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UNITED STATES ATOMIC ENERGY COMMISSION
RADIOACTIVE ISOTOPE TRACER TECHNIQUES
G. E. Boyd
Clinton National Laboratories \
This document consists of 7 pages.
Date Declassified: April 16, 1947
This document is for official use.
Its issuance does not constitute authority
for declassification of classified copleb
of the same or similar content and title
and by the same authorss.
Technical Information Division, Oak Ridge Directed Operatins r .-
Oak Ridge, Tennessee
'A C ',"';
RADIOACTIVE ISOTOPE TRACER TECHNIQUES*'
By G. E. Boyd
It is already evident that one of the most important by-products of the nuclear physics and chem-
istry.leading to the development of the atomic bomb will be the production of radioactive isotopes.
The method of radioactive indicators is a natural development of the early work of the Curies which
led to the discovery and separation of polonium and radium. As early as 1913, Paneth and Hevesy
made the first application of radioactive substances to chemical problems. The principles governing
the use of isotopes as "tracers" were all well worked out before 1930. There were severe limita-
tions in.the early work, however, because only the naturally occurring radioactive isotopes were
With-the discovery of artificial radioactivity in 1934 by the Curie-Joliots and with the perfection
by Lawrence of the cyclotron, the whole art gained a new momentum, and the use of isotopes as
tracers became a major field in applied nuclear physics during the middle and late 1930s. Only then
were radioisotopes of all the chemical elements discovered and made available, together with the
means for detecting them. Still more recently, the development of the uranium chain reactor, or
pile, has revealed new vistas for the mass production of these valuable, "fine" chemicals.
The fact that at least one usable radioisotope for every element is known (with the notable ex-
ceptions of nitrogen and oxygen) helps to emphasize the variety of potentialities latent in the applica-
tion of the method of isotopic tracers. A discussion of radioactive isotope tracer techniques is ap-
propriate to this symposium, for these procedures find their most frequent application on a bench
scale using the equipment and methods ordinarily employed in work at that level.
Before initiating a detailed discussion of tracer methodology, it is desirable to note four rather
distinct applications of isotopes to problems in chemistry and in chemical development. Radioactive
elements may be. used as "indicators," they may be used as "tracers," they may be used in radio-
autographic techniques, or, they may be used as sources of penetrating radiations.
Consider further the first two uses of radioisotopes, namely, as "indicators" and as "tracers"
for it is with these we shall be concerned. When an active isotope is added to a system together with
common, inactive isotopes in the same state of chemical combination, it may serve to "indicate"
the path of the compounds of the stable isotopes throughout the various changes the system may under-
go. If the initial ratio of the amount of the'active isotope to stable isotope remains constant through-
out, nq information other than the course of the element is revealed. Frequently, however, this ratio
is altered owing to the distribution of activity between two sources of inactive isotopes; then, some
type oilexchange process of possible significance may be disclosed. Hence, through the simple expe-
dient of "labeling" or "tagging" some of the components of a mixture, it becomes possible to "trace"
otherwise hidden chemical transformations, and hence to study many problems of basic importance in
both theoretical and applied chemistry.
In the span of time set apart for these remarks it is obvious it will not be.possible to discuss all
of the uses of all of the known radioisotopes, even though the field of applied radiochemistry has
*(Presented as an illustrated lecture before a meeting of the American Chemical Society. Slides
used with the lecture were not furnished for reproduction with this document.- A.E.C., T.I.D.)
MDDC 840 1
scarcely been more than scratched. Accordingly, the subject matter will be limited arbitrarily, and
the uses of only seven isotopes of the greatest immediate general importance will be indicated. Topics
to be considered briefly are: the production and properties of radioisotopes of hydrogen, carbon,
sulfur, phosphorous, and the halides, the labeling of chemical compounds with some of these radio-
isotopes, the design of experiments employing radioisotopes, the analysis for radioactivity, and, in
conclusion, some uses of compounds containing "tagged" atoms.
