Spectrophotometric Determination of Urea and Ammonia in Natural Waters with Hypochlorite and Phenol Robert Temple Emmet Naval Ship Research and Development Laboratory, Annapolis Division, Annapolis, Md. 21402
Colored compounds formed from urea and ammonia with absorbancy maxima at 454 and 630 mp, respectively, may be used to determine micromolar quantities of those compounds in natural waters. Optimal procedures yield molar absorbance indices of 4.4 X 103 for urea and 5.6 x lo3for ammonia in distilled water and of 8.0 x lo3 for urea and 3.6 x l o a for ammonia in seawater. The lower limits of detection of both compounds in seawater are 0.2 pg-atom nitrogen per liter, and both relative standard deviations in seawater are about 10% at a nitrogen concentration of 1fig-atom per liter.
Both the hypochlorite-phenol analyses in this paper produce general sample, blank sample and standard sample signals which develop rapidly in slightly alkaline aliquots of the sample. The signals are colored compounds absorbing strongly at 454 mp for urea and 630 mp for ammonia. Although the two signals will develop in a common reaction medium, the separate optimal procedures described improve the results in applications where extreme sensitivity is required. Possibly these methods could be applied to clinical problems. EXPERIMENTAL
BOTHIN SURFACE waters and in nutrient polluted waters, where urea and ammonia comprise a major portion of the dissolved combined nitrogen, analysis for those compounds would be useful ( I ) . At present there are two methods for the analysis of urea in natural waters. The more specific method described by Degens and Reuter involves chromatography and requires a lengthy procedure for the concentration of the sample (2). The more convenient method is the adaptation of the Fearon diacetyl-urea reaction (3-5) described by Newell, Morgan, and Cundy (6). However, the calibrations and blank determinations are not carried out in the natural sample where unexpected interferences might be encountered. Also, the red signal in this method develops slowly and the acid terminal solution is difficult to handle (7). In this paper a new urea method which avoids many of these difficulties is suggested for the routine analysis of natural waters. Several methods for determining ammonia in natural waters are reviewed by J. P. Riley (8). Because they require either delicate equipment, extra handling, or a separation of ammonia from the sample, they do not lend themselves to field use. More recently, Johnston’s adaptation (9) of the Prochhzkovh rubazoic acid method (IO) has been found satisfactory for routine use at sea (11). Newell’s adaptation of the hypochlorite-phenol ammonia method (12) is particularly sensitive; however, it is lengthy and best suited for analysis of dilute waters. The ammonia procedure herein described avoids interference from urea and the divalent cations in seawater because of the low terminal pH, 9 to 10, and is well suited for the routine, direct analysis of natural waters. (1) L. H. N. Cooper, J. Murine Biol. Assoc., 22, 183-204 (1937).
(2) E. T. Degens and H. J. Reuter, Inter. Ser. Monographs Eurfh Sci., 15, 377-402 (1964). (3) W. R. Fearson, Biochem. J., 33,902 (1939). (4) V. R. Wheatley, ibid., 43,m 4 0 2 (1948). (5) R. N. Beale and D. Croft, J. Clin. Pufhol., 14, 418-24 (1961). (6) B. S. Newell, B. Morgan, and J. Cundy, J. Murine Res., 24, 201-2 (1967). (7) . , W. B. Marsh. B. Fingerhut. and H. Miller. J. Clin. Chem..~.11, 624-7 (1965). (8) J. P. Riley and G . Skirrow, “Chemical Oceanography,” Vol. 11. Academic Press, New York, 1965, pp 368-9. (9) R.Johnston, Intern. Counc. Explor.Sea, N:10 (1965). (10) L. Proch6zkov6, ANAL.CHEM.,36, 865-71 (1964). (11) J. D. H. Strickland, Scripps Institute of Oceanography, La Jolla, Calif., personal communication, 1968. (12) B. S . Newell, J. Murine Biol. Assoc., 47, 271-80 (1967).
