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Anal. Chem. 1900, 60,684-687
Total Reflection Energy-Dispersive X-ray Fluorescence Spectrometry Using Monochromatic Synchrotron Radiation: Application to Selenium in Blood Serum P. A. Pella* Center for Analytical Chemistry, National Bureau of Standards, Gaithersburg, Maryland 20899
R. C. Dobbyn Institute for Materials Science and Engineering, National Bureau of Standards, Gaithersburg, Maryland 20899
Monochromatic synchrotron X-radiatlon at The Natlonal Synchrotron Llght Source (NSLS) has been lnvestlgated as an excitation source for the direct energy-dlsperslon X-ray fluorescence (EDXRF) spectrometric analysis of metals In solutlon at parts per bllllon (ppb) levels In the total reflection geometry. A minlmum detection llmlt (mdl) of 8 ppb was determined for selenium In human blood serum and In roposed NBSSRM 1598 bovine serum. The results show that tMs method Is sulflciently sensttive for analysls of Se In bCmd serum. Se Is present at about 30-100 ppb In human bl bod serum and about 40 ppb in NBS-SRY 1598. The lob ast concentratlon of selenium measured was 9.7 f 0.5 ppi In NBSSRM 1843b trace elements in water where a mdi of 3.6 ppb Se was obtalned. Mlnlmum detectlon llmlts were also calculated to compare relatlve sensltivltles of tube-excited secondary target conventlonal sources when Illuminating a large sample area wlth NSLS excltatlon when probhg a much smaller sample area.
natural levels (e.g., 30-100 ppb), is known. Because of the need for accurate measurement of selenium, we included human and bovine blood serum as test samples for determining the analytical sensitivity of the NBS facility at the NSLS. Selenium in pure solutions and in NBS-SRM 1643b (trace elements in water) was also measured for comparison. Minimum detection and quantitation limits, Le., as defined by Currie ( I O ) , were calculated to characterize instrumental performance with the intent of developing a sensitive analytical method for the certification of such elements at parts per billion levels in future NBS SRMs. Other samples were also measured a t the NSLS in a conventional geometry to compare minimum detection limits with those obtained by using a similar geometry in a commercial tube-excited secondary target X-ray spectrometer. Several experiments were performed a t the NSLS by using both conventional and glancing-angle geometries to compare small-area, high-brightness synchrotron excitation with large-area, lower brightness secondary target excitation.
Several papers (1-6) have recently appeared which describe the salient features of synchrotron X-radiation for the energy-dispersive X-ray fluorescence (EDXRF) spectrometric analysis of metals a t ng/g (ppb) levels. Because of the small size and natural collimation of synchrotron radiation exiting the storage ring, X-rays can be directed with high precision at small glancing angles to the specimen and are particularly effective for use in the total reflection geometry. In addition, the high degree of polarization, high intensity, and continuous tunability of the beam energy are contributing factors for obtaining good analytical sensitivity. The principle and application of total reflection geometry in trace XRF analysis has been discussed by several authors (7, 8). Even with conventional X-ray tubes, minimum detection limits (mdl) have been reported to be in the ppb range or below. In this technique, a primary X-ray beam impinges on a thin specimen mounted on an optically flat substrate (mirror), such as a Si crystal, a t angles of incidence just below the critical angle so that total reflection of the incident beam occurs. Since the primary beam does not appreciably penetrate the substrate surface, the resulting background contribution is substantially lowered. The purpose of the present work was to evaluate the capability of the NBS Materials Science facility at The National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory for performing total reflection EDXRF analysis with monochromatic excitation. It was of interest to conduct this evaluation with appropriate test samples. A recent publication (9) points out the importance of a blood serum reference material in which the selenium content, present at
