Anal. Chem. 1995, 67, 3261 -3264
Determination of Subnanomolar Concentrations of Nitrite in Natural Waters Robert J. Kieber* and Pamela J. Seatont Department of Chemistry and Marine Science Program, University of North Carolina at Wilmington, Wilmington, North Carolina 28403
Dissolved nitrite in natural waters was determined at subnanomolar levels by derivitization with 2,4-dinitrophenylhydrazine (2,4-DNPH) followed by liquid chromatography. The analysis is rapid, involves minimal sample preparation, and requires fairly standard laboratory equipment. The method has undetectable blanks and a detection limit of 0.1 nM with an average precision of 4%RSD at ambient natural water concentrations. The azide formed upon reaction of NOz- with 2,4-DNPH at levels typically found in coastal seawater is stable for at least 4 weeks when kept cold in the dark. The stability of the nitrite derivative is extremely useful because it allows for large numbers of samples to be collected and derivahd in relatively short time periods for later chromatographic analysis. Analytical results were verified by intercomparison with a standard, completely independent, colorometric technique in several natural water types. Applications to fresh, estuarine, and marine samples are also presented. The marine nitrogen cycle plays a crucial role in the chemistry and biology of the sea. Because nitrate is a critical limiting factor to primary productivity in the oceans, the dynamics of nitrogen cycling have been studied Nitrate is assimilated by phytoplankton, and “new” nitrate is recycled back to the euphotic zone from the deep ocean through bacterial nitrification of dissolved and particulate organic nitrogen in the water column and sediments. Nitrite is an obligatory and stable intermediate in both of these processes and is therefore a general indicator of biogeochemical processes that drive the nitrogen cycle. Trace amounts of nitrite in environmental samples can also indicate the extent of pollution and eutrophication of natural water^.^ Despite the significance of nitrite in the marine nitrogen cycle, outside of the primary and secondary nitrite maxima, little is known about the distribution of nitrite throughout the rest of the water column. Examination of spatial and temporal variations of nitrite in the ocean has been limited primarily by the inadequacy of existing analytical methods. Nitrite, and in many cases nitrate, cannot be detected at the low concentrations at which critical * Correspondence should be addressed to this author. FAX: (910) 3953013. E-mail:
[email protected]. + E-mail:
[email protected]. (1) Zafiriou, 0. C.; Ball, L. A,; Hanley, Q . Deep Sea Res. 1992.39 1329-1347. (2) Carpenter, E. J., Capone, D. G., Eds. Nitrogen in the marine environment: Academic Press: New York, 1983. (3) Kang, X.; Sharma, S. K; Taylor, G. T.; Muenow, D. W. Appl. Spectrosc. 1992. 46, 819-826. 0003-2700/95/0367-3261$9.00/0 0 1995 American Chemical Society
processes take place. Nitrite has been measured by ~olorimetry,4~~ chemiluminescence: fl~orometry,~ or Raman ~pectroscopy.~ Two predominant methods for analysis of nitrite in seawater, the colorometric and chemiluminescent techniques, do not provide sensitivities required for critical evaluation of subnanomolar levels of nitrite. The standard colorometric t e ~ h n i q u e ,which ~ is based on formation of a diazo dye from the reaction of nitrite with N-(lnaphthy1)ethylenediamine and sulfanilamide, has a detection limit of 30-50 nM, although 100 nM is considered to be a more realistic detection limit for shipboard determination of nitrite.6 This method is not sensitive enough to detect nitrite levels in over 99% of the ocean. An optimized version of the colorometric method involves preconcentration of larger samples and allows for a detection limit of 1-2 nM.8 However, this method is extremely susceptible to atmospheric contamination and is not suitable for rapid determination of nitrite. The chemiluminecsent technique for determination of nitrite in seawater has detection limits to the subnanomolar range (0.10.2 nM). This method, developed by Garside6 and modified by Za6riou and co-workers,’ is quite sensitive; however, it is plagued by several limitations. The method is dif6cult and time consuming and requires fairly sophisticated equipment. Erratic air contamination during sample collection is such a large source of error that zero blanks cannot be obtained and reproducibility is compromised. Because of these limitations, large sample sizes are necessary with subsequently lengthy analysis times. This is of particular concern in biologically active samples where there is a rapid turnover of nitrite by the biota. We have developed a simple and extremely sensitive method for rapid analysis of nitrite in natural waters. The analysis is based on the known reactiongJOof nitrite with 2 , 4 d i n i t r o p h e n y l h y d e to form an azide, which is separated from interfering substances and quantified by HPLC. Other attempts at using HPLC for the quanti6cation of fluorometric or colorometric derivatives of nitrite are unsuitable for natural waters because their detection limits are too The method presented here is extremely desirable for natural samples because it very rapid and simple, has undetectable blanks, and has the lowest detection limit reported. In addition, it is well suited for shipboard and field analyses because samples can be analyzed in real time or stored for later laboratory analysis. (4) Clesceri, L. S., Greenbert, A. E., Trussell, R. R., Eds. Standard Methodsfor the Examination of Water and Wastewater; Port City Press: Baltimore, 1989. (5) Verma, K. K.: Verma A. Anal. Lett. 1992,25, 2083-2093. (6) Garcide, C. Mar. Chem. 1982,11, 159-167. (7) Zhou, J. Y.;Prognon, P.; Dauphin C.: Hamon, M. HPLC Fluorescence Chromatogr. 1993,36, 57-60. (8) Wada. E.: Hattori, A. Anal. Chim. Acta 1971,56, 233-240.
