Talanta 70 (2006) 281285

Simultaneous determination of nitrite and nitrate in dew, rain, snow and lake water samples by ion-pair high-performance liquid chromatography
Yuegang Zuo a,b, , Chengjun Wang a , Thuan Van a
a b

Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, MA 02747, United States University of Massachusetts, Graduate School of Marine Sciences and Technology, 285 Old Westport Road, North Dartmouth, MA 02747, United States Received 23 January 2006; received in revised form 12 February 2006; accepted 13 February 2006 Available online 15 March 2006

Abstract A simple, fast, sensitive and accurate reversed-phase ion-pair HPLC method for simultaneous determination of nitrite and nitrate in atmospheric liquids and lake waters has been developed. Separations were accomplished in less than 10 min using a reversed-phase C18 column (150 mm 2.00 mm i.d., 5 m particle size) with a mobile phase containing 83% 3.0 mM ion-interaction reagent tetrabutylammonium hydroxide (TBA-OH) and 2.0 mM sodium phosphate buffer at pH 3.9 and 17% acetonitrile (ow rate, 0.4 mL/min). UV light absorption responses at 205 nm were linear over a wide concentration range from 100 g/mL to the detection limits of 10 g/L for nitrite and 5 g/L nitrate. Quantitation was carried out by the peak area method. The relative standard deviation for the analysis of nitrite and nitrate was less than 3.0%. This method was applied for the simultaneous determination of nitrite and nitrate in dew, rain, snow and lake water samples collected in southeast Massachusetts. Nitrate was found being present at 4.795.99 g/mL in dew, 1.202.63 g/mL in rain, 0.320.60 g/mL in snow and 0.120.23 g/mL in lake water. Nitrite was only a minor species in dew (0.620.83 g/mL), rain (<0.0050.14 g/mL), snow (0.0210.032 g/mL) and lake water (0.120.16 g/mL). High levels of nitrite and nitrate observed in dew water droplets may constitute an important source of hydroxyl radicals in the sunny early morning. 2006 Elsevier B.V. All rights reserved.
Keywords: Nitrite; Nitrate; Photolysis; Hydroxyl radicals; Dew; Rain; Snow; Lake water; Ion-pair; HPLC

1. Introduction Nitrite (NO2 ) and nitrate (NO3 ) are important components in atmospheric liquids and surface water because their photolysis signicantly enhance the photooxidation of natural and anthropogenic pollutants by formation of hydroxyl radicals (OH ), the most powerful oxidant in aqueous solution [15]. Nitrate and its conjugated acid (HNO3 ) are the end oxidative product of nitric oxide and other nitrogen species. As a major component of atmospheric aerosols, HNO3 /NO3 not only represents a signicant nitrogen ux from the atmosphere to surface water, but also contributes substantially to the acidity of precipitation [2,68]. In contrast to nitrate, nitrite is only a trace component in atmospheric water droplets. However, due to the high photolytic efciency of nitrite and its conjugated nitrous acid, HONO, the photolysis of these N(III) species has been considered a predominant source of OH rad-

Corresponding author. Tel.: +1 508 999 8959; fax: +1 508 999 9167. E-mail address: yzuo@umassd.edu (Y. Zuo).

icals in the low atmosphere, particularly, in the early morning when the concentrations of other hydroxyl radical sources, such as formaldehyde and ozone, are low [1,2]. Nitrite and HONO in condensed phases are also involved in the formation of carcinogenic nitrosamines [2,9]. Given the signicant inuence of nitrite and nitrate on human environment and health, it is important to monitor their concentration and examine the mechanisms involved in their production, transport and decomposition in atmospheric condensed phase and surface water. Although nitrate has been determined routinely in most rainwater surveys due to the concerns about the causes of acid rain and the eutrophication of surface water, measurements of nitrite in dew, rain, fog and cloud water are scarce [8,1013]. The commonly used methods for nitrite determination include the spectrophotometric detection based on the Griess reaction [12,14], in which nitrite is diazotized with sulfanilamide and then reacted with N-(1-naphthyl)ethylenediamine to form a colored product. For the measurement of total concentration of nitrite and nitrate, it is necessary to reduce the nitrate to nitrite with copperised cadmium [15]. Nitrate concentration is then

