Capillary Electrophoresis Micro X-ray Fluorescence: A Tool for

Apr 3, 2003 - A new tool was developed for separation and elemental detection by interfacing a simple capillary electrophoresis (CE) apparatus, constr...
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Anal. Chem. 2003, 75, 2048-2053

Capillary Electrophoresis Micro X-ray Fluorescence: A Tool for Benchtop Elemental Analysis Thomasin C. Miller,† Martha R. Joseph,‡ George J. Havrilla,*,† Cris Lewis,† and Vahid Majid§

Chemistry Division, Analytical Chemistry Sciences Group, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, Chemistry Division, Actinide Analytical Chemistry Group, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, and Department of Chemistry, Westminster College, South Market Street, New Wilmington, Pennsylvania 16172

A new tool was developed for separation and elemental detection by interfacing a simple capillary electrophoresis (CE) apparatus, constructed using a thin-walled fusedsilica capillary, with a benchtop energy-dispersive micro X-ray fluorescence (MXRF) system. X-ray excitation and detection of the separated analytes was done using an EDAX Eagle II micro X-ray fluorescence system equipped with a polycapillary Rh target excitation source and a SiLi detector. It was demonstrated that this prototype system could be used for the separation and detection of species containing two different metals from one another, specifically Cu and Co. Free Co could also be separated from Co bound to cyanocobalamin (vitamin B-12). Two organic compounds were also separated from one another, a large biological protein, ferritin, from a small biological organic, cyanocobalamin. Preliminary average detection limits obtained on this system were on the order of 10-4 M and compared favorably to those reported for the similar technique of CE-synchrotron XRF. CEMXRF allows for nondestructive, simultaneous, on-line, benchtop elemental analysis for chemical speciation applications. Capillary electrophoresis (CE) is a technique used for the separation of analytes in a complex mixture based on their different electrophoretic mobilities. Specifically, it has the advantages of high separation efficiencies, small reagent and sample volume requirements (nL), and rapid sample throughput.1 Due to its ability to separate analytes in aqueous solution under environmentally and biologically relevant pH and ionic strength conditions, CE has become exceedingly popular in environmental, clinical, and forensic analysis with applications in the areas of waste characterization, protein separation, screening of small molecules in biological matrixes such as blood serum, and DNA analysis.2,3 There has also been a large focus on adapting * Corresponding author. Tel: 1-505-667-9627. Fax: 1-505-665-5982. E-mail: [email protected]. † Analytical Chemistry Sciences Group, Los Alamos National Laboratory. ‡ Westminster College. § Actinide Analytical Chemistry Group, Los Alamos National Laboratory. (1) Baker, D. R. Capillary Electrophoresis; John Wiley & Sons: New York, 1995. (2) Dolnik, V.; Hutterer, K. M. Electrophoresis 2001, 22, 4163-4178. (3) Thormann, W.; Lurie, I. S.; McCord, B.; Marti, U.; Cenni, B.; Malik, N. Electrophoresis 2001, 22, 4216-4243.

