4.4.03
Preparation of Standard Curve
AOAC Official Method 968.07
Nitrogen (Nitrate and Nitrite)
in Animal Feed
Colorimetric Method
First Action 1968
Final Action 1970
A. Principle
Nitrate and nitrite are extracted with cadmium and barium
chloride solution. Bulk of soluble proteins are precipitated in
alkaline solution and clarified solution is passed through metallic Cd
col umn, re duc ing ni trate to ni trite. Ni trite is mea sured
colorimetrically. (Caution: Cd salts are toxic.)
B. Reagents and Apparatus
(a) Ni trate ni tro gen stan dard so lu tions.—(1) Stock
solution.—12 µg nitrate N/mL. Dissolve 0.867 g KNO3 in 1 L H2O.
Dilute 25 mL to 250 mL with H2O. (2) Working solutions.—0.6, 1.2,
1.8, 2.4, 3.0 µg N/mL. Dilute 5, 10, 15, 20, and 25 mL stock solution
to 100 mL with H2O.
(b) Extracting solution.—Dissolve and dilute 50 g CdCl2 and
50 g BaCl2 to 1 L with H2O. Adjust to pH 1 with HCl.
(c) Ammonium chloride buffer solution.—pH 9.6. Dissolve 50 g
NH4Cl in 500 mL H2O and adjust pH with NH4OH. Dilute to 1 L
with H2O.
(d) Sodium hydroxide solution.—2.5M. Dissolve 50 g NaOH in
500 mL H2O.
(e) Sul fa nil amide so lu tion.—0.5%. Dis solve 1.25 g
sulfanilamide in 250 mL HCl (1 + 1). Solution is stable 1–2 months.
(f) Coupling reagent.—Dissolve 0.5 g N-(1-naphthyl)ethylene-
diamine⋅HCl in 100 mL H2O. Store in glass-stoppered dark bottle in
refrigerator. Solution is stable several weeks.
(g) Salt solution.—Dissolve 100 g NaCl in 500 mL H2O. Add
50 mL buffer solution, (c), and dilute to 1 L with H2O.
(h) Re duc ti on tube.—25 mL buret or equiv a lent id
chromatographic tube with stopcock and reservoir.
C. Preparation of Columns
Prepare supply of metallic Cd by placing Zn rods into 500 mL
20% CdSO4 solution. After reaction for 3 h, discard solution and
scrape moss-like Cd growth from Zn rods. Place Cd in high-speed
blender, add 500 mL H2O, and blend 2 s. Wash fine metal particles
with H2O onto sieves, col lecting only 20–40 mesh size. Fill
reduction tube with H2O and add 2 cm plug of glass wool. Press
any trapped air from glass wool as it is pushed to bottom of
column with glass rod. Add Cd to depth of 10 cm, using minimum
of very gentle tapping. Wash column with 25 mL 0.10M HCl, two
25 mL portions H2O, and finally 25 mL buffer, B(c), diluted 1 +
9. Keep col umn covered with salt solution, B(g), when not in use.
Normally columns can be used repeatedly if kept under salt
so lu tion be tween anal y ses. When suc ces sion of highly
proteinaceous or other soluble organic containing materials are
treated, flow rate may decrease gradually. Repeating 25 mL 0.10M
HCl treatment may restore original flow rate; if not, prepare new
column. Reproducible flow rate is important. Actual rate can be
3–5 mL/min but once established, it must be identical (±0.1 mL) for
test solutions and standards.
D.
Prepare standard curve of 3, 6, 9, 12, and 15 µg nitrate–nitrite N
by pipetting 5.0 mL aliquots of working standard solutions into
30 mL beakers. Add 5 mL buffer solution, B(c), and 15 mL H2O,
mix well, and transfer quantitatively to reduction column, using
minimum H2O. Adjust flow rate through column to 3–5 mL/min.
Just as reservoir empties, add 15 mL salt solution, B(g). Collect
eluate, including salt wash, in 50 mL volumetric flask (total
volume of eluate should be ca 40 mL). Add 5 mL sulfanilamide
solution, B(e), mix, and let stand 3 min. Add 2 mL coupling
reagent, B(f), mix, dilute to volume with H2O, mix, and let stand
20 min for maximum color development. Color is stable ≥2 h.
Determine absorbance (A) in 1 cm cells at 540 nm against reagent
blank. Plot A against µg nitrate–nitrite N.
E. Extraction
(a) Low level nitrate samples (grains, meals, supplements,
etc.).—Wash 5.0 g finely ground test portion into 250 mL volumetric
flask. Add 100 mL extracting solution, B(b), and 100 mL H2O, and
mix. Let stand 1 h with occasional swirling. Add 20 mL 2.5M NaOH,
dilute to volume with H2O, mix, and filter immediately through rapid
paper. Pipet 10 mL buffer solution, B(c), into 100 mL volumetric
flask, dilute to volume with clear filtrate, and mix.
(b) Dry, high level ni trate prod ucts (dried plants, hays,
meals, etc.).—Weigh 5.0 g finely ground test por tion into
500 mL vol u met ric flask. Add 100 mL ex tract ing so lu tion,
B(b), and 300 mL H 2O, and mix. Let stand 1 h with oc ca sional
swirl ing, add 40 mL 2.5M NaOH, di lute to vol ume with H 2O,
mix, and fil ter im me di ately through rapid pa per. Pipet 10 mL
buffer so lu tion, B(c), into 100 mL vol u met ric flask, di lute to
vol ume with clear fil trate, and mix.
(c) Grasses, silages, and other wet materials.—Weigh 100 g test
portion into 1 gal. (3.8 L) capacity high-speed blender. Add 100 mL
extracting solution, B(b), and 800 mL H2O, including volume H2O
contributed by sample as determined in 934.01 (see 4.1.03) or 925.04B
(see 4.1.04). Homogenize 1 min, pour into 2 L beaker, and let stand 1 h.
Add 100 mL buffer solution, B(c) (total volume 1 L), mix well, and filter
through Whatman No. 42 paper, collecting portion of clear filtrate.
F. Determination
(a) Nitrate plus nitrite nitrogen.—Pipet 25 mL buffered extracts,
E(a) or (b), or 5 mL extract, (c), into reduction column and treat as in
D, beginning, “Adjust flow rate through column . . .”. Rinse column
with 30 mL H2O between test solutions to remove NaCl. Use portion
of buffered extracts with equivalent dilution and pH as reference
solution in determining A at 540 nm. Also determine nitrate–nitrite
in reagents and correct for this blank value. Calculate total
nitrate–nitrite N from standard curve.
(b) Ni trite ni tro gen.—Pipet aliquot clear test fil trate
(containing <15 µg nitrite) into 50 mL volumetric flask and di lute
with H2O to ca 40 mL. Mix well, add 5 mL sulfanilamide solution,
B(e), mix, and let stand 3 min. Add 2 mL coupling reagent, B(f),
and dilute to vol ume with H2O. Mix well and let stand 20 min for
maximum color development. Measure A in 1 cm cells against
test extract with equivalent dilution at 540 nm. Correct for nitrite
reagent blank.
(c) Nitrate nitrogen.—Calculate by difference between (a) and
(b) above.
 2005 AOAC INTERNATIONAL
G. Calculations

µg/g NO2–N and/or NO3–N = µg NO3–N found

× dilution factor/g test portion

Dilution factors for extracts: E(a), 11.1; (b), 22.2; (c), 200.
Reference: JAOAC 51, 763(1968).

 2005 AOAC INTERNATIONAL