GB/T 5413.32-1997 Determination of nitrate and nitrite in milk powder
Some standard content:
GB/T5413.321997
The nitrate and nitrite in milk powder mainly come from artificial adulteration in raw milk and cattle feed. The reduction and photometric analysis methods are widely used to determine their content internationally. This standard is equivalent to the cadmium column regeneration part of the International Dairy Federation standard IDF95A:1984 "Determination of nitrate and nitrite content in milk powder (cadmium reduction and spectrophotometry)" with slight modifications. The other contents are the same. This series of standards will replace GB5413-85 from the date of implementation. This standard is proposed by the China Light Industry Federation.
This standard is under the jurisdiction of the National Dairy Standardization Center. The responsible drafting unit of this standard: National Dairy Product Quality Supervision and Inspection Center. Participating drafting units of this standard: Food Hygiene Supervision and Inspection Institute of the Ministry of Health, Zhejiang Light Industry Research Institute, Harbin Morinaga Dairy Co., Ltd., Nestle (China) Investment Service Co., Ltd. The main drafters of this standard: Huang Min, Tian Jian, Liu Bo, Wang Yun, Wang Xinxiang. 355
1 Scope
National Standard of the People's Republic of China
Determination of nitrate and nitrite contents in milk powder
Milk powder-Determination of nitrate and nitrite contents This standard specifies the method for determining nitrate and nitrite by cadmium reduction and photometric analysis. This standard is applicable to the determination of nitrate and nitrite in milk powder. The detection limits of this method are: nitrate 1.5mg/kg; nitrite 0.2mg/kg. 2 Principle of the method
GB/T5413.32-1997
Replaces GB5413-85
Dissolve the milk powder sample in water, precipitate fat and protein, and then filter. Use copper-plated cadmium particles to reduce the nitrate in part of the filtrate to nitrite. Add sulfonamide and N-1-naphthyl-ethylenediamine dihydrochloride to the unreduced filtrate and the reduced filtrate to make it pink, and then use a spectrophotometer to measure its absorbance at a wavelength of 538nm. Compare the measured absorbance with the absorbance of the standard series of sodium nitrite solutions to calculate the nitrite content in the sample and the total amount of nitrite after nitrate reduction; the nitrate content can be calculated from the difference between the two. 3 Reagents
The water used for determination should be distilled water or deionized water that does not contain nitrate and nitrite. Note: In order to avoid small bubbles in the copper-plated cadmium column (4.10), the distilled water or deionized water used in the preparation of the column (5.1), the inspection of the column reduction ability (5.2) and the regeneration of the column (5.3) should preferably be freshly boiled and cooled to room temperature. 3.1 Copper-plated particles: diameter 0.3~0.8mm. It can also be prepared as follows. Place an appropriate amount of zinc rod in a beaker and immerse the zinc rod in a 40g/L cadmium sulfate (CdSO4·8H,O) solution. Within 24h., scrape off the spongy cadmium on the zinc rod. Take out the zinc rod and drain the excess solution in the beaker, leaving only enough solution to immerse it. Rinse the spongy cadmium rod with distilled water 2-3 times, then transfer the cadmium rod to a small stirrer and add 400mL 0.1mol/L hydrochloric acid. Stir for a few seconds to obtain particles of the desired size. Pour the particles in the stirrer together with the solution back into the beaker and let it stand for a few hours. During this period, stir several times to remove bubbles. Pour out most of the solution and immediately plate copper according to the method described in 5.1.1 to 5.1.8. 3.2 Copper sulfate solution: Dissolve 20g of copper sulfate pentahydrate (CuSO,·5H,0) in water and dilute to 1000mL. 3.3 Hydrochloric acid-ammonia buffer solution: pH 9.6-9.7. Dilute 75mL of concentrated hydrochloric acid Lp201.19g/mL with 600mL of water, with a mass fraction of about 38%. After mixing, add 135mL of concentrated ammonia EP200.91g/mL, with a mass fraction equal to 25% of fresh nitrogen water. Dilute to 1000mL with water and mix. Use a precision pH meter to adjust the pH to 9.60-9.70. 3.4 Hydrochloric acid solution: c(H+) is about 2mol/L. Dilute 160mL of concentrated hydrochloric acid (p201.98g/mL) to 1000mL with water. 3.5 Hydrochloric acid solution: c(H+) is about 0.1mol/L. Dilute 50mL of 2mol/L hydrochloric acid solution to 1000mL with water. 3.6 Solution for precipitating protein and fat:
3.6.1 Zinc sulfate solution: Dissolve 53.5g of zinc sulfate heptahydrate (ZnSO4·7H2O) in water and dilute to 100mL. 3.6.2 Potassium ferrocyanide solution: Dissolve 17.2g of potassium ferrocyanide trihydrate [K,Fe(CN)·3H2O] in water and dilute to 100mL. 3.7 EDTA solution: Dissolve 33.5g of disodium ethylenediaminetetraacetate dihydrate (Na2CroHuN,O,·2H2O) in water and dilute to 1000ml.
