(c) FERRICYANIDE MICROMETHOD OF HAGEDORN AND JENSEN

Hagedorn and Jensen [48] devised a method particularly for the determination of blood sugar, but which is capable of extension to the analysis of any material containing minute quantities of reducing sugar. The maximum weight of dextrose which can be determined is 0.385 mg.

When ferricyanide in alkaline solution is heated with a reducing sugar it is reduced to ferrocyanide, the amount of reduction being a measure of the amount of sugar taken. The quantity of reduced ferricyanide is determined as the difference between the total and that remaining after the reduction reaction. The determination depends upon the reaction

2H,Fe(CN).+2HI=2H,Fe(CN)6+I2,

which is reversible but can be made to run quantitatively from left to right by the precipitation of ferrocyanide as a zinc complex:

2K,Fe(CN).+3ZnSO,=K2Zn3[Fe(CN)8]2+3K2SO4.

The liberated iodine is then titrated with standard thiosulfate. The Hagedorn-Jensen method has been used extensively in blood analysis. It is introduced into this circular because of its probable general applicability to other materials than blood. It has the great advantage that ferrocyanide is stable in air and no back oxidation occurs. Miller and Van Slyke [49] point out the disadvantage of the determination of ferrocyanide by difference, and they describe a method of direct titration with ceric sulfate, the end point being determined in the presence of the indicator, Setopaline C (a trade name). However, ceric sulfate gave uncertain results with fructose, which curtails the general applicability of the modification. Shaffer and Williams [50] have described a modification in which the amount of reduction is determined by measuring the potential of the ferriferrocyanide electrode. This is extremely rapid and convenient, but is to be recommended only if the number of analyses is great enough to justify the labor of assembling the necessary apparatus. Van Slyke and Hawkins [51], using the Van Slyke-Neil apparatus [52], determined unreduced ferricyanide gasometrically by measuring the pressure of nitrogen evolved by the reaction

4K,Fe(CN),+N2H1+4NaOH=4K,NaFe(CN)。+4H2O+N2 and have shown that the method can be used rapidly and conveniently for the determination of sugar in both blood and urine. Folin [53] determined the ferrocyanide by a colorimetric measurement of the prussian blue found by addition of ferric chloride to the reaction mixture after completion of the reduction. He also described an effective method for the purification of ferricyanide. Hawkins [54], using the gasometric method, found that the reduction was proportional to concentration for all sugars analyzed except fructose, arabinose, and xylose, in which instances it was proportional up to 0.1-mg concentration. The relative reducing powers are glucose, 1.00; mannose, 1.014; galactose, 0.792; fructose, 0.986; arabinose, 0.949; xylose, 1.019; maltose, 0.725; and lactose, 0.726.

Reagents. (1) Dissolve 1.65 g of K,Fe(CN)., recrystallized and dried at 50° C, and 10.6 g of anhydrous Na2CO3. Fill to 1 liter and preserve in the dark.

(2) ZnSO4.7H2O, 10 g; NaCl, 50 g; and water to 200 ml. Immediately before use, add solid KI in the proportion of 2.5 g per 100 ml to the volume of solution required for the analyses. If added to the stock solution, a separation of iodine occurs with lapse of time. (3) Acetic acid, 3 ml of glacial acid in 100 ml.

(4) One gram of soluble starch in 100 ml of saturated NaCl solution. (5) Sodium thiosulfate 0.005 N. Dissolve 0.7 g of crystals and make to 500 ml. Standardize frequently by titration against 0.005 N KIO, (0.3566 g in 2 liters), adding for each 10 ml of iodate solution about 20 mg of KI and a few milliliters of dilute HCl.

Procedure.-Into large test tubes (30 by 90 mm) transfer a sugar solution containing less than 0.38 mg of dextrose, and add water to make a volume of 12 ml. Add accurately 2 ml of the alkaline ferricyanide solution and heat in a boiling-water bath for 15 minutes. Cool and add 3 ml of the potassium-iodide-zinc-sulfate solution and 2 ml of acetic acid. Titrate the liberated iodine, using a microburette and 2 drops of starch indicator.

Conduct a blank determination in the absence of sugar. Refer to table 99, p. 604.

Example.-A sample of sugar required 1.26 ml of thiosulfate, and the blank, 1.86 ml; 2.00 ml of standard iodate required 1.90 ml of thiosulfate. Each titer is multiplied by 2.00/1.90 to reduce it to a 0.005 N basis. Corrected titers are, respectively, 1.33 and 1.95 ml. In table 99, 1.33 ml is equivalent to 0.119 mg, and 1.95 ml to 0.008 mg of dextrose. Therefore, the corrected sugar content is 0.111 mg.

