Internal Standard Technique for Precise Isotopic Abundance Measurements in Thermal Ionization Mass Spectrometry SIR: K e have developed an internal standard technique which gives more than a 5-fold improvement in the precision of a n isotopic analysis made in a thermal ionization mass spectrometer. For certain problems, this type of instrument now can attain precisions comparable to those attained by simultaneous collection in gas mass spectrometers. Although the use of internal standards is well established in the field of general chemical analysis, i t has to our knowledge not been applied before in thermal ionization mass spectrometry. The technique disclosed here has application in the following fields: certain half-life determinations; geochemical age determinations; nuclear absorption cross sections and low isotopic burnup; and isotopic dilution analysis for the assay of uranium. To apply the technique, three or more isotopes of an element must be present so that two different isotopic ratios of the Same element can be compared in such a manner as to eliminate almo>t completely mass discrimination (instrument bias). The effects of mass discrimination are most completely t3liniinated when the A 3 1 mass ranges betn(>entwo different ratio pairs which are being compared are equal. The fleuibility of the internal standard technique to problems which are amenable to surfacc ionization mass spectrometry is clarified in the following discussion of its application to uranium isotope abundance measurements and to a new determination of the half-life of Cs137. Application to Uranium Isotope Abundance Measurements. I n mass spectrometric abundance measurement-. m a + discrimination tends to enhance by a small amount the measured abundxnce of a light isotope as compared n i t h a. heavy isotope of the same element. To a high degree mass di-crimination is eliminated b y using the difference between the observed and known values of a n accurately calibrated ratio of two isotopes to determine a correction factor Rhich is applied t o observed values of the ratios of other isotopes of the same element. h new correction factor is obtained for each analysis, which is made on a different thermal ionization filament.
The internal standard for uranium is a calibrated mixture of the nonnaturally occurring isotopes U233and U236 in approximately equal amounts. High isotopic purity of these isotopes is essential for accurate isotopic dilution measurements. T h e U233 plus U236 isotopic dilution spike is added to an unknown uranium sample and is mixed thoroughly before transfer to the thermal ionization filament. Since the AM's for the calibrated 233/236 ratio and unknown 235/238 ratio are equal, a n observed ratio of ratios, Ro = R51s/ R318,will almost exactly equal the true ratio of ratios, so that an unknown 235/238 ratio (relative to a standard) is measured as R5,s = ROR3/6. This result is obtained by assuming that no UZ36or UZ3*is present in the U233 plus is present UZ36spike and that no in the unknown uranium sample. 9 method has been devised by us for calibrating the 233/236 internal standard ratio against the 235/238 ratio of a uranium standard, and for correcting observed ratios of a mixture (unknown plus spike) for the presence of small amounts of P4, UZ35,and UZ3*in the spike. When U236is present in the unknown, the procedure is more complicated. I n this case, separate spiked and unspiked analyses must be made of the unknown uranium sample. We are proceeding with an evaluation of the precision of the method for uranium analysis. Measurements are being made with a two-stage mass spectrometer which has been described (6). A more general analysis of the internal standard technique will be presented for publication with the uranium results. The method is applicablp to the precise measurement of plutonium isotopic abundances by using the long-lived isotopes Pu242and Pu244as an internal standard. Application to Cs13' Half-Life Determination. I n view of the considerable disagreement among published values of the half-life of C S ' ~ i~t .is desirable to have an entirely independent method which is not subject to chemical weighing errors, stoichiometry, mass spectrometric isotopic dilution errors, or to instrument bias. We believe that, to a very high degree,
