have no effect on the fluorescence of 4-methyl umbelliferone over the time of assay (3-5 minutes). Because the lipase assay system used with 4-methyl umbelliferone heptanoate as substitute is a completely homogeneous, soluble one the effect of the inhibitor is not believed to be on the physical state of the substrate. This is in contrast to other lipase systems involving insoluble (emulsified) substrates in which the inhibitor does affect the physical state of the substrate (7). Interferences. Organophosphorus compounds such as Sarin and Systox inhibit lipase (I),and would thus interfere in any procedure for the determination of the chlorinated pesticides. In order to eliminate possible interference from inorganic ions, the pesticide can be extracted from the sample with methyl cellosolve prior to analysis. Mixtures of pesticides can be separated by thin layer chromatography and each (7) P. Desnuelle, Adcan. Enzymol. 23, 130 (1961).
inhibitor determined separately. Thus the specific analysis of mixtures of pesticides in samples of urine, crop materials, animal tissue, milk, etc. is possible. Current research in these laboratories is directed toward the use of different enzymes with simple separation techniques for the assay of complex mixtures of pesticides. Results of this study will be forthcoming. ACKNOWLEDGMENT The authors thank H. Beckman, Agricultural Toxicology Labs, for supplying a sample of the Sevin used in this study.
RECEIVED for review August 12, 1968. Accepted October 14, 1968. The financial assistance of the Office of Saline Water, U. S. Department of the Interior (Grant No. 14-010001-1337) and the United States Public Health Service (Contract PH 21-2016 to Louisiana State University School of Medicine) is gratefully acknowledged.
Application of Interrupted-Elution to Combustion Radio Gas Chromatography Fulvio Cacace and Giorgio Perez Laboratorio di Chimica Nucleare del C.N.R.-Istituto di Chimica Farmaceutica e Tossicologica, Unicersity of Rome, Rome, Italy
THECOMBUSTION of the effluents, followed by the reduction of the resulting water to molecular hydrogen, is a practice widely employed in the radio gas chromatography of tritiated compounds (1-10). Such a technique allows, in fact, the radioactivity detector to be operated at room temperature, outside the oven of the chromatograph, a feature which is most desirable in many cases. In addition, the combustion of the effluents largely eliminates the variations of the detector efficiency and background caused by the elution of certain types of organic substances (7, I / ) ,because these effluents are converted to inorganic gases, such as Hz, CO, N2, etc., and affect to a much smaller extent the operation of the radioactivity detector. With methods based on the continuous combustion of the effluents, the residence time of a given peak in the combustion furnace depends entirely on the chromatographic separation being carried out, and the result may be inadequate for a quantitative conversion. While in a properly constructed and operated unit, ocerall conversions of 97.8 to 99.5% have been measured (12) even under difficult conditions (high flow rates, (1) F. Cacace, R. Cipollini, and G. Perez, Science, 132,1253 (1960). (2) F. Cacace, Nucleonics, 19, 5, 45 (1961), (3) A. T,James and E. A. Piper, J . Chromatogr., 5,265 (1961). (4) J, W. Winkelman and A. Karmen, ANAL. CHEM.,34, 1067 (1962). (5) A . Karmen, I. McCaffrey, and R. L. Bowman, J . Lipid Res., 3, 372 (1962). (6) A. T . James and E. A . Piper, ANAL. CHEM.,35, 515 (1963). (7) A. Karmen, I. McCaffrey, J. W. Winkelman, and R. L. Bowman, ibid., p 537. (8) F. Cacace, R. Cipollini, and G. Perez, ibid., p 1348. (9) A. Karmen, J. Assoc. Ofic.Agr. Chemists, 47, 15 (1964). (10) A. Karmen, J. Gas Chromatogr., 5,502 (1967). (11) J. K. Lee, E. K. C. Lee, B. Musgrave, Y.N. Tang, J. W. Root, and F. S. Rowland, ANAL. CHEM.,34, 741 (1962). (12) F. Cacace, R. CipoIlini, G. Perez, and E. Possagno, Gazr. Chim. Ztal. 91, 804 (1961). 368
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relatively large samples, etc.), the possibility of isotopic fractionation during the combustion (13) makes it very desirable to achieve in all cases, and especially in the assay of tritiated compounds, a complete conversion. The difficulties which are encountered in the continuous combustion approach could be largely overcome by the application of the principle of interrupted elution (14), which allows sufficient time to ensure the complete conversion of any eluted peak, and permits an effective removal of any activity left in the combustion train before the conversion of the next peak is undertaken. The advantages of the static activity analysis allowed by the interrupted-elution radio gas chromatography in applications not requiring the combustion of the effluents have been discussed elsewhere (15). The present paper describes the application of the interruptedelution principle to the combustion radio gas chromatography of tritiated compounds. EXPERIMENTAL Apparatus. The apparatus used is a Model B Fractovap of Carlo Erba Co. (Milan, Italy), modified as shown in Figure 1. The carrier gas (He, A, Nz etc.) flows from the pressure regulator, 1, through the injection port, 2, and the column, 3, housed in the oven, 4. A two-way stainless steel stopcock, 5 , is inserted between the injection port and the column, and a three-way stainless steel stopcock, 6, is inserted between the column and the thermal conductivity detector, 7, whose outlet is connected
(13) R. F. Glascock, “Isotopic Analysis for Biochemists,” Academic Press, New York 1954, p 87. (14) R. P. W. Scott, I. A . Fowlis, D. Welti, and T. Wilkins, “Gas Chromatography 1966,” A. B. Littlewood, Ed., the Institute of Petroleum, London 1967, p 318. (15) F. Cacace and G. Perez, ANAL. CHEM.39, 1863 (1967).
