CN115452816A

CN115452816A - Method for detecting nitrite by ratio absorbance colorimetric method

Info

Publication number: CN115452816A
Application number: CN202211138239.6A
Authority: CN
Inventors: 赵生国; 汪春娟; 王新潮; 马鑫; 姬良亮; 王波; 张泰恒; 祁光增; 侍金梅; 彭波
Original assignee: Lanzhou Customs Of People's Republic Of China
Current assignee: Lanzhou Customs Of People's Republic Of China
Priority date: 2022-09-19
Filing date: 2022-09-19
Publication date: 2022-12-09

Abstract

The invention discloses a method for detecting nitrite by a ratio absorbance colorimetric method, which utilizes MnO ₂ The oxidase-like activity of the nano-particles under acidic conditions is to oxidize colorless 3,3', 5' -tetramethylbenzidine into a blue TMB oxidation product TMBox, and when nitrite is added into the reaction solution, the blue TMBox is oxidized by NO ₂ ^‑ Further oxidized to form a yellow diimine derivative, from which NO is established ₂ ^‑ Concentration of (b) to absorbance ratio A of two oxidation products ₄₅₂ /A ₆₅₂ The linear relation of (2) to realize the quantitative detection of the nitrite in the food. The ratiometric absorbance method provides greater sensitivity and selectivity than single wavelength photometry, while also allowing visual detection by the naked eye. The invention has the advantages of simple instrument, low cost, good selectivity and accurate and reliable result.

Description

Method for detecting nitrite by ratio absorbance colorimetric method

Technical Field

The invention relates to a method for detecting nitrite, in particular to a method for detecting nitrite by a ratio absorbance colorimetric method, which can be used for detecting nitrite in food and belongs to the fields of analytical chemistry and food detection technology.

Background

Nitrite is one of the most widely used industrial salts, is often used as a preservative and colorant in food processing, is more common in meat processing (such as sausage, ham and the like), can keep good color and proper flavor of meat products, and is widely used in daily life. The overuse of nitrite is closely related to human life and public health, the appearance of the nitrite is similar to that of table salt, and nitrite poisoning is often caused by eating by mistake, suicide, poison input and the like. A large amount of nitrite is taken by a human body for a short time, which causes hemoglobinemia, cyanosis, headache, dizziness, accelerated heart rhythm, nausea, vomit, abdominal pain, diarrhea and other symptoms, and leads to anoxic death in severe cases. Therefore, it is very important to establish a simple, fast and accurate method for detecting the nitrite content in food.

At present, various analytical methods have been developed to determine nitrite in food, such as ion chromatography, spectrophotometry, liquid chromatography-mass spectrometry, fluorescence, surface Enhanced Raman Spectroscopy (SERS), chromatography, electrochemical methods, etc., which have the outstanding characteristics of simple operation, fast response speed, etc., and can detect various anions such as nitrite with high sensitivity, but the high salt concentration in the matrix solution can seriously interfere with the determination result, and pretreatment purification and separation techniques must be used. For chromatography-mass spectrometry, nitrite typically needs to be derivatized before it can be determined, and the proper selection of column packing, the use of which is time consuming and involves a complex and expensive process. Spectrophotometry is the most widely used method in nitrite monitoring due to its advantages of accuracy, simple operation, low cost and the like, and traditional spectrophotometry based on Griess analysis is widely used, and the methods comprise some heavy nitrogen coupling processes, use of organic reagents and solvents with high toxicity and cause the formation of toxic byproducts. Therefore, the development of a spectrophotometric method with high selectivity, high sensitivity and environmental friendliness for detecting nitrite is very significant.

Disclosure of Invention

The invention aims to provide a ratio absorbance colorimetric method for detecting nitrite, which has the advantages of high selectivity, high sensitivity and simple and convenient detection process. Under visible light, the visible detection of nitrite can be realized.

