- Email: [email protected]
On-line X-ray fluorescence detection for capillary electrophoresis separations
Nuclear Instruments and Methods in Physics Research B 149 (1999) 177±181
On-line X-ray ¯uorescence detection for capillary electrophoresis separations M.C. Ringo a, M.S. Huhta a, G. Shea-McCarthy b, J.E. Penner-Hahn a, C.E. Evans b
a,*
a Department of Chemistry, University of Michigan, 930 North University Av., Ann Arbor, MI 48109-1055, USA Consortium for Advanced Radiation Sources, University of Chicago at Brookhaven National Laboratory, Building 725, Upton, NY 11973, USA
Received 8 July 1998; received in revised form 9 October 1998
Abstract In these investigations, capillary electrophoresis with on-line X-ray ¯uorescence detection (CE-XRF) has been demonstrated for the ®rst time. The insertion of a polyethylene sample cell between fused-silica capillary segments enabled continuous XRF detection during electrophoresis with minimal additional band broadening. Detection limits in the 10ÿ4 M range are currently feasible for the CE-XRF separation of metal complexes, and design advances will enhance detectability to the 10ÿ5 ±10ÿ6 M range, permitting studies of important environmental and biological samples. Ó 1999 Elsevier Science B.V. All rights reserved.
The study of metal interactions in biological and environmental systems remains an enduring challenge. X-ray ¯uorescence spectroscopy (XRF) oers considerable advantages because it enables the identi®cation and quantitation of dierent metals simultaneously [1]. In many cases, however, the biological and environmental eects of metals are dependent on their oxidation state and binding environments. Unfortunately, XRF alone cannot discriminate between the same metal in dierent environments. However, the distinction of metals with dierent speciation can be accomplished by combining XRF with techniques which separate
* Corresponding author. Tel.: (734) 763-8012; fax: 647-4050; e-mail: [email protected]
(734)
species prior to analysis. In this study, we demonstrate the successful coupling of XRF with capillary electrophoretic separation for the discrimination and analysis of transition-metal complexes with a common chelating agent. Capillary electrophoresis (CE) has shown considerable promise in the separation and analysis of biological and environmental samples [2]. In capillary electrophoresis, the application of an electric ®eld across an electrolyte-®lled capillary induces the migration of solutes. The electrophoretic mobility of each solute is a function of the charge and hydrodynamic radius of the solute as well as the viscosity of the electrolyte. As a result, small differences in the charge-to-size ratio of solutes can lead to spatial separation. Capillary electrophoresis possesses several advantages that make it a
0168-583X/98/$ ± see front matter Ó 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 5 8 3 X ( 9 8 ) 0 0 8 8 9 - 1
178
M.C. Ringo et al. / Nucl. Instr. and Meth. in Phys. Res. B 149 (1999) 177±181
powerful technique for analyzing biological and environmental mixtures. Very small volumes (nL) of an impure sample can be analyzed, and separation can be conducted in aqueous solution under environmentally and biologically relevant pH and ionic strength conditions. In addition to its use as a separation tool, fundamental binding information is also accessible using CE including oxidation state, stoichiometry, and binding constant determinations [3,4]. In previous studies of metal-containing complexes, metal speciation has been determined by coupling CE with inductively coupled plasma mass spectrometry (ICP±MS) [5]. While this elementspeci®c detection technique has provided sensitive and information-rich analysis, separating power is often compromised by the mixing required within the ICP region. Moreover, this technique is inherently limited to species that can be volatilized. X-ray spectroscopy provides a valuable alternative, overcoming many of the disadvantages of previous methods. Recent studies have successfully coupled proton-induced X-ray emission (PIXE) with capillary electrophoresis in the analysis of metalloproteins and metal complexes [6,7]. Unfortunately, radiolysis of the aqueous buer generates gas bubbles, interrupting the electrophoretic separation. As a result, implementation requires decoupling of the separation and detection processes. This is accomplished by ®rst electrophoretically separating the solutes of interest and subsequently moving solutes into the detection window by hydrodynamic pumping [7]. By contrast, X-ray ¯uorescence employs a lower-energy excitation source that will not radiolyze water, thus facilitating continuous, on-line detection. The feasibility of this new detection scheme was demonstrated using the experimental apparatus depicted in Fig. 1, which was operated at beamline X-26A of the National Synchrotron Light Source at Brookhaven National Laboratory (Upton, NY). A Si(1 1 1) monochromator was used to select 10 keV X-rays, which were partially focused with Kirkpatrick-Baez (KB) mirrors to a spot size of 30 lm by 40 lm. X-ray ¯uorescence detection was accomplished using a single-channel Si/Li detector positioned in a coplanar right-angle geometry with the X-ray beam (Fig. 1) and interfaced to a Vax
Fig. 1. Schematic diagram of capillary electrophoresis apparatus with X-ray ¯uorescence detection.
