Quantitative method for biuret in urea-based compound fertilizer and application of quantitative method

Abstract

The invention belongs to the field of analysis and detection, and discloses a method for quantifying biuret in a urea-based compound fertilizer. In order to solve the technical problems of low sensitivity, poor separation effect and low precision in the urea-based compound fertilizer, a formic acid-containing extracting agent is used for extraction, and a cation exchange solid-phase extraction column is used for purification; and then detecting the extracted biuret by using a differential refraction detector by using a formic acid aqueous solution with a concentration of 0.05-0.2% as a mobile phase and an XDB-C18 chromatographic column as a stationary phase. The method realizes effective separation and accurate quantification of the biuret in the urea-based compound fertilizer, has high recovery rate and precision and low detection limit and quantification limit, and can be used for monitoring the content of the biuret in the urea-based compound fertilizer.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and testing, and more particularly to a quantitative method for biuret in a urea-based compound fertilizer and application thereof. Background Art

[0002] Urea-based fertilizers, as an important nitrogen fertilizer, are widely used in agricultural production. However, the biuret content has always been one of the key factors affecting fertilizer quality and crop growth. A certain amount of biuret is inevitably produced during the urea production process. Excessive biuret content can be toxic to crops, inhibiting crop growth and even causing crop death. Therefore, various countries have established strict limit standards for the content of biuret in fertilizers. For example, the national standard "Limit Requirements for Toxic and Hazardous Substances in Fertilizers" (GB38400-2019) stipulates that when the total nitrogen content of a fertilizer is indicated, the biuret content must be tested and determined; the international standard ISO18643 also sets clear requirements for the determination of biuret content in urea-based fertilizers.

[0003] As for the detection method of biuret in different matrices, the main commonly used detection technologies are spectrophotometry, continuous flow analysis, and high-performance liquid chromatography. For example, relevant literature reports on the method of using spectrophotometry to detect biuret in compound fertilizers, water-soluble fertilizers, and compound fertilizers. There are also literature reports on the method of using spectrophotometric detection technology to measure biuret in seawater. Secondly, there are literature reports on the method of using high-performance liquid phase photodiode array detection to determine the content of biuret in humic acid urea, diesel vehicle exhaust, compound urea products, and compound fertilizers. Since urea-based compound fertilizers are often accompanied by a large amount of urea, the target substance biuret has weak ultraviolet absorption, and constant urea will cause significant interference to the detection of trace amounts of biuret.

[0004] Current existing technologies generally use ultraviolet detectors as a means of detecting biuret in fertilizers by high-performance liquid chromatography. However, biuret is a compound with weak ultraviolet absorption, and the detection sensitivity of ultraviolet detectors for this type of compound is relatively low, or derivatization is required.

[0005] Prior Art (Hojjatie&abrams: Journal of AOAC International, Vol. 97, No.3, 2014, Validation for the Determination of Biuret in Water-Soluble, Urea-Based Commercial Inorganic Fertilizer Materials, Urea Solutions, and Sulfur-Coated Urea Products by Reversed-Phase Liquid Chromatography: Single-Laboratory Validation of an Extension of AOAC Official MethodSM (2003.14) discloses a method for quantifying biuret in urea-based compound fertilizers. The method uses an amino column as the stationary phase, an 85% acetonitrile aqueous solution as the mobile phase, and a UV detector to separate and quantify biuret in samples such as liquid urea fertilizer and dry urea products. The method has a high detection limit (0.1 ppm level) and a low precision (0.92-14.34%). This may be due to the weak UV signal of biuret and the low sensitivity of detection using the UV detector, as well as the incomplete separation of urea and biuret. Summary of the Invention

[0006] In order to solve the technical problems of low detection sensitivity, poor separation effect and poor reproducibility in the prior art, the primary purpose of the present invention is to provide a method for quantifying biuret in urea-based compound fertilizer.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions: The present invention provides a quantitative method for protecting biuret in a urea-based compound fertilizer, comprising the following steps: extracting biuret from the urea-based compound fertilizer using a formic acid-containing extractant to obtain an extract; purifying the extract using a cation exchange solid phase extraction column, and then eluting with an ammonia solution to obtain a test solution; The test liquid is subjected to high performance liquid chromatography analysis, wherein the high performance liquid chromatography comprises the following conditions: using 0.05-0.2% formic acid aqueous solution as the mobile phase; using an XDB C18 chromatographic column as the stationary phase; and using a differential refractive index detector for detection.