The invention relates to a ratio absorbance colorimetric method and a method for visually detecting nitrite, which comprises the following steps:

(1) MnO of ₂ Nanoparticles (MnO) ₂ NPs) and an ethanol solution of 3,3', 5' -Tetramethylbenzidine (TMB) are added to a HAc-NaAc buffer solution with pH = 4.0, mixed, reacted at 20 to 30 ℃ (preferably 25 ℃) for 5 to 20min (preferably 10 min) to produce a blue oxidation product TMBox, and then nitrite standard solutions with different concentrations are added to obtain a series of mixed solutions (the nitrite concentrations in the mixed solutions are respectively 0.15 μ g/mL,0.4 μ g/mL, 0.8 μ g/mL,1.6 μ g/m, 2 μ g/mL,4 μ g/mL,6 μ g/mL,8 μ g/mL,10 μ g/mL, and 12.5 μ g/mL), and the mixed solutions are incubated with heating at 20 to 40 ℃ and preferably 37 ℃) for 10 to 30 min (preferably 20min to obtain a yellow diimine derivative. According to different nitrite concentrations, the reaction solution shows gradual color gradient change from blue, yellow green and yellow, different colors correspond to different nitrite concentrations, and a qualitative or semi-quantitative analysis standard colorimetric card is obtained after the color of the solution with the corresponding concentration is photographed (as shown in figure 4).

(2) After the reaction is finished, simultaneously measuring the absorbance value A of the solution at two wavelengths of 652 nm and 452 nm by using an ultraviolet visible spectrophotometer ₆₅₂ And A ₄₅₂ (ii) a Establishment of A ₄₅₂ /A ₆₅₂ The relation curve of the absorbance ratio and the nitrite concentration is the working curve (as shown in the figure) for detecting the nitrite4）；

The ratio A of absorbance when the concentration of nitrite solution is in the range of 0.15-12.5 mu g/mL ₄₅₂ /A ₆₅₂ The linear relation with the nitrite concentration, the linear equation of the working curve is:

I = 0.4775 + 0.0812 C

wherein: i represents the ratio A of absorbance at two wavelengths, 652 nm and 452 nm ₄₅₂ /A ₆₅₂ And C represents the concentration of nitrite in μ g/mL.

MnO ₂ NPs nano particles are used as a nano enzyme oxidant, and two oxidation products TMBox and diimine derivatives of TMB are used as a bicolor colorimetric sensor. TMB is subjected to two oxidation processes, mnO ₂ NPs nanoenzymes first oxidize TMB to blue TMBox products, which are NO-substituted ₂ ^- The oxidation is continued to generate yellow diimine derivatives, the maximum absorption wavelength of the blue oxidation product TMBox is 652 nm, and the maximum absorption wavelength of the yellow diimine derivatives is 452 nm. Measuring by using an ultraviolet-visible spectrophotometer, and simultaneously measuring the absorbance value A of the solution at two wavelengths of 652 nm and 452 nm ₆₅₂ And A ₄₅₂ Calculating the ratio A of the two ₄₅₂ /A ₆₅₂ A linear equation is obtained.

(3) MnO of ₂ Adding the water solution of the nano-particles and the ethanol solution of 3,3', 5' -tetramethylbenzidine into HAc-NaAc buffer solution for mixing, adding a sample to be tested containing nitrite into the mixed system, heating and incubating the mixture for 10 to 30 min (preferably 20 min) at 20 to 40 ℃ (preferably 37 ℃), and measuring the absorbance values A at two wavelengths of 652 nm and 452 nm after the reaction is finished ₆₅₂ And A ₄₅₂ Then A is added ₄₅₂ /A ₆₅₂ Substituting the ratio into a linear equation of the working curve, and calculating to obtain the concentration of the nitrite in the sample to be detected.

And during qualitative or semi-quantitative analysis, comparing the reacted reaction solution with the qualitative or semi-quantitative analysis standard colorimetric card, and then qualitatively or estimating the content of nitrite in the solution to be detected.

In the steps (1) and (3), the MnO ₂ The concentration of the nano-particles is 0.2 to 1.0 mg/mL; the 3,3', 5' -tetramethylmethaneThe concentration of the biphenylanilide is 0.1 to 0.5 mM.

