National Food Safety Standard
Determination of Synthetic Colour in Foods
食品安全国家标准
食品中合成着色剂的测定
1 Scope
This standard specifies the method for determination of synthetic colour (excluding aluminium lake) in beverages, integrated alcoholic beverages, hard candy, preserved fruits, starch jelly candy, marble chocolate and colored sugar coated products.
This standard is applicable to the determination of synthetic colour (excluding aluminium lake) in beverages, integrated alcoholic beverages, hard candy, preserved fruits, starch jelly candy, marble chocolate and colored sugar coated products.
2 Principle
Extract the synthetic colour in foods by polyamide adsorption method or liquid-liquid distribution, make it into water solution, inject it into high performance liquid chromatograph, and then carry out qualitative analysis according retention time and quantitative analysis by comparing with peak area after reversed-phase chromatography separation.
3 Reagents and Materials
Unless otherwise specified, the reagents adopted in this method are all analytically pure, and the water is Grade 1 water specified in GB/T 6682.
3.1 Reagents
3.1.1 Methanol (CH3OH): chromatographically pure.
3.1.2 N-hexane (C6H14).
3.1.3 Hydrochloric acid (HCl).
3.1.4 Glacial acetic acid (CH3COOH).
3.1.5 Formic acid (HCOOH).
3.1.6 Ammonium acetate (CH3COONH4).
3.1.7 Citric acid (C6H8O7·H2O).
3.1.8 Sodium sulfate (Na2SO4).
3.1.9 n-butyl alcohol (C4H10O).
3.1.10 Tri-n-octylamine (C24H51N).
3.1.11 Absolute ethanol (CH3CH2OH).
3.1.12 Ammonia water (NH3·H2O): with content of 20%~25%.
3.1.13 Polyamide powder (Nylon 6): passing through a 200μm (-mesh) sieve.
3.2 Reagent preparation
3.2.1 Ammonium acetate solution (0.02mol/L): weigh 1.54g of ammonium acetate, add water to 1 000mL, dissolve and filter via 0.45μm microfiltration membrane.
3.2.2 Ammonia solution: measure 2mL of ammonia water, add water to 100mL and mix well.
3.2.3 Methanol-formic acid solution (6+4, volume ratio): measure 60mL of methanol, 40mL of formic acid and mix well.
3.2.4 Citric acid solution: weigh 20g of citric acid, add water to 100mL, dissolve and mix well.
3.2.5 Absolute ethanol-ammonia water-water solution (7+2+1, volume ratio): measure 70mL of absolute ethanol, 20mL of ammonia solution (3.2.2), 10mL of water and mix well.
3.2.6 Tri-n-octylamine-n-butyl alcohol solution (5%): measure 5mL of tri-n-octylamine, add n-butyl alcohol to 100mL and mix well.
3.2.7 Saturated sodium sulfate solution.
3.2.8 Water of pH6: add citric acid solution into water to adjust the pH value to 6.
3.2.9 Water of pH4: add citric acid solution into water to adjust the pH value to 4.
3.3 Standard substances
3.3.1 Tartrazine (CAS: 1934-21-0).
3.3.2 New red (CAS: 220658-76-4).
3.3.3 Amaranth (CAS: 915-67-3).
3.3.4 Ponceau 4R (CAS: 2611-82-7).
3.3.5 Sunset yellow FCF (CAS: 2783-94-0).
3.3.6 Brilliant blue FCF (CAS: 3844-45-9).
3.3.7 Erythrosine (CAS: 16423-68-0).
3.4 Preparation of standard solutions
3.4.1 Standard stock solutions of synthetic colour (1mg/mL): accurately weigh 0.1g (to the nearest of 0.000 1) of tartrazine, sunset yellow FCF, amaranth, ponceau 4R, new red, erythrosine and brilliant blue FCF respectively of which the mass are converted into 100% as per their purity; put them into a 100mL volumetric flask, add water of pH6 to the scale and obtain water solution (1.00mg/mL).
3.4.2 Standard working solution of synthetic colour (50μg/mL): dilute the standard stock solution by 20 times with water right before use, filter with 0.45μm microfiltration membrane and obtain the synthetic colour solution of 50.0μg/mL.
4 Instruments and Apparatus
4.1 High performance liquid chromatograph: equipped with diode array or ultraviolet detector.
4.2 Balance: with sensibility of 0.001g and 0.000 1g.
4.3 Thermostat water bath kettle.
4.4 G3 sintered funnel.
5 Analytical Procedures
5.1 Specimen preparation
5.1.1 Fruit juice beverage as well as fruit juice, carbonated beverages with fruity flavor etc.: weigh 20g~40g (to the nearest of 0.001g) of them and put into a 100mL beaker. For samples containing carbon dioxide, carbon dioxide shall be removed by heating or ultrasound.
5.1.2 Integrated alcoholic beverages: weigh 20g~40g (to the nearest of 0.001g) of them and put into a 100mL beaker, add several small porcelain pieces and heat to remove ethanol.
