Nucleonics and Analytical Chemistry Ten Years After
Five of the papers presented at the Tenth Annual Summer
Symposium the Division sponsored of Analytical by Chemistry and Analytical Chemistry, Lafayette. Ind., June 13 to 15,1957
Preparation and Analysis of Tracer Compounds JOHN
R. CATCH
The Radiochemical Cenfre, Amersham, Buckinghamshire, England
b The preparation and analysis of tracer compounds are reviewed. Special attention is given to uses in general analysis and requirements that arise from these uses, which are often different from those of biological tracer work. Topics include the status of tracer compound preparation during the past 10 years, some unsolved problems and prospective developments, general approaches employed, their relative merits and their indebtedness to recently developed analytical methods, criteria of purity of tracer compounds, methods employed in their analysis and limitations of these methods, sources of error in the interpretation of tracer studies, and implications of multiple labeling. In some circumstances the molecular character of labeling and not merely its statistical distribution in various positions of the molecule may affect the behavior of tracer compounds.
T
years ago the use of radioactive and stable isotopes as tracers already had a distinguished and successful history. Following pioneer work with the limited range of naturally available radioisotopes, the application of stable isotopes to metabolic studies had reoriented the biologist’s concepts of metabolism. Recognition of the value of isotope dilution analysis arose, almost incidentally as it were, out of this work (30, 36, 38). Later the production of small quantities of radioisotopes by artificial means promoted their application to a greater range of biological studies (18, 29). At that time, therefore, the uses and prospective uses of tracers were well established and predictable, and radioisotopes were forthcoming in hitherto undreamed-of EN
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quantity and variety. The need for tracer isotopes in specific chemical forms was widely recognized, and the rather special synthetic problems were being attacked with energy and resourcefulness, particularly in the special case of the carbon isotopes. Achievement may be gaged from progress reviews (19) and from the fact that a textbook published in 1949 (10) has not yet been superseded as the standard work on its subject. I n 1947, nevertheless, the preparation of tracer compounds was confined to a few laboratories and was on a very limited scale. An organized production of tracer compounds developed, first by laboratories operating on behalf of the U.S. Atomic Energy Commission and later by commercial producers. Now a score of producers supply many hundreds of labeled compounds, often as part of a general provision of equipment and services for radioisotope work. ils a result relatively few tracer workers prepare labeled compounds for themselves from choice. Analysts have had little to do with this development, whose character has been determined by the predominantly biological demand. Applications to academic or technical chemical problems are relatively few, although often of notable quality. Applications to analysis are fewer still. The demand from biologists has been for finished tracer compounds and the supply has inevitably been directed towards compounds of general and fundamental rather than specific interest. The call for more specialized tracer compounds such as vitamins, hormones, and natural and synthetic drugs is more diffuse and less capable of supporting economic production and distribution. Biologists often require specific position labeling and high specific activities. B y con-
trast, the requirements for tracer compounds in general analysis are less exacting in almost every respect. The neglect of tracer methods by analytical chemists is surprising in view of their successful application to a number of problems, especially reverse dilution analysis (1, 2, 4, 7 , 9, 17, 20, 21, 23, 27, 31, 35, 37, 42, 43, 45, 46). EXaggerated estimates of the dangers and cost of tracer work are sometimes to blame, and an exhortation t o assess these rationally has been published (14). A more valid criticism of radioactive tracer methods is that the statistical error of measurement together with other sources of error (as in sample preparation) reduces the precision of such methods to perhaps one tenth, generally speaking, of that attainable in the best established analytical procedures. Tracer methods, however, particularly dilution and derivative methods, are especially valuable when existing analytical procedures fail badly from the complexity or intractable nature of the sample, or when they are used to apply a second-order correction to a conventional analytical procedure. PREPARATION
