401 FATS AND FIXED OILS
The following definitions and general procedures apply to fats, fixed oils, waxes, resins, balsams, and similar substances.
PREPARATION OF SPECIMEN
If a specimen of oil shows turbidity owing to separated stearin, warm the container in a water bath at 50 until the oil is clear, or if the oil does not become clear on warming, pass it through dry filter paper in a funnel contained in a hot-water jacket. Mix thoroughly, and weigh at one time as many portions as are needed for the various determinations, using preferably a bottle having a pipet dropper, or a weighing buret. Keep the specimen melted, if solid at room temperature, until the desired portions of specimen are withdrawn.
Determine the specific gravity of a fat or oil as directed under Specific Gravity 841.
Determine the melting temperature as directed for substances of Class II (see Melting Range or Temperature 741).
ACID VALUE (FREE FATTY ACIDS)
The acidity of fats and fixed oils in this Pharmacopeia may be expressed as the number of mL of 0.1 N alkali required to neutralize the free acids in 10.0 g of substance. Acidity is frequently expressed as the Acid Value, which is the number of mg of potassium hydroxide required to neutralize the free acids in 1.0 g of the substance. Unless otherwise directed in the individual monograph, use Method I.
Procedure Unless otherwise directed, dissolve about 10.0 g of the substance, accurately weighed, in 50 mL of a mixture of equal volumes of alcohol and ether (which has been neutralized to phenolphthalein with 0.1 N potassium hydroxide or 0.1 N sodium hydroxide, unless otherwise specified) contained in a flask. If the test specimen does not dissolve in the cold solvent, connect the flask with a suitable condenser and warm slowly, with frequent shaking, until the specimen dissolves. Add 1 mL of phenolphthalein TS, and titrate with 0.1 N potassium hydroxide VS or 0.1 N sodium hydroxide VS until the solution remains faintly pink after shaking for 30 seconds. Calculate either the Acid Value or the volume of 0.1 N alkali required to neutralize 10.0 g of specimen (free fatty acids), whichever is appropriate. Calculate the Acid Value by the formula:
56.11V × N/Win which 56.11 is the molecular weight of potassium hydroxide; V is the volume, in mL; N is the normality of the potassium hydroxide solution or the sodium hydroxide solution; and W is the weight, in g, of the sample taken.
If the volume of 0.1 N potassium hydroxide VS or 0.1 N sodium hydroxide VS required for the titration is less than 2 mL, a more dilute titrant may be used, or the sample size may be adjusted accordingly. The results may be expressed in terms of the volume of titrant used or in terms of the equivalent volume of 0.1 N potassium hydroxide or 0.1 N sodium hydroxide.
If the oil has been saturated with carbon dioxide for the purpose of preservation, gently reflux the alcohol-ether solution for 10 minutes before titration. The oil may be freed from carbon dioxide also by exposing it in a shallow dish in a vacuum desiccator for 24 hours before weighing the test specimens.
Procedure Prepare 125 mL of a solvent mixture consisting of equal volumes of isopropyl alcohol and toluene. Before use, add 2 mL of a 1% solution of phenolphthalein in isopropyl alcohol to the 125-mL mixture, and neutralize with alkali to a faint but permanent pink color. Weigh accurately the appropriate amount of well-mixed liquid sample indicated in the table below, and dissolve it in the neutralized solvent mixture. If the test specimen does not dissolve in the cold solvent, connect the flask with a suitable condenser and warm slowly, with frequent shaking, until the specimen dissolves. Shake vigorously while titrating with 0.1 N potassium hydroxide VS or 0.1 N sodium hydroxide VS to the first permanent pink of the same intensity as that of the neutralized solvent before mixing with the sample. Calculate the Acid Value as indicated in Method I.
The Ester Value is the number of mg of potassium hydroxide required to saponify the esters in 1.0 g of the substance. If the Saponification Value and the Acid Value have been determined, the difference between these two represents the Ester Value.
Procedure Place 1.5 g to 2 g of the substance in a tared, 250-mL flask, weigh accurately, add 20 mL to 30 mL of neutralized alcohol, and shake. Add 1 mL of phenolphthalein TS, and titrate with 0.5 N alcoholic potassium hydroxide VS until the free acid is neutralized. Add 25.0 mL of 0.5 N alcoholic potassium hydroxide VS, and proceed as directed under Saponification Value, beginning with Heat the flask and omitting the further addition of phenolphthalein TS. The difference between the volumes, in mL, of 0.5 N hydrochloric acid consumed in the actual test and in the blank test, multiplied by 28.05 and divided by the weight in g of the specimen taken, is the Ester Value.
