⚗️ Nitrite nitrogen and nitrate nitrogen in water — flow analysis (CFA/FIA) with cadmium reduction and the Griess reaction (sulfanilamide, N-(1-naphthyl)ethylenediamine, 520–560 nm)

Physicochemistry PN-EN ISO 13395

Overview

ISO 13395:1996 specifies automated procedures for determining nitrite nitrogen, nitrate nitrogen and the sum of both in waters by flow analysis — continuous (CFA) and injection (FIA) — with spectrometric detection. Working ranges: nitrite(N) — range II 0.01–0.1 mg/l and range I 0.1–1.0 mg/l; nitrite/nitrate(N) — range II 0.2–2 mg/l and range I 2–20 mg/l; each range requires its own calibration (at least 5 standards). The chemical reactions of the nitrite are identical with those of the manual procedure (ISO 6777). The 2019 regulation (Dz.U. 2019 poz. 1311 [Journal of Laws 2019, item 1311]) names PN-EN ISO 13395 (flow analysis, CFA and FIA, with spectrometric detection) as the reference method for nitrite nitrogen and nitrate nitrogen in wastewater. In Poland it is in force as PN-EN ISO 13395:2001 (Polish version).

General interferences: nitrite cannot be stabilised — it is analysed at once; particles above 0.1 mm block the tubing (membrane filtration), and smaller ones are removed by dialysis; an interfering organic matrix (compounds of high molar mass) is removed by in-line dialysis or by filtration through activated carbon (provided this does not change the concentrations); the sample's own absorption is compensated by a measurement without the reagents; samples with a salinity above 30 g/l are diluted. The reduction of nitrate requires a pH of 6.5–7.5 after mixing with the buffer (strongly acid, strongly alkaline or buffered samples are corrected before the measurement); surfactants above 10 mg/l interfere with the formation of the dye (dialysis). Waste containing cadmium, whether liquid or solid, is disposed of appropriately. In manual practice (PWr textbook "Chemia wody" [Water chemistry]) nitrite is determined colorimetrically with sulfanilic acid and α-naphthylamine at 540 nm (0.001–0.1 mg/dm³) or according to PN-EN 26777 with 4-aminobenzenesulfonamide (that is, sulfanilamide) and N-(1-naphthyl)-1,2-diaminoethane dihydrochloride in phosphoric acid (pH 1.9, 540 nm), and nitrate — after evaporation with phenoldisulfonic acid (410 nm).

Method principle

The sample is continuously mixed with a buffer solution (imidazole 0.25 mol/l — 17.0 g/l, pH 7.5 adjusted with HCl; alternatively ammonium chloride 85 g/l at about pH 7.5 — with the addition of copper sulfate 2.5 g/l: 100 µl per 100 ml, freshly prepared); the nitrate in the sample is reduced to nitrite by metallic cadmium; then the reagent with phosphoric acid is continuously admixed (5 g of sulfanilamide and 0.5 g of N-(1-naphthyl)ethylenediamine dihydrochloride + 50 ml of H₃PO₄ 1.71 g/ml in 500 ml; at least 1 week in an amber bottle; HCl may be used instead of H₃PO₄) — the nitrite originally present and that formed by the reduction diazotises the sulfanilamide in the acid medium to a diazonium salt, which is coupled with the N-(1-naphthyl)ethylenediamine to give a red dye. Nitrite(N) is determined with the cadmium reduction bypassed; nitrate(N) from the difference sum − nitrite. Reductor: cadmium granules of 0.3–1.5 mm (a column of e.g. 4.0 mm × at least 5 cm in FIA) or a cadmium tube (internal diameter 1.1 mm in CFA) — the reduction efficiency has to be at least 90 %, checked before each series by comparing the signals of nitrate and nitrite solutions of the same N concentration (2 or 20 mg/l); preparation of the granules: stirring with HCl 1 mol/l until they have a metallic lustre, rinsing, twice 2 min with CuSO₄ 20 g/l (the surface turns black), rinsing, packing without bubbles, activation three times with nitrate solution; cadmium tube: 5 ml of buffered CuSO₄ (5 min, twice), 20 ml of imidazole solution; the columns are stored in imidazole solution without bubbles. Solutions: nitrite(N) 100 mg/l (492.6 mg of NaNO₂ dried at 150 °C made up to 1000 ml; 2 weeks at 4 °C), solutions of 20 mg/l and 1 mg/l (freshly prepared); nitrate(N) 200 mg/l (144.4 mg of KNO₃ made up to 100 ml; 1 month) and 20 mg/l (freshly prepared); calibration standards according to tables 2 and 3 (e.g. NO₂-N 0.01–0.10 mg/l from 1–10 ml of the 1 mg/l solution made up to 100 ml; NO₃-N 0.2–2.0 mg/l from 1–10 ml of the 20 mg/l solution made up to 100 ml), prepared immediately before the measurement; polyethylene glycol dodecyl ether surfactant 30 % (1 ml/l of the carrier solutions, about 4 weeks); the carrier and buffer solutions C and B degassed (for FIA). FIA apparatus: reagent reservoirs, a low-pulsation pump, an injector of 10–300 µl, a cadmium reductor, tubing and coils of 0.5–0.8 mm of inert plastics, an optional dialysis cell with a cellulose membrane (after the injector; equal flows of donor and acceptor), a photometric detector with a flow cell at 520–560 nm, a recorder (evaluation of peak heights), an autosampler; CFA: an autosampler, a pump, a cadmium tube, a manifold with bubble segmentation (nitrogen is recommended; with air — degassing before the cadmium column), dialysis, a detector at 520–560 nm. Check of the system: a standard of 0.05 mg/l NO₂-N or 1 mg/l NO₃-N in the lower range has to give an absorbance of at least 0.04 per 10 mm. Reagent blank: instead of the buffer B and the reagent R1, water is passed through for 2 min — a change of absorbance above 0.015/10 mm means that the water or the reagents are contaminated. Samples: nitrite only — a glass or PE bottle, analysis at once; the sum or nitrate where the nitrite content is low — also PE/PTFE, acidification with HCl to about pH 2, 2–5 °C, analysis within 24 h (exceptionally −20 °C for up to 8 days after verification); filtration where particles above 0.1 mm are present.