workstation. The Si/Li detector output was plotted using a Canberra multiple-channel analyzer (MCA) program implemented in IDL. Emission spectra were collected for 1.0 s live time with a cycle time of ca. 4 s. The cobalt, copper, and zinc Ka intensities were integrated after subtraction of a linear background for each peak. The system was operated at a total incident count rate of approximately 3000 counts/s to avoid detector saturation. This gave peak windowed ¯uorescence counts rates of ca. 100 counts/s. Silica is largely opaque to 7±10 keV X-rays, with signal attenuation by the capillary of approximately 90%. This consideration prevents direct XRF measurements within the unmodi®ed fused-silica capillary. A series of static XRF measurements was performed to determine the best available sample cell material. Polyetheretherketone (PEEK) had unacceptably high transitionmetal background, while the available wall thickness of Te¯on tubing had unacceptably high absorbance. In contrast, polyethylene tubing showed negligible transition-metal background and was highly transparent. As shown in the inset to Fig. 1, a length of polyethylene tubing (580 lm i.d. ´ 965 lm o.d., Intramedic, Clay Adams, Parsippany, NJ) was used to bridge two fused-silica capillary segments (100 lm i.d. ´ 361 lm o.d., Polymicro Technologies, Phoenix, AZ) with an approximately 100 lm gap between the capillary segments. This sample cell/fused-silica capillary assembly was then mounted on a precision x±y±z translational stage and positioned using a visible-light microscope.
M.C. Ringo et al. / Nucl. Instr. and Meth. in Phys. Res. B 149 (1999) 177±181
The electrically driven migration of solutes through the X-ray beam was accomplished using a custom-built capillary electrophoresis instrument (Fig. 1). The electrophoretic buer solution was 20 mM sodium borate decahydrate (Sigma, St. Louis, MO) at pH 9.00, which contained 1.0 mM trans1,2-diaminocyclohexanetetraacetic acid (CDTA) (Fluka, Buchs, Switzerland) [8,9]. A high-voltage power supply (Model PS/EH30R03.0, Glassman High Voltage, Whitehouse Station, NJ) was used to apply a potential of +10.5 kV to a platinum electrode immersed in a 1-ml vial of buer solution. A second electrode was grounded and immersed in a second 1-ml vial of buer solution. These solutions were connected by the fused-silica/ sample-cell assembly which was also ®lled with running buer solution. An electric ®eld was applied across the fused-silica/sample-cell assembly, causing electrically driven ¯ow from the high-potential buer vial to the grounded buer vial. Although the capillary gap and stagnant buer solution adjacent to the electrophoresis channel (inset, Fig. 1) might have been expected to induce some mixing, these features have no eect on electrically driven ¯ow because the electrical connectivity is not aected by these modi®cations. Using this con®guration, X-ray ¯uorescence detection occurred at a distance of 67.4 cm from the capillary inlet, with a total capillary length of 128.6 cm. Metal cation solutes of Co2 , Cu2 , and Zn2 were chosen based on their biological and environmental importance. A solution that contained 1.0±1.3 mM of each metal nitrate (Sigma) as well as 5.5 mM of CDTA was prepared in borate buer (vide supra) and introduced onto the capillary using electrophoretic injection for 10 s at +10.5 kV. The large equilibrium constants for the complexation of each of these metals with CDTA [10] together with the excess CDTA in the sample solution ensured that essentially all of metal cations in the sample were present in the complexed form. In addition, Vitamin B12 (cyanocobalamin, Sigma) was added at a concentration of 1.1 mM. Vitamin B12 exhibits electrophoretic mobility under these conditions which was con®rmed to be statistically indistinguishable from mesityl oxide, a common void marker in capillary electrophoresis. There-
179
