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This standard replaces GB/T 5009.141-2003 Determination of Allura Red in Foods.
The following main changes have been made with respect to GB/T 5009.141-2003 (the previous edition):
——This standard is renamed as National Food Safety Standard - Determination of Allura Red in Foods;
——Grades and molecular formula of reagents are added;
——Standard product is added.
National Food Safety Standard
Determination of Allura Red in Foods
1 Scope
This standard specifies the determination method of allura red in soda water, hard candies, pastries and ice cream.
This standard is applicable to the determination of allura red in soda water, hard candies, pastries and ice cream.
2 Principle
Allura red is adsorbed by polyamide under acidic conditions and desorbed under alkaline conditions; separate by paper chromatography and compare with the standard for qualitative determination and quantitative determination.
3 Reagents and Materials
Unless otherwise specified, analytically-pure reagents and Class-I water (defined in GB/T 6682) are adopted for the purpose of this method.
3.1 Reagents
3.1.1 Methanol (CH3OH).
3.1.2 Petroleum ether: with boiling range of 30℃~60℃.
3.1.3 Sulfuric acid (H2SO4): guaranteed reagent.
3.1.4 Ethanol (CH3CH2OH).
3.1.5 Ammonia (NH3·H2O): with content of 20%~25%.
3.1.6 Citric acid (C6H8O7·H2O).
3.1.7 Sodium tungstate (Na2WO4·2H2O).
3.1.8 Butanone (C4H8O).
3.1.9 Sodium citrate (C6H5Na3O7).
3.1.10 n-butyl alcohol (C4H10O).
3.1.11 Sea sand.
3.1.12 Formic acid (HCOOH).
3.2 Preparation of reagents
3.2.1 Sulfuric acid solution (10%, volume fraction): measure 1 mL sulfuric acid, slowly pour it into 8 mL water, mix well, cool, scale the volume with water to 10 mL, and mix well.
3.2.2 Ethanol-ammonia solution: take 2 mL ammonia, and add 70% (volume fraction) ethanol to 100 mL.
3.2.3 Ethanol solution (50%, volume fraction): measure 50 mL absolute ethanol and 50 mL water and mix well.
3.2.4 Citric acid solution (200g/L): weigh 20 g citric acid, add water to 100 mL, dissolve and mix well.
3.2.5 Sodium tungstate solution (100 g/L): weigh 10 g sodium tungstate, add water to 100 mL, dissolve and mix well.
3.2.6 Ammonia solution (1%, volume fraction): measure 1 mL ammonia, add water to 100 mL and mix well.
3.2.7 Sodium citrate solution (2.5%, volume fraction): weigh 2.5 g citric acid, add water to 100 mL, dissolve and mix well.
3.2.8 Methanol-formic acid solution (6:4, volume fraction): measure 60 mL methanol and 40 mL formic acid and mix well.
3.2.9 Developer 1: butanone + propaneol + water + ammonia (7+3+3+0.5).
3.2.10 Developer 2: n-butyl alcohol + absolute alcohol + 1% ammonia solution (6+2+3).
3.2.11 Developer 3: 2.5% sodium citrate + ammonia + ethanol (8+1+2).
3.3 Standard product
Allura red (CAS: 25956-17-6).
3.4 Preparation of standard solutions
3.4.1 Preparation of allura red standard stock solution: accurately weigh 0.025 g allura red (accurate to 0.0001 g, the mass of converted pure product according to actual purity of allura red), dissolve in water and scale the volume to 25 mL, with the concentration of 1.0 mg/mL.
3.4.2 Allura red standard working solution (0.1 mg/mL): pipet 5.0 mL allura red standard stock solution into a 50 mL volumetric flask and dilute with water to 50 mL.
4 Instruments and Apparatus
4.1 Visible spectrophotometer.
4.2 Electronic balance: with sensibility of 0.001 g and 0.0001 g.
4.3 Micropipettor: 10 μL and 50 μL.
4.4 Developing tank.
4.5 Electric hair dryer.
4.6 Centrifuge.
4.7 Thermostat water bath.
5 Analysis Steps
5.1 Preparation of specimens
5.1.1 Soda water: heat the sample to decarbonate, weigh 10 g (accurate to 0.001 g) sample into a beaker, adjust the pH with 20% citric acid as acid value, add 0.5 g ~ 1.0 g polyamide to adsorb pigment, transfer all the polyamide for absorbing the pigment into a funnel for filtering, wash with acidic hot water with pH value of 4 for many times (about 200 mL) to wash out substances like sugar. If with natural pigment, wash with methanol-formic acid solution for 1~3 times (20 mL each time) until the washing liquid is colorless. Wash with 70℃ water for many times again until the effluent is neutral. In the washing process, stir sufficiently, desorb the pigment with fractions of ethanol-ammonia solution, collect all desorption solution, remove the ammonia on water bath, evaporate till about 2 mL, transfer into a 5 mL volumetric flask, wash the evaporation dish with 50% ethanol in several times, merge the washing solutions into a 5 mL volumetric flask, scale the volume with 50% ethanol to the scale. This solution is reserved for paper chromatogram.
