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3.3 Nitrogen Determination by Combustion Method

Protein (Crude) in Animal Feed: Combustion Method. (990.03) Official Methods of Analysis. 1990. Association of Official Analytical Chemists. 15th Edition.

This method is applicable for the determination of nitrogen in all types of forages.

Basic Principle:
Nitrogen freed by combustion at high temperature in pure oxygen is measured by thermal conductivity detection and converted to equivalent protein by appropriate numerical factor.

Any instrument or device designed to measure nitrogen by combustion may be used which is equipped to provide following conditions:

  • Furnace to maintain minimum operating temperature of 950oC for pyrolysis of sample in pure (99.9%) oxygen. Some systems may require higher temperature.
  • Isolation system to isolate liberated nitrogen gas from other combustion products for subsequent measurement by thermal conductivity detector. Device for converting NO2 products to N2 or measuring N as NO2 may be required and included in the system.
  • Detection system to interpret detector response as % nitrogen (weight/weight). May include features such as calibration on standard material, blank determination and barometric pressure compensation. Any required calibration must be based on theoretical % nitrogen in pure primary standard organic material such as NIST SRM Uric Acid 913 or EDTA.

Safety Precautions:

  • Follow manufacturer's recommendation for safe operation of instrument.
  • Secure compressed gas cylinders and use proper gas regulators.

Operate instrument according to manufacturer's instructions; following are generalized instructions.

  1. Turn furnaces on (or take off standby).
  2. Turn gas regulators to desired flow rate.
  3. Wait until furnaces have stabilized at desired temperature.
  4. Enter sample number on console.
  5. Enter other parameters as required by computer software.
  6. Enter appropriate N content of pure primary standard.
  7. Include two blanks and three dried or dessicated pure primary standards at the beginning of each run to calculate the calibration factor for determining N.
  8. Weigh samples and transfer to autosampler tray. Weigh a second subsample to determine laboratory dry matter.
  9. Run samples.


  • System must be capable of measuring nitrogen in feed materials containing 0.2 to 20% nitrogen.
  • Suitable fineness of grind is that which gives relative standard deviation (RSD) 2.0% for 10 successive determinations of nitrogen in mixture of corn grain and soybeans (2/3and 1/3) that has been ground for analysis. RSD, % = (standard deviation divided by mean %N) times 100. Fineness of grind (about 0.5 mm) required to achieve this precision must be used for all mixed feeds and other nonhomogeneous materials.

Calculation: Percent Nitrogen (N)

% N (DM basis) =% N (from analyzer output) Lab DM/100

Calculation: Percent Crude Protein (CP)

CP (DM basis)= % N (DM basis) X F

  • F = 6.25 for all forages and feeds except wheat grains
  • F = 5.70 for wheat grains

Quality Control:
Include a reagent blank, one sample of NIST SRM Uric Acid 913, and one or more quality control (QC) samples in each run, choosing QC samples by matching analyte levels and matrices of QC samples to the samples in the run. Include at least one set of duplicates in each run if single determinations are being made. Accuracy of system is demonstrated by making 10 successive determinations of nitrogen in nicotinic acid and 10 successive determinations in lysine.monohydrochloride. Means of determinations must be within 0.15 of the respective theoretical values, with standard deviations 0.15. Standard tryptophan may be substituted for lysine.monohydrochloride.

An acceptable average standard deviation among replicated analyses for crude protein ranges from about 0.10 for samples with 10% CP to 0.20 for samples with 20% CP, which results in warning limits (2s) ranging from 0.20 to 0.40 and control limits (3s) ranging from 0.30 to 0.60. Plot the results of the control sample(s) on a X-control chart and examine the chart for trends. Results outside of upper or lower warning limits, 2s (95 percent confidence limits), are evidence of possible problems with the analytical system. Results outside of upper or lower control limits, 3s (99 percent confidence limits), indicate loss of control and results of the run should be discarded. Two consecutive analyses falling on one side of the mean between the warning limits and the control limits also indicate loss of control.


<< 3.2 Nitrogen Determination by Kjeldahl (Block Digestion)

3.4 Crude Protein by Near Infrared Reflectance Spectroscopy >>

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