CN115128051B - Fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products - Google Patents
Fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products Download PDFInfo
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- WBLZUCOIBUDNBV-UHFFFAOYSA-N 3-nitropropanoic acid Chemical compound OC(=O)CC[N+]([O-])=O WBLZUCOIBUDNBV-UHFFFAOYSA-N 0.000 title claims abstract description 70
- 240000000111 Saccharum officinarum Species 0.000 title claims abstract description 37
- 235000007201 Saccharum officinarum Nutrition 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 32
- 238000001917 fluorescence detection Methods 0.000 title claims abstract description 17
- 150000003220 pyrenes Chemical class 0.000 claims abstract description 28
- 239000012086 standard solution Substances 0.000 claims abstract description 26
- 238000012417 linear regression Methods 0.000 claims abstract description 16
- 238000002835 absorbance Methods 0.000 claims abstract description 10
- 230000008569 process Effects 0.000 claims abstract description 7
- 238000002360 preparation method Methods 0.000 claims abstract description 5
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 30
- 239000000047 product Substances 0.000 claims description 17
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 16
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 15
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 13
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 claims description 10
- 238000001514 detection method Methods 0.000 claims description 10
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 claims description 10
- -1 pyrene formaldehyde hydrazone Chemical compound 0.000 claims description 10
- 239000007787 solid Substances 0.000 claims description 9
- 239000002904 solvent Substances 0.000 claims description 9
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 7
- SMQUZDBALVYZAC-UHFFFAOYSA-N salicylaldehyde Chemical compound OC1=CC=CC=C1C=O SMQUZDBALVYZAC-UHFFFAOYSA-N 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- 238000004440 column chromatography Methods 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 6
- 238000002189 fluorescence spectrum Methods 0.000 claims description 6
- 238000003860 storage Methods 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 5
- 230000035484 reaction time Effects 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
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- 238000010521 absorption reaction Methods 0.000 claims description 3
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- 238000006243 chemical reaction Methods 0.000 claims description 3
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- 239000012362 glacial acetic acid Substances 0.000 claims description 3
- 239000003208 petroleum Substances 0.000 claims description 3
- 238000001228 spectrum Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 2
- 239000011550 stock solution Substances 0.000 claims description 2
- 238000004140 cleaning Methods 0.000 claims 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 230000004044 response Effects 0.000 abstract description 3
- 238000001819 mass spectrum Methods 0.000 description 4
- 238000011084 recovery Methods 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
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- 239000005720 sucrose Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- YISPKQZWSIMXMX-UHFFFAOYSA-N 3-nitropropan-1-ol Chemical class OCCC[N+]([O-])=O YISPKQZWSIMXMX-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 101710138657 Neurotoxin Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000835 electrochemical detection Methods 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002581 neurotoxin Substances 0.000 description 1
- 231100000618 neurotoxin Toxicity 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 1
- 229940081974 saccharin Drugs 0.000 description 1
- 235000019204 saccharin Nutrition 0.000 description 1
- 239000000901 saccharin and its Na,K and Ca salt Substances 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6417—Spectrofluorimetric devices
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6417—Spectrofluorimetric devices
- G01N2021/6421—Measuring at two or more wavelengths
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Pathology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products, which comprises the following steps: (1) preparation of a pyrene derivative fluorescence sensor; (2) preparing a standard solution; (3) drawing a standard curve; the fluorescence value of the fluorescence intensity at 456nm and the 3-nitropropionic acid standard solution with the concentration in the range of 0.5-10.0 mu mol/L are in good linear relation, and the obtained linear regression equation is as follows: f 456nm =1040.57+33.13c; the absorbance at 456nm of the 3-nitropropionic acid standard solution with the absorbance in the range of 10.0-100.0 mu mol/L shows a good linear relationship, and the obtained linear regression equation is as follows: f 456nm =1325.93+9.39c. The fluorescence sensor for detecting 3-nitropropionic acid has the advantages of simple operation, quick response, high sensitivity, good selectivity and low cost in the process of detecting 3-nitropropionic acid simultaneously.
Description
Technical Field
The invention belongs to the technical field of electrochemical detection methods, and particularly relates to a fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products.
Background
3-Nitropropionic acid is a highly toxic neurotoxin, a metabolite of 3-nitropropanol produced by a variety of fungi, widely distributed in mildewed sugarcane. The good stability of 3-nitropropionic acid can lead the 3-nitropropionic acid to be preserved in the subsequent saccharin process and remain in sucrose, and the sucrose is one of the most common food additives in our daily life and forms a great threat to human health. Therefore, the establishment of the 3-nitropropionic acid content determination method which is simple and convenient to operate, low in cost, sensitive and rapid has important practical significance.
