National Research Council Institute for Ecosystem Study Verbania Pallanza - Italy

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1 Tot-N UV220 pag. 1 National Research Council Institute for Ecosystem Study Verbania Pallanza - Italy Water Chemistry Laboratory Analytical Methods for internal use - Gabriele TARTARI Total Nitrogen in Water A peroxodisulphate oxidation procedure followed by spectrophotometric determination PRINCIPLE The sum of nitrate nitrogen, nitrite nitrogen, ammonium nitrogen and organically bonded nitrogen, each determined separately, is designed total nitrogen. Elemental nitrogen dissolved in the water is not contained in this total. Ammonium, nitrite and organic nitrogen, are oxidised to nitrate using potassium peroxodisulphate in a buffer boric acid-sodium hydroxide buffer. The oxidation of the nitrogen compounds is performed in an autoclave at 120 C, resulting in a ph change of the buffer from 9.7 to 5.0. The resulting nitrate is determined by spectrophotometry at 220 nm. (Note: Organic substances which interfere at this wavelength are assumed to be completely oxidised in the peroxodisulphate digestion procedure) Absorbance (u A) nm Wavelength (nm) Fig. 1 - Absorption spectrum of a nitrate solution between 200 nm to 250 nm SCOPE AND APPLICATION This procedure details the determination of total nitrogen in natural freshwater, surface water, and drinking water, in the form of ammonium, nitrite, nitrate and organic nitrogen compounds capable of conversion to nitrate under the oxidative digestion procedure described.

2 Tot-N UV220 pag. 2 RANGE OF MEASURE AND REPEATABILITY Range of quantification, limit of detection (LOD), limit of quantification (LOQ) and repeatability expressed in relative standard deviation (R.S.D.). (All units are expressed as mg N L -1 ) Cell length Range LOD LOQ 1 cm R.S.D mg N L -1 Fig. 2 Repeatability data of a control sample obtained on different days from 1989 to 2003 Figure 2 shows repeatability data from control charts used in the CNR-ISE Hydrochemistry Laboratory since Each value is obtained from a series of at least 30 measurements performed on different days on the same control sample. REAGENTS I Oxidising solution In a 5 L volumetric flask dissolve 250 g of analytical reagent grade potassium peroxodisulphate (K 2 S 2 O 8 ) (CAS N maximum 0.001% nitrogen as impurity, Merck n recommended), 150 g of boric acid (H 3 BO 3 ) (CAS N ) and 70 g of sodium hydroxide pellets (NaOH) (CAS N ) in 4,500 ml of de-ionised water. Allow to cool to room temperature before diluting to the mark. Keep at room temperature, do not cool below 4 C. The solution is stable for 3-4 months. II Sulphuric acid 98% (CAS N ) (maximum % nitrogen as impurity) GLASSWARE Glassware must be washed using P free detergents or 25% sulphuric acid and rinsed thoroughly with deionised water. It is suggested that 50 ml bottles are used for the autoclave digestion procedure (Fig. 3), screw cap and o-ring gas proof to a temperature of 120 C (SCHOTT DURAN, blue cap GL32, maximum use temperature 140 C). Fig ml Duran bottle used for the autoclave oxidation at 120 C

