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(13) S. S. Krishnan and D. R. Crapper, Radiochem. Radioanal. Lett., ZO(4-5), 279 (1975). (14) P. I. Artyukhin, E. A. Startseva, Yu A. Sil'vanovich, D. N. Abakumov, Yo. L. Mltyakin, and A. G. Mokhnachev, Radiochimla, 13(4),681 (1971). (15) W. Bock Werthmann and W. Schulze, Proc. Intern. Conf. Modern Trends in Activation Analysis, College Station, Texas, 1961. (16) S.F. Mughabghab and D.I. Garber, Neutron Cross Sections, BNL 325 (1973). (17) W. J. C. Barteis, KAPL-336 (1950). (18) J. Dwork, P. L . Hofmann, H. Hurwitz, Jr., and E.F. Clancy. KAPL-1262 (1955). (19) R. G. Nisle, Nucleonics Data Sheet, 36, 86 (1960). (20) A. Sola, Nucleonics, 18, 74 (1960). (21) P. Morzek, Kernenergie, 5 , 839 (1962). (22) 0. T. Hogdahl, Radiochemical Methods of Analysis, IAEA, 1, 23 (1965). (23) J. Chernik and R. Vernon, Nucl. Sci. f n g . , 4, 649 (1958).
(24) A. A. Samadt, R. Grynszpan, and M. Fedoroff, Radiochem. Radioanal. Lett., 4, 171 (1974). (25) E. M. Lobanov and R. I. Khusnutdinov, Zh. Ami. Khim., 21(6), 743 (1966). (26) M. Fedoroff, J. Blouri, and G. Revel, Nucl. Instrum. Mefhods, 113, 589 (1973). (27) C. Loos-Neskovic, M. Fedoroff, and G. Revel, Radiochem. Radioanal. Lett., 26(1), 17 (1976). (28) L. A. Currie, Anal. Chem., 40, 586 (1968). (29) J. L. Debrun and J. N. Barrandon, J . Radioanal. Chem., 17, 291 (1973). (30) C. Loos-Neskovic, M. Fedoroff, and G. Revel, Radiochem. Radioanal. Lett., 36(1), 13 (1978).
RECEIVEDfor review February 5, 1979. Accepted April 30, 1979.
Determination of Bromine in Blood Serum by Neutron Activation Analysis and X-Ray Spectrometry M. S. Rapaport, M. Mantel," and R. Nothmann Nuclear Chemistry Department, Soreq Nuclear Research Centre, Yavne, Israel
A method is described for the nondestructive determination of bromine in blood serum by INAA followed by X-ray spectrometry. The use of magnetic fields for the elimination of the particles emitted by the blood matrix reduces the background and makes possible the accurate measurement of the bromine X-rays. An average value of 7.38 f 0.44 mg Br/L blood serum was obtained for the single person tested.