EXPERIMENTAL SECTION The instrumentation at the NBS beamlines (port X-23A) was designed at NBS for diffraction imaging, spectroscopy,small-angle scattering and energy-dispersive experiments, and diffractometry and has been described elsewhere (11). The X-ray beam energy is tunable from 5 to 20 keV with a band-pass of 0.01 % (at 8 keV), using a pair of asymmetrically cut, flat silicon (111)crystals. For the total reflection geometry, the X-ray beam was incident at glancing angles, which could be varied from parallel to the X-ray mirror surface to above the critical angle in arc second increments. The vertical and horizontal edges of the beam were controlled by slits which are part of the monochromator assembly. Thus, only the desired portion of the X-ray mirror supporting the specimen was illuminated. The incident and specularly reflected beams were continuously monitored with the aid of an X-ray vidicon camera as shown in Figure 1. In this way, precise location of the specimen was made possible and convenient. Radiographic features of the specimen droplets as small as 25 pm were easily observed. Sealed ampules containing frozen human blood serum were thawed at room temperature, and 30-pL samples were pipetted onto a Si X-ray mirror previously rinsed with high-purity concentrated nitric acid followed by high-purity water (12). The serum specimens were air-dried in a clean air hood overnight and then kept refrigerated until just before use. The specimen spot size was 3-5 mm in diameter. Two vials containing about 5 g each of proposed NBS-SRM 1598 Bovine Serum were each spiked with approximately the same amounts of an internal standard solution of Ge. One of these was also spiked with a standard solution of Se. The standard solutions were prepared from high-purity metals and oxides at least 99.99% pure. Thirty- to forty-microliter aliquots of the spiked bovine serum samples were pipetted onto Si mirrors and treated in the same manner as the human serum specimens. Twenty-microliter droplets of solutions containing 1 and 10 pg/g of Se, respectively, and NBS-SRM 164% were also
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This article not subject to U.S. Copyright. Published 1988 by the American Chemical Society
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pipetted on similar Si mirrors and allowed to dry. The Si mirrors containing the specimens were mounted on a precision micropositioning stage providing the required rotational and translational degrees of freedom. The Si(Li) detector had a resolution of about 165 eV at 5.89 keV. The detector was carefully placed at right angles to the incident X-ray beam and parallel to the plane of the storage ring to take advantage of the polarization of the X-rays in minimizing background due to Compton scattering. Radiographic images of selected- specimens, contained in the specularly reflected beam, were digitized and stored on video tape. Data acquisition was performed with a TN-2010 multichannel analyzer and the spectra stored on floppy disks. Counting times were generally 300 s or as otherwise specified. All measurements performed at the NSLS were made in air. Conventional and glancing angle geometry experiments were also performed at the NSLS for the purpose of comparing minimum detection limits with those obtained with conventional EDXRF by using a Ni secondary target emitter. The NBS EDXRF spectrometer (Kevex Model 0810) has been described elsewhere (13). The Si (Li) detector in the NBS spectrometer has a resolution of 160 eV at 5.89 keV. A 3-kW Tungsten target X-ray tube was used to excite the Ni secondary target. In experiments at the NSLS, the sample was placed at about 40" to the incident beam and 50" to the Si(Li) detector to simulate the geometry used in the EDXRF spectrometer (e.g., primary beam incident angle = 45"; characteristic radiation emergence angle = 4 5 O ) . The distance from the edge of the NSLS beam at the sample to the detector was 1.7 cm. Samples consisted of thin glass films of known composition, sputter deposited on polycarbonate and on a Si mirror, and known concentrations of manganese ion on cation-exchangeresin filters (14). Because of the high Compton scattering in air encountered when the EDXRF secondary target system is operated at high tube power with little collimation, measurements were made in vacuum to obtain optimum performance.
