d-Elution Radio Gas Chromatography Fulvio Cacace and Giorgio Perez Centro Nazionale di Chimica delle Radiazioni e Radioelementi del C.N.R.,Istituto di Chimica Farmaceutica dell, Unifiersitd, Rome, Italy MOSTOF THE RADIO gas chromatographic techniques largely employed in such fields as radiation chemistry, hot atom chemistry, chemical kinetics, and biochemistry (1-9) rely on the continuous radioassay of the effluents which offers considerable advantages in regard to resolving power, speed and convenience of the analysis, and possibility of detecting carrier-free compounds. However, the necessarily short residence time of the radioactive peaks within the sensitive volume of a flow detector poses a serious limitation on the inherent sensitivity of the analysis, and enhances the errors arising from the statistical fluctuations encountered in any radioactivity measurements. Moreover, any variation in the flow rate of the effluent affects the efficiency of the flow detectors and can be expected to introduce an error, unless a correction based on the continuous and precise measurement of the flow rate is applied. In view of the above considerations, it appears that the principle of interrupted elution, which has been successfully applied by Scott et al. (IO) to the determination of the infrared and mass spectra of gas chromatographic effluents, could be extremely useful in the analysis of labeled compounds. While retaining the convenience of the continuous analysis over the batch assay, interrupted elution permits the measurement of the radioactive peaks under static conditions ; this ensures smaller statistical fluctuations with a greatly improved sensitivity and eliminates the errors arising from variations in the flow rate. The present paper describes a simple method, based on the use of a modified gas chromatograph in conjunction with a static ionization chamber, for the interrupted elution radio gas chromatography of radioactive substances. EXPERIMENTAL
Materials. The high specific activity tritiated alkanes employed in the present work-Le., C2H6T, C3HiT, and isoC4HgT-were obtained by catalytic hydrogenation, over Pd black, of the corresponding alkenes with HT. The latter was prepared from gaseous tritium supplied by Commissariat a 1’Energie Atomique (France). The nitrogen used as the carrier gas was a research grade product from SOC. Rivoira (Torino, Italy). The inactive hydrocarbons employed for the preparation of the tritiated alkanes and for identification purposes were also research grade samples (1) J. P. Adloff, Chromatog. Rea., 4,24 (1962). (2) Ibid.,7, 53 (1965). (3) F. Cacace, Nucleonics, 19 ( 5 ) , 45 (1961). (4) F. Drawert and 0. Bachmann, Angew. Chem., 75,717 (1963). ( 5 ) A. T. James, “New Biochemical Separations,” A. T. James and L. J. Morris, Eds., Van Nostrand, London, 1964, p. 1. (6) A. Karmen, J. Assoc. Ofic.Agr. Chemists, 47 (l), 15 (1964). (7) H. W. Scharpenseel, Angew. CIzem., 73, 615 (1961). (8) H. W. Scharpenseel and K. H. Menke, Z . Anal. Chem., 180,
81 (1961). (9) H. W. Scharpenseel, “Tritium in the Physical and Biological Sciences,” Vol. I, International Atomic Energy Agency, Vienna, 1962. p. 281. (10) R. P. Scott, F. A. Fowlis, D. Welti, and T. Wilkins, “Gas Chromatography 1966,” A. B. Littlewood, Ed., The Institute of Petroleum, London, 1967, p. 318.
