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Soil Science Society of America Journal 66:1008-1011 (2002)
© 2002 Soil Science Society of America

DIVISION S-8—NOTES

A technique to facilitate diffusions for nitrogen-isotope analysis by direct combustion

K. K. Moran, R. L. Mulvaney* and S. A. Khan

Dep. of Natural Resources and Environ. Sci., Univ. of Illinois, 1102 S. Goodwin Ave., Urbana, IL 61801

* Corresponding author (mulvaney{at}uiuc.edu)


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 Materials and Methods
 Results and Discussion
 REFERENCES
 
A technique was developed that permits the use of boric acid-indicator solution in carrying out diffusions for inorganic 15N analysis of soil extracts by direct combustion, so as to eliminate the need for a separate quantitative determination. In this technique, the titrated sample is acidified with 2.5 M KHSO4 and subsequently treated with methanol to remove H3BO3. The NH+4-N is dissolved in 1 mL of deionized water, concentrated after transfer to a microcentrifuge tube, and freeze-dried in a tin capsule. Isotope-ratio analyses were accurate to within 5% when diffusions were performed on 100 to 1000 µg of NH+4-N (0.2–10 atom % 15N), whereas errors of 8 to 17% occurred with 25 to 50 µg of labeled N, even after correction for isotopic dilution by atmospheric NH3. The technique described permits 15N analysis of any form of N that can be recovered by H3BO3 diffusion of soil, soil extracts, soil hydrolysates, Kjeldahl digests, water, or wastewater.

Abbreviations: ANCA-MS, automated N/C analyzer-mass spectrometry • LSD, least significant difference • SD, standard deviation


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
Materials and Methods
 Results and Discussion
 REFERENCES
 
THE GROWING USE OF automated mass spectrometers for N-isotope analysis by direct combustion (Barrie et al., 1995a,b; Barrie and Prosser, 1996) has promoted the development of diffusion techniques for recovering inorganic forms of N from soil extracts and water. In these techniques, the sample is treated with MgO to liberate NH+4 as gaseous NH3, with or without addition of Devarda's alloy to reduce NO-3 and NO-2 to NH+4, and of sulfamic acid to destroy NO-2. Diffusion is usually carried out in a plastic specimen container, a screw-cap bottle, or a Mason jar, the NH3 liberated being collected in an acidified filter, or in a pair of such filters, suspended above the sample (Brooks et al., 1989; Jensen, 1991; Lory and Russelle, 1994; Herman et al., 1995; Stark and Hart, 1996; Goerges and Dittert, 1998; Khan et al., 1998) or sealed inside Teflon (DuPont, Wilmington, DE) tape for placement within the sample (Sørenson and Jensen, 1991; Stark and Hart, 1996).

Instead of acidified disks, NH3 can be collected in H3BO3-indicator solution if diffusions are performed in a wide-mouth Mason jar (Mulvaney et al., 1997), in which case, isotope-ratio analyses can be carried out in conjunction with a quantitative N determination. This approach has the advantage that both determinations can be done within a wide concentration range, owing to the volume of sample that can be accommodated (up to 100 mL), and the absorbing capacity of the H3BO3 solution (up to 4 mg of N). An especially useful option is to carry out diffusions with gentle heating on a hot plate, which dramatically reduces the diffusion period (Khan et al., 1997). Perhaps most importantly, the use of H3BO3 permits inorganic N and 15N analyses to be performed on complex samples such as wastewater, septic effluent, or manure extract (Mulvaney and Khan, 1999; Khan et al., 2000b), and also directly on soil without the need for extraction (Khan et al., 2000a), total N and 15N analyses of Kjeldahl digests (Stevens et al., 2000), and fractionation of hydrolyzable soil N (Mulvaney and Khan, 2001). The latter application facilitates 15N analysis of amino acid N and amino sugar N, and has led to a simple soil test for potentially mineralizable N that could be employed in 15N-tracer research (Khan et al., 2001).

The purpose of the work reported here was to develop a technique whereby H3BO3 diffusions can be performed to carry out 15N analyses by automated N/C analyzer-mass spectrometry (ANCA-MS). This technique was evaluated using (NH4)2SO4 solutions that ranged from 0.2 to 10 atom % in their concentration of 15N, through comparative studies involving direct analysis of these solutions and diffusions by the method of Khan et al. (1998), which uses acidified disks.


