4.2.11 tion procedures must be used in order to include N from nitrate.

)
AOAC Official Method 2001.11 The amount of protein in most materials is calculated by
Protein (Crude) in Animal Feed, multiplying % N by 6.25, because most proteins contain 16% N.
Forage (Plant Tissue), Grain, and Oilseeds The H2SO4 and NaOH used are in concentrated form and are
Block Digestion Method Using Copper Catalyst highly corrosive. Wear gloves and eye protection while handling
and Steam Distillation into Boric Acid the chemicals. Do not mix concentrated acid and NaOH directly.
First Action 2001 If chemicals are splashed on the skin or in the eyes, flush with
[Applicable to the determination of 0.5–50% Kjeldahl N (3– copious amounts of water. Seek medical attention. Do not breathe
300% equivalent crude protein) in forage, animal feed and pet the sulfur oxide fumes produced during digestion.
food, grain, and oilseeds, and applicable to the same matrixes as B. Apparatus
976.05 (4.2.05), 976.06 (4.2.06), 984.13 (4.2.09), 988.05 (4.2.03), (a) Digestion block.—Aluminum alloy block with adjustable
and 990.02 (4.2.07); the method does not measure oxidized forms temperature device for measuring and controlling block temperature
of N or heterocyclic N compounds.] (Tecator Digestion System 20, 1015 Digestor, Foss North
See Tables 2001.11A and B for the results of the interlaboratory America, 7682 Executive Dr, Eden Prairie, MN
study, expressed on a protein basis (N  6.25), supporting accep- 55344, USA;
tance of the method. +1-952-974- 9892, Fax: + 1 - 9 5 2 - 9 7 4 - 9823,
A. Principle info@fossnorthamerica.com; or equivalent).
The material is digested in H SO to convert the protein N to (b) Digestion tubes.—250 mL.
2 4
(c) Distillation units.—(1) For steam distillation.—Foss
Tecator 2200, or equivalent, to accept 250 mL digestion tubes and
(NH4)2SO4 at a boiling point elevated by the addition of K2SO4
500 mL titration flasks. (2) For steam distillation and
with a Cu catalyst to enhance the reaction rate. Ammonia is
autotitration.—Foss Tecator 2300, or equivalent.
liberated by alkaline steam distillation and quantified
(d) Titration flask.—500 mL graduated Erlenmeyer flask (for
titrimetrically with stan- dardized acid. Aluminum block heaters
collection and titration of distillate).
increase the efficiency of the digestion.
(e) Fume exhaust manifold.—With Teflon ring seals,
The digest must contain residual H2SO4 to retain the NH3.
connected to a water aspirator in a hooded sink.
Water is added manually or automatically to the digest to avoid
(f) Weighing paper.—Low N, Alfie Packers No. 201 (Alfie
mixing concentrated alkali with concentrated acid and to prevent
Packers, Inc., 8901 J St, Ste 10, Omaha, NE 68127, USA), or
the digest from solidifying. Concentrated NaOH is added to
Fisher 09-898-12A, 3 3 in. (76  76 mm), or equivalent.
neutralize the acid and make the digest basic, and the liberated NH3
is distilled into a bo- ric acid solution and titrated with a stronger (g) Pipetting dispenser.—25 mL, adjustable volume, attached
standardized acid, HCl, to a colorimetric endpoint. The same to a 5 pint (2.4 L) acid bottle.
endpoint detection system (e.g., indicator, wavelength) must be C. Reagents
used for the standardization of the HCl and for the analyte. (a) Sulfuric acid.—Concentrated, 95–98% H2SO4, reagent
The analyte is referred to as “crude” protein because the method grade.
determines N, a component of all proteins. In addition, N from
sources other than true protein is also determined. (Additional
diges-

Table 2001.11A Interlaboratory study results for the determination of crude protein by block digestion with a copper catalyst and
distillation into 4% boric acid
ID No. of labsa Mean, % RSDr, % RSDR, % HORRAT
Protein block 10(1) 40.19 0.45 0.76 0.333
Swine pellets 10(1) 37.04 0.47 0.60 0.256
Corn silage 11 7.10 1.64 2.16 0.726
Grass hay 11 7.11 1.94 1.94 0.650
Fish meal 11 64.67 0.73 0.98 0.460
Dog food 11 24.50 0.87 0.91 0.369
Chinchilla food 11 18.01 0.89 0.99 0.383
Albumin 10(1) 79.14 0.40 0.44 0.212
Birdseed 11 13.48 0.88 1.29 0.475
Meat and bone meal 11 50.06 1.90 1.90 0.857
Milk replacer 11 20.78 1.39 1.39 0.550
Soybeans 9(2) 38.76 0.49 0.54 0.236
Sunflower seeds 11 17.43 2.38 2.38 0.916
Legume hay 11 18.81 1.45 1.45 0.565
a
Each value is the number of laboratories retained after elimination of outliers; each value in parentheses is the number of laboratories removed
as outliers.

