JOURNAL OF CHEMICAL EDUCATION
HOW TO USE RADIOCARBON CHEMICALS IN TEACHING RICHARD H. ROBINSON' Fisher Scientific Company, piitsburgh, Pennsylvania
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recent public availability of safe, low-activity, relatively inexpensive radiocarbon compounds2 presents a fine opportunity to students and teachers everywhere for a variety of interesting and instructive applications. After acquiring a.few vials of radiocarbons, a counting chamber, and a scaler, what then? I t is the purpose of this article to describe for those who have not followed this interesting new field of research very closely, a few representative applications, easily set up in classroom or school laboratory, with the hope of arousing increased interest in the valuable lessons that tracer studies can teach. THE ISOTOPIC DILUTION METHOD
For the budding biochemical analyst, this technique offersmany possibilities for bypassing improbable quantitative chemical determinations of a desired compound in a mixture of similar compounds. It can also be used to shorten many other analytical methods whose only ohjectional feature is that they take too long. Figure 1 illustrates the simplicity of the fundamental method. A typical experiment might result in the following data: B 2000 c. p. m. C 60 mg. 500 c. p. m. 'Present address: John T. Ryan Memorial Labomtoriea, Mine Safety Appliances Company, Pittsburgh. 'Recently announced by the Fisher Scientific Company in Loboralo7y, 22, 154 (1953).
Then A
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2000 (60)
- 40
=
200 mg. of benzoic acid
500 in the crude mixture, an accurate quantitative determination without a quantitative chemical isolation. To clarify, one might think of the activity added at the beginning of the assay as having similar characteristics to those of a coupling reagent for a colorimetric determination. The activity, considered as a material quantity, mixes intimately with all of the inactive benzoic acid in the crude mixture to give the benzoic acid in the sample a certain specific activit,y in counts per minute per milligram. Then, if any benzoic acid at all can be isolated, it will have the same specific activity in counts per minute per milligram (the active benzoic acid has identically the same physical-chemical characteristics as the inactive twin, with, of course, the exception of activity). Now, knowing the total activity added as a "coupling agent" by the preliminary count, and the specificactivity of the isolated portion, it becomes a matter of simple ratio to calculate the weight of benzoie acid that would, when divided into the total activity, have the same specific activity as the isolate. Then the subtraction of the small weight of benzoic acid that must necessarily be added to the crude sample as the carrier of the activity, must he made to find the weight of inactive benzoic acid that was in the sample before the activity was added. AS A TRACER IN BIOLOGICAL RESEARCH
An interesting and easy experiment using carbon
JULY, 1955
371
A Crude mixture of organic compounds including some bensoie acid and benzoate
B Add one whole vial of beneoic acid-7-C1v(one microcurie) (40 mg.) after checking its activity; equilibrate C Acidify, extract acids with an immiscible solvent. Purify by recrystallizing the bensoic acid to a constant melting point. Weigh and record weight
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D Determine the activity in counts per minute under exactly thesame oonditions of weight,distancefrom thedetector, andarea as in the count of B Figure 1.
E Calculate beneoic acid in A as follows: wt. bensoie c. p. m. B (weight C) - 40 c. p. m. D
The Determination of Benroic Arid in
dioxide-CL4to follow the hreathing process in living green leaves canhe made with a minimum of equipment. A large green leaf on a living plant is treated and sampled as shown in Figure 2. The radioactive carbon dioxide is generated right in the Fisher vial from one microcurie of barium carhonateCI4 or sodium carbonate-C14, by the addition of a few drops of warm dilute acid. The leaf's surface is pressed against the mouth of the vial and its natural life processes are allowed to continue, but with the portion encompassed by the vial's mouth breathing a radioactive atmosphere. At the end of 15 minutes, circles of the leaf tissue can be removed from the body of the leaf with a brass corkborer of the same diameter as the vial. The first sample should he, of course, the area enclosed by the vial wall, followed by a concentric ring of samples from the immediately surrounding area. A count of the activity of each circle in the counting apparatus will demonstrate that 96 per cent of the total activity is in the first sample taken, while the sum of the activities from the circles surrounding the first sample will contain the other 4 per cent and will rapidly lose even that small amount. Repeating the sampling-counting process at much longer intervals of time on samples from the outer edges of the leaf, and from other leaves on the same stem, will demonstrate the speed with which the new, biosynthesized compounds in the leaf are transported to other parts of the plant. SOLID-LIQUID PHASE EQUILIBRIA
In the study of crystal growth or crystal contami-
=
.
comp1s. Mirt"n, by Isotopic Dilution
nation, and coprecipitation and adsorption phenomena, radioactive tracers can again add useful knowledge unobtainable by other means. One study, easily made in the school laboratory, is the rate of growth of sucrose crystals in a saturated solution in dilute alcohol. For this particular demonstration, a few hundred ml. of a saturated sugar solution is prepared in water and partially precipitated by the addiiion of a auantitv of alcohol. The ' suspended crystals are shaken vigor- ~ ~ ~ k 2 ; o D~~~~~ n.dioactiv. coxin ~i.i~= ously and allowed to settle. plant A 40-mg. quantity of sucrose-C14 is dissolved in a few ml. of an alcoholic solution of the same concentration as the supernatant in the larger hatch (this involves recording the volumes of water and alcohol used in the preparation of the larger solution). The "active" solution is then added and intimately mixed with the larger solution. An immediate aliquot of a few ml. of the supernatant is taken, completely precipitated with the addition of a small amount of ether, and filtered, and the sucrose crystals are recovered, dried, and counted for activity. The crystals in the main body of the solution will continue to grow at a slow rate, but in a few days another aliquot can he removed from the supernatant, treated in the manner described, and counted under
y2Tz ti:.