The Hydroxyl Value is the number of mg of potassium hydroxide equivalent to the hydroxyl content of 1.0 g of the substance.
PyridineAcetic Anhydride Reagent Just before use, mix 3 volumes of freshly opened or freshly distilled pyridine with 1 volume of freshly opened or freshly distilled acetic anhydride.
Procedure Transfer a quantity of the substance, determined by reference to the accompanying table and accurately weighed, to a glass-stoppered, 250-mL conical flask, and add 5.0 mL of PyridineAcetic Anhydride Reagent. Transfer 5.0 mL of PyridineAcetic Anhydride Reagent to a second glass-stoppered, 250-mL conical flask to provide the reagent blank. Fit both flasks with suitable glass-jointed reflux condensers, heat on a steam bath for 1 hour, add 10 mL of water through each condenser, and heat on the steam bath for 10 minutes more. Cool, and to each add 25 mL of butyl alcohol, previously neutralized to phenolphthalein TS with 0.5 N alcoholic potassium hydroxide, by pouring 15 mL through each condenser and, after removing the condensers, washing the sides of both flasks with the remaining 10-mL portions. To each flask add 1 mL of phenolphthalein TS, and titrate with 0.5 N alcoholic potassium hydroxide VS, recording the volume, in mL, consumed by the residual acid in the test solution as T and that consumed by the blank as B. In a 125-mL conical flask, mix about 10 g of the substance, accurately weighed, with 10 mL of freshly distilled pyridine, previously neutralized to phenolphthalein TS, add 1 mL of phenolphthalein TS, and titrate with 0.5 N alcoholic potassium hydroxide VS, recording the volume, in mL, consumed by the free acid in the test specimen as A, or use the Acid Value to obtain A. Calculate the Hydroxyl Value taken by the formula:
(56.11N / W)[B + (WA / C) T]in which W and C are the weights, in g, of the substances taken for the acetylation and for the free acid determination, respectively; N is the exact normality of the alcoholic potassium hydroxide; and 56.11 is the molecular weight of potassium hydroxide.
The Iodine Value represents the number of g of iodine absorbed, under the prescribed conditions, by 100 g of the substance. Unless otherwise specified in the individual monograph, determine the Iodine Value by Method I.
Method I (Hanus Method)
Procedure Transfer an accurately weighed quantity of sample, as determined from the accompanying table, into a 250-mL iodine flask, dissolve it in 10 mL of chloroform, add 25.0 mL of iodobromide TS, insert the stopper in the vessel securely, and allow it to stand for 30 minutes protected from light, with occasional shaking. Then add, in the order named, 30 mL of potassium iodide TS and 100 mL of water, and titrate the liberated iodine with 0.1 N sodium thiosulfate VS, shaking thoroughly after each addition of thiosulfate. When the iodine color becomes quite pale, add 3 mL of starch TS, and continue the titration with 0.1 N sodium thiosulfate VS until the blue color is discharged. Perform a blank test at the same time with the same quantities of the same reagents and in the same manner (see Residual Titrations 541). Calculate the Iodine Value from the formula:
[126.9(VB VS)N] / 10Win which 126.9 is the atomic weight of iodine; VB and VS are the volumes, in mL, of 0.1 N sodium thiosulfate VS consumed by the blank test and the actual test, respectively; N is the exact normality of the sodium thiosulfate VS; and W is the weight, in g, of the substance taken for the test. [noteIf more than half of the iodobromide TS is absorbed by the portion of the substance taken, repeat the determination, using a smaller portion of the substance under examination.]
Potassium Iodide Solution Dissolve 10.0 g of potassium iodide in water to make 100 mL. Store in light-resistant containers.
Starch Indicator Solution Mix 1 g of soluble starch with sufficient cold water to make a thin paste. Add, while stirring, to 100 mL of boiling water. Mix, and cool. Use only the clear solution.
