January 15, 1941
ANALYTICAL EDITION
reported a t that time, on samples having a dissolved oxygen content over 7.0 p. p. m., was 0.31 p. p. m. These observations were obtained on duplicates dosed in the laboratory. It is apparent that in this particular set of data there is no advantage in a preliminary acid-azide dose performed in the field, probably because the B. 0. D. of these samples was very low. With samples in higher B. 0. D. ranges this procedure probably would be of value in field work or where samples have to be transported for a considerable distance for laboratory examination. It has long been known t h a t various substances interfere with the Winkler procedure. Modifications have been devised to overcome these difficulties, one such being the azide modification for the destruction of nitrites, discussed in this paper. These modifications themselves possess limitations and should not be used indiscriminately. Certain examples of interference have been noticed. Some of these have been confirmed; others are indicated by preliminary data and merit further investigation. The azide method is not recommended for the determination of dissolved oxygen in suspensions of river mud ( 5 ) ,or in the presence of ferrous and/or ferric iron, or in raw undiluted sewage. Tentatively, pending further investigation, the azide method is not recommended in the presence of considerable amounts of copper or of sulfite wastes or for any industrial waste containing reducing or oxidizing materials.
Summary The azide and the Rideal-Stewart modifications of the Winkler method are equally efficacious for the determination of B. 0. D. in river pollution studies. Either may be expected to give reliable results. The azide reagent may be added in a preliminary step following acid or it may be added in combination with the
15
alkaline-iodide Winkler reagent. The first method affords greater flexibility, while the latter has manipulative advantages for routine work. The authors’ results on samples from a 267-mile stretch of the Ohio River, extending from 265 t o 532 river miles below Pittsburgh, and from tributaries in the area, indicate t h a t these methods will be in complete agreement. A preliminary treatment with acid and azide, whenever i t may be necessary or convenient t o store a sample for a short time, is advantageous in preventing oxidation. Such a procedure is superior to a complete Winkler treatment through the final acidification, when the titration is delayed. A preliminary acid-azide treatment will probably be useful in the determination of dissolved oxygen when the samples are to be transported for a considerable distance to a central laboratory.
Acknowledgment The authors wish to acknowledge the cooperation of W. W. Walker and M. E. Ettinger in the analytical work upon which this paper is based.
Literature Cited (1) Alsterberg, G.,Biochem. Z . , 159,36-47 (1925). (2) Barnett, G. R.,and Hurwitz, E., Sewage Works J., 11, 781-87 (1939). (3) Brandt, H.J., Ge8undh.-Ing., 60,557-9 (1937); Sewage Works J., 10,377-80 (1938). (4) Poindexter, G . G., Ibid., 11, 1025-9 (1939). (5) Ruchhoft, C. C.,and Moore, W. A., in press. (6) Ruchhoft, C. C.,Moore, W. A., and Placak, 0. R., IND. ENQ.CHEM.,Anal. Ed., 10, 701-3 (1938). PRESENTED before the Division of Water, Sewage, and Sanitation Chemistry a t the 99th hleeting of the American Chemical Society. Cincinnati, Ohio.
Determination of Levulose in the Presence of Dextrose and Sucrose. A Ferricyanide Method H. C. BECKER AND D. T. ENGLIS, Noyes Chemical Laboratory, University of Illinois, Urbana, Ill.
B
ECAUSE there are only slight differences in the rates of reaction of dextrose and levulose with most oxidizing agents, the determination of one sugar in the presence of the other presents a much more difficult problem than the determination of total reducing sugar. At the present time, the only chemical method for levulose in general use is Jackson and Mathews’ (3) modification of the method proposed by Kyns. In this procedure, it is necessary to introduce a factor to correct for the reducing action of the dextrose that is present. Fischl (2) described a procedure which uses a reagent composed of cupric sulfate, Rochelle salt, sodium carbonate, and disodium phosphate. He reported that, under the conditions specified, levulose has a reducing action 167 times as great as dextrose, although no analytical results are given to substantiate this. A method developed by Strepkov (6) was reported several years ago; the reagent used is a 0.005 M solution of potassium ferricyanide containing 80 grams of disodium phosphate per liter. It has been impossible, however, t o confirm his finding that dextrose has no reducing action at all under the specified conditions.
