- 1 - A simplified diffusion method for 15N analysis of dissolved ammonium *John Spoelstra1, 2, Marilla Murray2, 3, Richard J. Elgood2 NWRI Report Number 11-038 1National Water Research Institute, Environment Canada Canada Centre for Inland Waters 867 Lakeshore Road, P.O. Box 5050 Burlington, ON, L7R 4A6 2 Department of Earth and Environmental Sciences University of Waterloo 200 University Avenue West Waterloo, Ontario, N2L 3G1 3 Now with: Matrix Solutions Inc Suite 200, 150 – 13 Ave. SW Calgary, AB T2R 0V2 *Corresponding Author: Phone: (905) 336-6246 Fax: (905) 336-6430 Email: John.Spoelstra@ec.gc.ca Cite as: Spoelstra, J., M. Murray, and R. J. Elgood. 2011. A simplified diffusion method for 15N analysis of dissolved ammonium. National Water Research Institute, Report Number 11-038. Environment Canada. 16 pp. https://open-science.canada.ca/handle/123456789/1207 - 2 - TABLE OF CONTENTS 1 Method Overview ................................................................................................................... 3 2 Materials ................................................................................................................................. 5 3 Sample collection and preservation ........................................................................................ 5 4 Preparing Solutions ................................................................................................................. 6 4.1 Reagents ........................................................................................................................ 6 4.2 Solutions ....................................................................................................................... 6 4.3 15N-NH4 + Standards .................................................................................................... 7 5 Cleaning of glassware and equipment .................................................................................... 7 6 Pre-diffusion calculations ....................................................................................................... 7 6.1 Sample Volume ............................................................................................................. 8 6.2 KCl addition .................................................................................................................. 8 7 Preparing the PTFE traps ........................................................................................................ 9 8 Preparing the diffusions .......................................................................................................... 9 9 Collecting the acidified disks ................................................................................................ 10 10 Freeze drying the acidified disks .......................................................................................... 11 11 Isotopic analysis .................................................................................................................... 11 12 Correction of the isotope data ............................................................................................... 11 13 Recommended Reading ........................................................................................................ 13 14 Appendix A ........................................................................................................................... 14 - 3 - 1 METHOD OVERVIEW The δ15N-NH4 + method presented here is a modified version of the commonly used acidified disk diffusion method and is designed for water samples with an NH4 + concentration of 0.6 mg N/L or greater. Samples with lower NH4 + concentrations can be analyzed using greater sample volumes in larger diffusion jars but optimal conditions for diffusion (e.g., disk size, diffusion time, jar size) have not been finalized yet and will be described separately. In summary, dissolved NH4 + in the sample is converted to NH3 gas and subsequently trapped on an acidified quartz disk that is enclosed in a gas permeable, hydrophobic membrane. The diffusion apparatus consists of a 60mL wide-mouth jar with tetrafluoroethylene (TFE)-lined cap containing an acidified quartz disk enclosed