CN110749614B - Method for rapidly and quantitatively detecting organic phosphorus in soil - Google Patents

Abstract

The invention relates to a method for rapidly and quantitatively detecting organic phosphorus in soil, which is characterized in that a soil sample is added into 0.25M NaOH and 0.05M Na ₂ Extracting with EDTA under shaking in dark, centrifuging, collecting supernatant, filtering, collecting supernatant, and coolingFreezing to obtain freeze-dried powder; dissolving 50mg of freeze-dried powder in 2mL of paramagnetic ion solution, swirling, and standing to obtain liquid ³¹ P NMR samples, detected using a Bruker 600MHz liquid NMR spectrometer, were passed through the spectrometer ³¹ The chemical shift value of the P signal can determine the organic phosphorus form corresponding to the signal, and the relative abundance of the corresponding organic phosphorus or the organic phosphorus can be determined according to the relative proportion of the signal peak intensity, so that the content of the organic phosphorus can be calculated. With the usual liquids ³¹ Compared with the P NMR method, the invention shortens the detection time of the NMR experiment and improves the efficiency of the quantitative analysis of the organic phosphorus in the soil.

Description

A Rapid Quantitative Method for Detecting Organic Phosphorus in Soil

technical field

The invention relates to a method for rapidly and quantitatively detecting organic phosphorus in soil, belonging to the technical field of chemical detection.

Background technique

Soil contains inorganic phosphorus and organic phosphorus, of which organic phosphorus accounts for 35-65% of the total soil phosphorus. Understanding the dynamics of soil organic phosphorus is essential for in-depth understanding of the biogeochemical cycle of soil phosphorus and the establishment of efficient sustainable agricultural management measures is of great significance.

The inorganic phosphorus in the soil is mainly orthophosphate, and its content can be determined by spectrophotometry and ICP-OES. The content of organic phosphorus in the soil can be determined by acid extraction of inorganic phosphorus and total phosphorus extracted after digestion. At the same time, specific soil organic phosphorus forms can also be extracted and analyzed by chromatography, such as using chloroform, methanol and water or dichloromethane and methanol to extract phospholipids, followed by chromatographic separation and content determination, but this This method cannot determine the form of organic phosphorus, but can only determine the content of a specific form of organic phosphorus. There are various molecular forms of organic phosphorus in soil, and it is necessary to establish advanced analytical methods to analyze the form and composition of organic phosphorus in soil at the molecular level.

Liquid ³¹ P nuclear magnetic resonance (NMR) spectroscopy is the preferred method for the quantitative detection of soil organic phosphorus species.

Since the isotropic NMR signal of ³¹ P in the liquid NMR spectrum has good resolution and can reflect the structural information of specific organophosphorus compounds, organic phosphorus in soil is usually extracted by solution and then analyzed by one-dimensional liquid ³¹ P NMR. Analyze, identify and compare quantitatively the species of organophosphorus. Despite the great success of ³¹ P NMR in soil science, certain factors have hindered its wider application in this field. Due to the relatively low phosphorus content in the soil, ³¹ PNMR tests usually require a long scan time (about 16-20 h for a sample), and liquid ³¹ PNMR quantitative analysis of soil organic phosphorus extracts usually requires thousands of pulse scans (that is, repeated ³¹ P excitation-relaxation cycle) to obtain enough signal to produce high-quality spectra, which makes the ³¹ P NMR laboratory equipment for the determination of soil organic phosphorus strained and expensive.

Contents of the invention

The object of the present invention is to solve the deficiencies of the above prior art, and provide a method for rapid quantitative detection of organic phosphorus in soil, which can effectively shorten the detection time and improve the efficiency of quantitative analysis of organic phosphorus in soil.

Technical solutions

Liquid ³¹ P NMR quantitative analysis of soil organic phosphorus extracts usually requires thousands of pulse scans to obtain enough signal to generate high-quality spectra, and the interval between pulse cycles needs to be set long enough to allow all ³¹ P The nuclei are fully restored from the excited state to the equilibrium state, otherwise the NMR signal intensity will not be able to quantitatively reflect the information of all organophosphorus species. Therefore, the pulse interval also determines the experimental duration of the liquid ³¹ P NMR measurement to a large extent. The recovery process of excited ³¹ P nuclei in solution is mainly controlled by their spin-lattice relaxation (T ₁ ), and there are significant differences in the T ₁ time of different phosphorus compounds. Therefore, the inventor shortens the detection time by shortening the pulse interval, thereby improving the efficiency of quantitative analysis of soil organic phosphorus. The specific plan is as follows:

A method for rapid quantitative detection of organic phosphorus in soil, comprising the steps of:

(1) Weigh 4g (dry soil weight) soil sample, add 60mL NaOH and Na ₂ EDTA mixed solution, in the mixed solution, the concentration of NaOH is 0.25M, and the concentration of Na ₂ EDTA is 0.05M; Centrifuge, collect the supernatant and filter it through a 0.45 μm filter membrane, collect the filtered supernatant, and freeze to obtain a lyophilized powder;

