Analysis of Metal Ions by Sweeping via Dynamic Complexation and

Sweeping is defined as the picking and accumulating of analytes by a carrier ... The cation-selective exhaustive injection and sweeping capillary elec...
0 downloads 0 Views 319KB Size
Download PDF
Anal. Chem. 2003, 75, 6789-6798

Analysis of Metal Ions by Sweeping via Dynamic Complexation and Cation-Selective Exhaustive Injection in Capillary Electrophoresis Kentaro Isoo* and Shigeru Terabe

Graduate School of Science, Himeji Institute of Technology, Kamigori, Hyogo 678-1297, Japan

To improve the detection sensitivity of metal ions in capillary zone electrophoresis (CZE), a novel method that combines complex formation and on-line sample preconcentration by sweeping was developed. Sweeping is defined as the picking and accumulating of analytes by a carrier in the background solution, with which they have considerable affinity. In this sweeping method, using ethylenediaminetetraacetic acid as carrier, dynamic complexation to form a UV-absorbing chelate and on-line preconcentration occur simultaneously during a run. The technique was validated in terms of the limit of detection, reproducibility, and sensitivity enhancement. Detection responses of some divalent metal ions, in terms of peak heights, were improved from 60- to 180-fold, relative to conventional CZE which employed precapillary complexation. The limits of detection were in the range of (1.823.4) × 10-8 M. This method was applied to the analysis of trace metal ions in factory wastewater. Furthermore, sweeping in conjunction with sample stacking accompanying electrokinetic injection, cation-selective exhaustive injection (CSEI-sweeping), was also examined. Up to 140 000-fold improvement in detector responses for some divalent and trivalent metal ions was realized by CSEI-sweeping. The limits of detection were in the range (2.4-25.2) × 10-11 M. In general, determination of trace metal ions is performed by spectroscopic methods such as atomic absorption spectrometry (AAS), inductively coupled plasma atomic emission spectrometry (ICP-AES), or inductively coupled plasma mass spectrometry (ICPMS). Although these methods are useful and provide high detection sensitivity for many metals, they can supply information only on the total elemental contents of trace metals present in samples, and they need a large volume of sample solution, except for graphite furnace AAS. In chromatographic methods, ion chromatography (IC) is a popular analytical technique for ionic compounds,1 but it does not satisfy many requirements for sensitivity and resolution. Capillary electrophoresis (CE) has been utilized for the separation of organic and inorganic analytes because of its high efficiency and the diversity of separation modes available. Fur* Corresponding author. Phone: +81-791-58-0173. Fax: +81-791-58-0493. E-mail: [email protected]. (1) Sarzanini, C. J. Chromatogr., A 1999, 850, 213-228. 10.1021/ac034677r CCC: $25.00 Published on Web 11/15/2003

© 2003 American Chemical Society

thermore, it provides short analysis times and requires only small sample volumes for analysis. Therefore, it represents a promising technique for the analysis of metal species, as demonstrated by a number of groups.2-7 Nevertheless, CE has poor concentration sensitivity for on-capillary UV detection due to low sample injection volume, short optical path-length, and weak UV absorption of most metal ions. Although indirect UV detection has been employed,7,8 this method still suffers from low sensitivity. There are several techniques to enhance the detection sensitivity for CE analysis of metal ions. One is an off-line preconcentration method based on solid- or liquid-phase extraction prior to CE analysis.9,10 Recently, alternative detector formats with high sensitivity, such as electrochemical,11,12 MS,13-16 fluorescence,17,18 and chemiluminescence19,20 detectors, have been developed. However, these detectors are expensive, and the procedures are often tedious. Another method is chemical derivatization, either off-line21-37 or on-line.38-41 The first is based on precapillary complexation with an organic ligand. An excess amount of a strong ligand is added (2) Liu, B. F.; Liu, L. B.; Cheng, J. K. J. Chromatogr., A 1999, 834, 277-308. (3) Paca´kova´, V.; Coufal, P.; Tulı´k, K. J. Chromatogr., A 1999, 834, 257-275. (4) Chiari, M. J. Chromatogr., A 1998, 805, 1-15. (5) Timerbaev, A. R. Talanta 2000, 52, 573-606. (6) Timerbaev, A. R.; Shpigun, O. A. Electrophoresis 2000, 21, 4179-4191. (7) Vogt, C.; Klunder, G. L. Fresenius J. Anal. Chem. 2001, 370, 316-331. (8) Foret, F.; Fanali, S.; Nardi, A.; Boc¸ ek, P. Electrophoresis 1990, 11, 780783. (9) Rudnev, A.; Spivakov, B.; Timerbaev, A. Chromatographia 2000 52, 99102. (10) Kuban, P.; Buchberger, W.; Haddad, P. R. J. Chromatogr., A 1997, 770, 329-336. (11) Kappes, T.; Hauser, P. C. J. Chromatogr., A 1999, 834, 89-101. (12) Polesello, S.; Valsecchi, S. M. J. Chromatogr., A 1999, 834, 103-116. (13) Barnes, R. M. Fresenius J. Anal. Chem. 1998, 361, 246-251. (14) Deng, B. Y.; Chan, W. T. Electrophoresis 2001, 22, 2186-2191. (15) Li, J.; Umemura, T.; Odake, T.; Tsunoda, K. Anal. Chem. 2001, 73, 59925999. (16) Casiot, C.; Donard, O. F. X.; Gautier, M. P. Spectrochim. Acta, B 2002, 57, 173-187. (17) Zhu, R.; Kok, W. T. Anal. Chim. Acta 1998, 371, 269-277. (18) Church, M. N.; Spear, J. D.; Russo, R. E.; Klunder, G. L.; Grant, P. M.; Andresen, B. D. Anal. Chem. 1998, 70, 2475-2480. (19) Zhang, Y.; Gong, Z.; Zhang, H.; Cheng, J. Anal. Commun. 1998, 35, 293296. (20) Zhang, Y.; Cheng, J. J. Chromatogr., A 1998, 813, 361-368. (21) Jung, G. Y.; Kim, Y. S.; Lim, H. B. Anal. Sci. 1997, 13, 463-467. (22) Owens, G.; Ferguson, V. K.; Mclaughlin, M. J.; Singleton, I.; Reid, R. J.; Smith, F. A. Environ. Sci. Technol. 2000, 34, 885-891. (23) Baraj, B.; Martı´nez, M.; Sastre, A.; Aguliar, M. J. Chromatogr., A 1995, 695, 103-111. (24) Jen, J.-F.; Wu, M.-H.; Yang, T. C. Anal. Chim. Acta. 1997, 339, 251-257.

