Method for detecting content of urea in water sample by using high performance liquid chromatography

Abstract

The invention provides a method for detecting the content of urea in a water sample by using high performance liquid chromatography. The method comprises the following steps: preparing a urea standard solution; performing filter membrane filtration pretreatment on the to-be-detected water sample; carrying out high performance liquid chromatography analysis according to optimized chromatographic conditions, and calculating the urea content by adopting a standard curve method; wherein the chromatographic conditions are as follows: a C18 type reversed-phase chromatographic column is adopted, a mixed solution of acetonitrile and ultrapure water is adopted as a mobile phase, isocratic elution is performed, and the detection wavelength is 190-200nm. According to the method, the mobile phase and the chromatographic column are improved, the urea content in the water sample is directly and accurately detected, operation is easy and convenient, the detection time is short, good specificity and stability are achieved, the detection limit is lower than 0.6 microgram / L, the recovery rate ranges from 95% to 105%, the relative standard deviation is smaller than 2%, and the method can be effectively applied to detection and analysis of the urea content in various environment water samples.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of water quality monitoring, in particular to a method for detecting urea content in water samples by high performance liquid chromatography. BACKGROUND

[0002] Urea is a white crystal, soluble in water, and is a strong polar ultraviolet end absorption compound. Urea has a wide range of uses and there are various detection methods. Currently, the detection of urea is mostly based on the detection of total nitrogen content instead of urea content. This method is complicated to operate, has a high lower limit of detection, and the results cannot represent the actual content of urea. Other methods for detecting trace urea, such as using ultraviolet spectrophotometry-colorimetry to detect urea, have many interference factors, poor sensitivity and repeatability, and a high lower limit of detection.

[0003] Although the current high performance liquid chromatography method is simple to operate, time-consuming and accurate, the detection range is mostly in the ppm level. There are also methods for detecting urea content by using pre-column derivatization liquid chromatography or post-column derivatization-liquid chromatography, or using high performance liquid chromatography tandem mass spectrometry to improve the accuracy of determination and reduce the lower limit of detection. However, the above methods have complex sample pretreatment, difficulty in adapting derivatizing agents, differences in derivatization effect, and expensive detectors.

[0004] For urea monitoring in water environment, the existing technology often fails to achieve high sensitivity and high precision detection due to the low urea content in water. When using conventional liquid chromatography methods, the separation effect of urea chromatographic peaks and impurity chromatographic peaks is poor, resulting in inaccurate detection results. In addition, the selection of mobile phase composition and chromatographic conditions in the existing methods is not ideal, and it is difficult to simultaneously consider detection sensitivity and analysis efficiency.

[0005] Therefore, there is an urgent need in the field of water quality monitoring to develop a simple, accurate, efficient and low-cost urea detection method, especially an analysis method capable of detecting extremely low content of urea in water samples. SUMMARY

[0006] The present application aims to provide a method for detecting urea content in water samples by high performance liquid chromatography, to solve the problem of inaccurate detection results when using conventional liquid chromatography methods to detect extremely low content of urea in water samples.

[0007] To achieve the above technical purposes, the present application provides a method for detecting urea content in water samples by high performance liquid chromatography, comprising: preparing a urea standard solution; pretreating the water sample to be tested; analyzing the standard solution and the sample solution by high performance liquid chromatography; and The urea content is calculated by using a standard curve method; The chromatographic conditions are as follows: The C18 type reversed-phase chromatographic column is used; the mobile phase is a mixed solution of an organic phase and an aqueous phase, the organic phase is acetonitrile, and the aqueous phase is ultrapure water; the mobile phase is isocratic elution; and the detection wavelength is 190-200 nm.

[0008] Optionally, the length of the C18 type reversed-phase chromatographic column is 200-300 mm, the inner diameter is 4-5 mm, the filler particle size is 3-6 μm, and the temperature of the chromatographic column is 40-50℃.

[0009] Optionally, the volume ratio of acetonitrile to ultrapure water in the mobile phase is 2:98 to 10:90; preferably, the volume ratio of acetonitrile to ultrapure water in the mobile phase is 5:95.

[0010] Optionally, the flow rate of the mobile phase is 0.8-1.2 mL / min.

[0011] Optionally, the pretreatment step comprises filtering using a 0.22-0.45 μm filter membrane.

