Anal. Chem. 2000, 72, 4289-4295
Detection and Quantification of Metals in Organic Materials by Laser-SNMS with Nonresonant Multiphoton Ionization A. Schnieders* and A. Benninghoven†
Physikalisches Institut der Universita¨t Mu¨nster, Wilhelm-Klemm-Strasse 10, D-48149 Mu¨nster, Germany
We have shown that the sensitive detection and in favorable cases the quantification of metals in organic materials by laser-SNMS with nonresonant multiphoton ionization (NRMPI) is possible. As a model system, sputterdeposited submonolayer coverages of metals on polymer surfaces (polycarbonate, poly(vinylidene chloride), polyimide) were investigated. By use of these samples, relative sensitivity factors and detection limits of several metals (Be, Cr, Mn, Fe, Co, Ni, Mo, W) were determined using laser-SNMS with NRMPI. The relative sensitivity factors for this kind of sample show a high level of agreement with those for metals sputtered from alloys. The detection limits (∼1 ppm of a monolayer) are almost the same as for inorganic matrixes such as Si or GaAs. Laser-SNMS with NRMPI was also used for the determination of the elemental composition of the active centers of metalloproteins (namely, the purple acid phosphatases extracted from sweet potatoes and from red kidney beans). These results have shown the ability of laser-SNMS to detect metal atoms bound to organic macromolecules with an atom concentration as low as 1 ppm. In comparison to TOF-SIMS, laser-SNMS is more sensitive for metal detection in organic matrixes, since the secondary ion yields observed for these matrixes are reduced compared to matrixes optimized for high secondary ion emission, such as, for example, highly oxidized surfaces. The detection and quantification of metals in organic materials is important in a variety of biological and medical applications, since metals are essential for a large number of biochemical processes. Whenever nature has a difficult task to perform, a metal atom, a constellation, or a cluster of such atoms is invariably employed. In addition, many other elements at very small concentrations can play significant roles in nutrition, toxicity, and biological function. To understand the manner of action of all these systems, it is necessary to determine the active elements and to locate and quantify them in their natural environment. This paper therefore addresses two aspects of highly sensitive, quantitative detection of metals in and on organic matrixes, namely, the surface analysis of metals in the uppermost monolayer on an organic substrate and also the detection of trace metals bound to organic * Corresponding author: (e-mail)
[email protected]; (phone) +49251-8333611; (fax) +49-251-8333682. † E-mail:
[email protected]. 10.1021/ac000118y CCC: $19.00 Published on Web 08/11/2000
© 2000 American Chemical Society
macromolecules in near-surface regions. Because of their different natures, these two analytical tasks are well suited to determine the possibilities and limitations of laser-SNMS with nonresonant multiphoton ionization (NRMPI) for detection and quantification of metals in organic matrixes. Metals on Polymer Surfaces. As a model system for the surface detection of metals on organic materials, sputter-deposited submonolayer coverages of metals on polymer surfaces were investigated. Sputter deposition has been chosen as preparation technique1 because it can provide surface coverages of different metals in various combinations with surface densities ranging between 109 and 1015 cm-2. It guarantees a homogeneous distribution of metal atoms on the surface relative to the length scale of the primary ion beam diameter (in this investigation LPI ) 100 µm). The amount of deposited material can be easily controlled by the total sputtering ion dose applied to the sputter target, provided that sputter equilibrium was reached. By use of these systems, relative sensitivity factors and detection limits of several metals for laser-SNMS with NRMPI were determined. Metalloproteins. In the second part of this investigation, laserSNMS with NRMPI was used to determine the elemental composition of the active centers of metalloproteins, namely, the purple acid phosphatases (PAPs) extracted from sweet potatoes and from red kidney beans. Purple acid phosphatases2-5 are transition-metal-containing glycoproteins with an Fe(III)-M(II) center in their active site. Plant PAPs have been reported to be homodimeric glycoproteins with a molecular weight of ∼110 000 u. The PAP extracted from red kidney bean seeds (kbPAP)6 is the best-characterized plant PAP. It contains an Fe(III)-Zn(II) metal center in each subunit. Recently, the three-dimensional structure of kbPAP was determined.7,8 The molecule, with a cysteine bridge connecting the two subunits, was found to have (1) Schnieders, A.; Schro¨der-Oeynhausen, F.; Burkhardt, B.; Ko¨tter, F.; Mo¨llers, R.; Wiedmann, L.; Benninghoven, A. In Secondary Ion Mass Spectrometry SIMS X; Benninghoven, A., Hagenhoff, B., Werner, H. W., Eds.; John Wiley & Sons: Chichester, U.K., 1997; pp 649-652. (2) Doi, K.; Antanaitis, B. C.; Aisen, P. Struct. Bonding 1988, 70, 1-26. (3) Vincent, J. B.; Olivier-Lilley, G. L.; Averill, B. A. Chem. Rev. 1990, 90, 14471467. (4) Que, L., Jr.; True, A. E. In Bioinorganic Chemistry; Lippard, S. J., Ed.; Progress in Inorganic Chemistry 38; John Wiley & Sons: Chichester, U.K., 1990; pp 97-200. (5) Klabunde, T.; Krebs, B. Struct. Bonding 1997, 89, 177-198. (6) Beck, J. L.; McConachie, L. A.; Summors, A. C.; Arnold, W. N.; De Jersey, J.; Zerner, B. Biochim. Biophys. Acta 1986, 869, 61-68. (7) Stra¨ter, N.; Klabunde, T.; Tucker, P.; Witzel, H.; Krebs, B. Science 1995, 268, 1489-1492.