the method we have devised depends only on the precision of measuring a number ( R value) which is proportional to the decay of Cs137a t any given time during the experiment. Thus, the halflife determination is reduced to a precise relative measurement of the time rate of decay of Cs137. The precision required for this type of direct Ineasurement until noJT has been unattainable in thermal ionization mass spectrometry. The isotopic mixture which is being used for the half-life determination of Cs137 contained at time zero an approximately 1:1:1 proportion of the cesium isotopes 133, 135, and 137. Cesium-133 is stable and CsI35 has a 2 X IO6 years half life, so that for the purposes of this experiment it also can be considered as stable. An R value which reflects the decay of the intermediate-lived isotope Cs137 is defined by an observed ratio of ratios, 137,435 + 138/133. The invaria n t 135/133 ratio is a n internal standard which corrects for instrument bias. It is not necessary to know the absolute values of the ratios 136/133 and 137/135 a t time zero or at any other time during the experiment, Sufficient data are taken to ensure a precision of approximately &0.05YG( l a ) for a single determination of a n R value. TJ7e have been measuring the decay of Cs137 for approximately 1 year. Our preliminary value for the half-life of Cs137 is in good agreement with Farrar, Dasgupta, and Tomlinson (4),who measured the half life of this isotope as tl/z = 30.4 i: 0.4 year. Before publishing our value we need to obtain one or two more groups of points to reduce our precision error in the half life. One might argue that the twhnique could be unreliable because Csl3' 8decays into Ba137 and that these two masses interfere in the mass spectrometer. This is not a valid objection, as we observe with the V-filament (1) that cesium emits at about 700" C. and barium emits at about 1200' C. For example, a preliminary measurement of the half-life of Cs134, which decays to Ba134, agrees well with the published value (3); barium interference mould have resulted in too high a value. The longer one measures a half-life b y the internal standard technique, the more accurately known i t becomes. For VOL 34, NO. 6, MAY 1962
709
this reason we intend to continue gathering data on the half-lives of Csi37 and for several more years. ,1 complete report nil1 be made in a few months time.
to J. I,. Mewherter for preparing the uranium and cesium samples; and to J. E. Noonan for assistance in obtaining data. The ccsium samples were obtained from the Oak Ridge Sational Laboratory.
ACKNOWLEDGMENT
K c are indebted to IT.D. Davis, who first proposed the U233 plus L-236 internal standard several years ago; to 11. H. Studier at the AirgonneSational Laboratory for providing thc C'36 isotope;
LITERATURE CITED
(1) Dietz, L -4, KeL. Scz T r i s t , 30, 235 (1959). ( 2 ) Dietz, L .I., Pachucki, C. F., Shefield, J. C., Hance, A , R., Hanrahan, L R., .kNAL. CHEZI. 32, 1276 (1'360).
( 3 ) Easterday, H. T., Smith, 11. L., Xuclear Phys. 20, 155 (1960). (4)Farrar, H., Dasgupta, A. K., TomlinR , H,, J . them, 39, 681 i1961) L. A. I ~ I E T Z C. F. PACHUCKI G. A. LASI) Knolls Atomic Po\\ er Lkiboratory General Electric Co.
Schenectady, S . T. The Knolls ;itonnc Poner Laboratory is operated bj- the Chiera1 Nectric Co. for the U. d. Atomlc Energy Commission. RECEIVEDfor review l l a r c h 1, 1962. -4ccepted March 20, 1962.
Sulfuric Acid as a Reagent for the Spectrophotometric Determination of 20-Methylcholanthrene SIR: This study was conducted because of a need for a aimple, conclusive method for the deterniination of 20inethylcholanthrene (1ICA) in body tissue. Methods currently employed are hindered greatly by difficulty in application (2-4, 8-10, I S ) , or, as in the case of the nidely used spectrophotometric method ( 6 ) , by interference of other substances which are normally present. The use of concentrated sulfuric acid as a test for steroids is well known (5,12, 14). Bandow observed that sulfuric acid reacts IT ith 20-methylcholanthrene ( I ) , but the reagent has never been employed for the spectrophotometric determination of 1ICA. We have found that the addition of concentrated sulfuric acid to 20-methylcholanthrene in solution with benzene or hexane, or in solid form, causes the formation of a red complex. Concentrations greater than 1 X mg. of J I C A per cubic centimeter of solvent can be quantitatively determined. The complex is visible in concentrations greater than 1X mg. of N C A per cubic centimeter of solvent, and has characteristic
Table 1.
