the size of the ortho-alkyl substituents parallels changes in other constants derived from the same structural differences, as may be seen from the data in Table IV. Again, only limited comparable data are available. As to the application of the HFA technique to quantitative analysis, it can be concluded that although it is applicable to most phenols, caution should be exercised in systems where there is a bulky ortho substituent or a strong electronegative group at the meta or para position. In such systems, the equilibrium does not lie too far to the right. This, however, can be partially overcome by the
presence of a great excess of free HFA. For example, to reach 99% reaction in a substituted phenol whose K with HFA is 100,a 1.0 molar excess of HFA should be present. ACKNOWLEDGMENT
The author thanks George A. Ward and Robert D. Mair for many helpful discussions, and Robert R. Kohler for experimental assistance. Received for review May 16, 1973. Accepted November 16, 1973. Hercules Research Center Contribution No. 1614.
Elemental Analysis of Whole Blood Using Proton-Induced X-Ray Emmission R. C. Bearse,' D. A. Close, J. J. Malanify, and C. J. Umbarger Los Alamos Scientific Laboratory of the University of California, Los Alamos, N.M. 87544
A technique for the analysis of trace elements in whole blood has been developed using proton-induced X-ray emission. Samples of 0.1 ml whole blood from humans and mice were dried, weighed, and then ashed in a plasma asher. Targets were prepared by placing drops made from the ash and a 400-ppm Pd solution onto Formvar backings. The samples were irradiated with 2.25-MeV protons, and the X-rays analyzed in a nondispersive X-ray detector. The elements Fe, Cu, Zn, Se, and Rb were detected with a precision of 7, 18, 7, 50, and 19%, respectively, in human whole blood. The precision of the technique was determined by statistical analyses of two different sets of 27 samples. The system was shown to be linear for variations in elemental concentration. The accuracy for determination of Zn was found to be within 1 0 % by comparison to atomic absorption spectrometry.
Although the study of trace elements in biological systems has been pursued for many years ( I ) , the increased awareness of trace metal toxicity has created a new interest in improved measuring techniques. The recent introduction of high-resolution nondispersive X-ray detectors has spurred a great deal of activity in X-ray fluorescence trace analysis. Several groups (2-4) have reported that proton-induced X-ray emission has great potential in trace analysis because many elements can be detected simultaneously and only small samples (-100 mg) are needed. Most of these papers, however, have been of a general and exploratory nature, and few deal with the details of a specific system for making measurements of a particular sample type. Most notably, previous workers seem to have relied on counting statistics as a measurement of precision, an approach that we have eschewed.
'
Visiting Staff Member from the University of Kansas, Lawrence, Kan. 66045 ( 1 ) E. J . Underwood, "Trace Elements in Human and Animal Nutrition." 3rd ed.. Academic Press, New York, N . Y . , 1971. ( 2 ) T. B. Johansson. R. Akselsson, and S. A . E. Johansson, NucI. Instrum. Methods, 84, 141 ( 1 9 7 0 ) . ( 3 ) F. C. Young, M . L. Roush, and P. G . Bergman, Int. J . AppI. Radiat. Isotop., 24. 153 (1973) ( 4 ) C. J . Umbarger, R. C. Bearse, D. A. Close, and J . J . Malanify, Advan. X - R a y A n a l . , 16, 102 ( 1 9 7 3 ) .
The purpose of this paper is to present details of a method of measuring trace elements in small volumes (0.1 ml) of whole blood using proton-induced X-ray emission. Blood was chosen because it is a physiologically important fluid that is easily sampled. We will discuss the basic principles of the technique, the blood sampling and preparation techniques, the apparatus used in analyzing the samples, the preparation of the proton beam, and the precision and accuracy of our final procedure. The principles of X-ray emission are simple. A heavyion beam, in passing through a thin sample, removes inner shell electrons with extremely high probability. The filling of these vacancies by outer shell electrons produces X-rays whose energies are characteristic of the element in which it is produced, and the number of X-rays is proportional to the number of atoms of that element present in the sample. Since the yield of X-rays per atom is a smoothly varying function of atomic number and bomand since modern X-ray detectors are barding energy (j), capable of resolving K a X-rays from the elements adjacent in the periodic table (for 2 > l l ) ,many elements can be measured simultaneously. Because of the multiplicity of X-ray lines ( e . g . KP) from each element and because of the possibility that some elements may be several orders of magnitude more prevalent than others, it may not always be possible to measure the presence of all elements without some prior chemical separation. EXPERIMENTAL Sample Preparation. Capillary pipets. rinsed in heparin and air-dried, were used to draw 0.1-ml samples of whole blood from the sinus cavities of mice. Human blood samples were drawn into 5-ml syringes, potassium oxalate was added, and the samples were repipetted with a n Oxford 0.1-ml autopipet. The samples were pipetted directly into I-ml borosilicate glass beakers that had been previously weighed on a Torbal balance to a n accuracy of 0.2 mg. About 30 samples were processed a t one time. The filled beakers were arranged on a 7.6- by 15.2-cm borosilicate glass plate on the base of a bell-jar system. and all beakers were repeatedly filled with liquid nitrogen until the blood was thoroughly frozen. With the beakers still containing liquid nitrogen, the bell jar was sealed and the system evacuated. The system was pumped for several hours, and a n ultimate vacuum of about 0.06 (5) R . C . Bearse, D. A . Close, J . J . Malanify, and C. J. Umbarger. Phys.
Rev.. 170, 1269 ( 1 9 7 3 ) .
ANALYTICAL CHEMISTRY, VOL. 46, NO. 4 , APRIL 1974
499
Analyzing Magnet PDP-9lL 3ata Acquisltlon 8 Reductlon
1
Figure 1. Equipment schematic for proton-induced X-ray emission
Torr was achieved. The beakers were then removed from the bell jar and reweighed to obtain the dry weight of the whole blood. The weight reduction from wet to dry blood was about a factor of 4.8. The samples were placed in a Tracerlab LTA-302 asher on a 7.6- by 15.2-cm borosilicate glass plate which allowed the beakers to be on the diameter of the ashing chamber. The samples were ashed for 48-72 hours a t a power of 100 watts, a pressure of 0.9 Torr, and a n oxygen flow of 150-cm3/min. The purpose of ashing is to increase the concentration of the elements with 2 L 26 by removal of the elements H, C, N.and 0 which comprise the bulk of the blood mass. Targets with a higher concentration of trace elements can then be fabricated with a much smaller total mass. This decrease in mass reduces the amount of background due to secondary electron bremsstrahlung ( 4 ) , thus improving the detectability limit. It has been shown that plasma ashing is the method of choice to keep metal losses to a minimum (6). After ashing, the samples were removed from the chamber, and 0.1 ml of a solution of 6 parts 1.870 HC1 and 4 parts of 1000 ppm PdCI2 (for normalization purposes) was added to each sample using the autopipet. A new disposable tip was used for each sample, and dissolution was effected by rapid inhalation and expiration of the solution. using the pipet. Occasionally, small black specks appeared in the solution and, so long a s they were few and small, they did not seem to affect the accuracy of the analysis. A 0.02-ml autopipet was used to transfer t h a t amount of the sample to the surface of a Formvar foil (7) on which a still wet 0.02-ml drop of 3% "4OH solution had been placed. The ",OH neutralized the acidic blood solution which would otherwise have a t tacked the foil. The samples dried on the foils within a diameter of 5-6 m m . They were stored and transported in a desiccator and then transferred to the Los Alamos Scientific Laboratory (LASL) trace-analysis chamber (8) which had been modified to hold six 2.5- by 5-cm aluminum frames with 1.9-cm diameter holes. Larger frame holes were used so that the target and beam area could be increased to a reasonable and practical size (governed by the size to which the liquid drops dried) without stray beam striking the frame. Even a beam lo3 times smaller than the primary beam would cause an appreciable background spectrum upon hitting the frame because of the effectively infinite thickness of the frame. Formvar foils were used in this work because they were relatively easy to prepare in quantity, withstood the required beam intensity, and were tough enough to endure the abuse of target preparation and subsequent handling. Another important reason for using Formvar was that it can be made very thin. thus contributing insignificantly to the secondary electron bremsstrahlung continuum. A Formvar solution ( 7 ) was prepared which consisted of 16 grams of Formvar resin 15195E (Monsanto Polymers and Petrochemicals Co.). 200 ml of methyl benzoate, 480 ml of toluene, and 320 ml of ethanol. The foils were made by placing a drop of this solution onto the surface of doubly distilled water. When the edges of the film began to wrinkle. it was picked up onto one of the aluminum frames, with care t h a t double layers were not formed. These films were measured, by weighing and by the ener( 6 ) C. E . Gleit and W. D. Holland, Anal. Chern., 34, 1454 (1962). ( 7 ) R. J. Grader. R . W. Hill, C . W . McGoff, D. S. Saimi. and J. P. Stoering, Rev. Sci. instrum.. 42, 465 (1971). ( 8 ) E. J. Feldl and C. J, Urnbarger, Nuci. Instrum. Methods. 103, 341 (1972).
