Simultaneous Determination of Iron-55 and Stable Iron by Liquid Scintillation Counting Frank J. Cosolito, Norman Cohen, and Henry G . Petrowl Institute of Environmental Medicine, New York University Medical Center, New York, N . Y.
IRON-55,produced by activation during thermonuclear testing, is now present in all active biological systems which contain iron. Iron-55 decays exclusively by electron capture with a half-life of 2.94 years. At present it is usually measured either by gas-flow proportional counting ( I , 2) or by thin crystal NaI(T1) scintillation spectrometry (3). Both of these methods depend on the detection of the 5.9-keV x-ray which is characteristic of the manganese-55 daughter product. Recent data indicating the presence of iron-55 in man and the environment ( I , 4 , 5 ) indicated the need for an improved technique for measuring this radionuclide. For samples containing from 0 to 20 mg of stable iron, liquid scintillation counting is a simpler and more sensitive technique than methods invo!.ving electrodeposition of iron. Electrodeposition is more sensitive for samples containing greater than 20 mg of stable iron assuming of course that this much iron is available and is required to yield enough iron-55 to provide a reliable result. Other methods have been reported for the measurement of iron-55 by liquid scintillation counting (6, 7). Of these only one (7) has resulted in a counting efficiency comparable to that reported here; the procedure of Eakins and Brown, however, is considerably more laborious and does not include a method for the simultaneous determination of stable iron. The procedure reported in this paper can be performed rapidly and simply. Samples containing up to 50 mg of stable iron may be analyzed for iron-55. However, maximum sensitivity is achieved if the sample size is chosen so as to provide from 20 to 30 mg of stable iron. EXPERIMENTAL
Apparatus. A Nuclear-Chicago Mark I liquid scintillation counter was used in these experiments. All samples were counted in 25-ml polyethylene counting vials which can be purchased from Nuclear-Chicago Corp. Reagents. DI(2-ETHYLHEXYL)PHOSPHORIC ACID (EHPA), 1.5M in toluene. Prepare 1.5M EHPA by diluting 500 ml of pure EHPA with an equal volume of toluene. Wash this solution with an equal volume of 4 M nitric acid. Next, wash the solvent for 2 minutes with 500 ml of 4 M sodium hydroxide. Draw off the aqueous phase which will contain suspended ferric hydroxide. Wash the solvent twice for 1 Present address, Prototech Co., 50 Moulton St., Cambridge, Mass. (1) M. E. Wrenn and N. Cohen, Health Phys., 13,1075 (1967). (2) L. Hallberg and H. Brise, Intern. J . Appl. Rudiarion Isotopes, 9, 100 (1960). (3) A. Nakajima, T. Yamamoto, E. Sekiguchi, S.So, and Y . Yoshitoshi, Tohoku J. Exptl. Med., 85,21 (1965). (4) H. E. Palmer and T. M. Beasley, Science, 149,431 (1965). (5) T. Jaakkola, Dept. of Radiochemistry, Helsinki Univ., Finland, Annual Report, 1965. (6) J. R. Dern and W. L. Hart, J . Lab. Clin. Med., 57, 322 (1961). (7) J. D. Eakins and D. A. Brown, Intern. J. Appl. Radiation Isotopes 17, 391 (1966).