Before considering the first lantern slide, let us mention several known types of nuclear reac-
tions of current importance in the production of radioisotopes. Firstly, there are those reactions
which change the nuclear charge. These are the transmutation reactions. Examples may be found
in the transmutations effected by the bombardment of nuclei with energetic particles, and in the ::.. r
transmutations caused by radioactive decay in which an alpha or a beta particle is emitted. Secondly,
there are those nuclear reactions which change the nuclear mass number. The most important o ,:.
these is the capture or emission of neutrons. Finally, of course, there are those reactions which.
change both nuclear charge and nuclear mass number.
From the point of view of the production of radioisotopes, a most important quantity is the field
of a nuclear reaction. In many cases the amount of activity produced is quite small, either owfiig to
a small cross section for a desired nuclear reaction or to a small flux of bombarding particles. If
the cross section for a nuclear reaction be low then it is essential that the number of the bombarding
particles be very large. Considered merely as a source of neutrons alone, the chain reacting pile -
greatly exceeds all earlier devices.
In the first slide (Slide 1) we have listed some information about the isotopes to be considered'
You will note radioactive hydrogen, the long-lived, radioactive carbon, radioactive phosphorous, sul-
fur, and the radiohalides. In the second column are the compounds irradiated; in the third are the
nuclear reactions for the production of these isotopes in the graphite pile. The fourth and fifth col-
umns list the most recent and presumably the best values for the half-life and for the maximum
energy of the radiations emitted in the decay of the isotopes. It is of interest to-note the wide varia-
tion in the half-period. Also, note that all the isotopes being considered are either pure beta emitters,
or else emit a beta together with a gamma ray. Here again, the wide variation in the energies of the
beta emitters should be remembered. In the last column are given the maximum unit quantities avail-
able of these isotopes if they are obtained through the Isotopes Branch of the United States Atomic"
Energy Commission at Oak Ridge. Recently, the quantities of isotope procurable have increase, and
the cost per unit has been lowered.
Let us suppose we have received a shipment of radioisotopes, and that we desire to utilize'them
in a research problem. Sometimes, although rarely, we may merely add the isotope to the'system we
wish to investigate in the form in which it comes. Rather more frequently, however, it is necessary
to synthesize the radioactive atom into a desired compound which is then employed. In the second
slide (Slide 2) we have brought together information necessary for a preliminary consideration'of
(a) the choice of the synthetic procedure to be used, and (b) the type of instrumentation to be employed
in the assay of the radioactivity of the final reaction products. In the second column, the initial state
of combination of the radioisotope is given. The third column summarizes the available specific ac-
tivities, expressed in millicuries per gram of element. Very large values of the specific activity
signify that the element is mainly comprised of the single radioisotope indicated. The nearly coniplete
absence of the normal stable isotopes allows us to refer to them as "carrier-free" preparations.
Examples are afforded by phosphorous", sulfur", and iodine '.
The specific activity, being a directly determined quantity, is a most important Index in the use
of radioisotopes. In fact, the interpretation of experiments using radioisotopes as tracers is based on
the observed changes found in the specific activity; this is, of course, the consequence of the dilution
of active by inactive isotopes. Evidently, then, the permissible dilution during an experiment is of
importance. The concept of dilution ratio, defined as the ratio of the initial specific activity to the
limiting practical detectable specific activity, is one of great value in the planning of experiments
using isotopes. It obviously has relevance to the choice of the synthetic method and techniques em-
ployed, since these determine the initial specific activity of the compounds formed. It bears on the
choice of lastrumentatien since this sets the lower limit on the specific activity.
'In generail,ft l is'leslrable in applying radioisotopes to be able to work with as large a dilution
as possible. This, therefore, means synthesizing the radioisotope into a chosen compound in the
highest possible speclfit activity Now, when it is remembered that one millicurie of radiocarbon
produced by the pile-is presently associated with approximately 150 mg of BaCQ0, or about 10 milli-
moles of total carbon, it becomes understandable that the gcale of the synthesis usually falls in the
range of A9 to 50.millimoles.of desired compound.- Consequently, semimicro or.microchemical ex-
perimental techniques are employed.
In the carrying out of syntheses of radio-organic compounds it is frequently necessary to handle
small amounts of volatile radioactive preparations. Owing to the possibly not inconsiderable health
hazards Involved, it is mandatory that all operations be conducted inside efficient, high draft hoods.
