# Determination of Nitrogen Oxides with Rhodamine B by Fluorescence Quenching Method

Authors: Yan Pugen; Qiu Haiou; Lu Cuili; Tang Zhiyong
Journal: International Journal of Spectroscopy
 (2011)

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## Abstract

NO2 can be transformed into nitrite by the absorption of Rhodamine B solution, which will make fluorescence quenching of Rhodamine B in acidic medium. According to this mechanism, a new method for detecting nitrogen oxides in the air is developed by a fluorescence spectrophotometry. The influence of environmental media and interfering substances in the fluorescence intensity of system was studied. Under the optimal experimental conditions, the decrease of fluorescence intensity varies linearly with the concentration of NO2− over the range of 0.009~0.500 μg/ml with a correlation coefficient of 0.9992. The method is simple and has a lower limit of detection than the common methods. It can be a referee for the environmental evaluation.


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## Body

## 1. Introduction

Nitrogen oxides are one of the main pollutants as well as the evaluation criteria of the air quality. Nitrogen oxides in the atmosphere have harmful effects on humans mainly through the respiratory system, which may cause acute and chronic poison. Therefore, the study of analysis and detection methods for nitrogen oxides will be significant. There are many methods for the determination of nitrogen oxides such as ion chromatography [1], chemiluminescence [2, 3], fluorescence [4], and colorimetric microdetermination [5]. However, the method that nitrogen oxides in the air were determined with Rhodamine B by fluorescence quenching has not been reported.Nitrogen oxides can exist in various forms including the main forms of NO and NO2 in the atmosphere. In this research, the contents of NO and NO2 in the air sample were determined indirectly by measuring the content of nitrite and solution absorption-fluorescence spectrometry. There are many methods for the determination of nitrite such as spectrophotometry [6–8], fluorescence [9–14], chemiluminescence [15–17], polarography [18] and chromatography [19–22]. Among these methods, the fluorescence method has attracted much attention and been applied widely for the detection of nitrite for the high sensitivity, selectivity, low limit of detection and simple operation. According to the literature, NO2−-organic dyes and NO2−-KBrO3−-organic dyes are the main systems for the determination of nitrite by the fluorescence spectrometry.The common determination of nitrogen oxides is Saltzman method [23, 24]. However, the contents of NO and NO2 in the actual air sample are determined with Rhodamine B by fluorescence quenching with quite satisfactory results. The method in the text is simple and has a lower limit of detection, which provides a referee for the assessment of environment.


## 2. Experimental Section

### 2.1. Instruments and Reagents

An LS-55 fluorescence spectrophotometry instrument (PerkinElmer Limited) equipped with a 1 cm*1 cm quartz cell, an HHS electrothermal thermostatic water-bath pot (Shanghai Boxun Co.), air sampler (Wuhan Tianhong TH-110B type), porous glass plate absorbs bottles packed with 10 mL absorbing liquid for short time sampling.Nitrite working solution (5.0μg/mL) was prepared by diluting the stock solution (1.0 g/L) before use. Rhodamine B working solution (5.0 × 10−6 mol/L), sulfuric acid solution, sodium acetate solution (2.0 mol/L). Absorbing liquid was prepared as follows. 4 mL of sulfuric acid solution (0.1 mol/L) and 12.00 mL of Rhodamine B solution was added into a 100 mL volumetric flask and diluted with the distilled water. Oxidation liquid (0.16 mol/L) was prepared by dissolving 2.5286 g of potassium permanganate in 50 mL of water with slight fever, in which 50 mL of sulfuric acid solution (1 mol/L) 50mL was added. The prepared solution was stored in brown reagent bottle. All chemicals were of analytical grade and used without further purification. All solutions were prepared with ultrapure water.


