Soil Total Carbon and Nitrogen


see KBS029-prot01


Soil total carbon and nitrogen as well as the ratio of C:N are important attributes of soil fertility. Both total C and N are measured by a combustion method that does not differentiate between organic and inorganic forms of the elements. In most soils, inorganic N is a tiny fraction of total N so total N is synonymous with organic N. Inorganic C, on the other hand, can be a significant portion of total C in soils with carbonate minerals. Extensive testing of KBS surface soils has shown soil inorganic C to be non-detectable; thus total C in KBS surface soils is synonymous with total organic carbon (TOC).

However, carbonates may still be present in deep or recently limed soils, which must be tested for inorganic carbon and, if present, the carbonates removed via acid fumigation (described below) prior to total C analysis. Inorganic C can also be analyzed separately via the inorganic C protocol.

To measure total C and N, subsamples of dried, finely ground soil are weighed into small foil capsules that are combusted in an automated CHN analyzer that measures the amount of released CO2 and N2 by gas chromatography. Final values are expressed as the percentage of carbon or nitrogen in dry soil. If inorganic C has been confirmed to be absent, removed, or analyzed separately and subtracted, values can be expressed as total organic C.

Sampling frequency depends on experiment and research objectives. Since 2001, soils of the LTER Main Cropping System Experiment are analyzed for total C and N as part of the decadal deep soil core (whole profile) sampling program, usually in late fall of wheat years.


For the LTER deep core soil samples, a sieved soil sample from each treatment, sampling station, and soil depth is oven dried at 60 degrees C for at least 48 hours until no further mass loss occurs. Air dried soil can also be used. The sample is then pulverized to a fine powder using a swing mill pulverizer (similar to a ball mill) and placed into polyethylene vials for storage.

Prior to total CN analysis the presence of inorganic carbon is tested qualitatively on a separate sample by adding a few drops of acid (1N HCl) to a small quantity of pulverized soil in a small weigh boat or beaker. Fine bubbles (compare to a soil with no carbonate) indicate the presence of carbonate. If detected, then soils must be pre-treated with acid fumigation as described below to drive off inorganic C as CO2.


  • Pulverizer, such as SPEX SamplePrep 8530 Enclosed ShatterBox
  • Capsules (from Elemental Microanalysis: pressed, standard weight, 8 × 5 mm):
    • if no acid pre-treatment is needed, use tin capsules
    • for acid pre-treatment, use silver capsules
  • Forceps
  • Micro spatula, scoop shaped
  • Sample tray, typically 96-well plate
  • Electronic microbalance
  • Standards (acetanilide, atropine, or phenacetin*)
  • Blind standard (stored soil standard)
  • CHN analyzer

* Prior to 1990, cyclohexanone-2,4-dinitrophenylhydrazone was used as standard.

For carbonate pre-test:

  • small weigh boats or beakers
  • eye dropper or syringe
  • 1N HCl acid (~10 mL)

For acid pre-treatment if needed to remove carbonates:

  • Microliter pipette
  • Nanopure water
  • Dessicator
  • Concentrated hydrochloric acid (12M HCl)


Prepare soil for analysis:

  1. Put about 20g of sieved and well-mixed dry soil into a grinding container.
  2. Place one container in the center or three containers on the three outside pins in the shatterbox.
  3. Close the lid, set timer for 30 seconds and press start.
  4. Transfer ~7 mL of pulverized soil from the grinding container into a labeled 7mL plastic vial. Discard the remainder.

Test for carbonates if necessary:

  1. Put a few grams of pulverized soil onto a weigh boat or beaker.
  2. Add 2-3 drops of HCl.
  3. If no bubbling is noted, stir gently to confirm (compare to soil with no carbonates to ensure bubbling is from carbonate dissolution and not from the physical release of entrapped bubbles).
  4. Record the presence or absence of carbonates and discard sample.

Prepare sample (see below for additional acid fumigation steps as needed):

  1. Always use forceps to handle capsules. Fingers and hands are sources of C and N contamination.
  2. Place an empty capsule on the scale of the microbalance. Close the housing doors. Tare the capsule weight.
  3. Remove tin capsule and place on marble block. Using a scooping spatula, transfer 15-20 mg of the finely pulverized soil in the sample vial to the capsule. Weigh the capsule to ensure it contains an appropriate weight of sample material.
  4. Return capsule to marble block and use forceps to first roll back the free end of the capsule and then to fold the capsule into a small ball, making sure that no sample leaks from the sealed capsule.
  5. Place folded capsule on the scale, close the doors, and record the sample weight by pressing “print” on the balance instrument panel to link its mass via computer software to the selected cell on the data spreadsheet.
  6. Place the capsule in the sample tray well designated by the data spreadsheet (see below).
  7. Always clean the forceps, spatula and marble block with lab wipes between samples.

Prepare analytical sample run:

  1. Start the run sequence by preparing a “bypass” sample containing material with a C and N content anticipated for samples (well #A1). Record the well number for each sample on the data spreadsheet.
  2. Always clean the forceps, spatula and marble block with lab wipes between samples.
  3. Next add a “blank” consisting of an empty folded tin capsule (well #A2).
  4. Follow with four replicates of a known standard, increasing the weight of sample with each replicate (0.2-0.3mg; 0.5-0.6 mg; 1.0-1.2 mg; and 2.0-2.8 mg). These samples are used to calibrate the analyzer (wells #A3-A6).
  5. Follow with one blind soil standard, prepared as above for soil sample (well #A7).
  6. Continue run sequence with samples to be analyzed, preparing three analytical replicates for each sample. Include a blind standard approximately every 12 capsules (well #A8 onward).
  7. Store sample tray in desiccator until analyzed. Follow analyzer protocol.

Acid-fumigation pre-treatment to remove carbonates when present (after Harris et al. 2001):

  1. Prepare samples as above but with the following exceptions: a) use silver capsules instead of tin; b) transfer 40-60 mg of finely pulverized soil to the capsule; and c) leave capsule open, do not fold. Store sample tray in desiccator until ready to proceed.
  2. Add 90 microliters of nanopore water to each open capsule to wet the soil.
  3. Place sample tray in desiccator containing a beaker of concentrated (12M) HCl. Allow at least 6-8 hours for carbonate dissolution, but not more than 24 hours because extended exposure makes silver capsules brittle.
  4. Remove sample tray from desiccator and dry in 60 degree C oven until dry, at least 24 hours.
  5. Fold capsules as described above. Silver capsules can become brittle after drying; be careful not to lose sample when folding. If brittle, enclose silver capsule in a tin capsule to avoid potential loss of sample. Discard sample if loss occurs.

Harris, D., W. R. Horwath, and C. van Kessel. 2001. Acid fumigation of soils to remove carbonates prior to total organic carbon or carbon-13 isotopic analysis. Soil Science Society of America Journal 65:1853-1856.


None needed. The CHN analyzer reports C and N as the percentage of C or N in the dried sample (g C or N per 100 g sample).

Values reported in the KBS LTER Data Catalog are the average of analytical replicates. Total C is equivalent to total organic C (TOC) if inorganic C was not present in the sample or was removed via fumigation.

Analyzer Protocols

Carlo Erba NA1500 Series II Combustion Analyzer Protocol
Costech Elemental Combustion System CHNS-O (ECS 4010)Protocol

Date modified: Tuesday, Oct 24 2023