144. Table for the Determination of Maltose.—The copper and alkaline solutions employed for the oxidation of maltose are the same as those used for invert and milk sugars.

In the manipulation twenty-five cubic centimeters each of the copper and alkali solutions are mixed and boiled and an equal volume of the maltose solution added, which should not contain more than one per cent of the sugar. The boiling is continued for four minutes, an equal volume of cold recently boiled water added, the cuprous oxid separated by filtration and the metallic copper obtained in the manner already described. The weight of maltose oxidized is then ascertained from the table.

• Example. Weight of impure maltose taken, ten grams to a liter:
• Quantity used, twenty-five cubic centimeters:
• Weight of copper obtained 268 milligrams:
• Weight of maltose oxidized 237 milligrams:
• Weight of impure maltose taken 250 milligrams:
• Percentage of maltose in sample 94.8.

Table for Maltose.

• (A) = Milligrams of copper.
• (B) = Milligrams of maltose.

145. Preparation of Levulose.—It is not often that levulose, unmixed with other reducing sugars, is brought to the attention of the analyst. It probably does not exist in the unmixed state in any agricultural product. The easiest method of preparing it is by the hydrolysis of inulin. A nearly pure levulose has also lately been placed on the market under the name of diabetin. It is prepared from invert sugar.

Inulin is prepared from dahlia bulbs by boiling the pulp with water and a trace of calcium carbonate. The extract is concentrated to a sirup and subjected to a freezing temperature to promote the crystallization of the inulin. The separated product is subjected to the above operations several times until it is pure and colorless. It is then washed with alcohol and ether and is reduced to a fine powder. Before the repeated treatment with water it is advisable to clarify the solution with lead subacetate. The lead is afterwards removed by hydrogen sulfid and the resultant acetic acid neutralized with calcium carbonate.

By the action of hot dilute acids inulin is rapidly converted into levulose.

Levulose may also be prepared from invert sugar, but in this case it is difficult to free it from traces of dextrose. The most successful method consists in forming a lime compound with the invert sugar and separating the lime levulosate and dextrosate by their difference in solubility. The levulose salt is much less soluble than the corresponding compound of dextrose. In the manufacture of levulose from beet molasses, the latter is dissolved in six times its weight of water and inverted with a quantity of hydrochloric acid, proportioned to the quantity of ash present in the sample. After inversion the mixture is cooled to zero and the levulose precipitated by adding fine-ground lime. The dextrose and coloring matters in these conditions are not thrown down. The precipitated lime levulosate is separated by filtration and washed with ice-cold water. The lime salt is afterwards beaten to a cream with water and decomposed by carbon dioxid. The levulose, after filtration, is concentrated to the crystallizing point.[110]

146. Estimation of Levulose.—Levulose, when free of any admixture with other reducing sugars, may be determined by the copper method with the use of the subjoined table, prepared by Lehmann.[111] The copper solution is the same as that used for invert sugar, viz., 69.278 grams of pure copper sulfate in one liter. The alkali solution is prepared by dissolving 346 grams of rochelle salt and 250 grams of sodium hydroxid in water and completing the volume to one liter.

Manipulation.—Twenty-five cubic centimeters of each solution are mixed with fifty of water and boiled. To the boiling mixture twenty-five cubic centimeters of the levulose solution are added, which must not contain more than one per cent of the sugar. The boiling is then continued for fifteen minutes, and the cuprous oxid collected, washed and reduced to the metallic state in the usual way. The quantity of levulose is then determined by inspection from the table given below. Other methods of determining levulose in mixtures will be given further on.

Table for the Estimation of Levulose.

• (A) = Milligrams of copper.
• (B) = Milligrams of levulose.

147. Precipitation of Sugars with Phenylhydrazin.—The combination of phenylhydrazin with aldehyds and ketones was first studied by Fischer, and the near relationship of these bodies to sugar soon led to the investigation of the compounds formed thereby with this reagent.[112] Reducing sugars form with phenylhydrazin insoluble crystalline bodies, to which the name osazones has been given. The reaction which takes place is a double one and is represented by the following formulas:

• Dextrose. Phenylhydrazin. Dextrose-phenylhydrazone.
• C₆H₁₂O₆ + C₆H₅NH.NH₂ = C₆H₁₂O₅.N.NHC₆H₅  +  H₂O
• and C₆H₁₂O₅.N.NHC₆H₅ + C₆H₅NH.NH₂ =
• Phenyldextrosazone.
• C₆H₁₀O₄(N.NHC₆H₅)₂ + 2H₂O.

The dextrosazone is commonly called glucosazone. The osazones formed with the commonly occurring reducing sugars are crystalline, stable, insoluble bodies which can be easily separated from any attending impurities and identified by their melting points. Glucosazone melts at 205°, lactosazone at 200° and maltosazone at 206°.

The osazones are precipitated in the following way: The reducing sugar, in about ten per cent solution, is treated with an excess of the acetate of phenylhydrazin in acetic acid and warmed to from 75° to 85°. In a short time the separation is complete and the yellow precipitate formed is washed, dried and weighed. The sugar can be recovered from the osazone by decomposing it with strong hydrochloric acid by means of which the phenylhydrazin is displaced and a body, osone, is formed, which by treatment with zinc dust and acetic acid, is reduced to the original sugar. The reactions which take place are represented by the following equations:[113]

• Glucososone.
• C₆H₁₀O₄(N.NH.C₆H₅)₂ + 2H₂O = C₆H₁₀O₆ + 2C₆H₅N₂H₂
•  
•   Dextrose (Glucose).
• C₆H₁₀O₆ + H₂ = C₆H₁₂O₆.

