A vortex-assisted liquid–liquid microextraction (VALLME) method using isooctanol as extractant followed by spectrophotometry was developed for the extraction and determination of trace nitrite in water samples. The method is based on selective ion-pairing complex (I3 MG+) formation of triiodideanion I3 with cationic dye malachite green (MG) at pH 3.0, and its subsequent extraction in an organic solvent. The extracted organic solvent-rich phase is diluted with methanol, and its absorbance is measured against an analyte blank at 630 nm. The variables affecting VALLME efficiency were investigated, and a set of optimized conditions was obtained. Under the optimum conditions, the linear range of nitrite was from 1.0 to 100 ng mL−1. The relative standard deviations (n = 10) were 2.1–3.9% and the limit of detection was 0.5 ng mL−1 and was successfully applied to the determination of nitrite in environmental water.

INTRODUCTION

Nitrite has many applications in different industries such as food preservation, fertilizers, detergent, wood pulp, dye and synthetic fiber and hence could enter the environment causing serious pollution problems. Nitrite may also be produced in the distribution system through the activities of microorganisms on ammonia (Nagaraja et al. 2010). The increasing concentration of nitrite in groundwater, rivers, and lakes has caused serious hazards to public health and the environment. The continual ingestion of these ions can have serious implications for animal and human health (Shailaja et al. 2006; Deane & Woo 2007; Rodrigues et al. 2007; Kroupova et al. 2008). The fatal dose of nitrite ingestion as reported by the World Health Organization is between 8.7 μM and 28.3 μM (Silva & Mazo 1998). The maximum limit value of nitrite present in drinking water is 3 mg/L as recommended by the World Health Organization (WHO 2004). Determination of nitrite is important due to its harmful effects on human health. Thus, sensitive, selective, and precise methods are required for the determination of nitrite.

A number of methods have been described in the literature for nitrite determination that are based on such analytical methods as spectrophotometry and spectrofluorimetry (Lopez Pasquali et al. 2010; Mašić et al. 2015), ion-chromatography (Tirumalesh 2008), flow-injection analysis (Gamboa et al. 2009), and capillary electrophoresis (Bord et al. 2005; Somer et al. 2016), and electrochemical (Kalimuthu & John 2009; Ko et al. 2009; Zazoua et al. 2009; Zheng et al. 2009) techniques. Spectrophotometry is the most widely used method for nitrite determination due to its simplicity and excellent limit of detection (LOD). But there are some difficulties for the direct determination of nitrite by spectrophotometry since its accuracy and selectivity as well as sensitivity is not sufficient at a trace level and it is susceptible to the influence of the matrix. Therefore, in order to circumvent the low sensitivity of spectrophotometric methods, several sample preparation techniques such as solid-phase extraction (Miró et al. 2001; Chen et al. 2008; Zhang et al. 2009) or cloud point extraction (Afkhami et al. 2005a, 2005b) have been developed for separation and preconcentration of nitrite in aqueous matrices prior to its spectrophotometric detection. Vortex-assisted liquid–liquid microextraction (VALLME) is based on dispersion of low-density extraction solvent into water and is obtained through using vortex mixing, a mild emulsification procedure (Zaruba et al. 2016). The fine droplets can rapidly extract target analytes from solution samples due to the shorter diffusion distance and larger surface area. After centrifugation, the extraction solvent containing target analytes in the upper surface of the aqueous sample was used for the next step of instrumental analysis after the aqueous phase was removed. VALLME methods have been developed for extraction solvents at all kinds of preconcentration (Zhang & Lee 2012; Xue et al. 2015; Yang et al. 2015).

In this study, higher sensitivity and selectivity of nitrite determinations combining VALLME preconcentration with spectrophotometry at 630 nm have been achieved. In the application of the method, malachite green (MG+) was used as ion-pair reagent and isooctanol as extractant in the presence of excess iodide at pH 3.0. Several factors influencing the extraction efficiency of the VALLME such as extraction solvent, volumes of the extraction solvent, vortex time and salt addition were investigated. The feasibility of the proposed method is illustrated for the determination of nitrite in water samples.

