Nitrate & Nitrite Analysis
Nutrient Analyses on Microplate Reader
Reagents
Vanadium solution (VCl3)
In 100 ml volumetric flask
- 0.80 g vanadium (III) chloride in 50 ml of 1 M HCl
CAUTION: Work quickly because Cl3V powder will react with air!
Flush with N2, store in the dark at 4掳C, or freeze, should be blue.
2.0 % (w/v) Sulfanilamide solution (SULF)
In 100 ml volumetric flask
- 2 g Sulfanilamide
- Bring to volume with 5% (v/v) HCl
Flush with N2; may be stored in the dark at 4掳C for several months, and discard if colored.
0.1% (w/v) NEDD solution
In 100 ml volumetric flask
- 100 mg N-(1-naphthyl)-ethylenediamine dihydrochloride
- Bring to volume with DI water.
Flush with N2; may be stored in the dark at 4掳C for several months, and discard if colored.
Premix solutions just prior to analysis to a ratio of 2:1:1 (20 ml VCl3 : 10 ml SULF : 10 ml NEDD)
|
Standard Dilutions |
||
|
Concentration (ppm ) |
Standard (ml) |
1 M KCl (ml) |
|
0 |
0 |
10 |
|
1 |
0.25 |
9.75 |
|
2 |
0.5 |
9.5 |
|
4 |
1.0 |
9.0 |
|
8 |
2.0 |
8.0 |
|
16 |
4.0 |
6.0 |
|
24 |
6.0 |
4.0 |
|
40 |
10 |
0.0 |
Standard (40 ppm NO3)
In 100 ml volumetric flask
- 4 ml 1,000 ppm nitrate (NO3) standard
- Bring to volume in 1 M KCl.
Procedure
Soil Solution Extraction
- 10 g Oven-dried equivalent field fresh, sieved soil
- 20 ml 1M KCl
Shake for 1 hour and centrifuge (10 min @ 3000 rpm).
Ambient (initial):
- Add 100 碌l of reagent to 100 碌l of sample, or standard in clear 96-wellplate.
Incubated (or farm soils):
- Add 50 碌l KCl and 100 碌l of reagent to 50 碌l of sample, or standard in clear 96-wellplate.
Incubate at 37掳C and measure when color develops. Measure absorbance at 540 nm. Samples should turn pink; if dark magenta (OVRFLW), redo by adding only 10 碌l of sample with 90 碌l of KCl and 100 碌l of reagent.
Adapted from Miranda et al., 2001, Nitric Oxide-Biology and Chemistry
Created: 03/06/07 Revised: 10/10/11