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Figure 1. Schematic diagram of gas flow circuit €or interrupted elution radio gas chromatographic analysis from Societti Italiana Ossigeno (Milano, Italy), and were used without further purification. Apparatus and Procedure. The interrupted elution gas chromatograph described by Scott et al. (IO) is a specially designed, fully automatic unit, featuring a high-efficiency column, a pressure programmer, a time sequence system, etc. It was felt that a conventional gas chromatograph with relatively minor modifications could be adequate for the less stringent requirements of most radio gas chromatographic analyses. A Model D Fractovap of Societti Carlo Erba (Milano, Italy) was modified by inserting two valves, 2 and 6, of Figure 1, in the gas flow system, one just before the column, the other immediately following a 25-ml flow ionization chamber, 5, directly connected to the thermal conductivity (TC) detector, 4. The current of the ionization chamber was measured with a Model 510 B electrometer of Keithley Instruments Co. (Ohio, USA). The outlet of the chromatograph was connected, through a second three-ways valve, 8, to a 2-liter Borkowski-type flow ionization chamber, 9, operated at room temperature, whose current was measured with a Model 475 A vibrating reed electrometer of Victoreen Instruments Co. (Illinois, USA). The carrier and the counting gas were supplied to the gas chromatograph by the cylinders, 1 and 7, respectively, through suitable flow regulating devices. The output of the TC detector and the two electrometers was fed to separate potentiometric recorders. At the beginning of the chromatographic run, and in the intervals between the peaks, the effluents from the column were vented through the valves 6 and 8. Initially, the 2-liter ionization chamber was thoroughly flushed with a stream of the counting gas, whose flow was suspended when the elution was started. As soon as the TC cell, or the 25-1111 ionization chamber in the case of carrier-free compounds, indicated that an elution band began to emerge from the column, the stopcocks 6 and 8 were turned and the effluents were allowed to enter the ionization chamber, 9. When the elution of the active peak was completed, as indicated by the response of the 25 ml ionization chamber, valves 2 and 6 were closed, and the column was sealed at both ends. The radioactive peak eluted from the column was then quantitatively transferred into the ionization chamber 9 with a stream of the counting gas VOL. 39, NO. 14, DECEMBER 1967
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4 Figure 2. Radio gas chromatograms obtained from a conventional ( A ) and an interrupted elution ( B )analysis of the same radioactive sample. Activity in the smallest peak is on the curies order of 5 X
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from the tank 7, both stopcocks 8 and 10 were closed, and the static analysis of the radioactive band trapped in the ionization chamber was started. The transfer of the active peak into the chamber could be followed easily, because the ionization current initially increases to reach a maximum and constant value when the entire active “plug” has entered the sensitive volume. Because of the relatively large volume of the ionization chamber and the low flow rate of the counting gas, typically 100 ml per minute, no difficulty was experienced to quantitatively transfer the radioactive band into the ionization chamber before any loss could occur from the outlet 10. Control experiments, involving the use of a small (25 ml) flow ionization chamber connected at outlet 10, confirmed that no loss of activity from the large chamber 9 occurred, even during the trapping of broad elution bands. After a time sufficient to allow a satisfactory measurement of the radioactive peak, valves 8 and 10 were opened and the chamber was flushed with a flow (4 liters per minute) of the counting gas, until the ionization current dropped to its initial (background) value. This operation generally required 1 or 2 minutes. The flow of the carrier gas through the column was then restored by turning valves 2 and 6, in order to carry out the measurement of the next radioactive peak. The same procedure was used when a programmed temperature run was carried out, taking care to suspend the temperature increase during the interruption of the chromatographic development. The separation of the tritiated hydrocarbon mixture was performed with a 4-meter silica gel column whose temperature was programmed from 55” to 205” C at a rate of 4.5” C per minute. Owing to the high specific activity of the tritiated alkanes, and the very small size of the sample injected, the active peaks were not detected by the thermal conductivity cell, The 25-11-11 flow ionization chamber was therefore necessary to follow the elution of each active band from the column. Nitrogen was used in all the runs as the carrier gas, at flow rates ranging from 60 to 100 ml per minute and also as the counting gas. RESULTS AND DISCUSSION
