167. Cobaltous Nitrate Test for Sucrose.—Sucrose in solution may be distinguished from other sugars by the amethyst violet color which it imparts to a solution of cobaltous nitrate. This reaction was first described by Reich, in 1856, but has only lately been worked out in detail. The test is applied as follows:
To about fifteen cubic centimeters of the sugar solution add five cubic centimeters of a five per cent solution of cobaltous nitrate. After thoroughly mixing the two solutions, add two cubic centimeters of a fifty per cent solution of sodium hydroxid. Pure sucrose gives by this treatment an amethyst violet color, which is permanent. Pure dextrose gives a turquoise blue color which soon passes into a light green. When the two sugars are mixed the coloration produced by the sucrose is the predominant one, and one part of sucrose in nine parts of dextrose can be distinguished. If the sucrose be mixed with impurities such as gum arabic or dextrin, they should be precipitated by alcohol or basic lead acetate, before the application of the test. Dextrin may be thrown out by treatment of the solution with barium hydroxid and ammoniacal lead acetate. The reaction may also be applied to the detection of cane sugar in wines, after they are thoroughly decolorized by means of lead acetate and bone-black. The presence of added sucrose to milk, either in the fresh or condensed state, may also be detected after the disturbing matters are thrown out with lead acetate. The presence of sucrose in honey may also be detected by this process. The reaction has been tried in this laboratory with very satisfactory results. The amethyst violet coloration with sucrose is practically permanent. On boiling the color is made slightly bluish, but is restored to the original tint on cooling. Dextrose gives at first a fine blue color which in the course of two hours passes into a pale green. A slight flocculent precipitate is noticed in the tube containing the dextrose. Maltose and lactose act very much as dextrose, but in the end do not give so fine a green color. If the solutions containing dextrose, lactose and maltose be boiled, the original color is destroyed and a yellow-green color takes its place. The reaction is one which promises to be of considerable practical value to analysts, as it may be applied for the qualitive detection of sucrose in seeds and other vegetable products.[133]
168. The Dextrose Group.—In case the carbohydrate in question shows a right-handed rotation and the absence of sucrose is established by the polariscopic observation described above, the presence of the dextrose group may be determined by the following test.[134]
Five grams of the carbohydrate are oxidized by boiling with from twenty to thirty cubic centimeters of nitric acid of 1.15 specific gravity, and then at gentle heat evaporated to dryness with stirring. If much mucic acid be present, as will be the case if the original matter contained lactose some water is added and the mixture well stirred, and again evaporated to dryness to expel all nitric acid. The residue should be of a brown color. The mass is again mixed with a little water and the acid reaction neutralized by rubbing with fine-ground potassium carbonate. The carbonate should be added in slight excess and acetic acid added to the alkaline mixture, which is concentrated by evaporation and allowed to stand a few days. At the end of this time potassium saccharate has formed and is separated from the mother liquid by pouring on a porous porcelain plate. The residue is collected, dissolved in a little water and again allowed to crystallize, when it is collected on a porous plate, as before, and washed by means of an atomizer with a little aqueous spray until it is pure white and free of any oxalic acid. The residual acid potassium saccharate may be weighed after drying and then converted into the silver salt. The potash salt for this purpose is dissolved in water, neutralized with ammonia and precipitated with a solution of silver nitrate. The precipitate is well stirred, collected on a gooch and washed and dried in a dark place. It contains 50.94 percent of silver. All sugars which contain the dextrose group yield silver saccharate when treated as above described. Inulin, sorbose, arabinose and galactose yield no saccharic acid under this treatment, and thus it is shown that they contain no dextrose group. Milk sugar, maltose, the dextrins, raffinose and sucrose yield saccharic acid when treated as above and therefore all contain the dextrose group.
169. Levulose.—The levulose group of sugars, wherever it occurs, when oxidized with nitric acid, gives rise to tartaric, racemic, glycolic and oxalic acids, which are not characteristic, being produced also by the oxidation of other carbohydrates. A more distinguishing test is afforded by the color reactions produced with resorcin.[135] The reagent is prepared by dissolving half a gram of resorcin in thirty cubic centimeters each of water and strong hydrochloric acid. To the sugar solution under examination an equal volume of strong hydrochloric acid is added, and then a few drops of the reagent. The mixture is gently warmed, and in the presence of levulose develops a fire-red color. Dextrose, lactose, mannose and the pentoses do not give the coloration, but it is produced by sorbose in a striking degree, and also by sucrose and raffinose since these sugars contain the levulose group.
170. Galactose.—The galactose which arises from the hydrolysis of milk sugar is readily recognized by the mucic acid which it gives on oxidation with nitric acid.[136] The analytical work is conducted as follows: The body containing galactose or galactan is placed in a beaker with about sixty cubic centimeters of nitric acid of 1.15 specific gravity for each five grams of the sample used. The beaker is placed on a steam-bath and heated, with frequent stirring, until two-thirds of the nitric acid have been evaporated. The residual mixture is allowed to stand over night and the following morning is treated with ten cubic centimeters of water, allowed to stand for twenty-four hours, filtered through a gooch, and the collected matter washed with twenty-five cubic centimeters of water, dried at 100° and weighed. The mucic acid collected in this way will amount to about thirty-seven per cent of the milk sugar or seventy-five per cent of the galactose oxidized. Raffinose yields under similar treatment, about twenty-three per cent of mucic acid, which proves that the galactose group is contained in that sugar. Raffinose, therefore, is composed of one molecule each of dextrose, levulose, and galactose.
171. Invert Sugar.—The presence of a trace of invert sugar accompanying sucrose can be determined by Soldaini’s solution, paragraph [124], or by boiling with methyl blue.[137] Methyl blue is the hydrochlorate of an ammonium base, which, under the influence of a reducing agent, loses two atoms of hydrogen and becomes a colorless compound. The test for invert sugar is made as follows: The reagent is prepared by dissolving one gram of methyl blue in water. If the sugar solution is not clear, twenty grams of the sugar are dissolved in water clarified by lead subacetate, the volume completed to 100 cubic centimeters, and the solid matters separated by filtration. The filtrate is made slightly alkaline with sodium carbonate to remove the lead. A few drops of soda lye solution are then added and the mixture thrown on a filter. To twenty-five cubic centimeters of the filtrate a drop of the methyl blue solution is added, and a portion of the liquor heated in a test tube over the naked flame. If, after boiling for one minute, the coloration disappear, the sample contains at least 0.01 per cent of invert sugar; if the solution remain blue it contains none at all or less than 0.01 per cent. The test may also be made with the dilute copper carbonate solution of Ost described further on.
172. Compounds with Phenylhydrazin.—Many sugars may also be qualitively distinguished by the character of their compounds with phenylhydrazin. In general, it may be said that those sugars which reduce fehling solution form definite crystalline compounds with the reagent named. If a moderately dilute hot solution of a reducing sugar be brought into contact with phenylhydrazin acetate, a crystalline osazone is separated. The reaction takes place between one molecule of the sugar and two molecules of the hydrazin compound, according to the following formula:
C₆H₁₂O₆ + 2C₆H₅N₂H₃ = C₁₈H₂₂N₄O₄ + 2H₂O + H₂.
The hydrogen does not escape but combines in the nascent state with the excess of phenylhydrazin to form anilin and ammonia.