Until further investigations are made, it is to be recommended that the temperature interval 20° to 70° C be employed with the coefficient 0.03441, that is, 1 g of levulose in 100 ml of solution in a 200-mm column diminishes 0.03441° S for each degree rise of temperature. The mean expansion coefficient between these temperature limits is, according to Jackson and Mathews, 0.00044. Thus if the higher temperature is exactly 70°, the observed polarization must be multiplied by 1.022 before subtracting from the polarization at 20° C. The decrease in the corrected polarization divided by 0.03441 yields the number of grams of levulose in 100 ml of solution.

(d) GALACTOSE BY MUCIC ACID PRECIPITATION

Galactose is oxidized by nitric acid to yield about 75 percent of mucic acid. Under closely specified conditions of analysis, the quantity of recovered mucic acid is reproducible and can be related empirically to the quantity of galactose in the sample. The method is applicable to free galactose or to the combined galactose in compound sugars or in galactans. Certain glycosides containing galactose, for example, saponins, yield insoluble products upon hydrolysis. Such glycosides must first be hydrolyzed with sulfuric acid (2 to 5 percent) and the insoluble material separated by filtration [8].

van der Haar [8] has given detailed specifications for the analytical procedure. Transfer the weighed sample containing galactose to a beaker (12 cm in height and about 60 mm in diameter) and add sufficient sucrose to increase the weight of total sugar to 1.000 g. Add 60 ml of nitric acid (sp gr 1.15 at 15° C) and place the beaker in an inclined position in a boiling-water bath and with repeated agitation allow it to remain until the weight of the contents has diminished to somewhat less than 20 g (that is, 19.8 to 20). Cool, and add water to make the weight exactly 20 g. Add 500 mg of pure, dry mucic acid and allow to stand for 48 hours at approximately 15° C, during which time stir occasionally. During the last few hours, adjust the temperature to exactly 15° C. Filter the precipitated mucic acid with suction on a weighed Gooch crucible prepared with asbestos which has previously been treated with nitric acid. Wash the precipitate four times with 5 ml of a solution of mucic acid saturated at 15° C and finally with 5 ml of water. Dry the precipitate at 100° C to constant weight. Deduct 500 mg from the weight of the precipitate and refer the result to column 3 of table 103, p. 608.

Acree [9] states that the oxidation of galactose by nitric acid is accelerated by the oxides of nitrogen, hence if the nitric acid is too pure it is preferable to add a small quantity of nitrous acid or an alkali nitrite.

(e) DETERMINATION OF. MANNOSE AS PHENYLHYDRAZONE

While all reducing sugars are capable of forming hydrazones, the hydrazone of mannose is particularly insoluble and thus is suitable for its quantitative estimation. Bourquelot and Hérissey [10] prescribe the conditions of analysis. About 1 g of mannose dissolved in 16.6 ml of water is treated with a solution of 1.2 ml of phenylhydrazin and 1.2 ml of glacial acetic acid made up to 6 ml with water, and allowed to stand for 8 hours at a temperature not above 10° C. The hydrazone is collected on a Gooch crucible and washed with 15 ml

of ice water, 10 ml of absolute alcohol, and 10 ml of ether. The precipitate is dried in a vacuum over sulfuric acid. One gram of mannose yields theoretically 1.5 g of phenylhydrazone.

The hydrazone is soluble to the extent of 40 mg in 100 ml of solution and a small correction for this solubility increases the precision of analysis.

Pellet [11] has found the method suitable for the estimation of small amounts of mannose in cane molasses.

(f) DETERMINATION OF ARABINOSE AS DIPHENYLHYDRAZONE

Neuberg and Wohlgemuth [12] have made use of the high insolubility of arabinose diphenylhydrazone for estimating arabinose in the presence of other monosaccharides. Mannose or fucose in excessive quantities are apparently the only sugars which interfere with the selectivity of the analysis. The authors illustrate the method by the following example.

A solution (100 ml) containing dextrose, fructose, xylose, glucuronic acid, and 1.0066 g of arabinose was evaporated to 30 ml. The resulting solution was heated on a water bath for hour with 6 g of diphenylhydrazine in 50 ml of 96-percent alcohol with additions of very dilute alcohol or preferably with a reflux condenser. The solution was cooled and allowed to stand for 24 hours. The precipitated hydrazone was collected on a Gooch crucible, washed with 50 ml of 50-percent alcohol, dried, and weighed. Yield, 2.1143 g of hydrazone, equivalent to 1.0035 g of arabinose. Factor, 0. 4747.

