Surface Characterization of Hydroxyapatite and ... - ACS Publications

Hongbo B. Lu,† Charles T. Campbell,‡ Daniel J. Graham,† and Buddy D. Ratner†,§. Departments of Bioengineering, Chemistry, and Chemical Engine...
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Anal. Chem. 2000, 72, 2886-2894

Surface Characterization of Hydroxyapatite and Related Calcium Phosphates by XPS and TOF-SIMS Hongbo B. Lu,† Charles T. Campbell,‡ Daniel J. Graham,† and Buddy D. Ratner†,§

Departments of Bioengineering, Chemistry, and Chemical Engineering, University of Washington, Seattle, Washington 98195-1700

The surfaces of six biologically interesting calcium phosphate (CaP) phases (hydroxyapatite, dibasic calcium phosphate dihydrate, dibasic calcium phosphate, monobasic calcium phosphate, β-tribasic calcium phosphate, octacalcium phosphate) have been examined by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS). The intensity of an O(1s) shake-up satellite correlates with the phosphate oxygen content. Together with the Ca/P and O/Ca XPS peak ratios, this feature helps provide identification of the CaP phase(s) present in the surface of unknown samples and establish their mole fractions, as proven with a bone sample. Contributions from carbonate impurities can be quantified using its C(1s) peak at 279.9 eV and subtracted from the O(1s) line shape to aid identification. Principal component analysis (PCA) has been applied successfully to analyze TOF-SIMS spectra of these six CaP phases. Multivariate analysis can help differentiate these CaP phases using the first two PCs, which are dominated by the relative intensities of only a few key ions: PO3-, O-, Ca+, CaOH+, PO2-, and OH-. Hydroxyapatite (HAP) and related calcium phosphate (CaP) compounds have received considerable attention in the fields of biomaterials, chromatography, and biomineralization. Interest in hydroxyapatite as a biomaterial developed due to its close chemical resemblance to apatite,1,2 a natural mineral comprising most of the inorganic components in bones and teeth, and its spontaneous interfacial osteointegration when implanted.3,4 Hydroxyapatite thin-film coatings on medical implants have been shown to effectively improve bone-to-implant bonding and prevent fibrous capsule formation after implantation.5-11 Implants coated with hydroxyapatite thin films do not form fibrous tissues †

Department of Bioengineering. Department of Chemistry. § Department of Chemical Engineering. (1) LeGeros, R. Z. Prog. Cryst. Growth Charact. 1981, 4, 1-45. (2) deJong, W. F. Recl. Trav. Chim. 1926, 45, 445. (3) Hench, L. L. J. Am. Ceram. Soc. 1991, 74, 1487-1510. (4) Drummond, J. L. In Biomaterials in reconstructive surgery; Rubin, L. R., Ed.; The C. V. Mosby Co.: St. Louis, MO,1983; pp 103-108. (5) Thomas, K. A. Orthopedics 1994, 17, 267-278. (6) Duchenye, P.; Hench, L. L.; Kagan, A.; Martens, M.; Burseens, A.; Mulier, J. C. J. Biomed. Mater. Res. 1980, 14, 225-237. (7) Hench, L. L. In Bioceramics: Materials Characteristics Versus in vivo Behavior; Ducheyne, P., Lemons, J. E., Eds.; Annals of the New York Academy of Science: New York, 1988; Vol. 523, pp 54-61. ‡

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with existing bone, but rather an extremely thin, epitaxial bonding layer. However, numerous studies have achieved little clarification of the mechanisms of its special osteointegration property, partially due to a lack of analytical tools for characterizing accurately the physical and chemical properties of its surfaces. Since the primary interactions between the material and its biological host occur on a molecular level and in a very narrow interfacial zone, surface properties play a crucial role in directing the biological properties of hydroxyapatite and related calcium phosphates.12,13 To understand the interfacial interactions between the materials and biological environment, we face a challenge of material characterization when only the outer 1 nm is of interest. There are at least six different biologically important calcium phosphate phases: HAP, dibasic calcium phosphate dihydrate (DCPD), dibasic calcium phosphate (DCP), monobasic calcium phosphate (MCP), β-tribasic calcium phosphate (β-TCP), and octacalcium phosphate (OCP). Amorphous calcium phosphate (ACP) is also present in the body in reasonable quantities. The biological response to different calcium phosphate phases can be different in terms of bone-bonding ability or osteointegration.14-18 Thus, distinguishing and identifying the different phases are crucial for properly understanding their biological properties. Although for bulk materials, phase characterization and identification are possible,19 surface analysis still remains a challenge.20 (8) Moroni, A.; Caja, V.; Stea, S.; Visentin, M. In Bioceramics (Proceedings of the 6th International Symposium on Ceramics in Medicine, Philadelphia, PA, November 1993); Christiansen, D., Ducheyne, D., Eds.; ButterworthHeinemann Ltd.: Cambridge, U.K., 1993; Vol. 6, pp 239-244. (9) Suzuki, K.; Yokoyama, Y.; Harada, Y.; Kokubo, T. In Bioceramics (Proceedings of the 6th International Symposium on Ceramics in Medicine, Philidelphia, PA, November 1993); Christiansen, D., Ducheyne, D., Eds.; Butterworth-Heinemann Ltd.: Cambridge, U.K., 1993; Vol. 6. (10) Geesnik, R. G. T. Clin. Orthop. Relat. Res. 1990, 261, 39-58. (11) Miyakawa, S.; Ebihara, K.; Fukubayashi, T. In Bioceramics; Christiansen, D., Ducheyne, D., Ed.; Butterworth-Heinemann Ltd.: Cambridge, U.K., 1993; pp 423-430. (12) Kasemo, B.; Lausmaa, J. CRC Crit. Rev.: Biocompat. 1986, 2, 235-380. (13) Somasundaran, P.; Wang, Y. H. C. In Adsorption on and Surface Chemistry of Hydroxyapatite; Misra, D. N., Ed.; Plenum Press: New York, 1984; pp 129-150. (14) Nagano, M.; Nakamura, T.; Kokubo, T.; Tanahashi, M.; Ogawa, M. Biomaterials 1996, 17, 1771-1777. (15) Winter, M.; Griss, P.; de Groot, K.; Tagai, H.; Heimke, G.; Dijk, H. J. A. V.; Sawai, K. Biomaterials 1981, 2, 159-160. (16) Klein, C. P. A. T.; Driessen, A. A.; de Groot, K.; van den Hooff, A. J. Biomed. Mater. Res. 1983, 17, 769-784. (17) Yamaski, H.; Sakai, H. Biomaterials 1992, 13, 308-312. (18) Semmelink, J. M.; Klein, C. P. A. T.; Vermeiden, J. P. W.; Althuis, A. L. Biomaterials 1986, 7, 152-154. (19) LeGeros, R. Z. In Monographs in Oral Science; Mayers, H. M., Ed.; Karger: San Francisco, CA, 1991; Vol. 15, p 1. 10.1021/ac990812h CCC: $19.00

