Diffusion protocol for determining 15N/14N using mass spectrometry
By Amber Moody
Table of Contents:
Introduction
2
Diffusion questions
3-5
Supplies for entire process
6
Preparing Whatman #1 filters
7
Mixing 2.5M KHSO4
8
Mixing standard stock solution
9
Digestion/oxidation protocol
10
Diffusion set-up
11
Supplies
11
Sample preparation
11-12
Blank preparation
12
Standard preparation
12-13
Teflon trap preparation
13
Starting the diffusions
13-14
Harvesting the diffusions
14-15
Rolling disks for mass spectrometry
15-16
1
Introduction:
Nitrogen is present in soil extracts in the form of dissolved inorganic nitrogen (DIN:
ammonium (NH4+) and (NO3-)) and dissolved organic nitrogen (DON). We use the
following diffusion technique to determine the amount of nitrogen that is present in the
form of 15N isotope relative to 14N. To do this we diffuse nitrogen from solution onto a
filter paper that is subsequently dried, rolled in tin capsules, and run on the mass
spectrometer which will give us the proportion of 15N to 14N.
Diffusing NH4+: We increase the pH of the solution to greater than 13 using magnesium
oxide (MgO). The high pH of the solution pushes the following equation to the far right:
NH4+ + OH- ↔ NH3 + H2O and the ammonium is converted to ammonia. Ammonia is a
gas at temperatures above –33.5oC so it diffuses out of solution through the Teflon tape
of the acid trap and the highly acidic filter paper converts the ammonia back to
ammonium and the ammonium is “trapped” on the filter paper. We harvest the filter
paper after 4-7 days of diffusion and dry the filters in an acidic environment. The filter
papers are then rolled in tin capsules and run on the mass spectrometer to determine the
relative amounts of 15N to 14N.
Diffusing NO3-: Once we have diffused the ammonium, we can then diffuse the other
component of DIN – NO3-. To do this we want to make sure there is no more ammonium
left in solution, so you will increase the pH to greater than 13 and allow the bottles to be
open to the atmosphere allowing any remaining ammonia to escape. Afterwards we will
add another scoop of MgO (to make sure the pH is greater than 13) and a scoop of 0.4g
devardas alloy. The nitrate reacts with devardas alloy and is converted to ammonium
which is immediately converted to ammonia due to the high pH of the solution. Once it
is in the form of ammonia it follows the same route as described above.
Diffusing digested samples: By this point all of your samples should have been digested
(see digestion protocol) which converts all forms of nitrogen in solution including the
organic N into NO3-. Then we use NaOH to increase the pH above 13. We add
devarda’s alloy to the sample and the reaction proceeds as described for diffusion NO3-.
2
Diffusion Questions:
The following is a letter we wrote to a chemist, Dr. Tom Clausen, with questions about
some results we were getting on some diffusions and digestions we were doing. First is
the letter with the questions and then Dr. Clausen responds to our questions below that.
Dr. Clausen,
There are two general procedures that we have questions on.
The first is a diffusion procedure we are using on soil extractions in order to determine
how much 15N is in our soils. The method we use is based on a method outlined in Stark
and Hart, 1996 (I will send you this paper along with another that has to do with our
second topic). Before running our samples we set up a series of tests to determine how
much of the NH4+ and NO3- we can expect to recover. The tests were set up in 75 ml of
0.5M K2SO4 with a nitrogen spike that contained 30µg of N in the form of NH4+ and
30µg in the form of NO3-. The diffusions are done in two steps, first we diffuse the NH4+
by converting it to NH3 and then we diffuse the NO3- by first converting it to NH4+ and
then to NH3. To trap the NH3 from the first diffusion we added two small filter disks
encased in Teflon tape with 5µl of KHSO4 on each disk. We then added a scoop of
~0.2mg of MgO and capped the bottle and shook it once a day for seven days. After
seven days we harvested the Teflon traps and ran then disks on a CN analyzer to
determine how much of the 30µg of N we were able to trap. We then set up the same
solution for NO3- diffusion by adding a new Teflon trap, a scoop of Devarda’s alloy to
convert the NO3- to NH4+, and then an additional scoop of MgO. These were allowed to
diffuse for 7 days and then harvested and analyzed on a CN analyzer as well. For all of
these tests we only recovered ~30% of the nitrogen spike that we had added for both the
NH4+ and the NO3-. Published papers report a recovery rate of >90%. At this point we
set up another series of tests to determine why we did not get a better recovery. The short
of that story is that if we add ~8.4 grams of KCl to the sample we get recovery around
90%. Stark and Hart say they added the KCl to “prevent swelling of the acid trap.”