-
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Apparatus. Both the urea and ammonia procedures are carried out in 25-ml volumetric flasks, adding the reagents with 0.50-ml glass syringes. The syringes are equipped with needles of Teflon which extend beneath the meniscus to ensure reproducible addition and rapid mixing. Absorbances are determined in 5-cm cells with a Beckman Model D U spectrophotometer. Reagents. UREAAND AMMONIA.Although distilled water is sufficiently free from urea, the readily absorbed ammonia should be removed by resin treatment before the preparation of the ammonia distilled water blank. For use in both procedures, prepare a 2Sz NaOCl solution. Also prepare a 0.2% NaOBr solution which should contain 0.75 ml Brz and 2.0 g NaOH per liter. UREA. For use in the urea procedure, prepare 0.25N HC1 which should contain 0.3% MgClz.6H20; a borate buffer 0.3% KCI, solution which should contain 0.6$ 0.2% Hz02,and 0.25% NaOH, pH 9.6; 1.OM phenol in 9 5 z ethanol; and a solution of crystalline urea for use as a standard. AMMONIA.For use in the ammonia procedure, prepare 0.50N HC1; 4.OM phenol in 95% ethanol; 0.50N NaOH; and a solution of crystalline ammonium chloride for use as a standard. Note: To remove the red oxidation products from old phenol crystals, distill in the hood using a round-bottomed flask and a short air condenser. The Brf can best be handled with a syringe. Reagent grade chemicals should be used throughout, and if these reagents are kept in stoppered containers, they are stable for several months. Procedures. The equilibria in the urea and ammonia procedures are strongly pH dependent and through many tests, the optimal pH ranges of the reaction media have been established and are listed in Table I. It is simpler and more accurate to use a pH meter and Table I to reproduce the reaction conditions at the outset of a series of tests, rather than to rely on the exact addition of reagents to a sample of unknown composition. As a further guide to the procedures, the volumes of reagent and the essential operations are specified in the following directions for sensitive analysis of urea and ammonia in pH 5.5 to 8.5 natural water. Note: The term, X,has been used to denote the volume of HC1 reagent required to titrate 0.50 ml of NaOBr reagent to pH 7.5, Le., about 0.20 ml. For each method there are three procedures which yield general, blank and standard signals. UREA. The general urea signal is obtained by placing a 20.0-ml aliquot of the natural sample, dialysate or filtrate, which should be free from solid matter and at temperature
Table I. Optimal pH Ranges of Reaction Media Sample pH during critical steps Distilled water Seawater General sample Blank sample General sample Blank sample
Reagent addition A. Urea procedures NaOBr HCl HC1 NaOBr Buffer Phenol
11.2+ 7.3-7.9
+
B. Ammonia procedures NaOBr HC1 NaOH Phenol NaOH
9.8+ 7.2-7.8
7.3-7.9 8.0-8.6 8.7-9.1
8.0-8.6 8.7-9.1
7.2-7.8 7.9-8.3 8.3-8.6
7.9-8.3 8.3-8.6
7.1-7.6 7.6-8.0 8.3-8.9 10.1-10.5
10.6+ 7.1-7.6 7.6-8.0 8.3-8.9 10.1-10.5
7.0-7.5 7.5-7.9 8.1-8.7 9.1-9.5
9.4+ 7.0-7.5 7.5-7.9 8.1-8.7 9.1-9.5
Table 11. Absorbances of Urea and Ammonia Standard Solutions Absorbance in s(:awater in 5-cm cell pg-atom/l. 454 mu Absorbanceb in dist illed water in 5-cm cell of added urea-N or ammonia-Na 454 mfi 630 mp Standard sample Blank sample 630 mp 0.011~ f 0.0006d 0.033c f 0.001 0.063O f 0.001 0.062 f 0.0004 0.00 0.044 f 0.002 0.018c f 0.001 0.047 f 0.0007 0.07Y f 0.001 0.50 0.063 f 0.0008 0.052c f 0.001 0.025c f 0.001 0.059 f 0.0004 0.08@ f 0.002 1.00 0.063 f 0.0008 0.060 f 0.002 0.041 f 0.002 0.106" f 0.002 0.090 fO.0005 2.00 0.064 f 0.001 0.080 f 0.001 0.147 f 0.002 4.00 0.116 f 0.002 0.315 f 0.004 0.121c f 0.001 0.27@ f 0.002 10.0 0.064 f 0.0005 0.223 f 0.002 0.222c f 0.004 0.063 f 0.001 0.582c f 0.005 O.46lc f 0.002 0.393c f 0.003 20.0 1.03 f 0.01 0.838O f 0.006 0.065 f 0.001 0.425" f 0.003 40.0 0.663 f 0.006 0.8376 j=0.004 1.54c f 0.01 0.057 f 0.006 80.0 1.28 f 0.01 2.31 f 0.01 125.e 0.047 f 0.003 2.61 f 0.03 250." 4.94 f 0.02 500.e 9.13 f 0.09 1000.~ a 1.00 pg-atom N/1. = 1.40 X mg N. * All absorbances are determined with a Beckman model DU spectrophotometer. c These means are averages of seven or more determinations. All others are averages of three to six determinations, d The estimate of dispersion is plus-or-minus one standard deviation. These absorbances were obtained with a shorter cell and adjusted to a 5-cm light path.
