Presented in Fartial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University



The Ohio State University


Approved bys 0


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 In- o stitute of the Public Health Service are gratefully








A. ASHING...... 12










VI. SUMMARY...... 52


TABLE 0 Page







O Iv ©


















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.


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 impurities in high purity 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 by deuteron bombardment, and minute amounts of in following neutron activation. Small amounts of and 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 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. 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 water, of biologic­ al materials, ^ and of dioxide have been measured.The concentration in single 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.


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 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 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.

Prom equation (6) the weight of an element, X, In the unknown is given by the following equation:

Total counts/sec. from x in unknown = gm. x in unknown Total counts/sec.from Xin standard gm. x in standard

The sensitivity of the method varies with the half- life and the type of radiation emitted by the radioactive species, the flux available, the decay time, the period of irradiation, etc. Assuming reasonable values for these 15 0 quantities, Leddicotte and Reynolds estimated the following sensitivities of detection:

-11 -10 •9 1 x 10 1 x 10 1 x 10 £ europium holmium scandium dysprosium indium arsenic sodium iridium praesodymium lutecium copper tantalum manganese gallium terbium O rhenium gold thulium samarium tungsten o lanthanum ytterbium palladium

1 x 10o8 g .1 x 10~7 g barium nickel cerium silver cadmium osmium chromium strontium cesium phosphorus mercury tellurium potassium molybdenum thallium cobalt rubidium neodymium tin erbium platinum zirconium yttrium ruthenium germanium zinc o hafnium 15. G. W. Leddicotte and S. A. Reynolds, Oak Ridge National laboratory. Private Communication. They assumed conditions similar to those attainable in their laboratory.

TRACE ELEMENTS IN BIOLOGICAL TISSUES O Several rather recent papers in the fields of biology, dealing with trace elements and their accumulation by

specific tissues, have indicated their importance and lack

of knowledge of their functions. For example, Tobias and 13 Dunn reported gold present in the blood of a leukemic human patient; the®concentrations were 11 x 10~8 gm of gold/ gm of wet mass in white cells, 0.4 x 10~8 gm/gm

of wet mass in red cells, and 0.07 x 10 8 gm/gm of wet mass in plasma.

Work with systems shows that the presence of

specific inorganic metallic ions is essential even though

analyses of the naturally occuring systems by a standard

analytical techniques fails to indicate the presence of 16 these ions.


These considerations, and the ready applicability of

radioactivation analysis to problems of the type described

led to the study of the application of this analytical

16. J. B. Sumner and G. Fred Somers. Chemistry and Methods of . New York: Academic Press Inc.,1953. technique to the determination of trace elements in animal tissue.

Gold, arsenic and cobalt were chosen as the elements to be measured. ghis choice was made because of the in­ terest in these elements to biologists cited above, and because they all have which have convenient half-lives. They all have large capture cross-sections for thermal neutrons, and therefore it was hoped that they

°would be produced in measurable quantities by neutron ir­ radiation of the isotopes occuring naturally in the tissues of animals.

Specifically, the problem involved: (1) devising methods for separating arsenic, gold, and cobalt activities

from all others formed when an ashed specimen of the tissues was irradiated; (2) devising methods for verify2- ing the nature of the activities separated and detecting any radioactive contamination; (3) finding the optimum conditions for the quantitative determination of each of 9 the elements and evaluating the accuracy to be expected;

and (4) applying the procedure to some biological tissues.

The sensitivity for the detection of gold, cobalt and arsenic, assuming optimum, values for the neutron flux, bombardment time, decay time, and counting efficiency, are © given in Table I. The quantities given were calculated

from equation (5). The maximum neutron flux in the nuclear o

o 10 reactor at the Oak Ridge National Laboratory was taken 12 / 2, 17 to be 1 x 10 neutron/ cm*' sec J 0a period of irradia­ tion of seven days and a decay time of two days were O assumed. Thermal neutron capture cross-sections and the relative abundances of the stable isotopes were taken 18 from the National Bureau of Standards Circular 499. o The counting efficiency was assumed to be 10 per cent.

17. G. E. Boyd. Anal, dihem. 21, 355 (1949). 1®* Nuclear Data, NBS Circular 499. Washington, D.C. : U.S. Gov. Printing Office (1950). O

0 o

11 o



o Gold

Amt. present Disintegrations Counts gms. per sec. per sec. o

1 x 10“8 1.47 x 103 r.47 x 102 o lox 10"9° 1.47 x 102 14.7

1 x 10"10 1.47 1.47.


1 x 10“8 44.8 4.48


1 x 10"8 2.45 x 102 24.5

1 x 10“S 24.5 2.45

1 x 10"10 2.45 0.25


The two essential phases of the analytical procedure 9 are the ashing of the tissues before irradiation and o the separation after irradiation of the elements of inter­

est from the radioactive isotopes of other elements that

o are also produced in the course of irradiation. °


The samples were ashed previous to irradiation in

order to remove water vapor which would otherwise have

been released in the pile during Irradiation. It was,

therefore necessary to exercise extreme care to avoid con­

tamination of the samples both prior to ashing and during

the ashing procedure, since any arsenic, gold, or cobalt

introduced at this point would be activated and measured

along with material present in the tissue. The ash must

then be transferred to a suitable container which in turn

is packed in a standard irradiation can as0 supplied by

the Oak Ridge National Laboratory.

The following procedure was found to be adequate for

the types of tissue used. The samples are oxidized to

small white residues which dissolve In 3 N nitric

to give clear solutions. The method does not completely

oxidize fatty tissues.