(2) Take 50 mg of freeze-dried powder, dissolve in 2 mL of paramagnetic ion solution, vortex for 30 s and let stand for 2 min to obtain a liquid ³¹ P NMR sample;

The paramagnetic ion solution formula: 20-100mg/L Fe ³⁺ , 0.25M NaOH, 0.05M Na ₂ EDTA, 10% (v/v) D ₂ O;

(3) Add the liquid ³¹ P NMR sample into a 5mm NMR sample tube, use a Bruker 600MHz liquid nuclear magnetic resonance spectrometer to detect, and obtain a spectrum. The chemical shift value of the ³¹ P signal in the spectrum can determine the form of organophosphorus corresponding to the signal , according to the relative proportion of the signal peak intensity (area), the relative abundance corresponding to a certain or a certain type of organic phosphorus can be determined, thereby calculating its content.

Further, in step (1), the centrifugation speed is 10000rpm, and the time is 10min.

Further, in step (2), the formula of the paramagnetic ion solution: 50mg/L Fe ³⁺ , 0.25M NaOH, 0.05M Na ₂ EDTA, 10% (v/v) D ₂ O.

Further, in step (3), the setting parameters of the Bruker 600MHz liquid nuclear magnetic resonance spectrometer are: the detection frequency of ³¹ P is 242.98MHz, the detection temperature is 25°C, the pulse program is 90° pulse, the signal acquisition time is 0.845s, and the pulse The interval time is 0.1s.

Beneficial effect of the present invention: the present invention provides a kind of method for rapid quantitative detection of organic phosphorus in soil, this method shortens pulse interval by adding paramagnetic ion in ³¹ P NMR sample on the basis of liquid ³¹ P NMR quantitative analysis, This shortens the detection time of NMR experiments. Compared with the commonly used liquid ³¹ P NMR method, the efficiency of quantitative analysis of soil organic phosphorus can be greatly improved.

Description of drawings

Fig. 1 is the ³¹ P NMR spectrum of the soil sample that adopts embodiment 1 method to measure;

FIG. 2 is the ³¹ P NMR spectrum of the soil sample measured by the method of Comparative Example 1.

detailed description

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

Example 1

Three paramagnetic ion solutions were prepared with different Fe ³ concentrations: (20, 50, 100) mg/L Fe ³⁺ , 0.25M NaOH, 0.05M Na ₂ EDTA, 10% (v/v) D ₂ O. Then sodium phosphate, sodium pyrophosphate, phytic acid (25mM P) and DNA (1mg/mL) were dissolved in the paramagnetic ion solution, respectively, and added to a 5mm NMR sample tube using a Bruker 600MHz liquid NMR (242.98MHz, 25°C) to invert The recovery method (pulse program t1irpg) measures the T1 time _of phosphorus compounds (set 10 pulse intervals, each interval pulse cycle is 32, the signal acquisition time is 0.845s, and the relaxation interval is 50s), the test results See Table 1:

Table 1 Spin-lattice relaxation (T ₁ ) times of phosphorus compounds at different concentrations of paramagnetic ions

It can be seen from Table 1 that when the concentration of paramagnetic ions Fe ³ is 50 mg/L, the spin-lattice relaxation (T ₁ ) time of phosphorus compounds is shorter. Therefore, a paramagnetic ion Fe3 concentration ^of 50mg/L was selected for the experiment.

The soil sample for testing is a farmland black clay in Shuangyashan City, Heilongjiang Province (N46°48′20.3″, E134°01′13.9″), and the crop is soybean. Basic physical and chemical properties: pH 6.90, C 56g kg ^–1 , N 13g kg ^–1 , P 1.8g kg ^–1 .

A method for rapid quantitative detection of organic phosphorus in soil, comprising the steps of:

(1) Weigh 4g (dry soil weight) soil sample, add 60mL NaOH and Na ₂ EDTA mixed solution, the concentration of NaOH is 0.25M, the concentration of Na ₂ EDTA is 0.05M; after shaking and extracting in the dark for 16h, centrifuge (speed is 10000rpm, and the time is 10min), the collected supernatant is filtered through a 0.45 μm filter membrane, the filtered supernatant is collected, and freeze-dried powder is obtained after freezing;

(2) Take 50 mg of lyophilized powder, dissolve in 2 mL of paramagnetic ion solution, vortex for 30 s and let stand for 2 min to obtain a liquid ³¹ P NMR sample;

The paramagnetic ion solution formula: 50mg/L Fe ³⁺ , 0.25M NaOH, 0.05M Na ₂ EDTA, 10% (v/v) D ₂ O;