The MnO ₂ The preparation method of the NPs comprises the following steps: mixing MnCl ₂ Sequentially adding a bovine serum albumin solution into NaOH, stirring at room temperature to obtain a brown product, and dialyzing by using a dialysis membrane to obtain the product; the MnCl ₂ The concentration of the sodium hydroxide is 0.8 to 1.2 mol/L, the concentration of NaOH is 0.8 to 1.2 mol/L, and the concentration of bovine serum albumin is 15 to 25 mg/mL; stirring for 1 to 5 hours at room temperature; the dialysis membrane has a molecular weight of MW = 3.5 kDa and a dialysis time of 10 to 15 h.

The present invention performs a selective test of the assay method. When in detection, other common anions and cations and other common food additives with different concentrations are added, and the common anions and cations and other common food additives are measured to 0.42 mug/mL NO ₂ ^- The measured interference was used to determine the maximum interference factor for each ion, and the results are shown in Table 1. The larger the maximum interference multiple is, the more the coexisting ion pair detects NO ₂ ^- The smaller the effect of (c). The system can be demonstrated for NO by combining the ions determined in Table 1 ₂ ^- The detection selectivity is very good, and the anti-interference capability is very strong.

。

The invention uses MnO ₂ Nanoparticles (MnO) ₂ NPs) oxidase-like activity under acidic conditions, colorless 3,3', 5' -Tetramethylbenzidine (TMB) is oxidized to a blue TMB oxidation product (TMBox, λ) _max = 652 nm) when nitrite is added to the reaction solution, blue TMBox is replaced with NO ₂ ^- Further oxidation to give a yellow diimine derivative (. Lamda.) _max = 452 nm). Thereby establishing the absorbance ratio and NO of the two oxidation products ₂ ^- The concentration linear relation of the nitrite in the food can be realized.

The invention has the beneficial effects that:

compared with the traditional single-wavelength colorimetric method, the method for detecting the nitrite has the advantages of high selectivity, high sensitivity, accurate and reliable realization of quantitative detection of the nitrite, realization of visual detection, and rapidness and convenience in detection. The invention has simple instrument, low cost and accurate and reliable result, and provides a reliable method for detecting nitrite in food.

Drawings

FIG. 1 shows MnO prepared in example 1 of the present invention ₂ Scanning Electron Microscopy (SEM) images of NPs.

FIG. 2 shows MnO prepared in example 1 of the present invention ₂ Fourier infrared spectrogram of NPs.

FIG. 3 shows MnO prepared in example 1 of the present invention ₂ X-ray diffraction patterns of NPs.

FIG. 4 is a graph of the operation of the sample measurement of the various embodiments of the present invention.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

EXAMPLE 1 preparation of MnO ₂ NPs nanoenzymes

480 mu.L of MnCl ₂ (1 mol/L) and 1200. Mu.L NaOH (1 mol/L) were added sequentially to 20 mL BSA (20 mg/mL) solution, stirred at room temperature for 3 h to give a brown product, which was dialyzed for 12h using a dialysis membrane (MW = 3.5 kDa) against a dialyzing solution observed by SEM (see FIG. 1), and MnO was prepared ₂ The NPs have a crystal structure, uniform size and good dispersibility. As shown in FIG. 2, fourier infrared spectrum at 3313 cm ^-1 And 2945 cm ^-1 The bands of (A) are due to-OH and-NH on BSA ₂ Vibration in extension and contraction and is located at 1650 cm ^-1 And 1526 cm ^-1 The other two bands of (a) are due to-COOH and N-H bending vibrations. For MnO ₂ NPs at 523 cm ^-1 The characteristic peak of Mn-O is shown, and MnO is preliminarily proved ₂ The successful synthesis of NPs. Characteristic diffraction peaks at 2 θ = 18.2 °, 27.8 ° and 41.9 °, as shown in the X-ray diffraction pattern of fig. 3, with MnO ₂ The respective crystal planes (001) and (002) are well matched.

Example 2

In this example, the nitrite content of a canned luncheon meat was determined according to the method of the invention:

(1) Accurately weighing 5.0 g of luncheon meat, homogenizing with a mashing machine, pouring into a 100 mL beaker, adding 30 mL of water, ultrasonically leaching for 30 min, heating in a 75 ℃ water bath for 5 min, taking out, standing and cooling to room temperature. 15 mL of ethanol was added to the above solution and vortexed for 2 min, and finally the solution was transferred to a 50 mL volumetric flask and diluted to the mark with water. Standing for 30 min, centrifuging 10 mL of supernatant in a 15 mL centrifuge tube for 5 min (6000 rpm), wherein the supernatant is the solution to be detected for measuring the nitrite content.