Figure 1. Schematic diagram of apparatus for interrupted-elution radio gas chromatography
Table I. Reproducibility and Accuracy of Interrupted-Elution Analysis of Mixtures of Tritiated Compounds Mixture 1 Mixture 2 Cyclohexane n-Propanol Cyclohexane n-Propanol Run, No. activity A, rnpC activity B, mpC Ratio B/A activity A, mpC activity B, mpC Ratio B/A 0.966 154.5 150.0 1.04 1 84.3 87.3 208.5 213.0 0.98 2 105.0 113.2 0.928 3 102.0 111.0 0.919 397.5 382.5 1.04 435.0 432.5 1.01 4 93.7 102.7 0.912 5 138.7 150.7 0.920 557.0 555.0 1.oo 6 147.0 160.5 0.916 755.0 762.4 0.99 7 142.5 156.0 0.913 667.5 572.5 0.99 8 123.7 136.5 0.906 617.5 615.1 1.oo 9 123.0 141.7 0.868 570.0 550.0 1.04 10 121.5 126.0 0.964 600.0 615.0 0.98 11 235.5 252.7 0.932 700.0 707.5 0.99 12 120.0 129.0 0.930 775.0 675.0 1.15 13 156.7 169.5 0.924 645.0 637.7 1.01 14 59.2 66.7 0.888 692.5 677.5 1.02 15 154.5 168.7 0.916 672.5 667.5 1.01 16 145.5 151.5 0.960 632.5 602.5 1.05 17 139.5 144.0 0.969 675.0 675.1 1.00 18 160.5 177.0 0.907 652.5 632.5 1.03 19 115.5 120.0 0.963 607.5 627.5 0.97 20 141.0 148.5 0.949 647.5 619.8 1.04 21 166.5 177.0 0.941 627.5 600.0 1.05 22 171.7 185.2 0,927 ... ... ... Average ratio 0,928 Average ratio 1.02 Standard deviation 0.024 Standard deviation 0.04 Actual ratio 0.900 Actual ratio 1.04 Per cent error 3.10 Per cent error 1.93
Interruption period, min 0 10
20
30 40 50 70
100 120 150
200
Table 11. Loss of Resolution Caused by Different Interruption Periods Resolution&for different pairs of peaks n-Hexane n-Hexane n-Hexane n-Heptane rz-Heptane n-Heptane rz-Octane Toluene n-Octane Toluene 3.86 8.31 9.33 4.82 7.20 3.27 7.22 8.43 6.63 4.28 2.92 6.81 8.26 6.37 4.08 2.60 6.09 8.17 3.63 5.76 2.47 8.09 5.97 3.49 5.63 2.31 7.64 5.62 3.35 5.39 2.09 7.14 5.31 3.16 5.06 1.81 6.58 4.83 2.88 4.66 1.73 6.36 4.62 4.57 2.83 1.58 4.40 5.87 4.32 2.70 1.42 3.89 5.42 2.53 4.07
Resolution is calculated according to Expression 2 width.