Analytical Chemistry, Vol. 75, No. 24, December 15, 2003 6789

to the sample to form UV-absorbing complexes, which are subsequently separated by capillary zone electrophoresis42 (CZE) or micellar electrokinetic chromatography43 (MEKC). In incapillary complexation, a ligand is added to the running electrolyte, and the complexation occurs during the electrophoretic migration. Detection limits in the sub-ppb levels were obtained, and the resolutions of some metal ions having similar electrophoretic mobilities were improved. Recently, there has been considerable interest in developing on-line preconcentration techniques in CE. They offer several advantages over off-line preconcentration methods, such as simplicity, high enrichment factors, automation, and reproducibility. Furthermore, separation and preconcentration can be performed in a single capillary without modification of existing instruments. Most of these techniques are based on velocitydifference-induced focusing of large sample volumes, with stacking44-46 being the most popular format. Liu and Lee reported sub-ppb sensitivities for some metal ions with off-line complexation and stacking.47-49 Hirokawa and co-workers have improved detection sensitivity to sub-ppb levels for a number of lanthanide ions using transient isotachophoresis.50 Sweeping, which was introduced in MEKC by Quirino and Terabe,51,52 represents a promising technique for on-line preconcentration of analytes. Sweeping is defined as a phenomenon whereby analytes are picked up and accumulated by the pseudostationary phase (micelles) that penetrates the sample zone. The sample solution is prepared to have an electric conductivity similar to that of the micellarcontaining background solution (micellar-BGS),53 but devoid of the micelle. The sample solution is hydrodynamically injected into

the capillary to obtain plug lengths much longer than those obtained with conventional injections. Sweeping-MEKC has been performed using ionic or neutral surfactant micelles (e.g., anionic sodium dodecyl sulfate (SDS),51,52 cationic tetradecyltrimethylammonium bromide (TTAB) or cetyltrimethylammonium chloride (CTAC),54,55 neutral Brij 35 or Briji 5856) as pseudostationary phases. Recently, sweeping of some monosaccharides by borate complexation has been demonstrated.57 Up to 5000-fold enhancement in detector response has been reported for some organic analytes. Furthermore, a combination of sample stacking with electrokinetic injection (field-enhanced sample injection, FESI)44 and sweeping, referred to as cation-selective exhaustive injection and sweeping (CSEI-sweeping), has achieved almost a millionfold enhancement in detector response for some cationic hydrophobic analytes.58 Thus far, sweeping and CSEI-sweeping have been applied to organic compounds only. In this report, to develop a highly sensitive analytical method for metal ions, the sweeping principle was applied for the first time as an on-line preconcentration technique for metal ions in CZE. Ethylenediaminetetraacetic acid (EDTA) was used as the on-line complexing reagent and carrier for sweeping, conferring two effects: UV visualization and concentration. Furthermore, CSEI-sweeping via dynamic complexation with EDTA was used to analyze ultratrace metal ions in the ppt levels. To stabilize the amount of injected samples by FESI, a neutral capillary modified with poly(ethylene oxide) coating was employed. Some divalent and trivalent metal ions were used as test analytes for evaluation of the techniques.

(25) Motomizu, S.; Oshima, M.; Matsuda, S.; Obata, Y.; Tanaka, H. Anal. Sci. 1992, 8, 619-625. (26) Takayanagi, T.; Motomizu, S. Anal. Sci. 2002, 18, 1021-1025. (27) Iki, N.; Hoshino, H.; Yotsuyanagi, T. Chem. Lett. 1993, 701-704. (28) Timerbaev, A. R.; Semenova, O. P.; Fritz, J. S. J. Chromatogr., A 1996, 756, 300-306. (29) Timerbaev, A. R.; Semenova, O. P.; Bonn, G. K. Analyst 1994, 119, 27952799. (30) Fung, Y.-S.; Tung, H.-S. Electrophoresis 1999, 20, 1832-1841. (31) Fung, Y.-S.; Tung, H.-S. Electrophoresis 2001, 22, 2192-2200. (32) Chen, Z. L.; Naidu, R.; Subramanian, A. J. Chromatogr., A 2001, 927, 219227. (33) Liu, B. F.; Liu, L. B.; Cheng, J. K. J. Chromatogr., A 1999, 848, 473-484. (34) Timerbaev, A. R.; Semenova, O. P.; Buchberger, W.; Bonn, G. K. Fresenius J. Anal. Chem. 1996, 354, 414-419. (35) Saitoh, T.; Hoshino, H.; Yotsuyanagi, T. J. Chromatogr. 1989, 469, 175181. (36) Saitoh, T.; Hoshino, H.; Yotsuyanagi, T. Anal. Sci. 1991, 7, 495-497. (37) Haddad, P. R.; Macka, M.; Hilder, E. F.; Bogan, D. P. J. Chromatogr., A 1997, 780, 329-341. (38) Haumann, I.; Ba¨chmann, K. J. Chromatogr., A 1995, 717, 385-391. (39) Chen, Z. L.; Naidu, R. J. Chromatogr., A 2002, 966, 245-251. (40) Naujalis, E.; Padarauskas, A. J. Chromatogr., A 2002, 977, 135-142. (41) Regan, F. B.; Meaney, M. P.; Lunte, S. M. J. Chromatogr., B 1994, 657, 409-417. (42) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302. (43) Terabe, S.; Otuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 111-113. (44) Chien R. L.; Burgi, D. S. Anal. Chem. 1992, 64, 489A-96A. (45) Quirino, J. P.; Terabe, S. J. Chromatogr., A 2000, 902, 119-135. (46) Britz-Mckibbin, P.; Bebault, G. M.; Chen, D. D. Y. Anal. Chem. 2000, 72, 1729-1735. (47) Liu, W. P.; Lee, H. K. Anal. Chem. 1998, 70, 2666-2675. (48) Liu, W. P.; Lee, H. K. J. Chromatogr., A 1998, 796, 385-395. (49) Liu, W. P.; Lee, H. K. Electrophoresis 1999, 20, 2475-2483. (50) Hirokawa, T.; Okamoto, H.; Ikuta, N. Electrophoresis 2001, 22, 3483-3489. (51) Quirino, J. P.; Terabe, S. Science 1998, 282, 465-468. (52) Quirino, J. P.; Terabe, S. Anal. Chem. 1999, 71, 1638-1644. (53) Quirino, J. P.; Terabe, S. J. High Resolut. Chromatogr. 1999, 22, 367-372.