© Immediately after dissection from -She animals, the O tissue samples which weigh one to three grams are 0 placed in tared 10 ml. glass-stoppered weighing bottles. ° o O The bottles are weighed again immediately in order to obtain the wet tissue weights. The samples are then o 19 transferred to 125 ml. Vycor Srlenmeyer' flasks using

10 ml. of triply-distilled water for washing. These flasks are reserved for this purpose and are not used otherwise during the experiments.

The samples are then slowly heated by means of electric hot-plates. Four portions of of

10 ml. each are added; the solutions are boiled and evaporated nearly to dryness.after each addition. Ten ml. of 5 N nitric acid is added and the volume of solu­ tion is reduced to approximately one ml. A drop of con­ centrated hydrochloric acid is added. The solution is © then transferred dropwise onto a boat of thin aluminum foil using a fine glass dropper. The solution is evaporated to dryness0°between additions.

A casserole is lined with two sheets of aluminum foil and the small boat prepared by folding the foil is placed on the two sheets. The casserole is covered with a watch glass and is heated by means of an infra-red lamp placed

19. Vycor is a 96 per cent silica glass product which is very stable chemically to . Corning Glass Works, Corning, N. Y. o o o

14 o about one foot above the samples. After the sample has been transferred, the casseroles are placed in a drying oven set at 150°C for a few hours; this is approximately • the temperature of the nuclear reactor. The aluminum foil O is rolled around the boat forming a pellet 2-3 cm lojgg and

0.5 cm diameter. The pellet is loosely covered with an­ other small piece of foil which is labeled with a glass- marking pencil. The pellets are placed in the aluminum irradiation can. Seven or eight such pellets may be o irradiated simultaneously. A map is made of the location of the samples in the can to facilitate removing them with a minimum of exposure.

The aluminum for each sample exclusive of the final wrapper weighs from 0.10 to 0.15 g. A record is kept of the exact weight. o Throughout the manipulation, the foil is handled with tipped tweezers which are reserved for this purpose. The droppers used in transferring the solutions are carefully cleaned and are likewise reserved for this purpose.


Known quantities of arsenic, gold and cobalt are in­ cluded in each activation. For this purpose reagent grade

arsenious oxide, gold tubing, and cobalt wte’e are used.

% e o 20 The purity of the gold tubing is 99.9 per cent. The purity0 of the cobalt wire based on measurements of the emission spectrum is 99.99 per cent.21 The arsenious o ° oxide is 99.8 per cent pure according to manufacturer's o specification. One to five mg. of each of these elements

are weighed in duplicate with a micro-balance. The stand­

ards are wrapped in aluminum foil, labeled, and placed

in the irradiation cans in the same manner as the samples.


In order to measure the radiation from the individual O a elements of interest it is necessary to isolate them

from the radioactive mixture produced by irradiation of

all of the other elements present °in the ash of the tissues. o Table II isaUst the radioactive materials expected © to be present, together with the intensity of the radio­

activity to be expected from0each. An irradiation for

seven days at 10*^ thermal neutrons/cm^ sec is assumed, as

for Table I; the nuclear data are from the National Bureau 19 of Standards Circular and the composition of the ash is

20. E." N. York, Ohio State Univ., Private Communication. 21. We are indebted to Mr. E. D. Lougher and Mr. S. M. Lambert of Ohio State Univ. for the analysis of the cobalt wire. © « 16

° 22 taken from a standard text in physiological chemistry. O ° The separation from these other radioactive,species made O by carrier chemistry. ®

One of the most sensitive procedures used for detect­

ing traces of gold is the rhodanine method. Slightly

acidic solutions of p-diethylaminobenzylidenerhodanine

gives compound with gold which is a red-violet colloidal

suspension. Colorimetric measurements on the solutions 23 give a limit of detectability of 0.05mcgm. One to four

mg of gold in 25 ml. may be detected by the o-tolidine 24 method. Chloroauric acid converts o-tolidine into a

compound in acid.



22. Practical Physiological Chemistry, P. B. Hawk, B. L. Oser, and W. H. Sumraerson, p.987,451 (Philadelphia: ° The Blakinston Co., 12th ed. 1947). 23. E. B. Sandell. Anal. Chem. 20, 253 (1948). 24. W. S. Clabaugh. J. Research Natl. Bur. Standards , 36j_ 119 (1946).

o TABLE II ACTIVITIES FORMED BY NEUTRON ACTIVATION OF “TISSUE ASH o Element Est. wt. isotope Counts present in consid­ Ti Nfcr fo-Nd - e~M ) A per 1 g of tissue ered 2 t’ sec.

Ca 0.015 o Ca45 152d 1.36xl08 4.08xl05 4.04xl04 4.04xl03

P 0.01 p52 14.3d 4.37xl07 1.25xl07 9.33x106 9.33xl03 42 K 0.035 K 12.4h 3.59xl07 3.59xl07 2.48x106 2.48xl05 35 3 S 0.0025 s 87.Id 1.04x10 52 44.5 4.45 24 7 7 6 § i Na 0.0015 Na 14.8h 1.78x10 1.78x10 1.87x10 1.87x10 ■ 38 6 6 O Cl 0.0015 Cl 37m 5.58x10° 3.58x10 *~0 - o 27 6 Mg 0.0005 Mg 10.2m 1.11x10° 1.11x10 0 - . -5 Fe 4 x 10 Fe59 46d 4.36xl02 43 41.6 4.16 o -6 5 Mn 3 x 10 M^i56 2.6h 4.30xl05 4.30x10 4.3 0.43 -6 2 Cu 2 x 10 Cueen 64 12.8h 3.61x10'J 3.SlxlO4 2.58xl03 2.58x10 e o Cu66 5m 1.06x10 1.06x10 \Z< 0 - ^128 o I 4 x 10-7 25m 1.34xl04 1.34x104 ^0 o o o

1 8

These detection limits are compared with the theoretical limit using activation analysis as calculated in Table I of 2 x 10-10g.

