933
V O L U M E 20, NO. 10, O C T O B E R 1 9 4 8
Cuneo, J. F., and Switzer, R. L., IND.ENQ.CHEX.,ANAL.ED., 15,50% (1943). Davis, H. S.,and Daugherty, J. P., Ibid., 4,193 (1932). Davis, H. S.,and Schuler, R., J. Am. Chem. SOC.,52,721 (1930). Ewell, R. H., Znd. Eng. Chem., 32,77%(1940). Frank, R. L.,Ernmick, R. D., and Johnson, R. S., J. Am. Chem. SOC.,69,2313 (1947). Gregg, C. L., IND.ENQ.CHEM.,ANAL.ED.,17,725 (1945). Kaufmann, H., and Wassermann, b.,J. Chem. SOC.,1939,870. Krauze, V. P., Charskaya, K. N., and Korchrnarck, V. V., Sintet. Kauchuk (U.S.S.R.), 1936,Nos. 7-8,3-19. Leendertse, J. J., Tulleners. A. J., and W a t e r m a n , H. I., Rec. trao. chim., 52,515 (1933); 53,715 (1934). Mcblillan, W. A., I N D . ENQ.CHEM., A N A L . EO.,9,511 (1937). Matuszak, M. P., Ibid., 10,354 (1938). MoldavskiK, B., and Zharkova, V., J . Ga. Chem. (U.S.S.8). 14, 358 (1944). Korris, J. F.,and Joubert, J. M., J. Am. Chern. SOC.,49, 873 (1927). Podbielniak, W. J., IND. ENQ.CHEM.,ANAL.Eo., 13,639 (1941). Robey, R. F., unpublished observations. Robey, R. F., and Morrell, C. E., IND. ENQ.CHEM.,ANAL.ED., 14,%SO (1942). Robey, R. F., Morrell, C. E., and Vanderbilt, B. M., Oi2 Gur J., 40,No.37,41 (1942). Robey, R. F., Morrell, C. E., and Wiese, H. K., J . Am. Chm. SOC.,63,627 (1941). Robey, R. F.. Wiese, H. K., and Morrell, C. E., I n d . Eng. Chem., 36, 3 (1944). Thornton, V., and Herald, A. E., Ax.4~.CHEX.,20, 9 (1948). Tropsch, H., and Mattox, W.J., IND.ENQ.CEEX., ANAL.ED., 6,104 (1934). Uhrie. K.. Lvnch. E.. and Becker. H. C.. Ibid.. 18. 550 (1946). Wagner, C. D., Goldstein, T., and Peters, E.’D.; ANAL.CHEM., 19,103 (1947). Whitby, G. S., and Crozier, R. N., Can. J . Research, 6, 203 (1932). RECEIV~D March 23, 1948. Presented before the Division of Petroleum SOCIETY,New Chemistry a t the 112th Meeting of the ~ M E R I C A XCHEMICAL York, N. Y.
rather widely in the petroleum industry, has been found adequate for complete analysis. The 90 X 1.25 em. (36 X 0.5 inch) Hyper-cal column (17) requires from 250 to 1000 ml. and should be operated with a reflux ratio of a t least 25. Undoubtedly, any of a number of columns having equal or better efficiency can be used just as well, if means are provided for maintaining condenser temperatures a t 0’ and lower during most of the distillation period. Employing the specific columns mentioned above, the results presented in Table VI1 were obtained on synthetic mixtures which simulate the composition of CSfraction from certain common types of cracking operations. These figures show that the mean deviation of the determined value from synthesis is somewhat leas than 1% based on total sample. This deviation is in line with the error in vapor blend analysis cited earlier and the probable errors involved in the distillation step. Somewhat greater error, a total of probably about 1% based on total sample, would be expected when diolefins are present in significant amounts. ACKNOWLEDGMENTS
The authors extend thanks to D. M. Mason, W. J. Troeller, Jr., G. F. Zoeller, and others among their colleagues for suggestions and for work of a confirmational nature. Thanks are also due to Standard Oil Development Company for permission to publish this work and to M. R. Fenske for some of the pure hydrocarbons. LITERATURE CITED
(1) hltieri, V. J., “ G a s Analysis and Testing of Gaseous Materials,” New York. American Gas hssoc.. 1945. (2) Brown, R. A:, Taylor, R. C., Melpolder, F. W., and Young, W. S., AKALCHEY.,20, 5 (1948). (3) Craig, D., J. Am. Chem. SOC..65, 1006 (1943).