[0008] The present invention can effectively extract biuret from urea-based compound fertilizer by using a specific mixed solution to extract biuret and using a cation exchange solid phase extraction column for purification, and further adopts a formic acid aqueous solution, octadecylsilane bonded silica gel, and a differential refractive index detector to detect the extracted biuret, thereby achieving effective separation and accurate quantification of biuret in the urea-based compound fertilizer, with good recovery rate and precision as well as low detection limit and quantification limit.

[0009] Given biuret's weak UV absorption and poor UV detector response, the present invention utilizes a differential refractive index detector for detection. This detector, which detects based on refractive index, responds to nearly all compounds and is a versatile detector. However, while it responds to most compounds, it is often susceptible to matrix interference. Furthermore, differential refractive index detectors are more sensitive to experimental fluctuations, prone to baseline fluctuations, and often struggle to achieve high precision.

[0010] The present invention found that, with a differential refractive index detector, simple extraction without purification makes it difficult to obtain a biuret chromatographic peak free of matrix interference, and therefore fails to achieve good accuracy, precision, and sensitivity. Based on this, the present invention further purifies the extract using a cation exchange solid-phase extraction column, achieving good separation results and further improving the accuracy, precision, and sensitivity of the method.

[0011] Furthermore, to address the issue of differential refractive index detectors being susceptible to fluctuations, the present invention specifically optimizes the mobile phase. The refractive index of organic solvents differs significantly from that of water. Even slight changes in the proportion of organic solvents can cause dramatic fluctuations in the differential refractive index detector signal, leading to baseline drift. The present invention utilizes a pure water solution containing a small amount of formic acid as the mobile phase for isocratic elution. This, on the one hand, mitigates the signal effects of the difference between organic solvents and water. The relatively stable refractive index of water and the low, fixed formic acid concentration reduce the mobile phase's own interference with the baseline and improve baseline stability. On the other hand, the acidic mobile phase suppresses urea ionization, improving the signal and peak shape of the biuret chromatographic peak, thereby achieving a low detection limit and high precision. In particular, the present invention achieves excellent precision (RSD ≤ 5.3%) using a differential refractive index detector, which is exceptionally high for a general-purpose detector.

[0012] Preferably, the organic solvent in the formic acid-containing extractant is selected from methanol or acetonitrile, more preferably methanol. Preferably, the volume ratio of the organic solvent to water in the formic acid-containing extractant is 8-10:0-1, more preferably 8-10:1, and even more preferably 9:1. Preferably, the formic acid content of the formic acid-containing extractant is 0.05-2%, more preferably 0.5-2%, and even more preferably 1.0%. More preferably, the formic acid-containing extractant is a 1.0% formic acid aqueous solution in methanol.

[0013] Specifically, the ratio of the amount of the formic acid-containing extractant to the amount of the urea-based compound fertilizer is 10-20 mL: 0.5-2 g, preferably 15 mL: 1 g.

[0014] Specifically, the extraction temperature is 25~60 , further preferably 40 .

[0015] Specifically, the extraction time is 5 to 20 minutes, more preferably 10 minutes.

[0016] Specifically, the extraction means include ultrasound, vibration, or vortex, and ultrasound is more preferred.

[0017] Preferably, the cation exchange solid phase extraction column is selected from a sulfonic acid cation exchange solid phase extraction column, a carboxylic acid cation exchange solid phase extraction column or a phosphate cation exchange solid phase extraction column, and is further preferably a sulfonic acid cation exchange solid phase extraction column.

[0018] Specifically, the purification step includes: passing the extract through a cation exchange solid phase extraction column and discarding the filtrate. Preferably, the volume of the extract added is 2-6 mL, more preferably 4 mL.

[0019] Preferably, the purification step further includes activating the cation exchange solid phase extraction column using a mixed solution of methanol and aqueous formic acid. More preferably, the volume ratio of methanol to aqueous formic acid in the mixed solution is 8-10:1, and more preferably 9:1; the weight percentage of formic acid in the aqueous formic acid solution is 0.5%-2%, and more preferably 1.0%.

