31.2.03 AOAC Official Method 952.

04 Pectic Acid in Cacao Products

First Action 1952 Final Action

[Sweet chocolate, usually characterized by its color, may contain small amounts of milk solids. When in doubt, use method for milk chocolate, (c)]. (a) In sweet chocolate containing no milk solids.(1) Extraction of fat.Weigh, within 0.15 g, amount (1460 g) of well-mixed grated test sample containing 4.75.2 g dry, fat-free cacao, and place in one or two 250 mL centrifuge bottles. (If test portion is >50 g, distribute it ca equally between 2 bottles.) (Make determinations in duplicate.) Add 120 mL petroleum ether (bp 3065C), or ether, at ca 30C, to each bottle, shake thoroughly, centrifuge, and decant. Repeat extraction with another 100 mL solvent; then extract with 100 mL alcohol, decant, and discard extracts. (2) Extraction of color, tannins, etc.To each centrifuge bottle containing defatted sample, add (from graduate) 150 mL acidified 82% alcohol (10 mL HCl + 432 mL alcohol diluted to 500 mL with H2O) that has been warmed so that temperature of liquid in centrifuge bottle is 55C. Stopper, shake vig orously 2 min, centrifuge 68 min, decant, and discard supernate. Add 100 mL alcohol to residue in each bottle, shake, centrifuge as before, decant, and discard extracts. The residue is used to extract pectin. (3) Extraction of pectin.Measure 150 mL H2O in graduate, add ca 75 mL to 1 centrifuge bottle, stopper, shake vigorously to disperse residue thoroughly, decant into other centrifuge bottle containing remainder of test portion, and again shake vigorously until residue is thoroughly dispersed. Decant mixture into 500 mL wide-mouth Erlenmeyer, rinse mouth of bottle with ca 1 mL H2O from wash bottle, and complete transfer of residue from bottles with ca 45 and 30 mL successive portions of H2O remaining in graduate. Make mixture in flask just alkaline to litmus with NH4OH (1 + 1) (ca 0.7 mL; note volume used; see Note). Acidify with CH3COOH, add 0.5 mL excess, and then add 50 mL 2% ammonium oxalateH2O solution (w/v), using solution to wash down sides of flask. Pass glass stirrer, with 2.53 cm (11 14 in.) diameter loop (perpendicular to shaft on end), loosely through hole in rubber stopper or through glass tube of slightly larger diameter held in rubber stopper placed in mouth of flask. Attach shaft of stirrer to motor, or air rotor, to stir contents of flask continuously, immerse flask below level of contents in water bath held at 9092C, and stir moderately 3 h. If level of liquid in flask is appreciably reduced, add enough hot water to bring back to original level. Remove flask, cool to 45C, quantitatively transfer contents to 250 mL volumetric flask, dilute to volume with H2O at 45C, and add 1.5 mL excess to correct for volume of cacao solids. Mix contents well, pour into centrifuge bottle, and centrifuge at 1800 rpm ca 15 min. Decant supernate extract, which may be turbid or opalescent, into 400 mL beaker. Rinse any residue in flask into centrifuge bottle with alcohol and reserve this cacao residue for further treatment to estimate fat-free cacao in the product. Warm extract to 45C, pour into graduate, note volume (VmL), and return to beaker. Rinse graduate with two 5 mL portions H2O, and add to beaker. Cool in bath to 1517C, make alkaline to phenolphthalein (internal indicator) with 15% NaOH solution (w/v), and add 11 mL excess. (See Note.) Stir, and let stand in bath 20 min at 1517C.