© 2002 AOAC
INTERNATIONAL
Table 2001.11B Interlaboratory study results for the recovery of nitrogen from standard compounds by block digestion with a cop-
per catalyst and distillation into boric acid
Compound No. of labsa Theoretical yield, % N Avg. found, % N Avg. rec., % RSDR, % HORRAT
Acetanilid 10(0) 10.36 10.37 100.1 1.50 0.53
LysineHCl 10(0) 15.34 13.32 86.8 4.16 1.53
Tryptophan 10(0) 13.72 13.55 98.8 1.04 0.39
a
Each value is the number of laboratories retained after elimination of outliers; each value in parentheses is the number of laboratories removed
as outliers.

(b) Catalyst.—7.0 g K2SO4 + 0.8 g CuSO4. (Commercially (1) Dry feed, forage, cereal, grain, oilseeds.—Weigh 1 g test por-
avail- able in tablet form as 3.5 g K 2SO4 and 0.4 g CuSO4 per tion of ground, well-mixed test portion onto a tared, low N weighing
tablet.)
(c) Sodium hydroxide solution.—40% (w/w) NaOH, low N
(5 g N/g).
(d) Methyl red indicator solution.—Dissolve 100 mg methyl
red in 100 mL methanol.
(e) Bromocresol green indicator solution.—Dissolve 100 mg
bromocresol green in 100 mL methanol.
(f) Boric acid solution.—4% (w/v). Dissolve 400 g H3BO3 in
5–6 L hot deionized water. Mix and add more hot deionized water to
a volume of about 9 L. Cool to room temperature, add 100 mL
bromocresol green solution and 70 mL methyl red solution, and
dilute to a final volume of 10 L. Adjust to obtain a positive blank of
0.05–0.15 mL with 30 mL H3BO3 solution, using 0.1M NaOH (to
increase blank) or 0.1M HCl (to decrease blank). Commercially
available.
(g) Boric acid solution.—1% (w/v). (Optional trapping solution
for titrators that automatically begin titration when distillation be-
gins.) Dissolve 100 g H3BO3 in 5–6 L hot deionized water, mix,
and add more hot deionized water to a volume of about 9 L. Cool
to room temperature, add 100 mL bromocresol green solution and
70 mL methyl red solution, and dilute to a final volume of 10 L.
Commer- cially available.
(h) Hydrochloric acid standard solution.—0.1000M. Prepare
as in 936.15 (see A.1.06) or use premade solution of certified
specifica- tion range 0.0995–0.1005M, and use 0.1000M for
calculation. Com- mercially available.
(i) Reference standards.—Ammonium sulfate, tryptophan,
lysine·HCl, or glycine p-toluenesulfonic acid, for use as standard;
99.9%.
(j) Sucrose.—N-free.

D. Preparation of Analytical Sample

Grind dry laboratory sample to fineness of grind (ca 0.7–1 mm),
which gives a relative standard deviation (RSD) of 2.0% for 10
suc- cessive determinations of N in ground mixture of corn grain
and soy- beans (2 + 1). Fineness required to achieve this precision
must be used for all dry mixed feeds and other nonuniform
materials. Mix liquids to uniformity.
E. Determination
(a) Digestion.—Turn on block digestor and heat to 420C.
Weigh materials, as indicated below, recording each test portion
weight (W) to the nearest mg for weights of 1 g, and to the
nearest
0.1 mg for weights of <1.0 g. Do not exceed 1.2 g. For materials
with 3–25% protein, weigh approximately 1.0 g test portion; with
25–50% protein, approximately 0.5 g test portion; and >50% pro-
tein, approximately 0.3 g test portion.
© 2002 AOAC
INTERNATIONAL
paper. Fold paper around material and drop into a numbered
Kjeldahl tube.
(2) Liquid feed.—Weigh slightly >1 g test portion of well-
mixed analytical sample into a small tared beaker.
Quantitatively transfer to a numbered Kjeldahl tube with <20
mL deionized water. Alterna- tively, weigh slightly >1 g well-
mixed test portion into a small tared beaker. Transfer to a
numbered Kjeldahl tube and reweigh beaker. The differential
weight loss corresponds to the amount of test por- tion
actually transferred to the tube.
(b) Standards.—Perform quality control analysis and
analyses of standards with each batch. The standards available
from Hach Co. (PO Box 389, Loveland, CO 80539, USA; +1-
800-227-4224 or +1-
970-669-3050), Sigma (St. Louis, MO), J.T. Baker
(Phillipsburg, NJ), the National Institute of Standards and
Technology (NIST; Gaithersburg, MD) are listed in Table
2001.11C.
The various 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 nic- otinic acid p-
toluenesulfonate serve as a check on digestion effi- ciency
because they are difficult to digest.
Include a reagent blank tube containing a folded low N
weighing paper with each batch.
(c) Digestion.—Add 2 catalyst tablets to each tube. Add 12
mL H2SO4 to each tube, using pipetting dispenser; add 15 mL
for high fat materials (>10% fat). Mixtures may be held
overnight at this point. If mixture foams, slowly add 3 mL 30–
35% H2O2. Let reaction subside in perchloric acid fume hood
or in exhaust system.
Attach heat side shields to tube rack. Place fume manifold
tightly on tubes, and turn water aspirator on completely. Place
rack of tubes in preheated block. After 10 min, turn water
aspirator down until acid fumes are just contained within
exhaust hood. A condensation zone should be maintained
within the tubes. After bulk of sulfur ox- ide fumes are
produced during initial stages of digestion, reduce vac- uum
source to prevent loss of H2SO4. Digest additional 50 min.
Total digestion time is approximately 60 min.
Turn digestor off. Remove rack of tubes with exhaust still in
place, and put in the stand to cool for 10–20 min. Cooling can
be increased by using commercial air blower or by placing in
hood with hood sash pulled down to increase airflow across
tubes. When fuming has stopped, remove manifold, and shut
off aspirator. Remove side shields. Let tubes cool. Wearing
gloves and eye protection, predilute digests manually before
distilling. Carefully add a few milliliters of deionized water to
each tube. If spattering occurs, the tubes are too hot. Let cool
for a few more minutes. Add water to each tube to a to- tal
volume of approximately 80 mL (liquid level should be about
half way between the 2 shelves of the tube rack). This is a
convenient stopping point.
If digest solidifies, place tube containing diluted digest in
block digester, and carefully warm with occasional swirling
until salts dis-

