nitrate in water experiment

Analysis of the Nitrate Electrode Standard Concentrations

• List the standard concentrations (100.0, 10.0, 1.0, 0.8, 0.4, 0.32, 0.2, and 0.12) under "bottle #" on the lab sheet.
• Plot absorbance or mV readings for the 100-, 10-, and 1-mg/L standards on semi-logarithmic graph paper, with concentration on the logarithmic (x) axis and the absorbance or millivolts (mV) on the linear (y) axis. For the nitrate electrode curve, a straight line with a slope of 58 ñ 3 mV/decade at 25 C should result. That is, measurements of 10- and 100-mg/L standard solutions should be no more than 58 ± 3 mV apart.
• Plot absorbance or mV readings for the 1.0-, 0.8-, 0.4-, 0.32-, 0.2-, and 0.12-mg/L standards on semi-logarithmic graph paper, with concentration on the logarithmic (x) axis and the millivolts (mV) on the linear (y) axis. For the nitrate electrode, the result here should be a curved line since the response of the electrode at these low concentrations is not linear.
• For the nitrate electrode, recalibrate the electrodes several times daily by checking the mV reading of the 10-mg/L and 0.4-mg/L standards and adjusting the calibration control on the meter until the reading plotted on the calibration curve is displayed again.

APHA. 1992. Standard methods for the examination of water and wastewater. 18 th ed. American Public Health Association, Washington, DC.

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Nitrate/Nitrite determination in water and soil samples accompanied by in situ azo dye formation and its removal by superabsorbent cellulose hydrogel

• Research Article
• Published: 16 June 2020
• Volume 2 , article number  1225 , ( 2020 )

Cite this article

• B. U. Gauthama 1 ,
• B. Narayana   ORCID: orcid.org/0000-0001-8945-4516 1 ,
• B. K. Sarojini 2 ,
• Kabiru Bello 2 &
• N. K. Suresh 1  

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In the present work, an efficient method is developed for the spectrophotometric determination of nitrite and nitrate ions in water and soil samples by in situ formation of azo dye (Griess reagent) which showed λ max at 385 nm. The reaction condition and the concentration of reagents used are optimized. The molar absorptivity, Sandell’s sensitivity, detection limit and quantification limit of the method are found to be 3.22 × 10 4 L mol −1 cm −1 , 1.98 × 10 −6 µg cm −2 , 0.0030 µg mL −1 and 0.0092 µg mL −1 respectively with the linearity range up to 2.6 µg mL −1 . The formation of azo dye is confirmed by 1 H and 13 C Nuclear Magnetic Spectroscopy. The azo dye formed during the determination is effectively removed using custom made ecofriendly cellulose modified hydrogel in order to reduce organic load in the test samples. The structure, morphology and the thermal properties of hydrogel are determined by Fourier-transform infrared spectroscopy, scanning electron microscopy and thermogravimetric analysis respectively. The dye removal mechanism involves pseudo second order kinetics, adsorption is found to be spontaneous based on the thermodynamic parameters and it is befitting to Freundlich isotherm model.

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• Environmental Chemistry

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1 Introduction

The bioaccumulation of nitrogenous pollutants like nitrite and nitrate in water and soil pose hazard to humans and plants. Nitrite is also utilized as an additive agent in food industry and its mishandle might lead to excessive amount of nitrite in consumables which is unwholesome to the public health. Organic nitrogen undergoes biochemical oxidation to form nitrate and can be readily converted into the more toxic nitrite by microbial reduction. The WHO recommends 6.5 × 10 −5 M is to be the maximal allowed nitrite concentration in drinking water as per the norms of WHO [ 1 ].

Excess of nitrite in the aquatic ecosystem leads to the formation of algal blooms more and more which also the consequences of low oxygen level in the water, and it further leads to the increase in the temperature of that ecosystem leading to dead zone. Nitrite is an active ion in the nitrogen cycle, formed due to the imperfect oxidation of ammonia or reduction of nitrates. Nitrosamines are the potential carcinogen mostly formed due to nitrites [ 2 ]. Nitrite can enter the body as nitrate, can be converted into nitrite which effects the hemoglobin in delivering oxygen to the cells. A potentially fatal blue baby syndrome ( methemoglobinemia )is also caused in infants due to nitrites. So, it is a major environmental concern, determination and removal of nitrite from water body has large scope [ 3 , 4 ].

There are many methods and instrumental techniques such as polarography [ 5 ], voltammetry [ 6 ], fluorimetry [ 7 ], flow injection spectroscopy [ 8 ], bioamperometry [ 9 ], gas chromatography mass spectroscopy [ 10 ], kinetic methods [ 11 , 12 ] described for the detection of nitrite, nitrate ions. But not all the methods are suitable for routine trace determination because some methods reported are either require expensive instruments or has complicated procedures. Thus, a utilization of above said methods is limited. On comparing with other methods spectrophotometric determination method gained more attention. Thus, highly sensitive, selective spectrophotometric method plays a significant role in the detection of nitrite and nitrate [ 13 ].

This spectrophotometric determination method is based on the Griess technique [ 14 ]. This involves the formation of diazonium salt from an aromatic primary amine, which is later couples with a coupling agent to form an azo dye which imparts colour to the solution. Nitrite ion in acidic medium gets converted into nitrous acid which catalyzes diazonium salt formation. Azo dye is a colour imparting material and the dye itself causes detrimental effects like intervening in the photosynthesis cycle of plants. It also has the affinity to generate carcinogenic/mutagenic products. The adsorption is the preferable method for the dye removal, since its relatively fast, appropriate and easy to carry out [ 15 , 16 ].

Nature of adsorbent is the main effecting factor for the adsorption efficiency. Environmental safety, high adsorption capacity and low-cost productivity are the criteria for the ideal adsorbent. Biodegradable and biocompatible polysaccharide-based hydrogels induced interest in many researchers for testing them in application [ 17 ]. Hydrogel is the material which has an ability to absorb large amount of water in comparing with normal water absorbent material. They are insoluble in water and in most of the organic solvents which could be suitable for the liquid environment [ 18 ]. Their commendatory water sorption property makes them suitable for many fields such as cosmetics [ 19 ], sanitary napkins [ 20 ], tissue engineering [ 21 ], waste water treatment [ 22 ], biomedicine [ 23 ] and many more. Cellulose is the most available natural polymer at present, is one of the most ecofriendly non-dietary sources for the manufacture of variety of nature friendly materials such as paper products, biopolymers, biocomposites and so on. Notably its biodegradability, biocompatibility renewability and low preparation cost made researchers to focus more and more [ 24 ].

In the present study a method has been developed for the detection of nitrite and nitrate in water with high sensitivity, reproducibility with accuracy. Once the determination of nitrite and nitrate are carried out, the coloured solution which contains a hazardous organic pollutant dye was put into sewage without separation. To avoid this, present work involves the removal of dye using newly synthesized cellulose derived hydrogel namely poly (ATAC-co-NaAc) prepared using APS as initiator and MBA as crosslinker, grafted on the carboxy methyl cellulose backbone part. The optimized hydrogel was investigated for kinetics, isothermal and thermodynamic studies and adsorption–desorption studies in order to check the practicability of dye adsorption.

2 Materials and methods

2.1 instrumentation.

Spectrophotometric data were recorded using SHIMADZU UV-2550 double beam spectrophotometer (Shimadzu corporation, Japan) with 1 cm quarts cell. The infrared spectrum of the material was recorded on Bruker tensor 27 spectrophotometer. The functional group analysis of oven dried material was carried out using Attenuated Transmission Method with the frequency range of 400–4000 cm −1 . TGA analysis of the synthesized polymer was taken by thermogravimetric instrument (model: TGA 8000, Perkin Elmer). All samples (5–7 mg) injected with gas flow rate of 100 mL/min with heating rate of 10 °C/min at a temperature range of 25–600 °C under nitrogen atmosphere. Field emission scanning electron microscopy (FESEM) (JEOL 7600) was used for the determination of surface morphology of the CMC-g-poly (ATAC-co-NaAc). This sample analysis sputtered with gold up to 15 min and microgram were recorded at 15 kV accelerating current voltage. Nuclear Magnetic Resonance spectroscopy was used for the structural confirmation of synthesized dye and it was recorded in Bruker 400 MHz and 100 MHz for proton and carbon 13 spectra respectively.

