J. A. Merrigan J. B. Nicholas and E. P. Rack University o f Nebrasko
A Radiochemical Experiment in Gas Chromatography
Lincoln
Radiogas chromatography has become the most convcnient and extensively employed technique in qualitative and quantitative analyses of tagged organic materials. This is attributable to the relative simplicity, high speed, and sensitivity of gas chromatography. The basic principles and practice of gas chro-
by the injector, and into the separatory column. After being separated in thecolumn, thefractionsof aninjected sample enter the other side of the dual thermal conductivity cell where macroscopic amounts (0.01 ml injected solution) are detected. These detections are supplied by an ordinary galvanometer or strip chart recorder connected across the H E A T I N G OVEN RADIATION DETECTOR Wheatstonebrideecircuit. The deflections of the galvanomINJECTOR T.C. CELLS eter or recorder may be noted COLUMN HEATED FLOW M E T E R for qualitative identification of TO effluent fractions. Next the effluent flows through heated capillary tubing to a heated counting cell (Fig. 2) where microscopic mounts (=lo-'= g) of ra&oactive products are OALVANOYETER monitored by the gamma radiFlgura 1. Schematic diogrom of rodiogos chromatograph for identifying radioactive compounds produced as ation emitted. It i~important o conrequence of nuclear reastionr. that the counting cell be maintained at the same temmatography are available in various reviews (1, B, 9). FROM Evans and Willard successfully applied gas chromatogCOLUMN raphy to the separation of a2Br-labeledproducts resulb TO TRAPS ing from neutron irradiation of n-propyl bromide (4). Because of the increased use of radioisotopes and the general growth of gas chromatography as an analytical H E A T SHIELD tool, there is an immediate demand for introducing these techniques into the radiochemical curriculum. This article presents a method for building a radiogaschromatograph from materials generally available in a radiochemistry laboratory, and an experiment using this H E A T I N G ELEMENT chromatograph which illustrates Szilard-Chalmers (5) effects, energies associated with nuclear transformations, and characteristics of the chromatograph.
I
Apparatus
Figure 1 depicts schematically the basic features of the radiogas chromatograph. Helium carrier gas issues from a pressurized steel cylinder and passes through a pressureregulating valve, tubing, a chamber, one side of a dual thermal conductivity cell,
CAPACITY
-GLASS
TUBE
Figure 2. Counting cell which sits in tho well of scintillation detector for monitoring radioactive fractions from the
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perature or higher than that of the separatory column, so that radioactive fractions do not condense and adhere to the cell walls. If condensation occurs in the counting cell, the spectral trace will not return to the base line because of radioactivity remaining in the cell. To heat the effluent train, a nichrome wire may be coiled around the capillary tubing and insulated with asbestos tape. A continuation of the nichrome wire is sealed in the glass counting cell, and the entire wire is heated electrically to maintain a high temperature inside the effluent train and counting cell without damage to the NaI scintillation crystal. A stream of air may be circulated between the counting cell and scintillation crystal to prevent heat conduction to the detector. Signals from the radiation detector go through a ratemeter and are traced on a strip chart recorder. Electrical signals from the ratemeter may also be monitored by a scaler for integrating under activity peaks, We employed a Nuclear-Chicago single channel analyzer as the ratemeter and recorder, and connected a EuclearChicago Model 181B scaler to the analyzer as an integrator. After as sing by the radiation detector, the effluent fractions are collected in cold traps. The carrier gas passes through the traps into a buret soap-film flowmeter, similar to the one described by James and Martin ( S ) , where the rate of gas flow is determined. From this point it is channeled to a hood or bubble chamber to be sure that radioactive materials, which may have gotten through the cold traps, do not escape into the laboratory. The column, injector unit, warm-up chamber, thermal conductivity cell, and separatory column are all housed in an ordinary Boekel heating oven with a temperature control, similar to drying ovens used in any laboratory. The temperature of these components is identical, thus preventing condensation of effluent fractions in the thermal conductivity detector and hold-up by the injector apparatus which could cause distortion of spectral peaks. All components of the radiogas chromatograph, which come in contact with the carrier gas, are made of glass except the resistances in the thermal conductivity cell. Glass is used in an effort to present a relatively inert surface to the sample so as to cause little chemical exchange and/or decomposition. The conductivity cell consistsof two 150-watt light bulb filamentsstretched through 5 mm inside diameter glass tubing. Similar cells were described by Breunen and Remball (6). One filament is located in the gas stream before the separatory column, and the other after. Thus, a reference cell with merely carrier gas passing, and a detection cell t o detect separated fractions, are established and form two sides of a Wheatstone bridge circuit. Changes in resistance of the detection cell as effluent fractions pass, are monitored by a deflection on the galvanometer or peaks registered on the strip chart recorder wired across the Wheatstone bridge circuit. The injector unit consists of a glass tube with a serum cap on one end, and a ball joint (for connection with the separatory column) on the other. A hypodermic syringe containing the sample is forced through the 544
