CN117229159A - Nitrite detection method - Google Patents
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- CN117229159A CN117229159A CN202311197463.7A CN202311197463A CN117229159A CN 117229159 A CN117229159 A CN 117229159A CN 202311197463 A CN202311197463 A CN 202311197463A CN 117229159 A CN117229159 A CN 117229159A
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- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 title claims abstract description 105
- 238000001514 detection method Methods 0.000 title claims abstract description 74
- -1 3,3 '-dimethoxy-5, 5' -dimethylbenzidine Chemical compound 0.000 claims abstract description 68
- 238000011161 development Methods 0.000 claims abstract description 59
- 238000000034 method Methods 0.000 claims abstract description 37
- 238000002835 absorbance Methods 0.000 claims abstract description 30
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- 238000002156 mixing Methods 0.000 claims description 36
- CBMPTFJVXNIWHP-UHFFFAOYSA-L disodium;hydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].OP([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O CBMPTFJVXNIWHP-UHFFFAOYSA-L 0.000 claims description 24
- 239000007853 buffer solution Substances 0.000 claims description 17
- 239000000758 substrate Substances 0.000 claims description 9
- 239000011259 mixed solution Substances 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 4
- 239000003593 chromogenic compound Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 abstract description 21
- 235000013305 food Nutrition 0.000 abstract description 19
- 238000004445 quantitative analysis Methods 0.000 abstract description 7
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- 230000002378 acidificating effect Effects 0.000 abstract description 5
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- 238000004458 analytical method Methods 0.000 abstract description 3
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- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 32
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 28
- 239000002904 solvent Substances 0.000 description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 20
- 235000010288 sodium nitrite Nutrition 0.000 description 16
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- 150000004795 grignard reagents Chemical class 0.000 description 6
- WDFQBORIUYODSI-UHFFFAOYSA-N 4-bromoaniline Chemical compound NC1=CC=C(Br)C=C1 WDFQBORIUYODSI-UHFFFAOYSA-N 0.000 description 5
- 238000010561 standard procedure Methods 0.000 description 5
- HKOJYPPTIPJZAZ-UHFFFAOYSA-N 2-methoxy-6-methylaniline Chemical compound COC1=CC=CC(C)=C1N HKOJYPPTIPJZAZ-UHFFFAOYSA-N 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000012452 mother liquor Substances 0.000 description 4
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- ZOIORXHNWRGPMV-UHFFFAOYSA-N acetic acid;zinc Chemical compound [Zn].CC(O)=O.CC(O)=O ZOIORXHNWRGPMV-UHFFFAOYSA-N 0.000 description 3
- 238000004737 colorimetric analysis Methods 0.000 description 3
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- 239000000276 potassium ferrocyanide Substances 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 description 3
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- HVBSAKJJOYLTQU-UHFFFAOYSA-N 4-aminobenzenesulfonic acid Chemical compound NC1=CC=C(S(O)(=O)=O)C=C1 HVBSAKJJOYLTQU-UHFFFAOYSA-N 0.000 description 2
- 102000001554 Hemoglobins Human genes 0.000 description 2
- 108010054147 Hemoglobins Proteins 0.000 description 2
- 238000006161 Suzuki-Miyaura coupling reaction Methods 0.000 description 2
- IPWKHHSGDUIRAH-UHFFFAOYSA-N bis(pinacolato)diboron Chemical compound O1C(C)(C)C(C)(C)OB1B1OC(C)(C)C(C)(C)O1 IPWKHHSGDUIRAH-UHFFFAOYSA-N 0.000 description 2
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- HQBJSEKQNRSDAZ-UHFFFAOYSA-N 2,6-dimethoxyaniline Chemical compound COC1=CC=CC(OC)=C1N HQBJSEKQNRSDAZ-UHFFFAOYSA-N 0.000 description 1
- 206010007269 Carcinogenicity Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
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- LECSHJWIACEDPZ-UHFFFAOYSA-N ethane-1,2-diamine naphthalene hydrochloride Chemical compound C(CN)N.C1=CC=CC2=CC=CC=C12.Cl LECSHJWIACEDPZ-UHFFFAOYSA-N 0.000 description 1
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- XKLJHFLUAHKGGU-UHFFFAOYSA-N nitrous amide Chemical compound ON=N XKLJHFLUAHKGGU-UHFFFAOYSA-N 0.000 description 1
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- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention relates to a nitrite detection method, and belongs to the technical field of detection and analysis. The method is characterized in that under the acidic condition, 3 '-dimethoxy-5, 5' -dimethylbenzidine reacts with nitrite to change the color of the solution, qualitative analysis can be carried out through naked eyes, or semi-quantitative analysis can be carried out by comparing the reaction solution with a nitrite concentration color chart, or quantitative analysis can be carried out by measuring the maximum absorbance of the reaction solution through a spectrophotometer. The chromogenic molecule used in the method is 3,3 '-dimethoxy-5, 5' -dimethylbenzidine, is TMB derivative, reduces potential toxicity and carcinogenic mutation risk in the detection process, has high specificity and sensitivity to nitrite, has stable color development, and can meet the detection requirement of a large-scale sample. In addition, the method is simple to operate, and the used reagent is safe, easy to transport and store and suitable for home detection of food in daily life.
