A New System for the Spectrophotometric Determination of Trace Amounts of Nitrite in Environmental Samples

Apresentamos um método espectrofotométrico seletivo e rápido para determinação de nitrito. O princípio do método é a reação do nitrito com p-nitroanilina, em meio ácido, formando o íon diazônio. O íon complexa com 2-etoxi maleato de dietila ou etilenoacetato, em meio básico, formando pigmentos tipo azo, com absorção máxima a 439 e 465 nm, respectivamente. O método obedece a lei de Beer no intervalo de 0,5-15 μg mL para o nitrito com 2-etoxi maleato de dietila, e 0,2-12 μg mL para o nitrito com etileno acetato. A absortividade molar e a sensitividade de Sandell encontradas para os respectivos pigmentos azo foram 5,04×10 L molcm, 0,98×10 μg cm e 1,21×10 L mol cm, 0,98×10 μg cm . Foram avaliadas as condições ótimas de reação, bem como outros parâmetros analíticos. O método foi aplicado com sucesso na determinação de nitrito em várias amostras de água e solos.


Introduction
Nitrogen forms two environmental important oxyacids: nitrous acid and nitric acid.Corresponding nitrite and nitrate anions are common in the environment.In aquatic systems, nitrite is formed by biological oxidation of ammonium, and converted subsequently to nitrate.Nitrite is a characteristic pollutant. 1High concentrations of nitrite are present in industrial water, sewage and in biologically purified effluents and in polluted streams.The determination of nitrite is of considerable interest because of many reasons.Traces of nitrite and nitrate in drinking water may lead to methemoglobinemia in infants and with long term exposure is possible cancer risk.3][4] The reduction of nitrate to nitrite is possible in the stomach of infants, where low acidity allows the growth of nitrite-reducing microorganisms.Nitrite levels, when correlated with other forms of nitrogen in waters, provide indexes of organic pollution in water. 5General procedures for the determination of nitrites are usually based up on some form of diazotization reaction.AOAC official method of analysis for nitrite and nitrate determination was based on the diazocoupling reaction between sulfanilamide and N-(1-naphthyl) ethylenediamine hydrochloride. 6This method required careful control of acidity in each step of the process and generates carcinogenic 7 compound when naphthalene nucleus is present.Some of the reported methods for the determination of nitrite are kinetic, 8,9 chromatographic, 10 and flow injection. 11The spectrophotometric methods for the determination of nitrite are summarized in Table 1.Some method often involve carcinogenic materials for the J. Braz.Chem.Soc.determination of nitrite, as the naphthylamines and some others have non-linear calibration graph, lack of sensitivity, selectivity and involves rigorous experimental conditions.
The present work introduces new coupling agents, ethoxyethylenemaleic ester and ethylcyanoacetate for the selective and rapid spectrophotometric determination of nitrite in water and soil samples without involving complicated steps.p-Nitroaniline is diazotized in acidic medium, and coupled with ethoxyethylene maleic ester or ethyl cyanoacetate to give water-soluble colored dyes in alkaline medium having absorption maximum at 439 and 465 nm respectively.The method has been successfully applied to the determination of nitrite in various water samples and soil samples.

Experimental
Apparatus A Secomam Anthelie NUA 022 UV-VIS spectrophotometer with 1cm quartz cell was used for the absorbance measurements and a WTW pH 330, pH meter were used.

General procedure for the determination of nitrite
An aliquot of the sample solution containing 0.2-20 μg mL -1 of nitrite was transferred to a series of 10 mL calibrated flasks.To this solution 1 mL of 0.05% p-nitroaniline and 0.5 mL of 0.25 mol L -1 hydrochloric acid were added and the solution was shaken thoroughly for 2 minutes to allow the diazotization reaction to go to completion.Then, volumes of 1 mL of 2% ethoxyethylenemaleic ester or ethylcyanoacetate and 2 mL of 5 mol L -1 sodium hydroxide solutions were added and the contents were diluted to 10 mL using double distilled water and mixed well.After 5 minutes, the absorbance of the colored azo dyes was measured at 439 or 465 nm against the reagent blank. 195 mL of water sample (containing not more than 10 μg mL -1 of nitrite) was treated with 0.5 mL of 1 mol L -1 NaOH and 0.5 mL of 0.2 mol L -1 EDTA.The solution was mixed and centrifuged to remove any precipitate formed. Te centrifugate was transferred to a 10 mL standard flask and directly used for the color development by following the procedure described above.The concentration of the nitrite was established by reference with a calibration graph prepared using 0.2-20 μg mL -1 of nitrite in 10 mL standard flasks using distilled water.

Determination of nitrite in soil samples 19
About 1 g of soil sample was weighed (0.9931 g and 0.9894 g respectively) and placed in a 50 mL beaker and then extracted 4 times with 5 mL portions of 1% sodium carbonate.The extract was filtered and made up to 25 mL with distilled water.Suitable aliquot of the sample solution was directly used for the analysis of nitrite in soil samples.

