SAMPLING AND ANALYSS OF COMMERCIAL FATS AND OILS LOCS AOCS Official Method Cd 8b-90 Reapproved 2008 Peroxide Value Acetic Acid-Isooctane Method DEFINITION This method determines all substances, in terms of milliequivalents of peroxide per 1000 grams of est sample, that oxidize potassium iodide under the conditions of the test. The substances are generally assumed to be peroxides or other similar products of fat oxidation, SCOPE “Applicable to all normal fats and ols, including margarine, This method is highly empirical, and any var tion in the rest procedure may result in erratic results. Because this method gives erratic results at peroxide values 270, this method should not be used with the AOM test, AOCS Official Method Cal 12-57, with which peroxide values 270 may be encountered, APPARATUS 1, Pipet—0.5 mL, or other suitable volumetric apparatus capable of dispensing 0.5 mL of saturated potassium iodide (KA) solution, 2, Erlenmeyer lasks—with glass stoppers, 250 mL. 3. Burette—25 ml. or 50 ml, class A, graduated in 0.1 mL divisions. 4 Timer ‘5, Balance—top loading, 500 g capacity with 0.01 gram sensitivity REAGENTS 1. Acetic acid-isooctane solution—3:2, viv, prepared by mixing 3 volumes of reagent-grade glacial acetic acid (see Notes, Causion) with 2 volumes of reagent-grade isooctane (see Notes, Caution. 2, Potassium iodide (KD solution—saturated, prepared fresh each day analysis is performed by dissolving an excess of KL in recently boiled isilled water. Make certain the solution remains saturated during use, a indicated by the presence of undis- solved KI crystals. Store inthe dark when notin use. Test the saturated KI solution by adding 2 drops of starch solution to 0.5 mL of the KI solution in 30 mL of the acetic acid-sooctane solution. fa blue color is formed that requires more than 1 drop of 0.1 M sodium thiosalfate solution to discharge, discard che KI soluion and prepare fesh solution. 3. Sodium thiosulfite (Na,S,O, - 5H,O) solution—0.1 M, accurately standardized vs. porassinm dichromate primary stan- dard a follows: {@). Sodium thiosulfate solution 0.1 M, prepared by dsslvis wll. (b) The potassium dichromate (see Notes, Caution) primary standard should be finely ground, dred at 105°C for 2 hr and cooled in a desiccator, Weigh 0.16-0.22 g of potassium dichromate into a 500 ml- flask or bore by difference from a ‘weighing bord. Disolve in 25 ml. of water, add 5 ml of concentrated hylrochloric acid (35-37%), 20 ml of potassium iodide solution (15% solution, 15 g KI in 100 mL. water), and rotate to mix. Allow to stand for 5 min and then ade 100 mL of distilled water. Titrate with sodium thiosulfae solution, shaking continuously until yellow color has almost disap- peared, Add 1~2 ml of starch indicator and continue the tration, adding the thiosalfae solution slowly until the blue Color just disappears. The stength ofthe sodium thisulfae solution is expresed in terms ofits molarity 20,394 X mas of KC, tof sodium thine 24,9 g of sodium thiosulfate in distilled water and diluting Molatity of Na,S,O, solution 4, Sodium thos wolucion—0.01 M, accurately standard. This sluion may be prepared by accurately pipetng 100 mL of 0.1 M sodium thiosulfate into a 1000 ml. vohumetc ask and accurately dilating to vlume with ecenty boiled disiled wate. 5. Src indicator slution—tsted for sensitivity, prepared by making a paste with I g of starch (ee Nores, 1) and a small amount of cold disilled water. Add, while string, o 200 mL. of boiling water and bol fr afew seconds. Immediately Femove from heat and cool Salicylic aed (1.25 g/L) may be added vo preserve the indicator. Iflong storage is required, the solution must be kepe ina xefsigerator at 4-10°C, Feesh indicator must be prepared when che end point ofthe tation from blue co colores fils tobe sharp. Iestred under reigeration, the sarch solution should be stable for about 2-3 weeks, Test for sentir lace 5 tn. watch solution in 100 mo of water and add 0.05mi of freshly prepared 0.1 M KT solu- tion and one drop of 50 ppm chlorine slation made by diluting | mL ofa commercial 5 sodium hypochlrice (NaOCI) solucion to 1000 mL. The deep biue color produced mus be discharged by 0.05 mL. of O.1 M sodium thiosulfate. 