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This is a laboratory protocol that outlines methods of extracting, and analyzing NO3- from soil.

Guidelines for Extracting Soil NO3-

  1. Collect a representative soil sample from the site/plot of interest. Typically a ‘W’ or grid pattern is created, and numerous soil sub-samples are taken and combined. Typical 0-30 cm (0-12") AND/OR 30-60 cm (12-24"). Make sure combined soil sample is wellmixed, and then take or 2-3 adequate samples for analysis. For N analysis 15-20 g soil samples are adequate.

  2. To determine soil moisture content, take an ~ 5.0 g sample of field-moist soil samples (record the exact mass), and then dry them at 105℃ to constant volume (1-2 days), re-weigh and again record the dry mass. Calculate and record the moisture factor (moist soil (g)/oven dried soil (g)).
    Note: the maximum setting of the lab incubator is 70℃ and this is adequate for drying soil.

  3. To extract NO3- from soil, weigh out 2-3 ~ 5.0 g samples of field-moist soil (measure and record the exact mass of each sample) into 125 mL Erlenmeyer flasks, and add 50 mL deionized water. Do a blank sample as well (Erlenmeyer flask with no soil with 50 mL deionized water). Note: all flasks, stoppers, funnels, etc must be carefully washed and rinsed with deionized water prior to use.

  4. Stopper the flasks and shake for 30 min at 160 strokes per minute. This is normally done on a reciprocating shaker to ensure reproducibility - if doing this by hand - shake for one minute, let sit 2-3 mins, shake for one minute, etc, for 30 min.

  5. After 30 min, filter the extract through Whatman No. 42 filter paper. Record the total volume of the extract.

  6. Analyze soil extract for NO3-.

NO3--N analysis using HANNA Nutrient Analyzer

  1. Turn on the HANNA Nutrient Analyzer; the on/off switch is on the back left of the analyzer.

  2. Press the “Method” key, and use the up/down arrow keys to select the appropriate method. For soil extract analysis, choose the nitrate LR (low range) option. (Press the key below the screen to select the method.)

  3. Add 6 mL of filtered soil extract into the instrument cuvette and replace the cap.

  4. Place the cuvette in the holder (ensure the raised dot on the cap aligns with the mark near the holder), close the lid, and press the “zero” key (below the display screen); the instrument should read 0.0 mg/L.

  5. Remove your sample cuvette and add the complete contents of 1 packet of HI 93728-0. Shake vigorously up and down for 10 s, and then mix completely by gently inverting for 50 s.

  6. Reinsert the cuvette (same alignment as before) into the instrument and press the “timer” key; after 4 min 30 s the instrument will read the NO3- in units of mg/L (which equal ppm).

Note: Two units are possible, mg/L of NO3--N, or mg/L of NO3-. These are not the same, so record your units carefully. For example, 19.0 mg/L NO3-- N = 83.5 mg/L NO3-. Press one of the “arrow” keys and then the “chem frm” key (below the display screen) to switch from one unit to the other.

19.0 mg/L NO3--N x (62 g NO3- / mole ÷ 14.0 g N / mole) = 84.1 mg/L NO3-

NO3--N analysis using the Vernier Nitrate Ion Selective Electrode

  1. Plug NO3-ISE (ion selective electrode) into a Go-Link adaptor and then into the USB port of a computer with Logger Lite Software installed on it.

  2. For long-term storage, the NO3-ISE should be stored in the long-term storage vial. This vial should contain a small sponge kept moist with deionized water. The probe should not be touching the sponge.

  3. Before using the NO3-ISE probe, it should be soaked in the short-term storage vial for at least 30 min prior to use. This vial should contain a 50 or 100 ppm nitrate solution. The probe should not be in this solution for longer than 24 h.

  4. Note that the probe has a PVC membrane at the bottom of the probe and two little white reference contacts on the side of the probe about 2-3 cm up from the bottom of the probe. When calibrating the probe or when taking readings, both of these must be in contact with the solution, and you need to ensure that there are no bubbles trapped below the probe. The probe should not be resting on the bottom of the container.

