Anal. Chem. 2002, 74, 5820-5825
On-Line Concentration of Acidic Compounds by Anion-Selective Exhaustive Injection-Sweeping-Micellar Electrokinetic Chromatography Lingyan Zhu, Chuanhong Tu, and Hian Kee Lee*
Department of Chemistry, National University of Singapore, 3 Science Drive 3, Republic of Singapore 117543
An easy, simple, and highly efficient on-line preconcentration method for acidic compounds in capillary electrophoresis was investigated. It combined two on-line concentration techniques, field-amplified sample injection (FASI) and sweeping. A low-pH (2.5) background electrolyte was used to suppress the electroosmotic flow (EOF), obviating the need of a coated capillary, as well as to neutralize the weakly acidic analytes. After injection of a plug of water inside the separation capillary, negative voltage was applied to initialize FASI for a much longer time than usual. The anions experienced a high electric field and moved quickly to the boundary of the water and the low-pH nonmicellar electrolyte. When the anions encountered the low-pH electrolyte, they were neutralized and a focused sample zone was formed. Then both inlet and outlet vials were changed to those containing the lowpH micellar background electrolyte. As negative voltage was applied, the anionic micelles moved into the capillary, and sweeping and separation began. The novelty in the present procedure is that a low-pH buffer is used to suppress the EOF and also the ionization of the analytes, without need of any other additives or use of a coated capillary. This method afforded 100 000-fold improvement in peak heights for some phenoxy acidic herbicides. The detection limits for these compounds could be low as 100 pg/mL. As a high-resolution separation technique, capillary electrophoresis (CE) is an alternative method to HPLC for analysis of different organic anions and cations.1-4 Its advantages over other separation techniques include high efficiencies, short analysis times, and low sample consumption. On the other hand, CE suffers from low concentration sensitivity due to the small amount of sample loaded into the capillary and the short path length for online UV detection. Capillary zone electrophoresis (CZE) is the simplest mode in CE. It has been widely used in the separation of charged ions, including inorganic and organic ions.5-7 The (1) Monning, C. A.; Kennedy, R. A. Anal. Chem. 1994, 66, 280R-314R. (2) Albert, M.; Debusschere, L.; Demesmay, C.; Rocca, J. L. J. Chromatogr., A 1992, 757, 281. (3) MeLaughlin, G. M.; Hauffe, A. J. Chromatogr., A 1996, 744, 123. (4) Chien, R. L.; Burgi, D. S. Anal. Chem. 1992, 64, 1046. (5) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302.
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introduction of micellar electrokinetic chromatography (MEKC) by Terabe8 extended the use of CE; neutral molecules, which cannot be separated by CZE, can be easily separated under MEKC. Apart from its ability to separate neutral molecules, MEKC may improve the separation of charged solutes as well.9,10 The same shortcomings of CE also apply to MEKC. Sample stacking is the most widely used on-line concentration method in CZE. Generally speaking, the ions that are electrophoretically migrating through a low-conductivity solution toward a high-conductivity solution slow dramatically and stack at the boundary of the two buffer solutions.11 This method has been widely applied to the analysis of anions, cations, or their mixtures.12-18 Both hydrodynamic injection and electrokinetic injection can be used for sample stacking. The latter is usually referred to as field-amplified sample injection (FASI). It has proven to be a very effective on-line concentration technique and provides much higher sensitivity improvements than hydrodynamic injection.19,20 Sweeping is a different on-line concentration method that is applied in MEKC. It was first described by Quirino and Terabe.21 The sample is prepared in a matrix having a resistance similar to that of the background solution. Also, the matrix is void of the pseudostationary phase used. The concentration effect relies on the ability of analytes to partition into the psedudostationary phase in MEKC.21-23 The higher the affinity of the analyte toward the pseudostationary phase, the greater the concentrating effect. (6) Jorgenson, J. W.; Lukacs, K. D. Science 1983, 222, 266. (7) McCormick, R. M. Anal. Chem. 1988, 60, 2322. (8) Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 111. (9) Otsuka, K.;; Terabe, S.; Ando, T. J. Chromatogr. 