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03 white fumes clear bulb of flask (ca 10 min), swirl gently, and con-
AOAC Official Method 988.05 tinue heating additional 40 min. (Note: Reagent proportions, heat in-
Protein (Crude) in Animal Feed and Pet Food
put, and digestion time are critical factors—do not change.) Cool,
CuSO4/TiO2 Mixed Catalyst Kjeldahl Method
First Action 1988 cautiously add about 250 mL H2O, and cool to room temperature.
Final Action 1990 (Note: Add H2O as soon as possible to reduce amount of caking. If
AOAC–AACC Method excessive bumping occurs during distillation, increase dilution H2O
A. Principle from 250 mL to ca 300 mL.)
Test portion is digested in H2SO4, using CuSO4/TiO2 as catalysts, Prepare titration beaker by adding appropriate volume of acid
converting N to NH3 which is distilled and titrated. standard solution to amount of H2O such that condenser tip will be
sufficiently immersed to trap all NH3 evolved. Add 3–4 drops of in-
B. Reagents
dicator solution, B(c).
(a) Sodium hydroxide solution.—Dissolve ca 450 g NaOH pel- Add additional 0.5–1.0 g Alundum granules to cooled digestion
lets or flakes (low N) in H2O, cool, and dilute to 1 L; or use solution flask. Optionally, 2–3 drops of tributyl citrate may also be added to
with specific gravity ≥1.36. reduce foaming. Slowly down side of flask, add sufficient NaOH so-
(b) Boiling stones.—Alundum, 8–14 mesh (No. 1590-D18; lution, B(a), such that mixture will be strongly alkaline.
Thomas Scientific Co.). Immediatley connect flask to distillation apparatus, mix completely,
(c) Methyl red indicator.—1 g/100 mL. Dissolve 1 g methyl red and distill at ca 7.5-min boil rate until ≥150 mL distillate is collected
(sodium salt) in 100 mL methanol. in titration beaker.
(d) Hydrochloric standard solution.—0.5M, or sulfuric acid Titrate excess standard acid in distillate with NaOH standard solu-
standard solution.—0.25M. Prepare as in 936.15 (see A.1.06) or tion, B(e). Correct for blank determination on reagents. Calculate
890.01 (see A.1.14). percent nitrogen as follows.
(e) Sodium hydroxide standard solution.—0.1M. Prepare as in When standard HCl is used:
936.16 (see A.1.12).
After standardizing both acid and base by methods suggested in N, % =
(d) and (e), also check one against the other. In addition, check entire [(M acid )(mL acid ) − (mL bk )(M NaOH ) − (mL NaOH )(M NaOH )] ×1400.67
method by analyzing NIST Standard Reference material No. 194, test portion wt, mg
NH4H2PO4, certified 12.15% N, and a high purity lysine⋅HCl.
C. Apparatus When standard H2SO4 is used:
(a) Digestion.—Kjeldahl flasks with capacity of 500–800 mL.
(b) Distillation.—Digestion flask connected to distillation trap N, % =
by rubber stopper. Distillation trap is connected to condenser with [(M acid )(2)(mL acid ) − (mL bk )(M NaOH ) − (mL NaOH )(M NaOH )] ×1400.67
low-S tubing. Outlet of condenser tube should be <4 mm diameter. test portion wt, mg
D. Determination
Weigh 0.250–1.000 g test portion into digestion flask. Add 16.7 g
K2SO4, 0.01 g anhydrous CuSO4, 0.6 g TiO2, 0.3 g pumice, 0.5–1.0 g where mLNaOH = mL standard base used for titration; mLacid = mL
Alundum granules, and 20 mL H2SO4. (Add additional 1.0 mL standard acid used as trapping solution; mLbk = mL standard base
H2SO4 for each 0.1 g fat or 0.2 g other organic matter if test portion needed to titrate 1 mL standard acid minus mL standard base needed
weight is >1 g.) to titrate reagent blank carried through method and distilled into
1 mL standard acid; Macid = molarity of standard acid; MNaOH =
Include at least 1 flask with high purity lysine⋅HCl in each day’s
molarity of standard base. Calculate percent (w/w) crude protein,
run as check of correctness of digestion parameters. If recovery is
defined as 6.25 ×percent nitrogen, or 5.7 ×percent nitrogen for wheat
not complete, make appropriate adjustments.
To digest test portion, first adjust heat to bring 250 mL H2O at
25°C to rolling boil in 5 min. Add a few boiling chips to prevent Reference: JAOAC 70, 907(1987).
superheating. Then heat samples at this 5-min boil rate until dense Revised: March 1996