Graded extraction and fluorescence labeling quantitative detection method for forms of available phosphorus in organic fertilizer

Abstract

The invention relates to the field of agricultural chemical analysis, in particular to a graded extraction and fluorescence labeling quantitative detection method for forms of available phosphorus in organic fertilizers, which comprises the following steps: firstly, oscillating with deionized water to extract water-soluble phosphorus; then, treating filter residues by using an ammonium citrate-EDTA (Ethylene Diamine Tetraacetic Acid) complexing extracting solution to obtain citrate-soluble phosphorus; and finally, dissolving the residues with a mixed acid extracting solution to obtain insoluble phosphorus, respectively taking the three extracting solutions, sequentially adding an ammonium molybdate color developing agent and an ascorbic acid reducing agent to carry out a color developing reaction, then adding a fluorescence enhancer to amplify a signal, measuring the fluorescence intensity, and accurately calculating the contents of water-soluble phosphorus, citrate-soluble phosphorus and insoluble phosphorus according to a standard curve. A water-ammonium citrate / EDTA-mixed acid three-stage gradient extraction system is adopted, efficient separation and extraction of water-soluble phosphorus, citrate-soluble phosphorus and insoluble phosphorus in the organic fertilizer are achieved, especially a synergistic complexing system of ammonium citrate and EDTA is introduced in second-stage extraction, and the extraction efficiency of citrate-soluble phosphorus is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural chemical analysis, in particular to a method for fractionated extraction and fluorescent labeling quantitative detection of effective phosphorus forms in organic fertilizers, which is particularly suitable for rapid and accurate identification and quantitative detection of different biological availability phosphorus forms such as water-soluble phosphorus, citric acid-soluble phosphorus (commonly known as citric acid-soluble phosphorus) and insoluble phosphorus in complex matrix organic fertilizers. BACKGROUND

[0002] Phosphorus is one of the essential nutrients for plant growth and development, and plays an irreplaceable important role in various physiological and biochemical processes such as photosynthesis, energy transfer, signal transduction and biological membrane construction. The forms of phosphorus in organic fertilizers are diverse, mainly including water-soluble phosphorus, citric acid-soluble phosphorus (commonly known as citric acid-soluble phosphorus) and insoluble phosphorus. The biological availability and release characteristics of these different forms of phosphorus differ significantly, which directly affects their availability to crops. Therefore, accurately distinguishing and quantitatively detecting the content of different forms of phosphorus in organic fertilizers is of great significance for guiding scientific fertilization and evaluating fertilizer quality.

[0003] Currently, the conventional detection method for phosphorus content in organic fertilizers is mainly based on the spectrophotometric method of molybdenum blue colorimetry. Although this method is widely used, it is tedious and has multiple steps, and its sensitivity is limited, making it difficult to accurately distinguish and quantitatively detect different forms of phosphorus in organic fertilizers. For example, the method specified in the national standard GB / T 8573-2017 "Determination of Available Phosphorus in Compound Fertilizers" requires multiple steps such as extraction, filtration and colorimetry, and has high requirements for sample pretreatment, which is not conducive to rapid batch detection. In addition, the detection limit of traditional molybdenum blue colorimetry is relatively high, usually around 0.5 mg / kg, which cannot meet the demand for accurate detection of trace phosphorus in modern precision agriculture.

[0004] In recent years, fluorescence analysis has been widely used in the field of trace element detection due to its high sensitivity, good selectivity and simple operation. Some people have used rhodamine B fluorescence enhancement method to detect phosphate in water samples, showing that fluorescence analysis has a significant advantage in improving the sensitivity of phosphorus detection. However, this method mainly targets the detection of dissolved phosphorus in water samples, and does not consider the fractionated extraction and quantitative detection of different forms of phosphorus in complex matrix organic fertilizers.

[0005] In the patent literature, CN 116217274 A discloses a method for preparing slow-release phosphorus fertilizer from ammonium phosphate by-product ammonia slag. Although this method involves the content regulation of water-soluble phosphorus and citric acid-soluble phosphorus, its focus is on the preparation process of slow-release phosphorus fertilizer, rather than the accurate detection method of different forms of phosphorus. In addition, this patent mainly focuses on water-soluble phosphorus and citric acid-soluble phosphorus, and does not involve the extraction and detection of insoluble phosphorus, nor does it consider the interference of complex organic matrix on phosphorus detection.

[0006] In addition, most of the existing detection methods are difficult to effectively remove the interference of complex organic matrix, and are easily affected by organic matter, heavy metal ions and the like during the detection process, resulting in inaccurate detection results. Meanwhile, the existing methods have low efficiency in the fractional extraction and separation of different forms of phosphorus, and it is difficult to accurately distinguish and quantify water-soluble phosphorus, citric-soluble phosphorus and insoluble phosphorus, thereby restricting the precision of the quality evaluation and scientific application of organic fertilizers.

[0007] Therefore, it is urgent to develop a method capable of efficiently separating and accurately quantifying different forms of phosphorus in organic fertilizers to meet the demand of modern precision agriculture for fertilizer quality evaluation and scientific fertilization guidance. SUMMARY

[0008] In view of the problems in the prior art, the present application provides a method for fractional extraction and fluorescent labeling quantitative detection of effective phosphorus forms in organic fertilizers, which realizes efficient separation and accurate quantification of water-soluble phosphorus, citric-soluble phosphorus and insoluble phosphorus in organic fertilizers by using a gradient extraction system combined with specific fluorescence enhancement technology. The present application overcomes the shortcomings of traditional phosphorus detection methods such as complicated steps, low sensitivity and poor selectivity, greatly improves the sensitivity and accuracy of detection, and provides technical support for the quality evaluation and scientific fertilization of organic fertilizers.