"T",in a 25-ml volumetric flask containing a stirring magnet. Add 3.0 ml of distilled water. Begin to stir vigorously without a vortex and continue stirring. Add 0.30 ml of NaOCl reagent and follow with 0.50 ml of NaOBr reagent. After 1 to 2 more seconds, add (0.30 X) ml of HCl reagent. After 10 to 20 more seconds, add 0.50 ml of borate buffer reagent. After 1 to 2 more seconds, add 0.30 ml of phenol reagent and stir for 10 to 20 more seconds. After 10, but not more than 20 more minutes, compare the absorbance of the reacted sample at 454 mp with that of distilled water. The urea blank procedure is slightly different from the general procedure. The blank signal is obtained by adding 3.0 ml of distilled water to a duplicate aliquot of the sample at T f 2 "C in a flask. Begin to stir vigorously. Then add 0.50 ml of NaOBr reagent. After 10 to 20 minutes, add 0.30 ml of NaOCl reagent. Add (0.30 X ) ml of HC1 reagent and proceed exactly as in the general procedure. The urea standard procedure is identical to the general procedure except that the 3.0-ml addition of distilled water is replaced by 3.00 ml of urea solution of a known concentration chosen so that the concentration of added urea approximates the concentration in the original sample. The urea absorbance is a linear function of concentration through 1000 pg-atoms urea-N per liter as can be shown from the data summarized in Table 11. If any of the reagents are renewed or if a particular sample differs from the previously accepted urea blank and standard
+
+
by more than 4 OC or 2% salinity, separate blank and sample signals should be obtained. AMMONIA.The optimal ammonia procedure differs from the urea in pH variations as shown in Table I, in the rate of development and linearity of the signal, and in the blank procedure. To obtain the general ammonia signal, place a 20.0-ml aliquot of the natural sample, dialysate, or filtrate at temperature "T" in a 25-ml volumetric flask containing a stirring magnet. Add 3.0 rnl of ammonia-free water, begin to stir vigorously without a vortex and continue stirring. Add 0.40 ml of NaOCl reagent and follow within 10 seconds with 0.25 ml of HCl reagent. After 10 to 20 more seconds, add 0.20 ml of NaOH reagent. After 1 to 2 more seconds add 0.30 ml of phenol reagent, follow by 0.40 ml of NaOH and stir for 10 to 20 seconds. After 25 but not more than 35 minutes, compare the absorbance of the reacted solution at 630 mp with that of distilled water. As is shown in Figure 1 from the data in Table 11, the ammonia absorbance is a linear function of concentration only through 20 pg-atoms per liter. The ammonia blank signal is obtained by means of the following formula: B, = B~ - ( B ~ - B ~ ) B~ - El. B, represents the blank absorbance to be used in the calculation of the ammonia concentration. B1 represents the absorbance of solution I which consists of distilled water processed by the general ammonia procedure. B1 represents
+
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the average of the B, absorbances obtained for a given general ammonia procedure with contamination-free reagents. BZ represents the absorbance of reacted solution I1 which is an aliquot of the sample. Stir solution I1 with 0.20 ml of NaOBr reagent for 5 to 10 minutes and treat by the general ammonia procedure, adding 0.05 ml extra HCl reagent. Ba represents the absorbance of reacted solution I11 which is also an aliquot of the sample and is treated as solution I1 except that twice as much NaOBr reagent and extra HC1 are added. The ammonia standard signal is obtained by reacting an aliquot of the sample by the general procedure replacing the 3.0-ml addition of distilled water with 3.00 ml of ammonia standard solution of known concentration. This concentration should be chosen, and if an unusually high concentration of ammonia is found, the sample should be diluted to keep the signal on the linear portion of the Beer’s law curve. A separate ammonia blank and standard should be obtained for each sample which differs from the previously accepted blank or standard by more than 4 “C or 4% in salinity or if any of the reagents are renewed. SAMPLING.The sampling and storage of water for ammonia analysis is especially