The detection method of 3-nitropropionic acid reported in the current literature is mainly chromatography, wherein high performance liquid chromatography and high performance liquid chromatography-mass spectrometry are most common, and the detection method is also the detection method used in the current national standard, and has higher sensitivity and accuracy. However, chromatograph has high purchase and use cost, complex operation, and strong technical requirements for use and maintenance, is difficult to realize intelligent and low-cost rapid detection, and has no good popularity in agricultural product production bases and small and medium-sized enterprises.
Disclosure of Invention
Aiming at the technical problems that the method for detecting the 3-nitropropionic acid in the prior art needs to use high-cost and professional technicians and fixed detection places, almost needs to carry out complex pretreatment processes, has complex operation and time-consuming, and has no advantages in rapid screening of samples, the invention provides a fluorescence detection method for detecting the 3-nitropropionic acid in sugarcane and sugarcane products, and aims to obtain a method for detecting the 3-nitropropionic acid in the sugarcane and sugarcane products by using a fluorescence method, which has simple operation, low cost, sensitivity and rapidness.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
a fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products comprises the following operation steps:
(1) Preparation of pyrene derivative fluorescence sensor: dissolving 1-pyrene formaldehyde in tetrahydrofuran solution, stirring at room temperature to dissolve completely, adding hydrazine hydrate, stirring at room temperature, vacuum filtering the reaction product, washing with diethyl ether, and drying to obtain pyrene formaldehyde hydrazone; dissolving the obtained pyrene formaldehyde hydrazone and 4-tertiary butyl-2, 6-formylphenol in tetrahydrofuran, dropwise adding glacial acetic acid, reacting for 24 hours at normal temperature, filtering, concentrating the filtrate, separating and purifying by using a column chromatography, and standing to volatilize a solvent to finally obtain a pyrene derivative solid; dissolving pyrene derivative solid in dimethyl sulfoxide to prepare a storage solution of a pyrene derivative fluorescence sensor;
(2) Preparing a standard solution: taking water as a solvent, dissolving 3-nitropropionic acid solid in water to prepare mother liquor, respectively taking a certain amount of mother liquor, mixing and diluting to obtain a series of mixed standard solutions to be measured with different concentrations, namely 3-nitropropionic acid standard solutions;
(3) Drawing a standard curve: mixing the storage solution of the pyrene derivative fluorescence sensor obtained in the step (1) and the 3-nitropropionic acid standard solution obtained in the step (2) according to a certain proportion, standing for a certain time, performing fluorescence emission spectrum scanning within the absorption wavelength range of 375-700 nm, recording the spectrum, drawing a standard curve by using a fluorescence intensity value measured at 456nm, and calculating to obtain a linear regression equation; in the process of actually detecting the 3-nitropropionic acid, replacing the 3-nitropropionic acid standard solution with a sample to be detected, and respectively substituting fluorescence values measured at 456nm into the obtained linear regression equation according to the same operation to calculate the content of the 3-nitropropionic acid in the sample to be detected;
The fluorescence value of the fluorescence intensity at 456nm and the 3-nitropropionic acid standard solution with the concentration in the range of 0.5-10.0 mu mol/L are in good linear relation, and the obtained linear regression equation is as follows: f 456nm =1040.57+33.13c, correlation coefficient r=0.9930, where F 456nm is fluorescence intensity value at 456nm, and C is concentration of 3-nitropropionic acid;
the absorbance at 456nm of the 3-nitropropionic acid standard solution with the absorbance in the range of 10.0-100.0 mu mol/L shows a good linear relationship, and the obtained linear regression equation is as follows: f 456nm =1325.93+9.39c, correlation coefficient r=0.9904; the detection limit of the method is as follows: 0.32. Mu. Mol/L.
Preferably, the amount of 1-pyrene formaldehyde in the step (1) is 0.5756g (2.5 mmol) and the amount of tetrahydrofuran is 10mL.
Preferably, the volume percentage of the hydrazine hydrate in the step (1) is 25%, the dosage is 4.85mL (25 mmol), and the mass ratio of the 1-pyrene formaldehyde to the hydrazine hydrate is 1:10; the reaction time of the 1-pyrene formaldehyde and the hydrazine hydrate is 12 hours.