EXPERIMENTAL SECTION

6790

Analytical Chemistry, Vol. 75, No. 24, December 15, 2003

Apparatus. A Hewlett-Packard 3DCE System (Waldbronn, Germany) with a UV absorbance detector was used for all experiments. Electrophoresis and sweeping experiments were performed in fused silica capillaries of 50 µm i.d. and 375 µm o.d., obtained from Polymicro Technologies (Phoenix, AZ). A neutral capillary modified with poly(ethylene oxide) coating (µSiL-Wax Capillary) of 50 µm i.d. and 360 µm o.d., obtained from Agilent Technologies (Waldbronn, Germany), was used for CSEIsweeping. The capillary temperature was set at 25 °C. Detection wavelength was 200 nm. The conductivity of sample matrixes and separation solutions was measured with a Horiba ES-12 conductivity meter (Kyoto, Japan). The pH values of the solutions were measured and adjusted with the aid of a Beckman Φ 34 pH meter. For ICP-AES analysis, the ICP-AES system consisted of a Seiko Instruments model ICP-AES SPS-5000 (Chiba, Japan), equipped with a standard torch assembly. The radio frequency power was set at 1200 W, and the argon gas flow rates for the nebulizer, auxiliary, and plasma flow were controlled at 0.8, 1.0, and 16 L/min, respectively, whereas the sample flow was kept constant at 1.8 mL/min. Reagents and Solutions. The water used was purified with a Milli-Q system from Millipore (Bedford, MA). Ultrapure water (54) Kim, J. B.; Otsuka, K.; Terabe, S. J. Chromatogr. A 2001, 912, 343-352. (55) Kim, J. B.; Quirino, J. P.; Otsuka, K.; Terabe, S. J. Chromatogr. A 2001, 916, 123-130. (56) Monton, M. R. N.; Quirino, J. P.; Otsuka, K.; Terabe, S. J. Chromatogr. A 2001, 939, 99-108. (57) Quirino, J. P.; Terabe, S. Chromatographia 2001, 53, 285-289. (58) Quirino, J. P.; Terabe, S. Anal. Chem. 2000, 72, 1023-1030.

Figure 1. Sweeping of metal ions via complexation with EDTA under weakly acidic condition. (A) Large-volume injection of S prepared in a matrix having a conductivity similar to those of EDTA-BGS but devoid of the EDTA. (B) Application of voltage at negative polarity; EDTA emanating from the cathodic vial sweeps metal ions. (C) Injected metal ions are completely swept.

used for CSEI-sweeping experiments, TTAB, and AAS-grade metal nitrate and chloride standard solutions (1000 mg/L) were purchased from Wako (Osaka, Japan). EDTA was purchased from Dojindo (Kumamoto, Japan). Sodium acetate was purchased from Merck (Darmstadt, Germany). All reagents were used without further purification. Background solutions (BGSs) were prepared from stock solutions of sodium acetate and EDTA, and their pH values were adjusted to the required value with 0.01 M hydrochloric acid. Buffer solutions were filtered through a 0.45-µm filter (Nacalai Tesque) and degassed before use. Sufficient EDTA was added to stock solutions of metal ions to give 2 M excess in the final sample to prepare metal complexes prior to conventional CZE analysis. To prepare the sample solution for sweeping-CZE analysis, the metal stock solutions were diluted with buffers devoid of EDTA, but the conductivities of which were previously adjusted to approximate that of the EDTA-containing BGS (EDTA-BGS). Factory wastewater was pretreated simply by passing it through a 0.45-µm filter and adding sodium chloride to adjust its electric conductivity to that of the BGS. Sample solutions for CSEI-sweeping experiments were prepared by dilution of the stock solutions of metal ions in ultrapure water. General Electrophoresis Procedures. The new capillary was conditioned prior to use by rinsing with 1 M NaOH (20 min), followed by 0.1 M NaOH (5 min), methanol (5 min), purified water (10 min), and finally BGS (5 min). The sample solution (S) was then injected into the cathodic end of the capillary by pressure (50 mbar ) 5 kPa). Negative voltage was applied for sweeping and CZE. Between consecutive analyses, the capillary was rinsed