critical because of the possibility of airborne contamination and continued biological activity after the sample has been obtained. An ammonia sample should therefore be drained directly from the Nansen or Knudsen bottle into a twice-rinsed storage bottle, capped immediately and analyzed within the hour if stored at greater than 25 “C. If refrigerated below 5 “C, it may be stored for up to 12 hours. An ammonia sample should be filtered only with extreme caution to prevent contamination. A urea sample is not as susceptible to contamination but because urea is produced by some metabolic and decay processes and is decomposed by bacteria (1) at such a rate that in open ocean surface water a diurnal maximum occurs in the early morning hours (13), it should be analyzed as promptly as the ammonia sample. Filtration will not significantly inhibit the bacteria, but it will remove the urea producing organisms and prepare the sample for freezing. Because of the possibility of organism rupture producing anomalously high values, neither urea nor ammonia samples should be frozen without filtration (12). Urea and ammonia may be separated from colloidal samples by dialysis. CALCULATION. Within the stated ranges of linearity, the concentration of urea nitrogen and ammonia nitrogen may be computed by means of the following formula : Nsam
=
ABsm Astd
- Abik
- Aaam x
a/23(Netd).
NSam equals the sample concentration of urea or ammonia nitrogen
Natdequals the urea or ammonia nitrogen concentration of the standard sample A., equals the sample absorbance in an n-cm cell Ablkequals the blank absorbance in an n-cm cell Aatdequals the standard sample absorbance in an n-cm cell For example, if Ana,,, were 0.082 absorbance unit, A.M 0.129 absorbance unit, A b l k 0.038 absorbance unit, and NaM10 pg-atoms N per liter, then by the formula, N., would be 0.122 pg-atom per liter. DISCUSSION
In concentrated solutions of urea or ammonia, the color signals can be formed by adding the essential reagents, hypochlorite and phenol, in almost any proportion. In more dilute solutions such as natural waters, however, to maximize the precision and minimize the blank, the conditions must be (13) R. T.Emmet, unpublished work, 1968. 1650
ANALYTICAL CHEMISTRY
i
I
10
DISTILLED WATER
J
I 20
30
I
I
40
MICROGRAM ATOM AMMONIA PER LITER
Figure 1. Calibration curves for ammonia procedure in distilled water and seawater from data given in Table I1 more carefully controlled. The following remarks sketch the evolution of the recommended optimal procedures. Choice of Reaction Conditions. GENERALPROCEDURES. Under optimal chlorination conditions, a variable decrease in color intensity was noticed when a large surface area of the reacting solution was exposed to the air. When a small reproducible area was exposed as in the neck of a volumetric flask, the yield and precision increased markedly. When the phenol was added directly to the chlorinating solution, the blank signals were high and the urea signal was unstable. In the urea procedure, these problems were solved by adding a borate buffer prior to the phenol. With this addition, the pH was raised reproducibly and the rates of color development and fading became more manageable. In the ammonia procedure, an addition of NaOH prior to the phenol reduced the blank signal and a larger addition following the phenol increased the rate of signal development. When KCl and HzOzwere added with the borate buffer and MgClz with the urea HCl reagent, the rate of urea signal development increased and the precision was higher between replicate samples. BLANKPROCEDURES. Urea and ammonia are oxidized completely by OBr- in the cold (14). When equivalent OBrwas added with the other reagents and brought quickly to a lower pH, as in the general urea procedure, a standard urea signal is not diminished. The slight absorbance which is added to the reacting solutions by side reactions of the NaOBr reagent is equivalent in all three urea procedures and is subtracted to yield the ammonia blank signal. The ammonia blank procedure also utilizes the OBr- oxidation but because (14) C. A. Jacobson, “Encyclopedia of Chemical Reactions,” Vol. V, Reinhold Publishing Co., New York, 1956,pp 98 and 143.