500
A N A L Y T I C A L C H E M I S T R Y , VOL. 46, N O . 4, APRIL 1974
-1
L 2>
33
35
SC
45
Atomic Number
Figure 2. System detection efficiency with and without a titaniu m absorber gy loss of alpha particles (9). to be about 10 kg/cm2. The foils were prepared ahead of time. allowed to dry. and stored in a desiccator containing C a s 0 4 desiccant. Analysis Apparatus. The samples were bombarded with a 150-nA beam of protons from the LASL 3.i5-MV Van de Graaff. The beam was spread over a 1-cm by 1-cm area centered on the frame hole. Beam collimation was achieved by four sets of slits and a collimator as shown in Figure 1. It is important to note that the final beam definition was performed by carbon slits only 12 cm from the target. This reduced the stray beam without the production of hard X-ray background to which the detector would be sensitive. The X-rays were detected by a n Ortec Si(Li) detector having a resolution of 175 eV at 5.9 keV. In reaching the detector the X rays passed through a 0.013-mm Mylar foil in the X-ray chamber, a 0.025-mm sheet of Ti. a 0.71-mm Lucite absorber, and the 0.025-mm Be window of the detector. The Ti and Lucite were necessary to preferentially attenuate the large numbers of Fe Xrays produced in the whole blood samples. The efficiency of the detector relative to Pd was determined by preparing targets from known mixtures of various elements (Harleco. Inc.). The efficiency curve is shown in Figure 2. The pulses from the detector were amplified by a n Ortec 460 amplifier. set for 3-bsec shaping time. and sent to a Northern Scientific NS 624 ADC. The ADC fed the LASL A-1 PDP-S/L (10) computer system which acted as both analyzer and data reducer, Spectra of 1024 channels were accumulated such that the KB Xrays from Pd were just on scale. An example is shown in Figure 3. The yields of X-rays were determined by a computer program which added all channels in the region of interest and performed a linear interpolation subtraction for background. This program suffers in that it is necessary to have peaks completely separated to effect accurate analysis. This was a drawback only for the Cu determinations where the K n from Cu. which is weak. was very close t o the much stronger Zn Kru transition. The correction to the Zn due to the Cu interference was insignificant because of the smallness of the Cu intensity. The computer, which accepted data for a predetermined livetime. usually 500 seconds. also controlled two scalers. One recorded the beam current as measured with a n Ortec 439 Current Digitizer. and the second kept realtime in hundredths of a second. The second scaler allowed easy determination of system deadtime which was typically 2-370. The targets were prepared in such a way that the final areal density was between 0.1 and 1 mg/cm2. In this mass range, there is enough material to produce a sufficient counting rate for rapid (9) M . C. High, University of Kansas, Lawrence, Kan., private communication, 1973. ( 1 0 ) L . V . East, Proceedings of the 1973 Spring DECUS Symposium. Philadeiphia. Pa
3L
I
4
3
-
I
2
-
1
1
2
2
Beom Posltlon i m m
0
256
512
76 8
3
I
IO24
Channel
Figure 4. Blood target profile measurements showing relative Fe yield as well as Fe/Zn ratio as a functlon of position
Figure 3. Typical proton-induced X-ray spectrum for mouse blood analysis; yet the material is thin enough to make self-absorption corrections insignificant and to keep beam heating small enough to prevent target sublimation. Beam Preparation. It was expected that since the targets were dried from solution, they would be neither uniform nor homogeneous. T o investigate this point, the beam was defined to a 1- by 1-mm spot, using the final carbon slits. The target was then moved through this beam in 1-mm steps, and the emitted X-rays were analyzed. T h e concentration of Fe a n d the ratio of Fe to Zn a t each point across the target are shown for one such sample in Figure 4. It can be seen that the Fe concentration of the sample varies considerably over the width of the target. while the Fe/Zn ratio is more stable. Since the X-ray yield is proportional to t h e product of beam intensity and target thickness, and since the target is not uniform, it is necessary to make the beam uniform across the target area. Several schemes are available to accomplish this, and we investigated two. We attempted to homogenize the beam by passing it through a 0.0013-mm Ni foil and then accepting only part of the scattered beam on our 1- by 1-cm target area. T o get sufficient target currents we had to use a primary beam of about 2 PA, and this overheated the Xi foil, causing pinholes to develop. In addition, the amount of homogenization was not sufficient. The method finally adopted was a simple defocusing of the