,/p/- 7 : g f AfiXK-- ? ^ MDDC - 840 UNITED STATES ATOMIC ENERGY COMMISSION RADIOACTIVE ISOTOPE TRACER TECHNIQUES by 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 copie of the same or similar content and title and by the same author(s). Technical Information Division, Oak Ridge Directed Operations Oak Ridge, Tennessee 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 available. 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," ttiey 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, no 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 of exchange 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 t l 2 ] MDDC - 840 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 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 of 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 yield of a nuclear reaction. In many cases the amount of activity produced is quite small, either owing 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 increased, 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 complete absence of the normal stable isotopes allows us to refer to them as "carrier-free" preparations. Examples are afforded by phosphorous 32 , sulfur 35 , and iodine 131 . 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 MDDC - 840 [3 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 instrumentation since this sets the lower limit on the specific activity. In general, it is desirable 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 specific activity. Now, when it is remembered that one millicurie of radiocarbon produced by the pile is presently associated with approximately 150 mg of BaCQj, or about 10 milli- moles of total carbon, it becomes understandable that the scale of the synthesis usually falls in the range of 10 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 contain 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 and 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. Let us enumerate some possible types of 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 way of illustration of such procedures, consider the synthesis of radiocarbon-containing compounds. The oxidation state in which carbon" is obtained from a neutron induced transmutation reaction must first be considered. The main carbon 14 compound isolated thus far is carbon dioxide, together with possibly very small amounts of methyl alcohol and formaldehyde. It seems not improbable that quantities of radiocarbon may be produced combined as methane; also, sources of unsaturated hydrocarbons containing C w 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 the carboxyl 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, it will be recalled, an excess of carbon dioxide is employed. In this case, owing to the necessity of conserving C0 2 by getting the best possible yield of acid, an excess of Grignard reagent was employed. Under these circumstances, side reactions of the type indicated in the last two lines (as shown on the slide) may occur. The extent of this complication will depend on the time elapsing before the mixture is subjected to hydrolysis, and perhaps 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. 4] MDDC - 840 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 with, 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, Nystrom, and Schlesinger. This method, as well as several others, is being applied in carbon 14 syntheses by mem- bers of the Clinton Laboratories' radio-organic group, now under the direction of Dr. W. G. 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 isotopic 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 are taken. 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 en- 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 vacuum equipment. 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 use 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 techniques. The requirement of high initial specific activity and high yield together with the need for suitable precau- 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 effects of radiation from the radio- active nucleus, nor should the chemical behavior of the active atomic species be altered by its own 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 compound, as in carbon, hydrogen, etc., in organic compounds, the initial chemical state of the radioisotope is well-defined. With some radiospecies, 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 MDDC - 840 [5 in the system. Finally, the same considerations which lead to a limitation in the size of the synthetic operations also determine the scale on which the experiment wherein the labeled compound is used. Let us suppose we have designed our experiment 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 reaction products. First, we must separate the products containing the radioactivity from the re- action mixture. When very small quantities of reactions products are formed, a "carrier" technique may be used to accomplish their isolation. Thus, if minute amounts of radioactive methyl alcohol were formed in a complex mixture, a known amount of inactive alcohol is added and then separated, say, by distillation. The weight of the added "carrier" recovered is determined, and this figure makes possible the estimation of the recovery of radioactive methyl alcohol. Next, an aliquot of this pure active plus inactive alcohol is converted into an assay form so as to determine its specific activity. This brings us to a consideration of the special chemistry in the treatment of the analysis sam- ple (Slide 8). Here, we see that complex substances are converted to rather simple assay compounds either by combustion, or by some other oxidation technique. Standard microchemical equipment and techniques again find use. As indicated, in the case of radioactive carbon, either gaseous carbon di- oxide or else solid barium carbonate may be used as an assay form and with radioactive sulfur, either barium sulfate or benzidene sulfate. In principle, the choice between a gaseous, liquid, or solid assay compound depends upon the quantity and upon the energy of the beta particles emitted by the radio- isotope employed. As may be remembered from the first slide, with radiohydrogen, carbon, and sul- fur, these energies