Stable gold consists of 100 per cent abundance of isotope 197 which has a capture cijos s-section of 96 barns for thermal neutrons. 'The product from this excitation will be gold 198. Gold 198 passes to stable mercury 198 by emission of a beta particle with maximum energy of

0*97 Mev and a gamma-ray with energy of 0.411 Mev. Be­

cause of the simplicity of the decay scheme and the forma-',

tion of only one radioactive gold isotope, the decay curve o for gold separated from the tissue samples should follow

a simple logarithmic plot. These decay curves offer a

simple means of checking for radioactive contamination and

establishing the identity of the separated activity. The 9 high energies of the beta particles emitted by gold 198

permit measuring the activity by means of a Geiger-Muller



The procedure used for Separating gold from the tis­

sue ash is the extraction of auric chloride with ethyl

acetate from an oxidizing acid solution which is 6 N in

chloride ion. This is followed by plating the

gold from a basic aurocyanide solution. Other elements s which are extracted by ethyl acetate are iron, gallium, ©

molybdenum, and thallium. The half-lives of these elements

© o are iron 46.3 d.,°2.91 y; gallium, 14.2 h.; molybdenum,

68 h; thallium, 2.7 y.; 4.23 m., 3.1 m. Of these only the half-life of molybdenum is not sufficiently different from that of gold to be detected from decay curves.

Since information on the behavior of molybdenum under these conditions is not available, the separation was made in the presence of radioactive molybdenum and stable gold carrier. No molybdenum was recovered in the plated sample starting with 8667 counts per sec. Therefore, the separation procedure whould free gold from molybdenum contamination.

With the exception of the elements listed in Table II, other elements may be present only in very minute concen-

0 trations. Because of the lack of knowledge of the complete behvior of trace impurities, it is assumed that they may be expected to precipitate in the same manner that they would if macro quantities were presentj so that separations may be affected by means of non-isotopic carriers.


The following portion of the, chemical separations is conducted behind a concrete shield 3 inches thick that is

shoulder high. Rubber gloves are worn and standard pre­

cautions against exposure to radiation are observed.

Throughout the work the radiation dosage was measured by means of a film-badge dosimeter which was checked esrery e

o 20 o two weeks. All films were exposed less than 30 mrep. o The aluminum pellets containing the radioactivated

tissue ash are ^removed from the irradiation can with tongs,

the aluminum (labeled) foil wrapper is removed and the

pellets are placed in 125 ml. Erlenmeyer flasks. Gold

carrier is added (4.8 mg.) as gold chloride solution.

A total of 10 ml. of aqua regia is added in small portions.

Ten ml. of water is then added and the solution is trans­

ferred to a 100 ml. separatory funnel. After this solution

has cooled to room temperature, 20 ml. of ethyl acetate

is added. The mixture is then shaken for 30 seconds,

allowed to stand 10 minutes, and the aqueous is

removed. Ten ml. of 6 N hydrochloric acid is added to

the separatory funnel, the mixture is shaken for 30 seconds

and allowed to stand 5 minutes. The aqueous phase is again

drawn off and added to that removed previously. The wash-

3 ing with 10 ml. of 6 N hydrochloric acid is repeated in

the same manner. 'The ethyl acetate layer is placed in a

50 ml. beaker; the separatory funnel is rinsed with an

additional portion of ethyl acetate which is added to the

beaker. The organic fraction now contains the gold and o

little other radioactivity. Therefore it may be removed

from behind the shielding and handled with those pre­

cautions required for tracer quantities only. The beaker

is placed under an infra-red heat lamp and the solvent o 21

removed by evaporation. The residue is dissolved in 0.5

ml. of aqua regia with slight heating. Ten ml. of 1 N

sodium hydroxide is added, and the solution is transferred

to an electrolysis cell using distilled water for the O ° washes. The volume of tiie liquid is made to 20 ml. with

water and 2 ml. of 5 per cent potassium in 0.1 N

sodium hydroxide is added. The solution is electrolyzed

for 1 hour at a .current of 10 milliamps. p°er sq.

cm. An additional 2 ml. of the potassium cyanide solution

is added and the electrolysis continued for two hours. 25 Figure 1 is a photograph of a Tracerlab electrolysis cell

as adapted for electroplating the radiogold. The

advantage of the adaptation is that it makes much easier

plating from small volumes of solutions and that the cell

can be used more readily with any electrolysis apparatus.

The adaptation includes shortening the arms of the base to

3 in. and threading their entire length. Cell bodies are

then formed from sections of 24 mm glass tubing of any

desired length.

The gold is plated onto the copper disc 2cm in diam.

'The cell is dismantled and washed with water and with

acetone and then it is dried. The activity of the discs

is measured with a Geiger-Mueller tube.

25. Tracerlab Inc., Cleveland, Ohio. o


Figure 2 is a schematic diagram of the arrangement used for measuring the activities of the copper discs.

The background was 0.6 to 0.7 counfc&/sec. The tube window was 2.2 mg./sq. cm. of mica.

In every case the activity is followed through several half-lives in order to check for the presence of radioactive impurities.

The standards are processed in a similar manner.

After evaporation of the ethyl acetate, the residue is diluted and an aliquoit taken. A 3 N hydrochloric- nitric acid solution is used to make the dilution. Be­ cause the concentration of gold in the aliquoit used -5 for plating is approximately 10 g., the same amout of o gold carrier is added as was added to the sample.