A previous publication (1) reported the results of a study of the effect of temperature and the concentration of sodium carbonate and disodium phosphate in the ferricyanide reagent upon the rates of oxidation of the two sugars. Further con-
sideration of the reaction has shown that the basis for calculation and expression of the results of the previous paper is somewhat misleading. It was believed that a near-stoichiometric relationship prevailed between the reactants. As a consequence, when ferricyanide was in excess, i t was assumed that the quantity of sugar oxidized was proportional to the amount of reagent reduced. The fact that the amount reduced reached a maximum and constant value after the mixture had been heated on a steam cone for 10 minutes caused this value to be assigned as that of 100 per cent oxidation for the quantity of sugar taken. However, as will be shown, the equivalent relationships of the reactants change considerably with change in the composition of the reagent and the conditions of the reaction. Hence, the values in the previous paper for per cent of sugar oxidized are only “apparent” but the results as expressed show a correct relationship of the various effects, since the apparent per cent oxidation is directly proportional to the amount of reagent reduced. As the previous paper has shown, the reaction was very rapid a t boiling temperature, and no great selectivity could be obtained. At 50” C., however, the reaction was slower but showed a much greater difference in the oxidation rates of lewlose and dextrose. Since a temperature of 50” C. was
16
INDUSTRIAL A N D ENGINEERING CHEMISTRY
most selective, it was used in most of this later work. The effect of increasing the concentration of sodium carbonate in the reagent was to increase the rate of oxidation of both sugars. Disodium phosphate specifically decreased the reaction rate for dextrose without affecting that for levulose. The most satisfactory reagent was found to be one which contained high concentrations of these two salts along with the potassium ferricyanide. These characteristic effects have been standardized and adapted to a method for the quantitative estimation of levulose in the presence of dextrose and sucrose.
Experimental All the inorganic reagents used were of analytical grade, as were also the sucrose and dextrose. The purity of the levulose was determined by polarization and found to be 98.6 er cent. Analysis by the chemical method of Jackson and bathews checked the purity value indicated by the optical method. In addition to disodium phosphate and sodium carbonate, a number of other salts were subjected to a reliminary examination in an effort to find some which woupd be more selective. Sodium acetate, sodium citrate, borax, trisodium phosphate, and disodium arsenate were tried but none proved to be so satisfactory as the two used. Borax decreased the oxidation rate of both sugars but affected levulose to a much greater extent than dextrose. Sodium citrate did not show any valuable properties and is objectionable in the final estimation of ferrous ion, in that citrates are also oxidized by ceric sulfate a t room temperature and another reagent must be used a t this stage of the procedure. Trisodium phos hate was so alkaline that even small amounts decreased the serectivity of the reagent. Sodium acetate was not alkaline enough to replace sodium carbonate and did not show any specificity for either sugar. Disodium arsenate worked as well as disodium phosphate but showed no particular advantages. Sodium carbonate and disodium phosphate, then, appear to possess to a high degree the properties which are necessary for the levulose reagent and their use was continued in the final study.
Vol. 13, No. 1