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the shape of a twisted heart with overall dimension 4.0 nm by 6.0 nm by 7.5 nm. The two dinuclear metal centers are 3.5 nm apart and lie at the bottom of a broad pocket formed by both monomers. In solutions, they are exposed to the solvent. The class of plant PAPs includes also the PAP extracted from sweet potato tubers (spPAP).9,10 It resembles the kbPAP as to the spectroscopic data and the molecular properties (homodimeric glycoprotein with a molecular mass of ∼110 000 u, similar amino acid sequence). But the nature of the metal center in this enzyme has been controversial. Early reports11,12 on the spPAP indicated the presence of one Mn atom per dimer. Later reports13 from the same laboratory demonstrated that the Mn could be removed and that an Fe-substituted enzyme could be prepared. In contrast, reports from another laboratory14 on what appears to be the same enzyme have shown that the spPAP contains two Fe atoms per dimeric protein and that Mn is a contaminant that can be removed with no effect on activity. To clarify the controversial discussion about the metal content of spPAP and in order to understand the structural and biochemical properties of the enzyme Durmus et al.15 improved the purification procedure of Hefler and Averill14 and performed spectroscopic investigations on the enzyme. They isolated homogeneous spPAP with a yield of ∼2 mg from 20 kg of sweet potato tubers. For the metal detection, synchrotron radiation excited X-ray fluorescence spectroscopy (SR-XRF) was used. Independently of the SR-XRF measurement, a small amount (some microliters of a 10-4 M solution in H2O) of the same spPAP extraction was used for the laser-SNMS measurements described in this paper. kbPAP was used as reference sample in this investigation, because the composition of the active center of kbPAP is well known. The investigations of the PAPs show the ability of laser-SNMS to detect metal atoms bound to organic macromolecules with an atom concentration down to 1 ppm and extremely low sample consumption. The influence of the preparation on the detection probability was also investigated. EXPERIMENTAL SECTION Instrumentation. The measurements presented in this paper were performed in a reflectron-type time-of-flight mass spectrometer developed at the University of Mu¨nster (TOF III type16). Both techniques, laser-SNMS and TOF-SIMS, are implemented in this instrument. It is equipped with a 10-keV electron impact primary ion source, which is coupled to a 90°-deflection pulsing unit. For mass analysis of the secondary particles, a double-stage (8) Klabunde, T.; Stra¨ter, N.; Fro ¨hlich, R.; Witzel, H.; Krebs, B. J. Mol. Biol. 1996, 259, 737-748. (9) Uehara, K.; Fujimoto, S.; Taniguchi, T. J. Biochem. 1974, 75, 627-638. (10) Uehara, K.; Fujimoto, S.; Taniguchi, T.; Nakai, K. J. Biochem 1974, 75, 639-649. (11) Fujimoto, S.; Ohara, A.; Uehara, K. Agric. Biol. Chem. 1980, 44, 16591660. (12) Sugiura, Y.; Kawabe, H.; Tanaka, H.; Fujimoto, S.; Ohara, A. J. Biol. Chem. 1981, 256, 10664-10670. (13) Kawabe, H.; Sugiura, Y.; Terauchi, M.; Tanaka, H. Biochim. Biophys. Acta 1984, 784, 81-89. (14) Hefler, S. K.; Averill, B. A. Biochem. Biophys. Res. Commun. 1987, 146, 1173-1177. (15) Durmus, A.; Eicken, C.; Sift, B. H.; Kratel, A.; Kappl, R.; Hu ¨ ttermann, J.; Krebs, B. Eur. J. Biochem. 1999, 260, 709-716. (16) Schwieters, J.; Cramer, H.-G.; Heller, T.; Ju ¨ rgens, U.; Niehuis, E.; Zehnpfenning, J.; Benninghoven, A. J. Vac. Sci. Technol. 1991, A9, 2864-2871.
