Anal. Chem. 1985, 57, 1691-1694
1691
Energy Dispersive X-ray Fluorescence Spectrometry with Direct Excitation at Picogram Levels Claude Ruch
Siemens AG, Geratewerk Karlsruhe, Karlsruhe, West Germany Faramarz Rastegar, Robert Heimburger, Eddie A. Maier, and Maurice J. F. Leroy*
Laboratoire de Chimie Minbrale, Unit6 Associeb 405 du C.N.R.S.,Ecole Nationale Sup6rieure de Chimie de Strasbourg, Strasbourg, France
A Mo high-power X-ray tube with an optlcal polnt focus, followed by a thlck Mo fliter, lrradlates a small and thin sample conslstlng of 50 pL of llquld deposlted and evaporated on a 4 pm thlck polypropylene film. The X-rays emltted by thls sample are detected by a SI(L1) detector placed close to the sample. This arrangement allows one to reach, with a very convenlent routlne measurement, detection llmlts In the nanogram per mlllliter range with total mass at picogram levels. Already hundreds of samples have been measured under these condltlons. A more sophlstlcated sample preparatlon (thlnner fllm, very good posltlonlng of the sample, or more than 50 pL of solutlon) decreases the detection llmlts to plcogram per mllllllter levels, Le., plcogram of total mass.
A prototype has been built with the aim of determining trace elements in samples available in very small quantities. These samples are usually encountered in medicine or in biological experiments on mice. The method is also important for large samples when preconcentration techniques are required. In this case the high sensitivity of the apparatus and the very low limits of detection make possible a direct determination, and thus avoid the contamination risk of the preconcentration techniques ( I , 2). In order to avoid P b contamination of the sample in the spectrometer, we substituted Ta for P b in all X-ray protection devices. The manipulation of the sample consists only in the deposition of the solution to be analyzed on a polypropylene thin film. The purpose of this work is the determination of the apparatus characteristics regarding the limits of detection in multielemental trace metals analysis.
EXPERIMENTAL SECTION The Instrument. A schematic view of the Siemens prototype is shown in Figure 1. The X-ray tube and the Si(Li) detector are put on the same side of the sample plane, this also allows the measurement of solid samples and is a great advantage compared to the total reflection method measuring only thin samples. The arrangement could be modified by placing the X-ray source on one side of the sample and the detector on the other side so that the X-ray fluorescence would be measured by transmission through the sample. The arrangement would in some cases improve the detection limit for thin-film samples but would not allow the measurement of powders or solid samples. The X-ray power supply allows a high voltage of 60 kV and a power of 4 kW. The high stability of 0.1% permits good reproducibility over a period of many months (Siemens K 810). The fine structure X-ray tube (Siemens AGF.Mo 4ST) is a water-cooled high-power tube that is easily interchangeable, it has a Mo anode with four beryllium windows. The chosen optical point focus of 0.4 mm X 0.8 mm offers the best geometry for the irradiation of a small sample. The region of shadowy irradiation on the sample is reduced to a minimum and the signal to background ratio is a maximum for all measurements. The tube was set at 40 kV and 30 mA. 0003-2700/85/0357-169 1$01S O / O
The manual sample changer with interchangeable filters between the X-ray tube and the sample also has interchangeable collimators between the X-ray tube and the sample and between the sample and the Si(Li) detector; this allows an optimal measurement of samples of different sizes. A 150 pm thick Mo filter largely eliminates the X-ray continuum so that the irradiation of the sample is similar to that of a secondary target of Mo. The vacuum in the X-ray path is only necessary in order to avoid the scattering in air and the fluorescence of the different air components in the case of samples with very small total mass, like aqueous solutions. In this work all the measurements are done under vacuum. This improves by a factor of 2 the signal to noise ratio (the scattering of Mo photons is two times smaller). The 3 mm thick Si(Li) crystal has a 135 8, thick gold window (measured with a secondary target spectrometer before mounting (3))and an active area reduced from 12.5 mm2 to 3 mm2 to improve the peak to valley ratio; the tailing under the peak is coming essentially from an incomplete charge collection on the edge of the detector. An advantage of the small active area of the detector associated to the small irradiated area of the sample lies in the weak divergence of the Compton scattered Mo K a so that the Compton peak is narrow. The 7.5 pm thick beryllium window of the cryostat allows the measurement of light elements (Siemens F 7 ) (4). The pulsed optical feedback preamplifier with a shaping time of 10 p s (modified Nuclear Semiconductor NSI 513 amplifier) gives a fwhm of 145 eV for the Mn Ka energy (with a Tracor Northern TN 2000 multichannel analyzer). The whole instrument is installed in a room with regulated temperature within 1 O C to stabilize the electronics. The deconvolution of peak overlap with a least-squares fitting requires a iO.O1% stability of the whole electronics. Sample Preparation. A 50-pL portion of liquid sample is deposited with an Eppendorf pipet on the thin film and dried with air at room temperature, protected from dust contamination. Only tridistilled water is used for the preparation of solutions. The use of rubber gloves is avoided because of a high zinc contamination risk. Sample Support. A 4 pm thick polypropylene film (Bollor6) is stretched between two plastic rings, and the dust on the film is removed in a clean air stream. The electrostatic charge on the polypropylene film which attracts dust particles can be removed with a piezo stick normally used for cleaning records.
