CN110749614B - Method for rapidly and quantitatively detecting organic phosphorus in soil - Google Patents
Method for rapidly and quantitatively detecting organic phosphorus in soil Download PDFInfo
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- CN110749614B CN110749614B CN201911095589.7A CN201911095589A CN110749614B CN 110749614 B CN110749614 B CN 110749614B CN 201911095589 A CN201911095589 A CN 201911095589A CN 110749614 B CN110749614 B CN 110749614B
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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
Technical Field
The invention relates to a method for rapidly and quantitatively detecting organic phosphorus in soil, and belongs to the technical field of chemical detection.
Background
The soil contains inorganic phosphorus and organic phosphorus, wherein the organic phosphorus accounts for 35-65% of the total phosphorus in the soil, and understanding the change dynamics of the organic phosphorus in the soil has important significance for deeply understanding the biogeochemical cycle process of the phosphorus in the soil and establishing efficient sustainable agricultural management measures.
The inorganic phosphorus in the soil is mainly orthophosphate, the content of the inorganic phosphorus can be measured by a spectrophotometric method and an ICP-OES method, the content of the organic phosphorus in the soil can be estimated by subtracting the inorganic phosphorus extracted by acid from the total phosphorus extracted after digestion, meanwhile, the organic phosphorus in the specific soil can be extracted and analyzed by a chromatography method, for example, chloroform, methanol and water or dichloromethane and methanol are used for extracting phospholipid, and then, the chromatographic separation and the content measurement are carried out, however, the method can not determine the form of the organic phosphorus, and only can determine the content of the organic phosphorus in a specific form. The organic phosphorus in soil has various molecular forms, and the form and the composition of the organic phosphorus in the soil are analyzed on a molecular level, so an advanced analysis method needs to be established.
Liquid, method for producing the same and use thereof 31 P Nuclear Magnetic Resonance (NMR) spectroscopy is the first method to quantitatively detect the organophosphorus form in soil.
Due to liquid NMR spectrum 31 The isotropic nuclear magnetic signal of P has good resolution and can reflect the junction of specific organic phosphorus compoundsStructural information, so organic phosphorus in soil is usually extracted by a solution and then is subjected to one-dimensional liquid 31 P NMR analysis was performed, and the kind of organic phosphorus was identified and quantitatively compared. Although it is a matter of course 31 P NMR has enjoyed great success in soil science, but certain factors have prevented its wider use in this field. Due to the relatively low phosphorus content of the soil, 31 p NMR measurements usually require long scan times (about 16-20 h for a sample), liquid against soil organophosphorus extracts 31 P NMR quantitative analysis typically requires thousands of pulse scans (i.e., repeated) 31 P excitation-relaxation cycles) to obtain sufficient signal to produce a high quality map that allows for the determination of soil organophosphorus 31 P NMR machines are time intensive and cost prohibitive.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provide a method for rapidly and quantitatively detecting organic phosphorus in soil, which can effectively shorten the detection time and improve the efficiency of quantitative analysis of the organic phosphorus in the soil.
Technical scheme
Liquid for organic phosphorus extracting solution of soil 31 P NMR quantitative analysis typically requires thousands of pulse scans to obtain sufficient signal to produce a high quality map, and the time interval between pulse cycles needs to be set long enough that all 31 The P-nucleus is fully restored from the excited state to the equilibrium state, otherwise the nuclear magnetic resonance signal intensity will not quantitatively reflect the information of all organophosphorus morphologies. The pulse interval thus determines to a large extent the liquid 31 Experimental length of P NMR measurement. Excited in solution 31 The process of return of the P-nucleus to equilibrium is mainly influenced by its spin-lattice relaxation (T) 1 ) Control of, T of, different kinds of phosphorus compounds 1 There was also a significant difference in time. Therefore, the inventor shortens the detection time by shortening the pulse interval, thereby improving the efficiency of the organic phosphorus quantitative analysis of the soil. The specific scheme is as follows:
a method for rapidly and quantitatively detecting organic phosphorus in soil comprises the following steps:
(1) A4 g (dry weight) soil sample was weighed and 60mL NaOH and Na were added 2 Mixed solution of EDTA, naOH concentration in the mixed solution is 0.25M 2 The concentration of EDTA is 0.05M; oscillating and extracting for 16h in dark place, centrifuging, collecting supernatant, filtering with 0.45 μm filter membrane, collecting filtered supernatant, and freezing to obtain lyophilized 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 formulation of the paramagnetic ion solution is as follows: 20-100mg/L Fe 3+ 、0.25M NaOH、0.05M 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 passing the spectrum 31 The chemical shift value of the P signal can determine the organic phosphorus form corresponding to the signal, and according to the relative proportion of the signal peak intensity (area), the relative abundance of the corresponding organic phosphorus or a certain type of organic phosphorus can be determined, so that the content of the organic phosphorus can be calculated.
Further, in the step (1), the rotation speed of the centrifugation is 10000rpm, and the time is 10min.
Further, in the step (2), the formulation of the paramagnetic ion solution is as follows: 50mg/L Fe 3+ 、0.25M NaOH、0.05M Na 2 EDTA、10％(v/v)D 2 O。
Further, 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.
The invention has the beneficial effects that: the invention provides a method for rapidly and quantitatively detecting organic phosphorus in soil, which is implemented in liquid 31 Based on the quantitative analysis of P NMR 31 Paramagnetic ions are added into the P NMR sample to shorten the pulse interval, so that the detection time of the NMR experiment is shortened. With liquids in common use 31 Compared with the P NMR method, the method can greatly improve the efficiency of the quantitative analysis of the organic phosphorus in the soil.