Decant al ka line liq uid into two 250 mL cen tri fuge bot tles, distributing volume ca equally. Let drain, and rinse twice with 58 mL cold H2O, adding 1 rinsing to each bottle. Add 10 mL HCl to each bottle, with stirring, and then gradually add, with continued stirring, 40 mL alcohol. Add to each bottle 0.81.0 g mixture of Filter-Cel and Celite 545 (1 + 1). Stir, rinse rod, stopper bottles, shake well, and cen tri fuge 1012 min. De cant and discard supernates without disturbing sediment, and wash residues once by shaking contents of each bottle with 100 mL alcohol, centrifuge, and decanting. Add 75 mL H2O to one bottle, stopper, and shake well. Make slightly alkaline with few drops NH4OH (1 + 1) and shake again. Decant liquid into second bottle, stopper, shake again, make alkaline to litmus with NH4OH (1 + 1), and add 0.5 mL excess. Stopper, and shake thoroughly 11.5 min to dissolve pectic acid precipitate. (Drops of liquid clinging to lip of bottle may be washed into second bottle with small squirt of H2O from wash bottle; otherwise do not rinse at this point.) Filter, with suction, through hardened paper (Whatman No. 41-H or 54, or equivalent) on 11 cm Bchner. Let bottle drain well; then rinse bottle twice with 25 mL portions H2O, each containing 12 drops NH4OH (1 + 1), pour rinsings on filter, and wait for each rinse to drain through filter before adding another. Pour filtrate into 250 mL centrifuge bottle, let flask drain, and rinse twice with 5 mL H2O. (Use of bell jar permits filtration directly into centrifuge bottle.) Add 5 mL HCl to contents of centrifuge bottle, stir in 90100 mL alcohol, rinse rod with alcohol (do not add filter-aid), stopper, shake, and centrifuge 8 min at 15001800 rpm. Decant into beaker, retaining most of precipitate in bottle, and filter liquid through 15 cm Whatman No. 41-H paper, or equivalent, on fluted funnel. Pour precipitate and liquid remaining in centrifuge bottle onto filter paper and drain thoroughly. (Do not rinse.) Quantitatively transfer precipitate in bottle and on filter to 250 mL beaker, using total of 75 mL 6075C H2O. Cool beaker and contents in bath at 1517C and add, with stirring, 15% NaOH so lu tion (w/v) (also cooled) un til mix ture is al ka line to phenolphthalein (internal indicator). Add 3 mL excess and let stand in bath 15 min at 1517C. During this time, heat on steam bath 2 wash bottles, containing respectively, wash solutions: A, mixture of 200 mL H2O, 50 mL alcohol, and 20 mL HCl (1 + 2.5); B, 400 mL alcohol diluted to 950 mL with H2O. Remove beaker from bath, acidify contents with 10 mL HCl (1 + 2.5) while stirring, and dilute to 100 mL with H2O. (Estimate volume by comparison with 100 mL in similar beaker.) Add few glass beads, cover, bring contents to bp, and boil 5 min. Remove from heat; add 10 mL HCl, with stirring, and then 400 mg prepared asbestos (previously alkali- and acid-washed and ignited, and free of coarse particles). Stir 40 s, and immediately filter through Whatman No. 41-H paper, or equivalent, on 711 cm Bchner with very gentle suction. (Suction should be so gentle that it can hardly be felt when thumb is placed on rubber tube before attaching tube to flask; test suspension should filter in small steady stream, and filtrate should be clear or only slightly opalescent, with no immediate separation of precipitate.) Wash beaker and filter with three ca 25 mL portions wash solution A, and then with four or five ca 25 mL portions wash solution B to remove acid. (Washings should be clear and pass through filter readily. Ignore any appearance of precipitate in flask at this stage.) Place filter and precipitate on fairly large, short-stem funnel, and wash pectic acid precipitate and asbestos into Pt dish with hot water. Determine blank on 400 mg asbestos by adding it to hot acid
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solution, filtering, and drying in same manner as test suspension. Heat dishes on steam bath until asbestos and precipitate appear thoroughly dry. Dry test solids and blank in oven at 100C to constant weight (0.2 mg; ca 1 h), cool in desiccator, weigh, ignite, cool, and reweigh. Loss in weight of test solids - loss in weight of blank = weight of pectic acid in aliquot taken. This weight 250/volume extract taken (V) = weight pectic acid in test portion To obtain dry, fat-free cacao in test portion, add 100 mL alcohol to cacao residue reserved in centrifuge bottle, stopper, shake well, centrifuge, and decant. Again shake with 100 mL alcohol, rinse stopper, and wash down sides of bottle with alcohol from wash bottle; centrifuge and decant. Repeat extraction, using 100 mL ether, washing down sides, centrifuging, and decanting. Let residual ether evaporate. Using brush and spatula, quantitatively transfer residue to tared Al dish with cover; dry dish and contents 12 h in oven at 100C; cover dish, cool in desiccator, and weigh. Weight residue 1.9 = weight dry, fat-free cacao in test portion, Weight pectic acid 100/weight dry, fat-free cacao = pectic acid, % (b) In chocolate li quor, breakfast cocoa, cocoa, and low-fat cocoa.Place ca 15 g cocoa or 25 g choc olate liquor, prepared as in 970.20 (see 31.1.01), in centrifuge bottle. To remove most of fat, shake thor oughly with 100 mL pe tro leum ether (bp 3065C) or ether; centrifuge, decant, and repeat extraction with another 100 mL petroleum ether or ether. Shake residue with third portion solvent, and filter through Whatman No. 41-H or 54 paper, or equivalent, on 11 cm Bchner with gentle to moderate suction. (Ap ply vac uum and wet fil ter with sol vent be fore starting filtration.) Let residue suck dry, transfer to porcelain dish or casserole, grind gently with pestle to pul verize and mix, and transfer to Al dish with cover. Dry ca 45 min in oven at 100C, cover dish, and cool in desiccator. Weigh 5 g of the dry, fat-free residue into 250 mL centrifuge bottle, and proceed as in (a)(1), last sentence, beginning . . . then extract with 100 mL alcohol, . . . and continue as in (2) and (3) through next-to-last paragraph (directions for calculating weight pectic acid in test sample). (Weight pectic acid found/5) 100 = % pectic acid in dry, fat-free cacao (No estimation of dry, fat-free cacao is necessary, since weighed amount of dry, fat-free cacao is used for pectic acid determination.)

