⚗️ Ammonium nitrogen in water — direct Nesslerization (Nessler’s reagent, measurement of the yellow colour at 425 nm)

Physicochemistry PN-C-04576-4

Overview

Nesslerization is the oldest and still used method of the colorimetric determination of ammonium nitrogen. Nessler’s reagent in a strongly alkaline medium forms with ammonium ions a yellow product whose intensity is proportional to the concentration of ammonia; the measurement is carried out at 425 nm. The manufacturer’s procedure (Hach, method 8038, an adaptation of Standard Methods 4500-NH₃ B and C) covers the range 0 to 2.500 mg NH₃-N/l for water, waste water and sea water, whereby for waste water and sea water a prior distillation is required; the estimated limit of detection is 0.017 mg/l.

The course of the direct method: to 25 ml of sample and the same amount of deionised water (the blank) three drops each of mineral stabiliser (which complexes the hardness) and three drops each of polyvinyl alcohol as a dispersing agent are added, then 1.0 ml each of Nessler’s reagent; after a reaction time of one minute the absorbance is measured. The measurement has to be carried out not later than 15 minutes after the addition of the reagent.

The variant with distillation is described by the method EPA 350.2 ("Nitrogen, Ammonia — Colorimetric; Titrimetric; Potentiometric — Distillation Procedure"): the sample is buffered to pH 9.5 with a borate buffer so as to limit the hydrolysis of cyanates and of organic nitrogen compounds, and distilled into a solution of boric acid; the ammonia in the distillate is determined colorimetrically by Nesslerization (range 0.05–1.0 mg NH₃-N/l), titrimetrically with sulfuric acid 0.02 N against a mixed indicator (1.0–25 mg/l) or potentiometrically with an ammonia electrode (0.05–1400 mg/l).

Interferences: iron and sulfides cause turbidity with Nessler’s reagent at every level of concentration; hardness up to 500 mg/l CaCO₃ and 500 mg/l Mg (as CaCO₃) does not interfere, above that — more mineral stabiliser has to be added; sea water requires 1.0 ml of stabiliser, which lowers the sensitivity by about 30 % because of the high concentration of chlorides; glycine, aliphatic and aromatic amines, organic chloramines, acetone, aldehydes and alcohols give greenish or other discolourations or turbidity and require distillation. Residual chlorine is removed with sodium arsenite or thiosulfate. According to EPA, aromatic compounds, amines, volatile alkaline compounds and cyanides also interfere.

Preservation: samples are taken into clean glass or plastic bottles; in the presence of chlorine one drop of sodium thiosulfate 0.1 N is added for every 0.3 mg/l of Cl₂ in a litre of sample, they are acidified with sulfuric acid to pH ≤ 2 (at least 2 ml) and kept at 4 °C or below — preserved samples are stable for up to 28 days; before the analysis they are warmed to room temperature and neutralised with sodium hydroxide 5 N, the result being corrected for the volumes added.

Method principle

Nessler’s reagent (an alkaline solution of potassium tetraiodomercurate(II)) reacts with ammonium ions, forming a yellow product whose absorbance at 425 nm is proportional to the concentration of ammonium nitrogen. Before the reagent is added, the sample is treated with mineral stabiliser (which complexes the calcium and magnesium, preventing the precipitation of hydroxides in the alkaline medium of the reagent) and with polyvinyl alcohol, which stabilises the suspension of the coloured product and ensures a repeatable reading. A blank is carried out in parallel with deionised water — the reagent itself gives a faint yellow colour in it. For waste water, sea water and samples with organic substances a prior distillation is necessary: the sample is buffered with borate to pH 9.5 and distilled into a solution of boric acid, and the Nesslerization is carried out on the distillate. The calibration is checked by the standard addition method (standard ampoules of 50 mg/l NH₃-N, additions of 0.1, 0.2 and 0.3 ml to 25 ml samples — every addition should give a recovery close to 100 %) or with a standard solution of 1.0 mg/l NH₃-N.

Applications

• Drinking water and raw waters — ammonium nitrogen as an indicator of fresh contamination
• Municipal and industrial waste water (after distillation) — control of nitrification and of compliance with the conditions of the water permit
• Sea water and saline waters — after the addition of an increased dose of mineral stabiliser or after distillation
• Rapid screening determinations in the laboratories of treatment plants — a result within a few minutes of sampling
• Laboratories accredited according to PN-C-04576-4:1994 (direct Nesslerization)

Key parameters

Standard

Standard number

PN-C-04576-4:1994

Title (PL)

Woda i ścieki — Badania zawartości związków azotu — Oznaczanie azotu amonowego w wodzie metodą bezpośredniej nessleryzacji

Title (EN)

Water and waste water — Tests for nitrogen compounds — Determination of ammonium nitrogen in water by direct Nesslerization

Step-by-step procedure

1. Sampling and preservation

   Take the sample into a clean glass or plastic bottle. In the presence of chlorine add 1 drop of sodium thiosulfate 0.1 N for every 0.3 mg/l of Cl₂ in 1 l of sample. Preserve with sulfuric acid to pH ≤ 2 and keep at a temperature of 4 °C or lower (for up to 28 days). Before the analysis warm to room temperature and neutralise with NaOH 5 N; correct the result for the volumes added.

2. Decision on the distillation

   For waste water, sea water and samples with glycine, amines, organic chloramines, acetone, aldehydes or alcohols carry out a distillation: buffer to pH 9.5 with a borate buffer and distil into a solution of boric acid; work further on the distillate.

3. Preparation of the sample and of the blank

   Fill a 25 ml graduated cylinder with the sample (prepared or the distillate), and a second one with 25 ml of deionised water as the blank.

4. Stabilisation

   Into each cylinder add 3 drops of mineral stabiliser, stopper it and invert it several times; then add 3 drops each of polyvinyl alcohol (holding the bottle vertically) and mix again. For sea water add 1.0 ml of stabiliser.

5. Nesslerization

   Into each cylinder add with a pipette 1.0 ml of Nessler’s reagent, stopper it and mix by inversion. Start the reaction time — 1 minute. A yellow colour appears in the presence of ammonia (the reagent itself gives a faint yellow in the blank).

   ⏱ Time: 1 min

6. Measurement

   Pour the solutions into cells, zero the instrument with the blank and read the result at 425 nm in mg/l NH₃-N (or converted into NH₃ or NH₄⁺). Carry out the measurement not later than 15 minutes after the addition of the reagent.

7. Check of the accuracy

   Check by the standard addition method: to three 25 ml samples add 0.1, 0.2 and 0.3 ml of the standard of 50 mg/l NH₃-N and measure each — the recovery should be close to 100 %. Alternatively measure a standard solution of 1.0 mg/l NH₃-N.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Nessler’s reagent contains mercury — it is toxic and corrosive; pipette only with a bulb, work in gloves and goggles, avoid contact with the skin.
• Treat the solutions after the reaction and the unused reagent as mercury waste — collect them separately and hand them over to an authorised receiver; do not pour them into the drains.
• Sodium arsenite (removal of chlorine) is highly toxic — use it only where necessary, prefer sodium thiosulfate.
• Sulfuric acid for the preservation and sodium hydroxide 5 N for the neutralisation — corrosive substances, eye and hand protection.
• Distillation with a borate buffer: beware of overheating and of the carry-over of foam into the receiver; work under a fume hood.

Frequently asked questions