Part 52

The calculation of the results is conducted by means of the formulas given in the preceding paragraph. In the original paper describing this method, it was stated that its accuracy depended on the complete oxidation of the reducing sugar in a manner leaving no optically active products, and on the inactivity of the reagents used in respect to the dextrin present. These two conditions are not rigidly fulfilled, as is shown by Wilson. According to his data maltose leaves an optically active residue, which gives a somewhat greater right hand rotation than is compensated for by the diminished rotation of the dextrin. Wilson, however, confesses that the dextrin used contained reducing sugars, which would not be the case had it been prepared by the process of treating it with alkaline mercuric cyanid as above indicated. Upon the whole, the oxidation of the reducing sugar by a mercury salt gives results which, while not strictly accurate, are probably as reliable as those afforded by fermentation. The author has attempted to supplant both the oxidation and fermentation methods by removing the reducing sugars with a precipitating reagent, such as phenylhydrazin, but the methods are not sufficiently developed for publication.

=257. Removal of Dextrose by Copper Acetate.=—Maercker first called attention to the fact that Barfoed’s reagent (one part copper acetate in fifteen parts of water, and 200 cubic centimeters of this solution mixed with five cubic centimeters of thirty-eight per cent. acetic acid) reacts readily with dextrose, while it is indifferent to maltose and dextrins. Sieben’s method of removing dextrose is based on this fact. It is found that under certain conditions pure maltose does not reduce either the acidified or neutral solution of copper acetate, while dextrose or a mixture of dextrose and maltose does so readily. It is also shown that the fermentation residue under suitable conditions acts like maltose. Maltose solutions reduce the reagent after boiling four minutes while at 40°-45° they have no effect even after standing four days. The amount of copper deposited by dextrose, under the latter conditions, is found to depend to a certain extent on the amount of free acetic acid present, and as the solutions of copper acetate always contain varying quantities of acetic acid which cannot be removed without decomposition and precipitation of basic salt, the use of an absolutely neutral solution is impracticable. The reagent prepared according to Barfoed’s directions is almost saturated, but a half normal solution is preferable. Sieben proposes two solutions: I, containing 15.86 grams copper and 0.56 gram acetic anhydrid per liter; II, containing 15.86 grams copper and three grams acetic anhydrid per liter. The reduction of the dextrose is secured by placing 100 cubic centimeters of the solution in a bottle, adding the sugar solution, stoppering and keeping in a water-bath at 40°-45° two or three days. An aliquot portion is then drawn off and the residual copper precipitated by boiling with forty-five cubic centimeters of the alkali solution of the fehling reagent and forty cubic centimeters of one per cent dextrose solution, filtered and weighed as usual. The results show that either solution can be used, and that standing for two days at 45° is sufficient. One hundred cubic centimeters of the copper solution are mixed with ten cubic centimeters of the sugar solution containing from two-tenths to five-tenths gram of dextrose, as this dilution gives the best results. No reduction is found to have taken place when solutions containing five-tenths gram maltose or five-tenths gram fermentation residue are used. The data can not be compiled in the form of a table similar to Allihn’s, as it is impossible to obtain a solution of uniform acidity each time, and the solution will have to be standardized by means of a known pure dextrose solution and the result obtained with the unknown sugar solution properly diluted compared with this. This method of Sieben’s has never been practiced to any extent in analytical separations and can not, therefore, be strongly recommended without additional experience.

=258. Removal of Dextrin by Alcohol.=—By reason of its less solubility, dextrin can be removed from a solution containing also dextrose and maltose by precipitation with alcohol. It is impracticable, however, to secure always that degree of alcoholic concentration which will cause the coagulation of all the dextrins without attacking the concomitant reducing sugars. In this laboratory it has been found impossible to prepare a dextrin by alcoholic precipitation, which did not contain bodies capable of oxidizing alkaline copper solutions.

The solution containing the dextrin is brought to a sirupy consistence by evaporation and treated with about ten volumes of ninety per cent alcohol. After thorough mixing, the precipitated dextrin is collected on a filter and well washed with alcohol of the strength noted. It is then dried and weighed. If weaker solutions of dextrin are used, the alcohol must be of correspondingly greater strength. In the filtrate the residual maltose and dextrose may be separated and determined by the chemical and optical methods already described.

