1 Scope
This standard specifies the method for determining phosphates in foods.
This standard is applicable to the determination of phosphate, pyrophosphate, hexametaphosphate, trimetaphosphate and tripolyphosphate in foods.
2 Principles
Extract and purify the specimen by adopting corresponding methods, take potassium hydroxide solution as eluent, separate with anion exchange column and detect with electrical conductivity detector. Determine the nature with retention time and determine the quantity with the external standard method.
3 Reagents and Materials
Unless otherwise specified, guaranteed reagents and Class I water (defined in GB/T 6682) are adopted for the purposes of this method.
3.1 Reagents
3.1.1 Sodium hydroxide (NaOH).
3.1.2 Potassium hydroxide (KOH).
3.1.3 Methanol (CH3OH): chromatographically pure.
3.2 Reagent preparation
3.2.1 Sodium hydroxide solution (10mmol/L): weigh 0.4g sodium hydroxide, dissolve it with water and scale the volume to 1,000mL.
3.2.2 Sodium hydroxide solution (50mmol/L): weigh 2.0g sodium hydroxide, dissolve it with water and scale the volume to 1,000mL.
3.3 Standard products
3.3.1 Sodium phosphate (Na3PO4) standard solution (1,000 mg/L, water based).
3.3.2 Sodium pyrophosphate (Na4P2O7) standard solution (1,000 mg/L, water based).
3.3.3 Sodium hexametaphosphate [(NaPO3)6] standard solution (1,000mg/L, water based).
3.3.4 Sodium trimetaphosphate [(NaPO3)3] standard product: purity ≥98%.
3.3.5 Sodium tripolyphosphate (Na5P3O10) standard product: purity ≥98%.
3.4 Preparation of standard solution
3.4.1 Intermediate (stock) phosphate anion standard solution (100mg/L): pipet 17.3mL standard sodium phosphate solution and put it in a 100mL volumetric flask, dilute it to the scale with 10 mmol/L sodium hydroxide solution, and the phosphate anion content per liter of the solution is 0.1g.
3.4.2 Pyrophosphate anion standard stock solution (100mg/L): pipet 15.3 mL sodium pyrophosphate standard solution and put it in a 100mL volumetric flask, dilute it to the scale with 10 mmol/L sodium hydroxide solution, and the pyrophosphate anion content per liter of the solution is 0.1g.
3.4.3 Hexametaphosphate anion standard intermediate (stock) solution (100mg/L): pipet 12.9 mL sodium hexametaphosphate standard solution and put it in a 100mL volumetric flask, dilute it to the scale with 10 mmol/L sodium hydroxide solution, and the hexametaphosphate anion content per liter of the solution is 0.1g.
3.4.4 Trimetaphosphate anion standard intermediate (stock) solution (1,000mg/L): dry the sodium trimetaphosphate in 103℃±2℃ drying oven for 3h, cool it down to ambient temperature in the dryer, accurately weigh 0.132 g sodium trimetaphosphate standard products (to the nearest of 0.0001g), dilute it to 100mL with 10 mmol/L sodium hydroxide solution, and the trimetaphosphate anion content per liter of the solution is 1.0g.
3.4.5 Tripolyphosphate anion standard stock solution (1,000mg/L): dry the sodium tripolyphosphate in 103℃±2℃ drying oven for 3h, cool it down to ambient temperature in the dryer, accurately weigh 0.148 g sodium tripolyphosphate standard products (to the nearest of 0.0001g), dilute it to 100mL with 10 mmol/L sodium hydroxide solution, and the tripolyphosphate anion content per liter of the solution is 1.0g.
3.4.6 Standard curve working solution: pipet five kinds of phosphate anion standard stock solutions, and dilute them with 10 mmol/L sodium hydroxide solution so as to prepare series of standard solutions, with the concentrations of the phosphate anion standard stock solutions detailed in Table 1.
