A FROCEDURE FOR THE DETERMINATION OF ARSENIC, GOLD AND COBALT IN BIOLOGICAL TISSUES Dissertation Presented in Fartial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By BETTIE MeSPEDDEN DALE, B, S., M. S The Ohio State University 1954 Approved bys 0 ACKNOWLEDGMENTS The author wishes to thank Professor A. B. Garrett and Dr. W. G. Myers, who jointly directed this research, for their encouragement and advice* Grants from The Ohio Department of Health and the National Cancer In- o stitute of the Public Health Service are gratefully acknowledged. ii \ 878nO TABLE OF CONTENTS Page I. INTRODUCTION........................... 1 A. HISTORICAL DEVELOPMENT............ 1 B. THEORY OF ACTIVATION ANALYSIS 4 C. TRACE ELEMENTS IN BIOLOGICAL TISSUES.......................... 8 II. STATEMENT OF' THE PROBLEM.............. 8 III. ANALYTICAL PROCEDURES................. 12 A. ASHING. ...................... 12 B. PREPARATION OF STANDARDS.......... 14 C. SEPARATION OF GOLD................ 15 THEORY OF SEPARATION.............. 18 EXPERIMENTAL PROCEDURE........ ... 19 DISCUSSION OF RESULTS............. 25 D. SEPARATION OF ARSENIC............. 28 THEORY OF SEPARATION.............. 30 EXPERIMENTAL PROCEDURE............ 32 DISCUSSION OF RESULTS............. 33 E. SEPARATION OF COBALT.............. 34 THEORY OF SEPARA TE ON.............. 35 EXPERIMENTAL PROCEDURE. ........ 36 DISCUSSION OF RESULTS........ 38 IV. TISSUE ANALYSES . ... 38 V. DISCUSSION OF RESULTS’................ 50 VI. SUMMARY........................... 52 VII. AUTOBIOGRAPHY.......................... 54 ill LIST OP TABLES TABLE 0 Page I SENSITIVITY OF DETECTION OP GOLD ARSENIC AND COBALT.................... 11 II ACTIVITIES FORMED BY NEUTRON ACTIVATION CF TISSUE ASH ............. 17 III FLOW SHEET OF ANALYTICAL e PROCEDURE........... 59 IV TISSUE ANALYSES........... 40 V ERRORS IN THE DETERMINATION OP KNOWN QUANTITIES OF GOLD, ARSENIC, AND COBALT .............. 42 O O Iv © LIST OB1 FIGURES ° FIGURE 0 Page 1 APPARATUS FOR PLATING GOLD SAMPLES................................ 23 2 SCHEMATIC DIAGRAM OF .ARRANGEMENT FOR MEASURING ACTIVITIES WITH A GEIGER MUELLER TUBE.................. 24 3 DECAY SCHEME FOR ARSENIC-76.......... 43 4 DECAY SCHEME FOR COBALT-60........... 44 5 DECAY CURVES FOR SAMPLES CONTAINING RADIOACTIVE GOLD...................... 45 6 DECAY CURVES FOR SAMPLES CONTAINING RADIOACTIVE ARSENIC.................. 46 7 ABSORPTION IN ALUMINUM OF BETA PARTICLES EMITTED BY RADIOACTIVATED COBALT................................ 47 8 GAMMA-RAY SPECTRUM OF A SAMPLE CONTAINING RADIOACTIVE COBALT........ 48 9 GAMMA-RAY SPECTRUM OF A SAMPLE CONTAINING RADIOACTIVE COBALT........ 49 O © © V 9 A PROCEDURE FOR THE DETERMINATION OF ARSENIC, GOLD AND COBALT IN BIOLOGICAL TISSUES INTRODUCTION One of the more recent developments in analytical technique is the quantitative determination of minute or "trace” quantities by bombarding specimens with neutrons or charged particles and measuring the radioactivity in­ duced in the element of interest. This technique has come to be known as radioactivation analysis. HISTORICAL DEVELOPMENT Prior to 1945 several applications of the method had been described in which accelerated charged particles and neutrons were used to induce radioactivity. The advent in 1943 of nuclear chain reacting "piles" or reactors with high neutron fluxes made radioactivation analysis available as an extremely sensitive tool for detecting and assaying small quantities of many elements. 1 The technique was first developed by Hevesy and Levi, who analyzed rare earth mixtures for dysprosium (0.1 per cent) and europium (1 per cent) with the aid of a neutron 1. G. V. Hevesy and H. Levi.Kgl. Danske VidenskabT Selskab Math-fya. Medd. 15, 11 (19387. 2 source. At about the same time Seaborg and Livingood estimated gallium impurities in high purity iron samples as 6 p.p.m. from measurements of the radioactivity in­ duced in the gallium by bombardment with 6.4 Mev deuterons, They were also able to detect small amounts of iron in cobalt oxide by deuteron bombardment, and minute amounts of copper in nickel following neutron activation. Small amounts of sulfur and phosphorus were measured by activation with neutrons and deuterons respectively, to form radio- s active phosphorus-32. Other early applications included: the detection of less than 0.01 per cent copper in silver 3 by bombarding the silver with 16 Mev alpha particles; the detection of 10 p.p.m. of sodium in aluminum by use 4 of 3 Mev deuterons; the determination of iridium in 5 platinum and in rhodium by neutron activation; the deter- 6 mination of zirconium in hafnium; and the detection of as little as 0.05 per cent carbon in iron using one Mev pro- 7 tons and deuterons for the radioactivations. 2. G-. T. Seaborg and J. J. Livinggood. J. Am. Chem. Soc. 60, 1784 (1938). 3. L. D. P. King and W. J. Henderson. Phys. Rev. 56, 1169 (1939). 