Description
Technical Field
The invention belongs to the technical field of detection and analysis, and particularly relates to a detection method of nitrite.
Background
Nitrite is an industrial salt that is widely used as a preservative, an antibacterial agent, and a color fixative in food processing. Small amounts of nitrite intake do not affect human health, but exceeding intake (> 0.06 mg/kg/day) can cause two major hazards to humans: firstly, the combination of nitrite and hemoglobin can reduce the oxygen carrying capacity of hemoglobin in blood, thereby causing anoxic poisoning of human body; and secondly, excessive nitrite can react with amine substances in a human body to form nitrosamine with carcinogenicity, so that the risk of cancer is increased. Therefore, the accurate quantitative analysis of nitrite in food is a great guarantee for ensuring food safety and maintaining life health of people.
Heretofore, there have been various classical nitrite detection methods including chromatography, electrochemical methods, fluorescence analysis, capillary electrophoresis, and the like. However, the threshold for the above-described techniques is relatively high and requires a skilled technician to perform the analysis, and is therefore generally limited to a central laboratory. In recent years, the increasingly complex food supply chain greatly increases food safety risks and supervision difficulties, and conventional laboratory detection technology is also difficult to meet the monitoring requirements of the existing food. Therefore, new technologies that are easy to operate, rapid to analyze, and suitable for nitrite field detection will play an increasingly important role in food safety supervision.
Colorimetric method is one of the ideal signal reading technologies in the development of on-site detection technology, and nitrite colorimetric method based on Gris reagent is also determined as one of the national standard methods (GB 5009.33-2016) for nitrite detection, and is widely applied to the detection of nitrite in food. However, this technique uses toxic sulfanilic acid and naphthalene ethylenediamine hydrochloride during the detection process, and thus this method is potentially harmful to both the operator and the environment. Therefore, developing a relatively safe and stable chromogenic molecule, and constructing a new technique which is simple, convenient and quick and can realize colorimetric detection of nitrite in food is still of great significance to ensuring food safety.
Disclosure of Invention
In view of the above drawbacks, the present invention provides a novel nitrite detection method. Under the acidic condition, 3 '-dimethoxy-5, 5' -dimethyl benzidine reacts with nitrite to change the color of the solution, so that qualitative analysis can be carried out by naked eyes, or semi-quantitative analysis can be carried out by comparing the reaction solution with a nitrite concentration colorimetric card, or quantitative analysis can be carried out by measuring the maximum absorbance of the reaction solution by a spectrophotometer. The chromogenic molecule used in the method is a newly synthesized 3,3 '-dimethoxy 5,5' -dimethylbenzidine molecule, has high specificity and sensitivity to nitrite, has stable color development, and can meet the detection requirement of a large-scale sample. In addition, the method provided by the invention is simple to operate, and the used reagent is safe and easy to transport and store, so that the method is suitable for home detection of foods in daily life.
The technical scheme of the invention is as follows:
the first technical problem to be solved by the invention is to point out the use of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine in colorimetric detection of nitrite.
Further, the structural formula of the 3,3 '-dimethoxy-5, 5' -dimethylbenzidine is shown as formula I:
further, the 3,3 '-dimethoxy-5, 5' -dimethylbenzidine is used as a chromogenic substrate for nitrite detection.
The second technical problem to be solved by the invention is to provide a color development liquid for nitrite detection, wherein a substrate in the color development liquid is 3,3 '-dimethoxy-5, 5' -dimethylbenzidine.
Further, the pH range of the color development liquid is 2.0-4.0; preferably pH is 2.6-3.2; more preferably 2.8, 3.0, 3.2.
The third technical problem to be solved by the invention is to provide a qualitative detection method of nitrite, which comprises the following steps: firstly extracting nitrite in an object to be detected to obtain an extracting solution, then mixing the extracting solution with a developing solution, and standing until the color of the mixed solution changes to indicate that the object to be detected contains nitrite, thereby realizing qualitative analysis of the nitrite; wherein the substrate in the color development liquid is 3,3 '-dimethoxy-5, 5' -dimethylbenzidine.
Further, the color development liquid comprises 3,3 '-dimethoxy-5, 5' -dimethylbenzidine solution and disodium hydrogen phosphate-citric acid buffer solution, and the concentration of the 3,3 '-dimethoxy-5, 5' -dimethylbenzidine in the color development liquid is 0.4-0.7 mM.
Further, the pH range of the color development liquid is 2.0-4.0; preferably pH is 2.6-3.2; more preferably 2.8, 3.0, 3.2.
Further, the volume ratio of the extracting solution to the color developing solution is 1:4 to 1:6, preferably 1:4.