(3) Add the liquid ³¹ P NMR sample into a 5mm NMR sample tube, use a Bruker 600MHz liquid nuclear magnetic resonance spectrometer (the detection frequency of ³¹ P is 242.98 MHz, the detection temperature is 25 °C, the pulse program is 90° pulse, the signal acquisition time 0.845s, the number of pulse cycles is 128, and the pulse interval time is 50-0.1s) to detect, obtain the spectrum (Fig. 1), and identify its form by the chemical shift of the ³¹ P signal, and the 5.2ppm signal corresponds to orthophosphate ( orthophosphate), -4.1ppm signal corresponds to pyrophosphate (pyrophosphate), and the signal between 5-4ppm comes from phytic acid (phytic acid). The signal area was integrated by BrukerTopsin software, and the relative abundance of the three forms was calculated. The results showed that the relative abundance of the three forms of phosphorus was analyzed after shortening the pulse interval time to 0.1s by adding 50 mg/L Fe ³⁺ . The relative deviations from the test results (51.52%, 23.96%, 24.52%) using 50s are still within 5%. After the pulse interval is shortened to 0.1s, the test time of the sample liquid ³¹ P NMR is 121s, which is about one-third of the time compared with the currently commonly used 2s pulse interval (364s).

Comparative example 1

Soil sample is identical with embodiment 1,

A method for detecting organic phosphorus in soil:

(1) Add the soil sample to the extractant containing 0.25M NaOH and 0.05M Na ₂ EDTA (solid-to-liquid ratio 1:10), and shake and extract at 160rpm in the dark for 16h;

(2) Centrifuge at 10,000 rpm for 10 min after the extraction is completed, collect the supernatant and filter it through a 0.45 μm filter membrane for later use;

(3) Enrich the sample by freeze-drying;

(4) Enrich the sample with the complete dissolution of NaOH solution containing more than 10% D ₂ O, and complete the production of liquid ³¹ P NMR samples;

(5) The liquid ³¹ P NMR sample is added in the 5mm NMR sample tube, using Bruker 600MHz liquid nuclear magnetic resonance spectrometer (the number of pulse cycles is 25600, the pulse interval time is 2 and 0.1s, other parameters and test temperature are the same as in Example 1 ) to obtain the spectrum (Fig. 2). The morphology is identified by the chemical shift of the ³¹ P signal. The signal of 5.2ppm corresponds to orthophosphate, the signal of -4.1ppm corresponds to pyrophosphate, the signal of 5-2ppm corresponds to monoesters, and the signal of 19ppm Corresponds to phosphonates. The signal area is integrated by Bruker Topsin software, and the relative abundance of these three forms is calculated. The results show that there is a significant difference between the spectrum obtained by shortening the pulse interval time to 0.1s and the currently commonly used 2s. For the identification of phosphorus forms, use There is no obvious phosphonate signal in the spectrum at 0.1s. For the quantitative analysis of phosphorus species, the relative abundance deviation of each species between the 0.1s and 2s spectra has reached more than 10%.

Claims (2)

1. A method for rapidly and quantitatively detecting organic phosphorus in soil is characterized by comprising the following steps:

(1) A soil sample weighing 4g of dry soil was weighed, and 60mL of 0.25M NaOH and 0.05M Na were added ₂ Performing vibration extraction on the EDTA solution for 16h in a dark place, centrifuging, collecting supernatant, filtering the supernatant through a 0.45-micron filter membrane, collecting the filtered supernatant, and freezing to obtain freeze-dried powder;

(2) Dissolving 50mg of lyophilized powder in 2mL of paramagnetic ion solution, swirling for 30s, and standing for 2min to obtain liquid ³¹ A P NMR sample;

the formula of the paramagnetic ion solution comprises the following components: 50mg/L Fe ³⁺ 、0.25 M NaOH、0.05 M Na ₂ EDTA、10% (v/v) D ₂ O；

(3) Mixing liquid ³¹ Adding a P NMR sample into a 5mm NMR sample tube, detecting by using a Bruker 600MHz liquid nuclear magnetic resonance spectrometer to obtain a spectrum, and obtaining a standard spectrum through the spectrum ³¹ The chemical shift value of the P signal can determine the organic phosphorus form corresponding to the signal, and the relative abundance of the corresponding organic phosphorus or the organic phosphorus can be determined according to the relative proportion of the peak intensity or the area of the signal, so that the content of the organic phosphorus can be calculated;

in the step (3), the setting parameters of the Bruker 600MHz liquid nuclear magnetic resonance spectrometer are as follows: ³¹ the detection frequency of P is 242.98MHz, the detection temperature is 25 ℃, the pulse program is 90-degree pulse, the signal acquisition time is 0.845s, and the pulse interval time is 0.1s.

2. The method for rapidly and quantitatively detecting organic phosphorus in soil according to claim 1, wherein in the step (1), the rotation speed of the centrifugation is 10000rpm and the time is 10min.

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