in a polytetrafluoroethylene (PTFE) membrane. The required sample volume, which contains 12-30g of NH4 +-N, is pipetted into each diffusion jar. Deionized water (DI) is added to each jar so that the total volume is approximately the same (20mL). In order to prevent the PTFE traps from absorbing water and bursting, KCl is added to each diffusion jar to bring the KCl concentration of the solution to approximately 4M. Phenolphthalein pH indicator solution is added to each diffusion jar and the samples are then made basic by the drop-wise addition of a NaOH solution. Immediately after the solution turns pink (pH = 8-9), 2mL of tetraborate pH buffer solution is added to stabilize the pH at about 9.5. A PTFE trap is then placed in the diffusion jar and the jar is capped. The pH is buffered slightly higher than the pKa of NH4 + (9.25; Equation 1) to promote the conversion of NH4 + to NH3 gas without the risk of organic N hydrolysis that could occur at higher pH. Diffusion jars are placed on an orbital shaker for 10 days to ensure complete diffusion of the NH3 onto the acidified disks. Three internal δ15N-NH4 + isotope standards are processed with each batch of samples. Once diffusion is complete, the PTFE traps are removed and the quartz disks collected and placed in clean vials. The vials are frozen, freeze dried overnight and are then ready for isotopic analysis. δ15N values of the quartz disks are determined at the Environmental Isotope Lab, University of Waterloo, using a Carlo Erba 1108 Elemental Analyzer interfaced with a Thermo Instruments Deltaplus isotope ratio mass spectrometer (EA- IRMS). The precision associated with δ15N-NH4 + analysis of water samples is generally better than ±0.2‰.     9.25pKa,HNHNH (aq)g3aq4   (Eqn. 1) - 4 - Résumé La méthode δ15N-NH4 + présentée ici est une version modifiée de la méthode de diffusion sur disque acidifié couramment employée, et elle est conçue pour l’analyse des échantillons d’eau renfermant une concentration de NH4 + de 0,6 mg N/L ou plus. Pour les échantillons renfermant des concentrations plus faibles de NH4 +, on peut utiliser des volumes d’échantillons plus grands, dans des bocaux de diffusion plus grands, mais les conditions optimales de diffusion (taille du disque, taille du bocal) n’ont pas encore été déterminées, et elles feront l’objet d’une description distincte. En bref, le NH4 + en solution dans l’échantillon est converti en NH3 gazeux, puis il est piégé sur un disque en quartz acidifié inséré dans une membrane hydrophobe perméable aux gaz. Le dispositif de diffusion se compose d’un bocal à goulot large de 60 mL avec un couvercle garni de tétrafluoroéthylène (TFE) comportant un disque en quartz acidifié inséré dans une membrane de polytétrafluoroéthylène (PTFE). Le volume d’échantillon requis, qui contient entre 12 et 30 mg de NH4 +-N, est pipeté dans chaque bocal de diffusion. On ajoute de l’eau désionisée (ED) dans chaque bocal de manière à ce que le volume total soit approximativement le même dans chacun (20 mL). Pour empêcher les membranes de PTFE d’absorber de l’eau et de fendre, on ajoute du KCl dans chaque bocal de diffusion pour atteindre une concentration de KCl dans la solution d’environ 4 mol/L. Une solution d’indicateur de pH à la phénolphtaléine est ajoutée dans chaque bocal de diffusion, et les échantillons sont basifiés par l’ajout goutte à goutte d’une solution de NaOH. Dès que la solution devient rose (pH = 8 à 9), 2 mL d’une solution tampon au tétraborate est ajoutée pour stabiliser le pH à environ 9,5. Une membrane de PTFE est alors placée dans le bocal de diffusion, qui est ensuite fermé. Le pH est tamponné à une valeur légèrement supérieure au pKa du NH4 + (9,25; équation 1) afin de favoriser la conversion du NH4 + en NH3 gazeux en évitant le risque d’hydrolyse du N organique qui surviendrait à pH plus élevé. Les bocaux de diffusion sont placés dans un agitateur rotatif, où on les laisse 10 jours pour s’assurer que la diffusion du NH3 dans les disques acidifiés est complète. Trois étalons internes d’isotopes δ15N-NH4 + sont traités avec chaque lot d’échantillons. Lorsque la diffusion est complète, les membranes de PTFE sont retirées, et les disques en quartz sont prélevés puis placés dans des flacons propres. Les flacons sont congelés, lyophilisés pendant une nuit, après quoi on peut procéder à l’analyse des isotopes. Les valeurs de δ15N associées aux disques en quartz sont déterminées au laboratoire des isotopes environnementaux de l’Université de Waterloo, à l’aide d’un analyseur d’éléments Carlo Erba 1108 couplé à un spectromètre de masse pour l’analyse du rapport des isotopes (AE-SMRI). La précision de l’analyse du δ15N-NH4 + dans les échantillons d’eau est généralement meilleure que ± 0,2 ‰.     