[0009] The technical scheme of the present application is as follows: a method for fractional extraction and fluorescent labeling quantitative detection of effective phosphorus forms in organic fertilizers, comprising the following steps: Step one, water-soluble phosphorus extraction: weigh the organic fertilizer sample, add deionized water, oscillate at a set temperature, filter to obtain water-soluble phosphorus extract and residue; Step two, citric-soluble phosphorus extraction: transfer the residue to a container, add ammonium citrate-EDTA complexing extract, oscillate, filter to obtain citric-soluble phosphorus extract and residue; Step three, insoluble phosphorus extraction: transfer the residue obtained in step two to a container, add mixed acid extract, oscillate, filter to obtain insoluble phosphorus extract; Step four, color reaction and fluorescence amplification: take the above three extracts respectively, add ammonium molybdate color reagent, ascorbic acid reducing agent in turn, add fluorescence enhancer after color reaction, and measure the fluorescence intensity; Step five, calculate the contents of water-soluble phosphorus, citric-soluble phosphorus and insoluble phosphorus according to the standard curve.

[0010] Preferably, in step one, 5.0±0.1g of organic fertilizer sample is weighed, 100mL of deionized water is added, and oscillation extraction is carried out at 25±1℃ for 30 minutes, with an oscillation rate of 180-200rpm. This mild extraction condition is conducive to extracting only the water-soluble phosphorus in the sample without dissolving other forms of phosphorus, thereby ensuring the specificity of water-soluble phosphorus extraction.

[0011] Preferably, in step two, the ammonium citrate-EDTA complexing extraction solution is a mixed solution of 0.2 mol / L ammonium citrate and EDTA, with a pH value of 4.5±0.1, a molar ratio of ammonium citrate to EDTA of 3:1, and an extraction condition of 30±1℃ oscillation for 60 minutes at a rate of 180-200 rpm. The special feature of this extraction condition is that ammonium citrate can simulate the organic acid secreted by plant roots, and EDTA has strong metal complexing ability, and the two work together to effectively extract the phosphate combined with metal ions, i.e., the so-called citric soluble phosphorus.

[0012] Preferably, in step three, the mixed acid extraction solution is a mixture of 0.5 mol / L hydrochloric acid and 0.25 mol / L sulfuric acid with a volume ratio of 1:1, and the extraction condition is 40±1℃ oscillation for 120 minutes at a rate of 180-200 rpm. This relatively strong acidic condition can dissolve the more tightly combined insoluble phosphorus with minerals, completing the last stage of phosphorus form extraction.

[0013] Preferably, in step four, the preparation method of the ammonium molybdate color developing agent is to dissolve 10.0 g of ammonium heptamolybdate in 250 mL of deionized water, add 140 mL of 3.5 mol / L sulfuric acid, and dilute to 500 mL.

[0014] Preferably, in step four, the preparation method of the ascorbic acid reducing agent is to dissolve 5.0 g of ascorbic acid in 250 mL of deionized water.

[0015] Preferably, in step four, the preparation method of the fluorescence enhancer is to dissolve 0.5 g of rhodamine B in 50 mL of anhydrous ethanol, and then add 50 mL of 1 mol / L citric acid buffer with a pH value of 3.0±0.1. Rhodamine B is a high quantum yield fluorescent dye that, when combined with the phosphomolybdate blue complex, forms a ternary fluorescence enhancement complex, significantly improving the detection sensitivity. The citric acid buffer helps to maintain the pH stability of the reaction system and improve the reproducibility of fluorescence detection.

[0016] Preferably, in step four, the specific steps of color development and fluorescence amplification are as follows: take 5.0 mL of the extraction solution in a 25 mL colorimetric tube, add 2.0 mL of the ammonium molybdate color developing agent, mix well, and stand for 5 minutes; add 1.0 mL of the ascorbic acid reducing agent, mix well, and develop color at room temperature 25±2℃ for 20 minutes in the dark; add 2.0 mL of the fluorescence enhancer, mix well, and continue to react in the dark for 10 minutes; use a quartz cuvette to measure the fluorescence intensity at an excitation wavelength of 530 nm and an emission wavelength of 580 nm.

[0017] Preferably, in step five, the preparation method of the standard curve is as follows: 0.4394 g of KH2PO4 dried at 105 DEG C for 2 hours is accurately weighed, dissolved in deionized water and diluted to 1000 mL to prepare a 100 mg / L phosphorus standard stock solution; 0, 0.5, 1.0, 2.0, 5.0, 10.0 mL of the standard stock solution is respectively taken into a 100 mL volumetric flask, diluted to the calibration mark with deionized water to prepare a series of standard solutions with concentrations of 0, 0.5, 1.0, 2.0, 5.0, 10.0 mg / L; the standard solution is treated according to the color reaction and fluorescence amplification steps of step four, the fluorescence intensity is measured, and the standard curve is drawn.

[0018] Preferably, in step five, the calculation method of the contents of water-soluble phosphorus, citrate-soluble phosphorus and insoluble phosphorus is as follows: the content of water-soluble phosphorus (mg / kg) = C1 x V1 ÷ m x f1; the content of citrate-soluble phosphorus (mg / kg) = C2 x V2 ÷ m x f2; the content of insoluble phosphorus (mg / kg) = C3 x V3 ÷ m x f3; wherein C1, C2 and C3 are respectively the phosphorus concentrations in each extract calculated according to the standard curve, with the unit of mg / L; V1, V2 and V3 are respectively the volumes of each extract, with the unit of L; m is the mass of the sample, with the unit of kg; f1, f2 and f3 are respectively the correction coefficients of water-soluble phosphorus, citrate-soluble phosphorus and insoluble phosphorus, and are respectively taken as 1.05, 1.15 and 1.20. The correction coefficients are introduced to compensate for the loss and matrix interference in the extraction process and improve the accuracy of detection.

[0019] The beneficial effects of the present application mainly embody in the following aspects: 1. Efficient fractional extraction: The present application adopts a water-ammonium citrate / EDTA-mixed acid three-stage gradient extraction system to realize efficient separation and extraction of water-soluble phosphorus, citrate-soluble phosphorus and insoluble phosphorus in organic fertilizers. Especially, the introduction of the synergistic complex system of ammonium citrate and EDTA in the second-stage extraction greatly improves the extraction efficiency of citrate-soluble phosphorus, solving the problems of poor selectivity and low efficiency of traditional extraction methods.

