Norman 5. Rodin1
Northwestern University Medical School Chicago, Mnois
hcorporating Radioisotope Techniques into the Chemistry Curriculum
M o s t efforts that have been made by tenrhers of chemistry to acquaint college students with the value of radioisotopes have emphasized the radiochemical and physical aspects of these isotopes. This point can be seen by perusing the collection (1) of papers published in THIS JOURNAL on radioisotopes. The topics of decay, parent-daughter relationships, nuclear activation, and nuclear chemistry have been frequently applied to teaching experiments. Where the more chemical uses, such as isotopic dilution and radiometric analysis, have been described, most of the experiments have taught simply another aspect of ra.dioactivity rather than a fundamental chemical principle or fact.. This emphasis is neglectful of many other important applicat,ions of radioactivity and there is a need for experiments which bring these applications into the ordinary chemical curriculum. Such experiments cannot he designed as "extra" experiments, for there is no extra time in the student's or teacher's day. If the tearher is to substitute a radioactivity experiment for a presently accepted experiment, he must sacrifice the illustration of an important chemical topic. If, as in so many srhools, the applications of radioactivity are taught in a special, advanced course, only a few students will become familiar with these new principles and they will have to give up time that might be better spent in st,udying their main field of chemistry. The problem posed can be solved by developing experiments which include realistic radioisotope uses, but only as a secondary feature. The experiments mupt teach t2heimportant chemical subject matter, so that chemistry teachers can freely displace one or more standard experiments. For optimum realism and acceptability, t,he new experiments should be available in all levels of college chemistry. The principles of design cited above have been the basis for a new laboratory manual (S),which was cosponsored by t,he U. S. Atomic Energy Commission and Rnrlear-Chicago Corporation, and prepared by the Present address: Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan.
Sample copies of "Radioisotope Experiments for the Chemistry Curriculum" are available t o college and university teachers a t no charge on request to Nuclear-Chicago Corporation, 333 East Howard Avenue, Des Plaines, IIlinois. Multiple copies for student use may be purchased through Nuclear-Chicago or through the Office of Technf eal Services, U.S. Department of Commerce, Washington 25, D. C. at $2 per copy for the experiment manual and $1 per copy for the instructor nates.
344 / Journal of Chemical Education
writer aud a group of other teachers. An outline of the chemistry experiments in this manual is shoxn in the table. It can be seen that in many experiments the isotopic technique has simply replaced the more usual gravimetric, volumetric, or colorimetric techniques of analysis. Such an exchange will surely not cause any serious weakening of the student's training in these standard procedures, for the student receives multiple exposure to these methods anyway. I n the organic chemistry experiments, standard synthetic reactions have simply been modified to allow use of isotopic starting materials. I n some of the experiments, the peculiar properties of tracers allow us to demonstrate chemical principles that could not be demonstrated otherwise; however, such demonstrations need not lack for other important chemical reactions or properties. Typical Radioisotope Experiments (2) Introductory Chemistry: Determination of atomic weights, with S%ulfate. Chemistry of sulfur; thionulfate, sulfur, and sulfite. Secular equilibrium; Pb2'O-BiP14 Qualitative Analysis: Determincttion of a solubility product for calcium sulfate. Separation of ions by liquid/liquid extraction. Coprecipita1,ion phenomena; calrium and permanganate a i t h barium sulfate. Quantitative Analysis: Determination of phosphate in a phosphate rock by an isotopic yield method. Determination of the formation constant of a complex ion; calcium citrate vs a n ion exehanee resin. Determination of calcium; remoiial of interference hy ion exchange and chelatometric titration. Ovganic Chemi~try: Synthesis of S"-sulfanilamide. Synthesis of C"-acetylsillieyclie acid. Identification of arnines by acetylntion with CIA-acetic anhydride. Physical Chemistry: Dynamic nature of equilibria. between ions in solution and a salt. Exchange between a metal and ions in solution. Analysis of the phase diagram of a three component system; dimethylaniline, water, acetic acid-C". Demonstration of the adsorption isotherm with charcoal and acetic acid. Radioactive decay and growth; C e " < P P . Instmmental Analvszs:
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Ion exchange and paper chromatography; separation of C"glyeine and glutamic acid; isotopic dilution determination. Properties of enzymes, studied with an isotopic substrate made h i the student. Incormretion of C"-plycine into a protein fraction of mouse spleen; properties ofthe in viiro &em
Some of the readers may wish to see other topics in chemistry modified to include an isotopic approach, and might be willing to develop such experiments. The purpose of this paper is to describe the practical approaches and principles utilized in the manual in the hope that they will be useful to those interested in preparing their own isotope experiments. It is hoped also that some readers will be encouraged to try introducing such esperimenta into their own regular chemistry courses. Counting
Holders for steel filter discs have not been available commercially and no serious consi'deratiou of these discs has appeared in the literature. I t was therefore necessary to design a suitable filtering apparatus, which is sketched in Figure 1. The apparatus consists simply of a Biichner funnel which comes apart. The porous steel disc rests on the lower half of the funnel and the upper half (chimney) screws onto the lower half, holding the disc in place. To make the assembly leak-proof, t ~ Seoprene o washers are needed. The upper washer
Systems
Most teachers of chemistry do not have the interest or time to build t,heirown radioactivity instrumentation, and it is fortunate indeed that the Atomic Energy Commission is making available funds to qualified colleges for the purpose of buying good counting equipment and associated supplies. It is now possible for the interested rhemistry teacher to plan experiments around t,he use of accurate, reliable, standard scalers with sensitive thin-window Geiger tubes. High standards of quantitative performance, rather than rough qualitative work, can no~r-be produced by students who work carefully. The use of standard Geiger tubes of tbe thin, endwindovv type means that the radioactive samples (in most cases) must be spread out evenly on planchets. This general procedure gives a reasonable observed activity with a minimum of investment in radioisotopes. Simplicity
of Sample Preparation
The preparation of samples for counting with weak beta emitt,ers (the preferred type of isotope for student use) is a t,opic which deserves very careful attention. T'ariations in sample preparation are usually the major source of error in radioactivity work and the time required for many procedures of preparation is too great for teaching applications. The methods that seem simplest-evaporating a small volume of solution, or spreading a slurry of solidsare actually useful only for very small samples (measured with a micropipet) or strong beta emitters, such as P3?. The most generally useful method, where high precision is wanted, consists of filtering suspended radioactive particles onto a porous planchet. Of the various porous filtering materials that are used in this way, stainless steel discsz seem to the writer to be the simplest to use. The steel discs filter rapidly, cannot be broken (unlike sintered glass discs), and are so rigid that t,hey need not be mounted on a backing planchet (unlike paper or hlillipore discs). Thus the steel discs are the easiest and fastest to handle and are desirable not only for the inexperienced student but for the research worker. The main drawbacks to the use of steel discs are the high initial expense, the danger of internal radioactivity contamination, and the danger of corrosion. The contamination problem is minimized by the use of a precoat of diatomaceous filter aid, which keeps the radioactive solids from touching the metal surface. The corrosion problem is minimized by careful attention to the properties of stainless steel; the main limitation is lack of resistance to hydrochloric acid. ,
.
? Micro Metallic Division, Pall Corp., Glen Cove, New York. Porosity G, 1/16" thick.
Figure 1. Filter adapter for filtrationplating of radioactive rurpenrionr. A, upper port ( ~ h i m n e ~ B, l ; lower port; C, sintered steel disc; D, wosherrhoped Neoprene gasket; E, O-ring, synthetic rubber; F, two steel rods jammed into part B. The roar act os screw threads when the chimney is twisted; they tighten down on gasket D.