MATERIALS AND METHODS

Instrument

A UV–vis spectrophotometer model Tu-1810 (Beijing, China) equipped with 1 cm quartz cell was employed for absorbance measurements. A vortex mixer (Shanghai Hanuo Instrument Co. Ltd, XH-B, Shanghai, China) was used to vortex-mix solution samples. A centrifuge (Shanghai surgical instrument factory, 80-2, Shanghai, China) was used to accomplish the phase separation process.

Reagents and solutions

All solutions were prepared with analytical grade reagents and ultrapure water. Nitrite ions were provided by sodium nitrite that was dried at 110 °C for 4 hours. The stock nitrite solution had a concentration of 1,000 μg mL−1. It was prepared by dissolving 0.75 g of NaNO2 in water in a 500 mL standard flask. The standard working solutions used for establishment of the calibration curve were prepared before use by stepwise dilution of the stock solution with water. A solution of 1.0 × 10−4 mol L−1 of MG+ was prepared by dissolving 0.0183 g of dye (Sigma, St Louis, MO, USA) in water in a 500 mL standard flask. Sulfuric acid of 0.1 mol L−1 was prepared by diluting 1.4 mL of H2SO4 (Merck) to 500 mL in a volumetric flask. KI of 0.02 mol L−1 was prepared by dissolving 0.3320 g of potassium iodide (Merck) in water and diluting to 100 mL in a volumetric flask, and n-butanol, n-pentanol, n-hexanol, n-octanol and isooctanol were purchased from Alading (Guangzhou, China).

Sample collection

Different natural water samples were analyzed in this work for nitrite determination, i.e. tap water, lake water, and river water. Tap water was collected at the Faculty of Yunnan Jianniu Bio Technology Co., Ltd, of Kunming. River water and lake water were collected in Green Lake and Dianchi (Kunming). Water samples were filtered through What man No. 41 filter paper, stored at 5 °C to retard bacterial growth, and analyzed within 24 h by the recommended method (Pourreza et al. 2012).

VALLME procedure

Amounts of 5.5 mL of 0.02 mol L−1 of KI and 3 mL of 0.1 mol L−1 sulfuric acid were added to a 50 mL centrifuge tube. Then an aliquot of the nitrite (so that its concentration would be in the range of 1–100 μg L−1) and 0.8 mL of 1.0 × 10−4 mol L−1 of MG+ were added and thoroughly mixed. Then 1 mL of isooctanol was added and the pH of the mixture was adjusted to 3.0 and was made up to the mark with ultra-pure water. Centrifugal vials were vortex-mixed for 1 min, and centrifuged for 5 min at 4,000 rpm to complete phase separation. As Figure 1 shows, the samples A, B and C with different colors are those with yellow color, and the with green color, MG+ solution only, respectively. After the complex reaction between the MG+ solution and triiodide, the ion pair is easier to extract into the organic extraction solvent. The water phase was carefully removed, using a syringe with a long needle, methanol was added into the remaining organic phase up to 2 mL, and the absorbance was then measured at 630 nm.
Figure 1

Enriched organic extraction phase with VALLME. (A) I + 2NO2, (B) I + 2NO2 + MG+, (C) I + MG+.

Figure 1

Enriched organic extraction phase with VALLME. (A) I + 2NO2, (B) I + 2NO2 + MG+, (C) I + MG+.

RESULTS AND DISCUSSION

The enhancement factor (EF) was calculated from the following equation: 
formula

where EF, k1 and k2 are standard for enrichment factor, the ratio of slope without preconcentration and the ratio of slope after preconcentration, respectively.

Reaction mechanism

The method is based on the well-known oxidation–reduction reaction of nitrite by iodide ions in an acidic medium and selective ion-pairing complex formation of triiodide, I3 and cationic dye malachite green (MG+) for indirect spectrophotometric determination of trace nitrite in environmental water after separation and preconcentration with VALLME. The reaction between nitrite and iodide in an acidic medium gives rise to the formation of iodine (I2) and I3 (Equations (1) and (2)), which forms a colored ion pair with MG+. This colored ion pair can be extracted into an organic solvent (Equation (3)). The proposed scheme of the reaction was confirmed by the measurement of the absorption spectrum of the studied system in the conditions of the proposed method without addition of the dye (Figure 1). 
formula
1
 