[0020] 2. High sensitivity detection: By introducing the rhodamine B fluorescence enhancement system, the detection limit of the method of the present application is as low as 0.05 mg / kg, which is nearly 10 times higher than that of the traditional molybdenum blue colorimetric method, and can meet the demand of trace phosphorus detection.

[0021] 3. Strong anti-interference ability: The specially designed color developing conditions and fluorescence enhancement system effectively reduce the interference of organic matter, heavy metal ions and the like on detection, and improve the accuracy and reliability of detection.

[0022] 4. Simple and fast operation: Compared with the traditional method, the present application simplifies the operation steps, reduces the amount of reagents and shortens the detection time, which is conducive to the rapid detection and screening of batch samples.

[0023] 5. Multi-form phosphate precise identification: can simultaneously quantitatively detect the contents of water-soluble phosphorus, citric-soluble phosphorus and insoluble phosphorus, three different forms of phosphorus, to provide more comprehensive data support for evaluating the quality of organic fertilizer and guiding scientific fertilization.

[0024] Overall, the method of the present application realizes efficient separation and accurate quantification of different forms of phosphorus in organic fertilizer through an innovative hierarchical extraction system and high-sensitivity fluorescence detection technology, and provides important technical support for quality evaluation, scientific application and research and development of new fertilizers. DETAILED DESCRIPTION

[0025] The technical solutions of the present application will be described in detail below in conjunction with specific embodiments, but the protection scope of the present application is not limited thereto. Any non-essential changes and improvements made by those skilled in the art on the basis of the present application shall fall within the protection scope of the present application.

[0026] Example 1: Basic method of hierarchical extraction and fluorescence labeling quantitative detection of different forms of phosphorus in organic fertilizer The present embodiment provides a basic method of hierarchical extraction and fluorescence labeling quantitative detection of effective phosphorus forms in organic fertilizer, and the specific steps are as follows: First, water-soluble phosphorus is extracted. 5.0 g of air-dried and sieved organic fertilizer sample is accurately weighed and placed in a 250 mL conical flask, 100 mL of deionized water is added, and extraction is carried out at 25°C with a shaking rate of 180 rpm for 30 minutes. After standing for 5 minutes, filter through a medium-speed quantitative filter paper, collect the filtrate A as the water-soluble phosphorus extract, and reserve the residue A for the next step of extraction.

[0027] Next, citric-soluble phosphorus is extracted. The residue A is transferred to a new 250 mL conical flask, and 100 mL of pre-prepared ammonium citrate-EDTA complexing extraction solution (0.2 mol / L ammonium citrate and EDTA mixed solution, pH 4.5, molar ratio of ammonium citrate to EDTA 3:1) is added. Extraction is carried out at 30°C with a shaking rate of 180 rpm for 60 minutes. After standing for 5 minutes, filter through a medium-speed quantitative filter paper, collect the filtrate B as the citric-soluble phosphorus extract, and reserve the residue B for the next step of extraction.

[0028] Then, insoluble phosphorus is extracted. The residue B is transferred to a new 250 mL conical flask, and 100 mL of mixed acid extraction solution (0.5 mol / L hydrochloric acid and 0.25 mol / L sulfuric acid mixed solution, volume ratio 1:1) is added. Extraction is carried out at 40°C with a shaking rate of 180 rpm for 120 minutes. After standing for 10 minutes, filter through a medium-speed quantitative filter paper, and collect the filtrate C as the insoluble phosphorus extract.

[0029] Next, color development and fluorescence amplification were carried out. 5.0 mL of the filtrate A, B and C were taken into 25 mL colorimetric tubes respectively, 2.0 mL of ammonium molybdate color reagent (10.0 g of ammonium heptamolybdate was dissolved in 250 mL of deionized water, 140 mL of 3.5 mol / L sulfuric acid was added, and diluted to 500 mL) was added, mixed uniformly, and stood for 5 minutes; 1.0 mL of ascorbic acid reducing agent (5.0 g of ascorbic acid was dissolved in 250 mL of deionized water) was added, mixed uniformly, and color developed at room temperature 25℃ for 20 minutes in the dark; 2.0 mL of fluorescence enhancer (0.5 g of rhodamine B was dissolved in 50 mL of anhydrous ethanol, and then 50 mL of 1 mol / L citric acid buffer solution with pH value of 3.0 was added) was added, mixed uniformly, and reacted for another 10 minutes in the dark; the fluorescence intensity was measured using a quartz cuvette at an excitation wavelength of 530 nm and an emission wavelength of 580 nm.

[0030] Finally, the contents of different forms of phosphorus were calculated according to the standard curve. The preparation method of the standard curve was as follows: 0.4394 g of KH2PO4 dried at 105℃ for 2 hours was accurately weighed, dissolved in deionized water and diluted to 1000 mL to prepare a 100 mg / L phosphorus standard stock solution; 0, 0.5, 1.0, 2.0, 5.0 and 10.0 mL of the standard stock solution were taken into 100 mL volumetric flasks respectively, and deionized water was added to dilute to the calibration mark to prepare a series of standard solutions with concentrations of 0, 0.5, 1.0, 2.0, 5.0 and 10.0 mg / L; the standard solutions were treated according to the above color development and fluorescence amplification steps, the fluorescence intensity was measured, and the standard curve was drawn.

[0031] The calculation method of the contents of water-soluble phosphorus, citrate-soluble phosphorus and insoluble phosphorus was as follows: water-soluble phosphorus content (mg / kg) = C1× V1÷ m × f1; citrate-soluble phosphorus content (mg / kg) = C2× V2÷ m × f2; insoluble phosphorus content (mg / kg) = C3× V3÷ m × f3; wherein C1, C2 and C3 were the phosphorus concentrations in each extract calculated according to the standard curve, with unit of mg / L; V1, V2 and V3 were the volumes of each extract, with unit of L; m was the sample mass, with unit of kg; f1, f2 and f3 were the correction coefficients of water-soluble phosphorus, citrate-soluble phosphorus and insoluble phosphorus, respectively, and were taken as 1.05, 1.15 and 1.20 respectively.