Applications

• Treated and raw wastewater — nitrite and nitrate nitrogen (reference method under Dz.U. 2019 poz. 1311), control of nitrification/denitrification
• Surface water and groundwater — monitoring of agricultural nitrate (range up to 20 mg/l N)
• Drinking water — nitrite from 0.01 mg/l N and nitrate
• High-throughput laboratories — automatic FIA/CFA flow analysis with an autosampler
• Coloured or turbid samples and samples with surfactants — in-line dialysis; saline waters (above 30 g/l) after dilution

Key parameters

Standard

Standard number

PN-EN ISO 13395:2001 (ISO 13395:1996)

Title (PL)

Jakość wody — Oznaczanie azotu azotynowego i azotanowego oraz ich sumy metodą analizy przepływowej (CFA i FIA) z detekcją spektrometryczną

Title (EN)

Water quality — Determination of nitrite nitrogen and nitrate nitrogen and the sum of both by flow analysis (CFA and FIA) and spectrometric detection

Step-by-step procedure

1. Sample

   Wash the vessels with water and rinse them with the sample. Determine nitrite at once (a glass or PE bottle). For the sum or for nitrate (where the nitrite content is low) acidify with HCl to about pH 2, store at 2–5 °C and analyse within 24 h (exceptionally at −20 °C for up to 8 days after verification); filter where particles above 0.1 mm are present; dilute if the salinity is above 30 g/l; adjust the pH of strongly acid/alkaline samples so that after the buffer it is 6.5–7.5.

2. Cadmium reductor

   Granules: stir with HCl 1 mol/l until they have a metallic lustre, rinse with water, stir twice for 2 min with CuSO₄ 20 g/l (they turn black), rinse, pack the column without bubbles (glass wool), install it and activate it three times with nitrate solution, measuring the highest standard of the range (2 or 20 mg/l) until the results are stable. Tube: 2 × 5 ml of buffered CuSO₄ (5 min each), 20 ml of imidazole. Store in imidazole without bubbles.

3. Check of the reduction efficiency

   Before each series measure in turn a nitrate(N) solution and a nitrite(N) solution of the same concentration (2 mg/l for range II, 20 mg/l for range I); if the nitrate signal is below 90 % of the nitrite signal — repeat the preparation/activation of the reductor.

4. Setting up the system and the blank

   Assemble the system (FIA/CFA; with the reductor for the sum, without it for the nitrite), pass the reagents through for about 10 min (for the sum: 10 min without and 10 min with the reductor), zero the baseline and check the signal-to-noise ratio; check the system with a standard of 0.05 mg/l NO₂-N or 1 mg/l NO₃-N (A ≥ 0.04/10 mm); run a reagent blank (water instead of B and R1 for 2 min; a change above 0.015 A → replace the water/reagents).

5. Calibration

   Choose the working range and prepare, immediately before the measurement, at least 5 standards (tables 2–3: e.g. 1–10 ml of the 1 mg/l NO₂-N solution made up to 100 ml → 0.01–0.10 mg/l; 1–10 ml of the 20 mg/l NO₃-N solution made up to 100 ml → 0.2–2.0 mg/l); measure the standards and the blank under the same conditions as the samples; a separate calibration for each range and form.

6. Measurement and result

   Measure the samples (peak heights); for coloured samples also measure the signal without the reagents and subtract it; calculate NO₂-N and NO₂+NO₃-N from the calibration functions, and NO₃-N as the difference; report in mg/l N (conversion factors to the ions: N-NO₃ × 4.4267 = NO₃⁻, N-NO₂ × 3.2882 = NO₂⁻ according to the textbook). Dispose of the cadmium waste in accordance with the regulations.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Cadmium and its waste (solutions, spent granules) are toxic and carcinogenic — collect them separately and dispose of them in accordance with the regulations
• N-(1-naphthyl)ethylenediamine and sulfanilamide — avoid contact with the skin; phosphoric and hydrochloric acids — goggles, gloves
• Imidazole — irritant; copper sulfate — harmful to the aquatic environment
• Nitrogen cylinder for the segmentation — secure it, ventilate the room

Frequently asked questions