For the complete precipitation of dextrose as osazone Lintner and Kröber show that the solution of dextrose should not contain more than one gram in 100 cubic centimeters. Twenty cubic centimeters containing 0.2 gram dextrose should be used for the precipitation.[114] To this solution should be added one gram of phenylhydrazin and one gram of fifty per cent acetic acid. The solution is then to be warmed for about two hours and the precipitate washed with from sixty to eighty cubic centimeters of hot water and dried for three hours at 105°. One part of the osazone is equivalent to one part of dextrose when maltose and dextrin are absent. When these are present the proportion is one part of osazone to 1.04 of dextrose. Where levulose is precipitated instead of dextrose 1.43 parts of the osazone are equal to one part of the sugar.

Sucrose is scarcely at all precipitated as osazone until inverted.

After inversion and precipitation as above, 1.33 parts osazone are equal to one part of sucrose.

The reaction with phenylhydrazin has not been much used for quantitive estimations of sugars, but it has been found especially useful in identifying and separating reducing sugars. It is altogether probable, however, that in the near future phenylhydrazin will become a common reagent for sugar work.

Maquenne has studied the action of phenylhydrazin on sugars and considers that this reaction offers the only known means of precipitating these bodies from solutions where they are found mixed with other substances.[115] The osazones, which are thus obtained, are usually very slightly soluble in the ordinary reagents, for which reason it is easy to obtain them pure when there is at the disposition of the analyst a sufficient quantity of the material. But if the sugar to be studied is rare and if it contain, moreover, several distinct reducing bodies, the task is more delicate. It is easy then to confound several osazones which have almost identical points of fusion; for example, glucosazone with galactosazone. Finally, it becomes impossible by the employment of phenylhydrazin to distinguish glucose, dextrose or mannose from levulose alone or mixed with its isomers. Indeed, these three sugars give, with the acetate of phenylhydrazin the same phenylglucosazone which melts at about 205°. It is noticed that the weights of osazones which are precipitated when different sugars are heated for the same time with the same quantity of the phenylhydrazin, vary within extremely wide limits. It is constant for each kind of sugar if the conditions under which the precipitation is made are rigorously the same. There is then, in the weight of the osazones produced, a new characteristic of particular value. The following numbers have been obtained by heating for one hour at 100°, one gram of sugar with 100 cubic centimeters of water and five cubic centimeters of a solution containing forty grams of phenylhydrazin and forty grams of acetic acid per hundred. After cooling the liquid, the osazones are received upon a weighed filter, washed with 100 cubic centimeters of water, dried at 110° and weighed. The weights of osazones obtained are given in the following table:

With solutions twice as dilute as those above, the relative conditions are still more sensible, and the different sugars arrange themselves in the same order, with the exception of levulose, which shows a slight advantage over sorbine and acquires the first rank. From the above determinations, it is shown that levulose and sorbine give vastly greater quantities of osazones, under given conditions, than the other reducing sugars. It would be easy, therefore, to distinguish them by this reaction and to recognize their presence also even in very complex mixtures, where the polarimetric examination alone would furnish only uncertain indications.

It is remarkable that these two sugars are the only ones among the isomers or the homologues of dextrose, actually known, which possess the functions of an acetone. They are not, however, easily confounded, since the glucosazone forms beautiful needles which are ordinarily visible to the naked eye, while the sorbinosazone is still oily and when heated never gives perfectly distinct crystals.

This method also enables us to distinguish between dextrose and galactose, of which the osazone is well crystallized and melts at almost the same temperature as the phenylglucosazone. Finally, it is observed that the reducing sugars give less of osazones than the sugars which are not capable of hydrolysis, and consequently differ in their inversion products. It is specially noticed in this study of the polyglucoses (bioses, trioses), that this new method of employing the phenylhydrazin appears very advantageous. It is sufficient to compare the weights of the osazones to that which is given under the same conditions by a known glucose, in order to have a very certain verification of the probabilities of the result of the chemical or optical examination of the mixture which is under study. All the polyglucoses which have been examined from this point of view give very decided results. The numbers which follow have reference to one gram of sugar completely inverted by dilute sulfuric acid, dissolved in 100 cubic centimeters of water, and treated with two grams of phenylhydrazin, the same quantity of acetic acid, and five grams of crystallized sodium acetate. All these solutions have been compared with the artificial mixtures and corresponding glucoses, with the same quantities of the same reagents. The following are the results of the examination:

It is noticed that the agreement for each saccharose is as satisfactory as possible. Numbers obtained with the products of inversion are always a little low by reason of the destructive action of sulfuric acid, and in particular, upon levulose. This is, moreover, quite sensible when the product has to be heated for a long time with sulfuric acid in order to secure a complete inversion. It is evident from the data cited from the papers of Fischer, Maquenne, and others, that the determination of sugars by this method is not a very difficult analytical process and may, in the near future, become of great practical importance.

148. Molecular Weights of Carbohydrates.—In the examination of carbohydrates the determination of the molecular weights is often of the highest analytical value.

The uncertainty in respect of the true molecular weights of the carbohydrates is gradually disappearing by reason of the insight into the composition of these bodies, which recently discovered physical relations have permitted.

Raoult, many years ago,[116] proposed a method of determining molecular weights which is particularly applicable to carbohydrates soluble in water.

The principle of Raoult’s discovery may be stated as follows: The depression of the freezing point of a liquid, caused by the presence of a dissolved liquid or solid, is proportionate to the absolute amount of substance dissolved and inversely proportionate to its molecular weight.

The following formulas may be used in computing results: