Analysis of Biological, Clinical, and Environmental Samples Using Proton-Induced X-Ray Emission R. L. Walter and R. D. Willis Department of Physics and Triangle Universities Nuclear Laboratory, Duke University, Durham, N.C. 27700
W. F. Gutknecht Department of Chemistry, Duke University, Durham, N. C. 27706
J. M. Joyce Department of Physics, East Carolina University, Greenville, N.C. 27834
A 3-MeV beam of protons of 2- to 150-nanoampere intensity has been used to excite X-ray emission from a wide range of biological and environmental samples-e.g., human tissue, body fluids, soil extracts, leaves, coal, fly ash, ion-exchange membranes, and proteins. The X-rays have been detected using a Si(Li) solid state detector for the elements P (Z = 15) through Pb (Z = 82). Linear response has been demonstrated for the typical elements of Pb, Cu, Zn, Co, and Mn from 5 ng to greater than 2 pg. A lower limit of sensitivity of approximately 200 picograms in the irradiated area has been attained with the more responsive elements when they are deposited on very thin substrates. The proton-induced X-ray emission technique seems especially suited to rapid and economical multielement analyses for samples of clinical and environmental interest. Numerous examples of data obtained are included for critical evaluation.
X-Ray fluorescence (or X-ray induced X-ray emission) has been used as a n analytical tool for some time. Recent reviews (1-3) attest to the increasing application of this technique, especially in the area of environmental analysis. However, it is only in recent times that X-ray emission caused by particles, such as alpha particles and protons, has been used for analytical purposes. Birks et al. made a n early comparison of the production of characteristic X-rays by protons, electrons, and primary X-rays ( 4 ) . More recently Cahill (5) and Watson et al. (6) have used cyclotrons to generate 30- and 80-MeV alpha-particle beams, respectively, for producing characteristic X-rays for analytical purposes. Several researchers (7-9) have performed limited evaluations of proton beams L. S. Birks. Anal. Chem.. 44 (5), 557R-562R (1972). J . R . Rhodes, in "Energy Dispersion X-Ray Analysis: X-Ray and Electron Probe Analysis," J. D. Russ, Ed., Seventy-third Annual Meeting of the American Society for Testing and Materials, Toronto, Canada, June 21-26, 1970, ASTM Spec. Tech. Publ. 485, ASTM, Philadelphia, Pa., 1971, 243-285. Robert D. Giauque, Fred S. Goulding, Joseph M. Jaklevic. and Richard H. Pehl. Anal. Chem., 45 (4), 671-681 (1973). L. S. Birks, R. E. Seebold, A. P. Batt, and J. S. Grosso, J. Appl. PhyS.. 35,2578 (1964). Thomas A. Cahill, Report to the California Air Resources Board and Project Clean Air, University of California, Davis, Rep. No. UCDCNL 162, October 1972. R. L. Watson, J. R. Sjurseth, and R. W. Howard, Nucl. Instrum. Methods. 93, 69 (1971) J. A. Cooper, Nucl. Instrum. Methods. 106, 525-538 (1973). T. 6 . Johansson, R. Akselsson, and S. A. E. Johansson, Advan. X-Ray Anal. 15, 373-387 (1972); Nucl. Instrum. Methods, 84, 141 (1970). C. J. Umbarger, R. C. Bearse. D. A. Close, and J. J. Malanify, Advan. X-RayAnal.. 16, 102-110 (1973).
as excitation sources for similar analyses. These aforementioned articles and a recent comparative study of particle and photon-excited X-ray emission techniques (10) show quite clearly that the proton-induced X-ray emission technique has great potential as a rapid, efficient tool for multielement, trace analysis and that the optimum proton energy is about 3 MeV. The potential of the particle method arises, in part, from the fact that the cross section for characteristic X-ray production is quite large using particle excitation and that a single particle can give rise to several characteristic X-rays as it penetrates a sample. Of course, the ultimate sensitivity available depends not only on the excitation technique, but also on the nature of the sample being analyzed and the spectral background. The main source of background in the case of particle excitation is bremsstrahlung originating from within the target due to stopping of electrons which have been knocked out of the target atoms by the incident protons. This background, which consists of a broad continuum of Xrays with maximum intensity at the low energy end of the X-ray spectrum, sets the lower limit of detection. In the past few years, several reports (7-9, 11) have appeared in the literature which indicate possible applications of proton-induced X-ray emission analysis (PIXEA) for trace element studies. Discussions of the interferencefree levels of detection available with PIXEA have been presented in most of these reports. In this paper, a much wider set of PIXEA data for specimens of interest to environmentalists, clinicians, and research scientists is reported along with some indications of the usefulness of the technique and suggestions for improvements. A brief description of the arrangement employed in our laboratory and of some of the fundamental concepts behind PIXEA is also included for completeness and to aid in the discussions that follow. It is hoped that this style of presentation will elucidate more completely the practical limits of detection and the general problems encountered with PMEA, the primary purposes here being to aid analysts who are aware of the method but are uncertain as to the interpretation of ultra low limits of detection (7), and to provide data for numerous realistic samples with which comparisons can be made with the widely employed X-ray fluorescence met hod. The reader will note that the results of this work are tpically presented as raw X-ray spectra, rather than lists of limits of detection and/or abundances. The presenta(10) W . R. Smythe and N . F. Mangelson, asdescribed in report of Cooper (7). (11) D. M . Stupin, P. Fintz, A. Gallmann, H. E. Gove, and G. Guillaume; A. Pape and J. C. Sens, Rapport interne, CRN-LPNIN 7202, Centre de Recherches Nucleaires et Universite Louis Pasteur, Strasburg, France, October, 1972. A N A L Y T I C A L CHEMISTRY, VOL. 46, NO. 7, J U N E 1974
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PROTON BEAM STOP
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and Ag tion of raw X-ray data is preferable here in order to exhibit not only the typical elements found in the various samples but also signal-to-noise ratios and/or noise levels from which limits of detection for “missing” elements can be obtained. Since each sample type has different matrix problems, the authors believe this method of data presentation is not only best but quite necessary for proper evaluation.
INSTRUMENTATION Beam Generation and Characteristics. The proton beam used in this study was generated with the Triangle Universities Nuclear Laboratory 4-MeV Van de Graaff accelerator. Beam energies used in the work ranged from 2.5 to 3.0 MeV, and beam currents were 2 FA or less. The beam tliameter could be varied from 0.2 to 2 cm using a combination of a 1.0-micron thick nickel foil diffuser to provide a homogeneous beam density, and a graphite collimator system, as shown schematically in Figure 1. The beam that eventually impinged on the targets in most of the work was held constant a t about 0.8 cm in diameter and ranged from 2 to 150 nA. The entire accelerator system, including the chamber for holding the samples, is operated under moderately hard vacuum, usually about 5 x Torr. X-Ray Detection a n d Counting. The samples are placed on a rectangular target holder with detachable handles. This frame, shown schematically in Figure 1, holds up to 20 samples. The detachable rods which extend through vacuum-seal O-rings at the ends of the target chamber serve to move the various targets into the proton beam. The target chamber is easily isolated from the remainder of the accelerator vacuum system with a singlestroke, hand-operated vacuum lock. The time for replacing a target holder and-pulling a high vacuum can be less than 10 minutes. [During these early feasibility runs, it was not considered necessary to automate the sample changer mechanism or to devise a method for holding more than 20 samples. Several automatic changers have been described, some of which hold up to 100 samples (5, 12, 13)]. The targets are usually placed with their normal directed at an angle of about 35” relative to the horizontal proton beam axis. Those X-rays which are emitted by the sample in a direction vertically downward are detected with a 30 mm2 Si(Li) detector as shown in Figure 1. The X-rays pass out of the target chamber through a 25-micron Mylar window, and pass through about 1 cm of air before entering the 25-micron thick Be window of the des. August, P. Shapiro, C. M. Davisson. and w. l. McGarry, Meeting of the American Physical Society, Washington, D.C., April, 1973,No. EO 12. (13) 8. K. Barnes, L. E. Beghian, G. H. R. Kegel, S.C. Mathur, and P. W. Quinn, Meeting of the American Physical Society, Washington, D.C., April, 1973,No. EO 13. (12) C. D. Bond, L.
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ANALYTICAL CHEMISTRY, VOL. 46,, NO. 7, J U N E 1974
The full-scale counts for the ordinate are shown directly under the various regions of the spectrum
tector. An absorber or filter is generally placed in this air gap to reduce the amount of low energy X-rays, which are often produced in overabundance. For the measurements to be reported, either 0.1 mm of Mylar or 0.8 mm of polyethylene was employed as the absorber. Pulses from the detector, which are proportional to the X-ray energy, are amplified and are analyzed in an ADC which is interfaced to the Triangle Universities Nuclear Laboratory DDP-224 (Honeywell Corporation) computer. At the end of a run, the data are dumped onto magnetic tape, and also transferred to another location in memory. Simultaneously with recording the next X-ray data set, previously accumlated data can be stripped down for preliminary investigation. A second, off-line DDP-225 computer with an oscilloscope display and light pen unit is available for a more careful analysis at a later time. For instance, automatic computer routines for peak fitting and background fitting can be utilized and peak areas can be extracted for a series of elements in about 10 seconds. The spectral resolution is typically about 200 eV for the 5.89-keV Mn Kcu line. As there is some variation in the proton beam current with time, analytical accuracy requires one to count Xrays produced per quantity of charge impinging on the target rather than counts per unit time. The accumulated charge is measured by collecting the beam current which enters the target chamber during the analysis of a particular sample. Actual analysis requires 1 to 15 min per sample, depending on the nature of the sample being analyzed and the concentration levels at which the elements are to be determined. Figure 2 shows the characteristic spectra for emission of the K a and KP lines from Mn and Ag. (For convenience, these spectra were obtained with radioactive sources.) As long as self-absorption is negligible, the KP line is present in a fixed ratio to the K a for a particular element. The KP lines cause unavoidable interferences between neighboring elements over a large range of elements. This interference is one of the most serious problems in X-ray systems which incorporate a Si(Li) detector for X-ray energy analysis. The Ka-KP interference, for example, makes the direct recognition of Co extremely difficult in an energy dispersive system because of the considerably larger amount of Fe occurring in most of the samples of interest. Nevertheless, computer spectrum-analysis programs can be utilized to resolve these overlapping signals in most cases, and in particular, can be helpful in setting upper limits of concentrations for the undetected neighbors. The combined method of excitation and detection used here results in a system having the highest sensitivity for the KO X-rays for the elements from about P to Cd. For
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emission system to MYLAR ABSORBER 3.0 MeV
See text for discussion of "Absorbers" 1
the heavier elements, the L X-rays (three major linesLa, Lp, L r ) are more strongly excited. As the L X-rays are about seven times lower in energy than the K X-rays from the same element, additional interferences result. Two examples of such interferences are Ba L-lines overlapping T i and V K-lines and the P b La-line overlapping the As Ka-line. It should be pointed out here, as will be stated again in the discussion following, that certainly not all detectable elements occur in all samples, and thus these overlap problems are limited to a few cases. Also, as mentioned above, computational techniques can be used to resolve the overlap in many cases. Target Preparation. Several of the sample types examined were self-supporting. These included leaves, hair, insect wings, air filters, pelletized substances, cloth, and ion-exchange membranes. These materials are simply attached to the sample holder with a small amount of adhesive. Non-self-supporting samples such as urine, blood, various aqueous solutions, and tissue sections are deposited upon a thin sheet of plastic material which has been stretched over a 2.5-cm diameter graphite ring that can be readily clamped t o the target holder. Various plastics were evaluated for this backing material. These included cellulose acetate, polystyrene, Mylar (Dupont), Millipore (Millipore Corp.), Nuclepore (General Electric), and Formvar (Shawinigan Products Corp.). The best all-around material was found to be 8-micron porosity Nuclepore filter material. It showed a moderate bremsstrahlung and about the lowest contaminant level of the materials tested. Moreover, it was thermally stable in the proton beam and dehydrated deposits from dissolved or emulsified samples adhered well to it. This feature is true only to a lesser extent for whole blood which dries to form a brittle solid. Mylar having a thickness of about 3 micron produced about 40% as much bremsstrahlung but lacked good adhesive properties. The lowest bremsstrahlung background was obtained with Formvar, a thin film created by dropping a single drop of Formvar solution onto a surface of pure water. The solvent evaporates and the ultrathin plas-
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tic film floating on the water surface is picked up from underneath with a target ring. However, the Formvar backing is fragile and probably not very practical for wide usage. Solution samples were prepared by depositing 20-100 11 (usually in 20-11 increments) of solution on the plastic backing with a micropipet. These deposited solution samples were dried in a vacuum desiccator. Serious flaking occurred for the more crystalline substances, like residues from sea water, and certain enzymes, and deposits from ashed substances and whole blood. Thus the best quantitative analysis for numerous types of solutions may require packaging like placing a thin layer of polystyrene or polyethylene over the deposit. Granular or coarse samples like soil, fly ash, coal, ground orchard leaf, and lyophilized tissue were sandwiched between two layers of thin polystyrene or the 3micron Mylar. Some of these samples could be readily pelletized in a 25-ton press and these pellets (about 1 cm in diameter) were also mounted by the sandwiching method. In practice, only crude, relative measurements were obtained for the coarser substances because the variability and the granularity of the particulates limit the accuracy of a n absolute determination. RESULTS AND DISCUSSION Elemental Range. Up to 23 elements ranging from P ( Z = 15) to P b ( Z = 82) have been observed in the wide variety of samples studied. The energies of the X-rays detected vary from about 2 keV to 20 keV. The net response of the system to X-rays of varying energy is not linear, but peaked. In the current mode of operation, the K X-ray sensitivity peaks at about Z = 25 (-6 KeV). The sensitivity curve is shown in Figure 3, ANALYTICAL CHEMISTRY, VOL. 46, NO. 7, JUNE 1974
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MYLAR ABSORBER
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Doped as shown
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CHANNEL Figure 5. X-Ray emission spectra from blank and doped Nuclepore filters
200
2 50
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Cross-hatched areas indicate the impurities observed in the blank filter. The amounts of the impurity elements are listed under the corresponding peaks. Quantities listed above the curves correspond to amounts of elements deposited on second blank filter
where the number of X-rays detected per pg per &'is plotted as a function of atomic number. Here it can be seen that the K X-ray sensitivity is low at both low and high 2. At the low 2 end, the soft X-rays are absorbed predominantly by the absorber but also by the Mylar window of the vacuum chamber. At high 2, the X-ray excitation probability drops off rapidly, resulting in the reduced sensitivity exhibited in Figure 3. For 2 > 40, the decrease in detector efficiency further contributes to the decline in sensitivity. The targets for these and other standards were prepared by depositing 10- to 2 0 4 samples of appropriate solutions prepared with deionized, distilled water and reagent grade chemicals onto Nuclepore backing. Note here that these response curves have been obtained with the two different X-ray absorbers described earlier. These absorbers are placed over the detector face, and their primary purpose is to cut down the count rate at the low-energy end of the spectrum. Additional absorbers like A1 foil have also been used to attenuate the X-rays from moderately heavy elements which are present in large amounts when trace levels of heavier elements are of interest. Such is the case when analyzing for As and Pb in the presence of a high percentage of Fe, as in blood, coal, and fly ash. If the high counting rates caused by the dominant elements are not reduced by a filter, summation peaks will arise from two X-rays striking the detector in an interval less than the resolving time of the electronic system. Concentration Range. The mass range over which linear response could be obtained was checked for several metal ions over three orders of magnitude. Figure 4 shows the results of the studies with 5-ng to 2-pg deposits on Nuclepore. Based on these data and other results with heavier concentrations, it has been concluded that the system is linear from 5-ng to above 10-pg amounts for elements above Ca. Standardization, Quantitative results have been ob846
ANALYTICAL CHEMISTRY, VOL. 46, NO. 7 , J U N E 1974
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deposited on Formvar backing tained in two different ways thus far. The first is to use values read from Figure 3 as standard sensitivity coefficients. This is quite reasonable for less complex samples. To determine the quantity of a specific element in the beam, one determines the area under the peak of the "unknown" and compares it to the appropriate standard, as found in Figure 3. Backgrounds can be obtained through an analysis of blank substrates where appropriate, and by a fitting procedure which produces a smooth curve to represent the bremsstrahlung in the vicinity of the peak. The contribution from tails of nearby peaks or total overlap from other lines can be estimated by a complex fitting code to be reported elsewhere. If there is concern about matrix effects, the method used to circumvent problems from these effects is the standard addition of a known amount of an element. Samples Tested. Early experiments with the system indicated that many different types of samples could be handled since little or no sample preparation is necessary. Subsequently, a large number of sample types have been
Figure 7.
Comparison of three blank substrate materials
gathered together and analyzed. Following herewith are descriptions of some of the samples analyzed. Although it is not the purpose of this paper to discuss the scientific justification or merit of the analyses performed so far, occasionally some informative comments regarding the particular choices of samples will be given. The presentation of the data typically will show the number of X-rays observed us. channel number, which is proportional to energy. It is significant to note that a fourcycle log display is used. Blank Nuclepore. As stated above, Nuclepore is normally used as the backing for non-self-supporting sample materials. To establish the background spectrum arising from proton bombardment of this material, a number of Nuclepore filter membranes were tested. Some typical results are shown in Figure 5 . Here the solid line (with cross-hatched peaks) represents the spectrum from a blank Nuclepore and the dashed line represents the spectrum of a second Nuclepore filter upon which 5.8, 6.3, and 6.4 ng of Mn, Cu, and Pb, respectively, have been deposited. The quantities listed below the curves represent estimates of the amounts of each of these materials found in the blank Nuclepore. Formuar. Figure 6 shows a spectrum from 100 ng of P b deposited on a Formvar backing. These data were accumulated in a few minutes with a beam of about 100 nA. Fe, Cu and Zn, probably intrinsic to the Formvar, are present in the abundances shown. Dashed peaks suggest easily detectable signals representing small amounts of other elements which are absent here. Using slightly more intense beams, in less than 3 minutes of running time, Cu and Zn levels down to 200 picograms would be detectable, by the 3 u criterion. As stated previously, the Formvar is fragile and thus is practical only when maximum sensitivity is needed. Some other organic substrates which are metal-free might be found to be as sturdy and hence even more useful than Formvar. Three blank substrates are compared in Figure 7 : Nuclepore, 6-micron thick Mylar, and Formvar. The only detectable impurities in the Mylar were Zn and Cu. The reduced bremsstrahlung for a thin layer like Formvar is apparent.
Soil Extracts. Acidic, aqueous extracts of soil samples collected from several mining areas in North Carolina were analyzed. The solutions were prepared by shaking 10 grams of soil in 100 ml of solution for 12 hours and then filtering. Some of the PIXEA spectra are shown in Figure 8. Only 40 ,ul of the solution was employed to obtain the spectra shown here. A rather large number of elements can be seen, including Ba and Pb. Significant differences in the Zn:Cu ratio are clearly exhibited for the two geographical areas compared here. The quantities of each element were determined from the standard curves of Figure 3. Leaf Sections. The uptake of metals in plants has been investigated for a number of species by analyzing the metal content of leaves. One project concerns the two types of plants which have survived in the otherwise barren regions surrounding the mines from which the above soil samples were collected. The intent of the study is to determine if the plants have evolved some mechanism that permits growth in the toxic soil, as for example, the plants having developed a means to restrict heavy metal uptake. The only part of the plants studied so far has been the leaves, which conveniently are ideally suited to our method. For the work shown here, small pieces (1 cm x 3 cm) were glued to the target holder for analysis. Typical spectra are shown in Figure 9. As might be expected, several of the same elements (e.g., Mn, Zn, and Pb) that were found in the soil extracts also gave a strong signal in the leaf sections. These results indicate quite clearly that this X-ray emission technique would be extremely helpful for tracing inorganic nutrient transport through plants, as well as for studying the more general topics of plant nutrition, effects of changing climatic conditions, pesticides, etc. B o d y Fluids. Figure 10 shows two superimposed spectra resulting from the analysis of dried urine samples. The amount of urine used here was 40 pl; the signal could have been enhanced severalfold by increasing the number of aliquots deposited. Several preliminary studies have been investigated and the two cases shown here represent typical metal levels in urine except for the following features. Sample VI which was obtained from a patient with cancer ANALYTICAL C H E M I S T R Y , VOL. 46, NO. 7, J U N E 1974
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CHANNEL Figure 8. X-Ray emission spectra from
soil extracts deposited on Nuclepore backing
LEAF (COREOPSIS M A J O R ) GOLD H I L L , N.C.
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X-Ray emission spectra from leaf section using two different X-ray absorbers
of the prostate showed a large Fe and Zn content. Sample V which shows a high Hg signal was one of those collected for Leonard Goldwater (14) for his extensive study of mercury burdens in humans in the Cape Fear region of North Carolina. It is coincidental that the amounts of K, Br, and Rb are nearly indistinguishable in these two urine (14) L. Goldwater, Department of Community Health Science, Duke University, Durham, N.C.