Further, the time for the standing is 10 to 90 minutes, preferably 15 to 30 minutes.
Further, the method for extracting nitrite in the to-be-detected substance is a general method in the prior art.
The fourth technical problem to be solved by the invention is to provide a semi-quantitative detection method of nitrite, which comprises the following steps: firstly extracting nitrite in an object to be detected to obtain an extracting solution, then mixing the extracting solution with a developing solution, standing, and then comparing a developing result with a corresponding numerical value on a nitrite concentration color chart to semi-quantitatively analyze the nitrite; wherein the substrate in the color development liquid is 3,3 '-dimethoxy-5, 5' -dimethylbenzidine.
Further, the color development liquid consists of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine solution and disodium hydrogen phosphate-citric acid buffer solution, wherein the concentration of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine in the color development liquid is 0.4-0.7 mM, preferably 0.5mM.
Further, the pH of the color-developing solution is in the range of 2.6 to 3.2, preferably 3.0.
Further, the volume ratio of the extracting solution to the color developing solution is 1:4 to 1:6, preferably 1:4.
Further, the time for the standing is 15 to 30 minutes, preferably 15 minutes.
Further, the method for extracting nitrite in the to-be-detected substance is a general method in the prior art; the nitrite concentration color chart is a common color chart in the prior art.
The fifth technical problem to be solved by the invention is to provide a quantitative detection method of nitrite, which comprises the following steps: firstly extracting nitrite in an object to be detected to obtain an extracting solution, then mixing the extracting solution with a developing solution, standing to develop the color of the mixed solution, and then calculating the content of the nitrite in the object to be detected according to the following formula:
wherein X is the nitrite content (in NaNO) 2 In mg/kg), V refers to the total volume of the extract (mL), c refers to the concentration of nitrite in the mixture (μM), V 1 Refers to the volume (mL), V of the extracted solution mixed with the color development solution 2 Refers to the volume (mL) of the color development liquid taken, and m refers to the mass (g) of the test object.
Further, the concentration of nitrite in the mixed solution is calculated by the following formula: a=0.0146c+0.0104, wherein a is the maximum absorbance of the mixed solution, as measured by a spectrophotometer.
Further, the color development liquid consists of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine solution and disodium hydrogen phosphate-citric acid buffer solution, wherein the concentration of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine in the color development liquid is 0.4-0.7 mM, preferably 0.5mM.
Further, the pH of the color-developing solution is 2.6 to 3.2, preferably 3.0;
further, the volume ratio of the extracting solution to the color developing solution is 1:4-1:6, preferably 1:4;
further, the time of the standing is 15 to 30 minutes, preferably 15 minutes;
further, the method for extracting nitrite in the to-be-detected substance is a general method in the prior art.
The sixth technical problem to be solved by the invention is to provide a nitrite detection kit, which comprises a color development liquid and a colorimetric tube, wherein a substrate in the color development liquid is 3,3 '-dimethoxy-5, 5' -dimethylbenzidine.
The invention has the beneficial effects that:
1. the 3,3 '-dimethoxy-5, 5' -dimethylbenzidine used in the nitrite detection method is a TMB derivative molecule which is newly synthesized, and potential toxicity and carcinogenic mutation risks in the detection process are reduced.
2. The 3,3 '-dimethoxy-5, 5' -dimethylbenzidine used in the method of the present invention has high specificity and sensitivity to nitrite under acidic conditions, can detect nitrite as low as 0.2 mu M (detection limit is 0.2 mu M) by using a spectrophotometer, quantitates nitrite as low as 0.5 mu M (quantitates limit is 0.5 mu M), and has stable color development, and can be subjected to qualitative or semi-quantitative analysis by naked eyes.
3. The method provided by the invention does not need the assistance of large-scale instruments and equipment, does not need excessive supporting conditions, is simple to operate, and the used reagent is easy to transport and store, so that the method is suitable for home detection of foods in daily life.
Drawings
FIG. 1 is a scheme showing the synthesis of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine in example 1.
FIG. 2 shows the result of detection of 50. Mu.M sodium nitrite by 0.5mM 3,3 '-dimethoxy-5, 5' -dimethylbenzidine in example 1.
FIG. 3 shows the stability test results of the nitrite detection color in example 2.
FIG. 4 shows the color development of 0.25mM 3,3 '-dimethoxy-5, 5' -dimethylbenzidine in example 3 for detection of 25. Mu.M sodium nitrite at different pH conditions.
FIG. 5 shows the color development of 25. Mu.M sodium nitrite measured on 3,3 '-dimethoxy-5, 5' -dimethylbenzidine of example 4 at various concentrations.
FIG. 6 shows the color development results of the method of the invention in example 5 for detecting different ions.
FIG. 7 shows the color development results of the method of the present invention in example 6 when nitrite ions are detected in the presence of other ions.
FIG. 8 is a schematic diagram of a detection flow of nitrite in a food sample according to an embodiment of the present invention.
