(c) ALLIHN METHOD FOR THE DETERMINATION OF DEXTROSE [14, 15]

Copper sulfate solution.-Dissolve 34.639 g of CuSO4.5H2O in water and dilute to 500 ml.

Alkaline tartrate solution.-Dissolve 173 g of Rochelle salt and 125 g of potassium hydroxide in water and dilute to 500 ml.

Description of method.-Place 30 ml of the copper solution, 30 ml of the alkaline tartrate solution, and 60 ml of water in a beaker, and heat to boiling. Add 25 ml of the sugar solution, which must be so prepared as to contain not more than 0.25 g of dextrose, and boil for exactly 2 minutes, keeping the beaker covered. Filter immediately through asbestos without diluting and obtain the weight of copper by one of the methods described on pages 178-185. The corresponding weight of dextrose is found from the Allihn table (table 79, p. 584). Quisumbing and Thomas found that the maximum amount of copper was reduced at a concentration of caustic alkali of 0.8125 N. Soxhlet's copper mixture is 0.625 N, whereas Allihn's solution is 0.922 N with respect to potassium hydroxide, the calculated normality being referred in each instance to the final reaction mixture containing the sugar. That all three solutions are within the range of high sensitivity is shown by the fact that in spite of varied conditions of analysis they all reduce approximately the same weight of copper. Thus 100 mg of dextrose reduces respectively 201.5, 198, and 195 mg of copper.

(d) QUISUMBING AND THOMAS METHOD

The method of Quisumbing and Thomas [6] was devised after a careful study of the fundamental properties of the reaction had revealed the most favorable conditions for conducting the analysis. In order to obviate the autoreduction of the alkaline copper solution and the reduction of copper by sucrose, and to avoid variations in temperature caused by changes of barometric pressure, the reduction reaction was carried out in a water bath at 80° C. Preliminary experiments had shown that the maximum reduction occurs at an alkalinity of 1.6 N and at a ratio of 5 to 6 of sodium hydroxide to 1 of copper. Fifty milliliters of mixed reagent should thus contain 3.2 g of sodium hydroxide and from 0.525 to 0.630 g of copper. With greater concentrations of copper, the total reduction is decreased, while the autoreduction is increased. The concentration of Rochelle salt employed in the Soxhlet reagent was retained.

This method is capable of producing more accurate results than any other gravimetric method. No reduction of copper is caused by sucrose if the sample does not contain more than 400 mg. In its present form the method is limited to materials of low sucrose content. It should be possible, however, to extend the application of the method to samples of high sucrose content by determining the corrections at higher sucrose concentrations. In view of its independence of barometric pressure, the method is useful at high altitudes.

Copper sulfate solution.-Dissolve 41.2 g of CuSO4.5H2O and dilute to 500 ml. The crystals should be free from dust or partially dehydrated salt.

Alkaline tartrate solution.-Dissolve 173 g of crystallized Rochelle salt in water in a 500-ml flask, and add the calculated amount of sodium hydroxide solution containing 65 g of sodium hydroxide.

903232 O-50 - 13

Fill to a volume of 500 ml. The alkali solution is freed from carbonates in the manner described on page 169.

General procedure.-Measure accurately 25 ml each of the copper sulfate and alkaline tartrate solutions into a 400-ml Pyrex-glass beaker, the diameter of which is about 9 cm. Add 50 ml of sugar solution containing 50 to 150 mg of dextrose, levulose, or invert sugar; or 100 to 300 mg of lactose or maltose. Cover the beaker with a watch glass, and place the beaker in a water bath maintained at 80° C. After digesting for exactly 30 minutes, filter the cuprous oxide by suction through a mat of asbestos in a Gooch crucible. Wash the precipitate in the usual manner and determine the copper by one of the methods described on page 178.

From the weight of copper or cuprous oxide obtained, find the corresponding weight of reducing sugar from table 80, p. 586.

(e) BERTRAND METHOD

This method [16] has been but little used in the United States. It is more widely employed in Europe, more frequently in the pure chemistry of carbohydrates than in commercial analysis.

The reagents are prepared as follows:

(a) 40 g of pure CuSO4.5H2O dissolved to 1 liter.

(b) 200 g of Rochelle salt and 150 g of sodium hydroxide dissolved to 1 liter.

