V O L U M E 25, NO. 12, D E C E M B E R 1 9 5 3 where a‘ and CY’’ are the activities of an unfractionated aliquot of the mixture a t times ( t ’ At) and (t” 4- At), respectively; At is Supposed to be very small compared with the half life Of the parent. A ~ / A is ? calculated from Equation 4. Equation 4 is being used to study carrying of bismuth a t tracer level, tyith insoluble salts of lead; the precipitation of lead in the esperiniental case under study is complete and secular equilibrium misted before precipitation, so simplifications mentioned before :ire applied; the radioisotopes being used are RaD and RaEi.e., lead-210 and bismuth-210.
+
1925 ACKNOWLEDGMENT
The author wishes t’o thank Aron Kuppermann for stimulating Kirby for criticism and suggestions on the discussions, and H. 1%’. manuscript. LITERATURE CITED
(1) Kirby, H. w,, ;1s.9L, CHEM., 24, 1678 (1952). (2) Kirby, H, w., private communication. (3) Williams, R. R., Jr., “Principles of Nuclear Chemistry,” p. 129, New York, D. Van Nostrand Co., 1950. RECEIVED for review April 9, 1953.
Accepted August 24, 1953.
Copper-Ethylenediaminetetraacetic Acid Complex in Alkaline Solution Qualitative Comparisons with Benedict’s Reagent HARRY WAGREICH AND BENJAMIN HARROW Department of Chemistry, The City College of New York, New York, N. Y . ~ T H Y L E S E D I . ~ ~ ~ I N E T E T R ~ .acid ~ C E T(ethylenebisiminodiacetic IC
h acid) and its salts have recently been studied by pfeifierand
(9), Offermann (6), Schwarzenbach, Biedermann and Bangerter Klemm (41, and Schwarzenbach and Ackermann (81, as chelating agents for various cations, like cupric. zinc, calcium, magnesium, and nickel. Schwarzenbach and Ackermann (8)found the cupric ,on complex very stable. Plumb, hiartell, and Berscvorth ( r ) leached the same conclusion by spectrophotometric studies; a t values there is an insignificant amount of copper ions in high - pH . equilibrium with the complex. This chelating agent has the ability to chelate copper ion over a broad pH range. It therefore was of interest to see how the cupric ion complex R ith ethylenediaminetetraacetic acid would act with various organic compounds (reducing and n o n r e d d n g ) encountered in the biological chemistry laboratory, and to compare this action qualitatively with the action of these organic compounds on the Renedict qualitative reagent. SOURCES O F CHEMICALS
The complexing agents used in this investigation were kindly furnished by the Bersworth Chemical Co. and the Alrose Chemiral Co. Qualitatively, the reagents supplied by these companies gave similar results. The ethylenediaminetetraacetic acid supplied by the illrose Chemical Co. is described as “technically pure.” -411 other reagents met I C s specifications. PREPARATION O F REAGENTS
As it w a h planned to compare the action of a solution containing wpper ethylenediaminetetraacetic acid m-ith Benedict’s qualitative reagent, the same quantities of copper sulfate and sodium ( nrbonate were used in preparing both reagents. One such solution contained 100.0 grams of sodium carbonate, 17.3 grams of copper sulfate pentahydrate, 25.8 grams of the disodium salt of ethylencdiaminctetraacetic acid, and water to make 1000.0 ml. The quantity of ethylenediaminetetraacetic acid used was that found by experiment to dissolve the copper carbonate precipitate. The pH of this solution was approximately 10.2, while that of Benedict’s qualitative reagent was 10.1. All pH values were obtained with the Beckman Model H pH meter using a Type E glnss electrode. Solutions of copper ethylenediaminetctraacetic acid containing sodium hydroxide instead of sodium carbonate were prepared. The composition of one such solution was: 17.3 grams of copper zulfate pentahydrate, 25.8 grams of disodium ethylenediamine trtraacetate, 7.5 grams of sodium hydroxide, and water to make 1000.0 mi. The approximate pH was 11.0. As the tetritsodium salt of ethylenediamine tetraacetate was ‘ivailable (.llrose’s compound), a solution of this compound was irisde as follows: 17.3 grams of copper sulfate pentahydrate, 20.2 grams of tetrasodium ethylenediamine tetraacetate, 5.05 grams of sodium carbonate (or sufficient 1.W sodium hydroxide wlution), and water to make 1000.0 ml. pH = 10.1, inother modification was to take an equivalent quantity (27.5 grams) of the cupric complex of disodium ethylenediamine tetra-
acetate (Alrose’s compound) and add to it about 500 ml. of water. The pH was adjusted to about 11.4 with 1 M sodium hydroxide solution or saturated sodium carbonate solution. EXPERIMENTS ON REDUCING AND NONREDUCING COMPOCNDS
T o 5 ml. each of Benedict’s qualitative reagent and copper ethylenediaminetetraacetic acid in separate test tubes were added about 10 mg. of the compounds listed below. Each mixture was boiled in an actively boiling water bath for 5 minutes, and then removed to cool spontaneously.
