SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OLS ROCS& AOCS Official Method Ca 2e-84 | Reapproved 2009| Moisture Karl Fischer Reagent DEFINITION This method determines the actual water content of fas and oils by titration with Fischer reagent, which reacts quantitatively wich water. SCOPE Applicable to fats and oils that do not react with, and are soluble in, the reagents and that do not contain imparitcs leading to secondary eactions. Such impurities are alkaline compounds and peroxides, which react with the reagent and therefore show high results (see References, 1). Replaces Ca 2e-55. APPARATUS 1 Karl Fischer titration assembly—manual or aueomatic, with stirrer REAGENTS Note—Karl Fischer eeage (see Notes, 1} is availabe from most chemical supply houses. While the reagent may be prepared as indicated below, due tothe hazards associated with the solvents used in the reagent preparation iis recommended chat the Karl Fischer reagent be purchased commercially. See Notes, Caution regarding the hazards associated with the solvents used in the reagent preparation. 1 “Methanol—not containing more chan 0.05% (wt/wt) of water. IFthe reagent contains more than this quantity of water dry it by distillation from magnesium turnings activated with iodine. Collect the distillate in receiver protected from armo- spheric moisture by means ofa guard tubs Frced with desiccant (Reagents, 8). 2-Methoxyethanol (ethylene glycol monomethy ether)—not containing more than 0.05% (ort/wt) of water. Ifthe reagent contains more than this quantity of water, dey it by distillation, rejecting the fist portion of distillate, which concains the Pyridine—nor containing more than 0.05% (oct) of water. If the agent contains more than this quantity of water, dry ic by distillation, rejecting the first portion of distillae, which contains the water. Sample solvent—cither a mixture containing 4 parts by volume of the methanol (Reagents. 1) and 1 part by volume of the pyridine (Reagents 3) of, preferably, for determinations with compounds containing carbonyl groups, a mixture containing 4 pares by volume of the 2-methoxyerhanol (Reagents, 2) and 1 part by volume of the pyridine (Reagents, 3). In special cases, other solvents may be used, c.g, atic acid pyridine, ora mixture containing I partby volume ofthe methanol (Reagents, 1) and 3 parts by volume of chloroform. Sulfur dioxide—anhydrous liquified gas in cylinder Todine—reagent grade Karl Fischer reagent—stabilized single solution, available commercially or prepared as follows: {@) Place 670 ml. of the methanol (Reagents, 1) or the 2-methoxyethanol (Reagents, 2) in a dry brown glass lass, iced. with 2 ground-glass stopper and having a capaciey of slightly greater chan 1 C- (b) Add about 85 g of iodine. Stopper the flask and shake it occasionally until the iodine is completely dissolved. Then add approximately 270 ml. of the pyridine (Reagents, 3), stopper the ask again and mix thoroughly. Using he method described below, dissolve 65g of sulfur dioxide inthis solution, cooling to ensure that the temperature does not exceed 20°C. ‘Note—As the reaction is exothermic, its necessary to cool the Pask ftom the beginning ancl to maintain i at about °C, eg. by immersing it in an ice bath or in crushed earbon dioxide. (0 Replace the ground-glas stopper by an atcachmene for introducing sulfur dioxide, consisting of cork bearing a ther- mometer and a glass inlet tube (6 x 8 mm), reaching ro within 10 mm of the bottom of the flask, and a small capillary tube for connecting to the atmosphere {)_ lace the whole assembly withthe ice bath on a balance and weigh to the neatest 1 g (6) Connect the inlet tube co a cylinder of sulfur dioxide by means ofa flexible connection and a drying tube filled with the desiccant (Reagents, 8): geady open the tap on the cylinder. Adjust the rate of flow of the sulfur dioxide so that all the fas is absorbed without the liquid showing any sign of rising in the inlet tube. Maintain the equilibrium of the balance by gradually increasing che tare, and ensure thar the remperacure of the liquid does not exceed 20°C. Close the tap on the oylinder as soon as the increase in weight reaches 65 (© Immediately remove the flexible connection and reweigh the flask and its inlet artachment. The weight of dissolved sulfur dioxide should be between 60 and 70 g. A slight excess is noc harmful Stopper the flask, mix che sofucion, and leave for at least 24 hr before use. Infact, asthe result of imperfectly understood reactions that occur in the fresh reagent, the water equivalent ofthe reagene decreases rapidly to begin with and then much more slowly. This water equivalent Page 1 0f 3MAING AND ANANSR OF COMMIRCIAL FATSAND ONS \Ca 2e-84 * Moisture Karl Fischer Reagent is between 3.5 and 4.5 mg/mL. Ie must be determined daily if methanol has been used, but may be determined less frequently if2-methosyethanol has been used. Ic is posible co prepare Karl Fischer eeagent having a lower water content by diluting the solution prepared as described above with the sample solvent (Reagent, 4). (©) Store the reagent in the dark and protected from atmospheric moisture. It preferably should be stored in the reagent bottle. 