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separation technologies, such as CE, toward species-selective analysis for trace metals and metalloids in biological matrixes.4-6 By combining CE with an elemental detection technique, chemical speciation becomes possible by allowing the qualitative and quantitative evaluation of different forms of a specific element or range of elements in a complex matrix. CE-elemental methods also have the potential to successfully separate and detect target elemental species, such as inorganic ions or metal complexes, which cannot be analyzed by conventional molecular methods such as UV-visible absorbance or fluorometry. Some of the most common hybrid CE-elemental analysis techniques include CEEC (electrochemical detection), CE-PIXE (proton-induced X-ray emission), CE-SRXRF (synchrotron X-ray fluorescence), and CEICPMS (inductively coupled plasma mass spectrometry). EC detection7-9 lends itself well to miniaturization, which is suited for the small dimensions required for CE systems. Additionally, current EC instrumentation is relatively inexpensive, and many well-characterized electrode/analyte systems are available for direct adaptation to CE. Detection limits for electroactive compounds are in the nanomolar range. The major drawbacks to this technique are that it is limited to analysis of electroactive species, and the electrochemical detector must be isolated from the CE separation potential. This requires the detection to be decoupled from the CE separation, either through fracturing of the capillary or the use of end-column detection. Using protons for elemental excitation, CE-PIXE10-12 boasts simultaneous, elemental analysis of all elements Z > 13 with detection limits ranging between 10-7 and 10-5 M. Prior to analysis, the fused-silica capillary used for CE separation must be modified through etching so it is thin enough (∼10 µm) for ion beam penetration and excitation, resulting in a capillary that is extremely fragile and subject to breakage. Additionally, proton irradiation can cause radiolysis of the run buffer, which interrupts (4) Timerbaev, A. R. Analyst 2001, 126, 964-981. (5) Szpunar, J. Analyst 2000, 125. (6) Lobinski, R. Fresenius J. Anal. Chem. 2001, 369, 113-114. (7) Baldwin, R. P. Electrophoresis 2000, 21, 4017-4028. (8) Holland, L. A.; Lunte, S. M. Anal. Commun. 1998, 35, 1H-4H. (9) Timerbaev, A. R.; Shpigun, O. A. Electrophoresis 2000, 21, 4179-4191. (10) Vogt, C.; Vogt, J.; Wittrisch, H. J. Chromatogr., A 1996, 727, 301-310. (11) Vogt, J.; Vogt, C. Nucl. Instrum. Methods Phys. Res. Sect. B 1996, 108, 133135. (12) Wittrisch, H.; Conradi, S.; Rohde, E.; Vogt, J.; Vogt, C. J. Chromatogr., A 1997, 781, 407-416. 10.1021/ac0207269 CCC: $25.00

© 2003 American Chemical Society Published on Web 04/03/2003

Table 1. Selection of Capillary

a

fused-silica capillary

o.d. (µm)

TSP100170 TSP250350 TSP530660 TSP075375

164 362 666 363

dimensions i.d. (µm) 97 256 534 75

wall thickness (µm)

Cu

33.5 53 66 144

0.084 0.046 0.008 1.983

static detection limita (mg/mL) Zn Fe 0.154 0.072 0.021 0.488

0.088 0.009 0.010 0.935

Co 0.112 0.031 0.025 1.248

Based on sample volume assuming a 50-µm MXRF spot size, n ) 3, and spectral dwell time 10 s.

the charge separation within the capillary. This precludes the ability to perform on-line separation and detection. Furthermore, the technique requires a van de Graaff accelerator for excitation, which is not readily accessible. Conversely, CE-SRXRF13,14 allows for on-line simultaneous, multielemental analysis of elements Z > 17 with detection limits in the range of 10-4 M. It is also a nondestructive technique, allowing further chemistries or analyses to be performed on the isolated analytes. However, this work could not be performed through an ordinary fused-silica capillary, requiring the more complicated construction of a polyethylene window in the separation system for adequate excitation and detection of emitted characteristic X-rays. Additionally, potential users are limited in their access to this technology due to the use of a synchrotron light source to generate incident X-rays. CE-ICPMS (e.g., refs 4, 9, and 15-17) offers simultaneous, on-line multielemental detection as well as the lowest detection limits reported for a CE-elemental analysis hybrid ranging from 10-11 to 10-9 M. Isotope-specific detection is also possible. As with all plasma-based systems, the technique is destructive and has a delicate (in terms of balancing flows) interface between the capillary and the ICPMS detector. Unfortunately, ICPMS suffers from poor detection capabilities for species with severe isobaric overlaps with the original matrix and the Ar plasma gas (e.g., Ca, Fe, As, Se, S), in addition, halogenated species that require high ionization energy are often overlooked by ICPMS. The goal of this research was to develop a new tool for separation and elemental detection that would allow for the successful on-line, simultaneous elemental identification, quantitation, and speciation of environmentally and biologically relevant species while overcoming many of the drawbacks of the techniques described above. Micro X-ray fluorescence (MXRF) was chosen as a detection method to achieve these goals. Similar to conventional X-ray fluorescence, MXRF uses direct X-ray excitation, which is absorbed by the sample. Incident X-ray photons remove inner shell electrons from the elements in the sample. The vacancies produced are filled by outer shell electrons, which release X-ray photons during the transition. The emitted X-ray photons are characteristic of the elements present in the sample. MXRF employs polycapillary optics that are used in conjunction with the X-ray source. This focuses the source X-rays, resulting (13) Ringo, M. C.; Huhta, M. S.; Shea-McCarthy, G.; Penner-Hahn, J. E.; Evans, C. E. Nucl. Instrum. Methods Phys. Res. Sect. B 1999, 149, 177-181. (14) Mann, S. E.; Ringo, M. C.; Shea-McCarthy, G.; Penner-Hahn, J. E.; Evans, C. E. Anal. Chem. 2000, 72, 1754-1758. (15) Majidi, V.; Miller-Ihli, N. J. Analyst 1998, 123, 809-813. (16) Majidi, V.; Miller-Ihli, N. J. Analyst 1998, 123, 803-808. (17) Majidi, V. Microchem. J. 2000, 66, 3-16.

