GB/T 5009.97-2003 Determination of sodium cyclamate in foods

This standard specifies three methods for the determination of sodium cyclohexylaminosulfonate in food - gas chromatography, colorimetry, and thin layer chromatography. The gas chromatography and colorimetry methods of this standard are applicable to the determination of sodium cyclohexylaminosulfonate in beverages, preserved fruits and other foods; the thin layer chromatography method is applicable to the determination of sodium cyclohexylaminosulfonate in beverages, fruit juices, jams, and cakes. The detection limit of this standard is 4μg. GB/T 5009.97-2003 Determination of sodium cyclohexylaminosulfonate in food GB/T5009.97-2003 Standard download decompression password: www.bzxz.net

ICS67.040
National Standard of the People's Republic of China
GB/T5009.97—2003
Replaces GB/T13112—1991
Determination of sodium cyclamate in foodsIssued on August 11, 2003
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implementation on January 1, 2004
GB/T5009.97—2003
This standard replaces GB/T13112-1991 "Determination of sodium cyclamate in foods". Compared with GB/T13112--1991, this standard has been modified as follows: the Chinese name of the standard has been modified, and the Chinese name of the standard has been changed to "Determination of Sodium Cyclohexylaminosulfonate in Foods"; the structure of the original standard has been modified according to GB/T20001.4-2001 "Standard Writing Rules Part 4: Chemical Analysis Methods".
This standard is proposed and managed by the Ministry of Health of the People's Republic of China. This standard was drafted by the Guangzhou Municipal Health and Epidemic Prevention Station, the Guangdong Provincial Health and Epidemic Prevention Station, and the Food Hygiene Supervision and Inspection Institute of the Ministry of Health. The main drafters of the first and second methods of this standard are: Zhang Nuanmin, Jiang Yuebi, Lv Aosheng, Liang Liwen, and Liu Yanxia. The main drafters of the third method of this standard are: Yang Zuying, Cui Hong, Jiang Luozhang, Jiao Shuting, and Yi Zaiming. The original standard was first issued in 1991, and this is the first revision. 688
1 Scope
Determination of sodium cyclohexylaminosulfonate in foods GB/T 5009.97--2003
This standard specifies three methods for the determination of sodium cyclohexylaminosulfonate in foods: gas chromatography, colorimetry, and thin layer chromatography. The gas chromatography and colorimetry of this standard are applicable to the determination of sodium cyclohexylaminosulfonate in beverages, preserved fruits, and other foods; the thin layer chromatography is applicable to the determination of sodium cyclohexylaminosulfonate in beverages, fruit juices, jams, and cakes. The detection limit of this standard is 4μg.
Gas chromatography
Method 1
2 Principle
Sodium cyclohexylaminosulfonate reacts with nitrous acid in a sulfuric acid medium to generate cyclohexanol nitrite, which is qualitatively and quantitatively determined by gas chromatography.
3 Reagents
3.1 n-Hexane.
3.2 Sodium chloride.
3.3 Chromatographic silica gel (or sea sand).
3.4 ​​50g/L sodium nitrite solution.
3.5 100g/L sulfuric acid solution.
3.6 Standard sodium cyclohexylaminosulfonate solution (containing sodium cyclohexylaminosulfonate, 98%): Accurately weigh 1.0000g sodium cyclohexylaminosulfonate, add water to dissolve and dilute to 100mL. This solution contains 10mg sodium cyclohexylaminosulfonate per milliliter. 4 Instruments
4.1 Gas chromatograph: with hydrogen flame ionization detector. 4.2 Vortex mixer.
4.3 Centrifuge.
4.4 10μL micro syringe.
