Soil Ammonium-N and Nitrate-N Colorimetry Assay
Bench manual for extracting soil inorganic nitrogen with 1M KCl, then measuring ammonium-N and nitrate-N by colorimetric microplate assay.
Extractant1M KCl
Field soil3 g + 15 mL
Incubation soil0.3 g + 1.5 mL
Read650 / 540 nm
Before Starting
Chemical hazards
This protocol uses hydrochloric acid, sodium hydroxide, bleach, vanadium(III) chloride, and color reagents. Wear lab coat, gloves, and eye protection. Prepare acid and caustic reagents in an appropriate hood or approved chemical workspace.
Plate batching
Analyze all extracts from different sampling times for one sample on the same plate when possible. This reduces plate-to-plate bias.
Controls
Include two KCl blanks, one QC soil, standards, negative controls, and sample duplicates for each assay run.
Materials
Extraction Reagent
| Reagent | Vendor | Catalog # |
|---|---|---|
| Potassium chloride, 500 g | Fisher | P217-500 |
Ammonium Colorimetry Reagents
| Reagent | Vendor | Catalog # |
|---|---|---|
| Ammonium chloride, 500 g | Fisher | A661-500 |
| Sodium nitroprusside dihydrate, 25 g | MP Biomedicals | 152061 |
| Sodium salicylate, 99%, 250 g | ThermoFisher Scientific | A17056.30 |
| Sodium citrate dihydrate, 500 g | Fisher | BP327-500 |
| Sodium tartrate dihydrate, 500 g | Fisher | BP352-500 |
| Sodium hydroxide, 500 g | Fisher | S318-500 |
| Bleach-Rite, 1 pt | Current Technologies | 70590-2 |
Nitrate Colorimetry Reagents
| Reagent | Vendor | Catalog # |
|---|---|---|
| Potassium nitrate, 500 g | Fisher | P263-500 |
| Hydrochloric acid, 500 mL | Millipore Corporation | HX0608-1 |
| Vanadium(III) chloride, 97%, 5 g | Sigma-Aldrich | 208272-5G |
| Sulfanilamide, 100 g | Fisher | 04525-100 |
| N-(1-Naphthyl)ethylenediamine dihydrochloride, ACS, 25 g | Thermo Scientific | J63214.14 |
Equipment and Consumables
- VWR Reciprocal Shaker
- Centrifuge for 15 mL and 2 mL tubes
- 15 mL centrifuge tubes
- 2 mL tubes
- Funnels and medium-porosity filter paper, if filtering
- SpectraMax ABS Plus spectrophotometer
- Corning 96-well assay plate with lid, catalog #3788
- Pipettes and tips
- Cuvettes, if using the cuvette option
Extraction Calculator
| Sample type | Soil per sample | 1M KCl per sample | Total soil | Total 1M KCl |
|---|---|---|---|---|
| Field soil | 3 g | 15 mL | 0 g | 0 mL |
| Incubation soil | 0.3 g | 1.5 mL | 0 g | 0 mL |
Part A: Prepare Extraction Reagent and Stocks
~30-60 min
1M KCl Extractant
- Dissolve 74.55 g KCl in DI water.
- Bring to 1 L with DI water.
- Swirl and heat as needed until dissolved completely.
- Store at 4°C or room temperature. Shake well before use.
100 ppm Standards Stocks
- Prepare 100 ppm NH4-N stock: dissolve 382 mg NH4Cl in 1 L 1M KCl.
- Prepare 100 ppm NO3-N stock: dissolve 722 mg KNO3 in 1 L 1M KCl.
- Store standards frozen at −20°C in dark-colored bottles. Thaw as needed and shake well before use.
Part B: Extract Ammonium and Nitrate from Soil
~2 h active/extraction time, plus storage
Field Soil Extraction
- Weigh 3 g moist soil, sieved to 2 mm, into clean labeled 15 mL centrifuge tubes.
- Include a sample for moisture content determination.
- Add 15 mL 1M KCl to each soil sample.
- Include two KCl blanks with no soil and one QC soil.
- Cap tubes and invert or shake to mix.
- Shake horizontally on the VWR Reciprocal Shaker at 650 rpm for 1.5 h. Reverse tube orientation midway through shaking.
- Centrifuge the slurry for 10 min.
- Pipette supernatant into clean labeled 15 mL tubes without disturbing the pellet.
- Freeze extracts at −20°C until colorimetric analysis. Thaw and vortex or shake before use.
Incubation Soil Extraction
- Weigh 0.3 g moist soil, sieved to 2 mm if needed, into clean labeled 2 mL tubes.
- Add 1.5 mL 1M KCl to each tube.
- Include two KCl blanks with no soil and one QC soil.
- Cap tubes and invert or shake to mix.
- Shake horizontally on the VWR Reciprocal Shaker at 650 rpm for 1.5 h. Reverse tube orientation midway through shaking.