(c) In prod ucts con tain ing milk sol ids.(1) Re moval of fat.Weigh (0.2 g) test portion containing ca 5 g dry, fat-free liquor (60110 g milk chocolate, etc.), and distribute ca equally between two 250 mL centrifuge bottles. Add 120 mL petroleum ether or ether at 2530C, shake thoroughly, centrifuge, and decant. Add another 120 mL portion tepid solvent to each bottle, shake thoroughly, centrifuge, and decant. In same manner extract contents of each bottle with 100110 mL acetone. (2) Extraction of milk protein.Add enough acetone (ca 90 mL) to make total of ca 110 mL with acetone remaining in residue. (Estimate on basis that 12 original test portion of 75 g retains ca 20 mL acetone in residue of each bottle.) Stopper, and shake vigorously to disperse residue thoroughly. Quickly add 100 mL triethanolamine solution (90 mL triethanolamine diluted to 500 mL with H2O) to each bottle, stopper immediately, and shake well 2 min. Let stand ca 1 min for foam to rise; then centrifuge 1214 min at 15001800 rpm. Carefully decant and discard supernate without disturbing residue, and extract residue with 100120 mL mixture of acetone and the triethanolamine solution (110 + 100), centrifuging and decanting as before. Then add 100 mL 85% alcohol (v/v) to each bottle, shake, centrifuge, decant, and discard extract. To residue in each bottle add 1520 mL acidified 82% alcohol, (a)(2), stir, and add enough HCl to make residue acid to litmus. Continue as for sweet chocolate, beginning with (a)(2). Note: Volume of NH4OH used should be noted, excess avoided, and approximate concentration of NH3 determined. (Solution becomes much less concentrated on standing from loss of NH3 around stopper.) This is necessary because in first hydrolysis (saponification) of pectin, part of NaOH solution added (after solution of test portion has been made alkaline to phenolphthalein) is used up in replacing with Na the NH4 in NH4 salts present. The 11 mL 15% NaOH solution added furnishes excess of 45 mL of this solution over amount required to neutralize acid and replace NH4 with Na, provided 1.15 mL 7.5M NH4OH (= 2.3 mL 15% NaOH solution) is added to neutralize residual HCl. [Approximately 3.75 mL 15% NaOH solution is needed to replace with Na the NH4 in (NH4)2C2O4 solution used in extraction of pectin.] If >1.15 mL of the NH 4 OH so lu tion is needed to neu tral ize HCl in (a)(3), correspondingly increase volume of 15% NaOH solution used for saponification, but avoid excess of >56 mL. Reference: JAOAC 35, 650(1952). CAS-9046-40-6 (pectic acid)

2006 AOAC INTERNATIONAL