Fractionation extraction and fluorescent labeling quantitative detection method of effective phosphorus forms in organic fertilizer

Abstract

The present application relates to the field of agricultural chemical analysis, and particularly relates to a method for grading extraction and fluorescent labeling quantitative detection of effective phosphorus forms in organic fertilizer, first, water-soluble phosphorus is extracted by oscillation with deionized water; then, the filter residue is treated with an ammonium citrate-EDTA complexing extraction liquid to obtain citric-soluble phosphorus; finally, the residue is dissolved with a mixed acid extraction liquid to obtain insoluble phosphorus, three kinds of extraction liquids are taken respectively, color developing reagent of ammonium molybdate, ascorbic acid reducing agent are added in turn to carry out color developing reaction, then a fluorescent enhancer is added to amplify the signal, the fluorescence intensity is measured, and the contents of water-soluble phosphorus, citric-soluble phosphorus and insoluble phosphorus are accurately calculated according to a standard curve, a water-ammonium citrate / EDTA-mixed acid three-stage gradient extraction system is adopted to realize efficient separation and extraction of water-soluble phosphorus, citric-soluble phosphorus and insoluble phosphorus in the organic fertilizer, especially, the synergistic complexing system of ammonium citrate and EDTA is introduced in the second-stage extraction, thereby improving the extraction efficiency of citric-soluble phosphorus.

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. At the same time, the existing methods have low grading extraction and separation efficiency for 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 organic fertilizer quality evaluation and scientific fertilization.

[0007] Therefore, it is urgent to develop a method capable of efficiently separating and accurately quantifying different forms of phosphorus in organic fertilizer, so as to meet the needs 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 grading extraction and fluorescent labeling quantitative detection of effective phosphorus forms in organic fertilizer, which realizes efficient separation and accurate quantification of water-soluble phosphorus, citric-soluble phosphorus and insoluble phosphorus in organic fertilizer 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 quality evaluation and scientific fertilization of organic fertilizer.

[0009] The technical scheme of the present application is: a method for grading extraction and fluorescent labeling quantitative detection of effective phosphorus forms in organic fertilizer, comprising the following steps:

[0010] 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;

[0011] Step two, citric-soluble phosphorus extraction: transfer the residue to a container, add ammonium citrate-EDTA complexing extract, oscillate and extract, filter to obtain citric-soluble phosphorus extract and residue;

[0012] Step three, insoluble phosphorus extraction: transfer the residue obtained in step two to a container, add mixed acid extract, oscillate and extract, filter to obtain insoluble phosphorus extract;

[0013] 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;

[0014] Step five, according to the standard curve, calculate the contents of water-soluble phosphorus, citric-soluble phosphorus and insoluble phosphorus.

[0015] Preferably, in step one, 5.0±0.1 g of the organic fertilizer sample is weighed, 100 mL of deionized water is added, and the mixture is shaken for 30 minutes at 25±1°C at a shaking rate of 180-200 rpm. This mild extraction condition is conducive to extracting only water-soluble phosphorus from the sample without dissolving other forms of phosphorus, thereby ensuring the specificity of water-soluble phosphorus extraction.

[0016] 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 shaking for 60 minutes at 30±1°C at a shaking rate of 180-200 rpm. The special feature of this extraction condition is that ammonium citrate can simulate the organic acids secreted by plant roots, and EDTA has strong metal complexing ability, so that the two can work together to effectively extract phosphate combined with metal ions, i.e., the so-called citrate-soluble phosphorus.

[0017] Preferably, in step three, the mixed acid extraction solution is a mixed solution of 0.5 mol / L hydrochloric acid and 0.25 mol / L sulfuric acid, with a volume ratio of 1:1, and an extraction condition of shaking for 120 minutes at 40±1°C at a shaking rate of 180-200 rpm. This relatively strong acidic condition can dissolve more tightly bound insoluble phosphorus combined with minerals, thereby completing the last stage of phosphorus form extraction.

[0018] 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.

[0019] 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.

[0020] 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-enhancing 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.

[0021] Preferably, in step four, the specific steps of color reaction and fluorescence amplification are as follows: 5.0 mL of the extract is taken in a 25 mL colorimetric tube, 2.0 mL of ammonium molybdate color reagent is added, mixed uniformly, and left for 5 minutes; 1.0 mL of ascorbic acid reducing agent is added, mixed uniformly, and left for color development at room temperature 25±2℃ for 20 minutes in the dark; 2.0 mL of fluorescence enhancer is added, mixed uniformly, and left for further reaction in the dark for 10 minutes; the fluorescence intensity is measured using a quartz cuvette at an excitation wavelength of 530 nm and an emission wavelength of 580 nm.