### 2.2. Experimental Methods

#### 2.2.1. Air Sample Collection and Processing

Nitrogen oxides in the air were determined actually by measuring the contents of NO2 and NO. NO2 can be absorbed directly by the absorption solution while NO cannot be adsorbed. Therefore, to determine the amount of nitrogen oxides, NO must be oxidized firstly to NO2. In our research, NO was oxidized by 10.0 mL of acidic potassium permanganate solution and the volume of absorption solution was 10.0 mL. The scheme of air sampling device is shown in Figure 1.Figure 1
The scheme of air sampling device. (1) air inlet, (2) absorb bottle, (3) bottle of acidic potassium permanganate, (4) absorb bottle, (5) buffer bottle, (6) flowmeter, (7) Air sampler.The expression of concentration is given as follows:(1)CNOX=CNO2+CNO,(2)CNO2=(ΔF1-a)⋅Vb⋅f⋅V0,(3)CNO=(ΔF2-a)⋅Vb⋅f⋅k⋅V0,
where CNOX: concentration of nitrogen oxides in the air, mg/m3; CNO2: concentration of nitrogen dioxide in the air, mg/m3; CNO:  concentration of nitric oxide in the air, mg/m3; ΔF1, ΔF2: difference between the value of fluorescent signal of the first and the second branch absorption in bottle sample and the value of blank signal, respectively; b, a: slope (ΔF·mL/μg) and intercept of linear regression equation of standard curve, respectively; V: volume of the absorption solution, mL; V0: volume of the sample under standard state (101.325 kPa, 273 K), L; K: oxidation coefficient of NO→NO2, 0.68; and F: coefficient of Saltzman experiment, 0.88 (it is 0.77 when the content of NO2 in the air is higher than 0.720 mg/m3).


#### 2.2.2. Determination Method

1.00 mL of H2SO4 solution (0.1 mol/L), 3.00 mL of Rhodamine B solution (5.0×10−6 mol/L), and a certain amount of nitrite standard solution were added in sequence into a 25 mL colorimetric tube, which was diluted with water to total volume of 25 mL and shaken before it was placed in a water bath (60°C) for 45 minutes. Then, the colorimetric tube was taken out for cooling. 1.00 mL of sodium acetate solution (2.0 mol/L) was added further into the above solution and shaken well. The excitation and emission wavelengths were 555 nm and 576 nm, respectively. The fluorescence intensity of both sample solution (F) and corresponding blank solution (F0) was detected. The value of ΔF(F0– F) was recorded.


## 2.1. Instruments and Reagents

An LS-55 fluorescence spectrophotometry instrument (PerkinElmer Limited) equipped with a 1 cm*1 cm quartz cell, an HHS electrothermal thermostatic water-bath pot (Shanghai Boxun Co.), air sampler (Wuhan Tianhong TH-110B type), porous glass plate absorbs bottles packed with 10 mL absorbing liquid for short time sampling.Nitrite working solution (5.0μg/mL) was prepared by diluting the stock solution (1.0 g/L) before use. Rhodamine B working solution (5.0 × 10−6 mol/L), sulfuric acid solution, sodium acetate solution (2.0 mol/L). Absorbing liquid was prepared as follows. 4 mL of sulfuric acid solution (0.1 mol/L) and 12.00 mL of Rhodamine B solution was added into a 100 mL volumetric flask and diluted with the distilled water. Oxidation liquid (0.16 mol/L) was prepared by dissolving 2.5286 g of potassium permanganate in 50 mL of water with slight fever, in which 50 mL of sulfuric acid solution (1 mol/L) 50mL was added. The prepared solution was stored in brown reagent bottle. All chemicals were of analytical grade and used without further purification. All solutions were prepared with ultrapure water.


