🍎 Nitrates and nitrites (nitrates(V) and nitrates(III)) in milk powder, cheeses and caseins — cadmium reduction, spectrometry at 538 nm
In short
The sample is dispersed in warm water, the fat and the proteins are precipitated (zinc sulfate, hexacyanoferrate) and it is filtered; in part of the filtrate the nitrates are reduced to nitrites in a column with copperised cadmium, and the nitrites in both solutions are determined with sulfanilamide and N-(1-naphthyl)ethylenediamine dihydrochloride at 538 nm against standards of sodium nitrite. The test is performed according to PN-EN ISO 14673-1:2004; Measurement: 538 nm, cells with an optical path of 1–2 cm. The procedure comprises 8 steps; it is used for: Control of the content of nitrates and nitrites in ripened cheeses (the addition of saltpetre to the cheese milk or to the brine), Testing of milk powder, whey powder, caseins and caseinates, also for infant food, Official supervision of the limits of nitrates and nitrites in milk products.
At a glance
- Standard: PN-EN ISO 14673-1:2004
- Category: Food chemistry
- Procedure steps: 8
- Measurement: 538 nm, cells with an optical path of 1–2 cm
- Reductor: Granules of cadmium 0.3–0.8 mm, copperised with a solution of CuSO4 20 g/l; a check of the reducing capacity and regeneration of the column
- Standards: Sodium nitrite: a stock solution of 0.150 g/l (dried at 110–120 °C), a working one of 1 µg NO2−/ml; potassium nitrate: 1.468 g/l, a working one of 4.50 µg NO3−/ml, prepared on the day of use
Overview
The standard specifies a method of determining the content of nitrates (nitrates(V)) and nitrites (nitrates(III)) in milk and milk products by cadmium reduction and spectrometry. It concerns whole, partly skimmed and skimmed milk powder, hard, semi-hard and soft cheeses, processed cheeses, whey cheeses, caseins and caseinates and whey powder. The method may also be carried out with automatic apparatus — by segmented flow analysis (SFA, ISO 14673-2) or by flow injection analysis (FIA, ISO 14673-3) — which limits the contamination of the working places and of the waste water with cadmium. The edition ISO 14673-1:2004 (a joint ISO/IDF 189-1, in cooperation with AOAC) is a minor revision of the edition of 2001.
The content of nitrates is expressed in mg of NO3− ions per kg of product, that of nitrites — in mg of NO2− per kg. Potassium nitrate (saltpetre) is in the national cheese-making added to cheese milk (0.01–0.02 %) in order to inhibit coliform bacteria and butyric fermentation; in cheeses for consumption the level of nitrates must not exceed 50 mg NaNO3/kg (36.5 mg NO3−/kg), and that of nitrites 2 mg NaNO2/kg. In studies of Tykocin cheese (the Maritime University of Gdynia) the determinations were carried out according to PN-EN ISO 14673-1:2004: the nitrites with sulfanilamide and N-1-naphthylethylenediamine dihydrochloride at 538 nm, the nitrates after reduction in a cadmium column.
The preview of the standard covers the principle, the reagents, the apparatus and the preparation of the samples; the details of the preparation of the column, of the extraction, of the reduction and of the measurement (clauses 9–10) are described here at the level of the structure of the standard and of data from the literature — before implementation they have to be checked in the full text.
Method principle
A test portion is dispersed in warm water, the fat and the proteins are precipitated with solutions of zinc sulfate (535 g/l) and potassium hexacyanoferrate(II) (172 g/l) and it is filtered through a filter paper free of nitrates and nitrites. In a portion of the filtrate the nitrate ions are reduced to nitrite ions in a glass column filled with granules of cadmium (0.3–0.8 mm) coated with copper (from a solution of copper sulfate 20 g/l), whose reducing capacity is checked and regenerated. In portions of the unreduced filtrate and of the solution after the reduction a red colour is produced by the addition of sulfanilamide (solution II: 0.5 g in 100 ml with 5 ml of HCl) and of N-(1-naphthyl)ethylenediamine dihydrochloride (solution III: 0.1 g/100 ml) in a medium of hydrochloric acid, and the absorbance is measured at 538 nm in cells of 1–2 cm. The content of nitrites and the total content of nitrites after the reduction are read off from a calibration curve of solutions of sodium nitrite (the working solution 1 µg NO2−/ml); the content of nitrates is calculated from the difference. For checking the column a working solution of potassium nitrate of 4.50 µg NO3−/ml serves.