beam. A 6-pA beam was focused through the analyzing magnet and immediately intercepted in a beam cup (see Figure 1) and measured. T h e beam was then defocused within the accelerator so that the beam cup measured only 500 nA, the cup was removed, and the defocused beam was allowed to travel -8 meters to reach the target chamber. Typically, of the 500 nA in the cup, 200 nA reached the target area where it passed through the target and was collected in a n electrically insulated tube beyond the target chamber proper. This amount of beam is of optimum intensity, because it allows reasonable d a t a accumulation rates without deterioration of the samples. At beam currents above 300 nA, about 50% of the samples would be destroyed by the beam. To measure the beam profile, a 0.5-mm square Ni chip, 0.0005 m m thick, was placed on a Mylar foil in the center of the target frame. The chip was then moved through the beam in I - m m steps, care being taken to keep the beam from hitting the frame. The number of Ni X-rays is a direct measure of the beam intensity a t the Ni chip. The result of this measurement is shown in F'igure 5. It can be seen that, at least in the horizontal dimension, the beam is uniform to better than 10% over the 6-mm target diameter. T h e result obtained with the Xi diffuser is shown for comparison. It was assumed t h a t the vertical profiles were similar. Because of the techniques used for beam preparation, such a n assumption is justified. These results, taken with the information about target nonuniformity, imply that a limit of precision of a few per cent over and above counting statistics should be attainable with the defocused heam. Another source of variation in our results is due to the T i absorber. The X-rays arising from target positions a few millimeters off the central axis must, because of the shallower angle of passage through the Ti, traverse more attenuating material. For our absorber. and a n extreme case of 6 m m from the central axis, the absorption of Fe X-rays is 7% higher t h a n those X-rays passing
120,
a
I
I
-3
-2
I
I
I
I
I
3
4
20
-4
-I 0 I Position ( m m )
2
Figure 5. Beam intensity profile showing results for defocused as well as diffused beam perpendicular to the Ti. Since no two targets are exactly alikeLe., t h e mass distribution and centering are not the same-variations in the measured sample quantity of up to a few per cent can be expected to occur.
RESULTS AND DISCUSSION
To determine the precision of our method, two series of measurements were made. In the first, a 4-ml sample of oxalated whole human blood was repipetted into 27 beakers and targets were prepared as described above. The blood had 0.1 ml of potassium oxalate solution added to each 1 ml of whole blood. A subsequent measurement of the oxalate solution alone indicated a small Zn contamination which contributed less than 1% to the final Zn yield. Each sample was exposed to -150 nA of beam for 500 seconds of analyzer livetime. Several elements were readily seen, among them Fe, Cu, Zn, Se, and Rb, in addition to the Pd spike. Since the amount of Pd in the sample relative to the original blood weight (dry) is known, as is the efficiency of the detector system relative to Pd, it is a simple matter to determine the amount of each element present. The ppm of each element (dry weight) is simply 40N
PEW where E = efficiency of detection relative to Pd for X-ray of interest, W = dry weight of sample, N = number of counts in peak of interest, and P = number of counts in P d peak. The factor of 40 arises because 0.1 mg of the 400-ppm Pd spiking solution is used. The amount of material relaANALYTICAL CHEMISTRY, VOL. 46, NO. 4, APRIL 1974
501
Table I. Average Values and Relative Standard Deviations of Five Trace Elements Detected in Whole Blood Fe
Cu
Zn
Se
Rb
384 7%
1.2 18%
6.4 7%
0.2 50%
0.9 19%
394 5%
0.4 51%
4.0 10%
0.3 31%
3.3 14%
27 Human samples
Av, ppm Re1 std dev 27 Mouse samples Av, ppm Re1 std dev
Table 11. Comparison of the Results Obtained with Proton-Induced X-Ray Emission and with Atomic Absorption Spectrometry as well as with the Average Values of Anspaugh et aLa Concentration in park p e r million (wet weight) Fe
1
i0c
d
400
600
I r o n Added ( p p m )
Figure 6. Detection linearity showing Fe, Z n , and R b response as a function of iron concentration
SampleNo. 1 Sample No. 2 SampleNo. 3 Sample No. 4 Expected"
434 455 453 460 39OC
Cu
Zn
Znb
Se
Rb
0.77 0.72 0.59 0.51 1.00
5.9 5.5 6.8 6.8 7.4
5.1 5.8 8.4 7.2 7.4
0.09 0.25 0.25 (0.09 0.18
1.2 1.4 1.6 1.7 1.5
"Average values from Anspaugh et al., Reference 11. *Results from atomic absorption spectrometry, Reference 12. cSea level value. At altitude of 2.2 km, blood contains 10-15% more hemoglobin; hence, Fe concentration is expected to be higher.