In order to evaluate the performance of the interrupted elution technique, in comparison with a conventional radio gas chromatographic method, synthetic mixtures of tritiated alkanes were analyzed with the apparatus described in the previous section, as well as with a standard radio gas chromatograph featuring a 25-ml flow ionization chamber. To approach the conditions prevailing in the assay of carrier-free compounds, the gaseous samples subjected to the analysis were extremely diluted solutions of very high specific activity alkanes, prepared from the corresponding inactive alkenes and tritium hydride. A comparison of two typical radio chromatograms obtained in the analysis of such mixtures indicates that the static activity analysis using the interrupted elution technique provides, as expected, a much higher sensitivity (Figure Z), while the resolution of the radioactive peaks is not noticeably impaired. The apparatus used in the present work allowed the development of the chromatogram to be suspended for a period up to 20 minutes, in order to measure the radioactivity of a given
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Table I. Analysis of Tritiated Alkanes Mixtures by Interrupted Elution and Conventional Radio Gas Chromatography No. of Composition, Mixture No. determinations HT CzHjT Ca&T ~so-C~H~T la 24 1.80 f 0.07 0.96 i.0.04 21.4 i 0.60 75.8 i 0.70 1b 16 1.65 f:0.16 0.96 i 0.11 21.8 f 2.00 76.1 i 1.40 3.96 i 0.17 88.8 f:0.60 2a 17 7.26 i 0.35 3a 5 2.39 =t0.07 1.26 i 0.03 9 6 . 4 . i 0.10 4a 4 1.86 i 0.10 ... 21.8'20.03 76.4 i 0.20 11 64.60 i 0.90 35.40 ZIE 0.80 ... ... 5a 6a 15 ... 22.1 f:0.70 77.9 rt 0.80 Analyzed by interrupted elution gas chromatography with a 2-liter ionization chamber. b Analyzed by conventional radio gas chromatography with a 25-ml flow chamber. Q
peak with the static chamber, before the resolution was affected. It is likely that the counting time could be lengthened in the case of an isothermal separation, by making use in the gas chromatographic unit of a more elaborate gas circuit, similar for instance to that described by Scott et al. (10). The quantitative results of the radiometric analysis, summarized in Table I, clearly demonstrate the higher precision attainable when the separation is carried out with the interrupted elution technique; the data show a considerably smaller standard deviation than those obtained from conventional radio gas chromatography. While the present investigation has been restricted to the use of ionization chambers in both the interrupted elution and the conventional radio gas chromatographic analyses, the result of the comparison between the two methods is valid, in principle, even for detectors of a different type. It may be added, in this respect, that with the typical accuracy of most conventional radio gas chromatographic techniques, the standard deviation of the results is on the order of 5x or higher, even when an appropriate correction for the flow rate changes is applied (11). As a whole, the present work indicates that the interrupted elution technique applied to the radiometric analysis of labeled compounds offers distinct advantages over the conventional, continuous elution method. In the first place, the static radioactivity analysis made possible by interrupted (11) G. Stocklin, F. Cacace, and A. P. Wolf, 2.Anal. Chem., 194 406 (1963).
elution allows a much longer time for the activity measurement, which results in a much higher accuracy and sensitivity. In the second place, the errors associated with the variations of the flow rate in the conventional analysis are eliminated. Finally, the elimination of the need to integrate the areas of the radioactive peaks is a useful simplification, because the integration often introduces significant errors in the radiometric analysis. Among the disadvantages of the interrupted elution technique, one arises from the relatively large size of the detector (0.5 to 2 liters) necessary to ensure that even broad peaks are quantitatively trapped within the sensitive region. Because of the increase of the background with the volume of the detector, the conventional radio gas chromatographic method, which makes use of relatively small detectors (10 to 100 ml), is generally superior in this respect. On the other hand, a larger detector volume is often desirable to obtain a higher efficiency in the measurement of energetic p emitters. Another drawback of the interrupted elution technique is the longer time required for the chromatographic separation, which poses severe limitation on its extension to the analysis of short-lived isotopes, such as and l3N which are frequently used in the study of hot atom reactions. ACKNOWLEDGMENT
The authors thank G . Giacomello for his interest in the present work and D. Carrara for his skillful assistance.
RECEIVED for review June 7,1967. Accepted August 17,1967.
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