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Figure 1. Scheme of the sputter deposition device. The sample surface is positioned at a distance r from the sputter target and an angle θo relative to the normal of the target.
reflectron is used in order to focus the energy spread in second order. The instrument has some free ports for mounting of additional equipment, e.g., the sputter deposition device described in the following section. In the laser-SNMS mode, long primary ion pulses with a pulse width of ∼1 µs and high pulse currents of ∼100 nA are used. Suppression of the sputtered positive secondary ions is achieved by a repulsive potential, which is applied to the extractor during the primary ion pulse and the time delay for the laser pulse. The generated negative secondary ions are suppressed by the analyzer, which is held at a negative potential for the analysis of the positive photoions. Almost simultaneously with the laser pulse, the extraction voltage is switched on to accelerate the generated photoions over the extraction distance into the analyzer. Since up to several hundred photoions of one mass can be generated in one cycle in the laser-SNMS mode, the signal must be detected using an analog registration mode rather than by single-ion counting. In the TOF-SIMS mode, secondary ions are extracted into the mass analyzer by a static extraction field. Because of the smaller energy spread of secondary ions compared with that of photoions, which are generated in an extended volume in the extraction field between target and extractor, the achieved mass resolution is in general higher than in the laser-SNMS mode.17 TOF-SIMS measurements are recorded in the single-ion counting mode by a multistop time-to-digital converter (TDC). For postionization of the sputtered particles, the radiation of a standard excimer laser (Lambda Physik EMG 103 MSC) was focused by a single quartz lens (focus length f ) 250 mm) into the interaction volume. The laser focus was located as close to the target surface as possible (distance ∼250 µm) in order to maximize the portion of sputtered particles in the interaction volume. The wavelength λ ) 248 nm was chosen for NRMPI, since all elements relevant in this investigation are efficiently photoionized in a 2-photon process by 248 nm. Samples. Metals on Polymer Surfaces. The submonolayer coverages of metals on polymer surfaces were prepared by sputtering corresponding metals or alloys by 10-keV Ar+ ions with an angle of incidence θPI ) 45°. Details of the arrangement are given in Figure 1. The sputtering ion beam was scanned over an area of 0.5 mm2 on the sputter target to compensate for possible inhomogeneities. Sputter equilibrium was ensured by adequate presputtering (primary ion fluence FPI > 1 × 1017 cm-2) while the sample was shuttered from sputtered flux. (17) Schnieders, A.; Mo ¨llers, R.; Terhorst, M.; Cramer, H.-G.; Niehuis, E.; Benninghoven, A. J. Vac. Sci. Technol. 1996, B14, 2712-2724.
The surface density ϑ(Α) of sputter-deposited material is determined by the flux of sputtered metal atoms, the sticking probability of the metal atoms arriving at the surface, and the behavior of the metal atoms on the surface, including desorption or diffusion into deeper surface regions. Assuming sputter equilibrium, a cosine angular distribution d2N/dΩ2 of sputtered particles, and a uniform sticking probability of 1, the surface density ϑ(A) of sputter-deposited metals can be calculated by the following equation:
ϑ(A) ) a(A)c(A)NPIYs(θPI)
∫∫
ΩA
d2N dΩ2 dΩ2 A
1 cos(θ0) (1) ≈ a(A)c(A)‚NPI cos-f (θPI)Ys(θPI ) 0) π r2 where c(A) is the relative bulk concentration of metal A in the sputter target, NPI the number of sputtering primary particles, and Ys(θPI) the total sputter yield for bombardment at angle θPI. Values for Ys(θPI ) 0) are listed in the paper of Matsunami et al.18 Ω is the solid angle and A the exposed wafer area. The exponent f, which considers the influence of the angle of incidence of the primary particles, depends on the mass ratio mT/mP of target and projectile particles.19 For mT/mP the exponent f is ∼5/3, for mT/mP the exponent f decreases toward a value somewhat less than 1. Factor a in eq 1 corrects for deviations from the assumptions made above (sputter equilibrium, cosine angular distribution, and uniform sticking probability). In particular, it considers the more pronounced ejection of sputtered particles in the direction of the primary ions. The value of a has been determined by an independent analysis technique (total reflection X-ray fluorescence spectroscopy, TXRF) for the sputter deposition of metals on Si to be about a ) 2(1 ( 10%).17 It was not possible to determine a for the polymer samples with metal surface densities between some 108 and 1012 cm-2, because of the lack of an appropriate analysis technique with similar sensitivity and information depth as laser-SNMS provides for this kind of sample. Therefore, a was set to be equal to 2 for the calculation of the surface densities on the prepared samples. Neglecting the uncertainty of a, the calculated values for the surface densities using eq 1 are accurate within 10%. For this investigation, presputtered samples of pure Co and an alloy (Havar) were used as sputter targets. The composition (in at %) of Havar is Be 0.25%, C 0.96%, Cr 22.2%, Mn 1.7%, Fe 18.4%, Co 41.6%, Ni 12.8%, Mo 1.2%, and W 0.88%.20 Sputter equilibrium and therefore stoichiometric composition of the sputtered flux were ensured for the alloy. As substrates, three different polymer surfaces were used: polycarbonate, poly(vinylidene chloride), and polyimide. In detail, a series of polycarbonate samples with surface densities of Co varying between some 108 cm-2 and some 1012 cm-2 was prepared, as well as multicomponent targets by sputter deposition of the alloy on the three polymers. (18) Matsunami, N.; Yamamura, Y.; Itikawa, Y.; Itoh, N.; Kazumata, Y.; Miyagawa, S.; Morita, K.; Shimizu, R.; Tawara, H. At. Data Nucl. Data Tables 1984, 31, 1-80. (19) Sigmund, P. Phys. Rev. 1969, 184, 383-416; Phys. Rev. 1969, 187, 768. (20) Goodfellow GmbH Bad Nauheim, Germany, German catalogue, 1994/95.