RESULTS AND DISCUSSIONS It was found that the NBS beamline X-23-A3 at the NSLS is well-suited for performing total reflection XRF experiments because of the wide selection of monochromatic X-ray energy combined with rapid tunability and rapid data acquisition times. The fixed exit position of the monochromatized beam as a function of energy provides both excellent control of beam position and size for specimen excitation. Fine control of the rocking angle of the sample substrate permits variation of the incident beam angle below the critical angle and is important in order to obtain optimum sensitivity for any specimensubstrate combination. Minimum detection limits (mdl) were calculated for selenium in natural blood serum and in solutions deposited on Si (111)mirrors. The beam size was adjusted in each case to just encompass the size of the sample as determined from its real-time image. These data are presented in Figure 2 and calculated as a function of glancing angle below the critical angle of silicon. The narrow band-pass of the monochromatic X-rays allows selective excitation of Se (K edge at 12.66 keV)
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Flgure 1. Schematic diagram of NBS facility at the NSLS for total
Calculated minimum detection limits for selenium as a function of glancing angle, 0, below the critical angle, !!Ic. Upper curve: 13.0 keV excitation, t = 300 s; (A)15.0 human blood serum with (0) keV excitation, t = 300 s; ( X ) 13.3 keV excitation; t = 600 s. Lower curve: ( 0 )aqueous solution (Se = 10 ppm), 15.0 keV excitation, 1 = 300 s; (+) NBSSRM 1643b trace elements in water (Se = 9.7 ppb), 13.3 keV excitation, t = 3600 s, but mdi normalized to 300 s.
Flgure 2.
at 13.0 and 13.3 keV while a t the same time preventing the excitation of a relatively high concentration of Br (K edge at 13.475 keV) in the serum. A detection limit of 8 ppb for Se in serum corresponds to a quantitation limit of 24 ppb when using a counting time of 600 s. This represents, according to Currie (IO),that concentration level that can be quantitatively measured with a 10% relative standard deviation. The mdl calculated from aqueous Se solutions is 2 ppb and is significantly lower than the mdl calculated for serum. This is because of the considerably less Compton scatter contribution from the residue left after the drying of the aqueous solutions as compared to the serum samples. The lowest concentration of Se measured was 9.7 f 0.5 ppb in NBS-SRM 1643b (see Certificate of Analysis NBS-SRM 164313). A mdl of 0.6 ppb was calculated where the specimen was counted for 1 h. This corresponds to a mdl of 2 ppb for a 300 s counting time and is in good agreement with results from pure solutions (see Figure 2). The use of a human serum specimen containing 169 ppb Se as a standard and the analysis of a natural level human serum specimen containing 94 ppb Se gave a result of 88 ppb, which is in good agreement since it is difficult to reproduce specimen size. As mentioned by Iida, et al. (6), reliable quantitative analysis can be accomplished by using an internal standard technique since this method should, in principle, compensate for such variables as specimen size and primary beam intensity, which varies as a function of time when using synchrotron radiation. The concentration of Se in bovine serum NBS-SRM 1598 was measured by using Ge as an internal standard (see Figure 3) and a result of 42 ppb was obtained. This is well above the calculated quantitation limit of 24 ppb. Therefore, the proposed NBS-SRM 1598 would appear to be an excellent candidate for analysis by this technique. Although the detection and quantitation limits calculated here for Se at least for pure solutions are solely instrumental in nature, they can truly be representative of a complete method of analysis provided that several factors are properly controlled. Most important is the reproducibility of the blank. Fortunately, Se is not a commonly found element in typical candidate mirrors. Even routine optical polising of the Si mirrors did not increase the level of Se background. This, however, is not the case, for such elements as Fe, Cu, and Zn which are present in the mirrors at detectable levels. Apart from the blank reproducibility is the eternal question of the integrity of the sample itself. Again Se is an element which is least likely to be introduced into a sample as a contaminant as contrasted with Zn, which is often present
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Table I. Comparative Minimum Detection Limits for Manganese NSLS, ng/cm2 2.5"~~ 1.0'
NSLS, ng 0.15n'b
Ni target, ng/cmz Ni target, ng
3.6"~~ 2.6'1~ 4.6'
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Flgure 3. EDXRF spectra taken at the NSLS using total reflection geometry: (A) proposed NBS SRM 1598 bovine serum contains only Ge spike, Se peaklbackground = 1.54; (B) proposed NBS SRM 1598 bovine serum contains both Ge and Se spikes; (C) NBS SRM 1643b trace elements in water (Se = 9.7 ppb), Se peak/background = 3.3; (D) Si X-ray mirror only. Note, the energy scale for spectra A and B is not the same as for C and D.