(8) DETERMINATION OF URONIC ACIDS

The uronic acids, glucuronic and galacturonic, are widely distributed in both plants and animals. They play an important role in the carbohydrate metabolism of the cell wall. Dickson, Otterson, and Link [13] have found that free glucuronic acid is present within the cell of corn seedlings and that a polymerized glucuronic acid sometimes associated with the cellulose, comprises part of the pectinaceous substance of the cell and cell wall. Nanji, Patin, and Ling [14] found that purified pectin preparations contained from 70 to 73 percent of uronic acid anhydride. Browne and Phillips [15] showed that uronic acids comprised about 3 percent of sugar-cane bagasse and that sugar-cane juice contained from 0.1 to 0.6 percent (based on ash-free solids) of uronic acids, the variations depending upon the methods of maceration. In cane molasses the uronic acids were found concentrated to an average of about 2 percent. These authors believed that the uronic acids are derived from pectins which are extracted with the juice.

When a uronic acid is heated with hydrochloric acid, decarboxylation occurs with the formation of furfural and carbon dioxide according to the equation

C&H1007=CH2O2+CO2+3H2O.

The yield of furfural is less than the theoretical, while that of carbon dioxide is quantitative. In the absence of other reactions yielding carbon dioxide, a measure of the gas evolved serves for the quantitative determination of uronic acid.

Whistler, Martin, and Harris [16] in a study of the determination of uronic acids in cellulosic materials, found that under the drastic conditions employed in the analysis, carbohydrates free from uronic acids were slowly but regularly decomposed with the formation of carbon dioxide, necessitating the application of a correction.

The method of determination was originally devised by Lefèvre and Tollens [17]. Dickson, Otterson, and Link [13] further elaborated the method, measuring the carbon dioxide by absorption in barium hydroxide and titration of unreacted alkali.

The details of the method described here are those of Whistler, Martin, and Harris [16], who adapted the procedure specifically to the determination of uronic-acid groups in cellulosic materials. Their procedure can, however, be used without modification for any other material by selecting a weight of sample which will yield 30 to 40 mg of carbon dioxide.

FIGURE 39 (a).-Apparatus for determination of the rate of evolution of carbon dioxide from uronic acids or materials containing uronic acids during treatment with hydrochloric acid.

The apparatus is not drawn to scale. See original article for dimensions.

The apparatus is shown in figure 39 (a). Nitrogen, which is used as the carrier gas for the evolved carbon dioxide, enters the apparatus through an empty safety bottle, A. It next passes through an alkaline solution of pyrogallol, B. The inlet tube in this bottle is drawn out to a small orifice, which produces very fine bubbles. From B the gas passes through two absorption towers, C, filled with soda lime, into a second safety bottle, D, which is provided with a mercury manometer, E, and then enters the reaction flask, F, by way of a side arm whose outlet is 10 to 15 mm above the surface of the liquid in the flask. The size of the flask depends upon the type of material and the size of the sample to be analyzed. In most experiments a 500-ml reaction flask is suitable. From the reaction flask the gas passes through a 40-cm reflux condenser, G, and into a bubbling tower, H, containing approximately 60 ml of concentrated sulfuric acid. The sulfuric acid serves to remove interfering decomposition products which are carried over from the reaction flask. The gas next passes through the U-tube, I, which is filled with anhydrous copper sulfate to remove chlorine or hydrogen sulfide, then through the tube, J,

which contains phosphorus pentoxide, and finally through the carbon dioxide-absorption tube, K, containing Ascarite, backed by phosphorus pentoxide. The absorption tubes, K, are connected into the train by means of mercury-cup seals [18]. This type of connection makes possible a rapid exchange of the absorption tubes. Tube K is protected by a soda-lime tube, L, which is followed by a calibrated flowmeter, M, for estimating the rate of flow of nitrogen through the apparatus.

The reaction flask is immersed in a vessel containing about 16 liters of hydrogenated cottonseed oil. A bath temperature of 130° C was found to be optimum for maintaining a steady but gentle boiling of the reaction mixture. The bath is brought to the operating temperature by means of two electric immersion heaters, one of 500 and one of 1,000 watts. When the desired temperature is reached, the 500-watt heater alone is sufficient to maintain thermal constancy within ±0.2° C. The time required to raise the temperature of the bath to 130° C is approximately 50 minutes.