F
25
30
35
I 40
45
Atomic Number
Figure 7. Proton-induced X-ray emission detectability in whole blood (wet weight) as a function of atomic number. The curve corresponds to the results for a 150-nA proton beam bombardment for 500 seconds tive t o the wet weight can be determined from the average dry weight for all the 0.1-ml samples, assuming that indeed the average of all pipettings is 0.1 ml. By making the measurements relative to Pd, it is then unnecessary to know the total mass present in the target, a difficult determination. The peaks from Ca, K, and Ti (the last due to secondary fluorescence in the absorber) were not analyzed as they were very nonreproducible. The absolute amounts of Fe, Cu, Zn, Se, and Rb in the 27 human blood samples (wet weight) were determined and are shown in Table I. A statistical analysis was performed on these measurements, and the results are also shown in Table I. A second test was run, using samples from 27 different mice that were from a line inbred for over 60 generations. A typical spectrum from one such sample has been shown in Figure 3. The average values and the relative standard deviations of these values are included in Table I. T o determine the linearity of the technique with variation in elemental concentration, a set of nine identical blood samples was ashed and then spiked with a successively more dilute Fe-Pd solution. Targets were prepared and run in the usual way. Figure 6 shows the amount of Fe, Zn, and Rb detected as a function of the Fe concentration added to the samples. The results for Zn and Rb 502
ANALYTICAL CHEMISTRY, VOL. 46, NO. 4 , APRIL 1974
are consistent with their fixed concentration despite a widely varying Fe concentration. The Fe results are consistent with linearity. A least squares fit to the Fe data, assuming a quadratic function, was never more than 5% different than the fit obtained assuming linearity. The accuracy of the technique is primarily determined by the accuracy with which the Pd spiking solution is prepared and by the accuracy of the efficiency curve. The spiking solution was compared with several independently prepared Pd solutions and was a t the expected strength to within 6%. The efficiency curve was measured on several occasions using different targets, and all determinations agreed to within 7%. A further error arises if the self-absorption in the blood targets is both large and significantly larger than the self-absorption in the targets used to determine the efficiency curve. Our estimates of self-absorption effects rule this out. This last effect would decrease the measured amount of material in the blood. The first two effects may be in either direction. In Table 11, we compare our results with those of Anspaugh et al. (11). Considering that our blood samples were taken from subjec,ts living a t an altitude of 2 . 2 km, agreement is quite good. As a further eheck on the accuracy of the technique, five aliquots of four different human blood samples were analyzed for Zn content and were compared to results obtained by atomic absorption spectrometry (AAS) (12). The results are shown in Table 11. Since our expected systematic error is lo%, the agreement is reasonable. Although we are able to see only five elements, these are normal samples, and screening for abnormalities would be an important use of the technique (screening, for instance, for Cd or other industrial health hazards). Before this work can be undertaken, we must determine if (11) L. R . Anspaugh, W. L . Robison. W. H. Martin, and 0. A. Lowe, "Compilation of Published Information on Elemental Concentrations in Human Organs in Both Normal and Diseased States," UCRL 51013 (1971). (12) P. C. Stein, Los Alamos Scientific Laboratory, private communication. 1973.
toxic levels are detectable. The limit of detectability is generally assumed to be three times the standard deviation of the background. Using our results, one can infer the detectability limits as a function of atomic number, and these results are shown in Figure 7. By increasing the bombarding time by a factor of four, the detectability limit will be improved by a factor of two. An important facet of any measuring technique is the time needed for analysis. The cleaning and weighing of the beakers (twice) takes about 1 man-hour for 30 samples. The ashing time is about 72 hours; but although we worked with 30 samples a t a time, t h e asher would easily handle 100 samples, and larger ashers are available. Further. the asher loading time was only a few minutes. The Formvar backings could be produced quite rapidly. One man-hour per 60 foils is easily achieved. The production of targets from the ash takes about 2 minutes apiece, not counting the drying time of about 3 hours. The largest expenditure of time per sample was in the actual beam irradiation. We used bombardment times of 500 seconds. Although the beam cannot be increased without endangering the samples, decreasing the accumulation time by a factor of two would increase the relative standard deviations for Fe. Zn, and Rb to about 7, 7, and 2770, respectively. The precision of the Fe and Zn measurements are limited by target inhomogeneities and beam profile variations; the Rb results are primarily determined by counting statistics. It seems, then, that one person could perform a t least 40 assays per %hour day under production conditions.
