LITERATURE CITED (1) C. J. Pederson and H. K. Frensdorff, Angew. Chem., lnf. Ed. Engl.. 11, 16 (1972). (2) H. K. Frensdorff, J. Am. Chem. SOC.,93,4684 (1971). (3) R. M. Izatt. J. H. Ryfting. D. P. Nelson. B. L. Hayrnore, and J. J. Christensen, Science, 164, 443 (1969). (4) K. H. Wong. G. Konizer, and J. Srnid, J. Am. Chem. SOC.,92,666 (1970).
(5) H. K. Frensdorff, J. Am. Chem. SOC.,93,600 (1971). (6) R. M. Izatt, D. P. Nelson, J. H. Ryfting. B. L. Hayrnore, and J. J. Christensen, J. Am. Chem. SOC.,93, 1619(1971). (7) J. W. Mitchell, unpublished work.
RECEIVEDfor review August 23, 1974. Accepted December 18, 1974.
Determination of Oxygen- 18 in Water Contained in Biological Samples by Charged Particle Activation R. A. Wood, K. A. Nagy, N. S. MacDonald, S. T. Wakakuwa, R. J. Beckman, and H. Kaaz Laboratory of Nuclear Medicine and Radiation Biology and Department of Radiological Sciences, University of California, Los Angeles, CA 90024
A comparator activation method is described for the determination of oxygen-18 in water, using the nuclear reaction I80(p,n)l8F ( Tjn, 110 min). A measure of the 0.511-MeV annihilation radiation from 18F yields a measure of the l80. The method is simple, requiring no chemical processing of the water other than distillation. The only competing reactions in the distilled Hzi80 are the I60(p,a)l3N and I3C(p,n)l3N ( T i n , 10 min). These interferences are eliminated by storing the samples for 2 hr before counting. This method of I8O analysis in the Hzi80 yields precise results and is comparable in accuracy to mass spectrometry. Water samples as small as 1.5 1.11 can be analyzed for l80 content using this activation technique. The primary advantage of this method of I8O analysis in water over mass analysis and other activation techniques is the simplicity of sample preparation and radioassay, permitting the use of large numbers of samples in l80tracer studies.
In studies of oxygen metabolism in various biological systems, the use of stable l8O as a tracer has become increasingly important and has created a need for rapid analytical techniques. In many of these studies the final product for lS0analysis is HzisO. The method most commonly employed in the analysis of l8O in water is mass spectrometry (1-3). However, the need to isolate the oxygen of water as a simple gas (e.g., CO or COZ) prior to mass analysis, requires complex vacuum trains as well as a complicated gas collecting apparatus. Other methods that have been used for lS0analysis in water are density measurement techniques ( 4 , 5 ) . The difficulties of sample preparation and the need for relatively large samples limit the use of this procedure in most tracer experiments. Activation analysis methods have frequently been used as a practical alternative to mass spectrometry or density measurements. In activation methods, the desired stable isotope is made to undergo a suitable nuclear reaction. The resulting nuclide is then measured and yields a measure of the parent isotope. The principal objective of this study was to develop a technique for measuring the '*O content of small samples of water ( 1 2 0 pl). Because activation is particularly suited to the analysis of small samples, we investigated the use of this method. Radioactivation methods used to determine 1 8 0 in H2180 include: the measurement of neutron emission following CY646
ANALYTICAL CHEMISTRY. VOL. 47, N O . 4 , APRIL 1975
particle bombardment (6, 7); the counting of delayed neutrons of 4.14-sec "N produced from triton bombardment of lRO(8);the measurement of charged particles emitted from the bombardment of l80with protons or deuterons (9);and by thermal neutron activation of ' 8 0 to form 29-sec (10). These methods are excellent in that they show high specificity and can measure I S 0 in very small samples. The principal limitation of many of these methods is the need for rapid sample transfer systems and specialized counting equipment. A more practical nuclear reaction for determining I8O in Hz180 is the ls0(p,n) 110-min 18F.The principal advantages of this conversion are that the relatively high capture cross section (500 mb a t 5 MeV) permits high conversion fractions in short bombardment periods, and that the long half-life of the product nuclide permits multisample analysis, resulting in a significant reduction in the cost per determination. Methods using the (p,n) reaction have been utilized to detect enriched l8O in the course of photosynthesis (II), and to determine oxide film thickness on various metal surfaces (12).In this paper the (p,n) reaction for IaO analysis is applied to the determination of lSO in HzI8O using standard comparator activation techniques (13).