In fact, it is generally to be urged when dealing. with radioactive substances that as many of the ma-
nipulations as possible be performed within a hood. A vacuum line mounted inside a well-lighted
hood is a useful piece of equipment for both synthetic operations and for experimental purposes.
At Clinton Laboratories, for example, organic reactions are carried out as far as possible in
all glass high vacuum systems consisting of a glass manifold connected to a mercury diffusion pump
and to which various pieces of apparatus can be attached by standard taper joints. Attached perma-
nently to the system are a McLeod gauge, several manometers, a Toepler pump, and several reser-
voir bulbs which may aentain reference gaseous, mixtures used as standards. Vacuum line technique
is particularly advantageous in the distillation of small quantities of organic substances. Repeated
fractionation can be. made without loss anm the low pressure employed minimizes decomposition. Of
course, it goes almost.without saying that the synthetic method should be chosen and conducted so as
to realize as high a yield as possible based on the radioisotope.
Letus enumerate some possible types-pf synthetic procedures for the labeling of the desired
compound with a radioactive nucleus that have found employment. First, there is the direct synthesis
of the.radioisotope into the desired compound using conventional chemical methods known to give high
yield. Secondly, there are biosynthetic procedures which involve the isolation of metabolite from
organisms grown in tracer containing medium. Thirdly, there is synthesis by exchange where either
thermal or catalytic exchange reaction is utilized.
By wayr illustration of such procedures, consider the synthesis of radiocarbon-containing
compounds. :Thp oxidation state in which carbon" is obtained from a neutron induced transmutation
reaction must first be considered. The main carbon" compound isolated thus far is carbon dioxide,
together with possibly very small amounts of methyl alcohol and formaldehyde.
It seems sot Improbable that quantities of radiocarbon may be produced combined as methane;
also, sources of unsaturated hydrocarbons containing C"4 may be developed. Since, however, these
compounds. are currently unavailable, it is evident that the first step in getting radioactive carbon
into an organic molecule Is to reduce radiocarbon dioxide. Suppose that acetic acid with the radio-
carbon.placed-in theearboxyl group is desired. Since the preparation of acids.from Grignard re-
agents and carbon dioxide is a standard synthetic operation giving consistently good yields, it was
studied by L. B. Spector, of the Clinton Laboratories radio-organic group. The reactions employed
and a drawing of the carbonation apparatus is shown on the next slide (Slide 3). In the conventional
procedure, itwill be recalled, an excess of carbon dioxide is employed. In this 'ase, owing to the
stec6sity ot conserving CO. by getting the best possible yield of acid, an excess of Grignard reagent
was employed. Undei these circumstances, 'sfe reactions' of the type indicated in the last two lines
(as shown 6n the slide) may occur. The extent of this complication will depend on the time elapsing
before the mixture Is subjected to fydrolysis, and lierhaps also on the nature of the radical R. The
results of Spector's work, summarized in the next slide (Slide 4) indicated that a substantial improve-
ment in yield can be achieved by conducting the carbonation and hydrolysis as rapidly as possible.
Here, we note that the overall yield may fall as low as 70% despite the fact that the actual syn-
thesis gives a 90% yield based on carbon dioxide. Efficient isolation and purification methods are
also essential. In the special case of acetic acid some difficulties of this sort were met weit, es-
pecially in the separation of the acid from water on a small scale. An azeotropic distillation with
benzene was finally used.
Of course, other types of reducing agents may be employed and one of these is lithium alumi-
num hydride, whose use was described in the September meeting by Brown,' Finholt, Nystroi, and
Schlesinger. This method, as well as several others, is being applied in carbon" syntheses by mem-
bers of the Clinton Laboratories' radio-organic group, now under the direction of Dr. W. O. Brown.
We shall not discuss biosynthetic reduction methods except to remark that in some cases they
appear to offer a possibility when it is difficult to'introduce carbon dioxide, or cyanide, into a mole-
cule by the usual chemical procedure. The isotopie carbon, however, is usually distributed through-
out the molecule synthesized unless an isolated enzyme system catalyzing a single step can be em-
ployed. Also, there is usually a fairly high dilution of isotopic carbon unless special precautions ?