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samples. Figure 11 shows superimposed spectra of two more aliquots of sample V, but one was “doped” with 1 ppm Hg and the other, 1 ppm Pb. The Hg and Pb levels in the original urine sample were thus found to be about 0.4 ppm and less than 0.1 ppm, respectively. Note here that good reproducibility was indicated in that the Zn, Br, and Rb peaks in the two spectra are indistinguishable. In other urine studies, abnormally high amounts of P b and As were found in suspected instances of poisoning
URINE ( 4 ' 0 ~ 1 ) POLYETHYLENE ABSORBER
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Figur 10. X-Ray emission spectra of
CHANNEL urine samples deposited on Nuclepore
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from the respective metals. Other body fluids studied were blood and serum. High levels of P b and of Hg were noted in a few special blood samples. The serum studies have not been too profitable yet but much of the bodyfluids work is in a primitive stage. E P A Environmental Air Filters. One of our long-range goals for initiating trace element studies at our laboratory is to determine if the PIXEA method can surpass the conventional X-ray fluorescence technique for measuring air pollutants collected on various membrane filters. A num-
ber of tests have been conducted for the Environmental Protection Agency and PIXEA has proved to be moderately practical although final conclusions will await the analysis of some intercomparisons. For completeness, we show in Figure 12 a spectrum obtained from the analysis of a 24-hour air filter from Durham, N.C., supplied by EPA. A large number of metals can be clearly recognized. Elements of interest include Ca, Ni, Fe, Ti, Pb, and Br, the latter four being constituents of fuel oil, coal and/or gasoline. However, some air filters do pose one problem A N A L Y T I C A L CHEMISTRY, VOL. 46, N O . 7, JUNE 1974
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E P A NUCLEPORE A I R FILTER 2 4 HR; DURHAM,N.C. POLYETHYLENE ABSORBER Ti, BO
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ABSORBER MYLAR ABSORBER
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50
for quantitative analysis. The target material--i.e., the filter media itself-is usually rather durable and hence thick, often being a form of filter paper. In the past, air filtering devices often employed Whatman paper which has a rather high areal density, like 8-10 mg/cm2. This density causes a large loss in proton beam energy on passage through the specimen, resulting in a reduced X-ray yield. As the material deposited on the filter varies in concentration through the filter (with most being on the front surface), it is difficult to be sure of a constant relationship between beam energy and the percentage of ma850
ANALYTICAL CHEMISTRY, VOL. 46, NO. 7 , JUNE 1974
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terial on the filter available a t the energy. This uncertainty causes analysis difficulties and leads us to the conclusion that for our method, with 3 MeV-protons, it is important to use thin membranes (ideally of the order of 1 mg/ cm2) in air filtering devices. Concerning the topic of air filters, it is important to indicate one of the special applications of the current method which can be used to study metals deposited in small regions (Le., less that 1 mmz) on membranes. Eight-hour personnel monitors could be developed with a small aperture which would permit use of a portable air pump that
is much smaller than those required for other detection methods that are less sensitive to absolute amounts of deposit. Animal Tissue Sections. One of the most interesting sample types analyzed was animal tissue. Several forms were attempted-thin sections, thick slices, lyophilized tissue and ashed-tissue residues. Figure 13 shows the spectrum resulting from the direct analysis of a 30-micron thick section of human kidney. The P b signal corresponds to about 50 ppm Pb; Se a t about the 10 ppm level was also found in this particular sample. Thin sections have been obtained and analyzed for some nine other organs from the same body of a male who died of unknown causes. Included were the heart, spleen, lung, liver, adrenalin gland, brain, testes, and one unknown section. The significance of enhanced signals of Rb and As in the brain, Br in the lung, Se in the testes, and As in the heart has not been investigated yet. To increase the counting rate with tissue samples, analyses can be made using pelletized tissue or thick sections, so thick (Le., greater than 15 mg/cm2) that the protons stop in the sample. X-ray attenuation can be sizeable in this situation, and corrections are probably important for elements lighter than vanadium in biological samples. At present, only crude corrections for this effect have been made. For samples where the protons stop inside the specimen, a serious background can result from X-rays produced when the accumulated positive charge arcs over to the aluminum target holder. To eliminate this problem, a thin film of aluminum was laid across the backside of the pellets and placed in contact with the frame. Thick section studies have primarily been made with marine animals. In Figure 14, a spectrum is plotted for muscle from a carp which was taken from a local, clean stream at the point of a sanitary system outfall. The metal abundances shown in ppm (wet weight) in Figure 14 indicate that the muscle was quite clean. Data from atomic absorption analysis are shown in the lower left and the present data were normalized to the 6.2 ppm Fe level. Agreement is reasonable for Mn and Cu but the Zn signal observed by us indicates a level about two times higher. This difference