Anal. Chem. 1998, 70, 3969-3973
Determination of Natural Waters
15
N Nitrate and Nitrite in Spiked
Robert J. Kieber,* Lynn Bullard, and Pamela J. Seaton
Department of Chemistry and Marine Science Program, University of North Carolina at Wilmington, Wilmington, North Carolina 28403-3297
Dissolved 15N nitrate and 15N nitrite were determined by derivatization of 15NO2- with 2,4-dinitrophenylhydrazine (2,4-DNPH), followed by FT-IR spectrophotometric analysis of the resulting 15N azide. Samples were derivatized in the aqueous phase and partitioned into CHCl3 with a single extraction with greater than 98% efficiency. The 15N azide displays an asymmetric stretching band at 2089 cm-1 in a region of the infrared spectrum with relatively few interferences. 15N nitrate can be determined after initial reduction to 15N nitrite. The described method is quick and has minimal sample manipulations, undetectable blanks, and a low limit of detection of 0.5 µM, with an average precision in coastal seawater of 6% RSD for 15NO - and 9% RSD for 15NO -. Analytical results were 2 3 verified with a completely independent standard colorimetric technique, in both deionized and natural waters. The two methods produced nitrite concentrations that agreed to within a few percent with no statistical differences observed in the data up to 60 µM. The quantification of 15N was not affected by variable amounts of 14N nitrate typically found in natural waters. Additionally, there was greater than 98% recovery of an added 15N nitrite spike from a coastal seawater sample. Application of the analysis to nitrogen uptake studies is also presented. In one such experiment, the average uptake of 15NO - in coastal seawater was 13.6 µM day-1 between 3 day 3 and day 7. This 15N uptake corresponded to maximum chlorophyll a increases in the cultured seawater, suggesting the biota were actively assimilating the added nitrate. Nitrogen is a biolimiting element in many of the world’s coastal and estuarine natural waters. Nitrate is assimilated by phytoplankton, while 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 a stable intermediate in the assimilation and nitrification processes and therefore is an indicator of biogeochemical processes driving the nitrogen cycle.1-4 * Corresponding author: (phone) (910) 962-3865; (fax) (910) 962-3013; (e-mail)
[email protected]. (1) Harrison, W. G. Nitrogen in the Marine Environment: Use of Isotopes. In Nitrogen in the Marine Environment; Carpenter, E. J., Capnes, G., Eds.; Academic: New York, 1983; pp 763-807. (2) Kaplan, W. A. Nitrification. In Nitrogen in the Marine Environment; Carpenter, E. J., Capnes, G., Eds.; Academic: New York, 1983; pp 139-90. S0003-2700(98)00273-X CCC: $15.00 Published on Web 08/19/1998
© 1998 American Chemical Society
Because of its importance in controlling marine primary productivity, there has been much interest in the vertical transport and uptake of nitrogen by marine phytoplankton.1,5 Stable isotopes such as 15N are often employed as tracers to follow this transport and uptake of nitrogen through marine ecosystems. The most common method for quantification of particulate 14N and 15N ratios is mass spectrometry, which requires that all nitrogen species be converted to N2(g) for measurement.1,6-13 Although extremely difficult and time-consuming, dissolved 15N species could also be analyzed via mass spectrometry. However, all analytes must first be quantitatively removed from solution, dried, and converted to N2(g).1 Limitations of this aqueous-phase N isotope analysis include the expense of equipment, the complex series of sample manipulations involved (both isolation of the nitrogen species and conversion to N2), the extensive time requirements, and the necessity of large sample volumes.6,14 In addition, because of these sample and instrumentation requirements, 15N uptake studies cannot be carried out aboard ship or in the field, which imposes severe limitations on the versatility of these methods. Recently, a high-performance cation-exchange and fluorometric detection method for the determination of 14N/15N ratios of NH4+(aq) was developed.11-13 This analysis is useful because, unlike mass spectrometry, it uses small sample volumes, relatively low cost equipment, and minimal sample manipulations. However, the utility of this cation-exchange method is limited because it detects only NH4+, relies on quantification of unresolved peaks of 14NH4+ and 15NH4+, requires relatively long analysis times of (3) Sharp, J. H. The Distribution of Inorganic Nitrogen and Dissolved and Particulate Organic Nitrogen in the Sea. In Nitrogen in the Marine Environment; Carpenter, E. J., Capnes, G., Eds.; Academic: New York, 1983; pp 1-35. (4) Zafiriou, O. C.; Ball, L. A.; Hanley, Q. Deep-Sea Res. 1992, 39, 1329-47. (5) Ward, B. B.; Kilpatrik, K. A.; Renger, E. H.; Eppley, R. W. Limnol. Ocenaogr. 