is flat and fits tightly into the chimney so that it doesn't fall out on disassembly. I t presses down on the upper face of the steel disc and limits the edges of the radioactive deposit, thereby producing a clean rim to allo~rhandling. The lower washer is an O-ring and is needed to improve the suction and prevent leakage of iiltrate when the suction is off. Such a filtering assembly can be made in the school shop out of stainless steel. Approximately 6 students can.be served by one assembly, as the filtration step is quite fast. Fortunately, a similar filtering assembly is about to be offered commer~ially.~ To prepare a radioactive sample, one must first add a suspension of diatomaceous earth (Celite Analytical Filter Aid)4to the filter assembly and filter off the suspending liquid. The smooth precoat not only prevents contamination but also produces a smoother radioactive deposit. Next the suspension of radioactive material is added and its liquid phase is filtered off. This deposit must be washed with rinse liquid without resuspending the particles, in most cases. After the rinse liquid is pulled through, the filter is disassembled and the steel disc is removed, dried, and counted. Given this simple technique, one must consider the types of radioactive materials that can he counted. Ion exchange resins, in small mesh size and high crosslinkage, yield uniform but fragile deposits. They can be used to take up ionic radioactive compounds, even from a dilute solution. Adsorbents should be useful Xucleru-Chicago Carp., Des Plaines, Illinois. "ohns-Manville. J
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38, Number 7, July 1961
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similarly. Many water-soluble radioactive substances can be precipitated ~ 5 t ha water-miscible organic solvent, such as alcohol. Conversely, lipoidal substances can be precipitated out with water. Proteins can be precipitated with trichloracetic acid or alcohol. Polyvalent cations can be precipitated by appropriate anions, and vice versa. Even neutral substances may be precipitable with a complexing agent, such as urea or picric acid. It is a rare substance that cannot be converted into a suspension suitable for filtration on the steel disc. Two problems arise in choosing a method for preparing a radioactive suspension: (a) completeness of precipitation, and (b) production of a uniform, stable filter deposit. The first problem, when it arises, can often be evaded by deliberately adding a known amount of non-radioactive carrier, thereby bringing the total weight of deposit above a certain minimum. This minimum, which produces filter cakes that. are "infinitely thick," is such that additional yield does not, change the observed activity. Thus, variations in yield of precipitate or losses due to washing will not affect the observed activity. The second problem call he a nuisance and may call for some experimentation. For example, we found that lead hydroxide gives uniform deposits, but bismuth hydroxide yields deposits which curl and crack on drying. Using oxalate as the bismuth precipitaut gave us a satisfactory filter cake. The choice of rinse solvent can be important in preventing curling. These and other considerations are discussed elsewhere (8). Close attention should be paid to the appearance of the final radioactive deposits. If the isotope used is a weak emitter (C14, P5,Ca45)and the sample is not infinitely thick, any visible unevenness will yield a grossly inaccurate answer. Simplicity of Calculations
Another approach that seems useful is the elimination of many of the "technical" complications so common in radioisotope work: corrections for decay, selfabsorption, and coincidence losses. These corrections always decrease the precision of counting and are not always essential nor are they intrinsic to most chemical applications of radioisotopes. The problem of decay can be eliminated simply by having each student count a standard with his unknown, both samples being made up similarly from the same isotopic starting material. The coincidence correction appears only with samples showing activities over 4,000 cpm; activities higher than this are not necessary, are wasteful of radioactive materials, and tend to make laboratory contamination a greater danger. The problem of correcting for self-absorption can be solved by making all samples the same weight or infinitely thick. The former approach is rather restrictive and difficult to arrange except in a few experiments. The latter approach is usually easy to arrange and suffers primarily from the tendency to yield lower observed activities (due to radiation absorption by the extra carrier that may have to be added). Not only does use of infinitely thick samples eliminate correction curves, but it also makes unnecessary the weighing of t.he planchets and samples, and tends to give more accurate counting, since small irregularities in sample uniformity have little effect. 346 / Journal o f Chemical Education
The mathematics of calculating results from data with infinitely thick samples is not generally known. In t,he case of infinitely thin samples (samples so small that self-absorption is negligible) the observed activity is proportional to the weight of the labeled element or compound. However, in the case of infinitely thick samples, the activity is proportional to &heconcentration of the labeled elemeut or compound in the sample: A = k c . The latter point was illustrated in one of our experiments, in which the atomic weights of various metallic elements are determined by forming insoluble sulfate salts, starting with Sa2S3sOn. A portion of the student's sodium sulfate is precipitated from water with methanol, an infinitely thick sample is prepared on a steel filter planchet, and the activity is measured. Given the atomic weights of the elements in this salt, the student calculates the concentration of sulfur (22.5 %) and then the proportionality constant for his counting equipment. Thus, if the sodium sulfate standard shows an activity of 2,250 counts/min (cpm), the constant is 2,250/22.5 = 100. If a heavy metal sulfate (formed from another portion of the sodium sulfate) shows an activity of 1,500 cpm, the sulfur concentration must be 1,500/100 = 15.0%. From this figure and information about the valence of the metal, the student can readily calculate the atomic weight of the met,al. This method of analysis does contain a weakness, since the backscattering coefficients vary slightly with the average atomic number. Thus, comparison of lead sulfate with sodium sulfate gives an expectedly high activity due to the greater reflection of beta rays from the upper portion of the deposit. However, the effect is small and can be explained qualitatively to the st,udents when the experimental results are analyzed. When the experiment must yield information about a weight, rather than a concentration, the infinite thickness method of sample preparation requires that carrier be added both to the standard and to the unknown. This application is illustrated in an experiment which demonstrates the Freundlich equation for the physical chemistry student. The usual adsorption of acetic acid (radioactive) on charcoal is followed by determining the amount of acetic acid left unadsorbed. To measure the amount of acetic acid, we deliberately dilute it with unlabeled acetic acid, precipitate the silver salt, and filter and count this. A standard, known portion of C14 acetic acid is processed similarly, and the calculations are something like this: Standard: 0.4 millimole of C1+-acetic acid 1.2 millimoles of ordinary acetic acid yield an observed activity of 2,000 cpm. The con1.2) and centration of labeled acid in the mixture is 0.4/(0.4 the basic equation is A = kc, so 2,000 = k[0.4/(0.4 1.2)] and k = 8,000.
