In the existence of appropriate amount of disodium ethylenediaminetetraacetate (EDTA), precipitation would not occur in seawater and other natural waters even if the sample solution was adjusted to strong basicity, and the NH3-OPA-sulfite reaction at the optimal pH range could be used to determine ammonium in natural waters. Based on this, a modified
Ammonia nitrogen consists of ammonia (NH3) and ammonium (
The main analytical parameters of the typical reported OPA methods.
Samples | Technology | Reagent | Reaction temperature | Working range (nmol/L) | LOQ (nmol/L) | Reference |
---|---|---|---|---|---|---|
Standard solution | Manual | OPA, mercaptoethanol | Room temperature |
|
|
[ |
Fresh water | Flow injection | OPA, Na2SO3, |
85°C | 250–20000 | 250 | [ |
Fresh/saline waters | Flow injection | OPA, Na2SO3, tetraborate | 30°C | 250–50000 | 250 | [ |
Seawater | Manual | OPA, Na2SO3, tetraborate | Room temperature | ND–10000 | ND | [ |
Seawater | Gas diffusion | OPA, Na2SO3 | 70°C | ND–40000 | ND | [ |
Seawater | Flow injection | OPA, Na2SO3 | 65°C | 100–600 | 100 | [ |
Seawater | Autonomous batch analyzer | OPA, Na2SO3 | Room temperature | 200–1000 | 200 | [ |
Seawater | Multipumping analyzer | OPA, Na2SO3, tetraborate | 63.5–86.5°C | 13–1000 | 13 | [ |
Seawater | Solid extraction technology | OPA, Na2SO3, tetraborate | 75°C | 1.67–300 | 1.67 | [ |
ND means “no data.”
The fluorometric reaction of NH3-OPA-sulfite was found to be pH dependent by much reported work. The optimal pH was reported at 11 by Amornthammarong and Zhang in 2008 [
All the chemicals used in this study were of analytical grade, supplied by Aladdin Chemical Reagent Co., China, unless stated otherwise. All solutions were prepared in ultrapure water (resistivity 18.2 MΩ
All vessels used in the experiments were firstly soaked with 1 mol
20 mL of standard ammonium solution or sample solution with a concentration range of 0.032–15.0
Based on the method of [
The OPA-NH3-sulfite reaction may be affected by the parameters OPA and sodium sulfite concentrations, reaction time, and pH. These parameters had been optimized by much work [
0.25
Excitation (a) and emission (b) spectra of products of OPA-NH3-sulfite (pH 11.3) in the presence of EDTA.
The product had the maximum excitation wavelength (
It was reported that the OPA-NH3-sulfite reaction could be pH dependent and formed a fluorescent isoindole complex [
Effect of pH on the reaction of OPA-NH3-sulfite ((a) 0.25
The effect of OPA concentration on the fluorescence reaction was studied over the range 0.115–0.913 g/L (Figure
Effect of OPA concentration in the final solution on the reaction of OPA-NH3-sulfite ((a) 0.25
The effect of sodium sulfite in the range of 0–10 g/L is illuminated in Figure
Effect of sodium sulfite concentration in the final solution on the reaction of OPA-NH3-sulfite ((a) 0.25
In this work, OPA and sodium sulfite were found to be the main source of reagent blank. To decrease the reagent blank, OPA and sodium sulfite solution were mixed together according to the depiction in Section
EDTA is a strong metal ion complexing agent. Precipitation would not occur in seawater sample at the existence of appropriate number of EDTA even if the solution was adjusted to strong basicity. When 3 mL of EDTA-NaOH buffer solution (R2) was added in 20 mL seawater sample in this work, the pH of the solution could be adjusted in the range of 11.0–11.4 and precipitation could not appear. The concentration of EDTA in the final solution was 5.77 mmol/L. This dosage was also suitable for determining freshwater sample. To investigate the effect of EDTA, the FI of different concentration standard ammonium solution was separately determined in the existence or absence of EDTA. The results are listed in Table
The FI of different concentration standard ammonium solution in existence and absence of EDTA.