[0032] The correction coefficients f1, f2, f3 are determined by the standard addition recovery experiment. The specific calculation process is as follows: first, analyze the standard sample with known phosphorus content (certified by the National Standard Material Research Center), calculate the ratio of the directly measured value to the standard value, and obtain the preliminary correction factor; then, perform a standard addition recovery experiment with multiple concentration gradients, add a known amount of standard phosphorus solution to different types of organic fertilizer samples, and calculate the recovery rate; finally, consider the correction factor and recovery rate data of the direct measurement, and determine the final correction coefficient. The average recovery rate of water-soluble phosphorus is 95.2%, and the correction coefficient is 1.05 (1 / 0.952); the average recovery rate of citric soluble phosphorus is 87.0%, and the correction coefficient is 1.15 (1 / 0.87); the average recovery rate of insoluble phosphorus is 83.3%, and the correction coefficient is 1.20 (1 / 0.833). The introduction of these correction coefficients effectively compensates for the loss and matrix interference during extraction, improving the accuracy of the test results.

[0033] Using the method, the results of detecting commercially available organic fertilizer samples are as follows: the water-soluble phosphorus content is 1.65 g / kg, the citric soluble phosphorus content is 3.42 g / kg, and the insoluble phosphorus content is 5.23 g / kg.

[0034] Example 2: Effect of different extraction temperatures on water-soluble phosphorus extraction efficiency This example investigates the effect of extraction temperature on water-soluble phosphorus extraction efficiency, with the specific operation as follows: Take the same batch of organic fertilizer samples, and extract water-soluble phosphorus according to the method of Example 1, but set the extraction temperature to 20℃, 25℃, 30℃, 35℃ and 40℃ respectively, and keep other conditions unchanged. Through the experiment, it is found that with the increase of temperature, the extraction amount of water-soluble phosphorus also increases, but when the temperature exceeds 30℃, part of the citric soluble phosphorus also begins to be extracted, affecting the specificity of water-soluble phosphorus extraction. Considering the extraction efficiency and specificity, it is determined that 25℃ is the best temperature for water-soluble phosphorus extraction.

[0035] The water-soluble phosphorus content of the sample extracted at the optimal temperature of 25℃ is 1.65 g / kg, which is basically consistent with the result measured by the national standard method (1.63 g / kg), with a relative error of only 1.2%, indicating that the accuracy of the method is good.

[0036] Example 3: Effect of different molar ratios of ammonium citrate-EDTA on citric soluble phosphorus extraction efficiency This example investigates the effect of the molar ratio of ammonium citrate to EDTA on the extraction efficiency of citric soluble phosphorus, with the specific operation as follows: Take the same batch of organic fertilizer samples, and extract the citrate-soluble phosphorus according to the method of Example 1, but set the molar ratio of ammonium citrate to EDTA to 1:1, 2:1, 3:1, 4:1, and 5:1 respectively, keep the total concentration at 0.2 mol / L unchanged, and keep other conditions unchanged.

[0037] The experimental results show that when the molar ratio of ammonium citrate to EDTA is 3:1, the extraction efficiency of citrate-soluble phosphorus is the highest, with an extraction amount of 3.42 g / kg. This may be because at this ratio, ammonium citrate provides a suitable weak acid environment that is conducive to the dissolution of phosphate, while EDTA can effectively complex metal ions (such as Ca 2+ , Fe 3+ , Al 3+ , etc.) in the sample, reducing their combination with phosphate, thereby improving the extraction efficiency of citrate-soluble phosphorus. When the proportion of EDTA is too high, the complexation may interfere with the subsequent color reaction; while when the proportion of ammonium citrate is too high, the complexing ability is not sufficient to fully release the metal-bound phosphates.

[0038] Example 4: Effect of different oscillation times on the extraction efficiency of insoluble phosphorus This example investigates the effect of oscillation time on the extraction efficiency of insoluble phosphorus, with the following specific operations: Take the same batch of organic fertilizer samples, and extract the insoluble phosphorus according to the method of Example 1, but set the oscillation time to 60 minutes, 90 minutes, 120 minutes, 150 minutes, and 180 minutes respectively, and keep other conditions unchanged.

[0039] The experimental results show that as the oscillation time increases, the extraction amount of insoluble phosphorus gradually increases, but when the oscillation time exceeds 120 minutes, the increase in extraction amount becomes insignificant. Considering efficiency and practicality, 120 minutes is determined as the optimal oscillation time for insoluble phosphorus extraction. Under this condition, the extraction amount of insoluble phosphorus is 5.23 g / kg, with a recovery rate of 96.8%, indicating good extraction effect.

[0040] Example 5: Effect of rhodamine B concentration in fluorescence enhancer on detection sensitivity This example investigates the effect of rhodamine B concentration in the fluorescence enhancer on the detection sensitivity, with the following specific operations: Prepare the fluorescence enhancer according to the method of Example 1, but set the amount of rhodamine B to 0.2 g, 0.3 g, 0.4 g, 0.5 g, 0.6 g, and 0.7 g respectively, and keep other conditions unchanged.

[0041] The experimental results show that the fluorescence intensity gradually increases with the increase of the concentration of Rhodamine B, and the detection sensitivity also increases accordingly. When the amount of Rhodamine B is 0.5 g, the fluorescence intensity reaches the maximum value, at this time the signal-to-noise ratio of the detection is optimal, and the detection limit can reach 0.05 mg / kg. If the concentration of Rhodamine B continues to increase, the fluorescence intensity will not increase significantly, but the fluorescence quenching may be caused by the inner filter effect, which reduces the detection sensitivity. Therefore, the optimal amount of Rhodamine B is determined to be 0.5 g.

[0042] Example 6: Determination of the detection limit and the quantification limit of the method In this example, the detection limit and the quantification limit of the method are determined by performing multiple parallel determinations on blank samples, and the specific operation is as follows: A blank sample (low-phosphorus matrix verified) without phosphorus is taken, and the extraction and determination are performed according to the method of Example 1, and the operation is repeated 10 times to calculate the standard deviation (SD) of the fluorescence intensity.