O 0





Cardboard cover

Geiger tube

p i S i ? with radio- active gold

Aluminum sliding shelf

Wooden stand

FIGURE 2 Schematic Diagram of Arrangement for Measuring Activities with Geiger-Muller Tube o 25 o o DISCUSSION OF PROCEDURE

The efficiency of the gold separation procedure ° was checked using radioactive gold, sodium and phosphorus because sodium 24 and phosphorus 32 are the chief radio­ actives generated in the ashes of tissues as Indicated in

Table II. O The extraction step was checked by following the procedure as outlined above. Eight series of samples were run to° test the effects of aluminum, sodium, and phos­ phorus on the efficiency of the method of extraction.

Two typical runs show the following recoveries:

Run A (only gold present) Sample No. Percent recovery

I 92.9 II 90.2 III 89.8 IV 95.6

Run B (Gold, Aluminum, Sodium present) Sample No. Percent recovery

I 90.2 II 95.1 III 93.6 o The presence of as much as 0.5 g. of aluminum and 0.1 g. of sodium chloride showe°d no effect on the efficiency of the gold extraction.

A series of extractions were made in the presence © ® of radioactive phosphorus-32 in the form of phosphoric o

© 26 ° acid. The solutions contained 1280 counts/sec. of phosphoric-32 and 12.3 counts per second of gold-198. o The decay curves showed the presence of approximately

10 counts per second of phosphorus-32 and 11.6 counts ° per second due to gold-198. This made it evident that

a second step in the gold purification was necessary.

The use of to reduce the gold chloride

followed by filtration was considered. This proved to

be successful in effecting separation from phosphorus

activity as evidenced by decay curves. However, large

amounts of gold were left on the glass vessel. These

difficulties led to the investigation of the plating

procedure described above.

Determinations by this plating procedure using

333 counts per second of phosphorus-32 and stable gold

showed the recovery of 0.23, 0.28 and 0.30 counts per

second in the separated gold. The background is 0.6 to

0.7 counts per second.

The behavior of sodium during the gold separation

was investigated by using radioactive sodium—24. This

activity was obtained by bombardment of sodium chloride

with deuterons in the cyclotron. The gold extraction

was conducted as outlined, sulfur dioxide being used to

reduce the gold chloride. A series of samples contain­

ing stable gold and 933 counts per second of sodium-24 o showed the recovery of approximately 0.83 counts per second in each sample. This counting rate was too low to establish the identity of the active isotope. In addition to sodium it may be due to impurities of the target holder, impurities of the sodium chloride, or products of competing reactions; therefore no further precaution to separate from sodium ions was deemed to be necessary. It may be pointed out that the difference in half-life 'between sodium-24 (14.8 hours) and gold-198

(2.7 days) is sufficient easily to identify contamination in a separated gold sample by following the decay curve.

The efficiency of the plating procedure was then investigated. The effect of the pH of the plating solution on the recovery of gold was checked. The recovery of gold was complete in solutions more basic than 0.3 N in sodium hydroxide.

Since iron is extracted into ethyl acetate under the conditions described, the effect of the iron (III) in the plating solution was investigated. Concentrations -5 of 8.7 x 10 g. in Fe in the plating solution showed no

interference in the forming of the gold plate. This is twice the calculated concentration in a one gram sample 22 of human tissue, o The plating was investigated with both platinum and copper cathodes. The plate on platinum was much smoother, but the recovery on both surfaces was within the standard deviation of the counting error.

SEPARATION OF ARSENIC O Arsenic 75 is 100 per*cent abundant in normal ■ o arsenic; therefore, excitation with thermal neutrons gives only the radio isotope, arsenic-76.. The capture 0 ' e cross-section for thermal neutrons for this reaction is 19 4.2 barns . Arsenic-76 decays by complex beta and gamma emission to stable selenium-76. A decay scheme proposed 26 recently is given in Figure 2.

Since only arsenic-76 is formed and it decays directly to a stable nuclide, the logarithmic plot of the activity against time should be a straight line. A plot of the activity observed for the arsenic samples separated from the activated tissue will therefore serve to verify the activity as belong to arsenic-76 and to check for radioactive contamination.

26. N. Marty, J. Labeyrlque, H. Langevin. ComptT rend. 228, 1722 (1949). 27 h. As 76 -2 f -

0.4 O

, r\ 0.567

Stable Se 76

Figure 3. Decay Scheme for As 76

10.7 m. Co. 60 r, > 9 0 % 0.059

/32 0.3!


Stable Ni 60

Figure 4 Decay Scheme for Co 60 o 30

THEORY OP ARSENIC SEPARATION ® o • Traces of arsenic are usually detected colori- me iirically bousing the heteropoly0 molybdenum method or by the spot test method of Gutzeit. In the former case, arsenic (V) and ammonium molybdate are reacted and the product is reduced to give a highly colored compound.

® -6 The method is reported to detect as little as 1 x 10" g . 27 arsenic.

The Gutzeit method is based on the reduction of

arsenic compounds to which is reacted with paper

impregnated with mercuri'c bromide or chloride. A com-

parison of the brown stain formed with standards make it possible to estimate the quantity present. A refined —8 procedure permits detecting as little as 4 x 10 g. of 28 arsenic by the method. However, It is not as accurate

nor as precise as the molybdenum method.

As indicated In Table I, activation analysis will

theoretically permit the measurement of as little as

1 x 10“9g. of arsenic.

