perature, and add 5 ml of dilute ammonia solution (1+9, approx. 1.43 N). Again mix, and allow to stand for 15 minutes. Add a sufficient quantity of dilute acetic acid (approx. 1.43 N) exactly to neutralize the ammonia added and again mix. Add with gentle mixing 12.5 ml of zinc-acetate solution (21.9 g of Zn (C2H3O2)2.2H2O+3 ml of glacial acetic acid per 100 ml) and subsequently 12.5 ml of potassium-ferrocyanide solution (10.6 g per 100 ml). Adjust the temperature to 20° C and fill to 200 ml. In all operations avoid the formation of air bubbles. Mix thoroughly, allow to stand for a few minutes, and filter, rejecting the first 25 ml of filtrate.

Re

Determination.-Introduce 25 ml of milk serum (40 g of condensed milk in 200 ml, clarified with zinc ferrocyanide) into a thin-walled boiling tube (8 by 14 inches), add 70 ml of the copper solution, mix, cover with a watch glass, and immerse to the level of the liquid in the tube in a large water bath maintained at 80° C for 20 minutes. move, cool in running water, filter on asbestos with suction, wash the tube and filter rapidly a few times with freshly boiled water. Dissolve the cuprous oxide in the tube and filter in 20 ml of the ferric sulfate solution. Wash the asbestos pad with cold water and add the washings to the ferric sulfate filtrate. Titrate with 0.1 N permanganate to faint permanent pink.

Table 27 is applicable to 25 ml of the milk serum containing 5 g of condensed milk.

TABLE 27.-Determination of monose sugar in condensed milk

(e) IODOMETRIC DETERMINATION OF ALDOSES

In the presence of iodine in alkaline solution, aldose sugars under suitable conditions are oxidized quantitatively to their respective aldonic acids. The postulated reactions for this oxidation are

I2+2NaOH NaIO+NaI+H2O

RCHO+NaIO+NaOH+RCOONa+NaI+H,O.

(49)

(50)

Equations 49 and 50 may be combined to represent the reaction, which is usually written

RCHO+I2+3NaOH→RCOONa+2NaI+2H2O.

(51)

It was early recognized that the sodium hypoiodite may also react to form sodium iodate:

3NaIO

NaIO3+2NaI.

(52)

Since sodium iodate cannot oxidize the sugar in alkaline solution, some active iodine is lost from the sugar oxidation reaction, but all the unreduced iodine, that is, all that remains in the form of iodate or iodite, is recovered when the solution is acidified and titrated with standard thiosulfate, thus

NaIO+NaI+H2SO、→I2+Na2SO1+H2O

NaIO3+5NaI+3H2SO-312+3Na2SO,+3H2O

I2+2Na2S2O→→→→2NaI+Na2S408.

The difference between the total iodine added and the excess iodine as found by the thiosulfate titration represents the amount of iodine used up in the oxidation of the sugar according to eq 51.

Slator and Acree [69] showed that a check on the iodine value could be obtained by titrating with standard alkali the free acid left after completing the iodine titration.

HC1+NaOH→NaCl+H2O.

Sugar acid lactone+H2ORCOOH.
RCOOH+NaOH→RCOONa+H2O.

As shown by eq 51, for every two equivalents of iodine used, three equivalents of acid are produced. The final acidimetric titer should then be three halves as great as that of the iodine consumed.

In its earliest form, the method was first devised by Romijn [70], who used buffer salts, such as alkaline carbonates, bicarbonates, phosphates, and borax, to supply the alkalinity in order to avoid the overoxidation which he found occurred with caustic alkali. Later, many investigators who had used buffer salts found that extended periods of time were required to complete the oxidation of the sugar in these weakly alkaline media and that consistent results were not always obtainable. In the most widely used methods, caustic alkali is employed.

The difficulties which must be overcome in standardizing the analytical procedure can be summarized briefly. If the whole required volumes of alkali and iodine are admitted to the sample simultaneously, much of the iodine is transformed to iodate by eq 52. This may leave a deficiency of iodine for the oxidation reaction. If the iodine is present in too great excess, overoxidation can occur. Kline and Acree [71] showed that while the ratio of alkali to iodine consumed by the aldehyde group was 3:2, the ratio in the oxidation of sucrose or levulose proved to be 5:4, indicating that the primary alcohol group was slowly oxidized to a carboxyl group. Conceivably the overoxidation of an aldose proceeds similarly. The rate of addition of the reagents has an important bearing on the results. In the analysis of a mixture of aldose and ketose, such as dextrose and levulose, it is important that the levulose remain unoxidized. If too great an excess of alkali is present the levulose can undergo the Lobry de Bruyn-van Ekenstein rearrangement to dextrose and man

nose, which are rapidly oxidized by iodine. The time of reaction has undergone endless variation in the specifications, from 24 hours, according to Romijn, to 2 minutes, according to Kline and Acree. Observers agree, however, that in the presence of sodium hydroxide the reaction is rapid, and that short periods of time are sufficient. Space permits the inclusion of only four of the many procedures that have been described.

