of 0.7 ppb for stripping in stirred solution. The error in quiet solution would be considerably less. Electrode Calibration Curves. Quantitative results were obtained either by the method of standard addition (with a blank correction) or by means of a calibration curve when a large number of samples were to be analyzed. A separate calibration curve is required for each CGE because of small but significant differences in area between individual electrodes. These curves were prepared by determining electrode response for successive 1:2 dilutions of the most concentrated standard until the lead content of the final dilution was -1 ppb. Duplicate determinations on each dilution beginning with a blank on the reagents were analyzed by the method of least squares. The results for a typical calibration in the 0-100 ppb range were: lead (ppb) = 98.14 mC - 4.88, where mC is the integrator reading (millicoulombs), and the standard deviations (a) for 18 points were: u (single determination) = f1.8 ppb; a(s1ope) = *1.3 ppb/mC; and a(intercept) = h0.56 ppb. The intercept represents the contribution of background processes and the lead content of the demineralized laboratory-distilled water. When the solutions were prepared with reagent-grade sulfuric acid, the intercept increased to -9 ppb. The sensitivity of the method was estimated by means of the Student's test (11). If a large number of blank determinations, n, have been run such that the standard deviation, a , of the blank is known, then the equation for the difference between an unknown and a blank can be written, (11) W. J. Youden. "Statistical Methods for Chemists." John Wiley & Sons, New York, N.Y., 1951
t = - AS
.1/2
where t = value obtained from t Table with N = 1 degree of freedom, A S = difference between sample and blank determinations, and a = standard deviation of the blank. The average blank in this study was equivalent to 1.0 0.1 ppb lead. Thus, with t = 6.314 (0.1 confidence level), a difference of 0.9 ppb can be detected with 90% certainty. Interferences. Representative rain and surface water samples were analyzed by spark source mass spectrometry (SSMS) to determine possible sources of interference. No interferences were observed when those elements identified by SSMS were added to the test solutions a t concentrations five times greater than observed in the representative samples. Metal ions that form insoluble oxides a t the CGE in acid solution represent potential interferences. Likely candidates included nickel, iron, chromium, and manganese. Of these, only manganese could be oxidized to the insoluble manganese dioxide in dilute acid solution. By increasing the deposition potential to -1.8 V, the Mn2+ was oxidized to soluble MnOc-, and samples containing 0.4M dissolved ionic species, results were irreproducible and exhibited a large drift. The automated system is capable of continuous measurement of ammonia down to -10-5M provided only that purer reagents with very low ammonia blanks are used. Since our primary concern was the measurement of ammonia between 10-1 to lO-3M, we did not attempt to carry out measurements below 10- 4M. Urea Concentration Measurements in Aqueous Samples. Having established the feasibility of using an ammonia electrode for measuring ammonia levels in a flowing stream, we carried out urea concentration measurements based on the urease enzyme catalyzed hydrolysis of urea
Urea
+ H,O- urease
C0,’-
+ 2NH,+
Initially, urea concentrations were measured at various pH values to find the pH dependence of reaction 1. The results are illustrated in Figure 2 using phosphate buffer.
c
1
I---
B [
_L
/-=
dC
/ Figure 3. Typical urea concentration calibration curve
01
I
8
I
9
