to a thick sirup, from which crystalline gulose CaCl2.H2O or (gulose)2CaCl2.H2O crystallizes readily. The crystalline massecuite is diluted with a small quantity of ethyl alcohol before filtration, and the crystals are washed with ethyl alcohol. After further concentration the mother liquors give additional product. In all, about 70 g of gulose.CaCl2.H2O is obtained.

In 6.8-percent aqueous solution, a-d-gulose.CaCl2.H2O gives [a]20=+37.1°, changing to an equilibrium value of -10.0°.

amalgam.
NOTES

1 Best results are obtained with hard amalgam in pieces about 1⁄2 cm in diameter. 2 The reaction can be followed by using thymol-blue or congo-red paper as an outside indicator. Thymol-blue paper should turn pink and congo-red paper should turn purple.

3 In order to obtain gulose. CaCl2. H2O free from sodium salts, it is necessary to remove all sodium salts before adding the calcium chloride. This is most readily accomplished by the intermediate preparation of the phenylhydrazone.

REFERENCES

[1] H. S. Isbell, BS J. Research 5, 741 (1930) RP226.

10. LACTOSE (4-(8-d-GALACTOPYRANOSIDO)-d-GLUCOSE) Method. This disaccharide is available at a low price as a commercial product which is prepared from the whey obtained from the manufacture of cheese. Details of the method are described by Nabenhauer [1] and are included in the United States Dispensatory [2]. The usual commercial product is a-lactose.H2O, but the much more soluble anhydrous B-lactose is also available in large quantities. Methods for preparing ß-lactose are reviewed by Bell [3].

Recrystallization.-One kilogram of lactose hydrate is dissolved in 750 ml of hot water. After the addition of 20 g of a decolorizing carbon the hot solution is filtered. The clear filtrate is allowed to cool slowly with stirring. After several hours at room temperature the crystals which separate are collected on a filter and washed, first with a mixture of equal volumes of water and methyl alcohol, and then with undiluted methyl alcohol. The first crop of crystals is about 65 percent of the crude product. The remaining sugar is reclaimed by evaporating the mother liquor in vacuo.

Crystalline a-lactose hydrate in 7.6-percent aqueous solution gives [a]=+85.0° initially, changing in several hours to +52.6°.

REFERENCES

[1] F. P. Nabenhauer, Ind. Eng. Chem. 22, 54 (1930). [2] United States Dispensatory, 22d ed., p. 589 (1937). [3] R. W. Bell, Ind. Eng. Chem. 22, 51 (1930).

11. LACTULOSE (4-(8-d-GALACTOPYRANOSIDO)-d-FRUCTOSE) Method. [1] A solution containing 1,200 g of commercial lactose1 in 6 liters of water saturated with calcium hydroxide at 35° C is kept at approximately 35° C for several days or until the optical rotation shows no further change. Subsequently the solution is concentrated in vacuo until the weight is reduced to about 1,500 g. The resulting residue, which contains considerable crystalline lactose, is diluted with 1,500 ml of methyl alcohol and allowed to stand in a cold place for several days for the crystallization of any unchanged lactose. The crystalline lactose (about 800 g) is separated by filtration and washed with 450 ml of methyl alcohol. The filtrate is concentrated in vacuo to a sirup which is diluted with 600 ml of water and evaporated again to remove the alcohol. The alcohol-free solution is diluted with water to a volume of 2.5 liters, and the quantity of bromine necessary to oxidize the aldoses to aldonic acids is ascertained in the following manner:

A 5-ml sample of the soution is placed in a 200-ml Erlenmeyer flask and 5 ml of 0.1 N iodine is added from a burette; 7.5 ml of 0.1 N sodium hydroxide is then added dropwise. The addition of alkali and iodine is repeated until 30 ml of iodine and 45 ml of alkali have been added. The solution is acidified with 10 ml of 1 N hydrochloric acid and the excess iodine titrated with 0.1 N sodium thiosulfate. using starch indicator. The iodine titration of the sample is the difference between the quantity of 0.1 N iodine added and the back titration. If the volume of the sugar solution is 2.5 liters, the bromine in hydrogen equivalents is one twentieth of the iodine titration. of the 5-ml sample. Before adding the bromine, sufficient calcium carbonate is added to combine with the acids formed by the oxidation;