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EXPERIMENTAL SECTION Reagents and Standards. All chemicals were obtained from Fisher Scientitic (Fair Lawn, NJ) and were reagent grade or HPLC grade, unless otherwise noted. 2,4Dinitrophenylhydrazine (2,4 DNPH; Aldrich, Milwaukee, WQ was recrystallized twice from a 70:30 mixture of acetonitrile/water (v/v) followed by a final recrystallization from pure acetonitrile. The reagent was dried under vacuum and stored in the dark in airtight 30-mL Teflon vials. The derivatizing reagent was prepared in a 30-mL Teflon vial by dissolving 15 mg of recrystallized 2,4DNPH in a 15mL solution of concentrated HCl (-12 M), water, and acetonitrile in a ratio of 2:5:1 (v/v/v). Just prior to use, nitrite contamination contained within the 2,4DNPH reagent solution was removed with successive extractions with carbon tetrachloride (Mallinckrodt Inc., Paris, KT). CCl, (5 mL) was added to the reagent solution and shaken on a wrist-action shaker (Model 75, Burrell Corp., Pittsburgh, PA) for 5 min. The mixture was centrifuged at 2000 rpm (Model PR-6 centrifuge, International Equipment Co., Needham, MA) for 5 min in order to separate the phases. The organic layer was removed via a 5 m L pipet, and reagent solution was reextracted as described above. The purified reagent was prepared immediately prior to use. HPLC Instrumentation. The HPLC system consisted of an E-Lab Model 2020 gradient programmer and data acquisition system (OMS Tech, Miami, FL) installed in an IBMcompatible PC. An Eldex model A-60-S pump (Eldex Laboratories, San Carlos, CA), with an inert solenoid valve placed on its low-pressure side, was used to generate the mobile phase. A two-solvent mobile phase was employed: (A) 10%acetonitrile in water and @) 100% acetonitrile. Derivatized samples were injected into a Valco sixport injector (Valco Instruments, Houston, TX) with a 2000-pL sample loop. The 2,4dinitrophenylazide was separated isocraticly (60%A, 2 mL/min) on a radial compression module with an 8 mm i.d. x 10 cm C18 reversed-phase Radial-pak cartridge (Type 8NVC184, Millipore Corp., Waters Chromatography Division, Milford, MA) and detected by an Isco V4 variable-wavelength absorbance detector (Isco, Lincoln, NE) at 307 nm. The signal was stored and processed by the E-Lab data system. The column was at ambient temperatures. Standard Colorometric Determination of Nitrite. Nitrite was analyzed in natural samples by the standard colorometric test described in ref 4. The color reagent was prepared by dissolving 10.0 g of sulfanilamide in 800 mL of deionized water plus 100 mL of 85%phosphoric acid. N-(1-Naphthy1)ethylenediaminehydrochloride (1.0 g) was added to the above solution, and the volume was brought to 1L with deionized water. The reagent was stored at 4 "C in the dark. Filtered natural water samples or nitrite standard solutions (5 mL) were treated with the reagent (0.2 mL) in cuvette tubes. The color developed for 15 min, and the absorbance at 543 nm was recorded with a Spectronic 20. RESULTS Confirmation of Azide Structure. Nitrite reacts with 2,4DNPH to form 2,4dintrophenyl azide under acidic conditions. The azide has been extracted and purified from 2,4-DNPH-treated natural seawater samples and has been synthesized through the reaction of an aqueous solution of sodium nitrite with 2,4DNPH in sulfuric acid. The infrared spectra of both isolated and synthesized products are identical and show a very intense band at 2136 cm-l, characteristic of the azide functionality. The proton and carbon NMR spectra of the azide show only aromatic proton 3262 Analytical Chemistry, Vol. 67, No. 78, September 75, 7995
0
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TIME (min)
Figure 1. Formation of the azide derivative as a function of time in coastal seawater. Each data point represents the average of four samples. Errors bars represent one standard deviation based on n = 4.