FIG. 9 shows the color development results of detection of nitrite by 3,3', 5' -Tetramethylbenzidine (TMB) in comparative example 1.
FIG. 10 is a synthetic scheme of 3,3', 5' -tetramethoxybenzidine of comparative example 2.
FIG. 11 shows the color development results of the detection of nitrite by 3,3', 5' -tetramethoxybenzidine in comparative example 2.
Detailed Description
The detection method of nitrite disclosed by the invention comprises the following steps: under the acidic condition, 3 '-dimethoxy-5, 5' -dimethyl benzidine reacts with nitrite to change the color of the solution, so that qualitative analysis can be carried out by naked eyes, or semi-quantitative analysis can be carried out by comparing the reaction solution with a nitrite concentration color chart, or quantitative analysis can be carried out by measuring the maximum absorbance of the reaction solution by a spectrophotometer. The chromogenic molecule used in the method is a newly synthesized 3,3 '-dimethoxy-5, 5' -dimethylbenzidine molecule, has high specificity and sensitivity to nitrite, has stable color development, and can meet the detection requirement of a large-scale sample. In addition, the method provided by the invention is simple to operate, and the used reagent is safe and easy to transport and store, so that the method is suitable for home detection of foods in daily life.
The invention is further illustrated by the following examples. It should be noted that the examples given should not be construed as limiting the scope of the present invention, but rather as merely providing for the benefit of this disclosure.
Noteworthy are: 1) The 3,3 '-dimethoxy-5, 5' -dimethylbenzidine of example 1 and the 3,3', 5' -tetramethoxybenzidine of comparative example 2 are two TMB derivative molecules synthesized by the applicant of the present invention; 2) The detection kit in the embodiment is a nitrite detection kit prepared according to the method of the invention; 3) Comparative examples 3 to 32 were tested by the second method (Gris reagent colorimetry) of the nitrite detection national standard method (GB 5009.33-2016), and the test results were compared with those of examples 8 to 37; 4) Examples 8-37 and comparative examples 3-32 judge whether the measured nitrite content exceeds the standard according to the national limit standards for nitrite in food (GB 2762-2022, GB 2760-2014).
Example 1
(1) Preparation of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine: as shown in figure 1, firstly, 2-methoxy-6-methylaniline (1) is substituted to generate p-bromoaniline (2), 2 is esterified by bis (pinacolato) diboron, and then the esterification product (3) and the bromination product (2) are subjected to Suzuki-Miyaura reaction to generate 3,3 '-dimethoxy-5, 5' -dimethylbenzidine. The 3,3 '-dimethoxy-5, 5' -dimethylbenzidine in the following examples were prepared by this method.
(2) 3,3 '-dimethoxy-5, 5' -dimethylbenzidine for detecting nitrite: sodium nitrite solution (5 mM, pure water as solvent), 3 '-dimethoxy-5, 5' -dimethylbenzidine solution (50 mM, DMSO as solvent), disodium hydrogen phosphate-citric acid buffer (pH=2.5, pure water as solvent) were prepared separately. One cuvette was taken and sequentially added with 1960. Mu.L of disodium hydrogen phosphate-citric acid buffer solution and 20. Mu.L of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine solution, and after mixing, 20. Mu.L of sodium nitrite solution was added, mixing was started, and the absorbance of the reaction solution at 454nm was measured by a spectrophotometer at 1, 3, 5, 10, 20, 30, 45, 60 minutes, respectively, and the result is shown in FIG. 2.
Example 2
The nitrite detection color development liquid is prepared by the following method: 0.5mL of 50mM 3,3 '-dimethoxy-5, 5' -dimethylbenzidine solution is prepared by using DMSO as a solvent, 49.5mL of disodium hydrogen phosphate-citric acid buffer solution with pH of 2.8 is prepared by using pure water as a solvent, and then 50mL of colorless developing solution is obtained by uniformly mixing the two solutions, wherein the concentration of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine in the obtained developing solution is 0.5mM. The prepared developing solution is protected from light and stored at low temperature, the developing solution is taken out every other day for stability test, a colorimetric tube is taken, 1980 mu L of developing solution and 20 mu L of sodium nitrite national standard substance solution (200 mu g/mL) for food detection are sequentially added, uniformly mixed, the absorbance of the reaction solution at 454nm is measured by a spectrophotometer after 15 minutes of reaction, and the result is shown in figure 3. As can be seen from FIG. 3, the obtained nitrite detection color liquid can be stably stored for one month.