(2) Pipetting 1 mL of the above test solution into a 5 mL centrifuge tube, and adding 500. Mu.L of HAc-NaAc buffer solution with pH = 4.0, 300. Mu.L of TMB (0.4 mM) in ethanol, and 150. Mu.L of MnO in that order ₂ An aqueous solution of NPs (0.5 mg/mL) was made up to 2 mL with ultrapure water. The mixture was left at room temperature for 10min, then reacted in a thermostatic water bath at 37 ℃ for 20min, and the absorbance values at the wavelengths of 452 nm and 652 nm were measured with an ultraviolet-visible spectrophotometer, respectively.

（3）NO ₂ ^- Calculation of ion concentration: the obtained absorbance ratio A is measured ₄₅₂ /A ₆₅₂ Substituting the linear equation I = 0.4775 + 0.0812C of the working curve to calculate NO in the liquid to be detected ₂ ^- The concentration of the ions. The results are shown in Table 2, and NO in canned luncheon meat samples ₂ ^- The content is 26.78 mg/Kg. If only qualitative or semi-quantitative analysis is needed, the content of the nitrite in the liquid to be detected can be qualitatively or estimated by comparing with the qualitative or semi-quantitative analysis standard colorimetric card.

Example 3

In this example, the content of nitrite in the chicken sausage was determined according to the method of the invention:

(1) Accurately weighing 5.0 g of chicken sausage, homogenizing with a pounder, pouring into a 100 mL beaker, adding 30 mL of water, ultrasonically leaching for 30 min, heating in a 75 ℃ water bath for 5 min, taking out, standing and cooling to room temperature. 15 mL of ethanol was added to the above solution and vortexed for 2 min, and finally the solution was transferred to a 50 mL volumetric flask and diluted to the mark with water. Standing for 30 min, centrifuging 10 mL of the supernatant in a 15 mL centrifuge tube for 5 min (6000 rpm), wherein the supernatant obtained by centrifugation is the solution to be detected for measuring the nitrite content.

(2) Pipetting 1 mL of the above test solution into a 5 mL centrifuge tube, and adding 500. Mu.L of HAc-NaAc buffer solution with pH = 4.0, 300. Mu.L of TMB (0.4 mM) in ethanol, and 150. Mu.L of MnO in that order ₂ An aqueous solution of NPs (0.5 mg/mL) was made up to 2 mL with ultrapure water. The mixture was left at room temperature for 10min, then reacted in a thermostatic water bath at 37 ℃ for 20min, and absorbance values at wavelengths of 452 nm and 652 nm were measured with an ultraviolet-visible spectrophotometer, respectively.

（3）NO ₂ ^- Calculation of ion concentration: the obtained absorbance ratio A is measured ₄₅₂ /A ₆₅₂ Substituting the standard curve into a linear equation I = 0.4775 + 0.0812C to calculate to obtain NO in the liquid to be detected ₂ ^- The concentration of the ions. The results are shown in Table 2, NO in Chicken sausage ₂ ^- The content was 21.35 mg/Kg. If only qualitative or semi-quantitative analysis is needed, the content of the nitrite in the liquid to be detected can be qualitatively or estimated by comparing with the qualitative or semi-quantitative analysis standard colorimetric card.

Example 4

In this example, the nitrite content in cantonese sausage was determined according to the method of the invention:

(1) Accurately weighing 5.0 g of sausage, homogenizing with a pounder, pouring into a 100 mL beaker, adding 30 mL of water, ultrasonically leaching for 30 min, heating in a 75 ℃ water bath for 5 min, taking out, standing and cooling to room temperature. 15 mL of ethanol was added to the above solution and vortexed for 2 min, and finally the solution was transferred to a 50 mL volumetric flask and diluted to the mark with water. Standing for 30 min, centrifuging 10 mL of the supernatant in a 15 mL centrifuge tube for 5 min (6000 rpm), wherein the supernatant obtained by centrifugation is the solution to be detected for measuring the nitrite content.