9.25pKa,HNHNH (aq)g3aq4   (Éqn. 1) - 5 - 2 MATERIALS Diffusion jars: 60mL Wheaton wide mouth jars (Part No. W216908) Quartz filters: Whatman 4.7cm QMA filters (Part No. 1851-047). To make the diffusion disks from the quartz fiber filters, use a one-hole punch to cut 6mm disks into a clean ceramic dish. Bake the disks at 550ºC for 3 hours. Once baked and cooled, keep the disks in a clean vial. Always use clean tweezers to handle the diffusion disks. Vials: Fisherbrand, 1dr. vials with caps (Cat. No. 03-338A) PTFE Tape: T-TAPE from any hardware/plumbing store 3 SAMPLE COLLECTION AND PRESERVATION NH4 + is a biologically available form of N that is generally preferred over other forms of inorganic N. Consequently, sample preservation is necessary for accurate NH4 + concentrations and δ15N values. Since NH4 + can also exist as gaseous NH3 (Equation 1), basic samples should be acidified to prevent volatilization and loss of NH3. NH3 volatilization is a highly fractionating process that enriches the residual NH3/NH4 + in 15N. The volume of sample required for 15N-NH4 + analysis depends on the NH4 + concentration of the sample. To obtain an optimum N peak on the EA-IRMS system (Section 11), each diffusion disk should have about 30g NH4 +-N diffused onto it (see Section 6 for more details). A conservative target for sample collection would be 200g NH4 +-N (Equation 2), which will allow for multiple repeats of δ15N analysis if required. 1) Filter samples to 0.45μm during collection (e.g., in-line filter) or immediately after collection (e.g., syringe-tip filter). Vacuum filtration should not be used because it increases the chances of contamination of samples with atmospheric NH3 or loss of NH3 by volatilization. 2) Immediately after collection, acidify samples with HCl to a pH of approximately 5. Note: HCl used should be fresh since it can absorb NH3 from the atmosphere over time if stored in a container that is not airtight. 3) Once filtered and acidified, samples should be kept cold in the field and then stored frozen until further processing.       N/LmgNH Nmg2.0 N/Lmgion ConcentratNHSample NmgAmount NHTarget (L)RequiredVolumeSample 4 4 4      (Eqn. 2) - 6 - 4 PREPARING SOLUTIONS All solutions should be made with NH4 +-free denionized water (DI) and stored in tightly capped bottles. If desired, larger volumes of the solutions can be made by modifying the instructions given below. It is the responsibility of all users to read the MSDS sheets for each chemical used in this method and be familiar with the hazards and safe-handling procedures. 4.1 Reagents Table 1, Reagents required for the diffusion procedure. Compound Formula Molecular Weight (g/mol) Potassium Chloride KCl 74.551 Sodium Tetraborate Decahydrate Na2B4O7·10H2O 381.37 Sodium Hydroxide NaOH 39.998 Sulfuric Acid H2SO4 98.073 4.2 Solutions 4M KCl: Dissolve 238g of KCl in 800mL of DI. Acidify with 3 drops of concentrated HCl. 6.0M NaOH: Dissolve 48g NaOH in 200mL of DI. NaOH should be added slowly while solution is mixed using a magnetic stirrer to prevent excessive heating of the solution. 