polarization. Whole blood was diluted 20/1 with buffered K3Fe(CN)6 solution in this experiment, and increments of lactate were added to the solution to yield an internal standard. Measurement Technique. The time to reach a steady state current for these systems was 3 to 10 minutes. That this was due to the time required to set up a steady diffusional flux was shown by addition of reduced oxidant (ferrocyanide) in place of substrate (lactate). A similar time response was obtained in both experiments. This slow time response is due to the thickness of the diffusion and reaction layer. By decreasing the layer thickness, the response time of the device is decreased. This may be accomplished by incorporating the enzyme into the dialysis layer. When this technique was used with glucose oxidase, as also demonstrated by Hicks
and Updike (12), the response time with a well stirred solution was approximately 30 seconds. Finally, having proved linearity of the current-concentration relationship, we would anticipate measurement of unknowns to be accomplished by a single addition of substrate, or by running standard solutions periodically.
RECEIVED for review June 17, 1969. Accepted October 21, 1969. This work was supported by Public Health Service, Diabetes and Arthritis Control Program, and the Artificial Heart Program of NIH, NHI. (12) G. P. Hicks and S. J. Updike, ANAL.CHEM., 38,726 (1966).
Trace Determination of Uranium in Biological Material by Fission Track Counting B. Stephen Carpenter Institute for Materials Research, National Bureau of Standards, Washington, D. C . 20234
Conrad H. Cheek Department of Chemistry, Howard Unicersity, Washington, D.
THEAIM of this investigation was to develop a sensitive and selective method for the determination of trace quantities of uranium in biological materials such as blood, tissue, and botanical materials, The National Bureau of Standards is considering such biological materials as Standard Reference Materials. Several techniques are described in the literature for the determination of trace amounts of uranium by a-counting ( I ) , fluorometry (2, 3), and activation analysis (4). The last technique is especially attractive in that it offers a variety of methods for trace analysis which are fast and simple and have good sensitivity. These techniques consist of procedures for radiochemical separation of fission isotopes, counting the 74 KeV photon peak produced from 238U(n,y)239U, or studying the fission tracks produced in solid state detectors. Of these, the nuclear track technique seems to be the most favorable because there is no requirement for yield correction or for testing radiochemical purity by half-life measurement. Successful use of this technique to determine the trace quantities of uranium in geological and metallurgical samples (5) suggested its application to quantitative determination of the uranium content of biological materials. As energetic protons and heavier charged particles travel through matter, they produce along their paths cylindricalshaped regions which have been atomically and electronically disturbed. The lengths of these zones of radiation damage depend critically on the charge and mass of the traversing particle and on the chemical conposition of matter traversed (1) E. E. Campbell, B. M. Head, and M. F. Milligan, U.S. At. Energy Comm. Rept. LA-1920 (June 1955). (2) J. Hoffman, Biochem. Z., 313, 377 (1943). (3) W. F. Newman, Natl. Nucl. Energy Serv., Diu. VI. I, 11, 701
(1949). (4) W. D. Mackintosh and R. E. Jervis, Atoric Energy Commission of Canada, Ltd., Report AECL-481 or CRDC-704 (April 1957). ( 5 ) P. B. Price and R. M. Walker, Appl. Phys. Lett., 2, 23 (1963).
c. 20001 (6). The radiation damage is only transient in some materials, notably metals. In some other materials, particularly in insulators, the damage is essentially permanent. In 1959, Silk and Barnes demonstrated that the tracks of heavy charged particles in an insulator were readily observed by electron microscopy (7). Further work by Fleischer and co-workers showed that upon the application of the proper chemical reagents the tracks produced in the various insulating materials could be made observable by optical microscopy (8,9). The chemical reagent reacted much more rapidly along the sensitized tracks than with the undamaged areas of the insulating materials. The preferential chemical etching converted the tracks to much larger, very nearly cylindrical channels, which scattered light and thus appeared as dark lines in the field of an optical microscope. In addition, Fleischer and his co-workers found that proper choice of the insulator permitted selectivity in track registration (9, IO). For instance, alpha particles will produce tracks in both cellulose nitrate and cellulose acetate butyrate, but not in the polycarbonate plastic, Lexan (CleHL803)(IO), whereas heavy nuclides arising from the fission of uranium, thorium, bismuth, and other heavy elements will produce tracks in both the cellulose polymers and polycarbonate plastic. This is the reason Lexan was chosen as the solid state detector; it also has the capability to discriminate against low-energy charged particles and lightweight recoiling nuclei. It is insensitive to track production from high-energy photons, deuterons, and alpha particles, all of which are commonly produced in nuclear reactions. (6) R. L. Fleischer, P. B. Price, and R. M. Walker, Scieme, 149, 383 (1965). (7) E. C. H. Silk and R. S. Barnes, Phil. Mag., 4,970 (1959). (8) R. L.Fleischer and P. B. Price, J. Appl. Phys., 34, 2903 (1963). (9) R. M. Walker, P. B. Price, and R. L. Fleischer, Appl. Phys. Lett., 3,28 (1963). (IO) R. L. Fleischer, P. B. Price, R. M. Walker, and E. L. Hubbard, Phys. Rev., 133, A1443 (1964).