(2) Pipetting 1 mL of the above test solution into a 5 mL centrifuge tube, and adding 500. Mu.L of HAc-NaAc buffer solution with pH = 4.0, 300. Mu.L of TMB (0.4 mM) in ethanol, and 150. Mu.L of MnO in that order ₂ An aqueous solution of NPs (0.5 mg/mL) was made up to 2 mL with ultrapure water. Standing the mixture at room temperature for 10min, reacting in 37 deg.C constant temperature water bath for 20min, and measuring absorbance at wavelength of 452 nm and 652 nm with ultraviolet-visible spectrophotometerThe value is obtained.

（3）NO ₂ ^- Calculation of ion concentration: the obtained absorbance ratio A is measured ₄₅₂ /A ₆₅₂ Substituting the standard curve into a linear equation I = 0.4775 + 0.0812C of the standard curve, and calculating to obtain NO in the liquid to be detected ₂ ^- The concentration of the ions. The results are shown in Table 2, NO in Guangdong style sausage ₂ ^- The content is 15.24 mg/Kg.

Example 5

In this example, the nitrite content of mustard tuber was determined according to the method of the invention:

(1) Accurately weighing 5.0 g of preserved szechuan pickle, homogenizing with a pounder, pouring into a 100 mL beaker, adding 30 mL of water, ultrasonically leaching for 30 min, heating in a 75 ℃ water bath for 5 min, taking out, standing and cooling to room temperature. 2.0 g of activated carbon was added to the above solution for decolorization, and finally the solution was transferred to a 50 mL volumetric flask and diluted to the mark with water. Standing for 30 min, centrifuging 10 mL of the supernatant in a 15 mL centrifuge tube for 5 min (6000 rpm), wherein the supernatant obtained by centrifugation is the solution to be detected for measuring the nitrite content.

(2) Pipette 1 mL of the above test solution into a 5 mL centrifuge tube, and add 500. Mu.L of HAc-NaAc buffer solution with pH = 4.0, 300. Mu.L of TMB (0.4 mM) in ethanol, and 150. Mu.L of MnO ₂ An aqueous solution of NPs (0.5 mg/mL) was made up to 2 mL with ultrapure water. The mixture was left at room temperature for 10min, then reacted in a thermostatic water bath at 37 ℃ for 20min, and absorbance values at wavelengths of 452 nm and 652 nm were measured with an ultraviolet-visible spectrophotometer, respectively.

（3）NO ₂ ^- Calculation of ion concentration: the obtained absorbance ratio A is measured ₄₅₂ /A ₆₅₂ Substituting the standard curve into a linear equation I = 0.4775 + 0.0812C of the standard curve, and calculating to obtain NO in the liquid to be detected ₂ ^- The concentration of the ions. The results are shown in Table 2, NO in Hot pickled mustard tuber ₂ ^- The content is 8.21 mg/Kg.

Example 6

In this example, the nitrite content of the hot and spicy sauce chips was measured as follows:

(1) Accurately weighing 5.0 g of spicy sauce slices, homogenizing with a mashing machine, pouring into a 100 mL beaker, adding 30 mL of water, ultrasonically leaching for 30 min, heating in 75 ℃ water bath for 5 min, taking out, standing and cooling to room temperature. 2.0 g of activated carbon was added to the above solution for decolorization, and finally the solution was transferred to a 50 mL volumetric flask and diluted to the mark with water. Standing for 30 min, centrifuging 10 mL of the supernatant in a 15 mL centrifuge tube for 5 min (6000 rpm), wherein the supernatant obtained by centrifugation is the solution to be detected for measuring the nitrite content.

（3）NO ₂ ^- Calculation of ion concentration: the obtained absorbance ratio A is measured ₄₅₂ /A ₆₅₂ Substituting the standard curve into a linear equation I = 0.4775 + 0.0812C of the standard curve, and calculating to obtain NO in the liquid to be detected ₂ ^- The concentration of the ions. The results are shown in Table 2, NO in the spicy sauce tablets ₂ ^- The content was 6.74 mg/Kg.

