Determination—Weigh 1 to 15 g. of the prepared sample into an Erlenmeyer flask, using the smaller quantity in the case of dark-colored, high acid fats. Add 50 to 100 cc. hot, neutral alcohol, and titrate with N/2, N/4 or N/10 sodium hydroxide depending on the fatty acid content, using phenolphthalein as indicator. Calculate to oleic acid, except that in the case of palm oil the results may also be expressed in terms of palmitic acid, clearly indicating the two methods of calculation in the report. In the case of coconut and palm kernel oils, calculate to and report in terms of lauric acid in addition to oleic acid, clearly indicating the two methods of calculation in the report. In the case of fats or greases containing more than 0.1 per cent of soluble mineral matter, add to the percentages of free fatty acids as determined 10 times the percentage of bases in the soluble mineral matter as determined.[30] This addition gives the equivalent of fatty acids combined with the soluble mineral matter.
TITER.
Standard Thermometer—The thermometer is graduated at zero and in tenth degrees from 10° C. to 65° C., with one auxiliary reservoir at the upper end and another between the zero mark and the 10° mark. The cavity in the capillary tube between the zero mark and the 10° mark is at least 1 cm. below the 10° mark, the 10° mark is about 3 or 4 cm. above the bulb, the length of the thermometer being about 37 cm. over all. The thermometer has been annealed for 75 hrs. at 450° C. and the bulb is of Jena normal 16''' glass, or its equivalent, moderately thin, so that the thermometer will be quick-acting. The bulb is about 3 cm. long and 6 mm. in diameter. The stem of the thermometer is 6 mm. in diameter and made of the best thermometer tubing, with scale etched on the stem, the graduation is clear-cut and distinct, but quite fine. The thermometer must be certified by the U. S. Bureau of Standards.
Glycerol Caustic Solution—Dissolve 250 g. potassium hydroxide in 1900 cc. dynamite glycerin with the aid of heat.
Determination—Heat 75 cc. of the glycerol-caustic solution to 150° C. and add 50 g. of the melted fat. Stir the mixture well and continue heating until the melt is homogeneous, at no time allowing the temperature to exceed 150° C. Allow to cool somewhat and carefully add 50 cc. 30 per cent sulfuric acid. Now add hot water and heat until the fatty acids separate out perfectly clear. Draw off the acid water and wash the fatty acids with hot water until free from mineral acid, then filter and heat to 130° C. as rapidly as possible while stirring. Transfer the fatty acids, when cooled somewhat, to a 1-in. by 4-in. titer tube, placed in a 16-oz. salt-mouth bottle of clear glass, fitted with a cork that is perforated so as to hold the tube rigidly when in position. Suspend the titer thermometer so that it can be used as a stirrer and stir the fatty acids slowly (about 100 revolutions per minute) until the mercury remains stationary for 30 seconds. Allow the thermometer to hang quietly with the bulb in the center of the tube and report the highest point to which the mercury rises as the titer of the fatty acids. The titer should be made at about 20° C. for all fats having a titer above 30° C. and at 10° C. below the titer for all other fats. Any convenient means may be used for obtaining a temperature of 10° below the titer of the various fats. The committee recommends first of all a chill room for this purpose; second, an artificially chilled small chamber with glass window; third, immersion of the salt-mouth bottle in water or other liquid of the desired temperature.
UNSAPONIFIABLE MATTER.
Extraction Cylinder—The cylinder shall be glass-stoppered, graduated at 40 cc., 80 cc. and 130 cc., and of the following dimensions: diameter about 1-3/8 in., height about 12 in.
Petroleum Ether—Redistilled petroleum ether, boiling under 75° C., shall be used. A blank must be made by evaporating 250 cc. with about 0.25 g. of stearine or other hard fat (previously brought to constant weight by heating) and drying as in the actual determination. The blank must not exceed a few milligrams.
Determination—Weigh 5 g. (±0.20 g.) of the prepared sample into a 200-cc. Erlenmeyer flask, add 30 cc. of redistilled 95 per cent (approximately) ethyl alcohol and 5 cc. of 50 per cent aqueous potassium hydroxide, and boil the mixture for one hour under a reflux condenser. Transfer to the extraction cylinder and wash to the 40-cc. mark with redistilled 95 per cent ethyl alcohol. Complete the transfer, first with warm, then with cold water, till the total volume amounts to 80 cc. Cool the cylinder and contents to room temperature and add 50 cc. of petroleum ether. Shake vigorously for one minute and allow to settle until both layers are clear, when the volume of the upper layer should be about 40 cc. Draw off the petroleum ether layer as closely as possible by means of a slender glass siphon into a separatory funnel of 500 cc. capacity. Repeat extraction at least four more times, using 50 cc. of petroleum ether each time. More extractions than five are necessary where the unsaponifiable matter runs high, say over 5 per cent, and also in some cases where it is lower than 5 per cent, but is extracted with difficulty. Wash the combined extracts in a separatory funnel three times with 25-cc. portions of 10 per cent alcohol, shaking vigorously each time. Transfer the petroleum ether extract to a wide-mouth tared flask or beaker, and evaporate the petroleum ether on a steam bath in an air current. Dry as in the method for moisture and volatile matter. Any blank must be deducted from the weight before calculating unsaponifiable matter. Test the final residue for solubility in 50 cc. petroleum ether at room temperature. Filter and wash free from the insoluble residue, if any, evaporate and dry in the same manner as before. The Committee wishes to emphasize the necessity of thorough and vigorous shaking in order to secure accurate results. The two phases must be brought into the most intimate contact possible, otherwise low and disagreeing results may be obtained.