4.R. Sagane, M. Eguchi and J . Shigeta. J. Phys. Math. Soc. Japan lo, 383 (1942). 5. ft. Uopel and K. Dopel. Physikal. Z. 44, 261(1943). 6. A. Aten. Nederland. Tl.idschr. Natuurk. 10,257(1945). 7. M. V. Ardenne and P. Bernhard. Z. Physik.122, 740 (1944). © O 3 The great increase in the available neutron flux, occasioned 1>y the development of the nuclear reactor, O O brought about a corresponding increase in the sensitivity O of the method, and hence in the scope of its applications. Typical analyses are the determinations of gallium in iron meteorites, in quantities less than 0.01 p.p.m., and of palladium in quantities of a few parts per 8 9 million. The arsenic content of sea water, of biologic­ al materials, ^ and of germanium dioxide have been measured.The thallium concentration in single crystals 12 of potassium iodide has been measured. Human blood 13 samples have been analyzed for gold. Work is underway to determine the amount of cobalt, rubidium and iron in animal tissue.^ 8. Harrison Brown and Edward Goldberg. Science 109, 347 (1949). ° 9. A. A. Smales and B. D. Pate. The Analyst 77, 188 (1952). 10. A. A. Smales and B. D. Pate. The Analyst 77, 196 (1952). 11. A. A. Smales and B. D. Pate. In the press. 12. C. J. Delbecq, L.E. Glendenin, and P. H. Yuster. Anal. Chem. 25, 350 (1953). 13. C. A. Tobias and R. W. Dunn. Science 109, 109 (1949). 14. C. A. Tobias and H. L. Helwig. Private Communica­ tion. © THEORY OP ACTIVATION ANALYSIS When a sample is placed in a bombarding flux, the number of radioactive atoms produced per unit of time is given by the equation: dN*/dt = N^f - AN* (1) o where f is the bombarding flux in units of particles per sq. cm. per sec.; oris the isotopic cross-section for p reaction in units of cm per target atoms: N is the number of target atoms present in the sample; N-* is the number of radioactive atoms present in the sample; ^ is the decay constant of the radioisotope produced. Upon integration, one finds that the number of radio­ active atoms present after irradiation for a time, t, is: N* = No-fd - e”M )/\ . (2) Thus the activity A^. in disintegrations per unit of time, due to the irradiation of N atoms for a time, t* is: A t « = Na-f(l - ) (3) O or , -/t «. A.(. * A»o (l-e ) (4) ® where A ^ is the saturation activity, fo-N, produced by an infinitely long irradiation and the saturation factor (1 - e-^ ) varies from zero to one* ®In practical work the saturation activity is necessarily assumed to be constant. In assuming this constancy it obviously must be assumed that the flux and the energy of the bombarding particles are constant, since the cross-section depends on the energy of the bom­ barding particles. Also, the original number of target atoms N must not change appreciably. If these conditions are satisfied, equations (1) and (2) will describe the growth of a radioactive species throughout the bombardment time, t. The newly formed activity will decay with its characteristic half-life, so that at a time, t', after the end of the bombardment, the amount of radioactivity present will be J At, = A^e"^' (1 - e'^). (5) Equation (2) may be solved for the weight, w, of the target element In the sample to give the expression: w = A tM/fKcr (6.02 x 1023 )(1 - e " ^ ) (6) where M is the chemical atomic weight of the element being sought; K is the fractional abundance of the target isotope in the element. This equation then permits the determination of the weight of a given element in a sample by making radiochemical measurements only. Two methods of approach to activation analysis are used: (l} the absolute method and (2) the comparative method. In the absolute method, equation (6) Is solved for the weight of element being analyzed. This necessitates O knowing the bombarding flux, the period of irradiation, the half-life, the activation cross-section, the atomic weight of the element, and the absolute disintegration rate. The determination of disintegration rate from counting rate required knowledge of the counter efficiency and corrections for geometry of the counter set-up, ef­ fects of air absorption, window-absorption,self-absorption, backscattering, etc. In order to minimize the effect due to uncertainity in half-life the irradiation time should be long in terms of half-life and the decay period fol­ lowing irradiation should be as short as possible. In the comparative method, a standard sample con­ taining a known amount of the element sought is irradi­ ated along with the sample to be analyzed. The activity produced in the unknown is compared to that produced in the standard, and from this comparison the concentration o in the unknown is determined. Thus only the two counting rates are measured. The s tandard should have the same general composition © as the unknown and should be put through the same chemi­ cal separation if such is necessary. The time, t ’, at which the activities are measured must be the same for both samples.