4.5 Chromatographic conditions
4.5.1 Chromatographic column: 2m long, 3mm inner diameter, U-shaped stainless steel column. 4.5.2 Stationary phase: Chromosorb WAWDMCS 80-100 mesh, coated with 10% SE-30. 4.5.3 Determination conditions
Column temperature: 80℃; Vaporization temperature: 150℃; Detection temperature: 150℃. Flow rate: Nitrogen 40mL/min; Hydrogen 30mL/min; Air 300mL/min. 5 Sample location
5.1 Liquid sample: Shake well and weigh directly. Samples containing carbon dioxide are first heated to remove, and samples containing alcohol are adjusted to alkalinity by adding 40g/L sodium hydroxide solution, and then heated to remove in a boiling water bath to prepare samples. 5.2 Solid samples: Samples of preserved fruits and candied fruits are cut into pieces to prepare samples. 689
GB/T5009.97-2003
6 Analysis steps
6.1 Sample preparation
6.1.1 Liquid sample: Weigh 20.0g of sample into a 100mL stoppered colorimetric tube and place in an ice bath. 6.1.2 Solid sample: Weigh 2.0g of the shredded sample into a mortar, add a little chromatography silica gel (or sea sand) and grind until it is powdery, pour it into a 100mL volumetric flask through a funnel, rinse the mortar with water, and transfer the washing liquid to the volumetric flask. Add water to the scale, shake from time to time, filter after 1 hour, and obtain the sample. Accurately pipette 20mL into a 100mL stoppered colorimetric tube and place in an ice bath. 6.2 Determination
6.2.1 Preparation of standard curve: Accurately pipette 1.00mL of sodium cyclohexylaminosulfonate standard solution into a 100mL stoppered colorimetric tube, add 20mL of water. Place in an ice bath, add 5mL of 50g/L sodium nitrite solution, 5ml.100g/L sulfuric acid solution, shake well, place in an ice bath for 30min, and shake frequently, then accurately add 10mL of n-hexane and 5g of sodium chloride, shake the hook and place on a vortex mixer for 1min (or shake 80 times), wait for static stratification, aspirate the hexane layer and centrifuge in a 10mL stoppered centrifuge tube for centrifugal separation. Each mL of hexane extract is equivalent to 1mg of sodium cyclohexylaminosulfonate. Inject 1uL~5μL of the standard extract into the gas chromatograph, and draw a standard curve based on the response value. 6.2.2 The sample tube is operated according to 6.2.1 starting from "add 5mL 50g/L sodium nitrite solution...", and then the sample is injected with 1 uL~5μL, the response value is measured, and the corresponding content is found from the standard curve. 7 Result calculation
See formula (1).
X=m×10×1000
m X VX 1 000
Where:
X——the content of sodium cyclohexylaminosulfonate in the sample, in grams per kilogram (g/kg); m—the mass of the sample, in grams (g); V——the injection volume, in microliters (μL); 10—the amount of n-hexane added, in milliliters (mL); m1-the mass of sodium cyclohexylaminosulfonate in the test sample, in micrograms (μg). The calculation result is rounded to two significant figures.
8 Precision
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. Second Method Colorimetric Method
9 Principle
Sodium cyclohexylaminosulfonate reacts with sodium nitrite in a sulfuric acid medium to generate cyclohexanol nitrite, which is then diazotized with sulfonamide and coupled with ethylenediamine hydrochloride to generate a red dye. Its absorbance is measured at a wavelength of 550nm and compared with the standard for quantification. 10 Reagents
10.1 Chloroform.
10.2 Methanol.
10.3 Dialysis agent: Weigh 0.5g of mercuric chloride and 12.5g of sodium chloride in a beaker and dilute to 100mL with 0.01mol/L hydrochloric acid solution.
10.410g/L sodium nitrite solution.
10.5100g/L sulfuric acid solution.
10.6100g/L urea solution (freshly prepared or stored in refrigerator before use). 10.7100g/L hydrochloric acid solution.
GB/T5009.97—2003
10.810g/L sulfonamide solution: weigh 1g sulfonamide and dissolve it in 10% hydrochloric acid solution, and finally make up to 100mL. 10.91/L hydrochloric acid naphthylethylenediamine solution.
10.10 Sodium cyclohexylaminosulfonate standard solution: accurately weigh 0.1000g sodium cyclohexylaminosulfonate, add water to dissolve, and finally make up to 100mL. This solution contains 1mg sodium cyclohexylaminosulfonate per milliliter. Dilute the sodium cyclohexylaminosulfonate standard solution 10 times before use. This solution contains 0.1 mg sodium cyclohexylaminosulfonate per milliliter. 11 Instruments
11.1 Spectrophotometer.
11.2 Vortex mixer.
11.3 Centrifuge.
11.4 Dialysis paper.
Sample treatment
Same as Chapter 5.
13 Analysis steps
13.1 Extraction
13.1.1 Liquid sample: Weigh 10.0 g of sample into dialysis paper, add 10 mL of dialysis agent, and tie the dialysis paper tightly. Put it into a 200 mL wide-mouth bottle containing 100 mL of water, cover it, and dialyze for 20 to 24 hours to obtain the dialysate. 13.1.2 Solid sample: Accurately pipette 10.0 mL of the sample extract treated in "6.1.2" onto the dialysis paper, and perform the following operations as in "13.1.1".
