0.00 to 0.08 pg. vanadium calibration curve was 1 0 . 2 for samples 1 and 2 which contained, respectively, 0.8 and 6.8 pg. of vanadium per liter. Using a 0.0 to 1.0 pg. vanadium calibration curve, the standard deviations for samples 3 (18 pg. per liter) and 4 (92 pg. per liter) were 1 0 . 6 and 1 0 . 8 , respectively.
LITERATURE CITED
(1) Haffty, J., U . S.Geol. Survey WaterSupply Paper 1540-A,1 (1960). (2) Jarabin, Z . , Szarvas, P., Acta b'niv. Debrecen 7, 131 (1961); ( ' . A . 57, 9192e (1962). ( 3 ) Naito, H., Sugawara, K., Bull. Chem. Sac. Japan 30, 799 (1957); C . A . 52, 5706i (1958).
(4) Silvey, FV. D., Brennan, R., ANAL. CHEM.34,784 (1962). ( 5 ) Sugawara, K., Tanaka, M., Saito, H., Bull. Chem. Sac. Japan 26, 417 (1953); C.,4.49. (3842b(1958). RECEIVEDfor review Rlarch 9, 1964. Accepted April 27, 1964. Division of Water and Waste Chemistry, 147th Meeting, ACS, Philadelphia, Pa., April 1964. Publication approved by the Director, L.S. Geological Survey.
Fluorometric Method for the Determination of Urea in Blood JOSEF E. McCLESKEY Clinical Chemistry Branch, U . S. Naval Medical School, National Naval Medical Center, Bethesda, Md.
b It has been observed that the compound produced by the reaction of urea with diacetyl monoxime exhibits fluorescence. A study of this fluorescent property has resulted in the development of a quantitative procedure for the determination of urea. The variables studied include the method of deproteinization of blood and serum, the time of heating, the concentration of diacetyl monoxime and the effect of pH. Comparison studies with the AutoAnalyzer and recovery studies show the method to b e valid.
T
fop a simple and accurate method for quantitatively determining urea nitrogen in blood has provided impetus for the reevaluation of several urea nitrogen procedures. A variety of methods have been developed utilizing enzyme reactions to form ammonia with subsequent titration (3). Several objectionable features to these methods are evident, via., loss of ammonia and time consumed. Several methods have also been published which utilize the reaction of urea with diacetyl ( 4 , 6), diacetyl monoxime ( 5 , 7-10)] or a-isonitrosopropiophenone (1) in acid media to form a colored compound. Color intensities are then measured to yield urea concentration. The methods using diacetyl or diacetyl derivatives usually suffer from the fact that the color produced does not conform to Beer's Law and is photosensitive. Experimentation oriented toward develoliment of a blood urea nitrogen method with more desirable characteristics led to the investigation, in our laboratory, of the method of Richter and Lapointe (9). During the course of this work a fluorescent property of the ureaalpha diketone compound was disHE NEED
1646
ANALYTICAL CHEMISTRY
millilit,ers of water. Dilut'e to 100 ml. This reagent is stable for several months at' room temperature. DI~CETY ~ ~LO N O X I M E .Dissolve 5.0 grams of diacetyl monoxime (Eastman Organic Chemicals) in 500 ml. of distilled water. Add 150 grams of sodium chloride, 100 ml. of distilled water and shake well until dissolved. Dilut'e to 1000 ml. a n 3 filter. If stored in an EXPERIMENTAL amber bottle a t room temperature this The fluorescent properties of the comreagent is stable for about six weeks. pound formed by the reaction of urea Procedure. The recommended proand diacetyl monoxime were studied cedure for the fluorometric determinawith a n Aminco-Bowman spectrophototion of blood urea nitrogen is as fluorometer. The fluorescent peak was follows: observed a t a wavelength of 415 mp Add 0.2 ml. of whole blood or serum when the compound was activated a t an to 4.0 ml. of distilled water. Mix well optimal wavelength of 380 millimicrons. and allow to stand for 10 to 15 minutes. Reagents and Solutions. VREA Add 5.0 ml. of 30Y0 trichloroacetic acid NITROGENS TOCK STANDARD. 