[0022] Preferably, in step five, the preparation method of the standard curve is as follows: 0.4394 g of KH2PO4 dried at 105℃ 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, and 10.0 mL of the standard stock solution are respectively taken in a 100 mL volumetric flask, diluted to the mark with deionized water 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 are treated according to the color reaction and fluorescence amplification steps of step four, the fluorescence intensity is measured, and the standard curve is drawn.

[0023] Preferably, in step five, the calculation method of the contents of water-soluble phosphorus, citrate-soluble phosphorus, and insoluble phosphorus is 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 are the phosphorus concentrations in each extract calculated according to the standard curve, with the unit of mg / L; V1, V2, and V3 are the volumes of each extract, with the unit of L; m is the sample mass, with the unit of kg; f1, f2, and f3 are the correction coefficients of water-soluble phosphorus, citrate-soluble phosphorus, and insoluble phosphorus, respectively, and are taken as 1.05, 1.15, and 1.20, respectively. The correction coefficients are introduced to compensate for the loss and matrix interference in the extraction process and improve the accuracy of detection.

[0024] The beneficial effects of the present application mainly include the following aspects:

[0025] 1. Efficient fractional extraction: The present application adopts a water-ammonium citrate / EDTA-mixed acid three-stage gradient extraction system, realizing efficient separation and extraction of water-soluble phosphorus, citrate-soluble phosphorus, and insoluble phosphorus in organic fertilizers. Especially in the second-stage extraction, the synergistic complex system of ammonium citrate and EDTA is introduced, greatly improving the extraction efficiency of citrate-soluble phosphorus, solving the problems of poor selectivity and low efficiency of traditional extraction methods.

[0026] 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.

[0027] 3. Strong anti-interference ability: The specially designed color development conditions and fluorescence enhancement system effectively reduce the interference of organic matter, heavy metal ions and other factors on the detection, and improve the accuracy and reliability of the detection.

[0028] 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 beneficial to the rapid detection and screening of batch samples.

[0029] 5. Accurate identification of different forms of phosphorus: The method can simultaneously and quantitatively detect the contents of water-soluble phosphorus, citric-soluble phosphorus and insoluble phosphorus, which provides more comprehensive data support for evaluating the quality of organic fertilizers and guiding scientific fertilization.

[0030] Overall, the method of the present application realizes the efficient separation and accurate quantification of different forms of phosphorus in organic fertilizers through innovative hierarchical extraction system and high sensitivity fluorescence detection technology, which provides important technical support for quality evaluation, scientific application and research and development of new fertilizers. DETAILED DESCRIPTION

[0031] 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.

[0032] Example 1: Basic method for hierarchical extraction and fluorescence labeling quantitative detection of different forms of phosphorus in organic fertilizer

[0033] The present embodiment provides a basic method for hierarchical extraction and fluorescence labeling quantitative detection of effective phosphorus forms in organic fertilizer, and the specific steps are as follows:

[0034] First, water-soluble phosphorus is extracted. Accurately weigh 5.0 g of air-dried and sieved organic fertilizer sample, and place it in a 250 mL conical flask. Add 100 mL of deionized water, and oscillate at 180 rpm for 30 minutes at 25°C. After standing for 5 minutes, filter through a medium-speed quantitative filter paper, and collect the filtrate A as the water-soluble phosphorus extract. Retain the residue A for the next extraction.

[0035] Then, the extraction of citrate-soluble phosphorus was performed. The residue A was transferred into 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) was added. The extraction was performed at 30°C with a shaking speed of 180 rpm for 60 minutes. After standing for 5 minutes, the filtrate B was collected by filtering through medium-speed quantitative filter paper, and the residue B was reserved for the next extraction.

[0036] Then, the extraction of insoluble phosphorus was performed. The residue B was transferred into 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) was added. The extraction was performed at 40°C with a shaking speed of 180 rpm for 120 minutes. After standing for 10 minutes, the filtrate C was collected by filtering through medium-speed quantitative filter paper.