absorption curves in the visible und ultraviolet regions of the spectrum. The procedure described here utilizes this reaction, and any additional operations, such as those involving fractionation (7, I I ) , are eliminated. EXPERIMENTAL
Apparatus and Reagents. Cary Model 11 Recording spectrophotometer, n i t h 1.0-em. quartz cells. Becknian Xodel B spectrophotonieter. 20-1LIethylcholanthrene (n1.p. 178' to 180' C.), Distillation Products Industries, Rochester, S . Y., and blann Biochemicals, Kew York, S.Y. Sulfuric acid, reagent grade. Benzene, reagent grade, thiophciic free. Hexane, reagent grade. Procedure. 20-Met hylcholant hreiie was dissolved in hexane or benzene in a concentration of 1 X lo-' mg per cubic centimeter, and an equal volume of concentrated sulfuric acid was then added. The mixture was shaken vigorously for 2 to 3 minutes with occasional niiaing of the solvcnt and acid layers by pipet to cnsure completeness of the reaction. (-1 more simplified
Colorimetric Reactions of Compounds Tested With Sulfuric Acid and with Subsequent Addition of Water Reaction with Reaction upon Compound Classa Sulfuric Acid Addition of \Tater Light green solution Sone 3,4 Benzopyrene C Degradation* Sone 1,2,5,6-l)ibenzanthracene C Orange solutionc Sone Deoxycholic acid d Yellow-green solution Reddish black solution Estradiol S Degradation Sone Cortisone acetate 8 No reaction S o reaction Diethylstibrsterol S Yellow solution Sone Estriol S Light orange colorc Sone Estrone 8 a C = Carcinogen, S = Steroid S o reaction imniediatelr observed. HoiT-ever, upon standing overnight degradation n as observed. Orange color visibly distinguishable from that of the methylcholanthrene-sulfuric acid complex. Also spectrophotometric absorption peaks differ and no reaction occurs upon addition of water as does the methylcholanthrene complex.
710
ANALYTICAL CHEMISTRY
technique usctl in rccciit cspcriincnt.: involvcd lioiiiogc.iiizat,ioii of acid :uicl solvent layers using :t niotor-driven honiogeiiization tube. 'I'hc1 organic solvent layer \vxs rcnin\ul by a pipct. The acid layer was dilutcd to si coiiccntration of 4 X lo-' mg. y r cubic centimeter and the characteristic absorption spectrum was deteriiiinetl in t,he range of 3750 to GOO0 A. I n thc ultraviolet region, absorption spectra of concentrations of 1 x 10-l ant1 3 x 10-2 nig. per cubic centimeter of benzene and hexane werc detcmiined in the range of 270 to 350 nip. For quantitative determinations, absorbances a t 4170 -\.\wre nieasured on solutions of 1 X IO-l, 1 X 1x 1X sncl 1 X lo-" ing. per cubic cc~iit~imcter. To test the procc~iurcon biological tissue, tcn young female niicc (Swiss Webst,er aiid ICR Strains, Charles River Farms) were c w h given one intravenous injectioii of 0.5 !ng. of 20-methylcholanthreiie suspension in saline solution. Fi\-cx mice were sacrificed one day aftc'r injection aiid the other five after two days. Extracts were made of the lungs, liver, kidneys, and brain by honiogcnizing the respective organs in 5 nil. of bcnzenc for all1)roximately 2 to 3 iiiinutes and ccntrifuging a t 4000 r.1i.m. for 10 niinutrs to remove all solid matter. The ultraviolet spectra of the benzene solutions were then determined. (Spcctrophotometric determinations \yere performed on several of the extracts. Several were tested solely by the observation of the red color.) Thc same procedure was carried out on ten controls not 6' riven the injection. RESULTS AND DISCUSSION
,4 characteristic absorption curve \\-as obtained with peaks a t 5150 and 4170 -4. The molar absorptivities were, respectively, 4960 and 12570 in benzene solution. I n the ultraviolet region, the spectrophotometric curves obtained with benzene and hexane solutions showed
