Multielement Instrumental Neutron Activation Analysis of Biological Materials R. A. Nadkarni and G. H. Morrison Department of Chemistry. Cornell University. Ithaca. N. Y . 74850
During the past few years, there has been an increasing realization of the importance of trace element chemistry in biological systems. This awareness has been stimulated by the rising concern in industrial nations of the impact of man on his environment and its biological effect on him. Of primary significance is the role played by trace elements and whether or not they are beneficial to the biochemistry of man. Recently it has been recognized that a number of trace elements are required nutrients ( I ) . The essential trace elements for plant and animal life to date include Co, Cr, Cu, B, F, Fe, I, Mn, Mo, Se, and Zn, with Ni, Sn, and V possibly essential. The minor elements Na, K, Mg, Ca, P, S, and C1 are also of interest in life systems. To this list of important elements must be added those of toxicological concern, i . e . , Li, Be, Ba, Ni, Ag, Cd, Hg, As, Sb, Bi, Pb, and Br. It is quite possible that additional trace elements may be found to exercise essentiality or toxicity as further research is performed on their biological role. To expedite the study of these trace elements, comprehensive multielement analytical methods of high sensitivity in complex biological matrices are necessary. Neutron activation analysis together with high resolution y spectrometry has already shown considerable promise. Instrumental neutron activation analysis (INAA), i.e. without radiochemical separations, has been used to determine 15 to 20 elements in a variety of biological materials (2-10). A recent INAA study claims to have determined 40 trace elements; however, no data are presented (11). Using INAA and NAA together with group radiochemical separations and y spectrometry, Morrison and Potter (12) have determined 31 elements in biological samples. Other radiochemical group separation procedures have also been published (13, 2 4 ) . (1) E . J. Underwood. "Trace Elements in Human and Animal Nutrition," Academic Press. New York. N . Y . . 1971. (2) R. A . Nadkarni and W . D. Ehmann. J . Radioanal Chem. 3, 175 (1969). (3) R . A . Nadkarni. D. E. Flieder. and W . D. Ehrnann. Radiochim. Acta. 11. 9 7 (1969). (4) R . A . Nadkarni and W . D Ehmann, Radiochem. Radioanal. Lett. 2, 161 (1969). (5) R . A . Nadkarni and W . D. Ehmann, Radiochem Radioanal. Lett.. 4, 325 (1970) (6) R A Nadkarni and W D Ehrnann. Radrochem Radioanal Lett 6, 89 (1971). (7) R. A Nadkarni and W . D. Ehmann. "Trace Substances in Environmental Health," D. D. Hemphill IV. E d . , University of Missouri. Columbia. Mo.. 1971. p 4 0 7 . (8) F. Girardi. J. Pauly. E . Sabbioni. and G . Vos, "Nuclear Activation Techniques in Life Sciences." I . A . E . A . . Vienna. 1967, p 229. (9) W. A. Haller, L. A. Rancitelli, and J. A. Cooper, Battelle Northwest Labs. Report SA-1634. (10) D. McKown. M . Kay, D. Gray. A . Abu-samra. M . Eichor, and J. Vogt. ' Nuclear Methods in Environment Research," J. R. Vogt. T. F. Parkinson. and R. L. Carter. E d . . University of Missouri, Columbia. Mo.. 1971. p 150. (11) T. F. Budinger, J. R . Farwell. A. R . Smith, and H. Bichsel. lnt J . Appl Rad/at. lsotop., 23,49 (1972). (12) G . H . Morrison and N. M Potter. Ana/ Chem.. 4 4 , 839 (1972). (13) E . Steinnes, 0. R. Birkelund. and 0 . Johansen, J. Radioanal. Chem.. 9 , 267 (1971). (14) K . Samsahl. P. 0. Wester. and 0. Landstrorn. Ana/ Chem.. 40, 181 (1968)
The present study describes an INAA method capable of determining up to 36 elements, including many of the essential and toxicological trace elements. Since no chemical processing is involved, volatile elements such as As, Br, C1, Hg, Se, and S b are not lost in the method. Also, no chemical yields need be determined for each of the elements determined. The method uses a multielement biological standard during irradiation which is similar in matrix element composition to the samples analyzed. This minimizes differences in neutron self-shielding and absorption effects between the standard and samples, as well as eliminates the task of preparing a large number of synthetic standards.
