5. REFERENCES [1] A. P. Dubrunfaut, Compt. rend. 23, 38 (1846). [2] L. Pasteur, Ann. chim. phys. 31, 67 (1851) Compt. rend. 42, 347 (1856). [3] E. O. Erdmann, Dissertatio di saccharo lactico et amylaceo cited in Jahresbericht für 1855, p. 671. [4] T. M. Lowry and G. F. Smith, Rapports sur les hydrates de carbone, 10th Conference of the International Union of Chemistry, p. 79 (Liege, 1930). [5] C. Tanret, Compt. rend. 120, 1060 (1895); Bul. soc. chim. 15, 349 (1896). [6] E. F. Armstrong, J. Chem. Soc. 83, 1305 (1903). [7] W. Gabryelski and L. Marchlewski, Biochem. Z. 261, 393 (1933); see also L. Marchlewski and W. Urbanczyk, Biochem. Z. 262, 248 (1933). [8] J. K. Dale, BS J. Research 3, 459 (1929) RP 106. [9] H. S. Isbell, J. Am. Chem. Soc. 55, 2166 (1933). [10] H. S. Isbell and W. W. Pigman, J. Research NBS 20, 773 (1938) RP1104. [11] H. S. Isbell and W. W. Pigman, J. Research NBS 18, 141 (1937) RP969. [12] C. N. Riiber and J. Minsaas, Ber. deut. chem. Ges. 59, 2266 (1926). [13] G. F. Smith and T. M. Lowry, J. Chem. Soc. 1928, 666. [14] W. W. Pigam and H. S. Isbell, J. Research NBS 19, 189 (1937) RP1021. [15] H. S. Isbell, J. Am. Chem. Soc. 56, 2789 (1934). [16] F. P. Phelps and F. J. Bates, J. Am. Chem. Soc. 56, 1250 (1934). [17] W. C. Austin and F. L. Humoller, J. Am. Chem. Soc. 56, 1153 (1934). [20] C. S. Hudson, Z. physik. Chem. 44, 487 (1903). [24] C. N. Riiber, Saertrykk av Tidsskrift for kjemi og bergvesen, nr. 10 (1932) S. T. nr. 252. [25] N. A. Sørensen, Kgl. Norske Videnskab. Selskabs, Skrifter, No. 2 (1937). [26] F. P. Worley and J. C. Andrews, J. Phys. Chem. 32, 307 (1928). [27] H. S. Isbell, J. Research NBS 18, 505 (1937) RP990. [28] H. S. Isbell and W. W. Pigman, J. Research NBS 22, 397 (1939) RP1190. [29] C. S. Hudson, J. Am. Chem. Soc. 31, 66 (1909). [30] H. T. Brown and S. U. Pickering, J. Chem. Soc. 71, 756 (1897). [31] J. N. Brönsted and E. A. Guggenheim, J. Am. Chem. Soc. 49, 2554 (1927). [32] H. S. Isbell (heretofore unpublished work). [33] T. M. Lowry and I. J. Faulkner, J. Chem. Soc. 127, 2883 (1925). [34] H. S. Isbell, BS J. Research 5, 741 (1930) RP226. [35] H. S. Isbell and H. L. Frush (heretofore unpublished work). [36] H. S. Isbell and C. S. Hudson, BS J. Research 8, 327 (1932) RP418. [37] H. S. Isbell, BS J. Research 8, 615 (1932) RP441. [38] H. S. Isbell and W. W. Pigman, BS J. Research 10, 337 (1933) RP534. [39] F. Lippich, Biochem. Z. 248, 280 (1932). [40] H. H. Schlubach and V. Prochownick, Ber. deut. chem. Ges 62, 1502 (1929). [41] H. Ohle, Die Chemie der Monosaccharide und der Glykoside, p. 126 (J. F. Bergmann, Munich, 1931). XXX. METHODS FOR THE PREPARATION OF CERTAIN SUGARS The methods reported here are those ordinarily used in the laboratories of the National Bureau of Standards for the preparation of the various sugars. No attempt has been made to give a complete. bibliography or to record the contributions of the various workers in the field. Some of the methods are essentially as given in the reference cited, while others have been improved in various ways. 1. d-ALLOSE Method [1, 2, 3]. Fifty grams of purified d-ailonic lactone 2 and 500 ml of distilled water are placed in a 1.5-liter wide-mouthed 43 In chapters XXX and XXXI the superscript numbers refer to the numbered notes, and the numbers in brackets refer to literature reference numbers at the end of each section. 3 flask or beaker. The solution is stirred vigorously and cooled in an ice-and-salt bath so that a little ice forms inside the beaker (to be sure that the solution is actually at about 0° C). About 4 ml of dilute (10-percent) sulfuric acid is added, and then 2.5-percent sodium amalgam in 250-g portions, while dilute sulfuric acid is continuously dropped from a burette at such rate that the solution remains just barely acid to congo-red test paper. After the addition of 3 or 4 portions of amalgam during the course of approximately 1 hour, the sugar content reaches a maximum. The solution is poured off from the mercury and treated with enough sodium carbonate so that after standing about 1⁄2 hour (cold) the