261. The Copper Cyanid Process.—It has been found by Blyth that the copper cyanid process of Gerrard gives practically the same results in the determination of sugar in milk as are obtained by optical methods.[216] The milk for this purpose is clarified, by precipitating the casein with acetic acid in the following manner:
Twenty-five cubic centimeters of milk are diluted with an equal volume of distilled water, and strong acetic acid added until the casein begins to separate. The liquid is heated to boiling and, while hot, centrifugated in any convenient machine. The supernatant liquid obtained is separated by filtration and the solid matter thrown upon the filter and well washed with hot water. The filtrate and washings are cooled and completed to a volume of 100 cubic centimeters. This liquid is of about the proper dilution for use with the copper cyanid reagent. The percentage of sugar determined by this reagent agrees well with that obtained by the optical method, when no other sugars than lactose are present. If there be a notable difference in the results of the two methods other sugars must be looked for. The presence of dextrin may be determined by testing a few drops of the clear liquor with iodin, which, in the presence of dextrin, gives a reddish color. Other sugars are determined by obtaining their osazones. For this purpose the filtrate obtained as above should be concentrated until the volume is about thirty cubic centimeters. Any solid matter which separates during the evaporation is removed by filtration. The osazones are precipitated in the manner described in paragraph [147]. On cooling, the almost solid crystalline mass obtained is placed on a filter, washed with a little cold water, the crystals then pressed between blotting paper and dried at a temperature of 100°. The dry osazones obtained are dissolved in boiling absolute alcohol, of which just sufficient is used to obtain complete solution. The alcoholic solution is set aside for twelve hours and the separation of a crystalline product after that time shows that dextrose or invert sugar is present. Milk sugar alone gives no precipitate but only a slight amorphous deposit. The lactosazone is precipitated by adding a little water to the hot alcoholic solution and the crystals thus obtained should be dissolved in boiling absolute alcohol and reprecipitated by the addition of water at least three times in order to secure them pure. The osazones are identified by their melting points, paragraph [172]. The first part of this method does not appear to have any advantage over the optical process by double dilution ([p. 278]), and requires more time.
262. Sugars in Evaporated Milks.—In addition to the lactose normally present in evaporated milks the analyst will, in most cases, find large quantities of sucrose. The latter sugar is added as a preservative and condiment. By reason of the ease with which sucrose is hydrolyzed, evaporated milk containing it may have also some invert sugar among its contents. A method of examination is desirable, therefore, which will secure the determination of lactose, sucrose and invert sugar in mixtures. The probability of the development of galactose and dextrose during the evaporation and conservation of the sample, is not great. The best method of conducting this work is the one developed by Bigelow and McElroy.[217] The principle on which the method is based rests on the fact that in certain conditions, easily supplied, the sucrose and invert sugar present in a sample may be entirely removed by fermentation and the residual lactose secured in an unchanged condition. The lactose is finally estimated by one of the methods already described.
The details of the process follow:
On opening a package of evaporated milk, its entire contents are transferred to a dish and well mixed. Several portions of about twenty-five grams each are placed in flasks marked at 100 cubic centimeters. To each of the flasks enough water is added to bring all the sugars into solution and normal rotation is made certain by boiling. After cooling, the contents of the flasks are clarified by mercuric iodid in acetic acid solution. The clarifying reagent is prepared by dissolving fifty-three grams of potassium iodid, twenty-two grams of mercuric chlorid, and thirty-two cubic centimeters of strongest acetic acid in water, mixing the solutions and completing the volume to one liter. The clarification is aided by the use of alumina cream ([84]). The flask is filled to the mark, and the contents well shaken and poured on a filter. After rejecting the first portion of the filtrate the residue is polarized in the usual manner. Two or more separate portions of the sample are dissolved in water in flasks of the size mentioned, heated to 55°, half a cake of compressed yeast added to each and the temperature kept at 55° for five hours. The residue in each flask is treated as above described, the mercuric solution being added before cooling to prevent the fermentative action of the yeast, and the polarization noted.
By this treatment the sucrose is completely inverted, while the lactose is not affected. The percentage of sucrose is calculated by the formulas given in paragraph [94], using the factor 142.6. At the temperature noted the yeast exercises no fermentative, but only a diastatic action.
In each case the volume of the precipitated milk solids is determined by the double dilution method, and the proper correction made ([p. 278]). The lactose remaining is determined by chemical or optical methods, but it is necessary, in all cases where invert sugar is supposed to be present, to determine the total reducing sugars in the original sample as lactose. If the quantity thus determined and the amount of sucrose found as above are sufficient to produce the rotation observed in the first polarization, it is evident that no invert sugar is present. When the polarization observed is less than is equivalent to the quantity of sugar found, invert sugar is present, which tends to diminish the rotation produced by the other sugars. In this case it is necessary to remove both the sucrose and invert sugar by a process of fermentation, which will leave the lactose unchanged.
This is accomplished by conducting the fermentation in the presence of potassium fluorid, which prevents the development of the lactic ferments. For this purpose 350 grams of the evaporated milk are dissolved in water and the solution boiled to secure the normal rotation of the lactose. After cooling to 80°, the casein is thrown down by adding a solution containing about four grams of glacial phosphoric acid and keeping the temperature at 80° for about fifteen minutes. After cooling to room temperature, the volume is completed to one liter with water, well shaken and poured onto a filter. An aliquot part of the filtrate is nearly neutralized with a noted volume of potassium hydroxid. Enough water is added to make up, with the volume of potassium hydroxid used, the total space occupied by the precipitated solid, corresponding to that part of the filtrate, and if necessary, refilter. The volume occupied by the precipitated solids is easily determined by polarization and double dilution. The filtrate, obtained from the process described above, is placed in portions of 100 cubic centimeters each in 200 cubic centimeter flasks with about twenty milligrams of potassium fluorid in solution, and half a cake of compressed yeast. The yeast is broken up and evenly distributed, and the fermentation is allowed to proceed for ten days at a temperature of from 25° to 30°. At the end of this time experience has shown that all of the sucrose and invert sugar has disappeared, but the lactose remains intact. The flasks are filled to the mark with water and the lactose determined by chemical or optical methods. By comparing the data obtained from the estimation of the total reducing sugars before fermentation or inversion and the estimation of the lactose after fermentation, the quantity of invert sugar is easily calculated. The experience of this laboratory shows that invert sugar is rarely present in evaporated milks, which is an indication that the sucrose added thereto does not generally suffer hydrolysis. The mean percentage of added sucrose found in evaporated milks is about forty.
SEPARATION AND DETERMINATION
OF STARCH.
263. Occurrence.—Many bodies containing starch are presented for the consideration of the agricultural analyst. First in importance are the cereals, closely followed by the starchy root crops. Many spices and other condiments also contain starchy matters. In the sap of some plants, for instance sorghum, at certain seasons, considerable quantities of starch occur. In the analysis of cereals and other feeding stuffs, it has been the usual custom to make no separate determination of starch, but to put together all soluble carbohydrates and estimate their percentage by subtracting from 100 the sum of the percentages of the other constituents of the sample. This aggregated mass has been known as nitrogen-free extract. Recent advances in methods of investigation render it advisable to determine the starch and pentosan carbohydrates separately and to leave among the undetermined bodies the other unclassed substances, chiefly of a carbohydrate nature, soluble in boiling dilute acid and alkali.