fore, by monitoring the migration of Vitamin B12 , the ¯uid-¯ow rate was measured as 2.74 cm/min. The powerful advantages of this new method are evident from inspection of plots of ¯uorescence versus time during the separation of metal-containing solutes, shown in Fig. 2. Because both Vitamin B12 and the CoáCDTA complex contain cobalt, both solutes exhibit cobalt Ka ¯uorescence. However, unlike conventional XRF which convolutes the ¯uorescence of all compounds containing the same metal, CE-XRF is capable of distinguishing between complexes containing the same metal by separating them prior to analysis. Therefore, the amounts of Vitamin B12 and CoáCDTA can be assessed separately with no mutual spectral interference. Capillary electrophoresis with XRF detection also provides straightforward identi®cation and discrimination between dierent metals complexed with the same chelating agent. In contrast, most common capillary electrophoresis detectors, such as ultraviolet absorbance, are considerably less selective for dierent transition-metal complexes. Peak identi®cation for many CE methods is based on matching elution time with single-compound standards, and is consequently ambiguous in some cases. By contrast, CE-XRF provides ready identi®cation of all elements heavier than argon present in a sample. As indicated in Fig. 2, the electrophoretic mobilities of the cobalt, zinc, and copper complexes with CDTA are quite similar (3.16, 3.22, and 3.26 ´ 10ÿ4 cm2 Vÿ1 sÿ1 , respectively), yet they are clearly resolved into individual peaks by CE-XRF. By contrast, CE-UV can exhibit signi®cant overlap between electrophoretic peaks which makes quantitative analysis less precise. In order for the coupling of X-ray ¯uorescence with capillary electrophoresis to be fully complementary, it is important that XRF detection does not interrupt electrophoresis or degrade the separation. In the present studies, the electrophoretic mobilities of the solute peaks match the mobilities of metaláCDTA complexes measured under identical conditions using a conventional silica capillary and UV detection. The peak broadening that was observed for each solute during the CE-XRF separation was in good agreement with the peak
180
M.C. Ringo et al. / Nucl. Instr. and Meth. in Phys. Res. B 149 (1999) 177±181 Table 1 XRF signal-to-noise ratios and detection limits for the 90% con®dence level (one-tailed, N 1) of electrophoretic solutes. Conditions as stated in text
Fig. 2. X-ray ¯uorescence detection during separation of various metal complexes with CDTA, shown with 3-point smoothing. Electrophoretic buer: 20 mM borate with 1 mM CDTA; pH 9.00. Sample contains 1 mM of each metal and Vitamin B12 as well as 5 mM CDTA.
length variance predicted by diusional factors alone, resulting in 104 ±105 theoretical plates per meter for each solute. These results demonstrate that electrically driven ¯ow is not disturbed signi®cantly by the presence of the XRF sample cell.
Solute
Signal-to-noise ratio
Detection limit (´10ÿ4 M)
Vitamin B12 CobaltáCDTA CopperáCDTA ZincáCDTA
14 5.9 6.5 5.6
0.99 2.8 2.2 2.3
The signal-to-noise ratio attained for the separation of these metal-containing solutes is shown in Table 1, and resulted in detection limits ranging from 0.99 to 2.8 ´ 10ÿ4 M for each solute. The slightly increased signal for Zn may be attributed to more ecient Ka excitation relative to Cu and Co. Simultaneously, greater elastic scattering contributions within the Zn window increase the background noise, leading to no overall enhancement in Zn S/N relative to Cu and Co. Although this level of detectability does not currently match CE-ICPMS or CE-PIXE methods, signi®cant improvements are straightforward. These preliminary measurements used a relatively small detector (ca. 1 cm2 active area), which was placed approximately 3 cm from the sample to avoid detector saturation. Thus, less than 5% of the available ¯uorescence was detected. Utilizing larger and faster detectors that are commercially available, this yield could be improved at least ®ve-fold. In addition, the ¯ux available at the X-26A beamline (ca. 108 photons/s) is 3±4 orders of magnitude lower than that available from third-generation synchrotron sources. The combination of better detectors and brighter sources will permit the CEXRF analysis of nanomolar concentrations. Acknowledgements The authors thank the National Institutes of Health (GM-38047) for funding of this research, as well as the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory, which is supported by the U.S. Department of Energy. Stephanie E. Mann is thanked for her assistance with measurements.