A Saturated BUcrOse solution in 50% alcohol with an excess of crvstalline sucrose
B 40 mg., one microcurie, of sucrose-CL4is added a6 a few ml. of solution in 50% deohol
4 An immediate aliquot of the supernatant is pptd. with ether, crystals are filtered off, dried, and counted D Another diquot of the supernatant is treated as in C, 1 c.p.m.C-e.p.m.DX1OO crystals dried and counted E Same a6D >G %loss = sample wt. counted F Same as E i H Rate of suorose crystallization per unit of time =
+
% loss activity tlme between aliquots
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JOURNAL OF CHEMICAL EDUCATION
372
the same conditions of weight, area, and distance from the detector. After preparing a plot of loss in activity from aliquot to aliquot versus time a t which thealiquot is taken, and plotting many such aliquots, a glance will reveal the relative amounts of sucrose deposited on the crystalline surfaces from the radioactive supernatant, and also the rate of deposition for any specific time interval. By varying alcoholic concentration, temperature, and time, the optimum conditions for growing large crystals of sucrose can be established. The general method is diagramed in Figure 3.
active benzoic acid is dissolved in the aliquot and the solution is evaporated to dryness with gentle heating. The dry powder is spread out and smoothed to yield an even deposit. Now a 2-ml. aliquot of the petroleum ether phase is treated in the same manner, made to dissolve 100 mg. benzoic acid, dried, and spread in another dish. This is labeled sample B. Both of the dishes, A and B, are counted separately for a determination of their activities by being placed in identical positions in the scaler. Figure 4 shows a plan of the method and the calculation of results. These simple demonstrations3 are not just illustraLIQUID-LIQUID PHASE EXTRACTION SOLUBILITY tions of useless phenomena. Consider, for example, Since many chemical separations involve isolation of that the isotopic dilution method of analysis can be a the desired material-or removal of an undesired im- valuable tool t o the organic student later on in his adpurity-with an extraction of a solution of the sample vanced experimental work, whether it be method rewith an immiscible solvent, it is often necessary to study search on new analytical methods or a complicated biothe distribution of the chemical compound of interest chemical investigation. between the solution liquid and the extracting liquid. The researcher into life processes, possibly the medThe significant ratios of solute t o extracting liquid ical investigator, can go on from the simple leaf experican he explored by setting them up in the following ment to more complex tracer applications, such as folthree-component system and using a radioactive variety lowing the metabolism of a new drug or hormone. The of the compound of interest t o supply an easy method study on sucrose crystallization may inspire an emof measurement. We will base our example on the dis- bryonic sugar chemist to improve the technology of tribution of benzoic acid between a 50 per cent alcoholic sugar refining at some future date or-transferring the solution and petroleum ether. knowledge gained on sucrose crystals-to invent new To a 25-ml. separatory funnel, add 5 ml. of petroleum and better methods of growing pure sapphire, silicon, ether, 2.5 ml. of anhydrous ethyl alcohol, and 2.5 ml. or titanium crystals. of water. Twenty mg. of benzoic acid-CL4(having an "uch of this work had its infipiration in the following referactivity of 0.5 microcuries) is added, the funnel is stop- ences: BRADFOAD, J. R., radioisotope^ in Industry," Reinhold, pered, and it is shaken vigorously for 30 seconds. The New York, 1953; CALVIN,M., "The path of carbon in photolayers are allowed to separate completely. synthesis," Chem. Eng. News, 31, 1622 (1953); FRIEDLANDER, The bottom phase is the 50 per cent alcohol. It is G., A N D J. W. KENNEDY,''Intmduction to Radioehemistry," labeled A , and allowed to flow into a separate container. John Wiley & Sons, Ino., New York, 1949; KAMEN,M. D., Tracgrs in Biology," Academic Press, New York, A 2-ml. aliquot is placed in a micro evaporating dish "Radioactive 1951; KURANZ, J. L., Vice-president Nuclear Instrument and (the tiny metal pans found in some scalers might doChemical Corp., Chicago, private communiestions to author, and will save a later transfer). Now 100 mg. of iu- 1954. 20 mg. beneoio aeid-C" after equilibration in a mixture of equal volumes of 50% ethyl alcohol (5 ml.) and petrpleum ether (5 ml.)
1 2 ml. of the lower, alcohol layer A made to dissolve 100 mg. inert beneoic acid, the solution evaporated to dryness L Dry solids arranged in scaler and counted as sample A
1
1
2 ml. of the upper, petroleum ether layer made to dissolve 10 mg. inert beneoic acid then evaporated to dryness as in A ; sample B , 1 Sample B solids arranged in an identical manner to A and counted
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Benzoic aoid in alcohol = c. p. m. A Beneoic acid in ether c. p. m. B Per cent benzoie acid in alcohol phase =
1
Mg. benzoic acid per ml. in alcoholic phase = c. p. m. A 20 (mg. added) C . ~ . ~ . A + C . ~ . (~V O . L B- ~~I CS . )
1
Per cent benaoic acid in petroleum ether phase =
Mg. benzoic acid per ml. in petroleum ether phase c. p. m. B 20 c. p. m. A a , p. m. B 5
Fivl~" 4. A Typical Liquid-Liquid Distribution &le
+
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