might have resulted from using different regions of the muscle. The method of using thick sections could be powerful, when coupled with a calibration of the more abundant elements obtained from thin section irradiations. Other organs from the same carp, which were of lesser food value, were also analyzed, and the pollutant burden seemed very low in these also. Additional marine specimens collected from special regions on the West Coast were analyzed in cooperation with the National Institute for Environmental Health Sciences. Noteworthy levels of As, Hg, Pb, Sr, and Br were observed. (This project will be repeated in a more controlled manner to determine if geographical patterns of the metal levels exist.) Irradiation of a limited area of tissue-i.e., about 0.5 cm2-gives rise to a concern about representative sampling of a larger region or of the whole organ. This problem is magnified when one recalls that 3-MeV protons are only “effective” in producing X-rays to a depth of about 0.15 mm in organic substances like animal tissue. To obtain more meaningful results, one can produce more homogeneous samples by lyophilizing a reasonable amount of the sample, grinding it into finer particulates, and then pressing the powder into a thin, self-supporting wafer or pellet (which, incidentally, is ideally suited for proton irradiation). Placenta and blood, which are easily pelletized after lyophilizing, were investigated using this form of preparation. Sample spectra from placenta runs at both 2.5 and
CARP MUSCLE(Tt4lCK SECTION)
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CHANNEL
Figure 14. X-Ray emission spectrum from thick carp-muscle section Ppm values are shown for thisanalysis and for an atomic absorption analysis
3.0 MeV are displayed in Figure 15. Detectability limits for placenta prepared in this fashion appear to be better than 0.5 ppm of dry weight for elements between Mn and Zr. Some preliminary work was been done on ashed tissue with the aim of enhancing the signals from trace metals and a general decrease in the detectability limit of about 2 to 3 times appears possible by means of this method for removing organic compounds and concentrating the remaining metals. The enhancement obtained after wet ashing (15) at 300 “C was not as large as was expected because bremsstrahlung from the dominant remaining elements, like Mg, K, Ca, and P is appreciable. Ashing is, in fact, generally unattractive in that the expense and time involved greatly increase the overall cost of the present analysis and increase the possibility of introducing impurities. Proteins. Proton induced X-ray emission is ideally suited to small sample sizes and an obvious choice would be proteins or enzymes which often are costly to prepare, even in small amounts. The abundances of Mo, Fe, Co, Cu, and Zn in metallo-enzymes have been measured to study their purity and the cleanliness of the preparation method. Levels down to 2 ppm Fe and 1 ppm Mo were obtained with 50 mg of dismutase for only 1.5 p C of charge. Zon-Exchange Membranes. Another project under way is based on a n interest to study trace metals in streams by using ion-exchange membranes as integrating samplers. A direct analysis of thin ion-exchange membranes which have been pre-equilibrated with sought-for ions in dilute solution has been made. This approach has permitted the detection’ of lo-* molar lead in aqueous solutions. Exhibited in Figure 16 are the results for varying concentrations of P b in a liter of deionized water. The high P b concentration solutions were also doped with Ca at levels typical of local streams. A full report of this study is given elsewhere (16). Local streams, urine, milk, and beer have been studied by this approach, but quantitative reporting will require careful calibration to learn the effects of competition for uptake when the solutions are more heavily loaded than in our equilibration tests. (The region of the stream from where the carp of Figure 14 originated was found to be very low in “pollutant” metals.) (15) G. Middletonand R. E. Stuckey,Ana/yst (London), 79, 138 (1954). (16)C. H. Lochmuller, J. Galbraith. R. Walter, and J. Joyce, Anal. Lett.. 5,943-951 (1972). ANALYTICAL CHEMISTRY, VOL. 46, N O . 7, JUNE 1974
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CHANNEL Figure 15. X-Ray emission spectra from pelletized placenta at 2.5 and 3.0 MeV normalized to accumulated charge
ION EXCHANGE MEMBRANE POLYETHYLENE ABSORBER A - 2 0 p p m Pb , 4 p p m C o 8 - 2ppm P b , 4ppm C o
0
50
100
I50
200
CHANNEL Figure 16. X-Ray emission spectra from self-supporting ion exchange membranes equilibrated Ca
Water and Other Liquids. As the samples currently must be placed in a vacuum chamber, liquids like HzO, gasoline, and fuel oil (as well as urine and serum) are dried in a vacuum desiccator. Therefore, all that can be measured are the nonvolatile residues, which is hardly satisfactory for gasoline studies where a significant portion of the metals of interest may be evaporated. Fuel oil, at the other extreme, has such a low vapor pressure that it 852
ANALYTICAL CHEMISTRY,
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250 with
various concentrations of Pb and
remains liquid and appears to retain most of the metal content after many hours of pumping in a hard vacuum. Water studies have been performed on samples taken from the sea, the tap, and from deionizers. The presence of NaCl in sea water made it difficult to observe elements other than Br and Sr. A spectrum for an 80+l sample of condensed tap water is shown in Figure 17. The preparation involved evaporating 100 ml of HzO in a flask and
CONDENSED TAP WATER
80pJ
-
POLYETHYLENE ABSORBER
2.5 MeV
_----- 3.0MeV
( x 1.75) ( x 1.0)
