raffinose, and a group of optically active nonsugars consisting mainly of amino acids. The rotation of the latter group they called the N value. The presence of three unknown quantities required three equations for their solution. They found, in agreement with Paine and Balch [45] and with Zerban [46], that the rotation of the nonsugars became zero in strongly acid solution but was restored to its original value by neutralization. If, therefore, the invert polarization was observed in both acid and neutral solution, the difference between the two readings became a measure of N. This fact had previously been stated by Zerban, who observed that the difference between Jackson and Gillis methods II and IV was a measure of the optically active amino compounds, but he did not apply the principle to a quantitative method of analysis.

(1) Procedure.-Transfer 130 g of the sample, or its equivalent, to a 500-ml Kohlrausch flask, add the necessary basic lead acetate, and make to 400 to 450 ml with water. Deaerate under vacuum until all visible gas bubbles are removed, using a few drops of ether or amyl alcohol to break the foam, if necessary. Make to 500 ml at 20° Č, mix, and filter. Delead the filtrate with the minimum of powdered ammonium dihydrogen phosphate, and filter, using a little filter aid if desired. Polarize in a 200-mm tube to obtain the direct polarization, P.

Pipette 50 ml of the deleaded filtrate into each of two 100-ml Kohlrausch flasks. Add 15 ml of water and heat to 68° to 69° C in a 70° C water bath. Remove from bath, and immediately add 10 ml of hydrochloric acid (d20 1.1029). Allow to cool spontaneously for 2 hours, and then cool to 20° C. Make the one invert to 100 ml at 20° C, mix, filter if necessary, and polarize at 20° C in a 400-mm tube, the reading being the invert polarization, I. To the second invert, add 1 or 2 drops of 0.2 percent methyl red indicator solution and neutralize with 6.34 N ammonium hydroxide, adding the ammonia very slowly from a burette while constantly whirling the flask. Then add exactly 1 ml in excess. Make to 100 ml at 20° C, filter if necessary, and polarize in a 400-mm tube to obtain the neutralized invert polarization, I'.

The N value, sucrose, and raffinose are then calculated by the formulas

N=I'—I+K,

P'=P-N,

S=

0.514+ (0.321+0.00009S) 0.835+0.00009S R=0.54 (P'-S),

in which

N=polarizing effect of the optically active nonsugars,
P'true direct polarization of the raffinose and sucrose,
S percentage of sucrose,

R=percentage of raffinose,

K=neutralization correction=0.0047S+0.00017Sv, in which

is the number of milliliters of basic lead (55 Brix) added per 100 ml. It is satisfactory to use P instead of S.

NOTES. The correction, K, is required because the negative rotation of invert sugar is enhanced to a higher negative value upon neutralization with ammonia. It also includes the effect of ammonium acetate, which varies with the volume of

lead acetate added. The numerical values of K are conveniently tabulated in the original article.

Not more than a few drops of amyl alcohol should be added, since it is optically active.

Cover filters during filtration, and discard the first 10 to 15 ml of each filtrate. No more ammonium phosphate than necessary should be used, since 1 g per 100 ml depresses the direct polarization by 0.35°S.

The authors restrict the method to beet products subsequent to the carbonation stage. It proved applicable to such products in a wide geographical territory, centering in Colorado, but not to those from California. It cannot be applied to any mixture containing invert sugar.

The

With the application of the method thus restricted, the authors found excellent agreement between this and the double-enzyme method. It proved inexpensive and well adapted to routine analysis. The average value of Ñ for 28 samples of beet molasses from both Steffen and non-Steffen factories, proved to be -1.62 by the double-acid method, and 1.71 by the double-enzyme method. The average sucrose content agreed within 0.01 percent and raffinose within 0.06 percent. International Commission for Uniform Methods of Sugar Analysis in 1936 found the method too restricted in its application to justify adoption. It is to be noted, however, that the method of Osborn and Zisch is the first successful attempt to find the necessary third equation for the solution of the three unknown quantities in the composition of beet products, even though somewhat restricted in its application.

(f) METHODS OF OTHER NATIONS

Much effort has been devoted to the elimination of the effect of optically active amino acids on the Clerget analysis. These nitrogenous substances exhibit one rotatory power in a neutral or alkaline medium and a quite different one in acid medium. It was, therefore, recognized early that both direct and invert polarizations must be made in the same medium in respect to hydrogen-ion concentration. These efforts have been directed in two ways, namely to read both solutions in neutral solution or both in acid solution.