No significant differences were noticed betffeen the action of Benedict’s qualitative reagent and copper ethylenediaminetetraacetic acid complex with the exception of chloroform and chloral hydrate. With both reagents positive tests (yellon- t o reddish brown precipitates) were obtained with the following reducing compounds: glucose, galactose, maltose, lactose, fructose, arabinose, xylose, and ascorbic acid. Compounds which gave no reduction of Benedict’s qualitative reagent and copper ethylenediaminetetraacetic acid were: sucrose, raffinose, starch, urea, formaldehyde, egg albumin (three times crystallized), bovine albumin (three times crystallized), trypsin, casein, creatinine, uric acid, gelatin, cellulose, and thrombin. As for the difference in the action of chloroform and chloral hydrate, Benedict’s qualitative reagent gave slight but positive tests with each (1), rhile the copper ethylenediaminetetraacetic acid reagent containing sodium carbonate gave no precipitate with either compound. However, the copper ethylenediaminetetraacetic acid reagent containing sodium hydroxide gave a positive test with chloroform, but not with chloral hydrate. On standing, the tubes containing creatinine and Benedict’s qualitative reagent or copper ethylenediaminetetraacetic acid develop a whitish turbidity, but give no evidence of reduction. NATURE O F PRECIPITATE FORMED
The precipitate was filtered and n-as dissolved in dilute ammonia water. The solution was colorless initially, but gradually turned blue, starting from the air-liquid interface dowu as time progressed. Addition of hydrogen peroside caused the solution to turn completely to the characteristic blue of the copper-ammonia complex. Another precipitate was treated with phosphomolybdic acid according to the details given IJJ~Folin and Wu ( 2 ) ,yielding x blue solution. Another precipitate w-as added to dilute sulfuric acid. The presence of copper and copper sulfate was experimentall) determined ( 3 ) . The colors and the properties of the precipitates obtained nith both copper ethylenediaminetetraacetic acid and Benedict’s qualitative reagent were the same.
1926 Thus there is little doubt that the precipitate resulting from the action of reducing sugars on copper ethylenediaminetetraacetic acid is cuprous oxide. o m I n i u M CONDITIOIVS FOR REDUCTION OF COPPER ETHYLENEDIAMINETETRAACETIC ACID BY GLUCOSE SOLUTIONS
I n connection with the effect of pH on the amount of precipitation, solutions of the chelating agent were prepared by dissolving 17.3 grams of copper sulfate pentahydrate and 29.2 grams of ethylenediaminetetraacetic acid in water and making the solution up to 500 ml. A series of various pH values from 3.0 to 13.0 was obtained by adding different amounts of a saturated solution of sodium carbonate or 131 sodium hydroxide solution. Each aliquot was then diluted with water to 50 ml. To two test tubes containing 5 ml. of each copper ethylenediaminetetraacetic acid solution of different pH were added 8 drops of 0.2 and 0.4% glucose, respectively, and tests were made in the usual manner. S o reactions were observed in the solution with a pH less than 9, while solutions of pH 9 and 9.5 gave small quantities of greenish yellox precipitates. Because differences in the size of the precipitate were difficult to distinguish accurately, the procedure was varied at this point. Aimodified Folin-Wu method ( 2 ) was adopted in which 2 ml. of reagent were mixed with 1 ml. of 0.04% glucose solution, boiled for 5 minutes in a boiling tvater bath, and cooled in an ice bath. After cooling, 5 ml. of phosphomolybdic acid reagent were added and the volume of the solution was made up to 25 ml. Comparative readings of the per cent transnlittance were made in a Leitz Rouy photometer at 580 mp.
It can be generalized that the higher the pH the greater the extent of precipitation. In the case of copper ethylenediamine tetraacetic acid containing sodium carbonate, a suitable operating range was pH 10 to 10.5, with best results a t 10.5. A suitable pH range for reduction of copper ethylenediaminetetraacetic acid containing sodium hydroxide was found to be between 10.3 and 12.8. However in the range between pH 11.5 and 12.5 there n‘as only a slight change in sensitivity. -4pH of 12 was chosen as a working standard. In general, a t a pH of 10.0 to 10.5 precipitates obtained with the chelating agent in sodium carbonate solution are larger than in sodium hydroxide solution. Benedict (1) states that “the hydroxides of the alkali metals have a greater destructive action upon dextrose and various other carbohydrates than have the carbonates, and in accordance with this fact, a copper-containing solution in which the alkalinity is secured by sodium carbonate makes a more delicate and specific test for the detection of dextrose than does a copper solution which contains sodium hydroxide,’’ The precipitates formed Y,ith copper ethylenediaminetetraacetic acid in the presence of sodium hydroxide appear faster and are more reddish, lyhile precipitates formed in the presence of sodium carbonate take a longer time to form and settle more slowly. It was then considered of value to determine a t a satisfactory pH (10.5 for the solutions containing sodium carbonate and 12.0 for the sodium hydroxide solutions) that amount of tetrasodium ethylenediamine tetraacetate required to give optimum precipitation with the 17.3 grams of copper sulfate pentahydrate. Amounts of tetrasodium ethylenediamine tetraacetate varying between 20 and 60 grams per liter were used. Using 20 grams of tetrasodium chelating compound under the above conditions did not prevent the precipitation of either copper hydroxide or copper carbonate upon the addition of sodium hydroxide or sodium carbonate. The optimum amounts of the tetrasodium ethylenediamine tetraacetate found both in the sodium hydroxide and sodium carbonate solutions were between 40 and 45 grams per liter. This information n-as obtained by dissolving the precipitates in phosphomolybdic acid ( 2 ) and comparing the resulting blue solutions in a colorimeter. The copper ethylenediamine tetraacetate reagent was generally a deeper blue than Benedict’s qualitative reagent. The time for reaction in the boiling water bath was studied-5 to 6 minutes were necessary to complete the reaction.