0039-9140/$ see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2006.02.034

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calculated as the difference between the total inorganic nitrogen anions and nitrite. Several ow injection analysis (FIA) methods have been developed based on this spectrophotometric technique for the simultaneous determination of nitrite and nitrate [16,17]. Incomplete reduction and sample carryover have been a reported problem in the FIA analyses. The most frequently used methods for the simultaneous analysis of the nitrate and nitrite include the ion chromatography (IC) with suppressed conductivity detection [10,18,19], which requires a long analysis time if the sample contains other common anions, such as sulfate and phosphate. During the IC separation, nitrite could be partially oxidized due to the use of a mobile phase at the high pH value. Recently, capillary electrophoresis (CE) [11,20,21] is employed in the analysis of environmental and biologic samples such as rain water, human saliva and urine. In addition to traditional IC, ion-pair chromatographic methods have also been widely studied for the separation of common anions, including nitrite and nitrate [22,23]. Ion-pair HPLC methods offer, with respect to ion chromatography, advantages of relatively lower cost in both instrumentation and columns and can be advantageously employed in laboratories where only conventional HPLC systems are available. On the other hand, the theoretical column efciency of ion-pair HPLC is better than that of an IC column, assuming that a suitable ion-pair reagent is chosen. In ion-pair LC, more parameters, such as stationary phase material, counterion and the concentration, pH and ionic strength of eluent, and organic modier and the concentration, can be selected and thus provide further advantages to increase the selectivity and resolution and minimize possible interference. A comprehensive review on this technique has recently been published by Gennaro and Angelino [24]. However, little information is available on the analysis of atmospheric water samples using ion-pair HPLC. Recently, we have reported on an indirect photometric ionpair HPLC method for the analysis of sulfur and nitrogen anions in atmospheric liquids [13]. Because both nitrite and nitrate ions have large molar absorption coefcients in the short UV wavelength region, a direct spectrophotometric detection would generate a high selectivity and sensitivity for the measurement of these nitrogen species. In this paper, we describe a direct spectrophotometric ion-pair HPLC method for the simultaneous analysis of nitrite and nitrate in dew, rain, snow and surface water samples. 2. Experimental 2.1. Chemicals Standards of sodium nitrite was purchased from Baker Chemical Co. (Phillipsburg, NJ, USA) and sodium nitrate from Fisher Scientic (Fair Lawn, NJ). Tetrabutylammonium hydroxide (TBA-OH) titrant (0.4 M in water, HPLC grade) and sodium phosphate were obtained from Acros Organics (Geel, Belglum, NJ). Acetonitrile was supplied by Pharmco Products (Brookeld, CT, USA). Except where noted, all reagents were of analytical grade and all solution preparations were made using doubly distilled-deionized water.

2.2. Samples Snow, rain, and dew water samples were collected during the periods from January to June 2004, and from September 2005 to January 2006 with a Teon container or lm on the roof of Building Group-II or on the grass of the University of Massachusetts Dartmouth campus, North Dartmouth, MA. Lake water samples were collected from Buttonwood Park, New Bedford, and North Dartmouth, MA. After sampling, snow samples were stored at 20 C; rain, dew and lake waters at 4 C in the dark until used. The samples were centrifuged and ltered through 0.45 m membrane lters (Fisher Scientic brand) before HPLC analysis. Precautions have always been taken to minimize sample contamination. All sample containers, glassware and ltration devices were thoroughly cleaned with 0.1 M HCl solution and then nally with doubly distilled-deionized water. The blank chromatograms with the doubly distilled-deionized water have shown no nitrite peak and nitrate below 10 g/L. 2.3. HPLC analysis A Dionex high-performance liquid chromatograph (Dionex Corporation, Sunnyvale, CA, USA) equipped with a P680 HPLC pump, a UVD-170U spectrophotometer detector, a Gina 50 autosampler, and Chromeleon 6.60 software was used for all experiments. Some of HPLC analyses were conducted with Beckman liquid chromatograph equipped with a Model 125 dual solvent pump, a 168 photodiod array detector, a 508 autosampler and Gold Nouveau Software. The analytical column used was a Phenomenex C18 reversed-phase column (150 mm 2.00 mm i.d., 5 m particle size) guarded by a 10 mm C18 guard column. The anions of interest were separated using an isocratic elution program. The mobile phase was made up of 83% 3.0 mM TBAOH titrant and 2.0 mM sodium phosphate buffer at pH 3.9 and 17% acetonitrile organic solvent. The ow rate was 0.4 mL/min. Detection of nitrite and nitrate anions was carried out by direct UV absorbance at 205 nm. A series of 1.5 mL standard mixture solutions of nitrite and nitrate were prepared from individual standard stock solutions. Twenty microliters of water samples or standard solutions was directly injected onto the HPLC system. 3. Results and discussion 3.1. Chromatographic separation Charged surfactants have been widely used as mobile phase modiers to improve the partitioning characteristics of charged solutes in reversed-phase HPLC. Various studies have been made to identify the interactions that occur between charged surfactants and ionic solutes and two retention mechanisms have been proposed. The rst assumes ion-pair formation in the mobile phase prior to its adsorption on to the non-polar stationary phase, while the second assumes the hydrophobic surfactant ion is held on the surface of the hydrophobic stationary phase, and the ionic solute is retained by electrostatic attraction to the charged hydrophobic surfactants. In an earlier study, we developed a