## 2.2. Experimental Methods

### 2.2.1. Air Sample Collection and Processing

Nitrogen oxides in the air were determined actually by measuring the contents of NO2 and NO. NO2 can be absorbed directly by the absorption solution while NO cannot be adsorbed. Therefore, to determine the amount of nitrogen oxides, NO must be oxidized firstly to NO2. In our research, NO was oxidized by 10.0 mL of acidic potassium permanganate solution and the volume of absorption solution was 10.0 mL. The scheme of air sampling device is shown in Figure 1.Figure 1
The scheme of air sampling device. (1) air inlet, (2) absorb bottle, (3) bottle of acidic potassium permanganate, (4) absorb bottle, (5) buffer bottle, (6) flowmeter, (7) Air sampler.The expression of concentration is given as follows:(1)CNOX=CNO2+CNO,(2)CNO2=(ΔF1-a)⋅Vb⋅f⋅V0,(3)CNO=(ΔF2-a)⋅Vb⋅f⋅k⋅V0,
where CNOX: concentration of nitrogen oxides in the air, mg/m3; CNO2: concentration of nitrogen dioxide in the air, mg/m3; CNO:  concentration of nitric oxide in the air, mg/m3; ΔF1, ΔF2: difference between the value of fluorescent signal of the first and the second branch absorption in bottle sample and the value of blank signal, respectively; b, a: slope (ΔF·mL/μg) and intercept of linear regression equation of standard curve, respectively; V: volume of the absorption solution, mL; V0: volume of the sample under standard state (101.325 kPa, 273 K), L; K: oxidation coefficient of NO→NO2, 0.68; and F: coefficient of Saltzman experiment, 0.88 (it is 0.77 when the content of NO2 in the air is higher than 0.720 mg/m3).


### 2.2.2. Determination Method

1.00 mL of H2SO4 solution (0.1 mol/L), 3.00 mL of Rhodamine B solution (5.0×10−6 mol/L), and a certain amount of nitrite standard solution were added in sequence into a 25 mL colorimetric tube, which was diluted with water to total volume of 25 mL and shaken before it was placed in a water bath (60°C) for 45 minutes. Then, the colorimetric tube was taken out for cooling. 1.00 mL of sodium acetate solution (2.0 mol/L) was added further into the above solution and shaken well. The excitation and emission wavelengths were 555 nm and 576 nm, respectively. The fluorescence intensity of both sample solution (F) and corresponding blank solution (F0) was detected. The value of ΔF(F0– F) was recorded.


## 2.2.1. Air Sample Collection and Processing

Nitrogen oxides in the air were determined actually by measuring the contents of NO2 and NO. NO2 can be absorbed directly by the absorption solution while NO cannot be adsorbed. Therefore, to determine the amount of nitrogen oxides, NO must be oxidized firstly to NO2. In our research, NO was oxidized by 10.0 mL of acidic potassium permanganate solution and the volume of absorption solution was 10.0 mL. The scheme of air sampling device is shown in Figure 1.Figure 1
The scheme of air sampling device. (1) air inlet, (2) absorb bottle, (3) bottle of acidic potassium permanganate, (4) absorb bottle, (5) buffer bottle, (6) flowmeter, (7) Air sampler.The expression of concentration is given as follows:(1)CNOX=CNO2+CNO,(2)CNO2=(ΔF1-a)⋅Vb⋅f⋅V0,(3)CNO=(ΔF2-a)⋅Vb⋅f⋅k⋅V0,
where CNOX: concentration of nitrogen oxides in the air, mg/m3; CNO2: concentration of nitrogen dioxide in the air, mg/m3; CNO:  concentration of nitric oxide in the air, mg/m3; ΔF1, ΔF2: difference between the value of fluorescent signal of the first and the second branch absorption in bottle sample and the value of blank signal, respectively; b, a: slope (ΔF·mL/μg) and intercept of linear regression equation of standard curve, respectively; V: volume of the absorption solution, mL; V0: volume of the sample under standard state (101.325 kPa, 273 K), L; K: oxidation coefficient of NO→NO2, 0.68; and F: coefficient of Saltzman experiment, 0.88 (it is 0.77 when the content of NO2 in the air is higher than 0.720 mg/m3).


