DATE: SEPT 2004
Soil Inorganic Nitrogen
Nitrate Nitrogen (Colorimetric Method)
1. Application
In this procedure, nitrogen in the form of the nitrate ion (NO3 —N) is extracted from the
soil with water and measured colorimetrically after reaction with phenoldisulphonic acid.
2. Summary of Methods .
Water is used to extract NO3 —N, using 1 part soil to 5 parts water. Colloids are
precipitated with Ca++, and soluble organics are removed with activated charcoal. After
filtration, an aliquot of extract is reacted with phenoldisulphonic acid. The NO3 —N forms
a blue-colored complex, which is analyzed with a colorimeter.
3. Safety
Each chemical compound should be treated as a potential health hazard. The laboratory
is responsible for maintaining a current awareness file of OSHA regulations regarding the
safe handling of the chemicals specified in this method. A reference file of material
handling data sheets should be made available to all personnel involved in the chemical
analysis. In this procedure, fuming sulfuric acid is used to prepare the phenoldisulphonic
acid.
4. Interferences
Principles interferences are chloride and soluble organic compounds. Chloride is
precipitated with Ag2 SO4 . Colored organic compounds are co-precipitated with Cu(OH)2
by the addition of CuSO4 , followed by Ca(OH)2 .
5. Apparatus and Materials
5.1 Soil scoop calibrated to contain 10 g of light-colored silt loam.
5.2 Erlenmeyer flask, 125- ml
5.3 Graduate cylinder, 50- ml, 100- ml
5.4 Oscillating shaker
5.5 Measuring scoop, ½ tsp
5.6 Beaker, 150- ml
5.7 Funnel tubes
5.8 Hotplate
5.9 Pipette, 10- ml
5.10 Medicine dropper, 3- ml
5.11 Burette, 50- ml
5.12 Colorimeter or spectrophotometer
5.13 Colorimeter tubes, matched
6. Reagents.
6.1 CuSO4 solution, saturated: Add 210 g of CuSO4 .5H2 O to 100 ml of water.
6.2 Ag2 SO4 solution, saturated: Add 10 g of Ag2 SO4 to 100 ml of water.
6.3 Ca(OH)2 : finely ground powder
6.4 MgCO3 : finely ground powder
6.5 Activated charcoal: Heat in a muffle furnace at 500 o C for 1 hour to remove NO3 -.
6.6 Phenoldisulphonic acid: Dissolve 83 g pure phenol in 500 ml of concentrated
H2 SO4 . Dissolve until clear. (Check the H2 SO4 for NO3 - contamination by
dropping several crystals of phenol in several ml of the acid. The solution must
remain clear.) Add a 1-pint bottle of fuming H2 SO4 . (Use the fume hood!) Place in
a boiling water bath for two hours. Store in an amber bottle in a dark cabinet. This
reagent is extremely corrosive.
6.7 NH4 OH, 1:1: Mix equal volumes of concentrated NH4 OH and distilled water.
6.8 Stock standard nitrate solution, 500 ppm N: Dissolve 3.60 g KNO3 , dried at 105 °C,
in water and dilute to 1 liter with water.
6.9 Dilute standard nitrate solution, 20 ppm N: Dilute 20 ml of 500 ppm N to 500 ml
with water.
7. Methods
7.1 Place a 10-g scoop of soil into a 125- ml Erlenmeyer flask.
7.2 Add 50 ml of water by means of a graduate cylinder.
7.3 Add 2 drops of Ag2 SO4 and 3 drops of CuSO4 .
7.4 Shake 10 min on an oscillating shaker (or 30 min intermittently by hand).
7.5 Add ½ tsp of Ca(OH)2 ; shake thoroughly by hand and let stand 10 minutes.
7.6 Decant about 30 ml of the suspension into a 150- ml beaker.
7.7 Add ½ tsp of MgCO3 and swirl.
7.8 Add ½ tsp of activated charcoal; shake by hand and let stand 2 to 3 minutes.
7.9 Filter into funnel tubes.
7.10 Wash the 150- ml beakers employed in steps 7.6 – 7.9.
7.11 Pipette 10 ml of filtrate into the same 150-ml beaker, and evaporate to
dryness on a hotplate. The temperature of the hotplate should not be
high enough to permit spattering as the solution approaches dryness. The
sample must be completely dry.
7.12 Cool; then add 3 ml of phenoldisulphonic acid rapidly to the residue in the beaker.
Use a rapid delivery medicine dropper calibrated to deliver 3 ml. The reagent
should flood the bottom of the beaker rapidly to prevent formation and loss of
volatile nitrogen oxides.
7.13 Swirl; let stand until the residue is dissolved and the solution is clear.
7.14 Carefully add approximately 20 ml of distilled water.
7.15 Cool.
7.16 With a 50- ml burette in a fume hood, carefully add 1:1 NH4 OH until full yellow
color develops and then 3 ml in excess (approximately 15 ml total).
7.17 Transfer the sample to a 100-ml graduate cylinder and dilute to 99 ml with
water. Mix the solution by pouring back-and-forth from cylinder to
beaker several times. (A small amount of solution will remain as a film in
the beaker. Also, a graduate cylinder is calibrated “to deliver” rather
than “to contain” a given volume. A 100-ml graduate cylinder will contain
slightly more than 100 ml, the excess being retained as a film on the cylinder
walls when the cylinder is emptied. To compensate, the cylinder is filled to
only 99 ml. A volumetric flask should be used for precise work.)
7.18 Determine the NO3 —N using a colorimeter at 420 nm. Zero the colorimeter with a
reagent blank.
7.19 Prepare a standard curve by evaporating the volumes of 20 ppm NO3 —N solution
indicated in the table below to dryness in a 150- ml beaker, and proceed with steps
7.12 above.
NO3 —N equivalents of different volumes of standard 20 ppm NO3 —N solution.
Vol. of 20 ppm Final conc. NO3 —N equiv-
NO3 —N soln. of NO3 —N alent in soil*
ml ppm ppm
0 0.0 0
1 0.2 5
2 0.4 10
3 0.6 15
4 0.8 20
5 1.0 25
7 1.4 35
10 2.0 50
* NO3 —N equivalent in soil using wt. of soil
and solution volumes indicated in Methods.
8. Calculations
ppm NO3 —N in soil = ppm NO3 —N in final solution x 50 ml x 100 ml
10 g 10 ml
—
= ppm NO3 N in final soln. x 50
9. Quality Control
9.1 Laboratory Reagent Blank (LRB) – At least one LRB is analyzed with each batch of
samples to assess contamination from the laboratory environment. Contamination
from the laboratory or reagents is suspected if LRB values exceed the detection limit
of the method. Corrective action must be taken before proceeding.
9.2 Standard soil – One or more standard soils of known extractable NO3 —N content is
analyzed with each batch of samples to check instrument calibration and procedural
accuracy.
10. Reporting
Results are reported as ppm of nitrogen in the form of nitrate NO3 —N in soil.