Synthesis of a radioactive atom into a desired compound either by means of a double decomposi-
tion reaction or by exchange with a catalyst is illustrated by the synthesis of several- organic radio-
halides, carried out by J. W. Richter, and tabulated in the next slide (Slide 5). In the second reaction
it may be seen that a relatively good yield of Isopropyl radiobromide may be got by a double de- .
composition reaction between radioactive silver bromide and isopropyl iodide. -An illustration of:the
experimental bench equipment and technique employed in the synthesis of radiohalides using aluminum
halide catalysts is afforded by the next slide (Slide 6). Here the vacuum line for the reaction was :eh-
closed entirely in a high velocity hood. All subsequent isolation operations wherein the desired' com-
pound was separated from its reaction mixture and purified were also carried out-in suitably designed
Recently, a description of the synthesis of several organic radiosulfur compounds has been pub-
lished by Henriques and Margnetti. These data are presented on the next slide (Slide 7) to illustrate
the reactions employed, the yield and the specific activity obtained. We shall refer later to the uWie
to which these compounds were put.
In summary, the following remarks about the synthesis of "tagged" compounds seem appropriate:
First, generally, well-known reactions may be employed. It is desirable that they be carried out on
a millimole scale necessitating the use of vacuum line or suitable microchemical techatqnes. -The
requirement of high initial specific activity and high yield together with the need'for suitable precan-
tions for the protection of personnel carrying out these reactions must always be kept in'mind. :
Suppose that a suitably labeled compound is at hand, and that we are ready to add it to a system
for chemical study. At this point it is essential to consider some further aspects of the design of ex-
periments using radioisotopes. The addition of a radioactive substance is, of course, predicted upon
certain assumptions. The system must not be disturbed by the effect of radiation from the radio
active nucleus, nor should the chemical behavior of the active atomic species be altered by its oww
radiation. Generally, this kind of an effect is of negligible importance in an ordinary chemical study.
It is well known, of course, that radiation effects are of very great consequence in biological work
and for that reason sometimes seriously limit the initial quantities of radioactivity.
A second tacit assumption is that there is a chemical identity between the radioisotope and the
stable isotope present in the system. When the radioactive atom is bonded covalently in a cpmppqurnl
as in carbon, hydrogen, etc., in organic compounds, the initial chemical state of the radioisotope is
well-defined. With some radlospecies, however, especially in aqueous solutions, where several oxi-
dation or hydrolytic states may exist, it is possible for the radioactive atomic species to be in a
condition quite different from that assumed. If a particular exchange reaction is being studied, then
one must make sure that there are no other unrecognized competing exchange processes occurring
in the system. Finally, the same considerations which lead to a limitation in the size of the synthetic
operatAi albu1tdtMei ttlde the seale nm whtih theoapertment wherein the labeled compound is used.
S Let us 'uppo. e have desi-ned our expeiment correctly, that the reaction being studied has
taken place, and that now we must carry out the analysis for the distribution of radioactivity among
the.'reatin products. First, we most separate the products containing the radioactivity from the re-
actUln mixture When very sdall qdantitles.oft actions products are formed, a "carrier" technique
.may' be used-to accomplish their' solation.- 'Thus, if minute amounts of radioactive methyl alcohol
wer'eformef hita complex mixture, a known amount of inactive alcohol is added and then separated,
sayy,' 1dstillatidn." Thd weight eithe added 'earBier" recovered is determined, and this figure
makts possible t~e'estlmkitfea of the recovery of radioactive methyl alcohol. Next, an aliquot of this
pilke adtivew ts 'inAztVe alcohol t1soaverted into an assay form so as to determine.its specific
a c ti tity .' I. ; *, .
