Urea detection method based on urease-ion chromatography combination, urease protective agent and application of urease protective agent

Abstract

The invention discloses a urea detection method based on urease-ion chromatography combination, a urease protective agent and application of the urease protective agent, and belongs to the technical field of urea detection. The method specifically comprises the following steps: (1) adding a urease protective agent into a urea solution to be detected, shaking and uniformly mixing, and filtering; (2) adding a urease solution into the solution treated in the step (1), uniformly mixing, and then reacting under a water bath condition to decompose urea; and (3) detecting the solution obtained in the step (2) and the standard solution by using an ion chromatograph, and then calculating the content of urea. Through combination of multiple technologies, the detection operation process is remarkably simplified, meanwhile, the quantitative detection limit is reduced, and the sensitivity and accuracy of urea detection are greatly improved; besides, by adding the urease protective agent, the influence of interference substances such as metal ions and nitrogen-containing compounds is effectively eliminated, and the anti-interference capability of the method is remarkably enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of urea detection, and specifically relates to a urea detection method based on the combination of urease and ion chromatography, a urease protectant and its uses. Background Art

[0002] Urea, as a key raw material in the fields of agriculture, medicine, chemical industry, etc., is of irreplaceable importance. Currently, the production of urea still mainly uses the Bosch-Meiser method. However, the reaction conditions of this method are extremely harsh (150 - 200 °C, 150 - 250 bar), which not only consumes a large amount of energy but also causes a large amount of pollutant emissions. In recent years, electrocatalytic synthesis of urea has received extensive attention due to its mild reaction conditions and characteristics of being green and sustainable. Driven by renewable energy, electrocatalytic synthesis of urea uses carbon dioxide (CO 2 ) and abundant nitrogen sources (N 2 , NH 3 , NO x , NO 2 - , NO 3 - ) as raw materials to generate urea through an electrochemical reaction under normal temperature and pressure, which can not only effectively reduce greenhouse gas emissions but also realize the efficient utilization of carbon and nitrogen resources. Therefore, electrocatalytic synthesis of urea has important strategic significance in the fields of energy conversion and environmental protection.

[0003] Although significant progress has been made in the field of electrocatalytic synthesis of urea, the low urea yield and complex by-product separation pose severe challenges to the quantitative analysis of urea, seriously restricting the evaluation of urea production efficiency and the development and optimization of new catalysts. Currently, the urease method, as a commonly used urea quantitative analysis method, exposes many limitations in the practical application of electrocatalytic synthesis of urea: First, its operation is cumbersome, time-consuming, and the detection range is narrow, making it difficult to achieve high-throughput detection; second, urease is easily inactivated by the poisoning effect of metal cations in the electrolyte solution, resulting in insufficient stability and reliability in complex reaction systems.

[0004] Although a method for protecting the activity of urease by adding urease protectants was previously disclosed in CN 111826369 A, its application in the detection of electrocatalytic urea synthesis still has significant deficiencies. Specifically, the urease protectants in this patent are mainly designed for soil detection systems, where the urea content is relatively high, and some potential interferences can be ignored. However, in electrocatalytic synthesis systems, the urea concentration is usually low, and the interfering substances are more complex, resulting in a significant decline in its protective effect. In addition, some components of urease protectants may undergo side reactions with other components in the electrolyte solution, seriously interfering with the quantitative analysis of urea, and further screening and optimization are still needed. More critically, the existing urease protectants are costly, and their shielding ability against various interfering substances (such as metal ions, organic by-products, etc.) in complex reaction systems is limited, making it difficult to meet the requirements of efficient and accurate detection for large-scale electrocatalytic urea synthesis. In summary, although urease protectants have improved the stability of urease to a certain extent, there are still many limitations in their practical applications. Therefore, there is an urgent need to develop a urea quantification method with high efficiency, stability, a wide detection range, and strong anti-interference ability, which has important scientific significance and application value for promoting the further development of the electrocatalytic urea synthesis field. Summary of the Invention

[0005] Aiming at the above deficiencies in the prior art, the present invention provides a urea detection method based on the combination of urease and ion chromatography, a urease protectant and its uses, to solve the problems in the current field of electrocatalytic synthesis of urea, such as the detection method being easily interfered, inaccurate quantification, narrow detection range, and cumbersome operation, and provides technical support for the accurate quantification of urea in the field of electrocatalytic synthesis of urea.