RESULTS AND DISCUSSION Blank Film. Many commercial polymer films have metallic impurities and cannot be used for a multielemental analysis (5). A spectrum of our blank film is reported in Figure 2. The Au La and Lo lines are produced by the impact of the Mo radiation on the gold contact of the Si(Li) crystal. Reducing the thickness of the 135 8, gold layer would reduce the intensity of these lines. The iron peak may be attributed to impurities in the Be window of the Si(Li) cryostat. This peak is also reported in the literature about the spectrum of a high-purity carbon samples (6). We analyzed a 7.5 pm thick Be window that came from the same batch we used for our cryostat. Figure 3 confirms that Fe is the main impurity; the Ti peak is attributed to an impurity in the film. 0 1985 Arnerlcan Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 8, JULY 1985
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Flgure 1. Schematic of the X-ray spectrometer.
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100
-
100
200
thickness of
300
M o (pn)
Flgure 4. Variation of the Pb and Se detection limits in 50-pL monoelement samples with the thickness of the Mo filter.
Energy
(kev)
Flgure 2. Spectrum of the 4 p m thick polypropylene film used as sample support.
Energy
(ked
Flgure 3. Spectrum of a 7.5 pm thick Be window.
Optimization of the Filter Thickness. The purpose of the Mo filter between the X-ray tube and the sample is to reduce the Bremsstrahlung emission by the Mo anode of the tube in order to get a quasi-monoenergetic excitation and therefore improve the signal to background ratio. The best thickness of the filter changes with the sample mass. For massive solid samples like pellets, a 300 pm thick Mo filter gives a background due only to an incomplete charge collection in the Si(Li) crystal, the contribution of the scattered Bremsstrahlung on the sample being negligible. In that case, the background is in the order of magnitude of a secondary target setting (7). With samples of small mass, a 300 pm thick filter does not allow saturation of the amplifier with the 30% dead-time which is used for each measurement. With thinner filters the contribution of the Bremsstrahlung appears as tailing of the Mo Ka Compton peak (8). This tailing increases when the thickness of the filter decreases. Furthermore, the best thickness value is also dependent on the energy of the fluorescence line to be measured. The detection limits in aqueous solutions for P b and Se have been measured for different filter thicknesses (Figure 4). In these experiments the collimators were optimized for
the deposit of 50-pL sample solutions. Detection limits of 68 pg and 34 pg, respectively, for Pb and Se were found using 80 and 100 pm Mo filters, a 1000-s live counting time, and a 3~7confidence level. These values correspond to concentrations of 1.4 ng/mL in Pb and 0.7 ng/mL in Se for a 50-pL sample and can be routinely obtained. Several modifications can be made to improve the detection: The detection can be improved by increasing the quantity of deposit by drying successively 10 drops of 50 p L of solution (waiting each time for evaporation),but the solution must have a low matrix to obtain a detection limit of 70 pg/mL for Se. Another way of reducing the detection limit is by use of a 0.4-pm film for the 4 pm polypropylene film (9). This would divide the background by 10 and the detection limits by 10 (Figure 4) and would lead with 10 deposited drops to a detection limit for Se of 20 pg/mL. The detection limit in mass can be reduced by depositing 5 pL and by using a fine collimator. In this case, the best positioning of the sample is difficult to obtain, but the background is reduced by a factor 30 with a 4-pm film. A thinner film would give a theoretical detection limit of about 1 pg. Up to now we have not encountered solutions available only in 5-pL quantities and generally low concentrations are required rather than low mass. For all of the following measurements a 150 pm thick filter was chosen. These conditions are not the best for Se but they greatly improve the results in the 11-15 keV energy range. The intensities obtained with this filter are 2 orders of magnitude greater than that for the secondary target spectrometer (IO). Detection Limits. Figure 5 gives the X-ray excitation curves for infinite thin samples for measurement of the different elements with the commonly used conditions (50 pL, 150 pm Mo, 40 kV, 30 mA, 1000 s). The results of the identification routine of the Tracor 2000 software are reported on the Y axis. This program performs an automatic elemental identification of the registered spectra and calculates a quantity which is representative of the net counts accumulated in each recognized peak. These curves are useful for the control of the different standard solutions used in the laboratory. The detection limits with the routine conditions are shown in Figure 6 with a 3a confidence level. All the modifications discussed earlier for improving these values are valid. Matrix Influence. All results presented concern the ideal but artificial situation of elements soluble in tridistilled water.