Approved by the State Administration of Technical Supervision on May 28, 1997356
Implemented on September 1, 1998
GB/T5413.32--1997
3.8 Color solution 1: 450:550 volume ratio hydrochloric acid solution. Add 450mL of concentrated hydrochloric acid to 550mL of water, and put it into a reagent bottle after cooling.
3.9 Color solution 2: 5g/L sulfonamide solution. Add 5ml of concentrated hydrochloric acid to 75mL of water, then heat it on a water bath and use it to dissolve 0.5g of sulfonamide (NH,CH,SO,NH,). After cooling to room temperature, dilute with water to 100mL. Filter if necessary3.10 Color solution 3: 1g/L naphthylamine hydrochloride solution. Dissolve 0.1 g of N-1-naphthyl-ethylenediamine dihydrochloride (CiH,NHCHzCH,NH·2HC1) in water and dilute to 100 mL. Filter if necessary. Note: This solution should be prepared in small quantities, stored in a sealed brown bottle, and stored in a refrigerator at 2~5°C. 3.11 Sodium nitrite standard solution: The equivalent concentration of nitrite is 0.001 g/L. Dry sodium nitrite (NaNO,) at 110~～120°C to constant weight. After cooling, weigh 0.150 g, dissolve in a 1000 mL volumetric flask, and make up to volume with water. Prepare the solution on the day of use. In a 1000 mL volumetric flask, take 10 mL of the above solution and 20 mL of buffer solution (3.3), and make up to volume with water. 1 mL of this standard solution contains 1.00 μg of NOz. 3.12 Potassium nitrate standard solution, equivalent to a nitrate concentration of 0.0045 g/L. Dry potassium nitrate (KNO3) to constant weight at a temperature range of 110-120°C, weigh 1.4680 g after cooling, dissolve in a 1000 mL volumetric flask, and make up to volume with water.
On the day of use, take 5 mL of the above solution and 20 mL of buffer solution (3.3) in a 1000 mL volumetric flask and make up to volume with water. 1 mL of this standard solution contains 4.50 μg of NO3. 4 Instruments
All glassware should be rinsed with distilled water to ensure that there is no nitrate and nitrite. 4.1 Analytical balance.
4.2 Beaker: 100 mL.
4.3 Conical flask: 250 mL, 500 mL.
4.4 Volumetric flask: 100 mL, 500 mL and 1000 mL. 4.5 Pipette: 2 mL, 5 mL, 10 mL, 20 mL. 4.6 Pipette 2mL5mL, 10mL, 25mL. 4.7 Volumetric tube: select as needed.
4.8 Glass funnel: about 9cm in diameter, short neck. 4.9 Qualitative filter paper: about 18cm in diameter.
4.10 Reduction column: referred to as cadmium column, as shown in Figure 1. 4.11 Spectrophotometer: the measurement wavelength is 538nm, and a cuvette with a light path of 1 to 2cm is used. 4.12 pH meter, with an accuracy of ±0.01, calibrated with pH7 and pH9 standard solutions before use. 357
5 Operation steps
5.1 Preparation of copper-plated cadmium column
Water stop clamp
GB/T5413.32—1997
Glass wool
Figure 1 Nitrate reduction device
5.1.1 Place the cadmium particles (3.1) in a conical flask (4.3) (the amount of cadmium particles used is based on the required height of the cadmium column). 5.1.2 Add enough hydrochloric acid (3.4) to immerse the cadmium particles and shake for a few minutes. 5.1.3 Drain the solution and rinse it repeatedly with water in the conical flask until all the chloride is rinsed off. 5.1.4 Copper plating on the cadmium particles. Add copper sulfate solution (3.2) to the cadmium particles (about 2.5mL per gram of cadmium particles) and shake for 1 minute. 5.1.5 Pour out the liquid and immediately rinse the copper-plated cadmium pellets with water. Note that the cadmium pellets must be submerged in water at all times. Stop rinsing when there is no more copper precipitation in the rinse water.
5.1.6 Install a few centimeters of glass fiber at the bottom of the glass column used to hold the copper-plated cadmium pellets (see Figure 1). Pour water into the glass column and remove all bubbles.
