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Nitrogen compounds with environmental relevance frequently analyzed in wastewater are ammonia, nitrite, nitrate, and Kjeldahl nitrogen. Ammonia discharged to surface water can be nitrified in the aqueous Environment if nitrifying microorganisms are present. The nitrifying bacteria consume dissolved oxygen for this process, thus depleting the oxygen content of the surface water with the consequence of massive dying of fish. Moreover, if the pH of the surface water is in the alkaline range, NH3 is formed which is toxic towards fish. The nitrate ion represents a nutrient leading to eutrophication of surface water, and nitrite is toxic and can react with amines (formed e.g. from amino acids of proteins) to yield N-nitosoamines which represent powerful carcinogens. Kjeldahl nitrogen is a sum parameter of compounds containing the nitrogen atom with an oxidation number of ‑3 (ammonia, amines and many other organic nitrogen compounds). It thus comprises organic nitrogen compounds besides ammonia nitrogen. This is also an important nitrogen parameter, because organic nitrogen compounds can be metabolized to ammonia (this conversion can also take place in surface water). Analytical procedures for the mentioned important nitrogen parameters are given in detail e.g. in the "Standard Methods" (Greenberg et al. 1985). Their principles will be described briefly in this chapter.

As many wastewater analyses (not only for nitrogen compounds) are photometric procedures, a short information about photometry will be given. Photometry uses light as an analytical tool. As particular substances (analytes) absorb photones of different wavelengths to different extents, the wavelength (or colour) of the light applied for photometric analysis affects the specificity of the analytical procedure for a given analyte. The specificity can be increased by converting the analyte by reaction with certain reagents to form coloured products, because (besides the colour) also the reaction with a given reagent is specific for the analyte (other wastewater constituents would not react at all with the reagents used for conversion of a particular analyte). For example, ammonia can be converted to an intensely blue indophenol derivative by the following reactions:

The last reaction is catalysed by Mn2+ ions. For obtaining the blue product, an aliquot of the wastewater sample is mixed with a small volume of aqueous MnSO4 solution. Then the mixture is stirred and hypochlorous acid reagent and finally an alkaline aqueous phenol solution ("phenate reagent") is added. After 10 min the colour formation is complete for these particular reactions. The coloured product exhibits a maximum absorption at 630 nm (the complementary light causes the blue colour). The solution is transferred to a cuvette which is irradiated with light exhibiting a wavelength of 630 nm (satisfactory results are obtained in the 600 to 660 nm region for this analytical procedure) and an intensity of Io in a photometer. In the photometer, the intensity of the light entering (Io) as well as the light leaving the cuvette (I) is determined (by means of a photodiode or a photomultiplyer) as shown schematically in figure 6. The absorbance, i.e. log(Io/I), is linearly related to the indophenol concentration as given by the Beer-Lambert law:

absorbance = log(Io/I) = e×c×d

With the proportionality constant e (molar absorptivity or molar extinction coefficient), the length d of the way of the light through the cuvette (frequently 1 cm) and the molar concentration c of the coloured substance, resp. the concentration of the analyte in the sample (as one molecule of ammonia will yield one molecule of the coloured substance, the absorbance will also be linearly related to the ammonia concentration in the wastewater or in calibration solutions, resp.).

Figure 6: Principle of photometric analysis measuring the decrease of intensity of light that passes a solution of a substance (concentration: c) that absorbs light of the applied wavelength; I: intensity after passing the cuvette; Io: intensity of the light before passing the cuvette.

The colourless nitrite ion NO2- is also transformed to a coloured substance prior to photometric analysis. A standard method used for nitrite analysis suitable for determinations down to 1 µg NO2-N/l is the reaction of nitrite at pH 2 (formation of nitrous acid) with sulfanilic acid to give a diazonium salt which reacts with another reagent, (1-naphthyl)-ethylenediamine, in order to form a reddish purple azo dye that can be detected photometrically at 543 nm. For quantification the nitrite concentration in wastewater samples, standard solutions containing known nitrite concentrations are also analyzed in the same way. As for all the other analytical methods mentioned here, the exact procedure can be read in the "Standard Methods" (Greenberg et al. 1985).

As for other analytes, also for nitrate determination several analytical methods can be applied. Greenberg et al. (1985) describe the chromotropic acid method as one of the possible procedures. Two molecules of nitrate react with one molecule of chromotropic acid (4,5-dihydroxy-2,7-naphthalene sulfonic acid) and the absorbance of the product is measured at 410 nm. The method interferes with nitrite. The nitrite ion is destroyed by reaction with urea which is also added to the test assay.

The German standard procedure for nitrate analysis utilizes the reaction of nitrate with 2,6-dimethylphenol under acid conditions to form 4-nitro-2,6-dimethylphenol with an absorbance maximum at 324 nm.

As already mentioned, the Kjeldahl method determines nitrogen in the trinegative state. Thus, it does not account for nitrogen in compounds like azide, azine, azo, hadrazone, nitrate, nitrite, nitrile, nitro, nitroso, oxime, and semi-carbazone (Greenberg et al. 1985). In the Kjeldahl method, the amino nitrogen of many organic nitrogen compounds is transformed to (NH4)2SO4 in the presence of H2SO4, K2SO4, and HgSO4 (this acts as a catalyst for the conversion) by boiling the mixture of wastewater sample and reagent solutions in a flask until fumes are occurring. A mercury ammonium complex generated in this procedure is decomposed by the addition of sodium thiosulfate/sodium hydroxide reagent after digestion of the organic nitrogen compounds. Ammonia and ammonium are also present as (NH4)2SO4 after treatment. Finally, the flask used for digestion is connected to a steamed-out distillation apparatus, and the ammonia which has been generated from (NH4)2SO4 by addition of hydroxide solution is distilled to a receiving flask containing a boric acid solution. Afterwards the distilled ammonia is determined by acid/base titration. If it is to be analysed by the above-mentioned phenate method for ammonia determination, the receiving flask must contain sulfuric acid. If small sample volumes have to be analysed for Kjeldahl nitrogen, an all-glass micro-Kjeldahl distillation apparatus (figure 7) is used for distillation of formed (and original) ammonia.

Figure 7: Micro-Kjeldahl distillation apparatus; Greenberg et al. (1985).
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