with 0.1 M NaOH (4 min), followed by methanol (3 min), purified water (5 min), and then BGS (5 min) to ensure reproducibility. The new neutral capillary for CSEI-sweeping was conditioned prior to use by rinsing with purified water (10 min), followed by 0.1 M phosphoric acid (15 min), methanol (5 min), purified water (10 min), and finally BGS without EDTA (non-EDTA-BGS) (5 min). To ensure reproducibility between consecutive analyses, the capillary was flushed at ∼1 bar with 0.1 M phosphoric acid (5 min), methanol (2 min), purified water (3min), and finally the nonEDTA-BGS (4 min). Other conditions are mentioned in relevant figure captions or text. To approximate the injected plug length of the sample solution, the velocity of the liquid was determined by measuring the migration time (seconds) of 500 ppm metal ions which was brought to the detector at 50 mbar pressure. From the velocities that were obtained, the lengths of the zones were computed, given the injection time. RESULTS AND DISCUSSION (a) Sweeping via Dynamic Complexation with EDTA. Concentration Mechanism of Sweeping via Dynamic Complexation with EDTA. The sweeping-CZE via complexation with EDTA of a positively charged metal ion, M, is illustrated in Figure 1. EDTA forms a strong 1:1 complex with many metal ions, regardless of their charge. To avoid precipitation and to promote complexation reaction, weakly acidic conditions were chosen for BGS in this work. Under these conditions, the silanol groups of capillary surface are mostly protonated and cause a weak, cathodedirected electroosmotic flow (EOF). The sample solution of metal Analytical Chemistry, Vol. 75, No. 24, December 15, 2003

6791

ions (S) was prepared in a matrix that has an electric conductivity similar to that of BGS but devoid of EDTA. The capillary is filled with EDTA-BGS. The S is then hydrodynamically injected into the capillary to obtain a plug length much longer than the usual injection (Figure 1A). EDTA-BGS is found at both ends of the capillary. The metal ions found near the interface between S and the EDTA-BGS zone on the cathodic and anodic sides are represented by Mc and Ma, respectively. The vertical dotted line through Figure 1A-C indicates the starting position of Ma. Note that EDTA and the anionic metal-EDTA complex (M-EDTA) migrate toward the anode because the effective electrophoretic velocities of EDTA, vep(EDTA), and M-EDTA, vep(M-EDTA), are higher than that of EOF (veof). When voltage is applied under negative polarity (Figure 1B), EDTA in the cathodic vial will enter the capillary and react (sweep) with metal ions in the sample zone. The M-EDTA is accumulated as a concentrated zone (dark zone). The metal ions at Ma will not react with EDTA until such time when EDTA reaches them from the cathodic end (Figure 1C). Finally, completely swept analytes are separated by conventional CZE. The formation constant (K) between M and EDTA can be expressed by eqs 1 and 2,

M + EDTA a M-EDTA K)

[M-EDTA] [M][EDTA]

(1) (2)

Determination of the migration behavior of metal complexes has been discussed in several papers.59-62 The effective electrophoretic velocity of Mc, vep(Mc), in the presence of EDTA is given by eq 3, where vep(M) is effective electrophoretic velocity of M. Since K

vep(Mc) )

1 v (M) + 1 + K[EDTA] ep K[EDTA] v (M-EDTA) (3) 1 + K[EDTA] ep

is very large, 1/(1 + K[EDTA]) and K[EDTA]/(1 + K[EDTA]) are nearly equal to 0 and 1, respectively, and we can assume vep(Mc) is equal to vep(M-EDTA):

vep(Mc) ≈ vep(M-EDTA)

(3′)

When the magnitude of vep(EDTA) is larger than that of vep(M-EDTA), the length of the sample zone after sweeping with EDTA (lsweep(complex)) is given by eq 4,

lsweep(complex) ) d(EDTA) - d(M-EDTA)

(4)

where d(EDTA) (eq 5) and d(M-EDTA) (eq 6) are the distances traveled by the EDTA and M-EDTA, respectively (see Figure 1C), (59) Timerbaev, A. R.; Semenova, O. P.; Petrukhin, O. M. J. Chromatogr., A 2002, 943, 263-274. (60) Wang, T.; Li, S. F. Y. J. Chromatogr., A 1995, 707, 343-353. (61) Vogt, C.; Conradi, S. J. Chromatogr., A 1994, 294, 145-153. (62) Iki, N. Bunseki Kagaku 2002, 51, 495-505.

6792

Analytical Chemistry, Vol. 75, No. 24, December 15, 2003

d(EDTA) ) v(EDTA)tsweep; ν(EDTA) ) νep(EDTA) + νeof (5) d(M-EDTA) ) ν(M-EDTA)tsweep; ν(M-EDTA) ) νep(M-EDTA) + νeof (6) linj

tsweep )

(7)

v(EDTA) - v(M)

v(M) ) vep(M) + veof

(8)

where v(EDTA), v(M-EDTA), and v(M) are the migration velocities of EDTA, M-EDTA, and M, respectively, and tsweep is the time when EDTA reaches Ma. Algebraic manipulation of eqs 3-8 yields the equation for lsweep, defined in terms of injection plug length and electrophoretic velocities (eq 9):

lsweep(complex) ) linj

(

)

vep(EDTA) - vep(M-EDTA) vep(EDTA) - vep(M)

(9)