1989, 34, 493-513. (6) Ness, J. C.; Dugdale R. C.; Dugdale, V. A.; Goering J. J. Limnol. Oceanogr. 1962, 7, 163-9. (7) Strickland, J. D. H.; Parsons, T. R. A Practical Handbook of Seawater Analysis, 2nd ed.; Bulletin of the Fisheries Resources Board of Canada: Ottowa, ON, Canada, 1972. (8) Gilbert, P. M.; Lipschultz, F.; McCarthy, J. J.; Altabet, M. A. Limnol. Oceanogr. 1982, 27, 639-50. (9) Bronk, D. A.; Gilbert, P. M. Mar. Ecol. Prog. Ser. 1991, 77, 171-82. (10) Bronk, D. A.; Gilbert, P. M. Mar. Biol. 1993, 115, 501-8. (11) Gardner, W. S.; Herche, L. R.; St. John, P. A.; Seitinger, S. P. Anal. Chem. 1991, 63, 1838-43. (12) Gardner, W. S.; Cotner, J. B.; Herche, L. R. Mar. Ecol. Prog. Ser. 1993, 93, 65-73. (13) Gardner, W. S.; Bootsma, H. A.; Evans, C.; St. John, P. A. Mar. Chem. 1995, 48, 271-82. (14) Kendall, C.; Grim, E. Anal. Chem. 1990, 62, 526-9.
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45 min, and has a relatively high detection limit of 1-2 µM, which is unsuitable for tracer-level experiments. Stable N isotope uptake studies would be much more versatile and provide more reliable data if other dissolved nitrogen species, in addition to NH4+, could be determined accurately and if some of the limitations of the analysis discussed above could be avoided. The method described in this paper focuses on determination of aqueous-phase 15NO3- and 15NO2- and is based on the strong characteristic IR absorbance of the 2,4-dinitrophenyl azide formed from the reaction of nitrite with 2,4-dinitrophenylhydrazine (2,4DNPH). The asymmetric stretch of the azide appears as a doublet at ∼2100 cm-1, which is typical of nitro-substituted benzazides.15
Quantification of the two isotopes is possible because the more intense stretching frequency of the 14N azide is at 2133 cm-1 while the more intense band of the azide formed from the heavier isotope, 15NO2-, is at 2089 cm-1 (Figure 1). Additionally, the region between 2000 and 2150 cm-1 of the IR spectrum has very few interferences, providing undetectable blanks and submicromolar detection levels for 15N or 14N nitrite and nitrate. This method is also fast, with a total analysis time of less than 20 min, has minimal sample manipulations, and requires fairly standard instrumentation which can be easily adapted for shipboard or field use. The method has the added advantage that samples can analyzed immediately or can be derivatized and stored up to 1 month prior to analysis, because of the stability of the azide derivative.16 EXPERIMENTAL SECTION Reagents and Standards. All chemicals were obtained from Fisher Scientific (Fair Lawn, NJ) and were certified ACS or HPLC grade, unless otherwise noted. Deionized water (DI) was obtained from a Milli-RO6 Plus/Milli-Q Plus water purification system (Millipore, Milford, MA). 2,4-DNPH (Aldrich, Milwaukee, WI) was recrystallized twice from a 70:30 mixture of acetonitrile/water (v/v) followed by a final recrystallization from pure acetonitrile. The resulting crystals were dried under vacuum and stored dark in airtight 30-mL Teflon vials. The nitrite derivatizing solution was prepared in a 30-mL Teflon vial by dissolving 20 mg of the recrystallized 2,4-DNPH in 20 mL of solution containing concentrated HCl and water in a ratio of 2:6 (v/v). The derivatizing solution was cleaned of any nitrite contamination by three successive extractions with carbon tetrachloride (Aldrich, Milwaukee, WI). CCl4 (2 mL) was added to the derivatizing solution and shaken on a wrist action shaker for 5 min followed by centrifugation at 2000 rpm for 5 min in order (15) Liever, E.; Dftedahl, E. J. Org. Chem. 1959, 24, 1014-7. (16) Kieber, R. J.; Seaton, P. J. Anal. Chem. 1995, 67, 3261-4.
