The solution is filtered and concentrated in vacuo to about 2.5 liters. precipitated calcium sulfate is separated, and the filtered solution is concentrated to a sirup (n=1.48). The sirup is mixed with 1 liter of hot methyl alcohol; the insoluble residue is separated and washed with three 100-ml portions of hot methyl alcohol. The residue is discarded, and the alcoholic extract is purified by adding 600 ml of ether and separating the precipitate by filtration. The solution is concentrated to a thick sirup (n=1.50), which is taken up in 200 ml of hot absolute alcohol. The alcoholic solution is filtered and then mixed with 75 ml of phenylhydrazine. After standing for 1 or more days in the refrigerator, the crystalline hydrazone is collected on a filter, washed with absolute alcohol, and dried at 50° C. The hydrazone (100 g) is transferred to a flask containing 50 ml of benzaldehyde and 2 liters of hot water. The mixture is stirred and heated to about 90° C for about 1 hour, after which it is cooled to room temperature. After the addition of 10 g of a decolorizing carbon, the benzaldehyde phenylhydrazone is separated by filtration. The filtrate is extracted three times with 100-ml portions of ether to remove all of the benzaldehyde. The solution is then made acid with acetic acid and concentrated to a sirup of about 80 percent of total solids. This sirup is diluted with about 50 ml of absolute alcohol and seeded with crystalline fucose. After this solution has stood for 1 or more days in the refrigerator, the resulting crystals are separated and washed with absolute alcohol. The vield is about 30 g. Recrystallization. The sugar is dissolved in approximately an equal quantity of hot water, and after treatment with a decolorizing carbon, is filtered. The solution is made acid with acetic acid and concentrated in vacuo to a sirup of about 80 percent of total solids (n=1.490). This sirup is diluted with 2 ml of absolute alcohol for each gram of sugar and placed in the refrigerator for crystallization to take place. The resulting crystals are collected on a filter and washed with 95-percent alcohol. The mother liquors are concentrated and additional crystals are separated so that nearly all of the sugar is reclaimed. Better purification but lower yields are obtained by crystallizing the sugar from an aqueous solution contained in a flask which is slowly rotated. A solution containing approximately 78 percent of sugar by weight gives a satisfactory crystallization. In 4-percent aqueous solution l-fucose gives [a]=-152.6° initially, which changes in the course of several hours to -75.9°. NOTES The method is essentially that described by Tollens and coworkers [1] as modified by Clark [2] and by Hockett, Phelps, and Hudson [3]. ? The fermentation removes mannose and galactose, which are present in certain seaweeds [4]. REFERENCES [1] J. A. Widtsoe and B. Tollens, Ber. deut. chem. Ges. 33, 132 (1900); A. Günther and B. Tollens, Liebigs Ann. Chem. 271, 86 (1892). [2] E. P. Clark, BS Sci. Pap. 18, 527 (1922) S459; J. Biol. Chem. 54, 65 (1922). [3] R. C. Hockett, F. P. Phelps, and C. S. Hudson, J. Am. Chem. Soc. 61, 1658 (1939). [4] R. H. F. Manske, J. Biol. Chem. 86, 571 (1930). 323414°-42-31 6. d-GALACTOSE Method. [1] Fifteen hundred grams of lactose is dissolved in 3,750 ml of hot water containing 75 g of concentrated sulfuric acid. The solution is brought to a boil and then simmered for 2 hours. A thin paste of barium carbonate is then added to the hot solution until it reacts neutral to congo-red paper. The precipitate of barium sulfate is allowed to settle, after which as much as possible of the supernatant liquid is drawn off and filtered. Then the precipitate is placed on the filter and washed with water. The filtrate is concentrated under diminished pressure until it has a weight of about 1,650 g (n20 between 1.5120 and 1.5125). The very thick sirup is warmed to between 60° and 70° C, and 250 ml of ethyl alcohol is dissolved in it by vigorous shaking. The solution is then poured into a beaker or jar and the remaining sirup is washed from the flask with 500 ml of methyl alcohol. This is best done by adding the methyl alcohol to the flask portionwise and warming and shaking in a water bath. The whole solution is thoroughly mixed, seeded with some pure galactose crystals,1 and allowed to crystallize, preferably in a slowly rotating flask. The crystallization is generally complete in about 4 days, after which the crystals are filtered off, washed with a little methyl alcohol, and dried. The yield of the crude sugar is about 27 percent of the lactose taken.2, 3 4 Recrystallization.