## 2.2.2. Determination Method

1.00 mL of H2SO4 solution (0.1 mol/L), 3.00 mL of Rhodamine B solution (5.0×10−6 mol/L), and a certain amount of nitrite standard solution were added in sequence into a 25 mL colorimetric tube, which was diluted with water to total volume of 25 mL and shaken before it was placed in a water bath (60°C) for 45 minutes. Then, the colorimetric tube was taken out for cooling. 1.00 mL of sodium acetate solution (2.0 mol/L) was added further into the above solution and shaken well. The excitation and emission wavelengths were 555 nm and 576 nm, respectively. The fluorescence intensity of both sample solution (F) and corresponding blank solution (F0) was detected. The value of ΔF(F0– F) was recorded.


## 3. Results and Discussion

### 3.1. Excitation and Emission Spectrum

Rhodamine B is a kind of pinkish synthetic dye, which has intense fluorescence in solution and has been used commonly in fluorescent dye of cell, colored glass, and characteristic fireworks, and so forth. Figure2 shows the fluorescence emission spectra of different Rhodamine B systems. It can be found that the fluorescent intensity of Rhodamine B decreases with the addition of NO2− (curve 4) in the system of Rhodamine B + H2SO4 (curve 2). Curve 1 and 3 shows that the system can be activated when an appropriate amount of sodium acetate is added. In addition, there is a linear relationship between the concentration of NO2− and ΔF under certain conditions. It can be concluded that a method for the determination of NO2 and NO is developed by fluorescence spectrophotometry. The maximum excitation and emission wavelengths for the system are 555 nm and 576 nm, respectively.Figure 2
Fluorescence emission spectra. (1) Rhodamine B + H2SO4 + NaAc, (2) Rhodamine B + H2SO4, (3) Rhodamine B + H2SO4 + NO2− + NaAc, (4) Rhodamine B + H2SO4 + NO2-. Rhodamine B: 6.0×10-7 mol/L, H2SO4: 4.0×10-3 mol/L, NO2−: 0.4 μg/mL, NaAc: 0.08 mol/L, temperature: 60°C, time: 45 minutes.


### 3.2. Influence of Reaction Media

The influence of reaction media in ΔF was analyzed. Different acidic media was investigated, such as sulfuric acid, hydrochloric acid, acetic acid, phosphoric acid, and nitric acid. The highest sensitivity was detected in the sulfuric acid media. Therefore, sulfuric acid was used in this experiment. In addition, the effect of dosage of sulfuric acid (0.1 mol/L) was also studied in the range of 0.2~2.00 mL. It was found that the maximum value of ΔF could be obtained as the dosage was 1.00 mL. Then, 1.00 mL of sulfuric acid solution was used in the experiment.


### 3.3. Influence of Dosage of Rhodamine B

The influence of Rhodamine B solution (5.0 × 10−6 mol/L) in ΔF was also studied. Results indicated that fluorescent signal of the system increased gradually with the increased Rhodamine B dosage while the value of ΔF increased firstly and then decreased. The maximum value of ΔF was obtained when the dosage of Rhodamine B was 3.00 mL. Therefore, 3.00 mL of Rhodamine B was chosen as optimum dosage in the further studies.


### 3.4. Influence of Dosage of Activator

Different materials were used to study the influence of activator, such as sodium citric acid, citric acid, sodium acetate, oxalic acid, sodium phosphoric acid and sodium dodecyl sulfonic acid. Both the improved fluorescence intensity and value of ΔF were detected by using the sodium acetate. Furthermore, the largest value of ΔF could be obtained for the 1.00 mL of sodium citrate solution (2.0 mol/L). Hence, 1.00 mL of sodium citrate was chosen in the experiment.


### 3.5. Influence of Reaction Temperature

The influence of temperature on the value of ΔF was tested in the range of 25~80°C. It was found that the value of ΔF increased gradually with the increased temperature. Moreover, the value of ΔF was relatively constant in the range of 60°C-80°C. Then, the reaction temperature was performed at 60°C in the experiment.


### 3.6. Influence of Reaction Time

The variation of the value of ΔF was studied with the operation time. It was found that the value of ΔF increased fast with the prolonged reaction time in 40 minutes. The stable values of ΔF was observed with the further increased reaction time. The reaction time was chosen at 45 minutes in this experiment.