In the early experiments, Pellet sought to equalize the effect by acidifying the direct polarization with sulfur dioxide. The acidity of such a solution, however, is too weak to affect the rotation of the amino acids to the same degree as the hydrochloric acid of the invert polarization. Andrlik observed the direct polarization in the presence of hydrochloric acid to which urea was added in such quantity as to slow the hydrolysis of sucrose. This interesting expedient has caused much discussion but has not been put into general use.

A more promising method was proposed by Stanek [47], who inverted with hydrochloric acid in the usual way, but upon completion of the inversion added potassium citrate stoichiometrically equivalent to the hydrochloric acid, causing the formation of potassium chloride and citric acid. To the direct polarization was added the same mixture, it being determined that the citric acid, having but 1.72 percent of the inverting power of hydrochloric acid, produced no appreciable effect. upon the sucrose. Stanek found a Clerget divisor of 132.66 at 20° C. The official methods in Czechoslovakia specify the Stanek method, with the rounded-off divisor 132.6 for sucrose determinations in beet molasses.

Babinski and Ablamowicz [48] utilized the Stanek principle, but substituted sodium acetate for potassium citrate. The method was officially adopted in Poland, with a basic value of 131.46 for the divisor. Both Stanek and Babinski clarified the solutions by the addition of saturated (3-percent) bromine water. This is stated to give good clarification and rapid filtration. The precipitate amounts, with molasses, to about 0.1 g and, therefore, its influence on the con

centration of the sample is negligible. The objecton to its use is the disagreeable odor and corrosive effect of free bromine.

In order to make use of the Stanek-Babinsky principle, Schlemmer [49] studied the clarification with Aktivin or Chloramin-T, which reacts with water as follows:

In combination with sodium bromide, the oxygen set free releases two atoms of bromine from the sodium bromide. The released bromine clarifies the solution which, after filtration, remains clear for about 1⁄2 hour. The precipitate from beet molasses weighs about 0.15 g. No odor of bromine is appreciable.

The following solutions are required:

(a) Hydrochloric acid, 18.38 percent; d=1.092.

(b) Sodium acetate, 400 g; and potassium bromide, 50 g in 1 liter. (c) A solution containing about 15 percent of Chloramin-T.

(d) A mixture of solutions (a) and (b) in the ratio 20 ml of (b) to 10 ml. of (a).

Procedure for beet molasses.-Transfer 52 g of molasses to a 200-ml flask and fill to the mark at 20°. Mix thoroughly, and pipette two 50-ml portions to 100-ml flasks. To one add 30 ml of solution (d). Add 10 ml of solution (c). Adjust to 20°, mix, filter, and polarize at 20°. To the other solution add 10 ml of solution (a), invert according to Schrefeld's method (page 129), cool, and add 20 ml of solution (b). Add in 3 portions 10 ml of solution (c), make to volume at 20° C, filter, and polarize at 20°.

Schlemmer determined the value of the divisor for one-half-, onefourth-, and one-eighth-normal solutions of sucrose and, surprisingly enough, found no variation with concentration. He reported the values 131.98 for pure sucrose and 131.75 at 20° for final beet molasses. Apparently no measures are taken in these methods to evaluate the raffinose content of beet products.

Steuerwald [50] devised a method, extensively used in the Dutch East Indies, in which the inversion is carried out at room temperature by hydrochloric acid of such high concentration that the reaction is completed without the attention of the analyst within 2 or 3 hours. The direct polarization is observed in the usual manner.

For the invert polarization, measure 50 ml of the clarified filtrate with a 100-ml flask, and add 30 ml of hydrochloric acid of 1.1 sp gr (acid of 1.188 sp gr diluted with an equal volume of water). Set aside for 3 hours if the temperature is between 20° and 25° C or for 2 hours if above 25°. Dilute the solution to 100 ml and polarize at a carefully observed temperature. Calculate both polarizations in terms of the normal weight of the sample in 100 ml.

Calculate the percentage of sucrose by the formula

Jackson and Gillis [3, p. 168] showed that the high basic value of the Steuerwald divisor was consistent with their own and other values of the divisor if the effect of the acid is considered.