reaction mixture is still slightly alkaline. Dilute sulfuric acid is added until the solution is slightly acid to litmus; it is then evaporated in vacuo to a small volume. Alcohol is added until further addition causes no further precipitate, the solution is filtered, and the alcoholic filtrate evaporated to a thick sirup. Upon extraction of the sirup with hot absolute alcohol, the allose dissolves, leaving sodium allonate as a sticky gum. Evaporation of this alcoholic extract yields crystalline allose. The crude product may be purified by dissolving it in a little warm water, adding 3 volumes of warm methyl alcohol, and filtering through a little decolorizing carbon. The filtrate is allowed to cool and is seeded with crystalline allose. After standing for several hours, the crystalline product is separated. NOTES 1 The same procedure may be used for the preparation of altrose and other sugars. 2 The preparation of allonic lactone from ribose is described on page 527. 3 The solution should be kept cold and the acidity should be carefully watched. 4 The purpose of the sodium carbonate treatment is to convert all unreduced lactone to the sodium salt in order to facilitate its removal by alcohol. REFERENCES [1] F. P. Phelps and F. J. Bates, J. Am. Chem. Soc. 56, 1250 (1934). [2] W. C. Austin and F. L. Humoller, J. Am. Chem. Soc. 55, 2167 (1933). [3] W. C. Austin and F. L. Humoller, J. Am. Chem. Soc. 56, 1153 (1934). 2. I-ARABINOSE 2 Method. [1] Three kilograms of mesquite gum is dissolved in 11 liters of water.3 Two liters of a solution containing 370 ml of concentrated sulfuric acid is added, and the solution is kept for 7 hours at a temperature of 80° to 90° C. The hot solution is neutralized with about 700 g of calcium carbonate, and the insoluble material is removed by filtration. To the filtrate is added 1 kg of decolorizing carbon. After several hours the solution is filtered and the filtrate evaporated in vacuo to a volume of 4 liters. Twelve liters of hot ethyl alcohol is now added and the two phases are thoroughly mixed. The gummy material is allowed to settle out for several hours while the liquid cools. The supernatant liquid is separated by decantation. The gums are given a second and third extraction, each time with 6 liters of warm methyl alcohol. The alcoholic extracts are combined and concentrated in vacuo to a thin sirup which is allowed to crystallize. About 900 g of crude arabinose is obtained in the first crop and 250 g of additional material may be separated by concentrating the mother liquors, removing the gummy impurities by precipitation with hot ethyl alcohol and evaporating the alcoholic extract.7 Recrystallization.-Crude arabinose which contains gums may be purified in the following manner: 300 g of the sugar is dissolved by heating with 100 ml of water; the solution is mixed with 750 ml of hot ethyl alcohol and 15 g of a decolorizing carbon. The hot solution is filtered, and the carbon residue is washed with 150 ml of hot ethyl alcohol. The hot filtrate is allowed to cool, and accidental seeding with crystalline arabinose is avoided. As the solution cools it separates into two phases, the lower more sirupy layer of which contains most of the impurities. After the solution reaches room temperature and the sirupy phase has settled, the alcoholic layer is decanted. The residue is extracted as before with 300 ml of hot methyl alcohol, followed by 300 ml of hot ethyl alcohol. The combined alcoholic extract is evaporated in vacuo to a volume of approximately 600 ml. Sometime before this volume is reached, crystallization takes place in the distillation flask. When the evaporation has reached the desired stage, the sirup is seeded with crystalline arabinose, placed in the refrigerator, and allowed to stand until a satisfactory crystal growth is obtained. The first crystals are usually about 60 percent of the arabinose content. By concentrating the mother liquors, additional crystalline sugar is obtained. Relatively pure arabinose can also be recrystallized in the following manner: 300 