[0020] Preferably, the mass concentration of ammonia in the ammonia solution is 0.05-0.2%, more preferably 0.1%.

[0021] Preferably, the added volume of the ammonia solution is 2-6 mL, more preferably 2-3 mL, and further preferably 3 mL.

[0022] Preferably, the mobile phase is 0.1% formic acid aqueous solution.

[0023] Preferably, the inner diameter of the chromatographic column is 2-5 mm, preferably 4.6 mm; the column length of the chromatographic column is 100-300 mm, preferably 250 mm; the filler particle size of the chromatographic column is 3-7 m, preferably 5 m.

[0024] Chromatographic conditions such as ETC temperature, response time, acquisition rate, and column temperature also affect baseline fluctuations in the differential refractive index detector. This invention strictly controls these parameters within specific ranges, further minimizing their impact on the differential refractive index detector and achieving high-precision quantification.

[0025] Specifically, the flow rate of the mobile phase is 0.8-1.0 mL / min, more preferably 1.0 mL / min.

[0026] Specifically, the temperature of the chromatographic column is 35~45 , further preferably 35 .

[0027] Specifically, the ETC temperature of the differential refractive index detector is 30-40 , more preferably 35 ; The response time is 0.1~0.3s, more preferably 0.1s; the acquisition rate is 5~20Hz, more preferably 5Hz.

[0028] The present invention also protects the application of the method in monitoring the biuret content in urea-based compound fertilizer.

[0029] Compared with the prior art, the present invention has the following beneficial effects: The present invention achieves effective separation and accurate quantification of biuret in urea-based compound fertilizer, wherein the separation degree of biuret is 3.5, the spiked recovery is 94.6% to 108.9%, the relative standard deviation is no more than 5.3%, the method detection limit is 0.6 to 0.9 mg / kg (24 to 36 ppb), and the quantification limit is 1.5 to 2.5 mg / kg. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the recovery rate of biuret under different types of solid phase extraction columns and different extraction solvents.

[0031] Figure 2 This is a contour map of the biuret extraction effect under the interaction of four factors.

[0032] Figure 3 Select the volume for the extraction and eluent.

[0033] Figure 4 The chromatograms of samples under different chromatographic columns and mobile phases. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of this application more clearly understood, the present application is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0035] Below in conjunction with specific embodiment, content of the present invention is further described, but embodiment does not limit the present invention in any form.If not otherwise specified, the technical means used in the embodiment are conventional means well known to those skilled in the art.Unless otherwise specified, the reagent, method and equipment used in the present embodiment are conventional reagents, methods and equipment in the art.

[0036] Main instruments and reagents High performance liquid chromatograph: Agilent 1260, equipped with a differential refractive index detector (Agilent, USA); High performance liquid chromatography tandem mass spectrometer: Agilent1290 / 6475 (Agilent, USA); Electronic analytical balance: Model XS205DU, Mettler-Toledo International Trading Shanghai Co., Ltd.

[0037] Experimental water: purified water, Milli-Q type, (Merck, USA).

[0038] Ultrasonic cleaner: Model KQ-250E, Kunshan Ultrasonic Instrument Co., Ltd.

[0039] Standards: biuret and urea were purchased from BePure, with a purity of ≥98%; methanol (HPLC grade) and acetonitrile (HPLC grade) were purchased from TEDIA; potassium dihydrogen phosphate and formic acid (analytical grade, 99%, Xilong Scientific).

[0040] Urea-based compound fertilizer and raw material samples: urea-based compound fertilizer, compound fertilizer, calcium ammonium nitrate, etc., are sourced from enterprise entrustment and customs ports.

[0041] Example 1 A quantitative method for biuret in urea-based compound fertilizer 1. Sample pretreatment 1. Extraction Take 1.0 g (accurate to 0.001 g) of a representative uniform sample in a 40 mL screw-capped glass bottle, add 15 mL of a mixture of methanol / 0.1% formic acid aqueous solution (V / V=9 / 1) to dissolve it, and ultrasonically extract it at 40°C for 10 min. Transfer the extract to a 25 mL volumetric flask, then wash the glass bottle wall several times with the above methanol / formic acid aqueous solution (V / V=9 / 1) mixture, and adjust the volume to the scale for purification and separation.