CARBOHYDRATES IN MILK.

=259. The Copper Tartrate Method.=—The lactose in milk is readily estimated by the gravimetric copper method described in paragraph =143=. Before the application of the process the casein and fat of the milk should be removed by an appropriate precipitant, and an aliquot part of the filtrate, diluted to contain about one per cent of milk sugar, used for the determination. The clarification is very conveniently secured by copper sulfate or acetic acid, as described in the next paragraph. A proper correction should be made for the volume occupied by the precipitate and, for general purposes, with whole milk of fair quality this volume may be assumed to be five per cent. One hundred grams of milk will give a precipitate occupying approximately five cubic centimeters. In the analytical process, to twenty cubic centimeters of milk, diluted with water to eighty, is added a ten per cent solution of acetic acid, until a clear whey is shown after standing a few minutes, when the volume is completed to 100 cubic centimeters with water, and the whole, after thorough shaking, thrown on a filter. An aliquot part of the filtrate is neutralized with sodium carbonate and used for the lactose determination. This solution contains approximately one per cent of lactose. In a convenient part of it the lactose is determined and the quantity calculated for the whole. This quantity represents the total lactose in the twenty cubic centimeters of milk used. The weight of the milk is found by multiplying twenty by its specific gravity. From this number the percentage of lactose is easily found. In this process the milk is clarified by the removal of its casein and fat. Other albuminoids remain in solution and while these doubtless disturb the subsequent determination of lactose, any attempt at their removal would be equally as disadvantageous. The volume of the precipitate formed by good, whole milk when the process is conducted as above described, is about one cubic centimeter, for which a corresponding correction is readily made.

=260. The Official Method.=—The alkaline copper method of determining lactose, adopted by the Association of Official Agricultural Chemists, is essentially the procedure proposed by Soxhlet.

Dilute twenty-five cubic centimeters of the milk, held in a half liter flask, with 400 cubic centimeters of water and add ten cubic centimeters of a solution of copper sulfate of the strength given for Soxhlet’s modification of Fehling’s solution, page 129; add about seven and a half cubic centimeters of a solution of potassium hydroxid of such strength that one volume of it is just sufficient to completely precipitate the copper as hydroxid from one volume of the solution of copper sulfate. In place of a solution of potassium hydroxid of this strength eight and a half cubic centimeters of a half normal solution of sodium hydroxid may be used. After the addition of the alkali solution the mixture must still have an acid reaction and contain copper in solution. Fill the flask to the mark, shake and filter through a dry filter.

Place fifty cubic centimeters of the mixed copper reagent in a beaker and heat to the boiling point. While boiling briskly, add 100 cubic centimeters of the lactose solution, prepared as directed above, and boil for six minutes. Filter immediately and determine the amount of copper reduced by one of the methods already given, pages 149-155. Obtain the weight of lactose equivalent to the weight of copper found from the table on page 163.

=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. 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. 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.

=264. Separation of Starch.=—Starch being insoluble in its natural state, it is impossible to separate it from the other insoluble matters of plants by any known process. In bringing it into solution it undergoes certain changes of an unknown nature, but tending to produce a dextrinoid body. Nevertheless, in order to procure the starch in a state of purity suited to analytical processes, it becomes necessary to dissolve the starch from the other insoluble bodies that naturally accompany it. As has been shown in preceding paragraphs, there are only two methods of securing the solution of starch which fully meet the conditions of accurate analysis. These are the methods depending on the use of diastatic ferments and on the employment of heat and pressure in the presence of water. These two processes have been described in considerable detail in paragraphs =179-181=. It is important, in starch determinations, to remove from the sample the sugar and other substances soluble in water and also the oils, when present in large quantities, before subjecting it to the processes for rendering the starch soluble.