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Fig. 1. Chromatogram of nitrite and nitrate standard mixture. NO2 and NO3 concentration: 6.00 g/mL and injection volume: 20 L.

with a correlation coefcient of R2 = 0.998 while for nitrate: peak area = 5.414CNO3 + 1.074 with R2 = 0.999. The mean values of retention time for nitrite and nitrate determined in standard mixtures were: nitrite 5.093 0.006 min; nitrate 6.812 0.012 min, respectively. The relative standard deviation (R.S.D.) values of the retention times and peak areas were generally smaller than 1%, indicating that the separation method developed was very stable and had high reproducibility. The relative standard deviations for the intra- and inter-analysis of nitrite and nitrate are less than 3.0% and 4.2%, respectively. The detection limit measured as three times the background noise was 10 g/L NO2 and 5 g/L NO3 . These detection limits are signicantly lower than those reported for ion chromatography with a suppressor column or indirect photometric ion-pair HPLC. Increasing the injection volume of sample can further lower the detection limits. 3.3. Determination of nitrite and nitrate in dew, rain, snow and lake waters The described method was tested in several atmospheric water matrices with known amounts of nitrite and nitrate added, and the average percentage recovery was found to be 98101% for both analyte ions. Figs. 24 illustrate typical elution prole of dew, rain and lake water samples. Chromatograms of these water samples did not show interference with the same retention volume as nitrite or nitrate. Nitrite and nitrate anions were identied by matching retention times against those of standards and standard addition. In the chromatogram of lake water sample, besides nitrite and nitrate, there are two other peaks which might be due to small molecular weight organic acids but have not been identied. The concentrations of nitrite and nitrate in dew, rain, snow, and lake water samples are given in Table 1. Among all samples analyzed, the higher concentrations of both nitrite and nitrate were found in dew. This can have signicant effects on vegetation and the material surfaces, since dew provides a means to hold a liquid in place for a long time (overnight) and since the concentration may further increase to a very high value during evaporation in the morning. Both nitrite and nitrate

successful method for the simultaneous determination of common sulfur, and inorganic nitrogen anions in atmospheric water samples based on this second mechanism, in which the stationary phase was recoated about every another month depending on the number of samples analyzed and their matrix complexity [13]. In this work, the short chain TBA-OH was selected to form hydrophobic ion-pairs with analyte anions to be separated in a reversed phase C18 HPLC column and the ion-pairs were determined using a direct photometric method. Fluoride, chloride, sulfate and sulte are optically transparent, do not interfere the UV absorbance measurement and thus make the simultaneous determination of nitrite and nitrate with the above mentioned rst mechanism possible and easier. After systematic experiments on the several mobile phase variables, including the concentration of TBA-OH, the CH3 CN/H2 O ratio (from 5% to 50%), pH of the mobile phase and mobile phase ow rate, the separation conditions were optimized and described in Section 2. Fig. 1 shows the separation of a standard mixture of nitrite and nitrate. A good separation can be achieved in a short elution time less than 8 min. The chosen wavelength of 205 nm provides a higher sensitivity with a clean chromatogram than the wavelength of 254 or 225 nm employed in previous studies [25,26]. Nitrate in the mobile phase used in this study has a molar absorptivity of 852 L mol1 cm1 , and nitrite 401 L mol1 cm1 at 205 nm. Acetonitrile has a short absorption wavelength cut-off and was used as an organic solvent in mobile phase. 3.2. Quantitative analysis In atmospheric liquids the concentration of nitrite is usually below 1.00 g/mL, while nitrate has a much broader concentration range. Therefore, the calibration curve for the analysis for nitrite was made in the concentration range of 0.0010.0 g/mL, and for nitrate anion in the range of 0.001.00 102 g/mL. Calibration curves for both nitrite and nitrate were linear over the concentration ranges tested. For nitrite a typical calibration curve followed the equation: peak area = 4.098CNO2 0.001