[0006] To achieve the above object, the technical solution adopted by the present invention to solve its technical problems is as follows:

[0007] A urea detection method based on the combination of urease and ion chromatography, comprising the following steps:

[0008] (1) Add a urease protectant to the urea solution to be tested, mix evenly by shaking, and then filter;

[0009] (2) Add the urease solution to the solution treated in step (1), mix evenly, and then react to decompose urea under water bath conditions;

[0010] (3) Use an ion chromatograph to detect the solution obtained in step (2) and the standard solution, and then calculate the urea content.

[0011] Further, the concentration of the urease protectant in the urea solution to be tested in step (1) is 0.2 - 20 mg / mL.

[0012] Further, the shaking time in step (1) is 1 - 10 min.

[0013] Furthermore, the urease protectant comprises a reducing agent, a buffer solution, and a protecting component with a mass ratio of 1-8:1-5:1-5.

[0014] Furthermore, the reducing agent is zinc powder, dithiothreitol, glutathione, or ascorbic acid;

[0015] The buffer solution is phosphate, Tris hydrochloride, carbonate, or HEPES salt;

[0016] The protecting component is EDTA, citric acid, tartaric acid, or gluconic acid.

[0017] Furthermore, the volume ratio of the urease solution to the urea solution to be measured is 0.1-0.5:1-5.

[0018] Furthermore, the concentration of urease needs to be precisely controlled. If the concentration is too low, the decomposition of urea in the solution will be incomplete; if the concentration is too high, it may introduce unnecessary background interference and increase the detection cost. Based on this, in the present invention, the concentration of the urease solution is 1-20 mg / mL, and the urease activity is 1-3 U / mg.

[0019] Furthermore, the urea solution to be measured is the solution generated during the electrocatalytic synthesis of urea.

[0020] Furthermore, the chromatographic analysis conditions of the ion chromatograph in step (2) are as follows: The chromatographic column uses a Dionex IonPac C12A cationic chromatographic column, with a specification of 5 μm, 4*250 mm. The column temperature is 40°C, the mobile phase is a 5-20 mmol / L methanesulfonic acid solution, the flow rate is 1 mL / min, the injection volume is 1-20 μL of the solution to be measured, isocratic elution is performed, and the total elution time is 15 min.

[0021] Furthermore, in the present invention, the reaction time and temperature of urease need to be controlled. If the reaction time is too short or the temperature is too low, the decomposition of urea may be incomplete; if the reaction time is too long, the detection efficiency will be reduced; if the reaction temperature is too high, urease will be inactivated, interfering with the detection results. Based on this, in step (2) of the present invention, the reaction temperature is 20-40°C, and the reaction time is 10-40 min.

[0022] Furthermore, in step (3), the external standard method or the standard curve method is used to calculate the content of urea. The specific process is as follows:

[0023] A series of urea standard solutions with concentration gradients are prepared. With the concentration of the urea standard solution as the abscissa and the peak area of the target substance as the ordinate, a standard curve of urea concentration and peak area is plotted. According to the peak area of the target substance in the sample to be measured, substituting it into the standard curve, the content of urea in the sample solution is calculated.

[0024] A urease protectant, which comprises a reducing agent, a buffer solution and a protecting component with a mass ratio of 1-10:1-5:1-5;

[0025] The reducing agent is zinc powder, dithiothreitol, glutathione or ascorbic acid;

[0026] The buffer solution is phosphate, Tris hydrochloride, carbonate or HEPES salt;

[0027] The protecting component is EDTA, citric acid, tartaric acid or gluconic acid.

[0028] The above urea detection method, or the use of the urease protectant in detecting the urea content during the electrocatalytic synthesis of urea.

[0029] Advantages of the present invention:

[0030] 1. The present invention adopts the urease-ion chromatography coupling technology, simplifies the detection operation process, reduces the detection limit, and significantly improves the sensitivity and accuracy of urea detection, especially suitable for the analysis of electrocatalytic synthesis of urea solution.

[0031] 2. By adding a urease protectant composed of multiple components, the present invention effectively eliminates the influence of interfering substances such as metal ions and nitrogen-containing compounds, and significantly improves the anti-interference ability of the method.

[0032] 3. The present invention supports batch detection, has a wide detection range and strong applicability. It can not only be used in the electrocatalytic synthesis of urea system, but also be extended to the quantitative analysis of urea in the fields of agriculture, medicine and environmental monitoring. Description of the Drawings

[0033] Figure 1 It is the flow chart of the detection method of the present invention;

[0034] Figure 2 It is the coordinate diagram of the standard curve and the linear regression coefficient provided in Example 1 of the present invention;

[0035] Figure 3 It is the chromatogram of the standard urea solution provided in Example 1 of the present invention;

[0036] Figure 4 It is the chromatogram of the sample solution provided in Example 1 of the present invention;

[0037] Figure 5 It is the actual detection result of the sample solution provided in Example 1 of the present invention under the interference of metal ions. Detailed Embodiments

[0038] The specific embodiments of the present invention will be described below to facilitate those skilled in the art to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions made using the concept of the present invention are within the scope of protection.

