Inorganic N Isotopes from KCl Soil Extraction Protocol

INTRODUCTION

This protocol describes the process to measure ammonium (NH4+) and nitrate (NO3) in soil extractions using potassium chloride (KCl). Soil extractions (in KCl) are incubated in Erlenmeyer flasks with 2 acid-trap filter packets (one at a time, for 6 days each) and sealed to the atmosphere. During the first week of incubation, magnesium oxide (MgO) is used to convert dissolved ammonium (NH4+) to gas, which diffuses onto the filter pack. On day 7, NH4+ filter packs are removed, and samples are allowed to vent for 24 hours. During the second week of incubation, Devarda’s alloy is used to convert nitrate (NO3) to NH4+, and MgO is again used to convert NH4+ to a gaseous form, which diffuses onto the second filter pack. Filters are then packed for analysis on IRMS.

SAFETY 

Familiarize yourself with the MSDS for all chemicals prior to starting this protocol. Devarda’s alloy is highly flammable and incompatible with strong oxidizing agents. Store in the flammables cabinet beneath the fume hood in room 232. Ammonium chloride and potassium nitrate are both oxidizing agents that will cause eye irritation and may be harmful if swallowed. Potassium chloride, potassium bisulfate, and magnesium oxide will also cause eye and skin irritation. Familiarize yourself with these chemicals’ SOPs before beginning. While this protocol does not need to be conducted in the fume hood, always wear proper protective equipment, including nitrile gloves. Face masks may be recommended while weighing out chemicals to prevent accidental ingestion.

MATERIALS

  • Aluminum foil
  • Laboratory tape
  • Filter forceps
  • Laboratory scissors
  • TaegaSeal PTFE Tape, Premium Military Grade, 1” wide, red label
  • 20-mL scintillation vial
  • One clean 125 mL Erlenmeyer flask + no. 5 black butyl stopper per sample
  • 100-mL graduated cylinder
  • Parafilm, cut into 2×1 strips
  • Laboratory spatula
  • Alcohol (Ethanol or Acetone)
  • Kimwipes
  • Large-well stable isotope sample cassette
  • Small-well stable isotope sample cassette
  • 9 x 5 mm Tin stable isotope capsules
  • Scalpel

PREPARING REAGENTS AND SUPPLIES

Ashed MgO – Combust in muffle furnace at 650 °C for 4 hours in a glass low-form weighing bottle. Remove hot and place in desiccator to cool before recapping. Plan for 0.6 g MgO per sample.

Filters — Cut 1 cm diameter glass fiber (GF) filters “punches” from larger 47 mm GF filters using a sharp coring tool. Combust filter punches at 450 °C for 2 hours in a glass low-form weighing bottle. Remove hot and place in desiccator to cool before recapping. Store in a desiccator along with a 100 mL beaker containing ~20 mL 2.5 concentrated H2SO4 (acid trap). You will need two filter punches per sample + extras.

Alternatively, you may use pre-“punched” 10mm Absorbent Discs (filters), made by Elemental Microanalysis. Prepare these punches in the same way described above: combust at 450°C for 2 hours in a glass low-form weighing bottle. Remove hot and place in desiccator to cool before recapping. Store in a desiccator along with a 100 mL beaker containing ~20 mL 2.5 concentrated H2SO4 (acid trap).

2.5 M KHSO4 — Weigh 34.04 g of potassium bisulfate (KHSO4) and add to a 100 mL volumetric flask. Add roughly 50 mL of 18.2 MW (nanopure) water to dissolve KHSO4. Once dissolved, fill to mark and decant to labeled media bottle. Be sure to update the concentration using the actual mass.

2 N KCl – Weigh 149 g of potassium chloride (KCl), and add to a 1000 mL volumetric flask. Add 800 mL of 18.2 MW (nanopure) water to dissolve KCl. Once dissolved, fill to mark and decant to labeled media bottle.

0.101 mg N/mL KNO3 Standard – Weigh 72.9 mg of potassium nitrate (KNO3), and add to a 100 mL volumetric flask. Add roughly 50 mL of 18.2 MW (nanopure) water to dissolve KNO3. Once dissolved, fill to mark and decant to labeled media bottle. Be sure to update the concentration using the exact mass of KNO3 measured.