[0037] Next, the color reaction and fluorescence amplification were performed. 5.0 mL of filtrates A, B, and C were taken into 25 mL colorimetric tubes, respectively, and 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 the solution was diluted to 500 mL) was added. After mixing well, the solution was allowed to stand for 5 minutes. Then, 1.0 mL of ascorbic acid reducing agent (5.0 g of ascorbic acid was dissolved in 250 mL of deionized water) was added, and the solution was mixed well. The color development was carried out at room temperature (25°C) for 20 minutes in the dark. Then, 2.0 mL of fluorescence enhancer (0.5 g of rhodamine B was dissolved in 50 mL of absolute ethanol, and 50 mL of 1 mol / L citric acid buffer solution, pH 3.0, was added) was added, and the solution was mixed well. The reaction was continued for 10 minutes in the dark. Finally, the fluorescence intensity was measured using a quartz cuvette at an excitation wavelength of 530 nm and an emission wavelength of 580 nm.

[0038] Finally, the contents of different forms of phosphorus were calculated according to the standard curve. The standard curve was prepared as follows: 0.4394 g of KH2PO4 dried at 105°C 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 the 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 reaction and fluorescence amplification steps, the fluorescence intensity was measured, and the standard curve was plotted.

[0039] The calculation method of water-soluble phosphorus, citric-soluble phosphorus and insoluble phosphorus content is as follows: water-soluble phosphorus content (mg / kg) = C1 x V1 ÷ m x f1; citric-soluble phosphorus content (mg / kg) = C2 x V2 ÷ m x f2; insoluble phosphorus content (mg / kg) = C3 x V3 ÷ m x f3; wherein C1, C2, C3 are the phosphorus concentrations in each extract calculated according to the standard curve, with the unit of mg / L; V1, V2, V3 are the volumes of each extract, with the unit of L; m is the sample mass, with the unit of kg; f1, f2, f3 are the correction coefficients of water-soluble phosphorus, citric-soluble phosphorus and insoluble phosphorus, respectively, with the values of 1.05, 1.15 and 1.20.

[0040] The above correction coefficients f1, f2, f3 are determined by 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) to calculate the ratio of the directly measured value to the standard value, and obtain the preliminary correction factor; then, perform 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, comprehensively consider the correction factor of direct measurement and the recovery rate data to 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 in the extraction process, improving the accuracy of the detection results.

[0041] Using the method to detect commercially available organic fertilizer samples, the results are as follows: water-soluble phosphorus content is 1.65 g / kg, citric-soluble phosphorus content is 3.42 g / kg, and insoluble phosphorus content is 5.23 g / kg.

[0042] Example 2: Effect of different extraction temperatures on water-soluble phosphorus extraction efficiency

[0043] This example investigates the effect of extraction temperature on water-soluble phosphorus extraction efficiency, with the specific operation as follows:

[0044] 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.

[0045] The water-soluble phosphorus content of the sample extracted under the optimal conditions of 25℃ was 1.65g / kg, which was basically consistent with the result determined by the national standard method (1.63g / kg), with a relative error of only 1.2%, indicating that the accuracy of the method was good.

[0046] Example 3: Effect of different ratios of ammonium citrate-EDTA on the extraction efficiency of citrate-soluble phosphorus

[0047] This example investigates the effect of the molar ratio of ammonium citrate to EDTA on the extraction efficiency of citrate-soluble phosphorus, with the specific operation as follows:

[0048] Take the same batch of organic fertilizer samples, and extract 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 0.2mol / L unchanged, and keep other conditions unchanged.

[0049] 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 3.42g / 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 EDTA proportion is too high, the strong complexation may interfere with the subsequent color reaction; while when the ammonium citrate proportion is too high, the complexing ability is not sufficient to fully release the metal-bound phosphates.

[0050] Example 4: Effect of different shaking times on the extraction efficiency of insoluble phosphorus

[0051] This example investigates the effect of shaking time on the extraction efficiency of insoluble phosphorus, with the specific operation as follows:

[0052] Take the same batch of organic fertilizer samples, and extract insoluble phosphorus according to the method of Example 1, but set the shaking time to 60 minutes, 90 minutes, 120 minutes, 150 minutes and 180 minutes respectively, and keep other conditions unchanged.

[0053] The experimental results show that as the shaking time increases, the extraction amount of insoluble phosphorus gradually increases, but when the shaking time exceeds 120 minutes, the increase in extraction amount becomes not obvious. Considering efficiency and practicality, 120 minutes is determined as the optimal shaking time for insoluble phosphorus extraction. Under this condition, the extraction amount of insoluble phosphorus is 5.23g / kg, with a recovery rate of 96.8%, indicating good extraction effect.