ANALYTICAL CHEMISTRY, VOL. 57, NO. 8, JULY 1985
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Flgure 7. Influence of the NaCl concentration on the detection limit of Pb (measured in air).
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Flgure 5. Mo X-ray excitation curves.
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Flgure 8. Spectrum of 200 pL of tap water.
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Figure 6. Detectlon limits determined with multielement samples.
Figure 7 gives the influence of NaCl on the detection limit of Pb. Measurements are not performed under vacuum because of the high concentration of NaC1. In addition, under vacuum the films obtained with concentrated solutions are broken into separated particles. We observe that the detection limits are raised by a factor of 1.4 when the NaCl concentration is increased from 0 to 1%. In the total reflection method the detection limit for P b in
the same conditions is increased from 0.07 ng/mL to 30 ng/mL (factor 400) (11). In thick solid samples (e.g., trace elements in organic matrix), the detection limits obtained with our prototype are comparable to those reported for a spectrometer using secondary targets or the Bragg reflection in Cartesian geometry (12,13). These limits are twice lower with an apparatus using secondary targets with polarization (10, 14) or without polarization but with a ratio for the Compton Mo Koi to the background of 6000 in the 7-keV region (7). This will be precisely discussed in a future paper. Tap Water. Figure 8 presents the spectrum of 200 WLof tap water. The fittings in Tables I and I1 are obtained by using the method of known additions in tridistilled water and in tap water. The standard deviations of both fittings are 1 ng/mL; in Table I1 the negative value of X for Y = 0 gives a concentration of P b in tap water of 10 ng/mL.
CONCLUSION The instrument described earlier, measures trace elements without any preconcentration method in the multielemental mode with great reliability (15, 16). The thin film method
Anal. Chem.
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T a b l e I. Fitting of Pb in 50 p L of T r i d i s t i l l e d W a t e r "
entered Y
entered X
fitted X
7'0 error
68.0000 156.0000 301.oooO 397.0000 573.0000
10.0000 20.0000 40.0000 50.0000 75.0000
9.3259 20.6303 39.2569 51.5889 74.1977
6.7405 -3.1517 1.8575 -3.1779 1.0696
X = (5.90722X 10-l)+ (1.28459X lO-')Y residual variance correlation for fit
1.524095 54 0.99912704
X is the Pb concentration (ng/mL). T a b l e 11. Fitting of Pb in 200 p L of Tap W a t e r by t h e M e t h o d of Known Standard A d d i t i o n s with Sr a s I n t e r n a l
Standard"
entered Y
entered X
fitted X
% error
0.0020 0.0032 0.0048 0.0062 0.0097 0.0141 0.0178
0.0000 10.0000 20.0000 30.0000 50.0000 75.0000 100.0000
2.1486 9.5447 19.4063 28.0351 49.6073 76.7264 99.5312
4.5520 2.9683 6.5494 0.7854 -2.3019 0.4687
X = -(LO11782 X 10) + (6.16345X 103)Y residual variance correlation for fit
2.478317 24 0.999217 53
" X is the added Pb concentration (ng/mL). with infinitely thin samples excited under a 45O incident beam presents much lower matrix effects than the total reflection method. In addition, with our method, inhomogeneous sample depositions with or without internal standards do not affect the results of the analysis. A flexibility of the excitation is possible
by changing the anode material of the X-ray tube. Low cost automation of the whole procedure is possible.
