Anal. Chem. 1998, 70, 2475-2480
Transient Isotachophoretic-Electrophoretic Separations of Lanthanides with Indirect Laser-Induced Fluorescence Detection Matthew N. Church, Jonathan D. Spear, and Richard E. Russo
Lawrence Berkeley National Laboratory, M/S 70-193A, Berkeley, California 94720 Gregory L. Klunder,* Patrick M. Grant, and Brian D. Andresen
Forensic Science Center, Lawrence Livermore National Laboratory, P.O. Box 808 L-178, Livermore, California 94550
Indirect laser-induced fluorescence was used for the detection of several lanthanide species separated by capillary electrophoresis. Quinine sulfate was the fluorescent component of the background electrolyte, and r-hydroxyisobutyric acid was added as a complexing agent to enable the separation of analyte ions that have similar mobilities. The UV lines (333-364 nm) of an argon ion laser were used as the excitation source with a diode array detector for monitoring the fluorescent emission at 442 nm. Electrokinetic injections and transient isotachophoresis were implemented to stack the analyte ions into more concentrated zones. On-line preconcentration factors were determined to be ∼700 and resulted in limits of detection for La3+, Ce3+, Pr3+, Nd3+, Sm3+, and Eu3+ in the low-ppb range (6-11 nM). Many of the advantages of capillary electrophoresis (CE), such as high resolution and short analysis times, are derived from the use of small-diameter capillaries. However, the small capillary diameters also limit the path length for optical absorption detection methods commonly used and, therefore, limit the achievable sensitivity. Increasing the path length in the detection windows has been pursued through instrumental modifications such as the z-cell, bubble capillary, or extended path length cell, which offer improvements in the detection limits with minimal losses in resolution.1,2 More sensitive optical detection methods have been pursued, with fluorescence being the most popular as demonstrated by the numerous applications described in a recent review by Schwartz et al.3 Using direct fluorescence, detection limits in the attomole to zeptomole range and even single-molecule detection have been reported for PAHs and proteins.4,5 By combining a long-path bubble cell capillary with fluorescence detection, Sepaniak et al. demonstrated an additional 3-fold improvement in fluorescence sensitivity for 7-ethoxycoumarin, a metabolic enzyme marker.6 (1) Li, S. F. Y. Capillary Electrophoreisis: Principles, practice, and applications; Elsevier: New York, 1993. (2) Kaltenback, P.; Ross, G. A.; Heiger, D. N. Presented at HPCE 97, Anaheim, CA. (3) Schwartz, H. E.; Ulfelder, K. J.; Chen, F.-T. A.; Pentoney, S. L., Jr. J. Capillary Electrophor. 1994, 1, 36-54. (4) Nie, S.; Dadoo, R.; Zare, R. N. Anal. Chem. 1993, 65, 3571-3575. (5) Chen, D.; Dovichi, N. J. Anal. Chem. 1996, 68, 690-696. S0003-2700(97)01043-3 CCC: $15.00 Published on Web 05/09/1998
© 1998 American Chemical Society
Direct fluorescence detection of chelated metal ions has been accomplished by Swaile and Sepaniak.7 Using 8-hydroxyquinoline5-sulfonic acid (HQS), which fluoresces only when complexed, they were able to detect Mg2+, Ca2+, and Zn2+ in the ppb range. However, this approach is restricted only to species that form fluorescing complexes. More commonly, indirect fluorescence detection techniques were used for analyses of metal ions, although the number of studies has been rather limited and they have not addressed analysis of the lanthanides. Gross and Yeung used a quinine sulfate buffer system for the indirect detection of several group IA and group IIA metals.8 When using with electrokinetic injections, they were able to achieve detection at micronormal (femtomole) levels. Other work with Ce3+ and 18crown-6-ether as the indirect fluorescence carrier electrolyte reported detection of group IA and group IIA metals at lowmicromolar concentration levels.9 In addition to instrumental modifications, methods to concentrate analytes on the column have been reported to improve the analytical detectability in capillary electrophoresis. One common sample stacking technique can be accomplished by injecting the sample electrokinetically; however, this approach can result in biased sample loading.10 Other techniques, such as field-amplified sample injection (FASI) described by Chien and Burgi, have been used to concentrate the sample on-column up to 1000-fold.11-15 In this method, a leading plug of