3

4

this requires 75 g for each equivalent of bromine. The calculated quantity of bromine is then placed in a dropping funnel and added drop by drop over a period of several hours while the solution is mixed with a mechanical stirrer. After all of the bromine has been added, the mixture is allowed to stand for several hours. About 20 g of a decolorizing carbon is then added and the solution is filtered. The bromides in the filtrate are precipitated by mixing with finely powdered silver sulfate, using 160 g for each equivalent of bromine. The resulting precipitate is separated by filtration and washed with water. The bromide-free filtrate is then treated with hydrogen sulfide to precipitate the excess silver. The resulting silver sulfide is removed by filtration, and the filtrate is then evaporated to a volume of about 1,500 ml in vacuo to remove the excess hydrogen sulfide. The solution is neutralized with calcium carbonate, filtered and evaporated to a sirup of approximately 75 percent of total solids (n=1.477). The sirup is mixed with 1,200 ml of hot methyl alcohol and separated into methyl alcohol-soluble and alcohol-insoluble material. The alcoholinsoluble material is given a second and a third extraction with 300ml portions of methyl alcohol, and it is then discarded. The combined alcoholic extract is diluted with 900 ml of 95-percent ethyl alcohol, and the resulting amorphous precipitate is allowed to settle. The clear alcoholic solution is decanted, and the gummy residue is extracted with 100 ml. of methyl alcohol. The residue is discarded. The alcoholic solution is combined with the alcoholic extract previously obtained, and the mixture is then evaporated in vacuo to a sirup of about 85 percent of total solids (n=1.503). This sirup is mixed with 300 ml of methyl alcohol, and then absolute ethyl alcohol is added in sufficient quantity (about 200 ml) to saturate the sirup without giving two liquid phases. The alcoholic solution is seeded with crystalline lactulose and allowed to stand for crystallization. After several days the crystalline lactulose is separated by filtration. The mother liquor after concentration, extraction with methyl alcohol, and subsequent evaporation yields additional lactulose. A total of about 180 g of crystalline lactulose is obtained.

Recrystallization. One hundred grams of crude lactulose is dissolved in hot water and the solution is filtered, using a small quantity of a decolorizing carbon. The filtrate is evaporated to a thick sirup containing about 85 percent of total solids (n=1.503). The sirup is mixed with 200 ml of warm methyl alcohol and after cooling, the solution is seeded with crystalline lactulose. Crystallization takes place in the course of several hours, during which time the mixture is preferably kept in gentle motion. The crystals are collected on a filter and washed with methyl alcohol. About 75 g of crystalline lactulose is obtained in the first crop. The rest of the sugar is reclaimed by concentrating the mother liquor.

Pure lactulose melts at 160° C and in 4-percent aqueous solution gives [a]=-11.9° initially, changing in several hours to -50.7° [2].

NOTES

1 For the preparation of lactose see page 467.

2 In a typical experiment the optical rotatory change as observed in a 2-dm tube was as follows:

3 If the titration requires less than 4 ml of the thiosulfate, sufficient iodine and sodium hydroxide were not added and the analysis must be repeated, using more of the reagents.

For each equivalent, use 26 ml of bromine.

5 In large-scale preparations oxalic acid and lead carbonate can be used in place of silver sulfate. The amount of oxalic acid equivalent to the bromine used is added first and then an equivalent quantity of lead carbonate. The resulting insoluble salts are separated and any bromide which remains in the filtrate is removed by precipitation with silver sulfate, after which the procedure is the same as that given above.

REFERENCES

[1] E. M. Montgomery and C. S. Hudson, J. Am. Chem. Soc. 52, 2101 (1930). [2] H. S. Isbell and W. W. Pigman, J. Research NBS 20, 773 (1938) RP1104.