   Materials and Methods
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
Results and Discussion
 REFERENCES
 
Diffusion Methods
Diffusions with MgO were performed on aqueous solutions of (NH4)2SO4, from 10 mL of deionized water using H3BO3-indicator solution (Khan et al., 1997), or from 10 mL of ~4 M KCl using acidified disks (Khan et al., 1998). In both cases, a hot plate was used to accelerate recovery of NH+4-N.

Boric-Acid Transfer Technique
Following titrimetric determination of diffused NH+4-N, the H3BO3 sample in the petri dish was acidified by addition of 2.5 M KHSO4 (0.1 µL µg N-1), and then evaporated to dryness on a hot plate (~90°C). To remove H3BO3, 5 mL of anhydrous methanol was added, and the methanol remaining after formation of trimethyl borate [B(OCH3)3] was removed by heating to dryness at 90°C. Five milliliters of deionized water was then added, the petri dish was swirled gently to dissolve any (NH4)2SO4 adhering to the wall of the dish, and drying was repeated at 90°C. The (NH4)2SO4 in the dish was dissolved in 1 mL of deionized water and transferred with a 1000-µL mechanical pipettor to a 1.5-mL microcentrifuge tube (Fisher Scientific, Pittsburgh, PA), followed by complete drying in a dry bath incubator (Fisher Scientific no. 11-718-6 equipped with four no. 11-718-9 heating blocks, Fisher Scientific, Pittsburgh, PA) at ~90°C. A 200- or 1000-µL pipettor was used to dispense 110 to 800 µL of deionized water into the tube, so as to obtain solutions containing at least 0.2 g N L-1, and preferably 0.9 to 1.25 g N L-1. The tube was then heated for a few minutes to ensure complete dissolution of the (NH4)2SO4, and a 100-µL aliquot was transferred with a 200-µL pipettor to an 8 by 5 mm tin capsule (cat. no. ATD 1008, Alpha Resources, Stevensville, MI) supported in a well of a microplate (cat. no. 001-010-2205, Dynatech Laboratories, Chantilly, VA). The samples were frozen completely while the microplate was kept in a freezer overnight, followed by freeze-drying. The top of the tin capsule was then closed using forceps, and the capsule was compressed into a compact pellet (1–2 mm in diameter) by applying pressure with the thumb and forefinger. To prevent contamination, the capsule was only handled while wearing polyethylene gloves, and the forceps was immersed in 0.1 M H2SO4, and subsequently dried with a laboratory wiper after immersion in deionized water, prior to each use. The compressed pellet was returned to the microplate for subsequent shipment to the Stable Isotope Facility, University of California at Davis, for N-isotope analysis by ANCA-MS using an INTEGRA-CN Integrated Stable Isotope Analyser (PDZ Europa, Cheshire, UK).

Evaluation of Boric-Acid Transfer Technique
All analytical data reported were obtained for standard solutions of (NH4)2SO4 that provided a wide range in 15N concentration. These solutions were prepared to contain 1 g N L-1, by dissolving 15N-labeled and unlabeled reagent-grade (NH4)2SO4 in 100 mL of deionized water in a volumetric flask, such that the approximate concentrations of 15N were 0.2, 0.37, 0.5, 1, 2, 5, or 10 atom%. In each case, dilutions were performed to obtain additional concentrations of 100 and 250 mg N L-1. For two of the 15N concentrations studied (0.37 and 10 atom % 15N), solutions also were prepared that contained 25, 50, or 500 mg N L-1.

The accuracy and precision of N-isotope analyses using the H3BO3-transfer technique were evaluated for all 15N concentrations provided by the standard solutions, through comparison to analyses when these solutions were transferred directly to a tin capsule or diffused using acidified disks. Diffusions with H3BO3 were performed to recover 250 µg of NH+4-N after diluting 1 mL of standard solution to 10 mL with deionized water, followed by transfer of a 100-µg aliquot for N-isotope analysis. When acidified disks were used, 100 µg of NH+4-N was diffused following dilution of 1 mL of standard solution with 9 mL of 4 M KCl. In either case, there were three replicate diffusions, and analytical accuracy was evaluated relative to data collected by carrying out N-isotope analyses (three replicates) after freeze-drying 100 µL of the (NH4)2SO4 solutions (1 g N L-1) in tin capsules.