5.1.7 Load the copper-plated cadmium pellets into the glass column as quickly as possible, and keep them exposed to air for as short a time as possible. The height of the copper-plated cadmium pellets should be within the range of 15 to 20 cm.
1 Avoid leaving air between the pellets.
2 Be careful not to let the liquid level fall below the top of the copper-plated cadmium pellets. 5.1.8 Treatment of the newly prepared column. Pass a mixture of 750 ml of water, 225 ml of potassium nitrate standard solution (3.12), 20 ml of buffer solution (3.3) and 20 ml of LEDTA solution (3.7) through the glass column just loaded with cadmium pellets at a flow rate of no more than 6 ml/min, and then rinse the column with 50 ml of 358
water at the same flow rate.
5.2 Check the reducing capacity of the column
GB/T 5413.32—1997
This check should be carried out at least twice a day, usually at the beginning and after a series of measurements. 5.2.1 Use a pipette to transfer 20 mL of potassium nitrate standard solution (3.12) into the reservoir cup at the top of the reduction column, and then immediately add 5 mL of buffer solution (3.3) to the reservoir cup. Collect the eluent in a 100 mL volumetric flask. The flow rate of the eluent should not exceed 6 mL/min. 5.2.2 When the reservoir cup is about to be emptied, rinse the cup wall with about 15 mL of water. After the rinse water flows out, repeat the rinse with 15 mL of water. When the second rinse water flows out, fill the reservoir cup with water and let it flow through the column at the maximum flow rate. 5.2.3 When the eluent in the volumetric flask is close to 100 mL, remove the volumetric flask from under the column, dilute to the mark with water, and mix well. 5.2.4 Transfer 10 mL of the eluent into a 100 mL volumetric flask and add water to about 60 mL. Then follow 5.8.2, 5.8.3 and 5.8.4.
5.2.5 Based on the measured absorbance, the nitrite content (μg/mL) in the diluted eluent (5.2.4) can be found from the standard curve (5.8.5). Based on this, the column reduction capacity expressed as a percentage can be calculated (the reduction capacity is 100% when the NOz content is 0.067μg/mL). If the reduction capacity is less than 95%, the column needs to be regenerated. 5.3 Column Regeneration
After the column is used, or when the reduction capacity of the cadmium column is less than 95%, regenerate it according to the following steps. 5.3.1 Add about 5mL of EDTA solution (3.7) and 2mL of hydrochloric acid solution (3.5) to 100mL of water. Pass through the column at a speed of about 10mL/min.
5.3.2 When the mixed solution in the reservoir is emptied, rinse the column with 25mL of water, 25mL of hydrochloric acid solution (3.5) and 25mL of water in sequence. 5.3.3 Check the reducing power of the column. If it is less than 95%, regenerate it repeatedly. 5.4 Weighing and dissolving sampleswwW.bzxz.Net
5.4.1 Liquid milk sample: Measure 90mL of sample into a 500mL conical flask, rinse the sample cylinder with 22mL of 50-55℃ water several times, pour the rinse liquid into the conical flask and mix well. 5.4.2 Milk powder sample: Weigh 10g of sample in a 100mL beaker, accurate to 0.001g. Wash the sample into a 500mL conical flask with 112mL of 50-55℃ water and mix well.
5.4.3 Whey powder and infant formula powder produced with whey powder as raw material: Weigh 10g of sample in a 100mL beaker, accurate to 0.001g. Wash the sample into a 500ml conical flask with 112ml.50-55℃ water and mix well. Cover the conical flask with aluminum foil, boil the dissolved sample in boiling water for 15 minutes, and then cool to about 50°C. 5.5 Removal of fat and protein
5.5.1 Add 24mL zinc sulfate solution (3.6.1), 24mL potassium ferrocyanide solution (3.6.2) and 40mL buffer solution (5.3) in sequence. Shake while adding, and shake thoroughly after adding each solution. 5.5.2 Let stand for 15min to 1h. Then filter with filter paper (4.9) and collect the filtrate in a 250mL conical flask. 5.6 Reduction of nitrate to nitrite
5.6.1 Transfer 20mL of filtrate to a 100mL beaker, add 5mL buffer solution (3.3), shake well, pour into the storage cup at the top of the cadmium column, and pass through the column at a flow rate of less than 6mL/min. The eluent (liquid after passing through the column) is transferred to a 100mL volumetric flask. 5.6.2 When the liquid cup is almost empty, rinse the small beaker with 15mL of water and pour it into the liquid storage cup. After the rinse water flows out, repeat it with 15mL of water. When the second rinse water is almost finished, fill the liquid storage cup with water and pass it through the column at the maximum flow rate. 5.6.3 When the eluent in the volumetric flask is close to 100mL, take out the volumetric flask, dilute it with water, and mix it. 5.7 Determination
5.7.1 Transfer 20mL of eluent (5.6.3) and 20mL of filtrate (5.5.2) to a 100mL volumetric flask and add water to about 60mL. 5.7.2 First add 6mL of color solution 1 (3.8) to each volumetric flask, mixing while adding; then add 5mL of color solution 2 (3.9). Carefully mix the solutions and let them stand at room temperature for 5 minutes, avoiding direct sunlight. 5.7.3 Add 2 mL of color solution 3 (3.10), mix carefully, and let it stand at room temperature for 5 minutes, avoiding direct sunlight. Add water to the mark and mix well.