EXPERIMENTAL Apparatus. The proton accelerator used was a compact 91-cm multiparticle AVF medical cyclotron obtained from Cyclotron Corporation, Berkeley, CA. It is housed a t the University of California a t Los Angeles Medical Center. The 2- and 20-pl disposable glass capillary pipets were obtained from Drummond Scientific Company and are accurate to f0.25% of stated volume. The spinning wheel target assembly used in this study was modified from an assembly used by Watson e t al. ( 1 4 ) for the measurement of cross-sections in charged particle activations. Our wheel was designed to rotate samples through the cyclotron beam a t 800 RPM in order to reduce errors due to the nonhomogeneity and drift of the beam. The wheel contained three arc slots 1 inch across (Figure 1).Radial grooves were made across the arc slots 3.75' apart to accommodate the glass capillary pipets. A second aluminum disc was made to fit over the first to hold the pipets in place during hombardment, The wheel assembly was attached to an aluminum plate that was fitted with an electric motor, pulley, and belt to rotate the target wheel. An electrically insulated Faraday cup was also attached to the aluminum plate, positioned so as to collect the cyclotron beam after it passed through the target wheel. Two target wheels were constructed: one to hold eighty-one 20-pl capillary pipets and one to hold one hundred twenty-nine 2-pl pipets. The entire assembly was mounted on the end of the cyclotron beam tube.
'
Proton Bombardment. The cyclotron used in this study produced an external beam of 22.1-MeV protons, with controllable beam currents up to 50 pA. In the bombardments, the proton energy impinging on the capillary tubes was degraded to 8.5 MeV by an aluminum absorber plate, 0.072 in. thick. This absorber plate also served as the vacuum window. The beam was collimated to one inch in diameter by the insertion of a 1-inch aluminum collimator ten inches before the absorber plate. The target wheel was designed so that the entire beam passed through the l-inch arc slats containing the glass capillary pipets. The rotating samples were bombarded with a 5-pA beam of 8.5-MeV protons for an integrated fluence of 1 pAlhr as recorded by the Faraday cup. The total integrated fluence per water specimen was approximately 2 X pA hr/cm2. Higher beam currents resulted in cavitation of the water in the tubes due to excessive heat produced. Radioassay. Samples were counted using a 50-sample capacity Nuclear Chicago gamma rotomatic counting system. The detector assembly consists of a 2- X 2-inch NaI(T1) well crystal system in conjunction with a Canberra single-channel analyzer. The counting system was set up t o connt each vial for 20 seconds. T h e counting cycle was continued until each vial was counted at least 14 times. The collected decay points of each sample extended over at least 5 half-lives of 110-min 18F. Preparation of Standards. Two standardized H P 0 samples were obtained from the mass spectrometry facility a t UCLA. One contained 0.198% lSO and the other 1.201%; these were used as comparators throughout this study. In addition, B series of 9 standard mixtures with intermediate H2lS0 concentrations were prepared by mixing weighed portions of the 1.201% and the 0.198% H2180.A portion of each solution was returned for standardization on the mass spectrometer. Our stock solutions were stored in sealed 10-em3 syringe hattles a t 5 "C. To minimize the danger of oxygen exchange with air during sample preparation, I-ml syringes were used to withdraw samples for transfer to the pipets. General Procedure. Distilled water samples are drawn into glass mieracapillary pipets (either 2- or 20-pl size) until the tube is about % full. The pipet is then flame-sealed on both ends and centrifuged to move the water t o one