449
V O L U M E 26, NO. 3, M A R C H 1 9 5 4 obtained. Increasing the thiocyanate concentration beyond this value has no effect on the absorbancy. I n order to ensure sufficient thiocyanate when larger amounts of mercury and other ions were present, it was decided t h a t 20 ml. of a 5 7 , potassium thiocyanate solution would be used to prepare 50 ml. of solution in subsequent determinations. ACIDITY. The effect of making the solutions 0 . l X in respect to sulfuric, nitric, hydrochloric, and perchloric acid was investigated using 5 p.p.m. of mercury. When the perchloric acid was used, there was a precipitate of potassium perchlorate but the other acids did not affect the absorption spectra appreciably. The acidity of the solution was increased until it was 1.h’ in sulfuric acid without affecting the absorbancy readings a t 281 mp, K h e n no acid was added, the solutions had a p H of 5 to 6 and had absorption spectra identical to those shown in Figure 1. Higher concentrations of hydrochloric acid should be avoided because there is a tendency to form the mercuric chloride complex when an appreciable amount of chloride ion is present in highly acidic solution. DIVERSEIONS.The effect of certain diverse ions was studied using 5 p.p.m. of mercury. An error of less than 2.5y0 was considered negligible. One thousand parts per million of the following parts did not interfere: ammonium, acetate, cadmium, chloride, magnesium, manganese(II), molybdate, perchlorate, phosphate, potassium, sodium, sulfate, tungstate, and zinc. Those ions which interfered are listed in Table I. Extractability of Mercuric Thiocyanate Complex. I t was found that 1-butanol was a satisfactory extractant for the mercuric thiocyanate complex. Preliminary experiments indicated that the distribution constant for butanol-water ratio was about 15. Thus, two extractions with 20- to 23-ml. portions of butanol would be sufficient to remove all but a negligible amount of the mercuric thiocyanate complex. The effect of acidifying the solution prior to extraction v,-as investigated using 5 p.p.m. of mercury. Making the solution 0.1N in respect to sulfuric, hydrochloric, or nitric acid did not change the absorbancy values. The addition of perchloric acid caused potassium perchlorate to precipitate, The absorbancy index a t 286 mp for the mercuric thiocyanate complex in 1-butanol is smaller than the absorbancy index a t 281 nip for the mercuric thiocyanate in water, as indicated by comparison of curves 3 and 5 in Figure I. The mercuric thiocyanate complex in butanol was stable for over a 24-hour period. A study of the effect of diverse ions on the extractability of
mercuric thiocyanate was made using 10 p.p.m. of mercury A negligible error (leps than 2.5%) was found for 1000 p.p.m. of the following ions: acetate, chloride, perchlorate, phosphate, potassium, sodium, and tungstate. Those ions causing interference are listed in Table 11. RECOMMENDED GENERAL PROCEDURE
Sample. Weigh, or measure by volume, a sample containing a sufficient amount of mercury so that the resultant solution obtained following the necessary preparative treatment contains 0.1 to 3.0 mg. of mercury per 100 ml. Desired Constituent. Transfer a 25-ml. aliquot to a 50-ml. flask. Add 20 ml. of a 5y0 potassium thiocyanate reagent, dilute to the mark, and mix thoroughly. Measure the absorbancy at 281 mp using 1-cm. silica cells and a reagent blank solution. DISCUSSION
The ultraviolet spectrophotometric determination method for determining mercury has been proposed using the colorless mercuric thiocyanate complex which exhibits a characteristic absorbancy maximum a t 281 mp in aqueous solutions and a t 286 mp in 1-butanol extracts. An indication of the precision of the proposed method can be obtained from the following data. Using 5 p.p.m. of mercury in aqueous solutions of pH 6 a mean absorbancy value of 0.391 was obtained for 12 determinations. The standard deviation was 0.0057, or 1.5%. Using 10 p.p.m. of mercury in 1-butanol extracts a mean absorbancy value of 0.441 was obtained for 15 determinations. The standard deviation in this case was 0.009, or 2.0%. LITERATURE CITED
(1) Cholak, J., and Hubbard, D. 31.,IND.ENG.CHEM.,ANAL.ED., 18. 149 f1946) I --(2) Hibbard, D. &I., Ibid., 12, 768 (1940). \ - - - - ,
(3) Laird, F. W., and Smith, Sister Alonaa, Ibid., 10, 576 (1938). (4) . . Law, E. P..and Nelson, K. W., J . Assoc. Offic. Agr. Chemists, 25, 399 (1942). (5) Kolthoff, I. M., and Sandell, E. B., “Textbook of Quantitative Inorganic Analysis,” 3rd ed., p. 548, New York, Macmillan Co., 1952. (6) hfarkle, G. E., and Boltz, D. F., AXAL.CHEM.,25, 1261 (1953). (7) Merritt, C . , Hershenson, H. h l . , and Rogers, L. B., Ibid., 25, 572 (1953). (8) Richmann, H. J., IND. Esc. CHEM.,ASAL. ED., 11, 66 (1939). RECEIVED for review August 20, 1953. Accepted November 27, 1953. Presented before the Division of Analytical Chemistry at the 124th Meeting of the . k M P R I C A S C H E U I C A L SOCIETY, Chicago, 111.