water was introduced prior to electrokinetic injection of the sample to create a high-field zone to concentrate the ions. Isotachophoresis (ITP) has long been used as a separation technique and can be a very effective method of preconcentration using larger diameter capillaries and larger sample injections.16 Under true ITP conditions, the analyte zones are concatenated but not physically separated, and conductivity detection is commonly used. A recent review by Mazereeuw et (6) Cole, R. O.; Hiller, D. L.; Chwojdak, C. A.; Sepaniak, M. J. J. Chromatogr., A 1996, 736, 239-245. (7) Swaile, D. F.; Sepaniak, M. J. Anal. Chem. 1991, 63, 179-184. (8) Gross, L.; Yeung, E. S. Anal. Chem. 1990, 62, 427-431. (9) Baechmann, K.; Boden, J.; Haumann, I. J. Chromatogr. 1992, 626, 259265. (10) Huang, X.; Gordon, M. J.; Zare, R. N. Anal. Chem. 1988, 60, 375-377. (11) Chien, R.-L.; Burgi, D. S. Anal. Chem. 1992, 64, 489A-496A. (12) Chien, R.-L.; Burgi, D. S. Anal. Chem. 1991, 63, 2042-2047. (13) Chien, R.-L.; Burgi, D. S. J. Chromatogr. 1991, 559, 141-152. (14) Chien, R.-L.; Burgi, D. S. J. Chromatogr. 1991, 559, 153-161. (15) Burgi, D. S. Anal. Chem. 1993, 65, 3726-3729.
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al. summarizes several methods for performing analyte concentration and separation in a single capillary.17 Transient isotachophoresis (TITP) is an alternate method of sample stacking which can be used with the sample loaded on the column and then stacked. Establishing ITP conditions at the beginning of a run provides a method for on-column preconcentration; separation then proceeds by zone electrophoresis, thus allowing optical detection to be used. Coupling ITP and CE instrumentation can be rather complex; however, several reports have described how this technique can be achieved using a single capillary.17-20 In the present study, indirect laser-induced fluorescence was employed as a sensitive detection method for the analysis of lanthanides by CE. Quinine sulfate was used as the background electrolyte with R-hydroxyisobutyric acid (HIBA) added for the separation of the lanthanide ions. Further improvements in the analytical detection were achieved with on-column stacking by transient isotachophoresis, which provided preconcentration and lower detection limits. THEORY Separation of metal ions has been demonstrated with numerous electrolyte systems and complexing ligands added to separate species with very similar mobilities. For the lanthanides (Ln), HIBA has been shown to be an effective chelate for providing well-resolved separations.21,22 However, even the Ln-HIBA complexes have similar mobilities which would make separation difficult.23-25 Separations are based on the differences in the degree of complexation at a given HIBA concentration which results in a change in the average ionic mobility. The effective mobility, µeff, is a function of the electroosmotic mobility, µeo, and the electrophoretic mobility µep, of the species:
µeff ) µeo + µep
µep ) χM3+µM3+ + χML2+µML2+ + χML2+µML2+ - χML4-µML4- (4)
where χi is the mole fraction and µi is the mobility of the species. Vogt and Conradi have shown that such mutual stepwise equilibria are independent of metal concentration and depend only on the free ligand concentration, which in turn is determined by pH.21
HL T H+ + L-
(5)
[L-] ) KaC/([H+] + Ka)
(6)
where C is the total concentration of the ligand and [L-] is the concentration of the free ligand. This assumption is valid when the ligand concentration is significantly greater than the metal concentration and the pH is greater than or equal to the pKa.22 Vogt and Conradi demonstrated that an average complexation number can be used for mutual equilibrium and stepwise complex formation. Timerbaev described a model that related species mobilities to the fundamental physical constants of radius of hydration and charge.22 In this work, we used a model based on the work of Vogt and Conradi to approximate the mobilities of complexed metal ions to establish proper isotachophoretic conditions.21 Isotachophoresis can be used for on-column stacking of the analyte when the proper mobility conditions are established. The mobilities of the leading and tailing electrolytes must bracket that of the sample. The concentration within a zone can be approximated by the Kohlrausch regulating function:
(1)
HIBA forms weak complexes with the lanthanides with up to four ligands coordinated to each central cation.