end and eliminate bubbles. The pipets are mounted on an aluminum disc equipped with an electric motor t o rotate the disc. This apparatus is positioned in the beam ofa. cyclotron, the disc is set spinning, and the pipets are hombarded for an appropriate time with 8.5-MeV protons. T h e pipets are recovered and washed with acetone to remove external contamination. Both ends of the pipets are broken off and placed in a counting vial, the contents of the tubes are blown out on a millipore filter in the same vial, and the tubes are rinsed 10 times with various reagents (e.g., 6N "03, methanol, and acetone). The tubes are discarded, and the vials containing the samples are counted in a gamma counting system for about 5 half-lives of ISF.The data are analyzed for half-life and corrected to a common time for all Samples and standards in that run by nonlinear least squares regression procedures. Normalized counting data from unknown samples are converted to atom % lSO hy comparison with, data from the standard solutions measured concurrently. Accurate measurement of the water volume in pipets is not necessary, hecause the cyclotron beam is collimated to irradiate a segment of constant length in the center of the pipet. Even though the water in the pipets mixed by convection and diffusion during bombardment, induced 18F activity is not affected by differences in sample volume, hecause much less than 1% of the in the sample is actually converted to 18F.Hence, the total number of '80 atoms exposed to the cyclotron beam remains essentially constant during bombardment. The pre- and postbombardment processing time for fifty samples is approximately 4 hours. Biological Sample Preparation. Two techniques were used to obtain distilled water from samples. For small whole animals (insects), tissues, or large fluid samples (0.2 ml or more), a technique similar to that described by Vaughan and Boling (15)was used. Our apparatus consisted of two test tubes with 22-mm outer diameters and 24/40 ground glass joints, and an interconnecting piece of glass tubing with male 24/40 joints on bath ends. This connector contained a 90" bend. At the angle of the bend, a smaller diameter tube led to a stopcock, so that the apparatus could he evacuated by connecting the small tube to a vacuum pump. The sample was placed in one of the test tubes, the other one being empty, and the stopcock was closed to prevent room air from entering the apparatus. The end containing the sample was frozen by immersion in liquid nitrogen, and the apparatus was evacuated for one minute with a vacuum pump. Then the empty test tube was placed in the liquid nitrogen, and the tube containing the sample was allowed to
._
, ,a.YvL z,,,y HZ1*Osamples thrniinh the h c i m nl.rZII
llyy
o ~ ~ , , , uv,_, y
."
.,.ate
the
and froze in the cold tuhe. T h e distillation process could he speeded by warming the sample with one's hand or by shining a high intensity light on it. However, excessive heat from the light caused additional substances to vaporize, as evidenced by discolored water after distillation. Samples with this kind of Contamination usually bubbled in the cyclotron beam. Careful distillation was therefore necessary for successful ' 8 0 assay. Large distilled samples were stored in break-seal glass tubes pending analysis. Small samples (ca. 10-50 pl) were distilled with a technique similar to that used by Smith (16). Common glass Pasteur pipets served as the apparatus. Capillary tubes containing the sample fluid were placed into the large portion of the Pasteur pipet, and this end was flame-sealed at the constriction by melting the glass in a gas-oxygen flame and twisting off the large end of the pipet. Initially, we tried t o flame-seal the large end outside the eonstriction, hut about half of these seals developed cracks