6, Sodium lauryl sulfate (SDS)—298% [Aldrich Chemical (W. Milwaukee, WI, USA) or Mallinckrode (Paris, KY, USA)] Prepaze 10% solucion by dissolving 10 g SDS in 100 mL water. Page 1 0f 3SRPING AND ANATSIS OF COMMERGAL TATSAND GIS Cd 86-90 © Peroxide Value Acetic Acid-Isooctane Method PROCEDURE FOR FATS AND OILS 1, Weigh the test portion (Table 1) into a 250 mL Erlenmeyer flask with plas stopper and add 50 mL ofthe 3:2 acetic acid- isooctane solution. Swirl co dissolve the tet portion. Add 0.5 ml. of saturated KI solution using a suitable volumetric piper. 2 Allow the solution ro stand for exactly I min, thoroughly shaking the solution atleast three times during the 1 min, and then immediately add 30 mL of distilled water. 43. Titrae with 0.1.M sodium thiosulfate, adding it gradually and with constane and vigorous agitation (see Notes, 2). Continue the titration uni the yellow iodine color has almoxc disappeared Add 0.5 ml. of 10% SDS (Reagents, 6), and then add about 0.5 mL, of starch indicator solution. Continue the tration with constant agitation, especially near the end point, to liberate all of the iodine from the solvent layer. Add the thioslfie solution dropwise und the blue color just disappears (se Notes, 3 and 4). 4, Conduct a blank determination of the reagents daily. The blank titration must not exceed 0.1 mb. of the 0.1 M sodium thiosulfate solucion. PROCEDURE FOR MARGARINE 1. Male the test portion by heating wich constant stirsing on a hotplate st at low heat, orby heating in an air oven at 60-70°C, Avoid excess heating and particularly prolonged exposure of the ol ro temperatures above 40°C. 2, When completely melted, remove the test portion from the hor plate or oven and allow to settle in a warm place until the aqueous portion and most of the milk solids have settled ro the bortom, 3, Decane the ol into a clean beaker and filter through a Whatman no. 4 paper (or equivalent) into another clean beaker. Do snot reheat for filtration unless absolutely necessary: The test portion must be clear and brilliant. 4, Proceed as directed in Procedure for Fats and Oils, paragraphs 1-4, CALCULATIONS (S~B) x M x 1000 1. Peroxide value (mlliequivalents peroxide/1000 g est portion) = ‘mas of tex portion, g Where— ‘B= volume of titrant, mL of blank = volume of tant, mL of test portion M = Molarity of sodium thiosalfate solution PRECISION ‘The details of interlaboratory tess are given in Tables 2-4. The values presented may noc be applicable to matrices other than those presented and may nor be representative for other concentrations 1, Repeatability —The difference between two tes results on che same material, in the same laboratory under the same condi tions, should not exceed che repeatability valu, 2. Reproducibiliy—The difference between two test results on the same material, under the same conditions in different labo- ratores, should not exceed the reproducibility value, R. NoTes Caution Tsooctane is flammable and a ite risk. Explosive limits in ai are 1.1~6,0%. It is toxic by ingestion and inhalation. A properly ‘operating fume hood should be used when working with this solvent. ‘Acetic acid in the pure state is moderately coxic by ingestion and inhalation. Iti a strong irritant co skin and tissue. The ‘TLV in ai is 10 ppm. Porasum dichromate is toxic by ingestion and inhalation. There is sufficient