If the sample contains a considerable quantity of invert sugar it would seem probable that the Steuerwald method would yield high results for sucrose, since the effect of the acid is to increase greatly the negative rotation of original invert sugar in the invert polarization. This effect is uncompensated.

5. REFERENCES

[1] M. T. Clerget, Ann. chim. phys. [3] 26, 175 (1849).

[2] C. A. Browne, J. Assn. Official Agr. Chem. 2, 134 (1916).

[3] R. F. Jackson and C. L. Gillis, Sci. Pap. BS 16, 141 (1920) S375.

[4] A. Herzfeld, Z. Ver. deut. Zucker-Ind. 38, 699 (1888).

[5] J. Dammüller, Z. Ver. deut. Zucker-Ind. 38, 746 (1888).

[6] H. S. Walker, Sugar 17, 47 (1915).

[7] L. M. Tolman, Bul. Bur. Chem. 73, 73 (1903).

[8] L. G. L. Steuerwald, Int. Sugar J. 16, 82 (1914).

[9] R. F. Jackson and C. L. Gillis, Z. Ver. deut. Zucker-Ind. 70, 521 (1920).

[10] O. Schrefeld, Z. Ver. deut. Zucker-Ind. 70, 402 (1920).

[11] O. Spengler, K. Zablinsky, and A. Wolf, Z. Wirtschaftsgruppe Zuckerind. 86,

670 (1936).

[12] H. S. Walker, J. Ind. Eng. Chem. 9, 490 (1917).

[13] S. Arrhenius, Z. physik. Chem. 4, 230 (1889).

[14] O. Gubbe, Ber. deut. chem. Ges. 18, 2207 (1885).

[15] R. F. Jackson and E. J. McDonald, J. Assn. Official Agr. Chem. 22, 580 (1939). [16] C. Tuchschmidt, Z. Ver. deut. Zucker-Ind. 20,, 649 (1870).

[17] R. Gillet, Z Ver. deut. Zucker-Ind. 64, 271 (1914).

[18] Official and Tentative Methods of Analysis of the Association Official Agricultural Chemists, 3d ed. (1935).

[19] W. C. Vosburgh, J. Am. Chem. Soc. 43, 219 (1921).

[20] F. W. Zerban, J. Assn. Official Agr. Chem. 8, 384 (1925); 11, 167 (1928). [21] E. von Lippmann, Die Chemie der Zuckerarten, 11, 1188, (Vieweg u. Sohn, Braunschweig, 1904).

[22] R. F. Jackson and C. L. Gillis, Louisiana Planter 66, 380 (1921); Facts About Sugar 13, 10 (1921); Int. Sugar J. 23, 445 (1921).

[23] C. A. Browne, Louisiana Planter 66, 171 (1921); Facts About Sugar 12, 230 (1921).

[24] R. J. Brown, Ind. Eng. Chem. 17, 39 (1925).

[25] C. A. Browne, J. Assn. Official Agr. Chem. 2, 138 (1916).

[26] E. Saillard, Eighth Int. Cong. Applied Chem. Communication 25, 541 (1912). [27] E. Saillard, J. fab. sucre (May 22, 1912 and July 1, 1914); Z. Ver. deut. Zucker-Ind. 64, 841 (1914).

[28] F. W. Zerban and C. A. Gamble, Ind. Eng. Chem., Anal. Ed. 5, 34 (1933). [29] R. Creydt, Z. Ver. deut. Zucker-Ind. 37, 153 (1887).

[30] C. A. Browne and C. A. Gamble, J. Ind. Eng. Chem. 13, 793 (1921). [31] S. J. Osborn and J. H. Zisch, Ind. Eng. Chem., Anal. Ed. 6, 193 (1934). [32] F. W. Zerban and C. A. Gamble, Ind. Eng. Chem., Anal. Ed. 5, 34 (1933). [33] F. W. Zerban, J. Assn. Official Agr. Chem. 8, 384 (1925); 9, 166 (1926); 10, 183 (1927); 11, 167 (1928); 12, 158 (1929); 13, 188 (1930); 14, 172 (1931). [34] F. W. Zerban, Orig. Com. Eighth Int. Cong. Applied Chem. 8, 103 (1912). [35] J. A. Ambler, Int. Sugar J. 29, 439 (1927).