-The crude galactose is dissolved in an equal weight of hot water, and after the addition of a small quantity of a decolorizing carbon the solution is filtered. A few drops of acetic acid are added, and the solution is evaporated in vacuo to a sirup of 60 percent of total solids (n=1.442). The sirup is then seeded with a-d-galactose and kept in motion while crystallization proceeds. When a satisfactory crystal growth is obtained (in about 1 day), the crystals are separated either by filtration or by means of a centrifuge. The crystals are washed thoroughly with methyl alcohol. About 50 percent of the sugar is obtained in the first crop, and the remainder may be separated by concentration of the mother liquor. A more rapid recrystallization can be made by dissolving 200 g of galactose in 100 ml of water, filtering the solution with the addition of about 5 g of a decolorizing carbon, and adding 100 ml of methyl alcohol. The mixture is seeded and stirred while crystallization takes place. After several hours the crystalline galactose is separated by filtration and washed with methyl alcohol. About 75 percent of the original sugar is obtained in the first crop of crystals; the rest of the sugar is reclaimed by concentrating the mother liquor. In 5-percent aqueous solution, pure a-d-galactose has an initial specific rotation, [a]2+150.7°, which changes after a number of hours to the equilibrium value, +80.2°. NOTES 1 Contamination by dextrose seed must be avoided. 2 If the hydrolysis is not complete, unchanged lactose may be obtained. Also dextrose may occasionally crystallize simultaneously with the galactose. 3 Galactose may be prepared from plant gums, such as those from the western larch [2] and by partial fermentation of the hydrolysis products of lactose [3]. A review of methods for the preparation of galactose is given by Harding [4]. If the solution is not kept in motion, the crystals form a solid mass. REFERENCES [1] E. P. Clark, BS Sci. Pap. 17, 227 (1922) S416. [2] A. W. Schorger and D. F. Smith, J. Ind. Eng. Chem. 8, 494 (1916). U. S. Patent 1,358,129 and British Patent 160,776. [3] G. Mougne, Bul. soc. chim. biol. 4, 206 (1922). [4] T. S. Harding, Sugar 25, 175 (1923). 1 7. GENTIOBIOSE (6-(8-d-GLUCOPYRANOSIDO)-d-GLUCOSE) Method. [1] Gentiobiose is conveniently prepared from the sirupy residue left from the preparation of dextrose from starch. About 4.5 kg of this residue called "hydrol" is dissolved in 30 liters of water, and after the addition of 100 g of calcium carbonate and 2 cakes of baker's yeast the mixture is allowed to stand until fermentation is complete. About 750 g of a decolorizing carbon is added and the solution is filtered. The filtrate is evaporated under reduced pressure to a thick sirup of about 80 percent of total solids (n=1.490). The sirup is taken up with 8 liters of a hot mixture of methyl and ethyl alcohols (7 parts of methyl to 1 part of ethyl alcohol). The granular precipitate is extracted several times with methyl alcohol, and the combined extracts are again evaporated to a thick sirup (n=1.510). This is mixed with 2 liters of glacial acetic acid and evaporated in vacuo until the solvent ceases to distill. The sirup, weighing 1,300 g and contained in a 12-liter flask, is heated with 500 ml of technical acetic anhydride until a slow visible reaction starts. Twenty grams of anhydrous sodium acetate 2 is introduced, and the mixture is heated with stirring until the reaction becomes vigorous. The source of heat is then removed and care must be taken to keep the reaction from becoming too violent. When the reaction has slowed down, more