Note that the term d(EDTA) - d(M-EDTA) in eq 4 is replaced by d(M-EDTA) - d(EDTA) when the relative magnitude between vep(EDTA) and vep(M-EDTA) is reversed by changing the analytical conditions (pH values, etc.). From eq 9, the analytes which have closer vep(EDTA) and vep(M-EDTA) will result in narrower and more concentrated sample zones than the analytes which have larger difference between them. lsweep practically depends on the difference between the two velocities, vep(EDTA) and vep(M-EDTA). Figure 2 shows an electeropherogram obtained by sweepingCZE of Co(II) via dynamic complexation with EDTA. The EDTA vacancy length (see Figure 1C) shown in Figure 2 was calculated to be 20.9 cm using the effective migration velocity of EDTA under the given conditions. This is almost equal to the injection length of S, 20.4 cm (50 mbar for 300 s). This result indicates that Co(II) was completely swept by EDTA. A large volume of injected Co(II) was concentrated and detected at high sensitivity. The width of the swept zone, lsweep (see Figure 1C), was 3.5 mm. A more detailed explanation of lsweep will be given later. Optimization of Sweeping Conditions. Assuming that all metal ions are transformed to M-EDTA by EDTA, the migration order reflects differences in both the charge and the size of the anionic metal complexes. The effective electrophoretic mobilities of metal ions are dependent on the degree of complex formation; thus, their separation can also be influenced by the concentration of the complexing agent. Addition of a weak complexing reagent to the BGS is usually done to obtain larger differences in effective mobilities. Acetate, which has a weak complexing capability, was used as the electrolyte for the studies. The pH plays an important role in the formation of anionic complexes. Similarly, the conversion of metal ions to anionic complexes inside the capillary is highly dependent on the buffer pH and the concentration of EDTA. As EDTA is an ionizable compound (pKa1 ) 1.99, pKa2 ) 2.67, pKa3 ) 6.16, pKa4 ) 10.26), the fully deprotonated EDTA concentration ([Y4-]) in the BGS depends on the buffer pH. [Y4-] is favored with increasing pH; thus, the magnitude of interaction of metal complexation with

Figure 2. Concentrated metal ion after sweeping via complexation with EDTA. Conditions: EDTA-BGS, 1 mM EDTA in 30 mM sodium acetate (pH 5.5); sample solution, Co(II) in acetate buffer (pH 5.5) adjusted to the conductivity of the EDTA-BGS; injected length, 20.4 cm; concentration of Co(II), 100 ppb; applied voltage, -27 kV; capillary, 56.5 cm to the detector (65 cm total).

Figure 3. Effect of EDTA concentration on sweeping. Conditions: EDTA-BGS, 30 mM sodium acetate (pH 5.5) containing (A) 0.1, (B) 0.5, (C) 1, and (D) 3 mM EDTA; concentration of ions, 100 ppb; other conditions are the same as those in Figure 2.

EDTA is increased. However, increasing the pH influences not only metal hydrolysis but also the separation selectivity and EOF. When the EOF is increased, the migration time of the analyte is also increased, resulting in a broader peak and therefore reduced detection sensitivity. The effect of the pH of the BGS on the sensitivity and the separation selectivity in sweeping-CZE was examined in the pH range of 3.5-6.0. A 30 mM sodium acetate buffer containing 1 mM EDTA was used as the BGS, and 100 ppb (ca. 10-7 M) of Cu(II), Pb(II), Co(II), and Mn(II) were used as test analytes. At lower pH (e.g., 3.5), the peak areas and heights of all complexes were not reproducible because of the instability of EOF. The net migration velocities of all complexes toward the anode decreased with the increase in pH because the EOF increased. The peaks were significantly broadened at pH 6.0, and resolution was poor. The peak heights of the complexes were roughly constant in the pH 4.5-5.5 region. However, Cu(II) and Pb(II) complexes comigrated at pH 4.5. At pH 5.5, good sensitivity and resolution were obtained; hence, subsequent studies were carried out at this pH. The concentration of EDTA in BGS also influences the complex formation. The effect of the EDTA concentration on sweeping efficiency was investigated in the range 0.1-3 mM (Figure 3). At 0.1 mM, the separation of Cu(II), Pb(II), and Mn(II) complexes was incomplete because the peaks were broadened. When the EDTA concentration was increased to 0.5 mM, the peaks became sharp and well resolved. Results were further improved by increasing the concentration to 1 mM. Above this value, peak heights and peak areas did not increase significantly. These results indicate that 1 mM EDTA is enough to stabilize the metal complexes. Hence, subsequent studies were carried out at this concentration. It should be noted, however, that the difference in absorbance between EDTA-BGS and EDTA vacancy increased with increasing EDTA concentration because EDTA is UVabsorbing. The effect of sample matrix on sweeping efficiency was also investigated. The sample matrixes contained different concentrations of sodium acetate. The concentration of acetate in EDTA-

BGS and the injection length were kept at 30 mM and 20.4 cm, respectively. At high acetate concentration in the sample matrix, the conditional formation constants between metal ion and EDTA were decreased. In addition, a higher concentration of electrolyte is liable to cause excess joule heating. In the absence or at a very low concentration of acetate in the sample matrix, the resolution was poor. A good compromise between resolution and sweeping efficiency was found to be 30 mM acetate in the sample matrix, equal to the concentration of acetate in EDTA-BGS. The EOF direction is opposite to that of the electrophoretic migration of the metal complexes. At higher pH, the EOF will increase, resulting in longer migration times, broader peaks, poorer resolution, and lower sensitivity. To obtain optimum resolution within a reasonable time, the addition of a cationic surfactant (TTAB) to the BGS was examined. TTAB reverses the EOF, thus reducing migration times. Furthermore, it alters the surface charge on the capillary wall, and possibly prevents the interaction between metal ions and capillary wall that leads to increased dispersion. However, faster migration often results in poorer resolution. TTAB (0.4 mM) was found to give the best compromise between analysis time and resolution. The dependence of the peak heights of four metal complexes on injection time of S was examined. The peak heights of complexes increased when the injection time was increased from 50 to 400 s. Beyond 400 s, the peak heights of all complexes no longer increased, and peak broadening ensued. This was thought to be caused by sample overloading, or long lsweep. Conversely, peak areas still increased because they are directly related to the amounts of analytes that were injected. For 100 ppb concentration of metal ions, maximum sensitivity enhancement was obtained by injecting between 300 and 400 s. Sensitivity enhancement by sweeping was examined for different concentrations of Co(II) (Figure 4). Figure 4A shows an electropherogram of a usual injection (1.4 mm) of 25 ppm sample solution of Co(II)-EDTA complex, which was prepared by precapillary reaction. The sample solutions used for Figure 4B-E are dilutions of a 25 ppm sample. Despite there being no Analytical Chemistry, Vol. 75, No. 24, December 15, 2003

6793

Figure 4. Effect of Co(II) concentration on sweeping. Conditions: EDTA-BGS, 1 mM EDTA and 0.4 mM TTAB in 30 mM sodium acetate (pH 5.5); injection length, (A) 1.4 mm and (B-E) 25.3 cm; applied voltage, -30 kV; capillary, 61.5 cm to the detector (70 cm total).