in much smaller beam diameters (i.e., 10-50 µm) and high X-ray fluxes (∼107 photons/s) at the sample surface, increasing detection sensitivity and enhancing performance over conventional X-ray instruments.18 Unlike SRXRF, benchtop micro X-ray spectrometers are commercially available. In this study, a simple CE apparatus employing an unmodified fused-silica capillary was constructed and successfully interfaced with a benchtop micro X-ray spectrometer. The resulting instrument demonstrated the feasibility of MXRF as an elemental detection method for CE separation. EXPERIMENTAL SECTION Reagents. All chemicals were reagent grade unless noted. Deionized, distilled water was used to prepare solutions. Single element standards (10.00 mg/mL) of Co, Cu, Fe, and Zn in 4% HNO3 were obtained from High Purity Standards Inc. (Charleston, SC). Ammonium acetate was obtained from Mallinckrodt Baker Inc. (Phillipsburg, NJ). Sodium hydroxide (for pH adjustment) was obtained from Fisher Scientific Inc. Trizma (tris(hydroxymethyl)aminomethane and tris(hydroxymethyl)aminomethane hydrochloride), concentrated hydrochloric acid, and concentrated nitric acid (for pH adjustment), ferritin, and cyanocobalamin (vitamin B-12) were obtained from Sigma Chemical Inc. Selection of Fused-Silica Capillary. Elemental standards containing 0, 0.01, 0.05, 0.1, 0.5, 1.0, and 10 mg/mL copper in deionized water were prepared by dilution from a 10 mg/mL Cu2+ single-element high-purity standard. Each solution, in turn, was injected by capillary action into a small piece (i.e., 5 cm) of fusedsilica capillary, placed into the MXRF sample chamber, and analyzed for X-ray emission for each different concentration. It took ∼5 s to fill the capillary with solution by capillary action (∼0.39 µL). This process was performed for four different types of fused-silica capillary (TSP100170, TSP250350, TSP530660, and TSP075375; Polymicro Technologies, Phoenix, AZ), whose dimensions are listed in Table 1. Each experiment was performed in triplicate to ensure repeatability. Static detection limits of Cu within each type of capillary could be calculated from a calibration curve prepared from the emitted Cu X-ray intensities for each standard. The process was repeated for Co, Fe, and Zn. The Co K-L2-3 (KR) (6.92 keV), Cu K-L2-3 (KR) (8.04 keV), Fe K-L2-3 (KR) (6.40 keV), and Zn K-L2-3 (KR) (8.63 keV) X-ray emission lines were monitored to detect characteristic line emissions. It should be emphasized that for all experiments the capillaries were not altered in any way. The excitation X-ray beam was focused through the capillary polyimide coating and the fused-silica capillary wall. (18) Schields, P. J.; Gibson, D. M.; Gibson, W. M.; Gao, N.; Huang, H.; Ponomarev, I. Y. Powder Diffr. 2002, 17, 70-80.