formula
2
 
formula
3

Effect of pH

The pH plays a unique role in ion-pairing complex formation and subsequent extraction procedures in VALLME. Separation and preconcentration of nitrite with VALLME involve the previous formation of a stable complex, which needs to present sufficient hydrophobicity to be extracted into the small volume of the surfactant-rich phase. The pH is a critical factor affecting both the redox reaction between nitrite and excess iodide, and produced triiodide ion, I3 and ion pairing ligand, MG+ and the extractability of the ion-pairing complex into the organic-rich phase. Thus, the effect in the range pH 2.0–5.5 was studied for the extraction and determination of nitrite in Figure 2. From the results, maximum absorbance was obtained with a phthalate buffer system at pH 3.0. The cationic dye malachite green (MG+) is protonated at lower pH and the ion-pairing ligand is easily formed. Therefore, pH 3.0 was used as the optimal value for further studies.
Figure 2

Effect of pH.

Figure 2

Effect of pH.

Effect of KI concentration

The concentration of KI that was used to react with nitrite and I3 must be enough. The results shown in Figure 3 indicate that the highest absorbance for triiodide extracted into the organic phase was obtained when KI concentration was 5 × 10−3 mol L−1 in the presence of 50 μg L−1 NO2. Further experiments were performed by adding 5.5 mL of 0.02 mol L−1 KI to the 50 mL solution in order to achieve this concentration.
Figure 3

Effect of KI concentration.

Figure 3

Effect of KI concentration.

Effect of MG concentration

Different concentrations of MG+ as ion-pairing reagent in the range of (0.4–6) × 10−6 mol L−1 were studied for the influence of its concentration on the analytical response for determination of nitrite a at fixed concentration of 50 μg L−1 NO2 (Figure 4). If the concentrations of MG+ are too high, the absorbance of blank solution is strong, and if the concentrations of MG+ are too low, the ion-pairing complex forms incompletely. The results show that the absorbance of the extract increases with increasing MG concentration up to 2.0 × 10−6 mol L−1, and then the analytical signal changes only slightly. However, due to the lower value of the blank test, 1.0 × 10−6 mol L−1 was chosen for further studies.
Figure 4

Effect of malachite green concentration.

Figure 4

Effect of malachite green concentration.

Effect of the extraction solvent type

The selection of an appropriate extraction solvent is an important step in the development of novel extraction procedures. The extraction solvent should be immiscible with water, should have high extraction efficiency for the target to be analyzed and should be distinguished with a high signal-to-noise ratio. In the present work, n-butanol, n-pentanol, n-hexanol, n-octanol and isooctanol were all tested as extractants. The effect of the extraction solvents is shown in Figure 5. No emulsification was observed in the system when butanol was used as the extraction solvent. The recovery in Figure 6 indicates that isooctanol was the best extraction solvent for the extraction of nitrite. Based on these results, isooctanol was adopted as the extraction solvent in the following experiments.
Figure 6

Effect of organic extraction solvent types.

Figure 6

Effect of organic extraction solvent types.

Figure 5

Effect of isooctanol volume.

Figure 5

Effect of isooctanol volume.

Effect of extraction solvent volume

The preconcentration factor depends on the volume of isooctanol, so the effect of isooctanol volume was studied. Different volumes of isooctanol in the range of 0.5 to 1.5 mL were examined in 50 mL water samples. As can be seen in Figure 5, recovery gradually increased with an increase in isooctanol volume and showed the highest response at 1.0 mL. Below that volume, analyses cannot be extracted absolutely and it is very difficult to make phases separate. So, 1.0 mL of isooctanol was used in subsequent experiments.

Effect of vortex time

Application of vortex agitation shortens the extraction time as a result of the formation of fine droplets of extraction solvent, which leads to an increase in the interface boundary. The dispersion of the extraction solvent into the aqueous phase depends on the rotational speed of the vortex agitator and the vortex time. The speed of the vortex agitator was set at its maximum (nearly 3,000 rpm). The effect of vortex time was examined in the range from 5 to 45 s. No significant effect was found when the vortex time ranged from 10 to 45 s. Based on the above considerations, 20 s of vortex extraction time was chosen to guarantee complete extraction of the target substance.