T h e application of neutron activation to the determination of trace amounts of elements has been a standard technique for many years. Recently Mantel e t al. ( I ) substituted high resolution X-ray spectrometry for the generally used -,-ray spectroscopy and listed the elements which may be detected as well as the sensitivities obtainable ( I ) . The characteristic X-rays emitted following radioactivation are the result of electron capture or internal conversion processes. Advantage is then taken of the direct and unambiguous correlation between the elements and their K or L X-ray energies. Furthermore, with the use of solid state Si(Li) diodes, the X-ray lines of neighboring elements can be resolved and the interference of high energy (>50 keV) y rays eliminated. On the other hand, a disadvantage of the method is the interference of the p particles emitted from the sample and detected by the diode. These fl rays raise the background, substantially increase the dead-time of the detector and upset its resolution in the detection of X-rays. T h e use of magnetic fields to remove fl particles after neutron activation was first demonstrated in our laboratory (2-4). In the present work, this technique was applied to the determination of bromine in blood serum. Most of the conventional methods used are destructive and based on the radiochemical separation of bromine (5). Shenberg, Gilat, and Finston (6) were the first to recommend X-ray spectrometry for the nondestructive determination of bromine. Peisach e t al. (7, 8) applied this technique t o blood, but studied only synthetic samples. By using magnetic fields and specially prepared thin samples, the present method overcomes the shortcomings of 0003-2700/79/0351-1356$01.00/0
X-ray spectrometry, Le., the high background produced by the bremsstrahlung emitted from the irradiated sample and the self-attenuation of the X-rays in the sample, and so makes the nondestructive determination of bromine in blood possible. EXPERIMENTAL The experimental setup shown in Figure 1 consists essentially of a Si(Li) detector and an electromagnet. A Si(Li) diode (Seforad, Israel) of 100 mm2 area, 4-mm depletion depth and 1.0-mm thick beryllium window was coupled through its preamplifier and research amplifier (Model 1412, Canberra, U.S.A.) to a 4096channel-analyzer (Promeda, Elscint, Isarel). The resolution (FWHM) of the system for 6.4 keV Fe K X-rays was 310 eV. Samples to be counted were placed 27 mm from the beryllium window. An electromagnet, available at our laboratory, was redesigned to permit the introduction of the Si(Li) detector into the electromagnet as close as 22 mm from the poles. Magnetic fields of up to 13 kG (- 12-mm gap between poles) were obtained. The magnetic field was constantly monitored with a gaussmeter. Blood samples were taken from one person in the upright position. The plasma was separated by centrifugation and stored in a polyethylene container at 5 "C. Micropipets of 50 pL were used to transfer the blood plasma onto 0.00025-inch thick Mylar foils. The samples, 3-4 mm in diameter, were then dried and stored in a desiccator containing P205as desiccant. Before irradiation the samples were covered with Mylar foils of the same thickness, and placed in polyethylene containers. Standard bromine sources were prepared similarly from a solution of NH4Br (2.26 pg/50 pL in triple distilled water). Irradiations were carried out in the pneumatic tube of the IRR-1 reactor for periods varying from 5 to 10 min a t a thermal flux of -1 x ioi3 n cm-* s-'. R E S U L T S AND DISCUSSION Neutron activation of bromine (79Br,50.69%; "Br, 49.31 70) results in the production of ground states and isomeric states of 80Brand 82Br. The cross sections for the production of sCh"Br (t1,* = 4.42 h) and 82mBr(t1,* = 6.1 min) are 2.6 and 2.4 barns, respectively, and the two isomeric states decay practically completely through internal transitions (I.T.). These I.T. are M3 in character and decay by internal conversion (-99%), mainly of the K shell (-85% 1. 82mBrdecays directly to 82gBr, while in 8omBrdecay the M3 transition is followed by a n E1 C 1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 9, AUGUST 1979
500
1357
1
I 1 8
0
12
14
Energy (keV)
Figure 1. Schematic representation of the detector system
Br Y,
E r e ; ; . ,'kei,