[0054] Example 5: Effect of the concentration of fluorescent enhancer rhodamine B on the detection sensitivity

[0055] This example investigates the effect of the concentration of fluorescent enhancer rhodamine B on the detection sensitivity, with the following specific operations:

[0056] The fluorescent enhancer was prepared according to the method of Example 1, but the amount of rhodamine B was set at 0.2 g, 0.3 g, 0.4 g, 0.5 g, 0.6 g and 0.7 g, respectively, and other conditions remained unchanged.

[0057] The experimental results show that as the concentration of rhodamine B increases, the fluorescence intensity gradually increases, and the detection sensitivity also increases accordingly. When the amount of rhodamine B is 0.5 g, the fluorescence intensity reaches a maximum value, at which the signal-to-noise ratio of the detection is optimal, and the detection limit can reach 0.05 mg / kg. Further increasing the concentration of rhodamine B does not significantly increase the fluorescence intensity, but may cause fluorescence quenching due to the inner filter effect, reducing the detection sensitivity. Therefore, the optimal amount of rhodamine B is determined to be 0.5 g.

[0058] Example 6: Determination of the detection limit and the quantification limit of the method

[0059] This example determines the detection limit and the quantification limit of the method by performing multiple parallel determinations on blank samples, with the following specific operations:

[0060] Take a blank sample (low-phosphorus matrix verified) that does not contain phosphorus, and extract and determine it according to the method of Example 1. Repeat the operation 10 times, and calculate the standard deviation (SD) of the fluorescence intensity.

[0061] 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. The calculation shows that the detection limit of this method is 0.05 mg / kg, and the quantification limit is 0.15 mg / kg, which is much better than the detection limit of the traditional molybdenum blue colorimetric method (about 0.5 mg / kg).

[0062] The accuracy and reliability of the method were 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) were added to the sample with known concentration, and the recovery rate was between 95.8% and 102.3%, indicating that the method has good accuracy.

[0063] Example 7: Analysis application of different matrix samples

[0064] This example investigates the applicability of the method in different matrix organic fertilizer samples, with the following specific operations:

[0065] Five different sources of organic fertilizer samples were collected, including livestock and poultry manure fermentation fertilizer, crop straw compost, municipal solid waste compost, sludge compost and commercial organic-inorganic compound fertilizer, which were analyzed according to the method of Example 1.

[0066] The experimental results show that the method has good applicability to different matrix organic fertilizer samples. The relative errors of the determination results of different forms of phosphorus in each sample compared with the determination results of the national standard method are all within ± 5%, indicating the accuracy and reliability of the method. Especially for complex matrix municipal solid waste compost and sludge compost, due to its strong anti-interference ability, the method can still obtain accurate determination results, while the traditional method is often significantly interfered in these samples.

[0067] Example 8: Effect of pH value of citric acid buffer on fluorescence intensity

[0068] This example investigates the effect of pH value of citric acid buffer in the fluorescence enhancer on the fluorescence intensity, and the specific operation is as follows:

[0069] The fluorescence enhancer was prepared according to the method of Example 1, but the pH value of the citric acid buffer was adjusted to 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5 respectively, and other conditions remained unchanged.

[0070] 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, the pH value of 3.0 is determined as the best condition. Under this condition, not only the fluorescence intensity is high, but also the stable time of fluorescence signal can reach more than 30 minutes, which is beneficial to the continuous determination of batch samples.

[0071] Example 9: Verification of indoor reproducibility and inter-laboratory reproducibility

[0072] This example verifies the indoor reproducibility and inter-laboratory reproducibility of the method, and the specific operation is as follows:

[0073] 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 RSDs 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.

[0074] 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 showed that the inter-laboratory RSD of water-soluble phosphorus, citrate-soluble phosphorus and insoluble phosphorus contents were 3.8%, 4.2% and 4.7% respectively, all less than 10%, indicating that the inter-laboratory reproducibility of the method was good.

[0075] Comparative Example 1: Comparison of traditional molybdenum blue colorimetry and the method of the present application

[0076] This comparative example compares the differences between the traditional molybdenum blue colorimetry and the method of the present application in terms of detection sensitivity and anti-interference ability, and the specific operation is as follows:

[0077] Take the same batch of organic fertilizer samples, and analyze them by traditional molybdenum blue colorimetry (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.