Applications
- Control of the content of nitrates and nitrites in ripened cheeses (the addition of saltpetre to the cheese milk or to the brine)
- Testing of milk powder, whey powder, caseins and caseinates, also for infant food
- Official supervision of the limits of nitrates and nitrites in milk products
- Technological studies of the transformations of nitrates during the ripening of cheeses
- The reference method for the automatic methods SFA and FIA (ISO 14673-2 and -3)
Key parameters
| Parameter | Value |
|---|---|
| Measurement | 538 nm, cells with an optical path of 1–2 cm |
| Reductor | Granules of cadmium 0.3–0.8 mm, copperised with a solution of CuSO4 20 g/l; a check of the reducing capacity and regeneration of the column |
| Standards | Sodium nitrite: a stock solution of 0.150 g/l (dried at 110–120 °C), a working one of 1 µg NO2−/ml; potassium nitrate: 1.468 g/l, a working one of 4.50 µg NO3−/ml, prepared on the day of use |
| Units | mg NO3−/kg and mg NO2−/kg of product |
| National limits for cheeses | Nitrates ≤ 50 mg NaNO3/kg (36.5 mg NO3−/kg), nitrites ≤ 2 mg NaNO2/kg |
| Buffer | pH 9.6–9.7 (hydrochloric acid + ammonia) |
Standard
- Standard number
- PN-EN ISO 14673-1:2004
- Title (PL)
- Mleko i przetwory mleczne — Oznaczanie zawartości azotanów(V) i azotanów(III) — Część 1: Metoda z wykorzystaniem redukcji kadmem i spektrometrii
- Title (EN)
- Milk and milk products — Determination of nitrate and nitrite contents — Part 1: Method using cadmium reduction and spectrometry
Step-by-step procedure
-
Preparation of the cadmium column
Prepare (or use commercial) granules of cadmium of 0.3–0.8 mm, coat them with copper from a solution of CuSO4 20 g/l and fill the column, using freshly boiled and cooled water so as to avoid bubbles of gas. Check the reducing capacity with the working solution of potassium nitrate (4.50 µg NO3−/ml); where necessary regenerate it.
-
Preparation of the sample
Mix milk powder and whey in a closed container of a volume about twice as large as the sample. Sieve caseins and caseinates through a 500 µm sieve (50 g), where necessary comminute them without heating. Suspend cheeses and whey cheeses in a mixer in a vessel of 250–400 ml.
-
Extraction and deproteinisation
Disperse the test portion in warm water, precipitate the fat and the proteins with the solutions of zinc sulfate and potassium hexacyanoferrate(II), make up to volume and filter through a filter paper free of nitrates and nitrites.
-
Reduction of the nitrates
Pass a buffered portion of the filtrate (pH 9.6–9.7) through the column with copperised cadmium; collect the solution after the reduction.
-
Colour reaction
To portions of the unreduced filtrate and of the solution after the reduction add solution I (hydrochloric acid), solution II (sulfanilamide) and solution III (NEDD); a red colour is produced.
-
Measurement and calibration curve
Measure the absorbance at 538 nm in a cell of 1–2 cm against a blank; in parallel prepare a series of calibration solutions from the working solution of sodium nitrite (1 µg NO2−/ml) and plot a calibration curve.
-
Calculations
Read off from the curve the content of nitrites in the sample (mg NO2−/kg) and the total content of nitrites after the reduction; calculate the content of nitrates (mg NO3−/kg) from the difference, taking into account the test portion and the dilutions.