Table 1 Concentrations of Five Kinds of Phosphate Anion Standard Working Solutions In: mg/L
Series of standard curves 1 2 3 4 5
Orthophosphate 0.00 0.300 1.00 5.00 10.0
Pyrophosphate anion 0.00 0.300 1.00 5.00 10.0
Trimetaphosphate anion 0.00 0.300 1.00 5.00 10.0
Tripolyphosphate anion 0.00 0.300 1.00 5.00 10.0
Hexametaphosphate anion 0.00 1.00 3.00 15.0 30.0
4 Instruments and Apparatus
4.1 Ion chromatograph: including electrical conductivity detector, equipped with suppressor, gradient pump or eluent generator; high-capacity anion exchange column; and 100 μL quantitative loop.
4.2 Food grinder.
4.3 Ultrasonic cleaner: allowing for temperature control at 80℃, 60Hz.
4.4 Balance: with sensibility of 0.1mg and 1mg.
4.5 Centrifuge: rotation speed ≥8,000 r/min; temperature may be adjusted to 4℃.
4.6 0.45 μm water-based filter membrane (with syringe filter).
4.7 Purification column: including OnGuard II RP column, Ag column and Na column1), or equivalent column.
4.8 Syringe: 1.0 mL and 5.0 mL.
Note: prior to use, all the glass wares shall be soaked with 2 mol/L potassium hydroxide solution and water for 4 h in sequence respectively, after which they shall be washed with water for 3~5 times and dried in the air for standby.
5 Analysis Steps
5.1 Specimen preparation
5.1.1 Fish, meat and their products: take 500g fish, meat or their products from the edible part, pound it to pieces and mix it uniformly, and preserve it by freezing.
5.1.2 Vegetables and fruits: take 500g from edible part, wash, air dry, cut up and mix it uniformly, and make it into homogenate with tissue mixer for future use.
5.1.3 Milk powder, dairy product drinks or beverages: shake repeatedly and reverse the container so that the sample is mixed sufficiently, until the sample homogenizes.
5.1.4 Sample of solid grease or fat, etc.: take 500g sample and grind it into homogeneous slurry; excessive heat shall be avoided during grinding process so as to avoid moisture loss.
5.1.5 Coarse cereals, wheat flour and its product, jelly, chocolate, candy, puffed food, cooked nuts and seeds: take 500 g from the edible part and mix it well with grinder for future use.
5.1.6 Condiments: as for condiment in powder form, shake repeatedly and reverse the container to mix the sample sufficiently; as for solid condiment in large particle form, take 500 g and mix it uniformly by using grinder for future use.
5.1.7 Desserts made of cereals and starch, rice flour, formula foods and complementary foods for infant and young children: shake repeatedly and reverse the container to mix the sample uniformly until it homogenizes.
Note: in the process of specimen preparation, the sample shall be protected from being polluted.
5.2 Sample pretreatment
5.2.1 Sample extraction
5.2.1.1 Vegetables and fruits, jelly, chocolate and candy, coarse cereals, wheat flour and its product, milk powder, dairy product drinks or beverages: weigh 2.5g (to the nearest of 0.001g, the sampling quantity of the specimen may be adjusted properly) of specimen, wash it with 50 mmol/L sodium hydroxide solution and pour it into a 50mL colorimetrical cylinder, mix it uniformly and dilute it to the scale, carry out ultrasonic extraction at 80℃ for 30min, shake it once every other 5min, and keep the stationary phase decentralized completely. Cool it to the ambient temperature, filter the solution with filter paper; take the filtrate and centrifuge it at 4℃ at the speed of 8,000 r/min for 10 min, and take the supernatant for future use.