[0012] Optionally, the step of preparing the urea standard solution comprises: Accurately weighing the urea standard, and adding ultrapure water to obtain a urea standard stock solution; Respectively, different amounts of the standard stock solution are measured, and ultrapure water is added to obtain urea standard working solutions of different concentrations.

[0013] Optionally, the standard curve method draws a standard curve with the peak area of the urea standard solution as the ordinate and the concentration of the urea standard solution as the abscissa; the peak area of the target object detected in the sample solution is substituted into the standard curve to calculate the content of urea in the sample.

[0014] Optionally, the detection limit of the method is lower than 0.6 μg / L, and the quantification limit is lower than 2 μg / L.

[0015] The application further provides a method for detecting low-concentration urea in environmental water samples by high-performance liquid chromatography, comprising: Collecting water samples; Detecting the urea content in the water samples by using the above method.

[0016] Optionally, the environmental water samples include surface water, underground water, seawater, lake water, river water or wastewater.

[0017] Compared with the prior art, the application has at least the following beneficial effects: The present application realizes accurate detection of extremely low content of urea in water samples by optimizing the detection conditions of high performance liquid chromatography, in particular, selecting a chromatographic column, optimizing the composition of a mobile phase and a detection wavelength. The method is simple in operation, good in chromatographic peak shape, good in separation effect of urea and impurity peaks, short in detection time, and can be used as an accurate determination method of urea in water and other matrices.

[0018] The method of the present application has the advantages of low detection limit, fast analysis speed, high precision, etc. By using the optimized mobile phase ratio and chromatographic column temperature conditions, the chromatographic peak shape can be effectively improved, and the analysis sensitivity and accuracy can be improved. Compared with the prior art, the method is more simple in operation, does not need complex sample pretreatment or derivatization steps, and the detection lower limit can reach nanogram level, meeting the demand of extremely trace urea detection in environmental water samples, and providing a reliable technical means for water environment monitoring. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 A flow chart of the method for detecting urea content in water samples by high performance liquid chromatography in the embodiment one of the present application; Figure 2 A standard curve diagram in the embodiment two of the present application; Figure 3 A chromatogram of the same concentration standard in the embodiment two of the present application; Figure 4 A chromatogram of the sample solution in the embodiment two of the present application. DETAILED DESCRIPTION

[0020] A method for detecting urea content in water samples by high performance liquid chromatography will be described in more detail below with reference to the accompanying drawings, wherein preferred embodiments of the present application are represented, and it should be understood that the present application described herein can be modified by those skilled in the art, while still achieving the advantageous effects of the present application. Therefore, the following description should be understood as extensive knowledge for those skilled in the art, and not as a limitation on the present application.

[0021] The present application will be described in more detail in the following paragraphs with reference to the accompanying drawings. The advantages and features of the present application will be more apparent according to the following description. It should be noted that the drawings are very simplified and all use non-precise proportions, only to facilitate, clearly assist the purpose of illustrating the embodiments of the present application.

[0022] Embodiment one The embodiment of the present application provides a method for detecting urea content in water samples by high performance liquid chromatography, please refer to Figure 1 , including the following steps: S1: preparing urea standard solution.

[0023] Specifically, 0.010 g of urea standard substance (accurate to 0.0001 g) is accurately weighed into a 1000 mL volumetric flask, and ultrapure water is added to the calibration line, and then the volume is fixed, and mixed to obtain a standard stock solution of urea. Using a pipette, 50 μL, 100 μL, 200 μL, 400 μL, 600 μL, and 1000 μL of the standard stock solution are respectively taken into a 100 mL volumetric flask, and ultrapure water is added to the calibration line, and then the volume is fixed, and mixed to obtain a standard working solution of urea with different concentrations.

[0024] In one specific example, the standard stock solution can be stored at 2-8°C, and the effective period is 1 month; the standard working solution is prepared and used immediately.

[0025] In another specific example, the mass of the urea standard substance can be selected in the range of 0.005-0.020 g, and the concentration range of the urea standard working solution can cover the expected concentration range of the analysis sample.

[0026] S2: Pretreatment of the water sample to be tested.

[0027] 0.22-0.45 μm filter membrane is used for filtration.