Example 3
Sodium nitrite solution (5 mM, pure water as solvent), 3 '-dimethoxy-5, 5' -dimethylbenzidine solution (50 mM, DMSO as solvent) and disodium hydrogen phosphate-citric acid buffer (pure water as solvent) with pH gradient of 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6 were prepared. 8 colorimetric tubes were taken and added with 1980. Mu.L of the above pH gradient disodium hydrogen phosphate-citric acid buffer solution, respectively, 10. Mu.L of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine solution was added, followed by mixing, and then 10. Mu.L of sodium nitrite solution was added, followed by mixing, and time was kept for 15 minutes. After 15 minutes, the absorbance of the reaction solution at 454nm was measured by a spectrophotometer, and the result is shown in FIG. 4. As is clear from FIG. 4, the absorbance of the reaction solution was maximized at pH 3.0, and therefore pH 3.0 was selected as the optimal acidic condition for nitrite detection.
Example 4
Sodium nitrite solution (5 mM, pure water as solvent), 3 '-dimethoxy-5, 5' -dimethylbenzidine solution (50 mM, DMSO as solvent), disodium hydrogen phosphate-citric acid buffer (pH=3.0, pure water as solvent) were prepared separately. 8 cuvettes were added with 1990. Mu.L, 1986. Mu.L, 1982. Mu.L, 1978. Mu.L, 1974. Mu.L, 1970. Mu.L, 1966. Mu.L, 1962. Mu.L of disodium hydrogen phosphate-citric acid buffer, respectively, 0, 4. Mu.L, 8. Mu.L, 12. Mu.L, 16. Mu.L, 20. Mu.L, 24. Mu.L, 28. Mu.L of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine solution, and after mixing, 10. Mu.L of sodium nitrite solution was added, respectively, and mixing was continued for 15 minutes. After 15 minutes, the absorbance of the reaction solution at 454nm was measured by a spectrophotometer, and the result is shown in FIG. 5. As is clear from FIG. 5, as the concentration of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine increases, the absorbance of the reaction solution gradually increases, and when the concentration of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine reaches 0.5mM, the absorbance does not substantially increase any more, so that 0.5mM is selected as the optimal substrate concentration for detection of nitrite.
The final concentration c "(mM) of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine in the present invention is calculated by the following formula:
wherein c 'refers to the concentration (mM) of the 3,3' -dimethoxy-5, 5 '-dimethylbenzidine mother liquor, V' refers to the volume (mL) of the 3,3 '-dimethoxy-5, 5' -dimethylbenzidine mother liquor taken, and V "refers to the total volume (mL) of the solution. The 3,3' -dimethoxy-5, 5' -dimethylbenzidine mother liquor is prepared by using DMSO as a solvent, and the concentration c ' (mM) is calculated by the following formula:
wherein M 'refers to the mass (mg) of the 3,3' -dimethoxy-5, 5 '-dimethylbenzidine taken, M refers to the relative molecular mass (g/mol) of the 3,3' -dimethoxy-5, 5 '-dimethylbenzidine, and V' "refers to the total volume (mL) of the mother liquor.
Example 5
Ion selectivity. The selectivity of the process of the invention for nitrite ions was verified under optimal reaction conditions (reaction time 15 min, pH 3.0, 3 '-dimethoxy-5, 5' -dimethylbenzidine concentration 0.5 mM), and NO was detected 2 - With other 17 inorganic ions (Fe 3+ 、K + 、Ca 2+ 、Mg 2+ 、Na + 、Al 3+ 、Zn 2+ 、Cu 2+ 、Ni 2+ 、Mn 2+ 、NO 3 - 、SO 4 2- 、PO 4 3- 、I - 、F - 、Cl - 、Br - ) The comparison is included. Preparing the above ion solution (5 mM, pure water as solvent), 3 '-dimethoxy-5, 5' -dimethylbenzidine solution (50 mM, DMSO as solvent)Agent), disodium hydrogen phosphate-citric acid buffer solution (pH=3.0, pure water as solvent), 18 colorimetric tubes were taken and added with 1972 μl of disodium hydrogen phosphate-citric acid buffer solution and 20 μl of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine solution respectively, and after mixing, 8 μl of the above ion solution was added respectively, mixing was carried out, and time was counted for 15 minutes. After 15 minutes, the absorbance of the reaction solution at 454nm was measured by a spectrophotometer, and the result is shown in FIG. 6. From fig. 6, it can be seen that the presence of ions other than nitrite ions does not produce a significant change in the absorbance value of the solution. Thus, the process of the present invention is highly selective for nitrite and not responsive to other ions.