0.099 mg N/mL NH4Cl Standard – Weigh 37.8 mg of ammonium chloride (NH4Cl), and add to 100 mL volumetric flask. Add roughly 50 mL of 18.2 MW (nanopure) water to dissolve NH4Cl. Once dissolved, fill to mark and decant to labeled media bottle. Be sure to update the concentration using the exact mass of NH4Cl measured.

MAKING ACID TRAP PACKETS (DAY 0)

  1. Spread out aluminum foil over the lab bench. Use lab tape to secure each corner. Clean forceps and aluminum foil surface with alcohol and let dry.
  2. Cut ~2” strip of Teflon tape (1” width, red label) using alcohol-cleaned scissors.
  3. Place GF filter on one side of the Teflon tape strip and center top to bottom. Pipette 100 μL of 2.5 M KHSO4 onto filter (it must be completely absorbed by the filter).
  4. Fold the other half of the Teflon on top of GF filter using forceps. Work quickly.
  5. Seal Teflon pack holding acidified GF filter by rolling the open end of a 20-mL scintillation vial around the outside portion of the Teflon filter. To ensure that the filter pack remains stuck together, you can also create another ring around the filter with a smaller diameter glass. You should notice a thinning of the Teflon filters around the edge where it is sealed. Hold the filter pack up to the light to verify this.
  6. Place fresh filter packets into a clean glass weighing bottle. When done, flush with argon, and cap.

*** It is best to make up filter packets within a few days of use. If necessary, filter packets can be stored for several weeks, but it’s critical that they be tightly capped in an air-free environment. ***

DEFROST SOIL EXTRACTION SAMPLES (DAY 0)

  1. Remove soil extraction samples from freezer and allow them to defrost on a laboratory countertop overnight. We have the capacity to process 35 flasks at a time. This accommodates 29 samples, 3 standards, and 3 blanks per batch.

DIFFUSE AND CAPTURE AMMONIUM (NH4+)(DAY 1)

  1. Pre-label one clean 125 mL Erlenmeyer flask per sample with each sample’s unique identifier using label tape and a sharpie.
  2. Use an acid-washed 100-mL graduated cylinder to measure the total KCl volume of the first sample.
  3. Record the total KCl volume (in mL) on the datasheet.
  4. Pour the total KCl volume into the pre-labeled Erlenmeyer flask.
  5. Repeat steps 2-4 for each sample, rinsing the cylinder with DI water in between each sample.
  6. Working quickly, add 300mg of MgO and an acid-trap pack to the first sample.
  7. Stopper that flask with a 5 black butyl stopper and wrap the neck of the flask (where the flask and the stopper meet) in parafilm.
  8. Repeat steps 6-7 for each sample, standard, and blank.
  9. Place flasks on shaker table (room 235), and start shaking. Record date and time that shaking was initiated. Samples will shake continuously for the next 6 days.

PREPARE BLANKS AND STANDARDS (DAY 1)

  1. Fill six (6) flasks with 2N KCl. Match the volume to the volume of KCl from your samples (typically 45 – 65 mL).
  2. Mark three of the flasks as ‘Blanks’ as set aside for the next step.
  3. Mark the remaining three flasks as ‘STDs’. Pipette 505 µL of 0.099 mg N/L ammonium chloride (NH4Cl) standard into each flask. Next add 495 µL of 0.101 mg N/L potassium nitrate (KNO3) to each standard flask.

RETRIEVE AMMONIUM (NH4+) FILTERS (DAY 7)

  1. Pre-label a large-well stable isotope cassette with “NH4 Filters” and your batch identifier.
  2. Remove samples from shaker table.
  3. Unwrap the parafilm and remove the rubber stopper from your first sample. Take care to keep each stopper with its respective flask for re-use.
  4. Using a laboratory spatula and filter forceps, remove the acid-trap filter pack from the first sample. Rinse any clumps of MgO from the filter pack as you remove the filter from the flask.
  5. Place the filter pack in the large-well cassette, and record the well position (well ID) for that sample.
  6. Clean the spatula and forceps with alcohol and a Kim wipe in between each sample.
  7. Repeat steps 3-6 for each sample.
  8. When all filter packs have been removed, secure the lid of the large-well cassette using lab tape, and store it in the desiccator with an open vial of 2.5 M KHSO4 (to absorb any ammonium in the air).
  9. Return all Erlenmeyer flasks to the shaker table (sans parafilm and rubber stopper) and resume shaking overnight. This allows any remaining ammonium (NH4+) to vent to the atmosphere before continuing with nitrate (NO3) diffusion.