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Because their composition near the surface is very likely to be different from the bulk,21-25 information given by bulk analysis methods could lead to misinterpretation of structure/function relationships. In addition, the development of accurate characterization methods for CaP phases is also needed for the ultrathin films present during the initial nucleation and formation stage of CaP films on solid surfaces, when conventional bulk characterization methods are not applicable.20 For the above reasons, we apply here surface-sensitive analytical tools [X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS)] to study these calcium phosphates. Both XPS and TOF-SIMS have been applied previously in surface characterization of calcium phosphates.20,25-29 On the basis of those studies alone, one could not assign an unknown sample to one of these six calcium phosphate phases. Most of the XPS studies identified the observable core-level shifts of characteristic peaks, such as Ca, O, and P, of several of these calcium phosphate species. Because of instrument and material differences, there was often poor agreement between the results of the different groups.20,27,28 The use of SIMS to study CaP mineral surfaces is more recent than XPS.20,25 Major peaks in both positive and negative SIMS spectra for different CaP phases have been identified, and some quantitative analyses such as relative peak ratios have been used successfully for distinguishing most of the CaP phases.20 However, due to the intrinsic complexity of SIMS data, more sophisticated quantitative data-processing methods such as multivariate analysis may be needed to fully extract useful information for CaP phase identification. Multivariate analysis methods have been used with success to discriminate SIMS spectra of different samples such as copolymers,30 plasmadeposited films (PDF),31 and various diamond-like carbons.32 These methods bypass the need for complete physical understanding of the ion-surface interaction. In contrast to quantification based on a single peak or a few peaks, multivariate analysis is a full-spectrum methodology.33-36 In this paper, principal compo(20) Chusuei, C. C.; Goodmann, D. W.; Stipdonk, M. J.; Justes, D. R.; Schweikert, E. A. Anal. Chem. 1999, 71, 149-153. (21) Amrah-Bouali, S.; Rey, C.; Lebugle, A.; Bernache, D. Biomaterials 1994, 15, 269-272. (22) Li, Y.; Zhang, X.; de Groot, K. Biomaterials 1997, 18, 737-741. (23) Suetsugu, Y. J. Mater. Sci. 1996, 31, 4541-4544. (24) Brown, W. E.; Mathew, M.; Tung, M. S. Prog. Cryst. Growth Charact. 1981, 4, 59-87. (25) Leadley, S. R.; Davis, M. C.; Castro, R. G.; Barbosa, M. A.; Paul, A. J.; Watts, J. F. Biomaterials 1997, 18, 311-316. (26) Santos, J. D.; JHS, L. J.; Monterio, F. J. J. Mater. Sci.: Mater. Med. 1996, 7, 181-185. (27) Landis, W. J.; Martin, J. R. J. Vacuum Sci. Technol. A 1984, 2, 1108-1111. (28) Hanawa, T.; Ota, M. Appl. Surf.ace Sci. 1992, 55, 269-276. (29) Lebugle, A.; Sallek, B. In Hydroxyapatite and related materials; Brown, P. W., Constantz, B., Eds.; CRC Press, Inc.: Boca Raton, FL, 1994; pp 319329. (30) Botreau, M.; Duc, T. M. In 10th International Conference on Secondary Ion Mass Spectrometry SIMS X; Wiley: Chichester, 1997; p 313. (31) Chilkoti, A.; Schmierer, A. E.; Perez Luna, V. H.; Ratner, B. D. Anal. Chem. 1995, 67, 2883. (32) Nicholas, M.; Linton, R. W.; Friedman, R. M.; Rading, D.; Benninghoven, A. In 10th International Conference on Secondary Ion Mass Spectrometry SIMS X; Wiley: Chichester, 1997. (33) Beebe, K. R.; Kowalski, B. R. Anal. Chem. 1987, 59, 1007A. (34) Brown, S. D. Appl. Spectrosc. 1995, 49, 14A. (35) Brown, S. D.; Sum, S. T.; Despagne, F.; Lavine, B. K. Anal. Chem. 1996, 68, 21R. (36) Thomas, E. V. Anal. Chem. 1994, 66, 795A. (37) LeGeros, R. Z. Calcif. Tissue Int. 1985, 37, 194-197.