Figure 2. Detail of a laser-SNMS spectrum of a polycarbonate sample covered with metals in submonolayer surface densities. The sample was prepared by sputter deposition with surface densities ϑ given in Table 1. Static sputtering: 10 keV Ar+, primary ion fluence FPI ) 1 × 1013 cm-2. Postionization: λ ) 248 nm, IL ) 1010 W cm-2. Table 1. Metal Surface Densities T of the Multicomponent Samples Prepared by Sputter Deposition of Havara element A
surface density, ϑ(A)/cm-2
element A
surface density ϑ(A)/cm-2
Be Cr Mn Fe
4 × 109 3.4 × 1011 2.6 × 1010 2.9 × 1011
Co Ni Mo W
6.4 × 1011 2.0 × 1011 2.0 × 1010 1.5 × 1010
aThe values are calculated by using eq 1 setting a ) 2. The remaining uncertainty of all values is ∼10% (50% for Be).
Metalloproteins. The metalloproteins spPAP and kbPAP were usually prepared by spin-coating of some microliters of a 10-5 M solution in H2O on pure Si wafers. kbPAP was used for the investigation of the influence of the preparation, because its amount was not so limited as that of spPAP. These samples have been prepared by droplet preparation and spin-coating of different amounts of solutions with various concentrations (up to 1 µl of a 10-3 M solution). Si wafers and polycarbonate films were used as substrates. RESULTS Metals on Polymer Surfaces. Figure 2 shows a part of a typical laser-SNMS spectrum of a polycarbonate sample covered with metals in submonolayer surface densities in the range of 1011 cm-2 (Table 1). The spectrum is dominated by the atomic peaks of the sputter-deposited metals. They are clearly separated from molecular signals at the same integral mass. In general, the intensities of molecular signals are low because of photon-induced dissociation of the sputtered molecular particles into smaller fragments (CxHy with x, y ) 1, 2, 3) by the high laser intensity (IL ) 1010 W cm-2) required for nonresonant photoionization. To check the qualification of laser-SNMS for quantitative analysis of this kind of sample, a series of polycarbonate samples with different surface densities of Co ranging from 3 × 108 to 3 × 1012 cm-2 was analyzed. The measurements show a linear dependence between the detected photoion intensity I(Cox) and the surface density of Co, demonstrated in Figure 3. (The sign x is used to mark a photoion, which in general is positive in charge. Analytical Chemistry, Vol. 72, No. 18, September 15, 2000
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Table 3. Laser-SNMS Detection Limits Tmin for Metals on Polycarbonate and on Sia detection limit, ϑmin/cm-2 metals on polycarbonate
Figure 3. Linear correlation between the normalized Cox intensity and the Co surface density ϑ(Co). Table 2. Relative Sensitivity Factors S(Mx, Crx) of Metals M on Polymer Surfaces with Respect to Cr for Laser-SNMSa matrix element Be Cr Mn Fe Co Ni Mo W
polycarbonate
poly(vinylidene chloride)
polyimide
alloys
0.26 1 0.16 2.2 1.4 1.7 1.6 0.8
0.28 1 0.22 2.1 1.1 0.8 2.9 1.3
0.10 1 0.20 2.5 0.9 0.8 2.2 1.1
1 0.39 ( 0.05 3.8 ( 0.5 1.2 ( 0.2 0.9 ( 0.2 1.0 ( 0.1
a Static sputtering: 10 keV Ar+. Postionization: λ ) 248 nm, I ) L 1010 W cm-2. The relative sensitivity factors for alloys are determined for laser-SNMS bulk analysis.21 They vary in the given range for alloys with strongly different compositions. The uncertainty of the other values is ∼10% (50% for Be) presuming the accuracy of the standards.