Br- interference prevents the detection of ammonia in the reagents, additional operations are required. CRITICAL TIMEINTERVALS. In the general urea procedure, the 1- to 2-second interval between the additions of NaOBr and HC1 is necessary to prevent the alkaline OBr- from reacting with the sample urea. The lower limit of the 10- to 20-second interval, after the addition of HCl is required to reproducibly mix and chlorinate replicate urea samples. The upper limit of that interval is necessary to avoid decreasing the signal sensitivity. The 1- to 2-second interval before the addition of phenol is required to prevent the chlorination equilibria from reversing at the higher borate buffer pH (15). The time limits for the development and reading of the urea signal can be conveniently adjusted by varying the terminal PH. In the ammonia general procedure, the 10-second upper limit between the additions of NaOCl and HC1 prevents a reduction in sensitivity. The 10- to 20-second chlorination interval permits reproducible chlorination, and the 1- to 2-second interval before the phenol addition prevents the ammonia chlorination from being reversed (15). The limits for the development and reading of the ammonia signal are also pH dependent and permit adequate signal development while avoiding interference from urea, biuret, and allantoin, which give rise eventually to an absorbance at 630 mp. Signal Stability. Once the ammonia signal develops, it is stable. The rate of development, however, increases with increasing terminal pH, but so does the rate of conversion of urea-yellow to ammonia-blue. Therefore, in the analysis for ammonia in solutions containing both urea and ammonia, to avert urea interference in the ammonia signal, a minimal terminal pH is recommended and the absorbance at 630 mp should be obtained as soon as possible after maximal signal development. The rates of development and fading of the urea signal increased with increasing terminal pH, temperature, and salinity. In the recommended urea procedure at 25 “C after maximal development, the urea signal fades at rates of 1 to 2 z per hour in distilled water and 3 to 4z per hour in seawater. The urea blank signal never reaches a maximum, but its growth can be retarded by keeping the terminal pH within the suggested limits. Temperature and Salt Effects. The temperature of the reacting sample did not affect the magnitude of a urea signal, and the ammonia signal increased only slightly with decreasing temperature. The sensitivity of the urea signal increased 90% with increasing salinity from fresh water to 35% seawater. Tests showed that the increase was due to the presence of divalent cations. The sensitivity of the ammonia signal decreased 40% with the increasing salinity between fresh water and 35% seawater. Tests showed that the Br- ion caused the inhibition. Specificity of Reactions. Dilute distilled water solutions of several nitrogenous compounds were reacted by the general urea and ammonia procedures, and the results are summarized in Table 111. At 630 mp, ammonia is the sole primary source of absorbance in the group of compounds tested. The allantoin and biuret colored compounds are thought to be identical with yellow urea compound because of similarity of reaction and absorbance characteristics. Of all the amino acids tested, tyrosine gave the sole positive reaction. Distilled
(15) R. E. Corbett, W. S. Metcalf, and F. G. Soper, J. Chem. SOC., London, 1953, 1927-9.