M3+ + nL- T MLn3-n
(2)
βn ) [MLn3-n]/[M3+][L-]n
(3)
where βn are the overall complexation stability constants and equal to products of the stepwise stability constants, K1, K2, ..., Kn. The (16) Everaerts, F. M.; Beckers, J. L.; Verheggen, T. P. E. M. Isotachophoresis: Theory, Instrumentation, and Applications; Elsevier Scientific: New York, 1976. (17) Mazereeuw, M.; Tjaden, U. R.; Reinhoud, N. J. J. Chromatogr. Sci. 1995, 33, 686-697. (18) Krivankova, L.; Gebauer, P.; Thormann, W.; Mosher, R. A.; Bocek, P. J. Chromatogr. 1993, 638, 119-135. (19) Reinhoud, N. J.; Tjaden, U. R.; van der Greef, J. J. Chromatogr., A 1993, 653, 303-312. (20) Foret, F.; Szoko, E.; Karger, B. L. Electrophoresis 1993, 14, 417-428. (21) Vogt, C.; Conradi, S. Anal. Chim. Acta 1994, 294, 145-153. (22) Timerbaev, A. R. J. Capillary Electrophor. 1995, 2, 165-174. (23) Hirokawa, T.; Aoki, N.; Kiso, Y. J. Chromatogr. 1984, 312, 11-29. (24) Hirokawa, T.; Matsuki, T.; Takemi, H.; Kiso, Y. J. Chromatogr. 1983, 280, 233-247. (25) Hirokawa, T.; Kiso, Y. J. Chromatogr. A 1994, 658, 343-354.
2476 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
average electrophoretic mobility for the metal ion species can be represented as the sum of the mobilities of the individual species.
µi(µL + µLC) Ci ) CL µL(µi + µLC)
(7)
where µ is the mobility of an ion (i), leading electrolyte (L), or leading counterion (LC). For a given experimental system, the mobility term will be constant and typically fall in the range of 0.5-0.9.17 This equation strictly applies to a three-component system and the concentrating effect can be significant for dilute samples, especially compared to zone electrophoretic separations where diffusional broadening tends to dilute the sample. For true ITP conditions, the analyte zones are concatenated but not separated, and changes in conductivity are detected in order to discriminate between the zones. Hirokawa and Kiso used ITP with proton-induced X-ray emission (PIXE) as a direct and element-specific method of detection for separation of the Ln’s.23-25 However, such detection systems are not readily available. Although, optical detection with ITP has been used for the determination of some molecules, direct detection of metal ions is difficult due to their low molar absorptivities. Transient isotachophoresis is a method of sample stacking which combines ITP and zone electrophoresis in a single capillary.17,19 One method of achieving TITP is to establish localized ITP conditions by first injecting a small plug of the leading electrolyte, which has a higher mobility than the analyte of
0.1 M NaOH, a 1-min flush with deionized water, and a 2-min flush with the background electrolyte. The pH of the background electrolyte solutions ranged between 2.8 and 3.3 as determined by pH test strips with 0.3 pH unit resolution (EM Science, Gibbstown, NJ). Quinine sulfate was added as quinine bisulfate with no additional sulfuric acid added to the buffer to adjust the pH. In the TITP experiments, H2SO4 (10 mN) was used as the leading electrolyte with 80 mM tris(hydroxymethyl) aminomethane (TRIS) as the tailing electrolyte.
Figure 1. Schematic diagram of the transient isotachophoresis process: T, trailing electrolyte; L, leading electrolyte; A, B, analytes; BGE, background electrolytes. (A) Initial injection conditions; (B) localized ITP stacking; (C) transition between ITP and zone electrophoresis; (D) zone electrophoresis separation predominates.