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Figure 1. CEMXRF interface.

Figure 2. Top view of CE apparatus.

CEMXRF Coupling. All separations were performed using a custom-built capillary electrophoresis system with on-line MXRF detection. The CE apparatus and CEMXRF interface are shown in Figures 1 and 2. Figure 1 illustrates how the constructed CE apparatus conveniently sits within the MXRF sample chamber on the XYZ translational stage with the detection region placed underneath the X-ray source and SiLi detector. Figure 2 shows the top view of the CE apparatus. The CE apparatus is housed in a small 12.0 × 8.5 × 6.0 cm3 plastic box. Within the plastic housing, the fused-silica capillary is coiled between two buffer reservoirs each containing a platinum electrode. The electrode/ buffer/capillary system is electrically connected by a high-voltage power supply. The only part of the CE system that is exposed to the outside is the detection region. The system is equipped with an interlock to reduce electrical exposure risk to the operator. Also, the plastic housing further insulates the CE apparatus from the operator and the outside MXRF instrumentation, minimizing any exposure to electrical arcs or shorts. The first step in CEMXRF operation is to inject the sample mixture onto the column. A potential is then applied to the column, which causes the analytes to separate and move through the capillary according to their electrophoretic mobilities. The analytes travel through the capillary from anode to cathode with the smaller, more positively charged species moving through the column more quickly than the larger, more negatively charged species. As the separated species pass through the detection region of the CEMXRF apparatus, they interact with the incoming source X-rays and emit characteristic X-rays, which are detected by the SiLi detector. The spectra acquired during the CE separation are then converted into electropherograms, plotting intensity versus migration time for the analytes of interest. CEMXRF Analysis. All CE separations were performed using a Bertan model ARB-30 high-voltage power supply (Bertan High Voltage, Hicksville, NY). 2050

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An unmodified fused-silica capillary with polyimide coating (TSP100170, 164 µm o.d. × 97 µm i.d., wall thickness 33.5 µm, Polymicro Technologies Inc.) was used for all separations. This capillary was chosen for reasons explained later in the paper. The length of the capillary from anode to cathode was 70 cm with a length of 61 cm to the detection region. X-ray excitation and detection was achieved using an EDAX Eagle II micro X-ray fluorescence system equipped with a Rh target excitation source and a SiLi detector (EDAX, Mahwah, NJ). The X-ray source was fitted with a polycapillary focusing optic having a 50-µm nominal X-ray spot size (X-ray Optical Systems, Albany, NY). X-ray tube operating conditions were maintained at 40 kV and 1000 µA during all experiments. All separations were performed at ambient pressure. Spectra were acquired during CE separation at a rate of 0.1 s-1 (i.e., spectral dwell time, 10 s). When focused, the distance between the MXRF polycapillary and the CE sample capillary was 10 mm. Prior to daily analysis, the capillary was first conditioned by flushing with 1.0 M NaOH for 15 min. The basic solution was rinsed from the capillary by flushing the column with distilled, deionized water for 15 min and then flushing with separation run buffer for an additional 15 min. (Choice of run buffer depends on the given separation run after conditioning.) Between runs, the capillary was rinsed with 0.2 M NaOH for 4 min, deionized water for 4 min, and run buffer for 4 min. Each of the separations was performed in triplicate to ensure repeatability. (a) Separation of Co2+ and Cu2+. A sample containing 0.1 mg/mL Co2+ and 0.1 mg/mL Cu2+ in deionized water was prepared by dilution from concentrated standards prepared from the chloride salt and nitrate salt of Cu and Co, respectively. The sample was injected hydrodynamically by reducing the pressure to 380 mbar at the cathodic end of the capillary for 4 s (1.29 µL injected). A separation potential of 10 kV was applied to platinum electrodes (sampling end of capillary at positive potential and outlet end at ground) to facilitate sample separation with a 50 mM NH4Ac run buffer, pH 4.5. The Co K-L2-3 (KR) and Cu K-L2-3 (KR) X-ray emission lines were monitored to detect the separated metals. (b) Separation of Cyanocobalamin from Free Co2+. A sample containing 0.2 mg/mL Co and 14 mg/mL cyanocobalamin (∼0.6 mg/mL bound Co) in deionized water was prepared by dilution from a concentrated standard prepared from the chloride salt of Co and dissolution of the appropriate weighed amount of solid cyanocobalamin. The sample was injected hydrodynamically by reducing the pressure to 380 mbar at the cathodic end of the capillary for 4 s (1.29 µL injected). A separation potential of 10 kV was applied to platinum electrodes to facilitate sample separation with a 75 mM Trizma run buffer, pH 8.0. The Co K-L2-3 (KR) X-ray emission was monitored to detect free and vitamin B-12bound Co. (c) Separation of Ferritin-Bound Fe3+ from Cyanocobalamin-Bound Co2+. A sample containing 1.16 mg/mL ferritin and 14 mg/mL cyanocobalamin in deionized water was prepared from solid samples of cyanocobalamin and ferritin. The sample was injected hydrodynamically by reducing the pressure to 253 mbar at the cathodic end of the capillary for 4 s (0.80 µL injected). A separation potential of 9.5 kV was applied to platinum electrodes to facilitate sample separation with a 100 mM Trisma run buffer,