upon cooling. T h e small end of the pipet was connected t o a tuhe leading t o the vaeuum pump, and the system was sealed by closing a stopcock positioned in the vacuum pump line. The sample was then frozen by immersion in liquid nitrogen. It was necessary to close the system before freezing the sample, because otherwise the nitrogen gas in the pipet liquified, and room air (with its unlabeled water vapor) rapidly entered the pipet to fill the void. After the sample was frozen, the vacuum pump was turned on, the stopcock was opened, and the pipet was evacuated for 15-30 seconds. With the pump still running, the small part of the pipet was sealed near the end by melting i t in a flame. T h e evacuated pipet was then placed on its side on a microscope slide warmer set a t about 50 "C to heat the sample. The small end of the pipet extended over the edge of the slide warmer and was cooled by evaporation of water from tissue paper wicks, which were draped over the small ends of the pipets. The wicks hung down into water-filled beakers. Samples were left to distill over night. Pipets were later opened by scoring and breaking the pipet near the juncture of the small and large portions. We were able ta recover more than 9 pl of distilled water from 10-pl water samples. Smith (16) did not freeze and evacuate his pipets, hut simply placed a cork in the large end and flamesealed the small end. We attempted to distil samples in this manner,hut were not able to get the distillate to condense near the tip of the small end of the pipet, even after 4 days. Nonlinear Least Squares Regression Analysis. If there are m samples with unknown atom % IRO concentrations, and there is one comparator sample in which the atom % lSO concentration is known, let y., be the counting data for the ith unknown sample (i = 1, 2,. . . , rn) in the j t h counting period ti = 1,2, be the counting data on the known comparator. The j t h counting period on the ith sample (i = 0, 1, 2, . . . , n ) is the time interval over which the j t h counting data were collected, and it is denoted by the interval (t;j"',ti,'2'). It is assumed that a t any time t, the intensity of the counts is given by the decay function
f ( t H B R , ) = Ri exp- ln(Z)t/H)
+B
(1)
where exp(z) = e', H is the half-life in seconds, B is the background in countslsec and R i is the instantaneous count rate (without regard to background) a t relative time t = 0. Therefore, the exANALYTICAL CHEMISTRY, VOL. 47. NO. 4, APRIL 1975
.
647
__ -- -- -- - - __ -__ Table I. Comparison of Atom 7% lSO Determined in Replicate H2180 by Proton Activation and Mass Spectrometric Techniques The0 retical
Mass s p e c t r o m e t q , a
h0.
N
1 2 3 4 5 6 7 8 9 10 11
0.1945 0.2035 0.2420 0.2775 0.2925 0.3850 0.4760 0.5870 0.7435 0.9495 1.1850
Proton activation,' N- 4
2
0.0353 0.0318 f 0.0381 i 0.0063 + 0.0191 i 0,0000 i 0.0000 0.0381 i 0.0571 i 0.0444 i 0.0635 i
i
*
0.1982 0.2100 0.2507 0.2814 0.3031 0.4016 0.5058 0.6113 0.8079 1.0090 1.2190
i
0.0033
+ 0.0038 i 0.0030 i 0.0052 i
0.0053
* 0.0086
* 0.0121 0.0081 0.0127 0.0149 i 0.0115 5
i i
atom 1
lgo
0.1980 0.2081 0.2485 0.2787 0.2991 0.3986 0.4995 0.5994 0.7996 1.0020 1.2010
Mean f 95% confidence interval. __ -I
...
..-
pected value of yz,,E(y,,), is given by the integral of Equation 1 between the limits ( t L , ( l ' , t L , ( * ) )
Now, for all i = 1, 2, . . . , m, R , = W,R,/W,, where W , is the atoms % lSO in the unknown sample, and U', is the atoms % l8O in the comparator. Hence, for all the samples with an unknown atom % l8O, the coefficient of the first term of Equation 2 becomes WLR,JlIln(2)W,. The equations given above are nonlinear functions of the nt t 3 unknown parameters W , (i = 1 , 2 , . . . , m ) ,R,, H, and B, and these parameters are estimated using weighted nonlinear least squares (27).
