mother liquor, are safe from contamination by the air of the room. The speed of rotation of the basket is controlled by a rheostat in series with the motor.

In order to secure a proper distribution of the crystals and insure smooth running of the basket, the crystal mass is introduced while the machine is stationary or running at very low velocity. The speed is gradually increased as the mother liquor runs off, until finally the maximum speed of 3,000 revolutions per minute is attained.

The substance is then recrystallized in a similar manner, but a second filtration is accomplished through the layer of asbestos in a glass filter, shown in figure 98, connected directly to the boiling apparatus which supplies the necessary vacuum. This asbestos filter effectively removes the shreds of filter paper which are almost invariably in the filtrate after a filtration through paper. After the solution has passed this filter, it comes in contact with nothing but clean glass, and all manipulation of the solution or crystals is carried out under glass cabinets, to reduce possible contamination with dust. The airdried crystals are powdered to dust in an agate mortar, of which the pestle carries a shield to prevent contamination, and are placed in a vacuum desiccator over lime.

(e) MODIFICATION OF METHOD OF BATES AND JACKSON [5]

A modification of the method of Bates and Jackson has been tried at this Bureau. A 50 percent solution is clarified with alumina cream, filtered and boiled below 35° C as described above. The boiling is continued to a concentration of 70 to 73 percent of sugar. It is then poured into a crystallizing jar and precipitated by the addition of an equal volume of pure alcohol. The precipitate is separated centrifugally, washed with alcohol, and air-dried. This process is repeated. Before bottling, it is dried over lime in a vacuum.

The alcohol used for the precipitation of pure sugar should be highly purified with respect to acids or aldehydes. It is not essential that it be dry or free from other members of the alcohol group. The method of purification described by Dunlap [6] meets these requirements. Dissolve 1.5 g of silver nitrate in 3 ml of water, add to a liter of 95 percent alcohol in a glass-stoppered cylinder and shake. Dissolve 3 g of caustic potash in 10 ml of warm alcohol, and after cooling pour slowly into the alcoholic AgNO3 solution. Do not shake. Allow to stand overnight. Siphon off and distill.

(f) ANALYSIS

If sugar is to be used for the purpose of standardizing instruments, its purity should be ascertained. This is particularly true of samples required for polarimetric standards and for scientific data of all kinds. There are many sorts of impurities whose presence, if undetected, may lead to false conclusions in the interpretation of data. These impurities may be grouped into classes for the sake of convenience: (a) Soluble inorganic impurities, (b) soluble organic impurities which reduce alkaline copper, (c) soluble organic impurities which do not reduce alkaline copper, and (d) moisture.

Inorganic impurities may readily be recognized by an ash determination. To perform this, a quantity of the sugar should be weighed into a very carefully weighed platinum capsule and burned to a char.

The char should then be ignited in a muffle furnace at a low red heat, properly below 550° C, until the carbon has been consumed. The ash from a sample of properly purified sugar should not amount to 0.01 percent. It must be ascertained that the dish itself is of constant weight during a similar period of heating. Samples prepared at this Bureau have an ash content of less than 0.1 mg for a 5-g sample.

The estimation of very small quantities of reducing substances in the presence of the very large quantity of sucrose requires the employment of special methods. The usual methods in which copper sulfate is dissolved in caustic alkali are unsuitable because of the destructive action of the reagent upon the sucrose.

Bates and Jackson [5] have made a detailed study of the reducing action of pure sucrose on other alkaline copper reagents in which the concentration of the hydroxyl ion was diminished. They found that after several recrystallizations of the sucrose a minimum value for the reduced cuprous oxide was obtained with each of the reagents investigated, and that further recrystallizations failed to lower this value. It appeared, therefore, that either a constant quantity of reducing sugar was present, distributing itself in a constant ratio between crystals and mother liquor, or sucrose itself effected the slight reduction of the copper. From this indirect method they concluded that if any reducing substance other than sucrose itself was present in their purified samples it was of the order of 0.001 percent and entirely negligible for most purposes. Of the several methods studied, Bates and Jackson found a modification of the Soldaini method to yield the most satisfactory results. The modified method is as follows:

Dissolve 297 g of KHCO3 and 1 g of CuSO4.5H2O in water and dilute to 1 liter. The potassium bicarbonate should not contain much K2CO3. Transfer 50 ml of this solution to a beaker, boil for 1 minute, add a solution containing 10 g of the sugar, bring the whole to boiling, and continue the boiling for exactly 2 minutes. At the end of this period add 100 ml of cold, recently boiled distilled water and collect the precipitate on a Gooch-Monroe crucible or on a very closely packed asbestos mat. Under this procedure it has been shown that 10 g of pure sucrose produces 1.1 mg of Cu2O, while each 0.01-percent impurity of reducing substance, estimated as invert sugar, increases this precipitate by 1.9 mg.