Preferably, the mass ratio relationship of pyrene formaldehyde hydrazone and 4-tertiary butyl-2, 6-formylphenol in the step (1) is 1:1.1; the reaction time of pyrene formaldehyde hydrazone and 4-tert-butyl-2, 6-formylphenol was 24 hours.
Preferably, the eluent in the column chromatography in the step (1) is petroleum ether and ethyl acetate, and the volume ratio of the eluent to the ethyl acetate is 16:1, 12:1, 10:1,8:1,6:1 and 4:1 in sequence.
Preferably, in the step (1), a stock solution of a pyrene derivative fluorescence sensor having a concentration of 15. Mu. Mol/L is prepared.
Preferably, the volume ratio of the solvent dimethyl sulfoxide to water in the step (1) is 2:1.
Preferably, the holding time in step (3) is 30min.
Compared with the prior art, the invention has the following beneficial effects:
The fluorescence sensor for detecting 3-nitropropionic acid is a pyrene derivative, the preparation process is simple, and the process for detecting 3-nitropropionic acid has the advantages of simplicity in operation, quick response, high sensitivity, good selectivity and low cost.
Drawings
FIG. 1 is a mass spectrum of a pyrene derivative biosolid prepared by step (1) of the present invention.
FIG. 2 is a fluorescence spectrum chart and a standard curve chart of the method of the invention; wherein a is a fluorescence spectrum diagram of 3-nitropropionic acid with different concentrations in pyrene derivative fluorescence sensor solution; b is a standard curve of fluorescence intensity of 3-nitropropionic acid at 456nm at different concentrations and corresponding concentrations.
Detailed Description
The following detailed description, in conjunction with the accompanying drawings, describes in detail, but it is to be understood that the scope of the invention is not limited to the specific embodiments. The raw materials and reagents used in the examples were commercially available unless otherwise specified.
Example 1
A fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products comprises the following specific operation steps:
(1) Preparation of pyrene derivative fluorescence sensor: 0.5756g (2.5 mmol) of 1-pyrene formaldehyde is dissolved in 10mL of tetrahydrofuran solution, stirred at room temperature to be completely dissolved, hydrazine hydrate with the mass percent of 25 percent and the dosage of 4.85mL (25 mmol) is added, the mass ratio of the 1-pyrene formaldehyde to the hydrazine hydrate is 1:10, the mixture is stirred at room temperature for reaction for 12 hours, the product obtained by the reaction is decompressed, pumped, filtered, washed by diethyl ether and dried to obtain pyrene formaldehyde hydrazone; taking 0.2441g (1.0 mmol) of the obtained pyrene formaldehyde hydrazone and 0.2268g (1.1 mmol) of 4-tertiary butyl-2, 6-formylphenol, dissolving in 10mL of tetrahydrofuran, dropwise adding 100 mu L of glacial acetic acid, reacting for 24 hours at normal temperature, filtering, concentrating filtrate, separating and purifying by using a column chromatography, wherein the eluent of the column chromatography is petroleum ether and ethyl acetate, the volume ratio of the eluent is 16:1, 12:1, 10:1,8:1,6:1,4:1 in sequence, standing to volatilize a solvent, and finally obtaining pyrene derivative solid, and preparing the pyrene derivative for mass spectrum analysis, and the result is shown in figure 1; dissolving 0.0324g of pyrene derivative solid in 50mL of dimethyl sulfoxide to obtain 1.5X10 -3 mol/L pyrene derivative solution, gradually adding dimethyl sulfoxide into 1.5mL pyrene derivative solution (1.5X10 -3 mol/L) to dilute to 100mL, and preparing a storage solution of a pyrene derivative fluorescence sensor with the concentration of 15 mu mol/L; the volume ratio of the solvent dimethyl sulfoxide to water is 2:1;
(2) Preparing a standard solution: taking water as a solvent, dissolving 3-nitropropionic acid solid in water to prepare mother solution with the concentration of 3.0X10 -4 mol/L, respectively taking a certain amount of mother solution for mixing and diluting, and fixing the volume to obtain a series of 3-nitropropionic acid mixed standard solutions with the concentration of 1.5, 3.0, 6.0, 9.0, 12.0, 24.0, 30.0, 60.0, 90.0, 120.0, 180.0, 240.0 and 300.0 mu mol/L, namely 3-nitropropionic acid standard solutions;