nent analysis (PCA) was used as it reduces data to a form more suitable for visualization, cluster analysis, or quantification.33-36 We show here that the characterization of XPS O(1s) shakeup peaks provide useful information to supplement the Ca/P and O/Ca ratios for distinguishing these six calcium phosphate phases. This information can also be applied to the quantification of their mixtures on surfaces. The PCA of both positive and negative TOFSIMS spectra is shown to provide useful information for further discriminating and identifying different CaPs. To our knowledge, this is the first use of multivariate analysis of SIMS spectra for CaP mineral phase identification. METHODS AND MATERIALS 1. Materials. HAP and β-TCP standard materials were kindly provided by Dr. Ming Tung at the Pattenbarger Research Center of the National Institute of Standards and Technology (NIST). Other calcium phosphates were purchased from Spectrum (DCPD, DCP, MCP); OCP was synthesized following the recipe described in ref 37. The crystal structures of all the powders have been verified by X-ray diffraction analysis (XRD) prior to the surface analyses. For the OCP samples, XRD data showed that the majority of the powder was OCP with a small HAP impurity. The XRD spectrum had a very strong (100) peak at 2θ ) 4.73° which confirms the existence of OCP, while some characteristic peaks of HAP showed up in the range around 2θ ) 31.5-33.5°. Composition was difficult to quantify because of complicated peak overlaps. XRD peaks for other powders matched the JCPDS standards within experimental error. For both XPS and TOF-SIMS experiments, these sample powders were pressed onto a 0.5 cm × 0.5 cm soft indium foil substrate and blown with N2 to remove loose powder. Prior to use, the indium foils were cleaned by sonicating in acetone (∼10 min), wiping with Kimwipes EX-L paper (Kimberly-Clark), sonicating in methanol for 10 min, and then drying with N2. The samples were immediately transferred into the XPS and SIMS vacuum chambers after preparation. The pig cortical bone samples were prepared following the method of Weiner and Price38 with slight modification. They were broken into pieces smaller than 0.5 cm × 0.5 cm × 0.5 cm. They were defatted with acetone and treated with 2.6% sodium hypochlorite at room temperature for ∼2 h. The hypochlorite treatment was repeated with fresh solution for two 1 h exposures. Then the samples were washed with an ascending series of ethanol solutions (60, 75, 90, and 100%) sequentially, 30 min each. Finally, the samples were treated with xylene for 30 min and dried with N2. The samples were mounted onto the sample holders by double-sided tape for XPS and TOF-SIMS experiments. 2. XPS. The XPS experiments were done on Surface Science Instruments X-Probe and S-probe spectrometers using monochromatic Al KR X-ray sources (hν ) 1486.6 eV), hemispherical analyzers, and multichannel detectors. The binding energy (BE) scales for the samples were referenced by setting the C(1s) BE of adventitious carbon to 284.6 eV.39 (The charging was moderate as judged by the BE corrections of 4-6.6 eV needed.) The highresolution S(2p) and C(1s) spectra were acquired at a pass energy (38) Weiner, S.; Price, P. A. Calcif Tissue Int. 1986, 39, 365-375. (39) Wagner, C. D.; Riggs, W. M.; Davis, L. E.; F., M. J. Handbook of Standard Data For Use in X-Ray Photoelectron Spectroscopy; Perkin-Elmer Corp. Physical Electronics Div.: Norwalk, CT, 1979.

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of 50 eV. The XPS data were acquired at photodetector takeoff angles of 55°, where the takeoff angle is defined as the angle between the surface normal and the axis of the analyzer lens. Further details of the XPS analysis procedures are given elsewhere.40 3. TOF-SIMS. The TOF-SIMS data were acquired using a model 7200 Physical Electronics instrument (PHI, Eden Prairie, MN) with a 8 keV Cs+ primary ion source. Data were acquired over a mass range from m/z ) 0 to 500 for both positive and negative secondary ions. The ion beam was moved to a new spot on the sample for each spectrum. The total ion dose used to acquire each spectrum was less than 2 × 1012 ions/cm2. The area of analysis for each spectrum was 100 µm × 100 µm. The secondary ions were extracted into a two-stage reflectron timeof-flight mass analyzer and then postaccelerated and converted to charged pulses by a stacked pair of chevron-type multichannel electromultiplier plates. The mass resolution (m/∆m) of the secondary ion peaks was typically between 5000 and 9000. The mass scale for the positive secondary ions from the calcium phosphate powders was calibrated using the Ca+, CaO+, CaOH+, CH3+, and C2H3+ peaks. The mass scale for the negative secondary ions from the powders was calibrated by using the O-, PO-, PO2-, and PO3- peaks. The differences between the expected and observed masses for both positive and negative ions after calibration were less than 20 ppm. 4. Principal Component Analysis of SIMS data. The PCA was done using Pirouette software (version 1.21, Informetrix, Inc., Seattle, WA, 98121). The theory of PCA is well established. A comprehensive discussion of PCA is not within the scope of this study, and the interested reader is referred to appropriate references.33-36 The calibration set contained the m/z ) 0-100 regions of both the positive and negative ion spectra for each sample. To make a quantitative comparison between the spectra, the peak intensities were normalized. The positive and negative ion components of the combined spectra were separately normalized with respect to the sum of intensities of all selected peaks in the respective part of the spectrum (the selected positive ions are Ca+, CaO+, and CaOH+; the selected negative ions include O-, OH-, P-, O2-, HO2-, PO-, PO2-, and PO3-). This was done to eliminate systematic differences in the absolute intensities observed for the positive and negative spectra and false influence from possible contamination due to different synthesis and sample preparation processes.

Figure 1. XPS survey spectrum for a HAP reference sample. It shows that Ca, P, O and small amounts of contamination (such as C) were present in the mineral sample. This has characteristics of typical XPS spectra obtained for all the calcium phosphate samples studied.

RESULTS AND DISCUSSION 1. XPS Results. a. Calcium Phosphate Reference Samples. By taking survey scans, detail scans, and high-resolution XPS spectra for the six CaP powders (HAP, OCP, DCP, DCPD, MCP, β-TCP), the surface atomic ratios of Ca/P and O/Ca, and the intensity of a shake-up satellite feature associated with the O(1s) peak were investigated. Composition. A typical survey XPS spectrum from calcium phosphate is shown in Figure 1 (in this case for HAP). Besides the expected Ca, P, and O peaks, a small C(1s) peak was observed in all the samples examined. This C impurity had atomic

percentages varying from 5 to 17% for different samples. Most of this carbon was so-called “adventitious carbon” due to adsorption of impurity hydrocarbons. This peak does not affect the interpretation for our results, and, in fact, was actually used for BE calibration by setting its BE to 284.6 eV39 to correct for sample charging. Carbonate-type carbon was also observed in the C(1s) region for some of the samples. Carbonate is a common impurity found incorporated into various synthetic calcium phosphates during synthesis, due to the presence of CO2 in the air and solutions.41 It is a major substituent found in biological minerals as well.1 The only other impurity besides C detected in HAP and β-TCP samples was F with atomic percentages less than 2%. For other samples, Na, Mg, and K were observed with atomic percentages varying between 0 and 1% while F had concentrations up to 3%. The impurities were possibly due to contamination from powder synthesis or the sample preparation procedure. The powder layers were thick and continuous, so that no signal from the indium sample holder was ever observed. Ca/P and O/Ca Ratios. The Ca/P and O/Ca atomic ratios were calculated and are presented in Table 1. The direct values were calculated directly from the XPS data, using the atomic sensitivity factors from Scofield cross sections modified by the instrumental transmission function.40 The adjusted values, which are only slightly different from the direct values, were calculated on the basis of direct values after taking into account the carbonate impurity present in each sample. The methods for making these adjustments are explained in the Appendix. The adjusted surface Ca/P and Ca/O atomic ratios are plotted against the bulk stoichiometric ratios in Figures 2 and 3. These surface compositions are within 1-10% of the bulk ones, which means that the sample powders have surface stoichiometries similar to their bulk crystal compositions. O(1s) Shake-Up/Off Satellite Features. As we looked into the O(1s) peak in more detail, we found that, for all six calcium phosphate species studied, two characteristic shake-up/off satellite

(40) Castner, D. G.; Hinds, K.; Grainger, D. W. Langmuir 1996, 12, 50835086.