EXPERIMENTAL Samples and Standards. U.S. National Bureau of Standards Standard Reference Materials SRM-1571 Orchard Leaves and SRM-1577 Bovine Liver, a standard Kale sample prepared by H. J. M. Bowen of Reading University, England, and standard reference tobacco 1R1 prepared by the University of Kentucky Tobacco and Health Research Institute, Lexington, Ky., were used for analysis. Details of the preparation of the Orchard Leaves and Beef Liver standards have been described by the NBS (15) and of the kale standard by Bowen (16). The preparation of the particular tobacco standard used in this study has been described by Nadkarni and Ehmann (5). The samples were dried prior to analysis according to the recommendations of the respective suppliers noted above. One of the standards, Orchard Leaves, was selected as the irradiation standard and the other materials were analyzed as "unknowns." Orchard Leaves was selected as the standard since it has been well characterized for a large number of elements. For those elements of biological interest with no reported values, they were determined in Orchard Leaves using USGS standard diabase rock W-1 or synthetic preparations as standards. Irradiations. Two irradiations were performed on each sample. For information on shorter-lived nuclides (half-lives up to 15 hr), 200-mg samples were irradiated in the Cornell University TRIGA Mark I1 reactor's pneumatic facility for 1-2 min a t a thermal neutron flux of 2 X 10l2 n/cmz sec. For longer-lived nuclides, the 200-mg samples were irradiated for 80 hr a t a thermal neutron flux of 2 X n/cmz sec in the Georgia Tech Research Reactor, Atlanta, Ga. For the short irradiations, the samples were encapsulated in polyethylene vials. For longer irradiation, sealed highpurity quartz ampoules were used. After irradiation, the quartz ampoules were cleaned externally by boiling in aqua regia and washing with distilled water and acetone. A blank ampoule was irradiated every time. No flux corrections were necessary as no variations were observed with the sample size used. Counting and D a t a Processing. The irradiated samples and standard were counted with a 30-cm3 coaxial Ge(Li) detector and 4096 channel analyzer. The system resolution was better than 2.8 KeV (FWHM) and the peak-to-Compton ratio better than 15:1, both for the 1.332 MeV peak of W o . For short-lived isotopes with half-lives from 2.3 min to 15 hr, the counting time was progressively increased from 1 to 30 min. For isotopes with half-lives between 26.4 hr and 4.53 days, counting times of about l hr each were used. For all other longer-lived isotopes, counting times of 3-5 hr were used. The data from the analyzer were acquired on a magnetic tape which was then processed on an IBM 360/65 computer. The final computer output provided digital tables of data (15)
National Bureau of Standards Certificates of Analysis for SRMs 1571 and 1577.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 11, SEPTEMBER 1973
1957
Table I. Nuclear Data for Elements Determined in Biological Materials (77) YOabundance of parent 100 99.8 30.9 5.3 11.3 9.62 27.9 100 100 34.5 24.5 12.4 71.7 1.16 100 9.86 6.8 69.1 100 100 49.5 99.9 28.9 0.15 57.3 23.8 0.0033
a See the
Cross section, o 0.24 4.9 2.3 0.14 0.027 0.2 0.12 7.4 6.4 0.2 0.4 0.1 0.4 1.5 13.3 1.3 1.2 4.5 0.53 4.5 3 8.9 1.1 880 6 0.51 0.3