Figure 5. Analysis of four metal ions by sweeping-CZE via complexation with EDTA. Conditions: EDTA-BGS, 1 mM EDTA and 0.4 mM TTAB in 30 mM sodium acetate (pH 5.5); injection length, (A) 1.4 mm and (B) 27.2 cm; concentration of ions, (A) 25 ppm and (B) 50 ppb; applied voltage, -30 kV; capillary, 61.5 cm to the detector (70 cm total).

significant difference in peak heights between parts B and C of Figure 4, the peak area of the former is 4.7-fold larger than that of the latter. When the concentration of Co(II) was too high (Figure 4B), the peak shape deteriorated as a result of sample overloading. In contrast, the peak in Figure 4C was sharp. This result indicates that EDTA concentration in BGS is sufficient to sweep most of the Co(II) contained in the 1 ppm sample solution. An even better peak shape was obtained by reducing the injection time of S (e.g., 50 s or 3.2 cm) with 1 ppm sample solution. When the Co(II) concentration was below 1 ppm (Figure 4D-E), sharp peaks were obtained even at long injection time (300 s or 20.4 cm). These results suggest that an optimum injection time should be chosen according to the sample concentration. Analytical Performance of Sweeping-CZE. Figure 5 shows the separation of four metal ions in trace amounts by sweepingCZE via complexation with EDTA. The electropherogram in Figure 5A was obtained with precapillary complexation and conventional CZE analysis of metal ions. Figure 5B was obtained 6794

Analytical Chemistry, Vol. 75, No. 24, December 15, 2003

by sweeping-CZE analysis of a 500-fold diluted sample solution (relative to Figure 5A). As may be inferred from the Figures, from 100- to 180-fold improvements, in terms of peak height, were obtained. Table 1 summarizes the calibration lines, limits of detection (LODs), averages of migration time, percentage relative standard deviations (%RSDs), theoretical plate numbers (plates), and sensitivity enhancement factors in terms of peak height (SEFheight) obtained for eight divalent metal ions with sweeping-CZE under the same conditions as given in Figure 5B. The linearity was tested in the concentration range 0.05-1.0 ppm. Correlation coefficients of the studied divalent metal ions were in the range of 0.9939-0.9997, while the LODs (S/N ) 3) were in the range of 2.6-25 ppb. SEFheight values were calculated as the ratio of the peak heights obtained from sweeping and conventional CZE of metal complexes and correction by dilution factor. From 60- to 180-fold improvements in detector response were obtained.

Table 1. Calibration Line, LODs, %RSDs, and SEFheight for the Test Metal Ions in Sweeping-CZE via Dynamic Complexationa

calibration lineb equation of the line correlation coefficient LODs (S/N ) 3) ppb ×10-8 M RSD (%, n ) 4) migration timec peak height corrected peak aread plates (×105) SEFheighte

Pd(II)

Cu(II)

Zn(II)

Pb(II)

Ni(II)

Co(II)

Cd(II)

Mn(II)

y ) 5.07x + 0.33 0.9981

y ) 7.21x + 0.20 0.9985

y ) 7.81x + 0.13 0.9969

y ) 28.55x 0.10 0.9939

y ) 40.53x 0.84 0.9995

y ) 31.55x 0.23 0.9956

y ) 4.29x 0.22 0.9977

y ) 12.54x + 0.15 0.9997

21.2 19.9

14.9 23.4

13.7 21.0

3.8 1.8

2.6 4.5

3.4 5.8

25.0 22.2

8.6 15.7

0.6 (9.13) 2.6 6.6

0.8 (9.38) 3.2 4.5

0.7 (9.45) 5.3 5.2

0.5 (9.53) 4.8 5.9

0.4 (9.64) 5.8 5.0

0.6 (9.66) 2.6 4.1

0.4 (10.23) 9.0 10.1

0.7 (10.24) 3.1 4.4

0.6 60

8.2 130

5.4 120

3.9 120

3.9 130

2.8 160

1.1 80

1.0 180

a Concentration of metal ions, 0.1 ppm. Other conditions as in Figure 5. b Calibration line: peak height (mAU) ) slope × concentration (ppm) + y-intercept. Calibration concentration range, 0.05-1 ppm. c The values in parentheses are migration times. d Corrected peak area ) peak area/ migration time. e SEFheight ) (hsweeping-CZE/hconventional CZE) × dilution factor, where hsweeping-CZE is the peak height obtained by sweeping via dynamic complexation with EDTA, and hconventional CZE is the peak height obtained by conventional CZE with precapillary complexation.

Table 2. Predicted and Experimentally Determined lsweep Values lsweep/cma analyte

predictedb

experimentalc

Cu(ii) Pb(II) Co(II) Mn(II)

0.10 0.30 0.29 0.66

0.25 0.32 0.35 0.78

a Based on a plug length of 20.4 cm. b Calculated using eq 9. Experimental lsweep refers to the peak width after migration to the detector. Conditions as described in Figure 2.