The separation of arsenic from the tissue ash is

based upon the precipitation of arsenious in

highly acidic solution and the distillation of arsenious

~r 27. E. B. Sandell. Colorimetric Determination of Traces of . New York: Interscience Publishers Inc., 2nd ed. 1950. pp.178-180. 28. H. S. Satterlee and 0. Blodgett. Ind. Eng. Chem. Anal. Ed. 16, 400 (1944). 31 chloride. Its boiling point is 130°C. Ammonium iodide is used to reduce arsenic (V) to arsenic (III) before the first sulfide precipitation. During the distilla­ tion cuprous chloride is used as a reducing agent to prevent formation of the arsenic (V). o The reactions for precipitating As^S^ are:

2As + £- s 3S”£-^ A s2S3 or 2AsO“3+ 16H -h 5 As|s3 +■ 8HgO + 25

The reduction is according to:

2Gu’1^ AsO^3* 8H 2Cuf2+- As^-^-iHgO

The initial precipitation of arsenious sulfide may O coprecipitate antimony, silver, , mercury, tin,0 copper, , cadmium, thallium, molybdenum, german­ ium, selenium, tellurium, tungsten, ruthenium, rhodium, platinum, palladium, iridium, osmium, and rhenium if they are present in the tissue ash. Of these only german­ ium, selenium, and partially antimony will distill over with the arsenic chloride. Germanium decays with half- life of 11.4 days; selenium activities show half-lives of

6.5 x 104 y. and several of less than an hour; antimony activities have hal°f-lives of 2.8 days and 60 days.

These all differ markedly from the 26.8 h. of arsenic; therefore if they are separated with the arsenic activity they may be detected and identified. EXPERIMENTAL PROCEDURE o O

The first portion of this procedure is conducted O ® behind concrete shielding and the precautions outlined earlier are observed. A of ammonium iodide and arsenic carrier as arsenious acid (12 mg.) are added to the aqueous phase from the gold Extraction.

Hydrogen sulfide is passed into the solution and the cooled precipitate is allowed to settle for one hour or o longer. .The solution is filtered through a Selas porce­ lain filter crucible number 3010. °The precipitate is dissolved in 1-2 ml. of concentrated ammonium hydroxide

and the solution is transferred to a distilling flask.

The activity thus separated is. now present in ’’trace" o quantities so ..shielding is no longer necessary. The

distilling apparatus contains a condenser cooled with o water and a receiver cooled with ice-water. It is

equipped with ground-glass joints. A slow stream of

hydrogen chloride is passed" through the system through­

out the distillation. Five ml. of saturated solution of

cuprous chloride in hydrochloric acid is added to the

distilling flask, and ten ml. °of concentrated hydro­

chloric acid is placed in the receiver. Fifteen ml.

is distilled; 5 ml. of cuprous chloride solution is 0

added to the pot and the distillation is continued until

a total of 20 ml. has distilled. Hydrogen sulfide is ©


33 o

passed into £he distillate and the precipitate is o allowed to stand for one hour or longer. The arsenious

sulfide is filtered off with a Selas filter crucible # O as before. The precipitate is dissolved in concentrated o o „ ammonium hydroxide and the solution and washings are °

transferred to the distilling flask. The process is . 0 G repeated exactly as described above. The arsenic (III)

„in the distillate is precipitated as the sulfide. This

is filtered off with a Selas filter crucible. The sides

of the crucible are washed down carefully, the precipi­

tate is washed with small, portions of ethyl alcohol fol­

lowed by ether. The activity is measured in the filter

crucibles with a Geiger-Mueller tube. The measurements

are made for severa half-lives. The geometry of the

counting arrangement is the same as used for measuring

the gold which is illustrated in Figure 2. The dis- L o tance between the tube window and the sample is 5.5 cm.


In order to check the arsenic separation, tracer

arsenic was prepared by bombarding arsenious oxide with e deuterons in the cyclotron.

The entire procedure including the gold extraction

step, was carried out with radioactive arsenic. The re­

covery of arsenic was complete within the standard

© o

34 o deviation of the counting error. The pirification of the arsenic was carried out in the presence of stable arsenic and phosphorous-32 (250 counts per second). No ° phosphorous-32 was recovered in the precipitate. The • purification was also performed in the presence of active cobalt (30 counts per second). No eactivityc was

( ° o o The0reproducibility of the mounting0was checked'by • o precipitating arserfc sulfide containing active tracer, filtering and comparing activities. The results for the four samples in the series are: 34.5, 35.2, 35.9, and 55.1 counts per second. These fall within the standard deviation expected for the counting rate.

The vapors formed during the solution of the aluminum pellets were checked for activity since this was a pos­

sible source of loss of arsenic." No activity was found. ■ i o

G O SEPARATIOF OF'CGBALT (•; 0 Normal cobalt consists of a single isotope, namely ( cobalt 59. It shows a capthre cross-section of 22 barns

for thermal neutrons to give cobalt-60 on activation in

a nuclear reactor. This isotope decays with the emission m

of a beta particle and two gamma rays in cascade accord- pQ ing to Figure 3. The decay time for the proposed

29. J. J. Livingood and G. T. Seaborg. Phys. Rev. 60, 913 (1941). 9

O experiments is sufficiently large that the 10.7 min. O activity will not be observed. Therefore the energy 9 O of the radiation rather than the half-life will be used to verify the .separated activity as belonging to cobalt-

60 and to check for radioactive impurities. Measurements of half-life were used to check for short-lived contamin­ ation*. Both absorption and scintillation spectroscopy was used to characterize the radiation.


Traces of cobalt are usually detected colorimet- rically by means of the reaction between nitrose- R 30 —6 salt or the thiocyanate.~ As little as 1 x 10 g of cobalt may be determined by the former method. The thiocyanate method is slightly less sensitive.

