produced, for example, by the production method of the present invention described above.

In order to use urea-free water, the analyzer of this embodiment is provided with a urea-free water production unit 50 for producing urea-free water. As the urea-free water production unit 50, any configuration can be used as long as it can produce urea-free water with a urea concentration of 1 μg/L or less. In the example shown here, the urea-free water producing section 50 having the carrier on which urease is immobilized, that is, the immobilized enzyme 51 is provided as described above. The urea-free water production unit 50 has a columnar shape and accommodates an immobilized enzyme 51 therein. A carrier water pipe 23 is connected to the bottom of the urea-free water production unit 50 . Pure water is supplied to the upper part of the urea-free water production unit 50 through a pipe 52, and the pipe 52 is provided with a heat exchanger 53 to which cooling water is supplied. The flowing pure water is cooled to a predetermined temperature, for example 20° C., by the heat exchanger 53 . The heat exchanger 53 is provided to keep the temperature of the water flowing through the immobilized enzyme 51 below room temperature, for example, 20°C. may be set to

In the urea-free water production unit 50, pure water supplied through the pipe 52 passes through the immobilized enzyme 51, and urea in the pure water is hydrolyzed at that time. As a result, immobilized enzyme-treated water having a urea concentration of 1 μg/L or less is supplied from the urea-free water production unit 50 to the pipe 23 as carrier water, and is used in the quantification of urea by the FIA method.

If the communication between port X and port Y in the hexagonal valve 11 is expressed as (XY), then the hexagonal valve 11 is (1-2), (3-4), and (5-6). It is configured to switch between the first state and the second states (2-3), (4-5), and (6-1). In FIG. 1, the connections between ports in the first state are indicated by solid lines, and the connections between ports in the second state are indicated by dotted lines. In the first state, the carrier water flows through the pipe 23→port 6→port 5→pipe 24 and flows out from the sampling valve 10 to the downstream side. The sample water flows through sample water pipe 21 →port 2 →port 1 →sample loop 12 →port 4 →port 3 and is discharged from pipe 25 . When the first state is switched to the second state, the sample water flows in the sample water pipe 21→port 2→port 3 and is discharged from the pipe 25, and the carrier water flows in the pipe 23→port 6→port. 1→sample loop 12→port 4→port 5→piping 24, and flows out to the downstream side. At this time, the sample water that has already flowed in and filled the inside of the sample loop 12 in the first state flows from the port 5 into the pipe 24 prior to the carrier water, and flows downstream of the sampling valve 10. flow. The volume of sample water flowing in line 24 is defined by sample loop 12 . Therefore, by repeatedly switching between the first state and the second state, for example, by rotating the hexagonal valve 11 in the direction of the arrow in the drawing, a predetermined volume of sample water can be repeatedly fed into the pipe 24 . Switching between the first state and the second state can be performed at predetermined time intervals in consideration of the residence time required for the reaction and the time until urea is detected by the detector 32 . Moreover, it is also possible to switch by detecting that the sample water introduced into the detector 32 is discharged from the detector 32 . By automatically switching between the first state and the second state in this manner, urea can be continuously quantified.

This analyzer applies the FIA method to the colorimetric determination of urea using diacetylmonoxime. Therefore, a diacetylmonoxime acetic acid solution and a reagent solution containing antipyrine are used as reaction reagents for quantification of urea. In the following description, the diacetylmonoxime acetic acid solution and the antipyrine-containing reagent solution may be referred to as reagent A and reagent B, respectively. Here, the case of using an antipyrine-containing reagent solution as a reagent to be used in combination with diacetylmonoxime will be described, but the reagent to be used in combination with diacetylmonoxime is not limited to the antipyrine-containing reagent solution. Reagent A and reagent B are stored in reservoirs 41 and 42, respectively.

As already disclosed in Patent Document 2, the present inventors have found that after preparing these reagents, when they are kept at room temperature for a long period of time, for example, several days or more for continuous quantification of urea, the peak intensity in absorbance measurement and that this decrease in peak intensity can be prevented by refrigerating the reagents, particularly Reagent B. In order to perform stable quantification, it is preferable that the peak intensity in the absorbance measurement does not decrease. Reagent A is prepared by dissolving diacetyl monoxime in an acetic acid solution. When the refrigeration unit 40 is provided, the preparation itself is performed in the storage tank 41, or the reagent A is stored in the storage tank 41 after preparation. do. Similarly, reagent B, which is prepared by dissolving antipyrine in, for example, sulfuric acid, is prepared in reservoir 42, or reagent B is stored in reservoir 42 after preparation. The refrigerator unit 40 shields the storage tanks 41 and 42 from light and cools the storage tanks 41 and 42, thereby reducing the temperature of the reagent A and the reagent B in the storage tanks 41 and 42 to 20°C or less, preferably 3°C or more to 20°C. Thereafter, the temperature is more preferably maintained at 5°C or higher and 15°C or lower. Note that the storage tank 41 for storing the reagent A does not necessarily have to be arranged in the refrigerating section 40 as long as it can be stored in the dark. The refrigeration temperature of the reagent may be less than 5°C as long as the reagent does not precipitate crystals. In the "Hygiene Test Method" (Non-Patent Document 1), 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 crystals precipitate and return to room temperature. It states that refrigerated storage is not suitable because it does not redissolve. However, the present inventors have experimentally confirmed that the antipyrine sulfate solution prepared according to the "hygiene test method" does not crystallize even at 3°C. If a dilution operation is performed during the preparation of reagent A and reagent B, the water used for dilution should be urea-free water having a urea concentration of 1 μg/L or less, such as water produced according to the present invention. It is preferred to use urea-free water.

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 through the mixing section 43 . The pipe 26 is provided with a pump P2 for sending the reagent A into the pipe 24 at a predetermined flow rate. Similarly, one end of a pipe 27 is connected to the storage tank 42 , and the other end of the pipe 27 is connected to the pipe 24 through the mixing section 44 . The pipe 27 is provided with a pump P3 for sending the reagent B into 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 respect to the liquid flow in the pipe 24 . The other end of the pipe 24 is connected to the inlet of the reaction coil 31 provided inside the constant temperature reaction bath 30 . The reaction coil 31 causes a color reaction with urea and diacetylmonoxime in the presence of antipyrine inside it, and the length and the flow rate inside the reaction coil 31 determine the residence time required for the reaction. is appropriately selected according to the The reaction constant temperature bath 30 heats the reaction coil 31 to a temperature suitable for the reaction. .

A detector 32 is provided at the end of the reaction coil 31 , that is, at the outlet, for measuring the absorbance of the color produced in the liquid by the color reaction, with the liquid flowing out from the reaction coil 31 as the target. The detector 32 determines the peak intensity or peak area of the absorbance near the wavelength of 460 nm, for example. By using the absorbance when the carrier water is flowing as a baseline and obtaining a calibration curve from the absorbance of a standard solution with a known urea concentration, the concentration of urea in the sample water can be obtained from the absorbance of the sample water. At the outlet of the detector 32, a back pressure coil 33 is provided to apply back pressure to a pipe line extending from the pump P1 through the sampling valve 10, the pipe 24 and the reaction coil 31 to the detector 32. FIG. 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 . From the outlet of the back pressure coil 33, the effluent of this analyzer flows out.

When quantifying urea with the analyzer of this embodiment, it is necessary to prepare a calibration curve using a urea standard solution. Urea-free water, for example, urea-free water treated with an immobilized enzyme to a urea concentration of 1 μg/L or less, is also used to prepare the urea standard solution used to create the calibration curve.

According to the analyzer of the present embodiment, since flow injection analysis is performed using carrier water with a urea concentration of 1 μg/L or less, it is possible to accurately quantify the μg/L level of urea contained in the sample water. can be done.

Next, the present invention will be described in more detail by way of examples.

[Example 1]
340 mg of urease (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) having an enzymatic activity of 150 U/mg was dissolved in 80 mL of pure water to prepare an aqueous urease solution. An acrylic synthetic adsorbent Amberlite (registered trademark) XAD-7HP manufactured by Organo was used as a carrier. This aqueous urease solution was brought into contact with 20 g of the carrier at 25° C. for 4 hours to immobilize urease on the carrier so that the immobilized amount was 830 U/g-R, thereby obtaining an immobilized enzyme.

Pure water was passed through the immobilized enzyme prepared by the above method so that the SV (space velocity) was 2 h ⁻¹ , and the water that passed through the immobilized enzyme, that is, the immobilized enzyme-treated water was analyzed by LC/MS/MS. As a result of analysis, it was confirmed that the urea concentration was 1 μg/L or less, which is the lower limit of determination. As LC/MS/MS devices, an LC device (LC800) manufactured by GL Science and an MS/MS device (3200 Q TRAP) manufactured by AB SCIEX were used.

Since it was confirmed that the urea concentration in the immobilized enzyme-treated water was 1 μg/L or less, a urea standard solution was prepared using the immobilized enzyme-treated water as a dilution solvent. Special grade urea manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. was used as the urea used for preparing the urea standard solution. Seven types of urea standard solutions with urea concentrations of 0, 1, 2, 5, 10, 20 and 50 μg/L were prepared here.

Next, sample water whose urea concentration was separately confirmed to be 5 μg/L was passed through the immobilized enzyme under the conditions of 4° C. and SV 2h ⁻¹ , and the sample after passing through the immobilized enzyme Using water as the carrier water for the FIA device shown in FIG. 1, the previously prepared seven types of urea standard solutions were analyzed. Table 1 shows the analysis results. From Table 1, analysis was performed so that the urea concentration was approximately the same as the nominal concentration of each urea standard solution. From this, it was found that the urea concentration can be measured accurately by using immobilized enzyme-treated water as carrier water.