Method of Willstäter and Schudel [72].-To the aldose solution containing from 1 to 100 mg of sugar, add about twice (1.5 to 4 times) the volume of 0.1 N iodine required for oxidation. At room temperature and with effective stirring, add dropwise 1.5 as much 0.1 N NaOH as iodine (from alcohol-free sodium hydroxide) and allow the mixture to stand at room temperature for 12 to 15 minutes (for small amounts of sugar, 20 minutes). Add dilute sulfuric acid to slight acidity and titrate the excess of iodine with 0.1 N thiosulfate, using starch indicator. Deduct the excess iodine from the volume originally added. The amount of reducing sugar is calculated from the difference where 1 ml of 0.1 N I-9.00 mg of hexose or 7.50 mg of pentose.

Kline and Acree [71] have criticized the procedure of Willstäter and Schudel on the grounds that the great excess of iodine in general tends to cause overoxidation. Occasional low results are probably caused by the formation of iodate, which removes iodine from the reacting system. Apparently, as Goebel [73] showed, the rate of addition of alkali requires delicate adjustment. If added over a period of 2 to 4 minutes, correct results were obtained. If added more rapidly, the recovery was low.

Method of Kline and Acree [71].-Kline and Acree have devised a procedure by which any considerable excess of either alkali or iodine is avoided, thus eliminating the errors due to iodate formation and overoxidation.

Transfer to an Erlenmeyer flask a weighed sample or aliquot containing approximately 180 mg of aldohexose or 150 mg of aldopentose and neutralize exactly (phenolphthalein, 1 drop only). Add 5 ml of 0.1 N iodine from a burette; then add drop by drop from a burette 7.5 ml of 0.1 N NaOH. Repeat this process until 22 ml of iodine and 35 ml of alkali solution have been run in, the operation requiring about 5 to 6 minutes. Allow a 2-minute interval for the completion of the oxidation. Acidify with 0.1 N (or 0.2 N) HCl and titrate the liberated iodine with 0.1 N thiosulfate (starch indicator). Add 2 to 3 drops of phenolphthalein and titrate the excess acid with 0.1 N NaOH. Deduct the thiosulfate titer from the number of milliliters of iodine added and deduct the hydrochloric acid titer from the number of milliliters of alkali added. The results will be the number of milliliters of iodine and alkali, respectively, consumed by the oxidation reaction. Then 1 ml of iodine equals 9.0 mg of hexose or 7.5 mg of pentose, and 1 ml of alkali equals 6.0 mg of hexose or 5.0 mg of pentose.

NOTES.-The ratio of volumes of alkali to iodine should be 3:2 if the reaction is confined to the aldehyde group alone. A departure from this ratio shows that other groups or other substances, such as lignins, are being oxidized. If assurance is had that only sugars are oxidized, and if it is desired to diminish the labor of analysis, either one of the two titrations may be omitted.

The back titration should require about 2 ml of thiosulfate. If the titer exceeds this, it is probable that over oxidation has occurred; if less, an insufficiency of iodine has been added. It is therefore advisable in such instances to repeat the analysis and to use more or less of the reagents, as indicated by the trial analysis.

A small amount of the lactone of the sugar acid is found after acidification, which will result in a fading end point with phenolphthalein. However, when the titration is carried out in a stoppered flask and the alkali is added slowly, a pink color which persists for 1 minute or longer may be taken as the end point.

Lothrop and Holmes method for the determination of dextrose and levulose in honey by the iodine-oxidation method.-Lothrop and Holmes [74] have recommended a procedure which is essentially the same as that of Willstäter and Schudel but specifically adapted to the analysis of honey. They recognized that levulose was slightly oxidized under the conditions of analysis, but the amount of oxidation was found to be an approximately constant percentage (1.2) of the levulose present and permitted the application of an empirical correction. The oxidation of levulose was found to vary rapidly with temperature, and hence the correction applied is valid only for 20° C. Total reducing sugar was determined by the Munson and Walker method and calculated as dextrose. The results agreed with the analysis by the Lane and Eynon volumetric method. In the following detailed procedure, the italicized words have been added to the original text.