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minute with an equal volume of saturated ammonium carbonate solution and discard the wash solution. Finally, wash the solvent for 1 minute with 500 ml of 4M nitric acid and twice with 500 ml of water. The resulting solvent will be 1.5M in EHPA and free of iron. EHPA can be purchased from the Union Carbide Chemical Co. TOLUENELIQUID SCINTILLATION COUNTINGSOLUTION. 1,4-Bis-2 -(4 - methyl - 5 -phenyloxazolyl)benzene (dimethylPOPOP) and 2,5-diphenyloxazole (PPO) were obtained from Nuclear-Chicago Corp. Naphthalene, reagent grade recrystallized from alcohol and toluene, reagent grade, were obtained from Eastman. Prepare a scintillation soliition consisting of 50 mg of POPOP, 5 grams of PPO, and 50 grams of naphthalene dissolved in toluene to a final volume of 1 liter. Calibration Procedures. To separatory funnels containing from 1 to 50 mg of stable iron as FeCb, add a known amount of carrier-free iron-55 activity (approximately 100,000 dpm). The solution volume should be 16 ml and from 0.1 to 0.5M in hydrochloric acid. Extract each sample for 15 minutes with 5 ml of 1.5M EHPA. Transfer the samples to centrifuge tubes and centrifuge for 5 minutes for complete phase separation. Pour the samples back into their respective separatory funnels and draw off as much aqueous phase as possible. Save the aqueous phase for the determination of per cent iron recovery. Return the organic phases to centrifuge tubes and add 15 ml of scintillation solution to each separatory funnel. After rinsing the funnels, add the rinse solutions to the appropriate centrifuge tubes. Centrifuge the mixture for 5 minutes and decant the organic phase into counting vials. Count the samples in a liquid scintillation counting system as described below. Obtain channels ratios and counting efficiencies and prepare calibration curves. Counting Procedure. The experiment was performed using 2 channels of a Nuclear-Chicago Mark I liquid scintillation counter. Channel A was optimized for iron-55 using a standard containing 9 mg of stable iron. The optimum calibration settings obtained using our instrument were: attenuator A, 9.85; upper discriminator, 2.5 V; lower discriminator, 0.5 V. With a 2-V window, 100% relative counting efficiency was maintained and background was 15 cpm in glass vials and 6 cpm in polyethylene vials at 0" C. Channel C was optimized for the barium-133 external standard. Using the counting system described above, a freshly prepared sample was counted repeatedly to determine the count equilibration time at 0" C for samples in polyethylene vials. On the basis of consecutive 4 minute coilnts, stability was achieved after 45 minutes of adaption to temperature and darkness. Therefore, samples should be equilibrated for at least 1 hour before counting. When counting is done in polyethylene vials, the channels ratio decreases with time. This may be due to toluene loss via diffusion through polyethylene which has the effect of increasing concentration. To achieve maximum sensitivity, however, polyethylene counting vials should be used because of their low background. Channels ratios obtained from 1 to 4 hours after placing the samples in polyethylene vials are highly reproducible. Therefore, all samples should be counted and channels ratios measured within this period. If longer counting times are required for samples of low activity, appropriate changes must be made in the calibration procedure to take this into VOL 40, NO. 1, JANUARY 1968
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0 Liquid scintillation countirg A Gar - prqportional countjng
Table I. Tabulated Results
Stable iron added, mg 1
3 6 9 12 15 15 18 20 21 24 27 30 30 33 40 50
