41.1.28 AOAC Official Method 969.33 Fatty Acids in Oils and Fats

41.1.28 Table 969.
33 Determination of flask size and amount of

AOAC Official Method 969.33 reagent from approximate test sample size
Fatty Acids in Oils and Fats 0.5M NaOH BF3 Reagent
Preparation of Methyl Esters Test sample, mg Flask, mL mL mL
Boron Trifluoride Method 100–250 50 4 5
First Action 1969
250–500 50 6 7
AOAC–IUPAC Method 500–750 100 8 9
Codex-Adopted–AOAC Method* 750–1000 100 10 12
A. Principle
Glycerides and phospholipids are saponified, and fatty acids are
liberated and esterified in presence of BF3 catalyst for further analy- from methyl alcohol and flask by passing in stream of N2 few min-
sis by IR, 965.34E (see 41.1.36), or GC, 963.22F (see 41.1.29). utes. Methyl esters should be analyzed as soon as possible. If nec-
Method is applicable to common animal and vegetable oils and essary, heptane solution may be kept under N2 in refrigerator. For
fats, and fatty acids. Unsaponifiables are not removed, and if present prolonged storage, seal in ampule and store in freezer or add equiv-
in large amounts, may interfere with subsequent analyses. alent of 0.005% 2,6-di-tert-butyl-4-methylphenol (BHT). For IR
Method is not suitable for preparation of methyl esters of fatty ac- analysis, solvent removal must be as complete as possible; for GC,
ids containing major amounts of epoxy, hydroperoxy, aldehyde, 5–10% solution is suitable.)
ketone, cyclopropyl, and cyclopropenyl groups, and conjugated Precise weighing is not required. Test sample size need be known
polyunsaturated and acetylenic compounds because of partial or only to determine size of flask and amounts of reagents, according to
complete destruction of these groups. Table 969.33.
Sample ca 350 mg is preferred for GC.
B. Apparatus
(a) For fats and oils.—Add sample to flask and then add
(a) Reaction flasks.—50 and 125 mL flasks with outer standard methanolic NaOH solution and boiling chip. Attach condenser, and
taper joints. reflux until fat globules disappear (usually 5–10 min). Add BF3 solu-
(b) Condenser.—Water-cooled, reflux, with 20–30 cm jacket tion from bulb or automatic pipet through condenser and continue
and standard taper inner joint. boiling 2 min. Add 2–5 mL heptane through condenser and boil
C. Reagents 1 min longer. Remove heat, then condenser, and add ca 15 mL satu-
rated NaCl solution. Stopper flask and shake vigorously 15 s while
(a) Boron trifluoride reagent.—125 g BF3/L methyl alcohol.
solution is still tepid. Add additional saturated NaCl solution to float
Available commercially or prepare as follows: Weigh 2 L flask con-
heptane solution into neck of flask. Transfer ca 1 mL upper heptane
taining 1 L methyl alcohol. Cool in ice bath and with flask still in
solution into glass-stoppered test tube and add small amount anhy-
bath, bubble BF3 from cylinder through glass tube into methyl alco-
drous Na2SO4 to remove H2O. If necessary, dilute solution to con-
hol until 125 g is absorbed. Work in hood. BF3 must be flowing
centration of 5–10% for GC.
through glass tube before it is placed in and until it is removed from
To recover dry esters, transfer aqueous and heptane phases to
methyl alcohol to prevent liquid from being drawn into cylinder
250 mL separator. Extract with two 50 mL portions petroleum ether
valve system. Gas should not flow so fast that white fumes emerge
(bp 30–60°C) or hexane. Wash combined extracts with 20 mL por-
from flask. Reagent is stable 2 years. (Caution: Remove BF3 vapors
tions H2O until acid-free to methyl red indicator. Dry over anhydrous
with effective fume removal device. Avoid contact with skin, eyes,
Na2SO4, filter, and evaporate solvent under stream of N2 on steam
and respiratory tract.)
bath. If sample is <500 mg, reduce volumes of solvent and H2O.
(b) Methanolic sodium hydroxide solution.—0.5M. Dissolve 2 g
More volatile esters may be lost if evaporation is prolonged or if
NaOH in 100 mL methyl alcohol containing ≤0.5% H2O. White pre- stream of N2 is too vigorous. For IR spectroscopy, terminate evapo-
cipitate of Na2CO3 forming on long standing may be ignored. ration as soon as solvent is removed. For GC, method is applicable to
(c) Heptane.—Pure, as determined by GC. If fatty acids contain- fatty acids with ≥8 C atoms, if solvent is not completely removed.
ing ≥20 C atoms are absent in fat or oil, hexane may be substituted. (b) For fatty acids.—Add fatty acid to flask, then add BF3 solu-
(d) Methyl red solution.—0.1% in 60% alcohol. tion, and continue as in (a) with 2 min boiling under reflux.
(e) Nitrogen.—Containing <5 mg O2/kg.
Check new batches of reagents, particularly BF3, by preparing and References: Anal. Chem. 38, 514(1966).
chromatographing methyl esters of pure oleic acid. If extraneous IUPAC 2.301, 7th Ed.
peaks appear (in C20-C22 region with BF3), reject reagent. J. Am. Oil Chem. Soc. 45, 103(1968).
JAOAC 58, 396(1975); 62, 709(1979).
D. Preparation J. Chromatogr. 247, 63(1982).
(Caution: See Appendix B, safety notes on distillation and petro-
leum ether.) Revised: March 1997
(Work in hood. Wash all glassware immediately after use. If * Adopted as a Codex Reference Method (Type II) for acid hydrolysis, bo-
fatty acids containing >2 double bonds are present, remove air ron trifluoride of linoleate (in the form of glycerides) in special foods.
© 2000 AOAC INTERNATIONAL

41.1.28 AOAC Official Method 969.33 Fatty Acids in Oils and Fats

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41.1.28 Table 969.

33 Determination of flask size and amount of

© 2000 AOAC INTERNATIONAL

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