3970 Analytical Chemistry, Vol. 70, No. 18, September 15, 1998
Figure 1. Infrared asymmetric stretch band from (A) 40 µM 14N azide from 14NO2-, (B) 40 µM 15N azide from 15NO2-, and (C) 40 µM 14N azide plus 40 µM 15N azide, respectively.
to separate the phases. The CCl4 layer was removed and the reagent solution re-extracted twice more as described. The derivatizing solution was stable at room temperature for 1 week after initial preparation. On subsequent days after initial preparation, the derivatizing solution was re-extracted once just prior to use. 15N nitrate samples were prepared from K15NO (98%+ from 3 Cambridge Isotope Labs, Andover MA) while 15N nitrite samples were prepared by reduction of K15NO3, through a copper cadmium reduction column, as described below. Stock solutions of concentrated ammonium chloride/disodium ethylenediamine tetraacetic acid (NH4Cl/EDTA) were prepared by dissolving 13 g of NH4Cl and 1.7 g of EDTA in 100 mL of DI water. The pH of these solutions were adjusted to 8.5 with concentrated NH4OH. Sample Preparation. Nitrite in 20-mL samples was derivatized with 400 µL of the derivatizing solution in 30-mL Teflon vials at ambient temperatures. The azide formed was extracted into 2 mL of chloroform by shaking for 5 min followed by centrifugation for 5 min at 2000 rpm. The CHCl3 layer containing the azide was drawn out via syringe and placed into the IR cell for analysis. All work with CHCl3 should be performed in a well-ventilated area such as a laboratory fume hood. Samples were derivatized for
10 min prior to extraction and analysis or were derivatized and stored in the dark at 4 °C for subsequent analysis.16 Nitrate in samples was reduced to nitrite with a standard copper cadmium reduction column.17 The column was generated by washing 20 g of cadmium metal in 5% HCl solution followed by successive rinses with DI water and 50-75-mL aliquots of 2% CuSO4. An acid-washed buret, packed with a small swatch of acidwashed glass wool, was packed with the Cu-Cd mix and rinsed with 15-20 mL of dilute NH4Cl/EDTA solution. Samples were prepared for reduction by combining 44.5 mL of sample with 4.5 mL of NH4Cl/EDTA stock after which they were passed through the column at 1-2 mL min-1 with the first 5 mL discarded. The resulting nitrite solutions were derivatized and analyzed as described above. The column was rinsed between samples with 10 mL of NH4Cl/EDTA solution and stored at ambient temperature. Natural water samples used in the intercomparison study were collected from a coastal seawater location near Wrightsville Beach, NC (34°1′ N, 77°9′ W) and an oligotrophic site in the Gulf Stream (34.4′ N, 75°6′ W) using acid-washed high-density polyethylene (HDPE) bottles, which were rinsed with sample three times prior to collection and stored at 4 °C in the dark. Prior to analysis, natural samples were filtered through a 0.2-µm Nuclepore filter. NO3- Uptake Study. A coastal seawater sample was collected near Wrightsville Beach, NC, in a autoclaved 10-L Nalgene bottle which was rinsed 3 times with sample prior to collection. The sample was enriched with nutrients by adding f/2 media18 with the exception of 60 µM Na15NO3 in place of 0.88 mM Na14NO3. The culture was sampled for 15NO3- and chlorophyll a at various time intervals over a 7-day period. At each sampling time, three separate 20-mL aliquots were removed and filtered through glass fiber filters (GFF). The 15NO3- was reduced to 15NO2- and quantified as described above. The cells on the filters were treated with 5 drops of saturated MgCO3 and stored frozen for subsequent chlorophyll a analysis by standard techniques.7 FT-IR Instrumentation. The FT-IR system consisted of a Mattson Polaris (Model IR-10410), a Balston CO2/H2O filtering system, (Whatman International Lts., Lexington, MA), and a WinFIRST data acquisition system (Version 3.1) installed on an IBM-compatible