- Centrifuge at 10,000 × g for 10 min.
- Pipette supernatant into clean 2 mL tubes without disturbing the pellet.
- Freeze extracts at −20°C until colorimetric analysis. Thaw and vortex or shake before use.
Clear extract required
Soil particles in the extractant will cause spectrophotometry error. Transfer only clear supernatant.
Part C: Prepare Color Reagents
~30-90 min, depending on reagent status
Ammonium Reagents
- Prepare Reagent A: in 250 mL DI water, dissolve 0.125 g sodium nitroprusside, 32.5 g sodium salicylate, 25 g sodium citrate, and 25 g sodium tartrate.
- Store Reagent A in a dark bottle at 4°C. It is light-sensitive and stable for about one month. Discard if it becomes dark.
- Prepare Reagent B: in 250 mL DI water, dissolve 15 g sodium hydroxide and add 5 mL bleach.
- Store Reagent B at 4°C. It is stable for several months. Shake well before use.
Nitrate Reagents
- Prepare 0.5M HCl: add 16.66 mL concentrated HCl, 12M, to about 200 mL DI water, then bring to 400 mL with DI water.
- Prepare Griess reagent: in 200 mL 0.5M HCl, dissolve 0.4 g sulfanilamide and 0.02 g N-(1-naphthyl)-ethylenediamine dihydrochloride.
- Store Griess reagent protected from light at −20°C. Shake well before use.
- Prepare vanadium(III) chloride solution: in 200 mL 0.5M HCl, dissolve about 1 g vanadium(III) chloride.
- Aliquot VCl3 solution into 10 mL aliquots and freeze.
- On the day of the nitrate assay, thaw and mix Griess reagent and VCl3 solution at a 1:1 ratio.
Mixed nitrate reagent
If the mixed Griess/VCl3 reagent turns pinkish before use, treat it as contaminated and remake the reagent.
Standards
Prepare fresh working standards each time the assay is performed. Add KCl first, then add the solute listed below. Vortex thoroughly after each dilution.
Ammonium Standards
| Standard | Concentration (ppm NH4-N) | Solute | KCl |
|---|---|---|---|
| Standard 7 | 10 | 200 µL 100 ppm NH4-N stock | 1800 µL |
| Standard 6 | 7.5 | 750 µL Standard 7 | 250 µL |
| Standard 5 | 5 | 550 µL Standard 6 | 275 µL |
| Standard 4 | 2.5 | 400 µL Standard 5 | 400 µL |
| Standard 3 | 1.25 | 400 µL Standard 4 | 400 µL |
| Standard 2 | 0.625 | 400 µL Standard 3 | 400 µL |
| Standard 1 | 0.0625 | 45 µL Standard 2 | 405 µL |
Nitrate Standards
| Standard | Concentration (ppm NO3-N) | Solute | KCl |
|---|---|---|---|
| Standard 7 | 5 | 75 µL 100 ppm NO3-N stock | 1425 µL |
| Standard 6 | 3.75 | 712.5 µL Standard 7 | 237.5 µL |
| Standard 5 | 2.5 | 550 µL Standard 6 | 275 µL |
| Standard 4 | 1.25 | 400 µL Standard 5 | 400 µL |
| Standard 3 | 0.625 | 400 µL Standard 4 | 400 µL |
| Standard 2 | 0.0625 | 45 µL Standard 3 | 405 µL |
| Standard 1 | 0.00625 | 40 µL Standard 2 | 360 µL |
KCl blank
Frozen KCl may read differently from fresh KCl. When making fresh standards, use the same KCl as the negative control.
Plate Reagent Calculator
| Assay reagent | Per well | Total | Total + 10% |
|---|---|---|---|
| NH4 Reagent A | 100 µL | 0 µL | 0 µL |
| NH4 Reagent B | 100 µL | 0 µL | 0 µL |
| NO3 mixed reagent | 200 µL | 0 µL | 0 µL |
Part D: Colorimetric Microplate Assay
NH4: 1-3 h development · NO3: overnight or 6-8 h development
Ammonium Assay
- Load 50 µL extract, standard, or KCl control into each assigned well.
- Add 100 µL Reagent A to each well.
- Add 100 µL Reagent B to each well.
- Cover the plate and let color develop for 1-3 h at room temperature in the dark.
- Read absorbance at 650 nm on the SpectraMax ABS Plus.
Nitrate Assay
- Load 50 µL extract, standard, or KCl control into each assigned well.
- Add 200 µL freshly mixed Griess/VCl3 reagent to each well.
- Cover the plate and let color develop in the dark at room temperature.
- Develop overnight, or at least 6-8 h to reach maximum development.
- Read absorbance at 540 nm on the SpectraMax ABS Plus. Color is stable for about two days.
Part E: SpectraMax Reading
~10-20 min per plate
- Set up the microplate map before loading. Record the map in Excel or a lab notebook.