3 METHOD Tot-N UV220 pag. 3 Blanks Three blanks should be prepared per batch, using 25 ml of ultra-pure water in place of the test sample. Digestion procedure Pipette 25 ml of the sample into the 50 ml Duran (digestion) bottle and add 3.5 ml of Reagent I - Oxidising solution. Close the digestion bottles well and place in an autoclave at 120 C for 30 minutes (or furnace at C for 120 minute). Remove the bottles when the autoclave temperature is below 60 C and allow to cool to room temperature. Total nitrogen quantification Turn on the spectrophotometer and the UV lamp at least 15 minutes before use. To each 28.5 ml of digested sample, add 0.4 ml of Reagent II - Sulphuric acid 98% and shake before reading the absorbance of the solution in a 1 cm quartz cell at 220 nm. The spectrophotometer is zeroed by using an acidified ultra-pure water solution (about 28 ml of ultrapure water containing 0.4 ml of Reagent II - Sulphuric acid 98%). Subtract the blank mean absorbance from each sample absorbance and then calculate the concentration of nitrogen in the sample(s) from the calibration curve. CALIBRATION Stock standard 100 mg N L -1 (0.10 mg N ml -1 ) Prior to preparing the stock standard, dry the analytical reagent salt in an oven at 110 o C for at least 1 hour and allow to cool in a desiccator. In a 1L volumetric flask dissolve g NaNO 3 (CAS N ) (or corrected weight after allowance for the degree of purity of nitrogen) in ultrapure water and dilute to the mark. This standard may be stored in glass in a refrigerator (4 o C) for up to 3 months. Working standards 0.4 to 6.00 mg N L -1 Working standards are prepared fresh every day in ultra-pure water by pipetting an appropriate aliquot of stock standard (0.10 mg N ml -1 ) and making up to the stipulated volume according to the following scheme: mg N L -1 Vol. of Stock standard Volumetric flask (ml) µl µl µl ml ml ml ml ml ml 500

4 Tot-N UV220 pag. 4 Reference solution In a 1L volumetric flask, dissolve 50.4 g of potassium hydrogen sulphate (CAS N ) (KHSO 4 ), 30 g of H 3 BO 3 and 14 g of NaOH in 800 ml of ultra-pure water. Allow to cool to room temperature before diluting to the mark. This reference solution represents the samples after the autoclave digestion procedure, based on the assumption that 100% of all nitrogen containing compounds were oxidised to nitrate. Calibration procedure Pipette in triplicate 25 ml of each blank and working standard into freshly washed 50 ml Duran (digestion) bottles, taking care to go in an ascending order from the blank to the highest standard, rinsing the pipette on every change of solution. Finally, to each 50 ml bottle add 3.5 ml of reference solution. Turn on the spectrophotometer and the UV lamp at least 15 minutes before use. To each 28.5 ml ( ml) blank and working standard, add 0.4 ml of Reagent II - Sulphuric acid 98% and shake before reading the absorbance of the solution in a 1 cm quartz cell at 220 nm. The spectrophotometer is zeroed by using the acidified ultra-pure water solution. An example of a typical absorbance (u A), relative standard variation (R.S.D.) for each standards, regression equation and linear correlation coefficient (r) of calibration using 1 cm quartz cell, is given below: mg N L u A media R.S.D N mg N L -1 = 4.35 u A 0.03 r = Perform and check the calibration with all the standard solutions every one year. CALCULATION Calculate the mean absorbance value of the three blanks (normally ranging between and u A, cell with 1 cm optical path). Subtract the blank mean absorbance from each sample (or working standard) absorbance and then calculate the concentration of nitrogen in the sample(s) from the calibration curve. Measurement units are expressed as mg N L -1, correct to the second decimal place, if the above analytical procedure is strictly adhered too. REFERENCE Tartari, G.A. & R. Mosello Metodologie analitiche e controlli di qualità nel laboratorio chimico dell Istituto Italiano di Idrobiologia del Consiglio Nazionale delle Ricerche. Documenta Ist. ital. Idrobiol., 60: 160 pp. Valderrama J.C The simultaneous analysis of total nitrogen and total phosphorus in natural waters. Marine Chemistry, 10:

5 Tot-N UV220 pag. 5 A.P.H.A., A.W.W.A., W.E.F Standard Methods for the Examination of Water and Wastewater (Method 4500 N C, Persulfate method). Amer. Publ. Health Ass., Washington. A.P.H.A., A.W.W.A., W.E.F Standard Methods for the Examination of Water and - Wastewater (Method 4500 NO 3 B, Ultraviolet spectrophotometric method). Amer. Publ. Health Ass., Washington.