Figure 2. X-ray spectrum of a standard bromine sample (2.26 gg NH,Br) after neutron activation transition to 8ogBr. Since the fluorescence yield is 0.622, the number of K X-rays of Br emitted per atom of 80mBror 82mBr will be -0.5, If the irradiation and counting time are of the order of 10 min, most of the bromine X-rays will be due to the decay of 82mBr.*OgBr (ti,? = 17.4 min) and **gBr (tIf'*= 35.4 h) decay by 0-emission to 80Kr and "Kr, respectively; the amount of Kr K X-rays produced is insignificant. However, BogBrwhich is mostly produced directly from '9Br, with a cross section of 8.5 barns, decays 6% by electron capture t o *OSe?resulting in Se K X-rays which have an intensity calculated to be -0.1 of that of bromine X-rays. Experiments were carried out to determine the best experimental conditions. An irradiation time of 12.0 min, magnetic fields of 11.0 kG, 10.0-min delay between end of irradiation and start of counting, and 10.0-min counting time were found to be the optimum experimental conditions. A shorter delay between the end of irradiation and the start of counting would be desirable: however this was not possible since it takes 5 min to transfer the sample from the reactor building to our counting laboratory. The bromine in blood serum was determined by counting a standard sample, a blood sample, and again a standard Br sample. While one sample was counted, the other was irradiated. This procedure was followed to monitor a sudden momentary change in the reactor neutron flux. The amount of Br in blood serum was then determined by comparison with the average amount of bromine found in the two standard samples. The X-ray spectrum of a standard Br sample is
Figure 3. X-ray spectra of identical 50-fiL blood serum samples after neutron activation. (a) Without magnetic field, (b) with magnetic field shown in Figure 2, and its shape is consistent with the one predicted above. The net counts due to X-rays emitted by bromine were determined for the combined Br(Ka and KP) and Se(Kn and KP) X-rays. The X-ray spectra of irradiated blood serum were measured with and without a magnetic field. The results obtained are shown in Figure 3. I t may be seen that, without a magnetic field, the 3 particles emitted from the blood matrix completely mask the bromine X-rays. By applying the magnetic field a substantial part of the 3 particles is removed (3, 4 ) and the background lowered, so that the X-ray peaks may be accurately integrated. The net number of counts, determined in the same way as for the standard Br sample, was 2600 counts/sample (average value). Peisach et al. ( 7 , 8 ) ,the first to suggest the determination of bromine in blood plasma using X-ray spectrometry following neutron activation, reported results obtained from synthetic plasma solutions made of dilute solutions of NH4Br (8.8-61.3 ng Br/sample) doped with the correct amount of Na and C1 (-3 mg/L). No results were reported for human blood plasma. The authors remark that the high background due to the interference of the bremsstrahlung of P radiation increases the error of the method. But an important component of blood plasma, phosphorous (11.4 mg per 100 mL normal blood plasma (9)),which is a strong 0emitter, was not added to the synthetic solutions. Thus the background was much lower than that observed in the measurement of blood plasma. On the other hand, the p particles emitted by 32P are low in energy (&a = 1.71 MeV) and thus easily removed, up to 95%, by the magnetic field ( 4 ) . As shown in Figure 3, it seems that without the use of magnetic fields it is not possible to determine bromine in blood plasma by INAA. Another disadvantage of X-ray spectrometry, the self-attenuation of the X-rays in the sample, was overcome by preparing extremely thin samples. This was achieved by sandwiching the sample to be analyzed between two thin Mylar foils, as described above. Mylar foils were used because of their high mechanical strength, very low absorption coefficient for X-rays, and the insignificant (1% correction) amount of bromine they contain. Interference could be produced from neighboring elements in the periodic table which after neutron activation produce Br or Se X-rays. However, practically no interference is expected, since there are no other elements present in blood plasma which produce Br X-rays during their decay. Selenium produces Se X-rays but the amount of selenium in blood
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 9, AUGUST 1979
The result is also in agreement with the value of Barrette et al. (10)who determined the amount of bromine in freez,e-dried blood serum by proton and N particle induced X-rays to be 6.44 + 0.04 mg/L with a relative standard deviation of k2.9'70.
Table I. Precision of the Method for Determination of Br in Blood Serum Br found,
average,
mdL 7.10 6.42 7.74 7.14 7.57 7.32 8.19 6.94 7.96
mg/L
rel.
std.