This marking does not imply a further difference between a positive secondary ion and a photoion of the same structure but the different origin.) The double-logarithmic plot has a slope of m ) 0.97 ( 0.04. The Cox intensity had to be normalized on the intensity of the Cx3 fragment of the polycarbonate to correct for variations in the total intensities caused by different locations of the interaction volume above the sample, resulting in different geometrical yields YIV. The exact and reproducible positioning of the laser focus was difficult, because the polymer surfaces are easily stimulated to laser-induced desorption. The Cx3 fragment was chosen for normalization, because it originates from dissociative ionization of sputtered molecular particles and not from dissociative ionization of gas-phase particles in the residual gas. In Table 2, the relative sensitivity factors S(Mx, Crx) of eight metals M on different polymer substrates with respect to Cr are given. They are determined for static sputtering with 10-keV Ar+ primary ions and nonresonant postionization with λ ) 248 nm. For this investigation, the multicomponent samples prepared by sputter deposition of Havar were used. The surface densities of the metals on these samples are given in Table 1. The relative sensitivity factors do not strongly depend on the respective substrates. Typical variations are smaller than a factor of 2. The higher variation of the relative sensitivity factor of Be may be the effect of statistics. The surface density of Be on the 4292 Analytical Chemistry, Vol. 72, No. 18, September 15, 2000
metals on Si
element
laser-SNMS
TOF-SIMS
laser-SNMS
Be Cr Mn Fe Co Ni Mo W
2× 2 × 109 4 × 109 5 × 108 8 × 108 1 × 109 2 × 109 6 × 109
>5 × 3 × 1010 3 × 1010 3 × 1010 4 × 1010 5 × 1010 >5 × 1010 >5 × 1010
4 × 109 2 × 109 3 × 109 2 × 109 8 × 108 1 × 109 2 × 109 2 × 109
a
109
109
The uncertainty of the values is within a factor of 2.
samples was very low (ϑ(Be) ≈ 4 × 10-9 cm-2), resulting in low count rates with correspondingly poor statistics. For comparison, the respective relative sensitivity factors for bulk analysis of alloys21 are also given in Table 2. These values have been determined by evaluating spectra of four different alloys. They are in good agreement with the relative sensitivity factors determined for the detection of metals on polymer surfaces. Maybe the remaining differences in the relative sensitivity factors for the same metal on different substrates are artifacts caused by false assumptions concerning the value of a and therefore the surface densities on the samples. In Table 3, the detection limits (The detection limit ϑmin(A) is the minimum surface density of A that can be detected with an acceptable signal-to-noise ratio (in this investigation a factor of 3 above the background) of the eight constituent metals of Havar on a polycarbonate surface analyzed with laser-SNMS and TOFSIMS are presented. They are all limited by the statistical background in the spectra. For comparison, the detection limits of the same metals on Si with laser-SNMS17 are also given in Table 3. They are almost the same as those for metals on polycarbonate with the exception of Fe. The detection of Fe on Si is hampered by an insufficient separation of the Fex peak and the Six2 peak (mass resolution 3500 of the instrument used in the laser-SNMS mode). Comparing the laser-SNMS and TOF-SIMS detection limits for metals on polycarbonate, higher sensitivities of the postionization technique between 1 and 2 orders of magnitude are found for this kind of sample. In contrast to this, the detection limits of metals on Si with TOF-SIMS are in the same range as those achieved with laser-SNMS.17 Because of the use of sputter-deposited samples for the determination of the detection limits, the given detection limits are “worst case” values. For the calculation of the surface densities on the samples after sputter deposition, a sticking probability of the metal atoms of 1 and no diffusion into the bulk was assumed. If these assumptions are not valid, the correction factor a and therefore the surface densities of the metals on the standards would be lower than assumed with the consequence that the detection limits, which were determined by use of these standards, would decrease by the same factor. (21) Kampwerth, G.; Terhorst, M.; Niehuis, E.; Benninghoven, A. In Secondary Ion Mass Spectrometry SIMS VIII; Benninghoven, A., Janssen, K. T. F., Tu ¨ mpner, J., Werner, H. W., Eds.; John Wiley & Sons: Chichester, U.K., 1992; pp 563-566.
Figure 4. Detail of a laser-SNMS spectrum of spPAP prepared on Si. Preparation: 1 µL of a 1 × 10-5 M solution in H2O spin-coated on Si. Static sputtering: 10 keV Ar+, primary ion dose NPI ) 2 × 109, primary ion fluence FPI ) 2 × 1012 cm-2. Postionization: λ ) 248 nm, IL ) 1010 W cm-2.
Figure 5. Details of a laser-SNMS spectrum and a TOF-SIMS spectrum of kbPAP prepared on Si. Preparation: 40 µL of a 1 × 10-5 M solution in H2O spin-coated on Si. Static sputtering: 10 keV Ar+, primary ion dose NPI ) 1.6 × 109, primary ion fluence FPI ) 1.6 × 1012 cm-2. Postionization: λ ) 248 nm, IL ) 1010 W cm-2.