Clearly the KCl is doing more than just preventing swelling.
Our first question is what is the mechanism behind a higher recovery rate when KCl
is added?
Our second question is: Is there a chemical reason we should use MgO rather than
NaOH to get the pH high enough to do the diffusions? The same method calls for
NaOH to increase the pH when doing diffusions on samples the have been digested using
persulfate oxidation method.
When we set up our soil extraction samples for diffusion, we added a 10µg spike of NH4+
and 10µg of NO3- to the extract. We diffused the samples twice for NH4+, the second
time adding the KCl (to increase our recovery of NH4+). Then we set them up for the
NO3- diffusions by adding a third scoop of MgO and a scoop of Devarda’s alloy. We
were dismayed to find that almost all of the samples had exploded over the weekend.
3
The volumes of the samples, standards and blanks ranged from 25-100ml and everything
exploded except for our standards. Our standards consisted of 0.5M K2SO4, the N spike,
~0.6mg MgO, and 0.2mg Devarda’s Alloy. The blanks also exploded. They were set up
exactly the same as the standards, but they did not have the nitrogen spike. We were
hoping you would have some idea why these samples exploded?!?
When we are oxidizing organic samples, we use the following recipe to make up our
oxidizing reagent: 50g K2S2O8, 30g HBO4, and 100ml 3.75 NaOH in 1L water. This
solution is mixed 50:50 with our extracts and then placed in the autoclave for 30min.
We know that persulfate is used to oxidize organic matter to NO3- and CO2. We also
know that NaOH is used to increase the pH to a range that allows the analysis of NO3following the digestion. But we were wondering what is the function of the boric
acid? The other thing we would like to know about these persulfate oxidations is
why does the pressure increase so drastically (we have had a bottle of the oxidation
solution explode in a drying oven; we have since put much less of the solution in each
bottle to prevent the same from happening again, but are still curious as to why)?
I will send those two papers I mentioned earlier your way. Well, I hope that all of this
makes some sense to you. If anything is still not clear about our procedures, please feel
free to ask!
Thanks again!
Dr. Clausen’s response:
The background and questions you posed to me were very well thought out. It
really helped in my getting a grip on the problems on hand. Thanks for your extra
effort!
Question 1: How does KCl increase recovery of NH3? The answer to this
probably is associated with the ionic strength of the solution. Salts such as KCl
are often used to reduce the water solubility of a substance (a process we call
"salting out"). For instance, while acetone is completely soluble in water, it is
only slightly soluble in brine. I suspect the KCl in your system simply sped up
the diffusion process by reducing NH3/H2O associations and more consistent
results would have been obtained if more time had passed.
Question 2: Is MgO and NaOH use interchangeable? I suspect in this case they
are not. Granted they are both strong bases and are equal to the task of
neutralizing NH4+. MgO, however, is only slightly soluble in water and so the
concentration of OH- is very limited regardless of how much MgO you added.
This is not the case for NaOH. As an example, we use Mg(OH)2 ("milk of
magnesia" which is what MgO turns into once added to water) to neutralize
excess acids in stomachs. Liquid plumber (NaOH) would also work but the
potential concentration of OH- would make its use extremely dangerous!
4
I am not sure but I think if the OH- concentration was extremely high, you would
neutralize the KHSO4 on the paper disk before the diffusion process was
complete.