Iron re- Iron in Counting Channels covery, scintillant, efficiency, ratio Figure of merit mg (C/A) 99.2 0.992 27.17 6.17 10.96 31.70 99.4 2.98 26.17 5.77 23.77 5.35 99.2 5.95 57.49 99.4 8.95 21.71 4.94 78.98 93.78 99.3 11.91 19.37 4.59 97.6 14.63 18.43 4.42 109.60 115.02 97.6 14.63 19.34 4.57 96.3 17.33 17.68 4.27 124.55 121.34 92.7 18.54 16.10 4.02 134.69 94.5 19.84 16.70 4.07 121.25 94.1 22.58 13.21 3.43 119.25 97.7 26.38 11.12 3.21 95.8 28.74 10.97 2.92 128.16 97.2 29.16 10.74 2.94 127.31 117.97 92.1 30.39 9.55 2.79 97.9 39.16 6.71 2.13 106.81 92.2 46.10 3.20 1.31 59.97
z
z
account. If maximum sensitivity is not required, the above difficulties can be eliminated by calibrating and counting in glass vials. In addition, channel B, which had been previously optimized for tritium, had 90% of the relative counting efficiency of channel A for iron-55 because of the nearly identical energies of the iron-55 x-ray and the average energy of the tritium beta particle. Thus, for some applications where dual calibration may be inconvenient and where maximum sensitivity is not required, iron-55 samples may be counted along with tritium samples. RESULTS AND DISCUSSION Data are presented in Table I. A straight line adequately represents the relationship between stable iron content and channels ratio as well as stable iron content and counting efficiency. Hence, the results are fitted to a straight line by the method of least squares. The figure of merit which has been plotted vs. milligrams of stable iron in Figure 1 was computed from: Figure of merit =
(mg stable Fe) (% counting efficiency) dbackground (cpm)
Figure 1 indicates that maximum sensitivity for liquid scintillation counting is obtained with samples containing 20 to 30 mg of iron. To present a more complete evaluation, liquid scintillation counting was compared to the method of proportional counting of electrodeposited iron. Samples containing from 4 to 60 mg of stable iron were electrodeposited on 2-inch planchets. Following plating, the samples were
!I
.-
+ 0
"
IO
20
30
40
50
60
mg of Stable Iron Figure 1. Figure of merit plotted cs. mg of stable iron
counted in a low background (1.7 cpm) gas-flow proportional counter. Good iron deposits were produced in less than 2 hours of electrodeposition at 1 A through continuous monitoring and control of pH, within a range of 4 to 6, during plating. For comparison with liquid scintillation counting, the figure of merit, computed as above, is shown in Figure 1. The data clearly indicate that electrodeposition is a more sensitive technique for samples containing greater than 20 mg of stable iron. This results because quenching losses for samples containing more than 20 to 30 mg of iron exceed self-absorption losses in electrodeposited samples of comparable or greater mass. These quenching losses more than compensate for the favorable geometry that is obtainable in a liquid scintillation system vis-a-vis a 2a proportional counter. If one considers the fact that the half-thickness of a 5.9 keV x-ray in iron is 7.5 mg/cm2, then a sample containing up to 80 mg could be electrodeposited on a 2-inch diameter planchet without exceeding the half-value layer. APPLICATION TO ANALYSIS OF BIOLOGICAL SAMPLES For this counting procedure to be applicable to actual samples, a pure iron fraction must be separated by the application of the appropriate chemistry. The final state of this separated iron should be as a precipitate of ferric hydroxide.
Table 11. Analysis of Equal Aliquots of Normal Human Blood
Background, Stable iron, Specific activity, Method cpm Iron-55, pCi mg pCi/mg Electrodeposition and windowless 1.92 =t 0.07 9.97 =t 0.52 4.75" 2.10 f 0.11 proportional counting 1.54 =k 0.06 11.89 f 0.50 4.95 2.40 f 0.10 Solvent extraction and liquid 6.07 + 0.17 10.75 f 0.56 5.60* 1.92 f 0.10 scintillation counting 6.07 =k 0.17 10.55 f 0.58 5.30 1.99 f 0.11 ,, Determined gravimetrically. * Determined from channels ratio. The results were corroborated by analysis by atomic absorption spectrophotometry which yielded a result of 5.5 mg.