[0078] 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 substances are added, the determination results of the traditional molybdenum blue colorimetry show obvious deviation, with the highest relative error reaching more than 15%; while the method of the present application is less affected by the interference substances due to the use of fluorescence enhancement technology and optimized color development conditions, and the relative error is controlled within 5%.

[0079] In terms of detection sensitivity, the detection limit of traditional molybdenum blue colorimetry is 0.5 mg / kg, while the detection limit of the method of the present application is as low as 0.05 mg / kg, with a sensitivity improvement of nearly 10 times. This means that the method of the present application can detect lower concentrations of phosphorus, meeting the demand of modern precision agriculture for accurate detection of trace elements.

[0080] Comparative Example 2: Effect of different complexing agents on extraction efficiency of citrate-soluble phosphorus

[0081] This comparative example compares the effect of different complexing agents on the extraction efficiency of citrate-soluble phosphorus, and the specific operation is as follows:

[0082] Take the same batch of organic fertilizer samples, and extract citrate-soluble phosphorus using the following four kinds of extractants respectively: A. 0.2 mol / L ammonium citrate solution; B. 0.2 mol / L EDTA solution; C. 0.2 mol / L ammonium citrate and EDTA mixed solution (molar ratio 3:1); D. 0.2 mol / L ammonium citrate and DTPA mixed solution (molar ratio 3:1). Other conditions remain unchanged.

[0083] The experimental results show that the extraction efficiency of citrate-soluble phosphorus is the highest by using the mixed solution of ammonium citrate and EDTA (C scheme), and the extraction amount is 3.42 g / kg. In comparison, the extraction efficiency of using ammonium citrate (A scheme) or EDTA (B scheme) alone is obviously lower, and the extraction amounts are 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.

[0084] 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 the C scheme, and DTPA is relatively high in price, which is not suitable for routine detection.

[0085] Comparative Example 3: Comparison of different fluorescence enhancement systems

[0086] This comparative example compares the influence of different fluorescence enhancement systems on detection sensitivity, and the specific operation is as follows:

[0087] The following four kinds of fluorescence enhancement systems are used for phosphorus detection respectively: A. Rhodamine B system; B. Fluorescein system; C. Eosin system; D. No fluorescence enhancer (traditional molybdenum blue colorimetry). Other conditions remain unchanged.

[0088] 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, with a detection limit of 0.05 mg / kg, a linear range of 0.2-10.0 mg / L, a stable fluorescence signal for more than 30 minutes, and less influence from interfering substances. In comparison, 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 their stability is also not as good as that of the Rhodamine B system. The traditional molybdenum blue colorimetry without fluorescence enhancer (D scheme) has the highest detection limit of 0.5 mg / kg and the lowest sensitivity.

[0089] This result verifies the superiority of Rhodamine B as a fluorescence enhancer. The ternary fluorescence enhancement complex formed by Rhodamine B and phosphorus molybdenum blue complex not only has high fluorescence intensity, but also has good stability, which is an ideal fluorescence enhancement system.

[0090] Example 10: Field application verification

[0091] In order to verify the application value of the method in actual agricultural production, a field test is carried out, and the specific operation is as follows:

[0092] Three different types of organic fertilizers (fermented manure, straw compost and commercial organic-inorganic compound fertilizer) were selected, and the contents of different forms of phosphorus in them were determined by the method, and a fertilization scheme was designed accordingly. In a rice test field, three fertilization treatments were set: T1 (conventional fertilization, without considering phosphorus forms); T2 (fertilization amount adjusted according to total phosphorus content); and T3 (fertilization scheme optimized according to different forms of phosphorus content).

[0093] The test results show that the rice yield and quality of the T3 treatment are better than those of the T1 and T2 treatments. Compared with the T1 treatment, the rice yield of the 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 can help to optimize the fertilization scheme and improve the fertilizer utilization efficiency and crop yield.

[0094] Through the research of the above examples and comparative examples, the following conclusions can be drawn:

[0095] 1. The method for grading extraction and fluorescent labeling quantitative detection of effective phosphorus forms in organic fertilizer can efficiently separate and accurately quantify water-soluble phosphorus, citrate-soluble phosphorus and insoluble phosphorus in organic fertilizer, and the detection limit is as low as 0.05 mg / kg, which is much better than the traditional molybdenum blue colorimetric method.

