SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS AOCS Official Method Cd 14-92 lodine Value of Fats and Oils Cyclohexane-Acetic Acid Method _ EE e—>e—— DEFINITION The iodine value is a measute ofthe unsaturation of fats and oils and is expressed in terms of the number of centigrams of iodine absorbed per gram of test sample (% iodine absorbed). SCOPE Applicable to all normal fats and oils with iodine values in the range of 18-165 that do not contain conju- gated double bonds (see Notes, 1). ‘APPARATUS 1. Glass-stoppered iodine flasks—500 mL 2. Glass-stoppered volumetric lasks—1000 mL, for preparing standard solutions. 3, Dipee—25 mL, for accurately dispensing 25.0 mL. of Wijs solution. 4, Volumetric dispenser—20 mL, I mL. adjustability, for 10% potassium iodide (KI) solution. 5. Volumetric dispenser—2 mL, 1 mL adjustability fr starch solution. 6. Volumetric dispenser—50 mi, | ml. adjustability, for distilled wate. 7. 8 9. Repeater pipet—with filling flask, 20 ml, for cyclohexane. Analytical balance—accurate to 0.0001 g. Magnetic stiret. 10, Filter paper—Whatman no, 541, or equivalent. 11, Beakers—50 mL. 12, Horair oven, 13, Timer REAGENTS 1, Wijs solution—see Notes, 2 and Caution 2. Potassium iodide (KI) solution—100 g/L (10% solution), prepared by dissolving 100 g of reagent grade KI in 1000 mL of deionized water. 3. Cyctohexane—reagent grade (see Notes, 3 and Caution) 4. Glacial acetic acid —reagent grade (sce Notes, Caution). 5. Reagent for diluting test sample—prepared by mixing cyclohexane and glacial acetic acid, 1:1, v/s The absence of oxidizable matter in the reagent is verified by shaking 10 mL. of the reagent with I mL. of saturated aqucous potassium dichromate solution and 2 mL of concentrated sulfuric ace no green coloration should appear. Hydrochloric acid—reagent grade (see Notes, Caution). . Soluble starch solution—recently prepared, tested for sensitivity (see Notes, 4). Make a paste with 1 g of natural, soluble starch (see Notes, 5) and a small amount of cold distilled water. Add, while stcring, to 100 ml. of boiling water. Tesefor senitiviy—Place 5 mL of starch solution in 100 mL of water and add 0.05 mL of freshly prepared 0.1 M KI solu- tion and one drop of a50 ppm chlorine solution made by diluting 1 mL. ofa commercial 5% sodium hypochlorite (NaOCl) solution co 1000 mL. The deep blue color produced must be discharged by 0.05 mL of 0.1 N sodium thiosulfate 8, Potassium dichromate—reagent grade. The potassium dichromate is finely ground and dried to constant weight at about 110°C before using (see Notes, Caution). 9. Sodium thiosulfate (Na,S,0,'5H,0)—0.1 M, accurately standardized, prepared from reagent-grade Na,S,O5H1,O (sce Notes, 6) Standardization of sodium thiosulfate—Weigh 0.16-0.22 g of finely ground and dried potassium dichromate into a 500 mil. lask or botde by difference from a weighing borte, Distolve in 25 mL of water, add 5 mL of concentrated hydro- chlorie acid, 20 mL of potassium iodide solution and rotate to mix. Allow to stand for 5 min, and chen add 100 mL of Aiscilled water. Titrate with sodium thiosulfate solution, shaking continuously until the yeliow color has almost disappeared. ‘Add 1-2 mL of starch indicator and continue the titration, adding the thiorulfae solution slowly until the blue color just disappears. The strengch of the sodium thiosulfate solution is expressed in terms ofits molarity. 