2.2 Reagents and solutions used for spectrophotometric determination

Chemicals used were analytic grade, all solutions were prepared using double distilled water. NaNO 2 (0.1500 g) and KNO 3 (0.7200 g) were dissolved in water for the making of nitrite and nitrate solutions of 1000 µg mL −1 each in two different 100 mL standard flask respectively. Aqueous solutions of sulfanilic acid (0.5%), Resorcinol (0.5%), Hydrochloric acid (2.5 M), Sodium Hydroxide (2.5 M) and EDTA (2%) was prepared.

2.3 Materials used for the preparation of hydrogel

Ethanol and toluene used for the wax extraction were procured from Spectrochem India. For the cellulose extraction sodium hydroxide and sodium hypochlorite were used was obtained from Loba chem India. Ammonium persulfate (APS), isopropyl alcohol, acrylic acid, N, N-methylene-bis-acrylamide (MBA) and sodium monochloroacetate were obtained from Spectrochem India, whereas 3-acryloxyethyltrimethylammonium chloride (ATAC) and Sodium acrylate (SA) were obtained from Sigma Aldrich. Entire chemicals used were of analytical grade and were used as received without purification.

2.4 General procedure for the spectrophotometric detection of nitrite in water

To the series of 25 mL standard flask, added Aliquot of the nitrite sample of 0.2–30 µg mL −1 , added 1 mL each of sulfanilic acid and hydrochloric acid, shaken well for 2 min. Made to stand for 3 min at 0–5 °C for the diazotization reaction to conclude. Then added 1 mL each of resorcinol and sodium hydroxide. Distilled water used to make up to the mark. [ 25 , 26 ]. The schematic representation was given in the Scheme 1 .

Schematic representation of preparation dye

2.5 General procedure for spectrophotometric detection of nitrate in water

Aliquot of nitrate of volume 10 mL was added to the beaker and added 5 mL conc. HCl and 2 g of granulated Zn/NaCl mixture, it was stirred at 30 rpm for 20 min to convert nitrate into nitrite. It was filtered through No. 41 Whatman filter paper to standard flask and diluted to 100 mL. Rest of the procedure was same as procedure for the detection of nitrite in water [ 27 ].

2.6 General procedure for detection of nitrate/nitrite in soil sample

To a clean 50 mL beaker, 1.0 g of soil sample was transferred. Then it was extracted using 0.5% sodium carbonate solution 4 times with 5 mL each, Whatman filter paper No. 41 was used for the filtration. Appropriate aliquots of the sample were transferred to 25 mL standard flask and analyzed for nitrite and nitrate according to the above procedure. All gave negative results. Now known amount of nitrite and nitrate were added separately, then analyzed using the same procedure.

2.7 General procedure for the Preparation of CMC-g-poly (ATAC-co-NaAc)

Graft copolymerization via free radical mechanism was performed for the making CMC-g-poly (ATAC-co-NaAc) on CMC. APS helped to initiate the reaction and role of MBA as cross linker. The main raw material i.e. CMC was made in our laboratory as reported [ 28 ], the preparation of performed via copolymerization of poly (ATAC-co-NaAc) on CMC using (APS) and (MBA) as initiator and cross linker respectively. In a typical reaction, 0.5 g of CMC was taken in a beaker containing 20 mL of distilled water with stirring for 10 h to get clear solution. Subsequently varied quantity of APS, ATAC and SA were added to the mixture with uniform stirring. Furthermore, different amount of MBA (0.01-0.120 g dissolved in 2 mL of demineralized water) was added and stirring continued for 3 h. Finally, the reactants were irradiated with 100-watt microwave power for 60 s for gelation. The hydrogel obtained was allowed overnight, followed by treated with three-fold of acetone for 3 h for the extraction of homopolymer. The unreacted monomers were removed by washing with distilled water [ 29 ]. It was then dried in an oven at 50 ° C for 2 h and finally stored for further analysis. The percentage of grafting and its efficiency were evaluated using Eqs. ( 1 ) and ( 2 ) respectively.

where W 0 is the weight of CMC, W 1 represents weight of (ATAC-co- NaAc) and W 2 represents CMC-g-poly (ATAC-co-NaAc) after the extraction of homopolymer. The schematic representation of the reaction was given in the Scheme 2 .

Proposed reaction mechanism of CMC-g-poly (ATAC-co- NaAc)

2.8 General procedure for adsorption studies

A known quantity of CMC-g-poly (ATAC-co-NaAc) was placed in 200 mL of 100 mg L −1 dye solution. The solution was stirred for predetermined time at 100 rpm, at each time interval 3 mL of dye solution was taken out, centrifuged to remove solid particles and diluted to predefined concentration, analyzed in spectrophotometer. The sample were analyzed in the wave length range of 200–800 nm. The calibration curve and linear regression plot was obtained by converting λ max of each sample into its concentration. The amount of dye adsorbed can be evaluated by the Eq. ( 3 )

where q e (mg g −1 ) represents the adsorption potential at equilibrium, C o indicates initial dye concentration and C e represents equilibrium dye concentration (mg L −1 ) in aqueous solution, M (mg) and V (mL) represents the weight of the hydrogel and volume of dye solution taken respectively.

2.9 General procedure for dye recycling studies

Dye adsorption–desorption investigation were performed out to check the reusability of the polymer. On the experiment, 100 mL of dye (isolated azo dye) solution of 100 mg L −1 was taken in a beaker to which added 17.4 mg of hydrogel and allowed it for adsorption up to its maximum adsorption of 3600 min. Later the hydrogel was removed from the solution and desorption experiment was proceeded. The adsorbed hydrogel was immersed in 0.5 mol L −1 each of HCl and NaOH solution, saturated NaCl solution of 100 mL each for 2 h at room temperature, hydrogel was taken out from the solution and washed with water to ensure hydrogel was free from dye on its surface, finally it was dried at 50 °C [ 30 ]. UV–Vis spectrophotometer was used to find out the amount of dye desorbed. The desorption ratio was calculated using the Eq. ( 4 )

where C o was the initial concentration and C e was the equilibrium concentration in the adsorbed solution whereas C d was the dye concentration in the desorbed solution, V i and V d were the solution volume taken for adsorption and desorption respectively.

2.10 General procedure for the removal of azo dye from the real test samples

In order to remove the azo dye from the test samples, we used 2.6 µg mL −1 of nitrite test solutions as such with varied quantity of hydrogel to check the adsorption capacity of azo dye. Immediately after testing for nitrite and nitrate hydrogel was put into the test solutions for 20 h.

2.11 General procedure for the removal of excess of resorcinol

Since the dye formation involves resorcinol as a key raw material, there was a chance of presence of excess of resorcinol in the solution. The excess of unreacted resorcinol from the solution was removed by activated charcoal [ 31 ].

3 Results and discussions

3.1 study of reagent concentration.

The effect of variation of sulfanilic acid and resorcinol concentration on the colour intensity of the forming azo dye was studied using present method (Fig. S1 ESI*). From the study it was revealed that the volume of 1 mL of 0.5% each of sulfanilic acid and resorcinol solutions gave the maximum absorbance. A higher or lower concentration of the reagent showed less absorbance intensity and 1 mL each of both solutions was enough for the complete colour development.