RESULTS AND DISCUSSION This method of l8O assay was developed primarily to provide an easier and more precise way of measuring the concentration of ' 8 0 in body fluids of animals, in connection with tracer studies of animal physiology (18, 19). Because water, both as vapor in air and as liquid, is ubiquitous, and because isotopically-labeled water may evaporate more slowly than unlabeled water (201, the fewer times a sample must be opened and processed before analysis, the less chance for contamination. Thus, we introduced rat blood plasma and urine directly into capillary pipets for bombardment, without any prior treatment. Unfortunately, both fluids bubbled, and the plasma proteins solidified, rendering the determinations invalid. We concluded that samples must be distilled to obtain pure water for analysis. Isotopically-labeled water (containing either tritium, deuterium, or l8O) is heavier than normal water. Thus, labeled water can be expected to have different vaporization properties. In fact, the vapor pressure of tritiated water ranges from 77% (at 26 "C) to 90% (at 76 "C) of normal water (20). This suggests that labeled water evaporates more slowly, and there should be fractional evaporation of water during distillation. T o check for any unsuspected errors and to test the extent of fractional distillation. portions of tritiated water of known activity were subjected to both partial and complete distillation in both of our apparatus. In all cases, we were unable to demonstrate any difference between the activity of the standard water and the 648
A N A L Y T I C A L C H E M I S T R Y , VOL. 47, NO. 4, A P R I L 1975
activity of the partially or completely distilled water. Thus, fractionation or leakage does not appear to introduce errors with our techniques. Before biological samples of unknown lSO content could be processed, we investigated the precision and accuracy of the activation analysis method by using standardized solutions of known lSO content. Accuracy. The data in Table I show a comparison of l80 yields from the analysis of replicate H2180 samples by mass spectrometry and proton activation. Preparatory to analysis by mass spectrometry, the oxygen of the duplicate 1 to 1.5-pl H2lS0 samples was isolated as COS using the guanidine hydrochloride method (1). In the proton activation 1 per H2IS0 sample were anamethod, four 1 5 ~ aliquants lyzed for l80.The samples run by activation were analyzed statistically by considering four large sets of data, each consisting of one for each of the eleven atom % lSO standard HzIS0 solutions. Each set was run with an independent set of comparator data. Since the estimates of unknown atom % within any one run of a nonlinear least squares are correlated, this technique was used to ensure that the sample standard deviation, and hence the given confidence intervals, would be based on four independent estimates. The 95% confidence intervals (half width) were determined from the four independent estimates of the atom % l8O as follows:
where t is the 975th percentile of the t distribution with n - 1 = 3 degrees of freedom for activation and n = 2 for mass spectrometry and s is the estimated standard deviation of the estimates of the atom % I 8 0 . Comparisons could not be made in samples 6 and 7 because of zero variance in the replicate mass spectrometer data. Comparisons between the f ISO estimates from the two analyses methods to the theoretical concentrations of lSO in the samples show that the f 180 values from proton activation are significantly different from the theoretical ' 8 0 concentrations for samples 8 and 11 a t the 95% confidence level. The 3 lSO yields from mass spectrometry are not significantly different from the theoretical l8O values in samples 1-9 and 11. However, the 3 ' 8 0 value in sample 10 from mass spectrometry is significantly different at the 95% confidence level from the corresponding theoretical lSO value. I t should be noted that both the mass spectrometry data and the proton activation data differ significantly from the theoretical atom % l80more times (essentially one time out of nine for the mass spectrometry data and exactly two times out of eleven for the activation data) than one would expect a t the 95% confidence level. For that activation method, this may be explained by any one or all of the following: 1) Because weighing methods were used to prepare the standard solution, the error associated with the theoretical concentration of lSO in the samples could be as great as 3%; 2) the estimates of the atom % l8O in the proton activation method are all based upon a comparator with a known atom % 1 8 0 , and an error that will bias the results of the statistical analysis; 3) the confidence intervals as given in Table I for the proton activation method are not independent. Therefore, as one of these intervals tends to "miss" the true value of the atoms % l80,other intervals will also tend to miss the true value. Two sample t-tests were used to compare the values obtained by the two methods. All of the eleven samples, excepting samples 1, 2, and 4, were significantly different from each other. However, the maximum difference between the means of the mass spectrometry data and the proton activation data occurs in sample number 9 with a
~
~
~
Table 11. Precisions of lSO Assays from the Analysis of 1.5- and 15-pl Aliquants of H2180 Containing 0.198 Atom % ‘SO 15-1 ahquants 1.5-111 aliquants