Question 3: Why did some samples explode with the Devarda's alloy? I suspect
that the aluminum in Devarda's alloy reacted with
the water to form hydrogen gas:
2Al + 6H2O ---> 2Al+3 + 6OH-
+ 3H2
This reaction is catalyzed by both acids and bases (and hence many of our
aluminum cooking utensils have severe warnings about the use of certain
cleaners. I suspect that if the alloy was in contact with a sufficient amount of
MgO, there may be sufficient gas formation to break the container.
Question 4. What is the purpose of the boric acid? The Cabrera and Beare paper
stated that pH was an important factor in the recovery of nitrogen. While my
reading of their paper leaves some questions what would be the optimum pH, it
does appear that there is an optimum range and that extremely high pH values
should be avoided. Boric acid/ NaOH solutions would act as a basic buffer (pH
ca 10 +/-).
Question 5. What is going on with the pressure in the persulfate oxidations?
Persulfate solutions have limited stability and decomposes according to the
equation even at room temperature:
S2O8-2 + H2O ---> 2HSO4-1
+
1/2 O2
This reaction is sped up by heat (the Merck index suggests the process is rapid at
100oC) and metals other than stainless steel can apt to cause decomposition
(MSDS). Clearly this is a hazardous material that can cause significant pressures
in closed containers and the oxygen atmosphere can cause serious secondary
problems. The decomposition is also exothermic which means it will heat up as it
progresses leading to potential out of control reactions. The HSO4-1 that is
produced also catalyzes the reaction in unbuffered media leading to an
autocatalytic process (also dangerous).
I think it is essential that you use containers well designed for high pressures.
You may also want to invest in an explosion shield.
I hope these insights are helpful. This is really a little outside my realm of
expertise and so I very likely missed some issues.
Tom
5
Supplies for entire process (supplies for oxidation/digestion protocol not included):
Samples for diffusion
Ordering information
2.5M KHSO4 (see protocol)
7ml concentrated H2SO4 (low in nitrogen contamination)
50mL dI water
22g K2SO4
100mL volumetric flask
MgO (0.2g/sample – 0.4g/sample if diffusing for both NH4+ and NO3-)
Calibrated scoop for MgO (0.2g per sample)
KCl (8.4g/sample)
Small weigh boats (for weighing out KCl)
Devarda’s Alloy (0.4g/sample) finely ground (40 mesh)
EM science DX01252
Calibrated scoop for 0.4g Devarda’s Alloy (0.4g per sample)
125ml HDPE plastic bottles (1 per sample)
Fisher 02-895-1A
Teflon (PFTE) tape (8 cm per sample)
hardware supply store
New razor blade
Pre-leached 7mm diameter Whatman #1 filter paper discs (2 per sample-see protocol)
6 mmGlass beads – acid washed (2 per sample)
Fisher 11-312D
5-10µL pipette and tip
5mL pipette tip for sealing Teflon traps
Forceps
Beaker of dI water
Kimwipes
Stainless steel wire (1 per sample)
hardware supply store
Wire snips
hardware supply store
Paperclip (1 per sample and a couple large paperclips for storing discs)
Desiccator with drierite
Small beaker of concentrated H2SO4
Styrofoam block
Labeling tape
Extra-fine point sharpie
21mm tin discs
ElanTech – D1064
Ethanol
Eliza plate
Sample table
Gloves
Lab coat
Safety glasses
6
Protocol for preparing Whatman #1 filters for diffusions
Goal: Leach filter papers of nitrogen contamination.
Supplies (given for one filter paper):
Whatman #1 filter paper
Buchner funnel
Beaker
200ml 0.5M K2SO4
300ml dI water
aluminum foil
forceps
gloves
paper punch – punches 7mm holes
thumbtack
bent paperclip
clean container for storing filter disks
Method: Wear gloves at all times while preparing the filter paper to limit contamination.
Place filter in buchner funnel over an empty beaker. Pour 4-50ml aliquots of 0.5M
K2SO4 followed by 6-50ml aliquots of dI water through the filter paper. Dry the filter
paper in the oven. Put the filter paper on a clean piece of aluminum foil or in a clean
petri dish and cover loosely to limit dust contamination. Once the filter paper is dry,
punch out little circles from the filter paper using a clean paper punch over a clean
surface (deli papers or bench paper is good – wear gloves!). With a clean thumbtack
poke small holes in the center of each of the filter disks. One way to do this is to put a
cushion below your deli paper and punch through the filter disks. Store the filter disks on
a paperclip that has been bent open and store the paperclip in a clean specimen cup or
other container.