(3) Drawing a standard curve: using a fluorescence spectrophotometer of the 960MC type, the spectral scanning parameters were set as follows: the measurement mode is wavelength scanning, the scanning mode is emission mode, the excitation wavelength is 360nm, the scanning speed is 1000nm/min, the scanning interval is 1nm, and the excitation bandwidth and the emission bandwidth are 5nm; mixing the storage solution (15 mu mol/L) of the pyrene derivative fluorescence sensor obtained in the step (1) with the 3-nitropropionic acid standard solution obtained in the step (2) according to the volume ratio of 2:1, placing for 30min, carrying out fluorescence emission spectrum scanning within the absorption wavelength range of 375-700 nm, recording the spectrum, drawing a standard curve by utilizing the fluorescence intensity value measured at 456nm, and calculating to obtain a linear regression equation, wherein the concentration of the pyrene derivative fluorescence sensor is 10 mu mol/L, and the concentration of the 3-nitropropionic acid is 0.5, 1.0, 2.0, 4.0, 8.0, 10.0, 20.0, 40.0, 60.0, 80.0 and 100.0 mu mol/L respectively;
The fluorescence intensity of the fluorescence value at 456nm and the 3-nitropropionic acid standard solution with the concentration within the range of 0.5-10.0 mu mol/L are in good linear relation, and the obtained linear regression equation is as follows: f 456nm =1040.57+33.13c, correlation coefficient r=0.9930, where F456nm is fluorescence intensity value at 456nm, C is concentration of 3-nitropropionic acid; substituting the measured fluorescence value into the linear equation of fig. 2 (b);
The absorbance at 456nm of the 3-nitropropionic acid standard solution with the absorbance in the range of 10.0-100.0 mu mol/L shows a good linear relationship, and the obtained linear regression equation is as follows: f 456nm =1325.93+9.39c, correlation coefficient r=0.9904; the detection limit of the method is as follows: 0.32 mu mol/L;
In the actual detection process of 3-nitropropionic acid, taking 20g of peeled sugar cane sample, cutting into small pieces, stirring and uniformly mixing to prepare a sample, and storing at the temperature of-20 ℃ for later use; weighing 2g of a sample, adding 10mL of deionized water, performing ultrasonic extraction for 10min, centrifuging for 5min with 10000r/min, transferring the supernatant into another centrifuge tube, repeatedly extracting for 2 times with 10mL of deionized water, and combining the supernatants to obtain the sample to be detected; replacing the 3-nitropropionic acid standard solution in the step (3) with a sample to be detected, substituting the fluorescence values measured at 456nm into the obtained linear regression equation respectively according to the same operation, calculating the content of the 3-nitropropionic acid in the sample to be detected, calculating the corresponding concentration value of the 3-nitropropionic acid from the standard curve according to the measured fluorescence intensity, and calculating the standard adding recovery rate of the 3-nitropropionic acid, wherein the result is shown in the table 1:
TABLE 1 3-nitropropionic acid in sugar cane samples labeled recovery determination results
| Sample content (mu mol/L) | Scalar (mu mol/L) | Measured value (mu mol/L) | Recovery (%) |
| 0.0 | 1.00 | 1.14 | 114.0 |
| 0.0 | 10.00 | 10.63 | 106.3 |
| 0.0 | 40.00 | 44.31 | 110.8 |
From Table 1, the content of 3-nitropropionic acid in the sugarcane sample is 0, and the fluorescence detection method for 3-nitropropionic acid in the sugarcane sample provided by the invention has good practicability and accuracy.
Fig. 1 is a mass spectrum of the pyrene derivative prepared by the present invention, and from the mass spectrum shown in fig. 1, a proton peak appears at m/z= 433.19, corresponding to the theoretical value of m/z= 433.18, which indicates that the pyrene derivative is successfully synthesized in step (1).
FIG. 2a is a graph showing fluorescence spectra of different concentrations of 3-nitropropionic acid in pyrene derivative fluorescence sensor solutions, as shown, with increasing 3-nitropropionic acid concentration, fluorescence emission peak at 456nm is gradually increased; b is a calibration curve of fluorescence intensity of 3-nitropropionic acid with different concentrations at 456nm and corresponding concentrations, and as shown in the figure, the fluorescence intensity of the fluorescence value at 456nm and a 3-nitropropionic acid standard solution with the concentration in the range of 0.5-10.0 mu mol/L are in good linear relation, and the obtained linear regression equation is: f 456nm =1040.57+33.13c; the absorbance at 456nm of the 3-nitropropionic acid standard solution with the absorbance in the range of 10.0-100.0 mu mol/L shows a good linear relationship, and the obtained linear regression equation is as follows: f 456nm =1325.93+9.39c.