Example 6
Ion interference. The method of the invention was verified for its resistance to ionic interference under optimal reaction conditions (reaction time 15 min, pH 3.0, 3 '-dimethoxy-5, 5' -dimethylbenzidine concentration 0.5 mM), the above-mentioned inorganic ion (K) being selected + 、Ca 2+ 、Mg 2+ 、Na + 、Al 3+ 、Zn 2+ 、Cu 2+ 、Ni 2+ 、Mn 2+ 、NO 3 - 、SO 4 2- 、PO 4 3- 、I - 、F - 、Cl - 、Br - ) As interfering ions. Preparing the above ion solution (100 mM, pure water as solvent) and NaNO respectively 2 Solution (5 mM, pure water as solvent), 3 '-dimethoxy-5, 5' -dimethylbenzidine solution (50 mM, DMSO as solvent), disodium hydrogen phosphate-citric acid buffer solution (pH=3.0, pure water as solvent), taking a cuvette as blank group, sequentially adding 1972. Mu.L of disodium hydrogen phosphate-citric acid buffer solution, 8. Mu.L of LNaNO 2 Mixing the solution uniformly; then 16 colorimetric tubes are taken and added with 1952 mu L of disodium hydrogen phosphate-citric acid buffer solution and 8 mu LNaNO respectively 2 Mixing the solution and 20 μl of the 16 ion solutions, adding 20 μl of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine solution into 17 colorimetric tubes, mixing, and timing for 15 min. After 15 minutes, the absorbance of the reaction solution at 454nm was measured by a spectrophotometer, and the result is shown in FIG. 7. As is clear from FIG. 7, the coexistence of the remaining ions (1 mM) does not cause significant interference in detection of nitrite ions, and furthermore Fe 3+ (≤0.5 mM) can coexist with nitrite ions under EDTA masking without interfering with the assay. Therefore, the detection method has better anti-ion interference performance.
Example 7
Nitrite detection accuracy. 3,3 '-dimethoxy-5, 5' -dimethylbenzidine solution (50 mm, dmso as solvent), disodium hydrogen phosphate-citric acid buffer (ph=3.0, pure water as solvent) were prepared. One cuvette was taken and sequentially added with 1960. Mu.L of disodium hydrogen phosphate-citric acid buffer solution and 20. Mu.L of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine solution, and after mixing, 20. Mu.L of sodium nitrite national standard substance solution (200. Mu.g/mL) for food detection was added, and after mixing, the absorbance of the reaction solution at 454nm was measured by a spectrophotometer after 15 minutes of reaction, and the concentration of sodium nitrite therein was quantitatively analyzed, and the results are shown in Table 1. As can be seen from Table 1, the method of the present invention has higher accuracy in detecting nitrite.
Table 1 test results of the method of the invention in example 7 for detecting national Standard substance solutions
The operation flow of the method for qualitatively, semi-quantitatively and quantitatively detecting nitrite in food samples is shown in figure 8.
Examples 8 to 17
(1) Weighing 10 parts of crushed pickle samples, each 2g of the crushed pickle samples, soaking the 10 parts of pickle samples in clear water for 5 minutes, adding 0.5ml of 106g/L potassium ferrocyanide solution (sample extract I), uniformly mixing, adding 0.5ml of 220g/L zinc acetate solution (sample extract II), uniformly mixing, and standing for 10 minutes;
(2) Filtering to obtain filtrate, sucking 0.5mL of the filtrate by a suction pipe, adding the filtrate into a colorimetric tube, adding 2.0mL of color development liquid into the colorimetric tube, uniformly mixing, and standing for 15 minutes to develop the color of the colorless color development liquid; in this example and the examples described below, the concentration of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine in the color-developing solution was 0.5mM, and the pH of the color-developing solution was 2.8.
(3) After the completion of the color development, the absorbance at 454nm was measured by a spectrophotometerDegree, substituting the measured absorbance into formula a=0.0146c+0.0104, calculating the concentration c (μm) of nitrite, and reusingThe nitrite content (NaNO 2 The results are shown in Table 2, in mg/kg.
Examples 18 to 27
(1) Weighing 10 parts of crushed meat sausage samples, soaking the 10 parts of meat sausage samples in clear water for 5 minutes, adding 0.5ml of 106g/L potassium ferrocyanide solution (sample extract I), uniformly mixing, adding 0.5ml of 220g/L zinc acetate solution (sample extract II), uniformly mixing, and standing for 10 minutes;
(2) Filtering to obtain filtrate, sucking 0.5mL of the filtrate by a suction pipe, adding the filtrate into a colorimetric tube, adding 2.0mL of color development liquid into the colorimetric tube, uniformly mixing, and standing for 15 minutes to develop the color of the colorless color development liquid;
(3) After the completion of the color development, the absorbance at 454nm was measured by a spectrophotometer, and the measured absorbance was substituted into the formula a=0.0146c+0.0104 to calculate the nitrite concentration c (μm), which was then usedThe nitrite content (NaNO 2 The results are shown in Table 3, in mg/kg.
Examples 28 to 37
(1) Weighing 10 parts of crushed meat can samples, soaking the 10 parts of meat can samples in clear water for 5 minutes, adding 0.5ml of 106g/L potassium ferrocyanide solution (sample extract I), uniformly mixing, adding 0.5ml of 220g/L zinc acetate solution (sample extract II), uniformly mixing, and standing for 10 minutes;
(2) Filtering to obtain filtrate, sucking 0.5mL of the filtrate by a suction pipe, adding the filtrate into a colorimetric tube, adding 2.0mL of color development liquid into the colorimetric tube, uniformly mixing, and standing for 15 minutes to develop the color of the colorless color development liquid;
(3) After the completion of the color development, the absorbance at 454nm was measured by a spectrophotometer, and the measured absorbance was substituted into formula a=0.0146c+0.0104, the concentration of nitrite c (. Mu.M) is calculated and reusedThe nitrite content (NaNO 2 The results are shown in Table 4, in mg/kg.