3.2 Spectrophotometric study

The azo dye formed by the reaction of sulfanilic acid and resorcinol showed absorption maximum at 385 nm (Fig. 1 a) in the spectrophotometer with the orange red colour. It was documented in the literature that the azo dye (was also called as Chrysoine resorcinol) exhibits absorption maximum at 387 nm evidencing the successful diazotization and coupling reactions [ 32 ]. Diazotization and coupling reaction were found to be temperature dependent and it should be carried at 0–5 °C and at 25 °C respectively. There were no distinguishable changes in the colour up to 35 °C. Above 40 °C there was a decrease in the intensity of colour of the solution. The reagents, 0.5% sulfanilic acid (1 mL), 0.5% resorcinol (1 mL), 2.5 M solution of both hydrochloric acid (1 mL) and sodium hydroxide (1 mL) solution per aliquot amount of sample 0.2–30 µg mL −1 resulted in maximum absorbance.

a Absorption spectra of dye and reagent blank. b Absorption spectra at different concentrations. c Calibration curve

3.3 Validity of Beer–Lambert’s law

By measuring the absorbance values of various concentration of nitrite solution ranging from 0.2 to 30 µg mL −1 (Fig. 1 b). Beer’s law was studied and results shows in the plot of absorbance versus concentration (Fig. 1 c). From the plot it was evident that Beer’s law obeyed from 0.2 to 2.6 µg mL −1 of nitrite. The molar absorptivity, Sandell’s sensitivity, LOD (D L  = 3.3 σ/S) and LOQ (Q L  = 3.3 σ/S; where σ was the standard deviation of reagent blank (n = 5) and S was the slope of the calibration curve) of the method were 3.22 × 10 4 L mol −1 cm −1 , 1.98 × 10 −6 µg cm −2 , 0.0030 µg mL −1 and 0.0092 µg mL −1 respectively which was compared to be better than the reference method developed earlier at our lab [ 25 ]. Comparison of final results obtained in present method with other cited methods are given in the Tables 1 , 2 a, b, 3 .

3.4 Effect of interfering ions

The effect of diverse ions on the detection of nitrite/nitrate in the proposed method was studied with the fixed concentration of nitrite (2.6 µg mL −1 ) and nitrate (2.6 µg mL −1 ). The test result unveiled that Pb(II), Hg(II), Sn(II), Fe(III) found to interfere severely. The intensity of absorbance decreased substantially by the addition of these metal ions into the solution. The reaction of these metal ions with nitrite ions could be the reason for the early removal of nitrite ions from the solution thereby reducing the concentration of nitrite ions for diazotization. Anyhow, the tolerance limit of these ions was increased by adding 3 mL of 2% EDTA solution which preferentially chelates with these metal ions [ 42 ]. The tolerance limits of all tested ions are listed in the Table 4 . The change in intensity of absorbance was not more than ± 2% caused by the change in the amount of targeted ions.

3.5 The NMR analysis for the confirmation of formation of azo dye

Structure of the dye, 4-((2,4-dihydroxyphenyl)diazinyl)benzenesulfonic acid) was confirmed by 1 H and 13 C NMR spectra (Fig. 2 a, b). 1H NMR(400 MHz, DMSO–d 6 ): δ ppm, 6.36 (s, 1H, Ar–H), 6.51 (d,1H, Ar–H), 7.65–7.85 (m, 5H, Ar–H), 10.62 (s, 1H, Ar–OH), 12.38 (s, 1H, Ar–OH), 13 C NMR (100 MHz, DMSO–d 6 ) 102.989 (C–N), 109.21 (C–N), 121.07, 126.66, 129.84, 132.34 (aromatic C’s), 149.46 (CH), 150.36 (C–S), 156.53 (C–OH),163.17 (C–OH).

a 1 H NMR of 4-((2,4-dihydroxyphenyl)diazinyl)benzenesulfonic acid), b 13 C NMR of 4-((2,4-dihydroxyphenyl)diazinyl)benzenesulfonic acid)

3.6 FT-IR analysis of hydrogel

Structural confirmation through functional groups identification of CMC and CMC-g-poly (ATAC-co-NaAc) was followed by FT-IR spectroscopy and its typical FTIR spectra were shown in the Fig. 3 . IR spectra of CMC has all the normal peaks of cellulose includes peaks at 3332, 2914, 1626, 1026 cm −1 which were attributed to O–H, C–H, C=O, C–O stretching vibrations respectively. The same peaks observed in the backbone of CMC-g-poly (ATAC-co-NaAc). In addition to the above, CMC-g-poly (ATAC-co-NaAc) showed new peak at 1718 cm −1 corresponds to stretching vibrations of carbonyl groups of esters indicating the successful grafting [ 20 ]. The band at 1484 and 960 cm −1 corresponds to symmetric and asymmetric stretching vibrations of C–N bond in the 2-acryloxy ethyl trimethyl ammonium group which also another evident for the successful grafting. The one more evident for the successful grafting was shown by the peak at 1139 cm −1 , correlate with the C–O–C bond between carboxy cellulose back bone and alkyl group in the polymer [ 43 ].

FTIR spectra of graft copolymerized CMC-g-poly (ATAC-co-NaAc)

3.7 FESEM analysis

Surface morphology of CMC and CMC-g-poly (ATAC-co-NaAc) were analyzed by FESEM. Figure 4 shows that CMC has a rod like shape whereas CMC-g-poly (ATAC-co-NaAc) surface morphology has completely different indicating successful grafting [ 44 ].

SEM images of a CMC, b and c CMC-g-poly (ATAC-co-NaAc)

3.8 Thermogravimetric analysis

Thermogravimetric analysis was a complimentary technique from which one can get the idea about the composition and the thermal stability of the sample. Thermograms of the CMC and CMC-g-poly (ATAC-co-NaAc) were shown in the Fig. 5 . It was integrated like, CMC has 3 stages of decomposition whereas CMC-g-poly (ATAC-co-NaAc) has 4 stages of decomposition with proportionate weight loss up on increase in the temperature. Both CMC and CMC-g-poly (ATAC-co-NaAc) dissipated initially from 25 to 91 °C and 25 to 70 °C with the loss in 10% and 4% mass respectively which might be due to loss disappearance of moisture content in the sample. There was insignificant decrease in weight on increase in temperature up to 280 °C for CMC. In addition, the second decomposition stage from 280 to 340 °C with 70% of total weight which may correspond to the decomposition of hydroxyl and carbonyl group of the polymer, similarly, third decomposition stage started from 340 °C till 500 °C with 20% weight loss. The CMC-g-poly (ATAC-co-NaAc) showed its second decomposition curve in the range of 250–290 °C with 30% loss in weight corresponded to the decomposition of carbonyl and hydroxyl groups. Third and its successive decomposition stage observed in the range of 350–400 °C with 20% weight loss and 450–550 °C with 15% of weight loss respectively. From the curve, it was concluded that CMC has lost its 70% of its weight below 340 °C whereas CMC-g-poly (ATAC-co-NaAc) lost only 30% of its weight indicating its significant higher thermal stability than the parent cellulose. It can also be considered as one of the evidences for successful grafting of CMC [ 45 ].

TGA graph of CMC and CMC-g-poly (ATAC-co-NaAc)

3.9 Adsorption kinetics

Kinetic study gives an important information about the adsorption mechanism of dye between the adsorbent and adsorbate which was necessary to predict the adsorbent adsorption rate and time, was given in the Fig. 6 . The effect of contact time on the adsorption ability of CMC-g-poly with the dye. The adsorption of dye increased rapidly until reaching a constant value at a contact time of 3600 min. Pseudo first order (linear and nonlinear) and pseudo second order kinetics in the linear form were analyzed and were evidenced by Eqs. ( 5 ) and ( 6 ) respectively:

a The pseudo second-order linear fit. b Pseudo second-order non-linear fit. c Pseudo first order adsorption kinetics for dye adsorption onto CMC-g-poly (APTAC-co-DMA; pH = 5–6, Dye concentrations = 100 mgL −1 , adsorbent dose = 76.5 mg 100 mL −1 ). d Effect of contact time on adsorption of dye by CMC-g-poly(APTAC-co-DMA)

where k 1 and k 2 (g mg/min) are the rate constants, q e (mg/g) was the amounts of dye adsorbed at equilibrium contact time and q t (mg/g) was the adsorption at time t min, respectively.

The correlation coefficient (R 2 ) for the pseudo-second (Linear) order kinetic model was 0.9811 and was considerably more than those of pseudo-second (Non-Linear) model and pseudo-first order model with correlation coefficient 0.8698 and 0.9562 (Table 5 ). This means the adsorption mechanism is chemisorption. Most importantly, theoretical amount of adsorption was near to the experimental values of pseudo second order kinetics [ 46 , 47 , 48 ].