The methods for conversion of the simple chemical forms in which isotopes are primarily available into labeled compounds include chemical synthesis, less familiar methods such as isotopic exchange, biological synthesis, and “hot atom” or recoil methods. Chemical, biological, and exchange methods have been reviewed ( I O , 13, 15. 28, 33, 44) and a detailed treatment is not given here. The character of the work depends on the half life, radiation, and chemical character of the isotope. Carbon-14 synthesis is a relatively conventional and systematic enterprise. Chemically
it proceeds through one or another of ten or eleven priniary transformation products of carbon d i o d e , of which only half a dozen are important; hen-ever, each is the progenitor of an evergrowing family of derived labeled conipounds. Cheniical syntheses have the merits of control, efficiency, specificity of labeling, and sometimes high specific activity, but often they lack stercospecificity and are unable to provide uniform labeling should it he required. Cheniical synthesis remains the most gcncrally used source of tracer compouiidq. Biological methods are conspicuously deficient in control cf productf a i d yields, and rarely alloiv specific lahcling, hut they can pro\-ide uniform Ialwhng, naturally occurring stereoisonieric forms, and sometimes estreniely high specific activities. Plant leaws and algae have proved especially valuabli~ from their direct and efficient utilization of carbon dimide The e l t r x t i o n and purification of biological products tend to be tedious and timc-consuming. Exchange methods of labeling, generally in presence of a catalyst. arc particularly valuable for tritium, RS is also the related method of hydrogenating an unsaturated linkage. Tlic ease of these procedures determines the whole character of application of tritium as a tracer; labeling methods are often simpler and more empirical than with carbon-14, but the position of the introduced label needs to be carefullj- considered (25, 26). The advantngcs peculiar t o tritium-labeling are the evtremely high specific acti! ities which can lie reached and the low cost. Preparation of Iabelcd compounds of shorter-lived and cheaper isotopes is approached diffwently than for carbon14, as labor costs bulk higher and m a terial costs are lower; the problems of radiation screening, contamination. and decomposition by self-irradiation are more pronounced. “Hot atom” and recoil labeling methods only recently have attracted much attention, and they promise a t present an easy and valuable approach to tritium compounds a t low to moderate specific activities and carbon-14 compounds a t very lo^ specific activities (39. 40, 47-9). Some f o r m of recoil labeling give nominally “carrier-free” products (39) and it may prove possible to isolate these under favorable conditions ( 2 2 ) . Purification of the products of such syntheses may be expected to present considerable difficulty on occasion (47, 48). In preparation of labeled conipounds by all methods the separation and purification of the products are often made particularly difficult by the small scale (0.1 to 1.0 gram) of such synthesec. Recent developments in analytical methods, particularly chroniatographic methods, have contributed greatly to solving this preparative
problem. Many of these methods have been devised and developed by chemists working on biological subjects. In one respect the approach to labeled compounds has altered considerably in 10 years; the practice of labeling with an isotope chemically foreign to the molecule being traced (a common choice being iodine-131) is nom used less frequently and more critically than formerly. This form of tagging has great value n hen applied with care. It has become especially valuable for protein and polypeptide labeling in circumstances n-hen there is hardly any alternative, and detailed study has been given to the conditions under which it is valid (6, 16, 32). UNSOLVED PROBLEMS OF LABELING
There are certain materials lvhich as yet cannot be labeled in a truly representative wag. Complev mixtures which cannot be reproduced artificially, such as natural petroleum and coal tar and partially purified fractions derived from them. cannot be so labeled, although pure individual components may be labeled and studied separately. Even “hot atom” labeling n-ith tritium appears to discriminate between hydrogens in different positions in the molecule (47‘) and therefore, cannot be relied on to preserve any definite relation between the hydrogen or carbon content or the size of a molecule and the quantity of activity incorporated. This is an unfortunate limitation because of the importance of such mixtures as solyents and the interest shown small residual quantities of them-e.g., in polymers or paint films. Other complex natural products such as animal or vegetable fats and proteins are nominally susceptible to strictly valid labeling but often in practice will be prohibitively expensive to prepare or obtainable only a t a low specific activity, because of the small yields and the low levels of radiation which the producing organisms will tolerate (41). ANALYSIS