Spectrophotometric Determination of Ethanedial J. M. DECHARY, ERNEST KUN’, and H. C . PITOT Departments o f M e d i c i n e and Biochemistry, Tulane University, N e w Orleans, La.
Specific and sensitive analytical procedures were required for the determination of ethanedial and 2-oxopropanal in biochemical materials, particularly animal tissues. The present colorimetric methods for the determination of the a-dicarbonyl compounds as derivatives of 1,2-benzenediamine (the quinoxalines) do not meet the above requirements. A highly sensitive color reaction was obtained when ethanedial was heated with 2,3-diaminophenazine in 15.8N sulfuric acid solution. This reaction was made suitable for spectrophotometry by tetrazotizing the excess color reagent and reducing the tetrazonium salt thus formed with hypophosphorous acid. Based on this procedure, ethanedial was determined in amounts of 0.01 to 0.2 1
Present address, University of Wisconsin, Madison, Wis.
micromole. A possible mechanism, based on the formation of pyrazino[b]phenazine,is proposed for the color reaction. The method has been adapted for biochemical analysis and made specific for ethanedial and 2-oxopropanal by the use of 4.3N acetic acid instead of sulfuric acid.
T
HE continuation of biochemical studies on the metabolism of ethanedial and 2-oxopropanal in animal tissues (4, 5 ) is partly dependent on sensitive and specific analytical procedures. The procedures should permit the determination of the a-dicarbony1 compounds in the presence of other substances containing the carbonyl function which occur in biological material. The derivatives of a-dicarbonyl compounds with 1,a-benzenediamine, the quinoxalines, are reasonably specific. In acid so-
450
ANALYTICAL CHEMISTRY
lution these substances give yelloi\- colors, but with this reaction i t was not possible to distinguish between a variety of carbonyl containing compounds, By substituting an o-diamine of an extended conjugated structure for o-benzenediamine, a stable, dark blue color was obtained when ethanedial was heated n4th 2,3-diaminophenazine in strong sulfuric acid solution. This color reaction was made suitable for spectrophotometric analysis only after the required excess of 2,3-diaminophenazine (red-broJYn in acid solution) was converted into the straw-yellow phenazine by treatment Kith nitrous acid and subsequent reduction of the tetrazonium salt thus formed with hypophosphorous acid. 2-Oxopropanal and 2,3-butanedione react also with 2,3-diaminophenazine under these conditione, but the absorption spectra of the reaction products are sufficiently different to permit their identification, and if necessary, suitable modifications in the color reaction can be made xhich allon s the determination of ethanedial in their presence, I n the discussion whichfollowsa detailed description of two niethods ail1 be given. The first (procedure A) is carried out in 15.8N sulfuric acid solution with prolonged heating and is suitable for the spectrophotometric determination of the pure a-dicarbony1 compounds. The second method (procedure B) is carried out in 4.3Ar acetic acid solution with a short heating time. It is suitable for the analysis of ethanedial in biochemical material, such as tissue extracts and enzyme reaction mixtures, which contain substances converted by heat and strong sulfuric acid into interfering products. PROCEDURE 4.