M.C. Ringo et al. / Nucl. Instr. and Meth. in Phys. Res. B 149 (1999) 177±181
References [1] R. Jenkins, X-Ray Fluorescence Spectrometry, Wiley, New York, 1988. [2] P.D. Grossman, J.C. Colburn (Eds.), Capillary Electrophoresis: Theory and Practice, Academic Press, San Diego, CA, 1992. [3] E. Dabek-Zlotozynska, E.P.C. Lai, A.R. Timerbaev, Anal. Chim. Acta 359 (1998) 1. [4] K.L. Rundlett, D.W. Armstrong, J. Chromatogr. A 721 (1996) 173. [5] K. Sutton, R.M.C. Sutton, J.A. Caruso, J. Chromatogr. A 789 (1997) 85.
181
[6] J. Vogt, C. Vogt, Nucl. Instr. and Meth. B 108 (1996) 133. [7] H. Wittrisch, S. Conradi, E. Rohde, J. Vogt, C. Vogt, J. Chromatogr. A 781 (1997) 407. [8] S. Motomizu, S. Nishimura, Y. Obata, H. Tanaka, Anal. Sci. 7 (1991) 253. [9] A.R. Timerbaev, O.P. Semenova, J.S. Fritz, J. Chromatogr. A 756 (1996) 300. [10] A.E. Martell, R.M. Smith, Critical Stability Constants: Amino Acids, vol. 1, Plenum, New York, 1974, pp. 238± 239.
On-line X-ray ¯uorescence detection for capillary electrophoresis separations M.C. Ringo a, M.S. Huhta a, G. Shea-McCarthy b, J.E. Penner-Hahn a, C.E. Evans b
a,*
a Department of Chemistry, University of Michigan, 930 North University Av., Ann Arbor, MI 48109-1055, USA Consortium for Advanced Radiation Sources, University of Chicago at Brookhaven National Laboratory, Building 725, Upton, NY 11973, USA
Received 8 July 1998; received in revised form 9 October 1998
Abstract In these investigations, capillary electrophoresis with on-line X-ray ¯uorescence detection (CE-XRF) has been demonstrated for the ®rst time. The insertion of a polyethylene sample cell between fused-silica capillary segments enabled continuous XRF detection during electrophoresis with minimal additional band broadening. Detection limits in the 10ÿ4 M range are currently feasible for the CE-XRF separation of metal complexes, and design advances will enhance detectability to the 10ÿ5 ±10ÿ6 M range, permitting studies of important environmental and biological samples. Ó 1999 Elsevier Science B.V. All rights reserved.