g of arabinose is dissolved with 180 ml of water. The hot solution is mixed with 200 ml of hot methyl alcohol, and after the addition of 50 g of a decolorizing carbon the mixture is filtered rapidly while hot. The filtrate is diluted with 400 ml of methyl alcohol and 300 ml of ethyl alcohol, cooled to room temperature, seeded, and set aside to crystallize. After several days the resulting crystals are collected on a filter and washed with methyl alcohol. The yield is about 150 g. The sugar in the mother liquors is reclaimed by evaporating the liquors to a sirup (n=1.465), which, after mixing with about 5 volumes of methyl alcohol, yields additional crystalline sugar. In 4-percent aqueous solution, B-l-arabinose gives [a]=+190.6° initially, which changes in the course of several hours to +104.5°. NOTES 1 The method is that of Anderson and Sands [1] as modified by Isbell (heretofore unpublished work). 2 Mesquite gum is obtained from a plant (Prosopis juliflora and related species) widely distributed through the southwest. It may be purchased from the Martin Drug Co., Tucson, Ariz. Cherry gum may also be used with essentially the same procedure. 3 The gum requires about 24 hours to dissolve. Before starting the hydrolysis, it is well to filter the solution through cheese cloth in order to remove bark and other foreign matter. ↑ This must be done slowly with small portions of calcium carbonate. Foaming may be reduced by the addition of a commercial antifoam agent or capryl alcohol. 5 The exceptionally large quantity of decolorizing carbon is added to reduce foaming, which can be further reduced by the use of an antifoam agent such as capryl alcohol. 6 A mechanical stirrer is of considerable help in this procedure. 7 l-Arabinose has been prepared from a number of other sources: Cherry gum [2], wheat and rye bran [3, 4], peach gum [5], Australian black wattle gun [6], and beet pulp [7]. Harding [8] gives a review of the sources. REFERENCES [1] E. Anderson and L. Sands, J. Am. Chem. Soc. 48, 3172 (1926), also, Organic Syntheses 8, 18 (1928). [2] H. Kiliani, Ber. deut. chem. Ges. 19, 3029 (1886). [3] W. Stone and B. Tollens, Liebigs Ann. Chem. 249, 238 (1888). [4] E. Steiger and E. Schulze, Ber. deut. chem. Ges. 23, 3110 (1890). [5] W. Stone, Am. Chem. J. 12, 435 (1890). [6] W. Stone, Am. Chem. J. 17, 196 (1895). [7] E. Zitkowski, Am. Sugar Ind. 13, 98 (1911). [8] T. S. Harding, Sugar 24, 656 (1922). 3. CELLOBIOSE (4-(8-d-GLUCOPYRANOSIDO)-d-GLUCOSE) Method. The octaacetyl cellobiose is prepared by the action of acetic anhydride and sulfuric acid on cotton or filter paper according to the method of Klein [1]. Fifty grams of pure cotton is put in a precipitating jar (about 12 cm in diameter and 25 cm high), which is cooled in an ice-and-salt bath. Acetic anhydride, 140 ml, is added and the jar covered with a watch glass. Sixty milliliters of acetic anhydride is cooled in an ice-and-salt bath and 28 ml. of sulfuric acid slowly added. When this solution has cooled, it is poured onto the cold mixture of cotton and acetic anhydride and the whole mixture worked to a homogeneous mass by stirring with a glass rod. The cotton gradually disintegrates. The mass is transferred to a 2-liter Erlenmeyer flask and after vigorous shaking and occasional heating on the steam bath, a reddish solution is obtained." Several such batches combined are allowed to stand at room temperature for 2 or 3 days while crystallization takes place. The crystalline mass is not poured into water, as described by Klein, but filtered on a large-size Büchner filter having small holes, and fitted with a double layer of ordinary filter paper. It is filtered dry and washed with ether. The product is now stirred in a dish with 95-percent alcohol, again filtered by suction, and finally recrystallized from 95-percent alcohol. The yield may reach 40 to 50 percent of the theoretical. In a 2.5-percent concentration in chloroform the specific rotation of the pure substance is [al+41.5°, and the melting point is 229.5° C. The cellobiose octaacetate is deacetylated