The analysis of tracer compounds has certain features peculiar to it which are not paralleled in ordinary chemical analysis. One of these arises from the small size and considerable value of the batch; d i e n a fraction of a gram of product is worth many thousands of dollars it is not possible to take large and numerous replicate samples for analysis. Quantities dispatched to individual users are often 1 mg. or less. Control of such packaging can be properly achieved only by random sampling and analysis, but even now production rarely attains the level a t which this form of control is really effectire. At the Radiochemical Centre batch sam-
ples of 1 to 3 mg., nhich are accurately weighable on a semimicrobalance, are commonly taken for analysis. It is necessary to consider how representative such a saniple is likely to be. Even volatile organic substances can be sampled (from a vacuum manifold) in such quantities; the technique used (12’) demands some care and skill but gives remarkably reproducible results in esperienced hands. In this type of sanipling it is necessary to take a vapor sample and condense it; it is not permissible to condense a small part only from a large vapor volume. Sampling of aliquots of a solution of known concentration, d i e n it can be used, is the method of choice for accuracy, economy, and validity of the sample. Few isotopes are used for labeledcompound preparation a t isotopic abundances approaching 100% (deuterium is the most important exception) ; for carbon-14, isotopic abundances in labeled compounds only exceptionally reach 407, or thereabouts and often less than 1% in tracer work. Abundances for shorter-lived isotopes are many orders of magnitude lower. Tracer compounds are nearly always mixtures of chemically identical labeled and unlabeled molecules in the simplest case. Foreign molecules, either labeled or unlabeled. niay also be present. There is consequently no fixed relationship between the chemical purity, n hich may be defined as the proportion or percentage of niolecules or mass in the desired chemical form, and the radiochemical purity, which is usually expressed as the percentage of the total activity present in the desired chemical form. Multiple labeling, 1%-liichcommonly occurs 11-ith carbon-14, introducesa further complication-e.g.,photosynthetic glucose with statistically uniform labeling will contain 64 molecular species, even neglecting the presence of carbon-13 and isotopes of oxygen and hydrogen (11)-which is fortunately rarely of practical significance, although it gives rise to formidable problems of exact nomenclature. Analysis for chemical purity alone has a very limited significance for labeled compounds. I n tracer synthesis it is common practice to add pure “carrier” to improve the chemical purity at a stage when this will improve a reaction yield or simplify the isolation of a product. This destroys any relationship which may have existed betm-een chemical and radiochemical purity; to put it in another way, labeled impurities may be present a t a specific activity much higher than that of the major product or vice versa. Chromatographic methods of isolation of biological compounds frequently lead to products of high radiochemical purity, such as amino acids, which are nevertheless contaminated by irrelevant unlabeled impurities, derived from VOL. 29, NO. 12, DECEMBER 1957
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such sources as filter-paper sheets, chromatographic solvents, and ion exchange resins. Radiochemical Purity. For most tracer workers radiochemical purity is the only important criterion; fortunately so, because it is easier t o determine than chemical purity. Dilution analysis remains t h e most generally trustworthy and useful method. It is important to remember that it is a comparison between chemical purity of the carrier and radiochemical purity of the sample. The purity of the carrier must be known if the result is to be reliable. Another source of error is choice of an inappropriate derivative. For example, it is unsound to analyze labeled benzaldehyde by carrier dilution and oxidation to benzoic acid which is then purified, for labeled benzoic acid, if present in the sample (as is probable), will not show up in such an analysis. It may be necessary in special cases to consider whether the test of purification to constant specific activity has been rigorously enough applied (48),but this difficulty is not often encountered in ordinary practice. The limited precision of radioactive measurement limits the use of dilution analysis to determination of specific components, whether major or minor ones; small amounts of unspecified radioactive impurities will go undetected. Chromatographic analysis, especially paper sheet chromatography, is