AOAC OFFICIAL METHODS OF AN

ALYSIS
ANIMALFEED (2019)
Chapter 4. p. 34
digestion procedures must be used in order to include
N
4.2.11 nitrate.) The amount of protein in most materials is
AOAC Official Method2001.11 multiplying % N by 6.25, because most proteins contain
Protein (Crude) in Animal Feed, The H2S04 and NaOH used are in concentratedform
N
Forage (Plant Tissue), Grain, and Oilseeds highly corrosive. wear gloves and eye protectionwhile
Block Digestion MethodUsing Copper Catalyst the chemicals. Do not mix concentrated acid and NaOH
and Steam Distillation into Boric Acid directly.
chemicals are splashed on the skin or in the eyes, flush with If
First Action 2001 medical attention.
Final Action 2005 amounts of water. Seek Do not breathe
the
oxide fumes produced during digestion.
[Applicable to the determination of 0.5—50%Kjeldahl N (3—
300% equivalent crude protein) in forage, animal feed and pet food, B. Apparatus
grain, and oilseeds, and applicable to the same matrixes as 976.05 (a) Digestion block.—Aluminum alloy block with adjustable
(see 4.2.05), 976.06 (see 4.2.06), 984.13 (see 4.2.09), 988.05 (see temperature device for measuring and controlling blocktemperature
4.2.03), and 990.02 (see 4.2.07); the method does not measure (Tecator Digestion System 20, 1015 Digestor, Foss NorthAmerica,
oxidized forms of N or heterocyclic N compounds.]
Eden Prairie, MN, USA; Tel: +1-952-974-9892,Fax:
See Tables 2001.11 A and B for results ofthe interlaboratory study,
9823, [email protected]; or equivalent).
expressed on a protein bat is (N x 6.25), supporting acceptance of
(b) Digestion tubes.—250 mL.
the method.
(c) Distillation units.—(l) For steam distillations—Foss
Tecator
A. Principle 2200, or equivalent, to accept 250 mL digestion tubes and 500
The material is digested in H2S04 to convert the protein N to titration flasks. (2) For steam distillation and autotitration.—Fos
(NH4)2S04 at a boiling point elevated by the addition of K2S04 with Tecator 2300, or equivalent.
a Cu catalyst to enhance the reaction rate. Ammonia is liberated (d) Titration flask.—500 mL graduated Erlenmeyerflask(for
by alkaline steam distillation and quantified titrimetrically with collection and titration of distillate).
standardized acid. Aluminum block heaters increase the efficiency (e) Fume exhaust manifold.—With Teflon ring seals, connected
of the digestion. to a water aspirator in a hooded sink.
The digest must contain residual H2S04 to retain the NH;. Water (f) Weighing paper.—Low N, Alfie Packers No. 201(Alfie
is added manually or automatically to the digest to avoid mixing Packers, Inc., Omaha, NE, USA), or Fisher 09-898-12A,3 x 3 in.
concentrated alkali with concentrated acid and to prevent the digest (76 x 76 mm), or equivalent.
from solidifying. Concentrated NaOH is added to neutralize the (g) Pipetting dispenser.—25 mL, adjustable volume, attached
to
acid and make the digest basic, and the liberated NH* is distilled a 5 pint (2.4 L) acid bottle.
into a boric acid solution and titrated with a stronger standardized C. Reagents
acid, HCI, to a colorimetric endpoint. The same endpoint detection
(a) Sulfuric acid. —Concentrated, 95—98% reagentgrade.
system (e.g., indicator, wave length) must be used for the
standardization of the HCI and for the analyte. (b) Catalyst.—7.0 g K2S04 + 0.8 g CuSOa. (Commercially
available in tablet form as 3.5 g K.S04 and 0.4 g pertablet.)
The analyte is referred to as "crude" protein because the method
(c) Sodium hydroxide (w/w) NaOH, lowN
determines N, a component of all proteins. In addition, N from
sources other than true protein is also determined. (Additional ($5 pg N/g).

copper catalystand
Table 2001.11A. Interlaboratory study results for the determination of crude protein by block digestion with a
distillation into 4% boric acid
No. of labs" Mean. % RSD,. % RSDR. % HorRat
40.19 0.45 0.76 0.333
Protein block 10(1)
0.60 0.256
Swine pellets 10(1) 37.04 0.47
7.10 1.64 2.16 0126
Com silage 11
1.94 0.650
Grass hay 11 7.11
0.460
Fish meal 11 64.67 0.73 0.98
0.369
Dog food 11 24.50 0.87 0.91
0.383
Chinchilla food 11 18.01 0.89 0.99
0.212
Albumin 10(1) 79.14 0.40 0.44
0.475
Birdseed 11 13.48 0.88 1.29
0857
Meat and bone meal 11 50.06 1.90 1.90
0.550
Milk replacer 11 20.78 1.39 1.39
0.236
Soy beans 9(2) 38.76 0.49 0.54
0.916
Sunflower seeds 11 17.43 2.38 2.38
0565
Legume hay 11 18.81 1.45 1.45
outliers
a Each value is the number of laboratories retained after elimination of outliers; each value in parentheses is the number of laboratories removedas

@ 2019 AOAC INTERNATIONAL

 OFFICIALMETHODS OF ANALYSIS (2019) ANIMALFEED
Chapter 4, p. 35

2001.11B. Interlaboratory study rosults for tho rocovory of nitrogen from standard compounds by block digestion with a
distillation Into boric acid
coppgsat'lyst and
No. of labs" Theoretical yield. % N Avg. found. % N Avg. rec.. % HorRat
éompound
10(0) 10.36 10.37 100.1 1.50 0.53
Acetanilid
10(0) 15.34 13.32 86.8 4.16 1.53
Lysine•HCl
10(0) 13.72 13.55 98.8 1,04 0.39
Tryptophan
Each value is the number of laboratories retained after elimination of outliers; each value in parentheses is the number of laboratories removed as outliers.

