33.6.13 AOAC Official Method 947.

07 Fatty Acids (Water-Insoluble) (WIA) in Butter

(Polenske Number) Gravimetric Method First Action 1947 Final Action

Weigh 50 g prepared test portion, 938.05(see 33.6.02), into 250 mL centrifuge bottle, and add 10 mL H2O; if necessary, remelt in warm water (not steam) bath and add 50 mL ether. Shake until fat dissolves. Add 1M NaOH in ca 0.2 mL increments to mixture in centrifuge bot tle un til neu tral ized to de cided pink, us ing 10 drops phenolphthalein and shaking between additions of alkali. Then add 0.5 mL excess and shake again 2 min. During this and all subsequent shakings, carefully release pressure several times to avoid blowing out stopper and losing some of contents. (It is difficult to shake >1 bottle at a time because of greasy stoppers and pressure that develops.) (Caution: Exhaust pressure from centrifuge bottle carefully to prevent loss of contents.) Remove stopper and add 50 mL petroleum ether, shake few times, and centrifuge 5 min at ca 1200 rpm (longer if separation is not sharp). Set bottle on horizontal surface and siphon off ether-fat layer. (If ether layer, after centrifuge, is reddish, add 10 mL H2O, shake, and again centrifuge as before. If reddish tinge still persists in ether layer, add 25 mL ether, shake, and again centrifuge.) Wash aqueous layer remaining in centrifuge bottle by adding 25 mL ether; mix thoroughly by shaking several s, add 25 mL petroleum ether, and again mix by shaking. Centrifuge, siphon off ether layer as before, and repeat washing as above. If after any washing, basic red of phenolphthalein fades, add additional phenolphthalein and alkali to give decided red (not pink). Add 1 mL H2SO4 (1 + 1) to residue in centrifuge bottle and shake vigorously few seconds. Then add 5 mL 10% Na2WO42H2O solution (w/v) and again shake vigorously few seconds. (Mixture should be distinctly acid to Congo red paper; if it is not, add more H2SO4.) Now add 75 mL ether, shake violently 2 min, and centrifuge. Siphon off ether layer into 500 mL separator. Wash siphon inside and out with 75 mL ether so that washings drain into centrifuge bottle. Shake bottle violently 2 min, centrifuge, and si phon off ether layer into the sep a ra tor. (Dis re gard slight opalescence of ether layer.) Add 100 mL alcohol (1 + 1) to combined extracts in separator and neutralize in same manner as before with 1M NaOH to decided pink. Add 0.5 mL excess and shake vigorously 2 min more. Immediately add 25 mL H2O, mix by single inversion of separator, and let stand until aqueous layer is clear. (Separation usually occurs in few min; slow separation may sometimes be hastened by playing fine stream of H2O on ether surface. If volume of emulsion at interface is only ca 10 mL it may be included in subsequent extraction.) Drain aqueous layer into 600 mL beaker. Add 50 mL alcohol (1 + 1) and ca 10 drops phenolphthalein to separator and neutralize with 1M alkali, shaking vigorously ca 2 min. Add 50 mL H2O, mix by single inversion of separator, and let stand until aqueous layer is clear. Drain aqueous layer into the beaker. Then add 10 mL H2O to separator, mix by

single inversion, let separate until aqueous layer is clear, and drain into beaker. Place beaker containing combined extracts and washings on steam bath (or carefully heat on hot plate) to expel any ether. Evaporate to ca 25 mL (small fan is useful if foaming is serious). (Solution should remain decided red through all these operations and up to point where soaps are acidified.) Transfer to 250 mL beaker with ca 25 mL H2O. (As alternative, material may be evaporated to dryness on steam bath and residue dissolved in ca 50 mL H2O.) Dissolve 5 g anhydrous Na2SO4 in warm solution (volume must be 50 mL when Na2SO4 and H2SO4 are added), heating if necessary. Cool to 20C, stirring frequently to keep soaps from forming hard crust on surface. Make acid by adding H2SO4 (1 + 1) dropwise, using Congo red paper as indicator. Stir vigorously to effect thorough liberation of fatty acids, mashing all pink soap curds. Add ca 500 mg filter-aid and mix. Filter with suction on suitable filter crucible. Rinse beaker with three ca 15 mL portions H2O at 20C and transfer rinsings to crucible. Maintain suction several min after visible dripping ceases to dry precipitate. (Heavy precipitate can be sucked drier if cracks are plastered up with some of precipitate. Filtrate should be clear.) Substitute tared beaker or flask (weighed with similar vessel as counterpoise), containing few glass beads or grains of sand, for receiving flask of filtering apparatus. Extract acids with four ca 15 mL portions ether, breaking up precipitate with stirring rod between extractions and thoroughly mixing with the ether. Let ether drip through filter before applying suction. (Filter pad must not be disturbed.) Evaporate ether extract, which should be no more than faintly opalescent, on steam bath, and dry acids 1 h in 100C oven. Cool and weigh. Re port re sults as mg H2O-insoluble ac ids (WIA)/100 g butterfat. Dissolve weighed acids in 10 mL neutral benzene and titrate with 0.1M sodium ethylate [pre pared similarly to 0.05M sodium ethylate, 927.06A(b) (see 34.1.15)], using 10 drops phenolphthalein as indicator, until end point holds 1 min. (Neutralized alcohol instead of benzene and 0.1M NaOH instead of sodium ethylate may be used.) Compute mean molecular weight of fatty acids by dividing mg acids found by mL 0.1M alkali used for titration and multiplying by 10. (Mean molecular weight should be 290. When amount acids is <150 mg/100 g butterfat, mean molecular weight is without significance.) [Notes: To siphon off ether, use tube similar to delivery tube of ordinary wash bottle but with intake end bent up into U shape in opposite direction to outlet end, with opening 612 mm higher than bottom of U, cut off horizontally. (Avoid excessive constriction when bending.) Set delivery tube loosely enough in stopper that it can be raised or lowered. In operating, adjust opening of U bend to ca 3 mm above surface of aqueous layer and blow ether layer off by gently blowing through mouthpiece tube inserted in adjacent hole in stopper.] For filtration of fatty acids use coarse fritted glass crucibles overlaid with glass fiber filter with porosity <2 mm. References: JAOAC 30, 575(1947); 31, 739(1948); 32, 731(1949). Revised: March 1996
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