CN116973431A - Simultaneous determination of plants 15 Method for detecting N isotope abundance and nitrogen content - Google Patents
Simultaneous determination of plants 15 Method for detecting N isotope abundance and nitrogen content Download PDFInfo
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 title claims abstract description 169
- 229910052757 nitrogen Inorganic materials 0.000 title claims abstract description 85
- 238000000034 method Methods 0.000 title claims abstract description 48
- 239000000126 substance Substances 0.000 claims abstract description 27
- 238000001514 detection method Methods 0.000 claims abstract description 22
- 238000005303 weighing Methods 0.000 claims abstract description 5
- 241000196324 Embryophyta Species 0.000 claims description 35
- 238000004364 calculation method Methods 0.000 claims description 10
- 241000219000 Populus Species 0.000 claims description 9
- 229960002989 glutamic acid Drugs 0.000 claims description 8
- 238000001819 mass spectrum Methods 0.000 claims description 6
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-Glutamic acid Natural products OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 4
- 238000005103 elemental analyser isotope ratio mass spectroscopy Methods 0.000 claims description 4
- 239000011159 matrix material Substances 0.000 claims description 4
- 125000003338 L-glutaminyl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])C([H])([H])C(=O)N([H])[H] 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 5
- 239000000758 substrate Substances 0.000 claims 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 abstract description 15
- 230000008569 process Effects 0.000 abstract description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 abstract description 5
- 238000012360 testing method Methods 0.000 abstract description 5
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 4
- 238000004904 shortening Methods 0.000 abstract description 2
- 239000004220 glutamic acid Substances 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 150000003863 ammonium salts Chemical class 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000004949 mass spectrometry Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000000700 radioactive tracer Substances 0.000 description 3
- 238000004448 titration Methods 0.000 description 3
- 241000209094 Oryza Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000004821 distillation Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- CRWJEUDFKNYSBX-UHFFFAOYSA-N sodium;hypobromite Chemical compound [Na+].Br[O-] CRWJEUDFKNYSBX-UHFFFAOYSA-N 0.000 description 2
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 108010064851 Plant Proteins Proteins 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000013485 heteroscedasticity test Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 239000000618 nitrogen fertilizer Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000008121 plant development Effects 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 235000021118 plant-derived protein Nutrition 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
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- G16C20/70—Machine learning, data mining or chemometrics
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
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Abstract
The invention relates to a method for simultaneously measuring plants 15 The method for detecting the abundance and the nitrogen content of the N isotope comprises the following steps: weighing a sample, acquiring peak high intensities with m/z of 28 and 29 by using an elemental analysis-isotope ratio mass spectrometer, and outputting an isotope ratio delta; calculated based on isotope ratio delta 15 N isotope abundance E; and calculating the total peak high intensity y based on the peak high intensities with m/z of 28 and 29, substituting the calculated total peak high intensity y into a standard curve established by the mass of the nitrogen element obtained from the standard substance and the total peak high intensity y, and calculating the mass of the nitrogen element of the sample to obtain the nitrogen content of the sample. The invention can simultaneously measure the plants 15 The method has the advantages of improving the detection efficiency, shortening the detection period, avoiding the use of corrosive chemical reagents such as concentrated sulfuric acid, sodium hydroxide and the like in the detection process, along with high accuracy and precision of test data and the like.
Description
Technical Field
The invention belongs to the technical field of stable isotope abundance detection, and relates to a method for simultaneously measuring plant abundance 15 N isotope abundance and nitrogen content detection method.
Background
Nitrogen is one of the most important nutrient elements necessary for plant growth and development, and is an important component constituting plant proteins, and the nitrogen directly or indirectly affects metabolic activity and growth and development of plants in various aspects. In recent years, with the development of research fields such as agricultural nitrogen migration and conversion processes, 15 the N-labeled tracer technique plays an important role in the research of nitrogen circulation of an ecological system. 15 The N-mark tracing technology is to make a certain number of 15 N-labeled tracers (e.g 15 N-labelled ammonium salt, 15 N-labeled nitrate, etc.) is added to soil and the plants are analyzed after a period of time has elapsed since their absorption 15 And N is sent to. 15 The N-labeled tracer is widely applied to agricultural science, and is applied to various aspects on the scale of a land ecological environment system, such as research of analyzing sources, utilization rates, distribution and destination of nitrogen elements in plants, conversion, absorption and nitrogen loss of nitrogen fertilizer and the like, so that the nitrogen management capacity is improved. 15 N-tracer studies were based on 15 The change of the abundance and the nitrogen content of N isotopes researches the transportation and transformation rule of nitrogen in plant bodies, thus, the plant bodies 15 Accurate determination of the abundance and nitrogen content of the N isotope is particularly important.