To 20 ml of a solution containing 0.2 g of honey add 40 ml of 0.05 N iodine solution, using a 250 ml Erlenmeyer flask. Run in 25 ml of 0.1 N sodium hydroxide slowly and with continuous agitation, stopper, and allow to stand 10 minutes at a temperature of 20° C. Acidify with 5 ml of 2 N sulfuric acid and titrate at once with 0.05 N'sodium thiosulfate, using starch indicator. The weight of dextrose in grams (not corrected for reduction of iodine by levulose) is found by multiplying the milliliters of 0.05 N iodine reduced by 0.004502. Calculate as follows:

LA approximate percentage of levulose,

=

R = percentage of total reducing sugars calculated as dextrose
(Munson and Walker),

D-percentage of apparent dextrose (iodometric),
L =true percentage of levulose,
D=true percentage of dextrose.

Then introducing numerical values,

LA 1.081 (R-D1)
D=D,-0.012 LA
L=1.081 (R-D),

in which the factor 1.081 is the reciprocal of 0.925, the reducing ratio of levulose to dextrose at the nearly constant concentration of reducing sugars specified by the method."

(f) HINTON AND MACARA CHLORAMINE-T METHOD FOR THE DETERMINATION OF ALDOSES [75]

A solution of Chloramine-T behaves like sodium hypochlorite in producing hypoiodite and a certain amount of free iodine when excess potassium iodide is added. It is believed that the reaction takes place

through an intermediate hydrolysis of the Chloramine-T to hypochlorite. The reactions which appear to occur are indicated by the following equations:

C,H,SO,Na.NCl+H2O→C,H,SO,H.NH+NaClO

NaClO+KI→NaCl+KIO

KIO+KI+H2O→2KOH+I2 (to a small extent before acidi

fication).

The hypoiodite can be used for the oxidation of aldoses.

KIO+CH.OCHO=CHOCOOK+HI.

Upon acidification, the iodine equivalent of the unaltered ChloramineT, and any unused hypoiodite can be determined by means of thiosulfate.

C,H,SO2Na. NC1+2HC1+2KI→C,H,SO2H. NH+NaCl+2KCl+I2.

This oxidation proceeds more slowly than when alkaline iodine solution is used and hence it is more easily controlled.

Reagents.-0.05 N Chloramine-T solution containing 7.04 g/liter freshly prepared and protected from the light. Standard sodium thiosulfate solution, preferably slightly stronger than 0.05 N. Sodium hydroxide solution, 0.5 N.

Sodium hydroxide solution, 0.1 N.
Potassium iodide solution, 10 percent.
Soluble starch solution.

Procedure.-Pipette 20 to 25 ml of sugar solution containing about 0.1 g of sugar, into a 250-ml Erlenmeyer flask. Add 20 ml of 10-percent KI solution and 0.8 ml of 0.5 N sodium hydroxide followed by 50 ml of 0.05 N Chloramine-T solution. Close the flask with a rubber stopper and allow it to stand 1%1⁄2 hours in a water bath at 17.5° C. Acidify with 10 ml of 2 N HCl and titrate at once with standard sodium thiosulfate solution.

1.410 g of iodine-1 g of dextrose.
0.706 g of iodine-1 g of lactose.

.710 g of iodine-1 g of invert sugar.

Sucrose has little effect on the oxidation of aldoses except in mixtures where there is only a small amount of the aldose present. Levulose, although unaffected in neutral solution under the conditions given above, has an apparent iodine equivalent of 0.007. This method has been studied by the authors in its application to condensed milk.

9. REFERENCES

[1] H. Fehling, J. Pharmacol 3, 106 (1849); 106, 75 (1858).

[2] F. Soxhlet, Z. anal. Chem. 18, 20, 425 (1879).

[3] Lobry de Bruyn and W. A. van Ekenstein, Rec. trav. chim. 14, 156, 203 (1895); 16, 162, 259 (1897); 19, 1 (1900).

[4] J. U. Nef, Liebigs Ann. Chem. 357, 214 (1907); 376, 1 (1910); 403, 204, 338 (1912).

[5] M. L. Wolfrom and W. L. Lewis, J. Am. Chem. Soc. 50, 837 (1928).