___I
(jz 5 :) where t~ and
ri
n-Octane -To1uene 2.57 2.36 2.35 2.13 2.10 2.04 1.93 1.80 1.74 1.70 1.55
denote retention time of peaks J and i, and W J and wi their
VOL. 41, NO. 2, FEBRUARY 1969
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369
to the combustion tube, 8, made of No. 1720 Pyrex glass from Corning Glass Works (Corning, N. Y.).The tube, having a i.d. of 8 mm and a total length of 600 mm, is filled with copper oxide in the combustion section (340 mm) and with zinc dust, supported on C-22 Celite, in the reduction section (250 mm). The outlet of the combustion tube, which is heated with two separate electric furnaces, 9, is connected by means of a three-way glass stopcock, 10, to the 2.5-liter flow ionization chamber, 11, which is equipped with two stopcocks and mounted on a Model 610 B electrometer from Keithley Instrument, Inc. (Cleveland, Ohio). A cylinder of purge gas-for example, a N z / H ~mixture, 9 : l v/v-is connected through the pressure regulator, 12, to the stopcocks, 6 and 10. For the analysis of carrier-free high-specific activity compounds, the apparatus is equipped with a small volume (10-ml) flow ionization chamber, featuring an insulator made of Teflon and inserted in the chromatographic oven. Materials. The carrier gases and the Nz/H2 mixture are research grade products supplied by Societg Industria Ossigeno (Milan, Italy). The copper oxide is a reagent grade sample, especially prepared by Carlo Erba Co. (Milan, Italy) for the combustion of organic compounds; the zinc is a reagent grade product from Merck Co. (Darmstad, Germany) and the Celite has been supplied by Carlo Erba Co. The tritiated products analyzed have been prepared, according to standard procedures, from gaseous tritium with a stated purity greater than 95z, obtained from the Commissariat a I'Energie Atomique (France). Procedure. The apparatus is set for operation by adjusting the temperature of the furnaces to 715 i 20 'C (oxidation zone) and 420 i 10 "C (reduction zone). The flow rate of the carrier gas and the temperature of the chromatographic oven are regulated according to the requirements of the particular separation being carried out, and the background of the 2.5-liter ionization chamber is measured before injecting the sample. Once the elution of the first peak to be measured is completed, as indicated by the thermal conductivity detector or, in the case of carrier-free compounds, by the 10-ml ionization chamber, stopcocks 5 and 6 are turned in such a way as to isolate the column and interrupt the development of the chromatogram. A stream of the purge gas, Le., the N ~ / H mixture z from pressure regulator 12, is allowed to flow gently through stopcock 6, in order to displace the eluted peak into the combustion tube, where it is trapped by closing stopcocks 6 and 10. After a period of a few minutes, necessary to ensure the complete combustion of the peak, the combustion products are swept out of the reactor with a stream of purge gas, at a flow rate of about 15 ml per minute, by turning valves 6 and 10. In such a way, the radioactive hydrogen is quantitatively displaced into the ionization chamber 11, where it is trapped by turning both stopcocks of the detector. The right moment to trap the active plug is clearly indicated by the record of the ionization current, which reaches a maximum and constant value when all the radioactive gas is comprised within the sensitive volume. The timing of the trapping operation is not critical, as a considerable interval elapses between the moment a given peak has completely entered the ionization chamber and the moment a measurable radioactivity can be detected in the gaseous stream leaving the chamber, as indicated by suitable control runs. Once the static radioactivity determination has been carried out, the chamber is washed with the Nz/H2 mixture from valve 10, until the ionization current drops to its initial (background) value. The development of the chromatogram is now resumed, turning stopcocks 5 , 6, and 10, and the aDDaratus is ready for the measurement of the next peak. The interruption of the elution can be extended, under our conditions, for periods up to 20 minutes before a serious loss of resolution becomes apparent.