GLUCOSE A Direct Colorimetric Method f o r Determining Carbohydrates G. HARVEY BENHAM, Illinois Institute of Technology, Chicago 16, Ill. AND
JOHN E. DESPAUL, Quartermaster Food and Container Institute for the Armed Forces, Chicago 9, Ill. The molybdenum blue reaction is described as a quantitative method for the direct colorimetric determination of glucose. The method involves the formation of a heteropoly complex with molybdate and phosphate, and its subsequent reduction to molybdenum blue upon heating with reducing sugar. The intensity of color increases w i t h heating
R
EDUCISG agents in the presence of phosphates, silicates, or certain other acid radicals react with molybdates to produce the familiar substance known as molybdenum blue (8). Several observers have noted that reducing sugars react with ammonium molybdate to develop a blue color when heated and have utilized this reaction for qualitative and approximate quantitative tests for sugars (1). The conditions for a more sensitive colorinietric determination for reducing sugars based upon the molybdenum blue reaction have been established by Gilbert and Seapass ( 3 ) with ammonium molybdate and potassium dihydrogen phosphate. In an effort to apply this reaction to sugar analysis by means of the photoelectric colorimeter, the present authors have investigated these conditions (5) for glucose.
time. The reaction time chosen gives the maximum sensitivity with a minimum of interference from reducing disaccharides. The method is suitable for the determination of glucose in the presence of moderate amounts of sucrose and other di- and trisaccharides. This principle may be extended to the determination of other sugars. EARLIER APPLICATIONS
The molybdenum blue reaction has been used in the sugar industry to detect traces of sucrose by its reducing effect on molybdates in acid solution. Pinoff and Gude ( 7 ) found that fructose reacted much more quickly than aldoses, and that larger amounts of glucose or lactose were required to give the test than is the case with fructose. Dorfmuller ( 2 ) stated that ammonium molybdate may be substituted for a-naphthol to detect sugar in factory condensates and sweet waters. The molybdenum blue reaction has been modified by Matthews ( 6 ) , who made it applicable to approximate quantitative estirnations of sucrose in samples such as entrainment waters from sugar pans which contain a negligible quantity of interfering
934
ANALYTICAL CHEMISTRY
reducing substances. Matthews considered the test to be delicate and reliable and to give uniform results. Gilbert (3) hrts applied the reaction to determine sugars obtained from tung leaves and hydrolysis products of tung and potato starch. A qualitative study of the color reaction with ammonium molybdate and a number of sugars was made in 1944 by Lo and Chu (5). These investigators obtained blue colors by heating 5% solutions of glucose, galactose, fructose, maltose, lactose, and sucrose after treating each sugar with 3 ml. of 1% ammonium molybdate and 1 ml. of 3 S sulfuric acid.
/
P /
M G GLUCOSE0
Figure 1.
1
2
3
4
5
6
7
R
9
IO
in a Coleman Model 11 Universal spectrophotometer a t a wave length of 650 mp, using 19-mm. tubes. Precision. A standard curve for glucose was prepared and found to conform to the requirements of Beer's law (Figure 1 ) . Thirteen identical determinations gave a standard deviation of 1.23 for galvanometer readings, with a mean value of 58.59. Interference Due to Other Sugars. All monosaccharides yield a blue color under these conditions (Table I), but disaccharides and raffinose are affected to a very small extent. Only high concentrations of maltose cause appreciable interference. The intensity of color increases with increase in the heating time up t o about 4 hours of heating a t 100" C. for glucose. Results of numerous experiments pointed to a heating time of 30 minutes for the determination of glucose in the presence of di- and trisaccharides. Less heating time would reduce the sensitivity appreciably, while more heating time would increase interference from reducing disaccharides. Results with glucose-sucrose mixtures using up to 10 mg. of glucose are given in Table 11. It is evident that the presence of sucrose in glucose solutions does not appreciably affect the readings, unless the sucrose-glucose ratio and the total amount of sugar are both high. I t is the authors' practice to use a concentration such that the aliquot to be analyzed contains between 2 and 5 mg. of glucose. When the sucrose-glucose ratio does not exceed 2 to 1, the results obtained are valid for the glucose content of the sample. The amount of inversion by this procedure is almost negligible. When samples of hard candy manufactured from glucose and sucrose mere assayed for glucose, results with the molybdenum blue reaction (23.5%) gave excellent agreement Kith those obtained by the Munson and Walker gravimetric method (23.8y0)
Standard Curve for Determination of Glucose by Molybdenum Blue Reaction
Quantitative application of the molybdenum blue method by reaction of a carbohydrate directly with molybdate has largely been limited to the approximate estimation of sucrose in sugar factory wastes and condenser waters. Available molybdate methods for sucrose require that the sugar be hydrolyzed by acid to form glucose and fructose, and are, thus, in the heating time specified, dependent upon the fructose present. While the reaction obtained by Lo was positive for glucose, it was necessary to have up to a 5% solution of carbohydrate to develop the color upon heating a t boiling temperature. However, the conditions set for the experiment by Gilbert are more favorable. He used 5 ml. of sugar solution containing 20 to 50 mg. of glucose, and obtained a blue color upon boiling for 20 to 30 minutes in a water bath. Although Gilbert increased the sensitivity appreciably, it WEIS still far from the level of attainment possible for the molybdenum blue reaction for elements like tin, germanium, arsenic, or phosphorus, where the sensitivity is measured in parts per million. High sensitivity was also obtained by Heard and Sobel (4),who applied the reaction to the determination of reducing steroids. But, fundamentally, the molybdenum blue reaction for sugars showed possibilities, and it was undertaken in this project to develop the reaction as a quantitative colorimetric method for glucose.