1 MG. to precipkate the prot,ein. Mix well PER ML. Weigh out 2.14 grams of and centrifuge at 2500 r.p.m. for 15 urea (c.P., A.c.s.) and transfer to a minutes or, alt,ernat'ively, filter t'hrough 100-ml. volumetric flask. Add 50 ml. Whatman #40 filter paper. Pipet 2.0 of 0.Ol.V sulfuric acid and swirl to ml. of the protein-free filtrate into a dissolve. Then dilute to 100 ml. screw cap test t'ube (16 X 125 mm.). with 0.01,V sulfuric acid. .4dd 2.0 ml. of diacetyl monoxime and UREA NITROGENWORKINGSTAXD- 0.30 ml. of concentrated sulfuric acid. ARD, 40 MG. PER100 ML. Dilute 4 ml. Screw the cap down tightly and mix of the stock standard to 100 ml. with well. Heat the tube in a boiling water 0.01N sulfuric acid solution. bath for 15 minutes. After heat'ing, TRICHLOROACETIC ACID REAGENT, release the pressure by loosening t'he 30%. Dissolve 30 grams of reagent cap, mix well, and allow to cool to grade trichloroacetic acid in a few room temperature. Run a standard (40 mg. per 100 ml.) in the same manner by using 0.2 ml. of the working standard in place of blood or serum. Measure Table I. Typical Calibration Data for the fluorescence in the spectrophotoDetermination of Urea in Blood and fluorometer a t a wavelength of 415 mp Blood Serum with an activation wavelength of 380 Urea nitrogen Fluorometer mp, Use the 40 mg. per 100 ml. concn. mg./100 ml. reading standard to adjust the instrument to a predetermined fluorescence. An 8070 0 0 0.0 adjustment is used in t8his laboratory. 9 0 10.0 18 0 15.0 Read t,he results from a previously 20.0 26 0 prerared calibration curve. 37 0 25.0 51 0 30.0 RESULTS A N D DISCUSSION 64 5 35.0 40.0 80 0 Table I shows a typical calibration Each sample contained 2.0 ml. of diadata using the procedure described cetyl monoxime and 0.30 ml. of concenabove, but substituting standard urea trated sulfuric acid. nitrogen solutions for blood and serum covered. Subsequent work indicated that this fluorescence could be adapted to a quantitative procedure for urea. A more complete study of reagents and reagent concentrations provided an accurate, simple, and reproducible procedure for urea nitrogen in blood.
55-
1
Table II. Comparison of the Fluorescent Method with the AutoAndyzer
Relative error,
Mg./100 ml.
I
, , .M
.lo
, .I5
, 2 0
, .25
,
,
,
.30 -35
,
.LO
4 5
, .50
, .55
,
,
.60 .65
, , .70 *75
1
CONCD. HzSOIADDED, MI. Figure 1 .
Effect of sulfuric acid concentration on fluorescence
samples. These solutions are prepared by making appropriate dilutions of the working standard. The points on the calibration curve can be reproduced from day to day within experimental error (*I%) when the same reagents are used. However, when new reagents or standards are used a new calibration curve has to be prepared. The concentration of urea in blood found by this procedure was compared with the results of the AutoAnaEyzer (Technicon Instrument Co., Chauncey, N. Y.) under rout'ine conditions. Description and operat'ion of the AutoAnalyzer are discussed in reference (11). The agreement between the two methods was satisfactory and showed an average relative error of 0.617, when the ;iutoAnalyzer results were taken as valid. The data from the comparison studies are shown in Table 11. The reproducibility of this procedure is shown in Table 111. Each sample was run a minimum of four times with mean errors and standard deviations being calculated. The standard deviations for the series' shown range from 0.11 to 0.18 with an average of 0.13. Recovery studies were made by adding known amounts of urea to blood samples which had previolisly been analyzed by this method and by the duto.4nalyzer. These determinations were carried out in quadruplicate. Results are shown in Table IV. The sensitivity of this method is much greater than the colorimetric procedures. Using a 0.2-ml. sample the concentration of urea nitrogen is 1-5 pg./ml. of test solution (1.7 X 10-5N). However, a sample as small as 0.025 ml. may be used which yields a concentration in the final volume of test solution of 3 X 10-6.\f. Selection of Method of Deproteinization. Several methods of deproteinization of biological samples were tried. .\fter considering simplicity and time needed, the trichloroacetic
acid method was selected. I t was found t h a t by laking 0.2 ml. of blood in 4.0 ml. of water, followed by deproteinization with 5 ml. of 3070 trichloroacetic acid, a satisfactory filtrate could be obtained.