CONCLUSIONS This work shows that proton-induced X-ray emission analysis of whole blood samples is capable of precision and accuracy. The spectrum is not particularly rich because of the dominance of the Fe in the spectrum and the necessity of its strong attenuation which also attenuates other heavier elements but not as severely. It is reasonable to assume that developmental efforts similar to this one for measurements on other specific samples should meet with a t least equal success. ACKNOWLEDGMENT We are deeply indebted to a number of people whose enthusiastic cooperation made this work possible. We particularly thank Robert Keepin for his continued interest and support of this work. We thank Evan Campbell for several stimulating discussions and Pat Stein for the comparison measurements using AAS. Jean Lindsey cheerfully supplied many gallons of redistilled water. We thank Marty Holland and Jerry London for providing the mouse samples, and Joe Tafoya for providing the human blood samples. We also tender our appreciation to Colleen Burns for sample and foil preparation and to Ed Adams for his efficient Van de Graaff operation. One of us (R.C.B.) wishes to thank the staff of the Los Alamos Scientific Laboratory for their generous hospitality. Received for review August 10, 1973. Accepted November 15, 1973. Work performed under the auspices of the U.S. Atomic Energy Commission.
Substoichiometric Extraction of Cations with Mixtures of Hexafluoroacetylacetone and Tri-n-Octylphosphine Oxide in Cyclohexane J. W . Mitchell Bell Laboratories, Murray H i / / . N . J . 07974
Roland Ganges Stanford University. Stanford. Calif.
A new method has been developed for substoichiornetric extraction of cations by adduct-reactions in the presence of excess hexafluoroacetylacetone (H HFA) and of substoichiometric quantities of the neutral donor, tri-n-octylphosphine oxide (TOPO), in cyclohexane. The equilibria of adduct-extraction systems are discussed to show advantages over the conventional approach of reacting cations with substoichiometric amounts of chelating ligands. Distribution curves for the substoichiornetric extraction of Coz', C u z - , F e z + , M n " , Z n 2 - , F e 3 + , E u 3 r , and Lu3+, have been measured, and experimental results with relative standard deviations of 0.92, 1.8, and 3.1% tor the substoichiometric isolation of Z n z + , Cu", and Eu3+ are reported. The general utility of this extraction system for substoichiometric separation of cations following neutron activation is discussed.
Recently the utility of methods of separation in nuclear analyses has been enhanced by the development of sub-
st'oichiometric methods, which extend the application of radioisotope dilution to the determination of trace elements and also make it possible to perform quantitative neutron activation analyses without measurements of radiochemical yield. Such methods have been developed rapidly and applied frequently since Ruzicka and Stary first summarized their techniques in 1968 (1).In a second review, these authors discussed methods published during the period 1968 to 1970 ( 2 ) . Since this review, several additional solvent extraction methods have been reported (3-16). A general discussion of substoichiometric methods ( 1 ) J Ruzicka and J. Stary, "Substoichlometry in Radiochemical Analysis." Pergamon Press, New York, N.Y., 1968. (2) J. Stary and J. Ruzicka, Talanta. 18, 1 (1971). (3) F. Kukula and M. Simkova, J. Radioanal. Chem., 4, 271 (1970). (4) G . N. Btlimovich and N. N Churkina, J. Radioanal Chem.. 8, 53 (1971). (5) U V. Yakovlev and R. V . Stepanets. Zh. Anal. Khim.. 25, 578 (1970). (6) R. A . Nadkarni and B. C. Haldar. Anal. Chem., 44, 1504 (1972) (7) B M . Tejam and B. C Haldar, Radiochem. Radioanai. Lett.. 9 , 19 ( 1972).
A N A L Y T I C A L C H E M I S T R Y , VOL. 46, N O . 4, APRIL 1974
503