+
+
+
Unknown: z millimoles of labeled acetic acid 1.2 millimoles of ordinary acid yield an activity of 1,000 cpm. Then 1,OW = k[z/(z 1.2)]. The etudent knows k is 8,000 and simply solves for r (0.17 millimole).
+
+
Use of Small Amounts of Activity
It is difficult to determine from most published teaching experiments with radioisotopes just how much radioactivity is actually needed. In our experience, the amounts are ordinarily so high that the teacher must
get special authorization from the Atomic Energy Commission in order to obtain the isotopes. This is a serious limitation, and it becomes necessary as a result to design experiments which do not waste radioactivity. Fortunately, it is possible to purchase reasonable amounts of certain radioisotopes without special authorizndion from the Federal Government, these quantities being the so-called "generally-licensed" amounts. Any teacher can purchase a single paekage of a generally-licensed amount in the same way that he can purchase any other chemical reagent. Ten such packages may he in one's possession a t any moment. The actual quantity of radioactivity in a package depends on the isotope; the figures range from 0.1 microcurie (SrgO)to 50 microcuries (CL4,S39. (2.50 microcuries of tritium can be bought, but this cannot be counted effectively by student-grade equipment.) I n most research experiments involving radioisotopes, the procedure involves eounting only a very small fractionof the total amount of activity used, perhapsO.l% or less. The size of the generally-licensed paekage does uot permit such wastage. Consider the following calculations in this respect. If one has a single generallylicensed paekage of CI4,you have 50 microcuries or 50 X 2,200,000 = 110,000,000 dpm (disintegrations/minute) which are to be allocated to (say) 30 students. Each student therefore gets 3,700,000 dpm, which is to be divided up, perhaps,. between 3 samples. Then each sample will contain 1,200,000dpm. If this were counted as a very thin sample with a thin-window Geiger counter (efficiencyroughly 4'33, it would give an observed aet,ivity of 48,000 cpm per sample. Under these conditions, one could accept an experiment where 97% of the aetivity is wasted. If instead the samples are diluted to the point, where they weigh 1.50 mg and therefore give infinitely thick samples on the typical planehet, the efficiency will be somewhat lower (about 1/10) and the observed activity would be about 4,800 cpm. With such a system, we would not like to throw away mueh more than 80% of the starting activity. The amount of activity that should be counted by the student depends on the precision desired and the time available for counting. The standard deviation of a measurement is equal to the square root of the number of counts collected, so that a sample counted until 10,000 counts are recorded will have an intrinsic statistical variability of 100 counts, or 1%standard deviation. This is generally considered good precision, especially as the variability in sample preparation may be greater than this. However, the time needed to accumulate such a large number will depend on the actual aetivity. Thus, a sample exhibiting 10,000 cpm will require only one minute of counting; if the activity is 1,000 cpm, 10 minutes will be needed; if the aetivity is 100 cpm, 100 minutes will be needed. It is evident that the counting step can be a bottle-neck in a large class when good precision is desired. If possible, the counter should he left running for several days and the students allowed t.o count in their spare time. To reduce the variability arising from subtraction of the counter's background, this figure should he determined with a long count by the instructor. The experiment on atomic weight determination described ahove is ideal with respect to economy of act,ivity as t,here is virtually no waste a t all. One gener-
ally-licensed package should suffice for roughly 150 students. I n the adsorption experiment described above, the weights of acetic acid and charcoal are chosen so that a reasonable fraction of the acetic acid is left unadsorbed and available for eounting. When one uses an experiment requiring small amounts of activity, one not only avoids the need to get a personal authorization but also greatly reduces the problems of safety and contamination of the laboratory. I t must be recognized that no one, including the inexperienced student, is able to work without a certain number of spills in his life. It is therefore to be expected that a certain amount of radioactivity will end up on the floor, table, clothing, or Geiger counter. Obviously, the less aetivity given to each student, the less serious are the spills. Generally-licensed amounts of activity are ordinarily considered non-toxic even if the student should swallow the entire portion. I t should be pointed out that naturally-occurring radioisotopes can he bought readily in astonishingly high amounts of activity withoutany question of governmental authorization. Several companies sell an equilibrinm n ~ ~ x t n r ~ ~ o iI)inn~urh, l ~ ~ i n l ,m d po~loniulnnirrr~ft'>. nll rndinnctire f:~tomirw:nhr 2109. 