APPAR4TUS AhD REAGENTS
The Beckman DU spectrophotometer was used with cells of 1-cm. light path for the examination of the absorption spectra of the reaction products of ethanedial, 2-oxopropanal, and 2,3butanedione with 2,3-diaminophenazine. The Coleman Juniortype s ectrophotometer was used for the quantitative determination ofethanedial. The color reagent was prepared by dissolving crystalline 2,3diaminophenazine (21.0 mg.) in 50 to 60 ml. of 1 0 S sulfuric acid by gentle warming and stirring. After dissolution of the odiamine, the volume was made up to 100 ml. with 1 O W sulfuric acid. The color reagent, which contains 1 micromole of 2,3diaminophenazine per ml.. is stable for a t least 5 to 7 days wheq kept in the dark a t 4' C. The o-diamine was prepared according to the method of Steigman ( 7 ) and purified by the procedure of Fischer and Hepp (1). The brown-yellow crystals do not melt below 350' C. but the analysis for nitrogen, which requires 26.65%, gave 26.707,. Aqueous 10N sulfuric acid. Concentrated sulfuric acid, analytical reagent grade. Potassium nitrite solution, prepared by dissolving 20.0 mg. of the C.P. salt in 100 ml. of distilled water. The solution is chilled in an ice bath prior to use. Hypophosphorous acid (purified, 50% solution, Oldbury Electro-Chemical Co., 19 Rector St., S e w York 6, S. Y.).
justed, if necessary, to 1 ml. with 10N sulfuric acid. This is followed by the addition of 0.7 ml. of the color reagent and finally by 0.5 ml. of concentrated sulfuric acid. The reagent blank is identical with the test solutions except that 1 ml. of 10N sulfuric acid replaces the solution of the a-dicarbonyl compound. The test tubes are shaken well and immersed in a vigorously boiling water bath for 1 hour and then chilled in an ice-water bath. After temperature equilibration ( 5 t o 8 minutes), 1 ml. of the potassium nitrite solution is added and the tubes are shaken vigorously. Finally 1 ml. of the hypophosphorous acid solution is added, and the tubes are shaken and immersed again in the boiling n-ater bath for 1 hour. The blue color which is formed during the first heating period fades during this treatment but reappears in a few minutes after the second heating period is begun. The 2-hour heating time was found to be ample to develop maximum color which did not change for a t least 24 hours. RESULTS OBT4INED WITH PROCEDURE A
Absorption spectra of the reaction products of ethanctiial, 2-oxopropanal, and 2,3-butanedione n-ith 2,3-diaminophenazine, which were prepared under identical conditions, are presented in Figure 1. I n each case the Beckman s ectrophotomcter was adjusted t o 0.0 absorbancy with the yelyow phenazine blank. While the ethanedial reaction product ( I ) has maximum light absorption a t 570 and 600 mp, those of the other two a-dicarbony1 compounds, even in six to seven times higher concentration, absorb much less light in this region and have an absorption maximum a t 610 to 620 mp. I n contrast t o the ethanedial reaction product, those of 2-oxopropanal(II) and 2,Q-butanedione(III) exhibit absorption mavima also a t 450 and 440 mp, respectively. The absorbancies of the ethanedial reaction product in the concentration range of 0.02 to 0.2 micromole were determined a t 595 to 600 mp with the Coleman Junior-type spectrophotometer, the instrument having been adjusted first to 0.0 absorbancy with the yellow phenazine blank. The blue color was found to obey Beer's 1aTY over the concentration range chosen. INTERFERENCES
The following compounds did not interfere with the color reaction: oxoethanoic, hydroxyethanoic, ethanedioic, 2-oxupropanoic, and other a-keto acids; dl-2-hydroxypropanoic and formic acids; methanal and ethanal. Bemil and 1,2-naphthoquinone did not yield colored reaction products under the given conditions. 700
4
COLOR DEVELOPMENT
The color reaction is carried out in 16 X 150 mm. calibrated borosilicate glass test tubes. The solution of the a-dicarbonyl compound is pipetted into the test tubes and the volume is ad-
(0,lOOpM)
a=2-0xOpropanal
(0 669,~M)
E=2,3-Butanedione
(0.700,&)l
I\"
PREPARATION OF STAYDARD SOLUTIONS
The ethanedial standard solution was prepared by dissolving 10.6 mg. of the anhydrous sodium bisulfite compound (99.2% pure as determined by analysis for sulfur) in 200 nil. of 10AVsulfuric acid. This solution contains 0.2 micromole of ethanedial per ml. An aqueous solution of 2-oxopropanal was prepared according to the procedure of Xeuberg et al. (6) and analyzed by iodometric titration ( 3 ) . I t contained 112 micromoles per ml. -4stock solution of 2,3-butanedione, containing 10 micromoles per ml., was prepared by dissolving 86.0 mg. of the freshly distilled material (boiling point 88" c. a t 760 mm.) in 100 ml. of distilled water. When the aqueous solutions of 2-oxopropanal and 2,3-butanedione were diluted to the required volume with 1O.V sulfuric acid, the normality was essentially unchanged.