Drawings
FIG. 1 is a view of a soil sample measured by the method of example 1 31 A P NMR spectrum;
FIG. 2 is a graph of soil samples measured by the method of comparative example 1 31 P NMR spectrum.
Detailed Description
The invention is further described with reference to the following figures and detailed description.
Example 1
Taking different Fe 3 Concentration, three paramagnetic ion solutions were prepared: (20, 50, 100) mg/L Fe 3+ 、0.25M NaOH、0.05M Na 2 EDTA、10％(v/v)D 2 And O. Then sodium phosphate, sodium pyrophosphate, phytic acid (25 mM P) and DNA (1 mg/mL) were dissolved in paramagnetic ion solution, respectively, and added to a 5mM NMR sample tube using Bruker 600MHz liquid NMR (242.98MHz, 25 ℃) to reverse recovery method (pulse program T1 irpg) for T of phosphorus compound 1 The time was measured (10 pulse intervals were set, each pulse cycle was 32, the signal acquisition time was 0.845s, and the relaxation interval was 50 s), and the results are shown in table 1:
TABLE 1 spin lattice relaxation (T) of phosphorus compounds at different paramagnetic ion concentrations 1 ) Time of day
As can be seen from Table 1, taken together, the paramagnetic ion Fe 3 The spin lattice relaxation (T) of the phosphorus compound at a concentration of 50mg/L 1 ) The time is shorter. Therefore, 50mg/L of paramagnetic ion Fe is selected 3 The concentration was tested.
The test soil sample was field black clay (N46 ℃ 48 '20.3', E134 ℃ 01 '13.9') from Shuangyashan city of Heilongjiang province, and the crop plant was soybean. Basic physicochemical properties: pH 6.90, C56 g kg –1 、N 13g kg –1 、P 1.8g kg –1 。
A method for rapidly and quantitatively detecting organic phosphorus in soil comprises the following steps:
(1) A4 g (dry weight) soil sample was weighed and 60mL NaOH and Na were added 2 In the mixed solution of EDTA, the concentration of NaOH is 0.25M 2 The concentration of EDTA is 0.05M; shaking and extracting for 16h in dark place, centrifuging (rotation speed 10000rpm for 10 min), collecting supernatant, filtering with 0.45 μm filter membrane, collecting filtered supernatant, and freezing to obtain lyophilized powder;
(2) Dissolving 50mg of lyophilized powder in 2mL of paramagnetic ion solution, vortexing for 30s, and standing for 2min to obtain liquid 31 P NMR samples;
the formulation of the paramagnetic ion solution is as follows: 50mg/L Fe 3+ 、0.25M NaOH、0.05M Na 2 EDTA、10％(v/v)D 2 O；
(3) Mixing liquid 31 P NMR samples were loaded into 5mm NMR sample tubes using a Bruker 600MHz liquid NMR spectrometer ( 31 Detecting P at 242.98MHz,25 deg.C, 90 ° pulse, signal acquisition time of 0.845s, pulse cycle number of 128, and pulse interval time of 50-0.1s to obtain a spectrum (figure 1), and analyzing by using a standard method 31 The morphology was identified by chemical shift of the P signal, with 5.2ppm signal corresponding to orthophosphate (ortho phosphate), -4.1ppm signal corresponding to pyrophosphate (pyrophosphate), and 5-4ppm signal derived from phytic acid (phytic acid). The relative abundance of these three forms was calculated by integrating the signal region through Bruker Topsin software and the results show: by adding 50mg/L Fe 3+ The relative abundances of the three forms of phosphorus obtained by analysis after shortening the pulse interval time to 0.1s were 50.24%, 24.90% and 24.87%, and the relative deviation from the test results using 50s (51.52%, 23.96%, 24.52%) was still within the 5% range. After the pulse interval was shortened to 0.1s, the sample liquid 31 The test duration of P NMR is 121s, which is about one third as long as the currently commonly used 2s pulse interval (364 s).
Comparative example 1
The soil sample was the same as in example 1,
a method for detecting organic phosphorus in soil comprises the following steps:
(1) Soil samples were added to a soil sample containing 0.25M NaOH and 0.05M Na 2 Extraction of EDTATaking out the mixture (solid-liquid ratio is 1;
(2) Centrifuging at 10000rpm for 10min after extraction, collecting supernatant, and filtering with 0.45 μm filter membrane;
(3) Enriching the sample by freeze drying;
(4) Using a catalyst containing more than 10% of D 2 The NaOH solution of O is completely dissolved to enrich the sample, and the liquid preparation is completed 31 A P NMR sample;
(5) Mixing liquid 31 P NMR samples were added to 5mm NMR sample tubes and a spectrum was obtained using a Bruker 600MHz liquid NMR spectrometer (number of pulse cycles 25600, time between pulses 2 and 0.1s, other parameters and test temperature the same as in example 1) (FIG. 2). By passing 31 The morphology was identified by chemical shift of the P signal, with 5.2ppm signal corresponding to orthophosphates (orthophosphates), -4.1ppm signal corresponding to pyrophosphates (pyrophosphates), 5-2ppm signal corresponding to phosphomonoesters (monoesters), and 19ppm signal corresponding to phosphonates (phosphonates). The relative abundance of these three forms was calculated by integrating the signal region through Bruker Topsin software and the results show: the pulse interval time is shortened to 0.1s, the spectrum obtained by the method is obviously different from the spectrum obtained by 2s which is commonly used at present, and for phosphorus morphology identification, the spectrum using 0.1s has no obvious phosphonate signal. For quantitative analysis of phosphorus morphology, the relative abundance deviation of each morphology between 0.1s and 2s spectra reaches 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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