by contrast valuable for determination of such unspecified impurities even in very small quantities, and gives valuable accessory evidence if its limitations are borne in mind. Reliance on any one solvent system to effect a satisfactory separation is unwise, and use of a second and different system is not a complete safeguard. Some pure compounds tend to “streak” or “tail”; this tendency interferes with quantitative determination, and any allowance made for it is likely to be somewhat subjective. Other errors are more characteristic of tracer compound analysis. One is the loss of material from a spot by simple volatilization or by volatilization following a hydrolysis. The possible magnitude of such losses should not be judged only from the molecular weight, or volatility under ordinary laboratory conditions; it depends also on the mass of the compound involved. This will have practical significance if the labeled impurities present are a t a specific activity higher than that of the major component. The converse error, when the major component is substantially lost and the impurities (from their lower volatility) remain, has also been observed. Unstable compounds may be subject to decomposition on chromatography, which results in apparent impurities, all of which appear on radioactive detection whereas a con-
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ventional method of detection may not reveal them. Another possible source of error in paper chromatography of labeled conipounds is a physical “carrier effect’’ which has been observed-with leucine and isoleucine in a solvent system (tertamyl alcohol-diethylamine - water) which effects a fairly good separation of these two compounds. The application of a very small load of L-isoleucine-carbon-14 resulted in much “tailing” of the spot, and much activity appeared in the leucine position. bddition of a little carrier isoleucine greatly improved the definition of the spot, whereas carrier leucine did not. In the paper chromatography of labeled mixtures of much glucose with little fructose (or vice versa) a small amount of the minor component usually is found in the major one, even though the intermediate zone is free from activity. It separates on rechromatography but does not continue to appear indefinitely and is not thought to be the result of epimerization. Paper chromatography remains an invaluable method when its possible shortcomings are recognized. Experience has shown that, when it gives a significantly different (usually more favorable) result than dilution analysis, it is the dilution analysis which is commonly proved to be correct. For volatile substances gas chromatography, well established for determination of chemical purity and for preparative separation of labeled compounds, has been adapted to what is in effect an analysis for radiochemical purity (EJ), but as yet it seems not to have been applied successfully to compounds of soft-beta emitters for which it would be most useful. Other chromatographic methods are rarely used; the association of paper sheet chromatography with radioautography is so simple and convenient that it is always preferred in analysis of this kind. Chemical Purity. The analysis of labeled compounds for chemical purity is less important and only occasionally is exact information requested, and then more commonly for specific contaminants rather than chemical purity in the strict sense. However, the chemical purity of reagents often affects reaction yields, sometimes all out of proportion to the actual amounts of impurity present. I n tracer compound synthesis it is especially important to base yields on results with representative materials and not on consecutive yields obtained step by step with .shelf reagents. Melting points by methods using a hot stage under a low-power microscope are clean to carry out and economical of sample. Vapor pressures are measured readily in a vacuum manifold and are useful, but not a very reliable index of purity. Gas chroma-
tography is much more reliable, and it is possible to recover the sample if desired. Freezing points of liquids can be determined satisfactorily on the vacuum manifold. Only at lorn specific activities can boiling points or boiling ranges be determined. Analyses may be conducted on inactive batches prepared in exactly the same way; this is, of course, not wholly satisfactory but it is sometimes the only readily available expedient--e.g., in determination of refractive index-although no doubt a method of determination with complete recovery of a volatile sample could bp set up if it were thought worth while. Absorption spectrometry is adaptable to tracer samples, especially in the ultraviolet region. The sample size and preparation for infrared spectrometry are sometimes less convenient, Colorimetric methods using color reagents are often useful. Such determinations as acid and basic equivalents are made when appropriate on