13.2 Determination
13.2.1 Take two 50mL stoppered colorimetric tubes, add 10mL of dialyzate and 10mL of standard solution respectively, add 1mL of 10g/L sodium nitrite solution and 1mL of 100g/L sulfuric acid solution in an ice bath at 0-3℃, shake well and put in ice water and shake from time to time, place for 1h, take out and add 15mL of chloroform, place on a vortex mixer and vibrate for 1min. After standing, remove the upper layer. Add 15mL of water, vibrate for 1min, remove the upper layer after standing, add 10mL of 100g/L urea solution and 2mL of 100g/L hydrochloric acid solution, vibrate for 5min, remove the upper layer after standing, add 15mL of water, vibrate for 1min, remove the upper layer after standing, and accurately remove 5mL of chloroform into two 25mL colorimetric tubes. Take another 25mL colorimetric tube and add 5mL of chloroform as a reference tube. Add 15 mL of methanol and 1 mL of 10 g/L sulfonamide to each tube, place in ice water for 15 min, take out, return to room temperature, add 1 mL of 1 g/L ethylenediamine hydrochloride solution, add methanol to the scale, place at 15°C to 30°C for 20 min to 30 min, and measure the absorbance at a wavelength of 550 nm with a 1 cm colorimetric cup to obtain the absorbance A and A. 13.2.2 Take two 50mL stoppered colorimetric tubes and add 10mL water and 10mL dialysate respectively. Except for not adding 10g/L sodium nitrite, proceed as in item 13.2.1. Measure the absorbance As and A0. 14 Calculation of the result
See formula (2).
Where:
X×AA×100+10、
m×A.-A..
×１000×１000
X—the content of sodium cyclohexylaminosulfonate in the sample, in grams per kilogram (g/kg); m—the mass of the sample, in grams (g);
GB/T 5009.97—2003
V—dialysis fluid volume, in milliliters (mL); c—standard tube concentration, in micrograms per milliliter (μg/mL); A.—absorbance of standard solution;
Ao—absorbance of water;
A—absorbance of sample dialysate;
A.—absorbance of sample dialysate without sodium nitrite. The calculation result shall retain two significant figures.
15 Precision
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. . The third method is thin layer chromatography
16 Principle
After the sample is acidified, it is extracted with ether. The sample extract is concentrated and spotted on a polyamide thin layer plate. After development, the color is developed and compared with the standard according to the migration value of sodium cyclohexylaminosulfonate on the thin layer plate and the depth of the color spot. 17 Reagents
Isopropanol.
N-butanol.
Petroleum ether: boiling range 30℃~~60℃.
17.4 Ether (does not contain peroxide).
Ammonium hydroxide.
Absolute ethanol. ||t t||Sodium chloride.
Sodium sulfate.
6mol/L hydrochloric acid: add 50mL hydrochloric acid to a small amount of water, and then dilute with water to 100mL. 17.10
Polyamide powder (nylon-6): 200 mesh.
Cyclohexylaminosulfonic acid standard solution: accurately weigh 0.0200g cyclohexylaminosulfonic acid, dissolve it in a small amount of anhydrous ethanol, transfer it to a 10mL volumetric flask, and dilute to the scale. This solution is equivalent to 2mg cyclohexylaminosulfonic acid per milliliter. Re-prepare it after two weeks (melting point of cyclohexylaminosulfonic acid: 169℃～17 0℃). 17.12 Developing agent
17.12.1 n-Butanol-concentrated ammonia water-anhydrous ethanol (20+1+1. 17.12.2 Isopropanol-concentrated ammonia water-anhydrous ethanol (20+1+1). 17.13 Color developer: Weigh 0.040g of bromocresol purple and dissolve it in 100mL of 50% ethanol solution, and adjust the pH to 8 with 1.2mL of 0.4% sodium hydroxide solution.
18 Apparatus
Chromatography cylinder.
Glass plate: 5cmX20cm.
Micro syringe: 10μL.
Glass sprayer.
19 Analysis steps
19.1 Sample collection
GB/T5009.97—2003
19.1.1 Beverages and jams: Weigh 2.5g (ml) of the sample that has been mixed evenly (soft drinks need to be heated to remove carbon dioxide), place it in a 25mL stoppered measuring cylinder, add sodium chloride to saturation (about 1g), add 0.5mL 6mol/L hydrochloric acid to acidify, extract twice with 15 and 10mL ether, shake for 1min each time, let it stand and separate, use a dropper to filter the upper ether extract through anhydrous sodium sulfate into a 25mL volumetric flask, wash the anhydrous sodium sulfate with a small amount of ether, add ether to the scale, and mix well. Take 10.0mL of the ether extract twice and place it in a 10mL stoppered centrifuge tube, evaporate it in a water bath at about 40℃, add 0.1ml of anhydrous ethanol to dissolve the residue, and set aside. 19.1.2 Cake: Weigh 2.5g of cake sample, grind it, put it in a 25mL stoppered tube, extract it with petroleum ether 3 times, 20mL each time, shake it for 3min each time, discard the petroleum ether, let the sample evaporate (stir the sample in a fume hood to remove the petroleum ether), add 0.5mL of 6mol/L hydrochloric acid to acidify it, then add about 1g of sodium chloride, and follow the steps in 19.1.1 from "Extract twice with 15 and 10ml of ether." 19.2 Determination
19.2.1 Preparation of polyamide powder board: Weigh 4g of polyamide powder, add 1.0g of soluble starch, add about 14mL of water and grind until it is uniform, immediately pour it into a coater to make 6 thin layer boards with an area of ​​5cm×20cm and a thickness of 0.3mm, dry it at room temperature, dry it at 80℃ for 1h, take it out, store it in a desiccator and store it for later use. 19.2.2 Spotting: On the baseline 2 cm below the bottom of the thin layer plate, use a microsyringe to spot 4ul of sample solution in the middle of the plate, and spot 2μL and 3uL of cyclohexylaminosulfonic acid standard solution on both sides.