[0039] Example 1

[0040] A urea detection method based on the combination of urease and ion chromatography, which comprises the following steps:

[0041] (1) Add urease to deionized water and dissolve it thoroughly by ultrasonic treatment to prepare a urease solution with a concentration of 5 mg / mL for standby;

[0042] (2) Take 2 mL of the urea solution after electrocatalytic reaction (with a known concentration of 2 mg / L), add 10 mg of urease protectant, where the components of the urease protectant are 5 mg of zinc powder, 2 mg of phosphate, and 2 mg of EDTA. After shaking and mixing for 5 min, filter to obtain the urea solution to be measured;

[0043] (3) Take 0.1 mL of the urease solution prepared in step (1) and add it to the urea solution to be measured processed in step (2), and mix well; then place the mixed solution in a constant temperature water bath and react at 37 °C for 20 min to completely decompose urea into CO 2 and NH 3 molecules;

[0044] (4) Use an ion chromatograph to detect and analyze the solution in step (3) and the standard solution respectively; the chromatographic column uses a Dionex IonPac CS12A cation exchange chromatographic column with a specification of 5 μm, 4 * 250 mm, the column temperature is 40 °C, the flow rate is 1 mL / min, the mobile phase is 10 mmol / L methanesulfonic acid solution, the injection volume is 2 μL, isocratic elution, and the total elution time is 15 min;

[0045] (5) Prepare a series of urea standard solutions with concentration gradients and perform processing and detection according to the above steps (1) to (4); take the concentration of the urea standard solution as the abscissa and the peak area of the target substance as the ordinate to draw the standard curve of urea concentration and peak area. According to the peak area of the target substance in the sample to be measured, substitute it into the standard curve to calculate the content of urea in the sample solution.

[0046] Example 2

[0047] A urea detection method based on the combination of urease and ion chromatography, which comprises the following steps:

[0048] (1) Add urease to deionized water and dissolve it completely by ultrasonic treatment to prepare a 5 mg / mL urease solution for standby.

[0049] (2) Take 5 mL of the urea solution after electrocatalytic reaction (known concentration is 5 mg / L), add 20 mg of urease protectant, where the components of the urease protectant are 10 mg of dithiothreitol, 5 mg of carbonate, and 5 mg of citric acid. After shaking and mixing for 5 min, filter to obtain the urea solution to be tested.

[0050] (3) Take 0.1 mL of the urease solution prepared in step (1) and add it to the urea solution to be tested treated in step (2), and mix well. Then place the mixed solution in a constant temperature water bath and react at 40 °C for 20 min to completely decompose urea into CO 2 and NH 3 molecules.

[0051] (4) Use an ion chromatograph to detect and analyze the solution described in step (3) and the standard solution respectively; the chromatographic column uses a Dionex IonPac CS12A cationic chromatographic column with a specification of 5 μm, 4 * 250 mm, the chromatographic column temperature is 40 °C, the flow rate is 1 mL / min, the mobile phase is a 10 mmol / L methanesulfonic acid solution, the injection volume is 2 μL, isocratic elution, and the total elution time is 15 min.

[0052] (5) Prepare a series of urea standard solutions with concentration gradients and perform treatment and detection according to the above steps (1) - (4); use the concentration of the urea standard solution as the abscissa and the peak area of the target substance as the ordinate to plot the standard curve of urea concentration vs. peak area. According to the peak area of the target substance in the sample to be tested, substitute it into the standard curve to calculate the urea content in the sample solution.

[0053] Example 3

[0054] A urea detection method based on the combination of urease - ion chromatography, which includes the following steps:

[0055] (1) Add urease to deionized water and dissolve it completely by ultrasonic treatment to prepare a 5 mg / mL urease solution for standby.

[0056] (2) Take 3.5 mL of the urea solution after electrocatalytic reaction (known concentration is 3.5 mg / L), add 15 mg of urease protectant, where the components of the urease protectant are 8 mg of ascorbic acid, 4 mg of Tris hydrochloride, and 3 mg of tartaric acid. After shaking and mixing for 5 min, filter to obtain the urea solution to be tested.