- Turn on both the computer and the SpectraMax Plus.
- Open SoftMax Pro.
- Place the plate on the loading deck with A1 aligned to the A1 label.
- Connect to the instrument from the Home screen by selecting SpectraMax ABSPlus, choosing COM4: SpectraMax ABSPlus, and selecting OK.
- Create a new experiment.
- Open acquisition settings and set wavelength to 650 nm for NH4 or 540 nm for NO3. Set shake to 5 sec before plate reading.
- Gently wipe the plate with a KimWipe to remove dust or debris.
- Close the tray and click the green Read icon.
- Copy readings from SoftMax Pro and save the raw data file.
Calculation Notes
- Copy raw spectrophotometer readings into Excel.
- Create separate NH4 and NO3 standard curves in a calculation sheet.
- Make one curve for each standard replicate, one curve from averaged standard values, and one curve from all standard readings to check accuracy.
- Plot absorbance as x-axis and concentration in ppm as y-axis. Add trendline equation and R².
- Calculate slope with
=SLOPE(standard concentration range, standard absorbance range). - Calculate intercept with
=INTERCEPT(standard concentration range, standard absorbance range). - Calculate R² with
=RSQ(standard concentration range, standard absorbance range). R² should approach 1. - Calculate sample ppm from absorbance using the standard curve slope and intercept, then subtract blank concentration if appropriate.
- Convert ppm to mg/L. For these aqueous standards, 1 ppm = 1 mg/L.
- Calculate mass in the reaction well: concentration in mg/L × 50 µL × 10^-6 L/µL.
- Calculate total mass in the KCl extraction: mass in well × total extractant volume / extract volume loaded in the well.
- Normalize to soil mass and moisture convention used in the lab spreadsheet.
Rate calculations
| Metric | Formula |
|---|---|
| N mineralization rate | ((N-NH4 + N-NO3) end point − (N-NH4 + N-NO3) start point) / duration |
| Nitrification rate | (N-NO3 end point − N-NO3 start point) / duration |
QC soil
QC soil values should be within 2 standard deviations of the overall mean. Add QC values to the lab QC chart.
Cuvette Option
| Concentration range | Extract | Ammonium Reagent A | Ammonium Reagent B |
|---|---|---|---|
| 0-2 ppm NH4-N | 0.4 mL | 0.4 mL | 0.4 mL |
| 0-5 ppm NH4-N | 0.2 mL | 0.5 mL | 0.5 mL |
| 0-10 ppm NH4-N | 0.08 mL | 0.5 mL | 0.5 mL |
| 0-20 ppm NH4-N | 0.04 mL | 0.5 mL | 0.5 mL |
| 0-60 ppm NH4-N | 0.04 mL | 1.5 mL | 1.5 mL |
| Concentration range | Extract | Nitrate mixed reagent |
|---|---|---|
| 0-2 ppm NO3-N | 0.1 mL | 1 mL |
| 0-10 ppm NO3-N | 0.05 mL | 1 mL |
| 0-20 ppm NO3-N | 0.03 mL | 1.5 mL |
| 0-40 ppm NO3-N | 0.03 mL | 3 mL |
Bench Record
| Date | |
|---|---|
| Operator | |
| Project / incubation | |
| Sample IDs | |
| Soil type | Field / Incubation |
| Fresh soil mass | |
| KCl extraction volume | |
| Shaker speed and time | 650 rpm, 1.5 h |
| Extract storage | −20°C |
| Assay | NH4 / NO3 / Both |
| Plate ID | |
| Standard curve R² | |
| QC soil result | |
| Raw data file | |
| Calculation file | |
| Notes |
Bench Notes
Stop Points
Stop and ask before:
- Continuing if extracts are cloudy or contain soil particles.
- Reading plates if standards, controls, or duplicate layout is missing from the plate map.
- Using mixed nitrate reagent that is pinkish before sample addition.
- Changing the moisture correction or N conversion factor in the calculation spreadsheet.
- Accepting a standard curve with poor R² or obvious standard failure.
References
- Doane, T.A., and W.R. Horwath. 2003. Spectrophotometric Determination of Nitrate with a Single Reagent. Analytical Letters 36(12): 2713-2722.
- Forster, J.C. 1995. Soil nitrogen. In Alef K and Nannipieri P, eds. Methods in Applied Soil Microbiology and Biochemistry. Academic Press, San Diego, pp. 79-87.
- Mulvaney, R.L. 1996. Nitrogen: Inorganic Forms. In J.M. Bartels et al., eds. Methods of Soil Analysis. Part 3. Chemical Methods. SSSA Book Series no. 5. SSSA, Madison, Wisconsin, pp. 1123-1184.
- Verdouw, H., van Echteld, C.J.A., and Dekkers, E.M.J. 1978. Ammonium determination based on indophenol formation with sodium salicylate. Water Research 12: 399-402.