Prom Table I the limit of sensitivity for the measurement of cobalt by the method of activation analy­ sis is of the order of 1 x 10*"®g.

The procedure devised for separating cobalt from O the remaining activities is the precipitation of cobal- tous sulfide in very basic solution followed by its precipitation as the l-nitroso-2-naphtholate. In the cobaltous sulfide precipitate from very basic solution

50. F. Feigl. Spot Tests. N e w York:Nordemann Publishing Co., Inc., '1937, p. 82. of #sodium hydroxide may also be nickel sulfide and manganese sulfide. Nickel and manganese do not form

a salt with l-nitroso-2-naphthol in solution at pH 30 of 6; so that by adjusting the acidity before the

precipitation, a separation from these ions is effected.

These reactions are:

. Co+ 2 -+~ S^JL-=* CoS

and » x

°°'*s +- ( y y ° H — * °°f’cioH6 (B°i73

3 *


The filtrate of 40-50 ml. from the separation of

arsenic sulfide was stored for several months during

which the level of total activity had dropped enough

to allow handling as "trace” quantities.

Cobalt carrier as the chloride (1.1 mg.) is added

to the solutions, which are slowly evaporated to dry- <9 ness; the residue is treated with°5 ml. of aqua regia

and taken to dryness. The nitrate ion is destroyed by

treatment with three portions of 10 ml. each of hydro­

chloric acid. The residue is then dissolved in 20 ml.

of water; a small portion of hydrochloric acid is O o 31. James L. Dick. Dissertation, The Ohio State Univ. 1953, p.29. added If necessary to complete the dissolution. Suf­ ficient sodium hydroxide is added to raise the pH to 10 as measured by indicator paper. During this treatment « aluminum hydroxide precipitates and completely redis­ solves. Hydrogen sulfide is passed into the solution O to precipitat©,; cobalt sulfide, which is filtered off with a Selas filter crucible. The precipitate is re­ dissolved In 1-2 ml. of aqua regia. This solution is boiled to dryness and is treated with three portions of

5 ml. of hydrochloric acid. The residue is dissolved

In 20 ml. of water and the pH is adjusted to 6 using

0.1 N ammonium hydroxide; and 10 ml. of a saturated

alcoholic solution of l-nitroso-2-naphthol is added.

The precipitate is allowed to stand about four hours

and is filtered through a Selas filter crucible. . The

walls of the crucible are carefully washed with water;

the precipitate is dried in air. The activity of the

cobalt samples is measured on a Geiger-Mueller tube.

Absorption of the beta-rays by aluminum and the gamma

ray spectrum as measured with a scintillation spectro­

meter is used to characterize the activity. O The standards were diluted to 25 or 50 ml. and one

ml. aliquoits were used. These were handled in exactly

the same manner as the samples of each from the tissues DISCUSSION OP RESULTS o 9 The efficiency of the precipitation of cob<ous © sulfide under the conditions described was investigated 0 by® means of radioactive cobalt tracer. ©The activity c o © recovered was complete..

The precipitation with l-nitros.o-2-naphthol appear- © C n " . * : ‘ " ' ; '1 :®' ed 0to be 100 per cent by comparison of recovered activity- with an aliquolt of the standard solution, although

differences in scattering due to differences in sample

thickness change the counting rate. The precipitation

was repeated a second time and both the filtrates were

boiled to drynessJ neither showed any activity. Table III

is a flow sheet of the procedure for separating radio­

active gold, arsenic, and cobalt from samples of tissue 9 ash.“ o


o . ■ o The analytical stheme developed here was applied to

O ° tissue samples taken from experimental animals.. c ° Rats of the C. P. Wistar bearing lymphosar­

coma tumors, mice of© C3H® strain bearing C3HBA tumors, o ABC strain mice bearing/tunwrsA, and CFW strain mice

bearing sacroma 37 were used.

Seven bombardments of 6 or 7 samples each were

analyzed. The results of these analyses are shown in 00

Table III. © ©



Activated Tissue Ash

aqua regia e

(i»o( III) ,Au( III) > As (V )

Au jarrier£ eth^l acetate ext;rac-tion

Au(IIT ,Co( III )As (V) ,

ele c tropiating As carrier NH«I HVS

As. Co(III

distillation Ha OH CuCl HvS HCl Co carriers CoS^

iqua regia H*S ■ >(IID 4^4 - 1-nitroso- distillation 2-naphthol CuCl pH 6 HC1 Co(.C„, h an o o ), jsCli"


ASg.S %


o o

o o 40

TABLE IV TISSUE ANALYSES o Sample wt. Wt.Arsenic Wt. Gold Wt. Cobalt (g.) per g. wet per g. wet per g. wet tissue ‘ tissue tissue ° ° . (micrograms) (micrograms) ftmicrograms)

Rat-A spleen 1.91 1.56 0.101 1.95 lymph- nodes 0.0516 1.45 0.161 1.19 tumor 3.S8 2.60 0.199 1.19

Rat-B spleen 0.904 ■0.101 1.57 (normal animal) tumor 2.999 0.026 0.093 spleen 1.900 0.135 1.19° (tumor bearing an imal)

C5H Mice spleen 0.*789 0.467 tumor 0.646 V o 0.184 . ° - blood 0.998 0.214 —

ABC Mice-A o o spleen 0.845 • 0.461 <0.001 tumor 1.107 0.177 - blood 1.131 3.43 0.202 -

ABC Mice- B spleen 0.663 - - ^0. OO 1 tumor 1.023 - 0.0717 /I blood 0.823 0.808 l\ - o ’" O CFW Mice o spleen 0.865 - 0.0113 <0.00 1 X h tumor 0.508 - 0.0183 blood 1.001 0.319 -

o o

© o o


The spleen and lymph nodes of Rat-A were taken from #

an animal bearing a transplanted lymphosarcoma. All

samples of tissue from mice are from animals bearing

transplanted tumors. The arsenic standards bombarded

with the ashed tissue. samples sOf Rat-B decayed com­

pletely before the separation was completed. Other radio-

o activities of the arsenic samples marked with a dash were o too small to measure. Tissue samples from ABC-A and C3H e mice were dissected at the same time and tissue from

ABC-B and CFW mice were dissected at the'same time.