19.2.3 Development and color development: Place the spotted thin layer plate in a development tank pre-filled with a developing agent (17.12.1 or 17.12.2), with filter paper attached around the developing tank. When the front edge of the solvent spreads to more than 10cm, take it out and evaporate it in the air, spray the color developer, and the spot will be yellow and the background will be blue. The amount of cyclohexylaminosulfonic acid in the sample is compared with the depth of the standard spot for quantification (when using the developing agent 17.12.2, the relative transfer value of cyclohexylaminosulfonic acid is about 0.47, sorbic acid 0.73, benzoic acid 0.61, and saccharin 0.31). 20 Result calculation
See formula (3).
Wherein:
X=mlX1 000×1. 12 = 2. 8m×V101000
m × V2
X is the content of sodium cyclohexylaminosulfonate in the sample, in grams per kilogram (or grams per liter); Lg/kg (or g/L); m is the mass of cyclohexylaminosulfonic acid equivalent to the sample point, in milligrams (mg); m is the mass of the sample, in grams (g);
Vi is the volume of anhydrous ethanol added, in milliliters (mL); V is the volume of the sample during the measurement, in milliliters (mL); 10 is the volume of the ether extract absorbed during the measurement, in milliliters (mL); - the total volume of the sample ether extract, in milliliters (mL); 25 -
1.12 is the mass of 1.00g of cyclohexylaminosulfonic acid equivalent to sodium cyclohexylaminosulfonate, in grams (g). The calculation result shall retain two significant figures.
21 Precision
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 28% of the arithmetic mean. This standard can simultaneously determine ingredients such as sorbic acid, benzoic acid, and saccharin. (3)2 Take two 50mL stoppered colorimetric tubes, add 10mL water and 10mL dialysate respectively, except not adding 10g/L sodium nitrite, proceed as in item 13.2.1”, and measure the absorbance As and A0. 14 Nesting calculation
See formula (2).
Where:bzxz.net
X×AA×100+10,
m×A.-A..
×１000×１000
X—the content of sodium cyclohexylaminosulfonate in the sample, in grams per kilogram (g/kg); m—the mass of the sample, in grams (g);
GB/T 5009.97—2003
V—dialysis fluid volume, in milliliters (mL); c—standard tube concentration, in micrograms per milliliter (μg/mL); A.—absorbance of standard solution;
Ao—absorbance of water;
A—absorbance of sample dialysate;
A. —absorbance of sample dialysate without sodium nitrite. The calculated result shall retain two significant figures.
15 Precision
The absolute difference between two independent measurement results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. Third Method Thin layer chromatography
16 Principle
After the sample is acidified, it is extracted with ether. The sample extract is concentrated and spotted on a polyamide thin layer plate. After development, the color is developed and compared with the standard according to the migration value of sodium cyclohexylaminosulfonate on the thin layer plate and the depth of the color spot. 17 Reagents
Isopropanol.
N-butanol.
Petroleum ether: boiling range 30℃~~60℃.
17.4 Ether (peroxide-free).
Ammonium hydroxide.
Absolute ethanol.
Sodium chloride .
Sodium sulfate.
6mol/L hydrochloric acid: add 50mL of hydrochloric acid to a small amount of water, and then dilute to 100mL with water. 17.10
Polyamide powder (nylon-6): 200 mesh.
Cyclohexylaminosulfonic acid standard solution: accurately weigh 0.0200g of cyclohexylaminosulfonic acid, dissolve it in a small amount of anhydrous ethanol, transfer it to a 10mL volumetric flask, and dilute to the scale. This solution is equivalent to 2mg of cyclohexylaminosulfonic acid per milliliter. Re-prepare it after two weeks (melting point of cyclohexylaminosulfonic acid: 169℃～170℃). 17.12 Developing agent
17.12.1 n-Butanol-concentrated ammonia water-anhydrous ethanol (20+1+1. 17.12.2 Isopropanol-concentrated ammonia water-anhydrous ethanol (20+1+1). 17.13 Color developer: Weigh 0.040g of bromocresol purple and dissolve it in 100mL of 50% ethanol solution, and adjust the pH to 8 with 1.2mL of 0.4% sodium hydroxide solution.