In existing plants 15 The detection method of N isotope abundance and nitrogen content is separately and independently detected, the nitrogen content in plants is mainly tested by adopting a Kjeldahl nitrogen determination method, the detection is carried out according to an industry standard NY/T2419-2013 plant total nitrogen content determination automatic azotometer method, concentrated sulfuric acid is used for high-temperature digestion, then steps such as sodium hydroxide distillation and the like are added, and a hydrochloric acid standard titration solution is titrated to obtainTo the nitrogen content of the plants. 15 Testing of the abundance of N isotopes according to GB/T20622-2006 Stable isotopes 15 The method of N inorganic labeling compound is operated, and ammonium salt tested by Kjeldahl nitrogen method is reacted with sodium hypobromite to generate 15 N marked nitrogen enters mass spectrum detection and is calculated according to the formula in GB/T20622-2006 15 N isotope abundance.
The prior art can not accurately measure two indexes at the same time, 15 the independent test of the abundance and the nitrogen content of the N isotope has long detection period, and a large amount of corrosive reagents such as concentrated sulfuric acid, sodium hydroxide and the like are consumed.
Zhu Yun et al (Zhu Yun, lei Guoliang, lin Yanyu, chen Xiuling. Method for elemental analyzer-isotope mass spectrometry determination of total nitrogen content. J. University of Fujian university (Nature science edition), 2013,29 (01): 58-62+114) utilize elemental analyzer-isotope mass spectrometry to determine total nitrogen content in a chemical reagent, establishing a mass spectrometry receiving peak area as a function of sample total nitrogen content. The elemental analyzer is often used for taking a single compound with higher nitrogen content as a standard substance (a method used in papers), a standard curve is further established, plants are not basically in the range of the standard curve due to lower nitrogen content, and in a plant nitrogen content detection experiment, the variance of an error term is often increased along with the increase of the nitrogen content, namely the heteroscedasticity exists.
Disclosure of Invention
The invention aims to provide a method for simultaneously measuring plants 15 The detection method of the N isotope abundance and the nitrogen content can improve the detection efficiency and accuracy, shorten the detection period and the like.
The aim of the invention can be achieved by the following technical scheme:
simultaneous determination of plants 15 The method for detecting the abundance and the nitrogen content of the N isotope comprises the following steps:
weighing a sample, acquiring peak high intensities with m/z of 28 and 29 by using an elemental analysis-isotope ratio mass spectrometer, and outputting an isotope ratio delta;
calculated based on isotope ratio delta 15 N isotope enrichedA degree E;
and calculating the total peak high intensity y based on the peak high intensities with m/z of 28 and 29, substituting the calculated total peak high intensity y into a standard curve established by the mass of the nitrogen element obtained from the standard substance and the total peak high intensity y, and calculating the mass of the nitrogen element of the sample to obtain the nitrogen content of the sample.
Further, the method comprises the steps of, 15 the calculation formula of the N isotope abundance E is as follows:
wherein E is 15 N isotope abundance, atom% 15 N; delta is the isotope ratio output by the instrument, and the permillage is realized.
Further, the calculation formula of the total peak high intensity y is:
y＝I 28 +I 29 wherein y is the total peak high intensity, mV; i 28 、I 29 Peak intensities of 28 and 29 m/z, mV, were collected for EA-IRMS mass spectra, respectively.
Further, the standard substances are plant matrix standard substances and chemical standard substances. Still further, the plant matrix standard is poplar leaf (preferably GBW 10235 poplar leaf component analysis standard, nitrogen content of 0.95%) and the chemical standard is L-glutamic acid (nitrogen content of 9.52%).