[0028] In one specific example, 0.22 μm filter membrane is selected for filtration, and the specific pore size can be selected according to the turbidity and suspended solids content of the water sample. For water samples with high clarity, no filtration is required, which is a common means for those skilled in the art and will not be described in detail.

[0029] S3: Analysis of standard solution and sample solution by high performance liquid chromatography.

[0030] The high performance liquid chromatography conditions are as follows: Chromatographic column: Thermo Hypersil GOLD™ C18 chromatographic column, column length 250 mm, inner diameter 4.6 mm, and filler particle size 5.0 μm; Mobile phase: acetonitrile and ultrapure water in a volume ratio of 5:95; Flow rate: 1 mL / min; Elution mode: isocratic elution; Elution time: 5 min; Column temperature: 45°C; Detection wavelength: 195 nm; Injection volume: 10 μL.

[0031] In one specific example, the C18 type reversed-phase chromatographic column can be various brands of commercially available C18 chromatographic columns, the length can be selected in the range of 200-300 mm, the inner diameter can be selected in the range of 4-5 mm, and the filler particle size can be selected in the range of 3-6 μm.

[0032] In another specific example, the volume ratio of acetonitrile to ultrapure water in the mobile phase can be selected in the range of 2:98 to 10:90, preferably 5:95. The flow rate of the mobile phase can be adjusted in the range of 0.8-1.2 mL / min. The temperature of the chromatographic column can be set in the range of 40-50℃. The detection wavelength can be selected in the range of 190-200 nm.

[0033] In another specific example, the mobile phase can be filtered with a 0.45 μm filter membrane before use, and degassed by ultrasonic for 20 min to ensure that there are no bubbles and impurities in the mobile phase.

[0034] S4: Calculate the urea content by using the standard curve method.

[0035] The peak area of the urea standard solution is taken as the ordinate, and the concentration of the urea standard solution is taken as the abscissa to draw a standard curve; the peak area of the target substance detected in the sample solution is substituted into the standard curve, and the content of urea in the test sample is calculated according to the standard curve.

[0036] The chromatographic conditions adopted by the method provided in the embodiments of the present application make the retention behavior of urea in the chromatographic system stable, the peak shape symmetrical, and the interference substances in the water sample well separated. In particular, by selecting the ratio of acetonitrile to ultrapure water of 5:95 and the detection wavelength of 195 nm, the background interference can be effectively reduced while ensuring the sensitivity of urea detection. Through the simple filter membrane filtration pretreatment step, complex sample pretreatment is avoided, and the operation process is greatly simplified. The entire analysis process only takes 5 minutes, which saves a lot of time compared to the traditional method. The method does not need to use ion pair reagents or a derivatization step, which reduces the determination cost and eliminates the potential error introduced by the derivatizing agent. The method is simple, fast, economical and reliable, and provides an efficient solution for accurate determination of the urea content in water samples.

[0037] Example Two The present embodiment verifies the methodology of the high performance liquid chromatography method described in Example One, including linearity, detection limit, quantification limit, repeatability, precision verification, and comparison experiments one to three.

[0038] I. Linearity verification: Take the prepared 6 concentrations of standard working solution, detect according to the chromatographic conditions described in step S3 of Example One, draw a standard curve, and the peak area is shown in Table 1 below.

[0039] Table 1: Urea linearity result statistics table The peak area of the urea standard solution is taken as the ordinate, and the concentration of the urea standard solution is taken as the abscissa to draw a standard curve, as shown in Figure 2

[0040] ​Linear correlation coefficient R of standard curve 2 ≥0.995, the linear result is good and meets the requirements. The linear range of the method is 5-100 μg / L.

[0041] II. Verification of detection limit and quantitative limit: take a low concentration of standard working solution, repeat injection for 10 times, detect according to the chromatographic conditions described in S3 of Example 1, calculate the standard deviation of different determination results, and calculate the detection limit and the quantitative limit by using the standard deviation. The detection limit (LOD) value is three times the standard deviation, and the quantitative limit (LOQ) value is ten times the standard deviation. The results are shown in Table 2.

[0042] Table 2: Detection limit and quantitative limit result statistics table of urea The verification results show that the detection limit of the method is 0.55 μg / L, and the quantitative limit is 1.83 μg / L, both of which are lower than 0.6 μg / L and 2 μg / L.