(41) Pal’chik, N. A.; Grigor’eva, T. N.; Stolpovskaya, V. N.; Arkhipenko, D. K.; Moroz, T. N. Russ. J. Appl. Chem. 1997, 70, 1513-1516.

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Table 1. Direct Ca/P, O/Ca, and O(1s)II/O(1s)Total Ratios and Adjusted Ca/P, O/Ca, and O(1s)II/O(1s)Total Ratios after Correcting for the Presence of the Carbonates for the Standard Samples and the Bone Samples. (Carbonate O/ Total O Ratio, Needed for the Correction, Is Also Listed)

(Ca/P)direct (Ca/P)adjusteda (O/Ca)direct (O/Ca)adjustedb (O(1s)II/O(1s)total)direct (O(1s)II/O(1s)total)adjustedc total C atomic % C(1s)carbonate/C(1s)total χ ) carbonate O/total Od

HAP

β-TCP

OCP

MCP

DCP

DCPD

bone

1.48 ( 0.09 1.46 ( 0.09 2.76 ( 0.03 2.74 ( 0.03 0.065 ( 0.003 0.066 ( 0.003 10.5 ( 2.3 0.10 ( 0.03 0.055

1.35 ( 0.04 1.31 ( 0.04 2.72 ( 0.03 2.67 ( 0.03 0.072 ( 0.001 0.076 ( 0.001 13.9 ( 4.1 0.18 ( 0.06 0.15

1.24 ( 0.09 1.24 ( 0.09 3.4 ( 0.2 3.4 ( 0.2 0.053 ( 0.004 0.053 ( 0.004 14.9 ( 1.8 nde nd

0.48 ( 0.04 0.48 ( 0.04 10.38 ( 0.08 10.38 ( 0.08 0.008 ( 0.002 0.008 ( 0.002 16.6 ( 1.2 nd nd

0.93 ( 0.06 0.93 ( 0.06 4.3 ( 0.2 4.3 ( 0.2 0.037 ( 0.002 0.037 ( 0.002 9.0 ( 1.0 0.01 ( 0.01 0.0065

0.98 ( 0.04 0.98 ( 0.04 5.4 ( 0.3 5.4 ( 0.3 0.020 ( 0.001 0.020 ( 0.001 5.0 ( 0.3 nd nd

1.49 ( 0.02 1.42 ( 0.02 3.20 ( 0.07 3.10 ( 0.07 0.055 ( 0.002 0.056 ( 0.002 17.7 ( 1.0 0.21 ( 0.02 0.22

a Assuming that CO 2- substitutes for PO 3- only, and three CO 2- ions substitute for two PO 3- ions. The adjusted Ca/P ratio ((Ca/P) 3 4 3 4 adjusted) is then equal to (1 - (2/3)(χ/3))(Ca/P)direct. b Assuming that CO32- substitutes for PO43- only and three CO32- substitute for two PO43- ions. Thus, every nine carbonate oxygens detected actually count for eight phosphate oxygens. The adjusted O/Ca ratio ((O/Ca)adjusted) is then equal to (O/Ca)dir(1 - χ/9). c See Appendix for details on the calculation. d All the numbers reported are average values from more than three measurements. See text for detail information about how to obtain χ. e nd, not detectable.

Figure 2. Surface Ca/P atomic ratios (adjusted from direct XPS measurement by carbonate component) vs bulk Ca/P stoichiometric ratios of different calcium phosphate samples. Multiple measurements (3-9 repeated times) were performed for each data point. Dashed curve: 1:1 slope expected for bulklike surface composition.

Figure 3. Surface O/Ca atomic ratios (adjusted from direct XPS measurement by carbonate component) vs bulk O/Ca stoichiometric ratios of different calcium phosphate samples. Multiple measurements (3-9 repeated times) were performed for each data point. Dashed curve: 1:1 slope expected for bulk composition at the surface.

peaks (with fwhm of ∼7 and ∼6 eV, respectively) were always observed at ∼23.5 ( 1.5 (peak I) and ∼36 ( 1 eV (peak II) lower kinetic energy (KE) (higher binding energy) than the main O(1s) peak at ∼ 531.6 eV BE (Figure 4). Such satellites appear at the kinetic energy of the core photoelectrons minus a loss of energy

Figure 4. O(1s) shake-up satellite peaks for different calcium phosphate samples. It shows that the relative intensity of peak II decreases from the top spectrum to the bottom spectrum in the following order: β-TCP > HAP > OCP > DCP > DCPD > MCP.

associated with discrete excitation of valence electrons.42 Such XPS loss peaks have been used previously to distinguish different species of the same element. For example, Cu (II) can be distinguished from its other oxidation states43-45 and metallic Cs can be identified on the basis of a satellite structure below the core level.46 Similarly, we find that the intensity of the shake-up peaks below the O(1s) peak of the different calcium phosphate phases showed a distinguishable variation between samples (Figure 4). Therefore, these peak areas were obtained by subtracting a Shirley baseline47 from each of the peaks and (42) Ertl, G.; Kiipers, J. Low Energy Electrons and Surface Chemistry, 2nd ed.; VCH Verlagsgesellschaft mbH: Weinheim, Germany, 1985. (43) Strohmeier, B. R.; Leyden, D. E.; Field, R. S.; Hercules, D. M. J. Catal. 1985, 94, 514. (44) Gaarenstroom, S. W.; Winograd, N. J. Chem. Phys. 1977, 67, 3500. (45) McElhaney, R. D.; Castner, D. G.; Ratner, B. D. In Metallization of Polymers; Sacher, E., Pireaux, J.-J., Kowalczyk, S. P., Eds.; ACS Symposium Series 440; American Chemical Society: Washington, DC, 1990; p 370-378. (46) Rodriguez, J. A.; Clendening, D.; Campbell, C. T. J. Phys. Chem. 1989, 93, 5238-5248. (47) Shirley, D. A. Phys. Rev. B 1972, 5, 4709-4714.