Another method applicable to the estimation of very small quantities of reducing sugars in pure sucrose is that of Ofner, see page 193.

The soluble organic impurities, which do not reduce alkaline copper, are not so easily detected, but investigations at this Bureau have shown that recrystallization from aqueous solution or by precipitation with alcohol results, in every case examined, in a purification of the sucrose. This fact can only be shown by a polarimetric study.

In order to dry pure, powdered sucrose, care should be exercised not to subject the sample to a prolonged high temperature. Under the influence of an elevated temperature, sucrose undergoes a decomposition which is similar to a process of "caramelization." In table 44 is given the time at each temperature required to form "caramel" equivalent in reducing power to 0.01 percent of invert sugar. Caramel can be detected by its reducing action on the alkaline copper solution. Finely pulverized sugar can be dried at 70° C in a vacuum in 4 hours.

323414-42-27

The moisture remaining will in general amount to less than 0.003 percent.

TABLE 44.-Time required at various temperatures to form caramel equivalent to 0.01 percent of invert sugar

Inulin can be prepared most satisfactorily from dahlia tubers, or in somewhat lower yield from chicory, or in much lower yield from jerusalem artichokes. The most suitable time for the extraction is early autumn, but in the case of dahlias the large clumps of tubers are usually divided for replanting in the late spring and the "blind" tubers which are unsuitable for planting are then available for inulin extraction. Even as late as early summer inulin can be obtained from healthy tubers.

Comminute the tubers or roots in a food chopper or similar appliance and express the juice with a tincture press, using, if necessary, a small proportion of water to complete the extraction. Heat the juice to 60° or 70° C and add milk of lime to about pH 8. Filter and adjust the pH to 7.0 with oxalic acid. Heat to 70° to 80° C, add active carbon, and filter. Allow the filtrate to stand quiescent overnight, during which time the inulin separates in the form of small spheroids. Slightly increased yields can be obtained by placing the filtrate in a refrigerator and still greater yields by completely freezing it and allowing it to thaw at low temperature. Filter the separated inulin with suction and wash with abundant quantities of cold water.

The moist cake of inulin has at this point a dry-substance content of 35 to 40 percent. It can be purified by dissolving in hot water to a concentration of about 15 to 18 percent, treating with carbon, refiltering, and allowing to separate again by chilling. Filter and wash with cold water.

For many purposes it is a satisfactory procedure to spread the moist. inulin cake on a glass plate and allow it to dry in air. When dried in this way, it takes the form of hard hornlike masses which can be pulverized readily in a mortar or ball mill. The air-dried substance contains about 10 percent of water of hydration and a small quantity of inorganic impurities. The latter can be diminished to negligible proportions by repeating the processes of solution and chilling, or by electrodialysis.

To avoid the formation of hornlike masses, the moist inulin cake can be washed with 95 percent alcohol, followed by absolute alcohol. The substance, however, has a tendency to retain alcohol [7], which conceivably displaces in part the water of hydration.

A procedure described by Tollens and Elsner [8] is probably suitable for less pure materials than fresh dahlia juices. The juice is expressed

in the presence of calcium carbonate, fermented at 25° C. with bakers' yeast, defecated with lead acetate, and filtered. After removal of the excess lead with hydrogen sulfide, the filtrate is frozen and thawed to cause the separation of inulin.

Inulin crystallizes in the form of doubly refracting sphero-crystals whose crystalline structure is indicated by X-ray patterns [9]. In aqueous solution inulin has a specific rotation, [a]=-39 to -40 referred to anhydrous substance.

If heated in glycerine, or glycol, or even in water solution and precipitated from these solutions by alcohol, inulin occurs in a form soluble in cold water [7, 10]. This soluble form is also produced by the action of carbon dioxide [11].

(b) PREPARATION OF LEVULOSE

Levulose can be prepared from inulin, or invert sugar, or directly from the expressed juices of inulin-bearing plants. The sugar can, with some difficulty, be crystallized directly from hydrolyzed inulin, but from the two latter sources it must, after hydrolysis, be isolated by means of the insoluble calcium levulate, CaO.C6H12O6.xH2O.