[Comparative Example 1]
Analysis was performed under the same conditions as in Example 1, except that the immobilized enzyme was not used in the treatment of the carrier water in Example 1. Table 2 shows the results. As shown in Table 2, the standard solutions with urea concentrations of 0, 1, and 2 μg/L, respectively, could not be measured, and the analysis results of the standard solutions with urea concentrations of 5, 10, 20, and 50 μg/L, respectively, were They were 0, 5, 15 and 46 μg/L, which were generally 5 μg/L smaller than the original values. Further, when the urea concentration of the carrier water used was measured separately, 5 μg/L of urea was contained. It was found that urea cannot be measured accurately if the carrier water contains urea above a certain level.

[Example 2]
Sample water having a urea concentration of about 10 µg/L was passed through a column filled with an immobilized enzyme prepared in the same manner as in Example 1 at 20°C and SV 500 h ^-1 to obtain immobilized enzyme-treated water, When the urea concentration in this immobilized enzyme-treated water was measured in the same manner as in Example 1, the urea concentration was 1 μg/L or less (that is, the lower limit of quantification or less).

[Comparative Example 2]
A sample water having a urea concentration of about 10 μg/L was passed through a column filled with an immobilized enzyme prepared in the same manner as in Example 1 at 20° C. and SV 1000 h ⁻¹ to pass through the immobilized enzyme. When the urea concentration of water was measured in the same manner as in Example 1, the urea concentration was about 3 μg/L.

[Example 3]
The durability of the immobilized enzyme was investigated. Raw water having a urea concentration of about 5 μg/L was continuously passed through the column filled with the immobilized enzyme prepared in the same manner as in Example 1 under the conditions of 4° C. and SV 2h ⁻¹ . got water. The urea concentration in the treated water was measured in the same manner as in Example 1, and changes over time in the urea concentration were investigated. Also, the urea concentration in raw water was measured in the same manner. The results are shown in FIG. In FIG. 2, the sample water treated with the immobilized enzyme is indicated as "treated water". As shown in FIG. 2, in the sample water treated with the immobilized enzyme, the urea concentration was less than 1 μg/L even after 100 days from the start of water flow. The lower detection limit of urea in the measurement is 1 μg/L, and in the part where the urea concentration in the treated water fluctuates in the range of less than 1 μg/L in FIG. 2, urea is not actually detected. it is conceivable that. From the results shown in FIG. 2, it was found that urea-free water with a urea concentration of 1 μg/L or less can be produced over a long period of time according to the production method according to the present invention. This means that the required amount of water used for urea quantification, particularly carrier water, can be continuously generated by passing pure water through the immobilized enzyme, and urea can be continuously quantified over a long period of time. This means that the necessary carrier water can be supplied continuously.

10 sampling valve 11 sample loop 31 reaction coil 32 detector 33 back pressure coil 50 urea-free water production part 51 immobilized enzyme

Claims (10)

A urea quantification method for quantifying urea in sample water,
Urea-free with a urea concentration of 1 μg/L or less obtained by passing at least one of the water used for preparing the urea standard solution and the carrier water through a urea-free water production unit provided inside the cold storage. using water ,
The quantification method, wherein the urea-free water production unit comprises at least a carrier on which urease is immobilized, and the urea-free water is obtained by passing water through the carrier on which the urease is immobilized. The quantification method according to claim 1, wherein the carrier is at least one of a synthetic adsorbent, an ion exchange resin and an entrapping immobilization carrier. 3. The quantification method according to claim 1, wherein the amount of urease immobilized on the carrier is 300 U/g-R or more and 7500 U/g-R or less. The quantification method according to any one of claims 1 to 3 , wherein urea in the sample water is quantified based on at least one of flow injection method, liquid chromatography method and mass spectrometry method. The quantification method according to any one of claims 1 to 4, wherein the temperature of the water flowing through the urea-free water production unit is 4°C or higher and 20°C or lower. A urea analyzer for introducing a constant amount of sample water into a flow of carrier water and quantifying urea in the sample water,
Having a urea-free water production unit provided inside the cold storage ,
Treated water having a urea concentration of 1 μg/L or less obtained by passing through the urea-free water production unit is used as the carrier water,
The analysis device, wherein the urea-free water production unit includes at least a carrier on which urease is immobilized, and the treated water is obtained by passing water through the carrier on which the urease is immobilized. 7. The analyzer according to claim 6 , wherein said carrier is at least one of a synthetic adsorbent, an ion exchange resin and an entrapping immobilization carrier. 8. The analyzer according to claim 6 , wherein the immobilized amount of said urease on said carrier is 300 U/g-R or more and 7500 U/g-R or less. 9. The analyzer according to any one of claims 6 to 8, which is at least one of a flow injection analyzer, a liquid chromatography analyzer and a mass spectrometer. 10. The analyzer according to any one of claims 6 to 9, wherein the temperature of water flowing through said urea-free water production unit is 4°C or higher and 20°C or lower.

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