5.2.1.2 Puffed food, cooked nuts and seeds, desserts made of cereals and starch, rice flour, formula foods and complementary foods for infant and young children: Weigh 2.5g (to the nearest of 0.001g, the sampling quantity of the specimen may be adjusted properly) of specimen, wash it with 50 mmol/L sodium hydroxide solution and pour it into a 25mL colorimetrical cylinder, mix it uniformly and dilute it to the scale, carry out ultrasonic extraction at 80℃ for 30min, shake it once every other 5min, and keep the stationary phase decentralized completely. Cool it to the ambient temperature, filter the solution with filter paper; take the filtrate and centrifuge it at 4℃ at the speed of 8,000 r/min for 10 min, and take the supernatant for future use.
5.2.1.3 Grease, fat and condiment: weigh 1g (to the nearest of 0.001g, the sampling quantity of the specimen may be adjusted properly) of specimen, wash it with 50 mmol/L sodium hydroxide solution and pour it into a 50mL colorimetric tube, mix it uniformly and dilute it to the scale, carry out ultrasonic extraction at 80℃ for 30min, shake it once every other 5min, and keep the stationary phase decentralized completely. Cool it to the ambient temperature, filter the solution with filter paper; take the filtrate and centrifuge it at 4℃ and at 8,000 r/min for 10 min, and take the water phase clear liquid for future use.
5.2.1.4 Fish, meat and their products: weigh 2.5g (to the nearest of 0.001g, the sampling quantity of the specimen may be adjusted properly) of specimen, wash it with 50 mmol/L sodium hydroxide solution and pour it into a 100mL colorimetric tube, mix it uniformly and dilute it to the scale, carry out ultrasonic extraction at 80℃ for 30min, shake it once every other 5min, and keep the stationary phase decentralized completely. Cool it to the ambient temperature, filter the solution with filter paper; take the filtrate and centrifuge it at 4℃ and at 8,000 r/min for 10 min, and take the supernatant for future use.
1 Scope
2 Principles
3 Reagent and Materials
4 Instruments and Apparatus
5 Analysis Steps
6 Expression of Analysis Results
7 Precision
8 Others
Appendix A Conversion Coefficient of Polyphosphate Anion
Appendix B Ion Chromatograms for Five Kinds of Phosphate Anion Standard Solutions
1 Scope
This standard specifies the method for determining phosphates in foods.
This standard is applicable to the determination of phosphate, pyrophosphate, hexametaphosphate, trimetaphosphate and tripolyphosphate in foods.
2 Principles
Extract and purify the specimen by adopting corresponding methods, take potassium hydroxide solution as eluent, separate with anion exchange column and detect with electrical conductivity detector. Determine the nature with retention time and determine the quantity with the external standard method.
3 Reagents and Materials
Unless otherwise specified, guaranteed reagents and Class I water (defined in GB/T 6682) are adopted for the purposes of this method.
3.1 Reagents
3.1.1 Sodium hydroxide (NaOH).
3.1.2 Potassium hydroxide (KOH).
3.1.3 Methanol (CH3OH): chromatographically pure.
3.2 Reagent preparation
3.2.1 Sodium hydroxide solution (10mmol/L): weigh 0.4g sodium hydroxide, dissolve it with water and scale the volume to 1,000mL.
3.2.2 Sodium hydroxide solution (50mmol/L): weigh 2.0g sodium hydroxide, dissolve it with water and scale the volume to 1,000mL.
3.3 Standard products
3.3.1 Sodium phosphate (Na3PO4) standard solution (1,000 mg/L, water based).
3.3.2 Sodium pyrophosphate (Na4P2O7) standard solution (1,000 mg/L, water based).
3.3.3 Sodium hexametaphosphate [(NaPO3)6] standard solution (1,000mg/L, water based).
3.3.4 Sodium trimetaphosphate [(NaPO3)3] standard product: purity ≥98%.
3.3.5 Sodium tripolyphosphate (Na5P3O10) standard product: purity ≥98%.
3.4 Preparation of standard solution
3.4.1 Intermediate (stock) phosphate anion standard solution (100mg/L): pipet 17.3mL standard sodium phosphate solution and put it in a 100mL volumetric flask, dilute it to the scale with 10 mmol/L sodium hydroxide solution, and the phosphate anion content per liter of the solution is 0.1g.