This brings us to a. consideration of the special chemistry in the treatment of the analysis sam-
ple lide 8). Here, we see that complex subsptnces are converted to rather simple assay compounds
either.by, ~mbustion, or by some other oxidation technique. Standard microchemical equipment and
tehoLuesa again ind use. As indicated, in the case of radioactive carbon, either gaseous carbon di-
oxide or else,qolis barium carbonate may be used as ai assay form and with radioactive sulfur, either
bariuzp sulfate or benildene sulfate. In principle, the choice between a gaseous, liquid, or solid assay
compound .pends upon the quantity and upon the energy of the beta particles emitted by the radio-
isdtope employed. As may be remembered from the first slide, with radiohydrogen, carbon, and sul-
fur, these energies are indeed low, particularly in the 4irst instance. Therefore, if a small amount of
one of the foregoing isotopes is present in a large amount of water, barium carbonate, or barium
sulfae, respectively, the.beta radiations will be so largely absorbed within the sample as to inter-
fere wt the efficient detection of thq radioactivity. With radiocarbon activity, when measured with
a..icai eipd-w-indow Geiger-Mueller (GM) tube in the presence of 20 mg of BiCO, spread over an area
,of one square cm, only one in one hundred of disintegrations produces a count.
A few-general remarks about equipment for the detection and measurement of radioactivity are
in order. The methods most widely used at present are dependent upon the detection and amplification
of.thfionizatidn produced by the radiations' emitted in radioactive decay. As is known, the passage
of a charged particle through matter results in the formation of ions through inelastic collision. Since.
many- ion pairs may be formed by the passage of a single charged particle, the detection of a single
particle'is often possible with relatively simple apparatus. 'Two fundamentally different approaches
are employed in the determination of radioactivity. In one, an effect proportional to the total loniza-
tion Is measured, which gives the integratebeffect of many:particles. This is the principle involved
in the use of the eleetroscope and the Ion chamber. In the other approach, the effects of bursts of
ionization de to single particles aredetected' as in the-M tube, pulse ionization chamber, cloud
chamziber, phatograklic track,'and sctntillatlon methods.'
The assay. f energetic beta and gamma radiations, as with radiophosphorous and the radiohalides,
is relatively easy and can be dope conveniently with mica end-window CM tubes and accessory equip-
ment of the type shown in the next two slides (Slides 9 and 10). This type of equipment is now widely
available' andfurther remarks are not required. The convenient and accurate measurement of weak
beta emitters, particularly when they are present in low specific activity, requires the use of special
eqalpment and techniques, however. In the case of hydrogen it is necessary to place the radioisotope
inside a GM tube, or else an ionization chamber, owing to the extremely small energy of its beta
radiation. With radiocarbon anzasulfur there is greater latitude, and if one begins with high initial
specific activities, mid if the dilution is not large, standard measuring equipment of the type just
shown will prove quite satisfactory. As a result of an extensive investigation of the problem of the
assay of weak beta emitters present in low specific activity, however, C. J. Borkpwski and Dr. W. B.
Leslie of Clinton Laboratories hare come to the conclusion that the use of gas ionization chamber
techniques,. along wiP the dynamic condensor electrometer, offers the best overall advantage when
sensitivity, stability, wide operating range, and ase of measurement are: taken together, The next:
slide (Slide 11) shows the general assembly of this apparatus. The assay of carbon" using this equip-
ment is being carried out on a routine basis by res'eardh'wortkers 'a'ttinton LAoiatbrbe's.'
Let us now attempt to illustrate the method of radioisotopes by some arbitrarily selectedeaamn.
ples of the uses for compounds containing ."tagged" atoms, The first.of these, will be ueed to show .t
the employment of radioisotopes simply as analytical tools. Ass.sch,, they possess uniqkg advantages,
for they may be detected with enormous sensitivity, interference are absent sinee other.wipe.radie-.
activity is absent, and further, the measurement of them may be effected withcnveniece wnj.accu-
racy. Our example is taken from the excellent recent publication of Heartques andMargnetti. Cql-*n
sider the analysis of a complex mixture of organic sulfur, compounds, as for-example, ,wen dibequl
sulfide, dibenzylsulfone, and dibenzylsulfonate occur together in varying proportions. The propedufa
to be followed is just the reverse of the "carrier" technique mentioned earlier. Preparations of
these three compounds are synthesized codtainiig radiosufuri in knotn specific activity.''ihe,1 if
the mixture is to be analyzed for dibenzyl sulfide, a predetermined amount of the radioactive bom"';
pound is added. The unknown quantity of inactive dibenzyl'sulfide in this complex ~nixture'dilutes':1"'
the active compound, which is, of course, strictly identical chemically. Next, by'sbme'rtugh,'t I r'
method a small amount of dibenzyl sulfide is separated, purified, weighed, and its 'pecific acktivi*
determined. From the observed dilution, and the weight, it is possiblW to conmpfub the t'til aibtinft'
present in the initial mixture with good accuracy, as may be seen fro&i the nexf slide (Siildf'l).