Into a 150-ml Erlenmeyer flask transfer 20 ml each of solutions (a) and (b) and 20 ml of the sugar solution, which should contain not more than 100 mg of reducing sugars. Heat to boiling and keep at gentle ebullition for exactly 3 minutes. Filter through asbestos and determine the reduced copper as follows by Mohr's volumetric permanganate titration. Reference table 81 is given on page 587. Transfer the asbestos film to the beaker, add about 30 ml of hot water, and heat the precipitate and asbestos thoroughly. Rinse the crucible with 50 ml of a hot saturated solution of ferric sulfate in 20-percent sulfuric acid, receiving the rinsings in the beaker containing the precipitate. After the cuprous oxide is dissolved, wash the solution into a large Erlenmeyer flask, and immediately titrate with a standard solution of potassium permanganate. One milliliter of the permanganate solution (0.1573 N) should equal 0.010 g of copper. Standardize the permanganate against pure sodium oxalate.

(f) HERZFELD METHOD FOR DETERMINATION OF LESS THAN 1.5 PERCENT OF INVERT SUGAR IN SUCROSE

Many of the products of the cane- and beet-sugar industries, particularly in the later stages of the processes, contain small residual quantities of invert sugar which cannot be accurately determined by the Meissl and Hiller method. The Herzfeld method [17] is designed to apply to this range of concentrations. It therefore serves to supplement the Meissl and Hiller method (p. 175), which is serviceable at ratios of invert sugar to sucrose greater than 1% percent. In his fundamental research, Herzfeld determined the copper reduced by a 10-g sample of total sugar containing varying quantities of added invert sugar, collecting the precipitated copper in a thick-walled filter tube (12 by 2 cm) containing 1.5 cm of asbestos supported by a platinum cone. The cuprous oxide was burned at low heat to cupric

oxide in order to destroy precipitated organic matter and then reduced to copper in a stream of hydrogen. Herzfeld's procedure, in spite of its early date, thus corresponds to modern procedure. He made triplicate or quadruplicate determinations at seven ratios of invert sugar to sucrose and plotted a smooth curve through the determined points.

The method is well adapted to the determination of reducing sugar in the higher grades of commercial raw sugar. These in general require a preliminary clarification with normal lead acetate. This can be conveniently done by weighing out a 20-g sample, transferring to a 100-ml flask, adding normal lead acetate, making to volume, and filtering. To the filtrate, dust in sufficient dry sodium oxalate to precipitate the excess lead and filter. A volume of 50 ml of the filtrate will contain 10 g of the original sample.

An alternative method [18] is to dissolve 44 g of the sample and transfer to a 200-ml volumetric flask. Clarify with normal lead acetate, make to volume, and filter. Measure 100 ml of the filtrate into a 100- to 110-ml flask, add sufficient sodium sulfate to precipitate the excess lead, and filter. Take for analysis 50 ml of the filtrate.

Add the 50-ml solution thus prepared to 50 ml of mixed Soxhlet solution and heat rapidly to boiling. From the moment when the boiling becomes vigorous continue the boiling for exactly 2 minutes. Then add 100 ml of cold water freed from air by previous boiling and collect the precipitated copper by filtration and thorough washing. Determine the copper by any of the usual methods (p. 178). Refer the weight of copper to table 82.

It was recognized by Herzfeld that the influence of sucrose upon the amount of copper reduced was relatively considerable and variable. Much of the variability was ascribed to the variable degree of superheating of the sugar solution during the period of boiling. Vondrak [19] in a careful study showed that the boiling temperature of the reaction mixture rose to 104° to 105° C, but the addition of talcum or roughened glass beads diminished the boiling temperature to 102° C and, moreover, the analytical results were far more reliable than those obtained by the original method. He sought to improve the procedure by using glass beads, 5 to 6 mm in diameter, which had been roughened by vigorous shaking in a stout bottle for 10 minutes with 2 percent of coarse carborundum (No. 100). These beads can be used repeatedly if after each use they are again shaken with carborundum for 5 minutes.

The analysis is conducted as prescribed by Herzfeld, with the exception that five roughened glass beads are added to the reaction mixture. The reducing action of sucrose is diminished and consequently a revision of Herzfeld's table (table 83, p. 589) was required. Interpolation between adjacent values of copper yields accurate results.