Procedure Melt the sample, if it is not already liquid. [noteThe temperature during melting should not exceed the melting point of the sample by more than 10.] Pass 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 100 but should be completed within 5 minutes ± 30 seconds. The sample must be absolutely dry. All glassware must be absolutely clean and completely dry. After filtration, allow the filtered sample to achieve a temperature of 68 to 71 ± 1 before weighing the sample. Once the sample has achieved a temperature of 68 to 71 ± 1, immediately weigh the sample into a 500-mL iodine flask, using the weights and weighing accuracy noted in the accompanying table. [noteThe weight of the substance must be such that there will be an excess of iodochloride TS of 50% to 60% of the amount added, that is, 100% to 150% of the amount absorbed.] Add 15 mL of a fresh mixture of cyclohexane and glacial acetic acid (1:1), and swirl to dissolve the sample. Add 25.0 mL of iodochloride TS, insert the stopper securely in the flask, and swirl to mix. Allow it to stand at 25 ± 5, protected from light, with occasional shaking, for 1.0 or 2.0 hours, depending on the Iodine Value (IV) of the sample: IV less than 150, 1.0 hour; IV equal to or greater than 150, 2.0 hours. Then, within 3 minutes after the indicated reaction time, add, in the order named, 20 mL of Potassium Iodide Solution and 150 mL of recently boiled and cooled water, and mix. Within 30 minutes, titrate the liberated iodine with 0.1 N sodium thiosulfate VS, while stirring by mechanical means after each addition of thiosulfate. When the yellow iodine color has almost disappeared, add 1 to 2 mL of Starch Indicator Solution, and continue the titration with 0.1 N sodium thiosulfate VS until the blue color is discharged. Perform a blank test at the same time with the same quantities of the same reagents and in the same manner (see Residual Titrations 541). The difference between the volumes, in mL, of 0.1 N sodium thiosulfate consumed by the blank test and the actual test, multiplied by 1.269 and divided by the weight, in g, of the sample taken, is the Iodine Value.
The Peroxide Value is the number that expresses, in milliequivalents of active oxygen, the quantity of peroxide contained in 1000 g of the substance. [noteThis test must be performed promptly after sampling to avoid oxidation of the test specimen.]
Procedure Unless otherwise directed, place about 5 g of the substance, accurately weighed, in a 250-mL conical flask fitted with a ground-glass stopper. Add 30 mL of a mixture of glacial acetic acid and chloroform (3:2), shake to dissolve, and add 0.5 mL of saturated potassium iodide solution. Shake for exactly 1 minute, and add 30 mL of water. Titrate with 0.01 N sodium thiosulfate VS, adding the titrant slowly with continuous shaking, until the yellow color is almost discharged. Add 5 mL of starch TS, and continue the titration, shaking vigorously, until the blue color is discharged. Perform a blank determination under the same conditions. [noteThe volume of titrant used in the blank determination must not exceed 0.1 mL.] The difference between the volumes, in mL, of 0.01 N sodium thiosulfate consumed in the actual test and in the blank test, multiplied by 10 and divided by the weight, in g, of the specimen taken, is the Peroxide Value.
The Saponification Value is the number of mg of potassium hydroxide required to neutralize the free acids and saponify the esters contained in 1.0 g of the substance.
Procedure Place 1.5 g to 2 g of the substance in a tared, 250-mL flask, weigh accurately, and add to it 25.0 mL of 0.5 N alcoholic potassium hydroxide. Heat the flask on a steam bath, under a suitable condenser to maintain reflux for 30 minutes, frequently rotating the contents. Then add 1 mL of phenolphthalein TS, and titrate the excess potassium hydroxide with 0.5 N hydrochloric acid VS. Perform a blank determination under the same conditions (see Residual Titrations under Titrimetry 541). The titration also can be carried out potentiometrically. The difference between the volumes, in mL, of 0.5 N hydrochloric acid consumed in the actual test and in the blank test, multiplied by 56.1 and the exact normality of the 0.5 N hydrochloric acid VS, and divided by the weight in g of specimen taken, is the Saponification Value.
If the oil has been saturated with carbon dioxide for the purpose of preservation, expose it in a shallow dish in a vacuum desiccator for 24 hours before weighing the test specimens.
The term Unsaponifiable Matter in oils or fats, refers to those substances that are not saponifiable by alkali hydroxides but are soluble in the ordinary fat solvents, and to products of saponification that are soluble in such solvents.