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Figure 7. Orbital energy structure for a Ca-PO4- cluster calculated by semiempirical quantum mechanical computation. The energy regions (bands) are labeled according to their orbital types based on coefficients of the atomic orbitals from the basis set.

Figure 5. Gaussian curve fitting of O(1s) shake-up/off satellites. The background was subtracted by Shirley baseline for each peak, and the area was measured as the integrated electron counts per second under each Gaussian fitting curve.

Figure 6. XPS peak intensity ratio of peak II/total O(1s) peaks (including peak I, peak II, and main peak) plotted vs the stoichiometric atomic ratio of bulk phosphate oxygen/total oxygen for different calcium phosphate samples. The ratios have been adjusted for carbonate component as well. Multiple measurements (3-9 repeated times) were performed for each data point.

measuring the counts of emitted photoelectrons under each Gaussian-fitted curve (Figure 5). As shown in Table 1, we found that the integrated intensity of peak II relative to the total O(1s) intensity including the main core peak, peak I, and peak II [O(1s)II/O(1s)total] decreased for the different calcium phosphate phases in the order β-TCP > HAP > OCP > DCP > DCPD > MCP. Such shake-up peak intensity should be closely related to the local electronic environment around the oxygen atom, due to the orbital overlap necessary for efficient shake-up.42 As shown in Figure 6, the relative intensity of peak II [O(1s)II/O(1s)total] correlates strongly with the fraction of phosphate-type oxygen in the bulk of the samples. (We define phosphate-type oxygen here as those oxygen atoms within PO43-, HPO42-, or H2PO4- ions except these in the OH form.) It seems clear that peak II arises mainly from such phosphate oxygens only. Again, the O(1s)II/ O(1s)total values plotted in Figure 6 were adjusted slightly from the values directly measured from XPS spectra [e.g., (O(1s)II/ O(1s)total)adjusted in Table 1], due to a small contribution of carbonate impurity at peak II, as described in the Appendix. As shown in 2890 Analytical Chemistry, Vol. 72, No. 13, July 1, 2000

Figure 6, the MCP, DCPD, DCP, OCP, HAP, and β-TCP samples were distinguishable from one another on the basis of the relative intensity of the O(1s) shake-up (peak II) alone. Of all the phases, the OCP has the largest error bar, possibly because it had a small amount of HAP impurity, according to XRD. We were not able to find pure OCP samples from any commercial or research source, because of the instability of this phase. Taking this fact into consideration, pure (hypothetical) OCP is presumably at the lower end of the error bar of the experimental data and should be distinguishable from HAP. The relative intensity of this shakeoff/up feature is unique and consistent for every compound. It is independent of sample charging associated with nonconducting samples, complicating any methodology using the binding energies of P or O peaks to identify different phases. Thus, the shakeup satellite provides a new tool for identifying the surface phase of CaP present which supplements the Ca/P and O/Ca ratios. Semiempirical Calculations of the O(1s) Shake-Up/Off Features. To help identify which orbitals might be involved in these two shake-up/off features, we performed semiempirical quantum mechanical calculations (using the ZINDO program from MOPAC48,49) of the electronic structure of several phosphate and calcium phosphate clusters. Both the PO43- clusters and the CaPO4- clusters adopted C3v symmetry. Bond distances were optimized using both ZINDO and AM1 calculations of cluster energy. A typical result is shown in Figure 7 for one CaPO4cluster. As can be seen, a high density of occupied orbitals are grouped in a band at ∼30 eV below the vacuum level. This group of orbitals is composed mainly of O(2s) orbitals (as determined from the coefficients on the atomic orbital basis set). This band lies 40-50 eV below a high density of unoccupied orbitals which are dominated by P-O antibonding orbital character. Given that the energy separation of such orbitals is usually overestimated by ∼15% in ZINDO,50,51 the excitation energy between these orbitals is probably 34-42 eV. This matches well the excitation energy for loss peak II, 36 ( 1 eV (see Figures 4 and 5), which suggests that this peak is a shake-up of valence electrons from the O(2s) orbitals into unoccupied P-O antibonding orbitals. This is consistent with the “phosphate” character of this peak as indicated by Figure 6. Because of the local nature of the excitation around the O(1s) core, the excitation probably originates from the O(2s) orbitals rather than the nearly degenerate Ca(2p) orbitals that are not included in Figure 7. (48) Ridley, J.; Zerner, M. C. Theor. Chim. Acta 1976, 42, 223. (49) Ridley, J.; Zerner, M. C. Theor. Chim. Acta 1973, 32, 111. (50) Rodriguez, J. A.; Campbell, C. T. Surf. Sci. 1987, 183, 449-468. (51) Rodriguez, J. A.; Campbell, C. T. Surf. Sci. 1988, 206, 424-450.