[0043] III. Reproducibility verification: prepare the sample solution according to the steps in Example 1, repeat injection for 6 times according to the chromatographic conditions described in S3 of Example 1, and calculate the content of urea in the sample according to the standard curve. The results are shown in Table 3, wherein Figure 3 is the chromatogram of the standard sample of the same concentration, Figure 4 is the chromatogram of the sample solution.

[0044] Table 3: Reproducibility result statistics table of urea The relative standard deviation RSD value is less than 2%, which meets the requirements of the pharmacopoeia, indicating that the method for determining the content of urea in water by high performance liquid chromatography has good reproducibility.

[0045] IV. Precision verification: prepare three samples of low, medium and high concentrations respectively, prepare the sample urea standard working solution according to the steps in Example 1, inject one needle according to the chromatographic conditions described in S3, and calculate the content of urea in the sample according to the standard curve. Calculate the recovery rate based on the theoretical content of the prepared sample, and the recovery rate = measured content / theoretical content x 100%. The results are shown in Table 4.

[0046] Table 4: Precision detection result statistics table The direct recovery method is used to verify the precision, and the recovery rate is between 95%-105%, and the RSD value is less than 2%, which meets the requirements.

[0047] In addition, in order to further optimize the detection method of the present application, the selection basis of the optimal chromatographic conditions, the system comparison experiments of the mobile phase organic phase type, the mobile phase proportion and the column temperature were also carried out in this embodiment, which verified the scientificity and rationality of the selected chromatographic conditions.

[0048] V. Comparative Experiment One In order to determine the optimal mobile phase organic phase type, the comparison experiments of methanol and acetonitrile were carried out, the volume ratio of the organic system to ultrapure water was set to 5:95, 10 ug / mL standard stock solution was taken as the sample, and methanol: ultrapure water and acetonitrile: ultrapure water were used respectively according to the chromatographic conditions (except the mobile phase organic phase type) described in step S3 in the embodiment one, and 6 injections were repeated, and the results are shown in Table 5.

[0049] Table 5: Selection of mobile phase The results show that when acetonitrile is used as the organic phase, the peak area is larger and the relative standard deviation RSD is smaller, which indicates that the separation effect of acetonitrile as the organic phase is better and the repeatability is better.

[0050] VI. Comparative Experiment Two In order to optimize the mobile phase proportion, the influence of different acetonitrile volume ratios on the detection results was systematically investigated, the mobile phase A was acetonitrile and the mobile phase B was ultrapure water, and the acetonitrile volume ratio was adjusted to 2%, 5%, 10% and 15%. 10 ug / mL standard stock solution was taken as the sample, and detection was carried out according to the chromatographic conditions (except the mobile phase proportion) described in step S3 in the embodiment one. The results are shown in Table 6.

[0051] Table 6: Selection of mobile phase proportion The results show that when the acetonitrile volume ratio is 5%, the relative standard deviation RSD is the smallest (0.17%), the repeatability is the best, the peak area is moderate, and the separation effect is better.

[0052] VII. Comparative Experiment Three In order to determine the optimal column temperature condition, the influence of different temperature settings on the detection of urea was investigated, and the column temperature was set to 30℃, 35℃, 40℃, 45℃ and 50℃. 10 ug / mL standard stock solution was taken as the sample, and detection was carried out according to the chromatographic conditions (except the column temperature) described in step S3 in the embodiment one. The results are shown in Table 7.

[0053] Table 7: Selection of column temperature setting The results show that when the column temperature is in the range of 40℃-50℃, the peak area is relatively stable and larger.

[0054] In summary, the present embodiment proves that the method for detecting the content of urea in water samples by high performance liquid chromatography established by the present application has good performance indicators. The method verification results show that the linear relationship is good, the linear correlation coefficient R 2 ≥0.9996 in the range of 5-100 μg / L; the detection sensitivity is high, the detection limit is 0.55 μg / L, and the quantification limit is 1.83 μg / L, both reaching the nanogram level of detection; the repeatability is excellent, the relative standard deviation RSD is 1.79%, which is much smaller than the requirement of 2% of the pharmacopoeia; the precision is good, the recovery rates at different concentration levels are all between 95%-105%, and the RSD values are all less than 2%. In addition, the comparative experiment systematically optimizes the key chromatographic conditions such as the type of organic phase of the mobile phase, the proportion of the mobile phase, and the column temperature.