The study of metal interactions in biological and environmental systems remains an enduring challenge. X-ray ¯uorescence spectroscopy (XRF) oers considerable advantages because it enables the identi®cation and quantitation of dierent metals simultaneously [1]. In many cases, however, the biological and environmental eects of metals are dependent on their oxidation state and binding environments. Unfortunately, XRF alone cannot discriminate between the same metal in dierent environments. However, the distinction of metals with dierent speciation can be accomplished by combining XRF with techniques which separate
* Corresponding author. Tel.: (734) 763-8012; fax: 647-4050; e-mail: [email protected]
(734)
species prior to analysis. In this study, we demonstrate the successful coupling of XRF with capillary electrophoretic separation for the discrimination and analysis of transition-metal complexes with a common chelating agent. Capillary electrophoresis (CE) has shown considerable promise in the separation and analysis of biological and environmental samples [2]. In capillary electrophoresis, the application of an electric ®eld across an electrolyte-®lled capillary induces the migration of solutes. The electrophoretic mobility of each solute is a function of the charge and hydrodynamic radius of the solute as well as the viscosity of the electrolyte. As a result, small differences in the charge-to-size ratio of solutes can lead to spatial separation. Capillary electrophoresis possesses several advantages that make it a
0168-583X/98/$ ± see front matter Ó 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 5 8 3 X ( 9 8 ) 0 0 8 8 9 - 1
178
M.C. Ringo et al. / Nucl. Instr. and Meth. in Phys. Res. B 149 (1999) 177±181
powerful technique for analyzing biological and environmental mixtures. Very small volumes (nL) of an impure sample can be analyzed, and separation can be conducted in aqueous solution under environmentally and biologically relevant pH and ionic strength conditions. In addition to its use as a separation tool, fundamental binding information is also accessible using CE including oxidation state, stoichiometry, and binding constant determinations [3,4]. In previous studies of metal-containing complexes, metal speciation has been determined by coupling CE with inductively coupled plasma mass spectrometry (ICP±MS) [5]. While this elementspeci®c detection technique has provided sensitive and information-rich analysis, separating power is often compromised by the mixing required within the ICP region. Moreover, this technique is inherently limited to species that can be volatilized. X-ray spectroscopy provides a valuable alternative, overcoming many of the disadvantages of previous methods. Recent studies have successfully coupled proton-induced X-ray emission (PIXE) with capillary electrophoresis in the analysis of metalloproteins and metal complexes [6,7]. Unfortunately, radiolysis of the aqueous buer generates gas bubbles, interrupting the electrophoretic separation. As a result, implementation requires decoupling of the separation and detection processes. This is accomplished by ®rst electrophoretically separating the solutes of interest and subsequently moving solutes into the detection window by hydrodynamic pumping [7]. By contrast, X-ray ¯uorescence employs a lower-energy excitation source that will not radiolyze water, thus facilitating continuous, on-line detection. The feasibility of this new detection scheme was demonstrated using the experimental apparatus depicted in Fig. 1, which was operated at beamline X-26A of the National Synchrotron Light Source at Brookhaven National Laboratory (Upton, NY). A Si(1 1 1) monochromator was used to select 10 keV X-rays, which were partially focused with Kirkpatrick-Baez (KB) mirrors to a spot size of 30 lm by 40 lm. X-ray ¯uorescence detection was accomplished using a single-channel Si/Li detector positioned in a coplanar right-angle geometry with the X-ray beam (Fig. 1) and interfaced to a Vax
Fig. 1. Schematic diagram of capillary electrophoresis apparatus with X-ray ¯uorescence detection.
workstation. The Si/Li detector output was plotted using a Canberra multiple-channel analyzer (MCA) program implemented in IDL. Emission spectra were collected for 1.0 s live time with a cycle time of ca. 4 s. The cobalt, copper, and zinc Ka intensities were integrated after subtraction of a linear background for each peak. The system was operated at a total incident count rate of approximately 3000 counts/s to avoid detector saturation. This gave peak windowed ¯uorescence counts rates of ca. 100 counts/s. Silica is largely opaque to 7±10 keV X-rays, with signal attenuation by the capillary of approximately 90%. This consideration prevents direct XRF measurements within the unmodi®ed fused-silica capillary. A series of static XRF measurements was performed to determine the best available sample cell material. Polyetheretherketone (PEEK) had unacceptably high transitionmetal background, while the available wall thickness of Te¯on tubing had unacceptably high absorbance. In contrast, polyethylene tubing showed negligible transition-metal background and was highly transparent. As shown in the inset to Fig. 1, a length of polyethylene tubing (580 lm i.d. ´ 965 lm o.d., Intramedic, Clay Adams, Parsippany, NJ) was used to bridge two fused-silica capillary segments (100 lm i.d. ´ 361 lm o.d., Polymicro Technologies, Phoenix, AZ) with an approximately 100 lm gap between the capillary segments. This sample cell/fused-silica capillary assembly was then mounted on a precision x±y±z translational stage and positioned using a visible-light microscope.