18 Instruments
Chromatography cylinder.
Glass plate: 5cmX20cm.
Micro syringe: 10μL.
Glass sprayer.
19 Analysis steps
19.1 Sample collection
GB/T5009.97—2003
19.1.1 Beverages and jams: Weigh 2.5g (ml) of the sample that has been mixed evenly (soft drinks need to be heated to remove carbon dioxide), place it in a 25mL stoppered measuring cylinder, add sodium chloride to saturation (about 1g), add 0.5mL 6mol/L hydrochloric acid to acidify, extract twice with 15 and 10mL ether, shake for 1min each time, let it stand and separate, use a dropper to filter the upper ether extract through anhydrous sodium sulfate into a 25mL volumetric flask, wash the anhydrous sodium sulfate with a small amount of ether, add ether to the scale, and mix well. Take 10.0mL of the ether extract twice and place it in a 10mL stoppered centrifuge tube, evaporate it in a water bath at about 40℃, add 0.1ml of anhydrous ethanol to dissolve the residue, and set aside. 19.1.2 Cake: Weigh 2.5g of cake sample, grind it, put it in a 25mL stoppered tube, extract it with petroleum ether 3 times, 20mL each time, shake it for 3min each time, discard the petroleum ether, let the sample evaporate (stir the sample in a fume hood to remove the petroleum ether), add 0.5mL of 6mol/L hydrochloric acid to acidify it, then add about 1g of sodium chloride, and follow the steps in 19.1.1 from "Extract twice with 15 and 10ml of ether." 19.2 Determination
19.2.1 Preparation of polyamide powder board: Weigh 4g of polyamide powder, add 1.0g of soluble starch, add about 14mL of water and grind until it is uniform, immediately pour it into a coater to make 6 thin layer boards with an area of ​​5cm×20cm and a thickness of 0.3mm, dry it at room temperature, dry it at 80℃ for 1h, take it out, store it in a desiccator and store it for later use. 19.2.2 Spotting: On the baseline 2 cm below the bottom of the thin layer plate, use a microsyringe to spot 4ul of sample solution in the middle of the plate, and spot 2μL and 3uL of cyclohexylaminosulfonic acid standard solution on both sides.
19.2.3 Development and color development: Place the spotted thin layer plate in a development tank pre-filled with a developing agent (17.12.1 or 17.12.2), with filter paper attached around the developing tank. When the front edge of the solvent spreads to more than 10cm, take it out and evaporate it in the air, spray the color developer, and the spot will be yellow and the background will be blue. The amount of cyclohexylaminosulfonic acid in the sample is compared with the depth of the standard spot for quantification (when using the developing agent 17.12.2, the relative transfer value of cyclohexylaminosulfonic acid is about 0.47, sorbic acid 0.73, benzoic acid 0.61, and saccharin 0.31). 20 Result calculation
See formula (3).
Wherein:
X=mlX1 000×1. 12 = 2. 8m×V101000
m × V2
X is the content of sodium cyclohexylaminosulfonate in the sample, in grams per kilogram (or grams per liter); Lg/kg (or g/L); m is the mass of cyclohexylaminosulfonic acid equivalent to the sample point, in milligrams (mg); m is the mass of the sample, in grams (g);
Vi is the volume of anhydrous ethanol added, in milliliters (mL); V is the volume of the sample during the measurement, in milliliters (mL); 10 is the volume of the ether extract absorbed during the measurement, in milliliters (mL); - the total volume of the sample ether extract, in milliliters (mL); 25 -
1.12 is the mass of 1.00g of cyclohexylaminosulfonic acid equivalent to sodium cyclohexylaminosulfonate, in grams (g). The calculation result shall retain two significant figures.
21 Precision
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 28% of the arithmetic mean. This standard can simultaneously determine ingredients such as sorbic acid, benzoic acid, and saccharin. (3)2 Take two 50mL stoppered colorimetric tubes, add 10mL water and 10mL dialysate respectively, except not adding 10g/L sodium nitrite, proceed as in item 13.2.1”, and measure the absorbance As and A0. 14 Nesting calculation
See formula (2).
Where:
X×AA×100+10,
m×A.-A..