I= Ethonedial
440
t ,200
4
400
t
450
500 Wave
550
600
650
length in my.
Figure 1. Absorption Spectra of Reaction Products of Ethanedial, 2-Oxopropanol, and 2,3-Butanedione with 2,3-Diaminophenazine in Sulfuric Acid Solution
V O L U M E 26, N O . 3, M A R C H 1 9 5 4
'8001
451 to 4 ml. by the addition of 0.5 ml. of glacial acetic acid. The addition of the glacial acetic acid a t the end of the second heating period prevents the turbidity which occurs a t times in samples containing high concentrations of 2-oxopropanal.
Wave length = 600 QU (Beckman)
600
RESULTS OBT413ED WITH PROCEDURE B
With this modification of the general procedure neither the sugars nor the progenitors of ethanedial and 2-oxopropanal yielded color reaction products with absorption maxima a t 600 and 610 mp. 2,S-Butanedione and ascorbic acid did not yield colored products having absorption maxima a t 600 t o 620 mp, but there is evidence that enediols such as ascorbic acid reduce 2,3-diaminophenazine even in acid solution, since a marked increase in light absorption occurred between 420 and 480 mp but not above 600 mp. The ethanedial reaction product obtained in aqueous acetic acid solution has the same absorption spectra as that obtained in l5.8N sulfuric acid, while that obtained with 2-oxopropanal in acetic acid solution has an additional absorption maximum a t 710 to 720 mp. Figure 2 shows the standard curve obtained for ethanedial using procedure B.
284
568 752 11.36 Micromoles x IO-' of ethanedial Figure 2. Standard Curve for Ethanedial Obtained by Procedure B
DISCCSSION OzCH
-Z&O
Z,?-Diaminophena3u?eE t h a n e
h/ra3tnoIbl p h e ~ j t n e
Blue
c ~ a i i o wchromogam'
Figure
3.
Reaction of 2,3-Diaminophenazine Ethanedial
with
Hydroxyethanal 4ielded the blue-colored product of ethanedial(I) ; 1,3-dihydroxy-2-propanone,S-hydroxy-2-propanone, and 2,3-diliydroxypropanal yielded the blue-green colored product of 2-o\opiopanal(II). These colored products xvere identified by their absorption spectra. A varirty of hexoses, pentoses, and phosphor vlated sugars yielded interfrring substances with the appearance of absorption maxima betr? een 400 and 500 mp. Diketones such as 4,5-octadione and di-n-valery1-5,6-decanedione interfered on heating 3 or more hours and gave light green rolored pioducts with an absorption maximum a t 610 to 620 mp. The absorbances of these colored products in this region, if calculated for equimolar quantities, do not exceed 2 to 6% of the alnorbance of the ethanedial reaction product. Of particular interest from a biochemical viewpoint are the triorephosphates and 3-hydro\y-2-propanone phosphate which, upori iwlation from animal tissues as the barium salts, might be anal\ 7ei1 readilv by this procedure since they give the blue-green coloird product of 2-osopropanal. PROCEDURE B.
