CN110261377A - A kind of nitrite quick detection reagent and preparation method thereof - Google Patents
A kind of nitrite quick detection reagent and preparation method thereof Download PDFInfo
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- CN110261377A CN110261377A CN201910671928.5A CN201910671928A CN110261377A CN 110261377 A CN110261377 A CN 110261377A CN 201910671928 A CN201910671928 A CN 201910671928A CN 110261377 A CN110261377 A CN 110261377A
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- nitrite
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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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
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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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N2021/775—Indicator and selective membrane
Abstract
The invention discloses a kind of nitrite quick detection reagent, the concentration for being 1:1 including volume ratio is 1mg/mL aminoglucose hydrochloride solution and 3,3', 5,5'- tetramethyl biphenyl amine aqueous solutions that concentration is 16.64 μM.Under conditions of weak acid, nitrite and 3,3', 5, the amino of 5'- tetramethyl benzidine (TMB) reacts to form diazonium salt, diazonium salt and aminoglucose hydrochloride (GAH) coupling reaction form orange-yellow color compound, and shade is linearly related to nitrite concentration, and have selectivity well.The aminoglucose hydrochloride solution and 3,3' that the present invention uses, detection reagent of 5, the 5'- tetramethyl biphenyl amine aqueous solutions as nitrite avoid toxic chemicals and complicated operation sequence, and the reaction time is very fast, and selectivity is high, detects sensitive.
Description
Technical field
The present invention relates to a kind of nitrite quick detection reagents and preparation method thereof, belong to nitrite detection field.
Background technique
Nitrite is a kind of very common food additives and colour former, such as sausage, jam, pickled product, jerky etc.
There are the addition of various dose, mainly coloring and corrosion-resistant effect in food.Excessive nitrite can cause poisoning and dead
It dies, its adult toxic dose: 0.2~0.5g, lethal dose: 3g.Under the environment such as gastric acid nitrite with it is secondary in food
The reactions such as amine, tertiary amine and amide generate strong carcinogen N- nitrosamine, it has extremely strong carcinogenesis.Nitrite can also make blood
In the low Ferri-hemoglobin of normal oxygen carrying be oxidized to ferrihemoglobin, thus lose oxygen carrying capacity and cause histanoxia.This
Outside, nitrosamine can also enter in fetus body through placenta, have teratogenesis to fetus.Baby within 6 months is to nitrous acid
Salt is especially sensitive, and the baby for clinically suffering from " methemoglobinemia " is caused by the high food of edible nitrite concentration,
Symptom is anoxic, cyanosis occurs, or even dead.
Currently, the detection for nitrous acid Asia mainly uses photometry (colorimetric method), principle is: in acidic environment
Under, p-aminobenzene sulfonic acid is reacted with nitrite anions first generates diazonium compound, then occurs again with N- (1- naphthalene)-ethylenediamine
Coupling reaction forms aubergine azo-compound, and light has absorption maximum and absorbs luminous intensity and nitrite at 540nm wavelength
Content is positively correlated, and detection is limited to 0.06 μ g/mL.Although the method is reproducible, accuracy is high, used to amino
Benzene sulfonic acid and N- (1- naphthalene)-ethylenediamine all have toxicity, and N- (1- naphthalene)-ethylenediamine toxicity is very big and irritant,
It is unfavorable for testing staff's operation, and needs static waiting in reaction process, takes a long time.
Summary of the invention
The purpose of the present invention is to provide a kind of nonhazardous and reaction time short nitrite quick detection reagent and its
Preparation method.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of nitrite quick detection reagent, including volume ratio be 1:1 concentration be 1mg/mL glucosamine salt
The 3,3' that acid salt solution and concentration are 16.64 μM, 5,5'- tetramethyl biphenyl amine aqueous solution.
The principle of the detection reagent is: under conditions of weak acid, nitrite and 3,3', and 5,5'- tetramethyl benzidines
(TMB) amino reacts to form diazonium salt, and diazonium salt and aminoglucose hydrochloride (GAH) coupling reaction form orange-yellow chemical combination
Object.