Blanks were run on the dishes used in ashing, and

on the reagents. In addition a blank was run in each

bombardment along with two standards. Samples of aluminum

foil were included in two° bombardments. The arsenic in

the blanks set . the limit on the sensitivity of the

arsenic®determination. This arsenic was>apparently, in­

troduced in the nitric acid>aluminum foil or in the ashing

dishes. The aluminum foil was also analyzed for these

elements. It contained no detectable gold or cobalt, GLnticL _7 4.25 x 10 g. of arsenic per g. aluminum. Following are

typical decay curves for samples of radioactivated gold o separated from tissue ash and a standard (Figure 5) and © for samples of radioactivated arsenic separated from

tissue ash and arsenic standard (Figure 6). An illustra­

tive sample calculation for the amounts of gold and arsenic o

o o 42 in the tissue samples made by means of these curves is e given below. «

Figure 7 is absorption curves in aluminum for cobalt beta radiation. , Figures 8 and 9 are the gamma-ray spectra of cobalt samples as measured with a scintillation spectro- © meter. Figure 9 is of a typical sample recovered from tissue ash; Figure 8 is of a cobalt standard.

All the standards were run in duplicate; therefore a comparison of these standards serves to give the accuracy obtainable using this procedure for the chemical © separations. Table IV lists these results.



Bombardment terror terror terror in in in arsenic Gold Cobalt detn. detn. detn.

II - 4.3 IV 7.9 2.8 0.3 » V 12.0 7.0 5.1 VI 12.3 9i7 - * VII 5.8 0.6 9 3 o

Sample I is not included as the second standard contained tissue ash. One of the standards of Sample III ° was spilled. The arsenic used for Sample”II was too weak to measure. o ©

o Activity, counts/sec. 10,000 1,000 100 10 IUE 5 FIGURE o 12/ 53/ 3 /5 /9 2 1 = To T 0 = 100 12 /1/53/12=00 /1/53/12=00 e a Cre fr ape Con­ Sampled for Curves Decay ann Rdocie Gold Radioactive taining uv I standard,, - II x (4.63 CurveI0 uv I pen f C of spleen - I Curve mice gold) 1 0 300 200 :0 onCre II noon-Curve 2:00 ie (hours) Time uv I Curve lp gvs T gives Slope noon Curve ‘ slope n, gives ^ T - uv I Curve - IUE 5 FIGURE

j =


400 strain H 65h -43- "5

Activity, counts/sec. IOOO i o n 50 0 IUE 6 FIGURE 0 1/1/3 10:30 AM 12/17/53/ =T0 o 1/1/3 I:0 noon I2:30 12/ 19/53/ =To Curve II lp givesSlope ie (hours) Time 100 Arse n ic Radioactive Containing Samples for Curves Decay 27h =

Curve H - standard - H Curve Cre I Curve — Curve I - spleen - ABC- of I Curve strarn mice , run A run mice ,strarn

29 x IQ- x (2.94 Soe ie T= 27h = T gives °Slope



4 4-

Activity, counts / sec. O.l FGR 7 oFIGURE bopin n lmnm f ea Particles Beta of Aluminum in Absorption mte b Rdocie Cobalt Radioactive by Emitted 200 uv I » uo o rt A rat of Tumor -» I Curve uv I - Standard - II Curve 400 Mg. AI /sq. cm. - 5 - 5 - cm. Mg./sq. AI IUE 7 FIGURE uv I Curve uv E Curve 600

800 9

1000 © ~ Activity, counts/sec. 375 - 0 5 2 IUE 8 FIGURE am-a Setu o a ape Containing Sample a of Spectrum Gamma-Ray aiatv Cbl, tnad 76 x lO'g.cobalt) x (7.62 Standard Cobalt, Radioactive 0.2 0.4 IUE 8 FIGURE 0.6 Curve I nry (mev) Energy 0.8 o o o O -L b - Activity, counts /sec. 3.75 2.50 .25 IUE 9 FIGURE am a Setu o a ape otiig aiatv Cobalt Radioactive Containing Sample a of Spectrum Ray - Gamma 0.2 0.4 Sample Sample 6 Q .Epne sae ° scale, .(Expanded " pen f CF of W strain spleen - o nry (mev) Energy iUE o 9 FiGURE 0.8 o mice © 0


Sample Calculations: o

Gold Sample - e

Prom Figure 5: Activity of Sample is 297 counts/sec at 12/1/53/12:00 noon. Sample is ash of spleen weighing 0.790 g. s

Activity of Standard (4.63 x 10 ^ g gold) is 1283 counts/sec at 12/14/53/12:00 noon.

Time difference is 312 hj Calculation of gold standard activity at 12/l/53/l2:00 noon: L0.W3XJI$ , NQ = Ne - (1283)e 4?---- = (1283)e5*'52 =

= (1283)(27.6) = 3.73 x 104 counts/sec

Using equation 7, page 8 :

2.97 x 10^ - x 3.73 x 10^ 4.63 x 10 «

x s 3.68 x 10“7 g.