[0042] 2. Purification Pipette 1 mL of a mixture of methanol and 1.0% formic acid (v / v = 9 / 1) into an activated SCX solid-phase extraction cartridge. Once fully absorbed, add 4 mL of the extract. Once the solution has fully absorbed and passed through the cartridge frit, discard the filtrate and replace the receiving flask. Elute the target compound by adding 3 mL of 0.1% ammonia solution to the cartridge. Collect the eluate, purge with nitrogen to remove ammonia, and dilute to 1 mL with 1.0% formic acid. Filter and prepare for testing.

[0043] 2. High Performance Liquid Chromatography Analysis Chromatographic conditions: XDB-C18 reversed phase liquid chromatography column (4.6 mm × 250 mm, 5 m); the mobile phase was 0.1% formic acid in water; the mobile phase flow rate was 1.0 mL / min; the injection volume was 10 L; column temperature 35 .

[0044] Differential refractive index detection conditions: the flow cell volume is 50 L; ETC temperature is 35 , the response time is 0.1s and the acquisition rate is 5Hz.

[0045] 3. Methodological Validation 1. Evaluation of matrix effects In this study, considering that the complex matrix environment of compound fertilizer samples may interfere with the precision and accuracy of the analytical method, it is necessary to conduct an in-depth evaluation of the matrix effect of the method. To this end, according to the above optimized experimental conditions, the matrix blank samples of solid urea-based compound fertilizer and liquid urea-based compound fertilizer were processed to prepare blank matrix solutions. Subsequently, the blank matrix solutions and distilled water were used to prepare the concentration range of 1.0-50.0 g / mL standard working solutions were tested and analyzed, and matrix-matched standard curves and solvent standard curves were constructed. Matrix effects were quantitatively assessed by comparing and calculating the slope deviation between the matrix-matched standard curve and the solvent standard curve. The evaluation results showed that the matrix effect deviation (ME%) ranged from -10% to 10%, as shown in Table 1. These results indicate that under the analytical conditions of this study, the matrix effect had a minimal impact on the analytical results and was considered insignificant.

[0046] 2. Linear range and lower limit of quantification of the method Weigh an appropriate amount of biuret standard substance into a 10 mL brown volumetric flask filled with methanol, dilute to a concentration of 2000 g / mL single standard stock solution.

[0047] Pipette appropriate amount of the above standard stock solution into 10mL brown volumetric flask, dilute to volume with distilled water, and prepare a biuret series with a mass concentration of 0.5 g / mL, 1.0 g / mL, 2.0 g / mL, 5.0 g / mL, 10.0 g / mL, 20.0 g / mL, 50.0 g / mL standard working solution.

[0048] The standard series of biuret solutions were analyzed and a standard curve was drawn with biuret concentration as X-axis and peak area as Y-axis. The results showed that biuret concentration was 0.5-50.0 The linearity was good within the g / mL concentration range, with correlation coefficients ranging from 0.9973 to 0.9999.

[0049] The limit of detection (LOD) was determined using a signal-to-noise ratio (S / N) of 3, and its reliability was verified using the standard deviation (SMDL) of seven replicates multiplied by the coefficient T (n-1, 1-α = 0.99). The limit of quantification (LOQ) was determined using a S / N ratio of 10. The method's limit of detection for biuret was determined to be between 0.6 and 0.9 mg / kg, and the limit of quantification was between 1.5 and 2.5 mg / kg. The regression equations, correlation coefficients, and LQs for biuret under different matrix conditions are shown in Table 1.