1985, 348, 39-47. (10) Quang, C.;; Strasters, J. K.; Khaledi, M. G. Anal. Chem. 1994, 66, 16461653. (11) Chien, R. L.; Burgi, D. S. Anal. Chem. 1992, 64, 489A. (12) Chien, R.; Burgi, D. S. Anal. Chem. 1991, 63, 2042. (13) Gebauer, P.; Thormann, W.; Bocek, P. Electrophoresis 1995, 16, 2039. (14) Quirino, J. P.; Terabe, S. Anal. Chem. 1998, 70, 1893-1901. (15) Quirino, J. P.; Terabe, S. J. Chromatogr., A 1999, 850, 339. (16) He, Y.; Lee, H. K. Anal. Chem. 1999, 71, 995-1001. (17) Zhang, C. X.; Thormann, W. Anal. Chem. 1998, 70, 540. (18) D Deforce, D. L.; Ryniers, F. P. K.; Van den Eeckhout, E. G.; Lemiere, F.; Esmans, E. L. Anal. Chem. 1996, 68, 3575. (19) Zhang, C. X.; Thormann, W. Anal. Chem. 1996, 68, 2523-2532. (20) Zhu, L. Y.; Lee, H. K. Anal. Chem. 2001, 73, 3065. (21) Quirino, J. P.; Terabe, S. Science 1998, 282, 465-468. (22) Quirino, J. P.; Terabe, S. Anal. Chem. 1999, 71, 1638-44. (23) Otsuka, K.; Vincent, J. B.; Vigh, G. J. Chromatogr., A 1999, 36, 323-329. 10.1021/ac020381u CCC: $22.00
© 2002 American Chemical Society Published on Web 10/22/2002
FASI was combined with sweeping in MEKC mode to concentrate cations on-line, a procedure described as cation-selective exhaustive injection-sweeping (CESI-sweeping).24,25 Many molecules of cations from a very dilute solution are electrokinetically injected and focused by sample stacking for a long period of time. The resulting stacked zones are then focused for a second time by sweeping using the psedudostationary phase. Focused zones are separated by MEKC. The same idea was applied to concentrate anions by Kim et al.26 They used a polyacrylamide-coated capillary to suppress the electroosmotic flow (EOF) and a cationic surfactant to perform the separation. Around 1000-6000-fold increase in detection sensitivity was obtained. In this paper, we demonstrated a 100 000-fold increase in concentration detection response for negatively charged analytes in CE. This was accomplished by combining FASI with sweeping under a low-pH background electrolyte. An uncoated fused-silica capillary was used. Several phenoxy acidic herbicides were used as the analytes. The EOF was suppressed by the low pH, and normal anionic sodium dodecyl sulfate (SDS)-MEKC was used for separation. We call the procedure anion-selective exhaustive injection and sweeping in MEKC using SDS (ASEI-sweepingMEKC-SDS). Anionic compounds, which were prepared in water or in basic matrix, were introduced by FASI for a considerable time immediately after injection of a water plug. The analytes would be neutral when their anions moved to the boundary between the water and the low-pH nonmicellar BGE. After FASI, the inlet vial was changed to one holding a low-pH buffer containing micellar BGE, and a negative voltage was applied to begin the sweeping and separation in MEKC mode. Different aspects regarding the improvement in on-line analyte concentration and analytical performance are discussed here. EXPERIMENTAL SECTION Apparatus. The experiments were performed on a Prince CE system (Prince Technologies, Emmen, The Netherlands). A 70 cm × 50 µm i.d. bare fused-silica capillary (Polymicro Technologies, Phoenix, AZ) with a detector window at 55 cm from the inlet end was used for separation. The detector was a Lambda 1010 spectrophotometer (Bischoff, Leonberg, Germany). The detection wavelength was 210 nm. The data were collected and processed by a DaX data system (Prince Technologies). Conductance was measured using a conductivity meter (CM-115, Kyoto Electronics, Kyoto, Japan) Reagents and Solutions. A stock solution of SDS (AnalaR, BDH, Poole, England) was prepared at 200 mM. Urea, purchased from Merck (Darmstadt, Germany), was prepared as a 10 M stock solution. Phosphoric acid (H3PO4) was purchased from AnalytiCals (Carlo Erba, Milan, Italy). The BGE was prepared fresh daily by dilution of phosphoric acid, SDS, and urea stock solutions in ultrapure water made by a Nanopure system (Barnstead Thermolyne Corp., Dubuque, IA). 4-(2,4-Dichlorophenoxy) butyric acid (2,4-DB), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2-(2,4,5-trichlorophenoxy)propionic acid (Fenoprop), and 4-chlorophenoxyacetic acid (4-CPA) were bought from Aldrich (Milwaukee, WI). 2,4-Dichlorophenoxyacetic (24) Quirino, J. P.; Terabe, S. Anal. Chem. 2000, 72, 1023. (25) Quirino, J. P.; Iwai, K.; Otsuka, K.; Terabe, S. Electrophoresis 2000, 21, 2899-2903. (26) Kim, J. B.; Osua, K.; Terabe, S. J. Chromatogr., A 2001, 932, 129-137.