HalfHalf -width Set 1
0.1982 0.1949 0.2025 0.1955 0.1958 0.1997 0.1977 0.2003 0.1983 0.1997
Set 2
0.1942 0.1980 0.1938 0.1953 0.1967 0.1942 0.2034 0.1981 0.1954 0.1982
width
Set 3
Set 4
X
S
CIo.95
Set 1
Set 2
0.1977 0.1958 0.1949 0.1989 0.1957 0.1922 0.1976 0.1957 0.1937 0.2022
0.1999 0.1972 0.2019 0.1996 0.1988 0.1969 0.1991 0.1999 0.2028 0.2033
0.1975 0.1965 0.1983 0.1973 0.1967 0.1958 0.1994 0.1985 0.1976 0.2009
0.0024 0.0014 0.0046 0.0022 0.0014 0.0033 0.0027 0.0021 0.0040 0.0023
0.0038 0.0022 0.0073 0.0035 0.0022 0.0052 0.0043 0.0033 0.0067 0.0036
0.1983 0.1983 0.2005 0.1991 0.1923 0.1954 0.2000 0.1967 0.2004 0,1990
0.1983 0.1988 0.1933 0.1955 0.1966 0.1994 0.1974 0.1999 0.1954 0.1983
magnitude of 0.0644. and differences of this size do not have much meaning in a biological sense. It is interesting to note that the mean atom % lSO values as given by the proton activation method are always greater than the mean atom % I 8 0 values given by the mass spectrometer. Also, the mass spectrometer gave means which were always lower than the theoretical values, while the proton activation method yielded means which were always greater than the theoretical values. The proton activation method is dependent upon a “known” comparator value, and in this case the value was taken as 0.1980 atom % l80. However, the mass spectrometer data for sample 1 (the assumed 0.1980 atom % l 8 0 ) had a mean value of 0.1945. If 0.1945 were used in place of 0.1980 in the calculation of the atom % l80in the proton activation method, the maximum absolute differences between the means of the two methods is reduced to 0.05 with most of the differences of a magnitude of less than 0.022. The overall accuracy of the proton activation method will be no better than the calibrated accuracy of the H2I80 used as the comparator. In the case of the data presented in Table I, a small shift in the “known” value produces shifts in the estimates of the unknown atom % l8O. Precision. The data in Table I1 are 180 yields from the analysis of forty 1.5-pl and forty 15-p1 aliquants from a single H2I80 standard solution (0.198 atoms % I 8 0 ) as determined by the activation method. The 1.5-111 samples were bombarded with 8.5-MeV protons for an integrated fluence of 2 ~ A e h r Ten . additional aliquants of the same H2l8O were run with each group as comparators. For computer analysis, both groups of samples were divided into 4 sets of 10 samples each. The I8O contents of the samples within sets were determined using 2 independent sets of comparator data. To ensure that the mean l80estimates reported within each group would be based upon uncorrelated data, samples from corresponding sets within the groups were treated as replicates (taken horizontally in Table 11). The data were analyzed for differences at p = 0.05 using the t test. The 95% confidence intervals were calculated as previously described. There were no significant differences between replicates (horizontal means), between groups, or between the replicate means and the theoretical value of 0.198 atom %. A measure of the precision for the activation method can be inferred from the data reported in Tables I and 11. The maximum value of the standard deviation for the 1.5-p1 aliquants is 0.0046, while the maximum value for the 15-pl aliquants is 0.0031. The range (maximum estimate minimum estimate) of the estimates for the forty 1 . 5 - ~ ali1
Set
3
0.1976 0.1972 0.1984 0.1980 0.1953 0.1971 0.1949 0.1967 0.1993 0.1982
Set 4
0.1934 0.1964 0.1991 0.1985 0.1941 0.1982 0.1949 0.1980 0.1968 0.1996
X
0.1969 0.1977 0.1978 0.1978 0.1946 0.1975 0.1968 0.1978 0.1980 0.1988
S
CIO. 95
0.0024 0.0011 0.003 1 0.0016 0.0018 0.0016 0.0024 0.0015 0.0022 0.0006
0.0038 0.0018 0.0049 0.0025 0.0029 0.0025 0.0038 0.0024 0.0035 0.0010
quants is 0.2034 - 0.1931 = 0.0103, and the range for the forty 15-pl aliquants is 0.2004 - 0.1923 = 0.0081. Therefore, the precision as measured by the standard deviation is at most 0.0046, and as measured by the range it is at most 0.0103. Also, the estimates obtained from the 15-pl aliquants are slightly more precise than those obtained from the 1.5-pl samples. Similarly good precision has been obtained in the analysis of 3-, 5 - , and IO-pl samples. It should be noted that in Table I the precision of the estimates progressively worsens as the theoretical atom % 1 8 0 moyes further away from the “known” atom % I S 0 of the comparator. Based on the data of Table I, the experimentor, in order to assure a more precise estimate of the unknown atom % l80,should if at all possible try to keep the “known” atom % l80 value of the comparator samples within the range of the unknown atom % l80f 0.1. Selection of Target Container. We tested a number of metal and nonmetal tubings (Pt, Au, Pd, Cu, Ag, Al, polyethylene, and Tygon) before selecting glass as the target container. All the tubing, including glass, yielded relatively long-lived proton-induced radioisotopes. The direct counting of the sample contained in any of these tubings would result in a significant reduction in the sensitivity of the proton activation method. Since separation of the H2I80 is required before counting, glass i s the least objectionable because of ease of handling and because the glass capillary pipets can be obtained in sizes suitable for small samples.