Comparative example 1
The nitrite was detected using commercially available 3,3', 5' -Tetramethylbenzidine (TMB). Sodium nitrite solution (5 mM, pure water as solvent), TMB solution (50 mM, DMSO as solvent), disodium hydrogen phosphate-citric acid buffer (pH=2.5, pure water as solvent) were prepared separately. One cuvette was taken out, 1960. Mu.L of disodium hydrogen phosphate-citric acid buffer solution and 20. Mu.L of LTMB solution were sequentially added, and after mixing, 20. Mu.L of sodium nitrite solution was added, and the mixing was started, and the absorbance of the reaction solution at 448nm was measured by a spectrophotometer at 1, 3, 5, 10, 20, 30, 45, 60 minutes, respectively, as shown in FIG. 9.
As can be seen from a comparison of fig. 2 and 9: the absorbance of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine for detecting nitrite tends to be stable after 10 minutes, while the absorbance of 3,3', 5' -Tetramethylbenzidine (TMB) for detecting nitrite gradually decreases after 10 minutes, so that 3,3 '-dimethoxy-5, 5' -dimethylbenzidine is improved in color development stability for detecting nitrite as compared with TMB, and is advantageous for on-site detection of nitrite based on semi-quantitative analysis by naked eyes.
Comparative example 2
(1) Preparation of 3,3', 5' -tetramethoxybenzidine: as shown in FIG. 10, p-bromoaniline (5) is first produced by substitution reaction of 2, 6-dimethoxyaniline (4), and after 5 is esterified with bis (pinacolato) diboron, the esterification product (6) and the bromination product (5) undergo Suzuki-Miyaura reaction to produce 3,3', 5' -tetramethoxybenzidine. The 3,3', 5' -tetramethoxybenzidine in the comparative example described later was prepared by this method.
(2) 3,3', 5' -tetramethoxybenzidine detects nitrite: sodium nitrite solution (5 mM, pure water as solvent), 3', 5' -tetramethoxybenzidine solution (50 mM, DMSO as solvent) and disodium hydrogen phosphate-citric acid buffer (pH=2.5, pure water as solvent) were prepared separately. One cuvette was taken out, 1960. Mu.L of disodium hydrogen phosphate-citric acid buffer solution and 20. Mu.L of 3,3', 5' -tetramethoxybenzidine solution were sequentially added, and after mixing, 20. Mu.L of sodium nitrite solution was added, and mixing was started, and the absorbance of the reaction solution at 451nm was measured by a spectrophotometer at 1, 3, 5, 10, 20, 30, 45, 60 minutes, respectively, and the result is shown in FIG. 11.
As can be seen from a comparison of fig. 2 and 11: the absorbance of the 3,3', 5' -tetramethoxybenzidine for detecting nitrite is lower than that of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine, which is unfavorable for naked eyes to read, and the sensitivity of the 3,3', 5' -tetramethoxybenzidine for detecting nitrite is lower than that of the 3,3 '-dimethoxy-5, 5' -dimethylbenzidine.
Comparative examples 3 to 12
(1) Sucking 0.5mL of the filtrate obtained in examples 11-20 by a suction pipe respectively, adding the filtrate into a colorimetric tube filled with 2.0mL of Grignard reagent, uniformly mixing, and standing for 15 minutes to color the Grignard reagent;
(2) After the completion of the color development, absorbance at 545nm was measured by a spectrophotometer, and quantitative analysis was performed, and the results are shown in Table 2.
The results show that: the detection result of the invention is similar to the detection result of the national standard method, and the accuracy of the detection method of the invention is verified.
TABLE 2 detection results of Experimental examples 8-17 and comparative examples 3-12
Comparative examples 13 to 22
(1) Sucking 0.5mL of the filtrate obtained in examples 21-30 by a suction pipe respectively, adding the filtrate into a colorimetric tube filled with 2.0mL of Grignard reagent, uniformly mixing, and standing for 15 minutes to color the Grignard reagent;
(2) After the completion of the color development, absorbance at 545nm was measured by a spectrophotometer, and quantitative analysis was performed, and the results are shown in Table 3.
The results show that: the detection result of the invention is similar to the detection result of the national standard method, and the accuracy of the detection method of the invention is verified.
TABLE 3 detection results of Experimental examples 18-27 and comparative examples 13-22
Comparative examples 23 to 32
(1) Sucking 0.5mL of the filtrate obtained in examples 31-40 by a suction pipe respectively, adding the filtrate into a colorimetric tube containing 2.0mL of Grignard reagent, uniformly mixing, and standing for 15 minutes to color the Grignard reagent;
(2) After the completion of the color development, absorbance at 545nm was measured by a spectrophotometer, and quantitative analysis was performed, and the results are shown in Table 4.