Fig. 2. Chromatogram of a dew sample.

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undergo rapid photolysis under sunlight irradiation to form OH radicals: NO2 + H2 O + h OH + NO + OH NO3 + H2 O + h OH + NO2 + OH (1) (2)

Due to the high reactivities of hydrogen peroxide, organic peroxides and hydroxyl radicals, these photooxidants have been suggested to cause oxidative stresses in plants and be responsible for the forest decline observed in Europe, North American and Japan [2729]. Among all the oxidants, the OH radical is known to be the most reactive one. Nitrite and nitrate in dew and other wet precipitation could arise from the dissolution of HNO2 and HNO3 in the gas phase or in the atmospheric aerosols. The reactions of nitrogen oxides in water droplets can also be an important source:
Fig. 3. Chromatogram of a rain sample.

NO + NO2 + H2 O 2HNO2 2NO2 + H2 O HNO2 + HNO3

(3) (4)

A reaction between NO2 and bisulte in water droplets could account for the formation of considerable amount of nitrite. On the other hand, the nitrite deposited and/or formed in dew water can be recycled into the mixing layer, due to the high volatility of ammonium nitrite. In this sense, the wet surface can be considered to be an active sink during the night and a source of nitrite during early morning. Given the importance of nitrite and nitrate in the atmospheric gas and liquid phase chemistry and on the health, further studies on the sources, concentration variation, transformation and fate of these nitrogen species in dew and other atmospheric precipitation is urgently needed. 4. Conclusions The described direct photometric ion-pair HPLC method has been proved a rapid, sensitive, and accurate technique for the simultaneous determination of nitrite and nitrate. This method was successfully applied to the quantitative measurement of nitrite and nitrate in dew, rain, snow and natural surface waters. Among the samples analyzed, dew water contains high levels of nitrite and nitrate. Acknowledgements
0.15 0.14 0.18 0.09 0.08 0.05 0.01 0.01 0.02 0.02 0.01 0.02 0.01 0.01

Fig. 4. Chromatogram of a lake surface water sample.

Table 1 The concentrations of nitrite and nitrate found in atmospheric and lake water samples (g/mL) Sample Dew I Dew II Dew III Rain I Rain II Rain III Snow I Snow II Snow III Snow IV Lake water I Lake water II Lake water III Lake water IV
a

Date of collection 27 September 2005 27 September 2005 26 September 2005 2 October 2005 2 October 2005 28 May 2005 29 January 2004 18 January 2004 12 January 2006 12 January 2006 29 September 2005 29 September 2005 29 September 2005 29 September 2005

pH value 6.20 6.32 6.70 3.86 3.90 3.60 4.88 4.55 3.95 4.02 5.49 5.50 5.64 5.57

Nitrite 0.64 0.02 0.62 0.02 0.83 0.02 NDa ND 0.14 0.00 0.021 0.001 0.032 0.001 0.032 0.002 0.026 0.002 0.12 0.01 0.16 0.01 0.12 0.01 0.12 0.01

Nitrate 4.87 4.79 5.99 2.63 2.62 1.20 0.320 0.376 0.60 0.56 0.12 0.23 0.14 0.17

The authors would like to thank Dr. T. Wu, L. Zhang and J. Kang who performed some preliminary experiments. This work was partly supported by the National Science Foundation under grant ATM 9984755. References
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