Fluorescent method
duto-
Mean
Analyzer
error
24.1 f 0 . 1 18.1 f 0.1 26.8 f 0 . 2 22.3 f 0 . 0 33.6 f 0 . 1 10.1 f 0 . 1 26.8 f 0 . 2 7 . 9 rt 0 . 1 16.9 f 0 . 1 20.2 f 0 . 2 24 l r t 0 1 209fOl 7 0 f 0 0 131fOl 142fO2 18.0 f 0 . 2
24 18 27 22 34 10 27 8 17 20 24 21 7 13 14 18
+o. 1 +o. 1
23OfO2 292503 2 9 2 f 0 2 13OfO2
23 29 29 13
yo
41 55 74 36 1 17
0 0 0 1
-0.2 +0.3 -0.4 +O.l -0.2 -0.1 -0.1 +0.2 $0.1 -0.1
1 00 0 74
1 25
0 58 1 00 0 41 0 47 0 00 0 76 1 42
0.0
+o. 1 +0.2
0.0
o on
0 0
000
-02 -02 0 0
069 069 000
Average mean error = 0.13 Average relative error = 0.657'
Effect of Time of Heating at 100' C. T h e formation of the urea-alphadiketone compound increases with time while heating a t 100" C. (2,4). The time required to produce maximum formation, as reported by other workers, varies from 20 to 35 minutes. I t has been observed t h a t by heating a t 100' C. under a positive pressure the optimum heating time for maximum fluorescence is from 14 to 16 min.
Effect of Varying Monoxime Concentration.
Diacetyl
Table 111. Reproducibility of Determinations
Mg./100 ml.
Sample
#
Urea nitrogen
LZIean value
53
8.0
7 9
53 53
7.8
-53 __
8
n
7.8 7.8 20.8 21 . o 20 8 20 8 20 8 29 2 29 0 29 2 29 2 29 1 13 0 12 7 13 0 13 2 13 1
Mean error
Standard deviation
+O 1
0 11
+n , -
1 ~
-0.1 -0.1 -0.1 0.0 +0.2
Varying the amount of diacetyl monoxime used in a sample produced significant changes in fluorescence intensity. The fluorescence increased with addition of diacetyl monoxime u p to 2.0 ml. With addition of more than 2.0 ml., the fluorescence intensity decreased slightly. This can probably be a t tributed t o a quenching effect of the reagent itself. Based on this finding it was decided to use 2.0 ml. of diacetyl monoxime in the procedure.
65 65 65 82 82 82 82 82 83 83 83 83 83
Effect of Varying Acid Concentration. Varying amounts of concen-
Average Standard Deviation = 0.13
53
65 66 ~~
20.8
0.12
29.1
0.0 0.0 0.0 +o. 1
0.11
13.0
0.0 0.0
0.18
-0.1 +0.1 +o. 1 +0.3 0.0
+0.2 +o. 1
trated sulfuric acid were added t o
Table IV.
Recovery Studies
Mg./100 ml.
Sample value 13 13 18 9 9 9
0 0 1
2 2 2
Urea nitrogen added 10 20 10 10 20 18
0 0 0 0
n 1
Theoretical value 23 33 28 19 29 27
0 0
1 2 2 3
Urea nitrogen
Added urea N recovered
Recovered,
22 33 28 19 29 27
9 8 20 1
98 0 100 5 100 0
found 8 1 1 0 0
2
10 0 9 8
19 8 18 0
7c
98 0 90 0
99 5
These data are the average of at least four separate determinations on each sample
VOL. 36, NO. 8 , JULY 1964
1647
samples of a constant concentration of urea nitrogen, and the fluorescence was measured. As seen in Figure 1, the fluorescence increased as the amount of sulfuric acid increased. The maximum fluorescence intensity occurred after the addition of 0 . 3 ml. Further addition caused a decrease in the intensity of fluorescence. This was the amount of acid selected for use in the procedure. The specificity of this reaction for urea has been discussed in p r e v i o a publications (2, 10, 1 8 ) . These authors show that contribution by similar compounds such as uric acid, et'c., is relatively insignificant, due to their minute presence in these samples. ;1 preliminary effort to adapt this procedure to the Coleman Model 12C
LITERATURE CtTED
B., '4m. J . Clin. Path. 24, 981 (1954). (5) Frieaman, H. S., ANAL.CHEM.25, 662 (1953). ( 6 ) LeMar, R. L., Bootzin, D., Ibid., 29, 1233 11957). ( 7 ) satelson,' S., Scott, M. L., Beffa, C., Am. J . Clzn. Path. 21, 275 (1951). ( 8 ) Ormsby, A. A., J . Bzol. Chem. 146, 595 (1942). ( 9 ) Richter, H. J., Lapointe, Y. S., Clin. Chem. 5 , 617 (1959). (10) Rosenthal, H. L., AXAL.CHEM.27, 1980 (1955). (11) Skeggs, L. T., Am. J . Clin. Path. 28, 311 (1957). (12) Wheatley, V. R., Biochem. J . 43, 420 (1948).