The results show that: the detection result of the invention is similar to the detection result of the national standard method, and the accuracy of the detection method of the invention is verified.
TABLE 4 detection results of Experimental examples 28-37 and comparative examples 23-32
Claims (10)
1. The application of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine in colorimetric detection of nitrite is characterized in that the structural formula of the 3,3 '-dimethoxy-5, 5' -dimethylbenzidine is shown as formula I:
2. use of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine for colorimetric detection of nitrite according to claim 1, wherein the 3,3 '-dimethoxy-5, 5' -dimethylbenzidine is used as chromogenic substrate for detection of nitrite.
3. A color development liquid for nitrite detection is characterized in that,
the substrate in the color development liquid is 3,3 '-dimethoxy-5, 5' -dimethylbenzidine;
the pH range of the color development liquid is 2.0-4.0; preferably pH is 2.6-3.2; more preferably 2.8, 3.0, 3.2.
4. The qualitative detection method for nitrite is characterized by comprising the following steps: firstly extracting nitrite in an object to be detected to obtain an extracting solution, then mixing the extracting solution with a developing solution, and standing until the color of the mixed solution changes to indicate that the object to be detected contains nitrite, thereby realizing qualitative analysis of the nitrite; wherein the substrate in the color development liquid is 3,3 '-dimethoxy-5, 5' -dimethylbenzidine;
5. a qualitative detection method of nitrite according to claim 4, wherein,
the color development liquid comprises 3,3 '-dimethoxy-5, 5' -dimethylbenzidine solution and disodium hydrogen phosphate-citric acid buffer solution, wherein the concentration of the 3,3 '-dimethoxy-5, 5' -dimethylbenzidine in the color development liquid is 0.4-0.7 mM;
the pH range of the color development liquid is 2.0-4.0; preferably pH is 2.6-3.2; more preferably 2.8, 3.0, 3.2;
the volume ratio of the extracting solution to the developing solution is 1:4-1:6, preferably 1:4;
the time for the standing is 10 to 90 minutes, preferably 15 to 30 minutes.
6. The semi-quantitative detection method of nitrite is characterized by comprising the following steps: firstly extracting nitrite in an object to be detected to obtain an extracting solution, then mixing the extracting solution with a developing solution, standing, and then comparing a developing result with a corresponding numerical value on a nitrite concentration color chart to semi-quantitatively analyze the nitrite; wherein the substrate in the color development liquid is 3,3 '-dimethoxy-5, 5' -dimethylbenzidine.
7. A method for semi-quantitatively determining nitrite according to claim 6, wherein,
the color development liquid consists of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine solution and disodium hydrogen phosphate-citric acid buffer solution, wherein the concentration of the 3,3 '-dimethoxy-5, 5' -dimethylbenzidine in the color development liquid is 0.4-0.7 mM, preferably 0.5mM;
the pH range of the color development liquid is 2.6-3.2, preferably 3.0;
the volume ratio of the extracting solution to the developing solution is 1:4-1:6, preferably 1:4;
the time for the standing is 15 to 30 minutes, preferably 15 minutes.
8. The quantitative detection method of nitrite is characterized by comprising the following steps: firstly extracting nitrite in an object to be detected to obtain an extracting solution, then mixing the extracting solution with a developing solution, standing to develop the color of the mixed solution, and then calculating the content of the nitrite in the object to be detected according to the following formula:
wherein X is the nitrite content (in NaNO) 2 In mg/kg), V refers to the total volume of the extract (mL), c refers to the concentration of nitrite in the mixture (μM), V 1 Refers to the volume (mL), V of the extracted solution mixed with the color development solution 2 Refers to the volume (mL) of the color development liquid taken, and m refers to the mass (g) of the test object.
9. The method for quantitatively detecting nitrite according to claim 8, wherein,
the concentration of nitrite in the mixed solution is calculated by adopting the following formula: a=0.0146c+0.0104, wherein a refers to the maximum absorbance of the mixed solution, as measured by a spectrophotometer;
the color development liquid consists of 3,3 '-dimethoxy-5, 5' -dimethylbenzidine solution and disodium hydrogen phosphate-citric acid buffer solution, wherein the concentration of the 3,3 '-dimethoxy-5, 5' -dimethylbenzidine in the color development liquid is 0.4-0.7 mM, preferably 0.5mM;
the pH of the color development liquid is 2.6-3.2, preferably 3.0;
the volume ratio of the extracting solution to the developing solution is 1:4-1:6, preferably 1:4;
the time for the standing is 15 to 30 minutes, preferably 15 minutes.
10. The nitrite detection kit comprises a color development liquid and a colorimetric tube, wherein a substrate in the color development liquid is 3,3 '-dimethoxy-5, 5' -dimethylbenzidine.
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