=265. Desiccation of Amyliferous Bodies.=—The removal of sugars and oils is best secured in amyliferous substances after they are deprived of their moisture. As has already been suggested, the desiccation should be commenced at a low temperature, not above 60°, and continued at that point until the chief part of the water has escaped. The operation may be conducted in one of the ways already described (pp. 12-27). There is great difference of opinion among analysts in respect of the degree of temperature to which the sample should be finally subjected, but for the purposes here in view, it will not be found necessary to go above 105°. Before beginning the operation the sample should be as finely divided as possible, and at its end the dried residue should be ground and passed through a sieve of half a millimeter mesh.

=266. Indirect Method of Determining: Water in Starch.=—It is claimed by Block that it is necessary to dry starch at 160° in order to get complete dehydration. Wet starch as deposited with its maximum content of water has nine molecules thereof, viz., C₆H₁₀O₅ + 9H₂O. Ordinary commercial starch has about eighteen per cent of water with a formula of C₆H₁₀O₅ + 2H₂O.

The percentage of water may be determined by Block’s feculometer or Block’s dose-fécule. The first apparatus determines the percentage of anhydrous starch by volume, and the second by weight.

Block’s assumption that starch can absorb only fifty per cent of its weight of water is the basis of the determination.

A noted weight of starch is rubbed up with water until saturated, the water poured off, the starch weighed, dried on blotting paper until it gives off no more moisture and again weighed. Half of the lost weight is water, from which the original per cent of water can be calculated. This at best seems to be a rough approximation and not suited to rigorous scientific determination.

=267. Removal of Oil and Sugar.=—The dried, finely powdered sample, obtained as described above, is placed in any convenient extractor (=33-43=) and the oil or fat it contains removed by the usual solvents. For ordinary purposes, even with cereals, this preliminary extraction of the oil is not necessary, but it becomes so with oily seeds containing starch. The sugar is subsequently removed by extraction with eighty per cent alcohol and the residue is then ready for the extraction of the starch. In most cases the extraction with alcohol will be found sufficient. In some bodies, for instance the sweet potato (batata), the quantity of sugar present is quite large, and generally some of it is found. If not present in appreciable amount, the alcohol extraction may also be omitted. The sample having been prepared as indicated, the starch may be brought into solution by one of the methods described in paragraphs =179-181=, preference being given to the aqueous digestion in an autoclave. The dissolved starch is washed out of the insoluble residue and determined by optical or chemical methods =186-194=.

=268. Preparation of Diastase for Starch Solution.=—The methods of preparing malt extract for use in starch analysis have been described in paragraph =179=. If a purer form of diastase is desired it may be prepared by following the directions given by Long and Baker. Digest 200 grams of ground malt for twenty-four hours with three parts of twenty per cent alcohol. Separate the extract by filtration and to the filtrate add about one and a half liters of ninety-three per cent alcohol and stir vigorously. After the precipitate has subsided the supernatant alcohol is removed by a syphon, the precipitate is brought onto a filter and washed with alcohol of a strength gradually increasing to anhydrous, and finally with anhydrous ether. The diastase is dried in a vacuum over sulfuric acid and finally reduced to a fine powder before using. Thus prepared, it varies in appearance from a white to a slightly brownish powder. Made at different times and from separate portions of malt, it may show great differences in hydrolytic power.

=269. Estimation of Starch in Potatoes by Specific Gravity.=—A roughly approximate determination of the quantity of starch in potatoes can be made by determining their specific gravity. Since the specific gravity of pure starch is 1.65, it follows that the richer a potato is in starch the higher will be its specific gravity. The specific weight of substances like potatoes is conveniently determined by suspending them in water by a fine thread attached to the upper hook of a balance pan. There may be a variation of the percentage of other constituents in potatoes as well as of starch, and therefore the data obtained from the following table can only be correct on the assumption that the starch is the only variable. In practice, errors amounting to as much as two per cent may be easily made, and therefore the method is useful only for agronomic and commercial and not for scientific purposes. The method is especially useful in the selection of potatoes of high starch content for planting. The table is constructed on the weight in grams in pure water of 10000 grams of potatoes and the corresponding per cents of dry matter and starch are given. It is not always convenient to use exactly 10000 grams of potatoes for the determination, but the calculation for any given weight is easy.