products in course of preparation or making up for dispensing--e.g., volatile fatty acids are distributed conveniently for most purposes as salts. Rotations can usually be determined if the cell used allows clean filling and recovery of the solution. Even in circumstances when chemical and radiochemical purity are known to be related, many analyses (such as optical rotations) are not always sensitive enough to be of much use. A dilution analysis by addition of optically pure carrier followed by separation of a diastereoisomer or application of a biological method such as D-amino oxidase is a much more sensitive method of determining a small proportion of D- or L- form than is the conventional polarimetric method. Many other methods of chemical analysis might be applied to labeled compounds but do not, except in special cases, justify the effort involved. Uses of labeled compounds are so various and have such varied purity requirements that it is hardly possible to conduct analyses which will answer all possible queries on purity. The Analyst’s Requirements. The analyst’s requirements for labeled compounds are so different from the biologist’s t h a t present supply and distribution have done less to meet his needs. It seems probable t h a t they will be applied mostly to direct or reverse dilution analyses, including methods of checking the validity of established analytical procedures. The compounds required especially for dilution analysis will be specific and varied, but are likely to be those for which established methods are unreliable-for example, minor components of coal or other tars, petroleum, molasses, or other complex mixtures. The specific activities and quantities of activity needed in such work are very modest
An even more important point is that the position of labeling is not a t all critical so long as it is not subject to chemical exchange under the conditions of the analysis. Indeed, apart from this requirement, the position or distribution of labeling need not even be known. The chemical purity of the tracer compound will not be exacting; even for radiochemical purity the requirement is likely to be less exacting than the biologist’s. Often it will be necessary to know only the quantity of activity added in the chemical form being traced, and that the mass is small by comparison with the mass of carrier present in the sample which will dilute it. Even the presence of labeled impurities need not be disadvantageous, so long as it is known that they are subsequently separated in purifying the sample for final measurement. Compounds for dilution analysis may, therefore, be labeled by the most convenient isotope and by the most efficient route without much concern for the position of labeling; they may be prepared a t lorn specific activities, which means that synthetic work may be on the conventional laboratory scale and not in the more difficult centigram range; recoil methods of labeling are especially promising; radiological precautions will not be exacting and radiation decomposition will rarely occur. A single preparation will provide material for hundreds or even thousands of analyses. The analyst who must undertake preparation of a labeled compound for his own use faces fewer problems than the biologist. Reagents required for methods of analysis using a labeled derivative may be an exception in requiring high specific activities ( 3 ) . PITFALLS IN USE OF TRACER COMPOUNDS
Some possible sources of error in the analysis or use of tracer compounds have been mentioned, among them the variation of quantitative behavior of a chemical substance with the mass involved. The abnormal behavior of very low ionic concentrations has long been familiar--e.g., with naturally occuring radioelements-but has become more widely recognized since the increased use of artificial radioisotopes, many of them almost “carrier-free”; the chemical, physical, and biological behavior of iodine-131, for instance, varies considerably with the chemical concentration. Similar variations will appear with labeled compounds, and in labeling procedures. Attempts to use very low concentrations of elementary iodine in aqueous solution may be defeated by reduction of the iodine by small amounts of organic impurity in the water or from the apparatus. Unsaturated oils and the like are sometimes labeled by addition of iodine-131
to the double bond. Sometimes an attempt is made to reduce as much as possible the quantity of iodine, although for no apparent reason, because the label, even if it is added to the desired double bond, will nevertheless be chemically characteristic of the modified and not the original unsaturated molecule. There is a decided risk that the iodine will react preferentially with a more reactive minor impurity in the oil, leading to anomalous tracer behavior. I n the trace-labeling of proteins by iodine there is, however, a real need for minimizing the iodine content (32).