×１000×１000
X—the content of sodium cyclohexylaminosulfonate in the sample, in grams per kilogram (g/kg); m—the mass of the sample, in grams (g);
GB/T 5009.97—2003
V—dialysis fluid volume, in milliliters (mL); c—standard tube concentration, in micrograms per milliliter (μg/mL); A.—absorbance of standard solution;
Ao—absorbance of water;
A—absorbance of sample dialysate;
A. —absorbance of sample dialysate without sodium nitrite. The calculated result shall retain two significant figures.
15 Precision
The absolute difference between two independent measurement results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. Third Method Thin layer chromatography
16 Principle
After the sample is acidified, it is extracted with ether. The sample extract is concentrated and spotted on a polyamide thin layer plate. After development, the color is developed and compared with the standard according to the migration value of sodium cyclohexylaminosulfonate on the thin layer plate and the depth of the color spot. 17 Reagents
Isopropanol.
N-butanol.
Petroleum ether: boiling range 30℃~~60℃.
17.4 Ether (peroxide-free).
Ammonium hydroxide.
Absolute ethanol.
Sodium chloride .
Sodium sulfate.
6mol/L hydrochloric acid: add 50mL of hydrochloric acid to a small amount of water, and then dilute to 100mL with water. 17.10
Polyamide powder (nylon-6): 200 mesh.
Cyclohexylaminosulfonic acid standard solution: accurately weigh 0.0200g of cyclohexylaminosulfonic acid, dissolve it in a small amount of anhydrous ethanol, transfer it to a 10mL volumetric flask, and dilute to the scale. This solution is equivalent to 2mg of cyclohexylaminosulfonic acid per milliliter. Re-prepare it after two weeks (melting point of cyclohexylaminosulfonic acid: 169℃～170℃). 17.12 Developing agent
17.12.1 n-Butanol-concentrated ammonia water-anhydrous ethanol (20+1+1. 17.12.2 Isopropanol-concentrated ammonia water-anhydrous ethanol (20+1+1). 17.13 Color developer: Weigh 0.040g of bromocresol purple and dissolve it in 100mL of 50% ethanol solution, and adjust the pH to 8 with 1.2mL of 0.4% sodium hydroxide solution.
18 Instruments
Chromatography cylinder.
Glass plate: 5cmX20cm.
Micro syringe: 10μL.
Glass sprayer.
19 Analysis steps
19.1 Sample collection
GB/T5009.97—2003
19.1.1 Beverages and jams: Weigh 2.5g (ml) of the sample that has been mixed evenly (soft drinks need to be heated to remove carbon dioxide), place it in a 25mL stoppered measuring cylinder, add sodium chloride to saturation (about 1g), add 0.5mL 6mol/L hydrochloric acid to acidify, extract twice with 15 and 10mL ether, shake for 1min each time, let it stand and separate, use a dropper to filter the upper ether extract through anhydrous sodium sulfate into a 25mL volumetric flask, wash the anhydrous sodium sulfate with a small amount of ether, add ether to the scale, and mix well. Take 10.0mL of the ether extract twice and place it in a 10mL stoppered centrifuge tube, evaporate it in a water bath at about 40℃, add 0.1ml of anhydrous ethanol to dissolve the residue, and set aside. 19.1.2 Cake: Weigh 2.5g of cake sample, grind it, put it in a 25mL stoppered tube, extract it with petroleum ether 3 times, 20mL each time, shake it for 3min each time, discard the petroleum ether, let the sample evaporate (stir the sample in a fume hood to remove the petroleum ether), add 0.5mL of 6mol/L hydrochloric acid to acidify it, then add about 1g of sodium chloride, and follow the steps in 19.1.1 from "Extract twice with 15 and 10ml of ether." 19.2 Determination
19.2.1 Preparation of polyamide powder board: Weigh 4g of polyamide powder, add 1.0g of soluble starch, add about 14mL of water and grind until it is uniform, immediately pour it into a coater to make 6 thin layer boards with an area of ​​5cm×20cm and a thickness of 0.3mm, dry it at room temperature, dry it at 80℃ for 1h, take it out, store it in a desiccator and store it for later use. 19.2.2 Spotting: On the baseline 2 cm below the bottom of the thin layer plate, use a microsyringe to spot 4ul of sample solution in the middle of the plate, and spot 2μL and 3uL of cyclohexylaminosulfonic acid standard solution on both sides.
19.2.3 Development and color development: Place the spotted thin layer plate in a development tank pre-filled with a developing agent (17.12.1 or 17.12.2), with filter paper attached around the developing tank. When the front edge of the solvent spreads to more than 10cm, take it out and evaporate it in the air, spray the color developer, and the spot will be yellow and the background will be blue. The amount of cyclohexylaminosulfonic acid in the sample is compared with the depth of the standard spot for quantification (when using the developing agent 17.12.2, the relative transfer value of cyclohexylaminosulfonic acid is about 0.47, sorbic acid 0.73, benzoic acid 0.61, and saccharin 0.31). 20 Result calculation
See formula (3).