3.4.2 Pyrophosphate anion standard stock solution (100mg/L): pipet 15.3 mL sodium pyrophosphate standard solution and put it in a 100mL volumetric flask, dilute it to the scale with 10 mmol/L sodium hydroxide solution, and the pyrophosphate anion content per liter of the solution is 0.1g.
3.4.3 Hexametaphosphate anion standard intermediate (stock) solution (100mg/L): pipet 12.9 mL sodium hexametaphosphate standard solution and put it in a 100mL volumetric flask, dilute it to the scale with 10 mmol/L sodium hydroxide solution, and the hexametaphosphate anion content per liter of the solution is 0.1g.
3.4.4 Trimetaphosphate anion standard intermediate (stock) solution (1,000mg/L): dry the sodium trimetaphosphate in 103℃±2℃ drying oven for 3h, cool it down to ambient temperature in the dryer, accurately weigh 0.132 g sodium trimetaphosphate standard products (to the nearest of 0.0001g), dilute it to 100mL with 10 mmol/L sodium hydroxide solution, and the trimetaphosphate anion content per liter of the solution is 1.0g.
3.4.5 Tripolyphosphate anion standard stock solution (1,000mg/L): dry the sodium tripolyphosphate in 103℃±2℃ drying oven for 3h, cool it down to ambient temperature in the dryer, accurately weigh 0.148 g sodium tripolyphosphate standard products (to the nearest of 0.0001g), dilute it to 100mL with 10 mmol/L sodium hydroxide solution, and the tripolyphosphate anion content per liter of the solution is 1.0g.
3.4.6 Standard curve working solution: pipet five kinds of phosphate anion standard stock solutions, and dilute them with 10 mmol/L sodium hydroxide solution so as to prepare series of standard solutions, with the concentrations of the phosphate anion standard stock solutions detailed in Table 1.
Table 1 Concentrations of Five Kinds of Phosphate Anion Standard Working Solutions In: mg/L
Series of standard curves 1 2 3 4 5
Orthophosphate 0.00 0.300 1.00 5.00 10.0
Pyrophosphate anion 0.00 0.300 1.00 5.00 10.0
Trimetaphosphate anion 0.00 0.300 1.00 5.00 10.0
Tripolyphosphate anion 0.00 0.300 1.00 5.00 10.0
Hexametaphosphate anion 0.00 1.00 3.00 15.0 30.0
4 Instruments and Apparatus
4.1 Ion chromatograph: including electrical conductivity detector, equipped with suppressor, gradient pump or eluent generator; high-capacity anion exchange column; and 100 μL quantitative loop.
4.2 Food grinder.
4.3 Ultrasonic cleaner: allowing for temperature control at 80℃, 60Hz.
4.4 Balance: with sensibility of 0.1mg and 1mg.
4.5 Centrifuge: rotation speed ≥8,000 r/min; temperature may be adjusted to 4℃.
4.6 0.45 μm water-based filter membrane (with syringe filter).
4.7 Purification column: including OnGuard II RP column, Ag column and Na column1), or equivalent column.
4.8 Syringe: 1.0 mL and 5.0 mL.
Note: prior to use, all the glass wares shall be soaked with 2 mol/L potassium hydroxide solution and water for 4 h in sequence respectively, after which they shall be washed with water for 3~5 times and dried in the air for standby.
5 Analysis Steps
5.1 Specimen preparation
5.1.1 Fish, meat and their products: take 500g fish, meat or their products from the edible part, pound it to pieces and mix it uniformly, and preserve it by freezing.
5.1.2 Vegetables and fruits: take 500g from edible part, wash, air dry, cut up and mix it uniformly, and make it into homogenate with tissue mixer for future use.
5.1.3 Milk powder, dairy product drinks or beverages: shake repeatedly and reverse the container so that the sample is mixed sufficiently, until the sample homogenizes.