[0050] Table 1 Matrix effect evaluation, regression equation, and detection and quantification limits

[0051] 3. Recovery and precision test of the method Two blank matrix samples and samples prepared with distilled water were selected: ① liquid urea-based compound fertilizer, ② solid urea-based compound fertilizer, and ③ distilled water. According to the sample pretreatment method in step 1 above, 1.0 g of samples ① and ② were weighed and placed in a screw-capped glass bottle. Biuret standard solution was added to the sample. After extraction and purification, the final preparation was biuret spiked concentrations of 2.0, 10.0, and 50.0 The sample spike solutions were prepared at 50, 250, and 1250 mg / mL, corresponding to pre-treatment biuret concentrations of 50, 250, and 1250 mg / kg. Using the optimized method (HPLC analysis in step 2 above), recoveries at three fortification levels were measured for the two blank matrix-spiked samples and samples prepared with distilled water. Six replicates (six spiked extracts were prepared for each sample) were run to determine recoveries and calculate relative standard deviations. The results are shown in Table 2. The spiked recoveries of biuret at three concentrations prepared with distilled water ranged from 91.0% to 106.0%, with relative standard deviations (RSDs) no greater than 4.9%; the spiked recoveries of biuret in liquid urea-based compound fertilizer matrix ranged from 90.5% to 112.6%, with relative standard deviations (RSDs) no greater than 4.4%; and the spiked recoveries of biuret in solid urea-based compound fertilizer matrix ranged from 91.0% to 118.0%, with relative standard deviations (RSDs) no greater than 5.5%. Both the precision and accuracy met the requirements of general quantitative analysis.

[0052] Table 2 Recovery and relative standard deviation of biuret in different matrices

[0053] Comparative Example 1 Selection of extraction solvent Considering that urea-based compound fertilizers contain high concentrations of inorganic ammonium salts or nitrates, and the target compound biuret has good solubility in both organic and aqueous phases, to prevent high concentrations of nitrogen-containing inorganic salts from dissolving into the equipment and contaminating the chromatographic system and entering the flow cell, interfering with the matrix refractive index, extraction tests were performed using primarily organic solvents. Therefore, methanol, acetonitrile, a mixture of methanol / 1.0% formic acid in water (V / V = 9 / 1), an acetonitrile / 1.0% formic acid in water (V / V = 9 / 1), and a 0.1% formic acid in methanol solution were selected for comparative testing. The supernatant was filtered to prepare the sample for testing.

[0054] The biuret sample was spiked at a concentration of 20 The test was conducted on a urea-based compound fertilizer with a concentration of 100 g / mL. Except for the extraction solvent, the other contents of the test method were the same as those in Example 1.

[0055] The results are as follows Figure 1 The results show that under room temperature conditions, the extraction recovery rates of methanol and acetonitrile extraction solutions were both low; 0.1% formic acid in methanol and acetonitrile / 1.0% formic acid water (V / V=9 / 1) mixed solution had comparable extraction effects and better recovery rates; methanol / 1.0% formic acid water (V / V=9 / 1) mixed solution had the best extraction effect and the highest recovery rate. This may be because amino compounds such as biuret are weakly alkaline compounds, and the amino group ( ), which can be protonated to form cations under acidic conditions ( ) and thus its solubility is enhanced. After carrying a positive charge, it interacts with a strong cation exchange column, which is also beneficial for subsequent sample purification. Therefore, a mixture of methanol / 1.0% formic acid water (V / V=9 / 1) was finally selected as the extraction solvent.

[0056] Comparative Example 2 Selection of Solid Phase Extraction Column Considering the properties of biuret, several solid phase separation columns with different filler components were selected to purify the above extraction solutions. ) type stationary phase SCX cationic solid phase extraction cartridge, carboxylic acid (-COOH) type stationary phase CS12 solid phase extraction cartridge, phosphate ( The experiments were carried out on SPE separation cartridges with CS12A column and silica gel stationary phase.

[0057] The test was conducted using a urea-based compound fertilizer spiked with a biuret sample at a concentration of 20 μg / mL. Except for the extraction solvent, the other contents of the test method were the same as those in Example 1.

[0058] The results are as follows Figure 1 As shown in the figure, the silica gel type stationary phase extraction column has the worst separation effect and has no enrichment effect on the target compound; the carboxylic acid group (-COOH) type stationary phase solid phase extraction column and the phosphate group ( ) type stationary phase extraction column separation and enrichment effect is general, a small amount of biuret is eluted during the elution process, resulting in a low recovery rate of biuret in the later eluent, sulfonic acid group ( ) type stationary phase extraction cartridge had the worst effect, with the recovery rate remaining above 90%, which may be due to the difference between the carboxylic acid (-COOH) type stationary phase and the phosphate ( ) type stationary phase is more suitable for the separation and analysis of alkaline earth metals and transition metals, which have a retention and enrichment effect on metal ions, and the separation conditions are good at the weak alkaline range. Therefore, when a mixture of methanol / 1.0% formic acid water (V / V=9 / 1) is used as the extraction solution, the sulfonic acid group ( The SCX cation column with ) type stationary phase was used as the solid phase extraction column.