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serum cap and the needle is extended to the column packing where the sample is injected directly onto the column. The column packing consists of ground fire brick, 42/60 mesh, coated with silicone oil. Forty grams of silicone oil is dissolved in 250 ml of chloroform, 100 g of firebrick is suspended in the solution, and the chloroform is then volatilized under vigorous mechanical stirring in the preparation of the column packing. The packing is introduced into the coiled glass column, 4 mrn id and 2 m in length, and packed tightly using an electric massager. All glass components are connected by ground glass ball and socket joints. Although a well-type scintillation detector was used in our design, one could also use other types of radiation detectors if great enough efficiency for the activity to be monitored can be established. The activity of the effluent gas is recorded as a spectrum on a recorder for quantitative determinations. If only one radioactive nuclide is monitored, the area under each spectral peak is proportional to the amount of that radioactive product present in the sample as long as the flow rate of the carrier gas remains constant. If the temperature of the chromatograph is increased (as iu temperature programing) the flow rate decreases. Since the activity recorded depends on the time a fraction spends over the detector, the activity under a peak is inversely proportional to the flow rate. Thus, one can normalize one peak to another in a temperature programed spectrum by multiplying the areas under the peaks by the flow rate at that temperature. The Experiment
Nuclear transformations yield products with abnormally high kinetic energy and/or positive charge, e.g., a 82Bratom formed by s'Br(n,r)82Br reaction will have a recoil energy of approximately 230 ev if a single 6 Mev gamma ray is emitted after the neutron capture (7). Since this energy is much higher than normal chemical bond energies (5-10 ev), the recoil atom will break its bonds, disrupt the immediate vicinity, and finally combine with its surroundings after being slowed to reaction energies. If the bromine is present in a liquid organic system, part of the radioactive 82Br(36 hr) will become stabilized in organic combination. I n the neutron irradiation of a bromine system, 82Brm is also produced by the (n,y) reaction with =lBr. This unstable nuclide decays by isomeric transition to S2Br, i.e., 82Br" (I.T.)82Br. Thus, the 82Br-1abeled products in the system will result from (I.T.) as well as (n,y) reactions. The products following both of these r e actions have been shown to be similar in CCll and the hexanes (8). This experiment is designed to show the variety of products formed in thismanner, asillustrated by submitting a liquid sample of Br2plus CClaor hexane to a thermal neutron flux (such as in a neutron generator or small reactor), allowing short half-lifed 80Br (18 min) and 80Brm(4.5 hr) to decay out (about 48 hr), and running a radiogas chromatogram of the sample. Samples may be prepared by mixing equal volumes of Br, and CCL or hexane. If the neutron source available produces a thermal neutron flux of about 10LO n em-% sec-', a 1 min irradiation is sufficient to obtain a generous supply of activity in chromatographic peaks, even though only a small portion of the irradi-
ated sample is injected. The activity obtained in a neutron irradiation may be calculated from equations outlined by Williams, et al. (9). A 2-3 ml sample of the irradiated system is washed with 0.5 M Nad303 solution to extract all of the radioactive bromine combined as HBr* or Br2*. The resultant organic phase (approx. 1 ml) contains organically combined S2Br. This step is necessary to eliminate any exchange of radioactive bromine with organic bromides at the temperature of the chromatograph. After decay of the short half-life nuclides of bromine, a small portion of the sample is injected onto the column of the chromatograph. Since only a very small amount of material is tagged by the 82Br,the thermal conductivity cell does not detect any products. The radioactivity detector will react to the microscopic amounts of products by virtue of the decay of 82Brto 82Krwith the emission of beta and gamma rays. Typical spectra of radioactive products in CC14 and n-hexane are presented in Figures 3 and 4, respectively.