c

lsweep values obtained experimentally were compared with the values predicted by eq 9, as shown in Table 2. The experimentally obtained lsweep values are slightly larger than the predicted ones because of diffusion. Equation 9 predicts the lengths of zones at the point of completion of the sweeping step and does not take into account normal band broadening as the samples move to the detector. The latter is reflected in the experimentally obtained values; hence, they are understandably broader. The differences, however, are small enough for the two sets of values to be considered consistent. The same method was applied to the analysis of metal ions in a factory wastewater sample (Figure 6). The EDTA complexes with Cu(II), Zn(II), and Pb(II) were baseline separated, but the baseline is quite noisy due to unknown constituents in the sample matrix. Reproducibilities (%RSD, n ) 5) were less than 4.2 and 13.0% for migration time and peak height, respectively. A comparison of the concentrations of these metal ions, determined by sweeping-CZE and ICP-AES, is shown in Table 3. Comparable results were obtained by the two methods. These results clearly indicate the potential of sweeping for the analysis of trace metals contained in real matrixes after simple pretreatments. (b) Combination of Sweeping-CZE via Dynamic Complexation with EDTA and Field-Enhanced Injection. Concentration Mechanism of CSEI-Sweeping-CZE via Dynamic Complexation with EDTA. The main idea of this work is illustrated in Figure 7, which is similar to the CSEI-sweeping-

Figure 6. Electropherogram of factory wastewater by sweepingCZE via EDTA complexation; other conditions are the same as in Figure 5B and described in the text. Table 3. Comparison of Results for Metal Determinations in Factory Wastewater by Sweeping-CZE and ICP-AES metal cation

sweeping-CZEa (ppb)

ICP-AES (ppb)

Cu(II) Zn(II) Pb(II)

49 ( 5.5 60 ( 4.3 12 ( 1.1

44 67 10

a

Data are given as mean ( SD, n ) 5.

MEKC method reported by Quirino and Terabe.56 A poly(ethylene oxide)-coated capillary is initially filled with a solution similar to the BGS used in the sweeping-CZE experiments but that does not contain EDTA (non-EDTA-BGS). If EDTA is in the capillary during injection, EDTA will move toward the inlet end and interfere with the entry of metal ions into the capillary. Furthermore, sweeping will not occur in the resulting zone of electrokinetically injected metal ions. The capillary is initially filled with a non-EDTA-BGS (e.g., 40 mM acetate buffer), followed by short hydrodynamic injection of a water plug (3.2 mm) (Figure 7A). Analytical Chemistry, Vol. 75, No. 24, December 15, 2003

6795

Figure 7. Schematic diagrams of the CSEI-sweeping-CZE model. (A) The starting situation, conditioning of the poly(ethylene oxide)-coated capillary with a non-EDTA-BGS having high conductivity, and injection of a short water plug. (B) Electrokinetic injection for a long time at positive polarity (FESI) of metal ions prepared in a low-conductivity matrix or water, non-EDTA-BGS found in the outlet end, metal ions focused or stacked at the interface between the water zone and high-conductivity buffer void of EDTA. (C) EDTA-BGSs are placed at both ends of the capillary, followed by application of voltage at negative polarity that will permit entry of EDTAs from the cathodic vial into the capillary and sweep the stacked metal ions to narrower bands. (D) Separation of zones on the basis of CZE.

The S, prepared in a low-conductivity solution (or simply water), is then placed at the inlet end, and non-EDTA-BGS is placed at the outlet end. Thereafter, voltage is applied at positive polarity (negative at the outlet) in order to electrokinetically inject the metal ions into the capillary (Figure 7B) for a period much longer than usual (e.g., 150 s). This procedure creates long zones of metal ions, which have concentrations greater than that in the original. The metal ions migrate toward the cathode. After electrokinetic injection, the vials containing EDTA-BGS are placed at both ends of the capillary, and voltage is applied at negative polarity to focus the injected zone by sweeping (Figure 7C). Separation is then achieved via CZE of metal-EDTA complexes (Figure 7D). In Figure 7B, the metal ions enter the capillary through the water plug with high velocities. Once the metal ions cross the stacking boundary or interface between the water and non-EDTABGS zone having high conductivity, they will slow and focus at this interface. Note that the water plug provides a high electric field at the tip of the capillary in sample stacking by electrokinetic injection, which will eventually improve the sample stacking procedure. In Figure 7D, once voltage is applied at negative polarity with the EDTA-BGS in the inlet vial, EDTAs enter the capillary, migrate quickly across the low-conductivity water plug, and then stack at its interface with the non-EDTA-BGS. The stacked EDTAs then sweep the stacked metal ions. After sweeping is completed, the metal-EDTA complexes separate by CZE in the reverse migration mode. Optimization of CSEI-Sweeping. CSEI-sweeping, which combines sample stacking and sweeping, achieved almost a million-fold enhancement in detector response for some cationic 6796

Analytical Chemistry, Vol. 75, No. 24, December 15, 2003

hydrophobic organic analytes. Thus, CSEI-sweeping using dynamic complexation was considered to be a promising method for the analysis of metal ions. Three divalent ions, Pb(II), Co(II), and Mn(II), and a trivalent ion, Fe(III), were used as test analytes. The pH value of 4.8 was chosen for subsequent studies since adequate resolution for the four ions was obtained in preliminary experiments. In the FESI step, cationic analytes were carried into the capillary by electrophoresis with their positive charge and the EOF directed toward the cathode. Under acidic conditions, however, the amount of sample injection was not reproducible because of the unstable EOF. This is inconvenient for determination of samples. To obtain reproducible injections, a neutral capillary modified with poly(ethylene oxide) coating, in which the EOF was low, was used in subsequent studies. Figure 8 shows some electropherograms illustrating the effect of different non-EDTA-BGSs that were injected before the water plug in CSEI-sweeping-CZE. The non-EDTA-BGSs were sodium acetate buffers having conductivity values 1× (A), 1.8× (B), and 2.4× (C) compared to that of the EDTA-BGS. The FESI time was kept at 150 s. FESI efficiency is usually improved by increasing the difference in the field strengths between sample solution and BGS. However, as seen in the electropherograms, detection sensitivities decreased with increasing non-EDTA-BGS conductivity. This result indicates that sweeping efficiency is affected by the conditions around the analytes after the FESI step, as discussed in the section about the effect of sample matrix on sweeping. The effect of the FESI time on CSEI-sweeping-CZE for four metal ions in the range of 50-350 s was examined. Initially, SEFs

