32.1.09 to 100 mL.

(It may be necessary to redistill acetic acid and

AOAC Official Method 944.02 recrystallize NaC2H3O2 from H2O, depending on amount Fe present.)
Iron in Flour (g) Sodium acetate solution.—2M. Dissolve 272 g
Spectrophotometric Method NaC2H3O2⋅3H2O in H2O and dilute to 1 L.
First Action 1944 (h) Buffer solution, pH 3.5.—Dilute 6.4 mL 2M NaC2H3O2 solu-
Final Action tion, (g), and 93.6 mL 2M HC2H3O2 (120 g/L) to 1 L with H2O.
Revised First Action 1993
B. Preparation of Standard Curve
(Applicable to enriched, enriched self-rising, and phosphated
Construct 10-point standard curve, plus zero, preparing solutions
flours.)
containing 0.0 (zero), 2.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0,
40.0, and 45.0 mL, respectively, of final diluted Fe standard solu-
See Table 944.02 for the results of the interlaboratory study sup-
tion, A(c), plus 2.0 mL HCl, in 100 mL H2O.
porting acceptance of the method.
Alternatively, construct a 5-point curve (5.0, 15.0, 25.0, 35.0, and
A. Reagents 45.0 mL), plus zero, after correction for reagent blank.
Using 10 mL of each of these solutions, proceed as in C, begin-
(Note: Rinse all flasks, beakers, funnels, etc., with H2O before use
and filter all reagents to remove suspended matter.) ning “…add 1 mL H2NOH⋅HCl…”. Plot concentration against scale
reading.
(a) o-Phenanthroline solution.—Dissolve 0.1 g C. Determination
o-phenanthroline in ca 80 mL H2O at 80°C, cool, and dilute to
(a) By dry ashing.—Ash 5.00 g test portion in Pt, SiO2, or porce-
100 mL.
lain dish (ca 60 mm diameter, 35 mL capacity) as in 923.03 (see
(b) α,a-Dipyridyl solution.—Dissolve 0.1 g α,α-dipyridyl in
32.1.05). (Porcelain evaporating dishes of ca 25 mL capacity are sat-
H2O and dilute to 100 mL. [Reagents (a) and (b) kept in cool, dark isfactory. Do not use flat-bottom dishes of diameter >60 mm.) Cool
place will remain stable several weeks.] and weigh if percent ash is desired. Continue ashing until practically
(c) Iron standard solution.—0.01 mg Fe/mL. (1) Dissolve 0.1 g C-free. To diminish ashing time, or for test portions that do not burn
analytical grade Fe wire in 20 mL HCl and 50 mL H2O, and dilute to practically C-free, use one of following ash aids:
1 L. Dilute 100 mL of this solution to 1 L. Or (2) Dissolve 3.512 g Moisten ash with 0.5–1.0 mL Mg(NO3)2 solution or with
Fe(NH4)2(SO4)2⋅6H2O in H2O, add 2 drops HCl, and dilute to redistilled HNO3. Dry and carefully ignite in furnace, avoiding spat-
500 mL. Dilute 10 mL of this solution to 1 L. tering. (White ash with no C results in most cases.) Do not add these
(d) Hydroxylamine hydrochloride solution.—Dissolve 10 g ash aids to self-rising flour (products containing NaCl) in Pt dish be-
H2NOH⋅HCl in H2O and dilute to 100 mL. cause of vigorous action on dish. Cool, add 5 mL HCl, letting acid
(e) Magnesium nitrate solution.—Dissolve 50 g rinse upper portion of dish, and evaporate to dryness on steam bath.
Mg(NO3)2⋅6H2O in H2O and dilute to 100 mL. Dissolve residue by adding 2.0 mL HCl, accurately measured, and
(f) Acetate buffer solution.—Dissolve 8.3 g anhydrous NaC2H3O2 heat 5 min on steam bath with watch glass on dish. Rinse watch glass
(previously dried at 100°C) in H2O, add 12 mL acetic acid, and dilute and dilute residue solution to 100 mL with H2O. If necessary (undis-

Table 944.02 Interlaboratory study results for determination of iron, spectrophotometric method

