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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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-
Table I. D a t a for Regression Analysis of Method of Additions Technique for Nitrogen in Orchard Leaves
Amount of nitrogen added, 7%
0
1.86 3.72 5.58 7.43 a
Table 11. Nitrogen in Orchard Leaves Nixon-Baldwin CN
1
Average No. of tracks per frame
No. of frames counted per detector'"
Net tracks per frame
7.23 i. 0.518 11.75 i. 0.579 15.53 j~0.214 19.63 2= 0.217 24.58 i 0.189
42 42 42 42 42
6.23 i 0.52 10.75 i 0.58 14.53 f 0.21 18.63 f 0.22 23.58 =IC 0.19
Ten detectors were counted for each sampling position.
lets, 1.5 mm thick and 13-mm diameter and weighing 250 mg, were held tightly against the CN detectors with a small C-clamp and melted paraffin was poured over the sample and detector. After cooling, the paraffin holds the sample to the detector for irradiation in the NBS Reactor. The samples were irradiated for 10 seconds a t a thermal neutron flux of -1.2 X 1O'O n cm-2 sec-'. After irradiation, the detector-sample combinations were separated and the CN detectors were etched chemically. Two types of CN detectors were used; the amber Nixon-Baldwin CN was etched for 2 hours a t 25 "C in a 6.5N NaOH solution, and Kodak CN, type CA80-15, was etched for 24 hours at 25 "C in 6.5N NaOH. Following etching, the detectors were rinsed in distilled water, air dried, and mounted on microscope slides for viewing. The proton tracks, now visible with an optical microscope, were counted within an area of 6 X cm2 with the aid of an image analyzing microscopy system. The nitrogen concentration was then determined by a least squares analysis of the change in track density as a function of nitrogen added. Typical data obtained for a least squares analysis of a method of additions procedure are given in Table I. Bovine liver, NBS Standard Reference Material 1577, was also analyzed. The samples were freeze-dried for 24 hours and prepared in pellet form under the same conditions for irradiation as the orchard leaves. Instead of using the method of standard additions for this analysis, the orchard leaves were used as the standard and the track density from the liver compared directly with that of the orchard leaves. The same irradiation conditions were followed as with the orchard leaves. Since the sample density will affect the range of the protons in the sample and, consequently, the track d e n s i t y 4 e . , track density will decrease with increasing sample density-it is necessary that the sample and standard have essentially the same density. Prior to using the orchard leaves as a standard for the bovine liver, the relative densities were measured and found to be similar. After irradiation, separation, chemical etching, and mounting, the proton tracks were counted in a 2.83 X cm2 area with the image analyzing microscopy system. The resulting track densities were then compared with the track density of the standard and the nitrogen concentration in the liver was obtained. Accompanying each irradiation were blank CN detectors coated with paraffin. The paraffin was removed and the detectors etched and counted under the same conditions as the sample detector. The track density due to the nitrogen present in the CN, was subtracted from the track density of the samples. This "background" track density, contributed by the CN itself, limited the successful determination of nitrogen in various materials to concentrations of 20.5%. In order to lower this detection limit, a sensitive plastic other than CN, preferably not containing nitrogen, must be found. The results of the two types of CN used for nitrogen analysis in the orchard leaves are given in Table 11. The results of the determination of nitrogen in the bovine liver SRM are shown in Table 111. Also given in the tables are the values obtained by W. P. Schmidt of the Microchemical Analysis Section of the NBS Analytical Chemistry Division using the Kjeldahl method. The results are in close agreement.
DISCUSSION The results indicate that the nuclear track technique is a satisfactory method for determining nitrogen. The overall elapsed time is the same, or slightly longer, than the Kjeldahl method, there are fewer manipulations involved,
2 3 Average
70
by Weight
2.86 f 0 . 0 8 2.72 i 0.02 2.69 f 0.02 2.76 =t0.09
Kodak CN
1 2
3 Average Kjeldahl
2.65 2.72 2.74 2.70
f 0.09 i 0.05 i 0.07 i 0.09
2.755 i 0.038
Table 111. Nitrogen in Bovine Livera Sampling positions
3 5 12 13 20 23 Average Kjeldahl
Nitrogen found, c/o
10.39 i 1.11 10.77 i 1.11 11.03 i 1.17 10.89 i 1 . 1 3 10.84 i 1.31 11.00 i 1 . 1 1 10.82 i 0.24 10.59 i 0 . 0 4
'Samples compared with the Orchard Leaves. Standard (2.76 Nitrogen).
+ 0.09%
and the nuclear track method allows the determination of nitrogen by a fundamentally different technique. Also, there is no differentiation due to the chemical state of the nitrogen in the sample, therefore removing some of the uncertainties in Kjeldahl determinations in some complex protein samples. Consideration was given to other nuclear reactions and processes that would produce possible interfering proton tracks in the CN detectors. Most of the nuclei that undergo (n,p) reactions with thermal neutrons cafi be eliminated because either their cross sections, u , , ~ , or their isotopic abundances are too low. The greatest interference would come from 35Cl, and that was calculated to be less than 0.5% of the total number of tracks produced in the detector from either the liver or the orchard leaves. In both determinations, the number of proton tracks produced from the chlorine isotope are small and indistinguishable within the precision of the track counting statistics. Since the thermal neutron flux used had a cadmium ratio, Rrd, of 536 for copper ( 6 ) , there is a negligible problem with "knock on" protons produced in the hydrogeneous material during irradiation. Although the possibility for interference from nuclear reactions producing alpha particles exists [e.g., I7O, log, and 'jLi ( n , a ) reactions], alpha tracks are readily distinguishable optically as they are 3 to 4 X larger than proton tracks, and can be excluded from proton track counts. While an image analyzing microscope is very convenient for the analysis, the use of an optical microscope for obtaining the track density is possible, thereby making the technique available t o most laboratories with access to a nuclear reactor. Received for review November 27, 1973. Accepted April 3, 1974. Presented in part a t the Spring Meeting of the American Nuclear Society, Las Vegas, Nev., June 1972. (6) Nat. B u r . Stand. ( U . S . ) Tech. Note. 548, "Activation Analysis Section: Summary of Activities, July 1969 to June 1970," P. D. LaFleur and D. A . Becker, Ed., December 1970, Washington, D.C.
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