Figure 8. Effect of high-conductivity buffer on CSEI-sweeping-CZE. Conditions: non-EDTA-BGS, sodium acetate buffer (pH 4.8) having conductivity values of (A) 4.1, (B) 6.7, and (C) 8.8 mS/cm; EDTA-BGS, 1 mM EDTA in 40 mM sodium acetate (pH 4.8); sample solution, 1 ppb each of three metal ions in water; injection schemes, the capillary was filled with non-EDTA-BGS and then 3.4 mm of water, followed by 25 kV FESI of the sample solution for 150 s; sweeping and CZE voltage, -25 kV with the EDTA-BGS at the both ends of the capillary; capillary, 56.5 cm to the detector (60 cm total). Table 4. Calibration Line, LODs, %RSDs, and SEFheight for the Test Ions in CSEI-Sweeping-CZE via Dynamic Complexationa Pb(II)

Co(II)

Mn(II)

Fe(III)

lineb

calibration equation of the line correlation coefficient LODs (S/N ) 3) ppt ×10-11 M RSD (%, n ) 4) migration time peak height corrected peak areac

y ) 0.016x + y ) 0.018x + y ) 0.006x - y ) 0.005x 0.20 1.50 0.65 0.34 0.9919 0.9960 0.9956 0.9911 4.5 2.4

3.9 6.8

11.8 21.5

14.1 25.2

2.0 12.0 13.6

1.1 7.0 9.2

1.3 11.5 12.2

2.2 13.3 14.4

5.8 13.7

4.3 10.3

3.8 5.2

plates (×105) 9.2 SEFheightd (× 104) 8.7

a Concentration of metal ions, 0.5 ppb. Other conditions as described in Figure 9. b See Table 1. Calibration concentration range, 0.05-1 ppb. c See Table 1. d SEFheight ) (hCSEI-sweeping-CZE/hconventional CZE) × dilution factor, where hCSEI-sweeping-CZE is the peak height obtained by CSEI-sweeping-CZE via dynamic complexation with EDTA, and hconventional CZE is defined as in Table 1.

increased with increases in the FESI time, since the amount injected naturally increases with the increase in injection time. However, with very long injection times (e.g., 250 s), SEFheight ceased to increase, and the peaks became broad. This is explained by the fact that sweeping is limited by the sample zone length. Note that a longer FESI time results in longer sample zone lengths. In contrast, peak areas increased linearly with injection time up to 300 s, but beyond 300 s, no significant increase was noted. This is because of sample depletion during the very long FESI time (e.g., 350 s). Therefore, to maximize the effect of sweeping, the FESI time should be optimized. An important precaution in performing CSEI-sweeping-CZE is that fresh samples should always be used for each injection. Analytical Performance of CSEI-Sweeping-CZE. The analysis of a mixture of divalent and trivalent metal ions in the 30 ppt level, under optimized conditions, is shown in Figure 9. Although peak 3 was not fully resolved from a system peak, clear

Figure 9. Analysis of ultra-trace-level metal ions by CSEIsweeping-CZE via dynamic complexation. Conditions: non-EDTABGS, 40 mM sodium acetate (pH 4.8); EDTA-BGS, 1 mM EDTA in 40 mM sodium acetate (pH 4.8); sample solutions, 30 ppt in water; injection schemes, the capillary was filled with non-EDTA-BGS and then 3.4 mm of water, followed by FESI of the sample solution for 200 s at 25 kV; sweeping and CZE voltage, -25 kV with the EDTABGS at the both ends of the capillary; capillary, 56.5 cm to the detector (60 cm total).

identification and high-sensitivity detection were achieved for all metal ions. In this experiment, we have paid particular attention to preventing contamination in all steps of the procedure, because this is very important in the ultra-trace-level analysis of metal ions. For the conditions stated in Figures 9, calibration lines, LODs, %RSDs, plates, and SEFheight are summarized in Table 4. The linearity of the present CSEI-sweeping technique was checked for peak height against concentration. Calibration lines with good linearity were achieved. We obtained the LODs of the test analytes in the range from 3.9 to 14.1 ppt, or from 2.4 × 10-11 to 25.2 × 10-11 M, with UV detection. This sensitivity is comparable to those of ICP-AES and ICPMS, the standard techniques for metal ion analysis. Even under optimized conditions, poor reproducibilities in peak heights (7.0-13.3%) and corrected peak areas (9.2-14.4%) Analytical Chemistry, Vol. 75, No. 24, December 15, 2003

6797

were obtained. This is because the amount of the samples injected into the capillary was not reproducible with a very long FESI time, probably due to sample depletion. An internal standard might be useful. CONCLUSION In the present work, we have shown that EDTA, as a complexing agent, can be used for on-line sample preconcentration by sweeping in CZE. The results indicate that the combination of on-line EDTA complexation and sweeping is effective for analysis of trace levels of metal ions. About 60- to 180-fold sensitivity enhancements were obtained compared to the sensitivity of conventional CZE with precapillary complexation, and the limits of detection were less than 23.4 × 10-8 M with sweeping. Furthermore, CSEI-sweeping allows the detection of ultratrace levels of some metal ions (e.g., ppt levels). From 50 000- to

6798

Analytical Chemistry, Vol. 75, No. 24, December 15, 2003

140 000-fold sensitivity enhancements were obtained, and the LODs of the studied metal ions were in the range (2.4-25.2) × 10-11 M. These techniques were developed using EDTA as a representative complexing agent. However, the principles of these techniques must be applicable to other metal ions. Even more sensitive analyses can be realized using alternative complexing agents. ACKNOWLEDGMENT The authors are grateful to Dr. J.-B. Kim for fruitful discussions. Special thanks are also given to Dr. P. Britz-Mckibbin and Miss M. R. N. Monton for their help in preparing the manuscript. Received for review June 22, 2003. Accepted October 16, 2003. AC034677R