Further, the aminoglucose hydrochloride solution, which refers to, is dissolved in small organic molecule for aminoglucose hydrochloride
Acid, the small organic molecule acid not only can dissolve aminoglucose hydrochloride, weakly acidic reaction environment can be also provided for reagent.
The small organic molecule acid can be one of acetic acid, formic acid, propionic acid.
Further, described 3,3', 5,5'- tetramethyl biphenyl amine aqueous solutions refer to 3,3', 5,5'- tetramethyl benzidines are molten
In cosolvent, cosolvent facilitates the dissolution of 3,3', 5,5'- tetramethyl benzidines, and the overall stability of detection reagent can also be provided.
The cosolvent is one of dimethyl sulfoxide, acetone, ether, dimethylformamide.
The present invention also provides nitrite quick detection reagent preparation methods, include the following steps:
(1) it prepares aminoglucose hydrochloride solution: aminoglucose hydrochloride solution is dissolved in small organic molecule acid
And make the concentration 1mg/mL of aminoglucose hydrochloride solution；
(2) 3,3' is prepared, 5,5'- tetramethyl biphenyl amine aqueous solution: by 3,3', 5,5'- tetramethyl benzidine is dissolved in cosolvent
In and make 3,3', 5,5'- tetramethyl benzidine solution concentration is 16.64 μM；
(3) step (1) is prepared into aminoglucose hydrochloride solution and step (2) prepares 3,3', 5,5'- tetramethyl biphenyl
Amine aqueous solution carries out being mixed to form TMB-GAH detection reagent according to volume ratio 1:1.
By the TMB-GAH detection reagent of the nitrite quick detection reagent preparation method preparation provided by the invention
It is used in the detection of nitrite.
Under conditions of weak acid, nitrite and 3,3', the amino of 5,5'- tetramethyl benzidines (TMB) reacts to form weight
Nitrogen salt, diazonium salt and aminoglucose hydrochloride (GAH) coupling reaction form orange-yellow compound；Again by maximum absorption wave
Long absorbance carries out the assay of nitrite.
The present invention also provides a kind of nitrite rapid detection method, the TMB-GAH detection reagent and unknown concentration
Nitrite solution mixed according to volume ratio 2:1 after, at 451nm wavelength light have absorption maximum and absorb luminous intensity
It is positively correlated with content of nitrite.In acid condition, 3,3', 5,5'- tetramethyl benzidines are reacted with nitrite anions first
Diazonium compound is generated, coupling reaction then occurs with aminoglucose hydrochloride again, forms orange-yellow azo-compound,
Multiple measurement is made at 375~700nm, its absorption value has dropped 20% compared with 451nm absorption value at 400nm and 500nm
~40%, 430nm absorption value then decline 15% or so compared with 451nm absorption value.Therefore orange-yellow color product of the invention
Maximum absorption wavelength be determined as 451nm.
It is operated by experimental method, draws standard working curve.The result shows that content of nitrite is in 1~75 μM of model
Enclose interior one Beer law of obedience lambert, regression equation Y=0.01327X-0.0176, related coefficient R2=0.9944.With reagent
The standard deviation of blank determination calculates detection of the invention and limits LOD=0.73 μM.
The present invention is by using aminoglucose hydrochloride solution and 3,3', and 5,5'- tetramethyl biphenyl amine aqueous solution is as sub-
The detection reagent of nitrate, nontoxic, to operator's fanout free region, and the reaction time is very fast, saves the time.
Detailed description of the invention
Fig. 1 is the lab diagram of pH value and absorption light spectral wavelength of the invention.
Fig. 2 is standard curve schematic diagram of the invention.
Fig. 3 is influence of the aminoglucose hydrochloride solution of the present invention to absorbance.