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ANALYTICAL CHEMISTRY
I
> o E *
I
0
m
I
0
cv
I
0 A
I
1
Cyclohexane
n-Pr o pa n 01
Methanol
Figure 3. Loss of resolution caused by interruption of elution
> E
Cyclohexane
Methanol
nP:r
I
Top: Continuous elution Bottom: Same sample, elution interrupted for 20 minutes (arrow)
o pa no I
time
RESULTS AND DISCUSSION
The radio gas chromatographic analysis of a mixture containing two tritiated compounds, namely cyclohexane-1,2-T and n-propanol-2,3-T, is illustrated in Figure 2. The plot of ionization current cs. time exhibits three distinct regions, characterized by different slope. In the first region, the bulk of tritiated hydrogen produced by combustion of the peak enters the detector and causes a sharp increase of ionization current. When all such activity is transferred into the chamber, the ionization current tends to level off. In the second region, the purge gas sweeps the reaction tube, and the large excess of inactive hydrogen removes any tritium dissolved or adsorbed in the reagents and the walls. The additional activity entering the ionization chamber produces a further increase of the current. Finally, once the whole activity of the peak is introduced into the sensitive volume, a constant value of the ionization current is obtained, and the stopcocks of the chamber can be closed, t o carry out the activity measurement under static conditions. The quantitative results obtained in the analysis of two mixtures containing different proportions of tritiated cyclohexane and n-propanol are summarized in Table I, which illustrates the accuracy and precision attainable with the interrupted-elution technique. A drawback of the method described is the relatively short life (about 150 analyses) of the combustion tube, caused by the passage of the purge gas, containing HP, through both the oxidation and the reduction sections. On the other hand, the use of hydrogen was necessary t o eliminate the serious memory effect invariably observed when pure Nz, He, o r A were employed as the purge gas in the analysis of a variety of tritiated compounds, including molecular hydrogen, the lower alkanes, aromatic hydrocarbons, aliphatic alcohols, and ketones, aromatic esters, etc. Such memory effect, whose extent is largely independent of the nature of the radioactive compound, can be traced to the
contamination of the oxidizing (13, 16) and especially the reduction zone of the combustion tube, and is completely eliminated by the addition of 10 volume HBt o the inert gas. As an example, no activity could be detected in the analysis of a 0.1-mg sample of inactive n-propanol, even after a deliberate attempt to contaminate the combustion tube by burning a much larger (10-mg) sample of tritiated n-propanol, having a specific activity as high as 0.1 wC/mg. Another drawback of the interrupted-elution approach is the loss of resolution, which becomes increasingly serious as the interruption period is extended. An example is illustrated in Table 11, whose data refer t o the analysis of a fourcomponent mixture, carried out with a 2-m didecylphthalate column, operated at 110 “C with a Nz flow rate of 50 ml per minute. The loss of resolution caused by a 20-minute interruption of the elution is also illustrated in Figure 3, which refers t o the analysis of a cyclohexaneln-propanol sample. The observed loss of resolution is not an inherent disadvantage of the interrupted-elution approach, but is likely t o arise from the rather large (ca. 5 ml) “extra-column” volume of the combustion furnace and associated tubing, In fact, by use of suitable low dead-volume components, Scott et al., who first introduced the interrupted elution method, found very small losses of resolution, even for extended periods of interruption (14). Finally, the time required for the analysis is considerably longer than in the conventional flow technique, and could prevent the application of the interrupted-elution method t o certain short-lived nuclides. O n the other hand, the static activity measurement, permitted by the interruption of the elution, offers a number of advantages (13, including high sensitivity, elimination of (16) J. Bigeleisen, M. L. Perlman, and H. C. Prosser, ANAL.CHEM. 24, 1356 (1952). VOL. 41, NO. 2, FEBRUARY 1969
371
errors introduced in the conventional analysis by variations of the flow-rate, the possibility of extending the counting time, in order t o reduce the statistical uncertainty associated with the radioactivity measurement. Still more important, the much longer residence time of each eluted peak within the furnace ensures a complete combustion of the tritiated compounds. Finally, while an ionization chamber has been used in the present work, it appears that the method described can be
easily modified t o be used in connection with other types of radioactivity detectors. ACKNOWLEDGMENT The authors thank D. Carrara for his skillful assistance. RECEIVED for review March 26, 1968. Accepted September 4,1968.