— -S-j~..S.g.12 ---- a 4.67 x 10 g. gold/g. wet tissue. 0 • 1 90

Arsenic Sample: 0

Prom Figure 6 : Activity of Sample is 6.50 counts/sec. at 12/20/53/12:30 noon. Sample is ash of spleen weighing 0.845 g. 9 Activity of standard is 2467 counts/sec. at 12/20/53/12:30 noon. Weight of standard is 2.94 x 10”4 g.

Using equation 7:

6.50 - x 2467 2.94 x 10"4 —7 x s 7.76 x 10 g. arsenic

o ©


_»7 Blank gives 4.41 x 10 ' g. arsenic

(7.76 - 4.41) x [email protected] x 10“^ g. arsenic due to ash.

3.35 x 10 7 - „ •, ,->-7 j, / j. Q~g45------3.96 x 10 ' g. arsenic/g. wet tissue.

Cobalt Sample:

From Figure 7: Activity of sample is 20.8 counts/sec. Sample is ash of tumor weighing 3.88 g.

Activity of standard is 22*4 counts/sec Standard weighs 4.99 x 10"6g.

Using “equation 7:

20.8 x ___ 2274 4.99 x 10-6

x = 4.60 x 10"6

-4> .° g - d o " ----- = x 10"6 g* cobalt/g. wet tissue, o • oo


The sensitivity of the analysis for gold appears to approach the value predicted from theoretical calcula­ tions. The procedure could be improved by use of a more elegant arrangement for the extraction of the gold chloride. ® ' The limit of sensitivity of the determination of arsenic is set by the arsenic impurity present in the acid and possibly in the aluminum ashing dishes. An improve­ ment of this would necessitate changing the ashing pro­ cedure. One solution is to freeze-dry the tissue sample and"irradiate it in quartz capsules. It would of course be necessary to check the quartz for impurities and to establish a uniform quality for the quartz. It is O rumored that very high purity quartz will be available presently from the Corning Glass Company.

One improvement of the method used for mounting the cobalt samples for beta counting would probably increase the sensitivity of the procedure appreciably. Another possibility is to count gamma-rays by means of a scintil­ lation counter. This gives the same order of efficiency as a Geiger tube gives for beta rays.

The chemical separation outlined is extremely time consuming, in order to simplify the procedure and simul­ taneously to analyze for more elements one might turn to paper chromatography and scintillation spectroscopy. © 51 o More Information might be obtained faster on the activated ash by the use of a beta-gamrna coincidence scintillation spectrometer. By integration of the area under the photoelectric peak of a differential pulse height curve, quantitative determination of the gamma- emitters present may be calculated. Mixtures of the three components have been determined with an average 32 precision of + 7 per cent.

The next step in the choice of samples might be to analyze human tissues.

The reliability of the proposed procedure as cal- © culated in Table IV compares favorably with the 10-20 33 per cent predicted by Boyd for the method. O

32. R. E. Connaliy and M. B. Leboeuf. Anal. Chem. 25, 1095-1100 (1953). 33. G. E. Boyd. Anal. Chem. 21, 337 (1949).

© o © o 52 © SUMMARY ©

A procedure for determining the content of gold, arsenic and cobalt in biological tissue samples has been devised. The method was applied to tissues of rats and mice and a comparison between cancerous and normal tissues was made.

The procedure .used activation analysis. The material O being analysed was irradiated with neutrons from a nuclear reactor; the activities formed belonging to gold, arsenic, and cobalt were isolated and measured. The quantities were calculated by comparison of the counting rates of the separated activities to the counting rates of stand­ ards of the elements which were irradiated at the same time.

The gold activity was separated from the tissue ash by extraction of an acid solution with ethyl acetate after adding gold carrier. The was then plated from a O basic cyanide solution and the activity measured. Half- life measurements served to identify the activity as be­ longing to gold-198 and to check for radioactive contam­ ination. The error of the method in determining known quantities of gold was ^ 9 . 7 per cent or less.

The arsenic activity was separated from the tissue ash by precipitating as the sulfide.0 The precipitate was purified by distilling as . © 53 o Measurements of half-life were used to identify the

activity and to check for contamination. The error of

the method in determining known quantities of arsenic

was 4 12.3 per cent or less.

The cobalt activity was separated from ©the tissue

ash by precipitating, as the sulfide in a very basic©

solution. The separated cobalt was reprecipitated as the

l-nitroso-2-naphtholate which was mounted for counting.

Measurement of total activity with a Geiger tube was

used in msicing the calculations. The absorption in © aluminum and the scintillation spectra were used to

identify the activity as belonging to cobalt and'to

check for contamination. The error of the method in

the determination of known quantities of cobalt was

£,5.1 per cent.


© © © 0



I, Bettie MeSpedden Dale, was born in Wichita

Palls, Texas, July 7, 1923. I received ray secondary

education in the public schools of Wichita Palls. My

undergraduate training was obtained at Baylor University,

from which I received the degree Bachelor $f Science in

1946. During 1944, I was employed by the American Cyanamid ta> and Chemical Corp., Corpus Christi, Texas, as an analytical

chemist. Following graduation from Baylor University, I'

was employed by the Phillips Petroleum Co., Phillips,

Texas, as an assistant chemist. I entered The Ohio State

University in October of 1947 and received the degree

^Master of Science in 1950. Prom 1947 to 1950, I served

as a teaching assistant in the Chemistry Department of © The Ohio State University, and during 1950-1951 I was

employed as a Research Assistant with the University Re­

search Foundation. In 1952 I received a grant from the

Ohio Department of Health. That year, I was also appointed

as a Public Health Service Research Fellow#of the National

Cancer Institute, which I have held while completing the

requirements for the degree Doctor of Philosophy.