3.10 Adsorption isotherm

Adsorption isotherm were predominant for discern the adsorbent-adsorbate interaction. Freundlich and Langmuir adsorption models were the two major, important models demonstrating the association between the adsorbent and adsorbate. Freundlich model gave empirical relation between the solute concentration on the adsorbent surface to the solute concentration in the medium whereas Langmuir model gives a relation between monolayer of adsorbate molecules surrounding a homogeneous solid surface called adsorbent. These two isotherm models were denoted by the Eqs. ( 7 ) and ( 8 ) respectively.

where q e (mg g −1 ) was the amount of adsorbate at equilibrium adsorption and q m was the amount of adsorbent adsorbed on saturated monolayer. The K L and K F were the constants of Langmuir and Freundlich models respectively and C e (mg L −1 ) was the dye concentration in solution at equilibrium. The heterogeneity factor 1/n or n represents adsorption intensity. From the plot of log q e versus log C e and C e /q e versus C e , Freundlich and Langmuir adsorption models were demonstrated respectively (Fig. 7 ) [ 49 ].

a Freundlich. b Langmuir adsorption isotherms for the dye adsorption

It was evident that Freundlich isotherm model was fitted for the experimental value when compared with the Langmuir model since the correlation coefficient R 2 for Freundlich was 0.9962 on comparing with the correlation coefficient R 2 for Langmuir model. Freundlich adsorption isotherm model was depend on the hypothesis that adsorption was a multilayer process on the heterogeneous surface with irregular adsorption heat and affinity distribution [ 50 ]. If the n value was in between 1 and 10 then the adsorption was favorable and here it was 1.103. Remaining parameters were tabulated in the Table 6 .

3.11 Adsorption thermodynamics

The adsorption rate and adsorption feasibility also depend on temperature of adsorption and it was evaluated by the thermodynamic specifications namely change in standard Gibb’s free energy ΔG o , change in standard enthalpy ΔH° and change in standard entropy ΔS°. The spontaneity of adsorption depends on the Gibb’s free energy and it was determined by the Eq. ( 9 )

where K was thermodynamic equilibrium constant, R was the gas constant T was the temperature.

Thermodynamic constant K was affected by the change in temperature and it was given by the Eq. ( 10 )

By integrating and on rearrangements of the Eq. ( 10 ), we get

The change in the Gibb’s free energy was given by

Δ H ° and Δ S ° were determined from the intercept and slope of the graph lnK versus 1/T (Fig. 8 ). The calculated and theoretical thermodynamic parameters were reported in the Table 7 . The positive value of Δ H ° shows the adsorption process was endothermic in nature [ 51 ]. Theoretical and experimental Gibb’s free energy value were approximately nearer which reflects that the adsorption was feasible with negative value indicating the spontaneous adsorption.

Plot of ln K versus 1/T of adsorption of dye at different temperatures

3.12 Recycling studies-adsorption and desorption

The recycling ability of the newly synthesized hydrogel was analyzed via continues adsorption–desorption process of dye. The stability of the hydrogel also studied by collecting the data of adsorption–desorption cycle. For the adsorption, solution of 100 mgL −1 of dye with hydrogel was stirred at room 28° C until the equilibrium adsorption achieved which was followed desorption studies. The desorption experiment was conducted at three different conditions i.e. acidic (0.5 M HCl solution), neutral (1% NaCl solution) and basic (0.5 M NaOH solution) pH. In the basic medium, adsorption–desorption cycle was restricted to one, this might be due to the nature of the hydrogel, the result shown in the Fig. S2 (ESI*). From the figure, it was evident that in acidic medium was favorable for the recycling over neutral medium and desorption experiment attained equilibrium in 1 h. Afterwards it was regenerated by drying at 50 o C and reused for both adsorptive and desorptive studies. The adsorption–desorption cycle was carried out three times and there was a minimal decrease in the efficiency of hydrogel on multiple cycles. Therefore, the synthesized hydrogel has enough efficiency to use it several times with appreciable capacity [ 52 , 53 , 54 , 55 ].

3.13 Removal of dye from the test samples

Based on the above experiment, for the removal of azo dye from the test samples which was formed during the nitrite estimation, varied amount of hydrogel i.e. 20 mg, 40 mg and 70 mg were put into the test samples of 2.6 µg mL −1 of nitrite for 20 h. The results are shown in the Fig. S3 (ESI*). The figure shows that 20 mg and 40 mg of hydrogels were not adequate enough to remove azo dye completely whereas 70 mg of hydrogel was sufficient for the removal of 2.6 µg mL −1 azo dye completely.

3.14 Removal of surplus reagents

In the process of determining nitrite, resorcinol and sulfanilic acid were being used as reagents. There observed a peak of lesser intensity around 280 nm which could be attributed either resorcinol (λmax:273 nm) or sulfanilic acid (λ max: 290 nm) [ 56 ]. In order to remove these impurities, activated charcoal (100 mg) was added to the test sample priory treated with 70 mg of hydrogel and kept for equilibration for 12 h. The UV absorption spectrum depicted in Fig. S4 (ESI*) indicated absence of absorption peak (red line) attributing both for dye as well as impurities. Hence this was an attempt to detect nitrite/nitrate by in situ azo dye formation and removal of the pollutants and contaminants from the water body.

4 Conclusion

In summary, this paper presents an approach to determine the nitrite and nitrate present in soil and water with high sensitivity and selectivity using Griess reagent prepared by commercially available sulfanilic acid and resorcinol. This method of detection of nitrite and nitrate exhibits a wide linearity range and low detection limit compared to the previously reported results. In the determination process, the azo dye formed could be considered as secondary pollutant for the environment. Hence dye from the test solutions was effectively removed by adsorption using custom made ecofriendly superabsorbent hydrogel [graft copolymer CMC-g-poly (ATAC-co-NaAc)]. The 70 mg of the hydrogel successively removed dye with a concentration of 2.6 μg mL −1 . The dye removal mechanism involved pseudo second order kinetics, adsorption was found to be spontaneous based on the thermodynamic parameters and it was befitting to Freundlich isotherm model. In addition, the impurities like unreacted reagents resorcinol/sulfanilic acids were removed from the test solutions by equilibrating with activated charcoal for 12 h. Therefore, the present investigation uses the versatile Griess reagent to determine the primary pollutants effectively at the same time suggests an efficient method to remove secondary pollutant azo dye formed during the determination.

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The authors gratefully acknowledge BSR one time Grant to BN for purchase of chemicals. Authors thank DST-PURSE laboratory for TGA analysis and other facilities.

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Gauthama, B.U., Narayana, B., Sarojini, B.K. et al. Nitrate/Nitrite determination in water and soil samples accompanied by in situ azo dye formation and its removal by superabsorbent cellulose hydrogel. SN Appl. Sci. 2 , 1225 (2020). https://doi.org/10.1007/s42452-020-3016-5

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Nitrite Determination in Environmental Water Samples Using Microchip Electrophoresis Coupled with Amperometric Detection

Simone bernardino lucas.

1 Instituto de Química, Universidade Federal de Goiás, Goiânia 74690-900, GO, Brazil

Lucas Mattos Duarte

2 Instituto de Química, Departamento de Química Analítica, Universidade Federal Fluminense, Niterói 24020-141, RJ, Brazil

Kariolanda Cristina Andrade Rezende

Wendell karlos tomazelli coltro.

3 Instituto Nacional de Ciência e Tecnologia de Bioanalítica (INCTBio), Campinas 13083-861, SP, Brazil

Associated Data

Not applicable.

Nitrite is considered an important target analyte for environmental monitoring. In water resources, nitrite is the result of the nitrogen cycle and the leaching processes of pesticides based on nitrogenous compounds. A high concentration of nitrite can be associated with intoxication processes and metabolic disorders in humans. The present study describes the development of a portable analytical methodology based on microchip electrophoresis coupled with amperometric detection for the determination of nitrite in environmental water samples. Electrophoretic and detection conditions were optimized, and the best separations were achieved within 60 s by employing a mixture of 30 mmol L −1 lactic acid and 15 mmol L −1 histidine (pH = 3.8) as a running buffer applying 0.7 V to the working electrode ( versus Pt) for amperometric measurements. The developed methodology revealed a satisfactory linear behavior in the concentration range between 20 and 80 μmolL −1 (R 2 = 0.999) with a limit of detection of 1.3 μmolL −1 . The nitrite concentration was determined in five water samples and the achieved values ranged from (28.7 ± 1.6) to (67.1 ± 0.5) µmol L −1 . The data showed that using the proposed methodology revealed satisfactory recovery values (83.5–103.8%) and is in good agreement with the reference technique. Due to its low sample consumption, portability potential, high analytical frequency, and instrumental simplicity, the developed methodology may be considered a promising strategy to monitor and quantitatively determine nitrite in environmental samples.

