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
The methods described are applicable for determination of
nitrogen (N) in forages.
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
³1.36 (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
- 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
- Keep baking soda and vinegar handy in case of chemical
- 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.
- 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.
- 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
- Place flask on preheated burner (adjusted to bring 250 mL
water at 25oC to rolling boil in 5 min).
- 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.
- 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.
- 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.
- Add 2 to 3 drops of tributyl citrate or other antifoam
agent to digestion flask to reduce foaming.
- Add another 0.5 to 1.0 g alundum granules.
- 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
- 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.
- Remove digestion flask and titrating flask from unit,
rinsing the condenser tube with distilled water as the
flask is being removed.
- 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
Following is a list of acceptable standards available to
include in Kjeldahl runs:
|Theoretical Yield Standard
|Ammonium p-toluenesulfonate (Hach 22779-24)
|Glycine p-toluenesulfonate (Hach 22780-24)
|Nicotinic acid p-toluenesulfonate (Hach 22781-24)
|Lysine monohydrochloride (Sigma L-5626 or Aldrich
|Various ammonium salts Diammonium hydrogen phosphate
|Ammonium chloride (100% assay)
|Ammonium sulfate (100% assay)
|Ammonium dihydrogen phosphate (NIST SRM 194)
|Citrus leaves (NIST SRM 1572)
|Urea (NIST SRM 2141)
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
- 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
- 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
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.