CN102749325B - Application of 3,3',5,5'-tetramethyl benzidine in detecting benzoyl peroxide added to flour - Google Patents

Abstract

The invention discloses a new application of 3,3',5,5'-tetramethylbenzidine, which can be used for qualitative detection or quantitative detection of benzoyl peroxide added in flour. 3,3',5,5'-Tetramethylbenzidine reacts rapidly after contacting with benzoyl peroxide, and the resulting product has an obvious blue color, and the reaction sensitivity is high, and the lowest detectable amount in flour is 4.0mg/kg of benzoyl peroxide. In quantitative detection, the sample processing process is simple, and the color reaction can be realized without complicated and cumbersome steps, which saves detection time, and the detection results are stable and reliable, and 3,3',5,5'-tetramethylbenzidine is harmful to human Non-toxic and harmless to the environment.

Description

3, 3', 5, 5'-tetramethylbenzidine for the detection of benzoyl peroxide added to flour

technical field

The invention belongs to the field of food additive detection, in particular to the detection of benzoyl peroxide.

Background technique

Benzoyl peroxide (C ₁₄ H ₁₀ O ₄ , hereinafter referred to as BPO) is a commonly used whitening additive in the field of flour production and processing. Excessive benzoyl peroxide can cause β-carotene, vitamin A, A large amount of nutrients such as vitamin B and vitamin E are destroyed, and eating wheat flour containing benzoic acid generated by excessive benzoyl peroxide hydrolysis is very harmful to the liver, and it is easy to cause a variety of diseases, such as bleeding gums and skin purple spots. Each country has set its limit standard. my country's "Food Additive Edible Hygienic Standards" revised in 1997 stipulated that the maximum amount of benzoyl peroxide used was 60 mg/kg, and the Ministry of Health required a complete ban on adding benzoyl peroxide to flour since 2011.

Currently, there are two detection techniques for benzoyl peroxide: chromatography and spectrophotometry. Chromatography mainly includes gas chromatography, high performance liquid chromatography and capillary electrophoresis. These methods need to reduce BPO to benzoic acid first, and then rely on bulky or expensive precision instruments to complete. Spectrophotometric methods include iodometric method, iron powder reduction method, and hydroxylamine hydrochloride reduction method. The sample pretreatment steps of these methods are cumbersome, the operation time is too long and skilled experimental technicians are required. It is also reported that different types of chromogens such as N,N,N',N'-tetramethyl-p-phenylenediamine, N-ethyl-2-naphthylamine and N,N-dimethyl-p-phenylenediamine were used To convert BPO into colored substances, these color reactions not only need to use a constant temperature water bath to heat the reaction system, but also need to use a catalyst. In addition, due to the strong carcinogenic and teratogenic properties of anilines, the application of these methods is limited. The above methods for determining BPO have certain limitations in practical applications, especially in routine analysis. Therefore, it is necessary to find a new method with high sensitivity and good selectivity, and to establish a new green method that is easy to operate, sensitive and reliable, and can meet the requirements of BPO routine analysis.

3, 3', 5, 5'-tetramethylbenzidine (3,3',5,5'-tetramethylbenzidine, hereinafter referred to as TMB) is a new type of safe reducing agent, which has gradually replaced the strong carcinogen benzidine And other carcinogenic benzidine derivatives, used in clinical laboratory, forensic examination, criminal detection and environmental monitoring fields, so far, the application of this reagent in the field of food processing has not been reported.

Contents of the invention

The purpose of this invention is to provide a new application of 3, 3', 5, 5'-tetramethylbenzidine.

The use of 3, 3', 5, 5'-tetramethylbenzidine for qualitative detection or quantitative detection of benzoyl peroxide added in flour.

Further, the qualitative detection is to drop an ethanol solution of 3, 3', 5, 5'-tetramethylbenzidine into the flour, mix well, and judge whether the content of benzoyl peroxide in the flour is determined by the change of the color of the flour. More than 4.0 mg/kg:

(1) The color of the flour turns blue, indicating that the content of benzoyl peroxide in the flour exceeds 4.0 mg/kg;

(2) No change in flour color means that the flour does not contain benzoyl peroxide or the content of benzoyl peroxide is less than 4.0 mg/kg.

Further, the quantitative detection is carried out according to the following steps:

(1) Wash the flour with acetone and centrifuge for 2 to 3 times, and combine the centrifuged supernatant;

(2) The clear liquid obtained in step (1) was blown dry with nitrogen to obtain a white solid;

(3) Add 3, 3', 5, 5'-tetramethylbenzidine ethanol solution to the white solid, then add pH = 4.0 sodium dihydrogen phosphate buffer solution, constant volume, measure the absorbance of the solution at 652 nm ;

(4) Replace the white solid in step (3) with benzoyl peroxide standard substance, and draw the concentration-absorbance standard curve;

(5) The content of benzoyl peroxide in flour can be calculated from the absorbance in step (3) and the concentration-absorbance standard curve in step (4).

Calculation of benzoyl peroxide content:

Wherein, W is the content of benzoyl peroxide in the flour (mg/kg); C is the concentration of benzoyl peroxide in the solution of step (3) (mg/L); V is the constant volume of the solution of step (3) Volume (L); m is the mass of flour weighed (kg).