To further compare the use of H3BO3 and acidified disks for N-isotope analysis by ANCA-MS, diffusions were performed (six replicates) by each method on 1-mL aliquots of standard solutions containing 25, 50, or 100 µg of NH+4-N and ~0.37 or 10 atom % 15N, after dilution to 10 mL with either deionized water (H3BO3 method) or 4 M KCl (disk method). Additional diffusions were carried out using H3BO3 from 10 mL of deionized water containing 250 (three replicates), 500 (two replicates), or 1000 (1 replicate) µg of NH+4-N, such that six analyses were performed on ~100 µg of N in each case. Analytical accuracy was evaluated relative to mean values obtained previously in measuring 15N concentrations by direct transfer to tin capsules.

Measured values of atom% 15N involving diffusion were corrected to account for contamination by natural abundance NH+4-N derived from nonsample sources such as reagents or ambient NH3 using the isotope-dilution equation

[1]
where C is the corrected value for atom% 15N, B is the micrograms of background NH+4-N (presumably natural abundance) determined colorimetrically (six replicates) by the macroscale method of Rhine et al. (1998) after diffusion of controls, D is the micrograms of N liberated from the sample, and M is the measured (uncorrected) value of atom% 15N. Values for B, in the case of the H3BO3 method, were obtained using a reagent prepared without indicator and processed by the transfer technique described previously, followed by dilution to 25 mL with deionized water in a volumetric flask. For the disk method, control diffusions were performed from 10 mL of 4 M KCl rather than water, and both disks from a single diffusion were treated with deionized water in a 25-mL volumetric flask. In both cases, colorimetric analyses were performed on a 1-mL aliquot.

Statistical Analysis
Data from replicate 15N analyses were characterized by computing means and standard deviations. After using Eq. [1] to correct mean values for replicate diffusions, analytical accuracy was evaluated on the basis of a least significant difference (LSD) at the 0.01 probability level, or by computing a relative error (E) as

[2]
where C is a corrected mean and T is the corresponding value obtained by direct transfer.


   Results and Discussion
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
REFERENCES
 
The transfer technique described provides the option of utilizing H3BO3 diffusions for N-isotope analysis by ANCA-MS, in lieu of diffusions that use acidified disks. This option has long been preferred in our laboratory for carrying out automated Rittenberg analyses, because a quantitative N determination is easily performed by titration to eliminate the need for a separate colorimetric analysis and verify sample integrity before N-isotope analysis. Moreover, 15N analyses can often be replicated without performing additional diffusions, since the H3BO3 reagent used can absorb up to 4 mg of N.

To ensure that accurate and precise data are obtained by the transfer technique described, a study was conducted to compare 15N analyses of standard (NH4)2SO4 solutions (0.2 to 10 atom% 15N) by this technique with values obtained when the same solutions were transferred directly to tin capsules or diffused using acidified disks. The results (Table 1) show that, with four of the seven 15N concentrations studied, analyses by the H3BO3 method were more accurate than those using acidified disks, although significant improvement was observed in only one case. In comparison to analyses by direct transfer, significant underestimation occurred only at 5 atom% 15N with the H3BO3 method, and only at 10 atom% 15N with acidified disks.


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  		Table 1. Comparison of 15N analyses with or without diffusion by two methods.

 
For the utmost accuracy, controls should be processed with each batch of samples to account for contamination by ambient NH3, which is present throughout buildings with central heating and air conditioning, but varies considerably in concentration depending on the use of NH3-based cleaning solutions and floor waxes. The effect of such contamination will necessarily be most serious when a meager amount of N is processed by the transfer technique described, so processing should be done with an excessive amount of N whenever possible, followed by subsampling to carry out analyses by ANCA-MS. The benefit of the latter approach is clearly evident from Table 2, which summarizes data obtained in a study to compare 15N analyses following diffusions to recover different quantities of NH+4-N, using either H3BO3 or acidified disks. In both cases, analytical accuracy tended to improve with increase in the quantity of N diffused, and as expected, this trend was more apparent for enriched than for unenriched samples. Analyses involving the use of H3BO3 were most accurate when diffusions were performed on 250, 500, or 1000 µg of NH+4-N, and the data thereby obtained were more accurate than analyses involving diffusion of 25 to 100 µg of N by either the H3BO3 or disk method.