Figure 3. Electropherogram of the separation of 0.1 mg/mL Co2+ and 0.1 mg/mL Cu2+ in deionized water. Buffer conditions: 50 mM NH4Ac run buffer, pH 4.5. Separation conditions: 4-s hydrodynamic injection via suction at 1/2 ambient pressure (380 mbar) and 10-kV run potential. Spectral acquisition rate, 0.1 s-1.

Figure 4. Electropherogram of the separation of 0.2 mg/mL Co2+ and 14 mg/mL cyanocobalamin in deionized water. Buffer conditions: 75 mM Trizma run buffer, pH 8.0. Separation conditions: 4-s hydrodynamic injection via suction at 1/2 ambient pressure (380 mbar) and 10-kV run potential. Spectral acquisition rate, 0.1 s-1.

pH 8.0. The Co K-L2-3 (KR) and Fe K-L2-3 (KR) X-ray emission lines were monitored to detect the Fe-bound ferritin and vitamin B-12-bound Co. RESULTS AND DISCUSSION Selection of Fused-Silica Capillary. The choice of fusedsilica capillary is crucial both for adequate separation of sample analytes and for sufficient detection of characteristic emissions by MXRF. Table 1 lists the types of fused-silica capillaries that were tested for use in the CEMXRF. Fused-silica TSP075375 is routinely used for CE analysis with UV and fluorescence detection. This capillary has a 75-µm inner diameter with thick walls (144 µm). This capillary is ideal for CE separations because the smaller inner diameter produces less Joule heating, which can be detrimental to a separation, and the large wall thickness provides good heat dissipation.1 However, due to its small optical path length and relatively thick walls, it produces a very large Bremsstrahlung background. Consequently, it is not the best capillary for MXRF excitation and detection of characteristic emitted X-rays. As shown in Table 1, the detection limits of Co, Cu, Fe, and Zn are very high for this capillary. The detection limits are improved significantly for capillaries such as TSP250350 and TSP530660 that are thin walled and have much larger inner diameters. However, these capillaries are inadequate for efficient CE separation due to their large inner diameters. The optimum inner diameters for CE separation range from 50 to 100 µm. 1 From all the capillaries tested, TSP100170 has a very thin wall (33.5 µm) while providing an inner diameter (97 µm) that is compatible with both CE separation and MXRF excitation and detection. This capillary was used for all separations. There were no modifications made to the capillary. All MXRF detection was done on the capillary with the polyimide coating in place. Analyses by CEMXRF. The following separations were successfully performed and detected by the new CEMXRF system: different free metal ions, free metal from complexed metal, and separation of two complexed metals. Figure 3 shows the electropherogram of the separation of two free metals in solution, Co and Cu. As is evident from the plot, the Co peak appeared at 4.5 min and the Cu peak eluted at 6.8 min. The