Further, the washing in step (1) is to disperse the flour in acetone with a vortex oscillator, and then ultrasonically treat it.

3, 3', 5, 5'-Tetramethylbenzidine reacts rapidly after contacting with benzoyl peroxide, and the resulting product has a clear blue color, and the reaction has high sensitivity. Benzoyl peroxide can be detected at least 4.0 mg/kg in flour. It can simply and quickly detect benzoyl peroxide qualitatively; for quantitative detection, the sample treatment is simple, and the color reaction can be realized without complicated and cumbersome steps, which saves detection time, and the detection results are stable and reliable. The relative standard of three repeated tests The deviations are all less than 3.55%, and 3, 3', 5, 5'-tetramethylbenzidine is non-toxic to human body and harmless to the environment.

Description of drawings

Figure 1 shows the color reaction of 3, 3', 5, 5'-tetramethylbenzidine to different concentrations of benzoyl peroxide.

Figure 2 is the qualitative detection of benzoyl peroxide in flour with 3, 3', 5, 5'-tetramethylbenzidine.

Detailed ways

The present invention will be further described below by means of the accompanying drawings and examples.

Example 1

Weigh 4 portions of commercially available flour solids and place them in centrifuge tubes, 0.50 g each, and then add 0.0, 10.0, 20.0 and 40.0 μg of BPO standard substance to each portion, then add 2 mL of acetone, and use a vortex shaker ( Qilinbeier, QL-901) After mixing flour and acetone evenly, ultrasonic treatment for 5 minutes, high-speed centrifugation, take out the supernatant, add 1 mL of acetone to the centrifuged flour, ultrasonication, centrifugation, repeat twice, The supernatants obtained by centrifugation were combined, and the supernatants were blown dry with nitrogen gas at room temperature to obtain a white solid. Add 15 μL of 20 mmol/L TMB ethanol solution to the obtained white solid, then immediately add 20 mmol/L pH = 4.0 sodium dihydrogen phosphate buffer solution, and dilute to 3 mL to obtain a blue solution. After the insoluble substances were filtered with a 0.45 μm microporous membrane, the absorbance of the solution was measured at 652 nm with a spectrophotometer, and each part was measured in parallel three times, and the average value was taken. According to the standard curve of concentration-absorbance, the concentration of BPO was obtained.

Draw concentration-absorbance standard curve: Weigh 0.0, 2.0, 8.0, 14.0, 24.0, 36.0 and 48.0 μg BPO standard substances respectively, add 15 μL 20 mmol/L TMB ethanol solution to each BPO standard substance, and then immediately add 20 Mmol/L pH = 4.0 sodium dihydrogen phosphate buffer solution, dilute to 3 mL to obtain a series of standard solutions, measure the absorbance of the standard solutions at 652 nm with a spectrophotometer, and draw the concentration-absorbance standard curve.

Calculation of benzoyl peroxide content in flour:

Wherein, W is the content of benzoyl peroxide in the flour (mg/kg); C is the concentration of benzoyl peroxide in the solution of step (3) (mg/L); V is the constant volume of the solution of step (3) Volume (L); m is the mass of flour weighed (kg).

Experimental results:

.

Example 2:

Weigh 1 g of commercially available flour and divide it into two parts, place it on a clean glass plate, add 60 μg of BPO solid to one part, and mix well. Then, 15 μL of 20 mmol/L TMB ethanol solution was added dropwise to the two parts of flour, and mixed immediately, the flour without BPO was still white (left in Figure 2), and the flour with BPO was blue (right in Figure 2 ), gradually reducing the BPO content, and the minimum detection limit was 4.0 mg/kg.

Claims (1)

1.3,3', the purposes of 5,5'-tetramethyl benzidine, is characterized in that: for qualitative detection or the quantitative benzoyl peroxide that is added on flour that detects;

Described qualitative detection is to drip 3,3' in flour, and the ethanolic solution of 5,5'-tetramethyl benzidine, mixes, and is judged whether the content of benzoyl peroxide in flour exceedes 4.0 mg/kg by the variation of flour color:

(1) flour color becomes the blue content that represents benzoyl peroxide in flour and exceedes 4.0 mg/kg;

(2) in the unchanged expression flour of flour color, do not contain benzoyl peroxide or Determination of benzoyl peroxide in wheat lower than 4.0 mg/kg;

Described quantitative detection is to carry out according to following steps:

(1), by acetone washing, centrifugal treating for flour 2～3 times, merge the supernatant liquor of centrifugal gained;

(2) step (1) gained clear liquid dries up with nitrogen, obtains white solid;

(3) in white solid, add 3,3', the ethanolic solution of 5,5'-tetramethyl benzidine, then add the sodium dihydrogen phosphate buffer of pH=4.0, constant volume, utilizes spectrophotometer to measure the absorbance of solution in 652 nm places;

(4) with the white solid of benzoyl peroxide standard items alternative steps (3), draw concentration-absorbance typical curve;

(5) can be calculated the content of benzoyl peroxide in flour by the absorbance of step (3) and concentration-absorbance typical curve of step (4).