1. Introduction

Inorganic ions are widely considered as parameters for monitoring and controlling the quality of food samples [ 1 , 2 ], biological matrices [ 3 , 4 ], environmental samples [ 5 , 6 ], and clinical diagnoses [ 7 , 8 ]. Among the inorganic ions, nitrite has been studied to evaluate food quality. Cardoso and coworkers determined the presence of nitrite in sausage and ham samples, due to the use of this compound as a food preservative [ 9 , 10 ]. In biological matrices, nitrite is evaluated due to its relationship with the metabolic pathway of nitrous oxide and a series of other clinical diagnoses, depending on the fluid in which it is evaluated (urine, saliva, or blood) [ 10 , 11 , 12 ]. In the field of environmental analysis, the presence of nitrite in water samples comes from natural processes such as biological denitrification and acid rain, or it is due to contamination by industrial waste and other economic activities [ 13 , 14 ].

In recent years, fertilizers and agrochemicals based on nitrogen species have been increasingly used, and in the case of an over-application, the lixiviation process can transfer nitrite to rivers and lakes [ 15 , 16 , 17 ]. The consumption of water and food containing nitrite above the limit can cause its accumulation in the human body [ 18 ]. In high concentrations, nitrite may be associated with methemoglobinemia or “blue baby syndrome” [ 19 ], carcinogenic nitrosamines, gastric cancer, spontaneous intrauterine growth restriction, abortions, and birth defects of the central nervous system, among other diagnoses [ 20 , 21 , 22 ].

The quantification of nitrite in environmental water supply samples is an important procedure to monitor and control the increase of NO 2 − in water resources and avoid possible poisoning [ 18 , 23 , 24 ]. Nitrite can be determined by spectrophotometric measurements through different methodologies including nitrosation-based [ 25 ], catalytic [ 26 ], and Griess reaction [ 14 ] assays. The latter is the most widely used due to its simplicity and the low cost per analysis; however, bench-based standard methodologies require a large volume of reagents, generate a considerable amount of waste, and may be considered time-consuming [ 14 , 25 ].

Other techniques have also been employed for nitrite analysis like chemiluminescence, chromatography, electrochemistry, and capillary electrophoresis (CE) [ 27 ]. Chemiluminescence provides simplicity, a wide linear range, and low cost, but it also offers poor stability and reproducibility [ 28 , 29 , 30 , 31 , 32 ]. Chromatographic methods such as gas chromatography hyphenated with mass spectrometry detection (GC-MS) and high-performance liquid chromatography (HPLC) are explored more often due to their high selectivity and the potential to perform a faster analysis [ 33 , 34 , 35 , 36 ]; however, they require high-cost instrumentation, sample pre-treatment, and derivatization steps [ 25 ]. Electrochemical detection has also been explored for nitrite analysis, offering low-cost analyses with miniaturization capability. In addition, the use of modified electrodes helps to potentially increase sensitivity and selectivity [ 37 , 38 , 39 , 40 , 41 ]. Lastly, conventional CE systems have gained prominence among the available separation techniques due to their efficiency, resolution, and relatively short analysis times, when compared to chromatographic separations [ 7 ].

With the aim of employing a faster alternative approach for the detection of nitrite, portable microsystems have been described in the literature, including paper-based colorimetric assays [ 38 ]. Nevertheless, paper-based devices can suffer interference from other ions, as well as limiting the possibility of using some solvents due to their interaction with the platform material [ 10 , 17 , 42 , 43 ]. Another alternative is the use of microchip electrophoresis (ME) devices due to their potential for portability and high separation efficiency, short analysis time, reduced sample consumption, and low waste generation [ 44 ]. In addition, ME devices can be manufactured on different materials including glass, silicon, polymers, threads, 3D printing filaments toner, and paper-based platforms on paper or polymers [ 45 , 46 , 47 , 48 , 49 ].

Freitas and coworkers used ME with capacitively coupled contactless conductivity detection (C 4 D) to monitor inorganic species, including nitrite in aquarium and river water, achieving a limit of detection (LOD) of 4.9 µmoL −1 [ 50 ]. In addition to conductometric detection [ 50 , 51 , 52 ], amperometric detection (AD) can be used in association with chip-based systems, because it provides better selectivity and sensitivity [ 53 , 54 , 55 , 56 ]. For environmental samples, selectivity is an important feature to be considered, especially because the presence of other charged species in water samples, such as chloride, nitrate, and sulfate anions, can negatively affect the separation performance when conductivity detection modes are used [ 57 , 58 ]. To increase the selectivity and sensitivity of amperometric detection for nitrite determination, different groups have proposed the use of simple hybrid electrodes, nanotubes, and/or nanocomposites [ 22 , 24 , 59 , 60 ].

In this context, the present study proposes the use of ME–AD for the quantification of nitrite in environmental water samples. The methodology was developed using a commercial and portable instrument, which comprises a potentiostat, a high voltage power supply, and a microfluidic platform to assemble ME devices with integrated electrodes. The feasibility of the proposed methodology was demonstrated through nitrite determination in water samples from two types of ecosystems, a small aquarium, and a fish breeding dam. The obtained results were compared with the standard spectrophotometric method based on the Griess reaction.

2. Materials and Methods

2.1. reagents and solutions.

Histidine (His), sodium hydroxide (NaOH), sulphanilamide, and N-(1-naphtyl)-ethylenediamine dihydrochloride (NED) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Lactic Acid (HLat) was acquired from Cromoline (Diadema, SP, Brazil), and sodium nitrite (NaNO 2 ) and citric acid were supplied by NEON (Sao Paulo, SP, Brazil). Stock solutions of HLat, His, NaOH, and NaNO 2 were prepared at concentrations of 200, 100, 100, and 10 mmol L −1 , respectively. For the reference methodology using the Griess reaction, stock solutions of 4 mmol L −1 NED and 50 mmol L −1 sulphanilamide were used. All solutions were weekly prepared with ultrapure water (resistivity 18 MΩ cm) and filtered through nylon filters with 0.22 µm pore diameter. All experiments were performed at 23 ± 1 °C.

2.2. Samples

Water samples were collected from two types of ecosystems located in the city of Inhumas-GO: a small aquarium (16°22′24.9″ S 49°29′04.4″ W) and a fish breeding dam (16°20′16.8″ S 49°29′44.4″ W). Both types of samples were collected, filtered through nylon filters with 0.22 µm pore diameter, and stored in polymeric sterile tubes. No further pretreatment was necessary, and dilutions were made for quantification by the reference and the proposed methods.

2.3. Instrumentation

For the analytical procedures performed with amperometric detection, an HVStat system supplied by MicruX Technologies (Asturias, Spain) was used. This system consists of a portable instrument that includes a high voltage power supply integrated with a 165 × 150 × 85 mm 3 potentiostat ( Figure 1 A) and a platform ( Figure 1 B) that has been specifically developed for use by coupling the microchips with the detection system. This system also incorporates software (MicruX Manager) for instrumentation control, data acquisition, and processing.

Portable instrumentation for ME–AD analysis including: ( A ) HVStat system manufactured by MicruX Technologies (Asturias, Spain) with integrated bipotentiostat coupled with a high voltage power source, ( B ) microfluidic holder (model MCE-HOLDER-DC02), ( C ) layout of the hybrid microchip composed of SU-8/glass with integrated electrodes for electrochemical detection highlighting the positioning of electrodes at end-channel arrangement.

The electrophoresis microchip was composed of a hybrid SU-8/glass platform (38 mm long × 13 mm wide × 0.8 mm thick) ( Figure 1 C) containing injection and separation microchannels (50 µm width × 20 µm deep) designed in a cross-shaped arrangement. The total and effective separation microchannel lengths were 35 and 30 mm, respectively. Amperometric detection was performed using integrated Ti/Pt (50/150 nm) thin-film electrodes, where the working electrode (WE) was positioned at 20 µm from the separation channel extremity (end-channel mode). All three electrodes were spaced at 100 μm from each other.