Sumner units/ml is necessary a t 37 "C. At enzyme concentrations 1 5 mg/ml, however, a high background of ammonia is produced which limits the linear range of the calibration curve to concentration levels 1 5 x 10- 3M. Very little difference is observed when 0.025 to 0.1M buffer and 0.1 to 0.5M NaOH quenching solution is used. When the total ionic composition of the sample solution is unknown (e.g., as in serum samples), it is better to use buffer concentrations of LO.1M and a quenching reagent of 0.5M to ensure sufficient buffer capacity and also to swamp out variations in the total ionic concentration prior to electrode measurement. A summary of urea measurements in serum samples is shown in Table 11. Data are presented a t two different temperatures to show that the conditions of the enzymatic raction (ie.,p H 7.4, 1 mg urease/ml, and an incubation time of 20 minutes) were sufficient to bring about 100% conversion of urea to ammonia. Increasing the temperature from 37 to 50 "C did not further increase the analytical signal, thus showing that all the urea had been converted to ammonia under the aforementioned conditions. However, if one compares the relative error obtained at 37" to that obtained at 50 "C, the error is greater a t 50 "C. This is a consequence of larger drifts, greater difficulty in maintaining constant temperature a t the electrode sensor
I
PH
Figure 2. p H dependence of the u r e a s e catalyzed hydrolysis of urea at various concentrations ( A ) 1 0 - ' M urea: ( E ) 10-2M urea; ( C ) 10-3M urea; ( D )10-4M urea; ( E ) i v 5 M urea
There is a slight urea concentration dependence of the optimum pH. Nevertheless, it is clear that between the urea concentrations of 10-1 and 10-3M, a pH of 7.2-7.4 is near optimum. The literature lists various pH optima for the urea-urease reaction in the range of 6.0-7.5 (1-4). A typical urea calibration curve using synthetic aqueous solutions is shown in Figure 3. Linearity exists between the signal obtained (peak height) and a rather broad urea concentration range. Such measurements, therefore, would be suitable for blood urea estimation. Table I summarizes our repetitive measurements of urea concentrations a t two temperatures and at two enzyme concentration (activity) levels. Good precision is easily attainable in the physiological range of 10-1 to 10-3M urea. The Table also shows that precision remains good, even though there is incomplete conversion of urea to ammonia during some of the measurements. For 100% conversion, an enzyme reagent containing 2 1 mg urease/ml corresponding to 20.4
Table I. Reproducibility of Urea Concentration M e a s u r e m e n t s in Aqueous Samples Trials, Peak height, AE in mV Urea Concn, M
1
2
3
4
5
178.0 123.0 67.5 23.5
175.5 122.0 67.0 24.0
176.0 122.5 68.5 25.5
176.6 f 0 . 9 122.7 +C 0 . 4 67.7 i 0 . 8 24.1 i. 0 . 8
128.0 103.5 54.0 18.5
128.5 104.0 54.0 18.0
127.5 103.5 53.0 18.0
127.1 103.0 53.6 18.1
i 1.4 f 1.2 =k 0 . 6 f 0.2
183.0 127.0 71.0 30.5
184.0 128.0 73.0 30.5
184.5 128.5 75.0 34.0
184.3 128.2 73.4 31.0
I-t 0 . 8 i: 0 . 8 i. 1 . 6 f 1.8
169.0 117.5 64.5 23.5
169.0 116.5 63.5 23.0
168.0 116.0 63.0 23.0
169.0 117.0 64.0 23.2
=t 0 . 7
I l i
a) At 30 OC and using 1.0 mg/ml urease enzyme solution
10 -1 10 - 2 10 - 3 10 - 4
176.5 123.0 68.0 24.0
176.5 123.0 67.5 23.5
b) At 30 OC and using 0.2 mg/ml urease enzyme solution
10 -1 10 - 2 10 - 3 10 - 4
125.0 101.0 53.0 18.0
126.5 103.0 54.0 18.0
c) At 37 OC and using 1.0 mg/ml urease enzyme solution
10 -1 10 - 2 10 - 3 10 - 4
185.0 128.5 73.5 29.0
185.0 129.0 74.5 31.0
d) At 37 OC and using 0.2 mgjml urease enzyme solution
10 -1 10 -*
TO - 3 10 - 4
169.0 117.0 64.0 23.5
170.0 118.0 65.0 23.0
i 0.8 i 0.8 =k 0 . 3
A N A L Y T I C A L CHEMISTRY, VOL. 46, NO. 8, J U L Y 1974
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Table 11. Typical S e r u m Urea Determinations Peak height, AE in mV
At 37'C
Trial 1 2 3 4 5 f + S
Re1 std dev Found, M Taken, M Re1 error
138.0 138.5 139.0 138.0 138.0 138.3 i 0 . 4 0.3% 4.65 X lo-* 4.71 X -1.3%
122.0 121* 0 121.5 120.5 120.5 121.1 f 0 . 7 0.6% 2.16 X 2.36 x -8.5%
135.5 133.5 134.5 131.5 133.0 133.6 =k 1 . 5 1.1% 4.70 x 4.71 X -0.2%
119.0 117.0 117.5 115.0 116.5 117.0 i 1 . 4 1.2% 2.05 X 2.36 x -13.1%
1.18 X -6.8%
83.0 82.0 84.0 81.5 83.0 82.7 i 0 . 9 1.1% 4.25 x 10-3 4.71 x 10-3 -9.8%
66.5 66.5 68.5 66.0 67.0 66.9 i 0.9 1.3% 2.10 x 10-3 2.36 x 10-3 -11.0%
35.0 33.5 36.0 34.5 35.5 34.9 i. 0 . 9 2.6% 5.40 x 10-4 4.71 x 10-4 +14.7%
102.0 101.0 100.5 98.5 99.5 100.3 If 1 . 4
80.0 79.5 78.5 78.0 78.5 78.9 i 0 . 8