12. d-LYXOSE

Method. [1] A mixture of 225 g of calcium galactonate.5H2O2, 20 g of barium acetate monohydrate, and 10 g of ferric sulfate (crystalline) is added to 3 liters of boiling water. After the mixture is cooled to 35° C, 120 ml of 30-percent hydrogen peroxide 3 is added. A vigorous reaction begins in a few minutes. The solution is cooled so that the temperature does not exceed 50° C. When the solution turns dark brown, it is cooled to 40° C and a second 120-ml portion of hydrogen peroxide is added. After the ensuing reaction is complete, about 20 g each of a decolorizing carbon and of calcium carbonate are added and the solution is filtered. The filtrate is concentrated in vacuo to a sirup of about 80 percent of total solids (n=1.490). The sirup is mixed with 300 ml of methyl alcohol and then with 600 ml of hot ethyl alcohol. The resulting precipitate is separated by filtration and washed, first on the filter with 100 ml of ethyl alcohol; then it is transferred to a beaker and mixed with 200 ml of ethyl alcohol, after which it is returned to the filter and finally washed with another 100 ml of alcohol. The insoluble residue is discarded. The filtrate is evaporated in vacuo to a thick sirup (n=1.51, or about 90 percent of total solids), which is extracted with 200 ml of hot absolute alcohol. The residue in the distillation flask is extracted with three 200-ml portions of hot isopropyl alcohol. The residue is discarded. The alcoholic extracts are combined and allowed to stand at room temperature until the supernatant liquid is clear. The clear liquid is decanted from the sirupy residue, and after the addition of a few drops of acetic acid the solution is evaporated in vacuo to a sirup which is then seeded with B-d-lyxose and placed in a desiccator over calcium chloride for crystallization to take place. After several days the crystals are collected on a filter, washed with absolute alcohol, and dried. The yield is about 30 g. The mother liquor is concentrated in vacuo, the sirup is extracted with absolute alcohol, and the solution is evaporated to give a second crop.

Recrystallization. An aqueous solution containing a few milliliters of acetic acid and 100 g of crude lyxose is concentrated in vacuo at 40° C to a sirup of 90 percent of total solids. This sirup is mixed with 200 ml of hot absolute ethyl alcohol and the hot solution is filtered, using about 5 g of a decolorizing carbon. After cooling and seeding, about 60 g of crystalline lyxose is obtained.

Usually B-d-lyxose crystallizes more readily than a-d-lyxose, although either isomer may be obtained by the same procedure.

In 4-percent aqueous solution B-d-lyxose gives [a]2=-72.6° initially, changing in 1 or 2 hours to -13.8°.

NOTES

The method described is a considerable improvement over that originally described by Ruff and Ollendorf [2]. The sugar may also be prepared from d-xylose by application of the glycal method [3] or by the pyridine rearrangement from d-xylonic acid [4].

2 The method used for the preparation of calcium galactonate is similar to that given on page 524.

3 If the hydrogen peroxide is not fresh, it should be analyzed and the volume equivalent to that given should be used.

When considerable residue is obtained at this point, it should be extracted with absolute ethyl or isopropyl alcohol in order to recover the lyxose which is present.

5 In order to obtain a satisfactory crystallization it is necessary to keep the amount of water in the sirup as low as possible. If the product fails to crystallize, usually crystallization can be effected by evaporating the alcoholic solution a second time, dissolving the residue in absolute alcohol, and precipitating the impurities with isopropyl alcohol.

REFERENCES

[1] R. C. Hockett and C. S. Hudson, J. Am. Chem. Soc. 56, 1632 (1934).

[2] O. Ruff and G. Ollendorff, Ber. deut. chem. Ges. 32, 550 (1899); 33, 1798 (1900).

[3] H. Gehrke and F. Obst, Ber. deut. chem. Ges. 64, 1724 (1931).

[4] E. Fischer and O. Bromberg, Ber. deut. chem. Ges. 29, 581 (1896).

13. MALTOSE (4-(a-d-GLUCOPYRANOSIDO)-d-GLUCOSE)

The technical grade of maltose usually contains considerable quantities of dextrins, which must be removed by precipitation with alcohol in order that a pure product be obtained on recrystallization. If the technical maltose is not available, the following directions, which are essentially those given by Harding [1] and based on the method of Herzfeld [2], may be used to prepare maltose from starch.

Method. Twenty-five hundred grams of soluble starch is added to 20 liters of hot water and the mixture is stirred until complete solution has taken place. After the solution is cooled below 50° C,100 g of barley flour (or coarsely ground barley meal) is added and the solution allowed to stand for 20 hours. The unfiltered solution is then concentrated under reduced pressure to a volume of about 3 liters. This solution (or one prepared by dissolving 2,500 g of technical maltose in water and evaporating the solution to a volume of 3 liters) is mixed with 5.5 liters of alcohol and stirred for 1⁄2 hour with a mechanical stirrer. The insoluble material is allowed to settle and the alcoholic solution decanted. The gummy residue is mixed with 3 liters of alcohol. The mixture is allowed to settle, and the alcoholic solution is separated by decantation. The residue is given a third extraction with 2 liters of alcohol, the alcohol is separated, and the residue discarded.