7
Protocol for mixing 100ml 2.5M KHSO4 for use in diffusions:
Goal: Make acid for trapping NH4+ on filter discs.
Supplies:
7ml concentrated H2SO4 (low in nitrogen contamination)
~100ml dI water
22g K2SO4
100mL volumetric flask
lab coat
gloves
safety goggles
SAFETY NOTE: H2SO4 is a concentrated acid! Safety precautions for using acids
should be followed at all times! Wear full-length lab coat, acid-proof gloves, and safety
goggles at all times while handling the acid. Always add acid to water; never add water
to acid!!) Always carry acid in secondary container when transporting from one place to
anther. Do this protocol in the hood. Clean up all acid spills immediately!
Method: Add 7ml concentrated H2SO4 to 50mL dI water (always add acid to water, not
the other way around!). Add 22g K2SO4 into the acid mixture. Bring the solution up to
100mL volume with dI water in 100ml volumetric flask.
8
Protocol for mixing standard stock solution:
Stock solution for nitrogen in the form of NH4+
Prepare a 40mgN/ml stock solution or 40,000ppm of nitrogen in the form of NH4+. You
can serially dilute this stock solution for use in spiking your samples. With this solution
you will have 100µg of nitrogen in the form of NH4+ in 2.5µl of solution for making your
non-diffused standards (see table below for target µg of nitrogen and the corresponding
µl of stock you would pipette onto each filter disc).
You need 152.752g NH4Cl/L solution. Make this stock solution in 500ml or greater
batches. For 500ml stock solution dissolve 76.376g of NH4Cl and bring up to 500ml
volume with your blank solution (use 0.5M K2SO4 for soil salt extracts or persulfate
blank solution for soil digests) in a volumetric flask.
Stock solution for nitrogen in the form of NO3Prepare a 40mgN/ml stock solution or 40,000ppm of nitrogen in the form of NO3-. You
can serially dilute this stock solution for use in spiking your samples. With this solution
you will have 100µg of nitrogen in the form of NO3- in 2.5µl of solution for making your
non-diffused standards (see table below for target µg of nitrogen and the corresponding
µl of stock you would pipette onto each filter disc).
You need 288.74g KNO3-/L solution. Make this stock solution in 500ml or greater
batches. For 500ml stock solution dissolve 144.371g KNO3 in your blank solution (use
0.5M K2SO4 for soil salt extracts or persulfate blank solution for soil digests) and bring
up to 500ml volume in a volumetric flask.
Below is a table describing the amount of 40mg/ml stock solution you need to pipette
onto the filter discs to achieve your target amount of nitrogen for your non-diffused
standards.
Target ug N
Total µl
µl stock
(total for both discs) stock solution per filter disc
50
1.25
0.625
100
2.5
1.25
150
3.75
1.875
200
5.0
2.5
To make a 200ppm solution, take 1ml of the 40,000ppm stock solution and put it in a
200ml volumetric flask and then fill it up to the line with your blank solution (0.5M
K2SO4 or persulfate blank solution). To spike your samples with 50µg of nitrogen pipette
in 0.25ml of your 200ppm solution into each sample that you want to spike (generally
samples with less than 50µg of nitrogen). To make your standards, pipette in the
following amounts of your 200ppm stock solution into your bottle with blank solution:
Target µg nitrogen:
ml of 200ppm stock solution:
50µg
0.25ml
100µg
0.5ml
150µg
0.75ml
200µg
1.0ml
9
Digestion/Oxidation Protocol:
For 2 L persulfate oxidation solution:
33.6g NaOH (or 200ml 3.75M NaOH)
100g K2S2O8
60g Boric Acid
Mix the above ingredients and bring up to 2L volume with dI water. Store up to 1 wk in
Amber bottle.
Sample digestion: To digest samples mix by volume 1:1 sample:persulfate oxidation
solution in Teflon-lined, screw-capped test tubes or clamped serum vials. Autoclave the
samples for ½ hour.