0.2M NaOH: Dissolve 1.6g NaOH in 200mL of DI or add 6.66mL of 6.0M NaOH solution to 200mL of DI. 0.02M NaOH Wash Bath: Dissolve 8.0g NaOH in 10L of DI or add 33.33mL of 6.0M NaOH solution to 10L of DI. Tetraborate buffer: Dissolve 7.63g of Na2B4O7·10H2O in 200mL of DI. Add 0.6g of NaOH or 2.5mL of the 6M NaOH solution. This solution should be made while it mixes on a magnetic stirrer because it will take some time for the reagents to dissolve completely. The composition of the final solution will be 0.1M Na2B4O7 and 0.075M NaOH. 0.5M Sulfuric Acid: Slowly pipette 2.8mL of concentrated (95-98%) H2SO4 into 100mL of DI while stirring on a magnetic stirrer. Phenolphthalein pH indicator: Purchase or make 1% (w/v) phenolphthalein indicator solution in 60% (v/v) isopropyl alcohol (e.g., Fisher Scientific, Cat. No. 5625616). - 7 - 4.3 15N-NH4 + Standards Three internal 15N standards are diffused in duplicate or triplicate with each batch of samples. These (NH4)2SO4 standards, EGC-3, EGC-14, and EGC-15, were previously calibrated against international 15N standards and have 15N-NH4 + values of 0.60‰, 21.87‰, and 35.18‰, respectively. Each of the 15N standards is prepared by adding 5.7mg of the (NH4)2SO4 standard salt to 200mL of acidified 4M KCl solution. Standard stock solutions are kept refrigerated. 5 CLEANING OF GLASSWARE AND EQUIPMENT Since very small amounts of NH4 + are diffused onto each acidified disk, it is crucial that all glassware, reagents, and equipment be free of other NH4 +. All glassware and non-metallic equipment used in this method are cleaned using the following procedure: 1) Rinse items with tap water to remove particulates and residual solutions. 2) Soak items in lab soap bath overnight. 3) Remove items from soap bath, scrub with brush, and rinse off soap residue with cold tap water. 4) Place items in a 10% HCl bath overnight. 5) Remove items from HCl bath and place in a bath of fresh DI water for at least 15 minutes. 6) Remove items from DI bath, rinse with DI, then place in a 0.02M NaOH bath for at least one hour. 7) Remove items from NaOH bath and place in a bath of fresh DI water for at least 15 minutes. 8) Remove items from DI bath then rinse thoroughly with Milli-Q DI. 9) Place items on clean lab bench paper to air dry. Alternatively, items can be oven dried. 10) Once clean, keep all equipment used for the δ15N-NH4 + technique in designated bins with lids, separate from other glassware and supplies. 6 PRE-DIFFUSION CALCULATIONS Ideally, approximately 30 g of NH4 +-N is diffused onto each acidified quartz disk for optimal peak height on the mass spectrometer. In reality, as little as 12 g N will still provide sufficient response on the Deltaplus IRMS (Section 11) to determine accurate δ15N-NH4 + values. Therefore the lower concentration limit for this technique (using the 60mL diffusion jars) is 0.6 mg N/L. The salinity in each diffusion jar is adjusted to approximately 4M using KCl to reduce the tendency for condensation to form in the diffusion jars; to prevent the PTFE traps from - 8 - absorbing water and bursting; and increase the speed of the diffusion process. The final volume in each diffusion jar is approximately the same so there is no significant volume effect on diffusion times. A spreadsheet has been designed to calculate the amount of sample, KCl salt, DI, and 4M KCl solution to be added to each diffusion jar (Appendix A). The spreadsheet contains a “Read Me” tab that explains each of the calculations. The calculations are also explained in brief below. 6.1 Sample Volume 1) Using Equation 3, calculate volume of sample (Vs) that contains 30 g NH4 +-N required for each diffusion disk. 2) If Vs <= 20mL, the diffusion procedure can be carried out in the 60mL jars. Note that Equation 3 calculates the volume that contains the ideal N amount (30g N) not the minimum amount (12g N). 6.2 KCl addition i) If Vs <= 1mL, the sample is added to 20mL of 4M KCl solution in a 60mL jar. ii) If Vs > 1mL and <= 5mL, the sample is added to (20mL - Vs) of 4M KCl solution in a 60mL jar. iii) If Vs > 5mL and <= 20mL, the sample is added to (20mL - Vs) of DI in a 60mL diffusion jar. KCl(s) is then added to the jar to make the KCl concentration of the solution approximately 4M. Equation 4 is used to calculate the amount of KCl(s) to add to the sample + DI solution. Equation 4 takes into account the initial KCl concentration of the sample, which may not always be zero (e.g., 2M KCl soil NH4 + extracts).       N/LmgNH Nmg03.0 V N/Lmgion ConcentratNHSample NmgAmount NHTarget L)(in Required)(VVolumeSample 4 s 4 4 s                                        mol KClg 55.74 L KClmol 4 1000mL 1L (mL)V mol KClg 55.74 L KClmol [KCl] L KClmol 4 1000mL 1L (mL)V(g)add toKCl DI is                                    1000mL 1L mol KClg 55.74(mL)V4 L KClmol [KCl] L KClmol 4(mL)V DIis (Eqn. 3) (Eqn. 4) - 9 - 7 PREPARING THE PTFE TRAPS 1) Create a clean working surface by placing a piece of aluminum foil on the lab bench and wiping it with ethanol. 