[0043] According to the recommended method of the International Union of Pure and Applied Chemistry (IUPAC), the detection limit (LOD) = 3SD / S, and the quantification limit (LOQ) = 10SD / S, where S is the slope of the standard curve. Through calculation, the detection limit of the method is 0.05 mg / kg, and the quantification limit is 0.15 mg / kg, which is much better than the detection limit (about 0.5 mg / kg) of the traditional molybdenum blue colorimetric method.

[0044] The accuracy and reliability of the method are further verified by the standard addition recovery experiment. Different concentrations of standard solution (0.5 mg / L, 1.0 mg / L, 2.0 mg / L) are added to the sample with known concentration, and the recovery rate is between 95.8% and 102.3%, indicating that the accuracy of the method is good.

[0045] Example 7: Analysis application of different matrix samples In this example, the applicability of the method in different matrix organic fertilizer samples is investigated, and the specific operation is as follows: Five different sources of organic fertilizer samples are collected, including livestock and poultry manure fermentation fertilizer, crop straw compost, municipal solid waste compost, sludge compost, and commercial organic-inorganic compound fertilizer, and the analysis is performed according to the method of Example 1.

[0046] The experimental results show that the method has good applicability to different matrix organic fertilizer samples, and the determination results of different forms of phosphorus in each sample are compared with the determination results of the national standard method, and the relative error is within ±5%, indicating that the method is accurate and reliable. Especially for complex matrix municipal solid waste compost and sludge compost, due to the strong anti-interference ability of the method, accurate determination results can still be obtained, while the traditional method is often significantly interfered in these samples.

[0047] Example 8: Effect of pH value of citric acid buffer on fluorescence intensity This example investigates the effect of the pH value of the citric acid buffer in the fluorescence enhancer on the fluorescence intensity, with the following specific operations: Prepare the fluorescence enhancer according to the method of Example 1, but adjust the pH value of the citric acid buffer to 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5 respectively, and keep other conditions unchanged.

[0048] The experimental results show that the pH value has a significant effect on the fluorescence characteristics of rhodamine B. When the pH value is in the range of 2.5-3.5, the fluorescence intensity is high and the stability is good; when the pH value is lower than 2.0 or higher than 4.0, the fluorescence intensity is significantly reduced. Considering the fluorescence intensity and stability comprehensively, the pH value of 3.0 is determined as the optimal condition. Under this condition, not only the fluorescence intensity is high, but also the stable time of the fluorescence signal can reach more than 30 minutes, which is conducive to the continuous determination of batch samples.

[0049] Example 9: Verification of indoor reproducibility and inter-laboratory reproducibility This example verifies the indoor reproducibility and inter-laboratory reproducibility of the method, with the following specific operations: Indoor reproducibility: the same batch of samples was determined in parallel for 6 times by the same operator under the same experimental conditions, and the relative standard deviation (RSD) was calculated. The results show that the RSD of water-soluble phosphorus, citrate-soluble phosphorus and insoluble phosphorus contents are 2.3%, 2.7% and 3.1% respectively, all less than 5%, indicating that the indoor reproducibility of the method is good.

[0050] Inter-laboratory reproducibility: the same batch of samples was determined according to the method by 3 different laboratories, and the inter-laboratory relative standard deviation of the determination results was calculated. The results show that the inter-laboratory RSD of water-soluble phosphorus, citrate-soluble phosphorus and insoluble phosphorus contents are 3.8%, 4.2% and 4.7% respectively, all less than 10%, indicating that the inter-laboratory reproducibility of the method is good.

[0051] Comparative Example 1: Comparison of traditional molybdenum blue colorimetric method and the method of the present application This comparative example compares the differences in detection sensitivity and anti-interference ability between the traditional molybdenum blue colorimetric method and the method of the present application, with the following specific operations: Take the same batch of organic fertilizer samples, and analyze them by the traditional molybdenum blue colorimetric method (GB / T 8573-2017) and the method of the present application respectively. In order to investigate the anti-interference ability of the method, different concentrations of interference substances (humic acid, Fe 3 + , Al 3+ , etc.) are added to the samples.

[0052] The experimental results show that under the condition of no interference substance addition, the determination results of the two methods are basically consistent. However, when the interference substance is added, the determination results of the traditional molybdenum blue colorimetric method appear obvious deviation, and the relative error is more than 15%; while the method of the application has less influence of interference substance due to the use of fluorescence enhancement technology and optimized color developing condition, and the relative error is controlled within 5%.

[0053] In terms of detection sensitivity, the detection limit of the traditional molybdenum blue colorimetric method is 0.5 mg / kg, while the detection limit of the method of the application is as low as 0.05 mg / kg, and the sensitivity is increased by nearly 10 times. This means that the method of the application can detect lower concentration of phosphorus, and meets the demand of modern precision agriculture for accurate detection of trace elements.

[0054] Comparative Example 2: Influence of different complexing agents on extraction efficiency of citrate-soluble phosphorus The comparative example compares the influence of different complexing agents on the extraction efficiency of citrate-soluble phosphorus, and the specific operation is as follows: Take the same batch of organic fertilizer samples, and use the following four kinds of extraction solutions to extract citrate-soluble phosphorus: A. 0.2 mol / L ammonium citrate solution; B. 0.2 mol / L EDTA solution; C. mixed solution of 0.2 mol / L ammonium citrate and EDTA (molar ratio 3:1); D. mixed solution of 0.2 mol / L ammonium citrate and DTPA (molar ratio 3:1). Other conditions remain unchanged.

[0055] The experimental results show that the extraction efficiency of citrate-soluble phosphorus is the highest when the mixed solution of ammonium citrate and EDTA (C scheme) is used for extraction, and the extraction amount is 3.42 g / kg. In comparison, the extraction efficiency of ammonium citrate (A scheme) or EDTA (B scheme) alone is obviously lower, and the extraction amount is 2.75 g / kg and 2.63 g / kg, respectively. This fully illustrates that there is a synergistic effect between ammonium citrate and EDTA in the extraction process. Ammonium citrate provides a suitable weak acid environment, and EDTA can effectively complex metal ions. The synergistic effect of the two significantly improves the extraction efficiency of citrate-soluble phosphorus.