Persulfate Blank Solution: In an autoclave safe container mix by volume 1:1 persulfate
oxidation solution:0.5M K2SO4 and autoclave for ½ hr. Only fill the container half way
to allow for expansion during autoclaving – if you do not heed this warning your
container may EXPLODE!
10
Diffusion Set-Up
Supplies:
Diffusing for NH4+
KCl (8.4g/sample)
MgO (0.2g/sample)
Calibrated scoop for MgO (0.2g per sample)
Diffusing for NO3- (following diffusion for NH4+)
Devarda’s Alloy (0.4g/sample) finely ground (40 mesh)
MgO (0.2g/sample)
Calibrated scoop for 0.4g Devarda’s Alloy (0.4g per sample)
Calibrated scoop for MgO (0.2g per sample)
Diffusing digestions (nitrogen in form of NO3-)
10M NaOH – volume needed depends on pH of samples
Devarda’s Alloy (0.4g/sample) finely ground (40 mesh)
Calibrated scoop for 0.4g Devarda’s Alloy (0.4g per sample)
For all diffusions
Samples to diffuse
2.5M KHSO4 (see protocol)
125ml HDPE plastic bottles (1 per sample)
Teflon (PFTE) tape (8 cm per sample)
New razor blade
Pre-leached Whatman #1 filter paper disc (2 per sample)
Glass beads – acid washed (2 per sample)
5-10µL pipette with tip
5ml pipette tip for sealing Teflon traps
Forceps
Gloves
Lab coat
Safety glasses
Sample preparation:
Use volume of sample necessary to achieve ~50-175µg N in the sample container1. You
want at least 40mL of solution so that the sample does not become semi-solid after
adding MgO – if necessary increase the volume of your sample with 0.5M K2SO4 (or
persulfate blank solution if diffusing digested samples). The trapping capacity of a teflon
trap is 350ug N, and you should never exceed 50-60% of the capacity. If you have less
than 50µg nitrogen in solution then spike your sample to bring the concentration above
50µg N. When using the 120ml bottles, target 50ml-75ml of sample. The bottles build
up pressure during the diffusion for NO3- and some may leak. Therefore, it is necessary
1
The nitrogen in the container is on a weight basis (µg nitrogen) not in terms of concentration (ug/ml except for in standard stock solutions). There is an important difference between the two! We target a
specific total weight of nitrogen in solution not a concentration of nitrogen in solution.
11
to leave a good amount of headspace in the bottle to accommodate the pressure buildup
during the diffusion.
Blanks preparation:
You should have at least two types of blanks in your diffusions, sample blanks and nondiffused blanks. A third type of blank is necessary if you are diffusing various volumes of
samples (i.e. not all of your samples are the same volume and you have not brought all of
the samples up to the same volume) or if you have to spike your samples to increase the
nitrogen in solution.
Sample Blanks – sample blanks are blank solution you extracted with your
samples. Run these to figure out background 15N introduced during the extraction/sample
handling. Diffuse these blanks along with the rest of your samples. They should be
treated the same as the rest of your samples except in the case where you have to spike
your samples with nitrogen. In this case, do not add the spike to your blanks. Create
different blanks for the spikes to determine the 15N in the nitrogen spike (see spike blanks
below).
Non-diffused Blanks – these blanks are used to determine the amount of 15N in the
filter paper, and the KHSO4. To make a non-diffused blank pipette 10ul of KHSO4 on to
two filter discs (5µl onto each disc). Dry these in the desiccator and prepare for mass spec
by rolling in tin disc (see rolling below).
Volumetric Blanks - If you are diffusing different volumes of samples you should
make blanks that encompass your different volumes to determine variation of 15N
contamination associated with the volume of solution. To mix up these blanks, use a
background solution that is the same as your samples. For 0.5M K2SO4 salt extractions
use varying volumes of 0.5M K2SO4. For digested samples use persulfate blank solution
at varying volumes.
