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3.1 Nitrogen Determination by Kjeldahl (Rack)

Protein (Crude) Determination in Animal Feed: Copper Catalyst Kjeldahl Method. (984.13) Official Methods of Analysis. 1990. Association of Official Analytical Chemists. 15th Edition.

Protein (Crude) Determination in Animal Feed: CuSO4/TiO2 Mixed Catalyst Kjeldahl Method. (988.05) Official Methods of Analysis. 1990. Association of Official Analytical Chemists. 15th Edition.

The methods described are applicable for determination of nitrogen (N) in forages.

Basic Principle:
The Kjeldahl method is the standard method of nitrogen determination dating back to its development in the late 1800's. The method consists of three basic steps: 1) digestion of the sample in sulfuric acid with a catalyst, which results in conversion of nitrogen to ammonia; 2) distillation of the ammonia into a trapping solution; and 3) quantification of the ammonia by titration with a standard solution.

Kjeldahl flasks, 500 to 800 mL Kjeldahl digestion unit with fume removal manifold Kjeldahl distillation apparatus - Kjeldahl flask connected to distillation trap by rubber stopper. Distillation trap is connected to condenser with low-sulfur tubing. Outlet of condenser should be less than 4 mm diameter. Erlenmeyer flask, 500 mL Analytical balance, sensitive to 0.1 mg

Sulfuric acid, concentrated, 95-98%, reagent grade Sodium hydroxide, pellets, flakes, or 45% solution with specific gravity 1.36 (low N) dissolve 450 g in cool water and dilute to 1 L Potassium sulfate (K2SO4), anhydrous Copper sulfate (CuSO4), anhydrous Titanium dioxide (TiO2) Alundum, boiling stones, 8-14 mesh Pumice Methyl red indicator dissolve 1 g methyl red (sodium salt) in 100 mL methanol or ethanol Tributyl citrate (for antifoam) or paraffin or antifoam A or equivalent Lysine monohydrochloride, reagent grade, dried at 110OC for four hr Hydrochloric acid standard solution, 0.5 N Prepare by diluting 430.1 mL 36.5 to 38% HCl to 10 L with distilled water and standardize by method 3.1.1 Sodium hydroxide standard solution Prepare 0.1 N sodium hydroxide (NaOH) solution and standardize by method 3.1.2. After standardizing hydrochloric acid and sodium hydroxide, check one against the other by titrating one with the other and calculating normality.

Safety Precautions:

  • Handle acid safely: use acid resistant fumehood. Always add acid to water unless otherwise directed in method. Wear face shield and heavy gloves to protect against splashes. If acids are spilled on skin, immediately wash with large amounts of water.
  • Sulfuric acid and sodium hydroxide can burn skin, eyes and respiratory tract severely. Wear heavy rubber gloves and face shield to protect against concentrated acid or alkali. Use effective fume removal device to protect against acid fumes or alkali dusts or vapors. Always add concentrated sulfuric acid or sodium hydroxide pellets to water, not vice versa. Concentrated sodium hydroxide can quickly and easily cause blindness. If splashed on skin or in eyes, flush with copious amounts of water and seek medical attention.
  • Keep baking soda and vinegar handy in case of chemical spills.
  • The sulfur oxide fumes produced during digestion are hazardous to breathe. Do not inhale.
  • Digests must be cool before dilution water is added to avoid a violent reaction during which the acid can shoot out of the flask. Likewise, the diluted digest must be cool before sodium hydroxide is added to avoid a similarly violent reaction.

Procedure: Digestion

  1. Weigh approximately 1 g ground sample into digestion flask, recording weight (W) to nearest 0.1 mg. Include reagent blank and high purity lysine HCl as check of correctness of digestion parameters. Weigh a second subsample for laboratory dry matter determination.
  2. Add 15 g potassium sulfate, 0.04 g anhydrous copper sulfate, 0.5 to 1.0 g alundum granules, or add 16.7 g K2SO4, 0.01 g anhydrous copper sulfate, 0.6 g TiO2 and 0.3 g pumice. Then add 20 mL sulfuric acid. (Add additional 1.0 mL sulfuric acid for each 0.1 g fat or 0.2 g other organic matter if sample weight is greater than 1 g.)
  3. Place flask on preheated burner (adjusted to bring 250 mL water at 25oC to rolling boil in 5 min).
  4. Heat until white fumes clear bulb of flask, swirl gently, and continue heating for 90 min for copper catalyst or 40 min for CuSO4/TiO2 mixed catalyst.
  5. Cool, cautiously add 250 mL distilled water and cool to room temperature (less than 25oC). Note: If bumping occurs during distillation, volume of water may be increased to ca. 275 mL.


