GB 5009.244-2016National food safety standard - Determination of chlorine dioxide in food (English PDF)

3.1 Reagents

3.1.1 Glycine (C2H5NO2).

3.1.2 Ethylenediaminetetraacetic acid disodium (EDTA) (C10H14N2Na2O8).

3.1.3 Potassium iodide (KI). purity 99.5%.

3.1.4 Sodium chlorite (NaClO2).

3.1.5 Acetic acid (CH3COOH).

3.1.6 Sulfuric acid (H2SO4).

3.1.7 Anhydrous sodium sulfate (Na2SO4).

3.1.8 Sodium hydroxide (NaOH).

3.1.9 Potassium dihydrogen phosphate (KH2PO4).

3.1.10 N, N-diethyl-p-phenylenediamine (DPD).

3.1.11 Sodium thiosulfate (Na2S2O3).

3.1.12 Soluble starch.

3.1.13 Potassium dichromate (Cr2K2O7).

3.1.14 High-purity nitrogen.

3.2 Preparation of reagents

3.2.1 DPD colour developing agent (1 g/L). weigh 1 g of DPD (3.1.10) to dissolve in

chloride-free grade two water (3.2.2) containing 8 mL of sulfuric acid solution (1 + 3)

and 200 mg of EDTA; use chloride-free grade two water to dilute to 1,000 mL; store in

a brown glass bottle with stopper; store in a dark place.

3.2.2 Oxidizing chloride-free grade two water. add sodium sulfite (3.1.4) in grade two

water; reduce oxidizing chloride to chloride ion (no colour develops when checked

using DPD); carry out distillation. The water obtained is oxidizing chloride-free grade

two water.

3.2.3 Glycine solution (10%). take 10 g of glycine (3.1.1); use water to dissolve; make

up to 100 mL.

3.2.4 Sodium hydroxide solution (2 mol/L). weigh 80 g of sodium hydroxide; use water

to dissolve; make up to 1 L.

3.2.5 Sodium hydroxide solution (0.1 mol/L). weigh 4 g of sodium hydroxide; use water

to dissolve; make up to 1 L.

3.2.6 Sulfuric acid solution (1 mol/L). measure 54 mL of 98.4% sulfuric acid (3.1.6);

slowly add into about 300 mL of distilled water; after cooling, transfer to a volumetric

flask; use about 20 mL of distilled water to wash the container for 3 times; also transfer

the washings to the volumetric flask; use water to make up to 1 L.

3.2.7 Potassium dihydrogen phosphate [c(KH2PO4) = 0.1 mol/L]. use water to dissolve

13.61 g of potassium dihydrogen phosphate solution (3.1.9); make up to 1 L.

3.2.8 Phosphate buffer solution (pH = 6.5). take 500 mL of 0.1 mol/L KH2PO4 and 81

mL of 0.1 mol/L NaOH; use water to dilute to 1 L.

3.2.9 Sodium chlorite saturated solution. take an appropriate amount of sodium chlorite

(3.1.4) to place in a beaker; add a small amount of water; stir to make a saturated

solution (the solubility of sodium chlorite is quite high, so it shall be prepared in

accordance with the amount required).

3.2.10 Preparation of sodium thiosulfate standard solution [c(Na2S2O3) = 0.1000 mol/L].

weigh 16 g of anhydrous sodium sulfate (3.1.7); dissolve in 1 L of water; heat to boil

for 10 min; allow to cool; filter to use after two weeks' storage in dark.

3.2.11 Calibration of sodium thiosulfate standard solution [c(Na2S2O3) = 0.1000 mol/L].

weigh 0.15 g of benchmark potassium dichromate (3.1.13) dried to constant weight at

120°C, accurate to 0.000 1 g; place into an iodine flask; add 25 mL of water; after

dissolving, add 2 g of potassium iodide and 20 mL of 20% sulfuric acid solution; shake

up; store in dark for 10 min. Add 150 mL of water; use prepared sodium thiosulfate

standard solution (3.2.10) for titration; add 3 mL of starch indicating liquid (5 g/L)

(3.1.12) near the end; continue titration until the solution changes from blue into bright

green. Meanwhile, carry out blank test.

3.2.12 Starch indicator (5 g/L). weigh 5.0 g of starch to place into a beaker of 50 mL;

measure 1 L of distilled water; use several drops to make starch into a paste; then take

about 900 mL of water to heat to slightly boiling when pour the pasty starch in; use the

residual distilled water to wash the beaker of 50 mL for 3 times; pour the washings into

the beaker; then add 1 drop of 10% hydrochloric acid; slightly boil for 3 min.

3.2.13 Starch solution (1%). weigh 1 g of soluble starch; use a small amount of water

to make a paste; use 100 mL of boiling water to dissolve it; heat to boil until clarified.

3.3 Standard substances

3.3.1 Preparation of chlorine dioxide standard stock solution

Add 300 mL of pure water into bottle A; connect the glass at one end of bottle A to air

compressor and another glass tube to bottle B. Bottle B is a high-strength borosilicate

glass bottle with three glass tubes at the opening; the first one is inserted to 5 mm to

the bottom to introduce air; the second is connected with a graduated cylindrical

separating funnel with its lower end stretching to below the liquid level; the lower end

of the third is apart from the liquid level and the upper end is connected with bottle C.