by dissolving it in dry methyl alcohol and adding 1 ml of 0.5 N barium methylate 3 solution for each gram of sugar. After standing overnight in the refrigerator, the barium is removed by precipitation with an equivalent quantity of sulfuric acid and the barium-free solution is concentrated in vacuo to a thin sirup which on standing yields crystalline cellobiose. Anhydrous B-cellobiose melts at 225° C and gives [a] = +14.2° initially, which changes in the course of several hours to +34.6°. NOTES The simultaneous hydrolysis and acetylation of cellulose was described first by Franchimont [2]. Modifications of the original method are described in references [3, 4, 5, and 6]. 2 This part of the preparation requires some practice in order to obtain uniform results. Several batches may be prepared in this way and combined in the next step, but it is not advisable to work with larger amounts up to this point, as charring may result. 3 The preparation of the barium methylate solution and additional details for the method are given on page 493, REFERENCES [1] F. Klein, Z. angew. Chem. 25, 1409 (1912). [2] A. P. N. Franchimont, Ber. deut. chem. Ges. 12, 1941 (1879). [3] Z. H. Skraup, Ber. deut. chem. Ges. 32, 2413 (1899). [4] G. Zemplén, Ber. deut. chem. Ges. 59, 1258 (1926). [5] G. Braun, Organic Syntheses 17, 36 (1937). [6] C. C. Spencer, Cellulosechemie 10, 61 (1929). 4. 2-DESOXYGALACTOSE Method. [1, 2, 3] Fifty grams of recrystallized galactal1 is dissolved in 700 ml of 5-percent cold sulfuric acid, and the solution is kept in the refrigerator at 0° C for 24 hours, after which it is neutralized with 120 g of barium carbonate and the esters are saponified by heating to 60° C. The saponification usually requires about 48 hours of heating. Eight- or ten-gram portions of barium carbonate are added at intervals during this time. The solid material is separated by filtration, and the filtrate is evaporated in vacuo to a thick sirup which is taken up with 50 ml of absolute alcohol and allowed to crystallize. About 40 g of material is obtained." The crystals are purified by dissolving in water and evaporating the solution to a sirup, which is taken up with three volumes of methyl alcohol and allowed to crystallize, preferably in a slowly rotating flask. Desoxygalactose melts at 120° to 121° C. In 4-percent aqueous solution, desoxygalactose gives [a]=+40.8° initially, which decreases in 5 minutes to a minimum and then increases to +60.5° in about 30 minutes. NOTES 1 The preparation of galactal is described on page 532. 2 The preparation of 2-desoxyglucose and 2-desoxyrhamnose is given by Bergmann, Schotte, and Leschinsky [3], while that of 2-desoxyarabinose and desoxyxylose is described by Levene and Mori [4]. Several natural-occurring desoxy sugars are reported [5, 6, 7]. REFERENCES [1] H. S. Isbell and W. W. Pigman, J. Research NBS 22, 397 (1939) RP1190. [2] P. A. Levene and R. S. Tipson, J. Biol. Chem. 93, 644 (1931). [3] M. Bergmann, H. Schotte, and W. Leschinsky, Ber. deut. chem. Ges. 55, 158 (1922); 56, 1052 (1923). [4] P. A. Levene and T. Mori, J. Biol. Chem. 83, 803 (1929). [5] H. Kiliani, Ber. deut. chem. Ges. 38, 4040 (1905). [6] A. Windaus and L. Hermanns, Ber. deut. chem. Ges. 48, 88 (1915). [7] W. A. Jacobs and N. W. Bigelow, J. Biol. Chem. 96, 355 (1932). 5. I-FUCOSE (I-GALACTOMETHYLOSE) Method.'-[1, 2, 3] The seaweed, ascophyllum nodosum, is washed with tap water and any large shells are removed. It is then air-dried and ground to a fine powder. One kilogram of the dried seaweed is added to 8 liters of hot 4-percent sulfuric acid, and the solution is allowed to simmer for 3 hours. The liquid is separated by filtration and the residue is washed with hot water. The filtrate is neutralized with an excess of calcium carbonate, refiltered, and the calcium sulfate washed with water. The mixture is cooled to room temperature and fermented with baker's yeast acclimatized to ferment galactose. When the fermentation is complete, about 100 g of a decolorizing carbon is added; and after standing for several hours, the |