interest, in front of the sample. A tailing electrolyte, which has a slower mobility than the analytes of interest, is injected behind the sample (see (A) in Figure 1).18 Initially, the analyte ions, A and B, will stack as in ITP (B) according to the Kohlrausch function. As the leading electrolyte migrates into the buffer, the mobility requirements necessary for ITP are no longer present (C). Eventually, separation of the analyte zones occurs in an electrophoretic manner, and the zones can be detected optically (D). EXPERIMENTAL SECTION Instrumentation. A Hewlett-Packard capillary electrophoresis system (HP3DCE, Waldbronn, Germany) was used for all separations, data acquisition, and data analysis. The excitation source was an argon ion laser (Spectra-Physics model 171, Mountain View, CA) equipped with an all-lines UV mirror, which provided three primary laser lines at 333.6, 351.1, and 363.8 nm. The laser beam was focused in the center of the capillary to a 50-µm waist with a power density of ∼4100 W/cm2 (∼80 mW per 2 × 10-5 cm2 area inside the capillary). Scattering losses inside the instrument were not taken into account. An optical alignment interface centered the capillary in front of a 620 µm tall × 50 µm wide slit, see Figure 2. The fluorescence emission was collected at a 90° angle from the incident excitation beam with a focusing grating and dispersed onto a diode array detector with no additional filters. Quinine sulfate emission was measured at 442 nm with a 16-nm bandwidth. Software and hardware modifications to the commercial instrument were incorporated as necessary to acquire the fluorescence intensity from the diode array detector. The capillary columns were uncoated, fused silica, 50 cm long, 41.6 cm to the detector, and 50 µm inner diameter (Polymicro Technologies Inc., Phoenix, AZ). An optical window was created by removing a section of the polyimide cladding with a flame, followed by cleaning the area with methanol. Separations were performed using a constant voltage of 25 kV. Samples were injected either electrokinetically or hydrodynamically. Reagents. Standards were prepared from 1000 ppm atomic absorption stock standard solutions in 1% nitric acid, except Ce3+ which was in 5% nitric acid (Aldrich Chemical Co., Milwaukee, WI). Capillaries were initially flushed with 1.0 M NaOH for 4 min. Prerun capillary conditioning consisted of a 1-min flush with
RESULTS AND DISCUSSION Quinine sulfate has commonly been used as a fluorescent standard due to its high quantum efficiency of 0.55, which is relatively independent of excitation wavelength below 350 nm.26 Gross and Yeung8 demonstrated that quinine sulfate can be used as a background electrolyte (BGE) for the indirect fluorescence detection of groups IA and IIA metal ions. Separation of the lanthanides was not possible with quinine sulfate as the BGE without the addition of a complexing ligand. HIBA, when added to the quinine sulfate background electrolyte, enabled the separation of the lanthanides, as shown in Figure 3. These data show the effect on CE separation for increasing amounts of HIBA (from 4 to 12 mM) with a constant 0.3 mM quinine sulfate. The fluorescence intensities were slightly offset for clarity (4 mM HIBA by + 1.0 and 8 mM HIBA by +0.5 fluorescence intensity units); however, there was a trend to lower background fluorescence and reduced baseline noise for increasing HIBA concentration. Increasing amounts of HIBA significantly shifted the migration time of all the peaks; however, relative to the system peak, the analyte migration times exhibited much smaller shifts. The shift in migration time may be attributed to changes in the pH, which is consistent with the observations of Lukacs and Jorgenson, who noted a trend toward slower migration with decreasing pH in fused-silica capillaries.27 Peak separation and resolution improves with increasing HIBA, as would be expected due to shifting the equilibria in favor of the metal complexes. However, as the HIBA concentration was increased the pH also decreased, which will reduce the amount of the free ligand available for complexation. During these studies it was necessary to operate at low pH values (∼3.0) in order to maintain the quinine in the more fluorescent dication form.8 This pH range is less than optimum for HIBA since it is less than the pKa and results in significant changes in the amount of free ligand with small changes in pH. Measurements of pH in this study were approximate and therefore can only be useful to show analytical trends and not as exact values for calculations. Figure 4 shows the electropherograms that resulted from varying the quinine sulfate concentration with constant 8 mM HIBA. Again, the fluorescence intensities have been slightly offset for clarity (0.4 mM quinine by +0.25 fluorescence intensity units and 0.5 mM quinine by +0.5 fluorescence intensity units), although the trend to increased background fluorescence intensity with increasing quinine sulfate was evident. The leading system peak did not shift significantly, indicating it was primarily attributed to HIBA, the major component of the BGE. Increased quinine sulfate concentrations shiftedthe analyte peaks relative (26) Chen, R. F. Anal. Biochem. 1967, 19, 374-387. (27) Lukacs, K. D.; Jorgenson, J. W. J. High Resolut. Chromatogr. 1985, 8, 407411.
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Figure 2. Schematic of the optical system used for indirect, laser-induced fluorescence detection. The alignment interface (inset) maintains the capillary in the proper position for the slit and the diode array detector.