Ill -I
I02L Element
10
I tI
0 Figure 17.
Mn
Fe Ni
cu
Zn As Se Br Rb Sr Hg
Pb
ppb
3.1
11
3.6 230 305 < 0.5 .C 0.3 12 2.1 39 cl.5 6.3 I
1
50
IO0
I
I
I
2 00 CHANNEL
I
250
I50
300
X-Ray emission spectra from condensed tap water
The 2.5-MeV data have been multiplied by a factor of 1.75
dissolving the residue in 1 ml of acid solution. Data obtained from the 3-MeV run indicated detectability limits below 1 ppb for elements from Ti to Sr and around 1.5 ppb for Hg and Pb. Amounts of some of the metals observed are listed in Figure 17. Aerosol Sprays. Solid residues from aerosol sprays can be readily prepared for analysis by spraying a thin, relatively clean membrane like the 4-micron Mylar, and determining the weight of the material deposited by a difference weighing. The results of the analysis of one commercial (non-paint) product is displayed in Figure 18. The large percentage (The current federal regulation for the maximum P b content in the dried residue for paint is about 0.5%.) of P b was a surprise to the manufacturer and to the Federal Consumer Product Safety Commission, both of which are conducting further studies on such aerosols. Other products designed for the same general purpose showed a P b content below 40 ppm. Potential f o r Areal Scans. As the area of proton beams can be reduced to quite small dimensions, some scanning possibilities exist. To emphasize capabilities of the technique, we show the results of one other measurement where two regions of a green maple leaf which was speckled with circular brown spots were irradiated. The tree appeared to be healthy in other respects. The histograms shown in Figure 19 represent the elemental abundances observed for the different regions of the leaf. (The values of the lightest elements are too low because the absorption of their characteristic X-rays occurs to a significant, but undetermined, amount.) The size of the irradiated regions here were about 0.5 cm2. It is readily possible to reduce the beam area to less than 0.5 mm2 for a sharper scan but, in such a case, one must be concerned that too much power deposited in a small area would vaporize the sample. So, to do such a scan with our current method
TI Cr Mn
.IFe:
N I Cu
Zn
Pb
J.1
I
1 Se
Hg
Br
j . 1
1
i
Rb
i
1 1 AEROSOL
,:
r
Sr
II
DEPOSITED ON MYLAR
X-RAY ENERGY ( A R B I T R A R Y UNITS)
Figure 18. X-Ray spectrum from deposit of spray-can-aerosol indicating large Pb content
might require reducing the proton beam current which would necessitate a proportionate increase in measuring time. Yet, even with a beam size of about 1- to 2-mm diameter one can see uses in scanning, for example, tissue sections, or membranes from air filters which deposit particulates of graduated sizes in different positions on a single filter. DISCUSSION AND CONCLUSIONS The technique discussed here permits the detection of most elements of concern in clinical and environmental samples in the range of phosphorus to lead. The sensitivity available depends on both the atomic number of the element and also on the sample in which the element is found. In a sample such as whole blood, where large ANALYTICAL CHEMISTRY, VOL. 46,
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I-t
SUGAR M A P L E L E A F t B X I GREEN AREA (lcm2 1 0BROWN SPOT(lcm2 1
I nanogram 2: I part per million
I6O
I4O
Uncalibrated
4
x
C r c 50ng
I
TI
'200
NI
c
30 ng
-.8 n g
See 2ng Ass 6ng Rbc l o n g Moc I n g C d c 15ng Sn c 15ng H g c 6ng
5 1201
a
Jioo
I
K
r
Sr Rb I
Figure 19. Results of analysis of two regions of a leaf. Calibration for elements below K is uncertain
amounts of organic materials and iron are present, the background will be large and the limits of detection for other elements such as mercury and lead will be rather high. These limitations are minimal, on the other hand, for a simple aqueous solution deposited on organic backings as thin as the Formvar film employed here. The technique is generally sensitive to about the 1-ppm level in the deposited residue if no heavy element is present at levels above about 0.5%. Improvements in the technique and lowering of the detectability' limits are suggested throughout the paper. One consideration that is important is optimization of the proton bombarding energy. Data for most of the Figures was obtained at 3 MeV but one should note the two comparisons in Figures 15 and 17 where both 3- and 2.5-MeV spectra are displayed. When samples of moderate areal density are irradiated, the major source of background is bremsstrahlung from within the sample matrix. Lowering the proton energy reduces the average energy that can be transferred to the electrons of the matrix. As the bremsstrahlung is proportional to the electron energy, the noise level can be reduced in this way, but one faces a concurrent decrease in characteristic X-ray production. The primary effect of lowering the energy from 3.0 to 2.5 MeV is to reduce the characteristic X-ray yield for all elements by a factor of approximately 1.8 and to reduce the bremstrahlung in the region between Ca and Fe (K Xrays) by a factor of 4. (This factor is consistent with the proton-induced X-ray cross-section data reported by Umbarger et al. (8) when compensation is made for the energy loss in the Ni diffuser foil.) Note that in Figure 17, the 2.5-MeV counts have been multiplied by 1.75. In Figure 15, the usual counts per unit charge are shown; this figure emphasizes the overall decrease in counting rate. The apparent signal-to-noise improvement in lowering the energy is tempered by the decrease of about (1.8)1'2in statistical accuracy (for the same amount of accumulated charge). When analyzing for Al, Mg, P, and S, it is necessary to remove the Mylar absorber, leaving only the 25-pm window of the chamber. In fact, best quantitative results for A1 would require placing the Si(Li) detector inside the vacuum chamber so that the X-rays need to penetrate only the Be window across the detector housing. Unfortu854
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nately, X-ray absorption in the sample matrix can be excessive for these lighter elements. For brevity, a discussion comparing the current method to other analysis techniques will be omitted from the present paper. It is the authors intent to detail some of these comparisons in later reports when sufficient data have been accumulated to make critical, valid evaluations. However, it seems wise to tabulate some points, specific advantages as well as possible disadvantages, which call attention to the strengths and weaknesses of proton-induced X-ray emission analysis. Some of the features listed also apply, to a large extent, to those X-ray fluorescence systems which use energy dispersive systems. Advantages 1. Economical for multielemental analysis 2. High sensitivity in terms of absolute abundances 3. Moderately high sensitivity in terms of relative abundances 4. Extreme ease of sample preparation 5. Nondestructive in most cases 6. Rapid turnaround for obtaining results 7 . Surface analyses possible, i.e., can limit the effective proton penetration to less than a 50-micron depth 8. Small area scans permitted by restricting irradiated area to less than 0.5 mm2 Disadvantages 1. Need access to a suitably equipped proton-accelerator laboratory 2. Samples should be less than 5 mg/cm2 for best quantitative accuracy 3. Volatile components may be lost because of placement in vacuum or because of mild heating 4. Matrix effects can spoil absolute determination of the light element abundances 5 . Interelement interferences affect sensitivity limits 6. Liquid specimens must be dried As can be readily recognized, a few of the items listed under disadvantages may have no bearing on a particular type of sample. Furthermore, some of these problems may be circumvented in the future-e.g., by developing a thin window cell, which will operate in a vacuum system and which would permit entry of flowing liquid samples into the proton irradiation area.