  1. Prepare titration flask by adding appropriate volume (VHCl) accurately measured acid standard solution to amount of water so that condenser tip is immersed (try 15 mL acid and 70 mL water if undecided). For reagent blank, pipet 1 mL of acid and add approximately 85 mL water. Add 3 to 4 drops methyl red indicator solution.
  2. Add 2 to 3 drops of tributyl citrate or other antifoam agent to digestion flask to reduce foaming.
  3. Add another 0.5 to 1.0 g alundum granules.
  4. Slowly down side of flask, add sufficient 45% sodium hydroxide solution (approximately 80 mL) to make mixture strongly alkali. (Do not mix until after flask is connected to distillation apparatus or ammonia will be lost.)
  5. Immediately connect flask to distillation apparatus and distill at about 7.5 boil rate (temperature set to bring 250 mL water at 25oC to boil in 7.5 min) until at least 150 mL distillate is collected in titrating flask.
  6. Remove digestion flask and titrating flask from unit, rinsing the condenser tube with distilled water as the flask is being removed.


  1. Titrate excess acid with standard sodium hydroxide solution to orange endpoint (color change from red to orange to yellow) and record volume to nearest 0.01 mL (VNaOH). Titrate the reagent blank (B) similarly.


  • Reagent proportions, heat input and digestion time are critical factors - do not change.
  • Ratio of salt to acid (wt:vol) should be 1:1 at end of digestion for proper temperature control. Digestion may be incomplete at lower ratio; nitrogen may be lost at higher ratio. Each gram of fat consumes 10 mL sulfuric acid and each gram of carbohydrate consumes 4 mL sulfuric acid during digestion.
  • Catalyst mixtures are commercially available in powdered or tablet form. Dispensers are available for convenient delivery of powdered catalyst mixtures.
  • Check with local authorities for proper disposal procedures of copper containing waste solution.
  • Include a reagent blank and at least one sample of high purity lysine hydrochloride in each day's run as check of correctness of digestion parameters. If digestion is not complete, make appropriate adjustments. A standard, such as NIST Standard Reference Material No. 194, ammonium phosphate (NH4H2PO4), certified 12.15%N should also be included.

Following is a list of acceptable standards available to include in Kjeldahl runs:

Theoretical Yield Standard % nitrogen
Ammonium p-toluenesulfonate (Hach 22779-24) 7.402
Glycine p-toluenesulfonate (Hach 22780-24) 5.665
Nicotinic acid p-toluenesulfonate (Hach 22781-24) 4.743
Lysine monohydrochloride (Sigma L-5626 or Aldrich Gold Label) 15.34
Various ammonium salts Diammonium hydrogen phosphate (100% assay) 21.21
Ammonium chloride (100% assay) 26.18
Ammonium sulfate (100% assay) 21.20
Ammonium dihydrogen phosphate (NIST SRM 194) 12.15
Citrus leaves (NIST SRM 1572) 2.86
Urea (NIST SRM 2141) 46.63

The ammonium salts and glycine p-toluenesulfonate serve primarily as a check on distillation efficiency and accuracy in titration steps because they are digested very readily. Lysine and nicotinic acid are difficult to digest, therefore serve as a check on digestion efficiency.

Calculation: Percent Nitrogen (N)
%N (DM basis) =[(VHCl x NHCl) - (VBK x NNaOH) -(VNaOH x NNaOH)]/1.4007 X W X Lab DM/100

  • Where VNaOH = mL standard NaOH needed to titrate sample
  • VHCl = mL standard HCl pipetted into titrating flask for sample
  • NNaOH = Normality of NaOH
  • NHCl = Normality of HCl
  • VBK = mL standard NaOH needed to titrate 1 mL standard HCl minus B
  • B = mL standard NaOH needed to titrate reagent blank carried through method and distilled into 1 mL standard HCl
  • 1.4007 = milliequivalent weight of nitrogen x 100
  • W = sample weight in grams

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 high purity lysine hydrochloride, 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. 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 an 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. Nitrogen and Crude Protein Determination

3.1A Addendum to Nitrogen Determination... >>

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