M.C. Ringo et al. / Nucl. Instr. and Meth. in Phys. Res. B 149 (1999) 177±181
The electrically driven migration of solutes through the X-ray beam was accomplished using a custom-built capillary electrophoresis instrument (Fig. 1). The electrophoretic buer solution was 20 mM sodium borate decahydrate (Sigma, St. Louis, MO) at pH 9.00, which contained 1.0 mM trans1,2-diaminocyclohexanetetraacetic acid (CDTA) (Fluka, Buchs, Switzerland) [8,9]. A high-voltage power supply (Model PS/EH30R03.0, Glassman High Voltage, Whitehouse Station, NJ) was used to apply a potential of +10.5 kV to a platinum electrode immersed in a 1-ml vial of buer solution. A second electrode was grounded and immersed in a second 1-ml vial of buer solution. These solutions were connected by the fused-silica/ sample-cell assembly which was also ®lled with running buer solution. An electric ®eld was applied across the fused-silica/sample-cell assembly, causing electrically driven ¯ow from the high-potential buer vial to the grounded buer vial. Although the capillary gap and stagnant buer solution adjacent to the electrophoresis channel (inset, Fig. 1) might have been expected to induce some mixing, these features have no eect on electrically driven ¯ow because the electrical connectivity is not aected by these modi®cations. Using this con®guration, X-ray ¯uorescence detection occurred at a distance of 67.4 cm from the capillary inlet, with a total capillary length of 128.6 cm. Metal cation solutes of Co2 , Cu2 , and Zn2 were chosen based on their biological and environmental importance. A solution that contained 1.0±1.3 mM of each metal nitrate (Sigma) as well as 5.5 mM of CDTA was prepared in borate buer (vide supra) and introduced onto the capillary using electrophoretic injection for 10 s at +10.5 kV. The large equilibrium constants for the complexation of each of these metals with CDTA [10] together with the excess CDTA in the sample solution ensured that essentially all of metal cations in the sample were present in the complexed form. In addition, Vitamin B12 (cyanocobalamin, Sigma) was added at a concentration of 1.1 mM. Vitamin B12 exhibits electrophoretic mobility under these conditions which was con®rmed to be statistically indistinguishable from mesityl oxide, a common void marker in capillary electrophoresis. There-
179
fore, by monitoring the migration of Vitamin B12 , the ¯uid-¯ow rate was measured as 2.74 cm/min. The powerful advantages of this new method are evident from inspection of plots of ¯uorescence versus time during the separation of metal-containing solutes, shown in Fig. 2. Because both Vitamin B12 and the CoáCDTA complex contain cobalt, both solutes exhibit cobalt Ka ¯uorescence. However, unlike conventional XRF which convolutes the ¯uorescence of all compounds containing the same metal, CE-XRF is capable of distinguishing between complexes containing the same metal by separating them prior to analysis. Therefore, the amounts of Vitamin B12 and CoáCDTA can be assessed separately with no mutual spectral interference. Capillary electrophoresis with XRF detection also provides straightforward identi®cation and discrimination between dierent metals complexed with the same chelating agent. In contrast, most common capillary electrophoresis detectors, such as ultraviolet absorbance, are considerably less selective for dierent transition-metal complexes. Peak identi®cation for many CE methods is based on matching elution time with single-compound standards, and is consequently ambiguous in some cases. By contrast, CE-XRF provides ready identi®cation of all elements heavier than argon present in a sample. As indicated in Fig. 2, the electrophoretic mobilities of the cobalt, zinc, and copper complexes with CDTA are quite similar (3.16, 3.22, and 3.26 ´ 10ÿ4 cm2 Vÿ1 sÿ1 , respectively), yet they are clearly resolved into individual peaks by CE-XRF. By contrast, CE-UV can exhibit signi®cant overlap between electrophoretic peaks which makes quantitative analysis less precise. In order for the coupling of X-ray ¯uorescence with capillary electrophoresis to be fully complementary, it is important that XRF detection does not interrupt electrophoresis or degrade the separation. In the present studies, the electrophoretic mobilities of the solute peaks match the mobilities of metaláCDTA complexes measured under identical conditions using a conventional silica capillary and UV detection. The peak broadening that was observed for each solute during the CE-XRF separation was in good agreement with the peak
180
M.C. Ringo et al. / Nucl. Instr. and Meth. in Phys. Res. B 149 (1999) 177±181 Table 1 XRF signal-to-noise ratios and detection limits for the 90% con®dence level (one-tailed, N 1) of electrophoretic solutes. Conditions as stated in text
Fig. 2. X-ray ¯uorescence detection during separation of various metal complexes with CDTA, shown with 3-point smoothing. Electrophoretic buer: 20 mM borate with 1 mM CDTA; pH 9.00. Sample contains 1 mM of each metal and Vitamin B12 as well as 5 mM CDTA.