The fluorescence detection method has the characteristics of simple operation, low cost, quick response, short reaction time, good reproducibility and the like, and can be a reliable means for low-cost and quick detection of the 3-nitropropionic acid.
The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (8)
1. A fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products, which is characterized by comprising the following operation steps:
(1) Preparation of pyrene derivative fluorescence sensor: dissolving 1-pyrene formaldehyde in tetrahydrofuran solution, stirring to dissolve the 1-pyrene formaldehyde, adding hydrazine hydrate, stirring, filtering a product obtained by the reaction, cleaning and drying to obtain pyrene formaldehyde hydrazone; dissolving the obtained pyrene formaldehyde hydrazone and 4-tertiary butyl-2, 6-formylphenol in tetrahydrofuran, dropwise adding glacial acetic acid, reacting at normal temperature, filtering, concentrating the filtrate, separating and purifying by using a column chromatography, standing to volatilize a solvent, and finally obtaining a pyrene derivative solid; dissolving pyrene derivative solid in dimethyl sulfoxide to prepare a storage solution of a pyrene derivative fluorescence sensor;
(2) Preparing a standard solution: taking water as a solvent, dissolving 3-nitropropionic acid solid in water to prepare mother liquor, respectively taking a certain amount of mother liquor, mixing and diluting to obtain a series of mixed standard solutions to be measured with different concentrations, namely 3-nitropropionic acid standard solutions;
(3) Drawing a standard curve: mixing the storage solution of the pyrene derivative fluorescence sensor obtained in the step (1) and the 3-nitropropionic acid standard solution obtained in the step (2) according to a certain proportion, standing for a certain time, performing fluorescence emission spectrum scanning within the absorption wavelength range of 375-700 nm, recording the spectrum, drawing a standard curve by using a fluorescence intensity value measured at 456nm, and calculating to obtain a linear regression equation; in the process of actually detecting the 3-nitropropionic acid, replacing the 3-nitropropionic acid standard solution with a sample to be detected, and respectively substituting fluorescence values measured at 456nm into the obtained linear regression equation according to the same operation to calculate the content of the 3-nitropropionic acid in the sample to be detected;
The fluorescence value of the fluorescence intensity at 456nm and the 3-nitropropionic acid standard solution with the concentration in the range of 0.5-10.0 mu mol/L are in good linear relation, and the obtained linear regression equation is as follows: f 456nm =1040.57+33.13c, correlation coefficient r=0.9930, where F 456nm is fluorescence intensity value at 456nm, and C is concentration of 3-nitropropionic acid;
the absorbance at 456nm of the 3-nitropropionic acid standard solution with the absorbance in the range of 10.0-100.0 mu mol/L shows a good linear relationship, and the obtained linear regression equation is as follows: f 456nm =1325.93+9.39c, correlation coefficient r=0.9904; the detection limit of the method is as follows: 0.32. Mu. Mol/L.
2. The fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products according to claim 1, wherein: the amount of 1-pyrene formaldehyde in the step (1) was 0.5756g (2.5 mmol), and the amount of tetrahydrofuran was 10mL.
3. The fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products according to claim 1, wherein: the volume percentage of the hydrazine hydrate in the step (1) is 25%, the dosage is 4.85mL, and the mass ratio relationship of the 1-pyrene formaldehyde and the hydrazine hydrate is 1:10; the reaction time of the 1-pyrene formaldehyde and the hydrazine hydrate is 12 hours.
4. The fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products according to claim 1, wherein: in the step (1), the mass ratio relationship of pyrene formaldehyde hydrazone and 4-tertiary butyl-2, 6-formylphenol is 1:1.1; the reaction time of pyrene formaldehyde hydrazone and 4-tert-butyl-2, 6-formylphenol was 24 hours.
5. The fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products according to claim 1, wherein: the eluent of the column chromatography in the step (1) is petroleum ether and ethyl acetate, and the volume ratio of the eluent to the ethyl acetate is 16:1, 12:1, 10:1,8:1,6:1 and 4:1 in sequence.
6. The fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products according to claim 1, wherein: in the step (1), a stock solution of a pyrene derivative fluorescence sensor having a concentration of 15. Mu. Mol/L was prepared.
7. The fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products according to claim 1, wherein: in the step (1), the volume ratio of the solvent dimethyl sulfoxide to water is 2:1.
8. The fluorescence detection method for detecting 3-nitropropionic acid in sugarcane and sugarcane products according to claim 1, wherein: the placing time in the step (3) is 30min.
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