[0059] Comparative Example 3 Optimization of purification and elution parameters In order to obtain the optimal conditions for the extraction of the target compound biuret in urea-based compound fertilizer by the mixing ratio of the elution mixture and the eluent, an orthogonal test was conducted to select the optimal parameters. A solid urea-based compound fertilizer raw material sample with a determined target compound content was tested under the conditions described in Example 1. The volume concentration of 1.0% formic acid water in the elution mixture (A,%), the mass concentration of ammonia in the eluent (B,%), the extraction temperature (C, ), extraction time (D, min), as variable parameters, each variable was set to 3 levels, and multiple experiments with 4 factors and 3 levels were conducted on biuret according to the principle of central composite experimental design of response surface. The mass volume concentration of 1.0% formic acid water in the elution mixture was selected as 5, 10, and 20% for the experiment, the mass concentration of ammonia in the elution solution was selected as 0.1, 0.5, and 1.0% for the experiment, and the ultrasonic temperature was 25 , 40 , 60 The test was carried out with extraction times of 5, 10 and 20 min for recovery test.

[0060] The variance analysis of the experimental results showed that the biuret test model had a good fit, with an adjusted correlation coefficient of 0.9763, indicating that the predicted values ​​were well correlated with the measured values, and the model could explain 97.63% of the response value changes. The regression equation obtained by fitting was: R=98.74+2.22A-5.13B-0.1848C-0.1075D+1.41AB-0.8317AC+0.3589AD+ 0.42BC-0.5BD-0.1546CD-25.62A2-1.766B2-3.39C2-4.51D2 The experiment showed that the first-order term A of the biuret model had a significant effect, term B had a moderate effect, and term C and D had no significant effect. Therefore, the order of influence of single factors on the extraction of target substances was A>B>C≈D. That is, from the perspective of significant influence, the change in the volume concentration of 1.0% formic acid water in the elution mixture solution>the change in the mass concentration of ammonia in the elution solution>ultrasonic time≈ultrasonic temperature. The isotherm of the biuret with the four-factor interaction is shown in Figure 2 As can be seen from the figure, the volume concentration of 1.0% formic acid water in the elution mixture is between 10% and 14%, and the mass concentration of ammonia in the elution solution is about 0.1%, and the recovery rate can reach a high level. Therefore, the final choice is methanol / 1.0% formic acid water with a volume fraction of V / V = 9 / 1, and a temperature of 40 , and the ultrasonic extraction test was carried out with an extraction time of 10 min.

[0061] Using the above parameters, the volume of sample extract and 0.1% ammonia eluent was selected, and the concentration of biuret was 20 In the optimization test of urea-based compound fertilizer with a 1000-1000 mmol / L slurry, the volume of the sample extract added was 2, 3, 4, 5, and 6 mL, and the volume of the 0.1% ammonia eluent was 2, 3, 4, 5, and 6 mL. Except for the volume of the sample extract or the volume of the ammonia eluent, the rest of the test method was the same as in Example 1.

[0062] The results are as follows Figure 3As shown, the recovery rate of biuret increases rapidly when the sample volume is approximately 4-5 mL. Biuret is detected in more than 4 mL of filtrate. The recovery rate increases with the added volume, likely due to excessive sample volume and the penetration of excess target into the filtrate. When the 0.1% ammonia eluent volume is approximately 2-3 mL, the recovery rate of eluted biuret remains above 90%. Therefore, 4 mL of sample extract was selected as the added volume, and 3 mL of 1% ammonia eluent was selected as the eluent volume.