APPARATUS AYD REAGENTS
The Beckman Model DU spectrophotometer was used with cells of 1-cm. light path for all quantitative measurements. The color reagent was prepared by dissolving the 2,3-diaminophenazine (21.0 mg.) in 100 ml. of 8.5N acetic acid. Glacial acetic acid, Baker analyzed reagent. 8 . 5 5 acetic acid was prepared by mixing 1 volume of glacial acetic acid with 1 volume of distilled water. It is advisable to prepare 2 liters of this solution which should be used throughout the analyses. The potassium nitrite and the hypo hosphorous acid solutions are identical with those used in procefure A. Preparation of Standard Solutions. All standards (see proccdure .I) were prepared in aqueous solution. COLOR DEVELOPMENT
One-milliliter samples of ethanedial, not exceeding 0.15 micromole per ml. (or 2-oxopro anal, not exceeding 0.6 micromole per ml.) are treated with 1 mf: of the color reagent in calibrated test tubes and are then heated, together with the reagent blank, in a boiling water bath for exactly 10 minutes. After temperature equilibration in an ice-water bath, 1 ml. of the potassium nitrite solution and 0.5 ml. of the hypophosphorous acid solution are added and the tubes are heated for 20 minutes in the boiling water h t h . After cooling, the volumes of the solutions are made up
Khile the rate of development of the color of the ethanedial reaction product reaches 90% its maximum value even under mild conditions (procedure B w t h 10 minutes of heating), that of the 2-oxopropanal reaction product is much more dependent on the heating time and the acidity. Khen the color of the 2oxopropanal reaction product is developed by procedure B, 0.6 micromole gives an absorbance of 0.140 a t 610 mp, whereas if the acetic acid concentration is increased to 8.5-Y(the 2,3-diaminophenazine is dissolved in glacial acetic acid), the absorbance is 0.315 to 610 mp. Ethanedial and 2-ouopropanal can be determined in tissue extracts conveniently by procedure B and the above-mentioned differences between the color development and absorbances of their respective reaction products make it possible to set up experimental conditions which are suitable for analyses of the two compounds in mixtures. The presence of 2-ouopropanal can be determined by absorbance measurements a t 600, 610, and 715 mp. The appearance of an absorption maximum a t 715 mp indicates the presence of 2-oxopropanal. The small contribution of 2-oxopropanal to the absorbance of the ethanedial reaction product a t 600 mp is readily corrected for by subtracting the amount of 2-oxopropanal which is determined a t 715 mp. The part of the absorption spectrum below 560 mp is suitable for differential spectrophotometry only in the absence of interfering substances. MECH4NISM OF THE COLOR RE4CTlOY
I n general, the reaction products of a-dicarbonyl compounds containing two to four carbon atoms lvith 2,3-diaminophenazine give blue or blue-green colors in strong acid solutions. Upon neutralization the color fades, but the process is reversible. It is noteworthy that no blue color develops if the reaction of ethanedial with 2,3-diaminophenazine is carried out in concentrated sulfuric acid. Upon the addition of a few drops of water the intense blue color develops instantly. Ethanedial reacts readily with the color reagent, even under mild conditions, while the substitution of only one hydrogen atom bv a methyl group, as in 2-oxopropanal, greatly decreases the reactivity. I n order to obtain more direct evidence of the mechanism of rolor formation. the reaction product of ethanedial with 2,S-diaminophenazine was prepared by refluxing the components together in absolute ethyl alcohol in the presence of glacial acetic acid ( 2 ) . The resulting brown-yellow pyrazino[blphenazine Tvas thoroughly freed of excess ethanedial by washing with 95% ethanol and drying over phosphorous pentoxide in vacuum. This product gives an intense blue color in strong sulfuric acid or acetic acid solution. The absorption spectra of this blue compound is identical with that obtained by the analytical procedure. The pvrazino [b]phenazine was also recovered from the blue product by neutralization. It is suggested that the reaction sequence outlined in Figure 3 is probable. Similar pyrazinophenazine derivatives of 2-oxopropanal, 2,3butanedione, benzil. 1,2-naphthoquinone, and 6-carboxyisatin
ANALYTICAL CHEMISTRY
452 were prepared and will be discussed elsewhere. The first four of these derivatives give blue or blue-green colors in strong acid solution. apparently the specificity of the analytical procedure depends on the formation of the pyrazinophenazine under the given conditions. If the derivatives are isolated first ( 2 ) , many of them form highly colored products in strong acid solution. ACKIVOWLEDGMEh-T
This work was supported by research grants from the National Heart Institute and National Microbiological Institute. It is a pleasure to thank Otto Schales for samples of 2,3-butanedione, 4,5-nctadione, and the ethanedial bisulfite compound and for criticism of the manuscript, Thanks are due to Thomas B. Crum-
pler for his suggestions co_ncerning the preparation of the manuscript. A sample of hypophosphorous acid received from the Oldbury Electro-Chemical Co. is acknowledged with thanks. LITERATURE CITED
(1) Fischer, O., and Hepp, E., Ber., 22, 356 (1889). (2) Ibid., 23, 841 (1890). (3) Kuhn, R., and Hecksher, R., 2. physiol. Chem., 160, 116 (1926). (4) Kun, E., J. Biol. Chen., 187, 289 (1950). (5) Ibid., 194, 603 (1952). (6) Neuberg, C., Faiker, E., and Levite, A, Biochem. Z., 8 3 , 244 (1917). (7) Steigman, A., Brit. J . Phot., 93, 256 (1946). RECEIVED for review July 22, 1953. Accepted December 7, 1953.