6. COLORIMETRIC AND VISUAL METHODS

(a) POT METHOD OF MAIN FOR DETERMINATION OF REDUCING SUGARS IN RAW SUGARS AND SIMILAR PRODUCTS [55]

Main has devised a visual method for the determination of reducing sugars, which is capable of yielding results of high precision and of extending the range of quantitative estimation to very low percentages. The reaction is carried out in large resistant-glass test tubes, 150-mm length by 38-mm internal diameter and weighing 50 to 55 g. In order to avoid back oxidation by air, a series of floats is constructed of similar test tubes having slightly smaller diameter and which make a sliding fit into the others. The barrels of these floats are 100 mm long, and the upper end of each is drawn out to a taper, making a total length of about 170 mm.

The water bath is an ordinary oval iron kitchen pot, tinned inside, the capacity of which is 3 gallons. An overflow is fitted near the upper edge of the boiler, and hot water is added continuously through a "sight feed" to replace loss by evaporation. The temperature of the water must be maintained at the boiling point, for which a large ring gas burner is necessary. While in the water bath, the tubes are supported symmetrically by clips in a carrier.

Two alkaline copper reagents are employed: solution I, for concentrations of invert sugar extending up to 16 percent; solution II, for samples containing a maximum of 0.832 percent.

Solution I.-The usual Soxhlet modification of Fehling solution (p. 170). 10 ml of the mixed reagents are used for the analysis. The results are referred to in table 100, p. 605.

Solution II-A Soxhlet solution (A) as usual, containing 34.639 g of CuSO4.5H2O in 500 ml of Soxhlet solution; (B) 173 g of Rochelle

salt, 50 g of NaOH, and 14.647 g of K4Fe(CN), in 500 ml of solution. For solution II, mix 1 volume of (A), 1 volume of (B), and 2 volumes of 5 N NaOH. This mixed reagent was designated "L. F. S.", and the results are referred to in table 101.

In the extra-alkaline L.F.S. solution, the ferrocyanide, which is present in the ratio of 1 mole to 4 moles of cupric sulfate, has the function of combining with the reduced copper to form cuprous ferrocyanide and obviates the red coloration that tends to mask the end point as indicated by the methylene blue.

For the standardization of copper solution I, invert sugar is prepared according to the method of Lane and Eynon [36]. Prepare a standard invert-sugar solution by dissolving 9.5 g of pure sucrose, accurately weighed, in about 80 ml of water, adding 5.3 ml of hydrochloric acid (sp gr 1.16, or approximately 10 N), and completing the volume to approximately 100 ml. Allow this solution to stand at 22° to 25° C for 3 or 4 days and dilute to 1 liter. From this stock solution, pipette 50 ml into a 250-ml flask, neutralize with sodium hydroxide (approximately 2.5 ml of N NaOH), and complete to volume. This solution now contains 0.002 g of invert sugar per milliliter.

Transfer the following solutions to each of three tubes in the order stated: 10 ml of mixed Soxhlet solution (solution I); standard neutralized invert-sugar solution, 24.5, 25.0, and 25.5 ml, respectively; and 2 drops of 1-percent methylene blue.

Mix the contents of each tube by gentle rotation and insert the floats so that they rest on the liquid, care being taken not to entrap any air bubbles. Place the tubes in the carrier and immerse in the briskly boiling water for exactly 5 minutes. Then remove and inspect. In general, one of the three solutions will show complete reduction of copper, while the adjacent one will show a trace of blue. The volume of invert intermediate between these two is taken as equivalent to 10 ml of Soxhlet solution. The precision of standardization and also of analysis can be greatly increased by lessening the intervals between the volumes in the tubes. The mean between the last blue and the first red is always taken as the true result, unless the blue color is actually seen to fade in a tube on removing it from the pot at the end of the 5 minutes. In such case, the actual volume in that tube is taken as the correct figure.

As in other methods, some preliminary idea of the amount of invert sugar present in the sample must be ascertained by using a rough incremental titration or other guide.

For the estimation of small percentages of invert sugar, that is, less than 0.8 percent, L. F. S. solution (solution II) may be used. The table upon which the use of this solution is based overlaps table 100 for solution I, since the latter permits the estimation down to about 0.3 percent. The L. F. S. solution should be standardized against an invert-sugar solution containing 0.025 g per 100 ml; 37 ml of such a solution should decolorize 4 ml of L. F. S. in the presence of 2 drops of methylene blue.