[0096] 2. The synergistic complex system of ammonium citrate and EDTA is the key to extracting citrate-soluble phosphorus, and when the molar ratio of the two is 3:1, the extraction efficiency is the highest. This 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.

[0097] 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 phosphorus molybdenum blue compound not only has high fluorescence intensity, but also has good stability, which is suitable for accurate detection of trace phosphorus.

[0098] 4. The method has excellent anti-interference ability, and even in complex matrix containing humic acid, heavy metal ions and other interference substances, accurate determination results can still be obtained, and the relative error is controlled within 5%.

[0099] 5. The 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.

[0100] In summary, the organic fertilizer provided by the present application provides a method for hierarchical extraction and fluorescent labeling quantitative detection of effective phosphorus forms in the organic fertilizer, overcomes the defects of traditional methods such as complicated steps, low sensitivity and poor selectivity, realizes efficient separation and accurate quantification of different forms of phosphorus in the organic fertilizer, and provides important technical support for quality evaluation and scientific fertilization of the organic fertilizer.

[0101] The technical mechanism of the present application mainly embodies the following aspects:

[0102] 1. The mechanism of hierarchical extraction: the present application adopts a water-ammonium citrate / EDTA-mixed acid three-stage gradient extraction system 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 moderately strong combined phosphates, and needs to be extracted by using a suitable complexing agent; and the insoluble phosphorus is combined with minerals more closely and needs to be dissolved under acidic conditions. The hierarchical extraction system is designed based on this theory, and the separation of different forms of phosphorus is realized by gradually improving the dissolution ability of the extraction solution.

[0103] 2. The mechanism of ammonium citrate-EDTA synergistic complexation: there is an obvious synergistic effect between ammonium citrate and EDTA in the extraction of citrate-soluble 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, the citrate radical can form a soluble complex with metal ions, reducing the combination of metal ions with phosphate. EDTA is a strong metal complexing agent containing multiple carboxyl and amino groups, which can form stable metal-EDTA complexes and effectively chelate 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, and the extraction efficiency is the highest.

[0104] 3. The mechanism of phosphomolybdate blue color reaction: phosphate reacts with ammonium molybdate under acidic conditions to generate phosphomolybdate, which is reduced to blue phosphomolybdate blue under the action of a reducing agent (such as ascorbic acid). This reaction is the basis for phosphorus detection, but the traditional colorimetric method has limited sensitivity.

[0105] 4. Mechanism of fluorescence enhancement: The rhodamine B fluorescence enhancement system introduced in this 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 is combined with 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 process 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.

[0106] 5. Stabilizing effect of citric acid buffer system: Fluorescence detection is sensitive to pH, and this 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.

[0107] Through the synergistic effect of the above mechanisms, this 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.

[0108] Results of examples and comparative examples:

[0109] Table 1. Optimization results of different forms of phosphorus extraction conditions

[0110] 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%

[0111] Table 2. Performance comparison of different fluorescence enhancement systems

[0112] 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

[0113] Table 3. Determination results of phosphorus form content in different samples (g / kg)

[0114] 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

[0115] Table 4. Field test results

[0116] 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

[0117] Table 5. Effect of different interfering substances on two detection methods (sample: commercial organic-inorganic compound fertilizer)

[0118]

[0119] Table 6. Effect of different interfering substances on two detection methods (sample: sludge compost)

[0120]

[0121] As can be seen from Table 5 and Table 6, when various interfering substances are added, the determination results of the traditional molybdenum blue colorimetric method show obvious negative deviation, especially in the presence of high concentration of 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 of interfering substances, indicating that the method has excellent anti-interference ability. This is mainly because the method adopts rhodamine B fluorescence enhancement technology and optimized color developing conditions, effectively reducing the influence of interfering substances on detection.

[0122] 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 concentration of phosphorus, meeting the demand of modern precision agriculture for accurate detection of trace elements.

[0123] The application provides a method for grading extraction and fluorescent labeling quantitative detection of effective phosphorus forms in organic fertilizer, realizes efficient separation and accurate quantification of water-soluble phosphorus, citrate-soluble phosphorus and insoluble phosphorus in organic fertilizer by a three-stage gradient extraction system of water-ammonium citrate / EDTA-mixed acid combined with rhodamine B fluorescence enhancement technology.

[0124] The main advantages of the method include: (1) efficient grading extraction, especially the introduction of the 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 fertilizer.