DISCUSSION
This study treats of the application of the molybdenum blue reaction to the quantitative estimation of glucose in the presence of sucrose, and provides the nucleus for the further development of this test for determining other carbohydrates. Basically similar methods could be developed for the determination of any one of the reducing monosaccharides in the presence of disaccharides and other noninterfering substances. The possibility of applying this method to the determination of other carbohydrates like maltose, starch, and glycogen that are hydrolyzable to glucose is indicated. The further possibility of developing this method for the determination of the more reactive in the presence of the less reactive sugars is also suggested. This prob-
Table I. Transmittancy at 650 mw Developed with 1 Mg. of Sugar in 30 Minutes' Heating Time Using 19-mm. Tubes Galvanometer Readings Arabinose Galactose Levulose Glucose Xylose Mannose Maltose Lactose Sucrose Raffinose
44.5 50.5
61.5
76 n 71:5 75.5 95.8 99.3 98.8 99.5
EXPERIMENTAL
Reagents. Potassium dihydrogen phosphate, c.P., 0.02 M. Ammonium molybdate, c.P., 7.57%. Highest grade sugars. Procedure. The reaction was carried out a t the optimum pH of about 5.3 as follows: Into each of several 25-ml. volumetric flasks,5ml. of 0.02 A4 potassium dihydrogexlphosphate and 10 ml. of 7.5y0 ammonium molybdate were introduced. hmounts of the sugar under study varying from 1.0 to 10 mg. wereaddedandthe flasks were made up tovolume. Afterthesolution had beenmixed and the stoppers removed, the flasks were covered with a piece of sheet metal to prevent contamination from condensing steam, and heated in an autoclave, using open steam, a t 100' C. for exactly 30 minutes. They were cooled a t once in ice water to room temperature which stops the reaction. The color intensity was read
Table 11. Transmittancy at 650 r n p of Increasing Quantities of Glucose in Rlixtures of Glucose and Sucrose (Galvanometer readings at 650 mp using 19-mm. tubes for glucose-sucrose mixtures of percentage composition indicated) Glucose, 100% G 60% G 40% G 20% G 0% S 40% S 60% s 8O%s Jfg. 100 0 100 100 100 100 2 4 6 8 10
66.6 47.0 33.5 26.8 21 .o
63.2 44.3 31.0 26.6 18.0
64.2 45.9 32.6 26.4 17 3
62.3 42.4 33.7 25.8
17.7
64.7 45.8 30.2 22.2 18.2
V O L U M E 20, NO. 10, O C T O B E R 1 9 4 8 lem could be solved by reducing the heating time to the point wyhere the slon-er reacting monosaccharides are negative while the faster reacting monoqaccharides are positive. Finally, by heating a nii\tuie of sugars for various durations of time and measuring the subsequent color developed, binary and ternary mixtures of sugars might poqsiblv be determined by mathematical analv-is of the results obtained. LITERATURE CITED
(1) Bron-n, C. A,, and Zerban, F. W., “Physical and Chemical Methods of Sugar Analysis,” p. 651, Kew York, John Wile,- & Sons,
1941.
93s (2) Dorfmtiller, G., Deut. Zuckerind., 44, 574 (1919) , (3) Gilbert, S. G., U. S. Dept. Agr., Bur. Plant Industry, soil3 and
(4) (5) (6) (7) (8)
Agricultural Engineering, private communication to Leslie R. Hedrick, Illinois Institute of Technology, October 1946. Heard, R. D. H., and Sobel, H., J . Biol. Chem.. 165, 687 (1946). Lo, C., and Chu, L. J., IXD.ESG. CHEM.,-&SAL. ED., 16, 637 (1944). Matthem, N. W., Maryland Acad. Sci., Bull. 7, No. 3, 35 (1928). Pinoff, E., and Gude, K., Chem. Z t g . , 38, 625, 626 (1914). Woods, J. T., with Mellon, AT. G., ISD.ENG.CHEST., AKAL.ED., 13, 760 (1941).
RECEIVED February 9, 1948. Presented before the Division of Analytica and Micro Chemistry a t the 113th Meeting of the AXERICINCHEMICAL S O C I E I P , Chicago, 111.
Determination of Tetraethyl Pyrophosphate in Mixtures of Ethyl Phosphate Esters AT. N. DVORNIKOFF AND H. I,. l\JOKRTLL, Monsanto Chemical Company, S t . Louis, M o .
An analytical method f o r determining tetraethyl pyrophosphate in mixtures of ethyl phosphate esters involves selective hydrolysis of the higher polyphosphates and separation of tetraethyl pyrophosphate, triethyl phosphate, and a small amount of diethyl acid phosphate by benzene extraction. The diethyl ester is neutralized and the tetraethyl pyrophosphate is determined by hydrolysis with alkali, Trieth?1 phosphate does not interfere.
T
HE principal insecticidally active ingredient in mixtures of ethyl phosphates such as “hexaethyl tetraphosphate,”
technical tetraethyl pyrophosphate, etc., is generally recognized to be tetraethyl pyrophosphate:
(