6. Page 1 of 3SANIPLING AND ANALYSIS OF COMMERCIAL FATS AND O1S Cd 14-92 * lodine Value of Fats and Oils PROCEDURE 1, Mee che test sample, ifs not already liquid (the temperature during melting should not exceed the melting point of the test sample by more than 10°C), and filter through two pieces of filter paper to remove any solid impurities and the last traces of moisture. The filtration may be performed in an air oven at 80-85°C, but should be completed within 5 min + 30 sec. The test sample must be absolutely dry ‘Note—All glassware must be absolutely clean and completely dry 2. Aferfleation, allow the filtered test portion to achieve a temperature of 68-71 + 1°C before weighing the test portion, 3. Once the tes portion has achieved a temperature of 68-71 + 1°C, immediately weigh the test portion into a 500 ml. iodine fas, using the weights and weighing accuracy noted in Table 1 (see Notes, 7). 4. Add 15 mL of cyclohexane + glacial acetic acid (Reagents, 5) on top of the test portion and swiel ro ensure thatthe tet portion is completely dissolved 5. Dispense 25 ml. of Wijs solution using the pipet (Apparatus 3) into the Aask containing the test portion, stopper the flask, and swirl to ensure an intimate mixture. Immediately set the timer for 1.0 or 2.0 hr, depending on the iodine value ofthe test portion IV < 150, 1.0 hr; IV 2 150, 2.0 hr (see Notes, 8) Immediately store the flasks in the dark for the tequired reaction time at a temperature of 25 + 5*C. Remove the flasks from storage and add 20 ml. of KI solution, followed by 100 ml of dislled water (ee Notes,9 and 10). Thrace wth 0.1 M Na,S,O, solution, adding ic gradually and with constant and vigorous shaking (see Notes, 11). Continue the titration until the yellow color has almost disappeared. Add 1-2 ml. of starch indicator solution and continue the titration until the blue color juse disappears 9. Prepare and conduct atleast one blank determination with each group of test portions simultaneously and similar in al respects to the test portion CALCULATIONS 1. Iodine valu Where— B = volume of titrant, ml. of blank S = volume of titrant, mL of test portion M = molarity of Na,S,O, solution B-9 x Mx 12.69 ‘mas of test portion, g PRECISION 1. International Union of Pure and Applied Chemistry (TUPAC)/International Organization for Standardization (ISO) collaborative studies gave che statistical results shown in Table 2. NoTES Caution Wijs solution causes severe burns, and the vapors can cause lung and eye damage. Use of a fume hood is recommended. Wijs solution without carbon tetrachloride is available commercially ‘Cyclohexane is flammable and a dangerous fire risk, It is moderately toxic by inhalation and skin contact. The TLY in ait is 300 ppm. Glacial acetic acid in the pure state is moderately toxic by ingestion and inhalation. It sa strong irritant to skin and tissu. ‘The TLY in aris 10 ppm. Hydrochloric acid isa strong acid and will cause severe burns. Protective clothing should be worn when working with this acid, Itis toxic by ingestion and inhalation and is a strong iritant to eyes and skin. The use ofa properly operating fume hood is recommended, When diluting the acid, always add the acid to the water, never the reverse Potassium dichromate is toxic by ingestion and inhalation. There is suficent evidence in humans for the carcinogenicity of chromium [+6], in particular, lung cancer. Ic isa stong oxidizing agent and a dangerous fre risk when in contact with organic chemical. NUMBERED NOTES 1. When the iodine value is determined on materials having conjugated systems, the results nota measure of total unsaturation, bout rather is an empirical value indicative of the amount of unsaturation present. Reproducible results ate obtained that lord a comparison of total unsaturation. When the iodine value is required on fatty acids, see Method Ty 12-64. Because the preparation ofthe Wijs solution is time-consuming and involves the use of both hazardous and toxic chemicals, this solution may be purchased from a chemical supplier. Solutions are available which contain no carbon tetrachloride, and such solutions should be used. All Wijs solutions are sensitive ro temperature, moiscure and light. Store ina cool and dark place, and never allow co come to a temperature above 25-30°C. The laboratory preparation of Wijs solution is noted in ‘AOCS Official Method Cd 1-25. 