  5. Open the Logger Lite software. To calibrate the probe (this should be done prior to each analysis run), follow the following steps:​​

    1. click on “Experiment”;

    2. click “Calibrate Now”;

    3. enter the concentration of this standard in the edit box (e.g. 100 for 100 mg/L)

    4. place the probe in the high NO3- standard (e.g. 100 mg/L);

    5. wait for the voltage reading to stabilize (~ 1 min) and then click on “Keep”;

    6. remove probe from the high NO3- standard, rinse with deionized water, shake or gently wipe off excess water, and place in the low NO3- standard;

    7. enter the concentration of this standard in the edit box (e.g. 1 for 1 mg/L)

    8. wait for the voltage reading to stabilize (~ 1 min) and then click on “Keep”, and “Done”.

  6. To measure the NO3--N levels in samples, simply place the probe in each sample, allow the probe to stabilize, and take the reading. Remember that both the PVC membrane at the bottom of the probe and the reference contacts must be in contact with the solution. Try not to scrape the PVC membrane on the bottom of your beaker. Rinse probe with deionized water between each sample.

  7. When finished, rinse probe with deionized water and place it in the long-term storage vial.

Additional Notes on the Use of the Vernier Nitrate Ion Selective Electrode

  1. For optimal results when measuring low concentrations of nitrate ions, it is common to use an ion strength adjuster (ISA) solution. Adding an ISA ensures that the total ion activity is similar among different samples, and can improve accuracy when measuring low concentrations of ions.

    Nitrate ISA recipe: 2.0 M (NH4)2SO4 = 42.50 g (NH4)2SO4 /100 mL

    Add nitrate ISA to each calibration solution and sample at a ratio of 1 mL ISA : 50 mL total solution.

  2. Calibration solutions can be purchased, or made from solid NaNO3 salts. To make a solution containing 100 mg NO3--N /L:

    100 mg NO3--N /L x (1 g N/1000 mg N) x (1 mole N/14 g N) x (1 mole NaNO3/ 1 mole N) x (85.0 g NaNO3/1 mole NaNO3) = 0.607 g NaNO3 / L solution

    i.e. to make 1 L of a 100 mg NO3--N /L calibration solution, fill a 1 L volumetric flask 1/2 full of deionized water, add exactly 0.607 g NaNO3, dissolve and top up to exactly 1 L

    to make 1 L of a 1 mg NO3--N /L calibration solution, fill a 1 L volumetric flask 1/2 full of deionized water, add exactly 10 mL of 100 mg NO3--N /L and top up to exactly 1 L

Materials needed for soil nitrate extraction and analysis by nitrate ISE

  • soil corer
  • balance to weigh soil 
  • 250 mL flasks with stoppers
  • deionized water
  • 50 mL graduated cylinders
  • NaNO3 (to make your own calibration solutions)
  • Whatmann #42 filter papers
  • funnels
  • reciprocating shaker?
  • drying oven
  • laptop with Logger Lite software installed
  • Go!Link adaptor
  • nitrate ISE with short and long-term storage vials
  • calibration solutions (100 and 1 mg NO3--N /L)
  • (NH4)2SO4 (to make nitrate ISA)

Calculations: Converting the Concentration of N in a Soil Extract, to the Concentration of N in your Original Soil Sample

Calculations of the concentration of soil nitrate on a field-moist soil basis:

____ mg/L NO3--N x ____ mL soil extract (from step 5) x 1 L/1000 mL x 1/ ____ g moist soil (from step 3) x 1000 g/kg = ____ mg/kg NO3--N (or ppm NO3--N )


____ mg/L NO3- x ____ mL soil extract (from step 5) x 1 L/1000 mL x 1/ ____ g moist soil (from step 3) x 1000 g/kg = ____ mg/kg NO3- (or ppm NO3-)

Calculations of the concentration of soil nitrate on an oven-dried soil basis:

____ mg/kg NO3--N (or ppm NO3--N ) x ____ (moisture factor from step 2) = ____ mg/kg NO3--N (or ppm NO3--N)


____ mg/kg NO3- (or ppm NO3- ) x ____ (moisture factor from step 2) = ____ mg/kg NO3- (or ppm NO3-)