8, Aluminum sodium silicate—~anhydrous, in the form of granules of 1.7 mm diameter, for use as a desiccant. The granules may be regenerated by washing with water and drying at 350°C for at lease 48 he. Alternatively, activated silica gel may be used as a desiccant 9. Chloroform—reagent grade. DETERMINATION OF THE WATER EQUIVALENT OF THE KARL FISCHER REAGENT 1. Accurately weigh (o che nearest 0.0001 g) a drop of water, approximately 50 mg, and transfer co the citration vessel. For cis calibration of the reagent, 250 mg of crystalline sodium tartrate (Na,C,H,O,- 2H,0) may alo be used instead of a drop of ‘Add the stirring rod by means of a pair of eweezers and connect the titration vessel with the apparatus. ‘Add 20 mb, of methanol (Reagents 1) by means ofa pipet or a burette. Close the aperture and dissolve by stirring genely. ‘Note—The amount of solvent used must allow the electrode to dip 2~3 mm. 4, Tirate with the Karl Fischer reagent up to the electrometric end point. This poine is reached when the deflection remains constant for 30 sec after dropwise adding the Karl Fischer reagent. Carry out a blank ces. 5. For direct reading apparatus, the calibration Factor is inserted into the equipmene in accordance with manufacturer’ instruction, and a blank determination i not required. PROCEDURE 1, Weigh (co the nearest 0.01 g) 5-25 g of the test sample into the titration vessel, but make certain chat it does not contain more than 100 mg of water. Dilute the Karl Fischer solution in case of a low water content and/or poor solubility of the sample inorder to get a sufficiently high titration 2, Put into the titration vessel the stirring rod by means ofa pair of tweezers and connect it with the apparatus. Add, if neces- sary, exacly 10 mL. chloroform (Reagents, ) or another suitable solvent and dissolve the sample by string. Often solid fas do not dissolve completely. These are first dissolved in 10 ral. of dry chloroform and then 10 mL of methanol are added. 3. The amount of solvent used must allow the electrode to dip 2-3 mm. 4, Add 10 mb. of methanol and titrate with the Karl Fischer reagent up tthe eleetrometric end poine. 5. Carry outa blank test following the same procedure and using che same quantity of ll of the reagents as used for the detet= ination bue omitting the test portion. This is noc required for direct reading instruments CALCULATIONS 1, The water equivalent, T, ofthe Karl Fischer reagent in mg H,O/mL is given by the formula Where— ‘m, = mass. in mg, of the water added Vy = volume, in mL, of the Karl Fischer reagent used for the determination of che warer equivalent of the Kael Fischer reagent V, = volume, in mL, of the Karl Fischer eagent used for the blank eest 2. The water content of the test sample i given by the forrmula MW =W xT Percent HO (we = 100 Where— im, = mass, in mg, of the test portion Y, = volume, in mL, of the Karl Fischer reagent used for the determination (Procedure, 4) Vj = volume, in ml, ofthe Karl Fischer reagent used forthe blank test (Procedure, 5) 3, Take aba resule the arithmetic mean of two determinations. PRECISION 1. The difference berween the results of two differene determinations carried our simultaneously or in rapid succession by the same operator on the same sample shall nor exceed 0.64% relative error Page 2 of 3SAMPLING AND ANAIVSIS OF COMMERCIAL TATSAND OWS Ca 2e-84 * Moisture Karl Fischer Reagent, NOTES Caution Methanol is fammable and toxic. Avoid contact with eyes. Avoid breathing vapors. Use effective fume-removal device. Can react vigorously wich sodium hydroxide + chloroform, potassium hydroxide + chloroform, and perchloric acid. Pyridine is fammable and a dangerous fre risk. The explosive limits in air are 1,812.49. Iis toxic by ingestion and inhala- sion, The TLY is 5 ppm in air. The danger from crude pyridine is greater than from pure pyridine, the asociated homologs and ‘impurities being even more toxic than pyridine itself Chloroform is + known carcinogen. Ieisroxic by inhalation and has anesthetic propetcs. Avoid contac with the skin Prolonged inhalation or ingestion can lead to liver and kidney damage and may be fatal. It is nonflammable, but will buen on protonged exposure to lame or high temperature. The TLV is 10 ppm in air. A Fume hood should be used acall times when using chloroform, NUMBERED NOTES 1. Because ofits toxicity, pyridine is an undesirable constiuent of the Karl Fischer reagent. Both one- and ewo-component Karl Fischer reagents, based on aliphatic amines and heterocyclic compounds and containing no pyridine, are available commercially. REFERENCES 1, Reference for the collaborative study is Bernetti, R, S. |. Kochan, and J. J. Pienkowski, /. Asoc. Off Anal. Chem. 67:299 (1984), Page 3 0f