CN112595796B - Preparation method of p-aminoazobenzene standard substance - Google Patents
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Abstract
The invention relates to a preparation method of a para-aminoazobenzene standard substance, which adopts para-aminoazobenzene hydrochloride instead of para-aminoazobenzene as a raw material, adopts a three-time water washing method to remove most impurities in the para-aminoazobenzene to obtain the para-aminoazobenzene standard substance with the content of 99-99.5%, and provides a para-aminoazobenzene content detection method by using the standard substance, comprising the following steps of: preparing a standard storage solution of p-aminoazobenzene with the concentration of 1.0 mg/ml; preparing a standard working solution of the p-aminoazobenzene; sample solutions were prepared and tested. The invention has scientific and reasonable design and good selectivity, because the method is based on the special reaction of the aromatic primary amine, the secondary amine and the tertiary amine have no interference, the aniline is 40 times, the diazo aminobenzene is 20 times and the para-aminoazobenzene has no interference, the method has high accuracy and good reproducibility, the relative standard deviation RSD of the measurement is 0.068 percent, and the relative error is 0.14 percent.
Description
Technical Field
The invention belongs to the field of chemical synthesis, relates to a chemical detection method, and in particular relates to a preparation method of a p-aminoazobenzene standard substance.
Background
Para-aminoazobenzene, also known as 4-aminoazobenzene, para-phenylazoamine, is an azo dye intermediate, and has been widely used in the production of acid dyes and disperse dyes. Industrial grade para-aminoazobenzene is a pale grey crystal or powder, slightly soluble in water, and soluble in ethanol, diethyl ether, chloroform, benzene and oils.
Para-aminoazobenzene is used as an aromatic amine compound with carcinogenic action, and has been a widely accepted limiting substance in the detection industry at home and abroad for many years. Due to the structural specificity, under the condition of the traditional detection method, azo bonds in the structure can be broken to generate two non-cancerogenic aromatic amines of aniline and p-phenylenediamine, so that the target substance is difficult to detect. Thus, unlike the other 23 carcinogenic aromatic amines, the detection of p-aminoazobenzene in dyes has been controversial. The method and the standard for detecting the p-aminoazobenzene in a plurality of industries are released at home and abroad before and after 2008, and the problem is gradually solved.
The detection methods in the prior art all adopt a gas chromatography-mass spectrometry combined instrument method, are simple and rapid, have better reproducibility and sensitivity, and are more methods adopted by the existing detection mechanism. However, since the price of the instrument is relatively high, the popularity is not as high as that of liquid chromatography, the accuracy is not as high as that of spectrophotometry, and the application of the method is limited to a certain extent. It is necessary to study a simple and rapid method for determining the content of p-aminoazobenzene.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the method for detecting the content of the p-aminoazobenzene, which has the advantages of good selectivity, high accuracy and good repeatability, wherein the relative standard deviation RSD is measured to be 0.068%, and the relative error is measured to be 0.14%.
The invention solves the technical problems by adopting the following technical scheme:
a preparation method of a para-aminoazobenzene standard substance comprises the following steps:
weighing 5kg of industrial-grade para-amino azo, adding 2-5kg of hydrochloric acid, adding 3-5 liters of distilled water, stirring for 5-10 minutes, performing suction filtration, collecting precipitate, adding a small amount of distilled water, stirring and washing once;
adding the washed p-aminoazobenzene hydrochloride crystals into pure water at 60-80 ℃, adding a 40% sodium hydroxide aqueous solution of 3-4 kg under stirring, neutralizing to pH9-10, cooling, precipitating p-aminoazobenzene crystals, filtering, and collecting the crystals;
dissolving the collected crystals with hot ethanol, adding active carbon, stirring for 5-10 minutes, filtering while the mixture is hot, observing the color of the filtrate, cooling the filtrate if the color is qualified, precipitating crystals, and if the color of the filtrate is too dark or blackened, carrying out secondary decolorization, wherein the content of the p-aminoazobenzene can only be 98% after primary recrystallization;
in order to obtain the p-aminoazobenzene with higher content, a secondary ethanol recrystallization method is adopted, and the pure product with the purity of 99-99.5% can be obtained after drying at 60-80 ℃.