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Test report
Give the identification of the sample, the method with a reference to ISO 14673-1 | IDF 189-1, the result for the nitrates and the nitrites and any departures; when comparing with the limits, convert into NaNO3 and NaNO2.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Glass reduction column | According to Figure 1 of the standard, filled with copperised cadmium | — |
| Spectrometer | For measuring the absorbance at 538 nm, cells of 1–2 cm | — |
| Analytical balance | An accuracy of 1 mg, a reading of 0.1 mg | — |
| Laboratory mixer or homogeniser | Glass vessels of 250 or 400 ml for suspending cheeses; a comminuting device without excessive heating | — |
| Sieve 500 µm, funnels, filter papers, class B volumetric flasks, class A pipettes | A sieve of 200 mm diameter according to ISO 565; medium filter papers, about 15 cm, free of nitrates and nitrites | — |
| Water bath, drying oven 110–120 °C, magnetic stirrer | The drying oven for drying the standards to constant mass | — |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Cadmium, granules 0.3–0.8 mm | 7440-43-9 | Commercial or prepared from cadmium sponge deposited on zinc from a solution of cadmium sulfate 40 g/l; hand the used cadmium over as chemical waste |
| Copper(II) sulfate pentahydrate | 7758-99-8 | A solution of 20 g/l for copperising the cadmium |
| Hydrochloric acid | — | Concentrated (1.19 g/ml); a dilute solution of about 2 mol/l (160 ml/l), a working one of about 0.1 mol/l; solution I: 450 ml/l; a component of the buffer of pH 9.6–9.7 with ammonia of 0.91 g/ml |
| Zinc sulfate heptahydrate (CAS 7446-20-0) and potassium hexacyanoferrate(II) trihydrate | 14459-95-1 | Solutions of 535 g/l ZnSO4·7H2O (53.5 g/100 ml) and 172 g/l K4[Fe(CN)6]·3H2O (17.2 g/100 ml) for precipitating the fat and the proteins; EDTA 33.5 g/l |
| Sulfanilamide | 63-74-1 | Solution II: dissolve 0.5 g on a bath in 75 ml of water with 5 ml of concentrated HCl, make up to 100 ml |
| N-(1-naphthyl)ethylenediamine dihydrochloride | 1465-25-4 | Solution III: 0.1 g/100 ml; stable for up to 1 week in a dark bottle in a refrigerator |
Health and safety (OHS)
- Cadmium and its compounds are toxic and carcinogenic — work in gloves, under a fume hood; hand the used cadmium and the solutions over as chemical waste, consider the automatic methods (SFA, FIA) which limit the contact
- Concentrated hydrochloric acid and ammonia — a fume hood, goggles, gloves; prepare the solutions by carefully adding the acid to the water
- N-(1-naphthyl)ethylenediamine dihydrochloride and sulfanilamide — avoid raising dust and contact with the skin
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 14673-1:2004 — “Milk and milk products — Determination of nitrate and nitrite contents — Part 1: Method using cadmium reduction and spectrometry”.
How does this method work?
A test portion is dispersed in warm water, the fat and the proteins are precipitated with solutions of zinc sulfate (535 g/l) and potassium hexacyanoferrate(II) (172 g/l) and it is filtered through a filter paper free of nitrates and nitrites. In a portion of the filtrate the nitrate ions are reduced to nitrite ions in a glass column filled with granules of cadmium (0.
What is the measuring range and accuracy?
Measurement: 538 nm, cells with an optical path of 1–2 cm; Reductor: Granules of cadmium 0.3–0.8 mm, copperised with a solution of CuSO4 20 g/l; a check of the reducing capacity and regeneration of the column; Standards: Sodium nitrite: a stock solution of 0.150 g/l (dried at 110–120 °C), a working one of 1 µg NO2−/ml; potassium nitrate: 1.468 g/l, a working one of 4.50 µg NO3−/ml, prepared on the day of use; Units: mg NO3−/kg and mg NO2−/kg of product.
How long does the test take?
The procedure comprises 8 steps. The standard does not give a duration for every stage — the laboratory's own procedure decides.
What equipment is required?
Glass reduction column, Spectrometer, Analytical balance, Laboratory mixer or homogeniser, Sieve 500 µm, funnels, filter papers, class B volumetric flasks, class A pipettes, Water bath, drying oven 110–120 °C, magnetic stirrer.
Where is this test used?
Control of the content of nitrates and nitrites in ripened cheeses (the addition of saltpetre to the cheese milk or to the brine); Testing of milk powder, whey powder, caseins and caseinates, also for infant food; Official supervision of the limits of nitrates and nitrites in milk products; Technological studies of the transformations of nitrates during the ripening of cheeses; The reference method for the automatic methods SFA and FIA (ISO 14673-2 and -3).
What safety precautions apply?
Cadmium and its compounds are toxic and carcinogenic — work in gloves, under a fume hood; hand the used cadmium and the solutions over as chemical waste, consider the automatic methods (SFA, FIA) which limit the contact; Concentrated hydrochloric acid and ammonia — a fume hood, goggles, gloves; prepare the solutions by carefully adding the acid to the water; N-(1-naphthyl)ethylenediamine dihydrochloride and sulfanilamide — avoid raising dust and contact with the skin.
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number PN-EN ISO 14673-1.