2) The PTFE tape should be cleaned and dried ahead of time using the cleaning procedure described in Section 5 (without the lab soap step). 3) Cut the PTFE into 3cm sections, at least one for each diffusion jar. 4) Fold each piece in half, crease the fold, and reopen (Figure 1A). 5) Fold a pre-baked quartz filter disk in half to make a “tent” (Figure 1A). This step helps keep some air in the PTFE trap, which helps the trap stay afloat in the diffusion jar. 6) Place the filter disk on one half of the creased PTFE strip (Figure 1A). 7) Pipette 10L of 0.5M H2SO4 onto the filter disk. 8) Gently fold the PTFE strip over itself along the crease. Take care not to flatten the filter disk. 9) Seal the two halves of the PTFE trap together by firmly pressing the open end of a vial on the trap, with the acidified filter disk in the center (Figure 1B, 1C). 10) Place the trap in a clean, tightly capped container until ready to use. Storage tests indicate that PTFE traps can be stored in an airtight container for at least 2 months. Figure 1, PTFE acid traps prior to crimping (A), during closing with the mouth of a glass vial (B) and a finished trap (C). 8 PREPARING THE DIFFUSIONS 1) Create a clean working surface by using new bench paper or piece of aluminum foil. 2) Label each diffusion jar with a unique identifier (e.g., JS-EC-####). 3) Add the required amounts of KCl, sample, DI or 4M KCl solution to the diffusion jars. 4) Add two drops of the phenolphthalein indicator solution to each diffusion jar. 5) Cap each jar after the reagents are added. 6) Place jars on an orbital shaker and shake gently for 15min to dissolve and mix reagents. A B C - 10 - 7) While mixing gently by hand, add the 0.2M NaOH drop wise (e.g., by syringe) until the sample solution turns bright pink. 8) Immediately after the sample solution turns pink, add 2mL of tetraborate buffer solution (e.g., by repeater pipette or bottle-top dispenser), insert the PTFE trap, and cap the diffusion jar. 9) Repeat steps 7 and 8 for each diffusion. 10) Place diffusion jars on an orbital shaker on low speed to gently mix while diffusing. 11) Allow samples to diffuse on the shaker for at least 10 days to ensure complete collection of NH4 + onto the acidified filters. 9 COLLECTING THE ACIDIFIED DISKS 1) Label a clean 1dr vial for each diffusion jar. Rinse two pairs of tweezers with DI and dry with a clean Kimwipe. Always rinse the tweezers before handling EACH filter, even if it is from the same sample (i.e., the duplicate). 2) Take the diffusion jar from the shaker and remove the cap. 3) Using the clean tweezers, take the PTFE trap out of the diffusion jar. 4) Rinse the trap and tweezers with DI to remove the diffusion solution. 5) Using a second pair of tweezers, unfold the trap (Figure 2). 6) Place the acidified disk into a labeled 1dr vial using clean tweezers. Figure 2, Removing the acidified disk from the PTFE trap following diffusion. 7) Note any anomalies with the disk or solution (e.g., PTFE trap already open, disk wet, solution not pink). 8) Repeat steps 1 to 7 for each diffusion jar. - 11 - 9) Pour the sample solution into a labeled waste container. Rinse the diffusion jars with tap water and set aside for washing. 10 FREEZE DRYING THE ACIDIFIED DISKS 1) Stand the 1dr vials in a tray and place in a freezer for at least two hours. 2) Loosen the vial caps but do not remove completely. 3) Place tray in freeze dryer and dry overnight. 4) Once dry, remove the vials from the freeze dryer, cap tightly, and submit for δ15N analysis by EA-IRMS. 11 ISOTOPIC ANALYSIS The N isotopic composition of the diffusion disks containing the NH4 + as (NH4)2SO4 is determined at the Environmental Isotope Lab, University of Waterloo, using a Carlo Erba 1108 Elemental Analyzer interfaced with a Thermo Instruments Deltaplus isotope ratio mass spectrometer (EA-IRMS) (Thermo Fisher Scientific, Milan Italy). NH4 +-N isotope ratios are reported in delta (δ) notation relative to atmospheric N2. The precision associated with the δ15N- NH4 + analysis of samples is generally better than ±0.2‰. Duplicate samples that vary by more than 0.5‰ are rerun. 