and carbon signals. The UV maximum of the azide in acetonitrile is at 307 nm. All spectra were identical to spectral data reported in the literature?JO Extracted and synthetic 2,4diitrophenylazide analyzed by HPLC coeluted with the nitrite derivative formed upon 2,4-DNPH treatment of natural water samples. Derivatization Reaction. The time required for complete derivitization of nitrite by 2,4-DNPH was evaluated by following the derivitization to the azide as a function of time in coastal seawater collected from Wrightsville Beach, NC. One liter of the seawater was gravity filtered through a 47-mm glass fiber filter (type GF/F, Whatman International Ltd., Maidstone England) and four, 5mL aliquots per sampling time were withdrawn and derivatized by adding 50 mL of 2,4DNPH reagent in a 7-mLTeflon vial. The vial was shaken and the derivatization reaction allowed to proceed at ambient temperatures for varying time periods. A 2-mL aliquot was removed from each vial and analyzed by HPLC. The results presented in Figure 1 indicate the formation of the azide through reaction of nitrite by 2,4-DNPH is rapid, with complete derivitization after only 5 min at ambient temperatures. Blanks and Detection Limit. Nitrite is undetectable in blanks prepared in deionized water. A typical blank chromatogram is presented in Figure 2A The detector and integrator for this chromatogram were set to their most sensitive setting, and no peaks in the blank were observed in the area where the nitrite derivative elutes. Similar undetectable blanks have been obtained for samples taken on a variety of days and locations. The chromatogram obtained for a typical coastal seawater sample is presented in Figure 2B (20 nM nitrite) for comparison. Formaldehyde, present in many natural water samples, elutes -1 min after the nitrite azide derivative. The undetectably low background signal is an important component of this analysis because it allows for determination of extremely low levels of nitrite in natural waters. A conservative detection limit based on a signalto-noise ratio of 3 is 0.1 nM (0.0046 mg of NOz-/L) nitrite. Storage of Derivatized Nitrite Samples. In order to test for stability of the nitrite derivative, a 5WmL sample of coastal seawater was collected and filtered and 5mL aliquots were placed in 80 Teflon vials. 2,4DNPH (50 pL) was added to each; the samples were shaken and placed in storage in the dark at 4 "C. Four vials were subsequently removed per sampling day and (9) Dyall. L. IC Aust. J. Chem. 1986.39, 89-101. (10) Gromping, A. H. J.: Karst, 6.; Cammann, K J. Chromatogr. A
34 1-347.
1993,653,
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Figure 4. lntercomparison between the standard colorometric method4and the chromatographic determination of nitrite in a variety of natural waters. The line represents perfect agreement between results, not a statistical fit to the data: (+) Sargasso seawater, ( x ) Cape Fear Estuaty, (A)Greenfield Lake, and (W) coastal seawater.
Figure 2. HPLC chromatograms. (A) System blank prepared by adding 50 mL of purified 2,4-DNPH to 5 mL of deionized water with 2 mL injected into the HPLC system. (B) Direct injection of 2 mL of derivatized coastal seawater. The nitrite-2,CDNPH derivative elutes at 7.2 min (1) reagent; (2) azide derivative; (3) formaldehyde.
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Flgure 3. Test of storage procedure. Eighty coastal seawater samples were added to Telon vials and derivatized with 50 mL of 2,4-DNPH. Each time point represents the average concentration of nitrite observed in four samples on a given day.
