red indicator, pH 4.4; then to another 50 ml of the lead-free solution in a 100-ml volumetric flask, add the requisite quantity of acid and 5 ml of the invertase preparation, fill the flask with water nearly to 100 ml, and let stand overnight (preferably at a temperature not less than 20° C). Cool, and dilute to 100 ml at 20° C. Mix well and polarize at 20° C in a 200-mm tube. If there is doubt as to the completion of the hydrolysis, allow a portion of the solution to remain for several hours and again polarize. If there is no change from the previous reading, the inversion is complete. Carefully note the reading and temperature of the solution. If it is necessary to work at a temperature other than 20° C, which is permissible within narrow limits, complete the volumes and make both direct and invert readings at the same temperature. Correct the polarization for the optical activity of the invertase solution and multiply by 2. Calculate the percentage of sucrose by the formula in which S= 100 (P-I) 142.1+0.073 (m—13) — t/2' S percentage of sucrose, P direct reading, normal solution, I invert reading, normal solution, t=temperature at which readings are made, m=g of total solids from original sample in 100 ml of the invert solution. Determine with the refractometer the total solids as a percentage by weight, as directed on page 258, and multiply this figure by the density at 20° C, as obtained from table 113, p. 626. Rapid inversion at 55° to 60° C.-If more rapid inversion is desired, proceed as follows: Prepare the sample as directed under "direct reading," p. 157, and to 50 ml of the lead-free filtrate in a 100 ml volumetric flask add glacial acetic acid in sufficient quantity to render the solution distinctly acid to methyl red, pH 4.4. The quantity of acetic acid required should be determined before pipetting the 50-ml portion, as described in the preceding paragraph. Then add 10 ml of invertase solution, mix thoroughly, place the flask in a water bath at 55° to 60° C, and allow to stand at that temperature for 15 minutes with occasional shaking. Cool, add sodium carbonate until distinctly alkaline to litmus paper, dilute to 100 ml at 20° C, mix well, and determine the polarization at 20° C in a 200-mm tube. Allow the solution to remain in the tube for 10 minutes and again determine the polarization. If there is no change from the previous reading, the mutarotation is complete. Carefully note the reading and the temperature of the solution. Correct the polarization for the optical activity of the invertase solution and multiply by 2. Calculate the percentage of sucrose by the formula given above. If the solution has been rendered so alkaline as to cause destruction of sugar, the polarization, if negative, will in general decrease, since the decomposition of fructose ordinarily is more rapid than that of the other sugars present. If the solution has not been made sufficiently alkaline to complete mutarotation quickly, the polarization, if negative, will in general increase. As the analyst gains experience he may omit the polarization after 10 minutes, if he has satisfied himself that he is adding sodium carbonate in sufficient amount to complete mutarotation at once without causing any destruction of sugar during the period intervening before polarization. (2) SUCROSE AND RAFFINOSE BY POLARIZATION BEFORE AND AFTER TREATMENT WITH TWO ENZYME PREPARATIONS. Invertase solution (top-yeast extract).-Prepare as directed on page 147. This solution should be free from the enzyme melibiase. Its invertase activity should be at least as great as that used for the determination of sucrose in the absence of raffinose. Invertase-melibiase solution (bottom-yeast extract).-Prepare as directed on page 147, using bottom fermenting yeast (brewers' yeast) instead of bakers' yeast. The invertase activity should be at least as great as that from the top-yeast extract. Test the melibiase activity of the solution as follows. Add 2 ml of the solution to be tested to 20 ml of a weakly acid melibiose solution polarizing +20.0° S and allow to stand 30 minutes at about 20° C. Then add sufficient sodium carbonate to render the solution slightly alkaline to litmus paper. A preparation suitable for the overnight hydrolysis of solutions containing not more than 0.2 g of raffinose in 100 ml should hydrolyze 35 percent of the melibiose present under the conditions mentioned; a preparation suitable for the