CONCLUSIONS The proton activation method for the analysis of lSO in H2l80 is precise and is shown to be as accurate as similar analysis by mass spectrometry. In addition, the activation method for analysis in H2180 shows the following advantages over conventional mass analysis methods: requires only distillation of the HZ180 prior to analysis, as many as 129 (1.5-pl) or 81 (ls-pl) samples can be analyzed during any one run, and radioassay of the induced ISF can be done using simple gamma counting systems. Disadvantages in comparison to mass spectrometry are: a) the need for a proton accelerator which produces proton beams of =8 MeV, and b) the method is limited at present to the analysis of l8O in distilled water media.
ACKNOWLEDGMENT The authors thank F. B. Turner, H. Neely, R. Birdsall, and J. Takahashi for their valuable technical assistance during the course of this study and R. J. Beckman, Statistical Consultant, Los Alamos Scientific Laboratory, Los Alamos. NM. A N A L Y T I C A L CHEMISTRY, VOL. 4 7 , NO. 4 , APRIL 1975
649
LITERATURE CITED ( 1 ) P. D. Bover. D. J. Graves, C. H. Suelter and M. E. DemDsev, . . Anal. Chem., 33, 1906 (1961). (2) P. D. Boyer, A. D. Falcone and A/ B. Harrison, J. Biol. Chem., 215, 202
119551 ---, (3) A. B. Falcone. Anal. Blochem.. 2, 147 (1961). (4) I. Lander and i. R . Wilson, Aust. J. Chem.,l2, 613 (1959). (5) E. Krell. Glas. hstrum.-Tech., 5, 291 (1961). (6) S. Amiel and' P. A. Nir, Radiochem., Methods, Anal. Proc. Symp., Salzburg, Austria, 1, 293 (1965). (7) A. W. Rosenstein and A. Nir, Anal. Chem., 45, 1707 (1973). (8) S.Amiel and M. Peisach, Anal. Chem., 35, 323 (1963). (9) G. Amsei and D. Samuel, Anal. Chem., 39, 1689 (1967). (10) T. Kamemato, Nature(London), 203, 513 (1964). ( 1 1) Ingrid Fogelstrom-Finernanand Coworkers, lnt. J. Appl. Radiat. /sot., 2, 280 (1957). (12) B. Thompson, Anal. Chem., 33, 583 (1961). (13) R. Overman, H. Clark, "Radioisotope Production", McGraw-Hill, New York, NY, 1960, pp 392-5. \
(14) I. Watson, S.Waters, D. Bayley, and S. Siivester, Nucl. Instrum. Methods, 106, 231 (1973). (15) B. E. Vaughan and E. A. Boling, J. Lab. Clin. Med., 57, 159 (1961). (16) R. I. Smith, B i d . Bull., 139, 351 (1970). 117) R. H. Moore and R . K. Zeialer. LASL Ren. LA-2367 119601. il8j N. Lifson and R. McClintock, J. Theor. Biol,, 12, 46 (1966): (19) K. A. Nagy, Symposium on "Environmental Physiology of Desert Organisms", Tempe, AZ, 1974, N. F. Hadley, Ed., Dowden, Hutchison and Ross,Stroudsburg, PA, 1975. (20) L. E. Feinendegen, Tritium-labeled Molecules in Biology and Medicine", Academic Press, New York, NY, 1967, p 16.