TABLE I. EFFECTOF COMPOSITION OF REAGENT UPON LEVULOSE-FERRICYANIDE EQUIVALENCE (5 mg. of levulose heated for 10 minutes at 100" C.) 25 111. of Reagent Containing 4 Grams of KsFe(CN)a and Varying Amounts of NazHP0~.12HzOand NazCO; per Liter NazHP04.12Hz0, grams .. 80 80 250 250 24 NazCOI, gram8 24 150 24 150 Ce(SO4)zo KaFejCN)s 0.1460 0,1539 0.1596 0.1598 0.1619 reduced, milliequivalent Ce(S0r)t per mg. levulose, 0.0289 0.0307 0.0319 0.0320 0.0323 milliequivalent 25 M1. of Reagent Containing 24 Grama of NaaCOa and Varying Amounts of KsFe(CN)s per Liter KaFe(CN)s, grams Ce(SO4)zc KiFe (CN)s reduced, milliequivalent Ce(SOa)z,per mg. levulose, milliequivalent
4 7 0.1450 0.1509
0.0289
8 10 12 0.1518 0.1558 0.1569
24 0.1658
0.0302 0.0304 0.0314 0.0316
0.0332
TABLE 11. STANDARD LEVULOSE SERIES (10 ml. of sugar solution heated 60 minutes at 50' C. with 25 ml. of reagent) Ce(SOd2 e Average Deviation for Levulose KsFe(CN)a Reduced 28 Determinations Ma* Mdliequiualents Milliequivalent 4.9 0.226 0.001 9.9 0.450 0.002 ~ . ~ . ~ 0.874 0.002 19.7 1.296 29.6 0.003 1.700 0.002 39.4 2.089 0.002 49.3 2.460 0.007 59.2 2.813 0.007 69.0 3.152 78.9 0.008 88.7 3.477 0.008
Since the ferricyanide concentration is so important, it WBS thought that a large excess might improve the equivalent relation FACTORS AFFECTING LEVULOSE-FERRICYANIDE EQUIVA- of the reactants. The ferricyanide concentration was, therefore, increased from the usual 4 grams per liter to 20 and 50 grams per LENCE. The effect of variation in the concentration of the liter. As Figure 1 shows, the levulose-ferricyanide equivalence salts in changing the equivalence of the sugar oxidized and was definitely improved and the concentration range for the levdose increased ninefold. the ferrocyanide formed was established and the results are The high concentration of ferricyanide gave rise to two difficulshown in Table I. With higher salt concentrations more ferties. 1,lO-Phenanthroline, which had been used as the indicator rocyanide is formed in the oxidation of a fixed amount of in all the previous work, could not be used because it formed sugar. The same effect is obtained by increasing the conan orange colloidal suspension which gave no color change a t the end point. However, a 0.005 M solution of sodium diphenylamine centration of the potassium ferricyanide. sulfonate, prepared according to the directions of Sarver and Kolthoff (6).worked very satisfactorily and gave a good'color change-at the end point. The second difficulty was the formation of a light blue-green precipitate which proved to be cerous phosphate. At times, the precipitate was so heavy that it partially masked the end point, particularly if the titration was not carried out with sufficient rapidity. It was possible, by increasing the volume of the solution in the flask and carrying out the titration rapidly, to reduce greatly the amount of precipitate formed so that it did not interfere with the end point. The reagent finally chosen contained 50 grams of potassium ferricyanide, 225 grams of disodium phosphate dodecahydrate, and 150 grams of anhydrous sodium carbonate per liter. This reagent was stored in a black bottle and all light was excluded. Under these conditions, there was an initial aging period of 6 to 8 days during which the reagent changed by about 2 per cent, but it remained perfectly stable during the next 30 days, which was the longest period over which the tests were conducted. A vellow DreciDitate formed in the solution on standing. This precipitate may I . . . I . " 1 . . . 1 . . . be fltered off, or the delivery tube kept several 0.030 20 40 60 80 IO0 centimeters above the bottom of the bottle. Even m g of IevuIo~e large amounts of this precipitate do not, however, interfere with the determination. FIGURE 1. EFFECTOF FERRICYANIDE CONCENTRATION UPON LEVULOSEFERRICYANIDE EQUIVALENCE CONDITIONSFOR OXIDATIONPROCEDURE. Levulose heated at 50' C. for 75 minutes with 25 ml. of reagent containing 80 grams of The Specific Conditions for carrying out the NazHP04.12HtO and 150 grams of Ka?COa in addition to varying amounts of KsFe(CN)s
January 15, 1941
17
ANALYTICAL EDITION
oxidation were studied in order to keep the error below a definite limit.