64.0 63.0 63.0 62.0 62.8 62.9 i 0 . 7 1.1% 1.93 x 10-3 2.36 x 10-3 -18.2%
33.5 33.0 32.0 32.0 31.5 32.4 i 0 . 8 2.5% 5.10 x 10-4 4.71 x 10-3 +8.3%
105.0 105.0 106.0 103.5 104.5 104.8 i 0 . 9 0.9% 1 . 1 0 x 10-2
At 50 O C
Trial 1 2 3 4 5 a + s
Re1 std dev Found M Taken, M Re1 error
1.4% 1.00 x 10-2 1.18 X 1-2
-15.3%
and, also, loss of ammonia to the air bubble feed to waste a t this higher temperature. The values summarized in Table I1 were obtained in a routine manner with little attempt to maximize precision and accuracy. It is possible, however, to improve precision by minimizing the time lag between measurements to reduce drift effects and better accuracy could be attained by using standards whose concentrations bracket the concentration of the unknown sample. We have demonstrated the utility of the ammonia gas' electrode for the automated continuous flow analysis of urea in serum samples. The specificity of the enzyme urease coupled with the use of the ammonia gas sensing elec-
1.0%
4.05 x 10-3 4.71 x 10-3 -14.0%
trode, which is not subject to ionic interferences, provides a method with significant advantages for precise and accurate measurement of urea concentrations. The principle involved is generally applicable to all enzyme mediated reactions which are terminally productive of ammonia (e.g., arginine, creatinine, etc.), and should find useful applications in the clinical field. Received for review October 29, 1973. Accepted March 4, 19'74. We gratefully acknowledge the support of a grant from the National Institutes of Health. Ramon A. Llenado also gratefully acknowledges the support of an American Chemical Society Analytical Division Fellowship sponsored by the Procter and Gamble Company.
Nitrogen Determination in Biological Materials by the Nuclear Track Technique B. Stephen Carpenter and Philip D. LaFleur Activation Analysis Section. Analytical Chemistry Division. National Bureau of Standards. Washington. D.C. 20234
Alternatives to the classic Kjeldahl method ( I ) for nitrogen determination have been sought to speed analyses and to reduce uncertainties associated with the analysis of some complex protein samples. One alternative is automated instrumentation for the Kjeldahl and Dumas Technique. Another is neutron activation analysis in which material is irradiated with fast neutrons and the 13N activity produced from the nuclear reaction 14N(n,2n)13N (2-4) is counted. Carpenter and LaFleur ( 5 ) , in an earlier work demonstrated that proton tracks from the nuclear reaction (1) R. 8. Bradstreet, "The Kjeldahl Method for Organic Nitrogen," Academic Press, New York. N . Y . , 1965. (2) J. T. Gilmore and D. E. H u l l , Anal. Chem.. 34, 187 (1962). (3) M . R. C r a m b e s , S. S. Nargolwalla, and L. May, Trans. Amer. Nucl. S O C . . 10, 63 (1967). ( 4 ) D. E. Wood, Kaman Nucl. Rep.. KN6927(R) (1969). (5) 8.S. Carpenter and P. D. LaFleur, Int. J . Appi. Radia!. Isotopes. 23, 157 (1972).
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A N A L Y T I C A L CHEMISTRY, VOL. 46, NO. 8, JULY 1974
14N(n,p)14C,initiated by thermal neutrons, could be observed in cellulose nitrate (CN) using the nuclear track technique (NTT). In this paper we deal with the analytical application of that earlier work.
EXPERIMENTAL The number of proton tracks produced is a function of the nitrogen concentration. By adding known amounts of N to the sample, the method of standard additions can be employed and the original N content of the sample determined. Samples of orchard leaves, National Bureau of Standards Standard Reference Material 1571, were dried in a vacuum oven at 90 "C for 24 hours. The samples were then doped with 1.86%, 3.7270, 5.58% and 7.43% of added nitrogen with a solution of urea. Each sample was then mixed and re-dried in a vacuum oven a t 90 "C. After drying, the leaves were pressed into pellets using a pressure 13.8 X lo7 newtons m- * with a hydraulic press. Ten samples at each concentration level were analyzed to ascertain that the urea distribution was homogeneous throughout the orchard leaves. The homogeneity was better than f 5 % throughout the entire sample. The pel-
