256. The Oxidation Method.—The removal of the reducing sugars may be accomplished by oxidation instead of fermentation. The process of analysis is in all respects similar to that described in the foregoing paragraph, substituting oxidation for fermentation.[212] For the oxidizing agent mercuric cyanid is preferred, and it is conveniently prepared by dissolving 120 grams of mercuric cyanid and an equal quantity of sodium hydroxid in water mixing the solutions and completing the volume to one liter. If a precipitate be formed in mixing the solutions it should be removed by filtering through asbestos. For the polarization, ten grams of the sugars in 100 cubic centimeters is a convenient quantity. Ten cubic centimeters of this solution are placed in a flask of water marked at fifty cubic centimeters, a sufficient quantity of the mercuric cyanid added to remain in slight excess after the oxidation is finished (from twenty to twenty-five cubic centimeters) and the mixture heated to the boiling point for three minutes. The alkali, after cooling, is neutralized with strong hydrochloric acid and the passing from alkalinity to acidity will be indicated by a discharge of the brown color which is produced by heating with the alkaline mercuric cyanid. The heating with the mercury salt should be conducted in a well ventilated fume chamber.
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.[213] 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.[214] 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 1[43]. 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.[215]
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].