(f) EFFECT OF ACIDS AND BASES ON THE MUTAROTATION RATES

The acceleration of the mutarotation rates of the sugars by acids and bases has been the subject of many investigations. The early workers attributed the catalytic effect of acids and bases to the hydrogen and hydroxyl ions, but subsequent work has revealed that catalysis is not the exclusive property of the hydrogen and hydroxyl ions. Experiments by Lowry and coworkers, [4, p. 111,] by Brönsted and Guggenheim [31], and others have disclosed the catalytic activity of molecules of undissociated acids, of anions of weak acids, and of cations of weak bases. In general, amphoteric solvents are complete catalysts for the mutarotation, while hydrocarbons, chloroform, and carbon tetrachloride do not promote mutarotation.

It may be observed from the curve given in figure 108 that the velocity for the mutarotation of glucose in aqueous solutions does not change appreciably in the range from pH 2.5 to 6.5. However, in more

FIGURE 108-Variation of the mutarotation constants with pH [10].

strongly alkaline or acid solutions, the velocity rises rapidly with increasing concentrations of the hydrogen and hydroxyl ions. If carefully purified distilled water, free from carbon dioxide, is employed in mutarotation measurements, the velocity constants are substantially higher than those obtained in slightly acid solutions. Fortunately the region of minimum velocity falls in the pH range of distilled water containing carbon dioxide, such as the water ordinarily used for mutarotation measurements. Minute quantities of bases suffice to cause large alterations in the rate, and therefore it is difficult to obtain satisfactory velocity constants except in the presence of an acid-base buffer. Mutarotation measurements made with unbuffered solutions in nickel, brass, or copper tubes give substantially higher velocity constants than those made in glass tubes [32]. The higher constants appear to be due to an alteration in the acidity caused by the metallic oxide from the tubes which dissolves in the sugar solution.

Precautions are necessary in making mutarotation measurements to avoid accidental catalysis. In order that results from different sources be placed on a comparable basis, the use of 0.001 N potassium acid phthalate is recommended as a solvent for mutarotation measurements. According to Hudson, the catalytic activity of water in the mutarotation of glucose may be represented by the equation

k=0. 0096+0. 258 [H+]+9750 [OH-].

The equation shows that the alterations caused by the hydrogen and hydroxyl ions are proportional to their concentration but that there is a residual catalytic activity which is now considered to be due to undissociated water molecules.

In the mutarotation of glucose at pH 4.7, the contribution of the hydroxyl ion to the catalysis is about 40,000 times that of the hydrogen ion, and the residual catalytic effect of the water is nearly 500 times that of the combined catalytic effect of the hydrogen and hydroxyl ions. Lowry and Faulkner [33] showed that pure pyridine and pure cresol do not promote mutarotation; however, a mixture of 1 part of pyridine with 2 parts of cresol does accelerate the rate for the mutarotation of tetramethyl glucose to 20 times that obtained in water solution. Lowry explained this seemingly anomalous behavior by assuming that for the mutarotation to take place the solvent must possess acid and basic properties simultaneously. Pyridine and cresol, or pyridine and water, or water alone, can act both as an acid and as a base, but pyridine alone can act only as a base and cresol alone can act only as an acid. In this connection the acid and basic functions are used in the Brönsted sense, that is, the ability to give or to accept protons. The molecules of an undissociated acid can give, and the anions of a weak acid can accept, a proton, as follows:

The ammonium cation can give a proton, and ammonia can accept a proton as follows:

NH+ gives H+, leaving NH3

NH3 accepts H+, giving NH2+

Undissociated water accepts a proton and forms the H3O+ ion or gives a proton and forms the hydroxyl ion. The anions of highly ionized strong acids and metallic cations do not give or accept protons and hence do not exhibit catalytic activity. In harmony with this concept, calcium chloride, sodium sulfate, and other metallic salts of strong acids do not accelerate the mutarotation reaction, while salts of weak acids, like sodium acetate, have marked catalytic action. Isbell and Pigman [10] have shown that the mutarotation of levulose and the rapid mutarotation of galactose are extremely sensitive to the catalytic action of acids and bases. The extreme sensitivity of the pyranosefuranose interconversions to acid and basic catalysts furnishes a convenient means for distinguishing them from the alpha-beta pyranose interconversions.

(g) 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. initial change in rotation due to the new solvent is followed by a complex mutarotation reaction which apparently consists of the rapid

The

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 a

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 barium 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.