8/14/2019 Nitrate (NO3 N) protocol

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Environmental Engineering LaboratoryBy: Ngoc Thuan Le

Nitrate (NO3--N)-Brucine method

Sample storage condition: It should be determined promptly after sampling, store for up to

24h at 4oC, for longer storage, preserve with 2ml of conc H2SO4/L and store at 4oC. NOTE:

when sample is preserved with acid, NO3 and NO2

can not be determined as individual

species.

Apparatus

Water bath, Volumetric tubes, Spectrophotometer.

Procedure

1. Turn on water bath (hood)

2. Use pipet, place 10ml of sample to volumetric tubes.

3. Add 2ml solution of NaCl 30% (w/v)

4. Add 10ml H2SO4 4:1

5. Add 0.5ml solution of C23H26N2O4.2H2O (brucine) and HOSO2NH2 (sulfurin). Mixing

after adding.

6. Rinse tubes in the boiling water 20 minutes, color will be yellow.

7. Remove tubes from water bath, cooling in the air.

8. Bring volume to 25ml with DW.

9. Measure color photometrically at 410nm and compare with calibration curve, using

distilled water blank.

10. Calibration curve: N-NO3- solution 0.001mg/ml

Concentration range: 0, 1, 2, 4, 6, 8, 10ml (~0, 0.1, 0.2, 0.4, 0.6, 0.8, 1 mg/L)

Calculation

Reagents

- NaCl 30% (w/v): dissolve 30g NaCl with distilled water, bring volume to 100ml.

- H2SO4 4:1. 800ml conc. H2SO4 bring to 1000ml with H2O (note: use high quality

sulfuric acid to avoid nitrate contamination).

- Mixture of C23H26N2O4.2H2O (brucine) and HOSO2NH2 sulfurin (or sulfanilic

NH2C6H4SO2H) dissolved 1g brucine + 0.1g sulfurin in 3ml HCl (dissolve

completely first), bring to 100ml with dW.

- N-NO3- solution 0.001mg/ml.