This same band of occupied O(2s) orbitals lies 28-40 eV below a band of unoccupied orbitals of mainly Ca character (plus some oxygen character) in Figure 7. Again, since ZINDO overestimates such separation by ∼15%, the excitation energy between these orbitals is probably ∼23-34 eV. This range includes the excitation energy of 23.5 ( 1.5 eV for loss peak I (see Figures 4 and 5). This suggests that peak I is due to a shake-up excitation of O(2s) valence electrons into unoccupied Ca orbitals. This peak’s relative intensity in XPS was almost independent of the “phosphate oxygen” content and about the same for all the CaP samples, which is consistent with the lack of participation of phosphorusderived orbitals in such a mechanism. The small loss peak (shoulder) at ∼9-12 eV below the main O(1s) XPS peak (see Figures 4 and 5) may be similarly due to shake-up excitation of the O(2p) electrons into unoccupied Ca orbitals. b. Application of XPS Results for CaP Phase Differentiation. As shown in Figure 6, the MCP, DCPD, DCP, OCP, HAP, and β-TCP samples were distinguishable from one another on the basis of the relative intensity of the O(1s) shake-up (peak II) alone. By further combining all the information obtained from XPS, including the Ca/P ratio, P/Ca ratio, and O(1s) shake-up feature, it is possible to provide unique assignments to most of the calcium phosphate phases and even to estimate their relative amounts in surfaces containing mixtures of calcium phosphates. For example, DCP and DCPD have the same Ca/P ratio (Figure 2) but are clearly distinguished with the peak II intensity in Figure 6 and the O/Ca ratio in Figure 3. Similarly, β-TCP and HAP are overlapping with each other in their O/Ca ratios (Figure 3) but are well separated by their Ca/P ratio in Figure 2 and the O(1s) peak loss intensity in Figure 6. Also, the relative positions of the different calcium phosphates are different in these three figures. It was found in this study and many other reports that XPS Ca/P ratios of the different calcium phosphate phases were consistently lower than the bulk stoichiometric values,20,52 while the O/Ca ratios are consistently higher than their corresponding values. Chusuei et al. pointed out that this might be due to the instability of the powders and prolonged exposure of the powders to the X-ray source leading to selective ejection of the Ca substituent.20 Also, in many calcium phosphates, the original crystal structure can coexist with small amount of defects and impurities. Biological apatite is one of the examples of a HAP crystal structure, but usually with a deficiency of Ca based on the expected stoichiometry. Thus, phase differentiation depending only on Ca/P and O/Ca ratios can give misleading results for such minerals. Including the O(1s) shake-up feature discussed above as one of the phase differentiation parameters will help avoid such misinterpretation. Application to a Bone Sample. To illustrate the advantage of this multiparameter analysis method, we analyzed a cortical bone sample prepared by the method described in the Experimental Section. An XPS survey spectrum showed Ca, P, and O, as well as C, F, Na, and K (figure not shown). Table 1 lists the direct atomic ratios of Ca/P and O/Ca and the shake-up peak II relative intensities obtained from detailed XPS scans for each phase. (Each value is the average over several samples of that phase.) Biological minerals naturally contain various “impurities.” Carbonate (CO32-) is one such constituent commonly observed in bone that might

interfere with interpretation of the data. [The carbonate composition is reported as 5.8-7.4% (average wt %) in bones1,19,53]. Therefore, we also measured by XPS the percentage of carbonate in the bone sample and adjusted the Ca/P, O/Ca, and O(1s)II/ O(1s)total accordingly. Following the procedure described in the Appendix, we found that the carbonate oxygen was ∼22% of the total oxygen present. The adjusted Ca/P, O/Ca, and O(1s)II/ O(1s)total ratios are also listed in Table 1. The calculations are only valid when assuming that all the oxygen atoms in the samples are either carbonate oxygen or stoichiometric (e.g., HAP) oxygen. By comparing the adjusted Ca/P, O/Ca, and O(1s)II/O(1s)total atomic ratios of the bone with the values from the reference CaPs, we can now confidently identify the surface phases of the minerals in our crushed bone samples. No single CaP phase is consistent with all three observed values, so binary mixtures will be considered. The adjusted Ca/P ratio, 1.42 ( 0.02, indicates that the bone mineral samples are a mixture of HAP plus any for the other CaPs (see Figure 2). Due to the fact that more cation impurities, such as Na+ and K+, were observed in bone samples (∼3%) than the standard samples (∼0-1%), it is possible for the real Ca/P ratio to be even lower than the adjusted Ca/P obtained by only considering the effect of carbonate composition. However, such effects should be smaller than that from the carbonate due to their relatively lower percentages. The adjusted O/Ca ratio, 3.10 ( 0.07, is consistent with a mixture of HAP with any one of the other CaPs except β-TCP (see Figure 3). Thus, the bone is clearly a mixture of mainly HAP with minor amounts of another CaP, but the Ca/P and O/Ca XPS ratios alone cannot specify which other CaP. The adjusted shake-up peak intensity ratio, (O(1s)II/ O(1s)total)adjusted, from the bone samples is 0.056 ( 0.002, and it must also be included in order to limit the possibilities. To do this, however, requires a more quantitative analysis, which we performed. Briefly, the adjusted Ca/P, O/Ca, and O(1s)II/O(1s)total ratios measured for the bone were each equated to a linear combination of the form

(52) Tanahashi, M.; Matsuda, T. J. Biomed. Mater, Res. 1997, 34, 305-315.

(53) Tung, M. S. Personal communications, 1999.

Rbone ) χiRi + (1 - χi)RHAP where Ri is the measured ratio for species i (OCP, MCP, DCP, or DCPD) based on our analysis above, χi is the mole fraction of species i, and 1 - χi is the mole fraction of HAP. For the case that we assume the bone is a mixture of HAP with OCP, the equation becomes

Rbone ) χOCPROCP + (1 - χOCP)RHAP Three such equations can be obtained for the three adjusted ratios: Ca/P, O/Ca, and O(1s)II/O(1s)total. Each equation can then be solved for χOCP. These three equations can be obtained for each of the binary mixtures: HAP + OCP, HAP + MCP, HAP + DCP, and HAP + DCPD. For the correct binary mixture, the three equations should all give the same mole fractions, at least within their resulting error bars (propagated from the errors on the ratios Ri, given in Table 1). For HAP combined with either OCP or DCP, the resulting mole fractions were statistically indistinguishable and were limited to the range χOCP ) 0.48 ( 0.12 and χDCP ) 0.23 (

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0.02, respectively. For HAP combined with MCP or DCPD, the resulting mole fractions were statistically different. This proves that the bone is neither a binary HAP + MCP nor a HAP + DCPD mixture, but it could be HAP combined with either OCP or DCP in the mole fractions stated above. The above calculations show that the relative intensity of peak II in the O(1s) region can indeed help in identification and quantification of the calcium phosphate species on the surfaces. In agreement with the present results, OCP is a common phase observed by bulk analytical techniques to exist in bone along with HAP.24,54 2. TOF-SIMS Results. Both positive ion and negative ion TOF-SIMS spectra of the calcium phosphate powders in the m/z 0-500 range were acquired. Characteristic peaks with significant intensity (>0.4% of the largest peak) were observed only in the m/z range of 1-100 amu for both positive and negative spectra (see Figure 8 for an example). The characteristic peaks shown in the positive spectra include Ca+, CaO+, and CaOH+. Impurities such as Mg+, Na+, K+, and hydrocarbons showed up in the cation spectra as well. The signal intensities of impurities varied over a wide range depending on the powder species, consistent with XPS results. The characteristic peaks shown in the negative spectra include O-, OH-, P-, O2-, HO2-, PO-, PO2-, and PO3-. Impurities were observed as Cl- and F- in some of the samples. The most distinguishable patterns in the negative ion spectra are the intensity variations of the PO- (m/e ) 47), PO2- (m/e ) 63), and PO3- (m/e ) 79) peaks. These three peaks are present in all the sample materials, and their relative intensities changed between the different calcium phosphate samples. Chusuei et al.20 have reported similar results from their TOF cluster SIMS data (with Cs+, (CsI)2Cs+, (CsI)Cs+, and C60+ primary ions). Our observation