2.3 min 3.8 min 5.2 rnin 5.8 min 9.5 min 14.6 rnin 17.8 rnin 22 min 25 min 25 rnin 37 min 49 min 85 min 2.52 h r 2.56 h r 2.8 h r 12.4 h r 12.8 h r 15 hr 26.4 h r 35.3 h r 40.2 h r 53 hr 65 hr 67.2 h r 67 hr 4.53 days
2.60 0.10 72.2 100 4.31 0.14 35.2 0.31 29.8 42.8 67.8 100 0.87
2,100 8.8 0.9 7.4 17 11,000 10 1.1 4 3.3 0.04 13 30
6.74 12 18.7 27 27.8 31 45 45 46.6 60.2 71.4 83.8 120
days days days days days days days days days days days days days
48.9 48.7 100 100 47.8
0.46 3 28 19 5,900
243.7 253 2.07 5.26 12.2
days days years years years
Best y-ray used, MeV 1.78 1.434 1.039 0.32 1.013 0.307 1.836 0.312 0.443 0.15 1.64, 2.17 0.247 0.166, 1.43 1.481, 0.367 0.847, 1.811 0.388 1.524 1.348 1.369 0.657, 0.559 0.777, 0.555 0.487, 0.328 0.337, 0.528, 0.493 0.078, 0.069 0.686, 0.564 0.14 0.815, 0.16, 1.308 0.208, 0.133 0.496, 0.373 1.08 0.31 2 0.32 0.177, 0.198 0.482, 0.346 1.10, 1.291 0.28 1.691, 0.603 0.81, 0.865, 1.67 0.889, 1.12 0.265, 0.136, 0.401 1.114, 0.51 0.657 0.796, 0.605 1.333, 1.173 0.965, 1.408, 0.779
Best time for measurement 1-10 min 1-10 min 1-10 min 1-10 min 5-30 rnin 10-30 rnin 10-30 rnin 20-60 rnin 20-60 min 20-60 min 20-60 min 1 hr 30 min to 2 h r 30 rnin to 2 h r 30 min to 2 h r 30 rnin to 2 h r 6-12 h r 6-12 h r 6-12 h r 3-4 days 3-4 days 3-4 days 3-4 days 3-4 3-4 3-4 3-4
days days days days
2 weeks 2 weeks 2 weeks 3-4 weeks 3-4 weeks 3-4 weeks 3-4 weeks 3-4 weeks 3-4 weeks 3-4 weeks 3-4 weeks 3-4 weeks 3-4 weeks 3-4 3-4 3-4 3-4 3-4
weeks weeks weeks weeks weeks
text for details.
and calculated peak areas corrected for background. The peaks of interest were then corrected for decay when necessary.
RESULTS AND DISCUSSION Using the INAA method, it was possible to determine approximately 36 elements in biological materials. The radioisotopes used in the determinations are given in Table I, along with necessary nuclear information. Approximately 19 elements are determined after a 2-min irradiation and periodic counting from immediately after irradiation up to 12 hr. The remainder of the elements are determined after a long irradiation and counting after decay a t intervals of 3-5 days, 2 weeks, and 1 month. It should be emphasized here that although 36 elements are listed in Table I, it will not always be possible to deter1958
Half-life of daughter
mine all of them in all biological matrices. This clearly depends on the levels a t which the elements are present and the matrix in which they are present. In a highly mineralized sample like bone, the number of elements that can be determined is much less. In blood, the concentration level of the elements is much lower than in a less complex material like vegetable leaves, so that fewer elements can be determined. The y-rays listed in Table I represent interference-free (see below) lines for determination of those particular elements in most biological samples. Certain aspects of these considerations are discussed below. Cadmium, Th, and Mo can be determined through the daughter isotopes 115In, 233Pa, and IOlTc, respectively. Nickel can be determined better using the 58Ni (n,p) 58C0
ANALYTICAL CHEMISTRY, VOL. 45, NO. 11, SEPTEMBER 1973
Table II. Determination of Elements by INAA in NBS Standard Bovine Liver, Kale, and Tobacco NBS-SRM 1577 Bovine Liver Element, ppm NBS-SRM 1571 unless indicated Orchard Leaves
Foundb
Eu
...
0.24% (0.055)
0.25% 0.141 26.1 0.09 2.28 4.09
...
... ...
0.17% 0.15 23.1 0.06 2.49 4.43 0.02 0.43 1.04 103 0.17 0.07
118 0.167 0.069
401 2.05 260 2.14 4.31 2.53 0.06 2.3 4.25 489 0.22 0.098
0.28 0.006 0.15 31.7 ND 0.07 0.044