PC. The IR was programmed with the following parameters: 32 scans; resolution of 2.0 cm-1; iris at 6%. The IR cell (0.1 or 1.0 mm KCl, Wilmad, Buena, NJ) was filled with sample via syringe and placed in the FT-IR sample holders housed in the instrument. After 5 min of purging CO2 from the sample chamber, the IR spectrum was collected. The 14N azide peak absorbed at 2133 cm-1 while the 15N azide peak absorbed at 2089 cm-1 (Figure 1). All analyses were performed at ambient temperature with peak heights used for standard curve and sample analyses. Peak height was chosen over peak area to quantify the nitrite signal because it yielded better precision, especially at lower levels (10 µM and below) of the analyte. RESULTS AND DISCUSSION. Extraction. To determine the efficiency of the CHCl3 extraction of the azide formed from the reaction of dissolved nitrite with (17) Clesceri, L. S.; Greenberg, A. E.; Trussell, R. R. Standard Methods of the Examination of Water and Wastewater; Port City Press: Baltimore, 1989. (18) McLachlan, J. Growth Media-Marine In Handbood of Phydological Methods: Culture Methods and Growth Measurements; Cambridge: Cambridge, U.K., 1973; pp 25-31.
Table 1. Initial and Final Concentrations of Aqueous-Phase NO2- a initial NO2- (µM)
final NO2- (µM)
% extracted
20.20 29.90 40.20
0.20 0.50 0.60
99.0 98.3 98.5
a Values are single extractions of three different NO - samples with 2 each concentration representing the average of four replicates. Percent extracted was calculated relative to initial levels.
2,4-DNPH, an experiment was performed in which 20-mL samples of varying nitrite concentrations were derivatized for 10 min and extracted with 2 mL of CHCl3. The residual azide concentration remaining in the aqueous phase after extraction was determined by a previously described high-performance liquid chromatographic technique.16 This latter analysis was necessary because, although it cannot distinguish between N isotopes, it can accurately determine the very low concentrations of nitrite expected to remain in the aqueous phase after extraction. The percent nitrite extracted, calculated as the amount of azide in the chloroform relative to pre-extraction levels, was greater than 98% after a single extraction with CHCl3 (Table 1). Because this single extraction provided >98% extraction efficiency, provided 10-fold concentration of the azide, and required minimal sample manipulations, additional extractions of the aqueous phase were not required. Linearity and Precision. The response of the FT-IR detector to 15NO2- and 15NO3- was linear up to 40 µM nitrite and 60 µM nitrate. Higher concentrations were not analyzed because nitrate concentrations in natural waters are rarely higher. Samples containing significantly higher concentrations of nitrate or nitrite could be analyzed after simple dilution. Typical calibration curves for both analytes yield a linear equation y ) 5 × 10-4x with a y-intercept near zero and a correlation coefficient near 1.0. The precision of the FT-IR method was tested by multiple analyses of a coastal seawater sample spiked with 15NO2- and 15NO3-. For the nitrite spiked samples, fifteen 20-mL aliquots were removed, derivatized, and analyzed by FT-IR with a resulting RSD of 6%. An analogous study done with nitrate had an RSD of 9% with the larger RSD resulting from the additional step required to reduce nitrate to nitrite. Intercalibration and Accuracy. To verify analytical results obtained by the FT-IR method reported here, an intercomparison study was conducted with a standard colorimetric method for nitrite.17 In this latter analysis, 25-mL samples are treated with a sulfanilamide-N-(1-naphthyl)ethylenediamine colorimetric reagent which reacts with nitrite producing an azo dye that is quantified spectrophotometrically at 543 nm. Although this standard colorimetric procedure cannot differentiate between different isotopes of nitrogen, it is valid in this intercomparison study because the only source of nitrite was as 15NO2-. Natural water samples used in this intercomparison study were collected from a coastal seawater location near Wrightsville Beach, NC, and at an oligotrophic site in the Gulf Stream. Three variable additions of 15NO2- were added to deionized, Wrightsville Beach (WB) and Gulf Stream (GS) water for a total of nine samples. Four replicates were withdrawn from each sample and analyzed Analytical Chemistry, Vol. 70, No. 18, September 15, 1998