5.1.3 Hard candy, preserved fruits, starch jelly, etc.: weigh 5g~10g (to the nearest of 0.001g) crushed sample and put into a 100mL small beaker, add 30mL of water and dissolve warmly; if the pH value of sample solution is relatively high, add citric acid solution to adjust the pH value to about 6.
5.1.4 Marble chocolate and colored sugar coated products: weigh 5g~10g (to the nearest of 0.001g) of them and put into a 100mL small beaker, wash the pigments repeatedly with water until the marble chocolate is pigment-free and collect the pigment rinsing solution as the sample solution.
5.2 Pigment extraction
5.2.1 Polyamide adsorption method: add citric acid solution to adjust the pH value of sample solution to 6, heat it to 60℃; add a little water into 1g of polyamide powder to mix into mucus, pour it into the sample solution, stir for a while, wash for 3~5 times with water in 60℃ and pH4 after suction filtration with G3 sintered funnel. After that, wash for 3~5 times with methanol-formic acid mixed solution (samples containing erythrosine are treated with the method in 5.2.2), and then rinse to normal pH and desorb for 3~5 times with ethanol-ammonia water-water mixed solution until pigments are desorbed completely; collect the desorption solution, neutralize with acetic acid and evaporate to nearly dry, dissolve with water and scale the volume to 5mL. Filter the final solution with 0.45μm microfiltration membrane and put it into high performance liquid chromatograph for analysis.
5.2.2 Liquid-liquid distribution (applicable to samples containing erythrosine): put the prepared sample solution into the separating funnel, add into 2mL of hydrochloric acid, and 10mL~20mL of tri-n-octylamine-n-butyl alcohol solution (5%), extract after shaking, separate the organic phase, extract repeatedly until organic phases are colorless; and then combine organic phases, wash for twice with saturated sodium sulfate solution and 10mL for each time, separate organic phases, put into evaporation dish, concentrate to 10mL by heating in water bath, transfer to separating funnel, add 10mL of n-hexane, mix well and extract for twice or three times with ammonia solution (5mL for each time); after that, combine ammonia solution layer (containing water soluble and acidic pigments), wash for twice with n-hexane, neutralize the ammonia water layer by adding acetic acid, evaporate to nearly dry by heating in water bath and scale the volume to 5mL. Filter the final solution with 0.45μm microfiltration membrane and put it into high performance liquid chromatograph for analysis.
Foreword i
1 Scope
2 Principle
3 Reagents and Materials
4 Instruments and Apparatus
5 Analytical Procedures
6 Expression of Analysis Results
7 Precision
8 Others
Appendix A Standard Chromatogram of Colorant
National Food Safety Standard
Determination of Synthetic Colour in Foods
食品安全国家标准
食品中合成着色剂的测定
1 Scope
This standard specifies the method for determination of synthetic colour (excluding aluminium lake) in beverages, integrated alcoholic beverages, hard candy, preserved fruits, starch jelly candy, marble chocolate and colored sugar coated products.
This standard is applicable to the determination of synthetic colour (excluding aluminium lake) in beverages, integrated alcoholic beverages, hard candy, preserved fruits, starch jelly candy, marble chocolate and colored sugar coated products.
2 Principle
Extract the synthetic colour in foods by polyamide adsorption method or liquid-liquid distribution, make it into water solution, inject it into high performance liquid chromatograph, and then carry out qualitative analysis according retention time and quantitative analysis by comparing with peak area after reversed-phase chromatography separation.
3 Reagents and Materials
Unless otherwise specified, the reagents adopted in this method are all analytically pure, and the water is Grade 1 water specified in GB/T 6682.
3.1 Reagents
3.1.1 Methanol (CH3OH): chromatographically pure.
3.1.2 N-hexane (C6H14).
3.1.3 Hydrochloric acid (HCl).
3.1.4 Glacial acetic acid (CH3COOH).
3.1.5 Formic acid (HCOOH).
3.1.6 Ammonium acetate (CH3COONH4).
3.1.7 Citric acid (C6H8O7·H2O).
3.1.8 Sodium sulfate (Na2SO4).
3.1.9 n-butyl alcohol (C4H10O).
3.1.10 Tri-n-octylamine (C24H51N).
3.1.11 Absolute ethanol (CH3CH2OH).
3.1.12 Ammonia water (NH3·H2O): with content of 20%~25%.
3.1.13 Polyamide powder (Nylon 6): passing through a 200μm (-mesh) sieve.
3.2 Reagent preparation
3.2.1 Ammonium acetate solution (0.02mol/L): weigh 1.54g of ammonium acetate, add water to 1 000mL, dissolve and filter via 0.45μm microfiltration membrane.
3.2.2 Ammonia solution: measure 2mL of ammonia water, add water to 100mL and mix well.
3.2.3 Methanol-formic acid solution (6+4, volume ratio): measure 60mL of methanol, 40mL of formic acid and mix well.
3.2.4 Citric acid solution: weigh 20g of citric acid, add water to 100mL, dissolve and mix well.
3.2.5 Absolute ethanol-ammonia water-water solution (7+2+1, volume ratio): measure 70mL of absolute ethanol, 20mL of ammonia solution (3.2.2), 10mL of water and mix well.