Results and Discussion
The method involves the diazotization of p-nitroaniline, followed by the coupling of ethoxyethylenemaleic ester or ethylcyanoacetate in alkaline medium (Scheme 1).The absorption spectra of the azo dyes formed between p-nitroaniline-ethoxyethylenemaleic ester and p-nitroanilineethylcyanoacetate have absorption maxima at 439 and 465 nm respectively (Figure 1).

Effect of acid concentration and temperature on diazotization
Diazotization and coupling reactions were found to be temperature dependent.Diazotization was carried out at 0-5 ºC and coupling reactions wass carried out at room temperature.Above 50 ºC there is a decrease in intensity of the color.The effect of acidity on the diazotization reaction was studied in the range 0.1-0.5 mol L -1 hydrochloric acid, and constant absorbance was observed in this range.Above this range, a decrease in the absorbance was observed.The optimum acidity for the diazotization was fixed at 0.25 mol L -1 and minimum time for the complete diazotization was found to be 2 minutes.

The effect of the p-nitroaniline concentration
The effect of the p-nitroaniline concentration on the color intensity was studied using the proposed procedure and adding 1 mL of 1-4% solutions of p-nitroaniline in hydrochloric acid (0.25 mol L -1 ) to a series of nitrite solutions.The results showed that a 2% p-nitroaniline solution was sufficient for complete color development.Higher concentration did not enhance the absorbance further, and lower concentration did not give good results.

Effect of coupling agent
The effect of varying the concentration of coupling agent was studied using the proposed procedure and adding 0.2-2.0mL of 2% ethoxyethylenemaleic ester or 0.2-2.0mL of 2% ethylcyanoacetate to a series of nitrite solutions.It was found that maximum and stable color was formed with 1 mL of each ethoxyethylenemaleic ester (2%) or ethylcyanoacetate (2%) solution in a final volume of 10 mL.

Effect of sodium hydroxide concentration
The effect of sodium hydroxide concentration on the absorbance was studied; volumes from 0.1-1.0mL of 5 mol L -1 sodium hydroxide solutions were examined.The investigations showed that 0.4-0.5 mL of NaOH gave maximum absorbance and 0.5 mL of 5 mol L -1 NaOH solutions was chosen for the procedure.Other alkaline solutions were tried, but best results were obtained by using sodium hydroxide.
The stability of the reagents and also the reliability of the proposed method were tested by determining the concentration of 10 samples containing 2 μg mL -1 nitrite per 10 mL over a period of 6h, the mean absorbance was found to be 0.6 and relative standard deviation 1.5% respectively.The results showed no systematic error in the method and thus indicate its reliability.

Effect of interfering species
The effect of other ions on the determination of nitrite using the proposed method was studied for solutions containing 5 μg mL -1 nitrite.Metal ions forming hydroxides in alkaline medium such as Co(II), Cu(II), Hg(II), and Fe(III) were found to interfere, but a large number of these ions were masked with EDTA.Iron(III) was masked using sodium fluoride.The tolerance limit of the foreign ions is shown in Table 2 as the amounts that caused not more than ±2% changes in the values of absorbance during the determinations.

Applications
The proposed methods were applied to the determination of nitrite in water and soil samples.The water samples were collected from different sources, and were filtered before analysis.As the concentration of common pollutants in the natural water is generally far below the tolerance levels shown in Table 2, the method was applied directly to the determination of nitrite in natural water samples.The results obtained are compared with the standard sulfanilamide-NEDA 6 method (Tables 3 and 4).Statistical analysis of the results by t and F-tests showed no significant difference in accuracy and precision of the proposed and reference method. 6The reliability of the method to analyze real samples was checked by recovery experiments, which gave quantitative results with convenient reproducibility.

Figure 1 .
Figure 1.Absorption spectra of diazocouple of p-nitroaniline and ethoxyethylene maleic ester vs. reagent blank (a), Absorption spectra of diazocouple of p-nitroaniline and ethylcyanoacetate vs. reagent blank (b) and reagent blank vs. distilled water (c).

Table 2 .
Effect of diverse ions on the determination of nitrite (5 μg mL -1 )

Table 3 .
Determination of nitrite in different water and soil samples using diazocouple of p-nitroaniline with ethoxyethylenemaleic ester a Mean ± standard deviation (n=5); b tabulated t-value for 8 degrees of freedom at P(0.95) is 2.306; c tabulated F-value for(4, 4)degrees of freedom at P(0.95) is 6.39; d tap water gave no test for nitrite.

Table 4 .
Determination of nitrite in different water and soil samples using diazocouple of p-nitroaniline with ethylcyanoacetate Mean ± standard deviation (n=5); b tabulated t-value for 8 degrees of freedom at P (0.95) is 2.306; c tabulated F-value for(4, 4)degrees of freedom at P (0.95) is 6.39; d tap water gave no test for nitrite. a