ANALYTICAL CHEMISTRY The solutions containing the ethylenediamine tetraacetate appear to be very stable on standing. One solution 4 months old hen tested in the and another 15 months old gave good results i~ usual manner. However, Benedict’s solution shows no sign of reduction or other alteration upon heating for 24 hours in a boiling water bath ( I ) , whereas Fehling’s solution shows a marked precipitation of cuprous oxide after 3 hours’ heating (1) and the preparations containing ethylenediaminetetraacetic acid begin to form cuprous oxide in 2.5 to 3 hours when subjected to this treatment. When the amount of ethylenediamine tetraacetate was increased from 0.09 to 11.6 moles keeping the quantity of copper sulfate constant (0.07 mole), no precipitate appeared until 4.5 hours of heating. COWPARISON WITH BEIYEDICT’S REAGENT
The copper ethylenediamine tetraacetate reagent prepared with sodium carbonate a t a pH of 10.5 nas more sensitive than the complex containing sodium hydroxide solution a t the same pH for qualitative determination of glucose in aqueous solutions, but was less sensitive than Benedict’s reagent in the qualitative estimation of glucose in urine. With aqueous solutions of 0.12% dextrose both Benedict’s solution and copper ethylenediamine tetraacetate sodium carbonate gave visible precipitates of cuprous oxides, although the precipitate obtained with Benedict’s solution was larger. In urine the copper ethylenediamine tetraacetate sodium carbonate reagent did not give significant yellow precipitates until 0.5% glucose solution was added to normal urine. However, Benedict’s reagent can give a positive test with 0.1 to 0.2oj, glucose in urine. The lesser sensitivity of the ethylenediamine tetraacetate reagent compared with Benedict’s reagent for the determination of glucose in aqueous solutions could be due partly to the presence of a smaller concentration of cupric ions in the former case. Schwarzenbach and .4ckermann (8) found the copper ethylenediamine tetraacetate complex very stable, with a log K of 18.2, while Meites ( 6 ) obtained a value of 14.2 for the copper citrate complex. West and Todd (10) discussing the Fehling and Benedict tests state that these complexes dissociate sufficiently to provide a continuous supply of readily available cupric ions for oxidation. From this it would follow that the more stable complex in allowing fewer cupric ions to be present would result in less reduction. ACKhl OWLEDGMENT
The technical assistance of Jack Preiss, Marion Reiner, and Saul rllford is gratefully acknom-ledged. Thanks are due to the Alrose Chemical Co., Providence, R. I., and the Bersworth Chemical Co., Framingham, Mass., for their kindness in furnishing adequate quantities of ethylenediamine tetraacetate. LITERATURE CITED
Benedict, S. R., J. Biol. Chem., 5, 485-7 (1908-9). Folin, O., and Wu, H., Ibid., 41, 367 (1927). Fresenius, T. W., “Introduction to Qualitative Chemical Analysis,” p. 290, New York, John Wiley &- Sons, 1921. (4) Klemm, W., Z . anorg. Chem., 252, 225-6 (1944). Meites, L., J. Am. Chem. Soc., 72, 180 (1950). Pfeiffer, P., and Offermann, W., Ber., 75B, 1-12 (1942). Plumb, R. C., Martell, A. E., and Bersworth, F. C., J . Phus. and Colloid Chem., 54, 1208 (1950). Schwaraenbach, G., and Ackermann, H., Hela. Chim. Acta, 30, 1798-804 (1947). Schwarzenbach, G., Biedermann, W., and Bangerter, F., Ibid., 29, 811-18 (1946). West, E. S., and Todd, W. R.. “Textbook of Biochemistry,” p. 256, Kew York, Macmillan Co., 1951. R E C ~ I Vfor E Dreview J l a r c h 10, 1953. Accepted -4ugust 20, 1053. Investigation aided (in part) b y a grant from The City College Research Committee, The City College of Xew York, New Tork, S . Y .