ANALYTICAL CHEMISTRY, VOL. 42,
NO. 1, JANUARY 1970
121
Figure 1. Etched fission tracks in Lexan Further selectivity can he obtained by exposing the samples only to a thermal neutron flux. This eliminates interference by thorium which undergoes only fast neutron fission. Plutonium would interfere, hut is very unlikely to be present. The advantages which the nuclear track technique offers the analyst are sensitivity and selectivity. Depending upon the irradiation conditions, as little as 10-l2 gram of uranium can be detected with. an integrated thermal neutron flux of 1.1 X 1014 with a corresponding increase in sensitivity with increased exposure. EXPERIM
tutes of Health. Ammonium citrate of unknown uranium content had been added as a preservative and anticoagulant prior to receipt. The uranium content is reported for the modified blood as received. The human plasma was obtained from the National Naval Medical Center. The botanical material selected consisted of leaves of the white oak (Quercus alba) which were collected at the NBS-Washington site. The materials were freeze-dried to remove most of the water, weighed in silica boats, and ashed in an oxygen atmosphere in a low-temperature dry asher. The freeze-dried oak leaves were ground in a blender prior to weighing. After dry ashing, the residue was digested in nitric acid, filtered, and diluted to 50 ml. A 100-pl aliquot of the resulting solution was pipetted onto a previously marked 15 mm* area on a Lexan slide to facilitate relocating the sample area after irradiation. Each slide was 20 mm wide X 40 mm long X 0.25 mm thick. The solutions on the Lexan detectors were then evaporated to dryness in a laminar-flow clean bench. After drying, the samples were coated with a solution of collodion in ether. Samples were packaged in polyethylene envelopes to prevent external contamination. These samples were placed in plastic containers and irradiated in the Naval Research Laboratory reactor for 5 minutes at a thermal neutron flux of approximately 3.5 X 10" n.cm-s.s-'. After irradiation, the protective collodion layer was removed and the slides were etched for 45 minutes in 6.5N NaOH at 50 + 2 "C. This etching technique is similar to that used by Fleischer and co-workers in their studies of track production in various solid-state detectors (10). The slides were rinsed several times with distilled water and once with ethanol. The enlarged fission tracks, as shown in Figure 1, were then counted with the aid of an optical microscope. It is essential to count all the tracks in order to avoid errors due to non122
ANALYTICAL CHEMISTRY, VOL. 42,
uniformity of deposition of uranium on the detector. The number of tracks observed in the case of the actual samples was compared with the number of tracks produced in standards and blanks, irradiated under the same conditions, to determine the amount of uranium present. Calibration standards were prepared from NBS SRM No. 950-A Uranium Oxide, with a certified composition of 99.94% U,O,. The U308was dissolved in nitric acid to convert the oxide to U022+,which then was diluted to the desired volume. Various known quantities of uranium were spotted on Lexan slides and irradiated simultaneously to determine the dependence of the track production on the amount of uranium. The number of tracks produced was shown to be closely proportional to the weight of uranium over the range from 8.2 X lo-" to 6.2 X 10-o gram. This linearity establishes the basis of the comparative method of analysis. To determine the blank contribution of uranium from the reagents and environment, slides were prepared from the same reagents and in the same manner and laboratory environment in which the sample slides were prepared. These blank slides were irradiated and etched along with the sample slides and standard slides. The reagent blanks were found to contain 5.24 X 10-l2 to 2.35 X lo-" gram of uranium. RESULTS
Table I presents the results of determination of uranium in human whole blood. The uranium content is quoted in two ways. The values in column 6 are in units of parts per million (ppm) and refer to tbe freeze-dried blood, while the values in column 7 are in parts per billion (ppb) and refer to whole blood. According to Bowen ( I l ) , water comprises 85 Z of whole blood. The average of all the values in column 7 is 86.1 =t5.6 ppb, where the uncertainty is the standard deviation of the mean. Values of the uranium content of plasma are presented in Table I1 in the same manner as the values for blood in Table I. The uranium content of plasma is expressed in parts per billion after correcting for 9 4 Z water which was removed by freeze-dryiiig. The average value for whole plasma is 60.5 =k 12.0pph. ""A..,. +".+",-.,cylllnIL ^F *La Cnmn 1 -..1 -:.. .wL,LLc .L:t^ Values fv, +La LLLc yLLLLllyll. Llr IICCltC-uIIN oak leaves were widely scattered, ranging from 1.6 to 15.2 ppm ,and therefore are not tabulated.