Concentration of ammonium ( |
FI in existence of EDTA | FI in absence of EDTA |
|
||
0 | 62.871 | 74.994 |
0.125 | 253.749 | 235.408 |
0.250 | 444.613 | 414.782 |
0.375 | 589.166 | 551.247 |
0.500 | 760.241 | 690.585 |
|
||
The relationship between FI and |
FI = |
FI = |
|
||
Corresponding parameters | ||
|
0.9973 ( |
0.9970 ( |
The standard deviation of the intercept | 12.65 | 11.90 |
The standard deviation of the slope | 41.32 | 38.87 |
Under the optimal conditions chosen above, the typical calibration curves were determined according to Section
Calibration curves and the corresponding performances.
Method | Excitation/emission slit widths | Calibration curves |
|
|
Standard deviation of the intercept | Standard deviation of the slope | Linearity range ( |
---|---|---|---|---|---|---|---|
3 nm/5 nm | FI = |
6 | 0.9970 | 11.90 | 38.87 | 0.032–0.500 | |
The proposed method | 3 nm/3 nm | FI = |
6 | 0.9996 | 4.88 | 2.94 | 0.25–3.00 |
1.5 nm/1.5 nm | FI = |
8 | 0.9971 | 5.58 | 0.81 | 1.00–15.0 | |
|
|||||||
The classical method | 3 nm/5 nm | FI = |
6 | 0.9951 | 5.71 | 12.66 | 0.25–2.00 |
Under the same experimental environment as the proposed method, a calibration curve of the classical OPA method was determined according to Section
The reproducibility of the method was evaluated with 5 repetitive determinations of a 0.250
Fresh water samples, groundwater and mountain spring water, were collected at Yaoshan Scenic Area in Guilin. A surface seawater sample was collected from the South China Sea and aged for one year. In order to examine the recovery of the method, these three samples spiked with a series of concentration of ammonium (0, 0.125, 0.250, 0.500, and 1.000
The matrix spiked recovery.
Matrix | Matrix spiked curve | Corresponding calibration curve | The average matrix spiked recovery |
|
|||
Groundwater | FI = |
FI = |
101.60% |
|
|||
Mountain spring water | FI = |
FI = |
103.83% |
|
|||
Seawater | FI = |
FI = |
97.60% |
Two typical seawater samples obtained from the South China Sea were analyzed using the proposed method according to Section
Analytical results of the proposed method and classical OPA method.
Seawater sample | The proposed method ( |
The classical OPA method ( |
Calculated |
Critical |
---|---|---|---|---|
1 | 0.536 ± 0.008 ( |
0.560 ± 0.018 ( |
2.43 | 2.45 |
2 | 0.385 ± 0.006 ( |
0.360 ± 0.035 ( |
1.22 | 2.78 |
Huajian River is located in Guilin city and passes through Huajian Compus of Guilin University of Electronic Technology (GUET). Twenty-three surface water samples were collected from Huajian River at December 27, 2013, and filtered by 0.45
Intercomparison data with indophenol blue method.
The spatial variation of ammonium concentration in Huajian River.
A new modified OPA fluorometric analytical method was established to determine ultratrace concentrations ammonium in natural waters using EDTA-NaOH as buffer. In this method, the NH3-OPA-sulfite reaction at the optimal pH could be used to determine ammonium in natural waters. There was no significant statistical difference between the results obtained from the proposed method and classical OPA method. The results of the proposed method applied to determine the river water were agreed with that of indophenol blue method. Compared to the classical OPA method, the main merit of the proposed method was enhancing the sensitivity by increasing the amount of reaction production under the optimal pH condition. It could quantify nanomolar level ammonium without enrichment.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The work was financially supported by the National Natural Science Foundation of China (no. 41206077). The authors would like to thank the group of Professor Yuan Dongxing in Xiamen University for supplying the seawater samples.