12 CORRECTION OF THE ISOTOPE DATA A linear correction, based on the diffused standards within the run is applied to all samples in order to account for any blank effects resulting from the diffusion process. Typically, values of diffused isotopic standards are not significantly different from the same standard (NH4)2SO4 material run directly on the EA-IRMS as a pure salt (i.e., there is typically only a minor diffusion or blank effect) (Figure 3). A comparison of the amount of N expected on the quartz disk to the amount calculated from the EA-IRMS data is done as a check. A significant discrepancy between the two amounts can be due to several factors: 1) Inaccurate NH4 +-N concentration value for the original sample. 2) Contamination of glassware or reagents (more N on disk than expected). 3) Incomplete diffusion of NH3 onto the disk (less N on disk than expected and lower δ15N value because of faster diffusion rate of 14NH3). 4) A change in the NH4 +-N concentration caused by microbial activity or volatilization due to improper preservation or storage (e.g., if samples are not kept frozen during transit/storage). - 12 - Figure 3, Typical correction plot of diffused standards that is used to account for any blank or diffusion effect on sample δ15N values. y = 1.0291x - 0.3308 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 Machine d 15N value of diffused standards (‰) T ru e st a n d a rd d 1 5 N v a lu e (‰ ) - 13 - 13 RECOMMENDED READING Brooks, P.D., J.M. Stark, B.B. McInteer, and T. Preston. 1989. Diffusion method to prepare soil extracts for automated nitrogen-15 analysis. Soil Sci. Soc. Am. J. 53: 1707. Downs, M.R., Michener, R.H., Fry, B., Nadelhoffer, K.J. 1999. Routine measurement of dissolved inorganic 15N in precipitation and streamwater. Environmental Monitoring and Assessment 55: 211-220. Holmes, R.M., McClelland, J.W., Sigman, D.M., Fry, B., Peterson, B.J. 1998. Measuring 15N- NH4 + in marine, estuarine and fresh waters: an adaptation of the ammonia diffusion method for samples with low ammonium concentrations. Marine Chemistry 60: 235-243. Khan, S.A., R.L. Mulvaney, and P.D. Brooks. 1998. Diffusion methods for automated nitrogen- 15 analysis using acidified disks. Soil Science Society of America Journal 62:406-412. Khan, S.A., Mulvaney, R.L., Hoeft, R.G. 2001. A simple soil test for detecting sites that are nonresponsive to nitrogen fertilization. Soil Science Society of America Journal 65: 1751-1760. Khan, S.A., Mulvaney, R.L., Mulvaney, C.S. 1997. Accelerated diffusion methods for inorganic- Nitrogen analysis of soil extracts and water. Soil Science Society of America Journal 61: 936- 942. Lehmann, M.F., S.M. Bemasconi, and J.A. McKenzie. 2001. A method for the extraction of ammonium from freshwaters for nitrogen isotope analysis. Analytical Chemistry 73: 4717-4721. Moran, K.K., Mulvaney, R.L., Khan, S.A. 2002. A technique to facilitate diffusions for nitrogen- isotope analysis by direct combustion. Soil Science Society of America Journal 66: 1008-1011. Schleppi, P., Bucher-Wallin, I., Saurer, M., Jäggi, M., Landolt, W. 2006. Citric acid traps to replace sulphuric acid in the ammonia diffusion of dilute water samples for 15N analysis. Rapid Communications in Mass Spectrometry 20: 629-634. Sebilo, M., Mayer, B., Grably, M., Billiou, D., Mariotti, A. 2004. The use of the 'ammonium diffusion' method for 15N-NH4 + and 15N-NO3 - measurements: comparison with other techniques. Environmental Chemistry 1: 99-103. Sigman, D.M., Altabet, M.A., Michener, R., McCorkle, D.C., Fry, B., Holmes, R.M. 1997. Natural abundance-level measurement of the nitrogen isotopic composition of oceanic nitrate: an adaptation of the ammonia diffusion method. Marine Chemistry 57: 227-242. Sorensen, P., Jensen, E.S. 1991. Sequential diffusion of ammonium and nitrate from soil extracts to a polytetrafluoroethylene trap for 15N determination. Analytica Chimica Acta 252: 201-203. - 14 - 14 APPENDIX A Figure A1, Layout of the δ15N-NH4 + spreadsheet that calculates the amount of sample and reagents to use. - 15 - Figure A2, Layout of the δ15N-NH4 + spreadsheet showing the formulas used (continued on next page). - 16 -