Table 111. Absorbance Indices of Several Nitrogen Compounds Gram-atom nitrogen absorbance index in absorbance unitsa Cm cell X gram-atom nitrogen per liter 454 mp 630 mp
Compound Acetamideb+J UC U Allantoin 1 . 6 X loa U Ammonia (including NaOBr reagent) u U Ammonia (excluding NaOBr reagent) 0.35 x 103 5.6 X 108 1 . 6 x 103 U Biuret Tyrosine 1.0 x 103 U Urea 2.2 x 103 U All absorbances were read with a Beckman model DU spectrophotometer. All solutions comprise distilled water and 40 pg-atoms N/liter. “u” represents a low absorbance index which could not be distinguished from the blank. Negligible interference was also encountered at 454 mp and 630 mp using 40-pg-atom N/1. solutions of alanine, arginine, asparagine, aspartic acid, barbital, caffeine, citrulline, creatine, creatinine, cystiene, diphenyl urea, glutamic acid, glutathione, glycine, hippuric acid, histidine, isoleucine, leucine, lysine, methionine, methyl urea, monoethanolamine, phenylalanine, phenyl urea, n-propylamine, semicarbazide, serine, threonine, and uracil. Table IV. Absorbance of Distilled Water Solutions of Tyrosine pg atoms/l. of added tyrosine-N
0.0
25.0 50.0 100.
Absorbance at 375 mp in a 5-cm cella 0.032b f 0.001~ 0.290 f 0.002 0.582 f 0.002 1.136 f 0.006
a All readings were made with a Beckman model DU spectrophotometer. All means are averages of two or more determinations. The estimate of dispersion is plus-or-minus one standard deviation.
water-tyrosine solutions, which were reacted by the general urea procedure, excluding the NaOBr reagent, developed signals within two minutes that were stable for several hours. The results in Table IV show that the tyrosine signal at 375 mp has a molar absorbance index of 2.2 X lo8 and is linear through 100 pg-atoms nitrogen per liter. Both urea and ammonia interfere with the tyrosine signal, but when neither is present, the urea blank procedure produces a valid tyrosine blank signal. Br- inhibits the tyrosine and ammonia signals to the same extent. Precision of Urea and Ammonia Methods. The data given in Table I1 reflect the reproducibility of salt effect of urea and ammonia standard samples. At 1.0 pg-atom urea nitrogen per liter, the standard deviation of a set of seven standard seawater absorbances is equivalent to 0.1 pg-atom urea nitrogen per liter. The standard deviation of the set of nine seawater blank absorbances in the three most dilute groups is equivalent to 0.05 pg-atom urea nitrogen per liter, and the variances sum to 0.0125 pg-atom2 urea nitrogen per liter2. Therefore, the estimated standard deviation of the urea nitrogen detected at 1.0 pg-atom per liter is 0.11 pg-atom urea nitrogen per liter. At the 95 con-
z
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fidence level for a sample to be significantly different from a blank, it must contain urea equal to twice the standard deviation. The lower limit of detection of the analysis in seawater is thus 0.2 m a t o m urea nitrogen per liter and the estimated relative standard deviation is 10% at a urea nitrogen concentration of 1.O pg-atom per liter. Similarly, from the ammonia seawater data, for a sample to be significantly different from the blank at the 95% confidence level, it must contain at least 0.2 pg-atom ammonia nitrogen per liter. The relative standard deviation is 20%
at the ammonia nitrogen concentration of 0.5 pg-atom per liter.
for review February 11, 1969, Accepted July 11, 1969. The principal research was sponsored by the Naval Ship Systems Command ocean science program, Work was taken in part from a thesis by the author submitted in partial fulfillment of the M.A. degree in oceanography at The Johns Hopkins University. The opinions and assertions of the writer are his alone and do not necessarily reflect those of the Navy or the Naval establishment at large.
NOTES
Spectrophotometric Determination of Uranium(V1) with Z-(Z-Pyridylazo)-5-diethylaminophenol T. M. Florence, D. A.