Spike Blanks – You would want to make spike blanks if you spike any of your
samples. This is to determine the amount of 15N introduced in your spike. In three
bottles, add the right volume of your background solution (either 0.5M K2SO4 or
persulfate blank solution) and then add the same volume of spike that you added to your
samples. You can eliminate this blank if you prepare a diffused standard with the same
amount of N as you spiked with (see diffused standards below)
Standards preparation:
You should have two types of standards with varying amounts of nitrogen in your
diffusion: diffused standards and non-diffused.
Diffused Standards – Create a set of standards that encompass the amount of
nitrogen you have in your samples. To do this have blank 0.5M K2SO4 solution (or
persulfate blank solution if you are diffusing digested samples) at the appropriate volume
in your bottle (your most typical sample volume) and add appropriate amount of nitrogen
standard solution to achieve the desired µg of nitrogen in solution. A typical standard
array would have three replicates of a low-end standard of around 50µg nitrogen in
solution, another set with 100µg nitrogen in solution, and a third with nitrogen greater
than your highest amount of anticipated nitrogen. If you are diffusing for both NH4+ and
12
NO3- then you should add nitrogen in both forms into your initial standard solution. You
will diffuse for the NH4+ first and then the NO3-; for digested samples create standards
with NO3- only. Treat these the same as your samples.
Non-diffused Standard – These standards are used to determine fractionation that
occurs during the diffusion. These standards are not diffused, so there should be no
fractionation and you can compare these to the diffused standards. To prepare, take two
filter discs and spear them with a stainless steel wire and pipette 5µl KHSO4 on to each
disc and then pipette the desired amount of nitrogen standard in the appropriate form to
achieve the desired µg of nitrogen on the disc (use NH4Cl standard stock for NH4+ and
KNO3 standard stock for NO3-; see protocol for mixing up stock solution above). Each
filter disc can hold 7.5µl of fluid, 5µl of which are used by the acid, so you can pipette
about 2.5µl of standard solution onto each filter disc. The targeted nitrogen
concentrations should mimic those of your diffused standards and you should have three
replicates of each. Dry the filter discs in the desiccator and prepare for mass
spectrometry as described below.
Teflon trap preparation:
1. Prepare clean surface for making Teflon traps. Cover bench with either deli paper
or bench paper. Wear gloves throughout the process to reduce the amount of
sample contamination.
2. Cut 8cm strip of Teflon tape using a razor blade.
3. Place two filter disks on one half of the Teflon tape, spacing the disks about 5mm
apart.
4. Pipette 5µL of 2.5M KHSO4 onto each filter disk.
5. Using forceps, fold the other half of the Teflon tape over the filter disks. Gently
smooth the Teflon tape with a gloved finger.
6. Seal a circle around each filter disk using the wide end of the 5mL pipette tip by
pressing down around the disk but not twisting.
7. Store pre-made traps in a specimen cup until you are ready to start the whole set
of samples. Make all traps the same day you start the diffusions.
Starting the diffusion:
Diffusing for NH4+:
1. Add ~5.6g KCl to 50mL samples and ~8.4g KCl to 75mL samples.
2. Add two glass beads.
3. Quickly add scoop of 0.2g MgO and a Teflon trap to each sample.
4. Cap immediately.
5. Swirl and invert each sample 5 times.
6. Store sample upside down so that if the sample leaks, only the solution
will leak and the ammonia gas will be trapped in the headspace at the
top where it cannot leak. Store samples on a surface that will not be
affected if the samples leak – on a tray with a layer of absorbent bench
diaper to soak up any leaking solution. The solution is highly basic
(pH>13) so be careful when handling samples – wear protective gear.
13
7. Allow the sample to diffuse for 6 days at room temperature, inverting
each sample daily.
8. Harvest sample after 6 days of diffusion (see below for harvest
instructions).
Diffusing for NO3- (you have already diffused the sample for NH4+):
Michelle has tested for remaining NH4+ and has found it unnecessary to do
steps 1-4, so start at step 5.
1. Add 0.2g scoop of MgO and mix.
2. Open sample and allow any remaining NH3 to escape.
3. Mix and vent sample daily each day for 4 days.
4. At this point the sample should be free of NH3 and you can now
diffuse for NO3-.