Dissolve 10 g of sodium chlorite in 750 mL of pure water and pour into bottle B; load

3.3.2 Calibration of chlorine dioxide standard stock solution

Add 100 mL of pure water, 1 g of potassium iodide and 5 mL of acetic acid into an

iodine flask of 250 mL; shake the iodine flask to make potassium iodide fully dissolve.

Add 10.00 mL of chlorine dioxide standard solution before storing in dark for 5 min.

Use sodium thiosulfate standard solution of 0.1 mol/L (3.2.10) for titration until the

solution appears faint yellow; add 1 mL of starch solution; continue titration to the end.

Add the same amount of reagents in accordance with the above-mentioned

procedures (but only chlorine dioxide is not added); use sodium thiosulfate standard

solution for titration until blue disappears; record the amount of blank used.

The concentration of chlorine dioxide standard stock solution is calculated in

accordance with Equation (1).

where.

rho(ClO2) – the concentration of chlorine dioxide standard stock solution, in mg/mL;

c – the concentration of sodium thiosulfate standard solution, in mol/L;

V1 – the volume of sodium thiosulfate standard solution used for titration of chlorine

dioxide, in mL;

Separating funnel

V0 – the volume of sodium thiosulfate standard solution used for titration of blank, in

mL;

V2 – the volume of chlorine dioxide, in mL;

13.49 – the mass of chlorine dioxide, expressed in mg, which is equivalent to 1.00 mL

of sodium thiosulfate standard solution [c(Na2S2O3) = 0.1000 mol/L] (3.2.10).

3.4 Preparation of chlorine dioxide standard solution

4.1 Ultraviolet-visible spectrophotometer.

4.2 Homogenizer. >= 8,000 r/min.

4.3 Centrifugal machine. >= 10,000 r/min.

4.4 Conical flasks with stopper. 100 mL.

4.5 Filter membranes. 0.22 µm.

4.6 Pipettes. 1.0 mL, 2.0 mL, 5.0 mL and 10.0 mL.

4.7 Analytical balance.

5.1 Sample preparation

Take about 1 kg of representative sample from all sample product; use a masher to

mash sample completely and uniformly; divide sample into two equal parts; load them

in clean containers as test sample. Seal and label.

5.2 Sample preservation

Sample is preserved at below - 18°C. During the operating process of sampling and

preparation, prevent sample from being polluted or residue content changing.

5.3 Extraction

5.3.1 Fruits and vegetables

Weigh 1.00 g (accurate to 0.01 g) of sample to place into a centrifuge tube of 50 mL;

add 20 mL of phosphate buffer solution (pH 6.5) (3.2.8); carry out homogeneous

extraction for 3 min at 8,000 r/min; carry out refrigerated centrifugation in a high-speed

centrifugal machine for 10 min at 10,000 r/min; take out; use fibre filter paper to filter

into a colourimetric tube with stopper of 10 mL for the determination on a

spectrophotometer.

5.3.2 Livestock-Poultry Products and aquatic products

Weigh 1.00 g (accurate to 0.01 g) of sample to place into a centrifugal tube of 100 mL;

add 50 mL of phosphate buffer solution (pH 6.5) (3.2.8); carry out homogeneous

extraction for 3 min at 8,000 r/min; carry out refrigerated centrifugation in a high-speed

centrifugal machine for 10 min at 10,000 r/min; take out; use fibre filter paper to filter

into a colourimetric tube with stopper of 10 mL for the determination on a

spectrophotometer.

5.4 Determination

5.4.1 Plotting of standard curve

Add a certain amount of chlorine dioxide standard using solution to a series of 10 mL

stoppered colourimetric tubes to make the concentrations of all tubes equal to chlorine

dioxide standard solutions of 0.00 mg/L, 0.05 mg/L, 0.10 mg/L, 0.50 mg/L, 1.00 mg/L,

2.00 mg/L and 5.00 mg/L.

Add respectively 1.0 mL of phosphate buffer solution (3.2.8), 1.0 mL of DPD solution

(3.2.1) and 1.0 mL of glycine solution; make up to scale and shake up; within 60 s, use

cuvettes of 1 cm to measure the absorbance at 552 nm; plot standard curve using the

concentration of standard working solution as the abscissa and the response value

(absorbance value) as the ordinate. See Annex A.

5.4.2 Determination of chlorine dioxide in sample

Take 5 mL of filtrate to pour into a stoppered colourimetric tube of 10 mL; add 1 mL of

glycine solution (3.2.3) to the filtrate to mix up; add 1 mL of phosphate buffer solution

(3.2.8) and 1.0 mL of DPD solution (3.2.1); use water to make up to 10 mL and shake

up. Immediately within 60 s, use cuvettes of 1 cm to measure the absorbance at 552

nm; obtain the concentration of chlorine dioxide in accordance with the standard curve.

NOTE. The whole process shall be carried out in dark.