dev., 9%
LITERATURE CITED 7.38
M. Mantel and S. Arniel, Anal Chem., 44, 548 (1972). S. Amiel, M. Mantel, and 2.B. Alfassi, J . Radioanal. Chem.. 37, 189 (1977). 2.6. Alfassi, T. Izak-Biran, and M. Mantel, Nucl. Instrum. Methods, 151, 227 (1978). M. Mantel, 2.B. Alfassi, and S. Amiel, Anal. Chem., 5 0 , 441 (1978). M. M. Atallah and I. C. Geddes, B r . J . Anaesth.. 45, 134 (1973). C. Shenberg, J. Gilat, and H. L. Finston, Anal. Chem., 39, 780 (1967). M. Peisach. B. Maziere, C. Loc'h, D. Comar, and C. Kellershohn, J . Radioanal. Chem., 14, 269 (1974). M. Peisach, D. Comar, and C. Kellershohn. Radiochem. Radioanal. Left., 8, 267 (1971). "Biology Data W",Vol. 3, P. L. Akman and D. S. Darner, Ed., Federation of American Societies for Experimental Biology, Bethesda, Md., 1974, D 1752. M Barrette, G Lamoureux, E Lebel, R Lecornte, P Paradis, and S Monaro, Nucl Instrum Methods, 134, 189 (1976)
i7.45
plasma (11 wg/L) (IO) is so small compared with that of bromine that under the experimental conditions of the present method its contribution is negligible. The precision of the method was determined by the analysis of nine blood plasma samples. The average value was determined to be 7.38 & 0.44mg B r / L with a relative standard deviation of 1 7 . 4 % (Table I). This result is within the range mg/L listed in the "Biology Data Book" (91. of values :;3:
RECEIVED for review Kovember 17, 1978. Accepted January 29, 1979. This work was supported by a grant from the U.S.-Israel Binational Science Foundation, Jerusalem.
On Confidence in the Results of Learning Machines Trained on Mass Spectra J. A. Richards* School of Electrical Engineering, The University of New South Wales, P.O. Box 1 Kensington, New South Wales 2033, Australia
A. G. Griffiths D@ital Equipment Australia, R y . Lid., Brisbane, Queensland, Australia
particular learning machine. This is, of necessity, highly testing set dependent and is a problem which has not been treated to date although it is mentioned in passing by Soltzberg et al. The following is addressed specifically to this concept.
A measure is presented by which a user can assess the degree of confidence he can place on the results obtained by using a learning machine for the (multicategory) classification of mass spectra. Being based upon the weighted average of a set of backward probabilities, this measure is properly highly testing set dependent; its minimum value therefore is recommended as a reliable confidence indicator. Numerical results are presented to illustrate the significance of this indicator compared to measures often used to describe the integrity of results obtained with a learning machine.
A PRIORI PROBABILITIES The probabilities with which patterns from individual classes are likely to be presented to a learning machine are referred to as a priori probabilities, and are related directly to the so-called testing set composition (in this case a real testing set rather than an ad hoc set used usually to diagnose the machine characteristics). The influence of the a priori probabilities on the performance of learning machines has been considered by Rotter and Varmuza (1) in their expression
In the field of mass spectral pattern recognition, attention has been given recently to the task of deriving an index or measure by which various types of learning machine may be compared. Rotter and Varmuza (1) have approached this problem by utilizing the concept of the gain in information about the testing set provided by using the learning machine. This is an information theoretic approach and parallels the theory of information transmission in communication channels. Soltzberg et al. (2) point to the testing set dependence of the information gain derived by Rotter and Varmuza and then proceed to modify it to minimize this dependence. A problem associated with the need to devise a machine comparison index of these types is the desirability of establishing an indicator which will express the degree of confidence a user can place on the recommendations of a 0003-2700/79/0351-1358S01 OO/O
Z(A;B) = H ( A ) - H(A(B)
(1)
for the information gain provided by using a learning machine as against trying to guess pattern class membership a priori. In this A refers to the set of real categories whereas B refers to the classification made by the machine. H(A1 and H(AIB) are the information theoretic entropies ( 3 ) of the pattern classes before and after the application of the learning machine. These are related to the (remaining) uncertainty in the observer's knowledge about the classification of the patterns. Equation 1 has been treated extensively in the communications and information theory literature, especially with regard to maximization of Z(A;B)-often referred t o as mutual C
1979 American Chemical Society