Wherein:
X=mlX1 000×1. 12 = 2. 8m×V101000
m × V2
X is the content of sodium cyclohexylaminosulfonate in the sample, in grams per kilogram (or grams per liter); Lg/kg (or g/L); m is the mass of cyclohexylaminosulfonic acid equivalent to the sample point, in milligrams (mg); m is the mass of the sample, in grams (g);
Vi is the volume of anhydrous ethanol added, in milliliters (mL); V is the volume of the sample during the measurement, in milliliters (mL); 10 is the volume of the ether extract absorbed during the measurement, in milliliters (mL); - the total volume of the sample ether extract, in milliliters (mL); 25 -
1.12 is the mass of 1.00g of cyclohexylaminosulfonic acid equivalent to sodium cyclohexylaminosulfonate, in grams (g). The calculation result shall retain two significant figures.
21 Precision
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 28% of the arithmetic mean. This standard can simultaneously determine ingredients such as sorbic acid, benzoic acid, and saccharin. (3)12 Developing agent
17.12.1 n-Butanol-concentrated ammonia water-anhydrous ethanol (20+1+1. 17.12.2 Isopropanol-concentrated ammonia water-anhydrous ethanol (20+1+1). 17.13 Color developer: Weigh 0.040g of bromocresol purple and dissolve it in 100mL of 50% ethanol solution, and adjust the pH to 8 with 1.2mL of 0.4% sodium hydroxide solution.
18 Apparatus
Chromatography cylinder.
Glass plate: 5cmX20cm.
Micro syringe: 10μL.
Glass sprayer.
19 Analysis steps
19.1 Sample collection
GB/T5009.97—2003
19.1.1 Beverages and jams: weigh 2.5g (ml) of the well-mixed sample (soft drinks need to be heated to remove carbon dioxide), place it in a 25mL stoppered measuring cylinder, and add sodium chloride until saturated (about 1g), add 0.5mL 6mol/L hydrochloric acid to acidify, extract twice with 15 and 10mL ether, shake for 1min each time, let stand and separate, use a dropper to filter the upper ether extract through anhydrous sodium sulfate into a 25mL volumetric flask, wash the anhydrous sodium sulfate with a small amount of ether, add ether to the scale, and mix well. Take 10.0mL of ether extract twice and place it in a 10mL stoppered centrifuge tube, evaporate it in a water bath at about 40℃, and add 0.1ml of anhydrous ethanol to dissolve the residue 19.1.2 Pastry: Weigh 2.5g pastry sample, grind it, put it in a 25mL stoppered tube, extract it with petroleum ether for 3 times, 20mL each time, shake it for 3min each time, discard the petroleum ether, let the sample evaporate (stir the sample constantly in a fume hood to remove the petroleum ether), add 0.5mL 6mol/L hydrochloric acid to acidify it, and then add about 1g sodium chloride. The following operations are carried out according to 19.1.1 starting from "Extract twice with 15 and 10ml ether." 19.2 Determination
19.2.1 Preparation of polyamide powder plate: Weigh 4g polyamide powder, add 1.0g soluble starch, add about 14mL water and grind until it is uniform and appropriate, then pour into the applicator to make 6 thin layer plates with an area of ​​5cm×20cm and a thickness of 0.3mm. After drying at room temperature, dry at 80℃ for 1h, take out, store in a desiccator and store for future use. 19.2.2 Spotting: On the baseline 2cm below the thin layer plate, use a micro syringe to spot 4ul of sample solution in the middle of the plate, and spot 2μL and 3uL of cyclohexylamine sulfonic acid standard solution on both sides.
19.2.3 Development and color development: Place the spotted thin layer plate in a development tank pre-filled with a developer (17.12.1 or 17.12.2), with filter paper attached around the development tank. When the solvent front extends to more than 10 cm, take it out and evaporate it in the air. Spray the developer. The spots are yellow and the background is blue. The amount of cyclohexylaminosulfonic acid in the sample is quantitatively compared with the depth of the standard spot (when using the developer 17.12.2, the relative transfer value of cyclohexylaminosulfonic acid is about 0.47, sorbic acid 0.73, benzoic acid 0.61, and saccharin 0.31). 20 Result calculation
See formula (3).
Wherein:
X=mlX1 000×1. 12 = 2. 8m×V101000
m × V2
X is the content of sodium cyclohexylaminosulfonate in the sample, in grams per kilogram (or grams per liter); Lg/kg (or g/L); m is the mass of cyclohexylaminosulfonic acid equivalent to the sample point, in milligrams (mg); m is the mass of the sample, in grams (g);
Vi is the volume of anhydrous ethanol added, in milliliters (mL); V is the volume of the sample during the measurement, in milliliters (mL); 10 is the volume of the ether extract absorbed during the measurement, in milliliters (mL); - the total volume of the sample ether extract, in milliliters (mL); 25 -
1.12 is the mass of 1.00g of cyclohexylaminosulfonic acid equivalent to sodium cyclohexylaminosulfonate, in grams (g). The calculation result shall retain two significant figures.