RECEIVEDfor review July 8, 1974. Accepted December 23, 1974. These studies were supported by Contract AT (04-1) GEN-12 between the Atomic Energy Commission and the University of California.
Quantitative Multielement Analysis Using High Energy Particle Bombardment Patrick J. Clark, George F. Neal,' and Ralph 0. Allen Department of Chemisfry, University of Virginia, Charlottesville, VA 2290 7
Charged particles ranging in energy from 0.8 to 4.0 MeV are used to induce resonant nuclear reactions, Coulomb excitation (7-rays), and X-ray emission in both thick and thin targets. Quantitative analysis is possible for elements from Li to Pb in complex environmental samples, although the matrix can severely reduce the sensitivity. lt is necessary to use a comparator technique for the 7-rays while for X-rays an internal standard can be used. A USGS standard rock is analyzed for a total of 28 elements. Water samples can be analyzed either by nebulizing the sample doped with Cs or Y onto a thin Formvar film or by extracting the sample (with or without an internal standard) onto ion exchange resin which is pressed Into a pellet.
The bombardment of a target by protons or other heavy charged particles frequently results in the ejection of inner shell electrons and the subsequent emission of characteristic X-rays which can be used for qualitative and quantitative analysis. In a simple matrix as little as gram of an element can be detected ( I ) . These accelerated particles also take part in a variety of nuclear scattering and reaction processes. In the MeV energy range, the cross sections for these reactions, especially those between protons and light nuclei, exhibit sharp resonances associated with the formation of metastable compound nuclei. The narrowness of the resonances allows selective analysis of single elements in a complex matrix. By increasing the energy of the protons above the resonance energy to compensate for the energy loss with penetration depth, these resonance reactions also allow a measurement of depth distribution for the element of interest (2). Another type of nuclear excitation is due to the interaction of the Coulomb fields of the projectile and target nuclei, resulting in population of certain low-lying excited nuclear states with subsequent decay by y-ray emission (3). Present address, Department of Physics, University of Notre
Dame, South Bend, IN. 650
ANALYTiCAL CHEMISTRY, VOL. 4 7 , N O . 4, APRIL 1975
This paper will describe procedures used for elemental analysis of complex samples such as biological and geological materials by a combination of these proton excitation processes. The use of the energetically well-defined beam from a Van de Graaff accelerator allows measurement of the light elements one a t a time in the order of increasing resonance energy. The X-rays and Coulomb excited y-rays were measured at one proton energy. The problem of converting integrated peak areas for X-rays and y-rays to accurate precise elemental concentrations was an important aspect of this study.
THEORETICAL In the bombardment of a target with a charged particle beam, the probability of a nuclear or atomic reaction is expressed as a cross-section which is a function of the kinetic energy of the beam. When the electromagnetic radiation (X-rays and y-rays) emitted in these reactions is monitored, the yield ( Y = eventdunit charge) or count rate in a particular detector will depend upon the total cross section (@) = cm2/atom) for formation of the excited state a t the bombarding energy E, the product ( k = &kf) of the attenuation factors (kf)for the emitted radiation or the fraction of excited nuclear or atomic states which result in emission of the radiation being observed, the density of target nuclei ( N = particles/cm3) in the target material, the thickness (X = cm) of the target, the inverse of the ionization charge state of the particles in the beam ( I = beam particledunit charge), the efficiency ( 6 ) of the detector for the radiation energy being observed, and the fractional solid angle ( Q ) subtended by the detector. dY = ,VI& kU(E)dx
(1)
Charged particles with initial energy Eo readily lose energy in penetrating the target and emerge with energy Ef (Ef = 0 when the target is thick enough to stop the beam). This energy loss can be expressed as the areal stopping power S ( E )which is the rate of energy loss per unit of path length divided by the total target density ( p =~ Z,p, =