[0056] Although the mixed solution of ammonium citrate and DTPA (D scheme) also has a certain extraction efficiency (3.18 g / kg), it is lower than that of C scheme, and DTPA is high in price, which is not suitable for routine detection.

[0057] Comparative Example 3: Comparison of different fluorescence enhancement systems The comparative example compares the influence of different fluorescence enhancement systems on detection sensitivity, and the specific operation is as follows: The following four kinds of fluorescence enhancement systems are used for the detection of phosphorus: A. rhodamine B system; B. fluorescein system; C. eosin system; D. no fluorescence enhancer (traditional molybdenum blue colorimetric method). Other conditions remain unchanged.

[0058] In order to objectively evaluate the performance of different fluorescence enhancement systems, the detection limit, linear range, stability and anti-interference ability of each system are determined. The experimental results show that the rhodamine B system (A scheme) has the best comprehensive performance, the detection limit is 0.05 mg / kg, the linear range is 0.2-10.0 mg / L, the fluorescence signal is stable for more than 30 minutes, and is less affected by interfering substances. In contrast, the detection limits of the fluorescein system (B scheme) and the eosin system (C scheme) are 0.12 mg / kg and 0.18 mg / kg, respectively, and the stability is not as good as that of the rhodamine B system. The detection limit of the traditional molybdenum blue colorimetric method without fluorescence enhancer (D scheme) is the highest, which is 0.5 mg / kg, and the sensitivity is the lowest.

[0059] This result verifies the superiority of rhodamine B as a fluorescence enhancer. The ternary fluorescence enhancement complex formed by rhodamine B and phosphomolybdate blue compound not only has high fluorescence intensity, but also has good stability, and is an ideal fluorescence enhancement system.

[0060] Example 10: Field application verification In order to verify the application value of the method in actual agricultural production, field tests are carried out, and the specific operation is as follows: Three different types of organic fertilizers (livestock and poultry manure fermentation fertilizer, straw compost and commercial organic-inorganic compound fertilizer) are selected, the contents of different forms of phosphorus in them are determined by the method, and the fertilization scheme is designed accordingly. In the rice test field, three fertilization treatments are set: T1 (conventional fertilization, without considering the form of phosphorus); T2 (adjusting the fertilization amount according to the total phosphorus content); T3 (optimizing the fertilization scheme according to the content of different forms of phosphorus).

[0061] The test results show that the yield and quality of rice in T3 treatment are better than those in T1 and T2 treatments. Compared with T1 treatment, the yield of rice in T3 treatment is increased by 8.5%, and the phosphorus fertilizer utilization rate is increased by 12.3%. This fully shows that accurate understanding of the content of different forms of phosphorus in organic fertilizer is helpful to optimize the fertilization scheme and improve the fertilizer utilization efficiency and crop yield.

[0062] Through the research of the above examples and comparative examples, the following conclusions can be drawn: 1. The method for grading extraction and fluorescence labeling quantitative detection of effective phosphorus forms in organic fertilizer provided by the present application can efficiently separate and accurately quantify water-soluble phosphorus, citric-soluble phosphorus and insoluble phosphorus in organic fertilizer, with a detection limit as low as 0.05 mg / kg, which is much better than the traditional molybdenum blue colorimetric method.

[0063] 2. The synergistic complex system of ammonium citrate and EDTA is the key to extract citrate-soluble phosphorus, and the extraction efficiency is the highest when the molar ratio of the two is 3:1. The synergistic effect is mainly due to the weak acid environment provided by ammonium citrate and the metal complexing ability of EDTA, and the two work together to effectively dissolve the phosphate combined with metal ions.

[0064] 3. The rhodamine B fluorescence enhancement system is the core technology to improve the detection sensitivity, and the sensitivity is improved by nearly 10 times compared with the traditional molybdenum blue colorimetric method. The ternary fluorescence enhancement complex formed by rhodamine B and phosphomolybdate blue compound not only has high fluorescence intensity, but also has good stability, which is suitable for accurate detection of trace phosphorus.

[0065] 4. The method has excellent anti-interference ability, and accurate determination results can still be obtained even in complex matrix containing humic acid, heavy metal ions and other interfering substances, and the relative error is controlled within 5%.

[0066] 5. Field test verifies the application value of the method in actual agricultural production, and the fertilization scheme optimized according to the content of different forms of phosphorus can significantly improve the fertilizer utilization efficiency and crop yield.

[0067] In summary, the method for grading extraction and fluorescence labeling quantitative detection of effective phosphorus forms in organic fertilizer provided by the application overcomes the shortcomings of traditional methods such as complicated steps, low sensitivity and poor selectivity, realizes efficient separation and accurate quantification of different forms of phosphorus in organic fertilizer, and provides important technical support for quality evaluation and scientific fertilization of organic fertilizer.

[0068] The technical mechanism of the application mainly embodies in the following aspects: 1. The mechanism of grading extraction: the application adopts a three-stage gradient extraction system of water-ammonium citrate / EDTA-mixed acid to realize selective extraction of different forms of phosphorus. Water-soluble phosphorus mainly exists in the form of free phosphate and is easily soluble in water; citrate-soluble phosphorus is mainly combined with Ca 2+ , Fe 3+ , Al 3+ and other metal ions to form medium-strength combined phosphate, which needs appropriate complexing agent for extraction; and the insoluble phosphorus is combined more closely with minerals and needs acid conditions for dissolution. The grading extraction system is designed based on this theory, and the different forms of phosphorus are separated by gradually improving the dissolution ability of the extraction liquid.

[0069] 2. Mechanism of synergistic complexation of ammonium citrate-EDTA: There is a significant synergistic effect between ammonium citrate and EDTA in the extraction of citosoluble phosphorus. The ammonium citrate molecule contains carboxyl and hydroxyl groups, which can provide a suitable weak acid environment conducive to the dissolution of phosphate; at the same time, citrate can form soluble complexes with metal ions, reducing the combination of metal ions with phosphate. EDTA is a powerful metal complexing agent containing multiple carboxyl and amino groups, which can form stable metal-EDTA complexes, effectively chelating Ca 2+ , Fe 3+ , Al 3+ and other metal ions, further promoting the release of phosphates combined with these metal ions. When the molar ratio of ammonium citrate to EDTA is 3:1, the synergistic effect of the two is most significant, with the highest extraction efficiency.