^a Mean ± standard deviation of the sample measured 3 times

Example 7

The detection results of the method for lunch meat, chicken sausage, cantonese sausage and preserved szechuan pickle are compared with the detection results of the national standard spectrophotometry (GB 5009.33-2016). At a 95% confidence level, the method of the invention is not statistically significantly different from GB5009.33-2016 spectrophotometry, as compared to the t-test method. The method is proved to have higher accuracy and reliability, and is suitable for the quantitative analysis of the nitrite in the actual sample. The results are shown in Table 3.

^a Mean. + -. Standard deviation of 4 sample determinations

^b At the 95% confidence level, a degree of freedom of 3 corresponds to a t value of 3.18.

Claims

1. A method for detecting nitrite by a ratio absorbance colorimetry method comprises the following steps:

(1) MnO of ₂ Adding an aqueous solution of nanoparticles and an ethanol solution of 3,3', 5' -tetramethylbenzidine into a HAc-NaAc buffer solution with the pH = 4.0, mixing, reacting at 20-30 ℃ for 5-20min, then respectively adding nitrite standard solutions with different concentrations to obtain a series of mixed solutions, and heating and incubating at 20-40 ℃ for 10-30 min; according to different nitrite concentrations, the reaction solution shows gradual color gradient change from blue, yellow green and yellow, and a qualitative or semi-quantitative analysis standard colorimetric card is obtained after the color of the solution with corresponding concentration is photographed;

(2) After the reaction is finished, simultaneously measuring the absorbance value A of the solution at two wavelengths of 652 nm and 452 nm by using an ultraviolet visible spectrophotometer ₆₅₂ And A ₄₅₂ (ii) a Establishment of A ₄₅₂ /A ₆₅₂ The relation curve of the absorbance ratio and the nitrite concentration is a working curve for detecting the nitrite;

when the concentration of nitrite solution is in the range of 0.15-12.5 mu g/mL, the ratio of absorbance A ₄₅₂ /A ₆₅₂ The linear relation with the nitrite concentration, the linear equation of the working curve is:

I = 0.4775 + 0.0812 C

wherein: i represents the ratio A of absorbance at two wavelengths, 652 nm and 452 nm ₄₅₂ /A ₆₅₂ C represents the concentration of nitrite in units of μ g/mL;

(3) MnO of ₂ Adding the water solution of the nano-particles and the ethanol solution of 3,3', 5' -tetramethylbenzidine into HAc-NaAc buffer solution with pH = 4.0, mixing, adding a sample to be tested containing nitrite into a mixing system, heating and incubating the mixture at 20-40 ℃ for 10-30 min, and testing after the reaction is finishedAbsorbance values A at both wavelengths 652 nm and 452 nm ₆₅₂ And A ₄₅₂ Then A is added ₄₅₂ /A ₆₅₂ Substituting the ratio into a linear equation of the working curve, and calculating to obtain the concentration of the nitrite in the sample to be detected; and during qualitative or semi-quantitative analysis, comparing the reacted reaction solution with the qualitative or semi-quantitative analysis standard colorimetric card, and then qualitatively or estimating the content of nitrite in the solution to be detected.

2. The method of colorimetric absorbance ratio detection of nitrite according to claim 1 wherein: in the steps (1) and (3), the MnO is ₂ The concentration of the nano-particles is 0.2 to 1.0 mg/mL; the concentration of the 3,3', 5' -tetramethylbenzidine is 0.1 to 0.5 mM.

3. The method of colorimetric absorbance ratio detection of nitrite according to claim 1 wherein: in the step (1), the concentration range of the nitrite in the mixed solution is 0.15 to 12.5 mu g/mL.

4. The method of colorimetric absorbance ratio detection of nitrite according to claim 1 wherein: in the steps (1) and (3), the MnO is ₂ The preparation method of the NPs comprises the following steps: mixing MnCl ₂ Sequentially adding a bovine serum albumin solution into NaOH, stirring at room temperature to obtain a brown product, and dialyzing by using a dialysis membrane to obtain the sodium hydroxide solution; the MnCl ₂ The concentration of (A) is 0.8 to 1.2 mol/L, the concentration of NaOH is 0.8 to 1.2 mol/L, and the concentration of bovine serum albumin is 15 to 25 mg/mL; stirring for 1 to 5 hours at room temperature; the dialysis membrane has a molecular weight MW = 3.5 kDa and a dialysis time of 10 to 15 h.