in vial caps used for the encapsulation of the serum samples (15). We now plan to analyze a series of NBS-SRM 1598 bovine serum samples for selenium by using the internal standard technique. Assessment of any systematic errors will then be made by comparison of results obtained from other NBS analytical methods such as isotope-dilution mass spectrometry. C o m p a r i s o n with C o n v e n t i o n a l EDXRF. Since the NBS EDXRF spectrometer was of conventional design it was of interest to compare relative sensitivities with NSLS excitation in the same geometry. Although it would have been desirable to make this comparison with a standard thin film containing Se, we did not have such a sample. Instead, we chose to use NBS-SRM 1832 thin films deposited on polycarbonate and on Si mirrors since they are well-characterized with respect to elemental composition and are quite uniform. Manganese was selected for measurement because it can be efficiently excited at 7.50 keV at the NSLS, and also with a Ni secondary target (Ni K a = 7.47 keV) in the EDXRF spectrometer. Since the glass film on a polycarbonate substrate has low mass thickness, high tube power can be used to excite the secondary target and large solid angles can be employed without causing saturation of the X-ray detector. Because of the small size of the NSLS beam as compared to that from the secondary target, it is useful to express the mdl also in terms of absolute mass (see Table I). In Table I are presented comparative rndl for manganese in units of areal density (i.e., ng/cm2) which are more appropriate for purposes of this comparison. Although the mdl(2.6 ng/cm2) appear comparable for both NSLS and secondary target excitation experiments in the conventional geometry, the area of the beam intercepted by the sample in the secondary target EDXRF spectrometer is about 100 times greater than that intercepted by the sample a t the NSLS. Also, the detector aperture of the secondary target system has an effective area 5.7 times greater. Therefore, the mdl of 2.6 ng/cm2 is very close to being optimum for the secondary target system. In terms of the minimum detectability of mass, however, the mdl determined from the NSLS experiment was still about 800 times lower
"Thin glass film on polycarbonate;mass loading = 51.8 fig/cm2; Mn = 1.54 fig/cm2. *NSLS 7.50 keV, conventional geometry; detector-to-sample distance = 1.7 cm; beam area = 0.058 cm2; detector collimator = 3 mm. 'EDXRF with Ni secondary; W tube = 45 kV, 40 mA; detector-to-sample distance = 3.94 cm; beam area = 6.15 cm2; detector collimator = 3 mm. dSame as c above except detector collimator = 7 mm. 'Thin glass film on polycarbonate; mass loading = 27.6 pg/cm2; Mn = 0.83 pg/cm2; NSLS 7.50 keV, glancing angle geometry. /Thin glass film on Si (111)about 800 %, thick (27.6 fig/cm2); total reflection geometry; B / B , = 0.851. gSA-2 ion exchange paper; Mn = 0.064 fig/cm2;NSLS 7.50 keV; glancing angle geometry; t , = 600 s. hSame as g above except in conventional geometry. 'SA-2 paper as in g; EDXRF with Ni secondary; W tube = 30 kV, 30 mA. (i.e., 125/0.15) than that from the secondary target system. It should be pointed out also that the NSLS experiment was not optimized since it was more convenient to work in air (e.g., absorption of 7.50 keV primary X-rays over the distance employed at the NSLS was about 50%). One way to increase the solid angle in the NSLS experiment, since one cannot change the source size, is to irradiate the specimen at a glancing angle in essentially the same geometry as used in the total reflection experiment. With glancing angle excitation the mdl decreased by