5.1.2 Hard candies: weigh 10 g (accurate to 0.001 g) ground sample, add 30 mL water, heat the sample to dissolve; if the sample solution has relatively high pH value, adjust the pH value with citric acid solution (3.2.4) to about 4. Add 0.5 g ~ 1.0 g polyamide to adsorb pigment, transfer all the polyamide for absorbing the pigment into a funnel for filtering, wash with acidic hot water with pH value of 4 for many times (about 200 mL) to wash out substances like sugar. If with natural pigment, wash with methanol-formic acid solution for 1~3 times (20 mL each time) until the washing liquid is colorless. Wash with 70℃ water for many times again until the effluent is neutral. In the washing process, stir sufficiently, desorb the pigment with ethanol-ammonia solution in several times, collect all desorption solutions, remove the ammonia on water bath, evaporate till about 2 mL, transfer into a 5 mL volumetric flask, wash the evaporation dish with 50% ethanol in several times, merge the washing solutions into a 5 mL volumetric flask, scale the volume with 50% ethanol to the scale. This solution is reserved for paper chromatogram.
5.1.3 Pastries: weigh 10 g (accurate to 0.001 g) ground sample, add 30 mL petroleum ether to extract the fat for three times, blow dry with an electric blower, pour into a funnel, desorb the pigment with ethanol - ammonia solution, evaporate the desorption solution on water bath to 20 mL, add 1 mL sodium tungstate solution to precipitate the protein, vacuum for filtration, desorb the allura red on filter paper with ethanol - ammonia solution, remove ammonia on water bath, adjust the pH value to be acidic. Add 0.5 g ~ 1.0 g polyamide to adsorb pigment, transfer all the polyamide for absorbing the pigment into a funnel for filtering, wash with acidic hot water with pH value of 4 for many times (about 200 mL) to wash out substances like sugar. If with natural pigment, wash with methanol-formic acid solution for 1~3 times (20 mL each time) until the washing liquid is colorless. Wash with 70℃ water for many times again until the effluent is neutral. In the washing process, stir sufficiently, desorb the pigment with fractions of ethanol-ammonia solution, collect all desorption solutions, remove the ammonia on water bath, evaporate till about 2 mL, transfer into a 5 mL volumetric flask, wash the evaporation dish with 50% ethanol in several times, merge the washing solutions into a 5 mL volumetric flask, scale the volume with 50% ethanol to the scale. This solution is reserved for paper chromatogram.
5.1.4 Ice cream: weigh 10 g (accurate to 0.001 g) uniform sample into a beaker, add 20 g sea sand and 15 mL petroleum ether to extract the fat for two times, pour out the petroleum ether, remove the petroleum ether on 50℃ water bath, add ethanol - ammonia solution to desorb the allura red, pour the desorption solution into a 100 mL evaporation dish until the desorption solution is colorless. Remove the ethanol in the desorption solution on water bath, add 1 mL sulfuric acid (1+10) and 1 mL sodium tungstate solution to precipitate the protein when the volume is about 20 mL, place for 2 min, adjust the pH value with ethanol - ammonia solution to be alkaline, transfer the solution into a centrifugal tube to centrifuge for 15 min at a rotation speed of 5,000 r/min, pour off the supernatant, remove the ethanol on water bath, adjust the pH value with citric acid solution to be acidic, add 0.5 g ~ 1.0 g polyamide to adsorb pigment, transfer all the polyamide for absorbing the pigment into a funnel for filtering, wash with acidic hot water with pH value of 4 for many times (about 200 mL) to wash out substances like sugar. If with natural pigment, wash with methanol-formic acid solution for 1~3 times (20 mL each time) until the washing liquid is colorless. Wash with 70℃ water for many times again until the effluent is neutral . In the washing process, stir sufficiently, desorb the pigment with ethanol-ammonia solution, collect all desorption solutions, remove the ammonia on water bath, evaporate till about 2 mL, transfer into a 5 mL volumetric flask, wash the evaporation dish with 50% ethanol in several times, merge the washing solutions into a 5 mL volumetric flask, scale the volume with 50% ethanol to the scale. This solution is reserved for paper chromatogram.
Foreword i
1 Scope
2 Principle
3 Reagents and Materials
4 Instruments and Apparatus
5 Analysis Steps
6 Expression of Analysis Results
7 Accuracy
8 Other