Moreover, the technical indexes of the para-aminoazobenzene standard are as follows:
the content is more than or equal to 98 percent;
melting point range 125-126 ℃;
loss on drying of 0.28-0.3%
0.02% of burning residues.
The activated carbon is granular activated carbon, wooden activated carbon and has the granularity of 100-150 meshes.
A method for detecting the content of p-aminoazobenzene comprises the following steps:
preparing 1.0mg/ml p-aminoazobenzene standard stock solution
Weighing 50.00mg of p-aminoazobenzene standard substance with the content of more than or equal to 99.0%, dissolving in methanol in a 50ml volumetric flask, diluting to 50ml, and refrigerating in a refrigerator;
preparation of para-aminoazobenzene standard working solution
Respectively taking standard stock solutions of the p-aminoazobenzene according to gradient concentration in a 10ml volumetric flask, diluting to 10ml by using methanol as a solvent to prepare serial standard solutions with different concentrations, and placing the serial standard solutions in a refrigerator for refrigeration and preservation;
preparing a sample solution and detecting
Weighing 0.1g of sample, placing the sample in a conical flask, adding 20ml of sodium hydroxide solution, shaking to uniformly disperse the sample, adding 5g of sodium chloride, fully soaking and dissolving, shaking uniformly, adding 0.2g of sodium hydrosulfite, fully shaking, dissolving, placing in a shaking water bath at 40 ℃ for 30 minutes to crack the sample, cooling the sample to room temperature, transferring the sample into a separating funnel, adding 10ml of anhydrous diethyl ether, covering a bottle stopper, shaking with force, standing, and taking supernatant for detection after two phases are layered.
Moreover, the detection method comprises a high pressure liquid chromatography determination method and an ultraviolet spectrophotometry.
The high pressure liquid chromatography measurement method includes: the standard working solution is measured by adopting an Agilent chromatograph, a 48 column, the column length is 150mm, the inner diameter is 4.6mm, the particle diameter is 5.0 mu m, the infusion pump-flow range is 0.1-5.0ml/min, the sample injection amount is 10 mu l, the column temperature is 25-30 ℃, the standard working solution is measured, the chromatographic peak area and the concentration are used for drawing a standard working curve, the sample solution is subjected to three parallel measurement by using the chromatographic peak area external standard method, the average value of the parallel measurement results is calculated, and the absolute value difference of each parallel measurement result is not more than 10% of the arithmetic average value of the measurement results.
The detection wavelength of the high pressure liquid chromatography measurement method is 380nm.
Moreover, the high-pressure liquid chromatography measurement method adopts acetonitrile+water as a mobile phase, acetonitrile: water = 60:100.
moreover, the ultraviolet spectrophotometry has the following reaction formula:
Ph-N=N-Ph-NH2 + SO2 → Ph-N=N-Ph-NHSO2H
Ph-N=N-Ph-NHSO2H + HCHO → Ph-N=N-Ph-SO2CH2OH
the specific method comprises the following steps:
preparation of standard solution
Preparing 100ml of p-aminoazobenzene standard stock solution with the concentration of 1 mg/ml;
preparation of standard use solution for preparing the same
Taking a standard storage solution of p-aminoazobenzene, diluting the standard storage solution to a scale with ethanol in a 500ml volumetric flask, and preparing a standard use solution of 10.00 mug/ml;
preparation of sodium sulfite solution
Preparing 0.1% solution of analytically pure sodium sulfite with water, and taking 2.0ml;
preparation of formaldehyde solution
Preparing 3.6% formaldehyde aqueous solution by using water to analyze pure formaldehyde, and taking 1.0ml;
preparation of working curve for bathing
Adding 2ml of sodium sulfite solution, 2.00 ml of standard solution, 4.00 ml of standard solution, 6.00 ml of standard solution and 8.00ml of formaldehyde solution into a 50ml volumetric flask respectively, shaking uniformly, diluting with water to a scale, standing for 10 minutes, using a 2cm cuvette at 515nm, and measuring the absorbance by taking a reagent as a blank;
sample measurement
The sample was weighed to 0.1000g, diluted to 100ml, and 1ml was separated for measurement by the same working curve method.