evidence in humans forthe carcino-genicityof chro= ‘ium [+6] in particular, lung cancer. Ie is a song oxidizing agent and a dangerous fie risk when in contact with organic chemicals. NUMBERED NOTES 1. “Porato Starch for lodometry” is recommended, because this starch produces a deep blue color in the presence ofthe iodo- ‘ium ion. “Soluble Starch” is not recommended because a consistent deep blue color may not be developed when some Soluble starches interact with the iodonium ion. The following are suitable starches: Soluble Starch for lodometry, Fishet 5516-100; Soluble Porato Starch, Sigma $-2630; Soluble Porato Starch for lodometry, J.T. Baker 4006-04, 2. There isa 15~30 sec delay in neutralizing the starch indicator for peroxide values 70 meq/kg and higher. This delay is due tw the tendency of isooctane to float on the surface of an aqueous medium, and the time necessary to adequately mix the solvene in large volumes of aqueous titrant, thereby liberating the las traces of iodine. Based on collaborative study results (References,1,2), the recommendation is o use 0.1 M ticant for peroxide value ranges (10-150 meq/kg). Erratic results reported for this method, especially at higher peroxide values, appeat to be related co the isooctane floating on the surface of the aqueous layer (References, 3). Rapid mechanical stirring (ee, with magnetic sticter) and/or use of a surfactant, sch 25. sodium lauryl sulfate (Reagents, 6), is highly recommended. Page 2 of 3SANIPING AND ANAIVSIS OF COMMERCIAL FATS AND OFS Cd 8b-90 * Peroxide Value Acetic Acid-Isooctane Method 3. Ifthe titration sles han 0.5 ml using 0.1 M sodium thiosulfate, repeat the determination using 0.01 M sodium thiosulfate, ‘using vigorous agitation andlor surfactant for the reason stated in Notes, 2. Analysts may use 0.001 M sodium thiosulfate if full validation protocols ar followed. 4. The test should be carried out in diffuse daylight or in artificial light shielded ffom a direct light source (References, 4) REFERENCES 1. Brooks, D.D., and D.L. Bemer, looctane as an Alternative Solvent for Peroxide Value Determination. StadyT, poster pre= sentation at AOCS Annual Mecting, Baltimore, MD, April 23, 1990, 2. Collaborative study results published in JNFORM 1:884 (1990) 3. Brooks, D.D., S.K. Brophy, B. Hayden, and G.R. Goss, Alternative Solvents for Peroxide Value Determination, poster pre- sentation at AOCS Annual Meeting, Cincinnati, OH, May 10, 1989, 4. J Asoc. Off Anal. Chem. 75:507 (1992). 5. The fncernational Standards Organization (ISO) successfully completed an ince in 1996, The results are presented in ISO 3960. Table 1 ‘Mass of test portion and accuracy of weighing. «ional collaborative suudy ofthis method Eapected peroxide Mas of ese ‘Weighing value (meas) portion @) accuracy (2 owl 5.0020 £001 121020 200012 £001 201030 12008 £0.01 3010 50 080005 0.001 501090 05%003 0.001 Table2 ‘The results of international collaborative tests held between 1993 and 1999 (see References, 5). 7 ‘Coconut oil ‘Linola oil Lard Tallow Beef far Olive oil Palm stearin * 4 5 B n 0 16 16 Mean 134 Bat 2.89 40 122 24.10 927 Repeaabiiey * 007 016 018 079 0.36 098 073 RSD, 538 sua 634 S61 253 408 736 r 020 045 ost 2.20 1.00 275 204 Reproducibilcy ” 035 0.99 031 407 4d 392 243 RSD, 26.04 3176 10.65 2908 33.96 1626 26.18 R 0.99 2m 0.86 40 1160 1.98 680 Table 3 ‘Test on olive ol. Refined olive oil Olive oil Mean valve, meq/g waa 7.66 Relative repeatability sandand deviation, 1133 605 Repeatability Limit,» meq/g 423 130 Relative reproducibility andard deviation, 1248 1324 458 284 Table4 ‘Test on lard, tallow, and beet fat. ed Tallow Number of laboratories 1 " Number of aceptable results n n Mean vale, meq/g 49 67 Repeatability limit. mea/kg 04 1 Reproduetbiiey init, K meq/kg 24 57 Page 3 of 3