(g) MEISSL AND HILLER METHOD FOR DETERMINING INVERT SUGAR ADMIXED WITH SUCROSE IN ALL PROPORTIONS

At a very early period Meissl recognized the importance of correcting the precipitated copper for the reducing action of sucrose. Meissl and Wein determined the reducing powers of mixtures containing less than 10 percent of invert sugar. In order to extend the applicability of the principle, Hiller [20] determined the reducing action of invert sugar in the presence of widely varying quantities of sucrose and con

structed a table of factors by which the weight of copper should be multiplied in order to yield invert sugar correctly in the presence of all ratios of sucrose to invert sugar up to 99 percent. Hiller's table of factors (p. 589) illustrates well the effect of sucrose. Each column gives the factors (expressed in percent) for a constant weight of invert sugar in the presence of varying quantities of sucrose. In extreme cases an error of nearly 30 percent could be introduced by disregarding the effect of sucrose.

In order to select the proper factor, it is necessary to determine polariscopically the approximate sucrose content of the sample. The direct polarization in the presence of invert sugar is not a correct measure of sucrose but is sufficiently exact for the purpose, except in very low-grade products. If the sucrose is determined by the Clerget method, its percentage should be substituted for P, the direct polarization, in the formulas given below.

The Hiller factors were carefully determined, but not with the rigid specifications which are imposed at the present time. He used Fehling solution, boiled "2 to 3 minutes," and collected the precipitate on a paper filter, ignited, and reduced the oxide by hydrogen to copper. It is not essential that enough solution be taken to precipitate nearly all of the copper, but, in general, the results are more accurate at higher than at lower sugar concentrations.

Prepare a solution of suitable concentration of the material to be examined, clarify with neutral lead acetate, and remove the excess of lead with an alkali oxalate or sodium phosphate. Prepare a series of solutions in large test tubes by adding 1, 2, 3, 4, and 5 ml of this solution to each tube successively. Add 5 ml of mixed Soxhlet reagent to each, heat to boiling, boil 2 minutes, and filter. Note the volume of sugar solution that gives the filtrate lightest in tint but still distinctly blue. Place 20 times this volume of the sugar solution in a 100-ml flask, dilute to the mark, and mix well.

Transfer 50 ml of mixed Soxhlet reagent and 50 ml of the solution to a 250-ml beaker. Heat this mixture at such a rate that a period of approximately 4 minutes is required to bring it to the boiling point, and then boil for exactly 2 minutes. Add 100 ml of cold recently boiled water. Filter immediately through asbestos, and determine the copper by one of the methods described on page 178.

Let

Then

Cu the weight of copper obtained,

P=polarization of the sample (or sucrose by Clerget analysis), W the weight of the sample in the 50 ml of solution used for the determination,

F the factor obtained from Hiller's table.

sugars,

100-R-I, approximate percentage of invert sugar,

Cu F

W

-percentage of invert sugar.

The factor, F, for calculating copper to invert sugar, is found in table 84.

Example: The polarization of a sugar is 86.4, and 50 ml of solution containing 3.256 g of sample gave 0.290 g of copper.

By consulting the table, it will be seen that the vertical column headed 150 is nearest to Z, 145, and the horizontal column headed 95:5 is nearest to the ratio of R to I, 95.1:4.9. Where these columns meet there is found the factor 51.2, which enters into the final calculation:

(h) BROWN, MORRIS, AND MILLAR METHOD FOR REDUCING SUGAR IN MOLASSES [21]

The general method of these authors has been adapted by W. A. Davis to the determination of reducing sugar in cane and beet molasses. The method is extensively used in Great Britain. It is applied to solutions containing 1 g of molasses in 100 ml (not defecated). The Fehling solutions contain 34.639 g of copper sulfate crystals in 500 ml, and 173 g of Rochelle salt and 65 g of sodium hydroxide in 500 ml, respectively. Mix 25 ml of each of the two solutions in a 250-ml beaker of tall lipped form and heat in a gently boiling water bath for 6 minutes. Add 50 ml of the molasses solution (1 g in 100 ml), cover the beaker, and continue the heating for 12 additional minutes. Collect the cuprous oxide in a Gooch crucible, wash with 200 ml of boiling water, and finally with alcohol. Dry and oxidize to copper oxide by placing the Gooch crucible in a larger ordinary crucible in a tilted position, heat gently at first, and finally strongly over a Teclu burner (but not with a blast lamp). Correct the weight of copper oxide for the quantity (usually less than 1 mg) obtained in a blank determination.

For weights of copper oxide less than 0.245 g multiply the weight of copper oxide by the factor 81.5 to obtain directly the percentage of invert sugar in the sample. For weights greater than 0.245 g, use the factors given in table 20.

TABLE 20. Factors for calculating invert sugar from copper oxide