(d) Methylred indicator solution. —Dissolve 100 mg methyl red to a numbered Kjeldahl tube with <20 mL deionized water.
in 100 mL
methanol. Alternatively, weigh slightly >l g well-mixed test portion into
(e) Bromoctvsolgreen indicator solution.—Dissolve 100 mg a small tared beaker. Transfer to a numbered Kjeldahl tube and
hromocresolgreen in 100 mL methanol. reweigh beaker. The differential weight loss corresponds to the
(0 Boricacid solution.—4% (w/v). Dissolve 400 g H3B(m in amount of test portion actually transferred to the tube.
5-0L hotdeionizedwater. Mix and add more hot deionized water (b) Standards.—Perform quality control analysis and analyses
toa volumeof about 9 L. Cool to room temperature, add 100 mL of standards with each batch. The standards available from Hach
bromoctesolgreen solution and 70 mL methyl red solution, and co. (Loveland, CO, USA; Tel: +1-800-2274224 or +1-970-669-
diluteto a final volume of 10 L. Adjust to obtain a positive blank of 3050), Sigma (St. Louis, MO, USA), J.T. Baker (Phillipsburg, NJ,
0.05-0.15mL with 30 mL 1431303solution, using 0.1 M NaOH (to USA), the National Institute of Standards and Technology (NIST;
increaseblank) or 0.1 M HCI (to decrease blank). Commercially Gaithersburg, MD, USA) are listed in Table 2001.1 IC.
available. The various ammonium salts and glycine p-toluenesulfonate
(g) Boricacid solution.—l% (w/v). (Optional trapping solution serve primarily as a check on distillation efficiency and accuracy
for titratorsthat automatically begin titration when distillation in titration steps because they are digested very readily. Lysine
begins.)Dissolve 100 g H B03 in 5—6L hot deionized water, mix, and nicotinic acid p-toluenesulfonate serve as a check on digestion
andadd more hot deionized water to a volume of about 9 L. Cool efficiency because they are difficult to digest.
toroomtemperature,add 100 mL bromocresol green solution and Include a reagent blank tube containing a folded low N weighing
70mLmethylred solution, and dilute to a final volume of 10 L. paper with each batch.
Commerciallyavailable. (c) Digestion.—Add two catalyst tablets to each tube. Add
(h) Hydrochloricacid standard solution.—().1000 M. Prepare 12 mL to each tube, using pipetting dispenser; add 15 mL
as in 936.15(see A.l.06) or use premade solution of certified for high fat materials (>10% fat). Mixtures may be held overnight
specificationrange 0.0995—0.1005M, and use 0.1000 M for at this point. If mixture foams, slowly add 3 mL 30—35%H.O,. Let
calculation.
Commercially available. reaction subside in perchloric acid fume hood or in exhaust system.
(i) Reference standards. —Ammonium sulfate, tryptophan,
lysine•HCl,or glycine p-toluenesulfonic acid, for use as standard; Table 2001.11C. Standards
99.9%.
Approximate Theoretical
(j) Sucrose.—N-free. Standard weight, g yield, % N
D.Preparationof Analytical Sample 7.402
Ammonium p-toluenesulfonate 0.5
Grinddry laboratorysample to fineness of grind (ca 0.7—1mm), (Hach 22779-24)
whichgives a relative standard deviation (RSD) of f2.0% for Glycine p-toluenesulfonate 0.6 5.665
10successivedeterminations of N in ground mixture of corn grain (Hach 22780-24)
andsoybeans(2 + l). Fineness required to achieve this precision Nicotinic acid p-toluenesulfonate 0.2 4.743
mustbe used for all dry mixed feeds and other nonuniform (Hach 22781-24)
materials.Mix liquids to Lysine monohydrochloride 0.1 15.34
uniformity.
E. Determination (Sigma L-5626)
Acetanilide (Baker A068-05) 0.3 10.36
(a) Digestion.—Turn
on block digestor and heat to 4200C. Tryptophan (Sigma T 8659) 0.2 13.72
Weighmaterials,
as indicated below, recording each test portion
weight(W)to Ammonium salts
the nearest mg for weights of 21 g, and to the nearest
01 mgfor 0.2 21.21
weights of<l .0 g. Do not exceed 1.2 g. For materials Diammonium hydrogen phosphate
With3-25%protein, (100% assay)
test portion; with
25-50%protein, weigh approximately 1.0 g 26.18
approximately 0.5 g test portion; and >5()% Ammoniumchloride (100% assay) 0.2
protein,
approximately0.3 g test portion. Ammoniumsulfate (100% assay) 0.2 21.2
(l) Dryfeed,
forage, cereal, grain, oilseeds.—Weigh I g test 0.3 12.18
Portionof Ammonium dihydrogen phosphate
ground, well-mixed
weighingpaper. test portion onto a tared, low (NIST 200)
Fold paper around material and drop into a 2.86
numbered
Kjeldahl tube. Citrus leaves (NIST 1572) 1.0
(2) 0.1 46.63
Liquidfeed.—Weighslightly urea (NIST 2141)
analytical >l g test portion of well-mixed
sampleinto a
small tared beaker. Quantitatively transfer
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