Figure 3. Effect of complexing agent on the separation of lanthanides with 0.3 mM quinine sulfate and 4 (offset +1.0), 8 (offset +0.5), and 12 (no offset) mM HIBA. Approximate pH values were 3.2, 3.0, and 2.7, respectively. Hydrodynamic injections of 20 ppm each of (1) La3+, (2) Ce3+, (3) Pr3+, (4) Nd3+, (5) Sm3+, and (6) Eu3+.
to the system peak although the pH did not change significantly. Although sulfate forms mono- and dicoordinate complexes with the lanthanides, the stability constants are significantly lower than those for HIBA and would not be expected to have a significant influence (see Table 1).23,28 However, since the pH was greater than the pKa, appreciable sulfate in the BGE was present as free sulfate ions and potentially available for complexation with the Ln species. Mobilities of the sulfate complexes are not available in the literature. The migration times of each Ln peak relative to the system peak are plotted in Figures 5 and 6. The migration times for the data in Figure 3 are plotted in Figure 5 and do not exhibit significant changes relative to the system peak with increasing HIBA concentration. Assuming the migration time of the system peak was directly proportional to the electroosmotic flow, a free ligand model was used to approximate the average electrophoretic (28) de Carvalho, R. G.; Choppin, G. R. J. Inorg. Nucl. Chem. 1967, 29, 725735.
2478 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
Figure 4. Effect of quinine sulfate concentration on the separation of lanthanides with 8 mM HIBA and 0.3 (no offset), 0.4 (offset +0.25), and 0.5 (offset +0.5) mM quinine. Approximate pH values were 3.0, 3.1, and 3.2, respectively. Hydrodynamic injections of 20 ppm each of (1) La3+, (2) Ce3+, (3) Pr3+, (4) Nd3+, (5) Sm3+, and (6) Eu3+.
mobility. The effective mobility is inversely proportional to the migration time:
µ ) Ll/Vt
(8)
where L is the total length of the column, l is the length to the detector, V is the applied voltage, and t is the migration time. Theoretical results based on literature values of stability constants and mobilities are presented as reciprocal of mobility represented by the ×’s (right-hand y-axis values) in Figure 5.9,24,25 For the input parameters of 8 mM HIBA and pH 3.5, the trends in the calculated values compare favorably with the experimental data of 8 mM HIBA and a pH of 3.0. Similarly, Figure 6 shows the migration times relative to the system peaks corresponding to the separations for the data in Figure 4. The separations followed the same trend but were offset with increased amounts of quinine sulfate. Essentially, all the sulfate added will exist as the free dianion and will be available for complexation whereas only a small
Figure 5. Mobility of Ln ions, relative to the system peak in 4 mM (2), 8 mM (9), and 12 mM HIBA ([) with constant 0.3 mM quinine sulfate. Theoretical mobilities, (×, right y-axis) are for 8 mM HIBA at pH 3.5.
Figure 6. Mobility of Ln ions, relative to the system peak in 0.3, 0.4, and 0.5 mM quinine with constant 8 mM HIBA. Theoretical mobilities (×, right y-axis) are for 8 mM HIBA at pH 2.8. Table 2. Detection Limits
Table 1. Stability Constants Ln-Sulfate Complexes28 log β2 log β1
log β1 La Ce Pr
1.29 1.24 1.27
1.88
log β1 La Ce Pr Nd Sm Eu
Nd Sm Eu
2.99 3.09 3.24 3.29 3.42 3.51
1.26 1.30 1.37
Ln-HIBA Complexes23 log β2 log β3 5.15 5.44 5.56 5.66 6.0 6.19
6.2 6.36 6.79 7.08 7.64 8.11
log β2 1.79 1.91 1.96
log β4
8.83 8.56
(9)
The transfer ratio is the number of background molecules displaced for each analyte molecule, while the dynamic reserve is the background signal divided by the noise. Assuming a transfer ratio of 1, the concentration limit of detection was estimated to be 8.6 µM. The data obtained with the present instrumental setup are listed in the second and third columns of Table 2. These measurements indicate that we are close to the theoretical detection limits. Thus, improvements in analytical limits of detection would need to be accomplished by sample preconcentration prior to detection. Electromigration dispersion and diffusion contribute to the zone broadening and restrict (29) Yeung, E. S.; Kuhr, W. G. Anal. Chem. 1991, 63, 275A-282A.
injection size LODb (fmol)
TITP preconcn factorc
TITP LODd (nM)
La3+ Ce3+ Pr3+ Nd3+ Sm3+ Eu3+
2.8 (390) 4.4 (620) 4.8 (680) 5.6 (810) 6.3 (950) 10.1 (1540)
3.4 5.4 5.8 6.9 7.8 12.5
432 710 737 744 762 939
6.5 (0.90) 6.2 (0.87) 6.5 (0.92) 7.5 (1.08) 8.3 (1.25) 10.8 (1.64)
a Constant injection volume of 1.2 nL with changing concentration (7.2-144 µM). Limits of detection (LOD) determined by 3 × (std dev of background)/(slope of calibration curve). Values in parentheses in ppb. b Constant injection concentration of 20 ppm with changing injection volume (0.3-1.2 nL). LOD determined by 3 × (std dev of background)/(slope of calibration curve). c Maximum preconcentration factor for each ion, determined by comparison to a hydrodynamic injection of a 10 ppb analyte solution. d Calculated by dividing the concentration LOD (column 2) by the preconcentration factor (column 4). Values in parentheses in ppb.