GB/T5413.32--1997
5.7.4 Measure the absorbance of the above sample solution at a wavelength of 538 nm within 15 minutes, using the blank test liquid as a control. 5.8 Preparation of standard curve
5.8.1 Transfer (or use a burette to release) 0, 2, 4, 6, 8, 10, 12, 16 and 20 mL of sodium nitrite standard solution (3.11) to nine 100 mL volumetric flasks respectively. Add water to each volumetric flask to make the volume about 60 mL. 5.8.2 First add 6 mL of color solution 1 (3.8) to each volumetric flask, mixing while adding; then add 5 mL of color solution 2 (3.9). Carefully mix the solution and let it stand at room temperature for 5 minutes, avoiding direct sunlight. 5.8.3 Add 2 mL of color developing solution 3 (3.10), mix carefully, and let it stand at room temperature for 5 minutes, avoiding direct sunlight. Make up to the mark with water and mix well.
5.8.4 Within 15 minutes, measure the absorbance of the other eight solutions at a wavelength of 538 nm, using the first solution (without sodium nitrite) as a control.
5.8.5 Plot the measured absorbance against the mass concentration of nitrite. The mass concentration of nitrite can be calculated based on the amount of sodium nitrite standard solution added. The mass concentration of nitrite is the horizontal axis and the absorbance is the vertical axis. The mass concentration of nitrite is expressed in μg/100 mL.
6 Description of analysis results
6.1 Nitrite content
20000×c1
Nitrite content in sample (mg/kg)=mxV
Where: C1—concentration of NOz read from the standard curve according to the absorbance (5.7.4) of the filtrate (5.5.2), μg/100mL; m—mass of sample (mass of liquid milk sample is 90×1.030g), g; V,—volume (5.7.1)mL of the filtrate (5.5.2). Nitrite content in sample expressed as sodium nitrite W(NaNO,) - 1. 5 × W(NO,)
W(NO,)——nitrite content in sample expressed as sodium nitrite, mg/kg. Report the determination result with an accuracy of 0.1 rmg/kg. 6.2 Nitrate content
(100 000 X c2 - W (NO)
Nitrate content in sample (mg/kg) = 1.35×m XV
Wherein: c2~ — Nitrite ion concentration read from the standard curve according to the absorbance (5.7.4) of the eluent (5.6.3), μg/100mL:
m——mass of sample, g;
V2---—volume (5.7.1) of the eluent (5.6.3), mL; W(NO,) —
—Nitrite content calculated according to formula (1). If the reducing capacity of the column is taken into account,
Nitrate content in sample (mg/kg) = 1.35×Wherein: r — reducing capacity of the column after measuring a series of samples. The content of nitrate in the sample as sodium nitrate (100 000 × 2 - W (NO,)
W(NaNO,) = 1. 371 × W (NO;)W(NO,)—the content of nitrate in the sample, mg/kg; W(NaNO)—the content of nitrate in the sample as sodium nitrate, mg/kg. The test results are reported to an accuracy of 0.1 mg/kg. 360
（3）
.(4)
(5)
7 Allowable error
7.1 Repeatability
GB/T5413.32--1997
7.1.1 The difference between two nitrite results measured by the same analyst within a short time interval shall not exceed 1 mg/kg.
7.1.2 The difference between two nitrate results measured by the same analyst within a short time interval shall not exceed 3 mg/kg when the nitrate content is less than 30 mg/kg; and shall not exceed 10% of the average value of the results when the nitrate content is greater than 30 mg/kg. 7.2 Reproducibility
The difference between two nitrate results measured by two analysts in different laboratories on the same sample shall not exceed 8 mg/kg when the nitrate content is less than 30 mg/kg; and shall not exceed 25% of the average value of the results when the nitrate content is greater than or equal to 30 mg/kg.
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