3.2.6 Tri-n-octylamine-n-butyl alcohol solution (5%): measure 5mL of tri-n-octylamine, add n-butyl alcohol to 100mL and mix well.
3.2.7 Saturated sodium sulfate solution.
3.2.8 Water of pH6: add citric acid solution into water to adjust the pH value to 6.
3.2.9 Water of pH4: add citric acid solution into water to adjust the pH value to 4.
3.3 Standard substances
3.3.1 Tartrazine (CAS: 1934-21-0).
3.3.2 New red (CAS: 220658-76-4).
3.3.3 Amaranth (CAS: 915-67-3).
3.3.4 Ponceau 4R (CAS: 2611-82-7).
3.3.5 Sunset yellow FCF (CAS: 2783-94-0).
3.3.6 Brilliant blue FCF (CAS: 3844-45-9).
3.3.7 Erythrosine (CAS: 16423-68-0).
3.4 Preparation of standard solutions
3.4.1 Standard stock solutions of synthetic colour (1mg/mL): accurately weigh 0.1g (to the nearest of 0.000 1) of tartrazine, sunset yellow FCF, amaranth, ponceau 4R, new red, erythrosine and brilliant blue FCF respectively of which the mass are converted into 100% as per their purity; put them into a 100mL volumetric flask, add water of pH6 to the scale and obtain water solution (1.00mg/mL).
3.4.2 Standard working solution of synthetic colour (50μg/mL): dilute the standard stock solution by 20 times with water right before use, filter with 0.45μm microfiltration membrane and obtain the synthetic colour solution of 50.0μg/mL.
4 Instruments and Apparatus
4.1 High performance liquid chromatograph: equipped with diode array or ultraviolet detector.
4.2 Balance: with sensibility of 0.001g and 0.000 1g.
4.3 Thermostat water bath kettle.
4.4 G3 sintered funnel.
5 Analytical Procedures
5.1 Specimen preparation
5.1.1 Fruit juice beverage as well as fruit juice, carbonated beverages with fruity flavor etc.: weigh 20g~40g (to the nearest of 0.001g) of them and put into a 100mL beaker. For samples containing carbon dioxide, carbon dioxide shall be removed by heating or ultrasound.
5.1.2 Integrated alcoholic beverages: weigh 20g~40g (to the nearest of 0.001g) of them and put into a 100mL beaker, add several small porcelain pieces and heat to remove ethanol.
5.1.3 Hard candy, preserved fruits, starch jelly, etc.: weigh 5g~10g (to the nearest of 0.001g) crushed sample and put into a 100mL small beaker, add 30mL of water and dissolve warmly; if the pH value of sample solution is relatively high, add citric acid solution to adjust the pH value to about 6.
5.1.4 Marble chocolate and colored sugar coated products: weigh 5g~10g (to the nearest of 0.001g) of them and put into a 100mL small beaker, wash the pigments repeatedly with water until the marble chocolate is pigment-free and collect the pigment rinsing solution as the sample solution.
5.2 Pigment extraction
5.2.1 Polyamide adsorption method: add citric acid solution to adjust the pH value of sample solution to 6, heat it to 60℃; add a little water into 1g of polyamide powder to mix into mucus, pour it into the sample solution, stir for a while, wash for 3~5 times with water in 60℃ and pH4 after suction filtration with G3 sintered funnel. After that, wash for 3~5 times with methanol-formic acid mixed solution (samples containing erythrosine are treated with the method in 5.2.2), and then rinse to normal pH and desorb for 3~5 times with ethanol-ammonia water-water mixed solution until pigments are desorbed completely; collect the desorption solution, neutralize with acetic acid and evaporate to nearly dry, dissolve with water and scale the volume to 5mL. Filter the final solution with 0.45μm microfiltration membrane and put it into high performance liquid chromatograph for analysis.
5.2.2 Liquid-liquid distribution (applicable to samples containing erythrosine): put the prepared sample solution into the separating funnel, add into 2mL of hydrochloric acid, and 10mL~20mL of tri-n-octylamine-n-butyl alcohol solution (5%), extract after shaking, separate the organic phase, extract repeatedly until organic phases are colorless; and then combine organic phases, wash for twice with saturated sodium sulfate solution and 10mL for each time, separate organic phases, put into evaporation dish, concentrate to 10mL by heating in water bath, transfer to separating funnel, add 10mL of n-hexane, mix well and extract for twice or three times with ammonia solution (5mL for each time); after that, combine ammonia solution layer (containing water soluble and acidic pigments), wash for twice with n-hexane, neutralize the ammonia water layer by adding acetic acid, evaporate to nearly dry by heating in water bath and scale the volume to 5mL. Filter the final solution with 0.45μm microfiltration membrane and put it into high performance liquid chromatograph for analysis.
Contents of GB 5009.35-2016
Foreword i
1 Scope
2 Principle
3 Reagents and Materials
4 Instruments and Apparatus
5 Analytical Procedures
6 Expression of Analysis Results
7 Precision
8 Others
Appendix A Standard Chromatogram of Colorant
