(a) EFFECT OF THE SOLVENT AND OTHER SUBSTANCES ON THE EQUILIBRIUM STATE The optical rotations of the reducing sugars in different solvents vary widely. The variation results, in part from the fact that a given constituent will have different optical rotations in different solvents, and in part from the displacement of the equilibrium between the various sugar modifications. The effect of a change in the solvent on the equilibrium state can be ascertained most readily by observing the mutarotation which follows a change of solvent. For example when a concentrated aqueous solution of galactose is diluted with several volumes of alcohol, a nearly instantaneous change in optical rotation takes place. This is due to the difference in the optical rotations of the constituents because of the change in solvent. The initial change in rotation due to the new solvent is followed by a complex mutarotation reaction which apparently consists of the rapid and slow mutarotation reactions previously discussed. This complex mutarotation shows that in some manner the equilibrium between the various constituents is altered by a change in solvent. The effect of solvents on the equilibrium state has not been investigated very thoroughly. Apparently in some cases the solvent combines with the sugar, and may facilitate the separation of different modifications of the sugar. For example, d-glucose crystallizes from pyridine solutions in the form of a pyridine compound which contains the beta pyranose modification; while from water solutions at ordinary temperatures d-glucose crystallizes in the form of a hydrate which contains the alphapyranose modification. The existence of numerous solvated sugars and sugar derivatives shows that the solvent is not merely an inert medium in which the sugar is dissolved. Isbell [34] has shown that the position of the equilibrium between the various modifications of d-gulose in solution can be altered by the addition of calcium chloride, or by changing the concentration of d-gulose calcium chloride. The mutarotation which follows a change in the concentration of d-gulose calcium chloride indicates that in some manner the sugar equilibrium is displaced by the change in concentration. The addition of a salt to the system complicates the equilibrium conditions by forming molecular compounds with the various isomeric forms of the sugar, some of which compounds have been isolated. In dilute solution the compounds are largely dissociated into the sugar and calcium chloride. As shown by the curves given in figure 109, dilution of a concentrated solution of d-gulose CaCl2.H2O with water results in the formation of the more levorotatory modification of d-gulose, while dilution with alcohol results in the formation of the more dextrorotatory modification. Obviously the equilibrium existing in an alcoholic solution of d-gulose CaCl2.H.2O is different from the equilibrium existing in an aqueous solution. Similarly, the equilibrium of d-a-glucoheptose is shifted by calcium chloride in a manner comparable to the shift observed for d-gulose [35]. Another interesting example of the influence of calcium chloride on the equilibrium state is the formation of a calcium chloride compound of the furanose modification of d-mannose [8, 9]. A pure aqueous solution of d-mannose does not appear to contain an appreciable quantity of the furanose modification, but when calcium chloride is added to the mannose solution and the solution evaporated, d-mannofuranose calcium chloride crystallizes. 4. CHEMICAL METHODS FOR STUDYING THE EQUILIBRIUM STATE When a sugar is treated with methyl alcohol in the presence of hydrochloric acid, the alpha and beta methyl pyranosides and the alpha and beta methyl furanosides are formed. The furanosides are formed much more rapidly than the pyranosides, so that with short treatment at low temperatures, the furanosides predominate, whereas with longer treatment at higher temperatures, the pyranosides predominate. Obviously, on account of the equilibrium displacement, the formation of either the furanosides or the pyranosides does not give information concerning the equilibrium existing in the original solution. But under favorable conditions, by consideration of the mechanism of the reaction and of the rates at which the various modifications change one into another, one can interpret a chemical reaction in terms of the equilibrium state. The most extensive investigation of this character has been made on the oxidation of the sugars with bromine [36, 37, 38]. In slightly acid solution the pyranose sugars are oxidized rapidly to delta lactones (as shown by the results given in [9, 37]), whereas the furanose sugars are oxidized to gamma lactones. Since the oxidation is rapid in comparison with the rate at which one modification of the sugar changes to another, the identification of the oxidation products indicates which substances are present in the equilibrium solution. The alpha and beta sugars differ widely in their rates of reaction. The oxidation of the sugar in the equilibrium solution proceeds rapidly until the easily oxidizable beta modification is used up, and more slowly as the difficultly oxidizable alpha modification continues to be oxidized. By comparing the rates of oxidation of the sugar in the equilibrium solution with the rates for the alpha and beta modifications as determined separately, it is possible to calculate the proportions of each modification in the equilibrium solution. Table 62 gives results of work reported by Isbell [27, 37] and by Isbell and Pigman [11]. TABLE 62.-Oxidation of sugar solutions at 0° C with bromine water in the presence of barium carbonate ♣ The experimental details for the oxidation measurements are given on p. 173 of reference [11]. Rates of oxidation were determined in aqueous solutions containing 0.05 mole of sugar per liter, and approximately 0.08 mole of free bromine per liter, and buffered with barlum carbonate and carbon dioxide. Percentage calculated from Hudson's optical rotations derived from measurements of the initial and final solubilities at 20° C. The reaction of the sugars with hydrogen cyanide has been used for estimating the concentration of the open-chain modifications. It is assumed that the open-chain modifications of the sugars combine readily with hydrogen cyanide, whereas the ring modifications do not. Lippich [39] has shown that each sugar in the equilibrium state has an initial power for combining with hydrogen cyanide, which he considers a measure of the amount of the open-chain modification present in the equilibrium state. The reaction of the sugars with acetic anhydride in pyridine solution appears to give some information concerning the modifications present. Schlubach and Prochownick [40] found, for example, that the proportions of the pyrano- and furano-acetates of galactose, formed by acetylation of galactose dissolved in dry pyridine vary with temperature. The reaction of the sugars with acetone in the presence of copper sulfate also has been used to show the presence of furanose modifications [41]. In conclusion, further investigation of the reaction rates and the quantitative determination of the products is needed to give additional information concerning the equilibrium state. 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). [23] C. S. Hudson, Sci. Pap. BS 21, 268 (1926) $533. [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? 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. I-ARABINOSE 4 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 |