The real power of the technique appears to lie in the feature that it allows a multielemental analysis directlyi. e., without sample preparation-of many different types of samples. Other samples analyzed but not discussed include human hair, cloth, paper, other filter media, and even a section of a butterfly wing which showed the lead and bromine normally associated with automobile exhaust emissions. Computer analysis of the spectra is fast, permitting the determination of 20 elements in as little as 2 minutes on our off-line computer. Therefore, it does appear that proton excited X-ray emission shows real potential as a rapid, mass-screening or survey tool. This could apply to whole-body tissue analyses as well as analyses of pl samples of blood or urine from large populations being surveyed in environmental pollution studies. Note Added in Proof: Very recently a review article by Goulding and Jaklevic has appeared in Annual Reviews of Nuclear Science (1973) in which comparison is made between detectability limits for X-ray fluorescence and proton-induced X-ray emission systems. The calculations upon which they base their conclusions, i. e., bremsstrahlung flux and characteristic X-ray yields are in reasonable agreement with values that can be derived from our data shown here, e.g., in Figure 5. However, the 3 u detectability limits for Zn that can be determined from Figures 14 and 15 (where Zn is present a t a level of around 40 ppm dry weight) would be around 0.5 ppm (dry weight) for a run of about 5-min duration. These results do not contradict the stated value of a detectability limit of 14 ppm designated by
Goulding and Jakelevic for different beam and target conditions, but it certainly tempers their explicit comparisons and the implicit conclusions presented.
ACKNOWLEDGMENT We would like to acknowledge the generous assistance of Janette Stanford, Alan Larkin, and Mike Corcoran in preparing targets and in data accumulation and analysis. The botanical studies were made possible through contributions of J . Antonovics, and the ion-exchange studies through the efforts of C. H. Lochmuller and J. Galbraith. The cooperation of E. G. Bilpuch and H . W. Newson of TUNL is sincerely appreciated. Contributions from G. K. Klintworth, D. Paulson, and L. Goldwater of the Duke Medical Center, K. V. Rajagopalan of the Duke Biochemistry Department, and B. A . Fowler of the National Institute for Environmental Health Studies is gratefully recognized. Special gratitude is owed to S. M. Shafroth from the University of North Carolina for the use of his X-ray chamber and Si(Li) detector in the first running period and to A. B. Baskin for his efforts to provide us with a suitable, semi-automatic peak fitting code. Received for review July 27, 1973. Accepted February 4, 1974. This work was supported in part by the U S . Atomic Energy Commission and the U S , Environmental Protection Agency. Additional support for one of us (J.M.J.) from the North Carolina Board of Science and Technology is also gratefully acknowledged.
Cavity-Dumped Argon-Ion Laser as an Excitation Source in Time-Resolved Fluorimetry F. E. Lytle and M. S. Kelsey Department of Chemistry, Purdue University, Lafayette, Ind. 47907
Both the operational principles and the experimental aspects of cavity-dumping the continuous wave (CW) argon-ion laser are discussed. The technique is used to construct an excitation source for time-resolved fluorimetry having the advantages of a variable repetition rate (-10 MHz to single-shot), variable pulse width ("9 nsec to CW) and moderate peak power per unit band width (-12 W in the blue-green and 600 mW in the UV). Two classes of sample data are shown for molecules at trace (ppb) levels. The first type is intensity-vs-time at a fixed wavelength. This allows the calculation of the fluorescence lifetime. The second type is in tensity-vs.-wavelength at an arbitrary but constant time with respect to the excitation pulse peak. This allows the display of the fluorescence spectrum without interference from scatter and Raman lines. Finally, future instrumental advances are discussed that should allow much shorter pulse widths and greater wavelength selection.
A brief survey of the review literature (1-3) in the field of time-resolved fluorimetry indicates the tremendous advances made during the past decade in detector and am-
plifier technology. Concomitantly it has become apparent that the excitation source is now usually the restricting feature with respect to the lower achievable limits on concentration and lifetime. Therefore, continued enhancements in performance will require improved methods of generating short duration light pulses. The instrumentation reported in this paper is the first step in an overall program designed to explore solutions to this problem. Before designing an excitation source, it is necessary to decide whether the overall device should yield data based on phase delays or pulse decays. Although both techniques should yield identical results when pushed to their ultimate, some experiments are easier to perform with one arrangement or the other. As an example, phase delays can be used to determine extremely short relaxation rates (1) G. E. Peterson, "Fluorescent Lifetimes of Trivalent Rare Earths," in "Transition Metal Chemistry," Vol. I l l , R. L. Carlin, Ed., Marcel Dekker, New York, N.Y., 1966, pp 202-302. (2) J. B. Birks and I . H. Munro, "The Fluorescence Lifetimes of Aromatic Molecules." in "Progress in Reaction Kinetics," Vol. I V , G. Porter, Ed., Pergamon Press, New York, N.Y., 1967, pp 239-32. (3) W . R. Ware, "Transient Luminescence Measurements," in "Creation and Detection of the Excited State." A . A . Lamola, Ed., Marcel Dekker. New York. N.Y., 1971, pp 213-32.
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