The time of heating for amounts of invert sugar below 0.01 percent must be increased to 10 minutes, as shown in table 101, p. 606.

(b) DE WHALLEY METHOD FOR THE DETERMINATION OF INVERT SUGAR IN REFINED WHITE SUGARS [56]

Range, 0.001 to 0.015 percent, capable of extension to higher percentages by dilution with pure sucrose.

Select test tubes of white glass 6 by % inches having a uniform weight of about 9.4 to 9.6 g. Fit large rubber rings about the tops in order to support them in the water bath. The bath is of sheet copper, 7-inch cube with three holes 1 inch in diameter, the central hole being used for the test and the other two as steam vents. A constant water level is maintained 2 inches below the top of the bath. Heat is applied by a ring burner protected from draught. For accurate work, maintain a constant gas pressure of 3.5 to 3.75 inches of water by means of a pressure regulator.

Prepare accurately solutions of, (1) 0.20-percent methylene blue and (2) 3 N sodium hydroxide.

Grind the sample of sugar and transfer 7 g, weighed correctly within 0.05 g, to a clean, dry test tube. Add 6 ml of distilled water, 1 ml. of methylene blue (microburette or calibrated pipette), and 1 ml of the caustic soda solution.

Shake the test tube (closed with a rubber stopper) vigorously for 15 seconds, and immerse in the boiling water bath for exactly 2 minutes. Remove from the bath and compare immediately with the row of standard tubes.

Standards. Prepare a solution of 19.5 g of CuSO4.5H2O in boiled distilled water and make to 500 ml. Measure accurately the following volumes into 50-ml volumetric flasks and add to each 10 ml of ammonium hydroxide (sp gr 0.880; 32.9 percent of ammonium hydroxide by titration) and make to volume.

TABLE 26.-Standards for use with the de Whalley method

When a solution of reducing sugar is added to a boiling alkaline copper solution, the potential set up at a platinum electrode during the first part of the titration is represented by the expression

The concentration of cupric ions is governed by the composition of the alkaline tartrate solution and by the removal of copper in the form of insoluble cuprous oxide. The concentration of ouprous ions is determined by the solubility product of cuprous hydroxide. When

all the cupric ions have been removed the potential changes rapidly. This sudden change in potential indicates the end of the reaction between cupric ions and reducing sugar.

A micro application of the electrometric method has been made by Niederl and Müller [59]. They used alkaline copper solutions composed of (A) 3.95 g of CuSO,, 5H2O in 500 ml of solution; (B) 19.75 g of Rochelle salt and 7.4 g of NaOH in 500 ml of solution. For the analysis, they mixed equal volumes of A and B. Their apparatus consisted of two vessels, each containing an alkaline copper solution of the same concentration, in which was immersed a platinum electrode. The vessels were connected by an agar-potassium chloride bridge.

For analysis, 3 to 5 mg of substance is accurately weighed, dissolved in water (1 ml for each mg), and transferred to a microburette. One milliliter of the mixed copper reagent, containing 1 mg of copper is placed in each of the two vessels. A reading is taken on the potentiometer and again after the solution in one vessel is brought to boiling. A preliminary titration is carried out as follows: 0.1 ml of the sugar solution is added, the mixture boiled, and a reading taken again. Additions of the sugar solution are continued until 1.2 ml has been used. In the region of the end point the change of potential is very considerable. The maximum change per 0.01 ml marks the end point. The actual determination is carried out in the same way, but larger volumes of the sugar solution are used until the end point is approached.

Their results are as follows:

The following method, in which the end point is determined by the change of potential between two thick copper wires serving as electrodes, has been devised by Tryller [60]:

Reagents and apparatus.-(1) Soxhlet solution (I), 34.639 g of CuSO4.5H2O made to 500 ml of solution; (II), 173 g of Rochelle salt and 50 g of NaOH made to 500 ml of solution.

(2) Sodium sulfate for cell, 39.415 g of anhydrous Na2SO, made to 1 liter of solution.

(3) An electrode of thick copper wire (about 2 mm in diameter). (4) A cell of Pyrex-glass tubing (8 mm inside diameter with a plug (8 mm long) of plaster of paris at one end. The plug should be washed with the cell solution in which it should be stored when not in use.

(5) A dead-beat moving-coil galvanometer with central zero. (6) A press-key.

(7) Burette stand, 250-ml Pyrex flasks, burettes, etc.

Method. The arrangement consists of a galvanometer wired directly to the electrodes through a press-key. Any convenient