5.1.4 Sample of solid grease or fat, etc.: take 500g sample and grind it into homogeneous slurry; excessive heat shall be avoided during grinding process so as to avoid moisture loss.
5.1.5 Coarse cereals, wheat flour and its product, jelly, chocolate, candy, puffed food, cooked nuts and seeds: take 500 g from the edible part and mix it well with grinder for future use.
5.1.6 Condiments: as for condiment in powder form, shake repeatedly and reverse the container to mix the sample sufficiently; as for solid condiment in large particle form, take 500 g and mix it uniformly by using grinder for future use.
5.1.7 Desserts made of cereals and starch, rice flour, formula foods and complementary foods for infant and young children: shake repeatedly and reverse the container to mix the sample uniformly until it homogenizes.
Note: in the process of specimen preparation, the sample shall be protected from being polluted.
5.2 Sample pretreatment
5.2.1 Sample extraction
5.2.1.1 Vegetables and fruits, jelly, chocolate and candy, coarse cereals, wheat flour and its product, milk powder, dairy product drinks or beverages: weigh 2.5g (to the nearest of 0.001g, the sampling quantity of the specimen may be adjusted properly) of specimen, wash it with 50 mmol/L sodium hydroxide solution and pour it into a 50mL colorimetrical cylinder, mix it uniformly and dilute it to the scale, carry out ultrasonic extraction at 80℃ for 30min, shake it once every other 5min, and keep the stationary phase decentralized completely. Cool it to the ambient temperature, filter the solution with filter paper; take the filtrate and centrifuge it at 4℃ at the speed of 8,000 r/min for 10 min, and take the supernatant for future use.
5.2.1.2 Puffed food, cooked nuts and seeds, desserts made of cereals and starch, rice flour, formula foods and complementary foods for infant and young children: Weigh 2.5g (to the nearest of 0.001g, the sampling quantity of the specimen may be adjusted properly) of specimen, wash it with 50 mmol/L sodium hydroxide solution and pour it into a 25mL colorimetrical cylinder, mix it uniformly and dilute it to the scale, carry out ultrasonic extraction at 80℃ for 30min, shake it once every other 5min, and keep the stationary phase decentralized completely. Cool it to the ambient temperature, filter the solution with filter paper; take the filtrate and centrifuge it at 4℃ at the speed of 8,000 r/min for 10 min, and take the supernatant for future use.
5.2.1.3 Grease, fat and condiment: weigh 1g (to the nearest of 0.001g, the sampling quantity of the specimen may be adjusted properly) of specimen, wash it with 50 mmol/L sodium hydroxide solution and pour it into a 50mL colorimetric tube, mix it uniformly and dilute it to the scale, carry out ultrasonic extraction at 80℃ for 30min, shake it once every other 5min, and keep the stationary phase decentralized completely. Cool it to the ambient temperature, filter the solution with filter paper; take the filtrate and centrifuge it at 4℃ and at 8,000 r/min for 10 min, and take the water phase clear liquid for future use.
5.2.1.4 Fish, meat and their products: weigh 2.5g (to the nearest of 0.001g, the sampling quantity of the specimen may be adjusted properly) of specimen, wash it with 50 mmol/L sodium hydroxide solution and pour it into a 100mL colorimetric tube, mix it uniformly and dilute it to the scale, carry out ultrasonic extraction at 80℃ for 30min, shake it once every other 5min, and keep the stationary phase decentralized completely. Cool it to the ambient temperature, filter the solution with filter paper; take the filtrate and centrifuge it at 4℃ and at 8,000 r/min for 10 min, and take the supernatant for future use.
Contents of GB 5009.256-2016
1 Scope
2 Principles
3 Reagent and Materials
4 Instruments and Apparatus
5 Analysis Steps
6 Expression of Analysis Results
7 Precision
8 Others
Appendix A Conversion Coefficient of Polyphosphate Anion
Appendix B Ion Chromatograms for Five Kinds of Phosphate Anion Standard Solutions