acetic anhydride (500 ml) and sodium acetate (20 g) are added. The addition is repeated until a total of 4,000 ml of the acetic anhydride and 350 g of sodium acetate has been introduced.3 Heating is then continued for 1 more hour. The solution, after cooling, is poured into 15 liters of water. The insoluble phase is separated by decantation and given several washings with water. The combined water washings are extracted with chloroform to recover any dissolved or suspended product. After evaporation of the chloroform, the residue is combined with the water-insoluble portion and stirred a second time with water. After several hours the insoluble material is separated again and then dissolved in ether and kept at 0° C until crystals of gentiobiose octa-acetate form. This may require 1 or more weeks; hence it is advisable to seed the product with crystalline gentiobiose octa-acetate. Several days after seeding, the crystalline material is separated by filtration, washed with alcohol, and dried. The yield is about 100 g. The crude gentiobiose octa-acetate is recrystallized by dissolving in boiling ethyl alcohol, adding a decolorizing carbon, filtering, and allowing the solution to cool. The recrystallized material weighs 80 g and has a melting point of 193° to 196° C. The gentiobiose octa-acetate is deacetylated by the barium methylate method of Isbell. Eighty grams is dissolved in 6 liters of dry methyl alcohol with the aid of heating. The solution is cooled to room temperature, 100 ml of 0.6 N barium methylate in dry methyl alcohol is added, and the solution is kept at 0° C for 48 hours, after which the barium is precipitated by sulfuric acid or carbon dioxide and removed by filtration. The solution, free from barium, is concentrated under reduced pressure to a thick sirup which is taken up with methyl alcohol. Crystallization usually takes place in several hours. The crystals when separated weigh about 33 g, and about 15 g more are obtained from the mother liquors.5 Recrystallization.-Purification is accomplished by dissolving the material in water, treating with a decolorizing carbon, filtering, and concentrating to a heavy sirup (n=1.515) which is dissolved in methyl alcohol and allowed to crystallize. The isomer obtained in this way has the formula, a-gentiobiose .2CH3OH. The melting point is 86° C, and the initial rotation, [a]=+21.4°, changes to the equilibrium value, [a] =+8.7° (as alcoholate). NOTES "Hydrol" may be obtained from the concerns manufacturing dextrose. 2 Anhydrous sodium acetate is prepared by fusing the hydrate in an iron crucible. See p. 488, note 1. 3 The reaction takes place much less violently as the addition proceeds, and the materials may be added at short intervals after the third or fourth addition. The alcohol washings should be kept separate from the mother liquor, which usually does not yield any further material. 5 Gentiobiose may be prepared from gentian root [2, 3, 4] or synthesized by the method of Reynolds and Evans [5]. Methyl alcohol is the only alcohol which should be used, since the sugar crystallizes as the difficultly soluble methyl alcoholate. REFERENCES [1] H. Berlin, J. Am. Chem. Soc. 48, 2627 (1926). [2] C. S. Hudson and J. Johnson, J. Am. Chem. Soc. 39, 1272 (1917). [3] G. Zemplén, Z. physiol. Chem. 85, 399 (1913); Ber. deut. chem. Ges. 48, 233 (1915). [4] E. Bourquelot and H. Hérissey, Compt. Rend. 132, 571 (1901); 135, 290 (1902). [5] D. D. Reynolds and W. L. Evans, J. Am. Chem. Soc. 60, 2559 (1938). 8. 4-GLUCOSIDOMANNOSE, (4-(ß-d-GLUCOPYRANOSIDO)-d-MANNOSE) Method.-[1,2] Six grams of octaacetyl-4-glucosidomannose is dissolved in 150 ml of absolute methyl alcohol, and the solution treated with 5 ml of about 0.5 N barium methylate solution. After 24 hours the barium is removed by adding an equivalent quantity of sulfuric acid and filtering. The filtered solution is evaporated in vacuo to a thick sirup which is taken up with 10 ml of ethyl alcohol. About 2.4 g of crystalline 4-glucosidomannose separates in the first crop, and a small amount (0.2 g) is obtained from the mother liquor. The sugar crystallizes in the alpha modification as a monohydrate which melts at 137° C.4 3 In 4-percent aqueous solution, 4-(B-d-glucosido)-a-d-mannose hydrate gives [a] 29 +14.7° initially, which changes in the course of several hours to +5.9°. NOTES 1 The preparation of octaacetyl-4-glucosidomannose is given on page 501. 