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Table 2. (A) Concentration of 15NO2- in the Presence and Absence of Equimolar Concentrations of 14NO2- a 15NO - (µM) with 2 15NO - (µM) equimolar 14NO2- (µM) 2 20.8 ( 0.8 29.8 ( 0.7 39.8 ( 1.1
22.0 ( 0.4 32.8 ( 0.9 41.8 (0.3
(B) Concentration of 15NO2- Observed with Variable Concentrations of 14NO2- Addedb 15NO - (µM) 14NO - (µM) 2 2
Figure 2. Comparison of analytical results obtained by the FT-IR method and the standard colorimetric method in three different water matrixes; DI, BC, and GS. Each data point represents the average of four replicates. The line represents perfect agreement between results, not a statistical fit to the data.
for nitrite concentration by both the FT-IR method and the standard colorimetric analysis (Figure 2). The line in Figure 2 is not a statistical fit to the data but rather represents a line with a slope of unity passing through the origin which would result in the case of perfect agreement. In all cases, the disparity between the FT-IR and colorimetric methods is less than a few percent, which is well within analytical uncertainty. In addition, no statistical differences were revealed between the two methods for these nine samples (t-test; n ) 4; p < 0.001). Spike Recovery. A spike recovery test was performed to determine whether any positive or negative interferences on the quantification of the analyte by the FT-IR method were present. Three separate aliquots of DI and WB water were spiked with 25 µM 15NO2-. Three unspiked WB samples were also analyzed to quantify ambient levels of 15NO2- in the samples. The recovery of the nitrite by the FT-IR method was greater than 98%. In addition, the mean value for the samples prepared in DI water was statistically equivalent to the sample prepared in WB water, suggesting that no positive or negative interferences were present in the coastal seawater samples relative to the deionized water (t-test; n ) 3, p < 0.001). Comparison Studies. To determine whether the amount of 15NO - quantified by FT-IR was impacted by 14NO -, a comparison 2 2 study was performed between standard solutions of 15NO2- alone and solutions of 15NO2- plus equimolar 14NO2- (Table 2A). Although ∼2 µM more 15NO2- nitrite was observed in the presence of 14NO2- in these three samples, these differences were not statistically significant. In a second study, the concentration of 15NO2- was held constant (∼20 µM) while the concentration of 14NO2- was gradually increased from 0 to 60 µM. These concentrations are typical of what would be expected for nitrate in most coastal seawater samples. As in the case with equimolar concentrations, there were no statistical differences between 15NO - quantified with zero 14NO - addition and in the presence 2 2 of up to 60 µM 14NO2-. The results presented in Table 2 suggest that the quantification of 15NO2- is not dependent on ambient concentrations of 14NO2-, up to as much as 3 times the concentra3972 Analytical Chemistry, Vol. 70, No. 18, September 15, 1998
0.00 10 20.00 40.00 60.00
19.2 ( 0.5 19.3 ( 0.2 19.0 ( 1.5 19.6 ( 1.1 20.6 ( 1.1
mean std dev
19.50 0.63
a Each data point represents the average of four replicate samples. Each data point again represents the average of four replicate samples with the error representing ( one standard deviation from the mean.