[36] C. A. Browne and R. E. Blouin, Louisiana Sugar Expt. Sta. Bul. 91, 93 (1907). [37] C. S. Hudson and T. S. Harding, J. Ind. Eng. Chem. 7, 2193 (1915).

[38] F. W. Reynolds, Ind. Eng. Chem. 16, 169 (1924).

[39] L. Michaelis and H. Davidsohn, Biochem. Z. 35, 386 (1911).

[40] R. Willstätter and R. Kuhn, Ber. deut. chem. Ges. 56, 509 (1923).

[41] M. Adams and C. S. Hudson, J. Am. Chem. Soc. 60, 982 (1938).

[42] N. K. Richtmyer and C. S. Hudson, J. Am. Chem. Soc. 60, 983 (1938).

[43] H. S. Paine and R. T. Balch, J. Am. Chem. Soc. 49, 1019 (1927).

[44] F. W. Zerban, J. Am. Chem. Soc. 47, 1104 (1925).

[45] H. S. Paine and R. T. Balch, J. Ind. Eng. Chem. 17, 240 (1925).

[46] F. W. Zerban, J. Assn. Official Agr. Chem. 12, 158 (1929).

[47] V. Stanek, Z. Zuckerind. Böhmen 38, 429 (1914); Int. Sugar J. 16, 387 (1914).

[48] J. Babinski and W. Ablomowicz, Gez. Ceur. 1914-15, T44, S10, 147.

[49] J. Schlemmer, Z. Zuckerind. čechoslovak. Rep. 53, 13 (1928).

[50] L. Steuerwald, Arch. Suikerind. 21, 831 (1913). Int. Sugar J. 15, 489 (1913).

IX. CHEMICAL METHODS FOR THE DETERMINATION OF REDUCING SUGARS

1. THEORETICAL AND GENERAL

(a) INTRODUCTION

The history of the growth of reducing-sugar analysis begins in 1815, when Vogel showed that the reddish precipitate produced by boiling copper acetate with honey was not metallic copper, as had previously been supposed, but was cuprous oxide. From this small beginning the development was slow, with the major steps in progress decades apart. In 1841 Trommer found that, by making the copper solution alkaline, not only was a differentiation of sugars made possible, but the sensitivity was increased. In 1838 a French society offered a prize for a successful method of quantitative estimation of sugar, and an award of a portion of the prize was made in 1844 to Barreswil, who adapted Trommer's qualitative method to a quantitative method of analysis. He also showed that cane sugar could be determined by observing its reducing power before and after inversion.

In 1849 H. Fehling [1] worked out with great care the details of the method, giving some account of the stoichiometrical equivalents. Fehling believed that one molecule of glucose reduced five equivalents of copper, not recognizing that the reaction is quantitative only within narrow limits of concentration and time of reaction. Fehling's method proved satisfactory in respect to sensitivity and reproducibility of analysis, but the copper solution was unstable.

Soxhlet [2] effected still further improvements, utilizing the same reagents in the same proportions as Fehling, but preserving the copper solution and the alkaline tartrate solution in separate containers until required for analysis. This solution and method have been utilized up to the present day.

(b) REDUCING SUGARS IN ALKALINE SOLUTION

When glucose, levulose, or mannose is subjected to the action of dilute alkali in aqueous solution, the three sugars undergo a mutual conversion into each other until an equilibrium is established. The composition of this mixture is the same regardless of the sugar taken as the starting material. These relations were shown in a very striking manner by Lobry de Bruyn and van Ekenstein [3], who applied the same conditions to other sugars and found equilibria between galactose, talose, and tagatose, and in many other systems. This reaction is a perfectly general one and is of practical value for the conversion of readily available sugars into new sugars or into sugars of less common occurrence.

The mechanism of the reaction has been studied extensively. Nef [4] showed that a hexose in alkaline solution was converted into a 1,2-enediol (I). He accounted for the final products by assuming that, to this enediol, water can be added in three different ways: The hydroxyl may attach to the terminal carbon atom, yielding the aldehyde group; the hydrogen attaching to the second carbon atom

The reactions occurring in alkaline solution, and briefly indicated in this and succeeding paragraphs, are too involved to describe in detail. The purpose here is to show the nature of the reactions rather than their accurate course.