Johnson, and Yvonne J. Farrar Analytical Chemistry Section, Australian Atomic Energy Commission, Lucas Heights, N.S.W., Australia
A RECENT REVIEW (1) showed the wide application and rapid growth in popularity of heterocyclic azo dyestuffsin analytical chemistry. Some dyes of this class have proved particularly useful for the spectrophotometric determination of uranium. The first such dye, 1-(2-pyridylazo)-2-naphthol(PAN) was superseded by the water-soluble, and more sensitive reagent, 4-(2-pyridylazo) resorcinol (PAR) (2, 3). At pH 8 uranium (VI) forms an intense red color with PAR, the complex having a molar absorptivity of 3.87 X lo4 at 530 nm (4). Florence and Farrar ( 4 ) developed a mixed complexing solution containing (1,2-cyclohexylene-dinitrilo)tetra-acetic acid (CyDTA) sulfosalicylate, and fluoride which effectively masks most metals, including thorium, and prevents their interference in the PAR method for uranium. This method has been in use in our laboratories for over six years, and has proved reliable, accurate, and versatile. We have now synthesized the diethylaminophenol analog of PAR, 2-(2-pyridylazo)-5-diethylaminophenol (PADAP). This new reagent is almost twice as sensitive towards uranium (VI) as PAR, with a molar absorptivity of 7.61 X l o 4 at pH 8.2 and 564 nm. In the presence of the mixed complexing solution it is also even more selective than PAR, and because the color is developed in an acetone-water mixture, uranium can be determined directly in methyl iso-butyl ketone (MIBK) extracts, By a simple preliminary extraction of uranium into MIBK from a calcium nitrate-EDTA medium, the PADAP method can be made specific for uranium.
Dry ethyl ether (20 ml) and 2-aminopyridine (5 grams in 5 ml ether) were added while nitrogen was passed through the flask. The mixture was gently refluxed under nitrogen for 30 min, and iso-amyl nitrite (5 ml) added. Refluxing was continued for another hour. The precipitated sodium 2pyridyldiazoate was filtered, washed with ether, and dried under vacuum. The diazoate is unstable in air and should be used immediately, or refrigerated in sealed containers. Sodium 2-pyridyldiazoate (8.6 grams) was dissolved in cold ethanol (10 ml) and added to a solution of m-diethylaminophenol (10.0 grams) in cold ethanol (20 ml). The mixture was kept cold in an ice-bath, with COZpassing through the solution, while coupling took place. Carbon dioxide was passed until nearly all the ethanol had evaporated. The reddish reaction mixture was dissolved in 20 ml of 10M HC1 and diluted to 1 liter with water. Zinc chloride (25 grams dissolved in a little 1M HCl) was added and the pH of the mixture gradually raised to pH 4.5-5.0 by the addition of 5M NaOH, while stirring vigorously. The precipitated zinc salt of PADAP was filtered under suction, and washed with water followed by 50% ethanol. The product was dried in a vacuum oven at 70 "C. The crude product is suitable for use as an analytical reagent without further purification, because the impurities do not react with uranium or hinder color formation. The pure zinc complex was obtained by double recrystallization from an 8:l benzene-ethanol mixture. Found: C, 48.5%; H, 4.90%; N, 15.0%; Zn, 17.6%. Calcd. for ClsH17N40C1.Zn: C, 48.7%; H, 4.6%; N, 15.2%; Zn, 17.6%. A qualitative test for chloride was positive.
EXPERIMENTAL Reagents. Synthesis of PADAP (zinc complex). Sodium amide (2.1 grams, as a 50% suspension in toluene) was introduced into a dry 250-ml flask fitted with a reflux condenser. (1) R. G. Anderson and G. Nickless, Analyst, 92, 207 (1967). (2) W. J. Geary, G. Nickless and F. H. Pollard, Anal. Chim. Acta, 26, 575 (1962). (3) L. Sommer, V. M. Ivanov, and H. Novotna, Talanta, 14, 329 (1967). (4) T. M. Florence and Y.J. Farrar, ANAL.CHEM., 35, 1613 (1963). 1652
ANALYTICAL CHEMISTRY
H' 0 PADAP
The PADAP reagent solution was prepared by dissolving 0.05 grams of Zn-PADAP in 50 ml of acetone, and diluting to 100 ml with water. The solution is stable indefinitely. The complexing solution was made by suspending 50 grams