confirms their conclusion in this respect (but here with Cs+ ion excitation only). They used quantitative analysis of the relative PO3-/PO2- peak intensities in their SIMS data with (CsI)Cs+ as primary ions to successfully differentiate five out of the six of the CaP phases they studied. The PO3-/PO2- ratios we observed for our CaP compounds (using Cs+ primary ions) were as follows: HAP ) 0.51 ( 0.01, β-TCP ) 0.65 ( 0.03, OCP ) 0.73 ( 0.02, DCPD ) 1.15 ( 0.07, DCP ) 1.22 ( 0.05, and MCP ) 2.33 ( 0.11. As can be seen, these compounds can be just distinguished (within the error bars of 1 standard deviation) on the basis of this ratio. However, this ratio is so close for HAP, β-TCP, and OCP, and DCPD and DCP that they might not be distinguishable in routine analysis of real samples where impurities might complicate the spectra. The ratios are similar to those reported by Chusuei et al.20 for the four phases they also studied (β-TCP, HAP, OCP, DCPD) using (CsI)Cs+ primary ions, with the exception that their ratio for HAP was slightly higher than for β-TCP. The PO3-/PO2- ratio could distinguish all four of these phases both in their study and our present results. However, their distinction between OCP and TCP was somewhat clearer, possibly because they were using (CsI)Cs+ ions, which also allow them to use a much lower ion dose.20 Although it is also possible to use a few peaks in TOF-SIMS spectra such as Figure 8 as “fingerprints” for discrimination of CaP phases, it is desirable to have a more robust method which might be more applicable to mixtures of the phases. Therefore, PCA was used for analyzing these TOF-SIMS spectra. The peak intensities in the positive and negative spectra were normalized to the sum of all the intensities of the selected characteristic peaks in the respective spectra prior to PCA. It was observed that this normalization improved the PCA model to achieve better clustering of the data from the same samples. Other pretreatment methods, including mean centering, autoscaling, range scaling, and variance scaling,55 were tried in order to reduce data scatter and obtain the best differentiation of the CaP powders. Mean centering yielded the most clustered scores plot in this study. A description of the influence of different preprocessing and scaling methods on PCA data analysis for TOF-SIMS spectra can be found in ref 55. After normalization and mean centering, the peak intensity data in both the positive and negative TOF-SIMS spectra were analyzed by PCA. In our results, only two principal components are needed to explain ∼95% of the variation. Thus, the results are presented in scores and loadings plots33,35 associated with the first two PCs (parts a and b of Figure 9 for scores and loadings plots, respectively). At least two samples were analyzed for each phase. Each data point was associated with a label to designate a different sample phase or peak mass (ion), for scores or loadings plots, respectively. The scores plot allows data points with the same origin to be gathered into a point cluster, while the loadings plots reveal the ions whose intensities contribute to the two PCs.55 (The loadings value is the coefficient of that ion in the PC.) As shown in Figure 9a, the first two PCs can separate the different CaP phases into clusters in separate regions. As can be seen, MCP is easily differentiated from other CaP phases. OCP has relatively larger scatter, possibly due to the HAP phase existing in the synthesized powder. The discrimination between

(54) Tung, M. S.; Brown, W. E. Calcif. Tissue Int. 1985, 37, 329-331.

(55) Eynde, X. V.; Bertrand, P. Surf. Interface Anal. 1997, 25, 878.

Figure 8. Positive (a) and negative (b) TOF-SIMS spectra for a β-TCP sample. Similar characteristic peaks appeared in all the calcium phosphate samples studied, but with changes of relative peak intensities.

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Figure 9. Multivariate analysis (PCA) results of TOF-SIMS data obtained from different calcium phosphates samples. Score (a) and loading (b) plots of PC1 vs PC2 for all the samples. Phase names and peak positions are as labeled.

β-TCP and HAP is relatively difficult, but possible. The loadings of PC1 vs PC2 (Figure 9b) shows that the normalized intensities of PO3- (m/z ) 79), O- (m/z ) 16), Ca+ (m/z ) 40), CaOH+ (m/z ) 57), PO2- (m/z ) 63), and OH- (m/z ) 17) ions have significant coefficients in PC1 and PC2. Thus, good discrimination ability is possibly offered by comparing the relative intensities of these ions alone between CaP phases. More specifically, the PO3-, O-, Ca+, CaOH+, OH-, and PO2- ion intensities have high loadings (larger coefficients) in PC1, whereas PC1 loadings for the other CaP peaks are close to zero. On the other hand, PC2 has high loadings (coefficients) from the Ca+, CaOH+, PO3-, CaO+, and O- ions only. Other variables are located almost at (0,0) of the loadings plot, showing little contribution to either PC. By comparing the scores and loadings plots, we realized that certain individual peaks might be particularly useful for distinguishing some specific CaP phases, e.g., PO3- for MCP, Ca+ for DCP, and CaO+ for OCP. We actually did univariate analysis for these peaks (variation of single peak intensity for different samples at these m/z), and such univariate plots did show better differentiation power for separating the corresponding samples than other peaks (plots not shown). This demonstrates the power of PCA to elucidate trends in the data and identify key spectral features from a complex spectrum. After building a PCA model with a set of reference spectra from known CaP phases as we have done here, one could then apply this model to the spectra of an unknown CaP sample. However, because of the close clustering of some phases, positive identifications could become difficult, considering errors within the plots (which could in principle be reduced with more measurements). This SIMS/PCA analysis would be particularly powerful if combined with the three important XPS ratios mentioned above. It is not clear how well SIMS with PCA would work on a mixture of CaP phases, especially for mixtures where the phase domains are tiny, since matrix effects are very important in changing relative SIMS intensities. For such intimate mixtures, XPS should be preferred since XPS interactions are very insensitive to matrix effects. CONCLUSIONS Both XPS and TOF-SIMS can be used to differentiate the six different CaP phase reference samples with surface sensitivity.