In earlier investigations22 of metals on polymer surfaces, TOFSIMS detection limits for metals on polymers in the range between 1011 and 1012 cm-2 were found. In contrast to the results presented in this work, these limits were affected by the background in the spectrum, mainly caused by metastable decay of molecular ions. This was caused by a different reflectron design of the instrument (TOF-SIMS II, University of Mu¨nster)23 used in the work of Karen et al. Metalloproteins. Part of a laser-SNMS spectrum of spPAP prepared on Si is shown in Figure 4. The spectrum is dominated by element peaks in this mass region. Only a few molecular signals can be identified. In detail, Fe, Zn, and traces of Mn and Cu were detected. Similar spectra were acquired for kbPAP prepared in the same way. A spectrum of a pure reference Si wafer shows no Mnx, Fex, Cux, and Znx peaks. Therefore it could be concluded that spPAP contains an Fe-Zn core in its active sites like kbPAP. This result was confirmed by SR-XRF.15 The Mnx and Cux signal originated from contamination of the solution, which could also be detected by SR-XRF. Considering that the metals in the active sites of spPAP have an atom concentration of ∼100 ppm and the atomic signals have an integral intensity of ∼10 000 counts, detection limits of less than 1 ppm atom concentration for metals bound to organic macromolecules seem to be possible. This estimation is only valid if an appropriate clean preparation is possible, because minor metal contaminants of the substrate and the solution (here Mn and Cu) could result in intensities in the spectrum comparable to those of the analyte. The typical amino acid fragments expected in a mass spectrum achieved from a protein like spPAP only appear with low intensities. This is on one hand due to the low absorption of the characteristic functional groups of the amino acids at the applied laser wavelength λ ) 248 nm. If one is interested in information on the amino acids in the protein, the use of λ ) 193 nm would be a better choice.24 On the other handsand this is the more important reasonsthe low molecular intensities are due to photoninduced fragmentation in the intense laser field required for
nonresonant ionization of the elements. In the case of organometallic molecules, like the PAPs, multiphoton excitation leads, in general, first to fragmentation and then to ionization of the fragments especially of the metal atom.25 This fragmentation behavior and also the sputter-induced fragmentation are often of advantage for the elemental analysis of samples such as those investigated in this work, because of the lack of severe mass interferences. In Figure 5, details of a laser-SNMS spectrum and a TOF-SIMS spectrum of kbPAP prepared on Si are given. The spectra were acquired from the same sample under the same conditions, especially the same primary ion dose NPI. The different intensities of the 64Zn+ peak demonstrate the higher sensitivity (factor of ∼25) of laser-SNMS compared with TOF-SIMS for the detection of metals bound to organic macromolecules. The detection of metals with TOF-SIMS is mainly hampered by the low secondary ion yield achieved from these samples. To investigate the influence of the preparation on the secondary neutral emission, the yields of the components Fe and Zn in the active sites of kbPAP were determined for several preparation techniques. Figure 6 demonstrates the strong influence of the preparation on the achieved yields. The yields vary over a range of ∼2 orders of magnitude. For the same preparation procedure, the yields vary in the worst case by a factor of ∼2. The highest yields are achieved for spin-coating 1 µL of a 10-4 M solution on a Si substrate. The yields achieved for spin-coating the same amount of a more dilute solution (10-5 M) are correspondingly lower. The yields achieved for spin-coating a larger amount (40 µL) of a 10-5 M solution on a Si substrate are comparable with those achieved for spin-coating 1 µL of a 10-4 M solution. For droplet preparation on Si and preparation on an organic substrate, e.g., polycarbonate, the yields are the lowest. This behavior can be explained by assuming different secondary neutral yields for the different samples. Mo¨llers et al.24,26 have shown that the substrate has a significant influence on the sputter process of organic molecules. As long as the total surface coverage Θtot of organic molecules on a metal or Si surface is below a
(22) Karen, A.; Benninghoven, A. In Secondary Ion Mass Spectrometry SIMS IX; Benninghoven, A., Nihei, Y., Shimizu, R., Werner, H. W., Eds.; John Wiley & Sons: Chichester, U.K., 1994; pp 788-791. (23) Niehuis, E.; Heller, T.; Feld, H.; Benninghoven, A. J. Vac. Sci. Technol. 1987, A5, 1243-1246.
(24) Mo¨llers, R. Flugzeitmassenspektrometrische Untersuchungen zur Zersta¨ubung und Photoionisierung organischer Moleku ¨ le. Ph.D. Thesis, Westfa¨lische Wilhelms-Universita¨t Mu ¨ nster, Germany, 1996. (25) Gedanken, A.; Robin, M. B.; Kuebler, N. A. J. Phys. Chem. 1982, 86, 40964107.
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Figure 6. Dependence of the yields of the components Fe and Zn in the active sites of kbPAP on the preparation of the sample. For each preparation two spectra have been acquired in different areas of the sample. Static sputtering: 10 keV Ar+, primary ion dose NPI ) 2 × 109, primary ion fluence FPI ) 2 × 1012 cm-2. Postionization: λ ) 248 nm, IL ) 1010 W cm-2.