( l ! Archibald, R. M.,J . Bzol. Chem. 167, 007 (1945). ( 2 ) Beale, R. N., Croft, D., J . Clin. Path. 14, 418 (1961). ( 3 ) Day, H. G., Bernstorf, E., Hill, R. T., ANAL.CHEM.21, 1290 (1949). ( 4 ) Dickenman, R. C., Crafts, B., Zak,
RECEIVED for review January 21, 1964. Accepted April 21, 1964. The opinions and views expressed are those of the author and are not to be construed as official or necessarily reflecting those of the 3ledical Department of the 1Jnited States Savy or the Saval Service at large.
photofluorometer has thus far proved unsuccessful. This may be the result of poor resolution of the filter system which we have employed. K o r k is continuing on this modification in instrumentation. ACKNOWLEDGMENT
The author thanks T. E. Wheeler and R. I. Morgan for their interest and assistance during the course of this invest igat ion.
Polarographic Behavior of Silver in Cyanide Solutions ROBERT F. LARGE and EDWIN P. PRZYBYLOWICZ Research laboratories, Eastman Kodak
Co., Rochester, N. Y. I4650
b The polarographic behavior of aqueous solutions of the argentocyanide ion has been investigated. A combined anodic and cathodic wave with an E l l 2 of -0.18 volt vs. SCE was observed with a solution 1 .O X 1 OP3Min argentocyanide ion, buffered at a pH of 6.7 and containing no excess cyanide ion. The wave has been shown to be the result of the anodic dissolution of mercury in the presence of cyanide ion and the reduction of mercuric cyanide, both species having been produced by a rapid chemical reaction between mercury and the argentocyanide ion: Hg f 2 Ag(CN)2-$ Hg(CN)Z f 2 Ag
+ 2 CN-
The analytical utility of a previously reported method has been evaluated in the light of the mechanism presented in this work, with an extension to include the simultaneous polarographic determination of silver and mercury.
T
of silver ion has been described by a number of authors (2-4, 9, 13). With a dropping mercury electrode and the usual solution conditions, silver does not give a welldefined reduction wave, but rather a wave which merges with the anodic dissolution curve of mercury. Although the diffusion current may be measured and related linearly t o the concentration of silver. no distinct step-wave is observed. This results from the fact that silver ion is reduced at a potential H E POLAROGRAPHY
1648
ANALYTICAL CHEMISTRY
which is more positive than that for the dissolution of mercury. The simultaneous reduction of ions of other metals more noble than mercury, as well as species of mercury itself, constitutes a serious interference. As a result, polarography does not offer a selective measure of silver ion, and it3 application in this area has been limited. Complex formation has not proved successful in efforts to shift the polarographic wave away from the mercury dissolution curve. In such cases, the system usually contains an escess of the complesing agent, most of which form more stable complexes with mercury than with silver. Thus, the relative position of the polarographic wave for the silver complex, as compared with that of the anodic dissolution wave of mercury, cannot be altered and again no step-wave is observed. Recently, Dagnall and R e s t (3) have reported a procedure whereby a stepwave is obtained with solutions of the argentocyanide ion in which the excess complexing agent is masked through the use of a metal ion which forms a slightly less stable complex with cyanide ion than does silver. Nickel(I1) was chosen as the masking agent for this purpose since it forms a relatively stable cyanide compleh, and the excess nickel (11) would not constitute an interference polarographically. This use of masking reactions is novel in polarographic work and could well find application in other similar systems. However, our attempts to use the system described by Dagnall and
West led to unexpected results. Cnlike the results reported by these authors, a step-wave was observed for silver which was a composite anodiccathodic curve, the ratio of anodic to cathodic currents being approximately 1 : 1. It thus appeared that the electrochemical process was not simply the reduction of the argentocyanide ion, but a process of a more complex nature. This combined wave was observed previously by Bowers and Kolthoff ( 1 ) in studies of the induced reduction of colloidal silver bromide by the argentocyanide ion. However, the electrode processes were not completely characterized by these authors. An investigation was undertaken to define the nature of the observed wave, and to evaluate the analytical utility of the Dagnall and West procedure in view of these new developments. EXPERIMENTAL
Apparatus. All polarographic d a t a exclusive of t h a t associated with the tracer experiments were obtained with a Sargent Model XV Polarograph, equipped with a pen speed of 3 . 3 seconds for full-scale, deflection. T h e polarographic cells and auxiliary equipment used were those marketed by Metrohm Ltd. A silver-silver chloride reference electrode was employed. Solution contact was made through a saturated potassium nitrate salt bridge. The capillary employed had a drop time of 4.73 seconds and a rate of mercury flow of 1.86 mg. per second at an applied potential of -0.10