[0125] The method of the application has significant application value in actual agricultural production, by accurately understanding the content of different forms of phosphorus in organic fertilizer, the fertilization scheme can be optimized, the fertilizer utilization efficiency and crop yield can be improved, and important technical support is provided for modern precision agriculture.

Claims

1. A method for graded extraction and quantitative detection of available phosphorus forms in organic fertilizers using fluorescent labeling, characterized in that, Includes the following steps: Step 1, water-soluble phosphorus extraction: Weigh the organic fertilizer sample, add deionized water, shake and extract at the set temperature, and filter to obtain water-soluble phosphorus extract and filter residue; Step 2, extraction of citrate-soluble phosphorus: Transfer the filter residue to a container, add ammonium citrate-EDTA complex extract, shake to extract, and filter to obtain 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 extraction solution, shake to extract, and filter to obtain the insoluble phosphorus extract; Step 4, colorimetric reaction and fluorescence amplification: Take the above three extracts respectively, add ammonium molybdate colorimetric agent and ascorbic acid reducing agent in sequence, add fluorescence enhancer after colorimetric reaction, and measure fluorescence intensity; Step 5: Calculate the content of water-soluble phosphorus, citrate-soluble phosphorus, and sparingly soluble phosphorus based on the standard curve; In step two, the ammonium citrate-EDTA complex extraction solution 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 extraction conditions of shaking at 30±1℃ for 60 minutes at a shaking rate of 180-200 rpm. In step four, the fluorescence enhancer is prepared by dissolving 0.5g of Rhodamine B in 50mL of anhydrous ethanol, and then adding 50mL of 1mol / L citrate buffer, wherein the pH value of the citrate buffer is 3.0±0.

1. In step four, the specific steps for the colorimetric reaction and fluorescence amplification are as follows: Take 5.0 mL of the extract into a 25 mL colorimetric tube, add 2.0 mL of ammonium molybdate colorimetric reagent, mix well, and let stand for 5 minutes; Add 1.0 mL of ascorbic acid reducing agent, mix well, and develop color in the dark at room temperature (25±2℃) for 20 minutes. Add 2.0 mL of fluorescence enhancer, mix well, and continue the reaction in the dark for 10 minutes; Fluorescence intensity was measured using a quartz cuvette at an excitation wavelength of 530 nm and an emission wavelength of 580 nm.

2. The method for graded extraction and quantitative detection of available phosphorus forms in organic fertilizers according to claim 1, characterized in that: In step one, weigh 5.0±0.1g of organic fertilizer sample, add 100mL of deionized water, and extract by shaking at 25±1℃ for 30 minutes at a shaking rate of 180~200rpm.

3. The method for graded extraction and quantitative detection of available phosphorus forms in organic fertilizers according to claim 1, characterized in that: In step three, 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℃ for 120 minutes at a shaking rate of 180-200 rpm.

4. The method for graded extraction and quantitative detection of available phosphorus forms in organic fertilizers according to claim 1, characterized in that: In step four, the preparation method of the ammonium molybdate colorimetric agent is as follows: dissolve 10.0g of ammonium heptamolybdate in 250mL of deionized water, add 140mL of 3.5mol / L sulfuric acid, and dilute to 500mL.

5. The method for graded extraction and quantitative detection of available phosphorus forms in organic fertilizers according to claim 1, characterized in that: In step four, the ascorbic acid reducing agent is prepared by dissolving 5.0 g of ascorbic acid in 250 mL of deionized water.

6. The method for graded extraction and quantitative detection of available phosphorus forms in organic fertilizers according to claim 1, characterized in that: In step five, the method for preparing the standard curve is as follows: Accurately weigh 0.4394 g of KH2PO4 dried at 105℃ for 2 hours, dissolve it in deionized water and dilute it 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 standard stock solution into 100 mL volumetric flasks, 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. The standard solution was processed according to the colorimetric reaction and fluorescence amplification steps in step four, the fluorescence intensity was measured, and a standard curve was plotted.

7. The method for graded extraction and quantitative detection of available phosphorus forms in organic fertilizers according to claim 1, characterized in that: In step five, the calculation methods for the contents of water-soluble phosphorus, citrate-soluble phosphorus, and sparingly soluble phosphorus are 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; Where C1, C2, and C3 are the phosphorus concentrations in each extract calculated from the standard curve, in mg / L; V1, V2, and V3 are the volumes of each extract, in L; m is the sample mass, in kg; and 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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