The invention selects two different types of standard substances, one type is a compound (L-glutamic acid, nitrogen content is 9.52%), and the other type is a plant matrix standard substance (poplar leaf, nitrogen content is 0.95%). In the standard curve drawing process, the common least square fitting calculation is used to meet the Gaussian-Markov assumption, namely, the measurement error meets the conditions of zero mean, same variance and mutual independence. In the plant nitrogen content detection experiment, the variance of the error term is often increased along with the increase of the nitrogen content, namely, the variance exists, at the moment, the relative deviation of the calculation result of the low-nitrogen content plant sample is larger due to the adoption of the common least square method, the prediction result of the standard curve does not accord with the actual situation, and the situation can be effectively improved by adopting the weighted least square method.
Furthermore, when the standard curve is drawn, two standard substances with different masses are weighed, and the weight is 1/x 2 And when the method is used, the quality of the nitrogen element and the total peak high intensity are used for fitting by adopting a weighted least square method to establish a standard curve. The mass of the nitrogen element is the product of the mass of the corresponding standard substance and the nitrogen content, and the mass is obtained by weighing by means of a balance and the like.
Further, the formula of calculation of the nitrogen content of the sample is:
w sample ＝x sample /m sample wherein w is sample The nitrogen content of the sample,%; x is x sample Mg, which is the mass of the sample nitrogen element calculated by the standard curve; m is m sample Mg is the mass of the sample.
Compared with the prior art, the invention can simultaneously measure the plants 15 The method has the advantages of improving the detection efficiency, shortening the detection period, avoiding the use of corrosive chemical reagents such as concentrated sulfuric acid, sodium hydroxide and the like in the detection process, along with high accuracy and precision of test data and the like.
Detailed Description
The present invention will be described in detail with reference to specific examples. The present embodiment is implemented on the premise of the technical scheme of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection scope of the present invention is not limited to the following examples.
In the following examples, the conventional methods used were: the nitrogen content in one plant is mainly tested by adopting a Kjeldahl nitrogen determination method, and is tested according to an industry standard NY/T2419-2013 automatic nitrogen determination method for total nitrogen content determination of plants, concentrated sulfuric acid is used for high-temperature digestion, then steps such as sodium hydroxide distillation and the like are added, and the nitrogen content of the plant is obtained by titration of a hydrochloric acid standard titration solution. And two, a second step of, in the second step, 15 testing of the abundance of N isotopes according to GB/T20622-2006 Stable isotopes 15 The method of N inorganic labeling compound is operated, and ammonium salt tested by Kjeldahl nitrogen method is reacted with sodium hypobromite to generate 15 N marked nitrogen enters mass spectrum detection and is calculated according to the formula in GB/T20622-2006 15 N parityAbundance of the element.
The standard substances used were GBW 10235 poplar leaf component analysis standard substance (nitrogen content: 0.95) and USGS 40L-glutamic acid (nitrogen content: 9.52%).
Example 1:
15 in N-labeled rice 15 The method for measuring the N isotope abundance and nitrogen content comprises the following steps:
respectively weighing (accurate to 0.001 mg) 0.3, 0.6, 0.9 and 1.2mg of poplar leaf and USGS 40L-glutamic acid standard substance, respectively placing the ground samples into tin cups, packaging, EA-IRMS detection, mass spectrum acquisition with m/z of peak heights of 28 and 29, and outputting isotope ratio delta by an instrument, wherein the formula is utilizedCalculation of 15 N isotope abundance;
the poplar leaf and USGS 40L-glutamic acid standard substance are respectively weighed (0.001 mg accurate) to 0.3, 0.6, 0.9 and 1.2mg, and the weight is 1/x 2 When the method is used, the mass of the nitrogen element and the total peak high intensity are adopted to carry out fitting by adopting a weighted least square method to establish a standard curve, the total peak high intensity of the sample is substituted into the standard curve to calculate the mass of the nitrogen element, and then the mass of the sample is divided to calculate the nitrogen content of the sample, the result is shown in a table 1, the data in the table 1 show that the deviation of the method is less than or equal to 0.05 percent compared with the traditional method, and the method is obvious 15 The N isotope abundance and nitrogen content determination methods also have very high measurement accuracy.
