CardelúsMircoplate Determination of NO3- and NH4+ Updated Messing 8/2012
Step 1:
Weigh out ~ 25 mg of (new) Devarda’s alloy into each well (need to weigh out the
day of experiment)
Samples with Devarda’s allow need to incubate for 5 hours!
TRAY 1: Devarda’s alloy and 0.2 M H2SO4 (Conversion of Nitrate and Nitrate 
Ammonium)
1.
2.
3.
4.
~ 25 mg of Devarda’s alloy into each well
10 μL of 0.2 M H2SO4to each well
250 μL of sample into each well
incubate at room temperature for 5 hours
TRAY 2: Devarda’s allow, 0.2 M H2SO4, and 0.21 Sulfamic acid (decmposition of
nitrite with NH3SO3 followed by reduction with Devarda’s alloy)
1.
2.
3.
4.
ORDER IS IMPORTANT!!!
~25 mg of Devarda’s alloy into each well
10 μL of 0.2 M H2SO4
25 μL of 0.21 M NH3SO3 (which decomposes NO2- before reduction of
Devarda’s allow)
5. 250 μL of sample into each well
6. incubate at room temperature for 5 hours
TRAY 3: NH4+
1. add 150 μL of sample into each well (after trays 1 and 2 have incubated)
Color Development:
ALL TRAYS FOLLOWING INCUBATION OF TRAYS 1 AND 2:
1.
2.
3.
4.
5.
6.
7.
add 150 μL of reduced sample from trays 1 and 2 to new microplates
add 25 μL of citrate reagent to trays 1-3
add 50 μL salicylate-nitrprusside reagent to trays 1-3
add 25 μL hypochlorite reagent to trays 1-3
adjust volume to 275 μL (by adding 25 μL of DI H2O) in trays 1-3
incubate for 30 minutes at 350C in microplate reader
shake in microplate reader for 30 seconds then read at 650 nm
Calculations:
NO3- Concentrations = Tray 2 (NH4+ + NO3-) – Tray 3 (NH4+)
NO2- = Tray 1 (NH4+ + NO3- + NO2-) - Tray 2 (NH4+ + NO3-)
NH4+ = Tray 3 (NH4+)
1
CardelúsMircoplate Determination of NO3- and NH4+ Updated Messing 8/2012
Determination of inorganic N in soil and water extracts (Sims 1995)
Sims, G. K., T. R. Ellsworth R. L. Mulvaney. 1995. Microscale determination of
inorganic nitrogen in water and soil extracts. Community and Soil Science
Plan Annals 26:303-316.
Three measurements are required:
1. NH4+ + NO3- +NO2- (after treatment with Devarda’s alloy to reduce NO2- and
NO3- to NH4+)
2. NH4+ and NO3- (after decomposition of nitrite with NH3SO3 followed by
reduction with Devarda’s alloy)
3. NH4+
Reagents and Materials:



Devarda’s Alloy
0.2 M H2SO4 (sulfuric acid)
0. 21 M NH3SO3 (sulfamic acid)
Citrate Reagent
 5 g Trisodium citrate
 2 g NaOH pellets
 in 100 mL H2O
Salicate Reagent





7.813 g sodium salicylate
0.125 g sodium nitropusside
in 100 mL H2O
pH = 6-7 adjust with drops of NaOH
(can be stored for up to 4 weeks in refrigerator in a light-proof
container)
Hypochlorite Reagent





1 g Na3PO4 (sodium phosphate) OR 2.3 g Na3PO4*12H2O
2 mL of 2 M NaOH
10 mL of fresh commercial household bleach (0.7 M NaOCl)
bring to volume in 100 mL H2O
pH = 12-13 with NaOH
2
CardelúsMircoplate Determination of NO3- and NH4+ Updated Messing 8/2012
Standards:
NO3-:
use 1,000 ppm NO3- with 0.5 M K2SO4 as the carrier
solution
mg/L
uL NO3volume flask (mL)
0.025
1.3
0.05
1.3
0.1
2.5
0.5
12.5
1
25
1.25
31.3
1.5
37.5
50
25
25
25
25
25
25
NH4+
use 1,000 ppm NH4+ with 0.5 M K2SO4 as the carrier
solution
mg/L
uL NH4+
volume flask (mL)
0.025
1.3
0.05
1.3
0.1
2.5
0.5
12.5
1
25
1.25
31.3
1.5
37.5
50
25
25
25
25
25
25
NH4+ and NO3use 1,000 ppm NH4+ and 1,000 ppm NO3- with 0.5 M K2SO4 as the carrier
solution
mg/L
uL NH4+
uL NO3volume flask (mL)
0.05
1.3
1.3
0.1
2.5
2.5
0.5
6.25
6.25
1
12.5
12.5
1.25
15.65
15.65
1.5
18.75
18.75
50
50
25
25
25
25
3