2.4. Electrophoretic Procedure

The microchips were preconditioned and rinsed with 0.1 mol L −1 NaOH, ultrapure water, and running buffer for 30, 15, and 10 min, respectively. The procedure was performed with the aid of a simple vacuum system, at 560 mmHg. After the preconditioning stage, all the reservoirs and microchannels were filled with the running buffer solution.

The microchip was connected to the HVStat system while fixed in its holder and the baseline was stabilized. The sample was electrokinetically injected using the floating mode, where an electric potential was applied for enough time to fill the injection channel [ 61 , 62 , 63 ]. Following this, another electric potential was applied so that the sample volume at the intersection of the channels went to the detection zone. Finally, the detection potential was applied and the electropherogram was recorded. All experiments were performed at 23 ± 1 °C.

2.5. Reference Analytical Method

To assess the accuracy of the developed methodology, the samples were also analyzed according to the spectrophotometric procedure based on the Griess reaction, following the protocol of the Association of Official Analytical Chemists [ 64 ]. After the colorimetric reaction, measurements were carried out with a UV-Vis Spectrophotometer manufactured by FEMTO (São Paulo, SP, Brazil) at 530 nm.

3. Results and Discussion

3.1. detection potential optimization.

Hydrodynamic voltammograms were recorded to evaluate the best potential for nitrite detection during electrophoretic procedure. A standard solution of 200 µmolL −1 NO 2 − was then used as a model in electrophoretic runs and detected by applying potentials between 0.5 and 1.0 V to the working electrode (see the electropherograms presented in Figure S1A —available in the Supplementary Materials ). The signals were evaluated for the area, intensity, and peak width. In addition, the stability of the electric current was observed. Considering all these parameters, the potential of 0.7 V was defined as optimum and kept constant for the next steps due to providing the highest intensity and peak area, as shown in Figure S1B . Furthermore, it is important to mention that the baseline current remained stable for the analysis that was applying this detection potential. For a higher potential, a noticeable increase in the background current was observed ( Figure S1C ).

3.2. Optimization of the Running Buffer

According to the literature, the separation of the anionic species by ME devices is successfully performed when lactic acid is used on the running buffer [ 50 , 51 ]. Therefore, a buffer solution composed of lactic acid and histidine was prepared at different ratios to investigate the pH effect on nitrite determination. Th analyses were carried out in an acidic medium to favor nitrite oxidation [ 65 , 66 , 67 , 68 ]. For this purpose, the lactic acid concentration was fixed at 30 mmol L −1 and the histidine concentration ranged from 5 to 25 mmol L −1 (5 mmol L −1 increments), providing solutions with pH at 3.2, 3.6, 3.8, 4.1, and 4.5, respectively (a–e) ( Figure 2 A). An increase in the concentration of histidine led to an increase in the pH of the electrolyte and an improvement in peak shape. However, at 20 mmol L −1 of histidine, the peak area decreased, and the peak width had widened ( Figure 2 B), which negatively impacted the sensitivity and separation efficiency. Thus, the run buffer that was composed of 30 mmol L −1 lactic acid and 15 mmol L −1 histidine (pH = 3.8) was defined as optimum and kept constant once it also provided the lower relative standard deviation values.

( A ) Electropherograms showing the separation and detection of nitrite (50 µmol L −1 ) using a running buffer composed of 30 mmol L −1 lactic acid and histidine in the concentrations between 5 and 25 mmol L −1 , resulting in a pH from 3.2 to 4.5 (a–e). ( B ) Peak area and width versus buffer pH values. Injection: −800 V for 10 s, Separation: −1000 V for 60 s, Detection 0.7 V versus Pt.

3.3. Eletrophoretic Parameters

The sample was electrokinetically introduced into microchannels using the floating mode. To achieve the suitable injection time, a 50 µmol L −1 nitrite standard solution was used as model. For this purpose, injection time was varied from 1 to 20 s. As shown in Figure 3 A, the presence of nitrite was observed only for injection times longer than 3 s. As expected, the increase in the injection time caused a greater volume of the sample to be injected and, consequently, a higher signal was observed [ 63 ]. For injection times longer than 10s, a noticeable peak broadening with a consequent loss of peak symmetry was observed. Based on the data presented in Figure 3 B, the injection time of 10 s was then selected as ideal for the subsequent experiments.

( A ) Electropherograms showing the separation and detection of nitrite (50 µmol L −1 ) introduced into microchannels under different injection times: 1–20 s. ( B ) Area and peak width versus injection time. Running buffer: 30/15 mmol L −1 Lactic Acid/Histidine (pH = 3.8). Injection: −800 V, Separation: −1000 V for 60 s, Detection 0.7 V versus Pt.

The potentials used for electrokinetic control of solutions into microchannels were also optimized. The injection potential was varied from −300 to −1000 V, and separation from −500 to −1200 V ( Figure 4 A). The peak width and migration times presented similar behaviors, being inversely proportional to the application of potential. The peak area, as expected, was directly proportional to the increase in the applied potential ( Figure 4 B).

( A ) Electropherograms showing the separation and detection of nitrite (50 µmol L −1 ) under different injection/separation voltages. ( B ) Peak area and migration time versus injection/separation potentials. Experimental conditions are given in Figure 3 .

When applying −1200 V for the separation potential, despite obtaining the highest peak area and the shortest migration time, a slightly higher RSD value (8.6%, n = 3) was obtained. Therefore, −1000 V was defined for the separation potential since the difference in the area for both conditions was low, and under these conditions a lower RSD was obtained between the replicates ( Figure 4 B).

The effect of the difference in conductivity between the sample and the running buffer, in the detection, was also evaluated ( Figure S2 ). To achieve this, a fortified sample and a standard solution were prepared at the same concentration (30 μmolL −1 of NO 2 − ). The samples were diluted in water and a 10% v/v running buffer. When comparing the nitrite peak areas and intensities recorded under the two dilution procedures, it was observed that samples diluted in 10% v/v buffer presented a smaller standard deviation. The difference in the peak area in the fortified sample compared to that obtained in the standard solution was lower than 12.4%. This may be attributed to a biased electrokinetic injection and possibly to sample stacking. Therefore, a 10% v/v running buffer was used in all subsequent dilutions.

3.4. Method Validation

After experimental optimizations, the analytical performance of the proposed methodology was investigated by consecutive injections of a standard nitrite solution prepared at 100 μmol L −1 . For a sequence of three sequential injections, the relative standard deviation (RSD) values for migration time, peak area, and intensity ranged from 0.1 to 2.3% ( Table 1 ), thus suggesting satisfactory injection–to–injection repeatability in an intra-day comparison. In the same way, the nitrite analysis was also performed on three different days using two different commercial microchips of the same model (MCE-SU8-Pt001). In the inter-day comparison ( n = 3), the RSD values for the measured parameters varied from 3.3 to 15.6%. Lastly, for an inter-chip comparison and based on the recorded electropherograms, the RSD values calculated for migration time, peak area, and intensity varied between 3.4 and 11.8%. All the data obtained are summarized in Table 1 and the variations observed may be associated with slight changes on the electroosmotic flow mobility. Nevertheless, the comparison discussed herein demonstrates the potential of SU-8/glass devices for routine analysis employing a portable instrument, which may enable its use for in-field assays.

Summary of intra-day, inter-day, and inter-chip comparisons for the analysis of a 100 µmolL −1 nitrite standard solution ( n = 3).

In addition to the comparisons discussed above, the analytical performance was also investigated in terms of linear range and detectability levels. The developed method exhibited good linear behavior (R 2 = 0.999) in the concentration range between 20 and 80 μmol L −1 (Area = −7.123 + 0.692 × [NO 2 − ]; R 2 = 0.999). The limit of detection (LOD) was calculated based on the ratio between three times the standard deviation for the blank and the angular coefficient of the analytical curve and the value was 1.3 μmol L −1 . The LOD value achieved using the ME–AD system was compared to other reports published in the last five years involving miniaturized and conventional techniques, as summarized in Table 2 . In addition to the LOD, other features such as analysis time, tested sample, employed technique, and portability ware also included.