-
I 0
2.0If the ferricyanide reagent stands in contact with the sugar solution a t room temperature for 0 ' any appreciable length of time previous to heat0 ing, sufficient oxidation occurs to cause a serious Q ' error in the determination. To avoid this effect, e the reagent must be added to the sugar solution 1 0 ' o Levulose z just before the flask is placed in the bath. v . Dextrose In order that duplicate levulose estimations 9 shall not differ more than 0.1 mg., the temperax- I .o ture must be maintained at 50 * 0.05'' C. and v . the heating must be carried out for 60 minutes * 5 seconds. The 60-minute period was chosen 6 because, as shown in Figure 2, the curve levels off rapidly a t this point and it is not an inconveniently long time. 0 The volume of sugar solution must be exactly 10 ml. and the 25-ml. portion of the ferricyanide I reagent must be within *0.1 ml. of this volume. 15 30 45 60 75 90 The reaction vessel used was a 125-ml. ErlenMinutes of Heotlng meyer flask, but larger sizes can be used without affecting the results to an appreciable extent. AXD DEXTROSE FIGURE 2 . RATEO F OXIDATIOX O F LEVULOSE A strip of sheet lead made into a collar is fitted over the flask to prevent its tipping or floating in 50 mg. of sugar heated a t 50' C. with 25 ml. of reagent containing 50 grams of KaFe(CN)e, 225 grams of iYazHPOa.12Hz0, and 150 grams of Na2C03 the w t e r bath. The water level in the bath should extend 1 or 2 cm. above the level of the solution in the flask. After the 60-minute heating period, the flask is removed from STANDARD LEVULOSESERIES. d standard levulose series the bath and immediately cooled in cold A ater, and the solution \\.as made and all the above-mentioned precautions rrere obis carefully acidified with 60 ml. of 3 N sulfuric acid. The acid qerved. The results in Table 11are the average of 14 separate must be added slonlv and the flask shaken continuousl\r to Dredeterminations made in duulicate with three different batches vent smtterine bv "the sudden evolution of carbon 'bioxihel. The 1nhicator,-6 tb 8 drops of 0005 M sodium diphenylamine of reagent orer a period o f 30 days. These results can be sulfonate, 1s added and Solution titrated t o the appearance of a used mostreadily by plotting a graph on such a scale that red color with a standard ceric sulfate solution n hich is 0.1 S levulose can be estimated accurately to 0.1 mg. and ceric to 0 15 N . sulfate to 0.001 milliequiv&nt. The weight of levulose used is corrected to 100 per cent purity. TABLE 111. I~EDKCTSG ACTIOXOF DEXTROSE IN PRESEXCE OF LEVULOSE EFFECTOF DEXTROSEUPON APDextrose Dextrose PARENT LEVULOSE VALUESIN A MIXRatio of Taken EquivaLevulose Error in Divided Apparent lent t o TURE OF TWO SUGARS. The reducing t o Total Levulose Dextrose Ce(S0d)z Levulose 1 Mg of Corrected Levulose by Sugar Taken Levulose Found Taken Used Factor4 Found Levulose action of dextrose was investigated by R Mg. MQ. M . eq. M Q. .Wu. MQ. Me. R . using principles employed by Jackson Levulose and Xathews ( 3 ) . Weighed amounts Range Total Sugar, 38 t o 56 Per Cent of mixtures of levulose and dextrose 50 29.6 29.6 0.0 30.0 1.380 31.6 2.0 15.0 were dissolved in 10 ml. of water and 40 38.4 38.6 0.5 56.7 1.820 42.5 3.9 13.8 38 45.8 2.134 4.6 45.9 0.2 73.8 50.5 15.7 oxidized according t o the procedure 2.140 56 48.2 38.6 50.7 2.4 48.3 0.2 15.4 53 53.9 47.6 57.1 3.0 2.386 54.1 0.4 14.9 previously described. The difference 53 58.3 2.542 58.1 - 0.3 61.4 3.1 50.9 16.4 between the amount of levulose actually 2.956 50 69.0 68.8 - 0.3 73.1 4.3 70.0 17.1 taken and the apparent levulose indiRange Levulose Total Sugar, 30 t o 36 Per Cent cated by the milliequivalents of ceric 30 9.9 20.0 0.508 11.2 15.4 1.4 9.8 - 1.0 sulfate consumed represented the re32 25.2 52.9 1.254 28.6 15.6 3.6 25.0 0.8 ducing action of the dextrose present. 33 29.6 60.0 1.454 15.8 4.1 33.4 29.3 - 1.0 33 49.3 2,313 55.2 100.0 16.9 6.2 49.0 - 0.6 The amount of dextrose taken divided 33 56.6 2.623 113.0 16.4 7.0 63.5 56.5 0.2 36 66.6 2.985 116.3 15.9 7.2 73.9 66.7 0.1 by the difference in the two levulose 36 67.2 3.009 74.6 118.7 16.1 7.4 67.2 0.0 33 69.0 values gave a factor which represented 3.100 140.0 16.7 8.7 77.4 68.7 0.4 32 76.6 3.410 162.1 16.1 86.7 10.1 76.6 0.0 the number of milligrams of dextrose Levulose having the same reducing action as 1 Range Total Sugar, 23 t o 28 Per Cent mg. of levulose. Column 6 of Table 25 22.9 69.0 1.221 27.8 14.1 4.7 23 29.6 100.0 I11 gives the factors thus obtained for 1.577 36.4 14.7 6.9 24 38.1 117.0 1.957 45.9 15.1 8.1 different amounts and proportions of 28 43.4 112.6 2.153 51.0 14.8 7.0 23 46.1 151.0 2.345 56.0 15.3 9.4 levulose and dextrose, 25 49.3 150.0 2.428 58.3 16.7 9.3
-
~
~
25 78
69.0 87.3
210.0 24.8
3.261 3.474
82.2 88.7
15.9 17.7
13.0 1.5
Av. (of 24)
0.5
Levulose Range Total Sugar, Less t h a n 20 Per Cent ~
5
9 12 15 16 17 0
38.4 24.4 7.3 9.9 14.8 20.1
670.0 237.0 54.8 50.0 80.6 91.6
3.162 1.741 0.523 0.609 0.924 1.174
79.2 40.5 11.5 13.5 20.8 26.7
16.4 14.7 13.1 13.9 13.4 13.9
45.9 16.2 3.8 3.4 5.5 6.3
14.6 with less t h a n 40 mg. of levulose, 16.1 with more t h a n 40 mg. of levulose.