© 2002 AOAC
INTERNATIONAL
Table 2001.11C Standards
tration immediately after distillation starts, 1% H3BO3 may be sub-
Approxima Theoretic stituted.) Steam distill until 150 mL distillate is collected (180 mL
te al
Standard weight, g yield, % N total volume). Remove receiving flask. Titrate H3BO3 receiving so-
Ammonium p- lution with standard 0.1000M HCl to violet endpoint (just before the
0.5
toluenesulfonate (Hach solution goes back to pink). Lighted stir plate may aid visualization
22779-24)
7.402 of endpoint. Record milliliters of HCl to at least the nearest 0.05
Glycine p- mL. This is done automatically by using a steam distiller with auto-
toluenesulfonate matic titration. Follow the manufacturer’s instructions for operation
0.6
(Hach 22780-24)
of the specific distiller or distiller/titrator.
Nicotinic acid p- 5.665
toluenesulfonate (Hach F. Verification of Nitrogen Recovery
22781-24) Run N recoveries to check accuracy of procedure and equipment.
0.2
Lysine
monohydrochloride 4.743
(Sigma L-5626)
0.1 15.34
Acetanilide (Baker A068-05) 0.3 10.36 (a) Nitrogen loss.—Use 0.12 g (NH4)2SO4 and 0.67 g sucrose
Tryptophan (Sigma T 8659) 0.2 13.72 per flask. Add all other reagents as in E, and distill under same
Ammonium salts condi- tions as in E. Recoveries must be 99%.
Diammonium hydrogen 0.2 (b) Distillation and titration efficiency.—Distill 0.12 g
phosphate (100% assay) (NH4)2SO4, omitting digestion. Recoveries must be 99.5%.
21.21
Ammonium chloride (100% assay) 0.2 26.18 (c) Digestion efficiency.—Use 0.3 g acetanilide or 0.18 g
Ammonium sulfate (100% assay) 0.2 21.2 tryptophan, with 0.67 g sucrose per flask. Add all other reagents
as
Ammonium dihydrogen stated in E. Digest and distill under same conditions as used for a
0.3
phosphate (NIST 200) de- termination. Recoveries must be 98%.
12.18
Citrus leaves (NIST 1572) 1.0 2.86
G. Calculations
Urea (NIST 2141) 0.1 46.63
Kjeldahl (VS VB )  M 14.01
nitrogen, % =
W 10

solve. If distilling unit equipped with steam addition for equilibration Crude protein, % = % Kjeldahl N  F
is used, the manual dilution steps can be omitted. About 70 mL
deionized water is then automatically added during the distillation where VS = volume (mL) of standardized acid used to titrate a
cycle. test; VB = volume (mL) of standardized acid used to titrate reagent
(e) Distillation.—Place 40% NaOH in alkali tank of distillation blank; M = molarity of standard HCl; 14.01 = atomic weight of N;
unit. Adjust volume dispensed to 50 mL. Attach digestion tube W = weight (g) of test portion or standard; 10 = factor to convert
con- taining diluted digest to distillation unit, or use automatic mg/g to percent; and F = factor to convert N to protein.
dilution feature, if available. Place graduated 500 mL Erlenmeyer F factors are 5.70 for wheat, 6.38 for dairy products, and 6.25
titration flask containing 30 mL H3BO3 solution with indicator on for other feed materials.
receiving platform, and immerse tube from condenser below Reference: J. AOAC Int. (future issue).
surface of H3BO3 solution. (When an automatic titration system is
used that begins ti-

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