Comparison of the analytical performance of the proposed methodology for nitrite determination with other studies reported since 2017.

N/E: not specified. * Time of sample treatment not considered.

As shown in Table 2 , well-established analytical techniques have provided better LOD values. However, most of the examples exploring these techniques require bulky and costly instrumentation, which are restricted to a few research groups and are not compatible with portability, making their in-field use difficult [ 3 , 24 , 26 , 44 , 59 , 60 ]. Furthermore, when compared to paper-based devices [ 38 , 43 ], the portability of ME–AD has a noticeably lower appeal. On the other hand, ME–AD devices can be reused many times (estimated up to 1000 analyses) and, although not demonstrated in this study, they can allow selective analysis in the presence of other anionic species.

In comparison to the recent studies employing the ME–AD, our proposed methodology has provided one of the lowest LOD values that was achieved using unmodified electrodes, thus revealing attractive advantages over other methods which use modified electrodes and sample pretreatment steps [ 56 ]. Moreover, based on the examples using ME devices with portable instrumentation, the device explored in this study has offered the shortest analysis time. In view of the studies compared in Table 2 , the performance obtained through the proposed method as well as the previously discussed advantages make clear its potential for in-field analysis.

3.5. Environmental Water Analysis and Comparison with a Reference Methodology

Environmental samples of aquarium water (A) and fish breeding dam water (D) were analyzed and the recorded electropherograms are displayed in Figure 5 . Samples were diluted 50% ( v / v ) and then spiked with nitrite standard solution. Thus, samples labelled as A1, A2, and A3 correspond to the signal obtained for the analysis of aquarium water diluted at 50% ( v / v ) and fortified with 30, 40, and 60 μmol L −1 of NO 2 − , respectively. On the other hand, samples labelled as D1 refer to a sample of dam water diluted by 50% ( v / v ), while samples D2 and D3 are the same dam water diluted but fortified with 20 and 40 μmol L −1 of NO 2 − , respectively. All six samples (A1, A2, A3, D1, D2, and D3) were analyzed in triplicate by both the proposed and the reference methods. The aquarium samples (A) showed a lower concentration of nitrite due to the nitrogen cycle, presenting concentrations below the detection limit for both the proposed method and the reference method. Freitas and collaborators also found this nitrite profile in aquarium water samples in the first weeks (0 to 8) of operation; however, due to the formation and development of the ecosystem of a small aquarium, the conversion of species to nitrite is not very significant, and the aquarium where the samples were collected had recently been cleaned and reassembled [ 50 ].

Electropherograms showing the detection of nitrite in (A1–A3) aquarium water samples (A1–A3) and fish breeding dam water samples (D1–D3). Injection: −800 V for 10 s; Separation: −1000 V for 60 s. For the other conditions, see Figure 3 .

Reference analyses were carried out using a spectrophotometric procedure based on the Griess reaction. The obtained linear regression equation related to the analytical curve was: Absorbance = 0.002 + 0.037 × [NO 2 − ] (R 2 = 0.999). The original dam water sample (D) showed a NO 2 − concentration of 64.4 ± 0.6 μmol L −1 . Based on this, the final concentration of NO 2 − in samples D1, D2, and D3 were 32.2 ± 0.3; 52.2 ± 0.5, and 72.2 ± 0.6 μmol L −1 , respectively. The electropherograms obtained for the analysis of each of the samples are shown in Figure 5 . It was possible to observe the increase of the signal referring to the nitrite after the fortifications. There was also a difference in the profile of the signals obtained for nitrite according to the sample matrix. For aquarium samples (A1–A3) the nitrite peaks had a lower intensity and peak symmetry, resulting in wider signals. In the samples from dam water (D1–D3), the signals obtained were more intense and symmetrical.

This difference in the peak symmetry can be justified by the fact that the aquarium water was collected only one day after changing the filters. In addition, the aquarium fish population was entirely the same species, Kinguio (Carassius auratus) a small fish, while the dam water samples showed a population of larger fish. This difference between ecosystems affects the conductivity of the matrix that influences the peak shape, as already reported by Ollikainen and collaborators, who used the same instrumentation (HVStat) [ 69 ]. Using the nitrite peak area and the obtained linear regression from the quantification by the ME–AD, the nitrite concentration for the six samples was calculated. The results are presented in Table 3 .

Nitrite concentration values found in water samples using the reference and the proposed methodologies ( n = 3).

For all analyses, recoveries above 83.5% were achieved. Thus, the determination of nitrite by the ME–AD developed in this study is a promising methodology for analysis in environmental samples, with no detected matrix effect that changes the reliability of the determination.

4. Conclusions

In this study, a promising methodology using ME–AD for the quantification of nitrite in water samples was developed. The use of a portable instrument has offered a satisfactory analytical performance. The proposed methodology was optimized, and the proof-of-concept was successfully demonstrated through the determination of nitrite in environmental samples of water from aquariums and dams. The obtained results revealed a good agreement with the data recorded by the reference technique. Based on the achieved results, the methodology developed in a portable and compact instrument may emerge as powerful analytical tool for in-field analysis.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/mi13101736/s1 , optimization of the detection potential; effect of the medium conductivity on the electrophoretic performance.

Funding Statement

The authors gratefully acknowledge financial support from CNPq (grants 307554/2020-1 and 405620/2021-7), CAPES (grant 88887.192880/2018-00 and finance code 001), and INCTBio (grant 465389/2014-7). CAPES and CNPq are also recognized for the scholarships and researcher fellowship granted to the authors.

Author Contributions

Conceptualization, S.B.L., L.M.D., K.C.A.R. and W.K.T.C.; methodology, S.B.L. and L.M.D.; validation, S.B.L. and K.C.A.R.; formal analysis, S.B.L. and K.C.A.R.; investigation, S.B.L. and L.M.D.; data curation, L.M.D. and K.C.A.R.; writing—original draft preparation, S.B.L. and K.C.A.R.; writing—review and editing, K.C.A.R., L.M.D. and W.K.T.C.; resources; W.K.T.C.; supervision, L.M.D. and W.K.T.C.; project administration, W.K.T.C.; funding acquisition, W.K.T.C. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

Conflicts of interest.

The authors declare no conflict of interest.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Diffusion in liquids

In association with Nuffield Foundation

Demonstrate that diffusion takes place in liquids by allowing lead nitrate and potassium iodide to form lead iodide as they diffuse towards each other in this practical

In this experiment, students place colourless crystals of lead nitrate and potassium iodide at opposite sides of a Petri dish of deionised water. As these substances dissolve and diffuse towards each other, students can observe clouds of yellow lead iodide forming, demonstrating that diffusion has taken place.

This practical activity takes around 30 minutes.

• Eye protection
• White tile or piece of white paper
• Lead nitrate (TOXIC, DANGEROUS FOR THE ENVIRONMENT), 1 crystal
• Potassium iodide, 1 crystal
• Deionised water

Greener alternatives

To reduce the use of toxic chemicals in this experiment you can conduct the experiment in microscale, using drops of water on a laminated sheet, find full instructions and video here, and/or use a less toxic salt than lead nitrate, eg sodium carbonate and barium chloride. More information is available from CLEAPSS.

Health, safety and technical notes

• Read our standard health and safety guidance.
• Wear eye protection throughout.
• Lead nitrate, Pb(NO 3 ) 2 (s), (TOXIC, DANGEROUS FOR THE ENVIRONMENT) – see CLEAPSS Hazcard HC057a .
• Potassium iodide, KI(s) – see CLEAPSS Hazcard HC047b .
• Place a Petri dish on a white tile or piece of white paper. Fill it nearly to the top with deionised water.
• Using forceps, place a crystal of lead nitrate at one side of the petri dish and a crystal of potassium iodide at the other.
• Observe as the crystals begin to dissolve and a new compound is formed between them.

Source: Royal Society of Chemistry

As the crystals of potassium iodide and lead nitrate dissolve and diffuse, they will begin to form yellow lead iodide

Teaching notes

The lead nitrate and potassium iodide each dissolve and begin to diffuse through the water. When the lead ions and iodide ions meet they react to form solid yellow lead iodide which precipitates out of solution.

lead nitrate + potassium iodide → lead iodide + potassium nitrate

Pb(aq) + 2I – (aq) → PbI 2 (s)

The precipitate does not form exactly between the two crystals. This is because the lead ion is heavier and diffuses more slowly through the liquid than the iodide ion.