In addition to Ca/P and O/Ca peak ratios in XPS, the O(1s) shakeup intensity [relative to the total O(1s) intensity] can help to distinguish between different CaP phases and quantify their amounts when in mixtures. The combination of these three XPS ratios is particularly powerful in this respect, as shown with a bone sample. PCA of both the positive and the negative TOF-SIMS spectra can effectively differentiate the CaP phases using only the first two PCs, which are dominated by the relative intensities of a few ions: PO3-, O-, Ca+, CaOH+, PO2-, and OH-. The combination of XPS peak ratios and TOF-SIMS PCA analysis promises to be powerful in identifying surface structures of CaP, especially for ultrathin films that are not amenable to bulk analytical methods such as XRD. These should provide a direct and powerful way for further study of the effect of surface properties on the corresponding biological behavior of different CaP mineral phases. ACKNOWLEDGMENT We are grateful to Drs. Stephen Goledge, David G. Castner, and Stephen Porter for their valuable discussion and experimental help with XPS and TOF-SIMS. Dr. Bonnie Tyler is highly appreciated for her help with multivariate analysis. We also want to thank Dr. Ming Tung at the NIST research center for generously providing us the standard HAP and β-TCP samples, and Drs. Bart Kahr and Sihyvn Ham for help with the quantum calculations. This research has been funded by a graduate fellowship from the Center for Nanotechnology at the University of Washington (H.B.L.), NSF Engineering Research Center EEC 9529161 (B.D.R.), DOE-BES Chemical Science Division (C.T.C.), and NIH Grant RR01296. We thank Drs. Charles Chusuei and D. W. Goodman for supplying their manuscript before publication and helpful discussions. APPENDIX To apply XPS for identifying real CaP samples, it is necessary to consider the effect of some common substituents in CaP on the ratios discussed in the paper, i.e., Ca/P, O/Ca, and O(1s)II/ O(1s)total. Carbonate is one of such substituent that was present in the samples examined in this paper. Here we describe the measurements and calculations used to adjust the characteristic XPS ratios due to carbonate. Analytical Chemistry, Vol. 72, No. 13, July 1, 2000

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First, the fraction of the total surface carbon in each sample that is carbonate-type carbon was measured in XPS C(1s) spectra by discriminating carbonate carbon and hydrocarbon. By taking a high-resolution spectrum for a pure CaCO3 sample, it was found that there were two C(1s) peaks whose binding energies were separated by ∼4.7 eV. The peak at lower BE (284.6 eV) was small and attributed to hydrocarbon contamination on the sample, while the peak at higher BE (289.3 eV) was from carbonate carbon. It was obvious that these two types of C signals can be well differentiated. The hydrocarbon does not affect the relative atomic ratios of Ca/P, O/Ca, and O(1s)II/O(1s)total. But carbonate can change the Ca/P and O/Ca ratios (carbonate is thought to substitute for PO43- when incorporated into calcium phosphate minerals), and its oxygen contributes to both the O(1s)II and O(1s)total intensities. The separation of these two types of C(1s) peaks allows us to extract the carbonate oxygen from the total oxygen composition to obtain more appropriate Ca/P, O/Ca, and O(1s)II/O(1s)total values for surface characterization, using the C(1s) peak intensity ratio of carbonate-type C to total C: C(1s)carbonate/C(1s)total. Thus, the atomic percentage of carbonate carbon in the XPS probe depth was calculated by

% Ccarbonate ) % Ctotal(C(1s)carbonate/C(1s)total)

(2)

The χ values were then used for adjusting Ca/P, O/Ca, and O(1s)II and O(1s)total. For adjusting the Ca/P and O/Ca atomic ratios, it was assumed that CO32- ions substitute for PO43- ions only and that three CO32ions substitute for two PO43- ions. Then the adjusted surface

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(Ca/P)adjusted ) [1 - (2/3)(χ/3)](Ca/P)dir

(3)

(O/Ca)adjusted ) (8/9)χ(O/Ca)dir + (1 - χ)(O/Ca)dir ) (O/Ca)dir (1 - χ/9) (4) The (Ca/P)dir and (O/Ca)dir atomic ratios were those directly measured by XPS. The CaCO3 reference sample also showed a shake-up satellite in the O(1s) spectrum at the position of peak II, with an O(1s)II/ O(1s)total intensity ratio of 0.05. To extract the effect of carbonate oxygen from the total oxygen features to obtain more appropriate O(1s)II/O(1s)total values for the CaP samples, the O(1s)II/O(1s)total ratio for the pure CaCO3 sample was scaled by the fraction of oxygen that is carbonate-type oxygen (χ). Thus, in any of the samples where a carbonate component was observed, the effect of carbonate on the O(1s) peak II shake-up intensity should be calculated by the following equations:

[O(1s)II]carbonate ) 0.050[O(1s)total]carbonate

(5)

[O(1s)total]carbonate ) χ[O(1s)total]dir

(6)

(1)

where % Ctotal is the total atomic percentage of carbon in XPS. The atomic percentage of oxygen (% Ocarbonate) due to carbonate was taken to be 3 times the carbonate carbon percentage (three oxygen per one carbon in CO32-). The ratio of carbonate oxygen to total oxygen (χ) was calculated by dividing the carbonate oxygen atomic percentage by the total oxygen atomic percentage and is presented in Table 1:

χ ) % Ocarbonate/% Ototal

atomic ratios are given by

where χ was calculated for each sample as described above and listed in Table 1. Then, the adjusted O(1s)II/O(1s)total ratio is

[O(1s)II/O(1s)total]adjusted ) {[O(1s)II]dir [O(1s)II]carbonate}/{[O(1s)total]dir - [O(1s)total]carbonate} (7) Here, [O(1s)II]dir and [O(1s)total]dir were the intensities obtained from the XPS measurements directly. (Note: The calculations are only valid when assuming that all the oxygen atoms in the samples are either carbonate oxygens or stoichiometric oxygens.) Received for review July 22, 1999. Accepted December 9, 1999. AC990812H