-- ..-
DISCUSSION
The standard deviation ot the mean ior uranium in whole blood is considered to be satisfactory in view of the very small quantity of uranium in the samples, but the mean value is in poor agreement with Newman's (3) value of 14 pph, and in serious disagreement with Hoffman's (2) value of 0.1 ppb. Newman and Hoffman utilized the method of fluorescence spectrometry, and their results are based on only one and two determinations, respectively. However, the disagreement in the three results may be due to the history of the samples. For example, the amount of uranium in the citrate which had been added to our samples is unknown, hut it is considered unlikely that this caused the large discrepancy. The results for whole plasma exhibit considerably more scatter than the determinations for blood, and the reason for this is not known. The relative precision of 20%, however, is considered acceptable in the concentration range of the measurements. (11) 11. 1. M. Bowen, United Kingdom Atomic hrrgy Authority Research Croup Report, ACRF.21196(1963).
NO. 1. JANUARY 1970
Weight of sample irradiated, mg 0.294 0.242
Number of tracks observed 148 128 124 120 80 77 84
Table I. Uranium Content of Human Blood Grams Weight of uranium of uranium in sample, g Net per track (X ( X 10'0) tracks observed 130
Uranium content of freeze-dried sample, ppm
Uranium content of whole b!ood, ppbb
1.70 1.44 1.39 1.34 0.81 0.77 0.86
0.579 0.596 0.576 0.555 0.562 0.535 0.597
86.9 89.4 86.4 83.3 84.3 80.3 89.6
1.35
1.63 1.84 1.63
0.554 0.626 0.554
83.1 93.9 83.1
1.31 1.31
0.294
125 140 125
110 106 102 62 59 66 121 136 121
0.146
57 71 59
53 67 55
1.35
0.72 0.90 0.74
0.493 0.616 0.507
73.9 92.4 76.1
0.246
114 115 113
110 111 109
1.35
1.49 1.50 1.47
0.606 0.610 0.598
90.9 91.5 89.7
7382
7364
1.30
0.587
88.1
0.144
16.27
1.31
95.8
Obtained from simultaneously irradiated standards. Calculated after correcting for the 85 % water loss. Table 11. Uranium Content of Human Blood Plasma Grams Weight of uranium of uranium Net per track in sample, g track observed ( X 10'2)a tx 10'0)
Uranium content of freeze-dried sample, pprn
Uranium content of whole plasma, ppbb
3.64 3.10
1.18 1.01
70.8 60.3
1.31
1.99 3.07 2.82
0.85 1.31 1.20
51 .o 78.6 72.0
122 80 82
1.31
1.59 1.05 1.07
1.26 0.83 0.85
75.6 49.8 51 .O
314
310
1.35
4.19
1.27
76.2
0.202
140
122
0.77
0.94
0.82
49.2
0.228
153 139 149
149 135 145
1.35
2.01 1.82 1.96
0.88 0.80 0.86
52.8 48.0 51.6
Weight of sample irradiated, mg
Number of tracks observed
0.308
296 255
278 237
1.31
0.235
170 252 233
152 234 215
0.126
140 98 100
0.330
= Obtained from simultaneously irradiated standards. b
Calculated after correcting for the 94% water loss.
The very large scatter in the results for oak leaves is believed to be due to substantial variations in uranium content from leaf to leaf, nonuniform distribution of uranium within each leaf, and failure of the grinding to homogenize the stems, so that the necessarily small samples for dry ashing were not representative. The technique for sampling this material must be improved. The nuclear track technique is a sensitive analytical method for determining uranium in biological materials. The blood analysis, especially, illustrates the reproducibility the method offers the analyst. Modifications of the technique could lead to somewhat better precision in the blood analyses and to much better precision in the plasma and oak leaf measurements. This method requires that the sample size be very small if a low temperature asher is used. When very small fractions of the original weighed sample are irradiated, as in these experiments, it is particularly important to establish that a
representative sample has been selected for the analysis. The sensitivity and the detection limit of the method are dependent upon the integrated flux being used. Finally, the method is a relatively inexpensive means of uranium analysis, equipmentwise, provided the analyst has access to a reactor. The method presented here is in several ways similar to that used by Fleischer and Lovett (12) to measure uranium in water. The major difference is that they did not use the comparative technique and therefore required accurate knowledge of the neutron flux and reaction cross sections.
RECEIVED for review July 29, 1969. Accepted October 22, 1969. Work done in partial fulfillment for the M.S. degree of B.S.C., Howard University, December 1968. Experimental work was done at the National Bureau of Standards. (12) R. L. Fleischer and D. B. Lovett, Giochim. Cosmochim., 32, 1126 (1968).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 1, JANUARY 1970
123