Destruction of organic compounds by oxidation, hydrolysis, microbiological attack, or exposure to light all become proportionately more significant as the concentration is lowered or the sample size reduced; an exception is decomposition by self-irradiation, which (from reduced self-absorption) becomes less pronounced with small quantities. Isotope effects-that is to say, discrimination between labeled and unlabeled molecules arising from differences in the bond energies of the isotopes-are significant for the biological tracer worker only in exceptional circumstances and rarely will affect the analyst. Chromatography of carbonlabeled amino acids over ion exchange resins can result in slight separation of the labeled molecules (34, depending on the position of labeling, and with the increasing refinement of analytical methods it is not unlikely that this phenomenon, which invalidates the fundamental assumption of the tracer method, may be observed in other experiments. This example would reveal a chemical distinction (albeit a slight one) between doubly labeled molecules and an equivalent mixture of singly labeled molecules. A purely chemical distinction of this kind is in the formation of labeled recoil fragments from doubly labeled molecules (60),as in the case of methylamine-carbon-14 from ethane-1,2-C-I4. Such distinctions must occur also with double labeling with two different isotopes-e.g., carbon-14 and tritium or carbon-14 and sulfur-35. I n these cases molecules which are truly doubly labeled will leave labekd recoil fragments on disintegration, mostly carbon14 fragments, whereas a mixture of separately labeled molecules will not, Such an effect, unlike radiation decomposition, will be entirely unaffected by addition of an energy absorbing diluent. At isotopic abundances hitherto in common use it will be negligible, but compounds of carbon-14, tritium, and sulfur-35 are being prepared and used a t very high specific activities and double labeling a t these high levels can foreseeably give rise to significant quantities of active impurities. For methionine-S-35-1-C-14, which has been
studied ( I I ) , it is conceivable that the proportion of doubly labeled molecules might be as high as lo-‘ or higher, and within a year nearly all this proportion would be in the form of carbon-14labeled recoil fragments. Such quantities are perfectly detectable and begin to approximate in amount to the residual radiochemical impurities in a highly purified labeled compound. Another aspect of double-labeling technique arises when one of the labels is not strictly valid, as in the iodination of proteins. A protein may be labeled biologically with carbon-14 or sulfur-35, through a labeled amino acid or otherwise. This label will be valid. It may then be iodinated (24) but the simple assumption that, after any biological degradation resulting from the presence of an abnormal constituent (iodine), the carbon-14 or sulfur-35 activity is characteristic of the residual part of the molecule, will not be true unless substantially all the protein molecules have been modified; otherwise some of it represents normal uniodinated molecules. I n the example chosen, from the large molecular weight of the protein, this would require that the iodine concentration be very low, but it is just in this field that low iodine concentrations are often used. Most tracer experiments make certain assumptions about the chemistry of transformations employed in synthesis and degradation. Earlier studies of the Willgerodt reaction are an example of a false conclusion arising from failure to consider and test such assumptions (8). It has been observed that the Hofmann degradation of acetamide leads to appreciable oxidation of the methyl carbon to carbon dioxide. Chemists are aware that equations for chemical reactions (especially organic chemical reactions) are only approximations, and are accustomed to the concepts of yield and by-products, but in tracer work with specific labeling side reactions can lead to misinterpretations of results which are not always foreseen. Tracer studies with hydrogen isotopes will require more care than with carbon labeling, from the greater mobility of hydrogen atoms in organic molecules. FUTURE OF LABELED COMPOUNDS
There is no reason to doubt that carbon-14 labeling, with its versatility and positive character, will remain of the first importance in biological research work, but will be increasingly supplemented by tritium labeling. Exchange methods for tritium labeling still promise to be of more value than hot atom and recoil methods, but these also will certainly find extensive use. The scope of tritium labeling is inevitably somewhat restricted and it will be applied to specific rather than general VOL. 29, NO. 12, DECEMBER 1957
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biological problems. Users of tracer nicthods in the study and development of new pharmaceuticals will find more than the surprisingly small use that has been made of them so far, aiid no one can doubt that tracers can contribute much more usefully to diagnostic medicine than they yet have done. Applications so far have been fairly simple in character, and the possibilities inherent in such experiments as those a t the University of California Radiation Laboratory ( 5 ) have yet to be developed. Such developments are likely to make use of tritium and other cheaper and short-lived isotopes, wherever possible, rather than carbon-14. I n the study of chemical reaction mechanisms the uses of tracers are unique but limited, but in prepsratire chemistry gencrally the facility of analysis by tracer methods is so great that nonessential uses in the study of reaction yields and by-products will certainly increase when chemists generally are more familiar with the possibilities and have overcome some of their inhibitions about radioactive tracers. In normal analytical practice progress in using this new tool to advantage n-ill again depend on the enterprise of analytical chemists, who will need to acquire a realistic grasp of what tracers can aiid cannot do as a preliminary to finding more analytical problems nliich their unique powers can solve.
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>I.,’ and bthers, “Isotopic Carbon,” Wilev, Kew York, 1949. (11) Catch,