[0055] Example Three The present application provides a method for determining the content of urea in environmental water samples, which adopts the method described in Example One to determine the content of urea in different types of environmental water samples.

[0056] S1: Collecting water samples: Collect different types of environmental water samples such as surface water, groundwater, seawater, lake water, river water, and wastewater. Among them, the surface water sample is collected from a city park pond, the groundwater sample is collected from a monitoring well, the seawater sample is collected from a near-sea area, the lake water sample is collected from a freshwater lake, the river water sample is collected from a city river, and the wastewater sample is collected from the inlet of a sewage treatment plant. All water samples are immediately stored at 4°C after collection and analyzed within 24 hours.

[0057] S2: Water sample pretreatment: Pretreat the collected water samples according to the method described in S2 of Example One, which is not repeated here.

[0058] S3: Sample analysis: The pretreated water samples are analyzed by high performance liquid chromatography under the chromatographic conditions, and each sample is determined in triplicate. At the same time, the content of urea in each water sample is calculated by the standard curve method described in step S4 of Example One, and the influence of different matrix water samples on the determination results is small.

[0059] The content of urea in various types of environmental water samples can be accurately determined, and the relative standard deviation of the detection results is less than 2%, the matrix effect of each type of water sample on the determination results is small, and the present method is suitable for the determination of the content of urea in various types of environmental water samples such as surface water, groundwater, seawater, lake water, river water, and wastewater, showing good universality.

[0060] In summary, the application provides a method for detecting urea content in water sample by high performance liquid chromatography, through improvement of mobile phase and chromatographic column, direct and accurate detection of urea content is realized by using liquid chromatography, technical problems such as complicated operation, high detection lower limit and inaccurate results in prior art are solved, and the method has important application value in the field of urea content detection and analysis.

[0061] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application belong to the scope of the claims of the present application and their equivalents, the present application also intends to include these modifications and variations.

Claims

1. A method for detecting urea content in water samples using high performance liquid chromatography, characterized in that, include: Prepare a standard urea solution; Pre-treatment of the water sample to be tested; The standard solutions and sample solutions were analyzed by high performance liquid chromatography. as well as Urea content was calculated using the standard curve method; The chromatographic conditions were as follows: The chromatographic column used was a C18 reversed-phase column; the mobile phase was a mixed solution of organic and aqueous phases, wherein the organic phase was acetonitrile and the aqueous phase was ultrapure water; the mobile phase was isocratic elution; the detection wavelength was 190-200 nm.

2. The method according to claim 1, characterized in that, The C18 reversed-phase chromatographic column has a length of 200-300 mm, an inner diameter of 4-5 mm, a packing particle size of 3-6 μm, and a column temperature of 40-50℃.

3. The method according to claim 1, characterized in that, The volume ratio of acetonitrile to ultrapure water in the mobile phase is 2:98 to 10:90; preferably, the volume ratio of acetonitrile to ultrapure water in the mobile phase is 5:

95.

4. The method according to claim 1, characterized in that, The flow rate of the mobile phase is 0.8-1.2 mL / min.

5. The method according to claim 1, characterized in that, The pretreatment step includes filtration using a 0.22-0.45 μm filter membrane.

6. The method according to claim 1, characterized in that, The steps for preparing the urea standard solution include: Accurately weigh out the urea standard, add ultrapure water to dilute to volume, and obtain the standard stock solution of urea. Different amounts of standard stock solution were measured and diluted with ultrapure water to obtain urea standard working solutions of different concentrations.

7. The method according to claim 1, characterized in that, The standard curve method uses the peak area of ​​the urea standard solution as the ordinate and the concentration of the urea standard solution as the abscissa to plot the standard curve. Substitute the peak area of ​​the target analyte detected in the sample solution into the standard curve to calculate the urea content in the sample.

8. The method according to any one of claims 1-7, characterized in that, The detection limit of the method is less than 0.6 μg / L, and the quantitation limit is less than 2 μg / L.

9. A method for detecting low concentrations of urea in environmental water samples by high performance liquid chromatography, characterized in that, include: Collect water samples; The method described in any one of claims 1-8 is used to detect the urea content in a water sample.

10. The method according to claim 9, characterized in that, The environmental water samples include surface water, groundwater, seawater, lake water, river water, or wastewater.

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