PROTOCOL FOR DIFFUSING INORGANIC N FOR DETERMINING 15N/14N BY MASS SPECTROMETRY
5 May, 1998, S. E. Hobbie
Reference
Stark, J. M. and S. C. Hart. 1996. Diffusion technique for preparing salt solutions, Kjeldahl digests, and persulfate digests for nitrogen-15 analysis. Soil Sci. Soc. Am. J. 60: 1846-1855
Materials Needed
3/4" (12.5 mm) teflon (PTFE) tape (7-8 cm strip/sample)
ruler
filter paper (2 7-mm discs/sample)
preleach Whatman No. 1 filter paper by placing it in Buchner funnel; leach
with 4 50-ml aliquots of 2 M KCl, followed by 6 50-ml aliquots of DI water,
followed by oven drying.
paper punch
120-ml specimen cups (Fisher 14-375-148)
stainless steel wire
14 mm plastic culture tube (w/ rounded edges)
scoop for MgO (0.2 g)
scoop for Devarda's alloy (0.4 g)
forceps
dessicator
styrofoam blocks
tin capsules (5 x 8 mm)
pipette (5-10 µL)
gloves
new paper clips
microtiter plates
Chemicals Needed
2.5 M KHSO4 (10 µL/sample)
make by carefully adding 7 ml of conc. H2SO4 to 50-ml DI H2O; add 22 g K2SO4,
add more DI H2O; mix until salt is dissolved;
bring to 100 ml final volume.
Devarda's Alloy (0.4 g/sample, KCl only), finely ground (40-mesh)
MgO (0.2 g/sample)
concentrated H2SO4
concentrated NaOH (1:1 NaOH:H2O by weight)
Sample Preparation
Instrument Blanks
1. Impale filter disks onto steel wires.
2. Pipette 5 µL 2.5 M KHSO4 onto 7-mm filter disks. Place in a dessicator with conc. H2SO4 for 4 h.
3. After drying, wrap disks into tin capsules, 2 disks/capsule.
Samples: General Instructions (Read specific instructions, below, first): 30-40 samples per hour.
1. Before diffusing, measure out the volume of sample necessary to achieve:
20-100 µg N at 10-30 atom %.
100-200 µg N at 1-10 atom %.
(note: 1 ml of 0.5 M K2SO4 = 1.087135 g, 1 ml of 2 M KCl = 1.14912 g, 1 ml of Vitousek lab digest = 1.08632 g)
2. Wearing gloves, smooth out teflon tape on a non-slick surface (such as lab bench paper).
3. Pre-scewer filter discs in bulk by punching a hole in an overturned specimen cup, spearing filter paper disks in batches onto a stainless steel wire, and unloading them using a clean toothpick for storage into a clean specimen cup.
4. Place two pre-scewered filter disks on one-half of the teflon tape, 3 cm apart.
5. Pipette 5 µL of 2.5 M KHSO4 onto each disc. (trapping capacity is 350 µg N total; never exceed 50-60 % of this).
6. Fold the other half of the teflon tape over the first half, and smooth it out.
7. Press down in a concentric circle around each disc FIRMLY (but DO NOT TWIST) with the open end of a 14-mm plastic culture tube, to seal the teflon tape.
Batches of teflon tape with filter paper may be stored in a closed specimen cup until ready to add to diffusions.
8. Simultaneously place one teflon tape trap (containing 2 filter discs) and 1 scoop MgO (or stronger base, if diffusing digests) and/or devarda's alloy (see below) into specimen cup containing sample. Cap immediately. Swirl and invert 5 times.
9. Let samples diffuse for 6 days at room temperature (22°C), inverting daily to prevent the formation of competing acid trap droplets on the specimen cup walls. Make sure that the acid trap doesn't get stuck to the lid.
10. After diffusing, remove trap from sample with forceps, rinse with DI into a specimen cup, and place on blotting paper. Gently peel back teflon tape with forceps. Center filter disk on top of overturned specimen cup with hole in it. Impale disk on stainless steel wire; repeat with second disk, impaling onto same wire (if possible--sometimes it's easier to use separate wires for each disk); dry in dessicator with conc. H2SO4 for 4 h. After drying, wrap both disks in a 5 x 8 mm tin capsule.
Note: if leakage affects one of the traps, the other trap can still be used by itself, but you won't be able to tell if leakage has occurred. Therefore, it's very important not to let the filter paper disks touch each other until they are dry.
Digested K2SO4 Samples
1. Along with acid trap, add 7-8 ml of concentrated NaOH (1:1 NaOH:H2O by weight) instead of the MgO. This will get the pH high enough (14-15) to deaminate the NH4.
KCl Extracts
For samples to be diffused for 15NH4.
1. Along with acid trap, add 0.2 g scoop of MgO.
For sample to be diffused for 15NO3.
1. Add 0.2 g scoop of MgO. After mixing, remove lid and allow cup to sit open for 4 d. Cap and invert 5 times daily, opening lid after each mixing (or alternately, swirl the cup once daily).
2. After 5 d, add 0.4 g scoop Devarda's Alloy and 0.2 g scoop of MgO along with acid trap. Cap. Invert 5 times. Let sit 6 d, inverting daily.
Extraction/Digestion/Diffusion Blanks
1. Diffuse extraction blanks as though they were samples. Determine the mass of N diffused by adding up all the beams on the mass spec. output.
2. For KCl extracts, run 3 blanks for each batch of KCl used.
3. For digested K2SO4 extracts, run 1-2 blanks for each digestion rack.
4. Use the following equation to correct samples for dilution of the 15N/14N ratio by unenriched N from contamination.
Es = (EmMs+b - MbEb)/(Ms+b - Mb)
where Es = corrected enrichment of the sample, Em is the enrichment of the of the sample + blank as measured by mass spectrometry, Ms+b is the mass of N (sample + blank) recovered in the acid trap (µg), Mb is the mass of N recovered in the acid trap from the diffusion blank, and Eb is the enrichment in the blank (assumed to be 0.366 atom %, natural abundance). All N mass is calculated as if all N were 14N.
Standards: General Considerations
1. It's important to match the MASS of N in your sample with the mass of N in your standard. It's less important to match the atom %15N of your sample with that of your standard.
2. Make 2 types of standards: diffused standards and non-diffused standards.
3. For both, first make a 10,000 ppm (10,000 mg N/L) stock solution that is 1 atom % 15N. [e. g., 46.7174 g (14NH4)2SO4/L and 0.4438 g (15NH4)2SO4/L].
Non-Diffused Standards: Use the stock solution
1. Place 2 filter paper disks onto a stainless steel wire and place in styrofoam.
2. Pipette 5 µL of 2.5 M KHSO4 onto each disk.
3. Pipette in enough 10,000 ppm stock to give you the desired mass of N.
a. For standards to receive = 60 µg N, pipette half of the total volume of standard stock solution onto each disk.
b. For standards to receive = 50 µg N, pipette the entire volume of standard stock solution onto the top disk.
4. Dry in dessicator over conc. H2SO4 overnight and wrap in both disks into one tin capsule.
Diffused Standards: Dilute the stock solution by 10.
1. Make a 1,000 ppm (1,000 mg N/L) solution from the 10,000 ppm stock.
2. Measure out a 40 ml volume of 2 M KCl for each standard.
3. Pipette in enough 1,000 ppm standard to give you the desired mass of N.