Specific embodiment
Embodiment 1
Nitrite quick detection reagent provided in this embodiment include volume ratio be 1:1 concentration be 1mg/mL amino Portugal
The 3,3' that grape sugar HCI solution and concentration are 16.64 μM, 5,5'- tetramethyl biphenyl amine aqueous solution.
The principle of the detection reagent is: under conditions of weak acid, nitrite and 3,3', and 5,5'- tetramethyl benzidines
(TMB) amino reacts to form diazonium salt, and diazonium salt and aminoglucose hydrochloride (GAH) coupling reaction form orange-yellow chemical combination
Object.
The aminoglucose hydrochloride solution, which refers to, is dissolved in small organic molecule acid for aminoglucose hydrochloride, described to have
Machine small molecule acid not only can dissolve aminoglucose hydrochloride, and weakly acidic reaction environment can be also provided for reagent.It is described organic
Small molecule acid can be one of acetic acid, formic acid, propionic acid.
Described 3,3', 5,5'- tetramethyl biphenyl amine aqueous solutions, which refer to, is dissolved in cosolvent for 3,3', 5,5'- tetramethyl benzidines,
Cosolvent facilitates 3,3', 5,5'- tetramethyl benzidines, and the overall stability of detection reagent can also be provided.The cosolvent is
One of dimethyl sulfoxide, acetone, ether, dimethylformamide.
Embodiment 2
The present invention also provides nitrite quick detection reagent preparation methods, include the following steps:
(1) it prepares aminoglucose hydrochloride solution: aminoglucose hydrochloride solution being dissolved in acetic acid and makes amino
The concentration of glucosamine salt acid salt solution is 1mg/mL；
(2) prepare 3,3', 5,5'- tetramethyl biphenyl amine aqueous solution: by 3,3', 5,5'- tetramethyl benzidine is dissolved in diformazan Asia
In sulfone and make 3,3', 5,5'- tetramethyl benzidine solution concentration is 16.64 μM；
(3) step (1) is prepared into aminoglucose hydrochloride solution and step (2) prepares 3,3', 5,5'- tetramethyl biphenyl
Amine aqueous solution carries out being mixed to form TMB-GAH detection reagent according to volume ratio 1:1.
The content of nitrite is examined and determine
1, the determination of pH value and extinction spectrum wavelength
Raw material: 25 μM of sodium nitrite solutions, TMB-GAH detection reagent prepared by embodiment 2, pH buffer, photometer.
Step: taking 4 test tubes, and 25 μM of sodium nitrite solutions of 1mL are in vitro first added, in the TMB-GAH that 2mL is added
PH buffer is added in detection reagent, and making invisible spectro pH value is respectively 2,2.5,3 and 3.5.
Extinction spectrum: multiple measurement its absorption value and 451nm at 400nm and 500nm have been made at 375~700nm and have inhaled
Receipts value then declines 15% or so compared to 20%~40%, 430nm absorption value is had dropped compared with 451nm absorption value.Therefore this reality
The maximum absorption wavelength for applying the orange product of example is determined as 451nm, as shown in Figure 1, photometer when pH value is lower than 2.5
It absorbs clearly.
2, working curve and detection limit
11 test tubes are taken, and (1~11) is numbered to test tube, 1mL or less concentration is added in 1~No. 11 test tube respectively
75 μM, 60 μM, 50 μM, 40 μM, 30 μM, 20 μM, 10 μM, 7.5 μM, 5 μM, 2.5 μM and 1 μM of nitrite solution, then plus
The reagent for entering the preparation of 2mL embodiment adjusts zero point with 1cm cuvette with reagent blank.
Absorbance is surveyed after adjusting spectrometer wavelength 451nm, standard curve is done according to concentration.Calibration curve equation is Y=
0.01327X-0.0176, related coefficient R2=0.9944, as shown in Figure 2.Absorbance is measured using blank sample liquid 3 times
3 blank values are obtained, absorbance standard deviation is 0.0032, wherein 0.73 μM of detection limit.