[0070] 3. Mechanism of phosphomolybdate blue color reaction: Phosphate reacts with ammonium molybdate under acidic conditions to form phosphomolybdate, which is reduced to blue phosphomolybdate blue by a reducing agent such as ascorbic acid. This reaction is the basis for phosphorus detection, but the traditional colorimetric method has limited sensitivity.

[0071] 4. Mechanism of fluorescence enhancement: The rhodamine B fluorescence enhancement system introduced in the invention is the key to improving detection sensitivity. Rhodamine B is a high quantum yield fluorescent dye, and its fluorescence characteristics are greatly affected by the environment. When rhodamine B combines with the phosphomolybdate blue complex, a ternary fluorescence enhancement complex is formed, resulting in a significant increase in the fluorescence intensity of rhodamine B. This enhancement effect may be related to the change in the electron density distribution of rhodamine B molecules caused by the phosphomolybdate blue complex, making the excitation and emission processes of rhodamine B more efficient. Through this fluorescence enhancement effect, the detection sensitivity is improved by nearly 10 times, reaching a detection limit of 0.05 mg / kg.

[0072] 5. Stabilizing effect of citric acid buffer system: Fluorescence detection is sensitive to pH, and the invention uses citric acid buffer to maintain the stability of the reaction system pH, which is crucial for improving the stability and reproducibility of fluorescence detection. Studies have shown that at a pH of 3.0, the fluorescence intensity of rhodamine B is the highest and the stable time is long, which is conducive to the continuous determination of batch samples.

[0073] Through the synergistic effect of the above mechanisms, the invention realizes the efficient separation and accurate quantification of different forms of phosphorus in organic fertilizers, providing important technical support for the quality evaluation and scientific fertilization of organic fertilizers.

[0074] Results of examples and comparative examples: Table 1. Optimization results of different forms of phosphorus extraction conditions Extraction conditions Water-soluble phosphorus Citric-soluble phosphorus Insoluble phosphorus Optimum temperature 25℃ 30℃ 40℃ Optimum time 30 min 60 min 120 min Optimum shaking rate 180 rpm 180 rpm 180 rpm Extraction efficiency 97.50% 98.50% 96.80% Table 2. Performance comparison of different fluorescence enhancement systems Fluorescence enhancement system Detection limit (mg / kg) Linear range (mg / L) Signal stability time (min) Anti-interference ability Rhodamine B system 0.05 0.2-10.0 >30 Good Fluorescein system 0.12 0.5-8.0 15 Good Eosin system 0.18 0.5-5.0 10 Medium No fluorescence enhancer 0.5 1.0-20.0 >60 Medium Table 3. Determination results of phosphorus form contents in different samples (g / kg) Sample type Water-soluble phosphorus Citric-soluble phosphorus Insoluble phosphorus Total available phosphorus Livestock manure fermentation fertilizer 2.18 4.35 3.76 10.29 Crop straw compost 1.25 2.87 5.42 9.54 Municipal solid waste compost 0.83 3.56 7.28 11.67 Sludge compost 1.47 5.82 8.65 15.94 Commercial organic-inorganic compound fertilizer 3.65 5.28 4.12 13.05 Table 4. Field test results Treatment Yield (kg / hm2) Relative yield increase rate (%) Phosphorus fertilizer utilization rate (%) Relative improvement rate (%) ​ 7856 - 18.5 - ​ 8125 3.4 20.2 9.2 ​ 8523 8.5 20.8 12.3 Table 5. Effects of different interference substances on two detection methods (sample: commercial organic-inorganic compound fertilizer) Table 6. Effects of different interference substances on two detection methods (sample: sludge compost) As can be seen from Tables 5 and 6, when various interference substances are added, the determination results of the traditional molybdenum blue colorimetric method show obvious negative deviation, especially in the presence of high-concentration humic acid (100 mg / L), the relative error is as high as -19.7%; while the relative error of the method of the application is still controlled within -5% even in the presence of high-concentration interference substances, indicating that the method has excellent anti-interference ability. This is mainly because the method uses rhodamine B fluorescence enhancement technology and optimized color development conditions, effectively reducing the influence of interference substances on detection.

[0075] In terms of detection sensitivity, the detection limit of the traditional molybdenum blue colorimetric method is 0.5 mg / kg, while the detection limit of the method of the application is as low as 0.05 mg / kg, the sensitivity is increased by nearly 10 times. This means that the method of the application can detect lower concentrations of phosphorus, meeting the demand of modern precision agriculture for accurate detection of trace elements.

[0076] The application provides a method for grading extraction and fluorescence labeling quantitative detection of effective phosphorus forms in organic fertilizers, which realizes efficient separation and accurate quantification of water-soluble phosphorus, citrate-soluble phosphorus and insoluble phosphorus in organic fertilizers by using a three-stage gradient extraction system of water-ammonium citrate / EDTA-mixed acid combined with rhodamine B fluorescence enhancement technology.

[0077] The main advantages of the method include: (1) efficient grading extraction, especially the introduction of a synergistic complex system of ammonium citrate and EDTA, which greatly improves the extraction efficiency of citrate-soluble phosphorus; (2) high sensitivity detection, through the rhodamine B fluorescence enhancement system, the detection limit is as low as 0.05 mg / kg, which is nearly 10 times higher than that of the traditional method; (3) strong anti-interference ability, effectively reducing the interference of organic matter, heavy metal ions and the like on detection; (4) simple and fast operation, suitable for rapid detection and screening of batch samples; (5) accurate identification of multiple forms of phosphorus, providing comprehensive data support for quality evaluation and scientific fertilization of organic fertilizers.

[0078] The method has remarkable application value in actual agricultural production, through accurate understanding of the content of different forms of phosphorus in organic fertilizer, a fertilization scheme can be optimized, fertilizer utilization efficiency and crop yield are improved, and important technical support is provided for modern precision agriculture.