about 2.5 times. This is about the same mdl obtained when using the total reflection geometry (Le., 0.7 ng/cm2). Minimum detection limits measured from standards consisting of manganese ion on cation-exchange paper were significantly higher in the conventional geometry for both NSLS and secondary target experiments, as would be expected from the substantially higher Compton scattering due to the much greater mass thickness of the cation-exchange paper substrate compared to polycarbonate. This of course is a limiting factor in obtaining lower mdl. Glancing angle experiments at the NSLS using the same samples of manganese ion on paper again show improved detection limits compared to the conventional geometry because of the greater solid angle. These experiments emphasize the importance of a small, high-brightness X-ray source for performing ultratrace microanalysis in the total reflection geometry. This technique is similar in concept to other microtechniques where the important consideration in terms of sensitivity is the number of analyte atoms per unit volume of sample probed by the X-ray beam. Although this technique was examined in light of possible applications to nondestructive analysis, one should not lose sight of the fact that with appropriate sample dissolution, matrix separation, and concentration of the analyte in small volumes, the sensitivity can be greatly increased and the analysis of samples containing trace elements at subparts-per-billion levels should be possible providing always, of course, that the blank is acceptable. Registry No. Se, 7782-49-2;HzO, 7732-18-5. LITERATURE C I T E D (1) Sparks, C. J., Jr. Synchrotron Radiation Research; Winick, H., Doniach, S., Eds.; Plenum: New York, 1960; Chapter 14. (2) Knoechel, A,; Peterson, W.; Tolkiehn, G. Nucl. Instrum. Methods Phys. Res. 1983, 208,659-665. (3) Giifrich. J. V.; Skelton, E. F.; Qadri, S. B.; Kirkland. J. P.; Nagei, D. J. Anal. Chem. 1983, 55, 232-240. (4) Iida, A,; Sakurai. K.; Matsushita, T.; Gohshi, Y. Nucl. Instrum. Methods Phys, Res ., Sect. A 1985, 228,556-563.
Anal. Chem. 1988. 60, 687-691 (5) Jaklevic, J. M.; Giauque, R. D.. Thompson, A. C. Nucl. Instrum. Methods Fhys. Res., Sect. E 1985, E 1 0 / 1 1 , 303-308. (6) Iida, A.; Yoshinaga, A,; Sakurai, K.; Gohshi, Y. Anal. Chem. 1988, 58,394-397. (7) Aiginger, H.; Wobrauschek, P. I n Advances in X-Ray Analysis; Barrett, C. s., Predecki, P. K., Leyden, D. E., Eds.; Plenum: New York, 1985; Vol. 28, pp 1-10, ( 8 ) Knoth, J.; Schwenke, H. Fresenius’ Z.Anal. Chem. 1980, 301, 7-9. (9) Ihnat, M.; Wolynetz, M. S.;Thomassen, Y.; Verlinden, M. Pure Appl. Chem. 1986, 58, 1063-1076. (10) Currie. L. A. X-Ray Fluorescence Analysis of EnvironmentalSamples; Dzubay, T. G . , Ed.; Ann Arbor Sclence: Ann Arbor, MI, 1977; Chapter 25. (11) Spal, R.; Dobbyn, R. C.; Burdette, H. E.: Long, G. G.; Boettinger, W. J.; Kuriyama, M. Nucl. Instrum. Methods Phys. Res., Sect. A 1984, 222, 189-192. (12) Kuehner, E. C.; Alvarez, R.; Paulsen, P. J.; Murphy, T. J. Anal. Chem. 1972, 44, 2050.
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(13) Pella, P. A,; Myklebust, R. L.; Darr, M. M.; Heinrich, K. F. J. NBS Special Report No. 77-121 1; US. Government Printing Office: Washington, DC, June 1977. (14) Kingston, H.; Pella, P. A. Anal. Chem. 1981, 53,223-227. (15) Moody, J. R.; Epstein, M. S. “Container Caused Contamination of SRM’s in Serum and Urine”; NBS Report of Analysis, February 1984.
RECEIVED for review July 13, 1987. Accepted September 11, 1987. Certain commercial equipment, instruments, or materials are identified in this report to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.