0 1 1 1 ~the bismuth radiations are readily detected by a Geiger counter but nearly all the metal atoms in this mixture are lead. P33, despite its uncomfortably short half-life, makes a good isotope for student work. A generally-licensed package contains only 10 mierocuries, but the beta radiation is so penetrating that it is easy to prepare infinitely thin samples even though the weight of the samples is appreciable. 0.06 microcurie in a thin sample will give an ample reading. It is necessary to synchronize your experiment with the delivery schedule of your supplier, as the half-life is two weeks. Safety Precautions
In ordinary chemical work there is ordinarily a certain amount of spillage or drippage which is considered acceptable. I n ~ o r kwith radioactivity, even with student-size portions, it is wise to aim a t strictly quantitative procedures with a minimum of handling and eontact. Radioactive starting materials should be pipetted out in solution form rather than weighed out by the students, partly because pipetting is normally faster, hut mainly because there is less tendency toward spillage. We modified the sulfur-sulfite experiment of McCool and Hentz (4) in this way. Where they have the students weigh out powdered Sa5,we dispense a solution in carbon disulfide into the reaction flask and have each student precipitate his sulfur with acetone. After washing the sulfur with acetone and water, the student heats it with sodium sulfite. I n this modification the student also learns something extra about the solubility properties of sulfur as well as the usefulness of Pasteur pipets for handling small volumes of liquid. (Pasteur pipets are simply elongated eye-droppers.) If possible, solutions should not be poured, even with the aid of pouring rods. There is too mueh risk of get)ting radioactivity on the outside of the vessel. I t is better to transfer the liquid-if the volume is not too great-with a Pasteur pipet. The transfer can be made complete, if desired, with only a modest volume of rinse liquid, using the same pipet for the rinsing and transferring. Suspensions can also be transferred efficiently Volume 38, Number 7, July 1961
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this may, possibly with the aid of a nonionic detergent. We use Pasteur pipets for transferring radioactive slurries to the steel disc atering device, then for rinsing the inner wall of the chimney and the filter cake on the disc. I n order to avoid pouring, we also try to avoid volumetric flasks and graduated cylinders, substituting pipets where possible. Volumetric flasks are admittedly more accurate than pipets, hut often unnecessarily so. Pipets should not be operated by mouth vacuum, of course. A safety pipettor of some sort should always be used, even when the student pipets a non-radioactive solution (since the pipet top or his fingers may be contaminated). Commercially available safety pipettors work well, but are expensive and a bit of a nuisance to clean if the student should suck radioactive material into the pipettor. We prefer the extremely cheap and simple device described by Kolb (6),which we use routinely for pipetting organic solvents and other reagents also. Rodkey's pipettor, readily made from commercially available T-tubes, might be preferred (6). With any type of pipettor, it is imperative that the students be given an opportunity to practice its use (with water) during a prior lab period. Pipettors are very easy to use, but practice cannot be avoided.
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Journal of Chemical Education
Incidentally, it should be pointed out that pipets cannot be washed adequately by individual students. We strongly recommend having a student assistant collect the pipets, wash them in a commercial soakerrinser, and dry them in an oven. This little "luxury" is a great help in maintaining a high level of accuracy ~n isotopic work. It would indeed be a shame after all this effort and expense to give the students the false impression that radioisotopic techniques lead to inaccurate results. Literature Cited (1) A oolleetion of papers from the JOURNAL OF CHEMICAL EDUCATION related to training and experiments in radioactivity, presented by Nuclear-Chicago Corp., 333 East Howard Avenue, Des Plaines, Illinois. (2) RADIN,NORMAN S., Editor, "Radioisotope Experiments for the Chemistry Curriculum," Nuclear-Chicago Corp., Des Plaines, Illinois, 1960. (3) Technical Bulletin No. 10, available gratis from SuelenrChicago Corp. (4) McCoo~,W. J. AND HEKTZ, R. R., J. CHEM. EDUC.,32,329 (1955). (5) KOLB,J. J., Chemisl-Analusl, 47, 19 (1958). ( 6 ) RODKEY, 1'. L., Science, 118,488 (1953).