[0057] (3) Take 0.1 mL of the urease solution prepared in step (1) and add it to the urea solution to be measured after being treated in step (2), and mix well; then place the mixed solution in a constant temperature water bath and react at 20 °C for 20 min to completely decompose urea into CO 2 and NH 3 molecules;

[0058] (4) Use an ion chromatograph to detect and analyze the solution described in step (3) and the standard solution respectively; the chromatographic column uses a Dionex IonPac CS12A cationic chromatographic column with a specification of 5 μm, 4 * 250 mm, the column temperature is 40 °C, the flow rate is 1 mL / min, the mobile phase is 10 mmol / L methanesulfonic acid solution, the injection volume is 2 μL, isocratic elution, and the total elution time is 15 min;

[0059] (5) Prepare a series of urea standard solutions with concentration gradients, and perform treatment and detection according to the above steps (1) to (4); use the concentration of the urea standard solution as the abscissa and the peak area of the target substance as the ordinate to draw the standard curve of urea concentration and peak area. According to the peak area of the target substance in the sample to be measured, substitute it into the standard curve to calculate the urea content in the sample solution.

[0060] Comparative Example 1

[0061] Compared with Example 1, the difference is that sodium borohydride is used to replace zinc powder in the urease protectant, and the rest of the process is the same as that of Example 1.

[0062] Comparative Example 2

[0063] Compared with Example 1, the difference is that potassium hydroxide is used to replace phosphate in the urease protectant, and the rest of the process is the same as that of Example 1.

[0064] Comparative Example 3

[0065] Compared with Example 1, the difference is that: in step (3), the reaction is carried out at 50 °C in a constant temperature water bath for 20 min, and the rest of the process is the same as that of Example 1.

[0066] Comparative Example 4

[0067] Compared with Example 1, the difference is that zinc powder, a reducing agent, is not used in the urease protectant group. To control the experimental variables, 5 mg of inert component silica is added, and the rest of the process is the same as that of Example 1.

[0068] Comparative Example 5

[0069] Compared with Example 1, the difference is that the protectant EDTA is not used in the urease protectant group. To control the experimental variables, 2 mg of inert component silica is added, and the rest of the process is the same as that of Example 1.

[0070] Test Example 1

[0071] To fully demonstrate the superiority of this method in urea detection for electrocatalytic urea synthesis and the uniqueness of the components of the urease protectant, a comparative experiment was conducted using the national standard method (GB / T 18204.2-2014), the method of the present invention (Example 1), and the comparative example respectively. The results are shown in Table 1 as follows:

[0072] Table 1 Comparison of experimental results of the national standard method, this method, and the comparative example

[0073]

[0074] Note: RD is the relative deviation, RSD is the relative standard deviation, and the concentration is mg / L

[0075] The above results show that the urea detection method based on the combination of urease and ion chromatography is significantly superior to the traditional national standard method in terms of stability, accuracy, and repeatability. In addition, each component in the urease protectant plays an irreplaceable role, and its uniqueness and synergistic effect jointly ensure the high activity of urease and the stability of detection, providing reliable technical support for the precise quantitative analysis of urea in electrochemical reactions

[0076] Test Example 2. Verification of the effect of the urease-ion chromatography combined method constructed by the present invention

[0077] To fully prove that this method has high applicability and accuracy in actual urea detection, verification was carried out from four aspects: linear relationship, detection limit, repeatability, and accuracy

[0078] (1) Linear relationship: According to the same steps of Example 1, urea standard solutions with concentrations of 0-2 mg / L and 0-20 mg / L were respectively prepared. Through the urease-catalyzed reaction, NH 3 was generated, and the relationship between the concentration of NH 3 and the chromatographic signal was measured by ion chromatography. The results show that there is a good linear relationship between the urea concentration and the chromatographic peak area, and the correlation coefficient (R 2 ) is 0.9998 ( Figure 2 ), proving that this method has strong linear response characteristics in a relatively wide range. The detailed data are shown in Table 2

[0079] (2) Detection limit: According to the same steps of Example 1, blank deionized water was injected under the same chromatographic conditions, and the maximum baseline noise value was detected to be 0.005 mV within the range of 1-2 minutes at the main peak position. According to the calculation of the detection limit with 3 times the signal-to-noise ratio (S / N = 3), the quantitative limit concentration of this method for urea is 0.23 mg / mL, with a relatively low detection limit. The detailed data are shown in Table 2

[0080] Table 2 Standard Curve and Detection Limit of Urease-Ion Chromatography Coupled Urea Detection Method

[0081]

[0082] (3) Repeatability test: To evaluate the repeatability of this method, multiple repeated tests were carried out on the urea standard solution and experimental samples (see Figure 3 and Figure 4 respectively), and the analysis was carried out strictly according to the steps of Example 1. The results show that for both standard samples and experimental samples, the relative standard deviation (RSD) values of the measured urea concentration results are less than 2% (see Table 3 for details), meeting the requirements of the pharmacopoeia, indicating that the urease-ion chromatography coupling technology adopted in the present invention has excellent repeatability and is particularly suitable for the detection of urea concentration in the electrocatalytic synthesis of urea system.