A very-good agreement-was observed between.the weight-determined by sulfur" analysis,. and. .
the weight added to form a mixture of known composition. .:.,
Another use of radibisotopes is in the discovery and measurement 'ofphenomena accessibfdri't
by virtue of the existence of isotopes. The great ability of the hydrogen atoms of bbnzeri'at~ bo&dI;
temperatures when this compound is in the presence of a nickel catalyst was not suspecteAhtiltfWe'
demonstration of the rapid, reversible exchange of deuterium under thele same conditions 1 Po'lazyi.
The most interesting use, from an academic point of view, of isotopes is in the.study of the wechan-
isms of reactions. In many cases, the findings serve to confirm theories already supported by.it,- :.:
dependent experimental evidence; in some cases, new and unsuspected facts are uncqyered. The. %as
of radioisotopes in the study of the. oxidation of organic acids by potassium permanganate affords p -.
example in the latter category. On the next slide (Slide 13) we have summarized some of the data..
found by Allen and Ruben in their study of the omidation of fumaric acid. By labeling one of the carr.,
boxyl groups with radiocarbon, and one of the methene groups with radiohydrogen, or tritumn, andi,.
by determining the distribution of the activities in the end-products of the reaction, it.was possible':
to deduce some valuable information about this reaction. The carbon of the.formic acid was found
completely inactive; all of the C" being In the.carbon dioxide. On theL.other hand,,the water formed
was found to be free of tritium; all of the radioactive hydrogen was found ass .ciated with: he for io.
acid. From this we may conclude that the acid is derived from the methene grouping and that the
C- H bond is never broken. It is possible, further, tb eliminate dihydroxy-fumaric acid- s'artidler-
mediate, for otherwise the formic aqid would not have retained its original hydrogen'
A second, now classic example of the use of isotopes.in the study of reaction mechanisms, is, ...
presented on the next slide (Slide 14). Here, two -reactions, the racemization and the isotoRie C- ;-'
change reactions are indicated. By labeling iodide ion (the short-lived iodine'"8 was used) the rate,.,
of the isotopic exchange was measured and compared with the rate of racemization. If every e- -.
change were accompanied by an inversion, the two rate constants would be expected to be identical.
The data in the table show this to be the case. Questions of this type about the Walden-inqersion hape
been thus given a satisfactory answer. I ,,
In conclusion, the speaker would like to make some discursive retnarls touching ini a general'
way upon topics already mentioned. In so far as the production of radioisotopes is concerneii, If' "
seems likely that the trend will be increasingly towards limiting specific activities. Here,:'we Mean
that radioisotopes produced will be pure, or only very slightly diluted by stable or inactive isotopes.
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Certain anticipated developments in the uses of radioisotopes may be hazarded. At first, experl-
ments usibg them will be performed on a microcurle scale on laboratory bench tops and in hoods.
Later, as familiarity with them increases, they will be worked with on a millicurie scale so as to
permit experiments kith still larger dilutions. Ultimately, it is supposed that experiments will be
carried out on a curie level. Experimental techniques will tend, therefore, more and more toward
bench top remote control equipment placed in high draft hoods.
A.second anticipated development is in the gradual use of double or polyisotope tracer techniques
wherein several stable and/or radioisotopes may be employed. In principle, the use of isotopes as
tracers does not depend uniquely upon the nuclear property of radioactivity. The availability of both
active and inactive heavy isotopes will doubtless become comparable in the future, and convenient
means for assaying both of them are now at hand.
Radioisotopes will find their most important use in dealing with problems for which no other tools
Shave been found. They seem to offer promise in studies of the oxidation of hydrocarbons, with refer-
ence, particularly, to internal combustion engines and to lubricant oxidation; in the unraveling of the
mechanisms of hydrocarbon isomerization, cracking, and alkylation reactions, and in the determina-
Stion of the fate of catalysts or of inhibitors in polymerization reactions, to mention only a very few.
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