2 The barium methylate solution is prepared by adding 125 g of powdered barium oxide to 500 ml of absolute methyl alcohol. After standing for several hours, the solution is filtered. It should be kept in a bottle protected from moisture and carbon dioxide. 3 Since moisture or acids in the original sirup will decompose the barium methylate, it is well to test the deacetylated mixture to see if an excess of barium methylate is present. This may be done by diluting a few milliters with water and adding several drops of phenolphthalein solution. A definite red color indicates a sufficient excess. If the test is not positive, add more barium methylate solution to the deacetylation mixture and allow 24 hours more for the deacetylation to be completed. 4 Glucosidomannose has been made by the oxidation of cellobial with perbenzoic acid [3,4]. The method as applied to d-talose is given on page 479. REFERENCES [1] D. H. Brauns, J. Am. Chem. Soc. 48, 2776 (1926). [2] H. S. Isbell, BS J. Research 5, 1179 (1930) RP253; 7, 1115 (1931) RP392. [3] M. Bergmann and H. Schotte, Ber. deut. chem. Ges. 54, 1570 (1921). [4] W. N. Haworth, E. L. Hirst, H. R. L. Streight, H. A. Thomas, and J. Webb, J. Chem. Soc. 1930, 2636. 9. a-d-GULOSE.CaCl2.H2O Method. [1] Pure d-gulonic gamma lactone is reduced by sodium amalgam in a 6-liter stainless-steel pot equipped with a strong mechanical stirrer with broad metal paddle and cooled by a mixture of ice and salt. The lactone (90 g) is dissolved in 800 ml of water and 150 ml of 95-percent ethyl alcohol, and cooled to about -10° C. About 14 ml of 20-percent sulfuric acid is added to the solution, followed by 1,500 g of 3-percent sodium amalgam, which is added (in 1 portion) with vigorous stirring. Twenty-percent aqueous sulfuric acid is added to the mixture at such rate as to maintain the solution acid at about pH 3.2 After the sodium amalgam has reacted, the mixture is transferred to a separatory funnel and the mercury is separated. The solution is returned to the pot used for reduction, and after cooling to about -10° C, a second 1,500-g portion of sodium amalgam is added and the reduction continued while the solution is kept acid and cold as before. When the amalgam is spent, the mixture is again transferred to a separatory funnel and the mercury is separated. The cold solution is filtered to remove the crystalline sodium sulfate, and sodium hydroxide is added to the filtrate in sufficient quantity to neutralize the excess acid and to saponify any gulonic lactone. After the lactone is saponified, as shown by an alkaline reaction after standing for 15 minutes, the solution is acidified with dilute sulfuric acid and evaporated under reduced pressure until the formation of crystalline sodium sulfate causes difficulty with further evaporation. The concentrated liquor is mixed with 2 volumes of ethyl alcohol and allowed to stand for several hours, preferably in a refrigerator. The insoluble salts are separated from the alcoholic solution by filtration and then washed with methyl alcohol. The alcoholic solution is evaporated to a thick syrup. This is mixed with 500 ml of hot ethyl alcohol, which results in the precipitation of most of the salt in the form of an amorphous residue. Most of the sugar is found in the alcoholic solution; that which remains in the residue may be separated by dissolving the residue in water and reprecipitating the salt by the addition of alcohol. The combined alcoholic solutions are evaporated in vacuo to a heavy sirup. The crude gulose may be purified by means of its phenylhydrazone or the product may be converted to d-gulose calcium chloride without further purification. The calcium chloride compound is prepared by adding in aqueous solution approximately 1 molecular equivalent of calcium chloride. The aqueous solution is evaporated in vacuo |