acid (2,4-D) and 2-(2,4-dichlorophenoxy) propionic acid (Dichlorprop) were bought from Fluka AG (Buchs, Switzerland). 2-(4Chloro-2-methylphenoxy)butyric acid (Mecoprop) was from Accustandards (New Haven, CT). Phenoxy acidic herbicide standards were prepared in methanol as 1000 µg/mL stock solutions and then diluted to give working solutions at different concentrations with ultrapure water. All solutions were filtered through 0.45-µm filters prior to CE experiments. General Electrophoresis Procedure. The separation capillary was preconditioned prior to use with 1 M NaOH for 20 min, followed by 0.2 M NaOH for 20 min, then purified water for 20 min, and finally the nonmicellar BGE for 5 min. First, a certain plug length of water was hydrodynamically injected at the inlet. Thereafter, electrokinetic injection of the sample was performed at negative polarity with the nonmicellar BGS at the outlet end of the capillary. Negative voltage was then applied with the micellar BGE at both ends of the capillary for subsequent separation. The capillary was flushed using 1 M NaOH, 0.2 M NaOH, and water for 1 min, respectively, between runs. The nonmicellar buffer solution was 100 mM H3PO4/20% acetonitrile/1 M urea (pH 2.5). The micellar buffer used in this study was 25 mM H3PO4/75 mM SDS/20% acetonitrile/1 M urea (pH 2.5). RESULTS AND DISCUSSION ASEI-Sweep-MEKC Model. The main idea of this work is illustrated in Figure 1. The column is a bare fused-silica capillary that is initially conditioned with the low-pH nonmicellar electrolyte, which had a conductivity similar to the micellar BGE. The eletroosmotic flow was suppressed only by the low pH (2.5). A long plug of water was injected hydrodynamically into the capillary before sample injection (Figure 1a). The current was very low at this stage due to the water plug inside. Thereafter, the anionic sample prepared in water was injected electrokinetically into the capillary for a period of time much longer than usual for FASI (e.g., 10 min). With the application of negative voltage (the anode is at the outlet end), the anions moved rapidly into the capillary across the water plug toward the boundary of water and BGE and were neutralized when they entered the low-pH BGE zone (Figure 1c). A zone of neutral sample was created, which has much higher concentration than the original sample and is represented by the area A in Figure 1c. The water plug was moving out of the capillary from the inlet during the injection procedure because the EOF (toward the inlet) was controlled by the water plug (Figure 1d). This is indicated by the gradual increase of current. When the current was stable, the inlet and outlet ends of the capillary were then transferred to the vial containing the BGE, and a negative voltage (-20 kV, positive at the outlet) was applied (Figure 1e). The anionic micelles entered the sample zone, and sweeping and separation were then achieved via MEKC (Figure 1f). The dark bands in the figure represent plugs of analytes. The low pH of the electrolyte is essential for this preconcentration method. It plays two roles. By suppressing the EOF with low pH, the water plug would be pumped out of the capillary by the electroosmotic pump during the sample injection, and sweeping could be performed for a second preconcentration step. Also, Analytical Chemistry, Vol. 74, No. 22, November 15, 2002
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Figure 1. Schematic illustration of the anionic sample stacking mechanism of the ASEI-sweeping-MEKC model. Q represents phenoxy acid herbicide anion; (a) after filling the capillary with low-pH nonmicellar electrolyte, a water plug is injected into the capillary to provide the high electric field at the injection point; (b) negative voltage (-20 kV) is applied with positive electrode at the capillary outlet, and the sample is electrokinetically injected into the capillary. Due to the high electric field, the anions move rapidly toward the outlet. At the same time, the water plug is moving out of the inlet of the capillary; (c) when the sample anions enter the boundary of water and low-pH BGE, they are neutralized and cease moving. A focused sample zone is formed (shaded area A); (d) injection is halted and both vials at inlet and outlet are changed to low-pH micellar BGE; (e) negative potential -20 kV is applied to permit the micelles to enter the capillary and sweep the focused sample zone as a narrow band. The water plug continues to move out of the inlet; (f) subsequent separation is achieved under MEKC mode. The dark bands represent three components being separated.