monolayer equivalent, there is a linear relationship between the total surface coverage Θtot and the secondary neutral yield Yo. (The total surface coverage Θtot(A) is defined as the integral coverage of A in the near-surface region.) The highest yields are achieved for monolayer-equivalent coverages, whereas the yields decrease with further increase of the layer thickness and reach a plateau for multilayer coverages. This behavior is attributed to a sputter-induced matrix effect, i.e., the dependence of the sputter yield (the average number of desorbed particles per primary ion) on the substrate. Assuming a similar effect for the sputtering of the organic macromolecules, one can explain the trends for the yields. Then the preparation by spin-coating 1 µL of a 10-4 M solution would be assumed to result in a monolayer-equivalent preparation. The preparation on polycarbonate is comparable with a thick organic overlayer with a correspondingly low sputter yield. Comparing the relative intensities achieved for Fe and Zn depending on the preparation, one see variations in the relative sensitivity up to a factor of 10. Quantification of the metal content in the protein from these spectra is not possible; even a relative quantification is not possible. One reason is the surface sensitivity of laser-SNMS. In general, only particles from the uppermost monolayer could be desorbed by ion bombardment. Since the results shown in Figure 6 were acquired under static sputtering conditions, the arrangement of the macromolecules in the overlayer has a distinct influence on the composition of the sputtered flux. For some preparation techniques, it seems possible that the metal atoms are covered by the molecule to different degrees. Also, denaturation of the protein through contact of the molecule with the surface is possible. This could result in different binding forces of the two metals depending on the preparation technique. In that case, the different relative sensitivities could be explained by some kind of preferential sputtering. For the determination of the relative metal concentration in the protein, the complete removal of the protein layer seems to be appropriate. In this case, all material in the analyzed area is used for the analysis and the effects mentioned above such as, for example, preferential sputtering can be neglected. In Figure (26) Mo ¨llers, R.; Schnieders, A.; Kortenbruck, G.; Benninghoven, A. In Secondary Ion Mass Spectrometry SIMS X; Benninghoven, A., Hagenhoff, B., Werner, H. W., Eds.; John Wiley & Sons: Chichester, U.K., 1997; pp 943-946.
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Figure 7. Photoion intensities in dependence on the primary ion fluence FPI for the complete removal of a spPAP layer on Si. Preparation: 40 µL of a 1 × 10-5 M solution in H2O spin-coated on Si. Sputtering: 10 keV Ar+, sputtered area A ) 1 × 10-3 cm2; raster, 32 × 32 pixel. Analysis: 10 keV Ar+, primary ion dose NPI ) 2 × 109 per data point. Postionization: λ ) 248 nm, IL ) 1010 W cm-2.
7, the dependence of photoion intensities on the primary ion fluence FPI for the complete removal of a spPAP layer on Si is shown. The plots for the two components Fe and Zn in the active core do not show the same shape. Initially, there is a ratio of ∼5 between the Fex intensity and the Znx intensity. This ratio decreases as the amount of sputtered material increases and approaches 2 in the equilibrium after sputtering most of the layer. This behavior can also be explained by different degrees of coverage or by different binding forces of the two metals resulting in preferential sputtering. The integral intensities of Fex and Znx have a ratio of ∼2.7. Since the two metals have the same concentration in the sample, this value can be considered as the “bulk” relative sensitivity factor S(Fex, Znx) of Fe with respect to Zn for metals bound to organic molecules. DISCUSSION Features of Laser-SNMS with NRMPI. The measurements presented in this paper have demonstrated the possibility of detecting metals in organic materials with atom concentrations down to 1 ppm and extremely low sample consumption with laserSNMS, regardless of whether they are incorporated in the
structure of a large organic molecule or adsorbed on an organic substrate. The achieved yields are comparable to yields of corresponding elements on inorganic substrates and therefore high enough for elemental surface mapping with high lateral resolution (in the 100-nm range provided the concentrations are high eneough).27,28 The values of the relative sensitivity factors for the surface detection of metals on polymers show good agreement with those for metals sputtered from alloys. The detection limits are the same as for inorganic matrixes such as Si or GaAs. Whereas laser-SNMS is a sensitive analysis technique with low matrix dependence, the SIMS results show a more pronounced matrix effect when changing from an inorganic matrix to an organic one. The low matrix effect for laser-SNMS shows that the sensitivity of laserSNMS depends predominantly on the efficiency of ionization and not on the desorption process, since the matrix effect only influences the sputter process and not the postionization. In general, quantification of metals in organic materials with laser-SNMS is possible, e.g., for submonolayer surface coverages on polymers or in cases where all of the available material in nearsurface regions can be used for analysis. But in cases where one is restricted to static sputtering conditions, only a qualitative statement is possible, because for a real-world sample the surface structure and the type of bonds of the metal atoms to the matrix are not known and may have a considerable influence on their sputter yields. Comparison with Resonant Laser-SNMS. A further increase of