overnight hydrolysis of solutions containing not more than 0.65 g of raffinose in 100 ml should produce 50-percent hydrolysis of melibiose; and a preparation suitable for the overnight hydrolysis of solutions containing 0.65 to 1.3 g of raffinose in 100 ml should hydrolyze at least 70 percent of the melibiose present under the above condition. The polarizations that correspond to 35-, 50-, and 70-percent hydrolysis of a melibiose solution polarizing, before hydrolysis, +20° are +16.4°, +14.9°, and +12.9° S, respectively. Determination. In the analysis of sugar-beet products, weigh the quantity of material specified in table 19, transfer to a 300-ml volumetric flask, add the quantity of basic lead acetate solution indicated in the table, and dilute to volume at 20° C. Mix thoroughly and filter through fluted paper in a closely covered funnel, rejecting the first 25 ml of filtrate. When sufficient filtrate has collected, remove the lead from the solution by adding ammonium acid phosphate in as small excess as possible (see table 19). This condition is readily determined, after a little practice, by the appearance of the lead phosphate precipitate, which usually flocculates and settles rapidly in the presence of a slight excess of the salt. Mix well and filter, again rejecting at least the first 25 ml of the filtrate. Make a direct polarization in a 200-mm tube at 20° C, unless the solution contains an appreciable quantity of invert sugar, in which case pipette a 50-ml portion of the lead-free filtrate into a 100-ml flask, dilute with water to the mark, mix well, and polarize at 20° C, preferably in a 400-mm tube. This reading, calculated to the normal weight of 26 g in 100 ml and 200-mm tube length, is the direct reading (P) of the formula given in table 19 for polarization before inversion. Transfer two 50-ml portions of the lead-free filtrate to 100-ml flasks. To one add 5 ml of invertase solution (top-yeast extract), page 147, and to the other add 5 ml of invertase-melibiase solution (bottom-yeast extract), page 147, let stand overnight at atmospheric temperature (preferably not below 20° C), dilute to volume, mix well, and polarize at 20° C, preferably in a 400-mm jacketed tube. If a TABLE 19.-Quantity of sample and reagents required for clarification and deleading of beet sugar-house products Neutralize with acetic acid before adding basic lead acetate. Lime in solution will be precipitated partly by the phosphate, and it is necessary to add sufficient phosphate to complete the precipitation of both the lead and lime salts; hence no definite quantity can be specified. rapid hydrolysis is desired, add 10 ml of each of the enzyme solutions to the 50-ml portions of deleaded filtrate in 100-ml flasks, and place in a water bath at 50° to 55° C for 40 minutes. Then add sodium carbonate until the solution is slightly alkaline to litmus paper, dilute to volume at 20° C, mix well, and polarize at 20° C, preferably in a 400-mm tube. Correct the invert readings for the optical activity of the enzyme solution, and calculate the polarization to that of a normal-weight solution of 26 g in 100 ml; also calculate the reading to a 200-mm tube length, if necessary. Calculate the percentages of anhydrous raffinose and sucrose from the formula R=1.354 (A-B), S= in which (P−2.202A+1.202B) 100 132.12-0.00718[132.12—(P−2.202A+1.202B)]' R=percentage of raffinose, S percentage of sucrose, P=direct polarization, normal solution, A corrected polarization after top-yeast hydrolysis, normal solution, B=corrected polarization after bottom-yeast hydrolysis, normal solution. A and B are treated algebraically. (e) DOUBLE-ACID METHOD OF OSBORN AND ZISCH Osborn and Zisch [31], finding the double-enzyme method reliable in the analysis of beet products, but impracticable because of the expense of the enzymes and the difficulty of obtaining satisfactory preparations, sought to modify the methods of acid hydrolysis in such manner as to make them suitable for routine analysis. They pointed out that the direct polarization is the resultant rotation of sucrose, 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=1'-I+K, P'=P-N, S= 0.514+ (0.321+0.00009.S) 0.835+0.00009S R=0.54 (P'-S), in which N-polarizing effect of the optically active nonsugars, 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. 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. The 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 |