analyzed. This procedure was repeated at various time intervals throughout a 4-week period. The results are presented in Figure 3. The derivatized nitrite is stable over the Cweek period within analytical uncertainty (&la) of the method. In addition, the nitrite derivative in four samples placed in the dark in a freezer over the 4-week period was also stable. Linearity and Precision. The response of the detector is linear over the concentration range of 0.5-1000 nM, which covers the range of nitrite typically found in most natural waters. The correlation coefficient over the entire range was 0.999 (n = 7), which is typical of our routinely run calibration curves. The precision of the method was determined, at ambient levels by performing multiple injections (n = 4) of samples run for each water sample tested. In some instances, as is the case for coastal
seawater, this procedure was repeated many times. The average relative standard deviation of the analysis was 4%. Intercalibration and Spike Recovery. In order to verify the analytical results obtained by the HPLC analysis of nitrite reported here, an intercomparison study was performed. Samples from a variety of natural waters (including fresh, estuarine, coastal seawater, and oligotrophic seawater) were collected, filtered, spiked with NO*-, and analyzed for nitrite chromatographically and by a completely independent, standard colorometric a n a l ~ s i s . ~ The detection limit of the latter technique is -100 nM; therefore, to have sufficient NOz- several of the samples were spiked with a nitrite standard. The results of this study are presented in Figure 4. In all cases, the disparity between the chromatographic and colorometric analyses was less than a few percent, which is well within analytical uncertainty. The recovery of the nitrite in spiked samples by the HPLC analysis was 100%. DISCUSSION We have developed an extremely sensitive and relatively simple analysis for the determination of subnanomolar concentrations of nitrite in natural waters. The analytical results have been veriiied by intercalibration with a completely independent, standard, colorometric technique! Nitrite concentrations determined by both analyses from a wide array of natural water types agreed withii a few percent, well within analytical uncertainty. The HPLC analysis is not subject to any known interferences and has excellent precision with a relative standard deviation of 4% at concentrations of nitrite typically found in natural waters. When the derivatiziig agent is properly purified, nitrite is not detectable in blanks. Background contamination is one of the limiting factors reported by Zafariou et al.' in their chemiluminescent analysis of nitrite in seawater. An undetectable blank is also important because it ultimately dictates the limit of detection, which for the method reported here is 0.1 nM, the lowest reported for routine determination of nitrite in seawater . This detection limit is sufficiently low to determine nitrite even in the most oligatrophic open oceans.1,11J2 One of the greatest utilities of the nitrite analysis discussed in this paper is its ease of operation. A relatively inexpensive (11) Holligan, P. M.; Balch, W. M.; Yentsch C. M.J. Mar. Res. 1984,42, 10511073. (12) Bonilla, J.; Senior, W.; Bugden J. Geophys J. Res, 1993,98, 2245-2257.
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Table 1. Concentration of Nitrite (nM) in a Variety of Natural Waters.
water type
salinity (ppt)
av nitrite concn (nM)
Greenfield Lake Cape Fear Estuary coastal seawater
0.00 10.00 35
231 (n = 4) 331 (n = 4) 17.5 (n = 12)
Sampling sites were located near Wilmington, NC.
isocratic HPLC system can be outfitted for use in the field or aboard ship. The chemiluminescent technique requires more elaborate hardware and is more cumbersome in its Our HPLC analysis of nitrite is rapid, approximately eight samples per hour can easily be analyzed. Another valuable aspect of our method is that the derivatization process is rapid, resulting in negligible air contamination and correspondingly undetectable nitrite blanks. The derivatized nitrite at levels typically found in coastal seawater is also stable for several hours at ambient temperatures and up to 4 weeks when kept cold in the dark. The ability to store samples allows for collection of greater numbers of samples, as many as 100/h for later HPLC analysis. An entire (13) Nuccio. J. Masters Thesis, University of North Carolina at Wilmington. 1994.
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12-bottle CTD cast from a shipboard collection system has been collected and derivatized with replicates in under 1h.I3 This rapid processing of samples will greatly lessen alterations in nitrite concentrations which may occur during the time between analyses. We have used the HPLC analysis of nitrite by derivatization with 2,4DNPH to study nitrite distributions in natural waters collected near Wilmington, N C. The concentrations observed are reported in Table 1. Coastal seawater always contained the lowest NOz- concentrations, and estuarine samples collected from the Cape Fear River Estuary contained the highest levels. This agrees with nitrite levels reported in the literature.l,ll,lz ACKNOWLEDGMENT Financial assistance was provided by the Center for Marine Science Research Center at UNCW, Contribution 106, and by a Charles L. Cahill Award for faculty research and development. Shiptime on the RV Cape Hatteras was provided by NSF as a Duke-UNC Consortium cruise. Received for review February 20, 1995. Accepted June 21, 1995.@ AC950184D @
Abstract published in Advance ACS Abstracts, August 1, 1995.