the injection anions could be neutralized by the low pH electrolyte and immobilized as a sample zone. Study of the Stacked Zones. After electrokinetic sample injection, a sample zone formed in the capillary. We investigated the sample zone by pushing the sample out by pressure after sample injection over different injection durations. From Figure 2, it can be seen that most of the samples were focused at the boundary between the water and the nonmicellar electrolyte, illustrated by the positive peak 1: the higher the positive peak, the higher the sample concentration. A small part of the sample went into the nonmicellar BGE due to dispersion. Hence, in the nonmicellar BGE, a zone of sample molecules with nonhomogeneous concentration was formed. The injection duration was 0.5, 2.0, 6.0, and 10 min, respectively, at -20 kV in Figure 2A-D. Therefore, the length of the sample zone (L) and the focused sample concentration adjacent to the boundary increases with injection duration. To maintain good repeatability, the sample solution should be refreshed for each injection. The negative peaks in Figure 2A and B represent water plugs that had not been removed from the inlet end when the sample injection was halted. There was no negative peak present in Figure 2C and D, implying that when the injection was long enough, the water plug was removed completely. On the other hand, the peak in Figure 2D is much higher than that in 5822
Analytical Chemistry, Vol. 74, No. 22, November 15, 2002
Figure 2C. It can be concluded that even though the water plug had been removed from the capillary, the nonmicellar electrolyte was still retained in the capillary, and the focused sample concentration could be increased by further sample injection. However, in the latter case, the repeatability was unsatisfactory. To improve the repeatability of this method, it was advantageous to maintain a small water plug inside the capillary before the inlet vial was changed to one containing micellar BGE. Duration of Injection Time. As the stacked sample zone was produced by the electrokinetic injection, the length of sample zone was dependent on the injection duration. The longer the injection duration, the longer the sample plug zone. Consequently, the swept peak height should be higher but up to a point. As shown in Figure 3, for separate, fresh samples at a concentration of 100 ng/mL each, the peak height increased with injection duration up to 12 min. With the injection duration increased beyond 12 min, no increase in peak height was observed. At the concentration used for this experiment, all of the anions were loaded into the capillary by the 12-min injection. Thus, with injection duration beyond 12 min (e.g., 14-min injection as shown), there was no increase in peak height. Additionally, due to the long duration of electrokinetic injection, the concentration of the ions in the sample vial decreased progressively for repetitive injections, as shown in Figure 4. For
Figure 2. Study of the focused sample zone. After hydrodynamic injection of a 1.65-cm pure water plug, FASI was operated at -20 kV for a certain period of time. The focused sample zone was then pushed out by a pressure of 200 mbar. Sample concentration, 100 ng/mL; injection time, (A) 0.5, (B) 2.0, (C) 6.0, and (D) 10.0 min. L, length of sample zone.
Figure 3. Effect of injection duration on the peak height. Conditions: water plug (pure water), 3.31 cm; sample concentration, 100 ng/mL. Peak identification: (1) 2,4-DB; (2) 2,4,5-T; (3) fenoprop; (4) dichlorprop; (5) mecoprop; (6) 2,4-D; (7) 4-CPA.
this experiment, a 200-µL sample solution was repetitively injected, and the sample was thoroughly mixed after each injection. Figure 4 shows that the peak height decreased gradually, and no peak could be seen after five injections. Hence, to maintain good
Figure 4. Depletion of analyte anions in the sample solution after five consecutive runs of the ASEI-sweeping-MEKC procedure (using the peak height of 2,4,5-T as an example). Conditions: water plug length, 3.31 cm; injection time, 12 min at -20 kV.
repeatability, the sample solution should be refreshed for each analysis. Effect of the Water Plug. As mentioned by other groups,27-29 the water plug introduced before sample injection in FESI provides (27) Zhang, C. X.; Aebi, Y.; Thormann, W. Clin. Chem. 1996, 42, 1805. (28) Chien, R. L.; Burgi, D. S. J. Chromatogr. 1991, 559, 153-161.