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ANALYTICAL CHEMISTRY
Dissolve this precipitate in sufficient 0.3M hydrochloric acid to yield a final volume of 16 ml. If at this point the solution has even the slightest brown cast (due to basic iron complexes) concentrated hydrochloric acid should be added dropwise followed by stirring, until a clear straw-yellow solution is obtained. If this precaution is not taken, a brown iron species is extracted which causes severe quenching. To illustrate the applicability of this method for analyzing for iron-55 in biological materials, a blood sample was analyzed in duplicate by both this method and by electrodeposition. The results of these analyses are shown in Table 11. The electrodeposited samples were counted for 400 minutes each in a 2 P windowless proportional counter; 400-minute background counts were taken on each planchet prior to sample preparation. Each sample prepared for liquid scintillation counting was first equilibrated for 1 hour and then counted for 200 minutes. Next, each sample was automatically counted for 1 minute in the presence of the external standard for automatic computation of channels ratio. Background for the liquid scintillation counter had been previously determined for samples in the range of 1 to 15 mg of stable iron. It was found to be 6.07 f 0.17 cpm and independent of stable iron concentration. This procedure is currently being expanded to include the additional determination of iron-59 along with iron-55 and stable iron. Because iron-59 emits fairly energetic beta
particles, these events can be easily resolved in the third channel of a liquid scintillation counter. The ability to measure iron-55, iron-59, and stable iron simultaneously in the same sample would prove to be of great advantage in diagnostic iron function tests (8). It is likely that EHPA is applicable to liquid scintillation measurement of the specific activity of radionuclides other than those of iron, and this is presently under study. ACKNOWLEDGMENT
The authors thank Clifford Strehlow for his measurements of iron recovery, Naomi Harley for providing the calibrated iron-55 standard, and McDonald E. Wrenn for his informative discussions. RECEIVED for review April 12, 1967. Accepted October 6, 1967. This investigation, supported by a project grant from the United States Atomic Energy Commission, contract No. AT (30-1) 3086, is part of a core program supported by the U. S. Public Health Service, Bureau of State Services grant ES00014, and the National Cancer Institute grant CA06989. (8) W. C. Peacock, R. D. Evans, J. W. Irvine, Jr., W. M. Good, A. F. Kip, S. Weiss, and J. G. Gibson, J. Clin. Invest., 25, 605 (1946).
Rapid Gas Chromatographic Separation of Hydrocarbons over 200” C below Their Boiling Points Using Water as Liquid Phase Barry L. Karger and Arleigh Hartkopf Department of Chemistry, Northeastern University, Boston, Mass. 02115
VOLATILE SOLVENTS have not been used very often as liquid phases in gas-liquid chromatography (GLC) because they seem to contradict the very meaning of the term stationary phase. Even at room temperature, many common solvents such as water, ethanol, and benzene have vapor pressures high enough that they would be gradually removed from the column during chromatographic operation. This change in column characteristics with time need not, however, be an insurmountable problem. Kwantes and Rijnders (1) were the first to presaturate the carrier gas with solvent to overcome this depletion problem. They used a forecolumn containing the same solvent as the column being studied to presaturate the carrier gas. Since then, a number of other workers have utilized this and similar techniques (2-5). The use of volatile solvents as stationary phases in GLC merits further study. In comparison to the ill-defined poly(1) A. Kwantes and G. W. A. Rijnders in “Gas Chromatography
1958,” D. H. Desty, Ed., Butterworths, London, 1959. (2) 0. Grubner and L. Duskova, Collection Czech. Chem. Commun., 26, 3109 (1961). (3) L. H. Phifer and H. K. Plurnmer, ANAL. CHEM.,38, 1652 (1966). (4) R. E. Pecsar and J. J. Martin, Ibid.,p. 1661. (5) P. E. Barker and A. K. Hilmi, J. Gas Chromatog., 5 , 119 (1967).
meric liquid phases often employed, these simple volatile solvents should offer more controllable and understandable mechanisms of separation. Also, most solution data (including chemical reaction equilibria and kinetics) have been obtained in such solvents. This information can be put to use in separation problems, or, conversely, data obtained by GLC can augment other studies. In this paper we report the use of water as a liquid phase for the separation and rapid elution of high molecular weight n-alkanes at temperatures over 200 O C below their boiling points. EXPERIMENTAL
An F & M Model 810 gas chromatograph equipped for on-column injection and flame-ionization detection was used in this work. To eliminate the problem of solvent depletion, the carrier gas (He) was presaturated with water by inserting a thermostated fritted glass saturator between the flow controller and the injection port. Copper tubing, 1-meter or 3-meter X 0.25 inch, was packed with uncoated Chromosorb P, nonacid washed, 60/80 mesh (Johns-Manville). Water was added to the column packing by keeping the saturator 10” C above the column temperature. For steady-state operation both sections were kept at the same temperature. (Steady-state was assumed when solute retention times did not vary by more than 3 over a 12-hour period.) Average VOL 40, NO. 1, JANUARY 1968
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