Procedure Transfer about 5.0 g of the oil or fat, accurately weighed, to a 250-mL conical flask, add 50 mL of an alcoholic potassium hydroxide solution prepared by dissolving 12 g of potassium hydroxide in 10 mL of water and diluting this solution with alcohol to 100 mL, and heat the flask on a steam bath under a suitable condenser to maintain reflux for 1 hour, swirling frequently. Cool to a temperature below 25, and transfer the contents of the flask to a separator having a polytetrafluoroethylene stopcock, rinsing the flask with two 50-mL portions of water that are added to the separator (do not use grease on stopcock). Extract with three 100-mL portions of ether, combining the ether extracts in another separator containing 40 mL of water. Gently rotate or shake the separator for a few minutes. [noteViolent agitation may result in the formation of a difficult-to-separate emulsion.] Allow the mixture to separate, and discard the lower aqueous phase. Wash the ether extract with two additional 40-mL portions of water, and discard the lower aqueous phase. Wash the ether extract successively with a 40-mL portion of potassium hydroxide solution (3 in 100) and a 40-mL portion of water. Repeat this potassium hydroxide solution-water wash sequence three times. Wash the ether extract with 40-mL portions of water until the last washing is not reddened by the addition of 2 drops of phenolphthalein TS. Transfer the ether extract to a tared flask, and rinse the separator with 10 mL of ether, adding the rinsings to the flask. Evaporate the ether on a steam bath, and add 6 mL of acetone to the residue. Remove the acetone in a current of air, and dry the residue at 105 until successive weighings differ by not more than 1 mg. Calculate the percentage of unsaponifiable matter in the portion of oil or fat taken by the formula:
100(WR / WS)in which WR is the weight, in g, of the residue; and WS is the weight, in g, of the oil or fat taken for the test.
Dissolve the residue in 20 mL of alcohol, previously neutralized to the phenolphthalein endpoint, add phenolphthalein TS, and titrate with 0.1 N alcoholic sodium hydroxide VS to the first appearance of a faint pink color that persists for not less than 30 seconds. If the volume of 0.1 N alcoholic sodium hydroxide required is greater than 0.2 mL, the separation of the layers was incomplete; the residue weighed cannot be considered as unsaponifiable matter, and the test must be repeated.
SOLIDIFICATION TEMPERATURE OF FATTY ACIDS
Preparation of the Fatty Acids Heat 75 mL of glycerinpotassium hydroxide solution (made by dissolving 25 g of potassium hydroxide in 100 mL of glycerin) in an 800-mL beaker to 150, and add 50 mL of the clarified fat, melted if necessary. Heat the mixture for 15 minutes with frequent stirring, but do not allow the temperature to rise above 150. Saponification is complete when the mixture is homogeneous, with no particles clinging to the beaker at the meniscus. Pour the contents of the beaker into 500 mL of nearly boiling water in an 800-mL beaker or casserole, add slowly 50 mL of dilute sulfuric acid (made by adding water and sulfuric acid (3:1)), and heat the solution, with frequent stirring, until the fatty acids separate cleanly as a transparent layer. Wash the acids with boiling water until free from sulfuric acid, collect them in a small beaker, place on a steam bath until the water has settled and the fatty acids are clear, filter into a dry beaker while hot, and dry at 105 for 20 minutes. Place the warm fatty acids in a suitable container, and cool in an ice bath until they congeal.
Test for Complete Saponification Place 3 mL of the dry acids in a test tube, and add 15 mL of alcohol. Heat the solution to boiling, and add an equal volume of 6 N ammonium hydroxide. A clear solution results.
Procedure Using an apparatus similar to the Congealing Temperature Apparatus specified therein, proceed as directed for Procedure under Congealing Temperature 651, reading solidification temperature for congealing point (the terms are synonymous). The average of not less than four consecutive readings of the highest point to which the temperature rises is the solidification temperature of the fatty acids.
FATTY ACID COMPOSITION
Standard Solution Prepare an ester mixture of known composition containing the esters required in the individual monograph. This Standard Solution may contain other components. [noteEster mixtures are available commercially from Nu-Chek-Prep, Inc., P.O. Box 295, Elysian, MN 56028. Typical Nu-Chek-Prep ester mixtures useful in this test include Nu-Chek 17A and Nu-Chek 19A.] Nu-Chek mixture 17A has the following composition:
Nu-Chek mixture 19A has the following composition:
Test Solution [noteIf fatty acids containing more than 2 double bonds are present in the test specimen, remove air from the flask by purging it with nitrogen for a few minutes.] Transfer about 100 mg of the test specimen to a 50-mL conical flask fitted with a suitable water-cooled reflux condenser and a magnetic stir bar. Add 4 mL of 0.5 N methanolic sodium hydroxide solution, and reflux until fat globules disappear (usually 5 to 10 minutes). Add 5 mL of a solution prepared by dissolving 14 g of boron trifluoride in methanol to make 100 mL, swirl to mix, and reflux for 2 minutes. Add 4 mL of chromatographic n-heptane through the condenser, and reflux for 1 minute. Cool, remove the condenser, add about 15 mL of saturated sodium chloride solution, shake, and allow the layers to separate. Pass the n-heptane layer through 0.1 g of anhydrous sodium sulfate (previously washed with chromatographic n-heptane) into a suitable flask. Transfer 1.0 mL of this solution to a 10-mL volumetric flask, dilute with chromatographic n-heptane to volume, and mix.