length variance predicted by diusional factors alone, resulting in 104 ±105 theoretical plates per meter for each solute. These results demonstrate that electrically driven ¯ow is not disturbed signi®cantly by the presence of the XRF sample cell.
Solute
Signal-to-noise ratio
Detection limit (´10ÿ4 M)
Vitamin B12 CobaltáCDTA CopperáCDTA ZincáCDTA
14 5.9 6.5 5.6
0.99 2.8 2.2 2.3
The signal-to-noise ratio attained for the separation of these metal-containing solutes is shown in Table 1, and resulted in detection limits ranging from 0.99 to 2.8 ´ 10ÿ4 M for each solute. The slightly increased signal for Zn may be attributed to more ecient Ka excitation relative to Cu and Co. Simultaneously, greater elastic scattering contributions within the Zn window increase the background noise, leading to no overall enhancement in Zn S/N relative to Cu and Co. Although this level of detectability does not currently match CE-ICPMS or CE-PIXE methods, signi®cant improvements are straightforward. These preliminary measurements used a relatively small detector (ca. 1 cm2 active area), which was placed approximately 3 cm from the sample to avoid detector saturation. Thus, less than 5% of the available ¯uorescence was detected. Utilizing larger and faster detectors that are commercially available, this yield could be improved at least ®ve-fold. In addition, the ¯ux available at the X-26A beamline (ca. 108 photons/s) is 3±4 orders of magnitude lower than that available from third-generation synchrotron sources. The combination of better detectors and brighter sources will permit the CEXRF analysis of nanomolar concentrations. Acknowledgements The authors thank the National Institutes of Health (GM-38047) for funding of this research, as well as the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory, which is supported by the U.S. Department of Energy. Stephanie E. Mann is thanked for her assistance with measurements.
M.C. Ringo et al. / Nucl. Instr. and Meth. in Phys. Res. B 149 (1999) 177±181
References [1] R. Jenkins, X-Ray Fluorescence Spectrometry, Wiley, New York, 1988. [2] P.D. Grossman, J.C. Colburn (Eds.), Capillary Electrophoresis: Theory and Practice, Academic Press, San Diego, CA, 1992. [3] E. Dabek-Zlotozynska, E.P.C. Lai, A.R. Timerbaev, Anal. Chim. Acta 359 (1998) 1. [4] K.L. Rundlett, D.W. Armstrong, J. Chromatogr. A 721 (1996) 173. [5] K. Sutton, R.M.C. Sutton, J.A. Caruso, J. Chromatogr. A 789 (1997) 85.
181
[6] J. Vogt, C. Vogt, Nucl. Instr. and Meth. B 108 (1996) 133. [7] H. Wittrisch, S. Conradi, E. Rohde, J. Vogt, C. Vogt, J. Chromatogr. A 781 (1997) 407. [8] S. Motomizu, S. Nishimura, Y. Obata, H. Tanaka, Anal. Sci. 7 (1991) 253. [9] A.R. Timerbaev, O.P. Semenova, J.S. Fritz, J. Chromatogr. A 756 (1996) 300. [10] A.E. Martell, R.M. Smith, Critical Stability Constants: Amino Acids, vol. 1, Plenum, New York, 1974, pp. 238± 239.