Another experiment – a teacher demonstration providing an example of a solid–solid reaction  – involves the same reaction but in the solid state.

Additional information

This is a resource from the  Practical Chemistry project , developed by the Nuffield Foundation and the Royal Society of Chemistry. This collection of over 200 practical activities demonstrates a wide range of chemical concepts and processes. Each activity contains comprehensive information for teachers and technicians, including full technical notes and step-by-step procedures. Practical Chemistry activities accompany  Practical Physics  and  Practical Biology .

The experiment is also part of the Royal Society of Chemistry’s Continuing Professional Development course:  Chemistry for non-specialists .

© Nuffield Foundation and the Royal Society of Chemistry

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• Physical chemistry
• Reactions and synthesis

Specification

• Precipitation is the reaction of two solutions to form an insoluble salt called a precipitate.
• Motion of particles in solids, liquids and gases.
• Diffusion (Graham's law not required).

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AgBioResearch

Msu scientists study how to increase water-, nutrient-use efficiency in greenhouses, nurseries.

Jack Falinski <[email protected]> - September 18, 2024

Tom Fernandez, a professor in the Department of Horticulture, has conducted extensive research on how to efficiently irrigate plants in greenhouses and nurseries. His newest project examines how to effectively treat water after it’s used in production.

EAST LANSING, Mich. — Tom Fernandez , a Michigan State University professor in the Department of Horticulture , has spent much of his 25-year career at MSU studying how to effectively manage water in greenhouses and nurseries to increase water-use efficiency and reduce nutrient runoff.

With funding support from Project GREEEN — Michigan’s plant agriculture initiative based at MSU and supported by the Michigan Plant Coalition, Michigan Department of Agriculture and Rural Development , MSU AgBioResearch and MSU Extension — Fernandez has developed management strategies to ensure agricultural inputs such as fertilizers and pesticides aren’t washed away from their intended targets, harming the surrounding environment and diminishing water quality.

According to the U.S. Environmental Protection Agency, about a half million tons of pesticides, 12 million tons of nitrogen and 4 million tons of phosphorous fertilizer are annually applied to crops. The runoff of these inputs contributes to some of the leading strains on water quality.

In greenhouses and nurseries, it’s easy to overwater many plants because the containers they’re in allow water to easily drain. Fernandez has found that by applying water based on a plant’s daily water use, irrigation can be reduced between 30%-80% depending on the species, and growers can conserve water and reduce the runoff of nutrients from the potting mix.  

In addition to minimizing the runoff of nutrients from fertilizers, such as nitrates and phosphates, Fernandez has also examined how to lessen the movement of pesticides from the soil and nontarget areas. Pesticides are sprayed over the top of plants, so as a result, they hit unintended spaces such as the gaps between plants or the groundcover in greenhouses and nurseries. When irrigation is applied overhead, the pesticides in these spaces can move with the water and impact its quality.

Like how the movement of nutrients from fertilizer in the soil were reduced, Fernandez said applying less water to plants can help mitigate pesticides from moving in the soil and from nontarget surfaces. He also said that micro-irrigating individual pots using spray stakes, which fan water over single containers, proved to significantly reduce the surface runoff of pesticides.

“Time really is on our side when we’re thinking about both nutrients and pesticides,” Fernandez said. “The longer we keep them from getting into water systems, the more can happen to them biologically so they don’t cause a problem.”

With these strategies, Fernandez said there became a better understanding for how to irrigate container plants without promoting runoff. Since then, he’s taken on a new project: studying how to treat the water used in production by addressing the amount of nutrients and pesticides in it after application.

Beginning in 2018, Fernandez and Gemma Reguera , associate dean of faculty affairs and development in MSU’s College of Natural Science and professor in the Department of Microbiology, Genetics and Immunology , started to examine how nutrients from fertilizers interact with bioreactors, as well as to what extent bioreactors separate them from water used in greenhouses — an undertaking originally studied by Fernandez’s former doctoral student Damon Abdi, now an assistant professor of horticulture at Louisiana State University.

What do these bioreactors look like?

“They have a fancy name, but they’re really just big tubs of woodchips,” Fernandez said.

Fernandez said they originally developed a two-stage bioreactor system composed of woodchips, which convert nitrates into nitrogen gas, and heat-expanded clay, which gives the phosphorus from phosphates a large surface area to bind to when water runs through it.

Research showed that when water ran through the system, over 95% of nitrates could be taken out, and 80%-87% of phosphates could be broken down and removed. Fernandez and his team discovered that the activity occurred mostly in the woodchips, so the second stage of the bioreactor that implemented heat-expanded clay has since been discontinued.

This function of the bioreactor is critical for water that may not be reused in operations because it reduces the chance nutrients discharge into and contaminate the environment. However, many modern greenhouses and nurseries operate using closed-loop water systems where water is kept within the facility and oftentimes recycled in production. Fernandez said for water that’s recycled, he’s received inquiries about the potential to recycle nutrients in the bioreactor while reducing the presence of pesticides.

“Our partners wanted to remove the pesticides but keep the nutrients in the water because they’re paying for those — that’s fertilizer,” Fernandez said.

To keep the nutrients in water, water must travel through the bioreactor at a quicker pace. When it does so, there’s less time for the bioreactor to become anaerobic — a state without oxygen — preventing nutrients such as nitrates to be off-gassed.

After modifying the bioreactor to allow water to move through it at around a 4-hour pace instead of a 72-hour pace, which was roughly the amount of time it took for nutrients to be removed from water, Fernandez said his team has been able to recycle 90%-100% of the nutrients in water to be used again for production.

Fernandez and Reguera also observed that when pesticides ran through the bioreactor, they didn’t affect the functionality of the microorganisms working within the system. In fact, they found that — depending on the mobility of each pesticide in water — the bioreactor could reduce the total amount of pesticides in water anywhere between 30%-75%.

“In our lab experiments, we found if we went to a low retention time — the length in which water is kept within the bioreactor — we could keep the nutrients in the water stream and remove many of the pesticides,” Fernandez said.

Amy Upton, executive director of the Michigan Nursery and Landscape Association, said the data from Fernandez and his team’s research helps the greenhouse and nursery industries market their clean-water production, and the hands-on demonstrations offered by the team aid growers in evaluating and adopting these technologies.

“Water quality and security are critically important to the nursery and greenhouse industries,” Upton said. “Dr. Fernandez and his team’s research not only address quality and security, but also incorporate important aspects such as improved soilless substrates that optimize water and nutrient retention and proven at-scale treatment technologies that reduce pesticides and pathogens in water sources.

“Michigan’s nursery and greenhouse industries are appreciative of the support from Dr. Fernandez and his team, as well as the continued funding support from Project GREEEN.”

Jim Kells , coordinator of Project GREEEN, said the novel ability to manage nutrients in the water will help greenhouses and nurseries increase sustainability and efficiency.

“Through research supported by Project GREEEN, Dr. Fernandez has developed innovative systems to minimize water use and reduce pesticides in water while recycling valuable nutrients,” Kells said. “This research has the potential to reduce the environmental impact of greenhouse and nursery systems while increasing grower profitability.”

This will be the third year in which Fernandez and his team, including doctoral students Henry Gonzalez and Marcela Tabares , monitor how bioreactors perform within a large-scale greenhouse operation. Using 300- and 600-gallon water tanks as the bioreactor containers, Fernandez said they’re currently studying how pesticides degrade differently in anaerobic (without oxygen) and aerobic (with oxygen) conditions, hoping that the information gathered can further advance the degree to which pesticides can be removed from water.

“It’s really the first time I know of that anyone has used this type of system for bioreactors,” Fernandez said.

Michigan State University AgBioResearch scientists discover dynamic solutions for food systems and the environment. More than 300 MSU faculty conduct leading-edge research on a variety of topics, from health and climate to agriculture and natural resources. Originally formed in 1888 as the Michigan Agricultural Experiment Station, MSU AgBioResearch oversees numerous on-campus research facilities, as well as 15 outlying centers throughout Michigan. To learn more, visit agbioresearch.msu.edu .

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