Moreover, the ultraviolet spectrophotometry sample selection is between 1 and 100 mu g/50mL, the correlation coefficient is more than 99.5%, the molar absorptivity is more than or equal to 3.9x104, the relative standard deviation RSD is 0.068%, and the relative error is 0.14%.
The invention has the advantages and positive effects that:
1. according to the invention, a p-aminoazobenzene standard product is innovatively developed, p-aminoazobenzene hydrochloride is adopted as a raw material instead of p-aminoazobenzene, and p-aminoazobenzene is directly used for purification, so that the content of the obtained product reaches 98%, impurities such as aniline and diazoaminobenzene cannot be removed completely, and the requirements of the standard product cannot be met. According to the property of detecting the water-insoluble property of the p-aminoazobenzene hydrochloride, a three-time water washing method is adopted to wash out impurities such as aniline hydrochloride, nitrite and the like which are dissolved in water, and the method is simple and rapid and does not need special equipment. And then adopting a neutralization purification method to convert the p-aminoazobenzene hydrochloride into p-aminoazobenzene precipitate in an alkaline water solution, and removing aniline and other water-soluble impurities again.
2. According to the preparation method of the p-aminoazobenzene standard substance, impurities such as diazo aminobenzene which is insoluble in cold ethanol are removed in a cold ethanol solution according to the solubility of the p-aminoazobenzene in the ethanol. In order to ensure the color of the para-aminoazobenzene, i.e., to remove oxidation products, the process of the present invention employs activated carbon adsorption decolorization. The decoloring temperature is respectively kept at 60-80 ℃, and the quality and granularity of the used activated carbon are key for ensuring decoloring. In order to ensure thorough decolorization without affecting the filtration speed, the method adopts granular activated carbon, and wooden activated carbon with granularity of 100-150 meshes is adopted. And then carrying out three times of recrystallization by an ethanol recrystallization method.
3. The invention firstly adopts an ultraviolet-visible spectrophotometry to scan a methanol solution of a 4-aminoazobenzene standard substance, and discovers that the aminoazobenzene has two absorption peaks between 200nm and 400nm, namely 248 nm and 380nm, which are theoretical basis for measuring the aminoazobenzene by using a high-pressure liquid chromatography, and in order to improve the detection sensitivity, the HPLC detection wavelength adopts 380nm. 4. The process of the present invention attempts at several mobile phases: for example, methanol+water, acetonitrile+water, and the peak shape of the p-aminoazobenzene is found to be better in the mobile phase of acetonitrile+water, so that the method of the invention finally adopts acetonitrile+water as the mobile phase, acetonitrile: water = 60:100 is most effective.
5. The method adopts an Agilent chromatograph, 48 columns, the column length is 150mm, the inner diameter is 4.6mm, the particle size is 5.0 mu m, the infusion pump-flow range is 0.1-5.0ml/min, the sample injection amount is 10 mu l, the column temperature is 25-30 ℃, the gradient elution method is adopted, the elution is thorough, the standard curve method is adopted, the accuracy is high, the measurement result is repeated 10 times in the experiment, the relative standard deviation is 0.89%, and the detection requirement can be completely met.
6. Para-aminoazobenzene is an organic intermediate and is also an organic chemical reagent. The gas chromatography-mass spectrometry and the high-pressure liquid chromatography are limited in application due to high cost, and the invention provides another simple and rapid ultraviolet spectrophotometry which is low in instrument cost and convenient to popularize. The principle is that the excess reducing agents sodium sulfite and formaldehyde are adopted to reduce the p-aminoazobenzene and generate a stable mauve complex, and the ultraviolet spectrophotometry measurement can be carried out on the mauve complex with characteristic absorption peak (lambda=515 mu m) in the ultraviolet region.
The experimental aminoazobenzene complex has a maximum absorption peak at 515nm, so 515nm is selected as the measurement wavelength.
7. According to the ultraviolet spectrophotometry, through experiments, standard series sample selection is between 1-100 mug/50 mL, linearity is good, the correlation coefficient is larger than 99.5%, the consumption of the reducing agent and the complexing agent is related to stability of the sensitivity and the linear relation of the determination of the aminoazobenzene, and the method adopts 2.0mL of 0.1% sodium sulfite and 1.0mL of 3.6% formaldehyde, and has high sensitivity and molar absorptivity of more than or equal to 3.9x104.