sensitivity of laser-SNMS is expected by use of resonanceenhanced multiphoton ionization (REMPI) for postionization of the sputtered particles.29,30 This is due first to the better overlap of the interaction volume with the sputtered plume resulting in a correspondingly higher geometrical yield. Cross sections for resonant ionization are orders of magnitude larger than those for nonresonant ionization, and therefore, unfocused laser beams produce efficient excitation in a corresponding large interaction volume. A second reason for the higher sensitivity results from the high selectivity of resonant ionization and therefore from the absence of mass interferences and background in the spectra.31 The major drawback of this technique is the fact that it is only possible to detect those elements that are defined before the measurement, because the wavelengths of the lasers used for postionization have to be tuned to the respective transitions. Also, it is not possible to monitor more than a few elements, because each element has its own spectroscopy requiring the adjustment of the respective laser wavelengths, and up to now, each wavelength requires a single laser. Comparison with Synchrotron Excited X-ray Fluorescence Spectroscopy. Comparing the abilities of laser-SNMS for the (27) Terhorst, M.; Mo¨llers, R.; Niehuis, E.; Benninghoven, A. Surf. Interface Anal. 1992, 18, 824-826. (28) Kollmer, F.; Kamischke, R.; Ostendorf, R.; Schnieders, A.; Kim, C. Y.; Lee, J. W.; Benninghoven, A. In Secondary Ion Mass Spectrometry SIMS XII; Benninghoven, A., Bertrand, P., Migeon, H. N., Werner, H. W., Eds.; NorthHolland Elsevier Science Publishers B. V.: Amsterdam, in press. (29) Quinn, T. L.; Murphy, T. J.; Moore, L. J.; Arlinghaus, H. F.; Spaar, M. T.; Taylor, E. H.; Thonnard, N. In Resonance Ionization Spectroscopy 1990; Parks, J. E., Omenetto, N., Eds; Institute of Physics Conference Series 114; Institute of Physics: Bristol, 1991; pp 333-336. (30) Arlinghaus, H. F.; Spaar, M. T.; Switzer, R. C.; Kabalka, G. W. Anal. Chem. 1997, 69, 3169-3176. (31) Arlinghaus, H. F.; Whitaker, T. J.; Joyner, C. F.; Kwoka, P.; Jacobson, B.; Tower, J. In Secondary Ion Mass Spectrometry SIMS X; Benninghoven, A., Hagenhoff, B., Werner, H. W., Eds.; John Wiley & Sons: Chichester, U.K., 1997; pp 123-130.
detection of metals bound to organic macromolecules with those of SR-XRF,32 which is usually used to solve these questions, one has to consider the sensitivity and the means of quantification and localization of the techniques. For the determination of the metals in the active site of spPAP with SR-XRF, 140 µL of a 0.34 × 10-3 M solution was used,15 whereas for the detection of the metals with laser-SNMS only 1 µL of a 10-5 M solution was used. Both amounts were more than sufficient for the detection of the metals. The achieved spectra are comparable in terms of signalto-noise ratio, which determines the detection limits. In principle SR-XRF using a crystal monochromator has detection limits on the order of a few tens of ppb for an irradiated area of ∼1 mm2.32 Durmus et al. used a relatively highly concentrated solution for their SR-XRF measurement. The reason was that the same solution was used for the subsequent structure analysis33 with extended X-ray absorption fine structure analysis (EXAFS), which requires this concentration. The means of localization are of interest in a large number of applications, e.g., in the broad field of life sciences. Laser-SNMS offers the possibility of localizing elements in samples with high lateral and indepth resolution, because the desorbing primary ion beam can be focused down to below 100 nm, and the technique has a surface sensitivity in the range of the uppermost monolayer. SR-XRF can also be used for microarea analysis, but the radiation can only be focused to spot sizes down to some micrometers and the technique has in principle an information depth, which is orders of magnitude larger than that of SIMS and laser-SNMS. There are clear advantages for surface mass spectrometry with regard to sensitivity and localization. But there is a major disadvantage for the quantification of elements. With SR-XRF, quantification is relatively easy to do by use of standard sample solutions, whereas laser-SNMS is hampered in quantification just because of its surface sensitivity. Further advantages of SR-XRF come from the lower radiation damage during the analysis and the possibility of working under a low-vacuum condition. But the main disadvantages are the high costs and the sophisticated technique required to produce the synchrotron radiation. Outlook. Further investigations of the use of laser-SNMS and TOF-SIMS for the detection of metals in organic materials have to focus on the sample preparation and a better quantification. The goal is to have comparable standards and to use reliable preparation techniques to achieve reproducible high yields in order to make use of the high sensitivity of laser-SNMS and also to obtain more quantitative information about the samples. A better understanding of the sputter process in organic matrixes is also necessary. ACKNOWLEDGMENT The authors gratefully acknowledge support by A. Durmus and B. Krebs (Anorganisch-Chemisches Institut, University of Mu¨nster, Germany) for introducing us to the field of research on purple acid phosphatases and for providing the necessary samples. Received for review February 3, 2000. Accepted June 8, 2000. AC000118Y (32) Iida, A.; Gohshi, Y. In Handbook on Synchrotron Radiation; Ebashi, S., Koch, M., Rubenstein, E., Eds.; North-Holland Elsevier Science Publishers B. V.: Amsterdam, 1991; Vol. 4, pp 307-348. (33) Sift, B. H.; Durmus, A.; Meyer-Klaucke, W.; Krebs, B. J. Synchrotron Radiat. 1999, 6, 421-422.
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