33.3 24.3 7.7 10.1 15.3 20.4
-13.3 0.4
Av.
5.5 2.0 3.4 1.5 4.4
1 Since t h e completion of this paper, W. J . Shannon, who has been using the method, has made the following suggestions a8 improvements: Increase the size of t h e reaction flask to 200 ml. and introduce t h e sulfuric acid from a separatory funnel or other suitable device with a glass stopcock, so t h a t t h e flow may be regulated as the carbonate is neutralized a n d t h e danger of loss of solution minimized. Use ice in cooling t h e flask after the heating period.
INDUSTRIAL AND ENGINEERING CHEMISTRY
18
TABLEIV. EFFECT OF SUCROSE UPON LEVULOSE-DEXTROSE REDUCINQ RATIO Levulose Taken
Me. 0 0
Dextrose Taken XO. 0
Sucrose Taken
Ce(SOdz Used
Mg.
M.
0.002 0.006 0.349
eq.
Apparent Levulose Found Mg.
0.05 0.13 7.5
Dextrose Taken Divided by Corrected Factora Levulose 'VQ Xg.
.
... ...
0
0 100.0
210.0 700.0 210.0
49.3 49.3 45.1 51.5 22.0
100.0 0 148.6 0 102.7
210.0 210.0 970.7 1720.0 552.0
2.317 2.082 2.264 2.144 1.267
49.2 53.9 50.8 28.9
...
7.0
49.1 49.2 44.7 50.8 21.9
581.1 310.5 992 4
0.828 1.960 1.980
18.5 46.0 46.5
11.1 4.4 4.5
7.4 41.8 42.0
6.9
0.05 0.13 0.6
Error in Levulose Found MQ. 0.05 0.13 0.6 (7 " ,"
161.3 7.1 70.4 41.4 72.4 42.0 a 14.6 for less than 40
55.3
6.2
9.2
...
mg., 18.1 for more than 40 mg. of levulose.
I n general, this factor was not constant and tended to increase slightly with increasing amounts of levulose. To ensure greater accuracy, two factors were used for different amounts of levulose. When less than 40 mg. of levulose was present, the average factor was 14.6; and when more than 40 mg. were present, the factor was 16.1. The amount of dextrose actually taken was then divided by the proper factor, determined by the amount of levulose present, to give a subtractive correction which was applied to the apparent levulose value to obtain the true amount of levulose. The accuracy of the determination depends, not so much on the amount of levulose present, as on the ratio of levulose to dextrose. When the levulose present was more than 20 per cent of the total reducing sugar, the average error was 0.5 per cent; but when levulose represented less than 20 per cent of the reducing sugar, the error became much greater. This is to be expected, since the correction becomes large as compared to the amount of levulose present. SUCROSE EFFECT. The effect of this reagent on sucrose under the specified conditions was studied. When no other sugars were present, 700 mg. of sucrose had the Bame reducing action as 0.13 mg. of levulose. When mixtures of the three sugars were analyzed, levulose was determined with about the same accuracy as in the absence of sucrose. Sucrose may be present in amounts of 10 to 20 times that of the total reducing sugar without causing any appreciable error in the levulose determination. Some of the results obtained with sucrose present are given in Table IV. PROCEDURE AND CALCULATION FOR UNKNOWN SAMPLE.Exactly 10 ml. of the sugar solution, which contains not more than 90 mg. of levulose, or its equivalent in a levulose-dextrose mixture, are treated with 25 * 0.1 ml. of ferricyanide reagent and heated at 50 * 0.05" C. for 60 minutes * 5 seconds. The flask is removed from the bath, immediately cooled in cold water, and carefully acidified with 60 ml. of 3 N sulfuric acid, 6 t o 8 drops of 0.005 M sodium diphenylamine sulfonate indicator solution are added, and the ferrocyanide is titrated with a 0.1 N t o 0.15 N standard ceric sulfate solution. The milliequivalents of ceric sulfate required are referred to the graph plotted from the figures in Table I1 to obtain the apparent levulose value. The apparent levulose is subtracted from the total reducing sugar present, which has been previously determined by any standard method, such as that of Lane and Eynon (4), to give a figure for the apparent dextrose present. This apparent dextrose value divided by the proper factor gives a correction which is subtracted from the apparent levulose to give a new approximation. This new levulose value subtracted from the total reducing sugar gives a new dextrose value, which is again divided by the factor to give a second correction which is applied to the original apparent levulose. This calculation is repeated until successive values remain constant. Two, or sometimes three, approxi-