fraction of the HIBA will be present as the free ligand. This results in a linear relationship for the change in the migration with a change in sulfate concentration. Because numerous Ln complexes would form with both the sulfate and HIBA, generation of a complete model would be difficult even with all the necessary constants. Appropriate tailing electrolytes can be chosen based on their mobilities. Detection limits for indirect detection can be predicted as described by Yeung and Kuhr using the transfer ratio (TR) and the dynamic reserve, (DR).29
CLOD ) CBGE/(TR)(DR)
ion
concn LODa (µΜ)
achievable limits of detection with conventional hydrodynamic injections. In particular, electromigration dispersion was greatest for the heaviest lanthanides in the experimental mixture, Sm3+ and Eu3+. To minimize such broadening, on-column stacking of the sample by transient isotachophoresis was investigated. Initial ITP conditions were produced on-column by the following procedure. The capillary column was filled with the BGE (0.4 mM quinine sulfate and 8 mM HIBA) and a plug of the leading electrolyte, 10 mN H2SO4, was hydrodynamically injected. Sulfuric acid was chosen as the leading electrolyte over other strong acids which tend to quench the fluorescence from quinine. The sample was then injected either hydrodynamically or electrokinetically. The mobility ratio for uncomplexed Ln cations is approximately unity, and bias for electrokinetic injections would therefore be minimal. If analyte mobilities differ significantly, however, hydrodynamic injections should be used to minimize these biases. The final step in establishing the TITP separation was injection of the tailing electrolyte, 80 mM TRIS. Because the three solutions were injected in order of their mobility, their zones moved at the same speed and thus did not separate from each other. This behavior reduced diffusion while at the same time, the analytes begin to stack up in order of their mobility. Figure 7 shows the data from a transient isotachophoretic separation of six lanthanides present in the sample at 10 ppb each which were Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
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Figure 7. Electropherogram from indirect laser-induced fluorescence detection of a mixture containing 10 ppb of each ion. Electrokinetic injection at 30 kV for 10.0 s, leading electrolyte 10 mN H2SO4, tailing electrolyte 80 mM TRIS, and a running buffer consisting of 0.4 mM quinine sulfate and 8.0 mM HIBA.
injected electrokinetically. The large, leading system peak was similar to that observed in Figures 3 and 4. The leading electrolyte, H2SO4, increased the magnitude of this system peak, indicating some pH dependence on the magnitude of this system peak. A large peak due to the TRIS tailing electrolyte was also observed. The empirical concentration factors were calculated by comparing peak areas from conventional CE separations with those obtained under TITP conditions. The largest concentration effects were observed for Sm3+ and Eu3+. The tailing electrolyte appears to have limited the back diffusion of these species and stacked them into sharper more well-defined peaks.
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CONCLUSION We have demonstrated that the analytical detection limits for CE separations are greatly improved by combining sample concentration techniques with sensitive detection methods. The analytical detection limits for several of the lanthanides were determined to be in low-ppb range and required less than 1 µL of sample. These values compare favorably to those achieved by other techniques such as ICP emission spectroscopy where continuous flow rates of ∼1 mL/min are required. Future improvements in the present method can be made by using fluorescent dyes with higher quantum efficiencies in the BGE. This approach should result in improvements in the dynamic reserve while simultaneously allowing for a lower concentration of the BGE to be used. It would also be beneficial to find a fluorophor that was more compatible with the higher pH values (4.0-4.5) that facilitate the Ln-HIBA complexation. ACKNOWLEDGMENT The authors thank Fritz Bek and Klaus Witt of Hewlett-Packard Co. for their support with computer hardware and software, and Susanne Conradi for helpful discussions. This work was funded by the U.S. Department of Energy’s Nonproliferation and National Security Program, Office of Research and Development (NN-20) and was performed under the auspices of the Department of Energy by Lawrence Livermore National Laboratory under Contract W-7405-ENG-48.
Received for review September 22, 1997. January 23, 1998. AC971043+
Accepted