[0063] Comparative Example 4 Optimization of chromatographic conditions Urea-based compound fertilizers contain a high content of urea, which can interfere with the detection of trace amounts of biuret. Specifically, the chromatographic peaks of the constant substances may mask the chromatographic peaks of other substances with similar retention times, thereby affecting the accuracy of the test results. Since biuret and urea are polar compounds containing amino structures in their molecules, methanol / water, acetonitrile / water, methanol / potassium dihydrogen phosphate aqueous solution, methanol / ammonium acetate aqueous solution, and pure water were selected as mobile phases to compare the XDB-C18 chromatographic columns (2.1mm×100mm, 1.8 m), XDB-C18 chromatographic column (4.6 mm × 250 mm, 5 m), EC-C18 chromatographic column (4.6 mm × 250 mm, 5 m), ZORBAX NH2 chromatographic column (4.6 mm × 250 mm, 5 m), taking the actual concentration of sample biuret as 20 The test was carried out on a sample with a concentration of 100 g / mL. Except for the chromatographic column, the rest of the test method was the same as in Example 1.

[0064] Experiments show that, since the differential refractive index detector can detect almost most substances, there are many interferences in the chromatogram. Except for the XDB-C18 column, the separation effect of the other columns on biuret and interfering substance urea and other unknown impurities in urea-based compound fertilizers under the above mobile phase conditions is not ideal. The ZORBAX NH2 column is particularly poor, with many interferences in the chromatogram. When the mobile phase contains organic solvents, the separation effect of the XDB-C18 column is also not good. Only when the mobile phase is pure water, the separation effect of the XDB-C18 column with a column length of 250mm is relatively satisfactory. Figure 4 This demonstrates the separation performance of the XDB-C18 column. Furthermore, if potassium dihydrogen phosphate or ammonium acetate is added to the pure water mobile phase, the chromatographic peaks will tail, resulting in a wider peak width and affecting the detection sensitivity. However, if the mobile phase is modified with 0.1% formic acid aqueous solution, the chromatographic peak tailing phenomenon will not occur. Finally, the Eclipse XDB-C18 (4.6mm×250mm, 5 m) chromatographic column as analytical column and 0.1% formic acid aqueous solution as mobile phase, e.g. Figure 4 shown.

[0065] The above embodiments are preferred experimental methods of the present invention, but the implementation methods of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A quantitative method for biuret in urea-based compound fertilizer, characterized in that, The steps include: extracting biuret from the urea-based compound fertilizer using a formic acid-containing extractant to obtain an extract; purifying the extract using a cation exchange solid phase extraction column, and then eluting with an ammonia solution to obtain a test solution; The test liquid is subjected to high performance liquid chromatography analysis, wherein the high performance liquid chromatography includes the following conditions: using 0.05-0.2% formic acid aqueous solution as the mobile phase; using an XDB-C18 chromatographic column as the stationary phase; and using a differential refractive index detector for detection.

2. The quantitative method according to claim 1, characterized in that The organic solvent in the formic acid-containing extractant is selected from methanol or acetonitrile; and the volume ratio of the organic solvent to water in the formic acid-containing extractant is 8-10:0-1.

3. The quantitative method according to claim 1 or 2, characterized in that The formic acid content in the formic acid-containing extractant is 0.05-2%.

4. The quantitative method according to claim 1, characterized in that The cation exchange solid phase extraction column is selected from a sulfonic acid cation exchange solid phase extraction column, a carboxylic acid cation exchange solid phase extraction column or a phosphate cation exchange solid phase extraction column.

5. The quantitative method according to claim 1, characterized in that The mass concentration of ammonia in the ammonia solution is 0.05-0.2%.

6. The quantitative method according to claim 1, characterized in that The method also includes a step of activating the cation exchange solid phase extraction column before purification, wherein the activation adopts a mixed solution of methanol and formic acid aqueous solution.

7. The quantitative method according to claim 1, characterized in that The inner diameter of the chromatographic column is 2-5 mm, the column length of the chromatographic column is 100-300 mm, and the filler particle size of the chromatographic column is 3-7 m.

8. The quantitative method according to claim 1, characterized in that The flow rate of the mobile phase is 0.8-1.0 mL / min, and the temperature of the chromatographic column is 35-45 .

9. The quantitative method according to claim 1, characterized in that The ETC temperature of the differential refractive index detector is 30~40 , the response time is 0.1~0.3s, and the acquisition rate is 5~20Hz.

10. Use of the method according to any one of claims 1 to 9 in monitoring the biuret content in urea-based compound fertilizers.

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