Determination of Trace Metals in Marine Biological Reference Materials by Inductively Coupled Plasma Mass Spectrometry Diane Beamhemin,* J. W. McLaren, S. N. Willie, and S. S. Berman Analytical Chemistry Section, Chemistry Division, National Research Council of Canada, Ottawa, Ontario, Canada K I A OR9
Inductively coupled plasma mass spectrometry ( ICP-MS) was used for the analysls of two marine biological reference materlals (dogfish liver tissue (DOLT-1) and dogflsh muscle tissue (DORM-I)). The materlais were put Into solution by digestion In a nitric acld/hydrogen peroxide mixture. Thirteen elements (Na, Mg, Cr, Fe, Mn, Co, NI, Cu, Zn, As, Cd, Hg, and Pb) were then determined. Accurate results were obtained by standard addltlons or isotope dllution technlques for ail of these elements in DORM-I and for ail but Cr In DOLT-1.
Inductively coupled plasma mass spectrometry (ICP-MS) is a rapidly expanding technique, the development and many current features of which have been summarized in four recent review articles (1-4). Although the number of applications is increasing in many areas, there are still few of them devoted to the analysis of biological materials. While concentrations of many of the trace metals of interest in these materials are low enough to necessitate a minimum of dilution in the sample dissolution procedure, concentrations of certain concomitant elements (e.g. K, Na, Ca, and Mg) can be high enough to cause significant suppression or enhancement effects (5,6). ICP-MS appears to be more susceptible to interferences by such elements than would have been predicted on the basis of previous experience in inductively coupled plasma atomic emission spectrometry (ICP-AES) (3-5). While the mechanism of these effects is not yet fully understood, processes additional to those occurring in the ICP are clearly involved. The generic term “ion sampling effects” was recently proposed (7) to describe phenomena unique to ICP-MS which may be occurring in the interface region between the two ion sampling orifices (i.e. the “sampler” and the “skimmer”) and in the ion optics between the skimmer and the quadrupole mass filter. Furthermore, ICP-MS is also more susceptible than initially expected to isobaric interferences from polyatomic species arising from the plasma (8),the acids used in sample preparation (9,lO) or the sample itself (e.g. ref 10). Because of the low concentrations of many of the elements of interest in biological
materials, the dissolution procedure is an especially critical step. Three reports on the analysis of reference biological materials (11-13) by ICP-MS have appeared. Pickford and Brown (11) described the analysis of three NBS standard reference materials (orchard leaves, bovine liver, and oyster tissue) and two other bovine liver materials by ICP-MS and ICP-AES. They had problems of poor precision with ICP-MS compared to ICP-AES using external calibration (i.e. the use of reference solutions) only and recommended the use of isotope dilution. They also reported matrix suppression not seen in ICP-AES. Ward and co-workers (12) described the analysis of three reference materials to assess errors associated with the matrix, using holmium as an internal standard. They noted some problems of polyatomic isobaric interferences at low anal@ levels. Finally, Munro and co-workers (13)looked at two NBS reference materials and one other proposed reference material by ICP-MS (using Bi and Y as internal standards) and electrothermal atomization atomic absorption spectrometry (ETA-AAS). They noted that ICP-MS was less precise than ETA-AAS, especially a t the lower levels. However, none of these studies used either the method of standard additions or the isotope dilution technique which have been reported to be less susceptible to ion sampling effects than external calibrations (e.g. ref 14 and 15). The recent development of two marine biological reference materials (16) in this laboratory (a dogfish liver tissue with the acronym DOLT-1 and a dogfish muscle with the acronym DORM-1) provided an opportunity to assess the performance of ICP-MS in the analysis of two biological tissues which were characterized at the same time by a number of other methods, including atomic absorption spectrometry and neutron activation analysis. Trace elements were determined by isotope dilution ICP-MS whenever possible and additional analyses were performed by the method of standard additions.
EXPERIMENTAL SECTION Instrumentation. The inductively coupled plasma mass spectrometer used for this work was the ELAN 250 SCIEX Division of MDS Health Group, Ltd. (Thornhill, Ontario, Canada).
0003-2700/88/0360-0687$01.50/00 1988 American Chemical Society