Figure 3.
CCll
Gar chromotogram a t 1 20DC, flow rote 4 5 ml min-', of
- W r 2 system.
Compound identification was made b y soncurrenl galvanometer deflectionr ond activity peaks.
The activity peaks are identified by adding carriersconsisting of the compounds one might expect to be formed during the irradiation-and observing if the carriers emerge from the column concurrently with the radioactive products. Their emergence is monitored by the thermal conductivity cell. If a two-pen recorder is available, one pen may be connected across the Wheatstone bridge, and the other pen to the ratemeter to yield a trace of carriers and radiopeaks. Coucurrence of detections provides a tentative identification of the radioactive product peaks. A change of column and/or operating temperature should be made, and the same concurrence of detections monitored for positive identification of the products. The number of theoretical plates or "plate equiv* lents" of the chromatograph column is determined by the commonly accepted method (2) of measuring the
retention time from introduction of the sample, Tll, and the peak base measured at the points where the extended tangents intersect the base line, At (Fig. 3). The number of plates is given by the equation n = 16(T~/At)~
The height equivalent to a theoretical plate (HETP) is obtained by dividing the column length by the number of equivalents. The number and variety of products containing 82Br is indicative of the energy associated with nuclear transformations. The separation of these products by gas chromatography is a good method of obtaining labeled compounds of high specific activity. Radiogas chromatography on a preparatory scale can also be used to purify tagged chemicals which have undergone radiolytic self-decomposition while in storage.
Figure 4. Gar chromatogram of n-herone-Wr2 system. Temperature was varied a t a constant rote from 90°C to 160'C. Peak asignmenh: I l l CH&, CIHsBr, CaHiBr, CH2Brl, Cdi~Br,CSHIIB~; (21 sec. CsHtdr; (31 n-C.H,aBr; (41 polymers and polybromider.
Literature Cited
(1) BAYER, E., "Gas Chromatograpl~y,~) American Elsevier Puhlishing Co. h e . , New York, 1961. (2) P ~ c s o n ,R. L., Editor, "Principles and Practice of Gss Chromatography," John Wiley and Sons, Inc., London, 1959. (3) J.~MEs, A. T.,AND M.ARTIN, A. J. P., J. Bioehem., 50, 679 (1952). J. E., J. Am. Chem. Soc., 7 8 , (4) EVANS,J. B., and WILL:ARD, 2908 (1956). (5) SEIL~RD, L., AND CHALMERS, T. A., Nalu~e,134, 462, 494 ( 1-924) - .,. (6) BEENNEN, D., AND KEMBALL, C.,J. CHEM.EDUC.,33, NO.10, 490 (1956). ( 7 ) WILL.~RD, J. E., Nature, 19, NO. 10,61 (1961). (8) J. A,. NICHOLAS, J. B., AND RACK,E. P., Radio. . MERRIGAN. chimica kcla (In press). (9) WILLIAMS. R. R.. TIAMILL. W. H.. AND SCHULER. R. H., J. \
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