21 Precision
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 28% of the arithmetic mean. This standard can simultaneously determine ingredients such as sorbic acid, benzoic acid, and saccharin. (3)12 Developing agent
17.12.1 n-Butanol-concentrated ammonia water-anhydrous ethanol (20+1+1. 17.12.2 Isopropanol-concentrated ammonia water-anhydrous ethanol (20+1+1). 17.13 Color developer: Weigh 0.040g of bromocresol purple and dissolve it in 100mL of 50% ethanol solution, and adjust the pH to 8 with 1.2mL of 0.4% sodium hydroxide solution.
18 Apparatus
Chromatography cylinder.
Glass plate: 5cmX20cm.
Micro syringe: 10μL.
Glass sprayer.
19 Analysis steps
19.1 Sample collection
GB/T5009.97—2003
19.1.1 Beverages and jams: weigh 2.5g (ml) of the well-mixed sample (soft drinks need to be heated to remove carbon dioxide), place it in a 25mL stoppered measuring cylinder, and add sodium chloride until saturated (about 1g), add 0.5mL 6mol/L hydrochloric acid to acidify, extract twice with 15 and 10mL ether, shake for 1min each time, let stand and separate, use a dropper to filter the upper ether extract through anhydrous sodium sulfate into a 25mL volumetric flask, wash the anhydrous sodium sulfate with a small amount of ether, add ether to the scale, and mix well. Take 10.0mL of ether extract twice and place it in a 10mL stoppered centrifuge tube, evaporate it in a water bath at about 40℃, and add 0.1ml of anhydrous ethanol to dissolve the residue 19.1.2 Pastry: Weigh 2.5g pastry sample, grind it, put it in a 25mL stoppered tube, extract it with petroleum ether for 3 times, 20mL each time, shake it for 3min each time, discard the petroleum ether, let the sample evaporate (stir the sample constantly in a fume hood to remove the petroleum ether), add 0.5mL 6mol/L hydrochloric acid to acidify it, and then add about 1g sodium chloride. The following operations are carried out according to 19.1.1 starting from "Extract twice with 15 and 10ml ether." 19.2 Determination
19.2.1 Preparation of polyamide powder plate: Weigh 4g polyamide powder, add 1.0g soluble starch, add about 14mL water and grind until it is uniform and appropriate, then pour into the applicator to make 6 thin layer plates with an area of ​​5cm×20cm and a thickness of 0.3mm. After drying at room temperature, dry at 80℃ for 1h, take out, store in a desiccator and store for future use. 19.2.2 Spotting: On the baseline 2cm below the thin layer plate, use a micro syringe to spot 4ul of sample solution in the middle of the plate, and spot 2μL and 3uL of cyclohexylamine sulfonic acid standard solution on both sides.
19.2.3 Development and color development: Place the spotted thin layer plate in a development tank pre-filled with a developer (17.12.1 or 17.12.2), with filter paper attached around the development tank. When the solvent front extends to more than 10 cm, take it out and evaporate it in the air. Spray the developer. The spots are yellow and the background is blue. The amount of cyclohexylaminosulfonic acid in the sample is quantitatively compared with the depth of the standard spot (when using the developer 17.12.2, the relative transfer value of cyclohexylaminosulfonic acid is about 0.47, sorbic acid 0.73, benzoic acid 0.61, and saccharin 0.31). 20 Result calculation
See formula (3).
Wherein:
X=mlX1 000×1. 12 = 2. 8m×V101000
m × V2
X is the content of sodium cyclohexylaminosulfonate in the sample, in grams per kilogram (or grams per liter); Lg/kg (or g/L); m is the mass of cyclohexylaminosulfonic acid equivalent to the sample point, in milligrams (mg); m is the mass of the sample, in grams (g);
Vi is the volume of anhydrous ethanol added, in milliliters (mL); V is the volume of the sample during the measurement, in milliliters (mL); 10 is the volume of the ether extract absorbed during the measurement, in milliliters (mL); - the total volume of the sample ether extract, in milliliters (mL); 25 -
1.12 is the mass of 1.00g of cyclohexylaminosulfonic acid equivalent to sodium cyclohexylaminosulfonate, in grams (g). The calculation result shall retain two significant figures.
21 Precision
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 28% of the arithmetic mean. This standard can simultaneously determine ingredients such as sorbic acid, benzoic acid, and saccharin. (3)
Tip: This standard content only shows part of the intercepted content of the complete standard. If you need the complete standard, please go to the top to download the complete standard document for free.