3. Fresh cyclohexane should be used. Erratic results may be obtained if old cyclohexane is used. See Reagents, 5 fora test for the presence of oxidizable substances. Page 2 of- “ANPING AND ANALYSIS OF COMMERCIAL FATS AND OHS Cd 14-92 ¢ lodine Value of Fats and Oils 4, 10% starch solution may be purchased from a chemical supplier. 5. “Potato Starch for fodometry” is recommended, because this starch produces a deep blue color in the presence of the ioclonium 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, Fisher $516-100; Soluble Potato Starch, Sigma S-2630; Soluble Potato Starch for lodometry, JT. Baker 4006-04, 6. The sodium thiosulfate solution may be purchased from a chemical supplier. However i still must be accurately standardized in the laboratory. The weight of the test portion must be such that there will be an excess of Wijs solution of 50-60% of the amount added, i.e, 100=150% of the amount absorbed. 8. The indicated reaction times are those specified in the IUPAC Iodine Value Method 2.205 (References, 1) and were the reaction times used in the IUPACIISO validation study of the cyclohexane + acetic acid method, Previous AOCS versions of iodine value methods specified a reaction time of 0.5 hr, regardless of the iodine value, but noted that “a longer reaction time may be necessary for oils with high iodine value.” The longer reaction times appear to be particularly critical when cyclohexane is used asa replacement for carbon retachloride (References, 2). 9. If the reaction is not terminated within 3 min after the reaction time, the test portion must be discarded. 10. The ces portion must be cieated within 30 min of reaction termination, after which the analysis is invalid 11. Mechanical stirring is recommended for agitation during the addition of thiosulfate. REFERENCES 1. Standard Methods for the Analysis of Oi, Fats and Derivatives, International Union of Pure and Applied Chemiotry, th edn, Blackwell Scientific Publications, 1987, IUPAC Method 2.205, 2. AOCS collaborative study results using cyclohexane alone appear in J. Am. Oil Chem. Soc. 65745 (1988). 3. IUPAC collaborative study results using cyclohexane + glacial acetic acid (1:1) appear in Pure Appl. Chem. 62:25 (1990) and were reported in INFORM 1-484 (1990), Table Test Sample weights Tedine Value expected =a, g 2 0.003 3 3.000 520 1.000 21-50 0.400 51-100 0.200 101-150 0.130 151-200 0.100 Table 2 Statistical analysis of results obtained in IUPAC/ISO collaborative study (11 laboratories) of Wijs iodine value with carbon tetrachloride and cyclohexane + glacial acetic acid (1:1) solvents¢ Tea Sample _Mean vale RSD, RSD, SD, SD, i crc CHX CTC CTC cHX CTC cHX_Cr iX_CTC_CHX Sunflower 13361329 107 129257 17L 143 ATL 3442.28 Refined palm 53.1 53.0 023 029 051 048 012 016 027 036 Crude fish 109.1 108.5 060 050 155 1.00 065 054 169 1.09 Tang 14S 1634 124 085 188 154 204 139 310 252 Tallow (beef) 472 469 049 048 098 0.75 (023 022 046 035, Crudepalm 525 526 049 077 076 103 026 040 040 0.54 Used fing 37.7377 035 049 (042 089 (0.13 019 0.16 0.33 Palm kernel 182183, 0.06 005 0.18 0.23 0.01 0.01 0.03 0.04 Olive 823 822 027 063 066 0.94 022 051 055 0.77 Hsso1* 102.6 1023 050 079 LL 182 051 080 176 1.86 HsB02 47 748 061 054 132 075 045 040 099 0.56 HFO! 73.0728 G61 057 089 082 0.44 0.42 065 0.60 “ce References, 3, RSD, relative standard deviation, repeatabiliy; RSD, elaive eandard deviation, reproducibility; SD, standard deviation, repeatability SD, standard deviation, reproducibility; CTC, eabon terachloride; CH, cyloherane «glacial acetic acid (1:1); HSBO, hdrogenaed soybean oll HFO, hydrogenated fh ol Additional seady involving 17 laboratories Page 3 of 3