Study of interferences

In order to evaluate the selectivity of the proposed method, the possibility of interference by the various ions accompanying nitrite on the determination of 25 μg L−1 of nitrite was tested using the recommended procedure under the optimum conditions. Variation in the absorbance value of ±5% from that obtained in the absence of any interfering ions was taken as a sign of interference. The obtained results are summarized in Table 1. It was found that the commonly coexisting ions do not interfere in the determination of nitrite in water samples.

Table 1

Effect of interfering ions on the determination of 25 μg L−1 of nitrite

Interfering ionTolerance limit (μg mL−1)
K+, Na+, NH4+, Ca2+, Mg2+, HCO3, SO42−, PO43− 1,000 
Cl, F, Br, NO3, Zn2+, Cr3+ 500–800 
Fe2+, SCN, Al3+, acetate, citrate 100–300 
Ni2+, Co2+, CO32−, CN 30 
Cu2+, Fe3+ 
Interfering ionTolerance limit (μg mL−1)
K+, Na+, NH4+, Ca2+, Mg2+, HCO3, SO42−, PO43− 1,000 
Cl, F, Br, NO3, Zn2+, Cr3+ 500–800 
Fe2+, SCN, Al3+, acetate, citrate 100–300 
Ni2+, Co2+, CO32−, CN 30 
Cu2+, Fe3+ 

Analytical performance

The analytical performance of the VALLME system was evaluated in terms of LOD, linear range and reproducibility. The calibration was performed under optimum conditions in order to establish the working range. The calibration graph was linear in the range of 1–100 μg L−1 with an equation of the line A = 0.290C + 0.021 where A is the absorbance and C is the nitrite concentration in μg L−1. The relative standard deviations were 2.1–3.9% and the LOD based on three times the standard deviation of the blank (3Sb) was 0.5 ng mL−1 (n = 10).

Application

In order to validate the suitability of the proposed method it was applied to the determination of nitrite in water samples prepared as described above. Parallel determination was also carried out to validate the recommended method with the Griess assay, where nitrite is converted into a highly absorbing azo compound through a diazotization/coupling reaction and the absorbance at 540 nm is measured (Zhang et al. 2009). The results are presented in Table 2, and agreed with the Griess procedures in the t-test at the 95% confidence level.

Table 2

Analytical results for determination of nitrite in water environmental samples

SampleSpiked (μg L−1)Found (μg L−1)Recovery (%)Griess method (μg L−1)
Lake water 37.8 ± 1.5 – 38.4 ± 1.8 
10 43.2 ± 2.1 90.4  
20 53.2 ± 1.8 92.1  
50 83.6 ± 2.3 95.2  
River water 51.6 ± 3.9 – 52.9 ± 2.4 
10 54.1 ± 1.3 87.8  
20 66.3 ± 2.2 92.6  
50 102.6 ± 1.9 101.0  
Tap water 18.2 ± 2.5 – 17.5 ± 1.7 
10 25.4 ± 3.0 90.2  
20 35.1 ± 2.4 91.9  
50 69.8 ± 1.8 102.4  
SampleSpiked (μg L−1)Found (μg L−1)Recovery (%)Griess method (μg L−1)
Lake water 37.8 ± 1.5 – 38.4 ± 1.8 
10 43.2 ± 2.1 90.4  
20 53.2 ± 1.8 92.1  
50 83.6 ± 2.3 95.2  
River water 51.6 ± 3.9 – 52.9 ± 2.4 
10 54.1 ± 1.3 87.8  
20 66.3 ± 2.2 92.6  
50 102.6 ± 1.9 101.0  
Tap water 18.2 ± 2.5 – 17.5 ± 1.7 
10 25.4 ± 3.0 90.2  
20 35.1 ± 2.4 91.9  
50 69.8 ± 1.8 102.4  

CONCLUSION

The results of this study demonstrated the usefulness of the proposed VALLME/spectrophotometric method for quantitative extraction of trace nitrite in water samples. The proposed VALLME method gives very low LOD, and good precision and extraction of indirect nitrite as an ion-pairing complex with isooctanol, from its initial matrix after pretreatment with two reduction procedures. The method can be considered as an alternative tool to expensive, time-consuming, excess-solvent-consuming and experienced-user-requiring analytical techniques.

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