Product Standard curve version Mean, mg Fe/100g sr sR RSDr, % RSDR, %

Milk-based infant formulaa 5 1.48 0.13 0.21 8.48 14.20
10 1.47 0.13 0.19 8.96 13.21
Soy-based infant formulab 5 9.64 0.43 0.59 4.43 6.13
10 9.62 0.44 0.58 4.61 5.99
Nonfortified cereal 5 5.95 0.18 0.26 3.08 4.41
10 5.95 0.19 0.25 3.20 4.23
Fortified cereal 5 22.30 1.24 1.24 5.55 5.55
10 22.34 1.18 1.18 5.29 5.29
Enriched bread 5 5.25 0.64 0.64 12.14 12.14
10 5.25 0.63 0.63 12.09 12.09
Whole wheat flour 5 4.77 0.16 0.40 3.43 8.39
10 4.76 0.15 0.39 3.21 8.18
Enriched macaroni 5 4.54 0.12 0.27 2.71 5.96
10 4.53 0.14 0.27 3.10 5.87
Enriched egg noodles 5 4.10 0.08 0.15 2.05 3.54
10 4.10 0.09 0.13 2.10 3.24
a
Powder, low Fe.
b
Powder, Fe-fortified.

© 2000 AOAC INTERNATIONAL

solved particles visible in residue solution), filter diluted residue so- Pipet 10 mL into 25 mL volumetric flask, add 1 mL H2NOH⋅HCl
lution through ashless paper and discard first 15–20 mL filtrate. solution, rotate flask, and let stand 2–3 min. Add 9.5 mL 2M
Pipet 10 mL aliquot into 25 mL volumetric flask and add 1 mL NaC2H3O3 solution, A(g), and 1 mL o-phenanthroline solution, di-
H2NOH⋅HCl solution; let stand 5 min and then add 5 mL buffer lute to volume, and mix. Let stand ≥5 min and determine A in
solution, A(f), and 1 mL o-phenanthroline, A(a), or 2 mL spectrophotometer or photometer at ca 510 nm.
dipyridyl solution, A(b), and dilute to volume. Determine With self-rising flour, the 9.5 mL 2M NaC2H3O3 solution, A(g),
absorbance, A, in spectrophotometer or photometer at ca 510 nm. may be reduced to 8.0 mL. To determine exact amount of buffer
From reading, determine Fe concentration from equation of line solution, A(h), needed to adjust each digest to most desirable pH
representing standard points or by reference to standard curve for range, mix 10 mL aliquot of test solution with measured amount of
known Fe concentration. If further dilution is required to main- buffer solution, dilute with H2O to 25 mL, and determine pH either
tain sample absorbance reading below highest standard point on potentiometrically or colorimetrically.
curve, pipet smaller aliquot into 25.0 mL flask, dilute to 10.0 mL For colorimetric determination, add 5 drops bromophenol blue in-
with 2% HCl solution (v/v) and continue as described in C, para- dicator (grind 0.1 g powder with 1.5 mL 0.1M NaOH and dilute to
graph 3. Determine blank on reagents and make correction. Cal- 25 mL with H2O) to solution and compare color with that of equal
culate Fe in flour as mg/lb. volume of pH 3.5 buffer solution, A(h), also treated with 5 drops in-
(b) By wet digestion.—Transfer 10.00 g test portion to 800 mL dicator. Although color develops in range pH 2–9, avoid pH <3.0
Kjeldahl flask, previously rinsed with dilute acid, then with H2O; and preferably work at pH 3.5–4.5. With cereal products, 9.5 mL
add 20 mL H2O and mix; pipet 5 mL H2SO4 into flask and mix; add buffer solution, A(h), is satisfactory. With test portions high in Fe,
25 mL HNO3 and mix well. After 2–3 min, heat flask very gently at aliquot of 5 mL instead of 10 mL may be used with 4.8 mL buffer so-
brief intervals (to avoid foaming out of flask) until heavy evolution lution, A(h).
of NO2 fumes ceases. Continue to heat gently until material begins to Conduct digestion so as to avoid contamination with Fe and deter-
char; then cautiously add 2–3 mL HNO3 at intervals until SO3 fumes mine blank. After correction for blank, calculate as mg Fe/lb
evolve and colorless or very pale yellow liquid is obtained (453.6 g).
(60–65 mL HNO3 total in ca 2 h). Cool, add 50 mL H2O and 1 glass
References: JAOAC 27, 86, 396(1944); 28, 77(1945).
bead, and heat to SO3 fumes; cool, add 25 mL H2O, and filter quanti-
tatively through 11 cm paper into 100 mL volumetric flask; cool, and J. AOAC Int. 75, 384(1994).
dilute to volume. CAS-7439-89-6 (iron)

© 2000 AOAC INTERNATIONAL