SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS LOCS* AOCS Offical Method Ca 6b-53 Reapproved 2008 Unsaponifiable Matter DEFINITION ‘Unsaponifiable matter includes those substances frequently found dissolved in fats and ols that cannot be saponified by the usual caustic reatment, but are soluble in ordinary fa and ol solvents. Included in this rounds are higher aliphatic alcohols, sterols, pigments, and hydrocarbons. Applicable to fats and oils containing higher levels of unsaponifiable matter than usually found in normal tallows and greases. This method is especially suited for marine ois, but is also applicable ro vegetable oi deodorizer distillates and sludges. This method does not apply to feed-grade fats ‘APPARATUS 1. Extraction eylinder—graduated, with glass stopper capacity about 200 mL (see Notes, 1). 2! Erlenmeyer flasks—natrow mouch, 250 mil. eapacity, with $240 outer joint, 3. Separatory funnels—250 mL. 4 Glass siphon—see Procedure, 3 and Notes, 1 5. Condenser with S 9%» joint to fit Eelenmeyer flasks. Either water- or air-cooled condensers may be used 6. Beakers—250 mL. 7. Erlenmeyer Alasks, of flat-bottom extraction flasks—50 ml REAGENTS 1. Ethyl alcohol, 959—USSD formulas 30 and 3A are permitted (see Notes, Geto). 2. Aqueous potassium hydroxide (KOH), 30% by weight—prepared by dissolving 60 g of reagent-grade KOH in 40 mL of dlstilled water with cooling (See Notes, Caution) “Aqueous potassium hydroxide (KOH) solution, 0.5 M, prepared by dissolving 30 g of reagent grade KOH in water, cooling and diluting to I Titer (ee Notes, Caution) Sodium hydroxide (NaOH) solution, 0.02 M—accurately standardized. See AOCS Specification Hi 12-52. Phenolphthalein indicator solution—1.0% in 95% ethyl alcohol Diethyl ether—reagent grade, free ftom peroxides (see Notes, Catton), - Acetone —teagent grade (see Noes, Caution). PROCEDURE 1. Accurately weigh about 2.0-2.5 g 4 0.1 mg of well-mixed cest sample into a 250 mL Erlenmeyer flask with ground-glass joint. Add 25 mL of 95% ethyl aleohol and 1.5 mL of 50% KOH solution, Boil gently but steadily under reflux, with occa- sional swiding, for 30 min or until completely saponified. Complete saponification is essential. No loss of alcohol should jccur during saponification. 2. Transfer while warm to the extraction cylinder (see Notes, 1), using a total of 50 ml. of water. Wash the flask with 50 mL of diethyl ether and add to the cylinder. Cool the contents ofthe eylinder to room temperature (20-25°C). 3, Insert the stopper and shake vigorously for a least | min, and allow ro sercle until both layers are clear. Use a glass siphon to remove the upper layer as completely as possible without including any of the lower portion (see Notes, 1). Transfer the diethyl ether layer to 250 ml. separatory funnel. 4, Repeat the extraction two more times, using 50 mL portions of diethyl ether each time and shaking vigorously with each extraction (see Notes, 2). 5, Rotate te combined diethyl echer extracts gently with 20 mL of water. Violent agitation at this step may result in emulsions that are difficult to break. Allow the layers to separate complerely and draw off the lower aqueous layer. Wash the diethyl ‘ther layer vo more times, using 20 mL of water each time, shaking gently cach time and discarding the lower aqueous layer after separation. 6. Wash the combined extracts in the separatory funnel three times, using 20 mL. portions of 0.5 M KOH, shaking vigorously. Follow each alkali washing by washing wich 20 mL of water. [fan emulsion forms during this washing procedure, allow 0 separate as much as possible, discard the clear aqueous layer, and proceed with the next sep, leaving any emulsion in the separatory funnel with the diethyl ether layer. After the third washing with 0.5 M KOH, wash che diethyl ether with succes- sive 20 mL portions of water until the washings are no longer alkaline to phenolphthalein. 7. Transfer the diethylether extract roa tared beaker, rinsing the separatory funnel and its pouring edge with diethylether and adding the rinsings to the solution inthe beaker. Evaporate to dryness in a water bath, using a gentle stream of clean, dry nitrogen. When almos all of the dicthyl ether has been evaporated, add 2-3 ml. of acerone and remove all traces of solvent Page 1 of 3“SAMPINGAND ANADSS OF COMMERCIAL TARSAND OS with the aid of a stream of nitrogen. Complete the drying co constanc weight ina vacuum oven at 75-80°C and an internal pressure of not more than 200 mm of mercury. Cool in a desiccator and weigh. The result becomes “A” in the calculations. 8, After weighing, take up the residue in 2 ml. of diethylether and then add 10 rl. of 95% alcohol, containing phenolphtha- lein indicator and previously neutralized co the phenolphithalein end point. Tieace with 0.02 M NaOH to the same final color. Correct the weight of the residue (See Notes, 3) for free fatty acid content, using the following relationship: 1 mL of (0.02 M NaOH is equivalent to 0.0056 g of oleic acid. The grams of fatty acid determined by this titration become “B” in the calculations. A reagent blank correction should be determined. 