Determination of Oxygen in Refractory Oxides Carolyn S. MacDougall, Maynard E. Smith and Glenn R. Waterbury Unic'ersity of California, Los Alamos Scientific Laboratory, Los Alamos. N . M . 87544 THEDEPENDENCE of the properties of refractory oxide fuel materials upon the oxygen to metal atom ratio necessitated the development of a method for accurately determining this ratio. A review of reported methods is given by Marken, Walter, and Bones ( I ) . Some independent methods for measurement of oxygen in these oxide fuels include: reaction of the oxide with bromine trifluoride and measurement of the oxygen formed (2), and reaction of the oxide with carbon in an inert atmosphere o r vacuum and subsequent measurement by conductometry (3,4) or gravimetry (5,6) of the C O formed. Methods based upon the direct measurement of O2 are less empirical than those methods based solely on changes in sample weight, and the gravimetric determination of the off-gases offers the desired precision of measurement. Determination of the contents of metals in these oxides by titration methods (7) permits calculation of the critical oxygen t o metal atom ratio. I n basic concept, the method developed is similar t o previously reported methods (5, 6), but differs significantly in sample preparation, sample size, lack of crucible bath, automatic temperature programming, and the products formed in the inert gas fusion. EXPERIMENTAL Apparatus. The major features of the analytical train are shown in Figure 1 . The sample is heated in an AUC grade graphite crucible which is contained in a water-cooled fusedsilica furnace tube. Argon carrier gas, purified by passage over hot uranium turnings, sweeps the reaction products from the furnace through a desiccant tube into an absorption tube containing Ascarite to trap the COz. The effluent C O is oxidized over hot CuO and trapped in the second absorption tube. The Schiitze reagent in the second desiccant tube ensures complete oxidation of any CO from the CuO furnace and indicates inefficiency of the CuO furnace by turning brown. The 25-kW induction generator is equipped with a timed, motor-driven power control to raise the power level automatically from a preset minimum to a preset maxi~
(1) T. L.Marken, A. J. Walter, and R. J. Bones, At. Energy Research Establishment, R4608 (1964). (2) H. R. Hoeckstra and J. J. Katz, ANAL.CHEM.,25,1608 (1953). (3) E. J. Beck and E. E. Clark, fbjd.,33,1767-70 (1961). (4) H. L. MacDonnell, R. J. Prossman, and J. P. Williams, ibid., 35, 579 (1963). ( 5 ) H. T. Goodspeed and D. Pettis, U. S. At. Energy Comm. Rept, ANL-7264 (1967). (6) B. D. Holt and J. E. Stoessel, ANAL.CHEM.,36, 1320 (1964). (7) G. B. Nelson, K. S. Bergstresser, G. R. Waterbury, and C . F. Metz, Twelfth Conference on Analytical Chemistry in Nuclear Technology, Gatlinburg, Tenn., Oct. 8-10, 1968. 372
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mum within a desired time interval. Weighing, grinding, and pelletizing of the sample are done in an inert atmosphere enclosure similar to that designed by Smith (8). Procedure. All samples are ground and homogenized in a mixer-mill prior to preparation of the sample pellet. A quantity of oxide, containing approximately 120 mg of 0 2 , and 0.2 gram of SP-I graphite are accurately weighed and transferred to a stainless steel mixer-mill capsule. After a 2-minute mixing period, the sample is pelletized a t 8000-lb gauge pressure in a 1/4-inch pellet die, and then weighed. Proportionate loss of both oxide and graphite during pellet preoaration is assumed. This loss should be no greater than 10%. With the induction generator off and a positive pressure in the fused silica furnace, the pellet is placed in the crucible. When replacing the crucible and furnace caps, care must be taken to purge from the system any air trapped during this operation. The flow of Ar through the apparatus is adjusted to 100 ml/minute, and the automatic programmer raises the crucible temperature from 1000 to 2000 "C in 20 minutes, maintains the maximum temperature for 1 hour, and then turns off the power. The absorption tubes are weighed according to an established gravimetric procedure. Each day, a blank determination is made prior to a series of analyses by repeatidg the procedure using a spent pellet. The weight increase of either tube should not exceed 0.1 mg for the blank. RESULTS AND DISCUSSION The recently reported inert gas fusion methods for determining O2in oxides used a relatively small sample (10 to 250 mg) and a platinum bath in the reaction crucible. Under these conditions, CO was formed, and the amount of CO?was ne:ligible. In order to obtain representative samples of fuel element materials, quantities of oxides weighing not less than 1 gram are used. Mass spectrometric analysis of the effluent gases produced from reaction of 1 gram of sample with an excess of graphite in a dry crucible showed that appreciable amounts of CO?, as well as C O were produced. This COz must be measured and included in the calculations. The mixing of the graphite with the oxide and pelletizing provides intimate contact of the reacting species and eliminates the necessity of using a Pt bath. As the pellet contains an excess of carbon, the crucible is not appreciably attacked and can be used almost indefinitely. Removal of each pellet
(8) M. E. Smith, J. M. Hansel, and G. R. Waterbury, U. S. At. Energy Comm. Rept, LA-3344 (1965).