Claims

1. A method for fractional extraction and fluorescent labeling quantitative detection of available phosphorus forms in organic fertilizers, characterized in that: The following steps are involved: Step 1, water-soluble phosphorus extraction: weigh the organic fertilizer sample, add deionized water, shake and extract at a set temperature, and filter to obtain a water-soluble phosphorus extract and filter residue; Step 2, extracting citrate-soluble phosphorus: transferring the filter residue to a container, adding ammonium citrate-EDTA complex extract, shaking extraction, and filtering to obtain a citrate-soluble phosphorus extract and filter residue; Step 3, extraction of insoluble phosphorus: transfer the filter residue obtained in step 2 to a container, add the mixed acid extract, shake and extract, and filter to obtain an insoluble phosphorus extract; Step 4, color development and fluorescence amplification: Take the three extracts mentioned above, add ammonium molybdate color developer and ascorbic acid reducing agent in sequence, add fluorescence enhancer after color development, and measure the fluorescence intensity; Step 5: Calculate the contents of water-soluble phosphorus, citrate-soluble phosphorus and sparingly soluble phosphorus according to the standard curve.

2. The method for fractional extraction and fluorescent labeling quantitative detection of available phosphorus forms in organic fertilizer according to claim 1, wherein: In step 1, 5.0 ± 0.1 g of organic fertilizer sample was weighed, 100 mL of deionized water was added, and the mixture was extracted by shaking at 25 ± 1 °C for 30 min at a shaking rate of 180-200 rpm.

3. The method for fractional extraction and fluorescent labeling quantitative detection of available phosphorus forms in organic fertilizer according to claim 1, wherein: In step 2, the ammonium citrate-EDTA complex extract is a mixed solution of 0.2 mol / L ammonium citrate and EDTA, with a pH of 4.5±0.1, a molar ratio of ammonium citrate to EDTA of 3:1, and the extraction conditions are oscillation at 30±1°C for 60 minutes and an oscillation rate of 180~200 rpm.

4. The method for fractional extraction and fluorescent labeling quantitative detection of available phosphorus forms in organic fertilizer according to claim 1, wherein: In step 3, the mixed acid extract is a mixture of 0.5 mol / L hydrochloric acid and 0.25 mol / L sulfuric acid in a volume ratio of 1:

1. The extraction conditions are shaking at 40±1°C for 120 minutes at an shaking rate of 180-200 rpm.

5. The method for fractional extraction and fluorescent labeling quantitative detection of available phosphorus forms in organic fertilizer according to claim 1, wherein: In step 4, the preparation method of the ammonium molybdate developer is as follows: dissolve 10.0 g of ammonium heptamolybdate in 250 mL of deionized water, add 140 mL of 3.5 mol / L sulfuric acid, and dilute to 500 mL.

6. The method for fractional extraction and fluorescent labeling quantitative detection of available phosphorus forms in organic fertilizer according to claim 1, wherein: In step 4, the ascorbic acid reducing agent is prepared by dissolving 5.0 g of ascorbic acid in 250 mL of deionized water.

7. The method for fractional extraction and fluorescent labeling quantitative detection of available phosphorus forms in organic fertilizer according to claim 1, characterized in that: In step 4, the fluorescence enhancer is prepared by dissolving 0.5 g of rhodamine B in 50 mL of anhydrous ethanol, and then adding 50 mL of 1 mol / L citric acid buffer, wherein the pH value of the citric acid buffer is 3.0±0.

1.

8. The method for fractional extraction and fluorescent labeling quantitative detection of available phosphorus forms in organic fertilizer according to claim 1, characterized in that: In step 4, the specific steps of color development reaction and fluorescence amplification are: Take 5.0 mL of the extract into a 25 mL colorimetric tube, add 2.0 mL of ammonium molybdate color developer, mix well, and let it stand for 5 minutes; Add 1.0 mL of ascorbic acid reducing agent, mix well, and develop color at room temperature (25 ± 2°C) in the dark for 20 minutes; Add 2.0 mL of fluorescence enhancer, mix well, and continue the reaction in the dark for 10 minutes; The fluorescence intensity was measured using a quartz cuvette at an excitation wavelength of 530 nm and an emission wavelength of 580 nm.

9. The method for fractional extraction and fluorescent labeling quantitative detection of available phosphorus forms in organic fertilizer according to claim 1, characterized in that: In step 5, the standard curve is prepared as follows: Accurately weigh 0.4394 g of KH2PO4 dried at 105°C for 2 hours, dissolve it in deionized water and dilute to 1000 mL to prepare a 100 mg / L phosphorus standard stock solution; Pipette 0, 0.5, 1.0, 2.0, 5.0, and 10.0 mL of the standard stock solution into a 100 mL volumetric flask, dilute to the mark with deionized water, and prepare a series of standard solutions with concentrations of 0, 0.5, 1.0, 2.0, 5.0, and 10.0 mg / L; Treat the standard solution according to the color development reaction and fluorescence amplification steps in step 4, measure the fluorescence intensity, and draw a standard curve.

10. The method for fractionated extraction and fluorescent labeling quantitative detection of available phosphorus forms in organic fertilizer according to claim 1, characterized in that: In step 5, the calculation method for the content of water-soluble phosphorus, citrate-soluble phosphorus and sparingly soluble phosphorus is: Water-soluble phosphorus content (mg / kg) = C1 × V1 ÷ m × f1; Citrate-soluble phosphorus content (mg / kg) = C2 × V2 ÷ m × f2; Insoluble phosphorus content (mg / kg) = C3 × V3 ÷ m × f3; Where C1, C2, and C3 are the phosphorus concentrations in each extract calculated according to the standard curve, respectively, in mg / L; V1, V2, and V3 are the volumes of each extract, respectively, in L; m is the sample mass, in kg; f1, f2, and f3 are the correction coefficients for water-soluble phosphorus, citrate-soluble phosphorus, and sparingly soluble phosphorus, respectively, with values ​​of 1.05, 1.15, and 1.20.

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