### 3.7. Stability Test

The fluorescence intensity was recorded after the reaction was terminated for 4 hours. The decreased fluorescence intensity was observed in 2 hours. However, the value of ΔF was unchanged, which indicated that this system was comparatively stable. Therefore, the determination could still be carried out in 2 hours.


### 3.8. Working Curve and Limit of Detection

Under the optimal conditions described above, a linear relationship between the concentration of NO2− and the value of ΔF was obtained in the concentration range of 0.009 ~0.500 μg/mL of NO2−. The linear regression equation is (4)ΔF=49.042ρ(μg/mL)-1.098.The correlation coefficient (r) was 0.9992. The limit of detection was calculated to be 0.003 μg/mL by 3 Sb/k (Sb the standard deviation of blank, and k the slope of linear calibration). The relative standard deviation (n=10) was 3.23% for 0.25 μg/mL NO2−. The limit of detection of NO2 was 0.004 mg/m3as 30 L solution was sampled by 10 mL of absorbing solution.


### 3.9. Influence of Coexistent Ion

In order to examine the effect of foreign ions in the air on the determination of nitrite, a number of ions were investigated according to the recommended procedure. The maximum error was restricted to be ±5% for the 0.25μg/mL nitrite. The limits of the tolerant foreign ions (by times) were Cl− (10,000), CO32 − and SO42− (3000), PO43 − (900), Br −(700), NH4+ and F−(600), Formaldehyde and NO3 − (500), I− and SO32 − (110), and Pb2 + (21), respectively. It could be seen that most of ions, even in a large excess, did not interfere with the detection.


## 3.1. Excitation and Emission Spectrum

Rhodamine B is a kind of pinkish synthetic dye, which has intense fluorescence in solution and has been used commonly in fluorescent dye of cell, colored glass, and characteristic fireworks, and so forth. Figure2 shows the fluorescence emission spectra of different Rhodamine B systems. It can be found that the fluorescent intensity of Rhodamine B decreases with the addition of NO2− (curve 4) in the system of Rhodamine B + H2SO4 (curve 2). Curve 1 and 3 shows that the system can be activated when an appropriate amount of sodium acetate is added. In addition, there is a linear relationship between the concentration of NO2− and ΔF under certain conditions. It can be concluded that a method for the determination of NO2 and NO is developed by fluorescence spectrophotometry. The maximum excitation and emission wavelengths for the system are 555 nm and 576 nm, respectively.Figure 2
Fluorescence emission spectra. (1) Rhodamine B + H2SO4 + NaAc, (2) Rhodamine B + H2SO4, (3) Rhodamine B + H2SO4 + NO2− + NaAc, (4) Rhodamine B + H2SO4 + NO2-. Rhodamine B: 6.0×10-7 mol/L, H2SO4: 4.0×10-3 mol/L, NO2−: 0.4 μg/mL, NaAc: 0.08 mol/L, temperature: 60°C, time: 45 minutes.


## 3.2. Influence of Reaction Media

The influence of reaction media in ΔF was analyzed. Different acidic media was investigated, such as sulfuric acid, hydrochloric acid, acetic acid, phosphoric acid, and nitric acid. The highest sensitivity was detected in the sulfuric acid media. Therefore, sulfuric acid was used in this experiment. In addition, the effect of dosage of sulfuric acid (0.1 mol/L) was also studied in the range of 0.2~2.00 mL. It was found that the maximum value of ΔF could be obtained as the dosage was 1.00 mL. Then, 1.00 mL of sulfuric acid solution was used in the experiment.


## 3.3. Influence of Dosage of Rhodamine B

The influence of Rhodamine B solution (5.0 × 10−6 mol/L) in ΔF was also studied. Results indicated that fluorescent signal of the system increased gradually with the increased Rhodamine B dosage while the value of ΔF increased firstly and then decreased. The maximum value of ΔF was obtained when the dosage of Rhodamine B was 3.00 mL. Therefore, 3.00 mL of Rhodamine B was chosen as optimum dosage in the further studies.