b
Figure 3. Uptake of 15NO3- in a cultured coastal seawater sample. Chlorophyll a levels are also presented to indicate the growth stage of the natural phytoplankton assemblages.
tion of 15NO2- (60 µM 14NO2- vs 20 µM 15NO2-). On the basis of the signal-to-noise level from the IR spectra and the 1-mm path length cell used in these experiments, we estimate quantification would be possible with as low as 5 µM15NO2- in the presence of 60 µM14NO2-. 15NO - Uptake Study. A natural water sample, collected near 2 Wrightsville Beach, NC, was enriched with nutrients by adding f/2 media18 and 60 µM 15NO3-. The culture was sampled for 15NO - and chlorophyll a at various time intervals over a 7-day 3 period. The concentration of phytoplankton remained relatively stable (as measured by chlorophyll a) for the first 3 days (Figure 3). The concentration of 15NO3- also remained unchanged over this time interval. The exponential growth phase of the phytoplankton, as determined by chlorophyll a production, began on day 3. The 15NO3- had a direct, inverse profile relative to the chlorophyll a with concentrations declining as chlorophyll a increased, presumably due to uptake by the phytoplankton. The uptake rate of 15NO3- is 13.6 µM day-1 between day 3 and day 7. Detection Limit. The experiments described above were all performed using a 1.0-mm path length KCl cell. When a 10-mm cell was used, the detection limit of the FT-IR method, based on
2 times the signal-to-noise ratio, was 0.5 µM. To use this larger path length IR cell, sample size had to be increased to 40 mL followed by a 4.0-mL extraction with CHCl3. DISCUSSION We have developed the first sensitive, relatively simple, and rapid method capable of measuring both aqueous-phase 15NO2and 15NO3- in natural waters. The analytical results have been verified by intercalibration with a completely independent, standard colorimetric technique in three different water matrixes. No statistical differences in 15NO2- concentrations determined by both analyses were observed, and the data agreed to within a few percent, which was well within analytical uncertainty. This FT-IR method has no known interferences and has an excellent precision of 6% RSD 15NO2- and 9% RSD for 15NO3-. In addition, this method has undetectable blanks that allow for a low limit of detection of 0.5 µM, which is several times lower than the aqueous-phase 15NH4+ method currently in use. We are presently investigating solid-phase extraction techniques that would allow further preconcentration of the analyte. The goal of this phase in method development is to lower the detection limit sufficiently so ambient levels of 15NO2- in natural waters can be determined.
One of the greatest utilities of the method described in this paper is its ease of operation. A relatively simple FT-IR system can be outfitted for use in the field or aboard ship as well as in the laboratory. This is essential for reliable and versatile nitrogen uptake studies because it can provide real-time data while the study is in progress, rather than collection with analysis occurring much later, as with mass analyzers. A typical application of the method to natural water nitrogen isotope uptake studies with 15NO - is reported, where samples were collected and run within 3 1 h. Another valuable aspect of this method is that once the samples have been derivatized they can be stored, if required, for up to 1 month in the dark at 4 °C because of the stability of the azide derivative. A great number of samples, therefore, can be collected and derivatized within a very short amount of time. This decreases the chance of variations in nitrite concentrations prior to sample analysis. The method may also be extended to the analysis of other nitrogen species, such as 15NH4+ or dissolved organic 15N nitrogen, after their oxidation to nitrite using standard methods.7 Received for review March 10, 1998. Accepted July 10, 1998. AC9802731
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