*** Recommended: Assemble the necessary number of acid-trap filter packs for nitrate diffusion and capture (below), beginning on Day 8 (assembly instructions above). ***

DIFFUSE AND CAPTURE NITRATE (NO3) (DAY 8)

  1. Remove samples from shaker table.
  2. Working quickly, add 300 mg of MgO, 75 mg of Devarda’s alloy, and an acid-trap pack to the first sample. NOTE: It is helpful to use weigh boats on both scales in room 232 to simultaneously weigh out ~300 mg of MgO and ~75 mg of Devarda’s alloy.
  3. Stopper that flask with a 5 black butyl stopper and wrap the neck of the flask in parafilm. Rubber stoppers may be re-used as long as they are used on the same flask for both diffusion periods.
  4. Repeat steps 2-3 for each sample, standard, and blank.
  5. Return flasks to shaker table (room 235), and start shaking. Record date and time that shaking was initiated. Samples will shake continuously for the next 6 days.

RETRIEVE NITRATE (NO3) FILTERS (DAY 14)

  1. Pre-label a large-well stable isotope cassette with “NO3 Filters” and your batch identifier.
  2. Remove samples from shaker table.
  3. Unwrap the parafilm and remove the rubber stopper from your first sample.
  4. Using a laboratory spatula and filter forceps, remove the acid-trap filter pack from the first sample, taking care to rinse any clumps of MgO and Devarda’s alloy from the filter pack as you remove it from the flask.
  5. Place the filter pack in the large-well cassette, and record the well position (well ID) for that sample.
  6. Clean the spatula and forceps with alcohol and a Kim wipe in between each sample.
  7. Repeat steps 3-6 for each sample.
  8. When all filter packs have been removed, secure the lid of the large-well cassette using lab tape, and store it in the desiccator with an open vial of 2.5 M KHSO4 (to absorb any ammonium in the air).

PREPARE FILTERS FOR ANALYIS BY IRMS

**Each punch filter will need to be packed into a 9 x 5 mm tin sample isotope capsule for analysis by IRMS. Since isotope values will be based on the surface area of the punch filter (rather than sample mass), it is not necessary to use the microbalance. Packing samples may take place in room 232.

  1. Let filter packs dry in desiccator for 3-4 days following retrieval from Erlenmeyer flasks.
  2. Spread out aluminum foil over the lab bench. Use lab tape to secure each corner. Clean forceps, scalpel, and aluminum foil surface with alcohol and let dry.
  3. Use the scalpel to open the filter pack, and use forceps to extract the punch filter from the Teflon tape pocket.
  4. PRO TIP: Simply stuffing the punch filter in the tin capsule and attempting to fold the capsule will cause it to tear. Instead, use the forceps to carefully tear the punch filter into quarters, and place each quarter into the capsule.
  5. Use forceps to fold the tin capsule. Make sure no filter material has escaped or can escape. The folded capsule should be small enough to drop into small-well isotope cassette wells freely.
  6. Note the well ID for each sample.
  7. When all samples have been packed, secure the lip of the isotope cassette with label tape and store it in the desiccator with an open vial of 2.5 M KHSO4 (to absorb any ammonium in the air).

WASTE

When the final filter packs have been retrieved, the contents of each Erlenmeyer flask may be dumped into an empty waste jug with the following label:

Chemical Composition %
Potassium chloride (KCl) 1.9 L
Magnesium oxide (MgO) 21 g
Devarda’s alloy, granular 2.6 g

The approximate amounts described above reflect a full batch of samples (n = 35). When the waste jug is 4/5 full, submit an Online Chemical Waste Collection request.

CLEANING & RECYCLING

Erlenmeyer flasks and black butyl stoppers should be cleaned following the HEEL Glass- & Plastic-ware Cleaning protocol. Parafilm may be disposed of in the trash.

REAGENTS & SUPPLIES

REFERENCES

Sigman, D. M., M. A. Altabet, R. Michener, D. C. McCorkle, B. Fry, and R. M. Holmes. 1997. Natural abundance-level measurement of nitrogen isotopic composition of oceanic nitrate: an adaptation of the ammonia diffusion method. Marine Chemistry 57:227-242.

Holmes, R. M., J. W. McClelland, D. M. Sigman, B. Fry, and B. J. Peterson. 1998. Measuring 15N-NH4 in marine, estuarine and fresh waters: an adaption of the ammonium diffusion method for samples with low ammonium concentrations. Marine Chemistry 60:235-243.

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