(1) HYDROLYZED INULIN.35 TO 100 g of hydrated inulin add 400 g of water and agitate, preferably with a motor-driven stirrer, until the inulin is "swollen" to a uniform paste. Heat to nearly boiling and allow the inulin to dissolve completely, adding more water if necessary. Reduce the temperature to 60° C and make the solution 0.08 N with sulfuric acid. Maintain the solution at 60° for 21⁄2 hours, preferably following the hydrolysis by polariscopic observation of samples withdrawn from time to time. Neutralize by agitating vigorously with barium carbonate in excess or by cooling and titrating to exact neutrality with barium hydroxide, preferably leaving the solution very slightly acid (that is, acid to bromthymol blue). Filter with carbon and evaporate in a vacuum to a thick sirup. Add absolute alcohol and evaporate again to a sirup to remove the remaining water. Extract the sirup with several portions of hot absolute alcohol. Allow the combined extracts to stand for 12 to 24 hours and decant the solution from the sirupy residue which forms. Inoculate with levulose crystals and allow the crystallization to become complete (usually within 2 or 3 days). The yield of sugar is usually relatively small.

(2) PREPARATION OF INVERT SUGAR.-Dissolve 250 g of pure cane sugar in 846 ml of water and heat to 70° C in a water bath. Add 20 ml of 5 N hydrochloric acid, mix, and allow the solution to remain at this temperature for 35 minutes. Cool and neutralize the solution with dilute sodium hydroxide, avoiding local alkalinity. The solution whose volume can be measured now contains 263.16 g of invert sugar, one-half of which is levulose.

(3) HYDROLYSIS OF PLANT JUICES.-Juices extracted from inulinbearing plant tubers or roots contain from 12 to 20 percent of dry substance depending upon the composition of the original juice and upon the quantity of maceration or diffusion water used for extraction. The polysaccharides contained in the juice are hydrolyzed,

35 This method is essentially that of M. Hönig, S. T. Schubert, and L. Jesser [Monatsh. 8, 529 (1887) 9, 562 (1888); Ber. deut. chem. Ges. 20, 721 R and 21, 663 R]; and Wohl, [Ber. deut. chem. Ges. 23, 2084 (1890)] but modified by the substitution of more definite directions for the hydrolysis of inulin (Jackson and Goergen, BS J. Research 3, 29 (1929) RP79].

preferably with sulfuric acid. It has been found impracticable to supply exactly defined conditions for this hydrolysis, since varying types of juice require variations of conditions. These variations arise from the fact that the acid is in great part rendered inactive by the buffering action of certain impurities in the juice. Even when the pH of different types of juice is made apparently the same, differences in the rate of hydrolysis occur. It is, therefore, necessary to follow the hydrolysis by means of polariscopic or reducing-sugar analysis of samples withdrawn from time to time. Typical hydrolysis data are given in table 45, in which N is the normality of sulfuric acid calculated from the amount of acid actually added; CH, the normality of hydrogen ion calculated from the pH measurement (quinhydrone electrode); k, the velocity constant in terms of common logarithms and minutes; and "time 99 percent," the time required for 99-percent hydrolysis.

The last two measurements were made on the same juice, and it is evident that the velocity is not proportional to the apparent hydrogenion concentration, the explanation probably being that the pH measurement is subject to errors caused by impurities in the juice. No difficulty is encountered, however, if the hydrolysis is followed analytically. The time required for 99-percent hydrolysis can be diminished at will by raising the temperature of reaction.

When the hydrolysis is complete, the solution is cooled to room temperature or below and treated with milk of lime to pH 7 to 8 with vigorous agitation and finally filtered.

The filtered juice is analyzed for total reducing sugar and levulose. In precise work the Lane and Eynon titration and a method selective for levulose are used (see p. 205). In approximate work, which is satisfactory in most cases, the Lane and Eynon titration method and the direct polarization are used (see the Mathews formula, p. 223).

(4) PRECIPITATION OF CALCIUM LEVULATE [12].-Prepare a milkof-lime suspension, magnesia-free, of 18 to 20 percent calcium oxide content and calculate the volume or weight required to contain an amount of calcium oxide equal to 45 or 50 percent of the levulose in the juice. Dilute either the juice or milk of lime so that when the equivalent quantities are mixed, the levulose shall be approximately 6 percent of the total mixture.

The details of procedure for precipitating the calcium levulate have been directed to the end of obtaining relatively large discrete crystals rather than the fine interlacing needles produced in the original method of Dubrunfaut. The precipitation preferably is made in a vessel equipped with a motor-driven stirrer and immersed in a cold bath. The following procedure illustrates the principal features of the