8. The invention has scientific and reasonable design, the detection method comprises a high-pressure liquid chromatography determination method and an ultraviolet spectrophotometry, and the selectivity is good, because the method is based on the special reaction of aromatic primary amine, the secondary amine and tertiary amine have no interference, the aniline is 40 times, and the diazo aminobenzene is 20 times and has no interference to the aminoazobenzene. The method has high accuracy and good reproducibility. Based on the measurement results, the relative standard deviation RSD was measured to be 0.068%, and the relative error was measured to be 0.14%. A preparation method of the standard storage liquid of the para-aminoazobenzene is innovated, and the detection result shows that most impurities in the para-aminoazobenzene can be removed, so that the standard substance of the para-aminoazobenzene with the content of 99-99.5% can be obtained, and can be used as a standard for measuring the content of the para-aminoazobenzene.
Detailed Description
The invention is further illustrated by the following examples, which are intended to be illustrative only and not limiting in any way.
A method for detecting the content of p-aminoazobenzene comprises the following steps:
1. preparation of standard stock solution of para-aminoazobenzene (1.0 mg/ml)
50.00mg of p-aminoazobenzene standard (content. Gtoreq.99.0%) was weighed out, dissolved in methanol in a 50ml volumetric flask, and diluted to 50ml. The stock solution should be placed in a refrigerator for cold storage and has a valid period of 3 months.
The standard stock solution is used as a reference substance, the content of the standard stock solution is required to be 98.5-99%, and the content of the para-aminoazobenzene detected by the reagent grade is 96-98%, so that the standard stock solution cannot be used as the reference substance. Therefore, it is necessary to research a high-content para-aminoazobenzene detection as a standard substance for detecting a para-aminoazobenzene content measurement reference substance.
In the method, a p-aminoazobenzene standard product is innovatively developed in the experimental process, p-aminoazobenzene hydrochloride is adopted as a raw material instead of p-aminoazobenzene, and p-aminoazobenzene is directly used for purification, so that the content of the obtained product reaches 98%, impurities such as aniline, diazoaminobenzene and the like cannot be removed completely, and the requirements of the standard product cannot be met.
According to the invention, a three-time water washing method is adopted according to the property of detecting the water insoluble property of the p-aminoazobenzene hydrochloride. Firstly, impurities such as aniline hydrochloride, nitrite and the like dissolved in water are washed away, and the method is simple and quick and does not need special equipment.
And then adopting a neutralization purification method to convert the p-aminoazobenzene hydrochloride into p-aminoazobenzene precipitate in an alkaline water solution, and removing aniline and other water-soluble impurities again.
According to the solubility of the p-aminoazobenzene in ethanol, impurities such as diazoaminobenzene insoluble in cold ethanol are removed in a cold ethanol solution.
In order to ensure the color of the para-aminoazobenzene, i.e., to remove oxidation products, the process of the present invention employs activated carbon adsorption decolorization. The decoloring temperature is respectively kept at 60-80 ℃, and the quality and granularity of the used activated carbon are key for ensuring decoloring. In order to ensure thorough decolorization without affecting the filtration speed, the method adopts granular activated carbon, and wooden activated carbon with granularity of 100-150 meshes is adopted.
And then carrying out three times of recrystallization by an ethanol recrystallization method. The detection result shows that most impurities in the p-aminoazobenzene can be removed, and the p-aminoazobenzene standard substance with the content of 99-99.5% can be obtained and can be used as a standard for measuring the content of the p-aminoazobenzene.
The specific method comprises the following steps:
weighing 5kg of industrial-grade para-amino azo, adding 2-5kg of hydrochloric acid, adding 3-5 liters of distilled water, stirring for 5-10 minutes, performing suction filtration, collecting precipitate, adding a small amount of distilled water, stirring and washing once.
And (3) adding the washed p-aminoazobenzene hydrochloride crystals into pure water at 60-80 ℃, adding a 40% sodium hydroxide aqueous solution of 3-4 kg under stirring, neutralizing to pH9-10, cooling, precipitating p-aminoazobenzene crystals, filtering, and collecting the crystals.
The crystals collected are dissolved with a certain amount of hot ethanol. Adding a small amount of active carbon, stirring for 5-10 min, filtering while the filtrate is hot, observing the color of the filtrate, cooling the filtrate if the color is qualified, precipitating crystals, and if the color of the filtrate is too deep or blackened, carrying out secondary decolorization and carrying out primary recrystallization to obtain the p-aminoazobenzene with the content of 98%.
In order to obtain the p-aminoazobenzene with higher content, a secondary ethanol recrystallization method is adopted, and the pure product with the purity of 99-99.5% can be obtained after drying at 60-80 ℃.
The amino azobenzene standard prepared by the method has the technical indexes as follows:
the content is more than or equal to 98 percent
Pass sulfuric acid dissolution test
Melting Point Range (. Degree.C.) 125-126
Ethanol dissolution test is qualified
Loss on drying of 0.28-0.3%
0.02% of burning residue
Pass the aniline impurity test
Diazoaminobenzene impurity test is qualified
The prepared p-aminoazobenzene completely meets the index requirement through detection after the purification process.
2. Preparation of standard working solution of p-aminoazobenzene
According to the gradient concentration, respectively taking proper amounts of p-aminoazobenzene standard stock solution into a 10ml volumetric flask, diluting to 10ml by using methanol as a solvent, preparing a series of standard solutions with different concentrations, and placing the standard solutions into a refrigerator for refrigeration and preservation, wherein the effective period is 10 days.
3. Preparation of sample solutions
Weighing 0.1g of sample, placing the sample in an conical flask, adding 20ml of sodium hydroxide solution, shaking to uniformly disperse the sample, adding 5g of sodium chloride, fully soaking and dissolving, shaking uniformly, adding 0.2g of sodium hydrosulfite, and fully shaking and dissolving. The sample was lysed by placing it in a 40℃shaking water bath for 30 minutes. The sample was cooled to room temperature, transferred to a separatory funnel, added with 10ml of anhydrous diethyl ether, capped with a bottle stopper, shaken vigorously and then allowed to stand, after the two phases had separated, the supernatant was taken for testing.
4. Detection method
High-pressure liquid chromatography measurement method
The standard working solution is measured by adopting an Agilent chromatograph, a 48 column, the column length is 150mm, the inner diameter is 4.6mm, the particle size is 5.0 mu m, the infusion pump-flow range is 0.1-5.0ml/min, the sample injection amount is 10 mu l, the column temperature is 25-30 ℃, and the standard working curve is drawn by using the chromatographic peak area and the concentration. The sample solution was subjected to three replicates using the chromatographic peak area external standard method, and the average value of the replicates was calculated. The absolute difference of each parallel measurement should not be more than 10% of the arithmetic mean of the measurements.
The invention firstly adopts an ultraviolet-visible spectrophotometry to scan a methanol solution of a 4-aminoazobenzene standard substance, and discovers that the aminoazobenzene has two absorption peaks between 200nm and 400nm, namely 248 nm and 380nm, which are theoretical basis for measuring the aminoazobenzene by using a high-pressure liquid chromatography, and in order to improve the detection sensitivity, the HPLC detection wavelength adopts 380nm.
The process of the present invention attempts at several mobile phases: for example, methanol+water, acetonitrile+water, and the peak shape of the p-aminoazobenzene is found to be better in the mobile phase of acetonitrile+water, so that the method of the invention finally adopts acetonitrile+water as the mobile phase, acetonitrile: water = 60:100 is most effective.
Ultraviolet spectrophotometry of the second place
Para-aminoazobenzene is an organic intermediate and is also an organic chemical reagent. The gas chromatography-mass spectrometry and the high-pressure liquid chromatography are limited in application due to high cost, and the invention provides another simple and rapid ultraviolet spectrophotometry which is low in instrument cost and convenient to popularize. The principle is that the excess reducing agents sodium sulfite and formaldehyde are adopted to reduce the p-aminoazobenzene and generate a stable mauve complex, and the ultraviolet spectrophotometry measurement can be carried out on the mauve complex with characteristic absorption peak (lambda=515 mu m) in the ultraviolet region. The reaction formula is as follows:
Ph-N=N-Ph-NH2 + SO2 → Ph-N=N-Ph-NHSO2H
Ph-N=N-Ph-NHSO2H + HCHO → Ph-N=N-Ph-SO2CH2OH
the aminoazobenzene complex had a maximum absorption peak at 515nm, so 515nm was chosen as the measurement wavelength. According to the method, through experiments, standard series samples are selected between 1-100 mug/50 mL. The linearity is good, and the correlation coefficient is more than 99.5%. The amounts of reducing agent and complexing agent are related to the stability of the sensitivity of the aminoazobenzene assay and the linear relationship. The method adopts 2.0ml of 0.1% sodium sulfite and 1.0ml of 3.6% formaldehyde. The method has high sensitivity, and the molar absorptivity is more than or equal to 3.9x104.
The method has good selectivity. Because the method is based on the special reaction of aromatic primary amine, the secondary amine and tertiary amine have no interference, the aniline is 40 times, and the diazo aminobenzene is 20 times and has no interference to the aminoazobenzene. The method has high accuracy and good reproducibility. Based on the measurement results, the relative standard deviation RSD was measured to be 0.068%, and the relative error was measured to be 0.14%.
The specific method comprises the following steps:
1. preparation of standard solutions
1mg/ml stock solution, p-aminoazobenzene standard substance, ethanol as solvent. 100ml of a stock solution of 1mg/ml was prepared.
2. Preparation of Standard use solution
A certain amount of stock solution is sucked up, diluted to a scale with ethanol in a 500ml volumetric flask, and a standard use solution of 10.00 mug/ml is prepared.
3. Preparation of sodium sulfite solution
Analytically pure sodium sulfite was made up with water to a 0.1% solution.
4. Preparation of formaldehyde solution
Analytically pure formaldehyde was prepared as a 3.6% aqueous formaldehyde solution with water.
5. Preparation of working curves
In a 50ml volumetric flask, 2ml of sodium sulfite solution, 2.00 ml of standard solution, 4.00 ml of standard solution, 6.00 ml of standard solution, 8.00ml of formaldehyde solution, 1ml of formaldehyde solution, shaking uniformly, diluting to a scale with water, standing for 10 minutes, and measuring the absorbance of the sample at 515nm by using a 2cm cuvette and taking the reagent as a blank.
6. Sample measurement
The sample was weighed to 0.1000g, diluted to 100ml, and 1ml was separated for measurement by the same working curve method.
Note that: standard deviation s=0.067%
Relative standard deviation rsd=0.068%
Relative error r=0.14%
Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.
Claims (2)
1. A preparation method of a para-aminoazobenzene standard substance is characterized by comprising the following steps: the method comprises the following steps:
weighing 5kg of industrial-grade para-amino azo, adding 2-5kg of hydrochloric acid, adding 3-5 liters of distilled water, stirring for 5-10 minutes, performing suction filtration, collecting precipitate, adding a small amount of distilled water, stirring and washing once;
adding the washed p-aminoazobenzene hydrochloride crystals into pure water at 60-80 ℃, adding a 40% sodium hydroxide aqueous solution of 3-4 kg under stirring, neutralizing to pH9-10, cooling, precipitating p-aminoazobenzene crystals, filtering, and collecting the crystals;
dissolving the collected crystals with hot ethanol, adding active carbon, stirring for 5-10 minutes, filtering while the mixture is hot, observing the color of the filtrate, cooling the filtrate if the color is qualified, precipitating crystals, and if the color of the filtrate is too dark or blackened, carrying out secondary decolorization, wherein the content of the p-aminoazobenzene can only be 98% after primary recrystallization;
in order to obtain higher content of para-aminoazobenzene, the para-aminoazobenzene is subjected to secondary ethanol recrystallization, and dried at 60-80deg.C to obtain 99-99.5% pure product,
the technical indexes of the p-aminoazobenzene standard substance are as follows:
the content is more than or equal to 98 percent;
melting point range 125-126 ℃;
loss on drying of 0.28-0.3%
0.02% of burning residues.
2. The method for preparing the p-aminoazobenzene standard according to claim 1, wherein the method comprises the steps of: the activated carbon is granular activated carbon, wooden activated carbon and has granularity of 100-150 meshes.
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