System Suitability Solution Transfer about 20 mg each of stearic acid, palmitic acid and oleic acid to a 25-mL conical flask fitted with a suitable water-cooled reflux condenser and a magnetic stir bar, and proceed as directed for Test Solution, beginning with Add 5.0 mL of a solution prepared by dissolving.
Chromatographic System (see Chromatography 621) The gas chromatograph is equipped with a flame-ionization detector, maintained at a temperature of about 260, a splitless injection system, and a 0.53-mm × 30-m fused-silica capillary column bonded with a 1.0-µm layer of phase G16. The chromatograph is programmed to maintain the column temperature at 70 for about 2 minutes after injection, then to increase the temperature at the rate of 5 per minute to 240, and finally to maintain this temperature for 5 minutes. The injection port temperature is maintained at about 220. The carrier gas is helium with a linear velocity of about 50 cm per second.
Chromatograph the System Suitability Solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.87 for methyl palmitate, 0.99 for methyl stearate, and 1.0 for methyl oleate; the resolution, R, between methyl stearate and methyl oleate is not less than 1.5; and the relative standard deviation of the peak area responses for the palmitate and stearate peaks for replicate injections is not more than 6.0%. The relative standard deviation of the peak area response ratio of the palmitate to stearate peaks from these replicate injections is not more than 1.0%.
Procedure Separately inject equal volumes (about 1 µL) of the Standard Solution and the Test Solution into the chromatograph, record the chromatograms, identify the fatty acid ester peaks in the chromatogram of the Test Solution by comparing the retention times of these peaks with those obtained in the chromatogram of the Standard Solution, and measure the peak areas for all of the fatty acid ester peaks in the chromatogram obtained from the Test Solution. Calculate the percentage of each fatty acid component in the test specimen by the formula:
100(A/B)in which A is the area of the peak response obtained for each individual fatty acid ester component; and B is the sum of the peak areas of all of the peaks, excluding the solvent peak, in the chromatogram obtained from the Test Solution.
WATER AND SEDIMENT IN FIXED OILS
Apparatus The preferred centrifuge has a diameter of swing (d = distance from tip to tip of whirling tubes) of 38 to 43 cm and is operated at a speed of about 1500 rpm. If a centrifuge of different dimensions is used, calculate the desired rate of revolution by the formula:
The centrifuge tubes are pear-shaped, and are shaped to accept closures. The total capacity of each tube is about 125 mL. The graduations are clear and distinct, reading upward from the bottom of the tube according to the scale shown in the accompanying table.
Procedure Place 50.0 mL of benzene in each of two centrifuge tubes, and to each tube add 50.0 mL of the oil, warmed if necessary to re-incorporate separated stearin, and mixed thoroughly at 25. Insert the stopper tightly into the tubes, and shake them vigorously until the contents are mixed thoroughly, then immerse the tubes in a water bath at 50 for 10 minutes. Centrifuge for 10 minutes. Read the combined volume of water and sediment at the bottom of each tube. Centrifuge repeatedly for 10-minute periods until the combined volume of water and sediment remains constant for 3 consecutive readings. The sum of the volumes of combined water and sediment in the two tubes represents the percentage, by volume, of water and sediment in the oil.
The anisidine value is defined as 100 times the optical density measured in a 1-cm cell of a solution containing 1 g of the substance to be examined in 100 mL of a mixture of solvents and reagents according to the method described below. [noteCarry out the operations as rapidly as possible, avoiding exposure to actinic light.]
Test Solution A Dissolve 0.500 g of the substance to be examined in isooctane, and dilute with the same solvent to 25.0 mL.
Test Solution B To 5.0 mL of Test Solution A add 1.0 mL of a 2.5 g per L solution of p-anisidine in glacial acetic acid, shake, and store protected from light.
Standard Solution To 5.0 mL of isooctane add 1.0 mL of a 2.5 g per L solution of p-anisidine in glacial acetic acid, shake, and store protected from light.
Procedure Measure the absorbance of Test Solution A at 350 nm using isooctane as the blank. Measure the absorbance of Test Solution B at 350 nm exactly 10 minutes after its preparation, using the Standard Solution as the compensation liquid. Calculate the Anisidine Value from the expression: As is the absorbance of Test Solution B at 350 nm; Ab is the absorbance of Test Solution A at 350 nm; and m is the weight, in g, of the substance to be examined in Test Solution A.
TOTAL OXIDATION VALUE (TOTOX)
Total Oxidation Value is defined by the formula:
2PV + AV
in which PV is the Peroxide Value, and AV is the Anisidine Value.