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  		Table 2. Comparison of two diffusion methods for 15N analysis of different quantities of NH+4-N.

 
Further examination of Table 2 reveals that when diffusions were performed on 25 to 100 µg of labeled NH+4-N, 15N analyses were more accurate using acidified disks as opposed to H3BO3, and the difference increased progressively with decrease in the amount of N diffused. This trend was especially evident for uncorrected data, which can be attributed to the fact that diffusion disks were dried in a desiccator without exposure to ambient air. To reduce the effect of contamination by ambient NH3 during the transfer technique described, discretion should be exercised in selecting a location to carry out evaporations, and heating should not be prolonged once drying is complete.

Comparison of standard deviations reported in Tables 1 and 2 reveals that 15N analyses tended to be more precise by the H3BO3 method than by the disk method. In the case of Table 1, this difference is no doubt related to the fact that replicate data were obtained by diffusing 250 µg of N by the H3BO3 method, as compared to 100 µg when diffusions were performed using acidified disks. Table 2 provides evidence of the same trend, in cases where both methods were used to diffuse 25, 50, or 100 µg of NH+4-N.

Particularly when diffusions are performed on <=100 µg of N, care should be taken that the NH+4-N recovered is transferred as quantitatively as possible to a tin capsule, so as to maximize data quality during isotope-ratio analyses by ANCA-MS. Examination of the quantitative data reported in Table 2 shows that transfers in such cases by the technique described were usually more than 90% efficient, and compared favorably with transfer efficiencies achieved using acidified disks.

When diffusing >100 µg of N, care should be taken in transferring an appropriate aliquot from the microcentrifuge tube to the tin capsule, so as to provide sufficient N to generate reliable data but not an excessive amount that would vitiate isotope-ratio analyses by ANCA-MS. The former requirement is particularly critical with natural abundance or 15N-depleted samples, whereas the latter is most apt to occur with highly enriched samples.

To prevent volatilization of NH+4-N that would promote isotopic fractionation, H3BO3 samples must be acidified prior to concentration and transfer by the technique described. Acidification is accomplished using KHSO4 rather than H2SO4, so as to control corrosion of the tin capsule. No difficulties have been observed from the use of H2SO4 as a titrant to determine the quantity of NH+4-N collected during diffusion.

Freeze-drying is employed to avoid salt creep during final drying in tin capsules, which would lead to loss of sample N. To ensure that no such difficulty occurs, samples should be frozen completely before freeze-drying, and evacuation should be carried out using a high-speed vacuum pump.

The transfer technique described allows ANCA-MS to be employed in carrying out 15N analyses in conjunction with any of the Mason-jar diffusion methods that have been developed using H3BO3-indicator solution. These methods eliminate the need for a high KCl concentration to control condensation during diffusion of inorganic N and allow diffusions to be easily performed from water, if desired (Khan et al., 1997, 2000b; Mulvaney et al., 1997). More importantly, the use of H3BO3 in a petri dish reduces the diffusion period by providing a large area for absorption of NH3. The shorter period thereby achieved is crucial for controlling chemical interferences, with the result that analyses can be performed to recover inorganic N from complex samples such as wastewater, septic effluent, and manure extract (Mulvaney and Khan, 1999; Khan et al., 2000b); to fractionate hydrolyzable forms of soil N, including amino acid and amino sugar N (Mulvaney and Khan, 2001); and to determine inorganic or potentially available soil N without extraction (Khan et al., 2000a, 2001).


   ACKNOWLEDGMENTS
 
Appreciation is expressed to D. Harris of the Stable Isotope Facility, University of California at Davis, for carrying out the 15N analyses reported.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
 REFERENCES
 
This study was a part of Project ILLU-65-0326, Illinois Agric. Exp. Stn.

Received for publication May 28, 2001.


   REFERENCES
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 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results and Discussion
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