TABLE 1 15 In N-labeled rice 15 Determination of N isotope abundance and Nitrogen content
Example 2:
in plants 15 The method for measuring the N isotope abundance and nitrogen content comprises the following steps:
the poplar leaf and USGS 40L-glutamic acid standard substance are respectively weighed (0.001 mg accurate) to 0.3, 0.6, 0.9 and 1.2mg, and groundThe samples are respectively placed in a tin cup, wrapped, EA-IRMS detection is carried out, the mass spectrum acquisition m/z is the peak heights of 28 and 29, the isotope ratio delta is output by the instrument, and the formula is utilizedCalculation of 15 N isotope abundance.
The poplar leaf and USGS 40L-glutamic acid standard substance are respectively weighed (0.001 mg accurate) to 0.3, 0.6, 0.9 and 1.2mg, and the weight is 1/x 2 And when the method is used, a standard curve is established by fitting the mass of the nitrogen element and the total peak height by adopting a weighted least square method, the total peak height of the sample is substituted into the standard curve to calculate the mass of the nitrogen element, and then the mass of the sample is divided to calculate the nitrogen content of the sample. The results are shown in Table 2. The data in Table 2 shows that the deviation of the invention from the conventional method is less than or equal to 0.05%, and it can be seen that the invention 15 The N isotope abundance and nitrogen content determination methods also have very high measurement accuracy.
TABLE 2 in plants 15 Determination of N isotope abundance and Nitrogen content
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Claims (7)
1. Simultaneous determination of plants 15 The method for detecting the abundance and the nitrogen content of the N isotope is characterized by comprising the following steps of:
weighing a sample, acquiring peak high intensities with m/z of 28 and 29 by using an elemental analysis-isotope ratio mass spectrometer, and outputting an isotope ratio delta;
calculated based on isotope ratio delta 15 N isotope abundance E;
and calculating the total peak high intensity y based on the peak high intensities with m/z of 28 and 29, substituting the calculated total peak high intensity y into a standard curve established by the mass of the nitrogen element obtained from the standard substance and the total peak high intensity y, and calculating the mass of the nitrogen element of the sample to obtain the nitrogen content of the sample.
2. A simultaneous assay according to claim 1 in plants 15 A method for detecting the abundance and nitrogen content of N isotope is characterized in that, 15 the calculation formula of the N isotope abundance E is as follows:
wherein E is 15 N isotope abundance, atom% 15 N; delta is the isotope ratio output by the instrument, and the permillage is realized.
3. A simultaneous assay according to claim 1 in plants 15 The detection method of the N isotope abundance and the nitrogen content is characterized in that the calculation formula of the total peak high intensity y is as follows:
y＝I 28 +I 29 wherein y is the total peak high intensity, mV; i 28 、I 29 Peak intensities of 28 and 29 m/z, mV, were collected for EA-IRMS mass spectra, respectively.
4. A simultaneous assay according to claim 1 in plants 15 The method for detecting the abundance and the nitrogen content of the N isotope is characterized in that the standard substances are plant matrix standard substances and chemical standard substances.
5. A simultaneous determination of plants according to claim 4 15 The method for detecting the abundance and nitrogen content of the N isotope is characterized in that the plant substrate standard substance is poplar leaves, and the chemical standard substance is L-glutamic acid.
6. A simultaneous assay according to claim 1 in plants 15 The method for detecting the abundance and the nitrogen content of N isotopes is characterized in that when a standard curve is drawn, standard substances with different masses are weighed, and the weight is 1/x 2 And when the method is used, the quality of the nitrogen element and the total peak high intensity are used for fitting by adopting a weighted least square method to establish a standard curve.
7. A simultaneous assay according to claim 1 in plants 15 The detection method of the N isotope abundance and the nitrogen content is characterized in that the calculation formula of the nitrogen content of the sample is as follows:
w sample ＝x sample /m sample wherein w is sample The nitrogen content of the sample,%; x is x sample Mg, which is the mass of the sample nitrogen element calculated by the standard curve; m is m sample Mg is the mass of the sample.
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