are indeed low, particularly in the first instance. Therefore, if a small amount of one of the foregoing isotopes is present in a large amount of water, barium carbonate, or barium sulfate, respectively, the beta radiations will be so largely absorbed within the sample as to inter- fere with the efficient detection of the radioactivity. With radiocarbon activity, when measured with a mica end-window Geiger-Mueller (GM) tube in the presence of 20 mg of BaC0 3 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 the ionization 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 ioniza- tion is measured, which gives the integrated effect of many particles. This is the principle involved in the use of the electroscope and the ion chamber. In the other approach, the effects of bursts of ionization due to single particles are detected as in the GM tube, pulse ionization chamber, cloud chamber, photographic track, and scintillation methods. The assay of energetic beta and gamma radiations, as with radiophosphorous and the radiohalides, is relatively easy and can be done conveniently with mica end-window GM 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 and further remarks are not required. The convenient and accurate measurement of weak beta emitters, particularly when they are present in low speciiic activity, requires the use of special equipment 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 and sulfur there is greater latitude, and if one begins with high initial specific activities, and 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. Borkowski and Dr. W. B. Leslie of Clinton Laboratories have come to the conclusion that the use of gas ionization chamber techniques, along with the dynamic condensor electrometer, offers the best overall advantage when 6 ] MDDC - 840 sensitivity, stability, wide operating range, and ease of measurement are taken together. The next slide (Slide 11) shows the general assembly of this apparatus. The assay of carbon 14 using this equip- ment is being carried out on a routine basis by research workers at Clinton Laboratories. Let us now attempt to illustrate the method of radioisotopes by some arbitrarily selected exam- ples of the uses for compounds containing "tagged" atoms. The first of these will be used to show the employment of radioisotopes simply as analytical tools. As such, they possess unique advantages, for they may be detected with enormous sensitivity, interferences are absent since otherwise radio- activity is absent, and further, the measurement of them may be effected with convenience and accu- racy. Our example is taken from the excellent recent publication of Henriques and Margnetti. Con- sider the analysis of a complex mixture of organic sulfur compounds, as for example, when dibenzyl sulfide, dibenzylsulfone, and dibenzylsulfonate occur together in varying proportions. The procedure to be followed is just the reverse of the "carrier" technique mentioned earlier. Preparations of these three compounds are synthesized containing radiosulfur in known specific activity. Then, if the mixture is to be analyzed for dibenzyl sulfide, a predetermined amount of the radioactive com- pound is added. The unknown quantity of inactive dibenzyl sulfide in this complex mixture dilutes the active compound, which is, of course, strictly identical chemically. Next, by some rough, ready method a small amount of dibenzyl sulfide is separated, purified, weighed, and its specific activity determined. From the observed dilution, and the weight, it is possible to compute the total amount present in the initial mixture with good accuracy, as may be seen from the next slide (Slide 12). A very good agreement was observed between the weight determined by sulfur 35 analysis, and the weight added to form a mixture of known composition. Another use of radioisotopes is in the discovery and measurement of phenomena accessible only by virtue of the existence of isotopes. The great lability of the hydrogen atoms of benzene at room temperatures when this compound is in the presence of a nickel catalyst was not suspected until the demonstration of the rapid, reversible exchange of deuterium under these same conditions by Polanyi. The most interesting use, from an academic point of view, of isotopes is in the study of the mechan- isms of reactions. In many cases, the findings serve to confirm theories already supported by in- dependent experimental evidence; in some cases, new and unsuspected facts are uncovered. The use of radioisotopes in the study of the oxidation of organic acids by potassium permanganate affords an 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 oxidation of fumaric acid. By labeling one of the car- boxyl groups with radiocarbon, and one of the methene groups with radiohydrogen, or tritium, and 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 n being in the carbon dioxide. On the other hand, the water formed was found to be free of tritium; all of the radioactive hydrogen was found ass ciated with the formic 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, to eliminate dihydroxy -fumaric acid as an inter- mediate, for otherwise the formic acid 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 isotopic ex- change reactions are indicated. By labeling iodide ion (the short-lived iodine 128 was used) the rate of the isotopic exchange was measured and compared with the rate of racemization. If every ex- 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 inversion have been thus given a satisfactory answer. In conclusion, the speaker would like to make some discursive remarks touching in a general way upon topics already mentioned. In so far as the production of radioisotopes is concerned, it 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. MDDC - 840 [7 Certain anticipated developments in the uses of radioisotopes may be hazarded. At first, experi- ments using them will be performed on a microcurie 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 with 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 have 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- tion of the fate of catalysts or of inhibitors in polymerization reactions, to mention only a very few. nnHnif.,?.S'.T.X 0F florid* 3 1262 08910 5331