Vol. 13, No. 1
mations are sufficient to obtain constant values. In choosing the proper factor, approximately one fifteenth of the apparent dextrose must be subtracted from the apparent levulose figure to obtain an estimation of the actual levulose value, if the levulose present is close to 40 mg., or there is a large amount of dextrose.
Discussion
-0.4 -0.2 -0.9 -1.4 -0.5
This procedure is, like most sugar methods, an empirical one and, consequently, any deviation from the standard procedure will cause an 4.2 error in the results. The time and temperature 0.5 0.0 of hesting must be adhered to very closely. If the reagent is stored away from all light, it is stable for a t least 30 days after an aging period of 1 week. It is recommended that &e ;;agent be tested frequently against pure levulose if i t is kept for more than 30 days. If maltose or lactose is present, the method is not applicable since they both exert a reducing action of about the same magnitude as does dextrose. However, a rough approximation of the amount of levulose can be obtained by considering all the other reducing sugars to be dextrose and making the calculation on this basis. The formation of cerous phosphate precipitate is an objectionable feature. However, if the titration is carried out as rapidly as possible after it has been started, the interference is small. Another disadvantage is the use of two factors in the calculation of the amount of levulose, but it is felt that the increased accuracy more than compensates for the trouble involved. The chief advantage of the method lies in the direct procedure for iinding the amount of ferrocyanide formed. If a water bath is used, which will hold 12 flasks a t the same time, one operator can complete 18 determinations in duplicate during a 4-hour period. This is done by removing flasks from the bath a t &minute intervals, which give sufficient time for carrying out the titration and preparing the next sample.
Summary A method is described for the determination of levulose in the presence of dextrose and sucrose. The reagent used contains 50 grams of potassium ferricyanide, 225 grams of disodium phosphate dodecahydrate, and 150 grams of anhydrous sodium carbonate per liter. The oxidation is made a t 50" C. for 60 minutes. A maximum of 90 mg. of levulose, or its equivalent in a levulose-dextrose mixture, can be determined. Dextrose exerts a small, but definite, reducing action and a factor is introduced to correct for its presence. Sucrose has very little effect on the determination and can be present in large quantities without interfering appreciably. When the levulose present is 20 per cent or more of the total reducing sugar, an average accuracy of 0.5 per cent can be obtained. The error increases rapidly as the ratio of levulose to total reducing decreases below this value.
Literature Cited (1) Englis, D. T I and Becker, H. C., IND.ENG.CHEM.,Anal. Ed.,
11, 145 (1939). (2) Fischl, F., Chern.-Ztg., 57,393 (1933). (3) . _ Jackson, R. I?. and Mathews, J. A., Bur. Standards J . Research, 8,404 (1932). (4) Lane, J. H., and Eynon, L., J . SOC.Chem. I d . , 42,32T (1923). ( 5 ) Sarver, L. A., and Kolthoff, I. XI., J . Am. Chern. SOC.,53, 2902 (1931). (6) Strepkov, S. M., Biochem. Z . , 287, 33 (1936). FROM a portion of the doctorate thesis of H. C Becker,
1940