## 3.4. Influence of Dosage of Activator

Different materials were used to study the influence of activator, such as sodium citric acid, citric acid, sodium acetate, oxalic acid, sodium phosphoric acid and sodium dodecyl sulfonic acid. Both the improved fluorescence intensity and value of ΔF were detected by using the sodium acetate. Furthermore, the largest value of ΔF could be obtained for the 1.00 mL of sodium citrate solution (2.0 mol/L). Hence, 1.00 mL of sodium citrate was chosen in the experiment.


## 3.5. Influence of Reaction Temperature

The influence of temperature on the value of ΔF was tested in the range of 25~80°C. It was found that the value of ΔF increased gradually with the increased temperature. Moreover, the value of ΔF was relatively constant in the range of 60°C-80°C. Then, the reaction temperature was performed at 60°C in the experiment.


## 3.6. Influence of Reaction Time

The variation of the value of ΔF was studied with the operation time. It was found that the value of ΔF increased fast with the prolonged reaction time in 40 minutes. The stable values of ΔF was observed with the further increased reaction time. The reaction time was chosen at 45 minutes in this experiment.


## 3.7. Stability Test

The fluorescence intensity was recorded after the reaction was terminated for 4 hours. The decreased fluorescence intensity was observed in 2 hours. However, the value of ΔF was unchanged, which indicated that this system was comparatively stable. Therefore, the determination could still be carried out in 2 hours.


## 3.8. Working Curve and Limit of Detection

Under the optimal conditions described above, a linear relationship between the concentration of NO2− and the value of ΔF was obtained in the concentration range of 0.009 ~0.500 μg/mL of NO2−. The linear regression equation is (4)ΔF=49.042ρ(μg/mL)-1.098.The correlation coefficient (r) was 0.9992. The limit of detection was calculated to be 0.003 μg/mL by 3 Sb/k (Sb the standard deviation of blank, and k the slope of linear calibration). The relative standard deviation (n=10) was 3.23% for 0.25 μg/mL NO2−. The limit of detection of NO2 was 0.004 mg/m3as 30 L solution was sampled by 10 mL of absorbing solution.


## 3.9. Influence of Coexistent Ion

In order to examine the effect of foreign ions in the air on the determination of nitrite, a number of ions were investigated according to the recommended procedure. The maximum error was restricted to be ±5% for the 0.25μg/mL nitrite. The limits of the tolerant foreign ions (by times) were Cl− (10,000), CO32 − and SO42− (3000), PO43 − (900), Br −(700), NH4+ and F−(600), Formaldehyde and NO3 − (500), I− and SO32 − (110), and Pb2 + (21), respectively. It could be seen that most of ions, even in a large excess, did not interfere with the detection.


## 4. Determination of Samples

Gas samples were sampled with a speed of 0.2 L/min for 2.5 hours. The content of targets can be calculated according to the fluorescent intensity of the absorbing liquid in two absorb bottles. The method has been applied to determine the concentration of NOx in the atmosphere of a laboratory building and make the comparison with Saltzman method. The breakthrough point is that the limit of determination is lower than Saltzman method. The results are shown in Table 1.Table 1
Results of the determination of NOx in the air samples.
Place of sampleAverage concentration of NOx per hour (mg/m3)RSD (%)(n=6)Saltzman methodMethod recovery (%)Laboratory0.02973.40.0304104.7Outside of laboratory0.00957.40.008697.8A gymnastical zone0.00298.2–-91.5


## 5. Conclusion

In summary, this paper has established a new method of the concentration of NO2 and NO in the air detected by the fluorescence quenching method, determination results were lower than air quality standards stipulated in the standard (0.24 mg/m3). The method is simple and has a low limit of detection. It can be a referee for the environmental evaluation.



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*Source: 281931-2011-04-03.xml*