[0083] Table 3 Repeatability Test Results of Urease-Ion Chromatography Coupled Urea Detection Method

[0084]

[0085] (4) Accuracy test: To verify the accuracy of this method, a sample spiking recovery experiment was used for evaluation. Urea standard solutions with low, medium, and high concentrations were added to the sample solutions respectively, and each sample was measured 3 times repeatedly. The analysis steps were the same as those in Example 1. The results show that the recoveries and relative standard deviations (RSD) of all spiked samples meet the requirements, and the specific data are shown in Table 4 for details.

[0086] Table 4 Spiking Recovery of Urease-Ion Chromatography Coupled Urea Detection Method (n = 3)

[0087]

[0088] Test Example 3 Verification of the Anti-Interference Ability of the Detection Method Provided by the Present Invention against Metal Ions

[0089] To verify the anti-interference ability of this method, various interfering metal ions at a concentration of 10 mg / L (including Cu 2+ , Pd 2+ , Ag + , Pt 4+ and Au 3+ etc.) were added to the standard samples, and the urea concentration analysis was carried out according to the same steps as in Example 1. At the same time, the pretreatment process without step (2) (i.e., without adding urease protectant) was used as a control, and the results are shown in Figure 5 .

[0090] As Figure 5As shown, since the control group was not treated with the urease protectant, the results showed that it was unable to accurately quantify urea, indicating that the urease was inactivated due to the poisoning effect of metal ions. In contrast, the experimental group pretreated with the urease protectant (the method of the present invention) was able to accurately quantify urea. This result fully demonstrates that the urease protectant added during the pretreatment process significantly enhanced the anti-interference ability of the method, effectively eliminated the inhibitory effect of metal ions on urease activity, and ensured the accuracy and reliability of the detection results (the results are shown in Figure 5 ).

[0091] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A urea detection method based on urease-ion chromatography, characterized in that: The following steps are involved: (1) Add urease protective agent to the urea solution to be tested, shake and mix evenly, and then filter; (2) adding the urease solution to the solution treated in step (1) and mixing them evenly, and then reacting and decomposing urea in a water bath; (3) Using an ion chromatograph, the solution obtained in step (2) and the standard solution are detected, and the urea content is calculated.

2. The urea detection method based on urease-ion chromatography according to claim 1, characterized in that: In step (1), the concentration of the urease protective agent in the urea solution to be tested is 0.2-20 mg / mL.

3. The urea detection method based on urease-ion chromatography according to claim 1 or 2, characterized in that: The urease protective agent comprises a reducing agent, a buffer and a protective component in a mass ratio of 1-10:1-5:1-5.

4. The urea detection method based on urease-ion chromatography according to claim 3, characterized in that: The reducing agent is zinc powder, dithiothreitol, glutathione or ascorbic acid; The buffer was phosphate, Tris hydrochloride, carbonate, or HEPES salt; The protective ingredients are EDTA, citric acid, tartaric acid or gluconic acid.

5. The urea detection method based on urease-ion chromatography according to claim 1, characterized in that: The volume ratio of urease solution to urea solution to be tested is 0.1~0.5:1~5.

6. The urea detection method based on urease-ion chromatography according to claim 1 or 5, characterized in that: The concentration of the urease solution is 1~20 mg / mL, and the urease activity is 1~3 U / mg.

7. The urea detection method based on urease-ion chromatography according to claim 1 or 5, characterized in that: The urea solution to be tested is a solution produced in the process of electrocatalytic synthesis of urea.

8. The urea detection method based on urease-ion chromatography according to claim 1, characterized in that: In step (2), the reaction temperature is 20-40° C. and the reaction time is 10-40 min.

9. A urease protective agent, characterized in that It includes a reducing agent, a buffer and a protective component in a mass ratio of 1~8:1~5:1~5; The reducing agent is zinc powder, dithiothreitol, glutathione or ascorbic acid; The buffer was phosphate, Tris hydrochloride, carbonate, or HEPES salt; The protective ingredients are EDTA, citric acid, tartaric acid or gluconic acid.

10. Use of the urea detection method according to any one of claims 1 to 8, or the urease protective agent according to claim 9, in detecting urea content during electrocatalytic synthesis of urea.

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