ACKNOWLEDGMENT We are deeply grateful to the following persons from the Siemens Company: P. Glasow for his management in the realization of the high performing Si(Li) crystal, P. Banerjee for the management in the construction of the low noise electronics, N. Broll for his pioneering work in the direct excitation mode with a high power X-ray tube, A. Bayer for helpful discussions during more than 13 years in building the detector head and the spectrometer chamber, U. Jecht and M. Or6ans for their support to the outcome of the collaboration between the E.N.S.C.S. and Siemens Karlsruhe. LITERATURE CITED Robberrecht, H.; Van Grleken, R.; Shanl, J.; Barak, S. Anal. Chim. Acta 1982. 736. 285-291. Versiek, J.'; Barbier, F.; Cornelius, R.; Hoste, J. Talanta 1982, 29, 973-984. Ruch, C.; Broll, N., unpublished data, 1980. Charblt, P.; Ruch, C. Slemens Anal. Appl. Note 1974, No. 110. Russell, S. B.; Schulte, C. W.; Faiq, S.; Campbell, J. L. Anal. Chem. 1981, 53, 571-574. Van Espen, P.; Nullens, H.; Adams, F. C. 2.Anal. Chem. 1981, 285, 215-225. Charblt, P.; Ruch, C. Siemens Anal. Appl. Note 1974, Note 7 7 1 . Broil, N., private communication. Barrus, D. M.; Blake, R. L. Rev. Scl. Instrum. 1977, 4 8 , 116-117. Ruch, C. These de 3' Cycle, Unlversit6 Louis Pasteur, Strasbourg, France, 1980. Knoth, J.; Schwenke, H. Fresenius 2.Anal. Chem. 1980, 307, 7-9. Blsgard, K.; Laursen, J.; Nlelsen, B. X-Ray Spectrom. 1981, 10, 17-24. Wobrauschek, P., Aiginger, H. X-Ray Spectrom. 1983, 12, 72-76. Standzenleks, P.; Sebln, E. Nucl. Instrum. Methods 1979, 165, 63-65. Rastegar, F.; Maier, E. A.; Heimburger, R.; Christophe, C.; Ruch, C.; Leroy, M. J. F. Clin. Chem. (Winston-Salem, N.C.) 1984, 30, 1300- 1303. Maler, E. A.; Rastegar, F.; Helmburger, R.; Ruch, C.; Pelletler, A.; Maler, A.; Leroy, M. J. F. Clin. Chem. (Winston-Salem, N.C.), In press.
RECEIVED for review December 12,1984. Accepted March 18, 1985.
New Approach to Phase and Modulation Resolved Spectra Enrico Gratton
Physics Department, University of Illinois at Urbana-Champaign, 11 10 West Green Street, Urbana, Illinois 61801 David M. Jameson*
Pharmacology Department, University of Texas Health Science Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75235
Time domain fluorescence spectrometry offers a versatlle and powerful approach to the analysis of heterogeneous emittlng systems. I n this paper we describe a new approach, based on software, to the acqulsitlon of phase and modulatlon resolved spectra. Mlxtures of fiuorophores with dlfferent ilfetimes can be analyzed in real time to glve the Individual excitation or emisslon spectra. Examples of two- and threecomponent mixtures are given and comparlsons are made wlth the commercially avallable hardware approach.
Fluorescence spectrometry is presently being applied to increasingly complex systems in the physical, chemical, and
biological sciences. The degree of sample heterogeneity in such systems is often a fundamental consideration. Information on the nature and extent of heterogeneity is important whether the fluorophore is used to probe its molecular environment or whether a compositional description is sought for analytical purposes. An extensive research effort has been expended on the methodologies of analysis of heterogeneous emissions. The application of fluorescence spectrometry to the identification of petroleum oils and similar materials began with the work of Parker and Barnes (1). The original matrix analysis approach of Weber (2) has been greatly extended and developed into a powerful rank analysis method, the excitation-emission matrix or EEM (3). The EEM approach and the related
0003-2700/85/0357-1694$01.50/00 1985 American Chemical Society