The flask is placed in position in the oil bath so that the oil level is 3 to 4 mm lower than the liquid level inside the flask. This precaution is taken to prevent the baking of small bits of sample which may be splashed against the sides of the flask. Nitrogen, at the rate of about 10 liters per hour, is passed through the apparatus until the Ascarite tube, K, shows no further gain in weight. This operation requires about 30 minutes, during which time the temperature of the oil bath is slowly raised to 50° C. When the apparatus is free of carbon dioxide, both heating units are turned on and the temperature is brought to 130° C. This procedure is always carefully followed in order to assure the same preliminary heating for all samples. The point of zero time is taken as the time at which the bath reaches 130° C. At that time the Ascarite tube, K, is removed for weighing and a second weighed Ascarite tube inserted in its place. At the end of 1 hour the second tube is removed for weighing and replaced by the first. This process is repeated at intervals of 1 hour for the duration of the analysis. When analyses are made of pure uronic acids or materials rich in uronic acids, a small amount of carbon dioxide is evolved by the time the temperature of the bath reaches 130° C. In these cases, this amount is measured and added to that evolved during the first hour.

Since the rate of evolution of carbon dioxide is appreciably affected by variations in acid strength, it is essential that the same concentration (within 0.02 percent) of hydrochloric acid be used in all analyses. The acid should be accurately 12 percent, or 3.290 N.

To determine the correction for the carbon dioxide evolved by decomposition of carbohydrates other than uronic acids, weigh the Ascarite containers hourly until the rate of increase in weight is constant. The constant rate of evolution indicates that the carbon dioxide is being derived solely from the uronic acid-free carbohydrates. Calculate from the determined increase per hour the total weight of carbon dioxide which was evolved during the period (3 to 5 hours) before the rate became constant, and deduct the computed weight from the total evolved during this period. The weight of carbon dioxide times 4.00 equals the weight of uronic acid anhydride.

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3.

DETERMINATION OF TWO SUGARS IN A MIXTURE

(a) TWO SUGARS BY COMBINATION OF TWO POLARIMETRIC EQUATIONS

In many instances it is possible to polarize a sugar mixture under .conditions sufficiently different to emphasize some striking difference in the properties of the two sugars. The variation in specific rotation of the two individuals under the varied conditions must be known in order to substitute in the two corresponding equations. If x and y are the respective percentages of each sugar in the mixture, a and a' the known specific rotations of one of the sugars under the two varied conditions, and b and b' those of the second sugar,

in which [a], and [a]' are determined experimentally. The specific rotations can obviously be replaced by the saccharimetric constants. While it is possible theoretically to determine both constituents of a mixture by the procedure outlined, the method is most frequently used to determine one constituent selectively in the presence of an optically active impurity. Thus the Clerget method, which has been described in detail, is employed for the determination of sucrose in the presence of invert sugar. Theoretically, it should be possible to calculate the invert sugar also, but it is at present difficult to assign with confidence a definite value to the specific rotation of invert sugar, since our knowledge of the partial rotatory powers of the constituents of sugar mixtures is incomplete.

The principle of the method is used in the determination of mixtures of sucrose and raffinose by the Creydt raffinose formula, which would yield exact results for both constituents but for the complication that usually a third group of optically active substances, namely aminoacids, contaminates the product which is subjected to analysis.

Other examples of the use of two polarimetric equations have been cited in the description of the determination of levulose and invert sugar by polarization at two temperatures. In some instances the second constituent of the mixture can be determined by calculation from the residual rotation obtained by deduction of the rotation of the determined constituent from the observed rotation.

(b) TWO SUGARS BY COMBINED POLARISCOPIC AND REDUCTION EQUATIONS

(1) BROWNE FORMULAS.- A thorough study of the determination of two sugars in a mixture by a combination of polariscopic and reducing-power methods has been made by Browne [2] and [3, page 475]. Reducing sugars are determined by the Allihn method and polarizations, observed in a 200-mm column, are stated in terms of Ventzke sugar degrees. Browne showed that by the Allihn method the reducing power of a sugar mixture is a strictly additive property of the constituents. The assumption is made tacitly that the polarizing power is also additive.

If the reducing ratio of sugar A to dextrose is a and of sugar B is b, then in a mixture of a percent of A and y percent of B, the combined influence is

ax+by=R,

in which R is the percentage of total reducing sugars determined as dextrose.