9. Correct for any reagent blank by conducting the unsaponifiable matter procedure without any fat or oil present. The blank determined by this procedure becomes “C” in the calculations. CALCULATIONS 1. Unsaponifiable matter, A= B49 ‘mass of ext portion, g x 100 Where— ‘A= mass of residue, g B ~ mass offty aid, g C= mass of blank, g PRECISION For vegetable oil deodorier distillates and sludges— 1, Two single determinations performed in the same laboratory should nor differ by more than 2.6%. 2. Two single dererminations performed in different laboratories should not differ by more than 3.8%. For other fats and oils see Numbered Notes, 4 NoTES Caution Ethyl alcohol (ethanol) is flammable. Use a fume hood when heating or evaporating this solvent. Potassium hydroxide, lke all alkalies, can buen skin, eyes, and respiratory tract severely. Wear heavy rubber gloves and face shield co procect against concentrated alkali liquids. Use effective fume-remaval device or gas mask co protect respiratory tract against allali dusts or vapors. When working with extremely caustic materials, such as potassium hydroxide, always add pellets to water and not the reverse, Alkales are extremely exothermic when mixed with water. Take precautions to contain the caustic solution in the event thar the mixing container breaks from the extreme heat generate. Diethyl ether is highly flammable and isa severe fire and explosion hazard when exposed to heat or flame. It sa central ner- ‘vous ystem depressant by inhalation and skin absorption. Ic will orm explosive peroxides upon exposure to light. Handle empty containers, particularly those from which ether has evaporated, with extreme caution. Explosive limits in air are 1.854896, The ‘TLV is 400 ppm in ait. A fume hood should be used at all times when using diethylether. Acetone is highly flammable. Ic may form explosive peroxides with oxidizing agents. Use effective Fime-removal device, Do ‘not mix with chloroform. NUMBERED NOTES 1. Alternately, a 500 mL. separatory funnel may be substituted for the extraction ¢ inating eve need for the siphon. fa separatory Funnel is substituted, draw off the lower aqueous layer into another separatory furnel, retaining the diethyl ether extract inthe first Funnel. Repeat the diethyl ether extraction of the aqueous phase, as noted in Procedure, 4, combining all of the diethyl ether extracts in the first Furne 2. Some fats high in unsaponifiable matter, specially those of marine origin, may require more than three extractions for ‘complete removal ofthe unsaponifiable matter. This is best judged by making another extraction and separately evaporating this extract as noted in Procedure, 7 There should be no unsaponifiable matter inthis extrac. IF there is, dissolve in a small volume of diethyl ether and add back tothe combined extracts. Continue with the extractions until no unsaponifiable matter remains in the extract 3. The titration correction for extractable free fatty acids and other exteactable unsaponifiable impurities (both reported as oleic acid) will tend co increase as the crude nature ofthe sample increases. For example, highenergy fats (used in animal feeds), feed fats, tll oil, and foots would be expected co give a higher free fatty acid tiation than relatively pure refined, bleached and deodorized (RBD) oil. 4, 15 3596 recommends the use of samples up to 5.0 g and adjusting solution volumes 0 50 mL. KOHethanol and doubling the volumes of diethyl ether and washing solutions. Precision values using these conditions are presented in Tables 1-3. Page 2 0f 3TANPINGAND ANANSS OF COMERCIAL FAIS AND OHS Ca 6b-53 * Unsaponifiable Matter Tablet Test organized by FOSFA International in June 1995. Soybean oil A No, of pattiipating laboratories after eliminating outliers? 0 ‘Mean value, % (by mass) 058 Repeatability standard deviation, 5, % 0.025 Repeatabilicylimier (2.8 %,), % 43 Coefficient of variation of repeatabiliry. 9 08 Reproducibility standard deviation, 5, % 02 Reproducibility limic R (2.8 x), % 379 Coefficient of variation of reproducibility, 9 067 B 0 0.6) 0.07 0.027 39) 0.82 0.24 M7 Sample A is refined, bleached, deadorized soybean ol, Sample B is dried, ere vater-degummed soybean ol, ‘Collaborative rest involving 51 laboratories i 16 countries, Table 2 Test organized by the FOSFA International. Repeatability limit x (2.8% 5, % CCoeflicient of variation of repeaabili, % Reproducibility standard deviation, sq, % Reprodueialiry lime R (2.8%), 96 Coefficient of variation of reprodu 29% ‘Collaborative rst involving 43 laboratories in 17 countries sd by IUPAC between 1976 and 1997. Refined soybean oll Refined allow Crude rapeseed ofl No. of patticpating laboratories after eliminating outies* 10 10 10 ‘Mean value, % (by mass) 0.630 0.253 1.432 Repeatability standard deviation, s, 9% 0.032 0.089 50 Repeatability limic (2.8% 5), % 0.024 0.067 93 Ceefliient of variation ofrepeatabiley, % 0.068 os 7 Reproducibility standard deviation, sy. % 0.140 0397 223 Reproducibilcy mie R28 x 5), 9 0.154 0.435 609 Coefficient of variation ofreprodu 0.137 0389 96 AInxernational collaborative test involving 10 laboratories. Page 3 of 3