Figure 5. Electropherogram of the separation of 1.16 mg/mL ferritin and 14 mg/mL cyanocobalamin in deionized water. Buffer conditions: 100 mM Trisma run buffer, pH 8.0. Separation conditions: 4-s hydrodynamic injection via suction at 1/3 ambient pressure (253 mbar) and 9.5 kV run potential. Spectral acquisition rate, 0.1 s-1.

electropherogram of the separation of a free metal, Co, from the same metal bound in a complex, Co bound to cyanocobalamin (vitamin B-12), is shown in Figure 4. The positively charged free Co2+ peak had a migration time of 5.0 min with a fwhm of 1 min, and the neutral vitamin B-12 peak was detected at 10 min with a fwhm of 1.5 min. While cyanocobalamin is neutral under the experimental conditions and has no electophoretic velocity, it does travel through the capillary with the electoroosmotic flow toward the cathode. Figure 5 illustrates the electropherogram of the separation of two different metal complexes, Fe bound to ferritin and Co bound to cyanocobalamin. The ferritin and vitamin B-12 species were separated from one another with neutral vitamin B-12 peak detection at 6.3 min with a fwhm of 1 min and the negatively charged ferritin peak occurring after 10 min with a fwhm of 1.7 min. Please note that the migration time of vitamin B-12 is different in Figures 4 and 5 because of the differences in the ionic strength of the run buffer and a slight electrical potential difference. Dynamic detection limits for each of the different species studied in the above separations are shown in Table 2 (dynamic meaning these analytes were calibrated under CE separation conditions as opposed to static conditions where the sample was Analytical Chemistry, Vol. 75, No. 9, May 1, 2003

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Table 2. Comparison of Dynamic Detection Limits between CEMXRF and CE-SRXRF CEMXRF flux: ∼1 × 107 photons/s over X-ray energy range 0-40 keV analyte Cu(II) Co(II) vitamin B-12 ferritin a

detection limit (mg/mL), n ) 3

detection limit (M), n ) 3

0.032 0.021 0.38 3.1

5.0 × 10-4 3.6 × 10-4 2.3 × 10-3 8.5 × 10-7

CE-SRXRFa,b flux: 4 × 108 photons/s at X-ray energy of 10 keV analyte Cu-CDTA Co-CDTA vitamin B-12 Fe-CDTA Zn-CDTA

detection limit (M)a

detection limit (M)b

2.2 × 10-4 2.8 × 10-4 9.9 × 10-5 nac 2.3 × 10-4

1.0 × 10-4 1.5 × 10-4 1.0 × 10-4 1.5 × 10-4 1.3 × 10-4

Taken from ref 13. b Taken from ref 14. c Not available.