3, the relationship between absorbance and aminoglucose hydrochloride concentration
This experiment also measured were the relationship between absorbance and aminoglucose hydrochloride concentration, be respectively 0.5 by concentration,
1, the aminoglucose hydrochloride of 2 and 3mg/mL prepares TMB-GAH detection reagent according to embodiment 2, and the TMB-GAH of preparation is examined
25 μM of sodium nitrite solution coupling reactions of test agent and 1mL generate orange-yellow compound, and using photometer in wavelength
Absorbance is surveyed at 451nm, result as shown in figure 3, thus prove influence of the solubility to absorbance of aminoglucose hydrochloride compared with
It is small, therefore the application uses 1mg/mL.
Embodiment 3
The nitrite solution of the unknown concentration mixed with TMB-GAH detection reagent according to volume ratio 2:1 after
There is absorption maximum in the 451nm wavelength time and be positively correlated with content of nitrite, and standard curve according to fig. 2 calculates
The concentration of nitrite solution.
The above is only the preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto, any
The transformation and replacement carried out based on technical solution provided by the present invention and inventive concept should all be covered in protection model of the invention
In enclosing.
Claims (9)
1. a kind of nitrite quick detection reagent, it is characterised in that: the concentration for being 1:1 including volume ratio is 1mg/mL amino Portugal
The 3,3' that grape sugar HCI solution and concentration are 16.64 μM, 5,5'- tetramethyl biphenyl amine aqueous solution.
2. nitrite quick detection reagent according to claim 1, it is characterised in that: the aminoglucose hydrochloride
Solution, which refers to, is dissolved in small organic molecule acid for aminoglucose hydrochloride solution.
3. nitrite quick detection reagent according to claim 2, it is characterised in that: the small organic molecule acid is vinegar
One of acid, formic acid, propionic acid.
4. nitrite quick detection reagent according to claim 1, it is characterised in that: described 3,3', 5,5'- tetramethyls
Biphenyl amine aqueous solution refers to that, by 3,3', 5,5'- tetramethyl benzidine is dissolved in cosolvent.
5. nitrite quick detection reagent according to claim 4, it is characterised in that: the cosolvent is that diformazan is sub-
One of sulfone, acetone, ether, dimethylformamide.
6. a kind of nitrite quick detection reagent preparation method, it is characterised in that include the following steps:
(1) it prepares aminoglucose hydrochloride solution: aminoglucose hydrochloride solution is dissolved in small organic molecule acid and is made
The concentration of aminoglucose hydrochloride solution is 1mg/mL；
(2) 3,3' is prepared, 5,5'- tetramethyl biphenyl amine aqueous solution: by 3,3', 5,5'- tetramethyl benzidine is dissolved in cosolvent simultaneously
Make 3,3', 5,5'- tetramethyl benzidine solution concentration is 16.64 μM；
(3) step (1) is prepared into aminoglucose hydrochloride solution and step (2) prepares 3,3', 5,5'- tetramethyl benzidine is molten
Liquid carries out being mixed to form TMB-GAH detection reagent according to volume ratio 1:1.
7. the TMB-GAH detection reagent of nitrite quick detection reagent preparation method preparation according to claim 6 is answered
With in the detection of nitrite.
8. a kind of nitrite rapid detection method, it is characterised in that: the nitrous of the TMB-GAH detection reagent and unknown concentration
Acid salt solution mixed according to volume ratio 2:1 after in 375~700nm wave-length coverage light have absorption maximum and and nitrous acid
Salt content is positively correlated.
9. nitrite rapid detection method according to claim 8, it is characterised in that: the wavelength of the absorption maximum light
For the ultraviolet light of 451nm.
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| CN114798021A (en) * | 2022-04-18 | 2022-07-29 | 四川农业大学 | Three-dimensional paper-based micro-fluidic chip for rapidly detecting nitrite in water body and preparation method and application thereof |
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