Spectrophotometric Method for Determination of Urea G E O R G E W. WATT and JOSEPH D. CHRISP The University o f Texas, Austin, Tex.
The work described in this paper was done to provide a dependable method for the determination of urea in samples containing urea, hydrazine, semicarbazide, and ammonium ion. This method involves a spectrophotometric determination of urea and is based upon the yellow-green color produced when p-dimethylaminobenzaldehyde is added to urea in dilute hydrochloric acid solution. This system exhibits a transmittancy minimum at 420 mp and shows good agreement with Beer's law at urea concentrations up to 320 p.p.m. With the instrument and procedure used, a relative error of only 1% is realized over the optimum urea concentration range of 50 to 240 p.p.m. Interferences investigated include ammonium ion, hydroxylamine, hydrazine, and semicarbazide; the latter two interfere, but a procedure for their removal is given. Although developed for the analysis of a particular type of sample, this method should be readily adaptable to the determination of urea in a wide variety of samples.
I
S A recent communication, the present authors described a spectrophotometric method for the determination of hydrazine ( 4 ) that is based upon the use of p-dimethylaminobenxaldehyde to develop yellow-colored solutions having a transmittancy minimum a t 458 mp, In the course of certain studies in which this method was employed for the determination of hydrazine, it became necessary to analyze numerous samples not only for hydrazine, but also for semicarbazide and urea, all in the presence of appreciable concentrations of ammonium ion. Spectrophotometric determination of hydrazine followed by determination of hydrazine and semicarbazide by the iodate titration method of Jamieson (3) permitted the determination of semicarbazide by difference. However, the existing methods for the determination of urea were either clearly inapplicable or failed to give satisfactory results when used in the analysis of samples of known urea content. From an inspection of Figure 2 in the previous paper ( 4 ) , it is evident that the broad transmittancy minimum region exhibited by solutions containing urea and p-dimethylaminobenzaldehyde might serve as a basis for the development of a satisfactory method for the determination of urea. Further experiments have shown that this system exhibits a reproducible transmittancy minimum a t 420 mp, which has been utilized in the manner described below for the determination of urea in samples of the type already indicated.
EXPERIMENTAL
Apparatus. A Beckman Model DU spectrophotometer and matched Corm cells of 1.001-cm. light path were wed for all transmittancy measurements. The instrument was operated a t constant sensitivity using a slit width of 0.1 mm., corresponding to a nominal band width of 2.4 mw a t 420 mp. Materials. Urea (Baker's C.P. analyzed) was recrystallized twice from methanol, washed with diethyl ether, and dried in vacuo for 48 hours over anhydrous magnesium perchlorate; melting point, 132" C., corrected. Hydrazine dihydrochloride (Eastman No. 1117) and semicarbaxide hydrochloride (Eastman Xo. 226) were titrated by the Jamieson method ( S ) , and their purity was found to be 99.8 and 99.4'%, respectively. Potassium iodate (Merck +4CS reagent grade) and p-dimethylaminobenzaldehyde (Eastman No. 95) were used asreceived. All other materials employed in this work were reagent grade chemicals that were used without further purification. Standard Urea Solution. This solution was prepared by dissolving 0.4 gram of urea in distilled water and diluting to 100.0 ml. Aliquots of this solution were used to make up the various urea solutions from which different aliquots were used to develop colored urea solutions within the concentration range 16 to 480 p.p.m. Color Development. The color reagent used was the same as that employed in the method for the determination of hydrazine
o UREA,P.P.M.
l
80
o
t
R
t
/
0
h
1
400
4 t O WAVELENOTH. M)I
Figure 1. Spectral Curves for Urea with p-Dimethylaminobenzaldehyde