injected onto the capillary for calibration, but no separation was performed). If the dynamic detection limits of Cu and Co in Table 2 are compared to their static detection limits listed in Table 1, the dynamic detection limits are 3-5 times lower than the static detection limits. This is due to the fact that, in dynamic CE measurements, the injected sample is concentrated into migrating zones, while during static measurements the sample is spread out through the entire capillary. Therefore, the resulting intensity from the dynamic measurements will be much higher than for the static measurements for a given sample concentration, resulting in lower dynamic detection limits. The CEMXRF system presented in this paper is very preliminary and still has much room for improvement. The major sources of uncertainty in this technique are associated with injection volume and the ability to both measure the exact injection volume and reproducibly inject the same exact volume sample after sample. Manual injection methods were used with this preliminary CEMXRF setup. In the future, the use of an autosampler could be integrated with the instrumentation to reduce uncertainty based on sample injection. Peak definition and S/N is mainly limited by the low instrument sampling rate (10 s per spectrum) required by the preliminary CEMXRF system. This spectral dwell time is needed to adequately detect the sample signal above the high background produced by the silica capillary. Improvements in CEMXRF materials and detection technology should be able to decrease spectral dwell times, therefore increasing the sampling rate, which would enhance sample peak definition and increase S/N. For example, the use of a monochromatic X-ray source or simple filtering techniques may be able to significantly lower the background produced by the silica capillary so that higher spectral sampling rates can be used. Research is currently underway to improve upon the presented technology. Comparison of CEMXRF to CE-SRXRF. The CE-elemental detection hybrid technique most closely related to CEMXRF is CE-SRXRF. Table 2 compares the calculated dynamic detection limits for both of these separation methods. Notice that the detection limits for CEMXRF performed with a conventional thin fused-silica capillary are of a magnitude similar to those found by Mann et al.14 and Ringo et al.13 with a polyethylene detection window and SRXRF excitation. Even lower detection limits are achievable for ferritin with CEMXRF due to the very large iron core present in this molecule (e.g., 4500 Fe3+ ions in the ferritin core19). Interestingly, comparable detection limits are achievable even though the SRXRF source has a much higher flux, 4 × 108 2052 Analytical Chemistry, Vol. 75, No. 9, May 1, 2003

Figure 6. Spectrum of 1 mg/mL Co, Cu, Fe, and Zn detected in the static mode through fused-silica capillary (TSP100170, 164 µm o.d. × 97 µm i.d., wall thickness 33.5 µm). Rh X-ray tube conditions: 40 kV, 1000 µA.

photons/s, for monochromatic X-rays at 10 keV, while the MXRF source has a flux of 1 × 107 photon/s distributed over the entire Bremsstrahlung output of the Rh X-ray tube through the polycapillary optic ranging from 0 to 40 keV. The useful analytical ranges of excitation energies are those greater than the absorption edges of Co, Cu, and Fe. Thus only a fraction of the X-ray photon flux is useful in exciting the analytes of interest. These results show that CE separation and X-ray fluorescence detection can be adequately performed using a benchtop MXRF system. Even though the detection limits of the new CEMXRF system are comparable to those found for CE-SRXRF, the sensitivity of the method still does not compare with other, more established methods such as CE-ICPMS. The elevated detection limits are caused by high Bremsstrahlung background resulting from scatter by the silica capillary. Figure 6 shows a typical spectrum of analytes detected statically within the 33.5-µm wall thickness fusedsilica capillary used in all separations performed in this study. The small analyte peaks are easily identified but must compete with the presence of a very large background signal. MXRF systems equipped with a commercial monochromatic excitation source could greatly increase sensitivity for analytes of interest by eliminating X-ray scattering background under the analyte peaks. For example, a monochromatic Cu X-ray excitation source would enhance the vitamin B-12/ferritin detection by reducing the scatter under the Co K-L2-3 (KR) and Fe K-L2-3 (KR) peaks. Similarly, simple filtering techniques may allow for background elimination or signal enhancement. Research is currently underway to improve detection limits by these means. (19) O’Neil, M. J., Ed. The Merck Index, 13th ed.; Merck & Co, Inc.: Whitehouse Station, NJ, 2001.

This work demonstrates the utility of using MXRF for detection of metal complexed organic and biologically based molecules. This method has the potential for increasing the sensitivity of the detection on unmodified silica capillary tubing. Therefore, this is a simple way to separate metal-containing species. In addition, this detection scheme is nondestructive so the separated material can be recovered for additional characterization.

ACKNOWLEDGMENT T.C.M. was supported by the Director of Central Intelligence (DCI) Postdoctoral Research Fellowship Program. Received for review November 25, 2002. Accepted January 30, 2003. AC0207269

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