5. Add 0.4g Devarda’s Alloy, 0.2g scoop MgO, and a Teflon trap to each
sample.
6. Cap immediately.
7. Swirl and invert each sample 5 times.
8. Store samples upside down on a surface that will not be affected if the
samples leak – on a tray with a layer of absorbent bench diaper.
9. Allow the sample to diffuse for 6 days at room temperature, inverting
each sample daily.
10. Harvest Teflon trap after 6 days of diffusion (see below for harvest
instructions).
Diffusing digested samples (N in form of NO3-):
1. A stronger base is necessary with these samples. Add enough 10M
NaOH to bring all samples above pH13.
NOTE: 10M NaOH is a very strong base and safety precautions
should be followed when handling a strong base!!!
2. Test the pH of several random samples to make sure pH is >13.
3. Add two glass beads.
4. Add a scoop of 0.4g Devarda’s Alloy and a Teflon trap.
5. Cap immediately.
6. Swirl and invert each sample 5 times.
7. Store samples upside down on a surface that will not be affected if the
samples leak – on a tray with a layer of absorbent bench paper.
8. Allow sample to diffuse for 4 days, inverting each sample once daily.
9. Harvest Teflon trap after 4 days of diffusion (see harvest instructions).
Harvesting diffusions:
Goal: Retrieve and dry the filter discs.
Supplies:
Forceps
Beaker of DI water
Kimwipes
14
Stainless steel wire (1 per sample)
Wire snips (to cut wire)
Paperclip (1 per sample)
Desiccator with drierite
Small beaker of concentrated H2SO4
Styrofoam block
Labeling tape
Extra fine point sharpie
Gloves
Lab coat
Safety glasses
Method:
Note: Be aware you are handling extremely basic samples and safety precautions
should be followed at all times. The samples may have built up pressure during the
diffusion and so some samples may spray when opened.
1. Carefully open the sample – you may want to have a kim wipe in your hand as
you open the lid to absorb any spray that might be released when the
pressurized bottle is opened.
2. Fish out the Teflon trap with a pair of forceps.
3. Briefly swirl Teflon trap in a beaker of dI water (replace with fresh dI water
frequently).
4. Place trap on a new kim wipe
5. Gently dab trap dry with half of the kim wipe
6. Gently peel the Teflon trap open with the forceps.
7. Spear the filter disks through the pre-poked hole with a new stainless steel
wire. Use a new paperclip to ease the traps onto the wire. Be sure to soak up
any beads of solution with your filter papers. Both traps can go on the same
wire unless you suspect one of the filters has leaked in which case you should
put each filter disc on a separate wire and make a note in your lab book.
There are two ways to label the filters:
a. you can either tape a little tab of lab tape onto the wire and write your
label on the tape before you spear your discs, or
b. you can lay down strips of lab tape on the Styrofoam and stab the wire
with discs in the styrofoam and write the sample number below the
wire on the tape.
8. Dry samples for 4 hours in a desiccator with drierite and a small beaker of
concentrated H2SO4 in the bottom of the desiccator (this creates an acidic
environment and keeps the NH4+ trapped on the filters discs).
Rolling disks for mass spectrometry
Goal: Prepare filter discs for mass spectrometry.
Supplies:
21mm tin disks from ElanTech
15
forceps
kimwipes
ethanol
eliza plate
sample table (for recording what samples are in which well in eliza plate)
Method:
1. Take a wire with two filters and pull the filters off of the wire using a clean
pair of forceps.
2. Lay the filters in the center of a tin disk.
3. Fold 1/3 of the tin over the discs and the third from the other side like a little
burrito.
4. Fold in either end of the tin and then gently crunch the disk into a ball, being
careful not to tear the tin.
5. Place sample in an eliza tray well.
6. Write on your sample table what well the sample was placed in.
7. Repeat for all samples.
8. Tape the eliza lid onto the tray and label the tray keep a photocopy of your
sample table rubber banded to the tray.
Note: We already know the N concentration so it is not necessary to weigh the
filter papers. We are only interested in the 15N in solution.
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