RECIPES
Reagents and Standards for Inorganic N Extraction and Analysis
AMMONIUM ANALYSIS
Stock Ammonium Solution, (NH4)2SO4; FW 132.14
(100 ppm N => 50 mg N/ 0.5 L (NH4)2SO4) (store in refrigerator):
0.23585 g ammonium sulfate: Fisher # A938-500
500 ml ultrafiltered DI water
OR: 47.15 mg/100 ml
OR: 117.925 mg/250 ml
Reagent A: Sodium salicylate solution (store in refrigerator):
In 100 ml volumetric flask, dissolve in ~50ml DI H O on a stirplate:
2
6.8 g sodium salicylate: Fisher # S395-500
5 g sodium citrate: Fisher # BP327-1 (dehydrate)
5 g sodium tartrate: Fisher # BP352-500
0.025 g sodium nitroprusside (sodium nitroferricyanide): Fisher # S350-100 (caution: toxic)
-bring to 100ml with DI H O.
2
OR FOR 500 ml:
34 g sodium salicylate: Fisher # S395-500
25 g sodium citrate: Fisher # BP327-1 (dehydrate)
25 g sodium tartrate: Fisher # BP352-500
0.125 g sodium nitroprusside (sodium nitroferricyanide): Fisher # S350-100 (caution: toxic)
-bring to 500ml with DI H O.
2
Reagent B: Bleach/NaOH solution (solution is unstable; mix fresh every day):
0.5 ml bleach
24.5 ml 1.5M NaOH (Bring up to 25 ml in a volumetric flask with 1.5M NaOH)
1.5M NaOH (store in refrigerator):
6 g sodium hydroxide (Fisher #S318-1)
Bring to 100ml in a volumetric flask with DI H O
2
OR: 15 g NaOH in 250 ml dH2O
*According to Tad Doane, these reagents can be stored for at least several months in the refrigerator,
or indefinitely if kept frozen between uses.
**Also quantities do not have to be totally exact
NITRATE ANALYSIS *Make these solutions in a fume hood*
Stock nitrate solution (100ppm); KNO3; FW 101.09 (store at 4C no more than one month!)
0.3609 g potassium nitrate
500 ml ultrafiltered DI water
OR: 72.17 mg KNO3/100 ml dH2O
Saturated vanadium chloride solution (prepares enough for about 38 plates):
(This concentrated solution can be used for both KCl and K2SO4 extracts.)
Mix the following then set aside until the vanadium is prepared:
1000 mg sulfanilamide
50 mg NED
500 ml 1M HCl (83.3 ml concentrated HCl into dH2O; bring up to 1 L)
250 ml prepared Vanadium chloride (2 g vanadium (III) chloride in 250 ml 1.0M HCl)
PREPARED VANADIUM CHLORIDE TO STORE(prepares approximately 30 50ml aliquots):
DO NOT USE THIS DIRECTLY! USE IN ABOVE PREPARATION FOR FINAL USE IN
ASSAY!
**Vanadium chloride powder releases a chlorine gas when exposed to air. Prepare solution in a fume
hood, and work quickly and carefully. The compound as well as the solution should also be stored in the
dark as it breaks down in light. The solution should also be purged with nitrogen. Vanadium III chloride
is a corrosive – wear mask, gloves, goggles, and a lab coat.
25 g vanadium (III) chloride (Fisher # AC19700-0250 OR Sigma #208272-25G)
3.125 L 1.0M HCl.
Swirl the solution for 1-2 minutes. The solution should turn a blue to blue green color. Gravity-filter the
vanadium solution using a funnel that has a cotton ball in its neck, and pre-wet the cotton ball with 1.0M
HCl. Add the HCl/sulfanilamide/NED solution to the vanadium solution. Store in 20 ml polypropylene
vials that have been purged with nitrogen. Store in the freezer.
Store the solution in amounts that are reasonable for one time use (e.g., a 20ml
scintillation vial holds enough for 1.5 plates or a 40ml septa vial holds enough for 2
plates). To store the solution, partition it into opaque scintillation vials or rubber septa
vials that are covered in aluminum foil. Purge the solution with nitrogen before storing.
This should be done immediately upon making the solution as it is very susceptible to
oxygen.
When purging a scintillation vial, have nitrogen air blown into the vial to displace any
empty head space with nitrogen. When purging a septa vial, insert two needles into the
septa, one that is attached to a nitrogen source and another that has nothing attached. As
the nitrogen is pumped into the empty head space, the displaced air flows out through
the second needle; cycle nitrogen through the vial for about 3 minutes to ensure that it
has been purged. When using this method, try to limit the number of times insert a
needle into the rubber septa, as overused septas can leak. The vials can be stored in the
freezer for up to 1 year. If making this solution in a nitrogen box, you can use a Schlenk
flask to store the solution.
Previously, if KCl was used as the matrix, this solution was prepared:
3.3ml 2% sulfanilamide solution
3.3 ml 0.2% NED solution
400 ml dH O
2
50ml saturated vanadium chloride solution (2 g vanadium III chloride + 250 ml 1M HCl)
1M HCl **Remember to always add acid to water
83.3 mL of concentrated HCl
Bring to 1L with DI H O
2
2% sulfanilamide solution
0.2g sulfanilamide
10ml 1.0M HCl
0.2% NED solution
0.02g N-(1-naphthyl)-ethylenediamine dihydrochloride
10ml DI H O
2
STANDARD EXTRACTANTS/MATRIX SOLUTIONS:
2.0M KCl
149.1 g KCl
1 L DI H O
2
0.5M K SO (used in extractions for microbial biomass N)
2
4
87.13g K2SO4
1 L DI H O
2
How much (NH4)2SO4 is needed to create 100 ppm NH4+-N solution?
100 ppm = 100 mg NH4+-Nx
L soln
X
1g
1000 ml
X
1 mol NH4+N
14.00674 g