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Table 1. Performance of ASEI-Sweeping-MEKC Methoda
2,4-DB 2,4,5-T fenoprop dichloroprop mecoprop 2,4-D 4-CPA
SE (ht)b
linear range (ng/mL)
RSD (%) peak ht
RSD (%) migration time
LOD (ng/mL)
no. of theor plates
95 000 78 000 78 500 82 000 85 000 103 000 110 000
1.0-250 1.0-250 1.0-250 1.5-250 1.5-250 1.5-250 1.0-250
5.14 3.99 3.54 3.70 4.04 6.50 6.99
1.56 2.06 2.04 1.74 1.41 1.34 2.68
0.1 0.1 0.2 0.2 0.2 0.5 0.2
1.12 × 106 1.03 × 106 7.41 × 105 1.05 × 106 8.41 × 105 5.54 × 105 5.22 × 105
a Sample was prepared in pure water. Injection conditions: water plug length, 4.96 cm; sample injection duration, 12 min. b SE (ht), Sensitivity enhancement in terms of peak height ) dilution factor × peak height (present method)/peak height (0.50 cm) injection.
Figure 5. Effect of adding acetonitrile to the water plug on the stacking. Conditions: water plug length, 3.31 cm; sample injection time, 10 min; sample concentration, 100 ng/mL. Composition of water plug: (A) 100% water; (B) 50% acetonitrile/50% water.
a higher electric field at the tip of the capillary, which improves stacking efficiency. The water plug in the present procedure here played the same role. Due to their low conductivity, acetonitrile and methanol were added into the sample solution by some workers to improve the concentration sensitivity.30-31 (29) Zhang, J.; Fang, Y.; Hou, J. Y.; Ren, H. J.; Jiang, R.; Roos, P.; Dovichi, N. J. Anal. Chem. 1995, 67, 4589-93. (30) Shihabi, Z. K. J. Chromatogr., A 1999, 853, 3-9.
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In this study, acetonitrile was added into the water plug to see whether it helped to enhance the stacking efficiency. The effect is shown in Figure 5. With all other conditions the same, when pure water was used, the result is shown in Figure 5A. When 50% acetonitrile/50% water was used, Figure 5B results. The peak height in Figure 5B is much higher than in Figure 5A. However, a water plug containing as high as 50% acetonitrile is liable to produce air bubbles in the capillary due to the large Joule heating generated. Therefore, 40% acetonitrile/60% water was used as the water plug in subsequent experiments. For the purpose of simplicity, subsequent mention of “water plug” below refers to this mixed acetonitrile/water plug. Length of the Water Plug. As mentioned before, it was necessary to keep a small plug of water inside the capillary before sweeping in order to maintain good repeatability. Additional experiments were needed to optimize the length of the water plug. After injection of different lengths of the water plug, electrokinetic injection was made at -20 kV for 12 min. The sample was pushed out by pressure. The appearance of a small reverse peak implied that the water plug was long enough to stay in the capillary after electrokinetic injection. In this study, a 4.96-cm water plug was used as the optimum length. Figure 6 shows the preconcentration effect of this method. Note that the sample concentration that was used to generate Figure 6A was 10 µg/mL, while that for Figure 6B was 10 000 times lower, i.e., 1.0 ng/mL. The peak height of Figure 6B is clearly much higher than that of Figure 6A. The system peaks that appear before the target peaks are a result of the change in composition of the liquid passing by the detection window. Qualitative and Quantitative Analysis. With the 4.96-cm water plug and injection duration of 12 min, the repeatability, linear range, and detection limits were investigated. The sensitivity enhancement factor, limit of detection values, linearity, repeatability of peak heights, and migration times for six replicate runs were calculated and are summarized in Table 1. The presence of the water plug (40% acetonitrile/60% water) inside the capillary, and perhaps the long injection time, improved the repeatability of the peak height (RSD
